Bhi broth

Two A. butzleri isolates from human faeces, HC-1 and HC-2, were characterised here by means of genomic and in vitro assays. In the Caco-2 cell line infection assays, strains Salmonella enterica serovar Typhimurium LT2 CECT 722 (Spanish Culture Cell Type), Escherichia coli DH5α NCCB 2955 (Netherlands Culture Collection of Bacteria), and A. butzleri RM4018 (CCUG 30485, Culture Collection University of Gothenburg) were also included; S. Typhimurium LT2 as positive control for the adhesion and invasion, E. coli DH5α as positive control for adhesion and negative for invasion, and A. butzleri RM4018 as reference. This latter strain was also included as positive control in the motility and urease activity tests. In addition, E. coli DH5α strain was included as negative control in the urease test.
Arcobacter strains were routinely grown at 30 °C for 12–16 h in BHI broth (Oxoid, Basingstoke, UK) or for 24–48 h on Columbia agar base plates (Oxoid, Basingstoke, UK) supplemented with 5% defibrinated sheep blood (Liofilchem, Roseto degli Abruzzi, Teramo, Italy), under aerobic conditions. S. Typhimurium LT2 and E. coli DH5α were routinely grown at 37 °C for 12–16 h in BHI broth (Oxoid, Basingstoke, UK) or on Muller–Hinton (MH) agar (Oxoid, Basingstoke, UK), aerobically. Shaking (150 rpm) was applied when necessary.

Vancomycin resistance development was initiated by incubating a single susceptible VVE colony in 5 ml of BHI broth (Oxoid, Basingstoke, United Kingdom) overnight, followed by a 1:100 dilution into 5 ml of BHI broth containing 2 or 8 μg of vancomycin or teicoplanin/ml. With observation of growth every 12 h the first 2 days and every 24 h thereafter, emerged resistant mutants were diluted 10⁶-fold and plated on BHI agar containing 8 μg/ml vancomycin to obtain single colonies. All incubations were performed at 37°C. The vanA cluster structures of revertants were assessed by PCRs as described above.

Stool samples were streaked on Bile Esculin Azide Agar (BEAA) (Hardy Diagnostics, Santa Maria, USA) and incubated for 24 hours at 37°C. Plates were observed for appearance of characteristic colonies with dark halo center. Characteristic colonies were selected randomly for characterization and identified presumptively as Enterococci [12 ] by the following phenotypic tests: (a) Gram stains; only plates that yield Gram-positive cocci in pairs or short chains were studied further. (b) Catalase test; catalase test was performed on suspected colonies according to standard microbiological procedure [12 ] and only microbial growth which yield negative result for catalase production were considered further. (c) Growth in 6.5% NaCl; similar colonies from each plate were picked and inoculated in to brain heart infusion (BHI) broth (OXOID LTD, UK) containing 6.5% NaCl and incubated at 37°C for 24–48 hours, and growth in the medium were indicated by turbidity [13 ]. (d) Growth at 45°C; colonies were picked, inoculated into BHI broth, and incubated at 45°C for 24 hours, and growth in the medium were indicated by turbidity. An isolate fulfilling the above criteria were considered as an Enterococcus species [12 ]. For further identification, stock cultures were stored at BHI broth containing 50% glycerol (Biochemica Synthesis Service, Germany) at −20°C.