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International audience; The nature and fate of urban contaminants washed by stormwater events and accumulating in a detention basin (DB) were investigated. Relations between bacterial and chemical contaminants of trapped urban sediments, and field parameters were analyzed. Fecal indicators and some pathogens known to be environmentally transmitted (Nocardia, Pseudomonas aeruginosa, and Aeromonas caviae) were tracked, and their persistence investigated. Six sampling campaigns were carried out over 3 years, using five sites including a settling chamber (SC). Aerosolized bacteria at these sites were also monitored. Deposits in the basin were made of fine particles and their content in chemical pollutants was found highly variable. High polycyclic aromatic hydrocarbon (PAH) contents were measured but only three pesticides, over 22, were detected. Deposits were significantly contaminated by fecal indicator bacteria (FIB), P. aeruginosa, A. caviae, and by Nocardia. Only A. caviae showed significant numbers in aerosolized particles recovered over the detention basin. Nocardia spp. cells heavily contaminated the SC. The efficacy of the detention basin at reducing bacterial counts per rain event and over time were estimated. A slight drop in the counts was monitored for fecal indicators but not for the other bacterial groups. Hydrodynamic parameters had a strong impact on the distribution and features of the deposits. Multiple factors impacted the fate of FIB, P. aeruginosa, A. caviae, and Nocardia cells, but in a group dependent manner. Nocardia counts were found positively correlated with volatile organic matter. FIB appeared highly efficient colonizers of the DB.

Aims: To develop a qPCR approach for the detection of Pseudomonasaeruginosa in soil and manure and explore its efficacy and limitationscompared with that of a classical culture-dependent approach.Methods and Results: A Ps. aeruginosa ecfX qPCR assay was developed. Thisassay was optimized for soils of contrasting physico-chemical properties andevidenced a three-log dynamic range of detection [5 9 10 4 – 5 9 10 6 cells (g drywt soil) 1 ] in inoculated microcosms. Sensitivity was determined tobe around 5 9 10 4 cells (g drywt soil) 1 . In parallel, the minimum detectionlimit was estimated in the range of 10–100 CFU (g drywt soil) 1 using aculture-dependent approach based on the use of a selective medium (cetrimideagar base medium supplemented with nalidixic acid), coupled to ecfX geneamplification to confirm isolate identity. These soil samples led to the growthof abundant non-Ps. aeruginosa colonies mainly belonging to otherPseudomonas species but also some beta-Proteobacteria. These bacteria stronglyimpacted the detection threshold of this approach. Efficacy of these approacheswas compared for Ps. aeruginosa enumeration among manure and agriculturalsoil samples from various sites in France, Tunisia and Burkina Faso.Conclusions: The developed qPCR assay enabled a specific detection ofPs. aeruginosa in soil and manure samples. The culture-based approach wasusually found more sensitive than the qPCR assay. However, abundance ofnon-Ps. aeruginosa species among the indigenous communities able to grow onthe selective medium affected the sensitivity of this latter approach.Significance and Impact of the Study: This study describes the first specificand sensitive qPCR assay for the detection and enumeration of Ps. aeruginosain soil and manure and shows its complementarity with a culture-basedapproach.IntroductionPseudomonas aeruginosa is an opportunistic pathogen fre-quently responsible for nosocomial infections. It is themain pathogen associated with respiratory tract infectionin cystic fibrosis patients (Gilligan 1991; Nazaret et al.2009), and it can cause a wide variety of infectionsamong compromised hosts (Richard et al. 1994). Infec-tions in healthy individuals can also occur as keratitis(Song et al. 2000), otitis (Heslop and Ovesen 2006) andothers. Pseudomonas aeruginosa can be detected in humanand animal faecal samples (Lavenir et al. 2008) and has

International audience; 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 undetectable bacterial groups among sediments. These 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.

Bacterial thiopurine methyltransferases (bTPMTs) can favour resistance towards toxic tellurite oxyanions through a pathway leading to the emission of a garlic-like smell. Gene expression profiling completed by genetic, physiological and electron microscopy analyses was performed to identify key bacterial activities contributing to this resistance process. Escherichia coli strain MG1655 expressing the bTPMT was used as a cell model in these experiments. This strain produced a garlic-like smell which was found to be due to dimethyl telluride, and cell aggregates in culture media supplemented with tellurite. Properties involved in aggregation were correlated with cell attachment to polystyrene, which increased with tellurite concentrations. Gene expression profiling supported a role of adhesins in the resistance process with 14% of the tellurite-regulated genes involved in cell envelope, flagella and fimbriae biogenesis. Other tellurite-regulated genes were, at 27%, involved in energy, carbohydrate and amino acid metabolism including the synthesis of antioxidant proteins, and at 12% in the synthesis of transcriptional regulators and signal transduction systems. Escherichia coli mutants impaired in tellurite-regulated genes showed ubiquinone and adhesins synthesis, oxidative stress response, and efflux to be essential in the bTPMT resistance process. High tellurite resistance required a synergistic expression of these functions and an efficient tellurium volatilization by the bTPMT.

One hundred sixty-nine nonreplicate imipenem-resistant Pseudomonas aeruginosa strains isolated in a large hospital on the coastal region of Croatia were studied. The most active antibiotics were colistin and amikacin. Most of the isolates were multiresistant. The most prevalent serotype was O12, followed by O11. Six strains carried the bla VIM-2 gene located in a novel class 1 integron composed in its variable part of the bla VIM-2 - bla oxa-10 -Δ qacF-aacA4 genes. Metallo-β-lactamase-producing strains belonged to sequence types ST235 and ST111.

Polymerase chain reaction–based replicon typing represents a novel method to describe the dissemination and follow the evolution of resistance plasmids. We used this approach to study 26 epidemiologically unrelated Enterobacteriaceae and demonstrate the dominance of incompatibility (Inc) A/C or Inc N-related plasmids carrying some emerging resistance determinants to extended-spectrum cephalosporins and carbapenems.

An outbreak of Pseudomonas aeruginosa showing a multidrug-resistant (MDR) phenotype (including carbapenems, ceftazidime, cefepime, gentamicin, tobramycin, and fluoroquinolones) was observed, during a 5-month period, in a general intensive care unit of a large tertiary care and clinical research hospital in southern Italy. The outbreak involved 15 patients, with a total of 87 isolates, mostly from lower respiratory tract specimens. Analysis of isolates involved in the outbreak revealed production of metallo-β-lactamase (MBL) activity, and genotyping by pulsed-field gel electrophoresis of genomic DNA digested by SpeI revealed clonal relatedness among isolates. Molecular analysis of the MBL determinant showed the presence of a bla IMP-13 gene carried on a gene cassette inserted in a class 1 integron which also contained an aacA4 aminoglycoside resistance cassette encoding an AAC(6′)-Ib enzyme. The bla IMP-13 -containing integron and its genetic environment appeared to be similar to those found in P. aeruginosa isolates producing IMP-13 from a hospital in Rome. The bla IMP-13 gene was not transferable by conjugation and was apparently carried on the chromosome. The outbreak was coincidental with a shortage of nursing personnel, and resolution was apparently associated with reinstatement of nursing personnel and reinforcement of general infection control practices within the intensive care unit. To our best knowledge this is the first description of a nosocomial outbreak of relatively large size caused by an IMP-producing gram-negative pathogen in Europe.

International audience; The significance of wastewater treatment lagoons (WWTLs) as point sources of clinically relevant Pseudomonas aeruginosa that can disseminate through rural and peri-urban catchments was investigated. A panel of P. aeruginosa strains collected over three years from WWTLs and community-acquired infections was compared by pulsed field gel electrophoresis (PFGE) DNA fingerprinting and multilocus sequence typing (MLST). Forty-four distantly related PFGE profiles and four clonal complexes were found among the WWTL strains analyzed. Some genotypes were repeatedly detected from different parts of WWTLs, including the influent, suggesting an ability to migrate and persist over time. MLST showed all investigated lineages to match sequence types described in other countries and strains from major clinical clones such as PA14 of ST253 and "C" of ST17 were observed. Some of these genotypes matched isolates from community-acquired infections recorded in the WWTL geographic area. Most WWTL strains harbored the main P. aeruginosa virulence genes; 13% harbored exoU-encoded cytoxins, but on at least six different genomic islands, with some of these showing signs of genomic instability. P. aeruginosa appeared to be highly successful opportunistic colonizers of WWTLs. Lagooning of wastewaters was found to favor dissemination of clinically relevant P. aeruginosa among peri-urban watersheds.

Resistance to carbapenems is an emerging problem among gram-negative hospital pathogens. A transferable plasmid encoding the VIM-4 metallo-β-lactamase was detected in isolates of Klebsiella pneumoniae and Enterobacter cloacae obtained from a single patient under carbapenem therapy. Thus, enterobacteria appear to increasingly contribute to the spread of VIM-type enzymes.

The pathogenic strain Nocardia cyriacigeorgica GUH-2 was isolated from a fatal human nocardiosis case, and its genome was sequenced. The complete genomic sequence of this strain contains 6,194,645 bp, an average G+C content of 68.37%, and no plasmids. We also identified several protein-coding genes to which N. cyriacigeorgica 's virulence can potentially be attributed.

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Phenotypic analyses of antibiotic and metal resistance of a collection of 130 strains of Pseudomonas aeruginosa from various outdoor (i.e. soil, water, animals) and hospital (environment, patients, individuals with cystic fibrosis) settings were performed. Resistance was scored according to the origin of the strains and their likely exposure to antibiotics and chemicals. Most of the 76 outdoor strains showed a wild-type antibiotic resistance phenotype, i.e. resistance to minocycline and trimethoprim–sulfamethoxazole. Sixty percent of hospital strains showed a multiresistance phenotype (from 3 to 16 antibiotics) and confirmed that frequent exposure to antibiotics favored selection and maintenance of antibiotic resistance in P. aeruginosa . Twelve percent of outdoor strains naturally exposed to antiseptics and hydrocarbons showed significant resistance profiles, suggesting that chemical contaminants could contribute to selection of antibiotic resistance. For metal resistance, outdoor strains were more frequently resistant to zinc and cadmium, whereas hospital strains were more frequently resistant to mercury and copper. Differences in metal resistance between the 130 strains investigated were not related to previously characterized processes such as those implicating czcA , involved in cadmium, zinc, and cobalt resistance, or copA and copB , involved in copper resistance. Regulatory or new processes were likely to have contributed to the observed variations. Strains showing strong resistance to antibiotics were the least resistant to metals, and inversely. The lack of significant correlations between antibiotic and metal resistance suggests involvement of distinct processes that are rarely co-selected. The effects of the P. aeruginosa collection size and multi-factorial selective pressure on data sets are discussed.

International audience; In a context of environmental risk assessment of nuclear 79Se radionuclide, the impact of low Se-selenite concentrations (0.008 and 8 mg kg−1) on bacterial communities of two soils, a silty clay loam and a sandy soil, was investigated over a 6-month incubation time. This Se-selenite was partially labelled with 75Se. The state of the Se-impacted bacterial communities was analyzed through total bacterial counts, DNA fingerprints (ARISA profiles) and metabolic profiling (carbon substrate utilization patterns). Furthermore, the genetic diversity of bacterial populations involved in Se volatilization was evaluated by tpm (thiopurine methyltransferase gene) profiling. Emissions of 75Se and CaCl2- extractable 75Se were measured by γ-spectrometry and scintillation analysis. Se-selenite inputs changed transiently the substrate utilization patterns of bacterial communities but did not affect the other indicators. Se volatilization was at its highest level just after adding Se-selenite and for about 1 week. This volatilization was proportional to the added Se-selenite concentrations. It was 100-fold higher in silty clay loam, even though Se bioavailability was reduced in this soil. The soils were amended with crushed grass 3 months after the addition of Se-selenite. This organic amendment affected the organization of bacterial communities and increased the Se-volatilizing activities of both soils. Original soil organic carbon and bacterial diversity and activities seemed responsible for the different levels of Se emissions observed in soils. tpm lineages, encoding Se methyltransferases, were detected in both soils, confirming the broad distribution of tpm-harbouring bacteria and their probable role in the emissions of volatile Se. Five distinct groups of tpm were recorded per soil, with tpmI lineage being detected throughout the incubation period. This study demonstrates the ability of bacterial communities at volatilizing Se concentrations inferior to geochemical backgrounds and suggests that a probable transfer of nuclear Se will occur through volatilization after an environmental spill

International audience; Faecal carriage of Pseudomonas aeruginosa was investigated by selective plating and PCR identification test, among healthy captive snakes from zoological and private collections from France as well as from wild snakes from Guinea. P. aeruginosa faecal carriage among captive snakes was high (72 out of 83 individuals), but low among wild specimen (3 out of 23 individuals). Genetic diversity analyses of the isolates, based on SpeI-PFGE profiles, evidenced five dominant clones or clonal complexes spreading among snakes within a site and between sites and persisting over time. Similar clones or clonal complexes were detected from mouth swabs of the owners and from water and preys used to feed the snakes, evidencing various sources of snake colonization and the first cases of P. aeruginosa crosscontamination between snakes and owners. These observations led to the conclusion that P. aeruginosa behaves as an opportunistic species within snakes in captivity and that colonization and dissemination occurs consecutively to processes similar to those identified within the hospital. Antibiotic susceptibility testing showed that most isolates had a wild-type resistance profile except for one persistent clone isolated from both snakes and preys that harboured multiple antimicrobial resistance genes mediated by an integron carrying the qacH, aadB, aadA2 and cmlA10 cassettes, and a tetA(C)-carrying transposon. Biocides or antibiotics used in the zoological garden could have led to the acquisition of this integron.

National audience

Objectives: To characterize the antibiotic resistance regions of pCC416, a VIM-4- and CMY-4-encoding plasmid from clinical enterobacteria, and to elucidate its relation with the CMY-encoding plasmids widely diffused in Salmonella. Methods: The enterobacterial multiresistant plasmid pCC416 was derived from an Escherichia coli transconjugant and characterized. Conventional and long-range PCR assays were performed using primers specific for VIM-4- and CMY-4-encoding segments of pCC416. Amplicons were characterized by sequencing. blaVIM-4, blaMY-4 and IntI1-specific probes were prepared from PCR products and used for the identification of various pCC416 clones. VIM- and CMY-positive Bam HI and Sau 3AI fragments of pCC416 were cloned into pACYC184 and their sequences were determined by gene walking. Results: The pCC416 plasmid contained two distinct resistant loci carrying β-lactamase genes. The blaVIM-4 gene was part of an integron located in a complex, multidrug-resistant region of novel structure, interspersed with mobile elements or remnants thereof and being similar to various regions of other resistance plasmids. Nevertheless, a region in the 3′ end of this structure resembled the respective region found in a CMY-2-encoding plasmid from Salmonella. The blaCMY-4 gene was identified within an 11.3 kb region also related to the CMY-2-encoding plasmids. Conclusions: pCC416 probably evolved from an IncA/C2, CMY-encoding plasmid through acquisition of a VIM-encoding In4-type integron providing an example of accretion of resistance determinants in a single replicon. © The Author 2007.

Ten multidrug-resistant Pseudomonas aeruginosa strains producing VIM-1-like acquired metallo-β-lactamases (MBLs), isolated from four European countries (Greece, Hungary, Italy, and Sweden), were analyzed for genetic relatedness by several methodologies, including fliC sequence analysis, macrorestriction profiling of genomic DNA by pulsed-field gel electrophoresis (PFGE), random amplification of polymorphic DNA (RAPD), and multilocus sequence typing (MLST). The four approaches yielded consistent results overall but showed different resolution powers in establishing relatedness between isolates (PFGE > RAPD > MLST > fliC typing) and could usefully complement each other to address issues in the molecular epidemiology of P. aeruginosa strains producing acquired MBLs. In particular, the recently developed MLST approach was useful in revealing clonal relatedness between isolates when this was not readily apparent using RAPD and PFGE, and it suggested a common ancestry for some of the VIM-1-like MBL-positive P. aeruginosa strains currently spreading in Europe. The MBL producers belonged in three clonal complexes/burst groups (BGs). Of these, one corresponded to the previously described BG4 and included serotype O12 strains from Hungary and Sweden, while the other two were novel and included serotype O11 or nonserotypable strains from Greece, Sweden, and/or Italy. Comparison of the integrons carrying bla VIM-1 -like cassettes of various isolates revealed a remarkable structural heterogeneity, suggesting the possibility that multiple independent events of acquisition of different bla VIM -containing integrons had occurred in members of the same clonal lineage, although a contribution of integrase-mediated cassette shuffling or other recombination mechanisms during the evolution of similar strains could also have played a role in determining this variability.

The diversity of bacterial thiopurine methyltransferases (bTPMT) among five natural Se-methylating freshwaters was investigated by polymerase chain reaction (PCR) screenings and sequencings. DNA sequence analyses confirmed the cloned products' identity and revealed a broad diversity of freshwater TPMTs. Neighbour-joining (NJ) phylogenetic analyses combining these sequences, all GenBank entries closely related to these sequences and deduced TPMTs obtained in this work from selected gamma-proteobacteria showed TPMTs to form a distinct radiation, closely related to UbiG methyltransferases. Inside the TPMT phylogenetic cluster, eukaryote sequences diverged early from the bacterial ones, and all the bacterial database entries belonged to a subgroup of gamma-proteobacteria, with an apparent lateral transfer of a particular allele to beta-proteobacteria of Bordetella. The NJ phylogenetic tree revealed 22 bTPMT lineages, 10 of which harboured freshwater sequences. All lineages showed deep and long branches indicative of major genetic drifts outside regions encoding highly conserved domains. Selected residues among these highly variable domains could reflect adaptations for particular ecological niches. PCR lineage-specific primers differentiated Se-methylating freshwaters according to their 'tpm lineage' signatures. Most freshwater tpm alleles were found to be distinct from those available in the databases, but a group of tpm was found encoding TPMTs identical to an Aeromonas veronii TPMT characterized in this work.