Virulent and Multi-drug-Resistant Edwardsiella tarda Infection in Oscar Fish: Unveiling the Threat of Mass Mortality and AMR Dissemination.

The Oscar fish (Astronotus ocellatus) is among the most commonly domesticated and exported ornamental fish species from Kerala. The ornamental fish industry faces a significant challenge with the emergence of diseases caused by multi-drug-resistant bacteria. In the present study, six isolates were resolved from the diseased Oscar fish showing haemorrhages, necrosis, and loss of pigmentation. After phenotypic and genotypic characterization, the bacteria were identified as Edwardsiella tarda, Klebsiella pneumoniae, Enterococcus faecalis, Escherichia coli, Brevibacillus borstelensis, and Staphylococcus hominis. Experimental challenge studies in healthy Oscar fish showed that E. tarda caused 100% mortality within 240 h with 6.99 × 106 CFU/fish as LD50 and histopathology revealed the typical signs of infection. The pathogen was re-recovered from the moribund fish thereby confirming Koch's postulates. E. tarda was confirmed through the positive amplification of tarda-specific gene and virulence genes viz., etfD and escB were also detected using PCR. Antibiotic susceptibility tests using disc diffusion displayed that the pathogen is multi-drug-resistant towards antibiotics belonging to aminoglycosides, tetracyclines, and quinolones categories with a MAR index of 0.32, which implicated the antibiotic pressure in the farm. Plasmid curing studies showed a paradigm shift in the resistance pattern with MAR index of 0.04, highlighting the resistance genes are plasmid-borne except for the chromosome-borne tetracycline resistance gene (tetA). This study is the first of its kind in detecting mass mortality caused by E. tarda in Oscar fish. Vigilant surveillance and strategic actions are crucial for the precise detection of pathogens and AMR in aquaculture.

Investigation of antibiotic-resistant vibrios associated with shrimp (Penaeus vannamei) farms.

For the sustainable farming of disease-free and healthy shrimps, antimicrobial use is frequent nowadays in shrimp-cultured system. Considering the serious impact of global antimicrobial resistance (AMR), the present study was focused to investigate the prevalence of antimicrobial-resistant vibrios among infected shrimps (Penaeus vannamei) from two brackish water-cultured farms. Diverse species of vibrios viz. V. alginolyticus, V. parahaemolyticus, V. cholerae, V. mimicus, and V. fluvialis along with Aeromonas hydrophila, A. salmonicida and Shewanella algae were recovered from the shrimps on TCBS medium. Shannon-Wiener diversity index and H' (loge) were 1.506 and 1.69 for the isolates from farm 1 and farm 2, respectively. V. alginolyticus was found to be the most resistant isolate by showing multiple antibiotic resistance (MAR) index of 0.60 followed by V. mimicus (0.54) and V. parahaemolyticus (0.42). Among the 35 antibiotics of 15 different classes tested, tetracyclines, beta-lactams and cephalosporins were found as the most resistant antibiotic classes. All the isolates possessed a MAR index > 0.2 and the majority exhibited minimum inhibitory concentration (MIC) > 256 mcg/ml, thereby indicating the excess exposure of antibiotics in the systems. An enhanced altered resistance phenotype and a significant shift in the MAR index were noticed after plasmid curing. Public health is further concerning because plasmid-borne AMR is evident among the isolates and the studied shrimp samples are significant in the food industry. This baseline information will help the authorities to curb antimicrobial use and pave the way for establishing new alternative strategies by undertaking a multidimensional "One-Health" approach.

Antibiotic-resistant Enterobacteriaceae from diseased freshwater goldfish.

Goldfish farming gained more attention among the ornamental fishes in aquaculture industry. The occurrence of bacterial infections and further antimicrobial treatment lead to the major crisis of antibiotic resistance in aquaculture. We have isolated diverse enterobacteriaceae groups which affect the goldfish and identified their response towards 46 antimicrobials of 15 different classes. Thirteen significant bacterial isolates such as Edwardsiella tarda, Serratia marcescens, Klebsiella aerogenes, Proteus penneri, P. hauseri, Enterobacter cloacae, E. cancerogenus, E. ludwigii, Citrobacter freundii, E. coli, Kluyvera cryocrescens, Plesiomonas shigelloides and Providencia vermicola were recovered from the infected fish with the Shannon-wiener diversity index of 2.556. Multiple antibiotic resistance (MAR) index was found to be maximum for P. penneri (0.87) and minimum for C. freundii and E. cloacae (0.22), highlighting the hyper antibiotic selection pressure in the farm. The minimum concentration of antibiotics required to inhibit most of the resistant isolates was found to be > 256 mcg/ml. All the isolates were susceptible towards ciprofloxacin. Plasmid curing and further AMR tests could reveal the location of antibiotic resistance genes mainly as plasmids which determine the large extent of AMR spread through horizontal gene transfer. This study is the first of its kind to investigate the antimicrobial resistance profile of enterobacteriaceae recovered from goldfish, before and after plasmid curing.

Antimicrobial Resistance analysis of Pathogenic Bacteria Isolated from Freshwater Nile Tilapia (Oreochromis niloticus) Cultured in Kerala, India.

Aquaculture of popular freshwater species, Nile tilapia (Oreochromis niloticus), accounts for around 71% of the total global tilapia production. Frequent use of antibiotics for treating bacterial infections in tilapia leads to the emergence of antimicrobial resistance. To mitigate the issue, proper evaluation methods and control strategies have to be implemented. This study was aimed to analyze the antimicrobial resistance of bacterial isolates from the infected Nile tilapia cultured in freshwater. The recovered isolates were identified as Pseudomonas entomophila, Edwardsiella tarda, Comamonas sp, Delftia tsuruhatensis, Aeromonas dhakensis, A. sobria, A. hydrophila, A. lacus, Plesiomonas shigelloides and Vogesella perlucida through phenotypic and genotypic analyses. Using Primer-E software, Shannon Wiener diversity index of the isolates was determined as H' (loge) = 2.58. Antibiotic susceptibility test of the recovered strains through disk diffusion using 47 antibiotics, showed an elevated resistance pattern for Aeromonas hydrophila, Pseudomonas entomophila and Comamonas with higher multiple antibiotic resistance indexes (MAR index > 0.3). The minimum inhibitory concentration of antibiotics was > 256 mcg/ml for most of the resistant isolates. Meanwhile, all the recovered isolates were susceptible to amikacin, aztreonam, kanamycin, cefalexin, cefotaxime, levofloxacin, norfloxacin, piperacillin, and polymyxin-B.

Unravelling the menace: detection of antimicrobial resistance in aquaculture.

One of the major problems to be addressed in aquaculture is the prominence of antimicrobial resistance (AMR). The occurrence of bacterial infections in cultured fishes promotes the continuous use of antibiotics in aquaculture, which results in the selection of proliferated antibiotic-resistant bacteria and increases the possibility of transfer to the whole environment through horizontal gene transfer. Hence, the accurate cultivation-dependent and cultivation-independent detection methods are very much crucial for the immediate and proper management of this menace. Antimicrobial resistance determinants carrying mobile genetic transfer elements such as transposons, plasmids, integrons and gene cassettes need to be specifically analysed through molecular detection techniques. The susceptibility of microbes to antibiotics should be tested at regular intervals along with various biochemical assays and conjugation studies so as to determine the extent of spread of AMR. Advanced omic-based and bioinformatic tools can also be incorporated for understanding of genetic diversity. The present review focuses on different detection methods to unearth the complexity of AMR in aquaculture. This monitoring helps the authorities to curb the use of antibiotics, commencement of appropriate management measures and adequate substitute strategies in aquaculture. The long battle of AMR could be overcome by the sincere implementation of One Health approach. SIGNIFICANCE AND IMPACT OF THE STUDY: The use of antibiotics and increased antimicrobial resistance (AMR) are of major concerns in aquaculture industry. This could result in global health risks through direct consumption of cultured fishes and dissemination of AMR to natural environment through horizontal gene transfer. Hence, timely detection of the antimicrobial-resistant pathogens and continuous monitoring programmes are inevitable. Advanced microbiological, molecular biological and omic-based tools can unravel the menace to a great extent. This will help the authorities to curb the use of antibiotics and implement appropriate management measures to overcome the threat.

Diversity of antimicrobial-resistant pathogens from a freshwater ornamental fish farm.

The indiscriminate use of antimicrobials in aquaculture results in antibiotic selection pressure and proliferation of antimicrobial resistant (AMR) bacteria. Frequent assessment of antimicrobial resistance in aquaculture environment is inevitable so as to reduce the passage of clinically important AMR from aquatic to other environment. The present study analysed the antimicrobial resistance of pathogens associated with diseased koi carp and goldfish from an ornamental fish farm. Phenotypic and genotypic characterization of the recovered isolates from both fishes revealed significant pathogens in aquaculture such as Aeromonas, Edwardsiella tarda, Acinetobacter, Lactococcus, Citrobacter, Enterobacter and Comamonas. Shannon-Wiener diversity of koi isolates (2·359) was found to be higher than that of goldfish (1·864). Antibiotic susceptibility testing using disc diffusion with 47 antibiotics revealed significant resistance pattern of Acinetobacter, Comamonas, Klebsiella and Enterobacter from goldfish and Edwardsiella, Aeromonas, Lactococcus, Enterobacter and Acinetobacter from koi with higher multiple antibiotic resistance indexes (>0·3). The minimum inhibitory concentration of antibiotics for the major resistant isolates was found to be very high with >256 µg. All the isolates were susceptible to amoxicillin, kanamycin, cefepime, cefexime, cefotaxime, ceftazidime, doripenem, ciprofloxacin and norfloxacin, recommending their successful application in the farm. SIGNIFICANCE AND IMPACT OF THE STUDY: Antimicrobial resistance is a major threat faced in aquaculture industry. The current study provides baseline information regarding the antibiotic resistance patterns of diverse pathogens recovered from ornamental koi carp and goldfish. The higher MAR index of pathogens and greater MIC of antibiotics for the resistant isolates highlighted the intense use of antibiotics in aquaculture farm. The potential of the pathogens to exhibit resistance even towards the new generation antibiotics remind the need of prudent use of antibiotics and continuous monitoring and surveillance programmes.

Community composition of marine and brackish water ammonia-oxidizing consortia developed for aquaculture application.

To mitigate the toxicity of ammonia in aquaculture systems, marine and brackish water ammonia-oxidizing bacterial consortia have been developed and are used for activation of nitrifying bioreactors integrated to recirculating aquaculture systems. To shed more light on to these biological entities, diversity of both the consortia were analyzed based on random cloning of 16S rRNA gene and ammonia-oxidizing bacterial specific amoA gene sequences. The dendrograms of representative clones on the basis of amplified ribosomal DNA restriction analysis generated 22 and 19 clusters for marine and brackish water nitrifying consortia, respectively. Phylogenetic analysis demonstrated the presence of various autotrophic nitrifiers belonging to α-, ß- and γ-Proteobacteria, anaerobic ammonia oxidizers, heterotrophic denitrifiers, Bacteroidetes, and Actinobacteria. Distribution patterns of the organisms within the two consortia were determined using the software Geneious and diversity indices were investigated using Mega 5.0, VITCOMIC and Primer 7. The abundance of ammonia oxidizers was found in the order of 2.21 ± 0.25 × 109 copies/g wet weight of marine consortium and 6.20 ± 0.23 × 107 copies/g of brackish water consortium. Besides, marine ammonia-oxidizing consortium exhibited higher mean population diversity and Shannon Wiener diversity than the brackish water counterparts.