John Poté

Antibiotic-resistant soil bacteria in transgenic plant fields

Understanding the prevalence and polymorphism of antibiotic resistance genes in soil bacteria and their potential to be transferred horizontally is required to evaluate the likelihood and ecological (and possibly clinical) consequences of the transfer of these genes from transgenic plants to soil bacteria. In this study, we combined culture-dependent and -independent approaches to study the prevalence and diversity of bla genes in soil bacteria and the potential impact that a 10-successive-year culture of the transgenic Bt176 corn, which has a blaTEM marker gene, could have had on the soil bacterial community. The bla gene encoding resistance to ampicillin belongs to the beta-lactam antibiotic family, which is widely used in medicine but is readily compromised by bacterial antibiotic resistance. Our results indicate that soil bacteria are naturally resistant to a broad spectrum of beta-lactam antibiotics, including the third cephalosporin generation, which has a slightly stronger discriminating effect on soil isolates than other cephalosporins. These high resistance levels for a wide range of antibiotics are partly due to the polymorphism of bla genes, which occur frequently among soil bacteria. The blaTEM116 gene of the transgenic corn Bt176 investigated here is among those frequently found, thus reducing any risk of introducing a new bacterial resistance trait from the transgenic material. In addition, no significant differences were observed in bacterial antibiotic-resistance levels between transgenic and nontransgenic corn fields, although the bacterial populations were different.

Composition of bacterial and archaeal communities in freshwater sediments with different contamination levels (Lake Geneva, Switzerland)

The aim of this study was to compare the composition of bacterial and archaeal communities in contaminated sediments (Vidy Bay) with uncontaminated sediments (Ouchy area) of Lake Geneva using 16S rRNA clone libraries. Sediments of both sites were analysed for physicochemical characteristics including porewater composition, organic carbon, and heavy metals. Results show high concentrations of contaminants in sediments from Vidy. Particularly, high contents of fresh organic matter and nutrients led to intense mineralisation, which was dominated by sulphate-reduction and methanogenesis. The bacterial diversity in Vidy sediments was significantly different from the communities in the uncontaminated sediments. Phylogenetic analysis revealed a large proportion of Betaproteobacteria clones in Vidy sediments related to Dechloromonas sp., a group of dechlorinating and contaminant degrading bacteria. Deltaproteobacteria, including clones related to sulphate-reducing bacteria and Fe(III)-reducing bacteria (Geobacter sp.) were also more abundant in the contaminated sediments. The archaeal communities consisted essentially of methanogenic Euryarchaeota, mainly found in the contaminated sediments rich in organic matter. Multiple factor analysis revealed that the microbial community composition and the environmental variables were correlated at the two sites, which suggests that in addition to environmental parameters, pollution may be one of the factors affecting microbial community structure.

Effects of a sewage treatment plant outlet pipe extension on the distribution of contaminants in the sediments of the Bay of Vidy, Lake Geneva, Switzerland.

In 2001, the municipality of Lausanne extended the outlet pipe of the sewage treatment plant into the Bay of Vidy (Lake Geneva, Switzerland) as a measure to reduce bacterial water pollution and sediment contamination close to the lake beaches. The aim of the present study was to assess the impact of this measure. Lake bottom sediments were collected and analyzed for grain size, organic matter, organic carbon, nitrogen, phosphorus, heavy metals and hydrophobic organic compounds to evaluate their concentration and spatial distribution. Our results demonstrate that, compared to results obtained before the outlet pipe prolongation, the surface area of highly contaminated sediments was reduced by one third after the pipe extension. However, contaminant concentrations are still high and the accumulation of pollutants may represent a significant source of toxicity for benthic organisms. One concludes that contaminant reduction at the source will be necessary for a further improvement.

Local to regional scale industrial heavy metal pollution recorded in sediments of large freshwater lakes in central Europe (lakes Geneva and Lucerne) over the last centuries.

This research first focuses on the spatial and temporal patterns of heavy metals from contrasting environments (highly polluted to deepwater sites) of Lake Geneva. The mercury (Hg) and lead (Pb) records from two deepwater sites show that the heavy metal variations before the industrial period are primarily linked to natural weathering input of trace elements. By opposition, the discharge of industrial treated wastewaters into Vidy Bay of Lake Geneva during the second part of the 20th century, involved the sedimentation of highly metal-contaminated sediments in the area surrounding the WWTP outlet pipe discharge. Eventually, a new Pb isotope record of sediments from Lake Lucerne identifies the long-term increasing anthropogenic lead pollution after ca. 1500, probably due to the development of metallurgical activities during the High Middle Ages. These data furthermore allows to compare the recent anthropogenic sources of water pollution from three of the largest freshwater lakes of Western Europe (lakes Geneva, Lucerne, and Constance). High increases in Pb and Hg highlight the regional impact of industrial pollution after ca. 1750-1850, and the decrease of metal pollution in the 1980s due to the effects of remediation strategies such as the implementation of wastewater treatment plants (WWTPs). However, at all the studied sites, the recent metal concentrations remain higher than pre-industrial levels. Moreover, the local scale pollution data reveal two highly contaminated sites (>100 μg Pb/g dry weight sediment) by industrial activities, during the late-19th and early-20th centuries (Lake Lucerne) and during the second part of the 20th century (Vidy Bay of Lake Geneva). Overall, the regional scale pollution history inferred from the three large and deep perialpine lakes points out at the pollution of water systems by heavy metals during the last two centuries due to the discharge of industrial effluents.

Degradation and Transformability of DNA from Transgenic Leaves

ABSTRACT The fate of transplastomic (chloroplast genome contains the transgene) tobacco plant DNA in planta was studied when the plant leaves were subjected to decay conditions simulating those encountered naturally, including grinding, incubation with cellulase or enzymes produced by Erwinia chrysanthemi, and attack by the plant pathogen Ralstonia solanacearum. Direct visualization of DNA on agarose gels, gene extraction yield (the number of amplifiable aadA sequences in extracted plant DNA), and the frequency that recipient bacteria can be transformed by plant DNA were used to evaluate the quality and quantity of plant DNA and the transgene. These measurements were used to monitor the physical and biological degradation of DNA inside decaying plant tissues. Our results indicate that while most of the DNA will be degraded inside plant cells, sufficient DNA persists to be released into the soil.

Fate and transport of antibiotic resistance genes in saturated soil columns

Interest in the fate of microorganisms (genetically modified or not) added to soil for a range of applications (e.g., biological fertilizer, bioremediation) has led to the examination of the transport and dispersion of bacteria in soil and ground water environments. The fate of added microorganisms can also be related to the fate of their DNA or any DNA found within soil or groundwater. However, studies have not examined the movement and fate of plasmids themselves in water-saturated soils. Continuous-flow water-saturated soil columns were used to examine the environmental fate and movement of antibiotic resistance genes. The genes were located on a plasmid pLEP01, which was either introduced into the soil columns directly as a mixture of supercoiled and open circular forms or after linearization by restriction enzymes. Both quantitative and qualitative measurements were done throughout the study. Quantitative measurements included the total DNA and conservative tracer concentration in the column effluent and the transformation frequencies for E. coli DH 10B and Acinetobactersp. BD 413 in the presence of column effluent. Qualitative measurements included relative degradation of the introduced DNA by gel electrophoresis and the potential of effluent DNA to transform bacteria leading to the production of antibiotic resistant bacteria. The extent of DNA degradation was found to be proportional to its residence time in the soil column. In addition, tests on the biological activity (bacterial transformation) of the DNA demonstrated that this activity was roughly proportional to the remaining DNA after its degradation. Results suggest a potential for biologically active DNA to be transported over considerable distances in watersaturated soil and ground water.

Persistence and growth of faecal culturable bacterial indicators in water column and sediments of Vidy Bay, Lake Geneva, Switzerland

The aims of this study was to investigate the persistence and the growth of culturable bacterial indicators (CBI) including total coliforms (TC) and faecal coliforms represented by Escherichia coli, enterococcus (ENT), and aerobic mesophilic bacteria (AMB) in the surface sediments and the water column of Vidy Bay (Lake Geneva, City of Lausanne, Switzerland). The study was carried out for 60 d using microcosms containing Sewage Treatment Plant (STP) effluent and nonsterile water without CBI, as well as contaminated and non-contaminated sediments. The effects of water temperature and of organic matter associated with sediments on the survival of CBI in the sediments and the water column were observed. The number of CBI colonies in the contaminated sediments of Vidy Bay and in the STP effluent was almost identical in the order of 10(5)-10(7), 10(4)-10(6), 10(3)-10(5), and 10(4)-10(7) CFU/100 g sediment or/100 mL water for TC, E. coli, ENT, and AMB respectively. A degradation of CBI was observed in the sediments where organic matter content was low and in the water column at a temperature of 10 degrees C after 5 d of experimentation. In addition, a growth of CBI was observed in the sediment which is rich in organic matter at 20 degrees C. The results of this study indicate: (1) the higher concentrations of the CBI observed in different points in the water column of Vidy Bay may not be explained only by the recent contribution of the three potential sources of the Bay contamination including STP and the Chamberonne and Flon Rivers, but also by the persistence, removal from sediment and multiplication of CBI in the sediment and water column; (2) the sediment of Vidy Bay constitute a reservoir of CBI and can even support their growth; and (3) the CBI not only survive in sediments, but also can be remobilized and increased in the water column, therefore, it become a permanent microbiological pollution in Vidy Bay.

Accumulation of Clinically Relevant Antibiotic-Resistance Genes, Bacterial Load, and Metals in Freshwater Lake Sediments in Central Europe

Wastewater treatment plants (WWTP) receive the effluents from various sources (communities, industrial, and hospital effluents) and are recognized as reservoir for antibiotic-resistance genes (ARGs) that are associated with clinical pathogens. The aquatic environment is considered a hot-spot for horizontal gene transfer, and lake sediments offer the opportunity for reconstructing the pollution history and evaluating the impacts. In this context, variation with depth and time of the total bacterial load, the abundance of faecal indicator bacteria (FIB; E. coli and Enterococcus spp. (ENT)), Pseudomonas spp., and ARGs (blaTEM, blaSHV, blaCTX-M, blaNDM, and aadA) were quantified in sediment profiles of different parts of Lake Geneva using quantitative PCR. The abundance of bacterial marker genes was identified in sediments contaminated by WWTP following eutrophication of the lake. Additionally, ARGs, including the extended-spectrum ß-lactam- and aminoglycoside-resistance genes, were identified in the surface sediments. The ARG and FIB abundance strongly correlated (r ≥ 0.403, p < 0.05, n = 34) with organic matter and metal concentrations in the sediments, indicating a common and contemporary source of contamination. The contamination of sediments by untreated or partially treated effluent water can affect the quality of ecosystem. Therefore, the reduction of contaminants from the source is recommended for further improvement of water quality.

Antibiotic resistant bacteria/genes dissemination in lacustrine sediments highly increased following cultural eutrophication of Lake Geneva (Switzerland).

This study investigates faecal indicator bacteria (FIB), multiple antibiotic resistant (MAR), and antibiotic resistance genes (ARGs), of sediment profiles from different parts of Lake Geneva (Switzerland) over the last decades. MARs consist to expose culturable Escherichia coli (EC) and Enterococcus (ENT) to mixed five antibiotics including Ampicillin, Tetracycline, Amoxicillin, Chloramphenicol and Erythromycin. Culture-independent is performed to assess the distribution of ARGs responsible for, β-lactams (blaTEM; Amoxicillin/Ampicillin), Streptomycin/Spectinomycin (aadA), Tetracycline (tet) Chloramphenicol (cmlA) and Vancomycin (van). Bacterial cultures reveal that in the sediments deposited following eutrophication of Lake Geneva in the 1970s, the percentage of MARs to five antibiotics varied from 0.12% to 4.6% and 0.016% to 11.6% of total culturable EC and ENT, respectively. In these organic-rich bacteria-contaminated sediments, the blaTEM resistant of FIB varied from 22% to 48% and 16% to 37% for EC and ENT respectively, whereas the positive PCR assays responsible for tested ARGs were observed for EC, ENT, and total DNA from all samples. The aadA resistance gene was amplified for all the sediment samples, including those not influenced by WWTP effluent water. Our results demonstrate that bacteria MARs and ARGs highly increased in the sediments contaminated with WWTP effluent following the cultural eutrophication of Lake Geneva. Hence, the human-induced changing limnological conditions highly enhanced the sediment microbial activity, and therein the spreading of antibiotic resistant bacteria and genes in this aquatic environment used to supply drinking water in a highly populated area. Furthermore, the presence of the antibiotic resistance gene aadA in all the studied samples points out a regional dissemination of this emerging contaminant in freshwater sediments since at least the late nineteenth century.

A high-resolution historical sediment record of nutrients, trace elements and organochlorines (DDT and PCB) deposition in a drinking water reservoir (Lake Brêt, Switzerland) points at local and regional pollutant sources

The (137)Cs and (210)Pb dating of a 61-cm long sediment core retrieved from a drinking water reservoir (Lake Brêt) located in Switzerland revealed a linear and relatively high sedimentation rate (~1 cm year(-1)) over the last decades. The continuous centimeter scale measurement of physical (porewater and granulometry), organic (C(org), P, N, HI and OI indexes) and mineral (C(min) and lithogenic trace elements) parameters therefore enables reconstructing the environmental history of the lake and anthropogenic pollutant input (trace metals, DDT and PCBs) at high resolution. A major change in the physical properties of the lowermost sediments occurred following the artificial rise of the dam in 1922. After ca. 1940, there was a long-term up-core increase in organic matter deposition attributed to enhance primary production and anoxic bottom water conditions due to excessive nutrient input from a watershed predominantly used for agriculture that also received domestic effluents of two wastewater-treatment plants. This pattern contrasts with the terrigenous element input (Eu, Sc, Mg, Ti, Al, and Fe) which doubled after the rising of the dam but continuously decreased during the last 60 years. By comparison, the trace metals (Cu, Pb and Hg) presented a slight enrichment factor (EF) only during the second part of the 20th century. Although maximum EF Pb (>2) occurred synchronously with the use of leaded gasoline in Switzerland (between ca. 1947 and 1985) the Hg and Cu profiles exhibited a relatively similar trend than Pb during the 20th century, therefore excluding the alkyl-lead added to petrol as the dominant (atmospheric) source of lead input to Lake Brêt. Conversely, the Cu profile that did not follow the decrease registered in Pb and Hg during the last 10 years, suggests an additional source of Cu probably linked to the impact of agricultural activities in the area. In absence of heavy industries in the catchment, the atmospheric deposition of DDT and PCBs via surface runoff followed the historical emissions of POPs in Switzerland. Such result highlights the regional contamination of freshwater resources by the large-scale emission of toxic industrial chemicals in the 1960s and 1970s as well as the efficiency of the regulatory measures subsequently taken.