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A new plate medium for rapid presumptive identification and differentiation of enterobacteriaceae
International Journal of Food Microbiology, 14 (1991 ) 127-134 © 1991 Elsevier Science Publishers B.V. All rights reserved 0168-I605/91/$03.50
127
FOOD 00439
A new plate medium for rapid presumptive identification and differentiation of Enterobacteriaceae M. Manafi and M.L. R o t t e r Hygiene Institute, University of Vienna, Vienna,Austria (Received 17 January 1991; accepted 25 July 1991)
A new selective differential agar medium for rapid presumptive identification of Enterobacteriaceae from water and food samples is described (EMX ID afar). By a combination of fluorogenic and chromogenic substrates, the medium detects the presence of /3-~glucuronidase, O-D-galactosidese, ~8-D-xylosidase, tryptophane deaminase and H2S; additionally, cytochrome-oxidase and indole production can be demonstrated. This medium provides an inexpensive means for simple and rapid presumptive identification of E. co// and coliforms and for the differentiation within the /Oebs/el/a-Enterobacter and the Proteus-Providencia-Morganella group. Furthermore, it allows to distinguish between the H2S-positive Enterobacteriaceae Citrobacter freund~ Salmonella Spp., S. arizonae, Edward~lla, Proteus mirabilis, P. uulgaris and some oxidase.pmitive bacteria. Key words: Foods; Fiuorogenic; Chromogenic; Enterobacteriaceae; Culture medium; Rapid identification
Introduction Detection, differentiation, and enumeration of Enterobacteriaceae are of primary importance in the microbiological quality control of food and water. In particular, coliforms and Escherichia coli are used as indicator organisms. New techniques have been developed for simultaneous enumeration and rapid identification of enterobacteria. Some of them are based on the cleavage of fiuorogenic or chromogenic substrates by specific enzymatic activities (Manafi and Kneifel, 1990; Manafi et al, 1991). These methods allow fast identification even on primary isolation media without the need for time-consuming isolation procedures. This paper describes a new selective differential medium detecting simultaneously the production of ~8-D-glucuronidase (GUD), jS-D-galactosidase (GAL), Correspondence address: M. Manafi, Hygiene Institute, University of Vienna, Kinderspitalgasse 15, A-I095 Vienna, Austria.
128
/3-D-Xy.losidase (XYL), tryptophane-deaminase (TDA)and hydrogen sulfide (H,S). Furthermore, the production of indole (IND) as well as cytochrome-oxidase (OXI can be tested in a second step by addition of the appropriate chemicals to single colonies.
Material and Methods
Bacterial strains A total of 771 strains of Gram-negative rods was included in this study (Table II). All strains were fresh isolates from water and food specimens examined at the Hygiene Institute of the University of Vienna. The identification of pure cultures was carried out with the API 20E System (bio M&ieux S.A., Montalieu, Vercieu. France). Selective differential medium A selective medium was used developed for the detection of E. coli by indicating the production of GUD and Indole, using as a basal medium the commercially available Fluorocult-ECD agar TM (E. Merck AG, Darmstadt, F.R.G.), and supplemented with additional substrates. Fluorocult-ECD agar is composed of peptone (20 g), lactose (5 g), NaCI (5 g), K2HPO 4 (4 g), KH2PO n (1.5 g) bile salts (1.5 g), methylumbelliferyl-/3-D-glucuronide (MUG) (0.07 g), L-tryptophane (1 g), agar (15 g) and distilled water (985 ml). It was sterilized by autoclaving at 121°C for 15 min, cooled to 60 ° C, and placed in a holding water bath at 60 ° C. To this basal medium, 3 mi of each of the six following solutions were added aseptically. (i) The chromogenic substrate 5-bromo-4-chloro-3-indolyl-/3-D-galactopyranoside (XGAL) (Sigma Chemical Co., St. Louis, MO, U.S.A.) was dissolved (0.07 g) in a mixture of 2.5 ml ethanol (95%) plus 0.5 ml of 1 N NaOH as described by Watkins et al. (1988). The following substrates were dissolved each in 3 mi of distilled water. (ii) The chromogenic p-nitrophenyl-B-D-xylopyranoside (PNPX) (Serva Feinbiochemical, Heidelberg, F.R.G.) was dissolved 0.1 g, (iii) The fluorogenic substrate 4-trifluoromethylumbelliferyl-/3-t)-xylopyranoside (TMUX) (Lambda Probes & Diagnostics, Graz, Austria) was dissolved (0.07 g). (iv) 0.6 g L-Cysteine (E. Merck, F.R.G.), (v) 0.3 g sodium thiosulfate (E. Merck, F.R.G.) and (vi) 0.3 g ammonium iron-Ill-citrate (E. Merck, F.R.G.) were separately dissolved in 3 ml of distilled water. The solutions were sterilized by membrane filtration, and added aseptically to the basal medium. Ammonium iron-Ill-citrate has to be added as the last compound. It has to be mentioned that either solution ii (chromogenic substrate) or sollution iii (fluorogenic substrate) should be used. The pH was 7.0 :t: 0.1 and no pH adjustment was necessary. The medium was dispended in 20-ml portions into sterile Petri dishes. The final concentration of chromogenic and fluorogenic substrates amounted to MUG, 70 mg/l; XGAL, 70 mg/1; PNPX. 100 mg/l; and TMUX, 70 mg/l. The selective agar is designated as EMX ID-agar (ECD-MUG-XGAL identification agar).
12q TABLE I Possible substrates for the detection of/3-D-glucuronidaseand /3-D-galactosidaseand their reactions Enzyme/enzymesubstrate /3-~glucuronidase 4-methylumbelliferyl-/3-D-glucuronide
" ;. . . . .
p-nitrophenyl-/3-D-glucuronide 5-bromo-4-chloro-3-indolyl-~8-o-glucuronide #-D-galactosidase 4-methylumbelliferyl-/3-D-galactopyranoside o-nitrophenyl-/3-D-galaetolwyranoside 5-bromo-4-chloro-3-indolyl-/3-D-galactopyranoside
Reaction Blue fluorescence(365 nm) (Dahlen and Linde,1973) Yellowcoloration (Kilian and Biilow, 1979 Hansen and Yourassowsky,1984) Blue coloration (Frampton et al., 1988) Blue fluorescence(365 nm) (Berg and Fiksdal, 1988) Yellow-coloration (Le Minorand Ben Hamida,1962, Kilianand Billow,1976) Blue coloration (Manafiand Kneifel,1989)
Inoculation and incubation The plates were inoculated with bacterial suspensions in saline by streaking and stabbing and incubated overnight at 37 ° C.
Reading of results When XGAL is split by g-D-galactosidase, colonies appear light blue due to conversion of the liberated aglycone to indigo (Manafi and Kneifel, 1989). In the presence of /3-D-glucuronidase the fluorescing 4-methylumbelliferone (4MU) is split off from MUG. Colonies of E. coil are easily detectable under UV (365 nm) by their blue fluorescence (Hartmann, 1989). For a summary of the enzymatic substrates and reactions see Table I. Tryptophane-deaminase activity is recognized by the appearance of brownish colonies resulting from the oxidative deamination of tryptophane (Manafi and Kneifel, 1989). Hydrogensulfide production from cysteine and sodium thiosulfate is detected by a black precipitate along the stab inoculation. H2S reacts with the ferrous ions of ammonium iron-Ill-citrate. Depending on the substrate incorporated into the medium either yeiiow-coloured p-nitrophenole released from PNPX (Brisou et al., 1972) or under UV (365 nm) a greenish fluorescent 4-trifluoro-methylumbelliferone released from TMUX indicates the presence of/3-D-xylosidase (Manafi, unpublished data). These enzymatic activities are indicated without the addition of reagents after incubation. After having recorded the location and appearance of each colony, indole and cytochrome oxidase tests can be performed. For the Indole test one drop of Kovacs' indole reagent (a solution of p-dimethylaminobenzaldehyde in amyl alcohol) is added with a loop to the colony to be tested. The development of a pink coiour indicates a positive reaction. The Indole test for confirmation of E.
130
coil can be performed directly on the agar plate. For the oxidase test a drop of oxidase reagent (a fresh solution of 1% N,N-dimethyl-l,4-phenylenediammonium dichloride plus 6/% a-naphthol, E. Merck) is added with a loop to the colony tested. The development of clark blue coiour within 1 rain indicates a positive test. In the case that one single colony has to be tested for both oxidase and indole tests, this colony can be suspended in 0.3 ml of sterile water. Both tests may then be performed transferring bacterial suspension with a loop to paper strips which are impregnated with oxidase reagent or Kovacs' reagent. Results
The results of the enzymatic tests performed on various bacterial strains are presented in Table II. The majority of Escherichia coli and coliforms strains possessed /3-o-galactosidase and this produced light blue colonies. Of 101 strains of Citrobacter freundii 97% and all Salmonella arizonae strains gave blue colonies and a black precipitate in the agar. Salmonella spp. (only three were tested) and Edwardsiella (two isolates) formed white colonies with black precipitates. They were differentiated from each other by the indole test, Edwardsiella being indolepositive, Salmonella indole-negative. Under UV the typical blue fluorescence of 4-methylumbelliferone indicated the presence of E. coli. GUD activity was found in 98% of E. coli strains. Only five strains (2%) were negative. Excepting one strain of C. freundii and three strains of Yersinia enterocolitica, all other strains were GUD-negative. /3-t)-xylosidase activity was restricted to the genera Klebsiella and Enterobacter. Strains belonging to these genera produced blue colonies with a yellow halo when using PNPX or showed strong greenish fluorescence under UV when using TMUX. All isolates of K. oxytoca, K. pneumoniae, E. aerogenes, E. amnigenus, E. sakazakii and E. intermedium, 99% of E. cloacae and 56% of E. agglomerans were XYL-positive. Strains of Ki oxytoca were indole-positive, whereas the other members of the Klebsiella-Enterobacter group were not. All strains of the Morganella-Proteus-Providencia group developed brownish colonies, thus indicating tryptophane deaminase. In addition, P. vulgaris and P. mirabilis were characterized by a typical blackening in the agar plate along the stab inoculation. They could be distinguished from each other by the indole reaction, P. t'ulgaris being positive. All isolates of Pseudomonas spp., Aeromonas spp., Plesiomonas spp. and l,qbrio spp. (with the exception of V. metschnikouii) were oxidase-positive (dark blue colonies), and could easily be distinguished from coliforms. Discussion
The assessment of ~-o-galactosidase, indole and hydrogen sulfide production are of importance in the differentiation of members of the family Enterobacteriaceae.
131 TABLE II Results of various enzymatic reactions of test organisms on the EMX ID agar"
Organism according to API b 20 E
Number of strains tested
Proportion (.ffo),strams positive GAL.
GUD
Escherichia Escherichia Escherichia Escherichia
282 1 7 1
96 100 100 100
1 3 3 2 lOl 10
Klebsiella oxytoca Klebsiella pneumoniae Enterobacter cloacae Enterobacter agglomerans Enterobacter aerogenes Enterobacter amnigenus Enterobacter sakazakii Enterobacter intermedium
XYL
IND
TDA
98 0 0 0
0 0 0 0
93 100 0 0
0 0 0 0
0 0 0 0
0 0 0 0
100 100 0 0 lO0 90
0 0 0 0 1 0
0 0 0 0 0 0
0 0 0 100 0 90
0 0 0 0 0 0
0 100 100 100 97 0
0 0 0 0 0 0
12 38 76 16 4 30 14 1
91 100 98 100 100 1O0 100 100
0 0 0 0 0 0 0 0
100 100 98 56 100 100 100 100
100 0 0 0 0 0 0 0
0 0 0 0 0 0 0 0
0 0 0 0 0 0 0 0
0 0 0 0 0 0 0 0
Proteus mirabilis Proteus vulgaris Morganella morganii Providencia rettgeri
10 6 5 3
0 0 0 0
0 0 0 0
0 0 0 0
0 100 1O0 100
100 100 1O0 100
90 100 0 0
0 0 0 0
5erratia marcescens Serratia fonticola Serratia liquefaciens Hafnia alvei ButtiauxeUa agrestis Yer'dnia enterocolitica Acinetobacter spp.
10 11 13 22 8 47 3
100 1O0 76 63 100 38 0
0 0 0 0 0 1 0
0 0 0 0 0 0 0
0 0 0 0 0 61 0
0 0 0 0 0 0 0
0 0 0 0 0 0 0
0 0 0 0 0 0 0
2 1 5 4 4 8 5 2
100 100 100 100 100 0 0 0
0 0 0 0 0 0 0 0
0 0 0 0 0 0 0 0
50 100 60 100 100 0 0 0
0 0 0 0 0 0 0 0
0 0 0 0 0 0 0 0
0 100 100 100 100 100 100 100
coli adecarboxylata vulneris hermanii
Shigella sonnei Salmonella arizonae Salmonella spp. Edwardsieila spp. Citrobacter freund// Otrobacter diuersus
Vibrio metschnikovii V'tbrio vulnificus Aeromonas hydrophila A eromonas sobria Plesiomonas shigeiloides Pseudomonas aeruginosa Pseudomonas fluorescens Chromobacterium violaceum
H2S
OX
a EMX ID agar (ECD-MUG-XGAL Identification agar). b All species names given refer to the API 20E Manual. c GAL, g3-galactosidase; GUD, g3-glucuronidase; XYL,/3-~losidase; IND, tryptophanase; "IDA, tryptophane desaminase; H2S, hydrogen sulfide production; OX, cytochrome oxidase.
132 The positive fl-D-galactosidase reaction is characteristic of coliforms and could thus be used as a primary criterion for further identification tests. Among mesophilic Gram-negative bacteria, /3-o-glucuronidase is almost exclusively present in E. coli. It has, therefore, been widely used for the identification of E. coli from various clinical and environmental sources (Hartmann, 1989). This enzyme, which was first reported by Buchler et al. (1951), catalyses the hydrolysis of /3-D-glucopyranosiduronic derivatives into their corresponding aglycons and in D-glucuronic acid. Kilian and Biilow (1976, 1979) found that approx. 97% of E. coli strains but only some strains of Salmonella, Shigella and Yersmia, produce this enzyme. Some strains of E. coli isolated from human fecal specimens do, however, not posses GUD such as E. coli O157:H7 (Chang, 1989: Thompson et al., 1990). As far as known, other pathogenic E. coli posses GUD activity. Other Escherichia spp. appear to be negative for this enzyme (Rice et al., 1991). For completeness, it should also be mentioned, however, that some strains of flavobacteria (Petzel and Hartmann, 1986), staphylococci (Moberg, 1985), streptococci (R6d et al., 1974) and clostridia (Sakaguchi and Murata, 1983) have also been shown to produce GUD. But most of them would either not grow on this selective medium due to its content of bile salts or the incubation conditions or would be oxidase-positive (flavobacteria). The fluorogenic substrate 4-methylumbelliferyl-/3-D-glucuronide (MUG) have become common for the identification of E. coli in food and water bacteriology (Hartmann, 1989; Gauthier et al., 1991; Manafi et al., 1991). Since the fluorescence of 4-methylumbelliferone is known to be pH-dependent (Goodwin and Kavanagh 1950; Freier and Hartmann, 1987), the pH value of the growth media should be within the range of neutral to slightly alkaline; otherwise an alkaline solution needs to be added (Maddocks and Greenan, 1975, Freier and Hartmann, 1987). The disadvantage of incorporating MUG into agar is that fluorescence diffuses rapidly into the surrounding agar substrate and therefore the plates have to be read after overnight incubation usually. Several attempts have been made to simultaneously detect coliforms and E. coli in water, using o-nitrophenyl-/3-D-galactopyranoside (ONPG) and MUG (Edberg et al., 1988, 1989, 1990). However, it was observed that also chromogenic nitrophenolic substances such as ONPG or p-nitrophenol-/3-D-glucuronide (PNPG) diffuse easily through solid media. Therefore, these substrates cannot be used in solid media. The incorporation of 5-bromo-4-chloro-3-indolyl-/3-D-galactopyranoside (XGAL) both in solid and liquid media is more effective for detecting fl-D-galactosidase activity than are other chromogenic or fluorogenic substrates (Manafi and Kneifel, 1989). Coliform strains produce sharp blue colonies on the agar plate because of insolubility of the indigo dye which does not alter the viability of the colonies. Detection of /3-o-xyiosidase (XYL) has been used as a marker for the Klebsiella-Enterobacter group (Brisou et al., 1972; Kilian and Billow, 1976; Godsey et al., 1981) as XYL activity was found in a high percentage of the isolates. The results obtained in this study are in accordance with those of other researchers and confirm the close relationship between the genera Klebsiella and Enterobacter.
133 X Y L activity was detected with either chromogenic p-nitrophenyi-~-D-xylopyranoside (PNPX) or fluorogenic 4-trifluoromethylumbelliferyl-t3-D-xyiopyranoside (TMUX). The principal advantages of T M U X are its sensitivity and the sharp greenish fluorescence instead of a diffuse yellow zone around the colonies occurring with PNPX. Some strains of Pseudomonas produce a pigment that exhibits dull greenish fluorescence under long-wavelength UV irradiation. This may mimic the emission spectra of 4-methylurnbelliferone (4-MU) or 4-trifluoromethylumbelliferone (4T M U ) but the fluorescence signal is usually too small for visual detection. Strains such as these can be differentiated easily from Enterobacteriaceae by a positive oxidase test. The ability of the Morganella-Proteus-Providencia group to metabolize some amino acids and the production of a brown melanin-like pigment is a well-known property which was extensively described by Miiller (1985). This is in agreement with our results. The tryptophane-deaminase reaction permits a useful distinction of this group from other members of the Enterobacteriaceae family. In conclusion, the new selective indicator agar medium provides good presumptive identification of Enterobacteriaceae. The efficiency and rapidity of the detectable reactions make this medium a very useful tool in routine water and food microbiology.
References
Berg, J.D. and Fiksdal, I_ (1988) Rapid detection of total and fecal coliforms in water by enzymatic hydrolysis of 4-methylumbelliferone-tl-v-galactoside.Appl. Environ. Microbiol. 54, 2118-2122. Brisou, B., Richard, C. and Lenriot, A. (1972) lnt~r~.t taxonomique de la recherche de la #-xylosidase chez les Enterobacteriaceae. Ann. Inst. Pasteur 123, 341-347. Buchler, H.J., Kathnum, P.A. and Daisy, E.A. (1951) Studies on O-glueuronidase from Escherichiacoll. Proc. Soc. Exp. Biol. Med. 76, 672-676. Chang, G.W., Brill, J. and Lum, R. (1989) Proportion of 8-~glucuronidase-negative Escherichia col/in human fecal samples. Appl. Environ. Microbiol. 55, 335-339. Dahlen, G. and Linde, A. (1973) Screening plate method for detection of bacterial #-glucuronidase. Appl. Microbiol. 26, 863-866. Edberg, S.C., Allen, M.J., Smith, D.B. and National Collaborative Study (1988) National field evaluation of a defined substrate method for the simultaneous enumeration of total coliforms and Escher/ch/a co/i from drinking water: comparison with the standard multiple tube fermentation method. Appl. Environ. Microbiol. 54, 1595-1601, 3197. Edberg, S.C., Allen, MJ., Smith, D.B. and National Collaborative Study (1989) National field evaluation of a defined substrate method for the simultaneous detection of total coliforms and Escher/ch/a co/i from drinking water: comparison with presence-absence techniques. Appl. Environ. Microbiol. 55, 1003-1008. Edberg, S.C., Allen, MJ., Smith, D.B. and Kriz, N.J. (1990) Enumeration of total coliforms and Escherichia co/i from source water by the defined substrate technology.Appl. Environ. Microbiol. 56, 366-369. Frampton, E.W., Restaino, L. and Blaszko, N. (1988) Evaluation of the ~-glucuronidase substrate 5-bromo-4-chloro-3-indolyi-O-~glucuronide (X-GLUC) in a 24 hour direct plating method for Escherichia co//. J. Food Protect. 51,402-404.
134 Freier, T.A. and Hartmann. P.A. (1987) Improved membrane filtration media for enumeration of total coliforms and Escherichia coil from sewage and surface waters. Appl. Environ. Microbiol. 53. 1246-1250. Gauthier. M.J., Torregrossa, V.M.. Babelona, M.C., Cornax, R. and Borrego. J.J. (1991) An intercalibration study of the use of 4-methylumbelliferyl-~-D-glucuronide for the specific enumeration of Escherichia coil in seawater and marine sediments. Syst. Appl. Microbiol. 14. 183-189. Godsey, J.H., Matteo, M.R., Shen, D., Tolman, G. and Gohlke, J.R. (1981) Rapid identification of Enterobacteriaceae with microbial enzyme activity profiles. J. Clin. Microbiol. 13, 483-490. Goodwin, R.H. and Kavanagh, F. (1950) Fluorescence of coumarin derivatives as a function of pH. Arch. Biochem. Biophys. 27, 152-173. Hansen, W. and Yourassowsky, E. (1984) Detection of ,8-glucuronidase in lactose-fermenting members of the family Enterobacteriaceae and its presence in bacterial urine cultures. J. Clin. Microbiol. 20. 1177-1179. Hartmann, P.A (1989) The MUG (glucuronidase) test for Escherichia coil in food and water, In: A. Turano (Ed.), Rapid Methods and Automation in Microbiology and Immunology, Brixia Academic Press, Brescia, pp. 290-308. Kilian, M. and Billow, P. (1976) Rapid diagnosis of Enterobacteriaceae. I. Detection of bacterial glycosidases. Acta Pathoi. Microbiol. Scand. Sect. B 84, 245-251. Kilian, M. and Biilow, P. (1979) Rapid identification of Enterobacteriaceae. II. Use of/3-glucuronidase detecting agar medium (PGUA agar) for the identification of Escherichia coli in primary cultures of urine samples. Acta Pathol. MicrobioL Scand. Sect. B. 87, 271-276. Le Minor, L. and Ben Hamida, F. (1962) Avantages de la recherche de la fl-galactosidase sur celle de la fermentation du lactose en milieu complexe dans le diagnostic bact6riologique, en particulier des Enterobacteriaceae. Ann. Inst. Pasteur 102, 267-277. Maddocks. J.L. and Greenan, M.J. (1975) A rapid method for identifying bacterial enzymes. J. Clin. Pathol. 28, 686-687. Manafi. M. and Kneifel, W. (1989) A combined chromogenic-fluorogenic medium for the simultaneous detection of total coliforms and Escherichia coil in water. Zbl. Hyg. 189, 225-234. Manafi, M. and Kneifel, W. (1990) Rapid methods for differentiating Gram-positive from Gram-negative aerobic and facultative anaerobic bacteria. J. Appl. Bacteriol. 69. 822-827. Manafi. M., Kneifel, W. and Bascomb, S. (1991) Fluorogenic and chromogenic substrates used in bacterial diagnostics. Microbiol. Rev. 55, in press. Moberg, L j . (1985) Fluorogenic assay for rapid detection of Escherichia coil in food. Appl. Environ. Microbiol. 50, 1383-1387. Miiller, H.E. (1985) Production of brownish pigment by bacteria of the Morganella- Proteus- Prot'idencia Group Zbl. Bakt. Hyg. A 260, 428-435. Petzel, J.P. and Hartmann, P.A. (1986) A note on starch hydrolysis and/3-glucuronidase activity among Flacobacteria. J. Appl. Bacteriol. 61,421-426. Rice. E.W.. Allen, M.J., Brenner, D.J. and Edberg, S.C. (1991) Assay for fl-glucuronidase in species of the genus Escherichia and its application for drinking-water analysis. Appl. Environ. Microbiol. 57, 592-593. Rod, T.O., Haug, R.H. and Midtvedt, T. (1974) fl-Glucuronidase in the streptococci groups B and D. Acta Pathol. Microbiol, Seand. Sect. B 82, 533-536. Sakaguchi. Y. and Murata, K. (1983) Studies on the /3-glucuronidase production of Clostridia. Zbl. Bakteriol. I Orig. A 254, 118-122. Thompson, J.S.. Hodge, D.S. and Borczyk, A.A. (1990) Rapid biochemical test to identify verocytotoxin-positive strains of Escherichia coil serotype O157. J. Clin. Microbiol. 28, 2165-2168. Watkins, W.D., Rippey, S.R., Claret, C.R., Kelley-Reitz, D.J. and Burkhardt, W. Ill (1988) Novel compound for identifying Escherichia coil Appl. Environ. Microbiol. 54, 1874-1875.
127
FOOD 00439
A new plate medium for rapid presumptive identification and differentiation of Enterobacteriaceae M. Manafi and M.L. R o t t e r Hygiene Institute, University of Vienna, Vienna,Austria (Received 17 January 1991; accepted 25 July 1991)
A new selective differential agar medium for rapid presumptive identification of Enterobacteriaceae from water and food samples is described (EMX ID afar). By a combination of fluorogenic and chromogenic substrates, the medium detects the presence of /3-~glucuronidase, O-D-galactosidese, ~8-D-xylosidase, tryptophane deaminase and H2S; additionally, cytochrome-oxidase and indole production can be demonstrated. This medium provides an inexpensive means for simple and rapid presumptive identification of E. co// and coliforms and for the differentiation within the /Oebs/el/a-Enterobacter and the Proteus-Providencia-Morganella group. Furthermore, it allows to distinguish between the H2S-positive Enterobacteriaceae Citrobacter freund~ Salmonella Spp., S. arizonae, Edward~lla, Proteus mirabilis, P. uulgaris and some oxidase.pmitive bacteria. Key words: Foods; Fiuorogenic; Chromogenic; Enterobacteriaceae; Culture medium; Rapid identification
Introduction Detection, differentiation, and enumeration of Enterobacteriaceae are of primary importance in the microbiological quality control of food and water. In particular, coliforms and Escherichia coli are used as indicator organisms. New techniques have been developed for simultaneous enumeration and rapid identification of enterobacteria. Some of them are based on the cleavage of fiuorogenic or chromogenic substrates by specific enzymatic activities (Manafi and Kneifel, 1990; Manafi et al, 1991). These methods allow fast identification even on primary isolation media without the need for time-consuming isolation procedures. This paper describes a new selective differential medium detecting simultaneously the production of ~8-D-glucuronidase (GUD), jS-D-galactosidase (GAL), Correspondence address: M. Manafi, Hygiene Institute, University of Vienna, Kinderspitalgasse 15, A-I095 Vienna, Austria.
128
/3-D-Xy.losidase (XYL), tryptophane-deaminase (TDA)and hydrogen sulfide (H,S). Furthermore, the production of indole (IND) as well as cytochrome-oxidase (OXI can be tested in a second step by addition of the appropriate chemicals to single colonies.
Material and Methods
Bacterial strains A total of 771 strains of Gram-negative rods was included in this study (Table II). All strains were fresh isolates from water and food specimens examined at the Hygiene Institute of the University of Vienna. The identification of pure cultures was carried out with the API 20E System (bio M&ieux S.A., Montalieu, Vercieu. France). Selective differential medium A selective medium was used developed for the detection of E. coli by indicating the production of GUD and Indole, using as a basal medium the commercially available Fluorocult-ECD agar TM (E. Merck AG, Darmstadt, F.R.G.), and supplemented with additional substrates. Fluorocult-ECD agar is composed of peptone (20 g), lactose (5 g), NaCI (5 g), K2HPO 4 (4 g), KH2PO n (1.5 g) bile salts (1.5 g), methylumbelliferyl-/3-D-glucuronide (MUG) (0.07 g), L-tryptophane (1 g), agar (15 g) and distilled water (985 ml). It was sterilized by autoclaving at 121°C for 15 min, cooled to 60 ° C, and placed in a holding water bath at 60 ° C. To this basal medium, 3 mi of each of the six following solutions were added aseptically. (i) The chromogenic substrate 5-bromo-4-chloro-3-indolyl-/3-D-galactopyranoside (XGAL) (Sigma Chemical Co., St. Louis, MO, U.S.A.) was dissolved (0.07 g) in a mixture of 2.5 ml ethanol (95%) plus 0.5 ml of 1 N NaOH as described by Watkins et al. (1988). The following substrates were dissolved each in 3 mi of distilled water. (ii) The chromogenic p-nitrophenyl-B-D-xylopyranoside (PNPX) (Serva Feinbiochemical, Heidelberg, F.R.G.) was dissolved 0.1 g, (iii) The fluorogenic substrate 4-trifluoromethylumbelliferyl-/3-t)-xylopyranoside (TMUX) (Lambda Probes & Diagnostics, Graz, Austria) was dissolved (0.07 g). (iv) 0.6 g L-Cysteine (E. Merck, F.R.G.), (v) 0.3 g sodium thiosulfate (E. Merck, F.R.G.) and (vi) 0.3 g ammonium iron-Ill-citrate (E. Merck, F.R.G.) were separately dissolved in 3 ml of distilled water. The solutions were sterilized by membrane filtration, and added aseptically to the basal medium. Ammonium iron-Ill-citrate has to be added as the last compound. It has to be mentioned that either solution ii (chromogenic substrate) or sollution iii (fluorogenic substrate) should be used. The pH was 7.0 :t: 0.1 and no pH adjustment was necessary. The medium was dispended in 20-ml portions into sterile Petri dishes. The final concentration of chromogenic and fluorogenic substrates amounted to MUG, 70 mg/l; XGAL, 70 mg/1; PNPX. 100 mg/l; and TMUX, 70 mg/l. The selective agar is designated as EMX ID-agar (ECD-MUG-XGAL identification agar).
12q TABLE I Possible substrates for the detection of/3-D-glucuronidaseand /3-D-galactosidaseand their reactions Enzyme/enzymesubstrate /3-~glucuronidase 4-methylumbelliferyl-/3-D-glucuronide
" ;. . . . .
p-nitrophenyl-/3-D-glucuronide 5-bromo-4-chloro-3-indolyl-~8-o-glucuronide #-D-galactosidase 4-methylumbelliferyl-/3-D-galactopyranoside o-nitrophenyl-/3-D-galaetolwyranoside 5-bromo-4-chloro-3-indolyl-/3-D-galactopyranoside
Reaction Blue fluorescence(365 nm) (Dahlen and Linde,1973) Yellowcoloration (Kilian and Biilow, 1979 Hansen and Yourassowsky,1984) Blue coloration (Frampton et al., 1988) Blue fluorescence(365 nm) (Berg and Fiksdal, 1988) Yellow-coloration (Le Minorand Ben Hamida,1962, Kilianand Billow,1976) Blue coloration (Manafiand Kneifel,1989)
Inoculation and incubation The plates were inoculated with bacterial suspensions in saline by streaking and stabbing and incubated overnight at 37 ° C.
Reading of results When XGAL is split by g-D-galactosidase, colonies appear light blue due to conversion of the liberated aglycone to indigo (Manafi and Kneifel, 1989). In the presence of /3-D-glucuronidase the fluorescing 4-methylumbelliferone (4MU) is split off from MUG. Colonies of E. coil are easily detectable under UV (365 nm) by their blue fluorescence (Hartmann, 1989). For a summary of the enzymatic substrates and reactions see Table I. Tryptophane-deaminase activity is recognized by the appearance of brownish colonies resulting from the oxidative deamination of tryptophane (Manafi and Kneifel, 1989). Hydrogensulfide production from cysteine and sodium thiosulfate is detected by a black precipitate along the stab inoculation. H2S reacts with the ferrous ions of ammonium iron-Ill-citrate. Depending on the substrate incorporated into the medium either yeiiow-coloured p-nitrophenole released from PNPX (Brisou et al., 1972) or under UV (365 nm) a greenish fluorescent 4-trifluoro-methylumbelliferone released from TMUX indicates the presence of/3-D-xylosidase (Manafi, unpublished data). These enzymatic activities are indicated without the addition of reagents after incubation. After having recorded the location and appearance of each colony, indole and cytochrome oxidase tests can be performed. For the Indole test one drop of Kovacs' indole reagent (a solution of p-dimethylaminobenzaldehyde in amyl alcohol) is added with a loop to the colony to be tested. The development of a pink coiour indicates a positive reaction. The Indole test for confirmation of E.
130
coil can be performed directly on the agar plate. For the oxidase test a drop of oxidase reagent (a fresh solution of 1% N,N-dimethyl-l,4-phenylenediammonium dichloride plus 6/% a-naphthol, E. Merck) is added with a loop to the colony tested. The development of clark blue coiour within 1 rain indicates a positive test. In the case that one single colony has to be tested for both oxidase and indole tests, this colony can be suspended in 0.3 ml of sterile water. Both tests may then be performed transferring bacterial suspension with a loop to paper strips which are impregnated with oxidase reagent or Kovacs' reagent. Results
The results of the enzymatic tests performed on various bacterial strains are presented in Table II. The majority of Escherichia coli and coliforms strains possessed /3-o-galactosidase and this produced light blue colonies. Of 101 strains of Citrobacter freundii 97% and all Salmonella arizonae strains gave blue colonies and a black precipitate in the agar. Salmonella spp. (only three were tested) and Edwardsiella (two isolates) formed white colonies with black precipitates. They were differentiated from each other by the indole test, Edwardsiella being indolepositive, Salmonella indole-negative. Under UV the typical blue fluorescence of 4-methylumbelliferone indicated the presence of E. coli. GUD activity was found in 98% of E. coli strains. Only five strains (2%) were negative. Excepting one strain of C. freundii and three strains of Yersinia enterocolitica, all other strains were GUD-negative. /3-t)-xylosidase activity was restricted to the genera Klebsiella and Enterobacter. Strains belonging to these genera produced blue colonies with a yellow halo when using PNPX or showed strong greenish fluorescence under UV when using TMUX. All isolates of K. oxytoca, K. pneumoniae, E. aerogenes, E. amnigenus, E. sakazakii and E. intermedium, 99% of E. cloacae and 56% of E. agglomerans were XYL-positive. Strains of Ki oxytoca were indole-positive, whereas the other members of the Klebsiella-Enterobacter group were not. All strains of the Morganella-Proteus-Providencia group developed brownish colonies, thus indicating tryptophane deaminase. In addition, P. vulgaris and P. mirabilis were characterized by a typical blackening in the agar plate along the stab inoculation. They could be distinguished from each other by the indole reaction, P. t'ulgaris being positive. All isolates of Pseudomonas spp., Aeromonas spp., Plesiomonas spp. and l,qbrio spp. (with the exception of V. metschnikouii) were oxidase-positive (dark blue colonies), and could easily be distinguished from coliforms. Discussion
The assessment of ~-o-galactosidase, indole and hydrogen sulfide production are of importance in the differentiation of members of the family Enterobacteriaceae.
131 TABLE II Results of various enzymatic reactions of test organisms on the EMX ID agar"
Organism according to API b 20 E
Number of strains tested
Proportion (.ffo),strams positive GAL.
GUD
Escherichia Escherichia Escherichia Escherichia
282 1 7 1
96 100 100 100
1 3 3 2 lOl 10
Klebsiella oxytoca Klebsiella pneumoniae Enterobacter cloacae Enterobacter agglomerans Enterobacter aerogenes Enterobacter amnigenus Enterobacter sakazakii Enterobacter intermedium
XYL
IND
TDA
98 0 0 0
0 0 0 0
93 100 0 0
0 0 0 0
0 0 0 0
0 0 0 0
100 100 0 0 lO0 90
0 0 0 0 1 0
0 0 0 0 0 0
0 0 0 100 0 90
0 0 0 0 0 0
0 100 100 100 97 0
0 0 0 0 0 0
12 38 76 16 4 30 14 1
91 100 98 100 100 1O0 100 100
0 0 0 0 0 0 0 0
100 100 98 56 100 100 100 100
100 0 0 0 0 0 0 0
0 0 0 0 0 0 0 0
0 0 0 0 0 0 0 0
0 0 0 0 0 0 0 0
Proteus mirabilis Proteus vulgaris Morganella morganii Providencia rettgeri
10 6 5 3
0 0 0 0
0 0 0 0
0 0 0 0
0 100 1O0 100
100 100 1O0 100
90 100 0 0
0 0 0 0
5erratia marcescens Serratia fonticola Serratia liquefaciens Hafnia alvei ButtiauxeUa agrestis Yer'dnia enterocolitica Acinetobacter spp.
10 11 13 22 8 47 3
100 1O0 76 63 100 38 0
0 0 0 0 0 1 0
0 0 0 0 0 0 0
0 0 0 0 0 61 0
0 0 0 0 0 0 0
0 0 0 0 0 0 0
0 0 0 0 0 0 0
2 1 5 4 4 8 5 2
100 100 100 100 100 0 0 0
0 0 0 0 0 0 0 0
0 0 0 0 0 0 0 0
50 100 60 100 100 0 0 0
0 0 0 0 0 0 0 0
0 0 0 0 0 0 0 0
0 100 100 100 100 100 100 100
coli adecarboxylata vulneris hermanii
Shigella sonnei Salmonella arizonae Salmonella spp. Edwardsieila spp. Citrobacter freund// Otrobacter diuersus
Vibrio metschnikovii V'tbrio vulnificus Aeromonas hydrophila A eromonas sobria Plesiomonas shigeiloides Pseudomonas aeruginosa Pseudomonas fluorescens Chromobacterium violaceum
H2S
OX
a EMX ID agar (ECD-MUG-XGAL Identification agar). b All species names given refer to the API 20E Manual. c GAL, g3-galactosidase; GUD, g3-glucuronidase; XYL,/3-~losidase; IND, tryptophanase; "IDA, tryptophane desaminase; H2S, hydrogen sulfide production; OX, cytochrome oxidase.
132 The positive fl-D-galactosidase reaction is characteristic of coliforms and could thus be used as a primary criterion for further identification tests. Among mesophilic Gram-negative bacteria, /3-o-glucuronidase is almost exclusively present in E. coli. It has, therefore, been widely used for the identification of E. coli from various clinical and environmental sources (Hartmann, 1989). This enzyme, which was first reported by Buchler et al. (1951), catalyses the hydrolysis of /3-D-glucopyranosiduronic derivatives into their corresponding aglycons and in D-glucuronic acid. Kilian and Biilow (1976, 1979) found that approx. 97% of E. coli strains but only some strains of Salmonella, Shigella and Yersmia, produce this enzyme. Some strains of E. coli isolated from human fecal specimens do, however, not posses GUD such as E. coli O157:H7 (Chang, 1989: Thompson et al., 1990). As far as known, other pathogenic E. coli posses GUD activity. Other Escherichia spp. appear to be negative for this enzyme (Rice et al., 1991). For completeness, it should also be mentioned, however, that some strains of flavobacteria (Petzel and Hartmann, 1986), staphylococci (Moberg, 1985), streptococci (R6d et al., 1974) and clostridia (Sakaguchi and Murata, 1983) have also been shown to produce GUD. But most of them would either not grow on this selective medium due to its content of bile salts or the incubation conditions or would be oxidase-positive (flavobacteria). The fluorogenic substrate 4-methylumbelliferyl-/3-D-glucuronide (MUG) have become common for the identification of E. coli in food and water bacteriology (Hartmann, 1989; Gauthier et al., 1991; Manafi et al., 1991). Since the fluorescence of 4-methylumbelliferone is known to be pH-dependent (Goodwin and Kavanagh 1950; Freier and Hartmann, 1987), the pH value of the growth media should be within the range of neutral to slightly alkaline; otherwise an alkaline solution needs to be added (Maddocks and Greenan, 1975, Freier and Hartmann, 1987). The disadvantage of incorporating MUG into agar is that fluorescence diffuses rapidly into the surrounding agar substrate and therefore the plates have to be read after overnight incubation usually. Several attempts have been made to simultaneously detect coliforms and E. coli in water, using o-nitrophenyl-/3-D-galactopyranoside (ONPG) and MUG (Edberg et al., 1988, 1989, 1990). However, it was observed that also chromogenic nitrophenolic substances such as ONPG or p-nitrophenol-/3-D-glucuronide (PNPG) diffuse easily through solid media. Therefore, these substrates cannot be used in solid media. The incorporation of 5-bromo-4-chloro-3-indolyl-/3-D-galactopyranoside (XGAL) both in solid and liquid media is more effective for detecting fl-D-galactosidase activity than are other chromogenic or fluorogenic substrates (Manafi and Kneifel, 1989). Coliform strains produce sharp blue colonies on the agar plate because of insolubility of the indigo dye which does not alter the viability of the colonies. Detection of /3-o-xyiosidase (XYL) has been used as a marker for the Klebsiella-Enterobacter group (Brisou et al., 1972; Kilian and Billow, 1976; Godsey et al., 1981) as XYL activity was found in a high percentage of the isolates. The results obtained in this study are in accordance with those of other researchers and confirm the close relationship between the genera Klebsiella and Enterobacter.
133 X Y L activity was detected with either chromogenic p-nitrophenyi-~-D-xylopyranoside (PNPX) or fluorogenic 4-trifluoromethylumbelliferyl-t3-D-xyiopyranoside (TMUX). The principal advantages of T M U X are its sensitivity and the sharp greenish fluorescence instead of a diffuse yellow zone around the colonies occurring with PNPX. Some strains of Pseudomonas produce a pigment that exhibits dull greenish fluorescence under long-wavelength UV irradiation. This may mimic the emission spectra of 4-methylurnbelliferone (4-MU) or 4-trifluoromethylumbelliferone (4T M U ) but the fluorescence signal is usually too small for visual detection. Strains such as these can be differentiated easily from Enterobacteriaceae by a positive oxidase test. The ability of the Morganella-Proteus-Providencia group to metabolize some amino acids and the production of a brown melanin-like pigment is a well-known property which was extensively described by Miiller (1985). This is in agreement with our results. The tryptophane-deaminase reaction permits a useful distinction of this group from other members of the Enterobacteriaceae family. In conclusion, the new selective indicator agar medium provides good presumptive identification of Enterobacteriaceae. The efficiency and rapidity of the detectable reactions make this medium a very useful tool in routine water and food microbiology.
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