Pharmacokinetics and residues of florfenicol in Nile tilapia (Oreochromis niloticus) post-oral gavage.

In the study on Oreochromis niloticus, singular oral gavage of florfenicol (FFC) at 15 mg/kg biomass/day was conducted, mimicking approved aquaculture dosing. Samples of plasma, bile, muscle, intestine, skin, liver, kidney, gill, and brain tissues were collected at 0, 2, 3, 4, 6, 8, 12, 16, 24, 32, 48, 64, 96, and 128 hours (h) after oral gavage. LC-MS/MS analysis revealed FFC concentrations peaked at 12.15 µg/mL in plasma and 77.92 µg/mL in bile, both at 24 hours. Elimination half-lives were 28.17 h (plasma) and 26.88 h (bile). The residues of FFC ranked muscle>intestine>skin>liver>kidney>gill. In contrast, the residues of florfenicol amine (FFA) ranked kidney>skin>liver>muscle>gill>intestine>brain, particularly notable in tropical summer conditions. The minimum inhibitory concentration of FFC was elucidated against several bacterial pathogens revealing its superior efficacy. Results highlight bile's crucial role in FFC elimination. Further investigation, especially during winter when fish susceptibility to infections rises, is warranted.

Effects of environmentally relevant concentrations of florfenicol on the glucose metabolism system, intestinal microbiome, and liver metabolome of zebrafish.

Florfenicol, a widely used veterinary antibiotic, has now been frequently detected in various water environments and human urines, with high concentrations. Accordingly, the ecological risks and health hazards of florfenicol are attracting increasing attention. In recent years, antibiotic exposure has been implicated in the disruption of animal glucose metabolism. However, the specific effects of florfenicol on the glucose metabolism system and the underlying mechanisms are largely unknown. Herein, zebrafish as an animal model were exposed to environmentally relevant concentrations of florfenicol for 28 days. Using biochemical and molecular analyses, we found that exposure to florfenicol disturbed glucose homeostasis, as evidenced by the abnormal levels of blood glucose and hepatic/muscular glycogen, and the altered expression of genes involved in glycogenolysis, gluconeogenesis, glycogenesis, and glycolysis. Considering the efficient antibacterial activity of florfenicol and the crucial role of intestinal flora in host glucose metabolism, we then analyzed changes in the gut microbiome and its key metabolite short-chain fatty acids (SCFAs). Results indicated that exposure to florfenicol caused gut microbiota dysbiosis, inhibited the production of intestinal SCFAs, and ultimately affected the downstream signaling pathways of SCFA involved in glucose metabolism. Moreover, non-targeted metabolomics revealed that arachidonic acid and linoleic acid metabolic pathways may be associated with insulin sensitivity changes in florfenicol-exposed livers. Overall, this study highlighted a crucial aspect of the environmental risks of florfenicol to both non-target organisms and humans, and presented novel insights into the mechanistic elucidation of metabolic toxicity of antibiotics.

Fate of florfenicol and linezolid resistance genes and their bacterial hosts during two waste treatment models in swine feedlots.

Florfenicol resistance genes (FRGs) are widely present in livestock farms. The aim of this study was to evaluate the removal efficiencies of FRGs as well as the relationships between FRGs, mobile genetic elements (MGEs) and bacterial communities during the natural drying (ND) and anaerobic digestion (AD) processes of manure treatment in swine farms by combining bacterial isolation, quantitative PCR and metagenomic approaches. Solid manure showed a higher abundance of FRGs than fresh manure and was the main contamination source of fexA and fexB in ND farms, whilst biogas slurry displayed a lower abundance of FRGs than the wastewater in AD farms. Moreover, fresh manure and wastewater showed a high abundance of optrA, and wastewater was the main contamination source of cfr in both ND and AD farms. Both optrA/fexA-positive enterococci and cfr/fexA-positive staphylococci were mainly isolated along the farms' treatment processes. The cfr-positive staphylococci were highly prevalent in wastewater (57.14 % - 100 %) and may be associated with nasal-derived cfr-positive porcine staphylococci. An increased abundance of Enterococcus, Jeotgalibaca and Vagococcus in the bacterial community structures may account for the high optrA abundance in wastewater and Jeotgalibaca may be another potential host of optrA. Furthermore, the abundance of FRG-related MGEs increased by 22.63 % after the ND process and decreased by 66.96 % in AD farms. A significant correlation was observed between cfr and ISEnfa4, whereas no significance was found between optrA and IS1216E, although IS1216E is the predominant insertion sequence involved in the transfer of optrA. In conclusion, manure and wastewater represented independent pollution sources of FRGs in swine farms. Associated MGEs might play a key role in the transfer and persistence of FRGs. The AD process was more efficient in the removal of FRGs than the ND method, nevertheless a longer storage of slurry may be required for a complete removal.

Development of a monoclonal antibody-based time-resolved fluorescence immunochromatographic assay strip for sensitively detecting florfenicol residues in milk and eggs: Theoretical chemical insights into unexpected high specificity.

Florfenicol (FF), with its broad-spectrum antibacterial activity, is frequently abused in the livestock and poultry industries and has aroused the growing public concern. Owing to structural similarities and varying maximum residue limits between florfenicol and other chloramphenicol (CAP)-type antibiotics, including thiamphenicol (TAP) and chloramphenicol (CAP), there is an urgent need for a rapid and effective immunoassay method to distinguish them, in order to minimize the risk of false positives. Fortunately, a highly specific monoclonal antibody (mAb), named as SF11, has been developed using hybridoma technology. Molecular simulations have revealed that the mAb SF11's specificity in recognizing florfenicol stems from the π-π stacking interaction between florfenicol and the mAb SF11 binding pocket. Using this highly specific mAb, a sensitive time-resolved fluorescence immunochromatographic assay (TRFICA) strip for rapid florfenicol detection has been developed. Under optimal conditions, this TRFICA demonstrated good analytical performance for the detection of florfenicol in milk and eggs samples, with the half-maximal inhibition concentration (IC50) values of 1.89 and 2.86 ng mL-1, the limit of detection (LOD) of 0.23 and 0.48 ng mL-1, the cut-off values of 62.50 and 31.25 ng mL-1, and the testing time of approximately thirteen minutes. Spiked recoveries in the milk and eggs samples ranged from 104.7 % to 112.3 % and 95.3 % to 116.4 %, respectively, with no obvious cross-reactions with the other analogues observed. The TRFICA results correlated well with those of high-performance liquid chromatography tandem mass spectrometry (HPLC-MS/MS) for real samples, indicating that the developed TRFICA method was sensitive, accurate and adapted for the rapid determination of florfenicol in milk and egg samples.

Biochar reduces antibiotic transport by altering soil hydrology and enhancing antibiotic sorption.

The performance of biochar (BC) in reducing the transport of antibiotics under field conditions has not been sufficiently explored. In repacked sloping boxes of a calcareous soil, the effects of different BC treatments on the discharge of three relatively weakly sorbing antibiotics (sulfadiazine, sulfamethazine, and florfenicol) via runoff and drainage were monitored for three natural rain events. Surface application of 1 % BC (1 %BC-SA) led to the most effective reduction in runoff discharge of the two sulfonamide antibiotics, which can be partly ascribed to the enhanced water infiltration. The construction of 5 % BC amended permeable reactive wall (5 %BC-PRW) at the lower end of soil box was more effective than the 1 %BC-SA treatment in reducing the leaching of the most weakly sorbing antibiotic (florfenicol), which can be mainly ascribed to the much higher plant available and drainable water contents in the 5 %BC-PRW soil than in the unamended soil. The results of this study highlight the importance of BC's ability to regulate flow pattern by modifying soil hydraulic properties, which can make a significant contribution to the achieved reduction in the transport of antibiotics offsite or to groundwater.

Understanding the toxicity risk of antibiotic emissions of aquaculture from the perspective of fluctuations concentration.

Organisms are generally exposed to target contaminant with stable concentrations in traditional ecotoxicological studies. However, it is difficult to truly represent the dynamics and complexity of actual aquatic pollution for risk management. Contaminants may enter nearby aquatic systems in pulsed exposure, thus resulting in that aquatic organisms will be exposed to contaminants at fluctuating concentrations. Especially during the season of summer, due to the changes in displacement or periodic emissions of veterinary antibiotics in aquaculture, algal blooms occur frequently in surrounding waters, thus leading to eutrophication of the water. Florfenicol (FFC) is currently widely used as a veterinary antibiotic, but the aquatic ecological risks of FFC under concentration fluctuations are still unknown. Therefore, the acute exposure, chronic exposure and pulsed exposure effects of FFC on Microcystis aeruginosa were investigated to comprehensively evaluate the ecological risk of FFC and raise awareness of the pulsed exposure mode. Results indicated that the toxic effects of FFC on M. aeruginosa were dominated by exposure mode, exposure duration, exposure frequency, and exposure concentration. The maximum growth inhibition rate of the 10 µg/L FFC treatment amounted to 4.07% during chronic exposure of 18 days. However, the growth inhibition rate decreased from 55.1% to 19.31% when algae was exposure to 10 µg/L FFC during the first pulsed exposure (8 h). Therefore, when the concentration of FFC was equal under chronic and pulsed exposure, FFC exhibited greater toxicity on M. aeruginosa in short pulsed exposure than in continuous exposure. In addition, repetitive pulsed exposure strengthened the resistance of M. aeruginosa on FFC. The adaptive regulation of algae was related to the duration and frequency of exposure. Above results suggested that traditional toxicity assessments lacked consideration for fluctuating concentrations during pollutant emissions, thus underestimating the environmental risk of contaminant. This investigation aims to facilitate the standardization of pulsed exposure.

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.

Bacteriophage EPP-1, a potential antibiotic alternative for controlling edwardsiellosis caused by Edwardsiella piscicida while mitigating drug-resistant gene dissemination.

Edwardsiella piscicida causes significant economic losses to the aquaculture industry worldwide. Phage-based biocontrol methods are experiencing a renaissance because of the spread of drug-resistant genes and bacteria resulting from the heavy use of antibiotics. Here, we showed that the novel Edwardsiella phage EPP-1 could achieve comparable efficacy to florfenicol using a zebrafish model of Edwardsiella piscicida infection and could reduce the content of the floR resistance gene in zebrafish excreta. Specifically, phage EPP-1 inhibited bacterial growth in vitro and significantly improved the zebrafish survival rate in vivo (P = 0.0035), achieving an efficacy comparable to that of florfenicol (P = 0.2304). Notably, integrating the results of 16S rRNA sequencing, metagenomic sequencing, and qPCR, although the effects of phage EPP-1 converged with those of florfenicol in terms of the community composition and potential function of the zebrafish gut microbiota, it reduced the floR gene content in zebrafish excreta and aquaculture water. Overall, our study highlights the feasibility and safety of phage therapy for edwardsiellosis control, which has profound implications for the development of antibiotic alternatives to address the antibiotic crisis.

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.

Microfluidic-oriented green synthesis of pepsin-doped gold nanoparticles for colorimetric and photothermal dual-readout detection of food hazards.

Gold nanoparticles (AuNPs)-based immunochromatographic assay has gained popularity as a rapid detection method for food hazards. Synthesizing highly stable AuNPs in a rapid, simple and environmentally friendly manner is a key focus in this field. Here, we present a green microfluidic strategy for the rapid, automated, and size-controllable synthesis of pepsin-doped AuNPs (AuNPs@Pep) by employing glucose-pepsin as a versatile reducing agent and stabilizer. Through combining the colorimetric and photothermal (PoT) properties of AuNPs@Pep, both "signal-off" and "signal-on" formats of microfluidic paper analytical devices (PADs) were developed for detection of a small molecule antibiotic, florfenicol, and an egg allergen, ovalbumin. Compared to the colorimetric mode, a 4-fold and 3-fold improvement in limit of detection was observed in the "signal-off" detection of florfenicol and the "signal-on" detection of ovalbumin, respectively. The results demonstrated the practicality of AuNPs@Pep as a colorimetric/PoT dual-readout probe for immunochromatographic detection of food hazards at different molecular scales.

Whole-genome sequencing-based antimicrobial resistance and shedding dynamics of Escherichia coli isolated from calves before and after antimicrobial group treatments.

The fattening of calves is often associated with high antimicrobial use and the selection of antimicrobial resistance (AMR). The objective of this observational longitudinal study was to describe the AMR and strain dynamics, using whole-genome sequencing (WGS), of fecal Escherichia coli in a cohort of 22 calves. All calves received antimicrobial group treatments on Day (D) 1 (oxytetracycline, intramuscularly) and on D4 through D12 (doxycycline, in-feed). Additionally, eight calves received individual parenteral treatments between D7 and D59, including florfenicol, amoxicillin, marbofloxacin, and gamithromycin. Rectal swabs were collected from all calves on D1 (prior to treatment), D2, D9, and D82. The swabs were spread onto Enterobacterales-selective agar, and three E. coli colonies per plate were subjected to WGS. Out of 264 isolates across all calves and sampling times, 80 unique strains were identified, a majority of which harbored genes conferring resistance to tetracyclines, streptomycin, and sulfonamides. The diversity of strains decreased during the in-feed antimicrobial group treatment of the calves. On D82, 90% of isolates were strains that were not isolated at previous sampling times, and the median number per strain of AMR determinants to tetracyclines, florfenicol, ß-lactams, quinolones, or macrolides decreased compared to D9. Additionally, clonal dissemination of some strains represented the main transmission route of AMR determinants. In this study, WGS revealed important variations in strain diversity and genotypic AMR of fecal E. coli over time in calves subjected to group antimicrobial treatments. IMPORTANCE: The continued emergence and spread of antimicrobial resistance (AMR) determinants are serious global concerns. The dynamics of AMR spread and persistence in bacterial and animal host populations are complex and not solely driven by antimicrobial selection pressure. In calf fattening, both antimicrobial use and carriage prevalence of antimicrobial-resistant bacteria are generally recognized as high. This study provides new insights into the short-term, within-farm dynamics and transmission of AMR determinants in Escherichia coli from the dominant fecal flora of calves subjected to antimicrobial group treatments during the rearing period. The diversity of E. coli strains decreased over time, although, in contrast to previous observations in extended-spectrum ß-lactamase-producing Enterobacterales, the predominance of a few clones was not observed. The spread of AMR determinants occurred through the dissemination of clonal strains among calves. The median number per strain of AMR determinants conferring resistance to selected antimicrobials decreased toward the end of the rearing period.

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.

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.

Influence of Escherichia coli lipopolysaccharide-induced endotoxemia on plasma and tissue disposition of florfenicol after intramuscular administration in rabbits.

To assess the effects of the acute inflammatory response (AIR) induced by Escherichia coli lipopolysaccharide (LPS) on plasma and tissue disposition of florfenicol (FFC) and its metabolite florfenicol amine (FFC-a), after its intramuscular (IM) administration, twenty-two New Zealand rabbits were randomly distributed in two experimental groups: Group 1 (LPS) was treated with three intravenous doses of 2 µg LPS/kg bw, before an intramuscular dose of 20 mg/kg FFC twenty-four h after the first LPS or SS injection; Group 2 (Control) was treated with saline solution (SS) in equivalent volumes as LPS-treated group. Blood samples were collected before (T0) and at different times after FFC administration. Acute inflammatory response was assessed in a parallel study where significant increases in body temperature, C-reactive protein concentrations and leukopenia were observed in the group treated with LPS. In another two groups of rabbits, 4 h after FFC treatment, rabbits were euthanized and tissue samples were collected for analysis of FFC and FFC-a concentrations. Pharmacokinetic parameters of FFC that showed significantly higher values in LPS-treated rabbits compared with control rabbits were absorption half-life, area under the curve, mean residence time and clearance /F (Cl/F). Elimination half-life and mean residence time of FFC-a were significantly higher in LPS-treated rabbits, whereas the metabolite ratio of FFC-a decreased significantly. Significant differences in tissue distribution of FFC and FFC-a were observed in rabbits treated with LPS. Modifications in plasma and tissue disposition of FFC and FFC-a were attributed mainly to haemodynamic modifications induced by the AIR through LPS administration.

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.

A Florfenicol-Resistant Plasmid Shuttling Between Actinobacillus pleuropneumoniae and Glaesserella parasuis.

Porcine contagious pleuropneumonia, caused by Actinobacillus pleuropneumoniae, has resulted in significant economic losses to the swine industry. Although antibiotics are commonly employed to control this disease, their widespread use or misuse can lead to the development of antibiotic resistance in A. pleuropneumoniae. Consequently, it is crucial to conduct antimicrobial susceptibility testing on clinical isolates. In our study, we identified one strain of A. pleuropneumoniae with resistance to florfenicol and extracted a 5919 bp plasmid named pAPPJY, which confers florfenicol resistance. Sequence analysis revealed that the plasmid contains four open reading frames, namely rep, antioxin vbha family protein, floR, and a partial copy of lysr. Although a few variations in gene position were observed, the plasmid sequence exhibits a high degree of similarity to other florfenicol-resistant plasmids found in Glaesserella parasuis and A. pleuropneumoniae. Therefore, it is possible that the pAPPJY plasmid functions as a shuttle, facilitating the spread of florfenicol resistance between G. parasuis and A. pleuropneumoniae. In addition, partial recombination may occur during bacterial propagation. In conclusion, this study highlights the horizontal transmission of antibiotic resistance among different bacterial species through plasmids, underscoring the need for increased attention to antibiotic usage.

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.

Microfabrication of engineered Lactococcus lactis biocarriers with genetically programmed immunorecognition probes for sensitive lateral flow immunoassay of antibiotic in milk and lake water.

Micro/nanomaterials display considerable potential for increasing the sensitivity of lateral flow immunoassay (LFIA) by acting as 3D carriers for both antibodies and signals. The key to achieving high detection sensitivity depends on the probe's orientation on the material surface and its multivalent biomolecular interactions with targets. Here, we engineer Lactococcus lactis as the bacterial microcarrier (BMC) for a multivalent immunorecognition probe that was genetically programmed to display multifunctional components including a phage-screened single-chain variable fragment (scFv), an enhanced green fluorescent protein (eGFP), and a C-terminal peptidoglycan-binding domain (AcmA) anchored on BMC through the cell wall peptidoglycan. The innovative design of this biocarrier system, which incorporates a lab-on-a-chip microfluidic device, allows for the rapid and non-destructive self-assembly of the multivalent scFv-eGFP-AcmA@BMC probe, in which the 3D structure of BMC with a large peptidoglycan surface area facilitates the precisely orientated attachment and immobilization of scFv-eGFP-AcmA. This leads to a remarkable fluorescence aggregation amplification effect in LFIA, outperforming a monovalent 2D scFv-eGFP-AcmA probe for florfenicol detection. By designing a portable sensing device, we achieved an exceptionally low detection limit of 0.28 pg/mL and 0.21 pg/mL for florfenicol in lake water and milk sample, respectively. The successful microfabrication of this biocarrier holds potential to inspire innovative biohybrid designs for environment and food safety biosensing applications.

Pharmacokinetics of florfenicol in serum and seminal plasma in beef bulls.

The objectives of this study were to compare the serum and seminal plasma pharmacokinetic profiles of florfenicol (FLO) and florfenicol amine (FLA) after the administration of FLO either by IM or SC routes in beef bulls. Four clinically healthy Hereford bulls underwent a comprehensive physical exam, including breeding soundness examination, CBC, and chemistry profile panel. Bulls were healthy and classified satisfactory potential breeders. In one group (n = 2), a single dose of FLO was administered SC in the middle of the neck at a dose of 40 mg/kg of body weight. In the second group (n = 2), a single dose was administered IM in the muscles of the neck at a dose of 20 mg/kg. Concentrations of FLO and FLA in serum and seminal plasma were determined by ultra-high-performance liquid chromatography coupled to tandem mass spectrometry (UHPLC-MS/MS). Blood and semen samples were collected before the administration of FLO and at 12, 24, 36, 48, 72, 96, 120, 144, and 168 h after injection. The blood was collected from the coccygeal vessels, and semen was collected by electroejaculation. All samples were immediately refrigerated, processed within the first hour after collection, and finally stored at -80 °C. The mean level of total FLO in serum was higher when administered by the SC route (1,415.5 ng/mL) than by the IM route (752.4 ng/mL; P = 0.001). Differences were observed between the percentage of FLA in serum (1.8%; ranging from 1.3 to 2.9) and in seminal plasma (27.5%; ranging from 15.9 to 34.2; P = 0.0001). The mean level (±SD) of FLA was higher in seminal plasma compared to serum (467 ± 466 ng/mL and 18 ± 16 ng/mL, respectively; P = 0.001). The mean level of total FLO in seminal plasma was 1,454.8 ng/mL for the SC route and 1,872.9 ng/mL for the IM route without differences between the two routes (P = 0.51). Differences in the mean level of total FLO between serum and seminal plasma were detected (1,187 ± 2,069 ng/mL and 1,748 ± 1,906 ng/mL, respectively; P = 0.04). From the present investigation, it was concluded that FLO is a suitable antibiotic based on its pharmacokinetic attributes and may be employed for the treatment of bull genital infections when its use is indicated. To study the pharmacokinetics of FLO in seminal plasma, the analysis of FLA should be incorporated.

Impact of florfenicol dosing regimen on the phenotypic and genotypic resistance of enteric bacteria in steers.

The food animal sector's use of antimicrobials is heavily critiqued for its role in allowing resistance to develop against critically important antimicrobials in human health. The WHO recommends using lower tier antimicrobials such as florfenicol for disease treatment. The primary objective of this study was to assess the differences in resistance profiles of enteric microbes following administration of florfenicol to steers using both FDA-approved dosing regimens and two different detection methods. Our hypothesis was that we would identify an increased prevalence of resistance in the steers administered the repeated, lower dose of florfenicol; additionally, we hypothesized resistance profiles would be similar between both detection methods. Twelve steers were administered either two intramuscular (20 mg/kg q 48 h; n = 6) or a single subcutaneous dose (40 mg/kg, n = 6). Fecal samples were collected for 38 days, and E. coli and Enterococcus were isolated and tested for resistance. Fecal samples were submitted for metagenomic sequencing analysis. Metagenomics revealed genes conferring resistance to aminoglycosides as the most abundant drug class. Most multidrug resistance genes contained phenicols. The genotypic and phenotypic patterns of resistance were not similar between drug classes. Observed increases in resistant isolates and relative abundance of resistance genes peaked after drug administration and returned to baseline by the end of the sampling period. The use of a "lower tier" antimicrobial, such as florfenicol, may cause an increased amount of resistance to critically important antimicrobials for a brief period, but these changes largely resolve by the end of the drug withdrawal period.