Characterizing the development of biofilm in polyethylene pipes in the non-chlorinated Danish drinking-water distribution system.

In newly commissioned drinking-water polyethylene (PE) pipes, biofilm develops on the inner pipe surface. The microbial community composition from colonization to the establishment of mature biofilms is less known, including the effect on the distributed water quality. Biofilm development was followed through 1.5 years in PE-pipe side streams at two locations of a full-scale, non-chlorinated drinking-water distribution system (leaving a waterworks versus 5-6 km from a waterworks) along with inlet and outlet water quality. Mature biofilms were established after ∼8-9 months, dominated by Proteobacteria, Actinobacteria and Saccharibacteria (61-93% relative abundance), with a higher diversity (OTUs/Shannon Index/16S rRNA gene amplicon sequencing) in pipes in the far end of the distribution system. Comamonadaceae, and specifically Aquabacterium (>30% of reads), dominated young (∼1.5-month-old) biofilms. Young biofilms were linked to increased microbiological counts in drinking water (HPC/ATP/qPCR), while the establishment of mature biofilms led to a drop in HPC and benefited the water quality, highlighting the importance of optimizing commissioning procedures for rapidly achieving mature and stable biofilms.

Bacterial Human Virulence Genes across Diverse Habitats As Assessed by In silico Analysis of Environmental Metagenomes.

The occurrence and distribution of clinically relevant bacterial virulence genes across natural (non-human) environments is not well understood. We aimed to investigate the occurrence of homologs to bacterial human virulence genes in a variety of ecological niches to better understand the role of natural environments in the evolution of bacterial virulence. Twenty four bacterial virulence genes were analyzed in 46 diverse environmental metagenomic datasets, representing various soils, seawater, freshwater, marine sediments, hot springs, the deep-sea, hypersaline mats, microbialites, gutless worms and glacial ice. Homologs to 16 bacterial human virulence genes, involved in urinary tract infections, gastrointestinal diseases, skin diseases, and wound and systemic infections, showed global ubiquity. A principal component analysis did not demonstrate clear trends across the metagenomes with respect to occurrence and frequency of observed gene homologs. Full-length (>95%) homologs of several virulence genes were identified, and translated sequences of the environmental and clinical genes were up to 50-100% identical. Furthermore, phylogenetic analyses indicated deep branching positions of some of the environmental gene homologs, suggesting that they represent ancient lineages in the phylogeny of the clinical genes. Fifteen virulence gene homologs were detected in metatranscriptomes, providing evidence of environmental expression. The ubiquitous presence and transcription of the virulence gene homologs in non-human environments point to an important ecological role of the genes for the activity and survival of environmental bacteria. Furthermore, the high degree of sequence conservation between several of the environmental and clinical genes suggests common ancestral origins.

Occurrence and expression of bacterial human virulence gene homologues in natural soil bacteria.

The presence and in vitro expression of homologues to 22 bacterial human virulence determinants amongst culturable soil bacteria were investigated. About 25% of the bacterial isolates contained virulence gene homologues representing toxin (hblA, cytK2), adhesin (fimH), regulator (phoQ) and resistance (yfbI) determinants in pathogenic bacteria. The homologues of the toxin genes were found in Actinobacteria and Firmicutes (hblA), and in Firmicutes and Alpha- and Gammaproteobacteria (cytK2). The homologues to the type 1 fimbrial adhesin gene, fimH, and the L-Ara4N transferase gene, yfbI, were observed in Actinobacteria, Firmicutes and Gammaproteobacteria. The regulator gene, phoQ, was only found in Gammaproteobacteria. The presence of cytK2 in Alpha- and Gammaproteobacteria, fimH in Actinobacteria and Firmicutes, and hblA in Actinobacteria has not previously been described. A close sequence similarity (84-100%) was observed between the genes of environmental and clinical isolates, and expression assays suggested that the genes in some cases were expressed in vitro. The presence of functional virulence gene homologues underpins their importance for the survival of environmental bacteria. Furthermore, the high degree of sequence conservation to clinical sequences indicates that natural environments may be 'evolutionary cribs' of emerging pathogens.

Widespread occurrence of bacterial human virulence determinants in soil and freshwater environments.

The occurrence of 22 bacterial human virulence genes (encoding toxins, adhesins, secretion systems, regulators of virulence, inflammatory mediators, and bacterial resistance) in beech wood soil, roadside soil, organic agricultural soil, and freshwater biofilm was investigated by nested PCR. The presence of clinically relevant bacterial groups known to possess virulence genes was tested by PCR of 16S and 23S rRNA genes. For each of the virulence genes detected in the environments, sequencing and NCBI BLAST analysis confirmed the identity of the PCR products. The virulence genes showed widespread environmental occurrence, as 17 different genes were observed. Sixteen genes were detected in beech wood soil, and 14 were detected in roadside and organic agricultural soils, while 11 were detected in the freshwater biofilm. All types of virulence traits were represented in all environments; however, the frequency at which they were detected was variable. A principal-component analysis suggested that several factors influenced the presence of the virulence genes; however, their distribution was most likely related to the level of contamination by polycyclic aromatic hydrocarbons and pH. The occurrence of the virulence genes in the environments generally did not appear to be the result of the presence of clinically relevant bacteria, indicating an environmental origin of the virulence genes. The widespread occurrence of the virulence traits and the high degree of sequence conservation between the environmental and clinical sequences suggest that soil and freshwater environments may constitute reservoirs of virulence determinants normally associated with human disease.

Regulation of the IL-10/IL-12 axis in human dendritic cells with probiotic bacteria.

In this study, we have used monocyte-derived dendritic cells (DCs) to design a screening model for the selection of microorganisms with the ability to suppress DC-secreted IL-12p70, a critical cytokine for the induction of T-helper cell type 1 immune responses under inflammatory conditions. By the treatment of DCs with cocktails containing TLR agonists and proinflammatory cytokines, the cells increased the secretion of the Th1-promoting cytokine IL-12p70. Clinically used probiotics were tested for their IL-10- and IL-12p70-stimulating properties in immature DCs, and showed a dose-dependent change in the IL-10/IL-12p70 balance. Lactobacillus acidophilus NCFM(™) and the probiotic mixture VSL#3 showed a strong induction of IL-12p70, whereas Lactobacillus salivarius Ls-33 and Bifidobacterium infantis 35624 preferentially induced IL-10. Escherichia coli Nissle 1917 induced both IL-10 and IL-12p70, whereas the probiotic yeast Saccharomyces boulardii induced low levels of cytokines. When combining these microorganisms with the Th1-promoting cocktails, E. coli Nissle 1917 and B. infantis 35624 were potent suppressors of IL-12p70 secretion in an IL-10-independent manner, indicating a suppressive effect on Th1-inducing antigen-presenting cells. The present model, using cocktail-stimulated DCs with potent IL-12p70-stimulating capacity, may be used as an efficient tool to assess the anti-inflammatory properties of microorganisms for potential clinical use.