STAPHYLOCOCCI

DR. 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 lesions

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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 - Micrococcaceae Genus - Micrococcus and Staphylococcus Species - Staphylococcus aureus, S.citreus, S.albus etc.,

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Genus Staphylococci Divided into 2 sub-groups: Enzyme coagulase 1. Coagulase positive Staphylococci 2. Coagulase Negative Staphylococci (CONS)

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

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Genus Staphylococcus B.Coagulase negative Staphylococci: Staphylococcus albus (Staphylococcus epidermidis) - Rosenbach - 1884 Staphylococcus citreus – Passet – 1885 Staphylococcus saprophyticus Staphylococcus hominis, Staphylococcus capitus Staphylococcus hemolyticus

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STRUCTURE and PHYSIOLOGY Gram positive cocci in grape like clusters b’coz the cells divide along different planes and the daughter cells remain attached to one another Non-motile Facultative anaerobes Salt-tolerant and dessication tolerant (survival on fomites)

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Morphology Gram positive spherical cells, mostly arranged in irregular grape like clusters Polysaccharide capsule is rarely found on cells Peptidoglycan layer is the major structural component of the cell wall. Teichoic acid is present.

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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)

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Staphylococcus aureus Morphology: Spherical cocci 1µm 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 characteristics Grow readily on ordinary media Optimum: 37⁰C, pH 7.4-7.6 – Aerobe and Facultative anaerobe Colony 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 characteristics Pigment production is enhanced with 1% Glycerol monoacetate or milk Pigment: Lipoprotein allied to carotene Nutrient agar slope: Confluent growth – ‘Oil-paint apperance’ Blood agar: Hemolytic or non-hemolytic colonies

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Culture characteristics Most strains incubated with 20-25% CO2 are Hemolytic. Marked hemolysis on Rabbit or sheep blood Minimum hemolysis on horse blood agar Mac Conkey’s medium: Small pink lactose fermenting colonies. Liquid media: uniform turbidity

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Selective media Ludlam’s media- Lithium chloride and Tellurite Mannitol salt agar Milk salt agar or broth – 8-10% NaCl Baird – Parker agar Agar containing Polymyxin B Primary isolation: Sheep Blood Agar Plate (S-BAP)

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Biochemical reactions Indole test: Negative Urease test: positive Methyl Red test: positive Voge-Prauskuer test: positive Catalase test – positive Modified Oxidase test – Negative (It is positive for Micrococci)

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Biochemical reactions Urea hydrolysis test - positive Gelatin liquefaction test – positive Lipolytic – dense opacity on egg agar Phosphatase test – positive (Phenolphthalein diphosphate on nutrient agar – exposed to ammina vapour – turn pink color due to free Phenolphthalein)

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Staphylococcus aureus Coagulase positive, Ferment mannitol Clear hemolysis on Blood agar Golden yellow pigment Liquefy gelatin; Produce phosphatase Potassium tellurite medium: Black colonies Produce thermostable nucleases

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Penicillin resistane – 3 types1. Betalactamase production – Penicillinase A,B,C,D 2. Changes in bacterial surface receptors binding of beta

lactam antibiotics3. Development of tolerance to penicillin

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Pathogenicity and Virulence Cell Associated polymers:a. Polysaccharide peptidoglycanb. Teichoic acidc. Capsular polysaccharide

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Peptidoglycan Half of the cell wall weight is peptidoglycan Subunits 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)

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Teichoic acid Species-specific Phosphate containing polymers Bound 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 Fibronectin

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Capsule or polysaccharide slime layer Commonly believed to be found in-vivo Occasionally found when cultured in-vitro Eleven capsular serotypes identified in Staphylococcus aureus. Serotypes 5 and 7 – accounts for major infection.

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Capsule or polysaccharide slime layer Protects by inhibiting chemotaxis and phagocytosis by polymorphonuclear leukocytes; Also inhibits proliferation of mononuclear cells Facilitates the adherence of bacteria to catheters and other synthetic metrials

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Protein A Surface 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 Virulence Cell Surface proteins:a. Protein Ab. Clumping factor

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Cytoplasmic membrane Made up of complex proteins, lipids and small amount of carbohydrates Serves as osmotic barrier for the cell Provides anchorage for cellular biosynthetic and respiratory enzymes

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Pathogenicity and Virulence Extracellular enzymes:

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Coagulase and other surface proteins Coagulase Reacting Factor (CRF) in Plasma Clumping factor (Bound coagulase) on the outer surface Binds Fibrinogen – converts it into insoluble fibrin – Clumping or aggregates. Primary test in identifying Staphylococcus aureus Others: Collagen binding protein, Elastin binding protein, Fibronectin binding protein

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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 clots

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Staphylococcal enzymes (Extra cellular) Coagulase , Catalase Hyaluronidase Fibrinolysin Lipase, Nuclease Penicillinase Betalactamase

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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 Cephalosporins

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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 cells

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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 pneumonia

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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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Epidemiology•Primary parasites of humans•Colonise skin glands and mucus membranes•Human patients and carriers – potent source of infection•Animals and inanimate objects – less important•10-30% healthy population – nasal carriers•10% perineal carriers, 10% Hair carriers•5-10% Vaginal carriers (during menses – tampons - TSS)

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Staphylococcal carrier state•Starts early in life •Colonisation of umblical cord – neonates•Shedders: these carriers disseminate large numbers for prolonged period.•Cocci shed by patients- contaminate fomites like Hand kerchiefs, bed linen, blankets – persists for days-weeks

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Mode of infection A. Exogenous infection B. Endogenous infection Mode of transmission: A. Direct contact B. Indirect contact (fomites) C. Dust D. Droplet nuclei infection

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Nosocomial infection HEALTHCARE OR HOSPITAL ACQUIRED INFECTION MULTIDRUG RESISTANT BUGS MRSA- Methicillin resistant Staphylococcus Aureus Alteration of PBP 2a (Penicillin binding protein 2 to 2a) Mutation of Mec A gene – Short arm of Chromosome 6 CA-MRSA (20%); HA-MRSA (80%) - E-MRSA

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CHARACTERISTICS OF CA-MRSA and HA-MRSA---------------

FEATURES CA-MRSA HA-MRSA

Definition Community acquired MRSA Hospital acquired MRSA

Prevalence 20% 80%

Persons affected Young and healthy persons with no recent exposure to health care Previous contact with health care settings like hospitals, nursing homes, hemo-dialysis centers

Type of infection Mild to moderate severity causing skin and soft issue infection and more common

Severe invasive disease in hospitalized patients or by frequent contact with health care

Site infected Skin and soft tissues, lungs Blood stream, lung, surgical site, prosthetic implant

SCC type Type IV / Type V Type II

PFGE type USA 300, USA 400 USA100, USA 200

PVL gene Common Rare

Antibiotic resistance pattern

Susceptible to many antibiotics except betalactam antibiotics Multi drug resistance observed

SCC- 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 abusers

Skin trauma (lacerations, abrasions, tattooing etc)

Higher body mass index

Cosmetic body shaving

Physical contact with a person with draining lesion or a MRSA carrier

Incarceration

Previous skin infection with MRSA

Previous antibiotic use

Homosexuals

Military recruits and Mental asylum

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Lab diagnosis Samples to be collected; Transport and storage Isolation: BAP, MAC, NA; Selective media- Ludlam’s Nutrient agar – Golden yellow pigment Biochemical 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 MRSA Hand washing, Chlorheximide sprays, Mupirocin (topical) Vancomycin, Linezolid, Teicoplanin, Ceftabipirole, Ceftaroline (5th generation cephalosporins)

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Topical liniments

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Conrol measures 1. Isolation of patient with lesions 2. Detection among carriers of infection in Health care providers 3. Strict aseptic technique 4. Handwashing

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What is the five C’s 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 30µg antibiotic disc, where cefoxitin is the surrogate marker for oxacillin resistance

Minimum Inhibitory Concentration (MIC) determination of cefoxitin (Resistant if MIC ≥8µg/mL as per CLSI 2014) by E-test, Broth dilution method or automated system like Vitek-2 are employed

Oxacillin 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 infections Chlorhexidine, Bacitracin, Mupirocin ointment Note: Linezolid is used in case of Vancomycin resistance (VRSA/VISA) Teicoplanin is the choice for Linezolid resistant Staphylococcus aureus

Fifth 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 Micrococci MS-CONS MR-CONS S. epidermidis –stitch abscess, Endocarditis in drug addicts S. saprophyticus – UTI – Novobiocin resistant Hugh-Leifson test (Oxidative) Modified oxidase positive

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Staphylococcus aureus Antibotic Susceptibility Test: MSSA- Penicillin, Ampicillin, Amoxy clav, Cloxacillin, Ceftriaxone Cefoxitin – surrogate marker for MRSA Kirby – Bauer’s Disc diffusion method, MIC by E-strip method, Vitek Novobiocin sensitive, Bacitracin resistant, Furazolidone sensitive.

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Take home message Summary MSSA, MRSA, MS-CONS, MR-CONS Hand washing Diseases produced Lab diagnosis Culture – selective media Mec A gene detection by PCR Bacteriophage typing

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QUIZ Name three coagulase positive Staphylococci?

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QUIZ Name three coagulase positive Staphylococci? 1. Staphylococcus aureus 2. Staphylococcus intermedius 3. Staphylococcus hyicus

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Got it???

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THANK YOU