Microbiology: Disease and Therapy

• Module IV covers basic concepts of disease and therapy.
• The module explores microbiology.
• The notes are from August 2021.

Outline

• Overview and history of microbiology.
• Normal flora.
• Sterilization, disinfection, and antiseptics.

Learning Objectives:

• Microbiology and microorganisms must be defined.
• The effects of microorganisms must be understood.
• A description is needed of the history of microbiology and the germ theory of diseases.
• Differences between the benefits and harms of normal flora must be differentiated.
• Sterilization and disinfection must be explained.

Overview of Microbiology

• Focuses on microscopic organisms, too small for the naked eye.
• Studies a large, diverse group of microscopic organisms in single cells or clusters.
• Objects less than 1 mm need a microscope to be examined.
• Microbiology originates from Greek words: "micros" (small), "bios" (life), "logos" (science).

Microorganisms

• Microorganisms are too small to be seen with the naked eye, very diverse and found almost everywhere.

Medical Microbiology

• Studies pathogenic microbes and their role in human illness.
• Includes:
• Bacteriology: prokaryotes, single-celled
• Virology: Non-cellular, parasitic infectious agents.
• Mycology: Fungi, microscopic (molds & yeasts), macroscopic (mushrooms & puff balls).
• Protozoology: single-celled

Beneficial aspects of microorganisms

• Recycle vital elements (carbon, nitrogen, sulfur, hydrogen, oxygen).
• Nitrogen fixation.
• Photosynthesis.
• Recycle water in sewage treatment.
• Clean up toxic wastes through bioremediation.
• Manufacture food and drink.

Beneficial & harmful aspects of microorganisms

• Food spoilage
• Basis for bioterrorism
• Cause disease
  • Pathogen: micro-organism with the potential to cause disease.
  • Infection: invasion & multiplication of pathogens in an individual or population.
  • Disease: infection causes damage individual's vital functions or systems.
  • An infection does not always result in disease.

History of Microbiology

• Babylonians used yeast to make beer.
• Acetic acid bacteria made vinegar over 6000 years ago.
• About 2000 years ago, Romans thought diseases were caused by tiny animals.
• Real microbiology history starts from 1600s, when lenses and microscopes were invented.

Antony van Leeuwenhoek (1632-1723)

• Founding father of microbiology, microscopy, and zoology.
• Had no scientific training, but boundless curiosity.
• Used self-made lenses and microscopes to do observations.
• In 1677, he observed microorganisms in canal water, calling them animalcules, now known as protists.

Spontaneous generation vs biogenesis

• Spontaneous generation
• Living things arise from nonliving things.
• Held by Aristotle and philosophers from 300 BC to the 1700s.
• Decaying meat leads to maggots and flies.
• Grain leads to frogs.
• Biogenesis
• Living cells arise only from preexisting living matter.
• Animalcules arise from “germs” or “seeds” present in air.

Francesco Redi (1626-1697)

• Did experiments about spontaneous generation and biogenesis
• Placed decaying meat in jars to show maggots come from flies
• Conclusion: Maggots were offspring of flies and not spontaneous generation

John Needham (1731-1781)

• This experiment claimed a "life force" existed in inorganic matter which caused spontaneous generation
• Boiled soup briefly and placed in clean flasks with cork lids
• Led to microbial growth and a conclusion that they arose spontaneously from a life force

Lazzaro Spallanzani (1729-1799)

• Theorized Needham's organisms came from heat-resistant microbes
• Flasks boiled for 1-2 hours presented no growth
• Microbes were introduced to broth from the air

Louis Pasteur (1822-1895)

• Believed life originated from previous life
• Microorganisms and other life are present in the air and debunked arguments refuting this theory.

Germ Theory of Disease

• Certain diseases are caused by the invasion of the body by germs (microorganisms).
• 1876 - Robert Koch established "scientific principles" to show a cause and effect relationship between a microbe and a disease.
• These principles are known as Koch's postulates.

Koch's postulates

• The same microbe is always associated with a specific disease.
• The microbe must be isolated from the host with the disease and grown in pure culture.
• The specific disease must be reproduced when a pure culture of the bacteria is inoculated into a healthy susceptible host.
• The original microbe must be recovered from the experimentally infected host.

What Koch Established

• Koch established the etiology of these diseases:
  • 1. Cholera (fecal-oral disease) caused by Vibrio cholerae.
  • 2. Tuberculosis (pulmonary infection) caused by Mycobacterium tuberculosis.
  • 3. Anthrax (sheep and cattle) caused by Bacillus anthracis.

Exceptions to Koch's Postulates

• Exceptions to postulates are:
  • Growth on artificial media is impossible in some cases
    • Treponema pallidum (Syphilis)
    • Mycobacterium leprae (Leprosy)
    • All viruses cannot grow on artificial media
  • Ethical objections
    • No animal models for some disease

The Normal Flora (Microbiota)

• A mixture of microorganisms found at anatomical sites on within the body of a healthy person.
• Some microorganisms are found in association with humans or animals only.
• Others are found in the environment as well.
• The makeup of normal flora may be influenced by genetics, age, sex, stress, nutrition, and diet.

Normal Flora Types

• Two Types: 1) Resident and 2) Transient
• Resident flora
• If disturbed, it promptly reestablishes itself.
• Otherwise, other microorganisms may colonize, proliferate, and produce disease.
• More important.
• Transient flora
• Non-pathogenic or potentially pathogenic microorganisms inhabit skin or mucous membranes for hours, days, or weeks.
• Derived from the environment and does not produce disease.
• Does not establish itself permanently on the surface.
• Little significance.

Role of the Resident Flora

• Synthesizes and excretes vitamins: Enteric bacteria secrete Vitamin K and B12; lactic acid bacteria produce certain B-vitamins.
• Prevents colonization by pathogens: Competes for attachment sites or for essential nutrients, example, oral cavity, intestine, skin, and vaginal epithelium.
• May antagonize other bacteria: Intestinal bacteria produce nonspecific fatty acids and peroxides to highly specific bacteriocins, which inhibit or kill other bacteria.
• Stimulates the production of cross-reactive antibodies: Antibodies produced against components of the NF cross-react with certain related pathogens, preventing infection or invasion. Antibodies to NF are referred to as "natural" antibodies.

Harmful Effects of Normal Flora

• Potential pathogens (Parasites): Members of the normal flora may themselves produce disease under certain circumstances.
• Act as pathogens in tissue outside normal habitat: example, Normal flora of the intestine causes a UTI.
• Streptococcus viridans: Common in URT.
• When introduced to circulation following tooth extraction or tonsillectomy, may settle on deformed or prosthetic heart valve, producing infective endocarditis.
• Bacteroids: Residents of large intestine.
• If introduced to peritoneal cavity or pelvic tissue, they may cause bacteremia.
• Taking broad-spectrum antibiotics affects the balance of the normal microbial population, leading to:
  • Vaginitis in women caused by yeast.
  • Clostridium difficile: diarrhea or pseudomembranous colitis.
• They become pathogenic when the host resistance is lowered.
• Penicillinase producing organisms can aggravate infection.
• May cause confusion in diagnosis.

Relationship between normal flora and host

• Commensalism: One organism benefits, and the other is unaffected.
• Mutualism: Both organisms benefit.
• Parasitism: One organism benefits at the expense of the other.

Normal Flora of the Skin

• Staphylococcus epidermidis is the major inhabitant, making up more than 90% of the skin flora.
• Staphylococcus aureus inhabits the nose, perineum, and vulvar skin.
• Occurrence in nasal passages varies with age being greatest in newborns and less in adults.
• Additional bacteria on the skin include Micrococci, Diphtheroids, and Propionibacterium.
• Factors important in eliminating non-resident microorganisms from the skin are low pH, fatty acids, and lysozyme.
• Profuse sweating and washing/bathing can't eliminate normal resident flora or significantly change them.
• Benefits:
• Production of defensive antimicrobial peptides.
• Inhibit fungal growth.
• Wound healing.
• Inflammation control.
• Immune system development.
• Harms:
• Body odor.
• Acne.
• Opportunistic infections.

Normal flora oral cavity

• Birth: sterile mouth within 4-12 hours (lactobacilli, streptococci).
• Neonate (Streptococcus salivarius, staphylococci, Neisseriae, Moraxella catarrhalis.
• Teeth appear (Streptococcus mutans, Streptococcus parasanguis).
• Gingival crevice area (Anaerobic species, yeasts).
• Puberty (Bacteroides, spirochetes).
• 108 bacteria/mL of saliva; potentially >700 species Benefits
• Compete with pathogens for colonization sites
• Produce substances that inhibit pathogens
• Stimulate local immunity Harm
• Plaque formation and dental disease

Normal flora - respiratory tract

• Nasal passages and sinuses:
  • Firmicutes, Actinobacteria, and Proteobacteria (Staphylococcus epidermidis, viridans streptococci (VGS), Corynebacterium spp. (diphtheroids), Propionibacterium spp., and Haemophilus spp.
• The oropharynx:
  • Prevotella, Fusobacterium, Moraxella, and Eikenella, and some Candida fungal isolates.
• The pharynx:
  • Streptococcus, Haemophilus, and Neisseria.
• Lower Respiratory Tract
  • Usually sterile.
  • Ciliated epithelium.
  • Mucus blanket: entrapment.
  • Alveolar macrophages.
• If breached: opportunistic infections
• Benefits:
  • Produces bacteriocins against pathogenic microorganisms
  • Keeps the host system primed for invasion by pathogens.

Normal flora- Gastrointestinal tract

• benefits:
• Outcompete potential pathogens for space and nutrients through competitive exclusion
• Secrete bacteriocins which attack susceptible bacteria
• Aid in immune system development
• Ferment non-digestible residues
• Facilitate metabolite transformation and absorption
• Synthesize and excrete vitamins like K and B12 through enteric bacteria including lactic acid bacteria
• harms:
• have a relationship with inflammatory conditions
• aid in the transfer of antibiotic resistance genes
• can produce carcinogens by modifying chemicals in diets through their enzymes.
• The colon has the most bacteria with >1011 bacteria mL−1
• species include Alistipes, Anaerostipes, Bacteroides, Bifidobacterium, Clostridium cluster, Dorea, Eubacterium, Faecalibacterium, Parabacteroides, Roseburia, and Ruminococcus.
• The stomach has a lower count between 102 and 104 bacteria mL−1.
• species include Lactobacillus, Propionibacterium, Streptococcus, and Staphylococcus.
• The small intestine hosts 107 to 108 bacteria mL−1.
• Species include Escherichia, Bacteroides, Clostridium cluster XIVa, Lactobacillus, Streptococcus, and Veillonella species.

Normal flora. Genitourinary tract

• Upper urinary tract (kidneys, ureters, bladder) usually sterile
• Male anterior urethra same as skin - enteric and enterococcus
• Vagina: complex microbiota
• At birth: same as mother (PH 5)
• Neonate: same as skin+enteric+ strept (PH 7)
• At puberty: lactobacillus+same as skin+anaerobes+strep (PH 5)
• At menopause: return to prepuberty flora

Genitourinary Tract

• Benefits
• Competition with pathogens for colonization sites
• Production of lactic acid that inhibits pathogenic bacteria and yeasts

Sterilization, Disinfection, and Antiseptics: Definitions

• Sterilization: Destroys or removes all microbial life, including spores.
• Disinfection: Renders a substance or object non-infectious freeing an article from harmful microorganisms.
• Disinfectants: Kill microorganisms on inanimate objects or surfaces.
• Septic: Characterized by the presence of pathogenic microbes in living tissue.
• Antiseptic: Destroys or inhibits the growth of microorganisms on living tissue.
• Aseptic: Characterized by the absence of pathogenic microbes.
• Preservation: Prevents the multiplication of microorganisms in formulated products including pharmaceuticals and food.
• Germicide: Kills the vegetative forms of microorganisms, not necessarily spores.
• Sanitizer: Agent that kills 99.9% of microorganisms contaminating an area. Commonly used on inanimate objects.

Sterilization

• The complete destruction or removal of all microorganisms including spores.
• The recommended method for processing instruments and other items that will come in contact with the bloodstream or tissues under the skin.
• The efficiency of a sterilization process is often related to the degree of severity of treatment such as the duration of exposure to heat.

Factors Affecting Sterilization

• Type of microorganism present: some microbes are difficult to kill, others die easily.
• Number of microorganisms present: it is easier to kill one organism than many.
• Amount and type of organic material protecting the microorganisms: blood or tissue remaining on poorly cleaned instruments acts as a shield during sterilization.

Methods of Sterilization

• Two methods of sterilization: physical and chemical sterilization.

Physical Methods of Sterilization

• Heat
• Moist Heat
• Autoclaving (Steam under pressure)
• Boiling
• Tyndallization (Intermittent boiling)
• Sub-boiling Temperatures (Pasteurization)
• Dry Heat
• Radiation
• Filtration

Physical Methods of Sterilization

• A) Physical methods of sterilization
• Heat sterilization
• Most effective, widely utilized to kill microorganisms
• Heat sterilization
• Two kinds: Dry or wet/moist heat
• Dry heat is cheaper less and reliable relative to wet heat sterilization

Moist Heat

• Moist heat is more effective than dry heat.
• Moist heat causes denaturation of cell proteins.

Types of Moist Heat

• Autoclaving (Steam under pressure)
• Boiling
• intermittent boiling (tyndallization)
• pasturization

A) Moist Heat Treatments

• Autoclaving (Steam under pressure)
  • Pure steam sterilization is dependable
• An autoclave consists of a double walled chamber
• Chamber supplies steam under pressure to close the inter chamber in which goods are sterilized
• Done at 121 degree C at 15 pounds per square inch for 15 minutes
• Its a useful sterilization method for almost anything accept heat labile substances (i.e. plastic)
• Advantages:
  • Its one of the most effective sterilization practices
• Disadvantages:
  • Requires a sterilizer and continuous heat source for precise temp/time and pressure adhesion, sterilization can dull instruments, plastic wares cannot withstand high temps
• Autoclaving Requirements:
• (1) contact, (2) high temp, (3) correct time (4) sufficient time

B) Boiling

• Boiling at 100 degrees C will kill all endospores in 30 minutes
• Purifying drinking water for 5 minutes is adequate enough
• Giardia cysts are able to survive if too short of duration
• to kill endospores and sterilize the solution, it requires intermittent boiling

C) Tyndallization (intermittent boiling)

• Heat treatment occurs at 100 degrees C for 30 minutes for 3 days sequentially
• It sterilizes the autoclave and prevents it from being damaged or not sterilized to its full extent
• In 1st day- kills vegetative cells but NOT spores
• Spores germinate to vegetative cells with the help of heat induced endospores
• 2nd day- spores are induced by 2nd boiling and are killed
• 3rd day- traditional precaution

D)Sub boiling temperatures (pasturization)

• sterilizing causes adverse effects and may reduce microbial contamination ( in certain elements)
• A good example is pasturization, which is the use of mild heat to reduce the amount of microorganisms in a specific food or drink .
• There are two methods of pasturization
  • (1) Batch method (which has a low temperature - long time approach)
    • involves holding material at 62.8 degrees for 30 minutes
  • (2) Flash method ( has a high temperature - short time frame) -heat treatment at 71.7 C for 15 sections - It involves allowing flow to a material to exchange the heat, quickly cool it, and heat it again. - Is used in milk products

II. Dry Heat

• Microorganisms are killed with dry heat, or hot air at sufficiently high temperatures.
• Not as effective as moist heat: requires greater temperatures and longer exposures.
• Causes oxidation of the organic constituents of the cell by burning slowly.
• Types of Dry Heat Treatment
• A) Red hot
• B) Flaming
• C) Incineration
• D) Hot air ovens

A) Red hot

• It is done to loops and straight wires in microbiology labs
• Loops are put in a Bunsen burner to oxidize any potentially infectious component.
• Used in glass and metal objects.

B) Flaming

• Passing material through flame
• For example, flasks, loops, and spreaders

C) Incineration

• burn any organism and converted it in to ash to sanitize medical and other biohazardous waste

D) Hot Air Ovens

• require higher temperature and longer period of heating.
• Used to sterilize glass wares, metal instruments, grease, oils and lubricants (objects that will not melt during heating)
• Exposure time varies with exposure temperatures
  • 170°C for 1 hour
  • 160°C for 2 hours can be used to sterilize glasses and metals

Advantages for Hot Air Ovens

• Effective method, as dry heat reaches all surfaces of instruments
• Protective of sharps or instruments with a cutting edge (fewer problems with dulling of cutting edges)
• Do not leave chemical residue

Disadvantages for Hot Air Oven

• Plastic, rubber items cannot be sterilized, and require a source of electricity.
• Has temperature parameters that may have limitations for different equipment

RADIATION Overview

• energy content of radiation is inversely related to its wavelength; the shorter the wavelength has a greater the energy content.
• High-energy radiation includes gamma rays and UV light.
• These radiations kill microorganisms.
• There are two forms of radiation: ionizing radiation and non-ionizing radiation.
• Ionizing radiation: a radiation that ionizes molecules
• Non-ionizing radiation: radiation that does not ionize molecules

lonizing Radiation

• High - energy electron beams, y - rays and x - rays have sufficient energy to cause ionization of molecules.
  • E.g. H2O splits into OH- + H+ + electron (Hydroxyl radicals and Hydrogen ions respectively).
  • Hydroxyl radicals are highly reactive and destructive to normal cellular compounds like DNA and proteins.
• Advantage: able to penetrate packaging/products and sterilize

Non lonizing

• Radiations include UV Light
• light-waves are longer than x and gamma rays
• causes the molecule to react differently from non irradiated molecules, excitation of electrons causes to interact and absorb intracellular components
• damage occurs at 260 mm wavelengths
• creates exposure causing adjacent pyrimidines of a DNA stand to bonded together
• inhibits and alters and causes mutations or death
• Used at operating and sterile environments
• Because UV light has very limited source of penetration objects that lack depth at the surface are often killed.

3) Filtration

• Used to sterilize remove microorganisms from liquids that are prone to heat
• Examples, antibiotics, serum, amino acids, injectable drugs, and vitamins
  • Filters are made of ceramic, asbestos, cellulose ester

###B. Chemical Sterilization

• Antiseptic
• Should only be applicable for skin/mucous
• indicated for surgical scrubs and skin+ cervical vaginal preps
• ex. Mercurials, silver nitrate, iodine solution, Alcohols, detergents

Chemical sterilization

• Disinfectants - Is not to be used on skin or mucous members. Should be for cleaning objects and surfaces+ instruments such as chlorine and glutaraldehyde - Disinfecting supplies shouldn't be used for long duration

###Levels of Disinfection

• 1. High-level disinfection (HLD): Used for items involved with invasive procedures that cannot withstand sterilization procedures.
• Certain types of endoscopes and surgical instruments are made with plastic or other components
• Best with surface cleaning to remove organic matter precedes treatment.
• Liquids such as glutaraldehyde, hydrogen peroxide, peracetic acid, and chlorine compounds
• Eliminates bacteria, viruses, fungi and parasites but has limitations killing bacterial endospores

Intermediate Level Disinfection (ILD)

• Used for surfaces or instruments with unlikely contamination of bacterial spores and highly resistant organisms
• ex. alcohols, iodophors, Phenolic compounds
  • includes flexible fiber optic endoscopes, laryngoscopes, vaginal specula, and anesthesia breathing instruments
• does not use vegetative and bacteria with only spore killing ability