Eco-alternative treatments for Vibrio parahaemolyticus and V. cholerae biofilms from shrimp industry through Eucalyptus (Eucalyptus globulus) and Guava (Psidium guajava) extracts: A road for an Ecuadorian sustainable economy.

Understanding how environmental variables influence biofilm formation becomes relevant for managing Vibrio biofilm-related infections in shrimp production. Therefore, we evaluated the impact of temperature, time, and initial inoculum in the biofilm development of these two Vibrio species using a multifactorial experimental design. Planktonic growth inhibition and inhibition/eradication of Vibrio biofilms, more exactly V. parahaemolyticus (VP87 and VP275) and V. cholerae (VC112) isolated from shrimp farms were evaluated by Eucalyptus and Guava aqueous leaf extracts and compared to tetracycline and ceftriaxone. Preliminary results showed that the best growth conditions of biofilm development for V. parahaemolyticus were 24 h and 24°C (p <0.001), while V. cholerae biofilms were 72 h and 30°C (p <0.001). Multivariate linear regression ANOVA was applied using colony-forming unit (CFU) counting assays as a reference, and R-squared values were applied as goodness-of-fit measurements for biofilm analysis. Then, both plant extracts were analyzed with HPLC using double online detection by diode array detector (DAD) and mass spectrometry (MS) for the evaluation of their chemical composition, where the main identified compounds for Eucalyptus extract were cypellogin A, cypellogin B, and cypellocarpin C, while guavinoside A, B, and C compounds were the main compounds for Guava extract. For planktonic growth inhibition, Eucalyptus extract showed its maximum effect at 200 µg/mL with an inhibition of 75% (p < 0.0001) against all Vibrio strains, while Guava extract exhibited its maximum inhibition at 1600 µg/mL with an inhibition of 70% (p < 0.0001). Both biofilm inhibition and eradication assays were performed by the two conditions (24 h at 24°C and 72 h at 30°C) on Vibrio strains according to desirability analysis. Regarding 24 h at 24°C, differences were observed in the CFU counting between antibiotics and plant extracts, where both plant extracts demonstrated a higher reduction of viable cells when compared with both antibiotics at 8x, 16x, and 32x MIC values (Eucalyptus extract: 1600, 3200, and 6400 µg/mL; while Guava extract: 12800, 25600, and 52000 µg/mL). Concerning 72 h at 30°C, results showed a less notorious biomass inhibition by Guava leaf extract and tetracycline. However, Eucalyptus extract significantly reduced the total number of viable cells within Vibrio biofilms from 2x to 32x MIC values (400-6400 µg/mL) when compared to the same MIC values of ceftriaxone (5-80 µg/mL), which was not able to reduce viable cells. Eucalyptus extract demonstrated similar results at both growth conditions, showing an average inhibition of approximately 80% at 400 µg/mL concentration for all Vibrio isolates (p < 0.0001). Moreover, eradication biofilm assays demonstrated significant eradication against all Vibrio strains at both growth conditions, but biofilm eradication values were substantially lower. Both extract plants demonstrated a higher reduction of viable cells when compared with both antibiotics at 8x, 16x, and 32x MIC values at both growth sets, where Eucalyptus extract at 800 µg/mL reduced 70% of biomass and 90% of viable cells for all Vibrio strains (p < 0.0001). Overall results suggested a viable alternative against vibriosis in the shrimp industry in Ecuador.

Prevalence and antimicrobial susceptibility pattern of Vibrio cholerae isolates from cholera outbreak sites in Ethiopia.

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.

An essential host dietary fatty acid promotes TcpH inhibition of TcpP proteolysis promoting virulence gene expression in Vibrio cholerae.

Vibrio cholerae is a Gram-negative gastrointestinal pathogen responsible for the diarrheal disease cholera. Expression of key virulence factors, cholera toxin and toxin-coregulated pilus, is regulated directly by ToxT and indirectly by two transmembrane transcription regulators (TTRs), ToxR and TcpP, that promote the expression of toxT. TcpP abundance and activity are controlled by TcpH, a single-pass transmembrane protein, which protects TcpP from a two-step proteolytic process known as regulated intramembrane proteolysis (RIP). The mechanism of TcpH-mediated protection of TcpP represents a major gap in our understanding of V. cholerae pathogenesis. The absence of tcpH leads to unimpeded degradation of TcpP in vitro and a colonization defect in a neonate mouse model of V. cholerae colonization. Here, we show that TcpH protects TcpP from RIP via direct interaction. We also demonstrate that α-linolenic acid, a dietary fatty acid, promotes TcpH-dependent inhibition of RIP via co-association of TcpP and TcpH molecules within detergent-resistant membranes (DRMs) in a mechanism requiring the TcpH transmembrane domain. Taken together, our data support a model where V. cholerae cells use exogenous α-linolenic acid to remodel the phospholipid bilayer in vivo, leading to co-association of TcpP and TcpH within DRMs where RIP of TcpP is inhibited by TcpH, thereby promoting V. cholerae pathogenicity. IMPORTANCE: Vibrio cholerae continues to pose a significant global burden on health and an alternative therapeutic approach is needed, due to evolving multidrug resistance strains. Transcription of toxT, stimulated by TcpP and ToxR, is essential for V. cholerae pathogenesis. Our results show that TcpP, one of the major regulators of toxT gene expression, is protected from proteolysis by TcpH, via direct interaction. Furthermore, we identified a gut metabolite, α-linolenic acid, that stimulates the co-association of TcpP and TcpH within detergent-resistant membranes (also known as lipid-ordered membrane domains), thereby supporting TcpH-dependent antagonism of TcpP proteolysis. Data presented here extend our knowledge of RIP, virulence gene regulation in V. cholerae, and, to the best of our knowledge, provides the first evidence that lipid-ordered membranes exist within V. cholerae. The model presented here also suggests that TTRs, common among bacteria and archaea, and co-component signal transduction systems present in Enterobacteria, could also be influenced similarly.

Exploring extended-spectrum beta lactamase resistance in Vibrio parahaemolyticus and Vibrio cholerae within the tropical mangrove ecosystem of southwest India.

Multidrug-resistant pathogenic vibrios are a crisis of concern as they cause multiple illnesses, including gastroenteritis in humans and acute hepatopancreatic necrosis in aquaculture. In the current study, we investigated the prevalence of the beta-lactamase gene CTX-M-group 1 in Vibrio spp. (Vibrio cholerae and Vibrio parahaemolyticus) from the water and sediment of urban tropical mangrove ecosystems of Kerala, southwest India. A total of 120 isolates of Vibrio spp. were tested for antibiotic susceptibility to 14 antibiotics. In water, ampicillin resistance was very high in isolates of V. cholerae (94.1%, n = 17) and V. parahaemolyticus (89.1%, n = 46). 26.9% of V. parahaemolyticus and 14.2% of V. cholerae harbored the CTX-M-group 1 gene in water samples. Compared to V. cholerae, the CTX-M-group 1 gene was exclusively hosted by V. parahaemolyticus (49%) in sediment samples. A significant difference in the prevalence of the CTX-M-group 1 gene was observed among Vibrio spp. in both water and sediment samples (p < 0.05). The results revealed the presence of multidrug-resistant and beta-lactamase harboring Vibrio spp. in mangrove ecosystems, which may have evolved as a consequence of the misuse and abuse of broad-spectrum antibiotics as prophylaxis in human health care and aquaculture.

The alleviative effect of Bacillus subtilis-supplemented diet against Vibrio cholerae infection in Nile tilapia (Oreochromis niloticus).

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.

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.

Recent developments of agents targeting Vibrio cholerae: patents and literature data.

INTRODUCTION: Vibrio cholerae bacteria cause an infection characterized by acute diarrheal illness in the intestine. Cholera is sustained by people swallowing contaminated food or water. Even though symptoms can be mild, if untreated disease becomes severe and life-threatening, especially in low-income countries. AREAS COVERED: After a description of the most recent literature on the pathophysiology of this infection, we searched for patents and literature articles following the PRISMA guidelines, filtering the results disclosed from 2020 to present. Moreover, some innovative molecular targets (e.g., carbonic anhydrases) and pathways to counteract this rising problem were also discussed in terms of design, structure-activity relationships and structural analyses. EXPERT OPINION: This review aims to cover and analyze the most recent advances on the new druggable targets and bioactive compounds against this fastidious pathogen, overcoming the use of old antibiotics which currently suffer from high resistance rate.

Elevated concentrations of polymyxin B elicit a biofilm-specific resistance mechanism in Vibrio cholerae.

Vibrio cholerae can form biofilms in the aquatic environment and in the human intestine, facilitating the release of hyper-infectious aggregates. Due to the increasing antibiotic resistance, alternatives need to be found. One of these alternatives is antimicrobial peptides, including polymyxin B (PmB). In this study, we first investigated the resistance of V. cholerae O1 El Tor strain A1552 to various antimicrobials under aerobic and anaerobic conditions. An increased resistance to PmB is observed in anaerobiosis, with a 3-fold increase in the dose required for 50 % growth inhibition. We then studied the impact of the PmB on the formation and the degradation of V. cholerae biofilms to PmB. Our results show that PmB affects more efficiently biofilm formation under anaerobic conditions. On the other hand, preformed biofilms are susceptible to degradation by PmB at concentrations close to the minimal inhibitory concentration. At higher concentrations, we observe an opacification of the biofilm structures within 20 min post-treatment, suggesting a densification of the structure. This densification does not seem to result from the overexpression of matrix genes but rather from DNA release through massive cell lysis, likely forming a protective shield that limits the penetration of the PmB into the biofilm.

VP3 Phage Combined with High Salt Promotes the Lysis of Biofilm-Associated Vibrio cholerae.

Cholera, caused by pathogenic Vibrio cholerae, poses a significant public health risk through water and food transmission. Biofilm-associated V. cholerae plays a crucial role in seasonal cholera outbreaks as both a reservoir in aquatic environments and a direct source of human infection. Although VP3, a lytic phage, shows promise in eliminating planktonic V. cholerae from the aquatic environment, its effectiveness against biofilm-associated V. cholerae is limited. To address this limitation, our proposed approach aims to enhance the efficacy of VP3 in eliminating biofilm-associated V. cholerae by augmenting the availability of phage receptors on the surface of Vibrio cholerae. TolC is a receptor of VP3 and a salt efflux pump present in many bacteria. In this study, we employed NaCl as an enhancer to stimulate TolC expression and observed a significant enhancement of TolC expression in both planktonic and biofilm cells of V. cholerae. This enhancement led to improved adsorption of VP3. Importantly, our findings provide strong evidence that high salt concentrations combined with VP3 significantly improve the elimination of biofilm-associated V. cholerae. This approach offers a potential strategy to eliminate biofilm-formation bacteria by enhancing phage-host interaction.

Increased intracellular persulfide levels attenuate HlyU-mediated hemolysin transcriptional activation in Vibrio cholerae.

The vertebrate host's immune system and resident commensal bacteria deploy a range of highly reactive small molecules that provide a barrier against infections by microbial pathogens. Gut pathogens, such as Vibrio cholerae, sense and respond to these stressors by modulating the expression of exotoxins that are crucial for colonization. Here, we employ mass spectrometry-based profiling, metabolomics, expression assays, and biophysical approaches to show that transcriptional activation of the hemolysin gene hlyA in V. cholerae is regulated by intracellular forms of sulfur with sulfur-sulfur bonds, termed reactive sulfur species (RSS). We first present a comprehensive sequence similarity network analysis of the arsenic repressor superfamily of transcriptional regulators, where RSS and hydrogen peroxide sensors segregate into distinct clusters of sequences. We show that HlyU, transcriptional activator of hlyA in V. cholerae, belongs to the RSS-sensing cluster and readily reacts with organic persulfides, showing no reactivity or DNA dissociation following treatment with glutathione disulfide or hydrogen peroxide. Surprisingly, in V. cholerae cell cultures, both sulfide and peroxide treatment downregulate HlyU-dependent transcriptional activation of hlyA. However, RSS metabolite profiling shows that both sulfide and peroxide treatment raise the endogenous inorganic sulfide and disulfide levels to a similar extent, accounting for this crosstalk, and confirming that V. cholerae attenuates HlyU-mediated activation of hlyA in a specific response to intracellular RSS. These findings provide new evidence that gut pathogens may harness RSS-sensing as an evolutionary adaptation that allows them to overcome the gut inflammatory response by modulating the expression of exotoxins.

Globally Vibrio cholera antibiotics resistance to RNA and DNA effective antibiotics: A systematic review and meta-analysis.

BACKGROUND: Vibrio cholera (V. cholera) is a facultative pathogen that colonizes the small intestine and produces cholerae toxin as the primary virulence factor that causes cholera and fatal diarrhea in humans. In recent decades, V. cholera has emerged as a notorious multidrug-resistant enteric pathogen. This meta-analysis estimated the pooled proportion of V. cholera antimicrobial resistance against RNA and DNA effective antibiotics. METHOD: A systematic search was performed for relevant literature until 05 June 2021 in PubMed, Scopus, Embase, and Web of Science databases. Freeman-Tukey double arcsine transformation was performed to estimate weighted pooled resistance (WPR). RESULTS: The meta-analysis were included 164 articles. The WPR of V. cholera were as follows 76% [67,84] to furazolidone, 65% [29,94] to nitrofurantoin, 55% [44,66] to nalidixic acid, 10% [2,23] to rifampicin, 4%(0, 12) to novobiocin, 4% [2,6] to norfloxacin, 3% [1,4] to ciprofloxacin, 1%(0, 3) to sparofloxacin, 0%(0, 3) to levofloxacin, 0%(0, 2) to ofloxacin, 0%(0, 0) to gatifloxacin. CONCLUSION: V. cholera is a severe problem in Asia and Africa, especially in South Asian countries. The resistance patterns are various in geographical regions. novobiocin 0% (0, 0), and ofloxacin 0% (0, 1) in Africa, gatifloxacin 0% (0, 0), and levofloxacin 0% (0, 6) in Asia and ciprofloxacin 0% (0, 2) in North America are most effective antibiotis. The resistance rate to furazolidone, nalidixic acid, nitrofurantoin, and cephalothin has increased over the years. Monitoring antibiotic resistance and prescribing an appropriate antibiotic is vital to control resistance.

A commensal-encoded genotoxin drives restriction of Vibrio cholerae colonization and host gut microbiome remodeling.

SignificanceIn a polymicrobial battlefield where different species compete for nutrients and colonization niches, antimicrobial compounds are the sword and shield of commensal microbes in competition with invading pathogens and each other. The identification of an Escherichia coli-produced genotoxin, colibactin, and its specific targeted killing of enteric pathogens and commensals, including Vibrio cholerae and Bacteroides fragilis, sheds light on our understanding of intermicrobial interactions in the mammalian gut. Our findings elucidate the mechanisms through which genotoxins shape microbial communities and provide a platform for probing the larger role of enteric multibacterial interactions regarding infection and disease outcomes.

Nitric oxide stimulates type IV MSHA pilus retraction in Vibrio cholerae via activation of the phosphodiesterase CdpA.

Bacteria use surface appendages called type IV pili to perform diverse activities including DNA uptake, twitching motility, and attachment to surfaces. The dynamic extension and retraction of pili are often required for these activities, but the stimuli that regulate these dynamics remain poorly characterized. To address this question, we study the bacterial pathogen Vibrio cholerae, which uses mannose-sensitive hemagglutinin (MSHA) pili to attach to surfaces in aquatic environments as the first step in biofilm formation. Here, we use a combination of genetic and cell biological approaches to describe a regulatory pathway that allows V. cholerae to rapidly abort biofilm formation. Specifically, we show that V. cholerae cells retract MSHA pili and detach from a surface in a diffusion-limited, enclosed environment. This response is dependent on the phosphodiesterase CdpA, which decreases intracellular levels of cyclic-di-GMP to induce MSHA pilus retraction. CdpA contains a putative nitric oxide (NO)-sensing NosP domain, and we demonstrate that NO is necessary and sufficient to stimulate CdpA-dependent detachment. Thus, we hypothesize that the endogenous production of NO (or an NO-like molecule) in V. cholerae stimulates the retraction of MSHA pili. These results extend our understanding of how environmental cues can be integrated into the complex regulatory pathways that control pilus dynamic activity and attachment in bacterial species.

Vibrio cholerae was found in cultured bullfrog.

Bullfrog is one of the most important economic aquatic animals in China that is widely cultured in southern China and is a key breed recommended as an industry of poverty alleviation in China. During recent years, a fatal bacterial disease has often been found in cultured bullfrogs. The clinical manifestations of the diseased bullfrogs were severe intestinal inflammation and an anal prolapse. A bacterial pathogen was isolated from the diseased bullfrog intestines. The bacterium was identified as Vibrio cholerae using morphological, biochemical and 16S rRNA phylogenetic analysis. In this study, V. cholerae was isolated and identified in diseased bullfrogs for the first time, providing a basis for the diagnosis and control of the disease. Therefore, attention should be paid to the modes of transmission of V. cholerae from bullfrog and formulate reasonable safety measures.

Coumarins effectively inhibit bacterial α-carbonic anhydrases.

Coumarins are known to act as prodrug inhibitors of mammalian α-carbonic anhydrases (CAs, EC 4.2.1.1) but they were not yet investigated for the inhibition of bacterial α-CAs. Here we demonstrate that such enzymes from the bacterial pathogens Neisseria gonorrhoeae (NgCAα) and Vibrio cholerae (VchCAα) are inhibited by a panel of simple coumarins incorporating hydroxyl, amino, ketone or carboxylic acid ester moieties in various positions of the ring system. The nature and the position of the substituents in the coumarin ring were the factors which strongly influenced inhibitory efficacy. NgCAα was inhibited with KIs in the range of 28.6-469.5 µM, whereas VchCAα with KIs in the range of 39.8-438.7 µM. The two human (h)CA isoforms included for comparison reason in the study, hCA I and II, were less prone to inhibition by these compounds, with KIs of 137-948.9 µM for hCA I and of 296.5-961.2 µM for hCA II, respectively. These findings are relevant for discovering coumarin bacterial CA inhibitors with selectivity for the bacterial over human isoform, with potential applications as novel antibacterial agents.

Identification of Small Molecule Inhibitors of the Pathogen Box against Vibrio cholerae.

Antimicrobial resistance (AMR) has become a serious public and economic threat. The rate of bacteria acquiring AMR surpasses the rate of new antibiotics discovery, projecting more deadly AMR infections in the future. The Pathogen Box is an open-source library of drug-like compounds that can be screened for antibiotic activity. We have screened molecules of the Pathogen Box against Vibrio cholerae, the cholera-causing pathogen, and successfully identified two compounds, MMV687807 and MMV675968, that inhibit growth. RNA-seq analyses of V. cholerae after incubation with each compound revealed that both compounds affect cellular functions on multiple levels including carbon metabolism, iron homeostasis, and biofilm formation. In addition, whole-genome sequencing analysis of spontaneous resistance mutants identified an efflux system that confers resistance to MMV687807. We also identified that the dihydrofolate reductase is the likely target of MMV675968 suggesting it acts as an analog of trimethoprim but with a MIC 14-fold lower than trimethoprim in molar concentration. In summary, these two compounds that effectively inhibit V. cholerae and other bacteria may lead to the development of new antibiotics for better treatment of the cholera disease. IMPORTANCE Cholera is a serious infectious disease in tropical regions causing millions of infections annually. Vibrio cholerae, the causative agent of cholera, has gained multi-antibiotic resistance over the years, posing greater threat to public health and current treatment strategies. Here we report two compounds that effectively target the growth of V. cholerae and have the potential to control cholera infection.

Interplay between Sublethal Aminoglycosides and Quorum Sensing: Consequences on Survival in V. cholerae.

Antibiotics are well known drugs which, when present above certain concentrations, are able to inhibit the growth of certain bacteria. However, a growing body of evidence shows that even when present at lower doses (subMIC, for sub-minimal inhibitory concentration), unable to inhibit or affect microbial growth, antibiotics work as signaling molecules, affect gene expression and trigger important bacterial stress responses. However, how subMIC antibiotic signaling interplays with other well-known signaling networks in bacteria (and the consequences of such interplay) is not well understood. In this work, through transcriptomic and genetic approaches, we have explored how quorum-sensing (QS) proficiency of V. cholerae affects this pathogen's response to subMIC doses of the aminoglycoside tobramycin (TOB). We show that the transcriptomic signature of V. cholerae in response to subMIC TOB depends highly on the presence of QS master regulator HapR. In parallel, we show that subMIC doses of TOB are able to negatively interfere with the AI-2/LuxS QS network of V. cholerae, which seems critical for survival to aminoglycoside treatment and TOB-mediated induction of SOS response in this species. This interplay between QS and aminoglycosides suggests that targeting QS signaling may be a strategy to enhance aminoglycoside efficacy in V. cholerae.

Identification of Potent Natural Resource Small Molecule Inhibitor to Control Vibrio cholera by Targeting Its Outer Membrane Protein U: An In Silico Approach.

Vibrio cholerae causes the diarrheal disease cholera which affects millions of people globally. The outer membrane protein U (OmpU) is the outer membrane protein that is most prevalent in V. cholerae and has already been recognized as a critical component of pathogenicity involved in host cell contact and as being necessary for the survival of pathogenic V. cholerae in the host body. Computational approaches were used in this study to screen a total of 37,709 natural compounds from the traditional Chinese medicine (TCM) database against the active site of OmpU. Following a sequential screening of the TCM database, we report three lead compounds-ZINC06494587, ZINC85510056, and ZINC95910434-that bind strongly to OmpU, with binding affinity values of -8.92, -8.12, and -8.78 kcal/mol, which were higher than the control ligand (-7.0 kcal/mol). To optimize the interaction, several 100 ns molecular dynamics simulations were performed, and the resulting complexes were shown to be stable in their vicinity. Additionally, these compounds were predicted to have good drug-like properties based on physicochemical properties and ADMET assessments. This study suggests that further research be conducted on these compounds to determine their potential use as cholera disease treatment.

A periplasmic cupredoxin with a green CuT1.5 center is involved in bacterial copper tolerance.

The importance of copper resistance pathways in pathogenic bacteria is now well recognized, since macrophages use copper to fight bacterial infections. Additionally, considering the increase of antibiotic resistance, growing attention is given to the antimicrobial properties of copper. It is of primary importance to understand how bacteria deal with copper. The Cu-resistant cuproprotein CopI is present in many human bacterial pathogens and environmental bacteria and crucial under microaerobiosis (conditions for most pathogens to thrive within their host). Hence, understanding its mechanism of function is essential. CopI proteins share conserved histidine, cysteine, and methionine residues that could be ligands for different copper binding sites, among which the cupredoxin center could be involved in the protein function. Here, we demonstrated that Vibrio cholerae and Pseudomonas aeruginosa CopI restore the Cu-resistant phenotype in the Rubrivivax gelatinosus ΔcopI mutant. We identified that Cys125 (ligand in the cupredoxin center) and conserved histidines and methionines are essential for R. gelatinosus CopI (RgCopI) function. We also performed spectroscopic analyses of the purified RgCopI protein and showed that it is a green cupredoxin able to bind a maximum of three Cu(II) ions: (i) a green Cu site (CuT1.5), (ii) a type 2 Cu binding site (T2) located in the N-terminal region, and (iii) a third site with a yet unidentified location. CopI is therefore one member of the poorly described CuT1.5 center cupredoxin family. It is unique, since it is a single-domain cupredoxin with more than one Cu site involved in Cu resistance.

Dietary Isothiocyanates, Sulforaphane and 2-Phenethyl Isothiocyanate, Effectively Impair Vibrio cholerae Virulence.

Vibrio cholerae represents a constant threat to public health, causing widespread infections, especially in developing countries with a significant number of fatalities and serious complications every year. The standard treatment by oral rehydration does not eliminate the source of infection, while increasing antibiotic resistance among pathogenic V. cholerae strains makes the therapy difficult. Thus, we assessed the antibacterial potential of plant-derived phytoncides, isothiocyanates (ITC), against V. cholerae O365 strain. Sulforaphane (SFN) and 2-phenethyl isothiocyanate (PEITC) ability to inhibit bacterial growth was assessed. Minimum inhibitory concentration (MIC) and minimum bactericidal concentration (MBC) values indicate that these compounds possess antibacterial activity and are also effective against cells growing in a biofilm. Tested ITC caused accumulation of stringent response alarmone, ppGpp, which indicates induction of the global stress response. It was accompanied by bacterial cytoplasm shrinkage, the inhibition of the DNA, and RNA synthesis as well as downregulation of the expression of virulence factors. Most importantly, ITC reduced the toxicity of V. cholerae in the in vitro assays (against Vero and HeLa cells) and in vivo, using Galleria mellonella larvae as an infection model. In conclusion, our data indicate that ITCs might be considered promising antibacterial agents in V. cholerae infections.