Partial rpoB Gene Sequences Research Articles

Veillonella orientalis sp. nov., an anaerobic Gram-stain-negaitve coccus isolated from saliva of a Thai child

ObjectiveThe genus Veillonella is one of the major and important constitutions of the oral microbiome.A novel anaerobic, Gram-strain-negative coccus belonging to the genus Veillonella was isolated from the saliva of a Thai child coming from the east of Thailand. In the present study, the characterization of one strain, S12025-13T is described with the comparison of established species of the genus Veillonella and proposed a novel oral species of the genus Veillonella. MethodsStrain S12025-13T was conclusively identified using phenotypic, phylogenetic, genomic and biochemical approach. In phylogenetic analysis, multi-locus species trees (MLST) analysis was also conducted in this study. ResultsThe strain S12025-13T showed typical phenotypic characteristics of members of the genus Veillonella. Under anaerobic conditions, the strain produced acetic acid and propionic acid as metabolic end products in a trypticase-yeast extract-heamin medium containing 1% (w/v) glucose, 1% (w/v) fructose and 1% (v/v) sodium lactate. Sequencing of their 16S rRNA genes confirmed that it belongs to the genus Veillonella. Comparative analysis of the 16S rRNA, dnaK and rpoB gene sequences indicated that phylogenetically the strain comprised a distinct novel branch within the genus Veillonella. The novel strain showed 99.77, 97.29, and 99.02% similarity to partial 16S rRNA, dnaK and rpoB gene sequencing, respectively, to the type strains of the most closely related species. In MLST (selected 79 genes) analysis, strain S12025-13T formed a distinct taxon with robust bootstrap support (100%) within the genus Veillonella. Furthermore, strain S12025-13T shared the highest average nucleotide identity (ANI) value (95.97%) with the type strain of the most closely related species, “Veillonella faecalis” which is not validly published under the International Code of Nomenclature of Prokaryotes (ICNP). Likewise, strain S12025-13T showed the highest digital DNA-DNA hybridization (dDDH) value (65.00%) with the type strain of “V. faecalis”. In addition, strain S12025-13T formed a distinct branch in this clade with a bootstrap value of 72% with Veillonella nakazawae and 51% with “V. faecalis” related species of the genus Veillonella in the genome based phylogenetic tree. ConclusionThe novel strain could be discriminated from previously reported species of the genus Veillonella based on partial dnaK gene sequencing, MLST analysis, ANI and dDDH values, and genome-based phylogeny. Based on these observations, this strain represents a novel species, for which the name Veillonella orientalis sp., nov. is proposed. The type strain is S12025-13T (= JCM 33967T = CCUG 74596T).

Characterization of Erwinia amylovora isolates from the northern region of Saudi Arabia, including CRISPR genotyping

AbstractFire blight, caused by the bacterium Erwinia amylovora, poses a global threat to the pome fruit industry, leading to reduced yields and significant economic losses. A comprehensive countrywide survey was carried out to determine the possible E. amylovora distribution in pome fruit‐growing regions. During the period 2020–2023, 85 isolates characteristic of E. amylovora were obtained from pear across 23 locations in three main pear‐producing regions (Al‐Jouf, Hail and Tabuk) in northern Saudi Arabia (SA). Isolates were identified using biochemical, molecular and pathogenicity assays. Molecular characterization of Saudi strains was achieved using partial 16S rRNA and rpoB gene sequencing, rep‐PCR (specifically, BOX‐PCR) and CRISPR array analysis. The CRISPR genotyping in particular, using an existing scheme of three CRISPR arrays, CR1, CR2 and CR3, provided consistent patterns of the Saudi isolates with high similarity to those of E. amylovora strains from the UK and France, hinting at a possible origin of the fire blight infections from these countries. Some variation was observed in patterns between local strains, highlighting the importance of continued surveillance and genetic studies for effective disease management strategies. Identification of fire blight in three regions over several years should serve as a warning to the Ministry of Environment, Water and Agriculture in Saudi Arabia, prompting the initiation of a more comprehensive survey and an active disease management programme. This programme should include early detection and advice to growers on prevention and control measures. This is the first scientific study on the occurrence and genetic variability of E. amylovora in Saudi Arabia.

Common Features of Environmental Mycobacterium chelonae from Colorado Using Partial and Whole Genomic Sequence Analyses

Nontuberculous mycobacteria (NTM) are environmentally acquired opportunistic pathogens that cause chronic lung disease in susceptible individuals. While presumed to be ubiquitous in built and natural environments, NTM environmental studies are limited. While environmental sampling campaigns have been performed in geographic areas of high NTM disease burden, NTM species diversity is less defined among areas of lower disease burden like Colorado. In Colorado, metals such as molybdenum have been correlated with increased risk for NTM infection, yet environmental NTM species diversity has not yet been widely studied. Based on prior regression modeling, three areas of predicted high, moderate, and low NTM risk were identified for environmental sampling in Colorado. Ice, plumbing biofilms, and sink tap water samples were collected from publicly accessible freshwater sources. All samples were microbiologically cultured and NTM were identified using partial rpoB gene sequencing. From these samples, areas of moderate risk were more likely to be NTM positive. NTM recovery from ice was more common than recovery from plumbing biofilms or tap water. Overall, nine different NTM species were identified, including clinically important Mycobacterium chelonae. MinION technology was used to whole genome sequence and compare mutational differences between six M. chelonae genomes, representing three environmental isolates from this study and three other M. chelonae isolates from other sources. Drug resistance genes and prophages were common findings among environmentally derived M. chelonae, promoting the need for expanded environmental sampling campaigns to improve our current understanding of NTM species abundance while opening new avenues for improved targeted drug therapies.

Phylogenetic tree of Proteus spp. Based on partial rpoB gene sequence analysis

Due to the importance of the proposed rpoB gene as an alternative biomarker for microbial community studies, this study has focused on phylogenetic relationships among local Proteus clinical isolates. Fifty bacterial isolates were collected and identified phenotypically according to the culture, microscopic examination and biochemical tests. VITEK 2 compact system was used to confirm identification. Genotypic identification was performed after DNA extraction for 10 selected isolates and amplification with rpoB gene-specific primer and gel electrophoresis. The products were detected with a ( 1090 bp ) molecular size band, which was sent for Sanger sequencing using an ABI3730XL automated DNA sequencer, and data were analyzed and compared with standard sequences in GenBank.The isolates have been registered in the National Center for Biotechnology Information (NCBI) with accession numbers and named (HIMUS1, HIMUS2, HIMUS3, HIMUS4, HIUS5, HIMUS6, HIMUS7, HIMUS8, HIMUS9 and HIMUS10 ). The phylogenetic tree was constructed using partial (895 bp) rpoB gene sequences for those ten strains. Evolutionary distances were calculated using the method of Maximum Composite Likelihood with 1000 bootstrap replicates using GENEIOUS software. The sequences presented a similarity percentage ranging between (98.76% and 100%) when compared with the sequences of standard strains in NCBI. Keywords: rpoB gene, Proteus spp. Sequencing, Phylogenetic analysis

Paracholeplasma manati sp. nov. isolated from Florida manatees (Trichechus manatus latirostris).

Four novel strains of a member of the genus Paracholeplasma (OakleyT, Holly, Lorelei and Ariel) were isolated from skin of Florida manatees (Trichechus manatus latirostris). These strains were phenotypically and genetically characterized and compared with the known species of the genera Acholeplasma (A.), Alteracholeplasma (Al.), Haploplasma (H.), Paracholeplasma (P.) and Mariniplasma (M.). All the strains produced acid from glucose but did not hydrolyze arginine or urea. All were propagated in ambient air supplemented with 5±1 % CO2 at 35-37 °C using SP4-Z, Columbia and brain-heart infusion medium. Colonies on solid medium showed a typical fried-egg appearance and transmission electron microscopy revealed a typical mycoplasma-like cellular morphology. The results of phylogenetic analyses based on partial 16S rRNA, rpoB, gyrB and parE gene sequences and the whole proteome data indicated that the novel species is a unique species but phylogenetically closely related to Paracholeplasma vituli, Paracholeplasma morum and 'Paracholeplasma brassicae'. The average nucleotide identity and digital DNA-DNA hybridization values between strain OakleyT and the closely related species were significantly lower than the accepted thresholds for describing novel prokaryotic species at the genomic level. On the basis of the genomic, phenotypic and phylogenetic properties, the novel strains represent a novel species of the genus Paracholeplasma, for which the name Paracholeplasma manati sp. nov. with the type strain OakleyT (=NCTC 14352T =DSM 110686T) is proposed. The genomic DNA G+C content and complete draft genome size for the type strain are 38.35 % and 1 873 856 bp, respectively.

Streptococcus oriscaviae sp. nov. Infection Associated with Guinea Pigs.

ABSTRACTPet bite-related infections are commonly caused by the pet’s oral flora transmitted to the animal handlers through the bite wounds. In this study, we isolated a streptococcus, HKU75T, in pure culture from the purulent discharge collected from a guinea pig bite wound in a previously healthy young patient. HKU75T was alpha-hemolytic on sheep blood agar and agglutinated with Lancefield group D and group G antisera. API 20 STREP showed that the most likely identity for HKU75T was S. suis I with 85.4% confidence while Vitek 2 showed that HKU75T was unidentifiable. MALDI-TOF MS identified HKU75T as Streptococcus suis (score of 1.86 only). 16S rRNA gene sequencing showed that HKU75T was most closely related to S. parasuis (98.3% nucleotide identity), whereas partial groEL and rpoB gene sequencing showed that it was most closely related to S. suis (81.8% and 89.8% nucleotide identity respectively). Whole genome sequencing and intergenomic distance determined by ANI revealed that there was <85% identity between the genome of HKU75T and those of all other known Streptococcus species. Genome classification using concatenated sequences of 92 bacterial core genes showed that HKU75T belonged to the Suis group. groEL gene sequences identical to that of HKU75T could be directly amplified from the oral cavities of the two guinea pigs owned by the patient. HKU75T is a novel Streptococcus species, which we propose to be named S. oriscaviae. The oral cavity of guinea pigs is presumably a reservoir of S. oriscaviae. Some of the reported S. suis strains isolated from clinical specimens may be S. oriscaviae.IMPORTANCE We reported the discovery of a novel Streptococcus species, propose to be named Streptococcus oriscaviae, from the pus collected from a guinea pig bite wound in a healthy young patient. The bacterium was initially misidentified as S. suis/S. parasuis by biochemical tests, mass spectrometry. and housekeeping genes sequencing. Its novelty was confirmed by whole genome sequencing. Comparative genomic studies showed that S. oriscaviae belongs to the Suis group. S. oriscaviae sequences were detected in the oral cavities of the two guinea pigs owned by the patient, suggesting that the oral cavity of guinea pigs could be a reservoir of S. oriscaviae. Some of the reported S. suis strains may be S. oriscaviae. Further studies are warranted to refine our knowledge on this novel Streptococcus species.

Leptospira in Slaughtered Fattening Pigs in Southern Italy: Serological Survey and Molecular Typing.

Simple SummaryIn this study, serological and molecular assays in 55 pigs in Sicily were conducted in order to investigate Leptospira spp. prevalence and to carry out strain characterization. A seropositivity rate of 16.4% was determined; 3.64% of kidney samples tested positive for pathogenic Leptospiral DNA. Obtained data showed that Leptospira infection is common among pigs in southern Italy, confirming the importance of Leptospiral infection in pigs and reaffirming the potential role of these animals as a source of infection for humans (occupational risk) and other animals. Our study delivers a comprehensive overview based on up-to-date data to deepen the knowledge of swine leptospiral infections, characterize new potential emerging strains, and reinforce control measures able to reduce the infection risk in swine herds.Leptospirosis is a re-emerging zoonosis of worldwide significance; a wide spectrum of wild and domestic animal species act as natural or accidental hosts. Swine can act as maintenance or accidental hosts of pathogenic Leptospira spp. This study aimed at investigation of Leptospira spp. prevalence and diversity in slaughtered pigs in southern Italy (Sicily). In total, 55 samples of kidneys and blood were collected. Microscopic agglutination test and real-time PCR were performed to detect pathogenic and intermediately pathogenic Leptospira. Partial rpoB gene sequencing and multi-locus sequence typing (MLST) were performed to characterize Leptospira species. The analysis showed a seropositivity rate of 16.4%, with Australis representing the most frequently identified serogroup (63.64%); Pomona and Sejroe were detected with a prevalence of 27.27% and 9.09%, respectively. Pathogenic Leptospiral DNA was detected in 2 kidney samples (3.64%). Leptospira were identified through MLST as L. borgpetersenii serovar Tarassovi (serogroup Tarassovi). Obtained data confirmed the presence of Leptospira infection among pigs in southern Italy, suggesting that management of these animals may be considered an occupational risk for humans.

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 Zaradi2T (=ATCC BAA-1891T = DSM 22451T) is proposed.

Veillonella nakazawae sp. nov., an anaerobic Gram-negative coccus isolated from the oral cavity of Japanese children.

Two strains of previously unknown Gram-negative cocci, T1-7T and S6-16, were isolated from the oral cavity of healthy Japanese children. The two strains showed atypical phenotypic characteristics of members of the genus Veillonella, including catalase production. Sequencing of their 16S rRNA genes confirmed that they belong to genus Veillonella. Under anaerobic conditions, the two strains produced acetic acid and propionic acid as metabolic end-products in a trypticase-yeast extract-haemin medium containing 1 % (w/v) glucose, 1 % (w/v) fructose and 1 % (v/v) sodium lactate. Comparative analysis of the 16S rRNA, dnaK, rpoB and gltA gene sequences revealed that the two strains are phylogenetically homogeneous and comprise a distinct, novel lineage within the genus Veillonella. The sequences from the two strains shared the highest similarity, at 99.9, 95.8, 96.9 and 96.7 %, using the partial 16S rRNA, dnaK, rpoB and gltA gene sequences, respectively, with the type strains of the two most closely related species, Veillonella dispar ATCC 17748T and Veillonella infantium JCM 31738T. Furthermore, strain T1-7T shared the highest average nucleotide identity (ANI) value (94.06 %) with type strain of the most closely related species, V. infantium. At the same time, strain T1-7T showed the highest digital DNA-DNA hybridization (dDDH) value (55.5 %) with the type strain of V. infantium. The two strains reported in this study were distinguished from the previously reported species from the genus Veillonella based on catalase production, partial dnaK, rpoB and gltA sequences, average ANI and dDDH values. Based on these observations, the two strains represent a novel species, for which the name Veillonella nakazawae sp. nov. is proposed. The type strain is T1-7T (JCM 33966T=CCUG 74597T).

Pseudomonas brassicae sp. nov., a pathogen causing head rot of broccoli in Japan.

Phytopathogenic bacteria, MAFF 212426, MAFF 212427T, MAFF 212428 and MAFF 212429, were isolated from head rot lesions of broccoli (Brassica oleracea L. var. italica Plenck) in Hokkaido, Japan, and subjected to polyphasic taxonomic characterization. The cells were Gram-reaction-negative, aerobic, non-spore-forming, motile with one or two polar flagella, rod-shaped and formed pale yellow colonies. Results of 16S rRNA gene sequence analysis showed that they belong to the genus Pseudomonas with the highest similarity to 'Pseudomonas qingdaonensis' JJ3T (99.86 %), Pseudomonas laurentiana GSL-010T (99.22 %), Pseudomonas huaxiensis WCHPs060044T (99.01 %), Pseudomonas japonica NBRC 103040T (98.87 %) and Pseudomonas alkylphenolica KL28T (98.73 %). The genomic DNA G+C content was 63.4 mol% and the major fatty acids (>5 % of the total fatty acids) were summed feature 3 (C16 : 1 ω7c / C16 : 1 ω6c), C16 : 0, summed feature 8 (C18 : 1 ω7c / C18 : 1 ω6c) and C17 : 0 cyclo. Multilocus sequence analysis using the partial rpoD, gyrB and rpoB gene sequences and phylogenomic analyses based on the whole genome sequences demonstrated that the strains are members of the Pseudomonas putida group, but form a monophyletic, robust clade separated from their closest relatives. Average nucleotide identity (ANI) and digital DNA-DNA hybridization (dDDH) values corroborated their novel species status, with 88.39 % (ANI) and 35.8 % (dDDH) as the highest scores with 'P. qingdaonensis' JJ3T. The strains were differentiated from their closest relatives by phenotypic characteristics, pathogenicity on broccoli, and whole-cell MALDI-TOF mass spectrometry profiles. The phenotypic, chemotaxonomic and genotypic data showed that the strains represent a novel Pseudomonas species, for which the name Pseudomonas brassicae sp. nov. is proposed. The type strain is MAFF 212427T (=ICMP 23635T).

Corynebacterium godavarianum Jani et al. 2018 and Corynebacterium hadale Wei et al. 2018 are both later heterotypic synonyms of Corynebacterium gottingense Atasayar et al. 2017, proposal of an emended description of Corynebacterium gottingense Atasayar et al. 2017.

Seven strains of an unidentifiable Corynebacterium species recovered from blood cultures, urine or cerebrospinal fluid over 26 years, closest to but differentiated from Corynebacterium imitans by 16S rRNA gene and partial rpoB gene sequencing, were studied. In November 2017, Atasayar et al. described a blood culture isolate as Corynebacterium gottingense sp. nov., which had >99 % similarity by 16S rRNA gene sequencing to the Canadian strains. In January 2018, Jani et al. described Corynebacterium godavarianum sp. nov., recovered from the Godavari River, India, which also had >99 % similarity by 16S/rpoB sequencing to the Canadian strains and C. gottingense. In May 2018, Wei et al. described Corynebacterium hadale recovered from hadopelagic water; this too had >99 % similarity by 16S rRNA gene sequencing to C. gottingense, C. godavarianum and the Canadian strains. C. gottingense DSM 103494T and C. godavarianum LMG 29598T were acquired and whole genome sequencing was performed (not previously done). Results were compared with genomes from C. hadale (GenBank accession NQMQ01) and the Canadian isolates. We found that these ten genomes formed a single taxon when compared using digital DNA-DNAhybridization, average nucleotide identity using blastn and average amino acid identity criteria but exhibited some subtle biochemical and chemotaxonomic differences. Heuristically, we propose that C. godavarianum and C. hadale are later heterotypic synonyms of, and the Canadian isolates are identifiable as, C. gottingense. We provide an emended description of Corynebacterium gottingense Atasayar et al. 2017; genomes ranged from 2.48 to 2.69 Mb (C. gottingense DSM 103494T, 2.62 Mb) with G+C content of 65.1-65.6 mol% (WGS), recovered from clinical and environmental sites.

Mycoplasma nasistruthionis sp. nov. and Mycoplasma struthionis sp. nov. isolated from ostriches with respiratory disease

Twelve Mycoplasma (M.) strains isolated from the nose, the trachea, and the lung of ostriches (Struthio camelus) displaying respiratory disease were investigated. Analysis of 16S rRNA gene sequences placed five of these strains within the M. synoviae cluster, and seven strains within the M. hominis cluster of genus Mycoplasma, which was further confirmed by analyses of the 16S-23S rRNA intergenic spacer region, and partial rpoB gene and amino acid sequences. Genomic information as well as phenotypic features obtained by matrix-assisted laser desorption ionization time of flight (MALDI-ToF) mass spectrometry analysis and serological reactions indicated that the strains examined are representatives of two hitherto unclassified species of genus Mycoplasma, for which the names Mycoplasma nasistruthionis sp. nov., with type strain 2F1AT (= ATCC BAA-1893T = DSM 22456T), and Mycoplasma struthionis sp. nov., with type strain 237IAT (= ATCC BAA-1890T = DSM 22453T), are proposed.

Classification of Bisgaard's taxa 14 and 32 and a taxon from kestrels demonstrating satellitic growth and proposal of Spirabiliibacterium gen. nov., including the description of three species: Spirabiliibacterium mucosae sp. nov., Spirabiliibacterium pneumoniae sp. nov. and Spirabiliibacterium falconis sp. nov.

Avian Pasteurella-like organisms tentatively named taxon 14 of Bisgaard have been obtained from different lesions in birds including ducks, turkeys, pigeons, geese and peafowl. Taxon 32 of Bisgaard was first reported from lesions in pigeon hawks (Accipiter gentiles). The taxon isolated from kestrels (Falco tinnunculus) was V-factor dependent and originally reported as Haemophilus-like. The results of 16S rRNA gene sequence based phylogenetic analysis recently indicated that the taxa 14 and 32 and the kestrel taxon were located in a monophyletic group distantly related to [Pasteurella] testudinis with 92-93 % 16S rRNA gene sequence similarity. Comparison of 41 conserved protein sequences confirmed the monophyletic nature of the three taxa. Partial rpoB gene sequencing of 43 strains of taxon 14, taxon 32 and the kestrel taxon showed a relationship between taxon 14 and 32 of 88.2-90.0 % similarity. Within taxon 14, 93.3-100 % similarity was found, whereas the two strains of taxon 32 showed 99.8 % rpoB similarity. Sequencing of 16S rRNA genes of strains representing the rpoB diversity outlined showed more than 98 % similarity within taxon 14 and 99.4 % within taxon 32, while the kestrel strains showed 100 % 16S rRNA gene sequence similarity. A new genus, Spirabiliibacterium gen. nov., is proposed to include taxon 14, taxon 32 and the kestrel taxon. Phenotypically, members of the genus Spirabiliibacterium can be separated from members of the genera Aggregatibacter, Avibacterium and Volucribacter by maltose, oxidase and methyl red, respectively. Two or more phenotypic characters separate members of the genus Spirabiliibacterium from members of the remaining 27 genera of the family Pasteurellaceae.The G+C content of DNA ranged from 42.9 to 51.2 % (genome sequence) for members of the genus Spirabiliibacterium. The type strain of Spirabiliibacterium mucosae (taxon 14 of Bisgaard) is 20609/3T (=CCUG 16499T=DSM 111429T=HIM 913-3T). The type strain of Spirabiliibacterium pneumoniae is HPA106T (=CCUG 74731T=DSM 111430T). The type strain of Spirabiliibacterium falconis (kestrel taxon) is IPDH 2176T (=NCTC 11878T=CCUG 28587T).

Rapid and reasonable molecular identification of bacteria and fungi in microbiological diagnostics using rapid real-time PCR and Sanger sequencing

Analyses of short subunit gene sequences have been established for taxonomic classification and identification of bacteria and fungi. To produce partial bacterial ribosomal 16S rRNA and rpoB and fungal ribosomal ITS/LSU gene sequences for DNA sequencing, real-time PCR assays supplemented with the nucleic acid stain SYBR Green were created. Generation of PCR products was monitored based on amplification and melting curves. The PCR products were subsequently subjected to Sanger sequencing on demand for identification of bacteria and fungi in routine microbiological diagnostics within a period of two days.From a total of 78 bacterial isolates 40 (51%) or 67 (86%) could be identified at species level using only partial 16S rRNA or additionally rpoB gene sequences based on BLASTN (NCBI) database queries, respectively. Using partial 16S rRNA and rpoB gene sequencing unambiguous assignment was not possible for the closely related species of the Bacillus (B.) cereus group, Bordetella (B.) pertussis/ B. parapertussis/ B. bronchiseptica, Brucella spp., Enterobacter cloacae complex, Escherichia/ Shigella spp., Staphylococcus (S.) hyicus/ S. agnetis and Yersinia (Y.) pseudotuberculosis/ Y. pestis. However, partial rpoB gene sequencing succeeded in identifying 27 bacterial isolates at species level in addition to 16S rRNA gene sequencing.Regarding ITS/LSU gene sequencing, best results could be achieved by ITS gene sequencing followed by LSU gene sequencing, resulting in 32 (63%) and 21 (43%) of a total of 51 fungal isolates that could be identified at species level, respectively. Insufficient identification at species level was observed for the genera Apiotrichum, Aspergillus, Cladosporium, Cryptococcus, Microsporum, Nannizziopsis, Penicillium, Trichosporon, and Tolypocladium included in this study.The concept of this procedure is suitable for rapid and reasonable molecular identification of bacteria and fungi within two days and is therefore applicable in routine microbiological diagnostic laboratories.

Phylogenetic relationship of Ornithobacterium rhinotracheale isolated from poultry and diverse avian hosts based on 16S rRNA and rpoB gene analyses

BackgroundOrnithobacterium (O.) rhinotracheale is an emerging bacterial pathogen in poultry and not fully understood to date. Because of its importance particularly for the global turkey meat industry, reliable diagnostic and characterization methods are needed for early treatment and in future for better vaccine production. The host range of birds infected by O. rhinotracheale or carrying the bacterium in their respiratory tract has constantly increased raising important epidemiological and taxonomic questions for a better understanding of its diversity, ecology and transmission cycles. The purpose of this study was to introduce partial rpoB gene sequencing for O. rhinotracheale into routine diagnostics to differentiate strains isolated from poultry and more diverse avian hosts (i.e., birds of prey, corvids and pigeons) and to compare phylogenetic relationships with results from 16S rRNA gene analysis and multilocus sequence typing (MLST).ResultsPartial 16S rRNA gene analysis revealed a high level of homogeneity among the 65 investigated O. rhinotracheale sequences with similarity values ranging from 98.6 to 100% between sequences from non-galliform and poultry species. The corresponding rpoB gene sequences were heterogeneous and ranged in their similarity values from 85.1 to 100%. The structure of the rpoB tree was in strong correlation with previous MLST results revealing three main clusters A (poultry and birds of prey), B (poultry, birds of prey and corvids) and C (pigeons), which were clearly separated from each other.ConclusionsBy using partial sequences from a single gene, the rpoB gene analysis is in good agreement with MLST results with a slight decrease in resolution to distinguish more similar strains. The present results provide strong evidence that traditional phenotypic and genetic methods may not properly represent the heterogeneous group of bacteria classified as O. rhinotracheale. From housekeeping gene analyses, it is very likely that the genus Ornithobacterium includes additional species and partial rpoB gene sequencing can be recommended as fast, cost-effective and readily available method to identify strains and differentiate between O. rhinotracheale and Ornithobacterium-like bacteria.

Isolation and characterization of Corynebacterium spp. from bulk tank raw cow's milk of different dairy farms in Germany.

We detected Corynebacterium spp. in raw milk samples of three farms by means of a selective, tellurite-containing medium. The isolated strains were identified based on full 16S rRNA gene sequences and partial rpoB gene sequences as C. xerosis, C. variabile, C. lactis, C. callunae, C. confusum, C. glutamicum and C. crudilactis. The identification based on 16S rRNA and rpoB sequences was not reliable for isolates of C. xerosis. Chemotaxonomic markers of the isolates, fatty acids, acyl type of peptidoglycan, presence and length of mycolic acids, quinone patterns, and polar lipids, were in accord with the known characteristics of these species. Biochemical profiles, analyzed with the API Coryne system, were able to differentiate all groups, but were unable to identify the strains due to an inappropriate database for raw-milk associated corynebacteria. Most of the tested isolates showed a single-substance resistance against oxacillin, but three single isolates were classified as multidrug resistant.

Veillonella infantium sp. nov., an anaerobic, Gram-stain-negative coccus isolated from tongue biofilm of a Thai child.

A strain of a novel anaerobic, Gram-stain-negative coccus was isolated from the tongue biofilm of a Thai child. This strain was shown, at the phenotypic level and based on 16S rRNA gene sequencing, to be a member of the genus Veillonella. Comparative analysis of the 16S rRNA, dnaK and rpoB gene sequences indicated that phylogenetically the strain comprised a distinct novel branch within the genus Veillonella. The novel strain showed 99.8, 95.1 and 95.9 % similarity to partial 16S rRNA, dnaK and rpoB gene sequences, respectively, to the type strains of the two most closely related species, Veillonelladispar ATCC 17748T and Veillonellatobetsuensis ATCC BAA-2400T. The novel strain could be discriminated from previously reported species of the genus Veillonella based on partial dnaK and rpoB gene sequencing and average nucleotide identity values. The major acid end-product produced by this strain was acetic acid under anaerobic conditions in trypticase-yeast extract-haemin with 1 % (w/v) glucose or fructose medium. Lactate was fermented to acetic acid and propionic acid. Based on these observations, this strain represents a novel species, for which the name Veillonella infantium sp. nov. is proposed. The type strain is T11011-4T (=JCM 31738T=TSD-88T).

Lelliottia jeotgali sp. nov., isolated from a traditional Korean fermented clam.

Strain PFL01T was isolated from traditional Korean fermented clam, jogae-jeotgal, and characterized. The strain was a facultative anaerobic, Gram-stain-negative bacterium that was rod-shaped, motile and beige-pigmented. The phylogenetic sequence analysis based on the 16S rRNA gene from PFL01T revealed that it was closely related to Lelliottia nimipressuralis LMG 10245T and Lelliottia amnigena LMG 2784T with 99.3 and 99.3 % sequence identities, respectively. Multilocus sequence type analysis of concatenated partial aptD, gyrB, infB and rpoB gene sequences showed a clear distinction of strain PFL01T from its closest related type strains. The discrimination was also supported by unique repetitive extragenic palindromic PCR (Rep-PCR, ERIC-PCR) fingerprint patterns. In addition, results from average nucleotide identity analyses with other species were less than 85 %. vitek and API analyses revealed distinct characteristics from other species of Lelliottia. The cellular fatty acid profile of the strain consisted of C16 : 0, cyclo-C17 : 0, C16 : 1ω7c/C16 : 1ω6c and C18 : 1ω7c/C18 : 1ω6c as major components. The whole genome of PFL01T was 4.6 Mb with a G+C content of 55.3 mol%. Based on these results, strain PFL01T was classified as a novel species of the genus Lelliottia, for which the name Lelliottia jeotgali sp. nov. is proposed. The type strain in PFL01T (=KCCM 43247T=JCM 31901T).

Bifidobacterium catulorum sp. nov., a novel taxon from the faeces of the baby common marmoset (Callithrix jacchus).

In our previous study based on hsp60 PCR-restriction fragment length polymorphism and 16S rRNA gene sequencing, we stated that the bifidobacterial strains isolated from the individual faecal samples of five baby common marmosets constituted different phylogenetically isolated groups of the genus Bifidobacterium. In that study, we also proposed that these isolated groups potentially represented novel species of the genus Bifidobacterium. Out of them, Bifidobacterium aesculapii, Bifidobacterium myosotis, Bifidobacterium tissieri and Bifidobacterium hapali, have been described recently. Another strain, designated MRM 8.19T, has been classified as member of the genus Bifidobacterium on the basis of positive results for fructose-6-phosphate phosphoketolase activity and analysis of partial 16S rRNA, hsp60, clpC, dnaJ, dnaG and rpoB gene sequences. Analysis of 16S rRNA and hsp60 gene sequences revealed that strain MRM 8.19T was related to B. tissieri DSM 100201T (95.8 %) and to Bifidobacterium bifidum ATCC 29521T (93.7 %), respectively. The DNA G+C composition was 63.7 mol% and the peptidoglycan structure was l-Orn(Lys)-l-Ser. Based on the phylogenetic, genotypic and phenotypic data reported, strain MRM 8.19T represents a novel taxon within the genus Bifidobacterium for which the name Bifidobacterium catulorum sp. nov. (type strain MRM 8.19T=DSM 103154T=JCM 31794T) is proposed.

Species distribution, sequence types and antimicrobial resistance of Acinetobacter spp. from cystic fibrosis patients.

Acinetobacter spp. are important healthcare pathogens, being closely linked to antibiotic resistance and outbreaks worldwide. Although such species are rarely observed in patients with cystic fibrosis (CF), we describe the characteristics of 53 strains of Acinetobacter spp. isolated from the sputum of 39 Brazilian patients with CF. The species distribution was A. baumannii (n = 29), A. pittii (n = 13), A. nosocomialis (n = 8), A. seifertii (n = 1), A. soli (n = 1) and A. variabilis (n = 1) determined by partial rpoB gene sequencing. Sixteen strains (10 A. baumannii, 3 A. pittii and 3 A. nosocomialis) were multidrug-resistant (MDR) by disk diffusion test (30%) and eight MDR carbapenem-resistant A. baumannii strains harboured the bla OXA-23-like oxacillinase gene. Thirty-three sequence types (STs) were identified by multilocus sequence typing of which eight were novel (A. baumannii: 843, 844, 845, 847, 848; A. pitti: 643; A. nosocomialis: 862 and A. seifertii: 846); six STs (2 A. baumannii, 3 A. pittii and 1 A. nosocomialis) were found in more than one patient. Four strains of A. baumannii were assigned to two common clonal complexes (CCs), namely, CC1 (ST1, ST20 and ST160), and CC79 (ST79). This study underlines the extensive species diversity of Acinetobacter spp. strains in CF lung infections which may present difficulties for therapy due to significant antimicrobial resistance.