Conjugative multiple-antibiotic resistance plasmids in Escherichia coli isolated from environmental waters contaminated by human faecal wastes.

AIMS: To better understand the involvement of faecal contamination in the dissemination of antibiotic resistance genes, we investigated the genetic supports of resistances in nine multi-resistant Escherichia coli strains originating from human faecal contamination, and isolated from three different aquatic environments used for producing drinking water. METHODS AND RESULTS: Seven strains harboured at least one large plasmid that we have characterized (size, antibiotic resistance patterns, incompatibility group, capacity of autotransfer, presence of integron). Most of these plasmids were conjugative and carried numerous resistances. One of the plasmids studied, belonging to the IncP incompatibility group, was able to transfer by conjugation to Pseudomonas fluorescens and Aeromonas sp. Only two of the plasmids we studied carried class 1 and/or 2 integron(s). CONCLUSIONS: Conjugative plasmids isolated from multi-resistant E. coli strains explained most of the resistances of their host strains and probably contribute to the spread of antibiotic resistance genes coming from human faecal contamination. SIGNIFICANCE AND IMPACT OF THE STUDY: These results highlight the key role played by plasmids in the multi-resistance phenotype of faecal bacteria and the diversity of these genetic structures. Contaminated water, especially accidentally contaminated drinking water, could be a path back to humans for these plasmids.

Real-time PCR for quantification in soil of glycoside hydrolase family 6 cellulase genes.

UNLABELLED: Cellulose is the main structural component of the cell walls of higher plants, representing c. 35-50% of a plant's dry weight; after decomposition and transformation, and constituting a large part of soil organic matter. Telluric micro-organisms able to use cellulose as carbon and energy sources for growth are widely distributed in the environment, but the factors controlling the rate of cellulose degradation are not well understood. In this study, we have developed a quantitative real-time PCR (qPCR) primer set to quantify the glycoside hydrolase family 6 (GH6 family) cellulase genes in soil samples. The qPCR assays were linear over 8 orders of magnitude and sensitive down to 10 copies per assay. qPCR analysis of contrasted soil samples showed densities between 2·47 × 10(7) and 1·48 × 10(10) copies per gram of soil. Cloning and sequencing of the PCR products from environmental DNA confirmed both specific amplification (more than 96%) and the wide diversity targeted by the primer set, throughout nearly all the GH6 family, including sequences of bacteria and fungi. SIGNIFICANCE AND IMPACT OF THE STUDY: Telluric micro-organisms able to use cellulose as carbon and energy sources for growth are widely distributed in the environment, but the factors controlling the rate of cellulose degradation are not well understood. The objective of our study was to develop a qPCR for rapid quantification of GH6 cellulase genes in soil. This qPCR could be applied to study the potential for cellulose degradation in different soils in order to better understand the factors controlling the stability of the soil organic matter.

Diversity of Vibrio spp. isolated at ambient environmental temperature in the Eastern English Channel as determined by pyrH sequencing.

AIMS: To describe the diversity of the culturable mesophilic and potentially pathogenic vibrios isolated at 22 and 37°C on TCBS medium, in September 2009 from seawater and surface sediments. METHODS AND RESULTS: q-PCR assays previously selected for the identification of bacterial strains isolated at 37°C were used in combination with the partial sequencing of two housekeeping genes, pyrH and toxR, to identify 315 strains isolated at 22°C. The great majority of the 37°C strains was identified by q-PCR assays, (five of the six species) with the predominance of Vibrio alginolyticus (85·9%) and V. harveyi (10·7%). The human pathogens V. parahaemolyticus and V. cholerae were rarely detected (two strains each). The 22°C strains were successfully identified by the phylogeny analysis of pyrH and toxR genes, revealing 20 Vibrio species, with the predominance of the clam pathogen V. celticus (36·8%). The Splendidus and the Harveyi groups represented the main Vibrio group at 22°C (80%) and 37°C (99·5%), respectively. CONCLUSIONS: The combination of q-PCR assays and the sequencing of pyrH and toxR genes highlighted two different Vibrio communities at 22 and 37°C both dominated by pathogenic species for marine organisms. SIGNIFICANCE AND IMPACT OF THE STUDY: The sequencing of the pyrH gene revealed to be a valuable tool to identify environmental Vibrio spp. strains isolated at 22°C, as 92·3% of them were identified in this study.

Linking initial soil bacterial diversity and polycyclic aromatic hydrocarbons (PAHs) degradation potential.

The aim of this study was to understand the role of indigenous soil microbial communities on the biodegradation of polycyclic aromatic hydrocarbons (PAHs) and to determine whether PAHs degradation potential in soils may be evaluated by analysis of bacterial diversity and potential metabolisms using a metagenomics approach. Five different soils were artificially contaminated with seven selected PAHs and the most abundant bacterial taxa were assessed by sequencing the 16S rRNA gene, and linking them to PAH biodegradation efficiencies. A PICRUSt approach was then led to estimate the degradation potentials by metagenomics inference. Although the role of bacteria in PAHs degradation is not directly established here, the presence of a large number of bacteria belonging to the Betaproteobacteria class correlated to a higher degradation of LMW PAHs. A link with specific bacterial taxa was more difficult to establish concerning HMW PAHs, which seemed to require more complex mechanisms as shown by PICRUSt.

Recurrent recovery of Pseudomonas oryzihabitans strains in a karstified chalk aquifer.

Pseudomonas oryzihabitans is an uncommon pathogen that may cause catheter-associated infections, particularly in immunocompromised patients. Although it has been isolated from environment, the source of human infection is not well documented. In the present study, 14 isolates of P. oryzihabitans were recovered over a 28-month period from a karstified chalk aquifer, allowing to advance that distributed natural water could be a source of contamination. Microbiological analyses showed that the bacterium was mainly associated with suspended particulate matters. To investigate the clonality of P. oryzihabitans environmental isolates, 16S rRNA gene sequencing, antibiogram and randomly amplified polymorphic DNA (RAPD) typings were performed. Results demonstrated (i) the presence of at least three clones within the aquifer and (ii) that the presence of the bacterium in groundwater is not only the result of a biofilm bloom but also of an exogenous contamination.

Low effect of phenanthrene bioaccessibility on its biodegradation in diffusely contaminated soil.

This study focused on the role of bioaccessibility in the phenanthrene (PHE) biodegradation in diffusely contaminated soil, by combining chemical and microbiological approaches. First, we determined PHE dissipation rates and PHE sorption/desorption isotherms for two soils (PPY and Pv) presenting similar chronic PAH contamination, but different physico-chemical properties. Our results revealed that the PHE dissipation rate was significantly higher in the Pv soil compared to the PPY soil, while PHE sorption/desorption isotherms were similar. Interestingly, increases of PHE desorption and potentially of PHE bioaccessibility were observed for both soils when adding rhamnolipids (biosurfactants produced by Pseudomonas aeruginosa). Second, using 13C-PHE incubated in the same soils, we analyzed the PHE degrading bacterial communities. The combination of stable isotope probing (DNA-SIP) and 16S rRNA gene pyrosequencing revealed that Betaproteobacteria were the main PHE degraders in the Pv soil, while a higher bacterial diversity (Alpha-, Beta-, Gammaproteobacteria and Actinobacteria) was involved in PHE degradation in the PPY soil. The amendment of biosurfactants commonly used in biostimulation methods (i.e. rhamnolipids) to the two soils clearly modified the PHE sorption/desorption isotherms, but had no significant impact on PHE degradation rates and PHE-degraders identity. These results demonstrated that increasing the bioaccessibility of PHE has a low impact on its degradation and on the functional populations involved in this degradation.

Alternative techniques to HPCD to evaluate the bioaccessible fraction of soil-associated PAHs and correlation to biodegradation efficiency.

The total amount of polycyclic aromatic hydrocarbons (PAHs) in soils, given by exhaustive chemical extractions, does not relate directly to environmental risk, since only a fraction may be accessible to soil organisms. The rapid PAH desorbing fraction (Frap), which is weakly and reversibly sorbed to soils, is called the bioaccessible fraction, and can be estimated by non-exhaustive aqueous extractions. In order to better estimate Frap, different mild-extractants were tested, such as various cyclodextrins, surfactants and butanol. Their extractability performances were correlated to the Kd partition coefficients of seven PAHs obtained through sorption isotherms from five soils, but also to the PAHs molecular size and to the amounts of organic matter and of some clays (smectites and kaolinites). If hydroxypropyl-ß-cyclodextrin was actually a good extractant to assess PAH accessibility, the polymer of carboxymethyl-ß-cyclodextrin (pCMCD) was better (with a lower cost) to estimate the rapid mass transfer between soil particles and the soil solution, depending also on soil ageing. But Frap, estimated through pCMCD extractions, did not reflect the biodegradation of the PAHs after three months in soil microcosms. The chemical method underestimated the dissipation of 3-4 ring PAHs and overestimated that of 5-6 ring PAHs. So biodegradation was not only limited by PAHs mass-transfer, but also by biological factors, favoring the access of microorganisms to residual strongly sorbed fractions of 3-4 ring PAHs, and inhibiting the degradation of accessible but highly toxic 5-6 ring PAHs.

Correlations between PAH bioavailability, degrading bacteria, and soil characteristics during PAH biodegradation in five diffusely contaminated dissimilar soils.

The natural biodegradation of seven polycyclic aromatic hydrocarbons (PAHs) by native microorganisms was studied in five soils from Normandy (France) from diffusely polluted areas, which can also pose a problem in terms of surfaces and amounts of contaminated soils. Bioavailability tests using cyclodextrin-based extractions were performed. The natural degradation of low molecular weight (LMW) PAHs was not strongly correlated to their bioavailability due to their sorption to geosorbents. Conversely, the very low degradation of high molecular weight (HMW) PAHs was partly correlated to their poor availability, due to their sorption on complexes of organic matter and kaolinites or smectites. A principal component analysis allowed us to distinguish between the respective degradation behaviors of LMW and HMW PAHs. LMW PAHs were degraded in less than 2-3 months and were strongly influenced by the relative percentage of phenanthrene-degrading bacteria over total bacteria in soils. HMW PAHs were not significantly degraded, not only because they were less bioavailable but also because of a lack of degrading microorganisms. Benzo[a]pyrene stood apart since it was partly degraded in acidic soils, probably because of a catabolic cooperation between bacteria and fungi.