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Journal List > J Bacteriol Virol > v.54(3) > 1516088425

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Choi, Kang, Lee, and Jang: Increased Azithromycin Resistance Observed in Clinical Chlamydia trachomatis Isolates: An in vitro Induction Model
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J Bacteriol Virol. 2024;54(3):243-245.

DOI: https://doi.org/10.4167/jbv.2024.54.3.243

Increased Azithromycin Resistance Observed in Clinical Chlamydia trachomatis Isolates: An in vitro Induction Model

Yeon-Joo Choi1, 2, 3, †, Taeuk Kang1, 2, 3, †, Kwang-Jun Lee4, Won-Jong Jang1, 2, 3, *

1 Department of Microbiology, College of Medicine, Konkuk University, 120 Neungdong-ro, Gwangjin-gu, Seoul 05029, Republic of Korea Republic of Korea

2 Research Institute of Medical Science, College of Medicine, Konkuk University, 120 Neungdong-ro, Gwangjin-gu, Seoul 05029, Republic of Korea Republic of Korea

3 Institute of Biomedical Sciences & Technology, Konkuk University, 120 Neungdong-ro, Gwangjin-gu, Seoul 05029, Republic of Korea Republic of Korea

4 Division of Zoonotic and Vector Borne Disease Research, National Institute of Health, Korea Disease Control and Prevention Agency, Osong, Cheongju 28160, Republic of Korea Republic of Korea

†These authors equally contributed to this work

*wjjang@kku.ac.kr

Received 27 July 2024       Revised 13 September 2024       Accepted 19 September 2024

Copyright © 2024 Journal of Bacteriology and Virology

Journal of Bacteriology and Virology

(open-access, https://creativecommons.org/licenses/by-nc/4.0/):

This is an Open Access article distributed under the terms of the Creative Commons Attribution Non-Commercial License (https://creativecommons.org/licenses/by-nc/4.0/).

Abstract

Chlamydia trachomatis infections are increasing worldwide and constitute a significant threat to global public health. The number of chlamydial infection cases was estimated as 129 million in 2020. In addition, several countries have reported antimicrobial resistance in C. trachomatis, particularly against azithromycin, which is used for the first-line treatment of C. trachomatis infections. Despite such clinical significance, the molecular mechanisms of antimicrobial resistance acquisition are not comprehensively understood. In this study, antimicrobial resistance was induced in clinically isolated C. trachomatis via an in vitro model. We also attempted to detect single nucleotide polymorphism (SNP) in antimicrobial resistance-associated genes, such as 23S rRNA and rplD (L4). Resistance against azithromycin was induced in clinical C. trachomatis isolates. More importantly, an SNP causing an amino acid change on 23S rRNA was observed in azithromycin resistance-induced clinical isolates. This SNP (2058A<C, E686D) was previously reported to be associated with antimicrobial resistance. Our finding empirically proved that the abusive use of antimicrobials during C. trachomatis treatment may lead to the emergence of antimicrobial resistance. At the same time, the identified SNP on 23S rRNA can be used as a target to detect azithromycin resistance upon treatment failure in case of C. trachomatis infections. Continuous surveillance and investigations should be conducted to better comprehend the nature of C. trachomatis infections.

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Keywords: Azithromycin, Chlamydia trachomatis, Antibiotic resistance, 23S rRNA

BODY

Chlamydial infection caused by Chlamydia trachomatis, an obligatory intracellular bacteria, is a serious sexually transmitted infection (STI) from clinical and public health perspectives (1, 2). The World Health Organization estimated 129 million chlamydial infection cases in 2020 (1).
Despite the easy and low-cost treatment available for C. trachomatis infections, the high rate of asymptomatic infections causes delay in administering appropriate treatment (3). More importantly, the rise of antimicrobial resistance in C. trachomatis has been reported globally, limiting the use of currently available drugs, such as azithromycin (4, 5). The antimicrobial resistance of C. trachomatis is an emerging trend, yet the mechanisms of such antimicrobial resistance acquisition are poorly understood.
In this study, we used an in vitro model to induce azithromycin resistance in C. trachomatis collected from patients in the Republic of Korea. We confirmed the highest level of antimicrobial resistance that C. trachomatis can acquire against azithromycin. Lastly, we attempted to detect antimicrobial resistance-associated genes, 23S rRNA and rplD (L4), via polymerase chain reaction (PCR) before and after inducing antimicrobial resistance (6, 7, 8).
In this study, four clinically isolated C. trachomatis were used: C18-9, C18-10, C18-11, and S18-24. They were collected from female patients at the hospitals and clinics located at the Republic of Korea in 2018. The antimicrobial susceptibility test used in this study to determine the minimum inhibitory concentration (MIC) was described in a previous study (9). C18-9, C18-10, and C18-11 were azithromycin-resistant, with an MIC against azithromycin (Tokyo Chemical Inc., Japan) of 256 µg/ml. S18-24 was an antimicrobial-susceptible isolate, with a high mutation rate and an MIC of 125 ng/ml against azithromycin. Two reference C. trachomatis genotypes, A and D, were also included, with an MIC of 125 ng/ml against azithromycin.
Antimicrobial resistance against azithromycin was induced in vitro on S18-24. The clinical C. trachomatis isolate was inoculated into a McCoy cell for infection and cultured with azithromycin. The concentration of antimicrobials in the medium gradually increased, and C. trachomatis was recovered, as described in a previous study (6). The concentration range of azithromycin used in this study was 60 ng/ml to 64 µg/ml.
The induction of antimicrobial resistance against azithromycin in the S18-24 isolate increased the MIC from 125 ng/ml to 64 µg/ml. Subsequently, PCR and sequencing were performed to target 23S rRNA and rplD (L4) to detect any alterations as previously described (6). The results revealed that two azithromycin-resistant isolates and the azithromycin resistance-induced S18-24 had alterations in 23S rRNA. A nucleotide change in the form of 2058A<C was observed, which led to an E686D amino acid change. No alteration was observed in rplD (L4). The results are summarized in Table 1.
Table 1.

in vitro induction of antimicrobial resistance and mutations in antimicrobial resistance-associated genes

Isolate (Genotype) Azithromycin (ng/ml) 23s rRNA rplD (L4)
Before induction After induction Nucleotide change Amino acid change Nucleotide change Amino acid change
Reference C. trachomatis (A) 125 N/A - - - -
Reference C. trachomatis (D) 125 N/A - - - -
C18-9 (D/Ep6) 256 µg/ml N/A 2058A<C E686D - -
C18-10 (D/Ep6) 256 µg/ml N/A - - - -
C18-11 (D/Ep6) 256 µg/ml N/A 2058A<C E686D - -
S18-24 (D/Ep6) 125 64 µg/ml 2058A<C E686D - -

* MICs, minimum inhibitory concentrations; N/A, not avaliable.

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The prevalent C. trachomatis and its global emergence are a burden for public health. The emergence of antimicrobial-resistant C. trachomatis, particularly against azithromycin, the first-line drug used for C. trachomatis treatment, calls for serious caution (1, 4, 5). The clinical treatment outcome on the C. trachomatis infection depends mainly on antimicrobial resistance; therefore, the presence or absence of antimicrobial resistance is considered (4). However, only a few studies have been conducted on this subject. Our analysis revealed the in vitro induction of antimicrobial resistance in C. trachomatis and identified an SNP (2058A<C, E686D) on 23S rRNA. This SNP was previously reported as an antimicrobial resistance-associated SNP on 23S rRNA (7). This SNP may be used as a target in clinical settings to determine the molecular mechanisms underlying azithromycin resistance in cases where first-line treatment fails. Furthermore, the results indicate that abusively using antimicrobials for C. trachomatis treatment could lead to resistance. Continuous surveillance and investigations should be conducted to prevent C. trachomatis infections.
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CONFLICT OF INTEREST

The authors have no conflicts.
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ACKNOWLEDGMENTS

WJ Jang and YJ Choi were involved in research design and conceptualization. YJ Choi, TU Kang, and KJ Lee performed the experiments. YJ Choi and TU Kang analyzed the data and wrote the manuscript. All authors were involved in manuscript review and discussion.
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References

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8. Gomes C, Pons MJ, Magallon-Tejada A, Durand D, Lluque A, Mosquito S, et al. In vitro development and analysis of Escherichia coli and Shigella boydii azithromycin-resistant mutants. Microb Drug Resist. 2013;19(2):88-93.DOI: 10.1089/mdr.2012.0036.
9. Suchland RJ, Geisler WM, Stamm WE. Methodologies and cell lines used for antimicrobial susceptibility testing of Chlamydia spp. Antimicrob Agents Chemother. 2003;47(2):636-642.DOI: 10.1128/AAC.47.2.636-642.2003.
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