Data_Sheet_1_Characterization and Fitness Cost of Tn7100, a Novel Integrative and Conjugative Element Conferring Multidrug Resistance in Haemophilus influenzae.pdf

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.

本研究从挪威奥斯陆分离得到一株多重耐药（multidrug-resistant, MDR）流感嗜血杆菌（Haemophilus influenzae）Hi-228，该菌株对氨苄西林、头孢噻肟、氯霉素、庆大霉素及阿奇霉素均表现出表型耐药性。
该菌株属于2016至2017年的一次克隆暴发株系，该暴发涉及5株具有完全一致耐药型的序列型143（ST143）菌株。
Hi-228携带一株新型整合接合元件（integrative and conjugative element, ICE）Tn7100，该元件正是该菌株呈现此前未见报道的超强多重耐药表型的原因。
Tn7100携带以下耐药基因：blaTEM−1B、catA2、aac(6′)-Im、aph(2″)-Ib、mef(E)以及mel。其中后4种耐药基因此前在流感嗜血杆菌中未见报道或仅极罕见报道。
本研究针对该整合接合元件的遗传背景、转移机制、对表型药敏性的影响以及适合度成本展开了系统研究。
研究发现，Tn7100的整体结构与此前已报道的整合接合元件Tn6686高度相似，其携带的blaTEM−1B与catA2分别由Tn3与Tn10承载。
Tn7100与Tn6686的主要差异在于：Tn7100缺失tet(B)基因，但额外携带两组耐药基因对——aac(6′)-Im与aph(2″)-Ib，以及mef(E)与mel。
上述两组耐药基因对分别位于新型转座元件Tn7470与Tn7471之上，其中Tn7470与肠杆菌目（Enterobacterales）的一株质粒具有高度序列同源性，而Tn7471则与链球菌属菌株的整合接合元件序列相似度极高。
Tn7100可发生环化，且具备转移能力，但转移频率较低。
竞争性生长实验结果显示，获得Tn7100会降低菌株的生存适合度。
本研究证实，多重耐药菌株可通过克隆方式实现传播；同时流感嗜血杆菌的泛基因组（supragenome）所涵盖的可转移耐药基因范围正不断扩大，且存在跨属水平基因转移的证据。
本研究结果为解析流感嗜血杆菌的基因组动态变化提供了新视角，同时凸显了合理使用抗生素对于遏制该重要病原菌的耐药性传播及多重耐药菌株出现的重要意义。