Mycoplasma tauri sp. nov. isolated from the bovine genital tract

Since 2006, a Mycoplasma species unidentifiable to the species level has been regularly isolated from the semen and prepuce of apparently healthy bulls, and occasionally from cattle displaying inflammatory disease of the genital tract. Seven of these Mycoplasma isolates were subjected to a comprehensive taxonomic study. The strains investigated grew well in modified Hayflick’s medium and colonies on agar exhibited typical fried egg morphology and produced ‘film and spots’. Transmission electron microscopy revealed a cell morphology characteristic of mycoplasmas with spherically shaped cells bounded by a bi-layered cell membrane. The strains studied neither produced acid from sugar carbon sources nor did hydrolyse arginine or urea, and genome annotation indicated that organic acids (pyruvate, lactate) are used as energy sources. Phylogenetic analyses of 16S rRNA gene sequences, the 16S-23S intergenic spacer region, and partial rpoB gene and protein sequences placed the strains within the Mycoplasma (M.) bovis cluster of the Hominis group with M. primatum, M. agalactiae, and M. bovis being their closest relatives. Genomic information including whole-genome similarity metrics (ANIb, ANIm, TETRA, dDDH, AAI) and phylogenomics, proteomic features revealed by matrix-assisted laser desorption ionization time of flight (MALDI-ToF) mass spectrometry as well as serological reactions and polar lipid profiling strongly indicated that the strains examined were representatives of a hitherto unclassified species of genus Mycoplasma, for which the name Mycoplasma tauri sp. nov. with type strain Zaradi2^T (=ATCC BAA-1891^T = DSM 22451^T) is proposed.     

Determination of epidemiological relationships of Streptococcus agalactiae isolated from bovine mastitis

In the present study 79 streptococcal cultures isolated from subclinical mastitis of 54 cows from seven dairy farms (A-G) in Hesse, Germany, were comparatively investigated using conventional and molecular methods. The isolates could be identified as Streptococcus agalactiae, belonging to Lancefield's serological group B by determination of cultural, biochemical and serological properties and by polymerase chain reaction (PCR)-mediated amplification of species-specific parts of the 16S ribosomal DNA, the 16S-23S rDNA intergenic spacer region and the CAMP factor gene cfb. The investigated group B streptococci were further characterized serologically for specific polysaccharide and protein antigens. Serotyping the isolates revealed a predominance of surface protein antigen X, either alone or in combination with polysaccharide antigen Ia. This could be observed for 39 isolates of farms A, B and C. Six group B streptococci from farm E displayed the serotype pattern III/Rib, two isolates from farm G showed the serotype pattern Ib/calpha. The remaining cultures from farms D and F (n=32) were non-typable. The occurrence of protein Rib could be confirmed by PCR amplification of the gene rib. The two isolates with serotype pattern Ib/calpha also reacted positively for the cbeta-encoding gene bag. Additional properties which allowed a phenotypic characterization of the S. agalactiae were the degree of pigmentation, growth properties in fluid media and soft agar, the surface hydrophobicity, the ability to hemagglutinate rabbit erythrocytes and their resistance reactions to tetracycline and minocycline. The isolates of the seven farms showed identical or almost identical characteristics. The 79 group B streptococci were additionally investigated by macrorestriction analysis of their chromosomal DNA using the restriction endonucleases SmaI, ApaI and SalI. The restriction patterns obtained by pulsed-field gel electrophoresis displayed identical or closely related patterns for the cultures of the various farms. The pheno- and genotypic characteristics of the 79 group B streptococci of the present study revealed that a single S. agalactiae strain or at least closely related subtypes of this strain were responsible for the mastitis situation of the seven farms.     

Identification of lactoferrin-binding proteins in bovine mastitis-causing Streptococcus uberis

All strains of Streptococcus uberis evaluated bound to lactoferrin (Lf) in milk as detected by polyacrylamide gel electrophoresis and Western blotting. A biotin-avidin-based microplate binding assay and ELISA also revealed that these bacterial strains bound to purified Lf. Binding of bacteria of Lf was not inhibited by mannose and galactose, indicating that glycosidic domains of the Lf molecule were not involved in binding. Lf binding was also unaffected by bovine transferrin. Western blot analysis demonstrated that there were at least two bacterial proteins involved in Lf-binding. Lf binding by S. uberis could enable this bacterium to acquire iron necessary for its growth.     

Distribution of virulence-associated genes in Streptococcus uberis isolated from bovine mastitis

Streptococcus uberis is an important pathogen that has been implicated in bovine mastitis but the virulence factors associated with pathogenesis are not well understood. The aim of this work was to examine 11 putative and known virulence-associated genes by PCR in 78 S. uberis strains isolated from infected animals in Argentina. Additionally, the distribution of virulence patterns over various herds was determined. Not all genes were present in the strains but all of the detected virulence-associated genes were present in combination. Forty-seven (60.3%) isolates carried seven to 10 virulence-associated genes. Further analysis revealed 58 virulence patterns. Different patterns were found within the same herd and among herds, demonstrating that strains with different virulence patterns were able to cause mastitis. Despite the large number of strains with different virulence patterns, strains with identical patterns was found. Detection of virulence-associated genes in individual S. uberis strains isolated from infected animals revealed one to 10 virulence genes. This may indicate that other virulence factors could be involved. The present study reveals the occurrence and distribution of 11 virulence-associated genes among S. uberis isolates from bovine mastitis in various herds and contributes to a better understanding of the pathogenicity of this bacterium.     

Effect of milk on antibacterial activity of tetracycline against Escherichia coli and Staphylococcus aureus isolated from bovine mastitis

The susceptibility of mastitis-causing Escherichia coli and Staphylococcus aureus to two commonly used antibiotics, tetracycline and penicillin G, was tested in raw milk and in Muller–Hinton (MH) broth by introducing a pH indicator, bromocresol purple, which was shown to be a simple, sensitive, and rapid method. The minimum inhibitory concentration (MIC) of penicillin G in milk was the same as those in MH broth, whereas the MIC of tetracycline in milk was 4 to 32 times that in MH. An irreversible binding between tetracycline and large molecules of milk, which might be due to a hydrophobic interaction, was demonstrated by a dialysis test, suggesting the observed impairing effect was due to the action of milk on the tetracycline being tested. Further investigation revealed that much of the reduction of tetracycline’s activity in milk was attributable to the milk protein casein, while other heat-sensitive components in milk also play some roles.     

Flavobacterium circumlabens sp. nov. and Flavobacterium cupreum sp. nov., two psychrotrophic species isolated from Antarctic environmental samples

A taxonomic study of 24 Gram-stain-negative rod-shaped bacteria originating from the Antarctic environment is described. Phylogenetic analysis using 16S rRNA gene sequencing differentiated isolated strains into two groups belonging to the genus Flavobacterium. Group I (n = 20) was closest to Flavobacterium aquidurense WB 1.1-56^T (98.3% 16S rRNA gene sequence similarity) while group II (n = 4) showed Flavobacterium hydatis DSM 2063^T as its nearest neighbour (98.5–98.9% 16S rRNA gene sequence similarity). Despite high 16S rRNA gene sequence similarity, these two groups represented two distinct novel species as shown by phenotypic traits and low genomic relatedness assessed by rep-PCR fingerprinting, DNA-DNA hybridization and whole-genome sequencing. Common to representative strains of both groups were the presence of major menaquinone MK-6 and sym-homospermidine as the major polyamine. Common major fatty acids were C_15:0 iso, C_15:1 iso G, C_15:0 iso 3-OH, C_17:0 iso 3OH and Summed Feature 3 (C_16:1 ω7c/C_16:1 ω6c). Strain CCM 8828^T (group I) contained phosphatidylethanolamine, three unidentified lipids lacking a functional group, three unidentified aminolipids and single unidentified glycolipid in the polar lipid profile. Strain CCM 8825^T (group II) contained phosphatidylethanolamine, eight unidentified lipids lacking a functional group, three unidentified aminolipids and two unidentified glycolipids in the polar lipid profile. These characteristics corresponded to characteristics of the genus Flavobacterium. The obtained results showed that the analysed strains represent novel species of the genus Flavobacterium, for which the names Flavobacterium circumlabens sp. nov. (type strain CCM 8828^T = P5626^T = LMG 30617^T) and Flavobacterium cupreum sp. nov. (type strain CCM 8825^T = P2683^T = LMG 30614^T) are proposed.     

Mesorhizobium ventifaucium sp. nov. and Mesorhizobium escarrei sp. nov., two novel root-nodulating species isolated from Anthyllis vulneraria

Ten mesorhizobial strains isolated from root-nodules of Anthyllis vulneraria by trapping using soils from southern France were studied to resolve their taxonomy. Their 16S rDNA sequences were identical and indicated that they are affiliated to the genus Mesorhizobium within the group M. prunaredense/M. delmotii/M. temperatum/M. mediterraneum/M. wenxiniae and M. robiniae as the closest defined species. Their evolutionary relationships with validated species were further characterized by multilocus sequence analysis (MLSA) using 4 protein-coding housekeeping genes (recA, atpD, glnII and dnaK), that divides the strains in two groups, and suggest that they belong to two distinct species. These results were well-supported by MALDI-TOF mass spectrometry analyses, wet-lab DNA-DNA hybridization (≤58%), and genome-based species delineation methods (ANI < 96%, in silico DDH < 70%), confirming their affiliation to two novel species. Based on these differences, Mesorhizobium ventifaucium (STM4922^T = LMG 29643^T = CFBP 8438^T) and Mesorhizobium escarrei (type strain STM5069^T = LMG 29642^T = CFBP 8439^T) are proposed as names for these two novel species. The phylogeny of nodulation genes nodC and nodA allocated the type strains into symbiovar anthyllidis as well as those of M. metallidurans STM2683^T, M. delmotii STM4623^T and M. prunaredense STM4891^T, all recovered from the same legume species.     

Rhizobium ruizarguesonis sp. nov., isolated from nodules of Pisum sativum L

Four strains, coded as UPM1132, UPM1133^T, UPM1134 and UPM1135, and isolated from nodules of Pisum sativum plants grown on Ni-rich soils were characterised through a polyphasic taxonomy approach. Their 16S rRNA gene sequences were identical and showed 100% similarity with their closest phylogenetic neighbors, the species included in the ‘R. leguminosarum group’: R. laguerreae FB206^T, R. leguminosarum USDA 2370^T, R. anhuiense CCBAU 23252^T, R. sophoreae CCBAU 03386^T, R. acidisoli FH13^T and R. hidalgonense FH14^T, and 99.6% sequence similarity with R. esperanzae CNPSo 668^T. The analysis of combined housekeeping genes recA, atpD and glnII sequences showed similarities of 92-95% with the closest relatives. Whole genome average nucleotide identity (ANI) values were 97.5-99.7% ANIb similarity among the four strains, and less than 92.4% with closely related species, while digital DNA-DNA hybridization average values (dDDH) were 82-85% within our strains and 34-52% with closely related species. Major fatty acids in strain UPM1133^T were C18:1 ω7c / C18:1 ω6c in summed feature 8, C14:0 3OH/ C16:1 iso I in summed feature 2 and C18:0. Colonies were small to medium, pearl-white coloured in YMA at 28 °C and growth was observed in the ranges 8-34 °C, pH 5.5-7.5 and 0-0.7% (w/v) NaCl. The DNA G + C content was 60.8 mol %. The combined genotypic, phenotypic and chemotaxonomic data support the classification of strains UPM1132, UPM1133^T, UPM1134 and UPM1135 into a novel species of Rhizobium, for which the name Rhizobium ruizarguesonis sp. nov. is proposed. The type strain is UPM1133^T (=CECT 9542^T = LMG 30526^T).     

Proposal of Lysobacter pythonis sp. nov. isolated from royal pythons (Python regius)

The bacterial strains 4284/11T and 812/17 isolated from the respiratory tract of two royal pythons in 2011 and 2017, respectively were subjected to taxonomic characterization. The 16S rRNA gene sequences of the two strains were identical and showed highest sequence similarities to Lysobacter tolerans UM1^T (97.2%) and Luteimonas aestuarii DSM 19680^T (96.7 %). The two strains were identical in the sequences of the 16S-23S rRNA internal transcribed spacer (ITS) and partial groEL gene sequences and almost identical in genomic fingerprints. In the ITS sequence Ly. tolerans DSM 28473^T and in the groEL nucleotide sequence Luteimonas mephitis DSM 12574^T showed the highest similarity. In silico DDH analyses using genome sequence based ANIb and gANI similarity coefficients demonstrated that strain 4284/11^T represents a novel species and revealed Ly. tolerans UM1^T as the next relative (ANIb = 76.2 %, gANI = 78.0 %). Based on the topology of a core gene phylogeny strain 4284/11^T could be assigned to the genus Lysobacter. Chemotaxonomic characteristics including polyamine pattern, quinone system, polar lipid profile and fatty acid profile were in accordance with the characteristics of the genera Lysobacter and Luteimonas. Strains 4284/11^T and 812/17 could be differentiated from the type strains of the most closely related species by several physiological tests. In conclusion we are here proposing the novel species Lysobacter pythonis sp. nov. The type strain is 4284/11^T (= CCM 8829^T = CCUG 72164^T = LMG 30630^T) and strain 812/17 (CCM 8830) is a second strain of this species.     

Genomic and proteomic characterization of Staphylococcus aureus mastitis isolates of bovine origin

Staphylococcus aureus colonizes and infects humans as well as animals. In the present study, 17 S. aureus strains isolated from cows suffering from mastitis were characterized. The well-established multilocus sequence typing (MLST) technique and a diagnostic microarray covering 185 S. aureus virulence and resistance genes were used for genetic and epidemiological analyses. Virulence gene expression studies were performed by analyzing the extracellular protein pattern of each isolate on 2-D gels. By this way, a pronounced heterogeneity of the extracellular proteome between the bovine isolates has been observed which was attributed to genome plasticity and variation of gene expression. Merely 12 proteins were expressed in at least 80% of the isolates, i.e. Atl, Aur, GlpQ, Hla, LtaS, Nuc, PdhB, SAB0846, SAB2176, SAB0566, SspA, and SspB forming the core exoproteome. Fifteen extracellular proteins were highly variably expressed and only present in less than 20% of the isolates. This includes the serine proteases SplB, C, and F, and the superantigens SEC-bov, SEL and TSST-1. Compared to human isolates we identified at least six proteins with significantly different expression frequencies. While SAB0846 was expressed more frequently in bovine isolates, LytM, EbpS, Spa, Geh, and LukL1 were seen less frequently in these isolates.     

一株裂解性奶牛乳房炎源大肠杆菌噬菌体的分离鉴定及生物学特性分析

【目的】从新疆石河子地区奶牛粪样中分离裂解性大肠杆菌噬菌体(Escherichia coli phage),对其进行纯化及生物学特性分析。【方法】利用双层平板法从奶牛粪样中分离、纯化噬菌体,将纯化后的噬菌体浓缩液用醋酸双氧铀负染后通过透射电子显微镜观察其形态特征。对该噬菌体进行全基因组测序和遗传进化分析,同时测定噬菌体的宿主谱、最佳感染复数、一步生长曲线、热稳定性及酸碱稳定性。【结果】分离并纯化出一株裂解性噬菌体vB_EcoM_XJ2,噬菌斑圆形不透明,直径0.7 mm–1.2 mm;电镜显示其头部呈正多面体对称,有可伸缩性尾部;核酸类型为双链DNA,基因组大小为75.617 kb,G+C%含量为42.09%;其核酸序列与大肠杆菌噬菌体NJ01和vB_EcoP_SU10相似性高达94%。生物学特性研究显示该噬菌体能裂解多株临床分离的大肠杆菌;能耐受60°C左右高温,在pH 5.0–11.0范围内效价稳定;最佳感染复数为0.1,潜伏期为15 min,暴发期为95 min,裂解量约为10.6 PFU/cell。【结论】vB_EcoM_XJ2是一株在不同温度、不同酸碱性环境中有较强适应能力的裂解性肌尾科大肠杆菌噬菌体。     

Description of three new Alteromonas species Alteromonas antoniana sp. nov., Alteromonas lipotrueae sp. nov. and Alteromonas lipotrueiana sp. nov. isolated from marine environments,and proposal for reclassification of the genus Salinimonas as Alteromonas

In the course of a bioprospective study of marine prokaryotes for cosmetic purposes, four strains, MD_567^T, MD_652^T, MD_674 and PS_109^T, were isolated that 16S rRNA gene affiliation indicated could represent three new species within the family Alteromonadaceae. A thorough phylogenetic, genomic and phenotypic taxonomic study confirmed that the isolates could be classified as three new taxa for which we propose the names Alteromonas antoniana sp. nov., Alteromonas lipotrueae sp. nov. and Alteromonas lipotrueiana sp. nov. In addition, the consistent monophyletic nature of the members of the genera Alteromonas and Salinimonas showed that both taxa should be unified, and therefore we also propose the reclassification of the genus Salinimonas within Alteromonas, as well as new combinations for the species of the former. As the specific epithets profundi and sediminis are already used for Alteromonas species, we created the nomina nova “Alteromonas alteriprofundi” nom. nov. and Alteromonas alterisediminis nom. nov. to accommodate the new names for “Salinimonas profundi” and Salinimonas sediminis. Whole genome comparisons also allowed us to detect the unexpected codification of aromatic hydrocarbon biodegradative compounds, such as benzoate and catechol, whose activity was then demonstrated phenotypically. Finally, the high genomic identity between the type strains of Alteromonas stellipolaris and Alteromonas addita indicated that the latter is a junior heterotypic synonym of Alteromonas stellipolaris.     

Mesorhizobium intechi sp. nov. isolated from nodules of Lotus tenuis in soils of the Flooding Pampa,Argentina

Three symbiotic nitrogen-fixing bacteria (BD68^T, BD66 and BD73) isolated from root nodules of Lotus tenuis in lowland soils of the Flooding Pampa (Argentina), previously classified as members of the Mesorhizobium genus, were characterized in this study. Phylogenetic analysis of their 16S rRNA gene sequences showed a close relationship to M. japonicum MAFF 303099^T, M. erdmanii USDA 3471^T, M. carmichaelinearum ICMP 18942^T, M. opportunistum WSM 2975^T and M. jarvisii ATCC 33699^T, with sequence identities of 99.72%–100%. Multilocus sequence analysis of other housekeeping genes revealed that the three isolates belonged to a phylogenetically distinct clade within the genus Mesorhizobium. Strain BD68^T was designated as the group representative and its genome was fully sequenced. The average nucleotide identity and in silico DNA-DNA hybridization comparisons between BD68^T and the most related type strains showed values below the accepted threshold for species discrimination. Phenotypic and chemotaxonomic features were also studied.Based on these results, BD68^T, BD66 and BD73 could be considered to represent a novel species of the genus Mesorhizobium, for which the name Mesorhizobium intechi sp. nov. is hereby proposed. The type strain of this species is BD68^T (=CECT 9304^T = LMG 30179^T).     

Seminibacterium arietis gen. nov., sp. nov., isolated from the semen of rams

Two gram-negative, catalase- and oxidase-positive, bacillus-shaped bacterial strains were isolated from the semen of two rams. 16S rRNA gene sequencing demonstrated that both isolates represented a distinct subline within the family Pasteurellaceae with <95% sequence similarity to any recognized member of this family. Sequencing of rpoB and infB genes confirmed this finding with the semen isolates representing a new sub-line within the family Pasteurellaceae. The main cell fatty acids of strain DICM-00342^T were C_14:0, C_16:0, C_18:1ω7c and summed feature 3 (C_16:1ω7c/iso-C_15:0 2OH). Ubiquinone Q-8 was the major quinone and 1,3-diaminopropane was the predominat polyamine. Major polar lipids were phosphatidylglycerol and phosphatidylethanolamine. The new genus can be phenotypically distinguished from currently described genera of this family based on physiological traits and a combination of signature amino acids in the RpoB protein sequence. On the basis of these results we describe a new genus and species for which we propose the name of Seminibacterium arietis gen. nov., sp. nov. (DICM11-00342^T = CCUG 61707^T = CECT 8033^T).     

Three new asexual arthroconidial yeasts, Geotrichum carabidarum sp. nov., Geotrichum histeridarum sp. nov., and Geotrichum cucujoidarum sp. nov., isolated from the gut of insects

Twenty arthroconidial yeasts were isolated from the digestive tract of basidiome-feeding beetles and lepidopteran larvae. All of the yeasts reproduced only asexually by arthroconidia and some by endo- or blastoconidia as well. Based on the comparisons of sequences in ribosomal RNA genes and other taxonomic characteristics, the yeasts were identified as three unknown Geotrichum species. The three new species are described as Geotrichum carabidarum (NRRL Y-27727^T), G. histeridarum (NRRL Y-27729^T), and G. cucujoidarum (NRRL Y-27731^T). Phylogenetic analyses from ribosomal DNA sequences showed that members of the genus Geotrichum and related arthroconidial yeast taxa were divided into two major clades: (1) Dipodascus and Galactomyces with Geotrichum anamorphs including all the new species; and (2) Magnusiomyces with Saprochaete anamorphs. G. cucujoidarum formed a subclade with G. fermentans and Geotrichum sp. Y-5419, while the two closely related species, G. carabidarum and G. histeridarum, represent a new basal subclade in the clade of Geotrichum and its teleomorphs.     

Aspergillus huiyaniae sp. nov., a teleomorphic species in sect. Fumigati isolated from desert soil in China

Aspergillus huiyaniae, a new teleomorphic species isolated from desert soil in Xinjiang, China, was described and illustrated. Aspergillus huiyaniae is characterized by its yellowish white to pale yellow cleistothecia, broadly lenticular ascospores with two equatorial crests and irregularly ribbed to slightly reticulate convex surfaces, and subglobose to ovate or broadly ellipsoidal conidia with smooth walls. This species was supported further by the analyses of the β-tubulin, calmodulin and actin gene sequences.     

Corynebacterium aquatimens sp. nov., a lipophilic Corynebacterium isolated from blood cultures of a patient with bacteremia

An unknown lipophilic coryneform bacterium isolated from the blood cultures of a patient with bacteremia was characterized by phenotypic and molecular genetic methods. Chemical analysis revealed the presence of short chain mycolic acids consistent with the genus Corynebacterium. The DNA G+C content was 60.8mol%. Comparative 16S rRNA gene sequence analysis demonstrated that the isolate represents a new subline within the genus Corynebacterium. The closely phylogenetic relative of the unknown bacterium was found to be C. tuscaniense (97.8% sequence similarity). Partial rpoB gene sequence revealed that strain IMMIB L-2475(T) exhibited 13.5% sequence divergence with C. tuscaniense. The unknown bacterium was distinguished from C. tuscaniense by, DNA-DNA hybridization, cellular fatty acid profiles, MALDI-TOF analyses of cell extracts and biochemical tests. Based on the phylogenetic and phenotypic criteria, it is proposed that this bacterium be classified as new species, Corynebacterium aquatimens sp. nov., and is represented by strain IMMIB L-2475(T) (=DSM 45632(T)=CCUG 61574(T)).