Interspecies Transfer of the Penicillin-Binding Protein 3-Encoding Gene ftsI between Haemophilus influenzae and Haemophilus haemolyticus Can Confer Reduced Susceptibility to β-Lactam Antimicrobial Agents

Abstract

bMutations in ftsI, encoding penicillin-binding protein 3, can cause decreased ␤-lactam susceptibility in Haemophilus influenzae. Sequencing of ftsI from clinical strains has indicated interspecies recombination of ftsI between H. influenzae and Haemophilus haemolyticus. This study documented apparently unrestricted homologous recombination of ftsI between H. influenzae and H. haemolyticus in vitro. Transfer of ftsI from resistant isolates conferred similar but not identical increases in the MICs of susceptible strains of H. influenzae and H. haemolyticus. Haemophilus influenzae is the major human pathogen of the genus Haemophilus (1, 2), and infections are usually treated with ␤-lactam antimicrobial agents. Amino acid substitutions in penicillin-binding protein 3 (PBP3), encoded by the ftsI gene, can confer decreased susceptibility to ␤-lactams in strains lacking ␤-lactamase genes (3, 4). Genetically defined ␤-lactamase-negative ampicillin-resistant (gBLNAR) isolates carry either the N526K (Ubukata group II) or the R517H substitution in PBP3, while the additional substitutions S385T and/or L389F (Ubukata group III) further reduce susceptibility (3, 5-7). A range of other amino acid substitutions has been identified, but their significance is unclear (3-5, 8, 9).Whether the worldwide spread of gBLNAR isolates is caused by clonal dissemination or horizontal gene transfer is controversial (8,10,11). H. influenzae and Haemophilus haemolyticus are close relatives, (1) and putative transfer of ftsI between these two species is indicated by mosaic structures of ftsI from clinical and nasopharyngeal carriage isolates (12,13).(Part of these data was presented on a poster at the 2014 International Pasteurellaceae Conference, Prato, Italy, 15 May 2014.)The present study was undertaken to examine putative species barriers and delineate transformation events after ftsI transfer under standardized in vitro conditions. ftsI genes (1,833 bp) plus flanking regions from two gBLNAR H. influenzae strains and two gBLNAR H. haemolyticus strains were amplified by PCR (primers used for amplification and sequencing are listed in Table S1 in the supplemental material) and used for electroporetic transformation of susceptible strains of H. influenzae and H. haemolyticus (one representative each) as previously described (3). Characteristics of the donor and recipient strains are listed in Table 1, and overall similarity of ftsI and PBP3 are given in Table S2 in the supplemental material. Transformants were selected on chocolate agar (Columbia agar [Oxoid] supplemented with 5% horse blood and Vitox [Oxoid]) containing 0.5 g/ml ampicillin (AMP) and screened for the presence of the N526K substitution using real-time PCR as previously described (14). We observed intraspecies ftsI transformation frequencies between 4.2 ϫ 10 Ϫ7 and 6.7 ϫ 10 Ϫ7 with H. influenzae strain Rd as the recipient and between 7 ϫ 10 Ϫ5 and 1 ϫ 10 Ϫ4 with H. haemolyticus strain ATCC 33390T as the recipient; we observed interspecies ftsI transformation frequencies betwee...

Changes in the prevalence and biofilm formation of Haemophilus influenzae and Haemophilus parainfluenzae from the respiratory microbiota of patients with sarcoidosis

“…It may explain the acquisition of resistance or resistance gene exchange with other microorganisms. Besides, resistance and reduced susceptibility to beta-lactams mediated by altered PBPs is also important in many bacterial pathogens, including beta-lactamase negative H. influenzae [ 47 ]. For this reason, there are different events that may contribute to the emerge of resistance: the acquisition of resistance genes (e.g.…”

“…For this reason, there are different events that may contribute to the emerge of resistance: the acquisition of resistance genes (e.g. beta-lactamases) by conjugation or transformation; and inter-species recombination of the ftsI gene [ 47 , 48 ]. According to Gromkova et al [ 46 ], most efficient in transformation among H. parainfluenzae strains was biotype II, followed by biotype I.…”

Complete Deletion of the Fucose Operon in Haemophilus influenzae Is Associated with a Cluster in Multilocus Sequence Analysis-Based Phylogenetic Group II Related to Haemophilus haemolyticus: Implications for Identification and Typing

“…Recombination between the two species may not be rare (38,39) and may even involve rRNA genes. Comparative whole-genome analysis of H. influenzae and H. haemolyticus is warranted to elucidate the genomic differences between the two species (36,40).…”

Use of trans-complementation method to determine the effects of various ftsI mutations on β-lactamase-negative ampicillin-resistant (BLNAR) Haemophilus influenzae strains

“…In this study, a total of 7 H. in uenzae strains were used to transform with pLS88 plasmid and pADUTAS17 plasmid which contained wild type ftsI gene in pLS88 and the changes in sensitivity against to ampicillin and cefotaxime antibiotics were analyzed with and without trans-complementation with wild type ftsI gene. These strains were H. in uenzae RdΏBLNAR1 [7], H. in uenzae RdΏBLPACR4 [7], H. in uenzae RdΏBLPACR7 [7], H. in uenzae CF55 [2], H. in uenzae UTAS252 (Søndergaard et al, 2015), H. in uenzae ATCC49247, and H. in uenzae Rd strains (ATCC51907) (Table 1). We chose cloned strains (Rd transformed with mutated ftsI) because we have a baseline MIC (that of plain Rd) to see if the MIC of the cloned Rd BLNAR is close to totally reversed by trans-complementation.…”