Integrative and conjugative elements mediate transfer of antibiotic resistance genes in haemophilus influenzae through conjugation mechanisms / Ahmad Norasidi Mohd Raffie

Haemophilus influenzae is a distinct member of the Haemophilus spp. They are classified into serotypable (type a-f) and non-typeable strains. The widespread uses of a conjugate vaccine against the highly pathogenic H. influenzae type b (Hib) had successfully limited the diseases. However, other serotypes such as H. influenzae type a (Hia) and non-typeable H. influenzae (NTHi) had emerged and became more prominent pathogen over the last few decades, causing diseases almost similar to Hib. One of the virulence factors necessary for bacterial survival is antibiotic resistance, and it became too familiar nowadays, including amongst H. influenzae. Most commonly, bacterial resistance genes are harboured by plasmids, either in stable form or larger, chromosomally integrated plasmid known as integrative and conjugative elements (ICE). This study aimed to identify the ICE in antibiotic resistance NTHi and observe the transfer of ICE to Hia through the conjugation mechanism. Resistance to antimicrobial drugs was determined using a standard disk diffusion method involving ampicillin, tetracycline, and co-trimoxazole, and later confirmed by PCR detection of the related genes. ICE was also identified by using PCR-gel electrophoresis detection of the specific determinants in its sequence. The conjugative transfer of ICE between different strains was carried out using the cell mating technique and confirmed by transconjugant colony formation on selective agar. Whole-genome re-sequencing was done to analyze the genome of the transconjugant cells upon the acquisition of ICE. The analyses were carried out using several bioinformatics tools to compare the obtained genetic sequences with the available database. Overall, 8/14 (57%) of NTHi and 2/4 (50%) of Hia were resistant to at least one of the antibiotics tested, and related resistance genes were detected in >90% of the resistant strains. ICE was detected in 5/8 (63%) of those antibiotic resistance NTHi and none for Hia. Based on these findings, NTHi H620 strain and Hia H86 were selected as donor and recipient for cell mating, respectively. However, ICE’s conjugative transfer between these strains was experimentally unsuccessful, as indicated by the absence of transconjugants on the selective agar. Nevertheless, the experiment was remodelled by using a similar strain NTHi H152 as the recipient. As a result, transconjugant colonies were formed after overnight incubation, and thus, the colonies were harvested and proceeded with DNA extraction. PCR amplification detected ICE and the donor’s resistance gene (blaTEM-1) as well as the recipient’s resistance genes (dfrA1) in the transconjugant DNA. Upon bioinformatics analyses, it was evident that the transconjugant’s ICE originates from the donor strain, carrying the blaTEM-1 gene in its transposon. The mobilization of antibiotic resistance genes through cell-to-cell conjugation in prokaryotes is not uncommon and has been reported in numerous studies worldwide. Only these recent years, scientists were aware of the role of ICE in this process apart from stable, standalone plasmids’, which could explain the increasing cases of antibiotic-resistant bacteria these days. The unsuccessful transfer of ICE between NTHi and Hia in this study may be caused by a few underlying factors not fully understood yet. Nevertheless, future studies may investigate loopholes in this research to elucidate the actual mechanisms in resistance genes transfer and provide valuable insights in combating this problem.