Characterization of Vibrio cholerae isolates from 1976 to 2013 in Shandong Province, China

Abstract Cholera continues to be a serious public health issue in developing countries. We analyzed the epidemiological data of cholera from 1976 to 2013 in Shandong Province, an eastern coastal area of China. A total of 250 Vibrio cholerae isolates were selected for PCR analysis of virulence genes and pulsed-field gel electrophoresis (PFGE). The analysis of the virulence genes showed that the positive rates for tcpA and tcpI were the highest among strains from the southwest region, which had the highest incidence rate of cholera. Low positive rates for tcpA, tcpI and ctxAB among isolates from after 2000 may be an influencing factor contributing to the contemporary decline in cholera incidence rates. Spatiotemporal serotype shifts (Ogawa, Inaba, Ogawa, Inaba and O139) generally correlated with the variations in the PFGE patterns (PIV, PIIIc, PIa, PIIIb, PIIIa, PIb, and PII). O1 strains from different years or regions also had similar PFGE patterns, while O139 strains exclusively formed one cluster and differed from all other O1 strains. These data indicate that V. cholerae isolates in Shandong Province have continually undergone spatiotemporal changes. The serotype switching between Ogawa and Inaba originated from indigenous strains, while the emergence of serogroup O139 appeared to be unrelated to endemic V. cholerae O1 strains.

A rapid and reliable species-specific identification of clinical and environmental isolates of Vibrio cholerae using a three-test procedure and recA polymerase chain reaction.

Purpose: Vibrio cholerae, the cause of cholera, is one of the leading causes of morbidity and mortality in many developing countries. Most laboratories initially rely on biochemical tests for a presumptive identification of these strains, followed by a polymerase chain reaction (PCR)-based method to confirm their identification. The aim of this study is to establish a rapid and reliable identification scheme for V. cholerae using a minimal, but highly specific number of biochemical tests and a PCR assay. Materials and Methods: We developed a species-specific PCR to identify V. cholerae, using a housekeeping gene recA, and used that to evaluate the sensitivity and specificity of 12 biochemical tests commonly used for screening and / or presumptive identification of V. cholerae in the clinical and environmental samples. Results: Here we introduced a combination of three biochemical tests, namely, sucrose fermentation, oxidase test, and growth in trypton broth containing 0% NaCl, as also the PCR of the recA gene, for rapid identification of V. cholerae isolates, with 100% sensitivity and specificity. The established method accurately identified a collection of 47 V. cholerae strains isolated from the clinical cases (n = 26) and surface waters (n = 21), while none of the 32 control strains belonging to different species were positive in this assay. Conclusion: The triple-test procedure introduced here is a simple and useful assay which can be adopted in cholera surveillance programs for efficient monitoring of V. cholerae in surface water and fecal samples.

Reporte histórico: Primer Aislamiento de Vibrio cholera serogrupo O1 biovar El Tor serovar Inaba durante la epidemia de cólera en el Perú ‑ 1991 / Historical report: first isolation of Vibrio cholera serogroup O1 biovar El Tor serovar Inaba during the cholerae epidemic in Perú ‑ 1991

Hace 20 años apareció una enfermedad diarreica nueva en el Perú y el Laboratorio de Referencia de Enteropatógenos del Instituto Nacional de Salud, cumplió una labor destacada en el aislamiento e identificación rápida y oportuna del Vibrio cholerae. La enfermedad del cólera no se había presentado anteriormente, pero en la última semana de enero de 1991 se detectó un brote epidémico de diarrea aguda con deshidratación intensa y algunos casos de fallecidos. La epidemia afectó, al comienzo, varias localidades del litoral peruano. Equipos de trabajo de la Oficina General de Epidemiología y de los laboratorios del Instituto Nacional de Salud obtuvieron muestras fecales de pacientes con diarrea aguda procedentes de las ciudades de Chancay, Chimbote, Piura y algunos hospitales de Lima. Las muestras colectadas en el medio de transporte de Cary y Blair fueron procesadas en el Laboratorio Nacional de Referencia de Enteropatógenos (LANARE) del Instituto Nacional de Salud. De todas las muestras se aisló e identificó Vibrio cholerae serogrupo O1 biovar El Tor serovar Inaba que mostró ser sensible a la tetraciclina y a otros antibióticos. Esta investigación confirmó el primer brote epidémico de cólera en el Perú.

20 years ago, a new diarrheal disease was introduced in Peru and the Enteropathogens Reference Laboratory of the Instituto Nacional de Salud had an outstanding role in the isolation and rapid and timely identification of Vibrio cholerae. Cholera had not been seen before, but during the last week of January 1991 an outbreak of acute diarrhea was detected, presenting intense dehydration and some deaths. The epidemic affected, in the beginning, many locations of the peruvian coast. Some working teams of the General Office of Epidemiology and of the Instituto Nacional de Salud obtained fecal samples from patients with acute diarrhea coming from the cities of Chancay, Chimbote, Piura and some hospitals in Lima. The collected samples were transported on Cary and Blair media and processed in the National Reference Laboratory of Enteropathogens (LANARE) of the Instituto Nacional de Salud. Vibrio cholerae serogroup 01 biovar El Tor serovar Inaba was isolated from all the samples, it was sensible to tetracyclines and other antibiotics. This research confirmed the first outbreak of cholera in Peru.

Occurrence and composition of class 1 and class 2 integrons in clinical and environmental O1 and non-O1/non-O139 Vibrio cholerae strains from the Brazilian Amazon

This study identified and characterised class 1 and 2 integrons in clinical and environmental Vibrio cholerae O1 and non-O1/non-O139 strains isolated from the Brazilian Amazon. The aadA2 and aadA7 gene cassettes were found in class 1 integrons in two genotypes of environmental V. cholerae non-O1/non-O139. Empty integrons were found in strains from the Brazilian cholera epidemic. A class 2 integron was detected in one strain from the V. cholerae Amazonia lineage harbouring sat1 and aadA1 genes. All isolates were resistant to aminoglycosides, indicating aadA functionality. These findings suggest that environmental bacteria act as cassette reservoirs that favour the emergence of resistant pathogens.

Biotyping of Vibrio cholerae O1: time to redefine the scheme.

Considering the recent emergence of "hybrid biotype" and "El Tor variant", we propose to redefine the biotyping scheme for Vibrio cholerae O1 serogroup. The existing biotyping scheme has limitations and causes confusion as many of the hybrid biotype and El Tor variant strains have phenotypic and genetic changes. A revised biotyping scheme will play a significant role to understand the ecology, epidemiology and nature of infection of V. cholerae O1 strains in future.

Non-O1/non-O139 Vibrio cholerae septicemia with peritonitis.

A 67-year-old Thai female with alcoholic cirrhosis presented with fever and abdominal pain for 5 days. On examination, there was marked ascites with generalized abdominal tenderness. The result of ascitic fluid analysis showed yellow turbid fluid, a WBC count of 6,100 cells/mm3 with polymorphonucleocytes predominant. Blood cultures yielded non-O1/ non-O139 Vibrio cholerae. The patient improved gradually and recovered fully after 1 week of parenteral antibiotic.

Changing patterns of Vibrio cholerae in sevagram between 1990 and 2005.

PURPOSE: A retrospective analysis was done to note changes in prevalence, distribution of biotypes, serotypes, antibiotic susceptibility patterns and phage types of Vibrio cholerae isolated in Mahatma Gandhi Institute of Medical Sciences, Sevagram over a period of 16 years. METHODS: A total of 535 strains of V. cholerae were isolated from 10,406 stool samples and rectal swabs from January 1990 to December 2005. These comprised of serogroups O1 - 427 (79.89%), O139 - 86 (16.07%) and non O1, non O139 - 22 (4.11%). No classical V. cholerae was isolated. RESULTS: Vibrio cholerae serogroup O1 serotype Ogawa was the predominant isolate till 1992. During 1993, serogroup O139 became the main isolate; however, it completely disappeared during 1995-1996 only to reappear in 1997. Serotype Inaba in our area was conspicuous by its absence with only two strains being isolated till June 1999, but during July-December 1999, 11 out of 15 V. cholerae O1 isolates were El Tor Inaba. T4 was the predominant phage type till 1990, T2 during 1991-1994 and T27 (as per the new scheme) thereafter. Resistance to tetracycline varied between 2 and 17% for V. cholerae O1. CONCLUSIONS: The paper reports on the changing epidemiological markers of V. cholerae isolated from a rural hospital over a period of 16 years.

Aislamiento e identificación de especies pertenecientes a los géneros Aeromonas, Vibrio y Plesiomonas procedentes de muestras extra-intestinales en Cuba / Isolation and identification of species from the genera Aeromonas, Vibrio, and Plesiomonas from extraintestinal samples in Cuba

Las infecciones extra-intestinales producidas por los géneros Aeromonas, Vibrio y Plesiomonas presentan una elevada tasa de morbimortalidad en diferentes áreas geográficas. De enero 2002 a diciembre 2003 se recibieron en el Laboratorio Nacional de Referencia de Enfermedades Diarreicas Agudas del Instituto de Medicina Tropical Pedro Kourí, 95 cepas de bacilos gramnegativos, anaerobios facultativos, oxi-dasa positiva, procedentes de muestras extra-intestinales (hemocultivos, exudados óticos, pus de heridas, exudados conjuntivales, urocultivos, catéter, entre otras) remitidas de diferentes provincias del país. Se demostró la presencia de Aeromonas caviae, Aero-monas veronii bv sobria, Aeromonas jandaei, Vibrio cholerae no -O1, Vibrio vulnificus, Vibrio fluvialis y Plesiomonas shigelloides en las muestras estudiadas.

Extraintestinal infections caused by the genera Aeromonas, Vibrio and Plesiomonas have high morbidity and mortality rates in different areas of world. From January 2002 to December 2003, the National Reference Laboratory for Acute Diarrhoeal Diseases of the Pedro Kourí Tropical Medicine Institute received 95 gramnegative, facultative anaerobic, oxidase positive bacilli strains from different extraintestinal specimen (blood, ear exudates, infected wounds, conjunctive exudates, urine, and catheters, among others) sent by different provincial laboratories along the country. Aeromonas caviae, Aeromonas veronii bv sobria, Aeromonas jandaei, Vibrio cholerae no-O1, Vibrio vulnificus, Vibrio fluvialis and Plesiomonas shigelloides were the species most frequently found in the sample analysed.

Vibrios patogênicos em ostras (Crassostrea rhizophorae) servidas em restaurantes no Rio de Janeiro: um alerta para a Saúde Pública / Pathogenic Vibrios in oysters (Crassostrea rhizophorae) served at restaurants in Rio de Janeiro: a public health warning

Avaliaram-se 40 amostras de ostras (Crassostrea rhizophorae) servidas in natura em 15 restaurantes da Cidade do Rio de Janeiro, a fim de investigar a presença de Vibrio spp. As amostras de ostras foram analisadas e submetidas a enriquecimento em água peptonada alcalina adicionada de 1 e 3 por cento de NaCl, incubadas a 37°C por 24 horas. Em seguida, os cultivos foram semeados em agar tiossulfato citrato bile sacarose e as colônias suspeitas foram submetidas à caracterização bioquímica. Vibrio parahaemolyticus, Vibrio carchariae, Vibrio alginolyticus e Vibrio vulnificus representaram as principais espécies (> 60 por cento) isoladas a partir das ostras in natura.

Forty oyster samples (Crassostrea rhizophorae) served raw in 15 restaurants in the city of Rio de Janeiro were evaluated in order to investigate the presence of Vibrio spp. The oyster samples were analyzed and subjected to enrichment in alkaline peptone water with the addition of 1 and 3 percent NaCl and incubated at 37°C for 24 hours. Following this, the cultures were seeded onto thiosulfate citrate bile sucrose agar (TCBS) and the suspected colonies were subjected to biochemical characterization. Vibrio parahaemolyticus, Vibrio carchariae, Vibrio alginolyticus and Vibrio vulnificus were the main species (> 60 percent) isolated from raw oysters.

Antimicrobial resistance of clinical and environmental strains of Vibrio cholerae isolated in Lima-Peru during epidemics of 1991 and 1998

The susceptibility in vitro of 71 isolations of V. cholerae was evaluated: 24 of clinical origin and 47 strains of clinical and environmental origin collected in the epidemic of 1991 and during the outbreak epidemic of 1998 in Lima-Peru respectively. The biochemical and serological tests carried out established that 43 (60,6 percent) corresponded to the serogroup O1 Ogawa of the 1998 epidemic; 26 (36.6 percent) were of the serotype Inaba, being 24 of them isolated in 1991. Two strains did not belong to the serogroup O1. By means of disk diffusion method and Minimal Inhibitory Concentration (MIC), 15 strains with multi-resistance to antibiotics were determined, 10 of which were of clinical origin and 5 of natural origin, showing 9 antibiotypes with different resistance pattern. The evaluation of susceptibility in front of the vibriostatic agent O/129, demonstrated that 11.4 percent of the strains, collected in 1998, presented resistance to a concentration of 150 æg. A direct relationship among the resistance that presented the strains of clinical and environmental origin isolated in 1991 and 1998 was established as much for tetracycline, sulfa/trimethoprim and 0/129; 88.6 percent of the clinical strains of the year 1998 presented resistance to these three drugs, while 100 percent of clinical strains isolated in 1991 were sensitive to O/129 (150 µg), sulfa/trimethoprim and tetracycline. We conclude that V. cholerae O1 has increased its resistance to antimicrobial drugs of clinical use in the same way it is also losing susceptibility to the vibriostatic compound O/129 for what their use is not recommended for taxonomic purposes.

Structural organization of the transfer RNA operon I of Vibrio cholerae: differences between classical and El Tor strains.

Nine major transfer RNA (tRNA) gene clusters were analysed in various Vibrio cholerae strains. Of these, only the tRNA operon I was found to differ significantly in V. cholerae classical (sixth pandemic) and El Tor (seventh pandemic) strains. Amongst the sixteen tRNA genes contained in this operon, genes for tRNA Gln3 (CAA) and tRNA Leu6 (CUA) were absent in classical strains as compared to El Tor strains. The observation strongly supported the view that the above two pandemic strains constitute two different clones.

Diversity of Vibrio cholerae strains isolated in Delhi, India, during 1992-2000.

The National Institute of Communicable Diseases (NICD), Delhi, India, conducts a laboratory-based surveillance of cholera cases referred from the Infectious Disease Hospital, Delhi. The prevalence and antimicrobial susceptibilities of Vibrio cholerae O1, O139, and others, isolated from cholera patients for nine years, were analyzed to determine the changing trends in their isolation and drug-resistance patterns. In total, 29,196 stool samples or rectal swabs, collected during April 1992-December 2000, were included in this study. Of these, 13,730 (47%) were positive for V. cholerae: 11,091 for V. cholerae O1 (80.7%) and 1,943 (14%) for V. cholerae O139, and 696 (5%) were non-O1 and non-O139. V. cholerae O1 was the dominant serotype during 1992-1993, when V. cholerae O139 emerged as a new serotype but, thereafter, remained low from 1994 to 1999. Phenotypically, re-emerged V. cholerae O139 in 2000 displayed a difference compared to those that appeared in 1992-1993, in that the current O139 strains were sensitive to co-trimoxazole. Resistance to nalidixic acid and furazolidone was constantly high (100%) among strains of V. cholerae O1 and O139. All strains of V. cholerae were uniformly susceptible to chloramphenicol, tetracycline, amikacin, and norfloxacin. Molecular studies revealed different clones of V. cholerae O1 and O139 prevailing in the country with the re-emergence of V. cholerae O139 of a different clonality in Delhi in 2000, which is likely to play a critical role in temporal antigenic variation among the serogroups of V. cholerae.

Incidence of Vibrio cholerae serogroup O139 infection with low virulence in Hubli, Karnataka (India).

384 stool samples from patients with acute gastroenteritis were processed by standard culture techniques and antibiogram of V. cholerae was performed. Stool samples from 93 (24.22%) patients yielded V. cholerae, 58 (62.37%) of which were V. cholerae, El Tor O1 Ogawa, 31 (33.33%) V. cholerae O139 and 4 (4.30%) V. cholerae non O1 non O139. Of the culture proven cholera cases watery diarrhoea was observed in 79 (84.95%), vomiting in 57(61.29%), muscle cramps in 21 (22.58%) and sweating in 18 (19.35%). Majority of these patients presented with moderate dehydration 57 (61.29%). Mild dehydration was found in 19 (20.43%) and severe dehydration in 17 (18.28%). While majority of patients with O139 infection had mild to moderate dehydration 25 (80.65%), severe dehydration was more common with O1 infection 11 (64.71%). This study reflects the importance of monitoring the V. cholerae by serogrouping, antibiogram typing, which keep on varying constantly.

Bacteriological profile of cholera in Tamil Nadu (1980-2001).

BACKGROUND & OBJECTIVE: In addition to Vibrio cholerae O1, a recently emerged non-O1 serotype, V. cholerae O139 has also been found to be responsible for epidemics of cholera. In the context of the changing etiology of cholera, the occurrence of different serotypes, biotypes and phage types of V. cholerae in Tamil Nadu is presented. METHODS: Between 1980 and 2001, 26,502 stool/rectal swabs from patients suspected to have cholera were processed for the presence of V. cholerae at the King Institute of Preventive Medicine, Chennai (Madras), Tamil Nadu. Serotypes, biotypes and phage types (Basu & Mukerjee and new phage typing method) of the isolates were determined. Antimicrobial susceptibility determination was included from 1997 onwards. RESULTS: Of the 26,502 stool/rectal swabs tested, 6035 (22.8%) specimens yielded V. cholerae. 4,046 (67%) of them were O1 serotype; followed by 1529 (25.3%) O139 serotype and 448 (7.4%) non-O1 serotypes other than O139 were encountered. Twelve specimens yielded rough strains of V. cholerae. All V. cholerae O1 were the biotype ElTor and the predominant serotype was Ogawa (95.9%). After the initial explosive epidemic by O139 serotype in late 1992 and early 1993 occurrence of this serotype had declined. Resurgence of this new serotype was observed in 1997. O1 domination was observed during 1995 and 1996. The new phage typing scheme for V. cholerae O1 was able to differentiate the isolates into 22 types, type 27 (68.2%) being the predominant one followed by type 26 (12.3%). In Basu & Mukerjee scheme, the isolates were clustered between two types--type 2 (34%) and type 4 (61%). Further, about 5 per cent of them were untypeable. All but a few of the isolates irrespective of serotype, were susceptible to chloramphenicol, tetracycline, gentamicin and ciprofloxacin. 97 per cent of the O1 strains and 48.9 per cent of the O139 strains were resistant to cotrimoxazole. INTERPRETATION & CONCLUSION: V. cholerae O139 might have the ability to survive during interepidemic periods. Both V. cholerae O1 and O139--the two serotypes with epidemic potential seem to be firmly established in this region. This situation warrants constant surveillance.

A localised outbreak of Vibrio cholerae O139 in Kolkata, West Bengal.

BACKGROUND & OBJECTIVES: An explosive outbreak of diarrhoeal disease which occurred in the Baishnabghata, Patuli area of Kolkata Municipal Corporation during September 28 to October 12, 2000, was investigated by a team from the National Institute of Cholera and Enteric Diseases, Kolkata, to identify the causative agent and determine the antimicrobial susceptibility pattern. METHODS: Clinical and epidemiological data were collected from domiciliary cases and also from patients attending two medical camps that had been set up for the purpose. Stool and water samples were collected for isolation of diarrhoeagenic pathogens. RESULTS: A total of 710 cases of diarrhoea occurred with an attack rate of 7.1 per cent; majority were adults. All 6 faecal samples and 2 water samples collected, were positive for Vibrio cholerae O139. The strains were uniformly (100%) susceptible to the commonly used drugs for cholera such as tetracycline, norfloxacin, ciprofloxacin, co-trimoxazole and nalidixic acid but resistant (100%) to furazolidone and ampicillin. INTERPRETATION & CONCLUSION: This is the first localised outbreak of V. cholerae O139 in Kolkata since the devastating epidemic in 1992. Extensive chlorination of all water sources resulted in a dramatic decline of the outbreak. The appearance of resistance in V. cholerae O139 to furazolidone is a matter of great concern since this drug is used for the treatment of cholera in children and pregnant women.