607 Lecture Notes PDF

Lecture 2 Important concepts in Microbiology Application of microscopy in diagnostic microbiology - Rapid preliminary identification - Rapid final identification - Detection of different species - Detection of fastidious species Microbial growth Factors contributing to microbi...

Lecture 2 Important concepts in Microbiology Application of microscopy in diagnostic microbiology - Rapid preliminary identification - Rapid final identification - Detection of different species - Detection of fastidious species Microbial growth Factors contributing to microbial growth Factors: - Population size Enough # of bacteria will do things as a colony - Nutrient availability Other physical factors - Temperature usually at body temp. 35 – 37C - Oxygen - pH blood bacteria at pH 7, Gut – upper GI tract = acidic, lower GI tract = neutral - water - sugar Monosaccharide, polysaccharide, fructose, galactose, sucrose, glucose Temperature 37C - Pseudomonas aeruginosa - Bacillus subtilis - Staphylococcus aureus - Streptococcus pneumoniae - Enterococcus faecalis - Streptococcus pyogenes - Escherichia coli - Proteus mirabilis - Salmonella typhimurium - Pseudomonas aeruginosa – 5.5 - Bacillus subtilis – 5.5 - Staphylococcus aureus – 6.7 - Streptococcus pneumoniae – 7.8 - Enterococcus faecalis – 2.9 to 4.2 - Streptococcus pyogenes – 6.5 - Escherichia coli – 6.5 - Proteus mirabilis – 7.2 - Salmonella typhimurium – 3 to 4 E. faecalis & S. Typhimurium = stable at low pH (acidic condition) => allow to distinguish them from other species Water availability - Salt affects water activity (in the medium/substance) or osmotic pressure (to the cells) - Aw = 0.90 allows good microbial growth (ideal) - Fungi generally grow at Aw 0.70 - Sugar can also affect water activity or osmotic pressure. o The sugar that will distinguish two different species from each other + done in the lab = expected to know Microbial control - Sterilisation – Complete removal of ALL forms of life – i.e. Autoclave - Disinfection – kill harmful microbes (pathogens) - Sanitation – reduction of microbial count to a safe public health level Methods: Autoclave steam steriliser Autoclave 15 minutes per cycle Heat - Heat can kill microorganisms by denaturing their enzymes. For microbes to replicate, DNA should be retained - Heat resistance varies among microbes. Microbial cysts & spores are heat resistant. o *Microbial cyst: a resting or dormant stage of a microorganism - Thermal Death Time (TDT) o Minimal length of time for all bacteria in a particular liquid culture to be killed at a given temperature. UV - UV can inhibit the growth of microorganisms or kill microorganisms. - UV is a non-ionising radiation. - Causes mutations => cancer - Damages DNA by causing bonds to form between adjacent thymine bases, thus causing errors in replication. - Radiation is not very penetrating. - Organisms need to be directly exposed at a certain time period. Lecture 3 Human microflora + The concept of disease & infection Human Microflora ‘Normal Flora’ microorganisms that are frequently found in various body sites of a normal, healthy individual o Physiological state ▪ Antibiotics or medication designed to kill pathogens will block any of the normal flora ▪ Antifungal medication ▪ Suggestive ways? Probiotics – most of those on the market = aerobic probiotics => for the small intestine, upper digestive tract ∵ have much more oxygen in the small intestine Not useful in the lower digestive tract (large intestine) o Age ▪ Infant vs mature adult vs older people – distinct microbiomes ▪ This also changes as a baby grows ▪ Menopause can change the gut/vaginal microbiome morphological, physiological and genetic features allow them to: o Colonise and multiply at a particular site in the body ▪ Aerobes – small intestine/anaerobes – large intestine o Inhibit competing intruders inhibit pathogens from growing “colonisation resistance” ▪ Not much space or nutrients that pathogens could use ▪ Healthy flora can stimulate the immune system. The gut, and digestive tract = full of bacteria in a healthy person The nervous system, blood, and spinal fluid = do not want any bacteria Residents Normal microbial flora Transients Symbiotic microorganisms Commensal microorganisms Symbiotic: both getting something from each other Commensal: one species gains benefits while those of the other species neither benefit nor are harmed Parasitic: one getting from the other and harming them So where does the normal flora originate? - Amniotic fluid is sterile. - The initial seeding of the infant microbiome = from delivery o Vaginal delivery vs C-section – the initial setup of the gut microbiome is drastically different. AFTER birth, - Feeding method o Skin to skin vs bottle-fed o Breastmilk vs bottle in formula - Prematurity o Premature infants who are born before 36,37 weeks vs babies born with full-term Flora will be broadly similar to that of other individuals in the same age group and culture - Culture = diet, health status (chronic disease, autoimmune disease ) What factors determine the normal flora? Local physiological and environmental conditions Competition between microbes for nutrients o Important for: ▪ keeping out pathogens (colonisation resistance) Inhibition by microbial metabolites (e.g. volatile fatty acids, hydrogen peroxide) o Some microbial metabolites are toxic ▪ i.e. Clostridium difficile = hospital pathogen – causing severe diarrhoea that you can die from ▪ It comes in as a spore and won’t germinate if you have certain metabolites that are made from healthy gut ▪ Normal flora are also actively making things that prevent the growth of those pathogens Production of antibiotics What is the normal flora at different body sites? Staphylococcus epidermidis - Commensal at many sites, but not at some sites like blood, brain, and interstitial space – spaces between organs - Pathobiont = something that naturally normally lives there without causing any problem, but sometimes it can cause problems. o i.e. candida albcans – yeast. This can sometimes cause thrush in the throat. o Staphylococcus aureus on skin - Under normal conditions, optimal growth is due to normal flora. - Group B strep. is a problem during childbirth. o No need to swab if delivery is through C-section, only for vaginal delivery - In the human body, the colon or large intestine has the largest microbial population. It has been estimated that the number of microorganisms in stool specimens is about 1011 organisms per gram wet weight. - Bacteria can be grown on the dry surface of the skin. o The relatively dry surface of the skin is inhibitory to microbial growth. Moist regions of skin have higher numbers of normal flora. Pneumonia caused by s. pneumoniae / Intestine – E.coli = pathobiont / genital urinary tract = lactobacillus/ bladder = if something is present in the bladder, it means infection Gut microbiome – the more diverse it is, the healthier you are. Vaginal microbiome – not have a big diversity – Candida albicans – yeast, Gardnerella vaginalis - bacteria Skin, Vaginal, and gut microbiome What are the beneficial effects of the normal flora? Produce conditions that tent to block the establishment of exogenous pathogens Compromised defense systems also increase the risk of infection by normal flora - Vitamin deficiency - Diabetes, lymphoma and leukemia, chemotherapy autoimmune, metabolic diseases o Mess up the normal flora – a lot of GI issues – vomiting, diarrhoea Antibiotic therapy, especially with broad-spectrum antibiotics, can alter the normal flora Antibiotic-resistant microbes may proliferate and cause significant infections Notably in immune-compromised patients TB = pathogen – it can stay dormant in the lungs, so then at any point when you have an immune transplant or chemotherapy, they will check for the dormant TB because if you have a latent case and they suppress your immune system What is the role of the normal flora in disease? Many species of the normal flora are ‘opportunists’ o They will cause infection if they reach a site of the body in sufficient number Usually, no bacteria in the bladder Cardioendocarditis The concept of disease & infection Infection: pathogen inside the host Multiplication of the pathogen in the host Can result to little or no illness can be symptomatic/asymptomatic. Pathogen – causative microorganism Disease: Clinically apparent response or injury as a result of infection Manifestation of the infection into the symptoms With many communicable pathogens, infection is more common than disease o Infection without diseases is termed subclinical infection Don’t really feel sick = the most contagious o The host is referred to as a carrier o Asymptomatic carriers can be infectious to others designed to be infectious when people can move around What would be an exception where you wouldn’t need people to move around to spread the disease? Cholera – where the sheer volume of the bacteria comes out and goes into the water supply. Any kind of faecal-oral or contaminated food in water transmission so you don’t need a person or an animal to be moving around Types of infection Endogenous having an internal cause or origin – a small child wiping back to front => get a bladder infection Exogenous – having an external source/cause of origin i.e. cough. Nosocomial – hospital – acquired infections transmitted to patients by hospital personnel and other patients or arise from the patient’s own endogenous flora: SSI/VAP/MRSA Iatrogenic – resulting from the activity of a health care provider or institution: said of any adverse condition in a patient resulting from treatment by a physician, nurse, or allied health professional. May be an inescapable consequence, but also could be due to incompetence or carelessness. Opportunistic not always pathogenic – infection when a microorganism not commonly causing disease causes inflammation in immunocompromised or debilitated host Inflammation Initiation - damaged or invaded tissue Tissue response - chemical factors released (histamines, kinins, prostaglandins) that cause vasodilatation and increased permeability of blood vessels Leukocyte response - phagocytic cells engulf micro-organisms and damaged tissue resulting in blood clotting, pus and abscess formation o APCs => show them to adaptive immune system Resolution - tissue repair Cure - Return to healthy tissue Human defense mechanism Lines of defense: Skin and other barriers Innate immunity immediate immune response Adaptive immunity The immune system in action Aggressive mechanism of microbes Pathogenicity – ability to cause disease intrinsic to the pathogen Virulence – extent of pathogenicity how bad is the disease? Communicability – ability of microbes to spread Microbial toxins o Exotoxin – excreted extracellularly from intact bacterial cell (also produced on cell lysis) o Endotoxin – liberated only on cell lysis or death of bacterium ▪ Endotoxins are lipopolysaccharides in nature and are released from cell walls of lysed Gram- negative bacteria. ▪ LPS, flagella releases when bacteria dies => cause sepsis Reservoirs & sources of infection Can be endogenous Human Animals Food Water Soil Air Dust Fomites air falls on to the surface => transmission, so not many handles in the hospital Exogenous example: Malaria parasites = plasmodoim Route of transmission Can be: o Horizontal – direct or indirect person-to-person contact ▪ Including vectors o Vertical – from mother to fetus test amniotic fluid to check the transmission ▪ Right after the birth, test cord blood and placenta to check the transmission How to control and prevent infection? Control? Prevention? When? AFTER infection When? BEFORE infection Why? To minimize the spread Why? Keep people healthy How? Isolation How? Vaccination Medication Hygiene PPE/hygiene Cooking meat Epi/pH measures Wastewater treatment. diagnostics Prophylactics Vitamins & healthy diets Fx for coexisting diseases Good sleep + exercise Lecture 5 Human Pathogen 1 Staphylococcus species Brief history Robert Kock – Kock’s postulates: If two people have the same disease, you should find the same organism in those two people. If you isolate the organism and you give it to somebody else, it should cause the same disease. = Aligning cause with disease outcomes 1. The microorganism must be found in abundance in all organisms suffering from the disease but should not be found in healthy organisms. 2. The microorganism must be isolated from a diseased organism and grown in pure culture. 3. The cultured microorganism should cause disease when introduced into a healthy organism. 4. The microorganism must be re-isolated from the inoculated, diseased experiment host and identified as being identical to the original specific causative agent. Louis Pasteur Pasteurisation Alexander Ogston Taxonomy Do all these species cause human diseases? No Morphology & physiology Morphology Physiology Gram +ve cocci Non-motile Singly, pairs, tetrads, clusters (usually like this) Non-spore former Unencapsulated or have limited capsule Facultative anaerobe formation Ferments glucose anaerobically Teichoic acids in cell wall useful in diagnostics Produce enzyme catalase and coagulase only S. aureus form coagulase  Facultative anaerobe (could also be called facultative aerobe) Survive with or without oxygen Thrive with oxygen o Can ferment and oxidise glucose o Can use glucose no matter what Less oxygen down the tube o They don’t have a preference or a dominant one o BUT they congregate at the top o BECAUSE they make more ATP with oxygen than without oxygen Where are they found? Commensal microorganism Skin, skin glands, mucous membrane Mouth, intestine Genitourinary tract Upper respiratory tract Brain, Heart and Lungs, Digestive system (+ stomach), Respiratory system Skin and scalp – S. epidermidis Genitourinary – S. saprophyticus S. aureus can go anywhere – genitourinary, digestive system, Virulence The extent to which you cause a disease Pathogenicity Surface factors o Capsule ▪ Not on every Staph. o Surface protein A o Peptidoglycan & teichoic acids ▪ Structure of gram +ve cell wall Extracellular enzymes Things that go outside of the cell, secreted o Coagulase ▪ Destroys the coagulation factor in blood = preventing the clots ▪ S. aureus – it won’t tell if it’s pathogenic or not ▪ A toxin is what makes it highly virulent strain o Lipase ▪ Break down lipids/fats o Staphylokinase/fibrinolysin ▪ Break down fibrin = preventing the clots o Hyaluronidase ▪ Break down Hyaluronic acid to use it as a nutrient source o Nuclease ▪ Break down nucleic acids (DNA, RNA) Something that is not normally, particularly virulent but be in an immunocompromised person = opportunistic or pathobiont  Haemolysins o Alpha Green o Beta Clear o Gamma Alpha – Green - Break the blood down but don’t use iron from it Beta – Clear - Break the blood down and use iron from it Gamma – non-haemolytic Toxins Exotoxin – something that the bacteria makes and then secretes outside the cell Endotoxin – innate within the cell i.e. peptidoglycan or the capsule – can be toxic to the host, but they are not being secreted with the intention of being toxic The only problem is when the bacteria start to lyse, be degraded by the immune system, or die off Leucocidin Killing white blood cells – More associated with S.aureus o Exotoxin o Degradation of cytoplasm o Lysis of human polymorphonuclear cells (PMN) = neutrophils Exfoliants/Epidermolytic toxins Killing skin cells – More associated with S. epidermidis o ETA & ETB o Cause lysis of intercellular attachment between cells Might have exfoliants or Epidermolytic toxins in S. aureus that is infecting the skin BUT if it’s one that’s in the GI tract causing toxic shock, the problem is going to be more associated with toxic shock syndrome toxins or different exotoxins that some Staph. can have. Enterotoxins o Increase intestinal peristalsis Toxic shock syndrome toxin 1 o TSST-2 o Capillary leakage o Rash, high fever shock type o Can be life-threatening, fatal very quickly Case: Super max tampons where you wouldn’t have to change the tampons for two days o People started dying of shock ▪ It was BECAUSE it was a nice environment for S.aureus to grow => toxic shock ▪ Shock induced by exotoxins rather than induced by endotoxins (more like sepsis) S. epidermidis S. saprophyticus Adhesin so that it can stick onto the cell ESS-like substance Not ESS Extracellular slime substance (ESS) allow It to Urease use urea so happy in bladder make a biofilm Surface-associated protein (SSP) help it to Haemolysin break down haeme adhere Lipase Haemagglutinin break down haeme Protease Skin Bladder (don’t want it to go up to kidney) Staphylococcus aureus ~ “Golden cluster of grapes” Coccal Grows in clusters Gram +ve Facultative anaerobes Non-motile & No spores Catalase + (same for S. epidermidis & S. saprophyticus) Coagulase + (shown as “clumping up”) o Fibrinogen -> Fibrin A part of normal skin flora o BUT if more and more s.aureus is around on the skin, it starts to penetrate through tiny micro- fissures in the skin like you get with eczema 습진 as well as larger breaks in the skin like you might get after shaving o Troublesome in terms of causing wound infections o Low levels + intact skin = colonisation o High levels + breaks in skin = infection Infections S. aureus invading into the skin => localised skin infection = pimple => boil/furuncles => carbuncle Diffuse skin infection = superficial impetigo 농가진 (= infection of the epidermis) or Cellulitis (= infection of the dermis) – can spread over larger surfaces rapidly If the infection goes deeper, it develops into a subcutaneous abscess – a collection of pus that’s walled off and sometimes develops thin walls within it – called septations These abscesses can occur all over the body, including: Mouth – dental abscess, kidney, liver, spleen, brain Muscle – pyomyositis, bone – osteomyelitis, joint - septic arthritis In the bloodstream – septic thrombophlebitis (infected blood clot), Bacteremia = bacteria in the blood => widespread immune reaction => blood pressure ↓ => Hypotension & poor perfusion to various organs = “sepsis” S. aureus in the blood can get to various parts of the body Central nervous system => Bacterial meningitis/epidural abscess in the spine Lung => severe pneumonia Heart – grows on the heart valve in clumps “vegetation” => damaging the valves = infective endocarditis from surgery or dental work (infrequent) High-risk individual (immunocompromised or at risk for infective endocarditis) Treatment: Antibiotic prophylaxis Biofilm – a layer of “slime” within which the S.aureus live What? S. aureus has the ability to create biofilm on medical implants like indwelling intravenous catheters, prosthetic heart valves, and artificial joints. Where? It forms when a cluster of S. aureus adheres to a surface, either a natural one like the surface of a valve or an artificial one like the surface of a catheter. How? S. aureus starts to produce an extracellular matrix made of Exopolysaccharides (EPS) => surrounds the cells completely The cells that are surrounded by the gel-like layer of EPS can communicate with one another through biochemical signals and can even swap genetic information, including antibiotic resistance genes. S. aureus THRIVES but doesn’t divide RAPIDLY within the biofilms => Hard for antibiotics to penetrate => harder to get rid of biofilm infection=> often requires removal of growing surface Why? Antibiotics target: cell wall – The bacteria will only care if they are ACTIVELY making cell wall cell wall DNA replication, gene translation & transcription – this will only work when the bacteria are rapidly growing and dividing. Superantigens (toxins) 5 major toxins of S.aureus: 1. Toxic Shock Syndrome Toxin-1 (TSST-1) - Binds to MHC II receptors on antigen-presenting cells, stimulating excessive cytokine release (cytokine storm). - Effects: Causes fever, sunburn-like rash, low blood pressure, poor end-organ perfusion, potentially leading to death (toxic shock syndrome). 2. Panton-Valentine Leukocidin (PVL) - Creates pores in leukocytes, leading to cell necrosis and inflammation. - Effects: Can cause severe tissue damage, such as in necrotising pneumonia, leading to organ failure. 3. Haemolysin - Forms pores in erythrocytes (RBCs), releasing hemoglobin and iron. - Effects: Provides iron for bacterial metabolism. 4. Exfoliatin - Causes Staphylococcal Scalded Skin Syndrome (SSSS). - Effects: Leads to red, painful skin patches with fluid-filled blisters, usually resolving within weeks. 5. Enterotoxin - Stable in the environment, can survive cooking, causes food poisoning. - Effects: Results in vomiting and diarrhea within hours of ingestion; can cause toxic shock syndrome if it enters the bloodstream. Antibiotic Resistance in Staphylococcus aureus 1.Mechanisms of Resistance - Beta-lactamase Production: Enzyme that breaks down beta-lactam antibiotics. - mecA Gene: Encodes altered PBPs (penicillin-binding proteins) that beta-lactam antibiotics cannot bind to. 2. Types of Resistant Strains - Methicillin-Resistant Staphylococcus aureus (MRSA) - HA-MRSA: Healthcare-associated, found in hospitals and nursing homes. - CA-MRSA: Community-associated, linked to overuse of antibiotics in agriculture and general overprescription.  - Vancomycin-Intermediate Staphylococcus aureus (VISA) - Intermediate resistance to vancomycin. - Vancomycin-Resistant Staphylococcus aureus (VRSA) - Complete resistance to vancomycin. Treatment Strategies 1. Testing and Selection - Antibiogram: Used to determine resistance patterns and guide antibiotic selection. - Alternative Antibiotics for MRSA: Clindamycin, vancomycin, tetracyclines, trimethoprim-sulfamethoxazole, linezolid, tigecycline, daptomycin, quinupristin-dalfopristin. 2. Challenges - Rapid Resistance Development: Ongoing issue due to overuse of antibiotics. - Side Effects: Newer antibiotics like vancomycin come with problematic side effects. Identification in samples - Colony morphology S. aureus S. epidermidis S. saprophyticus - Usually large, smooth, - Relatively small, sometimes, - Large, entire, very glossy, entire, slightly raised, beta- adherent to medium, smooth, butyrous, more haemolytic usually non-haemolytic convex than colonies of - White to orange gold (gamma) other staphylococci, usually non-haemolytic (gamma) - White to yellow-orange The main difference is: Coagulase, haemolysis - Gram staining - Acid production - DNase test - Direct examination First thing when you get a sample = gram staining If the sample is gram-positive, what shape is it? Coccus Is it Staphylococcus or Streptococcus? The difference between them = the Catalase test Catalase +ve = Staphylococcus, Catalase -ve = Streptococcus Streptococcus: How to identify S. aureus? - Blood agar, beta haemolytic (clear halo around the colonies) - Coagulase test, S. aureus is coagulase +ve = make the latex agglutination clump up, forming the clots o Fibrinogen -> Fibrin Brief history Theodor Billroth Austrian surgeon, first described Streptococcus as a chain-forming coccus in 1877 Louis Pasteur isolated Streptococcus from saliva – pneumococcus 1881 Friedrich Rosenbach German scientist, described and differentiated species of Streptococcus; identified and named Streptococcus pyogenes 1884 Rebecca Lancefield, American microbiologist, serological classification of beta-haemolytic streptococci bacteria – Lancefield grouping 1933 Streptococcus species Enterococcus (= Streptococci Group D) – according to the Lancefield classification - Unencapsulated - Produces gas during fermentation of sugars - Typically non-haemolytic Gamma - Enterococcus faecalis - Enterococcus faecium How does the Lancefield grouping work? Look at the reaction in the sera of a person to the sugar on the outside of Streptococci. So, it has to have sugar on the outside for it to react. Some don’t have carbohydrate groups on the outside = not part of the classification = non- groups Streptococcus pneumoniae. Taxonomy of Streptococcus spp. Identifying the taxonomy: - DNA-DNA hybridisation - DNA-rRNA hybridization - 16S rRNA sequence - Other genetic techniques - Human clinical specimens Morphology & physiology Gram-positive Capsulated Non-motile - generally Spherical; ovoid to - True for some species lancet-shaped, cocci - Made of polysaccharide - Singly; in pairs or chain rather than clusters - Non-spore former - Facultative anaerobe - Homo fermentative o Glucose fermentation = lactic acid, with no gas formation - Catalase & oxidase negative Cell wall has: - Peptidoglycan - Carbohydrates tells what the Lancefield grouping is - Lipoproteins = fats + proteins - Surface proteins allow for inhibition Lancefield Grouping GAS: Flash-eating bacteria C is more for animals C, G, and F – do not cause as many issues in people, but they can cause them still. During vaginal delivery, you can get passage of GBS to the baby => leads to neonatal sepsis Enterococcus Not seen as much Streptococcus grouping Natural habitat Part of the human microflora Skin and mucous membranes Respiratory tract Gastrointestinal tract Genital tract Virulence Factors - Capsule - Surface protein - Haemolysins o 2 types: ▪ Streptolysin O (SLO) ▪ Streptolysin S (SLS) - Toxins - enzymes Group A Streptococci (GAS) Bacitracin sensitivity – for the diagnostic Bacitracin = A disc Bacitracin sensitive Definitive = obvious; diagnosis is done from the culture of the affected site Things to know: You treat streptococcus and Staphylococcus with penicillin – these are peptidoglycan binding protein inhibitors = Penicillin works best on gram-positive bacteria by inhibiting peptidoglycan production.  Moving on to alternatives when they are resistant. o All staphylococci are now resistant.  So moving on to a derivative of penicillin o Methicillin for Staphylococcus o Cephalosporins and macrolides for Streptococcus Methicillin-resistant Staph. aureus = MRSA Staph. aureus strains with intermediate resistance to vancomycin = vancomycin-intermediate staph aureus (VISA) The strains with complete resistance = vancomycin-resistant staph aureus (VRSA). Group B streptococci (GBS): Can get passage to the baby during the vaginal delivery Right before the vaginal delivery = GBS test Normal GBS colonisation = nothing wrong, but a problem during delivery => so get intrapartum (during childbirth) prophylactic antibiotic S. pyogenes & S. agalactiae differentiation: streaking on the blood agar & put A DISC Result: Both show beta haemolysis, BUT S. agalactiae = resistant, S. pyogenes = sensitive Treatment: Penicillin or penicillin derivative ==resistant==> Cefazolin & vancomycin Zone of inhibition shrink = resistant, Zone of inhibition remains = sensitive Non-grouped Optochin sensitive, Optochin = P disc  Rhinosinusitis = infection in nose and sinus Otitis = eye Pneumonia = lung Meningitis = CSF Treatment: Penicillin => Fluoroquinolones => Cephalosporins or Vancomycin Group D streptococci (GDS) Survive in more extreme conditions: High salt High pH High temperature Why? They have to survive in the gut – survive low pH in stomach and different pH in gut Bloodstream infection with bacteria, leading to sepsis = bacteraemia. Usually, Infections from abdominal surgery. Diagnosis/Treatment: Vancomycin-resistant enterococcus (VRE) = Linezolid, daptomycin or tigecycline Identification in samples - Colony morphology S. pyogenes S. agalactiae S. pneumoniae - 0.5 mm in diameter, - 1-2 mm in diameter, slightly - 1 mm in diameter, domed, smooth, entire, domed, mucoid, beta-haemolytic circular, glistening (young beta-haemolytic. (less obvious zone of colonies) - Translucent or transparent haemolysis) - Umbilicate; larger; more with smooth or matte - Yellow, orange or red mucoid (older colonies of surface pigment (anaerobic on capsulated); small and specific media) rough surface (unencapsulated strains) - Alpha haemolysis - Gram staining – direct examination - Streptococci Lancefield grouping - Salt tolerance; Bile-aesculin - CAMP Lecture 6 Human Pathogen Part 3 Three different types of bacteria: Primary pathogens (capable of causing disease) – Salmonella, Shigella, Yersinia, Klebsiella pneumoniae, Escherichia coli – Most of the species within the genus are considered pathogens, for some it’s just 1 or 2. Opportunistic Pathogens – under particular conditions Non-pathogenic General characteristics - Gram -ve rods with no specific arrangement - Non-spore forming - Aerobic and facultative anaerobic - Motile by peritrichous flagella or non-motile - Capsulated with ill-defined slime later or non-capsulated - Fimbriae or pili present is most species - Lactose fermenter - Catalase +ve - Reduce nitrate to nitrite - Grow on peptone or meat extract media without the addition of sodium chloride or other supplements. Antigenic structure O antigens – in the polysaccharide of cell wall (LPS) H antigens – flagellar protein Type 1 pilli – the most common one CFA, BFP, P- pili K antigen – polysaccharide capsule (only for some species) Not all have capsules Vi antigen – virulence ▪ Salmonella species only Should know this table: different diagnostic antigens whether or not they have pili, capsule, exotoxins, various pathogenic regions O, H, K – the most common ones Escherichia coli >150 serotypes All typed by O and H Example: O157:H7 (EHEC) Shigella – just looking at O for Serogroups 4 different species – S. dysenteriae, S.flexneri, S. boydi, S. sonnei Each have different serogroups: A – 10 types, B – 6 types, C – 15 types, D – just 1. Salmonella >2000 serotypes Typhoidal vs non-typhoidal are the main ones to distinguish between + Yersinia - Pseudotuberculosis – cause diseases similar to tuberculosis - Enteracalitica – diarrheal disease Epidemiology of Enterobacteriaceae Molecular pathogenesis of Enterobacteriaceae Fecal-oral spread – contaminated food goes into a person (Salmonella with typhi serotype & Shigella) or from an animal (Salmonella & E.coli) can come into a person Through Gastrointestinal tract Once it gets into the colon (generally) => different types of problems => get into the bloodstream (UTI), urinary tract, fever. Stays in the colon => causing diarrhea Escherichia - E.albertii - E.coli - E.fergusonii - E. hermannii - E. vulneris Escherichia coli - Gram -ve bacilli - Motile; facultative anaerobe Grow in both aerobic and anaerobic - this is how they cause diseases. Transmission: Fecal-oral transmission in general – contaminated stools => into water, food, unwashed hands => into the person orally => gets to intestines and stomach => cause illness It has to get out the lumen and into the cells in order to cause the symptoms. Shiga toxin +ve symptoms: bloody stools/diarrhea and fever, causing the bad excretion of the pathogen to come back out and restart the transmission cycle.  This is proteobacteria but not Enterobacteriaceae – the symptoms that it causes are very similar to Enterobacteriaceae. Campylobacter jejuni – jejunum is a part of the GI tract – where the name comes from Campylobacter spp. - Gram -ve - Slender, curved, Long spiral-shaped rods, maybe coccoid in really old culture (a bit more round) - Motile (single polar unsheathed flagellum at one or both ends) - Non-spore former - Microaerophilic & capnophilic Different to Enterobacteriaceae o Have to grow them at reduced oxygen and increased CO2 o Fastidious - Temperature – grows at 42°C – adapted to chickens with higher body temp. - Unable to use sugars either oxidatively or fermentatively; derive energy from amino acids; Not grow on MAC or NA; Should grow them on chocolate agar. - Virulence factors o Motility Allow them to burrow(dig) down through the mucus layer o Heat-labile enterotoxin = heat sensitive Transmission: Intestinal colonisation in Chicken – does not cause any problems to chickens. Not cooked enough, contaminated => eat => infection OR Contamination of faeces from chickens => water, other animals, particularly into milk => unpasteurised milk that’s contaminated with campylobacter Campylobacter spp. Comma-shaped (curved) - Common Cause: One of the most common causes of bacterial gastroenteritis worldwide. - Common Sources: Found in foods like poultry and unpasteurised milk. Anatomy and Physiology: - Cell Wall: Thin peptidoglycan cell wall; appears pink/red when gram-stained (gram-negative bacteria). - Motility: Has a flagellum at one end, making it motile. - Oxidase Test: Oxidase positive (can use oxygen to create ATP). - Growth Conditions: - Microaerophile (thrives in low oxygen environments). - Optimal temperature: 42°C. - Grows on blood agar varieties like Skirrow, butzler, and Campy-BAP. Chocolate agar (fastidious) Transmission: - Route: Fecal-oral transmission. - Common Carriers: - Birds (particularly in the gastrointestinal tract of poultry). - Cows (risk from unpasteurised milk). - Infected pets (notably puppies). Pathogenesis and Virulence Factors: - Attachment: Uses fimbri-like filaments and cell surface proteins like PEV-1 and CADF to attach to the mucosa of the small intestine and colon. - Invasion: - Drills into mucosa using its spiral shape and long flagella. - Releases cytotoxins like cytolethal distending toxins (CDT), causing cell damage and inflammation. - Complications: - Toxic Megacolon: Extensive inflammation can lead to colon dilation. - Bacteremia: Bacteria can enter the bloodstream. - Guillain-Barré Syndrome: The immune system produces antibodies that attack peripheral nerves, causing paralysis. - Reactive Arthritis: Autoimmune reaction causing joint inflammation. Symptoms: - Incubation Period: 1-7 days. - Initial Symptoms: Fever, muscle pain, malaise, headache. - Gastrointestinal Symptoms: - Crampy abdominal pain. - Watery diarrhea initially, which may turn bloody. - Bloody diarrhea is more common in children. - Complication Symptoms: - Toxic Megacolon: Bloating, tachycardia, loss of bowel sounds. - Guillain-Barré Syndrome: Ascending paralysis (starting at the feet and moving upwards). - Reactive Arthritis: Pain in large joints. Diagnosis: - Clinical Manifestations: Based on symptoms. - Stool Sample Examination: - Gram-negative, comma-shaped bacteria.  - Presence of white and red blood cells. - Cultures: Isolating the bacteria on specific media. Treatment: - General Treatment: Hydration and correcting electrolyte imbalances. - Severe Infection: Antibiotics like erythromycin. Distinguishing from Other Bacteria: - Gram Staining: Both Campylobacter and Enterobacteriaceae are gram-negative. - Microscopy: Campylobacter is comma-shaped, while Enterobacteriaceae are rod-shaped. - Motility: Campylobacter is motile (distinguishing it from non-motile Shigella). - Incubation Conditions: - Campylobacter grows in low oxygen and warmer temperatures. - Enterobacteriaceae do not have such specific growth requirements. - Growth Requirements: - Campylobacter requires specialised media (e.g., chocolate agar). - Enterobacteriaceae grow on a wider range of media. - Oxidase Test: - Campylobacter is oxidase positive. - Enterobacteriaceae are oxidase negative. Shigella spp. General Information: - Family: Enterobacteriaceae - Species: - Shigella dysenteriae - Shigella flexneri - Shigella boydii - Shigella sonnei - All species can cause shigellosis, a contagious intestinal infection, which can progress to dysentery. Anatomy and Physiology: - Gram-negative (appears red or pink). - Rod-shaped. - Facultative anaerobe (can survive with or without oxygen). - Motility: Non-motile (lacks flagella). - Non-spore former - Non-lactose fermenter. - Urease and oxidase negative. - H2S negative Identification: - Culture Medium: Grows on selective media like MacConkey agar. - Forms white, non-lactose fermenting, non-hydrogen sulfide-producing colonies. - Microscopy: Stool stains with methylene blue may show polymorphonuclear leukocytes. - PCR Testing: Direct identification of Shigella DNA. Pathogenesis: 1. Ingestion and Initial Infection: - Shigella is ingested and multiplies in the small intestine before passing into the colon. - Targets epithelial cells of the mucosal lining, particularly M-cells. 2. Invasion and Immune Response: - M-cells phagocytose Shigella and release it into mucosa-associated lymphoid tissues (MALT). - Macrophages in MALT gobble up Shigella, but Shigella induces apoptosis in macrophages. - Release of cytokines (e.g., interleukin-1-beta) triggers intense inflammation, attracting more immune cells, damaging epithelial cells, and causing ulcerations and abscesses. 3. Spread within the Host: - Disrupts cellular junctions, allowing Shigella to enter enterocytes. - Uses type III secretory system to inject proteins into enterocytes, inducing phagocytosis. - Inside enterocytes, Shigella escapes the phagosome into the cytoplasm, uses actin-based motility to move and spread infection. 4. Shiga Toxin (specific to Shigella dysenteriae serotype 1): - An AB toxin (two subunits: A and B). - B subunit binds to the host cell membrane; toxin engulfed into the phagosome. - Acidic environment in phagosome breaks disulfide bond, separating subunits. - A subunit diffuses into the cytoplasm, and cleaves 28S rRNA in ribosomes, halting protein synthesis and causing cell death.  - Can reach the bloodstream and cause hemolytic uremic syndrome (HUS). Symptoms: - Symptoms last about seven days. - infected person can be a carrier for infection up to 4 weeks after the onset of symptoms. - Typical Symptoms: - Severe abdominal and rectal cramping and pain. - High fever, vomiting, loss of appetite. - Watery, mucus-like diarrhea, potentially containing blood or pus. Bloody diarrhea - Complications: - Hemolytic Uremic Syndrome (HUS): Hemolytic anemia, kidney failure, thrombocytopenia. - Tonic-Clonic Seizures: Particularly in children. - Sepsis: Mostly in neonates, malnourished children, and people with S. dysenteriae serotype 1 infection. - Reactive Arthritis: Pain and swelling in knee joints. Transmission: - Fecal-oral transmission. - Sources: - Contaminated water or food. - Flies carrying stool particles. - Contact with contaminated objects or hands. - Sexual contact (occasionally). - Virulence: Extremely virulent; as few as 10 bacteria can cause an infection. - Survival: Can survive the acidic environment of the stomach. Diagnosis: - Fecal Sample: Cultured on selective media like MacConkey agar. - Stool Stains: Methylene blue may show polymorphonuclear leukocytes. - PCR Testing: For direct identification of Shigella DNA. Treatment: - General Treatment: Fluid and electrolyte replenishment. - Antibiotics: Beta-lactams (penicillins and cephalosporins), macrolides, quinolones. Salmonella typhoidal (Salmonella typhi) General Information - Family: Enterobacteriaceae - Species: Salmonella bongori and Salmonella enterica (with 6 subspecies) - Encapsulated gram-negative rod bacteria (appear red or pink on gram stain) - Characteristics: - Facultative intracellular pathogens - Motile with flagella - Non-spore former - Facultative anaerobes (can undergo respiratory and fermentative metabolism) - Can ferment glucose but not lactose - Oxidase negative - H2S positive - Specific to Humans: Only infects humans and causes enteric fever (typhoid fever) Pathogenesis - Ingestion and Targeting: - Reaches distal ileum of the small intestine - Targets epithelial layer of the mucosal lining - Adheres to and is phagocytosed by M cells - Spit into underlying Peyer's patches - Immune Evasion: - Vi capsular polysaccharide antigen protects against antibody tagging - Suppresses neutrophil recruitment - Induces recruitment of monocytes and macrophages - Can cause hypertrophy, necrosis, and potentially ileal perforation - Survival and Replication: - Phagocytosed by macrophages, forming a salmonella-containing vacuole - Type III secretion system injects effector proteins, preventing vacuole-lysosome fusion - Survives and replicates within the vacuole - Systemic Spread: - Uses macrophages to enter lymphatic vessels and systemic lymphatic circulation - Reaches reticuloendothelial tissues (liver, spleen, bone marrow, gallbladder) - Induces macrophage apoptosis, causing bacteremia - Can lead to sepsis - Complications: - Osteomyelitis in individuals with spleen issues - Chronic infection and carrier state (gallbladder reservoir) Transmission - Routes: Fecal-oral, poor sanitation, poor hygiene, contaminated water/food, particularly endemic to Asia, Africa, Latin America, and the Caribbean - Inoculum: Requires about 100,000 microorganisms to cause infection Symptoms - Onset: 1-2 weeks after infection - Duration: 4-6 weeks - Typical Symptoms: - High sustained fever - Abdominal pain - Constipation followed by diarrhea - Rose or salmon-coloured spots on chest and abdomen - Hepatomegaly and splenomegaly - Dehydration, weakness, headaches, and cloudy mental state in advanced cases Diagnosis - Methods: Blood culture, stool culture, intestinal secretions (vomit or duodenal aspirate), and gallbladder bile culture for carriers Treatment - Symptomatic Management: Fluid and electrolyte replenishment, NSAIDs for pain and fever - Antibiotics: Broad-spectrum antibiotics like fluoroquinolones or cephalosporins (ceftriaxone) - Carriers: Combination of antibiotics and gallbladder removal Prevention - Vaccines: Oral live attenuated vaccine and intramuscular vaccine containing VI capsular polysaccharide - Hygiene: Proper sanitation and hygiene practices Non-typhoidal Salmonella General Characteristics - Serotypes: Over 2,500 serotypes within subspecies enterica - Common Serotype: Salmonella enteritidis Pathogenesis - Ingestion and Targeting: - Reaches the distal ileum of the small intestine - Targets epithelial layer of the mucosal lining - Adheres to and is phagocytosed by M cells - Spit into underlying Peyer's patches - Immune Response: - Strong pro-inflammatory cytokine response - Recruitment of immune cells (especially neutrophils) - Causes enterocolitis (inflammation of the small intestine and colon) - Can lead to mucosal damage and ulcers - Potential Spread: - Usually limited to the mucosa and destroyed by local immune cells - In some cases, can become invasive and cause bacteremia Transmission - Reservoirs: Infected humans and animals (birds, reptiles, mammals, amphibians) - Routes: Fecal-oral, foodborne (contaminated raw/undercooked animal products, water, fruits, vegetables, peanut butter), direct contact with infected animals Symptoms - Onset: 24-48 hours after infection - Typical Symptoms: - Watery diarrhea (sometimes bloody) - Abdominal cramps - Nausea and vomiting - Headache, chills, low-grade fever - Dehydration Diagnosis - Methods: Stool culture on selective media, additional cultures for invasive infections Treatment - Symptomatic Management: Fluid and electrolyte replenishment, suppressing nausea, alleviating pain - Antibiotics: Typically not indicated for uncomplicated infections; used for invasive infections based on sensitivity of the strain Escherichia coli Characteristics: - Gram-negative: Thin peptidoglycan layer, stains pink with safranin dye. - Rod-shaped bacteria. - Catalase-positive: Produces catalase enzyme. - Lactose fermenter: Produces beta-galactosidase and cleaves lactose into glucose and galactose. Tests and Cultivation - Catalase Test: Adding hydrogen peroxide to a colony; the presence of catalase results in foaming. - Lactose Fermentation Test: Cultivation on lactose-containing media (e.g., phenol lactose), resulting in acid production and colour change from red to yellow. - Eosin Methylene Blue (EMB) Agar: Grows into black colonies with a greenish-black metallic sheen. Physical Properties - Encapsulation: Covered by a polysaccharide layer called a capsule. - Motility: Has flagella for movement. - Facultative Anaerobe: Can live with or without oxygen. Pathogenic Strains - Attachment: Uses fimbriae to attach to host cells. - Serotype Classification: Based on antigens: - Somatic (O) antigens: On the cell membrane. - Capsular (K) antigens: On the capsule. - Fimbrial (F) antigens: On the fimbriae. - Flagellar (H) antigens: On the flagella. Serotypes and Diseases - Examples: - K1 antigen: Causes neonatal meningitis. - O157:H7: Associated with hemorrhagic colitis, hemolytic uremic syndrome, and diarrheal outbreaks. - Numerous Serotypes: Up to 200 serotypes. Pathotype Classification - Pathotypes: Based on the disease-causing mechanism and virulence factors: 1. Shiga-like toxin-producing E. coli (STEC) - Produces Shiga toxin, causes bloody diarrhea, and can lead to hemolytic uremic syndrome (HUS). 2. Enterotoxigenic E. coli (ETEC) - Produces heat-labile and heat-stable enterotoxins, causing watery diarrhea. 3. Enteroinvasive E. coli (EIEC) - Invades and destroys intestinal epithelial cells, causing bloody diarrhea. 4. Enteropathogenic E. coli (EPEC) - Causes disease in children under two years old, leading to watery diarrhea. 5. Uropathogenic E. coli (UPEC) - Causes urinary tract infections (UTIs), including cystitis and pyelonephritis. Pathogenesis of Each Pathotype 1. Shiga-like Toxin-Producing E. coli (STEC) Attachment: STEC attaches to the host's intestinal cells using fimbriae. Toxin Production: Produces Shiga-like toxins (Stx1 and Stx2).  o Intestinal Injury: Toxins cause damage to the intestinal epithelium and blood vessels, resulting in inflammation. o Bloody Diarrhea: Inflammation and vessel damage lead to fluid and blood leaking into the intestinal lumen. Hemolytic Uremic Syndrome (HUS): o Systemic Toxin Release: Toxins enter the bloodstream, affecting the kidneys. o Kidney Damage: Toxins bind to endothelial cells in the glomeruli, causing apoptosis and gaps in the capillary walls, leading to proteinuria. o Inflammatory Response: Cytokines and chemokines are released, activating platelets and causing clot formation. o Thrombocytopenia: Decreased platelet count due to clot formation. o Hemolytic Anemia: Red blood cells are fragmented (schistocytes) as they pass through obstructed microvessels. o Ischemic Kidney Damage: Clots obstruct arterioles, leading to kidney ischemia, reduced filtration, and uremia. 2. Enterotoxigenic E. coli (ETEC) Attachment: ETEC uses fimbriae to adhere to the intestinal mucosa. Toxin Production: o Heat-labile Enterotoxin (LT): ▪ Mechanism: Activates adenylate cyclase, increasing cAMP, leading to chloride and water secretion. o Heat-stable Enterotoxin (ST): ▪ Mechanism: Activates guanylate cyclase, increasing cGMP, leading to electrolyte and water secretion. Outcome: Causes watery diarrhea without damaging the intestinal wall. 3. Enteroinvasive E. coli (EIEC) Attachment and Invasion: EIEC attaches to and invades intestinal epithelial cells. Intracellular Multiplication: o Invasion: Invades and multiplies within epithelial cells, causing cell destruction. Inflammatory Response: Triggers a strong inflammatory response, leading to widespread epithelial damage and bloody diarrhea. 4. Enteropathogenic E. coli (EPEC) Attachment: EPEC uses bundle-forming pili (BFP) to attach to intestinal epithelial cells. Effacement: o Mechanism: Injects effector proteins via a Type III secretion system, causing actin cytoskeleton rearrangement. o Effect: Leads to pedestal formation and microvilli effacement. Outcome: Impairs absorption, causing watery diarrhea, primarily in children under two years of age. 5. Uropathogenic E. coli (UPEC) Colonization: UPEC can colonize the periurethral area and ascend the urinary tract. Adhesion and Invasion: o Fimbriae: Uses type 1 fimbriae and P fimbriae to adhere to uroepithelial cells. o Invasion: Invades and replicates within bladder cells. Toxin Production: o Hemolysins: Produces alpha and beta hemolysins, causing lysis of urinary tract cells. Ascending Infection: o Cystitis: Infection of the bladder, causing dysuria and frequent urination. o Pyelonephritis: Can ascend to the kidneys, causing flank pain and more severe symptoms. Symptoms and Complications - General Symptoms: Diarrhea, abdominal cramps, vomiting. - STEC: Bloody diarrhea, low-grade fever, potential HUS with symptoms like body swelling, confusion, and jaundice. - ETEC: Watery diarrhea, possible fever, and bloating. - EIEC: Bloody diarrhea and chills. - EPEC: Severe dehydration in children, prolonged diarrhea leading to malnutrition. - UPEC: UTI symptoms like dysuria, urinary frequency, and possible flank pain if kidneys are affected. Diagnosis - General Diagnosis: - Gram Staining: On stool or urine sample. - Culture: On eosin methylene blue agar. - HUS Diagnosis: Identifying Shiga toxin in the blood. Treatment - Diarrhea: - Hydration and rest. - Antibiotics in severe cases (e.g., doxycycline, cotrimoxazole). - Hemolytic Uremic Syndrome (HUS): - Supportive care: Dialysis, corticosteroids, blood transfusions, plasmapheresis. - UTIs: - Antibiotics: Cotrimoxazole, nitrofurantoin, fluoroquinolones (e.g., ciprofloxacin). Lecture 7 Human Pathogen Part 4 Pseudomonas spp. Acinetobacter spp. Proteus spp. Chryseobacterium spp. NEED to know: taxonomy - the most important in terms of understanding who’s related to whom, General characteristics, Clinical importance important to know what the clinical manifestations are of those common bacteria i.e. Enterobacteriaceae as one group and within that, there’s different tests to narrow things down Pseudomonas aeruginosa – important medically Pseudomonas fluorescens – counter to P. aeruginosa Pseudomonas taxonomy: Human-associated species of clinical importance Focus on Pseudomonas aeruginosa Pseudomonas General characteristics: - Gram -ve rods - Motile by 1 or more polar flagella - Non-spore former - Strictly aerobic - Oxidase +ve - Catalase +ve Pseudomonas aeruginosa Distinct colony morphology - spreading and flat with serrated edges No smooth edge - often shows metallic sheen, Depending on the media - bluish-green, red, or brown Yellowy green on NA (dramatic and noticeable) - often β-haemolytic Not best test to identify Where can we find them? Everywhere, can’t get away from them entirely Virulence factors: This is how Pseudomonas causes infection. - Glycocalyx – which makes Mucoid substance o Good for Adhesion; biofilm formation – this in particular is very important for Pseudomonas - Pili - surface protrusions o Aid adhesion to the cell surface receptor on the host epithelial cell typically in the lungs - Neuraminidase enzymes important for binding o Facilitates pili binding - Endotoxin just part of the bacteria – but foreign substances to humans, causing immune system reacts o Lipopolysaccharides LPS – part of the cell wall o Causes symptoms of sepsis/fever/inflammation ▪ Enough of them bloodstream => shock, fever, o LPS Low toxicity - Exotoxin A – secreted toxins => lysis of the host cell, inhibit protein synthesis of the host cells o Specific exotoxins for Pseudomonas aeruginosa o Induce autolysis lysis of the host cells o Inhibits protein synthesis - Enterotoxin - Exoenzyme S o One of the enzymes that inhibits protein synthesis - Phospholipase C / Lecithinase / Haemolysin more exotoxins o destruction of cytoplasmic membrane of host cells o Inactivates opsonins host immune proteins fighting off the infection - Elastase important if you get Pseudomonas infection in the eye o plays role in corneal infection - Leukocidin inhibits WBCs o inhibits neutrophil and lymphocyte functions - Pyocyanins o Damage host tissues Acinetobacter General characteristics - Gram-negative - Short bacilli in pairs - Non-motile - Capsulated - Strictly aerobic - Oxidase - ve - Catalase + ve Where can we find them? Ubiquitous in the environment, all over the place, everywhere Acinetobacter sp. Clinical significance - Nosocomial infection - Catheter associate infection - Ventilator-associated - Wound/soft tissue infection - Endocarditis - THREAT in hospital Respiratory tract causing pneumonia - Bacteraemia bloodstream - Endocarditis - Meningitis - UTI urinary tract Natural resistance to the host response and can form a biofilm Anything that has a group activity = quorum sensing – so enough number of bacteria in one spot to turn something on. Quorum sensing: - Group activity – Quorum sensing is a way bacteria communicate with each other based on their population density. When enough bacteria are present, they can coordinate activities. - Gene expression regulation – this process involves adjusting gene expression in response to cell density. It allows bacteria to act collectively, which can enhance their survival and virulence. Immune system response – The human immune system responds to infections, but sometimes the response can be too strong, causing damage to the body’s own tissues. The part of the damage caused by infections can be due to the immune system’s overreaction rather than the bacteria themselves. Iron acquisition: - Iron extraction – Acinetobater spp. Can extract iron from their environment, which is crucial for their growth and survival. - Killing the host cells – by pulling iron from the host’s body, they can cuase damage to host cells. - Resistance to serum and complements – they are highly resistant to components of the immune system, such as serum (the liquid part of blood) and complements (a group of proteins that help fight infections). Environment - Survive in the environment – resistant to being dried out - Resistant to disinfectants and antibiotics (biofilm formation) For Pseudomonas, the worry is sepsis, the spread of infection through the bloodstream. For Acinetobacter, the worry is the infection in the heart or brain- endocarditis, meningitis Virulence factors – not much is known about the pathogenicity and virulence A. baumannii Nosocomial infection of importance - OmpA – outer membrane proteins - Pili assembly/fimbria - Biofilm associated proteins - Phospholipase D o resistance to human serum blood o epithelial cell evasion o pathogenesis - Phospholipase C o toxicity to epithelial cells Proteus mirabilis and Pseudomonas aeruginosa neutralizes opsonin. Differential Diagnosis - E. coli: A most common cause of UTIs, particularly uropathogenic E. coli (UPEC). - Clues for Proteus mirabilis include a patient's history of catheter use. Chryseobacterium General Characteristics Not so common, opportunistic  Gram-negative rods  Non-motile  Yellow pigment on BA distinctive – S. aureus can also be yellow on BA  Strictly aerobic  Oxidase +ve  Catalase +ve Where can we find them? All over the place Virulence Factors: - Protease breakdown proteins – damage tissues and evade the immune response - DNase breakdown DNA – assist in spreading the infection and evading immune defence. - Phosphatase removes phosphate groups from molecules o Regulating the activity of other enzymes by turning them on or off – this will be interfered o Phosphatase and kinases are two different enzymes that regulate the activity of other enzymes. ▪ Kinase – add phosphate groups to proteins or other molecules (phosphorylation) ▪ Phosphatases – remove phosphate groups from proteins or other molecules (dephosphorylation) Pseudomonas aeruginosa - Gram-negative rod bacterium. Ubiquitous in soil, homes, and hospitals. Characteristics: - Obligate aerobe (uses oxygen for metabolism). - Non-lactose fermenting. - Non-spore forming. - Catalase, citrate, and oxidase positive. - Encapsulated with a mucoid exopolysaccharide capsule. - Possesses flagellum for motility and pili for adhesion. - Multi-drug efflux pumps and various drug-resistance enzymes. Drug Resistance Mechanisms: 1. Efflux Pumps: Pump antibiotics out of the cell. 2. Beta-lactamases: Degrade beta-lactam antibiotics (e.g., penicillin). 3. Aminoglycoside-modifying enzymes: Inactivate aminoglycosides – a different class of antibiotics. 4. Biofilm Formation: - Encapsulation in mucoid layer. So the antibiotics cannot get to you - Dormant state inside the biofilm reduces the effectiveness of antibiotics targeting protein synthesis, cell wall synthesis, or DNA replication. Virulence Factors: - Endotoxin: Strong immune response causing host cell damage. - Biofilm Formation: Protects from immune cells and antibiotics. - Type 3 Secretion System (T3SS): Injects toxins into host epithelial cells. - Exotoxin A: targets ADP-ribosylation of EF2 (Elongation Factor 2), which activate ribosomal protein (protein synthesis) => cell death - Pyocyanin blue pigment: Generates reactive oxygen species, causing oxidative damage. => cell death - Phospholipase C: Degrades host cell membranes. Causing cell lysis - Pyoverdine green compound: Iron chelator aiding bacterial growth. Infections and Symptoms Common Infections: - Blood: Can lead to septic shock. - Skin: - Hot tub folliculitis (red pimply rash). - Ecthyma gangrenosum (blisters progressing to necrosis). - Wound infections (blue-green color, fruity smell). - Urinary Tract: Especially in catheter users. - Eyes: Conjunctivitis and eye pain, often with contact lens use. - Bones: Osteomyelitis, especially in diabetics. - Ears: Malignant external otitis (swimmer's ear). - Heart: Tricuspid valve endocarditis in IV drug users. - Lungs: Pneumonia in cystic fibrosis patients. Symptoms: - Systemic: Fever, elevated heart and respiratory rates, increased white blood cell count. - Localized: Specific to the infection site, e.g., skin rash, eye pain, ear pain. Diagnosis and Treatment Diagnosis: - Complete Blood Count (CBC). - Cultures of site of infection: Blood, sputum, skin. Treatment: - Self-limiting Cases: No treatment for mild infections like Pseudomonas folliculitis. - Serious Infections: Guided by cultures and antibiotic susceptibility testing. - Topical Antibiotics: For superficial infections. - Aerosolized Antibiotics: For lung infections. - Combination Therapy: Often needed due to multidrug resistance. - Examples: Beta-lactam with aminoglycoside or carbapenem with quinolones and aminoglycosides. Environmental Presence and Transmission Environmental Presence: - Survives on dry surfaces and inanimate objects for months. - Thrives in humid/wet conditions (e.g., hot tubs, contact lenses, catheters, medical ventilators). Transmission: - Contact with contaminated surfaces or aerosols. - Particularly affects high-risk individuals (e.g., those with cystic fibrosis, chronic diseases, immunodeficiencies, severe burns, deep wounds, IV drug users). Proteus mirabilis Overview - gram-negative bacillus. - Belongs to the family Enterobacteriaceae - Widely distributed in soil and water and part of the normal human intestinal flora. - Causes urinary tract infections (UTIs). Characteristics - Non-spore-forming and highly motile. - Facultative anaerobic (can survive in both aerobic and anaerobic environments). - Non-lactose fermenter and oxidase - ve. - Urease + ve (produces urease enzyme). Growth on Agar - Blood agar: Exhibits swarming growth, forming thin, filmy layers of concentric circles. - MacConkey agar: Forms smooth, pale, or colourless colonies; does not swarm. Biochemical Tests - Triple Sugar Iron (TSI) test: Produces hydrogen sulphide (H2S), which reacts with iron to form a black precipitate. Virulence Factors - Flagella: Confer motility; aids in swarming motility on surfaces, particularly urinary catheters. Swarming motility – especially in urinary catheters, it can differentiate from short swimmer cells into elongated swarm cells. These swarm cells express many flagella, allowing the bacteria to move and form multicellular rafts. - Fimbriae: Tiny projections used for attachment to uroepithelium cells. - Hemolysin: Creates tiny holes in cell membranes, causing cell damage. - ZapA protease: Destroys immunoglobulin A (IgA) and immunoglobulin G (IgG), neutralising the immune system's opsonins. Pseudomonas also neutralises opsonin. - Urease: Converts urea in urine to ammonia and carbon dioxide, leading to the formation of struvite stones. Pathogenesis in the Urinary Tract - Swarming motility aids in migration along catheter surfaces. - Ascends from the bladder to the kidneys, causing infections. - Struvite stones: Formed from phosphate, calcium, magnesium, and ammonium; can lead to staghorn renal calculi or kidney stones. - Causes urinary stasis, promoting bacterial multiplication and urinary alkalinisation. Infections Caused - Urethritis: Infection of the urethra. - Cystitis: Infection of the bladder. - Prostatitis: Infection of the prostate. - Pyelonephritis: Infection of the kidneys. - Struvite stones: May lead to xanthogranulomatous pyelonephritis, causing kidney destruction. Risk Factors - Common in elderly hospitalised patients. - Risk factors include urinary tract procedures (surgery, catheterisation), chronic kidney disease, neurogenic bladder, multiple prior UTIs, prior use of antibiotics, and sexual activity. Symptoms - Urethritis: Dysuria (pain during urination), pyuria (pus in the urine), increased urinary frequency. - Cystitis: Dysuria, urinary frequency, urgency, suprapubic pain, ammonia-smelling urine. - Prostatitis: Similar to cystitis; may also include fever, chills, swollen and tender prostate. - Pyelonephritis: Dysuria, urinary frequency, urgency, flank pain, fever, chills, nausea, vomiting. Diagnosis - Urine culture: Identifies Proteus mirabilis. - Urinalysis: Shows pyuria WBCs in urine, bacteriuria, and urinary pH above 7. - Complete blood count (CBC): Shows leucocytosis in pyelonephritis or prostatitis. Or increase in # of WBCs. - X-ray: Struvite stones appear radiopaque. Treatment - Cystitis: Trimethoprim-sulfamethoxazole (TMP-SMX), quinolones, or fosfomycin. - Pyelonephritis: Fluoroquinolones, cephalosporins, or gentamicin. - Struvite stones: Surgery may be required for removal. Lecture 8 Human Pathogen Haemophilus spp. Listeria spp. Clostridium spp. NEED to know: Taxonomy, General characteristics, virulence factors, clinical significance. Haemophilus spp. Taxonomy H. influenzae – causes influenza- like symptoms and also causes meningitis. H. parainfluenzae are two most common species General characteristics - Gram-negative - Pleomorphic small bacilli – sometimes almost coccal in shape, sometimes as short filaments - Non-motile - Non-spore former - Encapsulated or non-encapsulated Only the capsulated forms are called Type B – super virulent form So have HIB – H. influenzae Type B vaccine - Aerobic, facultatively anaerobic - Oxidase +ve, Catalase V Growth factor X & V H is a fastidious grower – needs some factors supplemented = Factor X & V; without these, it cannot grow Right – only grows around the disk – X + V Left – usually grows when factor V is supplemented because factor X is haem (already on it) Natural Habitat Non-encapsulated species – upper respiratory tract of humans (85%-adults) & other animals. Encapsulated – upper respiratory tract of children & adults (2% - 60%) Haemophilus influenzae Capsular serotype b –uncommon is healthy infants, children & rare in adults - common cause of infections Virulence factors: - capsule - pentose sugar + ribitol-phosphate Endo - Pili – adhesion to mucosal cells - Exotoxins IgA1 protease – evade mucosal resistance to infection, destroying IgA - Adhesins – adhesion to epithelial cells - Exotoxins Bacteriocin – haemocin this helps it fight off other bacteria so that it can have space to live. - Outer Membrane Proteins – OMPs Endo - Lipooligosaccharides - LOS Endo Clinical significance Haemophilus influenzae Haemophilus parainfluenzae - Meningitis - Endocarditis - Otitis meida eye - Secondary bacteraemia blood - Sinusitis throat - Urethritis - Epiglottitis throat - Pharyngitis & Laryngitis pharynx, larynx - Bacteraemia & Endocarditis blood, heart - Cellulitis tissue - Conjunctivitis eye Listeria spp. – 7 species L. monocytogenes General characteristics - Gram-positive coccobacilli - motile at room temperature 25 - Non-spore former - Facultatively anaerobic, facultative intracellular organisms can live in both inside or outside the cell - Oxidase - ve , catalase and aesculin + ve - Grow optimally at 35o C to 37o C, but growth also occurs at 4oC fridge - Usually β-haemolytic Unwashed lettuce, deli meats, pregnant–vulnerable Natural Habitat Can be everywhere: chicken, water – poor sanitation in the water system, soil, undercooked meat Virulence factors - Listeriolysin O (LLO) – inhibits macrophage-mediated antigen processing block the antigen presenting process = no signaling that there is an infection - Actin-assembly-inducing protein (ActA) the whole journey within the cell (moving from cell-cell) is done by recruiting actin – -filaments within a cell that gives eukaryotic cells their structure - the ActA allows the bacteria to gather up all of the actin fibres and use them to move from the cell to the next cell - Phospholipases C (PLCs) - Zinc metalloproteinase (Mpl) degrade proteins but requires zinc (metal) as a co-factor - Internalin A (InlA) - Internalin B (InlB) - Endotoxins-like material the components of cell walls Cell cycle of Listeria monocytogenes L. monocytogenes go into the cell through the engulfment – fully into the cells – finds the endosomal/lysosomal fusion – escape that. Instead of being broken down and digested into antigens and being put out on the cell surface, it gets out of that and recruits actin. It can either: 1. just stay in there and grow and divide. 2. Take those actins and shoot through into the next cell and do the same thing there. Clinical significance - Listeriosis Can look like gastroenteritis - Meningitis infection of CNS – spinal fluid and the brain - Septicaemia sepsis – too much toxins in the blood - Encephalitis another type of CNS infection Clostridium spp. 176 species 5 subspecies Pathogenic species Histotoxic clostridia – Clostridium tetani Causes tetanus Enterotoxigenic clostridia – Clostridium botulinum Causes botulism, the toxin from botulism = botox General Characteristics - Gram-positive may appear Gram-negative in older cultures - Straight or curved rods may appear singly, in pairs, or in chains - Spore former – Endospores so it looks like a gram -ve rod with gram +ve cocci in it - Motile – peritrichous flagella; except C. ramosum, C. innocuum - Non-encapsulated – except C. perfringens - Obligate anaerobes strict anaerobes Some Clostridium like C. difficile will die, or sporulate and the spores will survive. Some are oxygen tolerant, like C. perfringens – won’t die but not going to grow. - Catalase – ve - Saccharolytic can break down sugars and eat sugars - Proteolytic break down proteins Where can we find them? Lower intestinal tracts of humans and other animals because no oxygen Harmless saprophytes but some are pathogens Virulence factors: Exotoxins – varied among species C. perfringens – Type A, B, C, D, E the main types of toxins C. difficile – 2 exotoxins (Toxin A & B) = Antibiotic-associated diarrhoea (AAD) mostly hospital-acquired, can have community-acquired, associated with older people in nursing homes, recently taking antibiotics => perfused watery diarrhea that can be acute or progress into the long term chronic diarrhea that can be so severe that people waste away and die over a longer period of time – dying over months from diarrhea. Not like cholera – die in a few days. - can cause pseudomembranous colitis – inflammation of the colon – a sever inflammation of the inner lining of the large intestine. C. tetani – flagellar antigens (10 types), all of the antigens associated with flagella somatic; spore antigens; haemolysin haemolytic; tetanolysin; lyse the cells tetanus toxin (tetanospasmin) lead to the spasming tetanus two tetanus-causing toxins C. botulinum – botulinum toxin (neurotoxin) well-studied Clinical significance C. perfringens - Wound and soft tissue infections o Simple wound contamination o Cellulitis deeper tissues o Clostridial myonecrosis (gas gangrene) can cause GI issues as well C. tetani - Tetanus C. difficile - Enteric pathogens - Antibiotic-associate diarrhoea (AAD) - Antibiotic-associated colitis (AAC) - Pseudomembranous colitis (PMC) C. botulinum - Botulism Haemophilus influenzae Characteristics: - Gram-negative coccobacillus - Intermediate between round (coccus) and linear (bacillus). - Non-motile. - Facultative anaerobe. - Catalase and oxidase +ve Growth Requirements - Cultivation Medium: Chocolate agar, requires: - Factor X (hemin) - Factor V (NAD) - Alternative Growth Method: Blood agar with Staphylococcus aureus colonies to provide Factor V via red blood cell haemolysis. Classification - Encapsulated Strains: Six serotypes based on capsular antigens (a, b, c, d, e, f). - Unencapsulated Strains: Non-typeable, lacking a polysaccharide capsule. Virulence Factors Common to Both Strains: - Outer Membrane: Contains lipo-oligosaccharides (LOS) which inhibit mucociliary clearance. - IgA Protease: Destroys immunoglobulin A (IgA) in mucosal secretions. Encapsulated Strains: - Capsule: Major virulence factor, anti-phagocytic. - Pili and Adhesion Proteins: HMW1 and HMW2 help attach to epithelial cells. Unencapsulated Strains: - Phase Variation: Alters outer membrane oligosaccharides to evade the immune system. - Biofilm Formation: Protects bacteria from immune system and antibiotics. Diseases Caused - Encapsulated Strains (e.g., Haemophilus influenzae type B): - Epiglottitis: Fever, sore throat, dyspnea. - Meningitis: Fever, lethargy, irritability, vomiting, sore neck, altered mental status. - Cellulitis: Fever, warm tender area on the cheek or periorbital area. - Bacteraemia: Fever, chills, hypotension, tachycardia. - Septic Arthritis: Fever, pain, swelling, tenderness of joints. - Osteomyelitis: Fever, bone pain, weakness. - Unencapsulated Strains: - Otitis Media: Fever, ear pain, otorrhea. - Sinusitis: Fever, sinus tenderness, purulent nasal discharge. - Bronchitis: Inflammation of bronchi. - Pneumonia: Inflammation of the lungs, fever, chest pain, cough, shortness of breath. - Conjunctivitis: Eye redness, pain, burning. Epidemiology - Transmission: Respiratory droplets and secretions. - Colonisation Rates: - Non-encapsulated strains: 40-80% of children and adults. - Encapsulated strains: 3-5% of children aged 2-5 years. Risk Factors - Encapsulated Strains: - Children, especially unvaccinated. - Individuals with spleen issues (splenectomy, sickle cell disease). - People with malignancies or complement component deficiencies. - Those with acute viral infections, especially influenza virus. - Unencapsulated Strains: - Children and immunocompromised individuals (diabetes, malignancy, HIV). - People with chronic lung conditions (COPD, cystic fibrosis). Diagnosis - Sample Cultures: Blood, CSF, synovial fluid, pleural fluid, etc. - Serological Methods: Latex agglutination, enzyme immunoassay, coagglutination. - Specific Tests: - Epiglottitis: Laryngoscopy, X-ray showing "thumbprint sign." - Bronchopneumonia: Chest X-ray showing ground-glass opacities, bronchial wall thickening, consolidation. Treatment - Encapsulated Strains: - Ceftriaxone - Chloramphenicol - Unencapsulated Strains: - First-line: Amoxicillin ± clavulanate. - Alternatives: Second/third-generation cephalosporins, macrolides, fluoroquinolones. Listeria monocytogenes Characteristics: - Gram-positive, rod-shaped bacteria - Catalase +ve and oxidase -ve - Facultative intracellular pathogen: Can live both outside and inside host cells. - Non-spore forming and facultatively anaerobic: Survives in both aerobic and anaerobic environments. Discovery - Named "monocytogenes" due to increased monocyte levels in inoculated rabbits. Growth and Motility - Grows on blood agar medium, causing beta haemolysis (complete haemolysis) due to beta-haemolysin toxins. - Motility: - Extracellular Environment: Tumbling motility via flagella, active at 37°C and below. - Intracellular Environment: Actin-based motility using ActA protein to recruit actin filaments, propelling the bacteria forward. Pathogenesis - Foodborne; common sources include contaminated unpasteurised dairy products and cold deli meats. - Can survive at low refrigerator temperatures. Cellular Invasion 1. Attachment: Uses internalins to attach to host cell receptors like E-cadherin on goblet cells in the intestinal mucosa. 2. Internalisation: Invades host cells via endocytosis, forming an internalisation vacuole. 3. Escape from Vacuole: Releases listeriolysin O and phospholipases to degrade the vacuolar membrane. 4. Intracellular Replication: Multiplies by binary fission in the host cell cytoplasm. 5. Cell-to-Cell Spread: Propels to adjacent cells using actin-based motility, forming double-membrane vacuoles and repeating the invasion process. Disease Manifestations - Gastroenteritis: Limited to intestinal mucosa; symptoms include low-grade fever, diarrhea, vomiting. - Disseminated Listeriosis: High fever, muscle pain, additional symptoms depending on affected organs: - Liver Abscesses: Abdominal pain, jaundice. - Meningitis: Headache, stiff neck, altered mental status. - Neonatal Listeriosis: Fever, respiratory distress, granulomatosis infantiseptica (widespread, small, pale nodules). Populations at Risk - Immunocompromised Individuals: Neonates, elderly, pregnant individuals, adults with conditions like diabetes, malignancy, or HIV infection. Diagnosis - Culture Samples: Depends on symptoms: - Stool culture for gastroenteritis. - Blood culture for bacteremia. - Cerebrospinal fluid (CSF) culture for meningitis. - Amniotic fluid culture for trans-placental infection. - Imaging Studies: Ultrasound or CT scan to identify liver abscesses. Treatment - Gastrointestinal Listeriosis: Supportive care (hydration, rest). - Disseminated Listeriosis: Intravenous ampicillin combined with gentamicin. - Alternatives: Meropenem for those with contraindications to ampicillin (e.g., pregnant individuals, those allergic to ampicillin). Clostridium botulinum Characteristics: - Obligate anaerobes: Thrive in environments without oxygen, which means they grow and divide in the absence of oxygen but not necessarily that oxygen is toxic to them. - Spore Formation: Produce metabolically inert and resilient spores in stressful conditions, such as exposure to oxygen. - Gram-positive bacillus: Stains purple in a Gram stain, and appears rod-shaped under the microscope. Habitat - Natural Environment: Found in deep, compact soil where oxygen levels are low. Toxin Production - Botulinum Toxin: Causes botulism and comes in eight distinct types (A, B, C, D, E, F, G, H), varying in toxicity. Pathogenesis - Contamination: Spores can contaminate food during preparation processes that block out air, like canning and sausage making. - Gas Production: Bacteria metabolise sugars into short-chain fatty acids, creating gas (mainly carbon dioxide and hydrogen) that causes cans to bulge. Mechanism of Action 1. Ingestion: Consumption of contaminated food introduces botulinum toxin into the body. 2. Binding to Nerves: Toxin binds to nerves that use acetylcholine for muscle control. 3. Endocytosis: Neuron takes in the toxin via endocytosis, forming a vesicle in the cytoplasm. 4. Activation and Action: Toxin activates, exits the vesicle, and cleaves SNARE proteins. 5. Inhibition of Neurotransmitter Release: Without SNARE proteins, acetylcholine is not released, preventing muscle contraction and leading to flaccid paralysis. Clinical Manifestations - Early Symptoms: Affects muscles controlled by cranial nerves (facial muscles, eye movements, chewing, swallowing). - Autonomic Nervous System Effects: Can cause dry mouth, postural hypotension, nausea, vomiting, and constipation. - Progression: May lead to descending paralysis and potentially lethal flaccid paralysis if respiratory muscles are involved. - Symptoms: Double vision, drooping eyelids, inability to make facial expressions, difficulty swallowing. Diagnosis - Serum Analysis: Detection of botulinum toxin in the bloodstream. - Culture: Growing Clostridium botulinum in culture is difficult due to its anaerobic nature. Treatment - Supportive Care: Assist with breathing in affected patients. - Passive Immunization: - Trivalent Antitoxin: Covers toxins A, B, and E. - Heptavalent Antitoxin: Covers toxins A, B, C, D, E, F, and G. - Mechanism: Antitoxins capture free toxin molecules in the bloodstream before they damage neurons. Uses of Botulinum Toxin - Medical Applications: Types A and B are less toxic and used in treating various conditions: - Neuromuscular Conditions: Relax rigid muscles. - Hyperhidrosis: Reduce excessive sweating. - Achalasia: Treat esophageal spasm. - Cervical Dystonia: Reduce involuntary muscle spasticity. - Cosmetic Surgery: Used as Botox to smooth out wrinkles for a few months. Clostridium perfringens Characteristics: - Obligate anaerobes: Do not require oxygen to thrive and may be harmed by it due to lack of enzymes like catalase and superoxide dismutase. - Spore Formation: Produce spores in stressful conditions (e.g., exposure to oxygen) that are extremely resilient and can survive cooking. Growth and Laboratory Identification - Optimum Temperature: Have one of the fastest growth rates of any bacterium under optimal conditions. - Gram-positive, appearing purple and rod-shaped (bacilli) under the microscope. Foodborne Illness - Common Cause: Often called the "cafeteria germ" due to frequent outbreaks in food prepared in large quantities and kept warm for prolonged periods. - Contamination: Spores can contaminate food left out for a while, which then colonizes the gut upon ingestion. - Toxin Production: Produces Clostridium perfringens enterotoxin (CPE) within 24 hours of colonisation, targeting and destroying tight junctions in intestinal epithelial cells. - Heat Sensitivity: CPE is heat-labile and can be inactivated by cooking at 72°C or above. Symptoms of Foodborne Illness - Common Symptoms: Abdominal cramping, watery diarrhea, and vomiting. - Symptoms typically improve within a day without the need for antibiotics;