Resistance and genomic characterization of a plasmid pkh2101 harbouring erm(B) isolated from emerging fish pathogen Lactococcus garvieae serotype II in Japan.

The emergence of antibiotic-resistant pathogenic strains of Lactococcus garvieae serotype II isolated from fish in Japan has become a growing concern in recent years. The data on drug susceptibility and its associated resistance mechanism are limited. Therefore, the present study was conducted to determine the minimum inhibitory concentrations (MICs) of chemotherapeutic agents against 98 pathogenic strains of emerging Lactococcus garvieae serotype II isolated from fish from six different prefectures in Japan from 2018 to 2021. The tested strains were resistant to erythromycin, lincomycin and tiamulin. PCR amplification revealed the presence of erm(B) in all erythromycin-resistant strains, while a conjugation experiment confirmed that these strains carried erm(B) that could be transferred to recipient Enterococcus faecalis OG1RF with frequencies from 10-4 to 10-6 per donor cells. Nucleotide sequencing of the representative isolated plasmid pkh2101 from an erythromycin-resistant strain showed that it was a 26,850 bp molecule with an average GC content of 33.49%, comprising 31 CDSs, 13 of which remained without any functional annotation. Comparative genomic analysis suggested that pkh2101 shared the highest similarity (97.57% identity) with the plasmid pAMbeta1, which was previously isolated clinically from Enterococcus faecalis DS-5. This study provides potential evidence that the plasmid harbouring erm(B) could be a source of antibiotic resistance transmission in emerging L. garvieae infection in aquaculture.

The environmental effect on the seabed of an offshore marine fish farm in the tropical Pacific.

Marine aquaculture is expanding offshore, where the environmental interactions are not yet fully understood. We performed a benthic environmental assessment of an offshore fish farm on unconsolidated sediment. The physicochemical variables showed marked changes just under the fish farm, although the structure of the community and its bioturbation potential were not influenced. Under no or minimum influence from the fish farm, the physicochemical variables, including acid-volatile sulphides and redox, were notably different to those found in unaffected coastal areas. For this reason, classifications of the environmental status based on physicochemical variables should be adapted to offshore areas. Despite the low degree of impact detected, the organic matter carrying capacity should be carefully determined to avoid environmental drawbacks in terms of fine-grained offshore sediments. Offshore aquaculture could have a lower environmental impact than other types of aquaculture located closer to the coast, but further research is needed to obtain conclusive results.

The intestinal bacterial community of healthy and diseased animals and its association with the aquaculture environment.

Although increasing levels of attention have been targeted towards aquaculture-associated bacteria, the bacterial community of animal intestines and its relationship with the aquaculture environment need to be further investigated. In this study, we used high-throughput sequencing to analyze the bacterial community of pond water, sediment, and the intestines of diseased and healthy animals. Our data showed that Proteobacteria, Firmicutes, Cyanobacteria, and Bacteroidetes were the dominant taxa of bacteria across all samples and accounted for more than 90% of the total sequence. Difference analysis and Venn diagrams showed that most of the intestinal bacterial OTUs (operational taxonomic units) of diseased and healthy animals were the same as those of sediment and water, indicating that the aquaculture environment was the main source of intestinal bacteria. Compared with healthy animals, a considerable reduction of OTUs was evident in diseased animals. Welch's t test showed that the dominant bacterial taxa in sediment, water, and animal intestine were significantly different (p < 0.05) and each had its own unique dominant microorganisms. In addition, differences between the intestinal bacteria of healthy and diseased animals were represented by potential probiotics and pathogens, such as Bacillus, Vibrio, Oceanobacillus, and Lactococcus. Principal component analysis (PcoA) showed that a similar environment shaped a similar microbial structure. There was a large difference in the spectrum of intestinal bacteria in diseased animals; furthermore, the spectrum of intestinal bacteria in diseased animals was very different from the environment than in healthy animals. This study provides a theoretical basis for a relationship between the intestinal bacteria of healthy and diseased animals and the environment and provides guidance for environmental regulation and disease prevention in aquaculture areas.

Antibiotic resistance of Vibrio parahaemolyticus and Vibrio vulnificus in various countries: A review.

Vibrio parahaemolyticus and Vibrio vulnificus are the leading causes of seafood associated infections and mortality in the United States. The main syndromes caused by these pathogens are gastroenteritis, wound infections, and septicemia. This article reviewed the antibiotic resistance profile of V. parahaemolyticus and V. vulnificus in the United States and other countries including Italy, Brazil, Philippines, Malaysia, Thailand, China, India, Iran, South Africa and Australia. The awareness of antimicrobial resistance of these two pathogens is not as well documented as other foodborne bacterial pathogens. Vibrio spp. are usually susceptible to most antimicrobials of veterinary and human significance. However, many studies reported that V. vulnificus and V. parahaemolyticus showed multiple-antibiotic resistance due to misuse of antibiotics to control infections in aquaculture production. In addition, both environmental and clinical isolates showed similar antibiotic resistance profiles. Most frequently observed antibiotic resistance profiles involved ampicillin, penicillin and tetracycline regardless of the countries. The presence of multiple-antibiotic resistant bacteria in seafood and aquatic environments is a major concern in fish and shellfish farming and human health.

Occurrence, distribution, and risk assessment of antibiotics in a typical aquaculture area around the Dongzhai Harbor mangrove forest on Hainan Island.

The trees of the Dongzhai Harbor mangrove forest suffer from antibiotic contamination from surrounding aquaculture areas. Despite this being one of the largest mangrove forests in China, few studies have focused on the antibiotic pollution status in these aquaculture areas. In the present study, the occurrence, distribution, and risk assessment of 37 antibiotics in surface water and sediment samples from aquaculture areas around Dongzhai Harbor mangrove forests were analyzed. The concentration of total antibiotics (∑antibiotics) ranged from 78.4 ng/L to 225.6 ng/L in surface water (except S14-A2) and from 19.5 ng/g dry weight (dw) to 229 ng/g dw in sediment. In the sediment, the concentrations of ∑antibiotics were relatively low (19.5-52.3 ng/g dw) at 75 % of the sampling sites, while they were high (95.7-229.0 ng/g dw) at a few sampling sites (S13-A1, S13D, S8D). The correlation analysis results showed that the Kd values of the 9 antibiotics were significantly positively correlated with molecular weight (MW), Kow, and LogKow. Risk assessment revealed that sulfamethoxazole (SMX) in surface water and SMX, enoxacin (ENX), ciprofloxacin (CFX), enrofloxacin (EFX), ofloxacin (OFX), and norfloxacin (NFX) in sediment had medium/high risk quotients (RQs) at 62.5 % and 25-100 %, respectively, of the sampling sites. The antibiotic mixture in surface water (0.06-3.36) and sediment (0.43-309) posed a high risk at 37.5 % and 66.7 %, respectively, of the sampling sites. SMX was selected as an indicator of antibiotic pollution in surface water to assist regulatory authorities in monitoring and managing antibiotic pollution in the aquaculture zone of Dongzhai Harbor. Overall, the results of the present study provide a comprehensive and detailed analysis of the characteristics of antibiotics in the aquaculture environment around the Dongzhai Harbor mangrove system and provide a theoretical basis for the source control of antibiotics in mangrove systems.

[Exposure Level and Risk Impact Assessment of Pesticides and Veterinary Drugs in Aquaculture Environment].

To explore the exposure level of pesticides and veterinary drugs in an aquaculture environment and its impact on the ecological environment, this study took the aquaculture environment in Shanghai as an example, and samples of water, sediment, and inputs from 40 major aquaculture farms were collected from July to September 2022. The types and contents of pesticides and veterinary drugs were screened using high-performance liquid chromatography-electrostatic field orbital ion trap mass spectrometry, and the risk quotient (RQ) method was used to assess the ecological risk of pesticide contamination in water and sediment. The results showed that 13 drugs were screened out from 204 samples (72 samples of water, 72 samples of mud, and 60 samples of input), namely, chlorpromazine, carbendazim, thiophanate, diazepam, florfenicol, simazine, amantidine, diazepam, trimethoprim, ciprofloxacin, ofloxacin, mebendazole, and enrofloxacin. Among them, 12 species were found in water samples with concentrations ranging from 0.016 µg·L-1 to 2.084 µg·L-1. The concentrations of seven species in the mud samples ranged from 0.018 µg·kg-1 to 23.101 µg·kg-1. The results showed that there were four types of inputs, ranging from 1.979 µg·kg-1 to 101.940 µg·kg-1. Seven drugs were found in both water and sediment. The risk quotient (RQ) results showed that there were some high and middle risks in both water and sediment samples of aquaculture farms, and the ecological risks of carbendazim were the highest in both water and sediment samples of aquaculture farms; the RQ values were 3.848 and 1.580, respectively, indicating high risk. It is suggested to strengthen the control and management of exogenous pesticides and veterinary drugs in aquaculture environments to protect the ecosystem health of the aquaculture environment.

An application of waste algae biochar in aquaculture water to remove co-existed cadmium and PAHs and the corresponding mechanism.

Water quality in aquaculture farms is highly related to the quality of aquaculture products and the connected environment. Cadmium (Cd2+) and polycyclic aromatic hydrocarbons (PAHs) are two of the most common pollutants in the aquaculture water, while biochar derived from waste algae (Enteromorpha prolifera), namely BE, was applied in farms ponds to improve water quality. Firstly, the adverse environmental impact of BE was minor, while the concentrations of the heavy metal (Cd2+ in the present study) and PAHs (FLU, PHE, FLT and PYR) were removed with efficiencies of 49%, 88%, 90%, 91% and 88%, respectively. The ecological risk values (RQs) were reduced subsequently with a rate of 58 ± 11%. After dosing BE, the ecological risk values in all the studied ponds were lower than 1, indicating no ecological risk in the corresponding aquaculture environment. The sorption capacities (qm) of BE were 15, 12, 6.3, 0.41, 0.29 and 0.56 mg·g-1 for Cd2+, FLU, PHE, FLT, PYR and BaP, respectively. The sorption capacities were acceptable compared with those derived from other types of biomass. The removal mechanisms were partition (PAHs), complexation (Cd2+), π-π interaction (Cd2+ and PAHs), precipitation (Cd2+) and ion-exchange (Cd2+). Practically and theoretically, the algae biochar is applicable in the aquaculture environment, where Cd2+ and PAHs co-exist.

Emergence of blaIMI-2- and blaIMI-16-Producing Enterobacter asburiae in the Aquaculture Environment of Jiangsu, China.

Carbapenem-resistant Enterobacteriaceae strains have emerged as a serious threat to global public health. In recent years, blaIMI, a carbapenemase gene that drew less attention before, has been increasingly detected in both clinical and environmental settings. However, the environmental distribution and transmission of blaIMI, especially in aquaculture, require systematic investigation. In this study, the blaIMI gene was detected in fish (n = 1), sewage (n = 1), river water (n = 1), and aquaculture pond water samples (n = 17) collected from Jiangsu, China, demonstrating a relatively high sample-positive ratio of 12.4% (20/161). Thirteen blaIMI-2- or blaIMI-16-carrying Enterobacter asburiae strains were isolated from blaIMI-positive samples of aquatic products and aquaculture ponds. We also identified a novel transposon (Tn7441) carrying blaIMI-16 and a conserved region containing several truncated insertion sequence (IS) elements harboring blaIMI-2, all of which may play important roles in blaIMI mobilization. The occurrence of blaIMI-carrying Enterobacter asburiae in aquaculture-related water samples and fish samples highlights the risk of transmission of blaIMI-carrying strains through the food chain and the need for effective measures to prevent further dissemination. IMPORTANCE IMI carbapenemases have been detected in clinical isolates of many bacterial species with systemic infection and cause a further burden on clinical treatment in China, but their source and distribution are still unclear. The study systematically investigated the distribution and transmission of the blaIMI gene in aquaculture-related water bodies and aquatic products in Jiangsu Province, China, which is famous for its rich water resources and developed aquaculture industry. The relatively high prevalence of blaIMI in aquaculture samples and the identification of novel mobile elements harboring blaIMI enhance our knowledge of blaIMI gene distribution and highlight the public health risk and urgency of surveillance of aquaculture water systems in China.

Application of a nitrifying bacterium Pseudomonas sp. HIB_D to reduce nitrogen waste in the Litopenaeus vannamei cultivation environment.

Pseudomonas sp. HIB_D has been observed to have nitrification activity that can oxidize ammonia/ammonium to nitrite and nitrate. This bacterium was isolated from the aquaculture environment with Ancol Indonesian marine water sources. Pseudomonas sp. HIB_D was applied in the rearing water of Litopenaeus vannamei to decrease nitrogen pollution levels and support a sustainable cultivation environment. This present study was set up using a completely randomized design with four treatments and three replications, i.e., control (without bacterial application) and bacterial application with a cell density of 107, 108, and 109 CFU mL-1 in a volume of 100 mL for 90 L seawater treatment. Thirty-six individuals of 15-day-old postlarvae L. vannamei (PL 15) (a density of 120 PL m-2) were used in this study for an 8-week rearing period. Based on the water quality analysis, the ammonia level under the bacterial applications was lower than in the control after 8-week cultivation. Nitrate levels increased at week 6 and decreased at week 8, specifically in 109 CFU mL-1 treatment. L. vannamei with 109 CFU mL-1 bacterial application had the best production performance with a survival rate of 94.33 ± 2.78%, an absolute length gain of 10.59 ± 0.22 cm, an absolute weight gain of 8.16 ± 0.33 g, a specific growth rate of 14.12% ± 0.01% day-1, and a feed conversion ratio of 1.26 ± 0.03. The blood glucose level and total hemocyte count of shrimp under the bacterial application with a density of 109 CFU mL-1 showed the highest level (30.71 ± 13.9 mg dL-1) and the lowest level (15 × 106 cells mL-1), respectively. The results showed that all treatments performed better than the control, in which the 109 CFU mL-1 bacterial application was the best treatment.

Molecular characterization of carbapenem resistant E. coli of fish origin reveals the dissemination of NDM-5 in freshwater aquaculture environment by the high risk clone ST167 and ST361.

Aquatic environment can act as reservoir and disseminator of antimicrobial resistance and resistant pathogens. Novel high-risk carbapenem resistant E. coli (CREC) are continuously emerging worldwide; however, the occurrence of CREC in freshwater aquaculture environment is largely unexplored. To fill this gap, large scale sampling of freshwater pond sites and retail fish markets was done between Oct 2020 and Oct 2021 to investigate the CREC contamination in fish. The frequency of CREC contamination in the freshwater fish was 6.99% (95% CI: 3.78-10.20%). All the isolates were MDR and harbored carbapenemase encoding gene, blaNDM-5 along with other antimicrobial resistance genes (ARGs), blaTEM (64.7%), blaCTX-M-15 (35.3%), blaOXA-1 (5.9%), tet(A) (100%), sul1 (94.1%), qnrS (82.3%), cat1 (35.3%), and cat2 (23.5%). The isolates belonged to phylogroup C and showed low virulence gene profile. ERIC-PCR grouped the isolates into five clusters (I-V). The isolates of clusters I, II, and III were identified as ST167 (76.4%) and of cluster IV as ST361 (17.6%). This is the first report documenting the contamination of NDM-5 producing E. coli ST167 and ST361 of clinical/livestock lineage in freshwater fish from India. The blaNDM-5 was significantly associated with ARGs, tet(A), and sul1; and plasmid replicons, IncF, IncI1, and IncP, signifying the presence of blaNDM-5 and associated ARGs on these transferable plasmids. These findings were validated by the successful conjugal transfer of blaNDM-5 and associated ARGs into non-CREC strain (J53). Our study highlights the ability of CREC to disseminate antimicrobial resistance which has health implications and environmental concerns.

Microbiome analysis reveals microecological advantages of emerging ditchless rice-crayfish co-culture mode.

Ditchless rice-crayfish co-culture is an emerging model of rice-crayfish farming that circumvents the potential hazards of digging ditches in traditional rice-crayfish farming. However, due to the complex interactions among crayfish, ambient microbiota, and environmental variables, it is necessary to assess the differences in bacterial structure between ditchless and traditional rice-crayfish culture. In this study, the crayfish culture area in the Sichuan basin was selected as the study area, and the bacterial communities of two rice-crayfish culture systems were compared by high-throughput sequencing of 16S rDNA. The results showed that the ditchless system had lower water depth, higher dissolved oxygen, lower total ammonia nitrogen and lower morbidity. There are intuitive differences in the composition of environmental bacterial communities due to environmental changes, even if they are similar in composition at the phylum level. Microbiota in sediments from ditchless systems appear to produce less ammonia nitrogen. The abundance of the pathogens colonizing the intestine of ditchless crayfish was lower than ditched one, and the composition was similar to water. Ditch-farmed crayfish appear to be more susceptible to environmental microbes and have a more fragile intestinal structure. Water depth and dissolved oxygen are the main environmental factors that determine the distribution of microbiota. This study is the first to investigate the bacterial ecology of a ditchless rice- crayfish farming system. The results show that the ditchless rice-crayfish culture model has a more superior bacterial system than the traditional rice-crayfish culture.

Applicability of Equilibrium Sampling in Informing Tissue Residues and Dietary Risks of Legacy and Current-Use Organic Chemicals in Aquaculture.

Equilibrium sampling based on silicone polydimethylsiloxane (PDMS) has been used to determine the concentrations of freely dissolved hydrophobic organic compounds (HOCs) and assess the thermodynamic potentials for bioaccumulation of these compounds in the aquatic environment. This allows the use of PDMS-based sampling techniques in assisting conventional sampling and extraction methods for the determination of the concentrations of HOCs in aquaculture products. The present study is an ex situ demonstration of how well PDMS can inform the tissue residues and dietary risks of legacy or current-use organic chemicals in aquaculture species from farm ponds in eastern China. For legacy contaminants such as polybrominated diphenyl ethers (PBDEs, n = 10), good agreement between the predicted concentrations based on PDMS and the measured lipid-normalized concentrations was observed for 60% of the studied biota, including both pelagic and benthic species. For pesticides currently used, such as pyrethroid (PE) (n = 4) and organophosphate pesticides (OPPs, n = 7), the measured tissue residues were consistently higher than those predicted by PDMS, possibly caused by the continuous input from the surroundings. For the organochlorine pesticides (OCPs, n = 5), the only detected chemical was also underestimated. Adjusted by ingestion rates of aquaculture products and toxicology data, the target hazard quotients of these chemicals predicted from PDMS were generally comparable to those derived from measured concentrations in tissue because of the predominance of PBDEs. Overall, PDMS-based equilibrium sampling offered an alternative approach for the prediction of tissue residues and dietary risks of PBDEs. Moreover, it should be applied with caution for PEs, OPPs, and OCPs. Improving the application of PDMS for these chemicals in farm ponds warrants future study. Environ Toxicol Chem 2021;40:79-87. © 2020 SETAC.

Interactions of microplastics and antibiotic resistance genes and their effects on the aquaculture environments.

Microplastics (MPs) and antibiotic resistance genes (ARGs) have become the increasing attention and global research hotpots due to their unique ecological and environmental effects. As susceptible locations for MPs and ARGs, aquaculture environments play an important role in their enrichment and transformation. In this review, we focused on the MPs, ARGs, and the effects of their interactions on the aquaculture environments. The facts that antibiotics have been widely applied in different kinds of agricultural productions (e.g., aquaculture) and that most of antibiotics enter the water environment with rainfall and residual in the aquaculture environment have been resulting in the emergence of antibiotic resistance bacteria (ARB). Moreover, the water MPs are effective carriers of the environmental microbes and ARB, making them likely to be continuously imported into the aquaculture environments. As a result, the formation of the compound pollutions may also enter the aquatic organisms through the food chains and eventually enter the human body after a long-term enrichment. Furthermore, the compound pollutions result in the joint toxic effects on the human health and the ecological environment. In summary, this review aims to emphasize the ecological effects and the potential hazards on the aquaculture environments where interactions between MPs and ARGs results, and calls for to reduce the use of the plastic products and the antibiotics in the aquaculture environments.

Microbiome Analysis Reveals Microecological Balance in the Emerging Rice-Crayfish Integrated Breeding Mode.

The interaction between the microbial communities in aquatic animals and those in the ambient environment is important for both healthy aquatic animals and the ecological balance of aquatic environment. Crayfish (Procambarus clarkii), with their high commercial value, have become the highest-yield freshwater shrimp in China. The traditional cultivation in ponds (i.e., monoculture, MC) and emerging cultivation in rice co-culture fields (i.e., rice-crayfish co-culture, RC) are the two main breeding modes for crayfish, and the integrated RC is considered to be a successful rice-livestock integration practice in eco-agricultural systems. This study explored the ecological interactions between the microbial communities in crayfish intestine and the ambient environment, which have not been fully described to date. The bacterial communities in crayfish intestine, the surrounding water, and sediment in the two main crayfish breeding modes were analyzed with MiSeq sequencing and genetic networks. In total, 53 phyla and 1,206 genera were identified, among which Proteobacteria, Actinobacteria, Tenericutes, Firmicutes, Cyanobacteria, Chloroflexi, Bacteroidetes, Acidobacteria, RsaHF231, and Nitrospirae were the dominant phyla. The microbiota composition significantly differed between the water, sediment, and crayfish intestine, while it did not between the two breeding modes. We also generated a co-occurrence correlation network based on the high-confidence interactions with Spearman correlation ρ ≥ 0.75. In the genera co-correlation network, 95 nodes and 1,158 edges were identified, indicating significant genera interactions between crayfish intestine and the environment. Furthermore, the genera clustered into three modules, based on the different environments. Additionally, Candidatus_Bacilloplasma, g_norank_f_Steroidobacteraceae, Dinghuibacter, Hydrogenophaga, Methyloparacoccus, and Defluviicoccus had the highest betweenness centrality and might be important in the interaction between crayfish and the ambient environment. Overall, this study enhances our understanding of the characteristics of the microbiota in crayfish and their surrounding environment. Moreover, our findings provide insights into the microecological balance in crayfish eco-agricultural systems and theoretical reference for the development of such systems.

Antibiotic resistance of culturable heterotrophic bacteria isolated from shrimp (Penaeus vannamei) aquaculture ponds.

Shrimp aquaculture is one of the fastest growing food-producing avenues, where antibiotics usage has become an issue of great concern due to the development of antimicrobial resistance in bacteria. A total of 2304 bacterial isolates from 192 samples (sediment, water, shrimp, and source water) from Andhra Pradesh, India were screened. Antibiotic resistance of bacterial isolates was highest for oxytetracycline (23.4%) followed by erythromycin (12.7%), co-trimoxazole (10%) ciprofloxacin (9.6%), and chloramphenicol (6%), of which 11.9% isolates were multi-drug resistant. Bacterial isolates from shrimp (26.7%), water (23.9%), and sediment (19.6%) samples exhibited more resistance (p ≤ 0.05) towards oxytetracycline. Higher antibacterial resistance was observed from samples of southern Andhra Pradesh (locations L6 and L7). Gram negative bacteria were more prevalent (64%) and showed significantly (p ≤ 0.01) higher resistance. This study indicated the wider distribution of antibiotic-resistant bacteria in shrimp aquaculture ponds with potential risk to humans and the environment.

Determination of Peptide Antibiotics Residues in Sediment From Aquaculture Environment by High Performance Liquid Chromatography-Tandem Mass Spectrometry / 分析化学

A new method for the determination of peptide antibiotics in sediment from aquaculture environment by high performance liquid chromatography-tandem mass spectrometry was developed. The target analytes in sediments were ultrasonically extracted twice with citrate buffer solution and methol mixture (3∶ 4, V/ V), followed by complexation with 0. 5 g of Na2 EDTA, purification with 5 mL of methyl isobutyl ketone, and clean-up with HLB-SPE column. The analytes were separated on a MGII C18 column by gradient elution with 0. 1% formaic acid-0. 1% formaic acid acetonitrile as mobile phase, detected in multiple reaction monitoring (MRM) with electrospray ionization (ESI) under positive ion mode, and quantified by external standard method. The calibration curves were linear (R2 >0. 999) over a concentration range of 10 -10000μg / L for colistin and bacitracin and 4-4000 μg / L for virginiamycin M1 . The limits of detection (S / N = 3) were 5 μg / kg for colistin and bacitracin and 2 μg / kg for virginiamycin M1 . The limits of quantification (S / N=10) was 10 μg / kg for colistin and bacitracin and 4 μg / kg for virginiamycin M1 . At three spiked levels, the recoveries ranged from 79. 7% to 91. 6% (RSD=1. 9% -10. 8% ), showing high sensitivity, good reproducibility and wide applicability.