Nicolas Charbonnel

Eradication of microorganisms embedded in biofilm by an ethanol-based catheter lock solution

BACKGROUND Interdialytic locking of catheters with antimicrobial agents is frequently used for preventing catheter-related infections, often associated with biofilm formation. We determined the bactericidal effect of 60% ethanol (ETOH) versus a 46.7% trisodium citrate (TSC) solution on biofilm embedded in silicone catheters. METHODS Four- and 24-h biofilms of Staphylococcus aureus, S. epidermidis, Pseudomonas aeruginosa, Klebsiella pneumoniae and Candida albicans established in a microfermentor were exposed to ETOH and TSC for up to 24 h and the number of remaining viable microorganisms was determined. RESULTS ETOH 60% was significantly more effective than 46.7% TSC in rapidly eradicating sessile cells from all microorganisms tested. A 20-min ETOH 60% treatment completely eradicated the Gram-negative bacilli and C. albicans biofilms, which initially contained up to 10(8) and 10(5) cells, respectively. Gram-positive cocci biofilms only showed a significant 2.6-4.3 log reduction in the initial viable counts after 20 min of ETOH 60% treatment, with eradication occurring after 30 min. Confocal laser scanning microscopy observation of ETOH-treated biofilm showed sparse cells with respiratory activity. TSC 46.7% eradicated none of the tested microorganisms. In contrast, ETOH 60% totally eradicated planktonic cells, whereas TSC had significant bactericidal activity against K. pneumoniae, P. aeruginosa and C. albicans after 20 min, 1 and 24 h, respectively, but none on the Staphylococcus species. CONCLUSIONS This in vitro study demonstrates the superior antimicrobial activity of ETOH 60% in contrast to TSC 46.7% in eradicating biofilm formed on a silicon catheter. Hence, ethanol-based solution shows promise as a catheter lock solution.

The characterization of functions involved in the establishment and maturation of Klebsiella pneumoniae in vitro biofilm reveals dual roles for surface exopolysaccharides

The ability to form biofilm is seen as an increasingly important colonization strategy among both pathogenic and environmental Klebsiella pneumoniae strains. The aim of the present study was to identify abiotic surface colonization factors of K. pneumoniae using different models at different phases of biofilm development. A 2200 K. pneumoniae mutant library previously obtained by signature-tagged mutagenesis was screened in static and dynamic culture models to detect clones impaired at early and/or mature stages of biofilm formation. A total of 28 mutants were affected during late phases of biofilm formation, whereas 16 mutants displayed early adhesion defect. These mutants corresponded to genes involved in potential cellular and DNA metabolism pathways and to membrane transport functions. Eight mutants were deficient in capsule or LPS production. Gene disruption and microscopic analyses showed that LPS is involved in initial adhesion on both glass and polyvinyl-chloride and the capsule required for the appropriate initial coverage of substratum and the construction of mature biofilm architecture. These results give new insight into the bacterial factors sequentially associated with the ability to colonize an abiotic surface and reveal the dual roles played by surface exopolysaccharides during K. pneumoniae biofilm formation.

Dose-Dependent Immunomodulation of Human Dendritic Cells by the Probiotic Lactobacillus rhamnosus Lcr35

The response of the immune system to probiotics remains controversial. Some strains modulate the cytokine production of dendritic cells (DCs) in vitro and induce a regulatory response, while others induce conversely a pro-inflammatory response. These strain-dependent effects are thought to be linked to specific interactions between bacteria and pattern recognition receptors. We investigated the effects of a well characterized probiotic strain, Lactobacillus rhamnosus Lcr35, on human monocyte-derived immature DCs, using a wide range of bacterial concentrations (multiplicity of infection, MOI, from 0.01 to 100). DNA microarray and qRT-PCR analysis showed that the probiotic induced a large-scale change in gene expression (nearly 1,700 modulated genes, with 3-fold changes), but only with high doses (MOI, 100). The upregulated genes were mainly involved in immune response and identified a molecular signature of inflammation according to the model of Torri. Flow cytometry analysis also revealed a dose-dependent maturation of the DC membrane phenotype, until DCs reached a semi-mature state, with an upregulation of the membrane expression of CD86, CD83, HLA-DR and TLR4, associated with a down-regulation of DC-SIGN, MR and CD14. Measurement of the DC-secreted cytokines showed that Lcr35 induced a strong dose-dependent increase of the pro-Th1/Th17 cytokine levels (TNFα, IL-1β, IL-12p70, IL-12p40 and IL-23), but only a low increase in IL-10 concentration. The probiotic L. rhamnosus Lcr35 therefore induce a dose-dependent immunomodulation of human DCs leading, at high doses, to the semi-maturation of the cells and to a strong pro-inflammatory effect. These results contribute to a fuller understanding of the mechanism of action of this probiotic, and thus of its potential clinical indications in the treatment of either infectious or IgE-dependent allergic diseases.

Quorum sensing affects biofilm formation through lipopolysaccharide synthesis in Klebsiella pneumoniae

Biofilm formation by Klebsiella pneumoniae is modulated by quorum sensing through the synthesis of interspecies AI-2 autoinducers. We characterized in K. pneumoniae the genes homologous to those described in Escherichia coli involved in AI-2 transport, and created two isogenic mutants deleted of lsrCD and tqsA. The levels of extracellular AI-2 with lsrCD and tqsA knockout mutants showed increased and lowered concentrations of AI-2, respectively. The level of transcripts of luxS, the gene responsible for AI-2 synthesis, was increased in sessile cells of the tqsA mutant. In contrast, the expression of the AI-2 import regulator genes lsrR and lsrK was decreased. In addition, the two mutants lsrCD and tqsA formed biofilms with greater biomass but impaired architecture. Since exopolysaccharides play a main role in K. pneumoniae biofilm formation, we investigated their relationship with AI-2 synthesis. None of the mutations in luxS and the AI-2 transport systems affected the expression of three capsule polysaccharide-related genes (wzi, wza and wzx), but all induced an increase in the expression of two lipopolysaccharide (LPS)-synthesis-related genes, wbbM and wzm. AI-2 therefore seems to act as a regulator of biofilm formation and LPS synthesis in sessile K. pneumoniae cells.

Roles of Capsule and Lipopolysaccharide O Antigen in Interactions of Human Monocyte-Derived Dendritic Cells and Klebsiella pneumoniae

ABSTRACT In humans, Klebsiella pneumoniae is a saprophytic bacterium of the nasopharyngeal and intestinal mucosae that is also frequently responsible for severe nosocomial infections. Two major factors of virulence, capsular polysaccharide (CPS) and lipopolysaccharide (LPS) O antigen, are involved in mucosal colonization and the development of infections. These bacterial surface structures are likely to play major roles in interactions with the mucosal immune system, which are orchestrated by a network of surveillance based on dendritic cells (DCs). To determine the roles of K. pneumoniae CPS and LPS in the DC response, we investigated the response of immature human monocyte-derived DCs to bacterial challenge with a wild-type strain and its isogenic mutants deficient in CPS or LPS O-antigen production. As observed by flow cytometry and confocal laser microscopy, the rate of phagocytosis was inversely proportional to the amount of CPS on the bacterial cell surface, with LPS playing little or no role. The K. pneumoniae wild-type strain induced DC maturation with upregulation of CD83, CD86, and TLR4 and downregulation of CD14 and DC-SIGN. With CPS mutants, we observed a greater decrease in DC-SIGN, suggesting a superior maturation of DCs. In addition, incubation of DCs with CPS mutants, and to a lesser extent with LPS mutants, resulted in significantly higher Th1 cytokine production. Combined, our findings suggest that K. pneumoniae CPS, by hampering bacterial binding and internalization, induces a defective immunological host response, including maturation of DCs and pro-Th1 cytokine production, whereas the LPS O antigen seems to be involved essentially in DC activation.

Probiotics and Intestinal Colonization by Vancomycin-Resistant Enterococci in Mice and Humans

ABSTRACT We investigated the impact of probiotics on the intestinal carriage of vancomycin-resistant enterococci (VRE). Administration of Lactobacillus rhamnosus Lcr35 but not Escherichia coli Nissle reduced, although not significantly, the density of VRE colonization in a murine model. No effect of Lcr35 was observed in a double-blind placebo randomized study, involving nine patients.

A Tripartite Efflux Pump Involved in Gastrointestinal Colonization by Klebsiella pneumoniae Confers a Tolerance Response to Inorganic Acid

ABSTRACT The colonization of the gastrointestinal tract of patients by the opportunistic gram-negative bacillus Klebsiella pneumoniae generally occurs prior to the development of nosocomial infections. Mutant strain C-81 was isolated owing to its reduced capacity to colonize the digestive tract in a murine model following transposon mutagenesis (N. Maroncle, D. Balestrino, C. Rich, and C. Forestier, Infect. Immun. 70:4729-4734, 2002). Nucleotide sequence analysis showed that the transposon had inserted into the first open reading frame, eefA, of a three-gene locus (eefABC) whose homologue encodes a tripartite efflux pump in Enterobacter aerogenes (M. Masi, J. M. Pages, C. Villard, and E. Pradel, J. Bacteriol. 187:3894-3897, 2005), and this operon includes an additional short (183-bp) potential open reading frame, eefX, upstream of eefA. In vivo assays showed that a ΔeefA isogenic mutant strain normally colonized the gastrointestinal tract in single-strain tests but was significantly impaired in competition against wild-type strain LM21. Although the cecum was the compartment with the highest number of CFU, the ΔeefA mutant also was detected in the stomach in numbers smaller than those of the wild-type strain. The expression of this potential efflux pump could not be linked to any antimicrobial drug resistance phenotype, but it conferred on the bacteria an acid tolerance response to inorganic acid. The expression of the eef promoter region, measured via a lacZ reporter construction, was slightly induced by an acidic environment and also by hyperosmolarity but not by the presence of bile salts. These results suggest that an efflux pump can confer measurable ecological benefits on K. pneumoniae in an environment with high competition potential.

Pyrosequencing assessment of prokaryotic and eukaryotic diversity in biofilm communities from a French river

Despite the recent and significant increase in the study of aquatic microbial communities, little is known about the microbial diversity of complex ecosystems such as running waters. This study investigated the biodiversity of biofilm communities formed in a river with 454 Sequencing™. This river has the particularity of integrating both organic and microbiological pollution, as receiver of agricultural pollution in its upstream catchment area and urban pollution through discharges of the wastewater treatment plant of the town of Billom. Different regions of the small subunit (SSU) ribosomal RNA gene were targeted using nine pairs of primers, either universal or specific for bacteria, eukarya, or archaea. Our aim was to characterize the widest range of rDNA sequences using different sets of polymerase chain reaction (PCR) primers. A first look at reads abundance revealed that a large majority (47–48%) were rare sequences (<5 copies). Prokaryotic phyla represented the species richness, and eukaryotic phyla accounted for a small part. Among the prokaryotic phyla, Proteobacteria (beta and alpha) predominated, followed by Bacteroidetes together with a large number of nonaffiliated bacterial sequences. Bacillariophyta plastids were abundant. The remaining bacterial phyla, Verrucomicrobia and Cyanobacteria, made up the rest of the bulk biodiversity. The most abundant eukaryotic phyla were annelid worms, followed by Diatoms, and Chlorophytes. These latter phyla attest to the abundance of plastids and the importance of photosynthetic activity for the biofilm. These findings highlight the existence and plasticity of multiple trophic levels within these complex biological systems.

Anti-biofilm activity: a function of Klebsiella pneumoniae capsular polysaccharide.

Competition and cooperation phenomena occur within highly interactive biofilm communities and several non-biocides molecules produced by microorganisms have been described as impairing biofilm formation. In this study, we investigated the anti-biofilm capacities of an ubiquitous and biofilm producing bacterium, Klebsiella pneumoniae. Cell-free supernatant from K. pneumoniae planktonic cultures showed anti-biofilm effects on most Gram positive bacteria tested but also encompassed some Gram negative bacilli. The anti-biofilm non-bactericidal activity was further investigated on Staphylococcus epidermidis, by determining the biofilm biomass, microscopic observations and agglutination measurement through a magnetic bead-mediated agglutination test. Cell-free extracts from K. pneumoniae biofilm (supernatant and acellular matrix) also showed an influence, although to a lesser extend. Chemical analyses indicated that the active molecule was a high molecular weight polysaccharide composed of five monosaccharides: galactose, glucose, rhamnose, glucuronic acid and glucosamine and the main following sugar linkage residues [→2)-α-l-Rhap-(1→]; [→4)-α-l-Rhap-(1→]; [α-d-Galp-(1→]; [→2,3)-α-d-Galp-(1→]; [→3)-β-d-Galp-(1→] and, [→4)-β-d-GlcAp-(1→]. Characterization of this molecule indicated that this component was more likely capsular polysaccharide (CPS) and precoating of abiotic surfaces with CPS extracts from different serotypes impaired the bacteria-surface interactions. Thus the CPS of Klebsiella would exhibit a pleiotropic activity during biofilm formation, both stimulating the initial adhesion and maturation steps as previously described, but also repelling potential competitors.

Identification of Commensal Escherichia coli Genes Involved in Biofilm Resistance to Pathogen Colonization

Protection provided by host bacterial microbiota against microbial pathogens is a well known but ill-understood property referred to as the barrier effect, or colonization resistance. Despite recent genome-wide analyses of host microbiota and increasing therapeutic interest, molecular analysis of colonization resistance is hampered by the complexity of direct in vivo experiments. Here we developed an in vitro-to-in vivo approach to identification of genes involved in resistance of commensal bacteria to exogenous pathogens. We analyzed genetic responses induced in commensal Escherichia coli upon entry of a diarrheagenic enteroaggregative E. coli or an unrelated Klebsiella pneumoniae pathogen into a biofilm community. We showed that pathogens trigger specific responses in commensal bacteria and we identified genes involved in limiting colonization of incoming pathogens within commensal biofilm. We tested the in vivo relevance of our findings by comparing the extent of intestinal colonization by enteroaggregative E. coli and K. pneumoniae pathogens in mice pre-colonized with E. coli wild type commensal strain, or mutants corresponding to identified colonization resistance genes. We demonstrated that the absence of yiaF and bssS (yceP) differentially alters pathogen colonization in the mouse gut. This study therefore identifies previously uncharacterized colonization resistance genes and provides new approaches to unravelling molecular aspects of commensal/pathogen competitive interactions.