Discovery of bla OXA-199, a Chromosome-Based bla OXA-48-Like Variant, in Shewanella xiamenensis

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-48a 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.

Introduction bla OXA-48 , encoding the carbapenem-hydrolyzing enzyme OXA-48, was initially found in Klebsiella pneumoniae from Turkey [1] and has now been spread to other Enterobacteriaceae species in a few countries [2,3]. Several bla OXA-48 -like variants have been identified recently, including bla OXA-162 (GenBank Accession no. GU197550; one nucleotide different from bla OXA-48 ), bla OXA-163 (98.1% nucleotide identity with bla OXA-48 ) [4], bla OXA-181 (94.4% nucleotide identity with bla OXA-48 ) [5], bla OXA-204 (nucleotide sequence not available but encoding two amino acid substitutions compared with OXA-48) and bla OXA-232 (nucleotide sequence not available but encoding a single amino acid substitution compared with OXA-181) [3]. The bla OXA-48 gene was previously proposed as been derived from the chromosome-encoded bla OXA-54 of Shewanella oneidensis, but the two genes have only 84% nucleotide identity [6]. Through analyzing the complete genome sequences of a few strains belonging to various Shewanella species available in the GenBank, the bla OXA-48 -like genes are present on the chromosome of several Shewanella species with at least 80% identity to bla OXA-48 . Thus, the actual progenitor of bla OXA-48 may rather lie within a Shewanella species other than S. oneidensis. A Shewanella clinical strain previously isolated and characterized [7] and the type strain of Shewanella xiamenensis [8] were investigated for the presence of a bla OXA-48 -like gene.

Strains
Shewanella isolate WCJ25 was recovered from post-surgical abdominal drainages of a patient with pancreatitis and was identified as S. xiamenensis based on the close identity (99.6% for 16S rDNA gene and 98.5% for gyrB) between WCJ25 and the S. xiamenensis type strain S4 [7]. The S. xiamenensis type strain S4 was provided by Prof. Zhang Xiaobo, Zhejiang University.

Screening for bla OXA-48 -like Genes
PCR was used to screen bla OXA-48 -like genes and to obtain the complete sequence of the bla OXA-48 -like gene with primers listed in Table 1. PCR was conducted using the ExTaq mix (Takara,  Dalian, China) with the conditions being 94uC for 5 min, 30 cycles  (94uC for 30s, 52uC for 45 s, 72uC for 1 min) and a final  elongation step at 72uC for 7

Study on Genetic Context
The genetic context study of bla OXA-199 was investigated using inverse PCR. Genomic DNA of WCJ25, prepared using a commercial kit (Tiangen, Beijing, China), was restricted with AseI-or RsaI (Figure 2), self-ligated with T4 DNA ligase (New England Biolabs, Ipswich, NY, USA) and then used as a template for inverse PCR. The links between genetic elements were confirmed by overlapping PCR (Figure 2, primers listed Table 1). The genetic context of bla OXA-48 in the strain S4 was characterized by PCR mapping using that of bla OXA-199 as the template ( Figure 2). Primers were designed based on available sequences using the primer3 software (http://frodo.wi.mit.edu/primer3/) with the default settings. Inverse PCR, overlapping PCR and PCR mapping were also conducted using the ExTaq mix with the conditions being 94uC for 5 min, 30 cycles (94uC for 30s, 55uC for 45 s, 72uC for 5 min) and a final elongation step at 72uC for 7 min.
Amplicons were sequenced using an ABI 3730xl DNA Analyzer (Applied Biosystems, Foster City, CA) at the Beijing Genomics Institute (Beijing, China). Sequences were assembled using the SeqMan II program in the Lasergene package (DNASTAR Inc, Madison, WI) and similarity searches were carried out using BLAST programs (http://www.ncbi.nlm.nih.gov/BLAST/).
GenBank accession number. The genetic context of bla  in WCJ25 and that of bla OXA-48 in the strain S4 have been deposited in GenBank as JN704570 and JX644945, respectively.

Results and Discussion
S. xiamenensis is a newly-recognized species originally found in the coastal sea sediment in Xiamen, China [8] and has also been recovered from gutters in India very recently [10]. The identification of S. xiamenensis in India and two distant parts of China suggested that this species might be an underrecognized member of Shewanella with a wide geographical distribution.
The bla OXA-48 -like gene of strain S4 was confirmed as a variant of bla OXA-48 , designated bla OXA-48b here, which had four silent nucleotide differences from the bla OXA-48 variant (AY236073), designated bla OXA-48a here, found in the Enterobacteriaceae. Strain WCJ25 harboured a novel bla OXA-48 -like gene, designated bla OXA-199 by the b-lactamases numbering system available at www.lahey. org. The bla OXA-199 gene had five nucleotide differences from both bla OXA-48a and bla OXA-48b (99.4% identity), specifying the OXA-199 protein with three amino acid substitutions (H37Y, V44A and D153G) compared to OXA-48. During the process of this work, bla OXA-181 was identified in a S. xiamenensis isolate from India [10]. However, bla OXA-181 was significantly divergent from bla OXA-48b (94.7% identity, 42 nucleotide differences), bla OXA-48a (94.4% identity, 45 nucleotide differences) and bla OXA-199 (94.1% identity, 47 nucleotide differences). Based on a phylogenetic tree (Figure 1) constructed by the MEGA program, the results showed that the bla OXA-48 -like genes could be divided into three clusters among which bla OXA-48a , -48b , -162 , -163 , -181 and -199 were of a cluster different from the chromosome-encoded bla OXA-48 -like genes of Shewanella species other than S. xiamenensis. The bla OXA-48 -like genes of the same Shewanella species clustered together, suggesting that the divergence of the bla OXA-48 -like gene might reflect the phylogeny of Shewanella species. Genetic contexts of bla OXA-48b and bla OXA-199 were shown in Figure 2. The 26 bp sequence upstream and 63 bp downstream of bla OXA-199 were identical to those of bla OXA-48a and bla OXA-48b , also suggesting a common origin of these genes. Both bla OXA-48b and bla OXA-199 genes were adjacent to several genes upstream, i.e. an orf encoding the peptidase C15, sprT encoding a SprT-like protein of unknown function, endA encoding the endonuclease I and rsmE encoding a ribosomal RNA small subunit methyltransferase. Variants of these genes are also present adjacent to the bla OXA-48 -like gene in S. oneidensis MR-1 (AE014299), Shewanella sp. MR-4 (CP000446), Shewanella sp. MR-7 (CP000444) and Shewanella sp. ANA-3 (CP000469). The nucleotide identities of these genes among Shewanella species are shown in Figure 3. The insertion sequence ISEcp1 has been found upstream of bla OXA-181 [5] but was not detected upstream of bla OXA-48b and bla OXA-199 using long-range PCR.
As seen in the contexts of bla OXA-48a in K. pneumoniae strain 11978 (AY236073) [1], a putative lysR transcriptional regulator gene was located downstream of bla OXA-48b and bla  . The lysR gene was adjacent to an acc gene that encoded an acetyl-CoA carboxylase multifunctional enzyme at the other side. In K. pneumoniae 11978, the acc gene is truncated by the insertion of IS1999 (an insertion sequence also called IS10A) and two copies of IS1999 bracketing bla OXA-48a -lysR-accD formed a composite transposon, which could mobilize bla OXA-48a to different locations [1,11]. The lysR and acc genes are commonly present downstream of bla OXA-48 -like genes on chromosomes of Shewanella spp ( Figure 3). As mentioned above, genes located either upstream or downstream of the bla OXA-48 -like genes from different Shewanella spp. displayed variable degrees of identities, suggesting that these genes might have different mutation rates.
Based on the significant similarity among contexts of bla OXA-48a , bla OXA-48b and bla OXA-199 , it is reasonable to hypothesize that two copies of IS1999, one inserted at 26 bp upstream of a bla OXA-48like gene and another inserted in acc, could move bla OXA-48 -like-lysR-accD from the chromosome of S. xiamenensis to a plasmid. Such plasmid could have been transferred to Enterobacteriaceae later on resulting in the emergence of bla OXA-48 -like genes. Of note, bla OXA-48a and bla OXA-181 have always been found in distinct genetic contexts as bla OXA-48a is bracketed by two copies of IS1999 while bla OXA-181 is downstream of ISEcp1 [3]. In light of the distinct genetics and the significant nucleotide differences (94.4% identity) between bla OXA-48a and bla OXA-181 , it seems unlikely that the two genes derived from each other through mutations but had different origins from two Shewanella strains [3].

Conclusions
From the phylogenetic analysis performed in this study, it appears that bla OXA-48a might have originated from the bla OXA genes such as bla OXA-48b and bla OXA-199 on the chromosome of certain S. xiamenensis strains. The significant nucleotide differences (,95% identity) between bla OXA-181 and bla OXA-48b or bla OXA-199 might represent the divergence of the chromosome-encoded bla OXA-48 -like genes between different S. xiamenensis strains in different geographical regions and could also suggest that bla OXA-48a and bla OXA-181 were mobilized independently from different S. xiamenensis strains. The bla OXA-48a and bla OXA-181 determinants  appeared to have distinct origins and the emergence of bla OXA-48like genes in Enterobacteriaceae thus probably can not be attributed to a single mobilization event in the species S. xiamenensis but likely is a result of parallel or successive events occurring in multiple strains of S. xiamenensis.