LecA and LecB tetrameric lectins take part in oligosaccharide-mediated adhesion-processes of Pseudomonas aeruginosa. Glycomimetics have been designed to block these interactions. The great versatility of P. aeruginosa suggests that the range of application of these glycomimetics could be restricted to genotypes with particular lectin types. The likelihood of having genomic and genetic changes impacting LecA and LecB interactions with glycomimetics such as galactosylated and fucosylated calix[4]arene was investigated over a collection of strains from the main clades of P. aeruginosa. Lectin types were defined, and their ligand specificities were inferred. These analyses showed a loss of lecA among the PA7 clade. Genomic changes impacting lec loci were thus assessed using strains of this clade, and by making comparisons with the PAO1 genome. The lecA regions were found challenged by phage attacks and PAGI-2 (genomic island) integrations. A prophage was linked to the loss of lecA. The lecB regions were found less impacted by such rearrangements but greater lecB than lecA genetic divergences were recorded. Sixteen combinations of LecA and LecB types were observed. Amino acid variations were mapped on PAO1 crystal structures. Most significant changes were observed on LecBPA7, and found close to the fucose binding site. Glycan array analyses were performed with purified LecBPA7. LecBPA7 was found less specific for fucosylated oligosaccharides than LecBPAO1, with a preference for H type 2 rather than type 1, and Lewisa rather than Lewisx. Comparison of the crystal structures of LecBPA7 and LecBPAO1 in complex with Lewisa showed these changes in specificity to have resulted from a modification of the water network between the lectin, galactose and GlcNAc residues. Incidence of these modifications on the interactions with calix[4]arene glycomimetics at the cell level was investigated. An aggregation test was used to establish the efficacy of these ligands. Great variations in the responses were observed. Glycomimetics directed against LecB yielded the highest numbers of aggregates for strains from all clades. The use of a PAO1lecB strain confirmed a role of LecB in this aggregation phenotype. Fucosylated calix[4]arene showed the greatest potential for a use in the prevention of P. aeruginosa infections.

Rivers are often challenged by fecal contaminations. The barrier effect of sediments against fecal bacteria was investigated through the use of a microbial source tracking (MST) toolbox, and by Next Generation Sequencing (NGS) of V5-V6 16S rRNA gene (rrs) sequences. Non-metric multi-dimensional scaling analysis of V5-V6 16S rRNA gene sequences differentiated bacteriomes according to their compartment of origin i. e. surface water against benthic and hyporheic sediments. Classification of these reads showed the most prevalent operating taxonomic units (OTU) to be allocated to Flavobacterium and Aquabacterium. Relative numbers of Gaiella, Haliangium and Thermoleophilum OTU matched the observed differentiation of bacteriomes according to river compartments. OTU patterns were found impacted by combined sewer overflows (CSO) through an observed increase in diversity from the sewer to the hyporheic sediments. These changes appeared driven by direct transfers of bacterial contaminants from wastewaters but also by organic inputs favoring previously undetected bacterial groups among sediments. The NGS datasets appeared more sensitive at tracking community changes than MST markers. The human-specific MST marker HF183 was strictly detected among CSO-impacted surface waters and not river bed sediments. The ruminant-specific DNA marker was more broadly distributed but intense bovine pollution was required to detect transfers from surface water to benthic and hyporheic sediments. Some OTU showed distribution patterns in line with these MST datasets such as those allocated to the Aeromonas, Acinetobacter, and Pseudomonas. Fecal indicators (Escherichia coli and total thermotolerant coliforms) were detected all over the river course but their concentrations were not correlated with MST ones. Overall, MST and NGS datasets suggested a poor colonization of river sediments by bovine and sewer bacterial contaminants. No environmental outbreak of these bacterial contaminants was detected.

Pseudomonas aeruginosa (P.a) is one of the most critical antibiotic resistant bacteria in the world and is the most prevalent pathogen in cystic fibrosis (CF), causing chronic lung infections that are considered one of the major causes of mortality in CF patients. Although several studies have contributed to understanding P.a within-host adaptive evolution at a genomic level, it is still difficult to establish direct relationships between the observed mutations, expression of clinically relevant phenotypes, and clinical outcomes. Here, we performed a comparative untargeted LC/HRMS-based metabolomics analysis of sequential isolates from chronically infected CF patients to obtain a functional view of P.a adaptation. Metabolic profiles were integrated with expression of bacterial phenotypes and clinical measurements following multiscale analysis methods. Our results highlighted significant associations between P.a “metabotypes”, expression of antibiotic resistance and virulence phenotypes, and frequency of clinical exacerbations, thus identifying promising biomarkers and therapeutic targets for difficult-to-treat P.a infections

Aims To develop a qPCR approach for the detection of Pseudomonas aeruginosa in soil and manure and explore its efficacy and limitations compared with that of a classical culture-dependent approach. Methods and Results A Ps. aeruginosa ecfX qPCR assay was developed. This assay was optimized for soils of contrasting physico-chemical properties and evidenced a three-log dynamic range of detection [5 × 10⁴ – 5 × 10⁶ cells (g drywt soil)⁻¹] in inoculated microcosms. Sensitivity was determined to be around 5 × 10⁴ cells (g drywt soil)⁻¹. In parallel, the minimum detection limit was estimated in the range of 10–100 CFU (g drywt soil)⁻¹ using a culture-dependent approach based on the use of a selective medium (cetrimide agar base medium supplemented with nalidixic acid), coupled to ecfX gene amplification to confirm isolate identity. These soil samples led to the growth of abundant non-Ps. aeruginosa colonies mainly belonging to other Pseudomonas species but also some beta-Proteobacteria. These bacteria strongly impacted the detection threshold of this approach. Efficacy of these approaches was compared for Ps. aeruginosa enumeration among manure and agricultural soil samples from various sites in France, Tunisia and Burkina Faso. Conclusions The developed qPCR assay enabled a specific detection of Ps. aeruginosa in soil and manure samples. The culture-based approach was usually found more sensitive than the qPCR assay. However, abundance of non-Ps. aeruginosa species among the indigenous communities able to grow on the selective medium affected the sensitivity of this latter approach. Significance and Impact of the Study This study describes the first specific and sensitive qPCR assay for the detection and enumeration of Ps. aeruginosa in soil and manure and shows its complementarity with a culture-based approach.

Urban activities generate surface deposits over impervious surfaces that can represent ecological and health hazards. Bacteriome genetic structures of deposits washed off during rainfall events, over an urban industrial watershed, were inferred from 16 S rRNA gene (rrs) sequences generated by high throughput sequencing. Deposits were sampled over a 4 year-period from a detention basin (DB). Major shifts, matching key management practices, in the structure of these urban bacteriomes, were recorded. Correlation analyses of rrs similarities between samples and their respective concentrations in chemical pollutants, markers of human fecal contaminations (HF183) and antimicrobial resistances (integrons), were performed. Harsher environmental constraints building up in the older deposits led to an increase number of rrs reads from extremophiles such as Acidibacter and Haliangium. Deposits accumulating in the decantation pit of the DB showed an increase in rrs reads from warm blooded intestinal tract bacteria such as Bacteroides and Prevotella. This enrichment matched higher concentrations of Bacteroides HF183 genotypes normally restricted to humans. Bacteriomes of urban deposits appeared good indicators of human-driven environmental changes. Their composition was found representative of their origin. Soil particles and rain appeared to be major contributors of the inferred bacterial taxa recovered from recent deposits.

The invasion of aquifer microbial communities by aboveground microorganisms, a phenomenon known as community coalescence, is likely to be exacerbated in groundwaters fed by stormwater infiltration systems (SISs). Here, the incidence of this increased connectivity with upslope soils and impermeabilized surfaces was assessed through a meta-analysis of 16S rRNA gene libraries. Specifically, DNA sequences encoding 16S rRNA V5-V6 regions from free-living and attached aquifer bacteria (i.e., water and biofilm samples) were analysed upstream and downstream of a SIS and compared with those from bacterial communities from watershed runoffs and surface sediments from the SIS detention and infiltration basins. Significant bacterial transfers were inferred by the SourceTracker Bayesian approach, with 23 % to 57 % of the aquifer bacterial biofilms being composed of taxa from aboveground sediments and urban runoffs. Sediments from the detention basin were found more significant contributors of taxa involved in the buildup of these biofilms than soils from the infiltration basin. Inferred taxa among the coalesced biofilm community were predicted to be high in hydrocarbon degraders such as Sphingobium and Nocardia. The 16S rRNA-based bacterial community structure of the downstream-SIS aquifer waters showed lower coalescence with aboveground taxa (8 % to 38 %) than those of biofilms and higher numbers of taxa predicted to be involved in the N and S cycles. A DNA marker named tpm enabled the tracking of bacterial species from 24 genera including Pseudomonas, Aeromonas and Xanthomonas, among these communities. Several tpm sequence types were found to be shared between the aboveground and aquifer samples. Reads related to Pseudomonas were allocated to 50 species, of which 16 were found in the aquifer samples. Several of these aquifer species were found to be involved in denitrification but also hydrocarbon degradation (P. aeruginosa, P. putida and P. fluorescens). Some tpm sequence types allocated to P. umsongensis and P. chengduensis were found to be enriched among the tpm-harbouring bacteria, respectively, of the aquifer biofilms and waters. Reads related to Aeromonas were allocated to 11 species, but only those from A. caviae were recovered aboveground and in the aquifer samples. Some tpm sequence types of the X. axonopodis phytopathogen were recorded in higher proportions among the tpm-harbouring bacteria of the aquifer waters than in the aboveground samples. A significant coalescence of microbial communities from an urban watershed with those of an aquifer was thus observed, and recent aquifer biofilms were found to be significantly colonized by runoff-opportunistic taxa able to use urban C sources from aboveground compartments.