Cholera Research Articles

Using polymerase chain reaction cycle threshold (PCR Ct) values for predicting and managing cholera outbreaks: potential, challenges, and future directions

Abstract Background Cholera continues to be a major public health threat, especially in regions with poor sanitation and limited access to clean water. Accurate prediction and management of cholera outbreaks are essential to reduce mortality and morbidity. This review focuses on the potential of polymerase chain reaction (PCR) cycle threshold (Ct) values as an innovative tool for early detection and control of cholera outbreaks, specifically highlighting its utility in predicting outbreaks and guiding public health responses. Main body of abstract PCR Ct values offer a significant advantage in the early detection of Vibrio cholerae, the pathogen responsible for cholera, in both clinical and environmental samples. By providing quantitative data on bacterial load, lower Ct values indicate a higher concentration of the pathogen, signaling the potential for increased disease transmission. These values allow for more precise, real-time monitoring of cholera outbreaks and aid in targeting intervention strategies such as water sanitation improvements, vaccination campaigns, and antibiotic treatments. Moreover, the application of Ct values in environmental surveillance, particularly in monitoring water sources, offers a proactive approach to prevent the spread of cholera by identifying contamination risks before human cases arise. However, the implementation of PCR in low-resource settings faces significant challenges, including high costs, the need for advanced laboratory infrastructure, and a lack of technical expertise. Addressing these barriers through cost-effective innovations and capacity building initiatives is crucial for optimizing the use of PCR Ct values in cholera management. Conclusion PCR Ct values hold great promise for improving cholera outbreak prediction and control. Despite challenges in adopting this technology in resource-limited settings, the integration of Ct value monitoring into public health frameworks can enhance early detection efforts and contribute to more effective cholera management strategies.

Zambia's battle against cholera outbreaks and the path to public health resilience: a narrative review

ABSTRACT In Zambia, cholera has been a persistent public health concern for decades, mainly attributed to inadequate sanitation and restricted access to clean water in some parts of the country. The literature was collected from PubMed, Google Scholar, and public health organization websites, focusing on cholera outbreaks in Zambia since 2000. Key search terms included ‘cholera prevention’ and ‘Zambia outbreaks.’ A total of 30 articles were analyzed to assess public health strategies and identify gaps for future planning. Periodic outbreaks of cholera have characterized Zambia's history by significant fluctuations in case numbers and fatalities since 1977. Notably, the years 1993, 1999, 2003/2004, 2005/2006, 2010, 2017/2018, and the most recent outbreak from October 2023 to February 2024 have marked significant episodes in the country's struggle against this waterborne disease. This narrative review examines the recurrent outbreaks of cholera in Zambia to understand the recent outbreak's extraordinary spread and severity in the context of public health resilience. The most recent outbreak with staggering 19,719 cases and 682 deaths, underscores the unprecedented scale and severity of the current public health emergency. The findings highlight the need for a holistic public health approach that prioritizes resilience in disease prevention.

Barriers and Strategies for Rotavirus and Cholera Vaccine Uptake: A Systematic Review of Community-based Interventions in Bangladesh, India, and Pakistan

Background: Diarrhea is the second leading cause of death in children and poses a major threat to public health. It is mainly caused by pathogenic organisms of viral, bacterial, or fungal origin, two of which has been preventable via vaccination and has been identified as rotavirus and cholera. In 2020, India, Pakistan, and Bangladesh were among the top 10 South Asian countries with the highest mortality rates from childhood diarrhea. India and Pakistan are further listed by the WHO among the top five countries with the highest recorded mortality among children under five years old. Bangladesh has experienced the highest number of cholera epidemics. Vaccination is recommended as a potent preventive approach against rotavirus and cholera induced diarrhea and is considered the most cost-effective prevention method. However, despite their proven effectiveness, other factors appear to affect the impact of vaccination strategies in these countries. Therefore, this study aimed to explore these nonclinical factors. Methods: This study was conducted as a systematic review which involved the systematic search and selection of qualitative data from primary studies concerning the research topic which was developed using the SPIDER framework. The PRISMA tool was used to carry out this process, and the CASP was used to evaluate the methodological quality of each study. The combined data were then harmonized using meta-synthesis and analyzed thematically. Findings: Four themes emerged from the analysis of the study data: pre-vaccination strategy experience, post-vaccination strategy impact, opportunities for vaccination strategies, and threats to successful implementation of vaccination strategies. This formed the basis of the study discussion and expounded on the impact of vaccine strategies in the selected countries. Conclusion: The results of this study showed that providing the participants with correct information, vaccine education, willingness to learn despite poor knowledge, and access to vaccination are important solutions to the factors that surround the uptake and coverage of cholera and rotavirus vaccines in Bangladesh, India, and Pakistan as critical influencers of the prevalence of childhood diarrhea induced by rotavirus and cholera pathogens, respectively.

When Angels Misbehaved: Justiciability of the United Nations for the Cholera Outbreak in Hait

When Angels Misbehaved: Justiciability of the United Nations for the Cholera Outbreak in Hait

Retrospective analysis of a large-scale cholera outbreak in Sudan

Background: Cholera outbreak is a significant public health threat in some parts of Sudan. In 2023, there was a large-scale outbreak in Gadarif State. Aim: To analyse the 2023 cholera outbreak in Sudan and recommend prevention and control measures for the future. Methods: In this retrospective study we conducted an epidemiological analysis of data from suspected and confirmed cholera cases, aged ≥ 2 years, from 138 health centres in Gadarif State, Sudan. Results: A total of 1997 cholera cases were confirmed using rapid diagnostic test between 25 August and 17 December 2023. Most patients (99.2%) had severe watery diarrhoea, and vomiting was reported in over 73% of cases. Dehydration rates were similar for men and women (≈55–60%), average attack rate was 6.7 per 10 000 population. The attack rate varied by locality but not by gender across all age groups. The outbreak lasted 20 weeks and claimed 46 lives, giving a case fatality ratio of 2.6%. Public misconception about water chlorination substantially hindered control efforts. Conclusion: The prolonged period of the outbreak and the high case fatality ratio highlight the need for better laboratory and epidemiologic surveillance as well as better preparedness and response for future outbreaks, along with educational activities to address myths and misconceptions.

Epidemiological Investigation of a Cholera Outbreak in Nepal-India Border Communities: Public Health Implications

ObjectivesOn October 4, 2021, a cholera outbreak was reported in Kapilvastu district, one of the 26 districts in Nepal bordering India. This study examined the outbreak's characteristics, response efforts, and the challenges faced. MethodsA descriptive cross-sectional design was applied for the outbreak investigation. Data was gathered through a review of cases registered in three local health facilities and community-based active case searching. Descriptive analysis was conducted for data analysis. ResultsBetween 4 October and 2 December 2021, 1,570 cases were reported across 10 municipalities in Kapilvastu, with 84% of cases occurring in communities bordering India. Most affected were females (55%) and children aged 5-14 years (32%). A total of 39% cases were confirmed to have Vibrio cholerae O1 Ogawa serotype, which showed resistance to cotrimoxazole. Additionally, 453 cases of acute watery diarrhea were identified in the community, with 23% showing the signs of dehydration. Of 29 water samples, 17 (59%) were contaminated with fecal coliform. ConclusionCommunities along the Nepal-India border are highly vulnerable to cholera outbreaks, highlighting the urgent need for improved water, sanitation and hygiene practices. A coordinated approach to cholera surveillance and preparedness is crucial to prevent future outbreaks.

Environmental Vibrio cholerae Strains Harboring Cholera Toxin and Vibrio Pathogenicity Island 1, Nigeria, 2008-2015.

Analysis of clinical and environmental Vibrio cholerae O1 strains obtained during 2008-2015 in Nigeria showed that lineages Afr9 and Afr12 carrying cholera toxin and Vibrio pathogenicity island 1 can be isolated from water. Our findings raise concerns about the role of the environment in maintenance and emergence of cholera outbreaks in Nigeria.

Critical Evaluation of Neglected Consensus Risk Factors Causing the Recurrent Upsurge of Cholera Outbreaks in Some Regions in Cameroon: A Systematic Review

Background: Cameroon is a developing nation and has been enlisted amongst the several countries in sub-Sahara Africa situated in West and Central Africa to be experiencing the recurrent cholera outbreaks. The World Health Organization (WHO), Cameroon health authorities and others researchers have considered a few risk factors that are believed to be contributing to the current and on-going cholera outbreaks in Cameroon. These risk factors include the wide circulation of Vibrio cholerae in the country, limited access to safe-drinking water in some areas, a seasonal pattern of cholera re-occurrence and inadequate water, sanitation and hygienic (WASH) conditions, unstable political atmosphere that have resulted to internally displayed persons, very poor and vulnerable persons, refugees and humanitarian crises and continued unnecessary imposed lock-downs and road-blocks. However, despite these enormous efforts made by Cameroon health authorities so far and its partners over the past decades in responding and tackling all these risk factors, the recurrent outbreak of cholera in Cameroon still remained a great challenge and potential humanitarian crisis, an unendingly reality with bewilderment amid uncertainty, threatening and vulnerability to many affected regions. Aim: This systematic review seeks to critically evaluate, identify and classify some previously overlooked consensus risk factors driving cholera endemicity, pandemicity or epidemicity in Cameroon. By highlighting these neglected factors, this review hopes to inform evidenced -based intervention to finally combat the persistence recurrent upsurge and burden of future outbreaks of cholera in Cameroon. Methodology: The current Systematic reviews gathered information from various sources using different search engines provide a comprehensive overview of existing research. These include Academic Search Engines, Specialized Search Engines, Grey Literature, Primary Sources, Secondary Sources, Registers and Repositories, Open Access Sources, Hand Searching and Other Sources. Results: The current systematic review gathered information from diverse sources and identified and classified six groups of neglected consensus risk factors, along with their associated variables, contributing to cholera outbreaks in Cameroon. Additionally, the review assessed the level of intervention and preventive measures offered by the Cameroon health authorities and other stakeholders, categorizing them as either low or high, for both documented and undocumented strategies. All data sources were thoroughly referenced accordingly ensuring transparency and accountability. Conclusion: In spite of extensive research by the World Health Organization and Cameroonian health authorities identifying and documenting numerous risk factors contributing to Cameroon's ongoing cholera outbreaks, these efforts have not prevented or eradicated recurring episodes. Decades of enormous work and efforts by Cameroonian health authorities have been incapable to eradicate cholera. This systematic review unequivocally shows that without addressing the neglected consensus risk factors and variables, Cameroon could face a high likelihood of continued cholera outbreaks. Therefore, this review underscores the urgent need for Cameroon health authorities to adopt a multifaceted approach, addressing the identified neglected consensus risk factors and variables. Implementation of targeted interventions, strengthened health infrastructure, and community engagement are crucial to prevent future cholera outbreaks. Future research should focus on developing context-specific solutions, enhancing existing strategies, and evaluating the effectiveness of interventions.

The Costs and Cost-Effectiveness of a Two-Dose Oral Cholera Vaccination Campaign: A Case Study in a Refugee Camp Setting in Thailand

Oral cholera vaccination (OCV) campaigns are increasingly used to prevent cholera outbreaks; however, little is known about their cost-effectiveness in refugee camps. We conducted a cost-effectiveness analysis of a pre-emptive OCV campaign in the Maela refugee camp in Thailand, where outbreaks occurred with an annual incidence rate (IR) of up to 10.7 cases per 1000. Data were collected via health sector records and interviews and household interviews. In the base-case scenario comparing the OCV campaign with no campaign, we estimated the campaign effect on the cholera IR and case fatality rate (CFR: 0.09%) from a static cohort model and calculated incremental cost-effectiveness ratios for the outcomes of death, disability-adjusted life-years (DALYs), and cases averted. In sensitivity analyses, we varied the CFR and IR. The household economic cost of illness was USD 21, and the health sector economic cost of illness was USD 51 per case. The OCV campaign economic cost was USD 289,561, 42% attributable to vaccine costs and 58% to service delivery costs. In our base case, the incremental cost was USD 1.9 million per death averted, USD 1745 per case averted, and USD 69,892 per DALY averted. Sensitivity analyses that increased the CFR to 0.35% or the IR to 10.4 cases per 1000 resulted in a cost per DALY of USD 15,666. The low multi-year average CFR and incidence of the cholera outbreaks in the Maela camp were key factors associated with the high cost per DALY averted. However, the sensitivity analyses indicated higher cost-effectiveness in a setting with a higher CFR or cholera incidence, indicating when to consider campaign use to reduce the outbreak risk.

Recurrence of cholera epidemics in South Africa: inadequate sanitary facilities, poor environmental monitoring practices, and climate change as possible contributing factors

Cholera is an acute infectious disease caused by two toxin-producing strains, namely Vibrio cholerae O1, and Vibrio cholerae O139. Its recorded history stretches as far back as 1817, in Bengal, India, yet it continues to be a public health threat to this present day. Although cholera epidemics are now a relatively rare phenomenon in developed countries, frequent epidemics continue to be experienced in sub-Saharan Africa. Africa accounted for 63.33% of the 223 370 cases of and 4 159 deaths from cholera that were reported globally in 2020. Furthermore, of the 342 900 cholera cases and 3 304 cholera deaths reported globally in 2022, 40.28% of the cases and 77.85% of the deaths were in Africa. Inadequate sanitation infrastructure as well as inadequate provision of quality, safe drinking water in South Africa still creates conducive conditions for the transmission of cholera. In addition, climate change is increasingly becoming a risk factor towards the spread of V. cholerae pathogens in inland regions. To make recommendations on how South Africa, and potentially the Southern African region as a whole, can minimise the resurgence of cholera, this review addressed the following questions: Does South Africa have adequate sanitation infrastructure to curb the spread of cholera? Is there enough intentional surveillance of environmental water sources for vibrios as a cholera outbreak predictive tool? What is the impact of climate change on the resurgence of cholera in South Africa? And, what needs to be done to curb the resurgence of cholera in South Africa?

Cholera outbreak in diyala province

Background: Vibrio cholerae is a primary cause of severe dehydrating diarrhea and remains a serious public health problem in low- and middle-income countries (LMICs) with insufficient water, sanitation, and hygiene. Cholera remains a public health threat the worldwide, mainly in countries with poor access to safe water and sanitation facilities. According to estimates, there are 21,000–143,000 deaths and 1.3–4 million cases of cholera annual. Objective: This study aims to carry out an analysis of the available information on cholera outbreaks in Diyala province, 2022. Patients and Methods: A descriptive study conducted in the department of public health using the data of cases reported from eight sectors in province. The data was collected from the surveillance unit, communicable control division of the public health department in Diyala province, IRAQ in 2022. Results: The total number of confirmed cases of cholera was (n = 139), female (72) 52 % and male (67) 48%, death reported for two of these cases. According to the date of onset, the registration of cases began from July 19 to October 25, 2022. However, the case fatality ratio (CFR) was 1.4%, while the attack rate in the governorate is 7.6 per 100,000 populations during 2022, the highest attack rate in Khanaqin district was (77 cases) 46, while the lowest attack rate in Baldruze, Al-Mansuria, and Al-Khalis districts was (1 case) 0.3,0.6, and 0.8 respectively. The age mean of patients was (38.7 ± 17.14 years) and the occurrence of the disease is more incidence in the age group 30-44 and females more than males. Conclusion: Occurrence Cholera outbreak during year 2022 without confirmed cases recorded since 2015 and the occurrence of cholera cases in governorate is greater during the months of August and September and most of the cases were from Khanaqin district.

Social and Behavioural Change Communication Challenges, Opportunities and Lessons from Past Public Health Emergencies and Disease Outbreaks: A Scoping Review.

Background: Documentation of social behavioural change communication (SBCC) regarding challenges, opportunities and lessons drawn from past public health emergencies is worthwhile to inform priorities for future response efforts. Aim: The aim of this review is to scope the evidence on social behavioural change communication regarding challenges, opportunities and lessons drawn from Ebola, coronavirus disease 2019 (COVID-19), monkeypox and cholera outbreaks from studies published before March 2024, and suggest priorities for future response efforts. Methods: A Boolean strategy was used to search electronic databases for relevant published articles, complemented by relevant studies identified from reference lists. Results: The challenges, opportunities, lessons learnt and priorities for SBCC were consistent across study contexts, showing marked variations over time. The significance of technology, infodemic management, and behavioural data generation emerged more frequently and became increasingly important over time. Identified challenges were uptake hesitancy, limited capacity to undertake infodemic management, inadequate funding and human resources for SBCC, competing priorities, parallel or conflicting interventions due to inadequate coordination, difficulties evaluating SBCC programmes and missed opportunities for integration into routine programmes. Existing supportive structures for SBCC, strong political will and participation, as well as rapid information exchange enabled by technological advancement, represented opportunities for enhancing the effectiveness of SBCC programmes. Key lessons were that a multisectoral approach and coordination, partnership and active collaboration amongst stakeholders; building/strengthening trust, target population segmentation and localization of interventions, are important for enhancing the effectiveness of SBCC programmes. Political will, involvement and participation represent the core of social behavioural change (communication) interventions during a public health emergency. Conclusion: SBCC programming for future response to public health emergencies and disease outbreaks should consider the diverse assortment of benefits, threats/challenges and opportunities brought about by technology, infodemics and behavioural data generation to be more effective.

Fine-tuning of a CRISPRi screen in the seventh pandemic Vibrio cholerae

BackgroundVibrio cholerae O1 El Tor, the etiological agent responsible for the last cholera pandemic, has become a well-established model organism for which some genetic tools are available. While CRISPRi technology has been applied to V. cholerae, improvements were necessary to upscale it and enable pooled screening by high-throughput sequencing in this bacterium.ResultsIn this study, we present a genome-wide CRISPR-dCas9 screen specifically optimized for the N16961 El Tor model strain of V. cholerae. This approach is characterized by a tight control of dCas9 expression and activity, as well as a streamlined experimental setup. Our library allows the depletion of 3,674 (98.9%) annotated genes from the V. cholerae genome. To confirm its effectiveness, we screened for genes that are essential during exponential growth in rich medium and identified 369 genes for which guides were significantly depleted from the library (log2FC < -2). Remarkably, 82% of these genes had previously been described as hypothetical essential genes in V. cholerae or in a closely related bacterium, V. natriegens.ConclusionWe thus validated the robustness and accuracy of our CRISPRi-based approach for assessing gene fitness in a given condition. Our findings highlight the efficacy of the developed CRISPRi platform as a powerful tool for high-throughput functional genomics studies of V. cholerae.

Romania and the International Sanitary Conferences of the Late Nineteenth and Early Twentieth Centuries

This paper traces Romania’s participation in the International Sanitary Conferences of the late nineteenth and early twentieth centuries. Romania’s interest in international sanitary diplomacy was directly linked to the country’s geographical location and the role of the Danube in the spread of epidemics. The cholera outbreak that reached the Lower Danube in 1865 demonstrated that the river, which connected the Levant to Central Europe via steamship companies, was one of the main gateways through which epidemics spread to the continent. With their busy commercial links to other Black Sea ports, Sulina, Galați and Brăila were equally vulnerable to the importation of diseases that reached Ottoman or imperial Russian ports by land, i.e., via Persia or Central Asia. Such serious health concerns became even more important to the Romanian government after the Danube Delta and Dobrudja became part of independent Romania in 1878. The text follows the various interests that Romania had during these decades, defended by diplomats and doctors who worked together to protect public health from various epidemic, political or economic threats.

Pioneer of epidemiology and anesthesiology – John Snow (1813–1858)

IntroductionJohn Snow, a British physician of the 19th century, played a pivotal role in challenging prevailing medical beliefs during his time. His groundbreaking studies on the cholera epidemic led to the rejection of the miasma theory and the establishment of the link between contaminated water and the spread of the disease.AimThis study aims to explore the contributions of John Snow to the fields of anaesthesiology and epidemiology, focusing on his significant findings and methodologies.Material and methodsThis study utilizes a literature review approach to examine John Snow's contributions to anaesthesiology and epidemiology. Searches were conducted in databases using relevant keywords, focusing on peer-reviewed articles and authoritative sources. The data was qualitatively synthesized to extract key insights into Snow's methodologies and findings, aiming to provide a brief overview of his impact on medical science.Results and discussion:John Snow (1813-1858) is celebrated as a pioneer in epidemiology and anaesthesiology. He identified contaminated water as the source of the 1854 cholera outbreak in London, establishing the basis for modern epidemiology. Snow's innovative mapping of cholera cases was crucial in controlling the epidemic. He also advanced anaesthetic techniques, notably using chloroform on Queen Victoria during childbirth, improving the safety of anaesthesia. His work remains influential in both fields.Conclusions:John Snow's pioneering work significantly impacted the development of anaesthesiology and epidemiology. His insights into cholera transmission and anaesthetic agents, along with his seminal publication On Chloroform and Other Anaesthetic, continue to inspire advancements in medical science. Snow's legacy underscores the importance of evidence-based medicine and the pursuit of knowledge in addressing public health challenges.

Occurrence of Extended-spectrum β-lactamase (ESBL) and Carbapenemase-producing Escherichia coli isolated from Childhood Diarrhoea in Yaoundé, Cameroon

IntroductionExtended-spectrum β-lactamase (ESBL)-producing pathogenic E. coli is a global public health issue, especially in sub-Saharan African countries such as Cameroon. It contributes to increase significantly hospital length of stay, morbidity, mortality and economic costs because of treatment failures. This study aims at determining the resistance background and virulence profiles of ESBL-E. coli isolates among childhood diarrhoea during the cholera outbreak occuring in Yaoundé, Cameroon.Materials and methodsDuring a four-month periods, from March 1st to June 30th, 2023, a total of 84 stool samples were collected from 90 under five children presenting clinical signs of gastroenteritis and attending four hospitals in Yaoundé, Cameroon. Bacterial identification was done using API20E and antimicrobial susceptibility test was performed using the Kirby-Bauer disc diffusion method. After extraction, genomic DNA was subjected to conventional and multiplex polymerase chain reaction methods (PCRs) for detection of resistance and virulence genes. Statistical analysis was performed using Epi info™ (7.2.5.0). Statistical significance was considered at a p-value < 0.05.ResultsOut of 150 patients contacted, 90 patients were enrolled, 84 samples were collected, 52.38%(44/84) and 3.57%(03/84) were confirmed as extended-spectrum β-lactamase and carbapenemase-producing E. coli respectively. The risk factors were analyzed, and children who drank natural fruit juice (OR: 0.4, p-value: 0.03) were found to be significantly associated with ESBL-producing E. coli. The ESBL-producing E. coli isolates showed a high level of resistance to amoxicillin-clavulanic acid, cefotaxime, ceftazidime, cefepime, colistin, and tetracycline. The blaCTX−M was more prevalent ß-lactamase resistance gene. The tetracycline resistance genes tet(A) and tet(B) were also detected. The most important virulence genes detected were FimH (81.81%) and papA (79.54%).ConclusionThese findings suggest implementing routine surveillance and screening for antimicrobial resistance among children under five. Antimicrobial stewardship strategies (ASP) need to be implemented to curb the emergence and dissemination of ESBL-producing E. coli. In addition, a national surveillance program for antimicrobial resistance needs to be implemented at local and regional levels in order to reduce morbidity in Cameroon.

Burden of childhood diarrhea and cholera outbreaks in the Lake Tana Basin (Ethiopia): Review

Objectives: Diarrhoea is one of the leading causes of childhood morbidity and mortality, while cholera outbreaks are a major public health emergency insub-Saharan African countries. This study was aimed at investigating the link between climate changes and cholera outbreaks, and the burden of childhood diarrhoea in the Lake Tana Basin (Ethiopia). Methods: Research articles published in English were searched from Google scholar, PubMed, and Web of science and these were supplemented by a four-year secondary data of childhood diarrhoea and cholera outbreaks extracted from health management information system (HMIS) in 61 districts. Results: The mean prevalence of diarrhoea per 1000 children was 420 (95% CI:311.7; 528.6). The prevalence of childhood diarrhoea showed spatial and temporal variation hereby 16.4% (10/61) districts exhibited high prevalence (&gt;201/1000 children). The prevalence of diarrhoea was significantly higher in boys compared to girls (p = 0.001), while the incidence of cholera was significantly higher in females compared to males (p &lt; 0.001). Heavy precipitation and El-Niño events were linked to the episodes of cholera outbreaks. Conclusions: Health interventions should consider spatial and temporal variations of diarrhoea. The cholera outbreak preparedness needs to be aware during the events of climate changes in the hotspot areas. Keywords: Cholera; diarrhoea; climate change; Lake Tana Basin; Ethiopia.

Wastewater-based surveillance of Vibrio cholerae: Molecular insights on biofilm regulatory diguanylate cyclases, virulence factors and antibiotic resistance patterns

Vibrio cholerae is an inherent inhabitant of aquatic ecosystems. The Indian state of West Bengal, especially the Gangetic delta region is the highest cholera affected region and is considered as the hub of Asiatic cholera. V. cholerae were isolated from publicly accessible wastewater of Midnapore, West Bengal, India. Serotyping determined all isolates to be of non-O1/non-O139 serogroups. Moderate biofilm-forming abilities were noticed in most of the isolates (74.7 %) while, high biofilm formation was recorded for only 6.3 % isolates and 19 % of isolates exhibited low/non-biofilm-forming abilities. PCR-based screening of crucial diguanylate cyclases (DGCs) involved in cyclic-di-GMP-mediated biofilm signaling was performed. cdgH and cdgM were the most abundant DGCs among 93.7 % and 91.5 % of isolates, respectively. Other important DGCs, i.e., cdgK, cdgA, cdgL, and vpvC were present in 84 %, 75.5 %, 72 % and 68 % of isolates, respectively. Besides, the non-O1/non-O139 isolates were screened for the occurrence of virulence factor encoding genes. Moreover, among these non-O1/non-O139 isolates, two strains (3.17 %) harbored both ctxA and ctxB genes, which encode the cholera toxin associated with epidemic cholera. ompU was the most prevalent virulence factor, present in 24.8 % of isolates. Other virulence factors like, zot and st were found in 4.7 % and 9.5 % of isolates. Genes encoding tcp and ace were found to be PCR-negative for the isolates. Additionally, crucial virulence factor regulators, toxT, toxR and hapR were found to be PCR-positive in all the isolates. Antibiotic resistance patterns displayed further vulnerabilities with decreased sensitivity towards commonly used antibiotics with multiple antibiotic resistance index ranging between 0.37 and 0.62. The presence of cholera toxin-encoding multi-drug resistant (MDR) V. cholerae strains in environmental settings is alarming. High occurrence of DGCs are considered to encourage further investigations to use them as alternative therapeutic targets against MDR cholera pathogen due to their unique presence in bacterial systems.

MakC and MakD are two proteins associated with a tripartite toxin of Vibrio cholerae.

Pathogenic serotypes of Vibrio cholerae, transmitted through contaminated water and food, are responsible for outbreaks of cholera, an acute diarrheal disease. While the cholera toxin is the primary virulence factor, V. cholerae also expresses other virulence factors, such as the tripartite toxin MakABE that is secreted via the bacterial flagellum. These three proteins are co-expressed with two accessory proteins, MakC and MakD, whose functions remain unknown. Here, we present the crystal structures of MakC and MakD, revealing that they are similar in both sequence and structure but lack other close structural relatives. Our study further investigates the roles of MakC and MakD, focusing on their impact on the expression and secretion of the components of the MakABE tripartite toxin. Through deletion mutant analysis, we found that individual deletions of makC or makD do not significantly affect MakA expression or secretion. However, the deletion of both makC and makD impairs the expression of MakB, which is directly downstream, and decreases the expression of MakE, which is separated from makCD by two genes. Conversely, MakA, encoded by the makA gene located between makB and makE, is expressed normally but its secretion is impaired. Additionally, our findings indicate that MakC, in contrast to MakD, exhibits strong interactions with other proteins. Furthermore, both MakC and MakD were observed to be localized within the cytosol of the bacterial cell. This study provides new insights into the regulatory mechanisms affecting the Mak protein family in V. cholerae and highlights the complex interplay between gene proximity and protein expression.

Genomic evidence of two-staged transmission of the early seventh cholera pandemic

The seventh cholera pandemic started in 1961 in Indonesia and spread across the world in three waves in the decades that followed. Here, we utilised genomic evidence to detail the first wave of the seventh pandemic. Genomes of 22 seventh pandemic Vibrio cholerae isolates from 1961 to 1979 were completely sequenced. Together with 152 publicly available genomes from the same period, they fell into seven phylogenetic clusters (CL1–CL7). By multilevel genome typing (MGT), all were assigned to MGT2 ST1 (Wave 1) except three isolates in CL7 which were typed as MGT2 ST2 (Wave 2). The Wave 1 seventh pandemic expanded in two stages, with Stage 1 (CL1–CL5) spread across Asia and Stage 2 (CL6 and CL7) spread to the Middle East and Africa. Three non-synonymous mutations, one each, in three regulatory genes, csrD (global regulator), acfB (chemotaxis), and luxO (quorum sensing) may have critically contributed to its pandemicity. The three MGT2 ST2 isolates in CL7 were the progenitors of Wave 2 and evolved from within Wave 1 with acquisition of a novel IncA/C plasmid. Our findings provide new insight into the evolution and transmission of the early seventh pandemic, which may aid future cholera prevention and control.