PCR-amplified 16S and 23S rDNA restriction analysis for the identification of Acinetobacter strains at the DNA group level

The genus Acinetobacter is phenotypically rather homogeneous, but genotypicaliy heterogeneous. In this study, a simple method based on restriction analysis of a PCR-amplrfied large fragment (4.5 kb) of most of the ribosomal operon (16S and 23S ribosomal genes and the spacer in-between) was investigated. Sixty-seven collection strains belonging to the 20 DNA groups proposed until 1993 were studied. Using the enzyme Sau3AI, 25 DNA profiles were obtained. Strains belonging to DNA groups 1, 3, 6, TU13 and TU15 showed two profiles each, and DNA groups 4, 5 and 7 showed profiles with variants showing less intensive additional bands. The remaining 12 groups showed 12 different profiles. The profiles obtained were DNA-group-specific except for one profile which was shared between the unnamed DNA group 3 and a rarely encountered genotypicaliy related DNA group. These two DNA groups could be separated by using the enzyme Hinf1. Twenty-five additional clinical isolates previously characterized by standard DNA-DNA hybridization were selected in a double-blind fashion for identification at the DNA group level to check the reliability of the assay. All strains were correctly identified at the DNA group level. PCR-amplified 16S and 23S rDNA restriction analysis is both an accurate and rapid method for the identification of Acinetobacter at the DNA group level.     

Genotypic characterization of thermophilic bacilli: A study on new soil isolates and several reference strains

A genotypic study using amplified ribosomal DNA restriction analysis (ARDRA), random amplified polymorphic DNA fingerprinting (RAPD) and ribosomal spacer analysis (RSA) in comparison with DNA-DNA reassociation experiments was carried out with 85 thermophilic Bacillus isolates from uncultivated soil of 14 different geographical areas and seventeen reference strains representing defined thermophilic Bacillus species. This approach permitted the attribution of 51 % of the new isolates to the Bacillus thermoleovorans group and the identification of 40% of the new isolates as B. “thermodenitrificans”. Moreover, 2 strains were assigned to B. pallidus species and 1 isolate to B. thermosphaericus species. The remaining 6% of our thermophilic isolates from soil, constituting 2 DNA-DNA homology groups, are still unidentified. A detailed genotypic characterization of the heterogeneous species of B. thermoleovorans and B. stearothermophilus was also presented.     

Molecular characterization of carbapenem-non-susceptible Acinetobacter spp. in Japan: predominance of multidrug-resistant Acinetobacter baumannii clonal complex 92 and IMP-type metallo-β-lactamase-producing non-baumannii Acinetobacter species

We conducted an epidemiological study concerning carbapenem-non-susceptible clinical isolates of Acinetobacter spp. in Japan by molecular procedures including carbapenemase gene identification and amplified ribosomal DNA restriction analysis. Among 598 clinically isolated Acinetobacter spp. in 2007, 27 (4.5%) were non-susceptible to carbapenems. Most carbapenem-non-susceptible Acinetobacter baumannii (13/14) belonged to clonal complex (CC) 92, harbored bla (OXA-51-like) genes, including novel bla (OXA-206), downstream of ISAba1, and were recovered mainly from the Kanto region. Carbapenem-non-susceptible A. baumannii CC92 isolates were further divided by pulsed-field gel electrophoresis into two groups, one of which was characterized by the presence of bla (OXA-23). One A. baumannii CC276 isolate carried bla (IMP-1) and bla (OXA-58). Almost all non-baumannii Acinetobacter isolates (12/13), including Acinetobacter pittii (formerly Acinetobacter genomic species 3) and Acinetobacter nosocomialis (formerly Acinetobacter genomic species 13TU), produced IMP-type metallo-β-lactamases, and were recovered from various regions in Japan. This is the first report describing the nationwide molecular epidemiology of carbapenem-non-susceptible Acinetobacter spp. with genomic species-level identification in Japan.     

Diversity and phylogeny of plant growth-promoting bacilli from moderately acidic soil

The molecular diversity of aerobic endospore-forming bacteria, typically Bacillus and its derived genera, has been investigated in various environments. However, there have been few investigations concerning Bacillus in acidic soils. In this study, the genotypic diversity and phylogenetic relationships among plant growth-promoting (PGP) bacilli isolated from the rice rhizosphere growing in acidic soils of Kerala (pH varying from 6.3 to 6.8) were investigated. For assessing their biocontrol potential and PGP attributes, 115 isolates were randomly selected and 49 isolates that were positive for multiple traits were selected. Metabolic characterization of representative strains, using the Biolog GP2 (Gram Positive) MicroPlate(TM) , revealed a large versatility with respect to carbohydrate utilization. Amplified ribosomal DNA restriction analysis revealed 13 clusters at 65% similarity level, which consisted of 1-21 strains. 16S rDNA partial sequencing assigned all the isolates, except for one, to the Bacillus genus, with close relatedness to Bacillus humi, B. megaterium, B. drentensis, B. pocheonensis, B. aestuarii, B. arbutinivorans, B. niacini, and Brevibacterium casei. The Bacillus species with different metabolic capabilities, PGP abilities, and genetic diversity found in this study are likely to have ecological relevance.     

人参栽培土壤中细菌种群ARDRA研究体系的建立

目的:建立适用于开展人参栽培土壤微生物种群结构分析的ARDRA研究体系.方法:从基因组水平研究人参栽培土壤微生物种群多样性结构.结果:对人参栽培土壤样品分析发现,ARDRA体系不仅能高效区分土壤中不同的微生物类群,而且还可根据酶切谱带的差异统计出不同微生物类群在土壤总微生物中所占的比重.结论:建立的ARDRA研究体系能够用于人参栽培土壤微生物种群结构分析.     

阴道加德纳菌生物学分型、基因分型与细菌性阴道病相关性研究

目的探讨阴道加德纳菌(GV)的不同生物学型别和基因型别与细菌性阴道病(BV)的相关性。方法采用Biswas标准诊断BV;根据Briselden方法对GV进行生物学分型;运用ARDRA方法(扩增rDNA限制性分析)对GV进行基因分型。结果BV现患率为24.1%;BV患者的GV检出率(48.4%)高于非BV患者(13.1%),两者比较差异有显著性(P<0.001);共检测出7种型别,1,2,3,4型在BV患者的分布显著高于非BV患者(P<0.001),5型在非BV患者分布高于BV患者(P<0.05),未检出6型;将50株GV分为A,B,C三个基因型,没有发现基因型与BV有相关性。结论GV的不同生物型别与BV发病相关,未发现与BV发病相关的基因型别。     

ARDRA技术快速鉴别四种致病曲霉的实验研究

目的:建立检测侵袭性曲霉感染病原菌ARDRA方法。方法:用烟曲霉、黄曲霉、土曲霉和黑曲霉的标准株建立PCR技术和RFLP技术相结合的扩增rDNA限制性分析(ARDRA)方法,然后对16株临床株进行ARDRA分析。结果:ARDRA技术可以较好地鉴别引起侵袭性曲霉感染的四种常见菌种,从DNA提取到酶切分析结束可以在一个工作日中完成;16株临床株酶切图谱与对应标准株图谱相同。结论:ARDRA技术是一种能够快速鉴别侵袭性曲霉感染病原菌的有效方法。     

Archaeal diversity in acid mine drainage from Dabaoshan Mine, China

Three acid mine drainage (AMD) samples collected from Dabaoshan Mine (Guangdong Province, China) were studied. In addition to physicochemical analyses, the diversity and community structures of the archaeal communities in these samples were described at the genetic level by amplified ribosomal DNA restriction analysis (ARDRA). Nine different ARDRA patterns were obtained from 146 clones and were studied as operational taxonomic units (OTUs), which were re-amplified and sequenced. Sequence data and phylogenetic analysis showed that most of the clones belonged to the Thermoplasmatales, and that archaea belonging to the Sulfolobales were absent. Only 1 OTU attributed to Ferroplasma was found and was observed to be abundant in all 3 samples. Eight OTUs were related to 2 new undefined groups in the Thermoplasmatales. Of the 8 OTUs, the clones in 2 similar units were isolated from samples collected from an abandoned sulfide mine (Huelva, Spain) and those in 5 similar units were isolated from samples collected from a closed copper mine (Tonglushan, China). These diversities were characterized by the reciprocal of Simpson's index (1/D) and correlated with the concentrations of ferrous ions and toxic ions in the AMD samples. The high temperature of the sampling sites was one of the factors that could explain why archaea belonging to the Thermoplasmatales were abundant in the analyzed AMD samples while those belonging to the Sulfolobales were absent.