Arbitrarily primed PCR and sequencing of 16S rDNA for epidemiological typing and species identification of Burkholderia cepacia isolates from Swedish patients with cystic fibrosis reveal genetic heterogeneity.
To investigate whether arbitrarily primed (AP)-PCR and/or 16S rDNA sequencing could be used as rapid methods for epidemiological typing and species identification of clinical Burkholderia isolates from patients with cystic fibrosis (CF), a total of 39 clinical B. cepacia isolates, including 33 isolates from 14 CF patients, were fingerprinted. ERIC-2 primer was used for AP-PCR. The AP-PCR clustering analysis resulted in 14 different clusters at a 70% similarity level. The AP-PRC patterns were individual despite considerable similarities. To sequence rDNA, a broad-range PCR was applied. The PCR product included four variable loops (V8, V3, V4 and V9) of the 16S ribosomal small subunit RNA gene. The multiple sequence alignment produced 12 different patterns, 5 of them including more than one isolate. Heterogeneity of the bases in the V3 region, indicating the simultaneous presence of at least two different types of 16S rRNA genes in the same cell, was revealed in 10 isolates. Most of the CF patients were adults who had advanced disease at follow-up. Both the sequencing and the AP-PCR patterns revealed genetic heterogeneity of isolates between patients. According to the results obtained, AP-PCR could advantageously be used for epidemiological typing of Burkholderia, whereas partial species identification could effectively be obtained by sequencing of the V3 region of the 16S RNA gene.
Karpati
F
F
Stockholm Cystic Fibrosis Centre, Division of Paediatrics, Huddinge University Hospital, Stockholm, Sweden. ferenc.karpati@klinvet.ki.se
Giedraitis
V
V
Thore
M
M
Lindman
R
R
Monstein
H J
HJ
Hjelte
L
L
Jonasson
J
J
D000293:Adolescent; D000328:Adult; D001483:Base Sequence; D019117:Burkholderia / Q000145:classification / Q000235:genetics / Q000302:isolation & purification; D019121:Burkholderia Infections / Q000150:complications / Q000453:epidemiology*; D016956:Burkholderia cepacia / Q000145:classification / Q000235:genetics* / Q000302:isolation & purification; D003550:Cystic Fibrosis / Q000150:complications* / Q000235:genetics; D017931:DNA Primers; D004275:DNA, Ribosomal / Q000032:analysis / Q000235:genetics*; D005260:Female; D005798:Genes, Bacterial / Q000235:genetics; D005838:Genotype; D006801:Humans; D008297:Male; D008875:Middle Aged; D017720:Molecular Epidemiology / Q000379:methods; D008969:Molecular Sequence Data; D010802:Phylogeny; D016133:Polymerase Chain Reaction / Q000379:methods*; D015995:Prevalence; D012336:RNA, Ribosomal, 16S / Q000235:genetics*; D013183:Sputum / Q000382:microbiology; D013548:Sweden / Q000453:epidemiology
D017931:DNA Primers; D004275:DNA, Ribosomal; D012336:RNA, Ribosomal, 16S