GHENT UNIVERSITY FACULTY OF VETERINARY MEDICINE AZ …

GHENT UNIVERSITY

FACULTY OF VETERINARY MEDICINE

AZ Sint-Lucas

Academic year 2014- 2015

“Comparison of antimicrobial resistance profiles of commensal Escherichia coli obtained from

different animal, human and environmental niches.”

by

Karel Jacobs

Promoters:

Prof. Dr. J. Dewulf Research Report as part of

Dr. F. Boyen the Master’s Dissertation

Dr. A. Van den Abeele

© 2015 Karel Jacobs

Universiteit Gent, its employees and/or students, give no warranty that the information provided in this

thesis is accurate or exhaustive, nor that the content of this thesis will not constitute or result in any

infringement of third-party rights. Universiteit Gent, its employees and/or students do not accept any

liability or responsibility for any use which may be made of the content or information given in the thesis,

nor for any reliance which may be placed on any advice or information provided in this thesis..

1. Preface

This thesis would not have been possible without the participation of so many people at so

many different levels. First of all l thank the people who are not daily engaged in research

activities but that were willing to cooperate. Thanks to the farmers and dog owners, to supply

with the samples from their livestock or beloved pet. Thanks to the small animal veterinarians

who deliberately committed themselves to convince dog owners and particularly, thanks to the

new residents, nursery and executive staff of the nursing home ‘Veilige Have’. Without the

support of all these volunteers, I would have been nowhere.

Secondly I have to thank the staff of the two laboratories where the microbiological

examinations took place. Thanks to all the people of the bacteriology and mycology lab of my

faculty with special thanks to Dr. Filip Boyen, for all the brainstorming, the assistance during

my assays and the guidance in my research. Also, many thanks to all the people of the clinical

lab of AZ Sint-Lucas, for the time and dedication they put in the laboratory work, next to their

daily assignments. Special thanks to Dr. Annemie Van den Abeele. For her willingness to work

together with my faculty for my master’s dissertation and for her flexibility. Her punctuality and

dedication motivated me to set high standards for this thesis.

Many thanks to professor Dewulf, my promoter. He gave me the opportunity to develop a brand

new consortium for my research making adjustments where needed. He also gave me the

opportunity to participate at my first conference - he himself organized - which was one of the

highlights in my last year as a vet student. Next to the academic support, the student

association VDK also could count on his assistance he fully provided for our plans to build a

new student facility building at our faculty, and on the support and sympathy in general. In the

name of all members of the student association, thank you very much.

Finally, I would like to thank my friends and family. Thanks to my dear friend Boudewijn Catry,

for the academic support over the past years, but much more important, to be the decisive

factor in the best decision I ever took in my life, namely to start with Vet School. Many thanks

to my oldest friends from Leuven and my friends here in Ghent, for the many incredible

moments over the past 6 years. While mentioning this groups of friends, I cannot forget to

thank my student association VDK as well as my youth movement KLJ Nieuwrode. For all the

friendship, for all the opportunities and for all the things I learned. I will conclude with the most

important acknowledgement, in particular that of my parents. Their everlasting believe in me

and support in whatever I want to undertake: thanks, not only for the last 6 years, but for my

whole life.

2. Table of Contents

1. Preface

2. Table of Contents

3. Resume & Keywords ........................................................................................................................ 1

4. Introduction ..................................................................................................................................... 2

4.1 Antimicrobial resistance .......................................................................................................... 2

4.1.1 Transmission of antimicrobial resistance between animals and humans....................... 2

4.1.2 Transmission between human and animal due to (in)direct pathways .......................... 5

4.2 Research Question ................................................................................................................ 14

4.3 Objective of the research ...................................................................................................... 14

5. Materials and Methods ................................................................................................................. 15

5.1 Study design .......................................................................................................................... 15

5.1.1 Human ........................................................................................................................... 15

5.1.2 Environmental ............................................................................................................... 15

5.1.3 Animal ............................................................................................................................ 15

5.2 Study period .......................................................................................................................... 15

5.3 Subjects/Samples .................................................................................................................. 16

5.3.1 Human Samples ............................................................................................................. 16

5.3.2 Animal Samples ............................................................................................................. 16

5.4 Sampling ................................................................................................................................ 16

5.4.1 Human Samples ............................................................................................................. 16

5.4.2 Environment Samples .................................................................................................... 17

5.4.3 Animal Samples ............................................................................................................. 17

5.5 Preservation and transport of the samples ........................................................................... 19

5.6 Microbiology .......................................................................................................................... 19

5.6.1 Isolation & Purification .................................................................................................. 19

5.6.2 Identification ................................................................................................................. 19

5.6.3 Antimicrobial Susceptibility testing ............................................................................... 19

5.7 Data management and statistics ........................................................................................... 22

5.7.1 Ethical issues .................................................................................................................. 22

5.7.2 Interviews ...................................................................................................................... 22

5.7.3 Statistical methods ........................................................................................................ 22

6. Results ........................................................................................................................................... 25

6.1 Sampling and isolation ................................................................................................................ 25

6.2 Antimicrobial susceptibility ......................................................................................................... 27

6.1.1 The ARI Index on the level of Antimicrobial Classes ..................................................... 30

6.1.4 MDR (multi-drug resistance) ................................................................................................ 31

6.1.1 Antimicrobial resistance profiles ................................................................................... 33

6.1.2 Specific phenotypical patterns ...................................................................................... 33

6.2 Interviews .............................................................................................................................. 33

7. Discussion ...................................................................................................................................... 34

7.1 The strength of this study...................................................................................................... 34

7.2 The materials and methods applied ...................................................................................... 34

7.2.1 Advantages .................................................................................................................... 34

7.2.2 Limitations ..................................................................................................................... 35

7.3 Antimicrobial resistance ........................................................................................................ 35

7.4 Conclusion ............................................................................................................................. 37

8. References ..................................................................................................................................... 38

9. Annexes ......................................................................................................................................... 47

9.1 Annex 1: Instruction letter and form for broiler famers. ...................................................... 47

9.2 Annex 2: Form citizens of nursing home ............................................................................... 48

9.3 Annex 3: Dutch summary ...................................................................................................... 49

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3. Resume & Keywords

Since the first use of antimicrobial agents for the treatment of bacterial infections, the problem of

acquired resistance has been notified. In both veterinary and human medicine, the prevalence of

resistant strains rises year after year. This could be a public health risk, especially when there is

transmission of antimicrobial resistance from one species to another and in particular to human. The

process of interspecies transmission and the factors involved are poorly understood, in particular the

epidemiological link between environment, food producing animals and elderly that often suffer from

healthcare-associated infections. This research focused on investigating the resistance levels in

different ecological niches as livestock, surface water, nursing home residents and pets. Samples were

taken from mixed livestock manure, nursing home individuals and dog feces, and surface water in one

well demarcated area (Aalter, Belgium) and specified time period (2014-2015). E. coli were isolated and

purified with standard techniques and identified by MALDI-TOF. By using a combination of disk diffusion

and phoenix MIC-determination, resistant profiles were created. Results clearly showed that high levels

of resistance against critically classified antimicrobial classes such as aminoglycosides, tetracyclines,

fluoroquinolones, and 3rd and 4th generation cephalosporin, were seen in the broiler, human and pork

population. Further substantial similarities in resistant levels, multidrug resistance and the antimicrobial

resistant index between the poultry, swine and nursing home residents strongly suggested an

association between these ecological niches with consequence for public health. The findings in

respectively pig and broiler population of tigecycline and fosfomycine resistance, two so called last resort

antimicrobial classes that are not registered for veterinary use, was unexpected and furthermore

worrisome. Resistance to polymyxins (colistin) and carbapenems was not detected. Further research

that examines the transmission between these ecological niches should be encouraged. Additionally,

molecular investigations to identify the resistance determinants and mode of transmission that explain

especially the resistance against tigecycline and fosfomycine, is required.

Keywords: antimicrobial transmission - resistance profiles – ecological niche – indicator bacteria –

Escherichia coli

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4. Introduction

4.1 Antimicrobial resistance

Since the introduction of antimicrobial agents in medicine, the problem of antimicrobial resistance has

been acknowledged. Soon after the introduction of penicillin as an antimicrobial drug, researchers first

described resistance against penicillin for the target pathogens. For many antimicrobial agents that

came next on the market, the same problem occurred (Alanis et al., 2005). Because of a rise in invasive

medical procedures, the increase in immune compromised individuals, the excessive use as a result of

the population boom and an improved access to healthcare worldwide, the selection pressure by

antimicrobials and therewith the spread of resistance increased over the last decades. A recent

systematic review and meta-analysis, including 243 studies on the use of antimicrobials in human health,

concluded that there is a clear association between antimicrobial consumption and the subsequent

development of bacterial resistance (Bell et al, 2014). A recent study -combining published information

of 7 EU countries on animal husbandry- linked resistant bacteria from animal sources directly to

veterinary antimicrobial use. (Chantziaras et al., 2014).

The consequences of antimicrobial resistance (AR) are serious. The article ‘The bacterial challenge:

time to react’, describes a study executed by the European Centre for Disease Prevention and Control

ECDC and the European Medicines Agency (EMA), where the annual death toll caused by AR was

estimated to be 25100 persons for the European Union alone (Norrby et al., 2009). AR is causing

problems (= the burden) through 3 different mechanisms in both veterinary and human medicine:

infections caused by AR bacteria cause a longer duration of illness and higher rates of mortality.

Secondly, treatment costs of resistant infections are rising and at thirdly, procedures relying on effective

antimicrobial agents to prevent infections, cannot be carried out without creating an increased risk for

infection. (Laxminarayan et al., 2014) (Levin et al., 2014).

4.1.1 Transmission of antimicrobial resistance between animals and humans

In both veterinary and human medicine practices, the same conditions lead to the selection and spread

of resistant bacteria. Antimicrobial agents are heavily prescribed, mostly based on the risk of infection,

rather than on the documented presence of the infection itself and the natural microflora in both humans

and animals are exposed to many antimicrobials from different classes (Silbergeld et al., 2008). In animal

and human populations resistant bacteria are by consequence present and result in antibiotic resistance

transfer. Two major resistance pathways support this process: transmission of entire bacteria harboring

the resistance genes, and the specific transmission of the concerned resistance genes. This process

exists between bacteria from different origin and ecosystems and is here reviewed.

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Transmission of bacteria from animals to humans and vice versa

The bacterial residents of the gut of human and other vertebrates are similar. The composition of

bacterial flora differs mainly because of food habits, habitat, age, physical condition or disease status,

rather than animal species (Ley et al, 2008). Besides similar habitants of the intestines, bacteria

colonizing body surfaces or cavities of human and animals, also correspond between vertebrate

species. Therefore many bacteria can thrive on or in different animal species and/or on humans.

Interspecies transfer of bacteria can happen, and depending on the hazardous nature, they are referred

to differently. When a bacterial population is thriving without causing harm to the host, they are called

commensals. When the opposite is true and the bacteria produces disease, the population is called

pathogenic. The phrase facultative pathogen is used when disease is produced under certain conditions

or under changing circumstances. When a bacterial species is living as a commensal or pathogen in an

animal species and is pathogenic when transmitted to human, it is classified as a zoonotic agent.

Bacteria are often not host-specific i.e. they can colonize different species. When interspecies

transmission of bacteria occurs, the resistant genes they carry can also become disseminated. These

genes can subsequently cause harm if they are being transferred to other pathogenic bacteria or when

the resistant germs cause disease that is more difficult to treat.

Transmission of genes

Genes, including specific resistant genes, can be transferred from one bacterium to another. This

process, described as horizontal gene transfer (HGT), can lead to acquired resistance against

antimicrobial agents in a very fast way. Vertical transfer, the passing on of genetic material from one

generation to the next, occurs in general more slowly (Gogarten et al., 2005) since it relies on the

multiplication of the organism. Horizontal transfer sometimes exceeds phylogenetic borders, even

unrelated bacteria can exchange genes with each other if certain conditions are met. In practice, genes

from animal bacteria can be exchanged to germs living in the environment which causes the

development of a reservoir of resistant genes. Also, bacteria coming from animals can transfer their

genes to human bacteria and vice versa, for instance when commensals or pathogens are taken up by

humans or animals consuming contaminated food/water, they come in contact with indigenous flora and

possible exchange of genes can occur.

Basically, there are 3 major mechanisms for genetic transfer between bacteria.

First, genetic material can be transferred through mobile genetic elements (e.g. plasmids, transposons,

mobile genetic elements) from one bacterium to the other by direct contact. This process is called

‘conjugation’. These mobile genetic elements contain DNA that is given from a donor to an acceptor

strain if compatibility conditions are met, irrespective of a phylogenetic relation. On those mobile genetic

elements, besides other genes, resistant genes against one group or different groups of antimicrobial

agents can be present what leads to respectively mono-resistant or multi-resistant strains.

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Figure 1: Horizontal gene transfer between bacteria by conjugation (Source: Furuya et al., 2006)

A second mechanism of DNA exchange is transduction by bacteriophages. Bacteriophages are viruses

who infect bacteria. They attach to the surface and subsequently inject their DNA to the inside. A first

type of phages will inject their DNA and this will be translated directly to mRNA that leads to viral proteins

and other building materials for the development of new phages. Those so called lytic phages will cause

a burst of their host bacterium and new bacteriophages are spread in the surrounding environment. A

possibility is that DNA from the host cell is encapsulated in newly formed phages. When those phages

infect other bacteria, homologous recombination of that part of the DNA in to the chromosome of the

new bacterium may occur what causes transfer from genetic material from one bacteria to another.

Another type, lysogenic phages, will integrate their DNA into the DNA of the host. When that host

multiplies, the genetic material of the phage will multiply as well. In certain stress situations, the viral

DNA will free itself and new bacteriophages will be formed which on their turn will cause the burst of the

host cell. In this process, parts of bacterial DNA, neighboring the viral DNA can be incorporated into the

newly formed phages together with the viral DNA. In contrary with lytic phages, in this case, specific

DNA can be exchanged in contrary with any random peace of DNA. Transduction, caused by the

lysogenic phages is called ‘specific transduction’.

Figure 2: Horizontal gene transfer between bacteria by transduction. (Source: Furuya et al., 2006)

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Bacterial transformation is the third and last mechanism. This process is based on the intrinsic capacity

of some bacteria to collect DNA out of their environment and incorporate it in their own chromosomal

DNA by using homologue recombination. Those bacteria are called ‘competent’ and this process is

mainly described when strains are at the end of their exponential growing phase. Typically, this transfer

of DNA is only seen with linear DNA fragments and not with plasmids. The importance of this last

mechanism to transfer antimicrobial agent resistant genes is believed to be secondary to the first two

(Drlica et Perlin, 2011).

Figure 3: Horizontal gene transfer between bacteria by transformation. (Source: Furuya et al., 2006)

Intracellular transposons are entities able to exchange genetic material within one bacterial genome.

The transmission can occur from chromosomes to plasmids and the other way around, or between

chromosomes or plasmids reciprocally. The frequency of intracellular exchange can be influenced by

specific DNA regions called transposons. Neighboring DNA to those transposons are changing much

more often from one place to another, these genes are called ‘jumping genes’.

Transposons sometimes also include integrons. Those are regions inducing the placement of different

genes closely to each other (gene cassettes) and make sure that those genes are read correctly to

translate into RNA. Those integrons can often be carrier of different resistant genes, making the bacteria

multi resistant. Together with the first 3 mentioned intercellular mechanism, transposons and integrons

contribute also to the HGT. (Levy et al, 2004) (Mazel, 2006)

4.1.2 Transmission between human and animal due to (in)direct pathways

Transmission from animals to humans and vice versa can occur via two major pathways. Transmission

due to direct pathways is the result of direct contact between the animal and the human. Bacteria can

stick on the skin when people touch the animals, or when droplets of fluid are being released after

sneezing or coughing. When the skin surface is not intact, bacteria can enter through the skin or mucosa

into the body. Also, by swallowing or inhalation, scratch or bite wounds, germs can enter the body.

Transmission via the indirect pathways takes place through contact of humans to food from animal

origin, biological or inorganic vectors, through environment, through water, etc. (Colville et Berryhill,

2007).

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Figure 4: Outline drawing of transmission routes (source: own design)

Direct Pathways

Transmission through direct contact between animal and human is well documented with farmers who

live in close contact with their animals (Van den Bogaard et al., 2001), (Fey et al., 2000), (Voss et al.,

2005), (Wulf et al, 2008). Direct contact can lead to transfer of commensals or zoonoses from colonized

or infected animals. Also, other professions in animal husbandry such as veterinarians can be at risk for

transmission (Wulf et al., 2006). Research is mostly based on the difference in prevalence of resistant

strains between people handling animals and the general population.

Direct spread of Methicillin-Resistant Staphylococcus aureus (MRSA) between animal and human is

demonstrated worldwide. In a reflection paper of 2010, Catry et al. described the different characteristics

of MRSA transfer according to species. In livestock, a specific MRSA strain, CC398 (also known as

Livestock Associated-MRSA or ST398) is far more present than any other strain. This LA-MRSA is often

present in (pig) farmers and veterinarians worldwide. Two Dutch studies showed higher prevalence rates

of MRSA in both veterinarians (4,6%) and pig farmers (26%) compared to general population (Wulf et

al., 2006) (Voss et al., 2005). A surveillance study of professionals (farmers, veterinarians) handling pigs

in Denmark showed a higher prevalence rate as well (12, 5%) (Wulf et al, 2008).

For Gram-negative strains, research that investigated fecal samples from broilers, turkeys and their

farmers strongly support intense transmission of resistant E. coli bacteria and the transmission of

plasmids from the animals to their farmers. This research led to the discovery of E. coli carrying the

same resistance patterns for commonly used antimicrobial agents in turkeys and their farmers (Van den

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Bogaard et al., 2001. After genotyping with pulsed-field gel electrophoresis, two isolates from the

farmers were completely identical to the fecal strains from turkeys). A smaller study on the origin of a

ceftriaxone-resistant Salmonella enterica serotype Typhimurium that infected a 12 year old boy with

fever, abdominal pain and diarrhea was conducted using genotyping as well. The investigators showed

clear evidence that the strain was originating from ill cattle of a farm where the father of the patient was

farmer (Fey et al., 2000).

Direct transmission from pet to human and vice versa gained interest the last 2 decennia. Also pets can

act as a source of antimicrobial agent resistant strains (Guardabassi et al., 2004). A recent study

conducted in Japan showed that dog owners and their dogs can share the same identical E. coli strains.

Compared with a control population, the characteristics of E. coli isolates between dogs and the owners

where much more alike (Harada et al., 2012).

Ampicilline resistant Enterococcus faecium (AREF), a strain causing hospital associated infections

worldwide, is present in many animal species. A large cross-sectional and longitudinal study showed

high prevalence in resp. the UK (23%) and Denmark (76%). Molecular studies showed that a large part

of those isolates (76%) belonged to the same clonal complex (CC) that causes the worldwide pandemic,

CC17. One of the dog owners appeared to be carrier of the identical CC17, with indicates that dogs are

a possible reservoir and may play a role in the dissemination of the pathogen. Further research towards

the origin of the AREF strains, conducted by de Regt et al., 2012 and Tremblay et al., 2013 could not

figure out if the CC17 strains were first transferred from dogs to humans or the other way around.

Staphylococcus species can also be exchanged between pets and their owners or veterinarians. After

the statement that a gap in surveillance of Staphylococcus on small animals compared to food producing

animals existed (Guardabassi et al., 2004), further research has been executed and published. Isolates

of dogs and other pets were compared with isolates retrieved from hospital settings by molecular

techniques. The molecular typing of a significant number of methicillin resistant staphylococci isolates

from dogs by Malik et al., 2006, showed high similarities with strains causing healthcare-associated

infections. A much larger study, conducted in Germany by Strommenger et al., published in that same

year, stated as well that MRSA could been transferred between human and pets. Pet isolates showed

high similarity with the ST22 MRSA isolates that are widely spread in German hospitals after multilocus

sequence typing (MLST). Next, microbiological assays on strains isolated from pets and their veterinary

caregivers suggested same conclusions. Veterinary staff of a small animal hospital in the UK were

carriers of the same EMRSA-15 type that was found in the hospitalized dogs (Loeffler et al., 2005). An

Irish study showed similar strains between pet patients and small animal veterinarians witch were also

indistinguishable from the most common occurring type in the human inpatient (hospital) population

(O’Mahony et al., 2005).

Different case reports document the origin of the strains. In 2003 in the US, the case of a man with

diabetes that was victim of recurrent MRSA infections due to the nasal carriage of the bacterium of his

dog was described (Manian et al). Another report described a 31-year-old female nurse who became

MRSA carrier after a hospital outbreak in the Netherlands, and relapsed a month after the initial

treatment. The strain that colonized the nurse was also found on the body of her 1-year-old daughter

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and the family dog. A second attempt to eliminate the MRSA was in both cases successfully undertaken

by treating both humans and colonized dogs simultaneously. These events support the fact that pets

are at risk to become a carrier for MRSA when living in close contact with humans (Van Duijkeren et al.,

2004) and that MRSA can be transferred from human to animal, and vice versa.

It remains unclear if the transmission of MRSA happens by direct contact between human and pet or by

contact with the contaminated household environment, bringing us on the limit between direct and

indirect pathways. As concluded in a large study of Davis et al., 2012, strategies to prevent recurrent

infections should focus on both the treatment of the MRSA colonization of pets and the decontamination

of the household’s environments.

Indirect Pathways

Food-born transmission

Worldwide, the global meat production and consumption has been rising year after year because of the

increase in global population. (WHO, 2014). Next to the consumption of contaminated water, consuming

raw meat is estimated to be responsible for 320 000 clinical cases of zoonoses in the European Union

(EFSA, 2011). Because of their clinical importance, transmission of resistant zoonoses is also well

described. For example, Gupta et al., 2004, found fluoroquinolone-resistent Campylobacter strains on

different broilers in supermarkets all over the United States. Other research was conducted after the

outbreak of resistant clinical zoonoses; Holmberg et al., 1984, concluded that 18 persons were infected

by multidrug resistant Salmonella Newport due to eating burgers of beef. A year later, in 1985, a

Salmonella outbreak, this time Salmonella enteritidis, could be linked to the consumption of raw milk

establishing the presence of the same specific R plasmid (Tacket et al., 1985).

In Denmark, Salmonella enterica serotype Typhimurium DT104 was the cause of a clinical outbreak.

This strain was transferred from pork meat to humans and was resistant to ampicillin, chloramphenicol,

streptomycin, sulfonamides, tetracycline and fluoroquinolones (Mølbak et al., 1999). A large Belgian

study showed a high prevalence of that same S. Typhimurium DT104 on pork meat (7,4%) and in human

fecal isolates (13,2%). Upon today, this strain remains important for the community (Van Boxstael et al.,

2012). A more recent study, conducted by Nordstrom et al., 2013, linked urinary tract infections

outbreaks with a foodborne origin.

Both resistant Salmonella and E. coli have also been found on egg shells. The authors of the paper

stress the fact that the handling of eggs should be considered as a possible way of transferring resistant

genes and bacteria (Musgrove et al., 2006). Another research on E. coli, conducted in the US, showed

the presence of resistant strains on retail meat. The resistance pattern depended on the animal origin

of the species (Zhao et al., 2012). A risk assessment study, published that same year, tried to estimate

the risk of transfer of third generation cephalosporin resistant E. coli when eating a meal containing

chicken. In Belgium, 60 % of the broiler population carries this strain. The study concluded that there is

indeed a risk of exposure and that this risk mainly depends on the amount of cross contamination and

of the source of the meat but there is still too little known about the quantity of transfer of the resistant

genes from the E. coli of poultry origin to the indigenous human microbiota.

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Already in the late sixties, Smith et al., proved that E. coli, originating from animals, transferred their

resistant genes to resident E. coli in the human alimentary tract when there was a significant uptake of

colony forming units (Smith et al., 1969).

The environment

Figure 5: Outline drawing of indirect transfer via the environment. (source: own design)

Agriculture as a source of antibiotic resistant genes (ARG) Other than foodborne transmission, transmission through other indirect pathways, more precisely via

the environment, is much less understood and described. An early experimental landmark study

showed, after the application of antimicrobial agent-supplemented animal feed to chickens, the spread

of resistant plasmids between chickens and from chickens to humans who came in contact with the

chickens. This became clear when different types of E. coli became carrier of the same R plasmid and

substantiated the theoretical dissemination of resistant genes in the environment (Levy et al., 1976).

In literature, some events have been published that indicate indirect transfer of genetic material and

resistant bacteria between animal, environment and the community. First, when manure is spread over

agricultural lands, strains like Salmonella or Campylobacter and many other gut bacteria can survive for

weeks, even months in the soil, depending on the strain and environmental circumstances. Genes

leading to resistance are being disseminated and transferred to other soil bacteria and remain

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indefinitely present (Chee et al., 2008). Composting of manure before using it as a fertilizer for

agricultural land over a period of 5 weeks might be a way to reduce the total amount of colony forming

units (CFU’s) but resistant genes, e.g. for tetracycline (tet) or erythromycin (erm), will still be detectable

after the process (Sharma et al., 2009). A more recent study compared the processing techniques of

composting and lagoon treatment as a solution to kill resistant bacteria in swine manure. The composting

technique showed a much higher efficiency (decrease by 4 to 7 logs versus 1 to 2 logs) than the lagoon

treatment. Same as in the research done by Sharma et al., resistant bacteria and genes could not be

eradicated completely (Wang et al., 2012).

Secondly, the leaking of waste storage pits and the land-application of the manure could also lead to

contamination of surface and groundwater sources nearby. Sapkota et al. 2007, took water samples in

the surroundings of a swine feeding operation. Enterococcus spp., E. coli and other fecal coliforms could

be isolated and significant higher levels of erythromycin, tetracycline and clindamycin resistance were

present compare to the isolates from the samples taken upstream.

Purifying the waste water by water treatment plants is not sufficient to eliminate all the resistant bacteria

and genes. A study of Portugal showed that the water treatment of slaughterhouses only could reduce

the E. coli with a factor of 0,5 to 3 log (Da Costa et al., 2008). In the United States, water samples out

of rivers contained bacteria with plasmids carrying resistant genes towards β-lactam antimicrobial

agents as ampicillin, cefotaxime and ceftazidime, suggesting the presence of ESBL strains (Ash et al.,

2002).

The community and hospital settings as a source of antibiotic resistant genes Besides the animal population, antibiotic use in the human population has his effects on the spread of

ARG in the environment as well. When comparing the presence of vancomycin-resistant enterococci

(VRE) in urban sewage -both raw and treated- , surface water and hospital sewage, the researchers

were surprised by the high prevalence of VRE in the sewage of both hospitals and community (Iversen

et al., 2002),. The Enterococcus spp. could not be eliminated by the treatment plant. Concentration fell

from 103 to 104 per ml in raw sewage to 101 to 102 per mL in treated sewage and in surface waters. A

year later, a similar research towards the resistant patterns from E. coli isolates in 3 sewage plants and

the survival ratio of the E. coli bacteria after treatment has been published by Reinthaler et al. (2003).

Different levels of antimicrobial resistance were measured, depending on the source of the sewage.

Sewage, coming from a hospital had the highest levels of antimicrobial resistant. Again, in this case,

treatment of the sewage could only reduce the number of E. coli by 200-fold. Despite the purifying of

sewage water, resistant bacteria from the community and hospital settings are still released in the

environment. Another research even took it further. By not only investigating samples of wastewater and

surface water but also from drinking water derived from the surface water sources, they learned that

some resistant genes could be find in the drinking water network. This suggesting that resistant genes

could be transferred to indigenous drinking water bacteria and reenter people when drinking this water.

The vancomycin-resistent gene vanA and the β-lactam resistant gene ampC were present in the drinking

water samples. Only the mecA gene, encoding for methicillin resistance in staphylococci couldn’t been

retrieved (Schwartz et al., 2003).

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Wild animals as an indicator, a reservoir and a vector for dissemination Wild animals, like birds or mammals, can generally be assumed not to have been treated with

antimicrobial substances. They can be carrier of naturally resistant bacteria, e.g. Streptomyces spp. who

harbor resistant genes as a part of their natural protection mechanism, but when wildlife carries

substantial numbers of resistant bacteria, it is most likely transferred through contact with the

environment. Hence, they are very suitable to get a better idea of the scope of the dissemination of

antibiotic resistance in the environment. Worldwide, different research has been done.

In America, on the Mary land Eastern Shore of the United States, Enterococcus spp. and E. coli were

isolated out of fresh stools of Canadian Geese (Branta canadensis). The susceptibility towards 10

antimicrobial agents were tested and resistant profiles showed highest resistance towards cephalotin,

streptomycin, sulfathiazole and penicillin G, ampicillin, cephalotin, sulfathiazole respectively. No large

differences were noticed between seasons (Middleton et al., 2005).

On Europe’s mainland, in the Czech Republic, similar research on Escherichia coli isolates of black-

headed Gulls was conducted. Antimicrobial susceptibility towards eight antimicrobial agents was tested

by the disk diffusion method. Of the 257 isolates, 29,9% of them were resistant to one or more

antimicrobial agents with resistance towards tetracycline (19.1%) as the most prevalent (Dolejska et al.,

2007).

On the Iberian Peninsula, Portuguese researches found nearly two thirds of isolated E. coli out of fecal

samples of Iberian wolves resistant to one or more antimicrobial agents. Of the 19 tested drugs, again

tetracycline was the dupe, followed by ampicillin and streptomycin (Simões et al., 2012).

Guenther et al., 2010, not only isolated many Escherichia coli out of wild bird stool samples to test on

antimicrobial resistance, but subsequently compared the results with the published resistance patterns

observed for domesticated animals. Out of 187 isolates 4.8% were multi-resistant phenotypes, including

resistances against β-lactams, aminoglycosides, fluoroquinolones, tetracyclines and sulfonamides

which were also described in studies of livestock and companion animals in Germany and by extension

in Europe.

A lot has been published about the presence of the extended-spectrum β-lactamase harboring E. coli

specific in wild animal populations. In Poland, mallards, herring gulls and waterbirds, living on the Baltic

Sea Coast, showed a ratio of resp. 4.6 % (3/65), 11.1 % (3/27) and 5,2 % (3/58) of ESBL-producing E.

coli on the total amount of isolated E. coli (Literak et al., 2010). In Portugal, different studies showed an

even higher prevalence. ESBL-EC could be isolated out 8 of 77 fecal samples (10.4%) of wild boars

(Poeta et al., 2009). In common buzzards, 15.2 % (5/33) of the isolated E. coli were of the ESBL

phenotype, published a year later by Radhouani et al. ESBL-EC was present in 26.9 % (32/119) of fecal

samples of birds of prey from the Serra da Estrela Natural Reserve (Pinto et al., 2010) but the highest

prevalence was seen in the seagull population (32%) (Simões et al., 2010).

The dissemination of antimicrobial resistance into the environment goes further than the areas where

there are human activities or where strains are subject to selection pressure. Study’s showed the

presence of ARG on very remote locations. In the artic, some E. coli isolates from birds stool samples

were resistant to some of the 17 antimicrobial agents tested. Of the 97 isolates, 8 showed resistance of

which 4 were multi-resistant to 4 antimicrobials or more, mostly to ampicillin, sulfamethoxazole,

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trimethoprim, chloramphenicol and tetracycline. The authors did discussed the possibility of

spontaneous mutations or HGT with constitute environmental bacteria to explain the phenomenon but

concluded that most likely, the resistant genes were brought there by the migrating birds, stressing out

the complexity of the dissemination of antimicrobial resistance (Sjölund et al., 2008). A prove can be

seen in the high prevalence of acquired ARG in a very remote rural Bolivian Indian community. It is

known that preceding antibiotic use was very limited and exchange with the outside world was minimal

due to the remote and difficult-to-reach location. Of the 130 individuals, 67% were carriers of E. coli

resistant to one or more antimicrobial agents. Highest rates could be found for tetracycline (64%),

ampicilline (58%), trimethoprim sulfamethoxazole (50%) and chloramphenicol (41%).

Consequences of the antimicrobial resistant genes dissemination in clinical settings

As described above, problems because of the emergence of antimicrobial resistance in veterinary and

public health arise worldwide. When a new strain becomes resistant and causes hospital associated

untreatable infections, many research has been done to identify the source of the strain. Some research

suggests the link of a new hospital-associated bug with the environment and the veterinary sector.

Salmonella, Shigella isolated out of fecal samples from humans with diarrhea and E. coli that was

retrieved from urinary tract infections were carrier of a streptothricin-resistance gene. Because this class

of antimicrobials was only used to treat animals, this research provided strong arguments for

transmission (Witte et al., 2002).

More recently, other resistant genes are suspected as well to be transferred from animals. The discovery

of VRE in the animal population led to molecular analysis of the genes involved. Different researchers

couldn’t exclude the possible transfer of the vanA-gene. (Witte et al., 2000). Also, an experiment showed

the in vivo transfer of that same vanA-gene from an Enterococcus faecium of animal origin to E. faecium,

part of the indigenous microbiota when surpassing through the human intestines. The authors also warn

for possible spread to other E. faecium in the gut, especially with immunocompromised patients (Lester

et al., 2005).

Even more controversial is the discussion around the possible transfer from animals to humans of

Extended-spectrum cephalosporin-resistant Escherichia coli (ESCR-EC) and the newly arising

Carbapenemase-Producing Enterobacteriaceae (CPE) in clinical hospital settings.

A very recently published systematic review combining the results of 34 publications including

epidemiology studies and molecular studies concluded that there is clear evidence that a significant part

of the extra intestinal ESCR-EC infections are originating from animals, most likely poultry (Lazarus et

al., 2015). As referred to above, Extended-Spectrum β-lactamase (ESBL) possessing

Enterobacteriacae in wild animals indicates that these strains are wildly spread in the environment and

could support that theory.

CPE, the latest new chapter of the resistance story, is an emerging problem. Human clinical cases were

first reported in the early ’90 (Klebsiella pneumoniae) and today, mortality rates from invasive CPE

infections can mount up to 40 % (Paterson et al., 2015). Huang and his colleagues detected the

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presence of CPE in one-third of the 24 participating hospitals in a study in Belgium, suggesting that the

CPE’s are established and spread in some hospitals. Although there is not yet any published prove of

transfer from animals and the fact that carbapenems are not used in veterinary medicine, some medical

researchers are looking to the veterinary sector. Recent worrisome research found CPE present in

companion and farm animals, as well as in the environment. In Germany, the presence of a

carbapenemase-encoding gene in S. enterica was found in both poultry and pig farms. As indicated

before carbapenems are not licensed for the treatment of livestock animals, so the authors suggest that

the presence is the result of co-selection (Fischer et al., 2013). CPEs are also found in the environment.

Montezzi et al., 2014, collected water samples of the coast of recreational waters near Rio de Janeiro,

Brazil, that contained CPEs. A case in Germany described the presence of a CPE in an isolate from a

wild bird, a black kite. (Fischer et al., 2013)

Clearly, the common denominator in all the publications on the transmission through indirect pathways,

the process of transmission, the scope of the problem and the risk factors are poorly understood. In

particular the epidemiological link between environment, food producing animals and elderly that often

suffer from healthcare-associated infections with resistant microorganisms (Latour et al., 2012), needs

further investigations

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4.2 Research Question

How is the situation of the resistance levels towards the major classes of antimicrobials in following

ecological niches: livestock, surface water, nursing home residents, and pets?

Are there similarities between the antimicrobial resistance profiles of each ecological niche that indicate

potential routes of transmission between these niches?

Are the ecological niches harboring resistant Escherichia coli displaying resistance mechanisms similar

to those recently observed in hospital settings such as ESBL producing strains or CPE?

4.3 Objective of the research

The aim of this study was to investigate the rates of antimicrobial resistance among Gram-negative

bacteria between different ecological niches by monitoring the resistance in commensal E. coli. The

ecological niches include food-producing animals (dairy cattle, pigs and broilers), pets (dogs), surface

water and humans (residents of nursing homes – long term care facilities, LTCF).

Fecal Escherichia coli was chosen as Gram- indicator bacteria for following reasons. First, they easily

acquire antimicrobial resistance when exposed to antimicrobial agents. Also, they are assumed to be

present in all ecological niches. At last, they are commensals in both human, environmental and animal

reservoirs and could transfer resistance to other, sometimes pathogenic bacteria. Because of this last

reason, Escherichia coli has also a substantial clinical importance in both human and veterinary

medicine (DANMAP 2013).

To the best of our knowledge, investigating simultaneously the concerned different ecological niches in

one well demarcated area and in the same time period has not been done before. This narrow timely

and geographically study design ensures that niches can interact directly with each other. Comparing

the resistant patterns of different niches is therefore more valuable. We selected the town Aalter,

(Province of East Flanders, Belgium) with his diverse rural activities. Wherein this area all livestock

species of interest are held, and a large nursing home ‘Veilige Have’ is present. Also, the isolated E. coli

will undergo susceptibility testing for predominantly used veterinarian and human antimicrobial

compounds. The objective is to determine the antimicrobial resistance, present in the different ecological

niches and to determine resistance profiles. By comparing resistance profiles within and between each

sampled niche, we may find relevant associations that can lead to a further understanding of the

epidemiology (Harwood et al, 2000) and therefore interventions. In addition, by comparing the

antibiograms of E. coli from residents at admission with the antibiograms of E. coli coming from mixed

fecal samples during residency, we try to lean something more about the evolution of resistance of

commensals in nursing homes.

In summary, this observational study has as a main goal to contribute to a better understanding of the

dispersion of antimicrobial agent resistance in and between different ecological niches. This is done by

monitoring different ecological niches in the same geographical region and within the same time frame.

This may help us form a better idea of the potential public and environmental health risks associated

with the dispersion of antimicrobial resistance.

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5. Materials and Methods

5.1 Study design

This research was conducted in 2 geographical regions. Sampling occurred in the commune of Aalter.

The samples were subsequently brought to Ghent, where they were processed.

The rural city of Aalter (8.192 ha) houses farms with the different animals of interest, a large nursing

home and 2 small animal veterinary practices (commune of Aalter, 2015). Aalter is 30 km away from

Ghent, were all the samples are being processed. GIS-coordinates of Aalter: 51.083236 N, 3.447266 E.

5.1.1 Human

In this study, we worked together with the nursing home ‘Veilige Have’ to collect human samples. This

facility is situated in the center of Aalter and hosts approximately 400 residents.

5.1.2 Environmental

By using the satellite imaging (Google Maps) surface water in Aalter was located. Surface water was

regarded as suitable if it was easily accessible for humans, or if there was some sort of human activity

on or around the water. A total of 7 locations were selected for inclusion in the study.

5.1.3 Animal

By satellite imaging (Google Maps), 15 pig farms and 15 dairy cattle farms were located. For the broiler

farms a list from the city Aalter was obtained, containing 7 farms.

To sample dogs, 2 small animal veterinary clinics were involved in the study. The dog owners who

engaged to the research by letting their dog rectally sampled all lived in Aalter.

5.2 Study period

The human samples were collected in the period September 2014 to March 2015. Animal samples were

collected in the period December 2014 to March 2015. All environmental samples (Surface water) were

collected on one day, February 14th, 2015. All septic samples, out of the septic tanks were taken on

March 24th, 2015.

The bacteriological processing in the laboratory was started within 7 days after sampling. Collecting data

ended on April 15th, 2015. Statistical analysis was carried out in the second half of that same month (see

5.7.3.).

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5.3 Subjects/Samples

5.3.1 Human Samples

Individual fecal samples

The management of the nursing home kindly provided the samples that were simultaneously obtained

and processed in the scope of a surveillance study for MRSA in elderly, who were admitted at the nursing

home ‘Veilige Have’, Aalter.

In total, 44 individuals, 100% of the newly arrived people at ‘Veilige Have’, were sampled. Of those

people, 95 % comes was resident in Aalter, 5% was resident in neighboring communes.

Septic samples

Out of the 3 septic tanks of the nursing home ‘Veilige have’ in Aalter, 3 mixed fecal samples were taken.

5.3.2 Animal Samples

Farmers were fully informed about the course of, the purpose and the scientific benefits of the research.

They were kindly asked to participate in the study. The farmers themselves were ensured that their data

would be processed anonymously. They will be informed by (electronic) mail about the results of the

entire research and their specific situation, together with background information. After agreement to

participation, farmers were asked for their contact details and also, of all the animals present on the farm

and of the animals were mixed manure was taken from, identification numbers (tags) were written down.

The animal group included for sampling was always that group with the oldest mean age. The age was

also noted. Questions on other animal species present aside from the sampled livestock were asked to

determine if the resistome of one species on the farm could be detoriated by other animals in direct or

indirect contact.

5.4 Sampling

5.4.1 Human Samples

Individual fecal samples

Nursing home residents at initial admission were subjected to the surveillance study for MRSA. Perianal,

rectal and nasal swabs from each individual were taken by a registered nurse. The samples were

transported to the lab within the next 24 hours and an additional rectal swab was used to culture

Escherichia coli from.

The individuals were asked to participate in the research and by a written informed consent. Nurses

interviewed the elderly about a possible contact with animals.

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Septic Samples

Out of the 3 septic tanks of the nursing home ‘Veilige have’ in Aalter, 3 mixed fecal samples were taken,

similar to the way in which the samples of pigs and cows were taken out of the manure pit (see below).

All samples were taken on March 24, 2015.

5.4.2 Environment Samples

On each sample site, 2 samples of 500 mL were taken. All samples were taken on 26 February ’15

under moderate temperature conditions of 13° C.

5.4.3 Animal Samples

Sampling of the pigs

Two samples were taken from the manure pit at different locations. The farmer was asked to lead to the

manure pit of the piggeries were the oldest pigs of the farm were located. To take the samples, an own

‘manure collecting device’ was designed, analogously to a previous similar research (Casteleyn et al.,

2005). Sterile plastic containers of 100 mL were attached by a clamp at the end of the metal bar. For

every first sample a sterile plastic container was opened and with one fluent movement being dragged

through the manure. After being filled, the container was closed again and detached from the device by

opening the clamp. The same was done for the second sample afterwards. Before attaching the plastic

container, a specific code was given that would be linked with the farm details and the isolates with a

permanent marker. During the process, latex disposable gloves were worn. After the detachment of the

second plastic container, the manure collecting device was cleaned with tap water.

Figure 6: Manure Collecting device: left: clamp, upper right: lengthening piece

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Figure 7 Sterile plastic containers used for the manure samples

Figure 8 Container attached to manure collecting device

Sampling of the dairy cattle

By using the same method as with the porcine samples, the dairy cattle samples were taking of the

manure pit, draining the stables were lactating dairy cows are housed.

Sampling of the broilers

Because of the specific 6 weeks cycles of broiler chickens, unexpected visits as where done with dairy

cattle and porkers were impossible. The broiler farmers were contacted in advance, informed about the

research and asked for their collaboration. If the farmer agreed to cooperate, an appointment was made,

depending on when the next production cycle would start. Three sterile plastic containers of 100 ml, also

numbered with a specific code linked to their file, were dropped off in time, together with a letter with

instructions to the farmer of how to take and conserve the samples and a form with questions about the

quantity of the broiler population (see Annex 1: Instruction letter and form for broiler famers.). The

farmers were asked to take the samples during the 5th week of the cycle, one week before the broilers

were transported to the slaughterhouse. During the 5th week, contact by phone was made to friendly

remind the farmers to take the samples and to make an appointment to pick up the samples.

Dog samples

The 2 small animal practices in Aalter were contacted and asked for their participation. Both accepted

and convinced several dog owners of the usefulness of the research. Samples were taken by the

veterinarian who were asked only to sample dogs, older than 5 years in the period starting in From

December to January, Samples were taken by using dry swabs. From February onwards dry swabs

were chanced into commercial swabs with an agar (Copan, Italy).

Figure 9: Commercial swab with an agar (Copan, Italy)

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5.5 Preservation and transport of the samples

Samples of the dogs and the chickens were collected, resp. by the veterinarians and the broiler farmers

and they were asked to preserve the samples below 7° C (refrigerator). On appointment, the researcher

transported it to the microbiological laboratory of the AZ Sint-Lucas (Gent, Belgium) for bacteriological

investigations, ultimately 5 days later. Samples that were taken by the researcher, those of the other

farm animals, of the surface water and the septic samples, were cooled (< 7°C) and transported to the

laboratory (AZ Sint-Lucas) the same day or the day after.

5.6 Microbiology

5.6.1 Isolation & Purification

Ultimately 2 days after arrival the isolation and purification process of fecal or water samples was

initiated.

From fecal samples bacteria were isolated by plating them on a McConkey agar (Oxoid, Dardilly,

France). McConkey agar plates are selective media for coliform bacilli that grow as pink or red colonies

of 2-3 mm after overnight incubation under aerobic conditions. If present, the colonies were purified with

a sterile loop on a new McConkey agar. Monocultures were submitted to identification. From each

sample preferably one E. coli strain was isolated (Collins et Lyne, 2004).

Water samples were first filtered roughly to separate the debris. Secondly the water was filtered with aid

of the Sentino Microbiology Pump following the standard operating procedures described by Pall Life

Science (Sentino Microbiology Pump, Pall Life Sciences, Ann Arbor, USA)

5.6.2 Identification

To identify the isolated bacteria, the MALDI-TOF (matrix-assisted laser desorption/ionization-time of

flight mass spectrometry) mass spectrometry (MS) technique has been used with the MALDI biotyper

(Brüker, Billerica, United States) by using the standard operation procedures and IVD library, as

described by the manufacturer.

5.6.3 Antimicrobial Susceptibility testing

The goal was to test the most important veterinary and human antimicrobial agent classes with a limited

budget. We chose for the combination of the Automatic Susceptibility testing system ‘Phoenix’ for

determination by broth micro dilution of minimal inhibitory concentrations (MIC) and the additional

susceptibility testing of 6 other antimicrobial agents with the classic disk-diffusion method (Kirby-Bauer,

Figure 6).

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MIC-determination

In microbiology lab of the hospital ‘AZ Sint-Lucas’, all the routine susceptibility testing on clinical E. coli

is done by using the commercial NMIC-93 panel on the BD Phoenix system (BD Diagnostics, Sparks,

MD). This semi-automated method allows subsequently to determine the MIC concentrations for 25

antimicrobial classes by microbroth dilution (see below: Table 1). The MIC for a certain antimicrobial

agent of a bacterium is the lowest concentration of that antimicrobial agent that (visibly) inhibits bacterial

growth. Concentrations tested are twofold dilutions and the result is expressed as mg/L (or µg/mL). It is

regarded as the golden standard for clinical and epidemiological resistance determination. Interpretation

was done according to the EUCAST (European College for Antimicrobial Susceptibility Testing) clinical

breakpoints and assay conditions were done according to the manufactures’ guidelines.

Internal quality control was conducted by incorporating the reference strains E. coli ATCC 25922, E. coli

ATCC 35218, Pseudomonas aeruginosa ATCC 27853 en Klebsiella pneumonia ATCC 700603.

Disk-diffusion

Some classes of antimicrobial agents that are frequently used in animal husbandry are not included in

the Phoenix panel NMIC-93. For an additional panel of antimicrobial agents the Kirby-Bauer disk

diffusion method was applied with NeoSensitabs (Rosco, Taastrup, Denmark) following manufactures’

guidelines (Rosco, 2011). The EUCAST guidelines were followed to inoculate, incubate and to

implement internal quality control organisms. Two quality control strains were incorporated in each disk-

diffusion cycle (E. coli ATCC 25922 and ATCC 35218). Following antimicrobial agents were tested:

(dihydro)streptomycin (10µg); florfenicol (30µg), chloramphenicol (60µg), spectinomycin (200µg),

neomycin (120µg) and tetracycline (30µg). The inhibition zones were read after an incubation of 16 h.

Figure 10: A Mueller-Hinton plate with the 6 tested antimicrobials after incubation with a bacterial strain. Large inhibition zones (measured in mm) indicate susceptibility, whereas small inhibition zones indicate resistance round the antibiotic disks of interest.

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Breakpoints

For the 6 antimicrobial agents tested by using the disk diffusion method, the availability of a EUCAST

epidemiological cut-off value was checked ( www.eucast.org ). These values are preferred because they

exclude the variations of clinical breakpoints. For chloramphenicol 30µg disks only, such a cut-off value

existed. For florfenicol (30µg) and neomycine 120µg, the cut-off values were determined using an

algorithm developed in recent research (Callens et al., 2015), using the normalized resistance

interpretation method as conceptualised by Kronvall et al., 2010. For the remaining antimicrobial agents,

streptomycin (100µg), spectinomycin (200µg) and tetracycline (30µg), the clinical cut-offs as supplied

by the manufacturer of the disks (ROSCO) were used.

The NMIC-93 panel for MIC determination is constructed to test antimicrobial susceptibilities in a clinical

human setting. Therefore, the concentrations tested for each antibiotic is restricted to a narrow range

around the human EUCAST clinical breakpoints. As a consequence, this range was sometimes too

limited to use the EUCAST wild-type cut-offs for interpretation. In these events the human EUCAST

clinical breakpoints were used to categories the isolates as resistant or susceptible.

Table 1: Method and threshold values (cut-off) used per antimicrobial susceptibility testing of fecal E. coli of different origin.

method Applied cut-off Value

amikacin Mic-Determination Human EUCAST Clinical Breakpoint > 8 mg/L

amoxicillin-clavulanic acid Mic-Determination Human EUCAST Clinical Breakpoint > 8 mg/L

ampicillin Mic-Determination Human EUCAST Clinical Breakpoint > 8 mg/L

aztreonam Mic-Determination Human EUCAST Clinical Breakpoint > 1 mg/L

ceftazidime Mic-Determination Human EUCAST Clinical Breakpoint > 1 mg/L

cefepime Mic-Determination Human EUCAST Clinical Breakpoint > 1 mg/L

cefoxitine Mic-Determination Human EUCAST Clinical Breakpoint > 8 mg/L

ceftriaxone Mic-Determination Human EUCAST Clinical Breakpoint > 1 mg/L

cefuroxime Mic-Determination Human EUCAST Clinical Breakpoint > 8 mg/L

chloramphenicol Disk Diffusion Wild Type Cutoff EUCAST < 17 mm

ciprofloxacin Mic-Determination Human EUCAST Clinical Breakpoint > 0.5 mg/L

colistine / polymyxine Mic-Determination Human EUCAST Clinical Breakpoint > 2 mg/L

cotrimoxazole Mic-Determination Human EUCAST Clinical Breakpoint > 2 mg/L

ertapenem Mic-Determination Human EUCAST Clinical Breakpoint > 0.5 mg/L

florphenicol Disk Diffusion Callens et al., 2015 < 18 mm

fosfomycine Mic-Determination Human EUCAST Clinical Breakpoint > 32 mg/L

gentamicine Mic-Determination Human EUCAST Clinical Breakpoint > 2 mg/L

imipenem Mic-Determination Human EUCAST Clinical Breakpoint > 2 mg/L

levofloxacine Mic-Determination Human EUCAST Clinical Breakpoint > 1 mg/L

meropenem Mic-Determination Human EUCAST Clinical Breakpoint > 2 mg/L

neomycine Disk Diffusion Callens et al., 2015 < 24 mm

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norfloxacine Mic-Determination Human EUCAST Clinical Breakpoint > 0.5 mg/L

piperacilline-tazobactam Mic-Determination Human EUCAST Clinical Breakpoint > 8 mg/L

spectinomycin Disk Diffusion Rosco Clinical Cutoff < 20 mm

streptomycin Disk Diffusion Rosco Clinical Cutoff < 14 mm

temocilline Mic-Determination Human EUCAST Clinical Breakpoint > 16 mg/L

tetracycline Disk Diffusion Rosco Clinical Cutoff < 15 mm

ticarcillin Mic-Determination Human EUCAST Clinical Breakpoint > 8 mg/L

ticarcilline-clavulanic acid Mic-Determination Human EUCAST Clinical Breakpoint > 8 mg/L

tigecyclin Mic-Determination Human EUCAST Clinical Breakpoint > 1 mg/L

tobramycin Mic-Determination Human EUCAST Clinical Breakpoint > 2 mg/L

5.7 Data management and statistics

5.7.1 Ethical issues

The study protocol was submitted to and approved by the Commission of Medical Ethics of the hospital

‘AZ Sint-Lucas’, Ghent. The results of the persons participating in the survey were processed by using

a coding system where only the coordinating doctor of the nursing home, Dr. De Crop and Dr. Van den

Abeele of the AZ Sint-Lucas had access to.

As said above, the farmers who engaged themselves to this research, had been assured that the

information of the farms and the results were processed anonymously.

5.7.2 Interviews

The participating elderly were asked to fill in a questionnaire with the help of the nursing staff of ‘Veilige

have’. The individuals were asked about their contact with animals during the last year, due to

professional activities or as owner of pets at home or other reasons. (See: Annex 2: Form citizens of

nursing home)

5.7.3 Statistical methods

Antimicrobial classes To describe and discuss the results of the antimicrobial agent susceptibility testing, we used the

standardized international terminology and method, determined by Magiorakos et al., 2012, a result

of a joint initiative by the European Centre for Disease Prevention and Control (ECDC) and the American

Centers for Disease Control and Prevention (CDC). The main goal of the authors was to determine

guidelines to standardize the way antimicrobial susceptibility testing results should be interpreted and

to create definitions of multidrug-resistance (MDR), extensively drug-resistance (XDR) and pandrug-

resistance (PDR) for bacteria such as S. aureus, Enterococcus spp., Enterobacteriaceae, P. aeruginosa

23

and Acinetobacter spp. These bacterial genera/species were chosen because of their epidemiological

significance, the emerging antimicrobial resistance and the importance of these bacteria within the

healthcare system.

By this standardized method, an isolate is defined as non-susceptible to a tested antimicrobial agent

when it is tested resistant or intermediate (when using the Rosco cut-offs or the EUCAST clinical

breakpoints).

Before the paper of Magiorakos et al. (2012) was published, there was no standard approach to

determine types, classes or groups of antimicrobial agents that should be used when defining MDR,

XDR or PDR. The expert group therefore constructed ‘antimicrobial categories’ for each strain, also for

Enterobacteriaceae. This was done by first pooling the information of the ‘suggested agents‘ with FDA

(US Food & Drug Agency) clinical indications and the EUCAST’s Expert rules. Below, the table with

antimicrobial categories and agents to define MDR, XDR and PDR is given. In the fourth column, specific

species that are naturally resistant to the antimicrobials are listed. For describing the resistance

parameters, the corresponding antimicrobial agents or category should not be included. In case of

Escherichia coli, no exclusions are necessary.

Table 2: Enterobacteriaceae; antimicrobial categories and agents used to define MDR, XDR and PDR. (Magiorakos et al., 2012)

24

Antimicrobial resistant index (ARI) The antimicrobial resistance index (ARI) is the ratio of the number of antimicrobial agent classes against

which a germ shows resistance to the total amount of tested antimicrobial agent classes. Thereby, it can

vary from 0.00 (0%) (when the strain is susceptible to every tested antimicrobial agent) to 1.00 (100%)

(the strain is resistant to all tested antimicrobial agent classes). It is a straightforward way to describe

multidrug resistance (Manjusha et al., 2005) (Catry et al., 2005) (Vignesh et al., 2012).

The comparison of the ARI between the species was performed by means of one way ANOVA (analysis

of variance) and Sheffé post hoc test (SPSS 22.0).

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6. Results

6.1 Sampling and isolation

Out of all 44 human samples individual rectal samples, E. coli could be isolated.

All the farmers but one agreed to participate in the research. Of the 15 dairy farms and 15 pig farms

selected, 1 was wrongly regarded as a dairy farm and turned out to be a pig farm. Another farm turned

out to be housing both dairy cattle and pigs. In total, 14 populations of dairy cattle and 17 populations

of pigs were sampled. The farmer who did not agreed to participate was 1 of the 7 broiler farmers from

the city Aalter.

Table 3: Isolation rate per type of sample by ecological niche.

Sort samples E. coli isolated

Total samples

Success rate (%)

Human Samples 44 44 (44x1) 100%

Broiler Farm Samples 17 18 (6x3) 94%

Pig Farm Samples 30 34 (17x2) 88%

Dog Samples 11 17 (17x1) 65%

Septic Tank Samples 2 9 (3x3) 22%

Surface Water Samples 3 14 (7x2) 21%

From Table 2 it appears that isolation of E. coli from the septic tank samples was much more difficult

(2/9) than anticipated. This was also the case for the surface water (3/14) samples.

Figure 11: Visualization pig farms + yield isolation (E. coli strains isolated/samples)

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Figure 12: Visualization dairy farms + yield isolation (E. coli strains isolated/ samples)

Figure 13: Visualization broiler farms + yield isolation (E. coli strains/ samples)

Seventeen dog samples were taken. The first 5 were taken with a dry swab. Because of the low yield of

Escherichia coli isolation (1 out of 5), the dry swabs were replaced for the remainder of the study with

swabs with an agar. It was possible to isolate 10 E. coli out of 12 samples by this improved method. The

isolation success increased from 20% to 83%.

Because of the low yield of surface water (21%) and septic tank samples (14%), the results of the

susceptibility testing of these isolates were not included in the statistical analysis.

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6.2 Antimicrobial susceptibility

Each isolate was tested against a wide variety of antimicrobial substances. The proportion of the strains

of the ecological niches, resistant to an antimicrobial agent is listed in the following table.

Table 4: Proportion of resistance for each antimicrobial agent by ecological niche

Human Broiler Pig Dairy Cattle Dog

neomycin 97,7% 76,5% 90,0% 89,5% 90,9%

ampicillin 65,9% 94,1% 76,7% 5,3% 0,0%

ticarcillin 65,9% 94,1% 73,3% 5,3% 0,0%

ticarcillin-clavulanic acid 56,8% 58,8% 60,0% 5,3% 0,0%

tetracycline 45,5% 64,7% 50,0% 5,3% 0,0%

amoxycillin-clavulanic acid 40,9% 41,2% 43,3% 0,0% 0,0%

streptomycin 36,4% 76,5% 63,3% 15,8% 9,1%

norfloxacin 31,8% 52,9% 3,3% 0,0% 0,0%

ciprofloxacin 29,5% 17,6% 0,0% 0,0% 0,0%

co-trimoxazole 29,5% 70,6% 63,3% 0,0% 0,0%

levofloxacin 27,3% 17,6% 0,0% 0,0% 0,0%

chloramphenicol 20,5% 23,5% 36,7% 5,3% 9,1%

florphenicol 9,1% 0,0% 16,7% 5,3% 0,0%

tobramycin 9,1% 5,9% 0,0% 0,0% 0,0%

aztreonam 6,8% 17,6% 6,7% 5,3% 0,0%

ceftazidime 6,8% 11,8% 6,7% 0,0% 0,0%

cefepime 6,8% 17,6% 6,7% 5,3% 0,0%

cefuroxime 6,8% 17,6% 13,3% 10,5% 0,0%

ceftriaxone 6,8% 17,6% 6,7% 5,3% 0,0%

gentamicin 6,8% 5,9% 0,0% 0,0% 0,0%

spectinomycin 4,5% 58,8% 30,0% 5,3% 0,0%

amikacin 2,3% 0,0% 0,0% 0,0% 0,0%

cefoxitin 2,3% 0,0% 6,7% 0,0% 0,0%

colistin/polymyxin 0,0% 0,0% 0,0% 0,0% 0,0%

ertapenem 0,0% 0,0% 0,0% 0,0% 0,0%

fosfomycine 0,0% 5,9% 0,0% 0,0% 0,0%

imipenem 0,0% 0,0% 0,0% 0,0% 0,0%

meropenem 0,0% 0,0% 0,0% 0,0% 0,0%

piperacillin-tazobactam 0,0% 0,0% 0,0% 0,0% 0,0%

temocillin 0,0% 0,0% 0,0% 0,0% 0,0%

tigecycline 0,0% 0,0% 3,3% 0,0% 0,0%

28

Based on the antimicrobial classes (see 5.7.3), the 33 tested antimicrobial agents tested were divided.

A bacterial isolate was considered resistant to an antimicrobial class when it is ‘non-susceptible to at

least one agent in a category’. In the table below, resistance prevalence is shown for each ecological

niche.

Table 5: Percentage of resistance in E. coli from different origin against the 16 tested antimicrobial categories, Aalter, Belgium, 2014-2015.


Aminoglycos ides 97,7% 100,0% 93,3% 89,5% 90,9%

Penici l l ins 65,9% 94,1% 76,7% 5,3% 0,0%

Antipseudomonal penici l l ins + Beta-lactamase inhibi tors 56,8% 58,8% 60,0% 5,3% 0,0%

Tetracycl ines 45,5% 64,7% 50,0% 5,3% 0,0%

Penici l l ins + Beta lactamase inhibi tors 40,9% 41,2% 43,3% 0,0% 0,0%

Fluoroquinolones 31,8% 52,9% 3,3% 0,0% 0,0%

Folate pathway inhibi tors 29,5% 70,6% 63,3% 0,0% 0,0%

phenicols 20,5% 23,5% 36,7% 5,3% 9,1%

Non-extended spectrum cephalosporins ; 1st and 2nd gen. cephalosporins 6,8% 17,6% 13,3% 10,5% 0,0%

Extended-spectrum cephalosporins : 3rd and 4th gen. cephalosporins 6,8% 17,6% 6,7% 5,3% 0,0%

Monobactams 6,8% 17,6% 6,7% 5,3% 0,0%

Cephamycins 2,3% 0,0% 6,7% 0,0% 0,0%

carbapenems 0,0% 0,0% 0,0% 0,0% 0,0%

Glycylcycl ines 0,0% 0,0% 3,3% 0,0% 0,0%

Phosphonic acids 0,0% 5,9% 0,0% 0,0% 0,0%

Polymyxins 0,0% 0,0% 0,0% 0,0% 0,0%

29

Table 6: Percentage of resistance in E. coli from different origin against the 16 tested antimicrobial categories

There were many similarities between the human, broiler and pig population. In all three ecological

niches high levels of resistance were measured and most resistance was seen towards

aminoglycosides, penicillins (and beta-lactamase inhibitors) and tetracyclines at more or less similar

levels. Resistance towards the cephalosporins, and monobactams was seen in all 3 species at a low

rate and for the cephamycins, carbapenems, glycylcyclines, phosphonic acids and polymyxins,

0,0%

20,0%

40,0%

60,0%

80,0%

100,0%

120,0%

Resistance prevalence


30

resistance levels were very low to zero.

Although there are many similarities, some antimicrobial classes have divergent results when comparing

the 3 niches. The prevalence of resistance towards fluoroquinolones is very high in broiler isolates,

medium to high in human isolates and very low in pig isolates. Resistance towards folate pathway

inhibitors (sulphonamides – trimethoprim) was very common in the pig and broiler population, but much

lower in the human population and also, resistance prevalence towards phenicols was twice as high in

pig isolates compared to the human and broiler E. coli. Remarkable is the low but substantial prevalence

of resistance towards glycylcyclines and phosphonic acids in resp. the pig and broiler population.

In contrast with the human, broiler and pork population, the dairy cattle and dog population harbored

much less resistance. For all classes but aminoglycosides, resistance was very low (dairy cattle isolates)

to absent (canine/bovine samples)

6.1.1 The ARI Index on the level of Antimicrobial Classes

Descriptive

The ARI-index on the level of antimicrobial classes is given in the boxplots below (Figure 14: Boxplots

of the antimicrobial resistance index (ARI, The thicker black horizontal line shows the ARI-mean. The 2

lines at both ends of the box show the minimum and maximum values. The box itself is the

representation of the interquartile range (25 % to 75 % of the values).

For each population, the mean, standard deviation and the 95% confidence interval is given in the table below. Table 7: Descriptives of the ARI-Index for E. coli from different origin (%)

Mean Std. Deviation 95% Confidence Interval for Mean Minimum Maximum

Lower Bound Upper Bound

human 25,7% 15,2% 21,1% 30,3% 6,3% 62,5%

dog 6,3% 2,8% 4,4% 8,1% 0,0% 12,5%

dairy 8,2% 7,6% 4,6% 11,9% 0,0% 37,5%

broiler 35,3% 18,9% 25,6% 45,0% 6,3% 68,8%

pig 29,0% 12,7% 24,2% 33,7% 6,3% 56,3%

31

Figure 14: Boxplots of the antimicrobial resistance index (ARI, in %) for E. coli retrieved from different ecological niches, Aalter, Belgium 2014-2015.

Analytical

Overall a statistical significant difference between the ARI-index of the different ecological niche was

observed. In the post-hoc tests it was found that there was no significant difference between the human,

broiler and pig population and also, no significant difference could be found between the dairy cattle and

dog isolates. Yet, both the ARI of dairy and that of dogs were significantly different from the three other

species.

6.1.4 MDR (multi-drug resistance)

Descriptive

Many isolated strains show resistance to more than one antimicrobial class. Table 8 below shows the

proportion of the strains resistant to the corresponded number of antimicrobial classes. Isolates are

classified as multidrug resistant when they are resistant to at least 3 or more antibiotic classes by the

standardized method of Magiorakos et al., 2012. The horizontal red line segregates the MDR strains

from the non-MDR strains.

32

Table 8: Proportion of E. coli isolates resistant to one or more antimicrobial class.

# antimicrobial classes Human Broiler Pig Dairy cattle dog

0 0% 0% 0% 5% 9%

1 18% 6% 7% 79% 82%

2 16% 12% 7% 11% 9%

3 11% 18% 13% 0% 0%

4 9% 0% 20% 0% 0%

5 11% 18% 30% 0% 0%

6 18% 0% 7% 5% 0%

7 9% 18% 7% 0% 0%

8 2% 12% 3% 0% 0%

9 2% 6% 7% 0% 0%

10 2% 6% 0% 0% 0%

11 0% 6% 0% 0% 0%

12 0% 0% 0% 0% 0%

13 0% 0% 0% 0% 0%

14 0% 0% 0% 0% 0%

15 0% 0% 0% 0% 0%

16 0% 0% 0% 0% 0%

Figure 15: Proportion of E. coli isolates resistant to one or more antimicrobial class (Aalter, Belgium, 2014-2015)

In the table above, it is notable that the dairy cattle clearly harbors mostly strains with only resistance to

zero to two antimicrobial classes. For the human and broiler population, the number varies from 1 to

resp. 10 and 11 classes. The pig isolates form a normal distribution around 5 antimicrobial classes.

Broiler, Pig and Human population harbor resistant to larger numbers of antimicrobial classes, although

in respectively lower proportions. The highest level of multidrug resistance is shown in the broiler

population.

0%

10%

20%

30%

40%

50%

60%

70%

80%

90%

0 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16

Human Broiler Pig Dairy cattle dog

33

In the table below, the proportion of multidrug resistant strains are given for each ecological niche.

Table 9: Proportion multidrug resistant isolates

# multidrug resistant strains # strains % multidrug resistant strains

Human 29 44 65,9%

Broiler 14 17 82,4%

Pig 22 24 91,7%

Dairy Cattle 1 19 5,3%

Dog 0 11 0,0%

Multidrug resistant isolates are very low and absent in resp. dairy and dog population. The human,

broiler and pig population harbor increasing numbers of multidrug resistant strains.

6.1.1 Antimicrobial resistance profiles

Out of 121 isolates, 24 had a unique profile. The remaining 97 isolates could be classified in 23 groups

that harbored 2 or more isolates with the same resistance profiles. Three big clusters of resistance

profiles could be seen. The first cluster harbored 27 isolates, all resistant to aminoglycosides,

tetracyclines, penicillins and folate pathway inhibitors. All isolates of this cluster were coming from the

pig (11), broiler (9) or human (7) niche. The isolates were a mix from the human (10), Pig (7), Broiler (2)

and Dairy cattle (1) population. The second cluster harbored 20 isolates, all resistant to aminoglycosides

and penicillins but not to tetracyclines. A third and largest cluster could be formed by the isolates only

resistant to aminoglycosides. This cluster was formed by the collection of 1 broiler sample and resp. 2,

8, 9 and 14 pig, human, dog and dairy cattle samples.

6.1.2 Specific phenotypical patterns

In each ecological niche, extended-spectrum cephalosporin resistant E. coli are present. In dairy cattle,

only 5% of the strains have an ESBL-like phenotype. In both pig and human population, the proportion

is 7%. The broiler population rises above all with 18 % ESBL-like phenotypes.

In contrary with ESBL, is absent in all animal niches and in the human population.

6.2 Interviews

Of all 44 individual residents that were sampled, only 17 could be interviewed. Others did not want to

participate, suffered from mental disorientation (dementia) or were deceased at the moment of interview.

The number of participants was too low to be statistically significant.

34

7. Discussion

7.1 The strength of this study

As said in the introduction, most studies are limited in investigating the resistance levels and profiles of

only 1 or 2 species. In spite of the unachieved goal to isolate enough statistical relevant Escherichia (E.)

coli from all 6 ecological niches under investigation (broiler, dairy and pig farms, dogs, humans and

surface water), we were able to isolate enough strains of 5 species, including both animal and human

populations. Secondly, this study differs from most other previous studies because all samples were

taken within the same timeframe and in one well demarcated area, the town of Aalter. This ensures that

niches could interacted directly with each other, in line with the rationale that different ecological niches

do not stand alone but are communicating at the microbiological level (Laxminarayan et al., 2013)

(Schroeder et al., 2002).

The combination of testing a significant large amount of isolates from both human and animal population

to a high number of antimicrobials (n=31) used either in veterinary or in human medicine as well as in

both sectors, is also unique. The antimicrobial agents are covering all antimicrobial classes. Often in

surveillance studies, different ecological niches are tested to a limited amount of antimicrobial agents or

one single ecological niche is tested towards many antibiotics, e.g. in the surveillance program

DANMAP, 5 ecological niches (broilers, Danish broiler meat, imported broiler meat, pigs, Danish pork

and imported pork) are tested against 14 antimicrobial agents.

Finally, this is not the only study were both animal and human samples are processed and compared.

Here we included samples from elderly living in a long term care facility, a group that is much more

susceptible to the negative effects of multi-drug resistance (Latour et al., 2012).

7.2 The materials and methods applied

7.2.1 Advantages

The use of E. coli as the indicator for the presence of antimicrobial resistance in Gram-negative bacteria

is internationally adopted. As described in DANMAP (2013), E. coli is the most important commensal of

both animal and human reservoirs and they are ubiquitous. Also, they can acquire resistant genes as a

response to selective pressure and transfer those genes to pathogenic bacteria. The assessment of the

prevalence of antimicrobial resistance gives us a good idea of the selection pressure.

Presumptively identified Escherichia coli-like strains were confirmed by the MALDI-TOF MS technique.

This is a highly reliable, very quick and budget friendly technique that runs almost fully automatically

and is therefore not subject to human errors. Compared to the classic biochemical identification

technique (Quinn et al., 1994) MALDI-TOF has 2 other disadvantages. First, biochemical identification

is much more time consuming and secondly, depending on which biochemical steps are executed, the

identification is never completely accurate.

Antimicrobial susceptibility testing by using the combination of the NMIC-93 panel on the BD Phoenix

system together with the disk diffusion method had also several advantages. The NMIC-93 panel is

35

designed to use for routine clinical susceptibility testing. The technique is, like MALDI-TOF, quick and

budget friendly and less receptive to human errors because of its (partly) automated character. It can

test the susceptibilities for many different antibiotics (25) during the same run. Because of its

disadvantage, the antibiotics tested on the NMIC-93 panel are fixed and some important veterinary

antimicrobials (and classes) were not present, the range of antibiotics tested could be enlarged by

including a disk diffusion test. This is also a frequently used technique for the surveillance of

antimicrobial resistance. It is again budget friendly and easily to execute. Also, the antimicrobials tested

with this method can be chosen freely.

The determination of antibiotic resistance for all ecological niches has been done by the use of the same

2 techniques. Differences of resistance frequencies due to the used techniques in this research could

be reduced to zero. Therefore, the comparison of the resistance profiles between different niches was

more reliable.

7.2.2 Limitations

It was not always possible to isolate a sufficient number of E. coli to be representative for all ecological

niches under investigation. Out of the 14 surface water samples, only 3 E. coli could be isolated by the

applied technique. In some samples, other species such as Aeromonas spp., Rachnella spp., Klebsiella

spp. and Erwinia spp. could be isolated but in most samples, no bacteria were found. In further research,

the membrane filtration technique using mTEC Escherichia coli agar described by EPA (2002) could be

used to isolate more bacteria. Another option would be to include other Enterobacteriaceae and their

resistance profiles while taking natural resistance (see Fout! Verwijzingsbron niet gevonden.)

To test the samples on resistance towards 31 antibiotics, both broth microdilution (MIC-determination)

and disk diffusion were used. To interpret the results, many different cut-offs had to be applied because

of the use of different techniques and the lack of available cut-offs for some other antimicrobials. The

Phoenix panel NMIC-93, to determine the minimal inhibitory concentrations, is only designed for

concentrations around the clinical human breakpoints. For the six other antimicrobial agents, only for

one, chloramphenicol, a wild type cut-off was published by EUCAST, 2 cut-offs were determined by

Callens et al., 2015 (florphenicol and neomycine) and for 3 others, the Rosco Clinical cutoffs had to be

used. Preferably, and as described in the standardized terminology we used in this research (defined

by of Magiorakos et al., 2012) cut-offs of breakpoints by the CLSI, EUCAST or the FDA are used.

7.3 Antimicrobial resistance

The most notable in this study is the high similarity seen in resistance ratios between human, broiler and

pig intestinal flora. For almost all 16 tested antimicrobial classes, resistant ratios are more or less the

same. These results suggests that there might be some sort of link between the niches.

Next to the high similarity, the high levels of resistance in all three species is striking. The WHO (2011)

published a list of critically important antimicrobials for human medicine with the intention to inform

36

doctors and veterinarians to use critically important antimicrobials (CIA) with prudency. Antimicrobial

classes are listed as critical by 2 criteria. First, when it is sole, or one of limited available therapy to treat

a serious human disease. Secondly, a class is defined critical when it is used to treat human bacterial

infections, caused by organisms transmitted or infections that may acquire resistant genes from non-

human sources.

Resistance towards almost all tested antimicrobial classes listed as critically important is seen in all 3

niches and at a very high rate. These critically important classes include aminoglycosides, tetracyclines,

fluoroquinolones and 3rd and 4th generation cephalosporins.

In the poultry and pig population, the high rate of resistance towards some antimicrobial classes was to

be expected. Tetracycline for example is Belgians 3th most consumed antibacterial class in veterinary

medicine, stated by the BelVet-SAC National consumption report (2013). Same resistance levels of

isolates from livestock towards tetracyclines have been seen worldwide (Klein et al., 2003) (Schroeder

et al., 2002). The most and second most consumed antibiotic classes, β-lactam antibiotics and folate

pathway inhibitors (sulphonam & trimethoprim) are also resulting in high levels of resistance in these

two animal populations.

For other antimicrobial classes, rates were less expected. Despite the low use of aminoglycosides, the

resistance level measured is very high. Very unexpected and worrisome is the high rate of

fluoroqiunolones resistance in the poultry. Also, resistance towards the critically important 3th and 4 th

generation cephalosporins are seen. Fortunately, compared with other findings in other countries

(Fischer et al., 2013), there are no signs of the feared resistance towards the so called ‘last resort’

antimicrobial classes such as carbapenems and polymyxins (colistin) but remarkably, and suggesting

further molecular investigations to identify the resistance determinants and mode of transmission

involved, is the substantial number of tigecycline and fosfomycine resistant strains only found in resp.

the swine and broiler population. Tigecycline and fosfomycine are along with colistin and temocilline

among the last resort drugs frequently required to treat human infections with CPE (Jans et al., 2015).

Both are only authorized for human use. Co-selection, e.g. by other tetracyclines, might be an underlying

reason of this appearance. Previous research showed that resistance towards cephalosporins for

example could be co-transferred when selecting with chloramphenicol, sulfamethoxazole or tetracycline

as well (Rankin, 2002).

In the dairy cattle population, resistance is much less present. Dairy cattle is kept far less intensive then

pigs or broilers, and infection pressure is much lower. Therefore, lesser amounts of antibiotics is applied.

Also dairy farmers are much more cautious in applying antibiotics out of fear of residuals in the milk.

(Feyen et al., 2004). This results of low presence of resistance, are similar to earlier findings of Casteleyn

et al., 2005.

The dog population harbored also much less resistant strains. Only towards aminoglycosides, high rates

were seen. Of all populations sampled, the dog population was the one with the lowest amount of

isolates. Low resistance could be coincidental. A confounding factor could be that the 2 small animal

vets who convinced their clientele to participate were both very aware of the resistance problematic and

described themselves as very prudent appliers of antibiotics. Therefore, results may differ from the

37

Belgian situation.

When looking to the prevalence of multidrug resistance, antimicrobial resistance profiles and the ARI-

index, it is again possible to see the large difference between the human, broiler and pig population at

one side, and the dog and dairy population at the other. No statistically significant difference has been

found between the human, broiler and pig isolates. Of all three, the pig population harbors the largest

percentage of multidrug resistance isolates but when E. coli are classified as multidrug resistant, the

isolates of broilers are resistant to the largest numbers of antibiotic classes.

7.4 Conclusion

The results of this study showed that antimicrobial resistance was present in all ecological niches, at

high rates in the human, broiler and pig population and at low rates in the dairy cattle and dog population.

The red thread in this research is the similarity between the human, broiler and pig niche. When looking

to the resistance profiles, ARI, MDR and the figures of resistance ratios, it can be assumed that there is

a link between the ecological niches. However a direct causal relationship between the 3 species was

unintended by this epidemiological research. Nevertheless, the findings give a clear incentive to focus

on these 3 species when investigating transmission of antibiotic resistance in future research.

The results confirm the necessity of the surveillance of antimicrobial resistance. The collection of more

bacterial isolates from different sources out of the environment (e.g. soil and wild-life) has to be the goal

of following research, to attempt better the way these ecological niches are linked with each other. Also,

an extension to investigate resistant profiles of Gram-positive indicator bacteria and the genetic typing

of some isolates could be an added value in further research.

The implied link between these 3 ecological niches, all harboring high levels of resistance towards very

important classes, forms a high risk for public health. This research reaffirms overall that antibiotics,

especially those listed as critically or high importance, should be used with the highest prudency and

proves the weight of organizations trying to raise awareness with human and veterinary doctors about

the urgency of the antimicrobial crisis. Although there are no signs of the feared resistance towards the

so called ‘last resort’ antimicrobial classes such as carbapenems and polymyxins (colistin), low but

remarkable levels of resistance towards tigecycline and fosfomycine were found in pigs and broilers,

respectively. The presence of these levels should be unraveled in depth.

Finally, this research affirms the value of a good corporation between human and veterinary medicine.

This research is the result of when medical doctors and veterinarians join forces in a one health setting,

something that still happens far too little. The results point out that the resistance problematic exceeds

the phylogenetic borders and the only way to resolve it, is to face this problem together.

38

8. References

Alanis, A. J. (2005). Resistance to antibiotics: are we in the post-antibiotic era?. Archives of medical

research, 36(6), 697-705.

Ash, R. J., Mauck, B., & Morgan, M. (2002). Antibiotic resistance of gram-negative bacteria in rivers,

United States. Emerging infectious diseases, 8(7), 713-716.

Bell, B. G., Schellevis, F., Stobberingh, E., Goossens, H., & Pringle, M. (2014). A systematic review and

meta-analysis of the effects of antibiotic consumption on antibiotic resistance. BMC infectious diseases,

14(1), 13.

BelVet-SAC (2013). ‘Belgian Veterinary Surveillance of Antimicrobial Consumption National

consumption report 2013’. Internet Reference: http://www.belvetsac.ugent.be/pages/home/ (last

consulted on April, 28th , 2015)

Callens, B., Kronvall G., Boyen F., Haesebrouck F., Dewulf J. (2015). Normalized resistance

interpretation for antimicrobial resistance surveillance in fecal Escherichia coli from clinically healthy

pigs.

Casteleyn, C., Dewulf, J., Catry, B., de Kruif, A., & Maes, D. (2005). Antobiotic resistance in faecal

Escherichia coli isolates of hares, in comparison with domestic animals. In 1st Syposium of the Belgian

Wildlife Disease Society (pp. 24-24).

Catry, B., Haesebrouck, F., Vliegher, S. D., Feyen, B., Vanrobaeys, M., Opsomer, G., ... & Kruif, A. D.

(2005). Variability in acquired resistance of Pasteurella and Mannheimia isolates from the nasopharynx

of calves, with particular reference to different herd types. Microbial Drug Resistance, 11(4), 387-394.

Catry, B., Van Duijkeren, E., Pomba, M. C., Greko, C., Moreno, M. A., Pyörälä, S., ... & Torren-Edo, J.

(2010). Reflection paper on MRSA in food-producing and companion animals: epidemiology and control

options for human and animal health. Epidemiology and infection, 138(05), 626-644.

Chantziaras, I., Boyen, F., Callens, B., & Dewulf, J. (2014). Correlation between veterinary antimicrobial

use and antimicrobial resistance in food-producing animals: a report on seven countries. Journal of

Antimicrobial Chemotherapy, 69(3), 827-834.

Chee-Sanford, J. C., Mackie, R. I., Koike, S., Krapac, I. G., Lin, Y. F., Yannarell, A. C., ... & Aminov, R. I.

(2009). Fate and transport of antibiotic residues and antibiotic resistance genes following land

application of manure waste. Journal of environmental quality, 38(3), 1086-1108.

http://www.belvetsac.ugent.be/pages/home/

39

Commune of Aalter (2015). ‘Feiten en Cijfers’. Internet Reference: http://www.aalter.be/feiten-cijfers

(consulted on February, 2nd, 2015)

Collins et Lyne (2004). Microbiological Methods Eighth Edition. Chapter 26; Escherichia, Citrobacter,

Klebsiella and Enterobacter

Da Costa, P. M., Vaz-Pires, P., & Bernardo, F. (2008). Antimicrobial resistance in Escherichia coli isolated

in wastewater and sludge from poultry slaughterhouse wastewater plants. Journal of environmental

health, 70(7), 40-5.

Depoorter, P., Persoons, D., Uyttendaele, M., Butaye, P., De Zutter, L., Dierick, K., ... & Dewulf, J. (2012).

Assessment of human exposure to 3rd generation cephalosporin resistant E. coli (CREC) through

consumption of broiler meat in Belgium. International journal of food microbiology, 159(1), 30-38.

Damborg, P., Top, J., Hendrickx, A. P., Dawson, S., Willems, R. J., & Guardabassi, L. (2009). Dogs are

a reservoir of ampicillin-resistant Enterococcus faecium lineages associated with human infections.

Applied and environmental microbiology, 75(8), 2360-2365.

DANMAP 2013 (2013): Use of antimicrobial agents and occurrence of antimicrobial resistance in

bacteria from food animals, food and humans in Denmark. Internet reference:

http://www.danmap.org/~/media/Projekt%20sites/Danmap/DANMAP%20reports/DANMAP%202013/D

ANMAP%202013.ashx (last consulted on April, 25th, 2015)

Davis, M. F., Iverson, S. A., Baron, P., Vasse, A., Silbergeld, E. K., Lautenbach, E., & Morris, D. O.

(2012). Household transmission of meticillin-resistant Staphylococcus aureus and other staphylococci.

The Lancet infectious diseases, 12(9), 703-716.

de Regt, M. J., van Schaik, W., van Luit-Asbroek, M., Dekker, H. A., van Duijkeren, E., Koning, C. J., ...

& Willems, R. J. (2012). Hospital and community ampicillin-resistant Enterococcus faecium are

evolutionarily closely linked but have diversified through niche adaptation. PLoS One, 7(2), e30319.

Dolejska, M., Cizek, A., & Literak, I. (2007). High prevalence of antimicrobial‐resistant genes and

integrons in Escherichia coli isolates from Black‐headed Gulls in the Czech Republic. Journal of applied

microbiology, 103(1), 11-19.

Drlica et Perlin (2011) Antibiotic resistance; Understanding and responding to an emerging crisis. P. 95-

105

EFSA (2011). EFSA explains zoonotic diseases; Food-borne zoonoses. Internet Reference :

http://centaur.vri.cz/docs/EU2011/Food-borne_zoonoses.pdf (consulted February, 2nd, 2015)

http://www.aalter.be/feiten-cijfers

http://www.danmap.org/~/media/Projekt%20sites/Danmap/DANMAP%20reports/DANMAP%202013/DANMAP%202013.ashx

http://www.danmap.org/~/media/Projekt%20sites/Danmap/DANMAP%20reports/DANMAP%202013/DANMAP%202013.ashx

http://centaur.vri.cz/docs/EU2011/Food-borne_zoonoses.pdf

40

EPA (2002): Method 1603: Escherichia coli (E. coli) in water by membrane filtration using modified

membrane-thermotolerant Escherichia coli agar (modified mTEC). EPA-821-R-02-023. Office of Water,

Washington, D.C.

Fey, P. D., Safranek, T. J., Rupp, M. E., Dunne, E. F., Ribot, E., Iwen, P. C., ... & Hinrichs, S. H. (2000).

Ceftriaxone-resistant Salmonella infection acquired by a child from cattle. New England Journal of

Medicine, 342(17), 1242-1249.

FEYEN, B., CATRY, B., Opsomer, G., VANROBAEYS, M., & de Kruif, A. (2003). Streptococcus bovis as

indicator bacterium for antimicrobial resistance in cattle. Poster Abstracts 23rd World Buiatrics

Congress, Quebec, Canada (pp. 5–5)

Fischer, J., Rodríguez, I., Schmoger, S., Friese, A., Roesler, U., Helmuth, R., & Guerra, B. (2012).

Salmonella enterica subsp. enterica producing VIM-1 carbapenemase isolated from livestock farms.

Journal of Antimicrobial Chemotherapy, dks393.

Fischer, J., Schmoger, S., Jahn, S., Helmuth, R., & Guerra, B. (2013). NDM-1 carbapenemase-

producing Salmonella enterica subsp. enterica serovar Corvallis isolated from a wild bird in Germany.

Journal of Antimicrobial Chemotherapy, dkt260.

Furuya, E. Y., & Lowy, F. D. (2006). Antimicrobial-resistant bacteria in the community setting. NATURE

REVIEWS| MICROBIOLOGY, 4, 37.

Gogarten, J. P., & Townsend, J. P. (2005). Horizontal gene transfer, genome innovation and evolution.

Nature Reviews Microbiology, 3(9), 679-687.

Guardabassi, L., Schwarz, S., & Lloyd, D. H. (2004). Pet animals as reservoirs of antimicrobial-resistant

bacteria Review. Journal of Antimicrobial Chemotherapy, 54(2), 321-332.

Guenther, S., Grobbel, M., Lübke-Becker, A., Goedecke, A., Friedrich, N. D., Wieler, L. H., & Ewers, C.

(2010). Antimicrobial resistance profiles of Escherichia coli from common European wild bird species.

Veterinary microbiology, 144(1), 219-225.

Gupta, A., Nelson, J. M., Barrett, T. J., Tauxe, R. V., Rossiter, S. P., Friedman, C. R., ... & Angulo, F. J.

(2004). Antimicrobial resistance among Campylobacter strains, United States, 1997–2001. Emerging

infectious diseases, 10, 6..

41

Harada, K., Okada, E., Shimizu, T., Kataoka, Y., Sawada, T., & Takahashi, T. (2012). Antimicrobial

resistance, virulence profiles, and phylogenetic groups of fecal Escherichia coli isolates: A comparative

analysis between dogs and their owners in Japan. Comparative immunology, microbiology and

infectious diseases, 35(2), 139-144.

Hald, T., Lo Fo Wong, D. M., & Aarestrup, F. M. (2007). The attribution of human infections with

antimicrobial resistant Salmonella bacteria in Denmark to sources of animal origin. Foodborne

pathogens and disease, 4(3), 313-326.

Harwood, V. J., Whitlock, J., & Withington, V. (2000). Classification of antibiotic resistance patterns of

indicator bacteria by discriminant analysis: use in predicting the source of fecal contamination in

subtropical waters. Applied and Environmental Microbiology, 66(9), 3698-3704.

Huang, T. D., Berhin, C., Bogaerts, P., Glupczynski, Y., Caddrobi, J., Leroux, I., ... & Verhaegen, J.

(2013). Prevalence and mechanisms of resistance to carbapenems in Enterobacteriaceae isolates from

24 hospitals in Belgium. Journal of Antimicrobial Chemotherapy, dkt096.

Iversen, A., Kühn, I., Franklin, A., & Möllby, R. (2002). High prevalence of vancomycin-resistant

enterococci in Swedish sewage. Applied and Environmental Microbiology, 68(6), 2838-2842.

Jans, B., Daniel Huang, T. D., Bauraing, C., Berhin, C., Bogaerts, P., Deplano, A., ... & Glupczynski, Y.

(2015). Infection due to travel-related carbapenemase-producing Enterobacteriaceae, a largely

underestimated phenomenon in Belgium. Acta Clinica Belgica, 2295333715Y-0000000001.

Klein, G., & Bülte, M. (2003). Antibiotic susceptibility pattern of Escherichia coli strains with verocytotoxic

E. coli-associated virulence factors from food and animal faeces. Food microbiology, 20(1), 27-33.

Latour, K., Catry, B., Broex, E., Vankerckhoven, V., Muller, A., Stroobants, R., ... & Jans, B. (2012).

Indications for antimicrobial prescribing in European nursing homes: results from a point prevalence

survey. Pharmacoepidemiology and drug safety, 21(9), 937-944.

Lazarus, B., Paterson, D. L., Mollinger, J. L., & Rogers, B. A. (2015). Do Human Extraintestinal

Escherichia coli Infections Resistant to Expanded-Spectrum Cephalosporins Originate From Food-

Producing Animals? A Systematic Review. Clinical Infectious Diseases, 60(3), 439-452.

Laxminarayan, R., Duse, A., Wattal, C., Zaidi, A. K., Wertheim, H. F., Sumpradit, N., ... & Cars, O. (2013).

Antibiotic resistance—the need for global solutions. The Lancet infectious diseases, 13(12), 1057-1098.

42

Lester, C. H., Frimodt-Møller, N., Sørensen, T. L., Monnet, D. L., & Hammerum, A. M. (2006). In vivo

transfer of the vanA resistance gene from an Enterococcus faecium isolate of animal origin to an E.

faecium isolate of human origin in the intestines of human volunteers. Antimicrobial agents and

chemotherapy, 50(2), 596-599.

Levin, B. R., Baquero, F., & Johnsen, P. J. (2014). A model-guided analysis and perspective on the

evolution and epidemiology of antibiotic resistance and its future. Current opinion in microbiology, 19,

83-89.

Levy, S. B., Fitzgerald, G. B., & Macone, A. B. (1976). Spread of antibiotic-resistant plasmids from

chicken to chicken and from chicken to man.

Levy, S. B., & Marshall, B. (2004). Antibacterial resistance worldwide: causes, challenges and

responses. Nature medicine, 10, S122-S129

Ley, R. E., Lozupone, C. A., Hamady, M., Knight, R., & Gordon, J. I. (2008). Worlds within worlds:

evolution of the vertebrate gut microbiota. Nature Reviews Microbiology, 6(10), 776-788.

Literak, I., Dolejska, M., Janoszowska, D., Hrusakova, J., Meissner, W., Rzyska, H., ... & Cizek, A.

(2010). Antibiotic-resistant Escherichia coli bacteria, including strains with genes encoding the

extended-spectrum beta-lactamase and QnrS, in waterbirds on the Baltic Sea Coast of Poland. Applied

and environmental microbiology, 76(24), 8126-8134.

Loeffler, A., Boag, A. K., Sung, J., Lindsay, J. A., Guardabassi, L., Dalsgaard, A., ... & Lloyd, D. H. (2005).

Prevalence of methicillin-resistant Staphylococcus aureus among staff and pets in a small animal

referral hospital in the UK. Journal of Antimicrobial Chemotherapy, 56(4), 692-697.

Malik, S., Coombs, G. W., O'Brien, F. G., Peng, H., & Barton, M. D. (2006). Molecular typing of

methicillin-resistant staphylococci isolated from cats and dogs. Journal of Antimicrobial Chemotherapy,

58(2), 428-431.

Manian, F. A. (2003). Asymptomatic nasal carriage of mupirocin-resistant, methicillin-resistant

Staphylococcus aureus (MRSA) in a pet dog associated with MRSA infection in household contacts.

Clinical Infectious Diseases, 36(2), e26-e28.

Manjusha, S., Sarita, G. B., Elyas, K. K., & Chandrasekaran, M. (2005). Multiple antibiotic resistances

of Vibrio isolates from coastal and brackish water areas. American Journal of Biochemistry and

Biotechnology, 1(4), 201-206.

43

Magiorakos, A. P., Srinivasan, A., Carey, R. B., Carmeli, Y., Falagas, M. E., Giske, C. G., ... & Monnet,

D. L. (2012). Multidrug‐resistant, extensively drug‐resistant and pandrug‐resistant bacteria: an

international expert proposal for interim standard definitions for acquired resistance. Clinical

Microbiology and Infection, 18(3), 268-281.

Mazel, D. (2006). Integrons: agents of bacterial evolution. Nature Reviews Microbiology, 4(8), 608620.

Mølbak, K., Baggesen, D. L., Aarestrup, F. M., Ebbesen, J. M., Engberg, J., Frydendahl, K., ... &

Wegener, H. C. (1999). An outbreak of multidrug-resistant, quinolone-resistant Salmonella enterica

serotype Typhimurium DT104. New England Journal of Medicine, 341(19), 1420-1425.

Middleton, J. H., & Ambrose, A. (2005). Enumeration and antibiotic resistance patterns of fecal indicator

organisms isolated from migratory Canada geese (Branta canadensis). Journal of wildlife diseases,

41(2), 334-341.

Montezzi, L. F., Campana, E. H., Corrêa, L. L., Justo, L. H., Paschoal, R. P., da Silva, I. L. V. D., ... &

Picão, R. C. (2014). Occurrence of carbapenemase-producing bacteria in coastal recreational waters.

International journal of antimicrobial agents.

Musgrove, M. T., Jones, D. R., Northcutt, J. K., Cox, N. A., Harrison, M. A., Fedorka-Cray, P. J., & Ladely,

S. R. (2006). Antimicrobial resistance in Salmonella and Escherichia coli isolated from commercial shell

eggs. Poultry science, 85(9), 1665-1669.

Norrby R., 2009. The bacterial challenge: time to react: A call to narrow the gap between multidrug-

resistant bacteria in the EU and the development of new antibacterial agents. Internet reference:

http://ecdc.europa.eu/en/publications/Publications/0909_TER_The_Bacterial_Challenge_Time_to_Re

act.pdf (consulted on February 2nd , 1999)

Nordstrom, L., Liu, C. M., & Price, L. B. (2013). Foodborne urinary tract infections: a new paradigm for

antimicrobial-resistant foodborne illness. Frontiers in microbiology, 4.

O’Mahony, R., Abbott, Y., Leonard, F. C., Markey, B. K., Quinn, P. J., Pollock, P. J., ... & Rossney, A. S.

(2005). Methicillin-resistant Staphylococcus aureus (MRSA) isolated from animals and veterinary

personnel in Ireland. Veterinary microbiology, 109(3), 285-296.

Paterson, D. L. (2015, February). Carbapenemase-Producing Enterobacteriaceae. In Seminars in

respiratory and critical care medicine (Vol. 36, No. 1, pp. 74-84).

http://ecdc.europa.eu/en/publications/Publications/0909_TER_The_Bacterial_Challenge_Time_to_React.pdf

http://ecdc.europa.eu/en/publications/Publications/0909_TER_The_Bacterial_Challenge_Time_to_React.pdf

44

Pinto, L., Radhouani, H., Coelho, C., da Costa, P. M., Simões, R., Brandão, R. M., ... & Poeta, P. (2010).

Genetic detection of extended-spectrum β-lactamase-containing Escherichia coli isolates from birds of

prey from Serra da Estrela Natural Reserve in Portugal. Applied and environmental microbiology, 76(12),

4118-4120.

Poeta, P., Radhouani, H., Pinto, L., Martinho, A., Rego, V., Rodrigues, R., ... & Igrejas, G. (2009). Wild

boars as reservoirs of extended‐spectrum beta‐lactamase (ESBL) producing Escherichia coli of different

phylogenetic groups. Journal of basic microbiology, 49(6), 584-588.

Radhouani, H., Pinto, L., Coelho, C., Gonçalves, A., Sargo, R., Torres, C., ... & Poeta, P. (2010).

Detection of Escherichia coli harboring extended-spectrum β-lactamases of the CTX-M classes in fecal

samples of common buzzards (Buteo buteo). Journal of antimicrobial chemotherapy, 65(1), 171-173.

Rankin, S. C., Aceto, H., Cassidy, J., Holt, J., Young, S., Love, B., ... & Benson, C. E. (2002). Molecular

characterization of cephalosporin-resistant Salmonella enterica serotype Newport isolates from animals

in Pennsylvania. Journal of clinical microbiology, 40(12), 4679-4684.

Reinthaler, F. F., Posch, J., Feierl, G., Wüst, G., Haas, D., Ruckenbauer, G., ... & Marth, E. (2003).

Antibiotic resistance of E. coli in sewage and sludge. Water Research, 37(8), 1685-1690.

Rosco (2011). Rosco Guidelines for the Use of Neosensitabs, NEO-SENSITABS™ 2011. Internet

Reference :http://www.rosco.dk/default.asp?mainmenu=3&submenu=8&webmanage=Users%20Guide

%20Neo-Sensitabs (consulted March, 28th, 2015)

Sapkota, A. R., Curriero, F. C., Gibson, K. E., & Schwab, K. J. (2007). Antibiotic-resistant enterococci

and fecal indicators in surface water and groundwater impacted by a concentrated swine feeding

operation. Environmental Health Perspectives, 1040-1045.

Schroeder, C. M., Zhao, C., DebRoy, C., Torcolini, J., Zhao, S., White, D. G., ... & Meng, J. (2002).

Antimicrobial resistance of Escherichia coli O157 isolated from humans, cattle, swine, and food. Applied

and Environmental Microbiology, 68(2), 576-581.

Schroeder, C. M., Meng, J., Zhao, S., DebRoy, C., Torcolini, J., Zhao, C., ... & White, D. G. (2002).

Antimicrobial resistance of Escherichia coli O26, O103, O111, O128, and O145 from animals and

humans. Emerging infectious diseases, 8(12), 1409-1414.

Sharma, R., Larney, F. J., Chen, J., Yanke, L. J., Morrison, M., Topp, E., ... & Yu, Z. (2009). Selected

antimicrobial resistance during composting of manure from cattle administered sub-therapeutic

antimicrobials. Journal of environmental quality, 38(2), 567-575.

http://www.rosco.dk/default.asp?mainmenu=3&submenu=8&webmanage=Users%20Guide%20Neo-Sensitabs

http://www.rosco.dk/default.asp?mainmenu=3&submenu=8&webmanage=Users%20Guide%20Neo-Sensitabs

45

Silbergeld, E. K., Graham, J., & Price, L. B. (2008). Industrial food animal production, antimicrobial

resistance, and human health. Annu. Rev. Public Health, 29, 151-169.

Smith, H. W. (1969). Transfer of antibiotic resistance from animal and human strains of Escherichia coli

to resident E. coli in the alimentary tract of man. The Lancet, 293(7607), 1174-1176.

Simões, R., Ferreira, C., Gonçalves, J., Álvares, F., Rio-Maior, H., Roque, S., ... & da Costa, P. M. (2012).

Occurrence of virulence genes in multidrug-resistant Escherichia coli isolates from Iberian wolves (Canis

lupus signatus) in Portugal. European Journal of Wildlife Research, 58(4), 677-684.

Sjölund, M., Bonnedahl, J., Hernandez, J., Bengtsson, S., Cederbrant, G., Pinhassi, J., ... & Olsen, B.

(2008). Dissemination of multidrug-resistant bacteria into the Arctic. Emerging infectious diseases,

14(1), 70.

Strommenger, B., Kehrenberg, C., Kettlitz, C., Cuny, C., Verspohl, J., Witte, W., & Schwarz, S. (2006).

Molecular characterization of methicillin-resistant Staphylococcus aureus strains from pet animals and

their relationship to human isolates. Journal of Antimicrobial Chemotherapy, 57(3), 461-465.

Tacket, C. O., Dominguez, L. B., Fisher, H. J., & Cohen, M. L. (1985). An outbreak of multiple-drug-

resistant Salmonella enteritis from raw milk. Jama, 253(14), 2058-2060.

Turnidge, J., & Paterson, D. L. (2007). Setting and revising antibacterial susceptibility breakpoints.

Clinical microbiology reviews, 20(3), 391-408.

Van den Bogaard, A. E., London, N., Driessen, C., & Stobberingh, E. E. (2001). Antibiotic resistance of

fecal Escherichia coli in poultry, poultry farmers and poultry slaughterers. Journal of Antimicrobial

Chemotherapy, 47(6), 763-771.

Van Duijkeren, E., Wolfhagen, M. J., Box, A. T., Heck, M. E., Wannet, W. J., & Fluit, A. C. (2004). Human-

to-dog transmission of methicillin-resistant Staphylococcus aureus. Emerg Infect Dis, 10(12), 2235-7.

Vignesh, S., Muthukumar, K., & James, R. A. (2012). Antibiotic resistant pathogens versus human

impacts: a study from three eco-regions of the Chennai coast, southern India. Marine pollution

bulletin, 64(4), 790-800.

Voss, A., Loeffen, F., Bakker, J., Klaassen, C., & Wulf, M. (2005). Methicillin-resistant Staphylococcus

aureus in pig farming. Emerg Infect Dis, 11(12), 1965-1966.

ISO 690

46

Wang, L., Oda, Y., Grewal, S., Morrison, M., Michel Jr, F. C., & Yu, Z. (2012). Persistence of resistance

to erythromycin and tetracycline in swine manure during simulated composting and lagoon treatments.

Microbial ecology, 63(1), 32-40.

Wellington, E. M., Boxall, A. B., Cross, P., Feil, E. J., Gaze, W. H., Hawkey, P. M., ... & Williams, A. P.

(2013). The role of the natural environment in the emergence of antibiotic resistance in Gram-negative

bacteria. The Lancet infectious diseases, 13(2), 155-165.

WHO (2011). Critically Important Antimicrobials for Human Medicine. Internet reference :

http://www.who.int/foodsafety/publications/antimicrobials-third/en/ (last consulted on March, 30th, 2015)

WHO (2014). Availability and changes in consumption of animal products. Internet reference :

http://www.who.int/nutrition/topics/3_foodconsumption/en/index4.html (consulted on March 2nd, 2015)

Witte, W. (2000). Selective pressure by antibiotic use in livestock. International Journal of Antimicrobial

Agents, 16, 19-24.

Witte, W., Klare, I., & Werner, G. (2002). Molecular ecological studies on spread of antibiotic resistance

genes. Animal biotechnology, 13(1), 57-70.

Wulf, M. W. H., Sørum, M., Van Nes, A., Skov, R., Melchers, W. J. G., Klaassen, C. H. W., & Voss, A.

(2008). Prevalence of methicillin‐resistant Staphylococcus aureus among veterinarians: an international

study. Clinical microbiology and infection, 14(1), 29-34.

Wulf, M. W. H., Sørum, M., Van Nes, A., Skov, R., Melchers, W. J. G., Klaassen, C. H. W., & Voss, A.

(2008). Prevalence of methicillin‐resistant Staphylococcus aureus among veterinarians: an international

study. Clinical microbiology and infection, 14(1), 29-34.

Zhao, S., Blickenstaff, K., Bodeis-Jones, S., Gaines, S. A., Tong, E., & McDermott, P. F. (2012).

Comparison of the prevalences and antimicrobial resistances of Escherichia coli isolates from different

retail meats in the United States, 2002 to 2008. Applied and environmental microbiology, 78(6), 1701-

1707.

http://www.who.int/foodsafety/publications/antimicrobials-third/en/

http://www.who.int/nutrition/topics/3_foodconsumption/en/index4.html

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9. Annexes

9.1 Annex 1: Instruction letter and form for broiler famers.

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9.2 Annex 2: Form citizens of nursing home

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9.3 Annex 3: Dutch summary

Reeds vanaf de aanvang van het gebruik van antimicrobiële middelen als behandeling van bacteriële

infecties, is het probleem van verworven resistentie op de voorgrond getreden. Zowel in de dier- als

humane geneeskunde stijgt de prevalentie van resistentie jaar na jaar. Voornamelijk wanneer

antimicrobiële resistentie van de ene soort op de andere wordt overgedragen, in het bijzonder op de

mens, is dit mogelijk een belangrijk risico voor de volksgezondheid. Het mechanisme van resistentie

overdracht tussen verschillende diersoorten, alsook de oorzaken en de betrokken factoren hierbij, zijn

tot op heden nog steeds slecht begrepen, vooral dan het epidemiologisch verband tussen milieu,

voedselproducerende dieren en de oudere bevolking die vaak ziekenhuisinfecties vertoont. Deze

masterproef richt zich op het onderzoeken van de resistentie graad in verschillende ecologische niches

namelijk vee, oppervlaktewater, rusthuisbewoners en huisdieren. Stalen werden genomen van dierlijk

mengmest, stoelgang van rusthuisbewoners en honden en van oppervlaktewateren in een afgebakend

gebied (Aalter, België) en bepaalde tijdsperiode (2014-2015). Escherichia coli’s werden geïsoleerd en

opgezuiverd met standaardtechnieken en geïdentificeerd met behulp van MALDI-TOF. Door de

toepassing van een combinatie van disk diffusie en Phoenix MIC-bepaling werden resistentie profielen

gecreëerd. De resultaten tonen duidelijk aan dat een hoge mate van resistentie bestaat tegen de als

kritisch geclassificeerde antimicrobiële groepen in het bijzonder tegen aminoglycosiden, tetracyclines,

fluoroquinolonen en 3e en 4e generatie cefalosporines. Dit komt voor, zowel bij de vleeskuikens, de

varkenspopulatie, als bij de humane studie populatie. Verdere substantiële gelijkenissen in resistentie

niveau’s, multidrug resistentie en in de antimicrobiële resistentie index tussen het pluimvee, varkens en

rusthuisbewoners suggereren een sterk verband tussen deze ecologische niches met negatieve

gevolgen voor de volksgezondheid. De bevindingen in respectievelijk de varkens en vleeskuiken

populatie van tigecycline en fosfomycine resistentie, twee zogenaamde laatste redmiddel antimicrobiële

klassen die niet zijn geregistreerd voor diergeneeskundig gebruik, was onverwacht en bovendien

zorgwekkend. Resistentie tegen polymyxines (colistine) en carbapenems werd niet gevonden. Verder

onderzoek dat de transmissie tussen deze ecologische niches grondig uitklaart, moet worden

aangemoedigd. Bijkomend is moleculair onderzoek aangewezen om de resistentie determinanten en

de wijze van transmissie te identificeren, die de tigecycline en fosfomycine resistentie kunnen verklaren.

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