PROKARYOTES

crossm Draft Genome Sequences of Two Staphylococcus aureus Strains Isolated in Succession from a Case of Bacteremia Division of Microbiology, National Center for Toxicological Research, U.S. Food and Drug Administration, Jefferson, Arkansas, USAa; Division of Molecular and Genetic Toxicology, NCTR, U.S. Food and Drug Administration, Jefferson, Arkansas, USAb; Department of Internal Medicine, University of Arkansas for Medical Sciences, Little Rock, Arkansas, USAc; Department of Microbiology and Immunology, University of Arkansas for Medical Sciences, Little Rock, Arkansas, USAd

ABSTRACT Staphylococcus aureus strains MEH1 and MEH7 were successively isolated from the blood of a patient with recurrent bacteremia. The submitted draft genomes of strains MEH1 and MEH7 are 2,914,972 and 2,911,704 bp, respectively.

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taphylococcus aureus is a leading cause of nosocomial bacteremia that often leads to life-threatening diseases, such as osteomyelitis and infective endocarditis (1–7). S. aureus strains MEH1 and MEH7 were isolated 7 days apart from the blood of a bacteremic patient who had mitral and aortic valve endocarditis, L4-5 spinal diskitis, and osteomyelitis. These strains were sequenced to study the factors contributing to the persistence of the infection during the course of antibiotic treatment. Bacterial cell pellets were suspended in Tris-EDTA buffer (560 ␮L) with lysostaphin (1 ␮g/mL) and RNase (10 ␮g/mL) and incubated for 30 min at 37°C. Cell suspensions were further incubated with 30 ␮L of 10% SDS and 10 ␮L of proteinase K (20 mg/mL) (Thermo Fisher Scientific, Waltham, MA, USA) for 30 min. Five molar NaCl (100 ␮L) was added, vortexed before the addition of 80 ␮L of cetyltrimethylammonium bromide (10% wt/vol)–NaCl solution (4.1% wt/vol) (Sigma-Aldrich, St. Louis, MO, USA), and incubated at 65°C (10 min). To this, 700 ␮L of chloroform/isoamyl alcohol (24:1) (Sigma-Aldrich) was added and centrifuged at 9,400 ⫻ g for 5 min. To the upper aqueous layer an equal volume of phenol-chloroform/isoamyl alcohol (25:24:1, pH 6.7) (Sigma-Aldrich) was added and centrifuged for 10 min. DNA from the upper layer was precipitated with isopropanol at a ratio of 0.6:1. The DNA pellet obtained was washed with 70% ethanol, air-dried, and suspended in water. Genomic DNA libraries were independently dual-indexed (8 ⫻ 2) with the Nextera DNA library prep kit (Illumina, San Diego, CA, USA) as recommended by the manufacturer. Libraries were pooled and purified with a GeneRead size-selection kit (Qiagen, Valencia, CA, USA); the pooled DNA libraries were quantified with a Qubit dsDNA high-sensitivity assay kit (Thermo, Fisher Scientific) and analyzed with a high-sensitivity DNA chip (Agilent, Santa Clara, CA, USA). Libraries were paired-end (76 ⫻ 2) sequenced in a single dual-indexed (8 ⫻ 2) paired-end (76 ⫻ 2) NextSeq500 using a Mid Output (150 cycle) version 2 kit (Illumina) as recommended by the manufacturer. The resulting sequences were assembled into contiguous (contigs) fragments by the SPAdes version 3.6.2 genome assembler for Galaxy Tool version 1.2 with default parameters. The MEH1 and MEH7 draft genomes were determined to be 2,914,972 and 2,911,704 bp in length, distributed as 66 and 75 contigs, and based on average de novo assembly coverages of 110⫻ and 62⫻, respectively. The G⫹C content was 32.7% for both strains. The genome of MEH1 contains a total of 3,123 genes that include 2,977 coding genes, 4 rRNAs (5S, 16S, and 23S), 56 tRNAs, and 82 pseudogenes. The genome of MEH7 contains a total of Volume 5 Issue 23 e00259-17

Received 2 March 2017 Accepted 28 April 2017 Published 8 June 2017 Citation Basco MDS, Revollo JR, McKinzie PB, Agnihothram S, Kothari A, Saccente M, Hart ME. 2017. Draft genome sequences of two Staphylococcus aureus strains isolated in succession from a case of bacteremia. Genome Announc 5:e00259-17. https://doi.org/10.1128/ genomeA.00259-17. Copyright © 2017 Basco et al. This is an openaccess article distributed under the terms of the Creative Commons Attribution 4.0 International license. Address correspondence to M. D. S. Basco, [email protected], or M. E. Hart, [email protected].

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M. D. S. Basco,a J. R. Revollo,b P. B. McKinzie,b S. Agnihothram,a A. Kothari,c M. Saccente,c M. E. Harta,d

Basco et al.

3,142 genes that include 2,987 coding genes, 3 rRNAs (5S, 16S, and 23S), 65 tRNAs, and 83 pseudogenes. Annotations were performed using the NCBI Prokaryotic Genome Annotation Pipeline (8). Accession number(s). The genome sequences of S. aureus strains MEH1 and MEH7 have been deposited at DDBJ/EMBL/GenBank with the accession numbers MUIR00000000 and MUIS00000000, respectively. The versions described here are MUIR00000000.1 and MUIS00000000.1 for strains MEH1 and MEH7, respectively.

REFERENCES 1. Lowy FD. 1998. Staphylococcus aureus infections. N Engl J Med 339: 520 –532. https://doi.org/10.1056/NEJM199808203390806. 2. Ing M, Baddour L, Bayer A. 1997. Bacteremia and infective endocarditis: pathogenesis, diagnosis, and complications, p 331–354. In The staphylococci in human disease. Churchill Livingstone, New York, NY. 3. Mylotte JM, McDermott C, Spooner JA. 1987. Prospective study of 114 consecutive episodes of Staphylococcus aureus bacteremia. Rev Infect Dis 9:891–907. https://doi.org/10.1093/clinids/9.5.891. 4. Sanabria TJ, Alpert JS, Goldberg R, Pape LA, Cheeseman SH. 1990. Increasing frequency of staphylococcal infective endocarditis: experience at a university hospital, 1981 through 1988. Arch Intern Med 150:1305–1309. https://doi.org/10.1001/archinte.1990.00390180113021. 5. Jernigan JA, Farr BM. 1993. Short-course therapy of catheter-related

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Staphylococcus aureus bacteremia: a meta-analysis. Ann Intern Med 119: 304 –311. https://doi.org/10.7326/0003-4819-119-4-199308150-00010. 6. Musher DM, Lamm N, Darouiche RO, Young EJ, Hamill RJ, Landon GC. 1994. The current spectrum of Staphylococcus aureus infection in a tertiary care hospital. Medicine 73:186 –208. https://doi.org/10.1097/00005792 -199407000-00002. 7. Libman H, Arbeit RD. 1984. Complications associated with Staphylococcus aureus bacteremia. Arch Intern Med 144:541–545. 8. Angiuoli SV, Gussman A, Klimke W, Cochrane G, Field D, Garrity G, Kodira CD, Kyrpides N, Madupu R, Markowitz V, Tatusova T, Thomson N, White O. 2008. Toward an online repository of standard operating procedures (SOPs) for (meta) genomic annotation. OMICS 12:137–141. https://doi .org/10.1089/omi.2008.0017.

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ACKNOWLEDGMENTS We thank Ohgew Kweon, Division of Microbiology, National Center for Toxicological Research (NCTR), for his technical support and Nicole Emery, Department of Microbiology, UAMS, for assistance in preparing the strains. This work was supported by the NCTR of the U.S. Food and Drug Administration (FDA). Support for M. D. S. Basco was provided by the Office of Women’s Health (OWH) administered by the Oak Ridge Institute for Science and Education and NCTR through an interagency agreement between OWH and the FDA. We are indebted to John Sutherland, Kidon Sung, Ohgew Kweon, and Allen Gies for careful evaluation of the manuscript. The views presented in this publication do not necessarily reflect those of the FDA.

Draft Genome Sequences of Two Staphylococcus aureus Strains Isolated in Succession from a Case of Bacteremia.

Staphylococcus aureus strains MEH1 and MEH7 were successively isolated from the blood of a patient with recurrent bacteremia. The submitted draft geno...
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