PRAKTIKUMMIKROBIOLOGI INFEKSI SALURAN URINARIA (UTI) Aspek Penting Pemeriksaan Mikrobiologi Pada UTI : - Pengumpulan spesimen & penanganannya - Perlakuan spesimen and kultur - Interpretasi hasil Lab Mikrobiologi. Microbiology of Community-Acquired Urinary Tract Infection Dysuria-Pyuria Sydrome in Females Children Adults More frequent . Escherichia coli . Escherichia coli . Staphylococus saprophyticus (young, sexually active patient) Less Frequent . Other Enterobacteriaceae . Enterococci . Streptococcus agalactiae . Other Enterobacteriaceae . Enterococci Other Community-Acquired Infection Children Adults More frequent . Escherichia coli . Escherichia coli Less Frequent . Other Enterobacteriaceae . Enterococci . Other Enterobacteriaceae . Enterococci Microbiology of Nosocomially Acquired Urinary Tract Infection in Children or Adult Catheter-Associated Short-Term (< 30 –d) Catheterization More frequent . Escherichia coli Less Frequent . Other Enterobacteriaceae . Pseudomonas Spp. . Staphylococcus epidermidis Catheter-Associated Long-Term (> 30 –d) Catheterization More frequent . Providencia stuartii . Pseudomonas Spp. . Escherichia coli . Other Enterobacteriaceae Less Frequent . Staphylococcus epidermidis . Renal Tuberculosis spesimen diambil 3 hari berturut2 (pagi hari). • Pasien asimptomatik (2-3 spesimen). & 1 spesimen diambil setelah terapi 48 – 72 jam.GENERAL INFORMATION • Urine dpt terkontaminasi oleh Mo yang terdpt di uretra/ Periuretral Proliferasi Misleading • Pasien Simptomatik : 1 spesimen utk diagnosis. Pengumpulan spesimen Sampel urine diambil pd pagi hari. utk memperoleh bakteri tahan asam. fungi & Mo patogen lain. Midstream urine Mo yg tdp di bladder . • Urine dikumpulkan menggunakan tempat yg steril. & tahan bocor. .Continue… • Pediatric: diambil dr kantung urine kultur segera mungkin. • Hindari Kontaminasi • Pasien Simptomatik / Asimptomatik: Interpretasi kultur kuantitatif . dan perhitungan spesimen urine .Continue…. maka spesimen: disimpan di lemari es: tidak lebih 24 jam Inokulasi: medium primer sbl medium transport ditambahkan larutan pengawet • Perhitungan koloni dinyatakan Invalid jika spesimen tdk diproses selama 30 menit .SPECIMEN TRANSPORT • Segera (1-2 jam) tanam pd medium transport • Jika transportasi terlambat. Jika diduga (suspected) suprapubic bladder aspiration . Pasien Simptomatik. perhitungan dibawah < 100.000 CFU/ ml sering di validasikan dgn Σ leukosit pd spesimen • Kultur Anaerob sulit diperiksa.PROSES SPESIMEN • ≥ 100.000 CFU/ml Infeksi UTI. Continue … • Foley catheter tidak bisa di kultur • Sampel Urin kantung kateter dgn menggunakan syringe & jarum utk Aspirasi. melalui sampling port stl di desinfektan . . . Sampling Port Urine collection can be performed without needle Avoidance of needle stick injury . . Urine container criteria : Sterile. wide-necked. dry.TRANSPORT MEDIA Urine specimen is best collected Soon after a patient wakes when Organisms have had the opportunity to multiply over several hours. leak proof . Biochemical tests. Over night incubation Isolation of colonies. Over night incubation RESULT Urinary Tract infection Module’05 …. ..Laboratory examination of urine Quantitative (Colony counts) +1 +2 +3 +4 a urine sample is streaked on surface of Blood Agar plate ( for control) and medium Endo agar / Mc Conkey agar with a special loop calibrated to deliver a known volume. Drug susceptibility test. coli and coliform bacteria metabolize lactose with the production of aldehyde and acid. 4044 Selective culture medium for detection and isolation Of fecal Escherichia coli and coliform bacteria in Various material according to ENDO Sodium sulfide and fuchsin inhibit the growth of Gram positive bacteria. coli will giving the permanent greenish metallic sheen (Fuchsin sheen) . The aldehyde liberates fuchsin from the fuchsin-sulfide compound. E. E.Endo C agar cat. the fuchsin then colors the colonies red. No. Agar Endo . This agar is particularly recommended for the cultivation of pathogens which may be present in a variety of specimens such as urine. Briliant Green Agar and Brilliant Green bile broth. . and non selective medium such as Blood Agar. dairy product and biological specimens. Bismuth sulphite agar. faeces and wound swabs. MacConkey agar should be used in parallel with other selective indicator media such as Desoxycholate Citrate Agar. isolation and enumeration of coliform and intestinal pathogen in water.MacConkey Agar A differential medium for detection. • MCA (Mac Conkey Agar) • Escherichia coli (koloni merah muda) • Salmonella sp (koloni putih) . 000 Urinary Tract infection Module’05 ….000 +4 1000 100.000 >+2 250 25.000 +2 200 20.000 > +4 > 1000 > 100.000 +1 100 10..000 >+3 500 50.000 >+1 150 15.INTERPRETATION OF COLONY COUNTS Score Number of colonies Number of bacterial per ml < +1 50 5. Diagnostic significance Significant bacteriuria .000 +3 300 30. Gram Stain Escherichia coli Microscopic : • Gram negative rod . .L.E. This medium prevent the swarming phenomenon by having the low electrolyte content. lactobacilli and micrococci is also clearly elicited giving indicate and indication of the degree of contamination.C.D Medium (Cystine-Lactose-Electrolyte Deficient) This medium recommended for urinary bacteriology. may be present a problem in identifying the species responsible to UTI. The present of important contaminants such as diphtheroids. supporting the growth of all urinary pathogens. notably Proteus spp. Discrete colonies of motile and non motile bacteria may thus be urinary bacteriology where motile species. In laboratory C.Proteus This species causes urinary infection.. Urinary Tract System Module’05 ….E. Mostly Proteus strains produce a characteristic swarming growth over the surface of blood agar and several other culture media. especially following catheterization or cystoscopy.L. . posses urease which produces ammonia in the urine. Proteus culture have a distinctive smell. This raises the the pH and causes precipitation of phosphate crystals. leading to stone formation.D medium (electrolyte deficient) provide for plate culture of urinary organisms for prevents the swarming phenomenon Proteus spp. . Ureasa test Test the ability of microorganisms to metabolize urea and produce ammonia using urease (indicator: methyl red) Result : •Positive pink •Negative yellow . . NOVOBIOCIN TEST This test using for differentiated between Staphylococcus epidermidis and Staphylococcus saprophyticus from Urine specimens. Staphylococcus epidermidis (sensitive-Novobiocin). A. it causes cystitis in young women patients in general practice beside Escherichia coli Urinary Tract System Module’05 …. sometimes associated with catheterization. .. Staphylococcus saprophyticus (resistant-Novobiocin). occasionally cause of urinary infection in hospital. B. . Microscopic : • Gram positive cocci of uniform size • That occur characteristically in groups but also singly and in pairs .Gram Stain Staphylococcus sp. . Pseudomonas Pseudomonas aeruginosa: This species causes urinary infection. Urinary Tract System Module’05 …. . usually following catheterization or associated with chronic urinary infections.. This species is usually resistant to many antibiotics and is particularly associated with recurrent urinary tract infection in hospital patients. SABOURAUD DEXTROSE AGAR Sabouraud dextrosa agar is used to culture Candida albicans and other fungi . pH medium should be within the rage 5. candida albicans on sabouraud agar .4 – 5.8 at room temperature. Microscopic : • Gram positive .Gram Stain Candida sp. . .BIOCHEMICAL TEST Carbohydrate fermentation Urinary Tract infection Module’05 …. VP : Voges-Proskauer) .-D-galactopyramoside.Commercially identification The API 20Etm (produced by Biomerieux) system for bacteria such as coliform (ONPG : orthonitrophenyl. The organisms here is Escherichia coli. Immediately after inoculation b.Three API 20E strips : a. That in ( b) after addition of reagents to certain wells. After 24 hours incubation c. Here the first carbohydrate well (glucose) is also used for the nitrate reduction test . . H. Swab the surface of the plate three times.Antibiotic susceptibility tests Disc susceptibility test A. Urinary Tract System Module’05 …. B. Allow 3 to 5 minutes. touch the tops of 4 to 5 bacterial colonies of the same morphology on an agar plate culture. Using a forceps or multidisk dispenser plate antibiotic impregnated disk on surface of the agar. D. After overnight incubation. Match the density of the bacterial suspension to the 0. Transfer the bacteria to a tube containing 4 to 5 mL of Brain Heart Infusion broth or NaCl. Farland density standard. E. F. Using wire loop or Dacron-tipped swab. use a ruler or caliper to measure the diameter of incubation zones. Insert a cotton-tipped swab into the suspension.. C.5 Mc. Incubate at 35 0 C for 24 hours. rotating the plate 60 0 each time. Tamp the disks down firmly onto the surface of the agar. G. the swab firmly against the wall of tube to express excess fluid and streak the surface of dried Mueller-Hinton agar plate. . S (Sensitive) P C P C Staphylococcus aureus CN CN .Gentamycin (CN) : 12 .29 R (Resistant) .15 Chloramphenicol (C) : 12 .18 Penicillin (P) : 28 . I (Intermediate) . MIC result : Escherichia coli = 2 g . E-test strip .