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

Abstract

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...

Comprehensive antimicrobial susceptibility testing of a large collection of clinical strains of Aggregatibacter actinomycetemcomitans does not identify resistance to amoxicillin

“…Examples include the risk of creating an inoculum effect by use of too high concentration of inoculum (Gould, Heidecker, & LiPuma, ); the risk of overestimating the growth of the bacteria by extending the period of inoculation before interpretation (EUCAST & ESCMID, ; Matuschek, Brown, & Kahlmeter, ); and the risk of decreasing the antimicrobial concentration due to interactions between components in inappropriate media and the antimicrobial agent. For H. influenzae , it has been shown that the Etest generally overestimates MICs at lower ranges and underestimates MICs at higher range using broth microdilution as the gold standard (Skare et al, ).…”

Ampicillin resistance in <em>Haemophilus influenzae</em> from COPD patients in the UK

“…The clinical relevance of current breakpoints has been debated 11,26–28. The MIC of gBLNAR has been previously reported between 0.5 and 16 µg/mL,11,26,27 and the reduced reliability of gradient tests for H. influenzae sensitivity to aminopenicillins has been documented 29…”

“…Non-enzyme-mediated resistance occurs through mutations in penicillin-binding protein 3 (PBP3), with ftsI gene mutations identified as a cause of decreased β-lactam-binding affinity. This is termed β-lactamase-negative ampicillin resistance (BLNAR) 11. Disk diffusion and gradient tests are routinely used to evaluate the antibiotic sensitivity.…”

Impact of different amino acid substitutions in penicillin-binding protein 3 on beta-lactam susceptibility in Haemophilus influenzae

“…The most frequently found group of amino acid substitutions belonged to group II (19/53; 35.8%), which was also observed in studies from other European countries (3, 14, 20, 21, 36). We could not detect strains of the group III, which harbours high-level PBP3 alterations (11, 35).…”

“…In recent years, alterations in PBP3 leading to decreased binding affinity of certain beta-lactam antibiotics have emerged as a prevalent resistance mechanism in H. influenzae (35). In this study, we investigated the mutations in the ftsI gene encoding for PBP3 within a collection of clinical isolates from two Swiss hospitals.…”