Pandemic Vibrio parahaemolyticus O3:K6 spread, France.

To the Editor: Vibrio parahaemolyticus is a halophilic bacterium that occurs naturally in aquatic environments worldwide. It causes one of the most severe forms of gastroenteritis and is the leading cause of seafood-associated bacterial gastroenteritis in the world, often associated with the consumption of raw or undercooked seafood. Since 1996, the incidence of V. parahaemolyticus infections has increased dramatically. V. parahaemolyticus strains previously associated with only sporadic cases of gastroenteritis have caused large-scale outbreaks in North America and epidemics in India, Southeast Asia, and Japan (1). This increase in incidence appears to be related to the emergence of a new clone, belonging to the O3:K6 serovar, which has pandemic potential. This clone was named the new O3:K6 clone to distinguish it from strains belonging to this serovar isolated before 1996, which are less pathogenic. We report the first evidence for the presence in France and suggest the presence and persistence in French coastal areas of this pandemic O3:K6 serovar, which is indistinguishable from the O3:K6 clone isolated in Bangladesh in 1996.

We analyzed 13 clinical isolates of V. parahaemolyticus collected in France from 1997 to 2004 and sent to the National Reference Center (Table). All isolates were characterized by polymerase chain reaction (PCR) to detect the genes encoding the virulence-associated hemolysins, thermostable-direct hemolysin, and thermostatable-related hemolysin (2), and 2 other genetic markers, toxRS and orf8 (1,3). We also carried out molecular typing by various methods, including ribotyping, pulsed-field gel electrophoresis (PFGE), and arbitrarily primed PCR. Strains were initially identified by biochemical and cultural methods. Strain identities were confirmed by species-specific R72H PCR (4). The slide agglutination test was performed to determine whether the isolates belonged to the O3:K6 serovar. Two pandemic strains of the new O3:K6 clone and 1 strain of the old O3:K6 clone were included as external controls.

Five strains were identified as V. parahaemolyticus O3:K6 by slide agglutination test. With the exception of the strain referred to as old O3:K6 clone, all O3:K6 strains studied, whether isolated in France or included as controls, were positive for tdh, toxRS, and orf8 genes. Likewise, all strains, except the old O3:K6 clone, produced Bg/I rRNA gene restriction patterns identical to those of a major R4 ribotype previously described (5) and were genetically indistinguishable by arbitrarily primed PCR analysis. With the exception of the strain belonging to the old O3:K6 clone, the PFGE-typeable O3:K6 strains, despite having slightly different NotI patterns that reflect genetic rearrangement, clearly belonged to a single clone.

Until recently, V. parahaemolyticus caused only sporadic diarrhea and was never associated with a pandemic. The epidemiology of this organism changed abruptly after the new O3:K6 strains appeared in 1996. The spread of this serotype signaled the beginning of the first V. parahaemolyticus pandemic. Because the pathogenic V. parahaemolyticus O3:K6 isolates in France are derived from the new O3:K6 clone initially described in Bangladesh, this population likely was transported here in the same manner as V. parahaemolyticus O3:K6 was introduced into US coastal waters (6) and V. cholerae serogroup O1 was introduced into Gulf Coast waters in 1991 (7).

Epidemiologic information was collected from all patients with a standardized questionnaire concerning clinical history, symptoms, and seafood consumption. Responses indicated that some persons affected by V. parahaemolyticus O3:K6 had eaten local seafood harvested in uncontrolled areas. Furthermore, some had eaten seafood harvested in the same place several years apart. This provides evidence that pathogenic V. parahaemolyticus is present and suggests that it can persist in the French coastal environment.

The consumption of raw and lightly cooked seafood is increasing, as is the number of susceptible persons, which causes concern that the incidence of V. parahaemolyticus infections in Europe will increase. Monitoring this foodborne illness is difficult because only cases involving severe gastroenteritis are reported. Estimates are that only 1 in 20 cases of bloody diarrhea and only 1 in 38 cases of nonbloody diarrhea are reported in the United States (8). In France, the official surveillance authority estimated that the number of cases reported by the National Reference Center was representative of severe Vibrio infections. Making Vibrio isolations and infections reportable could help us estimate the true incidence of the disease and could improve the surveillance of V. parahaemolyticus infections.

Detecting the pathogenic V. parahaemolyticus O3:K6 in France, and previous results showing that pathogenic V. parahaemolyticus strains are present in French coastal areas at a higher frequency than was usually reported (9), may provide an early warning. Much effort is required to develop V. parahaemolyticus prevention strategies. Educating consumers about basic principles of food safety, particularly storage conditions, is an important component of prevention. Lack of continuous refrigeration from harvest to consumption may have contributed to these infections. The number of bacteria in seafood contaminated with only a small number of V. parahaemolyticus organisms can reach the infectious dose, thought to be [greater than or equal to] [10.sup.5] CFU per gram according to the Centers for Disease Control and Prevention (10), within a few hours when left in a warm place. Another component of prevention is the improvement of microbial surveillance by systematic testing for pathogenic V. parahaemolyticus isolates in the environment and in locally produced and imported seafood.

Table. Characteristics of the Vibrio parahaemolyticus O3:K6 strains
studied *

Strain no. CNRVC                         Date of      Source of
(source no.)             Origin         isolation    transmission

970136                   France         Oct 1997        Local
                    (Atlantic coast)                   oysters
980402                   France         Sep 1998      Shellfish
                      (southwest)
990346                   France         Aug 1999          -
                     (Mediterranean
                         coast)
30478                    France         Aug 2003        Local
                    (Atlantic coast)                  shellfish
30479                    France         Aug 2003          -
                    (Atlantic coast)
020468 (AN7410)        Bangladesh         1998
020469 (AO1851)        Bangladesh         1999
030085 (AQ4037)         Maldives          1985

                                        Detection of gene or phage
                                             sequences by PCR
Strain no. CNRVC
(source no.)             Origin         R72H    tdh    trh    toxRS

970136                   France          +       +      -       +
                    (Atlantic coast)
980402                   France          +       +      -       +
                      (southwest)
990346                   France          +       +      -       +
                     (Mediterranean
                         coast)
30478                    France          +       +      -       +
                    (Atlantic coast)
30479                    France          +       +      -       +
                    (Atlantic coast)
020468 (AN7410)        Bangladesh        +       +      -       +
020469 (AO1851)        Bangladesh        +       +      -       +
030085 (AQ4037)         Maldives         +       -      +       -

                                        Detection
                                        of gene or
                                          phage
                                        sequences
                                          by PCR
Strain no. CNRVC                                          Ribotype
(source no.)             Origin            orf8           profile

970136                   France             +           R4 ([dagger])
                    (Atlantic coast)
980402                   France             +                R4
                      (southwest)
990346                   France             +                R4
                     (Mediterranean
                         coast)
30478                    France             +                R4
                    (Atlantic coast)
30479                    France             +                R4
                    (Atlantic coast)
020468 (AN7410)        Bangladesh           +                R4
020469 (AO1851)        Bangladesh           +                R4
030085 (AQ4037)         Maldives            -         Rb-2 ([section])

Strain no. CNRVC                                PFGE            AP-PCR
(source no.)             Origin               profile           profile

970136                   France                P-1 a             AP-a
                    (Atlantic coast)
980402                   France                P-1 c             AP-a
                      (southwest)
990346                   France                P-1 c             AP-a
                     (Mediterranean
                         coast)
30478                    France         UT ([double dagger])     AP-a
                    (Atlantic coast)
30479                    France                  UT              AP-a
                    (Atlantic coast)
020468 (AN7410)        Bangladesh               P-1a             AP-a
020469 (AO1851)        Bangladesh               P-1b             AP-a
030085 (AQ4037)         Maldives                P-2              AP-b

* CNRVC, Centre National de Reference des Vibrions et du Cholera; PCR,
polymerase chain reaction; PFGE, pulsed-field gel electrophoresis;
AP-PCR, arbitrarily primed PCR.

([dagger]) R4 ribotype pattern as described previously (5).

([double dagger]) UT, untypeable: DNA was degraded before PFGE,
presumably by DNases.

([section]) According to our pattern designation.

Acknowledgments

We thank Alain Guenole for excellent technical assistance. Strains of the pandemic O3:K6 new clone and of the old O3:K6 clone were provided by G. B. Nair and by M. Nishibuchi, respectively. The acetyl aminofluorene (AAF)-labeled RNA probe and the anti-AAF mouse monoclonal antibody were prepared and purified by Jean-Luc Guesdon.

The Institut Pasteur and the Ministry of Health provided financial support.

References

(1.) Matsumoto C, Okuda J, Ishibashi M, Iwanaga M, Garg P, Rammamurthy T, et al. Pandemic spread of an O3:K6 clone of Vibrio parahaemolyticus and emergence of related strains evidenced by arbitrarily primed PCR and toxRS sequence analyses. J Clin Microbiol. 2000;38:578-85.

(2.) Nishibuchi M, Kaper JB. Thermostable direct hemolysin gene of Vibrio parahaemolyticus: a virulence gene acquired by a marine bacterium. Infect Immun. 1995;63:2093-9.

(3.) Myers ML, Panicker G, Bej AK. PCR detection of a newly emerged pandemic Vibrio parahaemolyticus O3:K6 pathogen in pure cultures and seeded waters from the Gulf of Mexico. Appl Environ Microbiol. 2003;69:2194-200.

(4.) Robert-Pillot A, Guenole A, Fournier JM. Usefulness of R72H PCR assay for differentiation between Vibrio parahaemolyticus and Vibrio alginolyticus species: validation by DNA-DNA hybridization. FEMS Microbiol Lett. 2002;215:1-6.

(5.) Bag PK, Nandi S, Bhadra RK, Ramamurthy T, Bhattacharya SK, Nishibuchi M, et al. Clonal diversity among recently emerged strains of Vibrio parahaemolyticus O3:K6 associated with pandemic spread. J Clin Microbiol. 1999;37:2354-7.

(6.) Daniels NA, Ray B, Easton A, Marano N, Kahn E, McShan A L, et al. Emergence of a new Vibrio parahaemolyticus serotype in raw oysters--A prevention quandary. JAMA. 2000;284:1541-5.

(7.) McCarthy SA, Khambaty FM. International dissemination of epidemic Vibrio cholerae by cargo ship ballast and other nonpotabte waters. Appl Environ Microbiol. 1994;60:2597 601.

(8.) Mead PS, Slutsker L, Dietz V, McGaig LF, Bresee JS, Shapiro C, et al. Food-related illness and death in the United States. Emerg Infect Dis. 1999;5:607-25.

(9.) Robert-Pillot A, Guenole A, Lesne J, Delesmont R, Fournier JM, Quilici ML. Occurrence of the tdh and trh genes in Vibrio parahaemolyticus isolates from waters and raw shellfish collected in two French coastal areas and from seafood imported into France. Int J Food Microbiol. 2004;91:319-25.

(10.) Centers for Disease Control and Prevention. Outbreak of Vibrio parahaemolyticus infections associated with eating raw oysters--Pacific Northwest, 1997. MMWR Morbid Mortal Wkly Rep. 1998;47:457-62.

Marie-Laura Quilici, * Annick Robert-Pillot, * Jessica Picart, * and Jean-Michel Fournier *

* Institut Pasteur, Paris, France

Address for correspondence: Marie-Laure Quilici, Unite du Cholera et des Vibrions, Centre National de Reference des Vibrions et du Cholera, Institut Pasteur, 25 rue du Dr Roux, 75 724 Paris Cedex 15, France; fax: [phone]; email:[e-mail]