| Abstract|| |
Aims: Staphylococcus aureus is a common pathogen causing a wide range of infections ranging from mild skin and soft tissue infections to severe, life-threatening infections. Accuracy in the detection of methicillin resistance is important to avoid treatment failures. The aim of this study was to compare the results of phenotypic and genotypic test methods to detect methicillin resistance and also to determine the antimicrobial susceptibilities. Materials and Methods: Two hundred and forty-two S. aureus strains isolated from skin and soft tissue samples were analyzed for methicillin resistance using oxacillin and cefoxitin disk diffusion (DD), oxacillin screen agar test, cefoxitin E-test, and mecA gene polymerase chain reaction (PCR). Results: 77 of 242 S. aureus isolates were mecA positive. Oxacillin, cefoxitin DD, oxacillin screen agar test and cefoxitin E-test exhibited sensitivities as 98.7%, 98.7%, 100%, 100%, and specificities as 96.9%, 97.5%, 96.9%, 97.5%, respectively. Conclusion: Results of oxacillin screen agar and cefoxitin DD test were in concordance with mecA gene PCR. Thus, it is determined that especially cefoxitin test can be an alternative to PCR in routine.
Keywords: Cefoxitin, mecA, methicillin-resistant Staphylococcus aureus, oxacillin screen
|How to cite this article:|
Demir T, Coplu N, Esen B. Comparative analysis of phenotypic and genotypic detection of methicillin resistance among Staphylococcus aureus. Indian J Pathol Microbiol 2016;59:314-7
|How to cite this URL:|
Demir T, Coplu N, Esen B. Comparative analysis of phenotypic and genotypic detection of methicillin resistance among Staphylococcus aureus. Indian J Pathol Microbiol [serial online] 2016 [cited 2019 Jun 19];59:314-7. Available from: http://www.ijpmonline.org/text.asp?2016/59/3/314/188103
| Introduction|| |
Methicillin-resistant Staphylococcus aureus (MRSA) has become an increasingly extensive pathogen worldwide with a prevalence varying throughout the countries in Europe, between <3 and over 70% according to the patient group and hospital settings.,,, Furthermore, MRSA strains cause therapeutic problems because of their ability to develop resistance to several classes of antimicrobials and MRSA is considered to have emerged from S. aureus through the acquisition of staphylococcal cassette chromosome mec, which carries the mecA gene for methicillin resistance.,,, The accurate and rapid determination of methicillin resistance has vital importance in the prognosis and control of infections, especially in hospital settings. Detection of resistance with phenotypic test methods such as broth dilution and disk diffusion (DD) testing require at least 24 h to perform, and difficulties can be seen in differentiating MRSA and borderline oxacillin-resistant S. aureus isolates due to heterogeneous expression in many strains.,,,, Moreover, phenotypic expression of methicillin resistance by using oxacillin DD test may change with environmental modifications such as incubation temperature or NaCl content of the medium, and this may affect the accuracy of the test method leading to misclassification of the resistant isolates as susceptible to methicillin.,,, Cefoxitin is a potent inducer of the mecA regulatory system ,, and was shown to be superior to oxacillin especially in low-level methicillin-resistant strains. CLSI has recommended cefoxitin DD method for the detection of methicillin resistance. Cefoxitin test results are easier to interpret, they do not require special requirements and are thus more sensitive for the detection of mecA mediated resistance than oxacillin results.,,, Oxacillin screen agar is also useful to detect methicillin resistance, as an additional test that can be used to confirm indeterminate results , although many borderline resistant strains will also grow on this medium. The gold standard for methicillin resistance detection is mecA polymerase chain reaction (PCR) analysis with a sensitivity of 100%.,
In this study, we aimed to compare the results of the tests used for methicillin resistance detection and to determine the antimicrobial susceptibilities to selected antimicrobials in S. aureus strains.
| Materials and Methods|| |
During the study, a total of 242 S. aureus strains isolated from skin and soft tissue infections from both outpatient and inpatients were included in this study. Isolates were identified on the basis of the colony and microscopic morphology, catalase, tube coagulase testing with rabbit plasma, deoxyribonuclease agar, and growth on mannitol salt agar. All isolates were stored in 16% glycerol broth at –80°C until use and were cultured twice onto brain heart infusion agar before incubation overnight at 37°C.
Phenotypic tests for methicillin resistance detection
Methicillin resistance was determined by Kirby–Bauer DD method using oxacillin 1 μg (Oxoid, England), cefoxitin 30 μg (Oxoid, England), and confirmed by oxacillin screen agar test supplemented with 4% NaCl and 6 μg/μl oxacillin (Sigma-Aldrich, USA) and cefoxitin E-test (AB Biodisc, Solna, Sweden) according to CLSI guidelines  and the manufacturer's instructions.
Detection of the mecA gene
Strains were grown on brain heart infusion agar at 37°C overnight and streaked twice from freezer storage prior testing. The mecA gene was detected by PCR as described by Murakami et al.S. aureus ATCC 43300 and S. aureus ATCC 25923 were used as positive and negative controls for PCR assay, respectively.
Antimicrobial susceptibility testing
Susceptibilities to antimicrobials including penicillin (P, 10 μg), gentamicin (GEN, 10 μg), tetracycline (TE, 30 μg), erythromycin (E, 15 μg), rifampin (RIF, 5 μg), mupirocin (MUP, 5 μg), clindamycin (CL, 2 μg), trimethoprim/sulfamethoxazole (SXT, 1.25/23.75 μg), linezolid (LIN, 30 μg), and quinupristin-dalfopristin (QD, 15 μg), teicoplanin (TEC, 30 μg) (Oxoid, England) were determined based on the guidelines of the CLSI. For fusidic acid (FA, 10 μg), zone diameter breakpoints of EUCAST. were used. S. aureus ATCC 25923 and S. aureus ATCC 43300 were used as susceptible and resistant control strains, respectively.
Comparisons were performed with SPSS software version 15.0 (SPSS, Inc., Chicago, IL, USA)using the Chi-square test or the Fisher's exact test. All hypotheses were two-tailed and were considered significant at the P < 0.05 level.
| Results|| |
Among 242 S. aureus isolates tested, 77 (31.8%) were mecA positive. Oxacillin was resistant not only in 76 mecA-positive isolates but also in five mecA-negative isolates. Much better results were obtained with the cefoxitin disk, giving four of the isolates as false resistant. All mecA positive and also five mecA negative isolates were resistant on oxacillin screen agar test [Table 1]. In six isolates discrepant results were observed at least in one test method [Table 2]. All but one isolates with differing results were resistant to methicillin by cefoxitin E-test with MIC values ranging between 4 and 256 mg/L. False resistance was detected for four isolates with cefoxitin E-test. Overall for the isolates tested, the highest sensitivity and specificity rates were observed with oxacillin screen agar/cefoxitin E-test and cefoxitin disk test/cefoxitin E-test, respectively [Table 3].
|Table 1: Results of the phenotypic tests of strains and correlation with mecA|
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|Table 2: Discrepant results between the phenotypic and genotypic test methods for methicillin detection|
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|Table 3: Evaluation of phenotypic test methods with mecA analysis by PCR|
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All isolates were susceptible to TEC, QD, and LIN. A total of 12 (4.9%) S. aureus isolates were susceptible to all antimicrobials tested. Of the 242 S. aureus isolates, 93.8% presented resistance to P, 46.7% to TE, 38.4% to RIF, 31.8% to GEN, 30.2% to E, 11.6% to CL, 2.5% to FA, 2.1% to MUP and 0.8% to SXT. Five (2%) isolates showed high-level MUP resistance. mecA positive strains were more resistant to all antimicrobials, except MUP, than the mecA negative isolates and were significantly more resistant to ERY, CLI, TET, GEN, and RIF compared to negative strains (P = 0.001) [Table 4].
|Table 4: Evaluation of the antimicrobial susceptibilities of methicillin resistant and susceptible S.aureus isolates (n=242)|
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| Discussion|| |
MRSA strains are responsible for a large proportion of infections both in hospitals and community settings with an increasing trend in antimicrobial resistance patterns. The accurate and rapid determination of methicillin resistance is crucial, especially in hospital environment. Phenotypic tests require modifications in inoculum size, NaCl concentration, medium contents, and incubation temperature to reach the required levels of sensitivity and specificity.,,, CLSI recommends mecA gene PCR analysis as the gold standard for MRSA diagnosis  but a limited number of laboratories can afford this method. Thus phenotypic susceptibility testing such as oxacillin and cefoxitin DD tests are still in use in most of the laboratories despite the inconsistent results.
In this study, we compared the results of oxacillin, cefoxitin DD and oxacillin screen agar test with mecA gene PCR analysis in 242 S. aureus strains isolated from skin and soft tissue infections. 77 (31.8%) isolates were mecA gene positive. Oxacillin and cefoxitin DD tests showed similar sensitivity results (98.7%), but specificity was higher in cefoxitin DD test (96.9% vs. 97.5%), consistent with the reports published previously.,,,,,, Several reports indicate that oxacillin often failed to detect low-level heterogeneous MRSA populations  and also should not be used in methicillin resistance detection due to lower specificity test results as 56–62%., Several reports indicate that cefoxitin disk is superior to oxacillin with a sensitivity 94–100% and specificity 96–100%,,,,,, and the former can be used as an alternative to PCR. Cefoxitin disk was shown to be useful even in low-level heterogeneous strains.
The use of oxacillin screen agar test containing 6 μg of oxacillin per ml, is useful for identifying MRSA, although many borderline resistant strains will also grow on this medium., The presence of resistance on an oxacillin screen agar generally means that the isolate is mecA positive. In this study, oxacillin screen agar test showed the most highest sensitivity (100%) with similar specificity with the other methods (96.9%). Oxacillin salt screen agar test can be used as a surrogate method to PCR with great sensitivity of 83–100% and specificity 85–100% reported in several studies,,,,,, despite some reports indicating lower specificity results as 62–75%.,
Six strains had discrepant results between at least one of the methods. All were resistant with FOX E-test. However, among the five mecA negative isolates giving false resistance with oxacillin screen agar and oxacillin disk test, one was susceptible to cefoxitin. Cefoxitin could detect only mecA-mediated resistance, however, non-mecA-mediated resistance is a rare occurrence, as evidenced by our study and this could be the reason for the varying results. Furthermore, discrepancies could be explained with a technical problem in PCR such as false colony selection for DNA extraction because of the mixture of MRSA and methicillin sensitive S. aureus isolates instead of a single colony in the first culture plate, loss or mutation in the gene. Technical problem could not be the reason due to using positive and negative control in each run, repeated DNA extraction and retesting. In some instance, mecA gene can be detected, while methicillin susceptible as phenotypically, representing heteroresistance, as shown in our study. This strain also showed growth on oxacillin screen test, probably due to hyperproduction of β-lactamase or altered ability to bind penicillin binding proteins. It is reported that this strains have the potential to become highly resistant if exposed to antistaphylococcal penicillins.
Empiric therapy options in S. aureus associated infections is likely to be related with MRSA prevalence in the community, risk factors, type of clinical lesion and severity. In this study, 93.8% of all S. aureus strains were resistant to penicillin. Glycopeptide and linezolid resistance was not detected. Although resistance to ERY and CLI is mediated by a similar mechanism, resistance rates showed the difference in MRSA isolates 63% and 28%, respectively. The low resistance rates for CLI could be the result of rare prescription of this drug. MRSA strains recovered from inpatients are often resistant to a wide range of antimicrobial agents including TE, macrolide, and aminoglycoside.,, In this study, overall among the antimicrobials tested, MRSA strains were more resistant to the majority of available antimicrobials tested except MUP, FA, and SXT leaving a limited choice for treatment. In contrast to increasing resistance to MUP and FA,,,, we found lower resistance rates both in resistant and susceptible strains as 1.3% versus 2.4% for MUP, 5.2% versus 1.2% for FA, respectively.
| Conclusion|| |
This study supports the evidence that either cefoxitin DD or oxacillin screen agar can be used as accurate surrogate markers for routine susceptibility testing instead of molecular detection methods and E-test for methicillin resistance. Appropriate use of antimicrobials, both in hospitals and outpatient settings, could help control the emergence of resistant strains and limit the acquisition of additional antimicrobial resistance genes in existing strains.
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Conflicts of interest
There are no conflicts of interest.
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Dr. Tulin Demir
Public Health Institution, National Microbiology Reference Laboratories, Ankara
Source of Support: None, Conflict of Interest: None
[Table 1], [Table 2], [Table 3], [Table 4]