Molecular characteristics and drug resistance of non-O1non-O139 Vibrio cholerae in Zhongshan City, Guangdong Province / 中国热带医学

@#Abstract: Objective　To investigate the molecular characteristics and drug resistance of non-O1/non-O139 Vibrio cholerae in Zhongshan City, and to provide laboratory basis for cholera prevention and control. Methods　The strains of non-O1/non-O139 Vibrio cholerae isolated from sporadic patients and aquatic products from 2015 to 2021 in Zhongshan city were collected. The identification and cluster analysis of the strains were analyzed by matrix assisted laser desorption/ionization time of flight mass spectrometry (MALDI-TOF-MS), the ctxA virulence gene of strains were detected by real-time fluorescence quantitative PCR, the cluster analysis of the strains was analyzed by pulsed-field gel electrophoresis (PFGE), and the drug resistance of the strains were analyzed by microbroth dilution method. Results　From 2015 to 2021, 33 strains of non-O1/non-O139 Vibrio cholerae were isolated from Zhongshan City, including 28 strains from sporadic patients and 5 strains from aquatic products. Through MALDI-TOF-MS identification, 33 strains of non-O1/non-O139 Vibrio cholera can be identified to the level of species, and the identification results were all Vibrio cholerae. Among 33 non-O1/non-O139 Vibrio cholerae strains, 1 strain carried the ctxA virulence gene. The drug-resistant strains accounted for 69.7% (23/33), and the multidrug resistant strains accounted for 18.2% (6/33). A total of 7 kinds of drug resistance spectrum were produced, including 3 kinds of multidrug resistant spectrum, and showed drug resistance to 8 antibiotics, among which the resistance rates to streptomycin, cefazolin and compound sulfamethoxazole were above 30%. The 33 strains of non-O1/non-O139 Vibrio cholerae were divided into 32 PFGE fingerprints with a similarity ranging from 61.7% to 100%. MALDI-TOF-MS cluster analysis divided 33 non-O1/non-O139 Vibrio cholerae strains into two clusters. Conclusions The results of molecular typing of non-O1/non-O139 Vibrio cholerae in Zhongshan City presented diversity, and no significant correlation was found between PFGE and MALDI-TOF-MS cluster analysis. The strains demonstrated various degrees of resistance to certain antibiotics, and there were multidrug-resistant and toxigenic strains. Therefore, it is necessary to alert to the harmfulness of non-O1/non-O139 Vibrio cholerae and enhance monitoring.

A rare and unusual cause of Vibrio cholerae non-O1, non-O139 causing spontaneous peritonitis in a patient with cirrhosis

@#Spontaneous bacterial peritonitis caused by Vibrio cholerae non-O1/ non-O139 is a rare phenomenon. V. cholerae is known as a common aetiology of epidemic diarrheal disease and rarely causes extra-gastrointestinal infections. In this report, a 52-year-old man presented to our hospital with a clinical scenario for chronic liver cirrhosis with low grade fever and loose stools. V. cholerae was isolated from peritoneal fluid culture, which was further confirmed as non-O1/ non-O139 strain by multiplex polymerase chain reaction. The patient was successfully treated with antimicrobial therapy and peritoneal drainage. This case represents the first isolation of V. cholerae non-O1/ non-O139 strain from peritoneal fluid.

Determinación de biopelículas y betalactamasas de espectro extendido en Vibrio cholerae no-O1, no-O139 aislados de pacientes con diarreas en Cuba / Determination of biofilms and extended-spectrum beta-lactamases in Vibrio cholerae non-O1, non-O139 isolates from patients with diarrhea in Cuba

. Vibrio cholerae no-O1, no-O139 es el tercer grupo de bacterias del género Vibrio que con más frecuencia producen diarreas. Sobrevive en los ambientes acuáticos, utilizando la formación de biopelícula como mecanismo de supervivencia que propicia la transmisión de la enfermedad diarreica. Desde 1977 se caracterizan aislados de V. cholerae con resistencia múltiple, y algunos de los mecanismos involucrados incluyen la producción de β-lactamasas de espectro extendido (BLEE). Este trabajo tuvo como objetivo determinar la formación de biopelícula en los aislados cubanos de V. cholerae no-O1, no-O139, causantes de enfermedad diarreica aguda (EDA), y detectar la producción de BLEE en aquellos con resistencia total e intermedia a ampicilina. Se realizó un estudio descriptivo de corte transversal, entre enero 2014 y junio 2015. Se estudiaron 55 aislados caracterizados previamente, que formaban parte del cepario del Laboratorio Nacional de Referencia de EDA del Instituto Pedro Kourí. Para la determinación fenotípica de BLEE se estudiaron 43, de los que ya se conocía su susceptibilidad a ampicilina. El 54,5 por ciento de los aislados resultaron positivos a la formación de biopelícula, y predominaron los clasificados como formadores moderados (46,6 por ciento ) y débiles (36,6 por ciento ). De los 34 resistentes a ampicilina, 26,5 por ciento resultaron positivos a la producción de BLEE. En el caso de los nueve aislados con resistencia intermedia a ampicilina, 44,4 por ciento resultaron positivos. Los resultados del presente estudio contribuyen al conocimiento sobre la capacidad que tienen de persistir en el ambiente y permiten profundizar sobre los mecanismos de resistencia a los antimicrobianos(AU)

Vibrio cholerae non-O1, non-O139 is the third bacterium group from the genus Vibrio most commonly causing diarrhea. It survives in aquatic environments, using the formation of biofilm as a survival mechanism facilitating the transmission of diarrheal disease. Multi-drug resistant V. cholerae isolates have been characterized since the year 1977, and some of the mechanisms involved include the production of extended-spectrum β-lactamases (ESBLs). The purpose of the study was to determine the formation of biofilm in Cuban isolates of V. cholerae non-O1, non-O139 causing acute diarrheal disease (ADD), and detect the production of ESBLs in those with total or intermediate resistance to ampicillin. A descriptive cross-sectional study was conducted from January 2014 to June 2015. The study sample was 55 previously characterized isolates obtained from the strain collection at the ADD National Reference Laboratory of Pedro Kourí Institute. For phenotypic determination of ESBLs, 43 were studied which were known to be susceptible to ampicillin. 54.5 percent of the isolates tested positive for biofilm formation, with a predominance of those classified as moderate (46.6 percent) and weak (36.6 percent) biofilm producers. Of the 34 isolates resistant to ampicillin, 26.5 percent were positive for ESBL production. Of the 9 with intermediate ampicillin resistance, 44.4 % were positive. The results of the present study contribute knowledge about their ability to persist in the environment, and provide insight into antimicrobial resistance mechanisms(AU)

Bacteriemia por Vibrio cholerae no-O1/no-O139 que porta una región homóloga a la isla de patogenicidad VpaI-7 / Bacteremia caused by Vibrio cholerae non-O1/non-O139 carrying a region homologous to pathogenicity island VpaI-7

Resumen Presentamos un caso de bacteriemia por Vibrio cholerae no-O1/ no-O139 en una mujer de 81 años con un cuadro de dolor abdominal, fiebre, vómitos, diarrea, coluria e ictericia, mientras visitaba una zona rural sin acceso a agua potable. La identificación se realizó por la técnica de espectrometría de masa MALDI-TOF, confirmándose una cepa no toxigénica no-O1/no-139. La caracterización molecular del aislado demostró la ausencia del gen de la toxina del cólera (CTX), y pilus TCP; sin embargo, presentó cinco de los seis genes de virulencia presentes en la isla de patogenicidad homóloga denominada VPaI-7 del V. parahaemolyticus (vcs N2+, vcs C2+, vcs V2+,toxR-, vspD+, T vopF+). Además, el aislado presentó los genes de virulencia hylA y rtxA. Este es el primer caso reportado en Chile de una cepa clínica de V. cholerae no-O1, no-O139 aislada de hemocultivos portador de un segmento homólogo de la isla de patogenicidad denominada VPaI-7 de V. parahaemolyticus, el cual codifica para un sistema de secreción tipo III (TTSS), que probablemente contribuye a su virulencia.

We report a case of V. cholerae non-O1 / non-O139 bacteremia in an 81-year-old woman with abdominal pain, fever, vomiting, liquid stools, choluria and jaundice, while visiting a rural area without access to potable water. The identification was made by the MALDI-TOF mass spectrometry technique and subsequently the non-toxigenic non-O1 / non-139 strain was confirmed in the national reference laboratory. The molecular characterization demonstrated the absence of the cholera toxin gene (CTX), and the TCP pilus, however, presented 5 of 6 virulence genes present in an island of homologous pathogenicity named VPaI-7 of V. parahaemolyticus (vcs N2 +, vcs C2 +, vcs V2 +, toxR-, vspD +, T vopF +) and in addition it was positive for hylAy rtxA virulence genes recognized outside the island. This is the first case reported in Chile of a clinical strain of V. cholerae non-O1, non-O139 isolated from blood culture that carries in its genome a homologous segment of the pathogenicity island named VPaI-7 of V. parahaemolyticus, which codifies for a type III secretion system (TTSS) that probably contributes to his virulence.

Analysis on the main virulence genes and molecular typing of non-O1/non-O139 Vibio cholerae in ;bloodstream infection / 中华传染病杂志

Objective To analyze the virulence genes and molecular typing of non-O1/non-O139 Vibio cholerae in bloodstream infection,and to provide the scientific basis for its diagnosis,treatment, prevention and controls.Methods Five Vibio cholerae strains were obtained from blood samples of five inpatients with sepsis in Ruian People ’s Hospital from 2012 to 2015 . Morphological examination, biochemical identification,drug sensitivity test and multilocus sequence typing (MLST)classification analysis of strains were conducted.Totally 17 virulence genes were detected by PCR amplification.Results These five suspected Vibrio cholerae isolates were confirmed as non-O1/non-O139 Vibrio cholerae . Drug susceptibility test showed that all the strains were sensitive to tetracycline,ciprofloxacin,piperacillin and tazobactam, meropenem, amikacin and gentamicin; one strain was resistant to trimethoprim/sulfamethoxazole;all were resistant to ampicillin.MLST analysis showed that all strains were new sequence types (ST),belonging to ST268,ST269,ST267,ST270 and ST271 ,and two novel alleles of RY03(mdh:60 and pyrC:86)were discovered.Virulence genes testing showed that the five strains were divided into 4 virulence genotypes:RY02 and RY04 (hlyA + toxR + hap + rtxA + nanH + vasH + vasA +vasK + ),RY01 (hlyA +toxR +hap +rtxA +nanH +vasH -vasA +vasK - ),RY03 (hlyA +toxR +hap +rtxA +nanH - vasH + vasA + vasK + ) and RY05 (hlyA + toxR + hap + rtxA + nanH + vasH - vasA - vasK - ). Conclusions Non-O1/non-O139 Vibrio cholerae can cause human bloodstream infection in immunocompromised patients.The pathogenic factors may be related to the virulence genes of hlyA, toxR,hap ,rtxA and T6SS.

Molecular analysis on non-O1 and non-O139 Vibrio cholerae isolates / 中华流行病学杂志

Objective According to results from the two-month consecutive surveillance program in Maanshan,six suspected cases of non-O1 non-O139 Vibrio (V.) cholerae infection,were found that called for identification of pathogens as well as molecular-epidemiological analysis to determine the aggregation of the epidemic situation.Methods Biochemical and serotype identification,hemolysis test,and drug sensitive test were used to detect the drug resistance spectrum.Real-time PCR and conventional PCR were used to detect the presence of V.cholerae specific genes,virulent genes and its related genes,including ompW,ctx,tcpA,toxR,hlyA,zot,ace,rstR and g ⅢCTX.Pulsed-field gel electrophoresis (PFGE) was used to analyze the molecular type of strains.Results All the six isolates of non-O 1 non-O 139 V.cholerae were identified by biochemical and serologic tests,and appeared to be β hemolytic.Twelve out of the 14 kinds of drugs showed 100％ sensitive.All isolates were positive of ompW gene by real-time PCR,but negative for ctx,tcpA,zot,ace,rstR and gⅢ CTK.Five of the six isolates were positive for toxR and hlyA,except for strain 1001434446.All strains had different PFGE types,but two strains had similar types.All strains had a low similarity compared to the toxigenic V.cholerae.Conclusion Six cases ofnon-O1 and non-O139 nontoxigenic V.cholerae infection appeared in the same period.Along with epide(m)iological information,we noticed that these cases had a sporadic nature,but frequently appeared in the same area.We got the impression that public health measurements should be strengthened,with special attention paid to those diarrhea outbreaks caused by non-O 1 /non-O 139 strains since V.cholerae had appeared in low incidence.

Gastroenteritis aguda causada por Vibrio cholerae no-O1, no-O139 que porta una región homologa a la isla de patogenicidad VpaI-7 / Acute gastroenteritis caused by a Vibrio cholerae non-O1, non-O139 strain harboring a genetic region homologous to the VpaI-7 pathogenicity island

Pathogenic Vibrio cholerae isolates, the etiologic agents of cholera, generally express one of two O antigens (O1 or O139). Most environmental isolates are nonpathogenic and are referred to as "non-O1, non-O139". However some V. cholerae non-O1, non-O139 strains are clearly pathogenic and have caused outbreaks or sporadic cases of gastroenteritis and extraintestinal infections in humans. We report a case of acute gastroenteritis by a V. cholerae non-O1, non-O139 harboring a genetic region homologous to a segment of the VpaI-7 V. parahaemolyticus pathogenicity island.

Cepas patogénicas de Vibrio cholerae, el agente causal del cólera, expresan generalmente uno de dos antígenos O (denominados O1 u O139). La mayoría de las cepas ambientales son no patogénicas y corresponden al tipo denominado "no-O1, no-O139". Sin embargo, algunas cepas de este tipo son claramente patogénas y han causado brotes de gastroenteritis e infecciones extra-intestinales en humanos. Se reporta un caso clínico de gastroenteritis aguda causado por una cepa de V. cholerae no-O1, no-O139 que contiene en su genoma una región homóloga a un segmento de la isla de patogenicidad VpaI-7 descrita previamente en V. parahaemolyticus.

Bacteriemia por Vibrio cholerae no-O1, no-O139 en un paciente en hemodiálisis crónica / Non-O1, non-O139 Vibrio cholerae bacteremia in a chronic hemodialysis patient

Vibrio cholerae no-O1, no-O139 es un agente poco frecuente como causal de bacteriemias y no hay informes que documenten su presencia en pacientes en hemodiálisis crónica. Se describe el caso de una paciente en hemodiálisis crónica que presentó un cuadro de sepsis, por lo cual inició un tratamiento con vancomicina y ceftacidima. Al cabo de seis horas y media de incubación en el sistema BACT/ALERT de hemocultivo, se evidenció la presencia de bacilos curvos gram negativos, posteriormente identificados como Vibrio cholerae mediante pruebas bioquímicas convencionales y el uso de los kits API 20 NE y VITEK 2. La evaluación del serogrupo y de la presencia de factores de patogenicidad, realizada en el laboratorio de referencia, determinó que el microorganismo hallado pertenecía al serogrupo no-O1, no-O139. No se detectó la toxina de cólera, tampoco el factor de colonización ni la toxina termoestable. El aislamiento presentó sensibilidad frente a ampicilina, trimetoprima-sulfametoxazol, ciprofloxacina, tetraciclina, ceftacidima y cefotaxima por el método de difusión con discos y por VITEK 2. La paciente cumplió 14 días de tratamiento con ceftacidima endovenosa, con evolución favorable.

Non-O1, and non-O139 Vibrio cholerae is an infrequent cause of bacteremia. There are no reports of such bacteremia in chronic hemodialysis patients. This work describes the case of a chronic hemodialysis patient that had an episode of septicemia associated with dialysis. Blood cultures were obtained and treatment was begun with vancomycin and ceftazidime. After 6.5 hours of incubation in the Bact/Alert system there is evidence of gram-negative curved bacilli that were identified as Vibrio cholerae by conventional biochemical tests, API 20 NE and the VITEK 2 system. This microorganism was sent to the reference laboratory for evaluation of serogroup and virulence factors and was identified as belonging to the non-O1 and non-O139 serogroup. The cholera toxin, colonization factor and heat-stable toxin were not detected. The isolate was susceptible to ampicillin, trimethoprim-sulfamethoxazole, ciprofloxacin, tetracycline, ceftazidime and cefotaxime by the disk diffusion method and the VITEK 2 system. The patient received intravenous ceftazidime for a 14 day- period and had a favorable outcome.

Caracterización de aislamientos de Vibrio cholerae no-O1, no-O139 asociados a cuadros de diarrea / Characterization of Vibrio cholerae non-O1 and non-O139 isolates associated with diarrhea

La infección por Vibrio cholerae, el agente causal del cólera, se trasmite al hombre por ingestión de agua y alimentos contaminados. Aunque son los serogrupos O1 y O139 los que habitualmente se asocian al cólera epidémico, los aislamientos de otros serogrupos también son causales de gastroenteritis e infecciones extra-intestinales. Durante el período 2003-2005, se investigó la presencia de V. cholerae en la materia fecal de niños con diarrea atendidos en el Hospital del Niño Jesús, Tucumán. Se recuperaron 34 aislamientos de V. cholerae no-O1, no-O139. Se determinaron sus perfiles de virulencia por PCR, la sensibilidad a los antimicrobianos y la diversidad genética por electroforesis en campo pulsado. Se obtuvieron ocho perfiles de virulencia, aunque ningún aislamiento fue positivo para la toxina colérica ni para la toxina termoestable. Cuatro aislamientos fueron positivos para el sistema de secreción de tipo tres. El 17,6% de los aislamientos fueron resistentes o de sensibilidad intermedia a ampicilina y el 5,9% fueron resistentes a trimetoprima-sulfametoxazol. Los aislamientos resultaron muy diversos: se hallaron 27 patrones distintos en 29 aislamientos tipificables por electroforesis en campo pulsado. A pesar de su baja incidencia, V. cholerae continúa siendo un agente causal de diarrea en niños, los que se ven afectados por una amplia variedad de cepas circulantes.

Vibrio cholerae, etiologic agent of cholera, is transmitted to humans by ingestion of contaminated food or water. Even though serogroups O1 and O139 are the ones usually associated to epidemic cholera, isolates from other serogroups also cause gastroenteritis and extraintestinal infections. During the period 2003-2005, presence of V. cholerae in stools was investigated in children with diarrhea that seaked assistance at the Niño Jesús Hospital in Tucumán. Thirty four isolates of V. cholerae non-O1, non-O139 were recovered. We characterized the isolates studying its virulence factors by PCR, antimicrobial susceptibility patterns and genetic diversity by pulsed-field gel electrophoresis. Eight virulence patterns were obtained although no isolate was positive for the cholera toxin or the thermostable toxin. Four isolates were positive for the type three secretion system. The 17.6% of the isolates were resistant or intermediate to ampicillin and 5.9% were resistant to trimethoprim-sulfamethoxazole. By SfiI-PFGE, all isolates were genetically very diverse, as 27 different patterns were identified in 29 typeable isolates by pulsed-field gel electrophoresis. Although it has a low incidence, V. cholerae continues to be a causative agent of diarrhea in children, who are affected by a variety of circulating strains of V. cholerae non-O1, non-O139.

A Case of Skin and Soft Tissue Infection Caused by Non-O1, Non-O139 Vibrio cholerae in a Patient with Liver Cirrhosis / 감염과화학요법

Non-O1, non-O139 Vibrio cholerae usually causes gastroenteritis and bacteremia. It can also cause skin and soft tissue infection but the incidence is very rare. Patients who have been reported to have skin and soft tissue infection caused by non-O1, non-O139 V. cholerae had liver cirrhosis or chronic hepatitis. We present here a case of skin and soft tissue infection caused by non-O1, non- O139 V. cholerae in a patient with liver cirrhosis in Korea. After treatment of cefotaxime, doxycycline and debridement, the wound was clinically improved. This case suggests that non-O1, non-O139 V. cholerae infection should also be considered in addition to V. vulnificus infection when skin and soft tissue infections occurs in patients with liver cirrhosis, especially if they have had seawater or seafood exposure.

A Case of Skin and Soft Tissue Infection Caused by Non-O1, Non-O139 Vibrio cholerae in a Patient with Liver Cirrhosis / 감염과화학요법

Non-O1, non-O139 Vibrio cholerae usually causes gastroenteritis and bacteremia. It can also cause skin and soft tissue infection but the incidence is very rare. Patients who have been reported to have skin and soft tissue infection caused by non-O1, non-O139 V. cholerae had liver cirrhosis or chronic hepatitis. We present here a case of skin and soft tissue infection caused by non-O1, non- O139 V. cholerae in a patient with liver cirrhosis in Korea. After treatment of cefotaxime, doxycycline and debridement, the wound was clinically improved. This case suggests that non-O1, non-O139 V. cholerae infection should also be considered in addition to V. vulnificus infection when skin and soft tissue infections occurs in patients with liver cirrhosis, especially if they have had seawater or seafood exposure.

Virulence factors of non-O1 non-O139 Vibrio cholerae isolated in Córdoba, Argentina / Factores de virulencia de Vibrio cholerae no-O1 no-O139 aislados en Córdoba, Argentina

V. cholerae non-O1 non-O139 serogroups isolated from clinical and environmental sources in Córdoba, Argentina, were analyzed for the presence and expression of virulence genes. Most of the strains studied contained the genes toxR and hlyA, but lacked ctxA, zot, ace, tcpA and stn. The culture supernatants were tested for hemolytic and cytotoxic activity. The enterotoxic potential of the strains was studied in a rabbit ileal loop assay and their genetic profiles were compared by PFGE. The environmental strains varied in their virulence phenotype and showed no-clonal relationships. The clinical strains were highly enterotoxic, hemolytic, proteolytic and showed indistinguishable PFGE profiles, although they differed in their cytotoxic activity. This is the first description, using cell culture and “in vivo” studies, of the virulence properties of non-O1 non-O139 V. cholerae from Argentina.

En este trabajo se analizó la presencia y expresión de genes de virulencia en V. cholerae no-O1 no-O139 de origen clínico y ambiental, aislados en Córdoba, Argentina. La mayoría de las cepas estudiadas contiene los genes toxR y hlyA, pero no ctxA, zot, ace, tcpA y stn. Se analizó la actividad hemolítica y citotóxica de estas cepas en los sobrenadantes de cultivo, así como su potencial enterotóxico en ensayos de asa ileal ligada de conejo. Además, los aislamientos fueron comparados por sus perfiles genéticos en PFGE. Las cepas del medio ambiente mostraron variación en su fenotipo de virulencia y no mostraron relación clonal. Las cepas clínicas fueron muy enterotóxicas, hemolíticas, proteolíticas y mostraron perfiles indistinguibles de PFGE, aunque mostraron diferencias en su actividad citotóxica. En este trabajo se describen por primera vez, utilizando ensayos de cultivo celular e “in vivo”, propiedades de virulencia de V. cholerae no-O1 no-O139 aislados en Argentina.

A Case of Vibrio cholerae non-O1/non-O139 Gastroenteritis / 대한임상미생물학회지

Vibrio cholerae is an autochthonous inhabitant of estuarine and seawater environment and is a facultative pathogen for humans. V. cholerae non-O1/non-O139 strains are associated with gastroenteritis, septicemia and/or extraintestinal infections. But the reported cases of gastroenteritis by non-O1/non-O139 serotype, are rare in Korea. The authors isolated V. cholerae non-O1/non- O139 strain from a stool of a 67 year-old-woman who had suffered from diabetes, hypertension and Alzheimer disease and analyzed presence of toxin genes by multiplex PCR method.