Characterization and Fitness Cost of Tn7100, a Novel Integrative and Conjugative Element Conferring Multidrug Resistance in Haemophilus influenzae

Abstract

A multidrug-resistant (MDR) strain of Haemophilus influenzae, Hi-228, with phenotypic resistance toward ampicillin, cefotaxime, chloramphenicol, gentamicin, and azithromycin, was isolated in Oslo, Norway. The strain was part of a clonal outbreak (2016–2017) comprising five ST143 strains with identical resistotypes. Hi-228 carries a novel integrative and conjugative element (ICE), Tn7100, contributing to this remarkable and previously unreported MDR profile. Tn7100 contains the following resistance genes: blaTEM−1B, catA2, aac(6′)-Im, aph(2″)-Ib, mef (E), and mel. The latter four are previously unreported or rarely reported in H. influenzae. In this study, we investigated the genetic environment, mechanisms of transfer, impact on phenotypic susceptibility, and fitness cost of this ICE. We found that Tn7100 has an overall structure similar to the previously described ICE Tn6686, with blaTEM−1B and catA2 carried by Tn3 and Tn10, respectively. The major difference between Tn7100 and Tn6686 is that Tn7100 lacks tet(B) but carries the resistance gene pairs aac(6′)-Im and aph(2″)-Ib and mef (E) and mel. The gene pairs are located on the novel transposable elements Tn7470 and Tn7471, which have high sequence identities to a plasmid in Enterobacterales and an ICE in streptococcal species, respectively. Tn7100 does circularize and is transferable, however, at a low frequency. Head-to-head competition experiments showed that uptake of Tn7100 reduces bacterial fitness. Our study shows that MDR strains are capable of clonal spread and that the H. influenzae supragenome comprises an increasingly wide range of transferable resistance genes, with evidence of transfer from unrelated genera. The findings offer a glimpse into the genome dynamics of H. influenzae, highlighting the importance of rational antibiotic usage to contain antimicrobial resistance and the emergence of MDR strains in this important pathogen.

Molecular epidemiology and antibiotic resistance profiles of invasive Haemophilus influenzae from Norway 2017–2021

“…A similar combination of nonbeta-lactam resistance genes was present in the genomes of two out of 15 Hia isolates (13.3%). These MDR profiles, to the best of our knowledge previously unreported in H. influenzae, suggest the presence of integrative conjugative elements (ICE) acquired through horizontal transfer (Hegstad et al, 2020;Johannessen et al, 2022). The molecular environment of the resistance genes and the presence of mobile genetic elements will be further investigated in a planned future study.…”

The alginate polymer OligoG alters susceptibility of biofilm-embedded non-typeableHaemophilus influenzaeto ampicillin and ciprofloxacin

“…For quantification of viable cells, the suspensions were diluted to 10 −8 and seeded quantitatively on Colombia agar plates (BBL Colombia agar, Becton Dickinson) with 15 mg/L NAD and 15 mg/L haemin. 8 The agar plates were incubated at 35°C in 5% CO 2 for 48 h, with subsequent determination of cfu/mL by colony counts.…”

“… 7 Since the year 2000, NTHi strains resistant to cefotaxime and other extended-spectrum cephalosporins have emerged in several geographical regions and constitute approximately half of the isolates in Japan, 5 , 6 and dissemination of MDR strains with co-resistance to clinically important non-β-lactams such as quinolones, trimethoprim/sulfamethoxazole, tetracyclines and/or macrolides restrict alternative therapeutic options. 6 , 8 , 9 …”

Horizontal Gene Transfer of Antibiotic Resistance Genes in Biofilms

“…There are just a handful of studies that investigated ICEs in biofilms. Nevertheless, multiple studies have demonstrated ICEs as the main reservoir for several resistance genes in biofilm-producing species [326][327][328][329][330].…”