Publications by authors named "Stephan Fuchs"

84 Publications

smORFer: a modular algorithm to detect small ORFs in prokaryotes.

Nucleic Acids Res 2021 Jun 14. Epub 2021 Jun 14.

Inst. Biochemistry and Molecular Biology, Department of Chemistry, University of Hamburg, 20146 Hamburg, Germany.

Emerging evidence places small proteins (≤50 amino acids) more centrally in physiological processes. Yet, their functional identification and the systematic genome annotation of their cognate small open-reading frames (smORFs) remains challenging both experimentally and computationally. Ribosome profiling or Ribo-Seq (that is a deep sequencing of ribosome-protected fragments) enables detecting of actively translated open-reading frames (ORFs) and empirical annotation of coding sequences (CDSs) using the in-register translation pattern that is characteristic for genuinely translating ribosomes. Multiple identifiers of ORFs that use the 3-nt periodicity in Ribo-Seq data sets have been successful in eukaryotic smORF annotation. They have difficulties evaluating prokaryotic genomes due to the unique architecture (e.g. polycistronic messages, overlapping ORFs, leaderless translation, non-canonical initiation etc.). Here, we present a new algorithm, smORFer, which performs with high accuracy in prokaryotic organisms in detecting putative smORFs. The unique feature of smORFer is that it uses an integrated approach and considers structural features of the genetic sequence along with in-frame translation and uses Fourier transform to convert these parameters into a measurable score to faithfully select smORFs. The algorithm is executed in a modular way, and dependent on the data available for a particular organism, different modules can be selected for smORF search.
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http://dx.doi.org/10.1093/nar/gkab477DOI Listing
June 2021

Towards the characterization of the hidden world of small proteins in Staphylococcus aureus, a proteogenomics approach.

PLoS Genet 2021 Jun 1;17(6):e1009585. Epub 2021 Jun 1.

University of Technical Sciences Braunschweig, Institute for Microbiology, Braunschweig, Germany.

Small proteins play essential roles in bacterial physiology and virulence, however, automated algorithms for genome annotation are often not yet able to accurately predict the corresponding genes. The accuracy and reliability of genome annotations, particularly for small open reading frames (sORFs), can be significantly improved by integrating protein evidence from experimental approaches. Here we present a highly optimized and flexible bioinformatics workflow for bacterial proteogenomics covering all steps from (i) generation of protein databases, (ii) database searches and (iii) peptide-to-genome mapping to (iv) visualization of results. We used the workflow to identify high quality peptide spectrum matches (PSMs) for small proteins (≤ 100 aa, SP100) in Staphylococcus aureus Newman. Protein extracts from S. aureus were subjected to different experimental workflows for protein digestion and prefractionation and measured with highly sensitive mass spectrometers. In total, 175 proteins with up to 100 aa (SP100) were identified. Out of these 24 (ranging from 9 to 99 aa) were novel and not contained in the used genome annotation.144 SP100 are highly conserved and were found in at least 50% of the publicly available S. aureus genomes, while 127 are additionally conserved in other staphylococci. Almost half of the identified SP100 were basic, suggesting a role in binding to more acidic molecules such as nucleic acids or phospholipids.
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http://dx.doi.org/10.1371/journal.pgen.1009585DOI Listing
June 2021

RegAB Homolog of Burkholderia pseudomallei is the Master Regulator of Redox Control and involved in Virulence.

PLoS Pathog 2021 May 28;17(5):e1009604. Epub 2021 May 28.

Friedrich Loeffler Institute of Medical Microbiology, University Medicine Greifswald, Greifswald, Germany.

Burkholderia pseudomallei, the etiological agent of melioidosis in humans and animals, often occupies environmental niches and infection sites characterized by limited concentrations of oxygen. Versatile genomic features enable this pathogen to maintain its physiology and virulence under hypoxia, but the crucial regulatory networks employed to switch from oxygen dependent respiration to alternative terminal electron acceptors (TEA) like nitrate, remains poorly understood. Here, we combined a Tn5 transposon mutagenesis screen and an anaerobic growth screen to identify a two-component signal transduction system with homology to RegAB. We show that RegAB is not only essential for anaerobic growth, but also for full virulence in cell lines and a mouse infection model. Further investigations of the RegAB regulon, using a global transcriptomic approach, identified 20 additional regulators under transcriptional control of RegAB, indicating a superordinate role of RegAB in the B. pseudomallei anaerobiosis regulatory network. Of the 20 identified regulators, NarX/L and a FNR homolog were selected for further analyses and a role in adaptation to anaerobic conditions was demonstrated. Growth experiments identified nitrate and intermediates of the denitrification process as the likely signal activateing RegAB, NarX/L, and probably of the downstream regulators Dnr or NsrR homologs. While deletions of individual genes involved in the denitrification process demonstrated their important role in anaerobic fitness, they showed no effect on virulence. This further highlights the central role of RegAB as the master regulator of anaerobic metabolism in B. pseudomallei and that the complete RegAB-mediated response is required to achieve full virulence. In summary, our analysis of the RegAB-dependent modulon and its interconnected regulons revealed a key role for RegAB of B. pseudomallei in the coordination of the response to hypoxic conditions and virulence, in the environment and the host.
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http://dx.doi.org/10.1371/journal.ppat.1009604DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8191878PMC
May 2021

Corrigendum: Genome-Wide Association Studies for the Detection of Genetic Variants Associated With Daptomycin and Ceftaroline Resistance in .

Front Microbiol 2021 27;12:686197. Epub 2021 Apr 27.

Department of Infectious Diseases, Robert Koch-Institute, Wernigerode, Germany.

[This corrects the article DOI: 10.3389/fmicb.2021.639660.].
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http://dx.doi.org/10.3389/fmicb.2021.686197DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8111692PMC
April 2021

Adaptation of Dinoroseobacter shibae to oxidative stress and the specific role of RirA.

PLoS One 2021 29;16(3):e0248865. Epub 2021 Mar 29.

Institute for Microbiology, Technische Universität Braunschweig, Braunschweig, Germany.

Dinoroseobacter shibae living in the photic zone of marine ecosystems is frequently exposed to oxygen that forms highly reactive species. Here, we analysed the adaptation of D. shibae to different kinds of oxidative stress using a GeLC-MS/MS approach. D. shibae was grown in artificial seawater medium in the dark with succinate as sole carbon source and exposed to hydrogen peroxide, paraquat or diamide. We quantified 2580 D. shibae proteins. 75 proteins changed significantly in response to peroxide stress, while 220 and 207 proteins were differently regulated by superoxide stress and thiol stress. As expected, proteins like thioredoxin and peroxiredoxin were among these proteins. In addition, proteins involved in bacteriochlophyll biosynthesis were repressed under disulfide and superoxide stress but not under peroxide stress. In contrast, proteins associated with iron transport accumulated in response to peroxide and superoxide stress. Interestingly, the iron-responsive regulator RirA in D. shibae was downregulated by all stressors. A rirA deletion mutant showed an improved adaptation to peroxide stress suggesting that RirA dependent proteins are associated with oxidative stress resistance. Altogether, 139 proteins were upregulated in the mutant strain. Among them are proteins associated with protection and repair of DNA and proteins (e. g. ClpB, Hsp20, RecA, and a thioredoxin like protein). Strikingly, most of the proteins involved in iron metabolism such as iron binding proteins and transporters were not part of the upregulated proteins. In fact, rirA deficient cells were lacking a peroxide dependent induction of these proteins that may also contribute to a higher cell viability under these conditions.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0248865PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8007024PMC
March 2021

Implementation of comparative detection approaches for the accurate assessment of sediment thickness and sediment volume in the Passaúna Reservoir.

J Environ Manage 2021 Jun 14;287:112298. Epub 2021 Mar 14.

Department of Aquatic Environmental Engineering, Institute for Water and River Basin Management, Karlsruhe Institute of Technology, Karlsruhe, Germany.

Siltation has significant economic and social impacts as it directly reduces the useable amount of water in reservoirs. Giving a solution to the issue of sedimentation is a complicated task and maybe one of the most important engineering and environmental challenges of the 21 century. The deposited volume and the distribution pattern of the sediment are often unknown and not easy to assess. The sedimentation process is highly dynamic, initially due to the hydrological conditions of the incoming rivers, but also due to common internal phenomena like resuspension or density currents. Sediment remediation measures such as mechanical sediment removal or flushing are planned based on the sediment thickness distribution and the overall sediment volume/mass. Often, the sediment thickness is calculated through topographic differencing between the pre-impoundment reservoir lake bottom and the actual lake bottom. However, it is common that the previous depth distribution map is not available or in insufficient quality. In this regard, alternative measurement techniques have to be taken into consideration. In this study, we assessed the best possible approach depending on the characteristics of the sediment and of the reservoir. We combined three different acoustic systems (a multibeam, a sub-bottom profiler, and a single beam dual frequency system) with sediment coring and dynamic free fall penetrometer measurements for an improved assessment of sediment stock and sediment distribution in the Passaúna Reservoir. Our results showed that topographic differencing could not be applied, as the data for the pre-impoundment lake bottom was insufficiently accurate. The parametric sub-bottom profiler could detect the sediment thickness in high accuracy, but significant limitations were recorded in areas with high gas contents. The dual-frequency echosounder derived the sediment thickness with a normalized mean absolute error of 56% due to the high volumetric gas content in the sediment. The dynamic free-fall penetrometer showed satisfying results compared to the other systems. The normalized mean absolute error was 22%, and sediment thickness could be detected in areas with up to 1.8 m of sediment. Sediment coring is also a reliable technique for sediment thickness determination. However, the results showed that if only traditional coring devices are used (gravity corer), the limited penetration depth of the equipment combined with sampling disturbances often prevent a correct assessment of the sediment thickness. The overall results of this study can help for an improved decision-making regarding reservoir management. The accurate assessment of sediment volume and distribution can reduce costs for sediment removal and assist in having a precise overview of the reservoir lifetime.
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http://dx.doi.org/10.1016/j.jenvman.2021.112298DOI Listing
June 2021

Effective treatment of the wastewater from ceramic industry using ceramic membranes.

Water Sci Technol 2021 Mar;83(5):1055-1071

Karlsruhe Institute of Technology: Karlsruher Institut fur Technologie, Karlsruhe, Germany E-mail:

Emissions of organic compounds, heavy metals and chemicals used in the ceramic industry cause significant organic and inorganic pollution of water. The effluent must be treated before it is discharged into a water body. International and EU laws control the chemical oxygen demand (COD) of the wastewater. Conventional technologies, such as sedimentation, flocculation and biological treatment, have lots of drawbacks, whereas membrane technologies give many benefits, as they are chemical-free and allow a reduction of the treatment steps. One-step wastewater nanofiltration with ceramic membranes of 450 Da cut-off is able to reduce the COD of ceramic wastewater to a sufficient level. However, the working time without cleaning is limited and the rejection of membranes can be significantly reduced due to fouling. Multistage filtration can be the solution. Filtration experiments with various combinations (MF, UF and NF) of ceramic membranes were performed at a laboratory scale with single-channel membranes and at pilot scale with 7-, 19- and 151-channel membranes in order to permanently reach the limit value of a COD below 80 mg/L and to increase the operating time. Four types of membranes were sequentially tested in the cross-flow mode: MF (200 nm pore size), UF (2,000 Da), NF (450 Da) and NF (200 Da). 5-day Biological Oxygen Demand (BOD) tests were performed in order to examine the wastewater biodegradability. The test results with single-channel membranes showed that in terms of the highest COD rejection and the highest permeability, the best combination was that of MF and UF membranes. Here, UF membranes were sufficient to reach the limit values. As for the multi-channel membranes, the combination of MF and NF (450 Da) was the best and the final COD concentration ranged from 11 to 48 mg/L. 5-day BOD bottle tests showed a COD/BOD ratio of 3.8, which opened up possibilities for combined treatment.
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http://dx.doi.org/10.2166/wst.2021.039DOI Listing
March 2021

Genome-Wide Association Studies for the Detection of Genetic Variants Associated With Daptomycin and Ceftaroline Resistance in .

Front Microbiol 2021 15;12:639660. Epub 2021 Feb 15.

Department of Infectious Diseases, Robert Koch-Institute, Wernigerode, Germany.

Background: As next generation sequencing (NGS) technologies have experienced a rapid development over the last decade, the investigation of the bacterial genetic architecture reveals a high potential to dissect causal loci of antibiotic resistance phenotypes. Although genome-wide association studies (GWAS) have been successfully applied for investigating the basis of resistance traits, complex resistance phenotypes have been omitted so far. For this especially refers to antibiotics of last resort like daptomycin and ceftaroline. Therefore, we aimed to perform GWAS for the identification of genetic variants associated with DAP and CPT resistance in clinical isolates.

Materials/methods: To conduct microbial GWAS, we selected cases and controls according to their clonal background, date of isolation, and geographical origin. Association testing was performed with PLINK and SEER analysis. By using analysis, we also searched for rare genetic variants in candidate loci that have previously been described to be involved in the development of corresponding resistance phenotypes.

Results: GWAS revealed MprF P314L and L826F to be significantly associated with DAP resistance. These mutations were found to be homogenously distributed among clonal lineages suggesting convergent evolution. Additionally, rare and yet undescribed single nucleotide polymorphisms could be identified within and putative candidate genes. Finally, we could show that each DAP resistant isolate exhibited at least one amino acid substitution within the open reading frame of . Due to the presence of strong population stratification, no genetic variants could be associated with CPT resistance. However, the investigation of the staphylococcal cassette chromosome (SCC) revealed various SNPs to be putatively linked with CPT resistance. Additionally, some CPT resistant isolates revealed no mutations, supporting the hypothesis that further and still unknown resistance determinants are crucial for the development of CPT resistance in .

Conclusion: We hereby confirmed the potential of GWAS to identify genetic variants that are associated with antibiotic resistance traits in However, precautions need to be taken to prevent the detection of spurious associations. In addition, the implementation of different approaches is still essential to detect multiple forms of variations and mutations that occur with a low frequency.
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http://dx.doi.org/10.3389/fmicb.2021.639660DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7917082PMC
February 2021

Genome-Based Analyses of Fitness Effects and Compensatory Changes Associated with Acquisition of -, -, and -Containing Plasmids in .

Antibiotics (Basel) 2021 Jan 19;10(1). Epub 2021 Jan 19.

Robert Koch Institute, Department Infectious Diseases, Division Nosocomial Pathogens and Antimicrobial Resistances, Wernigerode Branch, 38855 Wernigerode, Germany.

(1) Background: Resistance plasmids are under selective conditions beneficial for the bacterial host, but in the absence of selective pressure, this carriage may cause fitness costs. Compensation of this fitness burden is important to obtain competitive ability under antibiotic-free conditions. In this study, we investigated fitness effects after a conjugative transfer of plasmids containing various beta-lactamase genes transferred into . (2) Methods: Fourteen beta-lactamase-encoding plasmids were transferred from clinical donor strains to J53. Growth rates were compared for all transconjugants and the recipient. Selected transconjugants were challenged in long-term growth experiments. Growth rates were assessed at different time points during growth for 500 generations. Whole-genome sequencing (WGS) of initial and evolved transconjugants was determined. Results: Most plasmid acquisitions resulted in growth differences, ranging from -4.5% to 7.2%. Transfer of a single -carrying plasmid resulted in a growth burden and a growth benefit in independent mating. Long-term growth led to a compensation of fitness burdens and benefits. Analyzing WGS revealed genomic changes caused by Single Nucleotide Polymorphisms (SNPs) and insertion sequences over time. Conclusions: Fitness effects associated with plasmid acquisitions were variable. Potential compensatory mutations identified in transconjugants' genomes after 500 generations give interesting insights into aspects of plasmid-host adaptations.
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http://dx.doi.org/10.3390/antibiotics10010090DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7832316PMC
January 2021

Determination of antibiotic resistance genes in a WWTP-impacted river in surface water, sediment, and biofilm: Influence of seasonality and water quality.

Sci Total Environ 2021 May 7;768:144526. Epub 2021 Jan 7.

Institute of Functional Interfaces, Karlsruhe Institute of Technology (KIT), Germany.

Many pathogenic bacteria are adapted to live in aquatic habitats, which makes rivers possible sources and spread pathways of antibiotic resistance, since they usually receive effluents from wastewater treatment plants (WWTP), possibly containing antibiotic residues and also antibiotic-resistant bacteria. This study investigates different monitoring strategies to identify the occurrence of antibiotic-resistant bacteria in rivers. We analyzed the presence of 13 antibiotic resistance genes (ARGs) and seven gene markers for facultative pathogenic bacteria (FPB) with qPCR in sampling sites upstream and downstream of a small WWTP in Southern Germany. Five sampling campaigns were conducted from February to June 2019. Surface water, sediment, and biofilm samples were analyzed. The biofilm was collected from an artificial sampler placed in the river. bla, ermB, tetM, and sul1 genes were detected in all samples analyzed. The results showed there was a previous background in the river, but the WWTP and the water quality of the river influenced the concentration and occurrence of ARGs and FPB. Genes representing resistance against strong or last-resort antibiotics, such as mecA, bla, bla, and mcr-1, and multidrug resistance were also detected, mainly in samples collected downstream of the WWTP. Downstream of the WWTP, the occurrence of ARG and FPB correlated with ammoniacal nitrogen, while upstream of the WWTP correlated with turbidity, suspended solids, and seasonal factors such as UVA radiation and the presence of macrophytes. Biofilm samples presented higher abundances of ARGs and FPB. The biofilm sampler was efficient and allowed to collect biofilms from specific periods, which helped to identify seasonal patterns.
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http://dx.doi.org/10.1016/j.scitotenv.2020.144526DOI Listing
May 2021

A Multi-Omics Protocol for Swine Feces to Elucidate Longitudinal Dynamics in Microbiome Structure and Function.

Microorganisms 2020 Nov 28;8(12). Epub 2020 Nov 28.

Institute of Microbiology, University of Greifswald, Felix-Hausdorff-Str. 8, 17489 Greifswald, Germany.

Swine are regarded as promising biomedical models, but the dynamics of their gastrointestinal microbiome have been much less investigated than that of humans or mice. The aim of this study was to establish an integrated multi-omics protocol to investigate the fecal microbiome of healthy swine. To this end, a preparation and analysis protocol including integrated sample preparation for meta-omics analyses of deep-frozen feces was developed. Subsequent data integration linked microbiome composition with function, and metabolic activity with protein inventories, i.e., 16S rRNA data and expressed proteins, and identified proteins with corresponding metabolites. 16S rRNA gene amplicon and metaproteomics analyses revealed a fecal microbiome dominated by , , and Similar microbiome compositions in feces and colon, but not ileum samples, were observed, showing that feces can serve as minimal-invasive proxy for porcine colon microbiomes. Longitudinal dynamics in composition, e.g., temporal decreased abundance of and during the experiment, were not reflected in microbiome function. Instead, metaproteomics and metabolomics showed a rather stable functional state, as evident from short-chain fatty acids (SCFA) profiles and associated metaproteome functions, pointing towards functional redundancy among microbiome constituents. In conclusion, our pipeline generates congruent data from different omics approaches on the taxonomy and functionality of the intestinal microbiome of swine.
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http://dx.doi.org/10.3390/microorganisms8121887DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7760263PMC
November 2020

Survey of metaproteomics software tools for functional microbiome analysis.

PLoS One 2020 10;15(11):e0241503. Epub 2020 Nov 10.

University of Minnesota, Minneapolis, Minnesota, United States of America.

To gain a thorough appreciation of microbiome dynamics, researchers characterize the functional relevance of expressed microbial genes or proteins. This can be accomplished through metaproteomics, which characterizes the protein expression of microbiomes. Several software tools exist for analyzing microbiomes at the functional level by measuring their combined proteome-level response to environmental perturbations. In this survey, we explore the performance of six available tools, to enable researchers to make informed decisions regarding software choice based on their research goals. Tandem mass spectrometry-based proteomic data obtained from dental caries plaque samples grown with and without sucrose in paired biofilm reactors were used as representative data for this evaluation. Microbial peptides from one sample pair were identified by the X! tandem search algorithm via SearchGUI and subjected to functional analysis using software tools including eggNOG-mapper, MEGAN5, MetaGOmics, MetaProteomeAnalyzer (MPA), ProPHAnE, and Unipept to generate functional annotation through Gene Ontology (GO) terms. Among these software tools, notable differences in functional annotation were detected after comparing differentially expressed protein functional groups. Based on the generated GO terms of these tools we performed a peptide-level comparison to evaluate the quality of their functional annotations. A BLAST analysis against the NCBI non-redundant database revealed that the sensitivity and specificity of functional annotation varied between tools. For example, eggNOG-mapper mapped to the most number of GO terms, while Unipept generated more accurate GO terms. Based on our evaluation, metaproteomics researchers can choose the software according to their analytical needs and developers can use the resulting feedback to further optimize their algorithms. To make more of these tools accessible via scalable metaproteomics workflows, eggNOG-mapper and Unipept 4.0 were incorporated into the Galaxy platform.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0241503PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7654790PMC
December 2020

Computational strategies to combat COVID-19: useful tools to accelerate SARS-CoV-2 and coronavirus research.

Brief Bioinform 2021 03;22(2):642-663

Philipps-University Marburg, Germany.

SARS-CoV-2 (severe acute respiratory syndrome coronavirus 2) is a novel virus of the family Coronaviridae. The virus causes the infectious disease COVID-19. The biology of coronaviruses has been studied for many years. However, bioinformatics tools designed explicitly for SARS-CoV-2 have only recently been developed as a rapid reaction to the need for fast detection, understanding and treatment of COVID-19. To control the ongoing COVID-19 pandemic, it is of utmost importance to get insight into the evolution and pathogenesis of the virus. In this review, we cover bioinformatics workflows and tools for the routine detection of SARS-CoV-2 infection, the reliable analysis of sequencing data, the tracking of the COVID-19 pandemic and evaluation of containment measures, the study of coronavirus evolution, the discovery of potential drug targets and development of therapeutic strategies. For each tool, we briefly describe its use case and how it advances research specifically for SARS-CoV-2. All tools are free to use and available online, either through web applications or public code repositories. Contact:[email protected]
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http://dx.doi.org/10.1093/bib/bbaa232DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7665365PMC
March 2021

Genome sequences of two clinical isolates harboring the novel colistin-resistance gene variants - and -.

Gut Pathog 2020 3;12:40. Epub 2020 Sep 3.

Division of Nosocomial Pathogens and Antibiotic Resistance, Robert Koch Institute, Wernigerode, Germany.

Background: Colistin is still a widely used antibiotic in veterinary medicine although it is a last-line treatment option for hospitalized patients with infections caused by multidrug-resistant Gram-negative bacteria. Colistin resistance has gained additional importance since the recent emergence of mobile colistin resistance () genes. In the scope of a study on colistin resistance in clinical isolates from human patients in Germany we characterized the - gene variants.

Results: Our PCR-based screening for -carrying from German patients revealed the presence of -- genes in 60 isolates. Subsequent whole-genome sequence-based analyses detected one non-synonymous mutation in the - gene for two isolates. The mutations were verified by Sanger sequencing and resulted in amino acid changes Met1Thr (isolate 803-18) and Tyr9Cys (isolate 844-18). Genotyping revealed no relationship between the isolates. The two clinical isolates were assigned to sequence types ST155 (isolate 803-18) and ST69 (isolate 844-18). Both - variants were found to be located on IncX4 plasmids of 33 kb size; these plasmids were successfully conjugated into sodium azide resistant J53 Azi in a broth mating experiment.

Conclusions: Here we present the draft sequences of isolate 803-18 carrying the novel variant - and isolate 844-14 carrying the novel variant -. The results highlight the increasing issue of transferable colistin resistance.
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http://dx.doi.org/10.1186/s13099-020-00375-4DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7472697PMC
September 2020

A complete and flexible workflow for metaproteomics data analysis based on MetaProteomeAnalyzer and Prophane.

Nat Protoc 2020 10 28;15(10):3212-3239. Epub 2020 Aug 28.

Department of Infectious Diseases, Robert Koch Institute, Wernigerode, Germany.

Metaproteomics, the study of the collective protein composition of multi-organism systems, provides deep insights into the biodiversity of microbial communities and the complex functional interplay between microbes and their hosts or environment. Thus, metaproteomics has become an indispensable tool in various fields such as microbiology and related medical applications. The computational challenges in the analysis of corresponding datasets differ from those of pure-culture proteomics, e.g., due to the higher complexity of the samples and the larger reference databases demanding specific computing pipelines. Corresponding data analyses usually consist of numerous manual steps that must be closely synchronized. With MetaProteomeAnalyzer and Prophane, we have established two open-source software solutions specifically developed and optimized for metaproteomics. Among other features, peptide-spectrum matching is improved by combining different search engines and, compared to similar tools, metaproteome annotation benefits from the most comprehensive set of available databases (such as NCBI, UniProt, EggNOG, PFAM, and CAZy). The workflow described in this protocol combines both tools and leads the user through the entire data analysis process, including protein database creation, database search, protein grouping and annotation, and results visualization. To the best of our knowledge, this protocol presents the most comprehensive, detailed and flexible guide to metaproteomics data analysis to date. While beginners are provided with robust, easy-to-use, state-of-the-art data analysis in a reasonable time (a few hours, depending on, among other factors, the protein database size and the number of identified peptides and inferred proteins), advanced users benefit from the flexibility and adaptability of the workflow.
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http://dx.doi.org/10.1038/s41596-020-0368-7DOI Listing
October 2020

Comprehensive integrated NGS-based surveillance and contact-network modeling unravels transmission dynamics of vancomycin-resistant enterococci in a high-risk population within a tertiary care hospital.

PLoS One 2020 24;15(6):e0235160. Epub 2020 Jun 24.

Division of Nosocomial Pathogens and Antibiotic Resistance, Robert Koch Institute, Wernigerode, Germany.

Vancomycin-resistant E. faecium (VRE) are an important cause of nosocomial infections, which are rapidly transmitted in hospitals. To identify possible transmission routes, we applied combined genomics and contact-network modeling to retrospectively evaluate routine VRE screening data generated by the infection control program of a hemato-oncology unit. Over 1 year, a total of 111 VRE isolates from 111 patients were collected by anal swabs in a tertiary care hospital in Southern Germany. All isolated VRE were whole-genome sequenced, followed by different in-depth bioinformatics analyses including genotyping and determination of phylogenetic relations, aiming to evaluate a standardized workflow. Patient movement data were used to overlay sequencing data to infer transmission events and strain dynamics over time. A predominant clone harboring vanB and exhibiting genotype ST117/CT469 (n = 67) was identified. Our comprehensive combined analyses suggested intra-hospital spread, especially of clone ST117/CT469, despite of extensive screening, single room placement, and contact isolation. A new interactive tool to visualize these complex data was designed. Furthermore, a patient-contact network-modeling approach was developed, which indicates both the periodic import of the clone into the hospital and its spread within the hospital due to patient movements. The analyzed spread of VRE was most likely due to placement of patients in the same room prior to positivity of screening. We successfully demonstrated the added value for this combined strategy to extract well-founded knowledge from interdisciplinary data sources. The combination of patient-contact modeling and high-resolution typing unraveled the transmission dynamics within the hospital department and, additionally, a constant VRE influx over time.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0235160PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7314025PMC
September 2020

Micropollutant emissions from combined sewer overflows.

Water Sci Technol 2019 Dec;80(11):2179-2190

Karlsruhe Institute of Technology (KIT), Institute for Water and River Basin Management, Department of Aquatic Environmental Engineering, Gotthard-Franz-Str. 3, 76131 Karlsruhe, Germany E-mail:

In an extensive monitoring programme, event mean concentrations of 12 heavy metals, 16 polycyclic aromatic hydrocarbons (PAH), nine pesticides/biocides, three pharmaceuticals, three benzotriazoles, acesulfame, and DEHP (di-(2-ethylhexyl)phthalate) were measured at 10 combined sewer overflow (CSO) facilities throughout Bavaria, Germany, for more than 110 overflow events. A harmonised approach with large volume samplers was used to produce volume-proportional event composite samples. A wide range of event durations and volumes was covered successfully. All substances analysed were detected in CSO samples and the majority were quantified in more than 80% of the samples. Our results confirm that CSOs need to be considered in the debate on micropollutant emissions, and knowledge regarding their concentrations at a regional level needs to be solidified. Distinct substance-specific patterns can be observed in the variability between events and sites as well as in a correlation analysis of substance concentrations. These trends underline the need for differentiation of the substances by their predominant sources, pathways, and transport behaviours. Compared to wastewater treatment plants, CSOs are an important pollution source especially for ubiquitous, primarily stormwater-transported pollutants, including substances causing failure to achieve good chemical status of surface waters, such as the uPBT (ubiquitous, persistent, bioaccumulative and toxic) substances Hg and PAH.
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http://dx.doi.org/10.2166/wst.2020.035DOI Listing
December 2019

IS-Mediated Transfer of as the Main Route of Resistance Transmission During a Polyclonal, Multispecies Outbreak in a German Hospital.

Front Microbiol 2019 17;10:2817. Epub 2019 Dec 17.

Department of Infectious Diseases, Robert Koch-Institute, Wernigerode, Germany.

One of the most demanding challenges in infection control is the worldwide dissemination of multidrug-resistant (MDR) bacteria in clinical settings. Especially the increasing prevalence of carbapenemase producing Gram-negative pathogens poses an urgent threat to public health, as these enzymes confer resistance to almost all β-lactam antibiotics including carbapenems. In this study, we report a prolonged nosocomial outbreak of various NDM-1-producing species due to clonal spread and cross-species exchange of plasmids and possibly transposons. Between July 2015 and September 2017, a total of 51 carbapenemase-positive isolates were collected from 38 patients and three environmental sources in a single German hospital. Combining molecular typing methods and whole genome sequencing, the metallo-β-lactamase gene was found to be present in 35 isolates of which seven additionally carried the carbapenemase gene . Core genome MLST (cgMLST) revealed different clusters of closely related isolates of , , , or indicating clonal spread. The detailed reconstruction of the plasmid sequences revealed that in all outbreak-associated isolates was located on similar composite transposons, which were also very similar to Tn previously described for . In contrast to Tn, these structures were flanked by IS elements, which could facilitate horizontal gene transfer. Moreover, the identical plasmid was found to be shared by and isolates. Our results highlight the importance of detailed genome-based analyses for complex nosocomial outbreaks, allowing the identification of causal genetic determinants and providing insights into potential mechanisms involved in the dissemination of antibiotic resistances between different bacterial species.
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http://dx.doi.org/10.3389/fmicb.2019.02817DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6929489PMC
December 2019

[Occupational Diseases in General Practice].

Dtsch Med Wochenschr 2019 10 15;144(21):e160. Epub 2019 Nov 15.

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http://dx.doi.org/10.1055/a-1039-7359DOI Listing
October 2019

[Occupational Diseases in General Practice].

Dtsch Med Wochenschr 2019 10 21;144(21):1483-1486. Epub 2019 Oct 21.

This article shows an overview of occupational diseases. You can find a lot of these diseases in family practice. For treatment of occupational diseases specialists can be consulted. It is a big challenge to detect diseases caused by work influence. This is what you should do: inquire specifically about newly diagnosed illness after work. Ask about the current occupation and previous occupational activities. Already report the suspicion of an occupational disease to the competent authority. For every occupational disease, there is a leaflet with scientific basics, explanation of occupational stress and its impact on the body. Remedies and aids may not be prescribed by family doctors at the expense of the statutory health insurance.
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http://dx.doi.org/10.1055/a-0818-1469DOI Listing
October 2019

Emerging contaminants and antibiotic resistance in the different environmental matrices of Latin America.

Environ Pollut 2019 Dec 12;255(Pt 1):113140. Epub 2019 Sep 12.

Department of Hydraulics and Sanitation, Federal University of Parana, Av. Coronel Francisco Heráclito dos Santos, 210, 81531-980, Curitiba PR, Brazil; Capes Foundation, Ministry of Education of Brazil, Brasilia, 70040-020, DF, Brazil; Department of Chemistry and Biology, Federal Technology University of Paraná, Rua Deputado Heitor Alencar Furtado, 5000, 81280-340, Curitiba PR, Brazil.

This review aims to gather and summarize information about the occurrence of emerging contaminants and antibiotic resistance genes in environmental matrices in Latin America. We aim to contribute to future research by compiling a list of priority pollutants adjusted to the needs and characteristics of Latin America, according to the data presented in this study. In order to perform a comprehensive research and secure a representative and unbiased amount of quality data concerning emerging contaminants in Latin America, the research was performed within the Scopus® database in a time frame from 2000 to July 2019. The countries with higher numbers of published articles were Brazil and México, while most studies were performed in the surroundings of Mexico City and in Southern and Southeastern Brazil. The main investigated environmental matrices were drinking water and surface water. The presence of antibiotic resistance was frequently reported, mainly in Brazil. Monitoring efforts should be performed in other countries in Latin America, as well as in other regions of Brazil and México. The suggested priority list for monitoring of emerging contaminants in Latin America covers: di(2-ethylhexyl) phthalate (DEHP), bisphenol-A (BP-A), 4-nonylphenol (4-NP), triclosan (TCS), estrone (E1), estradiol (E2), ethinylestradiol (EE2), tetracycline (TC), amoxicillin (AMOX), norfloxacin (NOR), ampicillin (AMP) and imipenem (IMP). We hope this list serves as a basis for the orientation of the future research and monitoring projects to better understand the distribution and concentration of the listed emerging substances.
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http://dx.doi.org/10.1016/j.envpol.2019.113140DOI Listing
December 2019

Metaproteomics analysis of microbial diversity of human saliva and tongue dorsum in young healthy individuals.

J Oral Microbiol 2019 26;11(1):1654786. Epub 2019 Aug 26.

Interfaculty Institute for Genetics and Functional Genomics, Department of Functional Genomics, University Medicine Greifswald, Greifswald, Germany.

: The human oral microbiome influences initiation or progression of diseases like caries or periodontitis. Metaproteomics approaches enable the simultaneous investigation of microbial and host proteins and their interactions to improve understanding of oral diseases. : In this study, we provide a detailed metaproteomics perspective of the composition of salivary and tongue microbial communities of young healthy subjects. : Stimulated saliva and tongue samples were collected from 24 healthy volunteers, subjected to shotgun nLC-MS/MS and analyzed by the Trans-Proteomic Pipeline and the Prophane tool. : 3,969 bacterial and 1,857 human proteins could be identified from saliva and tongue, respectively. In total, 1,971 bacterial metaproteins and 1,154 human proteins were shared in both sample types. Twice the amount of bacterial metaproteins were uniquely identified for the tongue dorsum compared to saliva. Overall, 107 bacterial genera of seven phyla formed the microbiome. Comparative analysis identified significant functional differences between the microbial biofilm on the tongue and the microbiome of saliva. : Even if the microbial communities of saliva and tongue dorsum showed a strong similarity based on identified protein functions and deduced bacterial composition, certain specific characteristics were observed. Both microbiomes exhibit a great diversity with seven genera being most abundant.
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http://dx.doi.org/10.1080/20002297.2019.1654786DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6720020PMC
August 2019

PadR-type repressors controlling production of a non-canonical FtsW/RodA homologue and other trans-membrane proteins.

Sci Rep 2019 07 11;9(1):10023. Epub 2019 Jul 11.

FG11 Division of Enteropathogenic bacteria and Legionella, Robert Koch Institute, Burgstrasse 37, 38855, Wernigerode, Germany.

The Gram-positive bacterium Listeria monocytogenes occurs ubiquitously in the environment and infects humans upon ingestion. It encodes four PadR-like repressors, out of which LftR has been characterized previously and was shown to control gene expression in response to the antibiotic aurantimycin produced by other environmental bacteria. To better understand the PadR regulons of L. monocytogenes, we performed RNA-sequencing with mutants of the other three repressors LadR, LstR and Lmo0599. We show that LadR is primarily responsible for the regulation of the mdrL gene, encoding an efflux pump, while LstR and Lmo0599 mainly regulate their own operons. The lstR operon contains the lmo0421 gene, encoding a homolog of the RodA/FtsW protein family. However, this protein does not possess such functionality, as we demonstrate here. The lmo0599 operon contains two additional genes coding for the hypothetical trans-membrane proteins lmo0600 and lmo0601. A striking phenotype of the lmo0599 mutant is its impaired growth at refrigeration temperature. In light of these and other results we suggest that Lmo0599 should be renamed and propose LltR (listerial low temperature regulator) as its new designation. Based on the nature of the PadR target genes we assume that these repressors collectively respond to compounds acting on the cellular envelope.
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http://dx.doi.org/10.1038/s41598-019-46347-wDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6624303PMC
July 2019

Response of Microbial Communities and Their Metabolic Functions to Drying⁻Rewetting Stress in a Temperate Forest Soil.

Microorganisms 2019 May 13;7(5). Epub 2019 May 13.

Institute of Soil Research, Department of Forest and Soil Sciences, University of Natural Resources and Life Sciences Vienna (BOKU), Peter Jordan-Straße 82, 1190 Vienna, Austria.

Global climate change is predicted to alter drought-precipitation patterns, which will likely affect soil microbial communities and their functions, ultimately shifting microbially-mediated biogeochemical cycles. The present study aims to investigate the simultaneous variation of microbial community compositions and functions in response to drought and following rewetting events, using a soil metaproteomics approach. For this, an established field experiment located in an Austrian forest with two levels (moderate and severe stress) of precipitation manipulation was evaluated. The results showed that fungi were more strongly influenced by drying and rewetting (DRW) than bacteria, and that there was a drastic shift in the fungal community towards a more Ascomycota-dominated community. In terms of functional responses, a larger number of proteins and a higher functional diversity were observed in both moderate and severe DRW treatments compared to the control. Furthermore, in both DRW treatments a rise in proteins assigned to "translation, ribosomal structure, and biogenesis" and "protein synthesis" suggests a boost in microbial cell growth after rewetting. We also found that the changes within intracellular functions were associated to specific phyla, indicating that responses of microbial communities to DRW primarily shifted microbial functions. Microbial communities seem to respond to different levels of DRW stress by changing their functional potential, which may feed back to biogeochemical cycles.
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http://dx.doi.org/10.3390/microorganisms7050129DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6560457PMC
May 2019

Challenges and promise at the interface of metaproteomics and genomics: an overview of recent progress in metaproteogenomic data analysis.

Expert Rev Proteomics 2019 05 30;16(5):375-390. Epub 2019 Apr 30.

b VIB - UGent Center for Medical Biotechnology, VIB , Ghent , Belgium.

Introduction: The study of microbial communities based on the combined analysis of genomic and proteomic data - called metaproteogenomics - has gained increased research attention in recent years. This relatively young field aims to elucidate the functional and taxonomic interplay of proteins in microbiomes and its implications on human health and the environment. Areas covered: This article reviews bioinformatics methods and software tools dedicated to the analysis of data from metaproteomics and metaproteogenomics experiments. In particular, it focuses on the creation of tailored protein sequence databases, on the optimal use of database search algorithms including methods of error rate estimation, and finally on taxonomic and functional annotation of peptide and protein identifications. Expert opinion: Recently, various promising strategies and software tools have been proposed for handling typical data analysis issues in metaproteomics. However, severe challenges remain that are highlighted and discussed in this article; these include: (i) robust false-positive assessment of peptide and protein identifications, (ii) complex protein inference against a background of highly redundant data, (iii) taxonomic and functional post-processing of identification data, and finally, (iv) the assessment and provision of metrics and tools for quantitative analysis.
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http://dx.doi.org/10.1080/14789450.2019.1609944DOI Listing
May 2019

What keeps medical students healthy and well? A systematic review of observational studies on protective factors for health and well-being during medical education.

BMC Med Educ 2019 Apr 1;19(1):94. Epub 2019 Apr 1.

Department of General Practice, Carl Gustav Carus Faculty of Medicine, Dresden, Germany.

Background: Despite the growing evidence of a negative impact of medical school on students' health and well-being, little is known about protective factors for staying healthy and well during medical education. Therefore, a systematic review of peer-reviewed studies aiming to identify such predictors was conducted.

Methods: Medline, Embase, and PsychInfo were systematically searched by using preselected MeSH terms to identify English- and German-language peer-reviewed articles (observational studies) examining predictors for medical students' health and well-being, published between January 2001 and April 2018. Two authors independently selected abstracts reporting predictors for medical students' health and well-being. Further, two authors extracted information from the identified studies, needed for methodological quality assessment of the studies, as well as for comprehensive description of identified predictors.

Results: From 5013 hits in the database search, six observational studies met the inclusion criteria and were included in the final analysis. These studies were of heterogeneous design and quality. They featured a wide variety of health and well-being related outcomes and of its predictors. Lower levels of perceived stress, as well as lower levels of neuroticism were found to predict better health-related outcomes.

Conclusions: Further research, by using harmonized tools for the assessment of outcomes, as well as predictors, is needed to determine what keeps students healthy and well during medical education. Identifying protective factors is an essential prerequisite for the design of evidence-based health-promoting interventions.
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http://dx.doi.org/10.1186/s12909-019-1532-zDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6444449PMC
April 2019

Genomic and Functional Analysis of Emerging Virulent and Multidrug-Resistant Lineage Sequence Type 648.

Antimicrob Agents Chemother 2019 06 24;63(6). Epub 2019 May 24.

Institute of Microbiology and Epizootics, Free University Berlin, Berlin, Germany

The pathogenic extended-spectrum-beta-lactamase (ESBL)-producing lineage ST648 is increasingly reported from multiple origins. Our study of a large and global ST648 collection from various hosts (87 whole-genome sequences) combining core and accessory genomics with functional analyses and experiments suggests that ST648 is a nascent and generalist lineage, lacking clear phylogeographic and host association signals. By including large numbers of ST131 ( = 107) and ST10 ( = 96) strains for comparative genomics and phenotypic analysis, we demonstrate that the combination of multidrug resistance and high-level virulence are the hallmarks of ST648, similar to international high-risk clonal lineage ST131. Specifically, our , , and results demonstrate that ST648 is well equipped with biofilm-associated features, while ST131 shows sophisticated signatures indicative of adaption to urinary tract infection, potentially conveying individual ecological niche adaptation. In addition, we used a recently developed NFDS (negative frequency-dependent selection) population model suggesting that ST648 will increase significantly in frequency as a cause of bacteremia within the next few years. Also, ESBL plasmids impacting biofilm formation aided in shaping and maintaining ST648 strains to successfully emerge worldwide across different ecologies. Our study contributes to understanding what factors drive the evolution and spread of emerging international high-risk clonal lineages.
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http://dx.doi.org/10.1128/AAC.00243-19DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6535536PMC
June 2019

On the Coming End of Sociology.

Can Rev Sociol 2019 Feb 6;56(1):138-143. Epub 2019 Feb 6.

University of Virginia.

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http://dx.doi.org/10.1111/cars.12238DOI Listing
February 2019

A Core Genome Multilocus Sequence Typing Scheme for Enterococcus faecalis.

J Clin Microbiol 2019 03 27;57(3). Epub 2019 Feb 27.

Robert Koch Institute, Division of Nosocomial Pathogens and Antibiotic Resistances, Wernigerode, Germany.

Among enterococci, occurs ubiquitously, with the highest incidence of human and animal infections. The high genetic plasticity of complicates both molecular investigations and phylogenetic analyses. Whole-genome sequencing (WGS) enables unraveling of epidemiological linkages and putative transmission events between humans, animals, and food. Core genome multilocus sequence typing (cgMLST) aims to combine the discriminatory power of classical multilocus sequence typing (MLST) with the extensive genetic data obtained by WGS. By sequencing a representative collection of 146 strains isolated from hospital outbreaks, food, animals, and colonization of healthy human individuals, we established a novel cgMLST scheme with 1,972 gene targets within the Ridom SeqSphere software. To test the cgMLST scheme and assess the typing performance, different collections comprising environmental and bacteremia isolates, as well as all publicly available genome sequences from the NCBI and SRA databases, were analyzed. In more than 98.6% of the tested genomes, >95% good cgMLST target genes were detected (mean, 99.2% target genes). Our genotyping results not only corroborate the known epidemiological background of the isolates but exceed previous typing resolution. In conclusion, we have created a powerful typing scheme, hence providing an international standardized nomenclature that is suitable for surveillance approaches in various sectors, linking public health, veterinary public health, and food safety in a true One Health fashion.
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http://dx.doi.org/10.1128/JCM.01686-18DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6425188PMC
March 2019

Aurantimycin resistance genes contribute to survival of Listeria monocytogenes during life in the environment.

Mol Microbiol 2019 04 27;111(4):1009-1024. Epub 2019 Feb 27.

FG11 Division of Enteropathogenic Bacteria and Legionella, Robert Koch Institute, Burgstrasse 37, Wernigerode, 38855, Germany.

Bacteria can cope with toxic compounds such as antibiotics by inducing genes for their detoxification. A common detoxification strategy is compound excretion by ATP-binding cassette (ABC) transporters, which are synthesized upon compound contact. We previously identified the multidrug resistance ABC transporter LieAB in Listeria monocytogenes, a Gram-positive bacterium that occurs ubiquitously in the environment, but also causes severe infections in humans upon ingestion. Expression of the lieAB genes is strongly induced in cells lacking the PadR-type transcriptional repressor LftR, but compounds leading to relief of this repression in wild-type cells were not known. Using RNA-Seq and promoter-lacZ fusions, we demonstrate highly specific repression of the lieAB and lftRS promoters through LftR. Screening of a natural compound library yielded the depsipeptide aurantimycin A - synthesized by the soil-dwelling Streptomyces aurantiacus - as the first known naturally occurring inducer of lieAB expression. Genetic and phenotypic experiments concordantly show that aurantimycin A is a substrate of the LieAB transporter and thus, lftRS and lieAB represent the first known genetic module conferring and regulating aurantimycin A resistance. Collectively, these genes may support the survival of L. monocytogenes when it comes into contact with antibiotic-producing bacteria in the soil.
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http://dx.doi.org/10.1111/mmi.14205DOI Listing
April 2019