APPLIED AND ENVIRONMENTAL MICROBIOLOGY, JUlY 1987, p. 1556-1559 Vol. 53, No. 70099-2240/87/071556-04$02.00/0Copyright © 1987, American Society for Microbiology

Use of Sodium Dodecyl Sulfate-Polymyxin B-Sucrose Medium forIsolation of Vibrio vulnificus from Shellfish

RAYMOND G. BRYANT,* JOSEPHINE JARVIS, AND J. MICHAEL JANDAMicrobial Diseases Laboratory, California State Department of Health Services, Berkeley, California 94704

Received 1 December 1986/Accepted 6 April 1987

The differential and selective sodium dodecyl sulfate-polymyxin B-sucrose medium (SPS) of Kitaura et al. (T.Kitaura, S. Doke, I. Azuma, M. Imaida, K. Miyano, K. Harada, and E. Yabuuchii, FEMS Microbiol. Lett.17:205-209, 1983), which highlights alkylsulfatase activity, was evaluated for its potential use in the directisolation and enumeration of Vibrio vulnificus from shellfish. V. vulnificus was detected by this method in sixof nine shellfish samples collected from diverse geographic locales during the summer of 1986. Directenumeration of V. vulnificus at 7.0 x 102 to 2.2 X 104 CFU/g of shellfish was achieved on SPS agar. All sampleresults were confirmed in parallel examinations by using conventional glucose-salt-Teepol (Shell Oil Co.) brothand alkaline peptone water enrichment with plating onto thiosulfate-citrate-bile salts-sucrose agar. Addition-ally, alkylsulfatase activity was evaluated in vitro for 97 strains representing 14 Vibrio spp. V. vulnificus andVibrio cholerae-01 were the only species consistently found to possess this activity. The range of platingefficiencies for random V. vulnificus strains analyzed oh SPS was 11 to 74% (mean, 39%). The use of SPS showsgreat promise for the study of shellfish and other environmental sources for V. vulnificus.

Vibrio vulnificus is a pathogenic, halophilic marine vibriofirst described in 1976 by Hollis and colleagues (5) as alactose-fermenting Vibrio sp. phenotypically resembling Vi-brio parahaemolyticus. Since their article was published,numerous reports of human disease attributed to V.vulnificus, including bacteremia, wound infections, and mostrecently, gastroenteritis, have been described, and the orga-nism is now considered one of the major pathogenic specieswithin the genus (7, 11). Primary sepsis, often a life-threatening infection caused by V. vulnificus, principallyoccurs in immunocompromised individuals and has beenmost commonly associated with the consumption of contam-inated shellfish, particularly raw oysters (2, 6, 13).

V. vulnificus is a common inhabitant of coastal watersincluding the shellfish-harvesting areas of the United States.Concentrations of V. vulnificus in these habitats are gener-ally higher during the summer months, and recent studies byTison and Kelly (14) indicate that the potential virulence ofenvironmental and clinical isolates appears to be virtuallyidentical. Since the routine laboratory examination of shell-fish for coliforms and total mesophilic counts provides littleusable information that can predict vibrio concentrations ineach product, media designed to allow enumeration andidentification of specific pathogenic Vibrio spp. are required.The current vibrio isolation methods usually entail enrich-ment procedures with plating onto various modifications ofthiosulfate-citrate-bile salts-sucrose (TCBS) agar (15). How-ever, these methods can be time-consuming since an enrich-ment incubation is required, and a large number of similarcolonies must be picked and identified to detect all of thepathogenic vibrios that may be present. This study reportsthe evaluation of sodium dodecyl sulfate-polymyxin B-sucrose medium (SPS) as originally described by Kitaura etal. (9) for potential use in the differential isolation andenumeration of V. vulnificus in shellfish.

* Corresponding author.

MATERIALS AND METHODS

Strains and biochemical identification. Vibrio spp. recov-ered from clinical and environmental sources (48 strains)served as reference or control strains for these studies andincluded V. vulnificus ATCC 27562 and ATCC 29306, V.damsela ATCC 35083, V. metschnikovii ATCC 7708, and V.fluvialis NCTC 11327. Of these strains, 43 were kindlyprovided by E. Baron, R. Clark, R. Colwell, K. Gross, K.Harrington, S. W. Joseph, M. T. Kelly, T. Kiehn, J. Oliver,T. Overman, and K. Sandhu, while the remaining 5 isolateshave recently been recovered by one of us (J.M.J.) fromclinical specimens submitted to The Mount Sinai MedicalCenter, New York, N.Y. Five Vibrio cholerae-01 strainswere submitted to the Microbial Diseases Laboratory asreference cultures, and 44 Vibrio strains were recoveredfrom shellfish samples submitted to our laboratory eitherduring the course of this survey or over the preceding 2-yearperiod. All strains were identified to the species level byestablished criteria which included oxidase; indole; 1-galactosidase, and Voges-Proskauer activity; motility; theability to produce amino acid decarboxylases or dihy-drolases; carbohydrate fermentation; and growth in saltfreeand salt-supplemented nutrient broths (4).Media. SPS agar was prepared as previously described by

Kitaura et al. (9). Glucose-salt-Teepol and alkaline peptonewater (pH 8.5) enrichment broths, as well as TCBS agar(Eiken Chemical Co., Ltd., Tokyo, Japan) isolation plates,were prepared as described in the Bacteriological AnalyticalManual of the Federal Food and Drug Administration (15).Gelatin agar and heart infusion agar (HIA) were prepared aspreviously described (10).

Isolation of V. vulnificus from shellfish. Fresh oyster andclam samples were collected from retail outlets and whole-sale distributors in the San Francisco Bay area. The shellfishwere harvested from the Gulf Coast and Atlantic, Pacific,and Mexican coastal areas from June through September of

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TABLE 1. Isolation of V. v'ulnific,is from shellfish

Identification Shellfish type and Halo-positive No. or MPN' Other Vibrio spp.no. source SPC" colonies on of V. IfOtherficlVibolaeddirect SPS isolated (medium) isolated

S-i Oyster, Louisiana 1.3 x 106 Yes 1.0 x 104 (SPS) VC, VF, VAS-2 Oyster, Alabama 1.3 x 105 Yes 1.9 x 104 (SPS) VC, VP, VA, VANS-3 Clam, New York 9.5 x i03 No MPN, 11.8 VP, VF, VA, VANS-4 Clam, Mexico ND" No None VAS-S Oyster, East Coast 1.9 x 105 Yes 7.0 x 102 (SPS) VP, VF, VA

7.5 x 102 (TCBS)S-6 Oyster, East Coast 6.8 x 105 Yes 2.2 x 104 (SPS) VP, VFS-7 Oyster, East Coast 2.6 x 105 Yes 1.2 x 104 (SPS) VP, VF, VA, VFRS-8 Oyster, California 4.0 x 102 No MPN, 4.2 VA, VDS-9 Oyster, Maryland 2.8 x 104 Yes 3.0 x 103 (SPS) VP, VF, VA, VN

5.0 x 103 (TCBS)

aSPC, Standard plate count per gram.b Per gram.' VA, V. alginolyticus; VAN, V. angitillarum; VC, V. cholerae non-01; VD, V. darnsela: VF, V. fluvialis; VFR. V. fornissii: VN, V. natriegens; VP, V.

parahaemolyticus.d ND, Not done.

1986. All samples except those from Pacific sources were airshipped to San Francisco Bay area outlets.

Isolation of V. vulnificus from shellfish samples was ac-complished as follows. The exterior surfaces of oysters andclams were washed, the shells were aseptically opened, anda 25-g portion from each sample was homogenized in 225 mlof 3% NaCl diluent. Both TCBS and SPS plates were spreadinoculated with serial 10-fold dilutions (0.1 ml) of the samplehomogenate made in sterile 1% peptone water starting witha 1:100 (vol/vol) dilution for direct enumeration. Portions (1ml) of the original homogenate were also inoculated into 10ml of alkaline peptone water and glucose-salt-Teepol brothsfor enrichment in an abbreviated three-tube most-probable-number (MPN) determination. After overnight incubation at35°C, a loopful from each MPN enrichment tube showinggrowth was streaked onto TCBS agar. In addition, anenrichment culture of each sample was made by homogeniz-ing a second 25-g portion in alkaline peptone water andincubating it for 6 h at 35°C before streaking a loopful of theculture onto TCBS and gelatin areas. Whenever possible, aminimum of five colonies of each resulting colony type fromeach medium were picked. After identification of eachisolate by the method described above, the estimated countof each Vibrio sp. was determined. The overall bacterialquality of each shellfish sample was measured by standardplate count and fecal coliform methods as described previ-ously (12). Estimation of MPN values was by the formula ofThomas (1).

Sulfatase activity by Vibrio spp. A total of 97 Vibrio strainsrepresenting at least 14 species were individually streakedonto HIA containing 5% (vol/vol) sheep blood and wereincubated for 18 to 20 h at 35°C. After incubation, severalisolated colonies were removed with a sterile swab andradially inoculated (up to eight per plate) onto SPS agar.Plates were then incubated for 48 h at 35°C, and the presenceor absence of extracellular sulfatase activity (zone sizes andtype) was visually determined after 4, 24, and 48 h of growth.

Relative plating efficiency of V. vulnificus on SPS. Therelative plating efficiencies for 10 random strains of V.vulnificus (clinical, n = 5; environmental, n = 5) and the typestrain were determined. Briefly, each strain was grown onHIA supplemented with 5% sheep blood overnight, afterwhich a single colony was inoculated into physiologic salineand serially diluted in the same medium into an appropriate

range so that a 100-,ul sample yielded approximately 15 to150 colonies. This volume was then spread in duplicate ontoboth HIA and SPS plates. Plates were incubated for 24 h at35°C, after which total colony counts and colony size (for 10representative colonies) on the corresponding dilution forboth HIA and SPS were determined. The relative platingefficiency was expressed as (average number of coloniesrecovered on SPS/mean number recovered on HIA) x 100(%). Stock strain ATCC 7652 was plated onto 10 plates ofeach medium to obtain a more precise value for this strain.The student t test was applied to the data to determine thesignificance of this latter value.

RESULTS

The results of tests on nine shellfish samples for V.vulnificus and other Vibrio spp. are given in Table 1. Typicalopaque halo-positive colonies were observed on direct SPSenumeration plates from six of the nine samples examined,while two samples produced opaque halos on SPS agar onlyafter enrichment. The typical appearance of V. vulnificuscolonies on direct SPS enumeration plates after 24 h ofincubation (Fig. 1) as well as after prolonged (48-h) incuba-tion of a pure culture (Fig. 2) is shown. The details of thehalo structure around the colonies are also highlighted (Fig.2). The remaining sample was negative for V. vulnificus byboth direct and enrichment procedures. In two instances,colony isolation on TCBS agar was sufficient to allow acomparison of the estimated V. vulnificus counts on SPS andTCBS. In both instances, values obtained on each mediumdiffered by less than a factor of 2. Overall, of 31 halo-positivecolonies picked from SPS, about 60% were identified as V.vulnificus. The remaining isolates were found to be Pseu-domonas putrefaciens (37%) or Vibrio natriegens (3%). Thefecal coliform MPN per 100 g for all samples was less than230, except in two instances (S-1, 7,900; S-2, 1,100). Nocorrelation was found between the levels of V. vulnificus andthe presence of fecal coliforms in shellfish sampled, althoughspecimens containing increased levels of V. vulnificustended to have higher standard plate counts.The results of tests on 97 strains of various Vibrio spp. for

alkylsulfatase activity on SPS agar are given in Table 2.Visible opaque halos were formed after 24 h by 19 of 20 V.vulnificus strains and 5 of 5 V. cholerae-01 strains. The one

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FIG. 1. V. vulnificus colonies surrounded by opaque halos inmixed culture on direct SPS shellfish enumeration plate incubatedfor 24 h at 35°C.

negative V. vulnificus strain formed a precipitate, but onlyunder colonial growth. Visible opaque halos tended to formafter only 4 h for most of the V. vuilnficus strains, in contrastto the majority of other Vibrio spp. tested. Several randomlychosen V. vulnificuis strains were tested for arylsulfataseactivity in a separate assay and were negative. Although 24non-V. vulnificuis strains other than V. cholerae-01 formedhalolike zones on SPS, the halos were diffuse in most casesand did not resemble the typical opaque zones consistentlyformed around V. vulnificus. Only 6 non-V. vulnificus-V.

FIG. 2. V. vulnificus colonies (pure culture) on SPS agar incu-bated for 48 h at 35°C.

TABLE 2. Halo production on SPS for 97 Vibrio spp.

No.ith alos(%) % with opaqueVibrio sp. No. with halos at:tested

Diffuse Opaque 4 h 24 h 48 h

V. v'ulnificus 20 20 (100) 19 (95) 85 95 95V. parahaemolyticus 11 5 (45) 0V. alginolvtictis 12 0 0V. cholerae-01 5 0 5 (100) 100 100V. cholerae non-01 8 6 (75) 1 (12) 0 12 12V.flusvialis 8 5 (62) 0V. damsela 6 3 (50) 3 (50) 50 50 50V. furnissi 4 0 0V. mimicus 4 3 (75) 2 (50) 0 50 50V. hollisae 5 0 0V. anguillarurn 5 0 0V. aestuarianus 1 0 0V. diazotrophicus 1 0 0V. natriegens 1 1 (100) 1 (100) 0 0 100V. metschnikoViid 2 0 0Unidentified Vibrio 4 1 (25) 1 (25) 0 0 25

a ATCC 7708 failed to grow on SPS agar.bStrains currently not identifiable at the species level by standard biochem-

ical tests.

cholerae-01 strains were found to produce typical opaquezones after 24 h of incubation.The plating efficiency data for 11 V. vulnificus strains are

given in Table 3. The mean plating efficiencies for thedifferent strains ranged from 11 to 74%, with an overall meanof 39%. The plating efficiency value for ATCC 7652 wasstatistically significant (Student's t test; t = 13.65, P = 0.05,n = 10). Colony sizes on SPS ranged from 1.07 to 2.79 mm,which resulted in size ratios of 0.38 to 0.95 when comparedwith growth on HIA agar.

DISCUSSIONThe importance of environmental surveillance studies for

V. vulnificus and other pathogenic vibrios is now clear, giventhe increased incidence of reported cases of serious V.vulnificus infections (3, 11). Since most cases of V. vulnificusbacteremia are thought to arise because of prior consump-tion of raw oysters, levels of this vibrio in oyster samplesmay critically affect infective doses. Direct enumerationmethods for V. vulnificus may be preferred over enrichmentand MPN methods if the minimum infective dose is found tobe in the countable range (>100 bacteria per gram). This is

TABLE 3. Plating efficiency of SPS for 11 V. vulnificuis strainsIsolate no. Source" Colony size (mm)' Size ratio' RPE (%)iVV-1 Human 2.03 0.72 22VV-2 Oyster 1.63 0.57 63VV-3 Oyster 1.56 0.52 25VV-4 Clam 1.80 0.66 32VV-5 Stock strain 1.74 0.63 57VV-6 Human 1.09 0.38 44VV-7 Human 1.07 0.38 32VV-8 Oyster 1.89 0.94 19VV-9 Oyster 2.00 0.73 74VV-10 Human 2.79 0.95 11VV-11 ATCC 7652 2.43 0.87 48

" All human isolates were from blood.b Mean colony size on SPS agar.'Ratio of mean colony sizes (SPS/HIA).d RPE, Relative plating efficiency (SPS versus HIA). Mean relative plating

efficiency was 39%.

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apparently the case with V. parahaemolyticus, as recentlyreported by Karunasagar et al. (8). The results of our studyindicate that SPS medium can selectively isolate and allowenumeration of V. vulnificius directly from shellfish samplehomogenates. The relative plating efficiency is acceptable(48% for the American Type Culture Collection stockstrain), and typical halo-positive colonies are readily detect-able on SPS plates. Colony size for all strains tested was alsosatisfactory. Opaque halo production by Vibrio spp. otherthan V. vulnificus and V. cholerae-01 was largely limited toa few strains of V. cholerae non-01 and Vibrio mimicus,which are also of interest in the investigation of food-bornevibrio infections. In our experience, other halo-positivevibrios (such as damsela) are relatively rare in shellfish. P.putrefaciens was the only other organism that routinely grewand formed halo-positive colonies on SPS. Such isolateswere easily eliminated by their H2S production on triplesugar-iron agar. In addition, these preliminary studies sug-gest that the use of SPS has the distinct added advantage ofdetecting sucrose-fermenting strains of V. vulnificus, whichin our study accounted for 20% (n = 4) of the strains tested,all of which were from clinical sources. Since the biochem-ical reaction of V. vulnificus on SPS agar appears to be dueto a fairly specific straight-chain sulfatase, this differentialand selective medium may elicit an important biochemicalcharacteristic in the rapid identification of isolates of V.vulnificus in the clinical laboratory as well.The use of SPS for the direct enumeration of V. vulnificus

from shellfish and other environmental sources is promising.This study has confirmed the usefulness of SPS for theexamination of shellfish and extends the range of Vibrio spp.screened beyond those originally tested by Kitaura et al. (9).The relative plating efficiency of 11 V. vulnificus strains onSPS was also determined. Further studies should be per-formed with SPS to more comprehensively gauge its platingefficiency and to determine the range of reactions shown bya wider variety of aquatic organisms. The effect of variousincubation temperatures should also be investigated, andother chemical additives, including antimicrobial agents,could be investigated for incorporation into SPS agar toinhibit the growth of Pseudomonas spp. Further work is inprogress in our laboratory with SPS in the study of commer-cial shellfish supplies to gather more information on thefrequency and relative concentrations of V. vulnificus insuch products.

ACKNOWLEDGMENTS

We thank Barbara Portoni, Sharon Kurashige, and KimberlyHalsey for their excellent technical assistance in this work; Lu-AnneDodge for typing the manuscript; and the staff of the Food and DrugBranch, California State Department of Health Services, for obtain-ing samples.

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2. Blake, P. A., M. H. Merson, R. E. Weaver, and D. G. Hollis.1979. Disease caused by a marine vibrio: clinical characteristicsand epidemiology. N. Engl. J. Med. 300:1-5.

3. Bonner, J. R., A. S. Coker, C. R. Berryman, and H. M. Pollock.1985. Spectrum of vibrio infections in a Gulf Coast community.Ann. Intern. Med. 99:464-469.

4. Farmer, J. J., III, F. W. Hickman-Brenner, and M. T. Kelly.1985. Vibrio, p. 282-301. In E. H. Lennette, A. Balows, W. J.Hausler, Jr., and H. J. Shadomy (ed.), Manual of clinicalmicrobiology, 4th ed. American Society for Microbiology,Washington, D.C.

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6. Johnston, J. M., S. F. Becker, and L. M. McFarland. 1985.Vibrio vulnificus: man and the sea. J. Am. Med. Assoc. 253:2850-2853.

7. Johnston, J. M., S. F. Becker, and L. M. McFarland. 1986.Gastroenteritis in patients with stool isolates of Vibrio 'ulni-ficus. Am. J. Med. 80:336-338.

8. Karunasagar, I., M. N. Venugopal, I. Karunasagar, and K.Segar. 1986. Evaluation of methods for enumeration of Vibrioparahaemolyticus from seafood. Appl. Environ. Microbiol.52:583-585.

9. Kitaura, T., S. Doke, I. Azuma, M. Imaida, K. Miyano, K.Harada, and E. Yabuuchii. 1983. Halo production by sulfataseactivity of V. vulnificus and V. cholerae-01 on a new selectivesodium dodecyl sulfate-containing agar medium: a screeningmarker in environmental surveillance. FEMS Microbiol. Lett.17:205-209.

10. Madden, J. M., B. A. McCardell, and B. K. Boutin. 1984.Isolation and identification of Vibrio cholerae, p. 13.01-13.12.In Bacteriological analytical manual. Bureau of Foods, U.S.Food and Drug Administration, Washington, D.C.

11. Morris, J. G., Jr., and R. E. Black. 1985. Cholera and othervibrioses in the United States. N. Engl. J. Med. 312:343-350.

12. Neufeld, N. 1985. Procedures for the bacteriological examina-tion of seawater and shellfish, p. 37-63. In A. E. Greenberg andD. A. Hunt (ed.), Laboratory procedures for the examination ofseawater and shellfish, 5th ed. American Public Health Associ-ation, Washington, D.C.

13. Tacket, C. O., F. Brenner, and P. A. Blake. 1984. Clinicalfeatures and an epidemiological study of Vibrio vulnificus infec-tions. J. Infect. Dis. 149:558-561.

14. Tison, D. L., and M. T. Kelly. 1986. Virulence of Vibriovulnificus strains from marine environments. Appl. Environ.Microbiol. 51:1004-1006.

15. Twedt, R. M. 1984. Recovery of Vibrio parahaemolyticus andrelated halophilic vibrios, p. 12.01-12.08. In Bacteriologicalanalytical manual. Bureau of Foods, U.S. Food and DrugAdministration, Washington, D.C.

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