Novel single-nucleotide variations associated with vancomycin resistance in vancomycin-intermediate Staphylococcus aureus

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Open Access Full Text Article Novel single-nucleotide variations associated with vancomycin resistance in vancomycinintermediate Staphylococcus aureus This article was published in the following Dove Press journal: Infection and Drug Resistance Lee-Chung Lin 1 Shih-Cheng Chang 1,2 Mao-Cheng Ge 1 Tsui-Ping Liu 1 Jang-Jih Lu 1–3 1 Department of Laboratory Medicine, Chang Gung Memorial Hospital, Linkou, Taoyuan, Taiwan; 2Department of Medical Biotechnology and Laboratory Science, College of Medicine, Chang Gung University, Taoyuan, Taiwan; 3Department of Medicine, College of Medicine, Chang Gung University, Taoyuan, Taiwan Introduction Correspondence: Jang-Jih Lu Department of Laboratory Medicine, Chang Gung Memorial Hospital, Linkou, No. 5, Fu-Shin Street, Kweishan 333, Taoyuan, Taiwan Email Methicillin-resistant Staphylococcus aureus (MRSA) is a leading cause of nosocomial infection.1 Vancomycin is usually used to treat MRSA infections, but high-level vancomycin-resistant S. aureus (VRSA) has emerged mainly due to the acquisition of the vanA operon from Enterococci.2 There are also VRSA isolates with lower levels of vancomycin resistance, including vancomycin-intermediate S. aureus (VISA) and heterogeneous VISA (hVISA).3 The causes for the different levels of vancomycin resistance in MRSA are not completely clear.4,5 Identification of VISA and hVISA is conventionally done by determination of population analysis profile and area under the curve ratio (PAP/AUC). However, PAP/ AUC is very labor-intensive and not practical for clinical diagnosis. Transcriptome and proteome analyses of MRSA isolates have revealed a link between nucleotide sequence variations of several genes and vancomycin resistance.6,7 These genes include those of the two-component systems, such as vraRS, graRS or walRK,6,8,9 and those involved in cell wall synthesis, such as upps, fmtC and srtA.9–11 Singlenucleotide variations (SNVs) of genes related to antibiotic resistance have also been described.8,9,12,13 These SNVs may allow differentiation between vancomycin-sensitive S. aureus (VSSA) and VISA. 113 submit your manuscript | www.dovepress.com Infection and Drug Resistance 2018:11 113–123 Dovepress © 2018 Lin et al. This work is published and licensed by Dove Medical Press Limited. The full terms of this license are available at https://www.dovepress.com/terms. php and incorporate the Creative Commons Attribution – Non Commercial (unported, v3.0) License (http://creativecommons.org/licenses/by-nc/3.0/). By accessing the work you hereby accept the Terms. Non-commercial uses of the work are permitted without any further permission from Dove Medical Press Limited, provided the work is properly attributed. For permission for commercial use of this work, please see paragraphs 4.2 and 5 of our Terms (https://www.dovepress.com/terms.php). http://dx.doi.org/10.2147/IDR.S148335 Powered by TCPDF (www.tcpdf.org) Abstract: Prolonged vancomycin usage may cause methicillin-resistant Staphylococcus aureus to become vancomycin-intermediate S. aureus (VISA) and heterogeneous VISA (hVISA). Mechanisms of vancomycin resistance of VISA and hVISA are still unclear. In this study, analyses of nucleotide sequence variations in 30 vancomycin-sensitive S. aureus (VSSA), 41 hVISA and 16 VISA isolates revealed 29 single-nucleotide variations in 12 genes (fmtC, graR, graS, htrA, mecA, pbp2, pbp4, srtA, tcaA, upps, vicK and vraR) that are related to cell wall synthesis or the two-component system. Six of these 29 single-nucleotide variations were novel and resulted in the following amino acid changes: Q692E in FmtC; T278I, P306L and I311T in HtrA; and I63V and K101E in Upps. Since P306L and I311T in HtrA and I63V in Upps were present in the majority (76.7%–86.7%) of VSSA isolates, these three amino acid variations may not be associated with vancomycin resistance. The other three amino acid variations (T278I in HtrA, K101E in Upps and Q692E in FmtC) were present in the majority (87.5%–93.8%) of hVISA and VISA isolates, but only in a small number (22.9%–25.7%) of VSSA isolates, suggesting that they are associated with vancomycin resistance. Keywords: MRSA, VSSA, hVISA, VISA Dovepress Infection and Drug Resistance downloaded from https://www.dovepress.com/ by 219.100.37.240 on 05-Jun-2020 For personal use only. Lin et al Previous studies on hVISA and VISA strains have revealed 60 genes that may be associated with vancomycin resistance.4,9,10,14–23 We hypothesized that some nucleotide sequence variations in these genes correlate with the difference in vancomycin susceptibility of VSSA and VISA isolates. To test this hypothesis, we investigated the prevalence of SNVs of these 60 genes and found 29 SNVs (in 12 genes) that were more common in hVISA and VISA than in VSSA isolates. Among them, three novel amino acid sequence variations including Q692E in FmtC, T287I in HtrA and K101E in Upps proteins were found to be significantly associated with vancomycin resistance. Materials and methods Bacteria strains and growth conditions In total, 87 MRSA isolates were used in this study, including 72 from Chang Gung Memorial Hospital, 3 from National Taiwan University Hospital, 8 from Tri-Service General hospital (TSGH) and 2 each from Chi Mei Medical Center and China Medical University Hospital. These isolates were collected from 2009 to 2014. Detailed information of these isolates is presented in Table 1. All isolates were stored at −80°C and grown on tryptic soy agar plates at 37°C for 16 hours for the studies. Whole-genome sequencing and polymerase chain reaction (PCR) The whole genome of two VISA isolates (TSGH205 and TSGH243) was sequenced. Genomic DNA of each isolate was isolated and sonicated to generate fragments of 300–500 bp for construction of a DNA library, which was then subjected to Illumina next-generation sequencing. Raw sequence data generated were filtered and assembled for analysis using the CLC Genomics Workbench (QIAGEN, Venlo, the Netherlands). Genes selected for investigation of vancomycin resistance and PCR primers used to amplify these genes are shown in Table 2. PCRs were performed in a buffer containing 10 mM Tris-HCl (pH 8.0), 1.5 mM MgCl2 and 50 mM KCl under the following conditions: 5 minutes at 95°C, followed by 35 cycles of 30 seconds at 95°C for denaturation, 30 seconds at 55°C for primer annealing and 1 minute at 72°C for extension, and 5 minutes at 72°C for final extension. PCR products were verified by sequencing. Minimum inhibition concentration (MIC) E-tests E-test was performed to determine the vancomycin MIC of VSSA, hVISA and VISA isolates. Cells of an overnight culture of each isolate were suspended in 0.9% NaCl solution, adjusted to 0.5 McFarland u (...truncated)