The BCESM genomic region contains a regulator involved in quorum sensing and persistence in Burkholderia cenocepacia J2315.

AIM: In this study, we investigated the function of BCAM0257-8-9 located in the BCESM region of the Burkholderia cenocepacia J2315 genome. MATERIALS & METHODS: Differential RNA sequencing was used to determine transcription start sites. The phenotype of overexpression mutants was studied and a transcriptome analysis of the BCAM0258 overexpression mutant was performed. RESULTS: BCAM0257 and BCAM0258 were identified as belonging to an operon, positively regulated by BCAM0259. We found that this operon is involved in persistence and that BCAM0258 functions as a regulator influencing quorum sensing and activating pathways related to iron acquisition and biofilm formation. Overexpression of BCAM0257 increased virulence. CONCLUSION: The BCESM genomic region contains an operon that contributes to quorum sensing and is involved in persistence, biofilm formation and virulence. BCAM0257-8-9 is found in all sequenced B. cenocepacia ET12 genomes and these results may help explain why infections with strains of the B. cenocepacia ET12 lineage are difficult to treat.

Draft Genome Sequence of Methicillin-Resistant Staphylococcus epidermidis Strain ET-024, Isolated from an Endotracheal Tube Biofilm of a Mechanically Ventilated Patient.

Staphylococcus epidermidis strain ET-024 was isolated from a biofilm on an endotracheal tube of a mechanically ventilated patient. This strain is resistant to methicillin, and the draft genome sequence shares some characteristics with other nosocomial S. epidermidis strains (such as S. epidermidis RP62A).

Autoinducer-2 plays a crucial role in gut colonization and probiotic functionality of Bifidobacterium breve UCC2003.

In the present study we show that luxS of Bifidobacterium breve UCC2003 is involved in the production of the interspecies signaling molecule autoinducer-2 (AI-2), and that this gene is essential for gastrointestinal colonization of a murine host, while it is also involved in providing protection against Salmonella infection in Caenorhabditis elegans. We demonstrate that a B. breve luxS-insertion mutant is significantly more susceptible to iron chelators than the WT strain and that this sensitivity can be partially reverted in the presence of the AI-2 precursor DPD. Furthermore, we show that several genes of an iron starvation-induced gene cluster, which are downregulated in the luxS-insertion mutant and which encodes a presumed iron-uptake system, are transcriptionally upregulated under in vivo conditions. Mutation of two genes of this cluster in B. breve UCC2003 renders the derived mutant strains sensitive to iron chelators while deficient in their ability to confer gut pathogen protection to Salmonella-infected nematodes. Since a functional luxS gene is present in all tested members of the genus Bifidobacterium, we conclude that bifidobacteria operate a LuxS-mediated system for gut colonization and pathogen protection that is correlated with iron acquisition.

Involvement of toxin-antitoxin modules in Burkholderia cenocepacia biofilm persistence.

Biofilms are involved in the recalcitrance of infections due to the presence of persister cells. Although the molecular basis of persistence is still largely unknown, toxin-antitoxin modules (TA) are thought to play a role in this process. In this study, we investigated whether TA modules contribute to persistence toward antibiotics in Burkholderia cenocepacia J2315. Sixteen pairs of genes were identified based on their apparent similarity to TA modules. Overexpression of the putative toxins had various effects on growth, persistence, and biofilm formation. Toxins, whose overexpression resulted in growth inhibition, often increased the number of surviving persisters; in contrast, overexpression of putative toxins showing no effects on growth had no positive influence on the number of surviving persisters. Furthermore, the expression of the TA modules was compared between treated and untreated sessile and planktonic wild-type cultures. For 10 toxin-encoding genes, the expression was higher in untreated sessile cells than in untreated planktonic cells. Nine toxin-encoding genes were upregulated after treatment with tobramycin, but none after treatment with ciprofloxacin. These results indicate that most, but not all TA modules contribute to persistence in B. cenocepacia J2315 and that this contribution depends on the mode of growth and the antibiotic used.

Activity of disinfectants against multispecies biofilms formed by Staphylococcus aureus, Candida albicans and Pseudomonas aeruginosa.

Microbial biofilms are a serious threat to human health. Recent studies have indicated that many clinically relevant biofilms are polymicrobial. In the present study, multispecies biofilms were grown in a reproducible manner in a 96-well microtiter plate. The efficacy of nine commercially available disinfectants against Staphylococcus aureus, Candida albicans, and Pseudomonas aeruginosa in multispecies biofilms was determined and compared. The results showed that the direction and the magnitude of the effect in a multispecies biofilm depend on the strain and the disinfectant used and challenge the common belief that organisms in multispecies biofilms are always less susceptible than in monospecies biofilms.

Protease production by Staphylococcus epidermidis and its effect on Staphylococcus aureus biofilms.

Due to the resistance of Staphylococcus aureus to several antibiotics, treatment of S. aureus infections is often difficult. As an alternative to conventional antibiotics, the field of bacterial interference is investigated. Staphylococcus epidermidis produces a serine protease (Esp) which inhibits S. aureus biofilm formation and which degrades S. aureus biofilms. In this study, we investigated the protease production of 114 S. epidermidis isolates, obtained from biofilms on endotracheal tubes (ET). Most of the S. epidermidis isolates secreted a mixture of serine, cysteine and metalloproteases. We found a link between high protease production by S. epidermidis and the absence of S. aureus in ET biofilms obtained from the same patient. Treating S. aureus biofilms with the supernatant (SN) of the most active protease producing S. epidermidis isolates resulted in a significant biomass decrease compared to untreated controls, while the number of metabolically active cells was not affected. The effect on the biofilm biomass was mainly due to serine proteases. Staphylococcus aureus biofilms treated with the SN of protease producing S. epidermidis were thinner with almost no extracellular matrix. An increased survival of Caenorhabditis elegans, infected with S. aureus Mu50, was observed when the SN of protease positive S. epidermidis was added.

Bacteria that inhibit quorum sensing decrease biofilm formation and virulence in Pseudomonas aeruginosa PAO1.

In this study, we investigated the biotherapeutic potential of previously isolated quorum quenching (QQ) bacteria. Some of them produce and secrete small compounds that inhibit quorum sensing (QS), others quench QS by enzymatic degradation of N-acylhomoserine lactones (AHLs). The supernatant of cultures of these isolates was tested for inhibitory properties against P. aeruginosa PAO1 biofilms. Most isolates had a moderate effect on biofilm formation, as shown by viability staining and/or staining of the biofilm biomass. A substantial part of the isolates reduced P. aeruginosa elastase production in a concentration-dependent manner. Using Caenorhabditis elegans as an in vivo model system for virulence testing, we found that some of the isolates were able to increase survival of P. aeruginosa PAO1 and Burkholderia cenocepacia LGM16656-infected nematodes when co-administered with the pathogen. Altogether, these data indicate that some QQ bacteria, or the active compounds they produce, could be useful to attenuate virulence of P. aeruginosa PAO1 and possibly also other Gram-negative pathogens that use AHLs to regulate the production of virulence factors.

The presence of antibiotic-resistant nosocomial pathogens in endotracheal tube biofilms and corresponding surveillance cultures.

Mechanically ventilated patients often develop ventilator-associated pneumonia (VAP). Soon after intubation, a mixed biofilm harboring microbial pathogens is formed on the endotracheal tube (ET). It is believed that this biofilm contributes to the development of VAP. Unfortunately, the causative agent is often not known at the time VAP is suspected, and early therapy often relies on the identification of surveillance cultures (SC). It is thus important to know whether these SC can predict the microbial flora in ET biofilms. In this study, we compare the presence of a number of antibiotic-resistant nosocomial bacteria (Enterobacter aerogenes, Escherichia coli, Micrococcus luteus, Pseudomonas aeruginosa, Staphylococcus aureus, and Staphylococcus epidermidis) and of Candida albicans in cultures from ET biofilms and SC (i.e. sputum samples, nose swabs, and throat swabs) of 20 mechanically ventilated patients. Our data indicate that there is a good correlation between the presence of (antibiotic-resistant) pathogens in ET biofilms and SC.

Genomewide screening for genes involved in biofilm formation and miconazole susceptibility in Saccharomyces cerevisiae.

Infections related to fungal biofilms are difficult to treat due to the reduced susceptibility of sessile cells to most antifungal agents. Previous research has shown that 1-10% of sessile Candida cells survive treatment with high doses of miconazole (a fungicidal imidazole). The aim of this study was to identify genes involved in fungal biofilm formation and to unravel the mechanisms of resistance of these biofilms to miconazole. To this end, a screening of a Saccharomyces cerevisiae deletion mutant bank was carried out. Our results revealed that genes involved in peroxisomal transport and the biogenesis of the respiratory chain complex IV play an essential role in biofilm formation. On the other hand, genes involved in transcription and peroxisomal and mitochondrial organization seem to highly influence the susceptibility to miconazole of yeast biofilms. Additionally, our data confirm previous findings on genes involved in biofilm formation and in general stress responses. Our data suggest the involvement of peroxisomes in biofilm formation and miconazole resistance in fungal biofilms.

Biofilm-grown Burkholderia cepacia complex cells survive antibiotic treatment by avoiding production of reactive oxygen species.

The presence of persister cells has been proposed as a factor in biofilm resilience. In the present study we investigated whether persister cells are present in Burkholderia cepacia complex (Bcc) biofilms, what the molecular basis of antimicrobial tolerance in Bcc persisters is, and how persisters can be eradicated from Bcc biofilms. After treatment of Bcc biofilms with high concentrations of various antibiotics often a small subpopulation survived. To investigate the molecular mechanism of tolerance in this subpopulation, Burkholderia cenocepacia biofilms were treated with 1024 µg/ml of tobramycin. Using ROS-specific staining and flow cytometry, we showed that tobramycin increased ROS production in treated sessile cells. However, approximately 0.1% of all sessile cells survived the treatment. A transcriptome analysis showed that several genes from the tricarboxylic acid cycle and genes involved in the electron transport chain were downregulated. In contrast, genes from the glyoxylate shunt were upregulated. These data indicate that protection against ROS is important for the survival of persisters. To confirm this, we determined the number of persisters in biofilms formed by catalase mutants. The persister fraction in ΔkatA and ΔkatB biofilms was significantly reduced, confirming the role of ROS detoxification in persister survival. Pretreatment of B. cenocepacia biofilms with itaconate, an inhibitor of isocitrate lyase (ICL), the first enzyme in the glyoxylate shunt, reduced the persister fraction approx. 10-fold when the biofilms were subsequently treated with tobramycin. In conclusion, most Bcc biofilms contain a significant fraction of persisters that survive treatment with high doses of tobramycin. The surviving persister cells downregulate the TCA cycle to avoid production of ROS and at the same time activate an alternative pathway, the glyoxylate shunt. This pathway may present a novel target for combination therapy.

Synthesis and evaluation of thiazolidinedione and dioxazaborocane analogues as inhibitors of AI-2 quorum sensing in Vibrio harveyi.

Two focused libraries based on two types of compounds, that is, thiazolidinediones and dioxazaborocanes were designed. Structural resemblances can be found between thiazolidinediones and well-known furanone type quorum sensing (QS) inhibitors such as N-acylaminofuranones, and/or acyl-homoserine lactone signaling molecules, while dioxazaborocanes structurally resemble previously reported oxazaborolidine derivatives which antagonized autoinducer 2 (AI-2) binding to its receptor. Because of this, we hypothesized that these compounds could affect AI-2 QS in Vibrio harveyi. Although all compounds blocked QS, the thiazolidinediones were the most active AI-2 QS inhibitors, with EC(50) values in the low micromolar range. Their mechanism of inhibition was elucidated by measuring the effect on bioluminescence in a series of V. harveyi QS mutants and by DNA-binding assays with purified LuxR protein. The active compounds neither affected bioluminescence as such nor the production of AI-2. Instead, our results indicate that the thiazolidinediones blocked AI-2 QS in V. harveyi by decreasing the DNA-binding ability of LuxR. In addition, several dioxazaborocanes were found to block AI-2 QS by targeting LuxPQ.

Synthesis and evaluation of the quorum sensing inhibitory effect of substituted triazolyldihydrofuranones.

Acylhomoserine lactone (AHL) analogues in which the amide function is replaced by a triazole group were synthesized and evaluated for their effect on quorum sensing (QS) and biofilm formation in Burkholderia cenocepacia and Pseudomonas aeruginosa. In addition, the influence of the length of the acyl-mimicking chain was investigated. The compounds showed selectivity between two different AHL QS systems. 3-(1H-1,2,3-Triazol-1-yl)dihydrofuran-2(3H)-ones, in which the 4-substituent best resembled the acyl chain of the native AHL molecule exhibited significant QS agonistic and antagonistic activities. Replacing this aliphatic substituent by a phenyl-containing moiety resulted in active inhibitors of QS. The most active compounds showed biofilm inhibitory as well as biofilm eradicating activities in both test organisms.

Assessment of microbial diversity in biofilms recovered from endotracheal tubes using culture dependent and independent approaches.

Ventilator-associated pneumonia (VAP) is a common nosocomial infection in mechanically ventilated patients. Biofilm formation is one of the mechanisms through which the endotracheal tube (ET) facilitates bacterial contamination of the lower airways. In the present study, we analyzed the composition of the ET biofilm flora by means of culture dependent and culture independent (16 S rRNA gene clone libraries and pyrosequencing) approaches. Overall, the microbial diversity was high and members of different phylogenetic lineages were detected (Actinobacteria, beta-Proteobacteria, Candida spp., Clostridia, epsilon-Proteobacteria, Firmicutes, Fusobacteria and gamma-Proteobacteria). Culture dependent analysis, based on the use of selective growth media and conventional microbiological tests, resulted in the identification of typical aerobic nosocomial pathogens which are known to play a role in the development of VAP, e.g. Staphylococcus aureus and Pseudomonas aeruginosa. Other opportunistic pathogens were also identified, including Staphylococcus epidermidis and Kocuria varians. In general, there was little correlation between the results obtained by sequencing 16 S rRNA gene clone libraries and by cultivation. Pyrosequencing of PCR amplified 16 S rRNA genes of four selected samples resulted in the identification of a much wider variety of bacteria. The results from the pyrosequencing analysis suggest that these four samples were dominated by members of the normal oral flora such as Prevotella spp., Peptostreptococcus spp. and lactic acid bacteria. A combination of methods is recommended to obtain a complete picture of the microbial diversity of the ET biofilm.

Phytosphingosine-1-phosphate is a signaling molecule involved in miconazole resistance in sessile Candida albicans cells.

Previous research has shown that 1% to 10% of sessile Candida albicans cells survive treatment with high doses of miconazole (a fungicidal imidazole). In the present study, we investigated the involvement of sphingolipid biosynthetic intermediates in this survival. We observed that the LCB4 gene, coding for the enzyme that catalyzes the phosphorylation of dihydrosphingosine and phytosphingosine, is important in governing the miconazole resistance of sessile Saccharomyces cerevisiae and C. albicans cells. The addition of 10 nM phytosphingosine-1-phosphate (PHS-1-P) drastically reduced the intracellular miconazole concentration and significantly increased the miconazole resistance of a hypersusceptible C. albicans heterozygous LCB4/lcb4 mutant, indicating a protective effect of PHS-1-P against miconazole-induced cell death in sessile cells. At this concentration of PHS-1-P, we did not observe any effect on the fluidity of the cytoplasmic membrane. The protective effect of PHS-1-P was not observed when the efflux pumps were inhibited or when tested in a mutant without functional efflux systems. Also, the addition of PHS-1-P during miconazole treatment increased the expression levels of genes coding for efflux pumps, leading to the hypothesis that PHS-1-P acts as a signaling molecule and enhances the efflux of miconazole in sessile C. albicans cells.

Eradication of Propionibacterium acnes biofilms by plant extracts and putative identification of icariin, resveratrol and salidroside as active compounds.

Propionibacterium acnes is a Gram-positive bacterium that plays an important role in the pathogenesis of acne vulgaris. This organism is capable of biofilm formation and the decreased antimicrobial susceptibility of biofilm-associated cells may hamper efficient treatment. In addition, the prolonged use of systemic antibiotic therapy is likely to lead to the development and spread of antimicrobial resistance. In the present study we investigated whether P. acnes biofilms could be eradicated by plant extracts or their active compounds, and whether other mechanisms besides killing of biofilm cells could be involved. Out of 119 plant extracts investigated, we identified five with potent antibiofilm activity against P. acnes (extracts from Epimedium brevicornum, Malus pumila, Polygonum cuspidatum, Rhodiola crenulata and Dolichos lablab). We subsequently identified icariin, resveratrol and salidroside as active compounds in three of these extracts. Extracts from E. brevicornum and P. cuspidatum, as well as their active compounds (icariin and resveratrol, respectively) showed marked antibiofilm activity when used in subinhibitory concentrations, indicating that killing of microbial cells is not their only mode of action.

In vitro inactivation of endodontic pathogens with Nd:YAG and Er:YAG lasers.

Both Nd:YAG and Er:YAG lasers have been suggested as root canal disinfection aids. The aim of this in vitro study is to compare both wavelengths in terms of irradiation dose required for microbial inactivation, to quantify these irradiation doses and to investigate the influence of certain (laser) parameters on the antimicrobial efficacy. Agar plates containing a uniform layer of Enterococcus faecalis, Candida albicans or Propionibacterium acnes were mounted perpendicularly underneath the laser handpieces (5 mm spot). The Er:YAG laser was operated in single-pulse mode. Pulse energies of 40-400 mJ and pulse lengths of 100, 300, 600, and 1,000 µs were tested. After incubation at 37°C for 48 h, growth on the plates was scored. The pulse energy yielding complete absence of growth over the entire spot area was taken as the total inhibition threshold (TIT). TITs were determined for every species and pulse length. The Nd:YAG laser was operated with pulse trains because single pulses were ineffective. Output power was 15 W and frequency was 100 Hz. Spots were irradiated for 5-120 s. After incubation, the diameters of the inhibition zones were measured. For the Er:YAG laser, TITs varied between 100 and 210 mJ, and differed significantly between species and pulse lengths. Using Nd:YAG irradiation, TITs were around 5,300 J/cm(2) for C. albicans and 7,100 J/cm(2) for P. acnes. No inhibition was observed for E. faecalis. Er:YAG irradiation was superior to Nd:YAG in inactivating microorganisms on agar surfaces.

Isolation and identification of quorum quenching bacteria from environmental samples.

A large number of Gram-negative pathogens produce N-acylhomoserine lactones (AHLs) as signal molecules for quorum sensing (QS). This cell-cell communication system allows them to coordinate gene expression and regulate virulence. Therefore, strategies to inhibit QS are promising for the control of infectious diseases. The aim of the present study was to develop a high-throughput method for the isolation and identification of AHL-degrading bacteria from environmental samples. Samples were cultured in a microtitre plate in a minimal medium containing 1 mM N-(3-oxo-dodecanoyl)-L-homoserine lactone and 2 mM N-(3-oxo-hexanoyl)-L-homoserine lactone as the sole sources of carbon and nitrogen. Isolates growing on this minimal medium were subcultured and identified by partial 16S rRNA gene sequencing. Subsequently, the AHL-degrading capacity of each isolate was evaluated in the Pseudomonas aeruginosa QSIS2 biosensor assay, as such or after treatment with heat or proteinase K. The 16 samples tested yielded a total of 59 isolates which are, either alone or as part of a consortium, able to use AHL signal molecules as sole sources of carbon and nitrogen. Follow-up experiments have shown that in each sample there is at least one isolate with quorum quenching (QQ) activity, and that for all samples combined, 41 isolates have QQ activity. Furthermore, heat treatment did not fully inhibit QQ activity in all isolates. In some isolates, QQ activity was lost after proteinase K treatment, while others remained able to quench QS. Therefore, it is likely that some isolates produce and secrete (a) heat-stable, low molecular weight inhibitory compound(s).

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

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.

Structure-activity relationship of cinnamaldehyde analogs as inhibitors of AI-2 based quorum sensing and their effect on virulence of Vibrio spp.

BACKGROUND: Many bacteria, including Vibrio spp., regulate virulence gene expression in a cell-density dependent way through a communication process termed quorum sensing (QS). Hence, interfering with QS could be a valuable novel antipathogenic strategy. Cinnamaldehyde has previously been shown to inhibit QS-regulated virulence by decreasing the DNA-binding ability of the QS response regulator LuxR. However, little is known about the structure-activity relationship of cinnamaldehyde analogs. METHODOLOGY/PRINCIPAL FINDINGS: By evaluating the QS inhibitory activity of a series of cinnamaldehyde analogs, structural elements critical for autoinducer-2 QS inhibition were identified. These include an α,ß unsaturated acyl group capable of reacting as Michael acceptor connected to a hydrophobic moiety and a partially negative charge. The most active cinnamaldehyde analogs were found to affect the starvation response, biofilm formation, pigment production and protease production in Vibrio spp in vitro, while exhibiting low cytotoxicity. In addition, these compounds significantly increased the survival of the nematode Caenorhabditis elegans infected with Vibrio anguillarum, Vibrio harveyi and Vibrio vulnificus. CONCLUSIONS/SIGNIFICANCE: Several new and more active cinnamaldehyde analogs were discovered and they were shown to affect Vibrio spp. virulence factor production in vitro and in vivo. Although ligands for LuxR have not been identified so far, the nature of different cinnamaldehyde analogs and their effect on the DNA binding ability of LuxR suggest that these compounds act as LuxR-ligands.

What can be learned from genotyping of fungi?

Multiple genotyping studies have been carried out in order to clarify the epidemiology of fungal infections, more specifically to determine the sources, transmission routes, and colonization patterns of fungal isolates. In this review, the results obtained in genotyping investigations of Aspergillus isolates are summarized and discussed. Furthermore, we examine the epidemiologic studies of Candida albicans, Exophiala dermatitidis and Scedosporium apiospermum infections in patients with cystic fibrosis. Relative to Aspergillus fumigatus, colonization of the respiratory tract by multiple strains, and of deep organs by only a single strain were observed. On the other hand, the few studies which focused on other fungi isolated from patients with cystic fibrosis have suggested that colonization occurs primarily by a dominant genotype.