Improved Detection of mecA-Mediated β-Lactam Resistance in Staphylococcus lugdunensis Using a New Oxacillin Salt Agar Screen

Oxacillin resistance mediated by mecA in Staphylococcus lugdunensis is emerging in some geographic areas. We evaluated cefoxitin disk diffusion (DD) and a new oxacillin agar (supplemented with 2 μg/ml oxacillin and 2% sodium chloride) screen for the detection of mecA-mediated resistance in S. lugdunensis. A total of 300 consecutive, non-duplicated clinical S. lugdunensis isolates from diverse sources in Hong Kong in 2019 were tested. The categorical agreement and errors obtained between cefoxitin DD test, oxacillin agar screen and mecA PCR were analyzed. Isolates with discordant results were further tested by MIC, penicillin binding protein 2a (PBP2a) assays, population analysis and molecular typing. PCR showed that 62 isolates were mecA-positive and 238 isolates were mecA-negative. For cefoxitin DD results interpreted using S. aureus/S. lugdunensis breakpoints, the categorical agreement (CA) for two brands of Muller-Hinton agars, MH-II (Becton Dickinson) and MH-E (bioMérieux) were both 96.0%; MEs were both 0%; and VMEs were 19.4 and 12.9%, respectively. The new oxacillin agar reliably differentiated mecA-positive and mecA-negative isolates (100% CA) without any ME or VME results. The 8 isolates with false susceptibility in the cefoxitin DD testing had cefoxitin and oxacillin MICs in the susceptible range. The isolates showed heterogeneous oxacillin resistance with resistant subpopulations at low frequencies. All had positive PBP2a results and were typed as sequence type 27/SCCmec V. The findings highlight the inability of cefoxitin DD and MIC tests for reliable detection of some mecA-positive S. lugdunensis isolates.

The Phenotypic and Genetic Detection of the Methicillin-Resistant Staphylococcus aureus (MRSA)

Methicillin resistant strains of Staphylococcus aureus (MRSA) were identified shortly upon the introduction of methicillin into the clinical practice. The S. aureus samples were taken from patients hospitalized in General Hospital of Chania “Agios Georgios”. The strains were isolated from different pathological products, in the hospital laboratory. All isolates were tested using the cefoxitin disk diffusion, the oxacillin MIC methods and the PBP2’ latex agglutination test (bioMérieux), for the production of the Penicillin-Binding Protein 2a (PBP2a or PBP2’ protein). S. aureus ATCC 29213 was used as a negative control. We followed the detection of the mecA gene throughout the PCR method, as the standard “gold” method, in order to identify MRSA strains. Conventional methods for MRSA strains detection were compared with the PCR method. The antibiotic susceptibility testing was performed throughout the Kirby-Bauer disk diffusion method using antibiotic discs from Bioanalyse ltd. The mecA gene was found by PCR in 57.5 % of S. aureus strains, which allowed defining the isolated strains as MRSA strains. According to the oxacillin MIC values of the studied strains, 25 strains (53.2%) were identified as MRSA, 21 (44.68%) as MSSA and 1*strain (2.13%) as Borderline (BL) MRSA. The mecA gene is present in 24 of the MRSA strains with oxacillin MIC ≥ 4 being more common in strains showing the oxacillin MICs ≥ 256 (12/27). Adding the BL strains to the methicillin resistant strains, the rate of the MRSA strains increases to 26 (55.32%), the appropriate values of the MRSA strains percent, as determined by the PCR method (57.45%), which shows a concordance of 96.3% (26/27), between the results obtained by the two tests. Comparing the results obtained using the PCR method; with the oxacillin MICs and the PBP2’ latex agglutination test, concordant results were obtained for 89.36% of the strains (42/47) by oxacillin MICs and for 97.87% of the strains (46/47) by PBP2’ latex agglutination test. We conclude that the specificity of these methods is 100% for the mecA PCR method, 97.87% for the PBP2a latex method and 89.36% for the oxacillin MIC. The comparison of phenotypic methods (the PBP2a latex reaction, oxacillin MICs, the Cefoxitin disk diffusion test with the genotypic methods (the presence of the mecA gene), reveals that the PBP2a latex reaction has high sensitivity (97.87%), and can be used as an alternative method to PCR for the MRSA detection, in resource constraint settings.

Current status of oxacillin-susceptible mecA-positive Staphylococcus aureus infection in Shanghai, China: A multicenter study

The worldwide reported oxacillin-susceptible mecA -positive Staphylococcus aureus (OS-MRSA) represents a distinctly important challenge to detection and treatment of MRSA, but finite data on current status of OS-MRSA infection in Chinese hospitals are available. The present multicenter study carried out a battery of phenotypic susceptibility tests as well as diagnostic tests (PBP2a detection, mecA , and mecC PCR) for a collection of 956 clinical S. aureus isolates from 10 hospitals in Shanghai, molecular typing was performed for all identified OS-MRSA strains. OS-MRSA represented 1.8% (17/956) of total isolates and were commonly borderline oxacillin-susceptible (Oxacillin-MIC of 1 or 2 mg/L). 10 of 17 OS-MRSA were ST59 lineages, followed by ST965 (3/17). Unlike oxacillin-resistant MRSA that commonly exhibit a multidrug resistant (MDR) phenotype, OS-MRSA were less likely to be MDR and displayed MIC pattern remarkably differential from OR-MRSA. OS-MRSA showed oxacillin-inducible oxacillin resistance and the majority of them (15/17) were cefoxitin-resistant by cefoxitin disk diffusion. S. aureus with borderline susceptible oxacillin MICs (1 or 2 mg/L) should be confirmed by cefoxitin disk diffusion in clinical practice, whereas susceptible isolates reclassified by cefoxitin disk diffusion should still be subjected to PBP2a testing and (or) mecA (mecC) PCR. The presented study has characterized phenotypically and molecularly an atypical type of MRSA showing cryptic but inducible resistance to oxacillin, but its underlying mechanisms warrant further elucidation.

Evaluation of Oxacillin and Cefoxitin Disk Diffusion and Microbroth Dilution Methods for Detecting mecA-Mediated β-Lactam Resistance in Contemporary Staphylococcus epidermidis Isolates

ABSTRACT Methicillin (β-lactam) resistance in Staphylococcus epidermidis is mediated by the mecA gene, with resistance reported to be as high as 90%. The goal of this study was to evaluate oxacillin and cefoxitin disk diffusion (DD) and broth microdilution (BMD) methods for the detection of mecA-mediated β-lactam resistance in 100 human isolates of S. epidermidis (48 mecA-positive isolates and 52 mecA negative isolates). Oxacillin DD tests using the Clinical and Laboratory Standards Institute (CLSI) M100-S28 breakpoints for S. pseudintermedius/S. schleiferi accurately differentiated mecA-positive and -negative S. epidermidis isolates, with categorical agreement (CA) of 100% and no very major errors (VMEs) or major errors (MEs) identified. Likewise, oxacillin BMD and cefoxitin DD tests using the coagulase-negative Staphylococcus species (CoNS) breakpoints were highly reliable for detecting mecA-mediated β-lactam resistance in S. epidermidis isolates. For cefoxitin DD and BMD results interpreted using S. aureus/S. lugdunensis breakpoints, the CA was 97.6% and 96.2%, respectively. There were 4.9% VMEs for cefoxitin DD with 0% MEs, and 3.6% VMEs and 3.9% MEs for cefoxitin BMD. Oxacillin BMD using S. aureus/S. lugdunensis breakpoints yielded the highest VMEs at 17.4% and 90% CA. Our findings demonstrate that oxacillin DD tests using the CLSI M100-S28 breakpoints for S. pseudintermedius/S. schleiferi and oxacillin BMD and cefoxitin DD tests using the CoNS breakpoints reliably identified mecA-mediated β-lactam resistance in S. epidermidis. Using mecA PCR as the gold standard, the PBP2a SA culture colony test (Abbott Diagnostics) exhibited 100% sensitivity and specificity whereas 2 false negatives were identified using the PBP2ʹ latex agglutination test kit (Thermo Fisher Scientific) with sensitivity and specificity of 95.8% and 100%, respectively.

Ceftaroline Resistance by Clone-Specific Polymorphism in Penicillin-Binding Protein 2a of Methicillin-Resistant Staphylococcus aureus

ABSTRACT A total of 281 nonduplicated Staphylococcus aureus blood isolates were collected from January to May 2017 from eight hospitals in South Korea to investigate the epidemiological traits of ceftaroline resistance in methicillin-resistant S. aureus (MRSA). Cefoxitin-disk diffusion tests and the mecA gene PCR revealed that 56.6% (159/281) of the S. aureus isolates were MRSA, and most belonged to ST5 (50.3%, 80/281) and ST72 (41.5%, 66/281). Of the MRSA isolates, 44.0% (70/159) were nonsusceptible to ceftaroline (MIC ≥ 2 mg/liter), whereas all of the methicillin-susceptible S. aureus isolates were susceptible to the drug. Eight amino acid substitutions in penicillin-binding protein 2a (PBP2a), including four (L357I, E447K, I563T, and S649A) in the penicillin-binding domain (PBD) and four (N104K, V117I, N146K, and A228V) in the non-PBD (nPBD) of PBP2a, were associated with ceftaroline resistance. The accumulation of substitutions in PBP2a resulted in the elevation of ceftaroline MICs: one substitution at 1 to 2 mg/liter, two or three substitutions at 2 to 4 mg/liter, and five substitutions at 4 or 16 mg/liter. Ceftaroline resistance in MRSA might be the result of clone-specific PBP2a polymorphism, along with substitutions both in PBD and nPBD, and the elevated ceftaroline MICs were associated with the substitution sites and accumulation of substitutions.

Evaluation of Oxacillin and Cefoxitin Disk Diffusion and MIC Breakpoints Established by the Clinical and Laboratory Standards Institute for Detection of mecA-Mediated Oxacillin Resistance in Staphylococcus schleiferi

ABSTRACTStaphylococcus schleiferiis a beta-hemolytic, coagulase-variable colonizer of small animals that can cause opportunistic infections in humans. In veterinary isolates, the rate ofmecA-mediated oxacillin resistance is significant, with reported resistance rates of >39%. The goal of this study was to evaluate oxacillin and cefoxitin disk diffusion (DD) and MIC breakpoints for detection ofmecA-mediated oxacillin resistance in 52 human and 38 veterinary isolates ofS. schleiferi. Isolates were tested on multiple brands of commercial media and according to Clinical and Laboratory Standards Institute (CLSI) methods. Zone diameters and MIC values were interpreted using CLSI breakpoints (CLSI,Performance Standards for Antimicrobial Susceptibility Testing. M100-S27, 2017) forStaphylococcus aureus/Staphylococcus lugdunensis, coagulase-negative staphylococci (CoNS), andStaphylococcus pseudintermedius. Results were compared to those ofmecAPCR. Twenty-nine of 90 (32%) isolates weremecApositive. Oxacillin inhibition zone sizes and MICs interpreted byS. pseudintermediusbreakpoints reliably differentiatedmecA-positive andmecA-negative isolates, with a categorical agreement (CA) of 100% and no very major errors (VMEs) or major errors (MEs) for all media. For cefoxitin DD results interpreted usingS. aureus/S. lugdunensisand CoNS breakpoints, CA values were 85% and 75%, respectively, and there were 72% and 64% VMEs, respectively, and 0 MEs. For cefoxitin MICs interpreted usingS. aureus/S. lugdunensisbreakpoints, CA was 81%, and there were 60% VMEs and no MEs. Our data demonstrate that oxacillin DD or MIC testing methods using the currentS. pseudintermediusbreakpoints reliably identifymecA-mediated oxacillin resistance inS. schleiferi, while cefoxitin DD and MIC testing methods perform poorly.

Comparison of Different Phenotypic Approaches To Screen and Detect mecC-Harboring Methicillin-Resistant Staphylococcus aureus

ABSTRACT Similar to mecA, mecC confers resistance against beta-lactams, leading to the phenotype of methicillin-resistant Staphylococcus aureus (MRSA). However, mecC-harboring MRSA strains pose special difficulties in their detection. The aim of this study was to assess and compare different phenotypic systems for screening, identification, and susceptibility testing of mecC-positive MRSA isolates. A well-characterized collection of mecC-positive S. aureus isolates (n = 111) was used for evaluation. Routinely used approaches were studied to determine their suitability to correctly identify mecC-harboring MRSA, including three (semi)automated antimicrobial susceptibility testing (AST) systems and five selective chromogenic agar plates. Additionally, a cefoxitin disk diffusion test and an oxacillin broth microdilution assay were examined. All mecC-harboring MRSA isolates were able to grow on all chromogenic MRSA screening plates tested. Detection of these isolates in AST systems based on cefoxitin and/or oxacillin testing yielded overall positive agreements with the mecC genotype of 97.3% (MicroScan WalkAway; Siemens), 91.9% (Vitek 2; bioMérieux), and 64.9% (Phoenix, BD). The phenotypic resistance pattern most frequently observed by AST devices was “cefoxitin resistance/oxacillin susceptibility,” ranging from 54.1% (Phoenix) and 83.8% (Vitek 2) to 92.8% (WalkAway). The cefoxitin disk diffusion and oxacillin broth microdilution assays categorized 100% and 61.3% of isolates to be MRSA, respectively. The chromogenic media tested confirmed their suitability to reliably screen for mecC-harboring MRSA. The AST systems showed false-negative results with varying numbers, misidentifying mecC-harboring MRSA as methicillin-susceptible S. aureus. This study underlines cefoxitin's status as the superior surrogate mecC-positive MRSA marker.

Evaluation of different phenotypic methods for the detection of methicillin resistant Staphylococcus aureus and antimicrobial susceptibility pattern of MRSA

Background: Rapid and accurate detection of methicillin resistant Staphylococcus aureus (MRSA) is an important role of clinical microbiology laboratories to avoid treatment failure. The aim of this study was to compare conventional methods against the cefoxitin disc diffusion method to determine the best phenotypic method. Methods: Study was carried out in the Department of Microbiology, National Institute of Medical Sciences & Research, Jaipur (India), between July 2016 – December 2016. The methods included were Oxacillin E-test MIC, Oxacillin screen agar, Oxacillin disk diffusion, Cefoxitin disk diffusion and CHROMagar- MRSA methods. Antimicrobial susceptibility performed as per CLSI guidelines.Results: Out of 142 isolates of S. aureus, fifty three (37.32%) strains of MRSA were isolated from clinical specimen. E-MIC test was selected as gold standard method. The sensitivity and specificity of Oxacillin screen agar and CHROMagar-MRSA were same 98.07% and 97.80%, respectively. The sensitivity and specificity of oxacillin disk diffusion were 94.23% and 98.89%. Fifty three strains of S. aureus were MRSA by cefoxitin disk diffusion method and Oxacillin Ezy MIC test. The sensitivity and specificity of cefoxitin disk diffusion method and Oxacillin Ezy MIC method was 100% and 100% respectively. All isolates including MRSA were susceptible to Vancomycin and Linezolid. Conclusions: All phenotypic methods had high sensitivity and specificity for detection of MRSA. However, cefoxitin disk diffusion method in comparison to other methods had higher sensitivity and specificity.

Multicenter Evaluation of a Modified Cefoxitin Disk Diffusion Method and PBP2a Testing To PredictmecA-Mediated Oxacillin Resistance in Atypical Staphylococcus aureus

ABSTRACTPhenotypic variants ofStaphylococcus aureusthat display small colonies, reduced pigmentation, and decreased hemolysis and/or coagulase activity are periodically isolated by the clinical laboratory. Antimicrobial susceptibility testing (AST) of these isolates is complicated, because many do not grow on routine AST media, including Mueller-Hinton agar (MHA) and cation-adjusted Mueller-Hinton broth. This multicenter study evaluated cefoxitin disk diffusion for 37 atypicalS. aureusisolates (156 readings) with MHA supplemented with 5% sheep's blood (BMHA), usingmecAPCR as the reference standard. The correlation of two commercial PBP2a assays withmecAPCR was also assessed. Ten isolates were negative and 27 positive formecA. No major errors for cefoxitin were observed, but 19.5% very major errors (VMEs) were observed at 24 h of incubation, and 17.2% VMEs were observed at 48 h. The proportions of VMEs ranged from 14.7 to 23.0% at 24 h, and from 13.3 to 17.6% at 48 h, across three testing laboratories. PBP2a tests were performed from growth on BMHA and blood agar plates (BAP), with and without cefoxitin disk induction. The Alere PBP2a SA culture colony test sensitivities formecAwere 90.0% with uninduced growth and 97.4% with induced growth from BMHA. On BAP, sensitivity was 96.0% with induced growth. The sensitivities of the Oxoid PBP2′ latex agglutination test were 85.7% with uninduced growth and 93.9% with induced growth from BMHA and 95.9% with induced growth on BAP. On the basis of these data, we recommend that laboratories perform onlymecAPCR and/or PBP2a tests when requested to perform AST on atypical isolates ofS. aureus.

Evaluation of Oxacillin and Cefoxitin Disk and MIC Breakpoints for Prediction of Methicillin Resistance in Human and Veterinary Isolates of Staphylococcus intermedius Group

Staphylococcuspseudintermediusis a coagulase-positive species that colonizes the nares and anal mucosa of healthy dogs and cats. Human infections withS. pseudintermediusrange in severity from bite wounds and rhinosinusitis to endocarditis; historically, these infections were thought to be uncommon, but new laboratory methods suggest that their true incidence is underreported. Oxacillin and cefoxitin disk and MIC tests were evaluated for the detection ofmecA- ormecC-mediated methicillin resistance in 115 human and animal isolates of theStaphylococcus intermediusgroup (SIG), including 111Staphylococcuspseudintermediusand 4Staphylococcusdelphiniisolates, 37 of which weremecApositive. The disk and MIC breakpoints evaluated included the Clinical and Laboratory Standards Institute (CLSI) M100-S25Staphylococcus aureus/Staphylococcuslugdunensisoxacillin MIC breakpoints and cefoxitin disk and MIC breakpoints, the CLSI M100-S25 coagulase-negativeStaphylococcus(CoNS) oxacillin MIC breakpoint and cefoxitin disk breakpoint, the CLSI VET01-S2S. pseudintermediusoxacillin MIC and disk breakpoints, and the European Committee on Antimicrobial Susceptibility Testing (EUCAST)S. pseudintermediuscefoxitin disk breakpoint. The oxacillin results interpreted by the VET01-S2 (disk and MIC) and M100-S25 CoNS (MIC) breakpoints agreed with the results ofmecA/mecCPCR for all isolates, with the exception of one false-resistant result (1.3% ofmecA/mecCPCR-negative isolates). In contrast, cefoxitin tests performed poorly, ranging from 3 to 89% false susceptibility (very major errors) and 0 to 48% false resistance (major errors). BD Phoenix, bioMérieux Vitek 2, and Beckman Coulter MicroScan commercial automated susceptibility test panel oxacillin MIC results were also evaluated and demonstrated >95% categorical agreement withmecA/mecCPCR results if interpreted by using the M100-S25 CoNS breakpoint. The Alere penicillin-binding protein 2a test accurately detected allmecA-positive isolates, although for four isolates, cefoxitin induction was required prior to testing. These data demonstrate that the cefoxitin surrogate test does not reliably detect the presence ofmecAinS. pseudintermediusisolates and that laboratories should perform oxacillin disk or MIC tests of these isolates when they are encountered.