Mosaic penA Allele Research Articles

Molecular and Structural Analysis of Mosaic Variants of Penicillin-Binding Protein 2 Conferring Decreased Susceptibility to Expanded-Spectrum Cephalosporins in Neisseria gonorrhoeae: Role of Epistatic Mutations

Mutations in penicillin-binding protein 2 (PBP 2) encoded by mosaic penA alleles are crucial for intermediate resistance to the expanded-spectrum cephalosporins ceftriaxone and cefixime in Neisseria gonorrhoeae. Three of the ∼60 mutations present in mosaic alleles of penA, G545S, I312M, and V316T, have been reported to be responsible for increased resistance, especially to cefixime [Takahata, S., et al. (2006) Antimicrob. Agents Chemother. 50, 3638-3645]. However, we observed that the minimum inhibitory concentrations (MICs) of penicillin, ceftriaxone, and cefixime for a wild-type strain (FA19) containing a penA gene with these three mutations increased only 1.5-, 1.5-, and 3.5-fold, respectively. In contrast, when these three mutations in a mosaic penA allele (penA35) were reverted back to the wild type and the gene was transformed into FA19, the MICs of the three antibiotics were reduced to near wild-type levels. Thus, these three mutations display epistasis, in that their capacity to increase resistance to β-lactam antibiotics is dependent on the presence of other mutations in the mosaic alleles. We also identified an additional mutation, N512Y, that contributes to the decreased susceptibility to expanded-spectrum cephalosporins. Finally, we investigated the effects of a mutation (A501V) currently found only in nonmosaic penA alleles on decreased susceptibility to ceftriaxone and cefixime, with the expectation that this mutation may arise in mosaic alleles. Transfer of the mosaic penA35 allele containing an A501V mutation to FA6140, a chromosomally mediated penicillin-resistant isolate, increased the MICs of ceftriaxone (0.4 μg/mL) and cefixime (1.2 μg/mL) to levels above their respective break points. The proposed structural mechanisms of these mutations are discussed in light of the recently published structure of PBP 2.

Reduced susceptibility to ceftriaxone in Neisseria gonorrhoeae is associated with mutations G542S, P551S and P551L in the gonococcal penicillin-binding protein 2

Reduced susceptibility to extended-spectrum cephalosporins in Neisseria gonorrhoeae has, to date, been associated with three alterations: a mosaic penA allele encoding the penicillin-binding protein 2 (PBP2); A-del-mtrR, an adenine deletion in the mtrR promoter; and penB, comprising mutated alleles of PorBIb. In this study, we examined an association between reduced susceptibility to ceftriaxone and additional mutations in gonococcal PBP2. N. gonorrhoeae isolates (n = 76) exhibiting reduced susceptibility to ceftriaxone but lacking the mosaic penA sequence were investigated for A-del-mtrR and penB as well as substitutions at PBP2 501, 542 and 551 using a previously described real-time PCR approach. To further investigate PBP2 542 and 551 substitutions, we reanalysed penA sequence data from a previous study of 98 gonococci exhibiting a range of ceftriaxone MICs. Of 76 N. gonorrhoeae isolates exhibiting reduced susceptibility to ceftriaxone and lacking the mosaic penA sequence, a 501 (A501V or A501T) substitution was present in 9/76, a 542 substitution in 39/76 and a 551 substitution in 26/76 isolates. Reanalysis of 98 gonococcal isolates from a previous study showed that substitutions at PBP2 542 (G542S) and 551 (P551S or P551L) were significantly associated with raised MICs to ceftriaxone (P = 0.0186 and 0.001, respectively) and penicillin (P = 0.0231 and 0.0007, respectively). Our findings provide strong evidence for the involvement of PBP2 G542S and P551S/P551L in reduced susceptibility to ceftriaxone and to penicillin. Further studies are needed to investigate the precise and relative roles played by these mutations.

Real‐time PCR and subsequent pyrosequencing for screening of penA mosaic alleles and prediction of reduced susceptibility to expanded‐spectrum cephalosporins in Neisseria gonorrhoeae

Real-time PCR and subsequent pyrosequencing for screening of penA mosaic alleles and prediction of reduced susceptibility to expanded-spectrum cephalosporins in Neisseria gonorrhoeae

Neisseria gonorrhoeae isolates with reduced susceptibility to cefixime and ceftriaxone: association with genetic polymorphisms in penA, mtrR, porB1b, and ponA.

The recent emergence and transmission of Neisseria gonorrhoeae isolates with reduced susceptibility to expanded-spectrum cephalosporins such as cefixime and ceftriaxone have been reported. The aim of this study was to determine the correlation of different polymorphisms in the penA, mtrR, porB1b (penB), and ponA genes of N. gonorrhoeae with reduced susceptibility to cefixime and ceftriaxone. Eighteen gonococcal isolates with reduced cefixime and ceftriaxone susceptibility (Cef(i)) and two susceptible isolates were characterized using serovar determination, antibiograms, N. gonorrhoeae multiantigen sequence typing (NG-MAST), and sequencing of penA, mtrR, porB1b, and ponA alleles. For the Cef(i) isolates (n = 18), the MICs of cefixime and ceftriaxone ranged between 0.032 to 0.38 mug/ml and 0.064 to 0.125 mug/ml, respectively. These isolates were assigned five different serovars and six divergent NG-MAST sequence types. Eleven isolates (61%) with higher MICs of cefixime and ceftriaxone contained a nearly identical penA mosaic allele and previously described polymorphisms in mtrR (a single nucleotide [A] deletion in the promoter), penB (mutations in porB1b encoding loop 3 of PorB1b), and ponA (ponA1 polymorphism). The remaining seven Cef(i) isolates (39%), which had somewhat lower MICs of cefixime and ceftriaxone, contained an aspartic acid insertion (Asp-345a) in PBP 2 in conjunction with alterations of 4 to 10 amino acid residues in the C-terminal region of the transpeptidase domain of penA. In conclusion, an unambiguous association between penA mosaic alleles, in conjunction with genetic polymorphisms in mtrR, porB1b, and ponA, and greater reduced susceptibility to cefixime and ceftriaxone was identified.

Dynamics of the penA gene in serogroup C meningococcal strains.

The transpeptidase encoding region of the penA gene was sequenced in 44 meningococcal strains (41 serogroup C [23 characterized as serotype 2b and 18 as serotype 2a] and 3 serogroup B [B:2b:P1.2,5]). All strains were characterized by multilocus sequence typing and were determined to be susceptible or intermediate resistant to penicillin (Pen(s) or Pen(i), respectively). A high degree of homology was found among the penA alleles identified in the Pen(s) strains. All the Pen(i) C:2b strains, which belonged to 2 different clonal complexes, showed the same penA gene allele. This fact suggests that 1 of the clonal complexes acquired that allele, spreading it to the other by horizontal transfer. The same allele also was found in the B:2b strains studied, indicating that 1 of the Pen(i) C:2b strains underwent a capsular switching event. A different mosaic penA allele was identified in the Pen(i) C:2a strains, which belonged to the ET37 cluster.