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Journal of Clinical Microbiology, October 2003, p. 4666-4670, Vol. 41, No. 10
0095-1137/03/$08.00+0     DOI: 10.1128/JCM.41.10.4666-4670.2003
Copyright © 2003, American Society for Microbiology. All Rights Reserved.

Prediction of Decreased Susceptibility to Penicillin of Neisseria meningitidis Strains by Real-Time PCR

Paola Stefanelli, Alessandra Carattoli, Arianna Neri, Cecilia Fazio, and Paola Mastrantonio*

Laboratory of Bacteriology and Medical Mycology, Istituto Superiore di Sanitá, Rome, Italy

Received 17 March 2003/ Returned for modification 27 April 2003/ Accepted 6 July 2003

Sequence analysis of the penA gene, encoding penicillin-binding protein 2 (PBP2), in 30 penicillin-intermediate (PenI) Neisseria meningitidis strains showed altered gene sequences due to the translocation of exogenous DNA blocks derived from commensal neisseriae, which are known to have PBP2 proteins with decreased affinity for the antibiotic. In order to obtain a rapid and reproducible method for predicting the PenI phenotype, a real-time PCR assay was set up with primers and probes designed on the basis of the penA gene. The A->G mutation at codon 566, in the transpeptidase domain of the penA gene (which is present in the whole sample of 30 PenI strains and in all the 41 sequences of PenI meningococci isolated worldwide and has been deposited in the sequence databank), was chosen as a marker of penA translocations. Two hybridization probes were designed to distinguish the wild-type penA gene in penicillin-susceptible (PenS) meningococci from the mutated penA gene at codon 566 in PenI strains. Thermal analysis of probe hybridization revealed a melting temperature difference of at least 6°C between PenI and PenS strains. This real-time PCR protocol characterizes the penicillin phenotype of N. meningitidis in a few hours without DNA sequencing and is useful for rapid screening of the penicillin-intermediate genotype among meningococcal isolates.

* Corresponding author. Mailing address: Laboratory of Bacteriology and Medical Mycology, Istituto Superiore di Sanitá, V.le Regina Elena 299-00161, Rome, Italy, Phone: 390649902335. Fax: 390649387112. E-mail: pmastran{at}iss.it.

Journal of Clinical Microbiology, October 2003, p. 4666-4670, Vol. 41, No. 10
0095-1137/03/$08.00+0     DOI: 10.1128/JCM.41.10.4666-4670.2003
Copyright © 2003, American Society for Microbiology. All Rights Reserved.



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