Genetic diversity of penicillin-binding protein 2 genes of penicillin-resistant strains of Neisseria meningitidis revealed by fingerprinting of amplified DNA

Zhang, Q Y; Jones, D. M.; Nieto, Juan Antonio; Trallero, Emilio Pérez; Spratt, Brian G.

doi:10.1128/aac.34.8.1523

Cited by 68 publications

(31 citation statements)

References 11 publications

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“…The coding regions of the penA genes (from nucleotides 546 to 1746) of all the strains examined were amplified by using the primers and PCR conditions described by Zhang et al (15). The PCR products were further purified by use of QIAquick purification columns (Qiagen) for sequencing by the Sanger method (11a) with the ABI Prism DNA sequencer 377 (Perkin-Elmer).…”

Section: Methodsmentioning

confidence: 99%

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

et al. 2003

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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 A3G 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.

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Section: Methodsmentioning

confidence: 99%

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

et al. 2003

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“…In contrast to the PBP 2 genes (penA) of susceptible isolates, which are very uniform in sequence (14,17), those of penicillin-resistant isolates are very variable (17) and have mosaic structures consisting of regions that are essentially identical in sequence to the corresponding regions in penicillin-susceptible isolates alternating with regions that are highly diverged (14).…”

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confidence: 98%

Interspecies recombination between the penA genes of Neisseria meningitidis and commensal Neisseria species during the emergence of penicillin resistance in N. meningitidis: natural events and laboratory simulation

et al. 1994

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The penicillin-binding protein 2 genes (penA) of penicillin-resistant Neisseria meningitidis have a mosaic structure that has arisen by the introduction of regions from the penA genes of Neisseria flavescens or Neisseria cinerea. Chromosomal DNA from both N. cinerea and N. flavescens could transform a penicillin-susceptible isolate of N. meningitidis to increased resistance to penicillin. With N. flavescens DNA, transformation to resistance was accompanied by the introduction of the N. flavescens penA gene, providing a laboratory demonstration of the interspecies recombinational events that we believe underlie the development of penicillin resistance in many meningococci in nature. Surprisingly, with N. cinerea DNA, the penicillin-resistant transformants did not obtain the N. cinerea penA gene. However, the region of the penA gene derived from N. cinerea in N. meningitidis K196 contained an extra codon (Asp-345A) which was not found in any of the four N. cinerea isolates that we examined and which is known to result in a decrease in the affinity of PBP 2 in gonococci.

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“…The sequence of strain 3008 was 11.5% divergent with respect to that of N. meningitidis C311, 17 with 140 nucleotide substitutions leading to 37 amino acid substitutions. Neither deletions nor insertions were detected.…”

Section: Resultsmentioning

confidence: 99%

“…17 A region of 1.4-Kb was sequenced by the ABI PRISM dRhodamine Terminator Cycle sequencing kit and subsequently analyzed by capillary electrophoresis in an ABI 310 Analyzer (Perkin Elmer). Regions from transform ants MC2 and MC2-26 (first and second step transformants respectively) as well as the N. polysaccharea 3008 were sequenced.…”

Section: Polymerase Chain Reactionmentioning

confidence: 99%

Transfer of Penicillin Resistance BetweenNeisseriaein Microcosm

Orús

Viñas²

2000

Microbial Drug Resistance

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Horizontal gene transfer between commensal and pathogenic Neisseriae is the mechanism proposed to explain how pathogenic species acquire altered portions of the penA gene, which encodes penicillin binding protein 2. These changes resulted in a moderately penicillin-resistant phenotype in the meningococci, whose frequency of isolation in Spain increased at the end of the 1980s. Little has been published about the possibility of this gene transfer in nature or about its simulation in the laboratory. We designed a simple microcosm, formed by solid and liquid media, that partially mimics the upper human respiratory tract. In this microcosm, penicillin-resistant commensal strains and the fully susceptible meningococcus were co-cultivated. The efficiency of gene transfer between the strains depended on the phase of bacterial growth and the conditions of culture. Resistance of penicillin was acquired in different steps irrespective of the source of the DNA. The presence of DNase in the medium had no effect on gene transfer, but it was near zero when nicked DNA was used. Cellto-cell contact or membrane blebs could explain these results. The analysis of sequences of the transpeptidase domain of PBP2 from transformants, and from donor and recipient strains demonstrated that the emergence of moderately resistant transformants was due to genetic exchange between the co-cultivated strains. Finally, mechanisms other than penA modification could be invoked to explain decreased susceptibility. 99

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Genetic diversity of penicillin-binding protein 2 genes of penicillin-resistant strains of Neisseria meningitidis revealed by fingerprinting of amplified DNA

Cited by 68 publications

References 11 publications

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

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

Interspecies recombination between the penA genes of Neisseria meningitidis and commensal Neisseria species during the emergence of penicillin resistance in N. meningitidis: natural events and laboratory simulation

Transfer of Penicillin Resistance BetweenNeisseriaein Microcosm

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