vulnificus Biogroup 1

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1 JOURNAL OF CLINICAL MICROBIOLOGY, June 1983, p /83/ $02.00/0 Copyright ) 1983, American Society for Microbiology Vol. 17, No. 6 New Selective Plating Medium for Isolation of Vibrio vulnificus Biogroup 1 PHYLLIS R. BRAYTON,' PAUL A. WEST,1 ESTELLE RUSSEK,' AND RITA R. COLWELL'* Departments of Microbiology1 and Animal Sciences,2 University of Maryland, College Park, Maryland Received 17 December 1982/Accepted 3 March 1983 A new plating medium (VV agar) has been developed as an alternative to thiosulfate-citrate-bile salts-sucrose (TCBS) agar for the isolation of Vibrio vulnificus. Salicin (2% wt/vol) is employed as the source of carbohydrate, with potassium tellurite (0.0005% wt/vol), crystal violet ( % wtlvol), oxgall (0.8% wt/vol), and a ph of 8.6 to inhibit growth of gram-positive and gramnegative organisms other than V. vulnificus. Because strains of V. vulnificus do not strongly ferment salicin in VV agar, a ph indicator has not been included in the medium. Growth of V. vulnificus appears on VV agar as large grey colonies with black centers. Other non-vibrio strains which grow on the medium produce smaller colonies and fail to take up tellurite. VV agar has proved to be more effective than TCBS agar in inhibiting members of the Enterobacteriaceae as well as gram-positive cocci. Only Vibrio strains capable of utilizing salicin grow well on VV agar. Recovery and growth of V. vulnificus are superior on VV agar, compared with TCBS agar. Vibrio vulnificus is a recently described halophilic bacterium which has been recognized as a pathogen of humans and capable of causing lifethreatening infections (2, 6). Cases of septicemia have resulted from ingestion of seafood contaminated with this organism (1), and wound infections have followed exposure to seawater (3, 9). Ecological studies have indicated that V. vulnificus occurs naturally in marine and estuarine environments, and infections in humans appear to be fortuitous, after exposure to the aquatic environment (1-3, 8, 9, 15, 19). V. vulnificus was originally referred to as lactose-positive Vibrio (6) and, more recently, two biogroups have been proposed, with human pathogenic strains assigned to biogroup 1 (18). Thiosulfate-citrate-bile salts-sucrose (TCBS) agar is the most commonly used medium in studies of V. vulnificus (3, 7, 8, 15). However, strains of V. vulnificus have not been included in previous studies which evaluated the ability of different brands of TCBS agar to support the growth of Vibrio spp. as well as to inhibit growth of normal intestinal flora (14, 17). Recently, the growth of clinical and environmental strains of V. vulnificus was studied on four brands of TCBS agar (21). No brand was considered to provide adequate recovery and growth of this species compared with results on a non-inhibitory reference medium. This observation prompted us to develop an improved plating medium for V. vulnificus MATERIALS AND METHODS A new selective medium (VV agar) for V. vulnificus was developed by a stepwise modification of the composition of nutrients and inhibitory compounds originally developed for TCBS agar. Selectivity of VV agar was assessed by its ability to inhibit growth of bacteria, other than V. vulnificus, isolated from clinical and aquatic environments. Bacterial strains. Seven clinical strains, including strain ATCC 27562, and 22 environmental strains were used to evaluate the growth of V. vulnificus biogroup 1 on VV agar and TCBS agar (Table 1). Environmental strains were isolated in Maryland, Oregon, Florida, and Louisiana. The selectivity of VV agar for V. vulnificus was studied by using 200 strains (67 of V. cholerae, 50 of V. parahaemolyticus, 35 of V. fluvialis, 36 of decarboxylase-negative Vibrio spp., 8 of marine Vibrio spp., 2 of Plesiomonas shigelloides, and 2 of Aeromonas hydrophila) representing the family Vibrionaceae and isolated from the aquatic environment, as well as from clinical cases. The ability of VV agar and TCBS agar to inhibit growth of gram-positive and gram-negative organisms, other than V. vulnificus, was studied by using 66 clinical isolates obtained from the culture collection at Walter Reed Army Medical Center, Washington, D.C. (Table 2). All strains had been identified by recommended methods and schemes (12). Plating media. Cultures were stored at ambient temperature on T1N1 agar (tryptone [Difco], 10 g/liter; NaCl, log/liter; Bacto-Agar [Difco], 15 g/liter) and were subcultured at 5-day intervals. Strains of V. vulnificus were maintained on T1N1 agar containing, in addition, MgCl2 * 6H20 (2 g/liter) and KCI (1 g/liter) (T1N1+E agar). The batch number of TCBS agar

2 1040 BRAYTON ET AL. J. CLIN. MICROBIOL. TABLE 1. Characteristics of 29 strains of V. vulnificus used for comparing growth on VV agar and TCBS agar All strains positive Variable characteristicsa All strains negative Glucose fermentation Growth in 6% NaCl (24) Gram stain reaction Oxidase Growth at 42 C (19) Swarming Nitrate reduction Urease (3) Growth in 0% NaCl Motility H2S production [LIA] (1) Growth in 8% NaCl 0/129 sensitivity (50,ug/ml) Voges-Proskauer (1) Growth in 10% NaCl Catalase Ornithine decarboxylase (28) Gas from glucose Growth in 3% NaCl Tyrosine decomposition (3) Arginine dihydrolase Growth in 35 C Hemolysis of sheep blood (11) Acid from: ONPG reaction Luminescence (2) m-inositol Indole reaction (Kovacs) Acid from: D-Sorbitol Lysine decarboxylase L-Arabinose (1) Alginase Acid from: D-Mannitol (19) Growth on: Cellobiose D-Mannose (27) y-aminobutyrate Salicin Sucrose (2) Glutarate Trehalose Growth on: L-Leucine Amylase D-Galactose (28) Putrescine Casein hydrolysis D-Glucuronate (27) Ethanol Chitinase L-Glutamate (25) 1-Propanol Deoxyribonuclease L-Serine (7) Gelatinase Glycine (5) Lecithinase Sucrose (3) Tween 80 hydrolysis L-Arabinose (1) Growth on: Cellobiose D-Gluconate D-Mannose a Number of strains positive in parentheses. (Oxoid Ltd., Basingstoke, England) was 227/ Development of VV agar. After preliminary studies, the basal medium for the V. vulnificus-specific agar comprised (grams per liter): Bacto-Peptone (Difco), 2; oxgall (Difco), 8; Casamino Acids (Difco), 0.5; NaCl, 10; Bacto-Agar (Difco), 15; with a final ph of 8.6. The basal medium was autoclaved for 10 min at 15 lb/in2 and allowed to cool to 55 C before addition of a filtersterilized solution of salicin (Sigma Chemical Co.) to a final concentration of 2% (vol/vol). Modifications of the basal medium were investigated in the following stepwise procedure: (i) selection of a ph indicator dye from bromthymol blue, thymol blue, cresol red, cresol purple, curcumin, and phenol red; (ii) optimum concentration of NaCl, Mg2+, Ca2+, and K+ for growth; (iii) effect of Tween 80 (Sigma) on the growth of V. vulnificus; (iv) inhibitory properties of sodium thiosulfate and sodium citrate; and (v) inhibitory properties of potassium tellurite and crystal violet. The medium was modified at each stage before progressing to a subsequent stage. The final composition of VV agar and its method of preparation are listed in Table 3. Inoculation of plating media. For the stepwise development of VV agar, four clinical and four environmental strains of V. vulnificus were used. Strains were cultured and inoculated onto test plates, using the serial dilution and bias compensation inoculation procedure described by West et al. (21), except that strains were grown in T1N1+E broth and plates were examined after 24 and 48 h of incubation. For studies to compare the inhibitory properties of VV agar and TCBS agar, using organisms other than Vibrio spp., each strain was grown overnight at 35 C in TIN1 broth. Approximately 1,000,ul of each culture was used to inoculate plates of VV agar, TCBS agar, and TIN, agar. The inoculum was spread over each plate using a glass "hockey stick" and a rotary plater to achieve growth of discrete colonies after incubation at 35 C. The quality of growth on each medium was compared after incubation for 24 and 48 h. For the testing of the selectivity of VV agar with Vibrio strains, including V. vulnificus, a loopful of inoculum from the overnight TIN, broth was streaked onto agar plates which were incubated at 35 C and examined after 24 and 48 h. Comparison of VV agar and TCBS agar for growth of V. vulnificus. Twenty-nine strains of V. vulnificus were prepared and inoculated onto the media under test, according to the serial dilution and bias compensation procedure previously described (21). The only modifications were that T1N1+E broth replaced tryptic soy broth plus salt, T1NI +E agar replaced tryptic soy agar plus salt as the reference count medium, and plates were examined after 24 and 48 h of incubation. A ratio of one reference count plate to three test plates was used. Analysis of data. The mean and standard deviation of the colony counts on each half-plate of VV agar and TCBS agar, and the corresponding non-inhibitory reference agar plate, were enumerated. Ratios of counts on each inhibitory medium to the counts on the reference medium were compiled to represent the recovery rate for each of the 29 strains studied. The ratios were transformed into percentage recovery rates and normalized by a loglo transformation. A

3 VOL. 17, 1983 TABLE 2. V. VULNIFICUS PLATING MEDIUM 1041 Comparison of recovery of isolates taken from clinical specimens and plated onto VV agar and TCBS agar' Growth of strains on: Strainb Isolation siteb' VV agar TCBS agar 24 h 48 h 24 h 48 h Group D Streptococcus Wound (2); blood (1) (3) Streptococcus spp. (2) ND Staphylococcus aureus Wound (9); blood (3) (12) Staphylococcus epider- Wound (6); blood (2) midis (8) Shigella sonnei (2) Wound - Serratia marscesens (2) Wound (1); blood (1) + ± Salmonella sp. (1) ND Pseudomonas sp. (1) Wound Pseudomonas stutzeri Wound (1) Pseudomonas malto- ND + + phila (2) Pseudomonas aerugino- Wound sa (2) Providencia spp. (4) Feces (1); ND (3) Proteus vulgaris (1) Urine - ± + + Proteus rettgeri (1) Urine Proteus mirabilis (2) Wound Proteus sp. (1) Wound Klebsiella oxytoca (2) Wound (1); blood (1) - + Klebsiella pneumoniae Wound (4); blood (2) (6) Escherichia coli (3) Wound (2); blood (1) Enterobacter spp. (5) Wound (2); blood (3) Citrobacter sp. (1) Wound - Alcaligenes odorans (1) ND Alcaligenes sp. (1) Wound Acinetobacter spp. (2) ND a Symbols: +++, large colonies and count >80% of count on the reference agar; ++, small colonies and count 20 to 80%o of count on the reference agar; +, small colonies and count <20%o of count on the reference agar; ±, microcolonies present and <20% of count on the reference agar; -, no growth visible. b Numbers in parentheses indicate number of strains studied. c ND, Not determined. paired t test (16) was used to determine whether the differences between the counts and percentage recovery rates of V. vulnificus on VV agar and TCBS agar were statistically significant. RESULTS AND DISCUSSION The final composition and the method of preparation of VV agar are shown in Table 3. The composition of commercially available brands of TCBS agar was used in the initial developments of VV agar. Strains of V. vulnificus are unusual, compared with other organisms within the genus Vibrio, in being able to ferment salicin. Preliminary investigations, however, showed that strains of V. vulnificus failed to produce a fermentation reaction on media containing the quantities of peptone, yeast extract, ph indicators, and the inhibitory compounds in TCBS agar, but with salicin (2% vol/vol) replacing sucrose as the carbohydrate source. Production of alkaline products from peptone was believed to be the principal reason for failure to detect acid production from salicin. In addition, it was possible that V. vulnificus selectively and preferentially used the simple carbohydrates present in yeast extract. Accordingly, the amount of Bacto-Peptone in the medium was reduced to 2 g/liter, and Casamino Acids (0.5 g/liter) replaced yeast extract. All test strains grew satisfactorily on the basal medium of lower nutrient content. None of the ph indicator dyes was able to detect salicin-utilizing colonies of V. vulnificus, but a strong acid reaction was produced by colonies of Klebsiella spp. when cresol red (0.08 g/liter) was used. It appears that V. vulnificus is unable to ferment salicin strongly on media with relatively

4 1042 BRAYTON ET AL. TABLE 3. Composition and method of preparation of VV agar for isolation of V. vulnificus Component Amta Distilled water ml Bacto-Peptone (Difco) g Casamino Acids (Difco) g Oxgall (Difco) g NaCI g MgCl2 *6H20 KCI g g Crystal violet (0.15% [wt/vol] aqueous solution; Sigma) ml Bacto-Agar (Difco) g Salicin (20% [wt/vol] aqueous solution)b ml Tween 80 (10% [vol/vol] aqueous solution; Sigma)b ml Potassium tellurite (0.5% [wt/vol] aqueous solution; Fisher)b ml a Amount per liter of reconstituted medium. b Filter sterilized and added to medium cooled to 55 C after other components have been mixed, boiled, brought to ph 8.6 ± 0.1 with 2 M NaOH, and autoclaved for 10 min at 15 lb/in2. high or low nutrient concentrations, so a ph indicator dye is of little value in a selective medium. Nevertheless, the main selective feature of VV agar is the ability to grow salicinutilizing strains of V. vulnificus. Because V. vulnificus is a halophilic organism, our investigations also focused on the optimum concentration of electrolytes for growth. The requirement for Na+ was satisfied by 1% (wt/vol) NaCl. Some halophilic bacteria also require Mg2+, Ca2+, and K+ for growth (11). Therefore, MgCl2 * 6H20 (0.2% wt/vol) and KCI (0.1% wt/vol) were added to the medium, the result being slightly larger colonies compared with those on the medium containing only NaCl. The addition of CaCl2 * H20 (0.1% wt/vol and 0.01% wt/vol) did not further improve growth and produced a precipitate in the medium. Tween 80 (0.05% vol/vol) was added to the medium as a source of carbon and to assist in the uptake of other nutrients into cells (5, 20). Addition of Tween 80 resulted in faster growth of colonies and slightly larger colonies after incubation for 24 h. Compounds designed to inhibit growth of members of the Enterobacteriaceae and grampositive cocci were studied to determine their effects on the growth of V. vulnificus. Strains of Escherichia coli, Enterobacter aerogenes, Klebsiella pneumoniae, Proteus mirabilis, and Staphylococcus aureus were used as test strains. Sodium citrate has been used mostly in media designed to inhibit growth of members of the Enterobacteriaceae family (12). In the present study, the growth of V. vulnificus was severely inhibited by 0.5% (wt/vol) to 1.25% J. CLIN. MICROBIOL. (wt/vol) concentrations of sodium citrate. Sodium thiosulfate demonstrated no effect on the growth of V. vulnificus or other test strains at concentrations up to 1.25% (wt/vol). These observations lead us to conclude that the poor growth and recovery of V. vulnificus on TCBS agar result from the inhibitory concentration of sodium citrate in TCBS agar. The efficacy of potassium tellurite in inhibiting growth of the Enterobacteriaceae was investigated. Gohar (4) and Monsur (13) had previously recommended the use of tellurite for inhibiting coliforms in media designed to isolate V. cholerae. A concentration of % (vol/vol) potassium tellurite (Fisher Scientific Co.) in combination with oxgall (0.8% wt/vol) totally inhibited growth of the test strains, without compromising the growth of V. vulnificus. The final modification to the V. vulnificus agar (VV agar) was the addition of % (wt/vol) of crystal violet (Sigma Chemical Co.), primarily to provide a bluish-green tint to the medium for ease of colony recognition, but also to inhibit growth of gram-positive bacteria (10). Growth of V. vulnificus appeared on VV agar, after incubation for at least 24 h at 35 C, as large (2 to 4 mm diameter), light-grey, translucent colonies, raised and with a dark-grey or black center (Fig. 1). Colonies other than V. vulnificus appeared on VV agar as pinpoint, opaque colonies which failed to take up tellurite. Occasionally, non-vibrio colonies appeared as large white, opaque colonies which failed to fix tellurite. The merits of any selective medium are weighed according to its ability to isolate the organisms under study and to inhibit growth of undesirable bacteria. Significantly greater numbers of V. vulnificus colonies grew on VV agar than on TCBS agar when media were inoculated by means of a bias compensation method (Table FIG. 1. Typical growth of V. vulnificus biogroup 1 on VV agar after 24 h of incubation at 35 C. Bar = 5 mm.

5 VOL. 17, 1983 V. VULNIFICUS PLATING MEDIUM 1043 TABLE 4. Comparison of mean colony counts on VV agar and TCBS agar for growth of 29 strains of V. vulnificusa Mean colony counts of V. vulnificusc Strainb Source Reference TCBS agar VV agar medium (n = 2) (n =6) (n =6) E-216 Maryland ± ± 9.91 E-219 Maryland 79.5 ± ± ± 6.47 E-220 Maryland ± ± ± E-221 Maryland 86.0 ± ± ± 7.55 E-224 Maryland 29.5 ± ± ± 8.07 E-225 Maryland 57.5 ± ± ± 9.93 E-271 Maryland ± ± ± E-231 Florida ± ± ± 9.91 E-232 Florida ± ± ± 8.40 E-234 Florida 93.5 ± ± ± E-227 Oregon ± ± E-228 Oregon ± ± E-229 Oregon ± ± E-240 Oregon 80.5 ± ± ± E-236 Louisiana ± ± ± E-239 Louisiana 94.5 ± ± ± E-247 Louisiana 99.5 ± ± ± 7.50 E-250 Louisiana ± ± ± E-260 Louisiana ± ± ± E-261 Louisiana 91.5 ± ± ± E-263 Louisiana ± ± ± E-264 Louisiana ± ± ± C-178 Louisiana ± ± ± C-180 Louisiana ± ± ± C-181 Louisiana ± ± ± C-182 Louisiana ± ± ± C-183 Louisiana ± ± ± C-184 Louisiana 38.5 ± ± ± 8.36 C-425 ATCC ± ± ± a Significant difference (P < 0.001) between the mean colony count for 29 strains on VV agar and TCBS agar was detected by using a paired t test (16). b E, Environmental; C, clinical. c Mean ± standard deviation. 4). The recovery of V. vulnificus on VV agar and TCBS agar was also compared with that obtained on a non-inhibitory agar medium. From the data for 29 strains of V. vulnificus in Table 4, the mean percentage recovery rate and standard error recorded for VV agar (100.3 ± 2.82%) was significantly higher (P < by paired Student's t test) than the rate recorded for TCBS agar (84.9 ± 3.47%). Swabs from extraintestinal lesions and blood cultures are the primary clinical samples in which V. vulnificus is likely to be encountered. The superiority of VV agar, compared with TCBS agar, for inhibiting bacteria other than V. vulnificus likely to be found in these samples, has been demonstrated (Table 2). Significantly, the two major bacterial contaminants, Staphylococcus spp. and coliforms, likely to be found in association with V. vulnificus, were more inhibited on VV agar than on TCBS agar. Strains of V. cholerae failed to grow well on VV agar. However, the medium cannot be used for presumptive identification of V. vulnificus based on colonial morphology alone, since some strains of V. parahaemolyticus and V. fluvialis will grow on VV agar and will fix tellurite. Nevertheless, these organisms can be distinguished from V. vulnificus by the o-nitrophenyl-p-d-galactopyranoside (ONPG) reaction and the arginine decarboxylase test. The usefulness of TCBS agar for the isolation of V. cholerae and all other pathogenic vibrios from clinical and environmental samples is widely recognized. However, data from our previous study of different brands of TCBS agar suggest that it may not be very useful for isolation of the recently recognized pathogen V. vulnificus (21). Our new selective medium (VV agar) for V. vulnificus has been developed by using pure cultures, and the data reported here appear to be sufficiently promising to justify extensive field and clinical trials of VV agar.

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