scholarly journals Species-Specific PCR as a Tool for the Identification of Burkholderia gladioli

2000 ◽  
Vol 38 (1) ◽  
pp. 282-285
Author(s):  
Paul W. Whitby ◽  
Lauren C. Pope ◽  
Karen B. Carter ◽  
John J. LiPuma ◽  
Terrence L. Stull
Keyword(s):  
Cystic Fibrosis ◽  
Sensitivity And Specificity ◽  
Burkholderia Cepacia ◽  
Bacterial Species ◽  
23S Rrna ◽  
Computer Assisted ◽  
Accurate Identification ◽  
Specific Pcr ◽  
Burkholderia Gladioli ◽  
Species Specific

ABSTRACT Burkholderia gladioli colonizes the respiratory tracts of patients with cystic fibrosis and chronic granulomatous disease. However, due to the high degree of phenotypic similarity between this species and closely related species in the Burkholderia cepacia complex, accurate identification is difficult. Incorrect identification of these species may have serious repercussions for the management of patients with cystic fibrosis. To develop an accurate procedure for the identification of B. gladioli , a molecular method to discriminate between this species and other species commonly isolated from the sputa of patients with cystic fibrosis was investigated. The 23S ribosomal DNA was cloned from several clinical isolates of B. gladioli , and the nucleotide sequence was determined. Computer-assisted sequence comparisons indicated four regions of the 23S rRNA specific for this species; these regions were used to design three primer pairs for species-specific PCR. Two of the primer pairs showed 100% sensitivity and specificity for B. gladioli when tested against a panel of 47 isolates comprising 19 B. gladioli isolates and 28 isolates of 16 other bacterial species. One of the primer pairs was further assessed for species specificity by using a panel of 102 isolates obtained from the Burkholderia cepacia Research Laboratory and Repository. The species-specific PCR was positive for 70 of 74 isolates of B. gladioli and was negative for all other bacterial species examined. Overall, this primer pair displayed a sensitivity and specificity of 96% (89 of 93) and 100%, respectively. These data demonstrate the potential of species-specific PCR for the identification of B. gladioli .

scholarly journals Identification and Detection ofStenotrophomonas maltophilia by rRNA-Directed PCR

2000 ◽  
Vol 38 (12) ◽  
pp. 4305-4309 ◽  
Author(s):  
Paul W. Whitby ◽  
Karen B. Carter ◽  
Jane L. Burns ◽  
James A. Royall ◽  
John J. LiPuma ◽  
...  
Keyword(s):  
Burkholderia Cepacia ◽  
Direct Detection ◽  
Pcr Primers ◽  
Laboratory Culture ◽  
23S Rrna ◽  
Rrna Gene ◽  
Specific Pcr ◽  
23S Rrna Gene ◽  
Susceptible Populations ◽  
Species Specific

Stenotrophomonas maltophilia has recently emerged as an important nosocomial pathogen in immunocompromised patients, in transplant recipients, and in persons with cystic fibrosis (CF). While this organism is nonpathogenic in healthy individuals, it is increasingly associated with morbidity and mortality in susceptible populations. Recent studies have indicated that for approximately 10% of CF patients with moderate lung disease, S. maltophiliacan be cultured from respiratory tract secretions. Identification ofS. maltophilia can be problematic, and analysis of isolates from the Burkholderia cepacia Research Laboratory and Repository showed that several isolates presumptively identified asB. cepacia by clinical microbiology laboratories were in fact S. maltophilia. To overcome the problems associated with definitive identification, we developed species-specific PCR (SS-PCR) primers, designated SM1 and SM4, directed to the 23S rRNA gene, and tested their utility to accurately identify S. maltophilia directly from sputum. The SS-PCR was developed and tested against a panel of 112 S. maltophilia isolates collected from diverse geographic locations. To test for specificity, 43 isolates from 17 different species were analyzed. PCR with the SM1-SM4 primer pair and isolated genomic DNA as a template resulted in amplification of a band from all S. maltophilia isolates and was uniformly negative for all other species tested, yielding a sensitivity and a specificity of 100% for the SS-PCR. The utility of the SS-PCR to directly identify S. maltophilia in sputum was examined. Thirteen expectorated sputum samples from CF patients were analyzed by SS-PCR. Three samples were PCR positive, in complete concordance with the conventional laboratory culture. Thus, we have developed an SS-PCR protocol that can rapidly and accurately identifyS. maltophilia isolates and which can be used for the direct detection of this organism in CF patient sputum.

scholarly journals Species-Specific PCR for Identification of Common Contaminant Mollicutes in Cell Culture

2001 ◽  
Vol 67 (7) ◽  
pp. 3195-3200 ◽  
Author(s):  
Fanrong Kong ◽  
Gregory James ◽  
Susanna Gordon ◽  
Anna Zelynski ◽  
Gwendolyn L. Gilbert
Keyword(s):  
16S Rrna ◽  
Cell Cultures ◽  
Bacterial Species ◽  
Intergenic Spacer ◽  
16S Rrna Genes ◽  
Rrna Genes ◽  
23S Rrna ◽  
Rrna Gene ◽  
Specific Pcr ◽  
Species Specific

ABSTRACT Mycoplasma arginini, M. fermentans, M. hyorhinis, M. orale, and Acholeplasma laidlawii are the members of the class Mollicutes most commonly found in contaminated cell cultures. Previous studies have shown that the published PCR primer pairs designed to detect mollicutes in cell cultures are not entirely specific. The 16S rRNA gene, the 16S-23S rRNA intergenic spacer region, and the 5′ end of the 23S rRNA gene, as a whole, are promising targets for design of mollicute species-specific primer pairs. We analyzed the 16S rRNA genes, the 16S-23S rRNA intergenic spacer regions, and the 5′ end of the 23S rRNA genes of these mollicutes and developed PCR methods for species identification based on these regions. Using high melting temperatures, we developed a rapid-cycle PCR for detection and identification of contaminant mollicutes. Previously published, putative mollicute-specific primers amplified DNA from 73 contaminated cell lines, but the presence of mollicutes was confirmed by species-specific PCR in only 60. Sequences of the remaining 13 amplicons were identified as those of gram-positive bacterial species. Species-specific PCR primers are needed to confirm the presence of mollicutes in specimens and for identification, if required.

scholarly journals Use of selective medium for Burkholderia cepacia isolation in respiratory samples from cystic fibrosis patients

2002 ◽  
Vol 44 (4) ◽  
pp. 203-208 ◽  
Author(s):  
Luiz V.F. da SILVA FILHO ◽  
Luciana de F. VELLOSO ◽  
Christina N.O. BENTO ◽  
Edelyn GYTIN ◽  
Adriana F. TATENO ◽  
...  
Keyword(s):  
Cystic Fibrosis ◽  
Recovery Rate ◽  
Burkholderia Cepacia ◽  
Selective Medium ◽  
Sample Processing ◽  
Selective Media ◽  
Specific Pcr ◽  
Species Specific ◽  
The Impact

Burkholderia cepacia colonizes cystic fibrosis (CF) patients. We evaluated the impact of the use of a selective medium in the rate of B. cepacia recovery from respiratory samples of CF patients. During a 6-month period, respiratory samples were collected from 106 CF patients and cultivated on selective media including a B. cepacia selective medium. Confirmation of the identity of B. cepacia isolates was carried out by species specific PCR and determination of genomovar status performed by a sequential PCR approach. Results of B. cepacia isolation during this period were compared to the preceding two years, when the sample processing was identical except for the lack of the B. cepacia selective medium. B. cepacia was isolated in 11/257 (4.2%) of the samples using the selective medium, in contrast with the preceding two years, when it was isolated in 6/1029 samples (0.58%), p < 0.0001. Identity of all 11 isolates was confirmed by PCR and genomovar determination was accomplished in all but one isolate. These results suggest that the use of a selective medium increases recovery rate of B. cepacia from respiratory samples.

scholarly journals Lysogeny and bacteriophage host range within the Burkholderia cepacia complex

2003 ◽  
Vol 52 (6) ◽  
pp. 483-490 ◽  
Author(s):  
Ross Langley ◽  
Dervla T. Kenna ◽  
Peter Vandamme ◽  
Rebecca Ure ◽  
John R. W. Govan
Keyword(s):  
Host Range ◽  
Burkholderia Cepacia ◽  
Potential Role ◽  
River Sediments ◽  
Burkholderia Cepacia Complex ◽  
Potential Contribution ◽  
Closely Related Species ◽  
Burkholderia Gladioli ◽  
Life Threatening ◽  
Species Specific

The Burkholderia cepacia complex comprises a group of nine closely related species that have emerged as life-threatening pulmonary pathogens in immunocompromised patients, particularly individuals with cystic fibrosis or chronic granulomatous disease. Attempts to explain the genomic plasticity, adaptability and virulence of the complex have paid little attention to bacteriophages, particularly the potential contribution of lysogenic conversion and transduction. In this study, lysogeny was observed in 10 of 20 representative strains of the B. cepacia complex. Three temperate phages and five lytic phages isolated from soils, river sediments or the plant rhizosphere were chosen for further study. Six phages exhibited T-even morphology and two were lambda-like. The host range of individual phages, when tested against 66 strains of the B. cepacia complex and a representative panel of other pseudomonads, was not species-specific within the B. cepacia complex and, in some phages, included Burkholderia gladioli and Pseudomonas aeruginosa. These new data indicate a potential role for phages of the B. cepacia complex in the evolution of these soil bacteria as pathogens of plants, humans and animals, and as novel therapeutic agents.

scholarly journals Assessment of Clarithromycin Susceptibility in Strains Belonging to theMycobacterium abscessusGroup byerm(41) andrrlSequencing

2010 ◽  
Vol 55 (2) ◽  
pp. 775-781 ◽  
Author(s):  
Sylvaine Bastian ◽  
Nicolas Veziris ◽  
Anne-Laure Roux ◽  
Florence Brossier ◽  
Jean-Louis Gaillard ◽  
...  
Keyword(s):  
Cystic Fibrosis ◽  
Previous Treatment ◽  
Sensu Stricto ◽  
Mycobacterium Abscessus ◽  
23S Rrna ◽  
Rrna Gene ◽  
Drug Of Choice ◽  
Clarithromycin Resistance ◽  
Mycobacterium Bolletii ◽  
Species Specific

ABSTRACTClarithromycin was the drug of choice forMycobacterium abscessusinfections until inducible resistance due toerm(41) was described. BecauseM. abscessuswas split intoM. abscessussensu stricto,Mycobacterium massiliense, andMycobacterium bolletii, we looked forerm(41) in the three species and determined their clarithromycin susceptibility levels. Ninety strains were included: 87 clinical strains from cystic fibrosis patients (61%) and others (39%), representing 43M. abscessus, 30M. massiliense, and 14M. bolletiistrains identified on a molecular basis, and 3 reference strains. Clarithromycin and azithromycin MICs were determined by broth microdilution and Etest with a 14-day incubation period. Mutations inrrl(23S rRNA gene) known to confer acquired clarithromycin resistance were also sought.erm(41) was detected in all strains but with two deletions in allM. massiliensestrains. These strains were indeed susceptible to clarithromycin (MIC90of 1 μg/ml) except for four strains withrrlmutations.M. abscessusstrains harbored an intacterm(41) but had a T/C polymorphism at the 28th nucleotide: T28 strains (Trp10 codon) demonstrated inducible clarithromycin resistance (MIC90of >16 μg/ml), while C28 strains (Arg10) were susceptible (MIC90of 2 μg/ml) except for two strains withrrlmutations.M. bolletiistrains haderm(41) sequences similar to the sequence of the T28M. abscessusgroup, associated with inducible clarithromycin resistance (MIC90of >16 μg/ml).erm(41) sequences appeared species specific within theM. abscessusgroup and were fully concordant with clarithromycin susceptibility whenerm(41) sequencing was associated with detection ofrrlmutations. Clarithromycin-resistant strains, including the sixrrlmutants, were more often isolated in cystic fibrosis patients, but this was not significantly associated with a previous treatment.

scholarly journals Identification of Members of the Burkholderia cepacia Complex by Species-Specific PCR

2000 ◽  
Vol 38 (8) ◽  
pp. 2962-2965 ◽  
Author(s):  
Paul W. Whitby ◽  
Karen B. Carter ◽  
Kenneth L. Hatter ◽  
John J. LiPuma ◽  
Terrence L. Stull
Keyword(s):  
Burkholderia Cepacia ◽  
Growth Characteristics ◽  
Rrna Operon ◽  
Burkholderia Cepacia Complex ◽  
Pcr Analysis ◽  
Biochemical Tests ◽  
Specific Pcr ◽  
Dna Fragment ◽  
Species Specific ◽  
Phenotypic Tests

Definitive identification of the species in the Burkholderia cepacia complex by routine clinical microbiology methods is difficult. Phenotypic tests to identify B. multivorans andB. vietnamiensis have been established; more recent work indicates B. stabilis may also be identified by growth characteristics and biochemical tests. However, attempts to identify genomovars I and III have, thus far, proved unsuccessful. Previously, we demonstrated the utility of two primer pairs, directed to the rRNA operon, to specifically identify the B. cepacia complex in a PCR. One of these primer pairs, G1-G2, only amplified a DNA fragment from genomovars I and III and B. stabilis in a PCR with genomic DNA isolated from prototypical strains representing the five genomovars. Sequence analysis of the rRNA operon for all the genomovars indicated that this primer pair targeted a region shared by these isolates. Further analysis revealed a region of heterogeneity between genomovar III and B. stabilis internal to the amplified product of G1-G2. Primers designed to target this region were tested with prototypical strains following an initial amplification with the G1-G2 primer pair. New primers specific for the prototypical genomovar III and B. stabilis were designated SPR3 and SPR4, respectively. Analysis of 93 isolates representing 18 genomovar I, 13B. multivorans, 36 genomovar III, 11 B. stabilis, and 15 B. vietnamiensis isolates was performed. DNA from all isolates of genomovars I and III and B. stabilis was amplified by G1-G2. Genomovar III isolates yielded a product with SPR3/G1 while B. stabilis amplified with SPR4-G1. Genomovar I isolates were amplified by either SPR3-G1 or SPR4-G1, but not both. B. multivorans yielded a product with SPR3-G1 but not G1-G2, and B. vietnamiensis isolates were negative in all PCRs. Thus using an algorithm with G1-G2, SPR3-G1, and SPR4-G1 primers in a PCR analysis, genomovar III isolates can be separated from B. stabilis and the identity of B. multivorans and B. vietnamiensis can be confirmed.

scholarly journals Development of a Species Specific PCR for the Detection of Burkholderia gladioli

1999 ◽  
Vol 45 (4, Part 2 of 2) ◽  
pp. 357A-357A
Author(s):  
Paul W Whitby ◽  
Lauren C Pope ◽  
Terrence L Stull
Keyword(s):  
Specific Pcr ◽  
Burkholderia Gladioli ◽  
Species Specific

scholarly journals In Vitro Activity of Ceftolozane-Tazobactam and Other Antimicrobial Agents against Burkholderia cepacia Complex and Burkholderia gladioli

2017 ◽  
Vol 61 (9) ◽  
Author(s):  
Dale M. Mazer ◽  
Carol Young ◽  
Linda M. Kalikin ◽  
Theodore Spilker ◽  
John J. LiPuma
Keyword(s):  
Cystic Fibrosis ◽  
Burkholderia Cepacia ◽  
Antimicrobial Agents ◽  
Vitro Activity ◽  
Burkholderia Cepacia Complex ◽  
In Vitro Activity ◽  
Content Type ◽  
Burkholderia Gladioli

ABSTRACT We tested the activities of ceftolozane-tazobactam and 13 other antimicrobial agents against 221 strains of Burkholderia cepacia complex and Burkholderia gladioli. Most strains (82%) were cultured from persons with cystic fibrosis, and most (85%) were recovered since 2011. The ceftolozane-tazobactam MIC was ≤8 μg/ml for 77% of the strains. However, the MIC range was broad (≤0.5 to >64 μg/ml; MIC50/90, 2/32 μg/ml). Significant differences in susceptibility to some antimicrobial agents were observed between species.

scholarly journals Simultaneous Detection and Identification of Common Cell Culture Contaminant and Pathogenic Mollicutes Strains by Reverse Line Blot Hybridization

2004 ◽  
Vol 70 (3) ◽  
pp. 1483-1486 ◽  
Author(s):  
Hui Wang ◽  
Fanrong Kong ◽  
Peter Jelfs ◽  
Gregory James ◽  
Gwendolyn L. Gilbert
Keyword(s):  
Cell Culture ◽  
16S Rrna ◽  
Cell Line ◽  
Intergenic Spacer ◽  
23S Rrna ◽  
Pcr Assay ◽  
Intergenic Spacer Region ◽  
Specific Pcr ◽  
Specific Pcr Assay ◽  
Species Specific

ABSTRACT We have developed a reverse line blot (RLB) hybridization assay to detect and identify the commonest mollicutes causing cell line contamination (Mycoplasma arginini, Mycoplasma fermentans, Mycoplasma hyorhinis, Mycoplasma orale, and Acholeplasma laidlawii) and human infection (Mycoplasma pneumoniae, Mycoplasma hominis, Mycoplasma genitalium, Ureaplasma parvum, and Ureaplasma urealyticum). We developed a nested PCR assay with “universal” primers targeting the mollicute 16S-23S rRNA intergenic spacer region. Amplified biotin-labeled PCR products were hybridized to membrane-bound species-specific oligonucleotide probes. The assay correctly identified reference strains of 10 mollicute species. Cell cultures submitted for detection of mollicute contamination, clinical specimens, and clinical isolates were initially tested by PCR assay targeting a presumed mollicute-specific sequence of the 16S rRNA gene. Any that were positive were assessed by the RLB assay, with species-specific PCR assay as the reference method. Initially, 100 clinical and 88 of 92 cell culture specimens gave concordant results, including 18 in which two or more mollicute species were detected by both methods. PCR and sequencing of the 16S-23S rRNA intergenic spacer region and subsequent retesting by species-specific PCR assay of the four cell culture specimens for which results were initially discrepant confirmed the original RLB results. Sequencing of amplicons from 12 cell culture specimens that were positive in the 16S rRNA PCR assay but negative by both the RLB and species-specific PCR assays failed to identify any mollicute species. The RLB hybridization assay is sensitive and specific and able to rapidly detect and identify mollicute species from clinical and cell line specimens.

Sign in / Sign up

Export Citation Format

Share Document