Florfenicol Concentrations Research Articles

Subinhibitory concentrations of florfenicol increase the biofilm formation of Piscirickettsia salmonis.

Public health is facing a new challenge due to the increased bacterial resistance to most of the conventional antibacterial agents. Inadequate use of antibiotics in the Chilean aquaculture industry leads to the generation of multidrug resistance bacteria. Many fish pathogenic bacteria produce biofilm upon various sources of stress such as antibiotics, which provides several survival advantages for the bacterial life in community and can constitute a reservoir of pathogens in the marine environment. Being florfenicol a broad-spectrum antibiotic commonly used to treat infections in aquaculture, the aim of this study was to assess whether this antibiotic modulates in vitro the biofilm formation in several isolates of Piscirickettsia salmonis. Standard antibiotic-micro broth 96-flat well plates were used to determinate the minimal inhibitory concentration of florfenicol in eight different P. salmonis isolates. In vitro findings, with P. salmonis growing in the presence and absence of the antibiotic, exhibited a statistically significantly increase (p < .05) in biofilm formation in all the bacterial isolates cultivated with sub-MIC (defined as the half of the minimal inhibitory concentration in the presence of antibiotic) of florfenicol compared with controls (antibiotic-free broth). In conclusion, sub-MIC of florfenicol induced an increased biofilm formation in all P. salmonis isolates tested.

Comparative pharmacokinetics of florfenicol in heifers after intramuscular and subcutaneous administration.

Florfenicol was administered to five heifers intramuscularly at a dose rate of 20 mg/kg bwt and following wash-out period, subcutaneously at a dose rate of 40 mg/kg bwt. Blood plasma samples were collected from heifers before injection of florfenicol and up to 120 h after intramuscular (IM) injection and up to 264 h after subcutaneous (SC) injection. Florfenicol concentrations in plasma were measured by high-performance liquid chromatography with mass-spectrometric detection. Pharmacokinetics of florfenicol was estimated using non-compartment analysis. Mean maximum plasma concentration, area under the concentration-time curve and elimination half-life for florfenicol were 3.2μg/ml, 101.5μg × h/ml and 24.5h, respectively, after IM injection at 20 mg/kg bwt, and 2.7μg/ml, 194.5μg × h/ml and 103.8h, respectively, after SC injection at 40 mg/kg bwt. The obtained results indicated that both administration routes provided comparable bioavailability, whereas SC route was attributed with lower peak levels and markedly slower absorption of florfenicol from injection site. Both administration routes provided plasma florfenicol levels which are expected to be effective against prevalent infectious agents of cattle.

Florfenicol and Florfenicol Amine Quantification in Bull Serum and Seminal Plasma by a Single Validated UHPLC-MS/MS Method.

Florfenicol is a broad-spectrum antibiotic belonging to the amphenicols class that inhibits protein synthesis by binding to bacteria's ribosomal subunits. This drug is commonly used in veterinary medicine to treat bacterial infectious diseases in cattle, swine, poultry, and fish. The proposed method uses a quick protein precipitation with acetonitrile for the extraction of florfenicol and florfenicol amine in serum and seminal plasma, followed by analysis in UHPLC-MS/MS for their simultaneous quantification. A BEH C18 reversed-phase column was chosen for analyte separation, allowing to obtaining sharp and symmetrical peak shapes in a chromatographic run of just 3.5 min under programmed conditions. Two specific transitions were observed for each analyte, and florfenicol-d3 was used as the internal standard. The approach was fully validated in each matrix over ranges suitable for field concentrations of florfenicol and florfenicol amine, showing good linearity during each day of testing (R2 always >0.99). Excellent accuracy and precision were demonstrated, for both analytes, by calculated bias always within ±15% and CV% always below 15% at all QC levels tested. The satisfactory outcomes obtained during recovery, matrix effect, and process efficiency investigations in serum and seminal plasma confirmed the strength of the method for the quantification of target compounds. To our knowledge, this is the first LC-MS/MS-validated approach for the quantification of florfenicol and florfenicol amine in serum and seminal plasma and was successfully applied for the determination of their concentration-time profiles in bulls. This paves the way to understanding the pharmacokinetics of this antibiotic and its active metabolite in bull's seminal plasma, which will enable the design of more appropriate treatment protocols.

Impact of emerging pollutant florfenicol on enhanced biological phosphorus removal process: Focus on reactor performance and related mechanisms

Florfenicol (FF), an emerging pollutant antibiotic that is difficult to biodegrade, inevitably enters sewage treatment facilities with high level. To date, however, the performance and related mechanism of FF on enhanced biological phosphorus removal (EBPR) have not been reported. In order to fill this gap, this work investigated the potential impacts of FF on EBPR and revealed the relevant mechanisms. The effect of FF on EBPR was dose-dependent, that was, low dose had no effect on EBPR, while high FF concentration inhibited EBPR. Mechanism investigation showed that FF had no effect on anaerobic phosphate release, but reduced oxic phosphorus uptake. Three-dimensional Excitation-emission Matrix fluorescence spectroscopy and X-ray photoelectron spectroscopy analysis showed that FF affected the structure and components of activated sludge extracellular polymers (EPS). High content of FF stimulated sludge to secrete more EPS. High level of FF reduced the relative abundance of microorganisms responsible for biological phosphorus removal. Microbiological community structure analysis indicated 2.0 mg FF/L increased the relative abundance of Candidatus_Competibacter and Terrimonas from 9.22 % and 12.49 % to 19.00 % and 16.28 %, respectively, but significantly reduced the relative abundance of Chinophagaceae from 11.32 % to 0.38 %, compared with the blank.

The "steric-like" inhibitory effect and mechanism of doxycycline on florfenicol metabolism: Interaction risk.

Most of the studies on doxycycline (DOX) and florfenicol (FF) remain focused on the improvement of antimicrobial activity and antimicrobial spectrum, and there is no relevant report on whether there is interaction between the two drugs after the combination. This research study evaluated the effect of DOX on FF metabolism in vitro and its mechanisms. The findings of this study showed that DOX inhibits FF metabolism in two ways. Firstly, DOX significantly inhibits the expression of CYP3A29, leading to the slower metabolism of FF; secondly, DOX affects the binding of FF to R106 and R372 by competing for the R372 and/or by a "steric-like effect", thus slowing down FF metabolism, which may increase the residual concentration of FF in edible tissues of food producing animals.

Antibiotics in a seasonal ice-sealed reservoir: Occurrence, temporal variation, prioritization, and source apportionment

Antibiotics are prevalent in the aquatic environment as emerging contaminants. Their knowledge of seasonal ice-sealed reservoirs, however, is limited. The occurrence, temporal variation, and prioritization of twenty-three antibiotics in Shitoukoumen Reservoir during its ice-free and ice-sealed periods, as well as the source apportionment of the high-priority antibiotics, were investigated in this study. The results showed that florfenicol was the dominant antibiotic in Shitoukoumen Reservoir, with different median concentrations of 75.0 ± 6.5 ng L−1 and 7.0 ± 1.7 ng kg−1 in the water and ice, respectively. The concentrations of florfenicol, sulfaguanidine, and sulfamonomethoxine in the water of the reservoir water varied over time, but their monthly mass loads from inflow rivers were similar during ice-free and ice-sealed periods. This indicated that other factors, such as aquacultural practice, non-point source rain runoff, and the blocking effect of ice, determined the temporal variations of the three antibiotics and resulted in their relatively high concentrations during the ice-free period. High-priority antibiotics included erythromycin, florfenicol, ofloxacin, sarafloxacin, sulfaquinoxaline, thiamphenicol, and tylosin. Aquaculture was the primary source of high-priority antibiotics, accounting for 67.3 % and 59.4 % of the total high-priority antibiotic concentrations during ice-free and ice-sealed periods, respectively. The findings suggest that aquaculture, rain runoff, and ice blocking should all be considered as factors influencing antibiotic variations in a seasonal ice-sealed reservoir.

Analysis of florfenicol in pig plasma using a validated PPT-HPLC-DAD method

High performance liquid chromatography (HPLC) has proven to be an effective tool for examining the disposition kinetics of florfenicol (FF), a synthetic broad-spectrum antibiotic used to treat infectious diseases in veterinary medicine. Modification and optimisation of the protein precipitation (PPT) sample preparation procedure and HPLC with diode array detector (DAD) instrumental method were carried out to ensure conditions suitable for FF analyses in pig plasma samples. Stable supernatants with good plasma mean recoveries of FF (99.8% ± 0.7%RSD) were achieved using 1% v/v phosphoric acid solution in methanol and 10% w/v sodium chloride in aqueous solution. The PPT-HPLCDAD method’s detection limit of 0.004 μg/mL and quantification limit of 0.013 μg/mL provides high sensitivity for analyses of FF in plasma samples. In addition, the optimisation of method conditions resulted in shorter extraction and analysis time and less solvent consumption, which stresses the sustainability of this method in analytical chemistry. The optimised and validated PPT-HPLC-DAD method was applied in a comparative study of FF in pig plasma after administration of veterinary medicinal products. The study was conducted on fattening pigs following repeated intramuscular administration of two similar solutions for injection at a dose of 20 mg FF/kg bodyweight (test groups 1 and 2). The solutions for injection contained the same FF concentration, i.e., 300 mg/mL, but differed in excipients. The aim was to examine the influence of administrated solutions for injection on the extent of exposure to FF in pig plasma. The dynamics of kinetic profiles of FF in pig plasma from both treatments correspond to the FF kinetic profiles published in similar studies. However, differences were observed in the concentrations of FF, which were constant throughout the study, and statistical differences between the test groups were confirmed (P&amp;lt;0.05). Though these findings suggest the possible influence of excipients, a full comprehensive conclusion on the influence of administrated solutions for injection on FF exposure in pig plasma requires additional research.

Pharmacokinetics, optimal dosages and withdrawal time of florfenicol in Asian seabass (Lates calcarifer) after oral administration via medicated feed.

Asian seabass (Lates calcarifer) is an economically important fish in Asian and Australian markets, but few pharmacokinetic (PK) data of antimicrobial drugs in this species is available. The present study investigated the PK behaviour of florfenicol (FF) through medicated feed in Asian seabass cultured at 25°C. The serum and muscle/skin concentrations of FF and its metabolite florfenicol amine (FFA) were determined by the HPLC-FLD method and analysed by one-compartmental model. The optimal dosages were determined by pharmacokinetic-pharmacodynamic (PK-PD) approach and the linear regression analysis was used to determine the withdrawal time (WDT). The PK study following a single oral administration of 15 mg/kg FF via medicated feed revealed that the absorption half-life (t1/2Ka ), elimination half-life (t1/2K ), peak concentration (Cmax ), area under the concentration-time curve (AUC), volume of distribution (Vd/F) and clearance (CL/F) were 1.47 h, 8.07 h, 8.61 μg/ml, 146.41 h·μg/ml, 1.19 L/kg and 0.102 L/kg/h, respectively. The muscle/skin concentration-time profile was similar to that of the serum, suggesting well distribution but only a small fraction of FF was metabolized to FFA. The optimal dosage for a minimum inhibitory concentration of 2μg/ml was calculated as 13.38 mg/kg/day. The appropriate WDT after multiple oral medications with 15 mg/kg FF once daily for 7 days was determined as 8 days. Information obtained from the current study can potentially be applied for the treatment of bacterial diseases in farming Asian seabass.

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 Broad-Spectrum Antibiotic (Florfenicol) on Resistance Genes and Bacterial Community Structure of Water and Sediments in an Aquatic Microcosm Model.

This study evaluates the effects of a broad-spectrum antibiotic (florfenicol) on antibiotic resistance genes (ARGs) and bacterial community structure in aquatic environments. We constructed an indoor aquatic microcosm model, adding different concentrations of florfenicol (0.1, 1, 10, 100 mg L−1), and water and sediment samples were collected after 0, 7, 30, and 60 days. qPCR and 16S rDNA amplicon sequencing were used to study the changes in the ARGs and bacterial community structure of the collected samples. The results show that the inclusion of florfenicol resulted in an increased abundance of the floR and optrA genes. Adding 100 mg L−1 florfenicol to the water increased the abundance of optrA gene copies with the maximum on the Day 7, and increased the abundance of floR gene copies with the maximum on Day 30. Adding 100 mg L−1 florfenicol to the sediment increased the abundance of floR and optrA genes by one order of magnitude on Day 60. Meanwhile, the average number of operational taxonomic units (OTUs) in the water samples was 257, and the average number of OTUs in sediment samples was 823. The bacterial community diversity and richness in sediments were higher than those in water. The difference between the maximal and minimal values of the Shannon diversity index in the water and sediment samples was 4.36 and 1.95, respectively. The effect of florfenicol on the bacterial community structure in water was much higher than that in sediment. At 30 days, the diversity index and richness index of the florfenicol treatment groups with 1 and 10 mg L−1 concentrations began to increase; at 60 days, the diversity and richness indices of the 100 mg L−1 florfenicol treatment group began to increase. The samples at the same sampling time in the sediments clustered closer together. The results of this study provide a scientific basis for guiding the rational use of florfenicol in aquaculture, maintaining a healthy and stable microecological environment in aquaculture, and provide theoretical data for environmental ecological risk assessment and safety management caused by microbial resistance under the abuse of florfenicol.

PK/PD integration of florfenicol alone and in combination with doxycycline against Riemerella anatipestifer.

Riemerella anatipestifer (RA) is an important pathogen found in poultry. RA infection can kill ducks and lead to significant economic losses. Seven RA strains with different susceptibility phenotypes were chosen to study the pharmacokinetic/pharmacodynamic (PK/PD) integration of florfenicol (FF) alone and in combination with doxycycline (DOX). The checkerboard assay indicated that synergy [fractional inhibitory concentration index (FICI) ≤ 0.5] was detected in the CVCC3952 strain of RA and that additivity (FICI >0.5 to ≤ 1) was observed in other strains. Static time–kill curves showed that the bactericidal effect of FF against RA was produced at a FF concentration ≥4 MIC, and the antibacterial activity of FF against RA was enhanced from the aspects of efficacy and efficacy in combination with DOX. Dynamic time–kill curves indicated that FF elicited bactericidal activity against the CVCC3857 strain with a reduction ≥4.88 log10CFU/ml when the dose was ≥8 mg/L. However, a bactericidal effect was not achieved at the maximum administered dose of FF monotherapy (20 mg/L) for isolates with a MIC ≥4 μg/ml. The effect of FF against RA was enhanced upon combination with DOX. The combination of FF with DOX reduced the bacterial burden ≥4.53 log10CFU/ml for all strains with a MIC ≥4 μg/ml. Data were fitted to a sigmoidal Emax model. The PK/PD parameters of AUC24h/MIC (the area under the concentration–time curve over 24 h divided by the MIC) and %T >MIC (the cumulative percentage of time over a 24-h period at which the concentration exceeded the MIC) of FF for eliciting a reduction of 3 log10CFU/ml was 40.10 h and 58.71, respectively. For strains with a MIC ≤ 16 μg/ml, the magnitude of the AUC24h/MIC and Cmax/MIC required for a 3 log10CFU/ml of bacterial killing was 34.84 h and 4.74 in the presence of DOX at 0.5 MIC, respectively. These data suggest that combination of FF with DOX enhanced the activity against RA strains with various susceptibilities to FF and DOX.

Comparison of florfenicol depletion in dairy goat milk using ultra-performance liquid chromatography with tandem mass spectrometry and a commercial on-farm test.

Florfenicol is a broad-spectrum antibiotic commonly prescribed in an extra-label manner for treating meat and dairy goats. Scientific data in support of a milk withdrawal interval recommendation is limited to plasma pharmacokinetic data and minimal milk residue data that is limited to cattle. Therefore, a rapid residue detection test (RRDT) could be a useful resource to determine if milk samples are free of drug residues and acceptable for sale. This study compared a commercially available RRDT (Charm® FLT strips) to detect florfenicol residues in fresh milk samples from healthy adult dairy breed goats treated with florfenicol (40 mg/kg subcutaneously twice 4 days apart) with quantitative analysis of florfenicol concentrations using ultra-performance liquid chromatography with tandem mass spectrometry (UPLC-MS/MS). In addition, storage claims for testing bovine milk using the RRDT were assessed using stored goat milk samples. Milk samples were collected every 12 h for a minimum of 26 days. Commercial RRDT strips remained positive in individual goats ranging from 528 to 792 h (22–33 days) after the second dose, whereas, UPLC-MS/MS indicated the last detectable florfenicol concentration in milk samples ranged from 504 to 720 h (21–30 days) after the second dose. Results from stored milk samples from treated goats indicate that samples can be stored for up to 5 days in the refrigerator and 60 days in the freezer after milking prior to being tested with a low risk of false-negative test results due to drug degradation. Elevated somatic cell counts and bacterial colony were noted in some of the milk samples in this study, but further study is required to understand the impact of these quality factors on RRDT results.

Antimicrobial susceptibility profiles of Mycoplasma hyorhinis strains isolated from five European countries between 2019 and 2021.

Mycoplasma hyorhinis is an emerging swine pathogen bacterium causing polyserositis and polyarthritis in weaners and finishers. The pathogen is distributed world-wide, generating significant economic losses. No commercially available vaccine is available in Europe. Therefore, besides improving the housing conditions for prevention, antimicrobial therapy of the diseased animals is the only option to control the infection. Our aim was to determine the minimal inhibitory concentrations (MIC) of ten antimicrobials potentially used against M. hyorhinis infection. The antibiotic susceptibility of 76 M. hyorhinis isolates from Belgium, Germany, Hungary, Italy and Poland collected between 2019 and 2021 was determined by broth micro-dilution method and mismatch amplification mutation assay (MAMA). Low concentrations of tiamulin (MIC90 0.312 μg/ml), doxycycline (MIC90 0.078 μg/ml), oxytetracycline (MIC90 0.25 μg/ml), florfenicol (MIC90 2 μg/ml) and moderate concentrations of enrofloxacin (MIC90 1.25 μg/ml) inhibited the growth of the isolates. For the tested macrolides and lincomycin, a bimodal MIC pattern was observed (MIC90 >64 μg/ml for lincomycin, tulathromycin, tylosin and tilmicosin and 5 μg/ml for tylvalosin). The results of the MAMA assay were in line with the conventional method with three exceptions. Based on our statistical analyses, significant differences in MIC values of tiamulin and doxycycline were observed between certain countries. Our results show various levels of antimicrobial susceptibility among M. hyorhinis isolates to the tested antibiotics. The data underline the importance of susceptibility monitoring on pan-European level and provides essential information for proper antibiotic choice in therapy.

Effects of florfenicol on methane accumulation and changes in the structure of the prokaryotic community in a water–sediment system

Florfenicol has been widely used in the veterinary and aquaculture to control bacterial diseases because of its high efficacy, quick effect, and low cost. The water–sediment system has become an important sink for florfenicol, and the anaerobic environment of lake sediments is favorable for methane (CH4) production. Although antibiotics may impact methanogenesis under anaerobic conditions, the influence of florfenicol on CH4 accumulation in anaerobic water–sediment system remains uncertain. This study evaluated how florfenicol affects CH4 accumulation and the structure of the prokaryotic community in a water–sediment system. Anaerobic systems with different florfenicol concentrations (0, 0.2, 1, 5 and 10 mg/L) were incubated and CH4 accumulation, pH, total organic carbon content, degradation ratio of florfenicol, and structure of the prokaryotic community were monitored. It was found that CH4 accumulation raised in low florfenicol (0.2 and 1 mg/L) systems during the growth period, while CH4 accumulation declined in high florfenicol (5 and 10 mg/L) systems. In the first 13 d, 83.67–99.30 % of florfenicol degraded in different treatments. The addition of florfenicol also influenced the structure of the prokaryotic community of the sediments. Proteobacteria and Chloroflexi were dominant at the phylum level. The dominant taxa at the order level gradually changed from Methanomicrobiales to Methanobacteriales, and finally to Methanosarcinales, indicating the dynamic transformation of methanogens in the reactor. This study reveals the effects of florfenicol on CH4 production under anaerobic conditions and provides a theoretical basis for further research on the underlying mechanisms. The findings also provide some basic data on the impact of new pollutants on the global carbon cycle and greenhouse gas emission.

Pharmaceutical characterization and pharmacokinetics of florfenicol-loaded alginate dried beads in rabbits

The pharmacokinetic variables of a new formulation of florfenicol included in dried bean of alginate (FADBs), its acceptance as in food medication, and its relationship with theoretical minimum inhibitory concentration (MIC) values of the main pathogens in rabbits, are presented. FADBs sought to mask the unpleasant taste of florfenicol while enhancing sustained absorption in a day to facilitate and optimise its dosage in this species. The entrapment efficiency was determined to be 94-98% and 73.56±3.26% of drug loading. No reduction in food consumption was detected, nor selectivity when choosing from their usual food. The elimination half-life was 1.23 to 2.4 h slower than the one previously reported in the literature. Possible flip-flop pharmacokinetics is proposed for FADBs in rabbits, thus complying better with the key pharmacokinetics/pharmacodynamics (PK/PD) ratio of t≥MIC. Also, if a MIC2.0 μg/mL is taken as the cut-off point for florfenicol in rabbits, then ad libitum intake of FADBs in their standard diet is sufficient to maintain plasma concentrations of florfenicol above this level during the whole dosing interval of 24 h. Additionally, FADBs are a low-cost and attractive drug delivery system for the oral controlled release of florfenicol in rabbits.

In vitro susceptibility of Mycoplasma iowae isolates to antimicrobial agents

ABSTRACT Mycoplasma iowae, a potential re-emerging avian pathogen mainly affecting turkeys, has been reported from many parts of the world. Poor hatchability, embryonic death, joint and skeletal abnormalities, poor ossification, runting-stunting, poor feathering and airsacculitis may be observed in infected flocks. The reduction of the severity of clinical signs and short-term control of M. iowae are performed by antibiotic treatment. However, M. iowae develops resistance more rapidly and is considered to be more resistant to antimicrobials than other avian pathogenic mycoplasmas. The aim of the present study was to determine the in vitro susceptibility of 101 M. iowae isolates and strains to ten clinically important antimicrobial agents, and to analyse and compare the susceptibility patterns of isolates of various origins and from a wide time-period. The examined reference strains showed high susceptibility to all antimicrobials except for spectinomycin. Low concentrations of tiamulin, florfenicol and oxytetracycline inhibited the growth of the clinical isolates. Nevertheless, slow tendency of increasing minimum inhibitory concentration (MIC) values was observed over time in the case of the above mentioned agents, while MIC values of enrofloxacin showed relatively rapid changes. Spiramycin, erythromycin, tilmicosin, tylosin, lincomycin and spectinomycin did not inhibit the bacterial growth in most of the cases. Isolates originating from captive game birds showed similar susceptibility profiles to isolates from industrial turkey hosts. The widely detected low susceptibility of M. iowae isolates to macrolides, lincomycin and spectinomycin, and the increase of MIC values of frequently used antimicrobials against this pathogen, emphasize the importance of targeted antibiotic therapy. RESEARCH HIGHLIGHTS Antimicrobial susceptibilities of 101 Mycoplasma iowae isolates were determined. Minimum inhibitory concentrations were determined by broth micro-dilution method. Tiamulin, oxytetracycline and florfenicol showed low MIC values. Isolates rapidly adapted to antimicrobial pressure.

Fate and exposure risk of florfenicol, thiamphenicol and antibiotic resistance genes during composting of swine manure

Livestock manure is an important source of antibiotic resistance genes (ARGs) spreading to the environment, posing a potential threat to human health. Here, we investigated the dissipation of florfenicol (FF) and thiamphenicol (TAP), and their effects on the bacterial community, mobile genetic elements (MGEs), and ARGs during composting. The results indicated that FF and TAP dissipated rapidly in compost, with half-life values of 5.1 and 1.6 d, respectively. However, FF could not be completely removed during composting. The FF and TAP residues in manure could reduce the elimination of ARGs and MGEs during composting, and had a negative effect on the physicochemical factors of the compost. Significant correlations were found between floR and intI1, indicating that floR in manure may more easily diffuse to the soil environment. Meanwhile, the presence of FF in manure could increase the abundance of floR. Network analysis showed that Proteobacteria and Firmicutes were the dominant bacterial communities and important potential pathogen hosts carrying ARGs. The predicted environmental concentration of FF in the soil was over 100 μg kg−1, which indicates that FF poses a potential risk to the natural environment, and we verified this result through field experiments. The results showed that FF dissipated in the soil after it migrated from manure to soil. In contrast, TAP in manure posed lower environmental risk. This study highlights that changed in composting conditions may control the rate of removal of ARGs. Further studies are needed to investigate the best environmental conditions to achieve a faster degradation of FF and a more comprehensive elimination of ARGs during composting.

Antibiotics removal from aquaculture effluents by ozonation: chemical and toxicity descriptors

Antibiotics are often applied in aquaculture to prevent fish diseases. These substances can cause disturbances on receiving waters, when not properly eliminated from the aquaculture effluents. In this work, ozone (O3) was investigated as a possible oxidizing agent to remove fishery antibiotics from aquaculture effluents: florfenicol (FF), oxytetracycline (OTC), sulfadimethoxine (SDM), sulfamethoxazole (SMX), and trimethoprim (TMP). Batch experiments were performed using ultrapure water and aquaculture effluents spiked with a mixture of target antibiotics at relatively high concentrations (10 mg L-1 each). OTC, SMX and TMP were fully removed (< 30 min) regardless of the tested conditions, mainly by O3 direct attack. In contrast, FF was partially removed in 30 min (∼ 10 and 60%, in aquaculture effluents and ultrapure water, respectively), but only in the presence of hydroxyl radicals (HO•), the FF concentrations reaching levels below the detection limits in ultrapure water after 60 min. In the case of SDM, its degradation was highly influenced by the selected water matrix, but with removals always higher than 68%. In continuous-flow experiments applying more environmentally relevant antibiotic concentrations (100 ng L-1 each) and low O3 doses (1.5 mg L−1), ozonation highly removed (> 98%) all tested antibiotics from aquaculture effluents with a hydraulic retention time (HRT) of 10 min, except FF (68%). Although by-products were detected in treated samples, zebrafish (Danio rerio) embryotoxicity tests did not show a toxicity increase by applying this ozonation treatment. Ozonation is thus a possible solution to remove antibiotics from aquaculture effluents. Still, full-scale studies in aquaculture farms are needed, and generation of HO• may be favoured to readily oxidize the FF antibiotic.

Effect of Carbonyl Cyanide Chlorophenylhydrazone on Intrabacterial Concentration and Antimicrobial Activity of Amphenicols against Swine Resistant Actinobacillus pleuropneumoniae and Pasteurella multocida

Effects and mechanism of carbonyl cyanide chlorophenylhydrazone (CCCP) on antimicrobial activity of florfenicol (FF) and thiamphenicol (TAP) were investigated against amphenicol-resistant Actinobacillus pleuropneumoniae and Pasteurella multocida isolated from diseased swine. Broth microdilution and time-kill assays indicated that CCCP dose-dependently and substantially (4-32 fold MIC reduction) improved amphenicol antimicrobial activity. When combined with CCCP at the lowest literature reported dose (2-5 μg/mL), 85% FF resistant A. pleuropneumoniae and 92% resistant P. multocida showed significantly reduced FF MICs (≥ 4-fold). In contrast, none or few of the susceptible A. pleuropneumoniae and P. multocida had FF MICs reduction ≥ 4-fold. 90% FF resistant A. pleuropneumoniae and 96% resistant P. multocida carried the floR gene, indicating strong association with the FloR efflux pump. With CCCP, the intracellular FF concentration increased by 71% in floR+ resistant A. pleuropneumoniae and 156% in floR+ resistant P. multocida strains but not the susceptible strains. The degree of reduction in TAP MICs was found consistently in parallel to FF for both bacteria. Taken together, partially attributed to blockage of drug-efflux, the combination of FF or TAP with CCCP at sub-cytotoxic concentrations was demonstrated and showed feasibility to combat amphenicol-resistant A. pleuropneumoniae and P. multocida isolated from diseased swine.

Exploring the abundance and influencing factors of antimicrobial resistance genes in manure plasmidome from swine farms

Plasmids play a critical role in the dissemination of antimicrobial resistance genes (ARGs), however, a systematical understanding of ARGs originated from plasmids in swine production is currently lacking. Herein, quantitative polymerase chain reaction was applied to determine the prevalence of ten ARGs and the class1 integron gene intI1 of plasmid source in swine manure from 44 farms in Sichuan, Hubei and Hebei provinces, China. All assayed ARGs were observed in plasmid DNA samples, and the average absolute abundance of aac(6’)-Ib-cr, blaNDM, blaCTX-M, optrA, ermB, floR, mcr-1, qnrS, tetM, sul1 and intI1 were 7.09, 2.90, 4.67, 6.62, 7.55, 7.14, 4.08, 4.85, 7.16, 7.11 and 8.07 of 10 log copies/gram, respectively. IntI1 showed a high correlation (r > 0.8, P < 0.01) with the abundance of aac(6’)-Ib-cr and sul1 in swine manure. Moreover, the farm scale (i.e., herd population) and geographical location were not found to be critical factors influencing the absolute abundance of ARGs of plasmid DNA in swine farms. However, the concentrations of florfenicol, Cu, Zn, Fe, total phosphorus (TP) and total potassium (TK) demonstrated a significant correlation with the abundance of several ARGs. Particularly, Cu and Zn had high correlations with optrA and blaCTX-M, respectively. Our results demonstrated that antibiotics, heavy metals and environmental nutrients are likely jointly contributing to the long-term persistence of ARGs in swine production. This study provides insights into the abundance and influencing factors of ARGs from swine manure, which is of significance for assessing and reducing the public health risks in livestock production.