Genomic epidemiology of third-generation cephalosporin-resistant Escherichia coli from Argentinian pig and dairy farms reveals animal-specific patterns of co-resistance and resistance mechanisms.

Control measures are being introduced globally to reduce the prevalence of antibiotic resistance (ABR) in bacteria on farms. However, little is known about the current prevalence and molecular ecology of ABR in bacterial species with the potential to be key opportunistic human pathogens, such as Escherichia coli, on South American farms. Working with 30 dairy cattle farms and 40 pig farms across two provinces in central-eastern Argentina, we report a comprehensive genomic analysis of third-generation cephalosporin-resistant (3GC-R) E. coli, which were recovered from 34.8% (cattle) and 47.8% (pigs) of samples from fecally contaminated sites. Phylogenetic analysis revealed substantial diversity suggestive of long-term horizontal and vertical transmission of 3GC-R mechanisms. CTX-M-15 and CTX-M-2 were more often produced by isolates from dairy farms, while CTX-M-8 and CMY-2 and co-carriage of amoxicillin/clavulanate resistance and florfenicol resistance were more common in isolates from pig farms. This suggests different selective pressures for antibiotic use in these two animal types. We identified the ß-lactamase gene blaROB, which has previously only been reported in the family Pasteurellaceae, in 3GC-R E. coli. blaROB was found alongside a novel florfenicol resistance gene, ydhC, also mobilized from a pig pathogen as part of a new composite transposon. As the first comprehensive genomic survey of 3GC-R E. coli in Argentina, these data set a baseline from which to measure the effects of interventions aimed at reducing on-farm ABR and provide an opportunity to investigate the zoonotic transmission of resistant bacteria in this region. IMPORTANCE: Little is known about the ecology of critically important antibiotic resistance among bacteria with the potential to be opportunistic human pathogens (e.g., Escherichia coli) on South American farms. By studying 70 pig and dairy cattle farms in central-eastern Argentina, we identified that third-generation cephalosporin resistance (3GC-R) in E. coli was mediated by mechanisms seen more often in certain species and that 3GC-R pig E. coli were more likely to be co-resistant to florfenicol and amoxicillin/clavulanate. This suggests that on-farm antibiotic usage is key to selecting the types of E. coli present on these farms. 3GC-R E. coli and 3GC-R plasmids were diverse, suggestive of long-term circulation in this region. We identified the de novo mobilization of the resistance gene blaROB from pig pathogens into E. coli on a novel mobile genetic element, which shows the importance of surveying poorly studied regions for antibiotic resistance that might impact human health.

Serotype diversity and antimicrobial susceptibility profiles of Actinobacillus pleuropneumoniae isolated in Italian pig farms from 2015 to 2022.

Actinobacillus pleuropneumoniae (APP) is a bacterium frequently associated with porcine pleuropneumonia. The acute form of the disease is highly contagious and often fatal, resulting in significant economic losses for pig farmers. Serotype diversity and antimicrobial resistance (AMR) of APP strains circulating in north Italian farms from 2015 to 2022 were evaluated retrospectively to investigate APP epidemiology in the area. A total of 572 strains isolated from outbreaks occurring in 337 different swine farms were analysed. The majority of isolates belonged to serotypes 9/11 (39.2%) and 2 (28.1%) and serotype diversity increased during the study period, up to nine different serotypes isolated in 2022. The most common resistances were against tetracycline (53% of isolates) and ampicillin (33%), followed by enrofloxacin, florfenicol and trimethoprim/sulfamethoxazole (23% each). Multidrug resistance (MDR) was common, with a third of isolates showing resistance to more than three antimicrobial classes. Resistance to the different classes and MDR varied significantly depending on the serotype. In particular, the widespread serotype 9/11 was strongly associated with florfenicol and enrofloxacin resistance and showed the highest proportion of MDR isolates. Serotype 5, although less common, showed instead a concerning proportion of trimethoprim/sulfamethoxazole resistance. Our results highlight how the typing of circulating serotypes and the analysis of their antimicrobial susceptibility profile are crucial to effectively manage APP infection and improve antimicrobial stewardship.

Dechlorination Helps Defluorination: Insights into the Defluorination Mechanism of Florfenicol by S-nZVI and DFT Calculations on the Reaction Pathways.

Defluorination is essential to eliminate the antibiotic resistance and detrimental effects of florfenicol (C12H14Cl2FNO4S, FF), which is achievable by sulfidated nanoscale zerovalent iron (S-nZVI), yet a comprehensive understanding of the mechanism is lacking. Herein, we used experimental data and density functional theory calculations to demonstrate four dechlorination-promoted defluorination pathways of FF, depending on S-nZVI or not. FF was defluorinated in a rapid and then slow but continuous manner, accompanying a consecutive dechlorination to deschloro (dFF) and dideschloro FF (ddFF). Unexpectedly, the predominant defluorination occurs by spontaneous hydrolysis of ddFF to form the hydrolyzed byproduct (HO-ddFF), i.e., independent of S-nZVI, which is initiated by intramolecular attack from carbonyl O to alkyl F and is thus limited for FF and dFF owing to the diminished nucleophilicity by electron-withdrawing Cl. The removal of Cl also makes the reductive defluorination of ddFF by S-nZVI amenable. The other two minor but more rapid defluorination pathways occur in synergy with the dechlorination of FF and dFF, which are mediated by the reactive carbanion intermediates and generate HO-dFF and HO-ddFF, respectively. The reliability of these dechlorination-facilitated defluorination pathways was verified by the consistency of theoretical calculations with experimental data, providing valuable insights into the degradation of fluorinated contaminants.

A review on the antibiotic florfenicol: Occurrence, environmental fate, effects, and health risks.

Florfenicol, as a replacement for chloramphenicol, can tightly bind to the A site of the 23S rRNA in the 50S subunit of the 70S ribosome, thereby inhibiting protein synthesis and bacterial proliferation. Due to the widespread use in aquaculture and veterinary medicine, florfenicol has been detected in the aquatic environment worldwide. Concerns over the effects and health risks of florfenicol on target and non-target organisms have been raised in recent years. Although the ecotoxicity of florfenicol has been widely reported in different species, no attempt has been made to review the current research progress of florfenicol toxicity, hormesis, and its health risks posed to biota. In this study, a comprehensive literature review was conducted to summarize the effects of florfenicol on various organisms including bacteria, algae, invertebrates, fishes, birds, and mammals. The generation of antibiotic resistant bacteria and spread antibiotic resistant genes, closely associated with hormesis, are pressing environmental health issues stemming from overuse or misuse of antibiotics including florfenicol. Exposure to florfenicol at µg/L-mg/L induced hormetic effects in several algal species, and chromoplasts might serve as a target for florfenicol-induced effects; however, the underlying molecular mechanisms are completely lacking. Exposure to high levels (mg/L) of florfenicol modified the xenobiotic metabolism, antioxidant systems, and energy metabolism, resulting in hepatotoxicity, renal toxicity, immunotoxicity, developmental toxicity, reproductive toxicity, obesogenic effects, and hormesis in different animal species. Mitochondria and the associated energy metabolism are suggested to be the primary targets for florfenicol toxicity in animals, albeit further in-depth investigations are warranted for revealing the long-term effects (e.g., whole-life-cycle impacts, multigenerational effects) of florfenicol, especially at environmental levels, and the underlying mechanisms. This will facilitate the evaluation of potential hormetic effects and construction of adverse outcome pathways for environmental risk assessment and regulation of florfenicol.

Effect of UV pretreatment on the source control of floR during subsequent biotreatment of florfenicol wastewater.

UV photolysis has been recommended as an alternative pretreatment method for the elimination of antibacterial activity of antibiotics against the indicator strain, but the pretreated antibiotic intermediates might not lose their potential to induce antibiotic resistance genes (ARGs) proliferation during subsequent biotreatment processes. The presence of florfenicol (FLO) in wastewater seriously inhibits the metabolic performance of anaerobic sludge microorganisms, especially the positive correlation between UV irradiation doses and ATP content, while it did not significantly affect the organics utilization ability and protein biosynthetic process of aerobic microorganisms. After sufficient UV pretreatment, the relative abundances of floR from genomic or plasmid DNA in subsequent aerobic and anaerobic biotreatment processes both decreased by two orders of magnitude, maintained at the level of the groups without FLO selective pressure. Meanwhile, the abundances of floR under anaerobic condition were always lower than that under aerobic condition, suggesting that anaerobic biotreatment systems might be more suitable for the effective control of target ARGs. The higher abundance of floR in plasmid DNA than in genome also indicated that the potential transmission risk of mobile ARGs should not be ignored. In addition, the relative abundance of intI1 was positively correlated with floR in its corresponding genomic or plasmid DNA (p < 0.05), which also increased the potential horizontal transfer risk of target ARGs. This study provides new insights into the effect of preferential UV photolysis as a pretreatment method for the enhancement of metabolic performance and source control of target ARGs in subsequent biotreatment processes. KEY POINTS: • Sufficient UV photolytic pretreatment efficiently controlled the abundance of floR • A synchronous decrease in abundance of intI1 reduced the risk of horizontal transfer • An appreciable abundance of floR in plasmid DNA was a potential source of total ARGs.

Identification and florfenicol-treatment of pseudomonas putida infection in gilthead seabream (Sparus aurata) fed on tilapia-trash-feed.

The present study aimed to determine the major cause of the high mortality affecting farmed gilthead seabream (Sparus aurata) and controlling this disease condition. Fifteen diseased S. aurata were sampled from a private fish farm located at Eldeba Triangle, Damietta, fish showed external skin hemorrhages, and ulceration. Bacterial isolates retrieved from the diseased fish were identified biochemically as Pseudomonas putida and then confirmed by phylogenetic analysis of the 16 S rRNA gene sequence. P. putida was also isolated from three batches of tilapia-trash feed given to S. aurata. Biofilm and hemolytic assay indicated that all P. putida isolates produced biofilm, but 61.11% can haemolyse red blood cells. Based on the antibiotic susceptibility test results, P. putida was sensitive to florfenicol with minimum inhibitory concentrations ranging between 0.25 and 1.0 µg mL- 1, but all isolates were resistant to ampicillin and sulfamethoxazole-trimethoprim. Pathogenicity test revealed that P. putida isolate (recovered from the tilapia-trash feed) was virulent for S. aurata with LD50 equal to 4.67 × 107 colony forming unit (CFU) fish- 1. After intraperitoneal (IP) challenge, fish treated with 10 mg kg- 1 of florfenicol showed 16.7% mortality, while no mortality was recorded for the fish group that received 20 mg kg- 1. The non-treated fish group showed 46.7% mortality after bacterial challenge. HPLC analysis of serum florfenicol levels reached 1.07 and 2.52 µg mL- 1 at the 5th -day post-drug administration in the fish groups received 10 and 20 mg kg- 1, respectively. In conclusion, P. putida was responsible for the high mortality affecting cultured S. aurata, in-feed administration of florfenicol (20 mg kg- 1) effectively protected the challenged fish.

Influence of probiotic and prebiotic supplementation on intestinal microbiota and resistance to Edwardsiella ictaluri infection in channel catfish (Ictalurus punctatus) following florfenicol administration.

Enteric septicemia of catfish (ESC), caused by the gram-negative enteric bacteria Edwardsiella ictaluri, is a significant threat to catfish aquaculture in the southeastern United States. Antibiotic intervention can reduce mortality; however, antibiotic use results in an imbalance, or dysbiosis, of the gut microbiota, which may increase susceptibility of otherwise healthy fish to enteric infections. Herein, recovery of the intestinal microbiota and survivability of channel catfish in response to ESC challenge was evaluated following a 10-day course of florfenicol and subsequent probiotic or prebiotic supplementation. Following completion of florfenicol therapy, fish were transitioned to a basal diet or diets supplemented with a probiotic or prebiotic for the remainder of the study. Digesta was collected on Days 0, 4, 8 and 12, beginning on the first day after cessation of antibiotic treatment, and gut microbiota was characterized by Illumina sequencing of the 16S rRNA gene (V4 region). Remaining fish were challenged with E. ictaluri and monitored for 32 days post-challenge. Florfenicol administration resulted in dysbiosis characterized by inflated microbial diversity, which began to recover in terms of diversity and composition 4 days after cessation of florfenicol administration. Fish fed the probiotic diet had higher survival in response to ESC challenge than the prebiotic (p = .019) and negative control (p = .029) groups.

Mechanism of sturgeon intestinal inflammation induced by Yersinia ruckeri and the effect of florfenicol intervention.

The mechanism by which Y. ruckeri infection induces enteritis in Chinese sturgeon remains unclear, and the efficacy of drug prevention and control measures is not only poor but also plagued with numerous issues. We conducted transcriptomic and 16 S rRNA sequencing analyses to examine the differences in the intestinal tract of hybrid sturgeon before and after Y. ruckeri infection and florfenicol intervention. Our findings revealed that Y. ruckeri induced the expression of multiple inflammatory factors, including il1ß, il6, and various chemokines, as well as casp3, casp8, and multiple tumor necrosis factor family members, resulting in pathological injury to the body. Additionally, at the phylum level, the relative abundance of Firmicutes and Bacteroidota increased, while the abundance of Plesiomonas and Cetobacterium decreased at the genus level, altering the composition of the intestinal flora. Following florfenicol intervention, the expression of multiple apoptosis and inflammation-related genes was down-regulated, promoting tissue repair. However, the flora became further dysregulated, increasing the risk of infection. In conclusion, our analysis of the transcriptome and intestinal microbial composition demonstrated that Y. ruckeri induces intestinal pathological damage by triggering apoptosis and altering the composition of the intestinal microbiota. Florfenicol intervention can repair pathological damage, but it also exacerbates flora imbalance, leading to a higher risk of infection. These findings help elucidate the molecular mechanism of Y. ruckeri-induced enteritis in sturgeon and evaluate the therapeutic effect of drugs on intestinal inflammation in sturgeon.

Assessing the ecotoxicity of florfenicol exposure at environmental levels: A case study of histology, apoptosis and microbiota in hepatopancreas of Eriocheir sinensis.

The intensification of production practices in the aquaculture industry has led to the indiscriminate use of antibiotics to combat diseases and reduce costs, which has resulted in environmental pollution, posing serious threats to aquaculture sustainability and food safety. However, the toxic effect of florfenicol (FF) exposure on the hepatopancreas of crustaceans remains unclear. Herein, by employing Chinese mitten crab (Eriocheir sinensis) as subjects to investigate the toxic effects on histopathology, oxidative stress, apoptosis and microbiota of hepatopancreas under environment-relevant (0.5 and 5 µg/L), and extreme concentrations (50 µg/L) of FF. Our results revealed that the damage of hepatopancreas tissue structure caused by FF exposure in a dose-and time-dependent manner. Combined with the increased expression of apoptosis-related genes (Caspase 3, Caspase 8, p53, Bax and Bcl-2) at mRNA and protein levels, activation of catalase (CAT) and superoxide dismutase (SOD), and malondialdehyde (MDA) accumulation, FF exposure also induced oxidative stress, and apoptosis in hepatopancreas. Interestingly, 7 days exposure triggered more pronounced toxic effect in crabs than 14 days under environment-relevant FF concentration. Integrated biomarker response version 2 (IBRv2) index indicated that 14 days FF exposure under extreme concentration has serious toxicity effect on crabs. Furthermore, 14 days exposure to FF changed the diversity and composition of hepatopancreas microbiota leading remarkable increase of pathogenic microorganism Spirochaetes following exposure to 50 µg/L of FF. Taken together, our study explained potential mechanism of FF toxicity on hepatopancreas of crustaceans, and provided a reference for the concentration of FF to be used in culture of Chinese mitten crab.

Enhancement of dissolution rate and oral bioavailability of poorly soluble drug florfenicol by using solid dispersion and effervescent disintegration technology.

OBJECTIVE: Florfenicol(FF) is an excellent veterinary antibiotic, limited by poor solubility and poor bioavailability. SIGNIFICANCE: Here in, we aimed to explore the applicability of fast disintegrating tablets compressed from Florfenicol-loaded solid dispersions (FF-SD-FDTs) to improve the dissolution rate and oral bioavailability of Florfenicol. METHODS: Utilizing selecting appropriate preparation methods and carriers, the solid dispersions of Florfenicol (FF-SDs) were prepared by solvent evaporation and the fast disintegrating tablets (FF-SD-FDTs) were prepared by the direct compression (DC) method. RESULTS: The tablet properties including hardness, friability, disintegration time, weight variation, etc. all met the specifications of Chinese Veterinary Pharmacopeia(CVP). FF-SD-FDTs significantly improved drug dissolution and dispersion of FF in vitro compared to florfenicol conventional tablets (FF-CTs). A pharmacokinetics study in German shepherd dogs proved the AUC0-∞ and Cmax values of FF-SD-FDTs are 1.38 and 1.38 times more than FF-CTs, respectively. CONCLUSIONS: Overall, it can be concluded that FF-SD-FDTs with excellent disintegration and dissolution properties were successfully produced, which greatly improved the oral bioavailability of the poorly soluble drug FF, and the study provided a new idea for a broader role of FF in pet clinics.

Florfenicol urinary excretion and its potential for treating canine urinary tract infections.

The canine urinary excretion of florfenicol was evaluated to explore its potential for treating urinary tract infections. Nine healthy male intact purpose-bred Beagles and four healthy client-owned dogs each received a single oral dose of florfenicol 20 mg/kg (300 mg/mL parenteral solution) with food. All voluntary urinations were collected for 12 h. Although florfenicol is reportedly bitter tasting, 7/9 Beagles and 4/4 client-owned dogs completely ingested the florfenicol and were enrolled; salivation (n = 1) and headshaking (n = 3) were observed. The last measured urine florfenicol concentrations were variable: Beagles (0.23-3.19 mcg/mL), Pug (3.01 mcg/mL) English Setter (21.29 mcg/mL), Greyhound (32.68 mcg/mL), and Standard Poodle (13.00 mcg/mL). Urine half-life was similar for the Beagles and the Pug, 0.75-1.39 h, whereas the half-life was 1.70-1.82 h for the English Setter, Greyhound, and Standard Poodle. Larger breed dogs exceeded 8 mcg/mL florfenicol (wild-type cutoff) in their urine at 12 h, whereas the Beagles and Pug had <8 mcg/mL; it is unclear if this is an individual, breed, or size difference. These data suggest oral florfenicol may need to be administered q6-12h for canine urinary tract infections, but further data are needed (more enrolled dogs, multiple-dose regimens) before considering clinical trials or breed-specific differences.

Development of a QuEChERS-HPLC-FLD Procedure for the Simultaneous Detection of Residues of Florfenicol, Its Metabolite Florfenicol Amine, and Three Fluoroquinolones in Eggs.

A method utilizing high-performance liquid chromatography-fluorescence detection (HPLC-FLD) has been developed and refined for the simultaneous detection of florfenicol (FF) and its metabolite florfenicol amine (FFA) along with three fluoroquinolone (ciprofloxacin (CIP), enrofloxacin (ENR), and sarafloxacin (SAR)) residues in different parts of eggs (whole egg, egg yolk, and egg albumen). The QuEChERS ("Quick, easy, cheap, effective, rugged, and safe") procedure utilized 0.1 M disodium EDTA solution, water, and acetonitrile as extractants; sodium sulfate, sodium chloride, and trisodium citrate as dehydrating salts; and N-propylethylenediamine and C18 as adsorbents. A dual-channel FLD method was utilized to analyze the target compounds using an XBridge BEH C18 chromatographic column (4.6 mm × 150 mm, 5 µm). The mobile phase was employed isocratically using a solution of 0.01 M sodium dihydrogen phosphate, 0.005 M sodium dodecyl sulfate, and 0.1% triethylamine (pH 4.8) in combination with acetonitrile at a ratio of 65:35 (V/V). The limits of detection (LOD) and quantification (LOQ) of the analytes ranged from 0.03 to 1.5 µg/kg and from 0.1 to 5.0 µg/kg, respectively. The recoveries of the analytes in the blank egg samples ranged from 71.9% to 94.8% when reference standard concentrations of the LOQ, half of the maximum residual limit (MRL), MRL, and twice the MRL were added. The parameters of the presented protocol were validated and subsequently applied to the analysis of real samples, demonstrating the applicability and reliability of the method.

Intestinal Histopathological Aberrations in Oreochromis niloticus Juveniles upon Dietary Florfenicol Administration.

The aquaculture use of antibiotics can cause detrimental effects on fish organs and gut microbial dysbiosis. The impact of florfenicol (FFC) on fish intestinal histology, an approved antibiotic, remains unclear. This study aimed to investigate the effects of FFC on Oreochromis niloticus juveniles by administering FFC at 10 mg and 30 mg/kg biomass/day for 30 consecutive days to mimic long-term use. A dose-dependent reduction in feed intake, survival and biomass, with an upsurge in mortalities was observed. Even the therapeutic dose instigated mortalities on day 30 of FFC dosing (FD). Histopathological analysis revealed mild to moderate alterations, including loss of absorptive regions, epithelial degeneration, necrotized areas, intercellular enterocytic space and swollen laminar propria. Post-dosing, the observation of the detachment of lamina propria from the epithelium indicated imminent irritability. Goblet cells reduced drastically on day 30 FD, accompanied by an increase in intraepithelial lymphocytes. However, cessation of dosing for 13 days resulted in the reclamation of goblet cells and absorptive regions, indicating that the intestinal tissues underwent considerable repair after lifting antibiotic pressure. These findings suggested that O. niloticus can tolerate dietary FFC but emphasize the need for responsible use of antibiotics in aquaculture.

Prospecting of essential oils in combination with florfenicol against motile Aeromonas isolated from tambaqui (Colossoma macropomum).

Natural products with antimicrobial activity and their association with synthetic antimicrobials are a sustainable option in fish farming. The objective of this study was to determine antimicrobial activity, antibiofilm potential and synergism of five essential oils (EOs) with florfenicol against motile Aeromonas isolated from Amazonian Colossoma macropomum. As their major constituent, the EOs of the species of Aloysia triphylla, Croton cajucara (red and white morphotype), Cymbopongo citratus and Lippia gracilis present ß-pinene (22.1%), germacrene D (11.5%), linalool (23%), geranial (45.7%) and carvacrol (42.2%), respectively. The EOs of L. gracilis and C. citratus showed the best antimicrobial activities against the Aeromonas strains (5 mg mL-1). All EOs interfered with biofilm formation and consolidated biofilm. The EOs of A. triphylla, C. citratus and L. gracilis showed a synergistic effect with florfenicol, reducing the amount of the chemical into the water systems while treatment.

Ultra-sensitive detection of florfenicol by flow injection chemiluminescence immunoassay based on Nickel/Cobalt bimetallic metal-organic framework nanozymes.

The emergence and progress of metal-organic frameworks (MOFs) with high stability, large surface area, and abundant unsaturated active sites, once again promote the development of nanozymes, making nanozymes more advantageous to replace natural enzymes and will increase the applications of chemiluminescence immunoassay. In this study, a flow injection chemiluminescence immunoassay based on Ni/Co metal-organic framework (Ni/Co-MOF) nanozymes was developed, which can quickly and highly sensitively detect florfenicol (FF) in animal-derived food residues. Ni/Co-MOF0.75 nanospheres can not only form stable immune probes with antibodies but also act as nanozymes to efficiently catalyze H2O2 for amplifying the chemiluminescence signal of the luminol-H2O2 system. In addition, due to good biocompatibility and large specific surface area, carboxyl-modified resin beads are used as a suitable material for loading more coating antigens. Based on the principle of competitive immunity, FF standard solution will compete with coating antigen loaded on the carboxyl resin beads for the limited binding sites on the FF antibody. Under the best experimental conditions, the detection range of FF is 0.0001-1000 ng mL-1, and the detection limit (LOD) is 0.033 pg mL-1 (S/N = 3). Furthermore, this method has been successfully applied to the analysis of actual samples with satisfactory results, which will provide a certain reference for the detection of small molecules in food and environmental analysis.

Atlantic Salmon (Salmo salar) bacterial and viral innate immune responses are not impaired by florfenicol or tetracycline administration.

Antibiotics are used to treat bacterial infections in fish aquaculture, and these drugs can interact with immune cells/the immune system and potentially leave fish vulnerable to viral, fungal, parasitic, or other bacterial infections. However, the effects of antibiotics on fish immunity have largely been overlooked by the aquaculture industry. We tested, at 12 and 20 °C, whether tetracycline and florfenicol (the most commonly used antibiotics in commercial aquaculture), affected the Atlantic salmon's capacity to respond to bacterial or viral stimulation. Atlantic salmon were acclimated to 12 or 20 °C and fed with tetracycline or florfenicol (100 and 10 mg kg of body weight-1 day-1, respectively) medicated feed for 15 or 10 days, respectively. Thereafter, we evaluated their immune function prior to, and after, an intraperitoneal injection of Forte Micro (containing inactivated cultures of Aeromonas salmonicida, Vibrio anguillarum, Vibrio ordalii and Vibrio salmonicida) or the viral mimic polyriboinosinic polyribocytidylic acid (pIC). We measured the transcript expression levels of 8 anti-bacterial and 8 anti-viral putative biomarker genes, and the innate (leukocyte respiratory burst, plasma lysozyme activity and hemolytic activity of the alternative complement pathway) and cellular (relative number of erythrocytes, lymphocytes and thrombocytes, and granulocytes such as monocytes and neutrophils) responses to these challenges. Overall, we only found a few minor effects of either tetracycline or florfenicol on immune gene expression or function at either temperature. Although several studies have reported that antibiotics may negatively affect fish immune responses, our results show that industry-relevant dietary tetracycline and florfenicol treatments do not substantially impact the salmon's innate immune responses. Currently, this is the most comprehensive study on the effects of antibiotics administrated according to industry protocols on immune function in Atlantic salmon.

Antibacterial and antibiofilm activities and synergism with florfenicol from the essential oils of Lippia sidoides and Cymbopogon citratus against Aeromonas hydrophila.

AIMS: Aeromonas hydrophila is an opportunistic bacterium, with a high capacity for biofilm production, which can cause severe damage in aquaculture. The objective of this study was to identify the chemical compounds of the essential oils of Lippia sidoides (EOLS) and Cymbopogon citratus (EOCC), and to evaluate the biocidal, antibiofilm and synergistic action with the antimicrobial florfenicol of these essential oils (EOs) against A. hydrophila. METHODS AND RESULTS: The antibacterial activity of EOLS and EOCC was verified by the minimum bactericidal concentration and by the action of these EOs against both forming and consolidated biofilms. The synergistic activity of EOs with florfenicol was performed using the checkerboard technique. The main component of EOLS and EOCC was carvacrol (44.50%) and α-citral (73.56%), respectively. Both EOs showed weak inhibitory activity (≥3125.00 µg ml-1 ). Two bacterial isolates were able to produce biofilm, and EOLS and EOCC acted upon the bacterial isolates to prevent biofilm formation. A bactericidal effect was verified for EOLS in the previously consolidated biofilm for both isolates and for EOCC in only one of the isolates. In general, EOLS had a synergistic effect with florfenicol, while EOCF had an additive effect. CONCLUSIONS: Both EOs were able to interfere with biofilm formation and did not have an antagonistic effect in combination with florfenicol. The best results were found for EOLS, which showed a synergistic effect with florfenicol and the ability to interfere in the formation of consolidated biofilm. SIGNIFICANCE AND IMPACT OF THE STUDY: This study highlights the potential of EOLS and EOCC to interfere in biofilm and act in synergy with florfenicol to reduce the occurrence of A. hydrophila. Development of these compounds may contribute to the development of herbal medicines in aquaculture.

Microbiota composition and susceptibility to florfenicol and oxytetracycline of bacterial isolates from mussels (Mytilus spp.) reared on different years and distance from salmon farms.

Chilean aquaculture mainly produces salmonids and molluscs. Salmonid production has been questioned by its excessive use of antimicrobials. This study aimed to investigate the bacterial microbiota composition of Mytilus spp. cultivated near salmonid farms and to determine the minimum inhibitory concentration (MIC) to florfenicol and oxytetracycline of its culturable bacteria. Seven Mytilus farming sites classified according to their proximity to salmon farms as close (CSF) or distant (DSF) were sampled in two years. We analyzed Mytilus microbiota composition through culture-independent methods, and isolated culturable bacteria, and identified those isolates with MIC values ≥ 64 µg mL-1 to florfenicol or oxytetracycline. Results revealed that the alpha diversity was affected by sampling year but not by Mytilus farming site location or its interaction. Nevertheless, in 2018, we observed a significant negative correlation between the alpha diversity of Mytilus microbiota in each farm sites and the tonnes of florfenicol reported for each phytosanitary management area. We detected significant differences in beta diversity and relative abundance of specific bacterial taxa in Mytilus microbiota depending on the proximity to salmon farms and years. A higher proportion of isolates with MIC values ≥ 64 µg mL-1 to both antibiotics was detected in 2019 compared to 2018, but not significant differences were detected according to Mytilus farming site location. However, in 2019, isolates from CSF sites showed higher MIC values for both antibiotics than those from DSF. Bacterial genera corresponding to isolates with MIC values ≥ 64 µg mL-1 represented a low proportion of Mytilus microbiota identified with the culture-independent approach, reflecting the need to implement new methodologies in the study of antimicrobial resistance. These results suggest that the proximity to salmonid farms and sampling year influence the Mytilus microbiota and MIC values of their bacterial isolates; however, other environmental variables should be considered in further studies.

Low-dose florfenicol and copper combined exposure during early life induced health risks by affecting gut microbiota and metabolome in SD rats.

The potential health risks associated with simultaneous presence of residues of heavy metals and antibiotics in the environment and food have been of wide concern. However, the adverse health effects of combined heavy metal and antibiotic exposure at low doses remain unclear. In this study, the effects of combined exposure to florfenicol and copper at low doses during early life on toxicity, gut microbiota, drug resistance genes, and the fecal metabolome were investigated in Sprague-Dawley (SD) rats. The results showed that combined exposure induced inflammatory responses and visceral injury as well as faster weight gain compared with florfenicol or copper exposure alone. Alpha and beta diversity indices indicated that the composition of the gut microbiota and the abundance of bacteria related to energy intake and disease in the combined exposure group were significantly altered. The increase in resistance genes (floR, fexA) induced by florfenicol exposure was suppressed under combined exposure to florfenicol and copper. The fecal metabolome also demonstrated that metabolic pathways related to energy intake and liver injury were significantly affected in the combined exposure group. In conclusion, this study shows that combined exposure to florfenicol and copper during early life can pose a nonnegligible health risk even if the exposure concentration of florfenicol or copper is below the safe limit.

Effects of early florfenicol exposure on glutathione signaling pathway and PPAR signaling pathway in chick liver.

Florfenicol (FFC) is a common antibiotic for animals. The nonstandard and excessive use of FFC can cause veterinary drug residues in animals, pollute soil and marine environment, and even threaten human health. Therefore, it is necessary to study the toxicity and side effects of FFC on animals. Our previous studies have proved that FFC can cause liver injury in chicks, but there are few in-depth studies on the mechanism of FFC causing liver injury at the level of signaling pathway in chicks. Therefore, transcriptome and proteome sequencing were performed and combined analysis was performed. Sequencing results showed that 1989 genes and 917 proteins were significantly changed in chick livers after FFC exposure. These genes and proteins are related to redox, glutathione transferase activity and lipid metabolism. There are 9 significantly different genes and 7 significantly different proteins in glutathione signaling pathway. Oxidative stress may occur in the liver of chicks through the change of activation state of glutathione signaling pathway. And there are 13 significantly different genes and 18 significantly different proteins in PPAR signaling pathway. The changes of PPAR signaling pathway may induce lipid metabolism disorder in liver. The verification results of qPCR and PRM were consistent with the sequencing results. We also detected GSH-Px, GSH, GST, TG, TC and ANDP levels in liver. These changes of biochemical indicators directly confirmed oxidative stress and lipid metabolism disorders were occurred in the livers of chicks treated by FFC. In conclusion, FFC could induce liver injury in chicks by regulating the expression levels of significantly different genes and proteins in glutathione signaling pathway and PPAR signaling pathway.