Of the Acinetobacter spp., A. baumannii (genospecies 2) is the most clinically significant in terms of hospital-acquired infections worldwide. It is difficult to perform Acinetobacter-related taxonomy using phenotypic characteristics and routine laboratory methods owing to clusters of closely relate...
Of the Acinetobacter spp., A. baumannii (genospecies 2) is the most clinically significant in terms of hospital-acquired infections worldwide. It is difficult to perform Acinetobacter-related taxonomy using phenotypic characteristics and routine laboratory methods owing to clusters of closely related species. The ability to accurately identify Acinetobacter spp. is clinically important because antimicrobial susceptibility and clinical relevance differs significantly among the different genospecies. Based on the medical importance of pathogenic Acinetobacter spp., the distribution and characterization of Acinetobacter spp. isolates from 123 clinical samples was determined in the current study using four typically applied bacterial identification methods; partial rpoB gene sequencing, amplified rRNA gene restriction analysis (ARDRA) of the intergenic transcribed spacer (ITS) region of the 16~23S rRNA, the $VITEK^{(R)}$ 2 system (an automated microbial identification system) and matrix-assisted laser desorption/ionization-time of flight mass spectrometry (MALDI-TOF MS). A. baumannii isolates (74.8%, 92/123) were the most common species, A. nosocomialis (10.6%, 13/123) and A. pittii isolates (7.5%, 9/123) were second and third most common strains of the A. calcoaceticus-A. baumannii (ACB) complex, respectively. A. soli (5.0%, 6/123) was the most common species of the non-ACB complex. RpoB gene sequencing and ARDRA of the ITS region were demonstrated to lead to more accurate species identification than the other methods of analysis used in this study. These results suggest that the use of rpoB genotyping and ARDRA of the ITS region is useful for the species-level identification of Acinetobacter isolates.
Of the Acinetobacter spp., A. baumannii (genospecies 2) is the most clinically significant in terms of hospital-acquired infections worldwide. It is difficult to perform Acinetobacter-related taxonomy using phenotypic characteristics and routine laboratory methods owing to clusters of closely related species. The ability to accurately identify Acinetobacter spp. is clinically important because antimicrobial susceptibility and clinical relevance differs significantly among the different genospecies. Based on the medical importance of pathogenic Acinetobacter spp., the distribution and characterization of Acinetobacter spp. isolates from 123 clinical samples was determined in the current study using four typically applied bacterial identification methods; partial rpoB gene sequencing, amplified rRNA gene restriction analysis (ARDRA) of the intergenic transcribed spacer (ITS) region of the 16~23S rRNA, the $VITEK^{(R)}$ 2 system (an automated microbial identification system) and matrix-assisted laser desorption/ionization-time of flight mass spectrometry (MALDI-TOF MS). A. baumannii isolates (74.8%, 92/123) were the most common species, A. nosocomialis (10.6%, 13/123) and A. pittii isolates (7.5%, 9/123) were second and third most common strains of the A. calcoaceticus-A. baumannii (ACB) complex, respectively. A. soli (5.0%, 6/123) was the most common species of the non-ACB complex. RpoB gene sequencing and ARDRA of the ITS region were demonstrated to lead to more accurate species identification than the other methods of analysis used in this study. These results suggest that the use of rpoB genotyping and ARDRA of the ITS region is useful for the species-level identification of Acinetobacter isolates.
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제안 방법
, Seoul, Korea). After sequencing the DNA, the sequences were compared to available reference data in the Genbank database using Basic Local Alignment Search Tool (BLAST) to determine species identification.
Measurements were performed on VITEK® MS instrument equipped with both IVD 2.0 and RUO databases (bioMerieux, Marcy l'Etoile, France).
One-hundred and twenty three Acinetobacter clinical isolates were analyzed using ITS region genotyping. PCR amplification results for the ITS region are provided in Fig.
2 shows the PCR amplification results for the rpoB gene. One-hundred and twenty three Acinetobacter clinical isolates were analyzed using the rpoB gene genotyping (Supplementary data 1). Five Acinetobacter spp.
The rpoB gene amplification and sequencing was performed using a specific oligonucleotide primer set for genospecies identification of the clinical isolates (Table 1). PCR was performed using the following optimum conditions: initial denaturation at 94℃ for 5 min, followed by 35 cycles of 94℃ for 1 min (denaturation), 55℃ for 30 sec (annealing), and 72℃ for 90 sec (extension), and a final extension at 72℃ for 7 min.
성능/효과
isolates, collected from two general hospitals, in Busan, Korea, was evaluated in the current study. A. baumannii isolates (74.8%, 92/123) were the most common species, A. nosocomialis isolates (10.6%, 13/123) and A. pittii isolates (7.5%, 9/123) were the second and third common strains of ACB-complex, respectively (Table 2). A different Acinetobacter species distribution to that reported in other studies was determined in the current research.
The clinical isolates in the current study were identified using the VITEK®2 system in conjunction with the GN-ID card. All the isolates (n=123, 100.0%) were identified as A. baumannii (genospecies 2) (Table 2).
, 1995). ITS region of 16~23S rRNA gene was amplified for a total of 123 isolates belonging to the 5 species of the genus Acinetobacter and amplified product was restricted independently with a total of 5 different endonucleases (AluI, HhaI, HaeIII, MboI, and MSPI). Each enzyme generated up to 5 fragments per isolates.
In conclusion, our study demonstrated that the rpoB gene sequencing and ARDRA of ITS region of 16~23S rRNA seem to be very useful genotypic methods for the differentiation of Acinetobacter spp. at the species level.
One-hundred one isolates (Group A, 82.1%) were observed to be small in size (< 3 mm), smooth (the entire margin), round (circular form), and raised colonies on the MacConkey agar plates following incubation for 24 hrs.
1%) were observed to be small in size (< 3 mm), smooth (the entire margin), round (circular form), and raised colonies on the MacConkey agar plates following incubation for 24 hrs. Sixteen isolates (Group B, 13.0%) were seen to be of a medium size (3~5 mm), smooth (the entire margin), shiny, round (circular form), and characterized by slightly mucoid colonies. Six isolates (Group C, 4.
Three types of colonies were identified; (1) small size (< 3 mm), smooth, round shape and raised colonies (Group A), (2) medium size (3~5 mm), smooth, round shape and mucoid colonies (Group B), and (3) medium-large size (5~10 mm), rough, irregular shape and flat colonies (Group C).
As a result, 123 Acinetobacter spp. isolates were identified correctly: A. baumannii (genospecies 2), 92 isolates (74.8%); A. nosocomialis (genospecies 13TU), 13 isolates (10.6%); A. pittii (genospecies 3), 9 isolates (7.3%); A. soli, 6 isolates (4.9%) isolates; and A. calcoacetius (genospecies 1), 3 isolates (2.4%).
As a result of ITS region sequencing, the 5 Acinetobacter spp. were as follows: A. baumannii (genospecies 2), 92 isolates (74.8%); A. nosocomialis or A. calcoaceticus (genospecies 13TU or 1), 13 isolates (10.6%, 13 isolates were incorrectly identified as A. nosocomialis or A. calcoaceticus); A. calcoaceticus, A. pittii or A. baumannii (genospecies 1, 3 or 2), 12 isolates (9.7%, 12 isolates were incorrectly identified as A. calcoaceticus, A. pittii or A. baumannii); and A. soli or A. baumannii, 6 isolates (4.9%, isolates were incorrectly identified as A. soli or A. baumannii) isolates (Table 2). Thus, the findings using ITS region sequencing were relatively less accurate than those obtained using rpoB gene sequencing.
After analysis for ARDRA of ITS region, the 5 Acinetobacter spp. were identified correctly: A. baumannii (genospecies 2), 92 isolates (74.8%); A. nosocomialis (genospecies 13TU), 13 isolates (10.6%); A. pittii (genospecies 3), 9 isolates (7.3%); A. soli, 6 isolates (4.9%) isolates; and A. calcoacetius (genospecies 1), 3 isolates (2.4%).
Five Acinetobacter spp. were identified: A. baumannii (genospecies 2), 92 isolates (74.8%); A. nosocomialis (genospecies 13TU), 13 isolates (10.6%); A. calcoaceticus or A. pittii (genospecies 1 or 3), 12 isolates (9.7%, 12 isolates were incorrectly identified as A. calcoaceticus or A. pittii); and A. soli, 6 isolates (4.9%) isolates (Table 2).
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