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INTERNSHIP 2ND ROTATION:BACTERIOLOGY NOTESThis handout contains the ff.:  Gram-positive microorganisms, disease-associated, and differential tests  Gram-negative microorganisms, disease-associated, and differential tests  Enterobacteriaceae and biochemical tests  Culture and colony morphologyLeg...

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INTERNSHIP 2ND ROTATION: BACTERIOLOGY NOTES This handout contains the ff.:  Gram-positive microorganisms, disease-associated, and differential tests  Gram-negative microorganisms, disease-associated, and differential tests  Enterobacteriaceae and biochemical tests  Culture and colony morphology Legends:  (+): positive reaction  (-): negative reaction  (R): resistant  (S): susceptible  (V): variable  LF: Lactose fermenter  LLF: Late-lactose fermenter  NLF: Non-lactose fermenter  BAP: Blood Agar Plate  CAP: Chocolate Agar Plate  MSA: Mannitol Salt Agar  CAMP: Christie, Atkins, MunchPeterson Agar (and other culture media abbreviations)

GRAM-POSITIVE MICROORGANISMS 1. Staphylococcus aureus o Common bacterial pathogen o Normal flora in the axillae, inguinal, and perineal area o Considered to be an opportunistic pathogen o Produces Beta-lactamases o Resistant to penicillin

1 Bacteriology Internship Notes Rojo, K.F.

o Drug of choice: penicillinaseresistant penicillin drugs: oxacillin, cloxacillin, and methicillin

Disease-associated: - Food poisoning - Scalded skin syndrome (SSS) - Toxic shock syndrome toxin-1 (TSST-1) - Wound/skin infxns - Osteomyelitis - Nosocomial infxn - IV-drug user associated endocrditis  Virulence factors: - Enterotoxins B, A, D (food poisoning) - Epidermotoxins A, B (scalded skin syndrome) - TSST-1 (toxic shock syndrome toxin-1)  Identification: - Medium to large colonies - Smooth, butyrous, creamy appearance - Pigment: white to golden yellow - Edge: entire - Beta-hemolytic on BAP  Differential/Biochemical tests: - Catalase (+) - Coagulase (bound +) (free +) - DNAse (+): - Thermostable nuclease (+) - MSA: for growth and fermentation  Media used: - BAP - CNA - MSA (See culture and colony morphology on the latter part of this handout) 

o Has the ability to adhere to 2. Staphylococcus epidermidis o Normal flora of the skin and mucous membranes of humans and other animals. o Common in hospital machines o Associated with prosthetic valve endocarditis o Can cause UTI through catheter  Disease-associated: - Prosthetic valve endocarditis - Bacteremia and septicemia - UTI in catheter infxnx  Virulence factors: - Biofilm formation/Slime layer  Identification: - White, creamy, and raised colonies on BAP - Gamma-hemolytic on BAP  Differential/Biochemical Tests: - Coagulase (-) - DNAse (-) - CNA growth (+) - MSA growth, but NLF - Novobiocin (S)  Media used: - BAP - MSA (NLF) - CNA (See culture and colony morphology on the latter part of this handout)

3. Staphylococcus saprophyticus o Community-acquired UTI in sexually active women 2 Bacteriology Internship Notes Rojo, K.F.

epithelial cells of urinary tract and urethral cells.

 Disease-associated: - UTI in sexually active women - Catheter-associated UTI  Differential/Biochemical Tests: - Coagulase (-) - DNAse (-) - Gamma-hemolytic on BAP - MSA (V) - Novobiocin (R)  Media used: - BAP - MSA (See culture and colony morphology on the latter part of this handout)

4. Streptococcus pyogenes (Group A) o Most pathogenic o Acquired through person-toperson contact or aerosol droplets of coughing and sneezing o Resistant to drying o Fever-producing bacteria o Flesh-eating (F protein) o Sequelae: rheumatic heart disease and acute glomerulonephritis (M protein)  Disease-associated: - Pharyngitis & Tonsilitis “strep throat” - Scarlet Fever (scarlatina) - Skin infxns (cellulitis, erysipelas, impetigo) - Impetigo may lead to Necrotizing fasciitis - Rheumatic fever

- Acute glomerulonephritis (Bright’s dse) - Streptococcal TSS 

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Virulence factors: - M protein (attaches to the peptidoglycan; sequelae) - F protein (flesheating) - Hyaluronic acid (antiphagocytic; present in capsule) Extracellular Products: - Streptolysin O (O2 labile) - Streptolysin S (O2 stable) - Streptokinase (fibrinolysin that lyses blood clots; prevents fibrin barrier) - Streptodornase (degrades DNA and RNA) Identification: - White to gray color - Pinpoint, translucent, opaque, clear appearance; smoothglossy or roundmucoid - Beta-hemolytic on BAP Differential/Biochemical Tests: - Dick’s Test (+): erythema - Schultz-Charlton (+): blanching - Bacitracin (S) - SXT (R) - PYR (+) - CAMP (-) - Hippurate (-) - BEA (-)

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- VP (-)

5. Streptococcus agalactiae (Group. B) o Normal flora of the gastrointestinal tract and vaginal tract o Screening for pregnant women at 35-37 weeks of gestation is recommended o Mothers with GBS may transmit it through amniotic fluid during pregnancy or labor.  Virulence factors: - Capsule (sialic acid)  Disease-associated: - Pneumonia - Meningitis - Neonatal sepsis - Postpartum infxn - Osteomyelitis - UTI - Endocarditis - Bacteremia - Septic arthritis  Identification: - Medium, flat, and translucent or opaque colonies - Beta-hemolytic on BAP and CAMP  Differential/Biochemical Tests: - CAMP (+): arrowhead with S. au - Hippurate (+): purple - Bacitracin (R) - SXT (R) - PYR (-) - BEA (-)

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6. Streptococcus pneumoniae (No group) o Also known as Pneumococcus/Diplococcus o Normal flora of the upper RT of pre-schools (25-50%) o Common isolate both as a pathogen and normal flora of RT o Facultative anaerobe  Virulence factor: - Capsular polysaccharide (meningitis)  Disease-associated: - Atypical/Lobar pneumonia - Atypical Hemolytic Uremic Syndrome (HUS) *Typical HUS: EHEC (O1547:H7) - Meningitis (infants and elderly) - Otitis Media (3y/o below) - Bacteremia - Endocarditis - Perotinitis  Identification: - Lancet or bullet-shaped - Small, round, glistening, dome-shaped colonies with transparent and entire edge. - Flat colonies with depressed center. - Mucoid colonies indicate presence of capsule - Alpha-hemolytic  Differential/Biochemical Test: - Optochin/Taxo P (S) - Neufeld Quellung test (capsular) - Skin test: Francis Skin Test 4 Bacteriology Internship Notes Rojo, K.F.

- Co-agglutination test - Bile solubility test Media used: - BHI - TSA w/ 5% sheep blood - CAP - Sheep Blood Agar w/ 5-10% CO2 (incubate in a candle jar or CO2 chamber for 24hrs @ 35-37°C)

7. Viridans o Normal microbiota of the URT, female genital tract, and gastrointestinal tract o Opportunistic pathogens o Fastidious and some strains requires CO2 for growth. o More common in children and hematologic px o S. mutans (oral cavity; primary contributor to dental caries; common causative for bacteremia) o S. salivarus (bacteremia, endocarditis, and meningitis) o S. anginosus (oral and gastrointestinal normal microbiota) o S. mitis (oral cavity, GIT, and female genital tract) o S. sanguinis o S. constellatus o S. intermedius  Virulence factor: - Adhesin proteins (endocarditis) - Polysaccharide capsule - Cytolysin  Disease-associated: - Oropharyngeal commensals (Dental caries and gingivitis)

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Subacute bacterial endocarditis (associated with transient bacteremia) Meningitis Abscesses Osteomyelitis Empyema

Identification: - Small colonies and alphahemolytic (BAP); some isolates are beta or nonhemolytic Differential/Biochemical Test: - MSA (+) - Sorbitol fermenter - Urease (+) - VP (+) - BEA (-) - Optochin (R) - Bacitracin (R) - SXT (R) - Hippurate (-) - CAMP (-) - PYR (-) - Growth in 6.5% NaCl (-) Media used: - BAP - MSA

o o o o

E. avium E. calseliflavus E. gallinarum E. raffinosus

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Virulence factor: - Adhesin proteins - Serine protease - Gelatinase (E. faecalis) - Cytolysin (E. faecalis) Disease associated: - UTI - Bacteremia (hospitalacquired-dialysis px and undergone surgery) - Endocarditis (elderly px w/ prosthetic valves/valvular heart dse) - Enterococcal wound infxn and Sepsis (burn px) Identification: - 1mm-2mm colonies after 24hrs BAP, either alpha or non-hemolytic Differential/Biochemical Test: - Bacitracin (R) - SXT (R) - Optochin (R) - Hippurate (-) - CAMP (-) - PYR (+) - BEA (+) - Growth in 6.5% NaCl (+)

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8. Enterococcus (Group D) o Normal flora of the GIT, GUT, skin, and oral cavity. o It has the ability to grow in 40% bile and hydrolyzes esculin o Natural inhabitants of the intestinal tracts of both humans and animals o Can also be found in foods, plants, and oil. o E. faecalis (80-90% causes human enterococcal infxns; GIT infxn) o E. faecium (5-10%) o E. durans 5 Bacteriology Internship Notes Rojo, K.F.

GRAM-NEGATIVE MICROORGANISMS 1. BIOCHEMICAL TESTS ENTEROBACTERIACEAE

CULTURE AND COLONY MORPHOLOGY SOURCES: 1. Delost, M.D., (2015). Introduction to Diagnostic Microbiology for the Laboratory Sciences. 2. Mahon, C.R., (2015). Lehman, D.C., Manuselis, G., (5th edition). Textbook of Diagnostic Microbiology. 3. Old and personal notes from previous lectures and school

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