Genetic characterization and antimicrobial resistance patterns of Burkholderia pseudomallei isolates from animals and environment in Peninsular Malaysia

Burkholderia pseudomallei is the aetiological agent of an emerging and potentially fatal human and animal disease melioidosis. Much of the knowledge about this organism in Malaysia and elsewhere revolved around human clinical isolates and much less is known about animal and environmental isolates. The present study was conducted with the aim generating more information on the phylogenetic variability of the animal and environmental B. pseudomallei isolates and its relatedness to antibiotic resistance pattern and genes. The specific objectives are to determine the molecular characteristics of B. pseudomallei isolates from animals and the environment (soil and water) in Peninsula Malaysia, compare the phylogeny of these isolates to those from elsewhere in the world, determine the antimicrobial resistance pattern among animal and environmental isolates, determine the occurrence of antimicrobial resistance genes and compare to the resistance pattern observed and determine the association between the sequence types to the physical and chemical characteristics of environments (soil and water) where the isolates originated.A total of 113 Malaysian B. pseudomallei isolates from animals and farm environment (soil and water) were characterised by multilocus sequence typing (MLST). Eighteen alleles were recovered in this study, among which are novel allele 97 and 69 of gene locus ace and lepA respectively. The allelic combinations resolved the isolates into 12 distinct sequence types (STs) with five among which are novel STs; ST1130, ST1131, ST1338, ST1339 and ST1367. This study found no association between sources of isolates and ST whereby the STs recovered from animal cases co-cluster with those found in the environment and have also been previously reported in humans. The isolates were found to be highly clonal. Moreover, B. pseudomallei strains were recovered to have descended from a common ancestor clonal complex 48 (CC48) found regionally in Southeast Asia. Disc diffusion or Kirby Bauer and E-test minimum inhibitory concentration (MIC) antibiotics susceptibility tests were conducted on 111 B. pseudomallei isolates. Twelve (12), common commercially prepared antimicrobial discs: ceftazidime, ceftriaxone, meropenem, ticarcillin, aztreonam, trimethoprim-sulfamethoxazole (cotrimoxazole), ciprofloxacin, imipenem, chloramphenicol, gentamicin, tetracycline and doxycycline were used in the disc diffusion method. All (100%) of the B. pseudomallei showed susceptibility to chloramphenicol, imipenem and doxycycline while all the isolates in this study were resistant to gentamicin and ticarcillin, and 99% were resistant to aztreonam. There was no significant association between the source of isolates (whether from animals or the environment) to the occurrence of resistance to any of the 12 antibiotics by disc diffusion method (p> 0.05), however there was a significant association between the occurrence of resistance to meropenem (p<0.05) and cotrimoxazole (p<0.001) to the STs of B. pseudomallei isolates. Five antibiotics namely meropenem, imipenem, ceftazidime, trimethoprim-sulfamethoxazole and amoxicillin-clavulanic acid, recommended in both acute and eradication phases of melioidosis treatment were tested using E-test MIC method. The majority of isolates were susceptible to all the antibiotics tested, however the existence of few resistant strains to meropenem, cotrimoxazole, ceftazidime and co-amoxiclav was observed among these isolates. A statistically significant association was found between resistance to meropenem and the animal isolates (p