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ABSTRACT Accurate species-level identification of Enterobacter cloacae complex (ECC) is crucial for related research. The classification of ECC is based on strain-to-strain phylogenetic congruence, as well as genomic features including average nucleotide identity (ANI) and digitalized DNA-DNA hybridization (dDDH). ANI and dDDH derived from whole-genome sequencing have emerged as a reliable metric for assessing genetic relatedness between genomes and are increasingly recognized as a standard for species delimitation. Up to now, there are two different classification methods for ECC. The first one categorizes E. hormaechei, a species within ECC, into five subspecies (E. hormaechei subsp. steigerwaltii, subsp. oharae, subsp. xiangfangensis, subsp. hoffmannii, and subsp. hormaechei). The second classifies E. hormaechei as three species: E. hormaechei, “E. xiangfangensis,” “E. hoffmanii.” While the former is well-accepted in the academic area, the latter may have a greater ability to distinguish different species of ECC. To assess the suitability of these identification criteria for clinical ECC isolates, we conducted a comprehensive analysis involving phylogenetic analysis, ANI and dDDH value alignment, virulence gene identification, and capsule typing on 256 clinical ECC strains isolated from the bloodstream. Our findings indicated that the method of categorizing E. hormaechei into five subspecies has better correlation and consistency with the molecular characteristics of clinical ECC isolates, as evidenced by phylogenetic analysis, virulence genes, and capsule typing. Therefore, the subspecies-based classification method appears more suitable for taxonomic assignments of clinical ECC isolates.IMPORTANCEStandardizing taxonomy of the Enterobacter cloacae complex (ECC) is necessary for data integration across diverse studies. The study utilized whole-genome data to accurately identify 256 clinical ECC isolated from bloodstream infections using average nucleotide identity (ANI), digitalized DNA-DNA hybridization (dDDH), and phylogenetic analysis. Through comprehensive assessments including phylogenetic analysis, ANI and dDDH comparisons, virulence gene, and capsule typing of the 256 clinical isolates, it was concluded that the classification method based on subspecies exhibited better correlation and consistency with the molecular characteristics of clinical ECC isolates. In summary, this research contributes to the precise identification of clinical ECC at the species level and expands our understanding of ECC.

Objectives: We report a case of bacteremia with pyelonephritis in an adult male with an underlying disease caused by α-hemolytic streptococci. α-Hemolytic streptococci were isolated from blood, but it was challenging to identify its species. This study aimed to characterize the causative bacterium SP4011 and to elucidate its species. Methods: The whole-genome sequence and biochemical characteristics of SP4011 were determined. Based on the genome sequence, phylogenetic analysis was performed with standard strains of each species of α-hemolytic streptococci. Digital DNA-DNA hybridization (dDDH) and average nucleotide identity (ANI) values were calculated. Results: SP4011 showed optochin susceptibility and bile solubility, but did not react with pneumococcal omni antiserum. Phylogenetic analysis of the whole-genome sequence showed that SP4011 clustered with S. pneumoniae and S. pseodopneumoniae and was most closely related to S. pseodopneumoniae. Genomic analysis revealed that ANI and dDDH values between SP4011 and S. pseodopneumoniae were 94.0 % and 56.0 %, respectively, and between SP4011 and S. pneumoniae were 93.3 % and 52.2 %, respectively. Biochemical characteristics also showed differences between SP4011 and S. pseodopneumoniae and between SP4011 and S. pneumoniae. These results indicate that SP4011 is a novel species. Conclusion: Our findings indicate that SP4011 is a novel species of the genus Streptococcus. SP4011 has biochemical characteristics similar to S. pneumoniae, making it challenging to differentiate and requiring careful clinical diagnosis. This isolate was proposed to be a novel species, Streptococcus parapneumoniae sp. nov. The strain type is SP4011T (= JCM 36068T = KCTC 21228T).

The reemergence and spread of Xanthomonas translucens, the causal agent of bacterial leaf streak in cereal crops and wilt in turfgrass and forage species, is a concern to growers in the United States and Canada. The pathogen is seedborne and listed as an A2 quarantine organism by EPPO, making it a major constraint to international trade and exchange of germplasm. The pathovar concept of the X. translucens group is confusing due to overlapping of plant host ranges and specificity. Here, comparative genomics, phylogenomics, and 81 up-to-date bacterial core gene set (ubcg2) were used to assign the pathovars of X. translucens into three genetically and taxonomically distinct clusters. The study also showed that whole genome-based digital DNA-DNA hybridization unambiguously can differentiate the pvs. translucens and undulosa. Orthologous gene and proteome matrix analyses suggest that the cluster consisting of graminis, poae, arrhenatheri, phlei, and phleipratensis is very divergent. Whole-genome data were exploited to develop the first pathovar-specific TaqMan real-time PCR tool for detection of pv. translucens on barley. Specificity of the TaqMan assay was validated using 62 Xanthomonas and non-Xanthomonas strains as well as growth chamber-inoculated and naturally infected barley leaves. Sensitivity levels of 0.1 pg (purified DNA) and 23 CFUs per reaction (direct culture) compared favorably with other previously reported real-time PCR assays. The phylogenomics data reported here suggest that the clusters could constitute novel taxonomic units or new species. Finally, the pathovar-specific diagnostic tool will have significant benefits to growers and facilitate international exchange of barley germplasm and trade. [ABSTRACT FROM AUTHOR]

Objective: A multiplex gyrB PCR assay has been used to diagnose Acinetobacter baumannii. However, this assay has not been validated against the gold standard DNA-DNA hybridization assay, which is a laborious method. DNA-DNA hybridization assay is now replaced by whole genome sequence (WGS)-based methods. Two such methods are a k-mer-based search of sequence reads using the Kraken 2 program and average nucleotide identity (ANI). The objective was to validate the gyrB PCR assay with WGS-based methods.Subjects and Methods: We cultured 270 sequential A. baumannii isolates from the rectal swabs of 32 adult patients. The identity of the isolates was determined by gyrB PCR. The sequences of 269 isolates were determined by Illumina sequencing and the taxonomy was inferred by the Kraken 2 program and ANI.Results: All the 269 isolates were confirmed as A. baumannii by Kraken 2 and ANI.Conclusion: The gyrB PCR assay is now validated for easy identification of A. baumannii in comparison with gold standard WGS-based assays. [ABSTRACT FROM AUTHOR]

The pathovar-based taxonomy of the Xanthomonas translucens group is very confusing due to an overlap of plant host ranges and level of host specificity. Here, whole-genome sequence-based parameters (digital DNA-DNA hybridization and blast-based average nucleotide identity), phylogenomic, biochemical and phenotypical data were used to taxonomically analyse the 11 known pathovars of the X. translucens complex. This polyphasic approach taxonomically assigned the 11 pathovars of X. translucens complex into three distinct species, two of which are new: X. translucens , X. cerealis sp. nov. and X. graminis sp. nov. X. translucens consists of three pathovars: pv. translucens (=pv. hordei ), pv. pistaciae strain A ICMP 16316 PT and pv. undulosa (=pv. secalis ). X. cerealis sp. nov. encompasses the pv. cerealis strain LMG 679 PT and pv. pistaciae strain B ICMP 16317 PT with genome similarity of 92.7% (dDDH) and 99.0% (ANIb) suggesting taxonomically similar genotypes. The other new species, X. graminis sp. nov., consists of the remaining five designated pathovars (pv. graminis , pv. arrhenatheri, pv. poae , pv. phleipratensis and pv. phlei ) with highly variable dDDH and ANIb values ranging from 74.5 to 93.0% and from 96.7 to 99.2%, respectively, an indication of a very divergent taxonomic group. Only strains of pvs. phlei and phleipratensis showed the highest genomic similarities of 93.0% (dDDH) and 99.2% (ANIb), suggesting synonymic pathovars as both infect the same plant hosts. The dDDH and ANI data were corroborated by phylogenomics clustering. The fatty acid contents were similar but the type strain of X. graminis sp. nov. exhibited 20% less C 15 : 0 iso and 40% more C 17 : 0 iso fatty acids than the other species. Based on phenotypic, biochemical and whole-genome sequence data, we propose two new species, Xanthomonas cerealis sp. nov. and Xanthomonas graminis sp. nov. with type strains LMG 679 T (=NCPPB 1944 T ) and LMG 726 T (=NCPPB 2700 T ), respectively.

We report a case of bacteremia with pyelonephritis in an adult male with an underlying disease caused by α-hemolytic streptococci. α-Hemolytic streptococci were isolated from blood, but it was challenging to identify its species. This study aimed to characterize the causative bacterium SP4011 and to elucidate its species. The whole-genome sequence and biochemical characteristics of SP4011 were determined. Based on the genome sequence, phylogenetic analysis was performed with standard strains of each species of α-hemolytic streptococci. Digital DNA-DNA hybridization (dDDH) and average nucleotide identity (ANI) values were calculated. SP4011 showed optochin susceptibility and bile solubility, but did not react with pneumococcal omni antiserum. Phylogenetic analysis of the whole-genome sequence showed that SP4011 clustered with S. pneumoniae and S. pseodopneumoniae and was most closely related to S. pseodopneumoniae. Genomic analysis revealed that ANI and dDDH values between SP4011 and S. pseodopneumoniae were 94.0 % and 56.0 %, respectively, and between SP4011 and S. pneumoniae were 93.3 % and 52.2 %, respectively. Biochemical characteristics also showed differences between SP4011 and S. pseodopneumoniae and between SP4011 and S. pneumoniae. These results indicate that SP4011 is a novel species. Our findings indicate that SP4011 is a novel species of the genus Streptococcus. SP4011 has biochemical characteristics similar to S. pneumoniae , making it challenging to differentiate and requiring careful clinical diagnosis. This isolate was proposed to be a novel species, Streptococcus parapneumoniae sp. nov. The strain type is SP4011T (= JCM 36068T = KCTC 21228T). [ABSTRACT FROM AUTHOR]

Background and Objectives: DNA extraction is an important step of any molecular experiment. DNA could not be easily extracted from members of actinomycetes by the usual methods of lysis. Due to the low efficiency of the conventional DNA extraction methods, development of an effective technique for DNA extraction of actinobacteria in emergency cases seems to be necessary. Since most of the DNA extraction techniques and commercial kits are not efficient enough to extract DNA from actinobacteria, this study was conducted to improve an efficient method obtained from conventional one to extract DNA from this group of bacteria. Materials and Methods: DNA extraction was performed using five methods (an improved method, Invisorb Spin Plant Mini Kit, EZ-10 Spin Column, Sarrbrucken method (HZI, Germany) and Kirby Bauer's method). To evaluate the quantity and quality of extracted genomic DNA, UV absorbance of all samples and efficiency of polymerase chain reaction (PCR) were evaluated. Results: Overall, the results revealed that the highest quantity (up to 4000 ng/µl) and good quality of DNA was obtained using introduced DNA extraction method. Conclusion: Results indicated that recently introduced improved method was more efficient for extraction of DNA from actinobacteria for DDH (DNA-DNA hybridization) test and for those require the high concentration of DNA. [ABSTRACT FROM AUTHOR]

Background and Objectives: DNA extraction is an important step of any molecular experiment. DNA could not be easily extracted from members of actinomycetes by the usual methods of lysis. Due to the low efficiency of the conventional DNA extraction methods, development of an effective technique for DNA extraction of actinobacteria in emergency cases seems to be necessary. Since most of the DNA extraction techniques and commercial kits are not efficient enough to extract DNA from actinobacteria, this study was conducted to improve an efficient method obtained from conventional one to extract DNA from this group of bacteria. Materials and Methods: DNA extraction was performed using five methods (an improved method, Invisorb Spin Plant Mini Kit, EZ-10 Spin Column, Sarrbrucken method (HZI, Germany) and Kirby Bauer's method). To evaluate the quantity and quality of extracted genomic DNA, UV absorbance of all samples and efficiency of polymerase chain reaction (PCR) were evaluated. Results: Overall, the results revealed that the highest quantity (up to 4000 ng/µl) and good quality of DNA was obtained using introduced DNA extraction method. Conclusion: Results indicated that recently introduced improved method was more efficient for extraction of DNA from actinobacteria for DDH (DNA–DNA hybridization) test and for those require the high concentration of DNA.

Background: Subgingival dental plaque is an ecosystem playing a key role in supporting both oral health and systemic health. Menopause-related changes have the potential to disrupt its balance, which is crucial to postmenopausal well-being. Our study explored how circulating estradiol levels correlate with subgingival microbial composition using checkerboard DNA-DNA hybridization in premenopausal and postmenopausal women. We also demonstrated that combining this method with 16S ribosomal RNA (rRNA) sequencing insights remains valuable for examining subgingival ecology., Methods: We assessed 40 bacterial species in 77 premenopausal and 81 postmenopausal women using checkerboard DNA-DNA hybridization and measured serum estradiol with enzyme-linked immunosorbent assay (ELISA). Women were categorized by subgingival dysbiosis severity using a modified Subgingival Microbial Dysbiosis Index (mSMDI). Six women from each normobiotic and dysbiotic subgroup across premenopausal and postmenopausal women underwent 16S rRNA sequencing analysis., Results: DNA checkerboard analysis revealed that most observed variability in individual bacterial proportions is associated with periodontitis. Two species, Leptotrichia buccalis and Streptococcus constellatus, exhibited differences related to estradiol levels within the premenopausal group (p = 0.055 and p = 0.009, respectively). 16S rRNA sequencing confirmed the mSMDI's validity in categorizing normobiotic and dysbiotic states. Menopausal status was not associated with a dysbiotic shift in the subgingival microbiome despite significantly more attachment loss in postmenopausal compared to premenopausal women., Conclusions: Our results indicate that decreased estradiol levels or increased attachment loss during menopause are not associated with changes in species abundance or dysbiotic shifts in women. The mSMDI may be a useful tool for classifying subgingival ecology based on its normobiotic or dysbiotic inclination., (© 2024 American Academy of Periodontology.)

Accurate species-level identification of Enterobacter cloacae complex (ECC) is crucial for related research. The classification of ECC is based on strain-to-strain phylogenetic congruence, as well as genomic features including average nucleotide identity (ANI) and digitalized DNA-DNA hybridization (dDDH). ANI and dDDH derived from whole-genome sequencing have emerged as a reliable metric for assessing genetic relatedness between genomes and are increasingly recognized as a standard for species delimitation. Up to now, there are two different classification methods for ECC. The first one categorizes E. hormaechei , a species within ECC, into five subspecies ( E. hormaechei subsp. steigerwaltii, subsp. oharae, subsp. xiangfangensis, subsp. hoffmannii, and subsp. hormaechei ). The second classifies E. hormaechei as three species: E. hormaechei, "E. xiangfangensis," "E. hoffmanii." While the former is well-accepted in the academic area, the latter may have a greater ability to distinguish different species of ECC. To assess the suitability of these identification criteria for clinical ECC isolates, we conducted a comprehensive analysis involving phylogenetic analysis, ANI and dDDH value alignment, virulence gene identification, and capsule typing on 256 clinical ECC strains isolated from the bloodstream. Our findings indicated that the method of categorizing E. hormaechei into five subspecies has better correlation and consistency with the molecular characteristics of clinical ECC isolates, as evidenced by phylogenetic analysis, virulence genes, and capsule typing. Therefore, the subspecies-based classification method appears more suitable for taxonomic assignments of clinical ECC isolates., Importance: Standardizing taxonomy of the Enterobacter cloacae complex (ECC) is necessary for data integration across diverse studies. The study utilized whole-genome data to accurately identify 256 clinical ECC isolated from bloodstream infections using average nucleotide identity (ANI), digitalized DNA-DNA hybridization (dDDH), and phylogenetic analysis. Through comprehensive assessments including phylogenetic analysis, ANI and dDDH comparisons, virulence gene, and capsule typing of the 256 clinical isolates, it was concluded that the classification method based on subspecies exhibited better correlation and consistency with the molecular characteristics of clinical ECC isolates. In summary, this research contributes to the precise identification of clinical ECC at the species level and expands our understanding of ECC., Competing Interests: The authors declare no conflict of interest.

Bacterial leaf streak and black chaff diseases of wheat caused by Xanthomonas translucens pv. undulosa is becoming a major constraint to growers and trade since it is seedborne. Molecular tools for specific detection/differentiation of pv. undulosa are lacking. We report the development of a TaqMan real-time PCR for specific detection/identification of pv. undulosa targeting the recombination mediator gene ( rec F). Analysis of the complete rec F (1,117 bp) sequences identified the gene as a reliable phylogenetic marker for identification of pv. undulosa , differentiating it from the other pathovars; rec F-based sequence homology values among the 11 pathovars correlated well with genome-based DNA-DNA hybridization values. The discriminatory power of rec F to differentiate pv. undulosa from the other pathovars is due to nucleotide polymorphic positions. We used these nucleotide polymorphisms to develop a TaqMan PCR for specific detection of pv. undulosa . The specificity of the assay was validated using 67 bacterial and fungal/oomycete strains. The selected primers and the double-quenched FAM-labeled TaqMan probe were specific for the detection of 11 pv. undulosa / secalis strains. The 56 strains of other X. translucens pathovars ( n = 39) and non- Xanthomonas spp. ( n = 17) did not exhibit any detectable fluorescence. Also, greenhouse-inoculated and naturally infected wheat leaf samples showed positive reactions for the presence of pv. undulosa DNA but not healthy control plants. The TaqMan assay reliably detected as low as 1-pg DNA amount and 10 colony forming units of the target pathogen per reaction. This TaqMan assay could be useful to regulatory agencies with economic benefits to wheat growers., Competing Interests: The author(s) declare no conflict of interest.

A novel Gram-positive and endospore-forming bacterium assigned as strain SPB7T which is also a new source of a cyclic diketopiperazine (3S,6S)-3,6-diisobutylpiperazine-2,5-dione is described. A polyphasic (biochemical, phenotypic and genotypic) approach was used to clarify the taxonomic affiliation of this strain. The partial and complete 16S rRNA gene sequences revealed that strain SPB7T is a member of the Bacillus genus [showing high similarity (> 98.70%) with Bacillus spizizenii NRRL B-23049T, Bacillus tequilensis KCTC 13622T, Bacillus inaquosorum KCTC 13429T and Bacillus cabrialesii TE3T]. The maximum values for average nucleotide identity (ANI) and in silico DNA–DNA hybridization (GGDC, Formula 2) of strain SPB7T was obtained for twenty-five strains of Bacillus spizizenii (ANI 95.01–95.48% and GGDC 62.70–60.00%). The whole-genome phylogenetic relationship showed that SPB7T formed an individual and separated clade with the Bacillus spizizenii group. Principal cellular fatty acids identified in strain SPB7T were anteiso C15:0, anteiso C17:0, iso C15:0, iso C17:0, C16:0, C10:0 3OH and iso C17:1ϖ10c. Polar lipid profile showed presence of diphosphotidylglycerol, phosphatidylethanolamine, phosphatidylglycerol, two unknown phospholipids and five unknown lipids. Cells were rod shaped, catalase, oxidase-positive and motile. Growth occurred at 20–45 °C (optimal 35 °C), at pH 6.0–10.0 (optimal pH 8) and 0–10% (w/v) NaCl (optimal 2%). The phenotypic, biochemical, and genotypic traits of strain SPB7T strongly supported its taxonomic affiliation as a novel species of the Bacillus genus, for which the name Bacillus rugosus sp. nov. is proposed. The type strain is SPB7T (= NRRL B-65559T, = CICC 24827T, = MCC 4185T). [ABSTRACT FROM AUTHOR]

Species identification of Nocardia is difficult because of a complex and rapidly evolving taxonomy. In this study, gene sequencing (16S rRNA, gyrB, secA1, hsp65, rpoB), single 16S rRNA gene sequence phylogenetic analysis, and MALDI-TOF analysis were used to accurately identify 46 clinical Nocardia isolates to the species level. This provided a basis for establishing a routine method of multi-locus sequence analysis (MLSA) for molecular identification of Nocardia species. Genetic polymorphism analysis showed that MLSA was a powerfully discriminating method compared with the 16S rRNA gene to identify clinical Nocardia isolates. However, five-locus (gyrB-16S rRNA-secA1-hsp65-rpoB) MLSA led to misidentifications of all of the five Nocardia abscessus, which were confirmed by digital DNA-DNA hybridization (DDH) analysis. Interestingly, four strains identified as Nocardia beijingensis by a 16S rRNA gene phylogenetic tree may be novel species as suggested by DDH studies. For the purpose of achieving both accuracy and discrimination, the data of MLSA were reanalyzed. A three-locus MLSA with concatenated gyrB-16S rRNA-secA1 sequences was used to construct the phylogenetic tree with high accuracy and powerful discrimination. Therefore, a routine method of MLSA was developed to identify clinical Nocardia species. [ABSTRACT FROM AUTHOR]

Objectives: To assess the effects of an experimental 0.454% stannous fluoride (SnF2) dentifrice on the oral sulcular microbiota in patients with various stages of oral diseases using checkerboard DNA-DNA hybridization.Material and Methods: In the present one-month, single center, single product, prospective cohort trial, 37 adults (mean age 37.6) were assigned to one of four oral health condition cohorts with seven to 10 subjects each: 1. mild gingivitis, 2. marked generalized gingivitis to moderate periodontitis, 3. caries-prone and 4. treated moderate to advanced chronic periodontitis in supportive periodontal care. All four groups were asked to use the test dentifrice and a power toothbrush twice a day for one minute during a four-week test period. Before and after the trial period, Plaque Indices (PII, Silness and Löe, 1964) and Gingival Indices (GI, Löe and Silness, 1963) were recorded. Subgingival plaque samples were collected from all patients at Baseline, as well as after two and four weeks. These samples were analyzed for content of 40 bacterial species using checkerboard DNA-DNA hybridization.Results: As a result of the only one minute brushing with the stannous fluoride dentifrice, the mean PII at Baseline was significantly lower (p < 0.05) from the mean PII at four weeks. No statistically significant differences were found between premolar and molar mean values. Moreover, no statistically significant differences were found between the mean GI at Baseline and at four weeks. The microbiological analysis showed that at baseline subjects in groups 2 and 4 had significantly higher bacterial loads of bacteria than groups 1, and 3 (i.e. A. actinomyctemcomitans P. gingivalis, T. forsythia, and T. denticola. Over the study period, the total bacterial load did not change in groups 2, 3 and 4. In groups 1 and 3, however, an increase in the loads of Streptococci spp. were noticed (p < 0.05) including S. mitis, S. intermedius, and S. sanguis (p < 0.01) suggesting an increase in the presence of early colonizing and health associated bacteria.Conclusion: One minute brushing with a 0.454% stannous fluoride dentifrice did--after four weeks--not affect the subgingival microbial profiles in patients with moderate periodontitis and treated moderate to advanced periodontitis. However, the sulcular microbial profiles of mild gingivitis and caries-prone patients were affected, indicating a shift towards a gingival health associated microbiota in the sulcular region of patients not affected by attachment loss. RUNNING HEAD: Effect of stannous fluoride on sulcular microbiota. [ABSTRACT FROM AUTHOR]

Highly sensitive (high SBR) and highly specific (high SNP discrimination ratio) DNA hybridization is essential for a biosensor with clinical application. Herein, we propose a method that allows detecting multiple pathogens on a single platform with the SNP discrimination ratios over 160:1 in the dynamic range of 101 to 104 copies per test. The newly developed SWAT method allows achieving highly sensitive and highly specific DNA hybridizations. The detection and discrimination of the MTB and NTM strain in the clinical samples with the SBR and SNP discrimination ratios higher than 160:1 indicate the high clinical applicability of the SWAT.

A novel Gram-stain positive, aerobic, non-motile bacterial strain, designated Z1T, was isolated from a sample of petroleum-contaminated soil collected in Daqing, Heilongjiang province, China and characterised with a series of taxonomic approaches. The morphological and chemotaxonomic properties of the isolate were typical of those of members of the genus Rhodococcus. Phylogenetic analyses based on 16S rRNA gene sequences showed that strain Z1T belongs to the genus Rhodococcus and clustered with Rhodococcus maanshanensis DSM 44675T (99.2%, sequence similarity) and Rhodococcus tukisamuensis JCM 11308T (97.9%), respectively. However, the DNA–DNA hybridizations between strain Z1T and R. maanshanensis DSM 44675T and R. tukisamuensis JCM 11308T were both less than 70%. The optimal growth temperature and pH for strain Z1T were found to be at 28 °C and at pH 7.0. The peptidoglycan was found to contain meso-diaminopimelic acid; arabinose, galactose and glucose were detected as diagnostic sugars. The main polar lipids were identified as diphosphatidylglycerol, phosphatidylethanolamine, phosphatidylinositol, phosphatidylinositol mannoside and an unidentified lipid; MK-8(H2) was found as the major menaquinone. The major fatty acids were identified as C16:0, 10-methyl C18:0 and C18:1ω9c. Mycolic acids were found to be present. The G + C content of the genomic DNA was determined to be 66.7 mol%. Based on a comparative analysis of phenotypic and genotypic characteristics, in combination with DNA–DNA hybridization results, strain Z1T can be distinguished from the type strains of its two close neighbours as a novel species of the genus Rhodococcus, for which the name Rhodococcus daqingensis sp. nov. is proposed. The type strain is Z1T (= CGMCC 1.13630T = DSM 107227T). [ABSTRACT FROM AUTHOR]

An aerobic, Gram-negative, non-sporulating, motile, rod-shaped and lignin-degrading bacterial strain, Pseudomonas sp. CCA1, was isolated from leaf soil collected in Japan. This strain grew at 20-45 °C (optimum 20 °C), at pH 5.0-10.0 (optimum pH 5.0), and in the presence of 2% NaCl. Its major cellular fatty acids were C16:0 and summed feature 8 (C18:1ω6c and/or C18:1ω7c). The predominant quinone system was ubiquinone-9. Comparative 16S rRNA gene sequence analysis showed that Pseudomonas sp. CCA1 was related most closely to P. citronellolis NBRC 103043T (98.9%), but multilocus sequence analysis based on fragments of the atpD, gyrA, gyrB and rpoB gene sequences showed strain CCA1 to branch separately from its most closely related Pseudomonas type strains. DNA-DNA hybridization values between Pseudomonas sp. CCA1 and type strains of closely related Pseudomonas species were less than 53%. Based on its phenotypic, chemotaxonomic and phylogenetic features, we propose that Pseudomonas sp. CCA1 represents a novel species within the genus Pseudomonas, for which the name Pseudomonas humi sp. nov. is proposed. The type strain is CCA1 (= HUT 8136T = TBRC 8616T). [ABSTRACT FROM AUTHOR]

Taxonomic studies of strain PAGU 1678T, an obligately anaerobic, gram‐positive, spore‐forming bacterium isolated from biobreeding rat feces, were performed. This strain has been demonstrated to have the ability to exacerbate pathosis in a mouse model of dextran sulfate sodium‐induced ulcerative colitis. Phylogenetic analysis based on the 16S rRNA gene showed high homology with Paraclostridium bifermentans. To clarify the correct taxonomic position of strain PAGU 1678T, a comparative taxonomic study using P. bifermentans PAGU 2008T (═JCM 1386T) and the closely related bacterial species P. benzoelyticum PAGU 2068T (═LMG 28745T) was carried out. Despite the close similarity of 16S rRNA gene sequences, DNA‐DNA hybridization between strain PAGU 1678T and P. bifermentans PAGU 2008T was 60.03% on average, average nucleotide identity was 96.17%, and it was shown to have different genomic sequences. Biochemically, strain PAGU 1678T could be differentiated from P. bifermentans PAGU 2008T by H2S production. Furthermore, strain PAGU 1678T was characterized by the presence of two phospholipids with different polarity on polar lipid analysis. In addition, strain PAGU 1678T differed from P. bifermentans PAGU 2008T in findings on whole‐cell protein analysis and matrix‐assisted laser desorption/ionization‐time of flight mass spectrometry. On the basis of these biochemical and genetic characteristics, a novel subspecies of P. bifermentans with the name Paraclostridium bifermentans subsp. muricolitidis subsp. nov. is here proposed, with PAGU 1678T (═CCUG 72489T ═NBRC 113386T) as the type strain, which automatically creates P. bifermentans subsp. bifermentans subsp. nov. JCM 1386T (═ATCC 638T ═DSM 14991T). [ABSTRACT FROM AUTHOR]

A yellow pigmented, Gram-staining negative, motile and rod-shaped novel bacterial strain, designated MAH-14T was isolated from rhizospheric soil and was characterized using a polyphasic approach. The isolated strain was aerobic, oxidase and catalase were positive, optimum growth temperature and pH were 28-30 °C and 6.5, respectively. The novel strain is able to hydrolyze casein, starch, esculin, gelatin, L-tyrosine, DNA, tween 80, tween 20, L-arginine and 4-nitrophenyl-BD-galactopyranoside. On the basis of 16S rRNA gene sequence analysis, strain MAH-14T belongs to the genus Luteibacter and is most closely related to Luteibacter yeojuensis R2A16-10T (98.5%), Luteibacter anthropi CCUG 25036T (98.4%) and Luteibacter rhizovicinus LJ96T (98.3%). In DNA-DNA hybridization experiments, the DNA relatedness between strain MAH-14T and its closest phylogenetic neighbor was below 45.0%. The predominant respiratory quinone and the DNA G + C content of the novel strain were ubiquinone-8 and 63.5 mol%, respectively. The novel strain MAH-14T is able to produce flexirubin-type pigments. The major cellular fatty acids were C15:0 iso, summed feature 3 (C16:1 ω7c and/or C16:1 ω6c) and summed feature 9 (C17:1 iso ω9c and/or C16:0 10-methyl). The DNA-DNA hybridization results and results of the genotypic analysis in combination with chemotaxonomic and physiological data revealed that strain MAH-14T represented a novel species within the genus Luteibacter, for which the name Luteibacter pinisoli, is proposed. The type strain is MAH-14T (= KACC 19298T = CGMCC 1.16227T). [ABSTRACT FROM AUTHOR]

A Gram-stain negative, aerobic, short rod-shaped, motile by flagella bacterial strain (THG-N2.35T), was isolated from Pu’er tea. Growth occurred at 10-40 °C (optimum 28 °C), at pH 4-7 (optimum 7) and at 0-5% NaCl (optimum 1%). Based on 16S rRNA gene sequence analysis, the near phylogenetic neighbours of strain THG-N2.35T were identified as Paracoccus hibisci KACC 18632T (99.0%), Paracoccus tibetensis CGMCC 1.8925T (98.7%), Paracoccus beibuensis CGMCC 1.7295T (98.2%), Paracoccus aestuarii KCTC 22049T (98.2%), Paracoccus rhizosphaerae LMG 26205T (98.1%), Paracoccus zeaxanthinifaciens ATCC 21588T (97.1%), Paracoccus marcusii DSM 11574T (97.0%). Levels of similarity between strain THG-N2.35T and other Paracoccus species were lower than 97.0%. DNA-DNA hybridization values between strain THG-N2.35T and P. hibisci KACC 18632T, P. tibetensis CGMCC 1.8925T, P. beibuensis CGMCC 1.7295T, P. aestuarii KCTC 22049T, P. rhizosphaerae LMG 26205T, P. zeaxanthinifaciens ATCC 21588T, P.marcusii DSM 11574T were 47.5% (42.3%, reciprocal analysis), 36.1% (32.3%), 24.7% (22.1%), 19.2% (16.3%), 11.3% (8.8%), 11.1% (10.8%), 6.1% (5.8%), respectively. The DNA G+C content of strain THG-N2.35T was 62.3 mol%. The polar lipids were diphosphatidylglycerol, phosphatidyl-N-methylethanolamine, phosphatidylethanolamine, phosphatidylglycerol and phosphatidylcholine. The quinone was ubiquinone-10 (Q-10). The major fatty acids were C10:0 3OH, C16:0, C18:0 and C18:1ω7ϲ. On the basis of the phylogenetic analysis, chemotaxonomic data, physiological characteristics and DNA-DNA hybridization data, strain THG-N2.35T represent a novel species of the genus Paracoccus, for which the name Paracoccus pueri sp. nov. is proposed. The type strain is THG-N2.35T (= KACC 18934T = CCTCC AB 2016177T). [ABSTRACT FROM AUTHOR]

Solibacillus silvestris AM1 was the first strain from the genus to be reported for the production of a functional amyloid and its potential use as a surface active agent, a thermostable glycoprotein amyloid bioemulsifier BE‐AM1 capable of influencing environment and biofilm formation. Phylogenetic analysis based on 16S rRNA gene, molecular characterization studies on the basis of DNA‐DNA hybridization and chemotaxonomic fatty acid methyl ester (FAME) analysis showed that S. silvestris AM1 as a strain matches with the type strain S. silvestris HR3‐23. But strain AM1 differs from the type strain HR3‐23 in carbon substrate utilization studies along with amyloid bioemulsifier production ability with potential industrial and environmental applications. S. silvestris AM1 exhibited bioemulsifier production at wide range of factors like pH and NaCl concentrations, while temperature influenced the bioemulsifier production indirectly (since it affected the growth). Bioemulsifier production was observed even at oligotrophic conditions (0.5 mg ml−1) seen usually in its native environment. In this study, we have characterized the amyloid producing S. silvestris AM1 taxonomically and also analyzed 16S rDNA of 103 sequences of Solibacillus sp. available, which indicated the possibility of new species in this genus and can be studied for industrially and environmentally important biomolecules. [ABSTRACT FROM AUTHOR]

To gain a better understanding of how divergence occurs, and how taxonomy can benefit from studying natural populations, we isolated and examined 25 closely related Halorubrum strains obtained from different hypersaline communities and compared them to validly named species and other reference strains using five taxonomic study approaches: phylogenetic analysis using the 16S rRNA gene and multilocus sequencing analysis (MLSA), polar lipid profiles (PLP), average nucleotide identity (ANI) and DNA-DNA hybridization (DDH). 16S rRNA gene sequence could not differentiate the newly isolated strains from described species, while MLSA grouped strains into three major clusters. Two of those MLSA clusters distinguished candidates for new species. The third cluster with concatenated sequence identity equal to or greater than 97.5% was comprised of strains from Aran-Bidgol Lake (Iran) and solar salterns in Namibia and Spain, and two previously described species isolated from Mexico and Algeria. PLP and DDH analyses showed that Aran-Bidgol strains formed uniform populations, and that strains isolated from other geographic locations were heterogeneous and divergent, indicating that they may constitute different species. Therefore, applying only sequencing approaches and similarity cutoffs for circumscribing species may be too conservative, lumping concealed diversity into a single taxon. Further, our data support the interpretation that local populations experience unique evolutionary homogenization pressures, and once relieved of insular constraints (e.g., through migration) are free to diverge. [ABSTRACT FROM AUTHOR]

Since the pioneering work of van Leeuwenhoek in 1684, subsequently built upon by other renowned microbiologists Robert Koch, Willoughby Miller and GV Black, oral microbiology has developed innovative techniques to study the oral microflora (now termed the 'oral microbiome'). The advent of molecular techniques such as DNA-DNA hybridization, polymerase chain reaction and DNA sequencing has created an array of opportunities to construct a comprehensive picture of the diversity and composition of the oral microbiome. Approximately 700 oral bacterial species have been identified, of which 50% have yet to be cultivated, and some of these are known only by their signature DNA sequences. The synergism of ever-evolving culture-based and state-of-the-art culture-independent molecular techniques has facilitated in-depth understanding of the dynamics, acquisition and transfer of oral bacteria, along with their role in oral and general health and disease. Further research is needed to not only analyse but also to make sense of the ever-increasing volumes of data which these molecular techniques (especially high-throughput DNA sequencing) are generating, as well as why particular bacteria are present and what they are 'actually doing' there. This review presents a comprehensive literature search of oral microbiology-related methods currently used to study the oral microbiome. [ABSTRACT FROM AUTHOR]

Ehrlichia canis is an intracellular parasitic bacterium and arthropod-borne pathogen that receives growing attention, because it leads to increasing morbidity and mortality in animals. It does so by causing canine monocytotropic ehrlichiosis (CME). Infected canines may lack obvious clinical signs and stay in chronic stage. Herein we report a rapid screening method based on PCR assay combined with quartz crystal microbalance (QCM) to design a DNA sensor for detecting E. canis in early stages of infection. The test relies on DNA amplification of target nucleotide sequences via PCR followed by detecting DNA-DNA hybridization using QCM. The approach did not result in any cross-hybridization toward other blood bacteria or parasites in dogs, such as Anaplasma platys , Babesia canis and Trypanosoma spp, but turned out selective for the target species. The limit of detection of QCM was as low as 4.1 × 10 9 molecules/μl of 289 bp E. canis PCR products corresponding to 22 copy numbers/μl of E. canis . Furthermore, the technique is also simple, does not require complicated equipment and can in principle be reused. [ABSTRACT FROM AUTHOR]

Two closely related aerobic, Gram-negative rod shaped bacteria (strain W5 and W3) were isolated from Andaman Sea. Heterotrophic growth on marine agar was observed at 15-45 °C and pH 6-10. Strain W5 showed maximum 16S rRNA sequence similarity of 99.58% with Idiomarina marina JCM 15083. DNA fingerprinting analysis by ERIC-REP PCR, PFGE and MLSA revealed differences in banding patterns, also DNA-DNA hybridization values were well below 70% confirming W5 to be a new species. DNA G+C content was 46.7 mol%. Major fatty acids were iso-C15:0, iso-C17:0, iso-C17:1 ω9c, iso-C13:0 3OH, iso-C11:0 3OH and C16:0. Polar lipids included phosphatidylglycerol (PG) and phosphatidylethanolamine (PE) diphosphatidylglycerol (DPG) phospholipid (PL) two aminolipids (AL) and two unidentified lipids (L1-2). Q-8 is the predominant ubiquinone. On the basis of polyphasic taxonomic study, strain W5 is considered to be representative of a new species of the genus Idiomarina, for which the name Idiomarina andamanensis sp. nov. is being proposed. The type strain W5 (= LMG 29773 = JCM 31645) was isolated from Andaman Sea. [ABSTRACT FROM AUTHOR]

The genus Salinivibrio includes obligatory halophilic bacteria and is commonly isolated from hypersaline habitats and salted food products. They grow optimally between 7.5 and 10% salts and are facultative anaerobes. Currently, this genus comprises four species, one of them, S. costicola, with three subspecies. In this study we isolated and characterized an additional 70 strains from solar salterns located in different locations. Comparative 16S rRNA gene sequence analysis identified these strains as belonging to the genus Salinivibrio but could not differentiate strains into species-like groups. To achieve finer phylogenetic resolution, we carried out a MultiLocus Sequence Analysis (MLSA) of the new isolates and the type strains of the species of Salinivibrio based on the individual as well as concatenated sequences of four housekeeping genes: gyrB, recA, rpoA, and rpoD. The strains formed four clearly differentiated species-like clusters called phylogroups. All of the known type and subspecies strains were associated with one of these clusters except S. sharmensis. One phylogroup had no previously described species coupled to it. Further DNA–DNA hybridization (DDH) experiments with selected representative strains from these phylogroups permitted us to validate the MLSA study, correlating the species level defined by the DDH (70%) with a 97% cut-off for the concatenated MLSA gene sequences. Based on these criteria, the novel strains forming phylogroup 1 could constitute a new species while strains constructing the other three phylogroups are members of previously recognized Salinivibrio species. S. costicola subsp. vallismortis co-occurs with S. proteolyticus in phylogroup 4, and separately from other S. costicola strains, indicating its need for reclassification. On the other hand, genome fingerprinting analysis showed that the environmental strains do not form clonal populations and did not cluster according to their site of cultivation. In future studies regarding the classification and identification of new Salinivibrio strains we recommend the following strategy: (i) initial partial sequencing of the 16S rRNA gene for genus-level identification; (ii) sequencing and concatenation of the four before mentioned housekeeping genes for species-level discrimination; (iii) DDH experiments, only required when the concatenated MLSA similarity values among a new isolate and other Salinivibrio strains are above the 97% cut-off.

The assessment of bacterial diversity and bioprospection of the high-altitude lake Suraj Tal microorganisms for potent antimicrobial activities revealed the presence of two Gram-stain-variable, endospore-forming, rod-shaped, aerobic bacteria, namely IHBB 9852 T and IHBB 9951. Phylogenetic analysis based on 16S rRNA gene sequence showed the affiliation of strains IHBB 9852 T and IHBB 9951 within the genus Paenibacillus , exhibiting the highest sequence similarity to Paenibacillus lactis DSM 15596 T (97.8% and 97.7%) and less than 95.9% similarity to other species of the genus Paenibacillus . DNA-DNA relatedness among strains IHBB 9852 T and IHBB 9951 was 90.2%, and with P. lactis DSM 15596 T , was 52.7% and 52.4%, respectively. The novel strains contain anteiso-C 15:0 , iso-C 15:0 , C 16:0 and iso-C 16:0 as major fatty acids, and phosphatidylglycerol, phosphatidylethanolamine and diphosphatidylglycerol were predominant polar lipids. The DNA G+C content for IHBB 9852T and IHBB 9951 was 52.1 and 52.2 mol%. Based on the results of phenotypic and genomic characterisations, we concluded that strains IHBB 9852 T and IHBB 9951 belong to a novel Paenibacillus species, for which the name Paenibacillus ihbetae sp. nov. is proposed. The type strain is IHBB 9852 T (=MTCC 12459 T = MCC 2795 T = JCM 31131 T = KACC 19072 T ; DPD TaxonNumber TA00046) and IHBB 9951 (=MTCC 12458 = MCC 2794 = JCM 31132 = KACC 19073) is a reference strain. [ABSTRACT FROM AUTHOR]

The current prokaryotic taxonomy classifies phenotypically and genotypically diverse microorganisms using a polyphasic approach. With advances in the next-generation sequencing technologies and computational tools for analysis of genomes, the traditional polyphasic method is complemented with genomic data to delineate and classify bacterial genera and species as an alternative to cumbersome and error-prone laboratory tests. This review discusses the applications of sequence-based tools and techniques for bacterial classification and provides a scheme for more robust and reproducible bacterial classification based on genomic data. The present review highlights promising tools and techniques such as ortho-Average Nucleotide Identity, Genome to Genome Distance Calculator and Multi Locus Sequence Analysis, which can be validly employed for characterizing novel microorganisms and assessing phylogenetic relationships. In addition, the review discusses the possibility of employing metagenomic data to assess the phylogenetic associations of uncultured microorganisms. Through this article, we present a review of genomic approaches that can be included in the scheme of taxonomy of bacteria and archaea based on computational and in silico advances to boost the credibility of taxonomic classification in this genomic era. [ABSTRACT FROM AUTHOR]

The genus Salinivibrio includes obligatory halophilic bacteria and is commonly isolated from hypersaline habitats and salted food products. They grow optimally between 7.5 and 10% salts and are facultative anaerobes. Currently, this genus comprises four species, one of them, S. costicola, with three subspecies. In this study we isolated and characterized an additional 70 strains from solar salterns located in different locations. Comparative 16S rRNA gene sequence analysis identified these strains as belonging to the genus Salinivibrio but could not differentiate strains into species-like groups. To achieve finer phylogenetic resolution, we carried out a MultiLocus Sequence Analysis (MLSA) of the new isolates and the type strains of the species of Salinivibrio based on the individual as well as concatenated sequences of four housekeeping genes: gyrB, recA, rpoA, and rpoD. The strains formed four clearly differentiated species-like clusters called phylogroups. All of the known type and subspecies strains were associated with one of these clusters except S. sharmensis. One phylogroup had no previously described species coupled to it. Further DNA-DNA hybridization (DDH) experiments with selected representative strains from these phylogroups permitted us to validate the MLSA study, correlating the species level defined by the DDH (70%) with a 97% cut-off for the concatenated MLSA gene sequences. Based on these criteria, the novel strains forming phylogroup 1 could constitute a new species while strains constructing the other three phylogroups are members of previously recognized Salinivibrio species. S. costicola subsp. vallismortis co-occurs with S. proteolyticus in phylogroup 4, and separately from other S. costicola strains, indicating its need for reclassification. On the other hand, genome fingerprinting analysis showed that the environmental strains do not form clonal populations and did not cluster according to their site of cultivation. In future studies regarding the classification and identification of new Salinivibrio strains we recommend the following strategy: (i) initial partial sequencing of the 16S rRNA gene for genus-level identification; (ii) sequencing and concatenation of the four before mentioned housekeeping genes for species-level discrimination; (iii) DDH experiments, only required when the concatenated MLSA similarity values among a new isolate and other Salinivibrio strains are above the 97% cut-off. [ABSTRACT FROM AUTHOR]

Using 16S rRNA and rpoB gene sequencing, we identified 6 patients infected with Elizabethkingia bruuniana treated at E-Da Hospital (Kaohsiung, Taiwan) during 2005-2017. We describe patient characteristics and the molecular characteristics of the E. bruuniana isolates, including their MICs. Larger-scale studies are needed for more robust characterization of this pathogen. [ABSTRACT FROM AUTHOR]