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ID Core Curriculum: Urine Cultures

ID Core Curriculum: Urine Cultures. Melissa B. Miller, PhD February 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!

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ID Core Curriculum: Urine Cultures

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  1. ID Core Curriculum: Urine Cultures Melissa B. Miller, PhD February 1, 2008

  2. 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

  3. N. gonorrhoeae on TM +/- Lactose fermenation on MacConkey Culture-based methods • 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)

  4. 1 2 1 2 3 Culture-based methods • Quantitative • Semi-quantitative • Isolation

  5. 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)

  6. 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

  7. Urine Gram Stain

  8. 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

  9. 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

  10. 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

  11. 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

  12. 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

  13. Resident Flora of Human Urinary Tract • Genital tract flora- mixture of: • Lactobacillus • Alpha-hemolytic Streptococcus sp. • Diptheroids • CoNS • Gardnerella vaginalis • Yeast mixed urogential flora

  14. 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

  15. Urine Cultures

  16. Urine Cultures

  17. Indole + Abbreviated Identification • E. coli • Non-swarming, spot indole pos, oxidase neg • Hemolytic on SBA • Non-hemolytic on SBA and lactose positive (MacConkey or eosin methylene blue), PYR (pyrrolidonyl arylamidase) test positive • Non-hemolytic on SBA and lactose negative, rapid MUG (methylumbelliferyl-beta-D-glucoronidase) test positive

  18. AST and Emerging ResistanceBeta-lactamases • ESBL: E. coli, Klebsiella, P. mirabilis • 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 • Transferable plasmid containing other resistance genes • Predicted by ceftriaxone, ceftazidime, aztreonam • Disk diffusion or MIC testing of • Cefotaxime +/- clavulanic acid • Ceftazidime +/- clavulanic acid

  19. AST and Emerging ResistanceBeta-lactamases

  20. AST and Emerging ResistanceBeta-lactamases • AmpC: Enterobacter, Serratia, P. vulgaris, P. aeruginosa • 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

  21. AST and Emerging ResistanceBeta-lactamases

  22. Abbreviated Identification • Proteus spp. • Swarming growth • Indole • Negative: P. mirabilis/penneri • P. mirabilis: maltose neg, ornithine pos • P. penneri: maltose pos, ornithine neg • Positive: P. vulgaris

  23. Oxidase + 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

  24. Abbreviated Identification • 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

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

  26. 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

  27. 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 AST and Emerging ResistanceEnterococcus spp

  28. Abbreviated Identification • 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+) b-hem Enterococcus

  29. 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

  30. 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.’

  31. AST and Emerging Resistanceb-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 +

  32. AST and Emerging Resistanceb-hemolytic Streptococcus spp Phenotype Genotype Mechanism Erytho R Clinda S mef Efflux Erytho R Clinda Ri or Rc erm Methylase

  33. Abbreviated Identification • 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

  34. Abbreviated Identification • Candida CHROMagar

  35. 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.

  36. 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

  37. 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

  38. 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

  39. Now to the lab…

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