Helmut Bürgmann

Fundamentals of microbial community resistance and resilience.

Microbial communities are at the heart of all ecosystems, and yet microbial community behavior in disturbed environments remains difficult to measure and predict. Understanding the drivers of microbial community stability, including resistance (insensitivity to disturbance) and resilience (the rate of recovery after disturbance) is important for predicting community response to disturbance. Here, we provide an overview of the concepts of stability that are relevant for microbial communities. First, we highlight insights from ecology that are useful for defining and measuring stability. To determine whether general disturbance responses exist for microbial communities, we next examine representative studies from the literature that investigated community responses to press (long-term) and pulse (short-term) disturbances in a variety of habitats. Then we discuss the biological features of individual microorganisms, of microbial populations, and of microbial communities that may govern overall community stability. We conclude with thoughts about the unique insights that systems perspectives – informed by meta-omics data – may provide about microbial community stability.

Tackling antibiotic resistance: the environmental framework

Antibiotic resistance is a threat to human and animal health worldwide, and key measures are required to reduce the risks posed by antibiotic resistance genes that occur in the environment. These measures include the identification of critical points of control, the development of reliable surveillance and risk assessment procedures, and the implementation of technological solutions that can prevent environmental contamination with antibiotic resistant bacteria and genes. In this Opinion article, we discuss the main knowledge gaps, the future research needs and the policy and management options that should be prioritized to tackle antibiotic resistance in the environment.

Increased Levels of Multiresistant Bacteria and Resistance Genes after Wastewater Treatment and Their Dissemination into Lake Geneva, Switzerland

At present, very little is known about the fate and persistence of multiresistant bacteria (MRB) and their resistance genes in natural aquatic environments. Treated, but partly also untreated sewage of the city of Lausanne, Switzerland is discharged into Vidy Bay (Lake Geneva) resulting in high levels of contamination in this part of the lake. In the present work we have studied the prevalence of MRB and resistance genes in the wastewater stream of Lausanne. Samples from hospital and municipal raw sewage, treated effluent from Lausanne’s wastewater treatment plant (WTP) as well as lake water and sediment samples obtained close to the WTP outlet pipe and a remote site close to a drinking water pump were evaluated for the prevalence of MRB. Selected isolates were identified (16S rRNA gene fragment sequencing) and characterized with regards to further resistances, resistance genes, and plasmids. Mostly, studies investigating this issue have relied on cultivation-based approaches. However, the limitations of these tools are well known, in particular for environmental microbial communities, and cultivation-independent molecular tools should be applied in parallel in order to take non-culturable organisms into account. Here we directly quantified the sulfonamide resistance genes sul1 and sul2 from environmental DNA extracts using TaqMan real-time quantitative PCR. Hospital sewage contained the highest load of MRB and antibiotic resistance genes (ARGs). Wastewater treatment reduced the total bacterial load up to 78% but evidence for selection of extremely multiresistant strains and accumulation of resistance genes was observed. Our data clearly indicated pollution of sediments with ARGs in the vicinity of the WTP outlet. The potential of lakes as reservoirs of MRB and potential risks are discussed.

mRNA Extraction and Reverse Transcription-PCR Protocol for Detection of nifH Gene Expression by Azotobacter vinelandii in Soil

ABSTRACT The study of free-living nitrogen-fixing organisms in bulk soil is hampered by the great diversity of soil microbial communities and the difficulty of relating nitrogen fixation activities to individual members of the diazotroph populations. We developed a molecular method that allows analysis of nifH mRNA expression in soil in parallel with determinations of nitrogen-fixing activity and bacterial growth. In this study, Azotobacter vinelandii growing in sterile soil and liquid culture served as a model system for nifH expression, in which sucrose served as the carbon source and provided nitrogen-limited conditions, while amendments of NH4NO3 were used to suppress nitrogen fixation. Soil RNA extraction was performed with a new optimized direct extraction protocol that yielded nondegraded total RNA. The RNA extracts were of high purity, free of DNA contamination, and allowed highly sensitive and specific detection of nifH mRNA by a reverse transcription-PCR. The level of nifH gene expression was estimated by PCR amplification of reverse-transcribed nifH mRNA fragments with A. vinelandii-specific nifH primers. This new approach revealed that nifH gene expression was positively correlated with bulk nitrogen fixation activity in soil (r2 = 0.72) and in liquid culture (r2 = 0.84) and therefore is a powerful tool for studying specific regulation of gene expression directly in the soil environment.

Wastewater as a point source of antibiotic-resistance genes in the sediment of a freshwater lake.

Antibiotic-resistance genes (ARGs) are currently discussed as emerging environmental contaminants. Hospital and municipal sewage are important sources of ARGs for the receiving freshwater bodies. We investigated the spatial distribution of different ARGs (sul1, sul2, tet(B), tet(M), tet(W) and qnrA) in freshwater lake sediments in the vicinity of a point source of treated wastewater. ARG contamination of Vidy Bay, Lake Geneva, Switzerland was quantified using real-time PCR and compared with total mercury (THg), a frequently particle-bound inorganic contaminant with known natural background levels. Two-dimensional mapping of the investigated contaminants in lake sediments with geostatistical tools revealed total and relative abundance of ARGs in close proximity of the sewage discharge point were up to 200-fold above levels measured at a remote reference site (center of the lake) and decreased exponentially with distance. Similar trends were observed in the spatial distribution of different ARGs, whereas distributions of ARGs and THg were only moderately correlated, indicating differences in the transport and fate of these pollutants or additional sources of ARG contamination. The spatial pattern of ARG contamination and supporting data suggest that deposition of particle-associated wastewater bacteria rather than co-selection by, for example, heavy metals was the main cause of sediment ARG contamination.

A brief multi-disciplinary review on antimicrobial resistance in medicine and its linkage to the global environmental microbiota

The discovery and introduction of antimicrobial agents to clinical medicine was one of the greatest medical triumphs of the 20th century that revolutionized the treatment of bacterial infections. However, the gradual emergence of populations of antimicrobial-resistant pathogenic bacteria resulting from use, misuse, and abuse of antimicrobials has today become a major global health concern. Antimicrobial resistance (AMR) genes have been suggested to originate from environmental bacteria, as clinically relevant resistance genes have been detected on the chromosome of environmental bacteria. As only a few new antimicrobials have been developed in the last decade, the further evolution of resistance poses a serious threat to public health. Urgent measures are required not only to minimize the use of antimicrobials for prophylactic and therapeutic purposes but also to look for alternative strategies for the control of bacterial infections. This review examines the global picture of antimicrobial resistance, factors that favor its spread, strategies, and limitations for its control and the need for continuous training of all stake-holders i.e., medical, veterinary, public health, and other relevant professionals as well as human consumers, in the appropriate use of antimicrobial drugs.

New Molecular Screening Tools for Analysis of Free-Living Diazotrophs in Soil

ABSTRACT Free-living nitrogen-fixing prokaryotes (diazotrophs) are ubiquitous in soil and are phylogenetically and physiologically highly diverse. Molecular methods based on universal PCR detection of the nifH marker gene have been successfully applied to describe diazotroph populations in the environment. However, the use of highly degenerate primers and low-stringency amplification conditions render these methods prone to amplification bias, while less degenerate primer sets will not amplify all nifH genes. We have developed a fixed-primer-site approach with six PCR protocols using less degenerate to nondegenerate primer sets that all amplify the same nifH fragment as a previously published PCR protocol for universal amplification. These protocols target different groups of diazotrophs and allowed for direct comparison of the PCR products by use of restriction fragment length polymorphism fingerprinting. The new protocols were optimized on DNA from 14 reference strains and were subsequently tested with bulk DNA extracts from six soils. These analyses revealed that the new PCR primer sets amplified nifH sequences that were not detected by the universal primer set. Furthermore, they were better suited to distinguish between diazotroph populations in the different soils. Because the novel primer sets were not specific for monophyletic groups of diazotrophs, they do not serve as an identification tool; however, they proved powerful as fingerprinting tools for subsets of soil diazotroph communities.

Methane sources and sinks in Lake Kivu

Unique worldwide, Lake Kivu stores enormous amounts of CH 4 and CO 2 . A recent study reported that CH 4 concentrations in the lake have increased by up to 15% in the last 30 years and that accumulation at this rate could lead to catastrophic outgassing by ∼2100. This study investigates the present-day CH 4 formation and oxidation in Lake Kivu. Analyses of 14C and 13C in CH 4 and potential carbon sources revealed that below 260 m, an unusually high ∼65% of the CH 4 originates either from reduction of geogenic CO 2 with mostly geogenic H 2 or from direct inflows of geogenic CH 4 . Aerobic CH 4 oxidation, performed by close relatives of type X CH 4 -oxidizing bacteria, is the main process preventing CH 4 from escaping to the atmosphere. Anaerobic CH 4 oxidation, carried out by CH 4 -oxidizing archaea in the SO 4 2--reducing zone, was also detected but is limited by the availability of sulfate. Changes in 14C CH4 and 13C CH4 since the 1970s suggest that the amount of CH 4 produced from degrading organic material has increased due to higher accumulation of organic matter. This, as well as the sudden onset of carbonates in the 1960s, has previously been explained by three environmental changes: (1) introduction of nonnative fish, (2) amplified subaquatic inflows following hydrological changes, and (3) increased external inputs due to the fast growing population. The resulting enhancement of primary production and organic matter sedimentation likely caused CH 4 to increase. However, given the large proportion of old CH 4 carbon, we cannot exclude an increased inflow of geogenic H 2 or CH 4 . Copyright 2011 by the American Geophysical Union.

Simple Absolute Quantification Method Correcting for Quantitative PCR Efficiency Variations for Microbial Community Samples

ABSTRACT Real-time quantitative PCR (qPCR) is a widely used technique in microbial community analysis, allowing the quantification of the number of target genes in a community sample. Currently, the standard-curve (SC) method of absolute quantification is widely employed for these kinds of analysis. However, the SC method assumes that the amplification efficiency (E) is the same for both the standard and the sample target template. We analyzed 19 bacterial strains and nine environmental samples in qPCR assays, targeting the nifH and 16S rRNA genes. The E values of the qPCRs differed significantly, depending on the template. This has major implications for the quantification. If the sample and standard differ in their E values, quantification errors of up to orders of magnitude are possible. To address this problem, we propose and test the one-point calibration (OPC) method for absolute quantification. The OPC method corrects for differences in E and was derived from the ΔΔCT method with correction for E, which is commonly used for relative quantification in gene expression studies. The SC and OPC methods were compared by quantifying artificial template mixtures from Geobacter sulfurreducens (DSM 12127) and Nostoc commune (Culture Collection of Algae and Protozoa [CCAP] 1453/33), which differ in their E values. While the SC method deviated from the expected nifH gene copy number by 3- to 5-fold, the OPC method quantified the template mixtures with high accuracy. Moreover, analyzing environmental samples, we show that even small differences in E between the standard and the sample can cause significant differences between the copy numbers calculated by the SC and the OPC methods.

Does human activity impact the natural antibiotic resistance background? Abundance of antibiotic resistance genes in 21 Swiss lakes.

Antibiotic resistance genes (ARGs) are emerging environmental contaminants, known to be continuously discharged into the aquatic environment via human and animal waste. Freshwater aquatic environments represent potential reservoirs for ARG and potentially allow sewage-derived ARG to persist and spread in the environment. This may create increased opportunities for an eventual contact with, and gene transfer to, human and animal pathogens via the food chain or drinking water. However, assessment of this risk requires a better understanding of the level and variability of the natural resistance background and the extent of the human impact. We have analyzed water samples from 21 Swiss lakes, taken at sampling points that were not under the direct influence of local contamination sources and analyzed the relative abundance of ARG using quantitative real-time PCR. Copy numbers of genes mediating resistance to three different broad-spectrum antibiotic classes (sulfonamides: sul1, sul2, tetracyclines: tet(B), tet(M), tet(W) and fluoroquinolones: qnrA) were normalized to copy numbers of bacterial 16S rRNA genes. We used multiple linear regression to assess if ARG abundance is related to human activities in the catchment, microbial community composition and the eutrophication status of the lakes. Sul genes were detected in all sampled lakes, whereas only four lakes contained quantifiable numbers of tet genes, and qnrA remained below detection in all lakes. Our data indicate higher abundance of sul1 in lakes with increasing number and capacity of wastewater treatment plants (WWTPs) in the catchment. sul2 abundance was rather related to long water residence times and eutrophication status. Our study demonstrates the potential of freshwater lakes to preserve antibiotic resistance genes, and provides a reference for ARG abundance from lake systems with low human impact as a baseline for assessing ARG contamination in lake water.