Detailed analysis of metagenome datasets obtained from biogas-producing microbial communities residing in biogas reactors does not indicate the presence of putative pathogenic microorganisms

Biotechnology for Biofuels

Felix G Eikmeyer²  Antje Rademacher³  Angelika Hanreich³  Magdalena Hennig²  Sebastian Jaenicke¹  Irena Maus²  Daniel Wibberg²  Martha Zakrzewski¹  Alfred Pühler²  Michael Klocke³  Andreas Schlüter² 

[1] Computational Genomics, Center for Biotechnology, Bielefeld University, Bielefeld, D-33594, Germany

[2] Institute for Genome Research and Systems Biology, Center for Biotechnology, Bielefeld University, Bielefeld D-33594, Germany

[3] Department Bioengineering, Leibniz Institute for Agricultural Engineering Potsdam-Bornim, Potsdam, D-14469, Germany

关键词: Biogas;    Antibiotic resistance;    High throughput sequencing;    Virulence determinants;    Bacterial pathogens;    Anaerobic digester;    Metagenome analysis;   

Others  :  798106
DOI  :  10.1186/1754-6834-6-49

 received in 2012-11-05, accepted in 2013-03-12,  发布年份 2013

【 摘 要 】

Background

In recent years biogas plants in Germany have been supposed to be involved in amplification and dissemination of pathogenic bacteria causing severe infections in humans and animals. In particular, biogas plants are discussed to contribute to the spreading of Escherichia coli infections in humans or chronic botulism in cattle caused by Clostridium botulinum. Metagenome datasets of microbial communities from an agricultural biogas plant as well as from anaerobic lab-scale digesters operating at different temperatures and conditions were analyzed for the presence of putative pathogenic bacteria and virulence determinants by various bioinformatic approaches.

Results

All datasets featured a low abundance of reads that were taxonomically assigned to the genus Escherichia or further selected genera comprising pathogenic species. Higher numbers of reads were taxonomically assigned to the genus Clostridium. However, only very few sequences were predicted to originate from pathogenic clostridial species. Moreover, mapping of metagenome reads to complete genome sequences of selected pathogenic bacteria revealed that not the pathogenic species itself, but only species that are more or less related to pathogenic ones are present in the fermentation samples analyzed. Likewise, known virulence determinants could hardly be detected. Only a marginal number of reads showed similarity to sequences described in the Microbial Virulence Database MvirDB such as those encoding protein toxins, virulence proteins or antibiotic resistance determinants.

Conclusions

Findings of this first study of metagenomic sequence reads of biogas producing microbial communities suggest that the risk of dissemination of pathogenic bacteria by application of digestates from biogas fermentations as fertilizers is low, because obtained results do not indicate the presence of putative pathogenic microorganisms in the samples analyzed.

【 授权许可】

   
2013 Eikmeyer et al.; licensee BioMed Central Ltd.

【 图 表 】

Figure 1.

【 参考文献 】

• [1]Newell DG, Koopmans M, Verhoef L, Duizer E, Aidara-Kane A, Sprong H, Opsteegh M, Langelaar M, Threfall J, Scheutz F, van der Giessen J, Kruse H: Food-borne diseases — The challenges of 20 years ago still persist while new ones continue to emerge. Future Challenges to Microbial Food Safety Contributions resulting from a conference held in Wolfheze, the Netherlands. Int J Food Microbiol 2010, 139(S1):3-15.
• [2]Cork SC: Epidemiology of Pathogens in the Food Supply. In Zoonotic pathogens in the food chain. Edited by Krause DO, Hendrick S. Wallingford, Oxfordshire: CABI; 2011:21-58.
• [3]Brzuszkiewicz E, Thuermer A, Schuldes J, Leimbach A, Liesegang H, Meyer F, Boelter J, Petersen H, Gottschalk G, Daniel R: Genome sequence analyses of two isolates from the recent Escherichia coli outbreak in Germany reveal the emergence of a new pathotype: Entero-Aggregative-Haemorrhagic Escherichia coli (EAHEC). Arch Microbiol 2011, 12:883-891.
• [4]Mellmann A, Harmsen D, Cummings CA, Zentz EB, Leopold SR, Rico A, Prior K, Szczepanowski R, Ji Y, Zhang W, McLaughlin SF, Henkhaus JK, Leopold B, Bielaszewska M, Prager R, Brzoska PM, Moore RL, Guenther S, Rothberg JM, Karch H: Prospective genomic characterization of the german enterohemorrhagic Escherichia coli O104:H4 outbreak by rapid Next Generation Sequencing technology. PLoS One 2011, 7:e22751.
• [5]Altmann M, Wadl M, Altmann D, Benzler J, Eckmanns T, Krause G, Spode A, Ander Heiden M: Timeliness of surveillance during outbreak of shiga toxin-producing Escherichia coli infection, Germany, 2011. Emerg Infect Dis 2011, 10:1906-1909.
• [6]Milinovich GJ, Klieve AV: Manure as a Source of Zoonotic Pathogens. In Zoonotic pathogens in the food chain. Edited by Krause DO, Hendrick S. Wallingford, Oxfordshire: CABI; 2011:59-83.
• [7]Dahlenborg M, Borch E, Radstrom P: Development of a combined selection and enrichment PCR procedure for Clostridium botulinum types B, E, and F and its use to determine prevalence in fecal samples from slaughtered pigs. Appl Environ Microbiol 2001, 10:4781-4788.
• [8]Dahlenborg M, Borch E, Radstrom P: Prevalence of Clostridium botulinum types B, E and F in faecal samples from Swedish cattle. Int J Food Microbiol 2003, 2:105-110.
• [9]Boll C: Chronischer Botulismus. Tod aus der Biogasanlage. Wild und Hund 2011, 10:14-19.
• [10]Rodloff AC, Krüger M: Chronic Clostridium botulinum infections in farmers. Anaerobe 2012, 2:226-228.
• [11]Krüger M, Große-Herrenthey A, Schrödl W, Gerlach A, Rodloff A: Visceral botulism at dairy farms in Schleswig Holstein, Germany: prevalence of Clostridium botulinum in feces of cows, in animal feeds, in feces of the farmers, and in house dust. Anaerobe 2012, 2:221-223.
• [12]Pallen MJ, Wren BW: Bacterial pathogenomics. Nature 2007, 7164:835-842.
• [13]Dobrindt U, Hochhut B, Hentschel U, Hacker J: Genomic islands in pathogenic and environmental microorganisms. Nat Rev Microbiol 2004, 5:414-424.
• [14]Zhou CE, Smith J, Lam M, Zemla A, Dyer MD, Slezak T: MvirDB - a microbial database of protein toxins, virulence factors and antibiotic resistance genes for bio-defence applications. Nucleic Acids Res 2007, 35(supl. 1):D391-D394.
• [15]Liu B, Pop M: ARDB--Antibiotic Resistance Genes Database. Nucleic Acids Res 2009, 37(supl. 1):D443-447. Database issue
• [16]Goberna M, Podmirseg SM, Waldhuber S, Knapp BA, García C, Insam H: Pathogenic bacteria and mineral N in soils following the land spreading of biogas digestates and fresh manure. Appl Soil Ecol 2011, 49:18-25.
• [17]Massé D, Gilbert Y, Topp E: Pathogen removal in farm-scale psychrophilic anaerobic digesters processing swine manure. Bioresource Technol 2011, 2:641-646.
• [18]Watcharasukarn M, Kaparaju P, Steyer J, Krogfelt KA, Angelidaki I: Screening Escherichia coli, Enterococcus faecalis, and Clostridium perfringens as indicator organisms in evaluating pathogen-reducing capacity in biogas plants. Microb Ecol 2009, 2:221-230.
• [19]Rademacher A, Zakrzewski M, Schlüter A, Schoenberg M, Szczepanowski R, Goesmann A, Pühler A, Klocke M: Characterization of microbial biofilms in a thermophilic biogas system by high-throughput metagenome sequencing. FEMS Microbiol Ecol 2012, 3:785-799.
• [20]Hanreich A, Schimpf U, Zakrzewski M, Schlüter A, Benndorf D, Heyer R, Rapp E, Pühler A, Reichl U, Klocke M: Metagenome and metaproteome analyses of microbial communities in mesophilic biogas producing anaerobic batch fermentations indicate concerted plant carbohydrate degradation. Syst Appl Microbiol 2013. in Press
• [21]Krause L, Diaz NN, Edwards RA, Gartemann K, Kroemeke H, Neuweger H, Pühler A, Runte KJ, Schlüter A, Stoye J, Szczepanowski R, Tauch A, Goesmann A: Taxonomic composition and gene content of a methane-producing microbial community isolated from a biogas reactor. J Biotechnol 2008, 1–2:91-101.
• [22]Jaenicke S, Ander C, Bekel T, Bisdorf R, Droege M, Gartemann K, Juenemann S, Kaiser O, Krause L, Tille F, Zakrzewski M, Pühler A, Schlüter A, Goesmann A: Comparative and joint analysis of two metagenomic datasets from a biogas fermenter obtained by 454-pyrosequencing. PLoS One 2011, 1:e14519.
• [23]Schlüter A, Bekel T, Diaz NN, Dondrup M, Eichenlaub R, Gartemann K, Krahn I, Krause L, Krömeke H, Kruse O, Mussgnug JH, Neuweger H, Niehaus K, Pühler A, Runte KJ, Szczepanowski R, Tauch A, Tilker A, Viehöver P, Goesmann A: The metagenome of a biogas-producing microbial community of a production-scale biogas plant fermenter analysed by the 454-pyrosequencing technology. J Biotechnol 2008, 1–2:77-90.
• [24]Gerlach W, Stoye J: Taxonomic classification of metagenomic shotgun sequences with CARMA3. Nucleic Acids Res 2011, 14:e91.
• [25]Popoff MR, Bouvet P: Clostridial toxins. Future Microbiol 2009, 8:1021-1064.
• [26]Dohrmann AB, Baumert S, Klingebiel L, Weiland P, Tebbe CC: Bacterial community structure in experimental methanogenic bioreactors and search for pathogenic clostridia as community members. Appl Microbiol Biotechnol 2011, 6:1991-2004.
• [27]Finegold SM, Song Y, Liu C, Hecht DW, Summanen P, Kononen E, Allen SD: Clostridium clostridioforme: a mixture of three clinically important species. Eur J Clin Microbiol 2005, 5:319-324.
• [28]Deublein D, Steinhauser A: Biogas from waste and renewable resources. An introduction. Weinheim, Chichester: Wiley-VCH; 2010.
• [29]Jans C, Follador R, Lacroix C, Meile L, Stevens MJA: Complete genome sequence of the african dairy isolate Streptococcus infantarius subsp infantarius strain CJ18. J Bacteriol 2012, 8:2105-2106.
• [30]Kouguchi H, Watanabe T, Sagane Y, Sunagawa H, Ohyama T: In vitro reconstitution of the Clostridium botulinum type D progenitor toxin. J Biol Chem 2002, 4:2650-2656.
• [31]Tan KS, Wee BY, Song KP: Evidence for holin function of tcdE gene in the pathogenicity of Clostridium difficile. J Med Microbiol 2001, 7:613-619.
• [32]Tsuge H, Nagahama M, Nishimura H, Hisatsune J, Sakaguchi Y, Itogawa Y, Katunuma N, Sakurai J: Crystal structure and site-directed mutagenesis of enzymatic components from Clostridium perfringens iota-toxin. J Mol Biol 2003, 3:471-483.
• [33]Siezen RJ, Leunissen JA: Subtilases: the superfamily of subtilisin-like serine proteases. Protein Sci 1997, 3:501-523.
• [34]Valbuzzi A, Ferrari E, Albertini AM: A novel member of the subtilisin-like protease family from Bacillus subtilis. Microbiol (Reading Engl 1999, 145:3121-3127.
• [35]Graycar T: Proteolytic cleavage, reaction mechanism. In Encyclopedia of bioprocess technology. Fermentation, biocatalysis, and bioseparation. Edited by Flickinger MC. New York, NY: Wiley; 1999.
• [36]Tornero P, Conejero V, Vera P: Identification of a new pathogen-induced member of the subtilisin-like processing protease family from plants. J Biol Chem 1997, 22:14412-14419.
• [37]Welch RA: RTX toxin structure and function: A story of numerous anomalies and few analogies in toxin biology. Curr Top Microbiol 2001, 257:85-111.
• [38]Ficko-Blean E, Boraston AB: Cloning, recombinant production, crystallization and preliminary X-ray diffraction studies of a family 84 glycoside hydrolase from Clostridium perfringens. Acta Crystallogr F 2005, 61:834-836.
• [39]Adams JJ, Gregg K, Bayer EA, Boraston AB, Smith SP: Structural basis of Clostridium perfringens toxin complex formation. Proc Natl Acad Sci USA 2008, 34:12194-12199.
• [40]Robertson KP, Smith CJ, Am G, Rocha ER: Characterization of Bacteroides fragilis hemolysins and regulation and synergistic interactions of HlyA and HlyB. Infect Immun 2006, 4:2304-2316.
• [41]Welch RA, Pellett S: Transcriptional organization of the Escherichia coli hemolysin genes. J Bacteriol 1988, 4:1622-1630.
• [42]Hug I, Couturier MR, Rooker MM, Taylor DE, Stein M, Feldman MF: Helicobacter pylori lipopolysaccharide is synthesized via a novel pathway with an evolutionary connection to protein N-glycosylation. PLoS Pathog 2010, 6(3):e1000819.
• [43]Horwich AL, Fenton WA, Chapman E, Farr GW: Two families of chaperonin: physiology and mechanism. Annu Rev Cell Dev Biol 2007, 23:115-145.
• [44]Frees D, Savijoki K, Varmanen P, Ingmer H: Clp ATPases and ClpP proteolytic complexes regulate vital biological processes in low GC, Gram-positive bacteria. Mol Microbiol 2007, 5:1285-1295.
• [45]Nikaido H: Multidrug resistance in bacteria. Annu Rev Biochem 2009, 78:119-146.
• [46]Kuzin AP, Sun T, Jorczak-Baillass J, Healy VL, Walsh CT: Knox, JR: Enzymes of vancomycin resistance: the structure of D-alanine-D-lactate ligase of naturally resistant Leuconostoc mesenteroides. Struct Fold Des 2000, 5:463-470.
• [47]Roper DI, Huyton T, Vagin A, Dodson G: The molecular basis of vancomycin resistance in clinically relevant Enterococci: crystal structure of D-alanyl-D-lactate ligase (VanA). Proc Natl Acad Sci USA 2000, 16:8921-8925.
• [48]Altschul SF, Gish W, Miller W, Myers EW, Lipman DJ: Basic local alignment search tool. J Mol Biol 1990, 3:403-410.
• [49]Punta M, Coggill PC, Eberhardt RY, Mistry J, Tate J, Boursnell C, Pang N, Forslund K, Ceric G, Clements J, Heger A, Holm L, Sonnhammer ELL, Eddy SR, Bateman A, Finn RD: The Pfam protein families database. Nucleic Acids Res 2012, 40:D290-301. Database issue