Damien Balestrino

Global impact of mature biofilm lifestyle on Escherichia coli K‐12 gene expression

The formation of biofilm results in a major lifestyle switch that is thought to affect the expression of multiple genes and operons. We used DNA arrays to study the global effect of biofilm formation on gene expression in mature Escherichia coli K‐12 biofilm. We show that, when biofilm is compared with the exponential growth phase, 1.9% of the genes showed a consistent up‐ or downregulation by a factor greater than two, and that 10% of the E. coli genome is significantly differentially expressed. The functions of the genes induced in these conditions correspond to stress response as well as energy production, envelope biogenesis and unknown functions. We provide evidence that the expression of stress envelope response genes, such as the psp operon or elements of the cpx and rpoE pathways, is a general feature of E. coli mature biofilms. We also compared biofilm with the stationary growth phase and showed that the biofilm lifestyle, although sharing similarities with the stationary growth phase, triggers the expression of specific sets of genes. Using gene disruption of 54 of the most biofilm‐induced genes followed by a detailed phenotypic study, we validated the biological relevance of our analysis and showed that 20 of these genes are required for the formation of mature biofilm. This group includes 11 genes of previously unknown function. These results constitute a comprehensive analysis of the global transcriptional response triggered in mature E. coli biofilms and provide insights into its physiological signature.

Broad-spectrum biofilm inhibition by a secreted bacterial polysaccharide

The development of surface-attached biofilm bacterial communities is considered an important source of nosocomial infections. Recently, bacterial interference via signaling molecules and surface active compounds was shown to antagonize biofilm formation, suggesting that nonantibiotic molecules produced during competitive interactions between bacteria could be used for biofilm reduction. Hence, a better understanding of commensal/pathogen interactions within bacterial community could lead to an improved control of exogenous pathogens. To reveal adhesion or growth-related bacterial interference, we investigated interactions between uropathogenic and commensal Escherichia coli in mixed in vitro biofilms. We demonstrate here that the uropathogenic strain CFT073 and all E. coli expressing group II capsules release into their environment a soluble polysaccharide that induces physicochemical surface alterations, which prevent biofilm formation by a wide range of Gram-positive and Gram-negative bacteria. We show that the treatment of abiotic surfaces with group II capsular polysaccharides drastically reduces both initial adhesion and biofilm development by important nosocomial pathogens. These findings identify capsular polymers as antiadhesion bacterial interference molecules, which may prove to be of significance in the design of new strategies to limit biofilm formation on medical in dwelling devices.

Characterization of type 2 quorum sensing in Klebsiella pneumoniae and relationship with biofilm formation.

Quorum sensing is a process by which bacteria communicate by using secreted chemical signaling molecules called autoinducers. Many bacterial species modulate the expression of a wide variety of physiological functions in response to changes in population density by this mechanism. In this study, the opportunistic pathogen Klebsiella pneumoniae was observed to secrete type 2 signaling molecules. A homologue of luxS, the gene required for AI-2 synthesis in Vibrio harveyi, was isolated from the K. pneumoniae genome. A V. harveyi bioassay showed the luxS functionality in K. pneumoniae and its ability to complement the luxS-negative phenotype of Escherichia coli DH5alpha. Autoinducer activity was detected in the supernatant, and maximum expression of specific messengers detected by quantitative reverse transcription-PCR analysis occurred during the late exponential phase. The highest levels of AI-2 were observed in minimal medium supplemented with glycerol. To determine the potential role of luxS in colonization processes, a K. pneumoniae luxS isogenic mutant was constructed and tested for its capacity to form biofilms in vitro on an abiotic surface and to colonize the intestinal tract in a murine model. No difference was observed in the level of intestinal colonization between the wild-type strain and the luxS mutant. Microscopic analysis of biofilm structures revealed that the luxS mutant was able to form a mature biofilm but with reduced capacities in the development of microcolonies, mostly in the early steps of biofilm formation. These data suggest that a LuxS-dependent signal plays a role in the early stages of biofilm formation by K. pneumoniae.

Persistence of Colonization of Intestinal Mucosa by a Probiotic Strain, Lactobacillus casei subsp. rhamnosus Lcr35, after Oral Consumption

ABSTRACT The colonization by the probiotic Lactobacillus casei subsp. rhamnosus Lcr35 of the gastrointestinal tracts of mice and humans was studied. The mice were orally given 109 CFU of Lcr35 either once or three times at 24-h intervals. A 16S ribosomal nucleic probe used in hybridization assays detected Lcr35 in the feces of mice for up to 3 days after the feeding, at a level of 108 to 109 CFU/g of feces. In the human assay, 12 healthy volunteers were enrolled in a randomized trial and ingested Lcr35 at a dosage of 108 or 1010 or 1012 CFU every day for 7 days. Then, after a 3-week posttreatment period, there was a second intake period similar to the first one. Analysis of fecal samples showed significant increases in the number of lactobacilli during the first intake period, whatever the dose given. The greatest increases were observed in subjects harboring the lowest indigenous population of Lcr35-like bacteria. During the 3-week posttreatment period, the number of CFU slightly decreased over time, and an increase, although not a statistically significant one, was observed during the second test period. These findings suggest that Lcr35 is able to survive within the gastrointestinal tract.

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.

Identification of Klebsiella pneumoniae Genes Involved in Intestinal Colonization and Adhesion Using Signature-Tagged Mutagenesis

ABSTRACT Klebsiella pneumoniae is an opportunistic pathogen responsible for nosocomial infections that initially colonize the intestinal tract of patients. Signature-tagged mutagenesis was used to identify genes required for this function. A library of 2,200 mutants was analyzed for the inability of the mutants to survive in a murine model of intestinal colonization and to adhere to human intestinal cells (Int-407) in vitro. Twenty-nine attenuated mutants were selected for further analyses after competition assays against the wild-type strain. Whatever the screening model, most of the transposon insertions occurred in genes involved in metabolic pathways, membrane transport, DNA metabolism, transcriptional regulation, and unknown functions. Only one mutant was attenuated in both the murine colonization and the in vitro adhesion models, and the sequence disrupted by the transposon had homology to adhesin-encoding genes of Haemophilus sp.

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.

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.