Mobile-CRISPRi protocol optimization for Vibrionaceae.

Mobile clustered regularly interspaced palindromic repeats interference (Mobile-CRISPRi) is an established method for bacterial gene expression knockdown. The deactivated Cas9 protein and guide RNA are isopropyl ß-D-1-thiogalactopyranoside inducible, and all components are integrated into the chromosome via Tn7 transposition. Here, we optimized methods specific for applying Mobile-CRISPRi in multiple Vibrio species.

Cyclic olefin copolymer (COC) as a promising biomaterial for affecting bacterial colonization: investigation on Vibrio campbellii.

Cyclic olefin copolymer (COC) has emerged as an interesting biocompatible material for Organ-on-a-Chip (OoC) devices monitoring growth, viability, and metabolism of cells. Despite ISO 10993 approval, systematic investigation of bacteria grown onto COC is a still not documented issue. This study discusses biofilm formations of the canonical wild type BB120 Vibrio campbellii strain on a native COC substrate and addresses the impact of the physico-chemical properties of COC compared to conventional hydroxyapatite (HA) and poly(dimethylsiloxane) (PDMS) surfaces. An interdisciplinary approach combining bacterial colony counting, light microscopy imaging and advanced digital image processing remarks interesting results. First, COC can reduce biomass adhesion with respect to common biopolymers, that is suitable for tuning biofilm formations in the biological and medical areas. Second, remarkably different biofilm morphology (dendritic complex patterns only in the case of COC) was observed among the examined substrates. Third, the observed biofilm morphogenesis was related to the interaction of COC with the conditioning layer of the planktonic biological medium. Fourth, Level Co-occurrence Matrix (CGLM)-based analysis enabled quantitative assessment of the biomass textural fractal development under different coverage conditions. All of this is of key practical relevance in searching innovative biocompatible materials for pharmaceutical, implantable and medical products.

Probiotic potential of a novel endophytic Streptomyces griseorubens CIBA-NS1 isolated from Salicornia sp. against Vibrio campbellii infection in shrimp.

A novel endophytic Streptomyces griseorubens CIBA-NS1 was isolated from a salt marsh plant Salicornia sp. The antagonistic effect of S. griseorubens against Vibrio campbellii, was studied both in vitro and in vivo. The strain was validated for its endophytic nature and characterized through scanning electron microscopy, morphological and biochemical studies and 16SrDNA sequencing. The salinity tolerance experiment has shown that highest antibacterial activity was at 40‰ (16 ± 1.4 mm) and lowest was at 10 ‰ salinity (6.94 ± 0.51 mm). In vivo exclusion of Vibrio by S. griseorubens CIBA-NS1 was studied in Penaeus indicus post larvae and evaluated for its ability to improve growth and survival of P. indicus. After 20 days administration of S. griseorubens CIBA-NS1, shrimps were challenged with V. campbellii. The S. griseorubens CIBA-NS1 reduced Vibrio population in test group when compared to control, improved survival (60.5 ± 6.4%) and growth, as indicated by weight gain (1.8 ± 0.05g). In control group survival and growth were 48.4 ± 3.5% and 1.4 ± 0.03 g respectively. On challenge with V. campbellii, the S. griseorubens CIBA-NS1 administered group showed better survival (85.6 ± 10%) than positive control (64.3 ± 10%). The results suggested that S. griseorubens CIBA-NS1 is antagonistic to V. campbellii, reduce Vibrio population in the culture system and improve growth and survival. This is the first report on antagonistic activity of S. griseorubens isolated from salt marsh plant Salicornia sp, as a probiotic candidate to prevent V. campbellii infection in shrimps.

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.

Light-emitting plants development by inoculating of Vibrio campbellii RMT1 on the rhizospheric zone of Aglaonema cochinchinense.

The concept of utilizing light-emitting plants (LEPs) as an alternative to traditional electricity-based lighting has garnered interest. However, challenges persist due to the need for genetic modification or chemical infusion in current LEPs. To address this, researchers have investigated the interaction between plants and luminous bacteria, specifically Vibrio campbellii, which can efficiently be translocated into Aglaonema cochinchinense tissues through the roots to produce LEPs. This study concentrated on examining light intensity and enhancing luminescence by growing plants and spraying them with various media substances. The results indicated that V. campbellii successfully translocated into the plant tissue via the root system and accumulated a high number of bacteria in the stems, approximately 8.46 × 104 CFU/g, resulting in a light-emitting intensity increase of 12.13-fold at 48 h, and then decreased after 30 h. Interestingly, luminescence stimulation by spraying the growth medium managed to induce the highest light emission, reaching 14.84-fold at 48 h, though it had some negative effects on the plant. Conversely, spraying plants with CaCl2 on the leaves prolonged light emission for a longer duration (42 h after spraying) and had a positive effect on plant health, it maintained ion homeostasis and reduced-MDA content. This study highlights the potential of using V. campbellii and CaCl2 spraying for the future development of practical light-emitting plants.

Recovering high-quality bacterial genomes from cross-contaminated cultures: a case study of marine Vibrio campbellii.

BACKGROUND: Environmental monitoring of bacterial pathogens is critical for disease control in coastal marine ecosystems to maintain animal welfare and ecosystem function and to prevent significant economic losses. This requires accurate taxonomic identification of environmental bacterial pathogens, which often cannot be achieved by commonly used genetic markers (e.g., 16S rRNA gene), and an understanding of their pathogenic potential based on the information encoded in their genomes. The decreasing costs of whole genome sequencing (WGS), combined with newly developed bioinformatics tools, now make it possible to unravel the full potential of environmental pathogens, beyond traditional microbiological approaches. However, obtaining a high-quality bacterial genome, requires initial cultivation in an axenic culture, which is a bottleneck in environmental microbiology due to cross-contamination in the laboratory or isolation of non-axenic strains. RESULTS: We applied WGS to determine the pathogenic potential of two Vibrio isolates from coastal seawater. During the analysis, we identified cross-contamination of one of the isolates and decided to use this dataset to evaluate the possibility of bioinformatic contaminant removal and recovery of bacterial genomes from a contaminated culture. Despite the contamination, using an appropriate bioinformatics workflow, we were able to obtain high quality and highly identical genomes (Average Nucleotide Identity value 99.98%) of one of the Vibrio isolates from both the axenic and the contaminated culture. Using the assembled genome, we were able to determine that this isolate belongs to a sub-lineage of Vibrio campbellii associated with several diseases in marine organisms. We also found that the genome of the isolate contains a novel Vibrio plasmid associated with bacterial defense mechanisms and horizontal gene transfer, which may offer a competitive advantage to this putative pathogen. CONCLUSIONS: Our study shows that, using state-of-the-art bioinformatics tools and a sufficient sequencing effort, it is possible to obtain high quality genomes of the bacteria of interest and perform in-depth genomic analyses even in the case of a contaminated culture. With the new isolate and its complete genome, we are providing new insights into the genomic characteristics and functional potential of this sub-lineage of V. campbellii. The approach described here also highlights the possibility of recovering complete bacterial genomes in the case of non-axenic cultures or obligatory co-cultures.

Development of light-emitting Episcia lilacina leaf by applying Vibrio campbellii RMT1 and extending the glowing by CaCl2 and yeast extract.

Glowing Episcia lilacina was generated through foliar application of the bioluminescent bacterium Vibrio campbellii RMT1. Firstly, different nutrient formulas were tested, incorporating yeast extract and various inorganic salts, such as CaCl2, MgCl2, MgSO4, KH2PO4, K2HPO4, and NaCl, in order to enhance bacterial growth and light emission. The combination of 0.15% of yeast extract and 0.3% of CaCl2 in a nutrient broth (NB) + 1% NaCl medium extended light emission to 24 h and resulted in higher light intensity compared to other combinations of yeast extract and inorganic salts. The peak intensity reached approximately 1.26 × 108 relative light units (RLU) at 7 h. The optimal presence of inorganic salt ions likely contributed to enhanced light emission, while the yeast extract acted as a nutrient source. Secondly, the effect of proline on salt-induced stress symptoms was investigated by applying 20 mM proline to the glowing plant. Additionally, a 0.5% agar nutrient was spread on the leaves prior to bacteria application to support bacterial growth and penetration. Exogenous proline application led to a significant accumulation of proline in plant cells, resulting in decreased malondialdehyde (MDA) levels. However, the proline accumulation also reduced the light intensity of the bioluminescent bacteria. This study demonstrates the potential for generating light on a living plant using bioluminescent bacteria. Further understanding of the interaction between plants and light-emitting bacteria could contribute to the development of sustainably light-emitting plants.

Mutant-Dependent Local Orientational Correlation in Biofilms of Vibrio campbellii Revealed through Digital Processing of Light Microscopy Images.

Biofilms are key bacterial communities in genetic and adaptive resistance to antibiotics as well as disease control strategies. The mature high-coverage biofilm formations of the Vibrio campbellii strains (wild type BB120 and isogenic derivatives JAF633, KM387, and JMH603) are studied here through the unstraightforward digital processing of morphologically complex images without segmentation or the unrealistic simplifications used to artificially simulate low-density formations. The main results concern the specific mutant- and coverage-dependent short-range orientational correlation as well as the coherent development of biofilm growth pathways over the subdomains of the image. These findings are demonstrated to be unthinkable based only on a visual inspection of the samples or on methods such as Voronoi tessellation or correlation analyses. The presented approach is general, relies on measured rather than simulated low-density formations, and could be employed in the development of a highly efficient screening method for drugs or innovative materials.

Antibacterial activity and immunomodulatory role of a proline-rich antimicrobial peptide SpPR-AMP1 against Vibrio campbellii infection in shrimp Litopenaeus vannamei.

Antimicrobial peptides (AMPs) constitute one of the most promising sources of natural molecules used for the design of effective antimicrobial agents alternative to antibiotics. Previously, we have showed that a crab proline-rich AMP designated as SpPR-AMP1 is a potent AMP that exhibited antimicrobial activity against both Gram-positive and Gram-negative bacteria. Here, we demonstrated the importance of SpPR-AMP1 peptide in treating a virulent acute hepatopancreatic necrosis disease (AHPND) Vibrio campbellii VH-639 isolate and eliciting the innate immune response to counter the AHPND infection in shrimp Litopenaeus vannamei. SpPR-AMP1 exhibited a strong antimicrobial activity against V. campbellii VH-639 at MIC value of 0.195-0.39 µM. Scanning electron microscopy (SEM) revealed the membrane disruption potential of SpPR-AMP1 against the V. campbellii VH-639 cells. The in vivo effect of SpPR-AMP1 in shrimp L.vannamei was investigated and the results showed that SpPR-AMP1 was capable of modulating the innate immune response by stimulating the expression levels of AMP transcripts in shrimp hemocytes. Moreover, treatments with SpPR-AMP1 could promote the resistance of shrimp against V. campbellii VH-639 infection as demonstrated by a significant increase in shrimp survival rate and decrease in both the bacterial load and the expression levels of bacterial PirA and PirB toxin gene transcripts in the infected shrimp. These results suggest the potential of SpPR-AMP1 peptide with the combined antimicrobial and immunoenhancing capabilities as promising antimicrobial agent to treat V. campbellii VH-639 causing AHPND infection in shrimp aquaculture.

Selected essential oil components fail to induce an immunological response in Artemia but still protect against vibriosis.

In the present research, in order to screen out the best candidates from 12 different EOCs, we proposed three in vivo screening methods, namely the screening method of bioluminescence of V. campbellii associated with brine shrimp, regrowth performance of V. campbellii, and immune gene expression of brine shrimp without challenge. Our result showed that challenged with V. campbellii at 107 cells/mL, the survival of the brine shrimp at 48 h was significantly increased after treatment with the EOCs (at 0.0005%, v/v) of 4-allylanisole, R-(+)-limonene, S-(-)-limonene, (-)-terpinen-4-ol, (±)-citronellal, citral, trans-cinnamaldehyde and (+)-carvone, compared to the positive control group. Also, it was observed that the EOCs- of 4-allylanisloe, R-(+)-limonene, S-(-)-limonene, (-)-ß-pinene, geraniol, (±)-citronellal, citral, trans-cinnamaldehyde and (+)-carvone decreased significantly the in vivo bioluminescence of V. campbellii at 36 h after Vibrio exposure. The regrowth assay showed that independently from incubation time (1, 12 or 24 h), no difference was observed in the regrowth curve in all EOC treatment groups compared to the positive control group. The dscam gene expression in the (±)-citronellal group, and the sod gene in the citral group were observed to be significantly higher than in the negative control at 24 h, respectively. However, most of the immune genes were down-regulated in the EOC groups. Combining the survival data at 48 h with the bioluminescence result at 36 h, it was noted that the survival rate of brine shrimp was moderately correlated with in vivo bioluminescence of V. campbellii. The results indicate that the approach of determining in vivo bioluminescence of V. campbellii is a moderately reliable, fastest, and cheapest screening method for EOCs. As the regrowth performance assay of V. campbellii, and the immune genes expression assay of brine shrimp without challenge cannot predict Artemia survival properly, they cannot be used as screening methods for EOCs. Moreover, the immune genes expression assay is relatively slow, time-consuming and costly.

Genomic Analysis Reveals Adaptation of Vibrio campbellii to the Hadal Ocean.

The genus Vibrio is characterized by high metabolic flexibility and genome plasticity and is widely distributed in the ocean from euphotic layers to deep-sea environments. The relationship between genome features and environmental adaptation strategies of Vibrio has been extensively investigated in coastal environments, yet very little is known about their survival strategies in oligotrophic deep-sea. In this study, we compared genomes of five Vibrio campbellii strains isolated from the Mariana and Yap Trenches at different water depths, including two epipelagic strains and three hadopelagic strains, to identify genomic characteristics that facilitate survival in the deep sea. Genome streamlining is found in pelagic strains, such as smaller genome sizes, lower G+C contents, and higher gene densities, which might be caused by long-term residence in an oligotrophic environment. Phylogenetic results showed that these five Vibrio strains are clustered into two clades according to their collection depth. Indeed, hadopelagic isolates harbor more genes involved in amino acid metabolism and transport, cell wall/membrane/envelope biogenesis, and inorganic ion transport and metabolism through comparative genomics analysis. Specific macrolide export gene and more tellurite resistance genes present in hadopelagic strains by the annotation of antibiotic and metal resistance genes. In addition, several genes related to substrate degradation are enriched in hadopelagic strains, such as chitinase genes, neopullulanase genes, and biopolymer transporter genes. In contrast, epipelagic strains are unique in their capacity for assimilatory nitrate reduction. The genomic characteristics investigated here provide insights into how Vibrio adapts to the deep-sea environment through genomic evolution. IMPORTANCE With the development of deep-sea sampling technology, an increasing number of deep-sea Vibrio strains have been isolated, but the adaptation mechanism of these eutrophic Vibrio strains to the deep-sea environment is unclear. Here, our results show that the genome of pelagic Vibrio is streamlined to adapt to a long-term oligotrophic environment. Through a phylogenomic analysis, we find that genomic changes in marine Vibrio campbellii strains are related to water depth. Our data suggest that an increase in genes related to antibiotic resistance, degradation of macromolecular and refractory substrates, and utilization of rare ions is related to the adaptation of V. campbellii strains to adapt to hadal environments, and most of the increased genes were acquired by horizontal gene transfer. These findings may deepen our understanding of adaptation strategies of marine bacteria to the extreme environment in hadal zones.

Phloroglucinol shows prophylactic and metaphylactic effects against pathogenic stressors in Macrobrachium larvae.

Vibriosis caused by Vibrio campbellii and related species is amongst the major hindrance to the sustainable expansion of giant freshwater prawn Macrobrachium rosenbergii larviculture. Induction of heat shock protein Hsp70 is a natural response of stressed organisms that protect against many insults including vibriosis in aquaculture animals. Therefore, there is a great interest in searching for natural compounds that could induce Hsp70 in animals in a non-invasive manner. Previously, in a series of in vivo studies, we have shown that the phenolic compound phloroglucinol could induce Hsp70 in aquaculture organisms Macrobrachium and Artemia. This led to a significant increase in the resistance of the animals towards subsequent challenges with V. parahemolyticus. As V. parahaemolyticus belongs to the Harveyi clade similar to V. campbellii, our above findings triggered the hypothesis that phloroglucinol is a potential anti-microbial agent that could protect the freshwater prawn against V. campbellii infection. The results presented here provide evidence that the Hsp70-inducing compound phloroglucinol could induce both metaphylactic and prophylactic effects against infection stress mediated by V. campbellii. The wide-spectrum property of the compound to both prevent the occurrence and reduce the spread of V. campbellii infection in prawn larvae without affecting the larval growth makes it a potential natural agent for health management and V. campbellii-mediated disease control in freshwater prawn larvae. Overall results add new information about the functional properties of phloroglucinol and advance our knowledge of this compound as a potential antimicrobial agent for the sustainable production of giant freshwater prawns.

Essential Oils Improve the Survival of Gnotobiotic Brine Shrimp (Artemia franciscana) Challenged With Vibrio campbellii.

The halophilic aquatic bacterium Vibrio campbellii is an important aquatic pathogen, capable of causing vibriosis in shrimp and fish resulting in significant economic losses. In a previous work, essential oils (EOs) extracts from Melaleuca alternifolia, Litsea citrata, and Eucalyptus citriodora were found to inhibit the growth of V. campbellii in vitro. This study aimed to determine in vivo EOs' potential protective effect towards gnotobiotic brine shrimp Artemia franciscana, challenged with V. campbellii. The study showed that brine shrimp larvae supplemented with EOs of M. alternifolia (0.0008%) and L. citrata (0.002%) displayed significantly increased survival against V. campbellii. The results indicated that supplementation of these EOs increased the expression of immune-related genes (either in the presence or absence of the pathogen), probably contributing to enhanced protection. Furthermore, in vitro studies indicated that some EOs modulated the expression of virulence factors including swimming motility, biofilm formation, and gelatinase and lipase activity, while flow cytometry data and regrowth assay indicated that these EOs do not exhibit antimicrobial activity as V. campbellii grew at the tested concentrations [M. alternifolia (0.0008%) and L. citrata (0.002%)]. Our findings suggest that EOs extracted from M. alternifolia and L. citrata, can modulate virulence factor production and immunological responses and might hence become part of an intervention strategy to control vibriosis in a fish or shrimp aquaculture setting, a hypothesis that needs to be validated in the future.

Tocopherol and phytol possess anti-quorum sensing mediated anti-infective behavior against Vibrio campbellii in aquaculture: An in vitro and in vivo study.

Phytocompounds have long been well recognized in medicine and pharmacy. The natural compounds are frequently utilized as the fundamental resource in the development of novel therapeutic agents to treat bacterial infections. The rapid emergence of bacterial infections, particularly caused by Vibrio species, is seen as a serious concern for the development of aquaculture industries, resulting in substantial economic losses throughout the world. Notably, the presence of Vibrio campbellii in aquatic environments will be extremely problematic, leading to significant mortality in aquatic organisms. As a result, novel therapeutic agents are desperately needed to treat such diseases. This is the first research to demonstrate that plant-derived active compounds, tocopherol and phytol, are effective against V. campbellii infection in tomato clownfish. The findings showed that tocopherol and phytol significantly decreased the production of biofilm and virulence factors such as hemolysin, protease, lipase, hydrophobic index, and swimming motility in V. campbellii, without influencing the bacterial growth. In vivo experiments with tomato clownfish also proved that these phytocompound treatments significantly increased the survival rates of infected fishes by hindering the intestinal colonization of V. campbellii in tomato clownfish. Further, the disease protection efficacy against the pathognomonic sign of V. campbellii-infection was verified by histopathological investigation of the gills, gut, and kidney. Altogether, the results suggest that tocopherol and phytol could be promising therapeutic agents for the treatment of V. campbellii infections in aquaculture.

Amino acid divergence in the ligand-binding pocket of Vibrio LuxR/HapR proteins determines the efficacy of thiophenesulfonamide inhibitors.

The quorum-sensing signaling systems in Vibrio bacteria converge to control levels of the master transcription factors LuxR/HapR, a family of highly conserved proteins that regulate gene expression for bacterial behaviors. A compound library screen identified 2-thiophenesulfonamide compounds that specifically inhibit Vibrio campbellii LuxR but do not affect cell growth. We synthesized a panel of 50 thiophenesulfonamide compounds to examine the structure-activity relationship effects on Vibrio quorum sensing. The most potent molecule identified, PTSP (3-phenyl-1-(thiophen-2-ylsulfonyl)-1H-pyrazole), inhibits quorum sensing in multiple strains of V. vulnificus, V. parahaemolyticus, and V. campbellii at nanomolar concentrations. However, thiophenesulfonamide inhibition efficacy varies significantly among Vibrio species: PTSP is most inhibitory against V. vulnificus SmcR, but V. cholerae HapR is completely resistant to all thiophenesulfonamides tested. Reverse genetics experiments show that PTSP efficacy is dictated by amino acid sequence in the putative ligand-binding pocket: F75Y and C170F SmcR substitutions are each sufficient to eliminate PTSP inhibition. Further, in silico modeling distinguished the most potent thiophenesulfonamides from less-effective derivatives. Our results revealed the previously unknown differences in LuxR/HapR proteins that control quorum sensing in Vibrio species and underscore the potential for developing thiophenesulfonamides as specific quorum sensing-directed treatments for Vibrio infections.

The Polar Flagellar Transcriptional Regulatory Network in Vibrio campbellii Deviates from Canonical Vibrio Species.

Swimming motility is a critical virulence factor in pathogenesis for numerous Vibrio species. Vibrio campbellii DS40M4 is a wild-type isolate that has been recently established as a highly tractable model strain for bacterial genetics studies. We sought to exploit the tractability and relevance of this strain for characterization of flagellar gene regulation in V. campbellii. Using comparative genomics, we identified homologs of V. campbellii flagellar and chemotaxis genes conserved in other members of the Vibrionaceae and determined the transcriptional profile of these loci using differential RNA-seq. We systematically deleted all 63 predicted flagellar and chemotaxis genes in V. campbellii and examined their effects on motility and flagellum production. We specifically focused on the core regulators of the flagellar hierarchy established in other vibrios: RpoN (σ54), FlrA, FlrC, and FliA. Our results show that V. campbellii transcription of flagellar and chemotaxis genes is governed by a multitiered regulatory hierarchy similar to other motile Vibrio species. However, there are several critical differences in V. campbellii: (i) the σ54-dependent regulator FlrA is dispensable for motility; (ii) the flgA, fliEFGHIJ, flrA, and flrBC operons do not require σ54 for expression; and (iii) FlrA and FlrC coregulate class II genes. Our model proposes that the V. campbellii flagellar transcriptional hierarchy has three classes of genes, in contrast to the four-class hierarchy in Vibrio cholerae. Our genetic and phenotypic dissection of the V. campbellii flagellar regulatory network highlights the differences that have evolved in flagellar regulation across the Vibrionaceae. IMPORTANCE Vibrio campbellii is a Gram-negative bacterium that is free-living and ubiquitous in marine environments and is an important global pathogen of fish and shellfish. Disruption of the flagellar motor significantly decreases host mortality of V. campbellii, suggesting that motility is a key factor in pathogenesis. Using this model organism, we identified >60 genes that encode proteins with predicted structural, mechanical, or regulatory roles in function of the single polar flagellum in V. campbellii. We systematically tested strains containing single deletions of each gene to determine the impact on motility and flagellum production. Our studies have uncovered differences in the regulatory network and function of several genes in V. campbellii compared to established systems in Vibrio cholerae and Vibrio parahaemolyticus.

Isolation and characterization of marine bioluminescent bacteria for toxicity bioassays and biotechnological applications.

Toxic heavy metals pollution posed severe health hazards to the environment and biodiversity. Therefore, the development of rapid and non-invasive bioassays is in the race to monitor toxic chemicals using novel approaches. This study isolated and characterized an intense blue luminescence-producing marine bacteria, Vibrio campbellii STF1, for biosensing applications. Species-level identification of this strain was confirmed based on various phenotypic tests and multilocus sequence approach using 16s rRNA, toxR, and luxA gene sequence analysis. Fatty acid methyl ester analysis revealed the presence of three predominant fatty acids C15:0 anteiso (21.73%), C17:0 anteiso (11.27%), and C19:0 anteiso (9.08%) in STF1. Luciferase enzyme from V. campbellii STF1 was extracted, partially purified, and molecular masses (alpha subunit 40 kDa and beta subunit 37 kDa) were determined by SDS-PAGE gel for in vivo assays. MALDI-TOF-MS analysis of V. campbellii cells' protein extracts showed distinct mass spectral peaks at m/z of 2615, 3948, and 4232 da. V. campbellii STF1 is resistant to heavy metal lead, while other metals such as cadmium, copper, and mercury inhibited its growth and luminescence. Crude ethyl acetate extraction of V. campbellii demonstrated antibacterial activity against Shigella dysenteriae type 5 with a maximum inhibition zone of 27.0±1.0 mm.

Induction and Genome Analysis of HY01, a Newly Reported Prophage from an Emerging Shrimp Pathogen Vibrio campbellii.

Vibrio campbellii is an emerging aquaculture pathogen that causes luminous vibriosis in farmed shrimp. Although prophages in various aquaculture pathogens have been widely reported, there is still limited knowledge regarding prophages in the genome of pathogenic V. campbellii. Here, we describe the full-genome sequence of a prophage named HY01, induced from the emerging shrimp pathogen V. campbellii HY01. The phage HY01 was induced by mitomycin C and was morphologically characterized as long tailed phage. V. campbellii phage HY01 is composed of 41,772 bp of dsDNA with a G+C content of 47.45%. A total of 60 open reading frames (ORFs) were identified, of which 31 could be predicted for their biological functions. Twenty seven out of 31 predicted protein coding regions were matched with several encoded proteins of various Enterobacteriaceae, Pseudomonadaceae, Vibrionaceae, and other phages of Gram-negative bacteria. Interestingly, the comparative genome analysis revealed that the phage HY01 was only distantly related to Vibrio phage Va_PF430-3_p42 of fish pathogen V. anguillarum but differed in genomic size and gene organization. The phylogenetic tree placed the phage together with Siphoviridae family. Additionally, a survey of Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR) spacers revealed two matching sequences between phage HY01 genome and viral spacer sequence of Vibrio spp. The spacer results combined with the synteny results suggest that the evolution of V. campbellii phage HY01 is driven by the horizontal genetic exchange between bacterial families belonging to the class of Gammaproteobacteria.

Isolation and characterization of vibriophage vB_Vc_SrVc9: an effective agent in preventing Vibrio campbellii infections in brine shrimp nauplii (Artemia franciscana).

AIMS: This study describes the physicochemical and genomic characterization of phage vB_Vc_SrVc9 and its potential for phage therapy application against a pathogenic Vibrio campbellii strain. METHODS AND RESULTS: A lytic phage vB_Vc_SrVc9 against V. campbellii was isolated from shrimp farm sediment, and characterized physicochemical and genomically. The use of vB_Vc_SrVc9 phage increased the survival in brine shrimp Artemia franciscana and reduced presumptive V. campbellii to nondetectable numbers. Genomic analysis showed a genome with a single contig of 43·15 kb, with 49 predicted genes and no tRNAs, capable of recognizing and generating complete inhibition zones of three Vibrio sp. CONCLUSIONS: To our knowledge vB_Vc_SrVc9 is a lytic phage that could be used against Vibrio infections, reducing vibrio presence without any apparent impact over the natural microbiota at the family level in 28 libraries tested. SIGNIFICANCE AND IMPACT OF THE STUDY: vB_Vc_SrVC9 is a novel phage and ecofriendly alternative for therapeutic applications and biotechnological purposes because is stable at different environmental conditions, has the potential to eliminate several strains, and has a short latent period with a good burst size. Therefore, the use of phages, which are natural killers of bacteria, represents a promising strategy to reduce the mortality of farmed organisms caused by pathogenic bacteria.