1 Evolutionary Pathways and Trajectories in Antibiotic 2 Resistance 3 4 F. Baqueroa, J.L. Martínezb, V. F. Lanzaa,c, J. Rodríguez-Beltrána, J. C. Galána, A. 5 San Millánb, R. Cantóna, and T.M. Coquea 6 7 aDepartment of Microbiology, Ramón & Cajal University Hospital, Ramón & Cajal 8 Institute for Health Research (IRYCIS), Network Center for Research in Epidemiology 9 and Public Health (CIBERESP), 28034 Madrid, Spain. 10 bNational Center for Biotechnology (CNB-CSIC), 28049 Madrid, Spain 11 cCentral Bioinformatics Unit, Ramón & Cajal Institute for Health Research (IRYCIS), 12 28034 Madrid, Spain 13 14 Address correspondence to: Fernando Baquero ([email protected]) 15 16 17 18 19 20 1 21 SUMMARY ……………………………………………………………………… 12 22 INTRODUCTION……………………………………………………………… 13 23 RESISTANT BACTERIA AND RESISTANCE GENES………………………15 24 Resistant Bacteria and Unsusceptible Bacteria………………………………….18 25 The Antibiotic Resistome………………………………………………………….19 26 What is a Resistance Gene and How Does it Emerge?..........................................22 27 Emergence…………………………………………………………………… 22 28 The infinite universe of pre-resistance bacterial functions ………………...23 29 The limits of the operational definition of resistance gene………………... 26 30 Intrinsic Resistance Genes as Resilience genes………………………………... 27 31 EVOLUTION: UNITS, TOPOLOGIES AND TRAJECTORIES…………… 29 32 What does Evolution Mean when Applied to Antibiotic Resistance?............... 29 33 The Units of Evolution: Evolutionary Individuals…………………………… 31 34 Evolutionary Topology of Antibiotic Resistance Trajectories ……………... 33 35 Evolutionary Trajectories Interactions ………………………………… 35 36 The Question of Causality in Evolutionary Trajectories……………… 36 37 Are there Random Trajectories? Stochastic “Drift” Evolution……… 37 38 Dispersal and spatial structuration as sources of drift…………… 39 39 Fragmentation of infective populations, drift, and resistance…… 40 40 Drift, draft, and trajectories……………………………………… 41 2 41 Genealogical, Across-Network, and Spinning Trajectories………... 43 42 Diversifying and Unifying Evolution in Antibiotic Resistance……… 45 43 44 STEPS ALONG PATHWAYS: SOURCES AND FREQUENCIES OF 45 VARIATION ……………………………………………………………… 48 46 Phenotypic Variation: Bet-Hedging Adaptive Strategies……………… 48 47 Mutations Leading to Antibiotic Resistance …………………………… 51 48 Mutation rates ……………………………………………………………. 51 49 Mutation per species, gene, and day in a single host…………………… 52 50 Hypermutation in the evolution of antibiotic resistance………………. 54 51 Mutational events by insertion …………………………………………. 56 52 Polyploidy and Gene Amplification: from Adaptation to Neo-Functionalization...57 53 Horizontal Gene Transfer……………………………………………………… 60 54 The genes subjected to horizontal gene transfer………………………… 60 55 Gene capture by transposable elements…………………………………… 62 56 Genomes in turmoil: gene acquisition, gene loss…………………………… 62 57 Transferable resistance, recombination, and evolution…………………….. 63 58 59 DRIVERS OF VARIATION AND SELECTION SHAPING TRAJECTORIES 60 UNDER ANTIBIOTIC EXPOSURE ……………………………………………… 64 3 61 Stress and Antibiotics as Drivers of Genetic Variation…………………………… 64 62 Antibiotics as Drivers of Populational Variation ……………………………… 65 63 Selection for Resistant Non-Inheritable Phenotypes………………………… 67 64 Evolution of inducibility of antibiotic resistance mechanisms ………… 67 65 Selection of persistence and the evolution of antibiotic resistance …… 72 66 Selection of Antibiotic Resistance …………………………………………… 74 67 Antibiotic selective concentration gradients in time and space: concentration- 68 dependent selection and multivariate landscapes………………………………. 74 69 The “wicked problem” of modern antibiotic resistance and the history of 70 antimicrobial selective events …………………………………………………… 78 71 Genetic capitalism: resistance traits, global fitness, and evolvability tools... 81 72 Pharmacodynamics and selection of antibiotic resistance………………… 82 73 Strategies of antibiotic use and evolution of resistance; collateral sensitivity, 74 collateral damage………………………………………………………………….. 82 75 Resistance dynamics in the presence of diverse antimicrobials and antiresistance 76 strategies…………………………………………………………………………… 84 77 78 THE ECOLOGY AND TOPOLOGY OF EVOLUTIONARY TRAJECTORIES 79 OF ANTIBIOTIC RESISTANCE ………………………………………………… 85 80 Trajectories and Fitness Landscapes of Antibiotic Resistance………………… 85 81 Trajectories and Flows in Free-Energy Fitness Landscapes…………………... 87 4 82 Evolutionary Trajectories in Crumpled Landscapes…………………………... 88 83 Genotype by Environment Interactions: Environmental Merging and Coalescence 84 of Microbiotas……………………………………………………………………... 90 85 The Role of Environmental Heterogeneity ……………………………………... 91 86 Ecologically Cohesive Populations and Genetic Exchange Communities ……. 94 87 Antibiotic Resistance in Minority Populations………………………………… 95 88 Host-Environment Equilibrium as an Evolutionary Constraint: Evolutionarily 89 Stable Strategies ………………………………………………………………… 96 90 The Eco-Evolutionary Spaces of Gene Variation: Chromosomal Genes versus 91 Mobile Genetic Element Genes ………………………………………………… 97 92 93 EVOLUTIONARY TRAJECTORIES OF ANTIBIOTIC RESISTANCE GENES. 94 ………………………………………………………………………………………… 98 95 The Gene Space of Variation…………………………………………………………98 96 The gene mutational space ……………………………………………………... 98 97 Mutational cost and compensation: mutational robustness………………… 101 98 Antibiotic resistance mutations: fitness costs ……………………………… 102 99 Antibiotic resistance mutations: compensatory evolution………………... 104 100 Mutations competition, cooperation, and founder effects ……………… 105 101 Epigenetic epistasis shaping trajectories………………………………… 106 102 Epistasis and hidden genetic variation…………………………………… 109 5 103 Mutational Paths in Genes Involved in Antibiotic Resistance…………… 110 104 Mutational paths in target-resistance evolution ………………………. 110 105 Mutational paths in variant penicillin-binding protein mediated resistance. 111 106 Mutational paths in variant DNA topoisomerases ……………………… 113 107 Mutational paths and target gene conversion…………………………... 115 108 Mutational paths in evolution of detoxifying enzymes …………………. 116 109 Inactivating enzymes: the case of beta-lactamases …………………. 117 110 The case of aminoglycoside-inactivating enzymes…………………… 124 111 Mutational paths in efflux pumps ………………………………………. 125 112 Evolutionary trajectories of gene complexes…………………………... 126 113 Costs and Benefits of the Acquisition of Foreign Antibiotic Resistance genes and 114 Functions: the Question of Orthogonality……………………………………… 128 115 EVOLUTIONARY TRAJECTORIES OF MOBILE GENETIC ELEMENTS 116 HARBORING RESISTANCE GENES ………………………………………… 129 117 Ecology and Evolution of Mobile Genetic Elements …………………………... 130 118 Plasmids ………………………………………………………………………. 130 119 Transposable elements ……………………………………………………… 135 120 Insertion sequences and insertion sequence derivatives ……………… 136 121 The Tn3 superfamily …………………………………………………… 141 122 The Tn7 superfamily ……………………………………………………. 142 6 123 Tn5053/Tn402-like transposons ………………………………………… 143 124 Tn552-like elements ……………………………………………………… 144 125 Non-autonomous transposable elements …………………………………… 144 126 Genomic islands ……………………………………………………………… 145 127 Bacteriophages and phage-related particles ……………………………… 146 128 Flow of Mobile Genetic Elements and Antimicrobial Resistance Genes …… 149 129 The environmental context of antibiotic resistance gene flow …………... 149 130 Gene flow and DNA uptake proficiency…………………………………… 152 131 Gene flow and conjugation proficiency…………………………………….. 153 132 Gene flow and the acceptability to foreign genes: defence systems……… 153 133 Barriers between prokaryotic groups and antibiotic resistance gene flow.. 154 134 Barriers determined by interactions between mobile genetic elements…….156 135 Mobile genetic elements dispersal within species ……………………………157 136 Intracellular dynamics of mobile genetic elements………………………… 159 137 The intracellular “evolution laboratory” for antibiotic resistance……….. 161 138 139 The Ecogenetics of Antibiotic Resistance Transfer and Maintenance: Antibiotic 140 Resistance Genes in the Accessory Genome……………………………………… 161 141 Trajectories of accessory genome genes in Gammaproteobacteria…………... 162 142 Trajectories of accessory genome genes in Firmicutes………………………... 163 7 143 Evolutionary Kin Hindrances and Shortcuts: the Role of Relatedness………… 163 144 Facilitated recombination between gene families…………………………….. 164 145 Facilitated gene transfer among relatives: species and clones……………… 165 146 The eco-evolutionary advantages of relatedness: kin selection……………… 165 147 148 EVOLUTIONARY TRAJECTORIES OF ANTIBIOTIC-RESISTANT CLONES 149 AND SPECIES ……………………………………………………………………... 166 150 Evolutionary Dynamics of Resistant Clones ……………………………………... 169 151 The basic theoretical background: Red Queen, Stationary, and microbiota-on-a- 152 leash models……………………………………………………………………… 169 153 Clonal fluctuations and evolutionary rescue…………………………………… .171 154 The structure of clonal fluctuations…………………………………………… 173 155 New hosts invasion-driven genetic variation ……………………………... 173 156 Intra-clonal diversification within hosts………………………………… 174 157 Clonalization quashing, genetic and niche variation……………………. 174 158 Variation fostering cloud or bunch clonal selection……………………... 175 159 Clonal variation triggering community selection………………………... 175 160 Clonal diversity and antibiotic resistance………………………………... 176 161 Clonal fluctuations and antimicrobial resistance………………………... 177 162 Traveling clonal waves and antibiotic resistance………………………..