1. Can we cripple an antibiotic resistance system expressed by beta-lactam resistant Neisseria gonorrhoeae? S Chen, K Connolly, SM Zughaier, AE Jerse, WM Shafer Emory University School of Medicine, Atlanta, GA and Uniformed Services University of the Health Sciences
Penicillin:
Support from:
R37 AI (WMS)
U19 AI (AEJ)

4. T Neisseria gonorrhoeae and Antimicrobial Resistance:
“Urgent Threat” and “Super-Bug” Pathogen
Unemo and Shafer 2014

5. Fighting Back: Current Efforts Against N. gonorrhoeae
New drug discovery
Clinical trials underway
Vaccine efforts as a
parallel track
- Adjunctive therapeutics
- New combinations of
existing antibiotics

6. Why study mechanisms of resistance?
May help in design in newer antimicrobials and vaccines
Provides insights as to development of compensatory mutations that reverse fitness defects yet keep resistance
Informs on likely development of resistance to new antibiotics
We concentrate on multidrug efflux pumps

7. Gonococcal Efflux Pumps and Antimicrobial Substrates

8. Importance of the Mtr Efflux System
Contributes to resistance to host-derived antimicrobials and is necessary for in vivo survival as assessed using an experimental female mouse model of lower genital tract infection (Jerse et al ).
Required for chromosomally-determined resistance of gonococci to beta-lactams and other antibiotics (e.g., macrolides) (Veal et al & Golparian et al. 2015).
Might inhibitors of the Mtr pump or dampening of pump expression allow for a return of penicillin or increase longevity of currently antibiotics? Also, could this strategy also enhance natural host clearance of gonococci independent of antibiotics?

9. penB PmtrR FA6140 penA4 HO41 penA41 ponA Shafer, Folster &
Ceftriaxone
Antibiotic
Efflux
Outer
Membrane
PorB1B
Overexpression
Activates
PorB1B
Mutations
Decreased
Influx
penB
PmtrR
MtrCDE
Efflux
Pump
Decreased
Inactivation
FA6140
penA4
HO41
penA41
Shafer, Folster &
Nicholas 2009.
This slide summarizes in graphic form the various known resistance determinants and how resistance works. The structures here are either from GC—PBP 2—or are closely related to their GC homolgoues.
Remodeled
PBP 2
ponA
PBP 1
Cytoplasmic
Membrane

10. MtrR Regulation of the mtrCDE Efflux Pump-Encoding Operon
mtrF
mtrR
mtrC
mtrD
mtrE
250 bp GC P
MtrR
-35
-10
ATTATAAAAAAGACTTTTTATCCGTGCAATCGTGTATGTATAATGAAACCCATA
TAATATTTTTTCTGAAAAATAGGCACGTTAGCACATACATATTACTTTGGGTAT
-10
-35
13 bp IR and site of single bp deletion (T:A bp) found in 15-20% of clinical isolates that are antibiotic resistant

11. Regulation of mtrCDE ExpressionWT
bp∆
∆mtrR
mtrR::km
Hagman & Shafer J. Bacteriol.

12. H041: Japan, 2009
Ohnishi et al (2011)

13. Loss of MtrCDE Reverses Clinical Resistance
Outer
membrane
Inner
Periplasm
MIC X
Wendy Veal
2002
Magnus Unemo Ph.D. and lab
Orebro, Sweden

14. Expression of wild-type mtrR in HO41 reverses penicillin resistance
HTH
MS11: mtr120 (-10)
Inverted
Repeat
-35mtrC
-10mtrC
TATAAT
CAT....ATTATAAAAAAGACTTTTTATCCGTGCAATCGTGTATGTATATT....TATAAC....ATG
-10mtrR
-35mtrR
mtrR
mtrC
HO41
Shao-chun Chen Ph.D. expressed wild-type mtrR from a second site on the HO41 chromosome under an IPTG-inducible promoter: monitored resistance to beta-lactams as well as levels of MtrR and MtrE.

15. SC6 is HO41 mtrR+ anti-MtrR anti-MtrE MtrR decreases MtrE Levels kDa
FA19
SC6
IPTG-
IPTG+
HO41
FA19
SC6
IPTG-
IPTG+
kDa
204
130 92 38
MtrE 31
MtrR 18 7
SC6 is HO41 mtrR+

16. MIC
µg/ml

17. Use of CRO as “Proof of Principle”
Theoretical
30.5
16.4
28.1
20.1
SC6 = H041 mtrR+ rpsL
SC7= H041 rpsL
Kristie Connolly, Ph.D., USUHS Ann Eakin, Ph.D., NIAID
PK modeling was used to predict dosing regimen that will give plasma levels above the MIC of each strain for > 24 hrs.
Two regimens were predicted to be
effective against SC6 but not SC7
- 60 mg/kg, TID
- 120 mg/kg BID
Ceftriaxone; CRO

18. Sub-Inhibitory Doses of Cro Completely Clear H041 from Mice
within 48 Hours when H041 Expresses MtrR
n.s.
p 0.02
p <
CRO every 8 hours
CRO every 12 hours

19. Conclusions And The Future
Gonococci have a remarkable ability to develop resistance to antibiotics used in therapy and active surveillance systems are essential to detect changes in susceptibility
The MtrCDE drug efflux contributes significantly to such resistance
Transcriptional control systems modulate levels of efflux pump gene expressions and, as a consequence, levels of antibiotic resistance
Mutations that increase efflux pump gene expression can significantly impact clinical efficacy of antibiotics
Dampening efflux pump gene expression might allow for return of an old antibiotic (Pen) or allow for continued use of a current antibiotic (Cro)