Doxycycline pharmacokinetics and tissue depletion in striped catfish (Pagasianodon hypophthalmus) after oral administration.

The pharmacokinetics and residue depletion of doxycycline (DOX) in striped catfish (Pagasianodon hypophthalmus) after oral dosage were investigated. The pharmacokinetic experiment was conducted in an aquarium, while the experiment of residue depletion was performed in both an aquarium and earth ponds. Medicated feed was administered orally using the gavage method at a dosage of 20 mg/kg body weight. Blood, liver, and kidney from medicated fish samples were collected. In the depletion experiments, fish were fed medicated feed for five consecutive days at a dosage of 20 mg/kg body weight, with samples collected during and after medication. The concentrations of DOX were quantified using an LC-MS/MS system. The pharmacokinetics parameters of DOX in striped catfish included the absorption rate constant (ka), absorption half-life (T1/2abs), maximal plasma concentration (Cmax), time to maximal plasma concentration (Tmax), and area under the plasma concentration-time curve from time 0 to 96 h (AUC0-96 h) which were 0.12 h-1, 5.68 h, 1123.45 ng/mL, 8.19 h, and 25,018 ng/mL/h, respectively. Residue depletion results indicated that the withdrawal times of DOX in muscle (with skin) from fish kept in the aquarium were slightly longer than that in fish raised in earth ponds, corresponding to 194 degree-days compared with 150 degree-days. In conclusion, administration of DOX at the dosage of 20 mg/kg body weight can be used for treatment of bacterial infections in striped catfish, and a withdrawal time of 5 days at 29.4°C will ensure consumer food safety due to the rapid depletion of DOX from muscle and skin.

Genomic Insights into Edwardsiella ictaluri: Molecular Epidemiology and Antimicrobial Resistance in Striped Catfish (Pangasianodon hypophthalmus) Aquaculture in Vietnam.

Edwardsiella ictaluri is responsible for causing bacillary necrosis (BNP) in striped catfish (Pangasianodon hypophthalmus) in Vietnam. This study offers a comprehensive genomic characterization of E. ictaluri to enhance understanding of the molecular epidemiology, virulence, and antimicrobial resistance. E. ictaluri isolates were collected from diseased striped catfish in the Mekong Delta. The species was confirmed through PCR. Antimicrobial susceptibility testing was conducted using minimum inhibitory concentrations for commonly used antimicrobials. Thirty representative isolates were selected for whole genome sequencing to delineate their genomic profiles and phylogeny. All strains belonged to ST-26 and exhibited genetic relatedness, differing by a maximum of 90 single nucleotide polymorphisms. Most isolates carried multiple antimicrobial resistance genes, with the tet(A) gene present in 63% and floR in 77% of the genomes. The ESBL gene, blaCTX-M-15, was identified in 30% of the genomes. Three plasmid replicon types were identified: IncA, p0111, and IncQ1. The genomes clustered into two clades based on their virulence gene profile, one group with the T3SS genes and one without. The genetic similarity among Vietnamese isolates suggests that disease spread occurs within the Mekong region, underscoring the importance of source tracking, reservoir identification, and implementation of necessary biosecurity measures to mitigate spread of BNP.

Comparative genomic analysis of Aeromonas dhakensis and Aeromonas hydrophila from diseased striped catfish fingerlings cultured in Vietnam.

Introduction: Motile Aeromonas septicemia (MAS) is a burden for striped catfish (Pangasius hypophthalmus) farmers in Vietnam. MAS can be caused by several species of Aeromonas but Aeromonas hydrophila is seen as the leading cause of MAS in aquaculture, but recent reports suggest that A. dhakensis is also causing MAS. Methods: Here we investigated the bacterial etiology of MAS and compared the genomic features of A. hydrophila and A. dhakensis. We collected 86 isolates from diseased striped catfish fingerlings over 5 years from eight provinces in Vietnam. Species identification was done using PCR, MALDI-TOF and whole genome sequence (WGS). The MICs of commonly used antimicrobials was established. Thirty presumed A. hydrophila isolates were sequenced for species confirmation and genomic comparison. A phylogenetic analysis was conducted using publicly available sequences and sequences from this study. Results: A total of 25/30 isolates were A. dhakensis sequence type (ST) 656 and 5/30 isolates were A. hydrophila ST 251. Our isolates and all publicly available A. hydrophila isolates from Vietnam belonged to ST 251 and differed with <200 single nucleotide polymorphisms (SNP). Similarly, all A. dhakensis isolates from Vietnam belonged to ST 656 and differed with <100 SNPs. The tet(A) gene was found in 1/5 A. hydrophila and 19/25 A. dhakensis. All A. hydrophila had an MIC ≤2 mg/L while 19/25 A. dhakensis had MIC ≥8 mg/L for oxytetracycline. The floR gene was only found in A. dhakensis (14/25) which showed a MIC ≥8 mg/L for florfenicol. Key virulence genes, i.e., aerA/act, ahh1 and hlyA were present in all genomes, while ast was only present in A. dhakensis. Discussion: This study confirms previous findings where A. dhakensis was the dominating pathogen causing MAS and that the importance of A. hydrophila has likely been overestimated. The differences in antimicrobial susceptibility between the two species could indicate a need for targeted antimicrobial treatment plans. The lipopolysaccharide regions and outer membrane proteins did not significantly differ in their immunogenic potentials, but it remains to be determined with in vivo experiments whether there is a difference in the efficacy of available vaccines against A. hydrophila and A. dhakensis.

Bioconcentration and half-life of quinalphos pesticide in rice-fish integration system in the Mekong Delta, Vietnam.

In order to determine the distribution and enable the elimination of quinalphos, a popular active pesticide compound used in the Mekong Delta, an experiment was set up in a rice-fish integration system in Can Tho City, Vietnam. Fish was stocked into the field when the rice was two-months old. Quinalphos was applied twice in doses of 42.5 g per 1000 m2. Water, fish and sediment samples were collected at time intervals and analyzed by a Gas Chromatography Electron Capture Detector system. The results show that quinalphos residues in fish muscles were much higher than those of the water and the bioconcentration factor (logBCF) was above 2 for the fish. The half-life of first and second quinalphos applications were 12.2 and 11.1 days for sediment, 2.5 and 1.1 days for silver barb, 1.9 and 1.3 days for common carp, and 1.1 and 1.0 days for water, respectively.

Water metagenomic analysis reveals low bacterial diversity and the presence of antimicrobial residues and resistance genes in a river containing wastewater from backyard aquacultures in the Mekong Delta, Vietnam.

The environmental pathways for the dissemination of antibiotic resistance have recently received increased attention. Aquatic environments act as reservoirs or sources of antimicrobial-resistant bacteria, antimicrobial residues, and antimicrobial resistance genes (ARGs). Therefore, it is imperative to identify the role of polluted water in the dissemination of antimicrobial resistance. The aim of this study was to evaluate the antimicrobial residues, ARGs, and microbiota in the freshwater systems of the Mekong Delta. We selected 12 freshwater sites from aquacultures and rivers in Can Tho, Vietnam and analyzed them for 45 antimicrobial residues and 8 ARGs by LC/MS/MS and real-time PCR, respectively. A 16S rDNA-based metagenomic analysis was conducted to characterize the water microbiota. Residues of sulfamethoxazole (10/12) and sulfadimidine (7/12) were widely detected, together with the sulfa-resistance genes sul1 (11/12) and sul2 (9/12). Additionally, sulfamethoxazole residues and the ß-lactamase-resistance gene blaCTX-M-1 were detected in eight freshwater systems (8/12), suggesting that these freshwater systems may have been polluted by human activity. The metagenomic analysis showed that all the tested freshwater systems contained the phyla Proteobacteria, Actinobacteria, and Bacteroidetes, representing 64% of the total microbiota. Moreover, the Cai Rang River site (Ri-E), which is located at the merge point of wastewaters from backyard-based aquacultures, contained the genera Polynucleobacter, Variovorax, and Limnohabitans, representing more than 78.4% of the total microbiota. Bacterial diversity analysis showed that the Ri-E exhibited the lowest diversity compared with other regions. Principal coordinate analysis showed that the differences among water microbiotas in backyard-based aquacultures could be explained by the farmers' aquaculture techniques. In conclusion, this study demonstrated a collapse of bacterial diversity at the merge point of wastewaters from backyard-based aquacultures in the Mekong Delta.

Residues of 2-hydroxy-3-phenylpyrazine, a degradation product of some ß-lactam antibiotics, in environmental water in Vietnam.

Antibiotic-resistant bacteria have become a serious problem worldwide, caused in part by the excessive use and discharge of antibiotics into the environment. Ampicillin (ABPC) is a widely used antibiotic. However, this chemical rapidly decomposes in water containing divalent cations like Ca2+ and Mg2+, thus, detection of ABPC in environmental water is difficult. This study was carried out to evaluate the presence of 2-hydroxy-3-phenylpyrazine (HPP), one of the degradation products of ABPC and ß-lactam antibiotics with an ABPC substructure, in environmental water. An analytical method for HPP monitoring in environmental water was developed using liquid chromatography/tandem mass spectrometry. The analyte was extracted from water samples and enriched using a solid-phase extraction cartridge. The quantification limit was 1 ng L-1. The HPP recovery rates from spiked water samples of 25 and 125 ng L-1 were 84.1 and 86.1%, respectively. The method was then used to determine HPP residue levels in 98 environmental water samples from rivers, household ponds, and aquacultural ponds in Vietnam. HPP residues were detected in 60 samples. The HPP detection rates in rivers and household ponds were 42 and 79%, respectively. HPP was not detected in aquacultural ponds. HPP residue concentrations in the samples ranged from 1.3 to 413.3 ng L-1. The residue levels in rivers flowing through city centres were higher than levels in other sampling locations. The findings of this study suggest that HPP is a promising marker for assessing the discharge of ABPC and ß-lactam antibiotics with an ABPC substructure into the environment around sampling sites.

Identification and Antimicrobial Resistance of Bacteria Isolated from Probiotic Products Used in Shrimp Culture.

Probiotics are increasingly used in aquaculture to control diseases and improve feed digestion and pond water quality; however, little is known about the antimicrobial resistance properties of such probiotic bacteria and to what extent they may contribute to the development of bacterial resistance in aquaculture ponds. Concerns have been raised that the declared information on probiotic product labels are incorrect and information on bacterial composition are often missing. We therefore evaluated seven probiotics commonly used in Vietnamese shrimp culture for their bacterial species content, phenotypic antimicrobial resistance and associated transferable resistance genes. The bacterial species was established by 16S rRNA sequence analysis of 125 representative bacterial isolates. MIC testing was done for a range of antimicrobials and whole genome sequencing of six multiple antimicrobial resistant Bacillus spp. used to identify resistance genes and genetic elements associated with horizontal gene transfer. Thirteen bacterial species declared on the probiotic products could not be identified and 11 non-declared Bacillus spp. were identified. Although our culture-based isolation and identification may have missed a few bacterial species present in the tested products this would represent minor bias, but future studies may apply culture independent identification methods like pyro sequencing. Only 6/60 isolates were resistant to more than four antimicrobials and whole genome sequencing showed that they contained macrolide (ermD), tetracycline (tetL), phenicol (fexA) and trimethoprim (dfrD, dfrG and dfrK) resistance genes, but not known structures associated with horizontal gene transfer. Probiotic bacterial strains used in Vietnamese shrimp culture seem to contribute with very limited types and numbers of resistance genes compared to the naturally occurring bacterial species in aquaculture environments. Approval procedures of probiotic products must be strengthened through scientific-based efficacy trials and product labels should allow identification of individual bacterial strains and inform the farmer on specific purpose, dosage and correct application measures.

Quality of Antimicrobial Products Used in Striped Catfish (Pangasianodon hypophthalmus) Aquaculture in Vietnam.

Antimicrobial usage is common in Asian aquaculture. This study aimed to determine the quality of antimicrobial products used by Vietnamese striped catfish (Pangasianodon hypophthalmus) farmers. Twenty one antimicrobial products (11 products contained a single antimicrobial and 10 products contained a mixture of two different antimicrobials) commonly used by catfish farmers were obtained from so-called chemical shops located in the Mekong Delta, Vietnam. Ultra High Performance Liquid Chromatography Mass Spectrometry was used to analyze concentration of sulfonamides, trimethoprim, amoxicillin, cefalexin and ciprofloxacin whereas concentrations of florfenicol and doxycycline were analyzed by High Performance Liquid Chromatography with UV detection. Results revealed that only 4/11 products with a single antimicrobial and 2/10 products with a mixture of antimicrobials contained active substances within ±10% of the concentration declared on the product label. Two products with antimicrobial mixtures did not contain any of the declared antimicrobials. Comparing two batches, analysis of 11 products revealed that only one product contained a concentration of active compound that varied with less than 10% in both batches. Several product labels provided inadequate information on how to calculate therapeutic dosage and further stated withdrawal time despite lack of pharmacokinetic data on the antimicrobials in catfish. There is an urgent need to strengthen approval procedures and in particular regularly to monitor the quality of antimicrobials used in Vietnamese aquaculture.

Ecological risk assessment of the antibiotic enrofloxacin applied to Pangasius catfish farms in the Mekong Delta, Vietnam.

Antibiotics applied in aquaculture production may be released into the environment and contribute to the deterioration of surrounding aquatic ecosystems. In the present study, we assessed the ecological risks posed by the use of the antibiotic enrofloxacin (ENR), and its main metabolite ciprofloxacin (CIP), in a Pangasius catfish farm in the Mekong Delta region, Vietnam. Water and sediment samples were collected in a stream receiving effluents from a Pangasius catfish farm that had applied ENR. The toxicity of ENR and CIP was assessed on three tropical aquatic species: the green-algae Chlorella sp. (72 h - growth inhibition test), the micro-invertebrate Moina macrocopa (48 h - immobilization test), and the Nile tilapia (Oreochromis niloticus). The toxic effects on O. niloticus were evaluated by measuring the cholinesterase (ChE) and catalase (CAT) activities in the fish brain and muscles, respectively, and by considering feed exposure and water exposure separately. Ecological risks were assessed by comparing maximum exposure concentrations with predicted no effect concentrations for cyanobacteria, green algae, invertebrates and fish derived with available toxicity data. The results of this study showed that maximum antibiotic concentrations in Pangasius catfish farm effluents were 0.68 µg L(-1) for ENR and 0.25 µg L(-1) for CIP (dissolved water concentrations). Antibiotics accumulated in sediments down-stream the effluent discharge point at concentrations up to 2590 µg kg(-1) d.w. and 592 µg kg(-1) d.w. for ENR and CIP, respectively. The calculated EC50 values for ENR and CIP were 111000 and 23000 µg L(-1) for Chlorella sp., and 69000 and 71000 µg L(-1) for M. macrocopa, respectively. Significant effects on the ChE and CAT enzymatic activities of O. niloticus were observed at 5 g kg(-1) feed and 400-50000 µg L(-1), for both antibiotics. The results of the ecological risk assessment performed in this study indicated only minor risks for cyanobacteria communities, suggesting that residual concentrations of ENR and CIP after medication are not likely to result in severe toxic effects on exposed aquatic ecosystems. However, more studies should be performed by considering other antibiotic treatments used in Pangasius catfish production and the potential ecotoxicological effects of relevant antibiotic mixtures on sediment communities.

Effects of pre-slaughter stress on proteolytic enzyme activities and muscle quality of farmed Atlantic cod (Gadus morhua).

Farmed Atlantic cod were subjected to a combination of stressors in a holding tank before being killed, pre rigor filleted and stored in ice. At slaughter, a higher level of stress was confirmed by blood physiology analyses. This was further associated with significantly reduced muscle pH and somewhat elevated muscle collagenase-like activity in the stressed fish, whereas no differences in cathepsin-like activities were found. After 5 days of iced storage, the stressed fish had significantly lower water holding capacity, reduced hardness and yellowish colour compared to the control group, and no differences in the other parameters investigated. Independent of pre-slaughter stress, the activities of cathepsin B- and B/L-like enzymes increased and activities of cathepsin D/E- and collagenase-like enzymes decreased with storage.