Enterococcal DNA Profiling via Hybridization

Total DNA from enterococcal strains was isolated from overnight cultures grown in BHI, and 500 ng was spotted onto Hybond-N⁺ nylon membranes (Amersham). DNA was fixed by UV crosslinking with 70,000 µJ/cm². Membranes were washed in 2×SSC buffer [27] and blotted dry. Hybridization was carried out using the DIG-High Prime DNA Labeling and Detection Starter Kit I (Roche Diagnostics), per manufacturer's instructions. PCR products used for probes included amplified internal fragments of a putative bile acid hydrolase, cbh; capsule locus cpsF; cytolysin locus cylB; biofilm-related protein encoding esp; gelatinase, gelE; putative stress regulator, gls-24-like (EF0117); putative glycosyl hydrolase (EF0077); putative nuclease (EF0031); S. pneumoniae psaA Mn transporter homolog (EF0095); bifunctional aminoglycoside inactivating gene aac6′-aph2″; and the chloramphenicol acetyltransferase gene, cat. Each was amplified using primers listed Table S1. To sample genomes for the presence of portions of the E. faecalis pathogenicity island (PAI), genes from across the pathogenicity island were selected as shown in Fig S1; sampled regions of the PAI did not include the 5′ most region that contained high homology to plasmid pAM373, due to the extrachromosomal and highly variable nature of this DNA in isolates. Genes blaZ and ermB were detected by PCR using primer pairs ermB-1/ermB-2 and blaZ-1/blaZ-2, respectively. β-lactamase activity was confirmed by colorimetric assay. Due to the large diversity of tetracycline resistance determinants, genotyping for tetracycline resistance was performed only by PCR for the most common resistance determinants, tetL, and tetM[29] (link).