UNIT 4 microb

GPR bacillus sp

spore produced when stressed and clear on GS, cat pos, anthrax, food poisoning, aerobic,

GPR bacillus sp GS

can stain variable, confirm GS with KOH (GN lysed by 3% KOH releasing DNA, GN= viscous visible thread, GP= no thread bc wall too thick)

GPR bacillus sp growth

BAP or CHOC, 24hrs, ambient air or increased CO2, large flat beta motile= non anthrax, goal is to rule out anthrax and rule in as sp contaminant

GPR bacillus ANTHRAX

virulent org for human, bioterroism agent, nonmotile, gamma, antiphagocytic capsule, protective antigen binds and opens, edema factor (fluid accumulation), lethal factor (cell death)

GPR bacillus ANTHRAX prelim ID

gamma, large, gray flat irregular (medusa head)

large square ended rods, capsule, spore in nonclinical spec

non motile

GPR bacillus ANTHRAX lab safety

in hood, send to state health lab for ID confirmation

GPR bacillus cereus

beta hemolysis, production of entertoxins, emetic, diarrheal, motile, resistant to penicillin

GPR listeria monocytogenes

regularly shaped, no spore, cat +, bacteremia, meningitis, neonatal sepsis, aerobic, environment, food contaminant (lunch meats, dairy), grows at fridge temps (30-35 C or 4 C)

GPR listeria monocytogenes ID colonies

BAP, 24 hrs, small, ambient or CO2, round, smooth, tranclucent, BETA, GPCB

GPR listeria monocytogenes ID

motility test (tumbing wet mound, inverted umbrella), esculin hydrolysis +, hippurate hydrolysis +, 6.5 % nacl pos

GPR Erysipelothrix rhusiopathiae, Lactobacillus sp., Gardnerella vaginalis

regularly shaped, no spore, cat -, skin infection, normal flora, bacterial vaginosis, aerobic

GPR Erysipelothrix rhusiopathiae

cat -, no spores, non motile, H2S, VP-, zootonic pathogen (swine, poultry, fish), erysipeloid (skin disease), septicemia (endocarditis), diffuse, cutaneous infection

GPR Erysipelothrix rhusiopathiae colony

BAP, 5-10% CO2, 24 hrs- gamma pinepoint, 48+hrs- alpha large rough matte irregular, 35-37 C

GPR Erysipelothrix rhusiopathiae GS

variable, thin, pleomorphic rod, long filaments

GPR Gardnerella sp.

HUMAN INFECTION, vaginosis, 5-7% cO2, 35-37 C, under 24 hrs, BAP- gamma pinepoint, HBT- BETA, small gray opaque, GS variable thinner peptidoglycan short pleomorphic, R or CB

GPR corynebacterium sp. (diphtheroids)

irregularly-shaped, no spore, cat +, diphtheria, contaminants, aerobic, palisading, skin flora, non lipophilic and lipophilic, loeffler’s methylene ble stain positive

GPR corynebacterium dipheria

diptheria

GPR corynebacterium jekeium

opportunistic infection (septicemia, meningitis, pulmonary disease)

GPR corynebacterium urealyticum

UTI

GPR corynebacterium colonies

BAP, 24hrs, ambient air or CO2, dry, tiny, grey to white, irregular

GPR corynebacterium GS

club shaped, pleomorphic, irregularly staining due to metachromatic granules, palisade (L V chinese letters)

GPR nocardia sp, actinomyces sp, streptomyces sp

branching, filamentous, pulmonary, cervicofacial, opportunistic infection, aerobic

GPR nocardia colonies

variable, dry chalky, crumby, various colors, BETA, can spore from aerial hyphae

GPR nocardia colonies GS

beaded, branching, fine, intertwining filaments, partially acid fast

GPR streptomyces colonies

variable, glabrous or waxy, cream to brown black, white aerial hyphae

GPR streptomyces GS

branching with chains and spores, no fragmentation

GPR mycobacterium sp

acid fast (high levels of mycolic) bacillius, slender, curved or straight rods, TB, leprosy, NTM disease, aerobic

GNDC- aerobic sp

n. gonorrhoeae, n. meningitidis, n, lactamica, moraxella catarrhalis

aerobic GNDC virulence factors

transferrin receptors, LOS (lipo sca endotoxin), pili, capsule meningitidis, IgA protease, membrane proteins (I- porin, II- opacity, III- reduction modification)

GNDC aeobic growth

fastidious, enriched CHOC, MTM, capnophilic, colonies vary, GS coffee bean/ kidney, fat and short pairs, intracellular

GNDC aeobic ID tests

oxidase +, nitrate reduction, carb use, DNase, butyrate esterase, enzyme production by chromogenic substrates, immunoassays, MALDI-TOF, latex agglut, nuc acid amplification, GS

carb use CTA

cystine tryptic agar, glucose maltose lactose and sucrose, n. memingitides= pos glucose and maltose, n. goborrhoeae= pos glucose

DNase test

methyl green binds dna, extracellular exoenzymes hydrolysis dna, unbinds producing clear zone or halo around line, pos in s. aureus, neg in s. epi

butyrate esterase

tributyrin substrate hydrolysis, must be GNDC, ox +, hockey puck morphology, rub several colonies across the disk with a stick, read blue-green color after 2 min, pos= moraxella catarrhalis

enzyme production detected by chromogenic subs

isolates from enriched selective media (MTM), BactiNesseria card remel tests IB, PRO, GLUT, BGAL

BactiNesseria- moraxella catarrhalis

IB+ blue

BactiNesseria- n. lactamica

BGAL +, IB neg

BactiNesseria- n. gonorrhoeae

PRO + red

BactiNesseria- n. meningitidis

GLUT + red, PRO variable

multiple test systems

rapid tests for neisseria and moraxella sp, ox +, further test

immunoassays and MALDI-TOF

coag and florescent ab= n. gonorrhoeae, MALDI- identifies by protein signatures

latex agglut

direct specimen, csf, pos for n. meningitidis

nuc acid amplification

direct specimen, urine/genital, pos= n. gonorrhoeae

nested-pcr methods detect species in csf, blood, or resp

n. gonorrhoeae- colonies

enriched CHOC or MTM, capnophilic, small, grey/tan, translucent, raised, SLOW 24-48

MTM

selective choc, containing ab- vancomycin, colistin, nystanin, trimethoprom, selects for n. gon, n. mening, n. lactamica

vancomycin and lincomycin inhibits

GP

colistin inhibits

NG

nystatin, anisomycin, and amphotericin B inhib

yeast

trimethoprim inhib

proteus

n. gonorrhoeae- GS

intracellular urethral discharge diagnotic for men, colonies resemble GNR/GNCB so use penicillin disk and GS, GNR will elongate

N. gonor- nuc acid detection

urine, cervical, urethral, detects gon and chlamydia, likely co infection (STDs after HPV)

oxidase

detects cytochrome c using tetrameth-p-…, purple= pos

N. gonor ID

ox +, gluc pos, hydroxlypro PRO + yellow, coag, fluorescent, report within 7 days to state health dept

n. gonorrhoeae transport

immediatly, cold sensitive keep room temp, high humidity, susceptible to drying, use dacron or rayon swabs (calcium alginate and cotton inhib growth), delay use jembec, transgrow, gono-pak

n. gonorrhoeae presentation

genital, rectal, throat, synovial fluid or eyes use choc, must notify lab it is expected if from urine

n. meningitidis- colonies

BAP (blue-gray, smooth, convex, mucoid), CHOC maybe MTM (small, tan, smooth, mucoid, convex), room temp (sensitive), ASAP,

n. meningitidis- ID

ox +, glucose and maltose pos, PRO variable, GLUT pos, report asap to state health dept, bio safety cab level 2

n. meningitidis- virulence factors

pili (initial attachment), capsule (phagoc, 12 serotypes a b c w-235 and y), LOS endotoxin (tissue damage, inflamm), IgA1 protease (cleave IgA on mucosal increase invasion)

n. lactamica colonies and GS

normal flora of nasopha, blue-gray on BAP, small tan convex on CHOC/MTM, non pathogen grows on selec, GS not intracellular

n. lactamica vary colonies

cinerea= gray white, translucent, raised granular

succa- large gray white, opaque, wrinkled, dry irregular

subflava biovars- green yellow, smooth

n. lactamica- ID

ox +, glucose maltose and lactose pos, BGAL pos (breaks down lactose)

resp neisseria sp

non pathogenic but can be carriers of meningitidis, yellow colonies beta hem (n. subflava), normal flora so idc

sterile neisseria sp

MALDI for full ID

moraxella catarrhalis

aka branhamella, normal in upper resp, opportunistic in children and elderly, 3rd most common cause of otitis media and maxillary sinusitis

moraxella catarrhalis GS and colony

intracellular if from specimen not colony, BAP or CHOC- smooth, gray white, pink salmon color change, hockey puck (do not break when pushed by loop), Thayer martin variable

moraxella catarrhalis ID

ox +, cab neg, DNase pos, nitrate pos, nutyrate esterase pos (purple), IB pos

GNCB sp

Haemophilus sp, opportunistic normal flora of upper resp (h. ducreyi always pathogenic)

Haemophilus transmission

h. influenza type b and H. ducreyi person to person, exo source

other- normal flora gains access to sterile site, endogenous source

Haemophilus virulence factors

most invasive infection caused by encapsulated strains, antiphago

Haemophilus sp

fastidious, capnophilic, facultative anaerobe, required growth factors- v factor (NAD) and x factor (hemin), capsule and uncapsule

Haemophilus growth traditional and quad plates

CHOC- tannish, translucent, mousy odar (h. ducreyi- small grey yellow tan)

horse rabbit blood agar RTF w/ sheep blood- BETA, no NADase

satellitism- pinpoint orgs growing around bacteria and yeast due to NAD (V factor) production

Why is Chocolate agar (CHOC) preferred for the isolation of Haemophilus?

It contains both X and V factors released from lysed RBCs

Haemophilus colony morphology on BAP and MAC

no growth due to NADase in BAP and lac of nutrition in MAC

Haemophilus capsule stains

6 ag types (a-f), differentiate stereotypes by slide agglutination with antisera, type b (influenza, blood or csf) causes most invasive disease, no adherence factor

haemophilus un-encapsulated

un-typable, localized infections, invasive in elderly and immunocompromised

Haemophilus GS

tiny, school of fish or train tracks, csf pleomorphic GNCB, if capsule halo, if from colony also GNR, enhance with safrin, latex agglut (sens), EIA (specif)

Haemophilus influenza direct specimen detection

GS- intracellular tiny CNCB with capsule halo, presumptive diagnosis in csf using latex agglu for Hib ag, nested-pcr (common) in csf, blood, or resp

Haemophilus influenzae clinical cases

resp tract and viral infections (significant if many organisms from sputum culture with PMNs), meningitis (dramatic decrease Hib vaccine, leading cause serotyoe in unvax), otitis media, conjunctivitis (biogroup aegypticus)

Haemophilus ducreyi clinic present

not normal flora, chancroid/soft chancre std (contagious, painful genital ulcer, tropical areas), take base of lesion

Haemophilus ducreyi GS

GNCB, school of fish or train tracks

other Haemophilus sp clinic presentation

nonpatho (normal flora), bacteremia, endocarditis, rti, abbscesses

Haemophilus sp handling

isolated from blood, cerebral, ear, resp, conjuctivae swab, avoid drying and extreme temp, asap, CHOC, 5-10% CO2 moist, 24-48 hrs

Haemophilus ducreui collection

from base of lesion, enriched media, incubate 5-10% CO2 in moist environment for up to 5 days

Haemophilus growth factors

x or hemin- heat stable, in rbc released when lysed

v or NAD- heat liable, produced by some bacteria and yeast, in rbc yeast and potatoes

determining if x and v factor needed- Haemophilus

suspension of orgs, unsupplemented media, impregnated strips or disks with x v and xv factors, incubate 18-24 hrs 35 C 5-10% CO2

haemophilus quad plate

CO2 incubation, divided- X, V, XV, horse blood

quad with Haemophilus influenza and haemolyticus

grows in XV but not in just x or v

quad with Haemophilus parahaemolyticus and parainfulenzae

grows in V and XV not in X

quad with Haemophilus decreyi

growth in x and XV but not in V

prophyrin test

determines in org can synthesize x factor, substrate ala converted into porphobilinogen and porphyrin, 35 C for 4 hrs then add Kovac, pos = red orange with UV light-woods

porphyrin positive pros

reduc carryover problems, not needing x factor, pos in parainflu and parahaemolyt

h. influenza ID

cat +, ox +, XV, porphyrin -

h. haemolyticus

cat +, ox +, hemolysis in horse, XV, porphyrin -

h. parainfluenzae ID

cat V, ox +, V, porphyrin +

h. parahaemolyticus

cat +, ox +, hemolysis on horse, V, porphyrin +

h. ducreyi

cat -, ox -, sometimes hem on horse, X, porphyrin -