Search Results (131)

Wastewater-based epidemiology (WBE) has historically proven to be a powerful surveillance tool, particularly during the SARS-CoV-2 pandemic. Effective WBE depends on the sensitive detection of pathogens in wastewater. However, determining the process limit of detection (PLOD) of WBE through a comprehensive evaluation that accounts for pathogen concentration, nucleic acid extraction, and molecular analysis has rarely been documented. We prepared dilution series with known concentrations of S. Typhi, V. cholerae, rotavirus, and SARS-CoV-2 in surface water and wastewater. Pathogen concentration was performed using Nanotrap particles with the KingFisher™ Apex robotic platform, followed by nucleic acid extraction. Quantitative real-time PCR (qPCR) and digital PCR (dPCR) were used to detect the extracted nucleic acids of the pathogens. The PLODs and recovery efficiencies for each of the four pathogens in surface water and wastewater were determined. Overall, the observed PLODs for S. Typhi, V. cholerae, and rotavirus in surface water and wastewater were approximately 3 log₁₀ loads (2.1–2.8 × 10³/10 mL) using either qPCR or dPCR as the detection method. For SARS-CoV-2, the PLOD in surface water was 2.9 × 10⁴/10 mL with both RT-qPCR and dPCR, one log₁₀ higher than the PLODs of the other three pathogens. In wastewater, the PLOD for SARS-CoV-2 was 2.9 × 10⁴/10 mL using RT-qPCR and 2.9 × 10³/10 mL using dPCR. The mean recovery rates of S. Typhi, V. cholerae, rotavirus, and SARS-CoV-2 for dPCR in both surface water and wastewater were below 10.4%, except for S. Typhi and V. cholerae in wastewater, which showed significantly higher recoveries, from 26.5% at 4.6 × 10⁵/10 mL for S. Typhi to 58.8% at 4.8 × 10⁵/10 mL for V. cholerae. Our study demonstrated that combining qPCR or dPCR analysis with automated Nanotrap particle concentration and nucleic acid extraction using the KingFisher™ platform enables the sensitive detection of S. Typhi, V. cholerae, rotavirus, and SARS-CoV-2 in surface water and wastewater. Full article

The El Tor biotype of Vibrio cholerae caused the seventh cholera pandemic (7CP). Although V. cholerae variants of this biotype frequently emerge, studies on their microevolution and spatiotemporal transmission in epidemics caused by a single clone are limited. During the cholera outbreak in Sichuan Province, China, in the 1990s, strains belonging to phage type 6 (PT6) but resistant to typing phage VP5 due to a deletion mutation in ompW, which is the gene associated with the VP5 receptor were identified. In this study, we analyzed PT6 strains using genome sequencing to reveal the genomic and transmission characteristics of such a transient phage type in China’s cholera epidemic history. The findings revealed that the PT6 strains formed an independent clone during the four-year epidemic and emerged in wave 2. Most of them carried multiple CTX^classΦ genome copies on chromosome 2 (Chr. 2) and two copies each of RS1^ET and RS1-4** on chromosome 1 (Chr. 1). Frequent cross-regional transmission and local outbreaks within Sichuan Province, China, were revealed for this clone. A variety of spontaneous mutations in the ompW gene, conferring resistance to the VP5 phage, were observed under VP5 infection pressure, showing the incident mutation of OmpW for the survival adaptation of V. cholerae to phage pressure. Therefore, this genomic epidemiological revisit of these distinct phage-resistant phenotype strains reveals their clonal genetic structure, improves our understanding of the spread of V. cholerae by tracking their variation, and assists in epidemic source tracing and disease control. Full article

A key feature that differentiates Gram-positive and Gram-negative bacteria is the outer membrane, an asymmetric membrane composed of lipopolysaccharides, phospholipids, lipoproteins and integral proteins, including the outer-membrane proteins (OMPs). By being in direct contact with the extracellular milieu, the outer membrane and OMPs participate in multiple functions in Gram-negative bacteria, including controlling nutrient and molecule access to the cytoplasm, membrane vesicle formation and resistance to environmental stresses. OMPs have a characteristic barrel shape formed by antiparallel β-strands, with or without channels that allow diffusion of substrates through the outer membrane. The marine bacterium Vibrio cholerae is responsible for non-invasive gastroenteritis and cholera disease by consumption of contaminated water or food. Its OMPs, besides having a porin function, contribute to resistance to osmotic pressure and antimicrobial agents, intracellular signaling, adhesion to host cells and biofilm formation, amongst other functions. In this review, in addition to quickly reviewing the general structure of the outer membrane, the OMPs and how they reach the outer membrane, the functions attributed to these proteins are compiled. The mechanisms used by each of the described OMP to accomplish these functions in the marine pathogenic bacterium V. cholerae are discussed. Potential clinical and bioengineering applications of OMPs, such as diagnostic tools, vaccine development, and targeted antimicrobial or anti-virulence strategies are presented. What is known about the OMPs of V. cholerae is presented below. Full article

Background: Non-cholera Vibrio species are rare waterborne pathogens that can cause severe infections. Among these, few cases of Vibrio metschnikovii infections have been reported, especially in the gastrointestinal tract, with no cardiac tissue involvement as a result. Following the PRISMA checklist, we conducted a literature review, and thirteen articles for twenty-two cases overall were included: seven cases of sepsis (in three cases, the echocardiographic results were negative), seven cases of pneumonia, two skin infections, eleven cases of diarrhoea, and a gastroenteritis outbreak. This report documents the expanding clinical spectrum and the role played by V. metschnikovii in infective endocarditis. Case report: A 28-year-old male patient was referred to the cardiac surgery unit for urgent mitral valve replacement due to suspicion of infective endocarditis. Microbiological tests yielded negative results. Following recovery and discharge with antimicrobial therapy for 6 weeks, the patient experienced prosthesis detachment, necessitating re-hospitalisation for an emergency valve replacement. Vibrio metschnikovii was identified on the prosthesis valve through PCR and successfully treated with ciprofloxacin. However, a spontaneous rupture of the ascending thoracic aorta led to a neurological injury. Discussion: This case represents the first case of valve infection caused by Vibrio metschnikovii, characterised by diagnostic and therapeutic challenges and the involvement of the great vessels. Also considered in this case, for a disease with a median age of 58 years (11–83) and a male-to-female ratio of 2.2, were one male neonate and six cases for whom neither sex nor age was indicated. Excluding gastrointestinal cases, the septic forms are associated with high morbidity, although the single case described involved a young and healthy subject. Risk factors for the pathogen or predisposing/pathological conditions for endocarditis did not emerge. The routes and the time of infection could not be determined, deepening the possibility of occupational exposure via the patient’s position as a boat worker. Poor sensitivity to third-generation cephalosporins has been reported in the literature: the absence of an antibiogram does not allow for a comparison, although resolution was achieved with ciprofloxacin. Conclusion: The rising global incidence of non-cholera Vibrio infections, driven by environmental changes, calls for urgent research into the factors behind their pathogenicity and infection routes. Diagnostic complexities have emerged together with clinical severity. Full article

Traditional hazard identification techniques for Vibrio parahaemolyticus often neglect the distinction between viable and nonviable bacteria in aquatic products, leading to overestimated disease risks and uncertainties in risk assessments. To address this limitation, we developed an automated PMA pretreatment instrument that integrates dark incubation and photo-crosslinking into a unified workflow, allowing customizable parameters such as incubation time, light exposure duration, and mixing speed while maintaining stable temperatures (<±1 °C fluctuation) to preserve bacterial DNA integrity. Leveraging this system, a duplex qPCR assay was optimized for simultaneous quantitative detection of V. parahaemolyticus and V. cholerae in aquatic products and environmental samples. The assay demonstrated robust performance with 90–110% amplification efficiencies across diverse matrices, achieving low limits of detection (LODs) of 10¹–10² CFU/mL in shrimp farming environment water and 10²–10³ CFU/g in shrimp (Litopenaeus vannamei) and oyster (Crassostrea gigas). Notably, it effectively discriminated viable bacteria from 10⁶ CFU/mL(g) nonviable cells and showed strong correlation with ISO-standard methods in real-world sample validation. This integrated platform offers a rapid, automated solution for accurate viable bacterial quantification, with significant implications for food safety, pathogen surveillance, and risk management in aquatic industries. Full article

Consumption of contaminated drinking water is known to cause waterborne diseases such as diarrhoea, dysentery, typhoid, and hepatitis. This study applied whole-genome sequencing (WGS) to detect, identify, compare, and profile diarrhoeagenic pathogens (Vibrio cholerae, Shiga toxin-producing Escherichia coli, and Escherichia coli O157:H7) from 3168 water samples and 135 faecal samples (human and animal). Culture-based methods, MALDI-TOF mass spectrometry, and PCR were employed prior to WGS for identification of pathogens. Culture-based results revealed high presumptive prevalence of STEC (40.2%), V. cholerae (37.1%), and E. coli O157:H7 (22.7%). The MALDI-TOF confirmed 555 isolates with V. cholerae identified as Vibrio albensis. Shiga toxin-producing Escherichia coli (STEC) was more prevalent in wastewater (60%), treated water (54.1%), and groundwater (36.8%). PCR detected 46.4% of virulence genes from the water isolates and 66% of virulence genes from the STEC stool isolates. WGS also revealed STEC (92.9%) as the most prevalent species and found common virulence (e.g., hcp1/tssD1 and hlyE) and resistance (e.g., acrA and baeR) genes in all three types of samples. Five resistance and thirteen virulence genes overlapped among treated water and stool isolates. These findings highlight the diarrhoeagenic pathogens’ public health risk in water sources and underscore the need for better water quality monitoring and treatment standards. Full article

(1) Background: The COVID-19 pandemic highlights the critical necessity for the development of mucosal vaccines. (2) Objective: In this study, we aimed to develop mucosal vaccines based on the receptor-binding domain (RBD) of the SARS-CoV-2 Spike protein. (3) Methods: We engineered the RBD of the Spike protein by incorporating ten lysine residues (K10), thereby enhancing its positive charge under physiological conditions. (4) Results: Although this modification did not directly augment the immunogenicity of the antigen, its combination with the mucosal adjuvant cholera toxin B subunit (CTB) and administration via the pulmonary route in BALB/c mice resulted in the induction of robust neutralizing antibody titers. Antigen-specific antibody responses were observed in both serum and bronchoalveolar lavage fluid. Importantly, serum IgG antibody titers remained above 10⁴ six months following third immunization, suggesting the establishment of sustained long-term immunity. Additionally, the incorporation of five lysine residues (K5) into the RBD, in conjunction with CTB, significantly increased serum IgG and IgA antibody titers. (5) Conclusions: Adding poly-lysine to RBD and combining it with CTB can stimulate robust mucosal and humoral immune responses in mice. These findings offer valuable insights for the design of subunit mucosal vaccines. Full article

This study explored the effectiveness of plasma-activated water (PAW), generated by a newly developed compact generator, for decontaminating foodborne bacteria in oyster meats. The generator effectively produced PAW with antibacterial activity when the water passed through the plasma reactor in a single cycle. The temperature of the PAW produced by the developed device did not exceed 40 °C, enabling its direct application to biological tissues immediately after production and discharge from the plasma reactor. The effects of flow rates and post-discharge times on key reactive species—including hydrogen peroxide, nitrite, and nitrate—were analyzed, along with pH and temperature. Freshly produced PAW can completely inhibit both E. coli and S. aureus in vitro, with a 5-log reduction within 5 min of treatment. Application to oyster meats led to an 86.6% and 87.9% inactivation of V. cholerae and V. parahaemolyticus, respectively. These research findings indicate that PAW generated using the developed compact flow-through generator holds promise as a food safety solution for households. The fact that complete foodborne pathogen elimination was not achieved emphasizes the need for further optimization. Full article

Non-O1, non-O139 Vibrio cholerae (NOVC) extraintestinal infections are rare, but recently, several clinical incidents have been reported worldwide. Toxigenic V. cholerae is a well-known etiological agent of cholera, responsible for acute dehydrating watery diarrhea. Outbreaks occur in an epidemic seasonal pattern, particularly in countries with poverty and poor sanitation. Strains of NOVC are usually not involved in causing the epidemic or pandemic outbreaks seen with potential strains of V. cholerae serogroup O1 and O139. However, they can still cause severe sporadic cases of intestinal as well as extraintestinal infections. In this study, we investigated a case of extraintestinal infections associated with the NOVC serogroup isolated from a deep closed wound abscess. The isolate was screened for the presence of three major virulence genes, toxR, ctxA, and tcpA. The strain tested positive for the toxR gene encoding the regulatory protein and cholera toxin (ctx) gene and tested negative for the toxin-coregulated pilus (TCP) gene, which is essential for the colonization of the human intestine, causing the severe diarrheal disease cholera. To the best of our knowledge, this is the first case of extraintestinal infection caused by toxigenic Vibrio cholerae non-O1/non-O139 in a hospitalized patient in Saudi Arabia. Full article

In this paper, a stochastic continuous-time Markov chain (CTMC) model is developed and analyzed to explore the dynamics of cholera. The multitype branching process is used to compute a stochastic threshold for the CTMC model. Latin hypercube sampling/partial rank correlation coefficient (LHS/PRCC) sensitivity analysis methods are implemented to derive sensitivity indices of model parameters. The results show that the natural death rate $\left({\mu }_v\right)$ of a vector is the most sensitive parameter for controlling disease outbreaks. Numerical simulations indicate that the solutions of the CTMC stochastic model are relatively close to the solutions of the deterministic model. Numerical simulations estimate the probability of both disease extinction and outbreak. The probability of cholera extinction is high when it emerges from bacterial concentrations in non-contaminated/safe water in comparison to when it emerges from all infected groups. Thus, any intervention that focuses on reducing the number of infections at the beginning of a cholera outbreak is essential for reducing its transmission. Full article

The motility and chemotaxis of Vibrio cholerae, the bacterial pathogen responsible for cholera, play crucial roles in both environmental survival and infection. Understanding their molecular mechanisms is therefore essential not only for fundamental biology but also for infection control and therapeutic development. The bacterium’s sheathed, polar flagellum—its motility organelle—is powered by a sodium-driven motor. This motor’s rotation is regulated by the chemotaxis (Che) signaling system, with a histidine kinase, CheA, and a response regulator, CheY, serving as the central processing unit. However, V. cholerae possesses two additional, parallel Che signaling systems whose physiological functions remain unclear. Furthermore, the bacterium harbors over 40 receptors/transducers that interact with CheA homologs, forming a complex regulatory network likely adapted to diverse environmental cues. Despite significant progress, many aspects of these systems remain to be elucidated. Here, we summarize the current understanding to facilitate future research. Full article

Background: Cholera is a diarrheal disease prevalent in populations without access to clean water. Cholera is caused by Vibrio cholerae, which colonizes the upper small intestine in humans once ingested. A growing number of studies suggest that the gut microbiome composition modulates animal behavior. Zebrafish are an established cholera model that can maintain a complex, mature gut microbiome during infection. Larval zebrafish, which have immature gut microbiomes, provide the advantage of high-throughput analyses for established behavioral models. Methods: We identified the effects of V. cholerae O1 El Tor C6706 colonization at 5 days post-fertilization (dpf) on larval zebrafish behavior by tracking startle responses at 10 dpf. We also characterized the larval gut microbiome using 16S rRNA sequencing. V. cholerae-infected or uninfected control groups were exposed to either an alternating light/dark stimuli or a single-tap stimulus, and average distance and velocity were tracked. Results: While there was no significant difference in the light/dark trial, we report a significant decrease in distance moved for C6706-colonized larvae during the single-tap trial. Conclusion: This suggests that early V. cholerae colonization of the larval gut microbiome has a dampening effect on sensorimotor function, supporting the idea of a link between the gut microbiome and behavior. Full article

This study investigates the prevalence of Vibrio spp. in seafood from supermarkets and fish markets in Berlin, Germany. A total of 306 seafood samples, including shrimp and mussels, were bought from supermarkets between March 2023 and January 2024. Samples were analysed using the ISO standard method and multiplex PCR to identify V. parahaemolyticus, V. alginolyticus, V. cholerae and V. vulnificus. The results indicated an overall Vibrio spp. prevalence of 56%. Among the positive samples, the most prevalent species found was V. parahaemolyticus (58%), followed by V. alginolyticus (42%), V. cholerae non-O1/non-O139 (25%), and V. vulnificus (4%). Samples obtained from supermarkets exhibited a lower prevalence (50%) than those received from fish markets (91%). Virulence genes such as ctxA, tdh, or trh were not detected in the respective Vibrio species. Nevertheless, the high prevalence underscores the need and urgency of continuous seafood surveillance. Full article

The rise of antibiotic-resistant bacterial infections necessitates alternative therapeutic strategies, such as phage therapy. This study investigates the potential of phage vB_PmuM_CFP3 (CFP3) as a therapeutic agent against avian cholera caused by Pasteurella multocida (P. multocida). Phage CFP3 was isolated from the feces and wastewater of a laying hen farm and underwent comprehensive biological characterization, including host range, lytic activity, and environmental stability. Transmission electron microscopy revealed CFP3′s typical myovirus morphology, with a head diameter of approximately 60 nm and a tail length of about 120 nm. CFP3 demonstrated high stability across a pH range of 4–10 and temperatures of 30–40 °C, making it suitable for oral administration in poultry. The phage exhibited a latent period of about 90 min and an optimal multiplicity of infection (MOI) of 1. Despite its narrow host range, with a lysis rate of 28.2% against avian-derived type A P. multocida, CFP3′s specificity minimizes impact on non-target bacteria. Whole-genome sequencing revealed a 32,696 bp linear double-stranded DNA genome with 46 predicted open reading frames (ORFs) and no tRNA or antibiotic resistance genes, enhancing its safety profile. Phylogenetic analysis indicated a close evolutionary relationship with Haemophilus phages HP1, HP2, and Pasteurella phage F108. While CFP3 shows promise as a precision therapeutic tool, further in vivo studies are required to evaluate its efficacy and safety. Future research should focus on expanding the phage library, optimizing phage mixtures, and exploring synergistic effects with other antimicrobial strategies. This study provides foundational data supporting the development of CFP3 as a viable alternative to antibiotics for controlling avian cholera. Full article

Competition between bacterial species is a major factor shaping microbial communities. It is possible but remains largely unexplored that competition between bacterial pathogens can be mediated through antagonistic effects of bacterial effector proteins on host systems, particularly the actin cytoskeleton. Using Salmonella Typhimurium invasion into cells as a model, we demonstrate that invasion is inhibited if the host actin cytoskeleton is disturbed by actin-specific toxins, namely, Vibrio cholerae MARTX actin crosslinking (ACD) and Rho GTPase inactivation (RID) domains, Photorhabdus luminescens TccC3, and Salmonella’s own SpvB. We noticed that ACD, being an effective inhibitor of tandem G-actin-binding assembly factors, is likely to inhibit the activity of another Vibrio effector, VopF. In reconstituted actin polymerization assays and by live-cell microscopy, we confirmed that ACD potently halted the actin nucleation and pointed-end elongation activities of VopF, revealing competition between these two V. cholerae effectors. These results suggest that bacterial effectors from different species that target the same host machinery or proteins may represent an effective but largely overlooked mechanism of indirect bacterial competition in host-associated microbial communities. Whether the proposed inhibition mechanism involves the actin cytoskeleton or other host cell compartments, such inhibition deserves investigation and may contribute to a documented scarcity of human enteric co-infections by different pathogenic bacteria. Full article