The treatments of chronic osteomyelitis are difficult, time-consuming and relatively expensive due to the presence of bacterial biofilm that is highly resistant to antibiotics. This study aimed to assess synergistic antibacterial activities of gentamicin-Nigella sativa fusion towards the most common biofilm-bacteria in osteomyelitic infection. Briefly, a total 57 samples (prostheses, bones, tissues and swabs) were taken from 17 cases of osteomyelitic infection at Hospital Tengku Ampuan Afzan, Kuantan. The samples were processed, isolated species were identified, as well as biofilm identification and antibiotic sensitivity assays were performed. Fusion of gentamicin and N.sativa were formulated in 4 different types of emulsions (A, B, C, and D) consisting of constant 0.1% (w/v) gentamicin and different Nigella sativa oil concentrations from 32.5% to 46.6% (v/v). Antimicrobial activities of the emulsions were evaluated using disc diffusion assay and determination of minimum inhibitory concentration (MIC) and minimum bactericidal concentration (MBC). Then, the assessment of antibiofilm activities was carried out as pre- and post-biofilm assays. The pre-biofilm consists of biofilm formation inhibition and minimum biofilm inhibition concentration (MBIC). The post-biofilm assay was done to evaluate the effects of the emulsions on the biofilm, using biofilm penetration test and confocal laser screening microscope (CLSM) analysis. It was found that prosthesis (89%) and bone (66.7%) samples produce the most bacteria growth and Staphylococcus aureus (10 out of 16) was the most frequently identified. In the disc diffusion assay, significant synergistic effect of emulsions was seen only in resistant S.aureus (clinical isolate) (Tukey’s test p < 0.05). Additionally, emulsions MIC values were up to 10 times lower than gentamicin alone against all S.aureus while MBC values of emulsions were up to 3 times lower towards sensitive S.aureus (clinical isolate and control). No bactericidal activity was exhibited by all compounds tested on resistant S.aureus (clinical isolate). In pre-biofilm evaluation, there were significant differences in biofilm formation inhibition in comparison between these emulsions with N.sativa and gentamicin alone in both clinical isolate S.aureus (sensitive and resistant) (Tukey’s test p < 0.05). MBIC values of emulsions were up to 10 times lower than gentamicin against all S.aureus. In contrast, N.sativa alone was lesser than emulsions and gentamicin. For post-biofilm assessment, no significant difference in penetration rate was found between emulsions and gentamicin. As opposed to N.sativa which showed little penetration. In the CLSM analysis, only emulsion C was used. Results revealed that emulsion C significantly reduced the biofilm thickness compared to gentamicin and N.sativa alone (Tukey’s test p < 0.05). Furthermore, the surface percentage (%) of non-viable bacteria of emulsions is significantly higher than gentamicin and N.sativa alone (Tukey’s test p < 0.05). In conclusion, this new fusion of gentamicin-N.sativa have synergistic antimicrobial and antibiofilm properties towards different strains of S.aureus including resistant strains, thus, can be developed as a new, and customized, gram-positive-specific treatment for ostoemyelitic infection.

Leaves of Coleus blumei (two species), Coleus amboinicus (two species). Coleus aromaticus and Pagostemon cahlin from Lamiaceae family were collected from different localities, freeze dried and extracted with aqueous methanol. The biological activity in vitro, especially in relation to total phenolic & flavonoid contents, antioxidant and antimicrobial activities were studied. Total phenolic content was determined according to the Folin-Ciocalteu method whilist in antioxidant activity was assessed using 2,2-diphenyl-l-picrylhydrazyl (DPPH) method. The antimicrobial activity of the extract was determined by making use of macro dilution and disc diffusion methods on two gram positive bacteria, two gram negative bacteria and on fungus. Furthermore, the toxicity was also assessed for the extracts by performing acute toxicity test. The phenolic content among the six Lamiaceae leaves extract showed significant difference (p<0.05) in result ranging from 55.21 - 95.17 mg GAL/g of dried samples. Pogoslemon cablin (PCM) had the highest content of phenolic followed by C. blumei (CBPM). There were significant differences (p<0.05) of ICso value of six Lamiaceae leaves extract ranging from 10.5 - 34.1 U-g/ml. Among the species studies. Coleus amboinicus - Malaysia (CALM) and Pogoslemon cablin (PCM) showed high antioxidant activity compared to the other leaf extracts. All leaf extracts showed activity at least against one strain of bacteria and result showed significant difference (p<0.05) between activities on the five microorganism. On the contrary, all of the leaf extracts were not effective against C. albicans. The minimum inhibitory concentration (MIC) of all leaf extracts ranged from 1.0-2.0 mg/ml in inhibiting the growth of S. aureus., E. colt, P. aeruginosa and B.subtilis. The acute toxicity test using C. Blumei leaf extract showed that there wras no mortality of animals experimented recorded even at the highest dose level of 5000 mg/kg body weight and observed for the 24 hours after administration. This shows that Coleus blumei plant extract have no toxic effect in mice. This study shows that the extracts can be used as antioxidant and antimicrobial agent without having the toxic effect. ii