The identification of novel single nucleotide polymorphisms to assist in mapping the spread of Bacillus anthracis across the Southern Caucasus

Anthrax is common as a zoonotic disease in the southern Caucasus area including parts of Turkey and Georgia. In this region, population genetics of the etiological agent Bacillus anthracis comprises, where known, the major canonical single nucleotide polymorphism (canSNP) groups A.Br.Aust94 and A.Br.008/009 of the pathogen’s global phylogeny, respectively. Previously, isolates of B. anthracis from Turkey have been genotyped predominantly by multi locus variable number of tandem repeat analysis (MLVA) or canSNP typing. While whole genome sequencing is the future gold standard, it is currently still costly. For that reason we were interested in identifying novel SNPs which could assist in further distinguishing closely related isolates using low cost assay platforms. In this study we sequenced the genomes of seven B. anthracis strains collected from the Kars province of Eastern Anatolia in Turkey and discovered new SNPs which allowed us to assign these and other geographically related strains to three novel branches of the major A-branch canSNP-group (A.Br.) Aust94. These new branches were named Kafkas-Geo 1–3 and comprised isolates from the Kars region and the neighboring republic of Georgia suggesting a common ancestry. The novel SNPs identified in this study connect the population genetics of B. anthracis in the South Caucasus and Turkey and will likely assist efforts to map the spread of the pathogen across this region

Draft genome sequences of two Bulgarian Bacillus anthracis strains

Bacillus anthracis strains previously isolated from Bulgaria form a unique subcluster within the A1.a cluster that is typical for isolates from southeastern Europe. Here, we report the draft genome sequences of two Bulgarian B. anthracis strains belonging to the A branch (A.Br.) 008/009 canonical single nucleotide polymorphism (SNP) group of the major A branch

Machine Learning Algorithms for Classification of MALDI-TOF MS Spectra from Phylogenetically Closely Related Species Brucella melitensis, Brucella abortus and Brucella suis

(1) Background: MALDI-TOF mass spectrometry (MS) is the gold standard for microbial fingerprinting, however, for phylogenetically closely related species, the resolution power drops down to the genus level. In this study, we analyzed MALDI-TOF spectra from 44 strains of B. melitensis, B. suis and B. abortus to identify the optimal classification method within popular supervised and unsupervised machine learning (ML) algorithms. (2) Methods: A consensus feature selection strategy was applied to pinpoint from among the 500 MS features those that yielded the best ML model and that may play a role in species differentiation. Unsupervised k-means and hierarchical agglomerative clustering were evaluated using the silhouette coefficient, while the supervised classifiers Random Forest, Support Vector Machine, Neural Network, and Multinomial Logistic Regression were explored in a fine-tuning manner using nested k-fold cross validation (CV) with a feature reduction step between the two CV loops. (3) Results: Sixteen differentially expressed peaks were identified and used to feed ML classifiers. Unsupervised and optimized supervised models displayed excellent predictive performances with 100% accuracy. The suitability of the consensus feature selection strategy for learning system accuracy was shown. (4) Conclusion: A meaningful ML approach is here introduced, to enhance Brucella spp. classification using MALDI-TOF MS data.Peer Reviewe

Genotyping and phylogenetic placement of Bacillus anthracis isolates from Finland, a country with rare anthrax cases

Anthrax, the zoonotic disease caused by the gram-positive bacterium Bacillus anthracis, is nowadays rare in northern parts of Europe including Finland and Scandinavia. Only two minor outbreaks of anthrax in 1988 and in 2004 and one sporadic infection in 2008 have been detected in animals in Finland since the 1970’s. Here, we report on two Finnish B. anthracis strains that were isolated from spleen and liver of a diseased calf related to the outbreak in 1988 (strain HKI4363/88) and from a local scrotum and testicle infection of a bull in 2008 (strain BA2968). These infections occurred in two rural Finnish regions, i.e., Ostrobothnia in western Finland and Päijänne Tavastia in southern Finland, respectively

Use of Next Generation Sequencing to study two cowpox virus outbreaks

Background Between 2008 and 2011 about 40 cases of human cowpox were reported from Germany and France. Infections had been acquired via close contact to infected, young pet rats. An identical and unique sequence of the hemagglutinin gene was found in various cowpox virus (CPXV) isolates pointing to a common source of infection. In a second CPXV outbreak in cats in a small animal clinic in Germany in 2015, four out of five hospitalized cats showed identical hemagglutinin sequences and thus, a hospital-acquired transmission had been assumed. Next-Generation Sequencing was performed in order to re-investigate the outbreaks, as epidemiological data could not confirm all cases. Methods Homogenates of lesion material from rats, cats and humans were cultivated in cell culture. The genomes of four virus isolates, nine CPXVs from our strain collections and from DNA of three paraffin-embedded lesion materials were determined by Next Generation Sequencing (NGS). For phylogenetic analyses a MAFFT-alignment was generated. A distance matrix based on concatenated SNPs was calculated and plotted as dendrogram using Unweighted Pair Group Method with Arithmetic mean (UPGMA) for visualization. Results Aligning of about 200.000 nucleotides of 8 virus isolates associated with the pet rat outbreak revealed complete identity of six genomes, the remainder two genomes differed in as little as 3 SNPs. When comparing this dataset with four already published CPXV genomes also associated with the pet rat outbreak, again a maximum difference of 3 SNPs was found. The outbreak which lasted from 2008 till 2011 was indeed caused by a single strain which has maintained an extremely high level of clonality over 4 years. Aligning genomic sequences from four cases of feline cowpox revealed 3 identical sequences and one sequence which differed in 65 nucleotides. Although identical hemagglutinin sequences had been obtained from four hospitalized cats, genomic sequencing proved that a hospital-acquired transmission had occurred in only three cats. Conclusion Analyzing the rather short sequence of the hemagglutinin gene is not sufficient to conduct molecular trace back analyses. Instead, whole genome sequencing is the method of choice which can even be applied to paraffin-embedded specimens

Accumulation of mutations in antibody and CD8 T cell epitopes in a B cell depleted lymphoma patient with chronic SARS-CoV-2 infection

Antibodies against the spike protein of severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) can drive adaptive evolution in immunocompromised patients with chronic infection. Here we longitudinally analyze SARS-CoV-2 sequences in a B cell-depleted, lymphoma patient with chronic, ultimately fatal infection, and identify three mutations in the spike protein that dampen convalescent plasma-mediated neutralization of SARS-CoV-2. Additionally, four mutations emerge in non-spike regions encoding three CD8 T cell epitopes, including one nucleoprotein epitope affected by two mutations. Recognition of each mutant peptide by CD8 T cells from convalescent donors is reduced compared to its ancestral peptide, with additive effects resulting from double mutations. Querying public SARS-CoV-2 sequences shows that these mutations have independently emerged as homoplasies in circulating lineages. Our data thus suggest that potential impacts of CD8 T cells on SARS-CoV-2 mutations, at least in those with humoral immunodeficiency, warrant further investigation to inform on vaccine design

Geographical and temporal distribution of SARS-CoV-2 clades in the WHO European Region, January to June 2020

We show the distribution of severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) genetic clades over time and between countries and outline potential genomic surveillance objectives. We applied three genomic nomenclature systems to all sequence data from the World Health Organization European Region available until 10 July 2020. We highlight the importance of real-time sequencing and data dissemination in a pandemic situation, compare the nomenclatures and lay a foundation for future European genomic surveillance of SARS-CoV-2

Whole genome sequencing of Brucella melitensis isolated from 57 patients in Germany reveals high diversity in strains from Middle East

Brucellosis, a worldwide common bacterial zoonotic disease, has become quite rare in Northern and Western Europe. However, since 2014 a significant increase of imported infections caused by Brucella (B.) melitensis has been noticed in Germany. Patients predominantly originated from Middle East including Turkey and Syria. These circumstances afforded an opportunity to gain insights into the population structure of Brucella strains. Brucella-isolates from 57 patients were recovered between January 2014 and June 2016 with culture confirmed brucellosis by the National Consultant Laboratory for Brucella. Their whole genome sequences were generated using the Illumina MiSeq platform. A whole genome-based SNP typing assay was developed in order to resolve geographically attributed genetic clusters. Results were compared to MLVA typing results, the current gold-standard of Brucella typing. In addition, sequences were examined for possible genetic variation within target regions of molecular diagnostic assays. Phylogenetic analyses revealed spatial clustering and distinguished strains from different patients in either case, whereas multiple isolates from a single patient or technical replicates showed identical SNP and MLVA profiles. By including WGS data from the NCBI database, five major genotypes were identified. Notably, strains originating from Turkey showed a high diversity and grouped into seven subclusters of genotype II. MLVA analysis congruently clustered all isolates and predominantly matched the East Mediterranean genetic clade. This study confirms whole-genome based SNP-analysis as a powerful tool for accurate typing of B. melitensis. Furthermore it allows special allocation and therefore provides useful information on the geographic origin for trace-back analysis. However, the lack of reliable metadata in public databases often prevents a resolution below geographic regions or country levels and corresponding precise trace-back analysis. Once this obstacle is resolved, WGS-derived bacterial typing adds an important method to complement epidemiological surveys during outbreak investigations. This is the first report of a detailed genetic investigation of an extensive collection of B. melitensis strains isolated from human cases in Germany

The evolving SARS-CoV-2 epidemic in Africa: Insights from rapidly expanding genomic surveillance

INTRODUCTION Investment in Africa over the past year with regard to severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) sequencing has led to a massive increase in the number of sequences, which, to date, exceeds 100,000 sequences generated to track the pandemic on the continent. These sequences have profoundly affected how public health officials in Africa have navigated the COVID-19 pandemic. RATIONALE We demonstrate how the first 100,000 SARS-CoV-2 sequences from Africa have helped monitor the epidemic on the continent, how genomic surveillance expanded over the course of the pandemic, and how we adapted our sequencing methods to deal with an evolving virus. Finally, we also examine how viral lineages have spread across the continent in a phylogeographic framework to gain insights into the underlying temporal and spatial transmission dynamics for several variants of concern (VOCs). RESULTS Our results indicate that the number of countries in Africa that can sequence the virus within their own borders is growing and that this is coupled with a shorter turnaround time from the time of sampling to sequence submission. Ongoing evolution necessitated the continual updating of primer sets, and, as a result, eight primer sets were designed in tandem with viral evolution and used to ensure effective sequencing of the virus. The pandemic unfolded through multiple waves of infection that were each driven by distinct genetic lineages, with B.1-like ancestral strains associated with the first pandemic wave of infections in 2020. Successive waves on the continent were fueled by different VOCs, with Alpha and Beta cocirculating in distinct spatial patterns during the second wave and Delta and Omicron affecting the whole continent during the third and fourth waves, respectively. Phylogeographic reconstruction points toward distinct differences in viral importation and exportation patterns associated with the Alpha, Beta, Delta, and Omicron variants and subvariants, when considering both Africa versus the rest of the world and viral dissemination within the continent. Our epidemiological and phylogenetic inferences therefore underscore the heterogeneous nature of the pandemic on the continent and highlight key insights and challenges, for instance, recognizing the limitations of low testing proportions. We also highlight the early warning capacity that genomic surveillance in Africa has had for the rest of the world with the detection of new lineages and variants, the most recent being the characterization of various Omicron subvariants. CONCLUSION Sustained investment for diagnostics and genomic surveillance in Africa is needed as the virus continues to evolve. This is important not only to help combat SARS-CoV-2 on the continent but also because it can be used as a platform to help address the many emerging and reemerging infectious disease threats in Africa. In particular, capacity building for local sequencing within countries or within the continent should be prioritized because this is generally associated with shorter turnaround times, providing the most benefit to local public health authorities tasked with pandemic response and mitigation and allowing for the fastest reaction to localized outbreaks. These investments are crucial for pandemic preparedness and response and will serve the health of the continent well into the 21st century

DNA-microarrays for detection of relevant antibiotic resistance determinants in Bacillus anthracis and Yersinia pestis

Die Medikation von Personen nach Exposition oder zur Prophylaxe gegen hochpathogene Bakterien muss schnellstmöglich und effektiv erfolgen, wobei gemäß Empfehlungen Erreger-spezifische Antibiotika verabreicht werden. Bei B. anthracis sind dies Ciprofloxacin, Doxycyclin, Rifampin und Vancomycin; bei Yersinia pestis Ciprofloxacin, Tetracyclin, Streptomycin und Gentamicin. Da die Behandlung der infizierten Personen in der Regel im klinischen Alltag ohne vorherige Überprüfung der Resistenzeigenschaft des Erregers geschieht, wurde eine auf Microarray-Technology basierende Schnelldiagnostik entwickelt, die es ermöglicht innerhalb von 4 - 7 Stunden eine zuverlässige Aussage über vorhandene relevante genetische Resistenzdeterminanten zu treffen. Zu diesem Zweck wurden jeweils für Bacillus anthracis und Yersinia pestis ein Versuchsablauf entwickelt, der zunächst einen DNA-Anreicherungsschritt enthält, der mit einer auf den Erreger abgestimmten Multiplex-PCR realisiert wurde. Im weiteren Verlauf wird eine multiplexe enzymatisch-fluoreszente Markierungsreaktion angeschlossen und das erhaltene Produkt auf den entwickelten Microarrays hybridisiert. Mit diesen ist es nun möglich, sowohl klinisch relevante Resistenzgene nachzuweisen (tet(A-D); tet(K-O); vanA; vanB; aac3-Ia, -IIa, -IVa; ant3-Ia, -IIa; aph6’’-Id; aph3’’-Ib), als auch Resistent-vermittelnde Mutationen in den Genen gyrA, gyrB, parC bzw. rpoB. Die Evaluation der Microarrays wurde aus politischen und ethischen Gründen nicht mit resistenten B. anthracis oder Y. pestis – Stämmen durchgeführt. Die Microarrays konnten jedoch aufgrund der sehr nahen Verwandtschaft der beiden Organismen zur B. cereus ATCC10987 bzw. Y. pseudotuberculosis DSM8992 mit resistenten Isolaten dieser Spezies evaluiert werden. Dabei wurde sowohl die Detektion der Resistenzgene mit Misch-DNA resistenter Spezies simuliert, als auch die Bestimmung Resistenz-vermittelnden Punktmutationen mit zuvor isolierten und charakterisierten Mutanten geprüft. Im weiteren Verlauf wurden Kinetik und Reproduzierbarkeit der entwickelten Versuchsabläufe untersucht sowie eine Blind-Studie mit klinischen Yersinia enterocolitica Isolaten durchgeführt.The medication of persons after exposition or for prophylaxis against highly-pathogenic bacteria must take place as fast as possible and effectively. The following antibiotics are recommended: For B. anthracis are these Ciprofloxacin, Doxycyclin, Rifampin and Vancomycin; for Yersinia pestis Ciprofloxacin, Tetracyclin, Streptomycin and Gentamicin. Treatment of infected persons is usually realized in clinical diagnostic without previous examination of resistance of the infecting species. In front of this background an assay was developed based on microarray-technology, which enables the obtaining of reliable results of existing relevant genetic resistance determinants within 4 - 7 hours. Two separate assays for Bacillus anthracis and Yersinia pestis were developed, which harbour a DNA enrichment-step, -realized by a multiplex PCR compatible with the examined species-, subsequently a multiplex enzymatic fluorescent labelling and hybridization using the developed diagnostic microarrays. Using these microarrays it is possible to detect clinically relevant resistance genes (tet (A-D); tet (K-O); vanA; vanB; aac3-Ia, - IIa, - IVa; ant3-Ia, - IIa; aph6'' - Id; aph3'' - Ib) and resistance-mediating point-mutations in genes gyrA, gyrB, parC and/or rpoB in one single step. Because of political and ethical reasons evaluation of the microarrays was not performed using resistant B. anthracis- or Y. pestis - isolates. The microarrays could be evaluated because of a strong relationship of these two organisms to the non-highly-pathogenic organisms B. cereus ATCC10987 and Y. pseudotuberculosis DSM8992. The detection of both, resistance genes and resistance-obtaining point mutations, was performed using in this study isolated and well characterized mutants or mixture-DNA of resistant species. In further process kinetics and reproducibility of the developed assay were investigated as well as a blind panel was performed using clinical Yersinia enterocolitica-isolates