The epidemiology of non-typeable Haemophilus influenzae (NTHi) remains poorly understood. We therefore sought to determine the genetic relationship of 25 NTHi isolated from various states in Malaysia using multilocus sequence typing (MLST). The majority of isolates were obtained from sputum. There were 24 novel sequence types (STs). Eight isolates were single-locus variants, the remainder being singletons. Clustering was not based on clinical site of isolation or geographical origin. Despite the limited number of isolates examined in this study, we demonstrate that NTHi isolates in Malaysia are diverse and warrant further investigation. A C C E P T E D M A N U S C R I P T ACCEPTED MANUSCRIPT

Objective: This study aimed to determine the effects of the tocotrienol-rich fraction (TRF) on the regulations of phosphoinositide 3-kinases (PI3K)/Aktpathways related genes in preimplantation embryos induced by nicotine (Nic).Methods: Twenty-four female BALB/c mice were divided into four groups with Nic and TRF supplementation for 7 consecutive days. Animalswere superovulated before mating with fertile males. Plasma malondialdehyde, superoxide dismutase, catalase, and glutathione peroxidase weredetermined and analyzed accordingly. Embryos with two and eight blastomeres were assessed for gene expression analysis.Results: The levels of endogenous antioxidative enzymes for the group with TRF intervention and TRF only group showed no significant changes whencompared to the control group. The level of oxidative stress (OS) biomarkers was also significantly decreased when compared to the Nic-induced group.At 2-cell stage, Pten gene was significantly upregulated while Akt1, GSK3β, and Mapk1 were significantly downregulated almost similar to the baseline(control) in the Nic-induced mice. Intervention with TRF resulted in a significant downregulated of Pten gene followed by a significant upregulationof other genes. The same pattern was shown at the 8-cell stage.Conclusion: This showed that TRF evidently has OS protection capacity and it could be through modulating the PI3K/Akt signaling pathway.

Background: Nontypeable Haemophilus influenzae (NTHi) is known to be the causative agent of many chronic diseases such as otitis media, pneumonia, meningitis and sinusitis.Methods: In this work, a collection of 28 NTHi strains isolated from 12 different origin in Malaysia were examined on their antimicrobial susceptibility profile to 10 antibiotics using the diskdiffusion method. These strains were also analysed to determine for genotypic differences using a cost-effective typing method i.e. restriction fragment length polymorphisms (RFLP). In the RFLP analysis, all of the isolates were amplified by using 16S primers and digested with six restriction enzymes, namely EcoRI, HindIII, Hsp92I, BamHI, HaeIII and Hin1II.Results: Antimicrobial susceptibility test showed that 33% of the isolates were resistant to two or more of the tested antimicrobials. NTHi isolates demonstrated resistance most frequently to ampicilin (43.75%), trimethoprim-sulfomethoxazole (31.25%) and 6.25% each to tetracycline, streptomycin and rifampicin. With the exception of Hin1II, the results of the digestion with the other five restriction enzymes were not reproducible and thus, the results were excluded. Digestion with Hin1II produced between two to three fragments in each isolate. The DNA fragments of 50-200 bp size could be clearly resolved into two RFLP patterns among the 28 isolates. It was observed that the isolates fell into two clusters with genetic distances of >60% similarity. There were seven branches at F-value of ≥0.9, representing at a genetic distance of more than 90% similarity of which these branches can be referred to as clonal groups. This study demonstrated that 27 of 28 (96.4%) NTHi strains that were isolated from 12 distant locations had 100% similarity in their RFLP patterns. Few loci were detected and various other combinations of restriction enzymes for typing of NTHi would be required. Conclusion:This study showed the occurrence of antimicrobial resistance amongst NTHis isolated in Malaysia and although they were geographical-distantly isolated, they are genetically related.