Figures
Abstract
Introduction
blaOXA-48 is a globally emerging carbapenemase-encoding gene. The progenitor of blaOXA-48 appears to be a Shewanella species. The presence of the blaOXA-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 blaOXA-48-like genes and to obtain their complete sequences. A phylogenetic tree of the blaOXA-48-like genes was constructed. The genetic context of the blaOXA-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 blaOXA-48 variant, designated blaOXA-48b, with four silent nucleotide differences from the blaOXA-48 (designated blaOXA-48a) found in the Enterobacteriaceae was identified in strain S4. Strain WCJ25 had a new blaOXA-48-like variant, blaOXA-199, with five nucleotide differences from blaOXA-48a and blaOXA-48b. The OXA-199 protein has three amino acid substitutions (H37Y, V44A and D153G) compared with OXA-48. Both blaOXA-48b and blaOXA-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 blaOXA-199 but not of blaOXA-48b. The 26 bp sequences upstream and 63 bp downstream of blaOXA-48a, blaOXA-48b and blaOXA-199 were identical.
Citation: Zong Z (2012) Discovery of blaOXA-199, a Chromosome-Based blaOXA-48-Like Variant, in Shewanella xiamenensis. PLoS ONE 7(10): e48280. https://doi.org/10.1371/journal.pone.0048280
Editor: Paul Jaak Janssen, Belgian Nuclear Research Centre SCK/CEN, Belgium
Received: May 19, 2012; Accepted: September 21, 2012; Published: October 24, 2012
Copyright: © 2012 Zhiyong Zong. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.
Funding: This study was funded by internal funding from West China Hospital. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.
Competing interests: The author has declared that no competing interests exist.
Introduction
blaOXA-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 blaOXA-48-like variants have been identified recently, including blaOXA-162 (GenBank Accession no. GU197550; one nucleotide different from blaOXA-48), blaOXA-163 (98.1% nucleotide identity with blaOXA-48) [4], blaOXA-181 (94.4% nucleotide identity with blaOXA-48) [5], blaOXA-204 (nucleotide sequence not available but encoding two amino acid substitutions compared with OXA-48) and blaOXA-232 (nucleotide sequence not available but encoding a single amino acid substitution compared with OXA-181) [3]. The blaOXA-48 gene was previously proposed as been derived from the chromosome-encoded blaOXA-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 blaOXA-48-like genes are present on the chromosome of several Shewanella species with at least 80% identity to blaOXA-48. Thus, the actual progenitor of blaOXA-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 blaOXA-48-like gene.
Methods
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 blaOXA-48-like Genes
PCR was used to screen blaOXA-48-like genes and to obtain the complete sequence of the blaOXA-48-like gene with primers listed in Table 1. PCR was conducted using the ExTaq mix (Takara, Dalian, China) with the conditions being 94°C for 5 min, 30 cycles (94°C for 30s, 52°C for 45 s, 72°C for 1 min) and a final elongation step at 72°C for 7 min. The amplicons were purified using the OMEGA Cycle Pure kit (Norcross, GA, USA) and sequenced.
Phylogenetic Analysis of the blaOXA-48-like Genes
Sequences of blaOXA-48-like genes were retrieved from GenBank. The blaOXA-48-like genes and their accession numbers are blaOXA-48 (AY236073), blaOXA-54 (AY500173), blaOXA-55 (AY343493), blaOXA-162 (GU197550), blaOXA-163 (HQ700343), blaOXA-181 (JN205800) and those without assigned gene names on chromosomes of Shewanella spp., i.e., S. algae oxaSH (AY066004), S. baltica BA175 (CP002767), S. baltica OS117 (CP002811), S. baltica OS155 (CP000563), S. baltica OS185 (CP000753), S. baltica OS195 (CP000891), S. baltica OS223 (CP001252), S. baltica OS678 (CP002383), S. loihica PV-4 (CP000606), S. oneidensis MR-1 (AE014299), S. putrefaciens CN-32 (CP000681), S. putrefaciens 200 (CP000681), Shewanella sp. ANA-3 (CP000469), Shewanella sp. MR-4 (CP000446), Shewanella sp. MR-7 (CP000444) and Shewanella sp. W3-18-1 (CP000563). A phylogenetic tree of the blaOXA-48-like genes was constructed using the MEGA 4.0 program [9] using the neighbour-joining method and bootstrapping (value 100) (Figure 1).
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, blaOXA-181 has also been found in the Enterobacteriaceae [10]. It appears that blaOXA-48a, blaOXA-162 and blaOXA-163 have the S. xiamenensis origin.
Study on Genetic Context
The genetic context study of blaOXA-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 blaOXA-48 in the strain S4 was characterized by PCR mapping using that of blaOXA-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 94°C for 5 min, 30 cycles (94°C for 30s, 55°C for 45 s, 72°C for 5 min) and a final elongation step at 72°C for 7 min.
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 blaOXA-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 blaOXA-199, blaOXA-48b and blaOXA-48a are illustrated by broken lines. ISShes2 is inserted between blaOXA-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 blaOXA-48b in S. xiamenensis strain S4. Panel C, the genetic context of blaOXA-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.
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/).
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 blaOXA-48-like gene of strain S4 was confirmed as a variant of blaOXA-48, designated blaOXA-48b here, which had four silent nucleotide differences from the blaOXA-48 variant (AY236073), designated blaOXA-48a here, found in the Enterobacteriaceae. Strain WCJ25 harboured a novel blaOXA-48-like gene, designated blaOXA-199 by the β-lactamases numbering system available at www.lahey.org. The blaOXA-199 gene had five nucleotide differences from both blaOXA-48a and blaOXA-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, blaOXA-181 was identified in a S. xiamenensis isolate from India [10]. However, blaOXA-181 was significantly divergent from blaOXA-48b (94.7% identity, 42 nucleotide differences), blaOXA-48a (94.4% identity, 45 nucleotide differences) and blaOXA-199 (94.1% identity, 47 nucleotide differences). Based on a phylogenetic tree (Figure 1) constructed by the MEGA program, the results showed that the blaOXA-48-like genes could be divided into three clusters among which blaOXA-48a, -48b, -162, -163, -181 and -199 were of a cluster different from the chromosome-encoded blaOXA-48-like genes of Shewanella species other than S. xiamenensis. The blaOXA-48-like genes of the same Shewanella species clustered together, suggesting that the divergence of the blaOXA-48-like gene might reflect the phylogeny of Shewanella species.
Genetic contexts of blaOXA-48b and blaOXA-199 were shown in Figure 2. The 26 bp sequence upstream and 63 bp downstream of blaOXA-199 were identical to those of blaOXA-48a and blaOXA-48b, also suggesting a common origin of these genes. Both blaOXA-48b and blaOXA-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 blaOXA-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 blaOXA-181 [5] but was not detected upstream of blaOXA-48b and blaOXA-199 using long-range PCR.
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.
As seen in the contexts of blaOXA-48a in K. pneumoniae strain 11978 (AY236073) [1], a putative lysR transcriptional regulator gene was located downstream of blaOXA-48b and blaOXA-199. 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 blaOXA-48a-lysR-accΔ formed a composite transposon, which could mobilize blaOXA-48a to different locations [1], [11]. The lysR and acc genes are commonly present downstream of blaOXA-48-like genes on chromosomes of Shewanella spp (Figure 3). As mentioned above, genes located either upstream or downstream of the blaOXA-48-like genes from different Shewanella spp. displayed variable degrees of identities, suggesting that these genes might have different mutation rates.
An insertion sequence was inserted between blaOXA-199 and lysR, evidenced by the presence of 3 bp direct target repeats (DR) (Figure 2). This 1299-bp IS was 98.1% identical to ISShes2 of the IS3 family in nucleotide sequences and had 25-bp inverted repeat sequences (IR) with 23 bp perfectly matched (Figure 2). The ISShes2 element has also been seen in several Shewanella strains whose complete genome sequences are available at GenBank, including Shewanella sp. MR-4 (7 copies; CP000446), Shewanella sp. MR-7 (9 copies plus a truncated version; CP000444), Shewanella sp. ANA-3 (4 copies; CP000469), S. baltica OS195 (2 copies; CP000891), S. baltica OS678 (2 copies; CP002383) and S. baltica OS185 (1 copy; CP000753). Other Shewanella strains with complete genome sequences released, including S. baltica OS223 (CP001252), S. baltica BA175 (CP002767), S. baltica OS117 (CP002811), S. baltica OS155 (CP000563), S. woodyi ATCC 51908 (CP000961), S. oneidensis MR-1 (AE014299) and S. pealeana ATCC 700345 (CP000851) did not harbour ISShes2 but instead carried other insertion sequences sharing 65.7 to 85.9% nucleotide identity with ISShes2.
Based on the significant similarity among contexts of blaOXA-48a, blaOXA-48b and blaOXA-199, it is reasonable to hypothesize that two copies of IS1999, one inserted at 26 bp upstream of a blaOXA-48-like gene and another inserted in acc, could move blaOXA-48-like-lysR-accΔ from the chromosome of S. xiamenensis to a plasmid. Such plasmid could have been transferred to Enterobacteriaceae later on resulting in the emergence of blaOXA-48-like genes. Of note, blaOXA-48a and blaOXA-181 have always been found in distinct genetic contexts as blaOXA-48a is bracketed by two copies of IS1999 while blaOXA-181 is downstream of ISEcp1 [3]. In light of the distinct genetics and the significant nucleotide differences (94.4% identity) between blaOXA-48a and blaOXA-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 blaOXA-48a might have originated from the blaOXA genes such as blaOXA-48b and blaOXA-199 on the chromosome of certain S. xiamenensis strains. The significant nucleotide differences (<95% identity) between blaOXA-181 and blaOXA-48b or blaOXA-199 might represent the divergence of the chromosome-encoded blaOXA-48-like genes between different S. xiamenensis strains in different geographical regions and could also suggest that blaOXA-48a and blaOXA-181 were mobilized independently from different S. xiamenensis strains. The blaOXA-48a and blaOXA-181 determinants appeared to have distinct origins and the emergence of blaOXA-48-like 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.
Acknowledgments
The author is grateful to Chunhong Peng for collecting the strain WCJ25 and to Prof. Zhang Xiaobo, Zhejiang University for kindly providing the strain S4.
Author Contributions
Conceived and designed the experiments: ZZ. Performed the experiments: ZZ. Analyzed the data: ZZ. Contributed reagents/materials/analysis tools: ZZ. Wrote the paper: ZZ.
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