1 SUBMITTED 3 JAN 23 1 REVISION REQ. 27 FEB 23; REVISION RECD. 5 MAR 23 2 ACCEPTED 14 MAR 23 3 ONLINE-FIRST: MARCH 2023 4 DOI: https://doi.org/10.18295/squmj.3.2023.018 5 6 Molecular and Clinical Features of Heterogeneous Vancomycin 7 Intermediate Staphylococcus aureus in Tertiary Care Hospitals of South 8 India 9 Sreejisha M,1 Shalini Shenoy M,1 Suchitra Shenoy M,1 Dhanashree B,1 10 Chakrapani M,2 *Gopalakrishna Bhat K1 11 12 Departments of 1Microbiology and 2Medicine, Kasturba Medical College, Mangalore, 13 Manipal Academy of Higher Education, Manipal, Karnataka, India 14 *Corresponding Author’s e-mail: gkbhat999@gmail.com 15 16 Abstract 17 Objectives: This study aimed to detect heterogeneous vancomycin-intermediate 18 Staphylococcus aureus (hVISA) among methicillin resistant S. aureus (MRSA) isolated from 19 healthcare-associated infections and identify staphylococcal cassette chromosome mec 20 (SCCmec) types. Methods: Isolation and identification of MRSA were done using standard 21 bacteriological methods. Antimicrobial susceptibility testing was done using Kirby-Bauer disc 22 diffusion and macrolide-lincosamide-streptogramin B (MLSB) phenotypes identified using D 23 test. The minimum inhibitory concentration (MIC) of vancomycin was determined using agar 24 dilution. hVISA were confirmed by modified population analysis profile-area under the curve 25 (PAP-AUC) test. SCCmec types and Panton-Valentine leukocidin (pvl) were detected using 26 multiplex PCR. Results: Out of 220 MRSA stains, 14 (6.4%) were hVISA. None of the 27 MRSA isolate was vancomycin intermediate or resistant. All hVISA were susceptible to 28 linezolid and teicoplanin. Macrolide-streptogramin B (MSB) phenotype was present in 42.9% 29 hVISA. 92.9% hVISA strains had vancomycin MIC in the range 1-2 µg/mL. Majority of 30 hVISA and vancomycin susceptible MRSA were isolated from skin and soft tissue infections. 31 SCCmec III and IV were present in 50% and 35.7% hVISA respectively. 14.3% hVISA 32 harboured SCCmec V. Conclusion: The rate of hVISA among MRSA was 6.4%. MRSA 33 mailto:gkbhat999@gmail.com 2 strains should be tested for hVISA before starting vancomycin treatment. None of the isolates 34 was vancomycin intermediate or resistant. All the hVISA strains were susceptible to linezolid 35 and teicoplanin. The majority of hVISA were isolated from skin and soft tissue infections. 36 The majority hVISA harboured SCCmec III and IV. 37 Keywords: MRSA; Hospital infection; Molecular typing; Vancomycin 38 39 Advances in Knowledge 40 • To the best of our knowledge, this is the first report of heterogeneous vancomycin 41 intermediate Staphylococcus aureus (hVISA) infections in tertiary care hospitals of 42 coastal Karnataka, South India. 43 • This study showed high frequency of staphylococcal cassette chromosome mec 44 (SCCmec) types III and IV among hVISA. 45 Application to Patient Care 46 • Methicillin resistant Staphylococcus aureus (MRSA) isolated from clinical specimens 47 should be tested for the presence of hVISA before starting vancomycin treatment. 48 • Susceptibility of all hVISA strains to linezolid and teicoplanin. 49 50 Introduction 51 Methicillin resistant Staphylococcus aureus (MRSA) continues to be an important pathogen 52 that can cause a variety of healthcare-associated and community-associated infections.1 53 Although, vancomycin was the drug of choice for severe MRSA infections after its 54 introduction, the emergence of organisms with reduced susceptibility and complete resistance 55 has been a challenge in the treatment of such cases.2 MRSA with reduced susceptibility to 56 vancomycin includes heterogeneous vancomycin intermediate S. aureus (hVISA) and 57 vancomycin intermediate S. aureus (VISA), both first reported in Japan in 1997.3 The Clinical 58 and Laboratory Standards Institute (CLSI) defines VISA as S. aureus with vancomycin 59 minimum inhibitory concentration (MIC) 4-8 µg/mL.4 hVISA shows MIC of vancomycin in 60 the susceptible range (≤ 2 µg/mL) but, contains a subpopulation at the rate 10-5 to 10-6 with 61 vancomycin MIC in the intermediate range (4-8 µg/mL).5 The prevalence of hVISA and 62 VISA has increased worldwide from 4.68% and 2.05% in 2006 to 7.01% and 7.93% in 2014.6 63 A recent study from South India showed the prevalence of hVISA at 12.4%.7 64 65 3 Mutations of genes associated with the cell wall, thickened cell wall, slow growth, and 66 reduced autolysis are believed to be responsible for reduced susceptibility of hVISA/VISA 67 phenotypes to vancomycin.8 Mutations in the walKR (sensor protein kinase/regulator), graSR 68 (glycopeptide resistance–associated sensor/regulator), and vraSR (vancomycin resistance 69 associated sensor/regulator) and genes are considered important.2,9,10 Prolonged exposure to 70 vancomycin could induce these mutations.11 71 72 Vancomycin therapy has been shown to be ineffective for infections caused by hVISA.2 73 Therefore, detection of hVISA in the clinical specimens is essential before starting 74 vancomycin treatment. Detection of hVISA among MRSA is a challenge for clinical 75 microbiologists, because it exhibits vancomycin MIC in the susceptible range.2,5 The 76 antimicrobial susceptibility tests such as Kirby-Bauer disk diffusion, broth dilution, agar 77 dilution, and automated methods fail to detect hVISA.5 Screening tests such as macro E-test 78 (MET), vancomycin screen agar, and glycopeptide resistance detection (GRD) E-test vary in 79 their sensitivity and specificity.10,12 Population analysis profile-area under the curve (PAP-80 AUC), which is considered a reference method is labour intensive, expensive and 81 inappropriate for the routine clinical microbiology laboratories.12 82 83 Staphylococcal cassette chromosome mec (SCCmec) typing is being used for understanding 84 the epidemiology of MRSA infections. Healthcare-associated MRSA (HA-MRSA) normally 85 harbours SCCmec I, II and III. Whereas, community-associated MRSA (CA-MRSA) harbours 86 SCCmec IV, V and Panton-Valentine leukocidin gene (pvl).1,13 Panton- Valentine leukocidin 87 is an important virulence factor in CA-MRSA.13 Several recent studies have reported 88 overlapping of SCCmec types between HA-MRSA and CA-MRSA.14,15 Studies conducted in 89 Europe, USA, Australia and Japan have shown presence of SCCmec II, III, and IV among 90 hVISA.6 However, reports from India have shown predominance of SCCmec V in hVISA.10,16 91 Therefore, there are differences in the SCCmec types harboured by MRSA in different parts 92 of the world. The objectives of the present study were to determine the rate of hVISA among 93 MRSA isolated from healthcare-associated infections (HAIs) and to identify the SCCmec 94 types present in these strains. 95 96 Methods 97 4 The present cross-sectional study was conducted on nonrepetitive MRSA strains isolated from 98 patients admitted in four tertiary care hospitals attached to a private Medical College in 99 Coastal Karnataka South India during the period from February 2019 to March 2020. HAIs 100 were identified using Centers for Disease Control and Prevention (CDC) guidelines.17 101 102 Isolation and identification of S. aureus was done using standard bacteriological methods.18 103 Methicillin resistance was detected using cefoxitin (30 µg) disk diffusion method4 and 104 confirmed by detecting mecA gene using PCR.19 S. aureus ATCC 43300 and S. aureus ATCC 105 25923 were used as positive and negative controls respectively. Antimicrobial susceptibility 106 testing was done using the Kirby-Bauer disk diffusion. The following antibiotics (BD BBL™ 107 Sensi-Disc™ antimicrobial susceptibility test disks) were used: ciprofloxacin (5 µg), 108 clindamycin (2 µg), erythromycin (15 µg), gentamicin (10 µg), linezolid (30 µg), rifampicin 109 (5 µg), teicoplanin (30 µg) tetracycline (30 µg) and trimethoprim-sulphamethoxazole 110 (1.25µg/ 23.75 µg). Results were interpreted as per CLSI guidelines.4 S. aureus ATCC 25923 111 was used as the control. 112 113 Identification of macrolide lincosamide streptogramin B (MLSB) was done using D test. 4 114 Mueller-Hinton agar (MHA) (HiMedia laboratories, Mumbai, India) plates were lawn 115 cultured with test bacterial inoculum with turbidity matching McFarland 0.5 standard 116 (bacterial count 1.5 x 108 CFU/mL). Clindamycin (2 µg) and erythromycin (15 µg) disks 117 placed at a distance of 15 mm edge to edge on the inoculated plate. The plates were incubated 118 at 35°C for 16-18 h and the results were interpreted according to CLSI guidelines.4 119 120 The MIC of vancomycin to MRSA was determined using agar dilution method.4 MHA agar 121 plates with range of vancomycin (Sigma-Aldrich Corporation, St. Louis, US) concentrations 122 (32, 16, 8, 4, 2, 1, 0.5, 0.25 and 0.125 µg/mL) were prepared. Two to three colonies of the test 123 organism grown on blood agar plate were inoculated into Mueller-Hinton broth (HiMedia 124 laboratories, Mumbai, India) and incubated at 37°C for 4 to 6 h until the turbidity was 125 matched with McFarland 0.5 standard. The broth culture was diluted 10-1 to prepare the 126 working inoculum (1.5 x 107 CFU/mL). 2 µL was spot inoculated on each plate. The plates 127 were incubated at 35°C for 24 h and observed for growth. The minimum concentration of 128 vancomycin inhibiting the bacterial growth was considered as MIC and the results were 129 interpreted as per CLSI guidelines.4 MRSA isolates with MIC of vancomycin ≤ 2 µg/mL, 4-8 130 5 µg/mL and ≥ 16 µg/mL were considered VSSA, VISA and VRSA respectively.4 131 Enterococcus faecalis ATCC 29212, and S. aureus ATCC 29213 were used as vancomycin 132 susceptible controls. E. faecalis ATCC 51299 was vancomycin resistant control. 133 134 Screening of MRSA for hVISA was done using brain heart infusion agar (BHIA) (HiMedia 135 laboratories, Mumbai, India) containing 16 g/L pancreatic digestion of casein and 4 µg/mL 136 vancomycin.12 The test organisms were grown in brain heart infusion broth till the turbidity 137 matched with McFarland 0.5 and 2.0 standard. Four 10 µL drops from each suspension were 138 spot inoculated on BHI screen agar plates and allowed to dry for 10 minutes. The plates were 139 incubated at 35°C for 48 h and observed for bacterial growth. An isolate was considered 140 hVISA if at least one drop had 2 or more colonies.12 S. aureus ATCC 700698 (Mu3 strain of 141 hVISA) and S. aureus ATCC 29213 were used as a positive and negative controls 142 respectively. 143 144 Confirmation of hVISA was done using the modified population analysis profile- area under 145 the curve (PAP-AUC) method.20 In brief, the test and control (Mu3) were grown at 35°C for 146 4-6 hours in brain heart infusion broth, and the turbidity was matched with McFarland 0.5 147 standard. (1.5 x 108 CFU/mL). The broth culture was further diluted 10-4 to achieve viable 148 bacterial count of 104 CFU/mL and used for inoculation.5 A 10 µL bacterial inoculum 149 was spread on BHI agar plates with a range of vancomycin concentrations (16, 8, 4, 2, 1, 0.5, 150 0.25, and 0.125 µg/mL). The inoculated plates were incubated at 35°C for 48 h and colonies 151 were counted. The log10 number of colonies was plotted against the concentrations of 152 vancomycin and the area under the curve (AUC) was determined using GraphPad Prism 153 software version 9.0 (Graphpad Software USA).20 AUCtest /AUCMu3 ratio was calculated and 154 used for the confirmation of hVISA. MRSA strains with AUCtest/AUCMu3 ratio 0.9-1.3 were 155 considered hVISA [Figure 1] and strains with AUC ratio > 1.3 were considered VISA.5 Mu3 156 strain of hVISA (S. aureus ATCC 700698) and S. aureus ATCC 292l3 (VSSA) were used as 157 positive and negative controls respectively. 158 159 SCCmec types I-V and pvl in the test organisms were identified using multiplex PCR with 160 specific primers and controls.19,21 DNA was extracted using Qiagen DNA extraction kit as per 161 manufacturer’s instructions. The principle of present multiplex PCR performed was based on 162 a previous study by Zhang et al.19 Multiplex PCR kit was purchased from Qiagen, Hilden, 163 6 Germany. The list of primers used for the molecular detection and characterization of HA-164 MRSA isolates are listed in Table 1. 165 166 A 50 μL PCR mixture containing 25 μL multiplex master mix (Containing Taq DNA 167 polymerase, dNTPs and 1X Qiagen Multiplex PCR buffer with 6 mM MgCl2), 5 μL 10X 168 primer mix, 15 μL water and 5 μL DNA extract was prepared in 0.2 mL PCR tubes. Multiplex 169 PCR reaction was performed for 1 cycle of initial denaturation at 97°C for 5 minutes, 170 followed by 30 cycles of 30 seconds at 94°C, 30 seconds at 54°C and 90 seconds at 72°C, 171 with a final extension for 10 minutes at 72°C. The amplicons were analysed using 2% agarose 172 gel electrophoresis in 1X Tris-Acetate EDTA (TAE) buffer. The electrophoresis was carried 173 out at 120 V for 90 minutes, and the gel was stained with ethidium bromide staining solution 174 for 30 minutes. The gel was then visualized under an ultraviolet (UV) illuminator, and the 175 size of the bands was compared with the 100 base pair ladder (Bangalore Genei Private 176 Limited, Bengaluru, India). 177 178 Sensitivity and specificity analyses were done to evaluate the performance of vancomycin 179 agar screen. The data were analysed using the Statistical Package for the Social Sciences 180 (SPSS) version 29.0 (IBM Corp., Chicago, Illinois, USA). The rate of hVISA among MRSA 181 was expressed in percentage. The results were analysed using Fisher’s Exact test. P value of ≤ 182 0.05 was considered statistically significant. 183 184 This study had the approval of the Institutional Ethics Committee, Kasturba Medical College, 185 Mangalore. The isolates for the present study were collected from the clinical specimens 186 received at the laboratory for investigation. The samples were anonymized and the patient 187 details were not disclosed. Therefore, informed consent was not obtained in the present 188 investigation. 189 190 Results 191 Out of 220 nonrepetitive strains of MRSA isolated form healthcare associated infections, 14 192 (6.4%) were confirmed hVISA by PAP-AUC and the remaining 206 (93.6%) were 193 vancomycin susceptible. Vancomycin screen agar using both McFarland 0.5 and 2.0 standard 194 inoculum density detected hVISA in 21(9.5%) MRSA isolates. This included 14 isolates 195 confirmed by PAP-AUC. The sensitivity and specificity of the screening method were 100% 196 7 and 96.6% respectively. However, the end point (minimum 2 colonies) was clear in the 197 screening method using McFarland 2.0 standard inoculum. None of the isolates was VISA or 198 VRSA. Out of 14 hVISA, 10 (71.4%) and 4 (28.6%) were isolated from male and female 199 patient respectively. In case of 206 vancomycin susceptible MRSA, 133 (64.6%) and 73 200 (35.4%) were isolated from male and female patients respectively. The majority of hVISA 201 (6/14; 42.9%) were isolated from patients belonging to age group 61-70 years whereas 202 majority of vancomycin susceptible MRSA (48/206; 23.3%) were isolated from patients 203 belong to age group 41-50 years. 204 205 Out of 14 patients infected with hVISA, 11 (78.6%) were diabetic, 13 (92.9%) were 206 previously hospitalized, 8 (57.1%) received previous vancomycin treatment and 8 (57.1%) 207 underwent surgery previously. The majority of hVISA and vancomycin susceptible MRSA 208 were isolated from skin and soft tissue infection. 21.4% of hVISA and 10.7% of vancomycin 209 susceptible MRSA were isolated from cases of bacteremia [Table 2]. 210 211 Table 3 shows the antimicrobial resistance profile of the test organisms. Compared with 212 vancomycin susceptible MRSA more number of hVISA were resistant to antimicrobial agents 213 except trimethoprim-sulphamethoxazole. All the test organisms were susceptible to linezolid 214 and teicoplanin. More than 80.0% of the isolates were resistant to ciprofloxacin and 215 erythromycin. MSB phenotype was more common in both hVISA (6/14; 42.9%) and 216 vancomycin susceptible MRSA (82/206; 39.8%). 92.9 % hVISA had vancomycin MIC 217 ranging from 1 to 2 µg/mL [Table 4]. For both hVISA and vancomycin susceptible MRSA 218 MIC50 and MIC90 were 1 µg/mL and 2 µg/mL, respectively. 219 220 Results of SCCmec typing are presented in Table 5 and Figure 2. The majority of hVISA and 221 vancomycin susceptible MRSA carried SCCmec III and IV. There was no significant 222 difference between hVISA and vancomycin susceptible MRSA with regards to SCCmec type. 223 6.8% of vancomycin susceptible MRSA were nontypeable. pvl gene was detected in 2/14 224 (14.3%) hVISA and 57/206 (27.7%) of vancomycin susceptible MRSA isolates. 225 226 Discussion 227 In this study we present the prevalence and molecular features of hVISA in four tertiary care 228 hospitals of coastal Karnataka, south India. The hVISA phenotype was detected among 6.4% 229 8 of MRSA strains isolated from healthcare-associated infections. A recent systematic review 230 and meta-analysis has reported the rate of hVISA around the world.22 The hVISA phenotype 231 was reported in 82 studies on a total of 47,721 strains with an average prevalence of 4.6%. 232 This study showed that the prevalence of hVISA has increased over the last few years in 233 different parts of the world.22 Three previous studies from India have reported the prevalence 234 of hVISA ranging from 2 to 12.4%.7,23,24 The differences in the prevalence of hVISA could be 235 due to geographical area of the study, sample size, patient population and testing methods. 236 Increase in the rate of hVISA is a matter of concern. Further, since hVISA is considered as 237 the precursor stage of VISA,2,3 we may expect an increase in the rate of VISA in the future. 238 239 In this study there was no association between hVISA and type of infections. Factors such as 240 age, extended hospital stay, previous vancomycin treatment, diabetes mellitus, 241 instrumentation and surgery may increase in the risk of hVISA infections.2 In the present 242 study, more than 50 per cent of the patients infected with hVISA had risk factors such as 243 diabetes mellitus, previous hospitalization and vancomycin treatment. The clinical profile of 244 pvl positive cases was not different from the negative ones. 245 246 Vancomycin treatment of hVISA infections may result in persistence of infection, greater risk 247 of complications and treatment failure.2,25 Some researchers believe that hVISA arises as a 248 consequence of prolonged vancomycin treatment.25 Studies have demonstrated that area under 249 curve/MIC of vancomycin > 400 can bring about effective treatment.26 This can be achieved 250 if vancomycin MIC is ≤ 1 µg/mL. The European Committee on Antimicrobial Susceptibility 251 Testing (EUCAST) classifies S. aureus with vancomycin MIC >2 µg/mL as vancomycin 252 resistant.27 A previous study reported higher mortality among patients with hVISA infection 253 admitted in intensive care unit.28 In the present study, patients with hVISA deep infections 254 responded for vancomycin treatment. However, in cases where vancomycin toxicity 255 developed, vancomycin was replaced with teicoplanin. 256 257 Identification of hVISA phenotype among MRSA is difficult.2,12 The screening methods vary 258 in sensitivity, specificity and validity. Vancomycin screen agar method used in the present 259 study had sensitivity and specificity of 100% and 96.6% respectively. The PAP-AUC, which 260 is the reference method for the confirmation of hVISA is laborious.12 It may be difficult to 261 test all MRSA strains for hVISA. In this study, 92.9% of hVISA had vancomycin MIC 262 9 ranging from 1 to 2 µg/mL. Similar observations were made by other researchers too.10,29 263 Therefore, we suggest MRSA strains with MIC range 1-2 µg/mL could be chosen for 264 detection of hVISA phenotype. In critically ill patients with MRSA infection, hVISA 265 identification may have to be done upfront. In non-critical conditions, hVISA identification 266 may be carried out if clinical response is sub-optimal. 267 268 In this study, none of the MRSA was vancomycin intermediate or resistant. All hVISA and 269 vancomycin susceptible MRSA were susceptible to linezolid and teicoplanin. MSB phenotype 270 was most common followed by iMLSB (inducible clindamycin resistance). In routine disk 271 diffusion test, MRSA exhibiting inducible clindamycin appears resistant to erythromycin but 272 susceptible to clindamycin. If clindamycin is wrongly used for the treatment of infections 273 caused by such organisms, treatment failure occurs. Therefore, hVISA strains resistant to 274 erythromycin and susceptible to clindamycin should be subjected to D test to detect the 275 possibility of inducible clindamycin resistance. 276 277 In this study, the majority of hVISA harboured SCCmec III and IV. This in contrast to the 278 previous Indian studies which reported high frequency of SCCmec V among hVISA.7,10,16 279 Presence of hVISA harbouring SCCmec IV, V and pvl in the present study is suggestive of 280 entry of CA-MRSA into hospitals. This also shows that molecular differences between HA-281 MRSA and CA-MRSA is blurring. Although all hVISA strains in the present study could be 282 typeable, 6.8% vancomycin susceptible MRSA were nontypeable. It is possible that these 283 strains could harbour SCCmec types not included in the present study. A recent study from 284 South India also reported nontypeable strains among clinical isolates of MRSA.30 285 286 The present study had some limitations. It is difficult to draw general conclusions based on 287 investigations conducted on small number of hVISA. A larger sample size would have given 288 better understanding of hVISA infections. Multiplex PCR was designed for the detection of 289 SCCmec types I-V only. Additional genetic and molecular tests could have helped in better 290 understanding of the epidemiology hVISA. 291 292 Conclusion 293 The rate of hVISA among MRSA was 6.4%. MRSA strains should be tested for hVISA 294 phenotype before starting vancomycin treatment. Vancomycin agar screen with 4 µg/mL 295 10 vancomycin and McFarland 2.0 inoculum could be used for screening of MRSA for hVISA. 296 However, confirmation needs PAP-AUC. None of the isolates was vancomycin intermediate 297 or resistant. All hVISA strains were susceptible to linezolid and teicoplanin. The majority of 298 hVISA were isolated from skin and soft tissue infections. SCCmec III and IV were 299 predominant among hVISA and vancomycin susceptible MRSA. 300 301 Conflicts of Interest 302 The authors declare no conflict of interests. 303 304 Funding 305 This project was funded by the Department of Science and Technology, INSPIRE fellowship, 306 Government of India, Ministry of Science and Technology (Order No. DST/INSPIRE 307 fellowship/2018/1F180903, dated December 11, 2019) and Manipal Academy of Higher 308 Education (MAHE), Manipal, India 309 310 Authors’ Contribution 311 SM collected and organized data, performed the experiments, carried out the statistical 312 analysis of the results, and wrote the initial draft of the article. GBK conceived and designed 313 the study, reviewed the results, analysed and interpreted the data, wrote the initial and final 314 draft of the article, and supervised the study. SM and GBK acquired financial support for the 315 project and participated in the literature review, methods, and discussion. SSM (second 316 author), SSM, and GBK planning and execution of the research activity. SSM provided 317 logistic support and provided research materials. SSM (second author), SSM, DB, and CM 318 designed the study, analysed and interpreted the data, participated in the literature review, 319 methods, and discussion, participated in the final writing, and provided supervision. All 320 authors approved the final version of the manuscript. 321 322 Acknowledgments 323 The authors gratefully acknowledge the Department of Science and Technology (DST), 324 Ministry of science and technology, Government of India for sanctioning INSPIRE 325 Fellowship to Sreejisha M. The authors are thankful to the Head of the Institution and 326 Manipal Academy of Higher Education (MAHE), Manipal, India for providing the financial 327 assistance, materials and infrastructure to conduct the study. 328 11 329 References 330 1. Lakhundi S, Zhang K. 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J Med Microbiol 2019;68(5):720-27. 438 doi:10.1099/jmm.0.000977. 439 440 Table 1: Primer sequence, control strains with their respective genes used for multiplex PCR 441 and size of amplicon (base pair) post amplification 442 Genes Sequence Control strain Amplicon size (bp) mecA F- GTG AAG ATA TAC CAA GTG ATT R- ATG CGC TAT AGA TTG AAA GGA MRSA ATCC 43300 147 SCCmec I F- GCT TTA AAG AGT GTC GTT ACA GG R- GTT CTC TCA TAG TAT GAC GTC C MRSA NCTC 10442 613 SCCmec II F-CGTTGAAGATGATGAAGCG R-CGAAATCAATGGTTAATGGACC MRSA N315 398 SCCmec III F-CCATATTGTGTACGATGCG R-CCTTAGTTGTCGTAACAG ATCG MRSA 85/2082 280 SCCmec IVa F-GCCTTATTCGAAGAAACCG R-CTACTCTTCTGAAAAGCGTCG MRSA JCSC 4744 776 SCCmec IVb F-TCTGGAATTACTTCAGCTGC R-AAACAATATTGCTCTCCCTC MRSA JCSC 2172 493 F-ACAATATTTGTATTATCGGAGAGC 200 http://www.eucast.org/ 15 SCCmec IVc R-TTGGTATGAGGTATTGCTGG MRSA MR 108 SCCmec IVd F-CTCAAAATACGGACCCCAATACA R-TGCTCCAGTAATTGCTAAAG MRSA JCSC 4469 881 SCCmec V F-GAACATTGTTACTTAAATGAGCG R-TGAAAGTTGTACCCTTGACACC MRSA JCSC 4469 325 Pvl F-ATCATTAGGTAAAATGTCTGGACATGATCCA R-GCATCAAGTGTATTGGATAGCAAAAGC MRSA MR108 433 MRSA= Methicillin resistant Staphylococcus aureus; SCCmec= Staphylococcal cassette 443 chromosome mec; 444 445 Table 2: Isolation of heterogeneous vancomycin intermediate Staphylococcus aureus and 446 vancomycin susceptible methicillin resistant Staphylococcus aureus 447 Type of infections (Number) hVISA (N=14) Number (%) Vancomycin susceptible MRSA (N=206) Number (%) P value Surgical site infection (87) 4 (28.6) 83 (40.3) 0.385 Wound infection (63) 3 (21.4) 60 (29.1) 0.762 Bacteremia (25) 3 (21.4) 22 (10.7) 0.220 Abscess (18) 1 (7.1) 17 (8.3) 0.883 Cellulitis (6) 1 (7.1) 5 (2.4) 0.295 Osteomyelitis (6) 0 (0.0) 6 (2.9) 0.517 Carbuncle (5) 0 (0.0) 5 (2.4) 0.555 Gangrene (3) 1 (7.1) 2 (1.0) 0.054 Septic arthritis (2) 0 (0.0) 2 (1.0) 0.711 Umbilical site infection (2) 0 (0.0) 2 (1.0) 0.711 Necrotising fascitis (2) 0 (0.0) 2 (1.0) 0.711 Sepsis (1) 1 (7.1) 0 (0.0) 0.064 hVISA= Heterogeneous vancomycin intermediate Staphylococcus aureus; 448 MRSA= Methicillin resistant Staphylococcus aureus 449 450 Table 3: Antimicrobial resistance profile of heterogeneous vancomycin intermediate 451 Staphylococcus aureus and vancomycin susceptible methicillin resistant 452 Staphylococcus aureus 453 Antimicrobial agents hVISA (N=14) Number (%) resistant Vancomycin susceptible MRSA (N=206) Number (%) resistant P value Ciprofloxacin 14 (100.0) 179 (86.9) 0.227 16 Clindamycin 3 (21.4) 32 (15.5) 0.472 Erythromycin 13 (92.9) 173 (84.0) 0.701 Gentamicin 8 (57.1) 102 (49.5) 0.784 Linezolid 0 (0.0) 0 (0.0) - Rifampicin 6 (42.9) 11 (5.3) <0.001* Teicoplanin 0 (0.0) 0 (0.0) - Tetracycline 5 (35.7) 63 (30.6) 0.767 Trimethoprim- sulphamethoxazole 4 (28.6) 101 (49.0) 0.172 MLSB phenotypes iMLSB 4 (28.6%) 59 (28.6%) 1.000 cMLSB 3 (21.4%) 32 (15.5%) 0.472 MSB 6 (42.9%) 82 (39.8%) 1.000 *P value ≤ 0.05 statistically significant 454 cMLSB= Constitutive clindamycin resistance; hVISA= Heterogeneous vancomycin 455 intermediate Staphylococcus aureus; iMLSB= Inducible clindamycin resistance; MLSB= 456 Macrolide lincosamide streptogramins B; MSB= Macrolide streptogramins B; MRSA= 457 Methicillin resistant Staphylococcus aureus 458 459 Table 4: Minimum inhibitory concentration of vancomycin to heterogeneous vancomycin 460 intermediate Staphylococcus aureus and vancomycin susceptible methicillin resistant 461 Staphylococcus aureus 462 Vancomycin MIC (µg/mL) hVISA (N=14) Number (%) Vancomycin susceptible MRSA (N=206) Number (%) 0.125 0 (0.0) 0 (0.0) 0.25 0 (0.0) 5 (2.4) 0.5 1 (7.1) 55 (26.7) 1 8 (57.1) 93 (45.1) 2 5 (35.7) 53 (25.7) 4 0 (0.0) 0 (0.0) 8 0 (0.0) 0 (0.0) 16 0 (0.0) 0 (0.0) 32 0 (0.0) 0 (0.0) MIC50 a 1 µg/mL 1 µg/mL MIC90 b 2 µg/mL 2 µg/mL 17 hVISA= Heterogeneous vancomycin intermediate Staphylococcus aureus; MIC= Minimum 463 inhibitory concentration; MRSA= Methicillin resistant Staphylococcus aureus 464 aMIC50 = MIC value at which growth was inhibited in 50% of isolates; bMIC90 = MIC values 465 at which growth was inhibited in 90% of isolates 466 467 Table 5: Staphylococcal cassette chromosome mec types of vancomycin to heterogeneous 468 vancomycin intermediate Staphylococcus aureus and vancomycin susceptible methicillin 469 resistant Staphylococcus aureus 470 SCCmec types hVISA (N=14) Number (%) Vancomycin susceptible MRSA (N=206) Number (%) P value SCCmec I 0 (0.0) 0 (0.0) - SCCmec II 0 (0.0) 3 (1.5) 0.649 SCCmec III 7 (50.0) 73 (35.4) 0.389 SCCmec IVa 4 (28.6) 47 (22.8) 0.621 SCCmec IVb 0 (0.0) 0 (0.0) - SCCmec IVc 0 (0.0) 12 (5.8) 0.353 SCCmec IVd 1 (7.1) 20 (9.7) 0.752 SCCmec V 2 (14.3) 37 (18.0) 0.727 hVISA= Heterogeneous vancomycin intermediate Staphylococcus aureus; SCCmec= 471 Staphylococcal cassette chromosome mec; MRSA= Methicillin resistant Staphylococcus 472 aureus 473 474 475 476 18 477 Figure 1: Confirmation of hVISA using modified PAP-AUC 478 Mu3- hVISA reference strain (S. aureus ATCC 700698) 479 AUCtest = 9.750; AUCMu3 =10.50; AUCtest/AUCMu3 ratio = 0.93 (hVISA) 480 AUC= area under the curve; CFU= colony forming unit; MRSA= methicillin resistant 481 Staphylococcus aureus; hVISA= heterogeneous vancomycin intermediate Staphylococcus 482 aureus; PAP-AUC= population analysis profile-area under the curve 483 484 0 1 2 3 4 5 6 7 8 0 0.5 1 1.5 2 2.5 3 3.5 4 4.5 5 5.5 6 6.5 7 7.5 8 lo g 1 0 C F U /m L Vancomycin concentration (µg/mL) Mu3 (S.aureus 700698) hVISA 19 485 Figure 2: Gel electrophoresis of multiplex PCR for the detection of mecA, SCCmec types 1-V 486 and pvl gene 487 Lane 1: 100 bp DNA ladder; Lane 2: positive controls; lane 3: negative control (master mix 488 and nuclease-free water); Lane 4-8, 14, 15 and 18: Vancomycin susceptible MRSA isolates 489 positive for mecA, and SCCmec III; Lane 9, 10, 17, 19: Vancomycin susceptible MRSA 490 isolates positive for mecA, and SCCmec IVa; Lane 16: hVISA isolate positive for mecA, and 491 SCCmec IVa, Lane 11 and 12: Vancomycin susceptible MRSA isolates positive for mecA, 492 SCCmec V, and pvl; Lane 13: Vancomycin susceptible MRSA isolate positive for mecA, and 493 SCCmec V; Lane 20: Vancomycin susceptible MRSA isolate positive for mecA, SCCmec 494 IVc, and pvl 495 SCCmec= Staphylococcal cassette chromosome mec; hVISA= heterogeneous vancomycin 496 intermediate Staphylococcus aureus; pvl= Panton-Valentine leukocidin gene 497 498 SCCmec IVd (881bp) SCCmec IVa (776bp) SCCmec I (613bp) SCCmec IVb (493bp) pvl (433bp) SCCmec II (398bp) SCCmec V (325bp) SCCmec III (280bp) SCCmec IVc (200bp) mecA (147bp) 1 2 3 4 5 6 7 8 9 10 1 13 16 18 19 20 11 12 14 15 17