Urinary tract infections (UTIs) are the second most common infections, only after respiratory tract tinfections.

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1 Detection of fu Uropathogens by Using Chromogenic Mdi Media Farzaneh Azizmohseni, Director of Persian Type Culture Collection ((PTCC)

2 Introduction Urinary tract infections (UTIs) are the second most common infections, only after respiratory tract tinfections. Urinary tract infections are one of the most common causes of hospital admissions and clinic visits globally, making urine, the most frequent sample received for culture. Definitive diagnosis of urinary tract infection (UTI) is performed through quantitative urine culture Traditionally the presence of 100,000 or more bacteria/ml in urine has been considered to represent significant bacteriuria, indicating UTI

3 UTI Criteria Asymptomatic bacteriuria: Urine Culture 10 5 cfu/ml on 2 voided consecutive specimens (women) 10 5 cfu/ml on 1 clean-catch urine specimen (men) 10 2 cfu/ml on 1 catheterized urine specimen

4 UTI Criteria Symptomatic bacteriuria: Urine Culture 10 4 cfu/ml (pyelonephritis or fever with local GU symptoms) 10 3 cfu/ml (acute lower urinary tract symptoms) 10 5 cfu/ml (external catheter in men) 10 3 cfu/ml (aspirated indwelling catheter)

5 Diagnosis The diagnosis of urinary tract infections (UTIs) by performing urine cultures contributes greatly to the daily workload in a clinical microbiology laboratory. The laboratory diagnosis of urinary tract infection requires quantitative urine culture on standard agar media. The traditional approach to the detection of pathogenic bacteria in urine: Using a nonselective medium + selective medium

6 Urine Culture Blood agar (BA)+ MacConkey agar (MAC) or Eosin methylen blue agar (EMB) Cysteine lactose electrolyte-deficient (CLED) agar + MAC or EMB Peptone Beef extract Tryptone Lactose L-Cysteine Bromothymol blue Agar 4g/l 3g/l 4g/l 10g/l 128 mg/l 20 mg/l 15g/l ph 7.3± 0.2

7 Differentiation The key parameters for differentiation: 1-Lactose 2- ph indicator (MAC neutral red, EMB eosin & MB)

8 Improving the urine culture algorithms! Only 20 to 30% of urine samples result in significant growth a considerable amount of time is expended evaluating samples that do not have clinical utility. New methods or media with the potential to streamline urine culture processing are required. 1- Reducing technologist workload 2- Improving TATs 3- Reducing laboratory costs

9 Chromogenic media Chromogenic media are principally designed to target the pathogens with high specificity. Chromogen a chemical compound, itself without colour, that can be transformed into a coloured compound, or can react with another material to form a coloured compound Chromogenic media exploit enzyme substrates that release coloured dyes upon hydrolysis, thus resulting in pathogens forming coloured colonies that can easily be differentiated from commensal flora.

10 Chromogenic media An ideal chromogenic substrate should be hydrolysed to release a coloured product that remains highly localized on microbial colonies. This allows clear differentiation of microbes producing the target enzyme from those that do not. The chromogenic substrate should be designed for enzymes in the pathogen (Target organisms)

11 Background and principles of chromogenic media Chromogen Sugar component (e.g. galactose) Uptake Principles of Chromogenic agar Chromophore component (e.g. indoxyl derivative such as 5-bromo-4-chloro-3-indoxyl) y) E. coli Enzyme

12 Background and principles of chromogenic media Principles of Chromogenic agar E. coli

13 Principles of Chromogenic medium Br OH OH Cl Br Cl H HO H H H O OH H O NH β-galactosidase oxidation HN O NH 5-bromo-4-chloro-3-indoxyl-β-Dgalactopyranoside (X-Gal) OH OH O H HO H H O OH OH Br Cl H galactose H blue-turquoise chromophore

14 Why use Chromogen? Improve overall specificity of a diagnostic medium. Improve the sensitivity of identification of microorganisms in mixed culture Positive colonies are easy to identify. Reduce number of sub-cultures required. Promote more uniform interpretation of urine culture plates by less experienced bench technologists.

15 History Culture media for isolation of bacteria were first devised by Louis Pasteur, where liquid media like urine or meat broth were used. The earliest solid medium discovered was cooked cut potato by Robert Koch Agar was discovered by Frau Hasse, wife of one of the disciples of Robert Koch (late 19 century). Enriched, selective, differential, media were made and used dto support the growth of all type bacteria In I 1979 Kilian and Bu low described dthe first chromogenicmedium The chromogenic media wre commercialized from 1993

16 Current Chromogen situation The large scale synthesis of chromogens means compounds are affordable for use in culture media There are now chromogenic media available for almost all organisms of interest in both the Industrial and Clinical environments >30 chromogenic media formulations are currently available for the differentiation of E. coli and coliforms >10 formulations for Salmonella

17 Microbiology of UTI E.coli, is the most common pathogen, accounting for 60-90% of UTIs K. pneumoniae, Morganella spp. and Proteus spp. are the next most common P. aeruginosa is more common in the nosocomial setting Enterococcus spp. account for 10-16% of UTIs S. saprophyticus hti is a common cause of UTIs in young women (up to 11%)

18 Target Enzymes in Urine Cultures Bacteria Enzyme Substrate Color E.coli ß-Glucuronidase 6-Chloro-3-indoxyl- ß-D- glucuronide Pink - salmon E.coli ß-Galactosidase 6-Chloro-3-indoxyl-ßgalactopyranoside Pinksalmon PPM Group Tryptophan deaminase Brown Enterococci ß-Glucosidase 5-bromo-4-chloro-3- Indolyl-ß-D-glucopyranoside KECS Group ß-Galactosidase 6-Chloro-3-indoxyl-ßgalactopyranoside KECS Group ß-Glucosidase 5-bromo-4-chloro-3- Indolyl-ß-D-glucopyranoside Blue Purple

19 Chromogenic Substrate Should be hydrolysed to release a coloured product that remains highly localized on microbial colonies. High sensitivity Low toxicity Heat resistance M t i ll il bl h i di Most commercially available chromogenicmedia have indoxyl substrates and its halogenated derivatives

20 Test procedures Quality control procedures should be performed for all chromogenic media before use. The quality of the medium should be checked by using stable control strains. A dilution of the specimen on the plate (by using calibrated loops or) is inoculated on plates. Plates are incubated aerobically at 35 ± 2 C for not less than 20 to 24 h in an inverted position (agar-side up). Light exposure should be avoided After incubation colonies o should be investigated ed according to the guideline of the manufacturer instruction. Confirmatory tests may be applied.

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23 J. Clin. Microbiol. 2013, 51(4): ): , Manickam etal.

24 Specificity and Sensitivity From 70 to more than 90% have been reported in the literature. Chromogenic media could be allowed for the differentiation and identification of E. coli and enterococci without confirmatory testing, based on the criteria for identification established by the CLSI standard M35-A2, Abbreviated Identification of Bacteria and Yeast; Approved Guideline.

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