Staphylococci Quiz PDF

PREFINAL | PPT & RODRIGUEZ’S BOOK | BSMT 3-B LUSAY, CHLOE MARIZ L. STAPHYLOCOCCI ⇨ Genus I ▪ Derived from the Greek word...

PREFINAL | PPT & RODRIGUEZ’S BOOK | BSMT 3-B LUSAY, CHLOE MARIZ L. STAPHYLOCOCCI ⇨ Genus I ▪ Derived from the Greek word “staphle”, meaning ⇨ Family V: Staphylococcaceae “bunches of grapes” and “kokkos” that means ⇨ Order I: Bacillales “berry” ---- bacteria occurring in grapelike clusters or ⇨ Class III: Bacilli berry. ▪ Staphylococci are Gram-positive cocci, belong to the Difference between Staphylococcus and Micrococcus: family Micrococcaceae. Characteristics/Tests Micrococcus Staphylococcus ▪ Are normal inhabitant of the skin, mucous membrane, Morphology Large Gram-positive Gram-positive cocci, usually arranged in cocci in clusters, and intestine. tetrads or in pairs. sometimes in ▪ Are common isolates and responsible for several pairs and short suppurative types of infections. chains. ▪ Are catalase-producing bacteria; facultatively Clinical significance Usually considered Bacteria of this contaminants of clinical genus are of anaerobic except S. saccharolyticus which is an specimen; rarely primary clinical obligate anaerobe. implicated as cause of significance. ▪ Non-motile, non-spore-forming, and glucose infections. May cause fermenter. opportunistic infection in S. aureus is a an immunocompromised notorious ▪ Rare strains are fastidious requiring CO2, hemin, or host. pathogen. menadione for growth (small-colony variants with 1/10 the size of the wild type strains after 48 hours of S. saprophyticus causes incubation). honeymoon ▪ Microscopy: spherical cells that appear singly, in pairs, cystitis. and in clusters. Lysostaphin Not lysed with lysostaphin Lysed with ▪ Culture: BAP-colonies (4mm-8mm) on agar plate Sensitivity Test (resistant) lysostaphin (sensitive) appear creamy, white or light gold, and “buttery- Furazolidone Resistant to antibiotic Susceptible to looking”; other species have gravy colonies; some Sensitivity Test furazolidone antibiotic species are B-hemolytic, such as S. aureus. furazolidone Bacitracin Sensitivity Susceptible to 0.04 U of Resistant to 0.04 Test bacitracin U of bacitracin HISTORY Microdase Test Microdase positive Microdase › Robert Koch (1878) – first to see staphylococci in pus negative specimen Oxygen Only grow aerobically Facultative › Louis Pasteur (1880) – first to cultivate/culture in liquid requirements (obligate aerobe) anaerobic (can medium use either aerobic › Sir Alexander Ongston (1881) – named the bacteria respiration “staphylococcus”; staphle→bunch of grapes, and/or kokkos→berry fermentation depending on › von Recklinghausen (1871) – first observed in pus the availability of › Rosenbach (1884) – named S. aureus (golden yellow oxygen, does not colonies), S. albus (white colonies) solely depend on › Passet (1885) – S. citreus (lemon yellow colonies) aerobic respiration for growth). TAXONOMY Carbohydrate Utilize sugars oxidatively or Fermentative › Staphylococci are Gram-positive cocci, classified utilization not at all in the O/F test. into 2 families (as per 1986 edition of Bergey’s Manual of Systemic Bacteriology): Current Classification of Staphylococci ⇨ Micrococcaceae (GPC, catalase positive) ▪ 32 species ⇨ Streptococcaceae (GPC, catalase negative) ▪ 15 sub species › Micrococcaceae included 4 genera: Coagulase positive Coagulase negative ➔ Staphylococcus aureus ➔ Staph. epidermidis ⇨ Planococcus ➔ Staph intermedius ➔ Staph. hemolyticus ⇨ Micrococcus ➔ Staph pseudintermedius ➔ Staph saprophyticus ⇨ Stomatococcus ➔ Staph hyicus ➔ Staph. hominis Commensal ⇨ Staphylococcus ➔ Staph delphini ➔ Staph. capitis flora › Newer edition suggests that Staphylococci are: ➔ Staph lutrae ➔ Staph agnetis ⇨ Phylum: Firmicutes 1|P a g e ∇VII ∆VIII  PREFINAL | PPT & RODRIGUEZ’S BOOK | BSMT 3-B LUSAY, CHLOE MARIZ L. ➔ Some strains of Staph ➔ blood agar schleiferi ⇨ marked hemolysis on sheep blood agar ⇨ 20-25% Co2 ⇨ Rabbit/sheep blood Staphylococcus aureus ➔ Mac Conkey agar Commensal flora a. Pink colonies due to lactose fermentation MORPHOLOGY ➔ Liquid media ➔ Gram-positive, spherical cells, mostly arranged in a. Uniform turbidity irregular grape-like clusters. ➔ Selective medium ➔ Polysaccharide capsule is only rarely found on cells. a. Salt milk agar ➔ The peptidoglycan layer is the major structural b. Salt broth Commensal component of the cell wall. It is important inflora the c. Ludlam’s medium (Lithium chloride, Tellurite) pathogenesis of staphylococcal infections. Other important component of cell wall is teichoic acid. ➔ Protein A is the major protein component of the cell wall. It is located on the cell surface but is also released into the culture medium during the cell growth. A unique property of protein A is its ability to bind to the Fc part of all IgG molecules except IgG3. It is not an antigen-antibody specific reaction. ➔ Virulence factors: ⇨ Enterotoxins ⇨ Toxic Shock Syndrome Toxin 1 ⇨ Exfoliative toxin ⇨ Cytolytic toxins BIOCHEMICAL REACTIONS ⇨ Enzymes Catalase → Positive Indole → Negative ⇨ Protein A MR (Methyl Red) → Positive VP → Positive Additional info. for Staphylococcus aureus: Urease → Positive › Most virulent species; coagulase (+) Nitrate reduction → Positive › aureus means “gold” Mannitol fermentation → Positive › Culture: golden yellow pigment (lipochrome); Phosphatase production → Positive B-hemolytic—BAP › Can be cultivated with an added 7.5-10% NaCl (halophilic microorganism). RELATED INFECTIONS AND DISEASES › Chiefly responsible for the various skin, wound, 1. Toxin-induced cases → food poisoning, scalded and deep tissue infections. skin syndrome (SSS), and toxic shock syndrome CULTURE CHARACTERISTICS (TSS). a. Scalded skin syndrome (SSS) → an ➔ Grow on ordinary media. extensive exfoliative dermatitis that ➔ Temp. 10-42°C, optimum is 37°C occurs primarily in newborns and ➔ pH range 7.4-7.6 previously healthy children ➔ aerobes ▪ Epidermolytic toxin → toxin ➔ facultative anaerobes produced ➔ nutrient agar b. Toxic shock syndrome (TSS) → a rare a. large, circular, smooth, shiny, opaque, and but potentially fatal, multi-system easily emulsifiable colonies disease that is characterized by a b. yellow pigment which does not diffuse into sudden onset of fever, chills, vomiting, medium diarrhea, muscle aches, and rashes, c. pigment is carotene like lipoprotein which rapidly progresses to d. pigment production is best at 22°C or when hypotension and shock. glycerol monoacetate/milk is incorporated ✓ Associated with women who use into medium tampons ➔ nutrient slope a. oil paint appearance 2|P a g e ∇VII ∆VIII  PREFINAL | PPT & RODRIGUEZ’S BOOK | BSMT 3-B LUSAY, CHLOE MARIZ L. Pre-formed heat-resistant enterotoxin ▪ Impetigo → a very superficial skin infection common mediates staphylococcal food in children, usually produces blisters or sores on the poisoning (symptoms in 2-6 hours; face, neck, hands, and diaper area. usually self-limiting) ➔ Characterized by watery bristles, Exfoliative toxins A and B results in which becomes pustules and then staphylococcal scalded skin honey-colored crust. syndrome; usually in infants and 2. Deep Infections neonates. ▪ Osteomyelitis → inflammation of bone Ritter disease / SSS → produces ➔ Bacteria can get to the bone purulent material in the lesion 2. Bacteremia and sepsis ⇨ Via bloodstream 3. Urinary tract infection ⇨ Following an injury 4. Acute bacterial endocarditis 5. Cutaneous infections › Clinical features: pain, swelling, a. Folliculitis → mild inflammation of the deformity, defective healing, & in some hair follicle or sebaceous gland cases, pus follow b. Furuncles (Boils) → are large superficial › Diagnosis: X-ray, MRI, & bone aspirates abscesses which can be an extension of folliculitis ENZYMES AND TOXINS PRODUCED (VIRULENCE c. Carbuncles → develop from multiple FACTORS) furuncles which may advance into By Staph. aureus deeper tissues and cause fever and chills leading to systemic infection 1. Coagulase d. Impetigo → a superficial cutaneous ➔ Coagulates the fibrinogen in the plasma. infection commonly seen in children ➔ Promotes the formation of a fibrin layer around the and characterized by crusty lesions staphylococci abscess thereby protecting the and vesicles surrounded by a red bacteria from phagocytosis border. ➔ There are 2 types: e. Purulent abscess ⇨ Cell-bound coagulase or Clumping factor 6. Osteomyelitis → secondary to bacteremia → is bound to the cell wall and clots 7. Septic arthritis (children) human, rabbit, or pig plasma by directly 8. Food poisoning → enterotoxins A (78%), D (38%) converting fibrinogen into fibrin. and B (10%) ⇨ Unbound or Free coagulase → an extracellular enzyme that is not bound to 1. Cutaneous Infections the cell wall and causes clot formation ▪ Folliculitis → an inflammation of the hair follicles when bacterial cells are incubated with →a small red bump or pimple develops plasma. at infection sites of hair follicle 2. Hyaluronidase (Spreading-factor enzyme) ▪ Sty → a folliculitis affecting one or more hair follicles ➔ Enhances invasion and survival in the tissue. on the edge of the upper or lower eyelid ➔ Breaks down the hyaluronic acid that is present in ▪ Furuncle/Boils → a deep-seated infection, the intracellular ground substances of connective originating from folliculitis, (if infection extends from tissues, resulting in the spread of bacteria. follicle to neighbor tissue) ➔ Binds cells together and renders the intercellular ➔ Causes redness, swelling, & severe spaces passable to pathogen. pain ➔ Commonly found on the neck, 3. Staphylokinase (Fibrinolysin) armpit, and groin regions ➔ Causes fibrinolytic activities by dissolving fibrin clots ▪ Carbuncle → an aggregation of infected furuncles; 4. Lipase (Fat-splitting enzyme) may form large abscesses. ➔ Produced by both coagulase-positive and ➔ A large area of redness, swelling, coagulase-negative staphylococci. and pain, punctuated by several ➔ Essential for bacterial survival in sebaceous areas of sites of drainage pus. the body, and important in the formation of furuncles, carbuncles, and boils. 3|P a g e ∇VII ∆VIII  PREFINAL | PPT & RODRIGUEZ’S BOOK | BSMT 3-B LUSAY, CHLOE MARIZ L. 5. Deoxyribonuclease (Dnase) and Phosphatase sloughing of the epidermidis to produce a burn-like effect on the patient. ➔ Lowers the viscosity of exudates giving the ➔ Destroys the stratum granulosum and causes pathogen more morbidity and destroys DNA. scalded-skin syndrome (SSS) or Ritter’s disease. 6. B-lactamase 11. Toxic shock syndrome toxin 1 (TSST-1) / ➔ Breaks down penicillin and other B-lactam drugs. Enterotoxin F / Pyogenic exotoxin ➔ More than 90% of clinical staphylococci isolates are penicillin-resistant as a result of enzyme production. ➔ A chromosomal-mediated toxin and causes almost all cases of menstruation-associated TSS. 7. Enterotoxin (Heat-stable) ➔ Stimulates the production of a large number of ➔ Acts as neurotoxins that stimulate vomiting through cytokines that are responsible for the symptoms. vagus nerve, produced by the majority of Staph. 12. Protein A aureus isolates. ➔ Produced by 30-50% of isolates. ➔ Immunologically active substance found in the cell ➔ Stable heating at 100°C for 30 minutes. wall. ➔ Resistant to hydrolysis by gastric and jejunal ➔ Antiphagocytic by competing with neutrophils for the Fc portion of specific opsonin. enzymes. ➔ Examples of enterotoxins: A (78%), B, C1, C2, D, E, & G to J Differential Tests for Staph. aureus ➔ A, B, & D are responsible for food poisoning. 1. Coagulase test ➔ Enterotoxin B is associated with ➔ Best single criterion of pathogenicity of Staph. pseudomembranous enterocolitis. aureus. ➔ Signs and symptoms: diarrhea, abdominal pain, ➔ Reagent: Rabbit plasma and vomiting; no fever associated. ➔ Anticoagulant: Ethylenediaminetetraacetic acid (EDTA) 8. Leukocidin / Panton-Valentine Leukocidin ➔ Two methods: slide method & tube method (Cytolytic toxin) ➔ A type of exotoxin; lethal to PMNs. a. Slide method → used to screen catalase positive colonies, detects cell bound ➔ Attacks and kills WBCs, pore-forming exotoxin that coagulase or clumping factor. suppresses phagocytosis and responsible for ➔ Positive result: clot or coagulum necrotizing skin and soft tissue infections. formation within 30 seconds 9. Hemolysin (Cytotoxin) ➔ Other slide coagulase (+) organisms: Staph. lugdunensis and ➔ Causes anemia and make iron available for Staph. schleiferi microbial growth. ➔ Four types of hemolysins: b.Tube method → considered sensitive but ⇨ Alpha-hemolysin (a) → predominantly definitive method, detects extracellular or free coagulase. lysine produced by S. aureus; destroys ➔ Procedure: inoculate a tube RBCs, platelets, and macrophages, and containing plasma and incubate causes severe tissue damage. at 35°C ⇨ Beta-hemolysin (β) → destroys ➔ Positive result: clot or coagulum sphingomyelin and RBC around nerves; has formation after 1 to 4 hours of enhanced hemolytic activity on incubation incubation ➔ If no clot appears after 4 hours of at 35°C. incubation, the tube should be left ⇨ Gamma-hemolysin (γ) → less toxic than at room temp. for an additional 20 alpha and beta-lysins, produced by all S. hours of incubation. aureus strains that cause RBC injury in ➔ Other tube coagulase (+) culture and produces edematous lesions. organisms: Staph. hyicus, Staph. intermedius, S. delphini, and S. ⇨ Delta-hemolysin (δ) → destroys RBC and is schleiferi subsp. coagulans associated with the Panton-Valentine Notes to remember: leukocidin. › Citrate-utilizing organisms may yield false positive results; plasma containing EDTA rather 10. Exfoliatin serotypes A and B (Superantigens) / than citrate should be used. Epidermolytic toxins A and B ➔ A serin protease that divides the intracellular bridges of the epidermidis and causes extensive 4|P a g e ∇VII ∆VIII  PREFINAL | PPT & RODRIGUEZ’S BOOK | BSMT 3-B LUSAY, CHLOE MARIZ L. spectrum of antibiotics treatments, and exposure to nasal secretions. ➔ Can be controlled by proper isolation of the organism, rapid identification of the bacteria, hand hygiene, treatment of sources, and most importantly, a strict compliance to infection control programs. ➔ Chromogenic test: (+) mauve-colored colonies within 24 hours and confirmed within 48 hours ➔ Rapid tests: IDI-MRSA test and BD Gene Ohm assay (results w/in 2 hours) using nasal swab specimen 2. Mannitol fermentation test ➔ Three types of MRSA: ➔ Used to differentiate the pathogenic staph. from ⇨ Hospital-acquired MRSA / (HA) MRSA non-pathogenic ones; pathogenic staphylococci ⇨ Community-acquired MRSA / (CA) MRSA ferment mannitol and produce acid. ⇨ Health care associated community onset ➔ Culture medium: mannitol salt agar MRSA / (HACO) MRSA ➔ MSA (1% mannitol + 7.5% NaCl) is both a selective ➔ Positive Chromogenic test: change in color of MRSA and differential medium. colonies within 24-48 hours using CHROM agar against ➔ pH indicator: phenol red (will turn to yellow color) colorless colonies of non-MRSA ➔ Positive result: yellow-colored S. aureus colonies ➔ S. saprophyticus→ some strains also ferment ✓ if the color change = non-MRSA mannitol (resembles S. aureus on MSA) ✓ no color change = MRSA NOTES TO REMEMBER: › Resistance of Staphylococcus to penicillinase-resistant penicillin is due to an altered penicillin binding protein known as PBP2a or PBP2’ in the cell wall which is encoded by the mecA gene. › PBP2a has low affinity for binding all B-lactams drugs. › Altered PBP does not bind oxacillin, rendering the drug ineffective. Durham tube – to detect gas produced by › Isolates of oxacillin-resistant S. aureus are commonly microorganisms referred to as MRSA. 3. Tellurite glycine agar › Resistance of staphylococci to penicillinase-resistant ➔ This results in jet black colonies of S. aureus penicillin occurs in up to 80% of coagulase-negative 4. Polymyxin sensitivity test staphylococci and up to 50% of S. aureus. ➔ S. aureus is resistant to this test › Heteroresistant strains → all cells in the population 5. Voges-Proskauer (VP) test have the genetic element (mecA gene) for oxaxillin ➔ Differentiate S. aureus from S. intermedius resistance, but not all cells express this resistance by ➔ Positive result: acetoin/acethylmethyl carbinol virtue of PBP2a production. production → pink color › Borderline-oxacillin-resistant isolates → generally 6. Deoxyribonuclease (DNAse) test have MICs right above the breakpoint for oxacillin- ➔ Used to identify pathogenic species of susceptibility. staphylococci that produces DNAse. ➔ Culture medium: DNA-methyl green agar ➔ Positive result: clear/colorless zone around the test organism Coagulase negative Staphylococcus (CoNS) Additional info: ▪ S. epidermidis & S. saprophyticus a. 0.1 N HCl → agglutinate or precipitate protein b. 0.1% toluidine blue → (+) pink color = cell died ➔ They are coagulase-negative staphylococci (-) blue color = cell is alive ➔ Do not produce exotoxins ➔ Thus, they do not cause food poisoning or toxic shock syndrome Methicillin-resistant Staphylococcus aureus (MRSA) S. epidermidis ➔ Resistant to antibiotics such as methicillin, nafcillin, and ➔ Part of the normal flora of the skin and mucous oxacillin. membrane. ➔ Can be acquired after a prolonged stay in the ➔ Cause of hospital-acquired infections. hospital, close contact with individuals who are ➔ Involved in indwelling catheters, prosthetic materials, carriers of the organism, after-effects of a broad shunts, and surgery. 5|P a g e ∇VII ∆VIII  PREFINAL | PPT & RODRIGUEZ’S BOOK | BSMT 3-B LUSAY, CHLOE MARIZ L. ➔ Common cause of prosthetic heart valve ➔ Contains the mecA gene that codes for oxacillin endocarditis. resistance. ➔ Also causes nosocomial bacteremia; sepsis in ➔ Related infections include infective endocarditis, neonates, peritonitis in patients with renal failure; meningitis, septicemia, UTI, and skin and soft tissue cerebrospinal fluid shunt infections. infections. ➔ Often multiple antibiotic resistance →Methicillin Additional info: Additional information for S. epidermidis: › Can cause both community-associated and › Secretes poly-gamma-DL-glutamic acid that hospital-acquired infections. provides adherence to devices. › Can be more virulent and can clinically mimic S. › Has been known to cause various hospital- aureus infections. acquired infections. › Culture: small to medium-sized, non-hemolytic, Novobiocin susceptible CoNS Novobiocin resistant CoNS non-pigmented, white opaque, pin-head species species colonies → BAP ⇨ S. epidermidis ⇨ S. saprophyticus › Biochemical test: MSA (-); coagulase-negative ⇨ S. capitis ⇨ S. cohnii (CoNS) ⇨ S. haemolyticus ⇨ S. kloosii › Antimicrobial test: susceptible with 5 ug ⇨ S. hominis ⇨ S. xylosus Novobiocin (16-27mm) ⇨ S. lugdunensis ⇨ S. saccharolyticus S. saprophyticus ⇨ S. warneri ➔ Commonly isolated from animals and their carcasses. ➔ Coagulase and phosphatase negative Other Coagulase-negative Staphylococci: ➔ Urease and lipase positive ▪ S. warneri ➔ Resistant to the antibiotic Novobiocin, a characteristic ▪ S. capitis used in lab to distinguish it from S. epidermidis ▪ S. simulans ➔ Causes mainly UTI, particularly in sexually active young ▪ S. hominis women ▪ S. schleiferi ➔ 2nd most common cause of UTI, after E. coli in young women accounting for 10-20%. Resistant Genes Produced by Staphylococci ➔ Quinolones are commonly used in treatment of S. 1. erm genes saprophyticus UTI. ➔ Are class of enzymes inactivating genes, code for methylation of the 23s rRNA, Additional info: resulting to resistance to erythromycin › Associated with community-acquired UTI in and either inducible or constitutive young, sexually active females. resistance to clindamycin. › Adheres more effectively to the epithelial cells ➔ May not be detected in routine lining the urogenital tract than other CoNS. susceptibility testing. › Rarely found on other mucous membranes or skin ➔ Also confer cross resistance to the surfaces. macrolides (erythromycin) and › Culture: white opaque, pin-head slightly larger streptogramins (quinupristin) colonies; 50% of strains produce yellow pigment; 2. msr A gene non-hemolytic → BAP ➔ Codes for an efflux mechanism, that › Biochemical tests: MSA (-/+); coagulase-negative; results in resistance to erythromycin but susceptibility to clindamycin. DNAse negative › Urine culture: 10, 000 CFU/mL (significant findings) o 25 mm is susceptible and 24 mm is › ESLB-producing bacteria → should be considered resistant. resistant to all cephalosporins, penicillin, & ➔ All oxacillin resistant staphylococci must be aztreonam. reported as resistant to all B-lactam drugs. b. Double-Disk Diffusion Test (D Test) ➔ Used to detect inducible clindamycin resistance in staphylococci. ➔ An isolate of S. aures that is resistant to erythromycin but susceptible to clindamycin in a MIC test should be evaluated for inducible resistant to clindamycin by the D test. Procedure: 1. Place a 15 ug erythromycin disk and a 2 ug clindamycin disk 15 mm to 26 mm apart on a BAP with the isolate. 2. Incubate overnight at 35°C. Result: ▪ Positive → flattening of the one side (near erythromycin disk) of the clindamycin zone of inhibition, giving the appearance of “D zone” due to erm gene. ▪ Negative → without “blunting” indicates erythromycin resistant due to msr A gene. c. Macro E test ➔ For detection of heteroresistant vancomycn- intermediate S. aureus (VISA) since the test uses a higher conc. of the organism (1𝑥108 𝑏𝑎𝑐𝑡𝑒𝑟𝑖𝑎/𝑚𝐿) d. Broth Microdilution Test / Vancomycin Agar Screen ➔ Best method to detect either vancomycin-resistant S. aureus (VRSA) or VISA. e. Oxacillin-salt agar Plate ➔ Used to screen for MRSA in clinical samples, ➔ Used to differentiate MRSA isolates from hyperproducers of B-lactamase known as borderline oxacillin-resistant S. aureus (BORSA) strains, which won’t grow on this plate. ➔ Not recommended to screen CoNS. 8|P a g e ∇VII ∆VIII