Antibiotic resistance trends among Vibrio vulnificus and Vibrio parahaemolyticus isolated from the Chesapeake Bay, Maryland: a longitudinal study.

Antibiotics are often used to treat severe Vibrio infections, with third-generation cephalosporins and tetracyclines combined or fluoroquinolones alone being recommended by the US Centers for Disease Control and Prevention. Increases in antibiotic resistance of both environmental and clinical vibrios are of concern; however, limited longitudinal data have been generated among environmental isolates to inform how resistance patterns may be changing over time. Hence, we evaluated long-term trends in antibiotic resistance of vibrios isolated from Chesapeake Bay waters (Maryland) across two 3-year sampling periods (2009-2012 and 2019-2022). Vibrio parahaemolyticus (n = 134) and Vibrio vulnificus (n = 94) toxR-confirmed isolates were randomly selected from both sampling periods and tested for antimicrobial susceptibility against eight antibiotics using the Kirby-Bauer disk diffusion method. A high percentage (94%-96%) of V. parahaemolyticus isolates from both sampling periods were resistant to ampicillin and only 2%-6% of these isolates expressed intermediate resistance or resistance to third-generation cephalosporins, amikacin, tetracycline, and trimethoprim-sulfamethoxazole. Even lower percentages of resistant V. vulnificus isolates were observed and those were mostly recovered from 2009 to 2012, however, the presence of multiple virulence factors was observed. The frequency of multi-drug resistance was relatively low (6%-8%) but included resistance against antibiotics used to treat severe vibriosis in adults and children. All isolates were susceptible to ciprofloxacin, a fluoroquinolone, indicating its sustained efficacy as a first-line agent in the treatment of severe vibriosis. Overall, our data indicate that antibiotic resistance patterns among V. parahaemolyticus and V. vulnificus recovered from the lower Chesapeake Bay have remained relatively stable since 2009.IMPORTANCEVibrio spp. have historically been susceptible to most clinically relevant antibiotics; however, resistance and intermediate-resistance have been increasingly recorded in both environmental and clinical isolates. Our data showed that while the percentage of multi-drug resistance and resistance to antibiotics was relatively low and stable across time, some Vibrio isolates displayed resistance and intermediate resistance to antibiotics typically used to treat severe vibriosis (e.g., third-generation cephalosporins, tetracyclines, sulfamethoxazole-trimethoprim, and aminoglycosides). Also, given the high case fatality rates observed with Vibrio vulnificus infections, the presence of multiple virulence factors in the tested isolates is concerning. Nevertheless, the continued susceptibility of all tested isolates against ciprofloxacin, a fluoroquinolone, is indicative of its use as an effective first-line treatment of severe Vibrio spp. infections stemming from exposure to Chesapeake Bay waters or contaminated seafood ingestion.

Vertical dynamics of free-living and particle-associated vibrio communities in the eastern tropical Indian Ocean.

Members of the family Vibrionaceae (vibrios) are widely distributed in estuarine, offshore, and marginal seas and perform an important ecological role in the marine organic carbon cycle. Nevertheless, there is little knowledge about whether vibrios play ecological roles in the oligotrophic pelagic area, which occupies a larger water volume. In this study, we investigated the abundance, diversity, and composition of free-living and particle-associated vibrios and their relationships with environmental factors along the water depth in the eastern tropical Indian Ocean (ETIO). The abundance of vibrios in free-living fractions was significantly higher than that of particle-associated fractions on the surface. Still, both were similar at the bottom, indicating that vibrios may shift from free-living lifestyles on the surface to mixed lifestyles at the bottom. Vibrio-specific 16S rRNA gene amplicon sequencing revealed that Paraphotobacterium marinum and Vibrio rotiferianus were dominant species in the water column, and Vibrio parahaemolyticus (a clinically important pathogen) was recorded in 102 samples of 111 seawater samples in 10 sites, which showed significant difference from the marginal seas. The community composition also shifted, corresponding to different depths in the water column. Paraphotobacterium marinum decreased with depth, and V. rotiferianus OTU1528 was mainly distributed in deeper water, which significantly correlated with the alteration of environmental factors (e.g., temperature, salinity, and dissolved oxygen). In addition to temperature and salinity, dissolved oxygen (DO) was an important factor that affected the composition and abundance of Vibrio communities in the ETIO. Our study revealed the vertical dynamics and preferential lifestyles of vibrios in the ETIO, helping to fill a knowledge gap on their ecological distribution in oligotrophic pelagic areas and fully understanding the response of vibrios in a global warming environment.

Bacillus changyiensis sp. nov., isolated from coastal sediment.

Two Gram-stain-positive, facultatively anaerobic, motile, endospore-forming, rod-shaped bacteria, designated CLL-3-40T and CLL-7-23, were isolated from coastal sediment sampled in Changyi, Shandong Province, PR China. Phylogenetic analysis based on 16S rRNA gene sequences indicated that these strains were related to the genus Bacillus and close to six type strains of species within the Bacillus licheniformis group. In phenotypic characterization tests, strain CLL-3-40T could grow at 15-50 °C (optimum, 37 °C) and in media with pH 5-9 (optimum pH 7.0), and tolerate up to 12 % (w/v) NaCl. The fermentation broth supernatant extracted by ethyl acetate of strain CLL-3-40T could inhibit aquaculture pathogenic vibrios. The predominant cellular fatty acids of strain CLL-3-40T were anteiso-C15 : 0 (30.7 %) and iso-C15 : 0 (31.5 %); the peptidoglycan from cell-wall contained meso-diaminopimelic acid; the predominant quinone was menaquinone 7; and the major polar lipids were diphosphatidylglycerol, phosphatidylcholine, phosphatidylglycerol, phosphatidylethanolamine, an unidentified glycolipid and two unidentified phospholipids. The digital DNA-DNA hybridization values and average nucleotide identities among strains CLL-3-40T and CLL-7-23 and their close type strains were less than 21.9 and 48.4 %, respectively, thereby indicating that strain CLL-3-40T should represent a novel species of the genus Bacillus. The genomic DNA G+C contents were 38.4 mol% in strain CLL-3-40T and 38.3 mol% in strain CLL-7-23. The 12 biosynthetic gene clusters of strain CLL-3-40T were predicted based on results from the online server antiSMASH. Based upon the consensus of phenotypic and genotypic results, strain CLL-3-40T should be classified as representing a novel species of the genus Bacillus, for which the name Bacillus changyiensis sp. nov. is proposed. The type strain is CLL-3-40T (= MCCC 1A14857T=JCM 35755T).

Population response of intestinal microbiota to acute Vibrio alginolyticus infection in half-smooth tongue sole (Cynoglossus semilaevis).

Introduction: Vibriosis causes enormous economic losses of marine fish. The present study investigated the intestinal microbial response to acute infection of half-smooth tongue sole with different-dose Vibrio alginolyticus within 72 h by metagenomic sequencing. Methods: The inoculation amount of V. alginolyticus for the control, low-dose, moderate-dose, and high-dose groups were 0, 8.5 × 101, 8.5 × 104, and 8.5 × 107 cells/g respectively, the infected fish were farmed in an automatic seawater circulation system under a relatively stable temperature, dissolved oxygen and photoperiod, and 3 ~ 6 intestinal samples per group with high-quality DNA assay were used for metagenomics analysis. Results: The acute infections with V. alginolyticus at high, medium, and low doses caused the change of different-type leukocytes at 24 h, whereas the joint action of monocytes and neutrophils to cope with the pathogen infection only occurred in the high-dose group at 72 h. The metagenomic results suggest that a high-dose V. alginolyticus infection can significantly alter the intestinal microbiota, decrease the microbial α-diversity, and increase the bacteria from Vibrio and Shewanella, including various potential pathogens at 24 h. High-abundance species of potential pathogens such as V. harveyii, V. parahaemolyticus, V. cholerae, V. vulnificus, and V. scophthalmi exhibited significant positive correlations with V. alginolyticus. The function analysis revealed that the high-dose inflection group could increase the genes closely related to pathogen infection, involved in cell motility, cell wall/ membrane/envelope biogenesis, material transport and metabolism, and the pathways of quorum sensing, biofilm formation, flagellar assembly, bacterial chemotaxis, virulence factors and antibiotic resistances mainly from Vibrios within 72 h. Discussion: It indicates that the half-smooth tongue sole is highly likely to be a secondary infection with intestinal potential pathogens, especially species from Vibrio and that the disease could become even more complicated because of the accumulation and transfer of antibiotic-resistance genes in intestinal bacteria during the process of V. alginolyticus intensified infection.

Genome Characterization and Infectivity Potential of Vibriophage-ϕLV6 with Lytic Activity against Luminescent Vibrios of Penaeus vannamei Shrimp Aquaculture.

Shrimp aquaculture, especially during the hatchery phase, is prone to economic losses due to infections caused by luminescent vibrios. In the wake of antimicrobial resistance (AMR) in bacteria and the food safety requirements of farmed shrimp, aqua culturists are seeking alternatives to antibiotics for shrimp health management, and bacteriophages are fast emerging as natural and bacteria-specific antimicrobial agents. This study analyzed the whole genome of vibriophage-ϕLV6 that showed lytic activity against six luminescent vibrios isolated from the larval tanks of P. vannamei shrimp hatcheries. The Vibriophage-ϕLV6 genome was 79,862 bp long with 48% G+C content and 107 ORFs that coded for 31 predicted protein functions, 75 hypothetical proteins, and a tRNA. Pertinently, the vibriophage-ϕLV6 genome harbored neither AMR determinants nor virulence genes, indicating its suitability for phage therapy. There is a paucity of whole genome-based information on vibriophages that lyse luminescent vibrios, and this study adds pertinent data to the database of V. harveyi infecting phage genomes and, to our knowledge, is the first vibriophage genome report from India. Transmission electron microscopy (TEM) of vibriophage-ϕLV6 revealed an icosahedral head (~73 nm) and a long, flexible tail (~191 nm) suggesting siphovirus morphology. The vibriophage-ϕLV6 phage at a multiplicity of infection (MOI) of 80 inhibited the growth of luminescent V. harveyi at 0.25%, 0.5%, 1%, 1.5%, 2%, 2.5%, and 3% salt gradients. In vivo experiments conducted with post-larvae of shrimp showed that vibriophage-ϕLV6 reduced luminescent vibrio counts and post-larval mortalities in the phage-treated tank compared to the bacteria-challenged tank, suggesting the potentiality of vibriophage-ϕLV6 as a promising candidate in treating luminescent vibriosis in shrimp aquaculture. The vibriophage-ϕLV6 survived for 30 days in salt (NaCl) concentrations ranging from 5 ppt to 50 ppt and was stable at 4 °C for 12 months.

Crassostrea gigas oysters from a non-intensive farming area naturally harbor potentially pathogenic vibrio strains.

Farming intensification and climate change are inevitably linked to pathogen emergence in aquaculture. In this context, infectious diseases associated with vibrios span all developmental stages of the Pacific Oyster Crassostrea gigas. Moreover, virulence factors associated with pathogenicity spread among the vibrio community through horizontal gene transfer as part of the natural eco-evolutive dynamic of this group. Therefore, risk factors associated with the emergence of pathogens should be assessed before the appearance of mass mortalities in developing rearing areas. In this context, we characterized the vibrios community associated with oysters cultured in a non-intensive area free of massive mortalities located at Tongoy bay, Chile, through a culture-dependent approach. We taxonomically affiliated our isolates at the species level through the partial sequencing of the heat shock protein 60 gene and estimated their virulence potential through experimental infection of juvenile C. gigas. The vibrio community belonged almost entirely to the Splendidus clade, with Vibrio lentus being the most abundant species. The virulence potential of selected isolates was highly contrasted with oyster survival ranging between 100 and 30 %. Moreover, different vibrio species affected oyster survival at different rates, for instance V. splendidus TO2_12 produced most mortalities just 24 h after injection, while the V. lentus the most virulent strain TO6_11 produced sustained mortalities reaching 30 % of survival at day 4 after injection. Production of enzymes associated with pathogenicity was detected and hemolytic activity was positive for 50 % of the virulent strains and negative for 90 % of non-virulent strains, representing the phenotype that better relates to the virulence status of strains. Overall, results highlight that virulence is a trait present in the absence of disease expression, and therefore the monitoring of potentially pathogenic groups such as vibrios is essential to anticipate and manage oyster disease emergence in both established and under-development rearing areas.

Functional proteomics identify mannitol metabolism in serum resistance and therapeutic implications in Vibrio alginolyticus.

Serum resistance is recognized as one of the most important pathogenic traits of bacterial pathogens, and no control measure is available. Based on our previous discovery that pathogenic Escherichia coli represses glycine, serine, and threonine metabolism to confer serum resistance and that the reactivation of this pathway by exogenous glycine could restore serum sensitivity, we further investigate the mechanism underlying the action of glycine in Vibrio alginolyticus. Thus, V. alginolyticus is treated with glycine, and the proteomic change is profiled with tandem mass tag-based quantitative proteomics. Compared to the control group, glycine treatment influences the expression of a total of 291 proteins. Among them, a trap-type mannitol/chloroaromatic compound transport system with periplasmic component, encoded by N646_0992, is the most significantly increased protein. In combination with the pathway enrichment analysis showing the altered fructose and mannitol metabolism, mannitol has emerged as a possible metabolite in enhancing the serum killing activity. To demonstrate this, exogenous mannitol reduces bacterial viability. This synergistic effect is further confirmed in a V. alginolyticus-Danio rerio infection model. Furthermore, the mechanism underlying mannitol-enabled serum killing is dependent on glycolysis and the pyruvate cycle that increases the deposition of complement components C3b and C5b-9 on the bacterial surface, whereas inhibiting glycolysis or the pyruvate cycle significantly weakened the synergistic effects and complement deposition. These data together suggest that mannitol is a potent metabolite in reversing the serum resistance of V. alginolyticus and has promising use in aquaculture.

Predictive Intelligence for Cholera in Ukraine?

Cholera, an ancient waterborne diarrheal disease, remains a threat to public health, especially when climate/weather processes, microbiological parameters, and sociological determinants intersect with population vulnerabilities of loss of access to safe drinking water and sanitation infrastructure. The ongoing war in Ukraine has either damaged or severely crippled civil infrastructure, following which the human population is at risk of health disasters. This editorial highlights a perspective on using predictive intelligence to combat potential (and perhaps impending) cholera outbreaks in various regions of Ukraine. Reliable and judicious use of existing earth observations inspired mathematical algorithms integrating heuristic understanding of microbiological, sociological, and weather parameters have the potential to save or reduce the disease burden.

Exploring the dynamics of toxigenic environmental Vibrio mimicus and its comparative analysis with Vibrio cholerae of the southern Gangetic delta.

Vibrio mimicus and Vibrio cholerae are closely related species. Environmental V.mimicus were comparatively analyzed with V.cholerae, for the presence of virulence genes, antibiotic susceptibility, resistance genes, in-vitro hemolysis, and biofilm formation. Phylogenetic analysis was performed depending on toxin-gene disposition and isolation area. One V.mimicus isolate harbored ctxA, tcp El-Tor, toxT and toxS, whereas several strains contained incomplete copies of virulence cassettes and associated toxin genes. V.cholerae isolates harbored ctx, tcp and toxT genes, with a higher preponderance of hlyA, rtxA and toxR genes. V.mimicus were highly sensitive to amino/carboxy-penicillins, furazolidone & gentamycin, with quinolone & tetracycline resistance genes. V.cholerae isolates were sensitive to penicillins and cephalosporins, with 29% of the strains bearing the sxt gene. Phylogenetically, the apomorphic strains of both species were unique to the inland sites. V.cholerae has embodied an enormous public health burden globally but our findings emphasize the role of V.mimicus as an emerging etiological agent with similar epidemic potential.

An Integrative, Multiparametric Approach for the Comprehensive Assessment of Microbial Quality and Pollution in Aquaculture Systems.

As the aquaculture sector significantly expanded worldwide in the past decades, the concept of sustainable aquaculture has developed with the challenge of not only maximizing benefits but also minimizing the negative impacts on the environment assuring, at the same time, food security. In this framework, monitoring and improving the microbiological water quality and animal health are a central topic. In the present study, we evaluated the seawater microbiological quality in a mariculture system located in a Mediterranean coastal area (Northern Ionian Sea, Italy). We furnished, for the first time, a microbial inventory based on conventional culture-based methods, integrated with the 16S rRNA gene metabarcoding approach for vibrios identification and diversity analyses, and further implemented with microbial metabolic profiling data obtained from the Biolog EcoPlate system. Microbiological pollution indicators, vibrios diversity, and microbial metabolism were determined in two different times of the year (July and December). All microbial parameters measured in July were markedly increased compared to those measured in December. The presence of potentially pathogenic vibrios is discussed concerning the risk of fish disease and human infections. Thus, the microbial inventory here proposed might represent a new multiparametric approach for the suitable surveillance of the microbial quality in a mariculture system. Consequently, it could be useful for ensuring the safety of both the reared species and the consumers in the light of sustainable, eco-friendly aquaculture management.

Non-O1/non-O139 vibrios - occurrence not only in Europe in recent years.

Non-O1/non-O139 vibrios refer to all vibrios except toxin producing Vibrio cholerae serogroups O1 and O139. The prevalence of illness caused by non-O1/non-O139 vibrios steadily increases all over the world in the last 20 years, which is very probably related to global warming. These infections are reported year-round from tropical and subtropical climate zones, but they were also detected in the mild climate zone of the United States of America and Europe. In mild climate, they have markedly seasonal occurrence, typically peaking in May to October. A human can be infected after ingestion of contaminated food, especially seafood and fish, or water or while bathing. In Europe, non-O1/non-O139 vibrios were detected in the Baltic Sea, North Sea and Mediterranean Sea but also in ponds and rivers. Depending on the pathogen entry route, the clinical manifestation may appear as gastroenteritis, otitis, wound infection or severe up to fatal illness, predominantly in immunocompromised patients. There is no specific prevention. Non-specific prevention includes good personal and food handling hygiene practices and avoiding contact of unhealed wounds with sea or surface swimming water. Given the severity and increasing frequency of infections caused by non-O1/non-O139 vibrios, they should be considered in differential diagnosis of gastrointestinal and wound infections, especially in patients with a history of consumption of fish and seafood or with a history of contact of unhealed wounds with sea or other open swimming water.

Indian coastal waters: a concoction of sewage indicator bacteria! An assessment on recreational beaches.

Coastal water quality assessment is important to maintain a healthy environment for various uses including fisheries and recreation. Microbial populations are used as biological indicators of contamination to monitor water quality and are considered by the government to be one of the critical features for issuing safety guidelines. Different bacterial groups (pathogenic, vibrio and faecal) from five major recreational beaches of Chennai, India, were monitored for the assessment of coastal water quality. Faecal coliforms (FC) were high at all the beaches, with up to 4.2 × 105 CFU/mL and exceeding the normal standard limits of 100 CFU/100 mL set by the Central Pollution Control Board (CPCB) of India. Rainfall was found to have a role in the variability and distribution of indicator and pathogenic bacteria. The seasonal dry period witnessed elevated FC, while dilution in the wet period reduced Escherichia coli-like organisms (ECLO). High microbial counts were detected near the beach situated close to the river mouth, mainly due to discharges of untreated domestic sewage and industrial wastes. Similarly, the biological oxygen demand (BOD) was also high, 0.32 to 10.32 mg/L. Dissolved inorganic nitrogen (DIN) ranged from 2.21 to 134.53 µmol/L and inorganic phosphate (IP) ranged from 0 to 57.53 µmol/L. These values indicated the presence of significant untreated sewage in the coastal water. This study revealed that Chennai coastal waters carry high levels of faecal and pathogenic bacteria, detrimental for recreational and other contact activities. The quantitative and qualitative analyses will be useful for modelling and prediction of coastal water quality and management of other recreational beaches in India.

Prevalence and Characterization of Food-Borne Vibrio parahaemolyticus From African Salad in Southern Nigeria.

The demand for minimally processed vegetables (African salad) has increased partly due to its inclusion in ready-to-eat foods. Nevertheless, the associated risk of the presence of emergent foodborne pathogens, such as Vibrio parahaemolyticus might be underestimated. The present study was designed to isolate and characterize foodborne V. parahaemolyticus from minimally processed vegetables using culture-based methods and molecular approach. A total of 300 samples were examined from retail outlets between November 2018 and August 2019 from Southern Nigeria. The prevalence of vibrios from the overall samples based on the colonial proliferation of yellow, blue-green and/or green colonies on thiosulfate citrate bile salts sucrose agar was 74/300 (24.6%). An average of two green or blue-green colonies from respective plates was screened for V. parahaemolyticus using analytical profile index (API) 20 NE. Polymerase chain reaction further confirmed the identity of positive V. parahaemolyticus. The counts of V. parahaemolyticus ranged from 1.5 to 1,000 MPN/g. A total of 63 recovered V. parahaemolyticus were characterized further. The resistance profile of the isolates include ampicillin 57/63 (90.5%), cefotaxime 41/63 (65.1%), ceftazidime 30/63 (47.6%), amikacin 32/63 (50.8%), kanamycin 15/63 (23.8%), and oxytetracycline 16/63 (25.4%). The multiple antibiotic index ranged from 0-0.81. The formation of biofilm by the isolates revealed the following: strong formation 15/63 (23.8%), moderate formation 31/63 (49.2%), weak formation 12/63 (19.1%), and no formation 5/63 (7.9%). A total of 63/63 (100%), 9/63 (14.3%), and 20/63 (31.8%) of the isolates harbored the tox R gene, TDH-related hemolysin (trh) and thermostable direct hemolysin (tdh) determinants respectively. The isolates with O2 serogroup were most prevalent via PCR. Isolates that were resistant to tetracycline, kanamycin, and chloramphenicol possessed resistant genes. The presence of multidrug-resistant vibrios in the minimally processed vegetables constitutes a public health risk and thus necessitates continued surveillance.

-Blue mussel (Mytilus spp.) cultivation in mesohaline eutrophied inner coastal waters: mitigation potential, threats and cost effectiveness.

The EU-water framework directive (WFD) focuses on nutrient reductions to return coastal waters to the good ecological status. As of today, many coastal waters have reached a steady state of insufficient water quality due to continuous external nutrient inputs and internal loadings. This study focuses first on the current environmental status of mesohaline inner coastal waters to illustrate their needs of internal measures to reach demanded nutrient reductions and secondly, if mussel cultivation can be a suitable strategy to improve water quality. Therefore, nitrogen, phosphorus, chlorophyll a, and Secchi depth of nine mesohaline inner coastal waters in north east Germany were analyzed from 1990 to 2018. Two pilot mussel farms were used to evaluate their effectiveness as a mitigation measure and to estimate potential environmental risks, including the interactions with pathogenic vibrio bacteria. Further, estimated production and mitigation potential were used to assess economic profitability based on the sale of small sized mussels for animal feed and a compensation for nutrient mitigation. The compensation costs were derived from nutrient removal costs of a waste water treatment plant (WWTP). Results show that currently all nine water bodies do not reach the nutrient thresholds demanded by the WFD. However, coastal waters differ in nutrient pollution, indicating that some can reach the desired threshold values if internal measures are applied. The mitigation potential of mussel cultivation depends on the amount of biomass that is cultivated and harvested. However, since mussel growth is closely coupled to the salinity level, mussel cultivation in low saline environments leads to lower biomass production and inevitably to larger cultivation areas. If 50% of the case study area Greifswald Bay was covered with mussel farms the resulting nitrogen reduction would increase Secchi depth by 7.8 cm. However, high chlorophyll a values can hamper clearance rates (<20 mg m-3 = 0.43 l h-1 dry weight g-1) and therefore the mitigation potential. Also, the risk of mussel stock loss due to high summer water temperatures might affect the mitigation potential. The pilot farms had no significant effect on the total organic content of sediments beneath. However, increased values of Vibrio spp. in bio deposits within the pilot farm (1.43 106 ± 1.10 106CFU 100 ml-1 (reference site: 1.04 106 ± 1.45 106 CFU 100 ml-1) were measured with sediment traps. Hence, mussel farms might act as a sink for Vibrio spp. in systems with already high vibrio concentrations. However, more research is required to investigate the risks of Vibrio occurrence coupled to mussel farming. The economic model showed that mussel cultivation in environments below 12 PSU cannot be economic at current market prices for small size mussels and compensations based on nutrient removal cost of WWTPs.

Oceanic Hitchhikers - Assessing Pathogen Risks from Marine Microplastic.

As plastic debris in the environment continues to increase, an emerging concern is the potential for microplastic to act as vectors for pathogen transport. With aquaculture the fastest growing food sector, and microplastic contamination of shellfish increasingly demonstrated, understanding any risk of pathogen transport associated with microplastic is important for this industry. However, there remains a lack of detailed, systematic studies assessing the interactions and potential impacts that the attachment of human and animal pathogens on microplastic may have. Here we synthesise current knowledge regarding these distinct microplastic-associated bacterial communities and microplastic uptake pathways into bivalves, and discuss whether they represent a human and animal health threat, highlighting the outstanding questions critical to our understanding of this potential risk to food safety.

Type III Secretion 1 Effector Gene Diversity Among Vibrio Isolates From Coastal Areas in China.

Vibrios, which include more than 120 valid species, are an abundant and diverse group of bacteria in marine and estuarine environments. Some of these bacteria have been recognized as pathogens of both marine animals and humans, and therefore, their virulence mechanisms have attracted increasing attention. The type III secretion system (T3SS) is an important virulence determinant in many gram-negative bacteria, in which this system directly translocates variable effectors into the host cytosol for the manipulation of the cellular responses. In this study, the distribution of the T3SS gene cluster was first examined in 110 Vibrio strains of 26 different species, including 98 strains isolated from coastal areas in China. Several T3SS1 genes, but not T3SS2 genes (T3SS2α and T3SS2ß), were universally detected in all the strains of four species, Vibrio parahaemolyticus, Vibrio alginolyticus, Vibrio harveyi, and Vibrio campbellii. The effector coding regions within the T3SS1 gene clusters from the T3SS1-positive strains were further analyzed, revealing that variations in the effectors of Vibrio T3SS1 were observed among the four Vibrio species, even between different strains in V. harveyi, according to their genetic organization. Importantly, Afp17, a potential novel effector that may exert a similar function as the known effector VopS in T3SS1-induced cell death, based on cytotoxicity assay results, was found in the effector coding region of the T3SS1 in some V. harveyi and V. campbellii strains. Finally, it was revealed that differences in T3SS1-mediated cytotoxicity were dependent not only on the variations in the effectors of Vibrio T3SS1 but also on the initial adhesion ability to host cells, which is another prerequisite condition. Altogether, our results contribute to the clarification of the diversity of T3SS1 effectors and a better understanding of the differences in cytotoxicity among Vibrio species.

Resistome, Mobilome and Virulome Analysis of Shewanella algae and Vibrio spp. Strains Isolated in Italian Aquaculture Centers.

Antimicrobial resistance is a major public health concern restricted not only to healthcare settings but also to veterinary and environmental ones. In this study, we analyzed, by whole genome sequencing (WGS) the resistome, mobilome and virulome of 12 multidrug-resistant (MDR) marine strains belonging to Shewanellaceae and Vibrionaceae families collected at aquaculture centers in Italy. The results evidenced the presence of several resistance mechanisms including enzyme and efflux pump systems conferring resistance to beta-lactams, quinolones, tetracyclines, macrolides, polymyxins, chloramphenicol, fosfomycin, erythromycin, detergents and heavy metals. Mobilome analysis did not find circular elements but class I integrons, integrative and conjugative element (ICE) associated modules, prophages and different insertion sequence (IS) family transposases. These mobile genetic elements (MGEs) are usually present in other aquatic bacteria but also in Enterobacteriaceae suggesting their transferability among autochthonous and allochthonous bacteria of the resilient microbiota. Regarding the presence of virulence factors, hemolytic activity was detected both in the Shewanella algae and in Vibrio spp. strains. To conclude, these data indicate the role as a reservoir of resistance and virulence genes in the environment of the aquatic microbiota present in the examined Italian fish farms that potentially might be transferred to bacteria of medical interest.

Live Feeds Used in the Larval Culture of Red Cusk Eel, Genypterus chilensis, Carry High Levels of Antimicrobial-Resistant Bacteria and Antibiotic-Resistance Genes (ARGs).

The culture of red cusk eel Genypterus chilensis is currently considered a priority for Chilean aquaculture but low larval survival rates have prompted the need for the continuous use of antibacterials. The main aim of this study was to evaluate the role of live feed as a source of antibacterial-resistant bacteria in a commercial culture of G. chilensis. Samples of rotifer and Artemia cultures used as live feed were collected during the larval growth period and culturable bacterial counts were performed using a spread plate method. Rotifer and Artemia cultures exhibited high levels of resistant bacteria (8.03 × 104 to 1.79 × 107 CFU/g and 1.47 × 106 to 3.50 × 108 CFU/g, respectively). Sixty-five florfenicol-resistant isolates were identified as Vibrio (81.5%) and Pseudoalteromonas (15.4%) using 16S rRNA gene sequence analysis. A high incidence of resistance to streptomycin (93.8%), oxytetracycline (89.2%), co-trimoxazole (84.6%), and kanamycin (73.8%) was exhibited by resistant isolates. A high proportion of isolates (76.9%) carried the florfenicol-resistance encoding genes floR and fexA, as well as plasmid DNA (75.0%). The high prevalence of multiresistant bacteria in live feed increases the incidence of the resistant microbiota in reared fish larvae, thus proper monitoring and management strategies for live feed cultures appear to be a priority for preventing future therapy failures in fish larval cultures.

Shewanella algae and Vibrio spp. strains isolated in Italian aquaculture farms are reservoirs of antibiotic resistant genes that might constitute a risk for human health.

The aquatic environment can represent a reservoir of antimicrobial resistance genes. In the present study, phenotypical, biochemical and molecular techniques were used to screen a collection of marine strains isolated in Italian aquaculture farms to investigate their beta-lactam resistance profiles. The genome of 12 carbapenemase and/or beta-lactamase producing strains was sequenced and a phylogenetic analysis of the beta-lactamases found in their chromosomes was performed. Gene annotation and prediction revealed the presence of blaAmpC and blaOXA-55-like in all the Shewanella algae isolates whereas in Vibrio anguillarum and Vibrio parahaemolyticus strains, blaAmpC and blaCARB-19 were found, respectively. Multiple alignments of OXA-55-like and AmpC protein sequences showed different point mutations. Finally, comparisons between enzyme phylogeny and strain clusterization based on sampling sites and dates indicate the diffusion of specific Multi Drug Resistant (MDR) Shewanella algae clones along the Italian Adriatic coast.

New tetrodotoxin analogs in Brazilian pufferfishes tissues and microbiome.

While tetrodotoxin (TTX) is commonly found in pufferfish tissues, it is unclear if bacterial symbionts isolated from pufferfish tissues can produce TTX. In this investigation, UPLC qTOF-MS/MS analysis of tissue extracts obtained from Sphoeroides spengleri and Canthigaster figuereidoi identified TTX in their composition, indicating their consumption is unsafe. UPLC qTOF-MS/MS analysis coupled with Molecular Networking indicated new TTX analogs (methyl-TTX, TTX-acetate, hydroxypropyl-TTX and glycerol-TTX). Bacterial extracts from sixteen strains revealed a compound with a [M+H]+ ion at m/z 320.1088, identical to TTX. However, TTX itself was not detected in these cultures by UPLC-MS/MS. Neurotoxicity of Vibrio A665 purified fraction 2 (with precursor [M+H]+ ion at m/z 320.1088) was significant in human neural stem cells (hNSCs), but the Nav blockage activity was not confirmed by the veratridine/ouabain essays, indicating a possible difference in the mechanism of action between the bacterium A665 purified fraction 2 and TTX. Vibrios symbionts of pufferfish point out involving in the production of TTX precursors.