Lecture 4 Microbiology PDF

Lecture 4 Microbiology: Bacterial Infection & Pathogenesis 1  Content Normal human microbiota (Role of the resident microbiota, and locations in the human body) Virulence of bacteria, bacterial virulence factors and their...

Lecture 4 Microbiology: Bacterial Infection & Pathogenesis 1  Content Normal human microbiota (Role of the resident microbiota, and locations in the human body) Virulence of bacteria, bacterial virulence factors and their regulation (exotoxin, endotoxin, and their contribution to pathogenesis). Pathogenesis - types of bacterial infections, transmission, adherence , invasiveness, toxin production. Infection process (development, and outcomes) 2 3  Morobiota and Microbiome Although the terms “microbiota” and “microbiome” are often used interchangeably, Microbiota refers to the organisms that comprise the microbial community, whereas the Microbiome refers to the collective genomes of the microbes, which are composed of bacteria, bacteriophages, fungi, protozoa, and viruses that live inside and on the human body. The microbiota is now considered a human organ, with its own functions, i.e., modulating expression of genes involved in mucosal barrier fortification, angiogenesis, and postnatal intestinal maturation. The vast majority of microbial species present in microbiota give rise to symbiotic host–bacteria interactions that are fundamental for human health. 4 5  Relationships of microbiota with the humans Symbiotic - benefits the host Commensal – neutral to the host Parasitic – injuries the host The members of the normal flora may stay for a highly variable periods. Residents are strains that have an established niche at one of the many body sites, which they occupy indefinitely. Transients are acquired from environment and establish themselves briefly, but tend to be excluded by competition from residents or by innate or immune defense mechanism. The term carrier state is used when potentially pathogenic organism s are involved, however their implication of risk are not always justified. 6  Normal human microbiota Typical human body contains about 1012-1013 eukaryotic cells. However, most of the cells in our body are not our own, nor are they even human. They are microbial. Humans house about 1013-1014 microbial cells, at least 10 times more than eukaryotic cells. Almost every epithelial surface of our body contains a complex microbial community: skin, mouth, vagina, and gastrointestinal tract. Microbes can live even in the highly acidic environment of the stomach (pH=2). All these epithelial surfaces are populated by communities of microorganisms, collectively called microbiota, rather than by individual species, and often include hundreds and even thousands of different microbial members. Majority of these organisms are bacteria, though archaea, fungi, eukaryotic microorganisms, and viruses also take residence in different epithelial niches. Total weight of human microbiota varies from 1 to 3 kg (approximately 1%-3% of the total weight). 7 It's all in our gut (and on our skin): the Human Microbiome Revolution 8  Functions of microbiota Renewed interest in the human microbiota is associated with the recognition of the important relationships these microbes form with our bodies. Microbiota is essential for keeping us healthy and happy. Microbiota of the gut participate in host energy metabolism by breaking down complex polysaccharides in the diet, protect the host from pathogen invasion. Microbiota produces vitamins that we cannot make. Resident microbes also benefits the body through competition for resources or directly by inhibiting pathogen growth. Microbiota modulate the proper development and functioning of the human immune system, transform or excrete toxic substances, and help maintain epithelial homeostasis. Microbiota dysbiosis, defined as the perturbation of the normal microbial profile, has been linked to a number of human diseases including dental plaque, bacterial vaginosis, psoriasis, atopic dermatitis, and cystic fibrosis. 9  A Healthy Microbiome is a Healthy You Inside each and every one of us, we actually have our own microbiome. The human microbiome plays an extremely important role in the regulation of human bodily function. Helps digest food Protects against toxins Boosts the immune system Promotes skin health Influences mental health 10  A Healthy Microbiome is a Healthy You The human microbiome is extensive and highly variable, serving many functions in our bodies, the most notable of which are immune system support and aiding in proper digestion. Upsets in our bacteria can cause both of these things to fail. The most skin and internal microbiome we carry with us is wholly unimportant and generally unknown, however the fact is that it is a massive part of our livelihood. Bacterial cells outnumber our own cells by a factor of 10 to 1 i.e. the ratio between the numbers of human cells and those forming our microbes more than 10:1. While the research into the nature of our microbiomes is just beginning there has already been a large amount of evidence linking a healthy microbiome to a healthy, or at least healthier, person in general. 11 12  INTERSTING! Autistic populations have a unique microbiome consisting of more clostridial species. Half of all autistic children with gastrointestinal dysfunction were found to have the bacteria Sutterella which was completely absent in non-autistic children with gastrointestinal dysfunction. There is evidence that for some children with late-onset autism antibiotics can alleviate symptoms temporarily. 13  Gut microbiota and Mood Reading: http://allergiesandyourgut.com/tag/bacteroides-fragilis/ Very good news! An exciting new field of medicine is on the horizon: PSYCHOBIOTICS. PROBIOTICS are micro-organisms that have beneficial effects on the body when consumed 14  Colonization vs infection Colonization: establishment of a site of reproduction of microbes on a person without necessarily resulting in tissue invasion or damage. Infection: growth and multiplication of a microbe in or on the body of the host with or without the production of disease. Outcomes of exposure to a microorganism: 1. Transient colonization 2. Permanent colonization 3. Disease 4. Normal Flora and 5. Pathogenesis 15  Microbiota at different sites Sites that may be colonized: Respiratory tract and head: Blood, body fluids and Outer ear, tissues are sterile. Eye Nasopharynx Sterile sites: Oral cavity sinuses, Skin Oropharynx middle ear Lungs brain Gastrointestinal tract lower respiratory tract esophagus, stomach, (trachea, bronchiole, lungs) small intestine, bladder large intestine cervix Genitourinary system Vagina uterus Urethra Penis 16  Oral cavity Researchers have identified some 700 microbial species that inhabit the human mouth. Oral bacteria include: 1. Streptococcus viridans 2. Lactobacilli 3. Staphylococci (S. aureus and S. epidermidis) 4. Corynebacterium sp. 5. Bacteroides sp. 6. Streptococcus sanguis (dental plaque) 7. Streptococcus mutans (dental plaque) 8. Actinomyces sp Accumulating evidence suggests that the structure of this microbiome “is not haphazard or random. Among other things there is [a] first layer of microorganisms that allows for the attachment of the second-comers, the third-comers, the fourth, and so on, in a very hierarchical type of organization. “(http://www.the-scientist.com/?articles.view/articleNo/40600/title/The-Body-s-Ecosystem /) 17  Oral Microbiota Oral bacteria have been implicated in cardiovascular disease, pancreatic cancer, colorectal cancer, rheumatoid arthritis, and preterm birth, etc. 18 19  Normal microbiota of upper and low respiratory tract Upper respiratory tract 1.Staphylococcus epidermidis 2. Corynebacterium 3. Staphylococcus aureus 4. Neisseria sp. 5. Haemophilus sp 6. Streptococcus pneumoniae The lower respiratory tract (trachea, bronchi, and pulmonary tissues): Usually sterile. The individual may become susceptible to infection by pathogens descending from the nasopharynx e.g. H. influenzae S.pneumoniae 20  Lung microbiota There’s a constant flow into [the] lungs of aspirated bacteria from the mouth, but through the action of cilia, the cough reflex, and other cleansing responses, there’s also an outward flow of microbes, making the lung microbiome a dynamic community. The lung microbiome is about 1,000 times less dense than the oral microbiome, and about 1 million to 1 billion times sparser than the microbial community of the gut. Lungs are like small islands of clustered bacteria and wide stretches of unpopulated regions between them. That is in part because the lung lacks the microbe-friendly mucosal lining found in the mouth and gastrointestinal tract, instead harboring a thin layer of much-less-inviting surfactant to keep the respiratory organs from drying out, as well as ciliated cells that beat rhythmically to move debris and invading microbes. 21  Skin microbiota Propionobacterium Corynebacterium (diphteroides) Staphylococcus (coagulase-negative) 22  Normal microbiota of skin The most important sites are: 1.Axilla 2.Groin 3.Areas between the toes 23  Non-health related application of microbiome The microbiome has non-health related applications as well. The diversity of our skin microbiome is extensive, with a higher degree of diversity between individuals than that of our DNA. This uniqueness presents an interesting opportunity in the world of forensics. Since the skin microbiome is so diverse, it is actually a more reliable way of telling people apart than fingerprints – provided people live in the same general location during a crime. Microbe samples can be found on all sorts of surfaces where fingerprints might not be viable; and an individual microbiome can be matched to that person with a high degree of certainty. While it certainly won’t replace fingerprints, skin microbiome profiling can add a new layer of evidence, and help bring criminals to justice. 24  Skin microbiota Helococcus kunzii is a recently identified bacterium that is thought to be a nonpathogenic member of normal human skin flora and is rarely associated with skin infections. In the study though, the researchers report the isolation of the organism from an infected cyst on the breast of a 57-year-old immunocompromised woman. Finding provides further support for the opportunistic role of H. kunzii in causing infection in both immunosuppressed and healthy people (A.H. Chagla, A.A. Borczyk, R.R. Facklam, and M. Lovgren. 1998. Breast abscess associated with Helocococcus kunzii. Journal of Clinical Microbiology. 36:2377-2379.) 25 Intestinal microbiota 26 give us an information about the sex of the person 27  Genitourinary tract microbiota In women In men Lactobacillus spp. Lactobacillus Lactobacillus iners, L. crispatus, Streptococcus L. gasseri, or L. jensenii. Prevotella Fusobacterium These Lactobacillus sp. are thought to play key protective roles by lowering the environmental pH through lactic acid production and by producing various bacteriostatic and bacteriocidal compounds. However, the composition of the vaginal microbiome largely differs by age and ethnicity. Cause of vaginitis 28 When do we get colonized with normal microbiota? A human first becomes colonized by a normal flora at the moment of birth and passage through the birth canal. In uterus, the fetus is sterile, but when the mother's water breaks and the birth process begins, so does colonization of the body surfaces. Handling and feeding of the infant after birth leads to establishment of a stable normal flora on the skin, oral cavity and intestinal tract in about 48 hours. 29  Development of microbiota Originally, the intestine was thought to be sterile during fetal life, however the result of recent studies are changing these views. When present in the uterine compartment, some bacteria such as Ureaplasma spp. and Fusobacterium spp. appear to be the most significantly associated with negative pregnancy outcomes (e.g., prematurity). 30  Development of microbiota Upon delivery, the neonate is exposed to microbes from a variety of sources, including maternal vaginal, fecal, and skin bacteria. Initial colonization of the infant gut is highly influenced by the mother’s vaginal and fecal bacterial communities, which include facultative anaerobes such as streptococci and enterobacteriaceae. The first and most important phase of normal colonization occurs when the newborn fetus passes through the birth canal and ingests maternal vaginal and colonic microorganisms. 31 32  Development of microbiome The process of natural birth introduces the mother’s gut and vaginal microbes and shortly after birth, we’re exposed to the skin microbiome as well. This is a vital process for a healthy immune system, and, as it turns out there are much higher rates of allergies and asthma in children born through C-sections, a sterile process allowing the newborn to be exposed to only the mother’s skin microbiome (Pollan 2013) 33  Development of microbiota Infants delivered by cesarean section have a reduced number of bacteria compared with vaginally delivered infants, and colonization by bifidobacteria can be delayed by up to 6 months. The microbiota of vaginally delivered infants mirrors the mother’s vaginal and intestinal microbiota. These infants exhibit bacterial communities composed of prominent genera such as Lactobacillus, Prevotella, Escherichia, Bacteroides, and Bifidobacterium. 34  Role of microbiota in defense Intestinal mucosal barrier function can be defined as the capacity of the intestine to host the commensal bacteria and molecules, while preserving the ability to absorb nutrients and prevent the invasion of host tissues by resident bacteria. The dense communities of bacteria in the intestine are separated from body tissues by a monolayer of intestinal epithelial cells. The assembly of the multiple components of the intestinal barrier is initiated during fetal development and continues during early postnatal life. Collectively, the gut microbiota also influences tissue regeneration, permeability of the epithelium, vascularization of the gut, and tissue homeostasis. Pic from: http://journal.frontiersin.org/article/10.3389/fimmu.2012.00310/full 35  Factors disrupting microbiota Epidemiological studies have established, an association between the mode of delivery or the use of antibiotics and the occurrence of health disorders or diseases. The use of cesarean section delivery has markedly increased in the past 2 decades in a large number of middle- and high-income countries in the world, reaching an unprecedented level of 50.1% in Brazil in 2009. Although these operations can be lifesaving, both for mother and child, there is concern that increasing rates also may have short- and long- term deleterious effects. Studies suggested that children delivered by cesarean section could have increased risk later in life of atopy and allergies, asthma, and type 1 diabetes. 36  Factors influencing microbiota The makeup of the normal flora may be influenced by various factors, including genetics, age, sex, stress, nutrition and diet of the individual. Three developmental changes in humans, weaning, the Vaginal dryness eruption of the teeth, and the onset and cessation of ovarian functions, invariably affect the composition of the normal flora in the intestinal tract, the oral cavity, and the vagina, respectively. However, within the limits of these fluctuations, the bacterial flora of humans is sufficiently constant to a give general description of the situation 37 , weaning eruption of the teeth  Factors Affecting Normal Flora 1. Local Environment (pH, temperature, redox potential, O2, H2O, and nutrient levels…). 2. Diet 3. Age 4. Health condition (immune activity…) 5. Antibiotics,…..etc 38  Factors disrupting microbiota Antibiotic usage changes gut microbiota. For example, administration of broad-spectrum antibiotics significantly reduced the relative abundance of Bacteroides, with a concurrent increase in Firmicutes. The understanding of the dynamics and mechanisms that underlie functional changes in the microbiome in response to antibiotic treatments remains limited. The response depends on the type of antibiotics, length of dosing, and baseline microbiome. 39  Fecal bacteriotherapy Fecal bacteriotherapy, which is now officially and scientifically known as fecal micro biota transplantation (FMT) and is also referred to as fecal micro biota therapy, fecal transfusion, fecal transplant, stool transplant, fecal enema or human probiotic infusion (HPI), is a medical treatment for patients with pseudomembranous colitis (caused by Clostridium difficile), or ulcerative colitis that involves restoration of colon homeostasis by reintroducing normal bacterial flora from stool obtained from a healthy donor 40  Fecal transplantation Like an organ transplant, fecal microbiota transplantation begins with finding a donor, often a family member. The treatment team collects a fresh stool sample, at least 200 to 300 grams. The sample is mixed with salt water in a blender and filtered to remove particulate matter. It can be administered to the recipient through a colonoscopy, as an enema, or -- when the inflamed region is higher in the colon -- through a naso-gastric tube 41 42  Robert Koch’s postulates That the organism could be discoverable in every instance of the disease; That, extracted from the body, the germ could be produced in a pure culture, maintainable over several microbial generations. That the disease could be Koch's postulates are four criteria designed reproduced in experimental animals to establish a causative relationship between through a pure culture removed by a microbe and a disease - a sequence of numerous generations from the experimental steps that verified the germ organisms initially isolated; theory. He identified cause of anthrax, TB, and That the organism could he cholera retrieved from the inoculated animal and cultured a new. Developed pure culture methods 43  Pathogenicity A pathogen is a microorganism that is able to cause disease in a plant, animal or insect. Pathogenicity comes from Greek “patho” disease and “gene” – creation. Pathogenicity is a step-by-step ability to produce disease in a host organism. Virulence is a degree of pathogenicity Determinants of virulence of a pathogen are any of its genetic or biochemical or structural features that enable it to produce disease in a host. The relationship between a host and a pathogen is dynamic, since each modifies the activities and functions of the other. The outcome of such a relationship depends on: the virulence of the pathogen and the relative degree of resistance or susceptibility of the host, mainly due to the effectiveness of the host defense mechanisms. 44  Mechanisms of bacterial pathogenicity Invasiveness: the ability to invade tissues encompasses mechanisms for colonization (adherence and initial multiplication), production of extracellular substances which facilitate invasion (invasins) and ability to bypass or overcome host defense mechanism. 45 46  Number of invading microbes LD 50 - Lethal Dose of a microbes or toxin that will kill 50% of experimentally inoculated test animal within a certain period of time ID 50 - Infectious Dose required to cause disease in 50% of inoculated test animals Example: ID 50 for Vibrio cholerea 10 8 cells (100,000,000 cells) ID 50 for Inhalation Anthrax - 5,000 to 10,000 spores ID for E.coli O157:H7 - < 10 cells 47  How bacteria overcomes host defense? 1. Adherence - almost all pathogens have the means to attach to host tissue binding sites through adhesins and ligands 2. Capsules 3. Enzymes A. Leukocidins B. Hemolysins C. Coagulase D. Kinases E. Hyaluronidase F. Collagenase G. Necrotizing Factor 48 49  Adherance Surface molecules on a pathogen, called adhesins or ligands, bind specifically to complementary surface receptors on cells of certain host tissues. Adhesins and ligands are usually on Fimbriae Examples: Neisseria gonorrhoeae, ETEC (Entertoxigenic E. coli), Bordetella pertussis , etc. Adhesin/ligand Cell surface Cell receptor 50  Capsule –prevent phagocytosis Examples: Streptococcus pneumoniae Klebsiella pneumoniae Haemophilus influenzae Bacillus anthracis Streptococcus mutans Yersinia pestis Klebsiella pneumoniae 51  Enzymes Many pathogens secrete enzymes that contribute to their pathogenicity A. Leukocidins - Attack certain types of WBC’s 1. Kills WBC’s which prevents phagocytosis 2. Releases & ruptures lysosomes that contain powerful hydrolytic enzymes which then cause more tissue damage B. Hemolysins - cause the lysis of RBC’s Streptococci 1. Alpha Hemolytic Streptococci - secrete hemolysins that cause the incomplete lysis or RBC’s 2. Beta Hemolytic Streptococci - Secrete hemolysins that cause the complete lysis of RBC’s Action of Hemolysins 52  Enzymes C. Coagulase - cause blood to coagulate Blood clots protect bacteria from phagocytosis from WBC’s and other host defenses. Staphylococci - are often coagulase positive Negative Positive 53  Enzymes D. Kinases - enzymes that dissolve blood clots 1. Streptokinase - Streptococci Streptokinase - used to dissolve blood clots in the Heart (Heart Attacks due to obstructed coronary blood vessels) 1. Staphylokinase - Staphylococci - Helps to spread bacteria causes Bacteriemia E. Hyaluronidase - Breaks down Hyaluronic acid (found in connective tissues) It is known as “Spreading Factor” Use of Hyaluronidase - Mixed with a drug to help spread the drug through a body tissue In cosmetics Hyaluronidase is used to dissolve the skin fillers. 54  Enzymes F. Collagenase -Breaks down collagen (found in many connective tissues) Clostridium perfringens - Gas Gangrene Uses - to spread through muscle tissue Used in cosmetics to fight cellulitis, contacture 55  Enzymes G. Necrotizing Factor - Causes death (necrosis) to tissue cells “ Flesh Eating Bacteria” 56  Bacterial toxins Bacterial Toxins - Poisonous substances produced by microorganisms Toxins - primary factor of pathogenicity 220 known bacterial toxins 40% cause disease by damaging the Eukaryotic cell membrane Toxemia Toxins in the bloodstream 57  Toxins Toxigenesis: ability to produce toxins. Bacteria may produce two types of toxins: i. exotoxins and ii. endotoxins. Exotoxins are released from bacterial cells and may act at tissue sites remote from the site of bacterial growth. Endotoxins are cell-associated substance. (classic sense, endotoxin refers to the lipopolysaccharide component of the outer membrane of Gram-negative bacteria). 58  Toxins Endotoxins may be released from growing bacterial cells and cells that are lysed as a result of effective host defense (e.g. lysozyme) or the activities of certain antibiotics (e.g. penicillins and cephalosporins). Hence, bacterial toxins, both soluble and cell-associated, may be transported by blood and lymph and cause cytotoxic effects at tissue sites Some bacterial toxins may also act at the site of colonization and play a role in invasion. 59 60 61  Toxins Cytotoxins – kills cells Response to toxins Shigella If exposed to exotoxins: Vibrio antibodies against the toxin (antitoxins ) Neurotoxins –interfere with normal nerve impulses Exotoxins inactivated ( heat, Cl. tetani formalin or phenol) no longer cause disease, but stimulate Cl.botulinum the production of antitoxin Enterotoxins – affect cells lining Altered exotoxins - Toxoids to the gastrointestinal tract Toxoids - injected to stimulate E.coli the production of antitoxins Salmonella and provide immunity 62 63  Portals of entry of infection Ingestion (fecal-oral) Inhalation (respiratory) Trauma (e.g burn) Arthropod bite (zoonoses: mosquito, flea, tick, Tsetse fly) Sexual transmission Lathrogenic (needle stick, blood transfusion) Maternal-neonatal 64  Modes of infectious disease transmission Bacteria, fungi, virus… Ingestion: Salmonella, Shigella, Vibrio, Clostridium etc.. Inhalation: Mycobacterium, Mycoplasma, Chlamydia etc.. Trauma: Clostridium tetani Arthropod bite: Rickettsia, Yersinia pestis, etc. Sexual transmission: Neisseria gonorrhoeae, HIV, chlamydia, etc. Needle stick: Staphylococcus, HIV, HBV Maternal-neonatal: HIV, HBV, Neisseria 65 Modes of infectious disease transmission 66  Modes of infectious disease transmission Contact transmission Direct contact (person-to-person): syphilis, gonorrhearel herpes Indirect contact (formites): enterovirus infection, measles Droplet (less than 1 meter): whooping cough, strep throat Vehicle transmission Airborne: influenza, tuberculoses, chickenpox Water-borne (fecal-oral infection): cholera, diarrhea Food-borne: hepatitis, food poisoning, typhoid fever Vector transmission Biological vectors: malaria, plaque, yellow fever Mechanical vectors: E. coli diarrhea, salmonellosi 67  Extracellular versus Intracellular Parasitism Extracellular parasites - destroyed when phagocytosed. damaging tissues as they remain outside cells. inducing the production of opsonizing antibodies, they usually produce acute diseases of relatively short duration. Intracellular parasites can multiply within phagocytes. frequently cause chronic diseases. 68 Reading Your manual Chapters: 9. Pathogenesis of bacterial infection 10. Normal human microbiota 69 11. Normal Microbial Flora of the Human Body.................................... 197 Role of the Resident Flora 197 Normal Flora of the Skin 198 Normal Flora of the Mouth & Upper Respiratory Tract 198 Normal Flora of the Intestinal Tract 199 Normal Flora of the Urethra 200 Normal Flora of the Vagina 200 Normal Flora of the Conjuctiva 201 Review questions 70 71