Recent update on the prevalence of Vibrio species among cultured grouper in Peninsular Malaysia

Vibrio infections are common among marine fish and lead to serious problems in the aquaculture sector. This study reports a recent occurrence of Vibrio species (spp.) isolated from cultured groupers in Peninsular Malaysia using the gyrB and pyrH genes. A total of 147 Vibrio strains were successfully isolated from 77 (64%) groupers using culture method and subjected to gyrB and pyrH sequencing for species identification and confirmation. Results showed that 89% of Vibrio strains were identified and clustered to six groups of Vibrio spp., while 11% were not clustered to any Vibrio spp. using the gyrB sequences. Meanwhile, by analysis of the pyrH sequences all the 147 Vibrio strains (100%) were successfully identified and clustered into 11 groups of Vibrio spp., including the gyrB non‐identified strains. The pyrH gene provides a better resolution for identification of Vibrio spp. compared with the gyrB gene. Thus, the pyrH gene was more suitable for a rapid determination of Vibrio spp. distribution in Peninsular Malaysia. Using the pyrH gene, our study found higher prevalence of Vibrio vulnificus (33%), V. alginolyticus (24%) and V. parahaemolyticus (22%), followed by V. rotiferianus (5%), V. harveyi (3%), V. tubiashii (2%), V. campbellii (2%), V. ponticus (1%), V. diabolicus (1%), V. owensii (1%) and others Vibrio sp. (7%). Thus, the results of this study revealed that the occurrence of pathogenic vibrios among grouper fish is still high in Malaysian aquaculture. In addition, the pyrH gene was proved as a suitable marker for rapid identification of Vibrio species compared with the gyrB gene.     

Vibrios associated with Macrobrachium rosenbergii (De Man, 1879) larvae from three hatcheries on the Indian southwest coast

Surveys for bacteriological analysis of larval samples to isolate the associated vibrios were carried out during 1985–1992, 2001 and 2002 in three different hatcheries located on the southwest coast of India. Vibrio isolates were examined for their species diversity, virulence based on haemolysis in prawn blood agar, lipolysis, proteolysis and chitinolysis and antibiotic sensitivity. Vibrio cholerae was the predominant species in the apparently healthy larval samples, whereas V. alginolyticus and V. vulnificus dominated during disease and morbidity. No correlation was found between the hydrolytic properties and haemolytic activity of the vibrios associated with the larvae. All isolates were resistant to erythromycin and resistance to oxytetracycline, ampicillin and streptomycin sulphate was prevalent among the larger section of the Vibrio population. This suggested that antibiotic application may not be of much use to protect the larvae from vibriosis. This is the first report on the diversity of Vibrio species associated with Macrobrachium rosenbergii larvae and their virulence characteristics based on haemolysis in prawn blood agar.     

Antibiotic resistance and repetitive‐element PCR fingerprinting in Aeromonas veronii isolates

This study evaluated antibiotic resistance and the related genes in total 47 Aeromonas veronii isolates from pet fish, eel (Anguilla japonica) and koi (Cyprinus carpio) in Korea. In comparison with the antibiotic susceptibilities of isolates from eel and koi, those of pet fish were more resistant to ceftiofur, aminoglycosides, tetracycline and nitrofurantoin. And isolates from pet fish showed high prevalences of class 1 integron, quinolones and tetracycline resistance determinants than those from eel and koi. Repetitive‐element palindromic PCR (rep‐PCR) showed larger diversities among A. veronii isolates. Collectively, pet fish may be a reservoir for multiple clones of A. veronii involved in antibiotic resistance. In this aspect, imported fish in the aquaculture trade should be steadily and continually screened for bacterial antibiotic resistance and related genes.     

Antimicrobial activity of partially characterized analytes from Bacillus pumilus(B2)

This study was carried out to characterize the antimicrobial substance produced by the strain of Bacillus pumilus (B2) obtained from Novozymes Biologicals Inc. to compare its antimicrobial activity by agar well diffusion assay and bacteriocin activity assay via critical dilution method against seven different strains of Vibrio spp., specifically V. alginolyticus (A01), V. cholerae (C01), V. fluvialis (F01, F02), V herveyii (H), V. mimicus (M01), V. parahaemolyticus (P01) and V. vulnificus (V01, V02). All Vibrio spp. were isolated from the hemolymph and intestine of the white faeces disease‐infected moribund pacific white‐leg shrimp Litopenaeus vannamei (Boone 1931) and one strain (V. harveyi) from its diseased postlarva. The cell‐free neutralized supernatant (CFNS) of B2 showed moderate thermo‐stability being stable up to 70°C for 60 min with, however, reducing activity above 80°C for 20 min. B2 antimicrobials showed a stable activity within the pH ranging from 6 to 10 at room temperature and at 4°C, while residual antimicrobial activity of crude CFNS showed tolerance to salinity up to 7% of sodium chloride below 4°C. No B2 activity was obtained while exposed to proteolytic enzyme, such as proteinase k and pepsin, while its activity kept stable exposed to lipase. Initial B2 characterization for antimicrobial substance in CFNS revealed proteinaceous in nature owing to activity loss against proteolytic enzymes and no lipid moiety activity against lipase, which could be categorized as bacteriocin‐like inhibitory substance having potential application against several strains of Vibrio spp. in aquaculture.     

Plasmid‐mediated antimicrobial resistance in motile aeromonads from diseased Nile tilapia (Oreochromis niloticus)

For the sustainable farming of tilapia, proper maintenance of their health and adequate treatment for infections at appropriate time are inevitable. The indiscriminate use of antibiotics in aquaculture, as a part of treatment and as growth promoters, accelerates antimicrobial resistance (AMR) among the fish pathogens. In the present study, we have isolated diverse aeromonads from Nile tilapia and studied their antibiogram and plasmid profiling. Aeromonas hydrophila, Aeromonas veronii, Aeromonas sobria, Aeromonas dhakensis, Aeromonas caviae, Aeromonas jandaei and Aeromonas aquatica were isolated from infected tilapia (n = 150), and their Shannon wiener diversity index was calculated as 1.926. A. veronii was found to be the most multiple antibiotic‐resistant pathogen with the MAR index of 0.46, and A. aquatica was noticed as the least resistant isolate. The minimum inhibitory concentration of resistant antibiotics was shown as >256 mcg/ml for most of the isolates. The virulent genes such as aerolysin and hemolysin were identified in all the isolates except A. aquatica. The detection of class 1 integrons, plasmid profiling and plasmid curing studies confirmed that AMR exhibited by most of the Aeromonas species is of plasmid mediated. This challenges the risk of wide spread of AMR among the pathogens and subsequent treatment of the infection.     

Characterization of two bacteriophages for specific treatment of biofilm formed by a Vibrio sp. isolated from an abalone farm

Vibrio harveyi and related vibrios are potential pathogens causing luminous vibriosis in marine aquaculture systems. In this study, two lytic phages P4A and P4F isolated using Vibrio strains B4A and B4F as indicator bacteria, respectively, were isolated from seawater of an abalone farm. Vibrio strain B4F belongs to the Harveyi clade of the genus Vibrio and was found to cause mortality of abalones in laboratory microcosms. Both phages were able to lyse Vibrio strain B4F. Electron microscopy revealed that phage P4A had an icosahedral head while P4F possessed an elongated hexagonal head. Both phages belong to the family Siphoviridae with long non‐contractile tails. Restriction endonuclease analysis indicated that both phages were double‐stranded DNA viruses and the genome sizes of P4A and P4F were estimated to be about 49 and 44 kb respectively. One‐step growth curves revealed that these two phages exhibited distinct latent periods, exponential periods and burst sizes by infecting the same Vibrio strain B4F. Both phages were able to significantly reduce Vibrio population density in biofilm formed by Vibrio strain B4F on the surface of polyethylene film. It is suggested that these two phages may be promising candidates as biocontrol agents of infections caused by Vibrio strains belonging to the Haveyi clade in marine aquaculture systems.     

Occurrence of Vibrio cholerae and Vibrio parahaemolyticus among milkfish farms in Zanzibar

ABSTRACT

Fishing is among the main economic activities of the people of Zanzibar. Few fish dealers are transforming this sector into mariculture. Among the farmed fish is milkfish. Diseases are among the limiting factors in the development of the mariculture industry. Among other zoonotic diseases, vibriosis is caused by bacteria from the genus Vibrio. This study aimed to establish the occurrence of Vibrio cholerae and Vibrio parahaemolyticus among milkfish farms in Zanzibar. A total of 380 milkfish were sampled. Swabs were collected from gills, intestine, and kidney of each sampled milkfish. Preliminary identification of V. cholerae and V. parahaemolyticus was done by biochemical tests. PCR was run on 16S rRNA, outer membrane protein W, and collagenase genes to confirm Vibrio species, V. cholerae, and V. parahaemolyticus respectively. Almost one-third (32.1%) of all sampled milkfish were found to contain targeted Vibrio; 18% and 29.5% of the sampled milkfish were positive for V. cholerae and V. parahaemolyticus respectively.     

The high prevalence of pathogenic Vibrio harveyi with multiple antibiotic resistance in scale drop and muscle necrosis disease of the hybrid grouper,Epinephelus fuscoguttatus (♀) × E. lanceolatus (♂), in China

Scale drop and muscle necrosis disease with high mortality widely occurred recently in the hybrid grouper (Epinephelus fuscoguttatus ♀ × E. lanceolatus ♂), a crucial cultured marine fish species in China. In this study, 30 Harveyi clade isolates of 27 Vibrio harveyi strains were isolated from diseased hybrid groupers in the south‐east and north‐east coastal areas of China. A total of 22 V. harveyi strains were determined to be pathogenic, and most challenged fish died within 2 days of infection; surviving individuals exhibited scale drop and deep dermal lesions as naturally diseased fish. Although five typical virulence genes, including luxR, toxR_Vh, chiA, serine protease and vhh widely existed in V. harveyi, no obvious correlation was established between virulent strains and virulence genes harboured in them. Furthermore, multiple antibiotic resistance was widely exhibited in Harveyi clade strains, particularly for penicillins, polypeptides, lincomycins, acetylspiramycin, streptomycin, metronidazole and bacitracin. And the multiple antibiotic resistance indices were gradually decreased from southern to northern areas of China. This study demonstrated that the pathogenic V. harveyi with multiple antibiotic resistance is highly prevalent in hybrid grouper in China, which requires particular attention.     

Rapid and sensitive detection of Vibrio harveyi by loop‐mediated isothermal amplification combined with lateral flow dipstick targeted to vhhP2 gene

Vibrio harveyi is a causative agent of the Vibriosis or luminescent bacterial disease in worldwide aquaculture industry. A reliable assay for identification of V. harveyi infection is important to prevent the bacterial spread. In this study, biotinylated loop‐mediated isothermal amplification (LAMP) amplicons were produced by a set of four designed primers that recognized specifically the V. harveyi vhhP2 gene, encoding a putative outer membrane protein with unknown function, followed by hybridization with an fluorescein isothiocyanate (FITC)‐labelled probe and lateral flow dipstick (LFD) detection. A novel set of PCR primer was also designed specifically to vhhP2 gene and appear to be a species‐specific tool for V. harveyi detection. The optimized time and temperature conditions for the LAMP assay were 90 min at 65°C. The LAMP‐LFD and PCR methods accurately identified 22 isolates of V. harveyi but did not detect 16 non‐harveyi Vibrio isolates, and 34 non‐Vibrio bacterial isolates. The sensitivity of LAMP‐LFD for V. harveyi detection in pure culture was 1.1 × 10² CFU mL^?1 or equivalent to 0.6 CFU per reaction, while that of PCR was 6 CFU per reaction. For spiked shrimp sample, the sensitivity of LAMP was 1.8 × 10³ CFU g^?1 or equivalent to 5 CFU per reaction, while that of PCR was 50 CFU per reaction. In conclusion, the established LAMP‐LFD methods provided a valuable tool for rapid identification of V. harveyi and can be used to distinguish V. harveyi from V. campbellii.     

Identification and pathogenicity study of emerging fish pathogens Acinetobacter junii and Acinetobacter pittii recovered from a disease outbreak in Labeo catla (Hamilton, 1822) and Hypophthalmichthys molitrix (Valenciennes, 1844) of freshwater wetland in West Bengal,India

The disease outbreaks in aquaculture system of wetlands are the major cause of fish mortality. Among various bacterial septicaemic diseases, fish mortality caused by Acinetobacter spp. is recently reported in different fish species. Fish disease outbreak was investigated in a wetland of West Bengal, India to identify the aetiological factors involved. The moribund fish were examined and subjected to bacterial isolation. Two bacterial causative agents were identified as Acinetobacter junii and Acinetobacter pittii by biochemical characterization and 16S rRNA gene amplification. Both the isolates were oxidase‐negative, nitrate‐negative, catalase‐positive and indole‐negative. The molecular identification using 16S rRNA gene sequencing and phylogenetic tree analysis further confirmed the two Acinetobacter spp. with 97%–99% similarity. The antibiotic resistance patterns of these two bacteria revealed that both of them were resistant to β‐lactam, cefalexin, cephalothin, amoxyclav, cefuroxime, cefadroxil, clindamycin, vancomycin and penicillin. In addition, A. pittii was also resistant to other antibiotics of cephams group such as ceftazidime and cefotaxime. In the challenge experiment, both A. junii and A. pittii were found to be pathogenic with LD₅₀ of 1.24 × 10⁵ and 1.88 × 10⁷ cfu/fish respectively. Histopathological examination of gill, liver and kidney revealed prominent changes supporting bacterial septicaemia. The investigation reports for the first time on concurrent infection by A. junii and multidrug‐resistant (MDR)‐A. pittii as emerging fish pathogens to cause severe mortality in Labeo catla and Hypophthalmichthys molitrix in a freshwater wetland.     

Vibrogen‐2 vaccine trial in lumpfish (Cyclopterus lumpus) against Vibrio anguillarum

Lumpfish (Cyclopterus lumpus), a native fish of the North Atlantic Ocean, is utilized as cleaner fish to biocontrol sea lice infestations in Atlantic salmon aquaculture. However, bacterial infections are affecting cleaner fish performance. Vibrio anguillarum, the aetiological agent of vibriosis, is one of the most frequent bacterial infections in lumpfish, and effective vaccine programmes against this pathogen have been identified as a high priority for lumpfish. Vibrogen‐2 is a commercial polyvalent bath vaccine that contains formalin‐inactivated cultures of V. anguillarum serotypes O1 and O2, and Vibrio ordalii. In this study, we evaluated Vibrogen‐2 efficacy in lumpfish against a local isolated V. anguillarum strain. Two groups of 125 lumpfish were bath‐immunized, bath‐boost‐immunized at four weeks post‐primary immunization, and intraperitoneally (i.p.) boost‐immunized at eight weeks post‐primary immunization. The control groups were i.p. mock‐immunized with PBS. Twenty‐seven weeks post‐primary immunization, the fish were i.p. challenged with 10 or 100 times the V. anguillarum J360 LD₅₀ dose. After the challenge, survival was monitored daily, and samples of tissues were collected at ten days post‐challenge. Commercial vaccine Vibrogen‐2 reduced V. anguillarum tissue colonization and delayed mortality but did not confer immune protection to C. lumpus against the V. anguillarum i.p. challenge.     

Comparative genomic analysis of five high drug‐resistance Aeromonas hydrophila strains induced by doxycycline in laboratory and nine reference strains in Genbank

Aeromonas hydrophila is an opportunistic pathogen and the leading cause of fatal haemorrhagic septicaemia in fish and shellfish. Doxycycline, one of the second generation tetracyclines, has been used in fish farming to fight against infectious diseases caused by A. hydrophila due to its broad‐spectrum antimicrobial activity and lower cost. However, progressive increase in resistance of Aeromonas strains to doxycycline aroused serious concern. In this report, drug‐resistant A. hydrophilaAH10 strains were induced and selected by using a consecutive batch culture system in Mueller‐Hinton Broth (MHB) supplemented with varying concentrations of doxycycline. Five isolates (AH101‐105) were obtained from the bacterial culture induced by 25 μg/ml doxycycline for drug‐resistance analysis. Minimal inhibitory concentrations (MIC) values of all five isolates were 50 times higher than that of the parental strain AH10. All of them also displayed high‐level resistance to sulphonamides and amides. We sequenced five isolates and performed comparative genomic analysis of these draft genomes with nine A. hydrophila complete genomes from GenBank. Results showed that the pan‐genome of 14 strains contains 4,730 genes, 3,056 genes of which present in all strains. The drug‐resistance genes also showed significant difference in these genomes, which indicated dangers of indiscriminate use of antibiotics in aquaculture and the necessity of understanding the variation of antibiotic resistance of A. hydrophila. Pan‐genome analysis further revealed that no specific SNP (single nucleotide polymorphism) or InDel (insertion and deletion variation) was identified in any functional gene locus among the genomes of AH10 mutated strains, in contrast, significant CNVs (copy number variations) and SV (structure variations) for gene groups were identified in all the mutant genomes.     

Strain and dose infectivity of Vibrio parahaemolyticus: the causative agent of early mortality syndrome in shrimp

Diseases of shrimp have contributed to billions of dollars of economic loss in the aquaculture industry. Newly emerging strains of the bacterium Vibrio parahaemolyticus produce a condition in shrimp called early mortality syndrome or acute hepatopancreatic necrosis disease. Three different V. parahaemolyticus strains were evaluated for their respective pathogenicity on shrimp, Litopenaeus vannamei, when the bacterial strains were grown under various laboratory conditions prior to inoculating shrimp. For each trial, feed was inoculated with a known concentration of bacteria and then fed to the shrimp. The early mortality syndrome strain of V. parahaemolyticus was the most lethal resulting up to 100% mortality within 24 h after being introduced to shrimp via a single feeding. The other two strains of Vibrio, one isolated from the environment and the other from a human clinical case, resulted in 0% and 30% mortality within 96 h respectively. The concentration of the early mortality syndrome strain of V. parahaemolyticus that the shrimp were exposed to directly correlated with mortality rate, which allowed for lethal or sublethal short‐term disease challenge assays to be established. Infiltration of haemocytes was also evident in the midgut caeca of shrimp infected with the early mortality syndrome strain of V. parahaemolyticus, which has not been previously reported.     

Isolation and screening of probiotic candidates from marron,Cherax cainii (Austin, 2002) gastrointestinal tract (GIT) and commercial probiotic products for the use in marron culture

Six strains of bacteria including Bacillus mycoides (A10) and Shewanella species (A12) isolated from healthy marron intestine, Bacillus species (PM1), Bacillus subtilis (PM3), Bacillus sp. (PM4) and Bacillus sp. (AQ) from commercial probiotic products were investigated for probiotic potential in marron culture. Antibiotic susceptibility tests indicated PM3 and PM4 were susceptible to all nine antibiotics evaluated. A10, A12 and AQ were resistant to class penicillins, whereas PM1 was resistant to class penicillin and macrolides. All strains were non‐pathogenic for marron. Strong inhibition against Vibrio mimicus and Vibrio cholerae non‐01 was exhibited by PM4 and PM3. A10 inhibited V. mimicus at 72 h of growth, but not V. cholerae non‐01, whereas A12 inhibited V. cholerae non‐01 but not V. mimicus, and AQ showed no inhibition activity. A wide range of enzymes were produced by A10 and AQ using the API ZYM test. Protease enzymes were produced by PM3, PM4, AQ and PM1. In order of effectiveness, the following bacteria have probiotic potential: B. subtilis (PM3), Bacillus sp. (PM4) and B. mycoides (A10). Further study is required to determine the bacterium or any combination that gives a multibeneficial effect on marron.     

Comparative assessment of Vibrio virulence in marine fish larvae

Vibrionaceae infections are a major obstacle for marine larviculture; however, little is known about virulence differences of Vibrio strains. The virulence of Vibrio strains, mostly isolated from vibriosis outbreaks in farmed fish, was tested in larval challenge trials with cod (Gadus morhua), turbot (Scophthalmus maximus) and halibut (Hippoglossus hippoglossus) using a multiwell dish assays with single‐egg/larvae cultures. The strains differed significantly in virulence as some caused a high mortality of larva reaching 100% mortality after a few days, while others had no or only marginal effects on survival. Some Vibrio strains were pathogenic in all of the larva species, while some caused disease only in one of the species. Twenty‐nine of the Vibrio anguillarum strains increased the mortality of larvae from at least one fish species; however, pathogenicity of the strains differed markedly. Other Vibrio species had no or less pronounced effects on larval mortalities. Iron uptake has been related to V. anguillarum virulence; however, the presence or absence of the plasmid pJM1 encoding anguibactin did not correlate with virulence. The genomes of V. anguillarum were compared (D. Castillo, P.W. D'Alvise, M. Middelboe & L. Gram, unpublished data) and most of the high‐virulent strains had acquired virulence genes from other pathogenic Vibrio.     

Impact of a reduced water salinity on the composition of Vibrio spp. in recirculating aquaculture systems for Pacific white shrimp (Litopenaeus vannamei) and its possible risks for shrimp health and food safety

Tropical shrimp, like Litopenaeus vannamei, in land‐based recirculating aquaculture systems (RAS) are often kept at low water salinities to reduce costs for artificial sea salt and the amount of salty wastewater. Although these shrimp are tolerant against low salinities, innate immunity suppression and changes in the microbial composition in the water can occur. As especially Vibrio spp. are relevant for shrimp health, alterations in the species composition of the Vibrio community were analysed in water from six RAS, run at 15‰ or 30‰. Additionally, pathogenicity factors including pirA/B, VPI, toxR, toxS, vhh, vfh, tdh, trh, flagellin genes and T6SS1/2 of V. parahaemolyticus were analysed. The Vibrio composition differed significantly depending on water salinity. In RAS at 15‰, higher numbers of the potentially pathogenic species V. parahaemolyticus, V. owensii and V. campbellii were detected, and especially in V. parahaemolyticus, various pathogenicity factors were present. A reduced salinity may therefore pose a higher risk of disease outbreaks in shrimp RAS. Because some of the detected pathogenicity factors are relevant for human health, this might also affect food safety. In order to produce healthy shrimp as a safe food for human consumption, maintaining high water salinities seems to be recommendable.     

Chilled storage of banana shrimp (Fenneropenaeus merguiensis) spermatophores with supplementation of moringa (Moringa oleifera Lam.) extract

This study was designed to optimize the chilled storage method for banana shrimp (Fenneropenaeus merguiensis) spermatophores and evaluate the potential of moringa (Moringa oleifera Lam.) extract on the reduction in bacterial contaminants during spermatophore preservation due to the uncertainty of the broodstock. Spermatophores were suspended in five extenders: mineral oil, Ringer's solution, phosphate buffer, calcium‐free saline and 0.8% NaCl and stored at 2–4°C. During a 28‐day storage, spermatophores stored in mineral oil showed the highest sperm viability (89.1%) and intact morphology with a slight formation of hardened adhesive matrices. The effect of moringa extract was investigated on chilled spermatophores. Spermatophores were suspended in mineral oil (the control) and mineral oil containing either penicillin–streptomycin (0.1%) or moringa extract (0.1 mg/ml) during a 28‐day storage. Supplementation of moringa extract resulted in a significant increase (p < .05) in sperm survival, compared to the control, and a complete elimination of culturable Vibrio (Vibrio alginolyticus, Vibrio mimicus and Vibrio furnissii), Staphylococcus kloosii, Bacillus macerans, Listeria ivanovii, Corynebacterium paurometabolum and Corynebacterium bovis, in chilled spermatophores. Chilled storage of spermatophores in mineral oil containing moringa extract was a promising technique due to the inhibition of shrimp and human pathogens without the spermicidal effect on banana shrimp sperm.     

Vibrio harveyi (VirB11) recombinant vaccine development against vibriosis in orange‐spotted grouper (Epinephelus coioides)

Despite significant improvements in aquaculture to compensate wild catch, disease organisms have thrived in limiting its national and global potential. Using antibiotics, in a bid to remedy the havoc, has given rise to complications, attracting attention to disease prevention by immune enhancement against diseases. Grouper production has been inhibited for the threats of bacterial infection, particularly of Vibrio origin. Considering the rise in vibriosis cases, improved vaccines are necessary; moreover, recombinant vaccines, the choice for trial in the present experiment have been effective and more specific in improving immunity. The current work deals with grouper immune system enhancement with a recombinant vaccine developed from VirB11 gene in Vibrio harveyi. VirB11 was cloned in V. harveyi for recombinant vaccine development against vibriosis in orange‐spotted grouper (Epinephelus coioides). As indicated by the results, recombinant VirB11 protein showed effectiveness in conferring protection against vibriosis with observable specific antibody response in enzyme‐linked immunosorbent assay (ELISA) analysis; a significant increase (p < 0.05) in antibody levels was observed after a week and after 8 weeks post‐vaccination. From the weeks post‐vaccination, log2 (antibody titres) in the sera of vaccinated groups reached a peak of 14.2 at week 5 in the vaccinated group in comparison with a peak of approximately 5 and 2 in adjuvant and PBS controls. As indicated by the challenge results, 90% relative survival was observed in vaccinated group and 13% relative survival in control group I (adjuvant control). The cumulative performance of protein concludes VirB11 commendable for recombinant vaccine development.     

Determination and transferability of plasmid‐mediated antibiotic resistance genes of the bacteria isolated from rainbow trout

Antibiotic resistance and the presence of resistance genes (ARGs) were investigated in the bacteria isolated from rainbow trout (Oncorhynchus mykiss) from different trout farms located in Turkey. The most frequent types of antibiotic resistance were towards β‐lactams (cephalothin [70% of bacterial isolates], amoxicillin [63%], ampicillin [62%], ticarcillin [56%], aztreonam [51%]), macrolide [erythromycin, 68%] and sulphonamide [sulphamethoxazole, 51%]. Of bacterial isolates, 51% were multiple drug resistant (MDR), while 35% of the isolates were extensively drug resistant (XDR). None of isolates were pandrug resistant (PDR). The most common ARGs were ampC (36%) and sul1 (24%). The class 1 integron gene cassette was detected in 51% of the bacteria. There was a strong positive correlation between the antibiotic resistance rate and the presence of ARGs (r² = .932). Gene encodes bla_CTX‐M1, one of the extended spectrum beta‐lactamase enzymes, was first described in Aeromonas caviae, Photobacterium damselae, Pseudomonas luteola and Burkholderia cepacia. It was determined that 35% of the bacteria harboured at least one plasmid. Plasmid‐mediated ARGs were identified to be tetracyclines (tetA, tetB, tetC, tetD), sulphonamides (sul1, sul3) and β lactams (ampC, bla_pse). Thus, results suggest that ARG contamination situation deliberates resistance to tetracycline, aminoglycoside, chloramphenicol and sulphonamide. Therefore, the presence and activity of ARGs in fish and in environmental bacteria may play an important role in the spread of resistance genes among bacteria by transposition or integron gene cassettes.     

Virulence‐associated factors in Vibrio cholerae non‐O1/non‐O139 and V. mimicus strains isolated in ornamental fish species

During recent decades, ornamental fish have proven to be one of the fastest growing categories of pets in Europe. In this framework, we evaluated both the potential pathogenic and zoonotic risks caused by 53 Vibrio cholerae non‐O1/non‐O139 and a Vibrio mimicus strain isolated from ornamental fish species mostly originating from South‐East Asia countries between 2000 and 2015 in Italy. All the strains were firstly identified at species level by biochemical, phylogenetic and mass spectrometry (matrix‐assisted laser desorption ionization time of flight) methods, and then studied to reveal the presence of the main virulence and colonization‐associated factors, as ctxA, ace, zot, stn/sto, toxR, rtxA, hlyA and tcpA by multiplex and single endpoint PCR assays. Findings showed that 21 of 54 strains harboured at least one virulence factor with a predominance for the toxR⁺, rtxA⁺ and hlyAET⁺ genotype. Interestingly, the V. mimicus strain harboured the colonization factor and the CTX prophage receptor, tcpA, indicating the ability to capture and integrate it in its genome increasing its pathogenicity. Although these enterotoxins can sporadically cause gastroenteritis, the results highlight their probable involvement in causing severe implications for public health, suggesting the need for an European microbiological monitoring.