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

Abstract

Background: Healthy farm animals have been found to act as a reservoir of extended-spectrum β-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.

Fig. 1

Results of the microbiological studies (experiment I). a Results of microdilution assay, detection of MIC-values of 10 E. coli colonies per animal and sampling day (left red Boxplot: group A (n = 6 animals), right black boxplot: group B, n = 6 animals) after treatment of pigs; Ecoff: epidemiological cut-off (≤1 mg/L) ; cbp: clinical breakpoint (≥8 mg/L). b Overview about the number of examined CFU, number of detected CFU with MIC >1 mg Ceftiofur/L and number of swine with increased MICs. Starting at day 34 the numbers on the left side of each field in the table stands for the value of group A, whereas the second value stands for group B. c Results of agardilution assay; green box: MIC < 1 mg Ceftiofur/L, red box: MIC > 1 mg/L

Fig. 2

MIC-values of E. coli isolates collected by endo-agar plates by sedimentation (a) and by air pumps (b) in the stable after treatment of 6 animals of each group with 3 mg/kg b.w. i.m. (experiment I). a Determination of MIC-values from single colonies (n = 5) isolated from uncovered endo-agar plates via microdilution. Endo-agar plate 1 (location 1) was located in front of box A, whereas the second agar plate (location 2) was positioned besides box B. b MIC-values of E. coli isolates form the air of the stable after enrichment and agardilution. Left air pump was positioned at the side of box A and the second air pump was located close to box B

Fig. 3

Concentrations of DFC in sedimentation dust after treatment of six animals with 3 mg Ceftiofur/kgb.w. i.m. (experiment II). Samples of the sedimentation dust were collected at different locations of the barn (position 1: window ledge, position 2: feeding trough, position 3: resting place, position 4: ground between the boxes, position 5: opposite site of the boxes for the animals). DFC-concentrations were analyzed by mass spectrometry. The arrows indicated the time points of treatment

Fig. 4

Concentrations of DFC in the aerosol of the stable after treatment of six animals with 3 mg Ceftiofur/kg b.w. i.m. or 1 mg/kg b.w. i.m. (experiment II). Via air pumps located near to the animals at the feeding through and on the left side of the resisting place probes of the aerosol were sampled and the DFC content measured by mass spectrometry. For determination of the amount of the dust in the aerosol the filters were weighted before and after the 8 h sampling period