Molecular mechanisms of antibiotic resistance in Neisseria gonorrhoeae

Transcription

1 medicaldaily.com ppcorn.com Molecular mechanisms of antibiotic resistance in Neisseria gonorrhoeae Robert Nicholas University of North Carolina at Chapel Hill cdc.gov

2 Timeline of Antibiotic Resistance in N. gonorrhoeae 1987 Penicillin Not Recommended 2012 Cefixime Not Recommended 1945 Sulfonamide Resistance 1958 Penicillin Resistance 1977 Erythromycin Not Recommended 1977 Widespread Sulfonamide Resistance 1987 Widespread Spectinomycin Resistance 1987 Tetracycline Not Recommended 2009 Ceftriaxone Resistance 2007 Fluoroquinolones Not Recommended 1937 Sulfonamides 1949 Streptomycin/ Chloramphenicol 1961 Spectinomycin 1983 Azithromycin/ Cefixime 1991 Fluoroquinolones 1943 Penicillin 1952 Erythromycin 1962 Tetracycline 1989 Ceftriaxone

3 Modes of resistance in Neisseria gonorrhoeae Plasmid-mediated resistance Penicillin, Tetracycline Resistance occurs due to expression of a modifying protein or ribosome-protection protein TEM-1-like β-lactamase for Pen R TetM ribosomal-binding protein for Tet R β-lactamase does not hydrolyze ceftriaxone, so not important in cephalosporin resistance However, 1 aa change could convert bla gene into extendedspectrum β-lactamase Chromosomally mediated resistance Penicillin, Ceftriaxone, Tetracycline, Ciprofloxacin, Azithromycin, Spectinomycin Due to acquisition of chromosomal mutations by homologous recombination The most common form of resistance to penicillin, ciprofloxacin, tetracycline, and azithromycin All of the strains with decreased susceptibility to ceftriaxone are chromosomally mediated

4 pona pona pona Stepwise Transfer of Antibiotic Resistance Genes in Neisseria gonorrhoeae pona

5 Ceftriaxone Antibiotic Efflux Outer Membrane PenB PorB 1B Overexpression of MtrCDE Efflux Pump (mtr) Overexpression Activates PorB1B Mutations Decreased Influx Decreased Inactivation PBP 1 Mosaic WT PBP 2 (pena) Cytoplasmic Membrane

6 Genetics of Resistance-MICs of Stepwise Transformants 5X Resistant Susceptible 6X 2X 6X Overall increase: 400-fold Stepwise Resistance: Each step is a relatively small increase in resistance, but when multiplied overall, leads to a large increase in MIC FA19 = Susceptible wild-type strain FA6140 = Pen R clinical isolate

7 Emergence of Ceph I and Ceph R strains Strain MIC (µg/ml) Ceftriaxone Cefixime Date Isolated FA / H F Breakpoint for resistance is for both antibiotics

8 pena-mediated Resistance in the Gonococci The type of pena allele determines whether the strain is Pen R, Ceph I, or Ceph R The mtrr and penb determinants contribute additional resistance to β-lactam antibiotics and provide a general permeability barrier for antibiotics Ceph I and Ceph R strains likely have emerged by a single transformation event of a mosaic pena allele into existing Ceph S /Pen R strains

9 Clinical Ceph I Strains Harboring a Mosaic pena Allele Also Have mtrr, penb, and pona Alleles Lindberg et al Antimicrob Agents Chemother 51:2117

10 Mosaic pena alleles in Ceph I /Ceph R strains Wild Type N. gonorrhoeae N. meningiditis N. flavescens N. cinerea Mosaic pena allele Ito et al. (2005) AAC 49(1):

11 PBP2 mutations in β-lactam-resistant strains of N. gonorrhoeae Active site 13 new or different mutations compared to 35/ MIC PEN =4.0 µg/ml 5 mutations in PBP2 compared to FA19 35/02 MIC CFX =0.38 µg/ml 58 mutations in PBP2 compared to FA19 H041 MIC CFX =8 µg/ml 61 mutations in PBP2 compared to FA19

12 Of the 61 differences between PBP2 from FA19 and H041, what is the minimal set of mutations that increase resistance to the same level when incorporated into wildtype PBP2?

13 pena-mediated Resistance in the Gonococci The type of pena allele determines whether the strain is Ceph R The mtrr and penb determinants contribute additional resistance to β-lactam antibiotics and provide a general permeability barrier for antibiotics Ceph I and Ceph R strains likely have evolved by a single transformation event of a mosaic pena allele into existing Ceph S /Pen R strains The mosaic pena allele in Ceph I strains can accrue additional mutations, leading to Ceph R strains In some instances, a single additional mutation in a mosaic pena allele can lead to Ceph R Ala-501 mutations in a Ceph I pena allele generate a Ceph R strain

14 PBP2 F89 is essentially PBP2 35/02 with an A501P mutation Ala-501 Mutations in a Mosaic PBP2 Background FA19 Modeled Cefuroxime Breakpoint > 0.25 µg/ml FA6140 A501T

15 Meropenem α4 α2 α5 α8 α11 Thr501 β3-β4 loop β4 β3 β5

16 Contribution of non-pena resistance determinants on the MICs of different β-lactam antibiotics 50X 20X 4X 12X 20X 100X 600X 400X 400X Zhao et al. (2009) AAC, 53(9):

17 MtrCDE Efflux Pump Outer Membrane Cytoplasmic Membrane Member of the Resistance- Nodulation-Division (RND) Superfamily of Efflux Pumps The major efflux pump in N. gonorrhoeae involved in resistance Resistance occurs not by coding mutations but by promoter mutations that overexpress the pump Confers resistance to a wide range of compounds Detergents (e.g. Triton X-100 and spermicides) Hydrophobic agents (Crystal Violet, Erythromycin) Antibiotics Host Antimicrobial Peptides Shafer Lab, Emory University

18 Knockout of the MtrCDE Efflux Pump Markedly Decreases the MICs of β-lactam Antibiotics Veal et al. (2002) J Bact 184:5619; Golparian et al. (2014) AAC 58:3556

19 penb PorB1b

20 PenB mutations map to AA 120,121 of PorB 1b PIB LNSPLKNTGANVNAWESGKFTGNVLEISGMAQREHRY G120D/A121D G120K/A121X Loop 3 Olesky et al AAC; Olesky et al J Bacteriol

21 PenB mutations map to Loop 3 of PIB Loop 3 N. meningitidis porin PorB PDB 3A2R; Tanabe et al PNAS

22 Genetics of Resistance-penB is silent in the absence of mtrr Olesky et al J Bacteriol

23 Permeation of β-lactam antibiotics in whole cells Cross outer membranerate determining step nitrocefin Hydrolyzed by β-lactamase Increase in OD 480

24 Structural model of PorB1b

25

26 Conclusions Three resistance determinants (four including pona) encode altered forms of endogenous genes These determinants work in concert to increase the MIC above the breakpoint of the antibiotics The mtr and penb determinants work in concert to decrease the influx of antibiotics into the bacterium All future antibiotics will have to overcome this permeability barrier We are nearing a time in which we will have untreatable gonococcal infections

27 Future goals Identify new antibiotics that are active against antibioticresistant strains of N. gonorrhoeae My colleague Pei Zhou will talk about our efforts to develop an inhibitor of lipid A biosynthesis What additional mutations are there that compensate for the loss of fitness caused by the mosaic pena allele? We have identified at least two compensatory mutations that arise during competitive infections in the mouse model What is the mechanism by which mosaic PBP2 variants exclude β-lactam antibiotics (which are substrate analogs), but still retain essential PG transpeptidase activity?

28 UNC Acknowledgements USUHS Orebrö Univ Josh Tomberg Kate Newns Leah Vincent Ann Jerse Magnus Unemo UNC Emory MUSC Sam Kerr Yang Tan Alex Duncan Bill Shafer Chris Davies Funding: NIAID, NIGMS, AC STI CRC U19