Abstract
We report here the draft genome sequences of 11 heteroresistant vancomycin-intermediate Staphylococcus aureus (hVISA) strains from bloodstream infection. All strains harbor mutations in vraSR, graSR, walKR, and/or tcaRAB and are often implicated as the frequently mutated candidate genes in hVISA phenotypes.
GENOME ANNOUNCEMENT
Methicillin-resistant Staphylococcus aureus (MRSA) can cause serious community- and nosocomial-acquired infection. The use of vancomycin in the treatment of MRSA infection is challenged by the emergence of heteroresistant vancomycin-intermediate S. aureus (hVISA) and vancomycin-intermediate S. aureus (VISA). Several studies have reported the increasing frequency of hVISA/VISA-associated treatment failures in invasive infections (1, 2).
Here, we present the draft genome sequences of 11 hVISA strains isolated from bloodstream infections. All the isolates were found to have vancomycin MICs of 1 to 1.5 µg/ml. All these hVISA strains were confirmed with a population analysis profile-area under curve (PAP-AUC) method with the following PAP-AUC ratios: VB988, 1.03; VB9939, 0.92; VB16578, 0.96; VB20017, 1.0; VB44094, 0.97; VB44746, 1.33; VB46389, 1.03; VB35316, 0.96; BA43011, 1.28; Staphylococcus aureus strain 2016, 1.02; and VB1490, 1.0.
DNA isolation from pure cultures was performed using QIAamp DNA minikit (Qiagen, Germany). Whole-genome shotgun sequencing was performed using the Ion Torrent PGM system (Life Technologies, Inc.) with 400-bp chemistry. The raw data generated were assembled de novo using the assembler SPAdes version 5.0.0.0 embedded in Torrent suite server version 5.0.4. The genome sequence was annotated using PATRIC, the bacterial bioinformatics database and analysis resource (http://www.patricbrc.org) (3), and the NCBI Prokaryotic Genome Annotation Pipeline (PGAP) (http://www.ncbi.nlm.nih.gov/genome/annotation_prok/) (4). Downstream analysis was performed using the Center for Genomic Epidemiology (CGE) server (http://www.cbs.dtu.dk/services) and PATRIC. The resistance gene profile was analyzed using ResFinder 2.1 from the CGE server (https://cge.cbs.dtu.dk//services/ResFinder/) (5). The CRISPR finder (http://crispr.u-psud.fr/Server/) was used to detect and identify clustered regularly interspaced short palindromic repeat (CRISPR) and spacer sequences in the genome. The sequence type was determined for all the isolates in the allele order of arcc, aroe, glpf, gmk, pta, tpi, and yqil by comparing the sequences with S. aureus database maintained at the MLST website (http://saureus.mlst.net/).
The annotated genome size of MRSA isolates ranged from ~2.7 to ~2.9 Mb, with coverages of 33× to 88× (Table 1). The number of coding DNA sequences (CDSs) per genome ranged from 2,603 to 3,011. Genome annotation by PATRIC predicted a total of 41 to 60 tRNAs and five to 10 rRNAs in the sequenced isolates (Table 1). All the isolates were found to harbor various toxin and antimicrobial resistance genes. Further, none of the isolates were found to have CRISPR regions.
TABLE 1 .
Genome characteristics of hVISA isolates from bloodstream infection
| Isolate ID | Accession no. | Draft genome size (Mbp) | No. of CDSs | No. of contigs | No. of tRNAs | No. of rRNAs | Coverage (×) | Virulence genesa | Resistance genes | ST/SCCmec/spa typeb |
|---|---|---|---|---|---|---|---|---|---|---|
| VB9882 | MLQI00000000 | 2.77 | 2,692 | 132 | 60 | 10 | 79 | aur, scn, sak, seq, sen,seu, sei,sem, seo, seg, hlb, hlgB, hlgC, hlgA, lukS-PV, lukF-PV | aadD, acc(6′)-aph(2′′), mecA, blaZ, norA | 2371/I/t6827 |
| VB9939 | MLQK00000000 | 2.83 | 2,803 | 437 | 48 | 7 | 33 | aur, scn, seq, sem, seg, seo, sec3, sel, hlb, hlgB, hlgC, hlgA, lukS-PV, lukF-PV, sen,seu, sea/sep | ant(6)-la, aph (3′)-III, acc(6′)-aph(2′′), spc, mecA, blaZ, norA, mph(C), msr(A), dfrG | 772/V/t657 |
| VB16578 | MLQD00000000 | 2.79 | 2,667 | 143 | 59 | 9 | 61 | aur, scn, seq, sen,seu, sei,sem, seo, seg,sec3, sel, hlb, hlgB, hlgC, hlgA, lukS-PV, lukF-PV | ant(6)-la, aph(3′)-III, acc(6′)-aph(2′′), spc, mecA, blaZ, norA, mph(C), msr(A), dfrG | 772/V |
| VB20017 | MLQE00000000 | 2.85 | 2,916 | 420 | 41 | 5 | 34 | aur, scn, sak, seq, sen,seu, sei,sem, seo, seg, hlb, hlgB, hlgC, hlgA, lukS-PV, lukF-PV | aadD, acc(6′)-aph(2′′), mecA, blaZ, norA, ermC | 2371/V/ t6827 |
| VB44094 | MLQH00000000 | 2.77 | 2,683 | 110 | 57 | 10 | 79 | aur, scn, sak, seq, sen,seu, sei,sem, seo, hlb, hlgB, hlgC, hlgA, lukS-PV, lukF-PV | acc(6′)-aph(2′′), mecA, blaZ, norA, ermC | 22/IVc/t474 |
| VB44746 | MLQF00000000 | 2.84 | 2,818 | 265 | 50 | 8 | 41 | splA, splE, aur, scn, sak, lukD, lukE, hlb, hlgB, hlgC, hlgA,eta | blaZ, norA, ermC | 1290/IVh/t131 |
| VB46389 | MLQG00000000 | 2.80 | 2,700 | 235 | 50 | 7 | 42 | aur, scn, sea/sep, seg, seq, sen,seu, sei,sem, seo, seg,sec3, sel, hlb, hlgB, hlgC, hlgA, lukS-PV, lukF-PV | ant(6)-la, aph(3′)-III, acc(6′)-aph(2′′), mecA, blaZ, mph(C), msrA, norA, dfrG | 772/V/t458 |
| VB35316 | MLQC00000000 | 2.77 | 2,638 | 88 | 59 | 9 | 85 | splB, splA, splE, aur, scn, sak, lukD, lukE, seg, sen,seu, sei,sem, seo, hlb, hlgB, hlgC, hlgA | acc(6′)-aph(2′′), mecA, blaZ, norA, dfrG | 72/III/V/t2473 |
| VB43011 | MLQJ00000000 | 2.77 | 2,604 | 67 | 59 | 8 | 76 | splB, splA, splE, aur, scn, sak, sea/sep, seb, seq, sek,seh, lukD, lukE, hlb, hlgB, hlgC, hlgA | ant(6)-la, aph(3′)-III, blaZ, mphC, msrA, norA, | 1/V/t127 |
| Staphylococcus aureus strain 2016 | MLQA00000000 | 2.72 | 2,603 | 85 | 60 | 9 | 88 | splA, splE, aur, scn, sak, lukD, lukE, hlb, hlgB, hlgC, hlgA | blaZ, norA | 580/II/t4615 |
| VB1490 | MLQB00000000 | 2.97 | 3,011 | 143 | 58 | 8 | 64 | splB, splA, splE, aur, scn, sak, seq, sek, lukD, lukE, hlb, hlgB, hlgC, hlgA | ant(6)-la, aph(3′)-III, acc(6′)-aph(2′′), spc, mecA, blaZ, norA, ermA, tet(M) | 239/III/V/t037 |
PV, Panton-Valentine.
ST, sequence type; SCCmec, staphylococcal cassette chromosome mec element.
Infection with hVISA/VISA has been associated with high vancomycin MIC and poor clinical outcome. The most frequently mutated two-component system (TCS) determinants are vraSR, graSR, and walKR, and the rpoB gene (6). This chromosomal mutation leads to the upregulation of peptidoglycan biosynthesis and cell wall thickening and further prevents vancomycin from reaching its target (7). Multiple nonsynonymous mutations were seen in the vraSR, graSR, and walKR TCSs of all sequenced isolates. In addition, a mutation was seen in a teicoplanin resistance-associated (tcaRAB) operon. A mutation in the rpoB gene was not observed in any of the sequenced isolates.
Taken together, comparative genomic analysis of these sequenced isolates revealed preferential clustering of single nucleotide polymorphisms (SNPs) in hVISA candidate genes with high diversity across the loci of vraSR, graSR, walKR, and tcaRAB.
Accession number(s).
The draft genome sequences have been deposited in DDBJ/ENA/GenBank under the accession numbers as provided in Table 1.
ACKNOWLEDGMENT
We thank the institutional review board of Christian Medical College, Vellore, India, for approving the study.
Funding Statement
This research received no specific grant from any funding agency in the public, commercial, or not-for-profit sectors.
Footnotes
Citation Bakthavatchalam YD, Veeraraghavan B, Peter JV, Rajinikanth J, Inbanathan FY, Devanga Ragupathi NK, Rajamani Sekar SK. 2016. Novel observations in 11 heteroresistant vancomycin-intermediate methicillin-resistant Staphylococcus aureus strains from South India. Genome Announc 4(6):e01425-16. doi:10.1128/genomeA.01425-16.
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