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Journal of Global Antimicrobial Resistance 8 (2017) 169–171

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Journal of Global Antimicrobial Resistance journal homepage: www.elsevier.com/locate/jgar

Genome Note

Draft genome sequence of reduced teicoplanin-susceptible and vancomycin-heteroresistant methicillin-resistant Staphylococcus aureus from sepsis cases Yamuna Devi Bakthavatchalama , Balaji Veeraraghavana,* , Naveen Kumar Devanga Ragupathia , Priyanka Babua , Elakkiya Munuswamya , Thambu Davidb a b

Department of Clinical Microbiology, Christian Medical College, Vellore 632004, Tamil Nadu, India Department of Medicine (Unit II), Christian Medical College, Vellore 632004, India

A R T I C L E I N F O

Article history: Received 7 October 2016 Received in revised form 7 December 2016 Accepted 8 December 2016 Available online xxx Keywords: MRSA hVISA tcaA

A B S T R A C T

Here we report the whole-genome shotgun sequence of six methicillin-resistant Staphylococcus aureus (MRSA) showing reduced susceptibility to both vancomycin and teicoplanin. The typical Indian community-acquired MRSA (CA-MRSA) clone ST772-MRSA-V-t657 was the most common genotype (3/ 6; 50%), followed by ST672-MRSA-IV (2/6; 33%) and ST22-MRSA-IV (1/6; 17%). All strains harboured a mutation in the tcaRAB operon, vraSR, graSR and/or rpoB genes, which are frequently mutated determinants in a heteroresistant vancomycin-intermediate S. aureus (hVISA) phenotype. © 2017 Published by Elsevier Ltd on behalf of International Society for Chemotherapy of Infection and Cancer.

Methicillin-resistant Staphylococcus aureus (MRSA) causing nosocomial and community-acquired infections is a significant public health threat. Use of vancomycin in the treatment of MRSA infection is complicated by the emergence of vancomycinheteroresistant, vancomycin-intermediate and vancomycin-resistant S. aureus [1]. Here we present the draft genome sequences of six low-level teicoplanin-resistant MRSA that were heteroresistant to vancomycin isolates (VB9V352, VB12268, VB26276, VB23686, VBV169 and VB31683). All six isolates were recovered during 2015 from different patient with MRSA sepsis admitted to Christian Medical College, a 2600-bed tertiary level hospital in Vellore, South India. Except for isolate VB31683, which was a hospital-acquired MRSA, all isolates were found to be communityacquired MRSA. The length of vancomycin/teicoplanin usage in patients ranged from 3 days to 13 days (mean duration 8 days). Isolates were recovered during antimicrobial therapy from patients showing poor response to vancomycin/teicoplanin. Heteroresistant vancomycin-intermediate S. aureus (hVISA) was detected by population analysis profile—area under the curve

* Corresponding author. E-mail address: [email protected] (B. Veeraraghavan).

(PAP-AUC) method. The PAP-AUC profile ratios were between 0.92 and 1.04 with regard to the hVISA (Mu3) isolate. DNA was isolated from pure cultures using a QIAamp1 DNA Mini Kit (QIAGEN, Hilden, Germany). Whole-genome shotgun sequencing was performed using an Ion Torrent PGMTM System (Life Technologies, Waltham, MA) with 400-bp chemistry. The raw data generated were assembled de novo using SPAdes Genome Assembler v.5.0.0.0 (http://cab.spbu.ru/software/spades/) embedded in Torrent suite server v.5.0.4. The genome sequence was annotated using PATRIC, the bacterial bioinformatics database and analysis resource (http://www.patricbrc.org) [2] and the NCBI Prokaryotic Genome Automatic Annotation Pipeline (PGAAP) (http://www.ncbi.nlm.nih.gov/genomes/static/Pipeline.html). 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 analysed using ResFinder 2.1 from the CGE server (https://cge.cbs.dtu.dk//services/ResFinder/). Sequence types (STs) were determined for all of the isolates in the allele order of arcC, aroE, glpF, gmk, pta, tpi and yqiL by comparing the sequences with the S. aureus multilocus sequence typing (MLST) database (http://saureus.mlst.net/). The annotated genome size of the MRSA isolates ranged from ca. 2.7 Mbp to ca. 2.8 Mbp with coverage of 20–58 (Table 1). The number of coding DNA sequences (CDS) per genome ranged

http://dx.doi.org/10.1016/j.jgar.2016.12.008 2213-7165/© 2017 Published by Elsevier Ltd on behalf of International Society for Chemotherapy of Infection and Cancer.

170

Table 1 Genomic characterisation of reduced teicoplanin-susceptible and vancomycin-heteroresistant methicillin-resistant Staphylococcus aureus (MRSA) isolates recovered from bloodstream infections. Vancomycin/ teicoplanin MIC (mg/mL); PAP-AUC ratio a

Accession no.

Draft genome size (Mbp)

No. of CDS

No. of contigs

Coverage ()

Virulence genes

Resistance genes

Amino acid substitutions in tcaRAB/vraSR/graSR TCSs

ST/SCCmec/spa type

VBV9352

3/16; 0.98

LXWR00000000

2.82

2947

136

19.75

aur,scn, sem, sei, sea/sep, lukS-PV, lukF-PV, seu, hlgA, hlgC, hlgB, sen, hlb, seo, sec3, sel

aph(30 )-III, aac(60 )-aph(20 0 )-I, ant(6)-Ia, blaZ, mecA, norA, msr(A), mph(C), dfrG

tcaA: L218P, E230D, G312D tcaB; H6Y vraS: insertion of A at 686 leading to premature stop codon (non-functional protein) vraR: E59D, M81I graS: no mutation graR: insertion of T at 226 position leading to premature stop codon (non-functional protein) msrR: no mutation

ST772-SCCmec V-t657

VB12268

3/6; 1.02

LXWS00000000

2.88

3011

196

19.84

aur, ACME, sak, scn, lukE, lukD, sem, sei, seo, seu, hlb, sen, seg, hlgB, hlgC, hlgA

aph(30 )-III, ant(6)-Ia, blaZ, mecA, norA, msr(A), mph(C)

tcaA: L218P, E230D, G312D tcaB: I232L, K396R vraS: deletion of A at 243 leading to premature stop codon (non-functional protein) vraR: no mutation graS: T224I graR: no mutation msrR: no mutation

ST672-SCCmec IVa

VB26276

2/4; 0.98

LWMF00000000

2.82

2744

100

57.72

aur, scn, lukF-PV, lukS-PV, sea/sep, seg, sen, seu, sem, seo, hlgA, hlgC, hlgB, hlb

aph(30 )-III, aac(60 )-aph(20 0 ), ant(6)-Ia, blaZ, mecA, norA, msr(A), mph(C), dfrG

tcaA: L218P, E230D, G312D tcaB: H6Y vraS: no mutation vraR: E59D, M81I graS: no mutation graR: no mutation msrR: no mutation

ST772-SCCmec V-t657 c

VB23686

3/16; 1.04

MANS00000000

2.76

2628

93

58.34

aur, scn, sei, sem, seo, lukF-PV, lukS-PV, hlgB, hlgA, hlb, hlgC, sec3, sel, sea/sep, seg, sen, seu

aph(30 )-III, aac(60 )-aph(20 0 ), ant(6)-Ia, blaZ, mecA, norA, msr(A), mph(C), dfrG

tcaA: L218P, E230D, G312D tcaB: H6Y vraS: no mutation vraR: E59D, M81I graS: no mutation graR: no mutation msrR: V197I

ST772-SCCmec V-t657 c

VBV169

1.5/4; 0.97

LWMG00000000

2.85

2783

92

50.67

aur, sak, scn, lukE, lukD, sem, sei, seo, seu, hlb, sen, seg, hlgB, hlgC, hlgA

aph(30 )-III, aac(60 )-aph(20 0 )-I, ant(6)-Ia, blaZ, mecA, norA

tcaA: L218P, Y237H tcaB: I232L, K336R vraS: no mutation vraR: no mutation graS: T224I graR: no mutation msrR: no mutation

ST672-SCCmec IVd

b

b,d

Y.D. Bakthavatchalam et al. / Journal of Global Antimicrobial Resistance 8 (2017) 169–171

Isolate ID

MIC, minimum inhibitory concentration; PAP-AUC, population analysis profile–area under the curve; CDS, coding DNA sequences; TCS, two-component system; ST, sequence type; SCCmec, staphylococcal cassette chromosome mec; spa, staphylococcal protein A. a PAP-AUC profile ratios with regard to the hVISA (Mu3) isolate. b spa repeats were not matched and spa types were unassigned. c An additional SCC composite element ccrB3 and SCCHg (mercury) with integrated transposon Tn 554 in J1 region were arranged in tandem with no characteristic direct repeat (DR) sequences at the junction regions. d An additional ccrC locus was identified as SCC composite element at J3 region.

VB31683

3/32; 0.92

MANT00000000

2.77

2714

168

31.22

aur, scn, sak, sei, sem, seo, lukF-PV, lukS-PV, hlgB, hlgA, hlb, hlgC, sec3, sel, sea/sep, seg, sen, seu

aac(60 )-aph(20 0 ),blaZ, mecA, norA, erm(C)

tcaA: L218P, Y237H, G312D tcaB: T5 K, H6R, T75N, V105I, G288 V, V360I vraS: no mutation vraR: no mutation graS: S104L, E108D, E112D, K146E, Y156F, D218N, Y219H graR: D148Q msrR: no mutation

ST22-SCCmec IVc

b

Y.D. Bakthavatchalam et al. / Journal of Global Antimicrobial Resistance 8 (2017) 169–171

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between 2628 and 3011. All of the isolates were found to harbour various toxin and antimicrobial resistance genes. Interestingly, in this study reduced susceptibility to vancomycin and teicoplanin was observed in typical community-acquired MRSA (CA-MRSA) clone ST772-MRSA-V-t657 positive for Panton–Valentine (PVL) toxin. Remarkably, ST672-MRSA-IV was the clone of CA-MRSA found to be negative for PVL toxin. ST22-MRSA-IV (CA-MRSA clone) was identified as hospital-acquired MRSA from an immunocompromised leukemia patient, signifying CA-MRSA infiltration into the hospital. However, staphylococcal protein A (spa) typing failed to match the repeated region (spa gene) in ST672-MRSA-IV and ST22-MRSA-IV and thus spa types were unassigned. Potential rearrangement of the spa gene limits the application of spa typing in monitoring the evolution MRSA clones. Following prolonged exposure to vancomycin, hVISA can emerge in vivo or in vitro and these mutants are often found to harbour chromosomal mutations that lead to altered cell wall biosynthesis [3]. The most frequently mutated genes are twocomponent system (TCS) determinants vraSR, graSR and walKR as well as the rpoB gene [4]. All six isolates were found with mutations in at least one of these TCSs. Strains VBV9352 and VB12268 harboured an rpoB (H481Y) mutation, which is associated with rifampicin resistance [minimum inhibitory concentration (MIC) > 32 mg/mL]. Mutations in the teicoplanin resistance operon (tcaRAB) associated with teicoplanin resistance were seen in the annotated genome of MRSA isolates as described previously [5]. Comparative genomic sequence analysis of these sequences and other hVISA strains will help to understand the polymorphism associated with decreased vancomycin/teicoplanin susceptibility, which is hard to detect in the clinical laboratory. Accession number(s) The draft genome sequences have been deposited in DDBJ/ENA/ GenBank under the accession numbers provided in Table 1. Funding None. Competing interests None declared. Ethical approval Not required. References [1] Sievert DM, Rudrik JT, Patel JB, McDonald LC, Wilkins MJ, Hageman JC. Vancomycin-resistant Staphylococcus aureus in the United States, 2002–2006. Clin Infect Dis 2008;46:668–74. [2] Wattam AR, Abraham D, Dalay O, Disz TL, Driscoll T, Gabbard JL, et al. PATRIC, the bacterial bioinformatics database and analysis resource. Nucleic Acids Res 2014;42(Database issue):D581–91. [3] Howden BP, Davies JK, Johnson PD, Stinear TP, Grayson ML. Reduced vancomycin susceptibility in Staphylococcus aureus, including vancomycin-intermediate and heterogeneous vancomycin-intermediate strains: resistance mechanisms, laboratory detection, and clinical implications. Clin Microbiol Rev 2010;23:99–139. [4] Kato Y, Suzuki T, Ida T, Maebashi K. Genetic changes associated with glycopeptide resistance in Staphylococcus aureus: predominance of amino acid substitutions in YvqF/VraSR. J Antimicrob Chemother 2010;65:37–45. [5] Maki H, McCallum N, Bischoff M, Wada A, Berger-Bächi B. tcaA inactivation increases glycopeptide resistance in Staphylococcus aureus. Antimicrob Agents Chemother 2004;48:1953–9.