The need for timely establishment of diagnostic assays arose when Germany was confronted with the first travel-associated outbreak of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) in ...Europe. We describe our laboratory experiences during a large contact tracing investigation, comparing previously published real-time RT-PCR assays in different PCR systems and a commercial kit. We found that assay performance using the same primers and probes with different PCR systems varied and the commercial kit performed well.
From 2016 through the middle of 2017, the German Consiliary Laboratory on Diphtheria noted an increase in nontoxigenic Corynebacterium diphtheriae isolates submitted from cities in northern Germany. ...Many patients for whom epidemiologic data were available were homeless, alcohol or drug abusers, or both. After performing routine diagnostics and multilocus sequence typing (MLST), we analyzed isolates of sequence type (ST) 8 and previously submitted isolates by whole-genome sequencing. Results were analyzed for phylogenetic relationship by core genome MLST (cg-MLST) and whole-genome single-nucleotide polymorphism profiles. Next-generation sequencing-based cg-MLST revealed several outbreak clusters caused by ST8; the geographic focus was in the metropolitan areas of Hamburg and Berlin. To achieve enhanced analytical depth, we used additional cg-MLST target genes and genome-wide single-nucleotide polymorphisms. We identified patient characteristics and detected transmission events, providing evidence that nontoxigenic C. diphtheriae infection is a potential public health threat in industrialized countries.
Shiga toxin-producing
(STEC) and the STEC subgroup enterohemorrhagic
cause intestinal infections with symptoms ranging from watery diarrhea to hemolytic-uremic syndrome (HUS). A key tool for the ...epidemiological differentiation of STEC is serotyping. The serotype in combination with the main virulence determinants gives important insight into the virulence potential of a strain. However, a large fraction of STEC strains found in human disease, including strains causing HUS, belongs to less frequently detected STEC serovars or their O/H antigens are unknown or even untypeable. Recent implementation of whole-genome sequence (WGS) analysis, in principle, allows the deduction of serovar and virulence gene information. Therefore, here we compared classical serovar and PCR-based virulence marker detection with WGS-based methods for 232 STEC strains, focusing on less frequently detected STEC serovars and nontypeable strains. We found that the results of WGS-based extraction showed a very high degree of overlap with those of the more classical methods. Specifically, the rate of concordance was 97% for O antigens (OAGs) and 99% for H antigens (HAGs) of typeable strains and >99% for
,
, or
for all strains. Ninety-eight percent of nontypeable OAGs and 100% of nontypeable HAGs were defined by WGS analysis. In addition, the novel methods enabled a more complete analysis of strains causing severe clinical symptoms and the description of four novel STEC OAG loci. In conclusion, WGS is a promising tool for gaining serovar and virulence gene information, especially from a public health perspective.
Diphtheria toxin (DT) is produced by toxigenic strains of the human pathogen Corynebacterium diphtheriae as well as zoonotic C. ulcerans and C. pseudotuberculosis. Toxigenic strains may cause severe ...respiratory diphtheria, myocarditis, neurological damage or cutaneous diphtheria. The DT encoding tox gene is located in a mobile genomic region and tox variability between C. diphtheriae and C. ulcerans has been postulated based on sequences of a few isolates. In contrast, species-specific sequence analysis of the diphtheria toxin repressor gene (dtxR), occurring both in toxigenic and non-toxigenic Corynebacterium species, has not been done yet. We used whole genome sequencing data from 91 toxigenic and 46 non-toxigenic isolates of different pathogenic Corynebacterium species of animal or human origin to elucidate differences in extracted DT, DtxR and tox-surrounding genetic elements by a phylogenetic analysis in a large sample set.
Sequences of both DT and DtxR, extracted from whole genome sequencing data, could be classified in four distinct, nearly species-specific clades, corresponding to C. diphtheriae, C. pseudotuberculosis, C. ulcerans and atypical C. ulcerans from a non-toxigenic toxin gene-bearing wildlife cluster. Average amino acid similarities were above 99% for DT and DtxR within the four groups, but lower between them. For DT, subgroups below species level could be identified, correlating with different tox-comprising mobile genetic elements. In most C. diphtheriae, tox genes were located within known prophages. In contrast, in C. ulcerans diverse tox-including mobile elements could be identified: either prophages differing from C. diphtheriae prophages or an alternative pathogenicity island (PAI) described previously. One isolate showed a different, shorter tox-comprising putative PAI. Beyond the tox-overlapping elements, most isolates harbored a variety of additional prophages.
Our NGS data from 137 isolates indicate the existence of different genetic backgrounds of DT-mediated pathogenicity in different Corynebacterium species and evolution of once acquired pathogenicity features with the strains. Different groups of pathogenicity-related elements within C. ulcerans imply that tox transmission pathways between isolates may differ in the zoonotic species and contribute to their emerging pathogenic potential.
Matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF MS) has recently emerged as a powerful tool for the routine identification of clinical isolates. MALDI-TOF MS ...based identification of bacteria has been shown to be more rapid, accurate and cost-efficient than conventional phenotypic techniques or molecular methods. Rapid and reliable identification of food-associated bacteria is also of crucial importance for food processing and product quality. This review is concerned with the applicability of MALDI-TOF MS for routine identification of foodborne bacteria taking the specific requirements of food microbiological laboratories and the food industry into account. The current state of knowledge including recent findings and new approaches are discussed.
In this study mass spectrometry was used for evaluating extracted leptospiral protein samples and results were compared with molecular typing methods. For this, an extraction protocol for Leptospira ...spp. was independently established in two separate laboratories. Reference spectra were created with 28 leptospiral strains, including pathogenic, non-pathogenic and intermediate strains. This set of spectra was then evaluated on the basis of measurements with well-defined, cultured leptospiral strains and with 16 field isolates of veterinary or human origin. To verify discriminating peaks for the applied pathogenic strains, statistical analysis of the protein spectra was performed using the software tool ClinProTools. In addition, a dendrogram of the reference spectra was compared with phylogenetic trees of the 16S rRNA gene sequences and multi locus sequence typing (MLST) analysis.
Defined and reproducible protein spectra using MALDI-TOF MS were obtained for all leptospiral strains. Evaluation of the newly-built reference spectra database allowed reproducible identification at the species level for the defined leptospiral strains and the field isolates. Statistical analysis of three pathogenic genomospecies revealed peak differences at the species level and for certain serovars analyzed in this study. Specific peak patterns were reproducibly detected for the serovars Tarassovi, Saxkoebing, Pomona, Copenhageni, Australis, Icterohaemorrhagiae and Grippotyphosa. Analysis of the dendrograms of the MLST data, the 16S rRNA sequencing, and the MALDI-TOF MS reference spectra showed comparable clustering.
MALDI-TOF MS analysis is a fast and reliable method for species identification, although Leptospira organisms need to be produced in a time-consuming culture process. All leptospiral strains were identified, at least at the species level, using our described extraction protocol. Statistical analysis of the three genomospecies L. borgpetersenii, L. interrogans and L. kirschneri revealed distinctive, reproducible differentiating peaks for seven leptospiral strains which represent seven serovars. Results obtained by MALDI-TOF MS were confirmed by MLST and 16S rRNA gene sequencing.
Toxigenic Corynebacterium ulcerans can cause a diphtheria-like illness in humans and have been found in domestic animals, which were suspected to serve as reservoirs for a zoonotic transmission. ...Additionally, toxigenic C. ulcerans were reported to take over the leading role in causing diphtheria in the last years in many industrialized countries.
To gain deeper insights into the tox gene locus and to understand the transmission pathway in detail, we analyzed nine isolates derived from human patients and their domestic animals applying next generation sequencing and comparative genomics.
We provide molecular evidence for zoonotic transmission of C. ulcerans in four cases and demonstrate the superior resolution of next generation sequencing compared to multi-locus sequence typing for epidemiologic research. Additionally, we provide evidence that the virulence of C. ulcerans can change rapidly by acquisition of novel virulence genes. This mechanism is exemplified by an isolate which acquired a prophage not present in the corresponding isolate from the domestic animal. This prophage contains a putative novel virulence factor, which shares high identity with the RhuM virulence factor from Salmonella enterica but which is unknown in Corynebacteria so far. Furthermore, we identified a putative pathogenicity island for C. ulcerans bearing a diphtheria toxin gene.
The novel putative diphtheria toxin pathogenicity island could provide a new and alternative pathway for Corynebacteria to acquire a functional diphtheria toxin-encoding gene by horizontal gene transfer, distinct from the previously well characterized phage infection model. The novel transmission pathway might explain the unexpectedly high number of toxigenic C. ulcerans.
Severe necrotizing fasciitis was diagnosed in a 53-year-old man in Germany in 2012. Toxigenic Corynebacterium ulcerans was grown from a wound swab sample. One of the patient's 2 dogs was found to ...harbor a toxigenic C. ulcerans strain. Results of next generation sequencing of both isolates supported recent zoonotic transmission of this bacterial pathogen.
Toxigenic Corynebacterium ulcerans may cause both respiratory and cutaneous diphtheria in humans. As a zoonotic emerging pathogen it has been isolated from a wide variety of animals living in ...captivity, such as livestock, pet, zoo and research animals and additionally in a large number of different wild animals. Here we report the isolation of tox-positive C. ulcerans in four hedgehogs with cutaneous diphtheria and pneumonia, respectively.
Tularemia is a zoonotic disease caused by Francisella tularensis that has been found in many different vertebrates. In Germany most human infections are caused by contact with infected European brown ...hares (Lepus europaeus). The aim of this study was to elucidate the epidemiology of tularemia in hares using phenotypic and genotypic characteristics of F. tularensis.
Cultivation of F. tularensis subsp. holarctica bacteria from organ material was successful in 31 of 52 hares that had a positive PCR result targeting the Ft-M19 locus. 17 isolates were sensitive to erythromycin and 14 were resistant. Analysis of VNTR loci (Ft-M3, Ft-M6 and Ft-M24), INDELs (Ftind33, Ftind38, Ftind49, RD23) and SNPs (B.17, B.18, B.19, and B.20) was shown to be useful to investigate the genetic relatedness of Francisella strains in this set of strains. The 14 erythromycin resistant isolates were assigned to clade B.I, and 16 erythromycin sensitive isolates to clade B.IV and one isolate was found to belong to clade B.II. MALDI-TOF mass spectrometry (MS) was useful to discriminate strains to the subspecies level.
F. tularensis seems to be a re-emerging pathogen in Germany. The pathogen can easily be identified using PCR assays. Isolates can also be identified within one hour using MALDI-TOF MS in laboratories where specific PCR assays are not established. Further analysis of strains requires genotyping tools. The results from this study indicate a geographical segregation of the phylogenetic clade B.I and B.IV, where B.I strains localize primarily within eastern Germany and B.IV strains within western Germany. This phylogeographical pattern coincides with the distribution of biovar I (erythromycin sensitive) and biovar II (erythromycin resistance) strains. When time and costs are limiting parameters small numbers of isolates can be analysed using PCR assays combined with DNA sequencing with a focus on genetic loci that are most likely discriminatory among strains found in a specific area. In perspective, whole genome data will have to be investigated especially when terrorist attack strains need to be tracked to their genetic and geographical sources.
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