Search

UNLABELLED: Vibrio cholerae, the causative agent of the diarrheal disease cholera, poses an ongoing health threat due to its wide repertoire of horizontally acquired elements (HAEs) and virulence factors. New clinical isolates of the bacterium with improved fitness abilities, often associated with HAEs, frequently emerge. The appropriate control and expression of such genetic elements is critical for the bacteria to thrive in the different environmental niches they occupy. H-NS, the histone-like nucleoid structuring protein, is the best-studied xenogeneic silencer of HAEs in gamma-proteobacteria. Although H-NS and other highly abundant nucleoid-associated proteins (NAPs) have been shown to play important roles in regulating HAEs and virulence in model bacteria, we still lack a comprehensive understanding of how different NAPs modulate transcription in V. cholerae. By obtaining genome-wide measurements of protein occupancy and active transcription in a clinical isolate of V. cholerae, harboring recently discovered HAEs encoding for phage defense systems, we show that a lack of H-NS causes a robust increase in the expression of genes found in many HAEs. We further found that TsrA, a protein with partial homology to H-NS, regulates virulence genes primarily through modulation of H-NS activity. We also identified few sites that are affected by TsrA independently of H-NS, suggesting TsrA may act with diverse regulatory mechanisms. Our results demonstrate how the combinatorial activity of NAPs is employed by a clinical isolate of an important pathogen to regulate recently discovered HAEs. IMPORTANCE: New strains of the bacterial pathogen Vibrio cholerae, bearing novel horizontally acquired elements (HAEs), frequently emerge. HAEs provide beneficial traits to the bacterium, such as antibiotic resistance and defense against invading bacteriophages. Xenogeneic silencers are proteins that help bacteria harness new HAEs and silence those HAEs until they are needed. H-NS is the best-studied xenogeneic silencer; it is one of the nucleoid-associated proteins (NAPs) in gamma-proteobacteria and is responsible for the proper regulation of HAEs within the bacterial transcriptional network. We studied the effects of H-NS and other NAPs on the HAEs of a clinical isolate of V. cholerae. Importantly, we found that H-NS partners with a small and poorly characterized protein, TsrA, to help domesticate new HAEs involved in bacterial survival and in causing disease. A proper understanding of the regulatory state in emerging isolates of V. cholerae will provide improved therapies against new isolates of the pathogen.

Microbial biofilms represent an important lifestyle for bacteria and are dynamic three-dimensional structures. Cyclic dimeric guanosine monophosphate (c-di-GMP) is a ubiquitous signaling molecule that is known to be tightly regulated with biofilm processes. While measurements of global levels of c-di-GMP have proven valuable toward understanding the genetic control of c-di-GMP production, there is a need for tools to observe the local changes of c-di-GMP production in biofilm processes. We have developed a label-free method for the direct detection of c-di-GMP in microbial colony biofilms using matrix-assisted laser desorption/ionization mass spectrometry imaging (MALDI-MSI). We applied this method to the enteric pathogen Vibrio cholerae, the marine symbiont V. fischeri, and the opportunistic pathogen Pseudomonas aeruginosa PA14 and detected spatial and temporal changes in c-di-GMP signal that accompanied genetic alterations in factors that synthesize and degrade the compound. We further demonstrated how this method can be simultaneously applied to detect additional metabolites of interest from a single sample.

Biofilms are matrix-encased microbial communities that increase the environmental fitness and infectivity of many human pathogens including Vibrio cholerae. Biofilm matrix assembly is essential for biofilm formation and function. Known components of the V. cholerae biofilm matrix are the polysaccharide Vibrio polysaccharide (VPS), matrix proteins RbmA, RbmC, Bap1, and extracellular DNA, but the majority of the protein composition is uncharacterized. This study comprehensively analyzed the biofilm matrix proteome and revealed the presence of outer membrane proteins (OMPs). Outer membrane vesicles (OMVs) were also present in the V. cholerae biofilm matrix and were associated with OMPs and many biofilm matrix proteins suggesting that they participate in biofilm matrix assembly. Consistent with this, OMVs had the capability to alter biofilm structural properties depending on their composition. OmpU was the most prevalent OMP in the matrix, and its absence altered biofilm architecture by increasing VPS production. Single-cell force spectroscopy revealed that proteins critical for biofilm formation, OmpU, the matrix proteins RbmA, RbmC, Bap1, and VPS contribute to cell-surface adhesion forces at differing efficiency, with VPS showing the highest efficiency whereas Bap1 showing the lowest efficiency. Our findings provide new insights into the molecular mechanisms underlying biofilm matrix assembly in V. cholerae, which may provide new opportunities to develop inhibitors that specifically alter biofilm matrix properties and, thus, affect either the environmental survival or pathogenesis of V. cholerae.IMPORTANCECholera remains a major public health concern. Vibrio cholerae, the causative agent of cholera, forms biofilms, which are critical for its transmission, infectivity, and environmental persistence. While we know that the V. cholerae biofilm matrix contains exopolysaccharide, matrix proteins, and extracellular DNA, we do not have a comprehensive understanding of the majority of biofilm matrix components. Here, we discover outer membrane vesicles (OMVs) within the biofilm matrix of V. cholerae. Proteomic analysis of the matrix and matrix-associated OMVs showed that OMVs carry key matrix proteins and Vibrio polysaccharide (VPS) to help build biofilms. We also characterize the role of the highly abundant outer membrane protein OmpU in biofilm formation and show that it impacts biofilm architecture in a VPS-dependent manner. Understanding V. cholerae biofilm formation is important for developing a better prevention and treatment strategy framework.

Background: Cholera is a diarrheal disease recognized for being caused by toxin-producing Vibrio (V.) cholerae. This study aims to assess the vibriocidal and immunomodulatory properties of derived cell-free supernatants (CFSs) of Bifidobacterium (B.) bifidum and Lactobacillus (L.) acidophilus encapsulated in chitosan nanoparticles (CFSb-CsNPs and CFSa-CsNPs) against clinical multi-drug resistance (MDR) isolates of V. cholerae O1 El Tor. Methods: We synthesized CFSb-CsNPs and CFSa-CsNPs using the ionic gelation technique. The newly nanostructures were characterized for size, surface zeta potential, morphology, encapsulation efficacy (EE), stability in different pH values and temperatures, release profile, and in vitro cytotoxicity. The antimicrobial and antibiofilm effects of the obtained nanocomposites on clinical MDR isolates (N = 5) of V. cholerae E1 Tor O1 were investigated by microbroth dilution assay and crystal violet staining, respectively. We conducted quantitative real-time PCR (qRT-PCR) to analyze the relative gene expressions of Bap, Rbmc, CTXAB, and TCP in response to CFSb-CsNPs and CFSa-CsNPs. Additionally, the immunomodulatory effects of formulated structures on the expression of TLR2 and TLR4 genes in human colorectal adenocarcinoma cells (Caco-2) were studied. Results: Nano-characterization analyses indicated that CFSb-CsNPs and CFSa-CsNPs exhibit spherical shapes under scanning electron microscopy (SEM) imaging, with mean diameters of 98.16 ± 0.763 nm and 83.90 ± 0.854 nm, respectively. Both types of nanoparticles possess positive surface charges. The EE% of CFSb-CsNPs was 77 ± 4.28%, whereas that of CFSa-CsNPs was 62.5 ± 7.33%. Chitosan (Cs) encapsulation leads to increased stability of CFSs in simulated pH conditions of the gastrointestinal tract in which the release rates for CFSb-CsNPs and CFSa-CsNPs were reached at 58.00 ± 1.24% and 55.01 ± 1.73%, respectively at pH = 7.4. The synergistic vibriocidal effects observed from the co-administration of both CFSb-CsNPs and CFSa-CsNPs, as evidenced by a fractional inhibitory concentration (FIC) index of 0.57, resulting in a significantly lower MIC of 2.5 ± 0.05 mg/mL (p < 0.0001) compare to individual administration. The combined antibacterial effect of CFSb-CsNPs and CFSa-CsNPs on Bap (0.14 ± 0.05), Rbmc (0.24 ± 0.01), CTXAB (0.30 ± 0.09), and TCP (0.38 ± 0.01) gene expression was significant (p < 0.001). Furthermore, co-administration of CFSb-CsNPs and CFSa-CsNPs also demonstrated the potency of suppressing TLR 2/4 (0.20 ± 0.01 and 0.12 ± 0.02, respectively) gene expression (p = 0.0019) and reduced Caco-2 cells attached bacteria to 526,000 ± 51,46 colony-forming units/mL (11.19%) (p < 0.0001). Conclusion: Our study revealed that encapsulating CFSs within CsNPs enhances their vibriocidal activity by improving stability and enabling a controlled release mechanism at the site of interaction between the host and bacteria. Additionally, the simultaneous use of CFSb-CsNPs and CFSa-CsNPs exhibited superior vibriocidal potency against MDR V. cholerae O1 El Tor strains, indicating these combinations as a potential new approach against MDR bacteria. [ABSTRACT FROM AUTHOR]

This paper deals with the problem of the prediction and control of cholera outbreak using real data of Cameroon. We first develop and analyze a deterministic model with seasonality for the cholera, the novelty of which lies in the incorporation of undetected cases. We present the basic properties of the model and compute two explicit threshold parameters R0 and R0 that bound the effective reproduction number R0, from below and above, that is R0 ≤ R0 ≤ R0. We prove that cholera tends to disappear when R0 ≤ 1, while when R0 >1, cholera persists uniformly within the population. After, assuming that the cholera transmission rates and the proportions of newly symptomatic are unknown, we develop the EnKf approach to estimate unmeasurable state variables and these unknown parameters using real data of cholera from 2014 to 2022 in Cameroon. We use this result to estimate the upper and lower bound of the effective reproduction number and reconstructed active asymptomatic and symptomatic cholera cases in Cameroon, and give a short-term forecasts of cholera in Cameroon until 2024. Numerical simulations show that (i) the transmission rate from free Vibrio cholerae in the environment is more important than the human transmission and begin to be high few week after May and in October, (ii) 90% of newly cholera infected cases that present the symptoms of cholera are not diagnosed and (iii) 60.36% of asymptomatic are detected at 14% and 86% of them recover naturally. The future trends reveals that an outbreak appeared from July to November 2023 with the number of cases reported monthly peaked in October 2023. An impulsive control strategy is incorporated in the model with the aim to avoid or prevent the cholera outbreak. In the first year of monitoring, we observed a reduction of more than 75% of incidences and the disappearance of the peaks when no control are available in Cameroon. A second monitoring of control led to a further reduction of around 60% of incidences the following year, showing how impulse control could be an effective means of eradicating cholera. [ABSTRACT FROM AUTHOR]

The occurrence of Vibrio cholerae, Vibrio metschnikovii and Vibrio fluvialis, important aquatic human pathogens of genus Vibrio, were determined in the surface water of the Danube River in Bratislava. The isolates were identified by biochemical tests. Biochemical characteristics of environmental isolates could be slightly different from reference and clinical strains which is reflected in dissimilarity rate, which was in our case not sufficient. A molecular method called amplified ribosomal DNA restriction analysis was used for confirmation of phenotypic identification. The species identity of 19 out of 22 isolates was confirmed, in the case of 3 misidentified ones, the identity of the genus did not change. The identity of all V. cholerae isolates was confirmed by PCR using species-specific gene called ompW. Also, main virulence factors of these isolates were determined. None of the isolates possessed all three virulence factors necessary for the induction of cholera. [ABSTRACT FROM AUTHOR]

Bacteriocins are antimicrobial peptides produced by bacteria to prevent the growth of pathogens. Combining bacteriocins with metal nanoparticles, like silver nanoparticles (AgNPs), has developed into a viable strategy to get over bacteriocin limitations. In this study, bacteriocin BacZY05 was extracted from Bacillus subtilis ZY05 and purified using various techniques. The resulting purified bacteriocin was then combined with silver nanoparticles to form bacteriocin silver nanoconjugates (BacZY05-AgNPs). The physicochemical properties of the BacZY05-AgNPs were characterized using various analytical techniques. The mean diameter of the synthesized AgNPs was approximately 20–60 nm with an oval or spherical shape. The antimicrobial activity of the BacZY05-AgNPs was evaluated against several indicator strains by their zone of inhibition (ZOI), using the agar well diffusion method. Compared to bacteriocin (ZOI- 13 to 20 mm) and AgNPs (ZOI- 10–22 mm) alone, the antibacterial activity data demonstrated a 1.3-1.5-fold increase in the activity of bacteriocin-nanoconjugates (ZOI- 22 to 26 mm). For Staphylococcus aureus MTCC3103 and Klebsiella pneumoniae MTCC109, BacZY05-capped AgNPs exhibited the lowest minimum inhibitory concentration (MIC), measuring 10.93 µg/mL. For Salmonella typhi NCIM2501, the MIC was 28.75 µg/mL. The highest MIC value was 57.5 µg/mL for Escherichia coli DH5α and Vibrio cholerae MTCC3909. With BacZY05-capped AgNPs, the lowest minimum bactericidal concentration (MBC) of 28.75 µg/mL was observed for Staphylococcus aureus MTCC31003. In the cases of Salmonella typhi NCIM2501 and Klebsiella pneumoniae MTCC109 concentration was 57.5 µg/mL. Vibrio cholerae MTCC3909 and Escherichia coli DH5α had the highest MBC values at 115 µg/mL. [ABSTRACT FROM AUTHOR]

This study aimed to determine the contamination level, seasonal distribution and the persistence of Salmonella sp, Vibrio cholera, and E. coli in well waters located near municipal solid and liquid waste dumpsites in Zaria, Kaduna State, Nigeria. A total of 186 samples: well water samples (96), soils from waste dump sites (72) and waste water (18), were collected for this study. Samples were plated using freshly prepared molten plate count, Eosine methylene blue agars, poured and swirled properly for a uniform mixture. The presence of E. coli, Salmonella, and Vibrio sp were determined for the two seasons (dry and wet). The bacteria count revealed a significant difference between well waters, soil samples, liquid waste and the control (total plate count - p < 0.005; 0.0001, total coliform count - p < 0.005; 0.0021). All the sampled well water had coliform counts higher than the recommended standard. The counts were higher in the wet season than the dry season. Out of a total of 125 suspected isolates tested, 70 (56%) were positive for Vibrio cholerae, out of this, 38 (54.3%), 28 (40%) and four (5.7%) isolates were from well water, solid waste dumps and liquid waste respectively. Out of the 177 suspected E. coli isolates from well water, solid waste dumps and liquid waste tested, 162 (91.5%) were positive with 82 (50.6%) of the positive E. coli further confirmed to be E. coli 0157:H7. The results showed significant (p < 0.05) statistical difference between the wet and the dry seasons for all the water samples studied and between control and the other well water samples. Adequate treatments of well water, as well as public health education are highly recommended. [ABSTRACT FROM AUTHOR]

Cholera continues to cause many outbreaks in low and middle-income countries due to inadequate water, sanitation, and hygiene services. We describe a protracted cholera outbreak in Nairobi City County, Kenya in 2017. We reviewed the cholera outbreak line lists from Nairobi City County in 2017 to determine its extent and factors associated with death. A suspected case of cholera was any person aged >2 years old who had acute watery diarrhea, nausea, or vomiting, whereas a confirmed case was where Vibrio cholerae was isolated from the stool specimen. We summarized cases using means for continuous variables and proportions for categorical variables. Associations between admission status, sex, age, residence, time to care seeking, and outbreak settings; and cholera associated deaths were assessed using odds ratio (OR) with 95% confidence interval (CI). Of the 2,737 cholera cases reported, we analyzed 2,347 (85.7%) cases including 1,364 (58.1%) outpatients, 1,724 (73.5%) not associated with mass gathering events, 1,356 (57.8%) male and 2,202 (93.8%) aged ≥5 years, and 35 deaths (case fatality rate: 1.5%). Cases were reported from all the Sub Counties of Nairobi City County with an overall county attack rate of 50 per 100,000 people. Vibrio cholerae Ogawa serotype was isolated from 78 (34.8%) of the 224 specimens tested and all isolates were sensitive to tetracycline and levofloxacin but resistant to amikacin. The odds of cholera-related deaths was lower among outpatient cases (aOR: 0.35; [95% CI: 0.17–0.72]), age ≥5 years old (aOR: 0.21 [95% CI: 0.09–0.55]), and mass gathering events (aOR: 0.26 [95% CI: 0.07–0.91]) while threefold higher odds among male (aOR: 3.04 [95% CI: 1.30–7.13]). Nairobi City County experienced a protracted and widespread cholera outbreak with a high case fatality rate in 2017. [ABSTRACT FROM AUTHOR]

The article discusses the seventh cholera pandemic, which began in 1961 and highlights how cholera has historically linked different parts of the world and affected both rich and poor communities. Topics include the pathophysiology of cholera and the discovery of intestinal secretion, secretory diarrheas other than cholera, and evidence that chloride secretion was at the center of the pathophysiology of cystic fibrosis.

Cholera is a life-threatening gastrointestinal infection caused by a toxigenic bacterium, Vibrio cholerae. After a lull of almost 30 years, a first case of cholera was detected in Lebanon in October 2022. The outbreak lasted three months, with 8007 suspected cases (671 laboratory-confirmed) and 23 deaths. In this study, we use phenotypic methods and microbial genomics to study 34 clinical and environmental Vibrio cholerae isolates collected throughout this outbreak. All isolates are identified as V. cholerae O1, serotype Ogawa strains from wave 3 of the seventh pandemic El Tor (7PET) lineage. Phylogenomic analysis unexpectedly reveals the presence of two different strains of the seventh pandemic El Tor (7PET) lineage. The dominant strain has a narrow antibiotic resistance profile and is phylogenetically related to South Asian V. cholerae isolates and derived African isolates from the AFR15 sublineage. The second strain is geographically restricted and extensively drug-resistant. It belongs to the AFR13 sublineage and clusters with V. cholerae isolates collected in Yemen. In conclusion, the 2022-2023 Lebanese cholera outbreak is caused by the simultaneous introduction of two different 7PET strains. Genomic surveillance with cross-border collaboration is therefore crucial for the identification of new introductions and routes of circulation of cholera, improving our understanding of cholera epidemiology. Here the authors provide results from a genomic epidemiology study of a cholera outbreak in Lebanon, showing that it was caused by two Vibrio cholerae strains of serogroup O1 (El Tor biotype), namely an AFR15 sublineage related to South Asian isolates and extensively drug-resistant Yemeni AFR13. [ABSTRACT FROM AUTHOR]

Vibrio mimicus bacteria have caused sporadic cases and outbreaks of cholera-like diarrhea throughout the world, but the association of lineages with such events is unexplored. Genomic analyses revealed V. mimicus lineages carrying the virulence factors cholera toxin and toxin coregulated pilus, one of which has persisted for decades in China and the United States. [ABSTRACT FROM AUTHOR]

Bacterial illness causes detrimental impacts on fish health and survival and finally economic losses for the aquaculture industry. Antibiotic medication causes microbial resistance, so alternative control strategies should be applied. In this work, we investigated the probiotic-medicated diet as an alternative control approach for antibiotics in treating Vibrio cholerae infection in Nile tilapia (Oreochromis niloticus). One hundred eighty fish (50 ± 2.5 g Mean ± SD) were allocated into six groups in glass aquariums (96 L) in triplicate for 10 days. Groups 1 (G1), G2, and G 3 were intraperitoneally (IP) injected with 0.5 mL sterilized tryptic soy broth and fed on a basal diet, basal diet contained B. subtilis (BS) (1 × 10 5 CFU/ kg-1 diet), and basal diet contained trimethoprim-sulfamethoxazole (TMP-SMX) (1.5 g/kg-1 diet), respectively. Additionally, G4, G5, and G6 were IP challenged with 0.5 mL of V. cholerae (1.5 × 107 CFU) and received the same feeding regime as G 1 to 3, respectively. The results exhibited that the V. cholera-infected fish exhibited skin hemorrhage, fin rot, and the lowest survival (63.33%). Additionally, lowered immune-antioxidant biomarkers (white blood cells count, serum bactericidal activity, phagocytic activity, phagocytic index, and lysozymes) with higher lipid peroxidation marker (malondialdehyde) were consequences of V. cholerae infection. Noteworthy, fish-fed therapeutic diets fortified with BS and TMP-SMX showed a substantial amelioration in the clinical signs and survival. The BS diet significantly improved (P < 0.05) the immune-antioxidant indices of the infected fish compared to the TMP-SMX diet. The current findings supported the use of a BS-enriched diet as an eco-friendly approach for the control of V. cholerae in O. niloticus. [ABSTRACT FROM AUTHOR]

Bacterial contamination in fruits and vegetables cultivated in urban and peri-urban areas constitutes a serious public health risk. This paper investigates bacterial contamination in irrigation water of the Nairobi-Machakos counties interface, Kenya. Sixty-six irrigation water samples were tested for total coliforms, Escherichia coli, Shigella spp. Pseudomonas aeruginosa, Enterococcus faecalis, Vibrio cholerae, Salmonella typhi, BOD, COD, and pH. Results show a high load of bacterial pathogens in all samples except for Salmonella typhi, which tested negative. Based on Kenya's standards and WHO guidelines, the irrigation water samples are unfit for fruit and vegetable irrigation. Urgent and effective measures are required, including regular monitoring, sensitisation, and enforcement of phytosanitary and regulatory measures. [ABSTRACT FROM AUTHOR]

Background: Cholera is an acute infectious disease caused by ingestion of contaminated food or water with Vibrio cholerae. Cholera remains a global threat to public health and an indicator of inequity and lack of social development. The aim of this study was to assess the prevalence and antimicrobial susceptibility pattern of V. cholerae from cholera outbreak sites in Ethiopia. Methods: Across-sectional study was conducted from May 2022 to October 2023 across different regions in Ethiopia: Oromia National Regional State, Amhara National Regional State and Addis Ababa City Administration. A total of 415 fecal samples were collected from the three regions. Two milliliter fecal samples were collected from each study participants. The collected samples were cultured on Blood Agar, MacConkey Agar and Thiosulfate Citrate Bile Salt Sucrose Agar. A series of biochemical tests Oxidase test, String test, Motility, Indole, Citrate, Gas production, H2S production, Urease test were used to identify V. cholerae species. Both polyvalent and monovalent antisera were used for agglutination tests to identify and differentiate V. cholerae serogroup and serotypes. In addition, Kirby-Bauer Disk diffusion antibiotic susceptibility test method was done. Data were registered in epi-enfo version 7 and analyzed by Statistical Package for Social Science version 25. Descriptive statistics were used to determine the prevalence of Vibrio cholerae. Logistic regression model was fitted and p-value < 0.05 was considered as statically significant. Results: The prevalence of V. cholerae in the fecal samples was 30.1%. Majority of the isolates were from Oromia National Regional State 43.2% (n = 54) followed by Amhara National Regional State 31.2% (n = 39) and Addis Ababa City Administration 25.6% (n = 32). Most of the V. cholerae isolates were O1 serogroups 90.4% (n = 113) and Ogawa serotypes 86.4% (n = 108). Majority of the isolates were susceptible to ciprofloxacin 100% (n = 125), tetracycline 72% (n = 90) and gentamycin 68% (n = 85). More than half of the isolates were resistant to trimethoprim-sulfamethoxazole 62.4% (n = 78) and ampicillin 56.8% (n = 71). In this study, participants unable to read and write were about four times more at risk for V. cholerae infection (AOR: 3.8, 95% CI: 1.07–13.33). In addition, consumption of river water were about three times more at risk for V. cholerae infection (AOR: 2.8, 95% CI: 1.08–7.08). Conclusion: our study revealed a high prevalence of V. cholerae from fecal samples. The predominant serogroups and serotypes were O1 and Ogawa, respectively. Fortunately, the isolates showed susceptible to most tested antibiotics. Drinking water from river were the identified associated risk factor for V. cholerae infection. Protecting the community from drinking of river water and provision of safe and treated water could reduce cholera outbreaks in the study areas. [ABSTRACT FROM AUTHOR]

We report the discovery of a persistent presence of Vibrio cholerae at very low abundance in the inlet of a single wastewater treatment plant in Copenhagen, Denmark at least since 2015. Remarkably, no environmental or locally transmitted clinical case of V. cholerae has been reported in Denmark for more than 100 years. We, however, have recovered a near-complete genome out of 115 metagenomic sewage samples taken over the past 8 years, despite the extremely low relative abundance of one V. cholerae read out of 500,000 sequenced reads. Due to the very low relative abundance, routine screening of the individual samples did not reveal V. cholerae. The recovered genome lacks the gene responsible for cholerae toxin production, but although this strain may not pose an immediate public health risk, our finding illustrates the importance, challenges, and effectiveness of wastewater-based pathogen surveillance. [ABSTRACT FROM AUTHOR]

Malawi experienced its deadliest Vibrio cholerae (Vc) outbreak following devastating cyclones, with >58,000 cases and >1700 deaths reported between March 2022 and May 2023. Here, we use population genomics to investigate the attributes and origin of the Malawi 2022–2023 Vc outbreak isolates. Our results demonstrate the predominance of ST69 clone, also known as the seventh cholera pandemic El Tor (7PET) lineage, expressing O1 Ogawa (~ 80%) serotype followed by Inaba (~ 16%) and sporadic non-O1/non-7PET serogroups (~ 4%). Phylogenetic reconstruction revealed that the Malawi outbreak strains correspond to a recent importation from Asia into Africa (sublineage AFR15). These isolates harboured known antimicrobial resistance and virulence elements, notably the ICEGEN/ICEVchHai1/ICEVchind5 SXT/R391-like integrative conjugative elements and a CTXφ prophage with the ctxB7 genotype compared to historical Malawian Vc isolates. These data suggest that the devastating cyclones coupled with the recent importation of 7PET serogroup O1 strains, may explain the magnitude of the 2022–2023 cholera outbreak in Malawi. Malawi experienced a large cholera outbreak in 2022-2023 that was associated with high morbidity and mortality and distributed across all 29 districts of the country. This study describes the epidemiological and genomic features of the outbreak and attempts to understand the reasons for its severity. [ABSTRACT FROM AUTHOR]

Cholera is an infectious disease that is transmitted through contaminated water. The disease includes a long back history of epidemics. Despite the numerous hygiene and prevention techniques that have been developed for Cholera, outbreaks of cholera are still reported worldwide. The resolution to this issue lies in promptly identifying the area susceptible to cholera outbreaks, a matter that continues to perplex scientists and medical professionals. It has been reported that Vibrio is effective in nitrogen digestion because it contains the nasA gene. In this study, initially the impact of nutrients (nitrate and nitrite) on growth of Vibrio cholerae was determined, subsequently a relationship was developed between nutrient substrates and V. cholerae growth rate, using Monod model. Subsequently, the model was applied to large national river quality data set (2012–2014) developed by Central Pollution Control Board (CPCB) and a possible cholerae outbreak zone was predicted. This work will definitely help the policy makers to develop management strategy for keeping rivers safe from future cholera outbreak. [ABSTRACT FROM AUTHOR]

Seventh-pandemic Vibrio cholerae strains contain two pathogenicity islands that encode the DNA defense modules DdmABC and DdmDE. In this study, we used cryogenic electron microscopy to determine the mechanistic basis for plasmid defense by DdmDE. The helicase-nuclease DdmD adopts an autoinhibited dimeric architecture. The prokaryotic Argonaute protein DdmE uses a DNA guide to target plasmid DNA. The structure of the DdmDE complex, validated by in vivo mutational studies, shows that DNA binding by DdmE triggers disassembly of the DdmD dimer and loading of monomeric DdmD onto the nontarget DNA strand. In vitro studies indicate that DdmD translocates in the 5′-to-3′ direction, while partially degrading the plasmid DNA. These findings provide critical insights into the mechanism of DdmDE systems in plasmid elimination. [ABSTRACT FROM AUTHOR]

Background: The production of secondary metabolites in different strains of microalgae varies and is likely dependent on environmental conditions. Consequently, the production of bioactive substances as secondary metabolites occurs in microalgae to aid their survival in adverse environmental conditions such as salinity stress. Objectives: The purpose of this study is to investigate the antibacterial activity of the methanolic extract of Spirulina platensis algae cultivated under different salinity stresses against Yersinia rukeri, Escherichia coli, Salmonella sp., and Vibrio cholerae. Methods: The broth microdilution method was used to evaluate the growth inhibitory activity of the extracts against Yersinia ruckeri, Salmonella sp., Escherichia coli, and Vibrio cholerae bacteria. The minimum inhibitory concentration (MIC) and the minimum bactericidal concentration (MBC) were determined by this method, and the diameter of the inhibition zone was assessed through the well diffusion method. Results: In the treatment with algae extract grown under 3.5 ppt salinity stress, the concentrations of 50 and 25 mg/mL extract showed the largest inhibition zone diameter against Salmonella sp. compared to other bacteria. Under 7 ppt salinity stress, all studied concentrations of algal extract exhibited a higher inhibition zone diameter against V. cholerae compared to other bacteria. The results of comparing different concentrations of algae extract between the two salinity stresses of 3.5 and 7 ppt for each bacterium showed a significant difference (P < 0.05). As salinity increased, the diameter of the inhibition zone also increased for all bacteria. Conclusions: Our results showed an increase in the antibacterial activity of the methanolic extract of Spirulina platensis with higher salinity stress levels. Therefore, cultivating Spirulina algae in salt water can be a cost-effective and suitable method to produce more secondary metabolites for use in the pharmaceutical industry. [ABSTRACT FROM AUTHOR]

Understanding the biological functions and processes of genes, particularly those not yet characterized, is crucial for advancing molecular biology and identifying therapeutic targets. The hypothesis guiding this study is that the 3D proximity of genes correlates with their functional interactions and relevance in prokaryotes. We introduced 3D-GeneNet, an innovative software tool that utilizes high-throughput sequencing data from chromosome conformation capture techniques and integrates topological metrics to construct gene association networks. Through a series of comparative analyses focused on spatial versus linear distances, we explored various dimensions such as topological structure, functional enrichment levels, distribution patterns of linear distances among gene pairs, and the area under the receiver operating characteristic curve by utilizing model organism Escherichia coli K-12. Furthermore, 3D-GeneNet was shown to maintain good accuracy compared to multiple algorithms (neighbourhood, co-occurrence, coexpression, and fusion) across multiple bacteria, including E. coli , Brucella abortus , and Vibrio cholerae. In addition, the accuracy of 3D-GeneNet's prediction of long-distance gene interactions was identified by bacterial two-hybrid assays on E. coli K-12 MG1655, where 3D-GeneNet not only increased the accuracy of linear genomic distance tripled but also achieved 60% accuracy by running alone. Finally, it can be concluded that the applicability of 3D-GeneNet will extend to various bacterial forms, including Gram-negative, Gram-positive, single-, and multi-chromosomal bacteria through Hi-C sequencing and analysis. Such findings highlight the broad applicability and significant promise of this method in the realm of gene association network. 3D-GeneNet is freely accessible at https://github.com/gaoyuanccc/3D-GeneNet. [ABSTRACT FROM AUTHOR]

Over time, humanity has addressed microbial water contamination in various ways. Historically, individuals resorted to producing beer to combat the issue. Fast forward to the 19th century, and we witnessed a scientific approach by Robert Koch. His groundbreaking gelatine plating method aimed to identify and quantify bacteria, with a proposed limit of 100 colony‐forming units per millilitre (CFU/mL) to avoid Cholera outbreaks. Despite considerable advancements in plating techniques through experimentation with media compositions and growth temperatures, the reliance on a century‐old method for water safety remains the state‐of‐the‐art. Even though most countries succeed in producing qualitative water at the end of the production centres, it is difficult to control, and guarantee, the same quality during distribution. Rather than focusing solely on specific sampling points, we propose a holistic examination of the entire water network to ensure comprehensive safety. Current practices leave room for uncertainties, especially given the low concentrations of pathogens. Innovative methods like flow cytometry and flow cytometric fingerprinting offer the ability to detect changes in the microbiome of drinking water. Additionally, molecular techniques and emerging sequencing technologies, such as third‐generation sequencing (MinION), mark a significant leap forward, enhancing detection limits and emphasizing the identification of unwanted genes rather than the unwanted bacteria/microorganisms itself. Over the last decades, there has been the realization that the drinking water distribution networks are complex ecosystems that, beside bacteria, comprise of viruses, protozoans and even isopods. Sequencing techniques to find eukaryotic DNA are necessary to monitor the entire microbiome of the drinking water distribution network. Or will artificial intelligence, big data and machine learning prove to be the way to go for (microbial) drinking water monitoring? In essence, it is time to transcend century‐old practices and embrace modern technologies to ensure the safety of our drinking water from production to consumption. [ABSTRACT FROM AUTHOR]

Recent studies have demonstrated the ability of mucin O‐glycans to attenuate virulence in diverse, cross‐kingdom pathogens, sparking interest in their development as a novel therapeutic approach against infection. Although their virulence attenuating activity is evident, mucin glycans obtained from native sources comprise mixtures of several hundred distinct structures, and therefore the specific active glycan epitopes and molecular mechanisms of virulence attenuation remain unclear. Individual mucin glycan structures cannot be purified from native sources and are not amenable to automated synthesis; therefore, to further investigate the phenomena of virulence attenuation we have been developing convergent and scalable methods (>30 mg per target glycan) to assemble a mucin O‐glycan library in sufficient scale and purity to facilitate biological studies. Previously we described a method to obtain core 1 & core 2‐type mucin glycan derivatives that have since been used to identify active epitopes in Candida albicans and Vibrio cholerae. In the current study, we describe the expansion of our glycan library to include core 3 & core 4‐type derivatives, increasing the structural diversity of our platform for biological evaluation. [ABSTRACT FROM AUTHOR]

Vibrio parahaemolyticus, Vibrio vulnificus and non‐O1/non‐O139 Vibrio cholerae are the Vibrio spp. of highest relevance for public health in the EU through seafood consumption. Infection with V. parahaemolyticus is associated with the haemolysins thermostable direct haemolysin (TDH) and TDH‐related haemolysin (TRH) and mainly leads to acute gastroenteritis. V. vulnificus infections can lead to sepsis and death in susceptible individuals. V. cholerae non‐O1/non‐O139 can cause mild gastroenteritis or lead to severe infections, including sepsis, in susceptible individuals. The pooled prevalence estimate in seafood is 19.6% (95% CI 13.7–27.4), 6.1% (95% CI 3.0–11.8) and 4.1% (95% CI 2.4–6.9) for V. parahaemolyticus, V. vulnificus and non‐choleragenic V. cholerae, respectively. Approximately one out of five V. parahaemolyticus‐positive samples contain pathogenic strains. A large spectrum of antimicrobial resistances, some of which are intrinsic, has been found in vibrios isolated from seafood or food‐borne infections in Europe. Genes conferring resistance to medically important antimicrobials and associated with mobile genetic elements are increasingly detected in vibrios. Temperature and salinity are the most relevant drivers for Vibrio abundance in the aquatic environment. It is anticipated that the occurrence and levels of the relevant Vibrio spp. in seafood will increase in response to coastal warming and extreme weather events, especially in low‐salinity/brackish waters. While some measures, like high‐pressure processing, irradiation or depuration reduce the levels of Vibrio spp. in seafood, maintaining the cold chain is important to prevent their growth. Available risk assessments addressed V. parahaemolyticus in various types of seafood and V. vulnificus in raw oysters and octopus. A quantitative microbiological risk assessment relevant in an EU context would be V. parahaemolyticus in bivalve molluscs (oysters), evaluating the effect of mitigations, especially in a climate change scenario. Knowledge gaps related to Vibrio spp. in seafood and aquatic environments are identified and future research needs are prioritised. [ABSTRACT FROM AUTHOR]

The study investigated heavy metal residues and bacterial pathogens in blue crab (Callinectes amnicola), crayfish (Nematopaelemon hastatus), giant tiger prawn (Penaeus monodon), and Indian prawn (Fenneropenaeus indicus) from crude oil impacted Niger Delta waters. Standard methods of analysis were applied in all determinations. Results indicate that chromium, lead, and mercury were within regulatory acceptable levels. Total viable count (TVC) was below threshold, while Vibrio cholerae, Vibrio parahaemolyticus, and Salmonella typhimurium were isolated from all crustacean species. The distribution pattern of pathogens showed blue crab accumulated the highest population of pathogens. Consumption of crustaceans from the location exemplifies conflict of nutrient benefits and risks. [ABSTRACT FROM AUTHOR]

Aquatic environments, including their flora and fauna, are known reservoirs of pathogenic Vibrio species. This study aimed to determine the diversity and antimicrobial susceptibility of bacteria from water hyacinth (Eichornia crassipes) and surrounding waters in Lagos lagoons. Bacteria isolation, phenotypic, and genotypic analysis were conducted using standard microbiology and molecular methods..Sampled lagoons exhibited physicochemical parameters such as dissolved oxygen (4.76—6.91 mgL−1) and biochemical oxygen demand (80.5 – 242 mgL−1) that exceeded permissible levels, indicating pollution. A total of 41 bacterial isolates were recovered from water hyacinth samples and surrounding lagoon waters. Twelve (29.26%) were Vibrio strains, comprising 4 (9.8%) Vibrio cholerae, 3 (7.3%) V. paracholerae, 2 (4.9%) V. parahaemolyticus, 2 (4.88%) V. injensis and 1 (2.4%) V. mimicus. Of the 4 V. cholerae isolates, 1 belonged to the O1 serotype. Vibrio species were exclusively recovered from water samples. Phylogenetic analysis of the Vibrio strains isolated from different lagoons showed genetic relatedness. All 41(100%) bacteria isolates displayed pan sensitivity to meropenem, while the highest resistance rate 27(65.9%) was recorded for amoxicillin + clavulanic acid. Multiple antibiotic resistance index of 8/12 (66.7%) of multidrug resistant Vibrio isolates was > 0.2 (20%) which could imply a high risk source of contamination in the environment where they were isolated. tdh, trh, and t1 genes were detected in V. parahaemolyticus isolates. Although, water hyacinth in this study did not harbored Vibrio species, the lagoon remains a source of pathogenic drug resistant Vibrio species. Therefore, surveillance should be enhanced. [ABSTRACT FROM AUTHOR]

Growing interest in probiotics has spurred research into their health benefits for hosts. This study aimed to evaluate the probiotic properties, especially antibacterial activities and the safety of two Weissella confusa strains, W1 and W2, isolated from Khao-Mahk by describing their phenotypes and genotypes through phenotypic assays and whole genome sequencing. In vitro experiments demonstrated that both strains exhibited robust survival under gastric and intestinal conditions, such as in the presence of low pH, bile salt, pepsin, and pancreatin, indicating their favorable gut colonization traits. Additionally, both strains showed auto-aggregation and strong adherence to Caco2 cells, with adhesion rates of 86.86 ± 1.94% for W1 and 94.74 ± 2.29% for W2. These high adherence rates may be attributed to the significant exopolysaccharide (EPS) production observed in both strains. Moreover, they exerted remarkable antimicrobial activities against Stenotrophomonas maltophilia, Salmonella enterica serotype Typhi, Vibrio cholerae, and Acinetobacter baumannii, along with an absence of hemolytic activities and antibiotic resistance, underscoring their safety for probiotic application. Genomic analysis corroborated these findings, revealing genes related to probiotic traits, including EPS clusters, stress responses, adaptive immunity, and antimicrobial activity. Importantly, no transferable antibiotic-resistance genes or virulence genes were detected. This comprehensive characterization supports the candidacy of W1 and W2 as probiotics, offering substantial potential for promoting health and combating bacterial infections. [ABSTRACT FROM AUTHOR]

Vibrio cholerae and Vibrio parahaemolyticus are common pathogens linked to human gastroenteritis, particularly in seafood like shrimp. This study investigated the impact of lactic acid bacteria on V. cholerae and V. parahaemolyticus regarding the production of cadaverine, a concerning compound. V. cholerae NCCP 13589 and V. parahaemolyticus ATCC 27969 were significant producers of amines in experiments conducted using white-leg shrimp (Litopenaeus vannamei) and lysine decarboxylase broth. Notably, the Lactiplantibacillus plantarum NCIMB 6105 and Leuconostoc mesenteroides ATCC 10830 lactic acid bacteria strains demonstrated a pronounced antagonistic effect on the production of biogenic amines by these food-borne pathogenic bacteria. The presence of lactic acid bacteria led to a substantial reduction in cadaverine production in the lysine decarboxylase broth and shrimp extract. The co-culture of two lactobacilli species reduced the cadaverine production in V. cholerae and V. parahaemolyticus by approximately 77 and 80%, respectively. Consequently, the favorable influence of lactic acid bacteria in curbing cadaverine production by food-borne pathogens presents clear advantages for the food industry. Thus, effectively managing these pathogens could prove pivotal in controlling the biogenic amine levels in shrimp. [ABSTRACT FROM AUTHOR]

Aggregation behavior provides bacteria protection from harsh environments and threats to survival. Two uncharacterized proteases, LapX and Lap, are important for Vibrio cholerae liquid-based aggregation. Here, we determined that LapX is a serine protease with a preference for cleavage after glutamate and glutamine residues in the P1 position, which processes a physiologically based peptide substrate with a catalytic efficiency of 180 ± 80 M-1s-1. The activity with a LapX substrate identified by a multiplex substrate profiling by mass spectrometry screen was 590 ± 20 M-1s-1. Lap shares high sequence identity with an aminopeptidase (termed VpAP) from Vibrio proteolyticus and contains an inhibitory bacterial prepeptidase C-terminal domain that, when eliminated, increases catalytic efficiency on leucine p-nitroanilide nearly four-fold from 5.4 ± 4.1 × 104 M-1s-1 to 20.3 ± 4.3 × 104 M-1s-1. We demonstrate that LapX processes Lap to its mature form and thus amplifies Lap activity. The increase is approximately eighteen-fold for full-length Lap (95.7 ± 5.6 × 104 M-1s-1) and six-fold for Lap lacking the prepeptidase C-terminal domain (11.3 ± 1.9 × 105 M-1s-1). In addition, substrate profiling reveals preferences for these two proteases that could inform in vivo function. Furthermore, purified LapX and Lap restore the timing of the V. cholerae aggregation program to a mutant lacking the lapX and lap genes. Both proteases must be present to restore WT timing, and thus they appear to act sequentially: LapX acts on Lap, and Lap acts on the substrate involved in aggregation.

This paper deals with the problem of the prediction and control of cholera outbreak using real data of Cameroon. We first develop and analyze a deterministic model with seasonality for the cholera, the novelty of which lies in the incorporation of undetected cases. We present the basic properties of the model and compute two explicit threshold parameters R¯0 and R̲0 that bound the effective reproduction number R0, from below and above, that is R̲0≤R0≤R¯0. We prove that cholera tends to disappear when R¯0≤1, while when R̲0>1, cholera persists uniformly within the population. After, assuming that the cholera transmission rates and the proportions of newly symptomatic are unknown, we develop the EnKf approach to estimate unmeasurable state variables and these unknown parameters using real data of cholera from 2014 to 2022 in Cameroon. We use this result to estimate the upper and lower bound of the effective reproduction number and reconstructed active asymptomatic and symptomatic cholera cases in Cameroon, and give a short-term forecasts of cholera in Cameroon until 2024. Numerical simulations show that (i) the transmission rate from free Vibrio cholerae in the environment is more important than the human transmission and begin to be high few week after May and in October, (ii) 90% of newly cholera infected cases that present the symptoms of cholera are not diagnosed and (iii) 60.36% of asymptomatic are detected at 14% and 86% of them recover naturally. The future trends reveals that an outbreak appeared from July to November 2023 with the number of cases reported monthly peaked in October 2023. An impulsive control strategy is incorporated in the model with the aim to avoid or prevent the cholera outbreak. In the first year of monitoring, we observed a reduction of more than 75% of incidences and the disappearance of the peaks when no control are available in Cameroon. A second monitoring of control led to a further reduction of around 60% of incidences the following year, showing how impulse control could be an effective means of eradicating cholera.

Abstract Background Cholera is an acute infectious disease caused by ingestion of contaminated food or water with Vibrio cholerae. Cholera remains a global threat to public health and an indicator of inequity and lack of social development. The aim of this study was to assess the prevalence and antimicrobial susceptibility pattern of V. cholerae from cholera outbreak sites in Ethiopia. Methods Across-sectional study was conducted from May 2022 to October 2023 across different regions in Ethiopia: Oromia National Regional State, Amhara National Regional State and Addis Ababa City Administration. A total of 415 fecal samples were collected from the three regions. Two milliliter fecal samples were collected from each study participants. The collected samples were cultured on Blood Agar, MacConkey Agar and Thiosulfate Citrate Bile Salt Sucrose Agar. A series of biochemical tests Oxidase test, String test, Motility, Indole, Citrate, Gas production, H2S production, Urease test were used to identify V. cholerae species. Both polyvalent and monovalent antisera were used for agglutination tests to identify and differentiate V. cholerae serogroup and serotypes. In addition, Kirby-Bauer Disk diffusion antibiotic susceptibility test method was done. Data were registered in epi-enfo version 7 and analyzed by Statistical Package for Social Science version 25. Descriptive statistics were used to determine the prevalence of Vibrio cholerae. Logistic regression model was fitted and p-value

The aim was to assess the level of toxin production in Vibrio cholerae El Tor genovariants and to determine the localization of cholera toxin in vesicles.Materials and methods. The work is performed on typical strains and genovariants of V. cholerae El Tor, which were grown in AKI liquid nutrient medium and the one prepared according to J. Hyan recipe, providing for high toxin production under aeration conditions. The decontaminated supernatants of the studied strains served as a source for extraction of toxin preparations and membrane vesicles. The localization of cholera toxin inside or on the outer surface of vesicles was determined through polyacrylamide gel electrophoresis (PAGE), immunoblotting, GM1-ELISA, indirect uncompetitive ELISA, cell culture models CHO-K1, HuTu 80.Results and discussion. Vesicle preparations containing cholera toxin have been isolated from the supernatants of genovariants and typical V. cholerae El Tor with a high level of toxin production. After separation of the vesicles using PAGE, followed by immunoblot with a specific antitoxic serum, it has been found that cholera toxin retains the complete structure, including both subunits. Unlike CT secreted into the culture medium, vesicle-associated one does not bind to both the GM1 receptor of gangliosides sensitized on plates and on the surface of cell cultures, which indicates its absence on the outer surface of vesicles. The location of CT in the cavity of vesicles is also evidenced by their positive reaction with specific antitoxic antibodies after degradation of EDTA. The absence of the toxin on the outer surface of vesicles in typical strains and strains of V. cholerae El Tor genovariants excludes its binding with the GM1 receptor and suggests the possibility of their penetration into target cells via GM-independent pathways. The choice of the pathways by which the vesicle-associated toxin is transferred to host cells is probably determined by the location of the toxin, i.e. it is associated with the internal structures of vesicles or placement on their surface.

The aim was to determine the phylogenetic position and features of the genome organization of individual groups of Vibrio cholerae strains isolated in Siberia and the Far East under different epidemiological situations during the seventh cholera pandemic.Materials and methods. We examined 275 V. cholerae strains, isolated during epidemic complications and during the cholera-free period in Siberia and the Far East, with different profiles of the main genomic loci of pathogenicity. The genomes of 969 V. cholerae strains from GenBank were used for phylogenetic analysis. The phylogeny reconstruction was carried out through calculating the distances between strains based on the occurrence of k-mers. The search, analysis and visualization of the loci structure in mobile genetic elements in V. cholerae genomes were performed using the blastn and Prokka programs and the author’s R and Python scripts.Results and discussion. Strains of V. cholerae isolated in Siberia and the Far East have been included in three global phylogenetic lines – L2, L3, L4. The distribution of strains from Siberia and the Far East along phylogenetic lines corresponds to the epidemiological situation in which they were isolated. We have identified the differentiation of strains by groups consistent with the global waves of spread of the etiological agent of the seventh cholera pandemic. We also traced potential paths for the import of the cholera pathogen into the territory of the Russian Federation. It has been revealed that spontaneous mutants that lost cholera toxin genes during storage and cultivation on nutrient media belong to the L2 phylogenetic lineage as well as toxigenic El Tor vibrios. The structural analysis confirms the differences in their genome organization and strains that do not have a CTX prophage during primary PCR screening. We recommend a two-stage algorithm of phylogenetic analysis within the framework of genomic monitoring of cholera agent: the first stage is a simplified assessment based on the occurrence of k-mers for express epidemiological analysis; the second stage is an in-depth analysis of genomes using a complex of phylogenetic methods for the reconstruction of links in individual epidemic complications, to establish patterns of origin and time of divergence of the pathogen clones.

The aim of the review is to analyze the literature data on systems of resistance to lytic cholera phages in Vibrio cholerae strains. Cholera phages are both present in the water of open reservoirs and isolated together with the pathogen from cholera patients. The mechanisms of molecular protection of V. cholerae from phages are similar to these systems of other bacteria, act at all stages of phage infection and include the following stages: prevention of phage adsorption, degradation of phage nucleic acids and inhibition of the formation of phage particles. Blocking the interaction of a phage with a bacterial cell occurs as a result of modification of receptors and the production of extracellular polysaccharides that create a physical barrier between the phages and the cell surface. If the phage DNA does enter the cells, it is destroyed by restriction-modification enzymes, as well as by the adaptive immune system CRISPR-Cas. The most numerous are the mechanisms for blocking the formation of phage particles in cells. This process occurs with the participation of phage-inducible PLE islands, the BREX bacteriophage exclusion system and abortive Abi infection, including the cyclic oligonucleotide-based anti-phage signaling system (CBASS) and the toxin-antitoxin system. During Abi infection, cells infected with the phage self-destruct and die before mature phage particles are formed, which contributes to the preservation of the V. cholerae population. The molecular mechanisms of a number of anti-phage systems have not yet been fully elucidated, which indicates the need for further study of the phage-host relations.

The aim of the work was to determine the pheno- and genotypic features of the aquatic CTX+ strain of Vibrio cholerae isolated in 2023 and a comparative bioinformatic analysis of whole-genome sequencing data.Materials and methods. Whole-genome sequencing was performed on MiSeq (Illumina) and MinIon Oxford Nanopore Technologies (ONT) platforms; hybrid assembly of the whole genome was carried out using the Trycycler algorithm; assembly errors were eliminated by means of the Medaka algorithm and the Pilon program. Dendrogram construction and bioinformatics analysis were carried out with the help of the scipy and Graphviz packages, BioEdit, BLASTN, BLASTP, CARD, ICE Genotyper, and Vector NTI programs. The ability to produce cholera toxin was tested using the GM1ELISA.Results and discussion. The isolated strain was identified as V. cholerae O1 Ogawa, sensitive to most antibiotics. Based on the totality of genetic properties, it was classified as the first genovariant, distinct from the typical El Tor strains only by the presence of ctxB1 gene of classical type instead of ctxB3 of the El Tor type. It has been established that it contains a tandemly duplicated CTX prophage on the small chromosome and a tandem of two copies of RS1 prophage on the large chromosome. Thereat, the rstR gene of the CTX prophage belonged to the classical type, and the RS1 prophage – to the El Tor type. The remaining criteria of epidemic hazard – tcpAelt, rtxA1 and intact VSP-II did not differ from the prototypes. The genome of the strain carries the ICE element VchBan11, which contains the trimethoprim resistance gene dfrA1, and phenotypically the strain is resistant to this antibiotic. Under in vitro conditions, the strain did not produce cholera toxin, as shown by ELISA results. This may be due to the presence of a deletion within the toxR regulatory gene. Strains similar to the 2023 isolate are mainly attributed to the second wave of the seventh pandemic. Currently, they are almost replaced by new genovariants, but occasionally can emerge and even cause diseases. Therefore, their importation into Russian territory potentially pose a threat to public health.

The study presents the data on organization of laboratory testing of clinical and environmental samples within the framework of establishing the etiology of the acute intestinal infections outbreak, performed by the specialists of the joint SAET of the Rospotrebnadzor in Dolisie (Republic of the Congo) in the period of 07–24 July, 2023.Materials and methods. In order to identify the causative agents of cholera and other acute intestinal infections of bacterial and viral nature, 177 clinical and environmental samples were tested, as well as cultures on solid nutrient media and bacterial suspensions. A total of 1023 tests were carried out by polymerase chain reaction (PCR) and 305 – using bacteriological method.Results and discussion. The causative agent of cholera has not been detected in any of the samples tested. Using the PCR method, markers of acute intestinal diseases agents (Salmonella spp., Shigella spp., Campylobacter spp., Rotavirus A) have been identified in 23 clinical samples and 1 sample of bacterial suspension. No DNA/RNA of pathogens has been detected in environmental samples. During culture studies, Salmonella enterica serovar Typhi have been isolated from 8 clinical samples, and their antibiotic sensitivity has been determined. Applying whole-genome nanopore sequencing, using the MinIon platform (Oxford Nanopore Technologies, UK), nucleotide sequences of 4 S. Typhi isolates have been investigated and deposited in the international database NCBI GenBank (No. CP141260, CP141193, CP141194, CP141195). Additionally, the analysis of initially sterile samples (blood, peritoneal fluid, intraoperational samples) from the patients of General and Reference hospitals of Dolisie has resulted in the identification of 5 cultures of non-fermenting bacteria, and their antibiotic sensitivity has been determined.

Russian Research Anti-Plague Institute “Microbe” of the Rospotrebnadzor develops and implements methods for obtaining and purifying biologically active substances of the causative agents of plague, cholera, tularemia and anthrax, which are used in the design of preventive and diagnostic preparations.The aim of the review is to summarize and systematize the accumulated data on the isolation, purification and assessment of antigens and toxins of plague, cholera, tularemia and anthrax pathogens. Attention is also paid to the application and prospects for using the obtained antigens for the design of medical immunobiological preparations (MIBPs). The Institute “Microbe” is a reference center for plague, hence modern immunodiagnostic drugs are necessary and in demand. Antigen-level preventive drugs against the infections listed above are also the subject of study by the institute’s staff. The main stages of isolation, purification and analysis of antigens include the selection or construction of a suitable strain; cultivation, extraction, concentration and purification of antigens and toxins using biotechnological techniques that allow for obtaining and preserving the biologically active substance of interest to the researcher. To study the antigenic activity of purified antigens, laboratory animals are involved, the immune response is recorded and analyzed, and antiserum is obtained. This is followed by the stage of investigating the physicochemical and immunobiological properties of the isolated antigen preparations and drawing up an antigen profile. The characterized antigens are used for the design of preventive and diagnostic drugs.

Vibrio cholerae is the causative agent of cholera, an acute diarrheal disease that spreads locally and globally in epidemics and pandemics. Although it was discovered that fish harbor V. cholerae strains in their intestines, most investigations revealed non-toxic V. cholerae serogroups in fish. Due to the rarity of toxigenic V. cholerae serogroups, it is difficult to cultivate these strains from environmental samples. Hence, here we aimed to uncover evidence of the occurrence of toxigenic V. cholerae in the intestines and spleens of various fish species. By using molecular detection tools, we show that V. cholerae O1 and strains positive for the cholera toxin inhabit both healthy and diseased fish intestines and spleens, suggesting that fish may serve as intermediate vectors of toxigenic V. cholerae. No significant differences were found between the abundance of toxigenic V. cholerae (either O1 or cholera toxin positive strains) in the healthy and the diseased fish intestines or spleens. In conclusion, a variety of fish species may serve as potential vectors and reservoirs of toxigenic V. cholerae as they form a link between the other reservoirs of V. cholerae (chironomids, copepods, and waterbirds). Similarly, they may aid in the spread of this bacterium between water bodies. [ABSTRACT FROM AUTHOR]

Abstract Cholera continues to represent a major public health concern in Ethiopia. The country has developed a Multi-sectoral National Cholera Elimination Plan in 2022, which targets prevention and control interventions in cholera hotspots. Multiple methods to classify cholera hotspots have been used in several countries. Since 2014, a classification method developed by United Nations Children's Fund has been applied to guide water, sanitation and hygiene interventions throughout Sub-Saharan Africa based on three outbreak parameters: frequency, duration and standardized attack rate. In 2019, the Global Task Force on Cholera Control (GTFCC) proposed a method based on two parameters: average annual cholera incidence and persistence. In 2023, an updated GTFCC method for multisectoral interventions considers three epidemiological indicators (cumulative incidence, cumulative mortality and persistence,) and a cholera-case confirmation indicator. The current study aimed to classify cholera hotspots in Ethiopia at the woreda level (equivalent to district level) applying the three methods and comparing the results to optimize the hotspot targeting strategy. From 2015 to 2021, cholera hotspots were located along major routes between Addis Ababa and woredas adjacent to the Kenya and Somalia borders, throughout Tigray Region, around Lake Tana, and in Afar Region. The multi-method comparison enables decision makers to prioritize interventions according to a sub-classification of the highest-priority areas.

Background: In Addis Ababa, Ethiopia, open ditches along innner roads in residential areas serve to convey domestic wastewater and rainwater away from residences. Contamination of drinking water by wastewater through faulty distribution lines could expose households to waterborne illnesses. This prompted the study to assess the microbiological safety of wastewater and drinking water in Addis Ababa, identify the pathogens therein, and determine their antibiotic resistance patterns. Results Vibrio cholerae: O1, mainly Hikojima serotype, was isolated from 23 wastewater and 16 drinking water samples. Similarly, 19 wastewater and 10 drinking water samples yielded Escherichia coli O157:H7. V. cholerae O1 were 100% resistant to the penicillins (Amoxacillin and Ampicillin), and 51–82% were resistant to the cephalosporins. About 44% of the V. cholerae O1 isolates in this study were Extended Spectrum Beta-Lactamase (ESBL) producers. Moreover, 26% were resistant to Meropenem. Peperacillin/Tazobactam was the only effective β-lactam antibiotic against V. cholerae O1. V. cholerae O1 isolates showed 37 different patterns of multiple resistance ranging from a minimum of three to a maximum of ten antimicrobials. Of the E. coli O157:H7 isolates, 71% were ESBL producers. About 96% were resistant to Ampicillin. Amikacin and Gentamicin were very effective against E. coli O157:H7 isolates. The isolates from wastewater and drinking water showed multiple antibiotic resistance against three to eight antibiotic drugs. Conclusions: Open ditches for wastewater conveyance along innner roads in residence areas and underground faulty municipal water distribution lines could be possible sources for V. cholerae O1 and E. coli O157:H7 infections to surrounding households and for dissemination of multiple drug resistance in humans and, potentially, the environment. [ABSTRACT FROM AUTHOR]

Elephantorrhiza elephantina, a wild plant in southern Africa, is utilized in traditional medicine for various ailments, leading to its endangerment and listing on the Red List of South African Plants. To date, there have been no reports on bacterial endophytes from this plant, their classes of secondary metabolites, and potential medicinal properties. This study presents (i) taxonomic characterization of bacterial endophytes in leaf and root tissues using 16S rRNA, (ii) bacterial isolation, morphological, and phylogenetic characterization, (iii) bacterial growth, metabolite extraction, and LC-MS-based metabolite fingerprinting, and (iv) antimicrobial testing of bacterial crude extracts. Next-generation sequencing yielded 693 and 2,459 DNA read counts for the rhizomes and leaves, respectively, detecting phyla including Proteobacteria, Bacteroidota, Gemmatimonadota, Actinobacteriota, Verrucomicrobiota, Dependentiae, Firmicutes, and Armatimonodata. At the genus level, Novosphingobium, Mesorhizobium, Methylobacterium, and Ralstonia were the most dominant in both leaves and rhizomes. From root tissues, four bacterial isolates were selected, and 16S rRNA-based phylogenetic characterization identified two closely related Pseudomonas sp. (strain BNWU4 and 5), Microbacterium oxydans BNWU2, and Stenotrophomonas maltophilia BNWU1. The ethyl acetate:chloroform (1:1 v/v) organic extract from each isolate exhibited antimicrobial activity against all selected bacterial pathogens. Strain BNWU5 displayed the highest activity, with minimum inhibitory concentrations ranging from 62.5 µg/mL to 250 µg/mL against diarrhoeagenic Escherichia coli, Escherichia coli O157:H7, Salmonella enterica, antibiotic-resistant Vibrio cholerae, Staphylococcus aureus, Bacillus cereus, and Enterococcus durans. LC-MS analysis of the crude extract revealed common antimicrobial metabolites produced by all isolates, including Phenoxomethylpenicilloyl (penicilloyl V), cis-11-Eicosenamide, 3-Hydroxy-3-phenacyloxindole, and 9-Octadecenamide. [ABSTRACT FROM AUTHOR]

Ferrate (Fe(VI): HFeO4-/FeO42-), a potent oxidant, has been investigated as an alternative chemical disinfectant in water treatment due to its reduced production of disinfection by-products. In this study, we assessed the disinfecting ability of potassium ferrate against a variety of microorganisms, including waterborne pathogens, under varying pH and water temperature conditions. We presented CT values, a metric of ferrate concentrations (C) and contact time (T), to quantify microbial inactivation rates. Among the tested microorganisms, human adenovirus was the least resistant to ferrate, followed by waterborne bacteria such as Escherichia coli and Vibrio cholerae, and finally, the protozoan parasite Giardia duodenalis. We further investigated the impact of two pH values (7 and 8) and two temperatures (5 and 25 °C) on microbial inactivation rates, observing that inactivation rates increased with lower pH and higher temperature. In addition to showcasing ferrate's capacity to effectively inactivate a range of the tested microorganisms, we offer a ferrate CT table to facilitate the comparison of the effectiveness of various disinfection methods. [ABSTRACT FROM AUTHOR]

Les gastro-entérites sont le plus souvent d'origine virale, rotavirus et norovirus étant les virus les plus fréquemment en cause chez les jeunes enfants. Les PCR multiplex effectuées à partir des selles permettent de détecter des bactéries, des virus ou des parasites responsables ou non de la gastro-entérite. Si le profil étiologique de ces infections digestives a grandement bénéficié de la PCR, la présence de pathogènes potentiels ne justifie pas de traitement anti-infectieux en dehors de pathologies sous-jacentes. En effet, parmi les causes bactériennes, très peu nécessitent un traitement antibiotique en dehors des shigelloses, des formes graves de salmonellose et une partie des infections à Campylobacter sp. L'évolution de la résistance aux antibiotiques des salmonelles, shigelles et campylobacter est préoccupante dans le monde, limitant les options thérapeutiques. Les antibiotiques proposés dans ce guide sont en accord avec les recommandations communes de l'European Society of Pediatric Infectious Diseases et l'European Society of Pediatric Gastroenterology and Nutrition. L'azithromycine est préférentiellement utilisée pour traiter les infections à Shigella sp. ou à Campylobacter sp. La ceftriaxone et la ciprofloxacine sont recommandées pour traiter les salmonelloses nécessitant une antibiothérapie. Les traitements empiriques, sans identification bactérienne, ne sont pas indiqués en dehors d'un sepsis sévère ou chez des sujets à risque (drépanocytose par exemple). La prescription de métronidazole pour une amibiase intestinale aiguë ne doit être faite qu'après confirmation microbiologique. Gastroenteritis is usually due to viruses mainly Rotavirus and Norovirus. Among the bacterial causes, very few warrant systemic antibiotic treatment including Shigella , Vibrio cholerae , Campylobacter (only if diagnosed early) and severe cases of Salmonella infections. The antimicrobial treatments proposed in this guide are consistent with the latest recommendations of the European Society of Pediatric Infectious Diseases and the European Society of Pediatric Gastroenterology and Nutrition. Azithromycin is the preferred antibiotic for infections due to Shigella and Campylobacter. Ceftriaxone and ciprofloxacin are recommended for Salmonella infections that must be treated. Empirical treatment without bacteriological documentation should be avoided. The prescription of metronidazole for acute intestinal amebiasis should only be made after microbiological confirmation. [ABSTRACT FROM AUTHOR]

Purpose: Waterborne pathogens pose a significant threat to public health, emphasizing the continuous necessity for advancing robust detection techniques, particularly in preventing outbreaks associated with these pathogens. This study focuses on cholera, an infectious disease caused by Vibrio cholerae, serogroups O1 and O139, often transmitted through contaminated water and food, raising significant public health concerns in areas with poor sanitation and limited access to clean water. Methods: We developed a colorimetric biosensor using aptamer-functionalized gold nanoparticles to identify Vibrio cholerae O139 and address this issue. The detection mechanism relies on the color change of gold nanoparticles (AuNPs) from red to blue-purple induced by NaCl after the pathogen incubation and aptamer-target binding. Initial steps involved synthesizing and characterizing AuNPs, then exploring the impact of aptamer and NaCl concentrations on AuNP agglomeration. Optimization procedures for aptamer concentration and salt addition identified the optimal conditions for detection as 120 pM aptamers and 1 M NaCl. Results: The aptasensor demonstrated a robust linear relationship, detecting V. cholerae concentrations from 103 to 108 CFU/mL, with a limit of detection (LOD) of 587 CFU/mL. Specificity tests and accurate sample analyses confirmed the efficiency of the AuNPs aptasensor, showcasing its reliability and speed compared to traditional culture examination methods. Moreover, we extended the aptasensor to a paper-based sensing platform with similar detection principles. Conclusion: The change in color upon target binding was captured with a smartphone and analyzed using image processing software. The paper-based device detected the target in less than 2 min, demonstrating its convenience for on-field applications. [ABSTRACT FROM AUTHOR]

Aims: Epidemiological investigations have indicated low resistance toward nitrofuran in clinical isolates, suggesting its potential application in the treatment of multidrug-resistant bacteria. Therefore, it is valuable to explore the mechanism of bacterial resistance to nitrofuran. Results: Through phenotypic screening of ten multiple antibiotic resistance regulator (MarR) proteins in Vibrio cholerae, we discovered that the regulator VnrR (VCA1058) plays a crucial role in defending against nitrofuran, specifically furazolidone (FZ). Our findings demonstrate that VnrR responds to FZ metabolites, such as hydroxylamine, methylglyoxal, hydrogen peroxide (H2O2), β-hydroxyethylhydrazine. Notably, VnrR exhibits reversible responses to the addition of H2O2 through three cysteine residues (Cys180, Cys223, Cys247), leading to the derepression of its upstream gene, vnrA (vca1057). Gene vnrA encodes a novel nitroreductase, which directly contributes to the degradation of FZ. Our study reveals that V. cholerae metabolizes FZ via the vnrR–vnrA system and achieves resistance to FZ with the assistance of the classical reactive oxygen/nitrogen species scavenging pathway. Innovation and Conclusion: This study represents a significant advancement in understanding the antibiotic resistance mechanisms of V. cholerae and other pathogens. Our findings demonstrate that the MarR family regulator, VnrR, responds to the FZ metabolite H2O2, facilitating the degradation and detoxification of this antibiotic in a thiol-dependent manner. These insights not only enrich our knowledge of antibiotic resistance but also provide new perspectives for the control and prevention of multidrug-resistant bacteria. [ABSTRACT FROM AUTHOR]

This study examines the genetic contexts and evolutionary steps responsible for the formation of the widely spread transposon Tn6925 carrying blaTEM and aacC2e, which confers resistance to beta-lactam and aminoglycoside antibiotics in Gram-negative bacteria. The blaTEM-1 and aacC2e genes were found in several transposons. They were first observed within an IS26 bounded 3.7 kb transposon (Tn6925) on several Acinetobacter baumannii plasmids located within a 4.7 kb dif module. Truncated and expanded variations of Tn6925 were found across other A. baumannii plasmids, as well as in other Gram-negative bacteria (including Vibrio cholerae). Moreover, blaTEM-1 and aacC2e were in much larger resistance-heavy transposons including the ISAba1-bounded 24.6 kb (here called Tn6927), found in an A. baumannii chromosome. A novel ISKpn12-bounded transposon was also observed to contain blaTEM and aacC2e which was found interrupting Tn5393 along with an IS26 pseudo-compound transposon to form a 24.9 kb resistance island in an Acinetobacter pittii plasmid. Multiple mobile genetic elements are involved in the formation of transposon structures that circulate blaTEM and aacC2e. Among these, IS26 and ISAba1 appear to have played a major role in the formation and spread of these elements in the Acinetobacter species. [ABSTRACT FROM AUTHOR]

CRISPRi (Clustered Regularly Interspaced Palindromic Repeats interference) is a gene knockdown method that uses a deactivated Cas9 protein (dCas9) that binds a specific gene target locus dictated by an encoded guide RNA (sgRNA) to block transcription. Mobile-CRISPRi is a suite of modular vectors that enable CRISPRi knockdowns in diverse bacteria by integrating IPTG-inducible dcas9 and sgRNA genes into the genome using Tn7 transposition. Here, we show that the Mobile-CRISPRi system functions robustly and specifically in multiple Vibrio species: Vibrio cholerae, Vibrio fischeri, Vibrio vulnificus, Vibrio parahaemolyticus, and Vibrio campbellii. We demonstrate efficacy by targeting both essential and non-essential genes that function to produce defined, measurable phenotypes: bioluminescence, quorum sensing, cell division, and growth arrest. We anticipate that Mobile-CRISPRi will be used in Vibrio species to systematically probe gene function and essentiality in various behaviors and native environments. [ABSTRACT FROM AUTHOR]

Cells of Vibrio fischeri colonize the light organ of Euprymna scolopes, providing the squid bioluminescence in exchange for nutrients and protection. The bacteria encounter DNA-rich mucus throughout their transition to a symbiotic lifestyle, leading us to hypothesize a role for nuclease activity in the colonization process. In support of this, we detected abundant extracellular nuclease activity in growing cells of V. fischeri. To discover the gene(s) responsible for this activity, we screened a V. fischeri transposon mutant library for nuclease-deficient strains. Interestingly, only one strain, whose transposon insertion mapped to nuclease gene VF_1451, showed a complete loss of nuclease activity in our screens. A database search revealed that VF_1451 is homologous to the nuclease-encoding gene xds in Vibrio cholerae. However, V. fischeri strains lacking xds eventually revealed slight nuclease activity on plates upon prolonged incubation. This led us to hypothesize that a second secreted nuclease, ident ified through a database search as VF_0437, a homolog of V. cholerae dns, might be responsible for the residual nuclease activity. Here, we show that Xds and/or Dns are involved in essential aspects of V. fischeri biology, including natural transformation, aggregation, and phosphate scavenging. Furthermore, strains lacking either nuclease were outcompeted by the wild type for squid colonization. Understanding the specific role of nuclease activity in the squid colonization process represents an intriguing area of future research. [ABSTRACT FROM AUTHOR]

To sequence the whole genome of strains isolated from Fuzhou between 2018 and 2023, predict virulence genes, antimicrobial resistance genes, and sequence loci information, analyze the genetic relationships among different strains, and provide evidence for cholera prevention and control. Methods Whole genome sequencing was performed on 60 strains of Vibrio cholerae, and bioinformatics software was used for quality control, gene assembly, and prediction of the sequencing data. PubMLST, ResFinder, and VFDB databases were used to predict the multilocus sequence typing, antimicrobial resistance genes, and virulence genes of different strains. Combined with the NCBI database, the phylogenetic tree was constructed by the maximum likelihood method through the core cgSNP phylogenetic analysis and derecombination analysis. Results Based on the typing of 7 housekeeping genes, 60 Vibrio cholerae strains can be categorized into 16 known STs and 38 newly assigned STs. The clinical isolate H339 of serogroup O1 was identified as ST75. Serogroup O1 food isolates H13, H363, and H381 were all ST175. H263 and H357, the NOVC isolates, were both ST1218. H293 and H306 were ST1700. H311, H314, and H316 were all ST1699, with the remaining isolates displaying diversity. A total of 29 antimicrobial resistance genes were predicted, including aminoglycosides, β -lactams, quinolones, and folate pathway antagonists, with the majority of the strains carrying quinolones antimicrobial resistance genes. According to the VFDB prediction, all isolates had the virulence factors rtx and hlyA, 96.7% (58/60) of the strains carried the tlh genes, all serogroup O1 isolates carried tcp, zot, and ace genes, and all serogroup O1 clinical isolates carried ctxA genes. Phylogenetic tree analysis of the whole genome divided all strains into 20 branches, indicating high genomic divergence. Conclusions Thirty-eight new STs were identified. Genetic correlations were found among serogroup O1 food isolates, whereas serogroup O1 clinical isolates and serogroup O1 food isolates, as well as between serogroup O1 and NOVC strains, show distant phylogenetic relationships. There was diversity among the isolates. This study provides data support for the traceability of foodborne diseases. [ABSTRACT FROM AUTHOR]