The effect of environmental conditions on biofilm formation of Burkholderia pseudomallei clinical isolates

Abstract

Burkholderia pseudomallei, a Gram-negative saprophytic bacterium, is the causative agent of the potentially fatal melioidosis disease in humans. In this study, environmental parameters including temperature, nutrient content, pH and the presence of glucose were shown to play a role in in vitro biofilm formation by 28 B. pseudomallei clinical isolates, including four isolates with large colony variants (LCVs) and small colony variants (SCVs) morphotypes. Enhanced biofilm formation was observed when the isolates were tested in LB medium, at 30 °C, at pH 7.2, and in the presence of as little as 2 mM glucose respectively. It was also shown that all SVCs displayed significantly greater capacity to form biofilms than the corresponding LCVs when cultured in LB at 37 °C. In addition, octanoyl-homoserine lactone (C(8)-HSL), a quorum sensing molecule, was identified by mass spectrometry analysis in bacterial isolates referred to as LCV CTH, LCV VIT, SCV TOM, SCV CTH, 1 and 3, and the presence of other AHL's with higher masses; decanoyl-homoserine lactone (C(10)-HSL) and dodecanoyl-homoserine lactone (C(12)-HSL) were also found in all tested strain in this study. Last but not least, we had successfully acquired two Bacillus sp. soil isolates, termed KW and SA respectively, which possessed strong AHLs degradation activity. Biofilm formation of B. pseudomallei isolates was significantly decreased after treated with culture supernatants of KW and SA strains, demonstrating that AHLs may play a role in B. pseudomallei biofilm formation.

Figure 1. Effect of growth medium on biofilm formation by B. pseudomallei wild type and colony morphotypes.

Biofilm formation by B. pseudomallei (A) wild type isolates at 30°C and 37°C, (B) colony morphotypes at 37°C and (C) colony morphotypes at 30°C, in LB and MVBM. The results in Panel A are reported as the overall mean value for biofilm formation of 24 wild type isolates.

Figure 2. Effect of pH on biofilm formation by B. pseudomallei wild type and colony morphotypes.

Biofilm formation by B. pseudomallei (A) wild type isolates at 30°C and 37°C, (B) colony morphotypes at 37°C and (C) colony morphotypes at 30°C, in LB adjusted to different pH values.

Figure 3. Effect of glucose on biofilm formation by B. pseudomallei wild type and colony morphotypes.

Biofilm formation by B. pseudomallei (A) wild type isolates at 30°C and 37°C, (B) colony morphotypes at 37°C and (C) colony morphotypes at 30°C, in LB supplemented with glucose of different concentrations.

Figure 4. C. elegans killing assay.

C. elegans killing assay by B. pseudomallei of wildtype isolates (A), LCVs (B) and SCVs (C).

Figure 5. AHL identification of B. pseudomallei wild type via analytical TLC assay.

TLC analysis of short chain AHL production in wildtype and colony morphotypes of B. pseudomallei (A) and long chain AHL production in wildtype and colony morphotypes of B. pseudomallei (B). Lane 1: K96342, Lane 2: strain 1, Lane 3: strain 3, Lane 4: LCV TOM, Lane 5: LCV CTH, Lane 6: LCV OCY, Lane 7: LCV VL, Lane 8: SCV TOM, Lane 9: SCV CTH, Lane 10: SCV OCY and Lane 11: SCV VL.

Figure 6. AHL bioassay.

The results showed the quorum quenching activity of KW and SA isolates by degrading HHL (disappearance of purple pigmentation) over time. E. coli strain DH5α and PBS were included as negative controls.

Figure 7. B. pseudomallei AHLs treatment with Bacillus sp. culture supernatants.

Detection of AHL molecules in B. pseudomallei bioactive compound extracts prepared after treatment with Bacillus sp. culture supernatants. A and C, Bacillus sp. KW culture supernatant treated; B and D, Bacillus sp. SA culture supernatant treated. Panel A and B: Lane 1: K96342, Lane 2: strain 1, Lane 3: strain 3, Lane 4: LCV CTH, Lane 5: LCV VL, Lane 6: SCV TOM and Lane 7: SCV CTH. Panel C and D: Lane 1: K96342, Lane 2: strain 1, Lane 3: strain 3, Lane 4: LCV TOM, Lane 5: LCV CTH, Lane 6: LCV OCY, Lane 7: LCV VL, Lane 8: SCV TOM, Lane 9: SCV CTH, Lane 10: SCV OCY and Lane 11: SCV VL.

Figure 8. B. pseudomallei AHLs treatment with E. coli culture supernatants.

Detection of AHL molecules in B. pseudomallei bioactive compound extracts prepared after treatment with E.coli culture supernatants for short chain AHL production in wildtype and colony morphotypes of B. pseudomallei (A) and long chain AHL production in wildtype and colony morphotypes of B. pseudomallei (B). Lane 1: K96342, Lane 2: strain 1, Lane 3: strain 3, Lane 4: LCV TOM, Lane 5: LCV CTH, Lane 6: LCV OCY, Lane 7: LCV VL, Lane 8: SCV TOM, Lane 9: SCV CTH, Lane 10: SCV OCY and Lane 11: SCV VL.

Figure 9. Biofilm quenching assay at 37°C.

Inhibition of biofilm formation in B. pseudomallei (A) wild type isolates, (B) large colony morphotypes and (C) small colony morphotypes at 37°C in the presence of culture supernatants of quorum quenching Bacillus sp. isolates.

Figure 10. Biofilm quenching assay at 30°C.

Inhibition of biofilm formation in B. pseudomallei (A) wild type isolates, (B) large colony morphotypes and (C) small colony morphotypes at 30°C in the presence of culture supernatants of quorum quenching Bacillus sp. isolates.