atpE Mutation in Mycobacterium tuberculosis Not Always Predictive of Bedaquiline Treatment Failure

L. Fournier Le Ray, Alexandra Aubry, W. Sougakoff, M. Revest, jerome robert, Isabelle Bonnet, Nicolas Veziris, Florence Morel

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L. Fournier Le Ray, Alexandra Aubry, W. Sougakoff, M. Revest, jerome robert, et al.. atpE Mutation in Mycobacterium tuberculosis Not Always Predictive of Bedaquiline Treatment Failure. Emerging Infectious Diseases, 2022, 28 (5), pp.1062-1064. 10.3201/eid2805.212517. hal-03691441

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**atpE** Mutation in
*Mycobacterium tuberculosis*
Not Always Predictive of
Bedaquiline Treatment Failure

Laure Fournier Le Ray, Alexandra Aubry,
Wladimir Sougakoff, Matthieu Revest,
Jérôme Robert, Isabelle Bonnet, Nicolas Veziris,
Florence Morel

Author affiliations: Sorbonne Université, Institut National de la Santé et de la Recherche Médicale, Paris, France (L. Fournier Le Ray, A. Aubry, W. Sougakoff, J. Robert, I. Bonnet, N. Veziris, F. Morel); Hôpital Pitié-Salpêtrière, Assistance Publique–Hôpitaux de Paris, Sorbonne Université, Paris, France (A. Aubry, W. Sougakoff, J. Robert, I. Bonnet, F. Morel); Pontchaillou University Hospital, Rennes, France (M. Revest); Université de Rennes, Institut National de la Santé et de la Recherche Médicale, Rennes (M. Revest); Hôpital Saint-Antoine, Assistance Publique–Hôpitaux de Paris, Sorbonne Université, Paris (N. Veziris)

DOI: https://doi.org/10.3201/eid2805.212517

We report the emergence of an *atpE* mutation in a clinical *Mycobacterium tuberculosis* strain. Genotypic and phenotypic bedaquiline susceptibility testing displayed variable results over time and ultimately were not predictive of treatment outcome. This observation highlights the limits of current genotypic and phenotypic methods for detection of bedaquiline resistance.

Bedaquiline is one of the core drugs used to treat multidrug-resistant (MDR) tuberculosis (TB) and extensively drug-resistant TB (XDR TB) (1). Bedaquiline resistance is now part of the revised definition of XDR TB, and its incidence is rising alarmingly (2,3). Resistance to bedaquiline is mainly caused by mutations in *Rv0678* (*nmpR*), which encodes the repressor of the efflux pump MmpL5–MmpS5, usually leading to low-level resistance (4). Conversely, mutations in *atpE*, which encodes the target of bedaquiline, the c subunit of the ATP synthase, are rarely described in clinical strains (5) and are associated with high increase of MICs (4). Mutations in *pepQ* and *Rv1979c* are also reported, but their effect on bedaquiline susceptibility is unclear. We report a case of an *atpE* mutation in a bedaquiline-resistant clinical strain of *Mycobacterium tuberculosis* and discuss the performances of current methods for susceptibility testing (Appendix, https://wwwnc.cdc.gov/EID/article/28/5/21-2517-App1.pdf) and their clinical implications (6).

A 32-year-old man from Georgia received a diagnosis of bilateral cavitary lung MDR TB upon his

arrival in France in January 2020. Three consecutive treatment regimens of bedaquiline and clofazimine had failed. A fourth regimen combining bedaquiline, linezolid, cycloserine, clofazimine, delamanid, and amoxicillin/clavulanate + meropenem was initiated on arrival.

The first isolate from January 2020 (S1) was bedaquiline-resistant with a MIC one dilution above the breakpoint (MIC = 2 mg/L) and clofazimine-susceptible with a MIC close to the breakpoint (MIC = 1 mg/L). We detected 2 deletions (P129fs [15%] and G66fs [54%]) in Rv0678 (Figure).

At the end of March 2020, cycloserine was withdrawn because of phenotypic resistance, and bedaquiline, which had been stopped 1 month earlier, was resumed; the patient underwent a lobectomy. One month after, sputum microscopic examination and culture were still positive. The second isolate (S2) from April 2020 had an increased bedaquiline MIC (4 mg/L) but clofazimine MIC remained unchanged (1 mg/L). No mutation in Rv0678 was detected, but we observed an AtpE I66M (63%) substitution (Figure).

Two months later in June, the patient was sputum smear-negative but remained culture positive. Isolate S3 was susceptible to bedaquiline (MIC = 0.5 mg/L) and clofazimine resistant (MIC = 2 mg/L). A deletion was found in Rv0678 different from those identified in S1: deletion at position 293 (N98fs) (97%), whereas no mutation was identified in atpE (Figure). Verapamil and ethionamide were added and amoxicillin-clavulanate + meropenem was stopped. Finally, samples from September 2020 were culture negative, with regression of pulmonary lesions. The outcome was classified as treatment success in February 2021 after 13 months of treatment and was still favorable as of December 2021.

All 3 isolates shared the same spoligotype (SIT1) (Beijing lineage) and displayed only 3 single-nucleotide variants (SNVs) of difference by pairwise comparison. The SNVs were all nonsynonymous. Two SNVs were only recovered in strain S2, 1 corresponding to the AtpE: I66M substitution and 1 located in *Rv0243* (L136P substitution) encoding the acetyl-CoA acyltransferase FadA2 and probably implicated in lipid degradation. One SNV was only found in S1 in *Rv3909* (M683L substitution), encoding a protein of unknown function. No mutations were observed in *pepQ*, its promoter, or in *Rv1979c* (7).

As this case illustrates, identifying bedaquiline resistance in the laboratory and its effects on patient management appear complex. Over a 6-month period, we tested 3 *M. tuberculosis* isolates with different genotypic and phenotypic patterns regarding
<figure><img src="image_2.png" /><figcaption>**Figure.** Timeline of treatment regimen and microbiologic data for patient with multidrug-resistant tuberculosis before and after his arrival in France from Georgia. Timeline for each antibiotic indicates treatment (striped), phenotypic resistance (red), and susceptibility (green), as indicated in patient records. M, microscopic examination; C, culture.</figcaption></figure>

bedaquiline, exhibiting various MIC levels and muta-
tions in genes involved in bedaquiline resistance.
These isolates displayed only 3 SNVs by pairwise
comparison of their genomes, excluding a reinfection
by a new strain or a mixed infection.

Of note, mutations in *atpE* or *Rv0678* were found
only once and were not found at subsequent time-
points. Despite continuous bedaquiline treatment,
resistant strain S2 with the *atpE* mutation was not
selected, and the patient was cured. A previous in
vitro study suggested that, whereas *Rv0678* muta-
tions were dynamic over time, *atpE* mutations were
fixed once they appeared (8). This observation was
not confirmed by our clinical case. One possible ex-
planation for nonfixation of these mutations could
be the associated fitness cost. However, an in vitro
study did not show any fitness cost because of the
I66M substitution (9). Because fitness also depends
on the genetic background, the results of this in vi-
tro study might not be transposable here. Regard-
ing *Rv0678*, 2 mutations have been studied and did
not have fitness impact (E138G and R94Q) (4). Addi-
tional in vivo and epidemiologic studies would help
evaluate the fitness cost of such mutations. Anoth-
er explanation for the variability of genotypic and

phenotypic bedaquiline susceptibility over time
could be a spatial heterogeneity in the lesions as al-
ready described (10).

This case raises concerns about the ability of cur-
rent phenotypic and genotypic methods to detect
bedaquiline resistance. Further studies are needed
before relying on these methods for therapeutic deci-
sions. In the meantime, these data can help improve
the World Health Organization database of drug
resistance-related mutations (11). Overall, this case
underlines the complexity of bedaquiline-resistance
mechanisms and of the dynamics of mutation emer-
gence and selection.

Acknowledgments

We thank the technicians of NRC MyRMA for their
technical assistance.

We wish to honor the memory of our colleague, Wladimir
Sougakoff, who passed away in January 2022.

NRC MyRMA is supported by annual grant of Santé
Publique France. L.F.L.R. was supported by a grant for
Fondation du Souffle. The laboratory has received a
research grant from Janssen for study on bedaquiline
antimycobacterial activity.
## About the Author

Dr. Fournier Le Ray recently successfully defended a PhD thesis in microbiology. Her research was focused on the in vivo emergence of bedaquiline resistance.

## References

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2. World Health Organization. Meeting report of the WHO expert consultation on the definition of extensively drug-resistant tuberculosis, 27-29 October 2020 [cited 2021 Feb 5]. https://www.who.int/publications/i/item/ meeting-report-of-the-who-expert-consultation-on-the-definition-of-extensively-drug-resistant-tuberculosis

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4. Andries K, Villellas C, Coeck N, Thys K, Gevers T, Vranckx L, et al. Acquired resistance of *Mycobacterium tuberculosis* to bedaquiline. PLoS One. 2014;9:e102135. https://doi.org/10.1371/journal.pone.0102135

5. Peretokina IV, Krylova LY, Antonova OV, Kholina MS, Kulagina EV, Nosova EY, et al. Reduced susceptibility and resistance to bedaquiline in clinical *M. tuberculosis* isolates. J Infect. 2020;80:527-35. https://doi.org/10.1016/j.jinf.2020.01.007

6. World Health Organization. Technical manual for drug susceptibility testing of medicines used in the treatment of tuberculosis. 2018 [cited 2018 Oct 18]. https://apps.who.int/iris/handle/10665/275469

7. Battaglia S, Spitaleri A, Cabibbe AM, Meehan CJ, Utpatel C, Ismail N, et al. Characterization of genomic variants associated with resistance to bedaquiline and delamanid in naive mycobacterium tuberculosis clinical strains. J Clin Microbiol. 2020;58:1304-24. https://doi.org/10.1128/JCM.01304-20

8. Ismail N, Ismail NA, Omar SV, Peters RPH. In vitro study of stepwise acquisition of rv0678 and atpE mutations conferring bedaquiline resistance. Antimicrob Agents Chemother. 2019;63:e00292-19. https://doi.org/10.1128/AAC.00292-19

9. Huitric E, Verhasselt P, Koul A, Andries K, Hoffner S, Andersson DI. Rates and mechanisms of resistance development in *Mycobacterium tuberculosis* to a novel diarylquinoline ATP synthase inhibitor. Antimicrob Agents Chemother. 2010;54:1022-8. https://doi.org/10.1128/AAC.01611-09

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# Emerging Novel Reassortant Influenza A(H5N6) Viruses in Poultry and Humans, China, 2021

Wenming Jiang, Chunxia Dong, Shuo Liu, Cheng Peng, Xin Yin, Shaobo Liang, Lin Zhang, Jinping Li, Xiaohui Yu, Yang Li, Jingjing Wang, Guangyu Hou, Zheng Zeng, Hualei Liu

Author affiliations: China Animal Health and Epidemiology Center, Qingdao, China (W. Jiang, S. Liu, C. Peng, X. Yin, S. Liang, L. Zhang, J. Li, X. Yu, Y. Li, J. Wang, G. Hou, H. Liu); Chongqing Animal Disease Prevention and Control Center, Chongqing, China (C. Dong, Z. Zeng)

DOI: https://doi.org/10.3201/eid2805.212163

A novel highly pathogenic avian influenza A(H5N6) clade 2.3.4.4b virus was isolated from a poultry market in China that a person with a confirmed case had visited. Most genes of the avian and human H5N6 isolates were closely related. The virus also exhibited distinct antigenicity to the Re-11 vaccine strain.

Highly pathogenic avian influenza A(H5N1) virus emerged in China in 1996. H5 viruses have spread to Eurasia since 2003, Africa since 2005, and North America since 2014-2015. These viruses cause huge economic losses to the poultry industry and pose substantial threats to human health. By March 2022, a total of 75 confirmed cases of human infection with influenza A(H5N6) virus had been reported, including 48 cases in China since 2021 (https://www.who.int/teams/global-influenza-programme/avian-influenza/monthly-risk-assessment-summary).

On July 9, 2021, a human case of H5N6 infection was reported in Chongqing, China. One day later, we conducted an epidemiologic survey in the poultry market the patient had visited and collected swab samples from poultry. We identified the samples as H5N6 subtype by using H5- and N6-specific primers and probes. We propagated the virus in 10-day-old specific pathogen-free chicken embryos and designated the isolate as A/chicken/Chongqing/H1/2021(H5N6) (CK/CQ/H1). We sequenced the viral genome by using the Sanger method and deposited the sequences in GISAID (https://www.gisaid.org; accession nos. EPI1937512-9).

Phylogenetic analysis of the hemagglutinin (HA) genes showed that CK/CQ/H1 and A/Chongqing/

<footnote>Address for correspondence: Nicolas Veziris, Faculté de Médecine Sorbonne Université, 91, boulevard de l'hôpital, 75013 Paris, France; email: nicolas.veziris@sorbonne-universite.fr</footnote>