The Genome Sequence of a Type ST239 Methicillin-Resistant Staphylococcus aureus Isolate from a Malaysian Hospital

We report the genome sequence of a healthcare-associated MRSA type ST239 clone isolated from a patient with septicemia in Malaysia. This clone typifies the characteristics of ST239 lineage, including resistance to multiple antibiotics and antiseptics.


Introduction
Antibiotic resistance in S. aureus is a major concern, as an increasing number of infections are caused by methicillin-resistant S. aureus (MRSA). Figure 1 shows the phylogenetic position of S. aureus in relation to other staphylococci. In Malaysia, the incidence of MRSA-related infections is a cause of concern in hospitals country-wide. Health-associated MRSA (HA-MRSA) has been dominated by a few lineages in Southeast Asia, particularly ST239. Sequence type 239 is an international healthcare-associated (HA) MRSA lineage prevalent in Asia, South America and Eastern Europe, which includes EMRSA-1, -4, -7, and -11 and the Brazilian, Portuguese, Hungarian, and Viennese clones. Strains of type ST239 are typically resistant to multiple classes of antibiotics and antiseptics such as β-lactam antibiotics.

Classification and features
We have chosen a representative of an MRSA strain, termed MRSA PR01 isolated from a patient with septicemia, isolated from a hospital in Kuala Lumpur. Table 1 indicates general information gathered on MRSA PR01. The MRSA PR01 strain has been identified as sequence type 239 (ST239) by multilocus sequence typing (MLST). Initial disc susceptibility tests showed that the strain is resistant to β-lactam antibiotics oxacillin, ampicillin, cefuroxime, ceftriaxone, gentamicin, erythromy-cin, ciprofloxacin and co-trimoxazole.

Genome sequencing information Genome project history
This organism was selected for sequencing as a representative of MRSA infection in a local Malaysian hospital. The genome sequences of this organism were deposited in GenBank (WGS database). Sequencing, finishing and annotation were performed at the Pharmacogenomics Centre (PROMISE), UiTM. Table 2 presents the project information and its association with MIGS version 2.0 compliance [14].

Growth conditions and DNA isolation
MRSA PR01 was grown overnight under aerobic conditions in Tryptic Soy Broth at 37°C. DNA extraction was performed using MasterPure™ Gram Positive DNA Purification Kit (Epicentre, Madison, USA) as per manufacturer's instructions. The concentration and purity of resultant DNA was assessed by UV spectrophotometry (Nanodrop, Thermo Scientific). 5 µg of genomic DNA (A260/280 = 1.88) was used for library preparation.

Genome sequencing and assembly
The genome sequence was obtained using 104 Mb of paired-end (300 bp spacing) data from the Illumina GAIIx platform (Illumina, San Diego, CA) with 36-bp reads. Sequence data were assembled using CLCBio Genomics Workbench (CLC bio, Aar-hus, Denmark). One hundred and ninety-five contigs (N50: 13,272 bp) were generated, and were overlaid with the reference sequence Mu50 using OSLay. Fourteen supercontigs were gener-ated as a result. Gaps were closed using Sanger sequencing. haemolyticus strain CCM2737, X66100; and S. cohnii strain ATCC 49330, AB009936. The tree uses sequences aligned by the RDP aligner, and uses the Jukes-Cantor corrected distance model to construct a distance matrix based on alignment model positions without the use of alignment inserts, and uses a minimum comparable position of 200. The tree is built with RDP Tree Builder, which uses Weighbor [1] with an alphabet size of 4 and length size of 1000. The building of the tree also involves a bootstrapping process repeated 100 times to generate a majority consensus tree [2]. Staphylococcus lutrae (X84731) was used as an outgroup.

Genome properties
The MRSA PR01 genome consists of a 2,725,110bp circular chromosome with a GC content of 32.6% (Table 3). The MRSA PR01 genome contains 2668 CDs with 19 rRNA features (). A total of 1722 (64.5%) of protein coding genes were assigned to COGs, and a breakdown of the functional assignment of COG-assigned genes is shown in Table 4. Plasmid sequences were only partially sequenced. Figure 2 depicts genomic regions of interest found in the preliminary analysis of the MRSA PR01 genome. Initial analysis of the genome revealed several key features. This genome has a typical SCCmec type III cassette, containing cadmium resistance genes. SCCmec type III is a composite element that is comprised of SCCmec and SCCmercury. In the MRSA PR01 genome, like others, this region harbors ccrC, pI258 and Tn554 as well as the genes involved in cadmium resistance. The MRSA PR01 genome contains two pathogenicity islands, and several resistance features were identified such as the qacA gene, which confers resistance to antiseptics such as cationic biocides, quaternary ammonium salts, and diamidines via an exportmediated mechanism, and the norA gene which confers resistance to hydrophilic quinolones such as norfloxacin and ciprofloxacin. There were 9 regions defined as prophage regions by PHAST [17] with one complete prophage region.genes were identified in the genome. A total of 2,267 genes (72.66%) were assigned a putative function. The remaining genes were annotated as hypothetical proteins. The properties and the statistics of the genome are summarized in Table 3. The distribution of genes into COGs and KEGG functional categories is presented in Table 4.

Conclusion
This study is the first to report on the whole genome sequence of a Malaysian MRSA isolate. Preliminary analysis of the genome has highlighted the genetic determinants that are responsible for the organism to adapt easily to selective pressures. Further research is being conducted to pro-vide insight on the adaptive power of this healthcare-associated strain to attain high resistance to antibiotics. Nucleotide sequence accession numbers. This Whole Genome Shotgun project has been deposited at DDBJ/EMBL/GenBank under the accession ANPO00000000. The version described in this paper is the first version, ANPO01000000.  [19].   Strain AST-10 T is rod-shaped with size of 0.2-0.4 µm x 0.5-1.2 µm. It is an obligate anaerobe and occurs singly. The temperature range for growth is 15-35 o C, optimum at 30 o C. The pH range for growth is 6.5-8.5, optimum at 7.0-7.5. The salinity range for growth is 10-60 g L -1 , and optimum at 30 g L -1 . Strictly chemolithoautotrophic growth occurs with H2, HSor S2O3 2-as an electron donor and with nitrate as an electron acceptor. Nitrate is reduced to N2, and reduced sulfur compounds are oxidized into S 0 or SO4 2-(depending on molar ratio of S2O3 2-/NO3 -). The major cellular fatty acids are C14:0, C16:0, 2-OH C16:0, C16:1, C18:0, and C18:1, with C16:0 2-OH as a unique fatty acid different from other species in the genus Sulfurimonas.
The type strain AST-10 T = DSM 2096 T = JCM 18418 T , was isolated from coastal sediment at the Kai Tak Approach Channel connected to Victoria Harbour in Hong Kong, China. The GC content of the genome is 34.9%. The genome sequence has been deposited at DDBJ/EMBL/GenBank under accession number AUPZ00000000.