Gram Positives PDF

Medically Important Gram-Positive Cocci B.A. Buxton, Ph.D.  Learning Objectives List general microbiological characteristics of Staphylococcus spp., Streptococcus spp. & Enterococcus spp. – Catalase; coagulase; bacitracin; optochin; bile solubility – Classification system...

Medically Important Gram-Positive Cocci B.A. Buxton, Ph.D.  Learning Objectives List general microbiological characteristics of Staphylococcus spp., Streptococcus spp. & Enterococcus spp. – Catalase; coagulase; bacitracin; optochin; bile solubility – Classification system of Streptococcus spp. List clinical syndromes associated with Staphylococcus spp., Streptococcus spp. & Enterococcus spp. Describe major virulence factors associated with Staphylococcus spp., Streptococcus spp. & Enterococcus spp. and how they contribute to the manifestation of clinical disease by these organisms Describe the common diseases and major virulence factors associated with the Gram-positive rods Bacillus cereus, Bacillus anthracis, Corynebacterium diphtheriae, and Listeria monocytogenes Explain the sources, transmission & risk factors for the Gram-positive organisms discussed in this lecture  Organisms Discussed in This Lecture Gram-positive cocci Gram-positive rods – Staphylococci – Aerobic Staphylococcus aureus Bacillus Staphylococcus epidermidis – B. anthracis Staphylococcus – B. cereus saprophyticus Corynebacterium – Streptococci diphtherium S. pyogenes Listeria monocyogenes S. agalactiae – Anaerobic S. pneumoniae Clostridial species S. mutans and other oral strep. Viridans group streptococci Enterococcus sp. Gram-Positive Cocci Three genera – Staphylococcus – Streptococcus – Enterococcus Characteristics of Gram Positive Cocci Types of Hemolysis  Diagnostic findings of Medically Important Staphylococci Staphylococcus aureus – Most important pathogen – Coagulase-positive – Beta-hemolytic – Forms yellow colonies on manitol salt agar – Salt tolerant (halotolerant) Staphylococcus epidermidis – Coagulase- negative – Usually non-hemolytic – Forms white colonies on mannitol salt agar Staphylococcus saprophyticus – Coagulase-negative – Non-hemolytic  Sample Board-Style Question Expect this type of question on exams and quizzes A 67 year old man presented with fever and a tender, hot, swollen knee. Gram stain of the synovial fluid revealed a gram-positive cocci in clusters. Synovial fluid and blood cultures grew β-hemolytic, gram-positive cocci that were coagulase and catalase positive. What is the etiologic diagnosis? A. Staphylococcus aureus B. Staphylococcus epidermidis C. Streptococcus agalactiae D. Streptococcus pyogenes E. Enterococcus spp.  Epidemiology of S. aureus: Sources: People – Frequently colonizes external nares – Transiently on skin, oropharynx, perineum – 15% of normal healthy adults are persistent carriers (in nasopharynx) – 50-80% carriage in health care workers Transmission: – Spread from person to person Hand to hand contact Fomites – Inanimate objects contaminated with bacteria from skin – Staphylococci are very hardy in environment- survive heat, cold and dried states  Clinical Manifestations of Staphylococcus aureus Infections Staphylococcus aureus is associated with 2 major types of disease processes: Pyogenic – Pus forming – Infection leads to acute inflammatory response – Inflammatory process as well as bacterial growth and metabolism cause disease manifestations Toxigenic – Some strains secrete toxins which are responsible for disease manifestations  Pyogenic (aka suppurative) infections of S. aureus: overview Local staphylococcal infection leads to the formation of a collection of pus called an abscess. Early events characteristic of acute inflammation – Red, inflamed appearance – Rapid and extensive infiltration of neutrophils into site of infection Neutrophil chemotaxis in response to host and bacterial factors Too vigorous host inflammatory response can lead to tissue damage or sepsis  Pyogenic (aka suppurative) infections of S. aureus: overview Host response to focal inflammation is to surround the area within a fibrin capsule. Center of abscess is necrotic, consisting of dead cells including neutrophils, and dead and live bacteria and fluid  Suppurative Skin Infections: Impetigo – Superficial infection, crusty, pus-filled vesicles Boils and Furuncles – Large, painful, pus-filled cutaneous nodules (furuncles are larger than boils) Carbuncle – Coalescence of furuncles with extension into subcutaneous tissues and evidence of systemic disease (fever, chills, bacteremia) Folliculitis – Infection of hair follicles Cellulitis – Infection of the deep layer of skin  Suppurative Skin Infections Boil Carbuncle Folliculitis Cellulitis  S. aureus systemic pyogenic infections Endocarditis – Infection of heart valves – 50% mortality rate – Occurs on right side of heart in individuals who inject drugs Septic arthritis – Most common cause of joint infection Osteomyelitis – Infection of bone – Can occur secondary to trauma or following hematogenous dissemination to bone Pneumonia – Can develop after aspiration of oral secretions, or following hematogenous dissemination to lung  S. aureus Toxin-Mediated Diseases Toxin-mediated disease include – Food Poisoning – Toxic Shock Syndrome – Scalded skin syndrome – Bullous impetigo Some toxins are capable of non-specifically activating T cells to secrete large quantities of cytokines – These proteins are called superantigens Toxins include: – Enterotoxins (A-E, G-I)-cause vomiting when ingested in contaminated foods. SEA is most common – Staphylococcal toxin shock syndrome toxin (TSST)-1- causes life-threatening shock (hypotension) – Exfoliative toxin A- causes desquamation of skin  Staphylococcal Food Poisoning Most common type of food poisoning in US Results from contamination of food by human carrier Bacteria grow in food held at room temperature for hours S. aureus survive and grow in foods with high salt content (halotolerant) Only certain strains secrete enterotoxins Heating of food may kill bacteria but does not inactivate toxin  Toxic Shock Syndrome Due to some of the enterotoxins or TSST-1 Clinically similar to septic shock due to gram-negative organisms The toxins involved act as superantigens, activating multiple clones of T cells Following activation of T cells by toxins, massive quantities of pro-inflammatory cytokines are released causing fever, rash and hypotension which can lead to death  Bullous Impetigo Bullae- blisters of >5mm diameter Bullus impetigo – bullae form on skin ; can also involve buccal mucous membranes Bullae contain straw-colored fluid Occurs most commonly in infants Due to exfoliative toxins A and B which break down desmosomes Only certain strains produce exfoliative toxins FYI: S aureus phage group II type 71 is the predominant organism Mechanism of Exfoliative Toxin  Scalded skin syndrome More widespread version of bullous impetigo Mediated by exfoliative A and B toxins – Break down desmosomes Intense inflammation Widespread desquamation Super-infection with other bacteria or fungi can lead to sepsis and death  Brain Break Describe the disease associations of Staphylococcus aureus. Which associations are pyogenic and which are toxigenic? Describe the mechanisms of action of the toxins for toxigenic infections.  S. aureus Virulence Factors Factors enabling colonization of skin: – Lipases and glycerol esterases degrade skin lipids – Teichoic acid Facilitates adherence Factors that facilitate invasion – Fibrinolysin – Hyaluronidase – Lipase – Nucleases  S. aureus Virulence Factors Mechanisms to avoid phagocytosis and other immune mechanisms – Capsule Inhibits phagocytosis; facilitate adherence – Protein A Binds to Fc region of IgG Reduces binding of phagocytes via FcR Inhibits complement activation  Staphylococcal Protein A Inhibits Opsonization by Preventing Recognition of Rc Region by FcR FcR Staph aureus Neutrophil expressing FcR- cannot bind to Fc region of Ab, Because Fc region is bound by protein A  S. aureus Virulence Factors Mechanisms to avoid phagocytosis and other immune mechanisms – Catalase Converts hydrogen peroxide to water Counteracts neutrophil ability to kill bacteria via production of oxygen free radicals – Coagulase Clots plasma, converts fibrinogen to fibrin Bacteria can hide within the clot White cells, antibodies, even antibiotics do not penetrate clots well  S. aureus: Virulence factors Factors involved in inducing inflammation and cell death: – Peptidoglycan Endotoxin-like activity Attracts leukocytes – Cytotoxins Membrane damaging toxins that lyse eukaryotic cells including neutrophils – Pore-forming proteins (α, β, δ, γ toxins; aka hemolysins) » Made by almost all strains S. aureus: Virulence factors Toxins (superantigens) – Enterotoxins Staphylococcal enterotoxins A and B induce vomiting (staphylococcal food poisoning) – Toxic shock syndrome toxin (TSST-1) Cause of toxic shock syndrome Only certain strains produce TSST-1 – Exfoliative toxins A and B Causes scalded skin syndrome in neonates and bullous impetigo Causes breakdown of desmoglein-1 Only certain strains produce Many strains make β-lactamase – Enzyme that degrades antibiotics in penicillin family Some strains are methicillin resistant (MRSA) – Associated with resistance to penicillins and cephalosporins – Due to mutated PBP  S. aureus: Antibiotic β lactam ring Resistance Virtually all strains make β-lactamase (aka penicillinase) – Enzyme that degrades antibiotics in penicillin family (a relatively small number of antibiotics are affected by this bacterial enzyme) Methicillin resistant Staphylococcus aureus (MRSA) – Associated with resistance to most beta-lactam antibiotics (a huge number of antibiotics are in this group!) β-lactamase – Due to mutated PBP Methicillin Resistance is Due to Altered PBP Transpeptidase (aka Penicillin binding protein- PBP) carries out this reaction. Beta-lactam antibiotics target this enzyme. Mutation of PBP prevents binding of beta-lactam.  Other Strains of Staphylococci: S. epidermidis and S. saprophyticus Referred to as Coagulase-negative Staph Both are catalase positive, non-hemolytic and do not ferment mannitol – S. saprophyticus Novobiocin resistant – S. epidermidis Novobiocin-sensitive  Staphylococcus epidermidis Source – Colonizer of skin Disease Associations – Endocarditis – Infections of shunts and catheters – Infections of prosthetic joints Virulence factors – Ability to bind to foreign materials – Biofilm production  Staphylococcus saprophyticus Laboratory characteristics: novobiocin resistant coagulase-negative staph Sources – Has been isolated from a number of sources including gut of humans and various animals as well as environment – Colonizes the human GI tract (GIT) Colonization presumably following ingestion secondary to exposure to animals or ingestion of inadequately cooked food Disease Associations – 2nd most common cause of urinary tract (UT) infections in sexually active women (13-40 years of age) Young women more susceptible Sexual intercourse promotes spread from GIT to UT  Brain Break How do laboratory tests distinguish between Staphylococcus aureus, S. epidermidis and S. saprophyticus? The Streptococci Medically Important Streptococci S. pyogenes S. agalactiae S. pneumoniae S. mutans and other oral strep. Viridans group streptococci Enterococcus sp. – Considered by some to be included as Streptococci; others put them in separate group  Streptococci: Laboratory Classification Streptococci are classified on the basis of: – Patterns of hemolysis Alpha hemolytic Beta hemolytic Non-hemolytic – Serologic reactivity Referred to as Lancefield Groups Based on differences in cell wall carbohydrate antigens Useful only for differentiating β-hemolytic strains of streptococci Alpha hemolytic and non-hemolytic strains of streptococci lack group-specific cell wall antigens and cannot be classified by Lancefield’s grouping  Streptococcal Hemolysis Patterns Hemolysis on sheep red blood cell agar Incomplete: Alpha (green) Complete: Beta (clear) – S. pyogenes – S. agalactiae None: gamma (red) Streptococcal Serologic Groupings Based on antigenic differences carbohydrates of cell wall – Streptococcus pyogenes- Group A – Streptococcus agalactiae- Group B Which species is this? How would the laboratory determine this? Laboratory differentiation of streptococci  Sample Board-Style Exam Question A 47-year old moderately obese female was hospitalized with cellulitis. Blood cultures grew catalase negative, Gram-positive cocci as shown in the photographs. Disk A on the blood agar plate contains bacitracin. Which organism is causing her infection? A. Enterococcus spp B. Streptococcus agalactiae C. Streptococcus mutans D. Streptococcus pneumoniae E. Streptococcus pyogenes  Streptococcus pyogenes Group A β-hemolytic streptococci (GABHS) Many different strains (~150) based on antigenic differences in M protein Epidemiology (Source and transmission): – Strains transiently colonize oropharynx and skin Person-to-person transmission of different strains by respiratory droplets or direct contact with people or fomites Disease may follow acquisition of new strain not previously encountered  GABHS Pathogenesis-1. host responses S. pyogenes usually a secondary invader (following viral infection or disturbances in normal flora or break in skin) Normal host defenses include: – Intact physical barriers (mucosal and skin = 1ist line of defense) – Infection stimulates acute inflammation, recruits phagocytes to site of infection – Phagocytes= second line of defense Organism easily destroyed by neutrophils IgG against M protein important adaptive host defense (IgG acts as opsonin, facilitating phagocytosis) Too vigorous a host response can lead to tissue damage and sepsis  GABHS Pathogenesis-2 S. pyogenes Virulence Factors Capsule – Made of hyaluronic acid, a component of connective tissue – Helps hide Strep from host immune system- resembles host – Anti-phagocytic M protein – Protein protruding above cell surface – Adhesion, anti-phagocytic, degrades C3b, mediates internalization by host cells – Strains are based on antigenic differences in M protein Pyrogenic exotoxins (formerly called erythrogenic toxins) – 3 types: A,B and C; A is most important – Produced by strains infected with a lysogenic (temperate) phage – Superantigens- Associated with streptococcal toxic shock syndrome and scarlet fever Streptolysin O – Lyses cells, including phagocytes Streptokinase – Lyses blood clots- facilitates spread  Streptococcal M Protein Hair-like projections from cell wall Varies in amino acid sequence between strains of GABHS  GABHS Disease Associations Suppurative infections Toxin-mediated disease involving acute – Scarlet fever (result of toxin inflammatory secretion) response by host Can occur in some – Pharyngitis pharyngeal infections depending on strain of – Skin and Soft GABHS Tissue Infections – Streptococcal Toxic Shock Impetigo Syndrome (STSS) Cellulitis Immune-mediated diseases Erysipelas – Rheumatic fever – Necrotizing fasciitis Can lead to chronic – Bacteremia and rheumatic heart disease Sepsis (discussed in detail in M2) – Pneumonia – Post-streptococcal glomerulonephritis  Streptococcus pyogenes Disease Associations-1 Strep Throat impetigo Scarlet fever  Streptococcus pyogenes Disease Associations-2 Erysipelas and Cellulitis Erysipelas- involves upper layers of skin Cellulitis involves deeper Layers of skin, sub Q tissues  Streptococcus pyogenes Disease Associations-3 Necrotizing Fasciitis  GAS: immune-mediated sequelae Rheumatic fever – Sequelae of throat infections only (140F to kill spores; cooked food should be promptly refrigerated if not eaten immediately.  Clostridium botulinum Source: Soil and water organism Pathogenesis: Seven serotypes, each produce a distinct toxin with similar activities – Break down protein involved in vesicle transport and fusion with plasma membrane This process is essential for release of neurotransmitters – Neurotransmitters are stored within vesicles Disease Associations: – Foodborne botulism Consumption of home-canned foods containing preformed toxin – Infant botulism Consumption of spores leads to colonization of gut and release of toxin Associated with honey consumption in infants less than 1 yr age – FYI: Wound botulism – rare  Clostridium botulinum Heat-labile neurotoxin absorbed from gut, enters blood, taken up by axons – Similar in structure & function to the tetanus toxin – B- portion of toxin binds to receptors on cholinergic nerves – A-portion prevents release of acetylcholine by cleaving various proteins involved in release (see next slide) – Results in flaccid paralysis Botulism Toxin  Clostridium tetani Found in soil and intestinal flora of many animals Terminal spores – drumstick-like appearance Not common in the US – vaccine Still a problem in developing world Most cases in developing world involve neonates following umbilical cord contamination with dirt Organism produces disease through toxin elaboration- it is not invasive  Tetanospasm A-B toxin A-chain enters the CNS carried by retrograde axonal transport A chain is a peptidase released from postsynaptic dendrites, crosses the synaptic cleft and enters vesicles in the presynaptic terminals Breaks down synaptobrevin (aka VAMP in picture) in vesicles containing GABA neurotransmitter – Blocks the release of inhibitory neurotransmitters (GABA) Leads to unregulated excitation; spastic paralysis  Clostridium difficile Part of normal intestinal flora Nosocomial pathogen in patients on antibiotic therapy Many antibiotics eliminate normal flora while allowing overgrowth of C. difficile Responsible for antibiotic-dependent gastrointestinal disease – Ranges from a relatively mild diarrhea to severe pseudomembranous colitis Pathogenesis of C. difficile Organism is part of normal flora in animals and some humans, especially hospitalized patients Organism shed in feces and forms spores in environment Spores ingested from environmental sources Spores are found in hospital environments and are resistant to many disinfectants Antibiotics decrease other flora but do not harm spores Spores germinate, organism grows, releasing toxins leading to cell death, inflammation and fibrosis (pseudomembrane formation)  Clostridium difficile toxins Toxin A – Enterotoxin- induces hypersecretion of fluid from gut epithelial cells – Also attracts and activates neutrophils – Induces cytokine secretion from epithelial cells and leukocytes Toxin B – Induces depolymerization of actin – Loss of cell adhesion – Death of gut epithelial cells  Brain Break Close your notes and list the bacteria you have learned so far, along with their disease associations and pathogenesis.  Brief Summary Described the common diseases caused by the aerobic gram-positive rods Bacillus, Corynebacterium, Listeria – Bacillus anthracis- skin, gut, lung – Bacillus cereus- food poisoning (emetic and diarrheal forms) – C. diphtheriae- diphtheria (respiratory and myocarditis) – Listeria- meningitis, sepsis Discussed the source(s) of the bacteria – Soil organisms (Bacillus) – Colonizer of animals (Listeria) – Human pathogen (C. diphtheriae) Related the virulence factors of bacteria to the diseases they cause – Toxin production- B. anthracis, B. cereus, C. diphtheriae – Intracellular growth- L. monocytogenes  Brief Summary continued Clostridium are anaerobic, spore-forming, gram-positive rods which cause disease through secretion of toxins Neurological disease results from toxins secreted by C. tetani (spastic paralysis) and C. botulinum (flacid paralysis). Know the difference in clinical manifestations and cellular/molecular pathogenesis) Clostridium perfringens causes gas gangrene, due especially to secretion of alpha toxin (a lecithinase) C. difficile causes colitis following antibiotic therapy, due to toxins A and B which ultimately kill gut epithelial cells- know the role of both toxins in the pathogenesis of this disease. Be sure to now how one becomes infected! Actinomyces is a non-spore forming, anaerobic, filamentous, gram-positive rod primarily associated with jaw infections. They colonize the upper respiratory tract. Sulfur granules and pus tracts are a feature of jaw infections with this organism. Proprionibacterium acnes is a non-spore forming, anaerobic, gram-positive rod associated with acne  Brain Break Close your notes and list the bacteria you have learned so far, along with their disease associations and pathogenesis.