A high fidelity approach to assembling the complex Borrelia genome.

BACKGROUND: Bacteria of the Borrelia burgdorferi sensu lato (s.l.) complex can cause Lyme borreliosis. Different B. burgdorferi s.l. genospecies vary in their host and vector associations and human pathogenicity but the genetic basis for these adaptations is unresolved and requires completed and reliable genomes for comparative analyses. The de novo assembly of a complete Borrelia genome is challenging due to the high levels of complexity, represented by a high number of circular and linear plasmids that are dynamic, showing mosaic structure and sequence homology. Previous work demonstrated that even advanced approaches, such as a combination of short-read and long-read data, might lead to incomplete plasmid reconstruction. Here, using recently developed high-fidelity (HiFi) PacBio sequencing, we explored strategies to obtain gap-free, complete and high quality Borrelia genome assemblies. Optimizing genome assembly, quality control and refinement steps, we critically appraised existing techniques to create a workflow that lead to improved genome reconstruction. RESULTS: Despite the latest available technologies, stand-alone sequencing and assembly methods are insufficient for the generation of complete and high quality Borrelia genome assemblies. We developed a workflow pipeline for the de novo genome assembly for Borrelia using several types of sequence data and incorporating multiple assemblers to recover the complete genome including both circular and linear plasmid sequences. CONCLUSION: Our study demonstrates that, with HiFi data and an ensemble reconstruction pipeline with refinement steps, chromosomal and plasmid sequences can be fully resolved, even for complex genomes such as Borrelia. The presented pipeline may be of interest for the assembly of further complex microbial genomes.

Analysis of seven SARS-CoV-2 rapid antigen tests in detecting omicron (B.1.1.529) versus delta (B.1.617.2) using cell culture supernatants and clinical specimens.

PURPOSE: Omicron is rapidly spreading as a new SARS-CoV-2 variant of concern (VOC). The question whether this new variant has an impact on SARS-CoV-2 rapid antigen test (RAT) performance is of utmost importance. To obtain an initial estimate regarding differences of RATs in detecting omicron and delta, seven commonly used SARS-CoV-2 RATs from different manufacturers were analysed using cell culture supernatants and clinical specimens. METHODS: For this purpose, cell culture-expanded omicron and delta preparations were serially diluted in Dulbecco's modified Eagle's Medium (DMEM) and the Limit of Detection (LoD) for both VOCs was determined. Additionally, clinical specimens stored in viral transport media or saline (n = 51) were investigated to complement in vitro results with cell culture supernatants. Ct values and RNA concentrations were determined via quantitative reverse transcription polymerase chain reaction (RT-qPCR). RESULTS: The in vitro determination of the LoD showed no obvious differences in detection of omicron and delta for the RATs examined. The LoD in this study was at a dilution level of 1:1,000 (corresponding to 3.0-5.6 × 106 RNA copies/mL) for tests I-V and at a dilution level of 1:100 (corresponding to 3.7-4.9 × 107 RNA copies/mL) for tests VI and VII. Based on clinical specimens, no obvious differences were observed between RAT positivity rates when comparing omicron to delta in this study setting. Overall positivity rates varied between manufacturers with 30-81% for omicron and 42-71% for delta. Test VII was only conducted in vitro with cell culture supernatants for feasibility reasons. In the range of Ct < 23, positivity rates were 50-100% for omicron and 67-93% for delta. CONCLUSION: In this study, RATs from various manufacturers were investigated, which displayed no obvious differences in terms of analytical LoD in vitro and RAT positivity rates based on clinical samples comparing the VOCs omicron and delta. However, differences between tests produced by various manufacturers were detected. In terms of clinical samples, a focus of this study was on specimens with high virus concentrations. Further systematic, clinical and laboratory studies utilizing large datasets are urgently needed to confirm reliable performance in terms of sensitivity and specificity for all individual RATs and SARS-CoV-2 variants.

[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.

Replication kinetics and infectivity of SARS-CoV-2 variants of concern in common cell culture models.

BACKGROUND: During the ongoing Covid-19 pandemic caused by the emerging virus SARS-CoV-2, research in the field of coronaviruses has expanded tremendously. The genome of SARS-CoV-2 has rapidly acquired numerous mutations, giving rise to several Variants of Concern (VOCs) with altered epidemiological, immunological, and pathogenic properties. METHODS: As cell culture models are important tools to study viruses, we investigated replication kinetics and infectivity of SARS-CoV-2 in the African Green Monkey-derived Vero E6 kidney cell line and the two human cell lines Caco-2, a colon epithelial carcinoma cell line, and the airway epithelial carcinoma cell line Calu-3. We assessed viral RNA copy numbers and infectivity of viral particles in cell culture supernatants at different time points ranging from 2 to 96 h post-infection. RESULTS: We here describe a systematic comparison of growth kinetics of the five SARS-CoV-2 VOCs Alpha/B.1.1.7, Beta/B.1.351, Gamma/P.1, Delta/B.1.617.2, and Omicron/B.1.1.529 and a non-VOC/B.1.1 strain on three different cell lines to provide profound information on the differential behaviour of VOCs in different cell lines for researchers worldwide. We show distinct differences in viral replication kinetics of the SARS-CoV-2 non-VOC and five VOCs on the three cell culture models Vero E6, Caco-2, and Calu-3. CONCLUSION: This is the first systematic comparison of all SARS-CoV-2 VOCs on three different cell culture models. This data provides support for researchers worldwide in their experimental design for work on SARS-CoV-2. It is recommended to perform virus isolation and propagation on Vero E6 while infection studies or drug screening and antibody-based assays should rather be conducted on the human cell lines Caco-2 and Calu-3.

Molecular SARS-CoV-2 surveillance in Bavaria shows no Omicron transmission before the end of November 2021.

BACKGROUND: Five SARS-CoV-2 variants are currently considered as variants of concern (VOC). Omicron was declared a VOC at the end of November 2021. Based on different diagnostic methods, the occurrence of Omicron was reported by 52 countries worldwide on December 7 2021. First notified by South Africa with alarming reports on increasing infection rates, this new variant was soon suspected to replace the currently pre-dominating Delta variant leading to further infection waves worldwide. METHODS: Using VOC PCR screening and Next Generation Sequencing (NGS) analysis of selected samples, we investigated the circulation of Omicron in the German federal state Bavaria. For this, we analyzed SARS-CoV-2 surveillance data from our laboratory generated from calendar week (CW) 01 to 49/2021. RESULTS: So far, we have detected 69 Omicron cases in our laboratory from CW 47-49/2021 using RT-qPCR followed by melting curve analysis. The first 16 cases were analyzed by NGS and all were confirmed as Omicron. CONCLUSION: Our data strongly support no circulation of the new Omicron variant before CW 47/2021.

Outbreak of imported diphtheria with Corynebacterium diphtheriae among migrants arriving in Germany, 2022.

From July 2022, cases of imported diphtheria with toxigenic Corynebacterium diphtheriae remarkably increased among migrants arriving in Germany. Up to 30 September 2022, 44 cases have been reported to the national public health institute, all laboratory-confirmed, male, and mainly coming from Syria (n = 21) and Afghanistan (n = 17). Phylogeny and available journey information indicate that most cases (n = 19) were infected along the Balkan route. Active case finding, increased laboratory preparedness and epicentre localisation in countries along this route are important.

Prolonged SARS-CoV-2 RNA Shedding from Therapy Cat after Cluster Outbreak in Retirement Home.

We report a therapy cat in a nursing home in Germany infected with severe acute respiratory syndrome coronavirus 2 during a cluster outbreak in the home residents. Although we confirmed prolonged presence of virus RNA in the asymptomatic cat, genome sequencing showed no further role of the cat in human infections on site.

Reciprocal circulation pattern of SARS-CoV-2 and influenza viruses during the influenza seasons 2019/2020 and 2020/2021 in the Bavarian Influenza Sentinel (Germany).

The corona virus disease-2019 (COVID-19) pandemic began in Wuhan, China, and quickly spread around the world. The pandemic overlapped with two consecutive influenza seasons (2019/2020 and 2020/2021). This provided the opportunity to study community circulation of influenza viruses and severe acute respiratory syndrome-coronavirus-2 (SARS-CoV-2) in outpatients with acute respiratory infections during these two seasons within the Bavarian Influenza Sentinel (BIS) in Bavaria, Germany. From September to March, oropharyngeal swabs collected at BIS were analysed for influenza viruses and SARS-CoV-2 by real-time polymerase chain reaction. In BIS 2019/2020, 1376 swabs were tested for influenza viruses. The average positive rate was 37.6%, with a maximum of over 60% (in January). The predominant influenza viruses were Influenza A(H1N1)pdm09 (n = 202), Influenza A(H3N2) (n = 144) and Influenza B Victoria lineage (n = 129). In all, 610 of these BIS swabs contained sufficient material to retrospectively test for SARS-CoV-2. SARS-CoV-2 RNA was not detectable in any of these swabs. In BIS 2020/2021, 470 swabs were tested for influenza viruses and 457 for SARS-CoV-2. Only three swabs (0.6%) were positive for Influenza, while SARS-CoV-2 was found in 30 swabs (6.6%). We showed that no circulation of SARS-CoV-2 was detectable in BIS during the 2019/2020 influenza season, while virtually no influenza viruses were found in BIS 2020/2021 during the COVID-19 pandemic.

Corynebacterium rouxii, a recently described member of the C. diphtheriae group isolated from three dogs with ulcerative skin lesions.

Corynebacterium (C.) diphtheriae is one of the two etiological pathogens for human diphtheria with significant morbidity and mortality. Recently, members of its biovar Belfanti have been described as two novel species, C. belfantii and C. rouxii. The most important virulence factor and also the premise to cause diphtheria is the isolate's capacity to encode and express the diphtheria toxin (DT). In contrast to C. ulcerans, which represents a potentially zoonotic pathogen, C. diphtheriae (incl. the novel deduced species) has almost exclusively been found to comprise a human pathogen. We here report three rare cases of C. rouxii isolation from dogs suffering from disseminated poly-bacterial exsudative to purulent dermatitis and a traumatic labial defect, respectively. The isolates were identified as C. diphtheriae based on commercial biochemistry and matrix-assisted laser desorption/ionisation-time of flight mass spectrometry (MALDI-TOF MS) analysis. However, recently described specific spectral peaks were highly similar to spectra of C. rouxii, which was confirmed by whole genome sequencing. Further investigations of the dog isolates for the presence of DT by tox gene qPCR revealed negative results. The findings from this study point out that skin infections in companion animals can be colonized by uncommon and so believed human specific pathogens, thereby resembling the clinical signs of cutaneous diphtheria.

Vaccine breakthrough infection and onward transmission of SARS-CoV-2 Beta (B.1.351) variant, Bavaria, Germany, February to March 2021.

A breakthrough infection occurred in a fully Comirnaty (BNT162b2) vaccinated healthcare worker with high levels of neutralising antibodies with the SARS-CoV-2 B.1.351 (Beta) variant in February 2021. The infection was subsequently transmitted to their unvaccinated spouse. Sequencing revealed an identical virus in both spouses, with a match of all nine single nucleotide polymorphisms typical for B.1.351. To the best of our knowledge, no transmission of any variant of SARS-CoV-2 from a fully vaccinated person has been described before.

Detection of the new SARS-CoV-2 variants of concern B.1.1.7 and B.1.351 in five SARS-CoV-2 rapid antigen tests (RATs), Germany, March 2021.

SARS-CoV-2 variants of concern (VOC) should not escape molecular surveillance. We investigated if SARS-CoV-2 rapid antigen tests (RATs) could detect B.1.1.7 and B.1.351 VOCs in certain laboratory conditions. Infectious cell culture supernatants containing B.1.1.7, B.1.351 or non-VOC SARS-CoV-2 were respectively diluted both in DMEM and saliva. Dilutions were analysed with Roche, Siemens, Abbott, nal von minden and RapiGEN RATs. While further studies with appropriate real-life clinical samples are warranted, all RATs detected B.1.1.7 and B.1.351, generally comparable to non-VOC strain.

Toxigenic Corynebacterium diphtheriae-Associated Genital Ulceration.

In October 2016, an adolescent boy sought care for acute genital ulceration in Cologne, Germany. We presumed a sexually transmitted infection, but initial diagnostic procedures yielded negative results. He was hospitalized because swab samples from the lesion grew toxigenic Corynebacterium diphtheriae, leading to the diagnosis of possibly sexually transmitted cutaneous diphtheria.

Corynebacterium silvaticum sp. nov., a unique group of NTTB corynebacteria in wild boar and roe deer.

A total of 34 Corynebacterium sp. strains were isolated from caseous lymph node abscesses of wild boar and roe deer in different regions of Germany. They showed slow growth on Columbia sheep blood agar and sparse growth on Hoyle's tellurite agar. Cellular fatty acid analysis allocated them in the C. diphtheriae group of genus Corynebacterium. MALDI-TOF MS using specific database extensions and rpoB sequencing resulted in classification as C. ulcerans. Their quinone system is similar to C. ulcerans, with major menaquinone MK-8(H2). Their complex polar lipid profile includes major lipids phosphatidylinositol, phosphatidylinositol-mannoside, diphosphatidylglycerol, but also unidentified glycolipids, distinguishing them clearly from C. ulcerans. They ferment glucose, ribose and maltose (like C. ulcerans), but do not utilise d-xylose, mannitol, lactose, sucrose and glycogen (like C. pseudotuberculosis). They showed activity of catalase, urease and phospholipase D, but variable results for alkaline phosphatase and alpha-glucosidase. All were non-toxigenic, tox gene bearing and susceptible to clindamycin, penicillin and erythromycin. In 16SrRNA gene and RpoB protein phylogenies the strains formed distinct brancheswith C. ulcerans as nearest relative.Whole genome sequencing revealed the unique sequence type 578, a distinctbranch in pangenomic core genome MLST, average nucleotide identities <91%, enhancedgenome sizes (2.55 Mbp) and G/C content (54.4 mol%) compared to related species.These results suggest that the strains represent a novel species, for which wepropose the name Corynebactriumsilvaticum sp. nov., based on their first isolation from forest-dwellinggame animals. The type strain isKL0182T (= CVUAS 4292T = DSM 109166T = LMG 31313T= CIP 111 672T).

Rapid establishment of laboratory diagnostics for the novel coronavirus SARS-CoV-2 in Bavaria, Germany, February 2020.

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.

NGS-based phylogeny of diphtheria-related pathogenicity factors in different Corynebacterium spp. implies species-specific virulence transmission.

BACKGROUND: 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. RESULTS: 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. CONCLUSION: 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.

Genetic diversity and delineation of Salmonella Agona outbreak strains by next generation sequencing, Bavaria, Germany, 1993 to 2018.

BackgroundIn 2017, a food-borne Salmonella Agona outbreak caused by infant milk products from a French supplier occurred in Europe. Simultaneously, S. Agona was detected in animal feed samples in Bavaria.AimUsing next generation sequencing (NGS) and three data analysis methods, this study's objectives were to verify clonality of the Bavarian feed strains, rule out their connection to the outbreak, explore the genetic diversity of Bavarian S. Agona isolates from 1993 to 2018 and compare the analysis approaches employed, for practicality and ability to delineate outbreaks caused by the genetically monomorphic Agona serovar.MethodsIn this observational retrospective study, three 2017 Bavarian feed isolates were compared to a French outbreak isolate and 48 S. Agona isolates from our strain collections. The later included human, food, feed, veterinary and environmental isolates, of which 28 were epidemiologically outbreak related. All isolates were subjected to NGS and analysed by: (i) a publicly available species-specific core genome multilocus sequence typing (cgMLST) scheme, (ii) single nucleotide polymorphism phylogeny and (iii) an in-house serovar-specific cgMLST scheme. Using additional international S. Agona outbreak NGS data, the cluster resolution capacity of the two cgMLST schemes was assessed.ResultsWe could prove clonality of the feed isolates and exclude their relation to the French outbreak. All approaches confirmed former Bavarian epidemiological clusters.ConclusionEven for S. Agona, species-level cgMLST can produce reasonable resolution, being standardisable by public health laboratories. For single samples or homogeneous sample sets, higher resolution by serovar-specific cgMLST or SNP genotyping can facilitate outbreak investigations.

Whole genome sequencing suggests transmission of Corynebacterium diphtheriae-caused cutaneous diphtheria in two siblings, Germany, 2018.

In September 2018, a child who had returned from Somalia to Germany presented with cutaneous diphtheria by toxigenic Corynebacterium diphtheriae biovar mitis. The child's sibling had superinfected insect bites harbouring also toxigenic C. diphtheriae. Next generation sequencing (NGS) revealed the same strain in both patients suggesting very recent human-to-human transmission. Epidemiological and NGS data suggest that the two cutaneous diphtheria cases constitute the first outbreak by toxigenic C. diphtheriae in Germany since the 1980s.

Geographically Diverse Clusters of Nontoxigenic Corynebacterium diphtheriae Infection, Germany, 2016-2017.

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.

A de novo-designed antimicrobial peptide with activity against multiresistant Staphylococcus aureus acting on RsbW kinase.

Antimicrobial peptides are a promising complement to common antibiotics, development of resistance to which is a growing problem. Here we present a de novo-designed peptide, SP1-1 (RKKRLKLLKRLL-NH2), with antimicrobial activity against multiresistant Staphylococcus aureus (minimal inhibitory concentration: 6.25 µM). Elucidation of the mode of action of this peptide revealed a strong interaction with RsbW kinase (Kd: 6.01±2.73 nM), a serine kinase negatively regulating the activity of the transcription factor σB (SigB). SP1-1 binding and functional modulation of RsbW were shown in vitro by a combination of biochemical, molecular, and biophysical methods, which were further genetically evidenced in vivo by analysis of S. aureus ΔsigB deletion mutants. Intracellular localization of the peptide was demonstrated using nanometer-scaled secondary ion mass spectrometry. Moreover, microarray analysis revealed that transcription of numerous genes, involved in cell wall and amino acid metabolism, transport mechanisms, virulence, and pigmentation, is affected. Interestingly, several WalR binding motif containing genes are induced by SP1-1. In sum, the designed peptide SP1-1 seems to have multiple modes of action, including inhibition of a kinase, and therefore might contribute to the development of new antibacterial compounds, giving bacterial kinase inhibition a closer inspection.