Microbiology and Immunology Relevant to Dairy Safety and Human Health

A Critical Analysis of the Raw Milk Debate

Kevin Phillips BayDivision III, Spring 2011

Faculty Committee: Jason Tor, Associate Professor of Microbiology; Lynn Miller, Professor of Biology; and Chris Jarvis, Associate Professor of Cell Biology

A dissertation presented to the School of Natural ScienceHampshire College, Amherst, Massachusetts

In partial fulfillment of the requirements for the degree Bachelor of Arts

Table of Contents

Acknowledgements.....................................................................................................................1

Part I: Introduction to the Raw Milk Debate...........................................................................2

The Raw Milk Debate: What's at stake, what we know, and what we don't....................2

Political, Economic, and Emotional Issues Confound the Debate on Raw Milk.............2

Is There a Scientific Basis for Claims that Raw Milk Supports Health?.........................5

"Germ Theory" Dominates the Medical Paradigm of the 20th Century..........................10

Commensal Pathogens Highlight the Importance of Environment and Immunity..........12

Part II: The Enterococcus–A Probiotic Pathogen?.................................................................16

Characteristics and Identification of Enterococci...........................................................16

Habitats of Enterococci...................................................................................................17

Functionality of Enterococci in Dairy Fermentation......................................................19

Virulence Factors in Enterococci....................................................................................20

Regulation of Genes Encoding Virulence Factors..........................................................21

Diversity of Enterococcus spp. and Evolution of Virulence...........................................22

Gelatinase........................................................................................................................23

Capsule............................................................................................................................25

Biofilms...........................................................................................................................27

Aggregation Substance...................................................................................................29

Cytolysin.........................................................................................................................32

Horizontal Gene Transfer................................................................................................34

HGT, Virulence, and Antibiotic Resistance....................................................................36

Part III: Framing the Real Issues Behind the Raw Milk Debate..........................................40

What Makes an Enterococcus Pathogenic?....................................................................40

Dairy Foods and Issues of Hygiene................................................................................42

Farmstead Dairy and the Ecological Integration of Modern Communities....................48

Sources Cited (Parts I-III)........................................................................................................50

Part IV: Assessment of Gelatinase Activity in Enterococci Isolated from Local Milk.......58

Sources Cited (Part IV)............................................................................................................65

Appendix...................................................................................................................................66

Acknowledgements

I would like to offer thanks to my living family, particularly my father Keith and his wife Linda,

whose support of my pursuits has always kept me afloat. Also, to my deceased family, in particular my

mother Kathy, whose living memory has in so many ways inspired me to pursue an understanding of

what it means to be healthy.

Thanks to my friends, mod mates, and everyone in the Monday night potluck community. Thanks

to Claire Wiessbluth for helping me survive at Hampshire and find my way in the world. Thanks to

Maggie Grinnell for your loving support and perspective that has been invaluable in this past year.

Thanks to Dylan (Spring) for the light you have brought to my life through your music and friendship.

Many thanks to Luke William Gay, your persistent friendship and ability to challenge my perspectives

has been essential.

There are a number of NS faculty who are greatly deserving of thanks. Jason Tor, Lynn Miller,

and Chris Jarvis for your support, guidance, and overwhelming faith in my ability to direct my own

learning experience, even when I myself had doubts. Larry Winship for supporting my experience of

independently directed learning at Hampshire from the very beginning. Rayane Moreira for giving focus

and momentum to my fascination with the chemistry of life, and for cultivating a challenging academic

environment.

Thanks specifically to Jason for fostering the environment in which I could develop a strong

understanding of the relationship between food, bacteria, and health, and for gathering the cheese-lovers

of Hampshire together to form a community in which I was able to teach and be taught, to feed and be

fed, and to share in a feeling of togetherness with one common denominator: cultured milk.

Thanks to bare feet and dirt. Thanks to all the folks associated with FLPCI for reassuring me that

my dream of becoming an award winning cheesemaker is in fact a good idea. Thanks to Kate Clabby for

consistently engaging me in thought-provoking conversations about dairy. Thanks also to microbes

everywhere for continuing to do things that boggle my mind and giving me faith that what I can't

understand will always be central to my health and the health of the earth.

Last but certainly not least, thanks to Leslie Cox and the Dutch Belted cows at Hampshire

College Farm Center (especially Cookie) who aside from their zen-like stare, offered me an unbelievable

abundance of milk to marvel at and craft into creamy delights that could be shared with friends and

family as far away as Wisconsin. Milk is truly a sacred gift.

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Part I: Introduction to the Raw Milk Debate

The Raw Milk Debate: What's at stake, what we know, and what we don't

Modern dairy production and processing has changed significantly over the past 100 years

following technological advances that have made possible large-scale industrial agriculture and

centralized food production and distribution systems (Schmid, 2009, 203-206). In the past, it was

commonplace for minimally processed milk to be bottled on the farm and sold directly to the consumer.

Now, due to biased legislative regulations and a trend towards large-scale agriculture, small family

dairies have become increasingly rare. Farmers often have no choice but to sell their milk to large scale

processing and distribution facilities, and low milk prices have put increasing economic strain on dairy

farmers. Small-scale community and family farmers themselves express the concern that these pressures

put them in danger of being eliminated altogether (Ostrom and Buttel, 1999).

The demand for fresh minimally processed milk, however, has not gone away, and a slew of small-

scale, often unregulated farms using direct-to-consumer marketing have emerged to meet this demand,

including the demand for unpasteurized (raw) dairy products such as milk, cream, butter, and yogurt.

Pasteurization is simply a heat treatment process intended to eliminate the majority of the natural

microbiota present in milk, lowering the risk of illness outbreaks associated with milk-borne pathogens

(Gumpert, 2009, 17). The potential benefits and risks associated with consuming raw dairy products is a

hotly debated topic and an issue of increasing significance due to its involvement in a number of other

significant social, political, and health issues. These issues include disparities in power and resources;

the fundamental rights of farmers, consumers, and corporate entities; and economic, ecological, and

individual health. As a result of these complicating factors, much of the debate has been based on highly

biased interpretations of a relatively small number of controversial scientific studies. Confusion

surrounding the "true" benefits and risks associated with raw milk are amplified by the fact that the

diverse disciplines of nutrition, microbiology, biochemistry, and immunology have only begun to

unravel the mysteries of milk.

Political, Economic, and Emotional Issues Confound the Debate on Raw Milk

State and local government laws vary in regard to raw milk, but in recent years the FDA has taken

a strong stance against its production, sale, and consumption, arguing that it is inherently unsafe and

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provides no benefits over pasteurized milk (Gumpert, 2009, 103-104). Many state and local agricultural

regulators throughout the US have followed suit, and even in places where legislation allows for sale of

raw milk, fear based propaganda and strict regulations discourage the growth of the raw milk market. A

number of farms that produce raw dairy products for sale have been subject to stringent searches, sting

operations, and behaviors bordering on harassment by state and federal authorities despite the fact that in

most cases there is little to no evidence that the products produced on these farms pose a greater risk to

public health than any other food. The increased frequency of such interventions has prompted many

raw milk advocates to question the motivations of agencies such as the FDA cracking down on raw dairy

producers nationwide. Like any food, raw milk has the potential to carry food-borne pathogens,

especially if good manufacturing practices designed to decrease this risk are ignored. However, a critical

analysis of the various arguments for and against raw milk reveals that political, economic, and

emotional complications are really at the heart of what could be considered a decidedly biased and

irrational debate.

Federal guidelines for milk production are based on the Pasteurized Milk Ordinance (PMO), which

was updated last in 2007, and sets the national standards for production, processing, packaging and sale

of Grade "A" dairy products (FDA, 2007). These guidelines are an important part of food safety in the

highly automated and centralized food production system of the United States. Without them, it is likely

that negligence or ignorance regarding hygienic food production would lead to increased rates of acute

to severe cases of food-borne illness, but critical examination of this ordinance reveals that it does not

take account of consumer demands for minimally processed foods, and that its language and

interpretation is heavily influenced by the complicating factors involved in the raw milk debate.

First and foremost, these harm reduction guidelines apply only to milk intended for pasteurization,

and the document actually defines a "dairy farm" as a place with one or more lactating animal where

milk will be provided, sold, or offered for sale to a milk plant, receiving station or transfer station. That

is to say, unless you sell your milk to a processor, you don't even technically own a dairy farm according

to the PMO. The lengthy definition of "milk products" includes a wide variety of processed dairy foods,

but makes no mention of minimally processed alternatives such as non-homogenized, non-standardized,

low-heat vat pasteurized milk; nor (for obvious reasons) raw milk.

Other terms defined by the PMO bring up different issues. For example, the definition of "person...

include[s] any individual, milk plant operator, partnership, corporation, company, firm, trustee,

association or institution". This lumping together of individuals and corporate entities is indicative of the

PMO's attempt to set in place all-encompassing regulations, despite the fact that many of these

regulations are not ideal for most alternative models of dairy production. Taken in conjunction with their

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requirement that all dairy products be funneled through processing plants, it is clear that these

regulations are biased towards large-scale, centralized dairy distribution systems.

Finally, issue may be taken with the PMO's use of the term of "sanitization", which is defined as:

...the application of any effective method or substance to properly cleaned surfaces for the destruction of pathogens, and other microorganisms, as far as is practicable. Such treatment shall not adversely affect the equipment, the milk and/or milk product, or the health of consumers, and shall be acceptable to the Regulatory Agency.

This definition could be considered highly controversial from both a political and scientific standpoint.

First of all, it is important to look at the way the definition is concluded, that methods of sanitization

must be "acceptable to the Regulatory Agency". This essentially strips interpretive power from any

authority other than the FDA. A more in depth analysis of the effects of processing on milk included

later in this paper will show that the language preceding this statement is highly non-specific and the

scientific evidence supporting its conventional interpretation is inconclusive, or even contradictory.

Federal regulations disregard direct-to-consumer agricultural models as well as procedural

guidelines for hygienic milking practices on farms providing raw milk not intended for pasteurization,

and so state lawmakers as well as smaller localized political bodies and individual activists have stepped

in to fill these gaps (Schmid, 2009, 411-424). Alternative agricultural models such as cow-shares, in

which farmers and consumers organize to co-operatively distribute food within their own community,

allow conscientious individuals to opt out of regulated food systems. The observation that these markets

are then out of reach of the centralized dairy industry has led raw milk advocates to suggest that industry

profits are likely a significant motivator in the federal stance against raw milk, which is backed by

legislation that has been heavily influenced by industry lobbyists.

In spite of political considerations, though, the way that raw milk enflames the passions of parties

on both sides of the debate is indicative of the emotional issues that are at stake. For example, the

emergence of agricultural models that circumvent the authority of regulatory bodies may be personally

offensive to individuals employed by organizations such as the FDA and similar state-run organizations.

On the other hand, many consumers see interventions by regulatory bodies as an infringement of their

basic rights to consume the foods of their choice or to have access to foods they feel are healthy in what

some consider an increasingly over-processed and nutrient depleted food system.

In response to these concerns regarding consumer rights and increasing support for raw milk,

bureaucratic authorities have made statements that may be perceived as crass and personally offensive

by consumers, such as the FDA's John Sheehan, who coined the statement "drinking raw milk or eating

raw milk products is like playing russian roulette with your health" (Gumpert, 2009, 116). Such

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commentary has only exacerbated the turmoil surrounding this highly emotional issue. In an example

more deeply concerning to some consumers, a lengthy legal document prepared by FDA lawyers as part

of a suit filed by the Farm-to-Consumer Legal Defense Fund challenging the FDA ban on interstate

shipment of raw milk includes the statements "there is no absolute right to consume or feed children any

particular kind of food" and "there is no generalized right to bodily and physical health" (Sebelius and

Hamburg, 2010). The document is intended to refute claims that the ban infringes on fundamental

consumer rights, and these statements appear as headings a. and b. under section IV.–B.–4., entitled

"FDA's Regulations Do Not Infringe Upon Substantive Due Process Rights." The statements are then

followed by a detailed legal analysis defending their validity. Many consumers feel that the statements

go too far by restricting the free choices of responsible adults in the name of public health, but these

statements can actually be legally validated by the language of current federal regulations (Falkenstein,

2010).

Only time will tell whether increasing public awareness will result in legal reforms regarding the

rights of communities to produce and distribute foods outside the context of the centralized food

industry and its associated regulatory authorities. In the meantime, some community government bodies

have taken a more localized approach. Since March of 2011, several towns in Maine have voted on

ordinances that would make it unlawful for state or federal regulations to interfere with the rights of

their citizens to produce, process, sell, purchase, and consume local foods of their choice, and exempting

local food producers from licensure and inspection under the condition that their products are sold

directly to consumers for home consumption (Local Food and Community Self-Governance Ordinance,

2011). While the town of Brooksville, ME did not pass the ordinance, citing concerns that "it is

unenforceable" and "opens the town to potential liability issues and legal costs", three other towns

(Sedgewick, Penobscot, and Blue Hill) passed the ordinance unanimously (Gumpert, 2011[2 and 3]), a

powerful message to federal and state regulators that faithful consumers may not sit quietly on the

sidelines as interventions on private farm to consumer transactions threaten their trusted local food

sources.

Is There a Scientific Basis for Claims That Raw Milk Supports Health?

At the heart of the raw milk debate is the claim that it is fundamentally different from pasteurized

milk in its ability to support natural immunity and general health. Raw milk advocates expound the

miraculous health benefits of their favorite drink, citing anecdotal reports that it can help treat a variety

of serious medical issues including (but not limited to) arthritis, eczema, asthma, cancer, and diabetes

(Gumpert, 2009, 84-90). The same anecdotal reports often claim that pasteurized milk delivers no such

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benefits, and on the contrary often causes relapse into poor health, digestive issues, and lactose

intolerance. Finally, they also claim that raw milk has natural biological defenses that confer protection

against the growth of pathogenic organisms. But if these claims are to be considered true, the question

must be asked, what is it about raw milk that makes it so different from pasteurized milk?

In order to understand the fundamental differences between raw and pasteurized milk, it is

necessary to understand the various components of milk. Unfortunately, the composition of milk

remains a larger scientific mystery that one might imagine. Although milk may seem like a homogenous

white substance, the complexity and diversity of biological structures and the various ways they can

interact with the human body has only begun to be unravelled. Most people, if they consider milk on a

molecular level at all, would probably identify the main components as protein, fat, lactose, vitamins,

and minerals. In reality, there are many other bioactive components of milk (see figure 1) including

immunoglobulins, peptides, antimicrobial factors, hormones, growth factors, and approximately 70

indigenous enzymes (Silanikove, 2008).

Milk also contains living cells, including bacteria from contamination during milking, and active

immune cells. These living cells contribute to milk their own biological structures and secretions,

including potent immune modulating substances such as cytokines (Untalan et al., 2009). In addition to

membranes associated with bacterial and somatic cells, milk contains phospholipid membranes complete

with diverse bioactive membrane proteins. These include the milk-fat globule membrane (MFGM)

which contains proteins involved in immune functioning (Cavaletto et al., 2008) due in part to the

exocytosis of milk fat globules from lacteal cells, and the milk serum lipoprotein membrane vesicles, the

origin and function of which remains a mystery (Silanikove, 2008).

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Figure 1 - Image and text adapted from Silanikove (2008). Defines the 5 phases of milk and models their relationships and relative sizes.

All these diverse components are arranged in a highly organized fashion that can be grossly

divided into 5 distinct phases. These are displayed and defined clearly by Silanikove (2008) as shown in

Figure 1. While all the gross structural components have likely been identified, many of them have only

begun to be characterized. The functional contribution of most of these bioactive factors to milk and the

influence these may have on the health of dairy consumers is a highly complex topic that science has

barely begun to explore. It is beyond the scope of this paper to provide an in depth analysis on the

subject, but it remains a topic of great interest and a promising area of future research in dairy science.

Clearly, there is important information missing from the analysis of the biological activities of

milk. For this reason, it must be kept in mind that any scientific argument for or against the consumption

of milk (raw, pasteurized or otherwise processed) by a particular individual is vastly speculative, and

may well be a decision that is better informed by that individual's intuitive sense of which consumptive

patterns make them feel healthy. That being said, a hypothesis that raw milk may be beneficial to the

health of some individuals can be formed based on anecdotal reports, and some recent scientific findings

may support this hypothesis.

Research into the beneficial or deleterious effects of

pasteurization is heavily swayed by the agendas of its financiers,

and authorities such as the FDA vehemently deny the suggestion

that pasteurization may have an adverse effect on the health

benefits of milk, as exhibited by the chart in figure 2 taken from

the FDA's website. In an attempt to explain and validate the

deleterious effects of pasteurization, raw milk advocates most

frequently cite decreased bioavailability of nutrients,

denaturation of enzymes claimed to be vital to health, and the

elimination of naturally occurring beneficial bacteria that confer

a probiotic and immunoregulatory effect. While it is clear that

pasteurization does have some effects on these properties, these

effects are argued to be negligible (Cifelli et al., 2010).

It is true that there is not a cohesive and conclusive body of literature to support the claims of

raw milk advocates, perhaps in large part due to the fact that funding for such research is difficult to

obtain. The FDA's assertion that these claims have been conclusively refuted, however, is not accurate.

Research on this topic will probably continue to be muddled by the complicating factors of the raw milk

debate. On the other hand, research on human breast milk gives a very different and interesting

perspective on biological activities of milk, and although these results cannot be directly translated to

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Figure 2 - A side bar from the FDA's website addressing common misconceptions about pasteurization and raw milk (FDA, 2011).

consumption of cow or goat milk, they do present provocative conclusions that could inform potential

areas of interest in researching the influences of milk production and processing methods on consumer

health.

Breast milk is ubiquitously considered to be the ideal food for human infants. Some mothers,

however, may be unable to breast feed and for this purpose, "donor milk" is collected and provided at

milk banks, where it is often pasteurized to

avoid transmission of infectious disease by

microbes (Untalan et al., 2009). Viruses such

as human immunodeficiency virus (HIV) are

of particular concern (Tully et al., 2001).

While donor milk is regarded as a better

alternative than formula, the potential

deleterious effects of pasteurization on the

immune components of human milk has been

a subject of significant research. B- and T-cell

populations are entirely abolished by

pasteurization, and immunoglobulins and enzymes associated with the bacteriostatic properties of raw

milk are significantly affected as well. As a result of these effects, Tulley et al. (2001) note that

"microorganisms that could contaminate the milk after pasteurization will grow faster than they can in

raw milk". That raw milk has natural defenses against microbial growth is an argument often cited by

raw milk advocates. Although many advocates take the argument too far, asserting that raw milk actually

kills off pathogenic populations and that pathogens cannot grow in raw milk, it is clear that the natural

immune components of raw milk do have a

bacteriostatic effect.

More recently, the effect of

pasteurization on cytokines in donor milk has

been explored, and these results have shown

that pasteurization does have significant effects

on many of these potent immunomodulating

molecules (Ewaschuk et al., 2011; Untalan et

al., 2009). Ewaschuk et al. (2011) effectively

show the differential effect of pasteurization

on a wide variety of cytokines and other

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Figure 3 - Relative concentrations of various cytokines detected in raw (black) and pasteurized (white) breast milk (Unger, 2011).

Figure 4 - Relative concentrations of Heparin-binding-EGF-like growth factor (HB-EGF), Hepatocyte growth factor (HGF) and Granulocyte colony-stimulating factor (G-CSF) in raw (black) and pasteurized (white) breast milk (Unger, 2011).

molecular immune components. Their results are currently available in an advanced online publication,

but not through research databases. Some of their results, however, are ironically available through the

FDA website as part of a slide show recently prepared in support of donor milk by Sharon Unger, MD (a

Toronto physician and member of the Ewaschuk et al. research team). These results, displayed in figures

3 and 4, show an altered cytokine profile and significant reduction in hepatocyte growth factor (HGF).

Other immune components in human milk have been described as well, although the effect of

pasteurization on these has yet to be studied. Soluble forms of toll-like receptors (TLRs; pattern

recognition receptors that play a major role in identification of pathogens by the innate immune system)

as well as TLR co-receptor CD14 are found in high levels in human milk and modulate neonatal

microbial recognition (LeBouder et al., 2006). Additionally, nanovesicles such as milk serum lipoprotein

membrane vesicles (the "lost continent" of Silanikove, (2008)) have been isolated from human milk and

are suspected to have immunological effects, in part due to the fact that they can be taken up by

macrophages and contain functional RNA that can be delivered to other cells (Lässer et al., 2011).

Research on immune components in non-human milk does exist (Mehra et al., 2006; Trujillo et

al., 2007), but it is extremely limited. However, it is still very clear that in light of the results of studies

on human breast milk, the argument that pasteurization does not have a significant effect on the

biological properties of raw milk must be called into question. Additionally, the recently proposed

"hygiene hypothesis" suggests that excessive cleanliness may contribute to higher rates of health issues

such as allergy and asthma in developed geographic regions, and one review article notes that several

studies have shown unpasteurized milk to have "a protective effect... on the development of asthma, hay

fever, allergic sensitization, and atopic dermatitis" (von Mutius and Vercelli, 2010). The same authors

also suggest that homogenization may play a role in reducing milk's "asthma- and allergy-protective

effects" due to the disruption of the MFGM, which causes adsorption of allergenic proteins onto the

newly formed milk-fat globules as a result of their increased surface area. In fact, increasing rates of

asthma and allergic disease correlate more closely to the advent of homogenization than pasteurization

(Gumpert, 2009, 111).

Other authors have also suggested that practices aiming to eliminate the natural microbiota of

foods may decrease the ability of the GI microbiota to adapt to novel foods due to decreased frequency

of horizontal gene transfer (HGT) between natural food microbiota. These indigenous food microbes

have evolved the metabolic capacity to make best use of the resources in their environment, and GI

microbiota have the potential to acquire genes encoding these metabolic pathways (Sonnenburg, 2010).

This type of HGT was demonstrated by Hehemann et al. (2010), who observed that Bacteroides plebius

populations in Japanese individuals (who typically consume large quantities of seaweed in the form of

9

sushi) had acquired genes from the marine bacterium Zobellia galactanivorans that allowed them to

digest porphyran, a polysaccharide found in red algae. These genes were not found in the microbiota of

individuals from the US. These data lend some credence to the claim that raw milk may attenuate

symptoms of lactose intolerance, while pasteurized milk provides no such benefits.

There is scientific evidence warranting further research on the potential health benefits of

consuming minimally processed milk. Unfortunately, some statements made in defense of the safety of

raw milk are detrimental to the credibility of raw milk advocates (Blum, 2010). Extremist assertions

such as "the bacteria theory's a total myth" and "everything God designed is good for you" are easily

ridiculed by authors who perceive raw milk as nothing more than the latest health fad or foodie fetish.

These statements may also be perceived as personally offensive to victims of life threatening food-borne

illness and their families who have undergone highly traumatic experiences in which microbial

pathogens do play an important role. On both sides of the debate, oversimplification and

misunderstanding of the microbiological and physiological underpinnings of disease, and far-reaching

political, social, and emotional issues surrounding raw milk cultivate an environment that is not

conducive to rational assessment of the facts.

"Germ Theory" Dominates the Medical Paradigm of the 20th Century

Since the work of Louis Pasteur (for which the process of pasteurization is named) in the 19th

century that led to technologies with miraculous efficacy in controlling or nearly eradicating many

rampant infectious diseases of his time, including debilitating milk-borne illnesses such as tuberculosis

and diptheria, much of the world has taken for granted the idea that disease is caused by microbial

pathogens (Gumpert, 2009, 42-46). Germ theory, however, was not the only theory of pathogenesis at

the time, and some of Pasteur's contemporaries and co-researchers such as Claude Bernard and Elie

Metchnikoff proposed significant theories suggesting that the internal environment of the body as well

as the body's cellular defense systems are also important factors in susceptibility to illness.

The groundbreaking work of French-American microbiologist René Dubos, which has not

received due respect from many modern microbiologists, was largely dedicated to exploring the impact

of environmental conditions on microbial growth and pathogenesis. His first wife died of tuberculosis in

1942, which ignited in him a passionate desire to understand the reasons why the illness had developed

in her at that time (Encyclopedia.com, 2003). His investigation revealed that she had been infected with

tuberculosis as a child. Despite overcoming the illness in her youth, a latent infection remained. Dubos

was personally convinced that distress surrounding World War II and her concern for her family in

France had weakened her and allowed the latent infection to again take hold. Much of his subsequent

10

work was based on the theory that diverse environmental conditions such as poor nutrition, pollution,

psychological stress, and spiritual deprivation are all important etiological factors in human disease.

These ideas eventually brought him out of the lab to work with economically depressed communities

and to speak out on important social issues such as economic disparity and environmental injustice. A

1966 study by Dubos that was republished in 2005 elegantly displays the significance of quality of life

in resistance to infectious disease (Dubos et al., 2005).

Dubos was also highly influenced by the work of Pasteur, and notes that in spite of the focus of

Pasteur's experimental research, his conception of pathogenesis was actually much more sophisticated,

and took into account the significance of environment (Dubos, 1974). Although the pressing issues of

infectious disease and vaccination came to monopolize his research efforts, Dubos suggests that

Pasteur's early discoveries could just as easily have led him into a diversity of scientific fields including

microbial physiology or the effect of environmental conditions on disease resistance. Dubos provides

compelling evidence from Pasteur's early work to support these arguments, such as the simple

observation that the gut is lined with a multitude of microbial agents that only cause illness when the

body is weakened, or that most individuals do not develop post-surgical infections despite occasional

neglect of aseptic methods. Furthermore, in Pasteur's work on flacherie (an infectious disease in

silkworms), he observes the influence of environmental conditions on resistance to the disease. Dubos

states, "Pasteur considered that excessive heat and humidity, inadequate aeration, stormy weather, and

poor food were inimical to the general physiological health of the insects. As he put it, the proliferation

of microorganisms in the intestinal tract of worms suffering from flacherie was more an effect than a

cause of the disease."

These ideas reveal a much more complex picture of pathogenesis involving several variable

factors. In contrast, germ theory involves only one variable (the presence or absence of pathogenic

microbes) and is therefore more easily studied in a scientific context, and more easily understood by the

general public. In addition, it provided a quick fix to many serious medical issues at the time of its

emergence, further contributing to its popularity in both the medical and lay communities.

Assertions by raw milk advocates that pasteurization may have negative consequences to public

health and that bacterial contamination of raw milk is actually central to its health-giving properties

challenge the deeply held views of many parties (especially within the industrial, scientific, and medical

communities) regarding the origin of disease and what steps must be taken to prevent it. Yet even

Pasteur was not so dogmatic about germ theory, and his personal conception of pathogenesis was able

to account for the observation that not all exposures to pathogenic microbes result in illness. This

concept is exemplified by Gumpert (2009, 126), who reports on a 1987 case study of campylobacter

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associated with raw milk:

Raw milk proponents have another answer to the data and reports showing that people do become ill from raw milk. They argue that any dangers from infection by pathogens can be reduced significantly by regularly consuming raw milk, thereby building up immunity. They point to a 1987 case study of thirty-one freshmen fraternity pledges who, in the fall of 1982, went on a retreat to a large dairy farm owned by the parents of one member. Over the next ten days, nineteen of thirty-one students developed gastrointestinal illness and were found to have campylobacter, a common source of food poisoning. Three others without symptoms were also found to be infected. Interestingly, ten individuals who consumed the tainted milk and showed no signs of illness or infection–a few students and some farmhands–were found to be regular consumers of raw milk.

The authors of this study were able to directly correlate raw milk consumption with immunity to

Campylobacter jeujuni as well as levels of C. jeujuni specific antibodies (Blaser et al., 1987). This case

highlights the influence of individual variation and regular exposure to pathogens on immunity, as well

as the fact that individuals can be exposed to or even colonized by pathogenic organisms without

showing symptoms of illness. For these reasons, it provides provocative data in the debate on raw milk.

Although it is an isolated incident, and similar research has not been produced since, there are vast

bodies of scientific literature in the field of microbiology that can further inform the concepts brought to

light by this incident.

Commensal Pathogens Highlight the Importance of Environment and Immunity

It is only in recent years that technological advances have enabled scientific research to begin to

characterize the complex interactions involved in the pathogenesis of infectious disease. Bacterial

populations are transient and adaptable, and many of their phenotypic characteristics are highly

dependent on environmental conditions, which select for certain traits over time (Ehrlich et al., 2008), or

modulate genetic expression of existing populations (Hew et al., 2007). Ehrlich et al. (2008) make the

important observation that many virulence traits evolve under multiple evolutionary pressures, which

typically have nothing to do with host pathogenesis. The authors suggest it is "likely that many

pathogens did not initially evolve as pathogens, but simply take on this role as a result of a lack of

ability of the host to maintain homeostasis." The best examples of this type of evolution are the so-called

'commensal pathogens', which are typical members of the microbiota of normal healthy humans, but

under select circumstances can act as pathogens. These examples can give some insight into the

significant environmental factors associated with pathogenesis.

One such example is the bacterial genus Enterococcus, which encompasses a variety of species

that can occasionally act as pathogens, but are more commonly found as ubiquitous gastrointestinal

commensals, environmental colonizers, and food fermentors (Franz et al. 1999). Typically, enterococcal

12

infections occur in hospitals, where intrinsic and acquired antibiotic resistances harbored by many

strains of this genus give them a strong advantage over other bacteria (Gilmore and Ferretti, 2003).

Since enterococci are found ubiquitously in dairy products and other foods, and in especially high

numbers in traditional European aged raw milk cheeses, food safety concerns have been raised by the

scientific community (Franz et al., 2003). The turbulent political climate surrounding raw milk is also

the backdrop for the highly speculative debate regarding the safety of enterococci in foods. Research

directly addressing the potential risks of enterococci in raw milk and raw milk products have been

inconclusive, but continue in spite of overwhelming anecdotal evidence that their presence is not

problematic.

On the other hand, research into the probiotic capability of enterococci has been so successful

that probiotics such as Symbioflor® 1 (in which E. faecalis, the species of Enterococcus responsible for

90% of human infections (Domann et al., 2007) is the only bacterial species) have emerged for human

use. The instructions for use of Symbioflor® 1 indicate its use for "immunomodulation, chronically

recurrent infections of the upper respiratory passages, inflammations in the mouth, nose, pharynx, and

middle ear, colds, and disorders of the gastrointestinal function" (Instructions for Use Symbioflor® 1,

2002). The instructions also assert that there are no contraindications for its use nor any significant side

effects aside from isolated incidents of dry mouth, headache and stomach pain, and in the case that side

effects do occur, do not suggest discontinued use, but rather only a decrease in dosage. The product has

been on the market for over 50 years without any reported cases of infection (Vebø et al., 2010).

The vast majority of research associated with enterococci has focused on their emergence as

infectious agents in the hospital setting, where concerns surrounding their impact on health are more

than speculative (Fisher et al., 2009). There is a significant body of literature connecting the presence of

enterococci in foods and their roles in hospital acquired (nosocomial) infection and dissemination of

antibiotic resistance, and in one case a solid link between agricultural practices and antibiotic resistance

was established, resulting in far-reaching legislative and agricultural changes across Europe (van den

Bogaard et al., 2000). Critical examination of the two bodies of literature show that the complex

relationship between these two worlds is not well characterized, but that agricultural practices,

especially regarding hygiene and animal health, may be subjects of interest.

Unfortunately, studies on enterococci in food have not focused on agricultural practices and have

instead been preoccupied with characterizing the phenotypic traits associated with virulence (Semedo et

al., 2003; Lopes et al., 2006; Domann et al., 2007; Hew et al., 2007). Although life threatening cases of

enterococcal infection are rare, the very real ability of enterococci to infect hospital patients and cause

potentially fatal complications such as endocarditis (Chuang et al., 2009) stands in stark contrast to their

13

functional contribution to foods, their role as members of the commensal microbiota, and their ability to

confer a probiotic effect. For this reason, I suggest that the enterococci represent an example even more

perplexing than that of the commensal pathogens, and may more adequately be termed a 'probiotic

pathogen.'

Such apparent paradoxes have caused modern thinkers to entirely reconsider the scientific

underpinnings of what makes a pathogen pathogenic (Ehrlich et al., 2008). While the idea that pathogens

are not necessarily the root cause of disease may seem absurd to the average individual raised on the

dogma of germ theory, and government policy makers seem unwilling to even pay lip-service to its

potential validity, modern scientific research in microbiology and immunology lends support to this

newly emerging view. It may be unreasonable to claim that bacteria cannot cause illness, but it is also

unreasonable to claim that they are the sole agents of disease, as environmental, physiological and

immunological factors are now recognized to play a significant role in the pathology of most, if not all,

illnesses. An acute awareness of this neglected complexity has led some consumers to a radical line of

questioning that echoes the forgotten voices great microbiologists such as Dubos, and even Pasteur

himself, accounting for the other side of the story in examining the etiology of disease. The following

excerpt, which is contained in a legal document prepared defending accessibility of raw milk to Los

Angeles, CA consumers, is a good example of such questioning:

Are pathogens the instigators or the consequence of degenerative disease? Are they the cause or the cure? Is pointing the finger at microbes a distraction from true causes of disease? Is pollution of our food, water, air and medicine the predominant cause of disease, which then fosters bacterial growth? All hypotheses must be open to independent testing and researchers held accountable to the rules of evidence (Vonderplanitz and Douglass, 2001).

It may seem obvious that pathogens can cause disease, but generalized and oversimplified

assertions made on all sides of the raw milk debate reveal a significant gap in popular scientific

knowledge regarding dairy foods, microbiology, and pathogenesis. The general public is no longer

willing to accept the pretense that germ theory can solve all of our health problems, and rightfully so, as

the scientific community has known for some time that it is only one part of the highly complex and

diverse biological interactions that can lead to illness. Further study is necessary to develop more

inclusive models of pathogenesis, but it may be necessary to take a step back before we take a step

forward. By critical examination of current research in microbiology and immunology, we may be able

to distill more comprehensive and realistic models of pathogenesis. Such models will be of great value

in dispelling the misconceptions of the general public, of farmers, of regulatory authorities, of

legislators, and even of other scientists, so that we may all as a global community move forward to more

14

effectively and efficiently support the health of ourselves, our loved ones, and the social and ecological

systems we engage with every single day.

15

Part II: The Enterococcus–A Probiotic Pathogen?

Characteristics and Identification of Enterococci

The enterococci are Gram-positive cocci commonly found as commensal organisms in the

human gastro-intestinal (GI) tract (Tannock and Cook, 2002). They are also ubiquitous environmental

organisms and are associated with a number of fermented and processed food products (Hew, 2008).

They are facultative anaerobes displaying little to no catalase activity. Optimal growth occurs at 35C,

but growth can occur within a wide temperature range from 10 - 45C. As a result of adaptation to

environments such as fermented foods and the mammalian GI tract, enterococci can survive or grow in

challenging conditions. For example, they grow in 6.5% NaCl concentrations, and according to one

account can survive or grow in broth containing 27% NaCl (Huycke, 2002). They can also grow in up

to 40% bile salts (Facklam, et al., 2002), and in a range of acidity from pH 4.8-9.6 (Huycke, 2002). They

are reported to be highly thermotolerant and are known to withstand temperatures of 60C for up to 30

minutes (Ahmad et al., 2002). Other adverse conditions these organisms will resist include sodium

azide, detergents, sodium hypochlorite, heavy metals, ethanol, high oxidative stress, and prolonged

dessication (Huycke, 2002).

There are over 20 species currently included in the genus Enterococcus (Giraffa, 2003), but E.

faecalis, E. faecium, and to a lesser extent E. durans, are the most common to both the human GI tract

(Tannock and Cook, 2002) and dairy products (Wessels et al., 1988). Regardless of origin of isolation,

there is significant phenotypic heterogeneity within and between populations of Enterococcus, making

their identification difficult (Giraffa, 2003). Presumptive identification is relatively simple and can be

accomplished using selective growth media and basic phenotypic assays such as esculin hydrolysis

(Garg and Mital, 1991) and Gram staining. The most common species (including the most relevant dairy

organisms noted above) can then be differentiated from other Gram-positive, catalase-negative, homo-

fermentative cocci by their ability to grow at 10 and 45C, in 6.5% NaCl, in 40% bile, and at pH 9.6

(Franz et al., 2003). Other less common species, however, exhibit variation in these traits and may

require more extensive phenotypic characterization or the use of molecular methods to establish with

certainty their identity as enterococci.

Additionally, some traits that were historically used to identify enterococci are now less relevant

in modern taxonomy. For example, expression of Lancefield's group D antigen (a defining characteristic

of the enterococcal group or fecal streptococci) is not a defining characteristic of the modern genus

16

Enterococcus. Several enterococcal species do not express the group D antigen, while some bacteria

outside the genus Enterococcus, including some streptococci and leuconostocs do express the group D

antigen (Franz et al., 2003).

The enterococci were originally part of the genus Streptococcus, probably due to their

morphological similarity. In 1937, Sherman published a review classifying streptococci into 4 groups,

one of which he termed the enterococcal group. Other researchers suggested that the enterococcal group

might be sufficiently distinct as to form its own genus, but it was not until 1984, with the advent of

molecular methods to make taxonomical distinctions based on genetic similarity, that the genus

Enterococcus became formally recognized in the scientific community (Facklam et al., 2002).

Habitats of Enterococci

The enterococci are found in a diverse range of habitats including dairy products and other foods,

as well as in clinical and environmental contexts (Franz et al., 2003). E. faecalis and E. faecium are

broadly distributed and are common to most enterococcal habitats, while some of the less common

enterococci are associated with specific habitats or even specific hosts, such as E. asini, which is

specific to donkeys (Aarestrup et al., 2002). Most enterococci are considered to be of fecal origin and

are native to the GI tract of humans and many other mammals and birds.

Despite their ubiquitous nature as members of the commensal microbiota, they are typically kept

at very low levels in healthy hosts, and make up no more than 1% of the intestinal microbiota in the

average human adult (Tannock and Cook, 2002). In infants, however, their populations are much higher

and, along with lactobacilli and E. coli, enterococci form the dominant intestinal microbiota of neonates,

reaching levels of about 108 bacteria per gram of wet fecal matter of breast-fed children. Higher levels

are found in children fed infant milk formulations. A microbial succession is associated with changes in

diet from exclusive consumption of milk to the addition of solid foods. Obligate anaerobes such as

fusiforms and species of Bacteroides colonize the intestine and produce short-chain fatty acids, which

are inhibitory to facultative anaerobes under the conditions of the bowels, and contribute to the decline

in enterococcal populations.

The enterococci are occasionally pathogenic in animal hosts, and have been implicated in bovine

mastitis and some cases of diarrhea in animals such as rats, piglets, and poultry, although incidences of

enterococcal infection in animals receive less attention than human infections (Aarestrup et al., 2002).

This may be due in part to limited resources in veterinary clinics and difficulties in differentiating

enterococci from related organisms. As a result of these challenges, it is likely that many enterococcal

infections in animals are not reported. Human infection by enterococci, on the other hand, has become a

17

high profile issue in both the medical and scientific community. Enterococci are now considered

significant hospital acquired (nosocomial) pathogens (Upadhyaya et al., 2009), and special attention is

paid to their role in the acquisition and spread of antibiotic resistances in both clinical and agricultural

settings (Kak and Chow, 2002).

Interestingly, some strains of enterococci show a probiotic effect (Jansen et al., 1993), and

several commercially available probiotics contain strains of E. faecalis or E. faecium both for human

consumption and for use in animal feed (Franz et al., 1999). What factors contribute to enterococci

acting as probiotics as opposed to pathogens is debated and is currently a subject of scientific studies

(Domann et al., 2007; Veboe et al., 2010). As stated by Hew et al. (2007), "The ability of Enterococcus

to promote both health and illness at the same time is a contradiction that is currently not well

understood.” It is interesting to note that many of the same characteristics that make enterococci

potential pathogens are also important for inducing a probiotic effect. These traits include tolerance to

various adverse conditions that allow enterococci to survive in the digestive tract and the ability to

adhere to intestinal epithelial cells and effectively colonize the host intestine.

Enterococci make their way into the environment via fecal shedding (Aarestrup et al., 2002). The

use of untreated animal wastes as fertilizer may enhance the colonization of agricultural environments

by enterococci. They are often found in water samples and have been proposed as a reliable indicator of

fecal contamination. While even environmental enterococci are traditionally considered to be of fecal

origin, there are several species that appear to have adapted to the vegetative environment and

commonly colonize plants. These species are E. casseliflavus, E. mundtii, and E. sulfereus. Although

they are also found occasionally in the GI tract of animals, it is thought that they are only able to

colonize this environment transiently.

The ability of enterococci to survive on dairy equipment and in the dairy environment allows

them entry into milk, which is an ideal nutrient medium for them (Garg and Mital, 1991). Adaptation to

this niche in combination with their resistance to heat, salt, and low pH give them an edge in the cheese

environment, and they can be found during all stages of production and ripening for some types of

cheese (Manolopoulou et al., 2003). Indeed, microbiological analysis has shown enterococci to be a part

of the microbial ecology in many cheeses, particularly European artisanal cheeses, made from both raw

and pasteurized milk (Giraffa, 2003). They can colonize cheese either as contaminant during collection

and processing of milk, or sometimes by their presence in traditionally cultivated starters. Some

traditional starter cultures are made by pasteurizing raw milk and incubating it at 42 - 44C, a procedure

which strongly selects for thermophilic LAB such as enterococci and S. thermophilus.

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Functionality of Enterococci in Dairy Fermentation

The enterococci are classified as homo-fermentative lactic-acid bacteria (LAB) due to their

ability to produce L-lactic acid from carbohydrates, but their ability to colonize a variety of niches

reflects their capacity to produce a wide range of metabolites from diverse substrates. All species are

able to metabolize at least 13 sugars, and one or more species can metabolize an additional 31 sugars

(Huycke, 2002). Sugars, however, are not the only way the enterococci can extract energy from their

environment. They can also utilize such diverse energy sources as glycerol, lactate, citrate, α-keto acids,

arginine, and agmatine.

Several of these metabolic traits contribute to the functionality of enterococci in dairy ferments

including the ability to metabolize substrates that are abundant in milk, and the formation of metabolites

that lend desirable flavors to the finished product (Giraffa, 2003). For example, glycerol is a small

molecule used to bind fatty acids into triglycerides. The ability to metabolize this substrate, which is

abundant in milk fat, may provide an advantage to enterococci by allowing them access to an energy

source that may not be metabolized by competitors.

Although enterococci do produce lactic acid, they are typically much weaker acid producers than

other LAB, making them less suited as primary starter organisms (Giraffa, 2003). A diversity of other

metabolites, however, make them ideal for the development of flavor compounds during ripening,

especially due to their ability to grow in the highly inhibitory conditions of ripening cheese. Metabolism

of citrate, proteins, and lipids are traits that are well recognized as important for the ripening of cheese

and are all expressed by strains of enterococci to various degrees.

The diversity of metabolic pathways enterococci use to extract nutrients and the heterogeneity of

these metabolic traits within populations may help explain the purported functional aspects in fermented

foods. Cheese makers and connoisseurs often note the importance of "balance" in a good cheese

(Raskopf, 2010). If organisms involved in ripening have a limited number of potential metabolites

(which form the basis of flavor formation during ripening) then one flavor may become overpowering

and be considered a defect. As an extreme example, in the ripening of soft, bloomy rind cheeses,

Penicillium candidum or related molds typically produce ammonia. However, when this cheese is past

its peak, the pungent ammoniated rind can be considered undesirable. On the other hand, metabolic

diversity (such as is displayed by the enterococci) helps protect against any one metabolite or group of

metabolites becoming an overpowering off-flavor. In the same way, the presence of enterococci as part

of a diverse microbiota in fermented foods offers further protection against the dominance of one

metabolite. This may contribute to a balanced flavor palate exhibiting a diversity of subtle tastes, an

ideal in the world of cheese.

19

Another important functional characteristic of enterococci is the production of bacteriocins,

which are natural antibiotics produced by some kinds of bacteria (Giraffa, 2003). Enterococci are known

to produce a wide variety of bacteriocins, and these are sub-classified as 'enterocins.' These compounds

are relatively well characterized and have been shown to be compatible with significant starter LAB and

to be stable in the presence of rennet and under conditions of processing and fermentation such as

temperatures between 30 - 37C and low pH. Enterocins have the potential to inhibit the growth of more

vigorous pathogens such as Listeria monocytogenes and Staphylococcus aureus. The anti-Listeria

activity of enterocins is of particular relevance, as L. monocytogenes is one of the most significant

cheese-borne pathogens. Scientific studies support the use of enterococci as protective cultures, and

several experimental models have shown that enterococci can confer a protective effect on cheese

artificially inoculated with L. monocytogenes or S. aureus by inhibiting the growth of these pathogens.

The presence of enterococci in cheese, however, is not unequivocally good. Whether they

function as beneficial non-starter lactic acid bacteria (NSLAB) or as undesirable contaminants depends

largely on the microbial ecology of the cheese in question. Garg and Mital (1991) in a review on

enterococci in dairy products highlight this point by noting two significant studies. In one study, co-

culturing of E. faecalis subsp. liquefaciens with S. thermophilus significantly enhanced acid production

in the latter species. In an alternative example, the presence of high numbers of E. faecalis subsp.

liquefaciens in Swiss cheese inhibited propionibacteria, which are essential to the ripening of these types

of cheese, leading to retardation of the ripening process and formation of bitter flavors. Additionally,

recognition of the potential for enterococci to contribute to disease states either indirectly or as

opportunistic infectious agents, and the rapid development of antibiotic resistances within the genus has

raised questions regarding the safety of these organisms in food (Franz et al., 2003).

Virulence Factors in Enterococci

Due to the increasing significance of enterococci in hospital acquired infections, much scientific

research has focused on identifying traits that may contribute to their pathogenesis (Gilmore et al.,

2002). Although no single trait is ubiquitously present in pathogenic strains and absent from non-

pathogens, a number of traits have been identified that likely play a role in pathogenesis including

gelatinase, biofilm formation, polysaccharide capsule production, hemolysins, and enterococcal

aggregation substance. These traits, or "virulence factors" (Semedo et al., 2003), by definition are not

required for commensal functioning (as evidenced by their absence from some or all commensal

strains), and contribute to the severity of infection, which is usually assessed by experimental models of

infection that show the impact of knocking out a gene encoding a virulence factor (Gilmore et al., 2002).

20

These traits are often encoded on mobile genetic elements and can therefore be efficiently disseminated

throughout a population under the proper conditions.

Before the individual virulence factors are considered, it is important to understand how

virulence factors are conceptualized and take into account some complicating factors in modeling

virulence. Foremost, a strain cannot be defined as pathogenic by simply identifying the presence of

virulence factors or the genes encoding them. The textbook definition of pathogen is 'a disease causing

microorganism' (Madigan and Martinko, 2006, 3), but the presence of virulence factors in a strain of

Enterococcus does not necessarily guarantee that the strain will cause disease. This is clearly exhibited

by the presence of all of the classical virulence factors in at least some, if not most E. faecalis strains

isolated from the fecal matter of healthy Norwegian infants (Solheim et al., 2009). Genetic studies have

revealed that Symbioflor® 1, a commercially available probiotic strain of E. faecalis lacks several

virulence factors including hemolytic cytolysin, enterococcal surface protein (an adhesin), and

gelatinase; however, other virulence factors including aggregation substance, collagen adhesion protein,

ability to resist oxygen anions, and capsule formation are present in the probiotic strain (Domann et al.,

2007).

While many of the classical virulence factors are not needed to cause infection, some very basic

traits that are important for survival of enterococci in diverse niches are also important for pathogenesis.

These traits include resilience against various adverse environmental conditions needed to survive transit

through the gut, and production of adhesins allowing cells to colonize the intestinal epithelium (Hew et

al., 2007). The general hardiness of enterococci to environmental stress is advantageous while surviving

the process of cheesemaking just as much as it is advantageous while infecting a host (Giraffa, 2003).

Genes encoding adhesins are found in high proportions in populations of enterococci (Semedo et al.,

2003). They are likely advantageous in diverse environments and important for binding to both biotic

and abiotic surfaces. This has implications in both pathogenesis and in dairying. In the case of

pathogenic enterococci, adhesins that mediate binding to host cells are essential (Manley et al., 2007). In

a different scenario, adhesion to abiotic surfaces such as plastic and metal components of milking

equipment may allow enterococci entry into the milk, where they can thrive as a member of the dairy

microbiota (Giraffa, 2003).

Regulation of Genes Encoding Virulence Factors

Factors influencing genetic expression must also be considered, since an organism may present a

negative phenotype for a virulence factor even if it has the genetic potential to express that trait. Two

enterococcal mechanisms of gene regulation that are of particular significance are environmental sensing

21

and quorum sensing.

Hew et al. (2007) elegantly demonstrate that E. faecalis modulates expression of virulence genes

in response to various environmental conditions, and that some of these conditions may have

significance in food production and pathogenesis. The authors suggest that certain food processing

procedures could predispose a population of enterococci to express pathogenic traits. They also suggest

that enterococci may be able to sense the presence of host tissues and in response upregulate

transcription of traits that give them an edge in this environment (virulence factors). This type of

environmental sensing is typically mediated by two-component signal transduction systems that sense

extra-cellular conditions via membrane sensor kinases (Ma et al., 2008).

Quorum sensing (a mechanism of communication between bacterial cells) is mediated by a

similar process and appears to play an integral role in coordinating production of virulence factors

(Spoering and Gilmore, 2006). In this case, however, the critical environmental condition is cell-

density, because quorum sensing signaling molecules (typically small molecules or short-chain peptides)

are produced by the same bacterial cells capable of sensing them in their environment. As growth occurs

and cell density in a particular locality increases, a critical concentration of signaling peptide is

eventually reached, initiating significant changes in genetic expression. Since many enterococcal

virulence factors are regulated by quorum sensing (Hew et al., 2007), a critical cell density may be

requisite for pathogenesis. Considering the strict regulation of enterococcal populations in the human GI

tract (Tannock and Cook, 2002), it is possible that controlling population density of enterococci is a key

strategy in protecting against infection. Populations of enterococci in cheese, on the other hand, are less

controlled and are likely to reach cell densities required for expression of quorum mediated traits. The

implications of this, however, are unclear in terms of the risk associated with consumption of foods

containing large populations of enterococci.

Diversity of Enterococcus spp. and Evolution of Virulence

The classical virulence factors, which have been well described in E. faecalis, are much less

common in other species of Enterococcus, and although E. faecalis is responsible for the vast majority

of enterococcal infections, E. faecium is much more likely to be resistant to one or more antibiotics,

which is also significant to pathogenesis (Gilmore and Ferretti, 2003). Furthermore, less common

species of Enterococcus such as E. raffinosus, E. casseliflavus, and E. mundtii are occasionally known to

cause infection (Tannock and Cook, 2002). The latter two species just listed are native to plants, not the

GI tract of humans or even other animals, and the native habitat of E. raffinosus is unknown because it

has only been found in pathological materials.

22

That some infectious species of Enterococcus are native to plants suggests that a general

hardiness to environmental conditions may allow enterococci to persist as pathogens despite not being

specifically adapted to their host. There is some evidence to support this hypothesis. Hew et al. (2007)

performed a study in which virulence gene expression was assessed in response to various

environmental conditions. The results showed that stress related genes were typically upregulated during

exponential phase, and it is postulated that this is a preparation for entering the starvation stress induced

stationary phase, in which these genes will aid survival during nutrient sparsity. In particular, gls24

(which codes for a general stress protein) was upregulated during exponential phase in cells exposed to

almost all of the various environments, and the authors suggest that this may be a case in which food

processing conditions could contribute to the development of pathogens. As a general stress gene, gls24

may play important roles outside the context of virulence, but it has been shown to increase virulence in

multiple animal models.

It is important to stress that many pathogens evolve their pathogenic traits outside of their host

(Ehrlich et al., 2008), since this suggests that ecological and environmental conditions contribute to

pathogenesis. When conceptualizing enterococcal virulence, one must recognize that a virulence factor

may have evolved under multiple evolutionary pressures, especially considering the diverse habitats of

enterococci. A well known example of this type of evolution is E. coli 0157:H7. This human pathogen is

a native member of the bovine commensal microbiota and is not commonly transferred from person to

person, which suggests that its virulence may be subject to many evolutionary forces outside the host.

There is evidence to support this idea, at least in the case of shiga-like toxins, an important virulence

factor in E. coli. These toxins have been shown to help cells evade predation by the ubiquitous

bactivorous protozoan Tetrahymena pyriformis.

Examples like this show that microorganisms can be highly adaptable and that their multifaceted

mechanisms of survival can lend an advantage in numerous environments. While evidence strongly

suggests an etiological role for enterococcal virulence factors in pathogenesis, it is likely that these traits

perform multiple functions and are also advantageous outside the context of pathogenesis. The virulence

factors are common in clinical strains because they do lend an advantage in pathogenesis, but they are

not necessary for pathogenesis, nor do they ensure pathogenesis. Keeping that in mind, a more accurate

model of virulence can be conceptualized, and the significance of individual virulence factors can be

evaluated.

Gelatinase

Gelatinase is a multifaceted zinc metalloprotease enzyme produced by some strains of

23

enterococci that has been associated with virulence both in experimental infection models (Lopes et al.,

2006) and by prevalence in clinical isolates (Elsner et al., 2000), although conflicting data has called

into question its statistical association with clinical isolates (Semedo et al., 2003). The probiotic strain of

enterococcus marketed as Symbioflor® 1 does not produce gelatinase (Domann, 2007). Environmental

conditions can have an effect of gelatinase production, and Hew et al. (2007) found that brain heart

infusion (BHI) culture media upregulated transcription of gelE (the gene that encodes gelatinase). Dairy

enterococci commonly express gelatinase (Lopes et al., 2006), probably to extract nutrients from casein

and other mammalian proteins present in milk. To complicate accurate assessment of gelatinase

production, lab manipulation of culture isolates can cause loss of gelatinase activity.

This complication was clearly elucidated by Lopes et al. (2006) in their study on gelatinase

activity in dairy enterococci isolated from raw ewe's milk and cheese. It primarily addressed questions

regarding how conditions of culturing can influence the genetics, gene-expression, and activity of

gelatinase. They obtained 35 isolates and duplicated them, creating a total of 70 isolates. The duplicate

set of isolates underwent significantly more lab manipulation than the original set. Isolates were

screened for gelatinase activity. While in the original set, which experienced less manipulation, 33/35

isolates showed gelatinase activity, only 4/35 in the more manipulated duplicate set showed activity.

Isolates were then stored in glycerol at -80C for one year before a second set of experiments re-

assessed gelatinase activity and also collected genetic analysis to observe the presence and transcription

of gelE and the fsr operon, which contains 3 genes (fsrA, fsrB, and fsrC) that regulate expression of

various genes, including gelE. Freezing had a very large impact on gelatinase activity and only 4/70

isolates showed activity in the latter assay. The genetic data implicates deletions in the fsr operon in loss

of gelatinase activity, as at least one fsr gene was missing from 54 isolates. Some gelatinase negative

strains that do have an intact fsr operon, and further genetic testing suggests that post-translational

modification may be necessary to activate the enzyme.

This study highlights the significance of the fsr operon in regulation of gelatinase expression.

Gelatinase is probably the most thoroughly studied of the enterococcal virulence factors, and the

mechanism behind its regulation is well described (Hew, 2008). The most important thing to know about

regulation of fsr expression is that it is mediated in a quorom sensing manner. Production of gelatinase

biosynthesis activating pheromone (GBAP), an 11 amino acid cyclic lactone peptide, is encoded in the

C-terminus of the fsrB gene (Gilmore et al., 2002). This peptide has been shown to induce the

transcription of gelE and sprE (a co-transcribed serine protease), as well as to auto-induce its own

transcription creating a positive feedback loop that promotes gelatinase synthesis. According to Hew

(2008, 20), "studies have found that about 1,000 GBAP molecules are required per cell for the initiation

24

of gelE transcription."

Gelatinase is capable of hydrolyzing a variety of substrates including fibrin, fibrinogen, collagen,

casein, and its arbitrary namesake gelatin (Hew, 2008; Lopes et al., 2006). This suggests that gelatinase

is a multifunctional enzyme, and that it may be advantageous in more than one setting. It addition to it's

role in pathogenesis, it could be useful in extracting nutrients from casein when growing in milk. During

fermentation, especially the prolonged fermentation of aged cheeses, hydrolysis of casein by proteolytic

enzymes is an important functional aspect of enterococci contributing to flavor development (Giraffa,

2003). Considering its ability to hydrolyze casein, gelatinase has the potential to contribute to this flavor

development.

Gelatinase is considered an extra-cellular enzyme (Lopes et al., 2006), but it is also highly

hydrophobic (Makinen et al., 1989) a characteristic that could cause it to associate with the bacterial cell

surface, capsule, or substrate surfaces. Considering its ability to extract nutrients from the environment

(Lopes et al., 2006), and defend the cell against host immune response (Thurlow, 2009), close proximity

to the cell may be advantageous, allowing the cell to get the most benefit out of the enzyme.

Capsule

Of enterococcal serotypes A-D, only C and D produce a capsular polysaccharide (Thurlow et al.,

2009). This variable molecule forms a thick mucoid layer that surrounds the bacterium and is significant

to pathogenesis because it can help enterococci and other bacteria evade the immune response.

At least two mechanisms of immune evasion are described by Thurlow et al. (2009), both of

which rely on interfering with binding of molecular identity markers to receptors on the surface of

immune cells. In the first mechanism, capsular polysaccharide inhibits recognition of surface bound C3,

a protein present in the blood that is part of the complement system of innate immunity. The protein acts

somewhat like an antibody, but is less specific and binds to common pathogens marking them for

phagocytosis; however, the capsule interferes with recognition of C3 bound to encapsulated cells,

allowing them to resist opsonization by white blood cells, accounting for their enhanced survival in

serum and resistance to phagocytosis.

In addition to receptors that recognize proteins like C3, immune cells express pathogen

recognition receptors (PRRs) that recognize pathogen associated molecular patterns (PAMPs). For

Gram-positive bacteria, one of the most common PAMPs is lipoteichoic acid (LTA), which binds

specifically to immune cell receptors such as Toll-like receptor, allowing the recognition of pathogens

(Ginsburg, 2002). This binding induces production of cytokines, stimulating an immune response. If

LTA is covered by a layer of capsular polysaccharide, however, the immune response is attenuated. See

25

figure 5 for a detailed model of the organization of structures in the E. faecalis cell wall.

Thurlow et al. (2009) show experimentally that enterococcal capsules interfere with LTA binding

and alter cytokine production in cultured macrophages. Significantly, production of tumor necrosis

factor alpha (TNF-α), a transcription factor that induces expression of genes involved in the immune

response, was much higher when macrophages were exposed to unencapsulated strains as opposed to

encapsulated strains, which actually did not produce significantly greater TNF-α production than when

macrophages were not stimulated.

Coyette and Hannock (2002) note that capsule formation by enterococci is growth-phase

dependent, but a quorum sensing mechanism of regulation has not been identified. The synthesis of

capsular polysaccharides by Gram-positive organisms is, however, highly regulated (Hancock et al.,

2003), and quorum sensing regulates capsule formation in a strain of S. aureus via AI-2, a universal

bacterial signaling molecule (Zhao et al., 2010). This evidence, along with the prevalence of quorum

sensing mechanisms in regulation of other virulence genes, suggests that synthesis of capsular

26

Figure 5 - "Model for the organization of cell wall polymers in the cell wall of E. faecalis. The lipid-anchored lipoteichoic acid, also known as the streptococcal group D antigen, is shown protruding into the cell wall peptidoglycan. Shown anchored to N-acetylmuramic acid (MNAc) residues of the peptidoglycan are the integral cell wall teichoic acids and [a] hypothesized enterococcal species antigen. Anchored to the N-acetylglucosamine (GNAc) reisdues in the peptidoglycan and protruding out from the peptidoglycan is the serotype-specific capsular polysaccharide. (Coyette and Hancock, 2002)"

polysaccharide production is also influenced by quorum sensing mechanisms. Capsule production is

encoded by nine genes, cpsC-cpsK (Gilmore et al., 2002).

Capsules can be observed in enterococci using a simple india ink stain, which stains the

background of the slide but not the capsule (Bottone et al., 1998). Cell bodies can then be counter

stained with crystal violet, and the capsule appears as a transparent halo surrounding cells. This stain has

been used to detect capsules in isolates from patients with persistent urinary tract infections. Other

studies using india ink staining have found about 20% of isolates from the mammary secretions of

mastitic cows to be encapsulated (Coyette and Hancock, 2002). Like gelatinase production, detection of

capsule by india ink staining was also found to be highly dependent on culture and storage conditions

and about 80% of encapsulated strains had no identifiable capsule after storage in skim milk at -80C.

Studies specifically addressing the influence of environmental conditions on capsule production

in enterococci have been sparse as of yet, but initial findings suggest that capsule production is slightly

downregulated in both serum and urine as opposed to enriched microbiological media (Coyette and

Hancock, 2002). It is interesting to note that culture broth made from skim milk powder is used to

induce capsule production for capsule staining procedures (Smith and Hughes, 2010). Aside from

helping evade the immune system, capsules are thought to protect from desiccation, promote adhesion,

and biofilm formation. All these qualities either directly enhance virulence, or could potentially allow

virulent strains to persist in the environment and spread from one host to another. As a somewhat

counterintuitive twist in the story of enterococcal virulence, however, the probiotic strain Symbioflor® 1

does produce a capsule (Domann et al., 2007).

Biofilms

A biofilm is a population of cells encased in a hydrated mixture of polysaccharides, proteins, and

nucleic acids (Mohamed and Huang, 2007). The formation of biofilms is significant in both the dairy

and hospital environments. Since they allow adherence to abiotic surfaces, biofilms give enterococci the

potential to colonize surfaces such as milking equipment, allowing entry into milk, or surfaces in

hospitals allowing clinical strains adapted to human hosts to persist in the environment and spread.

Biofilm formation has been associated with the ability of Enterococci grow on various medical devices

such as silicone gastrostomy devices and intravascular catheters. Adherence to medical devices that are

inserted into patient bodies is a potential method of infection.

Of perhaps greater concern is the difficulty in eradicating bacterial biofilms, and the increased

resistance of biofilms to antibiotics, which can be 10-1000 times greater than that of planktonic bacteria

(Mohamed and Huang, 2007). Biofilms are no doubt one of the traits that allows enterococci to be such

27

hardy environmental organisms. They are also associated with pathogenesis, but prevalence and degree

of biofilm formation have been shown to vary, and one study found that 93% of clinical and faecal

enterococcal isolates produced biofilm (Mohamed et al., 2004). Despite variation, trends suggest that

biofilms are more common in E. faecalis than in E. faecium, and that biofilms may be significant in

enterococcal pathogenesis.

Biofilm formation is associated with gelatinase and is regulated by quorom sensing as shown by

recent studies investigating the role of DNA in biofilm structure (Thomas et al., 2009). Their findings

implicate gelatinase in a fratricidal mechanism of DNA release, lending evidence to a model of biofilm

formation in which gelatinase biosynthesis-activating pheromone (GBAP) mediates the production of

gelatinase via quorum sensing, which kills cells that do not respond to gelatinase regulating quorum

sensing oligopeptide GBAP, since they will also not express the gene SprE, which codes for a serine

protease that is co-transcribed with gelatinase and provides immunity to gelatinase mediated killing.

The killing action of gelatinase releases DNA into the environment where it can be used for

biofilm structure. It is interesting to note in this model that in order to provide immunity to a particular

cell, the extra-cellular protease SprE would provide a more reliable advantage if it associated with the

cell surface, as may be the case with hydrophobic gelatinase (Makinen et al., 1989), although evidence

to support this theory has not yet been gathered.

Various environmental conditions have been shown to influence biofilm formation in enterococci

(Mohamed and Huang, 2007). One of these factors is the presence of glucose, which reliably enhances

biofilm formation in several studies. Considering the environments of interest to this paper, it is

significant that glucose would be available both in milk (by hydrolysis of lactose) and in human tissues,

where it is used as a basic unit of energy. Of further significance to enterococcal infection, human serum

also enhances biofilm production.

Enterococcal surface protein (Esp), another purported virulence factor, is associated with biofilm

formation (Gilmore et al., 2002). The probiotic strain Symbioflor® 1 probably does not produce

biofilms since its genome does not code for gelatinase or Esp (Domann et al., 2007). The interconnected

functionality of virulence factors supports the idea that they are multi-functional traits. Looking at

biofilms through the multi-functional lens, it is significant to emphasize that biofilm formation is a

highly advantageous trait commonly expressed by bacteria to help cope with stressful environmental

conditions in a variety of settings, including pathogenesis.

On the other hand, exposure to 2-3% NaCl completely stops biofilm formation (Mohamed and

Huang, 2007), suggesting that salting of foods such as cheese may be a processing step that inhibits the

formation of biofilms and reduces virulence. In contrast to brain heart infusion, which upregulated

28

gelatinase production, Hew et al. (2007) found that in vitro environments mimicking food processing

conditions universally downregulated expression of gelatinase, which may be needed for biofilm

formation (Thomas et al., 2009). These data suggest that biofilm production is regulated in response to

environmental conditions, that enterococci may be able to sense host tissues, and that processing of food

may actually reduce virulence.

Aggregation Substance

Enterococcal aggregation substance (EAS) is a multifunctional surface protein expressed by

many enterococci. EAS is actually a group of related proteins sharing 90% sequence similarity with the

exception of a variable central domain where sequence similarity is only 30 to 40% (Chuang et al.,

2009). Although traditionally associated with E. faecalis, PCR amplification using primers designed for

agg (a gene that encodes for the most common EAS protein produced by E. faecalis) shows that highly

similar genes can be found in most if not all strains of Enterococcus (Semedo et al., 2003). This suggests

that homologous proteins exist in other species.

The various aggregation proteins are encoded on plasmids capable of sex-pheromone mediated

conjugative transfer (Wirth, 1994). This specialized quorum-mediated interaction requires both donor

cells and a recipient cells. Donor cells have the plasmid. Recipient cells lack the plasmid, but produce a

chromosomally encoded signal peptide

(sex-pheromone) that can be considered

similar to other quorum signal peptides

such as GBAP. When a critical

concentration is reached, the pheromone

induces transcription of genes encoding

EAS proteins, which subsequently coat

the surface of the donor cell (see figure 6).

The function of EAS proteins is

what makes this quorum sensing

mechanism unique. These proteins bind to

components of the bacterial surface such

as lipoteichoic acids and enterococcal

binding substance (EBS), genes for which

are encoded on the chromosome and

expressed by recipient cells (Wirth, 1994).

29

Figure 6 - Image produced using field emission scanning electron microscopy showing expression of surface proteins encoded on pCF10 pheromone responsive plasmid. Cells in both images carry a shuttle vector, but for cells on the left Asc10 and Sec10 (surface protein genes encoded on pCF10) have been spliced into the vector. These proteins are clearly visible as discrete globular entities on the surface of the cell. This image was retrieved from Clewell and Dunny (2002).

This binding causes the formation of cellular aggregates, or clumps, bringing donors and recipients in

close enough proximity to ensure the conjugative transfer of EAS encoding plasmids. Although there is

not yet research to support the hypothesis, clumping could have the added benefit of increasing local

concentrations of quorum signaling molecules and inducing the expression of other virulence factors and

otherwise advantageous quorum regulated traits. In this sense, EAS could be considered a meta-

virulence factor, promoting the proliferation of other virulence factors by forming aggregates of cells

with diverse traits and inducing the sharing of these traits. Harsh environmental conditions would then

drive the selection of only the most advantageous traits, enhancing adaptation.

EAS is perhaps the most well studied enterococcal virulence factor next to gelatinase, and many

studies have investigated its role in pathogenesis and its structural and functional properties. One such

study, performed by Chuang et al. (2009), assessed the virulence of chromosomally identical strains with

variations on the pCF10 plasmid. Variations included mutations of the prgB gene that encodes for Asc10

(mutations ranged from insertions and deletions of large sections of amino acid sequences to more subtle

alterations such as single amino acid substitutions), deletion of the prgB gene, and absence of the pCF10

plasmid. Virulence was assessed in a rabbit endocarditis model and was quantified based on the bacterial

load of infected tissue and weight of bacterial vegetative growths. The results showed that deletion of

the prgB gene resulted in greatly decreased virulence. Mutations of the prgB gene also resulted in

decreased virulence, with the more subtle changes such as amino acid substitutions resulting in some of

the greatest decreases in virulence. Interestingly, strains without the pCF10 plasmid did not show a

significant decrease in virulence compared to strains with wild-type pCF10, while strains with mutations

in the plasmid did show decreased virulence. The authors suggest that pCF10 may suppress the

expression of homologous, chromosomally encoded traits, or that proteins encoded on the plasmid may

coat the cell surface in such a way as to obscure other functional proteins, such that the presence of a

plasmid with non-functional proteins may inhibit the expression or functioning of otherwise significant

traits.

Gene transfer can still occur without EAS or EBS, but it is much less efficient in a liquid

environment, where conjugative transfer between these primarily non-motile cells is reliant on random

collisions. EAS/EBS deficient enterococci, however, still efficiently transfer genes on solid surfaces

(Clewell and Dunny, 2002). Biofilms, such as those that form on abiotic surfaces or on heart tissues

during endocarditis may speculatively be implicated in gene transfer in enterococci that do not express

EAS, but EAS has also been shown to enhance biofilm formation in experimental models of

endocarditis (Chuang et al., 2010). This could be a result of aggregation enhancing the efficacy of

quorum induced trait expression. While biofilms may facilitate gene transfer on medical equipment or

30

milking equipment, both serum and milk are liquid media, and EAS mediated clumping may be

important for enhancing genetic exchange and facilitating adaptation of enterococcal populations in

these environments.

In addition to their role in clumping and genetic exchange, EAS proteins also mediate adhesion

to eukaryotic cells including neutrophils (a type of immune cell), kidney cells, and intestinal cells

(Chuang et al., 2009). Their role in adhesion to eukaryotic cells was discovered when sequence

similarity was found between amino acid motifs in EAS and motifs in fibronectin, which mediates

binding between eukaryotic cells (Gilmore et al., 2002). Their ability to adhere to eukaryotic cells

suggests that these proteins may be important for mediating interactions between enterococcal cells and

host cells.

There is substantial evidence to support the role of EAS proteins in modulation of immune

function, which could contribute to pathogenesis (Gilmore et al., 2002). EAS proteins mediate binding

with immune cells such as neutrophils and macrophages, promoting phagocytosis of bacterial clumps.

EAS proteins also promote survival of cells after phagocytosis. Resistance to phagocytic killing is

strongly adaptive for pathogenic strains which must avoid the immune response both on mucosal

surfaces, or in serum and infected tissues.

EAS may also be expressed in response to environmental conditions such as exposure to serum,

and in contrast to scenarios in which EAS proteins attenuate the immune response, EAS and EBS

together can act as a 'super-antigen', stimulating T-cell proliferation and production of inflammatory

cytokines (Kayaoglu and Oerstavik, 2004). It is clear that EAS proteins can have varied effects on the

immune system, but that in general it provides an advantage to infectious populations.

EAS proteins are strongly implicated in pathogenesis due to their various interactions with host

tissues and fluids as well as their ability to act as a nexus for other virulence factors. Their association

with clinical isolates (Elsner et al., 2000) and ability to enhance virulence in experimental models

(Chuang et al., 2009) lend further support to this hypothesis. EAS proteins, however, appear to be very

common amongst isolates from diverse sources (Semedo et al., 2003) including strains isolated from

healthy infants (Solheim et al., 2009) and the probiotic strain Symbioflor® 1 (Domann et al., 2007). In

addition, the results of experimental models of pathogenesis sometimes suggest that EAS proteins may

not enhance virulence (Johnson et al., 2004).

It seems plausible that the probiotic efficacy of some strains of enterococci is due in part to

virulence traits like EAS protein and capsular polysaccharide that stimulate and/or modulate immune

function. Considering that the immune system is highly co-evolved with this ubiquitous organism, it

makes sense that exposure to enterococci in the right context may be useful or even necessary to

31

maintain balance in the immune system. Scientific research is beginning to show that the commensal

microbiota are essential in training the immune system and in maintaining healthy immune function

(Tlaskalova-Hogenova et al., 2004). It is likely that depending on environmental conditions, immuno-

modulating virulence factors like EAS protein can be involved in mechanisms that promote either

pathogenesis or healthy immune responses to enterococci or perhaps other microbes as well.

Cytolysin

Hemolysins are a common type of bacterial toxin that bind to the membrane of host cells forming

pores and resulting in cellular lysis (Bhakdi et al., 1996). Pore-forming toxins have been described as

"potent and versatile weapons with which invading microbes damage the host macroorganism," and they

are recognized as virulence factors in pathogens such as S. aureus and E. coli. They have also been

identified as virulence factors in enterococci, and studies on enterococci as early as 1921 utilized

hemolytic assays (Gilmore et al., 2002). Of the enterococcal hemolysins, the most well studied is

cytolysin, a unique hemolysin that also has bactericidal activity, perhaps due to distant relations to other

bacteriocins. This dual activity is a clear example of multifunctionality in enterococcal virulence traits.

Hemolytic activity can be assayed by culturing isolates with red blood cells (erythrocytes).

Isolates are grown on blood agar and colonies able to lyse erythrocytes are surrounded by a transparent

halo (Gaspar et al., 2009). Critical examination of this methodology, however, along with the advent of

genetic data, has revealed some discrepancies in this type of testing. These discrepancies may be due to

variables such as media contents, incubation conditions, or type of erythrocytes. In addition, genetic data

may conflict with phenotypic data because 8 different genes are required to express a positive

phenotype. In order to develop an accurate phenotypic assay for cytolysin, Gaspar et al. (2009) used

PCR specific to all 8 cytolysin genes to test the efficacy of various hemolysin assay procedures. The

researchers found that the assay was most accurate using Colombia blood agar supplemented with horse

erythrocytes and incubating anaerobically at 37C for 24-48 hours.

Some studies note a high incidence of hemolytic activity in clinical isolates (Ike et al., 1987;

Semedo et al., 2003). Others show the effect of its absence in experimental models of infection (Gilmore

et al., 2002). It may play a role in survival in the GI tract and translocation across intestinal epithelial

cells (IECs), and it is more certain to aid survival in the bloodstream. Cytolysin has killing action against

macrophages, PMNs, erythrocytes, and Gram-positive, but not Gram-negative, bacteria (Kayaoglu and

Oerstavik, 2004). It is easy to imagine a role in immune evasion considering its activity against

phagocytic immune cells, but in vitro experiments designed to test this hypothesis have so far been

inconclusive (Gilmore et al., 2002). The traditional perspective on hemolysins suggests that they may

32

increase fitness by releasing otherwise unavailable nutrients via cellular lysis. Lending support to an

etiological role of cytolysin in enterococcal pathogenesis, the probiotic strain Symbioflor® 1 does not

produce cytolysin (Domann et al., 2007).

Synthesis of cytolysin is complex, and requires the presence of 8 different genes (Gaspar et al.,

2009). These genes are typically encoded on plasmids, but occasionally are found on the chromosome

(Kayaoglu et al., 2004). The toxin itself actually consists of two separate peptides (CylLs" and CylLL"),

which are both required to elicit activity, and go through an elaborate modification process to achieve

their final active conformation (Gilmore et al., 2002). When first transcribed, the peptides are called

CylLs and CylLL. They are then post translationally modified within the cell to form CylLs* and CylLL*.

During secretion, a cysteine protease associated with cytolysin's membrane transporter cleaves leader

sequences from each of the peptides, at which point they are named CylLs' and CylLL'. Finally, the

peptides must be activated outside of the cell by proteolytic cleavage to form CylL s" and CylLL". A

membrane associated immunity protein is transcribed along with cytolysin to protect cells from their

own bactericidal toxin.

In addition to its pore-forming action, CylLs" also plays a role in quorum sensing, acting as an

auto-inducing peptide that regulates cytolysin expression via a novel mechanism that is not yet

characterized, but involves two regulatory components (CylR1 and CylR2) that suppress expression of

cytolysin at subthreshold levels of CylLs" auto-inducing peptide (Gilmore et al., 2002). Lending further

support to this model, Hew et al. (2007) found that cytolysin production correlates to cell density. The

presence of host cells also influences this regulation, as CylLL" binds preferentially to host cells, leaving

CylLs" free to function as an auto-inducer, stimulating high level cytolysin production (Coburn et al.,

2004).

Cytolysin is associated with EAS and pheromone mediated conjugative plasmids (Tanimoto,

1993), and Huycke et al. (1992) use a syrian hamster model show in vivo that pheromone-inducible

conjugation can effectively transfer hemolytic activity (along with antibiotic resistance) horizontally on

plasmids between E. faecalis strains. Genes for cytolysin and EAS are invariably located near one

another, such as in the case of pheromone responsive plasmids that encode for both (Gilmore et al.,

2002). Experimental models of pathogenesis also suggest that they may have a synergistic effect, as

strains expressing both traits were found to be 8 times more virulent than strains expressing either one

trait. This synergism may be due to bacterial clumping and its influence on quorum sensing; since

cytolysin, like the other virulence factors, is mediated by a quorum sensing mechanism, and requires a

critical local concentration of an inducer peptide in order to be expressed.

Gilmore et al. (2002) note that due to the nature of cytolysin auto-regulation, it is unlikely that

33

free bacterial cells in the bloodstream will produce high levels of cytolysin, since diffusion into vast

volumes of blood will inhibit auto-induction. However, microenvironments such as cellular aggregates,

biofilms, heart valve vegetations, and sites of intravascular coagulation may provide the conditions

necessary for local accumulation of auto-inducing peptide and high-level expression of cytolysin.

Curiously, isogenic mutants of the quorum sensing locus fsr (which is associated with biofilm

formation) show enhanced hemolytic activity in comparison to wild type cells (Pillar et al., 2003),

suggesting that while sex-pheromone mediated quorum sensing can enhance cytolysin expression,

GBAP mediated fsr quorum sensing actually attenuates this virulence factor. This relationship exhibits

the complexity of interactions between virulence factors, as their activities can be synergistic,

antagonistic, or independent of one another.

Horizontal Gene Transfer

Horizontal gene transfer (HGT) is the process by which mobile genetic elements are exchanged

between bacterial cells. Modern research in microbial genetics has elucidated the highly significant role

it plays in bacterial evolution, mediating processes such as "the rapid spread of antibiotic resistance

genes... [and] the evolution of pathogenic potential, metabolic diversity, and perhaps even the operon

structure of the genome itself " (Weaver et al., 2002). While it is not a virulence factor per se, its central

role in the evolution of bacterial populations make it a key factor in adaptation to the host environment

(as a commensal or as a pathogen), persistence in food production and health care environments, and

resistance to antimicrobial agents. Therefore, a basic understanding of HGT is requisite when discussing

enterococci and food safety.

Scientific research has identified three classes of HGT called transformation, transduction and

conjugation (Madigan and Martinko, 2006, 257-298). Transformation is the process by which free DNA

from lysed cells is randomly encountered by a recipient cell, taken up, and incorporated into

chromosome resulting in a genetic change. However, there are significant limitations on this type of

genetic exchange. First, because of the instability of long prokaryotic DNA molecules, outside the cell

they typically break into pieces about 10 kbp (about the length of 10 average genes). Although this is

enough to transfer significant traits (enterococcal cytolysin, for example, requires 8 genes for

expression), it does theoretically limit the amount of genetic data that can be transferred by this

mechanism. More significantly, not all cells have the capacity to 'be transformed'. A cell that is able to

take up free DNA and be transformed is said to be competent. Competence is regulated by special

proteins that uptake and process DNA. The percentage of cells that become competent and the length of

time that they remain competent during growth cycles differ widely between organisms. These factors

34

contribute to the relative inefficiency of transformation in comparison to other types of HGT.

Transduction occurs by incorporation of bacterial host DNA into the DNA of a bacteriophage

(bacterial viruses that commonly occur in microbial ecosystems), which can 'infect' other cells allowing

the transfer and incorporation of DNA into the new host (Madigan and Martinko, 2006, 257-298). These

phages, however, are typically not infectious in the traditional sense, since viral genes have been

replaced by bacterial genes. Transduced genes must be incorporated into the host chromosome or else

they will be lost because they cannot replicate independently.

Conjugation is the transfer of plasmids or chromosomally encoded transposable elements

through cell-to-cell contact, and is sometimes described as bacterial mating (Madigan and Martinko,

2006, 257-298). Plasmids are circular or linear segments of DNA that are not associated with the

chromosome. Plasmids range in size from 1 kbp to 1 Mbp in length, but most rolling circle replicating

(RCR) plasmids, which are the most significant in the context of gram-positive virulence and antibiotic

resistance, are less than 10 kbp in size, which is hypothesized to be the result of limitations imposed by

their mechanism of replication (Weaver et al., 2002).

Many bacteria, including enterococci, can contain multiple plasmids. In some cases, when a

plasmid that is taken up by a cell already containing a plasmid, the new plasmid will not be maintained

or replicated (Madigan and Martinko, 2006, 257-298). This is called incompatibility. Research has

identified that related plasmids sometimes cannot co-exist with one another within the same cell.

Groups of plasmids that cannot co-exist are called 'incompatibility groups'. Plasmids within an

incompatibility group exclude each other from replicating, but can coexist with plasmids from different

groups. This occurs because plasmids in the same incompatibility groups share mechanisms of

replication. They essentially compete for the resources required for replication, and only the plasmid that

confers the greatest adaptive advantage will remain. Plasmids are considered 'conjugative' if they encode

for their own transfer. When plasmid genes can be expressed by a recipient cell, it then becomes a donor,

which allows conjugative plasmid genetics to spread rapidly throughout populations. Plasmid encoded

traits include the ability to metabolize nutrients, bacteriocin production/resistance, virulence factors such

as hemolysins, and antibiotic resistances. A plasmid will typically persist in a population only if

selective pressure justifies the use of resources required to maintain it.

Transformation has not been a subject of discussion in the scientific literature, probably because

its inefficiency and inability to transfer large nucleotide sequences make its contribution to the transfer

of pertinent traits such as virulence factors and antibiotic resistances negligible. Although bacteriophage

based transduction likely does play a significant role in the transfer of these traits, this subject too has

not been the focus of scientific studies (Weaver et al., 2002). The conjugative transfer of mobile genetic

35

elements by plasmids and transposons, however, has received a lot of attention, and studies on this

subject have elucidated some unique and significant aspects of enterococcal evolution, ecology, and

pathogenesis.

HGT, Virulence, and Antibiotic Resistances

Enterococcal isolates typically possess from 1-7 plasmids (Palmer et al., 2010). Mobile genetic

elements in enterococci contribute to rapid adaptation and may also influence evolution through

mutation or hybridization of mobile elements. Many elements encoded on the chromosome are only

mobile via plasmid mediated horizontal transfer, and as noted above in discussions on EAS, species

specific pheromone responsive plasmids transfer by conjugation with great efficiency. Palmer et al.

(2010) speculate that the mechanism of HGT by pheromone-inducible conjugation "evolved to shuttle

niche specialization traits as E. faecalis strains from prey comingled with E. faecalis strains from

predators, allowing E. faecalis as a species to readily adapt to the dietary habits and other peculiarities of

particular hosts." Similar speculation could be extrapolated to suggest that this mechanism allows them

to adapt quickly to a variety of transient environmental factors, contributing to their ubiquitous nature

and their ability to colonize diverse niches. Whatever the evolutionary history of HGT in enterococci, in

the modern world its role in the transfer of antibiotic resistances has attracted much attention from the

scientific community and is at the heart of the debate on enterococci and food safety.

Experimental models show that conjugative plasmids containing virulence traits and antibiotic

resistances can occur during fermentation (Cocconcelli et al., 2003), although this occurs much more

efficiently in fermented sausages (10-3 transfers/recipient) than in cheese (10-6). For HGT to be a

significant factor in food safety, however, plasmids must also be able to transfer from populations in

food to populations in the consumer GI tract. Although a study by Huycke et al. (1992) exhibits the in

vivo capacity of E. faecalis to transfer a pheromone-inducible plasmid encoding erythromycin resistance

and hemolysin in the hamster GI tract, their experimental design does not reflect likely real-life

circumstances. Enterococcal overgrowth was induced by feeding antibiotics and subsequently

inoculating with 109-1010 CFU of streptomycin-spectinomycin resistant enterococci by orogastric

gavage. Three days later, hamsters were inoculated with 109 CFU of donor enterococci containing one of

three plasmids encoding erythromycin resistance and hemolysin production. Hamster stool was then

quantitatively tested for the presence of transconjugants (cells containing the donor plasmid). This

model was designed to mimic conditions experienced by hospital patients being treated with broad-

spectrum antibiotics that have little enterococcal activity, but even the authors note that the high levels

of inoculum used for orogastric gavage are unrealistic.

36

On the other hand, in traditional raw milk cheeses with relatively high levels of enterococcal

growth, populations after ripening generally range between 105-107 CFU/g (Franz et al., 1999),

indicating that in some cases, a 100 gram serving of cheese could provide as many as 109 CFU.

Therefore, the model used by Huycke et al. (1992) could be compared to a scenario in which a

hospitalized patient receiving antibiotic therapy eats a medium-large sized portion of cheese (aside from

differences in the anatomy of hamsters and humans and differences in environmental conditions of lab

animals and hospitalized humans). This suggests that cheese could potentially act as a carrier for

antibiotic resistances or virulence factors and that consumption of cheese by hospitalized patients

receiving broad-spectrum antibiotic therapy may increase the risk of enterococcal infection or facilitate

the spread of antibiotic resistances originating from agricultural environments.

These risks, however, may not be long term due to the fact that plasmids can be spontaneously

lost by a population in absence of selective pressures to justify energy expenditure on maintenance of

the plasmid (Madigan and Martinko, 2006, 279). For example, antibiotic resistance can be lost if no

antibiotics are present in environment. Indeed Huycke et al. (1992) find that transconjugants drop to

nearly undetectable levels within 7 days of inoculation, but more recent studies using similar methods

have produced conflicting results (Licht et al., 2002), one of which even demonstrates intergeneric

exchange of antibiotic resistance encoding plasmids from lactobacilli to enterococci (Jacobsen et al.,

2007). This study used a strain of Lactobacillus plantarum isolated from traditionally fermented

sausage.

The lactobacilli have not elicited the same concern regarding food safety in the scientific

literature as have the enterococci. They have a reputation as beneficial microorganisms with a protective

effect on fermented dairy products and possess the FDA designation "generally recognized as safe"

(GRAS) (Chung and Yousef, 2005) like most lactic acid bacteria. The Jacobsen et al. (2007) study,

however, suggests that even these benign organisms may play a role in compromising public health via

intergeneric dissemination of antibiotic resistances. This is a role which has also been attributed to the

enterococcus, which is not designated GRAS although it is a member of the lactic acid bacteria

(Fracalanzza et al., 2007). Safety concerns surrounding enterococci stem from its occurrence, if

infrequent, as an infectious agent, its capacity to harbor a number of intrinsic and acquired antibiotic

resistances, including resistance to penicillins, streptogramins, aminoglycosides, macrolides,

tetracyclines, ansamycins, phenicols, fluoroquinolones, nitrofurantoins, fosfomycins, and significantly,

the glycopeptides vancomycin and teicoplanin (Facklam et al., 2002).

Enhancing the level of concern is the ability of enterococci to mobilize its virulence and

resistance traits within its own genus and beyond. One of the most prevalent concerns regarding

37

horizontal transfer of enterococcal antibiotic resistances is the transfer of vancomycin resistance genes

to strains of methicillin-resistant Staphylococcus aureus (MRSA), a closely related but much more

virulent pathogen with a significantly larger impact on public health. Although the replication and

establishment of pheromone responsive plasmids is not observed in non-enterococcal hosts, pheromones

may induce the transfer of non-conjugative plasmids (Palmer et al., 2010). Although the exact

mechanism of transfer is unclear, 10 accounts in the US since 2002 document the transfer of

vancomycin resistance from enterococci to MRSA, and a 1992 study by Noble et al. shows the in vitro

transfer of vancomycin resistance from E. faecalis to S. aureus.

Since vancomycin represents the last line of

defense in antibiotic therapy of some infections

such as MRSA (Palmer et al., 2010), the

emergence of 'super-bugs' such as VRE and

vancomycin-resistant S. aureus (VRSA) is of

significant concern in the medical community,

and has prompted some to ask if we are not on

the verge of a re-emergence of untreatable and

fatal bacterial infections (Livermore, 2009).

Increases in prevalence of antibiotic resistance,

however, are associated with the frequency of

antibiotic use both in hospitals and agricultural

settings. For example, increased use of vancomycin as an antibiotic therapy in the US has been linked to

increasing rates of vancomycin resistance (Malani et al., 2002).

Similar phenomena have been observed with regard to casual antibiotic use in animal agriculture,

such as the high profile discovery that prevalence of VRE in european products of agriculture is

statistically correlated to the use of the aminoglycoside antibiotic avoparcin (which is cross-tolerant with

glycopeptides vancomycin and teicoplanin) as growth promoters (van den Bogaard et al., 2000). In

geographic regions where such practices are not authorized, VRE is not found in the food supply

(Fracalanzza et al., 2007), and following European bans on the use of avoparcin as a growth promoter,

rates of VRE have been found to decline sharply within the span of a decade (van den Bogaard et al.,

2000).

When populations of enterococci are exposed to antibiotics, the more antibiotic resistant variants

have a greater chance for survival, and this selective phenomenon fuels the evolution of resistant strains

38

Figure 7 - Electron micrograph image of S. aureus growing in a biofilm on the luminal surface of an indwelling catheter. An erythrocyte is also visible. Similar biofilms including both staphylococcal and enterococcal cells may be the site of intergeneric transfer of vancomycin resistance genes. Image retrieved from Stokowski (2008).

(Goldman, 2004). This is a straightforward concept backed up by modern understanding of microbial

evolution, yet despite pleas from the scientific community that the emergence of new antibiotic

resistance traits are "a cause for worldwide concern" (Moellering, 2010) and that "strong

epidemiological evidence [supports] a link between the use of antibiotics in human medicine and animal

husbandry and the emergence, spreading and persistence of resistant strains in animal products"

(Fracalanzza et al., 2007), irresponsible use of antibiotics remains a major global issue. The issue,

however, is becoming harder to ignore, and we may soon be forced re-evaluate protocols for the use of

antibiotics in both hospitals and agricultural settings if we hope to maintain the efficacy of antibiotic

therapy for when we really need it.

39

Part III: Framing the Real Issues Behind the Raw Milk Debate

What Makes an Enterococcus Pathogenic?

It is clear that the virulence traits cannot be considered in isolation, and that complex interactions

between these and other traits, environmental conditions, and host conditions all influence one another

in the evolution of an enterococcal population. The diverse distribution of adaptive traits in populations

of enterococci, along with the ability of these populations to efficiently exchange genetic material

through horizontal gene transfer probably contribute to their hardy, flexible nature, which allows them to

colonize a variety of niches and makes them ubiquitous organisms. Given specific permutations of

environmental conditions, these traits can work in concert to achieve a variety of goals, from surviving

on abiotic surfaces, to fermentation of dairy, to colonizing a host in either a commensal or pathogenic

manner.

An important question, however, remains inconclusive: do agricultural practices and food

processing practices provide the conditions in which virulent enterococci can emerge? Food production

environments have in the past been proposed as a stage for the evolution of virulence, as in the case of

the high profile pathogen E. coli O157:H7. Like the enterococcus, E. coli is typically a commensal

organism inhabiting the GI tract of humans and other animals. E. coli O157:H7, however, is a serotype

that has become a great concern to food safety due to the severity of illness that it causes in humans

(FDA, 2009). It has been suggested that the practice of feeding grain to cattle contributes to the

emergence or perpetuation of the pathogen in food products, although evidence to the contrary exists as

well (Hancock and Besser, 2006).

The fact that enterococci are not a cause of food-borne illness suggests that agriculture and

traditional processing practices such as fermentation do not fuel the evolution of particularly virulent

traits, and that their presence in cheese is not of great concern. Despite their inability to become

significant food-borne pathogens, their incidence as infectious agents in hospitals has been an important

subject of discussion in the medical and scientific communities. Scientific analysis of clinical isolates

suggests that certain strains of enterococci do possess enhanced virulence. Genetic analysis has led to

the identification of an enterococcal pathogenicity island, a group of over 100 genes including several

recognized virulence factors that can be mobilized for horizontal transfer as an integrated unit (Manson

et al., 2010). Phylogenetic analysis provides some evidence regarding the origin of these extra-virulent

strains, and the existence of a "hospital clade" of enterococci has been suggested (Ehrlich et al., 2008).

40

The hospital clade represents a lineage of enterococci that evolved to exploit a very specific, and

historically novel niche: the intensive care unit.

Mundy et al. (2000) note in a review on enterococcal virulence that most enterococci occur as

commensal organisms or environmental contaminants, and a relatively miniscule number undergo

natural selection as human pathogens. Since hospitals provide the unique conditions under which

selection for human pathogens can occur, it could be argued that this environment is a breeding ground

for the evolution and rapid horizontal transmission of virulence factors. However, I have argued in this

dissertation that many virulence factors emerge under multiple evolutionary pressures and are useful

outside the context of pathogenesis. In all likelihood, both scenarios occur, which can account for the

existence of virulence factors throughout diverse populations of enterococci as well as the increased

prevalence of virulence factors in clinical isolates.

At any rate, it appears that the clinical environment is much more significant than the agricultural

environment in the emergence of enterococci with enhanced virulence, although attenuated virulence

may be an intrinsic characteristic of all enterococci. From this perspective, enterococcal colonization of

the gut could be considered an unavoidable infection that impacts 100% of the population shortly after

birth. It is an infection, though, that is actually essential for health, and this is where a close examination

of the enterococcus really begins to turn traditional conceptions of virulence inside out. I propose that it

is not in spite of the presence of virulence factors, but because of them that this organism is essential for

health. Early and consistent challenge by enterococci with attenuated virulence may provide a safe

mechanism for training the innate immune system to recognize and control potentially invasive bacterial

pathogens.

Breast feeding could also be an integral aspect in encouraging growth of the proper enterococcal

populations and/or stimulating immune functioning in order to maintain a balanced microbiota.

Enterococcal populations in formula fed infants are on average 109.6 CFU/g of feces as opposed to breast

fed infants with an average of 106.3 CFU/g of feces (Tannock and Cook, 2002). Formula is designed

using processed cow's milk and supplemented to have a nutritional profile similar to human milk, but it

lacks many of the unique immune factors present in mother's milk. These factors likely play an

important role in establishing a balanced microbiota, especially in infants with developing immune

systems. Enterococci are also found in human milk and one study screening for virulence factors in

breast milk enterococcal isolates states:

The high concentration of enterococci in milk from healthy mothers strongly suggests that they may play an important biological role during the first months of life. Work is in progress to elucidate their potential to protect the newborn against infectious diseases and their role in the maturation of the infant gut-associated lymphoid tissue (Reviriego et al., 2005).

41

The authors could not find any virulence determinants in breast milk isolates, but virulence assays were

conducted 3 years after isolation of strains, and strains were routinely grown in brain heart infusion

broth. This suggests that virulence traits such as gelatinase could have been present in initial populations

and lost during lab handling, as has been shown in dairy enterococci (Lopes et al., 2006).

The use of enterococci as probiotics suggests they may have significant health benefits to adults

as well. Additionally, the current model of enterococcal infection discussed earlier in this dissertation

suggests enterococcal overgrowth is a crucial risk factor for enterococcal infection. Given these

observations as well as anecdotal reports of the health benefits of raw milk, an intriguing question arises.

Could unpasteurized cow's milk for adults act in a similar way to mother's milk for infants? As discussed

previously, scientific evidence to support or refute the health benefits of raw milk consumption is

lacking, but further studies on the interactions between the human gut microbiota, milk, and the immune

system could offer insight into this complex question.

Dairy Foods and Issues of Hygiene

Putting aside the potential immune benefits of exposure to low levels of bacteria, the

omnipresent risk of dairy products (or any food) causing food-borne illness can be drastically reduced

by using hygienic production and distribution practices. In this way, farms are much like hospitals,

where the single greatest tool for effective control of infectious disease is basic hygiene (Manchester,

2005). In the clinical setting, where patients are at high risk for enterococcal infection and strains with

enhanced virulence and antibiotic resistance are common, the spread of enterococci is a significant risk

factor for infection and the spread of antibiotic resistances. Controlling this transmission is an immense

challenge due to their ubiquitous nature. Enhancing the challenge is the fact that even when strict

standards are set in place, the tendency of some health care workers to resist compliance with standards

or to inaccurately report compliance makes monitoring and enforcing the standards difficult (Bay, L.M.,

MSN, RN, ACNS-BC, CCRN, Adult Health Clinical Nurse Specialist, personal correspondence).

In the dairy environment, effective implementation of hygienic standards is the single most

important factor in limiting the risk of food-borne illness outbreaks. Dairy producers, however,

encounter the same challenges as health care professionals: ubiquitous environmental contaminants and

difficulty enforcing protocols. This is why pasteurization is an important safety net when good hygiene

cannot be ensured, but for this same reason the practice of pasteurization may take the emphasis off of

strict hygienic standards. CDC data on the prevalence of Listeria monocytogenes in milk samples shows

that 5% of bulk milk samples prior to pasteurization are culture positive for the food-borne pathogen and

42

that 2% of pasteurized milk samples from over 700 U.S. dairy plants were culture positive for listeria

species, primarily L. monocytogenes (CDC, 1988). This calls into question both the hygienic standards

of dairy producers that intend to sell their milk to processing plants for pasteurization, as well as the

efficacy of pasteurization in protecting against food-borne illness.

Pasteurization is not the same as sterilization. It does not entirely eliminate any bacterial

populations, but only reduces them to levels that are very low and often undetectable using standard

microbiological plating methods. Although the most likely source of L. monocytogenes in pasteurized

milk is officially considered to be improper processing or post-pasteurization contamination, scientific

studies have raised questions regarding the efficacy of pasteurization in eliminating L. monocytogenes

(Doyle et al., 1987). Could remaining populations evolve high heat resistance just as many pathogens

have evolved resistances to multiple antimicrobial agents? This phenomenon has not yet been observed,

but the continuing efficacy of pasteurization as an effective food safety practice is challenged by the

theoretical possibility that pasteurization could select for pathogens with high heat tolerance, or that

heat-resistant microbes could, for any number of reasons, evolve pathogenic traits. While this scenario

may be unlikely, the chances of selecting for a novel trait are increased if pathogens are pooled together

and subsequently exposed to consistent selective pressures, as in both the case of injudicious

antimicrobial use in hospitals and compulsory pasteurization of bulk raw milk. Additionally, the effect of

heat shock on pathogenic populations may enhance their ability to survive stressful conditions, thus

enhancing virulence (Wong, 2010), although this hypothesis remains speculative.

Farmstead dairy products are by definition processed on the farm where the milk is produced.

This model affords some benefits to food safety as there is a shorter transportation and distribution chain

and the production and processing is monitored in a more personal way. Although they are found with

lower frequency than enterococci and other commensals such as fecal coliforms, food-borne pathogens

including L. monocytogenes, Salmonella, and Campylobacter are common residents of agricultural

environments (Pradhan et al., 2009). In light of this, the FDA's zero-tolerance policy on L.

monocytogenes might appear unrealistic, especially when applied to farmstead dairy operations. Other

countries have different policies, such as Canada, where up to 100 organisms per gram are allowed in

foods that have not been associated with an illness outbreak and do not foster the growth of L.

monocytogenes over a 10-day period of refrigeration (FDA, 2010). The Danish policy is much more

nuanced and includes several categories of food, but in raw ready-to-eat foods allows 2 of 5 samples to

contain between 10-100 organisms per gram, while no individual sample may exceed 100 organisms per

gram.

The zero-tolerance policy has been at the forefront of recent confrontations between the FDA and

43

farmstead cheesemakers such as the highly publicized case of Estrella Creamery in Washington State. In

this case, the FDA ordered the destruction of product representing countless hours of hard labor and

approximately $100,000 worth of sales (Neuman, 2010) based on the results of routine sampling which

identified L. monocytogenes in environmental samples and one product sample, despite the fact that no

illnesses have been associated with the farm's products. It is hard to say whether or not these tests

actually indicate a significant risk associated with the consumption of Estrella Creamery products. More

reliable data on the prevalence of pathogens in food and incidence of food-borne illness, as well as

continuing studies on the interaction between food-borne pathogens and the immune system are needed

to provide a realistic assessment of such risks.

While emphasizing the significance of hygiene in dairy environments is always of highest

priority, it must also be recognized that the production and processing of milk is not sterile even when

the most strict hygienic standards are applied. In cheese-making, these unavoidable environmental

contaminants are called non-starter lactic acid bacteria (NSLAB), and in spite of the addition of starter

cultures, the NSLAB will eventually become the dominant cultures responsible for the ripening of

cheese (Williams et al., 2002). The tenacity of the NSLAB is illustrated well by the following excerpt

from the text Fundamentals of Cheese Science:

Using aseptic conditions, it is relatively easy to produce curd free of NSLAB, but in our experience NSLAB always grow in such cheese, sometimes only after a long lag period (eg. 100 days). A cocktail of antibiotics (penicillin, streptomycin, and nisin) extends the lag period and reduces the final number of NSLAB (Fox et al., 2000).

The authors' language is initially confusing. While they claim to have produced "curd free of NSLAB",

the eventual growth of these organisms indicates that NSLAB were present in the initial curd, but

perhaps at levels so low as to be undetectable by typical microbiological techniques. Thus, even when

using modern materials such as milking machines, and metal or plastic implements that help maintain an

aseptic environment, milk collection and processing is not a sterile. Additionally, that NSLAB were able

to grow in the presence of multiple antibiotics indicates that these populations were diverse and included

individuals or populations harboring some degree of resistance to common antibiotics. As enterococci

are common members of the NSLAB in cheese and also typically harbor intrinsic and acquired

antibiotic resistances, it is likely that they are part of the tenacious populations described by Fox et al.

(2000).

It is also important to emphasize that the presence of enterococci in milk is not a reliable

indication of fecal contamination. Enterococci are considered a reliable index for the degree to which

water sources are contaminated with the feces of warm-blooded animals (EPA, 2002). To use this same

index for milk, however, is problematic, as fecal contamination is not likely the reason enterococci are

44

so common to dairy products. Enterococci, like other bacteria, are capable of colonizing abiotic surfaces

such as milking equipment and storage tanks, and surface contamination is well recognized as the

primary source of bacteria in modern milking systems (Akam et al., 1999). Cleaning of teats before

milking and use of milking systems that minimize exposure to the air render these sources of

contamination inconsequential. Regular cleaning of milking equipment minimizes surface

contamination, but very low levels of contamination will occur even under the most hygienic conditions.

These contaminants, however, will be of a defined and established microbial ecology so long as more

variable sources of contamination such as animal feces are not predominant.

One very interesting study summarized by Franz et al. (2003) provides evidence for the existence

of a defined dairy microbiota and gives some insight into the nature of this unique microbial ecology.

The study gives insight into sources of enterococcal contamination in raw milk cheddar cheese produced

on a small family farm in Ireland. The researchers used RAPD-PCR and PFGE to type enterococcal

isolates from dairy equipment, dairy products, and human fecal isolates. Their analyses showed that 3

clones (one of E. faecalis and two of E. casseliflavus) predominated in isolates from milk, cheese, and

human feces, and that the same clones were also isolated from bulk tanks and milking equipment. They

were able to show conclusively that enterococcal contamination was not of bovine fecal origin, as only

E. faecium and Streptococcus bovis strains were isolated from this source.

It is interesting to note that of the more diverse enterococcal populations colonizing the abiotic

surfaces of dairy equipment, only a specific subset were found in frequently in both dairy products and

human feces. It is also interesting to note that two of the strains were of the species E. casseliflavus,

which is considered native to plants and not the GI tract (Tannock and Cook, 2002). The researchers

speculate that the same enterococcal clones were found in the feces of the farmers because they

consistently ate the cheese containing these bacteria, not because the farmer's feces consistently

contaminated the cheese-milk. More studies of a similar nature are needed to better characterize the

nature of enterococcal contamination in traditional cheesemaking environments, but it is interesting to

consider that it is more likely a circular relationship than a linear one. The microbial ecology of the farm

environment, the processing environment, the farmers, and the consumers of the eventual dairy product

are all connected in complex ways that feed back on one another.

The microbial ecology associated with these relationships could be called 'the dairy microbiome',

and considering the integral connection between microbial ecologies and health, the factors influencing

the dairy microbiome could have far-reaching impacts on the health of both individuals and ecological

systems. The effects of different processing methods on this ecological relationship is another area

where continuing study is warranted, as it would appear that the act of processing milk products in any

45

way may fundamentally alter the ecology of its dairy microbiome.

Even in farmstead dairying, these relationships are highly complex. As dairy systems scale up,

become more centralized, and produce products that encounter a greater number of environments via

numerous production, processing, and distribution facilities, these relationship become even more mind-

boggling. While a circular relationship of some kind is still likely to exist, the impact of individual

actions are lost in a sea of multi-systemic interactions that in the modern age are not confined to

geographic localities, but are part of a global food system. Additionally, each environment encountered

by a dairy product has the potential to influence its hygienic quality, and as the number of check-points

increases, the likelihood that a product will encounter compromised hygiene at one or more of these

check-points and present an enhanced food safety risk is also increased.

Enterococci give interesting insight into the sources of bacterial contamination in dairy products.

This same issue, however, has been brought to the forefront of more significant political battles by

another gut-associated bacterium: coliforms. In 2006, six children in California became ill with E. coli

O157:H7 that was circumstantially linked to raw milk from Organic Pastures Dairy Co., the nation's

largest and most lucrative raw milk dairy (Gumpert, 2009, 4). Although the pathogen was never isolated

directly from any Organic Pastures (OP) products, the incident provoked considerable investigation into

OP dairy by state and federal authorities. It also ignited a political debate that would eventually result in

the proposal of AB 1735, a controversial California state law that would restrict the sale of raw milk

containing more than 10 coliforms per mL on the basis that levels higher than this would indicate that

the milk had been contaminated with feces (Gumpert, 2009, 187).

Like enterococci, coliforms can colonize surfaces and do not necessarily indicate fecal

contamination. Frustrated with publicly asserted accusations by the California Department of Food and

Agriculture (CDFA), California raw milk farmer Ronald Garthwaite exclaimed, "I am so sick and tired

of the CDFA telling people that our milk is contaminated with feces. It is not true. Our milk is not

contaminated with feces." As often seems to be the case when raw milk is the topic of discussion, this

exchange would scarcely seem out of place on an elementary school playground. The language of this

dissertation attempts to encompass the complexity of the microbiological world as it relates to dairy

safety and human health, from a molecular to an ecological level. In contrast, petty arguments remain at

the forefront of the discourse between farmers and regulators on highly significant topics such as food-

borne illness and fecal contamination of milk. This clearly indicates the inability of regulatory

authorities to apply basic concepts in dairy microbiology and engage in sophisticated discourse on the

subject.

In contrast to the idea that the presence of gut-associated bacteria in milk indicates a high risk of

46

that milk causing food-borne illness, enterococcal populations may confer protection against common

milk-borne pathogens such as L. monocytogenes through the production of bacteriocins (Giraffa, 2003;

Domann et al., 2007). Protection could occur on a more general level as well. With the application of

effective hygienic practices, the dairy microbiome becomes a mostly closed loop between the farmers

and the dairy products (with a minimal amount of milking and processing equipment in between),

environmental contaminants containing the occasional pathogen are kept to minimal levels and are

unable to compete with the predominant populations of the microbial ecosystem. If hygienic milk is

intentionally allowed to sour and is repeatedly used to inoculate fresh milk, the protective effect could

become yet more defined in subsequent cultured milk products. Occasionally, pathogens native to the

farm environment are likely to contaminate the milk at very low levels. Such low level exposure to

pathogens could over time cultivate enhanced immunity in consumers of raw dairy products.

Furthermore, since the pathogens that do make their way into the milk are the same ones that are

common to the local geographic region, raw milk may function as a tailor-made source of immunity that

confers the greatest benefits to local consumers.

Claims that pasture based dairy farms do not harbor pathogens or that raw milk cannot foster the

growth of pathogens are no more accurate than the claim that bacteria in milk are invariably indicative

of fecal contamination, and neither one of these extremist attitudes contributes to an earnest effort to

maintain public health. Human pathogens are inherent in natural ecosystems, especially those that

include animals. Hygienic milking practices and proper waste management are truly the best protection

against contamination by food-borne pathogens. Hygiene is easier to implement in small-scale pasture

based dairy farms due to the personalized nature of milking and even distribution of waste that occurs

naturally as cows are rotated throughout pastures. As opposed to being a health hazard, this waste is a

valuable and rapidly utilized fertilizer that leads to healthier pastures, healthier cows, and healthier milk.

Furthermore, conscientious raw milk advocates take hygiene very seriously, and detailed

resources on hygienic practices for the safe production of raw milk have been made openly available

(FTCLDF, 2008). Most raw dairy farmers are aware of the risk associated with unpasteurized milk and

are dedicated to producing a safe product, as both their reputations and the health of their consumers

(who they may know personally) are at stake. For these reasons, raw milk produced on small-scale

pasture based farms is typically of the highest hygienic quality. Nevertheless, it is still best for the

consumer to know the farmer, to have the opportunity to ask questions and/or directly experience the

conditions on the farm and the procedures implemented to minimize risk. Centralized dairy systems,

however, afford no such opportunity, and therefore the practice of compulsory pasteurization, even if

problematic and occasionally ineffective, is truly a necessity for consumer safety.

47

Farmstead Dairy and the Ecological Integration of Modern Communities

As exhibited by the circular nature of enterococcal contamination, it appears that dairy

enterococci are actually part of a unique ecosystem involving a diversity of microbes, plants, and

animals, in addition to human stewardship and dairy processing practices. It also appears that processing

milk on the farm using traditional fermentation practices can cultivate an intimate and integrated

relationship between the dairy microbiome, the farmer, and the consumers. Finally, it seems that

ecologically minded stewardship practices also contribute to the integrity of this relationship, and may

help to build ecological health, which then extends to the health of individuals participating in that

ecosystem.

Considering the most significant issues at stake in the raw milk debate, it could be highly

beneficial to move away from the emotionally charged term 'raw milk', and move towards the use of a

term such as 'farmstead milk' that more accurately reflects the values and ecological stewardship

practices supported by advocates of raw milk. Farmstead milk products could be raw, heat-treated,

cultured, or otherwise processed on the farm according to the needs and standards of local consumers.

Let us also keep in mind that milk could be boiled at home by the consumer. This accessible, low-tech

practice is the predominant mode of protection from pathogens in other parts of the world (Gumpert,

2011[1]), and actually offers greater protection by limiting the potential for post-heat-treatment

contamination.

Farmstead dairy offers a much more intimate integration between human beings and the

ecological systems they inhabit. This does not, however, suggest the plausibility of an idealistic world

filled with farmstead milk that is healthy for all people all the time, and two important caveats must also

be taken into account. First of all, individual heterogeneity in sensitivity to foods and susceptibility to

illnesses due to both environmental and inherited factors suggests that individuals must be conscious of

how their food choices impact their personal health, and not assume that the dietary choices of a healthy

individual will also benefit their own health. Secondly, it must be considered that the act of

consumption, or any other type of interaction with the outside world, brings with it some risk of

exposure to pathogens.

Individual heterogeneity and risk are fundamental attributes of biological and ecological systems

and their eradication does not seem likely nor on the whole desirable. In order to make an informed

decision, consumers must be conscious of the multitude of environmental and individual factors that

influence the potential health impacts of dairy consumption. These include (but are not limited to) the

agricultural practices of the farm from which the milk originated and the way the milk is subsequently

48

processed, epidemiological trends of infectious diseases relevant to the geographic region, and the

resilience afforded by an individual's immune system. The acquisition of such specific information relies

on access to local foods of which the agricultural production practices can be identified, decentralized

acquisition of and access to information on the health of local populations, and individual awareness of

one's own state of health. In contrast, the federal campaign against raw milk production decreases access

to local foods and offers highly centralized national epidemiological data, the statistical analysis of

which is questionable (Gumpert, 2009, 122-123). Furthermore, it encourages consumers to assume that

all foods are safe if they are endorsed by the FDA, produced by regulated and centralized food systems,

and available at authorized retail outlets. It does not serve to encourage the challenging and often

unpleasant process of personal self-reflection that is necessary to cultivate awareness of one's state of

health. Instead, it offers consumers a convenient, but highly ineffective cop-out. While such a campaign

may be perpetuated in the name of public health, it seems detrimental to its own cause in light of the

basic requirements for informed decision making.

Unfortunately, avoiding emotionally charged terminology on this topic is a much greater

challenge, as it is a topic that is inextricably connected to the very realistic human fear that good health

is transient, and could at any moment be taken away. That this fear is realistic, though, does not justify

its Orwellian exploitation by the interconnected systems of the dairy industry, the pharmaceutical

industry, and government regulatory authorities. There is an immense amount of individual profit to be

had in the business of centralized food distribution, and perhaps even more in protecting the public from

the perils of such a system. The pursuit of individual profit, however, is not a motive that contributes to

increased health on a population level. To be clear, making war with microbes and farmers is not a

productive approach to public health.

What is needed are not systems that allow a select few individuals to gain wealth, but systems

that allow the ecological community to maintain health. Observation of natural ecologies indicates that

such systems involve sacrifice, imperfection, and risk on an individual level. Current systems protect the

public from food-borne illness in the short-term, but in doing so perpetuate centralized systems of food

production and distribution that cannot fit within an ecological framework. This is ultimately the big

issue behind the raw milk debate, and the fact that many brush off raw dairy as simply the latest "foodie

fetish" is indicative both of public ignorance regarding the current state of global food systems, and the

inability of raw milk advocates to accurately articulate the various and complex issues that are actually

at stake.

Supporters of sustainable agriculture clearly cannot make the vastly idealistic argument that

returning to the ecological framework that will make pathogens disappear, nor that such a framework

49

will bring about the end of human suffering. Instead, we must find the words to articulate a much more

difficult and realistic argument. Although modern civilizations can lay claim to many great

accomplishments, particularly in the realm of individual freedom and prosperity, these accomplishments

have often been achieved by flouting a fundamental ecological truth. If we hope to establish a truly

sustainable human culture, we must integrate within ourselves the understanding that individual

sacrifice, imperfection, and risk, ubiquitous as they are to the human experience of suffering, are also

vital to ecological health.

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Part IV: Assessment of Gelatinase Activity in Enterococci Isolated from Local Milk

Abstract

The enterococci are Gram-positive, facultative anaerobes, and are ubiquitous colonizers of the

gastro-intestinal (GI) tract of humans and other animals, an assortment of processed and fermented

foods, as well as hospitals, farms, and other domestic and wild environments. Within the last 20 years,

they have emerged as significant hospital acquired infectious agents. Within the same time frame,

research has elucidated their presence in traditional foods, reaching high levels in some kinds of

traditional cheese with no apparent ill effects, and several strains have even been tested and marketed as

probiotics. The aim of this research was to isolate naturally occurring enterococci from local dairy

products (including both raw and pasteurized milk), and screen isolates for the production of the

virulence-related factor gelatinase, which was assayed based on the ability of isolates to hydrolyze

gelatin. Isolates were successfully obtained from raw milk, while levels of viable cells in pasteurized

milk were too low to do so. Gelatinase could not be identified in isolated strains, but previous research

suggests that lab handling may silence expression of this trait despite its presence in the original isolates.

The results of this study elucidate some significant issues and challenges in food microbiology research.

In addition, this field of research poses many provocative questions in relation to hygiene, health, and

the politics of food.

Introduction

Gelatinase is a hydrolytic enzyme that can degrade a variety of substrates including gelatin,

collagen, and casein (Lopes et al., 2006). It is considered to be a virulence factor in enterococci due to

its association with clinical isolates, but it also occurs in food isolates, probably due to its ability to

break down proteins and aid in nutrient extraction in a variety of environments. The dualistic nature of

enterococci as etiological factors in both health and disease is increasingly recognized in the scientific

community, and questions regarding the conditions influencing enterococcal virulence have been raised

(Hew et al., 2007).

Enterococci are common to dairy products and are found in particularly high levels in

unpasteurized dairy products (Lopes et al., 2006) such as traditional European raw milk cheeses, in

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which their presence is considered to have a beneficial effect on cheese ripening (Giraffa, 2003).

Enterococci have also been found in pasteurized milk (Fracalanzza et al., 2007). Contrary to their

beneficial effect on traditional cheeses, in pasteurized industrial cheeses their presence is considered an

indication of poor hygiene and is associated with undesirable effects on the sensory aspects of these

products (Giraffa, 2003). This suggests that pasteurization or factors associated with it may alter

attributes of enterococcal populations. More likely, pasteurization may have broad effects on the

microbial ecology that alter the influence of enterococcal populations. In the context of questions of

food safety, and considering the fact that the expression of virulence factors may be affected by food

processing conditions (Hew et al, 2007), it is important to characterize the effect of pasteurization on

virulence determinants such as gelatinase. Therefore, this study seeks to confirm the presence of

enterococci in local raw and pasteurized dairy products and to collect data regarding the prevalence of

gelatinase in enterococcal isolates from these populations. The goal of this research is to contribute to an

understanding of virulence factors in enterococcal populations from raw and heat-treated dairy products.

In an attempt to accomplish this goal, samples of raw and pasteurized milk were plated on

selective growth media to isolate presumptive enterococci. However, only raw milk samples yielded any

growth. In order to provide evidence that isolates were indeed enterococci, they were tested for their

ability to grow in the presence of bile salts, and to hydrolyze esculin, which are both characteristic traits

of the genus. Isolates were then stored on agar slants under refrigeration for later testing. After reviving

cultures in BHI broth, isolates were tested for their ability to grow in the presence of 6.5% NaCl

(another characteristic trait of the genus), and to hydrolyze gelatin.

Materials and Methods

Preparation of Growth Media

Slanetz and Bartley (SB) growth media (Oxoid) and Bile Esculin Azide (BEA) growth media

(Acumedia) were obtained for the purposes of this experiment and prepared according to manufacturer's

instructions. Detailed preparation protocols as well as a comparative analysis of media composition are

included in the appendix. All other media utilized Brain Heart Infusion (BHI; Oxoid) as a nutrient

source. BHI storage slants were made with 1 g agar in 80 mL medium (protocol detailed in appendix).

For NaCl assays, 3 grams of agar and 13 grams of NaCl were used for 200 mL of medium. For

gelatinase assays, 3 grams of agar and 6 grams of gelatin were used for 200 mL of medium.

Obtaining and Culturing Milk Samples

Milk was collected using a mobile milking machine that had been routinely sanitized by the

59

farmer. The milk was filtered into a metal container and then poured into a heat sterilized glass jar. The

jar was immediately closed and transported by foot directly to the lab (approx. a 15 minute walk) where

it was stored at 3C. After 3 days of refrigeration a serial dilution was prepared with the milk. 1 mL

aliquots of undiluted milk (0), 1/10 diluent (-1), 1/100 diluent (-2), and 1/1000 diluent (-3) were each

plated in duplicate on previously prepared 20 mL SB agar plates. Two plates were left empty to check

for contamination. Plates were then incubated at 37C and checked daily for growth until they were

removed on for observation after 70 hours of incubation. Detailed protocols can be found in the

appendix.

100 mL SB growth media was prepared for isolation of enterococci from pasteurized milk. Aside

from the difference in volume, the mixing protocol was the same as outline in the appendix. Pasteurized

milk was purchased from a local grocer on the day of the experiment. The milk was contained in a

factory sealed plastic bottle, was stored at 3C while in the lab, and was not opened until time of

sampling. 1 mL aliquots of pasteurized milk were deposited into pour plates in several evenly distributed

droplets using a sterile pipet tip. One plate was left empty to check for contamination. Cooled but still

liquid media was then poured into the plates. Plates were then incubated at 37C and checked for growth

daily until after 4 days it became apparent that no growth would appear.

Storing Cultures on BHI Agar Slants

BHI agar slants were prepared and cultures from BEA plates were streaked onto slants and stored

under refrigeration for storage. Detailed protocol is included in the appendix. Cultures were revived in

BHI broth for gelatinase and NaCl tolerance assays.

Assays for Esculin Hydrolysis, Gelatinase Hydrolysis, and NaCl Tolerance

For all assays, isolates were streaked onto agar plates containing their respective growth media

and amendments and incubated at 37C for 24-48 hours. A detailed protocol for streaking on BEA agar

plates is included in the appendix. All other assays utilize this streaking procedure aside from the fact

that in subsequent assays, each isolate was streaked on an individual plate, instead of splitting plates in

half as in the case of BEA. Criteria for assay interpretation are as follows: Isolates were considered

positive for esculin hydrolysis if a black halo appeared around colonies after incubation. Isolates were

considered positive for gelatinase if a clear halo appeared around colonies after incubation. Isolates were

considered NaCl tolerant if colonies appeared after incubation.

Results

60

Only undiluted samples displayed any growth, one of which grew 2 colonies while the other

grew 6. One of the 1/10 plates had a very small black dot that could potentially be a colony, but this is

unclear as it does not look similar to the colonies on the 0 plates. These colonies are a very deep red

color and are ringed in pinkish white. The shape of the colonies was diverse, ranging from relatively

uniform circles, to indented patterns reminiscent of flower petals, to more irregular splotchy shapes. One

colony showed a particularly interesting distinguishing feature. It had grown in a visible film of dried

milk solids and there appeared to be a semi-translucent halo ringing the colony, which could be a

preliminary indication of gelatinase activity. Attempted isolation of enterococci from pasteurized milk

using SB growth media yielded no results.

Substantial growth was apparent on BEA agar

plates 24 hours after inoculation with SB isolates.

Large black splotches appeared on all of the plates.

Some colonies were successfully isolated in more

dilute streaks and all of these were surrounded by

black halos. This indicates that all of the colonies on

the initial SB plates were capable of hydrolyzing

esculin. While this evidence is not conclusive, it does

support the hypothesis that colonies isolated from SB

agar are of the genus Enterococcus.

Only 7 of the 10 isolates showed turbidity after inoculation in BHI broth. Samples of broth from

all tubes were still plated, but indeed the 3 isolates that did not show turbidity also did not show growth

on agar, aside from two individual colonies on two separate gelatin agar plates, which were determined

to be contaminants. Of the isolates that were successfully recovered, all of them tolerated 6.5% NaCl.

However, no zones of clearance whatsoever were observed around any of the colonies growing on

gelatin agar. The results of all 3 assays are summarized in table 1.

Discussion

In order to interpret the results, an understanding of the composition and function of the media

used is necessary. A detailed list of the contents of the two media is included in the appendix as a

reference.

In some ways, SB agar and BEA agar are very similar in content, but there are distinct

differences as well. Both contain some form of enzymatically digested proteins and yeast extract, which

are typical staples in growth media as sources of nitrogen and various other nutrients. In SB medium,

61

Isolate Esculin 6.5% NaCl GelRME1 + + - RME2 no growth no growth contaminatedRME3 no growth no growth contaminatedRME4 + + - RME5 + + - RME6 + + - RME7 + + - RME8 no growth no growth no growthRME9 + + -

RME10 + + - Table 1: Raw Milk Enterococcus (RME) 1-10 were assayed for esculin hydrolysis, NaCl tolerance, and gelatinase activity. Positive assay (+), negative assay (-), inability to revive cultures after storage ("no growth"), and suspected contamination ("contaminated") are indicated.

tryptose is the main nitrogen source. Tryptose is considered superior to meat peptone as a single source

of nitrogen for fastidious organisms (Bacto™ Tryptose Product Information). This may be why BEA

medium contains two major protein sources. One of these is an enzymatic digest of casein, or milk

protein. While this may not be nutritionally sufficient for the diversity of fastidious organisms that

tryptose is, it makes sense that it would be used for culturing enterococci, which have metabolic

capabilities that are well adapted to the dairy environment. BEA medium also contains yeast enriched

meat peptone as a secondary source of nitrogen as well as a variety of other nutrients associated with

yeast. Perhaps most importantly are B-complex vitamins, which are abundant in yeast extract and are

essential nutrients for the growth of enterococci. SB medium contains yeast extract as a separate

addition. The total amount of protein and yeast extract in SB medium is 25 g/L, while BEA contains

34.5 g/L, making it a more nutrient rich medium.

SB medium also contains 2 g/L glucose, a common monosaccharide that is used by many

microorganisms as a source of carbon and energy. Using this specific monosaccharide and excluding

others might also help to select for certain organisms. BEA medium does not contain any sugars, but it

does contain both sodium citrate (1 g/L) and ferric ammonium citrate (0.5 g/L), which could potentially

serve as a source of carbon and energy, as enterococci are capable of metabolizing citrate. These salts

also perform other functions, though. They can act as a buffer to maintain the medium at a stable pH. SB

medium also includes 4 g/L of buffer salts in the form of di-potassium hydrogen phosphate.

Ferric ammonium citrate performs a unique function in BEA medium. It reacts with hydrolyzed

esculin forming a black pigment which can be observed as a halo around colonies. In this way colonies

that can hydrolyze esculin (a distinguishing feature of enterococci) can be differentiated from those that

do not. This is the main function that esculin serves in BEA medium. SB medium contains a visual

enhancer as well. Tetrazolium chloride (0.1 g/L) is added because it is reduced inside of bacterial cells

forming a red pigment. This coloration is apparent in the pink-dark red appearance of colonies that form

on this medium and makes them easier to see, especially if many need to be counted for enumeration.

The other main components of these media (besides agar, which is used simply to keep the medium

solid at room temperature) serve as environmental stressors or toxins to select for enterococci. Both

media contain sodium azide, which is highly toxic to aerobic organisms and helps select for enterococci,

which are facultative anaerobes. SB medium, however, contains 0.4 g/L; this is nearly twice as much as

BEA medium, which contains 0.25 g/L. Additionally, much of the sodium azide in BEA medium is

inactivated in the autoclave, where high temperature and pressure cause it to react with other

components in the medium. Thus, in reality, the active amount of sodium azide in BEA is significantly

less than that reported in its initial contents.

62

The heat susceptibility of sodium azide is the main reason why SB medium is gently heated

instead of being autoclaved, since SB medium is designed for enumeration of enterococci from water

samples, which could contain a vast diversity of microbes both aerobic and anaerobic. Therefore

stronger selection against aerobes might be beneficial. Interestingly, this is the only selective agent in the

medium. Perhaps the specificity and sparsity of nutrients in SB media acts sufficiently to select for

enterococci. In addition to sodium azide, BEA medium contains oxbile. This helps select for enteric

organisms, which must be able to tolerate environmental stressors in the GI tract such as bile salts and

acids. Finally, BEA medium contains 5 g/L of NaCl, which selects for enterococci due to their

remarkable salt tolerance. At approximately 7.2 and 7.1 respectively, these media have very similar pH

profiles, both being relatively neutral.

The results of the serial dilution suggest a very low level of enterococcal contamination in this

sample of fresh raw milk. The deep red coloration and white border of colonies is consistent with

expectations and indicates that these are likely colonies of Enterococcus species. A simple average of the

number of colonies suggests that there are approximately 4 CFUs of enterococci per mL of milk in this

sample. However, the number of colonies on both plates was so low that these estimates are not really

statistically valid. Furthermore, the variation in colony shape may suggest that one "colony forming

unit" could actually consist of two or more aggregated cells, especially given the tendency of

enterococci to from pairs, chains, and clumps.

That enterococci were undetectable in pasteurized milk samples suggests that low levels of

contamination in raw milk are reduced further by the process of pasteurization. This alone is an

interesting result, given the hypothesis that low level contamination of raw milk is in part responsible for

its health benefits. The capacity of pasteurization to reduce low level contamination to undetectable

contamination could therefore have a significant effect on its heath benefits. Reduced exposure to low

levels of enterococci may reduce the ability of the immune system to keep these ubiquitous infectious

agents in check, and pre-dispose individuals consuming pasteurized milk to enterococcal infection.

Colonies isolated from raw milk on SB agar and transferred to BEA agar did have the ability to

hydrolyze esculin. This is another positive indicator that enterococci were successfully isolated. In

routine enumerations, such as in water quality testing, these parameters are considered enough to assume

that the isolates are enterococci (Condalab, 2011). More in depth identification methods, such as

metabolic and serological assays, or protein-based and genetic fingerprinting, are used in scientific

studies to identify further to the species and strain level (Domig et al., 2003). Whether or not any of

these methods will be necessary will depend on future experimental goals.

The follow-up assay provides some interesting results as well. The ability of all isolates to

63

tolerate 6.5% NaCl is further evidence indicating their identity as enterococci, and suggests that they

would likely persist in cheeses produced with this milk. If aged sufficiently, enterococci could

potentially become a dominant organism. The results of the gelatinase assay did not indicate any

gelatinase activity in these isolates, but for now these results must be considered inconclusive. Although

the Gel+ phenotype is more prevalent in clinical strains than in food strains (Semedo et al., 2003), there

is strong evidence to suggest that enterococci in dairy products do produce gelatinase to some extent,

and that this expression my be silenced as a result of lab handling (Lopes et al., 2006). It is therefore

possible that some of the isolates obtained in this study had a Gel+ phenotype when they were initially

isolated, but lost this phenotype upon repeated sub-culturing and prolonged storage. This hypothesis is

supported by the observation that one of the colonies isolated on SB agar had grown in a film of dried

milk solids and formed a zone of clearance, which could be an indicator of proteinase activity,

potentially gelatinase. Finally, with regards to the results of this and other assays, the presence of

suspected contaminants on two separate gelatin agar plates suggests the potential for other unknown

contaminants, which must be considered when interpreting the results.

Gelatinase has been identified in both food and clinical isolates (Semedo et al, 2003), and is one

of the most well studied virulence factors in enterococci. Other virulence factors may provide more

promising areas of research. Although the production of capsular polysaccharide has been identified in

some enterococci, there is far less data in the primary literature on this subject. In particular, the

prevalence of capsular polysaccharide in food isolates does not appear to be addressed. Since capsule

production helps enterococci evade opsonization by phagocytes, a hypothesis could be formed that this

trait would be less common in isolates obtained from products made with pasteurized milk, as opposed

to raw milk, which still contains living phagocytes (Silanikove, 2008).

Even if this were the case, however, it does not necessarily suggest that enterococci from heat

treated cultured products are less dangerous. Although capsule is well defined as a factor that aids

immune evasion and therefore contributes to virulence, the expression of capsular polysaccharide by the

probiotic strain Symbioflor® 1 (Domann et al., 2007) indicates that capsule production and probiotic

effect are not mutually exclusive phenomena. On the contrary, it suggests that the presence of capsule in

naturally occurring populations of enterococci may actually be a boon to health. Further studies

regarding the interaction between encapsulated enterococci and the immune system both in vitro and in

vivo could be useful in characterizing the factors influencing the effect of enterococci on health.

Gelatinase, on the other hand, is absent from Symbioflor® 1, and there is evidence to suggest (as

argued in the preceding dissertation on enterococcal virulence) that capsule and gelatinase could have a

synergistic effect. Therefore, an interesting follow-up study might involve obtaining enterococcal

64

isolates from both raw and pasteurized dairy products that have been cultured at 37C and testing for the

prevalence of both capsule and gelatinase production. Points of interest in this study would include

comparing the prevalence of these traits in populations cultured in raw and pasteurized milk, and

analyzing the relationship between the two traits. Do the traits ever occur in the same isolates? If so, is

there any statistical correlation between the two traits? All of these questions could be answered with

more extensive follow-up studies, indicating the need for further research on the subject.

Sources Cited

Bacto™ Tryptose Product Information. Accessed on 4/27/2011 at http://www.bd.com/ds/technicalCenter/inserts/Bacto_Tryptose.pdf.

Condalab catalogue. Slanetz-Bartley Medium: For the Detection and Enumeration of Enterococci by the Membrane Filtration Technique. Accessed on 4/17/2011 at http://www.condalab.com/pdf/1109.pdf.

Domann, E., Hain, T., Ghai, R., Billion, A., Kuenne, C., Zimmermann, K., and Chakraborty, T. (2007). Comparative Genomic Analysis for the Presence of Potential Enterococcal Virulence Factors in the Probiotic Enterococcus faecalis Strain Symbioflor 1. International Journal of Medical Microbiology 297: 533-539.

Domig, K.J., Mayer, H.K., and Kneifel, W. (2003). Methods Used for the Isolation, Enumeration, Characterisation and Identification of Enterococcus spp. 2. Pheno- and Genotypic Criteria. International Journal of Food Microbiology 88: 165-188.

Fracalanzza S.A.P., Scheidegger, E.M.D., Santos, P.F., Leite, P.C., and Teixeira, L.M. (2007). Antimicrobial Resistance Profiles of Enterococci Isolated from Poultry Meat and Pasteurized Milk in Rio de Janeiro, Brazil. Memórias do Instituto Oswaldo Cruz 102(7): 853-859.

Giraffa, G. (2003). Functionality of Enterococci in Dairy Products. International Journal of Food Microbiology 88: 215-222.

Hew, C.M., Korakli, M., and Vogel, R.F. (2007). Expression of Virulence-Related Genes by Enterococcus faecalis in Response to Different Environments. Systematic and Applied Microbiology 30: 257-267.

Lopes, M.F.S., Simoes, A.P., Tenreiro, R., Marques, J.J.F., and Crespo, M.T.B. (2006). Activity and Expression of a Virulence Factor, Gelatinase, in Dairy Enterococci. International Journal of Food Microbiology 112: 208-214.

Semedo, T., Santos, M.A., Lopes, M.F.S., Marques, J.J.F., Crespo, M.T.B., and Tenreiro, R. (2003). Virulence Factors in Food, Clinical and Reference Enterococci: A Common Trait in the Genus? Systematic and Applied Microbiology 26: 13-22.

Silanikove, N. (2008). Milk Lipoprotein Membranes and Their Imperative Enzymes. From Bioactive Components of Milk. Bosze, Z. (ed). Springer: 143-161.

65

Appendix

Mixing SB agar selective growth medium for isolation and enumeration of enterococci Materials: latex gloves, mask, goggles, hot plate, stir bar, metal spatula, thermometer, 500 mL graduated cylinder, 500 mL beaker, bunsen burner, striker, Slanetz and Bartley (SB) pre-mixed selective growth media, scale, weigh paper, ultra-pure water (UPW), aluminum foil

For 200 mL SB growth medium:1. Put on gloves, a mask and goggles to avoid exposure to sodium azide. 2. Set up hot plate underneath a vented hood, turn on the hood. 3. Light the bunsen burner. 4. Gather stir bar, a metal spatula, a thermometer, a 500 mL graduated cylinder, and a 500 mL beaker. Briefly flame to sterilize. 5. Using the spatula, gently spoon 10.5 g of dry SB mixed media onto a scale with weigh paper. Take care to do this gently, producing as little dust as possible. 6. Dump the dry mix into a 500 mL beaker, taking care to cover the beaker for a moment with the weigh paper and a gloved hand while the dust settles. 7. Carefully transfer the beaker into the hood. 8. Measure 250 mL UPW in a graduated cylinder and pour into the beaker under the hood. 9. Stir to mix with the metal spatula. 10. Place the spatula on used weigh paper to minimize spread of toxic SB media. 11. Place the stir bar in the beaker and place the beaker on the hot plate. 12. Take a piece of aluminum foil and use it to cover the beaker. 13. Turn the heat on high and use the magnetic stir mechanism to create a vortex in the fluid. 14. Monitoring temperature with the thermometer, heat until there is no more visible undissolved powder and bubbles begin to appear at the bottom of the beaker (about 90C). 15. Remove from heat and let stand uncovered with a thermometer, checking the temperature periodically until the it reaches approximately 40C. 16. Arrange 10 petri plates in stacks of 2 or 3 underneath the hood. 17. Pour liquid SB media into petri plates, taking care to not lift the lids for very long.18. Make sure to leave a note indicating the toxicity of the media. 19. Allow to stand and cool until agar has firmly set.

Mixing Phosphate Buffered Saline (PBS) for serial dilutions of refrigerated raw milk Materials: NaCl, KCl, Na2HPO4 , KH2PO4, scale, weigh paper, ultra-pure water (UPW), HCl solution, NaOH solution, 500 mL graduated cylinder, 500 mL pyrex bottle with cap, pH meter, pipette

1. Using a scale and weigh paper, measure 8.00 g of NaCl, 0.20 g of KCl, 1.44 g of Na2HPO4, and 0.24 g of KH2PO4, mixing them together in a 500 mL pyrex bottle. 2. Measure 400 mL of UPW in a graduated cylinder and pour into the bottle to dissolve buffer salts. Use a plastic stir rod to break up chunks if needed. 3. Measure pH and adjust to 7.4 using a pipette and solutions of HCl or NaOH. 4. Pour buffered solution into a graduated cylinder and add UPW to 500 mL 5. Return buffered solution to the bottle. Cap, label, and store under refrigeration. Autoclave before use.

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Serial Dilution of Raw Milk from Hampshire College Farm Center Materials: phosphate buffered saline (PBS), 6 test tubes with caps, test tube rack, 50 mL beaker, micropipette, 1 mL sterile pipette tips, bunsen burner, striker, glass hockey stick spreader, 10 petri plates prepared with Slanetz and Bartley selective growth media, marker for labeling, ethanol, kimwipes, parafilm, raw milk (collected from HCFC and stored under refrigeration)

1. Place 6 test tubes each filled with 9 mL PBS in a rack and lightly place the caps on top. 2. Autoclave this along with a 50 mL beaker to sterilize. 3. Begin to prepare the workspace by lighting the bunsen burner. 4. Label tubes 3 tubes: -1, -2, -3. Label the other 3 tubes: -1*, -2*, -3*. 5. Label 5 SB agar plates: raw milk, -1, -2, -3, and control. Do the same for the other 5 plates, differentiating with * in the same fashion as the second set of tubes. 6. Arrange the plates in front of the test tube rack so that all are easily accessible. 7. Fill the beaker with refrigerated milk and place it next to the test tube rack. 8. Take the pipette with a sterile tip and transfer 1 mL raw milk from the beaker onto each of the corresponding petri plates. 9. Uncap and flame tube -1, then transfer 1 mL raw milk into the tube. Flame, recap, swirl to mix, and place in rack. 10. Repeat step 9 for tube -1*. 11. Discard the used pipette tip and replace with a new sterile tip. 12. Uncap and flame tube -1 and place in rack uncapped. 13. Transfer 1 mL diluent from tube -1 onto the corresponding petri plate. 14. Uncap and flame tube -2, then transfer 1 mL diluent from tube -1 into tube -2. Flame, recap, swirl to mix and place in rack. 15. Recap tube -1. 16. Repeat steps 11-15 using tubes -1* and -2*; tubes -2 and -3; and tubes -2* and -3*. 17. Repeat steps 11-13 using tubes -3 and -3*. 18. Surface sterilize the hockey stick spreader first with a kimwipe and ethanol, and then by briefly flaming and waiting a moment for it to cool. 19. Use the sterilized hockey stick to spread the sample over plates labeled raw milk and raw milk*. 20. Repeat steps 18 and 19 for plates -1 and -1*; plates -2 and -2*; plates -3 and -3*; and both control plates. 21. Seal petri plates with parafilm and incubate upside down at 37C for 70 hours.

Improved Raw Milk Serial Dilution Procedure Proposal Materials: see above procedure

1. Place 3 test tubes each filled with 9 mL PBS in a rack and lightly place the caps on top. 2. Autoclave this along with a 50 mL beaker to sterilize. 3. Begin to prepare the workspace by lighting the bunsen burner. 4. Label the tubes -1, -2, and -3. 5. Label 5 SB agar plates: 0, -1, -2, -3, and C. Label the other 5 plates: 0*, -1*, -2*, -3*, and C*. 6. Arrange the plates in front of the test tube rack so that all are easily accessible. 7. Fill the beaker with refrigerated milk and place it next to the test tube rack. 8. Take the pipette with a sterile tip and transfer 1 mL raw milk from the beaker onto petri plates 0 and 0*.

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9. Uncap and flame tube -1, then transfer 1 mL raw milk into the tube. Flame, recap, swirl to mix, and place in rack. 10.Discard the used pipette tip and with a sterile tip, transfer one mL of diluent from tube -1 to tube -2 using aseptic technique. 11. Repeat step 10 using tubes -2 and -3 to finish the dilution series. 12.Discard the pipette tip used for the dilution series and with a sterile tip transfer 1 mL of diluent from tube -3 onto both plate -3 and -3*. 13.Repeat step 12 for tubes -2 and -1, transferring onto their respective plates. 14.Surface sterilize the hockey stick spreader first with a kimwipe and ethanol, and then by briefly flaming and waiting a moment for it to cool. 15.Starting with plates C and C* (which contain no sample), and working up through the dilution series, use the sterilized hockey stick to evenly spread the samples over the agar media. 16.Seal petri plates with parafilm and incubate upside down at 37C for 70 hours.

Mixing Bile Esculin Azide agar for confirmation of suspected enterococcal colonies on SB mediumMaterials: gloves, goggles, bile esculin azide (BEA) premixed growth media, metal spatula, scale, weighing paper, hot plate, magnetic stir bar and magnetic rod for removal, 500 mL beaker, distilled water, aluminum foil, bunsen burner, striker, plastic petri plates

For 100 mL BEA growth media: 1. Assemble materials underneath a vented hood. 2. Weigh out 5.6 g premixed BEA media. Use a metal spatula to gently spoon the mix onto weighing paper. 3. Transfer the mix into a 500 mL beaker. 4. Use a volumetric flask to measure 100 mL of distilled water and add this to the beaker. 5. Use the metal spatula to stir and try to break up any large chunks. 6. Place the beaker on a hot plate and turn the heat on high. 7. Place a magnetic stir bar in the beaker and turn on the stir function to create a vortex. 8. Cover the beaker with aluminum foil. 9. Allow to heat to a boil, periodically turning off the stir function in order to check for the formation of bubbles indicating boiling temperature. 10.Continue heating at a boil for 1 minute or until there is no more undissolved powder remaining in the solution. 11. Remove the magnetic stir bar and replace aluminum foil. 12.Autoclave the beaker for 15 minutes at 121C. 13.Wait for the mixture to cool so it won't melt the plastic petri plates. 14.Before pouring the plates, light a bunsen burner. 15.Working around the flame, remove the aluminum foil from the beaker. Pour the molten media into 4 plastic petri plates, taking care not to lift the lids for very long. 16.Leave a note to indicate toxicity of the media. Allow to cool until set.

Streaking BEA agar plates to isolate colonies and confirm esculin hydrolysis Materials: previously prepared BEA agar plates, inoculating loop, enterococcal colonies previously cultured on SB agar, bunsen burner, striker, marker, parafilm

1. Set up the work station underneath the hood by assembling a bunsen burner, previously cultured SB agar plates, previously prepared BEA agar plates, and an inoculating loop.

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2. Use a marker to divide the BEA agar plates in half, drawing a line down the center. Label the plates with initials, date, and media type. 3. Light the bunsen burner and turn on the hood. 4. Wash hands. 5. Thoroughly flame the inoculating loop to sterilize and set aside to cool. 6. Remove parafilm from colonized SB agar plates. 7. Briefly flame inoculating loop to sterilize and wait a moment for it to cool. 8. Prod a colony with the inoculating loop, making sure some of the sample sticks to the loop. 9. Streak the sample over half of a BEA agar plate using the following procedure. 10.At the top of the semi-circular plate section, thoroughly smear the sample back and forth over a small area. 11. Briefly flame the loop to sterilize and allow to cool. 12.Pull the loop through a section of the previously smeared sample, then thoroughly smear this diluted sample back and forth over a small area. 13.Briefly flame the loop to sterilize and allow to cool. 14.Pull the loop through a section of the second smear, then utilizing the rest of the space available, gently streak the loop back and forth across the media, taking care NOT to overlap with any previously smeared sections. 15.Repeat steps 7-14 until all desired colonies from SB agar plates have been smeared on BEA agar plates. 16.Wrap BEA agar plates in a double layer of parafilm. 17.Incubate upside down for 24 hours at 37C.

Preparing and Streaking BHI Agar Slants to Store Enterococcal CulturesMaterials: 8 screw cap tubes, test tube rack, slant boards, brain heart infusion (BHI), agar, ultra-pure water (UPW), graduated cylinder, 500 mL beaker, hot plate, magnetic stir bar and magnetic rod for removal, scale, weighing paper, metal spatula, marker, tape, inoculating loop, bunsen burner, striker, previously prepared enterococcal cultures on BEA agar plates, parafilm

1. Weigh out 2.96 g BHI and 1 g agar and place in a 250 mL beaker.2. Add 80 mL UPW.3. Add a magnetic stir bar and place on a hot plate.4. Turn the hot plate on medium-high heat and initiate the stir mechanism to form a vortex.5. Allow to heat with agitation until agar is melted and BHI is completely dissolved.6. While you wait Arrange 8 screw cap tubes in a test tube rack.7. Using a graduated cylinder, measure 10 mL of UPW and pour it into the first screw cap tube.

Then use a marker to indicate approximately 10 mL of volume.8. Transfer this water into the next tube, again using a marker to indicate the 10 mL mark.9. Repeat step 9 until all the tubes have been marked and dispose of the 10 mL of water.10. Now remove the beaker from the hot plate and remove the magnetic stir bar.11. Decant approximately 10 mL of the liquid BHI agar into each of the 8 tubes.12. Loosely cap the tubes and autoclave to sterilize.13. Place the tubes on a slant board and allow to cool until fully set.14. Label the tubes (initial, date, 'BHI - enterococci').15. Assemble a bunsen burner, an inoculating loop, the cultured BEA agar plates, and the BHI agar

slants underneath the hood and light the bunsen burner.16. Thoroughly wash and dry hands.17. Thoroughly flame the inoculating loop to sterilize, then set aside to cool.18. Remove the parafilm from the BEA agar plates and place them in an accessible area.

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19. Briefly flame the first tube and remove the cap.20. Briefly flame the inoculating loop and allow to cool before using it to pick a colony from one

half of a BEA agar plate.21. Use the inoculating loop to streak the culture across the agar.22. Briefly flame the tube and recap. Place the tube back on the slant board.23. Repeat step 19-22 for subsequent tubes until all have been streaked.24. Thoroughly parafilm the tubes.25. Incubate for 24-48 hours at 37 C.26. Place in a labelled container (initials, date, 'potential pathogens') and store at 4 C.

Enterococcal Growth Media - Constituents

Slanetz and Bartley - Formula / Liter

Tryptose..................................................................................20 g An enzymatic digest of protein useful for culturing microbes that, like enterococci, can be difficult to grow due fastidious nutrient requirements. Yeast extract.............................................................................5 g Prepared from autolyzed yeast cells. Provides a variety of common microbial nutrients such as nitrogen, carbon, amino acids and vitamins. An especially good source of B-complex vitamins, some of which are essential to enterococcal growth. Glucose.....................................................................................2 g A common monosaccharide. Source of energy and carbon. Di-potassium hydrogen phosphate...........................................4 g Probably added as a pH buffer. May also serve as a nutrient source. Sodium Azide.........................................................................0.4 g Highly toxic to aerobic organisms, selects for anaerobic organisms. Tetrazolium chloride...............................................................0.1 g Reduction of this compound withing bacterial cells results in red coloration of colonies, making them easier to identify. Agar........................................................................................10 g Polysaccharide isolated from algal growth. Allows media to solidify at room temperature. Final pH 7.2 ± 0.2 at 25°C

Bile Esculine Azide - Formula / Liter

Enzymatic Digest of Casein....................................................25 g Nutrient source for bacterial growth. Since enterococci are commonly found in dairy environments, casein probably meets their nutritional needs quite well. Yeast Enriched Meat Peptone...............................................9.5 g Meat peptone is also an enzymatic digest of protein derived from animal tissues that serves as a nutrient source. It is likely enriched with yeast extract in order to provide essential nutrients such as B- complex vitamins. Oxbile........................................................................................1 gDehydrated fresh bile, selects for bile tolerant enteric organisms. Sodium Chloride........................................................................5 g Selects for NaCl tolerant organisms.Sodium Citrate..........................................................................1 g May be added as a pH buffer as well as a source of energy and carbon, as enterococci are able to

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metabolize citrate. Ferric Ammonium Citrate.......................................................0.5 g May be added as a pH buffer as well as a source of iron and citrate. Reacts with hydrolyzed esculin to produce the black halo useful for identifying colonies able to hydrolyze esculin. Esculin.......................................................................................1 g An organic compound found in plants. Esculin hydrolysis is a useful trait in differentiating enterococci from related organisms. A black halo is formed around colonies that hydrolyze esculin. Sodium Azide.......................................................................0.25 g Agar.........................................................................................14 g Final pH: 7.1 ± 0.2 at 25°C

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