Transcript Urine Cultures

ID Core Curriculum:
Urine Cultures
Melissa B. Miller, PhD
February 1, 2008
Culture-based methods
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Liquid media
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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
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Solid media
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Isolates can be quantitated
Sight-identification possible
Culture-based methods
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Non-selective media
Selective media
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Incorporate antimicrobial agents to inhibit flora while allowing
for the growth of a specific organism (i.e., Thayer-Martin for
GC)
Differential media
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N. gonorrhoeae
on TM
Incorporate one or more carbohydrates in the medium with a
pH indicator (i.e., MacConkey Agar)
+/- Lactose fermenation
on MacConkey
Culture-based methods
Quantitative
 Semi-quantitative
 Isolation
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Specimens Submitted From
Human Urinary Tract
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Acceptable specimens
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Clean catch, catheterized
Nephrostomy, indwelling
catheter/Foley , ileal conduit, and
cystoscopy specimens
Suprapubic aspirate, ureteral, kidney
specimens (Invasive – add broth
tube)
Gram Stain
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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
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Clean catch or catheterized urine
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Plate 1ul; 1 colony = 1000 organisms/ml
Clean catch: >105 orgs/ml
Cath urine: >104 orgs/ml
Suprapubic urine or patients w/
dysuria
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Plate 10ul; 1 colony = 100 organisms/ml
>103 orgs/ml
Processed aerobically and anaerobically
Urine Culture Set-up
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Suprapubic aspirates
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Acute dysuria
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10 ul
Sheep blood agar, MAC, anaBHI (brain heart
infusion, anaerobic)
10 ul
SBA, MAC
Add CHOC for post-prostatic massage
Routine urines
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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
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Gram positives take longer to grow
 May not be able to determine amount
of flora present
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ID of Normal Flora and
Potential Pathogens
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Judgment required
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Determine which organisms to look for
Variable depending upon specimen site
Determine what constitutes normal flora vs
potential pathogen
Extent of workup
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Contribution to unnecessary use of antibiotics
Contribution to emergence of resistant
organisms
Resident Flora of Human
Urinary Tract
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Sterile above urethra
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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
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Many organisms are same as found in genital
tract
Some transient colonizers are potential
pathogens
Resident Flora of Human
Urinary Tract
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Genital tract flora- mixture of:
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Lactobacillus
Alpha-hemolytic Streptococcus sp.
Diptheroids
CoNS
Gardnerella vaginalis
Yeast
mixed urogential flora
Most Common Pathogens
of Human Urinary Tract
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Community acquired
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E. coli is most frequent pathogen isolated
Klebsiella sp and other Enterobacteriaceae
Staphylococcus saprophyticus
Hospital acquired
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E. coli, Klebsiella, other Enterobacteriaceae
Pseudomonas aeruginosa
Enterococci and Staphylococci
Urine Cultures
Urine Cultures
Abbreviated Identification
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E. coli
Non-swarming, spot indole pos, oxidase neg
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1. Hemolytic on SBA
2. 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
Indole +
AST and Emerging Resistance
Beta-lactamases
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ESBL: E. coli, Klebsiella, P. mirabilis
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Mutant TEM-1, TEM-2, and SHV-1 b-lactamases
Hospital-acquired: Clinically relevant isolates
Resistant to all cephalosporins, including
cefotaxime, ceftazidime
• Cefoxitin may still be S
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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 Resistance
Beta-lactamases
AST and Emerging Resistance
Beta-lactamases
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AmpC: Enterobacter, Serratia, P. vulgaris,
P. aeruginosa
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Chromosomal cephalosporinase
May also be plasmid-mediated (Klebsiella)
Resistant to all b-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 Resistance
Beta-lactamases
Abbreviated Identification
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Proteus spp.
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Swarming growth
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Indole
• Negative: P. mirabilis/penneri
• P. mirabilis: maltose neg, ornithine pos
• P. penneri: maltose pos, ornithine neg
• Positive: P. vulgaris
Abbreviated Identification
P. aeruginosa
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Oxidase +
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
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CHROMagar Orientation
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Presumptive ID for some UTI pathogens
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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
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Enterococcus spp.
Cocci or coccobacilli in pairs and chains
 >1 mm colonies
 Non-hemolytic on SBA
 Catalase-negative
 PYR-positive
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(pyrrolidonyl-a-naphthylamide
hydrolysis)
AST and Emerging Resistance
Enterococcus spp
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E. faecalis
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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)
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vanA/vanB
vanC1, vanC2/C3
• E. gallinarum, E. casseliflavus, E. flavescens
• NOT true VRE
AST and Emerging Resistance
Enterococcus spp
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Vancomycin Resistant Enterococci (VRE)
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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
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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
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BD/GeneOhm VRE PCR awaiting FDA-approval
• VanB isolates likely to require confirmation
Abbreviated Identification
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S. agalactiae (GBS)
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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+)
b-hem Enterococcus
Urine Cultures
Reporting Issues
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Beta Hemolytic Streptococci (GAS/GBS)
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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
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Screening cultures- Group B Streptococcus
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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
b-hemolytic Streptococcus spp
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Group A and Group B
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Still universally susceptible to penicillin
Macrolide resistance on the rise
Clindamycin resistance
D-test • Constitutive
• Inducible
• D-Test
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Group B prenatal guidelines
D-test +
AST and Emerging Resistance
b-hemolytic Streptococcus spp
Phenotype
Genotype
Erytho R
Clinda S
mef
Efflux
erm
Methylase
Erytho R
Clinda Ri
or Rc
Mechanism
Abbreviated Identification
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Yeast
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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
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Candida CHROMagar
Abbreviated Identification
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Yeast
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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 trehalosepositive but will have different microscopic and
macroscopic features.
Urine Cultures
Reporting Issues
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Staphylococcus aureus
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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)
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Enterococci reported as part of mixed flora,
unless predominating
VRE reported to hospital epidemiology
Urine Cultures
Reporting Issues
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Staphylococcus saprophyticus
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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
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Corynebacterium urealyticum
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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 Cultures
Reporting Issues
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Aerococcus spp.
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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.
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Urogenital tract flora component; suggests poorly
collected specimen
If patient is symptomatic, consider recollection
Now to the lab…