Una vacuna de vesículas de membrana externa de Vibrio cholerae es aún una promesa: Caracterización proteómica / Outer membrane vesicle vaccine from Vibrio cholerae is still a promise: Proteomic characterization

Las epidemias de cólera afectan a un gran número de países africanos, asiáticos y del Caribe. Los cambios climatológicos y las constantes migraciones hacen que esta enfermedad se extienda, por lo que resulta necesario disponer de vacunas protectoras. En el presente trabajo se caracterizó una nueva vacuna de vesículas de membrana externa (VMEs) obtenidas de Vibrio cholerae O1 biotipo El Tor serotipo Ogawa cepa C7258, en el Instituto Finlay de vacunas (Cuba), a través de métodos proteómicos. Se identificaron 53 proteínas presentes en las VME (4 proteínas por banda electroforética) separadas por electroforesis unidimensional (1D) y digeridas con tripsina. Los fragmentos obtenidos fueron separados por cromatografía líquida de alta resolución (HPLC) acoplada a espectrometría de masa, secuenciados e identificados mediante bases de datos de proteínas Swiss-Prot y TrEMBL. El patrón proteico obtenido presentó algunas de las proteínas (12 proteínas citoplasmáticas y 5 proteínas de membrana externa) sugeridas dentro del proteoma de buena calidad para candidatos vacunales. Se estudiaron las mejores condiciones para la separación de las proteínas a través de electroforesis bidimensional. Las VME evaluadas cuentan con una composición fundamentada en proteínas necesarias para garantizar una respuesta inmune que proteja contra Vibrio cholerae O1 biotipo El Tor serotipo Ogawa.

Cholera epidemics affect a large number of African, Asian and Caribbean countries. The climate changes and the constant migrations cause this disease to spread, making it is necessary to obtain protective vaccines. In the present work, a new vaccine of outer membrane vesicles (OMV) from V. cholerae O1 El Tor biotype Ogawa serotype strain C7258 at Finlay Institute of vaccines (Cuba) was characterized by proteomic methods. A total of 53 proteins present in the OMV (approximate ratio of 4 proteins by electrophoresis band) were identified, separated by one dimension electrophoresis and digested by tripsin method. The fragments were separated by high performance liquid chromatography (HPLC) coupled to mass spectrometry, sequenced and identified, using Swiss-Prot and TrEMBL protein databases. The pattern showed some proteins (12 cytoplasmic proteins and 5 outer membrane proteins) suggested within the highest quality proteome for vaccine candidate. The best conditions for proteins separation by two dimension electrophoresis were studied. The OMV composition was based on proteins described to the immunity response and protection against V. cholerae O1 El Tor biotype Ogawa serotype.

As epidemias de cólera afetam um grande número de países africanos, asiáticos e caribenhos. As mudanças climáticas e as constantes migrações fazem com que esta doença se espalhe, portanto é necessário ter vacinas protectoras. No presente trabalho, uma nova vacina de vesículas de membrana externa (VMEs) obtidas de Vibrio cholerae 01 biotipo El Tor sorotipo Ogawa cepa C7258, no Instituto de Vacinas Finlay (Cuba), através de métodos proteômicos. Foram identificadas 53 proteínas presentes nas VME (4 proteínas por banda eletroforética) separadas por eletroforese unidimensional (1D) e digeridas com tripsina. Os fragmentos obtidos foram separados por cromatografia de alta resolução (HPLC) acoplada a espectrometria de massa, sequenciados e identificados usando bancos de dados de proteínas Swiss-Prot e TrEMBL. O padrão proteico obtido apresentou algumas das proteínas (12 proteínas citoplasmáticas e 5 proteínas de membrana externa) sugeridas dentro do proteoma de boa qualidade para candidatos vacinais. As melhores condições para a separação de proteínas através de eletroforese bidimensional foram estudadas. As VME avaliados possuem uma composição baseada em proteínas necessárias para garantir uma resposta imune que proteja contra Vibrio cholerae O1 biotipo El Tor sorotipo Ogawa.

Oral Cholera vaccine campaign in Sudan

A pre-emptive mass immunization campaign with oral cholera vaccines [OCV] has recently been carried out in Sudan in order to prevent spread of cholera amongst refugees escaping from South Sudan on account of war. The campaign, completed in two rounds, ended on 1st of September 2016

Inhibition of virulence potential of Vibrio cholerae by natural compounds.

The rise in multi-drug resistant Vibrio cholerae strains is a big problem in treatment of patients suffering from severe cholera. Only a few studies have evaluated the potential of natural compounds against V. cholerae. Extracts from plants like ‘neem’, ‘guazuma’, ‘daio’, apple, hop, green tea and elephant garlic have been shown to inhibit bacterial growth or the secreted cholera toxin (CT). However, inhibiting bacterial growth like common antimicrobial agents may also impose selective pressure facilitating development of resistant strains. A natural compound that can inhibit virulence in V. cholerae is an alternative choice for remedy. Recently, some common spices were examined to check their inhibitory capacity against virulence expression of V. cholerae. Among them methanol extracts of red chili, sweet fennel and white pepper could substantially inhibit CT production. Fractionation of red chili methanol extracts indicated a hydrophobic nature of the inhibitory compound(s), and the n-hexane and 90 per cent methanol fractions could inhibit >90 per cent of CT production. Purification and further fractionation revealed that capsaicin is one of the major components among these red chili fractions. Indeed, capsaicin inhibited the production of CT in various V. cholerae strains regardless of serogroups and biotypes. The quantitative reverse transcription real-time PCR assay revealed that capsaicin dramatically reduced the expression of major virulence-related genes such as ctxA, tcpA and toxT but enhanced the expression of hns gene that transcribes a global prokaryotic gene regulator (H-NS). This indicates that the repression of CT production by capsaicin or red chili might be due to the repression of virulence genes transcription by H-NS. Regular intake of spices like red chili might be a good approach to fight against devastating cholera.

Antimicrobials & cholera: are we stranded.

Antimicrobial resistance poses a major threat in the treatment of infectious diseases. Though significant progress in the management of diarrhoeal diseases has been achieved by improved hygiene, development of new antimicrobials and vaccines, the burden remains the same, especially in children below 5 yr of age. In the case of cholera, though oral rehydration treatment is the mainstay, antimicrobial therapy is mandatory at times to reduce the volume of stool and shorten the duration of the disease. Though for many pathogens, antimicrobial resistance emerged soon after the introduction of antibiotics, Vibrio cholerae remained sensitive to most of the antibiotics for quite a long period. However, the scenario changed over the years and today, V. cholerae strains isolated world over are resistant to multiple antibiotics. A myriad number of mechanisms underlie this phenomenon. These include production of extended-spectrum beta-lactamases, enhanced multi-drug efflux pump activity, plasmid-mediated quinolone and fluoroquinolone resistance, and chromosomal mutations. Horizontal transfer of resistance determinants with mobile genetic elements like integrons and the integrating conjugative elements (ICEs), SXTs help in the dissemination of drug resistance. Though all strains isolated are not resistant to all antibiotics and we are not as yet “stranded”, expanding spectrum of drug resistance is a definite cause for concern. Pipelines of discovery of new antibiotics are drying up as major pharmaceutical companies are losing interest in investing money in this endeavour, mainly due to the short shelf-life of the antibiotics and also due to the fast emergence of drug resistance. To address this issue, attempts are now being made to discover drugs which are pathogen specific and target their “virulence mechanisms”. It is expected that development of resistance against such antibiotics would take much longer. This review briefly focuses on all these issues.

Cholera outbreak in Baghdad in 2007: an epidemiological study

In 2007 there was an epidemic of cholera in Iraq with 4667 cases. The first case in Baghdad was diagnosed on 19 September 2007 and the last case on 13 December 2007. In all, 136 cases were reported [2.9% of the country total] in 6 of the 13 districts of Baghdad. The median age of the cases was 11 years [range = 0.3-71 years]. There were 3 deaths giving a case fatality rate of 2.2%. Bacteriological testing confirmed that the outbreak was caused by Vibrio cholerae 01, biotype El Tor, serotype Inaba. The strain was resistant to trimethoprimsulfamethoxazole, but sensitive to tetracycline and chloramphenicol. Efforts are needed in Baghdad to establish safe drinking-water and proper sanitation as limited availabilty of tap-water and sewage contamination probably contributed to the spread of the disease

Current status of anti-diarrheal and anti-secretory drugs in the management of acute childhood diarrhea.

Each year 1.8 million children die due to diarrheal diseases. Indiscriminate use of antibiotics has resulted in increasing resistance to commonly used antibiotics. Moreover the recent outbreaks of shigella and cholera have revealed multi-drug resistance strains. There is a need for review of recommended antibiotics for shigellosis. From recent data it emerges that fluoroquinolones should be the first line of therapy and cephalosporins to be used as the second line. Among the anti-cholera antibiotics, tetracyclines which were the drug of choice for adults, has the advantage of high sensitivity and low cost. Single dose doxycycline would have minimal side effects, hence can be the drug of choice even in children. We should not allow the business pressures to force usage of probiotics and racecadotril as their role in the management of acute diarrhea is yet to be established. Nitazoxanide has high efficacy against Cryptosporodial diarrhea only. Strict adherence to the recommendations for the management of acute childhood diarrhea is needed or else we dilute the effect of standard management.

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.

Sales de rehidratación oral: ¿es tiempo de cambiar la fórmula? / Rehydration solutions: is it time to change this formula?

En el presente artículo se exponen las razones fisipatológicas y las posibles ventajas de la utilización de sales de rehidrataciónoral de osmolaridad reducida para el tratamiento de las diarreas en relación a la solución estándar. Las mismas tienen lapropiedad de respetar la relación molar entre sodio y glucosa necesaria para un cotransporte eficiente, pero a su vez tienen laparticularidad de ofrecer una menor osmolaridad al tracto gastrointestinal que las SRO originales. Aunque no sin controversia,los ensayos clínicos en general demostraron menos vómitos, menores pérdidas fecales, menor duración de enfermedad y menornecesidad de suplementación intravenosa al utilizarlas. Se resumen también las nuevas recomendaciones vigentes de la OMSy UNICEF, que promueven el uso de las SRO de osmolaridad reducida y la mayor investigación de las mismas, especialmenteen los casos de cólera.

Características de la epidemia de cólera de 1998 en Ecuador, durante el fenómeno de "El Niño" / Characteristics of the cholera epidemic of 1998 in Ecuador during El Niño

Objetivos. Caracterizar el brote de cólera ocurrido en Ecuador en 1998 durante el fenómeno de "El Niño", presentar los datos sobre la resistencia de las cepas circulantes de Vibrio cholerae a los antimicrobianos y describir las medidas preventivas tomadas por las autoridades sanitarias para reducir el impacto de la enfermedad. Métodos. Los datos epidemiológicos provienen de los registros de la Dirección Nacional de Epidemiología del Ministerio de Salud Pública de Ecuador y del Instituto Nacional de Higiene y Medicina Tropical, y el informe final del Programa de Formación para la Lucha contra el Cólera y las Enfermedades Diarreicas (PROCED ALA 93/25). Se procedió a aislar, identificar y serotipificar V. cholerae en las muestras de heces de 10 por ciento de los pacientes con posible cólera identificados entre el 1 de enero y el 31 de diciembre de 1998. Los casos sospechados se definieron por la aparición súbita de diarrea acuosa, con o sin deshidratación, en zonas epidémicas. Las cepas aisladas se sometieron a un antibiograma estándar por el método de difusión, en el que se probaron los siguientes antibióticos: amoxicilina, tetraciclina, sulfametoxazol con trimetoprim, compuesto vibriostático O/129, ácido nalidíxico, eritromicina, norfloxacino, ciprofloxacino, gentamicina, cloranfenicol y colistina. Resultados. En 1998 se notificaron 3 755 casos en 17 de las 21 provincias del país, lo que corresponde a una tasa de incidencia de 53,96 por 100 000 habitantes. Treinta y siete pacientes fallecieron, lo cual supone una tasa de letalidad del 0,97 por ciento. Se aislaron 301 cepas de V. cholerae en las 637 muestras con sospechosa de cólera que se procesaron; todas correspondieron a V. cholerae O:1, El Tor, subtipo Ogawa. La totalidad de las cepas fueron sensibles a la tetraciclina y a las quinolonas, y 5,6 por ciento resistentes a la eritromicina. La única cepa resistente a la amoxicilina fue multirresistente. Las autoridades nacionales pusieron en práctica una serie de medidas preventivas en la comunidad y se fortaleció el sistema de vigilancia para reducir el impacto de la enfermedad. Conclusiones. Las medidas preventivas contribuyeron a reducir el impacto de la nueva epidemia de cólera en el Ecuador, tanto en términos de letalidad como de incidencia

Tetracycline in the treatment of severe cholera due to Vibrio cholerae O139 Bengal.

Vibrio cholerae O139 synonym Bengal, recognized in 1993, is the second member in the list of about 200 serogroups of V. cholerae with epidemic and pandemic potential. Although replacement of fluids and electrolytes remains the cornerstone in the management of cholera, antimicrobial therapy can significantly shorten the duration of diarrhoea, and reduce stool volume and requirements ofrehydration fluids. The role of antimicrobial therapy on the natural course of the disease caused by this relatively new pathogen has not been systematically assessed. A randomized, double-blind, placebo-controlled clinical trial was conducted to evaluate the efficacy of tetracycline in the treatment of adults with severe cholera due to V. cholerae O139 Bengal. Forty-three adult males with severe cholera were randomly allocated to receive either 500 mg of tetracycline (n=21) or placebo (n=22) for three consecutive days. Demographic and clinical characteristics of these patients on admission were comparable. Tetracycline therapy was associated with significantly reduced total median (inter-quartile range) stool volume [216.48 (90.18-325.22) mL/kg vs 334.25 (215.12-537.64) mL/kg; p=0.001], higher rates of clinical cure (81% vs 27%; p<0.001), and shorter median (inter-quartile range) duration of diarrhoea [32 (24-48) hours vs 80 (48-104) hours; p<0.001]. The mean +/- (SD) requirement of intravenous fluid was not significantly different between the two groups [146.42 +/- 42.12 mL/kg vs 150.44 +/- 27.21 mL/kg; p=0.70]. The median (inter-quartile range) duration of faecal excretion of V. cholerae O139 was significantly shorter in the tetracycline group than the placebo group [1(1-2) day vs 5 (3-6) days; p<0.001]. The results of the study indicate that tetracycline therapy is clinically useful in the treatment of severe cholera due to V. cholerae O139 Bengal.

Transition of drug susceptibilities of Vibrio cholerae O1 in Lao People's Democratic Republic.

The changes of drug susceptibilities of Vibrio cholerae O1 isolated during the past 7 years (1993-1999) in Lao PDR were investigated. The most noteworthy finding was the appearance of polymyxin B sensitive El Tor vibrios. Until 1996, the susceptibilities were almost as expected and cholera disappeared in 1997. When a cholera outbreak resurfaced in 1998, the susceptibilities of isolated V. cholerae O1 against tetracycline, sulfamethoxazol-trimethoprim, chloramphenicol and polymyxin B were quite different from those of previously isolated organisms. Minimum inhibitory concentrations (MICs) of tetracycline and chloramphenicol against the isolates in 1998 were about 16 times higher than those against the previous isolates, and the MICs of sulfamethoxazol-trimethoprim were about 256 times higher than those against the previous isolates, (trimethoprim 32 microg/ml: sulfamethoxazol 608 microg/ml). Eleven percent of the isolates (11/99) were as sensitive to polymyxin B as the classic cholera vibrios (MIC < 2 microg/ml). In 1999, the susceptibility pattern was almost the same as that in 1998 except for polymyxin B to which 58% of the isolates (21/36) became sensitive.

Comparison of the efficacy of tetracycline and norfloxacin in the treatment of acute severe watery diarrhea.

Antibiotic treatment appears to shorten the duration of diarrhea and eradicate Vibrio cholerae. The objective of this study was to compare the efficacy of tetracycline with norfloxacin therapy in patients (adults and children) with acute severe watery diarrhea caused by VC 01 and VC 0139. Patients (adults and children) with acute severe watery diarrhea admitted to Bamrasnaradura Infectious Disease Hospital, Thailand were randomized to receive either tetracycline (500 mg qid in adults and 12.5 mg/kg qid in children) or norfloxacin (400 mg bid in adults and 7.5 mg/kg bid in children) for 3 days each. The duration of diarrhea and the fecal shedding were comparable between two groups. Thirteen cases were treated with tetracycline and twelve cases with norfloxacin. The results showed the mean duration of diarrhea in tetracycline-treated and norfloxacin-treated groups were 1.31 and 1.25 days, respectively. The mean fecal shedding in tetracycline-treated and norfloxacin-treated group were 1.38 and 1.33 days, respectively. However, there were no statistically significant differences between two groups of both comparisons (p > 0.05). All isolates (VC 01 and VC 0139) in this study were susceptible to both antibiotics. Tetracycline therapy is as good as norfloxacin therapy for quick recovery and time for bacterial eradication in patients with acute severe watery diarrhea caused by Vibrio cholerae. Children aged less than 8 years should not use tetracycline therapy because of its toxic effects.

Investigation of the outbreak of cholera in Alleppey & Palghat districts, south India.

In May 1996, a massive outbreak of cholera occurred in Alleppey district of Kerala which spread to Palghat district by July 1996. Of the 575 patients hospitalized at the Alleppey Medical College hospital between May 1 and August 2, 1996, 30 deaths occurred with a case fatality rate of 5.2 per cent while of the 638 diarrhoea patients admitted at Agali PHC of Attapadi area in Palghat district, 30 (4.7%) deaths were recorded. Clinically, the patients had profuse watery diarrhoea with vomiting. The epidemic of cholera in Alleppey and Palghat districts was caused by V. cholerae O1 of the EITor biotype, Ogawa serotype which possessed both the ctxA and tcpA genes when examined by multiplex PCR. Gross contamination of water sources was incriminated as the cause of the epidemic.

Outbreaks of cholera in Nepal.

This paper presents the study of the etiological agents of diarrhea in children below 14 years of age, this study was conducted from May 1995 to April 1996. One thousand one hundred seven (1,107) children with acute diarrhea receiving Oral Rehydration Therapy (ORT) at National Kanti Children's Hospital were included in this study. Stool samples of these patients were investigated at the Microbiology Laboratory, Department of Microbiology, Institute of Medicine. None of the stool samples showed the growth of Vibrio cholerae 0139 synonym Bengal. In Nepal, V.cholerae could be isolated from June to November. From December to May, no cases of V. cholerae were detected. Therefore, we address to this incidence as outbreaks rather than endemic. Mixed infections along with V. cholerae were also seen in 29% of cholera patients. V. cholerae 01, Hikojima types were the major isolates in our study followed by Ogawa type. V. cholerae, Hikojima and Ogawa serotypes were associated with mixed infection in 16.1% and 12.9% of patients, respectively. These isolates were associated with Shigella, Salmonella and pathogenic E. coli.