Enterobacterial repetitive intergenic consensus sequences and the PCR to generate fingerprints of genomic DNAs from Vibrio cholerae O1, O139, and non-O1 strains.

Enterobacterial repetitive intergenic consensus (ERIC) sequence polymorphism was studied in Vibrio Cholerae strains isolated before and after the cholera epidemic in Brazil (in 1991), along with epidemic strains from Peru, Mexico, and India, by PCR. A total of 17 fingerprint patterns (FPs) were detected in the V. cholerae strains examined; 96.7% of the toxigenic V. cholerae O1 strains and 100% of the O139 serogroup strains were found to belong to the same FP group comprising four fragments (FP1). The nontoxigenic V. cholerae O1 also yielded four fragments but constituted a different FP group (FP2). A total of 15 different patterns were observed among the V. cholerae non-O1 strains. Two patterns were observed most frequently for V. cholerae non-01 strains, 25% of which have FP3, with five fragments, and 16.7% of which have FP4, with two fragments. Three fragments, 1.75, 0.79, and 0.5 kb, were found to be common to both toxigenic and nontoxigenic V. cholerae O1 strains as well as to group FP3, containing V. cholerae non-O1 strains. Two fragments of group FP3, 1.3 and 1.0 kb, were present in FP1 and FP2 respectively. The 0.5-kb fragment was common to all strains and serogroups of V. cholerae analyzed. It is concluded from the results of this study, based on DNA FPs of environmental isolates, that it is possible to detect an emerging virulent strain in a cholera-endemic region. ERIC-PCR constitutes a powerful tool for determination of the virulence potential of V. cholerae O1 strains isolated in surveillance programs and for molecular epidemiological investigations.

Detection of cholera (ctx) and zonula occludens (zot) toxin genes in Vibrio cholerae O1, O139 and non-O1 strains.

Vibrio cholerae O1 and V. cholerae non-O1 strains isolated from environmental samples collected in São Paulo, Brazil, during cholera epidemics and pre-epidemic periods were examined for the presence of toxin genes. V. cholerae O1 strains isolated from clinical samples in Peru and Mexico, and V. cholerae O139 strains from India were also examined for the presence of ctx (cholera toxin gene) and zot (zonula occludens toxin gene) by polymerase chain reaction (PCR). A modified DNA-extraction method applied in this study yielded satisfactory recovery of genomic DNA from vibrios. Results showed that strains of V. cholerae O1 isolated during the preepidemic period were ctx (-)/zot (-) whereas strains isolated during the epidemic were ctx (+)/zot (+). All V. cholerae non-O1 strains tested in the study were ctx (-)/zot (-), whereas all V. cholerae O139 strains were ctx (+)/zot (+). Rapid detection of the virulence genes (ctx and zot) can be achieved by PCR and this can serve as an important tool in the epidemiology and surveillance of V. cholerae.

Potentially pathogenic vibrios associated with mussels from a tropical region on the Atlantic coast of Brazil.

Mussels (Perna perna) harvested on the coast of Ubatuba, in three different stations in the State of São Paulo, Brazil, were examined for Vibrio spp. over a 1 year period. The ranges of most probable number (MPN 100 g-1) were: Vibrio alginolyticus (< 3-24,000), V. parahaemolyticus (< 3-24,000), V. fluvialis (< 3-1100), V. cholerae non-O1 (< 3-23), V. furnissii (< 3-30), V. mimicus (< 3-9) and V. vulnificus (< 3-3). The highest incidence was observed for V. alginolyticus (92-100%), followed by V. parahaemolyticus (67-92%), V. fluvialis (34-67%), V. vulnificus (8-17%), V. furnissii (0-17%), V. mimicus (0-17%) and V. cholerae non-O1 (0-8%). Tests for virulence factors were positive in 34.1% of the vibrios in the rabbit ileal loop and 31.7% in the Dean test. Positive results in the Kanagawa test were obtained with 0.51% of V. parahaemolyticus strains. The mean values (MPN 100 g-1) of faecal coliforms in mussels from the three regions varied from 1100 to 44,000, and seawater collected at the same stations gave average values for faecal coliforms in the range 18-3300 MPN 100 ml-1. These results highlight the potential risks of food poisoning associated with raw or undercooked seafood.

Distribution of uidA gene sequences in Escherichia coli isolates in water sources and comparison with the expression of beta-glucuronidase activity in 4-methylumbelliferyl-beta-D-glucuronide media.

The uidA gene, which encodes the beta-glucuronidase enzyme, was detected in 97.7% of 435 Escherichia coli isolates from treated and raw water sources by DNA-DNA hybridization; 92.4% of the strains expressed the translational product in 4-methylumbelliferyl-beta-D-glucuronide-containing media after reinoculation. Upon initial isolation from water samples, the minimal medium o-nitrophenyl-beta-D-galactopyranoside-4-methylum-belliferyl -beta-D-glucuronide preparations failed to detect more than 50% of the E. coli isolates that possessed uidA gene. Treated water gave the lowest recovery, with Colilert producing 26% positive samples and Coliquik producing 48% positive samples. There appears to be no relationship between the intensity of the autoradiographic signals of the uidA gene and the expression of beta-glucuronidase activity. Therefore, another variable such as physiological condition of the bacteria could be responsible for the nonexpression of the enzyme activity.

Detection of virulence factors in culturable Escherichia coli isolates from water samples by DNA probes and recovery of toxin-bearing strains in minimal o-nitrophenol-beta-D-galactopyranoside-4-methylumbelliferyl-beta-D-g luc uronide media.

A total of 449 Escherichia coli isolates in treated and raw water sources were submitted to DNA-DNA hybridization using seven different DNA probes to detect homology to sequences that code for Shiga-like toxins I and II; heat-stabile and heat-labile toxins, adherence factors EAF and eae, and the fimbrial antigen of entero-hemorrhagic E. coli. Fifty-nine (13%) of the isolates demonstrated homology with one or more specific DNA probes. More than 50% of the isolates in treated water were not recovered in MMO-4-methylumbelliferyl-beta-D-glucuronide media designed for detection of this indicator.