Jörg Rothgänger

Typing of Methicillin-Resistant Staphylococcus aureus in a University Hospital Setting by Using Novel Software for spa Repeat Determination and Database Management

ABSTRACT The spa gene of Staphylococcus aureus encodes protein A and is used for typing of methicillin-resistant Staphylococcus aureus (MRSA). We used sequence typing of the spa gene repeat region to study the epidemiology of MRSA at a German university hospital. One hundred seven and 84 strains were studied during two periods of 10 and 4 months, respectively. Repeats and spa types were determined by Ridom StaphType, a novel software tool allowing rapid repeat determination, data management and retrieval, and Internet-based assignment of new spa types following automatic quality control of DNA sequence chromatograms. Isolates representative of the most abundant spa types were subjected to multilocus sequence typing and pulsed-field gel electrophoresis. One of two predominant spa types was replaced by a clonally related variant in the second study period. Ten unique spa types, which were equally distributed in both study periods, were recovered. The data show a rapid dynamics of clone circulation in a university hospital setting. spa typing was valuable for tracking of epidemic isolates. The data show that disproval of epidemiologically suggested transmissions of MRSA is one of the main objectives of spa typing in departments with a high incidence of MRSA.

Automated DNA Sequence-Based Early Warning System for the Detection of Methicillin-Resistant Staphylococcus aureus Outbreaks

Background The detection of methicillin-resistant Staphylococcus aureus (MRSA) usually requires the implementation of often rigorous infection-control measures. Prompt identification of an MRSA epidemic is crucial for the control of an outbreak. In this study we evaluated various early warning algorithms for the detection of an MRSA cluster. Methods and Findings Between 1998 and 2003, 557 non-replicate MRSA strains were collected from staff and patients admitted to a German tertiary-care university hospital. The repeat region of the S. aureus protein A (spa) gene in each of these strains was sequenced. Using epidemiological and typing information for the period 1998–2002 as reference data, clusters in 2003 were determined by temporal-scan test statistics. Various early warning algorithms (frequency, clonal, and infection control professionals [ICP] alerts) were tested in a prospective analysis for the year 2003. In addition, a newly implemented automated clonal alert system of the Ridom StaphType software was evaluated. A total of 549 of 557 MRSA were typeable using spa sequencing. When analyzed using scan test statistics, 42 out of 175 MRSA in 2003 formed 13 significant clusters (p < 0.05). These clusters were used as the “gold standard” to evaluate the various algorithms. Clonal alerts (spa typing and epidemiological data) were 100% sensitive and 95.2% specific. Frequency (epidemiological data only) and ICP alerts were 100% and 62.1% sensitive and 47.2% and 97.3% specific, respectively. The difference in specificity between clonal and ICP alerts was not significant. Both methods exhibited a positive predictive value above 80%. Conclusions Rapid MRSA outbreak detection, based on epidemiological and spa typing data, is a suitable alternative for classical approaches and can assist in the identification of potential sources of infection.

Whole genome based Mycobacterium tuberculosis surveillance: A standardized, portable and expandable approach

ABSTRACT Whole-genome sequencing (WGS) allows for effective tracing of Mycobacterium tuberculosis complex (MTBC) (tuberculosis pathogens) transmission. However, it is difficult to standardize and, therefore, is not yet employed for interlaboratory prospective surveillance. To allow its widespread application, solutions for data standardization and storage in an easily expandable database are urgently needed. To address this question, we developed a core genome multilocus sequence typing (cgMLST) scheme for clinical MTBC isolates using the Ridom SeqSphere+ software, which transfers the genome-wide single nucleotide polymorphism (SNP) diversity into an allele numbering system that is standardized, portable, and not computationally intensive. To test its performance, we performed WGS analysis of 26 isolates with identical IS6110 DNA fingerprints and spoligotyping patterns from a longitudinal outbreak in the federal state of Hamburg, Germany (notified between 2001 and 2010). The cgMLST approach (3,041 genes) discriminated the 26 strains with a resolution comparable to that of SNP-based WGS typing (one major cluster of 22 identical or closely related and four outlier isolates with at least 97 distinct SNPs or 63 allelic variants). Resulting tree topologies are highly congruent and grouped the isolates in both cases analogously. Our data show that SNP- and cgMLST-based WGS analyses facilitate high-resolution discrimination of longitudinal MTBC outbreaks. cgMLST allows for a meaningful epidemiological interpretation of the WGS genotyping data. It enables standardized WGS genotyping for epidemiological investigations, e.g., on the regional public health office level, and the creation of web-accessible databases for global TB surveillance with an integrated early warning system.

Intuitive hypertext-based molecular identification of micro-organisms

Reliable identification of micro-organisms often requires several techniques, such as morphology and nutritional physiology. These methods are time consuming and can be ambiguous. With the use of sequence information, however, such as ribosomal RNA operons, every micro-organism— even those dead or uncultivated—can be identified by something it has in common with others, with the result that only one genotypical method is needed. Currently available sequence databases and analysis tools, such as the National Center for Biotechnology Information or Ribosomal Database Project are not very useful for rapid identification of clinically relevant micro-organisms. Amongst the drawbacks are low user-friendliness of analytical tools and poor quality of the data included in the databases. We developed therefore, a new intuitive user-interface hypertext based on available software (FASTA, CLUSTALW). For all new database entries, such as annotated sequences from culture-collection strains, a hypervariable ribosomal DNA region, no longer than one sequence run, was selected. This region is flanked by two strongly conserved nucleotide stretches which serve as an invariable annealing target for PCR and DNA-sequencing primers. The diagnostic procedure starts with a ribosomal DNA sequence from the specimen to be identified which is submitted to the query interface. From an interpreted similarity search, a species or genus name for the specimen in question is returned. If the first results are ambiguous or do not define to species level, hints for further molecular (internal transcribed spacer) and conventional differentiation are offered (“sequential and polyphasic” approach). An integrated phylogeny browser allows for the immediate control of the phylogenetic placement of the identified species. Further genus, species descriptions, and taxonomic information is created out of the database according to the search result on World Wide Web (WWW) pages. For quality control, sequence electropherograms of newly determined sequences are deposited on the WWW server. Because of the open hypertext-structure of the Ribosomal Differentiation of Medical Microorganisms (RIDOM) system, many context-sensitive links to other available WWW services are stablished thereby augmenting the information content (table). A report generator searches the internet and the RIDOM server for information regarding the determined species, and presents a one-page summary of

Diagnostics of Neisseriaceae and Moraxellaceae by Ribosomal DNA Sequencing: Ribosomal Differentiation of Medical Microorganisms

ABSTRACT Fast and reliable identification of microbial isolates is a fundamental goal of clinical microbiology. However, in the case of some fastidious gram-negative bacterial species, classical phenotype identification based on either metabolic, enzymatic, or serological methods is difficult, time-consuming, and/or inadequate. 16S or 23S ribosomal DNA (rDNA) bacterial sequencing will most often result in accurate speciation of isolates. Therefore, the objective of this study was to find a hypervariable rDNA stretch, flanked by strongly conserved regions, which is suitable for molecular species identification of members of the Neisseriaceae and Moraxellaceae. The inter- and intrageneric relationships were investigated using comparative sequence analysis of PCR-amplified partial 16S and 23S rDNAs from a total of 94 strains. When compared to the type species of the genera Acinetobacter, Moraxella, andNeisseria, an average of 30 polymorphic positions was observed within the partial 16S rDNA investigated (corresponding toEscherichia coli positions 54 to 510) for each species and an average of 11 polymorphic positions was observed within the 202 nucleotides of the 23S rDNA gene (positions 1400 to 1600).Neisseria macacae and Neisseria mucosa subsp.mucosa (ATCC 19696) had identical 16S and 23S rDNA sequences. Species clusters were heterogeneous in both genes in the case of Acinetobacter lwoffii, Moraxella lacunata, andN. mucosa. Neisseria meningitidis isolates failed to cluster only in the 23S rDNA subset. Our data showed that the 16S rDNA region is more suitable than the partial 23S rDNA for the molecular diagnosis of Neisseriaceae andMoraxellaceae and that a reference database should include more than one strain of each species. All sequence chromatograms and taxonomic and disease-related information are available as part of our ribosomal differentiation of medical microorganisms (RIDOM) web-based service (http://www.ridom.hygiene.uni-wuerzburg.de/ ). Users can submit a sequence and conduct a similarity search against the RIDOM reference database for microbial identification purposes.

Phylogenetic Analysis of Enterohemorrhagic Escherichia coli O157, Germany, 1987-2008

Clones of this organism persisted during the study period.

Ensuring backwards compatibility: traditional genotyping efforts in the era of whole genome sequencing.

When using next-generation whole genome sequencing (WGS), extraction of spa types from WGS data is essential for backwards compatibility with Sanger sequencing-based spa typing of methicillin-resistant Staphylococcus aureus (MRSA). We evaluated WGS-based spa typing with a 2×250 bp protocol in a diverse collection of 423 MRSA isolates using two pipelines that executed sequence quality-trimming and de novo assembly before spa typing. The SeqSphere(+) pipeline correctly typed 419 isolates (99.1%) whereas the CLCbio pipeline succeeded in 249 isolates (58.9%). In summary, WGS combined with an optimized de novo assembly enables nearly full compatibility with Sanger sequencing-based spa typing data.

Identification of Intermediate in Evolutionary Model of Enterohemorrhagic Escherichia coli O157

Single-nucleotide polymorphism typing found missing link between human strains in strain from deer.

Ridom TraceEdit: a DNA trace editor and viewer

Ridom TraceEdit is a cross-platform graphical DNA trace viewer and editor. TraceEdit displays the chromatogram files from Applied Biosystems automated sequencers and files in the Staden SCF format. Incorrect base calls can be edited and saved.

Harmonized Genome Wide Typing of Tubercle Bacilli Using a Web-Based Gene-By-Gene Nomenclature System

Background Global tuberculosis (TB) control is challenged by uncontrolled transmission of Mycobacterium tuberculosis complex (Mtbc) strains, esp. of multidrug (MDR) or extensively resistant (XDR) variants. Precise analysis of transmission networks is the basis to trace outbreak M/XDR clones and improve TB control. However, classical genotyping tools lack discriminatory power due to the high similarity of strains of particular successful lineages, e.g. Beijing or outbreak strains. This can be overcome by whole genome sequencing (WGS) approaches, but these are not yet standardized to facilitate larger investigations encompassing different laboratories or outbreak tracing across borders. Methods We established and improved a whole genome gene-by-gene multi locus sequence typing approach encompassing a stable set of core genome genes (cgMLST) and linked it to a web-based nomenclature server (cgMLST.org) facilitating assignment and storage of allele numbers. Findings We evaluated and refined a previously suggested cgMLST schema by using a reference strain set (n = 251) reflecting the global diversity of the Mtbc. A set of 2891 genes showed excellent performance with at least 97% of the genes reliably identified in strains of all Mtbc lineages and in discriminating outbreak strains. cgMLST allele numbers were automatically retrieved from and stored at cgMLST.org. Interpretation The refined cgMLST schema provides high resolution genome-based typing of clinical strains of all Mtbc lineages. Combined with a web-based nomenclature server, it facilitates rapid, high-resolution, and harmonized tracing of clinical Mtbc strains needed for prospective local and global surveillance.