Oxacillin-susceptible methicillin-resistant Staphylococcus aureus septic bursitis identified following clinical failure with β-lactam therapy: A case report

Abstract

Background:

Oxacillin-susceptible methicillin-resistant Staphylococcus aureus (OS-MRSA) is a phenotype of S. aureus that presents as methicillin susceptible on traditional screening tests but may have a resistant phenotype, leading to clinical failure.

Methods:

An isolate of S. aureus screened negative for methicillin resistance in a case of septic bursitis in a 75-year-old female. After failure to improve on cefazolin, a second aspirate was performed and sent for testing. The second sample strain retrieved had an additional PBP2a lateral flow performed, demonstrating the presence of mecA products.

Results:

Both S. aureus isolates proved to be identical on pulsed-field gel electrophoresis and had faint positive PBP2a bands on lateral flow, despite screening negative for methicillin resistance on oxacillin and cefoxitin testing. A point mutation was found in the mecA start codon, which could explain the OS-MRSA phenotype. Neither oxacillin nor cefoxitin screening tests were positive following a cefoxitin induction assay that was used locally to promote a methicillin-resistant phenotype in small-colony variant S. aureus (SCV).

Conclusions:

OS-MRSA is a rare but documented phenotype of S. aureus that has the potential to escape typical screening. Simple mutations may lead to reversal under selection pressure. Failure to respond to standard-of-care therapy for methicillin-sensitive S. aureus should prompt additional testing. Such methods could include a PBP2a immunoassay with high sensitivity. The patient recovered by using an antibiotic of a different class, trimethoprim-sulfamethoxazole, for which the isolate tested sensitive.

Introduction

Vitek 2 Antimicrobial Susceptibility Testing (Vitek 2 AST, BioMerieux Canada Inc, Saint-Laurent, Quebec, Canada) is an automated susceptibility testing system that can be used for Staphylococcus aureus. In addition to providing antimicrobial susceptibilities, the assay can screen for methicillin-resistant Staphylococcus aureus (MRSA) using oxacillin minimum inhibitory concentration (MIC; ≤2 µg/mL) and a cefoxitin disk diffusion (DD) screen (CLSI, Performance Standards for Antimicrobial Susceptibility Testing, 33rd edition, Clinical Laboratory Standards Institute, 2023). Identifying these isolates carries important therapeutic considerations for β-lactam antibiotics and misinterpretations can lead to potential treatment failure. According to the product insert for Vitek 2 AST, the very major error (VME) rate when screening both oxacillin and cefoxitin MICs for MRSA is 1.2% compared with mecA polymerase chain reaction (PCR) and 0.4% compared with cefoxitin DD assay, as described by CLSI M100 33rd edition. Clinical non-response to β-lactam antimicrobial therapy for septic bursitis prompted us to investigate a case of a falsely negative Vitek2 AST MRSA screen on a mecA-positive S. aureus isolate.

Case Presentation

A female in her 70s was referred to the Infectious Diseases Home Parenteral Therapy Program Clinic following a 24-hour admission to orthopaedics for left elbow olecranon septic bursitis. During her admission, fluid was aspirated from the infected bursa and sent for culture and susceptibility testing (AST). Initial results were consistent with methicillin-susceptible S. aureus (MSSA) and the patient was initially placed on ceftriaxone 2 g intravenous (IV) every 24 hours at the initial assessment, with follow-up scheduled on day 5. At follow-up, her left olecranon bursa remained persistently inflamed and swollen, a concern for treatment failure. The patient was then switched to cefazolin 2 g IV every 8 hours with reassessment in 2 days. Her bursa was aspirated for a second time and sent for culture, with the report from this second aspirate initially reported again as MSSA.

At the patient's follow-up appointment, it was noted that her second fluid aspirate had cultured MRSA. The patient was switched to trimethoprim-sulfamethoxazole DS 1 tablet twice per day and made a full recovery after completing 14 days of oral therapy. The ordering physician contacted the medical microbiologist to discuss the discrepant results and request further workup.

Methods

Workup of S. aureus from non-blood and non-central nervous system sites for this specific diagnostic laboratory relies on Vitek 2 AST for MRSA screening. Review of the workup of both cultures indicated that both isolates screened as MSSA on the Vitek 2 AST with an oxacillin MIC of 0.5 µg/mL and a negative cefoxitin screen. Due to clinical concerns, a PBP2a lateral flow (Abbott Laboratories, Abbott Park, Illinois, United States) was carried out on the isolate cultured from the second fluid aspirate. A faint line was observed on the expected position for a PBP2a result from a colony sweep, and per the instructions on the product insert and subsequent protocol interpretation, it was considered a positive test for MRSA (Figure 1).

Figure 1: Faint lines over the test strip for the Abbot Clearview PBP2a assay for “MSSA” and “MRSA” isolates.

The first fluid sample was retrieved from frozen cultures to test with the PBP2a lateral flow and to repeat the PBP2a on the second isolate. Both specimens were replanted onto denim-blue agar (DBA; Oxoid Limited, Basingstoke, United Kingdom), with the working hypothesis being that a mixed sample of MSSA and MRSA isolates was present. On the lateral flow assay the isolate from the first sample was also found to be weakly positive for PBP2a. The first sample had scant growth of blue colonies on DBA.

The second sample, which also yielded an S. aureus strain, was faintly blue over the inoculum but with no visible growth. Both susceptibility reports were amended to be reported as the MRSA phenotype for β-lactam agents, with all agents other than β-lactam testing sensitive per CLSI M100 33rd edition breakpoints. A DBA-positive isolate labelled “MRSA” and an off-white DBA-negative colony labelled “MSSA” were sent to the research laboratory for further workup.

Both isolates were screened for mecA via PCR and fragments were identified on gel electrophoresis. Both isolates demonstrated gene fragments for mupA, nuc, and smr in addition to mecA. Phenotypic screening for MRSA was performed using the CLSI cefoxitin DD assay. The MSSA isolate had a zone size of 26 mm and the MRSA zone size was 25 mm (Figure 2).

Figure 2: Cefoxitin disk diffusion assay showing the results of (A) “MSSA”- and (B) “MRSA”-labelled isolates compared with (C) control strain USA300, and (D) pulse field gel electrophoresis confirming identical strains.

Given the similarity in phenotypic and molecular characteristics, both strains were characterized with pulsed-field gel electrophoresis and revealed to be identical strains. Further typing revealed them to be spa t068 and SCCmec VI – belonging to clonal complex (CC) 8, most likely CMRSA10 (1). These findings were then confirmed via whole-genome sequencing along with other virulence genes (Table 1; Supplementary data).

Table 1:

Genomic characteristics of isolates compared with the closely related type strain CMRSA10 from McDougal et al (2003), with differences bolded

Strain	spa	mecA (start codon)	SCCmec	Multilocus sequence type
“MRSA”	t068 YHHGFMBQBLO	A > C (CTG)	VI	New, 3-3-1-1-16-4-3
“MSSA”	t068 YHHGFMBQBLO	A > C (CTG)	VI	New, 3-3-1-1-16-4-3
CMRSA10	t008 YHGFMBQBLO	ATG	IVa	ST8, 3-3-1-1-4-4-3

Further characteristics are available in the supplementary data

Notable findings through the Basic Local Alignment Search Tool (BLAST) noted that mecR1 was truncated and that mecA had a substitution A > C corresponding to the mecA start codon (Table 1; Supplementary data).

To reconcile the poor clinical response to ceftriaxone and cefazolin with the isolate's phenotype, we utilized a cefoxitin induction protocol that was validated for testing PBP2a induction on small-colony variant (SCV) S. aureus (2). Briefly, isolates were plated onto blood agar in 5% CO₂ for 18 hours with and without a 30 µg cefoxitin disk. Colonies from the zone edge were swept and tested on the ClearView PBP2a lateral flow assay and assessed for the qualitative strength of the PBP2a line. Furthermore, the zone edge isolates then underwent phenotypic MRSA testing with an oxacillin eTest and cefoxitin DD according to the CLSI M100, 33rd edition (Figure 3).

Figure 3: Oxacillin eTest results with an MIC of 0.38 µg/mL (left) and a cefoxitin disk diffusion test at 25 mm (right) after attempted induction with cefoxitin on blood agar in CO₂.

Discussion

Determination of the presence of mecA in S. aureus isolates in diagnostic microbiology labs is critical due to resistance to recommended first-line anti-staphylococcal agents (3). Very major errors on the Vitek 2AST are reported to be as low as 0.4%–1.2% when both oxacillin MIC and cefoxitin screening are used compared to cefoxitin DD or mecA PCR respectively. Here we sought to characterize whether this rare VME rate could have clinical treatment implications.

These isolates are collectively known as oxacillin-sensitive methicillin-resistant S. aureus (OS-MRSA), and they have been reported in the literature with some frequency for almost the last two decades (4–19). Prior studies have demonstrated up to 1.6% prevalence of these isolates that are missed by the Vitek2 2 AST screen, which is similar to the 1.2% reported by the manufacturer compared with mecA PCR (18). In a study by Liu et al, of 17 isolates producing a VME rate on their screen, 2 were also phenotypically sensitive to cefoxitin. Both oxacillin- and cefoxitin-sensitive isolates are rare, but they have been reported previously and resistance has been induced by a variety of methods (5,7,17,18).

To better assess the clinical relevance of induction testing, we utilized a protocol derived for diagnostic testing of the MRSA phenotype for SCV S. aureus. Our results indicate that basic induction methods in clinical labs fail to detect this variant, but there are still likely clinical treatment implications for patients (13,16). Based on assessments by the infectious disease physicians and orthopaedic surgeon, this patient had a poor response to first-line cefazolin anti-MSSA therapy and may have relapsed or progressed had her antimicrobials not been changed. Our case also serves to illustrate the need for clinical monitoring, re-aspiration, and re-culturing in the event of a lack of expected recovery to determine if there were microbiologic considerations.

Prior molecular sequencing of these isolates suggested mutations in mecA as the cause for phenotypic oxacillin sensitivity with inducible resistance (17,18), and our isolate had a single substitution in the mecA start codon. We posit this point mutation as the cause for phenotypic OS-MRSA that could be easily reversed under selection pressure.

Our findings in this case highlight the importance of following up traditional MRSA screening with PBP2a screening tests, such as the ClearView lateral flow or mecA PCR, when investigating persistent methicillin-sensitive S. aureus or clear failure to improve on appropriate therapy. Misdiagnosis from critical sites such as blood and central nervous system infections have little margin for error.

Funding Statement

No funding was received for this work.

Contributors:

Conceptualization, AJ Kipp, JM Conly; Methodology, AJ Kipp, JAM McClure, J Pitout, K Zhang, JM Conly; Validation, JAM McClure, J Pitout, K Zhang, JM Conly; Formal Analysis, AJ Kipp, JAM McClure, J Pitout, K Zhang, JM Conly; Investigation, AJ Kipp, JAM McClure, J Pitout, K Zhang, JN Powell, JM Conly; Resources, AJ Kipp, JAM McClure, J Pitout, K Zhang, JM Conly; Data Curation, AJ Kipp, JAM McClure, J Pitout, K Zhang, JM Conly; Writing – Original Draft, AJ Kipp; Writing – Review & Editing, AJ Kipp, JAM McClure, J Pitout, K Zhang, JN Powell, JM Conly; Visualization, AJ Kipp, JAM McClure, K Zhang; Supervision, JM Conly; Project Administration, AJ Kipp, JM Conly; Funding Acquisition, AJ Kipp, K Zhang, JM Conly.

Ethics Approval:

Ethics approval was not required for this article.

Informed Consent:

This patient consented to having the case history and microbiologic results provided in this case presentation.

Registry and the Registration No. of the Study/Trial:

N/A

Data Accessibility:

N/A

Funding:

No funding was received for this work.

Disclosures:

The authors have nothing to disclose.

Peer Review:

This manuscript has been peer reviewed.

Animal Studies:

N/A