ABSTRACT
We describe the development and application of a rapid and universal molecular technique for direct identification of multiple bacteria in clinical samples. Amplification of the 16S-23S rRNA spacer-region using universal primers led to fragment patterns distinct for different bacterial species and that were analyzed with fluorescent amplicon length analysis (FALA). 136 pure cultures of clinical isolates and 20 culture collection strains belonging to 22 different medically important species were used to create a primary database of fragments with sizes between 100 and 1000 bp. Subsequently, 127 respiratory samples were analyzed with culture-based techniques and via FALA of the 16S-23S rRNA spacer-region. Two DNA extraction methods were evaluated: Instagene (FALA-I) and Fastprept (FALA-P). Of the 127 samples, 26 culture-negative samples were also negative with FALA-P. Of 18 samples with growth of commensal oral flora, 10 gave a mixed oral flora pattern with FALA-P and 8 gave a negative result. For 54 samples with growth of a single bacterial species, FALA-P gave an identical result for 46. For 29 samples with growth of more than one bacterial species, identical results were obtained in 19 samples. False-negative results with FALA-P were mostly due to paucity (less than 10(3) CFU/ml) of bacteria (12 out of 18 false-negatives) or difficulties with homogenization of viscous samples (6 out of 18 false-negatives).With regard to identification of all significant pathogens of clinical samples tested, the sensitivity of FALA-P was 77% and its specificity was 100%. With FALA-I, the number of false-negative results was higher than with FALA-P due to less efficient extraction of DNA, particularly with Staphylococcal species. FALA-P allows rapid and direct identification of multiple species directly from clinical samples; pauci-cellular samples may give false-negative results.
Subject(s)
Bacteria/isolation & purification , Bacterial Infections/microbiology , DNA, Ribosomal Spacer/chemistry , Respiratory Tract Diseases/microbiology , Bacteria/genetics , Bacterial Infections/diagnosis , DNA, Bacterial/chemistry , DNA, Bacterial/genetics , DNA, Ribosomal Spacer/genetics , Electrophoresis, Capillary , False Negative Reactions , False Positive Reactions , Humans , Polymerase Chain Reaction , RNA, Ribosomal, 16S/chemistry , RNA, Ribosomal, 16S/genetics , RNA, Ribosomal, 23S/chemistry , RNA, Ribosomal, 23S/genetics , Respiratory Tract Diseases/diagnosis , Sensitivity and SpecificityABSTRACT
Of 233 erythromycin-resistant pneumococcal isolates collected in Belgium in 1999-2000, 89.7% carried the erm(B) gene, 6% the mef(A) gene, and 3.5%erm(B) plus mef(A). Two isolates contained neither erm(B) nor mef(A); one contained an erm(A) subclass erm(TR) gene, while the other contained an A2058G mutation in domain V of the 23S rRNA gene. Of 209 erm(B)-positive isolates, 191 had clindamycin MICs > 16 mg/L and 18 had MICs < or = 16 mg/L. Mef(A)-positive isolates all displayed the M resistance phenotype. Telithromycin remained active against erythromycin-resistant isolates, with the highest telithromycin MIC50 being found in mef(A)-positive isolates. No difference in the prevalence of different resistance mechanisms was observed compared to isolates collected in 1995-1997.
