bioRxiv preprint doi: https://doi.org/10.1101/2020.10.23.352104; this version posted October 23, 2020. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY 4.0 International license. 1 Evolution of Antibiotic Tolerance Shapes Resistance Development in Chronic 2 Pseudomonas aeruginosa Infections 3 4 Isabella Santi*, Pablo Manfredi*, Enea Maffei, Adrian Egli1,2, Urs Jenal 5 6 7 Affiliations 8 Biozentrum, University of Basel, Klingelbergstrasse 50, Basel, Switzerland 9 1 Division of Clinical Bacteriology and Mycology, University Hospital Basel, Basel, 10 Switzerland 11 2 Applied Microbiology Research, Department of Biomedicine, University of Basel 12 * These authors contributed equally to this work 13 14 For correspondence: [email protected] 15 16 17 18 19 20 21 22 Author Contributions 23 24 I.S., P.M. and U.J. designed the study; I.S., P.M., E.M and A.E. collected and 25 processed data; I.S. and P.M. performed the analyses; and I.S., P.M. and U.J. wrote 26 the paper. 27 Competing interests 28 29 The authors declare no competing financial interests. 30 31 32 33 34 1 bioRxiv preprint doi: https://doi.org/10.1101/2020.10.23.352104; this version posted October 23, 2020. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY 4.0 International license. 35 Abstract 36 The widespread use of antibiotics promotes the evolution and dissemination of 37 resistance and tolerance mechanisms. To assess the relevance of tolerance and its 38 implications for resistance development, we used in vitro evolution and analyzed in- 39 patient microevolution of Pseudomonas aeruginosa, an important human pathogen 40 causing acute and chronic infections. We show that the development of tolerance 41 precedes and promotes the acquisition of resistance in vitro and we present evidence 42 that similar processes shape antibiotic exposure in human patients. Our data suggest 43 that during chronic infections, P. aeruginosa first acquires moderate drug tolerance 44 before following distinct evolutionary trajectories that lead to high-level multi-drug 45 tolerance or to antibiotic resistance. Our studies propose that the development of 46 antibiotic tolerance predisposes bacteria for the acquisition of resistance at early 47 stages of infection and that both mechanisms independently promote bacterial 48 survival during antibiotic treatment at later stages of chronic infections. 49 50 51 Introduction 52 The great therapeutic achievements of antibiotics have been dramatically 53 undercut by the steady evolution of survival strategies allowing bacteria to overcome 54 antibiotic action 1,2. Although resistance plays a major role in antibiotic-treatment 55 failure, bacteria can use resilience mechanisms such as tolerance to survive antibiotic 56 treatment 3. Whereas experts and the public are well aware of problems related to 57 increasing resistance, pathogen tolerance is not common knowledge, despite of being 58 responsible for substantial morbidity and mortality 4. Resistance is generally drug 59 specific and can be enhanced genetically through mutations modifying the drug target 60 or by the acquisition of accessory genes such as efflux pumps 5. Such events lead to a 61 decrease of the effective antimicrobial concentration and an increase of the minimum 62 inhibitory concentration (MIC), which corresponds to the lowest drug concentration 63 needed to prevent pathogen growth. In contrast, tolerance can lead to persistent 64 infections despite a seemingly efficient treatment. In this case, a residual fraction of 65 pathogens can resume growth after treatment was stopped, leading to infection 66 relapses. For example, tolerance alters the kinetics of antibiotic killing without 67 affecting MIC, leading to prolonged treatment necessary for pathogen eradication. 68 Tolerance is thus measured by the minimum duration of killing of a specific fraction 2 bioRxiv preprint doi: https://doi.org/10.1101/2020.10.23.352104; this version posted October 23, 2020. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY 4.0 International license. 69 of the population 6. This phenotype has been related to non- or slow-growing bacteria 70 that are able to survive bactericidal antibiotics for extended times 7. Tolerance can be 71 adopted by all cells of a bacterial culture or only by a subpopulation called persisters 72 8. Bacterial populations with fractions of persisters are characterized by biphasic 73 killing during treatment with bactericidal agents, where an initial rapid killing phase is 74 followed by a phase of reduced killing 7. 75 Recent studies have shown that bacteria can rapidly evolve tolerance and 76 persistence when exposed to antibiotics in vitro 9-12, suggesting that both represent 77 successful strategies for bacteria to survive antibiotic treatment. In line with this, 78 treatment efficacy during chronic infections was shown to be lost progressively 79 without significant resistance development 13,14. Although challenging to diagnose 15, 80 tolerant variants exist among environmental bacteria 16 and clinical isolates of human 81 pathogens 17-19. Recently, it was proposed that antibiotic tolerance facilitates the 82 evolution of drug resistance in laboratory conditions 11,20. However, it is still unclear 83 if antibiotic tolerance plays a role in persistent infections and treatment failure 14,19,21 84 and if tolerance can facilitate resistance development in human patients 10,15,20. 85 Cystic fibrosis (CF) is the most common life-limiting, autosomal recessively 86 inherited disease in Caucasian populations with the primary cause of death being 87 respiratory failure resulting from chronic pulmonary infection 22. CF patients have 88 reduced lung clearance capacity leading to the development of lifelong chronic 89 infections caused by opportunistic bacterial pathogens such as Pseudomonas 90 aeruginosa. Over time, treatment efficacy gradually declines and increasing 91 inflammatory damage leads to fatal outcome 23. While infections are generally 92 initiated by non-host adapted (i.e. naïve) strains found in the environment 24-26, P. 93 aeruginosa undergoes significant microevolution during chronic infections of CF 94 lungs 13,27,28. Despite recurrent application of high doses of antibiotics, a significant 95 fraction of clinical isolates remains drug-sensitive 13,14. This argues that resistance 96 development may not fully explain long-term survival of pathogens in the lung and 97 that other strategies such as antibiotic tolerance contribute to the highly persistent 98 nature of such infections. 99 Here we show that a substantial fraction of P. aeruginosa isolates from CF 100 lungs has retained drug susceptibility, but has evolved various degrees of multi-drug 101 tolerance. We demonstrate that recurrent exposure to high concentrations of 102 antibiotics leads to the rapid development of tolerance, which generally precedes and 3 bioRxiv preprint doi: https://doi.org/10.1101/2020.10.23.352104; this version posted October 23, 2020. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY 4.0 International license. 103 boosts resistance development in P. aeruginosa. We show that tolerant strains display 104 population heterogeneity with slow-growing subpopulations and that tolerance- 105 mediated fitness costs can lead to the rapid loss of this phenotype after populations 106 have acquired high-level resistance. Based on phenotypic and genotypic analysis of 107 CF isolates, we propose that tolerance and resistance are alternative strategies 108 contributing to P. aeruginosa persistence during long-term chronic infections. 109 110 Results 111 P. aeruginosa develops multi-drug tolerance during chronic infections of CF 112 lungs. 113 To explore the role of antibiotic tolerance in patients, we analyzed a large set of 114 P. aeruginosa isolates (n=539) including strains from chronically infected CF patients 115 (n=472), isolates from acute infections (n= 58 strains from 58 patients) and non-host 116 adapted control strains from laboratory or environmental origins (n=9). CF patient 117 isolates were sequentially isolated from 91 patients from five to 61 years old. For each 118 strain we analyzed antibiotic resistance profiles (MIC) as well as the ability to survive 119 exposure to tobramycin and ciprofloxacin over time (Fig. 1a; Extended data Fig. 120 1a,b). We chose these antibiotics because they have different modes of action and 121 because they are used by clinicians to treat CF patients 29. Determination of MIC 122 breakpoints according to clinical standards 30 revealed that clinically resistant strains 123 were more common among isolates from chronic situations (22% tobramycin; 30% 124 ciprofloxacin) compared to isolates from acute infections (6% tobramycin; 17% 125 ciprofloxacin). When analyzing the survival of isolates with MIC values below the 126 clinical breakpoint 30 during exposure to high concentrations of tobramycin or 127 ciprofloxacin we observed large