[Assessment of available and currently applied typing methods including genome-based methods for zoonotic pathogens with a focus on Salmonella enterica]. / Bestandsaufnahme der verfügbaren und aktuell eingesetzten Typisierungsmethoden einschließlich genombasierter Verfahren von Zoonoseerregern am Beispiel von Salmonella enterica.

BACKGROUND: In recent years, whole genome sequencing (WGS) in combination with bioinformatic analyses has become state of the art in evaluating the pathogenicity/resistance potential and relatedness of bacteria. WGS analysis thus represents a central tool in the investigation of the resistance and virulence potential of pathogens, as well as their dissemination via outbreak clusters and transmission chains within the framework of molecular epidemiology. In order to gain an overview of the available genotypic and phenotypic methods used for pathogen typing of Salmonella and Shiga toxin-producing and enterohemorrhagic Escherichia coli (STEC/EHEC) in Germany at state and federal level, along with the availability of WGS-based typing and corresponding analytical methods, a survey of laboratories was conducted. METHODS: An electronic survey of laboratories working for public health protection and consumer health protection was conducted from February to June 2020. RESULTS AND CONCLUSION: The results of the survey showed that many of the participating laboratories provide a wide range of phenotypic and molecular methods. Molecular typing is most commonly used for species identification of Salmonella. In many cases, WGS-based methods have already been established at federal and state institutions or are in the process of being established. The Illumina sequencing technology is the most widely used technology. The survey confirms the importance of molecular biology and whole genome typing technologies for laboratories in the diagnosis of bacterial zoonotic pathogens.

Comparison of methods for economic and efficient tick and Borrelia DNA purification.

DNA purification is a critical step in the processing of samples for molecular diagnosis and/ or identification of pathogens via polymerase chain reaction (PCR). Especially when handling vectors like ticks, purifying the DNA always poses a challenge. In this study, we compared factors that may have an influence on DNA extraction namely commercially available DNA extraction kits vs alkaline hydrolysis for DNA extraction. The methods were applied to questing Ixodes (I.) ricinus ticks and Borrelia cultures of defined cell concentrations. A total of 69 questing I. ricinus ticks were collected. From 34 ticks, total DNA was extracted using a commercial DNA extraction kit. Thirty-five ticks were treated with 1.25% ammonium hydroxide (NH4OH). Six ticks from each batch were placed in 70% ethanol (EtOH) for one week prior to DNA extraction to see the effect of EtOH preservation on total DNA yield. DNA yield was estimated in field-collected ticks using conventional PCR targeting the Ixodes Cytochrome C oxidase (coi) gene and in cultured Borrelia isolates using quantitative real-time PCR (qPCR) targeting the FlaB encoding gene of Borrelia. Column DNA extraction yielded slightly better results than NH4OH treatment when tested in a PCR targeting a tick-specific coi gene (96% PCR-positive vs 86% PCR-positive results, respectively). EtOH preservation had a slightly negative effect on DNA yield and - again - slightly stronger PCR products were observed by commercial kit extraction. A Shapiro-Wilk test conducted revealed a significance-level of 90% for both the methods, indicating a normal distribution of the values generated by BioNumerics quantification. A two-sided t-test conducted revealed a significant (p < 0.01) mean difference between the methods. Similarly, qPCR on cultured specimen DNA of Borrelia burgdorferi sensu stricto (B. burgdorferi s.s.) (B31) with different concentrations revealed a better yield for kit extraction in comparison to NH4OH treatment; a difference of approximately 3 Ct-values was ascertained between extraction methods. A one-sided t-test showed a significant difference between the methods at lower concentration of Borrelia i.e. better extraction with a commercial kit at lower borrelial DNA concentration, while at higher concentration (106 cells per ml) the difference was not significant.

Whole genome sequencing identifies influenza A H3N2 transmission and offers superior resolution to classical typing methods.

OBJECTIVES: Influenza with its annual epidemic waves is a major cause of morbidity and mortality worldwide. However, only little whole genome data are available regarding the molecular epidemiology promoting our understanding of viral spread in human populations. METHODS: We implemented a RT-PCR strategy starting from patient material to generate influenza A whole genome sequences for molecular epidemiological surveillance. Samples were obtained within the Bavarian Influenza Sentinel. The complete influenza virus genome was amplified by a one-tube multiplex RT-PCR and sequenced on an Illumina MiSeq. RESULTS: We report whole genomic sequences for 50 influenza A H3N2 viruses, which was the predominating virus in the season 2014/15, directly from patient specimens. The dataset included random samples from Bavaria (Germany) throughout the influenza season and samples from three suspected transmission clusters. We identified the outbreak samples based on sequence identity. Whole genome sequencing (WGS) was superior in resolution compared to analysis of single segments or partial segment analysis. Additionally, we detected manifestation of substantial amounts of viral quasispecies in several patients, carrying mutations varying from the dominant virus in each patient. CONCLUSION: Our rapid whole genome sequencing approach for influenza A virus shows that WGS can effectively be used to detect and understand outbreaks in large communities. Additionally, the genomic data provide in-depth details about the circulating virus within one season.

Evaluation of six commercial DNA extraction kits for recovery of Burkholderia pseudomallei DNA.

Six commercially available DNA extraction kits, as well as thermal lysis and proteinase K DNA extraction were evaluated regarding bacterial inactivation, DNA yield and purity, and their use in a Burkholderia pseudomallei real-time PCR. While all methods successfully inactivated the bacteria, by measuring DNA purity and the level of detection by real-time PCR, the proteinase K method was the most sensitive.