RESUMEN
Multidrug-resistant pathogenic vibrios are a crisis of concern as they cause multiple illnesses, including gastroenteritis in humans and acute hepatopancreatic necrosis in aquaculture. In the current study, we investigated the prevalence of the beta-lactamase gene CTX-M-group 1 in Vibrio spp. (Vibrio cholerae and Vibrio parahaemolyticus) from the water and sediment of urban tropical mangrove ecosystems of Kerala, southwest India. A total of 120 isolates of Vibrio spp. were tested for antibiotic susceptibility to 14 antibiotics. In water, ampicillin resistance was very high in isolates of V. cholerae (94.1%, n = 17) and V. parahaemolyticus (89.1%, n = 46). 26.9% of V. parahaemolyticus and 14.2% of V. cholerae harbored the CTX-M-group 1 gene in water samples. Compared to V. cholerae, the CTX-M-group 1 gene was exclusively hosted by V. parahaemolyticus (49%) in sediment samples. A significant difference in the prevalence of the CTX-M-group 1 gene was observed among Vibrio spp. in both water and sediment samples (p < 0.05). The results revealed the presence of multidrug-resistant and beta-lactamase harboring Vibrio spp. in mangrove ecosystems, which may have evolved as a consequence of the misuse and abuse of broad-spectrum antibiotics as prophylaxis in human health care and aquaculture.
Asunto(s)
Antibacterianos , Pruebas de Sensibilidad Microbiana , Vibrio cholerae , Vibrio parahaemolyticus , beta-Lactamasas , India/epidemiología , Vibrio parahaemolyticus/genética , Vibrio parahaemolyticus/efectos de los fármacos , Vibrio parahaemolyticus/aislamiento & purificación , beta-Lactamasas/genética , beta-Lactamasas/metabolismo , Vibrio cholerae/genética , Vibrio cholerae/efectos de los fármacos , Vibrio cholerae/enzimología , Vibrio cholerae/aislamiento & purificación , Antibacterianos/farmacología , Ecosistema , Humedales , Sedimentos Geológicos/microbiologíaRESUMEN
OBJECTIVE: The genome of Vibrio cholerae has three paralog genes encoding for distinct pyruvate kinases. We were interested in elucidating whether they were expressed, and contributed to the pyruvate kinase activity of V. cholerae. VcIPK and VcIIPK were transformed and expressed in BL21-CodonPlus(DE3)-RIL strain, whereas VcIIIPK could not be transformed. Those studied did contribute to the pyruvate kinase activity of the bacteria. Therefore, our aim was to find an efficient transformation and commonly used over-expression heterologous system for VcIIIPK and develop its purification protocol. RESULTS: vcIpk, vcIIpk and vcIIIpk genes were transformed in six different BL21 expression strains. No transformants were obtained for the vcIIIpk gene using BL21(DE3), BL21(DE3)pLysS and BL21(DE3)CodonPlus-RIL strains. Reduced rates of cell growth were observed for BL21-Gold(DE3)pLysS and Origami B(DE3)pLysS. High efficiency of transformation was obtained for BL21-AI. Using this strain, VcIIIPK was purified but proved to be unstable during its purification and storage. Therefore, the transformation of vcIIIpk gene resulted in a toxic, mildly toxic or nontoxic product for these BL21 strains. Despite VcIIPK and VcIIIPK being phylogenetically related, the preservation of the proteins is drastically different; whereas one is preserved during purification and storage, the other is auto-proteolyzed completely in less than a week.
Asunto(s)
Expresión Génica , Piruvato Quinasa/metabolismo , Vibrio cholerae/enzimología , Genes Bacterianos , IsoenzimasRESUMEN
Biomimetic particles supporting lipid bilayers are becoming increasingly important to isolate and reconstitute protein function. Cholera toxin (CT) from Vibrio cholerae, an 87-kDa AB5 hexameric protein, and its receptor, the monosialoganglioside GM1, a cell membrane glycolipid, self-assembled on phosphatidylcholine (PC) bilayer-covered silica particles at 1 CT/5 GM1 molar ratio in perfect agreement with literature. This receptor-ligand recognition represented a proof-of-concept that receptors in general can be isolated and their function reconstituted using biomimetic particles, i.e., bilayer-covered silica. After incubation of colloidal silica with small unilamellar PC vesicles in saline solution, pH 7.4, PC adsorption isotherms on silica from inorganic phosphorus analysis showed a high PC affinity for silica with maximal PC adsorption at bilayer deposition. At 0.3 mM PC, fluorescence of pyrene-labeled GM(1) showed that GM(1) incorporation in biomimetic particles increased as a function of particles concentration. At 1 mg/mL silica, receptor incorporation increased to a maximum of 40% at 0.2-0.3 mM PC and then decreased as a function of PC concentration. At 5 microM GM(1), 0.3 mM PC, and 1 mg/mL silica, CT binding increased as a function of CT concentration with a plateau at 2 mg bound CT/m2 silica, which corresponded to the 5 GM(1)/1 CT molar proportion and showed successful reconstitution of receptor-ligand interaction.
Asunto(s)
Toxina del Cólera/química , Gangliósido G(M1)/química , Membrana Dobles de Lípidos/química , Fosfolípidos/química , Receptores de Superficie Celular/fisiología , Adsorción , Animales , Biomimética/métodos , Bovinos , Fenómenos Químicos , Química Física , Calor , Control de Infecciones , Liposomas/química , Lípidos de la Membrana , Tamaño de la Partícula , Fosfatidilcolinas/química , Pirenos/química , Receptores de Superficie Celular/aislamiento & purificación , Dióxido de Silicio/química , Vibrio cholerae/enzimologíaRESUMEN
The neuraminidase gene, nanH, is present in the O1, non-toxigenic Vibrio cholerae Amazonia strain. Its location has been assigned to a 150 kb NotI DNA fragment, with the use of pulsed-field gel electrophoresis and DNA hybridization. This NotI fragment is positioned inside 630 kb SfiI and 1900 kb I-CeuI fragments of chromosome 1. Association of the pathogenicity island VPI-2, carrying nanH and other genes, with toxigenic strains has been described by other authors. The presence of nanH in a non-toxigenic strain is an exception to this rule. The Amazonia strain nanH was sequenced (Genbank accession No. AY825932) and compared to available V. cholerae sequences. The sequence is different from those of pandemic strains, with 72 nucleotide substitutions. This is the first description of an O1 strain with a different nanH allele. The most variable domain of the Amazonia NanH is the second lectin wing, comprising 13 out of 17 amino acid substitutions. Based on the presence of nanH in the same region of the genome, and similarity of the adjacent sequences to VPI-2 sequences, it is proposed that the pathogenicity island VPI-2 is present in this strain.
Asunto(s)
Alelos , Neuraminidasa/genética , Vibrio cholerae/enzimología , Secuencia de Bases , Mapeo Cromosómico , Electroforesis en Gel de Campo Pulsado , Datos de Secuencia Molecular , Reacción en Cadena de la Polimerasa , Análisis de Secuencia de ADN , Vibrio cholerae/genéticaRESUMEN
The neuraminidase gene, nanH, is present in the O1, non-toxigenic Vibrio cholerae Amazonia strain. Its location has been assigned to a 150 kb NotI DNA fragment, with the use of pulsed-field gel electrophoresis and DNA hybridization. This NotI fragment is positioned inside 630 kb SfiI and 1900 kb I-CeuI fragments of chromosome 1. Association of the pathogenicity island VPI-2, carrying nanH and other genes, with toxigenic strains has been described by other authors. The presence of nanH in a non-toxigenic strain is an exception to this rule. The Amazonia strain nanH was sequenced (Genbank accession No. AY825932) and compared to available V. cholerae sequences. The sequence is different from those of pandemic strains, with 72 nucleotide substitutions. This is the first description of an O1 strain with a different nanH allele. The most variable domain of the Amazonia NanH is the second lectin wing, comprising 13 out of 17 amino acid substitutions. Based on the presence of nanH in the same region of the genome, and similarity of the adjacent sequences to VPI-2 sequences, it is proposed that the pathogenicity island VPI-2 is present in this strain.
Asunto(s)
Alelos , Neuraminidasa/genética , Vibrio cholerae/enzimología , Secuencia de Bases , Mapeo Cromosómico , Electroforesis en Gel de Campo Pulsado , Datos de Secuencia Molecular , Reacción en Cadena de la Polimerasa , Análisis de Secuencia de ADN , Vibrio cholerae/genéticaRESUMEN
Vibrio cholerae is an important human pathogen and the cause of cholera. Since genetic variation and antibiotic resistance of strains have implications for effective treatment of the disease, we examined the genetic diversity and antibiotic resistance profile in 92 clinical strains (serogroup O1) and 56 environmental strains (O1 antigen, 42 strains; non-O1 antigen, 14 strains) isolated in Brazil between 1991 and 1999. Clinical and environmental O1 strains showed greater drug resistance compared to environmental non-O1 strains. Nearly all clinical O1 strains were resistant to one or more antibiotics while half of the environmental O1 and non-O1 strains were resistant to one or more antibiotics. No plasmids or class 1 integrons were detected in the strains by PCR analysis. Multilocus enzyme electrophoresis analysis (MLEE) suggests most of the O1 strains belong to a single (South American) clone that is related but different to seventh-pandemic strains isolated from other parts of the world. Our results show that there is a close genetic relationship between clinical and environmental O1 strains and that many serogroups and the environment can be a reservoir for antibiotic resistance.
Asunto(s)
Antiinfecciosos/farmacología , Farmacorresistencia Bacteriana/genética , Vibrio cholerae/efectos de los fármacos , Vibrio cholerae/genética , Antiinfecciosos/uso terapéutico , Brasil/epidemiología , Cólera/tratamiento farmacológico , Cólera/epidemiología , Cólera/microbiología , Cartilla de ADN , ADN Bacteriano/análisis , Reservorios de Enfermedades , Electroforesis , Humanos , Reacción en Cadena de la Polimerasa , Vibrio cholerae/enzimologíaRESUMEN
The gene bla(CARB-9) was located in the Vibrio cholerae super-integron, but in a different location relative to bla(CARB-7). CARB-9 (pI 5.2) conferred beta-lactam MICs four to eight times lower than those conferred by CARB-7, differing at Ambler's positions V97I, L124F, and T228K. Comparison of the genetic environments of all reported bla(CARB) genes indicated that the CARB enzymes constitute a family of cassette-encoded beta-lactamases.
Asunto(s)
Penicilinasa/genética , Vibrio cholerae/enzimología , Vibrio cholerae/genética , beta-Lactamasas/genética , Secuencia de Aminoácidos , Argentina , Datos de Secuencia Molecular , Penicilinasa/clasificación , Secuencias Repetitivas de Aminoácido , beta-Lactamasas/clasificación , beta-Lactamasas/metabolismoRESUMEN
Since 1992 there have been seven major outbreaks of cholera in Argentina. Susceptibility analysis of 1,947 isolates (40% of reported cases) of Vibrio cholerae O1 biotype El Tor suggested the presence of extended-spectrum beta-lactamases (ESBLs) in 28 isolates. Because of their different susceptibility profiles, V. cholerae isolates M1502, M1516, M1573, and M3030 (all of which are of the Ogawa serotype) were selected for the present study. By susceptibility analysis, isoelectric focusing, and PCR-based restriction fragment length polymorphism analysis, CTX-M-type enzymes were identified in three isolates, whereas a PER-2-type enzyme, in addition to a TEM-1-like enzyme, was identified in the other isolate. The presence of these ESBLs in V. cholerae isolates resulted in MICs well below those commonly observed for members of the family ENTEROBACTERIACEAE: Genes that encode both ESBLs were transferred to Escherichia coli by conjugation, together with all determinants of resistance to non-beta-lactam antibiotics (gentamicin, kanamycin, and sulfamethoxazole for all isolates; amikacin and streptomycin for three isolates; trimethoprim, tetracycline, and chloramphenicol for two isolates). Plasmid profile analysis and Southern blotting revealed the presence of single plasmids of about 150 kb in the four V. cholerae isolates and their respective transconjugants and revealed that the plasmids harbored genes encoding CTX-M-type or PER-2-type ESBLs. These results strongly suggest the broad spread of these ESBLs among genera belong to families other than the ENTEROBACTERIACEAE:
Asunto(s)
Cólera/epidemiología , Brotes de Enfermedades , Plásmidos/genética , Vibrio cholerae/enzimología , beta-Lactamasas/genética , Adulto , Argentina/epidemiología , Cólera/microbiología , Conjugación Genética , Farmacorresistencia Bacteriana/genética , Femenino , Humanos , Lactante , Recién Nacido , Focalización Isoeléctrica , Masculino , Pruebas de Sensibilidad Microbiana , Reacción en Cadena de la Polimerasa , Polimorfismo de Longitud del Fragmento de Restricción , Vibrio cholerae/genética , Vibrio cholerae/aislamiento & purificación , beta-Lactamas/farmacologíaRESUMEN
A survey of pathogenic Vibrio cholerae O1 strains from the north of Brazil by using arbitrarily primed PCR fingerprints revealed a group of strains with similar fingerprint patterns that are distinct from those of the current El Tor epidemic strain. These strains have been analyzed by in vivo and in vitro techniques and the group has been denominated the Amazonian variant of V. cholerae O1.
Asunto(s)
Dermatoglifia del ADN/métodos , Variación Genética , Reacción en Cadena de la Polimerasa/métodos , Vibrio cholerae/clasificación , Animales , Técnicas de Tipificación Bacteriana , Secuencia de Bases , Brasil/epidemiología , Cólera/epidemiología , Cólera/microbiología , Toxina del Cólera/genética , ADN Ribosómico/genética , Diarrea/microbiología , Humanos , Datos de Secuencia Molecular , Antígenos O , Polisacáridos Bacterianos , Conejos , Vibrio cholerae/enzimología , Vibrio cholerae/genética , Vibrio cholerae/aislamiento & purificación , Virulencia/genéticaRESUMEN
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.