Growing Burkholderia pseudomallei in Biofilm Stimulating Conditions Significantly Induces Antimicrobial Resistance

Abstract

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 FindingsThe 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/SignificanceThe 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.

In situ hybridization to detect and identify Burkholderia pseudomallei in human melioidosis

“…22 We speculate that bacillary emboli (Figure 2c) or smaller clusters may be held together and protected against antibiotics and host immune response by a surrounding biofilm, a well-known phenomenon in B. pseudomallei infection. 23 Bone marrow infection, shown in one of our cases (case 5, Table 2), is consistent with bone marrow inflammation found in another series, 24,25 and the observation that bacteria could be cultured from bone marrow. 26 Morphologically intact bacteria were demonstrated in the wall of the mycotic aneurysm confirming B pseudomallei infection (case 7, Table 2).…”

Alteration of the Phenotypic and Pathogenic Patterns of Burkholderia pseudomallei that Persist in a Soil Environment

“…[10][11][12]35,[38][39][40] Even strains that originate from the same parental strain exhibit phenotypic diversity, particularly in colony morphology. 17,41 Both arginine deiminase and quorum sensing systems have been suggested to modulate the formation of colonies in response to different environmental conditions.…”

“…9 The general virulence determinants, such as extracellular enzymes (protease, phospholipase, and catalase), biofilm formation, swarming ability, and lipopolysaccharide (LPS) types of different B. pseudomallei isolates are diverse. [10][11][12] The expression of one or more of these virulence determinants is associated with changes in bacterial colony morphology. [13][14][15][16] In B. pseudomallei, wrinkled and dry colonies are commonly observed in human isolates, and these morphologies are associated with the degree of virulence in BALB/c mice.…”

Antimicrobial agents and Burkholderia pseudomallei: perspectives from Thailand

“…The very high minimum biofilm eliminating concentration explains the lack of success in the treatment of chronic manifestations of B. pseudomallei infection. In addition, the biofilm mutant strains are resistant to all antimicrobial agents when induced to form biofilm [26].…”

“…The possible drug-resistant mechanism of biofilm is still unclear. However, research found that stimulation of the bacteria to produce biofilm results in upregulation of some genes to be resistant against antimicrobial agents [26]. The protein expression study in B. pseudomallei wild-type and biofilm defective mutants in biofilm stimulating conditions, revealed a different protein expression under biofilm stimulating conditions.…”