At an age of four weeks healthy pigs (weight range from 8 to 9.2 kg) were kept after random allocation to two groups (each of six) in a stable for the first part of the experiment (experiment I) in which the development of resistant E. coli in treated and non-treated animals was investigated. The animals were fed with piglet starter food and had free access to water. Before starting the experiment rectal swab samples were taken from each animal and the presence of ESBL resistant E. coli were checked in the gut microbiome of the animals by an enrichment-procedure and agar dilution. In the second part of the experiment (experiment II) six female pigs weighing 10 to 11 kg were used.
All animal studies were conducted according to institutional guidelines for ethical care and use of animals for experimental and other scientific purposes. The study was registered by the Lower Saxony State Office for Consumer Protection and Food Safety (registry number: 33.12-42502-0-11/0338, Lower Saxony State Office for Consumer Protection and Food Safety). The registration procedure contains an approval of an internal ethic committee.
In the first part of the experiment the influence of ceftiofur on the intestinal microbiota of the pigs was investigated. For the experiments a barn with dimensions of 4.8 m × 1.9 m was divided in two boxes via a middle corridor (width 1.60 m). As result of this areal separation a direct contact of the grouped animals was excluded. To minimize points of contact between both animal groups, each box had its own stable equipment for cleaning and feeding. Protective clothing consisting of one-way overalls, gloves and over boots were used in addition to the working clothes for each entry in the stable. Afterwards the one-way articles were depolluted. To avoid a possible cross-contamination the handling and maintenance of the animals always start with the control group (group A).
The first group of animals (group A) served as antimicrobial-free control group and was not treated until day 45 of the experiment. To study the influence of a therapeutic dosage on the physiological enteric microbiota the swine of the second group (group B) were treated with 3 mg/kg/day ceftiofur hydrochloride on three consecutive days (day 1 to 3) and for a second time on days 29 to 31. For investigation of a possible impact of an antibiotic application on pre-exposed but formerly untreated animals the animals of the control group (group A) were treated on day 45 to 47 for the first time. Probes (faeces, dust and aerosol probes for MIC determination) were collected on the following days: 0, 5, 8, 14, 21, 28, 34, 37, 42, 50, and 53.
For collecting of dust of the stable filter pumps were used and in addition to that, samples were drawn by agar plates to observe the resistance situation of the environmental E. coli in the stable. Two of these pumps (flow range between 2.7 L/min and 3.625 L/min ) with sterile polycarbonate filters were placed at a distance of 30 cm from the treated group (group B) at the middle corridor. After running for 1 hour filter membranes from the pumps were transferred into 5 mL of Fluorocult®-LMX-broth (LMX-broth modified acc. to Manafi and Ossmer; Merck, Darmstadt, Germany) using a sterile tweezer. In addition two opened endo-agar plates (lactose-fuchsin-sulfite agar, Merck) were set on the brink of each box in the stable for 1 hour (height 1 m).
For the second part of the experiment (experiment II), only one box of the stable was used for six pigs. These experiments were performed in order to elucidate the input of ceftiofur residues into the stable due to biliary and renal excretion of animals treated with different dosage regimes. In addition, oral uptake of ceftiofur residues via air was simulated by a per os treatment with 3 mg/kg ceftiofur. To measure the excretion of DFC and its metabolites animals (n = 6) were treated in an interval of 2 weeks each time with different doses of ceftiofur (3 mg/kg, 1 mg/kg, and 0.3 mg/kg) i.m. as well as 3 mg/kg p.o. via feed three times every 24 h. For per os treatment the amount of ceftiofur was added to some sugar cubes and given to the animals.
Urine and faeces were collected separately on different days (day 0, 1–3, and 7, 9, 11). These samples were spontaneous excreted and placed into plastic tubes. In addition to that, sedimentation dust was collected from five different positions of the stable by using brand-new playing cards at the same days (see above). Four of these positions were close to the treated animals at the window ledge (position 1), partition grid (positions 2 and 3), and the ground beside the box (position 4). A distance of 1.60 m was between collecting position five and the box of the animals. To collect aerosols filter pumps were used for 8 h per sampling day by using polycarbonate as filter material. They were arranged at two positions close to the box. Dust and filters were placed into glass vials and covered with metal foil. All samples were stored light-protected at −20 °C until preparation.
Plasma samples of each treatment cycle were obtained directly before and at various time intervals after the injection of ceftiofur via syringe from the jugular vein. After i.m. injection blood samples were taken at 0, 24, and 48 h, whereas blood samples after per os application were collected at 0, 2, 4, 6, 24, 48, 50, 52, 54, 71, and 96 h. After centrifugation at 4 °C (3000 x g) for 10 min plasma was collected from blood samples and stored at −80 °C.
Ceftiofur (ceftiofur hydrochloride, ready to use product; Vétoquinol) was administered by intramuscular (IM) injection in the musculus trapezius of the neck. For measuring the concentration of DFC and DFC-metabolites in plasma ceftiofur sodium (ceftiofur sodium, sterile powder; Excenel®) was applicated.
Isolation and MIC-determination of non-type-specific E.coli
The detection of the minimal inhibitory concentration (MIC) followed the “performance standards for antimicrobial disk and dilution susceptibility tests for bacteria isolated from Animals”-M31-A2 of the Clinical and Laboratory Standards Institute . The method of microdilution was used. Microtiterplates containing serial dilutions of ceftiofur concentrations from 0.125 μg/mL up to 64 μg/mL (final concentration after inoculation) were made in the laboratory and stored in a freezer at −70 °C. Mueller-Hinton II broth was used for dilution.
Within 1 hour after sampling 1 g of faeces were homogenized in 9 mL of sterile physiological sodium-chloride solution, streaked onto endo-agar plates and incubated for 24 h at 37 °C.
For the examination of susceptibility, subcultures of at least ten single colonies, morphological typical for E. coli, were sub-cultured on Mueller-Hinton-agar. The susceptibility tests were always performed with colonies from overnight plates. An inoculum of 5 × 105 colony forming units (CFU)/mL per well, adjusted by densiotometry to 0.5 McFarland turbidity standard with Mueller-Hinton-II-broth, was transferred to microdilution plates. To protect the filled trays against dehydration during incubation at 37 °C for 20–24 h the plates were covered with an adhesive foil . For a positive control the reference strain ATCC 25922 was used and MICs were compared to the required MIC reference range (0.25 μg/mL to 1 μg/mL). Furthermore, a control for growth and a negative control were conducted for each colony. To determine CFU’s in inoculum suspensions a dilution of each applied inoculum was plated on Cystine-Lactose-Electrolyte Deficient (CLED)-agar. A number of 20–80 CFU  was set as reference range. The purity of the inoculum was currently checked. To confirm the presence of E. coli in the examined samples an aliquot of each CFU was transferred into Fluorocult® LMX Broth. A color change of the broth from yellow to blue, a blue fluorescence under long-wave UV light and a positive indole reaction with Kovacs reagent was regarded as highly specific for E. coli. Only bacterial colonies which fulfilled all these criteria were further evaluated and the MIC-values determined.
In addition to a quantitative examination with microdilution a qualitative determination was performed by agar dilution. A 1:10 dilution in Lysogeny broth LB-medium of each fecal sample and sampling day was incubated at 37 °C for 24 h and 10 μL of the culture was plated on endo-agar plates containing certain ceftiofur concentrations (0 μg/mL, 1 μg/mL, 2 μg/mL, 8 μg/mL). For confirmation of E. coli Fluorocult LMX broth in combination with Kovacs reagent was used.
Verification of ESBL producing E. coli
After selection of unsusceptible E. coli via agar dilution with cefotaxime containing MacConkey-agar (1 μg/mL), the species was confirmed using MALDI-TOF identification (MALDI Microflex ® LT and Biotyper ® database, Bruker Daltonics, Bremen, Germany). Identification of ESBL encoding genes of the CTX-M, TEM, SHV and CYM families was carried out by PCR according to the method published by Roschanski et al. .
Isolation and detection of E. coli in the environment
At the same day of sampling (see description of experiment I) uncovered endo-agar plates were placed in the stable. After 1 hour these plates were closed and incubated for 24 h at 37 °C. Five colonies per plate were selected and MIC’s determined by microdilution.
The filter membranes of the filter pumps were transferred in Fluorocult LMX broth and following an enrichment of the bacteria at 37 °C for 24 h the growth of E. coli was examined by color change, blue fluorescence and positive indole reaction. In the case of growth 10 μL of the suspension were plated on endo-agar containing different concentrations of ceftiofur (0 μg/mL, 0.5 μg/mL, 1 μg/mL, 2 μg/mL, 8 μg/mL). After incubation (37 °C for 24 h) the growth of CFU of E. coli was evaluated.
Analysis of desfuroylceftiofur (DFC) in the environment
Pretreatment of samples
To avoid excessive amounts of DFC all urine samples of the dosing days (1 to 3) were diluted 1:100.
To 1 mL of urine, 150 mg faeces, 4 mg dust and the filter from the air pumps 7 mL extraction solution [sodiumtetraborat-decahydrat (0.95 % w/v), dithiothreitol (0.23 % w/v)] were added. The samples were vortexed and placed in a water bath at 50 °C for 15 min. Each 3 min the tubes were inverted. Subsequently after adding of 1.5 mL derivatization solution [sodiumhydroxid solution (3.5 % w/v), iodoacetamid (5 % w/v)] to the sample, the dilutions were vortexed again and mixed gently in the dark at room temperature for 1 h. After adding of 250 μL acetic acid in water (20 % v/v) the samples were centrifuged for 10 min (4 °C, 3000 × g).
For solid phase extraction (SPE) all cartridges were cleaned, preconditioned with 2 mL of a solution of acetonitrile in water (1:1) and afterwards preconditioned using 2 mL bidest water. The following extraction protocol was used: the whole supernatant was applied to SPE columns and unbound sample components removed by washing with 3 mL bidest. After drying the column by applying vacuum for 15 s, the analytes were eluted using 1.5 mL of a solution of acetonitrile and bidest (1:1) into a test tube. The eluate was concentrated to a volume of 0.5 mL via SpeedVac® (Eppendorf, Hamburg, Germany), transferred to an autosampler vial and finally filled up to a volume of 1 mL with bidest.
The calibration of DFC was accomplished by pretreatment of ceftiofur sodium (Excenel®) according to the above mentioned protocol. For each specimen (plasma, urine, dust and faeces) matrix matched calibrations were prepared.
The amounts of DFC were analyzed with ultra-performance liquid chromatography (UPLC) in combination with tandem mass spectrometric detection (MS/MS). All detections were performed on an UltiMate 3000 RSLC (Thermo Fisher Scientific, Dreieich, Germany) coupled to a 5500 QTRAP mass spectrometer (AB Sciex, Darmstadt, Germany) equipped with an ESI Turbo Ion Spray source. For separation by liquid chromatography a Acquity BEH C18 separation column, (50 mm × 2.1 mm, 1.7 μm, Waters) connected to a VanGuard BEH Shield RP18 (1.7 μm) pre-column at 60 °C was used in gradient elution mode. The mobile phase consisted of eluent A (water, 0.1 % formic acid) and eluent B (acetonitrile, 0.1 % formic acid) at a flow rate of 900 μL/min. The gradient of Eluent B was: 0 min 5 %; 0 min 5 %; 1.5 min 20 %; 1.6 min 50 %; 2.1 min 50 %; 2.2 min 5 %; 3 min 5 %. The injection volume of each sample amounts to 5 μL.
For DFC quantification mass spectral data were acquired in positive ion electrospray ionization (ESI) mode using the multiple reaction monitoring (MRM) scan mode. Optimized ESI-MS/MS-dependent parameters were as following: ion spray voltage 5.5 kV, ion source gas 1, ion source gas 2, curtain gas, collision gas 40, 60, 20, medium (arbitrary units), respectively. The ion source was run at a temperature of 600 °C. The precursor ion for DFC was the ([M + H]+) ion of m/z 487.0. Characteristic product ions were at m/z 241.0, 227.0, and 210.0 completing the MRM-transitions for DFC quantification. Declustering potential, entrance potential and collision cell exit potential experiments were optimized and set to 120 V, 7 V, 13 V, respectively. By using weighted (1/x) least-squares regression analysis the calibration curves were obtained.
Results of the MIC determination by microdilution were analysed with Exact Fischer Test using Microsoft Excel software (Office 2007). Statistical significance was assumed at p-values of ≤ 0.05.