Josselin Bodilis

Both Cycloclasticus spp. and Pseudomonas spp. as PAH‐degrading bacteria in the Seine estuary (France)

Like other highly urbanized and industrialized estuaries, the Seine estuary (France) has, for decades, received high inputs of polycyclic aromatic hydrocarbons (PAHs). In order to estimate the bioremediation potentials and to identify the bacterial species involved in hydrocarbon degradation, we used microcosms containing seawater from the Seine estuary supplemented with either naphthalene, phenanthrene, fluorene or pyrene. In the microcosms enriched with naphthalene or phenanthrene, hydrocarbon biodegradation was significant within 9 weeks (43% or 46%, respectively), as shown by analyses in GC-MS. In similar microcosms incubated also with naphthalene or phenanthrene, analysis of the 16S rRNA gene sequences (DNA and cDNA) with denaturing gradient gel electrophoresis and clone libraries indicated that the PAH-degrading communities were dominated by Cycloclasticus spp., confirming their universal key role in degradation of low-molecular-weight PAHs in marine environments. However, in contrast to previous studies, we found that Pseudomonas spp. also degraded naphthalene and phenanthrene in seawater; this occurred only after 21 days, as was confirmed by real-time PCR. Although this genus has been abundantly described in the literature as a good PAH-degrading bacterial group in soil or in sediment, to our knowledge, this is the first evidence of a significant fitness in PAH degradation in seawater.

Phylogenetic relationships between environmental and clinical isolates of Pseudomonas fluorescens and related species deduced from 16S rRNA gene and OprF protein sequences.

The major surface protein of the genus Pseudomonas, OprF, is a non-specific porin that plays an important role in maintenance of cell shape, in growth in a low osmolarity environment, and in adhesion to various supports. The objectives of our study were (i) to carry out a comparative analysis of phylogenies obtained from the OprF protein and from the 16S rRNA gene in 41 isolates from various sources (water, soil, milk and the hospital) and (ii) to investigate the physiological characteristics correlated with the phylogeny of OprF. We report here an important incongruence between the phylogenies of the 16S rRNA gene and the OprF protein. Phylogenetic analysis of 16S rRNA genes grouped Pseudomonas fluorescens isolates into one cluster (termed fluorescens r-cluster) whilst the phylogeny of the OprF protein divided Pseudomonas fluorescens isolates into two quite distinct clusters (termed fluorescens 1 o-cluster and fluorescens 2 o-cluster) that may be related to the original habitat of the strain. The fluorescens 1 o-cluster contained the majority of non-rhizospheric soil isolates, while the fluorescens 2 o-cluster contained all our clinical isolates and most of the rhizospheric isolates (which are fixed to the roots). In order to check this correlation, we studied two physiological characteristics: the range of growth temperature and the capacity for non-specific adhesion to polystyrene. The temperature range study for strains did not explain the existence of the two o-clusters but it did confirm the capacity of certain P. fluorescens strains to grow at 37 degrees C. The adhesion capacities of the isolates in the two o-clusters seems to be correlated with ecological niche.

Variable copy number, intra-genomic heterogeneities and lateral transfers of the 16S rRNA gene in Pseudomonas.

Even though the 16S rRNA gene is the most commonly used taxonomic marker in microbial ecology, its poor resolution is still not fully understood at the intra-genus level. In this work, the number of rRNA gene operons, intra-genomic heterogeneities and lateral transfers were investigated at a fine-scale resolution, throughout the Pseudomonas genus. In addition to nineteen sequenced Pseudomonas strains, we determined the 16S rRNA copy number in four other Pseudomonas strains by Southern hybridization and Pulsed-Field Gel Electrophoresis, and studied the intra-genomic heterogeneities by Denaturing Gradient Gel Electrophoresis and sequencing. Although the variable copy number (from four to seven) seems to be correlated with the evolutionary distance, some close strains in the P. fluorescens lineage showed a different number of 16S rRNA genes, whereas all the strains in the P. aeruginosa lineage displayed the same number of genes (four copies). Further study of the intra-genomic heterogeneities revealed that most of the Pseudomonas strains (15 out of 19 strains) had at least two different 16S rRNA alleles. A great difference (5 or 19 nucleotides, essentially grouped near the V1 hypervariable region) was observed only in two sequenced strains. In one of our strains studied (MFY30 strain), we found a difference of 12 nucleotides (grouped in the V3 hypervariable region) between copies of the 16S rRNA gene. Finally, occurrence of partial lateral transfers of the 16S rRNA gene was further investigated in 1803 full-length sequences of Pseudomonas available in the databases. Remarkably, we found that the two most variable regions (the V1 and V3 hypervariable regions) had probably been laterally transferred from another evolutionary distant Pseudomonas strain for at least 48.3 and 41.6% of the 16S rRNA sequences, respectively. In conclusion, we strongly recommend removing these regions of the 16S rRNA gene during the intra-genus diversity studies.

Genome-wide analysis and literature-based survey of lipoproteins in Pseudomonas aeruginosa.

Pseudomonas aeruginosa is a Gram-negative opportunistic pathogen able to cause acute or chronic infections. Like all other Pseudomonas species, P. aeruginosa has a large genome, >6 Mb, encoding more than 5000 proteins. Many proteins are localized in membranes, among them lipoproteins, which can be found tethered to the inner or the outer membrane. Lipoproteins are translocated from the cytoplasm and their N-terminal signal peptide is cleaved by the signal peptidase II, which recognizes a specific sequence called the lipobox just before the first cysteine of the mature lipoprotein. A majority of lipoproteins are transported to the outer membrane via the LolCDEAB system, while those having an avoidance signal remain in the inner membrane. In Escherichia coli, the presence of an aspartate residue after the cysteine is sufficient to cause the lipoprotein to remain in the inner membrane, while in P. aeruginosa the situation is more complex and involves amino acids at position +3 and +4 after the cysteine. Previous studies indicated that there are 185 lipoproteins in P. aeruginosa, with a minority in the inner membrane. A reanalysis led to a reduction of this number to 175, while new retention signals could be predicted, increasing the percentage of inner-membrane lipoproteins to 20 %. About one-third (62 out of 175) of the lipoprotein genes are present in the 17 Pseudomonas genomes sequenced, meaning that these genes are part of the core genome of the genus. Lipoproteins can be classified into families, including those outer-membrane proteins having a structural role or involved in efflux of antibiotics. Comparison of various microarray data indicates that exposure to epithelial cells or some antibiotics, or conversion to mucoidy, has a major influence on the expression of lipoprotein genes in P. aeruginosa.

Comparative Genomics of Environmental and Clinical Stenotrophomonas maltophilia Strains with Different Antibiotic Resistance Profiles

Stenotrophomonas maltophilia, a ubiquitous Gram-negative γ-proteobacterium, has emerged as an important opportunistic pathogen responsible for nosocomial infections. A major characteristic of clinical isolates is their high intrinsic or acquired antibiotic resistance level. The aim of this study was to decipher the genetic determinism of antibiotic resistance among strains from different origins (i.e., natural environment and clinical origin) showing various antibiotic resistance profiles. To this purpose, we selected three strains isolated from soil collected in France or Burkina Faso that showed contrasting antibiotic resistance profiles. After whole-genome sequencing, the phylogenetic relationships of these 3 strains and 11 strains with available genome sequences were determined. Results showed that a strain’s phylogeny did not match their origin or antibiotic resistance profiles. Numerous antibiotic resistance coding genes and efflux pump operons were revealed by the genome analysis, with 57% of the identified genes not previously described. No major variation in the antibiotic resistance gene content was observed between strains irrespective of their origin and antibiotic resistance profiles. Although environmental strains generally carry as many multidrug resistant (MDR) efflux pumps as clinical strains, the absence of resistance–nodulation–division (RND) pumps (i.e., SmeABC) previously described to be specific to S. maltophilia was revealed in two environmental strains (BurA1 and PierC1). Furthermore the genome analysis of the environmental MDR strain BurA1 showed the absence of SmeABC but the presence of another putative MDR RND efflux pump, named EbyCAB on a genomic island probably acquired through horizontal gene transfer.

Abundance and diversity of copper resistance genes cusA and copA in microbial communities in relation to the impact of copper on Chilean marine sediments.

Microorganisms have developed copper-resistance mechanisms in order to survive in contaminated environments. The abundance of the copper-resistance genes cusA and copA, encoding respectively for a Resistance Cell Nodulation protein and for a P-type ATP-ase pump, was assessed in copper and non-copper-impacted Chilean marine sediment cores by the use of molecular tools. We demonstrated that number of copA and cusA genes per bacterial cell was higher in the contaminated sediment, and that copA gene was more abundant than cusA gene in the impacted sediment. The molecular phylogeny of the two copper-resistance genes was studied and reveals an impact of copper on the genetic composition of copA and cusA genes.

Occurrence of multi-antibiotic resistant Pseudomonas spp. in drinking water produced from karstic hydrosystems

Aquatic environments could play a role in the spread of antibiotic resistance genes by enabling antibiotic-resistant bacteria transferred through wastewater inputs to connect with autochthonous bacteria. Consequently, drinking water could be a potential pathway to humans and animals for antibiotic resistance genes. The aim of this study was to investigate occurrences of Escherichia coli and Pseudomonas spp. in drinking water produced from a karst, a vulnerable aquifer with frequent increases in water turbidity after rainfall events and run-offs. Water samples were collected throughout the system from the karstic springs to the drinking water tap during three non-turbid periods and two turbid events. E. coli densities in the springs were 10- to 1000-fold higher during the turbid events than during the non-turbid periods, indicating that, with increased turbidity, surface water had entered the karstic system and contaminated the spring water. However, no E. coli were isolated in the drinking water. In contrast, Pseudomonas spp. were isolated from the drinking water only during turbid events, while the densities in the springs were from 10- to 100-fold higher than in the non-turbid periods. All the 580 Pseudomonas spp. isolates obtained from the sampling periods were resistant (to between 1 and 10 antibiotics), with similar resistance patterns. Among all the Pseudomonas isolated throughout the drinking water production system, between 32% and 86% carried the major resistance pattern: ticarcillin, ticarcillin-clavulanic acid, cefsulodin, and/or aztreonam, and/or sulfamethoxazol-trimethoprim, and/or fosfomycin. Finally, 8 Pseudomonas spp. isolates, related to the Pseudomonas putida and Pseudomonas fluorescens species, were isolated from the drinking water. Thus, Pseudomonas could be involved in the dissemination of antibiotic resistance via drinking water during critical periods.

A Long-Branch Attraction Artifact Reveals an Adaptive Radiation in Pseudomonas

A significant proportion of protein-encoding gene phylogenies in bacteria is inconsistent with the species phylogeny. It was usually argued that such inconsistencies resulted from lateral transfers. Here, by further studying the phylogeny of the oprF gene encoding the major surface protein in the bacterial Pseudomonas genus, we found that the incongruent tree topology observed results from a long-branch attraction (LBA) artifact and not from lateral transfers. LBA in the oprF phylogeny could be explained by the faster evolution in a lineage adapted to the rhizosphere, highlighting an unexpected adaptive radiation. We argue that analysis of such artifacts in other inconsistent bacterial phylogenies could be a valuable tool in molecular ecology to highlight cryptic adaptive radiations in microorganisms.

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-β-cyclodextrin was actually a good extractant to assess PAH accessibility, the polymer of carboxymethyl-β-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.

Identification of a metagenomic gene cluster containing a new class A beta-lactamase and toxin-antitoxin systems.

Several reports mention the presence of antibiotic resistance genes in natural and polluted environments, but many studies are based on their detection via polymerase chain reaction (PCR amplification of known genes and not on an activity screening. We constructed a metagenomic fosmid bank from DNA isolated from a polluted river in Brussels, Belgium, the Zenne. A total of 120,000 clones were pooled and plated directly on solid media containing different antibiotics. Several clones were isolated which could grow in the presence of ampicillin. The DNA from several clones was extracted and subjected to restriction analysis and, based on their restriction pattern, two different clones were found. One of the clones was selected for further study as it showed a higher level of resistance to different β‐lactams antibiotics (ticarcilline and ceftazidime). To find out which gene is responsible for the resistance, an in vitro transposon mutagenesis was performed and clones having lost the resistance phenotype were analyzed via inverse PCR amplification. Several clones had an insert in a gene encoding a new type of β‐lactamase. The amplified fosmid DNA was fully sequenced revealing an insert of 41 kb containing 39 open reading frames (ORFs). Transposon insertions inactivating the resistance to β‐lactams were also found in the ORF upstream of the blaA gene, encoding an aminotransferase, suggesting a polar effect on the transcription of the gene downstream. In addition, other genes were found such as histidine biosynthesis genes, which were found to be scattered on the insert, a relA/spoT gene, and genes belonging to type II toxin–antitoxin system. This predicted system was experimentally validated in Escherichia coli using an inducible expression system.