Discovery of bla(OXA-199), a chromosome-based bla(OXA-48)-like variant, in Shewanella xiamenensis

Abstract

Introduction: bla(OXA-48) is a globally emerging carbapenemase-encoding gene. The progenitor of bla(OXA-48) appears to be a Shewanella species. The presence of the bla(OXA-48)-like gene was investigated for two Shewanella xiamenensis strains.

Methods: Strain WCJ25 was recovered from post-surgical abdominal drainages, while S4 was the type strain of S. xiamenensis. Species identification for WCJ25 was established by sequencing the 16S rDNA and gyrB genes. PCR was used to screen the bla(OXA-48)-like genes and to obtain their complete sequences. A phylogenetic tree of the bla(OXA-48)-like genes was constructed. The genetic context of the bla(OXA-48)-like gene in strain WCJ25 was investigated by inverse PCR using self-ligated AseI- or RsaI-restricted WCJ25 DNA fragments as template, while that in strain S4 was determined by PCR mapping using that in WCJ25 as template.

Results: A new bla(OXA-48) variant, designated bla(OXA-48b), with four silent nucleotide differences from the bla(OXA-48) (designated bla(OXA-48a)) found in the Enterobacteriaceae was identified in strain S4. Strain WCJ25 had a new bla(OXA-48)-like variant, bla(OXA-199), with five nucleotide differences from bla(OXA-48)a and bla(OXA-48b). The OXA-199 protein has three amino acid substitutions (H37Y, V44A and D153G) compared with OXA-48. Both bla(OXA-48b) and bla(OXA-199) were found adjacent to genes encoding a peptidase (indicated as orf), a protein of unknown function (sprT), an endonuclease I (endA), and a ribosomal RNA methyl transferase (rsmE) upstream and to transcriptional regulator gene lysR and an acetyl-CoA carboxylase-encoding gene downstream. In addition, the insertion sequence ISShes2 was found inserted downstream of bla(OXA-199) but not of bla(OXA-48b). The 26 bp sequences upstream and 63 bp downstream of bla(OXA-48a), bla(OXA-48b) and bla(OXA-199) were identical.

Conclusions: bla(OXA-48a), bla(OXA-48b) and bla(OXA-199) might have a common origin, suggesting that the bla(OXA-48a) gene found in the Enterobacteriaceae could have originated from the chromosome of S. xiamenensis.

Figure 1. Neighbour-joining tree of bla _OXA-48-like genes.

Constructed using the MEGA 4.0 program with bootstrap values and the bar of distance indicated. The host species and strains for the chromosome-encoded genes are indicated. Of note, bla _OXA-181 has also been found in the Enterobacteriaceae . It appears that bla _OXA-48a, bla _OXA-162 and bla _OXA-163 have the S. xiamenensis origin.

Figure 2. Genetic contexts of bla _OXA-199 and bla _OXA-48.

The orientations of insertion sequences are indicated using arrows and the IRs are depicted as poles. Amplicons and sizes for PCR mapping are shown. Panel A, the genetic context of bla _OXA-199. Restriction sites of enzymes that were used to generate DNA fragments as templates for inverse PCR are indicated. Common structures in the contexts of bla _OXA-199, bla _OXA-48b and bla _OXA-48a are illustrated by broken lines. ISShes2 is inserted between bla _OXA-199 and lysR, generating 3-bp DR (CCT). The acc gene was only partially sequenced. The gene encoding peptidase C15 is indicated as ‘orf’. Panel B, the genetic context of bla _OXA-48b in S. xiamenensis strain S4. Panel C, the genetic context of bla _OXA-48a in K. pneumoniae strain 11978 (AY236073). Two copies of IS1999 formed a composite transposon and was inserted into the tir gene (responsible for transfer inhibition), which is part of the IncFII plasmid backbone, generating 9-bp DR (CGTTCAGCA). Panel D, the alignment of right-hand IR (IRR) and left-hand IR (IRL) of ISShes2.

Figure 3. Genetic components surrounding bla _OXA-48-like genes in Shewanella strains WCJ25, S4, MR-1, MR-4 MR-7 and ANA-3.

Variants of the same gene are depicted in the same colour with nucleotide identities compared to the counterparts of WCJ25 being indicated underneath. Of note, the acc genes of strains MR-1, MR-4 MR-7 and ANA-3 are complete with 4554 bp in length but only 333 bp were included into the analysis in parallel with the available partial acc sequence of strains WCJ25 and S4.