We report sporadic cases of a severe gastroenteritis associated with Vibrio cholerae serogroup O141. Like O1 and O139 serogroup strains of V. cholerae isolated from cholera cases, the O141 clinical isolates carry DNA sequences that hybridize to cholera toxin (CT) gene probes. The CT genes of O1 and O139 strains are carried by a filamentous bacteriophage (termed CTX phage) which is known to use toxin-coregulated pili (TCP) as its receptor. In an effort to understand the mechanism of emergence of toxigenic O141 V. cholerae, we probed a collection of O141 clinical and environmental isolates for genes involved in TCP production, toxigenicity, virulence regulation, and other phylogenetic markers. The collection included strains isolated between 1964 and 1995 from diverse geographical locations, including eight countries and five U.S. states. Information collected about the clinical and environmental sources of these isolates suggests that they had no epidemiological association. All clinical O141 isolates hybridized to probes specific for genes encoding CT (ctx), zonula occludens toxin (zot), repetitive sequence 1 (RS1), RTX toxin (rtxA), the major subunit of TCP (tcpA), and the essential regulatory gene that controls expression of both CT and TCP (toxR). In contrast, all but one of the nonclinical O141 isolates were negative for ctx, zot, RS1, and tcpA, although these strains were positive for rtxA and toxR. The one toxigenic environmental O141 isolate was also positive for tcpA. Ribotyping and CT typing showed that the O141 clinical isolates were indistinguishable or closely related, while a toxigenic water isolate from Louisiana showed a distantly related ribotype. Nonclinical O141 isolates displayed a variety of unrelated ribotypes. These data support a model for emergence of toxigenic O141 that involves acquisition of the CTX phage sometime after these strains had acquired the pathogenicity island encoding TCP. The clonal nature of toxigenic O141 strains isolated from diverse geographical locations suggests that the emergence is a rare event but that once it occurs, toxigenic O141 strains are capable of regional and perhaps even global dissemination. This study stresses the importance of monitoring V. cholerae non-O1, non-O139 serogroup strains for their virulence gene content as a means of assessing their epidemic potential.The two most important virulence factors of Vibrio cholerae are cholera toxin (CT), a potent enterotoxin, and the toxincoregulated pilus antigen (TCP), an essential intestinal colonization factor (16). These virulence factors are encoded by accessory genetic elements which are nearly always present in clinical isolates of V. cholerae but are frequently absent in strains isolated from environmental sources, such as water or shellfish (6,16,17). The ctxAB genes are located on the CTX genetic element, which is composed of a 4.5-kb central core region flanked by one or more copies of a repetitive sequence (RS1 or RS2) (34). The core and RS2, a portion of the CTX element, are now known to corresp...

In this study, 176 clinical and environmental Vibrio cholerae strains of different O serotypes isolated inThailand from 1982 to 1995 were selected and studied for the presence of class 1 integrons, a new group of genetic elements which carry antibiotic resistance genes. Using PCR and DNA sequencing, we found that 44 isolates contained class 1 integrons harboring the aadB, aadA2, blaP1, dfrA1, and dfrA15 gene cassettes, which encode resistance to gentamicin, kanamycin, and tobramycin; streptomycin and spectinomycin; ␤-lactams; and trimethoprim, respectively. Each cassette array contained only a single antibiotic resistance gene. Although resistance genes in class 1 integrons were found in strains from the same epidemic, as well as in unrelated non-O1, non-O139 strains isolated from children with diarrhea, they were found to encode only some of the antibiotic resistance expressed by the strains. Serotype O139 strains did not contain class 1 integrons. However, the appearance and disappearance of the O139 serotype in the coastal city Samutsakorn in 1992 and 1993 were associated with the emergence of a distinct V. cholerae O1 strain which contained the aadA2 resistance gene cassette. A 150-kb self-transmissible plasmid found in three O1 strains isolated in 1982 contained the aadB gene cassette. Surprisingly, several strains harbored two integrons containing different cassettes. Thus, class 1 integrons containing various resistance gene cassettes are distributed among different V. cholerae O serotypes of mainly clinical origin in Thailand.

Results of a surveillance on cholera conducted with patients seen at the Children Hospital in Bangkok, Thailand from August 1993 to July 1995 are presented. Annually, isolation rates for Vibrio cholerae varied between 1.7 and 4.4% of patients with diarrhoea. V. cholerae O1 serotype Ogawa accounted for between 31 and 47% of patients cultured positive for V. cholerae, whereas the O139 serotype dominated in early 1994 after which it disappeared. Non-O1, non-0139 strains were isolated at similar rates as serotype O1 in 1993 and 1994, but accounted for 69% of V. cholerae culture positive specimens in 1995. However, the annual proportion of the isolation of non-O1, non-O139 strains showed little variation and remained low between 1.0 and 1.3%. Serotyping of 69 epidemiological unrelated non-O1, non-O139 strains produced 37 different O-serotypes. BglI ribotyping of serotypes containing more than two strains demonstrated a high degree of heterogeneity within and between serotypes, except seven serotype O37 strains which showed an identical ribotype suggesting clonality. None of the 69 strains hybridized with a cholera toxin probe and only two strains hybridized with a heat-stable enterotoxin probe. Susceptibility testing to 12 antibiotics showed that 40 of 69 (58%) non-O1, non-O139 strains were resistant to colistin, streptomycin and sulphisoxazole and 28 of 69 (41%) were multiple antibiotic resistant (MAR; > or = 4 antibiotics). Although 26 of 69 (38%) strains contained one or more plasmids, the plasmids were of low molecular weights and did not seem to encode antibiotic resistance. The results of the present study showed that a high proportion of heterogenous MAR V. cholerae non-O1, non-O139 strains were isolated from children at the hospital. With reference to the emergence of V. cholerae O139 in 1992, we suggest that non-O1, non-O139 strains should be monitored carefully to detect new serotypes with a possible epidemic potential, but also to determine the development and mechanism of antibiotic resistance.

The number of cholera cases and the mortality rates reported from different regions of Vietnam varied considerably in the period from 1979 to 1996, with between 2,500 and 6,000 cases reported annually from 1992 to 1995. Annual mortality rates ranged from 2.0 to 9.6% from 1979 to 1983 to less than 1.8% after 1983. Major cholera outbreaks were reported from the High Plateau region for the first time in 1994 and 1995; this is an area with limited access to health services and safe drinking-water supplies. All cases were associated with Vibrio cholerae O1. Using ribotyping, cholera toxin (CT) genotyping, and characterization of antibiotic susceptibility patterns and antibiotic resistance genes by PCR, we show that strains isolated after 1990 were clearly different from strains isolated before 1991. In contrast to strains isolated before 1991, 94% of 104 strains isolated after 1990 showed an identical ribotype R1, were resistant to sulfamethoxazole and streptomycin, and showed a different CT genotype. Furthermore, PCR analysis revealed that sulfamethoxazole-resistant strains harbored class I integrons containing a gene cassette ant(3")-1aencoding resistance to streptomycin and spectinomycin. This is, to our knowledge, the first report of class I integrons in V. cholerae. The development of cholera and the changes in the phenotypic and genotypic properties of V. cholerae O1 shown in the present study highlight the importance of monitoring V. cholerae O1 in Vietnam as in other parts of the world. In particular, the emergence of the new ribotype R1 strain containing class I integrons should be further studied.

Representative encapsulated strains of Vibrio vulnificus from market oysters and oyster-associated primary septicemia cases (25 isolates each) were tested in a blinded fashion for potential virulence markers that may distinguish strains from these two sources. These isolates were analyzed for plasmid content, for the presence of a 460-bp amplicon by randomly amplified polymorphic DNA PCR, and for virulence in subcutaneously (s.c.) inoculated, iron-dextran-treated mice. Similar percentages of market oyster and clinical isolates possessed detectable plasmids (24 and 36%, respectively), produced the 460-bp amplicon (45 and 50%, respectively), and were judged to be virulent in the mouse s.c. inoculation-iron-dextran model (88% for each). Therefore, it appears that nearly all V. vulnificus strains in oysters are virulent and that genetic tests for plasmids and specific PCR size amplicons cannot distinguish between fully virulent and less virulent strains or between clinical and environmental isolates. The inability of these methods to distinguish food and clinical V. vulnificus isolates demonstrates the need for alternative subtyping approaches and virulence assays.In the United States, Vibrio vulnificus is the leading cause of death associated with consumption of seafood (12, 15). Consumption of raw Gulf Coast oysters from April to November is responsible for nearly all of the cases. Although V. vulnificus is abundant in oysters during that time of year, cases are rare even in the high-risk group (i.e., those with preexisting liver disease or who are immunocompromised) (7). A major obstacle in developing effective control strategies is the inability to identify in the oysters V. vulnificus strains that are capable of causing human illness.Two major research needs identified at a 1994 V. vulnificus workshop sponsored by the Food and Drug Administration (FDA) were to develop methods to distinguish virulent V. vulnificus strains from avirulent strains and to determine the infectious dose (22). Since human volunteer studies with V. vulnificus are not ethical, a consensus approach was proposed to determine the infectious dose by relating disease frequency with exposure. It was also suggested that a collection of strains from oysters and human septicemia cases associated with oyster consumption should be characterized in various assays in an attempt to determine traits that may be linked to virulence. The Centers for Disease Control and Prevention (CDC), FDA, and various state departments of health collected approximately 75 well-characterized clinical strains from human septicemia cases with known sources of oysters consumed, patient histories, etc. A recent study of the abundance of V. parahaemolyticus and V. vulnificus in retail oysters by the Interstate Shellfish Sanitation Conference and the FDA generated a large collection of V. vulnificus cultures that is seasonally and geographically diverse and well defined (3). Molecular characterization and virulence assays of representative V. vulnificus isolates from these tw...