Virulence and resistance patterns of Vibrio cholerae non-O1/non-O139 acquired in Germany and other European countries.

Global warming has caused an increase in the emergence of Vibrio species in marine and estuarine environments as well as fresh water bodies. Over the past decades, antimicrobial resistance (AMR) has evolved among Vibrio species toward various antibiotics commonly used for the treatment of Vibrio infections. In this study, we assessed virulence and resistance patterns of Vibrio cholerae non-O1/non-O139 strains derived from Germany and other European countries. A total of 63 clinical and 24 environmental Vibrio cholerae non-O1/non-O139 strains, collected between 2011 and 2021, were analyzed. In silico antibiotic resistances were compared with resistance phenotypes according to EUCAST breakpoints. Additionally, genetic relatedness between isolates was assessed by two cgMLST schemes (SeqSphere +, pubMLST). Both cgMLST schemes yielded similar results, indicating high genetic diversity among V. cholerae non-O1/non-O139 isolates. Some isolates were found to be genetically closely related (allelic distance < 20), which suggests an epidemiological link. Thirty-seven virulence genes (VGs) were identified among 87 V. cholerae non-O1/non-O139 isolates, which resulted in 38 virulence profiles (VPs). VPs were similar between clinical and environmental isolates, with the exception of one clinical isolate that displayed a higher abundance of VGs. Also, a cluster of 11 environmental isolates was identified to have the lowest number of VGs. Among all strains, the predominant virulence factors were quorum sensing protein (luxS), repeats-in-toxins (rtxC/rtxD), hemolysin (hlyA) and different type VI secretion systems (T6SS) genes. The genotypic profiles revealed antibiotic resistance genes (ARGs) associated with resistance to beta-lactams, quinolones, macrolides, tetracycline, antifolate, aminoglycosides, fosfomycin, phenicols and sulfonamide. Carbapenemase gene VCC-1 was detected in 10 meropenem-resistant V. cholerae non-O1/non-O139 isolates derived from surface water in Germany. The proportion of resistance among V. cholerae non-O1/non-O139 species isolates against first line treatment (3rd generation cephalosporin, tetracycline and fluoroquinolone) was low. Empirical treatment would likely have been effective for all of the clinical V. cholerae non-O1/non-O139 isolates examined. Nevertheless, carbapenem-resistant isolates have been present in fresh water in Germany and might represent a reservoir for ARGs. Monitoring antimicrobial resistance is crucial for public health authorities to minimize the risks for the human population.

Phenotypic and genotypic discrimination of Francisella tularensis ssp. holarctica clades.

Francisella tularensis is the causative agent of tularemia, a zoonotic disease with a wide host range. F. tularensis ssp. holarctica (Fth) is of clinical relevance for European countries, including Germany. Whole genome sequencing methods, including canonical Single Nucleotide Polymorphism (canSNP) typing and whole genome SNP typing, have revealed that European Fth strains belong to a few monophyletic populations. The majority of German Fth isolates belong to two basal phylogenetic clades B.6 (biovar I) and B.12 (biovar II). Strains of B.6 and B.12 seem to differ in their pathogenicity, and it has been shown that strains of biovar II are resistant against erythromycin. In this study, we present data corroborating our previous data demonstrating that basal clade B.12 can be divided into clades B.71 and B.72. By applying phylogenetic whole genome analysis as well as proteome analysis, we could verify that strains of these two clades are distinct from one another. This was confirmed by measuring the intensity of backscatter light on bacteria grown in liquid media. Strains belonging to clades B.6, B.71 or B.72 showed clade-specific backscatter growth curves. Furthermore, we present the whole genome sequence of strain A-1341, as a reference genome of clade B.71, and whole proteomes comparison of Fth strains belonging to clades B.6, B.71 and B.72. Further research is necessary to investigate phenotypes and putative differences in pathogenicity of the investigated different clades of Fth to better understand the relationship between observed phenotypes, pathogenicity and distribution of Fth strains.

Isolation of Brucella inopinata from a White's tree frog (Litoria caerulea): pose exotic frogs a potential risk to human health?

Introduction: Cold-blooded hosts, particularly exotic frogs, have become a newly recognized reservoir for atypical Brucella species and strains worldwide, but their pathogenicity to humans remains largely unknown. Here we report the isolation and molecular characterization of a B. inopinata strain (FO700662) cultured from clinical samples taken from a captive diseased White's Tree Frog (Litoria caerulea) in Switzerland. The isolation of B. inopinata from a frog along with other reports of human infection by atypical Brucella raises the question of whether atypical Brucella could pose a risk to human health and deserves further attention. Methods: The investigations included histopathological analysis of the frog, bacterial culture and in-depth molecular characterization of strain FO700662 based on genome sequencing data. Results and Discussion: Originally identified as Ochrobactrum based on its rapid growth and biochemical profile, strain FO700622 was positive for the Brucella- specific markers bcsp31 and IS711. It showed the specific banding pattern of B. inopinata in conventional Bruce-ladder multiplex PCR and also had identical 16S rRNA and recA gene sequences as B. inopinata. Subsequent genome sequencing followed by core genome-based MLST (cgMLST) analysis using 2704 targets (74% of the total chromosome) revealed only 173 allelic differences compared to the type strain of B. inopinata BO1T, while previously considered the closest related strain BO2 differed in 2046 alleles. The overall average nucleotide identity (ANI) between the type strain BO1T and FO700622 was 99,89%, confirming that both strains were almost identical. In silico MLST-21 and MLVA-16 also identified strain FO700662 as B. inopinata. The nucleotide and amino acid-based phylogenetic reconstruction and comparative genome analysis again placed the isolate together with B. inopinata with 100% support. In conclusion, our data unequivocally classified strain FO700622, isolated from an exotic frog, as belonging to B. inopinata.

Genetic diversity and spatial distribution of Burkholderia mallei by core genome-based multilocus sequence typing analysis.

Burkholderia mallei is the etiological agent of glanders, a highly contagious and often fatal disease in equids. Due to the high genetic clonality of B. mallei, high-resolution typing assays are necessary to differentiate between individual strains. Here we report on the development and validation of a robust and reproducible core genome-based Multi Locus Sequence Typing Assay (cgMLST) for B. mallei, which is based on 3328 gene targets and enables high-resolution typing at the strain level. The assay was validated using a set of 120 B. mallei genomes from public databases and 23 newly sequenced outbreak strains from in-house strain collections. In this cgMLST analysis, strains from different geographic regions were clearly distinguished by at least 70 allele differences, allowing spatial clustering while closely related and epidemiologically related strains were separated by only zero to three alleles. Neither the different sequencing technologies nor the assembly strategies had an influence on the cgMLST results. The developed cgMLST is highly robust, reproducible and can be used for outbreak investigations, source tracking and molecular characterization of new B. mallei isolates.

The Needle in the Haystack-Searching for Genetic and Epigenetic Differences in Monozygotic Twins Discordant for Tetralogy of Fallot.

Congenital heart defects (CHDs) are the most common birth defect in human with an incidence of almost 1% of all live births. Most cases have a multifactorial origin with both genetics and the environment playing a role in its development and progression. Adding an epigenetic component to this aspect is exemplified by monozygotic twins which share the same genetic background but have a different disease status. As a result, the interplay between the genetic, epigenetic and the environmental conditions might contribute to the etiology and phenotype. To date, the underlying causes of the majority of CHDs remain poorly understood. In this study, we performed genome-wide high-throughput sequencing to examine the genetic, structural genomic and epigenetic differences of two identical twin pairs discordant for Tetralogy of Fallot (TOF), representing the most common cyanotic form of CHDs. Our results show the almost identical genetic and structural genomic identity of the twins. In contrast, several epigenetic alterations could be observed given by DNA methylation changes in regulatory regions of known cardiac-relevant genes. Overall, this study provides first insights into the impact of genetic and especially epigenetic factors underlying monozygotic twins discordant for CHD like TOF.

Complete Genome Sequence of Francisella tularensis subsp. holarctica Strain A271_1 (FDC408), Isolated from a Eurasian Beaver (Castor fiber).

Here, we report the complete genome sequence of Francisella tularensis subsp. holarctica strain A271_1, isolated from a Eurasian beaver (Castor fiber) in 2012 in the Berlin/Brandenburg region, Germany.

Zoonotic infection with swine A/H1avN1 influenza virus in a child, Germany, June 2020.

A zoonotic A/sw/H1avN1 1C.2.2 influenza virus infection was detected in a German child that presented with influenza-like illness, including high fever. There was a history of close contact with pigs 3 days before symptom onset. The child recovered within 3 days. No other transmissions were observed. Serological investigations of the virus isolate revealed cross-reactions with ferret antisera against influenza A(H1N1)pdm09 virus, indicating a closer antigenic relationship with A(H1N1)pdm09 than with the former seasonal H1N1 viruses.

Assessment of biorisk management systems in high containment laboratories, 18 countries in Europe, 2016 and 2017.

Europe-wide activities to improve biosafety and biosecurity performed within the frameworks of the European Union (EU)-funded Joint Actions EMERGE and QUANDHIP led to the development of an Integrated European Checklist for Laboratory Biorisk Management (ECL).To better understand different approaches shaping biorisk management (BRM) systems on an operational level in high containment laboratories, the ECL was used to map the implementation of BRM in 32 high containment laboratories in 18 countries in Europe. The results suggest that the BRM elements referring to standard microbiological working practices and the handling of infectious material were fulfilled particularly well. The elements safety exercises involving internal and external emergency responders, and appropriate decommissioning plans were not fulfilled particularly well. BRM in Biosafety Level (BSL) 4 laboratories handling Risk Group (RG) 4 viruses appear to vary among each other less than BSL3 laboratories handling RG 3 bacteria. It is important to agree on comparable regulations in Europe as high containment laboratories are indispensable for a safe, quick and effective response to public health threats. As high containment laboratories may also present a public health risk it is crucial to have robust BRM on organisational and operational levels.

Francisella tularensis Subspecies holarctica and Tularemia in Germany.

Tularemia is a zoonotic disease caused by Francisella tularensis a small, pleomorphic, facultative intracellular bacterium. In Europe, infections in animals and humans are caused mainly by Francisella tularensis subspecies holarctica. Humans can be exposed to the pathogen directly and indirectly through contact with sick animals, carcasses, mosquitoes and ticks, environmental sources such as contaminated water or soil, and food. So far, F. tularensis subsp. holarctica is the only Francisella species known to cause tularemia in Germany. On the basis of surveillance data, outbreak investigations, and literature, we review herein the epidemiological situation-noteworthy clinical cases next to genetic diversity of F. tularensis subsp. holarctica strains isolated from patients. In the last 15 years, the yearly number of notified cases of tularemia has increased steadily in Germany, suggesting that the disease is re-emerging. By sequencing F. tularensis subsp. holarctica genomes, knowledge has been added to recent findings, completing the picture of genotypic diversity and geographical segregation of Francisella clades in Germany. Here, we also shortly summarize the current knowledge about a new Francisella species (Francisella sp. strain W12-1067) that has been recently identified in Germany. This species is the second Francisella species discovered in Germany.

Induced pluripotent stem cells of patients with Tetralogy of Fallot reveal transcriptional alterations in cardiomyocyte differentiation.

Patient-specific induced pluripotent stem cells (ps-iPSCs) and their differentiated cell types are a powerful model system to gain insight into mechanisms driving early developmental and disease-associated regulatory networks. In this study, we use ps-iPSCs to gain insights into Tetralogy of Fallot (TOF), which represents the most common cyanotic heart defect in humans. iPSCs were generated and further differentiated to cardiomyocytes (CMs) using standard methods from two well-characterized TOF patients and their healthy relatives serving as controls. Patient-specific expression patterns and genetic variability were investigated using whole genome and transcriptome sequencing data. We first studied the clonal mutational burden of the derived iPSCs. In two out of three iPSC lines of patient TOF-01, we found a somatic mutation in the DNA-binding domain of tumor suppressor P53, which was not observed in the genomic DNA from blood. Further characterization of this mutation showed its functional impact. For patient TOF-02, potential disease-relevant differential gene expression between and across cardiac differentiation was shown. Here, clear differences at the later stages of differentiation could be observed between CMs of the patient and its controls. Overall, this study provides first insights into the complex molecular mechanisms underlying iPSC-derived cardiomyocyte differentiation and its transcriptional alterations in TOF.

Altered microRNA and target gene expression related to Tetralogy of Fallot.

MicroRNAs (miRNAs) play an important role in guiding development and maintaining function of the human heart. Dysregulation of miRNAs has been linked to various congenital heart diseases including Tetralogy of Fallot (TOF), which represents the most common cyanotic heart malformation in humans. Several studies have identified dysregulated miRNAs in right ventricular (RV) tissues of TOF patients. In this study, we profiled genome-wide the whole transcriptome and analyzed the relationship of miRNAs and mRNAs of RV tissues of a homogeneous group of 22 non-syndromic TOF patients. Observed profiles were compared to profiles obtained from right and left ventricular tissue of normal hearts. To reduce the commonly observed large list of predicted target genes of dysregulated miRNAs, we applied a stringent target prediction pipeline integrating probabilities for miRNA-mRNA interaction. The final list of disease-related miRNA-mRNA pairs comprises novel as well as known miRNAs including miR-1 and miR-133, which are essential to cardiac development and function by regulating KCNJ2, FBN2, SLC38A3 and TNNI1. Overall, our study provides additional insights into post-transcriptional gene regulation of malformed hearts of TOF patients.

Genetic Diversity and Spatial Segregation of Francisella tularensis Subspecies holarctica in Germany.

Francisella tularensis is an intracellular pleomorphic bacterium and the causative agent of tularemia, a zoonotic disease with a wide host range. Among the F. tularensis subspecies, especially F. tularensis subsp. holarctica is of clinical relevance for European countries. The study presented herein focuses namely on genetic diversity and spatial segregation of F. tularensis subsp. holarctica in Germany, as still limited information is available. The investigation is based on the analysis of 34 F. tularensis subsp. holarctica isolates and one draft genome from an outbreak strain. The isolates were cultured from sample material being that of primarily human patients (n = 25) and free-living animals (n = 9). For six of 25 human isolates, epidemiological links between disease onset and tick bites could be established, confirming the importance of arthropod linked transmission of tularemia in Germany. The strains were assigned to three of four major F. tularensis subsp. holarctica clades: B.4, B.6, and B.12. Thereby, B.6 and B.12 clade members were predominantly found; only one human isolate was assigned to clade B.4. Also, it turned out that eight isolates which caused pneumonia in patients clustered into the B.6 clade. Altogether, eight different final subclades were assigned to clade B.6 (biovar I, erythromycin sensitive) and six to B.12 (biovar II, erythromycin resistant) in addition to one new final B.12 subclade. Moreover, for 13 human and 3 animal isolates, final subclade subdivisions were not assigned (B.12 subdivisions B.33 and B.34, and B.6 subdivision B.45) because official nomenclatures are not available yet. This gives credit to the genetic variability of F. tularensis subsp. holarctica strains in Germany. The results clearly point out that the given genetic diversity in Germany seems to be comparably high to that found in other European countries including Scandinavian regions. A spatial segregation of B.6 and B.12 strains was found and statistically confirmed, and B.12 clade members were predominantly found in eastern parts and B.6 members more in western to southern parts of Germany. The portion of B.12 clade members in northeastern parts of Germany was 78.5% and in southwestern parts 1.9%.

Development and comparison of loop-mediated isothermal amplification and quantitative polymerase chain reaction assays for the detection of Mycoplasma bovis in milk.

Bovine mastitis is an economic burden for dairies worldwide. Mycoplasma species, and especially Mycoplasma bovis, are among the most important causative agents, and rapid, precise, and low-cost methods for Mycoplasma detection are urgently needed. For this purpose, loop-mediated isothermal amplification (LAMP) and quantitative PCR (qPCR) assays were developed and compared. The LAMP assay was designed and primer concentrations optimized to M. bovis oppD, encoding oligopeptide permease D. For qPCR, a Taqman assay (Applied Biosystems, Carlsbad, CA) targeting M. bovis gltX, encoding glutamate transfer RNA ligase, was optimized for primer concentration, annealing temperature, and DNA polymerase. Both assays were similarly sensitive, with a detection limit of approximately 104 to 105M. bovis cells/mL. Both assays were also successful in confirming M. bovis identity in laboratory culture suspensions and in bovine milk. The LAMP and qPCR assays combined with the MoBio DNA extraction kit (MoBio Laboratories Inc., Carlsbad, CA) resulted in the correct detection of 13 out of 13 M. bovis isolates and 14 out of 16 M. bovis-positive milk samples collected from commercial dairies in California. When combined with the PrepMan Ultra reagent (Applied Biosystems), the qPCR assay resulted in confirming 21 out of 21 M. bovis-positive milk samples. Comparison of the assays to milk containing either Mycoplasma arginini, Mycoplasma bovigenitalium, Mycoplasma californicum, M. alkalescens, or Acholeplasma laidlawii or milk lacking any detectable Mycoplasma species or relatives resulted in 3 out of 17 (LAMP with MoBio), 1 out of 17 (qPCR with MoBio), and 2 out of 36 (qPCR with PrepMan Ultra) false positives. Overall, the qPCR assay was more robust than LAMP and could be used on DNA recovered from milk prepared with the PrepMan Ultra reagent, a method that does not include a DNA purification step. The use of this qPCR method enables M. bovis detection in bovine milk in 40 to 55 min, and therefore provides new opportunities to accelerate and simplify M. bovis detection in unpasteurized milk to reduce the incidence of M. bovis mastitis outbreaks.

The Flagellar Regulon of Legionella-A Review.

The Legionella genus comprises more than 60 species. In particular, Legionella pneumophila is known to cause severe illnesses in humans. Legionellaceae are ubiquitous inhabitants of aquatic environments. Some Legionellaceae are motile and their motility is important to move around in habitats. Motility can be considered as a potential virulence factor as already shown for various human pathogens. The genes of the flagellar system, regulator and structural genes, are structured in hierarchical levels described as the flagellar regulon. Their expression is modulated by various environmental factors. For L. pneumophila it was shown that the expression of genes of the flagellar regulon is modulated by the actual growth phase and temperature. Especially, flagellated Legionella are known to express genes during the transmissive phase of growth that are involved in the expression of virulence traits. It has been demonstrated that the alternative sigma-28 factor is part of the link between virulence expression and motility. In the following review, the structure of the flagellar regulon of L. pneumophila is discussed and compared to other flagellar systems of different Legionella species. Recently, it has been described that Legionella micdadei and Legionella fallonii contain a second putative partial flagellar system. Hence, the report will focus on flagellated and non-flagellated Legionella strains, phylogenetic relationships, the role and function of the alternative sigma factor (FliA) and its anti-sigma-28 factor (FlgM).

Human Coprolites as a Source for Paleomicrobiology.

The paleomicrobiology of coprolites, which are fossilized fecal materials, has already yielded data about various organisms, including micro-eukaryotes, bacteria, and archaea, thus expanding our comprehension of ancient human dietary habits, gut microbiota, and intestinal and systemic infections. This mini-review briefly describes previous works and summarizes the main techniques used in handling coprolites and the findings obtained about ancient gut microbiota. Past intestinal and systemic infections are outlined.

Sequence-Specific Electrical Purification of Nucleic Acids with Nanoporous Gold Electrodes.

Nucleic-acid-based biosensors have enabled rapid and sensitive detection of pathogenic targets; however, these devices often require purified nucleic acids for analysis since the constituents of complex biological fluids adversely affect sensor performance. This purification step is typically performed outside the device, thereby increasing sample-to-answer time and introducing contaminants. We report a novel approach using a multifunctional matrix, nanoporous gold (np-Au), which enables both detection of specific target sequences in a complex biological sample and their subsequent purification. The np-Au electrodes modified with 26-mer DNA probes (via thiol-gold chemistry) enabled sensitive detection and capture of complementary DNA targets in the presence of complex media (fetal bovine serum) and other interfering DNA fragments in the range of 50-1500 base pairs. Upon capture, the noncomplementary DNA fragments and serum constituents of varying sizes were washed away. Finally, the surface-bound DNA-DNA hybrids were released by electrochemically cleaving the thiol-gold linkage, and the hybrids were iontophoretically eluted from the nanoporous matrix. The optical and electrophoretic characterization of the analytes before and after the detection-purification process revealed that low target DNA concentrations (80 pg/µL) can be successfully detected in complex biological fluids and subsequently released to yield pure hybrids free of polydisperse digested DNA fragments and serum biomolecules. Taken together, this multifunctional platform is expected to enable seamless integration of detection and purification of nucleic acid biomarkers of pathogens and diseases in miniaturized diagnostic devices.

Screening for single nucleotide variants, small indels and exon deletions with a next-generation sequencing based gene panel approach for Usher syndrome.

Usher syndrome is an autosomal recessive disorder characterized both by deafness and blindness. For the three clinical subtypes of Usher syndrome causal mutations in altogether 12 genes and a modifier gene have been identified. Due to the genetic heterogeneity of Usher syndrome, the molecular analysis is predestined for a comprehensive and parallelized analysis of all known genes by next-generation sequencing (NGS) approaches. We describe here the targeted enrichment and deep sequencing for exons of Usher genes and compare the costs and workload of this approach compared to Sanger sequencing. We also present a bioinformatics analysis pipeline that allows us to detect single-nucleotide variants, short insertions and deletions, as well as copy number variations of one or more exons on the same sequence data. Additionally, we present a flexible in silico gene panel for the analysis of sequence variants, in which newly identified genes can easily be included. We applied this approach to a cohort of 44 Usher patients and detected biallelic pathogenic mutations in 35 individuals and monoallelic mutations in eight individuals of our cohort. Thirty-nine of the sequence variants, including two heterozygous deletions comprising several exons of USH2A, have not been reported so far. Our NGS-based approach allowed us to assess single-nucleotide variants, small indels, and whole exon deletions in a single test. The described diagnostic approach is fast and cost-effective with a high molecular diagnostic yield.

Polyphasic analysis of a middle ages coprolite microbiota, Belgium.

Paleomicrobiological investigations of a 14(th)-century coprolite found inside a barrel in Namur, Belgium were done using microscopy, a culture-dependent approach and metagenomics. Results were confirmed by ad hoc PCR--sequencing. Investigations yielded evidence for flora from ancient environment preserved inside the coprolite, indicated by microscopic observation of amoebal cysts, plant fibers, seeds, pollens and mold remains. Seventeen different bacterial species were cultured from the coprolite, mixing organisms known to originate from the environment and organisms known to be gut inhabitants. Metagenomic analyses yielded 107,470 reads, of which known sequences (31.9%) comprised 98.98% bacterial, 0.52% eukaryotic, 0.44% archaeal and 0.06% viral assigned reads. Most abundant bacterial phyla were Proteobacteria, Gemmatimonadetes, Actinobacteria and Bacteroidetes. The 16 S rRNA gene dataset yielded 132,000 trimmed reads and 673 Operational Taxonomic Units. Most abundant bacterial phyla observed in the 16 S rRNA gene dataset belonged to Proteobacteria, Firmicutes, Actinobacteria and Chlamydia. The Namur coprolite yielded typical gut microbiota inhabitants, intestinal parasites Trichuris and Ascaris and systemic pathogens Bartonella and Bordetella. This study adds knowledge to gut microbiota in medieval times.

Viruses in a 14th-century coprolite.

Coprolites are fossilized fecal material that can reveal information about ancient intestinal and environmental microbiota. Viral metagenomics has allowed systematic characterization of viral diversity in environmental and human-associated specimens, but little is known about the viral diversity in fossil remains. Here, we analyzed the viral community of a 14th-century coprolite from a closed barrel in a Middle Ages site in Belgium using electron microscopy and metagenomics. Viruses that infect eukaryotes, bacteria, and archaea were detected, and we confirmed the presence of some of them by ad hoc suicide PCR. The coprolite DNA viral metagenome was dominated by sequences showing homologies to phages commonly found in modern stools and soil. Although their phylogenetic compositions differed, the metabolic functions of the viral communities have remained conserved across centuries. Antibiotic resistance was one of the reconstructed metabolic functions detected.