Masahiko Ehara

Antibiotic resistance conferred by a class I integron and SXT constin in Vibrio cholerae O1 strains isolated in Laos

ABSTRACT Changes in the drug susceptibility pattern were observed in Vibrio cholerae O1 isolated in the Lao Peoples Democratic Republic during 1993 to 2000. In this study, 50 V. cholerae O1 strains were selected during this period for studying the presence of class I integron and SXT constin. Twenty-four streptomycin-resistant strains out of 26 isolated before 1997 contained a class I integron harboring the aadA1 gene cassette. Twenty-four strains isolated after 1997 contained an SXT constin (a large conjugative element). Twenty of the strains were resistant to chloramphenicol, tetracycline, streptomycin, and trimethoprim-sulfamethoxazole, while four strains were susceptible to the antibiotic tested. The resistance genes included in the SXT constins were floR, tetA, strAB, and sulII, which encode resistance to chloramphenicol, tetracycline, streptomycin, and sulfamethoxazole, respectively. The antibiotic resistance gene cluster was found to be deleted in the four susceptible strains. SXTLAOS did not contain dfrA1 or dfr18, which confer resistance to trimethoprim in SXTET and SXTMO10, respectively. A hot spot region of SXTLAOS was sequenced, and we identified two novel open reading frames showing homology to sO24 (exonuclease) and sO23 (helicase) of the genomic island associated with the multidrug resistance region of Salmonella enterica serovar Typhimurium DT104. Analysis of SXTLAOS showed that there is a continuous flux of genes among V. cholerae SXT constins which should be carefully monitored.

Drug susceptibility and its genetic basis in epidemic Vibrio cholerae O1 in Vietnam.

The drug susceptibility and genes responsible for the drug resistance of Vibrio cholerae O1 isolated in Vietnam in 1995, 2000 and 2002 were studied. The strains isolated in 1995 were resistant to streptomycin and harboured the class I integron which contained the aadA1 gene responsible for streptomycin resistance. The strains isolated in 2000 were devoid of a class I integron but were multiple-drug resistant and harboured SXT constin, with several drug-resistant genes. The genes responsible for streptomycin resistance were strA and strB. The strains isolated in 2002 were sensitive to all drugs examined, and the organisms were devoid of both class I integron and SXT constin. Cholera outbreaks in the three periods examined (1995, 2000 and 2002) were apparently due to different categories of V. cholerae O1.

MOLECULAR ANALYSIS OF A FILAMENTOUS PHAGE (FSL) OF VIBRIO CHOLERAE O139

A filamentous bacteriophage from Vibrio cholerae O139 strain A1-4450 was isolated (fsl). The phage fsl had a ssDNA genome and dsDNA as a replicative form (RF) in lysogenic host cell. The DNA sequence of fsl RF was determined. It consisted of 6340 bp and had a G + C content of 43.5%. Fifteen possible ORFs were found in fsl. One of them, ORF384, was estimated to encode 384 amino acid residues (44.6 kDa) and had homologous regions with the zot gene of V. cholerae and gene I of the coliphage group. ORF104, located upstream of ORF384, was homologous to gene 93 protein of Pf3 (filamentous phage of Pseudomonas sp.) corresponding to gene VI of coliphage. Other than ORF384 and ORF104, the ORF81, ORF44, ORF29, and ORF193 were speculated to correspond to gene V, gene VII, gene IX, and gene III, respectively, in the order as reported on f1 phage.

Filamentous vibriophage fs2 encoding the rstC gene integrates into the same chromosomal region as the CTX phage [corrected].

The genome of the filamentous phage of Vibrio cholerae fs2 was found to contain rstC and rstB1 (truncated) genes downstream of ORF500. att-fs2-dir and att-fs2-rev sequences homologous to that of att-CTXphi were found between orf500 and rstC of the fs2 genome. This prompted us to search for the integration site of fs2 in the genomes of V. cholerae O1 and O139. The genome of fs2 was found to integrate downstream of attRS of the CTXphi phage, which integrated into chromosome I of V. cholerae O1 and O139. When infected with fs2, a fimbriate strain of V. cholerae O1 appeared to reduce fimbrial production in an adult rabbit ileal loop assay.

The protease from Vibrio cholerae nicks arginine at position 192 from the N-terminus of the heat-labile enterotoxin a subunit from enterotoxigenic Escherichia coli

It was examined where a protease purified from Vibrio cholerae might nick the heat-labile enterotoxin (LT) A subunit from enterotoxigenicEscherichia coli.LT was digested by the protease and contained a fragment which had the same mobility on SDS-PAGE as that of the Al fragment of LT digested by trypsin. The biological activity of LT by this protease was also identical to that of LT by trypsin. The amino acid sequence of the N-terminus of the A2-like fragment was Thr-Ser-Thr-Gly, which corresponded to the sequence from 193 to 196 of the A subunit.These data suggest that this protease, like trypsin, nicks arginine at position 192 from the N-terminus of the A subunit and that the biological activation of LT by this protease is similar to that by trypsin.

Pili of Vibrio cholerae O1 Biotype El Tor

Pili were found on the cell surface of non‐adhesive Vibrio cholerae O1 Biotype El Tor as well as the adhesive strain. Purified pili of the adhesive and non‐adhesive strains were morphologically, electrophoretically, and immunologically, indistinguishable from each other. The molecular weights of both pilin (subunit protein of the pilus) were about 16,000 daltons as determined by sodium dodecyl sulfate‐polyacrylamide gel electrophoresis. These 16 kDa pili are different from the pilus colonization factor, which is a 20.5 kDa protein, reported by Taylor et al. The 16 kDa pili of Vibrio cholerae O1 Biotype El Tor have hemagglutinating activity, but may have no role in colonization, because non‐adhesive strains also have such pili.

Morphological features of a filamentous phage of Vibrio cholerae O139 Bengal.

A filamentous phage was isolated from carrier strain AI‐1841 of Vibrio cholerae O139 Bengal and thus was termed fs phage. The phage was measured to be approximately 1 μm in length and 6 nm in width. One end of the phage was slightly tapered and had a fibrous appendage. The plaques developed on strain AI‐4450 of V. cholerae O139 were small and turbid. The phage grew in strain AI‐4450 and reached a size of 108 to 109 pfu/ml at 5 hr after infection without inducing any lysis of the host bacteria. The group of phages attached on rod‐shaped materials like fimbriae of this bacteria, with their fibrous appendages at the pointed end, were often found in the phage‐infected culture. The anti‐fimbrial serum effectively inhibited the infection of fs phage to the host strain AI‐4450. We thus concluded that the phage can be adsorbed on fimbriae with a fibrous appendage on the pointed end of the phage filament.

Immunogenicity of Vibrio cholerae O1 Fimbriae in Animal and Human Cholera

Parenteral immunization with either formalin‐fixed whole cells of the fimbriate Bgd17 strain or purified fimbriae protected against Vibrio cholerae O1 infection in rabbits, independent of biotype and serotype. Parenteral immunization of adult rabbits with purified fimbriae prior to V. cholerae O1 challenge resulted in a reduction of 2 to 3 orders of magnitude in the number of bacteria recovered from the small intestines of immunized rabbits in comparison to non‐immunized controls. IgG and IgA antibodies against fimbrillin of V. cholerae O1 were detected in the convalescent sera of patients with cholera; however, little fimbrial antigen was detected in the commercially available cholera vaccines when examined by polyclonal and monoclonal antibodies against fimbriae. These data suggest that fimbrial hemagglutinin is a major adhesin of V. cholerae O1 and that parenteral immunization with fimbriae generates a specific immune response in the gut that may serve as one means of mitigating subsequent V. cholerae O1 gut infection.

Studies on Novel Pili from Shigella flexneri

Pili were detected using electron microscopy in clinical isolates of Shigella flexneri which had been continuously subcultivated in liquid media. Morphologically, the pili appeared as thin, flexible, cylindrical structures of up to 2–5 μm in length and about 3–5 nm in diameter. Two strains showed mannose‐resistant (MR) hemagglutination to fresh fowl erythrocytes (type 4), and one to tannic acid‐treated horse erythrocyte (type 3) pili. These pili are novel and different from the mannose‐sensitive (MS) type 1 pili described by Duguid and Gillies.

Imported Dogs as Possible Vehicles of Vibrio Cholerae O1 Causing Cholera Outbreaks in Northern Vietnam

Strains of V. cholerae O1 were isolated from the sewage and a pond near the first patients house and also from domestic vegetables obtained at a neighboring market. From 24 October 2007 to 25 June 2009, 1,505 cases were confirmed positive for V. cholerae O1 (biotype El Tor, serotype Ogawa) in 22 cities and provinces in northern Vietnam. On May 8 and May 12, 2009, epidemic strains of V. cholerae O1 were isolated from dogs in slaughter houses in Hanoi and from dogs in cages in Thanh Hoa, respectively. Isolates of V. cholerae O1 in Laos and Thailand were found to be the same clone as those isolates from dogs, patients and environmental water samples in northern Vietnam. Although the transmission routes of cholera differed between the northern and southern provinces of Vietnam, the same clonality was observed among isolates from 2007 to 2010.