Evaluation of new medium with chromogenic substrates for members ...

JOURNAL OF CLINICAL MICROBIOLOGY, Jan. 1995, p. 199–201 0095-1137/95/$04.0010 Copyright q 1995, American Society for Microbiology

Vol. 33, No. 1

NOTES Evaluation of New Medium with Chromogenic Substrates for Members of the Family Enterobacteriaceae in Urine Samples H. KODAKA,1 M. ISHIKAWA,2 M. IWATA,2 F. KASHITANI,2 S. MIZUOCHI,3

AND

K. YAMAGUCHI1*

Department of Microbiology, School of Medicine, Toho University, 5-21-16,1 and Omori Hospital, Toho University, 6-11-1,2 Omorinishi, Otaku, Tokyo 143, and Nissui Pharmaceutical Co. Ltd., 1075-2, Hokunanmoro, Yuuki, Ibaraki 307,3 Japan Received 6 July 1994/Returned for modification 2 September 1994/Accepted 9 October 1994

A new medium containing 5-bromo-4-chloro-3-indolyl-b-D-glucuronide cyclohexylammonium salt (Glu agar) for Escherichia coli and a new medium containing 5-bromo-3-indolyl-b-D-galactoside (Gal agar) for b-galactosidase-positive members of the family Enterobacteriaceae were compared with MacConkey agar in a diagnostic trial with 3,562 urine specimens. The isolation rates of E. coli and b-galactosidase-positive Enterobacteriaceae were increased 8.4 and 19.5%, respectively. The sensitivities and specificities of Glu agar and Gal agar were 98.5 and 100% and 99.2 and 99.5%, respectively. Among the bacteria most commonly isolated from urinary tract infections are Escherichia coli and b-galactosidase-positive members of the family Enterobacteriaceae (3, 7, 8). The latter include members of the genera Cedecea, Citrobacter, Enterobacter, Escherichia, Hafnia, Klebsiella, Kluyvera, Rahnella, Salmonella III, Serratia, and Yersinia. Recently, chromogenic substrate assays based on the enzymatic activities of b-glucuronidase for the rapid identification of E. coli in urine (2), food (4, 15, 16), and water (6, 10, 16) and that of b-galactosidase for the coliform group in food (5) and water (12) have been assessed. However, 5-bromo-4-chloro-3-indolyl-b-D-glucuronide cyclohexylammonium salt (13, 15, 16) for E. coli and 5-bromo-3-indolyl-b-D-galactoside (5, 12) for b-galactosidase-positive Enterobacteriaceae have not been evaluated directly for use with urine samples. Therefore, we have assessed a new agar medium containing 5-bromo-4-chloro-3indolyl-b-D-glucuronide cyclohexylammonium salt (Glu agar) for rapid identification of E. coli and a new agar medium containing 5-bromo-3-indolyl-b-D-galactoside (Gal agar) for rapid identification of b-galactosidase-positive Enterobacteriaceae in urine samples. Urine samples. A total of 3,562 urine specimens were obtained from a total of 1,920 inpatients and outpatients at Toho University Omori Hospital in Tokyo, Japan. Midstream clean-void or catheterized urine specimens for culture were obtained in sterile plastic tubes. Random urine samples that were received in the laboratory were cultured immediately or were refrigerated for less than 24 h. Medium. A medium was developed primarily for rapid growth of Enterobacteriaceae. The medium contains the following ingredients per liter: Trypticase peptone (BBL), 10.0 g; Phytone peptone (BBL), 5.0 g; yeast extract (Difco), 5.0 g; NaCl, 5.0 g; K2HPO4, 2.5 g; KNO3, 1.0 g; sodium pyruvate, 1.0 g; sodium dodecyl sulfate (SDS) 0.2 g; and agar, 15.0 g (pH 7.0 6 0.1). The concentration of each chromogenic substrate was 0.01%. The medium was autoclaved for 15 min at 1218C. Glu

agar and Gal agar were supplemented with 5-bromo-4-chloro3-indolyl-b-D-glucuronide cyclohexylammonium salt and 5bromo-3-indolyl-b-D-galactoside (Wako Pure Chemical Industries, Ltd., Osaka, Japan), respectively. Urine samples in the plastic tubes were directly inoculated with a 0.001-ml calibrated loop (Nunc) onto Glu agar, Gal agar, MacConkey agar, and 5% sheep blood agar plates. After incubation at 368C for 24 h, bluish-green colonies on Glu agar and bluish-purple colonies on Gal agar were presumptively identified as E. coli and b-galactosidase-positive Enterobacteriaceae, respectively. Bacterial isolates were inoculated onto brain heart infusion agar plates and incubated at 368C for 24 h for confirming the purity of cultures. These isolates were inoculated onto triple sugar iron agar slants for the fermentation test and identified by ID TEST z EB-20 (Nissui Pharmaceutical Co. Ltd., Tokyo, Japan), which is the system for the identification of glucosefermentative gram-negative rods, according to the positive and negative reactions for the 20 biochemical tests and the motility test. The b-glucuronidase and b-galactosidase activities were confirmed further with the methylumbelliferyl-b-glucuronide reaction and o-nitrophenyl-b-D-galactopyranoside test, respectively. Glucose-nonfermentative gram-negative rods were identified by the Vitek System (bioMerieux-Vitek Japan, Ltd.). Columbia CNA agar (BBL) was used for the isolation and differentiation of gram-positive bacteria. Candida GS (Eiken Chemical Co., Ltd., Tokyo, Japan) was used for isolation of Candida spp. Organisms other than glucose-fermentative and -nonfermentative gram-negative rods were identified provisionally by their appearance on Gram stains and colonial morphology on 5% sheep blood agar; identification was confirmed by standard methods (1, 12). Of 1,920 patients examined, 1,231 (64.1%) yielded microorganisms in urine cultures. A total of 3,562 urine samples were tested, of which 1,273 (35.7%) showed significant growth (^103 CFU/ml). Table 1 shows the results of screening these samples on Glu agar, Gal agar, MacConkey agar, and 5% sheep blood agar. A total of 1,595 strains were isolated with blood agar. These included 344 (21.6%) E. coli strains and 619 (38.8%) b-galactosidase-positive Enterobacteriaceae strains. Of

* Corresponding author. Mailing address: Department of Microbiology, School of Medicine, Toho University, 5-21-16, Omorinishi, Otaku, Tokyo 143, Japan. Phone: (03) 3762-4151. Fax: (03) 5493-5415. 199

200

NOTES

J. CLIN. MICROBIOL.

TABLE 1. Organisms isolated from 1,273 urine specimens on three selective agar plates

a

Organism(s) (no.)

No. of isolates (no. of positive reactions) recovered with the following agar: MacConkey

b-galactosidase-positive Enterobacteriaceae Escherichia coli (344) Citrobacter spp. (1) C. diversus (6) C. freundii (19) Enterobacter aerogenes (8) E. agglomerans (3) E. amnigenus (1) E. cloacae (55) E. sakazakii (3) Hafnia alvei (2) Klebsiella spp. (15) K. oxytoca (12) K. pneumoniae (88) K. cryocrescens (1) Serratia spp. (2) S. fonticola (1) S. liquefaciens (4) S. marcescens (52) Yersinia frederiksenii (2) Total (619)

321 (306) 1 (1) 3 (0) 14 (14) 8 (6) 0 1 (1) 50 (42) 3 (2) 2 (0) 12 (11) 11 (10) 83 (83) 1 (1) 2 (0) 1 (0) 4 (2) 52 (19) 1 (0) 570 (498)

Gal

Glu

343 (340) 339 (334) 1 (1) 1 (0) 6 (6) 3 (0) 20 (20) 16 (0) 11 (11) 9 (0) 3 (3) 3 (0) 1 (1) 1 (0) 55 (55) 50 (0) 3 (3) 3 (0) 1 (1) 2 (0) 12 (12) 13 (0) 13 (13) 10 (0) 93 (93) 83 (0) 1 (1) 1 (0) 2 (0) 2 (0) 0 1 (0) 4 (4) 4 (0) 53 (52) 52 (0) 2 (2) 1 (0) 624 (618) 594 (334)

Other Enterobacteriaceae Morganella morganii (14) Proteus spp. (2) P. mirabilis (5) P. vulgaris (6) Providencia spp. (1) P. rettgeri (1) Total (29)

14 (0) 2 (0) 5 (0) 6 (0) 1 (0) 1 (0) 29 (0)

14 (0) 2 (0) 5 (0) 6 (0) 1 (0) 1 (0) 29 (0)

13 (0) 2 (0) 5 (0) 6 (0) 1 (0) 1 (0) 28 (0)

Other gram-negative species Aeromonas sobria (1) Acinetobacter spp. (7) Alcaligenes spp. (5) Flavobacterium spp. (1) Pseudomonas spp. (19) P. aeruginosa (136) Gram-negative rodsb (9) Total (178)

1 (0) 6 (0) 4 (0) 1 (0) 15 (0) 127 (0) 6 (0) 160 (0)

1 (1) 5 (0) 5 (0) 1 (0) 15 (0) 130 (0) 7 (0) 164 (1)

1 (0) 5 (0) 5 (0) 1 (0) 15 (0) 129 (0) 7 (0) 163 (0)

Gram-positive species (617)

2 (0)

5 (0)

5 (0)

Yeasts (152)

0

0

0

Grand total (1,595) a b

761 (498)

822 (619) 790 (334)

Number of isolates on blood agar. Unidentified nonfermenter.

790 strains isolated with Glu agar, all 334 of the b-glucuronidase-positive isolates were E. coli. No false-positive reactions were seen. Of the five false-negative strains (0.6%), all were methylumbelliferyl-b-glucuronide negative. The sensitivity and specificity of Glu agar for identification of E. coli directly from urine samples were 98.5 and 100%, respectively. Of the 822 organisms isolated with Gal agar, 619 were b-galactosidase positive; all but one of them were Enterobacteriaceae. The one false-positive strain was Aeromonas sobria. The three false-negative strains of E. coli were also o-nitrophenyl-b-D-galactopyranoside negative. The sensitivity and

specificity of Gal agar for identification of b-galactosidasepositive Enterobacteriaceae directly from urine samples were 99.2 and 99.5%, respectively. Of 570 b-galactosidase-positive Enterobacteriaceae colonies on MacConkey agar, 498 (87.4%) isolates were lactose-fermenting bacteria, and 72 (12.6%) isolates grew as colorless colonies. Of the gram-positive species, only a few enterococci and one coagulase-negative staphylococcus grew on Glu and Gal agar. None of the Candida species grew on these media. These results show that the selectivities of Glu and Gal agar were just as inhibitory for organisms other than gram-negative rods as MacConkey agar. Glu agar for E. coli and Gal agar for b-galactosidase-positive Enterobacteriaceae were compared with commonly used MacConkey agar for Enterobacteriaceae in the diagnostic trial with 3,562 urine specimens. The isolation rates of E. coli with Glu agar and b-galactosidase-positive Enterobacteriaceae with Gal agar were increased 8.4 and 19.5%, respectively. Edberg and Trepeta have reported that E. coli was represented by 82% of urinary isolates (3). Even if the isolation rate of E. coli (22.9%) in our study was relatively low, the most predominant isolate was E. coli. In the development of these media, the concentration of SDS was initially 0.1 g/liter. However, it was found that Staphylococcus warneri could grow on Glu agar as blue-green colonies because this bacterium produced b-glucuronidase and may be associated with urinary tract infections (9). Therefore, the SDS concentration was increased to 0.2 g/liter. The sensitivity value for E. coli by our results is 9.7% higher than that of Delisle and Ley (2), and the colony color of E. coli on Glu agar was clearer than that of E. coli on MacConkey agar. These results suggest that Glu agar is useful for the rapid identification of E. coli and Gal agar is useful for selection and isolation of b-galactosidase-positive Enterobacteriaceae. These are the most common bacteria in urinary tract infections. Therefore, we conclude that Glu and Gal media may help in interpretation of culture results and are useful for early medical treatment resulting from a rapid diagnosis made without complete identification of the microorganisms. Recently, various chromogenic substrates have been synthesized by Biosynth AG, Zurich, Switzerland. Therefore, it is possible that just one plate could be used for the differentiation of Enterobacteriaceae at the genus level by colony color caused by various enzyme activities (14). Further experiments on media for characterization of Enterobacteriaceae are being carried out. We are indebted to the staff of the Clinical Laboratory of Toho University Omori Hospital for their assistance. We thank Kazuki Horigome for helpful suggestions and Charles L. Hatheway (Centers for Disease Control and Prevention, Atlanta, Ga.) for critically reading the manuscript. REFERENCES 1. Barrow, G. I., and R. K. A. Feltham. 1993. Cowan and Steel’s manual for the identification of medical bacteria, 3rd ed. Cambridge University Press, London. 2. Delisle, G. J., and A. Ley. 1989. Rapid detection of Escherichia coli in urine samples by a new chromogenic b-glucuronidase assay. J. Clin. Microbiol. 27:778–779. 3. Edberg, S. C., and R. W. Trepeta. 1983. Rapid and economical identification and antimicrobial susceptibility test methodology for urinary tract pathogens. J. Clin. Microbiol. 18:1287–1291. 4. Frampton, E. W., L. Restaino, and N. Blaszko. 1988. Evaluation of the b-glucuronidase substrate 5-bromo-4-chloro-3-indolyl-b-D-glucuronide (XGLUC) in a 24-hour direct plating method for Escherichia coli. J. Food Prot. 51:402–404. 5. Hahn, G., and E. Wittrock. 1991. Comparison of chromogenic and fluorogenic substances for differentiation of coliforms and Escherichia coli in soft cheese. Acta Microbiol. Hung. 38:265–271.

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