X omat processor

Plasmid profiling was performed according to previously described methods [24] (link). Plasmid incompatibility (Inc) groups were determined by PCR with the following primers: HI1, HI2, I1-Iγ, X, L/M, N, FIA, FIB, W, Y, P, FIC, A/C, T, FIIAs, F, K, and B/O [25] (link).
Southern hybridization was performed as follows. Probes were prepared by PCR. Probes for bla_CTX-M-2 and bla_CTX-M-14 were prepared using a CTX-M consensus primer set [26] (link). The probe for bla_CMY-2 was prepared using primers described by Pérez-Pérez and Hanson [21] (link). These PCR products were labeled using a PCR DIG Labeling Mix (Roche Diagnostic, Tokyo, Japan) according to the manufacturer’s instructions. Plasmid DNA was separated by 0.8% (w/v) agarose gel electrophoresis at 100 V for 70 min. The DNA in the gel was transferred to a positive membrane (Roche Diagnostics) by the capillary method. Pre-hybridization (>30 min) and hybridization (>16 h) were performed using Easy Hyb solution (Roche Diagnostics) under high-stringency conditions, and digoxigenin (DIG) in the hybrids was detected using a DIG Luminescent Detection Kit (Roche Diagnostics) according to the manufacturer’s instructions. A hyper MP film (GE Healthcare Japan, Tokyo, Japan) was exposed to the membranes for 2 min at room temperature and developed in a Kodak X-Omat processor.

A DNA fragment containing the ST104 bacteriophage region was amplified from DT104 with the ST104F and ST104R primers (Table 1). The PCR product was purified using a QIAquick^® Gel Extraction Kit (Qiagen, Hilden, Germany) and labeled with digoxigenin (DIG)-11-deoxyuridine triphosphate by random priming using a DIG-High Prime DNA Labeling Kit (Boehringer GmbH, Germany), according to the manufacturer’s instructions. The labeled PCR product was then used as a probe for Southern hybridization analyses. After PFGE of agarose plugs, genomic DNA from S. enterica strains was transferred to positively charged membranes (Boehringer) by capillary action. Prehybridization (>30 min) and hybridization (>16 h) were performed in DIG Easy Hyb Solution (Boehringer) under high-stringency conditions, and hybrid detection was achieved with a DIG Luminescent Detection Kit (Boehringer), according to the manufacturer’s instructions. Hybridization products were detected by exposing Hyperfilm MP (Amersham International, Little Chalfont, UK) to the membranes for 1–10 min at room temperature. Films were developed in a Kodak X-Omat processor (Rochester, NY, USA).

Soft X-ray imaging was performed using a CMB-2 X-ray irradiation apparatus (SOFTEX). Mice were placed on X-ray films (FUJIFILM) and exposed to X-irradiation at 55 kV and 25 mA for 2 s. The irradiated films were developed using a Kodak X-Omat Processor (Kodak).