Carlos André Salles

Identification of Vibrio cholerae by enzyme electrophoresis

Zymovar analysis of 260 strains of Vibrio cholerae plus 3 reference strains of V. mimicus, using 13 structural loci, led to the grouping of strains in 73 zymovars (strain or group of strains sharing the same alleles). Effective separation of strains, distinction of V. cholerae strains from closely related V. mimicus and the detection of 2 vibrio strains, including one with two O1 serovars, in supposedly pure collection cultures, illustrate the potential of zymovar analysis in the identification of V. cholerae isolates. Two El Tor strains from USA, one CT+ and the other CT-, shared the same zymovar 71, while 127 typical El Tor strains belonged to zymovar 14.

Enzyme markers for Vibrio cholerae: identification of classical, El Tor and environmental strains.

Enzyme electrophoretic variants were studied in 49 strains of Vibrio cholerae using zymovar analysis. The following seven enzymes were selected for use: alanine dehydrogenase (ADH), isocitrate dehydrogenase (IDH), malate dehydrogenase (MDH), phosphoglucomutase (PGM), glucosephosphate isomerase (GPI), 6-phosphogluconate dehydrogenase (6PGDH) and glucose-6-phosphate dehydrogenase (G6PDH). The results indicated the presence of three main groups defined chiefly by their GPI and 6PGDH variants. The first group, defined by possessing the variants GPI-2 and 6PGDH-3, contained all the 01 serovar and E1T or biovar isolates from cholera cases. The second group, defined by possessing the variants GPI-3 and 6PGDH-2, contained all the 01 serovar and classical biovar isolates; the third group was heterogeneous and included the 01 serovar isolates from environmental sources as well as isolates of other serovars (the so called NAGs, non-agglutinable with 01 antisera or NCVs). It is thus now possible to separate the epidemic strains of 01 serovar from other members of this serovar isolated from the environment. Zymovar analysis deals with differences which are a direct expression of the genome and seems to be unaffected by gross phenotypic changes such as smooth-rough variation and phage resistance. It is a promising tool for investigating bacteriological and epidemiological questions, in particular the significance of an environmental reservoir of cholera.

Differentiation of environmental and clinical isolates of Vibrio mimicus from Vibrio cholerae by multilocus enzyme electrophoresis.

ABSTRACT In this study, we demonstrated that analyzed strains ofVibrio mimicus and Vibrio cholerae could be separated in two groups by using multilocus enzyme electrophoresis (MEE) data from 14 loci. We also showed that the combination of four enzymatic loci enables us to differentiate these two species. Our results showed that the ribosomal intergenic spacer regions PCR-mediated identification system failed, in some cases, to differentiate between V. mimicus and V. cholerae. On the other hand, MEE proved to be a powerful molecular tool for the discrimination of these two species even when atypical strains were analyzed.

Detection of Vibrio cholerae and V. mimicus heat- stable toxin gene sequence by PCR

Previously the heat-stable enterotoxin in Vibrio cholerae and V. mimicus has been detected by suckling mouse assay, a non-specific approach, and by DNA probes, a time-consuming method. This report describes a polymerase chain reaction (PCR) procedure for the detection of the stn (NAG-ST) and sto (O1-ST) gene sequences that is rapid and specific, allowing toxin gene molecular characterisation. A total of 34 V. cholerae and V. mimicus isolates was examined for ST and CT genes. The NAG-ST gene sequence was amplified in 13 of 22 non-O1/non-O139 V. cholerae and three of five V. mimicus strains. A new enterotoxin gene sequence pattern was found with MseI and TaqI restriction endonuclease PCR fragment digestion of two V. cholerae isolates, in addition to the pattern anticipated from the Genbank sequence, and found with the other ST+. These results show that ST-PCR detection is useful for the characterisation of V. cholerae and V. mimicus.

The distinction of pathogenic Vibrio cholerae groups using arbitrarily primed PCR fingerprints.

Pathogenic Vibrio cholerae strains were compared by fingerprinting with arbitrarily primed polymerase chain reaction (AP-PCR). They were O1 classical and El Tor strains and recent non-O1 Bengal strains. Ten oligonucleotides from a total of fifty-two tested gave distinctive patterns, and these strains were separated into four groups. A second technique, amplification of 16S/23S rRNA spacers with a pair of oligonucleotides, was also used. Various bands were obtained, and the result can be treated as an additional fingerprint with a different pattern for each of the groups. The method of AP-PCR fingerprinting is fast and sensitive. A test of the stability of the El Tor patterns was done with a set of strains isolated during the present Brazilian epidemics. Examples of AP-PCRs with non-O1 strains are given. A typing scheme is proposed in which oligo 1 is first used, and depending on the fingerprint obtained, additional oligonucleotides are used to confirm the classification of the strain. It is proposed that the AP-PCR technique be used for epidemiological studies, analysing strains reaching new locations or environmental isolates suspected of being pathogenic. It will be particularly helpful in cases in which traditional methods cannot clearly classify the strain.

The Zymovars of Vibrio cholerae: Multilocus Enzyme Electrophoresis of Vibrio cholerae

Zymovars analysis also known as multilocus enzyme electrophoresis is applied here to investigate the genetic variation of Vibrio cholerae strains and characterise strains or group of strains of medical and epidemiological interest. Fourteen loci were analyzed in 171 strains of non-O1 non-O139, 32 classical and 61 El Tor from America, Africa, Europe and Asia. The mean genetic diversity was 0.339. It is shown that the same O antigen (both O1 and non-O1) may be present in several genetically diverse (different zymovars) strains. Conversely the same zymovar may contain more than one serogroup. It is confirmed that the South American epidemic strain differs from the 7th pandemic El Tor strain in locus LAP (leucyl leucyl aminopeptidase). Here it is shown that this rare allele is present in 1 V. mimicus and 4 non-O1 V. cholerae. Non toxigenic O1 strains from South India epidemic share zymovar 14A with the epidemic El Tor from the 7th pandemic, while another group have diverse zymovars. The sucrose negative epidemic strains isolated in French Guiana and Brazil have the same zymovar of the current American epidemic V. cholerae.

The Amazonia Variant of Vibrio cholerae: Molecular Identification and Study of Virulence Genes

The pathogenic O1 Amazonia variant of Vibrio cholerae has been shown previously to have a cytotoxin acting on cultured Vero and Y-1 cells, and to lack important virulence factors such as the cholera toxin (Coelho et al. 1995a). This study extends the molecular analysis of the Amazonia strains, detecting the presence of the toxR gene, with a very similar sequence to that of the E1 Tor and classical biotypes. The outer membrane proteins are analyzed, detecting a variation among the group of Amazonia strains, with three different patterns found. As a by-product of this work a polymerase chain reaction fragment was sequenced, reading part of the sequence of the Lon protease of the Amazonia strains. This gene was not previously described in V. cholerae, but its sequence is present in the TIGR database specific for this species.