|Year : 2022 | Volume
| Issue : 1 | Page : 38-40
Discrepancy in methicillin sensitivity of Staphylococcus aureus between molecular and phenotypic assays
Pradnya Laxman Samant, Gaurav V Salunke, Sanjay K Biswas
Department of Microbiology, Tata Memorial Hospital, Homi Bhabha National Institute, Mumbai, Maharashtra, India
|Date of Submission||13-Mar-2022|
|Date of Acceptance||12-Apr-2022|
|Date of Web Publication||11-Jul-2022|
Pradnya Laxman Samant
Department of Microbiology, Tata Memorial Hospital, Homi Bhabha National Institute, Mumbai 400 012, Maharashtra
Source of Support: None, Conflict of Interest: None
Since the emergence of MRSA six decades ago, it continues to reign as a threatening pathogen in the health-care and community settings. Correct identification of Staphylococcus aureus as MRSA/MSSA is important to aid in antimicrobial stewardship. In the present study, S. aureus from a joint aspirate was identified as MRSA by molecular method; however, phenotypic method confirmed it as MSSA.
Keywords: Methicillin-resistant Staphylococcus aureus, molecular assays, methicillin-sensitive Staphylococcus aureus, phenotypic methods
|How to cite this article:|
Samant PL, Salunke GV, Biswas SK. Discrepancy in methicillin sensitivity of Staphylococcus aureus between molecular and phenotypic assays. J Acad Clin Microbiol 2022;24:38-40
|How to cite this URL:|
Samant PL, Salunke GV, Biswas SK. Discrepancy in methicillin sensitivity of Staphylococcus aureus between molecular and phenotypic assays. J Acad Clin Microbiol [serial online] 2022 [cited 2022 Aug 11];24:38-40. Available from: https://www.jacmjournal.org/text.asp?2022/24/1/38/350317
| Introduction|| |
Methicillin-resistant Staphylococcus aureus (MRSA) is one of the leading causes of hospital-acquired infections globally. These infections have caused increased duration of stay in the hospital with an apparent increase in morbidity, and thus, inadvertently put an additional burden on the health-care system. The detection of MRSA warrants for the implementation of contact isolation protocols to prevent potential outbreaks. Hence, the early detection of MRSA is essential. Real-time polymerase chain reaction and molecular methods are today preferred over conventional culture methods and susceptibility testing due to their short turn-around time. Molecular methods directly and correctly identify the organism and their resistant genes from the patient sample within a short span of time. These data thus guide the clinicians to start appropriate antimicrobial therapy. Nevertheless, interpretation of these results should be done with caution, especially in cases of a polymicrobial infection.
| Case Report|| |
A 54-year-old female patient, a case of metastatic chondroma of the right proximal femur, underwent a wide excision of the right proximal femur, with replacement and meshplasty and embolectomy from right superficial femoral artery. The patient was discharged on day five post-operatively. The patient subsequently came back on day 20, with complaints of continuous pain at the operated site and fever spikes for the past two days. Complete blood count revealed leucocystosis (with relative neutrophilia). Local examination showed evidence of flap necrosis for which she was taken to the operation theatre and debridement with meshplasty secondary to flap necrosis was performed. During the procedure, aspirate from the right hip joint was collected aseptically into BacT/Alert bottle and sent to the laboratory for microbiological investigations.,
The culture bottle flashed positive after six hours of incubation. 200 μL from the culture bottle was run on the Biomérieux Blood Culture Identification Syndromic panel (BioFire) targeting a set of Gram-positive bacteria, Gram-negative bacteria, yeasts and a set of antimicrobial genes. The syndromic panel detected the presence of Enterococcus faecalis, Acinetobacter baumannii and S. aureus. New Delhi Metallo-ß lactamase, mecA/mecC and mec right-extremity junction (MREJ) resistance genes were also detected, suggesting a multidrug-resistant (MDR) strain of Acinetobacter and MRSA strain.
The patient's sample was simultaneously plated on 5% sheep blood agar plate and MacConkey agar plate for the identification and antimicrobial susceptibility testing (AST). The culture plates showed growth of organisms (Gram-positive as well as Gram-negative organisms) after 24 h of incubation. The colonies were further processed for identification and AST by Vitek 2 Compact (Biomérieux, Marcy I'Etoile, France) automated system and also manually in accordance with the Clinical and Laboratory Standards Institute (CLSI). Vitek-2 confirmed the presence of E. faecalis (sensitive to Vancomycin, Teicoplanin and Linezolid), MDR A. baumannii (sensitive to tigecycline) and S. aureus. However, breakpoints for tigecycline for Acinetobacter infections are not given in CLSI guidelines or Food and Drug Administration guidelines. Vila et al. in their study treated MDR A. baumannii joint infections with debridement and tigecycline. However, the S. aureus strain was found to be methicillin sensitive (showing cefoxitin screen negative and sensitive to Vancomycin, Teicoplanin, Linezolid, Clindamycin and Daptomycin). The MIC of oxacillin for the S. aureus isolate by Vitek was 0.5 μg/mL. A disc diffusion test was also performed on Mueller–Hinton agar using bacterial suspension of S. aureus at a density equivalent to 0.5 McFarland standard and loaded with 30 μg of cefoxitin disc. It was found to be sensitive to cefoxitin, thus concluding the strain of S. aureus to be methicillin sensitive.
In view of the above discrepancy of results, the clinician started the patient on vancomycin for Gram-positive cover after being informed of the genotypic report. Apart from vancomycin, the patient was treated with tigecycline (for the Gram-negative cover) as per hospital policy. The patient improved symptomatically thereafter.
| Discussion|| |
The detection of mecA gene and MREJ is the golden standard for the identification of MRSA. However, the test is expensive and not within the scope of many clinical laboratories. Cefoxitin disc testing is fairly accurate and an easy method for detecting methicillin resistant strains of S. aureus. However, there can be discrepancies in the results between phenotypic and molecular methods, which have been discussed in the CLSI guidelines. The occurrence of rare micas positive S. aureus isolates that are susceptible to cefoxitin can be considered. The simultaneous presence of mecA positive Staphylococcus spp. (other than S. aureus) and MSSA resulting in false-positive MRSA molecular results on molecular platform cannot be ruled out. Strains harbouring unstable Staphylococcal cassette chromosome mec (SCCmec) insertions may lose mecA gene during subculture, thereby giving false-negative reports by phenotypic methods. Since the sensitivity of molecular methods being higher and specificity lower, occasional false-positive mecA results have been reported for direct blood culture molecular techniques. Finally, strains harbouring a SCCmec remnant lacking the mecA gene (mecA dropout) or mutant mecA allele may test positive in assays that target only SCC mec-orfX junctional regions.
In this case study, an argument can be made that the mecA detection by the molecular methods was due to the presence of Staphylococcus spp. other than S. aureus, which is not within the scope of the system. However, when the patient's sample was plated onto the culture media, no growth of Staphylococcus species other than S. aureus isolate was noted, thus ruling out the above possibility.
Although mecA is most commonly associated with Staphylococci,, the detection of a mecA homologue in Enterococcus hirae has led to the speculation that mecA also can occur in non-Staphylococcal genera, as mecA-containing Enterococci have been isolated from natural surface waters. Furthermore, Vitek 2 system does not test E. faecalis isolate for susceptibility to cefoxitin, and disc diffusion test to detect its susceptibility to cefoxitin (30 μg) was not performed as the same is not within the scope of CLSI.
Nonetheless, extrapolation of the above theory into Medical Bacteriology that E. faecalis contains mecA gene requires further research.
| Conclusion|| |
The above case report infers that although molecular techniques offer many advantages, they still have their own limitations. Their needs to be a validation data created to understand the compatibility of conventional phenotypic results with the findings by molecular tests. This will allow laboratories to follow with conviction their outcomes and guide the clinician to treat the patient suitably.
We acknowledge the expertise and technical help provided by Ms. Shamita Binod and Ms. Preethi Vijayan and the support by Tata Memorial Hospital, Parel-Mumbai.
Declaration of patient consent
The authors certify that they have obtained all appropriate patient consent forms. In the form, the patient(s) has/have given his/her/their consent for his/her/their images and other clinical information to be reported in the journal. The patients understand that their names and initials will not be published and due efforts will be made to conceal their identity, but anonymity cannot be guaranteed.
Financial support and sponsorship
Conflicts of interest
There are no conflicts of interest.
| References|| |
Lakshmi V. Culture of body fluids using the bact/alert system. Indian J Med Microbiol 2001;19:44-50.
] [Full text]
Tille PM. Bailey and Scott's Diagnostic Microbiology. 14th
ed. St. Louis, Missouri: Elsevier; 2017.
Vila A, Pagella H, Amadio C, Leiva A. Acinetobacter prosthetic joint infection treated with debridement and high-dose tigecycline. Infect Chemother 2016;48:324-9.
CLSI. Performance Standards for Antimicrobial Susceptibility Testing. 31st
ed. CLSI Supplement M100. USA: Clinical and Laboratory Standards Institute; 2021. p. 154.
CLSI. Appendix H. Using Molecular Assays for Resistance Detection. In: Performance Standards for Antimicrobial Susceptibility Testing. 31st
ed. CLSI Supplement M100. USA: Clinical and Laboratory Standards Institute; 2021. p. 274.
Hanssen AM, Kjeldsen G, Sollid JU. Local variants of Staphylococcal cassette chromosome mec in sporadic methicillin-resistant Staphylococcus aureus
and methicillin-resistant coagulase-negative Staphylococci: Evidence of horizontal gene transfer? Antimicrob Agents Chemother 2004;48:285-96.
Fitzgerald JR, Musser JM. The Molecular evolution of methicillin-resistant Staphylococcus aureus.
In: Fluit AC, Schmitz FJ, editors. MRSA: Current Perspectives. UK: Caister Academic Press; 2003. p. 137-58.
El Kharroubi A, Jacques P, Piras G, Van Beeumen J, Coyette J, Ghuysen JM. The Enterococcus hirae
R40 penicillin-binding protein 5 and the methicillin-resistant Staphylococcus aureus
penicillin-binding protein 2' are similar. Biochem J 1991;280 (Pt 2):463-9.
Kassem II, Esseili MA, Sigler V. Occurrence of mecA in nonstaphylococcal pathogens in surface waters. J Clin Microbiol 2008;46:3868-9.