Quantifying trade-offs between therapeutic efficacy and resistance dissemination for enrofloxacin dose regimens in cattle.

The use of antimicrobial drugs in food-producing animals contributes to the selection pressure on pathogenic and commensal bacteria to become resistant. This study aims to evaluate the existence of trade-offs between treatment effectiveness, cost, and the dynamics of resistance in gut commensal bacteria. We developed a within-host ordinary differential equation model to track the dynamics of antimicrobial drug concentrations and bacterial populations in the site of infection (lung) and the gut. The model was parameterized to represent enrofloxacin treatment for bovine respiratory disease (BRD) caused by Pastereulla multocida in cattle. Three approved enrofloxacin dosing regimens were compared for their effects on resistance on P. multocida and commensal E. coli: 12.5 mg/kg and 7.5 mg/kg as a single dose, and 5 mg/kg as three doses. Additionally, we explored non-FDA-approved regimes. Our results indicated that both 12.5 mg/kg and 7.5 mg/kg as a single dose scenario increased the most the treatment costs and prevalence of P. multocida resistance in the lungs, while 5 mg/kg as three doses increased resistance in commensal E. coli bacteria in the gut the most out of the approved scenarios. A proposed non-FDA-approved scenario (7.5 mg/kg, two doses 24 h apart) showed low economic costs, minimal P. multocida, and moderate effects on resistant E. coli. Overall, the scenarios that decrease P. multocida, including resistant P. multocida did not coincide with those that decrease resistant E. coli the most, suggesting a trade-off between both outcomes. The sensitivity analysis suggests that bacterial populations were the most sensitive to drug conversion factors into plasma ( ß ), elimination of the drug from the colon ( ϑ ), fifty percent sensitive bacteria (P. multocida) killing effect ( L s50 ), fifty percent of bacteria (E. coli) above ECOFF killing effect ( C r50 ), and net drug transfer rate in the lung ( γ ) parameters.

Extended period of selection for antimicrobial resistance due to recirculation of persistent antimicrobials in broilers.

OBJECTIVES: Antimicrobials can select for antimicrobial-resistant bacteria. After treatment the active compound is excreted through urine and faeces. As some antimicrobials are chemically stable, recirculation of subinhibitory concentrations of antimicrobials may occur due to coprophagic behaviour of animals such as chickens. METHODS: The persistence of three antimicrobials over time and their potential effects on antimicrobial resistance were determined in four groups of broilers. Groups were left untreated (control) or were treated with amoxicillin (unstable), doxycycline or enrofloxacin (stable). Antimicrobials were extracted from the faecal samples and were measured by LC-MS/MS. We determined the resistome genotypically using shotgun metagenomics and phenotypically by using Escherichia coli as indicator microorganism. RESULTS: Up to 37 days after treatment, doxycycline and enrofloxacin had concentrations in faeces equal to or higher than the minimal selective concentration (MSC), in contrast to the amoxicillin treatment. The amoxicillin treatment showed a significant difference (P ≤ 0.01 and P ≤ 0.0001) in the genotypic resistance only directly after treatment. On the other hand, the doxycycline treatment showed approximately 52% increase in phenotypic resistance and a significant difference (P ≤ 0.05 and P ≤ 0.0001) in genotypic resistance throughout the trial. Furthermore, enrofloxacin treatment resulted in a complete non-WT E. coli population but the quantity of resistance genes was similar to the control group, likely because resistance is mediated by point mutations. CONCLUSIONS: Based on our findings, we suggest that persistence of antimicrobials should be taken into consideration in the assessment of priority classification of antimicrobials in livestock.

Pharmacokinetic/pharmacodynamic relationships of enrofloxacin against Klebsiella pneumoniae in an in vivo infection model in young chicks.

Klebsiella pneumoniae is a serious pathogenic bacterium that poses a significant threat to young poultry and the cause of significant chick mortality and economic loss. We investigated the therapeutic efficacy of enrofloxacin in treating K. pneumoniae infections in chicks and employed an in vivo pharmacokinetic/pharmacodynamic (PK/PD) model. In vivo efficacy was evaluated using 6 multiple-dose groups (oral administration once a day for 3 d) and 2 single-dose groups (oral administration once only). The PK and PD parameters of plasma and lung were analyzed using PK/PD fitting analysis. K. pneumoniae administered intratracheally (108 CFU/mL in 0.4 mL saline) was used to establish a model for pulmonary infection. The plasma protein binding of enrofloxacin was 20.18%. Enrofloxacin displayed T1/2ß values of 4.78 ± 0.69 h and 4.78 ± 1.02 h in plasma and lung of infected chicks, respectively. When the dosage in the multiple-dose group was > 10 mg/kg, bactericidal activity was found and complete eradication was not achieved when the dosage was ≤ 40 mg/kg. When TMSW was set at 20%, the calculated dosage and bacterial reduction (E) based on plasma free drug data were 4.03 mg/kg and -1.982 Log10 CFU/mL, respectively. In the calculation of PK/PD parameters for reducing 3 Log10 CFU/mL and using Cmax/MIC, AUC72h/MIC and TMSW of free drug in plasma values at 9.479, 379.691, and 44.395%, respectively, the value of AUC72h/MIC based on the concentration of drug in lung was 530.800. According to the fitting correlation R2, the PK/PD fitting results of free drug in plasma were better. The corresponding enrofloxacin dosage for AUC72h/MIC of the plasma free drug concentration was 14.16 mg/kg. The administration regimen corresponding to these dosages was once daily for 3 d. This dosage regimen (14.16 mg/kg) was relatively high compared to the clinically recommended dosage in China (7.5 mg/kg) when treating infections caused by K. pneumoniae with MIC ≥ 0.125 µg/mL, so careful consideration is needed.

Interaction between Enrofloxacin and Three Essential Oils (Cinnamon Bark, Clove Bud and Lavender Flower)-A Study on Multidrug-Resistant Escherichia coli Strains Isolated from 1-Day-Old Broiler Chickens.

Avian pathogenic Escherichia coli (APEC) causes a variety of infections outside the intestine. The treatment of these infections is becoming increasingly difficult due to the emergence of multi-drug resistant (MDR) strains, which can also be a direct or indirect threat to humans as consumers of poultry products. Therefore, alternative antimicrobial agents are being sought, which could be essential oils, either administered individually or in interaction with antibiotics. Sixteen field isolates of E. coli (originating from 1-day-old broilers) and the ATCC 25922 reference strain were tested. Commercial cinnamon bark, clove bud, lavender flower essential oils (EOs) and enrofloxacin were selected to assess the sensitivity of the selected E. coli strains to antimicrobial agents. The checkerboard method was used to estimate the individual minimum inhibitory concentration (MIC) for each antimicrobial agent as well as to determine the interactions between the selected essential oil and enrofloxacin. In the case of enrofloxacin, ten isolates were resistant at MIC ≥ 2 µg/mL, three were classified as intermediate (0.5-1 µg/mL) and three as sensitive at ≤0.25 µg/mL. Regardless of the sensitivity to enrofloxacin, the MIC for cinnamon EO was 0.25% v/v and for clove EO was 0.125% v/v. All MDR strains had MIC values for lavender EO of 1% v/v, while drug-sensitive isolates had MIC of 0.5% v/v. Synergism between enrofloxacin and EO was noted more frequently in lavender EO (82.35%), followed by cinnamon EO (64.7%), than in clove EO (47.1%). The remaining cases exhibited additive effects. Owing to synergy, the isolates became susceptible to enrofloxacin at an MIC of ≤8 µg/mL. A time-kill study supports these observations. Cinnamon and clove EOs required for up to 1 h and lavender EO for up to 4 h to completely kill a multidrug-resistant strain as well as the ATCC 25922 reference strain of E. coli. Through synergistic or additive effects, blends with a lower than MIC concentration of enrofloxacin mixed with a lower EO content required 6 ± 2 h to achieve a similar effect.

Effects of four antibiotics on the photosynthetic light reactions in the green alga Chlorella pyrenoidosa.

Antibiotics are ubiquitously present in aquatic environments, posing a serious ecological risk to aquatic ecosystems. However, the effects of antibiotics on the photosynthetic light reactions of freshwater algae and the underlying mechanisms are relatively less understood. In this study, the effects of 4 representative antibiotics (clarithromycin, enrofloxacin, tetracycline, and sulfamethazine) on a freshwater alga (Chlorella pyrenoidosa) and the associated mechanisms, primarily focusing on key regulators of the photosynthetic light reactions, were evaluated. Algae were exposed to different concentrations of clarithromycin (0.0-0.3 mg/L), enrofloxacin (0.0-30.0 mg/L), tetracycline (0.0-10.0 mg/L), and sulfamethazine (0.0-50.0 mg/L) for 7 days. The results showed that the 4 antibiotics inhibited the growth, the photosynthetic pigment contents, and the activity of antioxidant enzymes. In addition, exposure to clarithromycin caused a 118.4 % increase in malondialdehyde (MDA) levels at 0.3 mg/L. Furthermore, the transcripts of genes for the adenosine triphosphate (ATP) - dependent chloroplast proteases (ftsH and clpP), genes in photosystem II (psbA, psbB, and psbC), genes related to ATP synthase (atpA, atpB, and atpH), and petA (related to cytochrome b6/f complex) were altered by clarithromycin. This study contributes to a better understanding of the risk of antibiotics on primary producers in aquatic environment.

Infection and antibiotic-associated changes in the fecal microbiota of C. rodentium ϕstx2dact-infected C57BL/6 mice.

Enterohemorrhagic Escherichia coli causes watery to bloody diarrhea, which may progress to hemorrhagic colitis and hemolytic-uremic syndrome. While early studies suggested that antibiotic treatment may worsen the pathology of an enterohemorrhagic Escherichia coli (EHEC) infection, recent work has shown that certain non-Shiga toxin-inducing antibiotics avert disease progression. Unfortunately, both intestinal bacterial infections and antibiotic treatment are associated with dysbiosis. This can alleviate colonization resistance, facilitate secondary infections, and potentially lead to more severe illness. To address the consequences in the context of an EHEC infection, we used the established mouse infection model organism Citrobacter rodentium ϕstx2dact and monitored changes in fecal microbiota composition during infection and antibiotic treatment. C. rodentium ϕstx2dact infection resulted in minor changes compared to antibiotic treatment. The infection caused clear alterations in the microbial community, leading mainly to a reduction of Muribaculaceae and a transient increase in Enterobacteriaceae distinct from Citrobacter. Antibiotic treatments of the infection resulted in marked and distinct variations in microbiota composition, diversity, and dispersion. Enrofloxacin and trimethoprim/sulfamethoxazole, which did not prevent Shiga toxin-mediated organ damage, had the least disruptive effects on the intestinal microbiota, while kanamycin and tetracycline, which rapidly cleared the infection without causing organ damage, caused a severe reduction in diversity. Kanamycin treatment resulted in the depletion of all but Bacteroidetes genera, whereas tetracycline effects on Clostridia were less severe. Together, these data highlight the need to address the impact of individual antibiotics in the clinical care of life-threatening infections and consider microbiota-regenerating therapies.IMPORTANCEUnderstanding the impact of antibiotic treatment on EHEC infections is crucial for appropriate clinical care. While discouraged by early studies, recent findings suggest certain antibiotics can impede disease progression. Here, we investigated the impact of individual antibiotics on the fecal microbiota in the context of an established EHEC mouse model using C. rodentium ϕstx2dact. The infection caused significant variations in the microbiota, leading to a transient increase in Enterobacteriaceae distinct from Citrobacter. However, these effects were minor compared to those observed for antibiotic treatments. Indeed, antibiotics that most efficiently cleared the infection also had the most detrimental effect on the fecal microbiota, causing a substantial reduction in microbial diversity. Conversely, antibiotics showing adverse effects or incomplete bacterial clearance had a reduced impact on microbiota composition and diversity. Taken together, our findings emphasize the delicate balance required to weigh the harmful effects of infection and antibiosis in treatment.

Induction of the mitochondrial pathway of apoptosis by enrofloxacin in the context of the safety issue of its use in poultry.

The overuse of antibiotics in both humans and livestock has led to the antibiotic resistance phenomenon which is now considered one of the biggest problems in the modern world. Some antibiotics used to control or prevent infections in livestock poultry were registered a long time ago, and as a result, data on the possible side effects of their use, both for birds and humans, are incomplete and should be updated. An example of such an antibiotic is enrofloxacin which has been widely used in poultry since 1989. Data in recent years have begun to indicate that this antibiotic induces the process of apoptosis in diverse types of eukaryotic cells. Unfortunately, such studies have never been conducted on chicken models even though it is in poultry that this antibiotic is most commonly used. Therefore, the purpose of this work was to investigate whether enrofloxacin induces apoptosis in chicken cells of the UMNSAH/DF-1 line and to study the molecular mechanism of its action. The results of these experiments indicated that enrofloxacin induces apoptosis in chicken cells but not in human HEK-293 and PC3 cells. This induction was accompanied by changes in the morphology and size of mitochondria, the process of apoptosome formation and activation of executive caspases, which clearly indicates the role of the mitochondrial pathway in the induction of apoptosis by enrofloxacin. This study is the first to show the toxicity of enrofloxacin against chicken cells and to demonstrate the exact mechanism of its action. The results presented in this work show the need to monitor the concentration of antibiotic residues in poultry foods as well as to study their impact on public health to guarantee consumer safety and prevent the phenomenon of antibiotic resistance in bacteria.

Synergism between molecules derived from Garcinia brasiliensis and antimicrobial drugs on field isolates of Mycoplasma hyopneumoniae.

Mycoplasma hyopneumoniae (M. hyopneumoniae) is one of the smallest free-living bacteria found in nature; it has an extremely small genome and lacks a cell wall. It is the main etiological agent of porcine enzootic pneumonia (EP), a chronic respiratory disease with worldwide distribution that causes significant losses in swine production. Due to the great economic impact caused by EP, new strategies for treating and controlling this agent are researched. The objective of this study was to verify the anti-M. hyopneumoniae activity of compounds derived from Garcinia brasiliensis and the synergism with the main antimicrobials used in the treatment of EP; this is the first study assessing the synergism between bioactive molecules and antimicrobial compounds in vitro against isolates of M. hyopneumoniae. The minimum inhibitory concentrations (MICs) of the antimicrobials tiamulin, valnemulin, and enrofloxacin, as well as the bioactive compounds guttiferone-A (Gut-A), 7-epiculsone (7-Epic), copper 7-epiculsone (7-Epic-Cu), and benzophenone, were determined. Subsequently, the interactions of antibiotics with the compounds were evaluated using the checkerboard method. Three field M. hyopneumoniae isolates were used, and the J strain was used as a control. The MIC values of the antimicrobials compared to the field isolates were equal to and lower than those of the reference strain J. Among the compounds used, 7-Epic-Cu showed the lowest MIC value. Synergistic association was observed for Gut-A with tiamulin and valnemulin, whereas 7-Epic and 7-Epic-Cu showed synergistic action with enrofloxacin. No synergistic effect was observed for benzophenone. Despite being an initial study, the results suggest that these combinations hold promise for the treatment of infections caused by M. hyopneumoniae.

Postantibiotic leukocyte enhancement-mediated reduction of intracellular bacteria by macrophages.

INTRODUCTION: Potentiation of the bactericidal activities of leukocytes, including macrophages, upon antibacterial agent administration has been observed for several decades and is summarized as the postantibiotic leukocyte enhancement (PALE) theory. Antibiotics-induced bacterial sensitization to leukocytes is commonly recognized as the mechanism of PALE. However, the degree of sensitization drastically varies with antibiotic classes, and little is known about whether and how the potentiation of leukocytes contributes to PALE. OBJECTIVES: In this study, we aim to develop a mechanistic understanding of PALE by investigating the immunoregulation of traditional antibiotics on macrophages. METHODS: Interaction models between bacteria and macrophages were constructed to identify the effects of different antibiotics on the bactericidal activities of macrophages. Oxygen consumption rate, expression of oxidases, and antioxidants were then measured to evaluate the effects of fluoroquinolones (FQs) on the oxidative stress of macrophages. Furthermore, the modulation in endoplasmic reticulum stress and inflammation upon antibiotic treatment was detected to analyze the mechanisms. At last, the peritoneal infection model was utilized to verify the PALE in vivo. RESULTS: Enrofloxacin significantly reduced the intracellular burden of diverse bacterial pathogens through promoting the accumulation of reactive oxygen species (ROS). The upregulated oxidative response accordingly reprograms the electron transport chain with decreased production of antioxidant enzymes to reduce internalized pathogens. Additionally, enrofloxacin modulated the expression and spatiotemporal localization of myeloperoxidase (MPO) to facilitate ROS accumulation to target invaded bacteria and downregulated inflammatory response to alleviate cellular injury. CONCLUSION: Our findings demonstrate the crucial role of leukocytes in PALE, shedding light on the development of new host-directed antibacterial therapies and the design of rational dosage regimens.

Revision of fluoroquinolone breakpoints used for interpretation of antimicrobial susceptibility testing of canine bacterial isolates.

The fluoroquinolone antimicrobial agents, enrofloxacin and marbofloxacin, were US Food and Drug Administration (FDA) approved in the United States for use in dogs in 1988 and 1999, respectively. There have been many advances since then concerning the pharmacokinetic-pharmacodynamic (PK-PD) evaluation of fluoroquinolones, and there are data available on the susceptibility of targeted pathogens since the original approval. Using this information, the Clinical and Laboratory Standards Institute (CLSI) Veterinary Antimicrobial Susceptibility Testing Subcommittee (VAST) revised its antimicrobial susceptibility testing breakpoints. The previous breakpoints (used in older editions of CLSI standards) for enrofloxacin in dogs were susceptible (S), ≤ 0.5 µg/mL, intermediate (I) 1-2 µg/mL, and resistant (R) ≥ 4 µg/mL. The new breakpoints are S ≤ 0.06 µg/mL for a dose of 5 mg/kg, 0.12 µg/mL for a dose of 10 mg/kg, 0.25 µg/mL for a high dose of 20 mg/kg, and R ≥ 0.5 µg/mL. The breakpoints of 0.12 and 0.25 µg/mL represent a new susceptible-dose dependent (SDD) category. For marbofloxacin, previous breakpoints were S, ≤ 1 µg/mL, I 2 µg/mL, and R ≥ 4 µg/mL. The new breakpoints are S ≤ 0.12 µg/mL for a dose of 2.8 mg/kg, 0.25 µg/mL for a dose of 5.5 mg/kg (SDD), and R ≥ 0.5 µg/mL. The new breakpoints will be published in the next edition of CLSI-Vet01(S) and deviate considerably from the prior breakpoints. Laboratories are encouraged to revise their testing standards. These changes will likely reduce the unnecessary use of these fluoroquinolones in dogs.

Pharmaceutical Design of a Formulation of Enrofloxacin-Alginate and its Strategic Dosage to Achieve Mutant Prevention PK/PD Ratios in Broiler Chickens.

INTRODUCTION: The comparative pharmacokinetics (PK) and PK/pharmacodynamics (PD) ratios of a new pharmaceutical design of enrofloxacin-alginate in dried beads (EADBs) and the reference enrofloxacin 10% solution was determined in broiler chickens. Also, the same parameters were determined after administering enrofloxacin with a double dosing scheme (through drinking water and as an in-feed medication of EADBs). 500 Arbor-Acres broiler chickens were randomly divided into five groups (n=100), adjusting in all cases, a dose of 10 mg/kg based on water and feed intake as follows: group EADBsad-lib receiving enrofloxacin through EADBs added to their feed as dressing; group EADBsbolus forcing the beads into the proventriculus using a semi-rigid gavage; group Enroad-lib dosed through their drinking water; group Enrobolus also administered into the proventriculus by gavage; group Enrow&f administering 5 mg/kg as EADBs in their feed, plus 5 mg/kg of enrofloxacin through their drinking water. METHODS: The PK parameters and the key PK/PD ratios were determined (Cmax/MIC and AUC0-24/MIC). Only group Enrow&f could achieve the PK/PD ratios regarded as mutant-prevention. RESULTS: This trial is the first one in which an in-feed medication of enrofloxacin, combined with water dosing, can result in PK/PD parameters superior to those obtained after administering the drug through drinking water at a dose of 10 mg/kg. CONCLUSION: Contrary to expectations, groups Enroad-lib and Enrobolus failed to achieve the desired PK/PD ratios when the breakpoint was established at 0.5 µg/mL but did so when MIC was set at 0.1 µg/mL. In contrast, EADBsbolus and Enrow&f achieved an adequate AUC0-24/MIC ratio for both MIC levels.

Effects of enrofloxacin's exposure on the gut microbiota of Tilapia fish (Oreochromis niloticus).

Enrofloxacin (ENFX) has a broad-spectrum antibiotic activity, which is widely used in aquaculture. The effect of different ENFX exposure ways on the gut microbiota of tilapia is unclear. This study was conducted to investigate the effects of ENFX exposure on the gut microbiota of tilapia fish (Oreochromis niloticus). Three methods of ENFX exposure were selected: injection (IEG), oral administration (OEG) and soaking (SEG). After 48 h of exposure period, the intestine of tilapia was collected for high-throughput sequencing. PCoA analysis revealed a distinct clustering of control group, and which was located rather far away from ENFX exposure groups. The dominant phyla in the gut microbiota of tilapia fish were Proteobacteria, Actinobacteriota, Fusobacteria and Firmicutes. Compared to the control group, phylum Fusobacteriota was increased in SEG and IEG while decreased in OEG. ENFX treatment led to a decline in Corynebacterium, Clostridium sensu stricto_3 and Bacillus in treated fish compared with control fish, accompanied by an increase in Akkermansia, Ralstonia and Romboutsia. IEG had the least effect on gut microbiota of tilapia because it retained more microbes among treatment groups. Alpha- diversity decreased the most in SEG, but retained more probiotics such as Cetobacterium and Akkermansia. We assessed the effect of enrofloxacin on tilapia by changes in intestinal flora. The result indicated that either exposure method significantly reduced the diversity of tilapia gut microbiota. It may provide basic data for the scientific use of ENFX in aquaculture.

Impact of intrauterine infusion of Platelets-Rich plasma on endometritis and reproductive performance of Arabian mare.

Equine endometritis is one of the most common causes of reproduction failure. To achieve better treatment outcomes, different diagnostic methods should be combined. In the current study, 39 repeat breeder mares were subjected to ultrasonography examination to detect excessive accumulation of intrauterine fluids and an abnormal oedema pattern, which revealed that 61.5% of mares were positive. Combined with endometrial cytology by low-volume uterine flush, 47.7% of smears contain neutrophils (more than 2-3 per HPF X100), and microbial culture. 92.3% of mares were infected with different bacterial isolates, such as Escherichia coli, Streptococci, Staphylococcus, Klebsiella pneumoniae, Klebsiella oxytoca, Citrobacter freundii, Providencia alcalifaciens, and Proteus mirabilis. All mares were given saline solution and gravity withdrawal before being given 20 IU of oxytocin (as ecbolic agents). Mares were divided into three groups; Group one (n = 15) received an intrauterine infusion of 20 mL of freshly prepared autologous platelet-rich plasma (PRP) 6 h after breeding, Group two (n = 15) was treated with three doses of systemic Enrofloxacin 5% during the estrus period, and Group three (n = 9) received only uterine lavage and 20 IU of oxytocin. PRP and Enrofloxacin resulted in a significant (p < .05) reduction in endometrial thickness (oedema; 5.05 and 6.74 mm, respectively) and disappearance of intrauterine fluids compared to the control (10.98 mm). Furthermore, PRP (days) and Enrofloxacin (17.89 days) reduced the days to the next oestrus compared to the control (18.58 and 17.89 vs. 21.19 days, respectively). Furthermore, the pregnancy rate improved to reach 70% in the PRP group and 60% in the Enrofloxacin group, while the control remained low at 22%. In conclusion, autologous PRP can be used as a low-cost alternative therapy for modulating the inflammatory process and effectively treating mares' endometritis.

Exposure to enrofloxacin affects the early development and metabolic system of juvenile American shad, as indicated by host metabolism and the environmental microbiome.

Enrofloxacin as a special fish medicine is widely used in aquaculture fishes in China. But the effect of enrofloxacin exposure to the gut of aquatic animals is still unclear. In our investigation, enrofloxacin (300 mg/kg feed) was experimentally exposed to the juvenile American shad for 7 days and monitored for alterations in metabolomic and transcriptomic responses. The results showed the similar subset of affected pathways (P-value < 0.05), but there were still many differences in the number of identified biomarkers (520 differentially expressed genes genes and 230 metabolites). Most gut metabolic profiles were related to oxidative stress, inflammation, and lipid metabolism. These multiomic results reveal the specific metabolic disruption by enrofloxacin altering many signaling pathways (P-value < 0.05), such as arginine and proline metabolism pathways, pyrimidine metabolism, the FoxO signaling pathway, and purine metabolism. In addition, the predicted functions of proteins analysis showed that enrofloxacin exposure in an aquaculture environment could prevent the occurrence of organic diseases, including Vibrio cholerae infection and bacterial toxins, in aquatic systems. This is the first research indicating that enrofloxacin affects the relationship between environmental microorganisms and intestinal metabolism, and a study of the ecotoxicity of enrofloxacin occurrences in the aquatic system is warranted.

Enrofloxacin-induced transfer of multiple-antibiotic resistance genes and emergence of novel resistant bacteria in red swamp crayfish guts and pond sediments.

Antibiotic resistance genes (ARGs) can be transferred from environmental microbes to human pathogens, thus leading to bacterial infection treatment failures. The aquaculture polluted by over-used antibiotics is considered as a notorious reservoir of ARGs. However, the origin, diachronic changes, and mobility of ARGs under antibiotic exposure in aquaculture systems remain elusive. Our findings showed that enrofloxacin application also increased the relative abundance of various ARGs in addition to quinolone-resistance genes and induced ARG dissemination in crayfish gut and sediment bacteria. Further investigation indicated that the transposase-mediated recombination was the major driver of horizontal gene transfer (HGT) of ARGs under antibiotic stress. Notably, enrofloxacin application also induced the generation of some metagenome-assembled genomes (MAGs) carrying multiple ARGs, which were identified as novel species. Additionally, Enterobacteriaceae constituted a mobile ARG pool in aquaculture. Therefore, aquaculture provides potential wide environmental pathways for generation and spread of antibiotic resistance. Our findings of ARG temporal variations and dissemination pattern in aquaculture with artificial use of antibiotics are critical to the management of antibiotic resistance, which is of great ecosystem and health implications.

Residual enrofloxacin in cattle manure increased persistence and dissemination risk of antibiotic resistance genes during anaerobic digestion.

Anaerobic digestion is a common approach to dispose and recycle livestock manures, and the agricultural application of anaerobic digestives represents an important pathway of spreading antibiotic resistance genes (ARGs) from livestock manures to soils. Enrofloxacin is a clinically important fluoroquinolone antibiotic with high residual concentrations in livestock manure, and propagation of fluoroquinolone resistance genes poses a huge risk to public health. Compared with other antibiotics, enrofloxacin is relatively durable in anaerobic digestion system. However, its effect on the persistence of ARGs during anaerobic digestion and its mechanism are not clear. In this study, we investigated effects of 0, 4, and 8 mg/L enrofloxacin on the abundance, persistence, and transferring risk of five plasmid-mediated fluroquinolone ARGs and five typic clinically important non-fluoroquinolone ARGs during cattle manure digestion. The responses of integrons and microbial communities to enrofloxacin were assessed to uncover the underlying mechanisms. All the ten detected ARGs were highly persistent in anaerobic digestion, among them seven ARGs increased over 8.2 times after digestion. Network analysis revealed that the potential hosts of ARGs were critical functional taxa during anaerobic digestion, which can explain the high persistence of ARGs. Residual enrofloxacin significantly increased the abundance of aac(6')-ib-cr, sul1, intI1, and intI2 throughout the digestion, but had no impact on the other ARGs, demonstrating its role in facilitating horizontal gene transfer of the plasmid-mediated aac(6')-ib-cr. The influence of enrofloxacin on microbial communities disappeared at the end of digestion, but the ARG profiles remained distinctive between the enrofloxacin treatments and the control, suggesting the high persistence of enrofloxacin induced ARGs. Our results suggested the high persistence of ARGs in anaerobic digestion system, and highlighted the role of residual enrofloxacin in livestock manure in increasing dissemination risk of fluroquinolone resistance genes.

Modified rougan decoction alleviates lipopolysaccharide-enrofloxacin-induced hepatotoxicity via activating the Nrf2/ARE pathway in chicken.

Liver injury plays a heavy burden on the chicken industry. Although modified rougan decoction is a prescription for the treatment of liver disease based on the classical prescription of rougan decoction (containing peony and licorice). However, the effect and mechanism of modified rougan decoction on the liver remain unclear. In this study, the effects of the water extracts (MRGD) and the alcohol precipitates of water extracts (MRGDE) against lipopolysaccharide-enrofloxacin (LPS-ENR)-induced hepatotoxicity were discussed in vivo and in vitro. The isolated hepatocytes and 128 one-day-old Hyline chickens were considered research objects. The indices of liver injury and oxidative stress were evaluated by hematoxylin and eosin (H&E) stained and the assay kits, and the nuclear erythroid 2-related factor 2 (Nrf2)/antioxidant responsive element (ARE) pathway was detected by the RT-PCR, western blot, and immunofluorescence tests. All data were analyzed using the IBM SPSS 20.0 software. In vivo, the structural integrity of the liver was maintained, AST, ALT, and MDA levels were decreased, and antioxidant enzymes were increased, confirming that the oxidative stress was reduced and liver injury was alleviated. Correspondingly, MRGD and MRGDE were observed to improve cell viability and decrease lactate dehydrogenase (LDH) in vitro, and the cell oxidative damage was reduced. In addition, the nuclear translocation of Nrf2 was improved significantly, and the mRNA and protein expression levels of the related genes were upregulated. In conclusion, MRGD and MRGDE can exert a protective effect against LPS-ENR-induced hepatotoxicity by activating the Nrf2/ARE pathway, which might be a potential therapeutic prescription for preventing or treating liver injury. Notably, no significant difference was found between the 2 extracts, suggesting that a depth extraction method did not always improve the efficacy of natural medicine. Our results provided new insights into finding effective hepatoprotective medicine.

Influence of Huangqin Decoction on the immune function and fecal microbiome of chicks after experimental infection with Escherichia coli O78.

Huangqin Decoction (HQD), a traditional Chinese medicine formula from the Shang Han Lun written by Zhang Zhongjing, has been used in China for nearly two thousand years. According to the traditional Chinese medicine and previous literature, HQD has the effect of clearing heat, removing toxins, relieving diarrhea and pain. Therefore, HQD was used to prevent or cure many diseases, such as inflammation, diarrhea, malaria, and other acute or chronic gastrointestinal diseases. The effect of HQD, one-herb-absent HQD treatments and enrofloxacin (ENR) on the average daily gain (ADG), mortality rates, visceral index and toll-like receptors (TLRs), inflammatory factors and intestinal microflora in E. coli O78-inoculated chicks were investigated. HQD supplementation increased ADG and reduced the mortality rates caused by E. coli challenge, decreased the heart, liver, bursa of Fabricius (BF) and spleen index. HQD supplementation decreased the serum lysozyme (LZM), IL-1ß, TNF-α, IL-10, IL-6 level, down-regulated the mRNA expression of TLR4, -5 and -15 in the spleen by E. coli challenged chicks, and up-regulated the mRNA expression of TLR4, -5 and -15 in BF. At the phylum level, HQD supplementation reversed the increase of Operational Taxonomic Unit (OTUs), decreased the relative abundance of harmful bacteria Proteobacteria, increased the relative abundance of probiotic bacteria Bacteroidetes and Firmicutes. At the genus level, HQD decreased the relative abundance of harmful bacteria Escherichia-Shigella and Pseudomonas. It means that HQD treatment reversed the change of the gut microbiota structure. Compared with HQD, HQD-DZ and HQD-HQ increased the mortality rates. HQD-HQ decreased the ADG and liver index. HQD-GC decreased the spleen index. All herb-absent increased the serum IL-6, but only the HQD-HQ and HQD-SY increased the serum TNF-α. All herb-absent did not activate the TLRs signaling pathways in spleen and BF of chicks. The harmful bacteria Escherichia-Shigella were increased in HQD-HQ and HQD-DZ treatments. HQD-DZ treatment also increased the level of Proteobacteria. The results showed that dietary supplementation with HQD, by down-regulating the mRNA expression of TLR4, -5 and -15 in the spleen, further decreasing the serum LZM and IL-1ß, TNF-α, IL-10, IL-6 level, improves the immune function and reverses the change of fecal microbiome in chicks challenged with E. coli. In herb-absent supplementation, the results showed that SY and DZ play a key role in reducing the levels of inflammatory factors and keeping fecal microbiome balance respectively. More importantly, HQ is indispensable in HQD, not only play a key role in reducing the level of inflammatory factors, but also in keeping the balance of fecal microflora.

Ornithobacterium rhinotracheale Isolated from Turkeys over a 20-Year Period Harbor Similar Antimicrobial Susceptibility Profiles and Multidrug Resistance.

Infections with Ornithobacterium rhinotracheale are causing respiratory diseases that require antibiotic treatment in poultry worldwide. In the field, this agent is known to often be resistant to many antimicrobials, complicating therapeutic interventions. Therefore, there is a clear need to monitor trends in resistance development. In the present study, antibiotic resistance profiles of 64 O. rhinotracheale strains isolated from diseased turkeys from 2002 to 2021 were investigated against 19 antimicrobial substances by the microdilution method. Susceptibility toward chloramphenicol, carbapenem, and sulfamethozaxole combination was found for all strains. Most isolates were also susceptible to penicillins (98%-100%), with the exception of oxacillin, cephalosporins (84%-100%), tetracycline (89%), and tylosin (88%). In the case of quinolones, 89% of isolates showed intermediate resistance to enrofloxacin, whereas 90% showed full resistance to nalidixic acid. Full resistance to the tested aminoglycosides and colistin was revealed for all strains. Eighteen different AMR profiles were elucidated; more than half of the isolates (53%) shared the same AMR profile. Similar susceptibility profiles of O. rhinotracheale isolates were found on the different farms, proving some stability over the years. All isolates were classified as multidrug resistant. Multiple outbreaks within a flock or in successive flocks within a farm comprised 46 O. rhinotracheale isolates. Here, occasional changes in susceptibility for some antimicrobial substances were observed. In general, most of the changes occurred in quinolones, followed by tetracycline switching mainly from intermediate resistance to full resistance and vice versa. The present surveillance provides actual data on effective antibiotic treatments in case of disease outbreaks and contributes to the One Health concept acknowledging the important link between animal and human health.

Los Ornithobacterium rhinotracheale aislados de pavos durante un período de 20 años albergan perfiles similares de susceptibilidad a los antimicrobianos y resistencia a múltiples fármacos. Las infecciones por Ornithobacterium rhinotracheale están causando enfermedades respiratorias que requieren tratamiento antibiótico en la avicultura en todo el mundo. En el campo, se sabe que este agente a menudo es resistente a muchos antimicrobianos, lo que complica las intervenciones terapéuticas. Por lo tanto, existe una clara necesidad de monitorear las tendencias en el desarrollo de resistencia. En el presente estudio, se investigaron los perfiles de resistencia a antibióticos de 64 cepas de O. rhinotracheale aisladas de pavos enfermos entre 2002 y 2021 frente a 19 sustancias antimicrobianas mediante el método de microdilución. Se encontró susceptibilidad a la combinación de cloranfenicol, carbapenem y sulfametozaxol para todas las cepas. La mayoría de los aislamientos también fueron susceptibles a las penicilinas (98 %­100 %), con la excepción de oxacilina, cefalosporinas (84 %­100 %), tetraciclina (89 %) y tilosina (88 %). En el caso de las quinolonas, el 89% de los aislados resultaron con susceptibilidad intermedia a la enrofloxacina, mientras que el 90% fueron resistentes al ácido nalidíxico. Todas las cepas revelaron resistencia a los aminoglucósidos y a la colistina probados. Se dilucidaron dieciocho perfiles diferentes de resistencia antimicrobiana; más de la mitad de los aislamientos (53%) compartían el mismo perfil antimicrobiano. Se encontraron perfiles de susceptibilidad similares de aislamientos de O. rhinotracheale en las diferentes granjas, lo que demuestra cierta estabilidad a lo largo de los años. Todos los aislamientos fueron clasificados como resistentes a múltiples fármacos. Los brotes múltiples dentro de una parvada o en parvadas sucesivas dentro de una granja comprendieron 46 aislamientos de O. rhinotracheale. Aquí, se observaron cambios ocasionales en la susceptibilidad a algunas sustancias antimicrobianas. En general, la mayoría de los cambios ocurrieron en las quinolonas, seguido por el cambio de tetraciclina principalmente de resistencia intermedia a resistente y viceversa. La vigilancia actual proporciona datos reales sobre tratamientos antibióticos efectivos en caso de brotes de enfermedades y contribuye al concepto de Una Salud que reconoce el vínculo importante entre la salud humana y animal.

Biological aspects of phage therapy versus antibiotics against Salmonella enterica serovar Typhimurium infection of chickens.

Phage therapy is a promising alternative treatment of bacterial infections in human and animals. Nevertheless, despite the appearance of many bacterial strains resistant to antibiotics, these drugs still remain important therapeutics used in human and veterinary medicine. Although experimental phage therapy of infections caused by Salmonella enterica was described previously by many groups, those studies focused solely on effects caused by bacteriophages. Here, we compared the use of phage therapy (employing a cocktail composed of two previously isolated and characterized bacteriophages, vB_SenM-2 and vB_Sen-TO17) and antibiotics (enrofloxacin and colistin) in chickens infected experimentally with S. enterica serovar Typhimurium. We found that the efficacies of both types of therapies (i.e. the use of antibiotics and phage cocktail) were high and very similar to one another when the treatment was applied shortly (one day) after the infection. Under these conditions, S. Typhimurium was quickly eliminated from the gastrointestinal tract (GIT), to the amount not detectable by the used methods. However, later treatment (2 or 4 days after detection of S. Typhimurium in chicken feces) with the phage cocktail was significantly less effective. Bacteriophages remained in the GIT for up to 2-3 weeks, and then were absent in feces and cloaca swabs. Interestingly, both phages could be found in various organs of chickens though with a relatively low abundance. No development of resistance of S. Typhimurium to phages or antibiotics was detected during the experiment. Importantly, although antibiotics significantly changed the GIT microbiome of chickens in a long-term manner, analogous changes caused by phages were transient, and the microbiome normalized a few weeks after the treatment. In conclusion, phage therapy against S. Typhimurium infection in chickens appeared as effective as antibiotic therapy (with either enrofloxacin or colistin), and less invasive than the use the antibiotics as fewer changes in the microbiome were observed.