Global challenge of -resistant

Sabine Pereyre

USC EA 3671 Mycoplasmal and chlamydial infections in humans INRA - University of Bordeaux -Bordeaux University Hospital

National French Reference Center for bacterial IST

ECCMID, April 24th 2018 ESCMID eLibrary © by author Disclosures

 Travel grants from – Diagenode, Belgium – SpeeDx, Australia – Hologic, USA

 My laboratory has received remuneration for contract work from – bioMérieux, France – Meridian Bioscience, Europe – Diagenode, Belgium – SpeeDx, Australia – Hologic, USA

ESCMID eLibrary 2 © by author M. genitalium

 STI agent responsible for – Non gonococcal urethritis, proctitis – Cervicitis, PID, preterm birth, spontaneous abortion – Carriage frequently asymptomatic

 Prevalence – Community-based population 1–3% – STI center population (high risk sexual behaviour) 4 – 38%

 Diagnostic – only NAAT tests – No serology – ≈ no culture  antimicrobial susceptibility testing hardly achievable ESCMID eLibrary 3 © by author M. genitalium and

 Intrinsic resistance to ATB targeting the cell wall (-lactams, fosfomycin, glycopeptides) and (mutation in rpoB gene)

 In vitro active antibiotics – and related antibiotics (MLSK) – Fluoroquinolones –

ESCMID eLibrary 4 © by author M. genitalium and tetracyclines

 Relative potency in vitro

MIC ranges (µg/ml)

Antibiotics M. genitalium M. hominis Ureaplasma spp.

Tetracyclines**

Doxycycline ≤0.060.01 -–0.31 0.1-2 0.02-1

Minocycline ≤0.01-0.2 0.03-1 0.06-1

MLS group

 BUT,Eryth rolowmyci neradication rate≤0.01 32->1 000 0.02-16

–RoxiMicrobiologicalthromycin cure of<0 doxycycline.01 : between>16 30 and 40 %0.1-2 – Not a first-line treatment ≤0.01-0.06 16->256 ≤0.004-2

Azithromycin ≤0.01-0.03 4->64 0.06-4

Pereyre, AntimicrobJosa. mChemotherycin . Vaccines, 3rd edition0.01, 2016;-0.02 Jensen Antimicrob0.05Agnets-2 Chemother 2014;0.03 -Jensen4 J Eur Acad Dermatol Venereol. 2016 ESCMID eLibrary 5 0.2-1 ≤0.008-2 0.2-64

Pristinamycin ≤0.01-0.02 0.1-0.5 0.1-1 Quinup risti n/ D alfo prist in ©0.05 by 0.03author-2 0.05-0.5

Telithromycin ≤0.015 2-32 ≤0.015-0.25

Solithromycin ≤0.000000063- 0.002-0.008 0.002-0.063 0.000125

Fluoroquinolones**

Ciprofloxacin 2 0.1-4 0.1-16

Ofloxacin 1-2 0.1-4 0.2-4

Levofloxacin 0.5-1 0.1-2 0.2-2

Moxifloxacin 0.03-0.06 0.06-0.125 0.125-1

Other agents

Chloramphenicol 0.5-25 4225 0.4-8

Gentamicin ND 2-16 0.1-13

M. genitalium and MLSK  Low MICs

Antibiotics M. genitalium M. hominis Ureaplasma spp.

MLSK group

Erythromycin ≤0.01 32->1 000 0.02-16

Roxithromycin <0.01 >16 0.1-2

Clarithromycin ≤0.01-0.06 16->256 ≤0.004-2

Azithromycin ≤0.01-0.03 4->64 0.06-4

Josamycin 0.01-0.02 0.05-2 0.03-4

Clindamycin 0.2-1 ≤0.008-2 0.2-64

Pristinamycin ≤0.01-0.02 0.1-0.5 0.1-1

Quinupristin/ 0.05 0.03-2 0.05-0.5

Telithromycin ≤0.015 2-32 ≤0.015-0.25

Solithromycin ≤0.000000063- 0.002-0.008 0.002-0.063 0.000125

ESCMID eLibraryPereyre, Antimicrob. Chemother. Vaccines, 3rd edition , 2016 6 © by author Acquired resistance to macrolides

• Point mutations in domain V of 23S rRNA - Single operon encoding 16S and 23S rRNA - A2058G/C/T, A2059G/C/T, A2062G/T (E. coli numbering)  high level resistance

2062

23S rRNA Domain V

ESCMID eLibraryPeptidyl transferase loop © by author  All M. genitalium-positive test should be followed up with an assay capable of detecting resistance- associated mutations

ESCMID eLibrary 8 © by author Detection of macrolide resistance-associated mutations

 Amplification and sequencing of 23S rRNA – Time-consuming, not adapted to routine

 Published in-house methods – FRET real-time PCR (Touati et al. J. Clin. Microbiol. 2014) – HRM (High Resolution Melting curve analysis) (Twin et al. PloS One 2012) – PCR and pyrosequencing (Salado-Rasmussen et al. Clin. Infect. Dis. 2014) – Taqman PCR using forward primers complementary to 23S mutations (Wold et al. J. Eur. Acas. Dermatol. Venereol. 2015), only if Ct<32. – Taqman PCR using 3 probes and subsequent endpoint fluorescence analysis (Kristiansen et al. J. Clin. Microbiol. 2016) – Single probe PCR and melting curve analysis (Gossé et al. J. Clin. Microbiol. 2016)

 Commercial kits – ResistancePlusTM MG kit (SpeeDx, Australia) : multiplex real-time PCR (Le Roy, J. Clin. Microbiol. 2017) – S-DiaMGRes kit (Diagenode, Belgium) : multiplex real-time PCR Detection of Mg and 5 mutations – Others expected ESCMID eLibrary 9 © by author Prevalence of macrolide resistance in M. genitalium10

100%(26/26)

56-61% 18% 4.6-6,7% 58% 41% 57% 53%

48% 35% 29%

No data 83% in MSM <10% 43-63% 10%-20% 9,8% 20%-40% 40-60% >60% 72-77%

Anagrius, PloS one 2013; Tagg, J. Clin. Microbiol. 2013; Pond, Clin. Inf. Dis. 2014; Salado-Rasmussen, Clin. Inf. Dis, 2014; Kikuchi, J. Antimicrob. Chemother. 2014; Hay, Sex. Transm. Dis. 2015; Gushin, BMC Infect. Dis. 2015; Nijhuis, J. Antimicrob. Chemother. 2015; Gesink, Can. Fam. Physician, 2016; Getman, J. Clin. Microbiol. 2016; Gossé, J. Clin. Microbiol. 2016; Shipitsina, Plos One, 2017; Basu, J. Clin. Microbiol. 2017; Tabrizi, J. Clin. Microbiol. 2017; Barbera, Sex. Transm. Dis. 2017; Dumke, Diagn Microbiol infect Dis, 2016; Coorevits, J. Glob. Antimicrob. Resist. 2017; Anderson, J. Clin.ESCMIDMicrobiol. 2017; Unemo, Clin. Microbiol. Infect.2017. eLibrary © by author Treatment studies : azithromycin (AZM) 1g

• Meta-analysis on the efficacy of AZM 1g for Mg treatment (Lau et al Clin. Infect. Dis. 2015)

100% 90% 83.5% 80% 67% 70% 60%  The efficacy of azithromycin 1 g has 50% 40% decreased 30% 20% 10% 0%

Pooled microbial cure rate (%) rate cure microbial Pooled Before 2009 Since 2009

• Selection of resistant mutants during AZM 1g treatment

• AZM 1g single dose is no more the 1st line treatment  Therapeutic failure if patient infected with a mutated strain

Björnelius Sex Transm Infect 2008; Manhart Clin Infect Dis 2013; Mena Clin Infect Dis 2009; Schwebke CID 2011, Sena J Infect Dis 2012; Stamm Sex Transm InfectESCMID 2007; Lau Clin Infect Dis 2015; Horner Clin Infect Dis 2015;eLibrary Jensen BMC Infect Dis 2015 © by author IMPORTANT MESSAGE : Rapid increasing prevalence of macrolide resistance most likely due to widespread use of azithromycin 1g single dose

Uncomplicated M. genitalium infection:

- In the absence of macrolide resistance-associated mutations Azithromycin 500 mg (day 1), then 250 mg (days 2-5)

 85% effective  Associated with lower risk of inducing AZM R (conflicting evidence)  Patients failing azithromycin 1g single dose cannot be treated successfully with extended 1.5 g AZM

12 Anagrius PLoS One 2013, Bjornelius Sex Transm Infect 2008, Falk J Antimicrob Chemother 2015, Gundevia STI 2015, Read, Clin Inf Dis 2017; Horner Sex ESCMIDTrans Ingfect 2018; Jernberg Sex Transm Infect 2008, JenseneLibrary Clin Infect 2009 © by author Uncomplicated M. genitalium infection:

- Macrolide-resistant M. genitalium infection - Second line treatment for persistent M. genitalium infection Moxifloxacin 400 mg/d - 7 to 10 days

Complicated M. genitalium infection (PID, epididymitis) Moxifloxacin: 400 mg - 14 days ESCMID eLibrary 13 © by author M. genitalium and fluoroquinolones

 Only moxifloxacin and sitafloxacin have low MICs

Antibiotics M. genitalium M. hominis Ureaplasma spp.

Fluoroquinolones

Ciprofloxacin 2 0.1-4 0.1-16

Ofloxacin 1-2 0.1-4 0.2-4

Levofloxacin 0.5-1 0.1-2 0.2-2

Moxifloxacin 0.03-0.06 0.06-0.125 0.125-1

Sitafloxacin (Japan) 0.125

Deguchi, J Infect Chemother 2014; Pereyre, Antimicrob. Chemother. Vaccines, 3rd edition, 2016 ESCMID eLibrary 14 © by author Fluoroquinolone resistance in M. genitalium

• Mutations in the bacterial target genes of fluoroquinolones - Most frequent mutations in parC (Topoisomerase IV) Primarily Ser83 and Asp87 - Rare mutations in gyrA (DNA gyrase)

• Many mutations have not been evaluated by in vitro MIC determination

• Molecular detection only: amplification and sequencing of target genes ESCMID eLibrary © by author Prevalence of fluoroquinolone resistance-associated mutations16

10.2% 6.2% 4.1% 20% 4.5% 10% 6% 13.6% 8% 31- 47%

No data <10% 12-19% 10%-20% 20%-40% 40-60% >60% 23%

Bissessor Clin Infect Dis 2015; Deguchi, Clin Infect Dis 2016; Dumke, DMID 2016; Kikuchi J Antimicrob Chemother 2014; Le Roy Emerg Infect Dis 2016; Pond Clin Infect Dis 2014; Shipitsina PLoS one 2017; Couldwell Int J STD and AIDS 2013; Gesink Can family Physian 2016; Tagg J Clin Microbiol 2013; Murray Emerg Infec Dis 2017; Barbera ESCMIDSex Transm infect 2017; Anderson, J Clin Microbiol 2017, UnemoeLibrary, Clin Microbiol Infect 2017. © by author M. genitalium fluoroquinolone treatment studies

 Meta-analysis on the efficacy of moxifloxacin for M. genitalium treatment – 17 studies, 252 patients

100 % 100 89 % 90 80 70 60 50 40 30 20 10 0

Before 2010 Since 2010 Pooled microbial cure rate (%) rate cure microbial Pooled

ESCMID eLibraryYi et al Int J STD AIDS 2017 © by author Dual class resistance

 Prevalence – Japan : 17-30% – Australia : 8.6 - 9.8% – Denmark : 4.2%

 Mainly due to successive treatment failures of macrolides then fluoroquinolones

 Enormous clinical implications – No other highly effective antibiotics available  What can we use as third line therapy?

Murray Emerg Infect Dis 2017, Kikuchi J Antimicrobiol Chemother 2014; Le Roy Emerg Infect Dis 2016; Degushi Clin Infect Dis 2016; Kikuchi J Antimicrobiol ESCMIDChemother 2014 eLibrary 18 © by author • Third-line treatment for persistent MG infection after AZM and MXF Doxycycline 100 mg x2 daily for 14 days Pristinamycin 1 g x4 daily for 10 days

ESCMID eLibrary © by author Pristinamycin

 MICs

– AZM-resistant strains have higher MIC90 (0.5 mg/l) than AZM-susceptible strains (MIC90=0.125 mg/l) – but MICs close to the breakpoint : 1 mg/l (French society for microbiology, not dedicated to mycoplasmas)  Maximum dose of 1g 4 times/day for 10 days was retained

 Evaluation in patients after azithromycin failure (2012-2016, Australia) – 114 persons : infection cured in 75%

 Option during pregnancy and in other situations where fluoroquinolones have failed or are contraindicated  Not registered in many countries

Renaudin et al. Antimicrob Agents Chemother. 1992; Bissessor et al. Clin Infect Dis 2015; Read et al. Emerg Inf Dis 2018. ESCMID eLibrary 20 © by author Therapeutic options

 Evaluating old antimicrobials

–  MICs around four-fold lower than that of doxycycline  Success of extended regimen 100 mg X2 for 14 days on 2 patients

– (-related AB)  1 clinical success after doxycycline, azithromycin and pristinamycin failure : 2g IM daily for 7 days  IM administration painful, limited availability, optimal dosage not established, monotherapy is not recommended

ESCMIDDegushi JeLibrary Infect Chemother 2017; Falk J Antimicrobiol Chemother 2016 21 © by author New therapeutic options

 New antimicrobials

– Sitafloxacin (fluoroquinolone)  Susceptible MICs in some moxifloxacin-resistant strains  Registered in Japan only

– Solithromycin (fluoroketolide)  In development for CA-pneumonia but potential hepatotoxicity (no FDA approval)  Cross resistance in strains harboring the A2058G mutation

– Gepotidacin (topoisomerase II inhibitor that inhibits DNA replication. Mechanism and target different from FQ)

 MIC90=0.032 (4-fold

Ito J Infect Chemother 2012; Hamasuna Antimicrob Agents Chemother 2009; Degushi J Infect Chemother 2015; Jensen Antimicrob Agents Chemother 2014; Waites, STI and VIH world congress, Rio, 2017 ESCMID eLibrary 22 © by author New therapeutic options

 New antimicrobials

– Zoliflodacin (spiropyrimidinetrione)  Arrests the cleaved gyrase complex -> inhibit DNA biosynthesis (accumulation of double- strand cleavages) Mode of action different from fluoroquinolone. No cross resistance  Potent on 12/13 AZM-R and Moxi-R strains (MICs ≤1 mg/l)  Promising candidate. Active against both N. gonorrhoeae and C. trachomatis

ESCMIDWaites, Antimicrob agents Chemother. 2015; Gouveia eLibrary, J. Antimicrob. Chemother. 2018 23 © by author New therapeutic options

 New antimicrobials

– (semi-synthetic )  Inhibition of protein synthesis (binding to the peptidyltransferase center of the 50S ribosomal subunit).  Phase 3 trials for treatment of community-acquired pneumonia  Active against macrolide- and moxifloxacin-resistant Mg

MIC90=0.063 mg/l (vs azithromycin >8 mg/l)  Promising candidate. Active against C. trachomatis and susceptible and resistant N. gonorrhoeae  Clinical trials are urgently needed

ESCMIDBradshaw et al. J Inf Dis 2017; Paukner et al. Antimicrob eLibraryAgents Chemother 2018 24 © by author New therapeutic strategy

 Sequential treatment Syndromic 1. Doxycycline 100 mg twice daily, 7 days treatment with

 Mean fall bacterial load : 2.9 log10 doxycycline to reduce Mg load 2. Test for macrolide resistance  If macrolide-susceptible Mg : azithromycin 2.5 g 5 days  If macrolide-resistant Mg : sitafloxacin 100 mg twice daily, 7 days Targeted therapy based on the – Microbiologic cure resistance profile  Macrolide susceptible : 95.7%  Macrolide resistant : 92.2 % Sex. Transm. Infect. 2018

ESCMIDRead et al, STI and HIV world congress eLibrary, Rio, July 2017; Horner, Sex Transm Infect 2018. © by author Conclusion

 Threat of untreatable M. genitalium – High prevalence of macrolide resistance worldwide – Alarming increasing fluoroquinolone and dual resistance – Rare other registered active antibiotics

 Superbug? New XDR bacteria? – Need to detect Mg and macrolide resistance at the same time – Need for new antimicrobial compounds and trials  Lefamulin? Zoliflodacin? – Need for trials of combinations of registered drugs

ESCMID eLibrary 26 © by author Bordeaux, France and its surroundings

ESCMID eLibrary27 © by author ESCMID eLibrary 28 © by author Risk factors for treatment failure

 Organism load – High organism loads are more likely to develop treatment failure and post-treatment resistance Walker Clin Infect Dis 2013; Guschin BMC Infect Dis 2015; Read Clin Infect Dis 2017

Walker Clin Infect Dis 2013

 Rectal infection – Macrolide resistance is more common in rectal infections

 100% macrolide-R in male rectal infection in an STI clinic in Australia (Tabrizi J Clin Microbiol 2017)

 80% macrolide-R in male rectal infection in HIV+ male in the USA (Dionne-Odom, Clin Infect Dis 2017) ESCMID eLibrary 29 © by author