A distinct Fusobacterium nucleatum clade dominates the colorectal cancer niche.

Fusobacterium nucleatum (Fn), a bacterium present in the human oral cavity and rarely found in the lower gastrointestinal tract of healthy individuals1, is enriched in human colorectal cancer (CRC) tumours2-5. High intratumoural Fn loads are associated with recurrence, metastases and poorer patient prognosis5-8. Here, to delineate Fn genetic factors facilitating tumour colonization, we generated closed genomes for 135 Fn strains; 80 oral strains from individuals without cancer and 55 unique cancer strains cultured from tumours from 51 patients with CRC. Pangenomic analyses identified 483 CRC-enriched genetic factors. Tumour-isolated strains predominantly belong to Fn subspecies animalis (Fna). However, genomic analyses reveal that Fna, considered a single subspecies, is instead composed of two distinct clades (Fna C1 and Fna C2). Of these, only Fna C2 dominates the CRC tumour niche. Inter-Fna analyses identified 195 Fna C2-associated genetic factors consistent with increased metabolic potential and colonization of the gastrointestinal tract. In support of this, Fna C2-treated mice had an increased number of intestinal adenomas and altered metabolites. Microbiome analysis of human tumour tissue from 116 patients with CRC demonstrated Fna C2 enrichment. Comparison of 62 paired specimens showed that only Fna C2 is tumour enriched compared to normal adjacent tissue. This was further supported by metagenomic analysis of stool samples from 627 patients with CRC and 619 healthy individuals. Collectively, our results identify the Fna clade bifurcation, show that specifically Fna C2 drives the reported Fn enrichment in human CRC and reveal the genetic underpinnings of pathoadaptation of Fna C2 to the CRC niche.

Characteristics of the Salivary Microbiota in Periodontal Diseases and Potential Roles of Individual Bacterial Species To Predict the Severity of Periodontal Disease.

The purposes of this study were to examine the compositional changes in the salivary microbiota according to the severity of periodontal disease and to verify whether the distribution of specific bacterial species in saliva can distinguish the severity of disease. Saliva samples were collected from 8 periodontally healthy controls, 16 patients with gingivitis, 19 patients with moderate periodontitis, and 29 patients with severe periodontitis. The V3 and V4 regions of the 16S rRNA gene in the samples were sequenced, and the levels of 9 bacterial species showing significant differences among the groups by sequencing analysis were identified using quantitative real-time PCR (qPCR). The predictive performance of each bacterial species in distinguishing the severity of disease was evaluated using a receiver operating characteristic curve. Twenty-nine species, including Porphyromonas gingivalis, increased as the severity of disease increased, whereas 6 species, including Rothia denticola, decreased. The relative abundances of P. gingivalis, Tannerella forsythia, Filifactor alocis, and Prevotella intermedia determined by qPCR were significantly different among the groups. The three bacterial species P. gingivalis, T. forsythia, and F. alocis were positively correlated with the sum of the full-mouth probing depth and were moderately accurate at distinguishing the severity of periodontal disease. In conclusion, the salivary microbiota showed gradual compositional changes according to the severity of periodontitis, and the levels of P. gingivalis, T. forsythia, and F. alocis in mouth rinse saliva had the ability to distinguish the severity of periodontal disease. IMPORTANCE Periodontal disease is one of the most widespread medical conditions and the leading cause of tooth loss, imposing high economic costs and an increasing burden worldwide as life expectancy increases. Changes in the subgingival bacterial community during the progression of periodontal disease can affect the entire oral ecosystem, and bacteria in saliva can reflect the degree of bacterial imbalance in the oral cavity. This study explored whether the specific bacterial species in saliva can distinguish the severity of periodontal disease by analyzing the salivary microbiota and suggested P. gingivalis, T. forsythia, and F. alocis as biomarkers for distinguishing the severity of periodontal disease in saliva.

Accuracy of ASTA MicroIDSys, a new matrix-assisted laser desorption/ionization time-of-flight mass spectrometry system, for the identification of Korean reference and clinical bacterial and yeast strains.

This study compared the accuracy of a new MALDI-TOF mass spectrometry system, ASTA MicroIDSys system, with that of MALDI Biotyper system for the identification of reference and clinical bacterial and yeast strains. The identification accuracy of the 2 systems was compared using a total of 406 strains comprising 142 aerobic and 180 anaerobic bacterial strains and 84 yeast strains. The genus and species identification rates were 98.0% and 89.4% using MicroIDSys and 96.1% and 89.4% using Biotyper, respectively. The species identification rates of MicroIDSys and Biotyper for aerobic bacteria were 93.0% and 97.2%, respectively, and those for anaerobic bacteria were 85.6% and 81.7%, respectively. The accuracy of yeast identification at the species level was 91.7% using MicroIDSys and 92.9% using Biotyper. These findings indicate that MicroIDSys could be useful for the accurate identification of bacteria and yeast in clinical microbiology laboratories.

Correction to: Genome-Based Reclassification of Fusobacterium nucleatum Subspecies at the Species Level.

The original version of this article contained errors in the description of novel species. These errors are corrected with this corrigendum.

Paenibacillus oralis sp. nov., Isolated from Human Subgingival Dental Plaque of Gingivitis Lesion.

A Gram-stain-negative, facultative anaerobic, spore-forming, motile, and rod-shaped bacterium, strain ChDC PVNT-B20T, was isolated from the human subgingival dental plaque of a gingivitis lesion. Phylogenetic analysis based on the 16S ribosomal RNA gene (16S rDNA) showed that the strain belonged to the genus Paenibacillus. BLAST analysis of 16S rDNA sequence of the strain displayed high identity to those of Paenibacillus faecis DSM 23593T (97.7% similarity) and Paenibacillus macerans ATCC 8244T (97.6% similarity). Draft genome of strain ChDC PVNT-B20T was composed of 8,112,407 bp. The DNA G+C content of the strain was 51.3 mol%. Average nucleotide identity values between strain ChDC PVNT-B20T and P. faecis DSM 23593T or P. macerans ATCC 8244T were 75.71% and 91.5%, respectively. Genome-to-genome distance values between strain ChDC PVNT-B20T and P. faecis DSM 23593T or P. macerans ATCC 8244T were 21.6% (19.3-24.0%) and 44.9% (42.3-47.4%), respectively. Major cellular fatty acids of strain ChDC PVNT-B20T were anteiso-C15:0 (43.4%), C16:0 (16.6%), iso-C16:0 (14.4%), and anteiso-C17:0 (12.4%). The sole respiratory quinone of the strain was menaqinone-7. Major polar lipids of the strain were phosphatidylglycerol (PG), diphosphatidylglycerol (DPG), and one unidentified glycolipid (GL). Minor polar lipids were one unidentified aminolipid (AL), one unidentified phospholipid (PL), and three unidentified lipids (L1-L3). Based on these results, strain ChDC PVNT-B20T is considered to represent a novel species of the genus Paenibacillus, for which the name Paenibacillus oralis sp. nov. is proposed. Type strain is ChDC PVNT-B20T (= KCOM 3021T = JCM 33462 T).

Streptococcus koreensis sp. nov., Isolated from Human Subgingival Dental Plaque of Periodontitis Lesion.

A novel facultative anaerobic, Gram-stain-positive coccus, strain JS71T, was isolated from the human subgingival dental plaque of a periodontitis lesion. Phylogenetic analysis based on the 16S ribosomal RNA gene (16S rDNA) revealed that the strain belonged to the genus Streptococcus. The 16S rDNA sequence had high similarity with Streptococcus rubneri DSM 26920T (98.6%), Streptococcus parasanguinis ATCC 15912T (98.5%), and Streptococcus australis CCUG 45919T (98.3%). The genome of strain JS71T was 2,009,592 bp in length. The DNA G+C content of the strain was 42.1 mol%. Average nucleotide identity values between strain JS71T and S. rubneri DSM 26920T, S. parasanguinis ATCC 15912T, and S. australis CCUG 45919T were 88.9%, 80.8%, and 92.4%, respectively. Genome-to-genome distance values between strain JS71TS. rubneri DSM 26920T, S. parasanguinis ATCC 15912T, and S. australis CCUG 45919T were 36.5% (34-39%), 26.3% (23.9-28.7%), and 48.0% (45.4-50.6%), respectively. The major fatty acids of the strain were C16:0 (39.7%), C18:1 ω6c/C18:1 ω7c (15.5%), and C18:0 (10.4%). Based on these results, strain JS71T (= KCOM 2890T = JCM 33454T) should be a novel species of the genus Streptococcus, for which the name Streptococcus koreensis sp. nov. is proposed.

Lautropia dentalis sp. nov., Isolated from Human Dental Plaque of a Gingivitis Lesion.

A novel Gram-stain-negative, motile, and facultative anaerobic coccus, strain ChDC F240T was isolated from human subgingival dental plaque of a gingivitis lesion. The phylogenetic analysis based on the 16S ribosomal RNA gene (16S rDNA) sequence showed that the strain belonged to the genus Lautropia. 16S rDNA of strain ChDC F240T had the highest similarity to that of Lautropia mirabilis ATCC 51599T (98.8%). Major cellular fatty acids of strain ChDC F240T were C16:0 (43.9%) and C16:1ω6C/C16:1ω7C (38.1%). Draft genome of the strain was 3,834,139 bp in length and the G+C content was 65.0 mol%. Average nucleotide identity and genome-to-genome distance values between strain ChDC F240T and L. mirabilis ATCC 51599 T were 81.99% and 28.50% (26.1-30.9%), respectively. These results reveal that strain ChDC F240T is a novel species within the genus Lautropia, for which the name Lautropia dentalis sp. nov. is proposed; type strain is ChDC F240T (= KCOM 2505T = JCM 33297T).

Streptococcus chosunense sp. nov., Isolated from Human Postoperative Maxillary Cyst.

A novel facultative anaerobic, non-spore forming, non-motile, and Gram-stain-positive coccus, designated strain ChDC B353T, was isolated from human postoperative maxillary cyst. The 16S ribosomal RNA gene (16S rDNA) sequence of the strain was most closely related to those of Streptococcus pseudopneumoniae ATCC BAA-960T (99.4%), Streptococcus mitis NCTC 12261T (99.3%), and Streptococcus pneumoniae NCTC 7465T (99.2%). The major fatty acids of the strain were C16:0 (43.2%) and C18:1 ω6c/C18:1 ω7c (20.2%). The genome of strain ChDC B353T was composed of 1,902,053 bps. The DNA G+C content of the strain was 40.2 mol%. Average nucleotide identity (ANI) values between strain ChDC B353T and S. pseudopneumoniae ATCC BAA-960T, S. mitis NCTC 12261T, and S. pneumoniae NCTC 7465T were 91.9%, 93.5%, and 91.3%, respectively. Genome-to-genome distance (GGD) values between strain ChDC B353T and S. pseudopneumoniae ATCC BAA-960T, S. mitis NCTC 12261T, or S. pneumoniae NCTC 7465T were 46.6% (44.0-49.2%), 53.2% (50.5-55.9%), and 46.0% (43.5-48.7%), respectively. The threshold values of ANI and GGD for species discrimination are 95-96% and 70%, respectively. These results reveal that strain ChDC B353T (= KCOM 1699T = JCM 33453T) is a novel species belonging to genus Streptococcus, for which a name of Streptococcus chosunense sp. nov. is proposed.

Lachnoanaerobaculum gingivalis sp. nov., Isolated from Human Subgingival Dental Plaque of a Gingivitis Lesion.

A novel Gram-stain-positive, obligately anaerobic, spore-forming rod, designated strain ChDC B114T, was isolated from a human dental plaque of a gingivitis lesion. The strain was characterized by polyphasic taxonomic analysis to identify it at the species level. The 16S ribosomal RNA gene (16S rDNA) sequence analysis revealed that the strain belongs to the genus Lachnoanaerobaculum. The percent similarity of the 16S rDNA of the strain was closest to the homologous gene sequence of Lachnoanaerobaculum orale N1T (98.5%) and Lachnoanaerobaculum saburreum CCUG 28089T (97.6%). The major fatty acids of strain ChDC B114T were C16:0 (30.7%), C14:0 (17.7%), iso-C19:0 (14.9%), and C17:0 2OH (12.0%). The draft genome of strain ChDC B114T was 3,097,953 bp in length. The G+C content of the strain was 35.9 mol %. Average nucleotide identity values between strain ChDC B114T and L. orale N1T and L. saburreum CCUG 28089T were 83.2% and 82.0%, respectively. Genome-to-genome distance values between strain ChDC B114T and L. orale N1T and L. saburreum CCUG 28089T were 26.8% (24.5-29.3%) and 26.30% (24.0-28.8%), respectively. Based on these results, strain ChDC B114T (= KCOM 2030T = JCM 33452T) should be classified as a novel species of genus Lachnoanaerobaculum, for which the name Lachnoanaerobaculum gingivalis sp. nov. is proposed.

Prevotella koreensis sp. nov., Isolated from Human Subgingival Dental Plaque of Periodontitis Lesion.

A novel Gram-negative, obligately anaerobic, non-motile, non-spore forming, and rod-shaped bacterium, designated strain JS262T, was isolated from human subgingival plaque of periodontitis lesion and was characterized by polyphasic taxonomic analysis. Comparison of 16S ribosomal RNA gene (16S rDNA) sequence revealed that the strain belonged to the genus Prevotella. The percent similarity of 16S rDNA of strain JS262T was closest to those of Prevotella buccae ATCC 33574T (89.1%) and Prevotella shahii JCM 12083T (88.9%). The major fatty acids of strain JS262T were C16:0 (29.2%), iso-C15:0 (19.2%), and anteiso-C15:0 (16.9%). Complete genome of strain JS262T was 2,691,540 bp in length and the G+C content was 43.9 mol%. Average nucleotide identity and genome-to-genome distance values between strain JS262T and P. buccae ATCC 33574T or P. loescheii DSM 19665T were > 70.4% and > 30.1%, respectively. On the basis of these data, a novel Prevotella species is proposed: Prevotella koreensis sp. nov. The type strain of P. koreensis is JS262T (= KCOM 3155T = JCM 33298T).

Streptococcus periodonticum sp. nov., Isolated from Human Subgingival Dental Plaque of Periodontitis Lesion.

A novel facultative anaerobic and Gram-stain-positive coccus, designated strain ChDC F135T, was isolated from human subgingival dental plaque of periodontitis lesion and was characterized by polyphasic taxonomic analysis. The 16S rRNA gene (16S rDNA) sequence of strain ChDC F135T was closest to that of Streptococcus sinensis HKU4T (98.2%), followed by Streptococcus intermedia SK54T (97.0%), Streptococcus constellatus NCTC11325T (96.0%), and Streptococcus anginosus NCTC 10713T (95.7%). In contrast, phylogenetic analysis based on the superoxide dismutase gene (sodA) and the RNA polymerase beta-subunit gene (rpoB) showed that the nucleotide sequence similarities of strain ChDC F135T were highly similar to the corresponding genes of S. anginosus NCTC 10713T (99.2% and 97.6%, respectively), S. constellatus NCTC11325T (87.8% and 91.4%, respectively), and S. intermedia SK54T (85.8% and 91.2%, respectively) rather than those of S. sinensis HKU4T (80.5% and 82.6%). The complete genome of strain ChDC F135T consisted of 1,901,251 bp and the G+C content was 38.9 mol %. Average nucleotide identity value between strain ChDC F135T and S. sinensis HKU4T or S. anginosus NCTC 10713T were 75.7% and 95.6%, respectively. The C14:0 composition of the cellular fatty acids of strain ChDC F135T (32.8%) was different from that of S. intermedia (6-8%), S. constellatus (6-13%), and S. anginosus (13-20%). Based on the results of phylogenetic and phenotypic analysis, strain ChDC F135T (= KCOM 2412T = JCM 33300T) was classified as a type strain of a novel species of the genus Streptococcus, for which we proposed the name Streptococcus periodonticum sp. nov.

Fusobacterium pseudoperiodonticum sp. nov., Isolated from the Human Oral Cavity.

In the present study, three strains (ChDC F213T, ChDC F251, and ChDC F267) were classified as novel species of genus Fusobacterium based on average nucleotide identity (ANI) and genome-to-genome distance (GGD) analysis and chemotaxonomic characterization. 16S rDNA sequences of strains ChDC F213T, ChDC F251, and ChDC F267 were highly similar to that of F. periodonticum ATCC 33693T (99.6, 99.4, and 99.4%, respectively). ANI and GGD values of the three isolates with F. periodonticum ATCC 33693T ranged from 92.5 to 92.6% and 47.7 to 48.2%, respectively. Considering that threshold of ANI and GGD values for bacterial species discrimination are 95-96% and 70%, respectively, these results indicate that the three isolates represent a novel Fusobacterium species. DNA G + C contents of the three isolates were 28.0 mol% each. Cellular fatty acid analysis of these strains revealed that C14:0, C16:0, and C16:1 ω6c/C16:1 ω7c were major fatty acids. Therefore, these three strains are novel species belonging to genus Fusobacterium. Strain ChDC F213T (= KCOM 1259T = KCTC 5677T = JCM 33009T) is the type strain of a novel species of genus Fusobacterium, for which a name of Fusobacterium pseudoperiodonticum sp. nov. is proposed.

Streptococcus gwangjuense sp. nov., Isolated from Human Pericoronitis.

A novel facultative anaerobic, Gram-stain-negative coccus, designated strain ChDC B345T, was isolated from human pericoronitis lesion and was characterized by polyphasic taxonomic analysis. The 16S ribosomal RNA gene (16S rDNA) sequence revealed that the strain belonged to the genus Streptococcus. The 16S rDNA sequence of strain ChDC B345T was most closely related to those of  Streptococcus mitis NCTC 12261T (99.5%) and Streptococcus pseudopneumoniae ATCC BAA-960T (99.5%). Complete genome of strain ChDC B345T was 1,972,471 bp in length and the G + C content was 40.2 mol%. Average nucleotide identity values between strain ChDC B345T and S. pseudopneumoniae ATCC BAA-960T or S. mitis NCTC 12261T were 92.17% and 93.63%, respectively. Genome-to-genome distance values between strain ChDC B345T and S. pseudopneumoniae ATCC BAA-960T or S. mitis NCTC 12261T were 47.8% (45.2-50.4%) and 53.0% (51.0-56.4%), respectively. Based on these results, strain ChDC B345T (= KCOM 1679T = JCM 33299T) should be classified as a novel species of genus Streptococcus, for which we propose the name Streptococcus gwangjuense sp. nov.

Antimicrobial Mechanism of Oleanolic and Ursolic Acids on Streptococcus mutans UA159.

Triterpenoid saponin derivatives oleanolic acid (OA) and ursolic acid (UA), but not betulinic acid (BA), were previously found to have strong antimicrobial activity against Streptococcus mutans. OA and UA inhibited the transcription of genes related to peptidoglycan biosynthesis, thereby preventing bacterial growth. However, it is not clear whether this is the only pathway involved in the antimicrobial activity of these compounds against S. mutans. Therefore, we used quantitative real-time PCR (qPCR) and microarray analyses to examine the expression of genes related to essential metabolic pathways in S. mutans UA159 following incubation with OA, UA, or BA. An oligonucleotide array consisting of 5363 probes was designed to survey 1928 of the 1963 genes in the genome of S. mutans UA159. Genes that showed >2-fold changes in expression in response to the treatment conditions were annotated, and selected target genes involved in central metabolism were analyzed by qPCR. Microarray analysis confirmed that the gene expression patterns of the OA- and UA-treated cells differed from that of the BA-treated culture, indicating differences in the antimicrobial mechanism. In particular, the expression of pfk and pykF, coding for glycolysis regulatory proteins phosphofructokinase and pyruvate kinase, respectively, were significantly decreased in the OA and UA groups (P < 0.05), as were genes involved in fatty acid and amino acid synthesis. In addition, the microarray analysis confirmed previous qPCR results showing that peptidoglycan synthesis is down-regulated in the OA- and UA-treated groups. OA and UA also appear to decrease the generation of organic acids by S. mutans UA159, which would have an anticaries effect. Overall, these findings suggest that OA and UA affect multiple genes involved in the central metabolism of S. mutans, with inhibition of glycolysis, fatty acid synthesis, amino acid synthesis, and peptidoglycan synthesis, all contributing to their antimicrobial activity.

Capnocytophaga endodontalis sp. nov., Isolated from a Human Refractory Periapical Abscess.

A novel Gram-negative, capnophilic, fusiform bacterium, designated strain ChDC OS43T, was isolated from a human refractory periapical abscess in the left mandibular second molar and was characterized by polyphasic taxonomic analysis. The 16S rRNA gene sequence revealed that the strain belongs to the genus Capnocytophaga, as it showed sequence similarities to Capnocytophaga ochracea ATCC 27872T (96.30%) and C. sputigena ATCC 33612T (96.16%). The prevalent fatty acids of strain ChDC OS43T were isoC15:0 (57.54%), C16:0 (5.93%), C16:0 3OH (5.72%), and C18:1 cis 9 (4.41%). The complete genome of strain ChDC OS43T was 3,412,686 bp, and the G+C content was 38.2 mol%. The average nucleotide identity (ANI) value between strain ChDC OS43T and C. ochracea ATCC 27872T or C. sputigena ATCC 33612T was >92.01%. The genome-to-genome distance (GGD) value between strain ChDC OS43T and C. ochracea ATCC 27872T or C. sputigena ATCC 33612T was 32.0 and 45.7%, respectively. Based on the results of phenotypic, chemotaxonomic, and phylogenetic analysis, strain ChDC OS43T (= KCOM 1579T = KCTC 5562T = KCCM 42841T = JCM 32133T) should be classified as the type strain of a novel species of genus Capnocytophaga, for which the name Capnocytophaga endodontalis sp. nov. is proposed.

Genome-Based Reclassification of Fusobacterium nucleatum Subspecies at the Species Level.

Fusobacterium nucleatum is classified as four subspecies, subsp. nucleatum, polymorphum, vincentii, and animalis, based on DNA-DNA hybridization (DDH) patterns, phenotypic characteristics, and/or multilocus sequence analysis (MLSA). The gold standards for classification of bacterial species are DDH and 16S ribosomal RNA gene (16S rDNA) sequence homology. The thresholds of DDH and 16S rDNA similarity for delineation of bacterial species have been suggested to be >70 and 98.65%, respectively. Average nucleotide identity (ANI) and genome-to-genome distance (GGD) analysis based on genome sequences were recently introduced as a replacement for DDH to delineate bacterial species with ANI (95-96%) and GGD (70%) threshold values. In a previous study, F. hwasookii was classified as a new species based on MLSA and DDH results. 16S rDNA similarity between F. hwasookii type strain and F. nucleatum subspecies type strains was higher than that between F. nucleatum subspecies type strains. Therefore, it is possible that the four F. nucleatum subspecies can be classified as Fusobacterium species. In this study, we performed ANI and GGD analyses using the genome sequences of 36 F. nucleatum, five F. hwasookii, and one Fusobacterium periodonticum strain to determine whether the four F. nucleatum subspecies could be classified as species using OrthoANI and ANI web-based softwares provided by ChunLab and Kostas lab, respectively, and GGD calculator offered by German Collection of Microorganisms and Cell Cultures. ANI values calculated from OrthoANI and ANI calculators between the type strains of F. nucleatum subspecies ranged from 89.80 to 92.97 and from 90.40 to 91.90%, respectively. GGD values between the type strains of F. nucleatum subspecies ranged from 42.3 to 46.0%. ANI and GGD values among strains belonging to the same F. nucleatum subspecies, subsp. nucleatum, subsp. polymorphum, subsp. vincentii, and subsp. animalis were >96 and >68.2%, respectively. These results strongly suggest that F. nucleatum subsp. nucleatum, subsp. polymorphum, subsp. vincentii, and subsp. animalis should be classified as F. nucleatum, F. polymorphum, F. vincentii, and F. animalis, respectively.

Oleanolic acid and ursolic acid inhibit peptidoglycan biosynthesis in Streptococcus mutans UA159.

In this study, we revealed that OA and UA significantly inhibited the expression of most genes related to peptidoglycan biosynthesis in S. mutans UA159. To the best of our knowledge, this is the first report to introduce the antimicrobial mechanism of OA and UA against S. mutans.

Oleanolic acid and ursolic acid inhibit peptidoglycan biosynthesis in Streptococcus mutans UA159

In this study, we revealed that OA and UA significantly inhibited the expression of most genes related to peptidoglycan biosynthesis in S. mutans UA159. To the best of our knowledge, this is the first report to introduce the antimicrobial mechanism of OA and UA against S. mutans.

Evaluation of four methods of assigning species and genus to medically important bacteria using 16S rRNA gene sequence analysis.

The four methods for assigning bacterial species are the Clinical and Laboratory Standards Institute (CLSI), modified CLSI (mCLSI), phylogenetic analysis (PA) and closest match (CM) methods, these are used to identify the genus and species using 16S rRNA gene sequence results. In this study, the results of identification by these four methods of 37 aerobic reference strains, 30 anaerobic reference strains, 15 Acinetobacter reference strains and 167 Acinetobacter clinical strains were compared. The rates of accurate identification to the species level using the CLSI, mCLSI, PA and CM methods were as follows: 24.3, 86.5, 86.5 and 89.2%, respectively, for the 37 aerobic reference strains; 73.3%, 96.7%, 90.0% and 93.3%, respectively, for the 30 anaerobic reference strains; 40.0%, 93.3%, 100% and 93.3%, respectively, for the 15 Acinetobacter reference strains; and 53.9%, 90.4%, 95.8% and 90.4%, respectively, for the 167 Acinetobacter clinical strains. The rates of accurate identification to the genus level using the CLSI, mCLSI, PA, and CM methods were as follows: 91.9%, 91.9%, 94.6% and 91.9%, respectively, for the 37 aerobic reference strains; 100%, 100%, 100% and 100%, respectively, for all of the 30 anaerobic reference strains, 15 Acinetobacter reference strains and the 167 Acinetobacter clinical strains. The mCLSI is the most practical and pragmatic method for identification of species based on 16S rRNA sequences for hospital, research or industry laboratories because it performs well and involves a simple procedure.