Unchalee Tattawasart

Growing Burkholderia pseudomallei in Biofilm Stimulating Conditions Significantly Induces Antimicrobial Resistance

Background Burkholderia pseudomallei, a Gram-negative bacterium that causes melioidosis, was reported to produce biofilm. As the disease causes high relapse rate when compared to other bacterial infections, it therefore might be due to the reactivation of the biofilm forming bacteria which also provided resistance to antimicrobial agents. However, the mechanism on how biofilm can provide tolerance to antimicrobials is still unclear. Methodology/Principal Findings The change in resistance of B. pseudomallei to doxycycline, ceftazidime, imipenem, and trimethoprim/sulfamethoxazole during biofilm formation were measured as minimum biofilm elimination concentration (MBEC) in 50 soil and clinical isolates and also in capsule, flagellin, LPS and biofilm mutants. Almost all planktonic isolates were susceptible to all agents studied. In contrast, when they were grown in the condition that induced biofilm formation, they were markedly resistant to all antimicrobial agents even though the amount of biofilm production was not the same. The capsule and O-side chains of LPS mutants had no effect on biofilm formation whereas the flagellin-defective mutant markedly reduced in biofilm production. No alteration of LPS profiles was observed when susceptible form was changed to resistance. The higher amount of N-acyl homoserine lactones (AHLs) was detected in the high biofilm-producing isolates. Interestingly, the biofilm mutant which produced a very low amount of biofilm and was sensitive to antimicrobial agents significantly resisted those agents when grown in biofilm inducing condition. Conclusions/Significance The possible drug resistance mechanism of biofilm mutants and other isolates is not by having biofilm but rather from some factors that up-regulated when biofilm formation genes were stimulated. The understanding of genes related to this situation may lead us to prevent B. pseudomallei biofilms leading to the relapse of melioidosis.

Novel lytic bacteriophages from soil that lyse Burkholderia pseudomallei

Burkholderia pseudomallei is a Gram-negative saprophytic bacterium that causes severe sepsis with a high mortality rate in humans and a vaccine is not available. Bacteriophages are viruses of bacteria that are ubiquitous in nature. Several lysogenic phages of Burkholderia spp. have been found but information is scarce for lytic phages. Six phages, ST2, ST7, ST70, ST79, ST88 and ST96, which lyse B. pseudomallei, were isolated from soil in an endemic area. The phages belong to the Myoviridae family. The range of estimated genome sizes is 24.0-54.6 kb. Phages ST79 and ST96 lysed 71% and 67% of tested B. pseudomallei isolates and formed plaques on Burkholderia mallei but not other tested bacteria, with the exception of closely related Burkholderia thailandensis which was lysed by ST2 and ST96 only. ST79 and ST96 were observed to clear a mid-log culture by lysis within 6 h when infected at a multiplicity of infection of 0.1. As ST79 and ST96 phages effectively lysed B. pseudomallei, their potential use as a biocontrol of B. pseudomallei in the environment or alternative treatment in infected hosts could lead to benefits from phages that are available in nature.

Antimicrobial resistance and genetic diversity of the SXT element in Vibrio cholerae from clinical and environmental water samples in northeastern Thailand

Multidrug resistance in V. cholerae has been increasing around the world including northeastern Thailand. The aquatic environment is a reservoir of V. cholerae and might be an important source of resistant strains. The aims of this study were to investigate the phylogenetic relationships of intSXT gene sequences from 31 clinical and 14 environmental V. cholerae O1 and non-O1/non-O139 isolates and 11 sequences amplified directly from environmental water samples. We also amplified class 1 integrons, the SXT elements (targeting the intSXT gene) and antimicrobial resistance genes directly from water samples. Phylogenetic analysis displayed two major distinct clusters (clusters 1 and 2). Most V. cholerae O1 (19/20, 95%) and non-O1/non-O139 isolates (8/11, 72.7%) from clinical sources, and all sequences obtained directly from water samples, belonged to cluster 1. Cluster 2 mostly comprised environmental non-O1/non-O139 isolates (10/12, 83.3%). We successfully amplified the SXT elements directly from17.5% of water samples. Associated resistance genes were also amplified as follows: sul2 (41.3% of water samples), dfrA1 (60%), dfr18 (33.8%), strB (70%) and tetA (2.5%). Class 1 integrons were not found in water samples, indicating that the SXT element was the major contributor of multidrug resistance determinants in this region. The SXT element and antimicrobial resistance genes could be transferred from clinical V. cholerae O1 to environmental V. cholerae non-O1/non-O139 was demonstrated by conjugation experiment. These findings indicate that there may have been cross dissemination and horizontal gene transfer (HGT) of the SXT element harbored by V. cholerae O1 and non-O1/non-O139 strains isolated from clinical and environmental water sources. Environmental water might be an important source of antimicrobial resistance genes in V. cholerae in this region. Direct detection of antimicrobial resistance genes in water samples can be used for monitoring the spread of such genes in the ecosystem.

Identification of the Burkholderia pseudomallei bacteriophage ST79 lysis gene cassette

To identify and characterize the lysis gene cassette from the bacteriophage ST79 that lyses Burkholderia pseudomallei.

Antimicrobial Resistance and Species Prevalence of Enterococcal Isolates in Srinagarind Hospital, Northeastern Thailand °"√¥◊àâÿ'ÿï' å◊— ∑'â'''å—'◊

A total of 300 clinical isolates of enterococci, collected from patients admitted at Srinagarind hospital, were investigated for antimicrobial resistance and species distribution. Antimicrobial susceptibility to 10 antimicrobial agents was performed by disc diffusion method. Screening for vancomycin resistance was performed by the agar plate method and minimal inhibitory concentration of vancomycin was determined for vancomycin resistant strains by microbroth dilution method. Enterococcus faecalis was found to be predominant species (58.7%) followed by E. faecium (35.7%) and the rest species (5.7%) including E. hirae, E. gallinarum, E. durans and E. dispar. The isolates were resistant to penicillin (51.3%), ampicillin (43.3%), high level gentamicin (57.7%), azithromycin (100%), chloramphenicol (16.3%), doxycycline (48%), quinupristin/dalfopristin (53%) and linezolid (8%). None of the isolates were resistant to teicoplanin. Five (1.7%) strains were resistant to vancomycin. The minimal inhibitory concentrations of vancomycin for vancomycin-resistant enterococci strains were 16∝g/ml. E. faecium was more resistant to penicillin, ampicillin and high level gentamicin than E. faecalis. Two hundred and seventy four (91.3%) strains showed multidrug resistance. Our study showed low prevalence of vancomycin resistance. However, there is a need to carry-out regular surveillance of antimicrobial resistance of enterococci to monitor changes in their patterns to prevent the spreading of resistant isolates.