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ORIGINAL ARTICLE
Year : 2004  |  Volume : 22  |  Issue : 3  |  Page : 153-158
 

Utility of urichrom II - A chromogenic medium for uropathogens


Department of Microbiology, Nizam's Institute of Medical Sciences, Punjagutta, Hyderabad - 500 082, Andhra Pradesh, India

Date of Submission03-Feb-2004
Date of Acceptance22-May-2004

Correspondence Address:
Department of Microbiology, Nizam's Institute of Medical Sciences, Punjagutta, Hyderabad - 500 082, Andhra Pradesh, India

 ~ Abstract 

PURPOSE: Chromogenic media are increasingly being used as versatile tools in early differentiation and identification of bacterial isolates from clinical specimens. We have evaluated the Urichrome II, a newly introduced chromogenic medium, for its utility as a primary isolation and identification medium for urinary microbial isolates. MATERIALS: This study included 5497 consecutively collected midstream and/or catheter catch urine samples obtained from patients attending several clinical services. They were inoculated on blood, MacConkey, cystine lactose electrolyte deficient, and Urichrom II agar plates for isolation of uropathogens. RESULTS: The Urichrom II medium allowed the growth, and primary identification, of the predominant uropathogens like E.coli, Klebsiella spp. and Enterobacter spp. (KES group). The different coloured colonies produced by the breakdown of the chromogenic substrates by the specific enzymes of the bacteria were very useful in the presumptive identification of these organisms even from mixed cultures by the colour differences of the colonies. The medium also supported growth and differentiation of gram positive organisms like Staphylococcus aureus and enterococci. CONCLUSIONS: Urichrom II can be used as a primary culture medium for predominant uropathogens like E.coli, KES group and enterococci. It is an easy to use primary screening medium that considerably reduces the daily workload and thus minimizes or limits the use of identification tests.

How to cite this article:
Lakshmi V, Satheeshkumar T, Kulkarni G. Utility of urichrom II - A chromogenic medium for uropathogens. Indian J Med Microbiol 2004;22:153-8


How to cite this URL:
Lakshmi V, Satheeshkumar T, Kulkarni G. Utility of urichrom II - A chromogenic medium for uropathogens. Indian J Med Microbiol [serial online] 2004 [cited 2019 Sep 17];22:153-8. Available from: http://www.ijmm.org/text.asp?2004/22/3/153/11208


Conventionally, blood agar, MacConkey agar (MAC) and cystine lactose electrolyte deficient medium (CLED) are used together for the processing of urine samples by culture.[1],[2] Over the last few years, several chromogenic media have been developed and commercialized, allowing more specific and direct differentiation of microorganisms on the primary plates.[3],[4],[5],[6],[7],[8],[9],[10] They not only minimize the need for further identification tests but also reduce the time required to report the results to the clinician to facilitate early initiation of antibiotic therapy.
Recently another chromogenic medium, the Urichrom agar II (International Microbio, Signes, France) an enzyme - substrate medium, was designed for isolation and presumptive identification of uropathogens. This study was initiated to evaluate the utility of Urichrom II as a primary medium for isolation and recovery of urinary pathogens, to study its utility for presumptive identification of uropathogens and to compare Urichrom II with the routinely used media (blood agar, MAC and CLED agar) in the recovery of the uropathogens.

 ~ Materials and Methods Top

Clinical samples
This study included 5497 consecutively collected midstream and/or catheter catch urine samples obtained from patients attending the various clinical services of Nizam's Institute of Medical Sciences, Hyderabad.
Culture media
All the culture plates (blood agar, MAC, CLED) were prepared in-house using commercially available dehydrated media (Hi Media Laboratories, Mumbai, India). They were prepared according to the manufacturer's recommendations and dispensed into petridishes, checked for sterility and stored at 2-8C till use.
Urichrom II
This is an enzyme substrate medium available as agar base and a chromogenic supplement in two separate bottles. The medium was prepared according to the manufacturer's instructions. The prepared plates can be stored at 2 to 8C for 1 month. Every fresh batch of medium was tested for its ability to support the growth of ATCC strains such as Staphylococcus aureus ATCC 25923, Enterococcus faecalis ATCC 29212,  Escherichia More Details coli ATCC 25922 and Pseudomonas aeruginosa ATCC 27853.
Inoculation of media and incubation
All urine samples were inoculated simultaneously on all four media with a calibrated loop (holding 10ml of sample) and incubated aerobically at 37C overnight and examined the next morning. A presumptive identification of the isolates was attempted based on the colony colours on the Urichrom II plates. The isolated microorganisms were identified to the species level as per the standard protocols.[11],[12]
Results
A total of 5497 urine samples were processed on all the four types of culture plates. One thousand eighty eight out of 5497 (19.79 %) urine samples were culture positive. While 1035 out of 1088 (95.12%) were unimicrobial, 53(4.87%) were polymicrobial cultures.
On Urichrom II, most of the urine isolates formed coloured colonies as shown in [Table - 1] and [Figure - 1]. A presumptive identification of the organisms could be done on the Urichrom II plates that were later confirmed by biochemical tests. However, in a few cases, the isolates formed cream coloured or pale colourless colonies because they probably failed to produce the enzymes at that point of time [Table - 2]. All these strains could be identified based on biochemical reactions[11],[12] only.
The colony counts on all the media were similar. The ability of Urichrom II to support the growth of the organisms was concurent with those on the conventional media, except in few cases as shown in [Table - 3].

 ~ Discussion Top

On solid culture media, the distinct colony morphologies and many other features such as pigmentation and haemolysis exhibited by the bacteria are used as features for identification. Solid culture media are of different types such as enriched, selective, indicator / differential media. In the latter, specific substances are incorporated in the medium that are specifically utilized by the growing bacteria .The metabolites thus produced bring out different changes in the medium that help to distinguish between one genus/species and the other.[2]
Blood agar is considered to be the optimal medium for the isolation of organisms from clinical samples, including urine.[1],[2] Though used universally as an enriched medium, it has several limitations.[5] It lacks the ability for primary differentiation between the gram negative colonies, thereby necessitating further identification tests and causing delay in the final result. It also completely fails to prevent swarming of the colonies of Proteus spp.
Though differentiation of lactose fermenters from nonlactose fermenters is possible on MAC, it does not support the growth of all organisms involved in UTI (e.g., Staphylococcus aureus   and Streptococcus spp.). Therefore, it cannot be used alone as a primary isolation medium.[5] CLED agar, introduced later, has proven to be useful as a primary medium and helped to reduce the plate burden. This medium has the advantage of supporting growth and differentiation of both the gram positive and gram negative isolates at the same time.[1],[5] It also has the advantage of inhibiting the swarming of Proteus spp.
Though the lactose fermenters can be differentiated from non lactose fermenters on both MAC and CLED agars, identification tests need to be performed to further differentiate between the lactose fermenters like the E.coli and the KES species.
Over the last few years several chromogenic media have been developed and commercialized allowing more specific and direct differentiation of microorganisms on the primary plate itself. They facilitate direct identification of the organisms and reduce the burden of biochemical characterization. Most of these media have been developed specifically for recovery and identification of one genus or species of organisms, e.g, BBL chromagar orientation and CPS ID2 agar for uropathogens, Glu agar for E.coli and Gal agar for Enterobacteriaceae, Chromagar for S.aureus and MRSA, Chromagar orientation with sodium azide or nalidixic acid and vancomycin for detection of enterococci and VRE, Chromagar for gram negative bacteria and enterococci.[5],[6],[7],[8],[9] A few of these media have been developed to isolate and identify majority of the organisms.
The principle of these chromogenic media is based on the fact that bacteria possess many enzymes for their physiological functions that help them to utilize substrates. They are either species specific or genus specific. This specific feature has been exploited in characterization of bacteria based on biochemical tests. Certain tests are considered as key biochemical identification tests for some bacteria and they have been used in the development of the chromogenic media. In such media, specific chromogenic substrates are specifically broken down by a bacterial enzyme thereby imparting a distinct colour to the growing bacterial colony that can be visually observed.[3],[4],[5],[6],[7],[9],[10]
Urichrom II was designed to isolate and identify uropathogens - mainly E.coli, Klebsiella spp. and enterococci. It has an advantage that it supports the growth of all uropathogens unlike MAC. E.coli produce the enzyme b glucuronidase that attacks b glucuronide chromogenic substrate and grow as distinct pink - burgundy colored colonies. The KES group produce the enzyme b glucosidase that attacks b glucoside chromogenic substrate and grow as distinct blue colonies.
An important aspect of Urichrom II was that it allowed an easy differentiation of the various species from mixed cultures [Figure:2], due to the specificity with respect to color of the colony, similar to the other chromogenic media studied.[4],[5],[6],[7],[9],[10] As in other studies,[10] E.coli was the most common isolate (51.7%) in our study. Urichrom II allowed a definite identification of E.coli without the need for further biochemical tests. Also the colony colour of E.coli was clearer and more distinct than that on MAC.[6]
However, the identification was only presumptive for the KES group. Further identification tests had to be done for definite speciation. Other workers also observed the same findings.[5],[10] Urichrom II also supports the direct identification of Proteus spp. with the addition of tryptophan deaminase reagent that forms the brown colour. The rest of the members of Enterobacteriaceae such  Salmonella More Details spp. grew as colourless colonies as they do not produce the specific enzymes. They required further identification by biochemical tests.[7]
The characteristic colony morphology of P. aeruginosa (pale/dark green or yellow green flat shining colonies) and Acinetobacter spp. (pale white to cream opaque dome shaped colonies) was very distinct on Urichrom II [Figure - 1] as on other chromogenic media.[5],[10]
Certain identification tests like the catalase, oxidase and indole test can be done directly from the colonies on Urichrom II agar. The colonies on Urichrom II can be used to perform antibiotic sensitivity without subculture onto another medium.[5],[7],[10] The recovery of the uropathogens on the Urichrom II plate correlated with that on the other plate media used. However, a few of the pathogenic isolates were recovered more frequently on Urichrom II as compared to the other media. This was also observed with other chromogenic media[5],[10] which might be attributed to the easy discrimination of different colonies facilitated by the characteristic color.[5] According to one study the isolation rate of E.coli on Glu agar and Enterobacteriaceae from Gal agar increased by 8.4% and 19.5% respectively.[6] Enterococci were isolated more frequently on Urichrom II as compared to the other media. Studies using a CPS ID2 agar and Chromagar also reported an increased rate of isolation of Enterococci.[5],[10]
There are however, certain exceptions to the rule that have been observed while using chromogenic media. We had similar experiences with Urichrom II. b glucuronidase negative strains of E.coli and b glucosidase negative strains of Klebsiella spp., and Enterobacter spp. appear colourless. Further identification of these colony variants was possible only by biochemical tests. In our study, 11% of isolates of E.coli failed to produce pink coloured colonies. In other studies, 0.7%-18.4%[5],[6],[7] isolates of E.coli failed to produce coloured colonies. We also observed seven isolates of b glucosidase negative Citrobacter spp. In one study using BBL Chromagar, three isolates of KES group, two isolates of  C.freundii  , and three isolates of S.saprophyticus produced colourless colonies.[5] In the same study, using CPS ID2 medium, colonies of six isolates of KES group, three isolates of C.freundii, and 13 isolates of S.saprophyticus were colourless.[5] In another study using Chromagar, 33% of C. freundii, 15% of  E.cloacae  , 26% of K.pneumoniae did not produce coloured colonies.[7] Similarly,  Salmonella More Details spp. produced colourless colonies. All these required further biochemical tests for identification.
The isolates of Morganella morganii produced cream coloured colonies instead of pale brown colony. According to the BBL study, one isolate of P. mirabilis produced rose coloured colonies, whereas two isolates of P. vulgaris produced blue and cream coloured colonies respectively.[5]
In conclusion, Urichrom II can be used as a primary culture medium for uropathogens like E.coli, KES group and enterococci, the predominant isolates involved in both community acquired and nosocomially transmitted UTI. Urichrom II is an attractive and easy to use primary screening medium that considerably reduced the daily workload and thus minimized or limited the use of identification tests.[4],[6],[7],[8],[9],[10] The rapid results obtained by using Urichrom II facilitate an early initiation of the appropriate antimicrobial therapy[4],[5],[6],[7],[8],[9],[10] by the treating clinician. 

 ~ References Top

1.Collee JG, Duguid JP, Fraser AG, Marmion BP. Laboratory strategies in the diagnosis of infectious syndromes. In: Practical medical Microbiology, 14th ed. Collee JG, Barrie MP, Fraser AG, Simmons A, Eds. (Churchill Livingston, New York) 1996:53-93.  Back to cited text no. 1    
2.Ananthanarayan R, Paniker CJK. Culture media. In: Textbook of Microbiology, 6th ed. Paniker CJK (ed.). (Orient Longman Ltd, Chennai, India) 2000:33-38.  Back to cited text no. 2    
3.Delisle GJ, Ley A. Rapid detection of E.coli in urine samples by a new chromogenic ? glucuronidase assay. J Clin Microbiol 1989;27:778-779.  Back to cited text no. 3    
4.Gaillot O, Wetsch M, Fortineau N, Berche P. Evaluation of Chromeagar S.aureus-A new chromogenic medium for isolation and presumptive identification of S.aureus from human clinical samples. J Clin Microbiol 2000;38:1587-1591.  Back to cited text no. 4    
5.Hengstler KA, Hammann R, Fahr AM. Evaluation of BBL Chromeagar orientation for detection and presumptive identification of urinary tract pathogens. J Clin Microbiol 1997;35:2773-2777.  Back to cited text no. 5    
6.Kodaka K, Ishikawa M, Iwata M, Kashitani F, Mizuochi S, Yamaguchi K. Evaluation of a new medium with chromogenic substrates for members of Enterobacteriaceae family in urine samples. J Clin Microbiol 1995;33:199-201.   Back to cited text no. 6    
7.Merlino J, Siarakas S, Robertson GJ, Funnell GR, Gottieb T, Braudbury R. Evaluation of Chromagar orientation for differentiation and presumptive identification of gram negative bacilli and Enterococcus spp. J Clin Microbiol 1996;34:1788-1793.  Back to cited text no. 7    
8.Merlino J, Siarakas S, Veal D. The development of new chromogenic screening test for the phenotypic identification and differentiation of Enterococci with application in the detection of vancomycin resistance. In: Medical Microbiology. Proffered paper. 1998:5.6.  Back to cited text no. 8    
9.Merlino J, Lezoi M, Braudbury R, Veal D, Harbour C. A new chromogenic medium for identification and detection of S.aureus and MRSA. J Clin Microbiol 2000;38:990-994.  Back to cited text no. 9    
10.Samra Z, Heifetz M, Jalmor J, Bain E, Bahar J. Evaluation of Chromagar in the detection of uropathogens. J Clin Microbiol 1998;36:990-994.  Back to cited text no. 10    
11.Collee JG, Miles RS, Watt B. In: Practical medical Microbiology, 14th ed. (Churchill Livingston, New York). 1996:131-151.  Back to cited text no. 11    
12.Garcia LS, Procop GW, Roberts GD, Thomson RB. Overview of conventional methods for bacterial identification. In: Diagnostic Microbiology, 10th ed. (Mosby, St. Louis). 1998:167-181.  Back to cited text no. 12    
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