Chemosensory Cell-Derived Acetylcholine Drives Tracheal Mucociliary Clearance in Response to Virulence-Associated Formyl Peptides.

Mucociliary clearance through coordinated ciliary beating is a major innate defense removing pathogens from the lower airways, but the pathogen sensing and downstream signaling mechanisms remain unclear. We identified virulence-associated formylated bacterial peptides that potently stimulated ciliary-driven transport in the mouse trachea. This innate response was independent of formyl peptide and taste receptors but depended on key taste transduction genes. Tracheal cholinergic chemosensory cells expressed these genes, and genetic ablation of these cells abrogated peptide-driven stimulation of mucociliary clearance. Trpm5-deficient mice were more susceptible to infection with a natural pathogen, and formylated bacterial peptides were detected in patients with chronic obstructive pulmonary disease. Optogenetics and peptide stimulation revealed that ciliary beating was driven by paracrine cholinergic signaling from chemosensory to ciliated cells operating through muscarinic M3 receptors independently of nerves. We provide a cellular and molecular framework that defines how tracheal chemosensory cells integrate chemosensation with innate defense.

Genome-wide association reveals host-specific genomic traits in Escherichia coli.

BACKGROUND: Escherichia coli is an opportunistic pathogen which colonizes various host species. However, to what extent genetic lineages of E. coli are adapted or restricted to specific hosts and the genomic determinants of such adaptation or restriction is poorly understood. RESULTS: We randomly sampled E. coli isolates from four countries (Germany, UK, Spain, and Vietnam), obtained from five host species (human, pig, cattle, chicken, and wild boar) over 16 years, from both healthy and diseased hosts, to construct a collection of 1198 whole-genome sequenced E. coli isolates. We identified associations between specific E. coli lineages and the host from which they were isolated. A genome-wide association study (GWAS) identified several E. coli genes that were associated with human, cattle, or chicken hosts, whereas no genes associated with the pig host could be found. In silico characterization of nine contiguous genes (collectively designated as nan-9) associated with the human host indicated that these genes are involved in the metabolism of sialic acids (Sia). In contrast, the previously described sialic acid regulon known as sialoregulon (i.e. nanRATEK-yhcH, nanXY, and nanCMS) was not associated with any host species. In vitro growth experiments with a Δnan-9 E. coli mutant strain, using the sialic acids 5-N-acetylneuraminic acid (Neu5Ac) and N-glycolylneuraminic acid (Neu5Gc) as sole carbon source, showed impaired growth behaviour compared to the wild-type. CONCLUSIONS: This study provides an extensive analysis of genetic determinants which may contribute to host specificity in E. coli. Our findings should inform risk analysis and epidemiological monitoring of (antimicrobial resistant) E. coli.

Horsing around: Escherichia coli ST1250 of equine origin harbouring epidemic IncHI1/ST9 plasmid with bla CTX-M-1 and an operon for short-chain fructooligosaccharides metabolism.

The relatedness of the equine-associated Escherichia coli ST1250 and its single- and double-locus variants (ST1250-SLV/DLV), obtained from horses in Europe, was studied by comparative genome analysis. A total of 54 isolates of E. coli ST1250 and ST1250-SLV/DLV from healthy and hospitalized horses across Europe [Czech Republic (n=23), the Netherlands (n=18), Germany (n=9), Denmark (n=3) and France (n=1)] from 2008-2017 were subjected to whole-genome sequencing. An additional 25 draft genome assemblies of E. coli ST1250 and ST1250-SLV/DLV were obtained from the public databases. The isolates were compared for genomic features, virulence genes, clade structure and plasmid content. The complete nucleotide sequences of eight IncHI1/ST9 and one IncHI1/ST2 plasmids were obtained using long-read sequencing by PacBio or MinION. In the collection of 79 isolates, only 10 were phylogenetically close (<8 SNP). The majority of isolates belonged to phylogroup B1 (73/79, 92.4%) and carried bla CTX-M-1 (58/79, 73.4%). The plasmid content of the isolates was dominated by IncHI1 of ST9 (56/62, 90.3%) and ST2 (6/62, 9.7%), while 84.5% (49/58) bla CTX-M-1 genes were associated with presence of IncHI1 replicon of ST9 and 6.9% (4/58) with IncHI1 replicon of ST2 within the corresponding isolates. The operon for the utilization of short chain fructooligosaccharides (fos operon) was present in 55 (55/79, 69.6%) isolates, and all of these carried IncHI1/ST9 plasmids. The eight complete IncHI1/ST9 plasmid sequences showed the presence of bla CTX-M-1 and the fos operon within the same molecule. Sequences of IncHI1/ST9 plasmids were highly conserved (>98% similarity) regardless of country of origin and varied only in the structure and integration site of MDR region. E. coli ST1250 and ST1250-SLV/DLV are phylogenetically-diverse strains associated with horses. A strong linkage of E. coli ST1250 with epidemic multi-drug resistance plasmid lineage IncHI1/ST9 carrying bla CTX-M-1 and the fos operon was identified.

Heartworms in Halichoerus grypus: first records of Acanthocheilonema spirocauda (Onchocercidae; Filarioidea) in 2 grey seals from the North Sea.

The assumed definitive host of the heartworm Acanthocheilonema spirocauda (Onchocerdidae; Filarioidea) is the harbour seal (Phoca vitulina). This filaroid nematode parasitizing in cardiac ventricles and blood vessel lumina of harbour seals (P. vitulina) has a low prevalence and seldom causes severe health impacts. The seal louse (Echinophthirius horridus) is the assumed intermediate host for transmission of A. spirocauda filariae between seals, comprising a unique parasite assembly conveyed from the terrestrial ancestors of pinnipeds. Although grey seals (Halichoerus grypus) are infected by seal lice, heartworm infection was not verified. Analysing a longterm dataset compiled over decades (1996­2021) of health monitoring seals along the German coasts comprising post mortem investigations and archived parasites, 2 cases of A. spirocauda infected male grey seals were detected. Tentative morphological identification was confirmed with molecular tools by sequencing a section of mtDNA COI and comparing nucleotide data with available heartworm sequence. This is the first record of heartworm individuals collected from the heart of grey seals at necropsy. It remains puzzling why heartworm infection occur much less frequently in grey than in harbour seals, although both species use the same habitat, share mixed haul-outs and consume similar prey species. If transmission occurs directly via seal louse vectors on haul-outs, increasing seal populations in the North- and Baltic Sea could have density dependent effects on prevalence of heartworm and seal louse infections. It remains to be shown how species-specificity of filarial nematodes as well as immune system traits of grey seals influence infection patterns of A. spirocauda.

Erysipelothrix anatis sp. nov., Erysipelothrix aquatica sp. nov. and Erysipelothrix urinaevulpis sp. nov., three novel species of the genus, and emended description of Erysipelothrix.

Seven genotypically distinct strains assigned to the genus Erysipelothrix were isolated in different laboratories from several animal sources. Strain D17_0559-3-2-1T and three further strains were isolated from samples of duck, pig and goose. The strains had >99 % 16S rRNA gene sequence similarity to each other and to strain VA92-K48T and two further strains isolated from samples of medical leech and a turtle. The closest related type strains to the seven strains were those of Erysipelothrix inopinata (96.74 %) and Erysipelothrix rhusiopathiae (95.93 %). Average nucleotide identity, amino acid identity and in silico DNA-DNA hybridization results showed that the strains represented two separate novel species. One further phylogenetically distinct strain (165301687T) was isolated from fox urine. The strain had highest 16S rRNA gene sequence similarity to the type strains of Erysipelothrix tonsillarum (95.67 %), followed by Erysipelothrix piscisicarius (95.58 %) and Erysipelothrix larvae (94.22 %) and represented a further novel species. Chemotaxonomic and physiological data of the novel strains were assessed, but failed to unequivocally differentiate the novel species from existing members of the genus. MALDI-TOF MS data proved the discrimination of at least strain 165301687T from all currently described species. Based on the presented phylogenomic and physiological data, we propose three novel species, Erysipelothrix anatis sp. nov. with strain D17_0559-3-2-1T (=DSM 111258T= CIP 111884T=CCM 9044T) as type strain, Erysipelothrix aquatica sp. nov. with strain VA92-K48T (=DSM 106012T=LMG 30351T=CIP 111492T) as type strain and Erysipelothrix urinaevulpis sp. nov. with strain 165301687T (=DSM 106013T= LMG 30352T= CIP 111494T) as type strain.

A dominant clonal lineage of Streptococcus uberis in cattle in Germany.

Bovine mastitis causes enormous economic losses in the dairy industry with Streptococcus uberis as one of the most common bacterial pathogens causing clinical and subclinical variations. In most cases mastitis can be cured by intramammary administration of antimicrobial agents. However, the severity of the clinical manifestations can vary greatly from mild to severe symtoms. In this study, a comparative genomic analysis of 24 S. uberis isolates from three dairy farms in Germany, affected by different courses of infection was conducted. While there were sporadic mild infections in farm A and B, a large number of infections were observed within a very short period of time in farm C. The comparison of virulence genes, antimicrobial resistance genes and prophage regions revealed no features that might be responsible for this severe course. However, almost all isolates from farm C showed the same, novel MLST profile (ST1373), thus a clonal outbreak cannot be excluded, whereby the actual reason for the particular virulence remains unknown. This study demonstrates the importance of extensive metagenomic studies, including the host genomes and the environment, to gain further evidence on the pathogenicity of S. uberis.

Corynebacterium rouxii, a recently described member of the C. diphtheriae group isolated from three dogs with ulcerative skin lesions.

Corynebacterium (C.) diphtheriae is one of the two etiological pathogens for human diphtheria with significant morbidity and mortality. Recently, members of its biovar Belfanti have been described as two novel species, C. belfantii and C. rouxii. The most important virulence factor and also the premise to cause diphtheria is the isolate's capacity to encode and express the diphtheria toxin (DT). In contrast to C. ulcerans, which represents a potentially zoonotic pathogen, C. diphtheriae (incl. the novel deduced species) has almost exclusively been found to comprise a human pathogen. We here report three rare cases of C. rouxii isolation from dogs suffering from disseminated poly-bacterial exsudative to purulent dermatitis and a traumatic labial defect, respectively. The isolates were identified as C. diphtheriae based on commercial biochemistry and matrix-assisted laser desorption/ionisation-time of flight mass spectrometry (MALDI-TOF MS) analysis. However, recently described specific spectral peaks were highly similar to spectra of C. rouxii, which was confirmed by whole genome sequencing. Further investigations of the dog isolates for the presence of DT by tox gene qPCR revealed negative results. The findings from this study point out that skin infections in companion animals can be colonized by uncommon and so believed human specific pathogens, thereby resembling the clinical signs of cutaneous diphtheria.

Streptococcus catagoni sp. nov., isolated from the respiratory tract of diseased Chacoan peccaries (Catagonus wagneri).

Novel catalase-negative, Gram-stain-positive, beta-haemolytic, coccus-shaped organisms were isolated from Chacoan peccaries that died from respiratory disease. The initial API 20 Strep profiles suggested Streptococcus agalactiae with acceptable identification scores, but the 16S rRNA gene similarity (1548 bp) to available sequences of streptococci was below 98 %. Next taxa of the genus Streptococcus, displaying highest similarities to the strains from this study, were S. bovimastitidis NZ1587T (97.5 %), S. iniae ATCC 29178T (97.5 %), S. hongkongensis HKU30T (97.4 %), S. parauberis DSM 6631T (97.1 %), S. penaeicida CAIM 1838T (97.1 %), S. pseudoporcinus DSM 18513T (97.0 %), S. didelphis DSM 15616T (96.6 %), S. ictaluri 707-05T (96.6 %), S. uberis JCM 5709T (96.5 %) and S. porcinus NCTC 10999T (96.4 %). All other Streptococcus species had sequence similarities of below 96.4 %. A sodA gene as well as whole genome-based core genome phylogeny of three representative strains and 145 available Streptococcus genomes confirmed the unique taxonomic position. Interstrain average nucleotide identity (ANI) and amino acid identity (AAI) values were high (ANI >96 %; AAI 100%), but for other streptococci clearly below the proposed species boundary of 95-96 % (ANI <75 %; AAI <83 %). Results were confirmed by genome-to-genome distance calculations. Pairwise digital DNA-DNA hybridization estimates were high (>90 %) between the novel strains, but well below the species boundary of 70 % for closely related Streptococcus type strains (23.5-19.7 %). Phenotypic properties as obtained from extended biochemical profiles and MALDI-TOF mass spectrometry supported the outstanding rank. Based on the presented molecular and physiological data of the six strains, we propose a novel taxon for which we suggest the name Streptococcus catagoni sp. nov. with the type strain 99-1/2017T (=DSM 110457T=CCUG 74072T) and five reference strains.

Streptobacillus canis sp. nov. isolated from a dog.

From a phlegmon in a dog an aerobic and facultatively anaerobic, indole-, oxidase- and catalase-negative, non-motile bacterium was isolated in 2019 in Germany that stained Gram-negative and showed a pleomorphic, rod-shaped, non-spore-forming appearance. Based on the results of 16S rRNA gene sequence analyses, strain IHIT1603-19T was assigned to the genus Streptobacillus with sequence similarities of 98.6, 98.0, 97.9, 97.1 and 94.4 % to the type strains of Streptobacillus felis, Streptobacillus notomytis, Streptobacillus ratti, Streptobacillus moniliformis and Streptobacillus hongkongensis, respectively. Strain IHIT1603-19T could also clearly be differentiated from other Streptobacillus species by rpoB, groEL and recA gene, nucleotide and amino acid sequence analyses as well as by core genome phylogeny. Regarding DNA-DNA relatedness, strain IHIT1603-19T demonstrated an average nucleotide identity of 83.00 and 82.28 % compared to S. felis 131000547T and S. moniliformis DSM 12112T, respectively. Chemotaxonomic and physiological data of strain IHIT1603-19T were in congruence with other closely related members of the family Leptotrichiaceae, represented by highly similar enzyme profiles and fatty acid patterns. MALDI-TOF MS analysis also proved suitable in unequivocally discriminating strain IHIT1603-19T from all currently described taxa of the genus Streptobacillus. On the basis of these data, we propose the novel species Streptobacillus canis sp. nov. with the type strain IHIT1603-19T (=DSM 110501T=CCUG 74118T=CIP 111795T). The G+C content of the DNA of the type strain is 26.6 mol%, genome size is 1.60 Mbp.

Differential expression of hemolysin genes in weakly and strongly hemolytic Brachyspira hyodysenteriae strains.

BACKGROUND: Swine dysentery (SD) is a diarrheal disease in fattening pigs that is caused by the strongly hemolytic species Brachyspira (B.) hyodysenteriae, B. hampsonii and B. suanatina. As weakly hemolytic Brachyspira spp. are considered less virulent or even non-pathogenic, the hemolysin is regarded as an important factor in the pathogenesis of SD. Four hemolysin genes (tlyA, tlyB, tlyC, and hlyA) and four putative hemolysin genes (hemolysin, hemolysin activation protein, hemolysin III, and hemolysin channel protein) have been reported, but their role in strong hemolysis is not entirely clear. Our study aimed to assess the transcriptional activity of eight (putative) hemolysin genes in a strongly hemolytic (B204) and a weakly hemolytic (G423) B. hyodysenteriae strain during non-hemolytic and hemolytic growth stages. RESULTS: Strongly and weakly hemolytic B. hyodysenteriae strains caused hemolysis on blood agar at different growth stages, namely during log phase (B204) and stationary/death phase (G423). During the lag, early log, late log (stationary phase in G423) and death phase (time points 1-4) strains differed in their hemolysin gene transcription patterns. At time point 1, transcription of the putative hemolysin gene was higher in B204 than in G423. At time point 2, tlyA and tlyC were upregulated in B204 during hemolysis. TlyB and hlyA were upregulated in both strains at all time points, but higher transcription rates were observed in the weakly hemolytic strain G423. The transcription activity of the hemolysin channel protein gene was quite similar in both strains, whereas the hemolysin activation protein gene was upregulated in the non-hemolytic stage of B204 at time point 4. Sequence analysis revealed deletions, insertions and single nucleotide polymorphisms in the G423 hlyA promoter, although without altering the transcription activity of this gene. CONCLUSION: Our data indicate a combined activity of TlyA and TlyC as the most probable underlying mechanism of strong hemolysis in B. hyodysenteriae. Further studies should verify if the expression of tlyA is upregulated by the putative hemolysin gene. Depending on their immunogenic potential TlyA and TlyC may serve as possible vaccine candidates, especially since vaccines for an effective control of swine dysentery are currently not available.

Marine mammals are natural hosts of Oceanivirga salmonicida, a bacterial pathogen of Atlantic salmon.

During 1992 and 1993, a bacterial disease occurred in a seawater Atlantic salmon Salmo salar farm, causing serious mortalities. The causative agent was subsequently named as Oceanivirga salmonicida, a member of the Leptotrichiaceae. Searches of 16S rRNA gene sequence databases have shown sequence similarities between O. salmonicida and uncultured bacterial clones from the digestive tracts of marine mammals. In the current study, oral samples were taken from stranded dolphins (common dolphin Delphinus delphis, striped dolphin Stenella coeruleoalba) and healthy harbour seals Phoca vitulina. A bacterium with growth characteristics consistent with O. salmonicida was isolated from a common dolphin. The isolate was confirmed as O. salmonicida, by comparisons to the type strain, using 16S rRNA gene, gyrB, groEL, and recA sequence analyses, average nucleotide identity analysis, and MALDI-TOF mass spectrometry. Metagenomic analysis indicated that the genus Oceanivirga represented a significant component of the oral bacterial microbiomes of the dolphins and seals. However, sequences consistent with O. salmonicida were only found in the dolphin samples. Analyses of marine mammal microbiome studies in the NCBI databases showed sequences consistent with O. salmonicida from the common dolphin, striped dolphin, bottlenose dolphin Tursiops truncatus, humpback whale Megaptera novaeangliae, and harbour seal. Sequences from marine environmental studies in the NCBI databases showed no sequences consistent with O. salmonicida. The findings suggest that several species of marine mammals are natural hosts of O. salmonicida.

Genomic and Functional Analysis of Emerging Virulent and Multidrug-Resistant Escherichia coli Lineage Sequence Type 648.

The pathogenic extended-spectrum-beta-lactamase (ESBL)-producing Escherichia coli lineage ST648 is increasingly reported from multiple origins. Our study of a large and global ST648 collection from various hosts (87 whole-genome sequences) combining core and accessory genomics with functional analyses and in vivo experiments suggests that ST648 is a nascent and generalist lineage, lacking clear phylogeographic and host association signals. By including large numbers of ST131 (n = 107) and ST10 (n = 96) strains for comparative genomics and phenotypic analysis, we demonstrate that the combination of multidrug resistance and high-level virulence are the hallmarks of ST648, similar to international high-risk clonal lineage ST131. Specifically, our in silico, in vitro, and in vivo results demonstrate that ST648 is well equipped with biofilm-associated features, while ST131 shows sophisticated signatures indicative of adaption to urinary tract infection, potentially conveying individual ecological niche adaptation. In addition, we used a recently developed NFDS (negative frequency-dependent selection) population model suggesting that ST648 will increase significantly in frequency as a cause of bacteremia within the next few years. Also, ESBL plasmids impacting biofilm formation aided in shaping and maintaining ST648 strains to successfully emerge worldwide across different ecologies. Our study contributes to understanding what factors drive the evolution and spread of emerging international high-risk clonal lineages.

Population structure and virulence gene profiles of Streptococcus agalactiae collected from different hosts worldwide.

Streptococcus agalactiae is a leading cause of morbidity and mortality among neonates and causes severe infections in pregnant women and nonpregnant predisposed adults, in addition to various animal species worldwide. Still, information on the population structure of S. agalactiae and the geographical distribution of different clones is limited. Further data are urgently needed to identify particularly successful clones and obtain insights into possible routes of transmission within one host species and across species borders. We aimed to determine the population structure and virulence gene profiles of S. agalactiae strains from a diverse set of sources and geographical origins. To this end, 373 S. agalactiae isolates obtained from humans and animals from five different continents were typed by DNA microarray profiling. A total of 242 different S. agalactiae strains were identified and further analyzed. Particularly successful clonal lineages, hybridization patterns, and strains were identified that were spread across different continents and/or were present in more than one host species. In particular, several strains were detected in both humans and cattle, and several canine strains were also detected in samples from human, bovine, and porcine hosts. The findings of our study suggest that although S. agalactiae is well adapted to various hosts including humans, cattle, dogs, rodents, and fish, interspecies transmission is possible and occurs between humans and cows, dogs, and rabbits. The virulence and resistance gene profiles presented enable new insights into interspecies transmission and make a crucial contribution to the identification of suitable targets for therapeutic agents and vaccines.

Acute Tetraplegia Caused by Rat Bite Fever in Snake Keeper and Transmission of Streptobacillus moniliformis.

We report acute tetraplegia caused by rat bite fever in a 59-year old man (snake keeper) and transmission of Streptobacillus moniliformis. We found an identical characteristic bacterial pattern in rat and human samples, which validated genotyping-based evidence for infection with the same strain, and identified diagnostic difficulties concerning infection with this microorganism.

Molecular study on Pasteurella multocida and Mannheimia granulomatis from Kenyan Camels (Camelus dromedarius).

BACKGROUND: Outbreaks of a Haemorrhagic Septicaemia (HS) like disease causing large mortalities in camels (Camelus dromedarius) in Asia and in Africa have been reported since 1890. Yet the aetiology of this condition remains elusive. This study is the first to apply state of the art molecular methods to shed light on the nasopharyngeal carrier state of Pasteurellaceae in camels. The study focused on HS causing Pasteurella multocida capsular types B and E. Other Pasteurellaceae, implicated in common respiratory infections of animals, were also investigated. METHODS: In 2007 and 2008, 388 nasopharyngeal swabs were collected at 12 locations in North Kenya from 246 clinically healthy camels in 81 herds that had been affected by HS-like disease. Swabs were used to cultivate bacteria on blood agar and to extract DNA for subsequent PCR analysis targeting P. multocida and Mannheimia-specific gene sequences. RESULTS: Forty-five samples were positive for P. multocida genes kmt and psl and for the P. multocida Haemorrhagic Septicaemia (HS) specific sequences KTSP61/KTT72 but lacked HS-associated capsular type B and E genes capB and capE. This indicates circulation of HS strains in camels that lack established capsular types. Sequence analysis of the partial 16S rRNA gene identified 17 nasal swab isolates as 99% identical with Mannheimia granulomatis, demonstrating a hitherto unrecognised active carrier state for M. granulomatis or a closely related Mannheimia sp. in camels. CONCLUSIONS: The findings of this study provide evidence for the presence of acapsular P. multocida or of hitherto unknown capsular types of P. multocida in camels, closely related to P. multocida strains causing HS in bovines. Further isolations and molecular studies of camelid P. multocida from healthy carriers and from HS-like disease in camels are necessary to provide conclusive answers. This paper is the first report on the isolation of M. granulomatis or a closely related new Mannheimia species from camelids.

Phylogenetic and comparative genomics of the family Leptotrichiaceae and introduction of a novel fingerprinting MLVA for Streptobacillus moniliformis.

BACKGROUND: The Leptotrichiaceae are a family of fairly unnoticed bacteria containing both microbiota on mucous membranes as well as significant pathogens such as Streptobacillus moniliformis, the causative organism of streptobacillary rat bite fever. Comprehensive genomic studies in members of this family have so far not been carried out. We aimed to analyze 47 genomes from 20 different member species to illuminate phylogenetic aspects, as well as genomic and discriminatory properties. RESULTS: Our data provide a novel and reliable basis of support for previously established phylogeny from this group and give a deeper insight into characteristics of genome structure and gene functions. Full genome analyses revealed that most S. moniliformis strains under study form a heterogeneous population without any significant clustering. Analysis of infra-species variability for this highly pathogenic rat bite fever organism led to the detection of three specific variable number tandem analysis loci with high discriminatory power. CONCLUSIONS: This highly useful and economical tool can be directly employed in clinical samples without laborious prior cultivation. Our and prospective case-specific data can now easily be compared by using a newly established MLVA database in order to gain a better insight into the epidemiology of this presumably under-reported zoonosis.

Caviibacter abscessus gen. nov., sp. nov., a member of the family Leptotrichiaceae isolated from guinea pigs (Cavia porcellus).

A pleomorphic, Gram-stain-negative, rod-shaped, indole-, oxidase- and catalase-negative, non-spore-forming, non-motile bacterium was originally isolated from the mandibular lymph node of a guinea pig and deposited as Streptobacillus moniliformis CCUG 39713 in 1998. A second strain, 151011837, was isolated from an identical lesion in a guinea pig in Germany in 2015. On the basis of 16S rRNA gene sequence analyses, these strains displayed highest sequence similarities with Sneathia sanguinegens NTS65407T (93.4%) and 'Sneathia amnii' Sn35 (93.2%), followed by Streptobacillus moniliformis DSM 12112T (91.3%), 'Streptobacillus ratti' OGS16 (91.2%), Streptobacillus notomytis AHL370-1T (91.0%), Streptobacillus hongkongensis HKU33T (90.9%) and Streptobacillus felis 131000547T (90.9%). Levels of sequence similarity with all other members of the family Leptotrichiaceae were <89%. Results of phylogenetic analyses of strains CCUG 39713T and 151011837, based on gyrB, groEL and recA nucleotide and deduced amino acid sequences, were highly similar, as the topologies of all trees were virtually identical. DNA relatedness values derived from average nucleotide identities calculated for comparisons between strain CCUG 39713T and the type strains of Sneathia sanguinegens and Streptobacillus moniliformis, respectively, were 72.05 and 70.42%. The genomes of CCUG39713T and 151011837 shared 99.57% average nucletide identity. The chemotaxonomic and physiological data for strains CCUG 39713T and 151011837 were in congruence with other closely related members of the family Leptotrichiaceae, with highly similar enzyme activities and fatty acid profiles. Matrix-assisted laser desorption ionization time-of-flight MS analysis was capable of clearly discriminating strains CCUG 39713T and 151011837 from all taxa of the family Leptotrichiaceae with validly published names. On the basis of these data, the novel taxon Caviibacter abscessus gen. nov., sp. nov. is proposed. The type strain of Caviibacter abscessus is CCUG 39713T (=DSM 101949T); 151011837 (DSM 101950) is an additional strain of the species.

Oceanivirga salmonicida gen. nov., sp. nov., a member of the Leptotrichiaceae isolated from Atlantic salmon (Salmo salar).

A pleomorphic, Gram-negative, rod-shaped, indole-, oxidase- and catalase- negative, non-spore-forming, non-motile bacterium was originally isolated in 1992 from moribund, seawater farmed Atlantic salmon with multifocal tissue necrosis. Strain AVG 2115T displayed considerable similarities with Streptobacillus moniliformis, one of the two etiological agents of rat bite fever, and has been stored as Streptobacillus sp. NCIMB 703044T. On the basis of 16S rRNA gene sequence analyses, this strain displayed >99 % sequence similarities with uncultured bacterial clones from the digestive tracts of marine mammals, followed by Sneathia sanguinegens CCUG 41628T (92.7 %), 'Sneathia amnii' Sn35 (92.5 %), Caviibacter abscessus CCUG 39713T (92.2 %), Streptobacillus ratti OGS16T (91.3 %), Streptobacillus notomytis AHL 370-1T (91.2 %), S. moniliformis DSM 12112T (91.0 %), Streptobacillus felis 131000547T (90.9 %) and Streptobacillus hongkongensis DSM 26322T (89.7 %). Sequence similarities to all other taxa were below 89 %. Phylogenetic analysis for strain NCIMB 703044T revealed highly similar results for gyrB, groEL and recA nucleotide and deduced amino acid sequence analyses independent of the employed treeing method. Average nucleotide identities (ANI) for complete genomes ranged from 66.00 % to 72.08 % between strain NCIMB 703044T and the type strains of Sebaldella termitidis, Leptotrichiabuccalis, Streptobacillus moniliformis, Sneathia sanguinegens and Caviibacter abscessus. Chemotaxonomic and physiological data of strain NCIMB 703044t were in congruence with closely related members of the family Leptotrichiaceae, represented by highly similar enzyme profiles and fatty acid patterns. MALDI-TOF MS analysis was capable to clearly discriminate strain NCIMB 703044T from all currently described taxa of the family Leptotrichiaceae. On the basis of these data we propose the novel taxon Oceanivirga salmonicida gen. nov. sp. nov. with the type strain AVG 2115T (=NCIMB 703044T) (=DSM 101867T). The G+C content is 25.4 %, genome size is 1.77 Mbp.

Streptobacillus ratti sp. nov., isolated from a black rat (Rattus rattus).

An indole-, oxidase- and catalase-negative, non-motile bacterium, strain OGS16T, was isolated from an oral swab of a feral black rat (Rattus rattus) in 2007 in Japan. It stained Gram-negative and had pleomorphic, rod-shaped, non-spore-forming cells. Based on 16S rRNA gene sequence analyses, strain OGS16T was assigned to the genus Streptobacillus, with 16S rRNA gene sequence similarities of 99.3, 99.0, 98.6 and 95.5% to the type strains of Streptobacillus moniliformis, Streptobacillus notomytis, Streptobacillus felis and Streptobacillus hongkongensis, respectively. Strain OGS16T could also be differentiated clearly from other species of the genus Streptobacillus by rpoB, groEL and recA nucleotide and deduced amino acid sequence analysis. DNA-DNA relatedness as obtained by average nucleotide identity was 89.10% between strain OGS16T and Streptobacillus moniliformis DSM 12112T. Chemotaxonomic and physiological data for strain OGS16T were congruent with results for other closely related members of the family Leptotrichiaceae, represented by highly similar enzyme profiles and fatty acid patterns. MALDI-TOF MS analysis also proved suitable in discriminating strain OGS16T unequivocally from all currently described taxa of the genus Streptobacillus. On the basis of these data, we propose the novel species Streptobacillus ratti sp. nov., with the type strain OGS16T (=JCM 31098T=DSM 101843T). The G+C content of the DNA of the type strain is 25.9 mol% and the genome size is 1.50 Mbp.

New polymorphisms within the variable number tandem repeat (VNTR) 7 locus of Mycobacterium avium subsp. paratuberculosis.

Variable number tandem repeat (VNTR) is a frequently employed typing method of Mycobacterium avium paratuberculosis (MAP) isolates. Based on whole genome sequencing in a previous study, allelic diversity at some VNTR loci seems to over- or under-estimate the actual phylogenetic variance among isolates. Interestingly, two closely related isolates on one farm showed polymorphism at the VNTR 7 locus, raising concerns about the misleading role that it might play in genotyping. We aimed to investigate the underlying basis of VNTR 7-polymorphism by analyzing sequence data for published genomes and field isolates of MAP and other M. avium complex (MAC) members. In contrast to MAP strains from cattle, strains from sheep displayed an "imperfect" repeat within VNTR 7, which was identical to respective allele types in other MAC genomes. Subspecies- and strain-specific single nucleotide polymorphisms (SNPs) and two novel (16 and 56 bp) repeats were detected. Given the combination of the three existing repeats, there are at least five different patterns for VNTR 7. The present findings highlight a higher polymorphism and probable instability of VNTR 7 locus that needs to be considered and challenged in future studies. Until then, sequencing of this locus in future studies is important to correctly assign the underlying allele types.(1).