Detection of N526K-substituted penicillin-binding protein 3 conferring low-level mutational resistance to  -lactam antibiotics in Haemophilus influenzae by disc diffusion testing on Mueller-Hinton agar according to EUCAST guidelines

Søndergaard, Annette; Petersen, Marianne Tidemann; Fuursted, Kurt; Nørskov‐Lauritsen, Niels

doi:10.1093/jac/dks070

Cited by 6 publications

(5 citation statements)

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“…Some cephalosporin discs, such as cefaclor, cefoxitin and cefuroxime (30 mg each), had been proposed for the detection of the ftsI mutation N526-K. 10 In the present study, the cefoxitin 30 mg disc (and a zone diameter breakpoint of 22 mm) was found to be useful for the detection of gBLPACR isolates (Table 1).…”

Section: Gblpacr Epidemiological Cut-offsmentioning

confidence: 54%

Evaluation of the EUCAST disc diffusion susceptibility testing method for Haemophilus influenzae based on the resistance mechanism to -lactam antibiotics

García-Cobos

Arroyo

Pérez-Vázquez

et al. 2012

Journal of Antimicrobial Chemotherapy

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Objectives: EUCAST developed an antibiotic susceptibility testing method for Haemophilus influenzae. We assessed the EUCAST testing method and EUCAST clinical breakpoints and newly proposed epidemiological cut-off values against H. influenzae clinical isolates with known molecular mechanisms of resistance to b-lactam antibiotics.Methods: In total, 89 clinical isolates were used: 30 were b-lactamase negative with PBP3 mutations (gBLNAR), 20 were b-lactamase positive without PBP3 mutations (gBLPAR), 15 were b-lactamase positive with PBP3 mutations (gBLPACR), and 24 were b-lactamase negative without resistance mechanism (gBLNAS). Twelve different b-lactam antibiotics and disc charges were tested.Results: None of the discs tested fully separated between gBLNAS and gBLNAR populations. According to EUCAST clinical zone diameter breakpoints, overall the best values of sensitivity and specificity were obtained with cefuroxime 30 mg and amoxicillin/clavulanic acid 2/1 mg discs for detection of gBLNAR and gBLPACR populations, although a previous b-lactamase test was needed. Other antibiotic discs could be suitable for epidemiological purposes, such us penicillin 10 U for separating gBLNAR isolates and cefoxitin 30 mg for detection of gBLPACR isolates. By Etest using the EUCAST method, the EUCAST MIC clinical breakpoints for ampicillin and amoxicillin/clavulanic acid showed high specificity, but low sensitivity, for the detection of genotypes with mutations in PBP3. Conclusions:The main genotypes of b-lactam-resistant H. influenzae can be separated by using the EUCAST disc diffusion method, although it should be noted that overlapping between populations with and without PBP3 mutations is common.

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Section: Gblpacr Epidemiological Cut-offsmentioning

confidence: 54%

Evaluation of the EUCAST disc diffusion susceptibility testing method for Haemophilus influenzae based on the resistance mechanism to -lactam antibiotics

García-Cobos

Arroyo

Pérez-Vázquez

et al. 2012

Journal of Antimicrobial Chemotherapy

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“…As PG1 is unsuitable for rPBP3 screening in bla-positive strains, we evaluated five disks with agents that are stable in the presence of beta-lactamase. The CXM5 disk categorized isolates according to resistance genotype with the highest accuracy (92.2%) and was superior to the previously evaluated CEC30, CXM30, AMC3, and AMC30 disks (29,31); however, CXM5 is currently not available from all manufacturers.…”

Section: Discussionmentioning

confidence: 81%

“…The PG1 disk recommended for screening by EUCAST (14) correctly categorized 95.5% of the isolates according to resistance genotype in the present study. Søndergaard et al found similar accuracy (96%) but lower sensitivity (91%) using the same method (29). Diverging results may be due to different interpretation of hazy growth (24); this phenomenon may vary with MHA from different manufacturers (unpublished data).…”

Section: Discussionmentioning

confidence: 94%

“…Using identical test populations, disk diffusion with current EUCAST breakpoints was noninferior (AMP2) or superior (AMC3) to Etest for categorization of susceptibility to aminopenicillins; with minor adjustments in zone breakpoints, both disks were superior to Etest. In a previous evaluation, Søndergaard et al tested 135 bla-negative H. influenzae isolates (33% rPBP3) and reported that six isolates were ampicillin resistant by both EUCAST disk diffusion (MH-F medium) and HTM BMD (MIC Ͼ 1 mg/liter), whereas 10% (i.e., 13 or 14 isolates) were resistant by BMD only (29). Thus, more than half (7/13 or 8/14) of the ampicillin-resistant isolates were falsely categorized as susceptible with the AMP2 disk, compared to 76.7% (current breakpoints) and 28.3% (adjusted breakpoints) in the present study.…”

Section: Discussionmentioning

confidence: 99%

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Haemophilus influenzae with Non-Beta-Lactamase-Mediated Beta-Lactam Resistance: Easy To Find but Hard To Categorize

et al. 2015

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Haemophilus influenzae is a major pathogen, and beta-lactams are first-line drugs. Resistance due to altered penicillin-binding protein 3 (rPBP3) is frequent, and susceptibility testing of such strains is challenging. A collection of 154 beta-lactamase-negative isolates with a large proportion of rPBP3 (67.5%) was used to evaluate and compare Etest (Haemophilus test medium [HTM]) and disk diffusion (EUCAST method) for categorization of susceptibility to aminopenicillins and cefuroxime, using MICs generated with broth (HTM) microdilution and clinical breakpoints from CLSI and EUCAST as the gold standards. In addition, the proficiency of nine disks in screening for the rPBP3 genotype (N526K positive) was evaluated. By Etest, both essential and categorical agreement were generally poor (<70%), with high very major errors (VME) (CLSI, 13.0%; EUCAST, 34.3%) and falsely susceptible rates (FSR) (CLSI, 87.0%; EUCAST, 88.3%) for ampicillin. Ampicillin (2 g) with adjusted (؉2 mm) zone breakpoints was superior to Etest for categorization of susceptibility to ampicillin (agreement, 74.0%; VME, 11.0%; FSR, 28.3%). Conversely, Etest was superior to 30 g cefuroxime for categorization of susceptibility to cefuroxime (agreement, 57.1% versus 60.4%; VME, 2.6% versus 9.7%; FSR, 7.1% versus 26.8%). Benzylpenicillin (1 unit) (EUCAST screening disk) and cefuroxime (5 g) identified rPBP3 isolates with highest accuracies (95.5% and 92.2%, respectively). In conclusion, disk screening reliably detects rPBP3 H. influenzae, but false ampicillin susceptibility is frequent with routine methods. We suggest adding a comment recommending high-dose aminopenicillin therapy or the use of other agents for severe infections with screening-positive isolates that are susceptible to aminopenicillins by gradient or disk diffusion. N ontypeable Haemophilus influenzae (NTHi) frequently causes acute otitis media, conjunctivitis, sinusitis, and respiratory tract infections, including pneumonia and exacerbations in chronic obstructive pulmonary disease and cystic fibrosis, and may also cause invasive disease (1). Beta-lactams are first-line drugs when systemic therapy is indicated, but resistance due to transferable beta-lactamase genes (bla TEM or bla ROB ) and/or substitutions in penicillin-binding protein 3 (PBP3), encoded by the ftsI gene, is common (1, 2). Strains with low-level PBP3-mediated resistance (low-rPBP3) possess the R517H (group I) or the N526K (group II) substitution, whereas strains with high-level resistance (high-rPBP3) are characterized by the additional substitution S385T (2, 3). The distinction is clinically important because high-rPBP3 strains express higher resistance to extended-spectrum cephalosporins (1, 2, 4-6). Group II low-rPBP3 is the predominating genotype in Australia (7), Europe (8, 9), and North America (6, 10), whereas high-rPBP3 strains predominate in Japan and Korea (6, 11).Susceptibility testing of H. influenzae has been characterized as "a tricky business" (12). Categorization of susceptibility to aminopenicillins (wi...

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“…A screening algorithm to identify rPBP3 strains in routine diagnostics based on disc diffusion with 1 U benzylpenicillin is suggested by the European Committee on Antimicrobial Susceptibility Testing (EUCAST) [ 10 ]. This screening algorithm has demonstrated high sensitivity and specificity in detecting rPBP3 isolates, although confirmatory testing of actual MIC levels is recommended to determine if screening-positive isolates are actually resistant [ 8 , 11 ]. Since current clinical breakpoints for aminopenicillins split the rPBP3 group, this implies that a subset of rPBP3 isolates are still considered susceptible [ 8 ] (Table 1 ).…”

Section: Introductionmentioning

confidence: 99%

A novel PBP3 substitution in Haemophilus influenzae confers reduced aminopenicillin susceptibility

Thegerström

Matuschek

et al. 2018

BMC Microbiol

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BackgroundIdentification and characterization of non-typeable Haemophilus influenzae (NTHi) with reduced susceptibility to β-lactam antibiotics due to mutations in penicillin binding protein 3 (PBP3) is a clinical challenge. We analyzed a blood isolate, NTHi93–57485, that was categorized as aminopenicillin resistant but lacked key amino acid substitutions in PBP3 that have previously been associated with reduced aminopenicillin susceptibility. The significance of an alternative amino acid substitution (Y528H) in this isolate was examined.ResultsSite-directed mutagenesis of a β-lactam susceptible H. influenzae (NTHi3655) was performed to introduce the Y528H substitution into wild-type ftsI (encoding for PBP3). Disc diffusion screening and broth microdilution determination of MICs for β-lactam agents were done with the NTHi3655-PBP3Y528H mutant and were compared with the NTHi3655 wild-type as well as the original clinical isolate NTHi93–57485. Introduction of the Y528H substitution in NTHi3655 resulted in positive screening for β-lactam resistance. MICs for aminopenicillins were increased in the mutant compared to the wild-type. However, the mutant remained susceptible to aminopenicillins according to EUCAST clinical breakpoints (assuming intravenous treatment) and the introduction of Y528H alone did not increase the resistance to the same level as NTHi93–57485. None of the isolates had frame shift insertions in the acrR gene regulating the AcrAB efflux pump.ConclusionsIn parallel to the previously well-described PBP3-substitutions R517H and N526K, we demonstrate that Y528H confers reduced aminopenicillin susceptibility.Electronic supplementary materialThe online version of this article (10.1186/s12866-018-1196-6) contains supplementary material, which is available to authorized users.

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Detection of N526K-substituted penicillin-binding protein 3 conferring low-level mutational resistance to -lactam antibiotics in Haemophilus influenzae by disc diffusion testing on Mueller-Hinton agar according to EUCAST guidelines

Cited by 6 publications

References 7 publications

Evaluation of the EUCAST disc diffusion susceptibility testing method for Haemophilus influenzae based on the resistance mechanism to -lactam antibiotics

Evaluation of the EUCAST disc diffusion susceptibility testing method for Haemophilus influenzae based on the resistance mechanism to -lactam antibiotics

Haemophilus influenzae with Non-Beta-Lactamase-Mediated Beta-Lactam Resistance: Easy To Find but Hard To Categorize

A novel PBP3 substitution in Haemophilus influenzae confers reduced aminopenicillin susceptibility

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