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 Table of Contents  
ORIGINAL ARTICLE
Year : 2017  |  Volume : 20  |  Issue : 2  |  Page : 196-200

Comparison of conventional and rapid methods for the detection of methicillin resistance in Staphylococcus aureus from blood cultures


Department of Microbiology, University College of Medical Sciences, Guru Teg Bahadur Hospital, New Delhi, India

Date of Web Publication14-Nov-2017

Correspondence Address:
Ashwani Kumar
Department of Microbiology, University College of Medical Sciences, Guru Teg Bahadur Hospital, New Delhi - 110 095
India
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DOI: 10.4103/tjmr.tjmr_48_16

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  Abstract 

Background: Considering the increasing rate of infections caused by methicillin-resistant Staphylococcus aureus (MRSA), reliable, accurate, and rapid testing for the detection of MRSA is essential for both initiation of antimicrobial therapy and infection control measures. Materials and Methods: Fifty clinical isolates of S. aureus from patients with bacteremia were confirmed bacteriologically and subjected to antimicrobial susceptibility testing, followed by the detection of methicillin resistance by oxacillin and cefoxitin disc diffusion methods, oxacillin agar screen test, and direct cefoxitin disc diffusion method from blood culture bottles. Results: On comparing the methods under study, 36% MRSA were detected by disc diffusion methods, whereas 44% and 46% were detected by oxacillin agar screen and direct cefoxitin disc diffusion blood culture, respectively. Conclusions: The emergence of MRSA underscores the need for programs to prevent the spread of antimicrobial-resistant organisms and control the use of antimicrobial drugs in health-care settings.

Keywords: Blood culture bottles, cefoxitin disc diffusion, methicillin-resistant Staphylococcus aureus, oxacillin agar screen test


How to cite this article:
Kumar A, Kashyap B, Mehndiratta A. Comparison of conventional and rapid methods for the detection of methicillin resistance in Staphylococcus aureus from blood cultures. Trop J Med Res 2017;20:196-200

How to cite this URL:
Kumar A, Kashyap B, Mehndiratta A. Comparison of conventional and rapid methods for the detection of methicillin resistance in Staphylococcus aureus from blood cultures. Trop J Med Res [serial online] 2017 [cited 2018 Dec 14];20:196-200. Available from: http://www.tjmrjournal.org/text.asp?2017/20/2/196/218218


  Introduction Top


Methicillin-resistant Staphylococcus aureus (MRSA) is an important etiological agent of hospital- and community-acquired infections. The first case of MRSA was reported in 1961.[1] The importance of MRSA as a nosocomial as well as a community-acquired pathogen is well documented. Emergence of MRSA worldwide has led to the overuse of glycopeptides antibiotics and to the emergence of vancomycin-resistant S. aureus. A distinctive feature of methicillin resistance is its heterogeneous nature, with the level of resistance varying according to the culture conditions and the β-lactam antibiotics being used. Methicillin resistance in S. aureus is based on the production of an additional penicillin-binding protein (PBP), PBP2 or PBP2a, which is mediated by the mecA gene. MRSA strains are frequently resistant to many different classes of antibiotics. Considering the increasing rate of infections caused by MRSA, reliable, accurate, and rapid testing for the detection of MRSA is essential for both antibiotic therapy and infection control measures. There are many traditional and commercial systems for the detection of MRSA in clinical microbiology laboratories. Most laboratories use disc diffusion methods for routine testing. The gold standard method for antimicrobial susceptibility testing has been the minimum inhibitory concentration (MIC) test determined by dilution methods. In the recent years, MIC methods have been replaced by molecular methods which detect mecA gene as a gold standard for determining classical methicillin resistance in S. aureus. However, the use of molecular methods for detection of MRSA is largely restricted to reference laboratories and is not utilized in many microbiology laboratories as a routine test.[1]

Conventional methods for the detection of MRSA include oxacillin disc diffusion, oxacillin MIC, and oxacillin screen agar methods. Recently, the Clinical and Laboratory Standards Institute (CLSI) recommended the use of cefoxitin disc diffusion method for MRSA detection (CLSI, 2008). Cefoxitin is a cephamycin-type antibiotic and has been described as an inducer of the PBP2a-encoding mecA gene. Various other methods for the detection of MRSA have been developed. The latex agglutination assay, developed by Denka Seiken Co., Japan, makes use of specific monoclonal antibodies directed toward the PBP2a antigen. In addition, CHROMagar™ is another method which utilizes a chromogenic medium for the identification of MRSA.[2]

The incidence of S. aureus bloodstream infections has significantly increased over the past few decades with tendency of these organisms to develop resistance to multiple antibiotics. The National Nosocomial Infections Surveillance System report states that >55% of the S. aureus isolates are resistant to methicillin, oxacillin, or nafcillin.[3] Strict clinical criteria and serial blood cultures are most important in sorting out these patients with septicemia, from cases of sample contamination, especially under situations such as prior antibiotic exposure, insulin-dependent diabetes, prolonged hospitalization with an underlying disease, urinary catheterization, and prior surgery.[4] Rapid characterization of staphylococci isolates from blood cultures as being methicillin resistant or sensitive is an important factor in the prompt and accurate treatment of bacteremia patients as the increase in Gram-positive organisms that are resistant to vancomycin threatens many accepted treatment regimens and justifies the empirical use of glycopeptides for bloodstream infections.[5]

Conventional methods of antibiotic resistance detection include the normal disc diffusion technique and oxacillin agar screen plate with newer methods rapidly emerging which include the direct cefoxitin disc diffusion test from blood culture bottles. The normal disc diffusion technique helps in detection of resistance by diffusing into the media, on which culture has been done. Estimation of the zone of inhibition gives an idea of the resistance or sensitivity. Some of the latest methods in MRSA detection are the polymerase chain reaction (PCR). Oxacillin screen agar involves oxacillin incorporation into the growth media at 6 μg/ml level and detection of any growth indicates resistance.

The newer method of direct cefoxitin disc diffusion method from blood culture bottles involves direct inoculation from broth saving the time that normally gets used up in culture and then subculture. Done after a presumptive Gram stain, it is a much faster and much affordable detection method in resource-constraint settings. Comparison however is needed for the assessment of the accuracy of these methods in MRSA detection to know the best method suitable to our setting where resources are limited. Skilled labor and availability of qualified doctors as a constraint add to the need for further research on such methods since the workload on health sector and the hospital setting are an ever increasing matter under which scenario rapidity of work is expected to help in a great deal.

In response to this concern, the CLSI has lowered the susceptibility and resistance breakpoints of vancomycin against S. aureus.[6] Compared with infections due to methicillin-susceptible S. aureus, infections due to MRSA are associated with poor clinical outcomes and increased healthcare costs.[7] Methicillin resistance is also widespread among coagulase-negative staphylococci commonly associated with human infections.[8]


  Materials and Methods Top


Place of work

The study was conducted from January 2015 to March 2015 in the Department of Microbiology, University College of Medical Sciences, Guru Teg Bahadur Hospital, Delhi.

Specimen

Fifty blood culture specimens from patients with bacteremia were included in the study.

Blood culture

All samples were inoculated in Bactec 9120 system (Becton Dickinson). A positive reading indicated the presumptive presence of viable organisms.

Microbiological processing

Isolation and identification

The organisms were isolated from the clinical specimen onto blood agar and MacConkey agar and incubated at 37°C after 24 h. On Gram staining, Gram-positive cocci about 0.5–1.0 μm in diameter in clusters, pairs, and occasionally short chains were noted.[9] This was differentiated from other Gram-positive cocci on the basis of motility, growth on NaCl agar, catalase, benzidine test, anaerobic acid from glucose, lysostaphin (200 μg/ml), bacitracin (0.04 disc). Species of coagulase-negative staphylococci were differentiated on the basis of colony pigment, staphylcoagulase, clumping factor, heat-stable nuclease, alkaline phosphatase, pyrrolidonyl arylamidase, ornithine decarboxylase, urease, B-galactosidase, acetoin production, novobiocin resistance, polymixin B resistance, acid aerobically from D-trehalose, D-mannitol, D-mannose, D-turanose, D-xylose, D-cellobiose, maltose, sucrose.[10]

Antimicrobial susceptibility pattern

Disc diffusion susceptibility by Kirby–Bauer method was performed on Mueller-Hinton agar (MHA) according to the recommendations of the CLSI (2013), with the following antibiotics: oxacillin - 1 μg, vancomycin - 30 μg, gentamicin - 10 μg, clindamycin - 2 μg, erythromycin 1–15 μg, co-trimoxazole - 25 μg, teicoplanin - 30 μg, amoxicillin - 10 μg, cephalexin - 30 μg, ciprofloxacin - 5 μg, rifampicin - 5 μg, tetracycline - 30 μg, linezolid - 30 μg, amoxicillin/clavulanic acid - 30 μg. Quantity control was achieved using standard strains of S. aureus ATCC 25923 (mecA negative) and S. aureus ATCC 43300 (mecA positive).[11]

Detection of methicillin resistance

Conventional disc diffusion method

All strains were tested with 1 μg oxacillin discs and 30 μg cefoxitin discs (Hi-Media) on MHA plates, for which a bacterial suspension adjusted to 0.5 McFarland was used. The zones of inhibition were interpreted according to the CLSI criteria

Oxacillin agar screen test

Inoculums' suspension was prepared by selecting colonies from overnight growth on MHA. The colonies were transferred to saline to produce turbidity matching 0.5 McFarland suspensions. Using calibrated loop, the suspension was spot inoculated on oxacillin agar screen plate consisting of MHA incorporating 6 μg/ml of oxacillin (Hi-Media) with 4% NaCl and incubated exactly for 24 h at 35°C. Growth in the inoculation area was considered to be resistant to methicillin.[12]

Direct cefoxitin disc diffusion test from blood culture bottles

Samples of blood cultures showing “Gram-positive cocci in clusters” on Gram staining were soaked on swab from the blood culture bottle using the same technique one would use in the preparation of a disc diffusion susceptibility test.[4],[11] The swab was then used to streak the entire surface of 100 mm tryptic soy blood agar plate, and subsequently, a cefoxitin disc was placed at the center of the plate and the plates were incubated overnight at 35°C in ambient air. The next day, cefoxitin zone size was measured and recorded.[2],[4],[11]


  Results Top


The antimicrobial susceptibility to various antibiotics is given in [Table 1]. The results of the three methods used to detect methicillin resistance are given in [Table 2]. Comparison of methods for detection of MRSA in the strains included in the study is given in [Table 3].
Table 1: Antimicrobial susceptibility pattern of the isolates under study (n=50)

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Table 2: Comparison of various methods used for detection of methicillin resistance

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Table 3: Frequency of methicillin sensitive and resistant strains of Staphylococcus aureus

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Of the 50 samples tested for MRSA by the above-said methods of detection, following observations were made. When tested using the conventional disc diffusion method, 18 out of 50 samples came out to be MRSA. With oxacillin screen method, 22 of 50 samples were MRSAs. In direct cefoxitin disc diffusion technique from broth bottles, 23 of 50 tested samples came out to be MRSAs. Percentage detection for MRSA by conventional disc diffusion method came out to be 36%, whereas it was 46% by the direct cefoxitin disc diffusion technique. Fifteen samples finally report as MRSA were detected by both conventional disc diffusion method and direct cefoxitin disc diffusion technique from broth bottles. However, eight samples that were MRSA by direct cefoxitin method were reported as sensitive by the conventional disc diffusion method.


  Discussion Top


The accurate and early determination of methicillin resistance is of key importance in the prognosis of infections caused by S. aureus.

A study comparing the methods for detection of methicillin resistant showed that the oxacillin disc diffusion test which was earlier used routinely is showing low specificity (56%), and among all phenotypic methods, cefoxitin disc diffusion and PCR alone have similar sensitivity and specificity.[10] In another study, the comparison between cefoxitin disc diffusion test, oxacillin screen agar, and PCR for the detection of MRSA showed that the direct cefoxitin disc diffusion test from blood culture bottles can be an alternative to PCR for detection of MRSA in resource-constraint settings.[9]

Our study has laid emphasis on the fact that the cefoxitin disc diffusion test can be the best method for routine detection of MRSA where molecular techniques are not available.[8] Direct cefoxitin disc diffusion technique from broth bottles is a much faster technique as time for reporting of MRSA resistance with direct cefoxitin method was about half the time taken by the conventional disc diffusion method, which takes a minimum of 2 days involving subculture from broth and subsequent disc diffusion. Media usage was also very nominal in terms of quantity showing very bright prospects of this method in the future. McNemar's test was used to compare the methods. There was no significant difference among the methods for testing methicillin resistance [Table 4].
Table 4: Statistical evaluation among various methods for detection of methicillin resistance

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  Conclusions Top


The emergence of methicillin resistance among S. aureus underscores the need for programs to prevent the spread of antimicrobial-resistant microorganisms and control the use of antimicrobial drugs in health-care setting. Although the numbers of isolates are less, this study provides evidence that direct cefoxitin testing can be used as an accurate surrogate marker in routine susceptibility testing. In addition, the results of various studies have shown 100% sensitivity and specificity as compared to mecA gene detection by PCR. Hence, it can be used as an alternative to the technically demanding PCR where financial constraints are existing.

Financial support and sponsorship

Nil.

Conflicts of interest

There are no conflicts of interest.

 
  References Top

1.
Odonkor Stephen T, Addo Kennedy K. Evaluation of three methods for detection of methicillin resistant Staphylococcus aureus (MRSA). Int J Biol Med Res 2011;2:1031-4.  Back to cited text no. 1
    
2.
Datta P, Gulati N, Singla N, Rani Vasdeva H, Bala K, Chander J, et al. Evaluation of various methods for the detection of meticillin-resistant Staphylococcus aureus strains and susceptibility patterns. J Med Microbiol 2011;60(Pt 11):1613-6.  Back to cited text no. 2
    
3.
National Nosocomial Infections Surveillance (NNIS) system report, data summary from January 1992-June 2001, issued August 2001. Am J Infect Control 2001;29:404-21.  Back to cited text no. 3
    
4.
Bennett K, Sharp SE. Rapid differentiation of methicillin-resistant Staphylococcus aureus and methicillin-susceptible Staphylococcus aureus from blood cultures by use of a direct cefoxitin disk diffusion test. J Clin Microbiol 2008;46:3836-8.  Back to cited text no. 4
[PUBMED]    
5.
Lee YC, Wu F, Dutta S, Della-Latta P. Rapid Detection of Methicillin-Resistant Staphylococcus aureus Directly from Blood Cultures Using Multiplex PCR. Abstract Interscience Conference on Antimicrobial Agents and Chemotherapy; 2000.  Back to cited text no. 5
    
6.
Tenover FC, Moellering RC Jr. The rationale for revising the Clinical and Laboratory Standards Institute vancomycin minimal inhibitory concentration interpretive criteria for Staphylococcus aureus. Clin Infect Dis 2007;44:1208-15.  Back to cited text no. 6
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7.
Cosgrove SE, Qi Y, Kaye KS, Harbarth S, Karchmer AW, Carmeli Y. The impact of methicillin resistance in Staphylococcus aureus bacteremia on patient outcomes: Mortality, length of stay, and hospital charges. Infect Control Hosp Epidemiol 2005;26:166-74.  Back to cited text no. 7
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8.
Hussain Z, Stoakes L, Massey V, Diagre D, Fitzgerald V, El Sayed S, et al. Correlation of oxacillin MIC with mecA gene carriage in coagulase-negative staphylococci. J Clin Microbiol 2000;38:752-4.  Back to cited text no. 8
[PUBMED]    
9.
Huebner J, Goldmann DA. Coagulase-negative staphylococci: Role as pathogens. Annu Rev Med 1999;50:223-36.  Back to cited text no. 9
[PUBMED]    
10.
Connie RM, Donald CL, Manuselis G. Text Book of Diagnostic Microbiology. 3rd ed. St. Louis, Missouri: Saunders: Imprint of Elsevier; 2007. p. 367-81.  Back to cited text no. 10
    
11.
CLSI. Performance Standards for Antimicrobial Susceptibility Testing, 15th Informational Supplement, M100-S15. Wayne, PA: Clinical and Laboratory Standards Institute; 2008.  Back to cited text no. 11
    
12.
Swenson JM, Williams PP, Killgore G, O'Hara CM, Tenover FC. Performance of eight methods, including two new rapid methods, for detection of oxacillin resistance in a challenge set of Staphylococcus aureus organisms. J Clin Microbiol 2001;39:3785-8.  Back to cited text no. 12
    



 
 
    Tables

  [Table 1], [Table 2], [Table 3], [Table 4]



 

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