unit 3 microbio

GPC species

staph, micro (tetrads, cat +, causes problems in lab), strep (in liquid or broth amplifies chains, cat neg), entero (cat neg)

gram pos cocci

part of indigenous (normal) microbial flora, skin and mucous membranes, opportunistic 2nd to enterobact in human infection

gram pos cocci transmission

found in every specimen, in fomites and in dirt/dust on floors and walls, spread by direct contact through skin or mucous membrane penetration (eating)

gram pos cocci virulence

suppurative/ pyogenic/ purulent defensive inflammation reaction to evade immune system, toxins and enzymes cause harm increasing pathogeny

GPC medias

SBA, CHOC, no growth MAC, if neg norm flora precent use CNA/PEA to select for GPC

staphylococcus general

pairs and clusters, aureus, epidermidis, saprophyticus, lugdunensis, normal flora of skin and mucous, opportunistic with enzymes and toxins, facultative anaerobes, fast growing 18-24 hrs, non motile, non spore forming

S. epidermidis

coag neg staph, s. epi.

staphylococcus growth characteristics

BAP CHOC CNA/PEA, not MAC, opaque, smooth, circular, gray-white, cream, yellow, nonhem (gamma), beta for S. aureus and S. hemolyticus

always do what test first

gram stain, then cat, neg = strep if pos do coag, pos= S. aureus if neg do microdase, pos = micrococcus if neg do novobiocin, R= saprophyticus if S some CNS

staph or microc catalase test

clean glass slide and smear colonies on, 3% H2O2 placed on colony, pos shows rapid production of bubbles (staph and micrococcus), neg (strep and enter), rbc from BAP give false pos with pseudo

slide coag

detects clumping factor fibrinogen to fibrin, organism mixed with plasma, angulation= pos = S. aureus, neg= CNS (coag neg staph)

tube coag

detects staphylocoagulase reacting with CRF thrombin like molecule, indirectly fibrogen to fibrin for clots, organism suspended in plasma and incubate for 4 hrs, pos= s. aureus, fibrinolysin can dissolve clot

rapid plasma latex agglutination

kits (staphaurex, staphTEX), uses plasma coated latex carrier particles to detect cluping with fibrinogen and Protein A with IgG Fc, high spec and sens, non selective media (not MSA), pos= aureus

novobiosin susceptibility

identifys coag neg staph in urine from young women with UT issues, place disc than incubate for 24 hrs, lawn- R no zone of inhib (idc who you are im gonna touch you)= saprophyticus, S (i care do not touch me) zone 16 mm, takes long time, multi test system,

PYR hydrolysis

Detects s. Aureus neg vs lug pos = bright red, Put the organism on the disc, reagent on top, L-pyrrolidonyl-a-naphthylamid, B-naphthlamide, DMAC

rapid ornithine decarboxylase

Tube method, incubate 2-4 hrs, Dark purple/violet= staph lug, pale yellow or grayish = neg

MALDI-TOF mass spec vs kit tests

PCR based vs non PCR

diff micrococcus from staphylococcus by

microdase, bactracin/taxo A, and glucose utilization/ OF

microdase test

Modified oxidase, opportunistic if only organism growing rapidly or reoccurring, within 30 sec pos= blue/purple= micrococcus, neg= no color staph, tetramethyl-p-phenylen dihydrochlor in DMSO

bacitracin/taxo A sus

S= 10 mm zone = micrococcus, R= growth up to disk= staph

glucose utilization or OF

oxidative- produce acid as yellow in O2= micrococcus, ferminatation- produce yellow acid with or without O2= staph (aureus)

S aureus

most clinically significant staph, in every specimen, opportunistic, nosocomial infections (HA), rapid ID, cat+, coag +, microdase -, OF ferm all yellow, bacitracin R

confirming tests for S aureus

coag neg latex but morph resembles aureus use tube, molecular methods, automated rapid multi test systems

S aureus toxins

exo, enter, and cyto, cause disease and promote spread

S aureus cellular components

Protein A- bind Fc IgG blocking phago and promotes adherence to host cells, capsule- inhib phag pro adher, clumping factor- binds fibrinogen allowing spread

s aureus enzymes cat coag fribronolysin

ca- inactivates peroxide and free radicals in phag cells

co- inhib phago and bactericidal activity, activate fibrin clot

f- breaks down fib clot

s aureus enzymes- hyaluronidase and B-lactam aka penicii

h- hydroly hyaluron acid in connect tissue allowing spread

B- hydroly B-lact ring of penicillins and cephalo causing resistance

S aureus colonies

opaque, raised, smooth, white-gold or cream, B hemo, larger than strep

S aureus MSA

selective and diff used to isolate from mixed flora, manitol is sole carb, pH indicatior, YELLOW

S epidermidis

contaminant in wound or blood cultures from improper collection or set up, opportunistic in immunocomp or device implant patients, cat+, Coag -, repeat or pure= infection

S epidermidis cellular components

PGA capsule promotes adhere with affinity for plastic, protects against high salt allowing colonization on skin

S epidermidis virulence factors

biofilm formation genes- biofilm development and maintenance, biofilm- inhibites antimicrobial peptides lymphocytes and neutrophils, involved with prostetics teflon plastic, requireing removal of device

S epidermidis biofilms steps

adherence/attachment to foreign body/ material/ living tissue

accumulation/aggregation- several layers of org and extracellular products

maturation/maintenance

detachment/disaggregation- evading immune system

S epidermidis colonies

opaque, round, small, smooth, raised, gray-white, gamma hem

S epidermidis after coag neg

microdase -, MSA pink, novobiocin S 16mm zone

S saprophyticus

cystitis in sexually active young women, UTI 2nd to E.coli, cat +, coag -

S saprophyticus colonies

opaque, smooth, round, white (yellowish), creamy, gamma

S saprophyticus after coag neg

microdase -, MSA pink, novobiocin R growth to disk= sap

S. lugdunensis

cat +, coag -, PYR pos, novo S zone, rap ornithine purple pos, opportunistic, for Endocarditis (heart valve replacement), septicemia, meningitis, skin & soft tissue infections, UTI, septic shock, repeat or pure= infection

S. lugdunensis colonies

opaque, round, smooth, glossy, very white, gamma, delayed B

S. lugdunensis after coag-

confirm with tube coag, PYR= red pos, novobiocin S zone 16mm, rapid orithine purple pos

micrococcus

cat +, coag-, microdase pos purple, OF oxidase only yellow in O2, bacitracin S 10mm zone, obligate aerobe in wounds (rare), cartenoid pigments yellow and pink, non motile, non spore forming, tetrads

micrococcus colonies

smooth, raised, opaque, rough or lacy in O2 incubator, white, bright yellow, pink

micrococcus after coag neg

microdase pos purple, OF oxidase only yellow in O2, bacitracin S 10mm zone

strep and enterococcus

GPC in pairs or chains, cat -, facultative anaerobes- capnophilic or aerotolerant (carbs metabolized by fermintation, end product= lactic acid no gas) , encapsulated

S. pyogenes group A GAS

fire throat, beta, rheumatic fever, scarlet fever, pharynitis, glomerulonephritic, bacitracin S, PYR +

S. agalactiae GBS

Beta, CAMP +, hippurate +, pregnancy, neonatal sepsis, meningitis, puerperal fever, pyogenic infections

S pneumo

alpha, pneumonia, meningitis, pyogenic infections, concave, optochin disk- S

enterococcus

faecalis and faecium, group D, alpha beta gamma, uti, pyogenic infection, 6.5% pos, PYR +, bile esculin pos

ID strep and enter order

1st hemo pattern, 2nd lancefield class, 3rd biochem ID (tests)

alpha hem on SBA test order

optochin disk- S = strep pnuem, R do bile esculin- neg = viridan strep pos do 6.5% nacl pyr- pos = enteroccoc, neg = group D strep

beta hem test order

bacitracin- S = group A pyogenes, R do CAMP- pos = group B agalactiae, neg= maybe group D or not A B or D

Beta alpha or gamma on BAP

bile esculin pos do 6.5% nacl- pos= enter pyr+, neg= group D strep pyr-

strep catalase

clean slide and smear colonies adding 3% h2o2, neg= no bubble= strep or enter

optochin p disk

ethylhydro hydrochl disk added to BAP, inoculate overnight with alpha strep colony lawn, S zone 14-16 mm= pneumoniae

bile solubility

suspend colony in sodium deoxycholate, autoclave (CLEAR) pos S pneum- bile salt or detergent cell wall lysis,

bacitracin/taxo for beta streps

S= zone 10mm group A, R= group B and D do CAMP

CAMP

aureus and group B perpendicular, incubate overnight 35C in CO2, arrowhead= pos confirming B

PYR strep

same as in staph, bright red for strep pyogenes, enterococcus and aerococcus

LAP strep

similar to PYR, red= pos= strep pyogenes, enterococcus, neg= aerococcus and leuconostoc

hippurate hydrolysis

pos= purple= group B, forms nenzoate and glycine

6.5% NaCl broth

pos= turb= enterococci, neg= no growth= strep

bile esculin

pos= black growth in 40% bile and hydrolyze= group D or enterococcus, neg= orange and non D strep

immunnoassays

direct specimens- throat swabs and culture, isolated colonies- agglutination and lancefield, ELISA, molecular- RT-PCR MALDI TOF Probes