German Strains Research Articles

Identification of New Microfoci and Genetic Characterization of Tick-Borne Encephalitis Virus Isolates from Eastern Germany and Western Poland.

(1) Background: Tick-borne encephalitis (TBE) is the most important tick-borne viral disease in Eurasia, although effective vaccines are available. Caused by the tick-borne encephalitis virus (TBEV, syn. Orthoflavivirus encephalitidis), in Europe, it is transmitted by ticks like Ixodes ricinus and Dermacentor reticulatus. TBEV circulates in natural foci, making it endemic to specific regions, such as southern Germany and northeastern Poland. Our study aimed to identify new TBEV natural foci and genetically characterize strains in ticks in previously nonendemic areas in Eastern Germany and Western Poland. (2) Methods: Ticks were collected from vegetation in areas reported by TBE patients. After identification, ticks were tested for TBEV in pools of a maximum of 10 specimens using real-time RT-PCR. From the positive TBEV samples, E genes were sequenced. (3) Results: Among 8400 ticks from 19 sites, I. ricinus (n = 4784; 56.9%) was predominant, followed by D. reticulatus (n = 3506; 41.7%), Haemaphysalis concinna (n = 108; 1.3%), and I. frontalis (n = 2; <0.1%). TBEV was detected in 19 pools originating in six sites. The phylogenetic analyses revealed that TBEV strains from Germany and Poland clustered with other German strains, as well as those from Finland and Estonia. (4) Conclusions: Although there are still only a few cases are reported from these areas, people spending much time outdoors should consider TBE vaccination.

Comparative Genomic Analysis of Staphylococcal Cassette Chromosome mec Type V Staphylococcus aureus Strains and Estimation of the Emergence of SCCmec V Clinical Isolates in Korea.

Staphylococcal cassette chromosome mec type V (SCCmec V) methicillin-resistant Staphylococcus aureus (MRSA) has been recovered from patients and livestock. Using comparative genomic analyses, we evaluated the phylogenetic emergence of SCCmec V after transmission from overseas donor strains to Korean recipient strains. Sixty-three complete MRSA SCCmec V genomes (including six Korean clinical isolates) were used to construct a phylogenetic tree. Single-nucleotide polymorphisms were identified using Snippy, and a maximum-likelihood-based phylogenetic tree was constructed using RAxML. The possible emergence of the most common ancestor was estimated using BactDating. To estimate mecA horizontal gene transfer (HGT) events, Ranger-dtl was applied to 818 SCCmec V strains using publicly available whole-genome data. The phylogenetic tree showed five major clades. German strains formed a major clade; their possible origin was traced to the 1980s. The emergence of Korean SCCmec V clinical isolates was traced to 2000-2010. mecA HGT events in Staphylococcus spp. were identified in seven strains. P7 (Hong Kong outbreak strain) served as the donor strain for two Korean sequence type (ST) 59 strains, whereas the other five recipient strains emerged from different SCCmec V donors. Most Korean SCCmec V strains may have emerged during 2000-2010. A unique MRSA SCCmec V strain, ST72 (a Korean common type of community-associated MRSA), was also identified. The genomic dynamics of this clone with a zoonotic background should be monitored to accurately understand MRSA evolution.

A comprehensive analysis of the genetic diversity and environmental adaptability in worldwide Merino and Merino-derived sheep breeds

BackgroundTo enhance and extend the knowledge about the global historical and phylogenetic relationships between Merino and Merino-derived breeds, 19 populations were genotyped with the OvineSNP50 BeadChip specifically for this study, while an additional 23 populations from the publicly available genotypes were retrieved. Three complementary statistical tests, Rsb (extended haplotype homozygosity between-populations), XP-EHH (cross-population extended haplotype homozygosity), and runs of homozygosity (ROH) islands were applied to identify genomic variants with potential impact on the adaptability of Merino genetic type in two contrasting climate zones.ResultsThe results indicate that a large part of the Merino’s genetic relatedness and admixture patterns are explained by their genetic background and/or geographic origin, followed by local admixture. Multi-dimensional scaling, Neighbor-Net, Admixture, and TREEMIX analyses consistently provided evidence of the role of Australian, Rambouillet and German strains in the extensive gene introgression into the other Merino and Merino-derived breeds. The close relationship between Iberian Merinos and other South-western European breeds is consistent with the Iberian origin of the Merino genetic type, with traces from previous contributions of other Mediterranean stocks. Using Rsb and XP-EHH approaches, signatures of selection were detected spanning four genomic regions located on Ovis aries chromosomes (OAR) 1, 6 and 16, whereas two genomic regions on OAR6, that partially overlapped with the previous ones, were highlighted by ROH islands. Overall, the three approaches identified 106 candidate genes putatively under selection. Among them, genes related to immune response were identified via the gene interaction network. In addition, several candidate genes were found, such as LEKR1, LCORL, GHR, RBPJ, BMPR1B, PPARGC1A, and PRKAA1, related to morphological, growth and reproductive traits, adaptive thermogenesis, and hypoxia responses.ConclusionsTo the best of our knowledge, this is the first comprehensive dataset that includes most of the Merino and Merino-derived sheep breeds raised in different regions of the world. The results provide an in-depth picture of the genetic makeup of the current Merino and Merino-derived breeds, highlighting the possible selection pressures associated with the combined effect of anthropic and environmental factors. The study underlines the importance of Merino genetic types as invaluable resources of possible adaptive diversity in the context of the occurring climate changes.

Diddensiella parasantjacobensis f.a., sp. nov., a yeast species from forest habitats.

Six conspecific yeast strains, representing an undescribed species, were isolated from rotten wood collected in different locations in Hungary and Germany and an additional one from fungal fruiting body in Taiwan. The seven strains share identical nucleotide sequences in the D1/D2 domain of the nuclear large subunit (LSU) rRNA gene. The Hungarian and Taiwanese isolates share identical internal transcribed spacer (ITS) sequences as well, while the two German isolates differ from them merely by three substitutions and four indels in this region. The investigated strains are very closely related to Diddensiella santjacobensis. Along their LSU D1/D2 domain they differ only by one substitution from the type strain of D. santjacobensis. However, in the ITS region of Hungarian and Taiwanese strains we detected 3.5 % divergence (nine substitutions and nine indels) between the undescribed species and D. santjacobensis, while the German strains differed by 13 substitutions and nine indels from D. santjacobensis. This ITS sequence divergence has raised the possibility that the strains investigated in this study may represent a different species from D. santjacobensis. This hypothesis was supported by comparisons of partial translation elongation factor 1-α (EF-1α) and cytochrome oxidase II (COX II) gene sequences. While no difference and 1-2 substitutions among the partial EF-1α and COX II gene sequences of the strains of the undescribed species, respectively, were detected; the undescribed species differ by about 4 % (36 substitutions) and 10 % (50-51 substitutions) from D. santjacobensis in these regions. Parsimony network analysis of the partial COX II gene sequences also separated the investigated strains from the type strain of D. santjacobensis. In this paper we propose Diddensiella parasantjacobensis f.a., sp. nov. (holotype: NCAIM Y.02121; isotypes: CBS 17819, DSM 114156) to accommodate the above-noted strains.

Simultaneous Coinfections with West Nile Virus and Usutu Virus in Culex pipiens and Aedes vexans Mosquitoes

The mosquito-borne zoonotic flaviviruses West Nile virus (WNV) and Usutu virus (USUV) are endemic in many European countries and emerged in Germany in recent years. Due to the increasing overlap of their distribution areas and their similar epidemiology, coinfections of WNV and USUV are possible. Indeed, coinfections in vertebrate hosts as a rare event have already been reported from some countries including Germany. However, it is largely unknown whether and to what extent coinfections could affect the vector competence of mosquitoes for WNV and USUV. For this purpose, the mosquito species Culex pipiens biotype pipiens, Culex pipiens biotype molestus, and Aedes vexans were orally infected in mono- and simultaneous coinfections with German strains of WNV and USUV. Mosquitoes were incubated for 14 days at 26°C, 85% relative humidity, and a 16 : 8 light-dark photocycle, before they were dissected and forced to salivate. The results showed a decrease in USUV susceptibility in Culex pipiens biotype pipiens, an increase in USUV susceptibility in Aedes vexans, and no obvious interaction between both viruses in Culex pipiens biotype molestus. Vector competence for WNV appeared to be unaffected by a simultaneous occurrence of USUV in all tested mosquito species. Coinfections with both viruses were only found in Culex mosquitoes, and cotransmission of WNV and USUV was observed in Culex pipiens biotype molestus. Overall, our results show that viral interactions between WNV and USUV vary between mosquito species, and that the interaction mainly occurs during infection and replication in the mosquito midgut. The results of this study confirm that to fully understand the interaction between WNV and USUV, studies with various mosquito species are necessary. In addition, we found that even mosquito species with a low susceptibility to both viruses, such as Ae. vexans, can play a role in their transmission in areas with cocirculation.

The detection and phylogenetic analysis of equine herpesviruses 1, 4 and 5 identified in nasal swab samples of asymptomatic horses from Serbia and Bosnia and Herzegovina.

Nasal swabs originating from 112 apparently clinically healthy and unvaccinated horses of different age, breed and from diverse rearing conditions from Serbia and Bosnia and Herzegovina were examined for the presence of equine herpesviruses 1, 4 and 5 using multiplex nested PCR (Mn‑PCR) and virus isolation. The detected viruses were subsequently characterised by gB gene nucleotide sequencing and their phylogenetic analysis was performed. The infections with EHV‑1, EHV‑4, and EHV‑5 in the examined horse populations are apparently chronic, subclinical and persistent, whilst the shedding of EHV‑1 and EHV‑5 was confirmed by their successful isolation. A connection was established between the finding of EHVs and rearing conditions since horses kept together in stables were positive for at least one EHV in contrast to animals held free grazing or individually. EHV‑5 was found most often in younger horses, however descending in frequency in animals up to 10 years of age. The phylogenetic analysis showed that the identified EHV strains group mostly with Turkish and German strains of respective viruses. A certain degree of genetic heterogeneity was determined regarding the identified EHV‑5 strains in contrast to EHV‑1 and EHV‑4.

Genomic and Micro-Evolutionary Features of Mammalian 2 orthobornavirus (Variegated Squirrel Bornavirus 1, VSBV-1).

Mammalian 2 orthobornavirus (VSBV-1) is an emerging zoonotic pathogen discovered in several exotic squirrel species and associated with fatal human encephalitis. The dynamics of VSBV-1 spread and evolution in its presumed natural hosts are unknown. Here, we present the phylogeny, micro-evolution, cross-species transmission and spread of VSBV-1 at a temporal and spatial resolution within the limits of animal husbandry. The results showed that VSBV-1 can be classified into six distinct groups and that the most recent common ancestor of the known German strains emerged at least 20 years ago. We here demonstrate that the genetic diversity of the VSBV-1 groups is shaped primarily by in situ evolution and most of the amino acid changes are deleterious polymorphisms removed by purifying selection. Evidence of adaptive evolution has been found in the G and L genes which might have an influence on transmission fitness. Furthermore, there was also evidence for some form of adaptive changes in the glycoprotein which suggests that many sites might be subjected to positive pressure evolving under episodic directional selection, indicating past occurrence of positive selection. Host switching events were detected as dominant evolutionary mechanisms driving the virus-host associations. Virus spread by animal trade followed by subsequent local micro-evolution in zoos and holdings is responsible for diversifying strains. Time-resolved phylogeny indicated that Prevost’s squirrels might be the original squirrel species carrying and seeding the virus in Germany. This study provides the first insight into the ecology and micro-evolutionary dynamics of this novel viral pathogen in the captive exotic squirrel population under artificial ecological conditions (zoos and animal husbandry) and co-housing of different squirrel species.

Antimicrobial Resistance and in silico Virulence Profiling of Aliarcobacter butzleri Strains From German Water Poultry.

Aliarcobacter butzleri is an emerging foodborne and zoonotic pathogen that is usually transmitted via contaminated food or water. A. butzleri is not only the most prevalent Aliarcobacter species, it is also closely related to thermophilic Campylobacter, which have shown increasing resistance in recent years. Therefore, it is important to assess its resistance and virulence profiles. In this study, 45 Aliarcobacter butzleri strains from water poultry farms in Thuringia, Germany, were subjected to an antimicrobial susceptibility test using the gradient strip diffusion method and whole-genome sequencing. In the phylogenetic analysis, the genomes of the German strains showed high genetic diversity. Thirty-three isolates formed 11 subgroups containing two to six strains. The antimicrobial susceptibility testing showed that 32 strains were resistant to erythromycin, 26 to doxycycline, and 20 to tetracycline, respectively. Only two strains were resistant to ciprofloxacin, while 39 strains were resistant to streptomycin. The in silico prediction of the antimicrobial resistance profiles identified a large repertoire of potential resistance mechanisms. A strong correlation between a gyrA point mutation (Thr-85-Ile) and ciprofloxacin resistance was found in 11 strains. A partial correlation was observed between the presence of the bla3 gene and ampicillin resistance. In silico virulence profiling revealed a broad spectrum of putative virulence factors, including a complete lipid A cluster in all studied genomes.

Sindbis Virus Strains of Divergent Origin Isolated from Humans and Mosquitoes During a Recent Outbreak in Finland.

Sindbis virus (SINV) is a mosquito-borne avian hosted virus that is widely distributed in Europe, Africa, Asia, and Oceania. Disease in humans is documented mainly from Northern Europe and South Africa and associated with genotype I. In 2018 under extremely warm climatic conditions, a small outbreak of 71 diagnosed SINV infections was recorded in Finland. We screened 52 mosquito pools (570 mosquitoes) and 223 human sera for SINV with real-time RT-PCR and the positive samples with virus isolation. One SINV strain was isolated from a pool (n = 13) of genus Ochlerotatus mosquitoes and three strains from patient serum samples. Complete genome analysis suggested all the isolates to be divergent from one another and related to previous Finnish, Swedish, and German strains. The study provides evidence of SINV strain transfer within Europe across regions with different epidemiological characteristics. Whether these are influenced by different mosquito genera involved in the transmission remains to be studied.

Rotavirus infection in a racing pigeon (Columba livia) in Great Britain during 2018

We report the first diagnosis of fatal rotavirus A (RVA) infection in a racing pigeon in Great Britain. In August 2018, a small group of racing pigeons (Columba livia) from...

Genomic Analysis and Antimicrobial Resistance of Aliarcobacter cryaerophilus Strains From German Water Poultry.

Aliarcobacter cryaerophilus (formerly Arcobacter cryaerophilus) is a globally emerging foodborne and zoonotic pathogen. However, little is known about the species’ genomic features and diversity, antibiotic resistance and virulence. In this study, 27 A. cryaerophilus strains from water poultry in Thuringia, Germany, were investigated using whole-genome sequencing. Four of these strains were sequenced using long- and short-read sequencing methods to obtain circularized genomes. The German strains belong to the A. cryaerophilus cluster I. Cluster I genomes exhibited a high degree of genetic diversity in which variable sites comprised 9.1% of the core genome. The German strains formed three subgroups that contained 2, 6, and 9 strains, respectively. The genomic analysis of cluster I revealed variable presence of mobile elements and that 65% of the strains lack CRISPR systems. The four circularized genomes carried a ∼2 Mbp chromosome and a single megaplasmid (size 98.1–154.5 Kbp). The chromosome was densely packed with coding sequences (∼92%) and showed inversions and shifts in the gene blocks between different strains. Antimicrobial resistance was assessed using a gradient strip diffusion method and showed that all 27 strains were resistant to cefotaxime and susceptible to erythromycin, gentamicin, and ampicillin. Sixteen strains were also resistant to ciprofloxacin, whereas 23 were resistant to streptomycin. The genetic prediction of antibiotic resistance identified numerous efflux pumps similar to those found in A. butzleri. All strains harbored two beta-lactamase genes which may explain the cefotaxime resistance. A correlation between the gyrA point mutation (Thr-85-Ile) and ciprofloxacin resistance was partially discovered in 15 out of 16 strains. In silico virulence profiling showed a wide range of virulence factors including a full chemotaxis system and most of the flagellar genes. In contrast to A. butzleri, no urease cluster was found. This study provides new insights into the genomic variability of A. cryaerophilus strains of cluster I. The different genetic makeup of these strains may contribute to the virulence of strains and the severity of the infections in humans.

Izozyme polymorphism of alpha-amylase in soft wheat

Aim. Study of alpha amylase polymorphism in winter wheat varieties. Methods. Isoenzymes of alpha-amylase were detected by electrophoretic protein separation in a polyacrylamide gel. The fall number on the Falling Number 1700 was determined. Results. Strains from various collections were explored: Institute of Plant Physiology and Genetics of the National Academy of Sciences of Ukraine, Kiev, The Plant Breeding and Genetics Institute – National Center of Seed and Cultivar Investigation of the National Academy of Agricultural Sciences of Ukraine, Odesa and german strains. The polymorphism of wheat grain alpha-amylase was detected by the method of electrophoretic protein separation. We searched associations between variants of alpha-amylase and resistance to pre-harvest germination, the falling number in the examined samples was determined. Conclusions. The frequency of the prevalence of variants for isoenzymes of alpha-amylase in soft wheat varieties was estimated. It was shown that genotypes containing the variant of the isoenzymes АbC are more resistant to pre-harvest germination. Among the studied varieties of PBGI NAAS and IPPG NAS of Ukraine, the variant of alpha-amylase AbC.&#x0D; Keywords: α-amylase, isoenzymes, soft wheat, electrophoresis, falling number.

The hypervirulent Coxiella burnetii Guiana strain compared in silico, in vitro and in vivo to the Nine Mile and the German strain

ObjectiveQ fever epidemic outbreaks have been reported in French Guiana and in The Netherlands. To determine whether the C. burnetii strains involved in these epidemics had a peculiar virulence pattern, we compared the pathogenicity of the Guiana and the German strain (a clone of The Netherlands strain), in silico, in vitro, and in vivo versus the Nine Mile strain. MethodThe pan-genomes of the Guiana (Cb175), German (Z3055), and the referent Nine Mile (RSA 493) C. burnetii strains were compared. In vitro, the growth rate and the morphological presentation were compared. In vivo (SCID and Balb/c mice), weight loss, histological lesions, C. burnetii bacterial load in deep organs, and serological response were reported according to each C. burnetii strain studied. ResultsThe Guiana strain had 77 times more missing genes and 12 times more unique genes than the German strain. The Guiana strain presented as large cell variants (LCVs) and led to the most pronounced fatality rate in SCID mice (100% at 4 weeks). The German strain presented as small cell variants (SCVs), and had an intermediate fatality rate (75% at 4 weeks). Both the Guiana and the German strains led to a significant higher serological response at 2 and 4 weeks post infection (p <0.05). ConclusionThe Guiana strain was the most virulent strain, followed by the German strain and the referent Nine Mile strain. Unique and missing genes could be implicated but further investigations are necessary to specify their role.

Detection of three novel atypical porcine pestivirus strains in newborn piglets with congenital tremor in southern China

Atypical porcine pestivirus (APPV) have been discovered in swine herds from three provinces in China, suggesting a wide distribution in China. This study reports the occurrence of three novel APPV strains in China. They were detected from newborn piglets with clinical signs of congenital tremors (CT) in Guangdong Province, China. The complete genomic sequences of three novel APPV strains exhibited only 80.5%–84.1% nucleotide sequences homology with other APPV reference sequences available in GenBank. Phylogenetic analysis showed that these novel APPV strains formed independent branch from the American, German, Netherlandish, Australian and other Chinese strains. These results will help us better understand the epidemiology and genetic characteristics of APPV in China.

Molecular diversity of "Candidatus Phytoplasma mali" strains associated with apple proliferation disease in Bulgarian germplasm collection.

A quarantine organism, "Candidatus Phytoplasma mali," is the causal agent of apple proliferation, one of the most important apple diseases in Europe. The genetic diversity of this pathogen in Central and Southern Europe has already been reported; however, almost no data exists from Eastern Europe. In this study, "Ca. P. mali" strains, which were identified in 14 apple trees from the Bulgarian germplasm collection, were characterized by restriction fragment length polymorphism (RFLP) and sequence analysis of four genomic loci. In total, nine distinct genetic lineages were recognized based on the combination of the following detected RFLP profiles: two profiles for the 16S-23S rDNA region (16SrX-A2, -A3), four profiles for the secY gene (one previously known: secY(X)-A, and three new: secY-C, secY-D, secY-E), three profiles for the rpl22-rps3 genes (rpX-A, rpX-B, rpX-F), and one profile for the nitroreductase- and rhodanese-like gene (AT-1). Phylogenetic analysis of the Bulgarian and other European "Ca. P. mali" strains based on 16S-23S rRNA gene sequences confirmed RFLP grouping, regardless of the phytoplasma origin. In a phylogenetic tree based on the secY data, only German strains formed separate clade from the other strains. The tree based on rp genes did not correspond to RFLP profiles. Unexpectedly, when using nitroreductase and rhodanese-like gene sequences, the Bulgarian strains clustered separately from the other European strains. Apart from the identification of different "Ca. P. mali" strains, the paper also recommends the unification of the rpX-subgroup nomenclature to avoid future confusions. Both aims of this paper provide valuable tools to understand the epidemiology of this quarantine pathogen.

Evaluation of Malus gene bank resources with German strains of Marssonina coronaria using a greenhouse-based screening method

Marssonina coronaria is considered a threat to organic apple production in Central Europe. Since the application of fungicides is limited in organic production, breeding for resistance seems to be a promising strategy to manage the disease in the future. In this study, an artificial inoculation method similar to procedures used in apple scab greenhouse screenings was developed using German strains of the pathogen M. coronaria for evaluating 110 Malus domestica cultivars. The strains were morphologically and molecularly characterized and confirmed as M. coronaria. Symptom development was significantly influenced by incubation method, conidia concentration and point of inoculation, but not by leaf position and different water types used for inoculation. The success of inoculation and spread of the fungus on infected leaves were confirmed by conventional PCR. Moreover, there was a difference in development of disease symptoms between inoculations with conidia from in vitro grown strains (in vitro inoculum) and conidia from diseased leaves from the orchard (field inoculum) in a time dependent manner. These differences which were first found with the susceptible cultivar ‘Topaz’ after artificial inoculation with in vitro and field inoculum were confirmed on 20 more apple cultivars inoculated with different in vitro grown strains. In summary, all tested cultivars, including 21 which are scab-resistant, developed symptoms of this disease. Results from three years of investigation indicate a decrease in virulence of M. coronaria strains, when cultivated on artificial culture (growth)-media. Hence field inoculum is recommended for artificial greenhouse screenings for the evaluation of disease resistance in Malus genetic resources.

Variability in the anatoxin gene clusters of Cuspidothrix issatschenkoi from Germany, New Zealand, China and Japan.

Anatoxin-a and homoanatoxin-a are neurotoxic cyanotoxins produced by benthic and planktonic cyanobacteria worldwide. These toxins are produced by the cyanobacterial genera Dolichospermum, Cuspidothrix, Phormidium, Oscillatoria, Tychonema and Cylindrospermum. In the present study the ana gene clusters (anaA-anaG; c. 21.1 kilobases) of two anatoxin producing Cuspidothrix issatschenkoi strains from Germany: (NIVA-CYA 711) and New Zealand (CAWBG02) were sequenced and compared with the ana gene clusters of two C. issatschenkoi strains from Japan (RM-6 and LBRI48) and one from China (CHABD3). All five ana gene clusters are characterized by the same gene order for anaA-anaG. Similarities were highest (99.56–99.57%) between German (NIVA-CYA 711), New Zealand (CAWBG02) and Chinese (CHABD3) strains. Similarities were lower (91.40–91.67%) when compared to the Japanese strains (RM-6 and LBRI48). Collectively, 2,037 variable sites (328 single nucleotide polymorphisms and 9 insertions/deletions, comprising 1,709 nucleotides) were found in the ana gene clusters of the German, New Zealand and Japanese strains compared to the Chinese strain (CHABD3). The ana gene clusters of the German (NIVA-CYA 711), New Zealand (CAWBG02) and Japanese (RM-6 and LBRI48) strains were characterized by 83, 84, 255 and 231 SNP’s compared to the Chinese strain (CHABD3), respectively. The anaE and anaF genes showed the highest variability in all five strains and are recommended as the best genetic markers for further phylogenetic studies of the ana gene cluster from C. issatschenkoi.

Sorbitol-Fermenting Enterohemorrhagic Escherichia coli O157:H- Isolates from Czech Patients with Novel Plasmid Composition Not Previously Seen in German Isolates.

Sorbitol-fermenting (SF) enterohemorrhagic Escherichia coli (EHEC) O157:H- strains, first identified in Germany, have emerged as important pathogens throughout Europe. Besides chromosomally encoded Shiga toxin 2a (the major virulence factor), several putative virulence loci, including the hly, etp, and sfp operons, encoding EHEC hemolysin, type II secretion system proteins, and Sfp fimbriae, respectively, are located on the 121-kb plasmid pSFO157 in German strains. Here we report novel SF EHEC O157:H- strains isolated from patients in the Czech Republic. These strains share the core genomes and chromosomal virulence loci encoding toxins (stx2a and the cdtV-ABC operon) and adhesins (eae-γ, efa1, lpfAO157OI-141, and lpfAO157OI-154) with German strains but differ essentially in their plasmids. In contrast to all previously detected SF EHEC O157:H- strains, the Czech strains carry two plasmids, of 79 kb and 86 kb. The 79-kb plasmid harbors the sfp operon, but neither of the plasmids contains the hly and etp operons. Sequence analyses demonstrated that the 79-kb plasmid (pSFO157 258/98-1) evolved from pSFO157 of German strains by deletion of a 41,534-bp region via homologous recombination, resulting in loss of the hly and etp operons. The 86-kb plasmid (pSFO157 258/98-2) displays 98% sequence similarity to a 92.7-kb plasmid of an extraintestinal pathogenic E. coli bloodstream isolate. Our finding of this novel plasmid composition in SF EHEC O157:H- strains extends the evolutionary history of EHEC O157 plasmids. Moreover, the unique molecular plasmid characteristics permit the identification of such strains, thereby facilitating further investigations of their geographic distribution, clinical significance, and epidemiology.IMPORTANCE Since their first identification in Germany in 1989, sorbitol-fermenting enterohemorrhagic Escherichia coli O157:H- (nonmotile) strains have emerged as important causes of the life-threatening disease hemolytic-uremic syndrome in Europe. They account for 10 to 20% of sporadic cases of this disease and have caused several large outbreaks. The strains isolated throughout Europe share conserved chromosomal and plasmid characteristics. Here we identified novel sorbitol-fermenting enterohemorrhagic E. coli O157:H- patient isolates in the Czech Republic which differ from all such strains reported previously by their unique plasmid characteristics, including plasmid number, composition of plasmid-carried virulence genes, and plasmid origins. Our findings contribute substantially to understanding the evolution of E. coli O157 strains and their plasmids. In practical terms, they enable the identification of strains with these novel plasmid characteristics in patient stool samples and thus the investigation of their roles as human pathogens in other geographic areas.

Molecular characterization of different equine-like G3 rotavirus strains from Germany

The genetic heterogeneity of rotaviruses constitutes a substantial burden to human and animal health. Occasional interspecies transmissions can generate novel virus strains in the human population. We detected equine-like G3P[8] strains in feces sampled from three children in Germany in 2015 and 2016, respectively. Thereof two showed a DS-1-like backbone. In one strain the NSP2 gene segment was of distinct genotype (G3-P[8]-I2-R2-C2-M2-A2-N1-T2-E2-H2). Phylogenetic analyses of the German strains showed a relation to other equine-like G3 rotaviruses circulating in different countries. The reconstruction of reassortment events in the evolution of novel equine-like G3 rotaviruses suggests an independent introduction of the three strains into the local human rotavirus population.

Diversity of Vibrio navarrensis Revealed by Genomic Comparison: Veterinary Isolates Are Related to Strains Associated with Human Illness and Sewage Isolates While Seawater Strains Are More Distant.

Strains of Vibrio navarrensis are present in aquatic environments like seawater, rivers, and sewage. Recently, strains of this species were identified in human clinical specimens. In this study, V. navarrensis strains isolated from livestock in Germany were characterized that were found in aborted fetuses and/or placentas after miscarriages. The veterinary strains were analyzed using phenotypical and genotypical methods and compared to isolates from marine environments of the Baltic Sea and North Sea. The investigated phenotypical traits were similar in all German strains. Whole genome sequencing (WGS) was used to evaluate a phylogenetic relationship by performing a single nucleotide polymorphism (SNP) analysis. For the SNP analysis, WGS data of two American human pathogenic strains and two Spanish environmental isolates from sewage were included. A phylogenetic analysis of concatenated sequences of five protein-coding housekeeping genes (gyrB, pyrH, recA, atpA, and rpoB), was additionally performed. Both phylogenetic analyses reveal a greater distance of the environmental seawater strains to the other strains. The phylogenetic tree constructed from concatenated sequences of housekeeping genes places veterinary, human pathogenic and Spanish sewage strains into one cluster. Presence and absence of virulence-associated genes were investigated based on WGS data and confirmed by PCR. However, this analysis showed no clear pattern for the potentially pathogenic strains. The detection of V. navarrensis in human clinical specimens strongly suggests that this species should be regarded as a potential human pathogen. The identification of V. navarrensis strains in domestic animals implicates a zoonotic potential of this species. This could indicate a potential threat for humans, as according to the “One Health” concept, human, animal, and environmental health are linked. Future studies are necessary to search for reservoirs of these bacteria in the environment and/or in living organisms.