Pseudomallei Biofilm Research Articles

Influence of carbonyl cyanide m-chlorophenyl hydrazone on biofilm dynamics, protease, and siderophore production by Burkholderia pseudomallei

Efflux pump inhibitors are a potential therapeutic strategy for managing antimicrobial resistance and biofilm formation. This article evaluated the effect of carbonyl cyanide m-chlorophenyl hydrazone (CCCP) on the biofilm growth dynamics and the production of virulence factors by Burkholderia pseudomallei. The effects of CCCP on planktonic, growing, and mature biofilm, interaction with antibacterial drugs, and protease and siderophore production were assessed. CCCP MICs ranged between 128 and 256 µM. The CCCP (128 µM) had a synergic effect with all the antibiotics tested against biofilms. Additionally, CCCP reduced (p < .05) the biomass of biofilm growth and mature biofilms at 128 and 512 µM, respectively. CCCP also decreased (p < .05) protease production by growing (128 µM) and induced (p < .05) siderophore release by planktonic cells (128 µM) growing biofilms (12.8 and 128 µM) and mature biofilms (512 µM). CCCP demonstrates potential as a therapeutic adjuvant for disassembling B. pseudomallei biofilms and enhancing drug penetration.

Burkholderia pseudomallei biofilm resists Acanthamoeba sp. grazing and produces 8-O-4′-diferulic acid, a superoxide scavenging metabolite after passage through the amoeba

Burkholderia pseudomallei, an etiological agent of melioidosis is an environmental bacterium that can survive as an intracellular pathogen. The biofilm produced by B. pseudomallei is crucial for cellular pathogenesis of melioidosis. The purpose of this investigation is to explore the role of biofilm in survival of B. pseudomallei during encounters with Acanthamoeba sp. using B. pseudomallei H777 (a biofilm wild type), M10 (a biofilm defect mutant) and C17 (a biofilm-complemented strain). The results demonstrated similar adhesion to amoebae by both the biofilm wild type and biofilm mutant strains. There was higher initial internalisation, but the difference diminished after longer encounter with the amoeba. Interestingly, confocal laser scanning microscopy demonstrated that pre-formed biofilm of B. pseudomallei H777 and C17 were markedly more persistent in the face of Acanthamoeba sp. grazing than that of M10. Metabolomic analysis revealed a significant increased level of 8-O-4′-diferulic acid, a superoxide scavenger metabolite, in B. pseudomallei H777 serially passaged in Acanthamoeba sp. The interaction between B. pseudomallei with a free-living amoeba may indicate the evolutionary pathway that enables the bacterium to withstand superoxide radicals in intracellular environments. This study supports the hypothesis that B. pseudomallei biofilm persists under grazing by amoebae and suggests a strategy of metabolite production that turns this bacterium from saprophyte to intracellular pathogen.

In vitro effect of the iron chelator deferiprone on the antimicrobial susceptibility and biofilms of Burkholderia pseudomallei

This study evaluated the effect of the iron chelator deferiprone (DFP) on antimicrobial susceptibility and biofilm formation and maintenance by Burkholderia pseudomallei. Planktonic susceptibility to DFP alone and in combination with antibiotics was evaluated by broth microdilution and biofilm metabolic activity was determined with resazurin. DFP minimum inhibitory concentration (MIC) range was 4–64 µg/mL and in combination reduced the MIC for amoxicillin/clavulanate and meropenem. DFP reduced the biomass of biofilms by 21 and 12% at MIC and MIC/2, respectively. As for mature biofilms, DFP reduced the biomass by 47%, 59%, 52% and 30% at 512, 256, 128 and 64 µg/mL, respectively, but did not affect B. pseudomallei biofilm viability nor increased biofilm susceptibility to amoxicillin/clavulanate, meropenem and doxycycline. DFP inhibits planktonic growth and potentiates the effect of β-lactams against B. pseudomallei in the planktonic state and reduces biofilm formation and the biomass of B. pseudomallei biofilms.

DNase I and chitosan enhance efficacy of ceftazidime to eradicate Burkholderia pseudomallei biofilm cells

Biofilm-associated Burkholderia pseudomallei infection contributes to antibiotic resistance and relapse of melioidosis. Burkholderia pseudomallei biofilm matrix contains extracellular DNA (eDNA) that is crucial for biofilm establishment. However, the contribution of eDNA to antibiotic resistance by B. pseudomallei remains unclear. In this study, we first demonstrated in vitro that DNase I with the administration of ceftazidime (CAZ) at 24 h considerably inhibited the 2-day biofilm formation and reduced the number of viable biofilm cells of clinical B. pseudomallei isolates compared to biofilm treated with CAZ alone. A 3–4 log reduction in numbers of viable cells embedded in the 2-day biofilm was observed when CAZ was combined with DNase I. Confocal laser-scanning microscope visualization emphasized the competence of DNase I followed by CAZ supplementation to significantly limit B. pseudomallei biofilm development and to eradicate viable embedded B. pseudomallei biofilm cells. Furthermore, DNase I supplemented with chitosan (CS) linked with CAZ (CS/CAZ) significantly eradicated shedding planktonic and biofilm cells. These findings indicated that DNase I effectively degraded eDNA leading to biofilm inhibition and dispersion, subsequently allowing CAZ and CS/CAZ to eradicate both shedding planktonic and embedded biofilm cells. These findings provide efficient strategies to interrupt biofilm formation and improve antibiotic susceptibility of biofilm-associated infections.

Burkholderia pseudomallei biofilm phenotypes confined but surviving in neutrophil extracellular traps of varying appearance.

Melioidosis is a fatal infectious disease caused by Burkholderia pseudomallei. Complications following treatment are usually due to antibiotic resistance and relapse is mainly caused by B. pseudomallei biofilm. Although the release of neutrophil extracellular traps (NETs) is crucial to capture and eliminate bacterial pathogens, to date response of NETs to B. pseudomallei biofilm is poorly understood. Here we compare the NETs produced by neutrophils in response to B. pseudomallei H777 (a biofilm-producing strain containing the bpsl0618 gene), a biofilm-defect strain lacking this gene (B. pseudomallei M10) and a bpsl0618 biofilm-complemented strain, B. pseudomallei C17, in which function of bpsl0618 was restored. Co-cultivation of these strains with healthy human neutrophils at MOI 10 with or without cytochalasin D demonstrated that H777 significantly resisted neutrophil-mediated killing and non-phagocytotic mechanisms compared to M10 (p < 0.0001). Three distinct morphotypes of NETs were seen: "aggregated", "spiky" and "cloudy". These were induced in different proportions by the different bacterial strains. All types of NETs were shown to confine all B. pseudomallei strains. Strains H777 and C17 could stimulate production of twice as much extracellular DNA (234.62 ng/mL and 205.43 ng/mL, respectively) as did M10 (111.87 ng/mL). Cells of H777 and C17 were better able to survive in the presence of neutrophil killing mechanisms relative to M10 (p < 0.0001) and NET formation (p < 0.0001 and 0.05). These findings suggest that NET stimulation was insufficient to eradicate B. pseudomallei H777 and C17 despite their possession of bpsl0618, a sugar-transferase gene associated with biofilm formation ability. Our findings demonstrate that B. pseudomallei biofilm phenotype may be a key factor in assisting pathogens to escape killing by neutrophils. This work provides a better understanding of how B. pseudomallei biofilm-associated infections induce and survive NET formation, resulting in bacterial persistence and increased severity of disease.

Chitosan biological molecule improves bactericidal competence of ceftazidime against Burkholderia pseudomallei biofilms

Biofilm-associated Burkholderia pseudomallei infections (melioidosis) are problematic because of reduced sensitivity to antibiotics and high frequency of relapse. Biofilm dispersal agents are essential to liberate the biofilm-encased cells, which then become planktonic and are more susceptible to antibiotics. This study aimed to evaluate the ability of deacetylated chitosan (dCS), an antimicrobial and antibiofilm biological macromolecule, to disrupt established biofilms, thus enabling ceftazidime (CAZ) to kill biofilm-embedded B. pseudomallei. We combined dCS with CAZ using a mechanical stirring method to generate dCS/CAZ. In combination, 1.25–2.5 mg ml−1 dCS/1–2 μg ml−1 CAZ acted synergistically to kill cells more effectively than did either dCS or CAZ alone. Notably, a combination of 5–10 mg ml−1 dCS with 256–512 μg ml−1 CAZ, prepared either by mechanical stirring (dCS/CAZ) or mixing (dCS + CAZ), drastically improved bactericidal activities against biofilm cells leading to a 3–6 log CFU reduction. Confocal laser-scanning microscope (CLSM) images revealed that 10 mg ml−1 dCS/512 μg ml−1 CAZ is by far the best formulation to diminish B. pseudomallei biofilm biomass and produces the lowest live/dead cell ratios of B. pseudomallei in biofilm matrix. Collectively, these findings emphasize the potential of novel therapeutic antibacterial and antibiofilm agents to fight against antibiotic-tolerant B. pseudomallei biofilm-associated infections.

Extracellular DNA facilitates bacterial adhesion during Burkholderia pseudomallei biofilm formation.

The biofilm-forming ability of Burkholderia pseudomallei is crucial for its survival in unsuitable environments and is correlated with antibiotic resistance and relapsing cases of melioidosis. Extracellular DNA (eDNA) is an essential component for biofilm development and maturation in many bacteria. The aim of this study was to investigate the eDNA released by B. pseudomallei during biofilm formation using DNase treatment. The extent of biofilm formation and quantity of eDNA were assessed by crystal-violet staining and fluorescent dye-based quantification, respectively, and visualized by confocal laser scanning microscopy (CLSM). Variation in B. pseudomallei biofilm formation and eDNA quantity was demonstrated among isolates. CLSM images of biofilms stained with FITC-ConA (biofilm) and TOTO-3 (eDNA) revealed the localization of eDNA in the biofilm matrix. A positive correlation of biofilm biomass with quantity of eDNA during the 2-day biofilm-formation observation period was found. The increasing eDNA quantity over time, despite constant living/dead ratios of bacterial cells during the experiment suggests that eDNA is delivered from living bacterial cells. CLSM images demonstrated that depletion of eDNA by DNase I significantly lessened bacterial attachment (if DNase added at 0 h) and biofilm developing stages (if added at 24 h) but had no effect on mature biofilm (if added at 45 h). Collectively, our results reveal that eDNA is released from living B. pseudomallei and is correlated with biofilm formation. It was also apparent that eDNA is essential during bacterial cell attachment and biofilm-forming steps. The depletion of eDNA by DNase may provide an option for the prevention or dispersal of B. pseudomallei biofilm.

BIOFILMS OF PATHOGENIC BURKHOLDERIA AND THEIR ROLE IN RESISTANCE TO ANTIBIOTICS

The review contains the current knowledge on the main issues of Burkholderia pseudomallei and Burkholderia mallei biofilm formation. The role ofknown structural elements of Burkholderia cells (flagella, type IV pili, LPS), as well as autotransporter adhesin proteins in the attachment of bacteria to surfaces, the formation of microcolonies and biofilm is described. The review also includes information of genetic regulatory mechanisms (QS-systems, RpoE-sigma factor, c-di-GMP, two-component signal transduction system), differentially expressed genes related to the formation of B. pseudomallei biofilm, role ofbiofilms in the virulence and resistance to antibiotics of pathogenic Burkholderia and their significance for the chronic processes and recurrent course of melioidosis and glanders.

Nitrate Sensing and Metabolism Inhibit Biofilm Formation in the Opportunistic Pathogen Burkholderia pseudomallei by Reducing the Intracellular Concentration of c-di-GMP.

The opportunistic pathogen Burkholderia pseudomallei is a saprophytic bacterium and the causative agent of melioidosis, an emerging infectious disease associated with high morbidity and mortality. Although melioidosis is most prevalent during the rainy season in endemic areas, domestic gardens and farms can also serve as a reservoir for B. pseudomallei during the dry season, in part due to irrigation and fertilizer use. In the environment, B. pseudomallei forms biofilms and persists in soil near plant root zones. Biofilms are dynamic bacterial communities whose formation is regulated by extracellular cues and corresponding changes in the nearly universal secondary messenger cyclic dimeric GMP. Recent studies suggest B. pseudomallei loads are increased by irrigation and the addition of nitrate-rich fertilizers, whereby such nutrient imbalances may be linked to the transmission epidemiology of this important pathogen. We hypothesized that exogenous nitrate inhibits B. pseudomallei biofilms by reducing the intracellular concentration of c-di-GMP. Bioinformatics analyses revealed B. pseudomallei 1026b has the coding capacity for nitrate sensing, metabolism, and transport distributed on both chromosomes. Using a sequence-defined library of B. pseudomallei 1026b transposon insertion mutants, we characterized the role of denitrification genes in biofilm formation in response to nitrate. Our results indicate that the denitrification pathway is implicated in B. pseudomallei biofilm growth dynamics and biofilm formation is inhibited by exogenous addition of sodium nitrate. Genomics analysis identified transposon insertional mutants in a predicted two-component system (narX/narL), a nitrate reductase (narGH), and a nitrate transporter (narK-1) required to sense nitrate and alter biofilm formation. Additionally, the results presented here show that exogenous nitrate reduces intracellular levels of the bacterial second messenger c-di-GMP. These results implicate the role of nitrate sensing in the regulation of a c-di-GMP phosphodiesterase and the corresponding effects on c-di-GMP levels and biofilm formation in B. pseudomallei 1026b.

Genome-scale analysis of the genes that contribute to Burkholderia pseudomallei biofilm formation identifies a crucial exopolysaccharide biosynthesis gene cluster.

Burkholderia pseudomallei, the causative agent of melioidosis, is an important public health threat due to limited therapeutic options for treatment. Efforts to improve therapeutics for B. pseudomallei infections are dependent on the need to understand the role of B. pseudomallei biofilm formation and its contribution to antibiotic tolerance and persistence as these are bacterial traits that prevent effective therapy. In order to reveal the genes that regulate and/or contribute to B. pseudomallei 1026b biofilm formation, we screened a sequence defined two-allele transposon library and identified 118 transposon insertion mutants that were deficient in biofilm formation. These mutants include transposon insertions in genes predicted to encode flagella, fimbriae, transcriptional regulators, polysaccharides, and hypothetical proteins. Polysaccharides are key constituents of biofilms and B. pseudomallei has the capacity to produce a diversity of polysaccharides, thus there is a critical need to link these biosynthetic genes with the polysaccharides they produce to better understand their biological role during infection. An allelic exchange deletion mutant of the entire B. pseudomallei biofilm-associated exopolysaccharide biosynthetic cluster was decreased in biofilm formation and produced a smooth colony morphology suggestive of the loss of exopolysaccharide production. Conversely, deletion of the previously defined capsule I polysaccharide biosynthesis gene cluster increased biofilm formation. Bioinformatics analyses combined with immunoblot analysis and glycosyl composition studies of the partially purified exopolysaccharide indicate that the biofilm-associated exopolysaccharide is neither cepacian nor the previously described acidic exopolysaccharide. The biofilm-associated exopolysaccharide described here is also specific to the B. pseudomallei complex of bacteria. Since this novel exopolysaccharide biosynthesis cluster is retained in B. mallei, it is predicted to have a role in colonization and infection of the host. These findings will facilitate further advances in understanding the pathogenesis of B. pseudomallei and improve diagnostics and therapeutic treatment strategies.

Promethazine improves antibiotic efficacy and disrupts biofilms of Burkholderia pseudomallei

Efflux pumps are important defense mechanisms against antimicrobial drugs and maintenance of Burkholderia pseudomallei biofilms. This study evaluated the effect of the efflux pump inhibitor promethazine on the structure and antimicrobial susceptibility of B. pseudomallei biofilms. Susceptibility of planktonic cells and biofilms to promethazine alone and combined with antimicrobials was assessed by the broth microdilution test and biofilm metabolic activity was determined with resazurin. The effect of promethazine on 48 h-grown biofilms was also evaluated through confocal and electronic microscopy. The minimum inhibitory concentration (MIC) of promethazine was 780 mg l−1, while the minimum biofilm elimination concentration (MBEC) was 780–3,120 mg l−1. Promethazine reduced the MIC values for erythromycin, trimethoprim/sulfamethoxazole, gentamicin and ciprofloxacin and reduced the MBEC values for all tested drugs (p<0.05). Microscopic analyses demonstrated that promethazine altered the biofilm structure of B. pseudomallei, even at subinhibitory concentrations, possibly facilitating antibiotic penetration. Promethazine improves antibiotics efficacy against B. pseudomallei biofilms, by disrupting biofilm structure.

Burkholderia pseudomallei resistance to antibiotics in biofilm-induced conditions is related to efflux pumps.

Burkholderia pseudomallei, the causative agent of melioidosis, has been found to increase its resistance to antibiotics when growing as a biofilm. The resistance is related to several mechanisms. One of the possible mechanisms is the efflux pump. Using bioinformatics analysis, it was found that BPSL1661, BPSL1664 and BPSL1665 were orthologous genes of the efflux transporter encoding genes for biofilm-related antibiotic resistance, PA1874-PA1877 genes in Pseudomonas aeruginosa strain PAO1. Expression of selected encoding genes for the efflux transporter system during biofilm formation were investigated. Real-time reverse transcriptase PCR expression of amrB, cytoplasmic membrane protein of AmrAB-OprA efflux transporter encoding gene, was slightly increased, while BPSL1665 was significantly increased during growth of bacteria in biofilm formation. Minimum biofilm inhibition concentration and minimum biofilm eradication concentration (MBEC) of ceftazidime (CTZ), doxycycline (DOX) and imipenem were found to be 2- to 1024-times increased when compared to their MICs for of planktonic cells. Inhibition of the efflux transporter by adding phenylalanine arginine β-napthylamide (PAβN), a universal efflux inhibitor, decreased 2 to 16 times as much as MBEC in B. pseudomallei biofilms with CTZ and DOX. When the intracellular accumulation of antibiotics was tested to reveal the pump inhibition, only the concentrations of CTZ and DOX increased in PAβN treated biofilm. Taken together, these results indicated that BPSL1665, a putative precursor of the efflux pump gene, might be related to the adaptation of B. pseudomallei in biofilm conditions. Inhibition of efflux pumps may lead to a decrease of resistance to CTZ and DOX in biofilm cells.

Burkholderia pseudomallei Biofilm Promotes Adhesion, Internalization and Stimulates Proinflammatory Cytokines in Human Epithelial A549 Cells.

Burkholderia pseudomallei is a Gram-negative bacterium that causes melioidosis. Inhalational exposure leading to pulmonary melioidosis is the most common clinical manifestation with significant mortality. However, the role of B. pseudomallei biofilm phenotype during bacterial-host interaction remains unclear. We hypothesize that biofilm phenotype may play a role in such interactions. In this study, B. pseudomallei H777 (biofilm wild type), B. pseudomallei M10 (biofilm mutant) and B. pseudomallei C17 (biofilm-complemented) strains were used to assess the contribution of biofilm to adhesion to human lung epithelial cells (A549), intracellular interactions, apoptosis/necrosis and impact on proinflammatory responses. Confocal laser scanning microscopy demonstrated that B. pseudomallei H777 and C17 produced biofilm, whereas M10 did not. To determine the role of biofilm in host interaction, we assessed the ability of each of the three strains to interact with the A549 cells at MOI 10. Strain H777 exhibited higher levels of attachment and invasion compared to strain M10 (p < 0.05). In addition, the biofilm-complemented strain, C17 exhibited restored bacterial invasion ability. Flow cytometry combined with a double-staining assay using annexin V and propidium iodide revealed significantly higher numbers of early apoptotic and late apoptotic A549 cells when these were infected with strain H777 (1.52%) and C17 (1.43%) compared to strain M10 (0.85%) (p < 0.05). Strains H777 and C17 were able to stimulate significant secretion of IL-6 and IL-8 compared with the biofilm mutant (p < 0.05). Together, these findings demonstrated the role of biofilm-associated phenotypes of B. pseudomallei in cellular pathogenesis of human lung epithelial cells with respect to initial attachment and invasion, apoptosis and proinflammatory responses.

Farnesol increases the susceptibility of Burkholderia pseudomallei biofilm to antimicrobials used to treat melioidosis.

The aim of this study was to analyse the in vitro activity of farnesol alone and combined with the antibacterial drugs amoxicillin, doxycycline, ceftazidime and sulfamethoxazole-trimethoprim against Burkholderia pseudomallei biofilms. Susceptibility was assessed by the broth microdilution test and cell viability was read with the oxidation-reduction indicator dye resazurin. The biofilms were evaluated through three microscopic techniques (optical, confocal and electronic microscopy). The minimum biofilm erradication concentration (MBEC) for farnesol was 75-2400 mmol l(-1). In addition, farnesol significantly reduced the MBEC values for ceftazidime, amoxicillin, doxycycline and sulfamethoxazole-trimethoprim by 256, 16, 4 and 4 times respectively (P < 0·05). Optical, confocal and electronic microscopic analyses of farnesol-treated B. pseudomallei biofilms demonstrated that this compound damages biofilm matrix, probably facilitating antimicrobial penetration in the biofilm structure. This study demonstrated the effectiveness of farnesol against B. pseudomallei biofilms and its potentiating effect on the activity of antibacterial drugs, in particular ceftazidime, amoxicillin, doxycycline and sulfamethoxazole-trimethoprim. The intrinsic antimicrobial resistance of B. pseudomallei is a serious challenge for the treatment of melioidosis. Thus, this paper reports the inhibitory potential of farnesol against B. pseudomallei biofilms, as well as highlights the favourable pharmacological interaction of farnesol with antibiotics tested, not only on cell viability, but also in the structural morphology of biofilms.

Correlation between biofilm production, antibiotic susceptibility and exopolysaccharide composition in Burkholderia pseudomalleibpsI, ppk, and rpoS mutant strains

Burkholderia pseudomallei is the cause of melioidosis, a fatal tropical infectious disease, which has been reported to have a high rate of recurrence, even when an intensive dose of antibiotics is used. Biofilm formation is believed to be one of the possible causes of relapse because of its ability to increase drug resistance. EPS in biofilms have been reported to be related to the limitation of antibiotic penetration in B. pseudomallei. However, the mechanisms by which biofilms restrict the diffusion of antibiotics remain unclear. The present study presents a correlation between exopolysaccharide production in biofilm matrix and antibiotic resistance in B. pseudomallei using bpsI, ppk, and rpoS mutant strains. CLSM revealed a reduction in exopolysaccharide production and disabled micro-colony formation in B. pseudomallei mutants, which paralleled the antibiotic resistance. Different ratios of carbohydrate contents in the exopolysaccharides of the mutants were detected, although they have the same components, including glucose, galactose, mannose, and rhamnose, with the exception being that no detectable rhamnose peak was observed in the bpsI mutant. These results indicate that the correlation between these phenomena in the B. pseudomallei biofilm at least results from the exopolysaccharide, which may be under the regulation of bpsI, ppk, or rpoS genes.

Global transcriptional analysis of Burkholderia pseudomallei high and low biofilm producers reveals insights into biofilm production and virulence.

BackgroundChronic bacterial infections occur as a result of the infecting pathogen’s ability to live within a biofilm, hence escaping the detrimental effects of antibiotics and the immune defense system. Burkholderia pseudomallei, a gram-negative facultative pathogen, is distinctive in its ability to survive within phagocytic and non-phagocytic cells, to persist in vivo for many years and subsequently leading to relapse as well as the development of chronic disease. The capacity to persist has been attributed to the pathogen’s ability to form biofilm. However, the underlying biology of B. pseudomallei biofilm development remains unresolved.ResultsWe utilised RNA-Sequencing to identify genes that contribute to B. pseudomallei biofilm phenotype. Transcriptome analysis of a high and low biofilm producer identified 563 differentially regulated genes, implying that expression of ~9.5 % of the total B. pseudomallei gene content was altered during biofilm formation. Genes involved in surface-associated motility, surface composition and cell wall biogenesis were over-expressed and probably play a role in the initial attachment of biofilms. Up-regulation of genes related to two component signal transduction systems and a denitrification enzyme pathway suggest that the B. pseudomallei high biofilm producer is able to sense the surrounding environmental conditions and regulate the production of extracellular polymeric substance matrix, a hallmark of microbial biofilm formation.ConclusionsThe transcriptome profile described here provides the first comprehensive view of genes that contribute to the biofilm phenotype in B. pseudomallei.Electronic supplementary materialThe online version of this article (doi:10.1186/s12864-015-1692-0) contains supplementary material, which is available to authorized users.

The Effect of Environmental Conditions on Biofilm Formation of Burkholderia pseudomallei Clinical Isolates

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 (C8-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 (C10-HSL) and dodecanoyl-homoserine lactone (C12-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.

Diffusion and activity of antibiotics against Burkholderia pseudomallei biofilms

The diffusion and activity of ceftazidime (CAZ), imipenem (IPM) and trimethoprim/sulfamethoxazole (TMP/SMX) against Burkholderia pseudomallei biofilms were comparatively tested using the high biofilm-producing strain B. pseudomallei 377 and the biofilm-defective mutant B. pseudomallei M6. Biofilms were generated by inoculation of bacteria on polycarbonate membranes placed on the surface of tryptic soy agar plates. The results showed that diffusion of TMP/SMX through B. pseudomallei biofilms was similar for both strains. However, diffusion of CAZ and IPM was significantly faster through strain M6 biofilm in comparison with strain 377 biofilm. The viabilities of strain 377 biofilm were significantly higher than those observed with strain M6 for all antibiotics challenged at 4h, suggesting that the biofilm-forming capacity may be involved in antibiotic susceptibilities in B. pseudomallei. These results re-emphasise the importance of biofilm for antibiotic resistance in B. pseudomallei.

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 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.