METHODS: A total of 345 wound swab samples were tested for bacterial pathogens. Acinetobacter baumannii was identified by culture and biochemical tests. Antimicrobial susceptibility pattern was determined by the disc diffusion method according to CLSI standards. Extended spectrum beta-lactamases were screened using the double disc synergy technique. Gene encoding AdeB efflux pump and NDM-1 were detected by Polymerase Chain Reaction (PCR).

RESULTS: A total 22 (6.37%) Acinetobacter baumannii were identified from 345 wound swab samples and 20 (91%) of them were multidrug resistant. High resistance rates to some antibiotics were seen namely, cefotaxime (95%), amoxyclavulanic acid (90%) and ceftriaxone (82%). All the identified Acinetobacter baumannii were sensitive to colistin and 82% to imipenem. Two (9%) ESBL producing Acinetobacter baumannii strains were detected. adeB gene was detected in 16 (80%) out of 20 multidrug resistant Acinetobacter baumannii. 4 (18%) of 22 Acinetobacter baumannii were imipenem resistant. NDM-1 gene was detected in 2 (50%) of the imipenem resistant strains of Acinetobacter baumannii.

RESULT: The screening of six physical conditions by Plackett-Burman Design has identified pH, inoculum size and incubation time as exerting significant effects on lipase production. These three conditions were further optimised using, Box-Behnken Design of Response Surface Methodology, which predicted an optimum medium comprising pH 6, 24 h incubation time and 2% inoculum size. T1 lipase activity of 2.0 U/mL was produced with a biomass of OD600 23.0.

Methods: Two complementary approaches, saturated transposon mutagenesis and spontaneous mutation induction with high concentrations of colistin and polymyxin B, were employed to select for mutations associated with resistance to polymyxins. Mutants were identified using transposon-directed insertion-site sequencing or Illumina WGS. A resistance phenotype was confirmed by MIC and further investigated using RT-PCR. Competitive growth assays were used to measure fitness cost.

Results: A transposon insertion at nucleotide 41 of the pmrB gene (EC958pmrB41-Tn5) enhanced its transcript level, resulting in a 64- and 32-fold increased MIC of colistin and polymyxin B, respectively. Three spontaneous mutations, also located within the pmrB gene, conferred resistance to both colistin and polymyxin B with a corresponding increase in transcription of the pmrCAB genes. All three mutations incurred a fitness cost in the absence of colistin and polymyxin B.

RESULTS: We attempted to identify genes that may be involved in biofilm formation in seven C. jejuni strains through construction of mutants using the EZ-Tn5 Transposome system. Only 14 mutants with reduced biofilm formation were obtained, all from one strain of C. jejuni. Three different genes of interest, namely CmeB (synthesis of multidrug efflux system transporter proteins), NusG (transcription termination and anti-termination protein) and a putative transmembrane protein (involved in membrane protein function) were identified. The efficiency of the EZ::TN5 transposon mutagenesis approach was strain dependent and was unable to generate any mutants from most of the strains used.

RESULT: The recipient transconjugants were resistant to erythromycin, cefpodoxime and were mecA positive. PCR amplification of mecA after mix culture plating on Luria Bertani agar containing 100 μg/mL showed that 75% of the donor and 58.3% of the recipient transconjugants were mecA positive. Additionally, 61.5% of both the donor cells and recipient transconjugants were mecA positive, while 46.2% and 41.75% of both donor and recipient transconjugants were mecA positive on LB agar containing 50 μg/mL and 30 μg/mL respectively.