Gram Positive Cocci

Family: Micrococcaeae includes genera ___, but Staphylococcus is now __.

Micrococcus, Rothia, Staphylococcus;

it’s own family = Staphylococcaceae

Staphylococceae general characteristics

• GPC

• single, pairs, tetrads, or clusters

• catalase + except Rothia

• col entire, smooth, opaque / white & butyrous growth

What test diff Staph from Strep? How does it work

catalase test

• reagent = 3% H2O2

• + → production of O2 and H2O (bubbles)

• - → no bubbles

Which genera are catalase + and -?

catalase + = Staphylococcus & Micrococcus

catalase - = Streptococcus & Enterococcus

What can cause a false +?

if pick up blood/rbc from agar → contains catalase → false +/bubbles

Which bacti is catalase + besides Staphylococcus? How to diff b/t other Staphylococceae?

Rothia mucilaginosa (sticky staph)

Rothia can’t grow in 5% NaCl

once catalase +, what test is done next to determine if S. aureus?

slide coagulase

• media = rabbit plasma w/EDTA

• rapid

• tests for coagulase enzyme (which alters fibrinogen → clot), specifically clumping factor/cell-bound coagulase

• if (-) → confirm w/ tube coag test

  • if (+) → S. aureus

why does slide coagulase test need negative control? what to do to if this occurs?

neg control checks for bacti that auto-agglutinate in saline → if auto-agglut → tube coag test

when is tube coagulase test done? what is it and what to look out for?

when (-) from slide coag test → will test for free coagulase

• inoculate loop of bacti into 0.5 mL of rabbit plasma → incubate 35C for 4 hr → observe at 4 hr mark in case of staphylokinase which lyses clot → if (-), incubate overnight

pos = clot formation or aggulatination

neg = none

Staph Latex / hemagglutination test

• detect what?

  • advantage/disadvantages?

detect: clumping factor, protein A (all S. aureus have virulence factors), and antigenic proteins

• rapid & more definitive, if + → call it S. aureus

• false - b/c some strains don’t produce enough of ^^^

• false + from certain Strep A, C, and G

  • get + from other Staph sp like S. lugdenesis, schleiferi, etc…

S. aureus

• infects where

• usually founds in __

• produces enterotoxins ___

in skin (furuncles, carbuncles, cellulitis, impetigo), pneumonia, meningitis, endocarditis

• thermostable enterotoxin (food poisoning): projectile vomiting & fast incubation time

• TSST-1 → toxic shock syndrome

• exfoliatin → scalded skin syndrome in kids

ID of S. aureus

• grows in 18-24h at 37C

• turns golden yellow w/ age

• typically beta-hem rxn

• coagulase +

S. aureus epidemiology

Most are resistant to __?

a significant nosocomial pathogen (hospital acquired)

95% resistant to penicilin due to Beta-lactamase or cell wall binding protein

MRSA stands for

what happens if pt is + for MRSA?

Methicillin/oxacillin resistant S. aureus due to mecA or mecC gene → codes for PBP2A (penicillin binding protein)

Why is cefoxitin disk better for detecting MRSA than oxacillin disk or agar?

What will it look like?

cefoxitin (= a cephalosporin, also has beta-lactam ring) → detects heteroresistant strains of MRSA (=don’t express Abx-R genes)

MRSA → cefoxitin-R → report 6mm ZOI

What does MRSA look like on chromogenic media?

Spectra MRSA → denim blue col

CHROMagar MRSA → mauve col

Methods to rapidly detect MRSA?

detection of mecA gene via PCR/probe, fluorescence tests, or slide latex agglutination

MRSA are resistant to ___ →

methicillin, oxacillin, nafcillin (semisynthetic penicillins) → other beta-lactam drugs are reported as resistant even if in vitro tests appear sensitive

What may be misID’d as S. aureus?

S. lugdunensis is misID’d b/c can look + on slide coag test (but is coag -) bc may have clumping factor and yellow pigment col

• confirm w/tube coag test that it’s coag -

• part of normal skin flora (toe or inguinal fold) → but can cause soft tissue infns

• AST interpretation reported like a S. aureus, NOT CNS

• ornithine decarboxylase +

Coagulase-negative Staph (CNS)

significant species and when to w/u?

Staph epidermidis - bad bc produce slime → adhere to catheters & prosthetics

S. saprophyticus - ID from novobiocin-R, UTI in young females

• only do w/u if multiple + BC or +BC AND +catheter tip

S. saprophyticus is resistant to ___.

novobiocin

< 16 mm ZOI

How to diff Micrococcus luteus from Staph spp.?

bright yellow col, coag neg

diff from Staph

• glucose oxidizer (Staph ferm glucose)

• bacitracin-S (Staph are R)

Streptococcus & related general traits

GPC in pairs and chains

facultative anaerobes

translucent, shiny, grey col

catalase -

What is the Lancefield Grouping system?

used for beta-hemolytic Streptococci

based on carbohydrate cell wall Ag → detected by kits via heat, chemical, enzyme

groups include:

• A and B - can use biochem tests to ID

• C, F, G - need serology tests to ID

  • D = Enterococcus?

Group A Strep aka __. Colony morph? Virulence mechanism?

Strep. pyogenes

tiny, beta-hemolytic, with larger zone of hemolysis

can be elongated cocci in chains

Streptolysin O (oxygen labile ie breaks down in O2) → if stab into agar → enhanced zone of beta-hemolysis

GAS clinical manifestations

spread via droplets or direct contact

most common cause of pharyngitis

impetigo, erysipelas

necrotizing fasciitis (got thigh swab from unknowns!)

strep toxic shock syndrome

after infection (sequelae) →

rheumatic fever: Ab from infn attack heart tissue

glomerulonephronitis - Ab/Ag complex deposits in kidneys

Media used for GAS?

Bacitracin → ?

PYR → ?

SXT or SSA

bAcitracin-S (A on disk is for group A strep)

PYR + (pink throat)

GAS rapid tests

use throat specimen

positives are conclusive, but neg must have culture

Group B Strep (Strep. agalactiae)

colony morph

found where?

clinical manifestations

larger col w/ narrow zone of beta-hemolysis (or diffuse zone)

normal flora of GI & genitourinary tracts

GBS is screened for in pregnant women in 3rd trimester → neonatal pneumonia, septicemia, meningitis

in adults: UTI, bacteremia, endocarditis, pneumonia, osteomyelitis

media for GBS?

Hippurate hydrolysis → ?

CAMP test → ?

use CNA & LIM broth (contain colistin & nalidixic acid)

hippurate hydrolysis + (hippy girls/bros like camping and gardening)

CAMP test + (like to camp in the vag/rectal area)

**hip gals (galactiae) camp positive

hippurate hydrolysis test

principle

steps

result

sodium hippurate → benzoic acid + glycine

glycine + ninhydrin → purple complex

inoculate bact in 0.5 mL distilled water in tube → add sodium hippurate disk → 35C for 2hr → 0.2 mL ninhydrin reagent

pos = blue/purple w/in 5 min

neg = no color change

Hippurate hydrolysis test is positive for

GBS, Listeria monocytogenes, Gardnerella vaginalis, Campylobacter jejuni

“Hippy Girls Like Camping & Gardening”

CAMP test

principle

steps

results

ID for GBS

free CAMP factor reacts w/Staphylococcal beta lysin to lyse rbcs

single straight streaks of test bact & CAMP S. aureus are made perpendicular to each other on SBA

pos = enhanced zone of beta hemolysis in shape of arrowhead pointing toward S.aureus

neg = no enhanced zone of hemolysis

Group C, F, and G Streptococcus

sources

clin manif?

how to ID?

still beta-hemolytic

normal flora of skin, nasopharynx, GI and genital tracts

infns in immunocompromised pt → similar infn as GAS and GBS, but no sequelae like GAS

ID w/kits

Group D Strep includes __

morphology

clinical significance

intrinsically resistant to __?

Enterococcus & Group, non-Enterococcus

grey-white, translucent, alpha or non-hemo, some rare beta-hemo

found in soil, food, water, normal flora of birds, animals, human (GI and female GU tract) → can cause UTI, bacteremia, endocarditis, mix wound infn

cephalosporins and aminoglycosides → need to treat w/multiple Abx for synergistic effect

How to distinguish Enterococcus spp vs Group D non-Enterococcus?

bile esculin

PYR

salt broth 6.5% NaCl

both have group D Ag

bile esculin → all group D + (black → dlack)

PYR → only Enterococcus + (pink)

salt broth → only Enterococcus + (purple or yellow growth)

bile esculin slant

reaction

results

40% bile → inhibits most G+ → used to diff Group D from non-group D strep

esculin → esculetin + dextrose → esculetin + ferric citrate → black pigment

pos = >1/2 slant turned black

neg = <1/2 slant black

PYR test

principle

steps

result

used to diff Enterococcus spp from Group D, non Enterococcus

PYR enzyme made by GAS & Enterococcus.

PYR -L pyrglutamyl aminopeptidase → free beta-naphthylamine —cinnamaldehyde → red complex

moisten substrate disk w/sterile water → touch colony to disk → incubate 2 min → add cinnamaldehyde

pos = red color on disk → GAS or Enterococcus spp.

neg = no color change

PYR + organisms

GAS → r/o Group B, C, F, G Strep, S. viridans

Enterococcus spp. → r/o S. gallolyticus

(some Staphylococcus strains, Micrococcus, Gemella, Lactococcus)

6.5% NaCl test

used to ID Enterococcus spp

inoculate and incubate overnight 35C

pos = growth (w or w/o change to yellow)

neg = no growth

Enterococcus faecalis

causes most human enterococcal infn

non-hemo

** Cali = huge population, most infns, non-hemo bc californians are chill → not resistant ie VRE

Enterococcus faecium

alpha-hemo

causes 10-15% enterococcal infn

more commonly vancomycin-R (VRE)

Enterococcus gallinarum/cassliflavus

motile

intrinisically vancomycin intermediate

Group D non Enterococcus aka

Streptococcus bovis (old name) → S. gallolyticus

assoc’d w/colon cancer

Strep pneumoniae

morphology

clin manif

GPC in pairs, lancet shaped, may have capsule

always alpha-hemo, older colonies may be donut-shaped (autolysing)

leading cause of bact meningitis and pneumonia; bacteremia, sinusitis, otitis media

ID’ing Strep pneumoniae

bile solubility

Optochin test

Quellung reaction

bile solubility → +, colony dissolves → can see alpha-hemo

Optochin → S

Quellung rxn (only for epidemiology) → capsule is enhanced for certain S. pneumo strains

Viridans Group Streptococcus

morphology

clinical signifance

typically alpha-hemo, but can be non or beta (viridans vAries)

butterscotch odor (sweet → viridans (veneers) → endocarditis)

opportunistic pathogens, normal flora of oral cavity, GI tract, female genital tract → poor dental care → bacteremia, endocarditis, meningitis

Viridans Group Strep ID

PYR and salt

bile esculin

optochin

bile solubility

all negative!

w/u only if multiple +BC

Abiotrophia spp aka ___

requires ___ to grow → grows on __ agar not SBA.

Will grow on SBA if?

PYR → ?

Granulicatella spp.

pyridoxal or thiol cmpds or cysteine; choc

linked to endocarditis (normal oral cavity flora)

beta-hemolytic Staph on SBA → B-hemo → sattelite streptococci in lysed region

PYR → +

other catalase - GPC