Chapter I: Introduction to Medical Microbiology PDF

Chapter I: INTRODUCTION TO MEDICAL MICROBIOLOGY A. Scope of Medical Microbiology B. Historical Background of Medical Microbiology C. Natural microflora of the human body D. Diagnostic/ clinical microbiology C. NATURAL MICROFLORA OF THE HUMAN BODY v Normal and characteristic microbial population...

Chapter I: INTRODUCTION TO MEDICAL MICROBIOLOGY A. Scope of Medical Microbiology B. Historical Background of Medical Microbiology C. Natural microflora of the human body D. Diagnostic/ clinical microbiology C. NATURAL MICROFLORA OF THE HUMAN BODY v Normal and characteristic microbial populations begin to establish themselves in an individual before birth (in utero) Ø placental microbiome= mostly Enterobacteriaceae and Propionibacterium these are found in the newborn’s intestine just before a woman gives birth, lactobacilli in her vagina multiply rapidly, and they become the predominant organisms in the newborn’s intestine C. NATURAL MICROFLORA OF THE HUMAN BODY v breathing and feeding= introduce more microorganisms to the newborn’s body from the environment v individual’s microbiome= changes rapidly during the first three years as the personal microbiome becomes established Ø after birth, E. coli and other bacteria acquired from foods, people, and pets begin to inhabit the large intestine these microorganisms remain there throughout life in response to altered environmental conditions, these may increase or decrease in number and contribute to health and disease C. NATURAL MICROFLORA OF THE HUMAN BODY v other usually harmless microorganisms establish themselves inside other parts of the normal adult body and on its surface Ø typical human body= contains 3 x 1013 body cells, and harbors an estimated 4 x 1013 bacterial cells Human Microbiome Project (2007)= analyzed microbiomes that live in and on the human body § goal is to determine the relationship between changes in the human microbiome and human health and disease § the human microbiome is very diverse C. NATURAL MICROFLORA OF THE HUMAN BODY v body’s normal microbiota= microorganisms that establish more or less permanent residence (colonize) but that do not produce disease under normal conditions Ø Historically were referred to as normal flora v transient microbiota= may be present for several days, weeks, or months and then disappear Ø Microorganisms are not found throughout the entire human body but are localized in certain regions C. NATURAL MICROFLORA OF THE HUMAN BODY v Factors that determine the distribution and composition of the normal microbiota: 1. NUTRIENTS microbes vary with respect to the types of nutrients they can use as an energy source accordingly, microbes can colonize only those body sites that can supply the appropriate nutrients these nutrients may be derived from dead cells, food in the gastrointestinal tract, secretory and excretory products of cells, and substances in body fluids. C. NATURAL MICROFLORA OF THE HUMAN BODY 2. PHYSICAL AND CHEMICAL FACTORS affect the growth of microbes and thus the growth and composition of the normal microbiota e.g. temperature, pH, available oxygen and carbon dioxide, salinity, and sunlight C. NATURAL MICROFLORA OF THE HUMAN BODY 3. HOST’S DEFENSES these defenses are extremely important against pathogens childhood exposure to microorganisms helps the immune system develop it has been proposed that insufficient exposure to microorganisms in childhood may interfere with the development of the immune system and may play a role in increasing rates of allergies and other immune disorder C. NATURAL MICROFLORA OF THE HUMAN BODY 4. MECHANICAL FACTORS certain regions of the body are subjected to mechanical forces that may affect colonization by the normal microbiota e.g. chewing actions of the teeth and tongue movements can dislodge microbes attached to tooth and mucosal surfaces; flushing action of urine removes unattached microbes; mucus traps microbes C. NATURAL MICROFLORA OF THE HUMAN BODY v Other factors: age geography health status personal hygiene disability living conditions hospitalization occupation stress lifestyle climate C. NATURAL MICROFLORA OF THE HUMAN BODY v Relationships between the Normal Microbiota and the Host 1. microbial antagonism or competitive exclusion involves competition among microbes consequence of this competition: § the normal microbiota protect the host against colonization by potentially pathogenic microbes by competing for nutrients, producing substances harmful to the invading microbes, and affecting conditions such as pH and available oxygen when this balance between normal microbiota and pathogenic microbes is upset, disease can result C. NATURAL MICROFLORA OF THE HUMAN BODY v Relationships between the Normal Microbiota and the Host 1. microbial antagonism or competitive exclusion E.g.: The normal bacterial microbiota of the adult human vagina maintains a local pH of about 4. The presence of normal microbiota inhibits the overgrowth of the yeast Candida albicans, which can grow when the pH is altered. If the bacterial population is eliminated by antibiotics, excessive douching, or deodorants, the pH of the vagina reverts to nearly neutral, and C. albicans can flourish and become the dominant microorganism there. This condition can lead to a form of vaginitis (vaginal infection). C. NATURAL MICROFLORA OF THE HUMAN BODY E.g.: In the large intestine, E. coli cells produce bacteriocins, proteins that inhibit the growth of other bacteria of the same or closely related species, such as pathogenic Salmonella and Shigella. A bacterium that makes a particular bacteriocin is not killed by that bacteriocin but may be killed by other ones. Bacteriocins are being investigated for use in treating infections and preventing food spoilage. C. NATURAL MICROFLORA OF THE HUMAN BODY E.g.: The normal microbiota of the large intestine effectively inhibit C. difficile, possibly by making host receptors unavailable, competing for available nutrients, or producing bacteriocins. However, if the normal microbiota are eliminated (for example, by antibiotics), C. difficile can become a problem. § This microbe is responsible for nearly all gastrointestinal infections that follow antibiotic therapy, from mild diarrhea to severe or even fatal colitis (inflammation of the colon). C. NATURAL MICROFLORA OF THE HUMAN BODY v Relationships between the Normal Microbiota and the Host 2. Commensalism one of the organisms benefits, and the other is unaffected § E.g. Staphylococcus epidermidis bacteria that inhabit the surface of the skin these bacteria live on secretions and sloughed off cells, and they bring no apparent benefit or harm to the host C. NATURAL MICROFLORA OF THE HUMAN BODY v Relationships between the Normal Microbiota and the Host 3. Mutualism a type of symbiosis that benefits both organisms § E.g. the large intestine contains bacteria, such as E. coli, that synthesize vitamin K and some B vitamins. These vitamins are absorbed into the bloodstream and distributed for use by body cells. In exchange, the large intestine provides nutrients used by the bacteria, allowing them to survive. C. NATURAL MICROFLORA OF THE HUMAN BODY v Relationships between the Normal Microbiota and the Host 4. Parasitism one organism benefits by deriving nutrients at the expense of the other § E.g. disease-causing bacteria Representative Normal Microbiota per Body Region 1. SKIN and MUCOUS MEMBRANE Ø Most of the microbes in direct contact with skin don’t become residents secretions from sweat and oil glands have antimicrobial properties keratin is a resistant barrier the low pH of the skin inhibits many microbes the skin has a relatively low moisture content 1. SKIN and MUCOUS MEMBRANE Ø moist areas= support much larger populations Ø sebum-rich areas= less diverse microbial communities sebum contains compounds that provide nutrition for certain microbes some of the lipids and fatty acids in sebum inhibit microbial growth Ø normal microbiota tends to inhibit transient-microbe colonization by producing antimicrobial substances and outcompeting other microbes that land on the surface of the skin helps to protect the skin from pathogenic infection 1. SKIN and MUCOUS MEMBRANE Ø eyes (conjunctiva) the conjunctiva, a continuation of the skin or mucous membrane, contains basically the same microbiota found on the skin tears and blinking eliminate some microbes or inhibit others from colonizing surface is colonized with coagulase- negative Staphylococci and organisms found in the nasopharynx (e.g., Haemophilus spp., Neisseria spp., viridans streptococci) 1. SKIN and MUCOUS MEMBRANE Ø eyes (conjunctiva) the conjunctiva, a continuation of the skin or mucous membrane, contains basically the same microbiota found on the skin tears and blinking eliminate some microbes or inhibit others from colonizing surface is colonized with coagulase- negative Staphylococci and organisms found in the nasopharynx (e.g., Haemophilus spp., Neisseria spp., viridans streptococci) 2. RESPIRATORY TRACT Ø upper respiratory tract mouth, oropharynx and nasopharynx § colonized with numerous organisms, with 10 to 100 anaerobes for every aerobic bacterium § most of the common organisms are relatively avirulent and are rarely associated with disease ü unless introduced into normally sterile sites (e.g., sinuses, middle ear, brain) 2. RESPIRATORY TRACT Ø lower respiratory tract larynx, trachea, bronchioles, and lower airways § generally sterile § transient colonization with secretions of the upper respiratory tract may occur 3. GASTROINTESTINAL TRACT Ø receives, moves, digests, and absorbs food and removes waste innermost surface of this tube is exposed to the environment Ø shifting conditions of pH and oxygen tension and differences in the microscopic anatomy of the GI tract cause variations in or distribution of the microbiota 3. GASTROINTESTINAL TRACT Ø areas that harbor appreciable permanent microbes: oral cavity, large intestine, and rectum oral cavity § houses more than 600 species § most common residents: aerobic Streptococcus species (S. sanguis, S. salivarius and S. mitis) § saliva normally has a high bacterial count (up to 5 × 109 cells per milliliter) 3. GASTROINTESTINAL TRACT Ø stomach= acid inhibits most microbes small numbers of lactobacilli and Helicobacter pylori (associated with stomach ulcers) can become established microbial population can dramatically change in patients receiving drugs that neutralize the production of gastric acids Ø small intestine= has a sparse population of lactobacilli and streptococci except for its terminal segment has flora more similar to that of the adjacent large intestine 3. GASTROINTESTINAL TRACT Ø large intestine= microbes have complex and profound interactions with the host cecum, colon and rectum= harbor a huge population of microbes (108−1011 per gram of feces) § constitute 30% or more of the fecal volume § even an individual on a long-term fast passes feces consisting primarily of bacteria 3. GASTROINTESTINAL TRACT Ø intestinal environment= favors strictly anaerobic bacteria (Bacteroides, Bifidobacterium, Fusobacterium, and Clostridium) many species ferment waste materials in the feces, generating vitamins (B12, K, pyridoxine, riboflavin, and thiamine) and acids (acetic, butyric, and propionic) of potential value to the host occasionally significant are bacterial digestive enzymes that convert disaccharides to monosaccharides or promote steroid metabolism Coliforms (e.g. E. coli, Enterobacter, and Citrobacter)= present in smaller numbers 3. GASTROINTESTINAL TRACT Ø antibiotic treatment= can rapidly alter the population, causing the proliferation of antibiotic- resistant organisms, such as Enterococcus, Pseudomonas, and fungi. C. difficile can also grow rapidly, leading to diseases ranging from diarrhea to pseudomembranous colitis. Ø exposure to other enteric pathogens e.g. Shigella, enterohemorrhagic E. coli and Entamoeba histolytica can also disrupt the colonic flora and produce significant intestinal disease 3. GASTROINTESTINAL TRACT Ø flatus some of the gas arises through the action of bacteria on dietary carbohydrate residues from vegetables such as cabbage, corn, and beans the bacteria produce an average of 8.5 liters of gas daily, but only a small amount is ejected in flatus Ø appendix= recent research is verifying its importance in replenishment of the normal microbiota 3. GASTROINTESTINAL TRACT Ø intestinal environment= favors strictly anaerobic bacteria (Bacteroides, Bifidobacterium, Fusobacterium, and Clostridium) many species ferment waste materials in the feces, generating vitamins (B12, K, pyridoxine, riboflavin, and thiamine) and acids (acetic, butyric, and propionic) of potential value to the host occasionally significant are bacterial digestive enzymes that convert disaccharides to monosaccharides or promote steroid metabolism Coliforms (e.g. E. coli, Enterobacter, and Citrobacter)= present in smaller numbers Distribution of microbes in the GI tract. Areas of the gastrointestinal tract that shelter major communities of resident microbes are highlighted in color. Noncolored areas do not harbor residents in significant numbers. 4. GENITOURINARY TRACT Ø anterior urethra and vagina= permanently colonized with microbes urethra § principal residents: nonhemolytic Streptococci, Staphylococci, corynebacteria, and occasionally, coliforms vagina § estrogen= important factor influencing changes in women ü stimulates the vaginal mucosa to secrete glycogen, which certain bacteria (primarily Lactobacillus species) ferment, thus lowering the pH to about 4.5) ü before puberty a girl produces little estrogen and little glycogen and has a vaginal pH of about 7= favor the establishment of diphtheroids, Staphylococci, Streptococci, and some coliforms ü as hormone levels rise at puberty, the vagina begins to deposit glycogen, and the microbiota shift to the acid- producing lactobacilli 4. GENITOURINARY TRACT Ø internal reproductive organs are kept sterile physical barriers such as the cervical plug and other host defenses kidney, ureter, bladder, and upper urethra= presumably kept sterile by urine flow and regular bladder emptying § shorter urethra in women (about 3.5 cm long)= frequently leads to urinary tract infections uterus= should also remain free of microorganisms v Maintenance of the Normal Microbiota Ø the microbes replace themselves naturally on a regular basis to maintain the types and numbers in their zones however, a number of changes can disrupt this balance: § broad-spectrum antibiotics § changes in diet § underlying disease Ø probiotic essentially involves introducing pure cultures of known microbes into the body through ingestion or inoculation growing trend in therapy D. DIAGNOSTIC/ CLINICAL MICROBIOLOGY v methods microbiologists use to identify bacteria: Ø phenotypic= includes a consideration of morphology (microscopic and macroscopic) as well as physiology or biochemistry Ø immunologic= entails analysis of the blood (serology) and other fluids Ø genotypic= genetic techniques  Specimen Collection v success of treatment depends on how specimens are collected, handled, and stored  Specimen Collection v general aseptic procedures should be used (including sample containers and other tools) to prevent contamination from the environment or the patient v nonsterile samples (e.g. urine, feces, and sputum) are especially prone to deterioration at room temperature Ø the overgrowth of normal microbiota in these samples could interfere with isolation of the pathogens Ø can also alter the numbers and proportions of cells, making analysis more difficult Ø many transport devices contain nonnutritive maintenance media, a buffering system, and an aerobic or sometimes anaerobic environment as necessary  Methods of Microbial Investigation 1. Inoculation Ø first stage in culturing Ø placing a sample into a container of medium that supplies nutrients for growth Ø methods: streak plate method spread plate technique pour plate or loop dilution Inoculation Methods Streak Plate Method Spread Plate Method Inoculation Methods Pour Plate Method  Methods of Microbial Investigation 2. Incubation Ø exposing the inoculated medium to optimal growth conditions for a few hours to days Ø encourages microbial growth microbe multiplies and produces a culture with macroscopically observable growth  Methods of Microbial Investigation 3. Isolation Ø separating individual microbes (pure culture) Ø achieving isolated colonies that can be readily distinguished from one another macroscopically 4. Inspection Ø observing cultures macroscopically for appearance of growth and microscopically for appearance of cells  Methods of Microbial Investigation 5. Information gathering Ø testing of cultures using procedures that analyze biochemical and enzyme characteristics, immunologic reactions, drug sensitivity, and genetic makeup 6. Identification Ø final determination of the types of microbes present in the original sample Ø accomplished by a variety of schemes  Procedures for Identifying Pathogens and Diagnosing Infections v Phenotypic Ø morphology (microscopic and macroscopic), physiology or biochemistry v immunologic Ø analysis of the blood (serology) and other fluids e.g. HIV testing= examination of a person’s blood for presence of antibody to the virus v genotypic Ø genetic techniques Ø more precise than with phenotypic methods  Rapid diagnostic test A rapid diagnostic panel uses a finger-prick that identifies antigens from different species of An oral home test rapidly blood drop to measure Plasmodium, the agent of tests for HIV antibodies in antibodies to hepatitis malaria, using a small drop saliva. B and C viruses and © Kathy Park Talaro HIV. Courtesy of of whole blood. Courtesy MedMira Inc. of Alere, Inc.  Phenotypic Characterization of Bacteria v Microscopic Morphology Ø cell shape and size Ø Gram stain reaction and acid-fast reaction Ø special structures (endospores, granules, and capsules) Ø electron microscope studies additional structural features such as the cell wall, flagella, pili, and fimbriae  v distinguish between Gram-negative Gram-stain and Gram-positive bacteria  Acid-Fast Stains v distinguish between acid-fast and acid-fast negative organisms v used to identify bacteria in the actinomycete genus Mycobacterium Ø some of which are pathogens M. leprae= causative agent of leprosy M. tuberculosis= causative agent of tuberculosis  Endospore Stain v a primary stain of malachite green is forced into the spore by steaming the bacterial emulsion Ø malachite green is water-soluble and has a low affinity for cellular material vegetative cells and spore mother cells can be decolorized with water (while the spores retain it) and counterstained with safranin  Bacterial Motility v Wet Mount Ø made by placing the specimen in a drop of water on a microscope slide and covering it with a cover glass Ø motility often can be observed at low or high dry magnification Ø viewing must be done quickly because of drying of the preparation  Bacterial Motility v Hanging Drop Ø allows longer observation of the specimen it doesn’t dry out as quickly Phenotypic Characterization of Bacteria v Macroscopic Morphology Ø appearance of growth texture, size and shape pigment of colonies speed of growth reactions to special types of selective and differential media  Selective Media v designed to enhance the isolation procedure by inhibiting growth of some organisms while encouraging the growth of others v differential media Ø contain indicators to expose differences between organisms Phenotypic Characterization of Bacteria v Physiological/ Biochemical Characteristics Ø tests for: fermentation of sugars capacity to digest or metabolize complex polymers production of gas presence of enzymes sensitivity to antimicrobial drugs  Oxidation–Fermentation Test v differentiate bacteria based on fermentative or oxidative metabolism of carbohydrates Ø oxidative organisms= oxidize the carbohydrate to CO2 and H2O Ø fermentative organisms= convert the carbohydrate to pyruvate which are then reduced to organic acids, gas, or alcohol  Fermentation Test v fermentative organisms Ø convert the carbohydrate to pyruvate which are then reduced to organic acids, gas, or alcohol  Catalase Test v identify organisms that produce the enzyme catalase Ø enzyme that converts hydrogen peroxide into water and gaseous oxygen  Antimicrobial Susceptibility Testing v frequently used in medicine to identify the antibiotic needed to treat an infection v can also be helpful in microbial identification Important Families and Genera of Pathogenic Bacteria I. Bacteria with gram-positive cell wall structure (Phyla Firmicutes and Actinobacteria) A. Cocci in clusters or packets that are aerobic or facultative Staphylococcus § members cause boils, skin infections Important Families and Genera of Pathogenic Bacteria B. Cocci in pairs and chains that are facultative Streptococcus § some species cause strep throat, dental caries C. Anaerobic cocci in pairs, tetrads, irregular clusters Peptostreptococcus § involved in wound infections Important Families and Genera of Pathogenic Bacteria D. Spore-forming rods Bacillus= anthrax Clostridium= tetanus, gas gangrene, botulism Important Families and Genera of Pathogenic Bacteria E. Non-sporeforming rods Lactobacillus, Listeria (food infection), Erysipelothrix (erysipeloid) Propionibacterium (involved in acne) Corynebacterium (diphtheria) Mycobacterium (tuberculosis, leprosy) Nocardia (lung abscesses) Actinomyces (dental infections) Streptomyces (important source of antibiotics) Important Families and Genera of Pathogenic Bacteria II. Bacteria with gram-negative cell wall structure (Phyla Proteobacteria, Bacteriodetes, Fusobacterium, Spirochaetes, Chlamydiae A. Aerobic cocci Neisseria (gonorrhea, meningitis) B. Aerobic coccobacilli Moraxella, Acinetobacter Important Families and Genera of Pathogenic Bacteria C. Anaerobic cocci Veillonella § involved in wound infections D. Aerobic rods Pseudomonas (pneumonia, burn infections) Legionella (Legionnaires’ disease) Important Families and Genera of Pathogenic Bacteria E. Facultative rods and vibrios Salmonella (typhoid fever) Shigella (dysentery) Yersinia (one species causes plague) Vibrio (cholera, food infection) Campylobacter (enteritis) Helicobacter (ulcers) Important Families and Genera of Pathogenic Bacteria F. Anaerobic rods Bacteroides, Fusobacterium (anaerobic wound and dental infections G. Helical and curviform bacteria Treponema (syphilis) Borrelia (Lyme disease), Leptospira (kidney infection) Important Families and Genera of Pathogenic Bacteria H. Obligate and facultative intracellular bacteria Ø Rickettsia (Rocky Mountain spotted fever) Important Families and Genera of Pathogenic Bacteria III. Bacteria with no cell walls (Class Mollicutes) Ø Mycoplasma (pneumonia), Ø Ureaplasma (urinary infection)  Flowchart key that separates genera of gram-positive and gram-negative bacteria often isolated from specimens