# Fluoroquinolone-resistant mycoplasma genitalium, southwestern France

Chloé Le Roy, Nadège Hénin, Sabine Pereyre, Cecile Bebear

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Chloé Le Roy, Nadège Hénin, Sabine Pereyre, Cecile Bebear. Fluoroquinolone-resistant mycoplasma genitalium, southwestern France. Emerging Infectious Diseases, 2016, 22 (9), pp.1677-1679. 10.3201/eid2209.160446. hal-02640928

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## References

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9. Woodford N, Wareham DW, Guerra B, Teale C. Carbapenemase-producing *Enterobacteriaceae* and non-*Enterobacteriaceae* from animals and the environment: an emerging public health risk of our own making? J Antimicrob Chemother. 2014;69:287-91. http://dx.doi.org/10.1093/jac/dkt392

Address for correspondence: Nicol Janecko, Public Health Agency of Canada, 160 Research Ln, Ste 103, Guelph, ON N1G 5B2, Canada; email: nicol.janecko@phac-aspc.gc.ca

# Fluoroquinolone-Resistant *Mycoplasma genitalium*, Southwestern France

Chloé Le Roy, Nadège Hénin, Sabine Pereyre, Cécile Bébéar

Author affiliations: University of Bordeaux, Bordeaux, France (C. Le Roy, N. Hénin, S. Pereyre, C. Bébéar); Institut National de la Recherche Agronomique, Villenave d'Ornon, France (C. Le Roy, N. Hénin, S. Pereyre, C. Bébéar); Bordeaux University Hospital, Bordeaux (S. Pereyre, C. Bébéar)

DOI: http://dx.doi.org/10.3201/eid2209.160446

**To the Editor:** *Mycoplasma genitalium* is a sexually transmitted bacterium involved in nongonococcal urethritis in men and associated with cervicitis and pelvic inflammatory disease in women. Azithromycin regimens have been commonly used as a first-line treatment of these conditions, but a recent increase in *M. genitalium* with azithromycin resistance has been described worldwide; in 2012, resistance in the organism was detected in France at a prevalence of 14% (1). In case of azithromycin failure, moxifloxacin is a second-line treatment; however, moxifloxacin treatment failures have also been reported and are associated with mutations in ParC or GyrA (2).

Prevalence of *M. genitalium* infection was ≈4% in 2013–2014 at Bordeaux University Hospital (Bordeaux, France). To evaluate the prevalence of fluoroquinolone resistance in *M. genitalium* in southwestern France, we examined the quinolone resistance-determining regions (QRDRs) of the *gyrA* and *parC* genes in *M. genitalium*-positive specimens obtained during 2013–2014. We retrospectively collected (from the Department of Bacteriology, Bordeaux University Hospital) 369 *M. genitalium*-positive urogenital specimens and DNA extracts from 344 patients. The *gyrA* and *parC* QRDRs were amplified and sequenced as described (3,4). We also assayed macrolide resistance-associated mutations using real-time PCR and melting curve analysis (1). To determine resistant genotypes A2058G or A2059G, we sequenced PCR products. Nucleotide positions in the 23S rRNA and amino acid positions in GyrA and ParC were identified according to *Escherichia coli* numbering.

From the 344 *M. genitalium*-positive patients, 200 specimens underwent complete analysis for the *gyrA* and *parC* genes, specimens from 221 patients were investigated for macrolide resistance, and specimens from 168 patients were examined for 23S rRNA, *gyrA*, and *parC* genes. Unsuccessful amplifications could be attributed to low bacterial loads of *M. genitalium* or to the degradation of frozen DNA during storage. Strains from 12/200 patients (6%; 95% CI 3.47%-10.19%) had QRDR mutations, with rates of 6.4% (6/93) for 2013 and 5.6% (6/107) for 2014. This prevalence is in accordance with the 4.5% moxifloxacin resistance described in the United Kingdom in 2011 (3) but lower than prevalences found in small numbers of strains in Japan and Australia during 2006–2014, which ranged from 10% to 47% (4–8).

Strains from 11 patients (patient nos. 6, 8, 12, 20, 23, 28–31, 46, 47) harbored alterations in the ParC QRDR (Table) at positions 80 (Ser→Asn or Ile) or 84 (Asp-84→Tyr or Asn). These mutations have been previously described for *M. genitalium* (4,6–8). In addition, 1 new amino acid alteration, Asn-96→Ser (strain from patient 20), was found in ParC. We detected a GyrA modification with the Ala-93→Thr transition in a strain from 1 patient (patient 3). These 2 amino acid changes were not
<table><caption>**Table.** Fluoroquinolone resistance-associated amino acid changes in GyrA and ParC and macrolide resistance-associated mutations in the 23S rRNA gene in *Mycoplasma genitalium*, France, 2013–2014*</caption><thead><tr><th rowspan="2">Patient no.</th><th rowspan="2">Date of collection</th><th rowspan="2">Patient sex</th><th rowspan="2">Specimen type</th><th rowspan="2">Mutation in the 23S rRNA gene</th><th colspan="2">Amino acid changes in</th></tr><tr><th>GyrA</th><th>ParC</th></tr></thead><tbody><tr><td>1</td><td>2013 Jan 9</td><td>F</td><td>Vaginal swab</td><td>A2058G/A2059G</td><td>NA</td><td>NA</td></tr><tr><td>2</td><td>2013 Feb 1</td><td>F</td><td>Vaginal swab</td><td>A2058G</td><td>–</td><td>–</td></tr><tr><td>3</td><td>2013 Feb 1</td><td>M</td><td>Urethral swab</td><td>A2058G/A2059G</td><td>Ala-93→Thr</td><td>–</td></tr><tr><td>4</td><td>2013 Feb 18</td><td>M</td><td>Urethral swab</td><td>A2058G/A2059G</td><td>–</td><td>–</td></tr><tr><td>5</td><td>2013 Feb 20</td><td>M</td><td>Urine</td><td>A2058G/A2059G</td><td>–</td><td>–</td></tr><tr><td>6</td><td>2013 Mar 11</td><td>M</td><td>Urine</td><td>A2058G/A2059G</td><td>–</td><td>Asp-84→Tyr</td></tr><tr><td>7</td><td>2013 Mar 28</td><td>F</td><td>Vaginal swab</td><td>A2058G/A2059G</td><td>NA</td><td>–</td></tr><tr><td>8</td><td>2013 Apr 3</td><td>M</td><td>Urine</td><td>Wild-type</td><td>–</td><td>Asp-84→Tyr</td></tr><tr><td>9</td><td>2013 Apr 8</td><td>F</td><td>Vaginal swab</td><td>A2058G/A2059G</td><td>–</td><td>–</td></tr><tr><td>10</td><td>2013 Apr 11</td><td>F</td><td>Vaginal swab</td><td>A2058G/A2059G</td><td>NA</td><td>NA</td></tr><tr><td>11</td><td>2013 Apr 16</td><td>F</td><td>Vaginal swab</td><td>A2058G</td><td>–</td><td>NA</td></tr><tr><td>12</td><td>2013 Apr 19</td><td>F</td><td>Vaginal swab</td><td>Wild-type</td><td>–</td><td>Ser-80→Asn</td></tr><tr><td>13</td><td>2013 May 21</td><td>M</td><td>Urethral swab</td><td>A2059G</td><td>–</td><td>–</td></tr><tr><td>14</td><td>2013 Jul 4</td><td>M</td><td>Urine</td><td>A2059G</td><td>–</td><td>–</td></tr><tr><td>15</td><td>2013 Jul 4</td><td>M</td><td>Urethral swab</td><td>A2059G</td><td>–</td><td>–</td></tr><tr><td>16</td><td>2013 Jul 19</td><td>M</td><td>Urine</td><td>A2059G</td><td>–</td><td>–</td></tr><tr><td>17</td><td>2013 Jul 19</td><td>M</td><td>Urine</td><td>A2058G</td><td>–</td><td>–</td></tr><tr><td>18</td><td>2013 Aug 9</td><td>F</td><td>Vaginal swab</td><td>A2059G</td><td>NA</td><td>–</td></tr><tr><td>19</td><td>2013 Sep 30</td><td>F</td><td>Vaginal swab</td><td>A2058G/A2059G</td><td>–</td><td>–</td></tr><tr><td>20</td><td>2013 Sep 30</td><td>F</td><td>Vaginal swab</td><td>A2058G/A2059G</td><td>–</td><td>–</td></tr><tr><td>21</td><td>2013 Oct 29</td><td>F</td><td>Vaginal swab</td><td>Wild-type</td><td>–</td><td>Asn-96→Ser</td></tr><tr><td>22</td><td>2013 Nov 22</td><td>F</td><td>Vaginal swab</td><td>A2058G/A2059G</td><td>NA</td><td>NA</td></tr><tr><td>23</td><td>2013 Nov 29</td><td>F</td><td>Vaginal swab</td><td>A2062T</td><td>–</td><td>–</td></tr><tr><td>24</td><td>2013 Dec 1</td><td>F</td><td>Vaginal swab</td><td>Wild-type</td><td>–</td><td>Asp-84→Tyr</td></tr><tr><td>25</td><td>2014 Jan 21</td><td>F</td><td>Vaginal swab</td><td>A2059G</td><td>–</td><td>–</td></tr><tr><td>26</td><td>2014 Jan 29</td><td>F</td><td>Vaginal swab</td><td>A2059G</td><td>–</td><td>–</td></tr><tr><td>27</td><td>2014 Jan 30</td><td>F</td><td>Vaginal swab</td><td>A2058G/A2059G</td><td>NA</td><td>–</td></tr><tr><td>28</td><td>2014 Feb 13</td><td>F</td><td>Vaginal swab</td><td>A2059G</td><td>–</td><td>–</td></tr><tr><td>29</td><td>2014 Feb 18</td><td>M</td><td>Urine</td><td>Wild-type</td><td>–</td><td>Ser-80→Ile</td></tr><tr><td>30</td><td>2014 Feb 24</td><td>F</td><td>Vaginal swab</td><td>Wild-type</td><td>–</td><td>Asp-84→Asn</td></tr><tr><td>31</td><td>2014 Mar 5</td><td>F</td><td>Vaginal swab</td><td>Wild-type</td><td>–</td><td>Asp-84→Asn</td></tr><tr><td>32</td><td>2014 Mar 14</td><td>M</td><td>Urine</td><td>NA</td><td>–</td><td>Ser-80→Asn</td></tr><tr><td>33</td><td>2014 Apr 3</td><td>F</td><td>Endocervical swab</td><td>A2058G</td><td>–</td><td>–</td></tr><tr><td>34</td><td>2014 Apr 7</td><td>M</td><td>Urethral swab</td><td>A2059G</td><td>–</td><td>–</td></tr><tr><td>35</td><td>2014 Jun 24</td><td>F</td><td>Vaginal swab</td><td>A2059C</td><td>–</td><td>–</td></tr><tr><td>36</td><td>2014 Jul 9</td><td>F</td><td>Endocervical swab</td><td>A2059G</td><td>NA</td><td>–</td></tr><tr><td>37</td><td>2014 Jul 25</td><td>F</td><td>Urine</td><td>A2058G/A2059G</td><td>NA</td><td>NA</td></tr><tr><td>38</td><td>2014 Aug 19</td><td>F</td><td>Endocervical swab</td><td>A2058G</td><td>–</td><td>–</td></tr><tr><td>39</td><td>2014 Aug 28</td><td>F</td><td>Vaginal swab</td><td>A2062T</td><td>–</td><td>NA</td></tr><tr><td>40</td><td>2014 Sep 24</td><td>F</td><td>Vaginal swab</td><td>A2058G/A2059G</td><td>–</td><td>–</td></tr><tr><td>41</td><td>2014 Oct 7</td><td>F</td><td>Vaginal swab</td><td>A2059G</td><td>–</td><td>–</td></tr><tr><td>42</td><td>2014 Oct 15</td><td>F</td><td>Vaginal swab</td><td>A2059G</td><td>–</td><td>–</td></tr><tr><td>43</td><td>2014 Oct 31</td><td>M</td><td>Urine</td><td>A2058G</td><td>–</td><td>–</td></tr><tr><td>44</td><td>2014 Nov 5</td><td>F</td><td>Vaginal swab</td><td>A2058G/A2059G</td><td>NA</td><td>–</td></tr><tr><td>45</td><td>2014 Nov 28</td><td>F</td><td>Vaginal swab</td><td>A2058G</td><td>–</td><td>–</td></tr><tr><td>46</td><td>2014 Dec 3</td><td>F</td><td>Vaginal swab</td><td>Wild-type</td><td>–</td><td>Asp-84→Asn</td></tr><tr><td>47</td><td>2014 Dec 3</td><td>M</td><td>Urethral swab</td><td>Wild-type</td><td>–</td><td>Asp-84→Asn</td></tr><tr><td>48</td><td>2014 Dec 4</td><td>M</td><td>Urine</td><td>A2059G</td><td>–</td><td>–</td></tr></tbody></table>

previously reported; however, mutations at the next positions (97 in ParC and 95 in GyrA) have been described for *M. genitalium* (4,7), and these positions are within the QRDRs, suggesting their involvement in fluoroquinolone resistance. As previously described, *M. genitalium* ParC alterations predominate over GyrA alterations.

None of the 12 patients with strain *parC* or *gyrA* mutations had a history of fluoroquinolone treatment. Six patients received no treatment; 4 patients received azithromycin

(1 g); 2 patients received extended azithromycin (1.5 g), 1 patient after azithromycin (1 g) failure, and 1 after receiving doxycycline for 7 days. Therapeutic outcomes were not available except for 1 patient, who experienced clinical failure after 2 azithromycin treatments.

Regarding macrolide resistance, 38 of 221 patients (17.20%; 95% CI 12.79%–22.72%) had *M. genitalium* with macrolide resistance-associated 23S rRNA mutations; prevalence was 17% (19/112) for 2013 and 17.4% (19/109)

<footnote>*A2058/A2059G indicates a macrolide-resistant (A2058G or A2059G) genotype. Positions in the 23S rRNA and in GyrA and ParC are identified according to *Escherichia coli* numbering. NA, not available; –, no amino acid change.</footnote>
for 2014. This prevalence is increasing compared to that described in France in 2012 (14%). We found 35 A→G substitutions at position 2058 or 2059, two A2062T mutations and one A2059C mutation (Table) (1,9). Notably, in patients 15 and 33, who were infected with strains with macrolide resistance-associated mutations, *M. genitalium* infection was unsuccessfully treated with azithromycin, with treatment failures after azithromycin (1 g) and extended azithromycin (1.5 g for 5 d), but moxifloxacin treatment was effective. Patient 15 had been treated 1 year earlier with azithromycin (1 g) for nongonococcal urethritis.

Among the 168 patients whose isolates were examined for the 23S rRNA, gyrA, and parC genes, strains from 2 patients (patients 3 and 6) had both macrolide- and fluoroquinolone-associated mutations (1.2%; 95% CI 0.33%-4.24%). Both patients received azithromycin (1 g), and patient 6 received additional azithromycin (1.5 g) after failure of azithromycin (1 g). Patient 6 experienced azithromycin failure again after the extended regimen. *M. genitalium* multidrug resistance is described in France at a prevalence of 1.2%, lower than prevalence described in Australia (7.5%) (7) and Japan (30.8%) (10).

In conclusion, *M. genitalium* fluoroquinolone resistance is emerging in France, with a prevalence of 6% in 2013–2014. Further, macrolide resistance also increased during this period, to a rate of 17.2%. Patients infected with *M. genitalium* strains containing both macrolide and fluoroquinolone resistance mutations associated with therapeutic failure raise concerns about untreatable *M. genitalium* infections.

## Acknowledgments

We thank Manon Zerbib and Manon Passard for technical assistance.

## References

1. Touati A, Peuchant O, Jensen JS, Bébéar C, Pereyre S. Direct detection of macrolide resistance in *Mycoplasma genitalium* isolates from clinical specimens from France by use of real-time PCR and melting curve analysis. J Clin Microbiol. 2014;52:1549–55. http://dx.doi.org/10.1128/JCM.03318-13

2. Couldwell DL, Tagg KA, Jeoffreys NJ, Gilbert GL. Failure of moxifloxacin treatment in *Mycoplasma genitalium* infections due to macrolide and fluoroquinolone resistance. Int J STD AIDS. 2013;24:822–8. http://dx.doi.org/10.1177/0956462413502008

3. Pond MJ, Nori AV, Witney AA, Lopeman RC, Butcher PD, Sadiq ST. High prevalence of antibiotic-resistant *Mycoplasma genitalium* in nongonococcal urethritis: the need for routine testing and the inadequacy of current treatment options. Clin Infect Dis. 2014;58:631–7. http://dx.doi.org/10.1093/cid/cit752

4. Shimada Y, Deguchi T, Nakane K, Masue T, Yasuda M, Yokoi S, et al. Emergence of clinical strains of *Mycoplasma genitalium* harbouring alterations in ParC associated with fluoroquinolone resistance. Int J Antimicrob Agents. 2010;36:255–8. http://dx.doi.org/10.1016/j.ijantimicag.2010.05.011

5. Deguchi T, Yasuda M, Horie K, Seike K, Kikuchi M, Mizutani K, et al. Drug resistance-associated mutations in *Mycoplasma genitalium*.

in female sex workers, Japan. Emerg Infect Dis. 2015;21:1062–4. http://dx.doi.org/10.3201/eid2106.142013

6. Bissessor M, Tabrizi SN, Twin J, Abdo H, Fairley CK, Chen MY, et al. Macrolide resistance and azithromycin failure in a *Mycoplasma genitalium*-infected cohort and response of azithromycin failures to alternative antibiotic regimens. Clin Infect Dis. 2015;60:1228–36. http://dx.doi.org/10.1093/cid/ciu1162

7. Tagg KA, Jeoffreys NJ, Couldwell DL, Donald JA, Gilbert GL. Fluoroquinolone and macrolide resistance-associated mutations in *Mycoplasma genitalium*. J Clin Microbiol. 2013;51:2245–9. http://dx.doi.org/10.1128/JCM.00495-13

8. Kikuchi M, Ito S, Yasuda M, Tsuchiya T, Hatazaki K, Takanashi M, et al. Remarkable increase in fluoroquinolone-resistant *Mycoplasma genitalium* in Japan. J Antimicrob Chemother. 2014;69:2376–82. http://dx.doi.org/10.1093/jac/dku164

9. Jensen JS, Bradshaw CS, Tabrizi SN, Fairley CK, Hamasuna R. Azithromycin treatment failure in *Mycoplasma genitalium*-positive patients with nongonococcal urethritis is associated with induced macrolide resistance. Clin Infect Dis. 2008;47:1546–53. http://dx.doi.org/10.1086/593188

10. Deguchi T, Kikuchi M, Yasuda M, Ito S. Multidrug-resistant *Mycoplasma genitalium* is increasing. Clin Infect Dis. 2016;62:405–6. http://dx.doi.org/10.1093/cid/civ898

Address for correspondence: Cécile Bébéar, USC EA 3671, Mycoplasmal and Chlamydial Infections in Humans, University of Bordeaux, Campus Bordeaux Carreire, 146 rue Léo Saignat, 33076 Bordeaux CEDEX, France; email: cecile.bebear@u-bordeaux.fr

# Possible Transmission of *mcr-1-Harboring Escherichia coli* between Companion Animals and Human

Xue-Fei Zhang, Yohei Doi, Xi Huang, Hong-Yu Li, Lan-Lan Zhong, Kun-Jiao Zeng, Yan-Fen Zhang, Sandip Patil, Guo-Bao Tian

Author affiliations: Sun Yat-Sen University Zhongshan School of Medicine, Guangzhou, China (X.-.F Zhang, X. Huang, L.-L. Zhong, K.-J. Zeng, Y.-F. Zhang, S. Patil, G.-B. Tian); Ministry of Education Key Laboratory of Tropical Diseases Control, Guangzhou (X.-F. Zhang, X. Huang, L.-L. Zhong, K.-J. Zeng, Y.-F. Zhang, S. Patil, G.-B. Tian); University of Pittsburgh Medical Center, Pittsburgh, Pennsylvania, USA (Y. Doi); Sun Yat-Sen University Memorial Hospital, Guangzhou (H.-Y. Li)

DOI: http://dx.doi.org/10.3201/eid2209.160464

**To the Editor:** Plasmid-mediated, colistin-resistance mechanism gene *mcr-1* was first identified in *Escherichia coli* isolates from food, food animals, and human patients in November 2015 (1). Reports on detection of *mcr-1* in *Enterobacteriaceae* from humans and food animals