Self-limiting nature of seasonal cholera epidemics: Role of host-mediated amplification of phage

Phage predation of Vibrio cholerae has recently been reported to be a factor that influences seasonal epidemics of cholera in Bangladesh. To understand more about this phenomenon, we studied the dynamics of the V. cholerae-phage interaction during a recent epidemic in Dhaka. Because the outbreak strain causing this epidemic was resistant to multiple antibiotics, including streptomycin, we used a selective medium containing streptomycin to monitor accurately the abundance of this strain in the environment. The changing prevalence in the environment of the epidemic V. cholerae O1 strain and a particular lytic cholera phage (JSF4) to which it was sensitive was measured every 48-72 h for 17 weeks. We also monitored the incidence of phage excretion in stools of 387 cholera patients during the epidemic. The peak of the epidemic was preceded by high V. cholerae prevalence in the environment and was followed by high JSF4 phage levels as the epidemic ended. The buildup to the phage peak in the environment coincided with increasing excretion of the same phage in the stools of cholera patients. These results suggest that patients toward the end of the epidemic ingested both JSF4 phage and the outbreak V. cholerae strain. Host-mediated phage amplification during the cholera epidemic likely contributed to increased environmental phage abundance, decreased load of environmental V. cholerae and, hence, the collapse of the epidemic. Thus, in vivo phage amplification in patients and subsequent phage predation in the environment may explain the self-limiting nature of seasonal cholera epidemics in Bangladesh.     

Phage types of Vibrio cholerae O1 and O139 in the past decade in India

Cholera has been a prevalent disease worldwide since the early 19th century. Vibrio cholerae O1 and O139 are the two serogroups that have been mainly implicated in causing cholera. This study reports the results of biotyping, serotyping and phage typing of V. cholerae O1 and O139 (1998-2007) strains received from different parts of India for the identification of the trends in the occurrence and spread of cholera in the country. However, there has been a notable steep decline in the occurrence of V. cholerae O139 strains over the past few years resulting in no strain of V. cholerae O139 being received from any part of India in 2007 and 2008. Of the total strains received, 79.1% were serotyped as Ogawa and the remaining 20.9% were found to be Inaba, which indicates that Ogawa was the predominant serotype. Almost 100% typeability was observed with the new scheme of V. cholerae O1, with type 27 being the dominant phage type and V. cholerae O139 strains were clustered into the predominant phage type T-1. From the phage typing and serotyping results, it can be concluded that V. cholerae O1 (T-27) and O139 (T-1) strains circulate throughout the country at any given time.     

The characterization of Vibrio cholerae isolated in Kenya in 1983

A total of 245 strains of Vibrio cholerae 01 and two strains of V. cholerae non-01 were isolated and collected from diarrhoeal patients in Homa Bay District Hospital and the other medical facilities in Nyanza Province, Kenya in 1983. The majority of V. cholerae 01 tested were Ogawa type (with the exception of nine Inaba type), biotype E1 Tor (except one untypable strain) and Celebes original type (except one cured type). Haemolytic activity to sheep red blood cells was detected in 75.5% of isolates. Out of 245 strains of V. cholerae 01, 184 were resistant to tetracycline, streptomycin and ampicillin. All were sensitive to chloramphenicol and nalidixic acid. Only one strain of V. cholerae 01 was sensitive to all five antimicrobial agents tested. An environmental cholera survey was done after the cholera outbreak subsided. Twenty strains of V. cholerae non-01 were isolated from water samples in Nyanza Province but none of V. cholerae 01 was isolated.     

Emergence of Vibrio cholerae O1 biotype El Tor serotype Inaba causing outbreaks of cholera in Orissa, India

A total of 431 rectal swabs, collected from acute diarrheal cases at a surveillance site and at different diarrheal outbreak areas of Orissa from May to October 2005, were bacteriologically analyzed. Out of 265 culture-positive samples, Vibrio cholerae O1 was isolated in 56 samples (20.8%), of which 37 were the Inaba serotype and 19 were the Ogawa. The antibiogram profile revealed that all the V. cholerae O1 Ogawa and Inaba serotypes were uniformly sensitive to ampicillin, chloramphenicol, gentamicin, ciprofloxacin, norfloxacin and tetracycline. The V. cholerae O1 Inaba serotypes were resistant to furazolidone and nalidixic acid, while the Ogawa strains were resistant to furazolidone, nalidixic acid and neomycin. The multiplex polymerase chain reaction (PCR) assay on some selected strains of both serotypes revealed that all the strains were positive for ctxA and tcpA genes showing biotype El Tor. The present study revealed the emergence of V. cholerae O1 biotype El Tor serotype Inaba, which caused sporadic outbreaks of cholera in 2005. The outbreaks of diarrheal disorders in one geographical area of the state (in the Pattamundai area, Kendrapara district) in 2005 were due to V. cholerae O1 Ogawa, whereas the other outbreaks in other areas (Puri, Khurda and Dhenkanal districts) from August to October 2005 were due to V. cholerae O1 serotype Inaba. This is the first report that an emergence of V. cholerae O1 serotype Inaba caused sporadic outbreaks of cholera in different parts of Orissa. Switching over of V. cholerae O1 Ogawa strains to Inaba, causing diarrheal outbreaks in Orissa, needs close monitoring.     

Epidemiology of antimicrobial resistant cholera in Kenya and East Africa

Strains of Vibrio cholerae O1, El Tor resistant to multiple antimicrobial agents, were isolated in Kenya between 1982 and 1985. Strains of serotype Ogawa were resistant to tetracycline, ampicillin, and trimethoprim/sulfamethoxazole. Resistance was mediated in all instances by a plasmid ca 100 mD of incompatibility group C. Based on analysis of restriction endonuclease digests, all Ogawa isolates had an identical resistance plasmid. This plasmid differed from plasmids in resistant V. cholerae O1 strains isolated in Tanzania, Nigeria, and Bangladesh. On Southern blot analysis of restriction endonuclease digests of chromosomal DNA using DNA probes there were no apparent differences between Kenyan V. cholerae O1 strains isolated before and after emergence of antibiotic resistance; however, a majority of El Tor strains isolated in other geographic areas had the same Southern blot pattern. Our data document the apparent endemicity of multiply antimicrobial resistant V. cholerae O1 strains in Kenya, and the persistence of a single unique resistance plasmid among isolates of serotype Ogawa.     

An El Tor cholera outbreak in Maldah district, West Bengal

An outbreak of cholera occurred in Maldah district, West Bengal during July-August 1998. Attack rate was 34/1000. Cases were more (59.3%) amongst adults (> 15 years.). V. cholerae 01 biotype E1 Tor serotype ogawa was isolated as a single pathogen from 52.9% (9/17 samples examined). All V. cholerae strains belonged to phage type 2 (Basu and Mukherjee scheme) and type 27 (new phage type scheme). The strains were resistant to co-trimoxazole, furazolidone, ampicillin, streptomycin and nalidixic acid.     

Modeling the role of bacteriophage in the control of cholera outbreaks

Cholera is a waterborne diarrheal disease that continues to plague the developing world. Individuals become infected by consuming water from reservoirs contaminated by virulent strains of the bacterium Vibrio cholerae. Epidemiological and environmental observations of a cholera outbreak in Dhaka, Bangladesh, suggest that lytic bacteriophage specific for V. cholerae may limit the severity of cholera outbreaks by killing bacteria present in the reservoir and in infected individuals. To quantify this idea and generate testable hypotheses, we analyzed a mathematical model that combines the epidemiology of cholera with the population dynamics of the bacteria and phage. Under biologically reasonable conditions, we found that vibriophage can ameliorate cholera outbreaks. If phage predation limits bacterial density before an outbreak, a transient reduction in phage density can disrupt that limitation, and subsequent bacterial growth can initiate a cholera outbreak. The severity of the outbreak depends on the density of phage remaining in the reservoir. If the outbreak is initiated instead by a rise in bacterial density, the introduction of phage can reduce the severity of the outbreak and promote its decline. In both situations, the magnitude of the phage effect depends mainly on vibrio growth and phage mortality rates; the lower the rates, the greater the effect. Our analysis also suggests that either bacteria in the environmental reservoir are hyperinfectious or most victims ingest bacteria amplified in food or drinking water contaminated by environmental water carrying few viable V. cholerae. Our theoretical results make a number of empirically testable predictions.     

O139群霍乱弧菌分子流行病学研究

目的分析2001-2006年广州地区霍乱暴发、散发事件及外环境监测中分离的O139群霍乱弧菌的致病相关基因型和PFGE型,追踪菌株的来源和变迁,探讨本地区O139群霍乱流行特点。方法采用多重PCR方法检测O139群菌株的4种致病相关基因,应用脉冲场凝胶电泳技术(PFGE)对菌株进行分子分型,采用软件Bio Numerics Version4.0对分型数据进行处理和分析。结果广州地区O139群菌株中存在2种致病相关基因型,即A型和C型,18株感染者相关菌株中,除1株外,均为致病相关基因A型;10株珠江水分离株均为致病相关基因C型。28株O139群霍乱弧菌分为20个不同的PFGE型,归为4个聚类群(Ⅰ、Ⅱ、Ⅲ、Ⅳ群)。珠江水中O139群菌株存在PFGE克隆型的多样性。O139群暴发中分离的菌株PFGE型相同或相近,O139群散发疫情中的病例分离株与部分暴发株也具有相同或相近的PFGE型。结论分子分型方法结合流行病学资料,可分析O139群霍乱菌株的流行特点,致病相关基因分型可代替噬菌体-生物分型来判断和区分O139群霍乱弧菌的流行株与非流行株,从而为霍乱的预防、控制和预警提供科学依据。     

Seasonal epidemics of cholera inversely correlate with the prevalence of environmental cholera phages

The relationship among (i) the local incidence of cholera, (ii) the prevalence in the aquatic environment of Vibrio cholerae, and (iii) bacterial viruses that attack potentially virulent O1 and O139 serogroup strains of this organism (cholera phages) was studied in Dhaka, Bangladesh. Over nearly a 3-year period, we found that significantly more environmental water samples contained either a phage or a phage-susceptible V. cholerae strain than both (P < 0.00001). The number of cholera patients varied seasonally during this period and frequently coincided with the presence of pathogenic V. cholerae strains in water samples that otherwise lacked detectable cholera phages. Interepidemic periods were characterized by water samples containing cholera phages but no viable bacteria. Our data support the conclusion that cholera phages can influence cholera seasonality and may also play a role in emergence of new V. cholerae pandemic serogroups or clones.     

Are the environmental niches of Vibrio cholerae O139 different from those of Vibrio cholerae O1 El Tor?

BACKGROUND: Vibrio cholerae are known to be normal inhabitants of surface water. However, the environmental niches of the different strains of cholera are not well known, and therefore, populations at risk for cholera outbreaks cannot be clearly identified. METHODS: This study identifies environmental risk factors for cholera caused by V. cholerae O1 El Tor and O139 and environmental niches of the two strains present in Matlab, a cholera endemic area of Bangladesh. The study year was 1993, the year that the O139 strain first appeared in the study area. Patients who had either strain of cholera identified in a laboratory were included in the study. A geographic information system was used to map the household locations of the patients, to describe the human sanitary environment and population density, and to address potential anthropogenic and environmental risk factors of the disease. Spatial point pattern and exploratory spatial data analysis techniques were used to define the environmental niches of the two cholera strains. RESULTS: The study suggests the niches of O1 El Tor and O139 strains of V. cholerae appear to be similar, based on common environmental risk factors. CONCLUSIONS: The results of this study support a theory that O1 El Tor could possibly be replaced by the newer O139 strain in the future.     

Genomic analysis of the Mozambique strain of Vibrio cholerae O1 reveals the origin of El Tor strains carrying classical CTX prophage

Cholera outbreaks in subSaharan African countries are caused by strains of the El Tor biotype of toxigenic Vibrio cholerae O1. The El Tor biotype is the causative agent of the current seventh cholera pandemic, whereas the classical biotype, which was associated with the sixth pandemic, is now extinct. Besides other genetic differences the CTX prophages encoding cholera toxin in the two biotypes of V. cholerae O1 have distinct repressor (rstR) genes. However, recent incidences of cholera in Mozambique were caused by an El Tor biotype V. cholerae O1 strain that, unusually, carries a classical type (CTX(class)) prophage. We conducted genomic analysis of the Mozambique strain and its CTX prophage together with chromosomal phage integration sites to understand the origin of this atypical strain and its evolutionary relationship with the true seventh pandemic strain. These analyses showed that the Mozambique strain carries two copies of CTX(class) prophage located on the small chromosome in a tandem array that allows excision of the prophage, but the excised phage genome was deficient in replication and did not produce CTX(class) virion. Comparative genomic microarray analysis revealed that the strain shares most of its genes with the typical El Tor strain N16961 but did not carry the TLC gene cluster, and RS1 sequence, adjacent to the CTX prophage. Our data are consistent with the Mozambique strain's having evolved from a progenitor similar to the seventh pandemic strain, involving multiple recombination events and suggest a model for origination of El Tor strains carrying the classical CTX prophage.     

Endemicity of cholera in Surabaya, Indonesia.

As the seventh pandemic of cholera is caused by V. cholerae biotype El Tor, the former criteria for endemicity of cholera need to be reconsidered as regards their applicability in areas that are infected with cholera. As the mortality rate of cholera nowadays can be reduced to a very low level due to modern methods of treatment, it is suggested that the infection rates of cholera should be taken into consideration as criteria of cholera endemicity, i.e. 1. Five years persistence of cholera cases in a given area. 2. Five percent infection rate among family contacts of cholera cases. 3. Minimum infection rate of 1% in a vicinity where cholera cases occur. It was also found that in such an endemic area it is very difficult to eliminate V. cholerae infection from a locality, even when all family contacts are treated with the full dose of tetracycline.     

Molecular epidemiology of Vibrio cholerae O-1 from outbreak and sporadic patients in Nagoya in 1989

Enterotoxigenic strains of Vibrio cholerae O-1 biotype eltor, isolated from three sporadic cases of cholera in Nagoya in 1989 and an outbreak of cholera in Nagoya in 1989 were analyzed for their similarities. All isolates of V. cholerae O-1 were indistinguishable in bacteriophage types, serovars, biovars and drug resistance patterns. Because the epidemiological investigation based on a primary structure of chromosomal DNA is more reliable, we isolated chromosomal DNA from these isolates and compared electrophoretic patterns of restriction endonuclease-digested DNA fragments. Furthermore, Southern hybridization of the cholera toxin gene was performed. Since no difference among six strains in these sporadic was observed, it was strongly suggested that both the independent cases and the outbreak of cholera were caused by the same strain.     

Genomic diversity of 2010 Haitian cholera outbreak strains

The millions of deaths from cholera during the past 200 y, coupled with the morbidity and mortality of cholera in Haiti since October 2010, are grim reminders that Vibrio cholerae, the etiologic agent of cholera, remains a scourge. We report the isolation of both V. cholerae O1 and non-O1/O139 early in the Haiti cholera epidemic from samples collected from victims in 18 towns across eight Arrondissements of Haiti. The results showed two distinct populations of V. cholerae coexisted in Haiti early in the epidemic. As non-O1/O139 V. cholerae was the sole pathogen isolated from 21% of the clinical specimens, its role in this epidemic, either alone or in concert with V. cholerae O1, cannot be dismissed. A genomic approach was used to examine similarities and differences among the Haitian V. cholerae O1 and V. cholerae non-O1/O139 strains. A total of 47 V. cholerae O1 and 29 V. cholerae non-O1/O139 isolates from patients and the environment were sequenced. Comparative genome analyses of the 76 genomes and eight reference strains of V. cholerae isolated in concurrent epidemics outside Haiti and 27 V. cholerae genomes available in the public database demonstrated substantial diversity of V. cholerae and ongoing flux within its genome.     

Plasmid mediated antibiotic resistance of Vibrio cholerae O1 biotype El Tor serotype Ogawa associated with an outbreak in Kolkata,India

ObjectiveTo determine the antibiotic resistance of Vibrio cholerae (V. cholerae) O1 biotype El Tor serotype Ogawa isolates involved in an outbreak of watery diarrhea in Kolkata, and to explore the role of plasmid in mediating antibiotic resistance.MethodsAntibiotic susceptibility and minimum inhibitory concentration (MIC) values of antibiotics for the isolated V. cholerae O1 Ogawa (n=12) were determined by disk diffusion and agar dilution methods, respectively, using ampicillin (Am), chloramphenicol (C), trimethoprim (Tm), tetracycline (T), erythromycine (Er), nalidixic acid (Nx), ciprofloxacin (Cp), amikacin (Ak) and cefotaxime (Cf). Plasmid curing of multidrug resistant (MDR) V. cholerae O1 Ogawa strains was done following ethidium bromide treatment. Following electrophoresis, the plasmid DNAs, extracted from the isolated MDR V. cholerae O1 Ogawa strains and their cured derivatives, were visualized and documented in ‘gel doc’ system.ResultsThe outbreak causing V. cholerae O1 Ogawa isolates were MDR as determined by disk diffusion susceptibility test, and MIC determination. The isolates showed three different drug resistance patterns: AmTmTErNx (for 6 isolates), TmTErCp (for 5 isolates), and AmTmNx (for one isolate), and showed uniform sensitivity to C, Ak and Cf. The loss of plasmids with the concomitant loss of resistance to Am, Tm, T and Er of the isolates occurred following ethidium bromide treatment.ConclusionsThe current findings suggest that the V. cholerae O1 Ogawa associated with the cholera outbreak were MDR, and resistance to Am, Tm, T and Er among the isolates were plasmid mediated.     

Acquisition of classical CTX prophage from Vibrio cholerae O141 by El Tor strains aided by lytic phages and chitin-induced competence

The El Tor biotype of Vibrio cholerae O1, causing the current seventh pandemic of cholera, has replaced the classical biotype, which caused the sixth pandemic. The CTX prophages encoding cholera toxin in the two biotypes have distinct repressor (rstR) genes. Recently, new variants of El Tor strains that carry the classical type (CTX(class)) prophage have emerged. These "hybrid" strains apparently originate through lateral gene transfer and recombination events. To explore possible donors of the CTX(class) prophage and its mode of transfer, we tested environmental V. cholerae isolates for the presence of CTX(class) prophage and mobility of the phage genome. Of the 272 environmental V. cholerae isolates tested, 6 were found to carry the CTX(class) prophage; all of these belonged to the O141 serogroup. These O141 strains were unable to produce infectious CTX(class) phage or to transmit the prophage to recipient strains in the mouse model of infection; however, the CTX(class) prophage was acquired by El Tor strains when cultured with the O141 strains in microcosms composed of filtered environmental water, a chitin substrate, and a V. cholerae O141-specific bacteriophage. The CTX(class) prophage either coexisted with or replaced the resident CTX(ET) prophage, resulting in El Tor strains with CTX genotypes similar to those of the naturally occurring hybrid strains. Our results support a model involving phages and natural chitin substrate in the emergence of new variants of pathogenic V. cholerae. Furthermore, the O141 strains apparently represent an alternative reservoir of the CTX(class) phage genome, because the classical V. cholerae O1 strains are possibly extinct.     

Seasonal, nontoxigenic Vibrio cholerae O1 Ogawa infections in the Eastern Region of Saudi Arabia.

BACKGROUND: Surveillance for Vibrio cholerae in the Eastern Region of Saudi Arabia has been ongoing since 1985 to detect and prevent local proliferation of imported cholera. In 1996 and 1997 the authors performed additional microbiologic and epidemiologic assessment of V. cholerae surveillance to better characterize a recurrent summertime pattern of V. cholerae infections in the Eastern Region of Saudi Arabia. METHODS: All health facilities routinely submitted stool or rectal swab specimens for isolation of V. cholerae from patients with gastroenteritis. In addition, specimens were taken from expatriate workers and household contacts of persons with confirmed V. cholerae infection. Forty-two isolates were evaluated for cholera enterotoxin by enzyme-linked immunosorbent assay, cholera toxin polymerase chain reaction, and Y1 adrenal cell assay; 12 isolates also were characterized by pulsed-field gel electrophoresis (PFGE). Interviews about potential exposures were done for all V. cholerae infections. RESULTS: Vibrio cholerae O1 serotype Ogawa biotype El Tor was identified in 113 gastroenteritis patients (6.0 per 100,000 population per year), 28 asymptomatic expatriate workers, and 16 of 982 household contacts of index patients. All symptomatic infected persons had mild illness that was not typical of cholera, and all 42 isolates evaluated were nontoxigenic. All 12 isolates evaluated by PFGE had an indistinguishable pattern (pattern 81). Infections appeared in late May, decreased in mid-July through August, increased again in September, and disappeared from December through April. Infections had a uniform geographic distribution and affected all ages. No linkage was identified between affected households, or between community cases and food-handlers or domestic servants. DISCUSSION: Surveillance in the Eastern Region of Saudi Arabia has identified a novel strain of nontoxigenic V. cholerae O1 Ogawa. This strain probably has a local environmental reservoir. Since cholera toxin is the primary virulence factor involved in the cause of cholera, assays for cholera toxin should be included in cholera surveillance.     

Seasonality and antimicrobial resistance pattern of Vibrio cholerae in a tertiary care hospital of North India

We retrospectively analysed the seasonal distribution of cholera and the antimicrobial resistance pattern of Vibrio cholerae isolates over a 5-year period from January 1998 to December 2002. Of 3213 stool specimens processed from 3213 patients with acute watery diarrhoea during this period, 431 samples (13.4%) were found positive for V. cholerae. There were 423 V. cholerae O1 biotype E1 Tor, 2 V. cholerae O139 and six isolates of non-O1 non-O139. The highest number of cholera cases occurred in May-June followed by July-August. Cases started appearing in April for all years except in the year 2002 when three cases occurred in the first week of March. A large number (90.25% strains) were resistant to at least one antibiotic.     

Seroepidemiological studies of El Tor cholera in Bangladesh: association of serum antibody levels with protection

In rural Bangladesh, family contacts of patients with cholera were studied prospectively to examine whether protection against colonization and disease due to Vibrio cholerae O1 was associated with circulating antibodies to V. cholerae. Family contacts (1,071) of 370 patients with cholera were visited daily for 10 days, cultured for V. cholerae, and queried about diarrhea. Sera collected on days 1 and 21 were assayed for vibriocidal antibodies, IgG and IgA antibodies to cholera toxin, and IgG antibodies to lipopolysaccharide (LPS). Vibriocidal titers of greater than or equal to 20 present in 50% of contacts by 20 years of age were associated with protection against both colonization and disease. An elevated level of IgG antitoxin was not associated with protection against colonization or disease but was the most sensitive indicator of recent symptomatic cholera and of immune response to the oral immunogen B subunit. IgG antibody to LPS and IgA antitoxin were of little value in predicting colonization or disease.     

Incomplete correlation of serum vibriocidal antibody titer with protection from Vibrio cholerae infection in urban Bangladesh

The serum vibriocidal antibody is the only recognized predictor of protection from cholera, but no seroepidemiological data have been gathered since the emergence of Vibrio cholerae O139. We assessed the association between the vibriocidal antibody titer and protection from cholera in an endemic setting. Although a higher baseline vibriocidal titer correlated with protection from V. cholerae O1, infection still developed in some contacts with very high titers. No association between baseline vibriocidal titer and protection from V. cholerae O139 infection was found. Our findings suggest that the vibriocidal antibody is an incomplete predictor of protection from V. cholerae infection.