Effects of intramuscularly administered enrofloxacin on the susceptibility of commensal intestinal Escherichia coli in pigs (sus scrofa domestica).

BACKGROUND: In the European Union, various fluoroquinolones are authorised for the treatment of food producing animals. Each administration poses an increased risk of development and spread of antimicrobial resistance. The aim of this study was to investigate the impact of parenteral administration of enrofloxacin on the prevalence of enrofloxacin and ciprofloxacin susceptibilities in the commensal intestinal E. coli population. METHODS: E. coli isolates from faeces of twelve healthy pigs were included. Six pigs were administered enrofloxacin on day 1 to 3 and after two weeks for further three days. The other pigs formed the control group. MIC values were determined. Virulence and resistance genes were detected by PCR. Phylogenetic grouping was performed by PCR. Enrofloxacin and ciprofloxacin were analysed in sedimentation samples by HPLC. RESULTS: Susceptibility shifts in commensal E. coli isolates were determined in both groups. Non-wildtype E. coli could be cultivated from two animals of the experimental group for the first time one week after the first administration and from one animal of the control group on day 28. The environmental load with enrofloxacin in sedimentation samples showed the highest amount between days one and five. The repeated parenteral administration of enrofloxacin to pigs resulted in rapidly increased MIC values (day 28: MIC up to 4 mg/L, day 35: MIC ≥ 32mg/L). E. coli populations of the control group in the same stable without direct contact to the experimental group were affected. CONCLUSION: The parenteral administration of enrofloxacin to piglets considerably reduced the number of the susceptible intestinal E. coli population which was replaced by E. coli strains with increased MIC values against enrofloxacin. Subsequently also pigs of the control were affected suggesting a transferability of strains from the experimental group through the environment to the control group especially as we could isolate the same PFGE strains from both pig groups and the environment.

Effects of ceftiofur treatment on the susceptibility of commensal porcine E.coli--comparison between treated and untreated animals housed in the same stable.

BACKGROUND: Healthy farm animals have been found to act as a reservoir of extended-spectrum ß-lactamase (ESBL)-producing Escherichia coli (E. coli). Therefore, the objective of the study was to determine the input of antimicrobial active ceftiofur metabolites in the stable via faeces and urine after intramuscular administration of the drug to pigs and the elucidation of the Escherichia coli ESBL resistance pattern of treated and untreated pigs housed in the same barn during therapy. METHODS: For determination of the minimal inhibitory concentration (MIC) the method of microdilutionaccording to the recommended procedure of the Clinical and Laboratory Standards Institute was used. Inaddition to that, a qualitative determination was performed by agar dilution. Unsusceptible E. coli speciesselected via agar dilution with cefotaxime were confirmed by MALDI-TOF and ESBL encoding genes wereidentified by PCR. The amounts of ceftiofur measured as desfuroylceftiofur (DFC) in the different probes (plasma, urine, faeces and dust) were analysed by UPLC-MS/MS. RESULTS: In a first experiment two groups of pigs (6 animals per group) were housed in the same barn in two separated boxes. One group (group B) were treated with ceftiofur according to the licence (3 mg/kg administered intramuscularly (i.m.) on three consecutive days, day 1-3). During a second treatment period (day 29-31) an increased rate of ESBL resistant E. coli was detectable in these treated pigs and in the air of the stable. Moreover, the second group of animals (group A) formerly untreated but housed for the whole period in the same stable as the treated animals revealed increased resistance rates during their first treatment (day 45-47) with ceftiofur. In order to investigate the environmental input of ceftiofur during therapy and to simulate oral uptake of ceftiofur residues from the air of the stable a second set of experiments were performed. Pigs (6 animals) were treated with an interval of 2 weeks for 3 days with different doses of ceftiofur (3 mg/kg, 1 mg/kg and 0.3 mg/kg i.m.) as well as with 3 mg/kg per os) and the renal and biliary excretion of ceftiofur as its active metabolite were measured in comparison to the plasma levels. In addition to that, probes of the sedimentation dust and the air of the stable were analysed for drug residues. CONCLUSION: The present study shows that treatment of several animals in a stable with ceftiofur influences the resistance pattern of intestinal Escherichia coli of the treated as well as untreated animals housed in the same stable. During therapy with the drug which was administered by injection according to the licence we detected nameable amounts of ceftiofur and its active metabolites in the dust and air of the stable.

Determination of fluoroquinolones in chicken feces - a new liquid-liquid extraction method combined with LC-MS/MS.

The application of antibiotics including fluoroquinolones to farming animals is widespread and may lead to the development of antibiotic resistance and other environmental effects. To calculate environmental loads and for a proper risk assessment it is necessary to determine the antibiotic concentration in feces. Therefore, a new liquid-liquid extraction method combined with HPLC-MS/MS for the detection of marbofloxacin, ciprofloxacin, enrofloxacin and difloxacin in chicken feces was developed. Recoveries ranged from 51.0% to 83.5%. LOQs were between 0.10 and 1.09µg/kg. Feces of chickens treated with an enrofloxacin dosage of 10mg/kg bodyweight revealed maximum enrofloxacin and ciprofloxacin concentrations of 61.3 and 18.8mg/kg. Both antibiotics could be detected in feces up to two days after the last application in notable amounts (∼1mg/kg). Thus, feces of recently medicated chickens should not be used as a fertilizer without any further processing.

Comparison of different solid-phase extraction materials for the determination of fluoroquinolones in chicken plasma by LC-MS/MS.

Fluoroquinolones are synthetic antibiotics which are frequently used in veterinary medicine e.g. for the treatment of poultry. Their specific importance is based on the fact that they are regarded as antibiotics of last resort because of their broad spectrum of action against Gram-negative and -positive bacteria. Here, a new and sensitive method for the simultaneous determination of four fluoroquinolones (marbofloxacin, ciprofloxacin, enrofloxacin and difloxacin) in chicken plasma by LC-MS/MS was developed. Solid-phase extraction was chosen for sample preparation because a selective sample clean-up is combined with an effective extraction. Various solid-phase extraction materials including polymer-based reversed-phase, silica-based reversed-phase and mixed-mode sorbents were compared. Selection criteria were analyte recovery, sample extract purity and economical aspects (analysis time and elution solvent volume). Best recoveries and minimized elution solvent volumes were achieved using polymeric reversed-phase cartridges. However, post-column infusion experiments revealed that the analysis is influenced by co-eluting matrix components. Hence, a combination of a mixed-mode anion-exchange cartridge and a mixed-mode cation-exchange cartridge was used as final extraction method. This method yield slightly lower analyte recoveries compared to polymeric-reversed-phase cartridges but exhibit no matrix effects. Recoveries of spiked chicken plasma ranged from 61.9% to 84.8% with an inter-day precision of generally less than 12%. LODs are between 0.03 and 0.05µg/L; LOQs are between 0.08 and 0.16µg/L. Maximum plasma concentrations of chickens medicated with an enrofloxacin dosage of 3mg/kg bodyweight were 38.9µg/L for enrofloxacin and 3.3µg/L for its main metabolite ciprofloxacin.

Effects of carry-over of fluoroquinolones on the susceptibility of commensal Escherichia coli in the intestinal microbiota of poultry.

Due to the frequent use of antibacterials in veterinary medicine as well as in human medicine the occurrence of antibacterial resistance rises worldwide. But independent of the usage of antimicrobials the microbiota from animals as well as from humans already harbour a diversity of resistance genes. As a consequence of manufacturing animal production the treatment of livestock in case of illness is carried out via feed or drinking water. This automatically implies several risks. It has been demonstrated that an antibiotic treatment of livestock via feed or drinking water cause an accumulation of antibiotics and their metabolites in the direct environment of animals. This can lead to a carry-over or rather a resumption of the antimicrobials and their metabolites. Thus, the aim of this study was to determine the influence of carry-over of enrofloxacin as a representative of the fluoroquinolones on the development of bacterial resistance of commensal E. coli in the intestinal microbiota of poultry. Therefore four different treatment groups were provided and the minimal inhibitory concentrations (MICs) of commensal E. coli were measured: One group acted as untreated control, another one was therapeutically treated with the recommended dosage. The third and fourth group were exposed to different "carry-over dosages" for three weeks, 3% of the recommended dosage were applied to the third and 10% to the fourth group. To determine the influence of a therapeutic treatment on a prestressed microbiota, both groups were treated with the recommended dosage for five days. The present study demonstrates that every kind of exposure of the commensal microbiota of poultry with enrofloxacin leads to an amplification and selection of resistant E. coli, which persist in the commensal microbiota. A long-term exposure of gut microbiota, which already harbour non-wild type E. coli, with high levels of carry-over of fluoroquinolones may lead to a development of high-level clinically resistant E. coli in the commensal microbiota. It has to be investigated to which extent antimicrobial leftovers occur in animal production.