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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.

ABSTRACT: 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 ?-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 ?g/mL) and cefixime (1.2 ?g/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.

SUBMITTER: Tomberg J

PROVIDER: S-EPMC2939205 | biostudies-literature | 2010 Sep

REPOSITORIES: biostudies-literature

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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.

Tomberg Joshua J Unemo Magnus M Davies Christopher C Nicholas Robert A RA

Biochemistry 20100901 37

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 in ...[more]

PMID: 20704258

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Project description:BACKGROUND: The spread of Neisseria gonorrhoeae strains with mosaic penA alleles and reduced susceptibility to extended-spectrum cephalosporins is a major public health problem. While much work has been performed internationally, little is known about the genetics or molecular epidemiology of N. gonorrhoeae isolates with reduced susceptibility to extended-spectrum cephalosporins in the United States. The majority of N. gonorrhoeae infections are diagnosed without a live culture. Molecular tools capable of detecting markers of extended-spectrum cephalosporin resistance are needed. METHODS: Urethral N. gonorrhoeae isolates were collected from 684 men at public health clinics in California in 2011. Minimum inhibitory concentrations (MICs) to ceftriaxone, cefixime, cefpodoxime and azithromycin were determined by Etest and categorized according to the U.S. Centers for Disease Control 2010 alert value breakpoints. 684 isolates were screened for mosaic penA alleles using real-time PCR (RTPCR) and 59 reactive isolates were subjected to DNA sequencing of their penA alleles and Neisseria gonorrhoeae multi-antigen sequence typing (NG-MAST). To increase the specificity of the screening RTPCR in detecting isolates with alert value extended-spectrum cephalosporin MICs, the primers were modified to selectively amplify the mosaic XXXIV penA allele. RESULTS: Three mosaic penA alleles were detected including two previously described alleles (XXXIV, XXXVIII) and one novel allele (LA-A). Of the 29 isolates with an alert value extended-spectrum cephalosporin MIC, all possessed the mosaic XXXIV penA allele and 18 were sequence type 1407, an internationally successful strain associated with multi-drug resistance. The modified RTPCR detected the mosaic XXXIV penA allele in urethral isolates and urine specimens and displayed no amplification of the other penA alleles detected in this study. CONCLUSION: N. gonorrhoeae isolates with mosaic penA alleles and reduced susceptibility to extended-spectrum cephalosporins are currently circulating in California. Isolates with the same NG-MAST ST, penA allele and extended-spectrum cephalosporin MICs have caused treatment failures elsewhere. The RTPCR assay presented here may be useful for the detection of N. gonorrheoae isolates and clinical specimens with reduced extended-spectrum cephalosporin MICs in settings where antimicrobial susceptibility testing is unavailable. In an era of increasing antimicrobial resistance and decreasing culture capacity, molecular assays capable of detecting extended-spectrum cephalosporin of resistance are essential to public health.

Project description:The recent emergence of Neisseria gonorrhoeae strains with decreased susceptibility to extended-spectrum cephalosporins is a major concern globally. We sequenced the genome of an N. gonorrhoeae multiantigen sequence typing (NG-MAST) ST1407 isolate (SM-3) with decreased susceptibility and resistance to oral extended-spectrum cephalosporins. The isolate was cultured in 2008 in San Francisco, CA, and possessed mosaic penA allele XXXIV, which is associated with an international clone that possesses decreased susceptibility as well as resistance to oral extended-spectrum cephalosporins globally. The genome sequence of strain NCCP11945 was used as a scaffold, and our assembly resulted in 91 contigs covering 2,029,064 bp (91%; >150× coverage) of the genome. Numerous instances of suspected horizontal genetic transfer events with other Neisseria species were identified, and two genes, opa and txf, acquired from nongonococcal Neisseria species, were identified. Strains possessing mosaic penA alleles (n = 108) and nonmosaic penA alleles (n = 169) from the United States and Europe (15 countries), cultured in 2002 to 2009, were screened for the presence of these genes. The opa gene was detected in most (82%) penA mosaic-containing isolates (mainly from 2007 to 2009) but not in any penA nonmosaic isolates. The txf gene was found in all strains containing opa but also in several (18%) penA nonmosaic strains. Using opa and txf as genetic markers, we identified a strain that possesses mosaic penA allele XXXIV, but the majority of its genome is not genetically related to strain SM-3. This implies that penA mosaic allele XXXIV was transferred horizontally. Such isolates also possessed decreased susceptibility and resistance to oral extended-spectrum cephalosporins. These findings support that genetic screening for particular penA mosaic alleles can be a valuable method for tracking strains with decreased susceptibility as well as resistance to oral extended-spectrum cephalosporins worldwide and that screening using only NG-MAST may not be sufficient.

Dataset Information

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.

Publications

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.

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