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

Poor efficacy of the combination of clarithromycin, amikacin, and cefoxitin against Mycobacterium abscessus in the hollow fiber infection model | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Short Report Poor efficacy of the combination of clarithromycin, amikacin, and cefoxitin against Mycobacterium abscessus in the hollow fiber infection model Etienne Vignaud, Sylvain Goutelle, Charlotte Genestet, Jérôme Guitton, and 8 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-5064595/v1 This work is licensed under a CC BY 4.0 License Status: Published Journal Publication published 30 Jan, 2025 Read the published version in Annals of Clinical Microbiology and Antimicrobials → Version 1 posted 8 You are reading this latest preprint version Abstract 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 Figures Figure 1 Figure 2 Figure 3 Introduction 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. Materials and methods 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 10 5 -10 6 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 (log 10 CFU/mL) and in relative values compared to D0, i.e. CFU count relative from baseline, (% D0 log 10 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 (log 10 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 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 log 10 CFU/mL, 95% confidence interval, 95%CI [-2.26; -0.08]) and D4 (-1.33 log 10 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 log 10 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 ). Discussion 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. Abbreviations CAMH cation-adjusted-Muller-Hinton CFU colony-forming unit D 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 NT growth control without antibiotic Declarations 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. References 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. Additional Declarations No competing interests reported. Cite Share Download PDF Status: Published Journal Publication published 30 Jan, 2025 Read the published version in Annals of Clinical Microbiology and Antimicrobials → Version 1 posted Editorial decision: Accepted 07 Jan, 2025 Reviews received at journal 07 Jan, 2025 Reviews received at journal 20 Dec, 2024 Reviewers agreed at journal 19 Dec, 2024 Reviewers agreed at journal 06 Dec, 2024 Reviewers invited by journal 06 Dec, 2024 Submission checks completed at journal 25 Nov, 2024 First submitted to journal 23 Nov, 2024 You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. 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Rousse, Hospices civils de Lyon","correspondingAuthor":false,"prefix":"","firstName":"Laura","middleName":"","lastName":"Lebouteiller","suffix":""},{"id":382138511,"identity":"ac3a792a-c18e-4d33-89fb-9039c79d9782","order_by":8,"name":"Albin Bernard","email":"","orcid":"","institution":"Institut des Agents Infectieux, Hôpital de la Croix Rousse, Hospices civils de Lyon","correspondingAuthor":false,"prefix":"","firstName":"Albin","middleName":"","lastName":"Bernard","suffix":""},{"id":382138512,"identity":"27e5c8fd-f255-46b6-9da1-731db8ae0918","order_by":9,"name":"Caroline Richet","email":"","orcid":"","institution":"Hôpital de la Croix Rousse, Hospices civils de Lyon","correspondingAuthor":false,"prefix":"","firstName":"Caroline","middleName":"","lastName":"Richet","suffix":""},{"id":382138513,"identity":"5605172b-d90f-40d3-81e2-e116dedb8cbe","order_by":10,"name":"Oana Dumitrescu","email":"","orcid":"","institution":"Institut des Agents Infectieux, Hôpital de la Croix Rousse, Hospices civils de Lyon","correspondingAuthor":false,"prefix":"","firstName":"Oana","middleName":"","lastName":"Dumitrescu","suffix":""},{"id":382138514,"identity":"17a3fb9a-f90e-4f27-bf52-899d84def399","order_by":11,"name":"Elisabeth Hodille","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAABLUlEQVRIie3Sv2qDQBzA8d8h6HLW9Xdc02e4ICQpSKVvEhGcOnTsVBRBl5asCfUhMmY0OHTxAVq6pAiZUkjGQP+dSWlKlUC2Uvzioj8/3J0I0NT0F9OID/ICARSAit4xaL4qn/c202UdUbYEvwhSoGlJsBySYS0pJ98EJMH+fmIoSrhcT6zrLujT2eISqcGKeSGhM+A+Ca+qhIUkYre5h6f+kdtO5MbYndc1SzJKUhLkVSIyEoEeZShS2uHlWcTThcr1d3TGD33t2a8SOyPh6i362BH7MVd5uYokJKghQn4xrkfpj1WQ7icoN8ZbucvGGTU3Z8Hc67AE0BwNnaCOGHFcrF4mZ4a4v2nPFq/WiRFnc1yA1RqgO60ju5Rf9+fbv+KQ7MNeb2pqavrHfQKii1xrQeZbngAAAABJRU5ErkJggg==","orcid":"","institution":"Institut des Agents Infectieux, Hôpital de la Croix Rousse, Hospices civils de Lyon","correspondingAuthor":true,"prefix":"","firstName":"Elisabeth","middleName":"","lastName":"Hodille","suffix":""}],"badges":[],"createdAt":"2024-09-10 12:36:11","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-5064595/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-5064595/v1","draftVersion":[],"editorialEvents":[{"content":"https://doi.org/10.1186/s12941-025-00776-w","type":"published","date":"2025-01-30T15:57:12+00:00"}],"editorialNote":"","failedWorkflow":false,"files":[{"id":69936574,"identity":"1f23720d-34c9-4e7d-90c1-53536a354466","added_by":"auto","created_at":"2024-11-26 19:24:30","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":75687,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eKinetics of antibiotic concentrations over time during 24 hours in the hollow-fiber system for the blood regimen.\u003c/strong\u003e CLA: clarithromycin (red point: point extrapolated from the first peak due to evening injection), AMK: amikacin, CFX: cefoxitin. Targets were peak concentration for CLA: 1.8 mg/L, peak concentration for AMK: 60 mg/L, steady-state concentration for CFX: 25 mg/L.\u003c/p\u003e","description":"","filename":"Figure1.png","url":"https://assets-eu.researchsquare.com/files/rs-5064595/v1/1044fd4f94e3f36469c6f701.png"},{"id":69936577,"identity":"c7186212-5ef3-429b-bceb-03a83b4d7e75","added_by":"auto","created_at":"2024-11-26 19:24:30","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":326848,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eMABS growth kinetics in the hollow-fiber system for 10 days.\u003c/strong\u003e In not treated condition (black), under the blood antibiotic regimen (red) and under the lung antibiotic regimen (blue). (a). Absolut count, log\u003csub\u003e10\u003c/sub\u003e CFU/mL. (b). in relative values compared to D0, % D0 log\u003csub\u003e10\u003c/sub\u003e CFU/mL. *, p \u0026lt; 0.05; **, p \u0026lt; 0.01 (comparison between blood regimen and not treated condition). *, p \u0026lt; 0.05; **, p \u0026lt; 0.01 (comparison between lung regimen and not treated condition). Mean ± SD of 3 independent experiments for each condition.\u003c/p\u003e","description":"","filename":"Figure2.png","url":"https://assets-eu.researchsquare.com/files/rs-5064595/v1/55a8005c7ca9db06970d1a02.png"},{"id":69936575,"identity":"8c4b848b-c554-48d7-a5a1-16654eb463c6","added_by":"auto","created_at":"2024-11-26 19:24:30","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":3024858,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eAntibiotic-tolerant or -persistent MABS growth kinetics in the hollow-fiber system for 10 days.\u003c/strong\u003e (a), (b), (c): Growth kinetics in absolute count (log\u003csub\u003e10\u003c/sub\u003e CFU/mL) of MABS total count (black), amikacin-tolerant or -persistent MABS (orange), cefoxitin-tolerant or -persistent MABS (blue) in the hollow-fiber system for 10 days, for the not treated condition (a), blood regimen (b) and the lung regimen (c), (d), (e): Growth kinetics of cefoxitin-tolerant or -persistent MABS (d) and amikacin-tolerant or -persistent MABS (e) expressed in proportion (%), in the hollow-fiber system for 10 days, for the not treated condition (black), blood regimen (red) and lung regimen (blue). *, p \u0026lt; 0.05, **, p \u0026lt; 0.01; (comparison between blood regimen and not treated condition). *, p \u0026lt; 0.05; (comparison between lung regimen and not treated conditions). Mean ± SD of 3 independent experiments for each condition.\u003c/p\u003e","description":"","filename":"Figure3.png","url":"https://assets-eu.researchsquare.com/files/rs-5064595/v1/114aa953361121a99317ccf0.png"},{"id":75351200,"identity":"882fb8ca-49d3-4bfc-9d9e-c09c8044addf","added_by":"auto","created_at":"2025-02-03 16:07:45","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":3702673,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-5064595/v1/375169f6-7e82-47d8-ab1d-bc43359f45d3.pdf"}],"financialInterests":"No competing interests reported.","formattedTitle":"Poor efficacy of the combination of clarithromycin, amikacin, and cefoxitin against Mycobacterium abscessus in the hollow fiber infection model","fulltext":[{"header":"Introduction","content":"\u003cp\u003e \u003cem\u003eMycobacterium abscessus\u003c/em\u003e 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 [\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e, \u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e]. A combination therapy is recommended and usually comprises amikacin, cefoxitin, and a macrolide agent [\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e, \u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e]. MABSC includes subspecies carrying the \u003cem\u003eerm\u003c/em\u003e(41) gene, conferring inducible resistance to macrolides (macrolides-iR) [\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e], which has been associated with poor clinical outcomes in pulmonary infections [\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e]. Recent guidelines of MABSC pulmonary infections treatment suggest using different macrolide-containing combinations depending on the presence of macrolides-iR [\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e], while extra-pulmonary infection guidelines have not been updated [\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e, \u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e]. 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 [\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e]. Nevertheless, no \u003cem\u003ein vitro\u003c/em\u003e 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 [\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eHerein, the aim was therefore to evaluate the efficacy of amikacin, cefoxitin, and clarithromycin combination against \u003cem\u003eMycobacterium abscessus\u003c/em\u003e (MABS) in a HFIM mimicking human blood and lung concentrations of macrolides.\u003c/p\u003e"},{"header":"Materials and methods","content":"\u003cp\u003eMinimal inhibitory concentrations (MIC) of MABS ATCC 19977 were determined by microdilution [\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e]. A hollow-fiber system (HFS), composed of a 20mL polysulfone fiber cartridge (FiberCell Systems\u0026reg;, New Market, MD, USA) connected to a central reservoir and a DUET pump (FiberCell Systems\u0026reg;), and containing Cation-adjusted-Muller-Hinton (CAMH) culture medium (Merck Millipore\u0026reg;, Burlington, MA, USA), was inoculated with MABS ATCC 19977 in exponential growth to achieve a 10\u003csup\u003e5\u003c/sup\u003e-10\u003csup\u003e6\u003c/sup\u003e 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 [\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e]. 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, Luitr\u0026eacute;-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.\u003c/p\u003e \u003cp\u003eAntibiotic concentrations achieved in the HFS were measured using an automated immunoassay on the Cobas platform (Roche, B\u0026acirc;le, 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.\u003c/p\u003e \u003cp\u003eSamples 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 (log\u003csub\u003e10\u003c/sub\u003e CFU/mL) and in relative values compared to D0, i.e. CFU count relative from baseline, (% D0 log\u003csub\u003e10\u003c/sub\u003e 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 (log\u003csub\u003e10\u003c/sub\u003e CFU/mL growing on antibiotic-supplemented media), and in relative values: \u003cspan class=\"InlineEquation\"\u003e\u003cspan class=\"mathinline\"\u003e\\(\\:\\%=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)}.\\:\\:\\)\u003c/span\u003e\u003c/span\u003eCefoxitin and amikacin MIC of strains growing on antibiotic-supplemented media were determined by microdilution [\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eAppropriate 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\u0026thinsp;\u0026lt;\u0026thinsp;0.05 was considered statistically significant.\u003c/p\u003e"},{"header":"Results","content":"\u003cp\u003eMABS 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 \u0026gt;\u0026thinsp;16mg/L on D14. The antibiotic concentration profiles achieved human concentration target values (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eOverall, none of the antibiotic regimens enabled HFS sterilization after D10 of treatment (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003e). 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 log\u003csub\u003e10\u003c/sub\u003e CFU/mL, 95% confidence interval, 95%CI [-2.26; -0.08]) and D4 (-1.33 log\u003csub\u003e10\u003c/sub\u003e CFU/mL, 95%CI [-2.16; -0.49]; Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003e (a)), but had no effect on CFU count relative from baseline, compared to NT (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003e (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 log\u003csub\u003e10\u003c/sub\u003e CFU/mL, 95%CI [-0.01; -2.72]; Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003e (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.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003e (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.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003e (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.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003e).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eThe emergence of MABS strains growing on antibiotic-supplemented media (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003e (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\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e).\u003c/p\u003e \u003cp\u003e \u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab1\" border=\"1\"\u003e \u003ccaption language=\"En\"\u003e \u003cdiv class=\"CaptionNumber\"\u003eTable 1\u003c/div\u003e \u003cdiv class=\"CaptionContent\"\u003e \u003cp\u003e\u003cb\u003eMIC (mg/L) to amikacin and cefoxitin for antibiotic-tolerant MABS.\u003c/b\u003e MIC for MABS growing on antibiotic-supplemented medium according to time and treatment (not treated, under the blood regimen, or under the lung regimen).\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"7\"\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c5\" colnum=\"5\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c6\" colnum=\"6\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c7\" colnum=\"7\"\u003e\u003c/div\u003e \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\"\u003e\u0026nbsp;\u003c/th\u003e \u003cth align=\"left\" colspan=\"3\" nameend=\"c4\" namest=\"c2\"\u003e \u003cp\u003eMIC (mg/L) Amikacin-tolerant\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colspan=\"3\" nameend=\"c7\" namest=\"c5\"\u003e \u003cp\u003eMIC (mg/L) Cefoxitin-tolerant\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eTime (Days)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eNot treated\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eBlood\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eLung\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003eNot treated\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003eBlood\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003eLung\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e2\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e8\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e8\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e8\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e3\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e2\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e8\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e8\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e8\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e4\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e8\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e8\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e8\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e5\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e2\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e2\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e8\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e8\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e8\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e7\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e2\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e2\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e8\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e8\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e8\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e8\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e4\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e4\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e8\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e8\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e8\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e9\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e4\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e4\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e8\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e8\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e8\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e10\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e4\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e4\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e8\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e8\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e8\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003ctfoot\u003e \u003ctr\u003e\u003ctd colspan=\"7\"\u003eMIC: minimal inhibitory concentration\u003c/td\u003e\u003c/tr\u003e \u003c/tfoot\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e \u003cp\u003eIn NT, the proportion of antibiotic-tolerant strains remained relatively stable over time; 0.01% of the inoculum for cefoxitin-tolerant strains (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003e (d)), and between 0.0001\u0026ndash;0.001% for amikacin-tolerant strains (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003e(e)).\u003c/p\u003e \u003cp\u003eBoth regimens led to an early emergence of cefoxitin-tolerant strains (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003e (b), (c)), which reached\u0026thinsp;\u0026gt;\u0026thinsp;0.1% of the inoculum at D1, a proportion significantly greater than in NT (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003e (d)). The proportion gradually decreased over time; at D10, there was no difference between NT and both regimens (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003e (d)). In all conditions, cefoxitin MIC of the cefoxitin-tolerant strains were stable (8 mg/L, Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eBoth 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\u0026thinsp;\u0026gt;\u0026thinsp;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.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003e (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\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e"},{"header":"Discussion","content":"\u003cp\u003eAs previously found in a HFIM mimicking lung exposure [\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e], 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 [\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e] appeared to prevent the development of amikacin-tolerance. This activity of high-concentration clarithromycin, in addition to its immunomodulatory effect previously described [\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e], 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.\u003c/p\u003e \u003cp\u003eFerro \u003cem\u003eet al\u003c/em\u003e. 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) [\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e]; 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 \u003cem\u003eserB2\u003c/em\u003e, 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].\u003c/p\u003e \u003cp\u003eHerein, 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 [\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e]. 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.\u003c/p\u003e"},{"header":"Abbreviations","content":"\u003cdiv class=\"DefinitionList\"\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eCAMH\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003ecation-adjusted-Muller-Hinton\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eCFU\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003ecolony-forming unit\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eD\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eday\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eHFIM\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003ehollow-fiber infection model\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eHFS\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003ehollow-fiber system\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eMABS\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003e \u003cem\u003eMycobacterium abscessus\u003c/em\u003e \u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eMABSC\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003e \u003cem\u003eMycobacterium abscessus\u003c/em\u003e complex\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eMacrolides-iR\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003einducible resistance to macrolides\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eMIC\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eminimal-inhibitory concentration\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eNT\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003egrowth control without antibiotic\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003c/div\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cem\u003eEthics approval and consent to participate\u003c/em\u003e\u003c/p\u003e\n\u003cp\u003eNot applicable.\u003c/p\u003e\n\u003cp\u003e\u003cem\u003eConsent for publication\u003c/em\u003e\u003c/p\u003e\n\u003cp\u003eNot applicable.\u003c/p\u003e\n\u003cp\u003e\u003cem\u003eAvailability of data and materials\u003c/em\u003e\u003c/p\u003e\n\u003cp\u003eThe datasets used and/or analysed during the current study are available from the corresponding author on reasonable request.\u003c/p\u003e\n\u003cp\u003e\u003cem\u003eCompeting interests\u003c/em\u003e\u003c/p\u003e\n\u003cp\u003eThe authors declare that they have no competing interests.\u003c/p\u003e\n\u003cp\u003e\u003cem\u003eFunding\u003c/em\u003e\u003c/p\u003e\n\u003cp\u003eThis work was supported by the internal \u0026ldquo;Jeunes Chercheurs\u0026rdquo; grant 2021 operated by the Hospices civils de Lyon.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cem\u003eAuthors\u0026apos; contributions\u003c/em\u003e\u003c/p\u003e\n\u003cp\u003eConception: SG, OD, EH\u003cstrong\u003e;\u0026nbsp;\u003c/strong\u003edesign 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.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cem\u003eAcknowledgements\u003c/em\u003e\u003c/p\u003e\n\u003cp\u003eWe thank Shanez Haouari (DRS, Hospices Civils de Lyon) for help in manuscript preparation.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n\u003cli\u003eTo 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.\u003c/li\u003e\n\u003cli\u003eLee 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\u0026ndash;46.\u003c/li\u003e\n\u003cli\u003eGriffith 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\u0026ndash;416.\u003c/li\u003e\n\u003cli\u003eDaley 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.\u003c/li\u003e\n\u003cli\u003eTortoli E, Kohl TA, Brown-Elliott BA, Trovato A, Le\u0026atilde;o 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\u0026ndash;9.\u003c/li\u003e\n\u003cli\u003ePasipanodya 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.\u003c/li\u003e\n\u003cli\u003eFerro 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\u0026ndash;6.\u003c/li\u003e\n\u003cli\u003eHoneybourne 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\u0026ndash;80.\u003c/li\u003e\n\u003cli\u003eRodvold 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\u0026ndash;402.\u003c/li\u003e\n\u003cli\u003eClinical and laboratory standards institute. M24 - Susceptibility testing of Mycobacteria, Nocardia spp., and Other aerobic actinomycetes. 3rd Edition. 2018.\u003c/li\u003e\n\u003cli\u003eSadouki 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\u0026ndash;9.\u003c/li\u003e\n\u003cli\u003eBase de donn\u0026eacute;es publique des m\u0026eacute;dicaments. R\u0026eacute;sum\u0026eacute; des caract\u0026eacute;ristiques du produit - ZECLAR 0,5 g, poudre pour solution \u0026agrave; diluer pour perfusion - [Internet]. [cited 2024 Jul 19]. Available from: https://base-donnees-publique.medicaments.gouv.fr/affichageDoc.php?specid=61433564\u0026amp;typedoc=R#RcpPropPharmacocinetiques\u003c/li\u003e\n\u003cli\u003eBase de donn\u0026eacute;es publique des m\u0026eacute;dicaments. R\u0026eacute;sum\u0026eacute; des caract\u0026eacute;ristiques 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\u0026amp;typedoc=R#RcpPropPharmacocinetiques\u003c/li\u003e\n\u003cli\u003eBase de donn\u0026eacute;es publique des m\u0026eacute;dicaments. R\u0026eacute;sum\u0026eacute; des caract\u0026eacute;ristiques 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\u0026amp;typedoc=R#RcpPropPharmacocinetiques\u003c/li\u003e\n\u003cli\u003eThe 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\u003c/li\u003e\n\u003cli\u003eDavey PG. The pharmacokinetics of clarithromycin and its 14-OH metabolite. J Hosp Infect. 1991;19 Suppl A:29\u0026ndash;37.\u003c/li\u003e\n\u003cli\u003eIsla A, Troc\u0026oacute;niz IF, de Tejada IL, V\u0026aacute;zquez S, Canut A, L\u0026oacute;pez JM, et al. Population pharmacokinetics of prophylactic cefoxitin in patients undergoing colorectal surgery. Eur J Clin Pharmacol. 2012;68:735\u0026ndash;45.\u003c/li\u003e\n\u003cli\u003eClinical and laboratory standards institute. M62 - Performance Standards for Susceptibility testing of Mycobacetria, Nocardia spp., and other aerobic actinomycetes. 1St Edition. 2018.\u003c/li\u003e\n\u003cli\u003ePrammananan 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\u0026ndash;81.\u003c/li\u003e\n\u003cli\u003eBalaban 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\u0026ndash;8.\u003c/li\u003e\n\u003cli\u003eBrauner A, Fridman O, Gefen O, Balaban NQ. Distinguishing between resistance, tolerance and persistence to antibiotic treatment. Nat Rev Microbiol. 2016;14:320\u0026ndash;30.\u003c/li\u003e\n\u003cli\u003eSulaiman JE, Lam H. Evolution of Bacterial Tolerance Under Antibiotic Treatment and Its Implications on the Development of Resistance. Front Microbiol. 2021;12:617412.\u003c/li\u003e\n\u003cli\u003eBernard 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.\u003c/li\u003e\n\u003cli\u003eHerbert S, Barry P, Novick RP. Subinhibitory clindamycin differentially inhibits transcription of exoprotein genes in Staphylococcus aureus. Infect Immun. 2001;69:2996\u0026ndash;3003.\u003c/li\u003e\n\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":false,"highlight":"","institution":"","isAcceptedByJournal":true,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"annals-of-clinical-microbiology-and-antimicrobials","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"cmam","sideBox":"Learn more about [Annals of Clinical Microbiology and Antimicrobials](http://ann-clinmicrob.biomedcentral.com/)","snPcode":"12941","submissionUrl":"https://submission.nature.com/new-submission/12941/3","title":"Annals of Clinical Microbiology and Antimicrobials","twitterHandle":"@BioMedCentral","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"em","reportingPortfolio":"BMC/SO AJ","inReviewEnabled":true,"inReviewRevisionsEnabled":true},"keywords":"Mycobacterium abscessus, hollow-fiber system, hollow-fiber infection model, clarithromycin, amikacin, cefoxitin, pulmonary infection, extra-pulmonary infection","lastPublishedDoi":"10.21203/rs.3.rs-5064595/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-5064595/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003e\u003cem\u003eBackground\u003c/em\u003e\u003c/p\u003e\n\u003cp\u003e\u003cem\u003eMycobacterium abscessus\u003c/em\u003e (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.\u003c/p\u003e\n\u003cp\u003e\u003cem\u003eObjectives\u003c/em\u003e\u003c/p\u003e\n\u003cp\u003eHerein, the aim was to evaluate the effect of the amikacin, cefoxitin, and clarithromycin combination against macrolides-iR MABS in a hollow-fiber infection model.\u003c/p\u003e\n\u003cp\u003e\u003cem\u003eMethods\u003c/em\u003e\u003c/p\u003e\n\u003cp\u003eThe hollow-fiber system was inoculated with \u003cem\u003eM.\u003c/em\u003e \u003cem\u003eabscessus\u003c/em\u003e subsp. \u003cem\u003eabscessus \u003c/em\u003etype 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.\u003c/p\u003e\n\u003cp\u003e\u003cem\u003eResults\u003c/em\u003e\u003c/p\u003e\n\u003cp\u003eUsing 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.\u003c/p\u003e\n\u003cp\u003e\u003cem\u003eConclusions\u003c/em\u003e\u003c/p\u003e\n\u003cp\u003eOur 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.\u003c/p\u003e","manuscriptTitle":"Poor efficacy of the combination of clarithromycin, amikacin, and cefoxitin against Mycobacterium abscessus in the hollow fiber infection model","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2024-11-26 19:24:25","doi":"10.21203/rs.3.rs-5064595/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"decision","content":"Accepted","date":"2025-01-07T10:48:46+00:00","index":"","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2025-01-07T10:27:20+00:00","index":"hide","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2024-12-20T08:35:15+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"272858512771711638595345587532382121352","date":"2024-12-19T15:51:56+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"205650299812048835286619204570898833781","date":"2024-12-06T18:28:22+00:00","index":"hide","fulltext":""},{"type":"reviewersInvited","content":"","date":"2024-12-06T17:37:16+00:00","index":"","fulltext":""},{"type":"checksComplete","content":"","date":"2024-11-25T06:41:11+00:00","index":"","fulltext":""},{"type":"submitted","content":"Annals of Clinical Microbiology and Antimicrobials","date":"2024-11-23T20:01:40+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"annals-of-clinical-microbiology-and-antimicrobials","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"cmam","sideBox":"Learn more about [Annals of Clinical Microbiology and Antimicrobials](http://ann-clinmicrob.biomedcentral.com/)","snPcode":"12941","submissionUrl":"https://submission.nature.com/new-submission/12941/3","title":"Annals of Clinical Microbiology and Antimicrobials","twitterHandle":"@BioMedCentral","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"em","reportingPortfolio":"BMC/SO AJ","inReviewEnabled":true,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"024f517b-041f-4589-afe5-b797dab4759d","owner":[],"postedDate":"November 26th, 2024","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"published-in-journal","subjectAreas":[],"tags":[],"updatedAt":"2025-02-03T16:00:18+00:00","versionOfRecord":{"articleIdentity":"rs-5064595","link":"https://doi.org/10.1186/s12941-025-00776-w","journal":{"identity":"annals-of-clinical-microbiology-and-antimicrobials","isVorOnly":false,"title":"Annals of Clinical Microbiology and Antimicrobials"},"publishedOn":"2025-01-30 15:57:12","publishedOnDateReadable":"January 30th, 2025"},"versionCreatedAt":"2024-11-26 19:24:25","video":"","vorDoi":"10.1186/s12941-025-00776-w","vorDoiUrl":"https://doi.org/10.1186/s12941-025-00776-w","workflowStages":[]},"version":"v1","identity":"rs-5064595","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-5064595","identity":"rs-5064595","version":["v1"]},"buildId":"qtupq5eGEP_6zYnWcrvyt","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}