Identification and whole-genome sequencing analysis of Vibrio vulnificus strains causing pearl gentian grouper disease in China.

Vibrio vulnificus is a pathogenic bacterium that causes disease in marine fish, affecting fish farming and human health worldwide. In May 2021, in the Bohai Bay region, a disease broke out in commercially farmed pearl gentian grouper (♀Epinephelus fuscoguttatus × â™‚Epinephelus lanceolatus), causing huge economic losses. The diseased fish had skin lesions, water accumulation in their abdomens, and showed tissue and organ damage. V. vulnificus biotype 2 has been reported in eels and other marine fish, but it is less reported in pearl gentian grouper. In this study, the pathogenic strain isolated from diseased fish was identified as V. vulnificus EPL 0201 biotype 2 on the basis of physiological and biochemical characteristics and the results of 16S rRNA gene and gyrB sequencing, virulence gene detection, and recursive infection experiments. To gain a comprehensive understanding of the pathogenicity and drug resistance of this strain, whole-genome sequencing was performed. Whole-genome analysis showed that the gene map of this strain was complete. The Virulence Factor Database annotation results showed that this strain had the key virulence factor genes vvhA and rtxA, which cause host disease. In addition, this strain had genes conferring resistance against cephalosporins, aminoglycosides, tetracyclines, and sulfonamides. Antimicrobial susceptibility testing confirmed the presence of these resistance genes identified in the genome. The results of this study show that V. vulnificus EPL 0201 biotype 2 is a multi-drug resistant strain with high pathogenicity.

Comparison between genome sequences of Chilean Tenacibaculum dicentrarchi isolated from red conger eel (Genypterus chilensis) and Atlantic salmon (Salmo salar) focusing on bacterial virulence determinants.

Tenacibaculum dicentrarchi is an emerging pathogen for salmonid cultures and red conger eel (Genypterus chilensis) in Chile, causing high economic losses not only in Chile but also to the global salmon industry. Infected fish show severe gross skin lesions that are sometimes accompanied by bone exposure. Despite pathogenicity demonstrated by Koch's postulates, no knowledge is currently available regarding the virulence machinery of T. dicentrarchi strains. Comparisons between the genome sequences of the eight T. dicentrarchi strains obtained from G. chilensis and Atlantic salmon (Salmo salar) provide insights on the existence of genomic diversity within this bacterium. The T. dicentrarchi type strain 3509T was used as a reference genome. Depending on the T. dicentrarchi strain, the discovered diversity included genes associated with iron acquisition mechanisms, copper homeostasis encoding, resistance to tetracycline and fluoroquinolones, pathogenic genomic islands and phages. Interestingly, genes encoding the T9SS membrane protein PorP/SprF were retrieved in all of the analysed T. dicentrarchi strains, regardless of the host fish (i.e. red conger eel or Atlantic salmon). However, the T6SS core component protein VgrG was identified in only one Atlantic salmon strain. Three types of peptidase genes and proteins associated with quorum sensing were detected in all of the T. dicentrarchi strains. In turn, all eight strains presented a total of 17 proteins associated with biofilm formation, which was previously confirmed through physiological studies. This comparative analysis will help elucidate and describe the genes and pathways that are likely involved in the virulence process of T. dicentrarchi. All or part of these predicted genes could aid the pathogen during the infective process in fish, making further physiological research necessary for clarification.

A novel multiplex PCR assay for rapid detection of virulent Aeromonas in cultured eels.

AIMS: Under intensive and stressful aquaculture conditions, cultured eels are highly susceptible to virulent Aeromonas sp. infections. To rapidly and simultaneously confirm Aeromonas isolate and its virulence, a two-tube multiplex PCR (mPCR) assay incorporating gyrB gene for genus-specific recognition and seven major virulence genes for virulence assessment was developed. METHODS AND RESULTS: Eight pairs of primers were designed and divided into two groups-gyrB, ahpA, epr and aerA in tube 1 and alt, act, ast and hlyA in tube 2. The optimized mPCR conditions were the same except for their final concentrations. The specificity of the mPCR was validated by the extracted DNA of 10 Aeromonas and 8 non-Aeromonas species, or mixed DNA templates. Detection limits were determined to be 200 copies per µl in tube 1 and 20 copies per µl in tube 2. The mPCR reproducibility was tested by both artificial challenge and clinical samples. CONCLUSIONS: The results showed this two-tube mPCR assay was rapid, specific, sensitive and reliable. SIGNIFICANCE AND IMPACT OF THE STUDY: To our knowledge, this is the first report to distinguish virulent Aeromonas isolates from nonvirulent ones by seven popular and major virulence genes at the genus-specific level. And it will be useful for large-scale screening of virulent Aeromonas sp. in cultured eels.

Effects of a vegetable-eel-earthworm integrated planting and breeding system on bacterial community structure in vegetable fields.

Agricultural production combined with planting and breeding, which can reduce chemical fertilizer and pesticide applications, reduce losses due to natural disasters, and improve the output and quality of agricultural products, is an important way to achieve green, circular and efficient production. To assess effects on soil bacterial community structure, a vegetable-eel-earthworm integrated planting and breeding platform (VEE-IPBP) combined with experiment planting was established at Chongming Island, Shanghai and compared to traditional planting. High-throughput sequencing to reveal soil bacterial community structure was performed on samples collected at 0, 3 and 6 years after implementation of the two models. Over time, the Shannon index first increased and then decreased in the VEE-IPBP system and was reduced by 3.2% compared to the traditional planting (In the same time and space scale, the single-degree planting method of dryland vegetables under mechanical cultivation is adopted) (p < 0.05). In contrast, Chao and Ace indices were increased by 2.4% and 3.2%. Thus, soil bacterial diversity was markedly different in the two planting models. The abundance of Proteus, Cyanophyta and Cyanophyta in soil increased after 6 years, and the proportion of Lysinibacillus increased significantly, contributing to improvement in soil disease resistance. Redundancy analysis (RDA) showed that the soil pH and water content were the main factors influencing the change in soil bacterial community structure in the two planting models, and the dominant species of soil bacteria were Lysobacter and Bacillus.

A Comparison of Genotypic and Phenotypic Methods for Analyzing the Susceptibility to Sulfamethoxazole and Trimethoprim in Edwardsiella piscicida.

In a study of 39 isolates of Edwardsiella piscicida made from Korean aquaculture sites, sul genes were detected in 16 isolates and dfr genes in 19. Ten isolates were shown to contain both sul and dfr genes. MIC and disc diffusion zones assays were performed to measure the phenotypic susceptibilities of the 39 isolates. Normalized resistance interpretation was applied to these data to categorize isolates as either fully susceptible or as manifesting reduced susceptibility. The standard CLSI protocols specify the use of a mixture of sulfamethoxazole/trimethoprim (20:1) in both MIC and disc diffusion tests. Using the CLSI MIC protocol, 100% of the isolates containing dfr genes, but only 75% of the isolates containing sul genes, were categorized as manifesting reduced susceptibility. Using the CLSI disc diffusion protocol, only 58% of the isolates containing dfr genes and 69% of those containing sul genes were categorized as manifesting reduced susceptibility. When the single agent trimethoprim was substituted for the combined mixture in both the MIC and disc diffusion protocols, 100% of the dfr-positive isolates were categorized as NWT. When the single-agent sulfamethoxazole was substituted, the analysis of the MIC characterized 100% and the disc zone data 94% of the sul-positive isolates as manifesting reduced susceptibility. It is argued that the use of trimethoprim and sulfamethoxazole as single agents in phenotypic susceptibility tests would provide more meaningful data than the currently recommended use of these two agents combined.

Edwardsiella tarda Bacteremia with Psoas and Epidural Abscess as a Food-borne Infection: A Case Report and Literature Review.

Edwardsiella tarda is commonly isolated from aquatic environments and a variety of animals. We present the first case of E. tarda bacteremia with psoas and epidural abscess. The patient was a 65-year-old woman with recurrent gastric cancer who had frequently consumed raw fish and grilled eel. She was successfully treated with antimicrobials and surgery. We also review reports published in English regarding E. tarda bacteremia in Japan and the experience at our hospital. On the basis of this review, we conclude that the major underlying disease leading to E. tarda bacteremia is malignancy and that the gastrointestinal tract is the most commonly affected organ. The overall mortality rate due to E. tarda bacteremia in our review was 38.1% (8/21). Although E. tarda bacteremia is rare, clinicians should be aware of this fatal food-borne infection.

Phylogenetic analysis of the pathogenic genus Aeromonas spp. isolated from diseased eels in China.

Analyses of 16S rRNA and housekeeping genes (HKGs) were valuated as identification markers for pathogenic Aeromonas isolated from diseased eels. The interrelationships of 32 Aeromonas strains which had been verified as pathogens to eels were studied using phylogenetic analysis with 16S rRNA and HKG sequences (cpn60, gyrB, rpoB and dnaJ) and identified by Biolog automatic microbiology analysis system (gene III). From the analysis of 5 genes, the mean gene divergences of 16S rRNA, cpn60, gyrB, rpoB and dnaJ in 32 isolates were 1.4 ± 0.2%, 7.1 ± 0.7%, 5.2 ± 0.5%, 2.2 ± 0.4% and 6.8 ± 0.5%, respectively. The results of comparative phylogeny between nucleotide based analyses (excluding the third codon position) of four HKGs with the sequences from 55 strains of Aeromonas (including 23 referenced strains of Aeromonas) showed cpn60 and dnaJ have higher discriminate power than gyrB and rpoB comparing with the taxonomical identification by Biolog system. In addition, amino acid sequences of concatenated cpn60-rpoB-gyrB is a good method for Aeromonas pathogens identification. This study showed analysis of HKG sequences can be used as an alternative method for sound identification of bacterial pathogens isolated from diseased eels in China.

An Enriched European Eel Transcriptome Sheds Light upon Host-Pathogen Interactions with Vibrio vulnificus.

Infectious diseases are one of the principal bottlenecks for the European eel recovery. The aim of this study was to develop a new molecular tool to be used in host-pathogen interaction experiments in the eel. To this end, we first stimulated adult eels with different pathogen-associated molecular patterns (PAMPs), extracted RNA from the immune-related tissues and sequenced the transcriptome. We obtained more than 2 x 10(6) reads that were assembled and annotated into 45,067 new descriptions with a notable representation of novel transcripts related with pathogen recognition, signal transduction and the immune response. Then, we designed a DNA-microarray that was used to analyze the early immune response against Vibrio vulnificus, a septicemic pathogen that uses the gills as the portal of entry into the blood, as well as the role of the main toxin of this species (RtxA13) on this early interaction. The gill transcriptomic profiles obtained after bath infecting eels with the wild type strain or with a mutant deficient in rtxA13 were analyzed and compared. Results demonstrate that eels react rapidly and locally against the pathogen and that this immune-response is rtxA13-dependent as transcripts related with cell destruction were highly up-regulated only in the gills from eels infected with the wild-type strain. Furthermore, significant differences in the immune response against the wild type and the mutant strain also suggest that host survival after V. vulnificus infection could depend on an efficient local phagocytic activity. Finally, we also found evidence of the presence of an interbranchial lymphoid tissue in European eel gills although further experiments will be necessary to identify such tissue.

The Fish Pathogen Vibrio vulnificus Biotype 2: Epidemiology, Phylogeny, and Virulence Factors Involved in Warm-Water Vibriosis.

Vibrio vulnificus biotype 2 is the etiological agent of warm-water vibriosis, a disease that affects eels and other teleosts, especially in fish farms. Biotype 2 is polyphyletic and probably emerged from aquatic bacteria by acquisition of a transferable virulence plasmid that encodes resistance to innate immunity of eels and other teleosts. Interestingly, biotype 2 comprises a zoonotic clonal complex designated as serovar E that has extended worldwide. One of the most interesting virulence factors produced by serovar E is RtxA13, a multifunctional protein that acts as a lethal factor for fish, an invasion factor for mice, and a survival factor outside the host. Two practically identical copies of rtxA13 are present in all biotype 2 strains regardless of the serovar, one in the virulence plasmid and the other in chromosome II. The plasmid also contains other genes involved in survival and growth in eel blood: vep07, a gene for an outer membrane (OM) lipoprotein involved in resistance to eel serum and vep20, a gene for an OM receptor specific for eel-transferrin and, probably, other related fish transferrins. All the three genes are highly conserved within biotype 2, which suggests that they are under a strong selective pressure. Interestingly, the three genes are related with transferable plasmids, which emphasizes the role of horizontal gene transfer in the evolution of V. vulnificus in nutrient-enriched aquatic environments, such as fish farms.

Comprehensive Analysis of a Vibrio parahaemolyticus Strain Extracellular Serine Protease VpSP37.

Proteases play an important role in the field of tissue dissociation combined with regenerative medicine. During the years new sources of proteolytic enzymes have been studied including proteases from different marine organisms both eukaryotic and prokaryotic. Herein we have purified a secreted component of an isolate of Vibrio parahaemolyticus, with electrophoretic mobilities corresponding to 36 kDa, belonging to the serine proteases family. Sequencing of the N-terminus enabled the in silico identification of the whole primary structure consisting of 345 amino acid residues with a calculated molecular mass of 37.4 KDa. The purified enzyme, named VpSP37, contains a Serine protease domain between residues 35 and 276 and a canonical Trypsin/Chimotrypsin 3D structure. Functional assays were performed to evaluate protease activity of purified enzyme. Additionally the performance of VpSP37 was evaluated in tissue dissociations experiments and the use of such enzyme as a component of enzyme blend for tissue dissociation procedures is strongly recommended.

Novel host-specific iron acquisition system in the zoonotic pathogen Vibrio vulnificus.

Vibrio vulnificus is a marine bacterium associated with human and fish (mainly farmed eels) diseases globally known as vibriosis. The ability to infect and overcome eel innate immunity relies on a virulence plasmid (pVvbt2) specific for biotype 2 (Bt2) strains. In the present study, we demonstrated that pVvbt2 encodes a host-specific iron acquisition system that depends on an outer membrane receptor for eel transferrin called Vep20. The inactivation of vep20 did not affect either bacterial growth in human plasma or virulence for mice, while bacterial growth in eel blood/plasma was abolished and virulence for eels was significantly impaired. Furthermore, vep20 is an iron-regulated gene overexpressed in eel blood during artificially induced vibriosis both in vitro and in vivo. Interestingly, homologues to vep20 were identified in the transferable plasmids of two fish pathogen species of broad-host range, Vibrio harveyi (pVh1) and Photobacterium damselae subsp. damselae (pPHDD1). These data suggest that Vep20 belongs to a new family of plasmid-encoded fish-specific transferrin receptors, and the acquisition of these plasmids through horizontal gene transfer is likely positively selected in the fish-farming environment. Moreover, we propose Ftbp (fish transferrin binding proteins) as a formal name for this family of proteins.

Pasteurella infection after muraenidae bite in an HIV infected patient.

We report a case of Pasteurella dagmatis wound infection in an immunocompromised HIV infected patient after bite by a marine carnivore in Caribbean Sea (Dominican Republic), presumably a muraenidae. Identification of the Pasteurella species from wound sampling was obtained twice by mass spectrometry and confirmed by 16S RNA sequencing.

MARTX of Vibrio vulnificus biotype 2 is a virulence and survival factor.

Vibrio vulnificus biotype 2 is a polyphyletic group whose virulence for fish relies on a plasmid. This plasmid contains an rtxA gene duplicated in the small chromosome that encodes a MARTX (Multifunctional, Autoprocessing Repeats-in-Toxin) unique within the species in domain structure (MARTX type III). To discover the role of this toxin in the fitness of this biotype in the fish-farming environment, single- and double-knockout mutants were isolated from a zoonotic strain and analysed in a series of in vivo and in vitro experiments with eel, fish cell lines and amoebae isolated from gills. Mice, murine and human cell lines were also assayed for comparative purposes. The results suggest that MARTX type III is involved in the lysis of a wide range of eukaryotic cells, including the amoebae, erythrocytes, epithelial cells and phagocytes after bacterium-cell contact. In fish, MARTX type III may act as a toxin involved in the onset of septic shock, while in mice it may promote bacterial colonization by preventing phagocytosis of bacterial cells. Moreover, this toxin could protect bacteria from predation by amoebae, which would increase bacterial survival outside the host and would explain the fitness of this biotype in the fish-farming environment.

An extracellular serine protease produced by Vibrio vulnificus NCIMB 2137, a metalloprotease-gene negative strain isolated from a diseased eel.

Vibrio vulnificus is a ubiquitous estuarine microorganism but causes fatal systemic infections in immunocompromised humans, cultured eels or shrimps. An extracellular metalloprotease VVP/VvpE has been reported to be a potential virulence factor of the bacterium; however, a few strains isolated from a diseased eel or shrimp were recently found to produce a serine protease termed VvsA, but not VVP/VvpE. In the present study, we found that these strains had lost the 80 kb genomic region including the gene encoding VVP/VvpE. We also purified VvsA from the culture supernatant through ammonium sulfate fractionation, gel filtration and ion-exchange column chromatography, and the enzyme was demonstrated to be a chymotrypsin-like protease, as well as those from some vibrios. The gene vvsA was shown to constitute an operon with a downstream gene vvsB, and several Vibrio species were found to have orthologues of vvsAB. These findings indicate that the genes vvp/vvpE and vvsAB might be mobile genetic elements.

Prevalence of genes encoding the type three secretion system and the effectors AexT and AexU in the Aeromonas veronii group.

The prevalence of important virulence factors, the type III secretion system (T3SS) and two T3SS-dependent toxins, AexT and AexU, was evaluated in the Aeromonas veronii group (AVG). Members of the AVG have a broad host range, including vertebrates and invertebrates, and form a variety of associations, spanning from pathogenic to mutualistic. Our AVG strain collection consists of human, duck, eel, and leech isolates. These isolates were examined for the presence of the T3SS, AexT, and AexU through PCR analysis. Two loci of the T3SS, ascV and ascF-ascG, and aexT and aexU were PCR amplified and sequenced from these isolates. All 20 environmental and clinical isolates possessed the T3SS and both effectors, which indicates a much greater prevalence than reported by previous studies. Sequence analysis of the C-terminal domains of aexT and aexU revealed a much higher nucleotide substitution rate for aexU (19.7%) when compared to aexT (4%). The lack of sequence variability among aexT homologs suggests that it has a conserved function among the AVG. The increased variation of the aexU sequence suggests the presence of different alleles, which indicates that it may serve different functions.

Vibrio vulnificus produces quorum sensing signals of the AHL-class.

Vibrio vulnificus is an aquatic pathogenic bacterium that can cause vibriosis in humans and fish. The species is subdivided into three biotypes with the fish-virulent strains belonging to biotype 2. The quorum sensing (QS) phenomenon mediated by furanosyl borate diester or autoinducer 2 (AI-2) has been described in human strains of biotype 1, and here we show that the luxS gene which encodes AI-2 is present in all strains of V. vulnificus regardless of origin, biotype or serovar. In this study, we also demonstrate that V. vulnificus produces QS signals of the acylated homoserine lactone (AHL) class (AI-1). AHLs were detected in strains of biotype 1 and 2 from water, fish and human wound infections but not in strains isolated from human septicaemic cases. The AHL compound was identified as N-butanoyl-homoserine-lactone (C(4)-HL) by both reporter strains and by HPLC-high-resolution MS. C(4)-HL was detected when AHL-positive strains were grown in low-nutrient medium [modified sea water yeast extract (MSWYE)] but not in rich media (tryptic soy broth or brain-heart infusion) and its production was enhanced when blood factors were added to MSWYE. C(4)-HL was detected in vivo, in eels infected with AHL-positive biotype 2 strains. No known AHL-related gene was detected by PCR or Southern blot suggesting that AHL-related genes in V. vulnificus are different from those found in other Gram-negative bacteria.

Spontaneous quinolone resistance in the zoonotic serovar of Vibrio vulnificus.

This work demonstrates that Vibrio vulnificus biotype 2, serovar E, an eel pathogen able to infect humans, can become resistant to quinolone by specific mutations in gyrA (substitution of isoleucine for serine at position 83) and to some fluoroquinolones by additional mutations in parC (substitution of lysine for serine at position 85). Thus, to avoid the selection of resistant strains that are potentially pathogenic for humans, antibiotics other than quinolones must be used to treat vibriosis on farms.

Microbial and histopathological study of the vibriosis caused by Vibrio vulnificus serovar E in eels: the metalloprotease Vvp is not an essential lesional factor.

Vibrio vulnificus biotype 2 serovar E (Bt2-serE) is a zoonotic pathogen that causes a haemorrhagic septicaemia in eels, called warm water vibriosis. The main objective of the present work was to study the onset of the eel vibriosis from the microbiological and histopathological viewpoint, as well as to ascertain the role of the protease Vvp as a lesional factor by comparing the histopathological lesions caused by the wild strain and its vvp deficient derivative. The wild-type strain was observed to attach to the gills, where it multiplied following saturation dynamics, subsequently invading the blood stream and reaching the internal organs. Here it reached population sizes that are notably lower than those associated with other fish septicaemia. Parallel to bacterial growth, there was a notable decrease in haematocrit values and haemoglobin concentration in blood as well as extensive haemorrhages in all the analysed organs. The main histopathological lesions were detected in the head kidney in the form of extensive necrosis affecting the haematopoietic tissue. Very few bacteria were visualized in the different organs, most of which were close to blood cells and capillary vessels, which is compatible with the results obtained in the microbiological study. The same lesions were produced when extracellular products (ECPs) were injected instead of bacteria or when the vvp-defective mutant or its ECPs were injected. The overall results suggest that the pathology caused by V. vulnificus in the eel is not caused by massive bacterial growth in the blood and internal organs but, rather, by the effect of potent toxic factors other than the metalloprotease, which have yet to be determined.

Variation of extracellular proteases produced by Vibrio vulnificus clinical isolates: genetic diversity of the metalloprotease gene (vvp), and serine protease secretion by vvp-negative strains.

Vibrio vulnificus is a causative agent of septicemia or wound infection in human and eel; however, the genetic variation between human and eel isolates has been reported. In the present study, the difference in the vvp gene encoding a tissue-damaging metalloprotease was investigated. The gene of strain E86 from a diseased eel (type B vvp) was 95.2% identical with that of strain L-180 from human blood (type A vvp). PCR using oligonucleotide primers designed to differentiate two types of the gene showed that eel avirulent strains (9 isolates) commonly carry type A vvp, whereas eel virulent strains (18 isolates) revealed significant genetic variation. The vvp genes from 12 strains including strain E86 were placed on type B while those from 3 strains were on type A. Other strains were found to be vvp-negative, but PAGE and amino acid sequencing analysis showed that they secreted a serine protease (VVA0302) instead of the metalloprotease. This protease is an orthologue of a toxic protease from Vibrio parahaemolyticus, a human pathogen causing wound infection as well as gastroenteritis. These findings suggest that, in addition to metalloprotease, the extracellular serine protease may contribute to pathogenicity of V. vulnificus.

Antibiotic resistance of motile aeromonads in indoor catfish and eel farms in the southern part of The Netherlands.

The prevalence and degree of antibiotic resistance in catfish and eel farms in the southern part of The Netherlands was examined using motile aeromonads as indicator bacteria. A total of 29 water samples were collected, originating from six catfish farms, one catfish hatchery and three eel farms, and were plated on an Aeromonas-selective agar with and without antibiotics. From each plate, one colony was screened for presumptive motile aeromonads and tested for antibiotic susceptibility. The prevalence of resistance was as follows: ampicillin and oxytetracycline 100%; sulfamethoxazole 24%; trimethoprim 3%; and ciprofloxacin and chloramphenicol 0%. The majority of samples showed a high degree of oxytetracycline resistance, implicating fish farms as a major reservoir of oxytetracycline resistance genes. This reservoir might form a risk for human health and has major consequences for the effectiveness of this antibiotic in the treatment of infectious diseases in fish.