9Mutations conferring resistance to one antibiotic can increase (cross resistance) or 10 decrease (collateral sensitivity) resistance to others. Drug combinations displaying 11 collateral sensitivity could be used in treatments that slow resistance evolution. 12However, lab-to-clinic translation requires understanding whether collateral effects 13 are robust across different environmental conditions. Here, we isolated and 14 characterized resistant mutants of Escherichia coli using five antibiotics, before 15 measuring collateral effects on resistance to other antibiotics. During both isolation 16 and phenotyping, we varied conditions in ways relevant in nature (pH, temperature, 17 bile). This revealed local abiotic conditions modified expression of resistance against 18 both the antibiotic used during isolation and other antibiotics. Consequently, local 19 conditions influenced collateral sensitivity in two ways: by favouring different sets 20 of mutants (with different collateral sensitivities), and by modifying expression of 21 collateral effects for individual mutants. These results place collateral sensitivity in 22 the context of environmental variation, with important implications for translation 23 to real-world applications. 24 exposed to the same antibiotic sometimes acquire collateral sensitivity to another 48 antibiotic, and sometimes do not 10,11 . This can be explained by different mutations, 49 which vary in their phenotypic effects on resistance, spreading in different replicate 50 populations 11,12,14,15 . However, we know from past work that phenotypic effects of 51 antibiotic resistance mechanisms also vary strongly depending on local 52 environmental conditions [16][17][18] . For example, bile can upregulate efflux pumps 19 , zinc 53 can reduce the activity of aminoglycoside degrading enzymes 20 , and high 54 temperature can modulate the effects of rifampicin-resistance mutations on growth 55 in the absence of antibiotics 21 . This raises the possibility that local environmental 56 conditions could influence both the emergence of collateral sensitivity (by affecting 57 which of the possible pathways to resistance are most strongly selected during 58 antibiotic exposure) and its expression (by modifying the phenotypic effects of 59 resistance alleles when bacteria are exposed to a second antibiotic). To date, 60 research on collateral sensitivity interactions has focused on testing many 61 combinations of antibiotics 5,6,9,14 , multiple strains 10 , or many replicate populations 62 for individual antibiotic combinations 11 . Therefore, the role of local abiotic 63 conditions in the emergence and expression of collateral sensitivity interactions 64 remains unclear. Answering this question would improve our understanding of the 65 robustness of collateral sensitivity across different populations and environments. 66This would in turn boost our ability to predict pathogen responses to treatment 67 regimens that exploit collateral sensitivity interactions. 68
69To address these gaps in our knowle...