JOURNAL OF CLINICAL MICROBIOLOGY, Aug. 1976, p. 175-179Copyright © 1976 American Society for Microbiology

Vol. 4, No. 2Printed in U.S.A.

Isolation ofVibrio parahaemolyticus from Fecal Specimens onMannitol Salt Agar

MARY M. CARRUTHERS* AND WILLIAM J. KABATVeterans Administration Lakeside Hospital and Northwestern University Medical Center, Chicago,

Illinois 60611

Received for publication 25 February 1976

Unless laboratories use an inhibitory medium, Vibrio parahaemolyticus willbe unrecognizable in fecal specimens. The use ofa medium exclusively for vibrioisolation, such as thiosulfate-citrate-bile salts-sucrose agar (TCBS), however,may not be considered economically justified in the United States. The isolationand recognition of V. parahaemolyticus is reported on mannitol salt agar (MS),a medium which is used for fecal specimens here. Eight Kanagawa-positive andtwo ofthree Kanagawa-negative strains of V. parahaemolyticus grew as well onMS as on TCBS and better than on a representative enteric medium, Hektoenenteric agar (HE). Twenty-two fecal specimens from 16 noninfected individualswere inoculated with known quantities of V. parahaemolyticus, and recovery ofthese vibrios was assessed on TCBS, MS, and HE. Recovery of vibrios from MSand TCBS was similar when inoculum size was 103 colony-forming units/ml orgreater. Recovery of vibrios from mixed culture was distinctly lower on HE. Thecolonial morphology of V. parahaemolyticus and several other bacteria on MS isillustrated.

Vibrio parahaemolyticus gastroenteritis israrely reported in the United States, but thewide distribution of this bacterium in ourcoastal waters (1) would suggest that the dis-ease may go unrecognized here. This is notsurprising, because media routinely used in theUnited States for isolation from fecal specimensdo not selectively support the growth of vibrios.The difficulty in isolation of a pathogen fromfecal culture without the use of inhibitory me-dia is well known. Bromothymol blue-teepoland thiosulfate-citrate-bile salts-sucrose(TCBS) agars are the media suggested (2) forisolation of vibrios from fecal specimens. How-ever, because bacteria other than vibrios growpoorly or not at all on these media, their rou-tine use may not be considered economicallyjustified in a geographic area where vibrio iso-lations are unusual.This report describes the recognition and iso-

lation of V. parahaemolyticus from fecal speci-mens on mannitol salt agar (MS). MS contains7.5% NaCl and is highly inhibitory to mostbacteria. It is widely used for the isolation ofstaphylococci from heavily contaminated speci-mens and for the differentiation ofStaphylococ-cus aureus from Staphylococcus epidermidis byfermentation of mannitol.Most strains of V. parahaemolyticus isolated

from the feces of patients with gastroenteritisproduce hemolysis on Wagatsuma agar and are

referred to as Kanagawa positive. On the otherhand, most V. parahaemolyticus strains iso-lated from raw seafood and marine sources failto cause such hemolysis and are referred to asKanagawa negative.

In this study, the ability or inability of bothKanagawa-positive and -negative strains of V.parahaemolyticus to grow on a number of dif-ferential media was determined, quantitativegrowth on four media was compared, and recov-ery of vibrios from fecal suspensions was deter-mined on MS and two other differential media.

MATERIALS AND METHODSBacterial strains. Kanagawa-positive strains of

V. parahaemolyticus studied were 33C 10, ATTC17802, AMB-4, M5242-1, 33518, 3106, and 7995. Allisolates except 3106 were originally isolated fromthe feces of patients with diarrhea during foodborneoutbreaks. Strain 3106 was isolated from the gallbladder of a patient with chronic cholecystitis.ATTC 17802 and 33C10 are Japanese isolates origi-nally supplied by R. Sakazaki (National Institute ofHealth, Tokyo, Japan). The other strains are Amer-ican isolates. Kanagawa-negative strains 5A52B,7A82K, and M5253J1 were originally isolated fromseafood. Strains 8700, 33C10, 5A52B, and 7A82Kwere kindly supplied by M. Fishbein ofthe Food andDrug Administration. Strain 33518 was supplied byM. L. Brown of the Illinois State Department ofPublic Health, M52421 and M5253J1 by R. W. Ryderof the Center for Disease Control, Atlanta, Ga., and

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176 CARRUTHERS AND KABAT

3106 and 7995 by H. M. Sommers of NorthwesternMemorial Hospital, Chicago, Ill.

Media. Media used were brain heart infusionbroth with NaCl added to 2.5% (BHI-NaCl broth),BHI agar with NaCl added to 2.0% (BHIA-NaCl),and TCBS. Hektoen enteric agar (HE), MS, sheepblood agar (BA), MacConkey agar (MAC), eosinmethylene blue agar (EMB), salmonella-shigellaagar (SS), bismuthsulfite agar (BS), brilliant greenbile agar (BGB), xylose-lysine deoxycholate agar(XLD), and deoxycholate agar (DC). EMB and HEwere obtained from Difco; the remainder of the me-dia was obtained from BBL. All media were pre-pared with deionized water according to the manu-facturer's directions and were used within 5 days ofpreparation. All media used in a single run wereprepared the same day.

Fecal specimens. Specimens were obtained frompatients hospitalized with non-gastrointestinalsurgical problems. The patients did not have gas-trointestinal symptoms nor were they receiving an-timicrobial therapy for at least 2 weeks before thetime the specimen was obtained. Twenty-two speci-mens from 16 individuals were studied.

(i) Growth of pure cultures of V. parahaemolyti-cus on differential media. All strains were exam-ined except 3106 and 7995, which were acquiredsubsequently. S. aureus and several members of thefamily Enterobacteriaceae were used as control bac-teria. Bacteria were grown in BHI broth (with NaCladded to 2.5% for vibrios; unmodified for controlbacteria) at 37°C for 18 h. Inoculations were made onBHIA-NaCl and on TCBS, HE, XLD, MAC, EMB,SS, BS, BGB, DC, and MS agars. A swab was dippedin the culture for inoculation of each plate, excessmoisture was removed, and a primary inoculationwas made. Plates were streaked for isolated coloniesand incubated at 37°C for 24 h. Growth in the finalstreak area was designated 4+; in the second streakarea, 3+; in the first streak area, 2+; and growthonly in the primary inoculation area was 1+.

(ii) Quantitative growth of V. parahaemolyticus.All isolates were grown in BHI-NaCl broth on arotating platform (model G2, New Brunswick Scien-tific Co.) at 100 oscillations/min at 37°C for 18 h. Thesurface of BHIA-NaCl, MS, HE, and TCBS plateswas inoculated by pipette with 0.1 ml of a suspen-sion containing 101 to 102 colony-forming units. Theinoculum was spread evenly by loop, and plateswere incubated at 37°C for 24 and 48 h. Colonies oneach plate were counted. Only series in which colonycounts on the noninhibitory medium were less than400 are included to allow comparison of growth onthe media used.

(iii) Recovery from fecal specimens inoculatedwith V. parahaemolyticus. Approximately 1 g ofeach fecal specimen was emulsified in 0.9 ml ofsterile saline by a Vortex mixer in a centrifuge tube,resulting in a total volume of 1.5 to 2 ml. Prelimi-nary inoculation was made on MS, HE, and TCBS toestablish that none of the individuals was excretingV. parahaemolyticus.

Kanagawa-positive strains 33518 and M5242-1and Kanagawa-negative strain M5253J1 were used.They were grown in BHI-NaCl broth shake cultureat 37°C for 18 h and diluted to 10-8 in normal saline.

A 0. 1-ml amount of 10-7 and 10-8 dilutions was inoc-ulated on BHIA-NaCl for quantitation, and 0.1 mlwas added to approximately 1.9 ml of fecal suspen-sion. The fecal suspension was emulsified by a Vor-tex mixer. A sterile cotton swab was dipped in thesuspension for each inoculation and pressed againstthe side of the tube, and TCBS, MS, and HE agarswere inoculated. The swab was used to streak ap-proximately one-quarter of the plate, and three sub-sequent isolation streaks were made by wire loop.

Preliminary experiments established that countson BHIA-NaCl and TCBS were identical at 24 and 48h. Therefore, colonies on these media were countedat 24 h only. Colonies on MS and HE plates werecounted at 24 h, the plates were reincubated for anadditional 24 h, and colonies were counted again at48 h. Colonies suspected of being vibrios were inocu-lated on TCBS and BA and in an Enterotube (RocheDiagnostics, Nutley, N.J.). Oxidase-positive bac-teria that grew on TCBS and had Encise II (RocheDiagnostics) numbers of 2,040, 2,240, 2,440, or2,640 were identified as V. parahaemolyticus.

Colonial morphology. V. parahaemolyticus andother bacteria isolated from feces were inoculated onMS and photographed at 24 and 48 h.

RESULTSMost V. parahaemolyticus strains grew on

TCBS, MS, HE, XLD, and MAC agars (Table1). No growth occurred on EMB, SS, BS, andBGB, since none of these media contain NaCl.When 0.5% NaCl was added, V. parahaemolyti-cus grew on BGB, whereas a few strains grewpoorly on EMB and BS. None grew on SS. Ofthe control bacteria, S. aureus grew on BHIAand MS only; the Enterobacteriaceae grew onall media except TCBS and MS.The results of inoculation of V. parahaemoly-

ticus on a noninhibitory medium (BHIA-NaCl),on a selective enteric medium (HE), and on twoinhibitory media, TCBS and MS, are listed inTable 2. Most strains were recovered in greaternumbers on MS than on TCBS. Recovery fromboth media was about half that from BHIA-NaCl. Recovery from HE was poor.Results of inoculation of fecal suspensions

containing varying numbers of V. parahaemo-lyticus are summarized in Table 3. With 10' to102 colony-forming units of V. parahaemolyti-cus per ml, recovery was poor, even on TCBS.At an initial inoculum of 102 to 103 colony-forming units/ml, vibrios were detected in 7 of16 specimens inoculated on TCBS and in noneof 16 specimens inoculated on MS. With largerinocula of 103 to 106 vibrios/ml, recovery ratesapproached 100% on TCBS and increased to 80and 88% on MS. Recovery rates were distinctlylower from HE.

Colonial morphology. Colonial morphologyis illustrated in Fig. 1, 2, and 3.V. parahaemolyticus colonies are convex,

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V. PARAHAEMOLYTICUS ON MANNITOL SALT AGAR

TABLE 1. Growth of V. parahaemolyticus on agar media

Growtha on:V. parahaemoly-

ticus strain BHIA- TCBS HE MAC EMB SS BS BGB XLD DC MSNaCi

ATTC 17802 3+ 2+ 2+ 1+ 0 0 0 0 2+ 0 1+AMB-4 4+ 3+ 2+ 2+ 0 0 0 0 3+ 2+ 2+5A52B 2+ 2+ 3+ 3+ 0 0 0 0 2+ 0 1+7A82K 2+ 1+ 0 0 0 0 0 0 0 0 1+33C10 3+ 2+ 2+ 2+ 0 0 0 0 2+ 1+ 1+8700 2+ 1+ 1+ 0 0 0 0 0 1+ 0 0M5253J 3+ 3+ 3+ 3+ 0 0 0 0 2+ 0 3+33518 3+ 2+ 2+ 1+ 0 0 0 0 2+ 0 1+a 0, No growth; 1 +, growth in primary streak area only; 2+, growth in above plus first streak area; 3

growth in above plus second streak area; 4+, growth in above plus third streak area.

TABLE 2. Growth of V. parahaemolyticus on TCBS, MS, and HE

Recovery rate (% of BHIA-NaCl control)Bacterium No. of tests on:

TCBS MS HE

Kanagawa-positive V. parahaemolyticusM5242-1 8 68 77 1533C10 3 28 55 0ATTC 17802 4 32 52 033518 6 32 52 10AMB-4 4 25 53 213106 2 43 46 177995 2 56 79 148700 4 37 51 1

Kanagawa-negative V. parahaemolyticusM5253J1 10 50 56 125A52B 3 3 34 07A82K 3 24 0 0

a Recovery rate, expressed as % = [number of colonies on TCBS, MS, or HE plate/number of colonies onnoninhibitory plate (BHIA-NaCl)] x 100. Percentage listed is average of the tests performed.

slightly irregular in shape, and have an undu-lant border. They are dull and semiopaque.Mannitol fermentation is apparent at 24 h.Proteus mirabilis colonies are circular,

smooth bordered, and low convex. They areglistening and mucoid.

Staphylococcal colonies have their typical ap-pearance.Bacillus subtilis colonies are similar to those

of V. parahaemolyticus but are larger, moremucoid, and more distinctively fermentativethan vibrios at each time interval.

Enterococcal colonies are tiny and more con-vex than vibrios. They are glistening and trans-lucent.

DISCUSSIONV. parahaemolyticus grows with distinctive

colonial morphology on MS, although suchgrowth is slower on MS than on TCBS or on anoninhibitory medium such as BHIA. MSplates should be held for 48 h before discarding

as negative, even though distinctive coloniesmay be visible at 24 h.

In this study, only staphylococci, enterococci,and bacillus species grew repeatedly from fecalsuspensions on MS. The P. mirabilis strainillustrated is the only enteric species that wasisolated on MS. Thus, any V. parahaemolyticuscolonies present are likely to be isolated andrecognizable on MS inoculated with fecal speci-mens.The number of vibrios excreted by patients

with V. parahaemolyticus gastroenteritis is un-known. A study of quantitative excretion inother enteric diseases (3) demonstrated thenumbers to be large. In Escherichia coli enteri-tis, patients were found to excrete an average of10 pathogenic E. colilg of feces. In shigella andsalmonella enteritis, patients excreted an aver-age of 107 pathogenic bacteria/g of feces. If thenumbers of bacteria excreted by patients withV. parahaemolyticus gastroenteritis are com-parable to numbers in these enteric diseases,

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178 CARRUTHERS AND KABAT J. CLIN; MICROBIOL.

TABLE 3. Isolation of V. parahaemolyticus from fecal suspensions

V.parahaemolyti- Inoculum concn Media recovery (no. of suspensions positive/no. examined)cus strain' (colony-forming

units/ml) TCBS MS HE

33518 101_102 2/4 0/4 0/4M5242-1 0/2 0/2 0/2M5253J1 1/4 0/4 0/4

Total 3/10 (30%)a 0/10 (0%) 0/10 (0%)

33518 102_103 4/7 0/7 1/7M5242-1 1/3 0/3 0/3M5243J1 2/6 0/6 0/6

Total 7/16 (44%) 0/16 (0%) 1/16 (6%)

33518 103-104 3/3 2/3 0/3M5242-1 3/3 1/3 0/3M5253J1 2/3 1/3 0/3

Total 8/9 (89%) 5/9 (56%) 0/9 (0%)

33518 104-105 11/11 10/11 1/11M5242-1 15/15 10/15 3/15M5253J1 18/18 15/18 2/18

Total 44/44 (100%) 34/44 (80%) 6/44 (14%)

33518 105_106 8/8 8/8 5/8M5242-1 12/12 10/12 8/12M5253J1 14/14 12/14 1/12

Total 34/34 (100%) 30/34 (88%) 14/34 (42%)a Percent positive.

FIG. 1. MS plate after 24 h of incubation.

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VOL. 4, 1976 V. PARAHAEM(

FIG. 2. MS plate aft

FIG. 3. Closer view of V. parahaemolyticus (left)4

chances of detection on MS appear excellent.MS is inferior to TCBS for the recovery of V.

parahaemolyticus from fecal specimens, andthe latter medium should be used when vibriosare suspected clinically. However, awareness ofthe ability of V. parahaemolyticus to grow onMS and of its colonial morphology may allowwider recognition of the bacterium in laborato-ries that do not use TCBS routinely.

9OLYTICUS ON MANNITOL SALT AGAR

ter 48 h of incubation.

and P. mirabilis (right) colonies (48-h incubation).

LITERATURE CITED

1. Barker, W. H., Jr. 1974. Vibrio parahaemolyticus out-breaks in the United States. Lancet 1:551-554.

2. Feeley, J. C., and A. Balows. 1974. Vibrio, p. 238-245.In E. H. Lennette, E. H. Spaulding, and J. P. Truant(ed.), Manual of clinical microbiology, 2nd ed. Ameri-can Society for Microbiology, Washington, D.C.

3. Thomson, S. 1955. The numbers of pathogenic bacilli infaeces in intestinal diseases. J. Hyg. 53:217-224.

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