Poor efficacy of the combination of clarithromycin, amikacin, and cefoxitin against Mycobacterium abscessus in the hollow fiber infection model

doi:10.21203/rs.3.rs-5064595/v1

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Poor efficacy of the combination of clarithromycin, amikacin, and cefoxitin against Mycobacterium abscessus in the hollow fiber infection model

https://doi.org/10.21203/rs.3.rs-5064595/v1

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Journal Publication

published 30 Jan, 2025

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Background

Mycobacterium abscessus (MABS) causes difficult-to-treat pulmonary and extra-pulmonary infections. A combination therapy comprising amikacin, cefoxitin, and a macrolide agent is recommended, but its antimicrobial activity and clinical efficacy is uncertain. Inducible resistance to macrolides (macrolides-iR) has been associated with poor clinical response in pulmonary infections, whilst for extra-pulmonary infections data are scarce.

Objectives

Herein, the aim was to evaluate the effect of the amikacin, cefoxitin, and clarithromycin combination against macrolides-iR MABS in a hollow-fiber infection model.

Methods

The hollow-fiber system was inoculated with M. abscessus subsp. abscessus type strain ATCC 19977 and treated during 10 days with the antibiotics combination. Two level of macrolide concentrations were evaluated mimicking the pharmacokinetics profiles of free (i.e. unbound) drug in blood and lung.

Results

Using blood concentrations, the combination failed to prevent bacterial growth. Using lung concentrations, the combination had a limited but significant effect on bacterial growth from day 2 to day 10. Moreover, increasing clarithromycin concentrations stabilized the amikacin-tolerance level: amikacin minimal inhibitory concentration of amikacin-tolerant strains increased over time using blood concentrations while it remained stable using lung concentrations.

Conclusions

Our finding confirms the low activity of the amikacin, cefoxitin, and clarithromycin combination against macrolide-iR MABS infection, and suggest the influence of clarithromycin concentrations on response. The low concentration of clarithromycin in blood may hamper efficacy for the treatment of extra-pulmonary MABS infection. Consequently, it should not be considered as an active molecule in the chosen antibiotic combination, as recently recommended for pulmonary infections.

Mycobacterium abscessus

hollow-fiber system

hollow-fiber infection model

clarithromycin

amikacin

cefoxitin

pulmonary infection

extra-pulmonary infection

Mycobacterium abscessus complex (MABSC) is a rapid-growing, antibiotic-resistant non-tuberculous Mycobacteria causing difficult-to-treat pulmonary and various extra-pulmonary infections, particularly in immunocompromised patients [1, 2]. A combination therapy is recommended and usually comprises amikacin, cefoxitin, and a macrolide agent [3, 4]. MABSC includes subspecies carrying the erm(41) gene, conferring inducible resistance to macrolides (macrolides-iR) [5], which has been associated with poor clinical outcomes in pulmonary infections [6]. Recent guidelines of MABSC pulmonary infections treatment suggest using different macrolide-containing combinations depending on the presence of macrolides-iR [4], while extra-pulmonary infection guidelines have not been updated [1, 3]. The efficacy of the amikacin, cefoxitin, and macrolide combination has also been questioned using a hollow-fiber infection model (HFIM) mimicking optimal pulmonary antibiotic concentrations [7]. Nevertheless, no in vitro model has ever mimicked extra-pulmonary antibiotic concentrations whereas clarithromycin is known to concentrate in epithelial lining fluid (at least 10-fold) and alveolar cells [8].

Herein, the aim was therefore to evaluate the efficacy of amikacin, cefoxitin, and clarithromycin combination against Mycobacterium abscessus (MABS) in a HFIM mimicking human blood and lung concentrations of macrolides.

Minimal inhibitory concentrations (MIC) of MABS ATCC 19977 were determined by microdilution [9]. A hollow-fiber system (HFS), composed of a 20mL polysulfone fiber cartridge (FiberCell Systems®, New Market, MD, USA) connected to a central reservoir and a DUET pump (FiberCell Systems®), and containing Cation-adjusted-Muller-Hinton (CAMH) culture medium (Merck Millipore®, Burlington, MA, USA), was inoculated with MABS ATCC 19977 in exponential growth to achieve a 10⁵-10⁶ CFU/mL density at day (D) 0. Then, HFS was treated during 10 days with an amikacin, cefoxitin, and clarithromycin combination. Two level of macrolide concentrations were evaluated, mimicking the pharmacokinetics profiles of free (i.e. unbound) drug in blood and lung. Clarithromycin (Mylan, Canonsburg, PA, USA) was injected twice a day to achieve a peak concentration of 1.8mg/L for the blood regimen [10] and 18mg/L (10-fold) for the lung regimen [8]. Amikacin (Viatris, Pittsburgh, PA, USA) was injected once a day in the HFS to achieve a peak concentration of 60mg/L [11] for both regimen. Cefoxitin (PANPHARMA, Luitré-Dompierre, France) was administered as a continuous infusion into the CAMH culture medium to obtain a steady-state concentration of 25mg/L [12] for both regimen. A growth control without antibiotic (NT) was also evaluated.

Antibiotic concentrations achieved in the HFS were measured using an automated immunoassay on the Cobas platform (Roche, Bâle, Switzerland) for amikacin, and high-performance liquid chromatography combined with mass-spectrometry for cefoxitin and clarithromycin. The 3 conditions (NT, lung regimen, and blood regimen) were performed 3 times in independent experiments.

Samples were obtained in HFS cartridge on at from D0, to D10 of treatment, and cultured on antibiotic-free CAMH agar (BioRad, Hercules, CA, USA) for bacterial count. Results were expressed in absolute values (log₁₀ CFU/mL) and in relative values compared to D0, i.e. CFU count relative from baseline, (% D0 log₁₀ CFU/mL) to account for variability in the initial D0 inoculum. Antibiotic tolerance was quantified by culturing on CAMH agar (Becton-Dickinson) containing 80mg/L of cefoxitin or 32mg/L of amikacin, and expressed in absolute values (log₁₀ CFU/mL growing on antibiotic-supplemented media), and in relative values: \(\:\%=100\:\text{X}\:\frac{\text{C}\text{o}\text{u}\text{n}\text{t}\:\text{o}\text{n}\:\text{a}\text{n}\text{t}\text{i}\text{b}\text{i}\text{o}\text{t}\text{i}\text{c}-\text{s}\text{u}\text{p}\text{p}\text{l}\text{e}\text{m}\text{e}\text{n}\text{t}\text{e}\text{d}\:\text{m}\text{e}\text{d}\text{i}\text{a}\:\text{a}\text{t}\:\text{D}\text{X}\:\left(\frac{\text{C}\text{F}\text{U}}{\text{m}\text{L}}\right)}{\text{T}\text{o}\text{t}\text{a}\text{l}\:\text{c}\text{o}\text{u}\text{n}\text{t}\:\text{a}\text{t}\:\text{D}\text{X}\:\left(\frac{\text{C}\text{F}\text{U}}{\text{m}\text{L}}\right)}.\:\:\)Cefoxitin and amikacin MIC of strains growing on antibiotic-supplemented media were determined by microdilution [9].

Appropriate tests were performed to compare bacterial growth and antibiotic-resistant subpopulation between NT and treatment conditions using the GraphPad Prism software, version 10.2.3. A p value < 0.05 was considered statistically significant.

MABS amikacin and cefoxitin MIC were 1 and 8 mg/L, respectively. MABS presented a clarithromycin-iR with increased MIC, from 0.12 mg/L on D3 to > 16mg/L on D14. The antibiotic concentration profiles achieved human concentration target values (Fig. 1).

Overall, none of the antibiotic regimens enabled HFS sterilization after D10 of treatment (Fig. 2). The blood regimen failed to produce a bactericidal or bacteriostatic effect over 10 days. It only had a limited but significant inhibitory effect on bacterial growth compared to NT at D3 (-1.17 log₁₀ CFU/mL, 95% confidence interval, 95%CI [-2.26; -0.08]) and D4 (-1.33 log₁₀ CFU/mL, 95%CI [-2.16; -0.49]; Fig. 2 (a)), but had no effect on CFU count relative from baseline, compared to NT (Fig. 2 (b)). The lung regimen also failed to kill bacteria. It exhibited an initial bacteriostatic effect but could not inhibit growth beyond 3 days. It had a limited but significant effect on growth inhibition compared to NT at D5 (-1.36 log₁₀ CFU/mL, 95%CI [-0.01; -2.72]; Fig. 2 (a)). This regimen significantly reduced CFU count relative from baseline compared to NT, from D2 to D10, with a maximum effect at D10 (-38.0%, 95%CI [-8.02; -67.9]; Fig. 2 (b)). Moreover, compared to NT, the reduction in the area under the curve of CFU counts relative from baseline was greater for the lung regimen (-65%) than for the blood regimen (-42%; Fig. 2 (b)). Finally, although the CFU counts were not significantly different between both regimens at D10 (in absolute and relative values), there was a trend toward greater CFU counts under the blood regimen, while they were stable under the lung regimen (Fig. 2).

The emergence of MABS strains growing on antibiotic-supplemented media (Fig. 3 (a), (b), (c)) was observed in all conditions and could be defined as tolerant and not resistant, since no strain reached the MIC defining the resistance clinical category [13] (Table 1).

Table 1

**MIC (mg/L) to amikacin and cefoxitin for antibiotic-tolerant MABS.** MIC for MABS growing on antibiotic-supplemented medium according to time and treatment (not treated, under the blood regimen, or under the lung regimen).
	MIC (mg/L) Amikacin-tolerant			MIC (mg/L) Cefoxitin-tolerant
Time (Days)	Not treated	Blood	Lung	Not treated	Blood	Lung
2	1	1	1	8	8	8
3	2	1	1	8	8	8
4	1	1	1	8	8	8
5	2	2	1	8	8	8
7	2	2	1	8	8	8
8	4	4	1	8	8	8
9	4	4	1	8	8	8
10	4	4	1	8	8	8
MIC: minimal inhibitory concentration

In NT, the proportion of antibiotic-tolerant strains remained relatively stable over time; 0.01% of the inoculum for cefoxitin-tolerant strains (Fig. 3 (d)), and between 0.0001–0.001% for amikacin-tolerant strains (Fig. 3(e)).

Both regimens led to an early emergence of cefoxitin-tolerant strains (Fig. 3 (b), (c)), which reached > 0.1% of the inoculum at D1, a proportion significantly greater than in NT (Fig. 3 (d)). The proportion gradually decreased over time; at D10, there was no difference between NT and both regimens (Fig. 3 (d)). In all conditions, cefoxitin MIC of the cefoxitin-tolerant strains were stable (8 mg/L, Table 1).

Both regimens resulted in a progressive emergence of amikacin-tolerant strains over time. Nevertheless, the proportion of amikacin-tolerant strains was greater under the blood regimen, reaching > 0.01% of the inoculum, which was significantly different from NT (at D4, D5, and D8). Under the lung regimen, the emergence of amikacin-tolerant strains was delayed (Fig. 3 (e)). Moreover, the amikacin-tolerance level was different between the two regimens: amikacin MIC of amikacin-tolerant strains increased over time for the blood regimen (2 and 4 mg/L at D5 and D8, respectively) while it remained stable under the lung regimen until D10 (1 mg/L; Table 1).

As previously found in a HFIM mimicking lung exposure [7], the present study confirmed that the amikacin, cefoxitin, and clarithromycin combination fails to produce a relevant antibacterial effect (bactericidal or bacteriostatic) against MABS using both blood and lung concentrations of clarithromycin. However, the lung regimen better inhibited MABS growth compared to the blood regimen. Moreover, increased clarithromycin concentrations achieved in lungs [8] appeared to prevent the development of amikacin-tolerance. This activity of high-concentration clarithromycin, in addition to its immunomodulatory effect previously described [4], may support the relevance of its use in the antibiotic combination against MABS pulmonary infections, even with clarithromycin-iR. However, new macrolide-containing drug combinations should be evaluated considering the poor efficacy of the present one.

Ferro et al. reported the emergence of antibiotic-resistant mutants in HFIM over time, notably an increase in cefoxitin-resistant mutants from D14 (although no MIC value was mentioned) [7]; we did not observe true antibiotic-resistance emergence but rather antibiotic-tolerance, defined as the ability of a subpopulation to survive exposure to a bactericidal drug concentration without an increase in the MIC [14], with a high proportion of the cefoxitin-tolerant subpopulation from D1 for both antibiotic regimens. It has been shown that tolerance often promotes the development of resistance [15]; it is thus possible that the development of the cefoxitin-tolerant subpopulation is the main factor favoring the emergence of the cefoxitin-resistant mutants. Moreover, it could be interesting to further explore the underlying mechanisms of antibiotic tolerance. A recently published study found that a mutation in serB2, a gene involved in L-serine biosynthesis, resulted in the increased emergence of MABS cross-tolerance to cefoxitin and moxifloxacin, through activation of a WhiB7-dependant adaptive stress response [16].

Herein, one of the main limitations was the duration of HFIM (10 days), which may not be sufficient to accurately consider the effects of antibiotic treatment on chronic lung infection. However, this duration may be relevant to study the efficacy of antibiotic treatment in more acute infections such as bacteremia, where the bactericidal effect must be rapid. In this case, we showed herein a very weak antibacterial effect of the amikacin, cefoxitin, and clarithromycin combination, insufficient to inhibit bacterial growth. In addition, there was no benefit in preventing antibiotic-tolerance. Thus, in the absence of increased clarithromycin concentration at the site of infection, the value of using clarithromycin in the antibiotic regimen against MABS with macrolides-iR should be critically reappraised. We think that the low concentration of clarithromycin in blood may hamper efficacy for the treatment of extra-pulmonary MABS infections and should not be considered as an active molecule in the chosen antibiotic combination, as recently recommended for lung infections [4]. Further investigations are urgently needed to find the best combination of antibiotics, which is rapidly and effectively bactericidal against MABS in invasive extra-pulmonary infections.

CAMH

cation-adjusted-Muller-Hinton

CFU

colony-forming unit

day

HFIM

hollow-fiber infection model

HFS

hollow-fiber system

MABS

Mycobacterium abscessus

MABSC

Mycobacterium abscessus complex

Macrolides-iR

inducible resistance to macrolides

MIC

minimal-inhibitory concentration

growth control without antibiotic

Ethics approval and consent to participate

Not applicable.

Consent for publication

Not applicable.

Availability of data and materials

The datasets used and/or analysed during the current study are available from the corresponding author on reasonable request.

Competing interests

The authors declare that they have no competing interests.

Funding

This work was supported by the internal “Jeunes Chercheurs” grant 2021 operated by the Hospices civils de Lyon.

Authors' contributions

Conception: SG, OD, EH; design of the work: EV, SG, AD, LL, AB, OD, EH; acquisition: EV, CG, JG, SC, CB, AD, LL, AB, CR, EH; analysis: EV, CG, JG, SC, AD, LL, AB, CR, EH; interpretation of data: EV, SG, CG, JG, SC, AD, LL, AB, CR, EH; drafted the work: EV, CG, EH; revised the work: SG and OD.

Acknowledgements

We thank Shanez Haouari (DRS, Hospices Civils de Lyon) for help in manuscript preparation.

To K, Cao R, Yegiazaryan A, Owens J, Venketaraman V. General Overview of Nontuberculous Mycobacteria Opportunistic Pathogens: Mycobacterium avium and Mycobacterium abscessus. J Clin Med. 2020;9:2541.
Lee M-R, Sheng W-H, Hung C-C, Yu C-J, Lee L-N, Hsueh P-R. Mycobacterium abscessus Complex Infections in Humans. Emerg Infect Dis. 2015;21:1638–46.
Griffith DE, Aksamit T, Brown-Elliott BA, Catanzaro A, Daley C, Gordin F, et al. An official ATS/IDSA statement: diagnosis, treatment, and prevention of nontuberculous mycobacterial diseases. Am J Respir Crit Care Med. 2007;175:367–416.
Daley CL, Iaccarino JM, Lange C, Cambau E, Wallace RJ, Andrejak C, et al. Treatment of nontuberculous mycobacterial pulmonary disease: an official ATS/ERS/ESCMID/IDSA clinical practice guideline. Eur Respir J. 2020;56:2000535.
Tortoli E, Kohl TA, Brown-Elliott BA, Trovato A, Leão SC, Garcia MJ, et al. Emended description of Mycobacterium abscessus, Mycobacterium abscessus subsp. abscessus and Mycobacteriumabscessus subsp. bolletii and designation of Mycobacteriumabscessus subsp. massiliense comb. nov. Int J Syst Evol Microbiol. 2016;66:4471–9.
Pasipanodya JG, Ogbonna D, Ferro BE, Magombedze G, Srivastava S, Deshpande D, et al. Systematic Review and Meta-analyses of the Effect of Chemotherapy on Pulmonary Mycobacterium abscessus Outcomes and Disease Recurrence. Antimicrob Agents Chemother. 2017;61:e01206-17.
Ferro BE, Srivastava S, Deshpande D, Pasipanodya JG, van Soolingen D, Mouton JW, et al. Failure of the Amikacin, Cefoxitin, and Clarithromycin Combination Regimen for Treating Pulmonary Mycobacterium abscessus Infection. Antimicrob Agents Chemother. 2016;60:6374–6.
Honeybourne D, Kees F, Andrews JM, Baldwin D, Wise R. The levels of clarithromycin and its 14-hydroxy metabolite in the lung. Eur Respir J. 1994;7:1275–80.
Rodvold KA, Gotfried MH, Danziger LH, Servi RJ. Intrapulmonary steady-state concentrations of clarithromycin and azithromycin in healthy adult volunteers. Antimicrob Agents Chemother. 1997;41:1399–402.
Clinical and laboratory standards institute. M24 - Susceptibility testing of Mycobacteria, Nocardia spp., and Other aerobic actinomycetes. 3rd Edition. 2018.
Sadouki Z, McHugh TD, Aarnoutse R, Ortiz Canseco J, Darlow C, Hope W, et al. Application of the hollow fibre infection model (HFIM) in antimicrobial development: a systematic review and recommendations of reporting. J Antimicrob Chemother. 2021;76:2252–9.
Base de données publique des médicaments. Résumé des caractéristiques du produit - ZECLAR 0,5 g, poudre pour solution à diluer pour perfusion - [Internet]. [cited 2024 Jul 19]. Available from: https://base-donnees-publique.medicaments.gouv.fr/affichageDoc.php?specid=61433564&typedoc=R#RcpPropPharmacocinetiques
Base de données publique des médicaments. Résumé des caractéristiques du produit - AMIKACINE VIATRIS 1 g, poudre pour solution injectable en flacon - [Internet]. [cited 2024 Jul 19]. Available from: https://base-donnees-publique.medicaments.gouv.fr/affichageDoc.php?specid=62516778&typedoc=R#RcpPropPharmacocinetiques
Base de données publique des médicaments. Résumé des caractéristiques du produit - CEFOXITINE PANPHARMA 1 g, poudre pour solution injectable (IV) - [Internet]. [cited 2024 Jul 19]. Available from: https://base-donnees-publique.medicaments.gouv.fr/affichageDoc.php?specid=69084488&typedoc=R#RcpPropPharmacocinetiques
The European Committee on Antimicrobial Susceptibility, EUCAST. Amikacin: Rationale for EUCAST Clinical Breakpoints [Internet]. 2024 [cited 2024 Nov 22]. Available from: https://www.eucast.org/fileadmin/src/media/PDFs/EUCAST_files/Rationale_documents/Amikacin_Rationale_Document_v_3.1_20240901.pdf
Davey PG. The pharmacokinetics of clarithromycin and its 14-OH metabolite. J Hosp Infect. 1991;19 Suppl A:29–37.
Isla A, Trocóniz IF, de Tejada IL, Vázquez S, Canut A, López JM, et al. Population pharmacokinetics of prophylactic cefoxitin in patients undergoing colorectal surgery. Eur J Clin Pharmacol. 2012;68:735–45.
Clinical and laboratory standards institute. M62 - Performance Standards for Susceptibility testing of Mycobacetria, Nocardia spp., and other aerobic actinomycetes. 1St Edition. 2018.
Prammananan T, Sander P, Brown BA, Frischkorn K, Onyi GO, Zhang Y, et al. A single 16S ribosomal RNA substitution is responsible for resistance to amikacin and other 2-deoxystreptamine aminoglycosides in Mycobacterium abscessus and Mycobacterium chelonae. J Infect Dis. 1998;177:1573–81.
Balaban NQ, Helaine S, Lewis K, Ackermann M, Aldridge B, Andersson DI, et al. Definitions and guidelines for research on antibiotic persistence. Nat Rev Microbiol. 2019;17:441–8.
Brauner A, Fridman O, Gefen O, Balaban NQ. Distinguishing between resistance, tolerance and persistence to antibiotic treatment. Nat Rev Microbiol. 2016;14:320–30.
Sulaiman JE, Lam H. Evolution of Bacterial Tolerance Under Antibiotic Treatment and Its Implications on the Development of Resistance. Front Microbiol. 2021;12:617412.
Bernard C, Liu Y, Larrouy-Maumus G, Guilhot C, Cam K, Chalut C. Altered serine metabolism promotes drug tolerance in Mycobacterium abscessus via a WhiB7-mediated adaptive stress response. Antimicrob Agents Chemother. 2024;68:e01456-23.
Herbert S, Barry P, Novick RP. Subinhibitory clindamycin differentially inhibits transcription of exoprotein genes in Staphylococcus aureus. Infect Immun. 2001;69:2996–3003.

No competing interests reported.

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Editorial decision: Accepted
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Poor efficacy of the combination of clarithromycin, amikacin, and cefoxitin against Mycobacterium abscessus in the hollow fiber infection model

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