Microbial Ecology and Controlling Microbial Growth

Module 5

Microbial Physiology

• Physiology: the study of the vital life processes of organisms

• Nutritional Requirements– Nutrients: chemical compounds used to

sustain life– Essential nutrients: compounds that an

organism must obtain from its environment

Energy Source

• Phototrophs: use light as energy source

• Chemotrophs: use either organic or inorganic chemicals as energy sources

Carbon Source

• Autotrophs: use CO2 as their sole source of carbon

• Heterotrophs: use organic compounds other than CO2 for their carbon source

**All medically important bacteria are chemoheterotrophs**

Metabolism and Enzymes

Metabolism: All the chemical reactions that occur in a cell

1. Enzymes are proteins that catalyze reactions.

2. Enzymes are specific for particular substrates

3. Enzymes are not altered during a chemical reaction.

Enzyme EfficiencyEnzymes need optimal conditions to work

properly.

1. Limited pH range

2. Limited temperature range

3. Optimal concentration of enzyme and substrate

Metabolism

• Metabolism= catabolism + anabolism

• Define:– Metabolite– Catabolism/ catabolic reaction– Anabolism/ anabolic reaction– ATP

Bacterial Genetics

• Genetics: the study of heredity

Define:GenotypePhenotype

**Genes direct ALL functions of the cell, providing it with its own particular traits and individuality.**

Mutations

• A change in the characteristics of a cells caused by a change in the DNA that is transmissible to the offspring

1. Beneficial mutations

2. Harmful and lethal mutations

3. Silent mutations

Mutagens

• Physical or chemical agents that affect the chromosome– X-rays, UV, radioactive substances

• Mutants: organisms that contain the mutation• Ames Test: a mutant strain of Salmonella is

used to learn if a chemical is a mutagen– Reversal of the organisms mutation demonstrates

that the chemical is mutagenic (and may therefore be carcinogenic)

Antibiotic Resistance

• R-factor: plasmid containing multiple genes for antibiotic resistance

• Recipient of a R-factor plasmid becomes a “superbug”

• Transduction, transformation, and conjugation are all used as tools by scientists to manipulate bacterial genomes

Factors that Affect Microbial Growth

• Availability of nutrient• Moisture vs. desiccation• Temperature• pH• Osmotic pressure• Tonicity (salinity)• Barometric pressure• Gaseous atmosphere

Nutrients and Moisture

• Nutrients provide energy

• Sources of C, O, H, N, P, S

• 25% of the naturally occurring elements are essential

• Cells are 70-95% water

• Desiccation– Produce spores/cysts

• Re-hydrate to “bring back to life”

Temperature

• Mesophiles (moderate)

• Thermophiles (high)

• Psychrophiles (low)

• What are the ranges for these bacteria?

• What is optimum temperature?

pH

• Acid & Alkaline• Neutral to slightly alkaline

(pH 7.0-7.4)• Acidophiles (pH 2-5)• Alkaphiles (pH >8.5)

• V. cholerae only human pathogen that grows well above pH 8

Osmotic Pressure and Salinity

• Osmotic pressure– Solutes and solvents

• Osmosis• Tonicity (hypertonic, isotonic, hypotonic)• Crenation (cell has crenated)• Plasmolysis• Hemolysis• Halophilic organisms

Barometic Pressure and Gaseous Atmosphere

• Barophiles

• Where do they live?

• Aerobic v. anaerobic

• What are the different conditions/names for bacteria that require different atmospheric environments?

Encouraging microbial growth in vitro

• Generation time (10 minutes – 24 hours)

• Culture media– Artificial media (synthetic media)– Chemically defined medium– Complex medium (brain-heart infusion)– Enriched media

Media

• Selective– MacConkey (inhibits

gm+)– PEA and CNA (inhibit

gm-)– Blood and Hb agars– Mannitol salt agar

(MSA), salt-tolerant

• Differential– MacConkey

(fermentation of lactose)

– MSA (fermentation of mannitol)

– Blood agar (hemolysis)

We can combine groups:Enriched and differential; selective and differential

Bacterial Growth in vitro

• Inoculation of culture media

• Sterile technique (contaminants)

• Incubation

• Pure culture

Bacterial Population Counts

• To determine degree of contamination– Turbidity (spectrophotometer)– Viable plate count (use of dilutions)

• Used in urine culture• 1ml= 220 colonies• 1:10,000 was used• 220 x 10,000= 2,200,00 bacterial/ml

Population growth curve

• Pure culture grown as constant temperature, samples collected at fixed intervals

• Four phases– A. Lag phase– B. Log phase– C. Stationary phase– D. Death phase

Obligate IC Pathogens in the lab

• Microbes that only grow in host cells

• Viruses

• Two groups of gm- bacteria– Rickettsia and chlamydia

• Inoculate into embryonated chicken eggs, lab animals or cell culture

Inhibiting Growth in vitro

• Sterilization: complete destruction of all microbes (cells, spores, viruses)

• Disinfection: destruction or removal or pathogens– Disinfectants: chemical substances, can not be used

on living tissue– Antiseptics: chemicals used to disinfect skin

• Sanitization: reduction of microbial populations to levels considered safe

Microbicidal Agents: Kill

• Germicides, biocides, microbicides: kill microbes

• Bactericides: specifically kill bacterial (but not endospores)

• Sporicides: kill endospores

• Fungicides and algicides

• viricides

Microbistatic Agents: inhibit

• Microbistatic: inhibits growth and reproduction

• Bacteriostatic agent: specifically inhibits metabolism and reproduction of bacteria

• Lyophilization: a process that freeze-dries organisms (does NOT kill, just prevents reproduction; storage for future use)

Sepsis and Asepsis

• Sepsis: presence of pathogens in blood or tissue

• What is sterile technique?

• Who was Joseph Lister?

• Asepsis: absence of pathogens in blood or tissue

• Antisepsis: prevention of infection

Physical Methods to Inhibit Growth

• Heat (time and temperature)– Thermal death point (TDP)– Thermal death time (TDT)

– How might pus, feces, vomitus, mucus, blood be protective to microbes during heating procedures?

Dry v. Moist Heat

• Dry• Metal, powder, oils,

waxes• 160-165 for 2 hours• 170-180 for 1 hour

• INCINERATION

• Moist• Faster and more

effective• Causes protein

coagulation• Boil 30 minutes

• AUTOCLAVE

Physical Methods

• Cold

• Desiccation

• Radiation

• Ultrasonic waves

• Filtration

• Gaseous atmophere

Chemical Methods

• Disinfectants– Prior cleaning– Organic load– Bioburden (type and level of contamination)– Concentration of disinfectant– Contact time for disinfectant– Physical nature of object– Temperature and pH

Ideal chemical antimicrobial agent

• Wide/broad spectrum

• Fast-acting

• Not affect by presence of organic matter

• Nontoxic to human tissue

• Should leave a residual film on surface

• Soluble in water and inexpensive

• Stable and odorless

How do disinfectants work?

• Destroy cell membranes

• Destroy enzymes or structural proteins

• Attack cell walls or nucleic acids

Controversy: Antimicrobial Agents

• Drug-resistance

• Animal feed

• Hand soaps, household sprays

• Immune system challenges

Microbial Ecology & Relationships

• Ecology – interaction of organisms with environment (living and nonliving)

• Symbiosis – interactions of 2 organisms living close together

• Mutualism – both organisms benefit• Parasitism – one benefits and the other is

harmed (pathogens and humans)– Synergy – 2 or more organism working together

• Examples:– Four (4) bacteria cause trench mouth– Vaginosis is caused by several working together

Indigenous Bacteria 500-100 species

10 trillion cells, 10x10 trillion microbes

• Fetus – initially sterile – exposure occurs during and after birth through openings and onto skin

• Blood, lymph, most internal tissue – sterile• Resident – normals –usually not pathogenic if they

stay in the correct system• Transients – may be washed away, killed by toxins

of other microbes, flushed by body excretions, or the site may not be suitable for growth – (E. coli toxin that kills any Salmonella or Shigella

passing thru gut

Indigenous Bacteria

• Skin– About 30 types– Most anaerobes – found in deep layers –

around hair follicles and oil glands– Examples

• Staph epidermis• Staph aureus• P. acnes – found in high numbers in acne

Indigenous Bacteria

• Ears and eyes– Outer ear and auditory canal – skin flora– Inner ear and middle – sterile

• Coughing and sneezing push microbes up Eustachian tubes to middle ear and cause infection

– Eyes – tears, mucus, and sebum clean eyes• Lysozyme – enzyme in tears that kills bacteria

Indigenous Bacteria

• Upper Respiratory Tract– Nose and pharynx

• Many microbes, most harmless, some opportunistic• Many residents can become pathogenic in a weakened

person- but only strep is treated in the carrier state• Neisseria meningitidis – normal that transiently colonizes the

area, and becomes invasive in a very small percentage of people – host risk factors include

– new colonization and lack of antibody protection– Concurrent colonization w a viral upper respiratory tract

infection that allows mucosal invasion– Certain congenital complement deficiency syndromes

Indigenous Bacteria

• Mouth– Aerobes and anaerobes– Anaerobes – gum margins, between teeth,

folds of tonsils– Bacteria on food in unclean mouth cause

tooth decay and gum disease– Alpha hemolytic strept most common– Lactobacillus produces acid that causes

cavities (dental caries)

Indigenous Bacteria

• Stomach– Usually stomach kills microbes passing thru it

because of low pH, but Helicobacter pylori live there. Cause stomach ulcers

• Duodenum – few bacteria – area of high pH

• Jejunum and ileum – more bacteria

Indigenous Bacteria

• Large Intestine– Most anaerobes– But most known about facultatives because are so

easy to grow and study in the lab (Family Enterobacteriaceae which includes E. coli)

– Enterics is the common word for intestinal bacteria– Coliforms refers to a special group of enterics– Water is checked for fecal coliforms (aerobic or

facultative anaerobic, gm-,lactose fermenting, non-endospore forming rods) when checking for contamination – easy test kits

– 50% fecal mass is bacteria

Indigenous Bacteria

• Microbes of GU tract– Kidneys, ureters, bladder – sterile– Urethra – cleaned by urination– Chlamydia, Neisseria, mycoplasmas introduced by

sex

• Vagina – before puberty and after menopause – more alkaline

• Childbearing years – acid – Lactobacillus is most prevalent

Microbial communities: Biofilms

• Complex assortment of organisms

• Secrete polysaccharides– Water channels to bring in food and get rid of

waste; organisms working together

• Examples: Dental plaque, some of the slippery coatings on rocks, can be found in many implants such as catheters

• Resistant to antibiotics and disinfectants