Rafael Szczepanowski

Prospective Genomic Characterization of the German Enterohemorrhagic Escherichia coli O104:H4 Outbreak by Rapid Next Generation Sequencing Technology

An ongoing outbreak of exceptionally virulent Shiga toxin (Stx)-producing Escherichia coli O104:H4 centered in Germany, has caused over 830 cases of hemolytic uremic syndrome (HUS) and 46 deaths since May 2011. Serotype O104:H4, which has not been detected in animals, has rarely been associated with HUS in the past. To prospectively elucidate the unique characteristics of this strain in the early stages of this outbreak, we applied whole genome sequencing on the Life Technologies Ion Torrent PGM™ sequencer and Optical Mapping to characterize one outbreak isolate (LB226692) and a historic O104:H4 HUS isolate from 2001 (01-09591). Reference guided draft assemblies of both strains were completed with the newly introduced PGM™ within 62 hours. The HUS-associated strains both carried genes typically found in two types of pathogenic E. coli, enteroaggregative E. coli (EAEC) and enterohemorrhagic E. coli (EHEC). Phylogenetic analyses of 1,144 core E. coli genes indicate that the HUS-causing O104:H4 strains and the previously published sequence of the EAEC strain 55989 show a close relationship but are only distantly related to common EHEC serotypes. Though closely related, the outbreak strain differs from the 2001 strain in plasmid content and fimbrial genes. We propose a model in which EAEC 55989 and EHEC O104:H4 strains evolved from a common EHEC O104:H4 progenitor, and suggest that by stepwise gain and loss of chromosomal and plasmid-encoded virulence factors, a highly pathogenic hybrid of EAEC and EHEC emerged as the current outbreak clone. In conclusion, rapid next-generation technologies facilitated prospective whole genome characterization in the early stages of an outbreak.

The metagenome of a biogas-producing microbial community of a production-scale biogas plant fermenter analysed by the 454-pyrosequencing technology

Composition and gene content of a biogas-producing microbial community from a production-scale biogas plant fed with renewable primary products was analysed by means of a metagenomic approach applying the ultrafast 454-pyrosequencing technology. Sequencing of isolated total community DNA on a Genome Sequencer FLX System resulted in 616,072 reads with an average read length of 230 bases accounting for 141,664,289 bases sequence information. Assignment of obtained single reads to COG (Clusters of Orthologous Groups of proteins) categories revealed a genetic profile characteristic for an anaerobic microbial consortium conducting fermentative metabolic pathways. Assembly of single reads resulted in the formation of 8752 contigs larger than 500 bases in size. Contigs longer than 10kb mainly encode house-keeping proteins, e.g. DNA polymerase, recombinase, DNA ligase, sigma factor RpoD and genes involved in sugar and amino acid metabolism. A significant portion of contigs was allocated to the genome sequence of the archaeal methanogen Methanoculleus marisnigri JR1. Mapping of single reads to the M. marisnigri JR1 genome revealed that approximately 64% of the reference genome including methanogenesis gene regions are deeply covered. These results suggest that species related to those of the genus Methanoculleus play a dominant role in methanogenesis in the analysed fermentation sample. Moreover, assignment of numerous contig sequences to clostridial genomes including gene regions for cellulolytic functions indicates that clostridia are important for hydrolysis of cellulosic plant biomass in the biogas fermenter under study. Metagenome sequence data from a biogas-producing microbial community residing in a fermenter of a biogas plant provide the basis for a rational approach to improve the biotechnological process of biogas production.

Detection of 140 clinically relevant antibiotic- resistance genes in the plasmid metagenome of wastewater treatment plant bacteria showing reduced susceptibility to selected antibiotics

To detect plasmid-borne antibiotic-resistance genes in wastewater treatment plant (WWTP) bacteria, 192 resistance-gene-specific PCR primer pairs were designed and synthesized. Subsequent PCR analyses on total plasmid DNA preparations obtained from bacteria of activated sludge or the WWTPs final effluents led to the identification of, respectively, 140 and 123 different resistance-gene-specific amplicons. The genes detected included aminoglycoside, beta-lactam, chloramphenicol, fluoroquinolone, macrolide, rifampicin, tetracycline, trimethoprim and sulfonamide resistance genes as well as multidrug efflux and small multidrug resistance genes. Some of these genes were only recently described from clinical isolates, demonstrating genetic exchange between clinical and WWTP bacteria. Sequencing of selected resistance-gene-specific amplicons confirmed their identity or revealed that the amplicon nucleotide sequence is very similar to a gene closely related to the reference gene used for primer design. These results demonstrate that WWTP bacteria are a reservoir for various resistance genes. Moreover, detection of about 64 % of the 192 reference resistance genes in bacteria obtained from the WWTPs final effluents indicates that these resistance determinants might be further disseminated in habitats downstream of the sewage plant.

Taxonomic composition and gene content of a methane-producing microbial community isolated from a biogas reactor.

A total community DNA sample from an agricultural biogas reactor continuously fed with maize silage, green rye, and small proportions of chicken manure has recently been sequenced using massively parallel pyrosequencing. In this study, the sample was computationally characterized without a prior assembly step, providing quantitative insights into the taxonomic composition and gene content of the underlying microbial community. Clostridiales from the phylum Firmicutes is the most prevalent phylogenetic order, Methanomicrobiales are dominant among methanogenic archaea. An analysis of Operational Taxonomic Units (OTUs) revealed that the entire microbial community is only partially covered by the sequenced sample, despite that estimates suggest only a moderate overall diversity of the community. Furthermore, the results strongly indicate that archaea related to the genus Methanoculleus, using CO2 as electron acceptor and H2 as electron donor, are the main producers of methane in the analyzed biogas reactor sample. A phylogenetic analysis of glycosyl hydrolase protein families suggests that Clostridia play an important role in the digestion of polysaccharides and oligosaccharides. Finally, the results unveiled that most of the organisms constituting the sample are still unexplored.

Occurrence of integron-associated resistance gene cassettes located on antibiotic resistance plasmids isolated from a wastewater treatment plant

The role of a municipal wastewater treatment plant as a reservoir for bacteria carrying antibiotic resistance plasmids was analysed. Altogether, ninety-seven different multiresistance plasmids were isolated and screened by PCR for the presence of class 1 integron-specific sequences. Twelve of these plasmids were identified to carry integrons. In addition, integron-specific sequences were found on plasmid-DNA preparations from bacteria residing in activated sludge and in the final effluents of the wastewater treatment plant. Sequencing and annotation of the integrons identified nineteen different gene cassette arrays, containing twenty-one different resistance gene cassettes. These cassettes carry genes encoding eight different aminoglycoside-modifying enzymes, seven dihydrofolate reductases, three beta-lactamases, two chloramphenicol resistance proteins and two small exporter proteins. Moreover, new gene cassettes and cassettes with unknown function were identified. Eleven gene cassette combinations are described for the first time. Six integron-associated gene cassette arrays are located on self-transmissible, putative broad-host-range plasmids belonging to the IncP group. Hybridisation analyses, using the integron-specific gene cassette arrays as templates and labelled plasmid-DNA preparations from bacteria of the final effluents as hybridisation probes, revealed that bacteria containing integron-specific sequences on plasmids are released into the environment.

Profiling of the metabolically active community from a production-scale biogas plant by means of high-throughput metatranscriptome sequencing.

Structural composition and gene content of a biogas-producing microbial community from a production-scale biogas plant fed with renewable primary products was recently analyzed by means of a metagenome sequencing approach. To determine the transcriptionally active part of the same biogas community and to identify key transcripts for the biogas production process, the metatranscriptome of the microorganisms was sequenced for the first time. The metatranscriptome sequence dataset generated on the Genome Sequencer FLX platform is represented by 484,920 sequence reads. Taxonomic profiling of the active part of the community by classification of 16S ribosomal sequence tags revealed that members of the Euryarchaeota and Firmicutes account for the dominant phyla. Only smaller fractions of the 16S ribosomal sequence tags were assigned to the phyla Bacteroidetes, Actinobacteria and Synergistetes. Among the mRNA-derived sequence tags from the metatranscriptome dataset, transcripts encoding enzymes involved in substrate hydrolysis, acidogenesis, acetate formation and methanogenesis could be identified. Transcripts for enzymes functioning in methanogenesis are among the most abundant mRNA tags indicating that the corresponding pathway is very active in the methanogenic sub-community. As a frame of reference for evaluation of metatranscriptome sequence data, the 16S rDNA-based taxonomic profile of the community was analyzed by means of high-throughput 16S rDNA amplicon sequencing. Processing of the obtained amplicon reads resulted in 18,598 high-quality 16S rDNA sequences covering the V3-V4 hypervariable region of the 16S rRNA gene. Comparison of the taxonomic profiles deduced from 16S rDNA amplicon sequences and the metatranscriptome dataset indicates a high transcriptional activity of archaeal species. Overall, it was shown that the most abundant species dominating the community also contributed the majority of the transcripts. In the future, key transcripts for the biogas production process will provide valuable markers for evaluation of the performance of biogas-producing microbial communities with the objective to optimize the biotechnology of this process.

High-quality genome sequence of Pichia pastoris CBS7435.

The methylotrophic yeast Pichia pastoris (Komagataella phaffii) CBS7435 is the parental strain of commonly used P. pastoris recombinant protein production hosts making it well suited for improving the understanding of associated genomic features. Here, we present a 9.35 Mbp high-quality genome sequence of P. pastoris CBS7435 established by a combination of 454 and Illumina sequencing. An automatic annotation of the genome sequence yielded 5007 protein-coding genes, 124 tRNAs and 29 rRNAs. Moreover, we report the complete DNA sequence of the first mitochondrial genome of a methylotrophic yeast. Fifteen genes encoding proteins, 2 rRNA and 25 tRNA loci were identified on the 35.7 kbp circular, mitochondrial DNA. Furthermore, the architecture of the putative alpha mating factor protein of P. pastoris CBS7435 turned out to be more complex than the corresponding protein of Saccharomyces cerevisiae.

Characterization of microbial biofilms in a thermophilic biogas system by high-throughput metagenome sequencing

DNAs of two biofilms of a thermophilic two-phase leach-bed biogas reactor fed with rye silage and winter barley straw were sequenced by 454-pyrosequencing technology to assess the biofilm-based microbial community and their genetic potential for anaerobic digestion. The studied biofilms matured on the surface of the substrates in the hydrolysis reactor (HR) and on the packing in the anaerobic filter reactor (AF). The classification of metagenome reads showed Clostridium as most prevalent bacteria in the HR, indicating a predominant role for plant material digestion. Notably, insights into the genetic potential of plant-degrading bacteria were determined as well as further bacterial groups, which may assist Clostridium in carbohydrate degradation. Methanosarcina and Methanothermobacter were determined as most prevalent methanogenic archaea. In consequence, the biofilm-based methanogenesis in this system might be driven by the hydrogenotrophic pathway but also by the aceticlastic methanogenesis depending on metabolite concentrations such as the acetic acid concentration. Moreover, bacteria, which are capable of acetate oxidation in syntrophic interaction with methanogens, were also predicted. Finally, the metagenome analysis unveiled a large number of reads with unidentified microbial origin, indicating that the anaerobic degradation process may also be conducted by up to now unknown species.

Bacterial Community Shift in Treated Periodontitis Patients Revealed by Ion Torrent 16S rRNA Gene Amplicon Sequencing

Periodontitis, one of the most common diseases in the world, is caused by a mixture of pathogenic bacteria and inflammatory host responses and often treated by antimicrobials as an adjunct to scaling and root planing (SRP). Our study aims to elucidate explorative and descriptive temporal shifts in bacterial communities between patients treated by SRP alone versus SRP plus antibiotics. This is the first metagenomic study using an Ion Torrent Personal Genome Machine (PGM). Eight subgingival plaque samples from four patients with chronic periodontitis, taken before and two months after intervention were analyzed. Amplicons from the V6 hypervariable region of the 16S rRNA gene were generated and sequenced each on a 314 chip. Sequencing reads were clustered into operational taxonomic units (OTUs, 3% distance), described by community metrics, and taxonomically classified. Reads ranging from 599,933 to 650,416 per sample were clustered into 1,648 to 2,659 non-singleton OTUs, respectively. Increased diversity (Shannon and Simpson) in all samples after therapy was observed regardless of the treatment type whereas richness (ACE) showed no correlation. Taxonomic analysis revealed different microbial shifts between both therapy approaches at all taxonomic levels. Most remarkably, the genera Porphyromonas, Tannerella, Treponema, and Filifactor all harboring periodontal pathogenic species were removed almost only in the group treated with SPR and antibiotics. For the species T. forsythia and P. gingivalis results were corroborated by real-time PCR analysis. In the future, hypothesis free metagenomic analysis could be the key in understanding polymicrobial diseases and be used for therapy monitoring. Therefore, as read length continues to increase and cost to decrease, rapid benchtop sequencers like the PGM might finally be used in routine diagnostic.

Unraveling the Chinese hamster ovary cell line transcriptome by next-generation sequencing.

The pyrosequencing technology from 454 Life Sciences and a novel assembly approach for cDNA sequences with the Newbler Assembler were used to achieve a major step forward to unravel the transcriptome of Chinese hamster ovary (CHO) cells. Normalized cDNA libraries originating from several cell lines and diverse culture conditions were sequenced and the resulting 1.84 million reads were assembled into 32,801 contiguous sequences, 29,184 isotigs, and 24,576 isogroups. A taxonomic classification of the isotigs showed that more than 70% of the assembled data is most similar to the transcriptome of Mus musculus, with most of the remaining isotigs being homologous to DNA sequences from Rattus norvegicus. Mapping of the CHO cell line contigs to the mouse transcriptome demonstrated that 9124 mouse transcripts, representing 6701 genes, are covered by more than 95% of their sequence length. Metabolic pathways of the central carbohydrate metabolism and biosynthesis routes of sugars used for protein N-glycosylation were reconstructed from the transcriptome data. All relevant genes representing major steps in the N-glycosylation pathway of CHO cells were detected. The present manuscript represents a data set of assembled and annotated genes for CHO cells that can now be used for a detailed analysis of the molecular functioning of CHO cell lines.