Rafael Szczepanowski

Publications of Rafael Szczepanowski

• Ion Torrent PGM sequencing for genomic typing of Neisseria meningitidis for rapid determination of multiple layers of typing information.

  Authors: Ulrich Vogel, Rafael Szczepanowski, Heike Claus, Sebastian Jünemann, Karola Prior, Dag Harmsen

  Journal of clinical microbiology. 03/2012;

  Neisseria meningitidis causes invasive meningococcal disease in infants, toddlers and adolescents worldwide. DNA sequence based typing has become the standard for molecular epidemiology of theNeisseria meningitidis causes invasive meningococcal disease in infants, toddlers and adolescents worldwide. DNA sequence based typing has become the standard for molecular epidemiology of the organism including multilocus sequence typing, analysis of genetic determinants of antibiotic resistance, and sequence typing of vaccine antigens. However, PCR of multiple targets and consecutive Sanger sequencing provides logistic constraints to reference laboratories. Taking advantage of the recent development of benchtop next generation sequencers (NGS) and of BIGSdb, a database accommodating and analyzing genome sequence data, we therefore explored the feasibility and accuracy of Ion Torrent Personal Genome Machine™ (PGM™) sequencing for genomic typing of meningococci. Three strains from a previous meningococcus B community outbreak were selected to compare conventional typing results with data generated by semiconductor chip based sequencing. In addition, sequencing of the meningococcal type strain MC58 provided information about the general performance of the technology. The PGM™ technology generated sequence information for almost all target genes addressed. The results were 100% concordant with conventional typing results with no further editing necessary. In addition, the amount of typing information, i.e. nucleotides and target genes analyzed, could be substantially increased by the combined use of genome sequencing and BIGSdb compared to conventional methods. In a near future, affordable and fast benchtop-NGS machines like the PGM™ might enable reference laboratories to switch to genomic typing on a routine basis. This will reduce workload and rapidly provide information for laboratory surveillance, outbreak investigation, assessment of vaccine preventability and antibiotic resistance gene monitoring.

• Prospective genomic characterization of the German enterohemorrhagic Escherichia coli O104:H4 outbreak by rapid next generation sequencing technology.

  Authors: Alexander Mellmann, Dag Harmsen, Craig A Cummings, Emily B Zentz, Shana R Leopold, Alain Rico, Karola Prior, Rafael Szczepanowski, Yongmei Ji, Wenlan Zhang, Stephen F McLaughlin, John K Henkhaus, Benjamin Leopold, Martina Bielaszewska, Rita Prager, Pius M Brzoska, Richard L Moore, Simone Guenther, Jonathan M Rothberg, Helge Karch

  PloS one. 01/2011; 6(7):e22751.

  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 deathsAn 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.