Prevalence of the Pandemic Genotype of Vibrio parahaemolyticus in Dhaka, Bangladesh, and Significance of Its Distribution across Different Serotypes

ABSTRACT Sixty-six strains of Vibrio parahaemolyticus belonging to 14 serotypes were isolated from hospitalized patients in Dhaka, Bangladesh, from January 1998 to December 2000. Among these, 48 strains belonging to four serotypes had the pandemic genotype and possessed the tdh gene. A marker (open reading frame ORF8) for a filamentous phage previously thought to correspond to the pandemic genotype was found to have a poor correlation with the pandemic genotype.

Vibrio parahaemolyticus, a seafood-borne pathogen, is the causative agent of gastroenteritis in humans. Unlike Vibrio cholerae, only two serogroups of which (serogroups O1 and O139) are involved in epidemic and pandemic diseases, V. parahaemolyticus gastroenteritis is a multiserogroup affliction, and as many as 75 different combinations of O and K serotypes of V. parahaemolyticus are recognized and known to be associated with gastroenteritis (7). Beginning in February 1996, a new clone of V. parahaemolyticus belonging to the O3:K6 serotype was responsible for a dramatic increase in the number of cases of diarrhea in Calcutta, India (1,12). An increase in the incidence of V. parahaemolyticus food poisoning observed during 1997 and 1998 was also ascribed to the increased incidence of food poisoning caused by the O3:K6 clone in Japan (15). Evidence supporting the hypothesis that the O3:K6 clone has recently emerged and has pandemic potential was presented by Matsumoto and coworkers (9).
The V. parahaemolyticus pandemic has spread into at least eight countries, and the emergence of two other serotypes (serotypes O4:K68 and O1:KUT [UT indicates untypeable]) possessing pandemic potential has been documented (3,9). Molecular biology-based analysis of these additional serotypes indicates that they may have diverged from the pandemic O3:K6 strains by alteration of the O:K antigens (4,9). The present study was performed to understand the status of the pandemic genotype of V. parahaemolyticus among hospitalized patients in Dhaka, Bangladesh.
Stool specimens were collected from patients enrolled in the 2% systematic routine surveillance system at the Clinical Research and Service Centre of the International Centre for Diarrhoeal Disease Research, Bangladesh (ICDDR, B). Stool specimens were plated directly, after enrichment in bile pep-tone broth, onto tauro-cholate-tellurite-gelatin agar (10). The colonies selected were screened for V. parahaemolyticus by a battery of biochemical tests elaborated previously (9). The commercially available V. parahaemolyticus antiserum kit manufactured by Toshiba Kagaku Kogyo Co., Ltd., Tokyo, Japan, was used for serological typing in accordance with the manufacturer's instructions.
PCR assays were performed to test for the presence of the species-specific toxR gene and also to test for the presence of the two known virulence genes, tdh and trh of V. parahaemolyticus, with primers for toxR, tdh, and trh, respectively, as described previously (8,14). The group-specific PCR (GS-PCR) for determination of the presence of the pandemic genotype was performed as described previously (9). PCR for open reading frame (ORF) ORF8 was performed in 20-l volumes containing 2.0 l of 10ϫ MgCl 2 -free PCR amplification buffer, 1.6 l of MgCl 2 (2.0 mM), 1.6 l of deoxynucleoside triphosphates (dNTPs; 2.0 mM each dNTP), 0.5 M (each) primers VP36RF8U and VP36RF8L (V. Laohaprertthisan, A. Chowdhury, U. Kongmuang, S. Kalnauwakul, M. Ishibashi, C. Matsumoto, and M. Nishibuchi, personal communication), 0.5 U of rTaq DNA polymerase (GIBCO), and 2.5 l of the template solution (supernatant of the boiled culture diluted 1:10). The amplification conditions were set at one cycle of 96°C for 5 min, followed by 25 cycles at 94°C for 1 min, 53°C for 1 min, and 72°C for 1 min, with a final extension of 72°C for 7 min (Perkin-Elmer, Norwalk, Conn.). The PCR products were electrophoresed in a 1% agarose gel and were visualized under UV light following staining with ethidium bromide. Chromosomal DNA was extracted and arbitrarily primed PCR (AP-PCR) was done as described previously (4). Southern blot hybridization with an rRNA gene probe was performed as described previously (5).
During the 3-year study period, 66 strains of V. parahaemolyticus were isolated from that many patients (Table 1). All patients from whom specimens were collected were symptomatic and were hospitalized and had the characteristic symptoms of V. parahaemolyticus infection. The total rate of isolation of V. parahaemolyticus during the 3 years of surveillance ranged from 0.8 to 1%, which is low compared to those for V. cholerae and diarrheagenic Escherichia coli, but the fact that the majority of the strains have acquired the pandemic genotype is ominous, in that isolation rates may increase in future, as has been seen in Calcutta (4, 12) and among travelers returning to Japan (15). All 66 strains of V. parahaemolyticus identified by a battery of biochemical tests were positive for the 368-bp specific toxR amplicon, thereby confirming the identities of the strains. Of the 66 strains, 60 strains were positive for the tdh gene but negative for trh, 4 strains carried only the trh gene, and 2 strains were negative for both the tdh gene and the trh gene but were isolated from hospitalized patients with acute diarrhea ( Table  2).
Of the 75 different O:K serotype combinations of V. parahaemolyticus currently recognized, 14 different serotypes were isolated during the study period, with O3:K6 (42.4%) being the dominant serotype, followed by O4:K68 (19.7%), O1:KUT (9.1%), and O5:KUT (6.1%) ( Table 1). On a yearly basis, the O4:K68 serotype dominated in 1998 (52.2%), while O3:K6 dominated in 1999 (78.3%) and 2000 (30%). Of the 66 strains of V. parahaemolyticus isolated in the 3 years, 48 strains were positive by the GS-PCR, indicating that these strains had the pandemic genotype ( Table 2). The number of strains bearing the pandemic genotype (GS-PCR positive) in each year was 18 in 1998, 19 in 1999, and 11 in 2000, indicating a decline in the rate of isolation of such strains in Dhaka. During the 3-year study period, four serotypes (serotypes O3:K6, O4:K68, O1: KUT, and O1:K25) had the pandemic genotype, and all these strains were positive only for the tdh gene, which is the usual pattern (9). One serotype, serotype O1:K25, isolated in Bangladesh in 1999 and 2000, was recorded for the first time to carry the pandemic genotype.
A recent report has revealed that ORF8 is a specific marker of the filamentous phage, phage f237, exclusively associated with pandemic strains of serotype O3:K6 strains of V. parahaemolyticus (11). In the present study, we examined all strains for ORF8 of phage f237 using specific primers ( Table 2). Of the 48 GS-PCR-positive strains, 39 were positive for the amplicon by the PCR for ORF8. Eight O3:K6 strains and one O4:K68 strain had positive results by the GS-PCR and negative results by PCR for ORF8. None of the GS-PCR-negative strains were positive for ORF8.
To understand if the strains which were ORF8 negative but GS-PCR positive belonged to the pandemic genotype, we examined a set of representative strains of O3:K6 and O4:K68 by AP-PCR and ribotyping. In the AP-PCR assay, seven O3:K6 strains (five GS-PCR-positive and ORF8-positive strains and two GS-PCR-positive and ORF8-negative strains) and four O4:K68 strains (three GS-PCR-positive and ORF8-positive strains and one GS-PCR positive and ORF8-negative strain) were analyzed with primers 1281 and 1283. All strains showed identical AP-PCR profiles. Similar results were obtained by ribotyping of these strains. All O3:K6 and O4:K68 strains had identical ribotype patterns, irrespective of the presence of ORF8. The reason for the identical AP-PCR and ribotype profiles of the GS-PCR-positive strains that were either ORF8 PCR positive or ORF8 PCR negative could relate to mutations in the primer annealing sequences. A recent study has shown that in ORF8 variants the entire phage genome is not lost since ORF10 was detected in these variants (Laohaprestthisan et al., personal communication).
Efforts to describe the bacterial factors of the pandemic O3:K6 strains led to the discovery that a related filamentous phage designated f237 is exclusively associated with the pandemic O3:K6 strains (11). Further analysis of the phage genome revealed that ORF8 was detected only in O3:K6 strains     (13), the repeated recovery of strains with identical or very closely related genotypes from patients with disease suggest that these changes are associated with increased fitness or, more specifically, transmissibility. The fact that the strains of V. parahaemolyticus belonging to O3:K6 isolated from 1996 onwards are more virulent than those isolated before 1996 (12) indicates that the O3:K6 serogroup is refurbishing its background genotype to a higher level of virulence. In this study, at least four different serotypes were seen to have the pandemic genotype. It thus appears overall that the enteropathogen is enriching its gene pool toward a higher level of virulence.