ID Core Curriculum: Urine Cultures

Melissa B. Miller, PhDFebruary 1, 2008

Culture-based methods Liquid media

Greater sensitivity; enrichment Must be sub-cultured to solid media for

identification in most instances Caveat: false positives!

• eliminate back up broths except for CSF and tissue

Solid media Isolates can be quantitated Sight-identification possible

Non-selective media Selective media

Incorporate antimicrobial agents to inhibit flora while allowing for the growth of a specific organism (i.e., Thayer-Martin for GC)

Differential media Incorporate one or more carbohydrates in the medium with a

pH indicator (i.e., MacConkey Agar)

N. gonorrhoeaeon TM

+/- Lactose fermenationon MacConkey

Culture-based methods

Culture-based methods Quantitative Semi-quantitative Isolation

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Specimens Submitted From Human Urinary Tract Acceptable specimens

Clean catch, catheterized Nephrostomy, indwelling

catheter/Foley , ileal conduit, and cystoscopy specimens

Suprapubic aspirate, ureteral, kidney specimens (Invasive – add broth tube)

Gram Stain

Sensitivity depends on the number of organisms in the specimen 1-2 bacteria/oil field (1000X) ≥ 105 CFU/ml

Specificity Depends on how morphologically unique an

organism appears microscopically Both also depend on laboratory competency

Urine Gram Stain

Urine Culture Quantitation Clean catch or catheterized urine

Plate 1ul; 1 colony = 1000 organisms/ml Clean catch: >105 orgs/ml Cath urine: >104 orgs/ml

Suprapubic urine or patients w/ dysuria Plate 10ul; 1 colony = 100 organisms/ml >103 orgs/ml Processed aerobically and anaerobically

Urine Culture Set-up Suprapubic aspirates

10 ul Sheep blood agar, MAC, anaBHI (brain heart

infusion, anaerobic) Acute dysuria

10 ul SBA, MAC Add CHOC for post-prostatic massage

Routine urines 1 ul SBA, MAC

Urine Cultures Plates initially read at 18-24 h All specimens plated after NOON of

the previous day, hold another overnight Gram positives take longer to grow May not be able to determine amount

of flora present

ID of Normal Flora and Potential Pathogens Judgment required

Determine which organisms to look for Variable depending upon specimen site Determine what constitutes normal flora vs

potential pathogen Extent of workup

Contribution to unnecessary use of antibiotics Contribution to emergence of resistant

organisms

Resident Flora of Human Urinary Tract Sterile above urethra

Urine normally sterile Must pass through contaminated regions

during specimen collection (noninvasive) Quantitative methods discriminate

contamination and colonization from infection Urine collected via invasive methods

(suprapubic aspiration) should be sterile Distal portion of urethra colonized

Many organisms are same as found in genital tract

Some transient colonizers are potential pathogens

Resident Flora of Human Urinary Tract Genital tract flora- mixture of:

Lactobacillus Alpha-hemolytic Streptococcus sp. Diptheroids CoNS Gardnerella vaginalis Yeast

mixed urogential flora

Most Common Pathogens of Human Urinary Tract Community acquired

E. coli is most frequent pathogen isolated Klebsiella sp and other Enterobacteriaceae Staphylococcus saprophyticus

Hospital acquired E. coli, Klebsiella, other Enterobacteriaceae Pseudomonas aeruginosa Enterococci and Staphylococci

Urine Cultures

Urine Cultures

E. coli Non-swarming, spot indole pos, oxidase neg

1. Hemolytic on SBA2. Non-hemolytic on SBA and lactose positive

(MacConkey or eosin methylene blue), PYR (pyrrolidonyl arylamidase) test positive

3. Non-hemolytic on SBA and lactose negative, rapid MUG (methylumbelliferyl-beta-D-glucoronidase) test positive

Abbreviated Identification

Indole +

AST and Emerging ResistanceBeta-lactamases

ESBL: E. coli, Klebsiella, P. mirabilis Mutant TEM-1, TEM-2, and SHV-1 -lactamases Hospital-acquired: Clinically relevant isolates Resistant to all cephalosporins, including

cefotaxime, ceftazidime • Cefoxitin may still be S

Transferable plasmid containing other resistance genes

Predicted by ceftriaxone, ceftazidime, aztreonam

Disk diffusion or MIC testing of• Cefotaxime +/- clavulanic acid• Ceftazidime +/- clavulanic acid

AST and Emerging ResistanceBeta-lactamases

AST and Emerging ResistanceBeta-lactamases

AmpC: Enterobacter, Serratia, P. vulgaris, P. aeruginosa Chromosomal cephalosporinase May also be plasmid-mediated (Klebsiella) Resistant to all -lactams Flattening of zones around ceftazidime and

piperacillin/tazobactam disks when in close proximity to cefoxitin disk

No CLSI confirmatory methods available May mask ESBL activity

AST and Emerging ResistanceBeta-lactamases

Proteus spp. Swarming growth

Indole• Negative: P. mirabilis/penneri

• P. mirabilis: maltose neg, ornithine pos• P. penneri: maltose pos, ornithine neg

• Positive: P. vulgaris

Abbreviated Identification

P. aeruginosa Oxidase-positive bacillus Typical smell (grapes) Colony morphology c/w P. aeruginosa:

metallic/pearlescent, rough, pigmented, mucoid Indole-negative (r/o Aeromonas) Realize P. aeruginosa isolates from CF patients may

appear atypical

Abbreviated Identification

Oxidase +

CHROMagar Orientation Presumptive ID for some UTI pathogens

• E. coli (dark rose to pink)• Enterococci (turquoise blue)• S. saprophyticus (light pink to rose)• S. agalactiae (light blue-green to light blue)• Proteus-Morganella-Providencia group (brown) • Klebsiella-Enterobacter-Serratia group (dark blue)

Issues and challenges• All except E. coli and enterococci require further ID

• Small E. coli colonies require spot indole • Poor growth of some gram-positive bacteria • Nonselective– other pathogens may or may not

produce color change

Abbreviated Identification

Enterococcus spp. Cocci or coccobacilli in pairs and chains >1 mm colonies Non-hemolytic on SBA Catalase-negative PYR-positive(pyrrolidonyl--naphthylamide hydrolysis)

Abbreviated Identification

AST and Emerging ResistanceEnterococcus spp

E. faecalis Ampicillin predicts imipenem susceptibility

(99% S) NOT true for E. faecium; AmpS/ImipenemR

strains due to increased production of PBP5, which has decreased affinity for imipenem

Quinupristin-dalfopristin (Synercid) resistant Vancomycin Resistant Enterococci (VRE)

vanA/vanB vanC1, vanC2/C3

• E. gallinarum, E. casseliflavus, E. flavescens• NOT true VRE

AST and Emerging ResistanceEnterococcus spp

Vancomycin Resistant Enterococci (VRE) Some institutions treat urine culture screens

for VRE same as rectal surveillance cultures Enterococcosel agar (8ug/ml vancomycin)

• Brownish-black to black halo; must confirm ChromID VRE (8ug/ml vancomycin)

• E. faecium (purple colonies) and E. faecalis (blue to blue-green colonies)

• In FDA-approval process; expected release from bioMerieux 2008

• Propose to read and report at 24 hr BD/GeneOhm VRE PCR awaiting FDA-approval

• VanB isolates likely to require confirmation

S. agalactiae (GBS) Cocci in pairs and chains Catalase-negative Narrow zone of beta-hemolysis on SBA Rapid hippurate hydrolysis test (beta strep only) OR Test for CAMP factor (spot or O/N) OR Typing by particle agglutination R/O beta hemolytic Enterococcus (PYR+)

Abbreviated Identification

-hem Enterococcus

Urine CulturesReporting Issues Beta Hemolytic Streptococci (GAS/GBS)

Routine urine cultures• Males any age, females <15 or >45• Work up and report per standard protocol if pure

culture or quantity ≥ mixed flora • Reporting options:

• Beta-hemolytic streptococci, morphology consistent with GAS or GBS (as appropriate)

• Rule in/out Group A and B only and report accordingly

• Beta-hemolytic Streptococci, not Group A or B

Urine Cultures Reporting Issues Screening cultures- Group B Streptococcus

Report ANY amount of GBS from women aged 15-45 years. This includes mixed and MUF urines; do not quantitate.

Patient is pregnant: Proceed with confirmatory ID Female, 15-45 y.o., not pregnant or unknown, site

read, report ‘Possible GBS present. If patient is pregnant please call … to request further identification.”

All males, females <15 or >45 y.o., report only if significant per normal urine protocol, sight read and report ‘Beta Hemolytic Streptococcus, morphology consistent with GBS.’

AST and Emerging Resistance -hemolytic Streptococcus spp

Group A and Group B Still universally susceptible to penicillin Macrolide resistance on the rise Clindamycin resistance

• Constitutive• Inducible

• D-Test

Group B prenatal guidelines D-test +

D-test -

AST and Emerging Resistance -hemolytic Streptococcus spp

Erytho RClinda S

Erytho RClinda Ri or Rc

Phenotype Genotype Mechanism

mef

erm

Efflux

Methylase

Yeast Candida albicans

• Microscopy required: oval, budding yeast• Colonies <48 h old on blood-containing medium with

“feet” or mycelial projections• Germ tube positive in <3 h

*C. dubliniensis fail to grow at 45°C.*C. tropicalis may have mycelial fringe after 24h that

must be differentiated from “feet.”*CHROMagar

Abbreviated Identification

Candida CHROMagar

Abbreviated Identification

Yeast Candida glabrata

• Microscopy required: small, oval to circular budding yeast (smaller than other Candida spp.)

• Morphology: small yeast on SBA • Trehalose-positive at 42°C

*Occasionally, other Candida spp. are trehalose-positive but will have different microscopic and macroscopic features.

Abbreviated Identification

Urine CulturesReporting Issues Staphylococcus aureus

Staphylococci reported as part of mixed flora unless predominating

MRSA reported to hospital epidemiology May be a diagnostic indicator of endocarditis

Vancomycin Resistant Enterococci (VRE) Enterococci reported as part of mixed flora,

unless predominating VRE reported to hospital epidemiology

Urine CulturesReporting Issues Staphylococcus saprophyticus

Coag-neg staph, resistant to novobiocin Routine AST not performed Infections respond to achievable urine concentrations

of antibiotics commonly used to treat acute, uncomplicated UTIs

• Trimethoprim/sulfamethoxazole, nitrofurantoin, or a fluoroquinolone

Corynebacterium urealyticum GPR, slow growing, strongly urease positive Alkaline-encrusted cystitis and urinary tract struvite

calculi Culture if alkaline urine and struvite crystals,

leukocytes, and erythrocytes present

Urine CulturesReporting Issues Aerococcus spp.

GPCs, alpha hemolytic colonies UTIs in elderly males with predisposing conditions such

as prostatic disease, diabetes or alcoholism Routine AST methods and standardized interpretive

criteria not available Predictably susceptible to penicillin, ampicillin,

tetracycline, and vanocmycin Resistant to sulfonamides

Gardnerella vaginalis or Lactobacillus spp. Urogenital tract flora component; suggests poorly

collected specimen If patient is symptomatic, consider recollection

Now to the lab…