Panacea Journal of Medical Sciences 2020;10(3):245–249 Content available at: https://www.ipinnovative.com/open-access-journals Panacea Journal of Medical Sciences Journal homepage: www.ipinnovative.com Original Research Article Detection of vancomycin susceptibility among methicillin resistant staphylococcus aureus in a tertiary care hospital Dimple Raina1,*, Sakeena2, Iva Chandola1, Reshmi Roy1 1Dept. of Microbiology, Shri Guru Ram Rai Institute of Medical and Health Sciences, Dehradun, Uttarakhand, India 2Dept. of Microbiology, Muzaffarnagar Medical College and Hospital, Muzaffarnagar, Uttar Pradesh, India A R T I C L E I N F O Article history: Received 19-06-2020 Accepted 01-07-2020 Available online 29-12-2020 Keywords: Staphylococcus aureus MRSA Vancomycin E-test Agar dilution CLSI A B S T R A C T Introduction: Staphylococcus aureus infections in current times have become challenging to treat because of advent of Methicillin Resistant Staphylococcus aureus (MRSA) strains which are concurrently resistant to a wide panel of drugs and posing a threat to clinicians and microbiologists globally. The optimal drug for treatment of such MRSA infections is vancomycin but strains with augmented Minimum Inhibitory concentration (MIC) for this drug also have surfaced. Objectives: To know the frequency of MRSA isolates in various clinical samples with their antimicrobial sensitivity patterns and to equate agar dilution and E-test methods for MIC determination of vancomycin to MRSA strains. Materials and Methods: A total of 50 non repeat clinical isolates of staphylococcus aureus isolates were collected from various clinical specimens and were tested for methicillin resistance using the cefoxitin disc diffusion test (30µ g). All MRSA isolates were tested for specific MIC by agar dilution and E-test methods. Results: 29 (58%) isolates were resistant to cefoxitin (MRSA). 13.8% isolates had MIC of 4µ g/ml for vancomycin (VISA) by both agar dilution and E-test methods. However by agar dilution method 25 (86.2%) isolates exhibited vancomycin MIC of ≤ 2 µ g/ml and by E-test 68.9% of the isolates showed MIC ≤ 2 µ g/ml. Conclusion: Multidrug resistant MRSA strains are on the rise and alternate drug of choice for these infections; vancomycin also is showing increased MIC so prudent use of this drug is advocated. E-test can detect MRSA strains with intermediate MIC values useful for detection of MIC creep so that vancomycin can be used rationally. © This is an open access article distributed under the terms of the Creative Commons Attribution License (https://creativecommons.org/licenses/by/4.0/) which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. 1. Introduction Staphylococcus aureus has become a huge concern because of its high morbidity, high mortality attributes and both community-acquired and nosocomial infections are associated with it. 1 The advent of methicillin-resistant Staphylococcus aureus (MRSA) strains has further made it challenging to treat Staphylococcus aureus infections. The only therapeutic alternatives in many cases are limited to glycopeptides such as vancomycin or teicoplanin. However, in recent times treatment failures for even vancomycin * Corresponding author. E-mail address: dr.dimpleraina@gmail.com (D. Raina). have been reported. Now there is an increasing body of evidence which suggests that a relationship exists between vancomycin MIC and clinical vancomycin failure, despite the fact that in vitro MRSA strains are absolutely susceptible (MIC 2 g/mL) to vancomycin. 2 Although majority of these strains have a vancomycin MIC within the susceptible limits, yet a gradual and progressive upsurge in vancomycin MIC which is also acknowledged as the "MIC creep" has been on the rise in recent years. 3 As a matter of fact, after arbitrating on the incessant rise in the cases of failed vancomycin therapy CLSI thereupon abridged the breakpoints of vancomycin from 4 mg/L to 2 mg/L for susceptible strains of Staphylococcus aureus https://doi.org/10.18231/j.pjms.2020.051 2249-8176/© 2020 Innovative Publication, All rights reserved. 245 https://doi.org/10.18231/j.pjms.2020.051 https://www.ipinnovative.com/ https://www.ipinnovative.com/open-access-journals https://www.ipinnovative.com/journal/PJMS https://creativecommons.org/licenses/by/4.0/ mailto:dr.dimpleraina@gmail.com https://doi.org/10.18231/j.pjms.2020.051 246 Raina et al. / Panacea Journal of Medical Sciences 2020;10(3):245–249 and for the resistant strains from 32 mg/L to 16 mg/L. Infections with MRSA isolates that reveal MIC creep can lead to poor prognostic outcomes, deferred therapeutic responses, amplified relapse rates, protracted hospital stay with consequent increased hospitalization costs and greater mortality rates. 4 This awareness of data of infections with MRSA strains that have increased vancomycin MICs can help in the early identification of patients who are at perils of vancomycin treatment failure so that alternate treatment options can be explored at the right time. 5 Therefore, the present study aims to approximate the vancomycin susceptibility patterns amongst Methicillin Resistant Staphylococcus aureus isolates in a tertiary care hospital. 2. Materials and Methods The study was conducted in the Department of Microbiology & Immunology, at Shri Guru Ram Rai Institute of Medical and Health Sciences & affiliated Shri Mahant Indiresh Hospital, Dehradun from November 2016 to April 2017. The study was approved by the institutional research board and ethics committee. A total of 50 non repeat clinical isolates of staphylococcus aureus were collected from diverse clinical specimens like pus, wound swab, blood culture, sputum, Broncho-alveolar lavage, pleural fluid and urine. Various attributes like colony characteristics, microscopic morphology, and biochemical reactions were used for preliminary detection of Staphylococcus aureus as per the standard protocol. Automated method; VITEK-2 (Biomerieux) was used for both identification and for determining the antibiotic susceptibility patterns. However manual method i.e. disc diffusion method based on mec A mediated oxacillin resistance and 30 µ g cefoxitin disk as a surrogate marker for oxacillin was also utilized for detecting MRSA strains. Reference Staphylococcus aureus strains; Staphylococcus aureus ATCC 25923 was used as negative and ATCC 43300 was used as positive control for quality maintenance and consistency of results. Isolates showing inhibition zone size ≥ 22 were considered as sensitive (MSSA) and ≤ 21 mm were considered as resistant (MRSA) as per CLSI criteria for zone diameter breakpoints of Staphylococcus aureus for cefoxitin.[6] CLSI guidelines were used to determine MIC of vancomycin by agar dilution method and by E-test method. ATCC strains; 25923 and ATCC 700698 of Staphylococcus aureus were incorporated within in all the test plates for quality and standardization. The least concentration of vancomycin that inhibited the visible growth of bacteria was considered as MIC of drug for that organism. Interpretation of MIC of vancomycin was done as per the CLSI guidelines for both agar dilution method and E-test strip method. Vancomycin MIC of ≤ 2 µ g/ml was considered as the breakpoint for vancomycin susceptible (VSSA) MRSA strains, 4-8 µ g/ml for vancomycin intermediate (VISA) strains and ≥16 µ g/ml for vancomycin resistant (VRSA) strains. 6 3. Results Over-all 50 Staphylococcus aureus strains were isolated from various clinical specimens of which pus specimens contributed for bulk of the isolates (78%). 29 (58%) isolates were resistant (zone diameter <21mm) to cefoxitin while 21 (42%) were sensitive (zone diameter >22 mm) to cefoxitin. Maximum number of MRSA isolates were recovered from Pus (75.8%) followed by 6.9 % each from blood, suction tip and tissue (Table 1). Allocation of patients on the basis of different sites from where MRSA was isolated is given in (Table 2). Majority of these isolates were recovered from the surgery wards (31%) followed by patients admitted in orthopedic wards (24.1%). Maximum number of isolates showed increased resistance to amoxicillin/clavulanic acid (89.7%) (apart from penicillin and cefoxitin for which 100% resistance was seen) (Table 3). However, linezolid sensitivity was observed in all MRSA isolates. By agar dilution method for determining vancomycin susceptibility among MRSA isolates, a total of 25 (86.2%) isolates were having MIC for vancomycin in the range of 0.5- 2 µ g /ml (≤ 2 µ g/ml) i.e. VSSA. By E-test method method 68.9% of the isolates had MIC ≤ 2 µ g/ml. 31% isolates reported a vancomycin MIC of >2 µ g/ml but <4 µ g/ml. 13.8% VISA isolates with MIC of 4µ g/ml were observed by both the methods. (Table 4). The concentrations of vancomycin that inhibited growth of 50% and 90% of the isolates were defined as MIC50 and MIC90 respectively. MIC50 and MIC90 of the study isolates were found to be 1 µ g /ml and 4 µ g /ml by both the methods respectively. Table 1: Distribution of MRSA isolates according to specimens (n=29) Clinical Specimen MRSA n Percentage (%) Pus 22 75.8% Blood 2 6.9% Suction Tip 2 6.9% Tissue 2 6.9% Urine 1 3.5% Total 29 100% 4. Discussion In the current study isolation of methicillin resistant Staphylococcus aureus was maximum from pus 22(75.8%) followed by 2(6.9%) from blood. Similar observations have been reported by Chaudhri CN et al in their study wherein maximum number of isolates were isolated from pus Raina et al. / Panacea Journal of Medical Sciences 2020;10(3):245–249 247 Table 2: Distribution of MRSA isolates according to Location/ site (n=29) Location/Site MRSA n Percentage (%) Surgery Ward 9 31% Orthopaedic Ward 7 24.1% OPD 5 17.2% Medicine Ward 2 6.9% Medicine HDU 2 6.9% Dermatology 1 3.5% Surgical ICU 2 6.9% Gynaecology/ Obstetrics 1 3.5% Total 29 100% Table 3: Trends of antibiotic resistance among MRSA isolates (n=29) Antibiotic Sensitive Intermediate Resistant n Percentage % n Percentage % n Percentage % Penicillin 0 0 0 0 29 100% Cefoxitin 0 0 0 0 29 100% Amoxycillin/Clavulanic acid 3 10.30% 0 0 26 89.70% Ciprofloxacin 6 20.6% 3 10.3% 20 68.9% Erythromycin 12 41.3% 0 0 17 58.6% Trimethoprim/Sulfamethoxazole 10 34.4% 2 6.8% 17 58.6% Clindamycin 16 55.1% 0 0 13 44.8% Tetracycline 12 41.3% 6 20.6% 11 37.9% Gentamycin 13 44.80% 6 20.70% 10 34.50% Linezolid 29 100% 0 0 0 0 Table 4: Comparative analysis of Vancomycin MIC by agar dilution and E-test methods (n=29) Vancomycin MIC µ g/ml Agar dilution n (%) E-test n (%) 0.25 - - 0.5 3(10.3) 4(13.8) 0.75 - 3(10.3) 1 13(44.8) 8(27.6) 1.5 - 3(10.3) 2 9(31.0) 2(6.9) 2.5 - 2(6.9) 3 - 2(6.9) 3.5 - 1(3.4) 4 4(13.8) 4(13.8) Total 29 29 (76.7%) followed by blood (3.9%) and also by Sreenivasulu Reddy P et al wherein most of the isolates were also isolated from pus (69%) followed by blood (9%). 7,8 Cutaneous and sub cutaneous infections caused by Staphylococcus aureus often manifest in the form of abscesses and are formed to restrain the focus of infection. 9 Maximum isolates were from the IPD (82.8%) whereas only 17.2% isolates were from OPD. This is in concurrence with Suryadevara VD who in her research work has reported 70% of the isolates from IPD and 30% isolates from OPD. 10 The reasons can be attributed to healthcare workers who are chronic carriers for these isolates, emergence of strains with amplified resistance sequences and extended hospital stay especially in ICUs. 58% isolates were resistant to cefoxitin which is in concurrence with other studies as by Sanjana et al in which the isolation rate of MRSA was 39.6%, Juayang et al (40.6%) and Arora et al (46%). 11–13 In a tertiary care center, the incidence of MRSA may be higher since the probability of the patient being put on antimicrobial drugs beforehand is quite elevated and thus onset of selective pressure may negotiate a critical role in acquisition of resistance to most of the frequently used drugs. 14 100% resistance was seen for penicillin and cefoxitin and similar observations have also been reported by Ramakrishna N. 15 Frequently prescribed antibiotics such 248 Raina et al. / Panacea Journal of Medical Sciences 2020;10(3):245–249 as ciprofloxacin erythromycin, clindamycin, gentamicin, tetracycline and co-trimoxazole all showed increased levels of resistance in our study as has also been reported from many parts of India particularly in association with MRSA. 10 Linezolid is one of the well-recognized alternative drugs for vancomycin and can be safely used for cutaneous and sub cutaneous infections, pneumonia, urinary tract infections and bacteremia caused by MRSA strains. 7 The current study shows that 68.9% of isolates had an MIC of 0.5 - 2µ g/ml whereas 31% isolates had an MIC >2µ g/ml by the E.-test method. Similar observations have been made by Sreenivasulu Reddy P et al wherein 82% isolates showed MIC between 0.5-2µ g/ml and 17% of isolates had an MIC of >2µ g/ml. 8 Tandel et al however reported 60.3% strains with an MIC of >2µ g/ml. 16 By the agar dilution method 86.2% isolates demonstrated MIC of 0.5 - 2µ g/ml. However, by both the methods 4 (13.8%) strains exhibited a vancomycin MIC of 4 µ g/ml and were therefore categorized as vancomycin intermediate (VISA). Kumari J et al have also reported 4.1% isolates with MIC >2 µ g/ml by both methods. 17 There are several researches that have established a correlation for MRSA strains with increased vancomycin MICs (>1 µ g/ml) and still within the context of susceptibility (≤ 2 µ g/ml) but unfortunately with proven clinical letdowns. 18,19 Determination of MIC of vancomycin by E-test method was constantly higher by a range of 0.5-1 µ g/ml than that worked out by agar dilution method. Such high MICs by E test as equated to agar dilution method have also been reported from other studies. 16 The important contributing factor could be the variances in the concentration gradient of vancomycin prepared in these tests. In agar dilution method the concentration of vancomycin is prepared in doubling dilutions or in geometric progression, whereas dilution of vancomycin concentrations is used in arithmetic progression in the E-test method so that the MICs for intermediate concentrations of the drug can also be determined. 20 Area under the vancomycin concentration curve-to- MIC ratio (AUC/MIC) is also a significant factor to establish the effectiveness of vancomycin therapy in MRSA infection therapeutics. 21 The odds of attaining this ratio is nearly 100% if MIC of vancomycin is ≤0.5 µ g/ml; and the likelihood reduces to practically 0% if the MIC of vancomycin is 2 µ g/ml. 22,23 MIC/AUC ratio can be considerably prejudiced by even a sole dilution alteration in the MIC which in turn can significantly influence the outcome of therapy. 21 If just one dilution difference is pertinent to envisage the clinical consequences of MRSA infections, then the MIC method is a vital component of this reckoning. 19 5. Conclusion For MRSA infections Vancomycin still is the drug of choice and therefore should be judiciously used in treatment of such resistant infections as also observed in our study that MRSA continue to be multi drug resistant leaving clinicians with limited therapeutic options. However, a silver lining in our study is that efficacy of Linezolid still incites hope for VISA and VRSA cases. MIC creep needs further evaluation to optimize treatment modalities hence early detection of such strains in high susceptibility range could minimize the risk of emergence of more aggressive VISA and VRSA infections. For determination of vancomycin MIC dilution methods still are the gold standard, yet E-tests could be considered to establish vancomycin MIC in the intermediary zones and for observing trivial MIC changes. 6. Acknowledgement We would like to express our heartfelt thanks to the central laboratory staff, Department of microbiology SGRRIM&HS for their unflinching support during the period of this study. 7. Source of Funding No financial support was received for the work within this manuscript. 8. Conflict of Interest The authors declare they have no conflict of interest. References 1. Abdulgader SM, Rijswijk A, Whitelaw A, Newton-Foot M. The association between pathogen factors and clinical outcomes in patients with Staphylococcus aureus bacteraemia in a tertiary hospital, Cape Town. Int J Infect Dis. 2020;91:111–8. doi:10.1016/j.ijid.2019.11.032. 2. Lina YC, Wangb JH, Lina KH, Hoc YL, Ho CM, M C. Methicillin-resistant Staphylococcus aureus with reduced vancomycin susceptibility in Taiwan. Tzu Chi Med J. 2018;30(3):135–40. 3. Chang W, Ma X, Gao P, Lv X, Lu H, Chen F. Vancomycin MIC creep in methicillin-resistant Staphylococcus aureus (MRSA) isolates from 2006 to 2010 in a hospital in China. Indian J Med Microbiol. 2015;33(2):262–6. doi:10.4103/0255-0857.148837. 4. Diaz R, Afreixo V, Ramalheira E, Rodrigues C, Gago B. Evaluation of vancomycin MIC creep in methicillin-resistant Staphylococcus aureus infections—a systematic review and meta-analysis. Clin Microbiol Infect. 2018;24(2):97–104. doi:10.1016/j.cmi.2017.06.017. 5. Manthan S, Geeta V, Summaiya M. Retrospective analysis of the distribution of vancomycin MIC Values among clinical isolates of methicillin-resistant Staphylococcus aureus in a Tertiary Care Hospital, Surat. NJIRM. 2017;8(2):5–8. 6. Clinical and Laboratory Standards Institute. Performance Standards for Antimicrobial Susceptibility Testing; Twenty – Seventh Informational Supplement. CLSI Document M100-S27. Wayne: CLSI; 2017. 7. Chaudhari CN. In vitro vancomycin susceptibility amongst methicillin resistant Staphylococcus aureus. Med J Armed Forces India. 2014;70:215–9. 8. Reddy SP, John MS, Devi VP, Reddy SPB. Detection of vancomycin susceptibility among clinical isolates of MRSA by using minimum http://dx.doi.org/10.1016/j.ijid.2019.11.032 http://dx.doi.org/10.4103/0255-0857.148837 http://dx.doi.org/10.1016/j.cmi.2017.06.017 Raina et al. / Panacea Journal of Medical Sciences 2020;10(3):245–249 249 inhibitory concentration method. Int J Res Med Sci. 2015;3(6):1378– 82. doi:10.18203/2320-6012.ijrms20150151. 9. Kobayashi SD, Malachowa N, DeLeo FR. Pathogenesis of Staphylococcus aureus Abscesses. Am J Pathol. 2015;185:1518–21. doi:10.1016/j.ajpath.2014.11.030. 10. Suryadevara VD, Basavaraju A, Vasireddy K. Prevalence of MRSA among clinical isolates and their antibiogram in a tertiary care hospital. J Evol Med Dent Sci. 2017;6(21):1667–9. doi:10.14260/jemds/2017/367. 11. Sanjana RK, Shah R, Chaudhary N, Singh YI. Prevalence and antimicrobial susceptibility pattern of methicillin-resistant Staphylococcus aureus (MRSA) in CMS-teaching hospital: a preliminary report. J Coll Med Sci Nepal. 2010;6:1–6. 12. Juayang AC, de los Reyes GB, de la Rama AJG, Gallega CT. Antibiotic Resistance Profiling ofStaphylococcus aureusIsolated from Clinical Specimens in a Tertiary Hospital from 2010 to 2012. Interdiscip Perspect Infect Dis. 2014;2014:898–909. doi:10.1155/2014/898457. 13. Arora S, Devi P, Arora U, Devi B. Prevalence of Methicillin- resistant Staphylococcus Aureus (MRSA) in a Tertiary Care Hospital in Northern India. J Lab Physicians. 2010;2(02):78–81. doi:10.4103/0974-2727.72154. 14. Pant ND, Sharma M. Carriage of methicillin resistant Staphylococcus aureus and awareness of infection control among health care workers working in intensive care unit of a hospital in Nepal. Braz J Infect Dis. 2016;20(2):218–9. doi:10.1016/j.bjid.2015.11.009. 15. Ramakrishna N, Murty DS, Reddy BK. Detection of Methicillin Resistance and Vancomycin Resistance among Clinical Isolates of Staphylococcus aureus in a Tertiary Care Hospital at Tirupati. Sch J App Med Sci. 2016;4(7B):2396–9. doi:10.21276/sjams.2016.4.7.19. 16. Tandel K, Praharaj AK, Kumar S. Differences in vancomycin MIC among MRSA isolates by agar dilution and E test method. Indian J Med Microbiol. 2012;30(4):453–5. doi:10.4103/0255-0857.103768. 17. Kumari J, Shenoy MS, Chakrapani M, Vidyalakshmi K, Gopalkrishna BK. Comparison Of E-test and agar dilution for determining minimum inhibitory concentration of vancomycin to Healthcare-Associated Methicillin-Resistant Staphylococcus aureus. Asian J Pharm Clin Res. 2016;9(4):189–91. 18. Aguado JM. High Vancomycin MIC and Complicated Methicillin Susceptible Staphylococcus aureus Bacteremia. Emerg Infect Dis. 2011;17(6):1099–102. 19. Wilcox M. Reporting elevated vancomycin minimum inhibitory concentration in methicillin-resistant Staphylococcus aureus: consensus by an International Working Group. Future Microbiol. 2019;14(4):345–52. 20. Kruzel MC, Lewis CT, Welsh KJ, Lewis EM, Dundas NE, Mohr JF, et al. Determination of Vancomycin and Daptomycin MICs by Different Testing Methods for Methicillin-Resistant Staphylococcus aureus. J Clin Microbiol. 2011;49(6):2272–3. doi:10.1128/jcm.02215- 10. 21. Prakash V, Lewis JS, Jorgensen JH. Vancomycin MICs for Methicillin-Resistant Staphylococcus aureus Isolates Differ Based upon the Susceptibility Test Method Used. Antimicrob Agents Chemoth. 2008;52(12):4528. doi:10.1128/aac.00904-08. 22. Dhand A, Sakoulas G. Reduced vancomycin susceptibility among clinical Staphylococcus aureus isolates (‘the MIC Creep’): implications for therapy. F1000 Med Rep. 2012;4:4–4. doi:10.3410/m4-4. 23. Mohr JF, Murray BE. Point: Vancomycin Is Not Obsolete for the Treatment of Infection Caused by Methicillin-Resistant Staphylococcus aureus. Clin Infect Dis. 2007;44(12):1536–42. doi:10.1086/518451. Author biography Dimple Raina, Associate Professor Sakeena, Tutor Iva Chandola, Associate Professor Reshmi Roy, Assistant Professor Cite this article: Raina D, Sakeena, Chandola I, Roy R. Detection of vancomycin susceptibility among methicillin resistant staphylococcus aureus in a tertiary care hospital. Panacea J Med Sci 2020;10(3):245-249. http://dx.doi.org/10.18203/2320-6012.ijrms20150151 http://dx.doi.org/10.1016/j.ajpath.2014.11.030 http://dx.doi.org/10.14260/jemds/2017/367 http://dx.doi.org/10.1155/2014/898457 http://dx.doi.org/10.4103/0974-2727.72154 http://dx.doi.org/10.1016/j.bjid.2015.11.009 http://dx.doi.org/10.21276/sjams.2016.4.7.19 http://dx.doi.org/10.4103/0255-0857.103768 http://dx.doi.org/10.1128/jcm.02215-10 http://dx.doi.org/10.1128/jcm.02215-10 http://dx.doi.org/10.1128/aac.00904-08 http://dx.doi.org/10.3410/m4-4 http://dx.doi.org/10.1086/518451 Introduction Materials and Methods Results Discussion Conclusion Acknowledgement Source of Funding Conflict of Interest