Vibrio cholerae and other vibrios associated with paddy field cultured prawns

Food Microbiology,

1991,8,203-208

Vibrio cholerae and other vibrios field cultured prawns G. Balakrish Nair*, and S. C. Pal

Rupak

K. Bhadra,

associated T. Ramamurthy,

with paddy A. Rameshl

Department of Microbiology, National Institute of Cholera and Enteric Diseases, Calcutta - 700 010, India and IDepartment of Environmental Conservation, Ehime University, Japan Received 23 July 1990 During a 6-month survey, 131 prawn samples belonging to five different species cultured in paddy fields were examined for the presence of Vibrio cholerae and related vibrios associated with human disease. Non-01 V. cholerae was the most common Vibrio species associated with the prawn samples followed by V, parahaemolyticus, V. mimicus, V. vulnificus and V. alginolyticus, in that order, V. cholerae non-01 appeared to constitute the normal microflora ofprawns and this association was not limited by salinity. Examination of a representative number of V. cholerae and V. parahaemolyticus isolates for established virulence factors revealed that none of the V. cholerae strains possessed genes that were homologous with cholera-toxin genes and none produced a heat stable enterotoxin while none of the V. parahaemolyticus isolates were Kanagawa phenomenon-positive and most of the strains were untypeable. The presence of such high numbers of vibrios in paddy field cultured prawn samples and its impact on the bacteriological quality of prawns destined for human consumption needs critical assessment.

Introduction Of the 30 species currently recognized in the genus Vibrio, nine have been found in association with human disease, particularly as etiologic agents of diarrhoea (Sakazaki and Shimada 19861. Seafoods have been increasingly incriminated in cholera outbreaks especially in the Western hemisphere. For some time it was thought that humans were the only reservoir of V cholerae, but a spate of recent reports especially from the United States (Colwell et al. 1981) and Australia (Bourke et al. 1986) seem to indicate that the natural aquatic envi* Corresponding author at: National Institute of Cholera and Enteric Diseases, P-33, CIT Road Scheme XM, Beliaghata, Calcutta - 700 010, India. 0740-0020/91/030203

+ 06 $02.00/O

ronment along with its flora and fauna may serve as reservoirs of this organism. In earlier studies conducted in the aquatic environs of Calcutta, we were unable to isolate the 01 serovar of I? cholerae (Nair et al. 1988a) or Kanagawa phenomenon-positive strains of I? parahaemolyticus (Nair et al. 1985, Sarkar et al. 1985) from environmental samples, indicating that the environmental isolates differ from strains of these species isolated from individuals with clinical infection. However, a more intense study on the environmental V; cholerae non-01 strains using a DNA probe containing sequences encoding the A subunit of cholera toxin (CT), revealed that a small percentage (0.5%) had homologous cholera toxin genes (Nair et al. 1988b). The present study is a continuation of our efforts to locate the 0 1991 Academic

Press Limited

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G. B. Nair et al.

possible sources of pathogenic the environment. Materials

vibrios in

and Methods

One-hundred and thirty-one specimens belonging to five different species of brackish water and fresh water prawns cultured in paddy fields (locally known as bhery culture) were sampled from June to November, 1985. Freshly caught prawn samples were harvested using standard equipment and immediately transferred to individual polythene bags. Samples were transported in portable ice chests (<5”(Z) and analysed in the laboratory within 4 h of collection. At the laboratory, sand and detritus adhering to the specimens were washed using sterile saline. A l-g portion taken from the caudal region of each sample was aseptically pulverized and introduced into 10 ml of alkaline peptone water and incubated for 18 h at 37°C. Subsequently, two loopfuls of the broth was plated on thiosulphate-citrate-bile salts-sucrose agar (TCBS; Eiken Chemical Co. Ltd., Japan). Both sucrose-positive and sucrose-negative colonies on TCBS were picked for further characterization. For rapid presumptive identification of V: cholerae, colonies resembling this organism on TCBS were inoculated into the multi-test screening medium (Nair et al. 1987). The preliminary screening test for suspect Vibrio isolates was the oxidase reaction. Organisms showing positive reaction in the oxidase test were further identified with API 20E diagnostic strips (Analytab Products Inc., Plainview, NY). The inoculum for the API strips was made by suspending colonies in 1% saline. The strips were incubated at 37°C for 18-24 h, after which reagents were added, and a seven-digit profile number was generated following the instructions of the manufacturer for test interpretation. Identifications were then made by finding the profile number in the appropriate API Analytical Profile Index. Further phenotypic characterization, if required, was performed on each isolate by methods published previously (Sakazaki and Shimada, 19861. Confirmed V: cholerae isolates were polyvalent 01 and serotyped using monospecific Inaba and Ogawa antisera prepared at our Institute. At all times, before initiating the serological analysis, the reactivity of the antisera was checked with standard clinical strains of V: cholerae 01. Determination of Kanagawa phenomenon

and

serotyping

of the confirmed V: parastrains were performed by standard procedures (Nair et al. 1985). A commercially procurable antisera kit for V: parahaemolyticus (Toshiba Kagaku Kogyo Co. Ltd., Japan) was used for the serological typing. The conventional suckling mouse assay was used as previously described (Takeda et al. 1979) to examine for production of heat stable enterotoxin by the I? cholerae isolates. v cholerae strains were also tested for hybridization with a gene probe for cholera toxin (CT). DNA hybridizations were performed on colonies in situ on nitrocellulose (Schleicher and Schuell Inc, Keene, NH) with a szP-labelled cholera toxin probe under conditions of high stringency (Kaper et al. 1981). The CT probe, found in the plasmid pCVD27 which contains a 554 bp XbaI-ClaI fragment encoding 94% of the gene encoding the A subnit ligated to EcoRI linkers and cloned into pBR325 (Kaper, J. B., pers. comm.1 was kindly supplied by Dr James Kaper, Center for Vaccine Development, MD, USA. For determination of haemolytic activity of the three strains of I? vulnificus, washed rabbit erythrocytes were diluted to a final concentration of 1% in 10 mM phosphate buffer (pH 7.01 containing 1.3% NaCl, mixed 1:l with culture supernatant, and incubated at 37°C for 1 h. The mixture was subsequently centrifuged at 1000 g for 5 min. The amount of released haemoglobin in the supernatant fluid was measured spectrophotometrically at 540 nm.

haemolyticus

Results and Discussion Of the 503 single colonies picked from TCBS which were positive in the oxidase reaction, 66% could be identified to the species level. Phenotypic characteristics of 225 and 89 strains were consistent with those of V: cholerae and V parahaemolyticus, respectively. Additionally, 14 strains were identified as V mimicus, three as V: vulnificus and one as V; alginolyticus, while the remaining 171 strains could not be identified using the battery of tests employed in this study. I? cholerae isolates could be recovered from the majority (81.7%) of samples

Vibrios Table 1. Incidence prawns. Prawn

species

of Vibrio No. of samples examined

cholerae

in paddy field cultured

and related

vibrios

in paddy

No. (%) of samples V cholerae

V mimicus

V parahaemolyticus

97

79 (81.4)

P indicus

11

10 (90.9)

2 (18.2)

Metapenaus brevicornis

3

l(33.3)

2 (66.7)

M. monoceros

2

l(50.0)

Macrobrachium rosenbergii

18

16 (88.9)

131

107 (81.7)

Total

3 (2.3)

205

cultured

positive

Penaeus monodon

3 (3.1)

field

prawns

30 (30.9)

35 (26.7)

V vulnificus 2 (2.1)

2 (1.5)

a Excerpted from Johnson et al. (1990b).

examined in this study (Table 1). Uniformly high isolation rates of V cholerae were observed in both the brackish water and freshwater prawn species. The salinity of the brackish water and freshwater areas from where the prawns were harvested was 7.9-10.3% and
brackish waters and are not necessarily associated with sewage contamination (Bashford et al. 1979, Kaper et al. 1979, Nair et al. 1980). Further, it has also been documented that vibrios constitute the predominant flora in the’ digestive tracts of prawns (Yasuda and Kitao 19801, fish (Sera et al. 1974) and mollusts (Kueh and Chan 1985). All strains of VI cholerae isolated in this study failed to agglutinate in the 0 group 1 antisera and would not be considered to have the potential of producing epidemics of cholera in humans. However, raw oysters and other shell fish have previously been associated with outbreaks of gastroenteritis caused by non-01 V cholerae (Hughes et al. 1978, Wilson et al. 1981). A representative (one strain from each prawn sample yielding a positive isolation) of 107 strains of I.? cholerae non-01 and 35 strains of V parahaemolyticus isolated in this study were therefore examined in detail for their ability to produce certain established virulence factors commonly associated with strains of these

206

G. B. Nair et al.

species isolated from individuals with clinical infection. None of the 107 strains of V; cholerae non-01 recovered in this study hybridized with the cholera toxin gene probe which suggests that none of the strains produce cholera toxin. About one-third (31.7%) of the VI cholerae non-01 strains examined produced a factor in the culture supernatant that was active in the suckling mice assay. However, the activity in all supernatants was lost upon heating at 100°C for 5 min, indicating that this factor was not similar to the heat stable enterotoxin reported previously (Arita et al. 1986). The fluid accumulation in the suckling mice observed in this study is probably due to the ElTor-like haemolysin of V cholerae non-01 which is reportedly active in the suckling mice (Ichinose et al. 19871. Similarly, none of the 35 V parahaemolyticus isolates produced the thermostable direct haemolysin which is a marker of virulence (Sakazaki et al. 1968) indicating that all the strains were Kanagawa-negative. Most (85%) of the strains of I? parahaemolyticus recovered were untypeable. Two strains each belonged to K29 and K3 while one strain belonged to K28. These K types are not associated with diarrhoeal illness in this part of the country (Nair et al. 1985). Only three strains of the lactose-positive Vibrio, I? vulnificus could be isolated from two prawn samples in the study (Table 1). Despite the very low incidence, the presence of V: vulnificus in prawn samples poses a significant risk since this species is a human pathogen capable of producing highly lethal blood stream infections and destructive wound infections (Blake et

al.

19801. Additionally, unlike V and ?? parahaemolyticus, 900/o of the environmental V vulnifkus strains produce in vitro virulence factors comparable to those produced by clinical V vulnificus isolates (Tison and Kelly 1986). The three strains of V: vulnificus isolated in this study produced haemolysin. However, it would be premature to comment on the virulence of the three isolates since the in vivo virulence test in ICR mice was not performed. From the public health standpoint, the detection of such a high percentage of V cholerae non-01 and V parahaemolyticus in paddy field cultured prawns is a cause of concern. However, from a rationalistic viewpoint, it would be impossible to eliminate these vibrios from seafoods since both V. cholerae and V parahaemolyticus are autochthonous to brackish waters. In the case of microorganisms, searching for the pathogenic isolates would be like looking for the proverbial ‘needle in the haystack’ since the pathogenic strains would be one among the millions of innocuous vibrios naturally associated with seafoods. Efforts in the next few years should be invested in developing methods which could differentiate and detect the elusive pathogens associated with fish and shellfish destined for human consumption. This would permit an unequivocal assessment of the bacteriological quality of seafoods. cholerae

Acknowledgements We thank Mr T. Dutta and Mr B. K. Das, Marine Products Export Development Authority, Calcutta who kindly provided us the prawn samples. The excellent technical assistance of Mr R. B. Bose throughout the study is gratefully acknowledged.

Vibrios

in paddy field cultured

prawns

207

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