Genome Sequence of a Carbapenem-Resistant Strain of Ralstonia mannitolilytica

Ralstonia mannitolilytica, a Gram-negative aerobic bacterium, is an opportunistic human pathogen that is becoming more common in cases of nosocomial infections. We report for the first time the whole-genome sequence analysis of R. mannitolilytica strain MRY14-0246, which carries the intrinsic OXA-443/OXA-22-like and OXA-444/OXA-60-like β-lactamase genes and is resistant to meropenem.

belonging to the genus Ralstonia, is prevalent in water supplies and is becoming more common in cases of nosocomial infections (1). R. mannitolilytica is closely related to Ralstonia pickettii and had previously been named "Pseudomonas thomasii" and R. pickettii biovar 3/"thomasii" (1,2). In 2001, R. mannitolilytica was classified as a novel species in the genus Ralstonia, based on 16S rRNA gene sequence analysis (2). R. mannitolilytica is known as an opportunistic human pathogen, possibly associated with cystic fibrosis (3), and the clinical isolates frequently exhibit resistance to imipenem (4). However, little is known about drug resistance and virulence phenotypes of R. mannitolilytica because its genetic basis is uncertain.
In this report, we announce the first draft genome sequence of R. mannitolilytica. R. mannitolilytica strain MRY14-0246 was recovered from a patient's urine in a medical institution in Japan and was resistant to meropenem, according to the MIC determined using the Vitek2 system and an Etest (bioMérieux) and applying the recommended breakpoints described by the CLSI (5). Whole-genome shotgun sequencing of strain MRY14-0246 was performed using the Illumina HiSeq 2500 pyrosequencing platform (500-to 750-bp insert size). Paired-end reads (2 ϫ 150 bp) were assembled de novo using CLC Genomics Workbench version 7.5.1 (Qiagen). The draft genome sequence of strain MRY14-0246 consisted of 48 contigs, yielding total sequences of 4,671,011 bp with N 50 contig sizes of 328,267 bp. The mean GϩC content was 65.8%. A total of 4,357 coding DNA sequences were annotated by the RAST server version 2.0 (http://rast.nmpdr.org). The 16S rRNA gene sequence of strain MRY14-0246 almost matched that of the R. mannitolilytica type strain LMG 6866 T (GenBank accession number AJ270258) (99.8%). Strain MRY14-0246 carried two novel class D ␤-lactamase gene variants, which we named bla OXA-443 and bla OXA-444 (accession numbers LC030178 and LC030179, respectively).
The bla OXA-443 and bla OXA-444 genes in strain MRY14-0246 are encoded in contigs 8_1 and 10_1 (accession numbers BBUP01000016 and BBUP01000018), respectively, both of which are parts of the chromosome with no transposable element, suggesting that these oxacillinase genes are intrinsically speciesspecific in R. mannitolilytica. R. pickettii produces two resident oxacillinases named OXA-22 and OXA-60 (6, 7). Unlike OXA-22, which is a narrow-spectrum oxacillinase, OXA-60 is an extendedspectrum oxacillinase with carbapenem-hydrolyzing properties (6). The OXA-443 and OXA-444 proteins exhibit close similarities to OXA-22 and OXA-60 (86.0% and 90.3% amino acid identities), respectively, suggesting that OXA-444/OXA-60-like ␤-lactamase could contribute to carbapenem resistance in strain MRY14-0246. Bacterial pathogens frequently use protein secretion systems to interact with their hosts. The type III secretion system (T3SS) and type VI secretion system (T6SS) are known as major virulence factors of the plant pathogen Ralstonia solanacearum (8,9). Strain MRY14-0246 does not contain the T3SS gene cluster, whereas the strain contains the T6SS gene cluster and two hcp and four vgrG translocator genes. T6SS delivers effectors into neighboring organisms, including bacteria and hosts, leading to cytotoxicity and cell death of targets (10). Hence, T6SS could be an important virulence determinant in R. mannitolilytica. A more detailed report of strain MRY14-0246 will be included in a future publication.
Nucleotide sequence accession numbers. The whole-genome shotgun projects of R. mannitolilytica strain MRY14-0246 have been deposited at DDBJ/EMBL/GenBank under the accession number BBUP00000000. The version described in this paper is the first version, BBUP00000000.1.

ACKNOWLEDGMENTS
We thank Kumiko Kai, Yoshie Taki, and Hiromi Toyoizumi for technical assistance and Karen Bush for helping with the nomenclature of the OXA-443 and OXA-444 ␤-lactamase genes. We are grateful to the participating medical institution for providing the strain and clinical information.