Jean-Michel Monier

Large-Scale Metagenomic-Based Study of Antibiotic Resistance in the Environment

Antibiotic resistance, including multiresistance acquisition and dissemination by pathogens, is a critical healthcare issue threatening our management of infectious diseases [1-3]. Rapid accumulation of resistance phenotypes implies a reservoir of transferable antibiotic resistance gene determinants (ARGDs) selected in response to inhibition of antibiotic concentrations, as found in hospitals [1, 3-5]. Antibiotic resistance genes were found in environmental isolates, soil DNA [4-6], secluded caves [6, 7], and permafrost DNA [7, 8]. Antibiotics target essential and ubiquitous cell functions, and resistance is a common characteristic of environmental bacteria [8-11]. Environmental ARGDs are an abundant reservoir of potentially transferable resistance for pathogens [9-12]. Using metagenomic sequences, we show that ARGDs can be detected in all (n=71) environments analyzed. Soil metagenomes had the most diverse pool of ARGDs. The most common types of resistances found in environmental metagenomes were efflux pumps and genes conferring resistance to vancomycin, tetracycline, or β-lactam antibiotics used in veterinary and human healthcare. Our study describes the diverse and abundant antibiotic resistance genes in nonclinical environments and shows that these genes are not randomly distributed among different environments (e.g., soil, oceans or human feces).

Is resistance futile? Changing external resistance does not improve microbial fuel cell performance.

Microbial fuel cells (MFCs) show promise as an alternative to conventional batteries for point source electricity generation. A better understanding of the relationship between the microbiological and electrical aspects of fuels cells is needed prior to successful MFC application. Here, we observed the effects of external resistance on power production and the anodic biofilm community structure. Large differences in the external resistance affected both power production and microbial community structure. After the establishment of the anodic microbial community, change in external resistance (from low to high and vice versa) changed the anodic microbial community structure, but the resulting community did not resemble the communities established at that same external resistance. Different microbial community structures, established under different external resistances, resulted in similar power production, demonstrating the flexibility of the MFC system.

Metagenomic mining for microbiologists

Microbial ecologists can now start digging into the accumulating mountains of metagenomic data to uncover the occurrence of functional genes and their correlations to microbial community members. Limitations and biases in DNA extraction and sequencing technologies impact sequence distributions, and therefore, have to be considered. However, when comparing metagenomes from widely differing environments, these fluctuations have a relatively minor role in microbial community discrimination. As a consequence, any functional gene or species distribution pattern can be compared among metagenomes originating from various environments and projects. In particular, global comparisons would help to define ecosystem specificities, such as involvement and response to climate change (for example, carbon and nitrogen cycle), human health risks (eg, presence of pathogen species, toxin genes and viruses) and biodegradation capacities. Although not all scientists have easy access to high-throughput sequencing technologies, they do have access to the sequences that have been deposited in databases, and therefore, can begin to intensively mine these metagenomic data to generate hypotheses that can be validated experimentally. Information about metabolic functions and microbial species compositions can already be compared among metagenomes from different ecosystems. These comparisons add to our understanding about microbial adaptation and the role of specific microbes in different ecosystems. Concurrent with the rapid growth of sequencing technologies, we have entered a new age of microbial ecology, which will enable researchers to experimentally confirm putative relationships between microbial functions and community structures.

Metagenomic exploration of antibiotic resistance in soil

The ongoing development of metagenomic approaches is providing the means to explore antibiotic resistance in nature and address questions that could not be answered previously with conventional culture-based strategies. The number of available environmental metagenomic sequence datasets is rapidly expanding and henceforth offer the ability to gain a more comprehensive understanding of antibiotic resistance at the global scale. Although there is now evidence that the environment constitutes a vast reservoir of antibiotic resistance gene determinants (ARGDs) and that the majority of ARGDs acquired by human pathogens may have an environmental origin, a better understanding of their diversity, prevalence and ecological significance may help predict the emergence and spreading of newly acquired resistances. Recent applications of metagenomic approaches to the study of ARGDs in natural environments such as soil should help overcome challenges concerning expanding antibiotic resistances.

Visual Evidence of Horizontal Gene Transfer between Plants and Bacteria in the Phytosphere of Transplastomic Tobacco

ABSTRACT Plant surfaces, colonized by numerous and diverse bacterial species, are often considered hot spots for horizontal gene transfer (HGT) between plants and bacteria. Plant DNA released during the degradation of plant tissues can persist and remain biologically active for significant periods of time, suggesting that soil or plant-associated bacteria could be in direct contact with plant DNA. In addition, nutrients released during the decaying process may provide a copiotrophic environment conducive for opportunistic microbial growth. Using Acinetobacter baylyi strain BD413 and transplastomic tobacco plants harboring the aadA gene as models, the objective of this study was to determine whether specific niches could be shown to foster bacterial growth on intact or decaying plant tissues, to develop a competence state, and to possibly acquire exogenous plant DNA by natural transformation. Visualization of HGT in situ was performed using A. baylyi strain BD413(rbcL-ΔPaadA::gfp) carrying a promoterless aadA::gfp fusion. Both antibiotic resistance and green fluorescence phenotypes were restored in recombinant bacterial cells after homologous recombination with transgenic plant DNA. Opportunistic growth occurred on decaying plant tissues, and a significant proportion of the bacteria developed a competence state. Quantification of transformants clearly supported the idea that the phytosphere constitutes a hot spot for HGT between plants and bacteria. The nondisruptive approach used to visualize transformants in situ provides new insights into environmental factors influencing HGT for plant tissues.

Strategy for In Situ Detection of Natural Transformation-Based Horizontal Gene Transfer Events

ABSTRACT A strategy is described that enables the in situ detection of natural transformation in Acinetobacter baylyi BD413 by the expression of a green fluorescent protein. Microscale detection of bacterial transformants growing on plant tissues was shown by fluorescence microscopy and indicated that cultivation-based selection of transformants on antibiotic-containing agar plates underestimates transformation frequencies.

Microbial fuel cell anodic microbial population dynamics during MFC start-up

In order to optimize energy production in MFCs, a better understanding of anodic communities is essential. Our objective was to determine the taxonomic structure of the bacterial communities present at the surface of the anode during the formation and development of electro-active biofilms in MFCs inoculated with fresh primary clarifier overflow. Quantitative microbial community dynamics were evaluated as a function of time and electrical performance using 16S rRNA gene-based phylogenetic microarrays and flow cytometry. Results show that the bacterial community stabilized partially but not completely when voltage output was stable. Geobacter appeared to be the predominant genus, whose growth was associated with voltage, while some other genus still developed or declined after the voltage stabilization. Flow cytometry revealed that some genus showing a decreasing proportional fluorescence intensity over time were still actively respiring bacteria, and thus, active albeit minor members of the biofilm. Finally, this study shows that anodic biofilm selection and maturation is still occurring after more than 20 days of operation and over ten days after voltage is stabilized.

Integrity and Biological Activity of DNA after UV Exposure

The field of astrobiology lacks a universal marker with which to indicate the presence of life. This study supports the proposal to use nucleic acids, specifically DNA, as a signature of life (biosignature). In addition to its specificity to living organisms, DNA is a functional molecule that can confer new activities and characteristics to other organisms, following the molecular biology dogma, that is, DNA is transcribed to RNA, which is translated into proteins. Previous criticisms of the use of DNA as a biosignature have asserted that DNA molecules would be destroyed by UV radiation in space. To address this concern, DNA in plasmid form was deposited onto different surfaces and exposed to UVC radiation. The surviving DNA was quantified via the quantitative polymerase chain reaction (qPCR). Results demonstrate increased survivability of DNA attached to surfaces versus non-adsorbed DNA. The DNA was also tested for biological activity via transformation into the bacterium Acinetobacter sp. and assaying for antibiotic resistance conferred by genes encoded by the plasmid. The success of these methods to detect DNA and its gene products after UV exposure (254 nm, 3.5 J/m(2)s) not only supports the use of the DNA molecule as a biosignature on mineral surfaces but also demonstrates that the DNA retained biological activity.

Comparative phylogenetic microarray analysis of microbial communities in TCE-contaminated soils

The arrival of chemicals in a soil or groundwater ecosystem could upset the natural balance of the microbial community. Since soil microorganisms are the first to be exposed to the chemicals released into the soil environment, we evaluated the use of a phylogenetic microarray as a bio-indicator of community perturbations due to the exposure to trichloroethylene (TCE). The phylogenetic microarray, which measures the presence of different members of the soil community, was used to evaluate unpolluted soils exposed to TCE as well as to samples from historically TCE polluted sites. We were able to determine an apparent threshold at which the microbial community structure was significantly affected (about 1ppm). In addition, the members of the microbial community most affected were identified. This approach could be useful for assessing environmental impact of chemicals on the biosphere as well as important members of the microbial community involved in TCE degradation.

Long-term persistence and bacterial transformation potential of transplastomic plant DNA in soil

The long-term physical persistence and biological activity of transplastomic plant DNA (transgenes contained in the chloroplast genome) either purified and added to soil or naturally released by decaying tobacco leaves in soil was determined. Soil microcosms were amended with transplastomic tobacco leaves or purified plant DNA and incubated for up to 4 years. Total DNA was extracted from soil and the number of transgenes (aadA, which confers resistance to both spectinomycin and streptomycin) was quantified by quantitative PCR. The biological activity of these transgenes was assessed by transformation in the bacterial strain Acinetobacter sp. BD413(pBAB2) in vitro. While the proportion of transgenes recovered increased with the increasing amount of transplastomic DNA added, plant DNA was rapidly degraded over time. The number of transgenes recovered decreased about 10,000 fold within 2 weeks. Data reveal, however, that a small fraction of the plant DNA escaped degradation. Transgene sequences were still detected after 4 years and transformation assays showed that extracted DNA remained biologically active and could still transform competent cells of Acinetobacter sp. BD413(pBAB2). The approach presented here quantified the number of transgenes (based on quantitative PCR of 50% of the gene) released and persisting in the environment over time and provided new insights into the fate of transgenic plant DNA in soil.