Learning from mistakes: Taq-polymerase contaminated with b-lactamase sequences provides false findings of Streptococcus pneumoniae containing TEM
Abstract number: 1733_951
Koncan R., Valverde A., Morosini M., Cantón R., Cornaglia G., Baquero F., del Campo R.
Background: In 2004 an Asiatic group published in a local journal an article entitled ``Study on the molecular epidemiology of b-lactamase TEM gene in isolated Streptococcus pneumoniae'' (PMID: 15769331). In this article the authors referred about S. pneumoniae isolates carrying TEM genes, never described before. Furthermore, the authors described new TEM sequences (TEM-129) which were included in the GenBank data base under the numbers AY452662 and AY392531. The aim of this work was to explore the reasons for such abnormal findings.
Methods: We used total DNA extracted from a S. pneumoniae collection of 24 isolates with MIC values for penicillin of 24 mg/mL. The strains were serotyped, PFGE-typed, and the PBP2x, 2b and 1a were characterised. Total DNA from TEM-4 harbouring Klebsiella pneumoniae F40 and S. pneumoniae R6 strains were used as positive and negative controls. To evaluate the presence of TEM genes, PCR experiments with specific primers were performed with two different Taq-polymerase enzymes: FastStart (12032945001 from Roche) and Taq-Core (Qbiogene, MpBIO). PCR products were further sequenced. Hydrolysis assays using nitrocefin as subtract were also performed in crude extracts of S. pneumoniae isolates.
Results: Using the FastStart polymerase, a clear positive amplification corresponded to the bla-TEM size were observed in all strains tested, including the negative control. With this enzyme, amplifications occurred also in absence of pneumococcal DNA. Four different amplicons were sequenced, which corresponded to the ESBL TEM-116 previously described. When the other Taq-polymerase was used, positive amplifications were not detected in any of the isolates, except the positive control. Nitrocefin test was consistently negative in all isolates.
Conclusion: Caution should be required to accept data about the presence of TEM-b-lactamases based only on PCR-based results in the absence of controls that might reveal TEM sequences contamination of amplification reagents.
|Session name:||European Society of Clinical Microbiology and Infectious Diseases|
|Location:||ICC, Munich, Germany|
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