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Studies on occurrence of Vibrio parahaemolyticus in fin fishes and shellfishes from different ecosystem of West Bengal

M Nithya Quintoil*, K Porteen and A K Pramanik*

Division of Veterinary Public Health, Indian Veterinary Research Institute, Izatnagar, UP-243122, India
*Department of Veterinary Public Health, West Bengal University of Animal and Fishery Sciences, Kolkata-700037, India
porteen@rediffmail.com


Abstract

In the present study occurrence of Vibrio parahaemolyticus in fin fishes and shellfishes collected from different ecosystem namely marine, brackish and fresh water were investigated. A total of 162 samples were collected and subjected to battery of standard biochemical test for identification of Vibrio parahaemolyticus, out of which 38 samples were found to be positive. To detect the pathogenicity of the identified isolates Kanagawa reaction was performed. Among 38 isolates, 5 were found to be positive for Kanagawa reaction. Antibiogram results showed that all the strains were sensitive to chloramphenical and cotrimoxazole.

Keywords: Antibiotic resistance, fin fishes, pathogenicity, shell fishes, Vibrio parahaemolyticus


Introduction

Today's world is witnessing the resurgence in the consumption of fish due to the new awareness about its low cholesterol, fat content and good quality of animal protein. Fish and other seafoods are among the most important protein rich foods for human beings especially in a country like India .Vibrios of seafood origin have attracted increasing attention from time to time as it is found to be one of the important causes of food poisoning in man. The majority of outbreaks have also been epidemiologically traced to the consumption of fishes and shellfishes originating from warm coastal waters.

Vibrio parahaemolyticus is compatible with a marine or brackish aquatic environment adjusting well to the broad range of salinities and this commonly found on shellfishes and all varieties of finfishes that are traditionally taken from marine and shore areas. Foods, particularly seafoods in which small or limited numbers of organism are often present may become heavily contaminated by time temperature abuse, a major cause of microbial replication and contamination (Syndman and Gorfach 1991). The short generation time of 12 min for Vibrio parahaemolyticus permits the organisms to accumulate in millions in a few hours. Environmental strains of Vibrio parahaemolyticus are typically not human pathogens. However these strains caused diseases in shrimps, oysters, mussels etc (Montilla et al 1994)

Water resources and its distribution system have the chances of pollution with various microorganisms, hospitals, farms and domestic sewages. The fishes and shellfishes living in this water sources are at risk of acquiring the antibiotic resistance. This may cause serious health hazards to human and animals especially in countries where the sanitary and hygiene measures are not up to the mark.

Keeping in view the above points the present study was envisaged to study the occurrence of Vibrio parahaemolyticus in fishes and shellfishes of different ecosystems and to compare its enteropathogenicity and antibiotic resistance profile.


Materials and method

Samples

A total of 162 samples comprising fin fishes and shellfishes from different ecosystems were purchased from retail fish markets of west Bengal, India.

Method of collection

Immediately after collection, the samples were placed in the sterile polythene bags and brought to the laboratory in icebox within 2-3h for the isolation of organism. Fish samples from far away places were brought to the laboratory by dissecting a portion of the fish and inoculating in Carry Blair Transport Medium. The fishes and shellfishes for the study are commonly consumed by the people of west Bengal.

Sample processing and enrichment
Fin fishes

The whole fin-fishes were aseptically cut into small pieces. About 10g of the fish sample was weighed aseptically and transferred into 100ml of Alakaline Peptone Water (APW) containing 3% NaCl.

Shell fishes

The shellfishes were also aseptically cut into small pieces and about 10g of the sample was aseptically transferred into 100ml APW. The samples in Carry Blair Transport medium were also transferred into 100ml APW.

The APW inoculated with finfish and shellfish specimens were incubated at 37°C for 6-8h enrichment of Vibrio parahaemolyticus (Elliot et al 1992).

Isolation of Vibrio paraahemolyticus

For selective isolation of vibrio parahaemolyticus a loopful of culture from APW after 6-8h enrichment was streaked onto Thiosulfate citrate bile salt sucrose agar (TCBS) and incubated at 37 °C for 24 h.

The characteristically large colonies (3-4mm) with light blue or green centers on TCBS were regarded as presumptive Vibrio parahaemolyticus and further subjected to standard biochemical tests (Alsina and Blanch 1994) for confirmation.

Detection of pathogenicity
Kanagawa test

The pathogenecity of vibrio parahaemolyticus has been related to its ability to cause β-haemolysis on a special high salt medium called Wagatsuma Agar known as Kanagawa Phenomenon or Reaction. The Kanagawa reaction is caused by a heat stable direct haemolysin (Honda et al 1988). It was found that the ability of Vibrio parahaemolyticus to produce a positive Kanagawa reaction is closely related with human pathogenecity (Miyamato et al 1969). The Kanagawa reaction was formed on Wagatsuma agar using 2 % human RBCs. Loopfuls of overnight grown culture of Vibrio parahaemolyticus isolates were spot inoculated onto Wagatsuma agar plates and incubated at 37 °C for 24 h. The β-haemolysis of human RBCs after 24 h incubation period is interpreted as a positive Kanagawa reaction (Beauchat 1982).

In this study identified Vibrio parahaemolyticus isolates were subjected to Kanagawa phenomenon to identify their enteropathogenic potential.

Antibiogram

The occurrence of antibiotic resistant Vibrio parahaemolyticus is associated with indiscriminate or uncontrolled use of antibiotics to contain fish or shrimp diseases, faecal and industrial pollution of water bodies (Watkins and Cabelli 1985), and impertinent use of antibiotics for treating the infection in human and animals especially cases of diarrhea. The drug resistant Vibrio parahaemolyticus may transfer its resistant genes to flora of gut making them also drug resistant. In this study all identified 38 isolates were screened for their sensitivity to six potential antibiotics viz. chloramphenical (30µg), ciprofloxacin (5µg), cotrimoxazole (25µg), nitrofurantoin (300µg), gentamycin (10µg), oxytetracycline (30µg), by agar disc diffusion method (Bauer et al 1966).

Young cultures of bacteria from Tryptone Soy Broth with 1% sodium chloride (TSAS) were inoculated on to the surface of TSAS using sterile cotton swabs. The inoculums was allowed to dry for 5 min. Antibiotic impregnated discs were then placed aseptically on to the inoculated agar plates at 15mm away from the edge at equal distance and sufficiently separated from each other to avoid overlapping of the zone of inhibition. The plates were incubated for 20-24 h at 35°C and the diameter of zone of inhibition in mm was measured.

Interpretation of sensitivity was based on the zone size interpretation chart given by manufacturer of antibiotic impregnated discs (Table 1). The resistance profiles and resistance patterns for the above six antibiotics were determined for the antibiogram data.

Table 1.   Zone interpretation chart

Antibacterial agents

Disc content, g

Zone of inhibition, mm

R≤

I

S≥

Chloramphenical

30

12

13-17

18

Ciprofloxacin

5

15

16-20

21

Cotrimoxazole

25

10

11-15

16

Nitrofurantoin

300

14

15-16

17

Gentamycin

10

12

13-14

15

Oxytetracycline

30

14

15-18

19

Statistical analysis
To understand the significant difference in multiple antibiotic resistance among different ecosystems the data were analyzed by Chi-Square Test (χ²)


Results and discussion

Systemic bacteriological examination of a total of 162 samples of marine, brackish water, freshwater fin fishes and shellfishes resulted in the isolation of vibrio parahaemolyticus from 38 samples (2.46%) (Table 2).

Table 2.   Isolation of Vibrio parahaemolyticus from marine, freshwater and brackish water fishes

Nature of sample

Number of samples examined

Number of samples positive for Vibrio parahaemolyticus

Percentage

I. Marine

 

 

 

i) Finfish

40

10

25.00

ii) Shrimp

19

7

36.84

II. Freshwater

 

 

 

i) Finfish

25

3

12.00

ii) Prawn

17

3

17.65

III. Brackish water

 

 

 

i) Finfish

20

4

20.00

ii) Shrimp

21

5

23.80

iii) Crab

20

6

30.00

Total

162

38

23.46


Of the total of 59 marine samples comprising 40 fin fishes and 19 shrimps, 17 (28.8%) were found to be positive for vibrio parahaemolyticus i.e. 10 from fin fishes and 7 from shellfishes. Forty two samples comprising 25 fin fish and 17 prawns of freshwater origin yielded 6 positive samples (14.3%). The break up was 3 each for fin fishes and prawns. Analysis of brackish water fin fishes (20), shrimps (21)and crabs (20) yielded 15 Vibrio parahaemolyticus positive samples, 4 from fin fishes, 5 from shrimps and 6 from crabs.

The highest incidence of Vibrio parahaemolyticus was from marine shrimps (36.8 %), followed by brackish water crabs (30%) marine fishes (25%), brackish water shrimps (23.8%), brackish water fin fishes (20%), freshwater prawns (17.6%) and freshwater fin fishes (12%).The present isolation rate in marine fishes was 28.8% which is slightly lower than the reports by Dileep et al (2003). Although Vibrio parahaemolyticus is halphilic it was isolated from 14.3% of  freshwater fishes. The results are in agreement with the findings of Sarkar et al (1985) who have isolated Vibrio parahaemolyticus from a variety of freshwater fishes in Kolkata. The incidence of Vibrio parahaemolyticus from brackish water fishes was found to be 24.6% which is slightly less than reported by Natarajan et al (1980) and Nair et al (1980).

Being halophilic organism the occurrence of Vibrio parahaemolyticus in the coastal area in not uncommon. But it was interesting to note the occurrence of this halophile in the freshwater environs. Analysis of the data in Table 3 of the present study revealed that the organism was prevalent in freshwater fishes to the extent of 14.3%. Similar findings were reported by Nair et al (1975) and Sarkar et al (1985).

Table 3.   Kanagawa reaction among Vibrio parahaemolyticus in different Ecosystem

Ecosystem

No. of samples

examined

No. of samples positive for

Vibrio parahaemolyticus

Percentage

 

Marine

17

4

23.5

Freshwater

6

0

0.00

Brackish water

15

1

6.00

Total

38

5

13.2

The results of the present study indicated that irrespective of their origin, fin fishes and shellfishes provide an ideal substrate for the survival and proliferation of Vibrio parahaemolyticus. Fishes and  perhaps other aquatic animals are the main reservoir of Vibrio parahaemolyticus in the freshwater environs. This finding s derives support from the observation of Sarkar et al (1985) who reported that the occurrence of Vibrio parahaemolyticus strains in freshwater ecosystem was primarily related to their association with a biological host, particularly fishes.

Detection of pathonenicity
Kanagawa reaction

Out of 38 identified environmental Vibrio parahaemolyticus strains 5 showed positive Kanagawa reactions. Of these three (3) Kanagawa positive isolates were from marine sources and other 2 from brackish water source. All the freshwater Vibrio parahaemolyticus isolates were Kanagawa negative Table 3. These results are in confirmation with the findings of Sarkar et al (1985) who reported that none of the freshwater isolates was Kanagawa positive so also in this study.

The isolation of Kanagawa positive strains of vibrio parahaemolyticus from food samples is significant because it is generally believed that Kanagawa positive strains are encountered only in case of gastroenteritis. It is assumed that Kanagawa positive strains contain a thermostable direct haemolysin (TDH), which is responsible of gastroenteritis syndrome by vibrio parahaemolyticus (Miyamato et al 1969). About 85% of the strains showing this phenotype possessed both the gene, which is responsible for TDH production. But gastroenteritis cases due to Kanagawa negative strains of Vibrio parahaemolyticus also have been reported by Teramoto et al (1969) and Zen-Yoji et al (1970).

The incidence of Kanagawa positive strains of Vibrio parahaemolyticus in the brackish water and marine eco systems stresses the need for hygienic handling of sea foods at every stage. Refrigeration or freezing is the most important method for preventing multiplication of this organism because even Kanagawa negative strains poses health hazard to man. Honda et al (1988) identified a TDH- related haemolysin (TRH) from Kanagawa negative strains of Vibrio parahaemolyticus and this TRH was immunologically similar but not identical to TDH. Therefore it is now evident the Kanagawa negative strains of Vibrio parahaemolyticus also produce some toxic materials which may play some role in the pathogenicity.

Antibiogram

A total of 38 strains from environmental samples were subjected to antibiotic susceptibility using 6 different antibiotics. The higher percentage of antibiotic resistance was found in freshwater ecosystem followed by brackish water and marine ecosystem. None of the strains were resistant to chloramphenical and co-trimoxazole.The order of antibiotic resistance among Vibrio parahaemolyticus isolates was as follows:

Ciprofloxacin≥oxytetracycline>nitrofurantoin>gentamycin>cotimoxazole=chloramphenical.

The high incidence of resistance of resistance among Vibrio parahaemolyticus isolates probably indicated the excessive use of these particular antibiotics so also oxytetracycline.

The multiple antibiotic resistance (MAR) among Vibrio parahaemolyticus is presented in Table 4.

Table 4.   Multiple antibiotic resistance in Vibrio parahaemolyticus

Source of samples

Total number of isolates

Multiple antibiotic resistance (MAR)

Numbers

Percentage

Brackishwatera

15

6

40.0

Freshwaterb

6

3

50.0

Marinea,b

17

1

5.88

a,b, :       Ecosystems sharing common superscripts exhibited significant difference in MAR (P<0.05)

a : c2 = 45.9; P<0.05

b : c2 = 6.01; P<0.05

The MAR was seen among 26.3% of the isolates. The incidence of MAR was more in brackish water followed by freshwater ecosystem probably the result of discharge of Kolkata metropolitan waste in to these ecosystems than in marine environs. There existed significant differences (P<0.05) in MAR between marine and brackish water ecosystem and also between marine and freshwater ecosystems. The differences in MAR between brackish water and freshwater ecosystem were insignificant (P>0.05).  The significance of MAR in different ecosystem was studied by Kaspar et al (1990) and the results of the present study are in agreement with their study.

The indiscriminate use of antibiotics for the therapeutic and other domestic purpose poses potential hazard to human being. There appears to be great danger due to the occurrence of resistance against ciprofloxacin, and oxytetracycline as they are most commonly used antibiotics.


Conclusions

References

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Received 23 April 2006; Accepted 27 October 2006; Published 1 January 2007

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