Effective Microbial Growth Control: Methods and Prevention

Posted on Feb 21, 2025 in Biology

Microbial Growth Control

Microbial death rate

Bacterial populations are at a constant log rate.

Effectiveness of Antimicrobial Treatment

• Number of microbes: An increase in microbes means it takes longer to eliminate the entire population.
• Environmental influences: The presence of organic matter inhibits the action of chemical antimicrobials. Microbes in surface biofilms are difficult for biocides to reach effectively due to temperature-dependent chemical reactions. Disinfectants sometimes work better in warm conditions. Fats and proteins are protective; therefore, microbes have a higher survival rate. Heat is more effective in acidic conditions.
• Time of exposure: Extended exposure is required to affect resistant microbes or endospores.
• Microbial characteristics: Affect the choice of chemical or physical method.

Actions of Microbial Control Agents

• Alteration of membrane permeability: Damage to proteins or fats of the plasma membrane causes cellular contents to leak into the surrounding medium and interferes with growth.
• Damage to proteins and nucleic acids: Breakage of bonds by heat or chemicals causes protein denaturation. Damage to nucleic acids by heat, radiation, or chemicals means the cell cannot replicate or perform its functions.

Physical Methods of Microbial Control

Heat

Heat preserves canned goods and is used to sterilize lab media, glassware, and hospital equipment. It kills microorganisms by denaturing enzymes.

• Thermal Death Point (TDP): The lowest temperature at which a microorganism will be killed in 10 minutes.
• Thermal Death Time (TDT): The minimum time to kill at a given temperature.
• Decimal Reduction Time: The time (in minutes) in which 90% of the bacterial population is killed at a given temperature.

Moist Heat Sterilization

Denatures proteins by breakage of hydrogen bonds. Boiling kills vegetative forms of bacterial pathogens, almost all fungi, viruses, and spores in 10 minutes. Free-flowing (unpressurized) steam is the same temperature as boiling water, but endospores and viruses are not killed as quickly. Some spores survive boiling for >20 hours, so boiling is not reliable. Boiling at high altitudes kills most pathogens, but reliable sterilization requires temperatures above boiling.

Autoclave

Steam under pressure is the preferred method unless damage by heat or moisture is a concern. Higher pressure means higher temperature. 100°C at 1 atm above sea level, 15 psi yields a temperature of 121°C. Most effective when the organism is contacted by steam directly or contained in a small volume of primarily water. Steam at 15 psi (121°C) will kill endospores in 15 minutes, except for prions. Similar to a pressure cooker, it is used for instruments, equipment, syringes, and items that can withstand high temperature and pressure.

Steam Sterilization

The surface of solids requires contact with steam. Used for glassware and bandages. Foil is impervious to steam and therefore not used to wrap sterile material. Steam must flow vigorously out of the valve in the lid for several minutes to carry with it all the air before the pressure cooker is sealed. Important for preventing botulism with improper canning.

Pasteurization

Eliminates pathogenic microbes and lowers microbial numbers to prolong shelf life. Thermoduric (heat-resistant) bacteria survive pasteurization but are unlikely to cause disease or spoil milk. Less effective in viscous or fatty substances. Milk is pasteurized at 63°C for 30 minutes.

• HTST (High-Temperature Short-Time): 72°C for 15 seconds, through a heat exchanger, lowers bacterial count.
• UHT (Ultra-High Temperature): Stored without refrigeration. Milk is sprayed through a nozzle, temperature reaches 140°C in seconds, then vacuumed airtight. Tastes different, fats are emulsified, and some organisms survive.

Dry Heat Sterilization

Kills by oxidation. Direct flaming for loops, incineration for paper cups, hot air for a long time and high temperature.

• Equivalent Treatment: Hot air: 170°C for 2 hours; Autoclave: 121°C for 15 minutes.

Cold

Refrigeration slows metabolic rate and prevents reproduction or toxin production. Deep freezing (between -50°C to -95°C) keeps microbes dormant, not killed. Slow freezing is more harmful because ice crystals form and disrupt cell/molecular structure of bacteria. Slower thawing is more damaging as part of the freeze-thaw cycle.

High Pressure

Applied to liquid suspensions and transferred throughout the sample, altering shape and inactivating endospores. Can be used for fruit juice (keeps color, taste, nutrients).

Dessication

Absence of water prevents microbial growth/reproduction. Lyophilization (freeze-drying) preserves microbes (coffee, fruit in dry cereal). Water is removed by vacuum at -54°C to -72°C.

Osmotic Pressure

High concentrations of salt and sugar keep food by creating a hypertonic environment (water leaves the cell), denying moisture. Used for curing meats and fruits. Mold/yeast are capable of growing in low moisture (fruits/grain can spoil).

Filtration

Sterilizes heat-sensitive material/solutions (antibiotics, vaccines) by removing/lowering microbes. Separates bacteria from liquid by passing liquid/gel through a membrane filter.

• High-Efficiency Particulate Air (HEPA) Filters: Remove all microorganisms larger than 0.3 μm in diameter.
• Membrane Filters: Made of cellulose, 0.1 mm thick, with pores 0.22 μm-0.45 μm intended for bacteria. Filters with small pores (0.01 μm) retain viruses/proteins.

Radiation

• Ionizing Radiation: Gamma rays, X-rays, high-energy electron beams, wavelength <1 nm, carries much more energy. Gamma rays penetrate deeply but may require hours. High-energy beams have lower penetration but require few seconds. Ionizing particles create mutations which kill. Used for food preservation with low ionizing radiation.
• Nonionizing Radiation: Wavelength >1 nm. UV damages DNA of exposed cells by causing bond formation with adjacent bases in the DNA chain, therefore inhibiting replication. UV at 260 nm kills microorganisms and can also control microbes in the air. UV lamps are used in hospital rooms and to disinfect vaccines and other medical products. Must be directly exposed, no covers.
• Microwave: No direct effect on microorganisms. Heat moisture contained in foods and kills vegetative pathogens. Solid food heats unevenly due to uneven moisture distribution. Food parasites may not be affected.

Microbial Contamination of Foods

Food storage depends on the food item: raw, cooked, processed, type (meat, fish/seafood, fresh fruit/veg, dairy, egg/egg product).

Factors for Bacterial Growth

• Food: High protein/starch content.
• Temperature: 4-60°C (Danger Zone).
• Time: Reproduce every 20 minutes.
• Moisture: Aw >0.85. Most require a minimum Aw of 0.91, Staph >0.86, yeast 0.88, mold 0.82.
• Common Foods: (Most) Bread, fruit, cheese, meat, jam, (Least) cracker unless high fat. Jam has high sugar and low water, so no growth. Different Aw requires different time to inactivate: 0.995 at 60°C = 0.18 min, 0.94 at 60°C = 4.3 min.
• Acidity: pH 4.6-7.0. <5 reduces growth except for molds, 7 supports growth. Most bacteria = 5.5-8, yeasts = 4.0-6.5, molds = 4.5-6.8.
• Oxygen: Presence or absence.

Food Spoilage and Microbial Contamination

Relationship between food spoilage and quantitative extent of microbial contamination:

• Raw milk: Sour at 10³-10⁶
• Vacuum-packed meats: Off odor at 10⁶-10⁷
• Aerobically-stored meats/vegetables: Off odor at 10⁷-10⁸
• Almost all food, aerobically stored meats: Slime at 10⁸-10⁹
• Structural change and decomposition >10⁹

• Internal Cooking Temperature: Medium rare 71°C, not recommended for elderly/kids. Pork 71°C; heat resistant.
• Chain of Infection: Contamination, moisture, temperature, time.

Foodborne Illness

• Infection: Consumption of living microorganisms, quicker onset, symptoms within 1-12 hours.
• Staph: Infected food handler, cooked high-protein food, heat-stable.
• Bacillus: Cooked rice.
• Clostridium: Produce, soil, home food.
• E. coli: Ground beef, meat, raw milk.
• Intoxication: Consumption of microbial toxins instead of microbes themselves, slower onset, symptoms 6-72 hours.
• Salmonella: Chicken, raw eggs, meat, dairy, 3 days later, diarrhea/vomiting.
• Shigella: Fecal contamination, salad, milk, water, bloody stool.
• Campylobacter: Chicken, raw meat, milk, water, bloody stool.

80/20 Rule

80% of problems are caused by improper cooling, advance prep (12 hours), infected person, inadequate reheating, improper hot storage. 20% are the vital few.

HACCP (Hazard Analysis and Critical Control Points)

Internationally recognized system to help ensure the manufacture of safe food and enhance food safety throughout the food chain.

Designed to prevent, reduce, or eliminate potential biological, chemical, or physical food safety hazards, including those caused by cross-contamination. During the development of a HACCP system, potential hazards are identified and control measures are implemented at specific points in the manufacturing process.

• Control Point: Any point, step, or procedure at which biological, physical, or chemical factors can be controlled.
• Critical Control Point: A point, step, or procedure in the product-handling process where controls can be applied and a food safety hazard can be prevented, eliminated, or reduced to acceptable levels (e.g., cooking step for elimination, chilling step for reduction).

7 Standardized Principles

1. Conduct a hazard analysis process of identifying the hazards that might affect a particular product in a specific processing operation, collecting and evaluating information on the hazards and conditions leading to their presence, and deciding which are significant to food safety and must be addressed by the HACCP plan.
2. Potentially Hazardous Foods: Perishable food or drink, cooked or raw animal products, meat, dairy, eggs, pies, pastries, cheese, sliced meats, custards, desserts, mayonnaise, salad dressings, fish/shellfish, soy products, cooked vegetables & starches, beans, rice, pasta, potatoes, raw seed sprouts, alfalfa, mung bean, preserves, jams, jellies, garlic and herbal oils.
3. Foodservice Establishments; Sources of Foodborne Microorganisms: Humans (nose & throat, hands, mucosal secretions, fecal matter, clothing, gloves, hat), foods of animal origin (poultry, meat, eggs, fish/shellfish), foods of plant origin (soils, water).
4. Principle 2: Determine the Critical Control Points (CCP) is a point, procedure in the food manufacturing process at which a control measure can be applied and is essential to prevent, eliminate, or reduce a food safety hazard to an acceptable level. Determining the CCPs involves identifying wherein the processing operation the hazards addressed in the HACCP plan can be prevented, reduced, or eliminated.
5. Principle 3: Establish Critical Limits. Critical limits are criteria that separate safe product from unsafe product. Critical limits must be established for each CCP. Critical limits must be clearly defined & measurable. Limit Time Danger Zone; 4-60°C, no more than a combined total of 4 hours for all procedures; thawing, preparation time, cooling, reheating.
6. Principle 4: Establish Monitoring Procedures. Monitoring is the process of conducting a planned sequence of observations or measurements to determine if a CCP is under control. For each CCP, monitoring procedures must be implemented and documented to ensure that the critical limit is being met.
7. Principle 5: Establish Corrective Actions. Corrective actions are predetermined activities that are taken when CCP monitoring results indicate that a deviation has occurred and there is the potential that unsafe food has been, or will be, produced. For each CCP there must be planned, written corrective actions. The objectives of taking corrective actions are to regain control of the hazard, to determine the disposition of the affected product and to prevent a reoccurrence of the problem.
8. Principle 6: Establish Verification Procedures. Verification is the application of methods, procedures, tests and other evaluations, in addition to monitoring, to determine conformance with the HACCP plan. Verification confirms that the HACCP plan is operating effectively and according to written procedures.
9. Principle 7: Establish Record Keeping and Documentation Procedures. HACCP plans must be documented. The required monitoring & verification records must be complete and accurate.

Sprouted Seeds

Increasing in popularity in Canada, favored for their nutritional value, but linked to outbreaks of foodborne illness.

Foodborne Outbreaks Associated with Seed Sprouts

• 1995-1999 alfalfa Salmonella >300 Canada-wide
• 2000 -2005 mung bean Salmonella >100 Canada-wide
• 1996 radish E. coli O157:H7 >6000 Japan
• 2011 fenugreek E. coli O104:H4 >9000 Germany

Microbial Contamination of Sprouted Seeds

Contamination most likely originates from seeds usually for field planting & not for raw consumption in the field or during harvesting, storage, and transportation. Germination process in sprout production involves keeping seeds warm and moist for 4 to 7 days. Low levels of microbial contaminants on seeds quickly reach levels high enough to cause illness.

CFIA Code of Practice for the Hygienic Production of Sprouted Seeds

Code recommends control of pathogens to occur in 2 areas: during seed production and during sprout production. Sprout manufacturers should control food hazards through a system based on Hazard Analysis Critical Control Point (HACCP) principles. A HACCP type program will reduce the risk of unsafe food by taking preventive measures to assure the safety and suitability of food at appropriate stages in the operation by controlling food hazards.

Section 8. Control of Sprouting Operations

Section 8.1 Control of Food Hazards

• Identify hazards that may be associated with sprouts and the sprouting process.
• Identify any steps in their operations which are critical to control the safety of sprouts.
• Implement effective control procedures at those steps by establishing critical limits.
• Monitor control procedures to ensure their continuing effectiveness.
• Have procedures in place for dealing with deviations that may occur with the critical limits.
• Verify control procedures periodically and whenever the operations change.
• Maintain records as specified in section 9 of this Code.

Section 9. Documentation & Records

Written records that accurately reflect product information and operational controls should be available to demonstrate the adequacy of the manufacturing activities.

Records should:

• Be available on demand.
• Be legible, permanent, accurate and be signed and dated by the individual(s) responsible.
• Include written procedures, controls, limits, monitoring results and subsequent follow-up documents.

Records must include:

• Seed sources and lot numbers.
• Water analysis results.
• Sanitation checks.
• Pest control monitoring.
• Sprout lot codes.
• Sprout and spent irrigation water analysis results.
• Production volumes.
• Storage temperature monitoring.
• Product distribution.
• Consumer complaints.

Records should be retained for at least 1 year for each lot of sprouts.

Listeria Contamination of Processed Meat Product

Maple Leaf Foods, Aug 2008

• Listeriosis outbreak
• 56 confirmed cases (41 in Ontario)
• 20 deaths

Listeria

• Foodborne bacteria that can grow at refrigerator temperatures.
• Found in soil, water, decaying vegetation, intestinal tract of animals.
• Infections result in flu-like illness, diarrhea, upset stomach.
• Healthy individuals rarely seriously ill.

Listeriosis can cause serious health problems, even death, in certain high-risk groups: pregnant women, newborns, elderly, individuals with weakened immune systems. Infection may spread to the nervous system, causing headache, stiff neck, confusion, loss of balance, convulsions.

An infected pregnant mother may experience only a mild, flu-like illness, yet the infection can still severely affect her unborn baby leading to miscarriage, premature labor, the delivery of a low-birthweight infant, and a wide range of health problems for a newborn or even infant death.

Listeria cannot be completely eliminated from the environment. It is important that surfaces in direct contact with product, such as slicers, must be 100% free of contamination. Contamination was most likely the result of a combination of factors, including: the potential for equipment to harbor organic material, employee and product movement, physical factors.

CFIA Response

Investigation has not confirmed the source of contamination. Aware of potential contributing factor related to inadequately cleaned meat slicing equipment. CFIA advice to all operators of federally registered establishments using meat slicing equipment is to use appropriate measures to address this potential risk factor as follows:

1. At the next scheduled line sanitation, disassemble and perform a systematic and thorough aggressive cleaning and sanitation procedure upon such equipment including all internal non-electronic parts. Inform the CFIA inspector of all details of this exercise.
2. Perform Listeria environmental sampling of contact surfaces and re-sanitize such equipment prior to reuse. Inform the CFIA inspector of the results of this exercise upon receipt of the results.
3. If warranted, and in consultation with the CFIA inspector, review the standard cleaning and sanitation procedures for such equipment to ensure that internal working parts are being suitably cleaned and disinfected on an ongoing basis. This includes the introduction of routine Listeria environmental sampling of contact surfaces for such equipment, if such testing is not already being done.

Different Types of Water

• Drinking (potable) water: Has levels of microorganisms/chemicals low enough not to be a health concern.
• Recreational water: Fresh or marine, swimming pools, beaches, sport/leisure.

Microbial Contamination of Water

Low levels of microbial contamination require concentration, culture, and detection OR collecting a large volume sample, concentrating, and detecting.

Waterborne Bacteria

Indicator Bacteria

Waterborne pathogens may be difficult to culture or detect, may not have adequate laboratory equipment or containment level, expensive and specialized tests. Quick, reliable, and relatively affordable tests for water quality monitoring are required. Indicator bacteria provide a suitable alternative.

Recovery and Detection of Bacteria from Water Samples

• Collection: 100-200 ml grab sample.
• Water Quality Tests: Monitor fecal indicator bacteria (rather than actual pathogens).
• Total coliforms
• E. coli

Total Coliforms

• Inhabit the intestines of warm-blooded mammals.
• Several genera of Enterobacteriaceae e.g., Escherichia, Klebsiella, Enterobacter & Citrobacter.
• Occur naturally in soil, vegetation, and water.
• Usually non-pathogenic.
• Presence indicates possible presence of pathogens or fecal pollution.
• Less reliable as a fecal pollution indicator but preferred as an indicator of treatment adequacy in drinking water supply systems.
• No detectable total coliform bacteria per 100 ml in drinking water.
• 1000 total coliform bacteria per 100 ml in recreational water.

Total Coliform Tests

• Total Coliform Fermentation Technique – Most Probable No. (MPN); statistical test provides an estimation of contamination.
• Total Coliform Membrane Filter Technique. When the amount of bacteria present is low – e.g., drinking water. Filter a larger volume (e.g., 100 ml) of water through a membrane. Specially formulated screen filters made of cellulose acetate, pore sizes on these filters can be regulated and microorganisms are trapped on the surface of screen filters.

Filter a known volume of water through a 0.45 μM membrane filter. Incubate filter on agar (Endo-type) containing lactose at 35°C for 24h. Count red colonies with a metallic sheen as coliforms. Not suitable for high turbidity.

Total Coliform Fermentation Technique

• Based on lactose fermentation.
• Serially dilute water sample.
• Add 5 aliquots of dilutions to 5 tubes of lauryl tryptose broth.
• Incubate at 35°C for 48h.
• Check tubes for growth – color change.
• Confirm using brilliant green lactose bile broth fermentation tubes.
• Calculate coliform density in terms of Most Probable Number (MPN)/100mL.
• Detects all aerobic and facultative anaerobic, Gram-negative, nonspore-forming, rod-shaped bacteria that ferment lactose with gas and acid formation within 48h at 35°C.
• Inverted Durham tubes trap the gas produced.

Fecal Coliforms

• Escherichia mainly.
• Lesser extent Klebsiella and Enterobacter.
• E. coli – indicator of fecal pollution.

Portion of coliforms capable of forming gas in EC (tryptose lactose bile salts) medium or blue colonies on m-FC (enriched lactose) broth within 24h at 44.5°C. Differentiates between coliforms found in the feces of warm-blooded animals and those from other environmental sources.

• No detectable E. coli per 100 mL in drinking water.
• 100 E. coli per 100 mL in recreational water (e.g., swimming and bathing beaches).

Fecal Coliforms Colilert (ONPG-MUG) Test Total Coliform and E. coli

• Coliforms b-galactosidase break down orthonitrophenyl-β-D-galactopyranoside (ONPG).
• Release yellow ONP indicator.
• Colilert Absence/Presence test.
• E. coli β-glucuronidase hydrolyzes 4-methyl umbelliferyl-β-D-glucuronide (MUG).
• Release fluorescent umbelliferone derivative.
• Very sensitive – 1 CFU/100 ml.
• Colilert Absence/Presence test.

USEPA Total Coliform & E. coli Membrane Filter Method

Incubate membrane filter on MI (enriched lactose) agar at 35°C for 24h. MI agar contains 4-(MUGal)&(IBDG). Count fluorescent colonies as total coliforms. Count blue colonies as E. coli.

Protozoal & Viral Contamination of Drinking Water

• No provincial or federal legislation maximum acceptable concentrations.
• At least a 3-log reduction in and/or inactivation of protozoan cysts and oocysts.
• At least a 4-log reduction and/or inactivation of viruses should be achieved by treatment technologies.
• Monitoring is not mandatory or routine.
• Detection is sometimes carried out during waterborne disease outbreaks.

Recovery and Detection of Protozoa & Viruses from Water

Collection: Filter >150L through yarn-wound or foam cartridges with 1.0 micron or smaller pore sizes.

Recovery and Detection of Protozoans from Water Samples

• Elution: Wash filters in detergent solution to elute trapped parasites.
• Concentration: Use immuno-magnetic separation (IMS) beads with specific binding reagents (antibodies) or overlay centrifuged pellets on density gradients.
• Detection: Stain parasites using immunofluorescence and enumerate with UV microscopy.

Protozoans in Surface Water

Cysts are environmentally hardy, resistant to chlorination, small (microns) difficult to filter.

Waterborne Protozoans

Toxoplasma Oocyst

No IFA stain or IMS beads available for detection in water. Routine tests not carried out. Use light, phase contrast or DIC microscopy.

Waterborne Amoeba

Routine tests not carried out. Use stains, light, phase contrast or DIC microscopy.

Drinking Water Sources

• Groundwater: wells, aquifers, springs.
• Surface water: lakes, rivers, creeks.

Drinking Water Treatment

Multiple Barrier Approach:

• Source water protection.
• Effective treatment.
• Coagulation, flocculation, and sedimentation.
• Filtration – membrane, sand.
• Disinfection – chlorination, ozonation, UV irradiation.
• Well-maintained distribution system.
• Routine verification of water quality.
• Public education.

Unfiltered Surface Water

• Improve watershed protection.
• Longer holding times in settling ponds/reservoirs.
• Use filtration – membrane or slow-sand.
• Use ozonation or UV irradiation.

Filtered Surface Water

• More effective flocculation / coagulation / sedimentation during high turbidity.
• Improve monitoring of treatment procedure.
• Optimize backwash procedure.

Emergency Water Treatment

• Boil water for at least 2 minutes or more at higher elevations.
• Use bottled water which meets federal health and safety standards.
• Filter water through absolute pore size of 1 micron or smaller.

Drinking Water Contamination

• Groundwater – wells, aquifers, springs.
• Sewage overflow.
• Shallow well.
• Surface water – lakes, river, creeks, (FILTERED).
• Filter backwash water recycling.
• Water filtration equipment malfunction.
• High turbidity – inadequate flocculation / coagulation.

Walkerton, May 2000

,50 residents,eoli in treated municipal water supply


1,346 reported cases
of gastroenteritis
CLINICAL DETAILS
 174 presumptive E. coli O157
 167 confirmed E. coli O157:H7
 116 confirmed Campylobacter jejuni
 66 hospitalized
 27 hemolytic uremic syndrome
 7 deaths 
Comparison of illness onset dates, rainfall
measurements and municipal well water
flows April – June 2000
 Location of Walkerton municipal wells 
WALKERTON Human Factors
Public Utilities Commission Operators
 Manager failed to disclose high levels of TC & E. coli
contamination in water supply in a timely manner
 Improper operating practices
 Unchlorinated and inadequately chlorinated well water was
being used
 Chlorine residuals improperly monitored and records
fabricated
 Sampling locations for microbiological tests were
mislabelled
 Poorly trained, did not understand the significance of
microbiological contamination of drinking water supply

WALKERTON
Environmental Factors
 Well 5 – total coliforms and E. coli present (at
>300 CFU/100ml & 120 CFU/100ml in treated
sample on May 23)
 Wildlife – all negative
 Livestock farms within 4 km radius
 11/13 farms with Campylobacter spp.
 2/13 farms with both E. coli O157:H7 &
Campylobacter jejuni
 1 of these farms adjacent to Well 5
 Likely origin – cattle manure
 Heavy rains & flooding
 Shallow well contaminated by surface water
 Water treatment system overwhelmed
 E. coli O157:H7 and Campylobacter spp. in
neighbouring farms
 The primary source of contaminants – manure
spread on a farm near Well 5 in late April 2000.
 DNA typing of animals and manure on the farm
showed that E. coli O157:H7 and Campylobacter
strains on farm matched strains prevalent in
human outbreak cases.
Canadian Waterborne
Outbreaks
 Sources of Contamination (Fecal)
 Livestock,wlkerton, ON: 2000 E. coli O157:H7
 Human North Battleford, SK: 2001 Cryptosporidium
hominis
 Wildlife Victoria, BC: 1995 Toxoplasma gondii ,creston, BC: 1992 Giardia lamblia

Cryptosporidiosis Outbreak
North Battleford, Saskatchewan
April 2001
Stirling et al, Can Commun Dis Rep. (2001) 27(22):185
 Cryptosporidiosis Outbreak
 Epidemiological estimate 5800-7100 affected
 Water filtration plant operating sub-optimally
 Cryptosporidium oocysts present in finished
drinking water

Comparison of epidemiological curve with
 Anti-diarrheal drug sales
 % settling in solids contact unit
 surface water treatment plant finished
water turbidity
Dye plume flow downstream from sewage
to water treatment plants 
2 drinking water source
– Ground water
– Surface water
 Spatial modelling results show that
 Residents in part of city supplied with
water from the surface water treatment
plant was 2 – 4X more likely to become ill
with cryptopsoridiosis
Contamination source,Was it from the sewage treatment plant ?
 Or from further upstream ?

Molecular Epidemiological
investigation  2 major human pathogenic species of Cryptosporidium,
 C. hominis : infects humans
 C. parvum : infects both humans and animals
ie. zoonotic


Source of contamination can be attributed by
identifying the species of Cryptosporidium present
in water samples and also in outbreak cases 
All outbreak cases were infected with C. hominis
 2 different sub-types were isolated from clinical
specimens
 Contamination was most likely derived from > 1
human source
 North Battleford surface drinking water most
probably contaminated by human sewage
 Likely not from agricultural activities further
upstream
Toxoplasmosis Outbreaks in BC
OUTBREAK MOST LIKELY CAUSE
 1995 Victoria Drinking water contaminated
by cougars
 100 cases  residents in area supplied by
Humpback Reservoir
Giardiasis Outbreaks in BC
OUTBREAK MOST LIKELY CAUSE
 1992 Creston Drinking water
contaminated by beaver
 1995 Revelstoke Drinking water
contaminated by beaver 
Drinking Water Contamination
UNFILTERED SURFACE WATER
1. Unprotected watershed
2. Multi-use watershed
3. High surface runoff –
heavy rain or snow melts
4. High turbulence/turbidity

Chemical Methods of
Microbial Control
Chemical agents
 Most reduce microbial populations to safe
levels
 Few achieve sterility
 No single disinfectant appropriate for all
circumstances
 Selection of the right chemical agent is
important
Factors related to
effective disinfection
 Concentration of disinfectant
– Dilute exactly as specified by manufacturer
 Nature of material being disinfected
– Organic material present may interfere
– pH of medium affects activity of disinfectant
 Time
– Contact period required with disnifectant
Methods used to evaluate
disinfectants
AOAC (American Official Analytical
Chemist) Use-dilution test
1. Metal rings dipped in test bacteria are dried
at 37°C for a short time.
2. Dried cultures are placed in disinfectant for
10 min at 20°C.
3. Rings are transferred to culture media to
determine whether bacteria survived
treatment.
Methods used to evaluate
disinfectants
 Disk-diffusion
method
–Disk soaked wit
disinfectant & placed
on agar plate with
lawn of bacteria


– Incubate and
examine zone of
inhibition of growth
Disk-diffusion method
 Chlorine (as bleach) & quaternary ammonium
compounds are more effective against Gram
positive bacteria
 Pseudomonas is only affected by bleach
Types of Disinfectants
Phenol & Phenolics
 Phenol: >1% v/v
 Phenolics: Lysol
 Bisphenols:
Hexachlorophene, Triclosan
–Disrupt plasma membranes
–Remain active in organic
matter
–Stable and persist for long
periods of time
–Use for pus, saliva & feces
–Effective against Gram
positive Staphylococci and
Streptococci bacteria and
mycobacteria 
Biguanides
 Biguanides: Chlorhexidine
– Disrupt plasma membranes
– Combined with detergents and alcohol in surgical
hand scrubs
– Low toxicity to skin
– Biocidal to most bacteria and fungi
– Not effective against mycobacteria, endospores
and protozoan cysts Ong ENH 222 Lecture 9 3


Types of Disinfectants
Halogens
Iodine
 Exact mode of action unknown, probably combines with
amino acids of enzymes and cellular proteins
 Effective against all types of bacteria, many endospores,
various fungi and some viruses.
 Available
– in aqueous alcohol solution as tincture or
– as an iodophor in combination with an organic molecule
from which iodine is released slowly eg. Betadine
 Used mainly for skin disinfection and wound teatment
 Iodine tablets for treating camping water
Types of Disinfectants
Halogens
Chlorine
 Exact mode of action unknown, strong oxidizing agent that
prevents cellular enzyme system from functioning
 Used as a gas or in water as hypochlorous acid (HOCl), Liquid form of compressed gas used for disinfecting drinking
water and swimming pools
 Choride compounds, sodium hypochlorite (NaOCl) bleach
eg. Chlorox used as household disinfectant
 Chloramines contain both chlorine and ammonia
–Glassware and eating utensil sanitizer
–Municipal drinking water treatment–Toxic to fish


Types of Disinfectants
Alcohols
Ethanol,
isopropanol
Denature proteins,
dissolve lipids, disrupt
membranes
 Acts before evaporating
leaving no residue
 Kill bacteria and fungi
but not non-enveloped
viruses or endospores
 Not effective in wounds,
cause coagulation of
protein under which
bacteria grow 
Types of Disinfectants
Heavy metals
Silver, mercury, copper and zinc
 Oligodynamic action by small amounts
 Denature proteins
 Silver with other anti-microbial drugs effective for burns,
wounds and catheters
 Mercury more toxic and used for
mildew control in paints
 Copper sulphate used as algicides
reservoirs, ponds and swimming pools
 Zinc chloride common ingredient in
mouthwashes
 Zinc oxide most widely used antifungal Ong ENH 222 Lecture 9 4
Types of Disinfectants
Surface-active agents
or surfactants
Soap & water
 Not antiseptic
 Emulsifier
 Cleans by washing away oil and dead cells

Acid-anionic (negatively charged)detergents,

 Reacts with plasma membranes
 Sanitizer for dairy utensils and equipment
 Wide spectrum includes thermoduric bacteria
 Non-toxic, non-corrosive and fast acting
Types of Disinfectants
Quaternary Ammonium
Compounds (Quats)
Positively charged cationic detergents
 Denature proteins, disrupt plasma membrane
 Bactericidal especially Gram positives
 Fungicidal, amoebicidal and virucidal against enveloped
viruses
 Do not kill endospores or mycobacteria
 Certain Gram negatives can survive and grow actively
 Zephiran
Mr. Clean
 Cepacol
cetyl
pyridimium
chloride
Types of Disinfectants
Aldehydes
 Inactivate proteins by cross-linking with functional
groups (–NH2
, –OH, –COOH, –SH)
 Used by morticians in enbalming
 Formaldehyde
– Gas is excellent disinfectant
– More commonly available as formalin (37% aqueous
solution)


Glutaraldehyde
– Used to disinfect hospital instruments
– 2% solution (Cidex)
 tuberculocidal, virucidal after contact for 10 min
 sporicidal after 3 – 10 hr
Types of Disinfectants
Gaseous sterilants
 Chemicals that sterilize in closed chamber
 Ethylene oxide
– Denature proteins
– Kills all microbes and endospores
– Lengthy exposure period of 4 –18 hrs
– Toxic and explosive so mixed with nonflammable gas such as CO2 or nitrogen
– Used for sterilizing hospital supplies and
equipment
Types of Disinfectants
Peroxygens (Oxidizing Agents)
 Ozone (O3)
– Highly reactive form of oxygen
– Generated by passing oxygen through high
voltage electrical discharges
– Helps neutralize taste and odour, used with
chlorine in water treatment 
(H2O2 Contact lens disinfection 
– Common antiseptic in households and hospitals
– Not suitable for open wounds as it slows down healing– Quickly broken down by catalase present in human cells – Effective on inanimate objects and surfaces especially against aerobes and facultative anaerobes – Increasing use in food industry todisinfect packaging material by passing through hot h2o2

Benzoyl peroxide
– Over-the-counter medication for acne
– Useful for treating wounds infected with
anaerobes
 Peracetic acid
– One of the most effective liquid chemical
sporicides
– Short disinfection times 5 – 30 mins
– Used for food-processing and medical
equipment as it leaves no toxic residues and is
minimally affected by organic matter
Microbial
Characteristics
and Microbial
Control
Factors that prevent
chemical disinfection
 Lipopolysaccharide layer
and porins of Gram
negative bacteria
 Lipid-rich cell wall of
Mycobacteria
 Bacterial endospores
 Fungal spores
 Protozoan (oo)cysts
 Non-enveloped viruses
 Prions
Types of Disinfectants
Chemical food preservatives
Organic acids & salts
Interferes with metabolism & plasma membranes
 Sorbic acid or potassium sorbate, benzoic acid or
sodium benzoate, and calcium propionate
–Control molds and bacteria in foods eg. cheese, soft drinks –Considered safe in foods


Sodium nitrate and nitrite

 –Prevents endospore germination
–Preserves red meat colour by reacting with blood
components
–Added to processed meat products eg. ham, bacon, hot
dogs & sausages
–React with amino acids to form carcinogenic nitrosamines
Types of Disinfectants
Chemical food preservatives
Antibiotics
 Nisin which is tasteless, easily digested and nontoxic prevents growth of endospore-forming
spoilage bacteria
 Natamycin anti-fungal & antibiotic approved for
use in foods eg. cheese
Antibiotics
 1928: Fleming
discovered penicillin,
produced by a mold
Penicilliumnotatum.
 1940: Howard Florey
and Ernst Chain
performed first
clinical trials of
penicillin.
 Sources of antibiotics
come from other
microorganisms Antibiotic
Sources
 Streptomyces
– Filamentous
bacteria found in
soil 
Most antibiotic producing
microbes have some kind of
sporulation process 
the Action of Antibiotics
 Bactericidal or bacteriostatic
 Does not affect human cells


Broad-spectrum antibiotics can destroy
normal flora in gut and other parts of the
body
 Yeast overgrowth can result
 Antibiotic resistance can also develop
Antibiotics: Inhibitors
of Cell Wall Synthesis
 Penicillin prevents bacterial cell wall peptidoglycan
synthesis
Penicillins 
ß-lactam ring in purple
area  Highly reactive ring
interferes with bacterial
cell wall synthesis
Destroyed by stomach acid not methicillin 
Cephalosporins
– 2nd, 3rd, and
4th generations
more effective
against gramnegatives
– Resistant to
penicillinase
– Cefotaxime,cefixime
Polypeptide antibiotics
 Bacitracin
– Interferes with cell wall
peptidoglycan synthesis
– Topical application for
skin infections
– Against gram-positives,nonorally taken,

 Vancomycin
– Glycopeptide
– Important “last line”
against methicillin
resistant S. aureus 
Isoniazid (INH)
– Inhibits mycolic acid synthesis in
mycobacteria
– Usually administered with other
drugs to prevent development of
drug resistance


Ethambutol
– Inhibits incorporation of mycolic
acid 
Chloramphenicol
– Binds 50S subunit,
inhibits peptide
bond formation
– Adverse side
effects eg.
suppresses
formation of blood
cells leading to
aplastic anaemia
– Broad spectrum 
Aminoglycosides
– Changes shape of 30S
subunit
– Streptomycin,
neomycin, gentamicin
– Broad spectrum and
effective against
Pseudomonas
infections in cystic
fibrosis 
Tetracyclines
– Binds 30S subunit
– Broad spectrum
– Also effective against
intracellular bacteria
– Absorbed by metal
ions eg. Ca
– Discolours teeth in
children
– Liver damage in
pregnant women 
Streptogramins
– Binds 50S subunit,
inhibits translation
– Cyclic peptides
– Gram-positives
– pristinamycin
 Macrolides
– Gram-positives
 Binds 50S, prevents
translocation Oxazolidinones – New class of FDA approved antibiotics in 2001 (after 25yr)
– Vancomycin resistant bacteria,Linezolid ,Gram-positives, Binds 50S subunit, prevents
formation of 70S ribosome

Antibiotics: Injury to the
Plasma Membrane
 Polymyxin B
– Injures plasma membranes
– Effective against gram negative bacteria such
as Pseudomonas
– Topical
– Combined with bacitracin and neomycin in
over-the-counter preparation.
Antibiotics: Inhibitors
of Nucleic Acid Synthesis
 Rifamycin
– Inhibits mRNA synthesis
– Able to penetrate tissues and reach
therapeutic levels in CSF and
abcesses
– Effective in treating tuberculosis
caused by intracellular Mycobacteria
located in macrophages 
Quinolones and fluoroquinolones
– Inhibits DNA gyrase needed for DNA
replication
– Nalidixic acid effective in treatment of
urinary tract infections
– Ciprofloxacin is broader spectrum used
in treatment of anthrax
Antibiotics
Competitive Inhibitors
 Sulphonamides (sulphur drugs)
– Structural similarity to para-aminobenzoic
acid (PABA) a folic acid precursor
– Inhibits folic acid synthesis
– Sulphamethoxazole with Trimethoprim
– Broad spectrum used for
treating Pneumocystis pneumonia infection in AIDS

Disk-Diffusion Test
 A.k.a Kirby-Bauer
test
 Filter disks soaked
with known
concentrations of
antibiotics
 Larger zone of
inhibition indicates
greater sensitivity
 Bacteria reported as
sensitive,
intermediate or
resistant
E Test:
Advanced Diffusion Method to
Determine the Minimal Inhibitory
Concentration 
Plastic coated
strips contain
gradient of
antibiotic
concentrations
Inhibitory Concentrations
of Antibiotics
 MIC: Minimal inhibitory concentration.
– The lowest antibiotic concentration that
prevents visible bacterial growth
– Does not distinguish between bacteriostatic or
bactericidal
 MBC: Minimal bactericidal concentration.
– MLC minimal lethal concentration
– Determined using broth dilution tests

DNA
Replication
Double-stranded“parental” DNA unwinds H bonds between nucleotides break 
New H bonds form with new daughter strands synthesized by DNA polymerase

Transcription 
DNA is transcribed to make RNA (mRNA, tRNA,
and rRNA). Transcription proceeds in the 5′  3′ direction  mRNA acts as intermediate between DNA and translation

Translation
Genetic Code 
3 nucleotides code for 1 amino acid. Each set of 3 nucleotides is specific for 1 particular amino acid 
Degenerate code due to alternative codons for an amino acid Translation in Bacteria Prokaryotes have no nuclear membranes
Translation can begin
before transcription is
complete Mutation
A change in the genetic material ,Mutations may be neutral, beneficial, or
harmful. Mutagen: Agent that causes mutations 
spontaneous mutations: Occur in the absence of a mutagen. Point Mutation
Single base substitution
or single nucleotide
polymorphism (SNP)
Change in one base Non-synonymous or
missensemutation
Results in amino acid
substitution
– Haemoglobin in sickle cell anaemia
– GAG for Glu replaced with GTG for Val

Point
Mutation 
Nonsense mutation Results in a nonsense codon or
stop codon, May be lethal if protein coded for is essential Frameshift
Mutation  Insertion or deletion of one
or more nucleotide pairs ,Translation is out of frame,equence downstream of mutation(s) is changed
– Huntingdon’s disease
– Huntingtin protein has a polyglutamine region  coded by a gene that
has multiple –CAGrepeats
– No. of repeats varies
Mutagens,Ionizing radiation (X rays
and gamma rays) – formation of ions that
eact with nucleotides and the deoxyribosephosphate backbone  UV radiation causes thymine dimers
– Skin cancers, Light-repair enzymes separates thymine dimers The Frequency of Mutation, Spontaneous mutation rate = 1 in 109
ie. 10–9 replicated base pairs or 1 in 106
ie. 10–6 replicated genes  Mutagens increase rate to between 10–5 and 10–3 per replicated gene
Genetic Recombination 
Exchange of genes between two DNA
molecules,Crossing over occurs when 2 chromosomes break and rejoin  Contributes to genetic diversity ,Beneficial mutations,Salmonella flagellarproteins ,Integration of fragments of DNA into chromosomes, Works best when donor and recipient cells are closely related

regulation of gene xpression:mechanisms to prevent synthesis of bacteria that is not needed.constitutive ezymes constantly produced at a fixed rate.repression;inhibits gene expression and decreases synthesis of enzymes.repressors block ability of RNA polymerase to initiate transcription from repressed gene, default is on.induction turns on transcription of a gene, the genes reqd for lactose metabolism in ecoli,b-galctosidase splits glucose & galactose,defalt is off.operon gene expression;consits of promoter,operator,and regulated by reg gene. inducible(based on ecol lactose cat);repressor active,operonoff.the repressor pr binds with operator, preventing transcription.repressor inactive, operon on.when the inducer allolactase binds to the repressor pro,inactivated repressor cantblock transcripttion.genes transcribed,resulting in production of enzymes needed for lac catabolism.Repressible(trytphanproduction);repressor inactive, and transcription and translation proceed, leading to tryptophan production.represor acrive, operon off,when tryp binds to repressor,activated repressor binds with operator, preventing transcription.

 Competent recipient cell is in a physiological state that can take up the donor DNA, Competence results from cell wall alterations that make it permeable to large DNA molecules. Genetic Transfer ,Vertical gene transfer: Occurs during reproduction between generations of
cells. Horizontal gene transfer: The transfer of genes between cells of the same generation.Plasmids
 Dissimilation plasmids: Encode enzymes for catabolism of unusual compounds eg. hydrocarbons in Pseudomonas  Pathogenicity islands: Toxins in E. coli
 Resistance factors: Encode antibiotic resistance
Plasmids: Resistance Factors
 very serious in
antibiotic treatment
of infectious diseases
 resistance genes
– mercury
– sulphonamides
– streptomycin
– chloramphenicol
– tetracycline
 can be transferred between a number of enteric species, including Escherichia, Klebsiella, Salmonella Plasmids: Resistance Factors
Bacteroides fragilis plasmids that encode clindamycin resistance Transposons Small segments of DNA that can move from one region of DNA to another , Contain insertion sequences for cutting and resealing DNA(transposase),

Complex transposons carry other genes, Kanamycin resistance.Transposons
Insertion of a transposonin a plasmid or chromosome (targetDNA) Transposase cuts DNA, leaving sticky
ends Vancomycin resistance transfer from Enterococcus faecalis to
Staphylococcus aureus via transposon Antibiotic Resistance Misuse of antibiotics that selects for resistance mutants includes:
– Using outdated or weakened antibiotics
– Using antibiotics for the common cold and other
inappropriate conditions
– Use of antibiotics in animal feed
– Failure to complete the prescribed regimen
– Using someone else’s leftover prescription
Mechanisms of Antibiotic
Resistance
1. Enzymatic destruction of drug
• Beta-lactamases are bacterial penicillinases that cleave
beta-lactam ring of the penicillin molecule
• MRSA (methicillin-resistant Staphylococcus aureus)
Methicillin 1
st semi-synthetic penicillin
• VRE (vancomycin resistant Enterococcus) Ong ENH 222 Lecture10 13
Mechanisms of Antibiotic
Resistance
2. Prevention of penetration of drug
• Tetracycline resistance
3. Alteration of drug’s target site
• Macrolide resistance

4. Rapid ejection of the drug
• Rapid efflux proton pumps makes malaria
parasite chloroquine resistant

Effects of Combinations of
Drugs
 Synergism occurs when the effect of two
drugs together is greater than the effect
of either alone
– Amoxicillin a broader spectrum semi-synthetic
penicillin
– Clavulanic acid a beta-lactamase inhibitor
 Antagonism occurs when the effect of two
drugs together is less than the effect of
either alone
– Penicillin and tetracycline

action of antimicrobial drugs.bactericidal(kill),bacteristatic(prevent microbe growth).

1.inhibition of cell wall synthesis;penecillions,cephalosporins,bacitranin,vancomycin.

2. inhibition of prosynthesis;cloamphenicol,erythromycin,tetracyclins,streptomycin.

3.injury to plasma membrane;polymyxin B

4.inhibtion of synthesis of essential metabolites; sulfanilamide, trimethoprim

microbial characteristicsresistance; most;prions,endospores,mycobacteria,cysts,vegetative protozoa,gram(-),fungi,viruses,gram+,viruses with lipid envelopes(least)

bacteria:simple,single-celled(unicellular),not enclosed in nucleur membrane(prokary),bacillus(rodlike),spira,starshaped,may form cluster,oillar,chains.enclosed in cell walls largely composed of CHO/PRO complex called peptodoglycan.reproduce by dividing into 2 equal cells;binary fission, some manufacturefood via phtosynthesis and some get from organic substance. asexual reproduction, clones.archae;consist of prokary cells,cell wall lacks peptidoglycan, found in extreme environments;methanogens;produce methane,halophiles;salty environment,thermophile;live in hot. eukarya;organisms whose cells have a distint nucleus containing DNA material surronded by envelope,nucleur membrane. fungi;uni or multicellular, chitin,yeast,mold,form mycelia composed of hyphae that intertwince.protozoa; unicellular,move by flagella,amoeba,variety of shapes.algae;phtosynthetic,sexual/asexual,unicellular,cellwall composed of CHO cellulose, blugreen make txins, fresh/saltwater,soil and plants. need light,water,co2,for food and growth. produce o2&co2.viruses;small,acellular,contain core of DNA/RNA surronded by procoat.parasites;multicellular.