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Analysis of 16S rRNA (rRNA) genes provides a central means of taxonomic classification of bacterial species. Based on presumed sequence identity among species of the Bacillus cereus sensu lato group, the 16S rRNA genes of B. anthracis have been considered unsuitable for diagnosis of the anthrax pathogen. With the recent identification of a single nucleotide polymorphism in some 16S rRNA gene copies, specific identification of B. anthracis becomes feasible. Here, we designed and evaluated a set of in situ, in vitro, and in silico assays to assess the unknown 16S state of B. anthracis from different perspectives. Using a combination of digital PCR, fluorescence in situ hybridization, long-read genome sequencing, and bioinformatics, we were able to detect and quantify a unique 16S rRNA gene allele of B. anthracis (16S-BA-allele). This allele was found in all available B. anthracis genomes and may facilitate differentiation of the pathogen from any close relative. Bioinformatics analysis of 959 B. anthracis SRA data sets inferred that abundances and genomic arrangements of the 16S-BA-allele and the entire rRNA operon copy numbers differ considerably between strains. Expression ratios of 16S-BA-alleles were proportional to the respective genomic allele copy numbers. The findings and experimental tools presented here provide detailed insights into the intra- and intergenomic diversity of 16S rRNA genes and may pave the way for improved identification of B. anthracis and other pathogens with diverse rRNA operons. IMPORTANCE For severe infectious diseases, precise pathogen detection is crucial for antibiotic therapy and patient survival. Identification of Bacillus anthracis, the causative agent of the zoonosis anthrax, can be challenging when querying specific nucleotide sequences such as in small subunit rRNA (16S rRNA) genes, which are commonly used for typing of bacteria. This study analyzed on a broad genomic scale a cryptic and hitherto underappreciated allelic variant of the bacterium’s 16S rRNA genes and their transcripts using a set of in situ, in vitro, and in silico assays and found significant intra- and intergenomic heterogeneity in the distribution of the allele and overall rRNA operon copy numbers. This allelic variation was uniquely species specific, which enabled sensitive pathogen detection on both DNA and transcript levels. The methodology used here is likely also applicable to other pathogens that are otherwise difficult to discriminate from their less harmful relatives.

In July 2018, brucellosis was diagnosed in a German patient without a travel history to regions endemic for Brucella. Microbiological analysis, including whole-genome sequencing, revealed Brucella suis biovar 1 as the etiologic agent. Core-genome-based multilocus sequence-typing analysis placed the isolate in close proximity to strains originating from Argentina. Notably, despite a strong IgM response, the patient did not develop Brucella-specific IgG antibodies during infection. Here, we describe the clinical course of infection, the extensive epidemiological investigations, and discuss possible routes of transmission.

Cases of pox-like lesions in horses and donkeys have been associated with poxviruses belonging to different genera of the family Poxviridae. These include the orthopoxviruses vaccinia virus (VACV), horsepoxvirus (HPXV) and cowpoxvirus (CPXV), as well as a potentially novel parapoxvirus and molluscum contagiosum virus (MOCV). However, with the exception of VACV, HPXV and CPXV, the genomic characterization of the causative agents remains largely elusive with only single short genome fragments available. Here we present the first full-length genome sequence of an equine molluscum contagiosum-like virus (EMCLV) directly determined from skin biopsies of a horse with generalized papular dermatitis. Histopathological analysis of the lesions revealed severe epidermal hyperplasia with numerous eosinophilic inclusion bodies within keratinocytes. Virions were detected in the lesions in embedded tissue by transmission electron microscopy. The genome sequence determined by next- and third-generation sequencing comprises 166 843 nt with inverted terminal repeats (ITRs) of 3473 nt. Overall, 20 of the predicted 159 ORFs have no equivalents in other poxviruses. Intriguingly, two of these ORFs were identified to encode homologues of mammalian proteins involved in immune signalling pathways, namely secreted and transmembrane protein 1 (SECTM1) and insulin growth factor-like family receptor 1 (IGFLR1), that were not described in any virus family so far. Phylogenetic analysis with all relevant representatives of the Poxviridae suggests that EMCLV should be nominated as a new species within the genus Molluscipoxvirus.

We are currently facing a pandemic of COVID-19, caused by a spillover from an animal-originating coronavirus to humans occuring in the Wuhan region, China, in December 2019. From China the virus has spread to 188 countries and regions worldwide, reaching the Sahel region on the 2nd of March 2020. Since whole genome sequencing (WGS) data is very crucial to understand the spreading dynamics of the ongoing pandemic, but only limited sequence data is available from the Sahel region to date, we have focused our efforts on generating the first Malian sequencing data available. Screening of 217 Malian patient samples for the presence of SARS-CoV-2 resulted in 38 positive isolates from which 21 whole genome sequences were generated. Our analysis shows that both, the early A (19B) and the fast evolving B (20A/C) clade, are present in Mali indicating multiple and independent introductions of the SARS-CoV-2 to the Sahel region.

Summary Background In December, 2019, the newly identified severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) emerged in Wuhan, China, causing COVID-19, a respiratory disease presenting with fever, cough, and often pneumonia. WHO has set the strategic objective to interrupt spread of SARS-CoV-2 worldwide. An outbreak in Bavaria, Germany, starting at the end of January, 2020, provided the opportunity to study transmission events, incubation period, and secondary attack rates. Methods A case was defined as a person with SARS-CoV-2 infection confirmed by RT-PCR. Case interviews were done to describe timing of onset and nature of symptoms and to identify and classify contacts as high risk (had cumulative face-to-face contact with a confirmed case for ≥15 min, direct contact with secretions or body fluids of a patient with confirmed COVID-19, or, in the case of health-care workers, had worked within 2 m of a patient with confirmed COVID-19 without personal protective equipment) or low risk (all other contacts). High-risk contacts were ordered to stay at home in quarantine for 14 days and were actively followed up and monitored for symptoms, and low-risk contacts were tested upon self-reporting of symptoms. We defined fever and cough as specific symptoms, and defined a prodromal phase as the presence of non-specific symptoms for at least 1 day before the onset of specific symptoms. Whole genome sequencing was used to confirm epidemiological links and clarify transmission events where contact histories were ambiguous; integration with epidemiological data enabled precise reconstruction of exposure events and incubation periods. Secondary attack rates were calculated as the number of cases divided by the number of contacts, using Fisher's exact test for the 95% CIs. Findings Patient 0 was a Chinese resident who visited Germany for professional reasons. 16 subsequent cases, often with mild and non-specific symptoms, emerged in four transmission generations. Signature mutations in the viral genome occurred upon foundation of generation 2, as well as in one case pertaining to generation 4. The median incubation period was 4·0 days (IQR 2·3–4·3) and the median serial interval was 4·0 days (3·0–5·0). Transmission events were likely to have occurred presymptomatically for one case (possibly five more), at the day of symptom onset for four cases (possibly five more), and the remainder after the day of symptom onset or unknown. One or two cases resulted from contact with a case during the prodromal phase. Secondary attack rates were 75·0% (95% CI 19·0–99·0; three of four people) among members of a household cluster in common isolation, 10·0% (1·2–32·0; two of 20) among household contacts only together until isolation of the patient, and 5·1% (2·6–8·9; 11 of 217) among non-household, high-risk contacts. Interpretation Although patients in our study presented with predominately mild, non-specific symptoms, infectiousness before or on the day of symptom onset was substantial. Additionally, the incubation period was often very short and false-negative tests occurred. These results suggest that although the outbreak was controlled, successful long-term and global containment of COVID-19 could be difficult to achieve. Funding All authors are employed and all expenses covered by governmental, federal state, or other publicly funded institutions.

The autosomal recessive genetic disorder Nijmegen breakage syndrome (NBS) is characterized by a defect in DNA double-strand break repair protein nibrin and chromosome instability associated with a high predisposition to cancer. Here we hypothesized that impaired nibrin/MRE11/RAD50 telomere maintenance complex may also affect telomere length and modulate the cancer phenotype.Telomere length was studied in blood from 38 homozygous and 27 heterozygous individuals, in one homozygous fetus, and in sex NBS lymphoblastoid cell lines (all with the founder mutation c.657_661del5), and in three humanized Nbs mice, using qPCR, TRF and Q-FISH.Telomere lengths were markedly but uniformly reduced to 20-40% of healthy controls. There was no correlation between telomere length and severity of clinical phenotype or age of death. By contrast, individual patients with very short telomeres displayed long survival times after cancer manifestation. Mildly accelerated telomere attrition was found in older NBS heterozygotes. In the NBS-fetus, the spinal cord, brain and heart had the longest telomeres, skin the shortest. Humanized Nbs mice (with much longer telo-meres than those in human beings) did not show accelerated telomere attrition.Our data clearly show that NBS is a secondary telomeropathy with unique features. Te- lomere attrition in NBS may cause genetic instability and contribute to the high cancer incidence in NBS. On the other hand, short telomeres may prevent an even worse pheno-type when a tumor has developed. These data may help to understand the high cancer rate in NBS and also the bifunctional role of telomere shortening in cancerogenesis.Author SummaryDNA damage is harmful because it leads to mutations in genes that initiate or accelerate cancerogenesis. The devastating consequences of DNA damage are manifested in diseases with non-functional repair pathways such as Nijmegen breakage syndrome (NBS). A common feature of these diseases is a high tumor incidence. However, cancer incidence varies and is not clear why it is highest for NBS. In a previous study, we have shown that the underlying nebrin mutation not only leads to defective DNA repair but also to higher degree of oxidative stress that generates further DNA lesions. Nibrin may play also an important role in protecting chromosome ends, the telomeres, from inap-propriate DNA repair. Therefore we examined the telomere length in NBS and show markedly reduced values in affected patients but not in NBC mice (with much milder phenotype and longer telomeres). Telomere attrition contributes to genetic instability and may thus contribute to the high cancer incidence in NBS. Individual patients with very short telomeres, however, displayed long survival times after cancer manifestation. Thus, short telomeres may also prevent an even worse phenotype when a tumor has developed. These data are fundamental to understanding the high cancer rate in NBS and also the bifunctional role of telomere shortening in cancer.

The zoonosis anthrax caused by the bacterium Bacillus anthracis has a broad geographical distribution. Active enzootic areas are typically located away from central and northern Europe where cases of the disease occur only sporadically and in limited numbers. In contrast, a few out of the 64 districts of Bangladesh are hyper-endemic for anthrax and there the disease causes major losses in live-stock. In this study we genotyped eight strains of B. anthracis collected from the districts of Sirajganj and Tangail in 2013. All these strains belonged to canSNP group A.Br.001/002 Sterne differing only in a few of 31 tandem-repeat (MLVA)-markers. Whole genome sequences were obtained from five of these strains and compared with genomic information of B. anthracis strains originating from various geographical locations. Characteristic signatures were detected defining two “Bangladesh” clusters potentially useful for rapid molecular epidemiology. From this data high-resolution PCR assays were developed and subsequently tested on additional isolates from Bangladesh and Central Europe. Remarkably, this comparative genomic analysis focusing on SNP-discovery revealed a close genetic relationship between these strains from Bangladesh and historic strains collected between 1991 and 2008 in The Netherlands and Germany, respectively. Possible explanations for these phylogenetic relationships are discussed.

Coxiella burnetii is a pathogen causing Q fever in domestic animals and humans. Seabirds have been implicated as possible reservoirs of this bacterium in the Arabian Gulf and in the Western Indian Ocean. Recently, Coxiella species closely related to C. burnetii was detected from ticks collected from oil rigs used as roosting areas by Socotra Cormorants (Phalacrocorax nigrogularis) in the western Arabian Gulf. We collected ticks from the largest breeding colony of Socotra Cormorants in the United Arab Emirates on the eastern extreme of the species' breeding range to determine the prevalence of C. burnetii and evaluate its role as a wild reservoir. All ticks were identified as Ornithodoros muesebecki and genomic DNA was extracted from larval and nymph/adult tick pools. Multiplex PCR tests were performed targeting three C. burnetii specific genes. C. burnetii was not detected although a Coxiella-like endosymbiont was identified that was closely related to Coxiella symbionts from Ornithodoros capensis ticks. Because domestic and wild ungulates are the primary source of C. burnetii, we suggest that the presence of free-ranging, native and non-native ungulates in some off-shore islands in the Arabian Gulf could disseminate C. burnetii to seabirds. More comprehensive studies on seabird colonies are needed to better understand the diversity and prevalence of Coxiella symbionts and to establish if C. burnetii is endemic on some of these islands.

We report the draft genome sequence of clindamycin-resistant Bacillus safensis strain Ingolstadt isolated from a patient with bacterial colonization after heart surgery. The draft genome comprises 3.75 Mbp and harbors 3,793 predicted protein-encoding genes and a small plasmid.

The causative agent of Q fever, Coxiella burnetii, is a query agent occurring naturally all over the world. We studied 104 German Coxiella burnetii strains/DNA samples obtained between 1969 and 2011 using a 14 microsatellite marker Multiple-locus variable-number of tandem repeat (VNTR) analysis (MLVA) technique. We were able to divide our collection into 32 different genotypes clustered into four major groups (A-D). Two of these (A and C) formed predominant clonal complexes that covered 97% of all studied samples. Group C consisted exclusively of cattle-associated isolates/DNA specimens, while group A comprised all other affected species including all sheep-derived strains/DNA samples. Within this second cluster, two major genotypes (A1, A2) were identified. Genotype A2 occurred in strains isolated from ewes in northern and central Germany, whereas genotype A1 was found in most areas of Germany. MLVA analysis of C. burnetii strains from neighbouring countries revealed a close relationship to German strains. We thus hypothesize that there is a western and central European cluster of C. burnetii. We identified predominant genotypes related to relevant host species and geographic regions which is in line with findings of the Dutch Q fever outbreak (2007-2010). Furthermore three of our analyzed German strains are closely related to the Dutch outbreak clone. These findings support the theory of predominant genotypes in the context of regional outbreaks. Our results show that a combination of 8 MLVA markers provides the highest discriminatory power for attributing C. burnetii isolates to genotypes. For future epidemiological studies we propose the use of three MLVA markers for easy and rapid classification of C. burnetii into 4 main clusters.

Due to their economic relevance, the study of plant pathogen interactions is of importance. However, elucidating these interactions and their underlying molecular mechanisms remains challenging since both host and pathogen need to be fully genetically accessible organisms. Here we present milestones in the establishment of a new biotrophic model pathosystem: Ustilago bromivora and Brachypodium sp. We provide a complete toolset, including an annotated fungal genome and methods for genetic manipulation of the fungus and its host plant. This toolset will enable researchers to easily study biotrophic interactions at the molecular level on both the pathogen and the host side. Moreover, our research on the fungal life cycle revealed a mating type bias phenomenon. U. bromivora harbors a haplo-lethal allele that is linked to one mating type region. As a result, the identified mating type bias strongly promotes inbreeding, which we consider to be a potential speciation driver. DOI: http://dx.doi.org/10.7554/eLife.20522.001, eLife digest Fungi cause many diseases in plants, and reduce the yield of important crops like wheat, corn and rice – all of which belong to the family of grasses. Much research into how disease-causing fungi infect plants will look at a given fungus that infects a specific plant in order to understand plant diseases in general. Over the years, scientists have generated suites of research tools to study these pairs of fungi and plants. However, many of these organism pairs (often called “model pathosystems”) have drawbacks when it comes to research in the laboratory, either on the side of the fungus or the side of plant. Brachypodium is a small grass that grows quickly and, unlike crop plants, it grows well in the laboratory. These characteristics make Brachypodium a promising model organism for studying many aspects of plant biology. Recently, a fungus called Ustilago bromivora – which is related to a fungus that infects corn – was reported to infect Brachypodium. This raised the question: could this fungus and this small grass become a new model pathosystem? Rabe, Bosch et al. set out to answer this question and now provide a toolkit that will help to establish U. bromivora and Brachypodium as a new model pathosystem. In all of U. bromivora’s close relatives, two compatible strains must meet and mate before the fungus can infect the plant; first Rabe, Bosch et al. confirmed that this is also the case for U. bromivora. Studying the life cycle of the U. bromivora fungus also unexpectedly revealed that while both mating partners are needed to infect the plant, only one of the strains survives outside of the plant after the infection. This phenomenon, referred to as a “mating type bias”, has been described for a few other related fungi. Next, Rabe, Bosch et al. conducted a genetic screen and identified two compatible strains that can grow without the plant as yeast-like cells. This means that these cells can be manipulated genetically, and indeed protocols to grow and genetically engineer the fungus and plant to address different research questions are included in the toolkit as well. Other new tools include the complete genetic sequence of the fungus with all its genes annotated, and a dataset of which genes are active in U. bromivora growing yeast-like in liquid culture versus those active when the fungus grows as a pathogen inside the plant. Together these new tools and datasets will provide a foundation to study different aspects of the interactions between grasses and disease-causing fungi. This in turn may lead to new methods to reduce fungal growth and reduce yield losses caused by fungal diseases in crop plants. Finally, the discovery that U. bromivora shows a mating type bias could provide a starting point for future studies into sexual reproduction in fungi and how new species arise. DOI: http://dx.doi.org/10.7554/eLife.20522.002

Coxiella burnetii strain AuQ01 was isolated from the serum of an Australian acute Q fever patient and represents the first whole genome from this historical Q fever country. This new genome shows distinct differences from existing genomic data and will enhance the understanding of this query pathogen.

The acute disease antigen A (adaA) gene is believed to be associated with Coxiella burnetii strains causing acute Q fever. The detailed analysis of the adaA genomic region of 23 human- and 86 animal-derived C. burnetii isolates presented in this study reveals a much more polymorphic appearance and distribution of the adaA gene, resulting in a classification of C. burnetii strains of better differentiation than previously anticipated. Three different genomic variants of the adaA gene were identified which could be detected in isolates from acute and chronic patients, rendering the association of adaA positive strains with acute Q fever disease disputable. In addition, all adaA positive strains in humans and animals showed the occurrence of the QpH1 plasmid. All adaA positive isolates of acute human patients except one showed a distinct SNP variation at position 431, also predominant in sheep strains, which correlates well with the observation that sheep are a major source of human infection. Furthermore, the phylogenetic analysis of the adaA gene revealed three deletion events and supported the hypothesis that strain Dugway 5J108-111 might be the ancestor of all known C. burnetii strains. Based on our findings, we could confirm the QpDV group and we were able to define a new genotypic cluster. The adaA gene polymorphisms shown here improve molecular typing of Q fever, and give new insights into microevolutionary adaption processes in C. burnetii.