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

2021 ◽  
Vol 12 ◽  
Author(s):  
Pak-Leung Ho ◽  
Ying-Hang Law ◽  
Melissa Chun-Jiao Liu ◽  
Andes Lau ◽  
Man-Ki Tong ◽  
...  
Keyword(s):  
Population Analysis ◽  
Sequence Type ◽  
Penicillin Binding Protein ◽  
Becton Dickinson ◽  
Staphylococcus Lugdunensis ◽  
Low Frequencies ◽  
Oxacillin Resistance ◽  
Reliable Detection ◽  
Cefoxitin Disk ◽  
Cefoxitin Disk Diffusion

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.

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

2016 ◽  
Vol 55 (2) ◽  
pp. 485-494 ◽  
Author(s):  
Shelley A. Miller ◽  
James Karichu ◽  
Peggy Kohner ◽  
Nicolynn Cole ◽  
Janet A. Hindler ◽  
...  
Keyword(s):  
Staphylococcus Aureus ◽  
Clinical Laboratory ◽  
Disk Diffusion ◽  
Diffusion Method ◽  
Disk Diffusion Method ◽  
Content Type ◽  
Oxacillin Resistance ◽  
Mueller Hinton ◽  
Cefoxitin Disk ◽  
Cefoxitin Disk Diffusion

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.

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

2019 ◽  
Author(s):  
Junlan Liu ◽  
Tianming Li ◽  
Ni Zhong ◽  
Xing Wang ◽  
Jie Jiang ◽  
...  
Keyword(s):  
Staphylococcus Aureus ◽  
Multicenter Study ◽  
Multidrug Resistant ◽  
Disk Diffusion ◽  
Current Status ◽  
Oxacillin Resistance ◽  
Important Challenge ◽  
Underlying Mechanisms ◽  
Cefoxitin Disk ◽  
Cefoxitin Disk Diffusion

Abstract 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.

scholarly journals 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

2017 ◽  
Vol 56 (2) ◽  
Author(s):  
H. K. Huse ◽  
S. A. Miller ◽  
S. Chandrasekaran ◽  
J. A. Hindler ◽  
S. D. Lawhon ◽  
...  
Keyword(s):  
Opportunistic Infections ◽  
Performance Standards ◽  
Inhibition Zone ◽  
Disk Diffusion ◽  
Testing Methods ◽  
Coagulase Negative Staphylococci ◽  
Content Type ◽  
Oxacillin Resistance ◽  
Cefoxitin Disk ◽  
Cefoxitin Disk Diffusion

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.

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

2018 ◽  
Vol 62 (9) ◽  
Author(s):  
Hyukmin Lee ◽  
Eun-Jeong Yoon ◽  
Dokyun Kim ◽  
Jung Wook Kim ◽  
Kwang-Jun Lee ◽  
...  
Keyword(s):  
Staphylococcus Aureus ◽  
Amino Acid ◽  
Methicillin Resistant Staphylococcus Aureus ◽  
Binding Protein ◽  
Amino Acid Substitutions ◽  
Penicillin Binding Protein ◽  
Methicillin Resistant ◽  
Content Type ◽  
Cefoxitin Disk ◽  
Cefoxitin Disk Diffusion

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.

Antimicrobial Susceptibility Testing for Staphylococcus lugdunensis

2021 ◽  
Author(s):  
Joanne S.K. Teh ◽  
Ioanna Pantelis ◽  
Xiao Chen ◽  
Tania Sadlon ◽  
Kelly Papanaoum ◽  
...  
Keyword(s):  
Antimicrobial Susceptibility ◽  
Susceptibility Testing ◽  
Resistant Isolate ◽  
Disc Diffusion ◽  
Broth Microdilution ◽  
Major Error ◽  
Staphylococcus Lugdunensis ◽  
Zone Diameter ◽  
Oxacillin Resistance ◽  
Zone Edge

Evaluation of penicillin and oxacillin susceptibility testing was conducted on two hundred Staphylococcus lugdunensis isolates. Disc diffusion with penicillin 1 IU (P1, EUCAST) and penicillin 10 IU (P10, CLSI) was compared with nitrocefin discs (Cefinase®) and automated broth microdilution (Vitek2®). Oxacillin susceptibility was extrapolated from cefoxitin 30μg disc diffusion (FOX) and compared with Vitek2®. Reference methods were blaZ and mecA PCR. Penicillin zone diameter and zone edge correlated with blaZ in all except two P10 susceptible isolates (VME; very major error) and one P1 resistant isolate (ME). One hundred and forty-eight isolates were blaZ -negative of which one hundred and forty-six and one hundred and forty-nine isolates were susceptible by P1 and P10 respectively. One hundred and twenty-seven isolates were penicillin susceptible by Vitek2®. Vitek2® overcalled resistance in twenty-one blaZ -negative, twenty P1 and twenty-two P10 susceptible isolates (Vitek2® ME rate, 14.2%). Two mecA -positive isolates were oxacillin resistant by FOX and Vitek2® (categorical agreement). However, eighteen FOX susceptible, mecA -negative isolates tested resistant by Vitek2®. In conclusion, Vitek2® over-estimated penicillin and oxacillin resistance compared with disc diffusion and PCR. Disc diffusion with zone edge interpretation was more accurate and specific than automated broth microdilution for S. lugdunensis in our study.

scholarly journals A Low-Affinity Penicillin-Binding Protein 2x Variant Is Required for Heteroresistance in Streptococcus pneumoniae

2014 ◽  
Vol 58 (7) ◽  
pp. 3934-3941 ◽  
Author(s):  
Hansjürg Engel ◽  
Moana Mika ◽  
Dalia Denapaite ◽  
Regine Hakenbeck ◽  
Kathrin Mühlemann ◽  
...  
Keyword(s):  
Streptococcus Pneumoniae ◽  
Abc Transporter ◽  
Binding Protein ◽  
Population Analysis ◽  
Resistance Testing ◽  
Penicillin Binding Protein ◽  
Secondary Resistance ◽  
Content Type ◽  
Abc Transporter Genes

ABSTRACTHeteroresistance to penicillin inStreptococcus pneumoniaeis the ability of subpopulations to grow at a higher antibiotic concentration than expected from the MIC. This may render conventional resistance testing unreliable and lead to therapeutic failure. We investigated the role of the primary β-lactam resistance determinants, penicillin-binding protein 2b (PBP2b) and PBP2x, and the secondary resistance determinant PBP1a in heteroresistance to penicillin. Transformants containing PBP genes from the heteroresistant strain Spain23F2349in the nonheteroresistant strain R6 background were tested for heteroresistance by population analysis profiling (PAP). We found thatpbp2x, but notpbp2borpbp1aalone, conferred heteroresistance to R6. However, a change ofpbp2xexpression was not observed, and therefore, expression does not correlate with an increased proportion of resistant subpopulations. In addition, the influence of the CiaRH system, mediating PBP-independent β-lactam resistance, was assessed by PAP onciaRdisruption mutants but revealed no heteroresistant phenotype. We also showed that the highly resistant subpopulations (HOM*) of transformants containing low-affinitypbp2xundergo an increase in resistance upon selection on penicillin plates that partially reverts after passaging on selection-free medium. Shotgun proteomic analysis showed an upregulation of phosphate ABC transporter subunit proteins encoded bypstS,phoU,pstB, andpstCin these highly resistant subpopulations. In conclusion, the presence of low-affinitypbp2xenables certain pneumococcal colonies to survive in the presence of β-lactams. Upregulation of phosphate ABC transporter genes may represent a reversible adaptation to antibiotic stress.

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