Vibrio Fluvialis Research Articles

The TorRS two component system regulates expression of TMAO reductase in response to high hydrostatic pressure in Vibrio fluvialis.

High hydrostatic pressure (HHP) regulated gene expression is one of the most commonly adopted strategies for microbial adaptation to the deep-sea environments. Previously we showed that the HHP-inducible trimethylamine N-oxide (TMAO) reductase improves the pressure tolerance of deep-sea strain Vibrio fluvialis QY27. Here, we investigated the molecular mechanism of HHP-responsive regulation of TMAO reductase TorA. By constructing torR and torS deletion mutants, we demonstrated that the two-component regulator TorR and sensor TorS are responsible for the HHP-responsive regulation of torA. Unlike known HHP-responsive regulatory system, the abundance of torR and torS was not affected by HHP. Complementation of the ΔtorS mutant with TorS altered at conserved phosphorylation sites revealed that the three sites were indispensable for substrate-induced regulation, but only the histidine located in the alternative transmitter domain was involved in pressure-responsive regulation. Taken together, we demonstrated that the induction of TMAO reductase by HHP is mediated through the TorRS system and proposed a bifurcation of signal transduction in pressure-responsive regulation from the substrate-induction. This work provides novel knowledge of the pressure regulated gene expression and will promote the understanding of the microbial adaptation to the deep-sea HHP environment.

Biotransamination of Furan-Based Aldehydes with Isopropylamine: Enzyme Screening and pH Influence.

Furan-based amines are highly valuable compounds which can be directly obtained via reductive amination from easily accessible furfural, 5-(hydroxymethyl)furfural (HMF) and 2,5-diformylfuran (DFF). Herein the biocatalytic amination of these carbonyl derivatives is disclosed using amine transaminases (ATAs) and isopropylamine (IPA) as amine donors. Among the different biocatalysts tested, the ones from Chromobacterium violaceum (Cv-TA), Arthrobacter citreus (ArS-TA), and variants from Arthrobacter sp. (ArRmut11-TA) and Vibrio fluvialis (Vf-mut-TA), afforded high levels of product formation (>80 %) at 100-200 mM aldehyde concentration. The transformations were studied in terms of enzyme and IPA loading. The pH influence was found as a key factor and attributed to the imine/aldehyde equilibrium that can arise from the high reactivity of the carbonyl substrates with a nucleophilic amine such as IPA.

Co-Immobilization of a Multi-Enzyme Cascade: (S)-Selective Amine Transaminases, l-Amino Acid Oxidase and Catalase.

An enzyme cascade was established previously consisting of a recycling system with an l-amino acid oxidase (hcLAAO4) and a catalase (hCAT) for different α-keto acid co-substrates of (S)-selective amine transaminases (ATAs) in kinetic resolutions of racemic amines. Only 1 mol % of the co-substrate was required and l-amino acids instead of α-keto acids could be applied. However, soluble enzymes cannot be reused easily. Immobilization of hcLAAO4, hCAT and the (S)-selective ATA from Vibrio fluvialis (ATA-Vfl) was addressed here. Immobilization of the enzymes together rather than on separate beads showed higher reaction rates most likely due to fast co-substrate channeling between ATA-Vfl and hcLAAO4 due to their close proximity. Co-immobilization allowed further reduction of the co-substrate amount to 0.1 mol % most likely due to a more efficient H2 O2 -removal caused by the stabilized hCAT and its proximity to hcLAAO4. Finally, the co-immobilized enzyme cascade was reused in 3 cycles of preparative kinetic resolutions to produce (R)-1-PEA with high enantiomeric purity (97.3 %ee). Further recycling was inefficient due to the instability of ATA-Vfl, while hcLAAO4 and hCAT revealed high stability. An engineered ATA-Vfl-8M was used in the co-immobilized enzyme cascade to produce (R)-1-(3-ethoxy-4-methoxyphenyl)-2-(methylsulfonyl)ethanamine, an apremilast-intermediate, with a 1,000 fold lower input of the co-substrate.

Vibrio fluvialis: An Emerging Pathogen for Intestinal and Extraintestinal Infections

The genus Vibrio is a ubiquitous group of bacteria belonging in the family Vibrionaceae. More than 100 species have been discovered in the genus Vibrio and about 14 of them have been reported to cause several human infections. Vibrio fluvialis, a member of this group is widely distributed in the aquatic environment, mostly in the seas, brackish waters and coastal zones.1 V. fluvialis is a pathogenic bacterium more commonly found in coastal areas. It causes diarrhea and enterocolitis and a significant number of extra intestinal infections. Due to increasing number of infections, it is considered as an emerging pathogen.1 Though this pathogen can be easily isolated by conventional staining and culture methods along with the biochemical properties, its identification is considered as a challenging problem due to its close phenotypic similarity of this species either with other vibrios and Aeromonas spp. By using automated BD Phoenix M50 techniques, it has become easier to identify V. fluvialis from clinical and different environmental samples. In this system biochemical properties of different 45 substrates are used to observe their fermentation reactions. Automated identification and antimicrobial susceptibility test systems are essential for modern clinical microbiology laboratories for rapid identification and proper selection of effective antibiotics. Several virulence factors of V. fluvialis have been identified.2 The rule and mechanisms of action of these virulent factors causing pathogenesis and infection are yet to be clarified. In this review article, the epidemiology, identification, pathogenicity, intra- and extra-intestinal clinical features and management of V. fluvialis infections have been focused. KYAMC Journal Vol. 14, No. 01, April 2023: 48-53

Distribution, Prevalence, and Antibiotic Susceptibility Profiles of Infectious Noncholera Vibrio Species in Malaysia.

The noncholera Vibrio spp. which cause vibriosis are abundantly found in our water ecosystem. These bacteria could negatively affect both humans and animals. To date, there is a paucity of information available on the existence and pathogenicity of this particular noncholera Vibrio spp. in Malaysia in comparison to their counterpart, Vibrio cholera. In this study, we extracted retrospective data from Malaysian surveillance database. Analysis was carried out using WHONET software focusing noncholera Vibrio spp. including Vibrio parahaemolyticus, Vibrio vulnificus, Vibrio fluvialis, Vibrio alginolyticus, Vibrio hollisae (Grimontia hollisae), Vibrio mimicus, Vibrio metschnikovii, and Vibrio furnissii. Here, we report the first distribution and prevalence of these species isolated in Malaysia together with the antibiotic sensitivity profile based on the species. We found that V. parahaemolyticus is the predominant species isolated in Malaysia. Noticeably, across the study period, V. fluvialis is becoming more prevalent, as compared to V. parahaemolyticus. In addition, this study also reports the first isolation of pathogenic V. furnissii from stool in Malaysia. These data represent an important step toward understanding the potential emergence of noncholera Vibrio spp. outbreaks.

Comparative genomics revealed that Vibrio furnissii and Vibrio fluvialis have mutations in genes related to T6SS1 and T6SS2

The type VI secretion system (T6SS) is important for interbacterial competition and virulence in Vibrio species. It is generally agreed that T6SS provides a fitness advantage to Vibrios. Some Vibrio species possess one, while others possess two T6SSs. Even within the same Vibrio species, different strains can harbor a variable number of T6SSs. Such is the case in V. fluvialis, an opportunistic human pathogen, that some V. fluvialis strains do not harbor T6SS1. This study found that Amphritea, Marinomonas, Marinobacterium, Vibrio, Photobacterium, and Oceanospirillum species have genes encoding V. fluvialis T6SS1 homologs. The cladogram of T6SS1 genes suggested that these genes appeared to be horizontally acquired by V. fluvialis, V. furnissii, and some other Vibrio species, when compared with the species tree. Codon insertions, codon deletions, nonsense mutations, and the insertion sequence are found in many genes, such as clpV1, tssL1, and tssF1, which encode structure components of T6SS1 in V. furnissii and V. fluvialis. Codon deletion events are more common than codon insertion, insertion sequence disruption, and nonsense mutation events in genes that encode components of T6SS1. Similarly, codon insertions and codon deletions are found in genes relevant to T6SS2, including tssM2, vgrG2 and vasH, in V. furnissii and V. fluvialis. These mutations are likely to disable the functions of T6SSs. Our findings indicate that T6SS may have a fitness disadvantage in V. furnissii and V. fluvialis, and the loss of function in T6SS may help these Vibrio species to survive under certain conditions.

Screening of host gut-derived probiotics and effects of feeding probiotics on growth, immunity, and antioxidant enzyme activity of hybrid grouper (Epinephelus fuscoguttatus♀ × E. lanceolatus♂)

In recent years, the widespread use of antibiotics in intensive grouper mariculture has resulted in the ineffectiveness of antibiotic treatment, leading to an increasing incidence of diseases caused by bacteria, viruses, and parasites, causing serious economic losses. Hence, it is crucial to develop alternative strategies to antibiotics for healthy and sustainable development of the mariculture industry. Here, we aimed to screen host gut-derived probiotics and evaluate its effects on growth and immunity of grouper. In this study, 43 bacterial strains were isolated from the intestine of the hybrid grouper (Epinephelus fuscoguttatus♀ × E. lanceolatus♂), and a potential probiotic strain G1-26, which can efficiently secrete amylase, protease, and lipase, was obtained using different screening mediums. Based on 16S rDNA sequencing, the potential probiotic strain G1-26 was identified as Vibrio fluvialis. The results of a biological characteristic evaluation showed that V. fluvialis G1-26 could grow at 25–45 °C, pH 5.5–7.5, salinity 10–40, and bile salt concentration 0–0.030%, and produce amylase, lipase, and protease under different culture conditions. Additionally, V. fluvialis G1-26 is sensitive to many antibiotics and does not exhibit aquatic biotoxicity. Subsequently, hybrid groupers were fed diets containing V. fluvialis G1-26 at different concentrations (0, 106, 108, and 1010 CFU/g) for 60 d. The results showed that V. fluvialis G1-26 at 108 CFU/g did not significantly affect the growth performance of the hybrid grouper (P > 0.05). V. fluvialis G1-26 supplementation at 108 and 1010 CFU/g significantly promoted the relative expression of immune-related genes in hybrid groupers (TLR3, TLR5, IL-1β, IL-8, IL-10, CTL, LysC, TNF-2, and MHC-2) and improved the activities of alkaline phosphatase, acid phosphatase, total superoxide dismutase, and total protein in the liver. In conclusion, V. fluvialis G1-26, a potential probiotic strain isolated from the intestine of the hybrid grouper, can be used as an effective immunopotentiator at an optimal dose of 108 CFU/g diet. Our results provide a scientific basis for the development and utilization of probiotics in the grouper mariculture industry.

Bacteria of Zoonotic Interest Identified on Edible Freshwater Fish Imported to Australia

Previous research has shown that freshwater edible fish imported into Australia are not compliant with Australian importation guidelines and as a result may be high risk for bacterial contamination. In the present study, the outer surface of imported freshwater fish were swabbed, cultured, confirmatory tests performed and antimicrobial patterns investigated. Channidae fish (Sp. A/n = 66) were contaminated with zoonotic Salmonella sp./Staphylococcus aureus (n = 1/66) and other bacteria implicated in cases of opportunistic human infection, these being Pseudomonas sp. (including P. mendocina and P. pseudoalcaligenes (n = 34/66)); Micrococcus sp. (n = 32/66); Comamonas testosteroni (n = 27/66) and Rhizobium radiobacter (n = 3/66). Pangasiidae fish (Species B/n = 47) were contaminated with zoonotic Vibrio fluvialis (n = 10/47); Salmonella sp. (n = 6/47) and environmental bacteria Micrococcus sp. (n = 3/47). One sample was resistant to all antimicrobials tested and is considered to be Methicillin Resistant S. aureus. Mud, natural diet, or vegetation identified in Sp. A fish/or packaging were significantly associated with the presence of Pseudomonas spp. The study also showed that visibly clean fish (Sp. B) may harbour zoonotic bacteria and that certain types of bacteria are common to fish groups, preparations, and contaminants. Further investigations are required to support the development of appropriate food safety recommendations in Australia.

Effects of different composting methods on antibiotic-resistant bacteria, antibiotic resistance genes, and microbial diversity in dairy cattle manures.

Composting is a common practice used for treating animal manures before they are used as organic fertilizers for crop production. Whether composting can effectively reduce microbial pathogens and antibiotic resistance genes remain poorly understood. In this study, we compared 3 different dairy manure composting methods-anaerobic fermentation (AF), static compost (SC), and organic fertilizer production (OFP)-for their effects on antibiotic-resistant bacteria, antibiotic resistance genes, and microbial community diversity in the treated manures. The 3 composting methods produced variable and distinct effects on antibiotic-resistant bacteria, zoonotic bacteria, and resistance genes, some of which were decreased and others of which showed no significant changes during composting. Particularly, SC and OFP reduced chloramphenicol resistance gene fexA and opportunistic pathogen Vibrio fluvialis, whereas AF significantly reduced tetracycline resistance gene tetB and opportunistic pathogens Enterococcus faecium and Escherichia fergusonii. The compositions of microbial communities varied significantly during the composting processes, and there were significant differences between the 3 composting methods. In all 3 composts, the dominant phyla were Firmicutes, Proteobacteria, Bacteroidetes, and Actinobacteria. Interestingly, Firmicutes, Proteobacteria, and Bacteroidetes remained stable in the entire AF process, whereas they were dominated at the beginning, decreased at the early stage of composting, and rebounded at the later stage during SC and OFP. In general, SC and OFP produced a more profound effect than AF on microbial community diversities, pathogens, and dominant species. Additionally, Enterococcus aquimarinus was isolated from AF for the first time. Phylogenetic Investigation of Communities by Reconstruction of Unobserved States function prediction analysis indicated that the genes related to membrane transport and amino acid metabolism were abundant in the 3 composts. The metabolism of amino acids, lipids, and carbohydrates increased as composting progressed. The biosynthesis of antibiotics was enhanced after fermentation in the 3 composting methods, and the increase in the SC was the most obvious. These results reveal dynamic changes in antibiotic-resistant bacteria, antibiotic resistance genes, microbial community composition, and function succession in different dairy manure composts and provide useful information for further optimization of composting practices.

Detection of pathogenic Vibrio species and antibiogram activity in Asian Seabass (Lates calcarifer) in Tumpat, Kelantan.

Some of Vibrio species is well known as pathogenic bacteria in aquaculture and the marine industry. Its infection is able to generate a massive outbreak and affect the fish population, especially for net caged fish such as seabass. This study was conducted to investigate the prevalence of Vibrio spp. isolated from seabass (Lates calcarifer) in Sri Tujuh Lagoon, Tumpat, Kelantan. Then, to determine the antibiotic resistance in Vibrio isolates. Polymerase chain reaction (PCR) was used to detect Vibrio species using specific primer VR169 and VR744 with estimation base pair size band, 597 bp and further identified by sequencing. On the other hand, antibiotic susceptibility tests were continued by using 13 types of antibiotics; kanamycin (K30), chloramphenicol (C30), neomycin (N10), ampicillin (AMP10), nitrofurantoin (F300), tetracycline (TE30), streptomycin (S10), norfloxacin (NOR10), ciprofloxacin (CIP5), nalidixic acid (NA30), gentamicin (CN10), doxycycline (DO30) and sulfamethoxazole (SXT100). As a result, 14 Vibrio isolates were identified, including Vibrio fluvialis (n=6), Vibrio parahaemolyticus (n=3), Vibrio harveyi (n=2) and each isolate for Vibrio vulnificus, Vibrio alginolyticus and Vibrio spp. The results showed that all isolates were sensitive to most antibiotics except ampicillin, neomycin and streptomycin. The MAR index value was ranging from 0 to 0.31. This study demonstrates the prevalence of Vibrio spp. in seabass and the report on multidrug resistance strains that could be of concern to the fish farmers. In addition, data from this study can be further used in fish disease management plans.

BACTERIAL FLORA OF CATFISH (Clarias gariepinus) FROM FISH PONDS IN LAGOS STATE, NIGERIA

Fish is a very important component of human diet. A total of 64 samples of Clarias gariepinus were collected from two fish ponds each in Yaba, Ijegun, Ikorodu and Epe in Lagos State, Nigeria. The bacterial flora of the fishes were determined quantitatively and qualitatively. The mean total viable count ranged from log10 7.6 cfu/g to log10 7.9 cfu/g. The bacterial load on the skin and in the intestine were generally higher than in the gills. Salmonella count ranged between log10 7.2 cfu/g and log10 7.6 cfu/g and it was highest in the intestine, followed by skin and gills. Coliform count was higher in the intestine and on the skin than the gills with range values of log10 7.6 cfu/g) to log10 7.9 cfu/g. Staphylococcal count was highest on the skin than the intestine and gills. The range of count was log10 7.2 cfu/g in the gills to log10 7.6 cfu/g on the skin. The bacteria isolated were identified based on cultural, morphological and biochemical characterization using Analytical Profile Index and they include Pseudomonas aeruginosa, Klebsiella pneumoniae, Salmonella typhi, Citrobacter amalonaticus, Vibrio fluvialis, Serratia odorifera, Acinetobacter baumannii, Proteus penneri, Burkholderia cepacia, Enterobacter pyrinus, Salmonella enteritidis, Edwardsiella tarda, Escherichia coli, Pseudomonas putida, Bacillus megaterium, Staphylococcus aureus, Bacillus mycoides, Corynebacterium pilosum, Bacillus subtilis, Micrococcus roseus, Bacillus cereus, Bacillus polymyxa, Micrococcus leteus and Corynebacterium kutscheri. The occurrence of pathogenic bacteria may constitute public health hazard

Metagenomic and genomic characterization of heavy metal tolerance and resistance genes in the rhizosphere microbiome of Avicennia germinans in a semi-arid mangrove forest in the tropics.

Mangroves are often exposed to heavy metals that accumulate in the food chain, generate toxicity to mangrove plants and affect microbial diversity. This study determined the abundance of genes associated with resistance and tolerance to heavy metals in the rhizosphere microbiome of Avicennia germinans from a semi-arid mangrove of La Guajira-Colombia by metagenomics and genomics approach. Twenty-eight genes associated with tolerance and 49 genes related to resistance to heavy metals were detected. Genes associated with tolerance and resistance to Cu, especially cusA and copA, were the most abundant. The highest number of genes for tolerance and resistance were for Zn and Co, respectively. The isolate Vibrio fluvialis showed the ability to tolerate Cu, Ni, Zn, and Cd. This work used a complementary approach of metagenomics and genomics to characterize the potential of mangrove microorganisms to tolerate and resist heavy metals and the influence of salinity on their abundance.

TssI2-TsiI2 of Vibrio fluvialis VflT6SS2 delivers pesticin domain-containing periplasmic toxin and cognate immunity that modulates bacterial competitiveness

ABSTRACT Vibrio fluvialis is a halophilic Gram-negative bacterium regarded as an emerging unusual enteric pathogen of increasing public health concern. Our previous work has identified two type VI secretion systems (T6SSs) in V. fluvialis, VflT6SS1, and VflT6SS2, and the latter is functional in mediating interbacterial competitiveness. However, its antibacterial effectors remain to be clarified. In this work, we focused on a new potential effector/immunity pair TssI2/TsiI2. Bioinformatics analysis revealed that the C-terminal domain of TssI2 belongs to a widespread family of pesticin, and its antibacterial toxicity and corresponding protection by TsiI2 were proved via bacterial killing assays, and their action sites were localized to the periplasm of bacterial cells. The interaction of TssI2 and TsiI2 was demonstrated by the bacterial adenylate cyclase two-hybrid, protein pull-down and isothermal titration calorimetry assays. Site-directed mutagenesis demonstrated that, in addition to Glu-844, Thr-863, and Asp-869, which correspond to three reported residues in pesticin of Yersinia pestis, additional residues including Phe-837, Gly-845, Tyr-851, Gly-867, Gln-963, Trp-975, and Arg-1000 were also proved to be crucial to the bactericidal activity of TssI2. Muramidase/lysozyme-related peptidoglycan (PG) hydrolase activities of TssI2 and its variants were validated with permeabilized Escherichia coli cells and purified PG substrate. Based on sequence homologies at C-terminals in various V. fluvialis isolates, TssI2 was subdivided into five clusters (12–22% identity among them), and the antibacterial activities of representative effectors from other four Clusters were also confirmed through periplasmic over-expression in E. coli host. Two selected cognate immunities were proved to confer protection against the toxicities of their effectors. Additionally, TsiI2, which belongs to Cluster I, exhibited cross-protection to effector from Cluster V. Together, current findings expand our knowledge of the diversity and consistency of evolved VgrG effectors in V. fluvialis and on how VflT6SS2 mediates a competitive advantage to gain a better survival.

The first report of Vibrio fluvialis isolated from a clinical sample in Iran.

Vibrio fluvialis is a Gram-negative, bacillus-shaped, curved bacterium known as an emerging pathogen. There are reports of outbreaks caused by this bacterium worldwide. Iran, especially Qom province, is an endemic region for gastrointestinal diseases caused by Vibrio species. So, the aim was to isolate V. fluvialis from clinical and environmental samples. During six months, 363 clinical and surface water samples were evaluated. The samples were cultured on specific media, and all incubated for 24 hours at 37°C. Suspicious colonies were evaluated by Gram staining and biochemical tests. The BD Phoenix automated microbiology system was used for the final confirmation of the isolated bacteria. Evaluation of antibiotic resistance of isolated strains was also performed according to CLSI standard. Eight cases (2.2%) of V. fluvialis, including seven from surface water samples (87.5%) and one from clinical samples (12.5%), were isolated. Based on antimicrobial susceptibility testing, all V. fluvialis isolates were susceptible to amikacin, gentamicin, trimethoprim/sulfamethoxazole, ciprofloxacin, tetracycline, ceftazidime, and chloramphenicol. High-level resistance to ampicillin and amoxicillin/clavulanate was also observed. V. fluvialis-infected patient had a mild fever, watery diarrhea, vomiting, nausea, and abdominal cramps that were manifested after drinking contaminated water or eating contaminated vegetables. The patient's symptoms recovered without antibiotic therapy after four days, resulting in self-limiting disease. The current study is the first human case of V. fluvialis infection isolated in Iran. Therefore, monitoring of water and food samples should be done routinely.

Culture-dependent examination of the bacteriological quality of ready-to-eat African salads in Enugu metropolis, Nigeria and antibiotic resistance profile of associated bacteria

This study investigated the bacteriological quality of ready-to-eat (RTE) African salads in Enugu metropolis, Enugu, Nigeria. A total of 10 samples of African salad were purchased from 10 different vendors in Enugu metropolis. The samples were purchased from Agbani Road, Ogbete, Mayor, Uwani, Kenyatta, Achara Layout, Obiagu and Timber. Isolation and enumeration of bacterial isolates were done using Nutrient agar, Eosin Methylene Blue (EMB) agar, Thiosulphate-citrate-bile salts-sucrose (TCBS) agar, Salmonella-Shigella Agar (SSA) and MacConkey agar, following standard methods. Identification of the bacterial isolates were done through biochemical tests and the Analytical Profile Index (API 20E) test kit. The antibiotic sensitivity of the bacterial isolates was also done using the Kirby Bauer disc diffusion method. Total culturable heterotrophic count was above 300 colonies across the samples. The highest bacterial counts recorded on EMB, SSA and TCBS across the samples were 6.3 × 106 CFU/g, 7.4 × 106 CFU/g and 1.21 × 107 CFU/g respectively. The identities of the organisms were; Salmonella spp., Staphylococcus aureus, Escherichia coli, Vibrio mimicus, Vibrio fluvialis, Vibrio cholerae, Vibrio parahaemolyticus and Vibrio hollisae. The prevalent organism across the samples was Vibrio spp. The antibiotic sensitivity test suggested that Vibrio spp. was resistant to Ampiclox and Amoxycillin but sensitive to Erythromycin, Pefloxacin and Septrin. From this study, it was discovered that consumers of RTE African salad from majority of the vendors across Enugu metropolis are at risk of severe food poisoning.

In Vitro and In Silico Screening of Anti-Vibrio spp., Antibiofilm, Antioxidant and Anti-Quorum Sensing Activities of Cuminum cyminum L. Volatile Oil.

Cuminum cyminum L. essential oil (cumin EO) was studied for its chemical composition, antioxidant and vibriocidal activities. Inhibition of biofilm formation and secretion of some virulence properties controlled by the quorum sensing system in Chromobacterium violaceum and Pseudomonas aeruginosa strains were also reported. The obtained results showed that cuminaldehyde (44.2%) was the dominant compound followed by β-pinene (15.1%), γ-terpinene (14.4%), and p-cymene (14.2%). Using the disc diffusion assay, cumin EO (10 mg/disc) was particularly active against all fifteen Vibrio species, and the highest diameter of growth inhibition zone was recorded against Vibrio fluvialis (41.33 ± 1.15 mm), Vibrio parahaemolyticus (39.67 ± 0.58 mm), and Vibrio natrigens (36.67 ± 0.58 mm). At low concentration (MICs value from 0.023–0.046 mg/mL), cumin EO inhibited the growth of all Vibrio strains, and concentrations as low as 1.5 mg/mL were necessary to kill them (MBCs values from 1.5–12 mg/mL). Using four antioxidant assays, cumin EO exhibited a good result as compared to standard molecules (DPPH = 8 ± 0.54 mg/mL; reducing power = 3.5 ± 0.38 mg/mL; β-carotene = 3.8 ± 0.34 mg/mL; chelating power = 8.4 ± 0.14 mg/mL). More interestingly, at 2x MIC value, cumin EO inhibited the formation of biofilm by Vibrio alginolyticus (9.96 ± 1%), V. parahaemolyticus (15.45 ± 0.7%), Vibrio cholerae (14.9 ± 0.4%), and Vibrio vulnificus (18.14 ± 0.3%). In addition, cumin EO and cuminaldehyde inhibited the production of violacein on Lauria Bertani medium (19 mm and 35 mm, respectively). Meanwhile, 50% of violacein inhibition concentration (VIC50%) was about 2.746 mg/mL for cumin EO and 1.676 mg/mL for cuminaldehyde. Moreover, elastase and protease production and flagellar motility in P. aeruginosa were inhibited at low concentrations of cumin EO and cuminaldehyde. The adopted in-silico approach revealed good ADMET properties as well as a high binding score of the main compounds with target proteins (1JIJ, 2UV0, 1HD2, and 3QP1). Overall, the obtained results highlighted the effectiveness of cumin EO to prevent spoilage with Vibrio species and to interfere with the quorum sensing system in Gram-negative bacteria by inhibiting the flagellar motility, formation of biofilm, and the secretion of some virulence enzymes.

Bdellovibrio lyse multiple pathogenic bacteria and protect crucian carp Carassius auratus gibelio from Aeromonas veronii infections

Bdellovibrio are obligate predators of Gram-negative bacteria that could parasitize other bacteria and cause their lysis. It has been considered as a potential probiotic and may replace antibiotics. In order to better explore the inhibitory activity of Bdellovibrio against common aquatic pathogens and its potential use in aquaculture, the Aeromonas hydrophilia was used as the host bacteria for culturing Bdellovibrio. An absolute qPCR detection system with specific primers were also established for the amount determination of Bdellovibrio sp.. The morphological characteristics of the cultured Bdellovibrio sp. were detected by scanning electron microscope. By detecting absorbance at OD600nm and double layer plates plaque experiments, the Bdellovibrio sp. exhibited a certain lysis effect on the selected aquatic pathogens including Aeromonas veronii, Aeromonas hydrophil ia, Vibrio fluvialis, Escherichia coli, Vibrio anguillarum, Vibrio cholerae and Citrobacter freundii. Adding the cultured Bdellovibrio sp. to water could significantly reduce the mortality rate of Carassius auratus caused by the infections with A. vironii. Meanwhile, the amount of Bdellovibrio sp. on intestines and gills of C. auratus could increase over time after treatment, indicating Bdellovibrio may colonized in fish. The present study revealed that Bdellovibrio may play important roles as an alternative to antibiotics in protecting aquatic animals from pathogenic bacteria. These results will provide a certain theoretical basis for the application of Bdellovibrio in aquaculture.

Draft-genome analysis provides insights into the virulence properties and genome plasticity of Vibrio fluvialis organisms isolated from shrimp farms and Turag river in Bangladesh.

Vibrio fluvialis is an opportunistic waterborne and seafood-borne enteric pathogen capable of causing severe diarrhea leading to death. This pathogen is endemic to Bangladesh, a country which is a major producer of cultured shrimp and wild-caught prawns. In this study, we carried out whole-genome sequencing of three V. fluvialis organisms isolated from shrimp farm and river sediment showing strong pathogenic characteristics in vivo and in vitro and compared their genomes against other V. fluvialis and related pathogenic species to glean insights into their potential as pathogens. Numerous virulence-associated genes including hemolysins, cytolysins, three separate Type IV pili, Types II and VI secretion systems, biofilm, and the V. cholerae pathogenesis regulating gene, toxR, were identified. Moreover, we found strain S-10 to have the propensity to acquire antibiotic resistance genes through horizontal gene transfer. These findings indicate that shrimp farms and rivers could be potential sources of V. fluvialis organisms which are an infection threat of public health concern.

Accurate Identification of Diverse N-acyl Homoserine Lactones in Marine Vibrio fluvialis by UHPLC-MS/MS.

Vibrio fluvialis is a marine opportunistic pathogen that frequently causes diseases in aquatic animals and humans. V. fluvialis can produce quorum sensing signaling molecules to coordinate cell density-dependent behavioral changes, including N-acyl homoserine lactone (AHL), which acts as a vital mediator of virulence-associated gene expression. Currently, several AHL molecules in V. fluvialis have been detected via biological and physicochemical methods, although different detection approaches have generated diverse AHL profiles. Here, we describe the AHL-producing bacterium, V. fluvialis BJ-1, which was isolated from marine sediments from the East China Sea. V. fluvialis BJ-1 could stimulate AHL-mediated β-galactosidase synthesis of the biosensor Agrobacterium tumefaciens NTL4 (pZLR4) but couldnot induce violacein production in the AHL reporter strain, Chromobacterium violaceum CV026. This bacterial isolate exhibited strong AHL-producing activity atlowcelldensity; however, the AHL activity declined when populationdensity remained at high levels. Analysis of the AHLs by Ultra-High-Performance Liquid Chromatography tandem Mass Spectrometry demonstrated that V. fluvialis BJ-1 produced five different AHL signaling molecules, including two linear chain AHL products (C8- and C10-HSL), and three β-carbon-oxidative AHL products (3-O-C8-, 3-O-C10- and 3-O-C12-HSL). Significantly, the present study is the first to accurately define the AHL profile of marine V. fluvialis. In future, the coupling of UHPLC to ESI-MS/MS is expected to be utilized for the accurate determination of AHL profiles in marine Vibrio.

In-silico analysis of bioactive compounds extracted from seaweed Amphiroa anceps on the pathogenicity of bacteria

Screening of biochemical compounds in marine algae is a gateway to understand the bioactive principles of antimicrobial activity and may be a significant tool in developing environment friendly herbicides and in drug discovery. In this study, the antibacterial activity of the seaweed Amphiroa anceps extracted with methanol and ethanol was determined against clinical isolates namely Vibrio cholerae 01 ogawa, Vibrio cholerae O139, Vibrio fluvialis, Vibrio parahaemolyticus, V. parahaemolyticus 81, V. parahaemolyticus O3:K6, salmonella enteritidis type 5, Enterotoxigenic E. coli, Bacillus cereus by disc diffusion method at different concentrations. Ethanolic extract showed the highest activity against V. parahaemolyticus, V. parahaemolyticus 81 and V. cholerae. The FTIR results showed the presence of major functional groups like amine, hydrogen bonded alcohols, alkanes and aromatic rings. Four major bioactive compounds were selected based on GC-MS data and used as ligands to dock in-silico against proteins of pathogenic bacteria that were used in antibiotic studies.