Penicillin resistance in the intestinal spirochaete Brachyspira pilosicoli associated with OXA-136 and OXA-137, two new variants of the class D β-lactamase OXA-63

Penicillin resistance mediated by β-lactamase activity has been reported previously in the anaerobic intestinal spirochaete Brachyspira pilosicoli, and a novel class D β-lactamase (OXA-63) hydrolysing oxacillin was described recently in a resistant human strain from France. In the current study, 18 B. pilosicoli strains from Australia and Papua New Guinea were tested for ampicillin and oxacillin susceptibility, and investigated for the presence of the class D β-lactamase gene bla_OXA-63 using PCR. PCR products were amplified from seven human and four porcine strains that were penicillin resistant, but not from seven penicillin-sensitive strains. Sequence analysis of the whole gene amplified from seven of the resistant strains from humans and pigs revealed only minor nucleotide differences among them, but there were significant differences compared with bla_OXA-63. The predicted amino acid sequence of the enzyme from all seven strains had the same key structural motifs as the previously reported OXA-63, but two variants with 94-95% identity with OXA-63 were identified. OXA-136 had an additional amino acid and 12 other consistent amino acid substitutions compared with OXA-63. OXA-137 had the same differences compared with OXA-63 as OXA-136, but had an additional amino acid substitution at position 1 6. No structures consistent with integrons or transposons were found in the nucleotide sequences in the vicinity of bla_OXA-136 in partially sequenced B. pilosicoli strain 95/1000, and the GC content (25.2 mol%) of the gene was similar to that of the whole genome. The gene encoding OXA-136 from B. pilosicoli strain Cof-10 conferred penicillin resistance on Escherichia coli. This study shows that penicillin resistance in human and porcine B. pilosicoli strains from Australia is associated with the production of two variants of OXA-63, and that susceptible strains lack the genes encoding OXA-63 or the variants.