Sequencing of Neisseria meningitidis penA gene: the key to success in defining penicillin G breakpoints.
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ABSTRACT:
SUBMITTER: Arreaza L
PROVIDER: S-EPMC310191 | biostudies-literature | 2004 Jan
REPOSITORIES: biostudies-literature
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Sequencing of Neisseria meningitidis penA gene: the key to success in defining penicillin G breakpoints.
Arreaza Luisa L Salcedo Celia C Alcalá Belén B Uría María José MJ Abad Raquel R Enríquez Rocío R Vázquez Julio A JA
Antimicrobial agents and chemotherapy 20040101 1
Testing of susceptibility to penicillin G by E-test and sequencing of an internal fragment of the penA gene were done for 43 meningococcal strains. Those strains for which the MIC was >/=0.094 micro g/ml showed mosaic alleles, so 0.094 micro g/ml is suggested as the penicillin G intermediate breakpoint when E-test is used. ...[more]
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Project description:Clinical isolates of Neisseria meningitidis with reduced susceptibility to penicillin G (intermediate isolates, Pen(I)) 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 Pen(I) phenotype. The deduced amino acid sequence of the corresponding PBP2 comprised five amino acid residues that were always altered. This correlation was not complete for rare alleles, suggesting that other mechanisms may also be involved in conferring reduced susceptibility to penicillin. Evidence of mosaic structures through events of interspecies recombination was also detected in altered alleles. A new website was created based on the data from this work (http://neisseria.org/nm/typing/penA). These data argue for the use of penA sequencing to identify isolates with reduced susceptibility to penicillin G and as a tool to improve typing of meningococcal isolates, as well as to analyze DNA exchange among Neisseria species.
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Project description: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 beta-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 microg/ml for penicillin G and an MIC of >0.064 microg/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.
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Project description: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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| S-EPMC298417 | biostudies-other
Project description:Neisseria meningitidis causes a life-threatening invasive meningococcal disease (IMD). Isolates resistant to antibiotics, such as penicillin, ceftriaxone, and ciprofloxacin that are recommended for the treatment of IMD patients and their close contacts have been serious public health concerns globally. However, susceptibility profiles to critically important antibiotics and the genetic characteristics of isolates possessing antibiotic resistance are extremely limited as IMD incidence is low in Japan. We assessed the susceptibility profiles of 87 randomly selected, sterile site-derived N. meningitidis strains isolated from hospitals nationwide, recovered between April 1998 and March 2018 in Japan, to seven antibiotics. As a result, we demonstrated, for the first time, that the isolates remained highly susceptible to ceftriaxone, meropenem, azithromycin, ciprofloxacin, chloramphenicol, and rifampin, but not to penicillin. We then characterized the genetic relatedness of six penicillin- and/or ciprofloxacin-resistant isolates obtained in this study with global 112 genomes using core-genome phylogenetic analysis. These results provide the first evidence that invasive lineages such as a penicillin-resistant serogroup W, sequence type (ST)-11 clonal complex (CC), and a ciprofloxacin-resistant serogroup B/C, ST-4821 CC that is considered as a global threat, have been sporadically identified in Japan. Our findings highlight the need to monitor antibiotic resistance in clinical isolates of N. meningitidis, thereby preventing the spread of antibiotic-resistant invasive lineages and maintaining effective treatment for IMD patients and their close contacts. IMPORTANCE Although antibiotics such as penicillin and ceftriaxone can treat invasive meningococcal disease (IMD), the emergence and spread of antibiotic-resistant Neisseria meningitidis have become a global concern. To provide effective treatment, including chemoprophylaxis to IMD patients and their close contacts, we highlighted the importance of recognizing the antibiotic resistance and genetic features of N. meningitidis isolates.
| S-EPMC9241714 | biostudies-literature
Project description:Objectives:Neisseria meningitidis is rarely penicillin resistant. We describe WGS analysis of a penicillin-resistant N. meningitidis collected from a case of invasive meningococcal disease. Methods:Serogrouping, serotyping and serosubtyping were performed with specific antibodies. ?-Lactamase was detected by nitrocefin. MICs were determined by Etest and agar dilution. Sequencing of N. meningitidis genomes was done on the Illumina MiSeq platform and genome data were analysed using the Bacterial Isolate Genome Sequence Database (BIGSdb) on the PubMLST Neisseria website (https://pubmlst.org/neisseria/). Transformation was used to confirm the genetic basis of the penicillin resistance. Results:An N. meningitidis blood isolate from a female patient in her mid-50s with a painful and septic left shoulder was found to have penicillin MIC values of 3-12?mg/L. The isolate was typed as Y: 14, 19: P1.- and ST3587, and was weakly ?-lactamase positive. WGS analysis identified a full-length copy of the ?-lactamase gene blaROB-1, which was contained on a 1719?bp insert with a G?+?C content of 41.7% (versus a G?+?C content of N. meningitidis of 51.7%), suggesting that the blaROB-1 gene came from a different bacterial species. A GenBank analysis of the blaROB-1 gene insert found 99.77% identity with a DNA segment found in plasmid pB1000' from Haemophilus influenzae. Transformation of a penicillin-susceptible strain with the blaROB-1 gene conferred ?-lactamase activity and penicillin resistance. Conclusions:N. meningitidis serogroup Y, ST3587 can carry and express the blaROB-1 gene, leading to penicillin resistance. It is highly likely that the N. meningitidis isolate acquired the blaROB-1 gene from H. influenzae.
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Project description:Neisseria gonorrhoeae strains with reduced susceptibility to cefixime (MICs, 0.25 to 0.5 micro g/ml) were isolated from male urethritis patients in Tokyo, Japan, in 2000 and 2001. The resistance to cephems including cefixime and penicillin was transferred to a susceptible recipient, N. gonorrhoeae ATCC 19424, by transformation of the penicillin-binding protein 2 gene (penA) that had been amplified by PCR from a strain with reduced susceptibility to cefixime (MIC, 0.5 micro g/ml). The sequences of penA in the strains with reduced susceptibilities to cefixime were different from those of other susceptible isolates and did not correspond to the reported N. gonorrhoeae penA gene sequences. Some regions in the transpeptidase-encoding domain in this penA gene were similar to those in the penA genes of Neisseria perflava (N. sicca), Neisseria cinerea, Neisseria flavescens, and Neisseria meningitidis. These results showed that a mosaic-like structure in the penA gene conferred reductions in the levels of susceptibility of N. gonorrhoeae to cephems and penicillin in a manner similar to that found for N. meningitidis and Streptococcus pneumoniae.
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Project description:The ponA gene encoding penicillin-binding protein 1 (PBP 1) from Neisseria gonorrhoeae was cloned by a reverse genetic approach. PBP 1 was purified from solubilized membranes of penicillin-susceptible strain FA19 by covalent ampicillin affinity chromatography and used to obtain an NH2-terminal amino acid sequence. A degenerate oligonucleotide based on this protein sequence and a highly degenerate oligonucleotide based on a conserved amino acid motif found in all class A high-molecular-mass PBPs were used to isolate the PBP 1 gene (ponA). The ponA gene encodes a protein containing all of the conserved sequence motifs found in class A PBPs, and expression of the gene in Escherichia coli resulted in the appearance of a new PBP that comigrated with PBP 1 purified from N. gonorrhoeae. A comparison of the gonococcal ponA gene to its homolog isolated from Neisseria meningitidis revealed a high degree of identity between the two gene products, with the greatest variability found at the carboxy terminus of the two deduced PBP 1 protein sequences.
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Project description:A rise in invasive diseases due to Neisseria meningitidis C:2b:P1.5 with decreased penicillin susceptibility occurred in Italy during the last 2 years. Real-time PCR identified the Peni phenotype, and the penA sequence revealed the mosaicism of the gene. Molecular analyses assigned the isolates to a single emergent clone of the hypervirulent A4 cluster.
| S-EPMC478501 | biostudies-literature
Project description: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 degrees 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.
| S-EPMC254320 | biostudies-literature