Reoccurring Bovine Anthrax in Germany on the Same Pasture after 12 Years.

The zoonotic disease anthrax, caused by the endospore-forming bacterium Bacillus anthracis, is very rare in Germany. In the state of Bavaria, the last case occurred in July of 2009, resulting in four dead cows. In August of 2021, the disease reemerged after heavy rains, killing one gestating cow. Notably, both outbreaks affected the same pasture, suggesting a close epidemiological connection. B. anthracis could be grown from blood culture, and the presence of both virulence plasmids (pXO1 and pXO2) was confirmed by PCR. Also, recently developed diagnostic tools enabled rapid detection of B. anthracis cells and nucleic acids directly in clinical samples. The complete genome of the strain isolated from blood, designated BF-5, was DNA sequenced and phylogenetically grouped within the B.Br.CNEVA clade, which is typical for European B. anthracis strains. The genome was almost identical to BF-1, the isolate from 2009, separated only by three single nucleotide polymorphisms (SNPs) on the chromosome, one on plasmid pXO2 and three indel regions. Further, B. anthracis DNA was detected by PCR from soil samples taken from spots in the pasture where the cow had fallen. New tools based on phage receptor-binding proteins enabled the microscopic detection and isolation of B. anthracis directly from soil samples. These environmental isolates were genotyped and found to be identical to BF-5 in terms of SNPs. Therefore, it seems that the BF-5 genotype is currently the prevalent one at the affected premises. The area contaminated by the cadaver was subsequently disinfected with formaldehyde.

Impact of a Novel PagR-like Transcriptional Regulator on Cereulide Toxin Synthesis in Emetic Bacillus cereus.

The emetic type of foodborne disease caused by Bacillus cereus is produced by the small peptide toxin cereulide. The genetic locus encoding the Ces nonribosomal peptide synthetase (CesNRPS) multienzyme machinery is located on a 270 kb megaplasmid, designated pCER270, which shares its backbone with the Bacillus anthracis toxin plasmid pXO1. Although the ces genes are plasmid-borne, the chromosomally encoded pleiotropic transcriptional factors CodY and AbrB are key players in the control of ces transcription. Since these proteins only repress cereulide synthesis during earlier growth phases, other factors must be involved in the strict control of ces expression and its embedment in the bacterial life cycle. In silico genome analysis revealed that pCER270 carries a putative ArsR/SmtB family transcription factor showing high homology to PagR from B. anthracis. As PagR plays a crucial role in the regulation of the protective antigen gene pagA, which forms part of anthrax toxin, we used a gene-inactivation approach, combined with electrophoretic mobility shift assays and a bacterial two-hybrid system for dissecting the role of the PagR homologue PagRBc in the regulation of cereulide synthesis. Our results highlight that the plasmid-encoded transcriptional regulator PagRBc plays an important role in the complex and multilayered process of cereulide synthesis.

Ultrasensitive Detection of Bacillus anthracis by Real-Time PCR Targeting a Polymorphism in Multi-Copy 16S rRNA Genes and Their Transcripts.

The anthrax pathogen Bacillus anthracis poses a significant threat to human health. Identification of B. anthracis is challenging because of the bacterium's close genetic relationship to other Bacillus cereus group species. Thus, molecular detection is founded on species-specific PCR targeting single-copy genes. Here, we validated a previously recognized multi-copy target, a species-specific single nucleotide polymorphism (SNP) present in 2-5 copies in every B. anthracis genome analyzed. For this, a hydrolysis probe-based real-time PCR assay was developed and rigorously tested. The assay was specific as only B. anthracis DNA yielded positive results, was linear over 9 log10 units, and was sensitive with a limit of detection (LoD) of 2.9 copies/reaction. Though not exhibiting a lower LoD than established single-copy PCR targets (dhp61 or PL3), the higher copy number of the B. anthracis-specific 16S rRNA gene alleles afforded ≤2 unit lower threshold (Ct) values. To push the detection limit even further, the assay was adapted for reverse transcription PCR on 16S rRNA transcripts. This RT-PCR assay was also linear over 9 log10 units and was sensitive with an LoD of 6.3 copies/reaction. In a dilution series of experiments, the 16S RT-PCR assay achieved a thousand-fold higher sensitivity than the DNA-targeting assays. For molecular diagnostics, we recommend a real-time RT-PCR assay variant in which both DNA and RNA serve as templates (thus, no requirement for DNase treatment). This can at least provide results equaling the DNA-based implementation if no RNA is present but is superior even at the lowest residual rRNA concentrations.

Mutant Strains of Escherichia coli and Methicillin-Resistant Staphylococcus aureus Obtained by Laboratory Selection To Survive on Metallic Copper Surfaces.

Artificial laboratory evolution was used to produce mutant strains of Escherichia coli and methicillin-resistant Staphylococcus aureus (MRSA) able to survive on antimicrobial metallic copper surfaces. These mutants were 12- and 60-fold less susceptible to the copper-mediated contact killing process than their respective parent strains. Growth levels of the mutant and its parent in complex growth medium were similar. Tolerance to copper ions of the mutants was unchanged. The mutant phenotype remained stable over about 250 generations under nonstress conditions. The mutants and their respective parental strains accumulated copper released from the metallic surfaces to similar extents. Nevertheless, only the parental strains succumbed to copper stress when challenged on metallic copper surfaces, suffering complete destruction of the cell structure. Whole-genome sequencing and global transcriptome analysis were used to decipher the genetic alterations in the mutant strains; however, these results did not explain the copper-tolerance phenotypes on the systemic level. Instead, the mutants shared features with those of stressed bacterial subpopulations entering the early or "shallow" persister state. In contrast to the canonical persister state, however, the ability to survive on solid copper surfaces was adopted by the majority of the mutant strain population. This indicated that application of solid copper surfaces in hospitals and elsewhere has to be accompanied by strict cleaning regimens to keep the copper surfaces active and prevent evolution of tolerant mutant strains.IMPORTANCE Microbes are rapidly killed on solid copper surfaces by contact killing. Copper surfaces thus have an important role to play in preventing the spread of nosocomial infections. Bacteria adapt to challenging natural and clinical environments through evolutionary processes, for instance, by acquisition of beneficial spontaneous mutations. We wish to address the question of whether mutants can be selected that have evolved to survive contact killing on solid copper surfaces. We isolated such mutants from Escherichia coli and methicillin-resistant Staphylococcus aureus (MRSA) by artificial laboratory evolution. The ability to survive on solid copper surfaces was a stable phenotype of the mutant population and not restricted to a small subpopulation. As a consequence, standard operation procedures with strict hygienic measures are extremely important to prevent the emergence and spread of copper-surface-tolerant persister-like bacterial strains if copper surfaces are to be sustainably used to limit the spread of pathogenic bacteria, e.g., to curb nosocomial infections.

Improved Discrimination of Bacillus anthracis from Closely Related Species in the Bacillus cereus Sensu Lato Group Based on Matrix-Assisted Laser Desorption Ionization-Time of Flight Mass Spectrometry.

Discrimination of highly pathogenic bacteria, such as Bacillus anthracis, from closely related species based on molecular biological methods is challenging. We applied matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF MS) to a collection of B. anthracis strains and close relatives in order to significantly improve the statistical confidence of identification results for this group of bacteria. Protein mass spectra of 189 verified and diverse Bacillus strains of the Bacillus cereus sensu lato group were generated using MALDI-TOF MS and subsequently analyzed with supervised and unsupervised statistical methods, such as shrinkage discriminant analysis (SDA) and principal-component analysis (PCA). We aimed at identifying specific biomarkers in the protein spectra of B. anthracis not present in closely related Bacillus species. We could identify 7, 10, 18, and 14 B. anthracis-specific biomarker candidates that were absent in B. cereus, B. mycoides, B. thuringiensis, and B. weihenstephanensis strains, respectively. Main spectra (MSP) of a defined collection of Bacillus strains were compiled using the Bruker Biotyper software and added to an in-house reference library. Reevaluation of this library with 15 hitherto untested strains of B. anthracis and B. cereus yielded improved score values. The B. anthracis strains were identified with score values between 2.33 and 2.55 using the in-house database, while the same strains were identified with scores between 1.94 and 2.37 using the commercial database, and no false-positive identifications occurred using the in-house database.

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

BACKGROUND: 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. RESULTS: The isolates were genetically characterized by PCR-based methods such as multilocus variable number of tandem repeat analysis (MLVA) and whole genome-sequence analysis (WGS). Phylogenetic comparison of the two strains HKI4363/88 and BA2968 by chromosomal single nucleotide polymorphism (SNP) analysis grouped these organisms within their relatives of the minor canonical A-branch canSNP-group A.Br.003/004 (A.Br.V770) or canonical B-branch B.Br.001/002, respectively. Strain HKI4363/88 clustered relatively closely with other members of the A.Br.003/004 lineage from Europe, South Africa, and South America. In contrast, strain BA2968 clearly constituted a new sublineage within B.Br.001/002 with its closest relative being HYO01 from South Korea. CONCLUSIONS: Our results suggest that Finland harbors both unique (autochthonous) and more widely distributed, common clades of B. anthracis. We suspect that members of the common clades such as strains HKI4363/88 have been introduced only recently by anthropogenic activities involving importation of contaminated animal products. On the other hand, autochthonous strains such as isolate BA2968 probably have an older history of their introduction into Finland as evidenced by a high number of single nucleotide variant sites in their genomes.

Restoration of growth by manganese in a mutant strain of Escherichia coli lacking most known iron and manganese uptake systems.

The interplay of manganese and iron homeostasis and oxidative stress in Escherichia coli can give important insights into survival of bacteria in the phagosome and under differing iron or manganese bioavailabilities. Here, we characterized a mutant strain devoid of all know iron/manganese-uptake systems relevant for growth in defined medium. Based on these results an exit strategy enabling the cell to cope with iron depletion and use of manganese as an alternative for iron could be shown. Such a strategy would also explain why E. coli harbors some iron- or manganese-dependent iso-enzymes such as superoxide dismutases or ribonucleotide reductases. The benefits for gaining a means for survival would be bought with the cost of less efficient metabolism as indicated in our experiments by lower cell densities with manganese than with iron. In addition, this strain was extremely sensitive to the metalloid gallium but this gallium toxicity can be alleviated by low concentrations of manganese.

Inactivation of bacterial and viral biothreat agents on metallic copper surfaces.

In recent years several studies in laboratory settings and in hospital environments have demonstrated that surfaces of massive metallic copper have intrinsic antibacterial and antiviral properties. Microbes are rapidly inactivated by a quick, sharp shock known as contact killing. The underlying mechanism is not yet fully understood; however, in this process the cytoplasmic membrane is severely damaged. Pathogenic bacterial and viral high-consequence species able to evade the host immune system are among the most serious lethal microbial challenges to human health. Here, we investigated contact-killing mediated by copper surfaces of Gram-negative bacteria (Brucella melitensis, Burkholderia mallei, Burkholderia pseudomallei, Francisella tularensis tularensis and Yersinia pestis) and of Gram-positive endospore-forming Bacillus anthracis. Additionally, we also tested inactivation of monkeypox virus and vaccinia virus on copper. This group of pathogens comprises biothreat species (or their close relatives) classified by the Center for Disease and Control and Prevention (CDC) as microbial select agents posing severe threats to public health and having the potential to be deliberately released. All agents were rapidly inactivated on copper between 30 s and 5 min with the exception of B. anthracis endospores. For vegetative bacterial cells prolonged contact to metallic copper resulted in the destruction of cell structure.

European multi-centre study to establish MIC and zone diameter epidemiological cut-off values for Bacillusanthracis.

OBJECTIVES: Bacillus anthracis clinical breakpoints, representing a systematic approach to guide clinicians in selecting the most appropriate antimicrobial treatments, are not part of the guidance from the European Committee on Antimicrobial Susceptibility Testing (EUCAST). This is because defined distributions of MIC values and of epidemiological cut-off values (ECOFFs) have been lacking. In this study, a Europe-wide network of laboratories in collaboration with EUCAST, aimed at establishing standardized antimicrobial susceptibility testing methods, wild-type MIC distributions, and ECOFFs for ten therapeutically relevant antimicrobials. METHODS: About 335 B. anthracis isolates were tested by broth microdilution and disc diffusion methodologies. MIC and inhibition zone diameters were curated according to EUCAST SOP 10.2 and the results were submitted to EUCAST for ECOFFs and clinical breakpoint determination. RESULTS: Broth microdilution and disc diffusion data distributions revealed putative wild-type distributions for the tested agents. For each antimicrobial agent, ECOFFs were defined. Three highly resistant strains with MIC values of 32 mg/L benzylpenicillin were found. MIC values slightly above the defined ECOFFs were observed in a few isolates, indicating the presence of resistance mechanisms to doxycycline, tetracycline, and amoxicillin. DISCUSSION: B. anthracis antimicrobial susceptibility testing results were used by EUCAST to determine ECOFFs for ten antimicrobial agents. The MIC distributions were used in the process of determining clinical breakpoints. The ECOFFs can be used for the sensitive detection of isolates with resistance mechanisms, and for monitoring resistance development. Genetic changes causing phenotypic shifts in isolates displaying slightly elevated MICs remain to be investigated.

Spanish Outbreak Isolates Bridge Phylogenies of European and American Bacillus anthracis.

The geographical origin of a major present-day phylogenetic group (A branch WNA; A.Br.WNA) of American Bacillus anthracis is controversial. One hypothesis postulated that the anthrax pathogen reached North America via a then-existing land bridge from northeastern Asia thousands of years ago. A competing hypothesis suggested that B. anthracis was introduced to America a couple of hundred years ago, related to European colonization. The latter view is strongly supported by genomic analysis of a group of French B. anthracis isolates that are phylogenetically closely related to the North American strains of the A branch A.Br.WNA clade. In addition, three West African strains also belong to this relationship group. Recently, we have added a Spanish strain to these close relatives of the WNA lineage of American B. anthracis. Nevertheless, the diversity of Spanish B. anthracis remains largely unexplored, and phylogenetic links to European or American relatives are not well resolved. Here, we genome sequenced and characterized 29 new B. anthracis isolates (yielding 18 unique genotypes) from outbreaks in west central and central Spain in 2021. Applying comparative chromosomal analysis, we placed the chromosomes of these isolates within the established phylogeny of the A.Br.008/009 (A.Br.TEA) canonical SNP group. From this analysis, a new sub-clade, named A.Br.11/ESPc, emerged that constitutes a sister group of American A.Br.WNA.

Draft genome sequence of Pseudomonas sp. strain M47T1, carried by Bursaphelenchus xylophilus isolated from Pinus pinaster.

The draft genome sequence of Pseudomonas sp. strain M47T1, carried by the Bursaphelenchus xylophilus pinewood nematode, the causative agent of pine wilt disease, is presented. In Pseudomonas sp. strain M47T1, genes that make this a plant growth-promoting bacterium, as well as genes potentially involved in nematotoxicity, were identified.

Draft genome sequence of Serratia sp. strain M24T3, isolated from pinewood disease nematode Bursaphelenchus xylophilus.

Here we report the draft genome sequence of Serratia sp. strain M24T3, which is associated with pinewood nematode Bursaphelenchus xylophilus, the causative agent of pine wilt disease. Serratia sp. strain M24T3 has been identified as a bionematocide for B. xylophilus in vitro, and multiple genes potentially involved in virulence and nematotoxity were identified.

Draft genome sequence of Pseudomonas psychrotolerans L19, isolated from copper alloy coins.

We report the draft genome sequence of Pseudomonas psychrotolerans strain L19, isolated from a European 50-cent copper alloy coin. Multiple genes potentially involved in copper resistance were identified; however, it is unknown if these copper ion resistance determinants contribute to prolonged survival of this strain on dry metallic copper.

Genome sequence of the moderately halotolerant, arsenite-oxidizing bacterium Pseudomonas stutzeri TS44.

We present the draft genome sequence of Pseudomonas stutzeri TS44, a moderately halotolerant, arsenite-oxidizing bacterium isolated from arsenic-contaminated soil. The genome contains genes for arsenite oxidation, arsenic resistance, and ectoine/hydroxyectoine biosynthesis. The genome information will be useful for exploring adaptation of P. stutzeri TS44 to an arsenic-contaminated environment.

Draft genome sequence of Bacillus anthracis BF-1, isolated from Bavarian cattle.

Bacillus anthracis BF-1 was isolated from a cow in Bavaria (Germany) that had succumbed to anthrax. Here, we report the draft genome sequence of this strain, which belongs to the European B2 subclade of B. anthracis. The closest phylogenetic neighbor of strain BF-1 is a strain isolated from cattle in France.

Draft genome sequence of Bacillus anthracis UR-1, isolated from a German heroin user.

We report the draft genome sequence of Bacillus anthracis UR-1, isolated from a fatal case of injectional anthrax in a German heroin user. Analysis of the genome sequence of strain UR-1 may aid in describing phylogenetic relationships between virulent heroin-associated isolates of B. anthracis isolated in the United Kingdom, Germany, and other European countries.

Characterization of a dipartite iron uptake system from uropathogenic Escherichia coli strain F11.

In the uropathogenic Escherichia coli strain F11, in silico genome analysis revealed the dicistronic iron uptake operon fetMP, which is under iron-regulated control mediated by the Fur regulator. The expression of fetMP in a mutant strain lacking known iron uptake systems improved growth under iron depletion and increased cellular iron accumulation. FetM is a member of the iron/lead transporter superfamily and is essential for iron uptake by the Fet system. FetP is a periplasmic protein that enhanced iron uptake by FetM. Recombinant FetP bound Cu(II) and the iron analog Mn(II) at distinct sites. The crystal structure of the FetP dimer reveals a copper site in each FetP subunit that adopts two conformations: CuA with a tetrahedral geometry composed of His(44), Met(90), His(97), and His(127), and CuB, a second degenerate octahedral geometry with the addition of Glu(46). The copper ions of each site occupy distinct positions and are separated by ∼1.3 Å. Nearby, a putative additional Cu(I) binding site is proposed as an electron source that may function with CuA/CuB displacement to reduce Fe(III) for transport by FetM. Together, these data indicate that FetMP is an additional iron uptake system composed of a putative iron permease and an iron-scavenging and potentially iron-reducing periplasmic protein.

Crystal structures of multicopper oxidase CueO bound to copper(I) and silver(I): functional role of a methionine-rich sequence.

The multicopper oxidase CueO oxidizes toxic Cu(I) and is required for copper homeostasis in Escherichia coli. Like many proteins involved in copper homeostasis, CueO has a methionine-rich segment that is thought to be critical for copper handling. How such segments function is poorly understood. Here, we report the crystal structure of CueO at 1.1 Šwith the 45-residue methionine-rich segment fully resolved, revealing an N-terminal helical segment with methionine residues juxtaposed for Cu(I) ligation and a C-terminal highly mobile segment rich in methionine and histidine residues. We also report structures of CueO with a C500S mutation, which leads to loss of the T1 copper, and CueO with six methionines changed to serine. Soaking C500S CueO crystals with Cu(I), or wild-type CueO crystals with Ag(I), leads to occupancy of three sites, the previously identified substrate-binding site and two new sites along the methionine-rich helix, involving methionines 358, 362, 368, and 376. Mutation of these residues leads to a ∼4-fold reduction in k(cat) for Cu(I) oxidation. Ag(I), which often appears with copper in nature, strongly inhibits CueO oxidase activities in vitro and compromises copper tolerance in vivo, particularly in the absence of the complementary copper efflux cus system. Together, these studies demonstrate a role for the methionine-rich insert of CueO in the binding and oxidation of Cu(I) and highlight the interplay among cue and cus systems in copper and silver homeostasis.

Mechanisms of gold biomineralization in the bacterium Cupriavidus metallidurans.

While the role of microorganisms as main drivers of metal mobility and mineral formation under Earth surface conditions is now widely accepted, the formation of secondary gold (Au) is commonly attributed to abiotic processes. Here we report that the biomineralization of Au nanoparticles in the metallophillic bacterium Cupriavidus metallidurans CH34 is the result of Au-regulated gene expression leading to the energy-dependent reductive precipitation of toxic Au(III)-complexes. C. metallidurans, which forms biofilms on Au grains, rapidly accumulates Au(III)-complexes from solution. Bulk and microbeam synchrotron X-ray analyses revealed that cellular Au accumulation is coupled to the formation of Au(I)-S complexes. This process promotes Au toxicity and C. metallidurans reacts by inducing oxidative stress and metal resistances gene clusters (including a Au-specific operon) to promote cellular defense. As a result, Au detoxification is mediated by a combination of efflux, reduction, and possibly methylation of Au-complexes, leading to the formation of Au(I)-C-compounds and nanoparticulate Au(0). Similar particles were observed in bacterial biofilms on Au grains, suggesting that bacteria actively contribute to the formation of Au grains in surface environments. The recognition of specific genetic responses to Au opens the way for the development of bioexploration and bioprocessing tools.

In-Depth Analysis of Bacillus anthracis 16S rRNA Genes and Transcripts Reveals Intra- and Intergenomic Diversity and Facilitates Anthrax Detection.

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.