Conjugal transfer of co-trimoxazole resistance gene from non-typeable haemophilus influenzae to haemophilus influenzae type B / Siti Yatimah Mohamad

Haemophilus influenzae is one of the pathogens that reside in human nasopharynx. Amongst all the types of H. influenzae, type b (Hib) is the most invasive while non­ typeable H. influenzae (NTHi) is an opportunist. Co-trimoxazole (SXT) is one of the drugs of choice to treat respiratory infections due to H. influenzae. However, this antibiotic has lost its potency as there are multiple reports that showed the emergence of SXT-resistant H. influenzae strains worldwide. A previous report in Malaysia showed that many strains of NTHi were resistant to SXT. When both strains ofNTHi and Hib co-exist in the nasopharynx, there is a possibility that SXT-resistant genes from NTHi can be transferred through conjugation. Therefore, this study intents to elucidate the mechanism of transfer of SXT resistance genes from NTHi to Hib when they are in close contact. Three methods of in-vitro mating (filter, liquid and solid) were performed to transfer SXT-resistant genes from NTHi (strain H152) to an SXT-sensitive Hib (strain H582). Attempts were made by varying the donor-to-recipient ratio (1: 1 and 1: 10). However, all three mating methods used failed to produce any transconjugants. In order to understand the reason of the inability of H 152 to transfer the SXT-resistant genes to H582, the DNA of both strains was sent for whole genome re-sequencing. Comparative analysis of amino acid sequences of both strains revealed that H152 contained 11 amino acids mutation in dihydrofolate reductase (DHFR) and insertion of five amino acids in dihydropteroate synthase (DHPS) that contributed to the SXT­ resistance. In addition, the attachment site attP, the DNA region for site-specific recombination, was present only in H582 but not in H152. Type IV secretory pathway (VirB4) components required for conjugation were only present in H582 and absent in H 152. As a conclusion, NTHi strain H 152 used in this study was not able to transfer the SXT-resistant gene to H582 due to lack of gene components for conjugation.