Staphylococci - Staphylococcus aureus - dr.somesh 2015 - Bacteriology - Microbiology -

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STAPHYLOCOCCI DR. SOMESHWARAN RAJAMANI, MD

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STAPHYLOCOCCI

STAPHYLOCOCCIDr. Someshwaran rajamani, md

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IntroductionGram positive cocci arranged in grape-like clustersUbiquitous in natureMost common cause of suppurative lesions in humansPotential pathogen due to its ability to develop antibiotic resistance1871 - Von Recklinghausen first observed Staphylococci in human pyogenic lesionsJanuary 28, 20152

Introduction (Cont.)1880 - Pasteur obtained liquid cultures of the cocci from pus and produced abscess by inoculating into rabbits1880 - Sir Alexander Ogston (Scottish surgeon) named StaphylococciGreek word: Staphyle - Bunch of grapes, Greek word: kokkos - grain or berryPathogenic Staphylococci produce golden yellow coloniesNon-pathogenic Staphylococci white colonies

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Genus Staphylococcus - Gram positive cocci:In singles, pairs or irregularly as grape-like clusters- Facultative anaerobe- Catalase positive bacteria- Comprises 40 species and subspecies today

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Genus Staphylococcus

Family - MicrococcaceaeGenus - Micrococcus and StaphylococcusSpecies - Staphylococcus aureus, S.citreus, S.albus etc., January 28, 20155

Genus StaphylococciDivided into 2 sub-groups: Enzyme coagulase1. Coagulase positive Staphylococci2. Coagulase Negative Staphylococci (CONS)January 28, 20156

Genus Staphylococcus A.Coagulase positive Staphylococci:Staphylococcus aureus - Rosenbach 1884Staphylococcus intermediusStaphylococcus hyicus

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Genus Staphylococcus B.Coagulase negative Staphylococci:Staphylococcus albus (Staphylococcus epidermidis) - Rosenbach - 1884Staphylococcus citreus Passet 1885Staphylococcus saprophyticusStaphylococcus hominis, Staphylococcus capitusStaphylococcus hemolyticusJanuary 28, 20158

STRUCTURE and PHYSIOLOGYGram positive cocci in grape like clusters bcoz the cells divide along different planes and the daughter cells remain attached to one anotherNon-motileFacultative anaerobesSalt-tolerant and dessication tolerant (survival on fomites)January 28, 20159

MorphologyGram positive spherical cells, mostly arranged in irregular grape like clustersPolysaccharide capsule is rarely found on cells Peptidoglycan layer is the major structural component of the cell wall. Teichoic acid is present.January 28, 201510

Morphology (Cont.)Protein A is the major protein component of the cell wall. Released into the culture medium during the cell growth.Unique property of Protein A Ability to bind to Fc part of IgG3 (Not an antigen-antibody specific reaction)January 28, 201511

Staphylococcus aureusMorphology: Spherical cocci 1m in diameter, arranged in a grape like clusters (Cluster formation- cell division in 3 planes with daughter cells in close proximity)Non motile, nonsporing, mostly small amount of capsular material seen in non-capsulated bacteria

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Culture characteristicsGrow readily on ordinary mediaOptimum: 37C, pH 7.4-7.6 Aerobe and Facultative anaerobeColony morphology: Large 2-4mm, circular, convex, smooth, opaque an easy emulsifiable.Most strains produce golden yellow colonies on nutrient agar, some also white, orange, yellow pigments.

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Culture characteristicsPigment production is enhanced with 1% Glycerol monoacetate or milkPigment: Lipoprotein allied to carotene Nutrient agar slope: Confluent growth Oil-paint apperanceBlood agar: Hemolytic or non-hemolytic coloniesJanuary 28, 201514

Culture characteristicsMost strains incubated with 20-25% CO2 are Hemolytic.Marked hemolysis on Rabbit or sheep bloodMinimum hemolysis on horse blood agarMac Conkeys medium: Small pink lactose fermenting colonies.Liquid media: uniform turbidity

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Selective mediaLudlams media- Lithium chloride and TelluriteMannitol salt agarMilk salt agar or broth 8-10% NaClBaird Parker agarAgar containing Polymyxin BPrimary isolation: Sheep Blood Agar Plate (S-BAP)January 28, 201516

Biochemical reactionsIndole test: NegativeUrease test: positiveMethyl Red test: positiveVoge-Prauskuer test: positiveCatalase test positiveModified Oxidase test Negative (It is positive for Micrococci)January 28, 201517

Biochemical reactionsUrea hydrolysis test - positiveGelatin liquefaction test positiveLipolytic dense opacity on egg agarPhosphatase test positive (Phenolphthalein diphosphate on nutrient agar exposed to ammina vapour turn pink color due to free Phenolphthalein)

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Staphylococcus aureusCoagulase positive,Ferment mannitolClear hemolysis on Blood agarGolden yellow pigmentLiquefy gelatin; Produce phosphatasePotassium tellurite medium: Black coloniesProduce thermostable nucleasesJanuary 28, 201519

Penicillin resistane 3 typesBetalactamase production Penicillinase A,B,C,D Changes in bacterial surface receptors binding of beta lactam antibioticsDevelopment of tolerance to penicillin

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Pathogenicity and VirulenceCell Associated polymers:Polysaccharide peptidoglycanTeichoic acidCapsular polysaccharideJanuary 28, 201521

PeptidoglycanHalf of the cell wall weight is peptidoglycanSubunits of peptidoglycan is N-Acetyl muramic acid (NAM) and N-Acetyl Glucosamine (NAG)Unlike gram negative cell wall Gram positive cell wall has many cross-linked bridging layers which makes the cell wall more rigid (RIGID CELL WALL)January 28, 201522

Teichoic acidSpecies-specific Phosphate containing polymersBound covalently to peptidoglycan layer or through lipophobic linkage to the cytoplasmic membrane (Lipo-teichoic acid)It mediates attachment to mucosal surfaces through its specific binding to FibronectinJanuary 28, 201523

Capsule or polysaccharide slime layerCommonly believed to be found in-vivoOccasionally found when cultured in-vitroEleven capsular serotypes identified in Staphylococcus aureus.Serotypes 5 and 7 accounts for major infection.

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Capsule or polysaccharide slime layerProtects by inhibiting chemotaxis and phagocytosis by polymorphonuclear leukocytes; Also inhibits proliferation of mononuclear cellsFacilitates the adherence of bacteria to catheters and other synthetic metrialsJanuary 28, 201525

Protein ASurface of Staphylococcus aureus but not CONS is specially coated with Protein A covalently linked to Peptidoglycan layer.Has a unique affinity for binding Fc receptor of Immunoglobulin IgG. Protein-A detection is one of the specific test to detect Staphylococcus aureus.Protein-A coated Staphylococci used as non-specific carrier of antibodies directed against other antigens like Streptococci (Serology: Co-agglutination test).

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Pathogenicity and VirulenceCell Surface proteins:Protein AClumping factorJanuary 28, 201527

Cytoplasmic membraneMade up of complex proteins, lipids and small amount of carbohydratesServes as osmotic barrier for the cellProvides anchorage for cellular biosynthetic and respiratory enzymesJanuary 28, 201528

Pathogenicity and VirulenceExtracellular enzymes:

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Coagulase and other surface proteinsCoagulase Reacting Factor (CRF) in PlasmaClumping factor (Bound coagulase) on the outer surfaceBinds Fibrinogen converts it into insoluble fibrin Clumping or aggregates.Primary test in identifying Staphylococcus aureusOthers: Collagen binding protein, Elastin binding protein, Fibronectin binding proteinJanuary 28, 201530

Staphylococcal enzymes- Coagulase: Triggers blood clotting- Hyaluronidase: breaksdown Hyaluronic acid enables the bacteria to spread between cells- Staphylokinase: Dissolves fibrin threads in blood clots allows to free itself from blood clotsJanuary 28, 201531

Staphylococcal enzymes (Extra cellular)Coagulase , CatalaseHyaluronidaseFibrinolysinLipase, NucleasePenicillinaseBetalactamaseJanuary 28, 201532

Staphylococcal enzymes- Lipases: Digests lipids aloow them to grow on skin surfaces and in cutaneous oil glands- Beta lactamase: breaks down penicillin resistant to beta lactam antibiotics like penicillins and CephalosporinsJanuary 28, 201533

Toxins of S. aureus Alpha toxin, beta toxin, delta toxin, gamma toxin, Panton-Valentine toxin, Exfoliative toxins A,B (Exfoliative dermatitis / Staphylococcal Scalded skin syndrome SSSS, Food poisoning preformed toxin 2-6 hours, self limiting)Super antigens: A.Enterotoxins- 8 (A-E, G-I), B.Toxic Shock Syndrome Toxin (TSST-1) super antigen activates a number of T cellsJanuary 28, 201534

Staphylococcal diseasesSkin and soft tissue infections: Folliculitis, furuncle (boil), Abscess (Particlarly breast abscess), wound infection, carbuncle, impetigo, paronychia, less often cellulitis.Musculoskeletal: Osteomyelitis, Arthritis, Bursitis, PyomyositisRespiratory: Tonsillitis, pharyngitis, sinusitis, otitis, broncho-pneumonia, lung abscess, empyema, rarely pneumoniaJanuary 28, 201535

Staphylococcal diseasesCentral nervous sytem: Abscess, meningitis, intracranial thrombophlebitisEndovascular: Bacteremia, Septicemia, Pyemia, EndocarditisUrinary: Uncommon un UTI, S.saprophyticus females common (Novobiocin resistant)

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Bacteriophage typing (Staphylococcus aureus)International basic set of phages for typing Staphylococcus human (origin)Group I - 29, 52, 52A, 79, 80Group II - 3A, 3C, 55, 71Group III - 6, 42E, 47,53, 54, 75, 77, 83AGroup IV - NILGroup V - 94, 96Not allocated - 81, 95

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EpidemiologyPrimary parasites of humansColonise skin glands and mucus membranesHuman patients and carriers potent source of infectionAnimals and inanimate objects less important10-30% healthy population nasal carriers10% perineal carriers, 10% Hair carriers5-10% Vaginal carriers (during menses tampons - TSS)

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Staphylococcal carrier stateStarts early in life Colonisation of umblical cord neonatesShedders: these carriers disseminate large numbers for prolonged period.Cocci shed by patients- contaminate fomites like Hand kerchiefs, bed linen, blankets persists for days-weeksJanuary 28, 201540

Mode of infectionA. Exogenous infectionB. Endogenous infectionMode of transmission:A. Direct contactB. Indirect contact (fomites)C. Dust D. Droplet nuclei infectionJanuary 28, 201541

Nosocomial infectionHEALTHCARE OR HOSPITAL ACQUIRED INFECTIONMULTIDRUG RESISTANT BUGSMRSA- Methicillin resistant Staphylococcus AureusAlteration of PBP 2a (Penicillin binding protein 2 to 2a)Mutation of Mec A gene Short arm of Chromosome 6CA-MRSA (20%); HA-MRSA (80%) - E-MRSAJanuary 28, 201542

CHARACTERISTICS OF CA-MRSA and HA-MRSA---------------FEATURESCA-MRSAHA-MRSADefinitionCommunity acquired MRSAHospital acquired MRSAPrevalence 20%80%Persons affectedYoung and healthy persons with no recent exposure to health carePrevious contact with health care settings like hospitals, nursing homes, hemo-dialysis centersType of infectionMild to moderate severity causing skin and soft issue infection and more commonSevere invasive disease in hospitalized patients or by frequent contact with health careSite infectedSkin and soft tissues, lungsBlood stream, lung, surgical site, prosthetic implantSCC typeType IV / Type VType IIPFGE typeUSA 300, USA 400USA100, USA 200PVL geneCommonRareAntibiotic resistance patternSusceptible to many antibiotics except betalactam antibioticsMulti drug resistance observedSCC- Satellite Cassette Chromosome, PFGE- Pulsed field Gel Electrophoresis, PVL- Panton Valentine Leukocidin---------

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What are the risk factors for MRSA infections?

Injection drug abusersSkin trauma (lacerations, abrasions, tattooing etc)Higher body mass indexCosmetic body shavingPhysical contact with a person with draining lesion or a MRSA carrierIncarcerationPrevious skin infection with MRSAPrevious antibiotic useHomosexualsMilitary recruits and Mental asylum

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Lab diagnosisSamples to be collected; Transport and storageIsolation: BAP, MAC, NA; Selective media- LudlamsNutrient agar Golden yellow pigmentBiochemical Tests to be performed:Catalse, Gram stain, Tube Coagulase, Mannitol fermentation, Phosphatase, I, MR, VP, Urea hydrolysis

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Gram stain

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Nasal carriers

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AST: Cefoxitin screen for MRSA

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Treatment of MRSAHand washing, Chlorheximide sprays, Mupirocin (topical) Vancomycin, Linezolid, Teicoplanin, Ceftabipirole, Ceftaroline (5th generation cephalosporins)January 28, 201549

Topical liniments

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Conrol measures1. Isolation of patient with lesions2. Detection among carriers of infection in Health care providers3. Strict aseptic technique4. HandwashingJanuary 28, 201551

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What is the five Cs strategy suggested by CDC for MRSA prevention?

1. Avoid Crowding;2. Avoid Contact (skin-to-skin); 3. Protect Compromised skin (cuts and scrapes); 4. Clean Contaminated items and surfaces; 5. Prevent Lack of cleanliness

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Which is the national and international reference center for bacteriophage typing of Staphylococcus aureus?

- National reference center for Staphylococcal phage typing is Maulana Azad medical college, New Delhi, India.- International reference center for Staphylococcal phage typing is Centers for Disease Control and Prevention (CDC), Atlanta, United States of America.

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What method would you employ to diagnose MRSA in the laboratory?Disc diffusion by Kirby Bauer method with cefoxitin 30g antibiotic disc, where cefoxitin is the surrogate marker for oxacillin resistance Minimum Inhibitory Concentration (MIC) determination of cefoxitin (Resistant if MIC 8g/mL as per CLSI 2014) by E-test, Broth dilution method or automated system like Vitek-2 are employedOxacillin screen agar is employed to detect oxacillin sensitivity. If resistant (MRSA),Vancomycin screen agar is used to identify resistance to Vancomycin.Polymerase chain reaction (PCR) and DNA probes for mecA gene detection and confirmation of MRSA

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What is the drug of choice and alternatives available for treating MRSA?Vancomycin is effective against life threatening MRSA infectionsChlorhexidine, Bacitracin, Mupirocin ointmentNote:Linezolid is used in case of Vancomycin resistance (VRSA/VISA)Teicoplanin is the choice for Linezolid resistant Staphylococcus aureusFifth generation cephalosporins like Ceftaroline, Ceftabipirole have anti-MRSA activity and are also effective against Vancomycin Resistant Enterococci (VRE)

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What is the role of Maggot therapy/Bio-surgery for MRSA?Maggot Debridement Therapy (MDT) for chronic infected wounds by introduction of sterile medicinal larvae of specific necrotic tissue eating common blow fly or green bottle is a suitable bio-surgery aid for MRSA.

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What is the clinical significance of MRSA infections with regards to antibiotic selection? MRSA is resistant to beta lactam antibiotics like penicillins,cephalosporins and other beta lactam antibiotics.

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CONS MicrococciMS-CONSMR-CONSS. epidermidis stitch abscess, Endocarditis in drug addictsS. saprophyticus UTI Novobiocin resistantHugh-Leifson test (Oxidative)Modified oxidase positive

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Staphylococcus aureusAntibotic Susceptibility Test:MSSA- Penicillin, Ampicillin, Amoxy clav, Cloxacillin, CeftriaxoneCefoxitin surrogate marker for MRSA Kirby Bauers Disc diffusion method, MIC by E-strip method, VitekNovobiocin sensitive, Bacitracin resistant, Furazolidone sensitive.January 28, 201560

Take home message SummaryMSSA, MRSA, MS-CONS, MR-CONSHand washingDiseases producedLab diagnosisCulture selective mediaMec A gene detection by PCRBacteriophage typing

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QUIZName three coagulase positive Staphylococci?January 28, 201562

QUIZName three coagulase positive Staphylococci?1. Staphylococcus aureus2. Staphylococcus intermedius3. Staphylococcus hyicusJanuary 28, 201563

Got it???

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THANK YOUJanuary 28, 201565