COMPARISON OF CEFOXITIN DISC DIFFUSION TEST,OXACILLIN SCREEN AGAR,AND PCR FOR mecA GENE FOR DETECTION OF MRSA

Cefoxitin is a potent inducer of the mecA regulatory system. It is being recommended for detection of methicillin resistance in Staphylococcus aureus (MRSA) when using disk diffusion testing. The aim of our study was to evaluate the efficacy of cefoxitin disc diffusion test to characterize MRSA and compare it with oxacillin agar screening and detection of mecA gene by PCR. Materials and Methods: Fifty strains of S. aureus isolated from clinical samples were used in the study. Routine antibiotic susceptibility testing was performed including oxacillin disk. Oxacillin screen agar plates with 4% NaCl and 6 µg/ml of oxacillin were inoculated and interpreted as per standard guidelines. Cefoxitin disc diffusion test was performed using 30 µg disc and zone sizes were measured. PCR for amplification of the mecA gene was performed. Results: Out of the 50 isolates, 28 were found to be methicillin resistant by oxacillin disc diffusion test, 30 were resistant by oxacillin screen agar method, and 32 were resistant with cefoxitin disc diffusion. For these 32 isolates mecA gene was positive. Conclusion: Results of cefoxitin disc diffusion test is in concordance with the PCR for mecA gene. Thus, the test can be an alternative to PCR for detection of MRSA in resource constraint settings.     

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

BackgroundOxacillin-susceptible mecA-positive Staphylococcus aureus (OS-MRSA) represents an important issue, as its oxacillin susceptibility has contributed to misidentification by conventional susceptibility tests and consequently potential therapeutic failure, but limited data on the current status of OS-MRSA infection in Chinese hospitals are available.MethodsThis multicenter study performed a battery of susceptibility tests and diagnostic tests for 956 S. aureus isolates from 10 hospitals, including automated susceptibility testing on VITEK 2, broth microdilution, disk diffusion, and detection of PBB2a, mecA gene and mecC gene. For all identified OS-MRSA, multi-locus sequence typing (MLST), together with spa typing, SCCmec typing and PVL detecting, was carried out.ResultsOS-MRSA, most of which were from pediatric inpatients, represented 1.8% (17/956) of total isolates. Of these 17 OS-MRSA, 10 were ST59, followed by ST965 (3/17), and 11 carried SCCmec type IV, while 5 carried SCCmec type V, but only one was Panton–Valentine leucocidin (PVL)-positive, also, 16 had one or two point mutations within mecA promoter. OS-MRSA had inducible oxacillin resistance and significantly lower MDR (Multi-Drug Resistant) rate. We observed that the VITEK 2 system exhibited some deficiency in OS-MRSA detection, whereas cefoxitin disk diffusion was shown to be a reliable and cost-saving alternative and should be supplemented in detecting S. aureus with borderline oxacillin susceptible MICs.ConclusionThis study has characterized phenotypically and molecularly OS-MRSA in China, and provided insights into more effective management of OS-MRSA.     

Development of Oxacillin Resistance in a Patient with Recurrent Staphylococcus aureus Bacteremia

Whole-genome sequencing was used to compare longitudinal isolates of Staphylococcus aureus that developed resistance to oxacillin (MIC up to 16 μg/ml). The mecA gene was absent. A novel 5-bp TATCC frameshift insertion in a gene encoding an ABC transporter similar to that of the teichoic acid translocation ATP-binding protein TagH and a 3-bp GCT nonframeshift insertion in the pdhA pyruvate dehydrogenase gene were detected in the oxacillin-resistant isolates.     

Evaluation of discrepancies between oxacillin and cefoxitin disk susceptibility for detecting methicillin-resistant <Emphasis Type="Italic">Staphylococcus aureus</Emphasis>

The Clinical Laboratory Standards Institute recommends that if both cefoxitin and oxacillin are tested against Staphylococcus aureus and either result is measured as resistant, the organism should be reported as oxacillin resistant. This indicates that discrepancies may be present between oxacillin and cefoxitin sensitivities in S. aureus. In this study, we aimed to investigate the discrepancy between oxacillin and cefoxitin susceptibility in S. aureus clinical isolates. Of 10,980 S. aureus isolates recovered from 2005 to 2010, 27 (0.3%) isolates with discordant results between oxacillin and cefoxitin were collected. Fourteen (oxacillin diameters 10–12 mm) of the 27 strains were susceptible (MICs = 0.5–2 μg/ml) and 13 (6–13 mm) were resistant (4–>256 μg/ml) to oxacillin. The cefoxitin MICs of 14 oxacillin-susceptible and 13 oxacillin-resistant strains ranged between 4 and 8 and 8 to 32 μg/ml, respectively. Discrepancies were present between oxacillin and cefoxitin in S. aureus, and these strains should be further tested for oxacillin MICs and for the mecA gene or β-lactamase activity.     

Impact of EUCAST ceftaroline breakpoint change on the susceptibility of methicillin-resistant Staphylococcus aureus isolates collected from patients with complicated skin and soft-tissue infections

ObjectivesIn 2018, the European Committee on Antimicrobial Susceptibility Testing (EUCAST) introduced an intermediate breakpoint for ceftaroline against Staphylococcus aureus. The objective of this study was to compare data on resistance to ceftaroline among methicillin-resistant S. aureus (MRSA) isolates using versions 7.1 (March 2017) and 8.0 (January 2018) of the EUCAST breakpoints.MethodsParticipating centers were located in Africa, Asia, Europe, Oceania and South America. Isolates were collected from patients with complicated skin and soft-tissue infections and were cultured from integumentary sources. Methicillin resistance among S. aureus was confirmed locally using the oxacillin method. The CLSI broth microdilution method was used to measure ceftaroline MICs at the central laboratory. Versions 7.1 and 8.0 of the EUCAST breakpoints were used to interpret MIC data.ResultsBetween 2015 and 2016, 9559 isolates of S. aureus were collected, of which 5566 (58.2%) isolates were MRSA. Overall, the lowest rate of MRSA was in Asia (56.5%; 705/1247) and the highest rate was in Oceania (62.7%; 299/477). Using version 7.1 of the EUCAST breakpoints, 4.5% (250/5566) of all MRSA isolates were resistant to ceftaroline and when version 8.0 of the breakpoints was applied, 4.2% (235/5566) of MRSA were in the intermediate category and 0.3% (15/5566) of all isolates were considered resistant.ConclusionsBy applying version 8.0 of the EUCAST breakpoints, the majority of MRSA isolates that were resistant are now in the intermediate category for ceftaroline. Ceftaroline resistance among MRSA now appears rare.     

Comparison of screening methods to identify methicillin-resistantStaphylococcus aureus

Screening methods to identify methicillin-resistantStaphylococcus aureus (MRSA) were compared using 96 isolates representing 17 distinct clones. The sensitivity of four commercial agglutination tests was determined in comparison to the tube coagulation test, and the results related to the presence of the coagulase gene. The broth screening test, agar dilution test and disc diffusion test were carried out, and the results related to the presence of themecA gene. Mannitol salt agar and Iso-Sensitest agar with varying salt supplements were used. All agglutination tests had high rates of detection ofStaphylococcus aureus (95.8–99.0%). Resistance in mecA gene-positiveStaphylococcus aureus isolates was correctly detected by the oxacillin broth test, the agar dilution test and the disc diffusion test on mannitol salt agar, whereas on Iso-Sensitest agar detection rates were lower (between 68.5% and 94.4%, depending on the salt supplement). Incubation of the Iso-Sensitest plates for 48 hours significantly improved the rate of detection of resistance, but increased the major error rate up to 71.4%.MecA genepositiveStaphylococcus aureus isolates not detected by the disc diffusion test on Iso-Sensitest agar had significantly lower oxacillin minimal inhibitory concentration values and were significantly less resistant to a variety of antibiotics. Thus, mannitol salt agar might be a suitable medium for use in the disc diffusion and agar dilution test to detect resistance to oxacillin inStaphylococcus aureus.     

Detection of Oxacillin-Susceptible mecA-Positive Staphylococcus aureus Isolates by Use of Chromogenic Medium MRSA ID

Reports of oxacillin-susceptible mecA-positive Staphylococcus aureus strains are on the rise. Because of their susceptibility to oxacillin and cefoxitin, it is very difficult to detect them by using routine phenotypic methods. We describe two such isolates that were detected by chromogenic medium and confirmed by characterization of the mecA gene element.     

Analogs of Eap Protein Are Conserved and Prevalent in Clinical Staphylococcus aureus Isolates

Map and Eap are secreted Staphylococcus aureus proteins that interact with various extracellular matrix molecules. PCR analysis using map primers yielded positive reactions in 97.9% of S. aureus isolates but not in Staphylococcus epidermidis isolates. Cloning and sequencing of the conferring genes revealed a high degree of overall homology combined with size variability of the gene product due to various repeat numbers and early translation termination in a poly(A) region. Thus, Map and Eap may provide a potential novel tool for S. aureus identification and typing.     

Coagulase gene-based typing of Staphylococcus aureus from mastitic cattle and goats from arid region in India

Staphylococcus aureus is an important pathogen and causes mastitis and many other infections in animals as well as in humans. In the present study, S. aureus isolates were investigated for any variations based on coa gene polymorphism and AluI restriction fragment length polymorphism of coa amplicons. Thirty S. aureus isolates obtained from cattle and goats with clinical mastitis were identified by their phenotypic properties and then confirmed genotypically by PCR where all the isolates produced species-specific amplicon of 1,250?bp. Amplification of the coa gene produced products of three different sizes (600, 680, or 850?bp), one specific to each isolate. The strains of S. aureus were common to both cattle and goat, but the predominance of S. aureus isolates with coa gene amplicon of 680?bp was observed in cattle and 600?bp coa gene amplicon in goats. No difference in the restriction fragment length polymorphism (RFLP) patterns of coa amplicons with AluI endonuclease was obtained in isolates from cattle or goats, and there was limited strain variations in the region under study. The coa amplicons and RFLP patterns of isolates were similar to those recorded elsewhere.     

Persistent Staphylococcus aureus Isolates from Two Independent Cases of Bacteremia Display Increased Bacterial Fitness and Novel Immune Evasion Phenotypes

Staphylococcus aureus bacteremia cases are complicated by bacterial persistence and treatment failure despite the confirmed in vitro susceptibility of the infecting strain to administered antibiotics. A high incidence of methicillin-resistant S. aureus (MRSA) bacteremia cases are classified as persistent and are associated with poorer patient outcomes. It is still unclear how S. aureus evades the host immune system and resists antibiotic treatment for the prolonged duration of a persistent infection. In this study, the genetic changes and associated phenotypic traits specific to S. aureus persistent bacteremia were identified by comparing temporally dispersed isolates from persistent infections (persistent isolates) originating from two independent persistent S. aureus bacteremia cases with the initial infection isolates and with three resolved S. aureus bacteremia isolates from the same genetic background. Several novel traits were associated specifically with both independent sets of persistent S. aureus isolates compared to both the initial isolates and the isolates from resolved infections (resolved isolates). These traits included (i) increased growth under nutrient-poor conditions; (ii) increased tolerance of iron toxicity; (iii) higher expression of cell surface proteins involved in immune evasion and stress responses; and (iv) attenuated virulence in a Galleria mellonella larva infection model that was not associated with small-colony variation or metabolic dormancy such as had been seen previously. Whole-genome sequence analysis identified different single nucleotide mutations within the mprF genes of all the isolates with the adaptive persistence traits from both independent cases. Overall, our data indicate a novel role for MprF function during development of S. aureus persistence by increasing bacterial fitness and immune evasion.     

Evaluation of Mannitol Salt Agar for Detection of Oxacillin Resistance in Staphylococcus aureus by Disk Diffusion and Agar Screening

Mannitol salt agar was evaluated for detection of oxacillin resistance in 136 Staphylococcus aureus isolates. All mecA-positive isolates (n = 54) were correctly categorized as oxacillin resistant by the disk diffusion test (1-μg disk; zone diameter, <16 mm); the specificity was 97.6%. Agar screening (2 μg of oxacillin per ml) revealed a sensitivity of 98.1% and a specificity of 95.1%.     

Methicillin-resistant Staphylococcus aureus nasal carriage and infection among patients with diabetic foot ulcer

PurposeTo evaluate the prevalence of methicillin-resistant Staphylococcus aureus (MRSA) nasal carriage in patients with diabetic foot ulcer (DFU) in Taiwan, and to assess the concordance between colonizing and clinical MRSA isolates from the patients.MethodA total of 354 nasal specimens were collected from 112 to 242 diabetic patients with and without foot ulcer, respectively. MRSA clinical isolates from DFU wound cultures were collected for comparison.ResultsNasal carriage rate of S. aureus and MRSA was similar between diabetic patients with and without foot ulcer (15.2% vs. 16.9% for S. aureus and 5.4% vs. 1.7% for MRSA). Nasal S. aureus colonization was an independent predictor for wound S. aureus infection (Odds ratio [OR]: 5.33, 95% confidence interval [CI]: 1.61–17.59), so did nasal MRSA colonization (OR: 19.09, 95% CI: 2.12–171.91). The levels of glycated hemoglobin, and the usage with immunosuppressant agent were associated with S. aureus nasal colonization while oral hypoglycemic agent usage a protective factor. Sequence type 59/staphylococcal chromosome cassette mec IV or V, the local endemic community-associated clone, accounted for 42% and 70% of the clinical and colonizing isolates, respectively. Six of 10 patients with paired colonizing and clinical isolates, either MRSA or methicillin-sensitive S. aureus, had a genetically identical strain from a single patient.ConclusionLess than one-fifth of patients with DFU have nasal S. aureus, including MRSA, colonization; however, the colonization is significantly associated with S. aureus diabetic foot infection. Screening for S. aureus colonizing status in DFU patients might have a potential clinical implication.     

EUCAST disc diffusion criteria for the detection of mecA-Mediated β-lactam resistance in Staphylococcus pseudintermedius: oxacillin versus cefoxitin

ObjectivesUntil recently, the European Committee on Antimicrobial Susceptibility Testing (EUCAST) recommended the cefoxitin disc to screen for mecA-mediated β-lactam resistance in Staphylococcus pseudintermedius. A recent study indicated that cefoxitin was inferior to oxacillin in this respect. We have re-evaluated cefoxitin and oxacillin discs for screening for methicillin resistance in S. pseudintermedius.MethodsWe included 224 animal and human S. pseudintermedius isolates from Europe (n = 108) and North America (n = 116), of which 109 were mecA-positive. Disc diffusion was performed per EUCAST recommendations using 30-μg cefoxitin and 1-μg oxacillin discs from three manufacturers and Mueller–Hinton agar from two manufacturers.ResultsCefoxitin inhibition zones ranged from 6 to 33 mm for mecA-positive S. pseudintermedius (MRSP) and from 29 to 41 mm for mecA-negative S. pseudintermedius (MSSP). The corresponding oxacillin zone intervals were 6–20 mm and 19–30 mm. For cefoxitin 16% (95% CI 14.8–18.0%) of the isolates were in the area where positive and negative results overlapped. For oxacillin the corresponding number was 2% (1.6–2.9%). For oxacillin a breakpoint of susceptible (S) ≥ 20 mm and resistant (R) <20 mm resulted in only 0.4% and 1.1% very major error and major error rates respectively.ConclusionsThis investigation confirms that the 1-μg oxacillin disc predicts mecA-mediated methicillin resistance in S. pseudintermedius better than the 30-μg cefoxitin disc. For a 1-μg oxacillin disc we propose that 20 mm should be used as cut off for resistance, i.e. isolates with a zone diameter <20 mm are resistant to all β-lactam antibiotics except those with activity against methicillin-resistant staphylococci.     

Association of Borderline Oxacillin-Susceptible Strains of Staphylococcus aureus with Surgical Wound Infections

Staphylococcus aureus isolates which produce type A staphylococcal β-lactamase have been associated with wound infections complicating the use of cefazolin prophylaxis in surgery. To further evaluate this finding, 215 wound isolates from 14 cities in the United States were characterized by antimicrobial susceptibility and β-lactamase type and correlated with the preoperative prophylactic regimen. Borderline-susceptible S. aureus isolates of phage group 5 (BSSA-5), which produce large amounts of type A β-lactamase and exhibit borderline susceptibility to oxacillin, comprised a greater percentage of the 120 wound isolates associated with cefazolin prophylaxis than they did of the 95 isolates associated with other prophylactic regimens (25% versus 12.6%, respectively; P < 0.05). In contrast, methicillin-resistant S. aureus isolates were distributed evenly between the two groups (8.3% versus 11.6%, respectively). In vitro assays demonstrated that cefazolin was hydrolyzed faster by BSSA-5 strains than by other β-lactamase-producing, methicillin-susceptible strains (1.54 versus 0.50 μg/min/10⁸ CFU, respectively; P < 0.0001). These data demonstrate that BSSA-5 strains are a distinct subpopulation of methicillin-susceptible S. aureus which frequently cause deep surgical wound infections. Cefazolin use in prophylaxis is a risk factor for BSSA-5 infection.     

Evaluation of the mecA femB Duplex Polymerase Chain Reaction for Detection of Methicillin-Resistant Staphylococcus aureus

 This study systematically evaluated a recently described duplex polymerase chain reaction test for methicillin-resistant Staphylococcus aureus with 25 different German epidemic strains of methicillin-resistant Staphylococcus aureus and 66 staphylococci other than methicillin-resistant Staphylococcus aureus, including 17 different coagulase-negative staphylococcal species and subspecies, that were either oxacillin susceptible or oxacillin resistant. The results were compared with those of conventional cultural identification and susceptibility testing. Of the 91 isolates tested, all 25 confirmed strains of methicillin-resistant Staphylococcus aureus were identified correctly. None of the remaining strains of methicillin-susceptible Staphylococcus aureus was misidentified as methicillin-resistant Staphylococcus aureus. It was concluded that the duplex polymerase chain reaction appears to offer a time-saving and accurate method of detection of methicillin-resistant Staphylococcus aureus.     

Transmission events and antimicrobial susceptibilities of methicillin-resistant Staphylococcus argenteus in Stockholm

ObjectivesStaphylococcus argenteus has been increasingly reported since the species was defined as a novel staphylococcal species in 2015. This study aims to investigate genetic epidemiological links and antimicrobial susceptibilities of methicillin-resistant S. argenteus isolates recovered in Stockholm.MethodsSixteen methicillin-resistant S. argenteus isolates were identified from a collection of methicillin-resistant Staphylococcus aureus in Stockholm 2007–2018, by using whole-genome sequencing and matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS). The genomes of the isolates were investigated by pulsed-field gel electrophoresis, single-nucleotide polymorphism (SNP)-based phylogeny, k-mer analysis, core-genome multi-locus sequence typing (cgMLST), resistance traits and virulence factors. The MICs of 19 antimicrobial agents for each isolate were determined by using the broth microdilution method.ResultsOf the 16 isolates, seven, seven and two isolates were assigned to ST1223, ST2250 and ST2793, respectively, with the S. aureus MLST-scheme. Analyses based on SNPs and cgMLST revealed a likely clonal spread of methicillin-resistant S. argenteus in 2007. Four isolates were found to be resistant to non-β-lactams in antimicrobial susceptibility testing.ConclusionsA transmission event of methicillin-resistant S. argenteus in family was identified by this study. Among our limited number of isolates, non-β-lactam resistance was detected, which highlights the necessity of a continued surveillance on this emerging pathogen. S. argenteus could be correctly identified by MALDI-TOF MS with the updated database, enabling its detection also in clinical laboratories.     

Is There a Need to Revise the Antibiotic Concentration in Clinical and Laboratory Standards Institute-Recommended Oxacillin Screen Agar?

Introduction: In routine diagnostic microbiology laboratories, Clinical and Laboratory Standards Institute (CLSI) recommends the use of cefoxitin disc, in addition to oxacillin screen agar (OSA) of 6 μg/ml for the detection of methicillin-resistant Staphylococcus aureus (MRSA), whereas minimum inhibitory concentration values of oxacillin for S. aureus are ≤2 μg/ml (susceptible) and ≥4 μg/ml (resistant). Hence, the study was carried out to evaluate the ability of screen agar with lower concentrations of oxacillin to identify the isolates of MRSA and to compare this with cefoxitin disc diffusion (CDD). Materials and Methods: Six hundred and seventy-six isolates of S. aureus were screened for methicillin resistance by OSA with 2 μg/ml and 4 μg/ml and 6 μg/ml of oxacillin concentration as well as CDD. Polymerase chain reaction for mecA gene was carried out for all isolates which grew on OSA 2, 4 and 6 μg/ml regardless of their cefoxitin susceptibility. Latex agglutination test for penicillin-binding protein 2a was performed for the isolates which grew on OSA 2 and or 4 μg/ml but not on OSA 6 μg/ml. Results: Eight per cent of MRSA isolates was missed by using OSA 6 μg/ml, when compared with other methods. Sensitivities of OSA 2 μg/ml, OSA 6 μg/ml and CDD were found to be 100%, 92.5% and 97.5%, respectively, and specificities for the same were found to be 100%, 100% and 98%, respectively. As per FDA criteria, categorical agreement for OSA 2 μg/ml was found to be 100% in comparison with the reference broth microdilution method. No major and very major discrepancies were documented. Conclusion: Similar findings on a larger and more heterogeneous collection of isolates may indicate the need to revise the concentration of OSA to 2 μg/ml for the detection of MRSA.     

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

Staphylococcus pseudintermedius is a coagulase-positive species that colonizes the nares and anal mucosa of healthy dogs and cats. Human infections with S. pseudintermedius range 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 of mecA- or mecC-mediated methicillin resistance in 115 human and animal isolates of the Staphylococcus intermedius group (SIG), including 111 Staphylococcus pseudintermediusand 4 Staphylococcus delphini isolates, 37 of which were mecA positive. The disk and MIC breakpoints evaluated included the Clinical and Laboratory Standards Institute (CLSI) M100-S25 Staphylococcus aureus/Staphylococcus lugdunensis oxacillin MIC breakpoints and cefoxitin disk and MIC breakpoints, the CLSI M100-S25 coagulase-negative Staphylococcus (CoNS) oxacillin MIC breakpoint and cefoxitin disk breakpoint, the CLSI VET01-S2 S. pseudintermedius oxacillin MIC and disk breakpoints, and the European Committee on Antimicrobial Susceptibility Testing (EUCAST) S. pseudintermedius cefoxitin disk breakpoint. The oxacillin results interpreted by the VET01-S2 (disk and MIC) and M100-S25 CoNS (MIC) breakpoints agreed with the results of mecA/mecC PCR for all isolates, with the exception of one false-resistant result (1.3% of mecA/mecC PCR-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 with mecA/mecC PCR results if interpreted by using the M100-S25 CoNS breakpoint. The Alere penicillin-binding protein 2a test accurately detected all mecA-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 of mecA in S. pseudintermedius isolates and that laboratories should perform oxacillin disk or MIC tests of these isolates when they are encountered.