Abstract
Here, we report the draft genome sequences of four nosocomial methicillin-resistant Staphylococcus aureus strains (PPUKM-261-2009, PPUKM-332-2009, PPUKM-377-2009, and PPUKM-775-2009) isolated from a university teaching hospital in Malaysia. Three of the strains belong to sequence type 239 (ST239), which has been associated with sustained hospital epidemics worldwide.
GENOME ANNOUNCEMENT
Genotyping using pulsed-field gel electrophoresis (PFGE) of methicillin-resistant Staphylococcus aureus (MRSA) strains circulating in our university teaching hospital revealed the presence of a dominant clone (designated pulsotype B), together with a pulsotype C, whose PFGE profile is only a one-band difference from that of pulsotype B (Xin-Ee Tan et al., submitted for publication). To understand better the biology and evolution of these two strains, we selected two representative strains from each pulsotype (B and C) for whole genome sequencing. The strains PPUKM-261-2009 and PPUKM-377-2009 are representative of pulsotype B, while PPUKM-332-2009 and PPUKM-775-2009 are representative of pulsotype C. Staphylococcal cassette chromosome mec (SCCmec) typing (1) results revealed that PPUKM-261-2009, PPUKM-332-2009, and PPUKM-775-2009 are of SCCmec type III-SCCmercury, which harbors ccrA3B3, ccrC, and class A mec complex, while PPUKM-377-2009 is of SCCmec type IV, harboring ccrA2B2 and class B mec complex. Multilocus sequence typing (MLST) results (2) showed that PPUKM-261-2009, PPUKM-332-2009, and PPUKM-775-2009 belong to sequence type 239 (ST239), which is widely associated with hospital epidemics (3) and has been reported to be the dominant clone in another Malaysian hospital (4). Conversely, PPUKM-377-2009 is from a new sequence type that has a close match to ST1178.
Whole genome sequencing for all strains was performed with an Ion personal genome machine (PGM) sequencer (Life Technologies, Carlsbad, CA). After sequencing, reads were filtered and trimmed prior to de novo assembly using Genome Sequencer (GS) assembler version 2.70. Noncoding rRNAs and tRNAs were predicted using RNAmmer 1.2 server and tRNAscan-SE v1.23 programs, respectively. The protein-coding gene models were predicted using the ab initio-based gene predictors Glimmer v3.02 and Genemark.hmm v2.10b, while gene annotations were carried out by BLASTp searched against the NCBI nonredundant (NR), Swiss-Prot, Gene Ontology (GO), and Kyoto Encyclopedia of Genes and Genomes (KEGG) databases. As validation, the assembled sequences were compared with the MRSA reference genomes of N315 (carrying the ccrA2B2 gene) (5) and TW20 (carrying the ccrA3B3 gene) (6).
For the de novo assembly, 638,248 to 923,828 clean reads spanning 115,836,594 to 180,374,605 bases were assembled and aligned. Ninety-seven to 125 contigs were generated, which totaled 2,956,301, 3,019,328, 2,851,702, and 3,000,990 bp for PPUKM-261-2009, PPUKM-332-2009, PPUKM-377-2009, and PPUKM-775-2009, respectively, with >95% (96.57%, 96.65%, 95.1%, and 96.12%, respectively) coverage for all assemblies when compared against reference genomes. The G+C contents were similar for all 4 strains (32.6% for PPUKM-261-2009, PPUKM-332-2009, and PPUKM-775-2009; 32.7% for PPUKM-377-2009). About 2,000 proteins were annotated for all strains using Swiss-Prot, while GenBank NR annotation predicted a larger number of proteins (>2,800) for each strain, which requires further validation through experiments (7). All strains had 2 copies of 5S rRNAs and 1 copy each of 16S and 23S rRNAs, whereas the numbers of predicted tRNAs were in the range of 28 to 53. Gene ontology (a functional classification) showed similar distributions for all 4 strains, which is owed to their close relatedness as visualized through PFGE typing. Further studies of the sequenced genomes are currently under way.
Nucleotide sequence accession numbers.
The draft sequences of PPUKM-261-2009, PPUKM-332-2009, PPUKM-377-2009, and PPUKM-775-2009 have been deposited in DDBJ/EMBL/GenBank with the accession no. AMRB00000000, AMRC00000000, AMRD00000000, and AMRE00000000, respectively. The versions described in this article are the first versions: AMRB01000000, AMRC01000000, AMRD01000000, and AMRE01000000.
ACKNOWLEDGMENTS
This work was supported by a Young Researcher Grant awarded to H.M.N. from Universiti Kebangsaan Malaysia (UKM-GGPM-TKP-068-2010).
We thank Rosniza Mohamed Hussain, Zamzureena Mohd. Rani, and Mohd. Ridhwan Abd Razak for their help in performing MLST.
Footnotes
Citation Neoh H-M, Mohamed-Hussein Z-A, Tan X-E, B Raja Abd Rahman RMF, Hussin S, Mohamad Zin N, Jamal R. 2013. Draft genome sequences of four nosocomial methicillin-resistant Staphylococcus aureus (MRSA) strains (PPUKM-261-2009, PPUKM-332-2009, PPUKM-377-2009, and PPUKM-775-2009) representative of dominant MRSA pulsotypes circulating in a Malaysian university teaching hospital. Genome Announc. 1(1):e00103-12. doi:10.1128/genomeA.00103-12.
REFERENCES
- 1. Kondo Y, Ito T, Ma XX, Watanabe S, Kreiswirth BN, Etienne J, Hiramatsu K. 2007. Combination of multiplex PCRs for staphylococcal cassette chromosome mec type assignment: rapid identification system for mec, ccr, and major differences in junkyard regions. Antimicrob. Agents Chemother. 51(1):264–274 [DOI] [PMC free article] [PubMed] [Google Scholar]
- 2. Enright MC, Day NP, Davies CE, Peacock SJ, Spratt BG. 2000. Multilocus sequence typing for characterization of methicillin-resistant and methicillin-susceptible clones of Staphylococcus aureus. J. Clin. Microbiol. 38(3):1008–1015 [DOI] [PMC free article] [PubMed] [Google Scholar]
- 3. Smyth DS, McDougal LK, Gran FW, Manoharan A, Enright MC, Song JH, de Lencastre H, Robinson DA. 2010. Population structure of a hybrid clonal group of methicillin-resistant Staphylococcus aureus, ST239-MRSA-III. PLoS One 5(1):e8582. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 4. Ghaznavi-Rad E, Nor Shamsudin M, Sekawi Z, Khoon LY, Aziz MN, Hamat RA, Othman N, Chong PP, van Belkum A, Ghasemzadeh-Moghaddam H, Neela V. 2010. Predominance and emergence of clones of hospital-acquired methicillin-resistant Staphylococcus aureus in Malaysia. J. Clin. Microbiol. 48(3):867–872 [DOI] [PMC free article] [PubMed] [Google Scholar]
- 5. Kuroda M, Ohta T, Uchiyama I, Baba T, Yuzawa H, Kobayashi I, Cui L, Oguchi A, Aoki K, Nagai Y, Lian J, Ito T, Kanamori M, Matsumaru H, Maruyama A, Murakami H, Hosoyama A, Mizutani-Ui Y, Takahashi NK, Sawano T, Inoue R, Kaito C, Sekimizu K, Hirakawa H, Kuhara S, Goto S, Yabuzaki J, Kanehisa M, Yamashita A, Oshima K, Furuya K, Yoshino C, Shiba T, Hattori M, Ogasawara N, Hayashi H, Hiramatsu K. 2001. Whole genome sequencing of meticillin-resistant Staphylococcus aureus. Lancet 357(9264):1225–1240 [DOI] [PubMed] [Google Scholar]
- 6. Holden MT, Lindsay JA, Corton C, Quail MA, Cockfield JD, Pathak S, Batra R, Parkhill J, Bentley SD, Edgeworth JD. 2010. Genome sequence of a recently emerged, highly transmissible, multi-antibiotic- and antiseptic-resistant variant of methicillin-resistant Staphylococcus aureus, sequence type 239 (TW). J. Bacteriol. 192:888–892 [DOI] [PMC free article] [PubMed] [Google Scholar]
- 7. Schnoes AM, Brown SD, Dodevski I, Babbitt PC. 2009. Annotation error in public databases: misannotation of molecular function in enzyme superfamilies. PLoS Comput. Biol. 5(12): e1000605. [DOI] [PMC free article] [PubMed] [Google Scholar]