First Characterization of Heterogeneous Resistance to Imipenem in Invasive Nontypeable Haemophilus influenzae Isolates

Abstract

This study describes the first two reported invasive nontypeable Haemophilus influenzae (NTHI) isolates (strains 183 and 184) with heterogeneous resistance to imipenem. For both isolates, Etest showed imipenem MICs of >32 g/ml. When the two strains were examined by the quantitative method of population analysis, both strain populations were heterogeneously resistant to imipenem and contained subpopulations growing in the presence of up to 32 g of imipenem/ml at frequencies of 1.7 ؋ 10 ؊5 and 1.5 ؋ 10 ؊7 , respectively. By pulsed-field gel electrophoresis analysis, the two isolates appeared to be genetically closely related. The sequencing of the ftsI gene encoding penicillin-binding protein 3 (PBP 3) and comparison with the sequence of the imipenem-susceptible H. influenzae strain Rd identified a pattern of six amino acid substitutions shared between strains 183 and 184; an additional change was unique to strain 183. No relationship between mutations in the dacB gene encoding PBP 4 and imipenem resistance was found. The replacement of the ftsI gene in the imipenem-susceptible strain Rd (for which the MIC of imipenem is 0.38 to 1 g/ml) with ftsI from strain 183 resulted in a transformant for which the MIC of imipenem ranged from 4 to 8 g/ml as determined by Etest. The Rd/183 transformant population showed heterogeneous resistance to imipenem; it contained subpopulations growing in the presence of up to 32 g of imipenem/ml at a frequency of 3.3 ؋10؊8 . The presence of additional resistance mechanisms, such as the overexpression of the AcrAB efflux pump, was investigated and does not seem to be involved. These data indicate that the heterogeneous imipenem resistance phenotype of our NTHI clone depends largely on the PBP 3 amino acid substitutions. We speculated that bacterial regulatory networks may play a role in the control of the heterogeneous expression of the resistance phenotype.Despite the decrease in the incidence of invasive Haemophilus influenzae type b (Hib) disease as a result of the widespread use of Hib conjugate vaccines, H. influenzae remains a significant cause of invasive disease among adults, especially patients with underlying predisposing conditions (26). In adults, invasive H. influenzae infections, such as meningitis and septicemia, have been associated mainly with nontypeable H. influenzae (NTHI) (3, 5). The treatment of these life-threatening infections can be severely affected by antibiotic resistance. For years, ampicillin (alone or in combination with chloramphenicol) was regarded as the drug of choice in the treatment of invasive H. influenzae disease, but since the emergence of ampicillin-resistant isolates, expanded-spectrum cephalosporins have been extensively used. Carbapenems are considered a useful alternative to expanded-spectrum cephalosporins in treating invasive infections caused by ampicillin-resistant H. influenzae isolates (4). Until now, carbapenems have maintained a high level of activity against H. influenzae. Rare imipenem-or meropenem-resistant H. influenzae isolates h...

Antimicrobial Heteroresistance: an Emerging Field in Need of Clarity

“…In Enterobacter species, mutation of ampD, which is involved in the regulation of production of a class C ␤-lactamase, at rates as high as 10 Ϫ4 to 10 Ϫ6 , resulted in a heterogeneous population of bacterial cells with differing levels of ␤-lactam resistance (135). Heteroresistance of invasive nontypeable H. influenzae to imipenem depended in part on the penicillin-binding protein PBP3, encoded by ftsI, on PBP4, encoded by dacB, or on the AcrAB efflux system, with a potential role of regulatory networks in the control of the heterogeneous expression of the resistance phenotype (35). In B. cenocepacia, an ornithine decarboxylase homologue and YceI, a small conserved protein, played a role in heteroresistance to ceftazidime (6).…”

“…Chemical communication of antibiotic resistance from the more resistant members of the population protecting less resistant bacteria is the major concern of such bacterial populations. tion, with 2-fold antibiotic increments, and by use of spread plate techniques for CFU counting (3,4,6,8,14,16,18,19,(25)(26)(27)(28)(29)(30)(31)(32)(33)(34)(35)(36)(37)(38)(39)(40)(41). Counting of CFU by dropping smaller aliquots is as efficient as spread plate techniques (6,42).…”

Carbapenems: Past, Present, and Future

“…Expression of PBPs in P. aeruginosa, A. baumannii, P. mirabilis, and Rhodococcus equi is decreased, resulting in carbapenem resistance (57, 68, 71, 150, 154). In addition, amino (6,17,31,56,100,109,134,150,158,165,184,185,218).…”

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

“…DNA extracts were eluted in 100 μl elution buffer. Amplification of ftsI gene was performed by using the conventional PCR assay described by Cerquetti et al (31). PCR products were purified with ExoSAP-IT® (USB Corporation, Cleveland, OH, USA) purification kit.…”

“…PCR products were purified with ExoSAP-IT® (USB Corporation, Cleveland, OH, USA) purification kit. The sequencing of the PCR amplicon was performed by Microsynth AG (Balgach, Switzerland) using Sanger sequencing (sequencing primer frw : 5’-GCGGATAAAGAACGAATTGC-3’ (14), sequencing primer rev : 5’-CTGGATAATTCTGTCTCAGA-3’ (31)). Sequences were aligned with Lasergene SeqMan Pro (DNASTAR, Madison, WI, USA) and translated into amino acid sequences using the ExPASy translate tool (http://web.expasy.org/translate/).…”