Ecological Relationship Between Vibrio parahaemolyticus ...

APPLiED AND ENVIRONMENTAL MICROBIOLOGY, Sept. 1978, p. 500-505 0099-2240/78/0036-0500$02.00/0 Copyright i) 1978 American Society for Microbiology

Vol. 36, No. 3

Printed in U.S.A.

Ecological Relationship Between Vibrio parahaemolyticus and Agar-Digesting Vibrios as Evidenced by Bacteriophage Susceptibility Patternst JOHN A. BAROSS,1* JOHN LISTON,2 AND RICHARD Y. MORITA1 School of Oceanography and Department ofMicrobiology, Oregon State University, Corvallis, Oregon 97331,1 and College of Fisheries, University of Washington, Seattle, Washington 981952

Received for publication 27 June 1978

Twenty bacteriophages active against Vibrio parahaemolyticus and agar-digesting vibrios, isolated from oysters (Crassostrea gigas) and Dungeness crab (Cancer magister) and by induction of a lysogenic agar digester, were tested as to their host range. These phages were specific for V. parahaemolyticus and various agar-digesting vibrios, and interspecies lysis occurred only between these two groups. V. alginolyticus, V. anguillarum and related species, V. cholerae, and a group of marine psychrophilic and psychrotrophic vibrios were not affected. No correlation was observed between the 0 and K serotypes of V. parahaemolyticus strains and bacteriophage susceptibility patterns, and 7 of 28 strains of V. parahaemolyticus were not lysed by any of the phages. Only two of the phage isolates were capable of lysing all susceptible V. parahaemolyticus strains. No correlation was observed between the inter- and intraspecies genetic relatedness (DNA homologies) of V. parahaemolyticus and agar-digesting vibrios and susceptibility patterns to different bacteriophages. Some of the phages were capable of plaque formation on V. parahaemolyticus as well as on some strains of agardigesting vibrios that were separated by 70 to 80% differences in their DNA homologies. The possible ecological significance of these vibrio bacteriophages, particularly those having a wide host range, is discussed.

Various species of Vibrio reside in near-shore marine environments, particularly associated with indigenous animals. The ecological role(s) of this physiologically versatile group of organisms is inferred but not substantively known. Among these vibrios there exist different species capable of growth from less than 00C to greater than 450C and in salt concentrations from less than 0.5% to greater than 10% (4, 5, 7, 11, 15). Rapid changes in numbers and species composition of vibrios occur continuously in estuarine environments, resulting from tidal periodicities, seasonal fluctuations in water temperature, dissolved-oxygen levels, and concentrations of organic nutrients (5, 10, 11). The population of vibrios within the gut of estuarine animals is consistently high but is variable in species composition, reflecting changing environmental conditions. During the summer months, when the water temperature frequently exceeds 300C, a mesophilic and psychotrophic population, consisting in part of Vibrio anguillarum, V. alginolyticus, and V. parahaemolyticus, predominates (5, 10, 11). V. parahaemolyticus and V. t Technical paper ment Station.

no.

alginolyticus are incapable of survival at temperatures below their minimum for growth and thus are rarely found during the winter months. Likewise, the psychrophilic vibrios that predominate during the winter disappear during the summer. Interposed within this gut population of vibrios is an equally abundant and apparently diverse population of vibrio bacteriophages (5, 6). Bacteriophages were consistently isolated for some strains of V. parahaemolyticus and a group of agar-digesting vibrios but rarely for the most commonly occurring inshore species, V. anguillarum and V. alginolyticus. V. parahaemolyticus phages were found to increase in numbers parallel to increases in the mesophilic vibrio population, and populations exceeding 106 phages/g of oyster were frequently encountered

(5).

In this report, evidence is presented indicating that V. parahaemolyticus can share some bacteriophages with a genetically diverse and presently unclassified group of agar-digesting vibrios isolated from marine mollusks and sediments. MATERIALS AND METHODS Source ofbacterial strains. V. parahaemolyticus K and Sak strains were supplied by R. Sakazaki (Na-

4879, Oregon Agricultural Experi-

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tional Institute of Health, Tokyo, Japan) and R. R. Colwell (Department of Microbiology, University of Maryland, College Park). V. cholerae strain NIH 35A3 was supplied by R. R. Colwell; V. cholerae strains Ogawa 5596 and El tor 8457 were provided by E. S_ Boatman (University of Washington, Seattle). V. parahaemolyticus T strains and V. alginolyticus V strains were supplied by H. ZenYoji (Tokyo Metropolitan Research Laboratory of Public Health, Shinjuku-ku, Tokyo, Japan). V. anguillarum strains 2.1 and V-2911 were obtained from E. J. Ordal (University of Washington, Seattle). V. marinus ATCC 1538, V. metchnikovii ATCC 7708, and V. ichthyodermis NCMB 407 were obtained from the Oregon State University culture collection. Antarctic psychrophiles were isolated from Eltanin cruise no. 46, 1971. All other Vibrio strains, including agar digesters and additional strains of V. parahaemolyticus, V. alginolyticus, and V. anguillarum, were isolated throughout the course of this study from various samples of marine fauna, sediment, and seawater obtained from Washington and Oregon. Bacteriophage isolation and preparation of phage stocks. Vibrio bacteriophages were isolated from marine samples as previously described (5, 6). Twenty bacteriophage isolates were selected for typing purposes primarily because they formed clear plaques on lawns of their respective host strain. The source, original host Vibrio strain, and methods for isolation of these phages are shown in Table 1. The initial isolation of bacteriophage for the prep-

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aration of stocks involved the stabbing of a single plaque with a wire loop, which was then inoculated into 10 ml of phage broth (peptone [Difco], 15 g; tryptone [BBL], 8 g; yeast extract [Difco], 1 g; NaCl, 25 g; distilled water, 980 ml; pH adjusted to 7.2 with NaOH; after autoclaving, 10 ml each of 0.1 M MgSO4 and 0.1 M CaCl2 was aseptically added to the medium) containing an early-log-phase culture of the host bacterial strain. The seeded tubes were incubated without aeration for 24 h at 22°C for mesophiles and for 72 h at 8°C for psychrotrophs and psychrophiles. After incubation, the contents of the tube was centrifuged at 12,500 x g for 15 min at 5°C, using a Sorvall RC-2B refrigerated centrifuge. The phage titer was then assayed by using the agar overlay technique as a test for homogeneity of plaque morphology. In the determination of bacteriophage titers, the phage cultures were diluted in phage buffer (Na2HPO4, 9.5 g; KH2PO4, 3.0 g; NaCl, 25 g; distilled water, 980 ml; pH adjusted to 7.2; after autoclaving, 10 ml each of 0.1 M MgSO4 and 0.1 M CaCl2 was aseptically added to the medium) and plated on an agar medium containing phage broth supplemented with 0.5% soluble starch (Baker) and 1.5% agar (PS agar), using the soft-agar technique. The soft-agar overlay was prepared with phage buffer supplemented with 0.7% agar. High-titer stocks of bacteriophage were prepared by seeding a log culture of the host strain with phage at a multiplicity of infection of approximately 1. The culture was incubated with aeration at 22°C for 12 to

TABLE 1. Source of bacteriophages used in phage typing of V. parahaemolyticus strains Original host vibrio Phage designaMethod of isolation Source strain

tion

K3a K3b

C5 10 19 21 22 24 26 27 31 35 WAL KL

Pacific oyster (C. gigas)a Market shucker oyster (C. gigas) Market sample of Dungeness crab meat (Cancer magister) Pacific oyster (C. gigas) Pacific oyster (C. gigas) Pacific oyster (C. gigas) Pacific oyster (C. gigas) Pacific oyster (C. gigas) Pacific oyster (C. gigas) Pacific oyster (C. gigas) Pacific oyster (C. gigas) Pacific oyster (C. gigas) C. gigas from Seabeck, Wash.d Clam (Venerupsis japonica) from Seabeck, Wash. Pacific oyster (C. gigas) Pacific oyster (C. gigas) C. gigas from Big Beef, Wash. C. gigas from Big Beef, Wash. C. gigas from Big Beef, Wash. Lysogenic agar-digesting vibrio

K3b

Enrichment Enrichment

K3

Enrichment

K4b

Enrichment Enrichment Enrichment Direct platingc Direct plating Enrichment Enrichment Direct plating Enrichment Enrichment Enrichment

K4 K4 K4 K4 K4 K4 K4 K4 K4 K4 K4

Enrichment TC1nE2Ae Enrichment TC11E2A Direct plating POYA52' POYA52 Direct plating Enrichment PA34' K4 Induction with mitomycin C TC,1E2A Wash. obtained from were C. Pacific of the Purdy, oyster gigas Samples b Vibrio strains K3 and K4 are Japanese strains of V. parahaemolyticus. 'The bacteriophages isolated by direct plating were the most abundant phages from these samples. d The clam and oyster samples from Seabeck, Wash., were involved in an outbreak of gastroenteritis presumed to be caused by V. parahaemolyticus. e Vibrio strains POYA52, and PA34 are psychrotrophic agar digesters.

P4A P4B 5214r 5214s P34 4-TC

TCn1E2A,

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BAROSS, LISTON, AND MORITA

18 h. The lysates were clarified by centrifugation at 12,500 x g for 15 min at 5°C. The bacteriophage supernatant was then centrifuged at 40,000 x g for 40 min at 5°C, using an International model B-60 centrifuge. The phage pellet was then resuspended in phage buffer and recentrifuged at 12,500 x g for 30 min at 5°C to remove remaining bacterial debris. The phage stocks were stored frozen at -20°C. Bacteriophage typing of Vibrio species. Twenty vibrio bacteriophage cultures were used for the typing of V. parahaemolyticus and related Vibrio spp. Cultures of vibrios were plated either on PS agar or LibX agar (4), using the agar overlay technique. The plates were placed on a cardboard template which was subdivided into 20 squares. A 3-mm-diameter wire loop was used to transfer the phage onto one of the designated areas on the plate. Bacteriophage titers sufficient to give confluent lysis were used (approximately 105 phage particles/ml). The plates were incubated for 12 to 24 h at 22°C. DNA base composition and hybridization. Various vibrio isolates were cultured for DNA extraction in Trypticase soy broth (BBL) with 2% added NaCl at 25'C and harvested in the late log phase as determined by optical density. Sodium lauryl sulfate at a final concentration of 1% was used to lyse the cells. After an initial phenol extraction, the DNA was purified by using the procedure of Marmur (12). The DNA base compositions were determined by their melting point by using the procedure of Marmur and Doty (13). DNA-DNA hybridizations were carried out by using the Anderson and Ordal (2) modification of the Denhart procedure (8). The reassociation temperature used was 63°C.

RESULTS Previous studies have established that shellfish harbor high titers of bacteriophage that can lyse specific strains of vibrios, including V. parahaemolyticus and various agar-digesting species. A set of 20 representative bacteriophages was isolated, using V. parahaemolyticus strains K3 and K4 and the agar digesters POYA52, PA34, and TCiiE2A. All of these phages were isolated from mulluscan and crustacean shellfish except phage 4-TC, which was obtained by induction of the agar digester TCjLE2A with mitomycin C (Table 1). The host specificity of these phages was tested with representative species of vibrios and members of the Enterobacteriaceae. These phages were found to be specific for V. parahaemolyticus and for some undelineated marine agar-digesting species, and 21 of 36 strains of V. parahaemolyticus and 10 of 10 strains of agar digesters tested were lysed by one or more of these phages. It should be noted that none of the confirmed strains of V. parahaemolyticus isolated from environmental and market samples from Washington and Oregon was susceptible to these bacteriophages. None of the 15 strains of V. alginolyticus, 7 strains of

APPL. ENVIRON. MICROBIOL.

V. anguillarum, 3 strains of V. cholerae, and 1 strain each of V. ichthyodermis, V. metchnikovii, and V. marinus was lysed by any of these 20 bacteriophages. Only one of the 19 psychrophilic and psychrotrophic vibrio isolates tested was susceptible to phage. It is interesting that the one susceptible psychrotrophic vibrio was isolated from the Pacific oyster (Crassostrea gigas), whereas psychrophiles isolated from open ocean waters or from the Arctic and Antarctic environmental samples were not lysed by these phages. None of the seven species of Enterobacteriaceae tested was susceptible to vibrio bacteriophages. Originally, the primary purpose for isolating V. parahaemolyticus phages was to investigate the possibility that a bacteriophage typing system, such as exists for V. cholerae, could be developed that would allow rapid identification of environmental and clinical strains of V. parahaemolyticus. Twenty-eight strains of V. parahaemolyticus representing 8 0 serotypes and 27 K serotypes were tested for susceptibility to the 20 bacteriophage isolates. These data are presented in Table 2 and show a wide variation in phage susceptibilities among the V. parahaemolyticus strains. In general, the lytic patterns among the different V. parahaemolyticus serotypes definitely suggest that most of these bacteriophage strains are different. Seven V. parahaemolyticus strains were not lysed by any of the phages. There appeared to be no relationship between susceptibility to one or more phages and specific 0 or K serotype, and, in some cases, V. parahaemolyticus strains such as ATCC 17802 (K 1) and SAK 4 (K 4) were not lysed by any of the phages even though different strains having identical 0 and K antigens such as SAK 1 and K4 were lysed by 13 and 19 phage strains, respectively. Phage strains 31 and K3B were found to have the widest lytic spectrum, and all phage-susceptible V. parahaemolyticus strains were lysed by these phages. None of the strains of V. parahaemolyticus was lysed by phage P34, an agar-digesting vibrio phage, whereas 16 strains of V. parahaemolyticus were suseptible to one or more of the other phage strains that originated from agar-digesting vibrios. Only 5 V. parahaemolyticus strains (Sak 2, K 3, K 4, Sak 9, and Sak 13) were lysed by all 19 virulent phage strains, and two strains, Sak 26 and T3991-1, were only susceptible to phages 31 and K3B. The observed ability of these phages to lyse both agar-digesting Vibrio spp. and V. parahaemolyticus suggested that these two groups of vibrios might be genetically closely related. A selected group of 10 agar-digesting vibrios that

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TABLE 2. Relationship between the antigenic types of V. parahaemolyticus and specific phage susceptibility Antigenic type

Strain designation 0

1 2

3

1 1 25 26 2

5 6 8 9

ATCC 17802 Sakl Sak 25 Sak 26 Sak2

3 3

Sak3 K-3

4 5 6

Sak4

7 7 14 30 4

4

Lytic phages

K

8

Sak5 Sak6 Sak7 T3991-1 Sak 14 Sak 30 K-4

Sak8 Sak 9

11 12 13

Sak 11 T3937-1 Sak 13

13 NDa

T3980-1 T3960-1 Sak 34 Sak17 T3979-1 Sak 18 Sak 20 Sak 23

34 17 15 18 20 23

No susceptibility C5, 10, 24, 26, 27, 31, 35, K3A, K3B, KL, P4A, P4B, 4-TC 31, 35, K3B, P4A, P4B 31, K3B C5, 10, 19, 21, 22, 24, 26, 27, 31, 35, K3A, K3B, K1, WAL, P4A, P4B, 4-TC, 5214r, 5214s 10, 19, 21, 24, 27, 31, 35, K3B, K1, P4A, P4B, 4-TC C5, 10, 19, 21, 22, 24, 26, 27, 31, 35, K3A, K3B, KL, WAL, P4A, P4B, 4-TC, 5214r, 5214s No susceptibility No susceptibility C5, 10, 19, 21, 22, 24, 26, 35, K3A, K3B, Kl, P4A, P4B, 5214r, 5214s 31, K3B, P4A, P4B 31, K3B 19,24,31, K3B, KL, 4-TC No susceptibility C5, 10, 19, 21, 22, 24, 26, 27, 31, 35, K3A, K3B, KL, WAL, P4A, P4B, 4-TC, 5214r, 5214s No susceptibility C5, 10, 19, 21, 22, 24, 26, 27, 31, 35, K3A, K3B, KL, WAL, P4A, P4B, 4-TC, 5214r, 5214s C5, 24,31, K3A, K3B, WAL, KL 19, 31, K3B C5, 10, 19, 21, 22, 24, 26, 27, 31, 35, K3A, K3B, KL, WAL, P4A, P4B, 4-TC, 5214r, 5214s C5, 22, 24, 31, 35, K3A, K3B, KL, P4A, P4B, 4-TC C5, 10, 19, 21, 22, 24, 31, 35, K3B, KL, P4A, P4B, 4-TC C5, 19, 21, 24, 31, 35, K3B, KL No susceptibility C5, 10, 19, 21, 24, 26, 27, 31, 35, K3B, P4A, P4B, 4-TC No susceptibility C5, 10, 19, 26, 27, 31, 35, K3A, K3B, KL, 4-TC 19, 27, 31, 35, K3B, 4-TC

a ND, Not determined.

shared common phages with V. parahaemolyticus were analyzed as to their DNA base composition and to their genetic relatedness as determined with DNA hybridization as an index. These data are summarized in Table 3. All of the agar-digesting vibrios had base compositions comparable to V. parahaemolyticus (45 to 47% guanine plus cytosine). The percent DNA homologies between these agar digesters and V. parahaemolyticus ATCC 17802, however, varied from marginally related (20 to 30%) to significantly related (58 to 68%). The latter value is comparable to the genetic relatedness noted between V. alginolyticus and V. parahaemolyticus (2, 14). None of the agar-digesting vibrios showed a broad phage susceptibility pattern such as those seen with some strains of V. parahaemolyticus. Also, there was no common lytic phage for all agar-digesting vibrios, and 12 of the 20 phages lysed one or more of these vibrios. It is interesting that seven of the virulent phages

were originally isolated with V. parahaemolyticus as the host strain, whereas several of the agar-digesting vibrio phages, such as P4A, P4B, and P34, could only form plaques on the original host strain. The phage 4-TC was incapable of lysing any of the agar digesters even though it was isolated from the agar digester TCjjE2A. Conversely, phages 5214r and 5214s are capable of lysing quite diverse strains of the agar digesters in addition to six strains of V. parahaemolyticus, representing a variation in DNA homologies of 80%.

DISCUSSION It is assumed that the primary function of bacteriophages in nature is to lyse and thus diminish populations of specific bacteria. The facts that lysogeny and transduction have been demonstrated in the laboratory and that increasing numbers of bacterial phenotypes have been attributed to bacteriophage conversions (3) do

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BAROSS, LISTON, AND MORITA

TABLE 3. Relationship between V. parahaemolyticus and various strains of marine agar-digesting vibrios as determined by DNA homologies and bacteriophage susceptibility

Agar-digest-g

Source

Vibrio strain

80AC 80A3 APAW3 PA34 3310

TC0,E2A POAgW POAgW6

POYA52 79A8

ATCC 17802

Pacific oyster (Puget Sound)c Pacific oyster (Puget Sound) Seawater (Puget Sound) Seawater (Puget Sound) Fish gut (Puget Sound) Sediment (Puget Sound) Pacific oyster (market sample) Pacific oyster (market sample) Pacific oyster (Puget Sound) Seawater (Puget Sound) V. parahaemolyti-

+% Homology to V. parahaemolyticus °)% G+Cb ATCC 17802 Lytic phages

Tma (0) T^

88.5

46.8

52

19, 21, 22

88.4

46.6

32

19, WAL, KL

88.2

46.1

23

5214r, 5214s

88.2

46.1

23

P34

NDd

ND

37

19

87.8

45.1

ND

P4A, P4B, 5214r, 5214s

88.0

45.6

68

5214r, 5214s

88.2

46.1

60

5214r, 5214s

87.9

45.4

55

5214r, 5214s

88.4

46.6

58

21, 22, 27, K3b

88.5

46.8

100 (80-100)e

cus b

Tm, Melting point. G+C, Guanine plus cytosine.

C Pacific oyster specie was C. gigas, including the Kumamoto variety of C. gigas.

d ND, Not determiined. e Intraspecies variation noted in the homologies among 70 strains of V. parahaemolyticus as reported by Staley and Colwell (14).

not add substantially to our understanding of the ecological signifiance of these bacteriophage phenomena. Generally, phages are species or sometimes strain specific, and their origin is believed to be lysogenic or pseudolysogenic strains of the same species. Indeed, lysogenic bacteria are quite common in nature (3, 9), and in experiments where phage and susceptible host strains were mixed, phage-resistant strains eventually arose (1). It can be assumed, therefore, that bacteriophages are somehow associated with most of the species of vibrios found in marine environments. The high incidence of vibrio bacteriophages and the presence of a diverse group of Vibrio species occurring simultaneously in shellfish lend substance to this claim (5). The specific Vibrio spp. from which V. parahaemolyticus bacteriophages originated are not known with certainty. The relative scarcity and low incidence of V. parahaemolyticus in Pacific Northwest estuarine samples precludes this species as a principal source of these phages. Moreover, none of the Pacific Northwest strains of V. parahaemolyticus tested was found to be susceptible to these phages. Similarly, Pacific Northwest isolates of V. alginolyticus and V.

anguillarum, the most commonly occurring species found in near-shore marine environments during the summer, were also found to be resistant to these phages. Induction experiments, already carried out with some of the U.S. and Japanese phage-resistant isolates of V. parahaemolyticus and V. alginolyticus, have indicated that lysogeny in these species is a common characteristic. The fact that some V. parahaemolyticus bacteriophages could lyse a diverse group of agardigesting vibrios and vice versa does provide evidence that some of these phages might have originated from Vibrio species that are remotely related to V. parahaemolyticus, such as various psychrophilic and psychrotrophic species, for example. The original host vibrio isolate for phages P4A, P4B, and 4-TC was the psychrophilic agardigesting vibrio TCjjE2A. These phages also lysed V. parahaemolyticus strains, but only at temperatures below 300C (6). Also, the fact that oysters harbor a large population of bacteriophage that lyse V. parahaemolyticus and a genotypically diverse group of agar digesters suggests an ecological relationship between these two groups of vibrios. These agar digesters were


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infrequently isolated from shellfish, but when present, they occurred in high numbers (>105/g of oyster). Moreover, the agar digesters isolated from any single oyster sample were found to consist of a genotypically diverse group ofvibrios that included both psychrotrophs and mesophiles. It is apparent that in some instances the ability to digest agar among vibrios is probably a transient characteristic linked to bacteriophages which can be transmitted to different Vibrio species in situ (6). The precise roles of bacteriophage in the ecological relationship between marine vibrios are not known. However, the ability of a genetically diverse group of vibrios to share common phages does indicate that in addition to regulating the species composition and numbers of organisms, bacteriophages may be the determinant factor in explaining the variability of some phenotypic characteristics observed in marine vibrios, such as human and marine animal pathogenicity. Induction, lysis, and bacteriophage conversion in near-shore marine vibrio populations might be coordinated with the natural fluctuations in water temperature and salinity and in the levels and kinds of organic nutrients (5). ACKNOWLEDGMENTS We express our gratitude to R. Anderson for performing some of the homology experiments. This investigation was supported in part by National Science Foundation grant OCE77-07820.

LITERATURE CMD 1. Anderson, E. S. 1957. The relations of bacteriophages to bacterial ecology, p. 189-217. In R. E. 0. WilLiams and C. C. Spicer (ed.), Microbial ecology, Cambridge University Press, London. 2. Anderson, R. S., and E. J. Ordal. 1972. Deoxyribonucleic acid relationships among marine vibrios. J. Bacteriol. 109:696-706. 3. Barksdale, L., and A. B. Arden. 1974. Persisting bacteriophage infection, lysogeny and phage conversions.

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Annu. Rev. Microbiol. 28:265-299. 4. Baross, J. A., F. J. Hanus, and R. Y. Morita. 1974. The effects of hydrostatic pressure on uracil uptake, ribonucleic acid synthesis and growth of three obligately psychrophilic marine vibrios, Vibrio alginolyticus and Escherichia coli, p. 180-202. In R. R. Colwell and R. Y. Morita (ed.), Effect of the ocean environment on microbial activities. University Park Press, Baltimore, Md. 5. Baross, J. A., J. Liston, and R. Y. Morita. 1978. Incidence of Vibrio parahaemolyticus bacteriophages and other vibrio bacteriophages in marine samples. Appl. Environ. Microbiol. 36:492-499. 6. Baross, J. A., R. Y. Morita, and J. Liston. 1974. Some implications of genetic exchange among marine vibrios, including Vibrio parahaemolyticus, naturally occurring in the pacific oyster, p. 129-137. In T. Fujino, G. Sakaguchi, R. Sakaxaki, and Y. Takeda (ed.), First International Symposium on Vibrio parahaemolyticus. Saikon Publishing Co. Ltd., Tokyo. 7. Colwell, R. R., J. Kaper, and S. W. Joseph. 1977. Vibrio cholerae, V. parahaemolyticus, and other vibrios: occurrence and distribution in Chesapeake Bay. Science 198:394-396. 8. Denhardt, D. T. 1966. A membrane-filter technique for the detection of complementary DNA. Biochem. Biophys. Res. Commun. 23:641-646. 9. Dhillon, T. S., E. K. S. Dhillon, H. C. Chau, W. K. Li, and A. H. C. Tsang. 1976. Studies on bacteriophage distribution: virulent and temperate bacteriophage content of mammalian feces. Appl. Environ. Microbiol. 32:68-74. 10. Keneko, T., and R. R. Colwell. 1975. Incidence of Vibrio parahaemolyticus in Chesapeake Bay. Appl. Microbiol. 30:251-257. 11. Liston, J., and J. Baross. 1973. Distribution of Vibrio parahaemolyticus in the natural environment. J. Milk Food Technol. 36:113-117. 12. Marmur, J. 1961. A procedure for the isolation of deoxyribonucleic acid from microorganisms. J. Mol. Biol. 3:208-218. 13. Marmur, J., and P. Doty. 1962. Determination of the base composition of deoxyribonucleic acid from its thermal denaturation temperature. J. Mol. Biol. 5:109-118. 14. Staley, T. E., and R. R. Colwell. 1973. Deoxyribonucleic acid reassociation among members of the genus Vibrio. Int. J. Syst. Bacteriol. 23:316-332. 15. Vanderzant, C., C. A. Thompson, Jr., and S. M. Ray. 1973. Microbial flora and level of Vibrio parahaemolyticus of oysters (Crassostrea virginica), water, and sediment from Galveston Bay. J. Milk Food Technol.

36:447-452.