Microbial control lecture reference

Biol 3400 Tortora et al Chap 7&20

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Control of Microbial Growth

Why control microbial growth

• To destroy pathogens and prevent their transmission

• To reduce and eliminate microorganisms responsible for the contamination of water, food and

other substances

• Many industries rely on the ability to control microbial growth

I. Terminology • All of these processes are aimed at reducing microbial populations (i.e., microbial load)

Sterilization

• Elimination of all viable cells, spores and acellular entities (viruses, viroids, prions)

• The viable cells, spores and acellular entities are killed, removed or inactivated

• Sterilant: chemical agent used to sterilize an object

Disinfection

• Killing, inhibition or removal of microorganisms that may cause disease

• A treatment with the primary goal of destroying potential pathogens

• May not eliminate all microorganisms but usually substantially reduces microbial numbers

• Disinfectants: agents, usually chemical, used in disinfection and are normally used on

inanimate objects.

Sanitization

• Related to disinfection but reduces microbial population to levels considered safe by public

health standards – also minimizes risk of disease transmission

• An inanimate object is usually cleaned and partially disinfected with a sanitizer

Antisepsis

• Prevention of infection or sepsis

• Antiseptics: chemical agents applied to living tissues to prevent infection by killing or

inhibiting pathogen growth

• Antiseptics are generally less toxic than disinfectants because it is important that they do not

destroy too much tissue

Chemotherapy

• The use of chemical agents to kill or inhibit the growth of microorganisms within host tissue


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A variety of chemicals can be used to eliminate or reduce the numbers of microbes – these are

often referred collectively as antimicrobial agents. Specific suffixes are used to indicate how

these agents work.

Bactericidal - "cidal" - to kill

Bacteriostatic - "static" - to inhibit; if the agents are removed then growth will resume.

Bacteriolytic – “lytic” – to lyse

suffixes can be applied to all types of agents

e.g., germicide, fungicide, algicide, viricide

fungistatic

II. The Pattern of Microbial Death • Analogous to population growth, population death is an exponential process: plotting the log

(population) vs time will produce a straight-line plot (Figure 7.1)

• There is a dilemma associated with determining when a microbe is dead (i.e., VBNC and

viruses) • It is important to assess the effectiveness of various treatments (i.e., quality control). A

variety of methods exist, some of which are described below or later on in these notes

1. Direct Assessment

i) Bacterial killing curves

• Plot log %survival vs a measure of the sterilizing agent

e.g., log % viable Salmonella cells vs [singlet oxygen]

ii) Time-dose-rate relationship

• The effect of the treatment depends both on concentration used and exposure time.

• In order to kill all the cells in a particular culture, one can hold the time constant and vary

the dose or keep the dose constant and vary the time.

e.g., A high dose for a short time will have the same effect as a low dose for a longer period

of time

iii) Death point

• Treatment dose necessary to sterilize the system in a given amount of time

e.g., Thermal death point = temperature necessary to sterilize a culture in 10 min.

iv) Death time

• Time necessary to sterilize a system with a particular treatment

e.g., Time in min. necessary to sterilize the culture when a particular temperature is applied

(Thermal death time)


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v) Decimal reduction time (D value) (Fig 7.1)

• Exposure time at a given temperature needed to reduce the number of viable microbes by

90% (1 log).

• Most precise way to characterize heat sterilization

• Plot of log (number of viable cells) vs. time of heating (min)

• Death rate increases with increasing temperature

• z value in the change in temperature required to reduce the D value to 1/10 of its value

2. Indirect Assessment

i) Sterility indicators

• Use certain bacterial endospores

• The most durable life forms known.

e.g., Geobacillus stearothermophilus spores are capable of surviving 5 min in an autoclave

(121°C; 15 psi)

III Conditions Influencing the Effectiveness of Antimicrobial Agents The efficacy of antimicrobial agents is affected by at least 6 factors

1. Population size – larger populations require a longer time to die

2. Microbial characteristics – endospores are more resistant than vegetative cells to antimicrobial

agents; there are also differences in susceptibility between species (Figure 7.11)

3. Concentration or intensity of an antimicrobial agent, e.g. 70% ethanol is more effective than

95% ethanol – killing activity is enhanced by water

4. Duration of exposure – the longer a population is exposed to a antimicrobial agent, the more

microbes are killed

5. Temperature – higher temperature generally enhance the activity of an antimicrobial agent

6. Local environment – The local environment may enhance or diminish the activity of an

antimicrobial agent (e.g., heat is more effective at an acidic pH; organic matter can protect

cells from heat treatments and chemical disinfectants

IV. Actions of Microbial Control Agents Antimicrobial agents kill or inhibit growth of a microbe in several ways

1. Damage to the membrane lipids or proteins cause cellular leakage that has a significant impact

on growth

2. Proteins are central to the functioning and structure of cells. Many antimicrobial agents target

key enzymes, transport or structural proteins. Agents that affect protein conformation (i.e.,

disrupt disulfide bridges or hydrogen bonds) inactivate enzymes or transport proteins.

3. Other antimicrobial compounds damage nucleic acids thereby preventing a cell from

replicating or carrying out normal metabolic functions such as enzyme synthesis.


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V. Physical Methods of Controlling Microbial Growth Note: Each method has it limitations, advantages and disadvantages

1. Heat

• All microbes have a maximum and a minimum temperature for growth

• Almost all macromolecules lose their structure (i.e., denature) and ability to function at very

high temperatures

• Moist or dry heat is used.

Moist heat is more effective than dry heat because

• It penetrates cellular structures better

• It facilitates unfolding of proteins, degrading DNA and disrupting membranes

• It causes hydrogen bond rearrangement

Dry heat is basically an oxidative process that

• denatures proteins and DNA

• disrupts membranes

The choice of heat treatment and temperature depends upon material being treated.

• Too high a temperature may destroy a food product or render a medium useless.

• Wet heat may cause metal instruments to rust

• The presence of certain compounds (e.g., protein, sugars and fats) may increase the resistance

of cells to heat

e.g., Microbial death is more rapid at acidic pH

High concentrations of sugars, proteins and fats decrease heat penetration

Dry cells and endospores are more resistant than wet cells

a. Autoclaving (Fig 7.2)

• Exposure of materials to 121°C at 15 psi (1.1 kg/cm2) produces high temperature steam

necessary to kill endospores

• This processes sterilizes materials

• Duration depends on size and bulk of object – heat must be transferred to interior

b. Canning

• Uses heat under pressure to sterilize and hermetic sealing to drive out oxygen

c. Pasteurization

• used to kill harmful organisms in food or beverages and to prevent spoilage.

• This process does not sterilize; sterilization would destroy desirable properties of many foods

and beverages

Two pasteurization processes that achieve the same results (more or less)

o Low temperature - hold (LTH, also known as bulk pasteurization) - 62.8°C for 30 min


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o High temperature - short time (HTST, also known as flash pasteurization) 71.7°C for 15s.

This method is preferable for milk because it alters the taste less, kills heat resistant

microbes more effectively and can be done on a continuous flow bases

d. Ultra High Temperature sterilization (UHT)

• Sterilizes food and other products

• 141°C for 4 - 15 s; allows for a continuous flow system

e. Dry Heat

• often requires much higher temperatures and longer exposure times (e.g., 170°C for 2 h)

• Extreme temperature may damage materials that can resist lower temperature (121°C)

• Other examples include the flaming of an inoculating loop and incineration of combustible

materials

2 Low Temperature

• Refrigeration (0 to 7°C) and freezing

• Does not kill all microbes but inhibits growth

3. Radiation

• Forms of electromagnetic radiation

i) Microwaves

• works partially due to heating

ii) Ultraviolet radiation

• 220 to 300 nm in wavelength

• This radiation is energetic enough to cause modifications or strand breaks in DNA that may

result in disruption of genetic material and death of the organism

• low energy, low penetrating power - surface treatment

iii) Ionizing radiation

• high energy, high penetrating power that produces electrons, hydoxyl radicals, and hydride

radicals ! breaks hydrogen bonds, oxidizes double bonds, destroys ring structures, degrade

and alter biopolymers

• Standard for biological applications is absorbed radiation dose measured as

Rad = 100 erg/g (where 1 joule = 10,000,000 erg)

Gray (Gy) = 100 rad

• A dose providing a 10 fold reduction in numbers is referred to as a D10. A standard killing

dose is 12 D10 equivalents

• Sensitivity to radiation differs for different microbes

Application of radiation

• 60

Co and 137

Cs sources

• medical supplies and food industry (spices and fresh meat)


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4. Filtration

• Applicable to liquids and gases

• Remove microbes - does not kill them

• Types include depth, membrane and nucleopore (Fig 7.4)

e.g., Biocontrol hoods & laminar flow hoods - High efficiency particulate air (HEPA) filters are a

type of depth filter – remove 99.97% of 0.3!m particles

5. Desiccation

• Removal of water - microbes can't grow

• Dried fruit, meat and fish

• lyophilization - freeze dried food

VI. Chemical Methods of Controlling Microbial Growth

Classes of Antimicrobial Agents

A. Inhibition of microbial growth on inanimate objects or on the exterior of animate objects

i. Sterilants

• A sterilant destroys all forms of microbial life including endospores

• “cold sterilization” often needed for sterilizing and decontaminating heat sensitive materials

e.g., ethylene oxide, formaldehyde, H2O2, sodium chlorite (bleach)

ii. Disinfectants

• kill or inhibit growth of microbes but not necessarily endospores.

e.g., ethanol, cationic detergents, chlorine, phenol

iii. Sanitizers

• A disinfectant that is used to reduce numbers of bacteria to levels judged to be safe by public

health officials.

iv. Antiseptics

• Agents that kill or prevent growth of microbes on living tissues

e.g., Alcohol, phenol, iodine, crystal violet

Alcohol - dehydrates and denatures proteins and disrupts membranes. Alcohol works best

when slightly diluted (70 to 90%). If alcohol is used full strength, then it dehydrates cells

before proteins can be denatured.

v. Preservatives

• agents that prevent microbial growth in foods and other products.

e.g., organic acids (propionic acid, lactic acid, citric acid, sorbic acid), NO3-, NO2-, NaCl


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Assessment of Disinfectant Efficacy

1. Phenol coefficient test – a standard amount of test organisms (Salmonella typhi and

Staphyloccous aureus) are added to a dilution series of i) the disinfectant of interest and ii) phenol.

The dilutions are placed in a water bath at 20 or 37°C. Samples are withdrawn at 5 minute

intervals and inoculated into growth medium. The inoculated medium is incubated for two or

more days. The highest dilution that kills the bacteria after a 10 minute exposure, but not after 5

minutes, is used to calculate the phenol coefficient.

• Phenol coefficient = reciprocal for the appropriate dilution of the disinfectant being tested

reciprocal for the appropriate dilution of phenol

• A value > 1 means the disinfectant is more effective than phenol

2. Use-dilution test – cylinders of metal or glass are dipped in standardized cultures of the test

organism, removed and allowed to dry. The dried cultures are placed in a solution of the

disinfectant and left for 10 minutes at 20°C. The cylinder is then transferred to a suitable growth

medium and monitored for growth.

3. Disk diffusion method (Agar-diffusion method) – See below for details

B. Inhibition of microbial growth in vivo

Chemotherapeutic Agents

• Chemical compounds that can be used internally for control of microbes

• Synthetic agents and Antibiotics = Antimicrobial agents

An antibiotic is a chemotherapeutic compound produced by microorganisms.

To be useful in therapy, a chemotherapeutic agent must have selective toxicity

e.g., high toxicity towards the microbe but low toxicity towards the host.

Selection of Chemotherapeutic Agents

• Sensitivities of target microorganism

• Side effects of antibiotic

• Longevity of antibiotic (e.g., biotransformations)

• Chemical properties of antibiotic- affect targeting of antibiotic in the body

• Selectivity - broad spectrum vs narrow spectrum of activity (Table 20.2)


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Assessment of Chemotherapeutic Agent Efficacy

i) Agar Diffusion Method (Kirby-Bauer test)

• designed for assessing the effectiveness of antibiotics - sensitivity of microbes

• Lawn of bacterium in question & disks impregnated with antimicrobial agent

• measure clear zone - zone of inhibition

• Standardized zone sizes are a measure of sensitivity for chemotherapeutic agents

S- susceptible (Ampicillin - 14 mm or more)

R - resistant (Ampicillin - 11 mm or less)

I - Intermediately sensitive (Ampicillin - 12-13 mm)

• Size of zone depends on a number of factors including amount of antimicrobial agent added to

the disc, solubility, and effectiveness of agent

• Conclusion - whether an antimicrobial agent has the potential to control a particular

microorganism

• This test is limited to use with rapidly growing bacteria

• It is a standardized test not designed for assessing the effectiveness of antibiotics against

filamentous fungi, anaerobes or slow growing bacteria, although it is possible to modify test in

order to assess these organisms.

ii) Minimum Inhibitory Concentration (MIC, also known as tube dilution technique)

• Dilutions of antibiotics or other chemical agents are tested for ability to inhibit growth of a

microbe in liquid culture

• MIC is the lowest concentration of an agent that completely inhibits the growth of the test

organism

• Determines the minimum amount of antimicrobial agent that must be present at the site of

infection to inhibit growth of the pathogen.

• Affected by the nature of the test organism, inoculum size, medium composition and other

growth parameters (incubation time, temperature, pH, aeration…)

• aids in selection of antibiotic and dosage (required to know how antibiotic distributes).

Physicians generally add in a safety margin - 10 times MIC

• Medium from tubes that do not show growth (i.e., have higher antimicrobial concentration

than the MIC) can be used to inoculate broth or on agar plates free of the antimicrobial agent.

If growth occurs then the drug was not bactericidal and the minimum bactericidal

concentration (MBC) can be determined.

VII. A look at chemotherapeutic agents and their mode of action

Antimicrobial agents can be categorized based on a number of key characteristics including

• Source (synthetic or natural products)

• Mode of action (Fig 20.2)

• Target microorganism

• Chemical structure


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A. Synthetic antimicrobial agents

1. Growth factor analogs – structural similarity to growth factors

Examples,

- Sulfa drugs – sulfonamide – analog of p-aminobenzoic acid (part of folic acid).

Treatment with sulfamethaxazole/trimethoprim results in sequential blocking of the

folic acid biosynthetic pathway (Fig 20.13). Resistance requires a double mutation in

the same microorganism

- Isoniazid – inhibits mycolic acid synthesis – The genus Mycobacterium has a cell wall

different from other bacteria. It contains mycolic acid that influences the staining

properties of mycobacteria. A number of important pathogens are found in this genus,

including those that cause tuberculosis (Mycobacterium tuberculosis) and leprosy

(Mycobacterium leprae).

- Nucleic acid analogs – often substituted with fluorine or bromine

2. Quinolones

• Not growth factor analogs - interact with DNA gyrase and topoisomerase II (involved in DNA

replication – packaging DNA)

• Effective against both gram-positive and gram-negative bacteria

e.g., Nalidixic acid

Fluoroquinolone derivatives – norfloxacin and ciprofloxacin are more soluble than

Nalidixic acid

B. Antibacterial Antibiotics

1) Cell Wall Inhibitors

a) Prevent formation of peptide cross linkages by binding to transpeptidase.

"-Lactams

• Characterized by "-lactam ring

• Transpeptidases are also known as penicillin binding proteins (PBP; FtsI)

• Account for over half of the antibiotics produced and used worldwide

Ineffective against

• bacteria which lack peptidoglycan cell walls - Mycoplasmas

• resting or dormant cells

"-lactam rings (Figure 20.6)

e.g., Penicillin - Penicillium chrysogenum

Cephalosporin - Cephalosporium spp.


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b) Bind directly to peptide portion of peptidoglycan

• block action of transpeptidase

e.g., Vancomycin – a glycopeptide that is not transported across the outer membrane of

many Gram negative cells

2) Protein Synthesis Inhibitors (Fig 20.4)

Aminoglycosidses - amino sugars bonded by glycosidic linkages to other amino sugars

• Streptomycin, Kanamycin, Neomycin – produced by members of the genus Streptomyces

• Not effective against anaerobes or Gram positive bacteria

• Bind to small subunit (30S) of the 70S ribosome – prevent protein chain initiation

• Not widely used today

Macrolides – multi-member lactone ring connected to sugar moieties (Fig 20.12)

• erythromycin, tylosin - Streptomyces

• act on the 50S ribosomal subunit - prevents peptide bond formation

• >10% of world production

Tetracyclines - four fused cyclic rings – naphthacene ring system (Fig 20.11)

• Broad spectrum – Gram-positive and Gram negative bacteria

• Bind to 30S subunit - block the A-site (aminoacyl site) – prevent protein elongation

• produced by Streptomyces

Chloramphenicol – simple structure makes this antibiotic inexpensive to synthesize (Fig 20.10)

• inhibits formation of peptide bonds in the nascent polypeptide by reacting with the 50S

subunit

• Produced by Streptomyces

Rifamycin

• block protein synthesis at the transcriptional level - binds to RNA polymerase "-subunit

• Best known example is rifampin

3). Membrane Function Inhibitors

• Polymyxin - Bacillus polymyxa - changes membrane structure and causes leakage

• Daptomycin – cyclic lipopeptide that binds to cell membrane and induces rapid depolarization

of membrane potential

4). DNA scission

• Phleomycin/bleomycin/zeocon

• Broad spectrum antibiotics that are affectcive against prokaryotes and eukaryotes

• Cleave DNA


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C. Antifungal Antimicrobial Agents

• Problems associated with host toxicity

1) Ergesterol inhibitors

• Polyene - affinity for membrane sterols such as ergesterol - disrupt membrane structure

e.g., Nystatin, Amphotericin B (Fig 20.14)

2) Ergesterol synthesis inhibitors

• Azoles (Fig 20.15) and allyamines

• Inhibit ergesterol biosynthesis - alter membrane permeability

e.g., Ketoconazole

3) Cell wall synthesis

• Polyoxins - interfere with chitin biosynthesis

• Echinocandins – interfere with 1,3 "-D glucan synthase – results in incomplete cell wall

4) Inhibit microtubule assembly

• Griseofulvin is produced by Penicillium spp.

• Prevents microtubule assembly, interfering with mitosis and thereby inhibiting fungal

reproduction

D. Antiviral Agents

• Problems associated with host toxicity

1) Nucleoside/nucleotide analogs (Fig 20.16)

• Acyclovir - Herpes simplex - blocks viral DNA replication

• Zidovudine (nucleoside analog) and tenofovir (nucleotide analog) - blocks DNA production

(reverse transcription) – effective against retroviruses (HIV) – RNA virus that converts

2) Non-nucleoside reverse transcriptase inhibitors

• Bind directly to the reverse transcriptase and inhibit activity

e.g., nevirapine

3) Protease inhibitors

• HIV requires protease activity in the maturation process

• Research has identified peptides that bind in the protease active site and inhibit protease

activity

• Atazanavir and indinavir and saquinavir are protease inhibitors of

4) RNA polymerase inhibitors

e.g., Rifamycin

5) Neuraminidase inhibitors

• Neuraminidase is an enzyme on viral surface that is important in releasing virions from host cell

e.g., Tamiflu (oseltamivir) – inhibitor of influenza neuraminidase


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6) Interferon – low molecular weight proteins

• prevent viral replication by stimulating the production of antiviral proteins that prevent further

viral infection

• no clinical use because interferon must be present in relatively high local concentrations

VIII. Antibiotic Resistance

• Antibiotic producers must protect themselves from the antibiotics that they produce

1. Mechanisms of Antibiotic resistance (Figure 20.20)

• The organism lacks the structure that antibiotic acts upon

• Impermeable to antibiotic

• Decreased transport - "-lactams

• Active secretion - Efflux pump - pumps out antibiotic - Tetracycline resistance

• Production of modifying enzymes

hydrolytic enzymes - "-lactamase

addition of chemical groups - acetylation or phosphorylation of macrolides

• Modification of site of antimicrobial action - ribosomal modification - streptomycin rpsL

• Production of alternate molecules that can replace those disrupted by antibiotics

2. Genetic Basis of Antibiotic Resistance

• Antibiotic resistance is coded at the chromosomal or the plasmid level (R-plasmids)

• Resistance at the chromosomal level almost always arises due to modification of the target of

the antibiotic

• Plasmid resistance often results from the production of enzymes that inactivate, prevent uptake

or actively pump out the antibiotic


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3. Spread of antibiotic resistance

Overuse and improper use of antibiotics

• Treatment of disease

• Intensive Livestock production

How did antibiotic resistant bacteria come about?

Sources of Genetic Variation

Mutation

high growth rates - production of large populations

spontaneous mutation 10-7

to 10-9

/generation

Recombination

• transformation

• transduction

• conjugation

Transfer of antibiotic resistance determinants via

• R-plasmids

• Transposons

• Integrons

Microbial control lecture reference

Health & Medicine

Transcript of Microbial control lecture reference