Roseomonas Research Articles

Application of broad-spectrum PCR amplification and direct sequencing for identification of the infrequent bacterial cultures from clinical sources, targeting the bacterial 16S rRNA gene with universal primes

Objectives To identify the infrequent strains in clinical isolates by broad-spectrum PCR amplification and direct sequencing targeting the bacterial 16S rRNA gene. Methods Total 48 clinical isolates and 7 false-positive blood culture samples were collected from 7 different hospitals or institutions from December 2010 to September 2011.The bacterial 16S rRNA gene were amplified and sequenced by universal prime sets of 27f-1492r and 27f-1525r，and MicroSeq 500 16S rRNA gene kit.The homology analysis was used by the Basic Local Alignment Search Tool, and comparing to gene sequence of the type strain.provided by the List of Prokaryotic names with Standing in Nomenclature.The criteria for the bacterial identification was interpreted according to the Clinical and Laboratory Standards Institute （CLSI） MM18-A. Results All of the 48 cultured strains were succeeded amplifying and sequencing the targeted 16S rRNA genes.According to the criteria of CLSI MM18-A, total 35 strains were specified to the species level, 11 strains were specified to the genus level, and the other 2 strains were specified to possible novel genus and species.Combining the analysis the sequence of other housekeeping gene with the results of biochemical results, total 42 strains can be specified to the species level, including some clinical important pathogens, such as Streptobacillus, Capnocytophaga, Nocardia, Mycobacterium, Roseomonas and Campylobacter.Two false-positive blood culture samples were managed to amplify 16S rRNA genes and finally identified as Streptococcus pneumoniae.We also identified one novel subspecies of Campylobacter fetus, and some new valid-published species, such as Acinetobacter parvus,Mycobacterium phocaicum,Roseomonas mucosa and Halomonas johnsoniae. Conclusions The 16S rRNA gene sequence based identification has unique advantages over the phenotypic methods.It is universal to almost of all the bacteria, and can provide the genetic classified information. It is very suitable for the clinical infrequent and special bacterial cultures, such as the slow-growing, fastidious, or un-cultured bacteria.（Chin J Lab Med,2012,35:612-619） Key words: RNA，ribosomal，16S; Polymerase chain reaction; Sequence analysis; Bacteriological techniques

Identification and characterization of one Roseomonas strain

Objective To identify one runny mucoid-like Gram-negative bacteria with pink pigment isolated from clinical pus sample. Methods The pus sample was aseptically extracted from a deep lesions of one patient, then stored in Amies medium at room temperature for transportation. One sheep blood plate and one chocolate plate were used to detect the possible pathogens from the specimens. After inoculation, the plates were placed in a humidified incubator with 5% CO2 at 35 ℃. To identify the obtained isolates, we used the commercial Vitek2 and API systems, combining some traditional morphological examination and classical biochemical and physiological characteristics. For pure cultures, the cellular fatty acids were extracted, methylated, and determined by gas chromatography method. The 16S rRNA gene was amplified and sequenced by a commercial broad-spectrum PCR primers. The phylogenetic tree based on 16S rRNA gene was constructed by Mega 4.1 software using the neighbour-joining methods with 1 000 bootstrap replications. Results One runny mucoid-like Gram-negative bacterium, named K8756, was isolated both on sheep blood and chocolate plates after 72 h incubation. The API 20NE profile was 1245045 after a 3-day culture, which would be identified as Ochrobactrum anthropi with a good confidence of 98% probability. It was identified as Ralstonia pickettii and Bordetella bronchiseptica by VITEK 2 GN kits. However, further comparative 16S rRNA gene sequences showed that strain K8756 was closely related to the valid published Roseomonas mucosa MDA 5527 with 100% identity. Colonial morphologic features, phenotypic characteristics and major cellular fatty acid composition were also with high similarity to Roseomonas mucosa. Conclusions Strain K8756( = GIMCC 1.0030 ) is identified as Roseomonas mucosa by the polyphasic phenotypic and genotypic characteristics. The comparative analysis based on 16S rRNA gene sequences is a useful method for identifying the problematic and newly named bacteria. Key words: RNA,ribosomal,16S; Sequence analysis,RNA

Isolation and identification of Roseomonas strain from the ticks

objective To identify the three strains of pink-pigmented bacteria isolated from ticks.Methads 16S rDNA sequence analysis,16S rDNA similarity searching,bacterial morphological,physiological and biochemical tests and fatty acids analysis were used.Results The bacterial strains were pinkpigment,gram negative,strictly aerobic coccobacillus.They grow optimally at 37℃,while not in the presence of≥5% NaCl.The tests showed positive result for urease,D-glucose,citrate,UV absorption,and catalase.The predominant fatty acids were C16:0,C17:0,C18:1 ωgc and C19:0 cyc.Comparative 16S rDNA sequences analysis revealed that the strains were closely related to Roseomonas cervicalis and could reach up to 99.1% identity.Conclusion Based on phenotypic,biochemical and phylogenetic data,we propose these three isolates represent distinct species in genus Roseomonas,of which ticks may serve as a potential vector. Key words: Roseomonas; Tick; Isolation; Identification

Bacteremia due to Roseomonas spp.

Bacteremia due to Roseomonas spp.