Microbial Ecology and Controlling Microbial Growth

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Microbial Ecology and Controlling Microbial Growth

• Module 5

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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

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Energy Source

• Phototrophs use light as energy source
• Chemotrophs use either organic or inorganic
  chemicals as energy sources

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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

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Metabolism and Enzymes

• Metabolism All the chemical reactions that occur
  in a cell
• Enzymes are proteins that catalyze reactions.
• Enzymes are specific for particular substrates
• Enzymes are not altered during a chemical
  reaction.

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Enzyme Efficiency

• Enzymes need optimal conditions to work properly.
• Limited pH range
• Limited temperature range
• Optimal concentration of enzyme and substrate

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Metabolism

• Metabolism catabolism anabolism
• Define
• Metabolite
• Catabolism/ catabolic reaction
• Anabolism/ anabolic reaction
• ATP

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Bacterial Genetics

• Genetics the study of heredity
• Define
• Genotype
• Phenotype
• Genes direct ALL functions of the cell,
  providing it with its own particular traits and
  individuality.

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Mutations

• A change in the characteristics of a cells caused
  by a change in the DNA that is transmissible to
  the offspring
• Beneficial mutations
• Harmful and lethal mutations
• Silent mutations

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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)

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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

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Factors that Affect Microbial Growth

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

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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

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Temperature

• Mesophiles (moderate)
• Thermophiles (high)
• Psychrophiles (low)
• What are the ranges for these bacteria?
• What is optimum temperature?

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pH

• Acid Alkaline
• Neutral to slightly alkaline (pH 7.0-7.4)
• Acidophiles (pH 2-5)
• Alkaphiles (pH gt8.5)
• V. cholerae only human pathogen that grows well
  above pH 8

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Osmotic Pressure and Salinity

• Osmotic pressure
• Solutes and solvents
• Osmosis
• Tonicity (hypertonic, isotonic, hypotonic)
• Crenation (cell has crenated)
• Plasmolysis
• Hemolysis
• Halophilic organisms

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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?

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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

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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
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Bacterial Growth in vitro

• Inoculation of culture media
• Sterile technique (contaminants)
• Incubation
• Pure culture

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Bacterial Population Counts

• To determine degree of contamination
• Turbidity (spectrophotometer)
• Viable plate count (use of dilutions)
• Used in urine culture
• 1ml 220 colonies
• 110,000 was used
• 220 x 10,000 2,200,00 bacterial/ml

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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

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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

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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

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Microbicidal Agents Kill

• Germicides, biocides, microbicides kill microbes
• Bactericides specifically kill bacterial (but
  not endospores)
• Sporicides kill endospores
• Fungicides and algicides
• viricides

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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)

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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

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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?

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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

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Physical Methods

• Cold
• Desiccation
• Radiation
• Ultrasonic waves
• Filtration
• Gaseous atmophere

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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

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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

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How do disinfectants work?

• Destroy cell membranes
• Destroy enzymes or structural proteins
• Attack cell walls or nucleic acids

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Controversy Antimicrobial Agents

• Drug-resistance
• Animal feed
• Hand soaps, household sprays
• Immune system challenges

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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

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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

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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

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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

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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

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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)

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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

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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

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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

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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