Genome Sequences of Four Clinical Staphylococcus aureus Strains withDiverse Drug Resistance Profiles Isolated from Diabetic Foot Ulcers

Thokur Sreepathy Murali,a Bobby Paul,a Hersh Parikh,b Rana Pratap Singh,b Shettigar Kavitha,a Manoj K. Bhat,a

Kapaettu Satyamoorthya

Department of Biotechnology, School of Life Sciences, Manipal University, Manipal, Indiaa; Invitrogen Bioservices Pvt., Ltd., Gurgaon, Indiab

Staphylococcus aureus is a major pathogen associated with diabetic foot ulcer infections. To gain insight into their pathogenicityand virulence potential, we report draft genome sequences of four strains of Staphylococcus aureus, isolated from diabetic footulcers, showing profiles with various degrees of resistance to common antibiotics.

Received 21 February 2014 Accepted 27 February 2014 Published 20 March 2014

CitationMurali TS, Paul B, Parikh H, Singh RP, Kavitha S, Bhat MK, Satyamoorthy K. 2014. Genome sequences of four clinical Staphylococcus aureus strains with diverse drugresistance profiles isolated from diabetic foot ulcers. Genome Announc. 2(2):e00204-14. doi:10.1128/genomeA.00204-14.

Copyright 2014 Murali et al. This is an open-access article distributed under the terms of the Creative Commons Attribution 3.0 Unported license.

Address correspondence to Kapaettu Satyamoorthy, ksatyamoorthy@manipal.edu.

Infections in diabetic foot ulcers are among the major comorbidconditions leading to mortality in diabetic individuals (1, 2). Toelucidate the pathogenic potential of bacterial communities asso-ciated with infected diabetic foot ulcers, we carried out culture-dependent isolation of bacteria from infected wound beds. Sam-ples were obtained from patients visiting a tertiary hospital,Kasturba Medical College, Manipal, India, and a total of 357 dia-betic ulcer samples were collected. From these samples, we iso-lated 940 bacterial strains, and these were further analyzed usingtraditional and molecular techniques to assess the diversity.Though an abundance of Gram-negative bacilli and Gram-positive cocci were obtained, Staphylococcus aureuswas frequentlyisolated from the wound samples. Staphylococcus aureus is consid-ered themajor pathogen associatedwith diabetic foot ulcers and isalso one of the most adaptable human pathogens, known to causea wide range of diseases (3). Though worldwide methicillin-resistant Staphylococcus aureus (MRSA) strains are a major causeof hospital-acquired infections, there are also reports of emer-gence of MRSA and methicillin-susceptible S. aureus (MSSA)from other environmental settings (4). Extensive structural fea-tures, including specific surface components that aid in adhesionto host tissues and an array of secreted proteins/toxins expressedby S. aureus strains, play major roles in enhancing the virulencepotential of these organisms (5, 6). The strains obtained weretested for their sensitivity to amikacin, ampicillin, amoxyclav, cip-rofloxacin, chloramphenicol, erythromycin, gentamicin, lin-ezolid, vancomycin, and cefoxitin. We selected four strains withprofiles showing various degrees of resistance (resistance to one to

nine antibiotics tested) to the above-listed antibiotics for whole-genome sequencing, and the draft genome sequences are reportedhere. Genomic DNAs from the S. aureus strains were isolated us-ing the phenol-chloroformmethod, and whole-genome sequenc-ing was performed with the Ion Torrent Personal Genome Ma-chine (Life Technologies, CA) per the manufacturers guidelines.A total of 1,624,573 to 2,469,063 reads were obtained, with cover-ages of 59.3 to 83.97 (Table 1). De novo assembly was per-formed using the MIRA-4 assembler (7), which yielded 59, 58,125, and 74 contigs for Staphylococcus aureus strain MUF168, S.aureus strain MUF256, S. aureus strain MUF270, and S. aureusstrainMUF475, respectively. The GC contents for the four strainsranged from 32.66 to 32.78%. The functional annotation of theassembled genomes was performed by the Prokaryotic GenomeAnnotation Pipeline v2.0 (NCBI) for deposition with the Ge-nome database. A total of 2,433 to 2,728 coding sequences(CDS) were predicted for these four strains (Table 1). All fourof the genomes harbored genes coding for beta-lactamase,acyl carrier protein, chloramphenicol resistance, teicoplaninresistance-associated membrane protein TcaB, fibronectinbinding proteins A and B, polysaccharide intracellular adhesinbiosynthesis proteins, siderophore staphylobactin biosynthesisproteins, staphostatin A, staphylocoagulase, and staphyloki-nase.

Nucleotide sequence accession numbers. The draft genomesequences have been deposited at GenBank under the accessionnumbers AZQR00000000, AZSE00000000, AZSF00000000, andAZSG00000000.

TABLE 1 Staphylococcus aureus genome assembly and annotation statistics

Strain No. of contigs Fold coverage N50 (bp) Consensus length (bp) GC content (%) No. of CDS Accession no.

MUF168 59 67.67 96,843 2,752,244 32.78 2,433 AZQR00000000MUF256 58 69.54 87,410 2,783,966 32.66 2,433 AZSE00000000MUM270 125 83.97 52,369 2,834,436 32.76 2,728 AZSF00000000MUM475 74 59.3 128,108 2,852,107 32.67 2,620 AZSG00000000

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ACKNOWLEDGMENTS

The study was supported by the TIFAC-CORE in Pharmacogenomics,Indo-Australia Biotechnology Fund, Department of Biotechnology, Gov-ernment of India, and Manipal University, Manipal, India.

We thank Ganesh Kumar and Divya for help rendered in sample pro-cessing.

REFERENCES1. Dowd SE, Sun Y, Secor PR, Rhoads DD, Wolcott BM, James GA,

Wolcott RD. 2008. Survey of bacterial diversity in chronic wounds usingpyrosequencing, DGGE, and full ribosome shotgun sequencing. BMCMi-crobiol. 8:43. http://dx.doi.org/10.1186/1471-2180-8-43.

2. Dowd SE, Wolcott RD, Sun Y, McKeehan T, Smith E, Rhoads D. 2008.Polymicrobial nature of chronic diabetic foot ulcer biofilm infections deter-mined using bacterial tag encoded FLX amplicon pyrosequencing (bTEFAP).PLoS One 3:e3326. http://dx.doi.org/10.1371/journal.pone.0003326.

3. David MZ, Daum RS. 2010. Community-associated methicillin-resistantStaphylococcus aureus: epidemiology and clinical consequences of anemerging epidemic. Clin. Microbiol. Rev. 23:616687. http://dx.doi.org/10.1128/CMR.00081-09.

4. Song JH, Hsueh PR, Chung DR, Ko KS, Kang CI, Peck KR, Yeom JS,

Kim SW, Chang HH, Kim YS, Jung SI, Son JS, So TM, Lalitha MK,Yang Y, Huang SG, Wang H, Lu Q, Carlos CC, Perera JA, Chiu CH,Liu JW, Chongthaleong A, Thamlikitkul V, Van PH; Study ANSORPGroup. 2011. Spread of methicillin-resistant Staphylococcus aureus be-tween the community and the hospitals in Asian countries: an ANSORPstudy. J. Antimicrob. Chemother. 66:10611069. http://dx.doi.org/10.1093/jac/dkr024.

5. Lffler B, Hussain M, Grundmeier M, Brck M, Holzinger D, Varga G,Roth J, Kahl BC, Proctor RA, Peters G. 2010. Staphylococcus aureusPanton-Valentine leukocidin is a very potent cytotoxic factor for humanneutrophils. PLoS Pathog. 6:e1000715. http://dx.doi.org/10.1371/journal.ppat.1000715.

6. Hair PS, Echague CG, Sholl AM, Watkins JA, Geoghegan JA, Foster TJ,Cunnion KM. 2010. Clumping factor A interaction with complement fac-tor I increases C3b cleavage on the bacterial surface of Staphylococcus aureusand decreases complement-mediated phagocytosis. Infect. Immun. 78:17171727. http://dx.doi.org/10.1128/IAI.01065-09.

7. Chevreux B, Wetter T, Suhai S. 1999. Genome sequence assemblyusing trace signals and additional sequence information, p 4556. Com-puter science and biology: proceedings of the German Conference onBioinformatics. Research Centre for Biotechnology (GBF), Braun-schweig, Germany.

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Genome Sequences of Four Clinical Staphylococcus aureus Strains with Diverse Drug Resistance Profiles Isolated from Diabetic Foot UlcersNucleotide sequence accession numbers. ACKNOWLEDGMENTSREFERENCES