Hans Nelis

In vitro and in vivo model systems to study microbial biofilm formation.

Biofilm formation is often considered the underlying reason why treatment with an antimicrobial agent fails and as an estimated 65-80% of all human infections is thought to be biofilm-related, this presents a serious challenge. Biofilm model systems are essential to gain a better understanding of the mechanisms involved in biofilm formation and resistance. In this review a comprehensive overview of various in vitro and in vivo systems is presented, and their advantages and disadvantages are discussed.

Quorum sensing inhibitors increase the susceptibility of bacterial biofilms to antibiotics in vitro and in vivo

ABSTRACT Although the exact role of quorum sensing (QS) in various stages of biofilm formation, maturation, and dispersal and in biofilm resistance is not entirely clear, the use of QS inhibitors (QSI) has been proposed as a potential antibiofilm strategy. We have investigated whether QSI enhance the susceptibility of bacterial biofilms to treatment with conventional antimicrobial agents. The QSI used in our study target the acyl-homoserine lactone-based QS system present in Pseudomonas aeruginosa and Burkholderia cepacia complex organisms (baicalin hydrate, cinnamaldehyde) or the peptide-based system present in Staphylococcus aureus (hamamelitannin). The effect of tobramycin (P. aeruginosa, B. cepacia complex) and clindamycin or vancomycin (S. aureus), alone or in combination with QSI, was evaluated in various in vitro and in vivo biofilm model systems, including two invertebrate models and one mouse pulmonary infection model. In vitro the combined use of an antibiotic and a QSI generally resulted in increased killing compared to killing by an antibiotic alone, although reductions were strain and model dependent. A significantly higher fraction of infected Galleria mellonella larvae and Caenorhabditis elegans survived infection following combined treatment, compared to treatment with an antibiotic alone. Finally, the combined use of tobramycin and baicalin hydrate reduced the microbial load in the lungs of BALB/c mice infected with Burkholderia cenocepacia more than tobramycin treatment alone. Our data suggest that QSI may increase the success of antibiotic treatment by increasing the susceptibility of bacterial biofilms and/or by increasing host survival following infection.

Cinnamaldehyde and cinnamaldehyde derivatives reduce virulence in Vibrio spp. by decreasing the DNA-binding activity of the quorum sensing response regulator LuxR

BackgroundTo date, only few compounds targeting the AI-2 based quorum sensing (QS) system are known. In the present study, we screened cinnamaldehyde and substituted cinnamaldehydes for their ability to interfere with AI-2 based QS. The mechanism of QS inhibition was elucidated by measuring the effect on bioluminescence in several Vibrio harveyi mutants. We also studied in vitro the ability of these compounds to interfere with biofilm formation, stress response and virulence of Vibrio spp. The compounds were also evaluated in an in vivo assay measuring the reduction of Vibrio harveyi virulence towards Artemia shrimp.ResultsOur results indicate that cinnamaldehyde and several substituted derivatives interfere with AI-2 based QS without inhibiting bacterial growth. The active compounds neither interfered with the bioluminescence system as such, nor with the production of AI-2. Study of the effect in various mutants suggested that the target protein is LuxR. Mobility shift assays revealed a decreased DNA-binding ability of LuxR. The compounds were further shown to (i) inhibit biofilm formation in several Vibrio spp., (ii) result in a reduced ability to survive starvation and antibiotic treatment, (iii) reduce pigment and protease production in Vibrio anguillarum and (iv) protect gnotobiotic Artemia shrimp against virulent Vibrio harveyi BB120.ConclusionCinnamaldehyde and cinnamaldehyde derivatives interfere with AI-2 based QS in various Vibrio spp. by decreasing the DNA-binding ability of LuxR. The use of these compounds resulted in several marked phenotypic changes, including reduced virulence and increased susceptibility to stress. Since inhibitors of AI-2 based quorum sensing are rare, and considering the role of AI-2 in several processes these compounds may be useful leads towards antipathogenic drugs.

Prevalence of vulvovaginal candidiasis and susceptibility to fluconazole in women

In this study a new method of membrane filtration using a panel of florigenic enzyme substrates to identify and differentiate four major Candida species within 9 to 12 hours was tested for its ability to diagnose vulvovaginal candidiasis. The study subjects were 612 women who were recruited from consecutive patients who attended a gynecological clinic at Ghent University Hospital during a 14-week period. Some subjects had symptoms of vaginitis, but others did not. Each subject underwent a clinical examination including a wet vaginal smear and a vaginal swab, which was placed in transport gel for delivery to the laboratory. The florigenic process involves the formation of a microcolony on a membrane filter over a 9- to 11-hour incubation period. The membrane is cut into five segments, which are impregnated with a buffered solution of a florigenic 4-methylumbilliferyl substrate. After a further incubation period of 30 minutes, during which cleavage of enzyme substrates takes place, the microcolonies are examined under long-wavelength ultraviolet light resulting in blue or orange fluorescence. A positive yeast culture was obtained from the vaginal swabs of 123 of the 612 women (20.1%). Sixty-five of the patients had symptoms of vaginitis (vulvar pruritus and vaginal discharge), and 547 were asymptomatic. Of the 65 symptomatic women, 60% (39 of 65) had a positive KOH wet smear, and 40% (26/65) had a negative KOH result. More than two thirds of the isolated species were C. albicans (68.3%); C. glabrata was found in 16.3%, C. parapsilosis was isolated in 8.9%, and C. humicola, C. krusei, and C. lusitaniae were found in 1.6%, 0.8%, and 0.8%, respectively. The florigenic technique agreed 100% of the time with chromogenic analysis performed as a comparative standard. The highest number of isolated yeasts was found in women with clinical symptoms of candidiasis and a positive KOH test (29 of 35 >1000 CFU/ml). Conversely, women with clinical symptoms and negative KOH test had low numbers of isolated yeasts. When the subjects were divided into subgroups, pregnant women were the most likely, and postmenopausal women with no hormonal replacement therapy were the least likely, to have a positive yeast culture (33 of 103, 32%, P =.045; 15 of 119, 12.6%, P =.003, respectively). Yeast was found in 26.9% of postmenopausal women not taking oral contraceptives (22 of 114), and 14.4% and 14.0% of premenopausal women taking oral contraceptives (16 of 111) or with an intrauterine device (8 of 57), respectively. The rate of yeast colonization was significantly related to the amount of estrogen in oral contraceptives, with the higher concentrations more likely to have positive yeast growth (P =.05). Similarly, yeast colonization in pregnant women grew significantly as pregnancy progressed (P =.05). Twenty-six of the 123 yeast isolates were resistant to fluconazole when subjected to in vitro sensitivity testing.

Evaluation of species-specific recA-based PCR tests for genomovar level identification within the Burkholderia cepacia complex.

The Burkholderia cepacia complex presently comprises nine genomovars: B. cepacia (genomovar I), B. multivorans (genomovar II), B. cepacia genomovar III, B. stabilis (genomovar IV), B. vietnamiensis (genomovar V), B. cepacia genomovar VI, B. ambifaria (genomovar VII), B. anthina (genomovar VIII) and B. pyrrocinia (genomovar IX). Strains of each genomovar can colonise the respiratory tract of cystic fibrosis (CF) patients. However, the majority of infections in CF patients are caused by B. multivorans and B. cepacia genomovar III isolates. Accurate genomovar-level identification is best achieved through a polyphasic approach combining phenotypic and genotypic analyses. In the present study, the sensitivity and specificity of recA-based genomovar specific primer pairs were evaluated with a collection of 508 B. cepacia complex isolates representing all nine genomovars. The assays for the identification of B. multivorans (sensitivity and specificity, 100%), B. cepacia genomovar III (sensitivity, 92%; specificity, 100%), and B. ambifaria (sensitivity and specificity, 100%) were the most efficient. However, the B. cepacia genomovar I assay lacked sensitivity (72%) and cross-reacted with all B. pyrrocinia isolates examined. Several new recA RFLP types were also revealed within the B. cepacia complex. One of these profiles was shared by a clinical and an environmental B. cepacia-like isolate and by the B. ubonensis type strain. The latter organism is a recently described soil bacterium. Its relationship to the various B. cepacia complex genomovars needs further study.

Enrichment of live food with essential fatty acids and vitamin C: effects on milkfish (Chanos chanos) larval performance

The effects of essential fatty acids (EFA) and vitamin C-enriched live food on growth, survival, resistance to salinity stress and incidence of deformity in milkfish larvae reared in tanks were investigated. Larvae were either fed rotifers cultured on Chlorella sp. and newly hatched Artemia nauplii (control), highly unsaturated fatty acid (HUFA)-enriched rotifers and Artemia nauplii or HUFA+vitamin C-enriched rotifers and Artemia nauplii. Milkfish growth in outdoor nursery ponds was also assessed to compare with growth in indoor tanks. Milkfish fed rotifers/Artemia enriched with HUFA (32–48 mg dry weight, DW) or HUFA+vitamin C (33–45 mg DW) exhibited significantly (P 0.05) among the treatment groups. Forty-day-old milkfish fed HUFA+vitamin C-enriched live food had significantly lower (P<0.05) incidence of opercular deformity (mainly cleft branchiostegal membrane) (8.4–14.7%) compared with those given HUFA-enriched (15.8–23.5%) or unenriched (27.3–33.5%) live food. Results demonstrated the effect of HUFA enrichment in enhancing milkfish larval growth and resistance to salinity stress but not overall survival. Moreover, HUFA and ascorbate supplementation decreased but did not totally eliminate incidence of opercular deformity in milkfish larvae.

Use of quorum sensing inhibitors to interfere with biofilm formation and development in Burkholderia multivorans and Burkholderia cenocepacia

Burkholderia cepacia complex strains are opportunistic pathogens causing life-threatening infections in cystic fibrosis patients. B. cepacia complex strains are resistant to many antimicrobial agents and commonly produce biofilms in vitro and in vivo. This contributes to their virulence and makes Burkholderia infections difficult to treat. Recently, the quorum sensing (QS) system of Burkholderia spp. has been found to affect their biofilm-forming ability, making it an attractive target for antimicrobial therapy. However, detailed information about the anti-biofilm effect of these compounds is still lacking. In the present study, we evaluated the anti-biofilm effect of several known QS inhibitors. The effect on Burkholderia spp. biofilm formation was examined using crystal violet, resazurin and SYTO9 staining, confocal laser scanning microscopy as well as plating. When used at subinhibitory concentrations, several compounds interfered with biofilm formation by Burkholderia spp. Our results suggest that the QS inhibitors affect later stages of biofilm formation and detachment.

Real-time PCR expression profiling of genes encoding potential virulence factors in Candida albicans biofilms: identification of model-dependent and -independent gene expression

BackgroundCandida albicans infections are often associated with biofilm formation. Previous work demonstrated that the expression of HWP1 (hyphal wall protein) and of genes belonging to the ALS (agglutinin-like sequence), SAP (secreted aspartyl protease), PLB (phospholipase B) and LIP (lipase) gene families is associated with biofilm growth on mucosal surfaces. We investigated using real-time PCR whether genes encoding potential virulence factors are also highly expressed in biofilms associated with abiotic surfaces. For this, C. albicans biofilms were grown on silicone in microtiter plates (MTP) or in the Centres for Disease Control (CDC) reactor, on polyurethane in an in vivo subcutaneous catheter rat (SCR) model, and on mucosal surfaces in the reconstituted human epithelium (RHE) model.ResultsHWP1 and genes belonging to the ALS, SAP, PLB and LIP gene families were constitutively expressed in C. albicans biofilms. ALS1-5 were upregulated in all model systems, while ALS9 was mostly downregulated. ALS6 and HWP1 were overexpressed in all models except in the RHE and MTP, respectively. The expression levels of SAP1 were more pronounced in both in vitro models, while those of SAP2, SAP4 and SAP6 were higher in the in vivo model. Furthermore, SAP5 was highly upregulated in the in vivo and RHE models. For SAP9 and SAP10 similar gene expression levels were observed in all model systems. PLB genes were not considerably upregulated in biofilms, while LIP1-3, LIP5-7 and LIP9-10 were highly overexpressed in both in vitro models. Furthermore, an elevated lipase activity was detected in supernatans of biofilms grown in the MTP and RHE model.ConclusionsOur findings show that HWP1 and most of the genes belonging to the ALS, SAP and LIP gene families are upregulated in C. albicans biofilms. Comparison of the fold expression between the various model systems revealed similar expression levels for some genes, while for others model-dependent expression levels were observed. This suggests that data obtained in one biofilm model cannot be extrapolated to other model systems. Therefore, the need to use multiple model systems when studying the expression of genes encoding potential virulence factors in C. albicans biofilms is highlighted.

Superoxide Dismutases Are Involved in Candida albicans Biofilm Persistence against Miconazole

ABSTRACT We investigated the cellular mechanisms responsible for the occurrence of miconazole-tolerant persisters in Candida albicans biofilms. Miconazole induced about 30% killing of sessile C. albicans cells at 75 μM. The fraction of miconazole-tolerant persisters, i.e., cells that can survive high doses of miconazole (0.6 to 2.4 mM), in these biofilms was 1 to 2%. Since miconazole induces reactive oxygen species (ROS) in sessile C. albicans cells, we focused on a role for superoxide dismutases (Sods) in persistence and found the expression of Sod-encoding genes in sessile C. albicans cells induced by miconazole compared to the expression levels in untreated sessile C. albicans cells. Moreover, addition of the superoxide dismutase inhibitor N,N′-diethyldithiocarbamate (DDC) to C. albicans biofilms resulted in an 18-fold reduction of the miconazole-tolerant persister fraction and in increased endogenous ROS levels in these cells. Treatment of biofilms of C. albicans clinical isolates with DDC resulted in an 18-fold to more than 200-fold reduction of their miconazole-tolerant persister fraction. To further confirm the important role for Sods in C. albicans biofilm persistence, we used a Δsod4 Δsod5 mutant lacking Sods 4 and 5. Biofilms of the Δsod4 Δsod5 mutant contained at least 3-fold less of the miconazole-tolerant persisters and had increased ROS levels compared to biofilms of the isogenic wild type (WT). In conclusion, the occurrence of miconazole-tolerant persisters in C. albicans biofilms is linked to the ROS-detoxifying activity of Sods. Moreover, Sod inhibitors can be used to potentiate the activity of miconazole against C. albicans biofilms.

Fungicidal activity of miconazole against Candida spp. biofilms

OBJECTIVES Although azole antifungals are considered to be fungistatic, miconazole has fungicidal activity against planktonic Candida albicans cells, presumably associated with the induction of reactive oxygen species (ROS) production. Only few data are available concerning the effect of miconazole against sessile C. albicans cells. In the present study, the fungicidal activity of miconazole against in vitro-grown mature Candida biofilms, and its relationship with the induction of ROS and ROS-dependent apoptosis were examined. METHODS The effect of miconazole on mature biofilms formed by 10 C. albicans strains and 5 strains from other Candida species was evaluated by plate counting and measuring the level of ROS induction. MIC tests were performed in the absence and presence of ascorbic acid, a quencher of ROS. The apoptotic population in C. albicans cells was determined using annexin-Cy3. RESULTS Miconazole showed a significant fungicidal effect against all mature Candida biofilms tested and caused elevated ROS levels, both in planktonic and sessile cells. Addition of ascorbic acid drastically reduced these levels. While ROS quenching decreased the susceptibility to miconazole of planktonic cells of most Candida strains, no reduced fungicidal activity of miconazole against biofilms was observed. Miconazole did not cause a significant increase in apoptosis. CONCLUSIONS ROS levels increased in all Candida biofilms upon addition of miconazole. However, ROS induction was not the only factor that underlies its fungicidal activity, as quenching of ROS did not lead to an enhanced survival of biofilm cells. ROS-induced apoptosis was not observed in C. albicans cells after miconazole treatment.