Investigation of potentially pathogenic Vibrionaceae in Saint-Louis city, Senegal.

Introduction: as cholera, due to toxigenic bacteria Vibrio cholera (serogroups O1 and O139), is a major public health threat in Africa, the aim of this work was to investigate potentially pathogenic Vibrionaceae bacteria firstly from human stool samples, and secondly from various environmental water points of Saint-Louis city in Senegal. Methods: a hospital-based study was conducted between 2013 and 2015. Stool samples were taken and cultured from daily incoming patients or hospitalized for acute diarrhea at Saint-Louis´ regional hospital. For environment, a monthly longitudinal sampling from January to October 2016 was carried out at 10 sites in the city. We used total DNA extracted from APW (alkaline peptone water) broth solutions and on suspect bacterial colonies to run PCR Multiplex targeting specific DNA fragments to detect Vibrio genus and specific species. In case of positivity, a simplex PCR was performed to test for cholera toxins Ctx, and V. parahaemolyticus TRH and TDH. Results: for 43 patients screened, bacterial culture was positive in 6% of cases but no strain of V. cholerae or other Vibrio sp. was isolated. PCR on 90 APW solutions were positive for Vibrio sp.(n = 43), V. cholera(n = 27), V. mimicus(n = 16), V. parahaemolyticus(8), V. alginolyticus(n = 4), and V. vulnificus(n = 2). Unlike for those on suspected colonies which were positive for a majority of V. parahaemolyticus (n = 40) and V. cholerae non-O1 / O139 (n = 35). Six strains of V. parahaemolyticus carried TRH gene, 3 of which expressed simultaneously virulence TRH and TDH genes. For physicochemical parameters, all temperatures varied similarly according to a unimodal seasonality, as well as salinity. Conclusion: despite the presence of natural populations of Vibrionaceae, even toxigenic ones, was noted in water environment, along with favorable habitat conditions that could play a role in transmission of Vibriosis in the Saint Louis population, we did not isolate any of them from patients screened at the hospital.

Non-O1 and non-O139 Vibrio cholerae Septicemia and Cellulitis: a Case Report.

Non-O1 and non-O139 Vibrio cholerae (NOVC) are serogroups that do not produce cholera toxin and are not responsible for epidemics. Even though rarely encountered in clinical practice, they can cause a spectrum of different conditions ranging from mild gastrointestinal syndrome to extraintestinal diseases, of which bacteremia and wound infections are the most severe. Risk factors for severe disease are cirrhosis, neoplasms, and diabetes mellitus. The mortality rate of NOVC bacteremia in hospitalized patients ranges from 24 to 61.5%. Incidence of NOVC infections is still rare, and consensus recommendations on treatment are not available. We report a case of NOVC bacteremia associated with severe cellulitis in an immunocompetent 75-year-old man who had eaten raw seafood in a location by the northern Adriatic Sea (Italy). Twenty-four hours after intake, he developed a high fever and vomiting. Afterwards, he started noticing the appearance of cellulitis in his right leg, which worsened in a matter of hours. The patient had a history of compensated type 2 diabetes mellitus. NOVC was isolated from both blood cultures and the leg ulcer. The non-O1, non-O139 serogroup was confirmed, and the detection of the cholera toxin gene was negative. Both tests were performed by the Reference National Laboratory of Istituto Superiore di Sanità (ISS). Multiple antimicrobial regimens were administered, with complete recovery. In conclusion, considering the severity of NOVC-associated manifestations, it is of pivotal importance to reach etiological diagnosis for a target antimicrobial therapy and to consider V. cholerae infection in the differential diagnosis in the presence of risk factors and potential exposure.

Molecular mechanism of plasmid elimination by the DdmDE defense system.

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.

Microbiology: Murder as a solution to promiscuity?

Strain-specific pili enable Vibrio cholerae bacteria to adhere to each other and form aggregates in liquid culture. A new study focuses on strains with less specific, promiscuous pili and suggests a role for contact-dependent bacterial killing in shaping the composition of these aggregates.

Evaluation of humoral and cellular immune responses against Vibrio cholerae using oral immunization by multi-epitope-phage-based vaccine.

INTRODUCTION: Cholera is a severe gastrointestinal disease that manifests with rapid onset of diarrhea, vomiting, and high mortality rates. Due to its widespread occurrence in impoverished communities with poor water sanitation, there is an urgent demand for a cost-effective and highly efficient vaccine. Multi-epitope vaccines containing dominant immunological epitopes and adjuvant compounds have demonstrated potential in boosting the immune response. MATERIAL AND METHODS: B and T epitopes of OMPU, OMPW, TCPA, CTXA, and CTXB proteins were predicted using bioinformatics methods. Subsequently, highly antigenic multi-epitopes that are non-allergenic and non-toxic were synthesized. These multi-epitopes were then cloned into the pCOMB phagemid. A plasmid M13KO7ΔpIII containing all helper phage proteins except pIII was created to produce the recombinant phage. Female Balb/c mice were divided into three groups and immunized accordingly. The mice received the helper phage, recombinant phage or PBS via gavage feeding thrice within two weeks. Serum samples were collected before and after immunization for the ELISA test as well as evaluating immune system induction through ELISpot testing of spleen lymphocytes. RESULTS: The titer of the recombinant phage was determined to be 1011 PFU/ml. The presence of the recombinant phage was confirmed through differences in optical density between sample and control groups in the ELISA phage technique, as well as by observing transduction activity, which demonstrated successful production of a recombinant phage displaying the Vibrio multi-epitope on M13 phage pIII. ELISA results revealed significant differences in phage antibodies before and after inoculation, particularly notable in the negative control mice. Mice treated with multi-epitope phages exhibited antibodies against Vibrio cholerae lysate. Additionally, ELISpot results indicated activation of cellular immunity in mice receiving both Vibrio and helper phage. CONCLUSION: This study emphasizes the potential of multi-epitope on phage to enhance both cellular and humoral immunity in mice, demonstrating how phages can be used as adjuvants to stimulate mucosal immunity and act as promising candidates for oral vaccination.

A Label-Free Approach for Relative Spatial Quantitation of c-di-GMP in Microbial Biofilms.

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.

Interactions between pili affect the outcome of bacterial competition driven by the type VI secretion system.

The bacterial type VI secretion system (T6SS) is a widespread, kin-discriminatory weapon capable of shaping microbial communities. Due to the system's dependency on contact, cellular interactions can lead to either competition or kin protection. Cell-to-cell contact is often accomplished via surface-exposed type IV pili (T4Ps). In Vibrio cholerae, these T4Ps facilitate specific interactions when the bacteria colonize natural chitinous surfaces. However, it has remained unclear whether and, if so, how these interactions affect the bacterium's T6SS-mediated killing. In this study, we demonstrate that pilus-mediated interactions can be harnessed by T6SS-equipped V. cholerae to kill non-kin cells under liquid growth conditions. We also show that the naturally occurring diversity of pili determines the likelihood of cell-to-cell contact and, consequently, the extent of T6SS-mediated competition. To determine the factors that enable or hinder the T6SS's targeted reduction of competitors carrying pili, we developed a physics-grounded computational model for autoaggregation. Collectively, our research demonstrates that T4Ps involved in cell-to-cell contact can impose a selective burden when V. cholerae encounters non-kin cells that possess an active T6SS. Additionally, our study underscores the significance of T4P diversity in protecting closely related individuals from T6SS attacks through autoaggregation and spatial segregation.

Mobile-CRISPRi as a powerful tool for modulating Vibrio gene expression.

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.IMPORTANCEThe genetic manipulation of bacterial genomes is an invaluable tool in experimental microbiology. The development of CRISPRi (Clustered Regularly Interspaced Palindromic Repeats interference) tools has revolutionized genetics in many organisms, including bacteria. Here, we optimized the use of Mobile-CRISPRi in five Vibrio species, each of which has significant impacts on marine environments and organisms that include squid, shrimp, shellfish, finfish, corals, and multiple of which pose direct threats to human health. The Mobile-CRISPRi technology is easily adaptable, moveable from strain to strain, and enables researchers to selectively turn off gene expression. Our experiments demonstrate Mobile-CRISPRi is effective and robust at repressing gene expression of both essential and non-essential genes in Vibrio species.

Plasmid targeting and destruction by the DdmDE bacterial defence system.

Although eukaryotic Argonautes have a pivotal role in post-transcriptional gene regulation through nucleic acid cleavage, some short prokaryotic Argonaute variants (pAgos) rely on auxiliary nuclease factors for efficient foreign DNA degradation1. Here we reveal the activation pathway of the DNA defence module DdmDE system, which rapidly eliminates small, multicopy plasmids from the Vibrio cholerae seventh pandemic strain (7PET)2. Through a combination of cryo-electron microscopy, biochemistry and in vivo plasmid clearance assays, we demonstrate that DdmE is a catalytically inactive, DNA-guided, DNA-targeting pAgo with a distinctive insertion domain. We observe that the helicase-nuclease DdmD transitions from an autoinhibited, dimeric complex to a monomeric state upon loading of single-stranded DNA targets. Furthermore, the complete structure of the DdmDE-guide-target handover complex provides a comprehensive view into how DNA recognition triggers processive plasmid destruction. Our work establishes a mechanistic foundation for how pAgos utilize ancillary factors to achieve plasmid clearance, and provides insights into anti-plasmid immunity in bacteria.

Slings and arrows: sRNAs mediate intragenomic competition.

Bacterial genomes are littered with exogenous: competing DNA elements. Here, Sprenger et al. demonstrate that the Vibrio cholerae prophage VP882 modulates host functions via production of regulatory sRNAs to promote phage development. Alternatively, host sRNAs inhibit the VP882 lytic phase by specifically regulating phage genes.

Structural insights in to the atypical type-I ABC Glucose-6-phosphate importer VCA0625-27 of Vibrio cholerae.

Sugar phosphates are potential sources of carbon and phosphate for bacteria. Despite that the process of internalization of Glucose-6-Phosphate (G6P) through plasma membrane remained elusive in several bacteria. VCA0625-27, made of periplasmic ligand binding protein (PLBP) VCA0625, an atypical monomeric permease VCA0626, and a cytosolic ATPase VCA0627, recently emerged as hexose-6-phosphate uptake system of Vibrio cholerae. Here we report high resolution crystal structure of VCA0625 in G6P bound state that largely resembles AfuA of Actinobacillus pleuropneumoniae. MD simulations on VCA0625 in apo and G6P bound states unraveled an 'open to close' and swinging bi-lobal motions, which are diminished upon G6P binding. Mutagenesis followed by biochemical assays on VCA0625 underscored that R34 works as gateway to bind G6P. Although VCA0627 binds ATP, it is ATPase deficient in the absence of VCA0625 and VCA0626, which is a signature phenomenon of type-I ABC importer. Further, modeling, docking and systematic sequence analysis allowed us to envisage the existence of similar atypical type-I G6P importer with fused monomeric permease in 27 other gram-negative bacteria.

Epidemiological and genetic characterization of multidrug-resistant non-O1 and non-O139 Vibrio cholerae from food in southern China.

This study reports a comprehensive epidemiological and genetic analysis of V. cholerae strains, specifically non-O1/non-O139 serogroups, isolated from animal-derived food samples in Guangdong province from 2015 to 2019. A total of 21 V. cholerae strains were obtained, which exhibited high resistance rates for nalidixic acid (57.14 %, 12/21), ampicillin (33.33 %, 7/21), and ciprofloxacin (19.05 %, 4/21). The quinolone resistance-related gene, qnrVC, was prevalent in 80.95 % (17/21) of the isolates. Additionally, chromosomally mediated quinolone-resistance mutations, including mutations in GyrA at position 83 (S83I) and ParC at position 85 (S85L), were detected in 47.62 % of the isolates. The combination of target mutation and qnrVC genes was shown to mediate resistance or intermediate resistance to ciprofloxacin in V. cholerae. Furthermore, an IncC-type conjugative plasmid carrying thirteen antibiotic resistance genes, including genes conferring resistance to two clinically important antibiotics, cephalosporins and fluoroquinolones, was identified in the shrimp-derived strain Vc516. While none of our food isolates harbored the toxigenic CTX- and TCP-encoding genes, they did possess genes encoding toxins such as HlyA and Autoinducer-2. Notably, some V. cholerae strains from this study exhibited a close genetic relationship with clinical strains, suggesting their potential to cause human infections. Taken together, this study provides a comprehensive view of the epidemiological features and genetic basis of antimicrobial resistance and virulence potential of V. cholerae strains isolated from food in southern China, thereby advancing our understanding of this important pathogen.

Determining the Young's Modulus of the Bacterial Cell Envelope.

Bacteria experience substantial physical forces in their natural environment, including forces caused by osmotic pressure, growth in constrained spaces, and fluid shear. The cell envelope is the primary load-carrying structure of bacteria, but the mechanical properties of the cell envelope are poorly understood; reports of Young's modulus of the cell envelope of Escherichia coli range from 2 to 18 MPa. We developed a microfluidic system to apply mechanical loads to hundreds of bacteria at once and demonstrated the utility of the approach for evaluating whole-cell stiffness. Here, we extend this technique to determine Young's modulus of the cell envelope of E. coli and of the pathogens Vibrio cholerae and Staphylococcus aureus. An optimization-based inverse finite element analysis was used to determine the cell envelope Young's modulus from observed deformations. The Young's modulus values of the cell envelope were 2.06 ± 0.04 MPa for E. coli, 0.84 ± 0.02 MPa for E. coli treated with a chemical (A22) known to reduce cell stiffness, 0.12 ± 0.03 MPa for V. cholerae, and 1.52 ± 0.06 MPa for S. aureus (mean ± SD). The microfluidic approach allows examination of hundreds of cells at once and is readily applied to Gram-negative and Gram-positive organisms as well as rod-shaped and cocci cells, allowing further examination of the structural causes behind differences in cell envelope Young's modulus among bacterial species and strains.

Genetic interaction mapping reveals functional relationships between peptidoglycan endopeptidases and carboxypeptidases.

Peptidoglycan (PG) is the main component of the bacterial cell wall; it maintains cell shape while protecting the cell from internal osmotic pressure and external environmental challenges. PG synthesis is essential for bacterial growth and survival, and a series of PG modifications are required to allow expansion of the sacculus. Endopeptidases (EPs), for example, cleave the crosslinks between adjacent PG strands to allow the incorporation of newly synthesized PG. EPs are collectively essential for bacterial growth and must likely be carefully regulated to prevent sacculus degradation and cell death. However, EP regulation mechanisms are poorly understood. Here, we used TnSeq to uncover novel EP regulators in Vibrio cholerae. This screen revealed that the carboxypeptidase DacA1 (PBP5) alleviates EP toxicity. dacA1 is essential for viability on LB medium, and this essentiality was suppressed by EP overexpression, revealing that EP toxicity both mitigates, and is mitigated by, a defect in dacA1. A subsequent suppressor screen to restore viability of ΔdacA1 in LB medium identified hypomorphic mutants in the PG synthesis pathway, as well as mutations that promote EP activation. Our data thus reveal a more complex role of DacA1 in maintaining PG homeostasis than previously assumed.

Fuzzy recognition by the prokaryotic transcription factor HigA2 from Vibrio cholerae.

Disordered protein sequences can exhibit different binding modes, ranging from well-ordered folding-upon-binding to highly dynamic fuzzy binding. The primary function of the intrinsically disordered region of the antitoxin HigA2 from Vibrio cholerae is to neutralize HigB2 toxin through ultra-high-affinity folding-upon-binding interaction. Here, we show that the same intrinsically disordered region can also mediate fuzzy interactions with its operator DNA and, through interplay with the folded helix-turn-helix domain, regulates transcription from the higBA2 operon. NMR, SAXS, ITC and in vivo experiments converge towards a consistent picture where a specific set of residues in the intrinsically disordered region mediate electrostatic and hydrophobic interactions while "hovering" over the DNA operator. Sensitivity of the intrinsically disordered region to scrambling the sequence, position-specific contacts and absence of redundant, multivalent interactions, point towards a more specific type of fuzzy binding. Our work demonstrates how a bacterial regulator achieves dual functionality by utilizing two distinct interaction modes within the same disordered sequence.

Small RNAs direct attack and defense mechanisms in a quorum sensing phage and its host.

Many, if not all, bacteria use quorum sensing (QS) to control collective behaviors, and more recently, QS has also been discovered in bacteriophages (phages). Phages can produce communication molecules of their own, or "listen in" on the host's communication processes, to switch between lytic and lysogenic modes of infection. Here, we study the interaction of Vibrio cholerae with the lysogenic phage VP882, which is activated by the QS molecule DPO. We discover that induction of VP882 results in the binding of phage transcripts to the major RNA chaperone Hfq, which in turn outcompetes and downregulates host-encoded small RNAs (sRNAs). VP882 itself also encodes Hfq-binding sRNAs, and we demonstrate that one of these sRNAs, named VpdS, promotes phage replication by regulating host and phage mRNA levels. We further show that host-encoded sRNAs can antagonize phage replication by downregulating phage mRNA expression and thus might be part of the host's phage defense arsenal.

Effect of physicochemical and microbiological factors on the development of viable but non-culturable and resuscitation states of Vibrio cholerae.

BACKGROUND: Vibrio cholerae can endure harsh environmental conditions by transitioning into viable but non-culturable (VBNC) form and resuscitate upon return of appropriate conditions. METHOD: In this study, we assessed the impact of physicochemical and microbiological factors, on the development of low temperature-induced VBNC state and subsequent recovery by temperature upshift. RESULTS: In estuarine water, Vibrio cholerae exhibits a slower decline in culturability over a period of 77 days as compared to 10 days in fresh water. When variable cell numbers from different growth phases were used for VBNC induction, it was observed that the higher inoculum size (106-107 cfu ml-1) from the late log phase culture appears to be crucial for entering the VBNC state. Conversely, starved cells could enter the VBNC state with an initial inoculum of 104-105 cfu ml-1, followed by resuscitation as well. The addition of glucose, GlcNAc and mannitol differentially affects progression into VBNC, while the addition of tryptone, yeast extract and casamino acid facilitated early entry into the VBNC state and shortened the length of the recovery period. CONCLUSION: Altogether these findings demonstrated that the ionic strength of water, inoculum size and the availability of nutrients played distinct roles during VBNC induction and resuscitation.

Phage predation, disease severity, and pathogen genetic diversity in cholera patients.

Despite an increasingly detailed picture of the molecular mechanisms of bacteriophage (phage)-bacterial interactions, we lack an understanding of how these interactions evolve and impact disease within patients. In this work, we report a year-long, nationwide study of diarrheal disease patients in Bangladesh. Among cholera patients, we quantified Vibrio cholerae (prey) and its virulent phages (predators) using metagenomics and quantitative polymerase chain reaction while accounting for antibiotic exposure using quantitative mass spectrometry. Virulent phage (ICP1) and antibiotics suppressed V. cholerae to varying degrees and were inversely associated with severe dehydration depending on resistance mechanisms. In the absence of antiphage defenses, predation was "effective," with a high predator:prey ratio that correlated with increased genetic diversity among the prey. In the presence of antiphage defenses, predation was "ineffective," with a lower predator:prey ratio that correlated with increased genetic diversity among the predators. Phage-bacteria coevolution within patients should therefore be considered in the deployment of phage-based therapies and diagnostics.

Genomic characteristics of clinical non-toxigenic Vibrio cholerae isolates in Switzerland: a cross-sectional study.

STUDY AIMS: Although non-toxigenic Vibrio cholerae lack the ctxAB genes encoding cholera toxin, they can cause diarrhoeal disease and outbreaks in humans. In Switzerland, V. cholerae is a notifiable pathogen and all clinical isolates are analysed at the National Reference Laboratory for Enteropathogenic Bacteria and Listeria. Up to 20 infections are reported annually. In this study, we investigated the population structure and genetic characteristics of non-toxigenic V. cholerae isolates collected over five years. METHODS:  V. cholerae isolates were serotyped and non-toxigenic isolates identified using a ctxA-specific PCR. Following Illumina whole-genome sequencing, genome assemblies were screened for virulence and antibiotic resistance genes. Phylogenetic analyses were performed in the context of 965 publicly available V. cholerae genomes. RESULTS: Out of 33 V. cholerae infections reported between January 2017 and January 2022 in Switzerland, 31 were caused by ctxA-negative isolates. These non-toxigenic isolates originated from gastrointestinal (n = 29) or extraintestinal (n = 2) sites. They were phylogenetically diverse and belonged to 29 distinct sequence types. Two isolates were allocated to the lineage L3b, a ctxAB-negative but tcpA-positive clade previously associated with regional outbreaks. The remaining 29 isolates were placed in lineage L4, which is associated with environmental strains. Genes or mutations associated with reduced susceptibility to the first-line antibiotics fluoroquinolones and tetracyclines were identified in 11 and 3 isolates, respectively. One isolate was predicted to be multidrug resistant. CONCLUSIONS:  V. cholerae infections in Switzerland are rare and predominantly caused by lowly virulent ctxAB-negative and tcpA-negative strains. As V. cholerae is not endemic in Switzerland, cases are assumed to be acquired predominantly during travel. This assumption was supported by the phylogenetic diversity of the analysed isolates.

In Vivo Microrheology Reveals Local Elastic and Plastic Responses Inside 3D Bacterial Biofilms.

Bacterial biofilms are highly abundant 3D living materials capable of performing complex biomechanical and biochemical functions, including programmable growth, self-repair, filtration, and bioproduction. Methods to measure internal mechanical properties of biofilms in vivo with spatial resolution on the cellular scale have been lacking. Here, thousands of cells are tracked inside living 3D biofilms of the bacterium Vibrio cholerae during and after the application of shear stress, for a wide range of stress amplitudes, periods, and biofilm sizes, which revealed anisotropic elastic and plastic responses of both cell displacements and cell reorientations. Using cellular tracking to infer parameters of a general mechanical model, spatially-resolved measurements of the elastic modulus inside the biofilm are obtained, which correlate with the spatial distribution of the polysaccharides within the biofilm matrix. The noninvasive microrheology and force-inference approach introduced here provides a general framework for studying mechanical properties with high spatial resolution in living materials.