Gassner agar

From September 2014 to March 2015 nasal and rectal swabs as well as composite fecal samples from the corresponding stables (n = 45) were taken from cattle (Bos taurus) in Hesse (coordinates 50°39′58″N 8°35′28″E), Germany. Cattle breeds included Holstein-Frisian, Angus, Hereford, Swiss-Brown, Pinzgauer and Vogelsberger Rotes Höhenvieh. The samples were cultured on blood agar (blood agar base by Merck Chemicals, Darmstadt, supplemented with 5% sheep blood) and on Gassner agar (Oxoid, Wesel, Germany). Screening for carbapenem-non-susceptible Acinetobacter spp. was done by using Mueller-Hinton agar plates (Oxoid, Wesel, Germany) containing 2 mg/L and 4 mg/L meropenem (Sigma-Aldrich, Munich, Germany), respectively. Colonies with suspected reduced susceptibility to carbapenems were initially identified at the species level using matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS; Bruker Daltonics, Bremen, Germany). Species identification was verified by multiplex PCR targeting different portions of the gyrB gene and by 16S rRNA gene sequence analysis [13 (link)].

Sixty-four frozen tissue samples from 50 animals (intestines (with fecal content) (n = 46), liver (n = 6), lung (n = 9), and kidney (n = 3)) were used to attempt cultivation of E. coli. To compensate for sub-lethal injuries of the bacteria due to the freezing process of organs at -80°C, all samples were initially incubated over night at 37°C in brain heart infusion (BHI) broth (Oxoid, Germany). BHI broth was streaked on Columbia blood agar (5% blood), Gassner agar and Chrom orientation agar (Oxoid, Germany) and incubated over night at 37°C. Purple colonies from Chrom orientation agar were confirmed as E. coli by conventional biochemical tests as described previously [24 (link)]. Ability of haemolysis of corresponding colonies was assessed on blood agar. Where biochemical tests revealed ambiguous results, bacterial species were identified with the Api 20E test system (Biomérieux, Germany). One E. coli isolate per sample was picked and used for further analyses, except in cases E. coli colonies showed two various morphologies on Gassner agar. Then two E. coli isolates per sample were taken. All isolates were stored at -80°C in BHI broth with 10% glycerol until further use.

Isolates of C. ulcerans were obtained during routine bacteriological investigations following skin swabbing (case no. 3–8, 10; Table 1) or post mortem examinations (animal no. I, VII; Table 1) from moribund and dead water rats between July 2013 and February 2014. Following full gross examination, tissue specimens of skin, mesenteric lymph node, lung, liver, kidney, intestine, brain, and conspicuous lesions were fixed in 10% neutral buffered formalin and embedded in paraffin. Sections (3 μm) were cut and routinely stained with haematoxylin and eosin. Additionally, sections of skin were stained with GMS, PAS and ZN. Native tissue samples were processed for bacterial culture. Briefly, organ samples and marginal areas of abscesses were flame sterilized and the surface of a fresh cut was directly inoculated onto culture media. Agar plates were incubated for up to 48 hours at 37°C using aerobic (Columbia agar with 5% sheep blood and Gassner agar; all Oxoid, Wesel, Germany), capnophilic and anaerobic conditions (Schaedler; Oxoid), respectively.