Failure VSSA .256 .256 43 VSSA VSSA 52 VSSA VSSA 43 0.5 .256 .256 ` 42 VSSA .256 .256 0.6 III t037 ST239 F VSSA 0.5 2 0.5 2 0.4 III t037 ST239 F 0.6 III t030 ST239 A Severe peumonia 17 10781694 VAN, MEM, FEP,Infection cleared LEV, AMK 0.5 2 2 0.6 III t030 ST239 A 0.5 2 256 0.7 III t030 ST239 B Severe peumonia 11 VAN, SCF Infection cleared 0.5 1 256 0.5 III t030 ST239 B Severe peumonia 0.5 2 256 0.5 III t030 ST239 C 18 VAN, SCF Infection cleared 0.5 2 256 0.5 III t030 ST239 C Coronary heart disease, 16 peumonia 0.7 III t030 ST239 A TEC, IPM, CAZ, Infection cleared MFX VSSA 0.5 2 0.25 2 0.5 III t030 ST239 AVSSA-pair11 (104d)V-F-sputumV-R-sputumVSSA-pair12 (18d)V-F-sputumV-R-sputumVSSA-pair13 (143d)V-F-sputumV-R-sputumVSSA-pair14 (15d)V-F-abdominal fluid VSSAV-R-abdominal fluid VSSAVSSA-pair15 (20d)V-F-sputumV-R-sputumabinterval (day) of the two isolates. Defined by population analysis profile (PAP) rea under the curve (AUC) method (PAP-AUC) (see Materials and Methods). NA, did not carry SCCmec. d The duration of Vn exposure represents the total length of time that the patient received vancomycin.The VSSA isolate of each pair obtained prior to beginning vancomycin / teicoplanin therapy. e VAN, vancomycin; TEC, teicoplanin; OXA, oxacillin; LEV, levofloxacin; MFX, moxifloxacin; CIP, ciprofloxacin; IPM, imipenem; MEM, meropenem; AMK, amikacin; FEP, cefepime; CRO, ceftriaxone; CAZ, ceftazidime; CXM, cefuroxime; SCF, cefoperazone-sulbactam; TZP, piperacillin-tazobactam; RIF, rifampin; SXT, sulfamethoxazole-trimethoprim; LZD, linezolid. doi:10.1371/journal.pone.0066880.tcThe Comparative Proteomics of hVISAThe Comparative Proteomics of hVISATable 2. List of primers for real-time quantitative reverse transcriptase ?PCR.Primer Name 16S rRNA-F 16S rRNA-R isaA-F isaA-R msrA2-F msrA2-R asp23-F asp23-R gpmA-F gpmA-R ahpC-F ahpC-RSequence GGCAAGCGTTATCCGGAATT GTTTCCAATGACCCTCCACG TGGCTCAACGTACTGGTGTT GACCCCAACCTGGCATAGTT King the top 100 proteins identified in the first step of analysis TGCAACATTAGCAGGAGGATG GCATGACCGCCACTATAACC CTTTACGGAAGATTTTAGGTGCTGA CAAGGTGTATCTGTTGAAGTTGGTG TCAGACTTTCGGAATAAGGCATC ACCTGCTGAAACCGAAGAACA CACGGCCAATTCCGTCA TGACCCATCACAAACAATCACTCLength (bp) 20 20 20 20 21 20 25 25 23 21 17NCBI Accession No. NC_NP_375681.NP_374474.NP_375295.NP_375527.NP_373615.doi:10.1371/journal.pone.0066880.tMass Spectrometry for 2D Gel Protein IdentificationGel plugs containing differentially expressed proteins were excised using a ProXcision robot (Perkin Elmer Inc., Wellesley, MA, US) and subjected to matrix-assisted laser desorption ionization-time of flight/time of flight (MALDI-TOF/TOF) analysis (Bruker Daltonics, Leipzig, GER). Gel plugs were placed in 96-well plates, and then washed with 25 mM NH4HCO3 (pH 8.0). Gel plugs were pre-frozen at ?0uC for 1 h, and then digested with trypsin (Promega, WI, USA). After extraction from the gel into 50 acetonitrile/0.1 formic acid, peptides were lyophilized in a speed vacuum and resuspended in 10 mL of 0.1 formic acid solution. Peptide MS/MS spectra were obtained by MALDI-TOF/TOF (Bruker Daltonics). The resulting MS/MS spectra were interpreted using Mascot and searched against eubacterial proteins in the National Center for Biotechnology Information protein database. Results showing MASCOT score 75 and confidence level 95 were considered reliable [23].Real-Time Quantitative Reverse Transcriptase CR (qRT?PCR)Total RNA was extracted from each sample using the RNeasy Kit (Qiagen, CA, USA). Complementary DNA (cDNA) was generated from total RNA using a random primer Finafloxacin hexamer.Failure VSSA .256 .256 43 VSSA VSSA 52 VSSA VSSA 43 0.5 .256 .256 ` 42 VSSA .256 .256 0.6 III t037 ST239 F VSSA 0.5 2 0.5 2 0.4 III t037 ST239 F 0.6 III t030 ST239 A Severe peumonia 17 10781694 VAN, MEM, FEP,Infection cleared LEV, AMK 0.5 2 2 0.6 III t030 ST239 A 0.5 2 256 0.7 III t030 ST239 B Severe peumonia 11 VAN, SCF Infection cleared 0.5 1 256 0.5 III t030 ST239 B Severe peumonia 0.5 2 256 0.5 III t030 ST239 C 18 VAN, SCF Infection cleared 0.5 2 256 0.5 III t030 ST239 C Coronary heart disease, 16 peumonia 0.7 III t030 ST239 A TEC, IPM, CAZ, Infection cleared MFX VSSA 0.5 2 0.25 2 0.5 III t030 ST239 AVSSA-pair11 (104d)V-F-sputumV-R-sputumVSSA-pair12 (18d)V-F-sputumV-R-sputumVSSA-pair13 (143d)V-F-sputumV-R-sputumVSSA-pair14 (15d)V-F-abdominal fluid VSSAV-R-abdominal fluid VSSAVSSA-pair15 (20d)V-F-sputumV-R-sputumabinterval (day) of the two isolates. Defined by population analysis profile (PAP) rea under the curve (AUC) method (PAP-AUC) (see Materials and Methods). NA, did not carry SCCmec. d The duration of Vn exposure represents the total length of time that the patient received vancomycin.The VSSA isolate of each pair obtained prior to beginning vancomycin / teicoplanin therapy. e VAN, vancomycin; TEC, teicoplanin; OXA, oxacillin; LEV, levofloxacin; MFX, moxifloxacin; CIP, ciprofloxacin; IPM, imipenem; MEM, meropenem; AMK, amikacin; FEP, cefepime; CRO, ceftriaxone; CAZ, ceftazidime; CXM, cefuroxime; SCF, cefoperazone-sulbactam; TZP, piperacillin-tazobactam; RIF, rifampin; SXT, sulfamethoxazole-trimethoprim; LZD, linezolid. doi:10.1371/journal.pone.0066880.tcThe Comparative Proteomics of hVISAThe Comparative Proteomics of hVISATable 2. List of primers for real-time quantitative reverse transcriptase ?PCR.Primer Name 16S rRNA-F 16S rRNA-R isaA-F isaA-R msrA2-F msrA2-R asp23-F asp23-R gpmA-F gpmA-R ahpC-F ahpC-RSequence GGCAAGCGTTATCCGGAATT GTTTCCAATGACCCTCCACG TGGCTCAACGTACTGGTGTT GACCCCAACCTGGCATAGTT TGCAACATTAGCAGGAGGATG GCATGACCGCCACTATAACC CTTTACGGAAGATTTTAGGTGCTGA CAAGGTGTATCTGTTGAAGTTGGTG TCAGACTTTCGGAATAAGGCATC ACCTGCTGAAACCGAAGAACA CACGGCCAATTCCGTCA TGACCCATCACAAACAATCACTCLength (bp) 20 20 20 20 21 20 25 25 23 21 17NCBI Accession No. NC_NP_375681.NP_374474.NP_375295.NP_375527.NP_373615.doi:10.1371/journal.pone.0066880.tMass Spectrometry for 2D Gel Protein IdentificationGel plugs containing differentially expressed proteins were excised using a ProXcision robot (Perkin Elmer Inc., Wellesley, MA, US) and subjected to matrix-assisted laser desorption ionization-time of flight/time of flight (MALDI-TOF/TOF) analysis (Bruker Daltonics, Leipzig, GER). Gel plugs were placed in 96-well plates, and then washed with 25 mM NH4HCO3 (pH 8.0). Gel plugs were pre-frozen at ?0uC for 1 h, and then digested with trypsin (Promega, WI, USA). After extraction from the gel into 50 acetonitrile/0.1 formic acid, peptides were lyophilized in a speed vacuum and resuspended in 10 mL of 0.1 formic acid solution. Peptide MS/MS spectra were obtained by MALDI-TOF/TOF (Bruker Daltonics). The resulting MS/MS spectra were interpreted using Mascot and searched against eubacterial proteins in the National Center for Biotechnology Information protein database. Results showing MASCOT score 75 and confidence level 95 were considered reliable [23].Real-Time Quantitative Reverse Transcriptase CR (qRT?PCR)Total RNA was extracted from each sample using the RNeasy Kit (Qiagen, CA, USA). Complementary DNA (cDNA) was generated from total RNA using a random primer hexamer.
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