Use Of Antimicrobials In Aquaculture Research Articles

Molecular characterization of Aeromonas hydrophila detected in Channa marulius and Sperata sarwari sampled from rivers of Punjab in Pakistan.

Aeromonas hydrophila is one of the major pathogenic bacteria responsible for causing severe outbreaks at fish farms and is also a major global public health concern. This bacterium harbors many virulence genes. The current study was designed to evaluate the antidrug and virulence potential of A. hydrophila by amplifying its antimicrobial resistance and virulence genes using PCR and examining their effects on fish tissues and organs. A total of 960 fish samples of Channa marulius and Sperata sarwari were collected from four sites of the rivers of the Punjab, Pakistan. A. hydrophila isolates were subjected to biochemical identification and detection of virulence and antimicrobial resistance (AMR) genes by PCR. We retrieved 181 (6.46%) A. hydrophila isolates from C. marulius and 177 (6.25%) isolates from S. sarwari. Amplification through PCR revealed the incidence of virulence genes in 95.7% of isolates in C. marulius and 94.4% in S. sarwari. Similarly, amplification through PCR also revealed occurrence of AMR genes in 87.1% of isolates in C. marulius and 83.9% in S. sarwari. Histopathological examination revealed congestion (5.2%) and hepatocyte necrosis (4.6%) in liver, lamellar fusion (3.3%) and the presence of bacterial colonies (3.7%) in gills, fin erosion (6%), and the presence of biofilms (3.5%) in tail fins of infected fish. Phylogenetic tree analysis of 16S rRNA and gyrB gene of A. hydrophila revealed 100% and 97% similarity, respectively, with 16S rRNA gene and gyrB of A. hydrophila isolated in previous studies. The results of antimicrobial susceptibility testing showed that all isolates demonstrated resistance to sulfamethoxazole, ampicillin, neomycin, and norfloxacin, while susceptibility to gentamicin, chloramphenicol, and tetracycline, and intermediate resistance was observed against cefotaxime. The results concluded that examined fish samples were markedly contaminated with virulent and multidrug strains of A. hydrophila which may be of a potential health risk. The study emphasizes the responsible antimicrobial use in aquaculture and the urgent need for effective strategies to control the spread of virulence and antimicrobial resistance genes in A. hydrophila.

Restrictions on antimicrobial use in aquaculture and livestock, Viet Nam.

Restrictions on antimicrobial use in aquaculture and livestock, Viet Nam.

Setting epidemiological cut-off values for Vibrio harveyi relevant to MIC data generated by a standardised microdilution method.

The lack of internationally harmonised criteria for interpreting the data generated by standardised susceptibility testing methods presents a serious obstacle for the development of prudent use of antimicrobials in aquaculture. The data required to set epidemiological cut-off values for minimum inhibitory concentrations for antibiotic agents against Vibrio harveyi was determined using a standard microdilution method that specified the use of cation-adjusted Mueller Hinton broth and incubation at 28°C for 24 to 28 h. In total, 120 observations were made in 4 independent laboratories from 109 unique isolates. The aggregated data from these laboratories were analysed by the normalised resistance method and by ECOFFinder to calculate epidemiological cut-off values. The data for chloramphenicol, meropenem and sulfamethoxazole were not considered as suitable for analysis. The data for ampicillin indicated that this species is innately resistant to this agent. No acceptable ranges for quality control strains have been set for ceftazidime and, therefore, only provisional cut-off values could be generated for this agent. The epidemiological cut-off values were, however, calculated for the other 6 agents. These values were ≤0.5 µg ml-1 for enrofloxacin, ≤1 µg ml-1 for florfenicol, oxolinic acid and oxytetracycline, ≤4 µg ml-1 for gentamicin and ≤0.5/9.5 µg ml-1 for trimethoprim/sulfamethoxazole. Evidence is presented demonstrating that the data for these 6 antimicrobial agents was of sufficient quantity and quality that they could be used by the relevant authorities to set internationally harmonised, consensus epidemiological cut-off values for V. harveyi.

Characterization of Vibrio parahaemolyticus isolated from stool specimens of diarrhea patients in Nantong, Jiangsu, China during 2018-2020.

Vibrio parahaemolyticus is the leading cause of acute seafood-associated gastroenteritis worldwide. The aim of this study was to investigate the presence of virulence genes, biofilm formation, motor capacities and antimicrobial resistance profile of V. parahaemolyticus isolates isolated from clinical samples in Nantong during 2018–2020. Sixty-six V. parahaemolyticus strains isolated from stool specimens of diarrheal patients were examined. The PCR results showed that there were two tdh+trh+ isolates, four tdh-trh- isolates and sixty tdh+trh- isolates, accounting for 3.0%, 6.1% and 90.9%, respectively. All the tdh carrying isolates manifested the positive reactions for the Kanagawa phenomenon (KP) test. Most of the isolates harbored at least one of the specific DNA markers of ‘pandemic group’ strains, suggesting that the dominant isolates of V. parahaemolyticus in Nantong might belong to the new O3: K6 or its serovariants. All tdh+ isolates possessed the Vp-PAI genes, but no tdh-trh- isolates carried the T3SS2 genes. All isolates were biofilm producers and had relatively strong motor capacities. In addition, the V. parahaemolyticus isolates were resistant to ampicillin (98.5%), cefuroxime (75.6%), cefepime (66.7%), piperacillin (59.1%) and ampicillin/sulbactam (50.0%), but sensitive to ciprofloxacin (100.0%), levofloxacin (100.0%), trimethoprim-sulfamethoxazole (98.5%), gentamicin (98.5%), amikacin (97%), meropenem (71.2%), and ceftazidime (56.1%). Multidrug-resistant isolates in clinical might be related to the inappropriate use of antimicrobials in aquaculture.

Use of Probiotic Bacteria and Bacteriocins as an Alternative to Antibiotics in Aquaculture.

In addition to their use in human medicine, antimicrobials are also used in food animals and aquaculture, and their use can be categorized as therapeutic against bacterial infections. The use of antimicrobials in aquaculture may involve a broad environmental application that affects a wide variety of bacteria, promoting the spread of bacterial resistance genes. Probiotics and bacteriocins, antimicrobial peptides produced by some types of lactic acid bacteria (LAB), have been successfully tested in aquatic animals as alternatives to control bacterial infections. Supplementation might have beneficial impacts on the intestinal microbiota, immune response, development, and/or weight gain, without the issues associated with antibiotic use. Thus, probiotics and bacteriocins represent feasible alternatives to antibiotics. Here, we provide an update with respect to the relevance of aquaculture in the animal protein production sector, as well as the present and future challenges generated by outbreaks and antimicrobial resistance, while highlighting the potential role of probiotics and bacteriocins to address these challenges. In addition, we conducted data analysis using a simple linear regression model to determine whether a linear relationship exists between probiotic dose added to feed and three variables of interest selected, including specific growth rate, feed conversion ratio, and lysozyme activity.

Typology of interventions for antimicrobial use and antimicrobial resistance in aquaculture systems in low- and middle-income countries

Indiscriminate antimicrobial use (AMU) in aquaculture to treat and prevent diseases is common and can lead to the emergence of antimicrobial-resistant micro-organisms, potentially impacting public health and connected ecosystems. This study aimed to develop a typology to classify and characterise interventions to reduce AMU in aquaculture and identify points of action. Seventeen aquaculture and animal health professionals in Asian and African countries were interviewed to gather information on characteristics of interventions in different contexts to develop a typology. Seven types of interventions were defined: (i) legislation and regulations; (ii) industry rules and standards; (iii) voluntary instruments; (iv) commercial technology and alternatives to antimicrobials; (v) on-farm management; (vi) learning and awareness-raising; and (vii) activities with co-benefits. Types were based on intervention function, scope of implementation, implementer, compulsion, strength of the intervention, AMU/antimicrobial resistance (AMR) objective and stakeholder to influence. For each type, examples were described and discussed. The most common interventions to address AMU and AMR were legislative and regulatory frameworks and voluntary instruments, including National Action Plans. Interventions addressing AMU/AMR specifically were scarce. Other interventions focused on indirect effect pathways to AMU and AMR reduction aiming to improve good aquaculture practices, disease prevention and improved management. Monitoring and evaluation of these interventions were found to be rare, only present for interventions driven by development projects and international agencies. The presented typology of existing strategies and interventions addressing AMU/AMR in aquaculture systems can guide evaluation of AMR-sensitive interventions that promote responsible AMU, and informs the design and implementation of future interventions.

Cytotoxicity and antimicrobial activity of synthetic peptides alone or in combination with conventional antimicrobials against fish pathogenic bacteria.

This study aimed to evaluate the in vitro cytotoxicity and efficacy of synthetic host defence peptides (HDPs), alone or in combination with florfenicol (FFC), oxytetracycline (OTC) or thiamphenicol (TAP), against different pathogenic bacteria isolated from diseased fish. Solid-phase synthesis, purification and characterization of several HDPs were performed manually, using the fluorenylmethyloxycarbonyl protecting group in different resins and via high-performance liquid chromatography-mass spectrometry, respectively. The in vitro cytotoxicity and antimicrobial activity of HDPs, FFC, OTC and TAP against Nile tilapia red blood cells (RBCs) and relevant fish pathogenic bacteria (Aeromonas, Citrobacter, Edwardsiella, Streptococcus, Lactococcus and Vibrio) was determined using the haemolysis assay and broth microdilution method, respectively. The checkerboard assay was used to evaluate the synergy between the most active HDPs and other antimicrobials against the tested strains. MUC 7 12-mer, FFC, OTC and TAP were not cytotoxic to Nile tilapia RBCs, in all tested concentrations. LL-37, (p-BthTX-I)2 and Hylin-a1 were not cytotoxic at concentrations up to 78·13, 19·53 and 9·77μgml-1 , respectively. HDPs demonstrated potent antimicrobial activity (minimum inhibitory concentration ≤31·25µgml-1 ) against Aeromonas jandaei (KR-12-a5), Citrobacter freundii (Kr-12-a5; (p-BthTX-I)2 ; LL-37; and Hylin a1), Streptococcus agalactiae (Hylin a1; (p-BthTX-I)2 and LL-37), Lactococcus garviae (Hylin a1), and Vibrio fluvialis (KR-12-a5). The combinations of (p-BthTX-I)2 with TAP and LL-37 with FFC showed synergistic activity against C. freundii (fractional inhibitory concentration index of 0·25 and 0·50, respectively). Synthetic HDPs have the potential as a good treatment option for bacterial diseases in aquaculture. The in vivo effectiveness of synthetic HDPs such as KR-12-a5; LL-37; (p-BthTX-I)2 and Hylin a1 can be tested alone or in combination with conventional antimicrobials as a treatment option to reduce the use of antimicrobials in aquaculture.

Temporal Resistome and Microbial Community Dynamics in an Intensive Aquaculture Facility with Prophylactic Antimicrobial Treatment.

Excessive use of antimicrobials in aquaculture is concerning, given possible environmental ramifications and the potential contribution to the spread of antimicrobial resistance (AR). In this study, we explored seasonal abundance of antimicrobial resistance genes and bacterial community composition in the water column of an intensive aquaculture pond stocked with Silver Carp (Hypophthalmichthys molitrix) prophylactically treated with sulfamethoprim (25% sulfadiazine; 5% trimethoprim), relative to an adjacent unstocked reservoir. Bacterial community composition was monitored using high-throughput sequencing of 16S rRNA gene amplicons in eight sampling profiles to determine seasonal dynamics, representing principal stages in the fish fattening cycle. In tandem, qPCR was applied to assess relative abundance of selected antimicrobial resistance genes (sul1, sul2, dfrA1, tetA and blaTEM) and class-1 integrons (int1). Concomitantly, resistomes were extrapolated from shotgun metagenomes in representative profiles. Analyses revealed increased relative abundance of sulfonamide and tetracycline resistance genes in fishpond-03, relative to pre-stocking and reservoir levels, whereas no significant differences were observed for genes encoding resistance to antimicrobials that were not used in the fishpond-03. Seasons strongly dictated bacterial community composition, with high abundance of cyanobacteria in summer and increased relative abundance of Flavobacterium in the winter. Our results indicate that prophylactic use of sulfonamides in intensive aquaculture ponds facilitates resistance suggesting that prophylactic use of these antimicrobials in aquaculture should be restricted.

Global trends in antimicrobial use in aquaculture

Globally aquaculture contributes 8% of animal protein intake to the human diet, and per capita consumption is increasing faster than meat and dairy consumption. Reports have documented antimicrobial use in the rapidly expanding aquaculture industry, which may contribute to the rise of antimicrobial resistance, carrying potential consequences for animal-, human-, and ecosystem-health. However, quantitative antimicrobial use across a highly diversified aquaculture industry is not well characterized. Here, we estimate global trends in antimicrobial use in aquaculture in 2017 and 2030 to help target future surveillance efforts and antimicrobial stewardship policies. We estimate antimicrobial use intensity (mg kg−1) for six species groups though a systematic review of point prevalence surveys, which identified 146 species-specific antimicrobial use rates. We project antimicrobial use in each country by combining mean antimicrobial use coefficients per species group with OECD/FAO Agricultural Outlook and FAO FishStat production volumes. We estimate global antimicrobial consumption in 2017 at 10,259 tons (95% uncertainty interval [UI] 3163–44,727 tons), increasing 33% to 13,600 tons in 2030 (UI 4193–59,295). The Asia–Pacific region represents the largest share (93.8%) of global consumption, with China alone contributing 57.9% of global consumption in 2017. Antimicrobial consumption intensity per species group was: catfish, 157 mg kg−1 (UI 9–2751); trout, 103 mg kg−1 (UI 5–1951); tilapia, 59 mg kg−1 (UI 21–169); shrimp, 46 mg kg−1 (UI 10–224); salmon, 27 mg kg−1 (UI 17–41) and a pooled species group, 208 mg kg−1, (UI 70–622). All antimicrobial classes identified in the review are classified as medically important. We estimate aggregate global human, terrestrial and aquatic food animal antimicrobial use in 2030 at 236,757 tons (95% UI 145,525–421,426), of which aquaculture constitutes 5.7% but carries the highest use intensity per kilogram of biomass (164.8 mg kg−1). This analysis calls for a substantial scale-up of surveillance capacities to monitor global trends in antimicrobial use. Current evidence, while subject to considerable uncertainties, suggests that for some species groups antimicrobial use intensity surpasses consumption levels in terrestrial animals and humans. Acknowledging the fast-growing nature of aquaculture as an important source of animal nutrition globally, our findings highlight the urgent need for enhanced antimicrobial stewardship in a high-growth industry with broad links to water and ecosystem health.

Aquaculture management practices associated with antimicrobial residues in Southwestern Nigeria

Aquaculture production has grown rapidly in Nigeria but with unbridled use of antibiotics which predisposes fish consumers to the risks of antimicrobial residues and resistance. A cross sectional survey of antimicrobial use in aquaculture and occurrence of their residues in African Catfish (Clarias gariepinus) samples from Southwestern states of Nigeria were carried out. The results showed that majority of the farmers lacked knowledge of drug residues in aquatic animals, and the food safety and public health implications on consumers. Frequently administered antibiotics include tetracyclines (27.5%), chloramphenicol (22.0%) and gentamicin (18.4%). About 84.7% of tested fish had residues with variable prevalence which were not statistically significant. Risk factor analysis indicated that fish farmers with tertiary and secondary education were more likely to produce fish that contained antimicrobial residues than those with primary education [odds ratio (OR) = 1.91, confidence interval (CI) = 1.24–5.99 and OR = 1.25, CI = 1.08–5.27, respectively] while fish from farms managed by men were about three times more likely to contain residues (OR = 3.25, 95% CI = 1.95–9.35) than those managed by women. Hence, most of the fish from Southwestern Nigeria were indiscriminately dosed with different antibiotics that predispose consumers to the risks of antimicrobial residues with food safety and antimicrobial resistance consequences. Implementation of good fishery management practices and farmers' education are advocated to reduce dependence on antimicrobials.

Antimicrobial resistance genes in bacteria from animal-based foods.

Antimicrobial resistance is a worldwide public health threat. Farm animals are important sources of bacteria containing antimicrobial resistance genes (ARGs). Although the use of antimicrobials in aquaculture and livestock has been reduced in several countries, these compounds are still routinely applied in animal production, and contribute to ARGs emergence and spread among bacteria. ARGs are transmitted to humans mainly through the consumption of products of animal origin (PAO). Bacteria can present intrinsic resistance, and once antimicrobials are administered, this resistance may be selected and multiply. The exchange of genetic material is another mechanism used by bacteria to acquire resistance. Some of the main ARGs found in bacteria present in PAO are the bla, mcr-1, cfr and tet genes, which are directly associated to antibiotic resistance in the human clinic.

Freshwater salmon aquaculture in Chile and transferable antimicrobial resistance.

Large amounts of antimicrobials are used in salmonid aquaculture in Chile. Most are used in marine aquaculture, but appreciable amounts are also employed in freshwater aquaculture. Much research and many publications have examined transferable antimicrobial resistance in bacteria isolated from marine salmon farms, but much less attention has been paid to this area in freshwater salmon farming. A recent paper by Domínguez et al. (2019) has as least in part remedied this situation. We now comment on some of its interpretations and have attempted to point out its areas of strength and weakness in light of the published scientific literature. Seen in this setting, the important results presented by Domínguez et al. (2019) underline the need for increased awareness of the challenge to animal and human health posed by excessive use of antimicrobials in aquaculture.

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 >256μg. 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.

Integrative and Conjugative Elements-Positive Vibrio parahaemolyticus Isolated From Aquaculture Shrimp in Jiangsu, China.

The development of multidrug- and toxin-resistant bacteria as a result of increasing industrialization and sustained and intense antimicrobial use in aquaculture results in human health problems through increased incidence of food-borne illnesses. Integrative and conjugative elements (ICEs) are self-transmissible mobile genetic elements that allow bacteria to acquire complex new traits through horizontal gene transfer and encode a wide variety of genetic information, including resistance to antibiotics and heavy metals; however, there is a lack of studies of ICEs of environmental origin in Asia. Here, we determined the prevalence, genotypes, heavy metal resistance and antimicrobial susceptibility of 997 presumptive strains of Vibrio parahaemolyticus (tlh+, tdh–), a Gram-negative bacterium that causes gastrointestinal illness in humans, isolated from four species of aquaculture shrimp in Jiangsu, China. We found that 59 of the 997 isolates (5.9%) were ICE-positive, and of these, 9 isolates tested positive for all resistance genes. BLAST analysis showed that similarity for the eight strains to V. parahaemolyticus was 99%. Tracing the V. parahaemolyticus genotypes, showed no significant relevance of genotype among the antimicrobial resistance strains bearing the ICEs or not. Thus, in aquaculture, ICEs are not the major transmission mediators of resistance to antibiotics or heavy metals. We suggest future research to elucidate mechanisms that drive transmission of resistance determinants in V. parahaemolyticus.

Effects of oxytetracycline residues in Thai Koi (Anabas testudineus Bloch) collected from Sylhet, Bangladesh

The present study was conducted to determine the levels of oxytetracycline residues in Thai Koi (Anabas testudineus Bloch) collected from some local fish markets of Sylhet Sadar Upazilla, Bangladesh during March to August, 2016. For the purpose of the study, 24 fresh Thai Koi fish samples were randomly collected from four (4) local markets of Sylhet Sadar Upazilla. Concentration of oxytetracycline residues in collected fish samples were determined by High Performance Liquid Chromatography (HPLC) at the Food Toxicology Laboratory of Institute of Food Science and Technology, Bangladesh Council of Scientific and Industrial Research, Dhaka. The results showed that 9 (37.50%) samples with detectable amount of oxytetracycline residues (mean = 42.30±3.00 ppb) were found in Thai Koi. Oxytetracycline residues level below detection limit was also reported in 15 (62.50%) samples of Thai Koi during this study. The detected residues of oxytetracycline in this fish sample did not exceed the maximum residue limit (MRL) 100 ppb recommended by the European Commission. Though residue level of oxytetracycline is lower than the MRL but long term exposure could be hazardous for human health. For that reason, control of antibiotic usage in aquaculture, regular residue monitoring, legislations and regulations for the use of antimicrobials in aquaculture and food safety education are utmost things in this regard.

Assessing concentration of antibiotics in tissue during oral treatments against piscirickettsiosis

The use of antimicrobials in aquaculture is increasingly being scrutinized. In Chile, piscirickettsiosis accounts for approximately 90% of the total volume of antibiotics used in marine aquaculture. Treatment failures are frequently reported, but there is limited information on why this occurs. Fish producers have started assessing the level of antibiotics in fish tissues during and immediately after in-feed treatments to determine if they are adequately medicating their fish. In this study, we evaluated the probability of finding antibiotic concentrations in muscle tissue above the minimum inhibitory concentration for 90% of the P. salmonis isolates (MIC90) recently tested in Chile, for two antibiotics commonly used in aquaculture. We found that the proportion of fish with antibiotic concentrations above the MIC90 varied, depending on the product used, species, day of sample collection, and size category of fish within a cage. The proportion of fish above the MIC90 was lower in fish treated with florfenicol than in fish treated with oxytetracycline. Using a mixed-effects logistic model, we modeled the probability of antibiotic concentrations above MIC90 when fish were treated with florfenicol. Our model suggested lower probabilities of having concentrations above MIC90 in Atlantic salmon than in rainbow trout when samples were collected 14 days after the treatment started compared to 7 days, and in the smaller fish within a cage. We discuss these findings and hypothesize about potential issues with treating large populations of fish with in-feed antimicrobials.

Tackling water pollution: real-time monitoring of residual antimicrobials concentration in aquaculture with microwave spectroscopy

To prevent the improper use of antimicrobials in aquaculture and to assist the food safety law enforcement, this paper reports on a bespoke electromagnetic (EM) wave sensing method for real-time in situ monitoring of residual antimicrobials concentrations in water samples. The antimicrobials solutions were tested in bespoke microwave cavity. Transmitted and reflected power signals were analysed in GHz frequency range and these were dependent on both: the type of antimicrobials present in water and on their concentration.

Evaluation of antimicrobials residues in farmed gilthead seabream (Sparus aurata) after administration through medicated feed

The use of antimicrobials in aquaculture is a well-known fact and merits the focus of the scientific community. In the present study, five drugs (oxytetracycline, sulfadiazine, trimethoprim, oxolinic acid and flumequine) were selected to assess their retention in muscle tissues from gilthead seabream (Sparus aurata). Fish were placed in 150 L tanks at 18 °C, and fed for 7 days with experimental diets containing two concentrations of each antimicrobial (ranging from 5.51 to 131.16 mg kg−1). Edible tissues were then analyzed through a validated multi-class quantification method (UHPLC-MS/MS). The results indicate that sulfadiazine concentrations were the highest immediately after the feeding period and decreased towards day 3. Flumequine was only detected on the first day with concentrations below the MRL. Both trimethoprim and oxolinic acid concentrations were below the MRLs 3 days after the feeding period was over (oxolinic acid was not detected in muscle samples at day 14 for prophylaxis and day 28 for both treatments). Oxytetracycline residues in muscle tissues were the highest through time, with concentrations above the MRL for 7 days (Cday7 of 111.2 and 157.2 μg kg−1 for both dosages). Results suggest that these antimicrobials can be present in gilthead seabream muscle samples for longer periods than previously reported, when realistic conditions are tested. With the exception of oxytetracycline, concentrations were below the MRLs established 3 days after the feeding trial was over meaning that adverse effects related to human consumption are not likely. Nevertheless, allergic reactions or resistance to antimicrobials can be developed if low concentrations of such compounds are ingested on a frequent basis, as is the case of the Mediterranean diet.

UPLC-MS/MS determination of florfenicol and florfenicol amine antimicrobial residues in tilapia muscle.

Despite the benefits to fish farmers, the use of antimicrobials in aquaculture has concerned consumers and competent authorities. The indiscriminate use of such substances promotes the emergence of resistant microorganisms, decreases the effectiveness of treatments, and causes possible toxic effects in humans. In Brazil, florfenicol is the only antimicrobial registered for use in aquaculture and is often used in tilapia in cage creation. Thus, this study aimed to develop a method for determination of florfenicol residues and its metabolite florfenicol amine in tilapia fillet by UPLC-MS/MS. Analytes were extracted with ethyl acetate, followed by liquid-liquid partition clean-up with hexane and SPE. The sorbents C18, phenyl and HLB-Oasis were evaluated by SPE. Phenyl sorbent showed the best results, and the extraction conditions were optimized in the sample matrix with fractional factorial design 24-1. The analytes were separated on a C18 chromatographic column (50×2.1mm×1.7μm) using water (A) and acetonitrile (B) as mobile phase at a flow rate of 0.3mLmin-1 with a linear gradient (in% B): 0-2.0min: 20%; 2.0-2.5min: increase to 90%; 2.5-3.5min: 90%; 3.0-3.5min: decrease to 20%; 4.0-5.0min: 20%. The analytes were monitored in a MS/MS triple quadrupole system by MRM mode with transitions at m/z 356.1>336.1 (florfenicol) and m/z 248.1>130.1 (florfenicol amine). The optimized method was validated obtaining LOQ values of 3 and 25ngg-1 for florfenicol and florfenicol amine, respectively, precision between 20 and 36%, absolute extraction efficiency between 38 and 80%, and adequate linearity. The method was applied to samples intended for human consumption, and within the 15 evaluated samples, only one showed florfenicol residue at 30ngg-1, which is below the maximum residue limit established in Brazil.

Multi-Drug Resistance Mediated by Class 1 Integrons in Aeromonas Isolated from Farmed Freshwater Animals.

Aeromonas is regarded as an important pathogen of freshwater animals but little is known about the genetics of its antimicrobial resistance in Chinese aquaculture. The aim of this study was to investigate the presence of integrons and characterize multidrug resistant Aeromonas spp. isolated from diseased farmed freshwater animals. These animal samples included fish, ornamental fish, shrimp, turtles, and amphibians which were collected from 64 farms in Guangdong province of South China. One hundred and twelve Aeromonas spp. isolates were examined for antimicrobial resistance phenotypes and the presence of class 1 integron sequences. Twenty-two (19.6%) of these isolates carried a class 1 integron comprising six different gene insertion cassettes including drfA12-orfF-aadA2, drfA12-orfF, aac(6′)-II-blaOXA-21-cat3, catB3, arr-3, and dfrA17. Among these, drfA12-orfF-aadA2 was the dominant gene cassette array (63.6%, 14/22) and this is the first report of aac(6′)-II-blaOXA-21-cat3 in an Aeromonas hydrophila isolate from a Chinese giant salamander (Andrias davidianus). All the integron-positive strains were resistant to more than five agents and 22 contained other resistance genes including blaCTX-M-3, blaTEM-1, aac(6′)-Ib-cr, and tetA. All integron-positive isolates also contained mutations in the quinolone resistance determining regions (QRDR). Our investigation demonstrates that freshwater animals can serve as a reservoir for pathogenic Aeromonas strains containing multiple drug-resistance integrons. This data suggests that surveillance for antimicrobial resistance of animal origin and a prudent and responsible use of antimicrobials in aquaculture is necessary in these farms.