The antifungal efficacy of essential oils in combination with chlorhexidine against Candida spp.

This study determined the chemical components of three essential oils (cinnamon oil, clove oil and lemongrass oil) by gas chromatography and mass spectroscopy. The in vitro antifungal activity of chlorhexidine (CHX) combined with essential oils was then assessed against planktonic Candida albicans ATCC10231, Candida krusei (STCK 1) and Candida tropicalis (STCT 1) and C. albicans biofilms using broth microdilution and chequerboard assays. The results demonstrated that CHX combined with either clove oil, cinnamon oil or lemongrass oil exhibited synergistic effect against planktonic C. albicans at FICI of 0.500, 0.375 and 0.312, respectively. Additive effects were recorded for combinations tested against C. tropicalis and C. krusei. Synergistic effects were observed for clove or cinnamon oil combined with CHX (FICI 0.500 and 0.375, respectively) against sessile C. albicans in biofilm, whereas the combinations of lemongrass oil and CHX showed only additive effect (FICI 1.062). In conclusion, the combination of CHX with either clove oil or cinnamon oil may prove useful as an alternative antifungal treatment for oral Candida spp

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

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

D-LL-31 enhances biofilm-eradicating effect of currently used antibiotics for chronic rhinosinusitis and its immunomodulatory activity on human lung epithelial cells.

Chronic rhinosinusitis (CRS) is a chronic disease that involves long-term inflammation of the nasal cavity and paranasal sinuses. Bacterial biofilms present on the sinus mucosa of certain patients reportedly exhibit resistance against traditional antibiotics, as evidenced by relapse, resulting in severe disease. The aim of this study was to determine the killing activity of human cathelicidin antimicrobial peptides (LL-37, LL-31) and their D-enantiomers (D-LL-37, D-LL-31), alone and in combination with conventional antibiotics (amoxicillin; AMX and tobramycin; TOB), against bacteria grown as biofilm, and to investigate the biological activities of the peptides on human lung epithelial cells. D-LL-31 was the most effective peptide against bacteria under biofilm-stimulating conditions based on IC50 values. The synergistic effect of D-LL-31 with AMX and TOB decreased the IC50 values of antibiotics by 16-fold and could eliminate the biofilm matrix in all tested bacterial strains. D-LL-31 did not cause cytotoxic effects in A549 cells at 25 μM after 24 h of incubation. Moreover, a cytokine array indicated that there was no significant induction of the cytokines involving in immunopathogenesis of CRS in the presence of D-LL-31. However, a tissue-remodeling-associated protein was observed that may prevent the progression of nasal polyposis in CRS patients. Therefore, a combination of D-LL-31 with AMX or TOB may improve the efficacy of currently used antibiotics to kill biofilm-embedded bacteria and eliminate the biofilm matrix. This combination might be clinically applicable for treatment of patients with biofilm-associated CRS

Biofilm-forming capacity of 50 <i>B. pseudomallei</i> isolates.

<p>Biofilm-forming capacity of 50 <i>B. pseudomallei</i> isolates.</p

Biofilm-forming capacity of <i>B. pseudomallei</i> mutants and their wild types.

<p>Biofilm-forming capacity of <i>B. pseudomallei</i> mutants and their wild types.</p

The response of <i>B. pseudomallei</i> mutants and their wild type to antimicrobial agents.

<p>Susceptibility of <i>B. pseudomallei</i> 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.</p

Time-lapse of biofilms under flow conditions.

<p><i>B</i>. <i>thailandensis</i> E264 biofilm after treated with 16×MIC of CAZ in MVBM and 0.1×MVBM under flow condition using Bioflux microfluidics platform with flow rate 0.5 dyn/cm² at 37 °C for 16 h. Black arrows indicated the starting time when changing of bacterial morphology was observed after treated with CAZ. The scale bar indicates 50 μm. Experiments were performed three times and representative examples are shown. The movies were shown in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0194946#pone.0194946.s001" target="_blank">S1</a>–<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0194946#pone.0194946.s004" target="_blank">S4</a> Figs.</p

LPS profiles of planktonic, shedding planktonic and biofilm cells of <i>B. pseudomallei</i> isolates during changing their antimicrobial susceptibility.

<p>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).</p