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 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.

Figure 1. The response of B. pseudomallei planktonic and biofilm cells to antimicrobial agents.

Susceptibility of planktonic and biofilm cells of B. pseudomallei isolates to doxycycline (DOX; A), ceftazidime (CTZ; B), imipenem (IMN; C) and trimethoprim/sulfamethoxazole (TMP/SMX; D) were shown. The cut off (---) indicates the resistant lines.

Figure 2. The response of B. pseudomallei mutants and their wild type to antimicrobial agents.

Susceptibility of B. pseudomallei mutants and their wild types to doxycycline (DOX; A), ceftazidime (CTZ; B), imipenem (IMN; C), and trimethoprim/sulfamethoxazole (TMP/SMX; D) were shown. The cut off (---) indicates resistant lines. The astericks (*) refer to resistant strains.

Figure 3. LPS profiles of planktonic, shedding planktonic and biofilm cells of B. pseudomallei isolates during changing their antimicrobial susceptibility.

LPS profiles of rough type isolate, A16 (Panel A) and smooth type A LPS isolate, 5-19, (Panel B), during planktonic status cultured in MHB medium in lane 1, MVBM medium in lane 2, shedding planktonic status in lane 3, and 2-day biofilm-formed status in lane 4. The LPS profile of smooth type B isolate, U882b (Panel C) obtained from its planktonic status in MVBM medium (lane 1), shedding planktonic (lane 2), and 2-day biofilm-formed status (lane 3). (Panel D) LPS profile of 365a isolate which was resistant to CTZ during planktonic status cultured in MHB medium (lane 1) and MVBM medium (lane 2), and 2-day biofilm-formed status (lane 3).

Figure 4. AHL synthesis from planktonic and biofilm-formed B. pseudomallei.

The 50 B. pseudomallei isolates were divided into 3 groups: low (n = 16), moderate (n = 20) and high (n = 14) biofilm producing isolates. The amounts of AHL were determined in culture supernatants of the planktonic (open bar) and 2-day biofilm-formed (solid bar) by using a bioluminescence assay with the reporter strain (E. coli JM 109 containing pSB401). Data were expressed as counts per second and shown as mean±SE of each groups. The asterisks (*) represent a significant difference (p<0.05).