BACKGROUND: Recently, genotypic identification of anaerobes is emerging as an alternative to the phenotypic method. In this study, we evaluated the performance of Vitek 2, API 20A and 16s rRNA gene sequencing for the identification of anaerobic bacteria. METHODS: A total of 35 anaerobe reference strains were identified using Vitek 2, API 20A and 16s rRNA gene sequencing. We evaluated the performance of three methods on the basis of the accurate identification rates. RESULTS: The Vitek 2, API 20A and 16s rRNA gene sequencing identified 54.3, 15.4, and 94.3% of test strains correctly at the species level and identified 77.1, 42.3, and 100% at the genus level, respectively. Results of the McNemar's test showed that there was a significant difference between each of the three identification methods in species level identification (P value<0.05). CONCLUSION: 16s rRNA gene sequencing showed better performance than Vitek 2 or API 20A for anaerobic bacteria. Considering its excellent performance, 16s rRNA gene sequencing may be useful for accurate identification of anaerobic bacteria that cannot be correctly identified by phenotypic methods.
Bacteria, Anaerobic* ; Genes, rRNA*

Aims: Pyrodinium bahamense var. compressum is one of the principal causal agents of harmful algal blooms (HABs) in the coastal waters of Sabah, Malaysia. Seafood and aquaculture products tainted with lethal concentrations of the principal neurotoxin, saxitoxin, have been implicated in mortality and morbidity. The bacteria-algae association may play a key role in paralytic shellfish toxin (PST) production during a toxic bloom event. The production of PST during a harmful bloom is unclear and research on the bacterial diversity associated with Sabah P. bahamense is scarce. The present study examined the cultivable bacteria diversity associated with P. bahamense through 16S ribosomal RNA (rRNA) gene sequence analysis. Methodology and results: The V3-V4 region of the 16S rRNA gene sequence was amplified and used to identify bacterial populations associated with P. bahamense var. compressum. A total of 62 isolates were successfully isolated, belonging to three different phyla, which were Proteobacteria; 55 (89%), Bacteroidetes; 6 (10%) and Actinobacteria; 1 (1%). Out of 55 Proteobacteria, 27 isolates were gamma-Proteobacteria (Marinobacter salsuginis) and 28 of the isolates were alpha-Proteobacteria; Mameliella atlantica (13), Roseibium denhamense (10) and Roseibium hamelinense (5). The remaining bacteria isolates from the phyla Bacteroidetes and Actinobacteria were identified as Muricauda lutimaris (6) and Micrococcus luteus (1), respectively. Conclusion, significance and impact of study The analysis of the bacterial 16S rRNA gene revealed multiple bacterial taxa associated with the toxic P. bahamense var. compressum bloom. The findings of the present work will pave the way for further studies aimed at isolating and characterizing genes involved in the saxitoxin biosynthesis in the associated bacteria.
Bacteria--metabolism ; Genes, rRNA

Species identification is an important item to characterize unidentified bacterial pathogens in developing and managing bacterial resources. In this study, unidentified pathogens based on the results of an automated identification system were identified using matrix assisted laser desorption ionization-time of flight mass spectrometry (MALD-TOF MS) and 16S rRNA gene analysis for development of national resources in the National Culture Collection for Pathogens (NCCP) in Korea. A total of 437 unidentified strains from branch banks of the NCCP were collected, and 16S rRNA and dnaJ gene sequencing, as well as MALDI-TOF MS analysis were performed to identify bacterial species. The mass spectra extracted were analyzed. Twelve strains exhibiting less than 98.65% similarity in 16S rRNA gene were selected as the primary candidates for novel species, and 21 strains exhibiting 98.65~99.0% similarity in 16S rRNA gene were selected as possible candidates for novel species. Among them, strain 32, belonging to Dermabacter sp., was finally selected as a possible strain representing a novel species and 14 unidentified bacterial strains using automated phenotypic identification system were newly registered at NCCP. The present study showed that unidentified pathogens using the automated phenotypic identification system were efficiently identified using the combination of MALDI-TOF MS and 16S rRNA gene analysis, and developed to the national resources in NCCP.
Genes, rRNA* ; Korea ; Mass Spectrometry

Microbacterium oleivorans is a gram-positive, coryneform rod bacterium. The pathogenic potential of the Microbacterium species has recently been reported to be increasing. Microbacterium comprises approximately 50 species. The differences in regards to the biochemical characteristics of Microbacterium species are unclear, and is why molecular investigations (e.g., using 16S rRNA gene sequencing) are the best method to identify the species. We report a case of bacteremia that was caused by Microbacterium oleivorans in a 4-year-old boy, who had no specific medical history. This represents the first report of M. oleivorans bacteremia in Korea.
Bacteremia ; Genes, rRNA ; Korea ; Preschool Child

The three components of taxonomy are classification, nomenclature and identification. Traditionally, bacterial classification and identification were performed based on the morphology and the biochemical data of the bacteria. In newer theories, or so-called natural concepts, the relationships between bacteria are based on the overall similarities of both the phenotypic and genotypic characteristics. The polyphasic taxonomy, or current taxonomy, describes the integration of all of the available genotypic, phenotypic, and phylogenetic information into a consensus type of general-purpose classification. When routine identification methods that are based on the biochemical tests fail, alternative procedures such as complete 16s rRNA gene sequence analysis are required. Although the results of 16s rRNA gene sequence analysis have not been fully discriminatory to differentiate closely related species, they may guide the additional analyses that are required for species identification.
Bacteria ; Consensus ; Genes, rRNA ; Sequence Analysis

Four Gram-positive cocci were isolated from the cerebrospinal fluid or blood of four different patients, but they could not be identified by an automated conventional identification system, so they were identified using cellular fatty acid (CFA) composition analysis and 16S rRNA gene sequencing analysis. Of these, two strains (SMC-A2662 and SMC-A5889), which were previously supposed to be Rothia dentocariosa according to the API Coryne system, were identified as Rothia aeria by the 16S rRNA gene analysis. SMC-A608, which was unidentified by both the VITEK2 and API Coryne systems, was identified as Rothia mucilaginosa. The one remaining SMC-2244T was distinguished from the other Rothia species by its biochemical profile, its CFA composition and its 16S rRNA gene sequence. Phylogenetic analysis showed that it was closely related to Rothia nasimurium but the 16S rRNA gene sequence dissimilarity of 1.8% was enough to differentiate it from R. nasimurium. Based on both the phenotypic and phylogenetic evidence, we propose a new species name for this bacterium, Rothia arfidiae sp. nov. The results of this study show that several Rothia species were isolated from human and we have identified them using 16S rRNA gene sequences.
Genes, rRNA ; Gram-Positive Cocci ; Humans

A mycoparasite, Scytalidium parasiticum sp. nov., isolated from the basidiomata of Ganoderma boninense causing basal stem rot of oil palm in Johor, Malaysia, is described and illustrated. It is distinct from other Scytalidium species in having smaller asci and ascospores (teleomorphic stage), longer arthroconidia (anamorphic stage), hyaline to yellowish chlamydospores, and producing a fluorescent pigment. The phylogenetic position of S. parasiticum was determined by sequence analyses of the internal transcribed spacers and the small-subunit ribosomal RNA gene regions. A key to identify Scytalidium species with teleomorphic stage is provided.
Ganoderma* ; Genes, rRNA ; Hyalin ; Malaysia* ; Sequence Analysis

For several days, there was a series of mortalities of chub mackerel (Scomber japonicus) that were reared for public exhibition in a private aquarium in Seoul, Korea. As part of the diagnosis of the dead fish, a bacterial isolate from the kidney was cultured, identified, and confirmed to be Vibrio (V.) harveyi using Vitek System 2 and 16S rRNA gene sequencing. Phylogenetic analysis was also performed by the neighbor-joining method. As a result, the V. harveyi isolated from chub mackerels of a private aquarium in Korea, called as SNUVh-LW1, was clustered in the same group with V. harveyi ATCC33843.
Cyprinidae* ; Diagnosis ; Genes, rRNA ; Kidney ; Korea ; Mortality ; Perciformes* ; Seoul ; Vibrio*

A sixty-seven-year-old man was admitted to a hospital with symptoms of high fever and chill. Bacterial isolates were obtained from sputum and blood. These isolates were identified as Klebsiella terrigena by API 20E (BioMerieux, Marcy-l'Etoile, France). K. terrigena is very rarely isolated from humans and no case of K. terrigena bacteremia has been reported yet. We analyzed partial 16S rRNA gene sequences of these isolates. The 16S rRNA gene sequences were matched with that of Klebsiella pneumoniae (ATCC 13886). 16S rRNA gene sequencing has been recently introduced in clinical laboratories for unidentified organisms by conventional biochemical tests. For the precise identification of bacteria rarely causing clinical infection, it might be considered to use genotypic methods, such as 16S rRNA gene sequencing.
Bacteremia ; Bacteria ; Fever ; Genes, rRNA ; Humans ; Klebsiella pneumoniae* ; Klebsiella* ; Sputum

BACKGROUND: Antimicrobial resistance is considered as the primary reason for eradication failure of Helicobacter pylori. Resistance to clarithromycin is mostly due to the point mutation in H. pylori 23S rRNA gene. The aims of this study were to determine the primary resistance rate to clarithromycin and metronidazole and to examine the mechanism of clarithromycin resistance in H. pylori isolates. METHODS: Seventy-nine strains were isolated from 73 patients within about five years. The susceptibility of H. pylori isolates to clarithromycin and metronidazole were tested by E-test and broth dilution test. To detect point mutations in the 23S rRNA gene, PCR-RFLP (restriction fragment length polymorphism) was performed. Mutations in clarithromycin-resistant strains also were analyzed by direct sequencing. RESULTS: The resistance rate to clarithromycin (>1 mg/L) and metronidazole (>8 mg/L) were 5.1% and 54.4%, respectively. Annual metronidazole-resistant rates were 43.7% (7/16) in 1996-1997, 61.1% (11/18) in 1998, 55.6% (5/9) in 1999, and 55.6% (20/36) in 2000. Annual clarithromycin- resistant rates were 6.3% (1/16) in 1996-1997, 0% (0/18) in 1998, 11.1% (1/9) in 1999, and 5.6% (2/36) in 2000. Two of 4 clarithromycin-resistant isolates contained the A2144G mutation. One isolate contained A2143G mutation. One isolate possibly contained T2183C mutation. Different strains, isolated separately from antrum and body in 6 patients, showed same susceptibility to clarithromycin. However, different strains in two patients showed different susceptibility to metronidazole. CONCLUSION: No significant increase of resistantce rate to both clarithromycin and metronidazole were found within recent five years. Resistance of H. pylori to clarithromycin was caused by A2144G and A2143G mutation mainly and by T2183C mutation possibly.
Clarithromycin* ; Genes, rRNA ; Helicobacter pylori* ; Helicobacter* ; Humans ; Metronidazole ; Point Mutation