WGS analysis of a penicillin-resistant Neisseria meningitidis strain containing a chromosomal ROB-1 β-lactamase gene (original) (raw)
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Target Gene Sequencing To Characterize the Penicillin G Susceptibility of Neisseria meningitidis
Antimicrobial Agents and Chemotherapy, 2007
Clinical isolates of Neisseria meningitidis with reduced susceptibility to penicillin G (intermediate isolates, PenI) harbor alterations in the penA gene encoding the penicillin binding protein 2 (PBP2). A 402-bp DNA fragment in the 3′ half of penA was sequenced from a collection of 1,670 meningococcal clinical isolates from 22 countries that spanned 60 years. Phenotyping, genotyping, and the determination of MICs of penicillin G were also performed. A total of 139 different penA alleles were detected with 38 alleles that were highly related, clustered together in maximum-likelihood analysis and corresponded to the penicillin G-susceptible isolates. The remaining 101 penA alleles were highly diverse, corresponded to different genotypes or phenotypes, and accounted for 38% of isolates, but no clonal expansion was detected. Analysis of the altered alleles that were represented by at least five isolates showed high correlation with the PenI phenotype. The deduced amino acid sequence of...
Antimicrobial Agents and Chemotherapy, 2006
In recent decades, the prevalence of Neisseria meningitidis isolates with reduced susceptibility to penicillins has increased. The intermediate resistance to penicillin (Pen i) for most strains is due mainly to mosaic structures in the penA gene, encoding penicillin-binding protein 2. In this study, susceptibility to -lactam antibiotics was determined for 60 Swedish clinical N. meningitidis isolates and 19 reference strains. The penA gene was sequenced and compared to 237 penA sequences from GenBank in order to explore the total identified variation of penA. The divergent mosaic alleles differed by 3% to 24% compared to those of the designated wild-type penA gene. By studying the final 1,143 to 1,149 bp of penA in a sequence alignment, 130 sequence variants were identified. In a 402-bp alignment of the most variable regions, 84 variants were recognized. Good correlation between elevated MICs and the presence of penA mosaic structures was found especially for penicillin G and ampicillin. The Pen i isolates comprised an MIC of >0.094 g/ml for penicillin G and an MIC of >0.064 g/ml for ampicillin. Ampicillin was the best antibiotic for precise categorization as Pen s or Pen i. In comparison with the wild-type penA sequence, two specific Pen i sites were altered in all except two mosaic penA sequences, which were published in GenBank and no MICs of the corresponding isolates were described. In conclusion, monitoring the relationship between penA sequences and MICs to penicillins is crucial for developing fast and objective methods for susceptibility determination. By studying the penA gene, genotypical determination of susceptibility in culture-negative cases can also be accomplished.
Antimicrobial Agents and Chemotherapy, 2006
In recent decades, the prevalence of Neisseria meningitidis isolates with reduced susceptibility to penicillins has increased. The intermediate resistance to penicillin (Pen i ) for most strains is due mainly to mosaic structures in the penA gene, encoding penicillin-binding protein 2. In this study, susceptibility to -lactam antibiotics was determined for 60 Swedish clinical N. meningitidis isolates and 19 reference strains. The penA gene was sequenced and compared to 237 penA sequences from GenBank in order to explore the total identified variation of penA. The divergent mosaic alleles differed by 3% to 24% compared to those of the designated wild-type penA gene. By studying the final 1,143 to 1,149 bp of penA in a sequence alignment, 130 sequence variants were identified. In a 402-bp alignment of the most variable regions, 84 variants were recognized. Good correlation between elevated MICs and the presence of penA mosaic structures was found especially for penicillin G and ampicillin. The Pen i isolates comprised an MIC of >0.094 g/ml for penicillin G and an MIC of >0.064 g/ml for ampicillin. Ampicillin was the best antibiotic for precise categorization as Pen s or Pen i . In comparison with the wild-type penA sequence, two specific Pen i sites were altered in all except two mosaic penA sequences, which were published in GenBank and no MICs of the corresponding isolates were described. In conclusion, monitoring the relationship between penA sequences and MICs to penicillins is crucial for developing fast and objective methods for susceptibility determination. By studying the penA gene, genotypical determination of susceptibility in culture-negative cases can also be accomplished.
Journal of Clinical Microbiology, 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 Pe...
Journal of Antimicrobial Chemotherapy, 2001
We studied polymorphism of penA (which encodes penicillin-binding protein 2) in 13 strains of Neisseria meningitidis susceptible to penicillin (pen S ) and 12 strains with reduced susceptibility to penicillin (pen I ). These strains differed in geographical origin. Serological and genetic typing showed that they were highly diverse and belonged to several genetic lineages. Restriction analysis and DNA sequencing of penA showed that all pen S strains had the same penA allele regardless of genetic group, whereas pen I strains harboured various penA alleles. Transformation with amplicons of penA and genomic DNA from several pen I strains conferred the pen I phenotype on a pen S strain. Thus, reduction in susceptibility to penicillin is directly related to changes in penA and analysis of penA polymorphisms could be used as a reliable tool for characterizing meningococcal strains in terms of their susceptibility to penicillin.
Journal of Bacteriology, 1994
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 exa...
European Journal of Clinical Microbiology & Infectious Diseases, 1993
A random selection of Neisseria rneningitidis strains isolated in Sweden in the period 1981-1990 were investigated for plasmid carriage and susceptibility to antimicrobial agents commonly used for treatment and prophylaxis of meningococcal disease. The MICs were determined by the agar dilution method for penicillin V, penicillin G, rifampicin, sulfadiazine, erythromycin and tetracycline. In 13 % of the invasive strains the MIC of penicillin V was _> 0.5 mg/l which may cause concern regarding the usefulness of penicillin V in prophylaxis. In strains isolated from the urogenital tract the MICs of penicillin V and penicillin G were higher than in the invasive strains. In about 82 % of the strains isolated in 1987-1988 the MIC of tetracycline was >_ 0.5 mg/l whereas no such strains were found in 1981-1982. Plasmids were found in 2 of 119 invasive strains, in I of 50 strains from the respiratory tract and in I of 19 strains from the urogenital tract. The plasmid sizes were L3, 2.6, 25 and 40 Mda. None of these strains were beta-lactamase producing and no relation to a high degree antibiotic resistance was observed. Strains of Neisseria meningilidis are normally susceptible to many of the antibiotics presently used (1), including beta-lactams, rifampicin, chloramphenicol, tetracyclines, quinolones and macrolides. These organisms have an inherent decreased susceptibility to trimethoprim, vancomycin and polymyxin B. The emergence of meningococcal strains resistant to sulphonamides in the USA in the early 1960s (2) served as an example of the unwanted biological side-effects of the use of antimicrobial agents. At present the relative susceptibility of meningococci to sulphonamides is mainly used as an epidemicological marker. Sulphonamides can, however, still be considered for use in prophylaxis of smaller groups of people providing that the invasive index strain is susceptible, i.e. the MIC of sulfadiazine is < 10 mg/l. In the 1980s an increasing number of reports were published of meningococci with decreased susceptibility to penicillin G (MIC > 0.16 mg/l) (3-15). Such strains have been isolated in England and Wales since 1983 (3, 4), in Spain since 1985 (5-12) and in South Africa since 1987 (13).