Microbiology: Disease and Therapy

Module IV: Basic Concepts of Disease and Therapy

Microbiology

• Microbiology studies microscopic organisms that are too small to see with the naked eye
• These organisms exist as single cells or in clusters
• Objects smaller than about 1 mm require a microscope for examination
• The word "microbiology" comes from Greek words: "micros" (small), "bios" (life), and "logos" (science), meaning the study of microscopic life

Overview of Microbiology

• Microorganisms are very diverse and can be found almost everywhere
• Medical microbiology focuses on pathogenic microbes and their role in human illness
• Bacteriology studies prokaryotes, specifically single-celled organisms
• Virology studies non-cellular, parasitic infectious agents
• Mycology studies fungi, including microscopic molds and yeasts, and macroscopic mushrooms and puff balls
• Protozoology studies single-celled organisms

Beneficial Aspects of Microorganisms

• Recycling vital elements like carbon, nitrogen, sulfur, hydrogen, and oxygen
• Nitrogen fixation
• Photosynthesis
• Recycling water via sewage treatment
• Cleaning up toxic wastes through bioremediation
• Manufacturing food and drink

Harmful Aspects of Microorganisms

• Food spoilage
• Use as a basis for bioterrorism
• Causing diseases
• A pathogen is a micro-organism that can cause disease
• Infection is the invasion and multiplication of pathogens
• Disease occurs when an infection damages vital functions / systems
• Infection will not always result in a disease

History of Microbiology

• Babylonians used yeast to make beer
• Acetic acid bacteria used make vinegar over 6000 years ago
• Romans believed tiny animals caused diseases about 2000 years ago
• The real development of microbiology began in the 1600s with crude lenses and microscopes
• Antony van Leeuwenhoek (1632-1723) founding father of microbiology, microscopy and zoology

Antoni van Leeuwenhoek's Discoveries and Beliefs

• In 1677, observed canal water through his microscope and discovered microorganisms
• He called the microorganisms "animalcules" (small animals), now defined as protists
• Had no scientific training
• He used home-made lenses and microscopes of his own design to examine things

Spontaneous Generation vs. Biogenesis

• Spontaneous generation describes how living things arise from nonliving things
• A view held by Aristotle and other philosophers from 300 BC to the 1700's
• Examples include decaying meat turning into maggots and flies, or grain turning into frog
• Biogenesis describes how living cells arise from preexisting living matter
• examples include animalcules arising from “germs” or “seeds” present in air

Key Figures in the Debate over Spontaneous Generation

• Francesco Redi (1626-1697) showed that maggots came from flies, not from decaying meat
• John Needham and his experiment: nutrient broth placed in flasks, boiled, and then sealed
• His experiment led to microbial growth and the conclusion: microbes arose spontaneously in broth from a "life force"
• Lazzaro Spallanzani (1729-1799) said Needham's organisms came from heat-resistant microbes
• Spallanzani's experiment: nutrient broth placed in flask, sealed, then boiled
• Spallanzini's experiment led to no microbial growth, concluding microbes were introduced into these flasks from the air

Louis Pasteur’s Contributions:

• Louis Pasteur (1822-1895) believed life only originated from previous life and demonstrated that microorganisms are present in the air.
• He disproved spontaneous generation with the swan-neck flask experiment
• Invented pasteurization

Pasteur's Experiment

• Pasteurs experiments consisted of two parts
• The broth in the flasks was boiled to sterilize it, when cooled, it remained free from contamination
• In the second part, the broth in the flasks was boiled and the neck was broken off, the broth became contaminated

Other contributions from Louis Pasteur

• Fermenting fruit can produce alcohol, and microbes are responsible
• Different microbes produce different tastes of wine
• Pasteurization is a method that removes undesired microbes from juice without affecting its quality
• The correct temperature for heating juices for pasteurization is 62.8°C for half an hour
• Isolated anthrax from blood of cattle, sheep and human
• He developed vaccine (a killed or attenuated microbe to induce immunity) against rabbis from the brains and spinal cord of rabbit

Further Advances in Microbiology

• John Tyndall (1820-1893) demonstrated that dust carried germs, and broth remained sterile in the absence of dust
• Martinus Willium Beijerinck (1851 – 1931) developed the enrichment technique to isolate various groups of bacteria
• Walther Hesse & Fannie E. Hesse (1883) used agar instead of gelatin for preparing media
• Joseph Lister (1878) developed a pure culture technique, referred to as the growth of mass cells of the same species in a vessel

Alexander Fleming and Penicillin

• In 1928, Alexander Fleming developed Penicillin from mold, specifically the Penicillium notatum species

The Germ Theory of Disease

• Certain diseases are caused by the invasion of the body by germs (microorganisms)
• In 1876, Robert Koch established the "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 Listed

• The same microbe is always associated with a specific disease
• The microbe must be isolated from the host
• 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

Diseases Koch Established the Etiology Of

• Koch established the etiology of 3 diseases: Cholera, Tuberculosis and Anthrax
• Cholera is a fecal oral disease, Vibrio cholerae
• Tuberculosis is a Pulmonary infection, Mycobacterium tuberculosis
• Anthrax affects Sheep and cattle, Bacillus anthracis

The Exceptions to Koch's Postulates

• Growth on artificial media is impossible in some cases
• Certain species, Treponema pallidum cause Syphilis
• Leprosy is caused Mycobacterium leprae
• All viruses cannot grow on artificial media
• Ethical objections exist: not possible for infecting subjects with infectious agents
• No animal models for some disease

The Normal Flora (Microbiota)

• These are mixture of microorganisms regularly found at any anatomical site on /within the body of a healthy person
• Some of these microorganisms are found in association with humans/animals only
• Others are found in the environment as well
• The makeup of the normal flora may be influenced by various factors, with genetics, age, sex, stress, nutrition and diet of the individual being primary

Normal Flora Types:

• The two types of normal flora are Resident and Transient
• Resident flora reestablishes itself promptly if disturbed
• If disturbed, other microorganisms may colonize, proliferate and produce disease
• Resident flora is often more important that transient flora
• Transient flora are non-pathogenic or potentially pathogenic microorganisms that inhabit the skin or mucous membranes for hours/days/weeks
• Transient flora are derived from the environment and does not produce disease or establish itself permanently on the surface
• Transient flora is of minimal significance

Role of the Resident Flora

• Synthesizes and excretes vitamins, including Vitamin K and Vitamin B12
• Enteric bacteria helps secrete Vitamin K and Vitamin B12
• Lactic acid bacteria produce certain B-vitamins
• Prevents colonization by pathogens by taking key attachment sites or nutrients
• A example of this protection takes place in the oral cavity, intestine, skin, and vaginal epithelium
• May antagonize other bacterial by generating nonspecific fatty acids and peroxides to highly specific bacteriocins

More Functions of the Resident Flora

• Stimulates the production of cross reactive antibodies
• Antibodies produced against components of the NF cross react with certain related pathogens, and prevent infection or invasion.
• Antibodies to NF are referred to as "natural" antibodies

Normal Flora and Harmful Effects

• Potential pathogens (Parasites) are members of the normal flora that may produce disease under certain circumstances
• In tissue outside their normal habitant they may become pathogenic
• E.g when normal flora of the intestine cause a UTI
• Streptococcus viridans is common in the URT
• May settle on deformed or prosthetic heart valves
• In cases like this, it will produce infective endocaritis if there is circulation following tooth extraction or tonsillectomy

The Harmful Effects of Bacteroids

• Bacteroids are bacteria within the large intestine that results in bacteremia when introduced to the peritoneal cavity or the pelvic tissue
• Taking broad spectrum antibiotics affect the balance of the normal microbial population and may lead to vaginitis in women due to yeast
• Clostridium difficile may occur which causes diarrhea or pseudomembranous colitis
• When the host resistance is lowered they become pathogenic
• Pencillinase producing organisms can aggravate infection if normal flora is present and confused during diagnosis

Relationship Between Normal Flora and Host

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

Factors that Influence The Human Microbiota

• Diet
• Age
• Baby feeding method
• Birth delivery process
• Antibiotics

Normal flora of the skin

• Staphylococcus epidermidis which is the major inhabitant making up more than 90% of th skine flor
• Staphylococcus aureus
  • Occurrence varys from Nose, perineum, vulvar skin
  • Occurrence in nasal passages varies with age, with the greatest concentration in newborns and less in adults
• Micrococci, Diphtheroids, Propionibacterium

Factors to Eliminate the Skin

• Low pH
• Fatty acids (sebaceous secretions)
• Lysozyme
• Neither profuse sweating nor washing and bathing can eliminate or significantly modify the normal resident flora

Normal Flora Benefits and Harms

• Benefit
  • Production of defensive antimicrobial peptides
  • Inhibit fungal growth
  • Wound healing
  • Inflammation control
  • Immune system development
• Harm
  • Body odor
  • Acne
  • Opportunistic infection

Normal Flora in the Oral Cavity

• Birth is sterile within 4-12 hours, with lactobacilli, streptococci, and other species quickly colonizing
• Neonates have Streptococcus salivarius, staphylococci, Neisseriae, and Moraxella Catarrhalis
• When teeth appear Streptococcus mutans and Streptococcus parasanguis are most prominent
• Gingival crevice areas have specific anaerobic species
• Puberty results in increases in Bacteroides and spirochetes

Oral Flora: Benefits and Harms

• Benefit
  • Compete with pathogens for colonization sites
  • Produce substances that inhibit pathogens
  • Stimulate local immunity
• Harm
  • Plaque formation and dental disease

Disinfection, Sterilization and Antiseptics:

• Sterilization is a process eliminating all spores and microbial life
• Disinfection used when all the harmful and infectous organisms are eliminated
• Disinfectants are used on inanimate objects and surfaces that kill microoganisms
• Septic refers to the presence of pathogens in living tissue
• Antisepsis are products containing biocide that is used to inhibit infection / growth of microorganisms

More Microbiology Terminology

• Aseptic - characterized by the absence of pathogenic microbes
• Preservation - the prevention of multiplication of microorganisms in formulated products, including pharmaceuticals and foods
• Germicide: - a chemical agent that kills the vegetative forms of microorganisms, but not necessarily their spores
• Sanitizer: - an agent that kills 99.9% of microorganisms contaminating an area
• These agents are commonly used on inanimate objects.

Sterilization Techniques

• The complete destruction or removal of all microorganisms and spores
• Recommended for processing instruments and the bloodstream under the skin
• Length of exposure to heat will affect the degree of treatment

Factors effecting Sterilization

• The type of microorganism present will vary, some die easily while others are very difficult to kill
• Number of microogranism will affect the results as is much easier to kill one organism than it is much easier to kill many
• Microogranisms that are protected by Blood or tissue are more liekly to survive on poorly cleaned instruments that acts as a shield

How to sterlize

• There are two methods of sterilization namely, Physical and Chemical sterilization
• Physical Sterilization comes in these forms
  • Heat
  • Radiation
  • Filtration

Physical methods of Sterilization

• Heat A) Moist Heat B) Dry Heat
• Radiation is key
• Filtartion is also required

Heat Sterilization

• Most effective and widely utalized
• Heat can be dry or wet
• Dry heat is less reliable, but cheaper that Wet
• Capacity to resist is Enormous
• The mai effect of heat is degridation particuarly affecting the proteins

Different Kinds Of Heat

• Moist heat more effective than dry heat
• Moist heat will denature
• Moist heat allows Low temperature and shorter exposure time than oxidation of proteins as in dry heat.

SubTypes Of Moist Heat

• Autoclaving (Steam under pressure)
• Boiling
• Tyndallization (Intermittent boiling)
• Sub-boiling Temperatures (Pasteurization)

A) Autoclaving:

• Pressure of pure steam, most dependable method to apply to moist heat
• Consists of double - walled chamber
• Steam circulates within jacket and supplied pressure in closed chamber
• Steam temperature rises atmospheric pressure, and the time of exposure diminishes
• Should be done at 121 degrees with 15 pounds pressure for 15 minutes

Steam Sterilization Required

1. Adequate contact
2. Sufficiently high temperature
3. Correct time
4. Sufficient moisture

• Most commonly used effective method of sterilization
• Cycle time is shorter that Dry heat or chemical
• However this process Reqeuals sterilizer with strict adherence time. temperature and preddure setting
• Cut cutting edges of isrtruments
• Platic items cannot with stand

B) Boiling:

• 100 C For 30 min will kill everything but endospores
• purifting water at 100 c 5 min is adaquete
• Giardia cryts are still live
• Sterilization will also occur

C) Tyndallization:

• Heat treament at 100 30 min each day for 3 days straight
• Used when auto claving would cause damege
• The first day klls vegetative cells but not spores
• 3rd day is traditional for precaultion

D) Pasteurization

• Sterlizaion can adverisly effects foods
• A Good exmaple: is Pasteurization
• Heat helps with microorganisms
• Batch Method low and long
  • Temp is 62.8 for 30 min Flash MEthod 71.7 for 15 s, material flows through excharger

II. Dry Heat

• If there are sufficiently high temperatures, the microorganisms will die
• It is less effective then moist heat
• Oxidation will allow for burn to happen
• There are 4 Types
  1. Red HOT
  2. Flaming 3)Incinerantion
  3. Hot air Ovens
     • Longer and higher than moist
     • Instruments can be sterilized
     • Temp varries

Ovens for Dry HEat

• Is efficitive method to reach all surface of instruments
• Its protective
• Doesnt leave as any chemical residue

Disadvantages

• Items cannot be be sterilized Dry Heat are too slow to penetrate materials
• Require continuous heat and electricity

Radiation

• Energy and radiation are inversely related to the wave length
• gamma and UV have heigher and high energy
• this is how they will kill cells

Two Forms of Radiation

• Ioniting causes molecels to ionize Non ionized do not

i) Ionizing

• High and electrion such as rays will sufice for ionization H2O split into Hydroxyl radicals
• Able to protect content and packaging

ii) Non-Ionizing

Radiations such as UV

• Light is longer
• UV excited electrode and molecule to react
• UV will be absorbed intracellularly, specifically damaging the DNA
• the greatest bactericidal action occurs at wavelengths of about 260 nm
• After exposure, Pyrimidine dimmers form when two adjacent UV cause mutations

Filtration

To remove microogranisms in liquid used with HEAT

• Types Antibitoics Serum Injecatble Drugs VIT
• Material used Celluose ceramics

B) Chemical Steralization

Mercuriral with antiseptitics such as Alcs

• can treat skin to kill micro organisms

Chemical Sterilization: Disinfectants

• Use instruments to kill microoranims
• Such as cholrinie Levels of disinfections
• The levels of disinfections include High, Intermediate, Low

Hight Level of Desinfection

Invasive prodcuees not steril Surigcal instruments

• Has to be cleaning surface
• moist heat glutaralhyde. Elimainate bacteria.

Intermediate Level Disinfection

• Clean surivsves with hihgly resistant organisms
• Includes flexible scops . . . Inactibates bacertia fungal

Levels Disenfction 2

• Treaty non-criotical
• blood pressure, ecg and step.
• kills most but M. Tuberuc

Methods of Desinfection in Table

• Heat
• Moist Heat is best
• High -Intermaida Liquid - All over

Disenfection: Phenols/Alcohol Mode of Action

Phenol known as Cabolic Acid

• One fo the fiest checmials
• Mode action alters membrane causing likage
• Inactivation occurs

Phenol Implements

Aques sus kills cell

• toxic Odourous Germicidal soaps Alcooll dentanute 70% Isopropyl - Alcohol use for skin

Disifenction Aldyhydes:

Glutadehlhides for baccreri afungi virus

• Two kinds
• Formalyhyde
• Gkurtadhlide
• used electorn . . microscope
• has cancer

DisifecectionHaloge

halgoens killl agents that destroy cells idones rapid is antiseptic Chlorines bacteria water

• Oxygen destroy cells
• used in water

Disifection: Metal

• Heavy metal Lead Zilcer and copper action in Protein

• Silver is dsinfetct in baby eyes. Murury- wonds a infeciton Cupper algicude in waster Zinc - athletes foo

Oxxidfizing

active yeast viruss.

• High contne will sporicdal hydrogen perxiod is widel used.