Gram positive cocci. Learning outcomes: The student should be able to -list the medically important genera of gram-positive cocci -describe the microscobic.

Gram positive cocci

Learning outcomes: The student should be able to -list the medically important genera of gram-positive cocci -describe the microscobic appearance of the important genera of gram-positive cocci For Stapylococcus, Streptococcus and Enterococcus -list the important biochemical properties -list the important species -list the important virulance factors(toxins and enzymes) -list the main routes of transmission -list the important clinical manifestations and describe the pathogenesis -list the important properties used in laboratory diagnosis -list the most important antibiotic resistance properties

Staphylococcus-Streptococcus- Enterococcus

Gram-positive cocci Aerobic: Aerobic: -catalase positive: Staphylococcus,Micrococcus… -catalase negative: Streptococcus, Enterococcus… Anaerobic: Anaerobic:Anaerococcus,Peptostreptococcus…

Gr + cocci~ 21 genera Common features  Spherical shape  Gram stain reaction  Absence of endospores  Catalase activitysubdividing  Catalase positive generaStaphylococcus Micrococcus Stomatococcus Alloiococcus  Catalase negative generaStreptococcus Enterococcus

Catalases Enzymes: that catabolize hydrogen peroxide into water and oxygen gas. If a drop of a peroxide solution is placed on a catalase-producing bacterial colony: - bubbles will appear when the oxygen gas is formed. Catalase:important virulans factor:H2O2 microbicidal and its degradation limits the ability of neutrophils to kill.

Staphylococcus Gram positive Grow patterncluster of grapes single cells, pairs, short chains Nonmotile Aerobic or facultatively anaerobic Catalase-positive Grow in 10 % NaCl medium

Staphylococcus Present on the skin and mucous membranes of humans Present on the skin and mucous membranes of humans Nose is the main site of colonization for S. aureus. Nose is the main site of colonization for S. aureus. 40 species 40 species

Staphylococcus Important pathogen Important pathogen Skin,soft tissue,bones,urinary tract, opportunistic infections Skin,soft tissue,bones,urinary tract, opportunistic infections S.aureus S.aureus S.epidermidis S.epidermidis S.haemolyticus,S.lugdunensis S.haemolyticus,S.lugdunensis S.saprophyticus S.saprophyticus

Staphylococcus S.aureus: S.aureus: -coagulase positive :clot forms (When a colony of S. aureus is suspended in plasma, coagulase binds to a serum factor, and this complex converts fibrinogen to fibrin, resulting in the formation of a clot.) (When a colony of S. aureus is suspended in plasma, coagulase binds to a serum factor, and this complex converts fibrinogen to fibrin, resulting in the formation of a clot.) Coagulase negative staphylococci Coagulase negative staphylococci

Most common Staph. causing diseases S. aureus S. epidermidis S. saprophyticus S. capitis S. haemolyticus Produce coagulase Coagulase negative staphylococcus

Methicillin-resistant S. aureus (MRSA) Beta-lactamase resistant penicillins such as methicillin Beta-lactamase resistant penicillins such as methicillin Contain mecA gene Contain mecA gene is notorious for producing serious infections in is notorious for producing serious infections in  hospitalized patients and  more recently outside the hospital in previously healthy children and adults.

Micrococcus genus Organisms in this genus is originally placed into six genera, with Micrococcus, Kocuria, and Kytococcus most commonly colonizing the human skin surface. Organisms in this genus is originally placed into six genera, with Micrococcus, Kocuria, and Kytococcus most commonly colonizing the human skin surface. These cocci resemble staphylococci and can be confused with the coagulase-negative staphylococci. These cocci resemble staphylococci and can be confused with the coagulase-negative staphylococci. Differ in that, they grow only aerobically

Micrococcus genus Although these bacteria may cause opportunistic infections in some patients, Although these bacteria may cause opportunistic infections in some patients, their isolation in clinical specimens usually represents clinically insignificant contamination. their isolation in clinical specimens usually represents clinically insignificant contamination.

Stomatococcus Alloiococcus Stomatococcus mucilaginosus  Only species in this genus  Infection in the immunocompromised host Alloiococcus otitidis  Only species in this genus  Aerobic Gr +  Disease??

Staphylococcus and their diseases S. aureus  Toxin-mediated Food poisoning Toxic shock syndrome, scalded skin syndrome Kawasaki syndrome: unkown etiology that may be caused by certain strains of S. aureus  Cutaneous Impetigo Folliculitis Furuncles Carbuncles, wound infections

Staphylococcus and their diseases S. aureus  Others Bacteremia Endocarditis, Pneumoniae Empyema Osteomyelitis Septic arthritis

Staphylococcus and their diseases S. epidermidis Bacteremia Endocarditis, Surgical wounds Urinary tract infections Opportunistic infections of catheters, shunts, prostetic devices and peritoneal dialysates

Staphylococcus and their diseases S. saprophyticus  Urinary tract infections  Opportunistic infections

Staphylococcus/physiology & structure Capsule Peptidoglycan Teichoic acids Protein A Coagulase and other sutface proteins Cytoplasmic membrane

Capsule and Slime Layer: polysaccharide capsule polysaccharide capsule protects the bacteria by inhibiting phagocytosis of the organisms by polymorphonuclear leukocytes (PMN) protects the bacteria by inhibiting phagocytosis of the organisms by polymorphonuclear leukocytes (PMN)

Capsule and Slime Layer: binds the bacteria to : binds the bacteria to :  tissues and  foreign bodies such as catheters, grafts, prosthetic valves and joints, and shunts

Peptidoglycan and Associated Enzymes The enzymes that catalyze construction of the peptidoglycan layer are called penicillin-binding proteins and are the targets of penicillins and other beta-lactam antibiotics. The enzymes that catalyze construction of the peptidoglycan layer are called penicillin-binding proteins and are the targets of penicillins and other beta-lactam antibiotics. Bacterial resistance to methicillin and related penicillins is mediated by acquisition of a gene (mecA) that codes for a novel penicillin-binding protein, PBP2', that is not bound by penicillins but retains its enzymatic activity Bacterial resistance to methicillin and related penicillins is mediated by acquisition of a gene (mecA) that codes for a novel penicillin-binding protein, PBP2', that is not bound by penicillins but retains its enzymatic activity

mecA gene methicillin-resistant S. aureus (MRSA) strains methicillin-resistant S. aureus (MRSA) strains previously restricted to hospital-acquired infections previously restricted to hospital-acquired infections are now present in the community and are responsible for the majority of staphylococcal infections. are now present in the community and are responsible for the majority of staphylococcal infections.

Peptidoglycan endotoxin-like activity endotoxin-like activity activation of complement activation of complement production of interleukin-1 from monocytes, and aggregation of PMN (a process responsible for abscess formation). production of interleukin-1 from monocytes, and aggregation of PMN (a process responsible for abscess formation).

Staphylococcus/teichoic acids Teichoic & lipoteichoic acid Mediate the attachment of staphs to mucosal surfaces Poor immunogens ( except when bound to peptidoglycan )

Staphylococcus/pathogenesis & immunity Toxins & Enzymes

Staphylococcus/toxins Alpha toxin Beta toxin Delta toxin Gamma toxin & Panton-Valentine Leukocidin Exfoliative toxins Enterotoxins Toxic shock syndrome toxin-1

Staphylococcus/toxins  Toxinimportant mediator of tissue damage in staphylococcal diseases toxic for many cells (erythrocytes, leukocytes, hepatocytes,..) produced by most strains of S. aureus

Staphylococcus/toxins  Toxinsphingomyelinase C toxic for many cells role in tissue destruction & abscess formation produced by most strains of S. aureus

Staphylococcus/toxins  Toxinwide spectrum of cytolytic activity detergent-like activity produced by most strains of S. aureus

Staphylococcus/toxins  Toxin & Panton-Valentine Leukocidin composed of two polypeptide chains toxic for many cells

Staphylococcus/toxins Exfoliative toxinsstaphylococcal scalded skin syndrome (SSSS) exfoliative dermatitis two types: ETA & ETB produced by 5 to 10 % of all S. aureus strains

Staphylococcus/toxins Enterotoxins8 serologically distinct enterotoxins exist (A-E, G-I) stable to heating resistant to gastric & jejunal enzymes produced by 30 to 50 % of all S. aureus strains are superantigens

Staphylococcus/toxins Toxic shock syndrome toxin-1 heat & proteolysis resistant all S. aureus responsible for menstruation-associated TSS produce TSST-1 50 % of the strains responsible for other forms of TSS produce TSST-1 superantigens

Staphylococcus/enzymes Coagulase Catalase Hyaluronidase Fibrinolysin Lipases Nuclease Penicillinase

Staphylococcus/enzymes Coagulase  S. aureus  2 forms Bound free

Staphylococcus/enzymes Catalase  “All staphylococci produce catalase”  H 2 O 2 (toxic)H 2 O O 2

Staphylococcus/enzymes Hyaluronidase  Hydrolyzes hyaluronic acidsfacilitates the spread of S. aureus in tissues  > 90 % of S. aureus produce hyal uro nid as e

Staphylococcus/enzymes Fibrinolysine  Staphylokinase  All S. aureus  Dissolve fibrine clots

Staphylococcus/enzymes Lipases  All S. aureus & 30 % of the CNS produce several different lipases Nuclease  A marker for S. aureus Penicillinase (  -lactamase)

Staphylococcus/IDENTIFICATION Microscopy Culture:  Nutritionally enriched agar media with sheep blood  Selective media (e.g., mannitol-salt agar)(Chapman agar)  Aerobic and anaerobic  Large, smooth colonies Identification  Positive coagulaseS. aureus

Disease Hospital- and community-acquired infections with MRSA are a significant worldwide problem

Epidemiology Normal flora on human skin and mucosal surfaces Organisms can survive on dry surfaces for long periods Person-to-person spread through direct contact or exposure to contaminated fomites (e.g., bed linens, clothing) Risk factors include presence of a foreign body (e.g., splinter, suture, prosthesis, and catheter), previous surgical procedure, and use of antibiotics that suppress the normal microbial flora

MRSA: Vancomycin, trimethoprim-sulfamethizole, clindamycin, linezolid, daptomycin, or quinupristin-dalfopristin The focus of infection (e.g., abscess) must be identified and drained Treatment is symptomatic for patients with food poisoning (although the source of infection should be identified so that appropriate preventive measures can be enacted) Proper cleansing of wounds and use of disinfectant help prevent infections Thorough handwashing and covering of exposed skin help medical personnel prevent infection or spread to other patients

Coagulase-Negative Staphylococci Biology, Virulence, and Disease Catalase-positive, coagulase-negative, gram- positive cocci arranged in clusters Relatively avirulent, although production of a "slime" layer can allow adherence to foreign bodies (e.g., catheters, grafts, prosthetic valves and joints, shunts) and protection from phagocytosis and antibiotics Infections include subacute endocarditis, infections of foreign bodies, and urinary tract infections

Epidemiology Normal human flora on skin and mucosal surfaces Organisms can survive on dry surfaces for long periods Person-to-person spread through direct contact or exposure to contaminated fomites, although most infections are with the patient's own organisms Patients are at risk when a foreign body is present The organisms are ubiquitous, so there are no geographic or seasonal limitations

Streptococcus Gram positive Grow patternpairs, chains Most species are facultatively anaerobes Some grow only in atmosphere enhanced with CO 2(capnophilic growth) Nutritional requirements are complex BBlood, serum “Catalase-negative”

Streptococcus Classification is complicated  3 different schemes are used C carbohydrate(serologic typing) 1.Lancefield groupings according to serologic properties- C carbohydrate(serologic typing) (A-W) 2.Hemolytic patterns:  &  hemolysis 3.Biochemical properties

Lancefield groupings Grouping: C-carbohydrate

S. pyogenes (group A streptococci)  Pharyngitis,  scarlet fever,  pyoderma,  erysipelas,  cellulitis,  necrotizing fasciitis,  streptococcal toxic shock syndrome,  bacteremia,  rheumatic fever,  glomerulonephritis

S. agalactiae (group B)  Neonatal infections (meningitis, pneumoniae, bacteremia)  Urinary tract infections  Amnionitis,  Endometritis  Wound infections

Streptococcus pyogenes/physiology & structure Spherical cocci Form short (clinical specimen) or long chains (liquid media) Grow on enriched blood agar media White colonies1-2 mm with large zones of  -hemolysis Encapsulated strainsmucoid Basic structure in the cell wall is peptidoglycan as Staphsgroup spec. Ag

Streptococcus pyogenes/physiology & structure Type specific proteins  M proteinmajor type-specific protein associated with virulent streptococci 2 polypeptide chains 1. Highly conserved among all group A streptococci 2. Responsible for the antigenic variability>100 serotypes  T protein (trypsin-resistant)secondary Usefull when bacteria fail to express the M protein

Streptococcus pyogenes/physiology & structure Other cell surface components  M-like proteins  Lipoteichoic acid  F protein Capsule

Streptococcus pyogenes/ Pathogenesis&Immunity Virulence of S. pyogenes  The ability of the bacteria to adhere to the surface of the host cells Invade into the epithelial cells Avoid opsonization & phagocytosis Produce a variety of toxins & enzymes

Streptococcus pyogenes/ Pathogenesis&Immunity Pyrogenic exotoxins (Streptococcal pyrogenic exotoxin, Spes)  Produced by lysogenic strains  Superantigens  Responsible for the streptococcal toxic shock syndrome

Streptococcus pyogenes/ Pathogenesis&Immunity Streptolysin S & O  Streptolysine Slyse erythrocytes leukocytes platelets responsible for  -hem.  Streptolysine Olyse erythrocytes leukocytes platelets antibodies are formed againstASO test

Streptococcus pyogenes/ Pathogenesis&Immunity Streptokinases  A & B  Lyse blood clots  Used in medicine Deoxyribonucleases  A to D  Depolymerase free DNA in pusreduce viscosity C5a peptidase Hyaluronidase,diphosphopyridine nucleotidase

S.pyogenes Most common cause of bacterial pharyngitis Most common cause of bacterial pharyngitis Life threatening disease caused by these flesh-eating bacteria Life threatening disease caused by these flesh-eating bacteria

Late complications Rheumatic fever Rheumatic fever Acute glomerulonephritis Acute glomerulonephritis

S.pyogenes Sensitive to penicillin Sensitive to penicillin erythromycin erythromycin

Streptococcus pyogenes: group A Streptococcus streptococcal pharyngitis Rapid antigen test:group antigen Culture (gold standart: the most sensitive and specific Two swabs

Rapid antigen test Sensitivity :58-96% Spesificity :63-100%

Diagnosis Antistreptolysin O (ASO) test:  for confirming rheumatic fever or glomerulonephritis associated with streptococcal pharyngitis Anti-Dnase B test:  for glomerulonephritis associated with pharyngitis or  soft-tissue infections

Streptococcus agalactiae/IDENTIFICATION Microscopy Culture:  Readily grow on a nutritionally enriched medium  Large colonies   -hemolysis may be absent selective broth medium with antibiotics Identification  Preliminary identification(+) CAMP test, hydrolysis of hippurate

Other beta-hemolytic streptococci Group C, F and G are most commonly associated with human disease 2 species of particular importance  S. anginosus  S. dysgalactiae

Viridans Streptococci  -hemolytic and nonhemolytic streptococci Produce green pigment on blood agar media Require complex media supplemented with blood products and inc. @ 5-10 % CO 2 atmsp.

Streptococcus pneumoniae Encapsulated, Gr + coccus Lancet-shaped cells, in pairs or short chains  -hemolytic Can grow only on enriched media (with blood products) Catalase (-) Capsule, classified accr. to polysaccharides  90 serotypes  Capsular polysaccharides are used in vaccines Teichoic acidC polysaccharide (CRP)

S. pneumoniae / diseases Pneumoniae Meningitis Sinusitis Otitis media Bacteremia

S. pneumoniae / diseases Children and the elderly are at greatest risk for meningitis People with hematologic disorder (e.g., malignancy, sickle cell disease) or functional asplenia are at risk for fulminant sepsis Although the organism is ubiquitous, disease is more common in cool months

Streptococcus pneumoniae/ Pathogenesis&Immunity The disease manifestations are caused primarily by the host response to infection

Streptococcus pneumoniae/ Pathogenesis&Immunity Colonization & migration  S.pn colonizes the oropharynx  Can spread to the lungs, paranasal sinuses, middle ear, blood stream By means of: Surface protein adhesins, secretory IgA (sIgA) protease, pneumolysin

Streptococcus pneumoniae/ Pathogenesis&Immunity Tissue destruction  Mobilization of inflamatory cellscharacteristic of pneumococcal infections Teichoic acid Peptidoglycan fragments Pneumolysin H 2 O 2 production Phosphorylcholin Phagocytic survival  Capsule  Pneumolysin mediated suppression of the phagocytosis

Streptococcus pneumoniae/ IDENTIFICATION Microscopy  Lancet-shaped, Gr (+) diplococci, unstained capsule (Gram stain with “quellung” reaction) Culture:  Enriched supplemented medium with blood  Selective medium with gentamicin Identification  Bile solubility test  Optochin

Streptococcus pneumoniae Diagnosis Microscopy is highly sensitive, as is culture, unless the patient has been treated with antibiotics Antigen test for pneumococcal C polysaccharide is sensitive with CSF (meningitis) but not with urine (meningitis, pneumonia, other infections) Nucleic-acid-based tests are not commonly used for diagnosis Culture requires use of enriched-nutrient media (e.g., sheep blood agar); organism highly susceptible to many antibiotics, so culture can be negative in partially treated patients Isolates identified by catalase (negative), susceptibility to optochin, and solubility in bile

Streptococcus pneumoniae Penicillin is the drug of choice for susceptible strains, although resistance is increasingly common Immunization is recommended for all children younger than 2 years of age and for adults at risk for disease

Enterococcus Most frequently isolated & most commonly responsible for human disease with streptococcus among gram-positive cocci “enteric cocci” Possess the group D cell wall antigen 16 species in the genus E. faecalis & E. faecium are most commonly isolated

Enterococcus/ physiology & structure They can not be differentiated from S. pneumoniae in microscope Facultatively anaerobic Optimal growth temp.= 35 o C (10 o C to 45 o C) White, large colonies on blood agar (after 24h) Nonhemolytic (or  or  -hemolysis)

Enterococcus/ Pathogenesis&Immunity Are commensal with limited potential for causing disease Do not possess toxins Cannot avoid being engulfed & killed by phagocytic cells BUT, Cause Serious Disease

Enterococcus/ Pathogenesis&Immunity Virulence factors  Adhesive factors  Bacteriocins Inherently resistant to many antibiotics

Enterococcus/ Clinical Diseases Can cause life-threatening infections One of the most feared nosocomial pathogens10% of all nos. infct. Most commonly involved sites  Urinary tract  Blood stream A sever complication: endocarditis(following bacteremia)

Enterococcus/ Laboratory diagnosis Grow readily on nonselective media Resemble S. pneumoniae  Differentiationresistant to optochin don’t dissolve when exposed to bile hydrolyze PYR pyrolidonyl-  -naphthylamide

Enterococcus Epidemiology Colonizes the gastrointestinal tracts of humans and animals; spreads to other mucosal surfaces if broad spectrum antibiotics eliminate the normal bacterial population Patients at increased risk include those hospitalized for prolonged periods and treated with broad-spectrum antibiotics (particularly cephalosporins, to which enterococci are naturally resistant)

Enterococcus Therapy for serious infections requires combination of an aminoglycoside with a cell-wall-active antibiotic (penicillin, ampicillin, or vancomycin); newer agents include linezolid, quinupristin/dalfopristin, and selected fluoroquinolones Antibiotic resistance to each of these drugs is becoming increasingly common, and infections with many isolates (particularly E. faecium) are not treatable with any antibiotics

Vancomycin resistant enterococci(VRE) The resistance in E. faecium to aminoglycosides and vancomycin is particularly troublesome, because it is mediated by plasmids and can be transferred to other bacteria.

Treatment, Prevention, and Control Newer antibiotics have been developed specifically to treat enterococci resistant to vancomycin. They include linezolid, quinupristin/dalfopristin, and selected fluoroquinolones. Unfortunately, resistance to linezolid is steadily increasing, quinupristin/dalfopristin has no activity against E. faecalis (the most commonly isolated enterococcal species), and the fluoroquinolones have poor activity against vancomycin-resistant enterococci.

Prevention, and Control It is difficult to prevent and control enterococcal infections. Careful restriction of antibiotic therapy and the implementation of appropriate infection-control practices (e.g., isolation of infected patients, use of gowns and gloves by anyone in contact with patients) can reduce the risk of colonization with these bacteria, but the complete elimination of infections is unlikely.

Hospital Infection MRSA (nose) VRE (rectal swab) Rapid detection by real-time PCR

Gram positive cocci. Learning outcomes: The student should be able to -list the medically important genera of gram-positive cocci -describe the microscobic.

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Gram positive cocci. Learning outcomes: The student should be able to -list the medically important genera of gram-positive cocci -describe the microscobic.

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Presentation on theme: "Gram positive cocci. Learning outcomes: The student should be able to -list the medically important genera of gram-positive cocci -describe the microscobic."— Presentation transcript:

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