Original article / research
| Year :
2022 |
Month :
July
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Volume :
11 |
Issue :
3 |
Page :
MO01 - MO04 |
Full Version
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Comparison of Vancomycin MICs by Broth Microdilution Method, E-Test and Vitek 2C among MRSA Isolates in Tertiary Care Centre, Hyderabad, India
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Bushra, Kanne Padmaja, Sukanya Sudhaharan, Vijay Dharma Teja
1. Junior Resident, Department of Microbiology, Nizam's Institute of Medical Sciences, Hyderabad, Telangana, India.
2. Associate Professor, Department of Microbiology, Nizam's Institute of Medical Sciences, Hyderabad, Telangana, India.
3. Associate Professor, Department of Microbiology, Nizam's Institute of Medical Sciences, Hyderabad, Telangana, India.
4. Head, Department of Microbiology, Nizam's Institute of Medical Sciences, Hyderabad, Telangana, India.
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Correspondence
Address :
Bushra, Kanne Padmaja, Sukanya Sudhaharan, Vijay Dharma Teja,
Dr. Kanne Padmaja,
Department of Microbiology, Nizam's Institute of Medical Sciences, Hyderabad, Telangana, India.
E-mail: kannepadmaja@gmail.com
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| | | ABSTRACT |  | : Introduction: The most important pathogen isolated from Skin and Soft Tissue Infections (SSTIs) is a gram positive organism, Staphylococcus aureus (S. aureus). Wide range of emerging Methicillin Resistant Staphylococcus aureus (MRSA) infections is leading to global threat causing Community Acquired-MRSA (CA-MRSA) or Hospital Acquired MRSA (HA-MRSA). Minimum Inhibitory Concentration (MIC) is done to ensure that antibiotics are chosen efficiently in the clinical settings by Vitek 2, Epsilometer-test (E-test) and Broth Microdilution (BMD) method.
Aim: To determine the clinical spectrum of MRSA and comparison of vancomycin MICs obtained by E-test, Vitek 2C and BMD method.
Materials and Methods: This was a cross-sectional study conducted at Nizam’s Institute of Medical Sciences, Hyderabad, Telangana, India, during the period of November 1st 2019 to December 31st, 2020 from Inpatient Department (IPD) and Outpatient Department (OPD). Among 464 SSTIs, 132 were S. aureus, out of which 38 isolates were MRSA. Identification and sensitivity of the isolates such as pus, wound swabs and tissues were identified through Vitek 2 compact system. Statistical analysis of the demographic and clinical characteristics was represented as frequency and percentages.
Results: Among the total 464 SSTIs, S. aureus were 132 of which MRSA were 38 (29%) and Methicillin sensitive Staphylococcus aureus (MSSA) were 94 (71%) with male predominance of 29 (76.3%) with MRSA. Most of the patients were in the age group of 21-30 years (26%). Amongst the total 38 patients analysed, 25 were from IPD and 13 were from OPD. Around 23/38 were wound swabs (60%) 12/38 of pus (31.5%) and 3/38 (8%) were tissues. The predominant risk factor observed was Surgical Site Infections (SSI) in 19 cases (50%) followed by prior antibiotic therapy in 17 (44.7%) cases. The median duration of hospitalisation was 31.5 days. Vancomycin susceptibility by all three methods with an MIC range of 0.5-2 μg/m by all three methods, except for one isolate where the MIC was >32 ug/mL by Vitek 2C and 8 ug/mL by E-test, which was sensitive by BMD with an MIC of 0.25 μg/mL.
Conclusion: Implementing infection control practices and controlling the risk factors will help in management of MRSA infections. Drug resistance to glycopeptides can be avoided by regular screening of vancomycin creeps by different susceptibility methods in order to avoid treatment failures.
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| Keywords
: Community acquired Staphylococcus aureus, Epsilometer test, Hospital acquired infections, Methicillin resistant Staphylococcus aureus, Minimum inhibitory concentration, Skin and soft tissue infections |
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| DOI and Others
: DOI: 10.7860/NJLM/2022/53225.2627
Date of Submission: Nov 10, 2021
Date of Peer Review: Jan 14, 2022
Date of Acceptance: Feb 19, 2022
Date of Publishing: Jul 01, 2022
AUTHOR DECLARATION:
• Financial or Other Competing Interests: None
• Was Ethics Committee Approval obtained for this study? No
• Was informed consent obtained from the subjects involved in the study? No
• For any images presented appropriate consent has been obtained from the subjects. No
PLAGIARISM CHECKING METHODS |
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INTRODUCTION |
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The most important bacterial pathogen, S. aureus results in causing various systemic infections such as bacteremia, endocarditis, osteomyelitis, meningitis, sepsis, SSTIs, pneumonia and septic arthritis. The emergence of MRSA as a nosocomial pathogen leads to wide range of MRSA infections resulting in CA-MRSA or HA-MRSA (1). Indiscriminate and inappropriate use of vancomycin results in the emergence and spread of resistant MRSA isolates in the health care settings resulting in poor clinical outcomes. The risk factors for MRSA infections are prolonged hospital stay, indiscriminate use of antibiotics, use of invasive medical devices, contact with healthcare workers, suppressed immune system, long-term use of antibiotics, overcrowding and unhygienic conditions (2).
First drug of choice for treating MRSA infections is vancomycin (Glycopeptide). Due to indiscriminate use of vancomycin to treat MRSA, it was observed that several strains with variable susceptibility have emerged showing discrepancies in-vitro that have resulted in treatment failure leading to morbidity and worsening of clinical outcomes (3). There is an alarming increase in the prevalence of MRSA infections with 28% in 2017 to 35.1% in 2019 (4).
The MIC is done to ensure that antibiotics are chosen efficiently in the clinical settings (5). The MIC breakpoint of vancomycin used by the Clinical and Laboratory Standards Institute (CLSI) is <2 ug/mL. Creep of vancomycin MIC from 1-2 μg/mL is widely reported leading to suboptimal clinical outcomes (6). Due to indiscriminate use of vancomycin to treat MRSA several strains with variable susceptibility have emerged and are reported worldwide with suboptimal clinical outcomes and creeping MIC values (6). By introducing BMD method testing, it allows the option of providing quantitative (MIC) results. It is necessary to perform standard susceptibility testing methods to detect higher MICs.
Automated systems do not accurately detect susceptibility to vancomycin. Henceforth, many laboratories should assess exact MICs by reference method such as BMD method or alternative methods, such as E-test for which this study was undertaken. As per our knowledge, BMD method is a reference method as suggested by CLSI for detection of MICs in comparison with the MICs of Vitek 2 and E-test which was done only in few Indian studies (7). Therefore, the present study was done to analyse the clinical spectrum of MRSA infections and to compare vancomycin susceptibility by three methods.
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Material and Methods |
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This was a cross-sectional study conducted at Nizam’s Institute of Medical Sciences, Hyderabad, Telangana, India, during the period of November 1st 2019 till December 31st of 2020 from IPD and OPD patients. Among 464 SSTIs, 132 were S. aureus, out of which 38 isolates were MRSA. Identification and sensitivity of the isolates such as pus, wound swabs and tissues were identified through Vitek 2 compact system.
Inclusion criteria: S. aureus (Methicillin resistant) isolated from different purulent samples were included.
Exclusion criteria: S. aureus (Methicillin sensitive) isolated from different purulent samples were excluded.
All the samples received over a period of one year were included in the study for this reason the sample size was not calculated.
Procedure
Preliminary gram staining from was done for all 464 samples and they were inoculated on chromogenic agar and 5% sheep blood agar (biomeriux, Marcy’l Etoile, France) and Mannitol Salt Agar (MSA) and incubated in aerobic conditions at 37°C for 18-24 hours. Culture showing growth of gram positive cocci were subjected to routine biochemical testing such as catalase test, slide and tube coagulase test (Table/Fig 1). Identification and antimicrobial susceptibility was performed using Vitek 2- compact system. The gram positive panels used were identification of gram positive organism and its sensitivity panel P628. Vancomycin susceptibilty was compared by three methods Vitek 2C, E-test and BMD method.
Epsilometer-strip test (E-test): Lawn culture of the bacterial colony was done on Mueller Hinton Agar (MHA) media with density of 0.5 McFarland. Place the vancomycin E-strip and incubate for 24 hours at 37°C. The test was read by a zone of inhibition intersecting the vancomycin MIC strip at graded concentrations of antibiotic (7).
Broth microdilution test: The BMD method was used for determination of the MICs of vancomycin and vancomycin potency of 950 μg/mg was used to prepare 1 mg/mL of stock solution. Further steps are followed according to the CLSI (7).
American Type Culture Collection (ATCC) standard strains 25923 (MSSA) and 43300 (MRSA) were used as Quality Controls (QC). Antibiotic susceptibility was interpreted as per CLSI guidelines (M100, 37th edition 2020 (7) were as follows:
MIC of <2 ug/mL for Vancomycin susceptible S. aureus (VSSA)
MIC of 4-8 μg/mL for Vancomycin intermediate S. aureus (VISA)
MIC of ≥16 μg/mL for Vancomycin resistant S. aureus (VRSA)
STATISTICAL ANALYSIS
Statistical analysis of the demographic and clinical characteristics was represented as frequency and percentages.
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Results |
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Among 464 SSTIs, 132 were S. aureus of which 38 (29%) S. aureus found to be MRSA, 94 (71%) were MSSA and majority of patients were in the age group of 21-30 years as shown in (Table/Fig 2) with males:female ratio of 3.2:1. Previous history of surgeries was the major risk factor observed in 19/38 cases (50%) followed by long-term usage of antibiotics in 17/38 (44.7%) cases as shown in (Table/Fig 3) with median duration of hospital stay was observed to be 31.5 days.
The different sites and wounds from where MRSA were isolated is shown in (Table/Fig 4), (Table/Fig 5). Of the clinical samples, MRSA was predominantly isolated from wound swabs shown in (Table/Fig 5). Vancomycin susceptibility was performed by Vitek 2C, E-test and BMD. Of the 38 MRSA isolates, 26 (68%) were susceptible to vancomycin with 0.5 μg/mL of MIC by Vitek 2C and E-test.
Vancomycin MIC with Vitek 2C showed 0.5 μg/mL (68%) and 1-2 μg/mL (28.9%), E-Test showed 0.5 μg/mL (68%) and 1-2 μg/mL (18.4%) whereas with BMD 0.5 μg/mL (55.2%) and 1-2 μg/mL (5.2%) were observed. One of the isolate was resistant to vancomycin with an MIC >32 μg/mL by Vitek 2C and intermediate susceptibility to vancomycin with MIC of 8 μg/mL by E- test, which was found susceptible by BMD method with an MIC of 0.25 μg/mL as shown in the (Table/Fig 6).
Majority of the outpatients 13/38 (34.2%) were treated with oral linezolid at a dosage of 600 mg for a period of 7 -10 days. Patients who were admitted 25/38 (65.7%) were treated with intravenous vancomycin 1g for a period of 7-10 days. The wound was healthy in 36/38 (94.7%) patients at the time of discharge with an advise to follow-up. Out of two patients 2/38 (5.2%), one with Road Traffic Accident (RTA) with MRSA wound infection and sepsis and other patient with craniotomy site wound infection had a fatal outcome.
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Discussion |
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Among 464 SSTIs, 132 were S. aureus of which 38 (29%) S. aureus found to be MRSA, 94 (71%) were MSSA and majority of patients were in the age group of 21-30 years as shown in (Table/Fig 2) with males:female ratio of 3.2:1. Previous history of surgeries was the major risk factor observed in 19/38 cases (50%) followed by long-term usage of antibiotics in 17/38 (44.7%) cases as shown in (Table/Fig 3) with median duration of hospital stay was observed to be 31.5 days.
The different sites and wounds from where MRSA were isolated is shown in (Table/Fig 4), (Table/Fig 5). Of the clinical samples, MRSA was predominantly isolated from wound swabs shown in (Table/Fig 5). Vancomycin susceptibility was performed by Vitek 2C, E-test and BMD. Of the 38 MRSA isolates, 26 (68%) were susceptible to vancomycin with 0.5 μg/mL of MIC by Vitek 2C and E-test.
Vancomycin MIC with Vitek 2C showed 0.5 μg/mL (68%) and 1-2 μg/mL (28.9%), E-Test showed 0.5 μg/mL (68%) and 1-2 μg/mL (18.4%) whereas with BMD 0.5 μg/mL (55.2%) and 1-2 μg/mL (5.2%) were observed. One of the isolate was resistant to vancomycin with an MIC >32 μg/mL by Vitek 2C and intermediate susceptibility to vancomycin with MIC of 8 μg/mL by E- test, which was found susceptible by BMD method with an MIC of 0.25 μg/mL as shown in the (Table/Fig 6).
Majority of the outpatients 13/38 (34.2%) were treated with oral linezolid at a dosage of 600 mg for a period of 7 -10 days. Patients who were admitted 25/38 (65.7%) were treated with intravenous vancomycin 1g for a period of 7-10 days. The wound was healthy in 36/38 (94.7%) patients at the time of discharge with an advise to follow-up. Out of two patients 2/38 (5.2%), one with Road Traffic Accident (RTA) with MRSA wound infection and sepsis and other patient with craniotomy site wound infection had a fatal outcome.
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Conclusion |
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Vancomycin is the drug of choice in treating MRSA infections. MRSA isolates with higher and susceptible range, are to be identified which has treatment failures with vancomycin. Automated systems such as Vitek 2C and E-test do not provide precise MIC values. BMD is the preferred method which has given MIC values in a susceptible range when compared to other methods. The outcome of MRSA S. aureus infections should take into account the method used for determining the vancomycin susceptibility.
Declaration
The authors have presented the study in MICROCON 2020 https://linkinghub.elsevier.com/retrieve/pii/S0255085721043085
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| |
| | 1. | Kala Yadhav ML, Panicker GJ. Prevalence and antibiogram of methicillin resistant staphylococcus aureus isolates at a tertiary care hospital in bangalore, south india. Int J Cur Res Rev. 2014;6(17):37-40.
[ Google Scholar] | | 2. | Siddiqui AH, Koirala J. Methicillin Resistant Staphylococcus aureus. Stat Pearls Publishing; 2021.
[ Google Scholar] | | 3. | Hsu DI, Hidayat LK, Quist R, Hindler J, Karlsson A, Yusof A, et al. Comparison of method-specific vancomycin minimum inhibitory concentration values and their predictability for treatment outcome of meticillin-resistant Staphylococcus aureus (MRSA) infections. Int J Antimicrob Agents. 2008;32(5):378-85.
[ Google Scholar] | | 4. | Lohan K, Sangwan J, Mane P, Lathwal S. Prevalence pattern of MRSA from a rural medical college of North India: A cause of concern. J Family Med Prim Care. 2021;13(10):752-57.
[ Google Scholar] | | 5. | Tarai B, Das P, Kumar D. Recurrent challenges for clinicians: Emergence of methicillin-resistant Staphylococcus aureus, vancomycin resistance, and current treatment options. J Lab Physicians. 2013;5(2):71-78.
[ Google Scholar] | | 6. | Soriano A, Marco F, Martinez JA, Pisos E, Almela M, Dimova VP, et al. Influence of vancomycin minimum inhibitory concentration on the treatment of methicillin-resistant Staphylococcus aureus bacteremia. Clin Infect Dis. 2008;46(2):193-200.
[ Google Scholar] | | 7. | CLSI ref. CLSI. Performance Standards for Antimicrobial Susceptibility Testing. 31st ed. CLSI supplement M100. Clinical and Laboratory Standards Institute; 2021.
[ Google Scholar] | | 8. | Sathish JV, Wadekar MD. Prevalence of MRSA in clinical samples and their antibiotic sensitivity pattern. Int J Curr Microbiol Appl sci. 2017;6(7):2338-43.
[ Google Scholar] | | 9. | Abdullahi N, Iregbu KC. Methicillin-Resistant Staphylococcus aureus in a Central Nigeria Tertiary Hospital. Ann Trop Pathol. 2018;9(1):6-10.
[ Google Scholar] | | 10. | Kumari N, Mohapatra TM, Singh Y. Prevalence of Methicillin-Resistant Staphylococcus aureus (MRSA) in a tertiary-care hospital in eastern Nepal. J Nepal Med Assoc. 2008;47(170):53-56.
[ Google Scholar] | | 11. | Adhikari R, Pant ND, Neupane S, Neupane M, Bhattarai R, Bhatta S, et al. Detection of Methicillin Resistant Staphylococcus aureus and Determination of minimum inhibitory concentration of vancomycin for Staphylococcus aureus isolated from pus/wound swab samples of the patients attending a tertiary care hospital in Kathmandu, Nepal. Can J Infect Dis Med Microbiol. 2017;2017:219153.
[ Google Scholar] | | 12. | Reema HA, Saldanha Dominic RM. Prevalence and antimicrobial susceptibility pattern of clinical isolates of Methicillin-resistant Staphylococcus aureus in a tertiary care hospital in Mangalore. J Int Med Dent. 2016;3(3):134-39.
[ Google Scholar] | | 13. | Maina EK, Kiiyukia C, Njeri Wamae C, Waiyaki PG, Kariuki S. Characterization of methicillin-resistant Staphylococcus aureus from skin and soft tissue infections in patients in Nairobi. Kenya. Int J Infect. 2013;17(2)115-19.
[ Google Scholar] | | 14. | Kshetry AO, Pant ND, Bhandari R, Khatri S, Shrestha KL, Upadhaya SK, et al. Minimum inhibitory concentration of vancomycin to methicillin resistant Staphylococcus aureus isolated from different clinical samples at a tertiary care hospital in Nepal. Antimicrob Resist Infect Control. 2016;21(5):27.
[ Google Scholar] | | 15. | Gupta S, Mishra B, Thakur A, Dogra V, Loomba PS, Jain M, et al. Risk factors associated with MRSA. Southern African Journal of Infectious Diseases. 2018;33(3):76-79.
[ Google Scholar] | | 16. | Dubert M, Pourbaix A, Alkhoder S, Mabileau G, Lescure F-X, Ghodhbane W, et al. Sternal Wound Infection after Cardiac Surgery: Management and Outcome. PLoS ONE. 2015;10(9):0139122.
[ Google Scholar] | | 17. | Jiménez-Martínez E, Cuervo G, Hornero A, Ciercoles P, Gabarrós A, Cabellos C, et al. Risk factors for surgical site infection after craniotomy: a prospective cohort study. Antimicrob Resist Infect Control. 2019;8:69. DOI https://doi.org/10.1186/ s13756-019-0525-3
[ Google Scholar] | | 18. | Mehta M, Dutta P, Gupta V. Antimicrobial susceptibility pattern of blood isolates from a teaching hospital in north India. Jpn J Infect Dis. 2005;58(3):174-76.
[ Google Scholar] | | 19. | Ranjan K, Arora D, Ranjan N. An Approach to Linezolid and Vancomycin against Methicillin Resistant Staphylococcus aureus. Webmed Central MICROBIOLOGY. 2010;1(9):WMC00590.
[ Google Scholar] | | 20. | Mouton JW, Jansz AR. The DUEL study: A multi-center in vitro evaluation of linezolid compared with other antibiotics in the Netherlands. Clin Microbiol Infect. 2001;7(9):486-91.
[ Google Scholar] | | 21. | Sader HS, Rhomberg PR, Jones RN. Nine-hospital study comparing broth microdilution and Etest method results for vancomycin and daptomycin against methicillin-resistant Staphylococcus aureus. Antimicrob Agents Chemother. 2009;53(7):3162-65.
[ Google Scholar] | | 22. | 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):453-55.
[ Google Scholar] | | 23. | Rossatto FC, Proença LA, Becker AP, Silveira AC, Caierão J, D'Azevedo PA. Evaluation of methods in detecting vancomycin MIC among MRSA isolates and the changes in accuracy related to different MIC values. Rev Inst Med Trop Sao Paulo. 2014;56(6):469-72.
[ Google Scholar] | | 24. | Himani CA, Madan M, Pandey A, Thakuria B. Detection of Vancomycin Susceptibility in MRSA isolates by micro broth dilution, E Test and Vitek 2 methods. J Clin Diagn Res. 2015;9(10):01-04.
[ Google Scholar] | | 25. | Anitha TK, Rao MR, Shankaregowda R, Mahale RP. Evaluation of vancomycin minimum inhibitory concentration in the clinical isolates of Methicillin Resistant Staphylococcus aureus (MRSA). J Pure Appl Microbiol. 2019;13(3):1797- 801.
[ Google Scholar] | | 26. | Choo EJ, Chambers HF. Treatment of Methicillin-Resistant Staphylococcus aureus Bacteremia. Infect Chemother. 2016;48(4):267-73.
[ Google Scholar] | | 27. | Delaney JA, Schneider-Lindner V, Brassard P, Suissa S. Mortality after infection with methicillin-resistant Staphylococcus aureus (MRSA) diagnosed in the community. BMC Med. 2008;31(6):2. [ Google Scholar]
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