Evidence of horizontal gene transfer and environmental selection impacting antibiotic resistance evolution in soil-dwellingListeria

22 23 Soil has been identified as a n important reservoir of antibiotic resistance genes (ARGs) 24 and there is a need to understand how corresponding environmental changes influence 25 their emergence, evolution, and spread. As a soil-dwelling bacteri al genus containing 26 important pathogens, Listeria, including L. monocytogenes , the causative agent of 27 listeriosis in humans, could serve as a key model for establishing this understanding. 28 Notably, acquired antibiotic resistance among L. monocytogenes isolated from foods and 29 the environment has been observed in some regions over the past decade. Here we 30 characterized ARGs using 594 genomes representing 19 Listeria species that we 31 previously isolated from soils across the United States. Among the five putatively 32 functional ARGs identified, lin, which confers resistance to lincomycin, was the most 33 prevalent, followed by mprF, sul, fosX, and norB. ARGs were found to be predominant in 34 Listeria sensu stricto species and species more closely related to L. monocytogenes 35 tended to harbor more ARGs. Notably, lin, fosX, and norB showed evidence of recent 36 horizontal gene transfer (HGT) across species, likely through transformation as opposed 37 to conjugation and transduction, while mprF and sul appear to have undergone positive 38 selection. In addition, soil properties and surrounding land use were identified as the most 39 important factors associated with ARG richness and genetic divergence, respectively. 40 Using machine learning, we demonstrated that the presence of ARGs can be predicted 41 from environmental variables with good accuracy (mean auROC of 0.76). Collectively, our 42 data suggest that recent HGT and environmental selection played a vital role in the 43 acquisition and diversification of ARGs in the soil environment. 44 45

antibiotic resistance, Listeria, soil, horizontal gene transfer, environmental 46 selection 47 .CC-BY-NC-ND 4.0 International licenseavailable under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity.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 The copyright holder for this preprintthis version posted June 27, 2024. ; https://doi.org/10.1101/2024.06.27.600992doi: bioRxiv preprint

48 49 Soil is a natural reservoir of antibiotic-resistance genes (ARGs), including ARGs that have 50 been encountered in human pathogens 1,2, playing a pivotal role in the emergence, 51 evolution, and dissemination of microbial antibiotic resistance across diverse 52 ecosystems3,4. Listeria is a soil -dwelling genus of bacteria 5 that includes pathogenic 53 members, such as L. monocytogenes and L. ivanovii. L. monocytogenes causes listeriosis 54 in vulnerable human populations with a notable fatality rate of 20-30%6,7, while L. ivanovii 55 rarely causes listeriosis in humans and is primarily a pathogen of ruminant animals 8. 56 Listeria can be broadly divided into two groups: sensu stricto and sensu lato, based on 57 the relatedness of species to L. monocytogenes, with sensu stricto species being more 58 closely related9. The standard treatment for listeriosis is a combination of penicillin and 59 aminopenicillins (ampicillin or amoxicillin) 9 or ampicillin and gentamicin 10. While the 60 incidence of resistance among clinical L. monocytogenes to these antibiotics, fortunately, 61 remains low at present , intrinsic resistance to cephalosporins exists , and increased 62 resistance to penicillin, trimethoprim, and rifampicin has been observed 9,11–14. 63 Furthermore, high rates of antibiotic resistance in L. monocytogenes have been observed 64 in food -related environments 15, possibly due to prolonged exposure to sublethal 65 concentrations of antimicrobial agents in food processing and agriculture settings 16,17, 66 such as poultries 15,18 and fresh produce factories 19,20. Since Listeria can be transmitted 67 from soils directly to humans,21 or indirectly, via the food production chain22, they could be 68 a key model for understanding how ARGs carried by soil microbes can be potentiated into 69 human pathogens. Establishing a fundamental understanding of the ecological and 70 evolutionary drivers of antibiotic resistance among soil-dwelling Listeria could help to 71 better interpret current and future trends in antibiotic resistance patterns observed in 72 clinical and food isolates. However, most studies of ARGs in Listeria have primarily 73 focused on food-related and clinical isolates of L. monocytogenes18,23–25, resulting in an 74 incomplete understanding of the dynamics of ARGs in Listeria in the natural environment. 75 76 Previous studies indicate that environmental factors, including nutrient availability, 77 temperature, pH, and exposure to natural or anthropogenic chemicals, can exert selective 78 pressure favoring antibiotic resistance26,27. Genes essential for metabolism and behavior, 79 including ARGs, have been observed to undergo positive selection (PS) to adapt to 80 varying environments 28,29. Apart from PS, environmental pressures can expedite 81 horizontal gene transfer (HGT), a pivotal pathway for the evolution of new resistant 82 strains30. HGT typically occurs through three mechanisms, transformation (i.e., the uptake 83 of free DNA from the environment), conjugation (i.e., the direct transfer of genetic material 84 from one bacterium to another through physical contact typically encoded by plasmids or 85 transposons), and transduction (i.e., transfer of genetic material via viruses, such as 86 prophages)31. Existing evidence suggests that the acquisition of ARGs among L. 87 monocytogenes is mediated by HGT. For example, the acquisition of tetracycline and 88 trimethoprim resistances in L. monocytogenes has been experimentally linked to 89 transposons like Tn916 -Tn1545 and Tn6198 32,33. Furthermore, a multi -drug resistant 90 plasmid in L. monocytogenes can be naturally acquired by other closely related bacteria 91 .CC-BY-NC-ND 4.0 International licenseavailable under a (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 The copyright holder for this preprintthis version posted June 27, 2024. ; https://doi.org/10.1101/2024.06.27.600992doi: bioRxiv preprint through transformation, thereby conferring resistance34. Despite the important role of the 92 environment and HGT on antibiotic resistance, the prevalence of ARGs, the extent of HGT 93 and PS acting on them, and the influence of environmental factors on the distribution and 94 evolution of ARGs in soil-dwelling Listeria species remains largely unknown. 95 96 To bridge these knowledge gaps, we comprehensively examined the distribution of ARGs 97 and associated HGT, PS, and environmental factors using in -depth population genetics 98 analyses in a unique nationwide whole -genome dataset for 594 soil -dwelling Listeria 99 strains representing 19 species, including L. monocytogenes. We identified five putatively 100 functional ARGs (i.e., with coverage of > 80% and no premature stop codon detected), lin, 101 mprF, sul, fosX, and norB, predominantly found among Listeria sensu stricto species. HGT 102 of ARGs across Listeria species appeared to be mediated by transformation, rather than 103 conjugation and transduction. We also revealed evidence of environmental selection 104 acting on the richness and genetic divergence of ARGs, with machine learning models 105 applied to predict the carriage of ARGs from environmental variables. This study yields 106 new insights into the dynamics of antibiotic resistance in soils and suggests that 107 environmental disturbance may facilitate the emergence and spread of ARGs among 108 Listeria species. 109 110

111 112 Prevalence and spatial distribution of ARGs among soil-dwelling Listeria 113 114 We identified seven distinct ARGs in soil-dwelling Listeria: lin, mprF, sul, fosX, norB, dfrD, 115 and mphB, with the first five being functional and the latter two being non-functional (Fig. 116 1a). Specifically, lin confers resistance to lincomycin, mprF to defensin, daptomycin, and 117 gallidermin, sul to sulfamethoxazole, fosX to fosfomycin, norB to fluoroquinolones and 118 nalidixic acid, dfrD to trimethoprim, and mphB to erythromycin, telithromycin, quinupristin, 119 pristinamycin IA, and virginiamycin S 35–37. Among the functional ARGs, lin was most 120 prevalent among Listeria isolates (82.66%) followed by mprF (82.32%), sul (81.14%), fosX 121 (60.77%), and norB (58.42%) (Fig. 1a). 122 123 Overall, high richness of functional ARGs was consistently observed in all sensu stricto 124 species, especially L. monocytogenes, L. innocua, L. marthii, L. farberi, L. cossartiae, and 125 L. swaminathanii, but not in sensu lato species (Fig. 1b). While ARGs were present in 126 sensu lato species, nearly all of them were non-functional and the overall prevalence was 127 lower than sensu stricto species (Fig. 1c). Specifically, lin, mprF, and sul were consistently 128 present in all strains of sensu stricto species (n = 491, Supplementary Fig. 1a), with each 129 being functional in 100.00%, 99.59%, and 98.17% of these strains, respectively (Fig. 1b). 130 Functional fosX was found in all sensu stricto species, except for L. seeligeri, L. immobilis, 131 and L. ivanovii. Functional norB was found in all sensu stricto species, except for L. 132 welshimeri (Fig. 1b). Among sensu lato species, functional ARGs were only detected in 133 fosX in one L. booriae strain and one L. rocourtiae strain (Fig. 1b). Notably, ARG richness 134 in Listeria species was highly correlated with their genetic similarity to L. monocytogenes 135 .CC-BY-NC-ND 4.0 International licenseavailable under a (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 The copyright holder for this preprintthis version posted June 27, 2024. ; https://doi.org/10.1101/2024.06.27.600992doi: bioRxiv preprint for both present (Spearman correlation ρ = 0.88, P = 1.2e-06) (Supplementary Fig. 1b) 136 and functional ARGs (Spearman correlation ρ = 0.88, P = 1.3e-06) (Fig. 1d). This indicates 137 that species more closely related to L. monocytogenes tend to manifest a higher richness 138 of ARGs. 139 140 Subsequently, we mapped the distribution of richness for both present ARGs 141 (Supplementary Fig. 1c) and functional ARGs (Fig. 1e). Notably, eastern regions of the 142 United States exhibited higher ARG richness compared to the western regions (Fig. 1e, 143 Supplementary Fig. 1c). The geographic signal of ARG richness appeared to be driven 144 by the distribution of species, given that sensu stricto species were more prevalent in the 145 eastern regions, especially L. monocytogenes, which harbor a high richness of ARGs (Fig. 146 1e, Supplementary Fig. 1c). 147 148 149 150 Fig. 1 | ARG profiles among soil-dwelling Listeria and their na:onal distribu:on. a Prevalence 151 of both present (blue) and func4onal (red) ARGs across genomes. b Propor4on of func4onal ARGs 152 among different Listeria species. c Richness of both present (blue) and func4onal (red) ARGs in 153 different Listeria species. d Correla4on between gene4c similarity to L. monocytogenes and 154 average richness of func4onal ARGs. Gene4c similarity was calculated based on pairwise ANI 155 between different Listeria species and L. monocytogenes. ρ represents the Spearman’s rank 156 correla4on coefficient. e Richness of func4onal ARGs among Listeria genomes across the United 157 States. Circles and crosses indicate genomes with and without ARGs, respec4vely, and are color-158 coded by species. Circle size is propor4onal to the ARG richness calculated in each genome. 159 160 161 .CC-BY-NC-ND 4.0 International licenseavailable under a (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 The copyright holder for this preprintthis version posted June 27, 2024. ; https://doi.org/10.1101/2024.06.27.600992doi: bioRxiv preprint Evidence of HGT and PS of ARGs among soil-dwelling Listeria 162 163 To offer insights into the origin and HGT of the five functional ARGs (i.e., lin, mprF, sul, 164 fosX, and norB), a gene tree was constructed for each ARG, depicted in Figs. 2a-e. If 165 these genes were introduced through speciation, we anticipate a tree topology aligning 166 with the phylogenetic relationships of Listeria species shown in Fig. 3a. However, if HGT 167 occurred, the gene tree would not form distinct clusters based solely on species 168 classification. Based on this assumption, the gene trees suggest that lin (Fig. 2a), fosX 169 (Fig. 2b), and norB (Fig. 2c) likely undergo HGT in certain species, particularly within the 170 sensu stricto species, while mprF (Fig. 2d) and sul (Fig. 2e) may have arisen through the 171 conventional process of speciation. For example, for lin (Fig. 2a), we observed clades that 172 include a mix of L. marthii and L. cossartiae strains, as well as L. innocua and L. farberi 173 strains. Similarly, fosX (Fig. 2b) displayed a comparable pattern among sensu stricto 174 species, with clades containing a mix of L. marthii and L. cossartiae strains, as well as L. 175 welshimeri and L. monocytogenes strains. Notably, the apparent HGT of fosX between L. 176 welshimeri and L. monocytogenes strains serves as an example of gene transfer between 177 pathogenic (L. monocytogenes) and non-pathogenic (L. welshimeri) species. Regarding 178 norB (Fig. 2c), evidence of HGT was observed between L. innocua and L. farberi strains, 179 as well as among L. monocytogenes (pathogenic), L. innocua (non-pathogenic), and L. 180 seeligeri (non-pathogenic) strains. 181 182 183 Fig. 2 | Maximum likelihood gene tree for func:onal ARGs detected among Listeria isolates. a-184 e depicts the gene trees for a lin, b fosX, c norB, d mprF, and e sul, respec4vely, where evidence 185 a d e b clin mprF sul fosX norB L7-1855 lin 761077 L7-1073 lin 827674 L7-1854 lin 173060 L7-0910 lin 330398 L7-0705 lin 802623 L7-1860 lin 578300 L7-0066 lin 703834 L7-0095 lin 73672 L7-1615 lin 19803 L7-1192 lin 756016 L7-1291 lin 169317 L7-1248 lin 740109 L7-1014 lin 818441 L7-1185 lin 777161 L7-0905 lin 820483 L7-1199 lin 318353 L7-1197 lin 326292 L7-0713 lin 550938 L7-1005 lin 761690 L7-0545 lin 144657 L7-0361 lin 313690 L7-1263 lin 804477 L7-1251 lin 71530 L7-1011 lin 78498 L7-1648 lin 793918 L7-1239 lin 210581 L7-0717 lin 823800 L7-1201 lin 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fosX 17577 L7-0601 fosX 80522 L7-0535 fosX 17577 L7-1282 fosX 17552 L7-0719 fosX 17553 L7-1255 fosX 17553 L7-1215 fosX 17553 L7-1197 fosX 80003 L7-0713 fosX 79987 L7-1247 fosX 79987 L7-1585 fosX 17553 L7-1642 fosX 17652 L7-0717 fosX 80001 L7-1535 fosX 17553 L7-1253 fosX 11194 L7-1616 fosX 84241 L7-1648 fosX 17532 L7-1180 fosX 80003 L7-1623 fosX 17552 L7-1201 fosX 80371 L7-1612 fosX 17552 L7-1584 fosX 79970 L7-1590 fosX 79970 L7-1707 fosX 17550 L7-1710 fosX 80002 L7-0393 fosX 79989 L7-0100 fosX 17601 L7-1718 fosX 79973 L7-1705 fosX 80016 L7-0036 fosX 80092 L7-1810 fosX 79971 L7-1600 fosX 18477 L7-1291 fosX 18476 L7-0737 fosX 18474 L7-1855 fosX 80778 L7-1851 fosX 473 L7-1233 fosX 80005 L7-1227 fosX 17552 L7-1846 fosX 80747 L7-1220 fosX 17552 L7-1203 fosX 17542 L7-1209 fosX 17542 L7-1499 fosX 17552 L7-0480 fosX 17551 L7-1862 fosX 17553 L7-1860 fosX 17556 L7-0066 fosX 17702 L7-1787 fosX 80003 L7-1239 fosX 80004 L7-1245 fosX 80002 L7-1041 fosX 17554 L7-1257 fosX 80003 L7-0095 fosX 80053 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fosX 473L7-1591 fosX 6239L7-1587 fosX 6239L7-0106 fosX 81321L7-0192 fosX 17555L7-1303 fosX 17552L7-0387 fosX 17552L7-1320 fosX 17553L7-1275 fosX 17552L7-0693 fosX 80748L7-1251 fosX 17552 L7-0082 fosX 17602L7-1542 fosX 17552L7-1785 fosX 17552L7-1107 fosX 75810L7-1625 fosX 86911L7-1624 fosX 140125L7-0007 fosX 17603L7-0291 fosX 17556L7-0304 fosX 86901L7-0285 fosX 17558L7-0297 fosX 85614L7-0313 fosX 17555L7-0317 fosX 86896L7-0794 fosX 18502L7-0279 fosX 17555L7-0789 fosX 66402L7-0795 fosX 17583L7-0280 fosX 17558L7-0312 fosX 18475L7-0288 fosX 85612L7-0303 fosX 86899 L7-0125 fosX 17581 L7-0796 fosX 17558 L7-0309 fosX 86900 L7-0311 fosX 17581 L7-1067 fosX 17560 L7-1062 fosX 57436 L7-1059 fosX 88320 L7-1699 fosX 84305 L7-1693 fosX 84305 L7-0083 fosX 85635 L7-0091 fosX 17591 L7-0072 fosX 17698 L7-1665 fosX 17614 L7-1669 fosX 50625 L7-1663 fosX 82360 L7-1675 fosX 85565 L7-1670 fosX 85564 L7-1743 fosX 85558 L7-1661 fosX 86865 L7-1726 fosX 17641 L7-1651 fosX 17611 L7-0013 fosX 17602 L7-1723 fosX 17682 L7-1725 fosX 17682 L7-1735 fosX 85789 L7-1750 fosX 3298 L7-1755 fosX 85114 L7-1800 fosX 17620 L7-1758 fosX 84481 L7-0419 fosX 17565 L7-1765 fosX 85139 L7-1737 fosX 85899 L7-0403 fosX 17565 L7-1808 fosX 17587 L7-0845 fosX 17566 L7-1749 fosX 56166 L7-1734 fosX 17564 L7-1731 fosX 17587 L7-1751 fosX 17609 L7-1319 fosX 866855 L7-0094 fosX 104533 L7-1025 norB 269412 L7-0683 norB 242157 L7-0669 norB 236932 L7-0415 norB 268846 L7-0921 norB 39603 L7-0323 norB 259182 L7-0325 norB 247957 L7-1018 norB 264091 L7-0960 norB 242048 L7-0973 norB 239653 L7-0640 norB 266936 L7-0862 norB 108873 L7-0829 norB 263201 L7-0854 norB 231817 L7-0677 norB 255691 L7-1637 norB 259857 L7-0958 norB 307696 L7-1146 norB 267332 L7-0139 norB 235166 L7-1735 norB 265485 L7-1731 norB 6282 L7-1141 norB 251736 L7-1165 norB 272898 L7-0843 norB 11709 L7-1139 norB 313615 L7-0112 norB 73944 L7-1725 norB 246103 L7-1751 norB 144406 L7-1317 norB 265368 L7-1315 norB 265180 L7-1635 norB 186705 L7-0845 norB 57359 L7-0419 norB 268224 L7-1723 norB 270723 L7-1800 norB 256897 L7-1749 norB 266054 L7-1752 norB 269567 L7-1808 norB 245317 L7-0403 norB 254827 L7-1737 norB 182102 L7-1755 norB 74251 L7-1765 norB 265677 L7-0763 norB 275788 L7-0755 norB 276639 L7-1393 norB 5901 L7-0641 norB 265800 L7-0205 norB 264408 L7-0222 norB 264710 L7-1367 norB 264051 L7-1027 norB 212660 L7-0625 norB 249327 L7-0624 norB 235255 L7-0407 norB 258961 L7-0437 norB 153665 L7-0355 norB 155164 L7-1750 norB 256594 L7-0411 norB 262629 L7-0339 norB 272222 L7-0830 norB 5879 L7-1145 norB 230341 L7-0647 norB 238507 L7-0639 norB 267458 L7-0201 norB 280063 L7-0213 norB 255772 L7-0627 norB 264335 L7-1375 norB 229576 L7-0666 norB 266117 L7-0144 norB 273566 L7-0197 norB 238058 L7-0148 norB 233036 L7-1035 norB 267882 L7-0593 norB 116676 L7-1401 norB 57511 L7-0145 norB 242859 L7-0214 norB 270749 L7-0221 norB 269138 L7-0626 norB 270932 L7-0631 norB 230367 L7-0678 norB 266844 L7-0672 norB 264763 L7-0633 norB 266896 L7-0623 norB 266897 L7-0157 norB 214351 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135226 L7-1173 norB 5687 L7-1418 norB 27915 L7-0471 norB 247639 L7-1431 norB 124463 L7-0443 norB 215425 L7-0235 norB 205977 L7-0451 norB 281675 L7-0379 norB 206861 L7-0347 norB 196751 L7-0801 norB 221048 L7-0327 norB 196750 L7-1423 norB 124995 L7-0463 norB 193563 L7-0239 norB 221994 L7-1432 norB 224291 L7-0479 norB 286423 L7-0809 norB 205290 L7-0827 norB 227227 L7-0466 norB 286871 L7-1444 norB 198510 L7-1326 norB 184425 L7-0777 norB 274858 L7-0251 norB 193563 L7-0013 norB 214851 L7-0609 norB 207116 L7-0602 norB 207116 L7-1336 norB 11839 L7-0261 norB 206561 L7-0983 norB 215131 L7-0427 norB 239351 L7-0001 norB 286049 L7-0543 norB 222401 L7-0521 norB 216557 L7-0487 norB 120186 L7-1175 norB 154405 L7-1361 norB 6326 L7-0819 norB 5680 L7-1332 norB 193423 L7-0337 norB 202728 L7-1415 norB 205071 L7-0495 norB 13613 L7-0230 norB 244607 L7-1675 norB 127263 L7-1726 norB 228109 L7-1663 norB 251710 L7-1661 norB 256749 L7-1665 norB 282924 L7-1651 norB 228985 L7-1699 norB 236230 L7-1693 norB 248017 L7-0072 norB 236394 L7-0091 norB 36210 L7-0083 norB 71309 L7-1062 norB 260902 L7-1067 norB 255647 L7-1059 norB 254127 L7-0280 norB 246529 L7-0007 norB 216197 L7-0789 norB 204533 L7-0795 norB 204533 L7-0125 norB 148759 L7-0288 norB 120712 L7-1624 norB 261477 L7-0304 norB 154676 L7-0311 norB 215030 L7-0291 norB 246431 L7-0285 norB 250379 L7-0297 norB 249236 L7-0309 norB 246579 L7-0279 norB 253986 L7-0313 norB 223198 L7-0312 norB 226044 L7-0317 norB 250402 L7-0794 norB 241309 L7-1625 norB 252542 L7-0303 norB 240943 L7-0796 norB 209850 L7-0373 norB 256322 L7-0367 norB 256507 L7-0523 norB 248410 L7-0530 norB 251598 L7-0369 norB 255759 L7-0368 norB 292206 L7-0515 norB 226113 L7-0349 norB 258974 L7-0331 norB 255704 L7-0329 norB 254718 L7-0503 norB 259057 L7-0504 norB 241880 L7-0529 norB 255959 L7-0350 norB 242219 L7-0020 norB 253334 L7-0233 norB 245526 L7-0229 norB 170636 L7-0253 norB 249410 L7-1425 norB 241698 L7-1434 norB 258395 L7-0478 norB 235656 L7-1435 norB 254231 L7-1447 norB 247223 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36466L7-1134 norB 335333 L7-1123 norB 7704 L7-1459 norB 187957L7-1580 norB 187960L7-1453 norB 188434L7-0723 norB 270836L7-1455 norB 262777 L7-0198 norB 21896L7-0934 norB 215442L7-0948 norB 215304L7-0177 norB 203650 L7-0920 norB 7348L7-1153 norB 21718 L7-0922 norB 7347L7-0898 norB 21718L7-1072 norB 22123L7-0181 norB 203650 L7-1626 norB 7378L7-1037 norB 332860 L7-1363 norB 7348L7-0191 norB 22372 L7-0842 norB 7347L7-1465 norB 155822 L7-0904 norB 137809 L7-1630 norB 331461 L7-0559 norB 200152 L7-1348 norB 183683 L7-0579 norB 173920 L7-0185 norB 332545 L7-0573 norB 22108 L7-1342 norB 205160 L7-1358 norB 137872 L7-0748 norB 92156 L7-0199 norB 183755 L7-0207 norB 183894 L7-0783 norB 183659 L7-0586 norB 366290 L7-1357 norB 205878 L7-0567 norB 199605 L7-0580 norB 64420 L7-1356 norB 22144 L7-0171 norB 36470 L7-1343 norB 329286 L7-0186 norB 325692 L7-0617 norB 169690 L7-1560 norB 202275 L7-0888 norB 206614 L7-1510 norB 182371 L7-1558 norB 196597 L7-1578 norB 160412 L7-1485 norB 202289 L7-1554 norB 176397 L7-1515 norB 180328 L7-1517 norB 190625 L7-1519 norB 180222 L7-1509 norB 180594 L7-0121 norB 197895 L7-1049 norB 153061 L7-0973 mprF 10770 L7-0960 mprF 10770 L7-1018 mprF 87211 L7-0669 mprF 85114 L7-0145 mprF 10770 L7-0678 mprF 10769 L7-0649 mprF 10803 L7-0623 mprF 10770 L7-0633 mprF 85780 L7-0144 mprF 10771 L7-0205 mprF 85770 L7-1145 mprF 85929 L7-0624 mprF 85424 L7-0843 mprF 85927 L7-0641 mprF 10770 L7-1367 mprF 10769 L7-0222 mprF 10769 L7-0863 mprF 86707 L7-0866 mprF 10771 L7-0880 mprF 10771 L7-1383 mprF 10793 L7-0874 mprF 10771 L7-0763 mprF 10770 L7-0755 mprF 85781 L7-1025 mprF 10792 L7-0213 mprF 10771 L7-1751 mprF 10815 L7-1101 mprF 83866 L7-1093 mprF 10771 L7-1315 mprF 10770 L7-0830 mprF 10770 L7-0535 mprF 10771 L7-0817 mprF 85698 L7-1027 mprF 85418 L7-0625 mprF 86699 L7-0868 mprF 19466 L7-0593 mprF 10771 L7-1750 mprF 7896 L7-1734 mprF 10770 L7-1735 mprF 90386 L7-1737 mprF 90496 L7-0845 mprF 10772 L7-1808 mprF 10794 L7-0419 mprF 10771 L7-1758 mprF 89079 L7-1800 mprF 10826 L7-1393 mprF 10793 L7-0677 mprF 84502 L7-0221 mprF 85136 L7-0148 mprF 10770 L7-0155 mprF 85774 L7-0672 mprF 10803 L7-1439 mprF 10770 L7-1026 mprF 87206 L7-1146 mprF 10771 L7-0958 mprF 10772 L7-0647 mprF 86055 L7-0639 mprF 86036 L7-0631 mprF 85137 L7-0626 mprF 85137 L7-0131 mprF 10771 L7-0661 mprF 83852 L7-0323 mprF 10770 L7-1139 mprF 84502 L7-1129 mprF 10767 L7-1165 mprF 10812 L7-1141 mprF 84503 L7-1637 mprF 86059 L7-0407 mprF 10815 L7-0683 mprF 10771 L7-1635 mprF 10815 L7-0112 mprF 84820 L7-0410 mprF 10814 L7-0115 mprF 10864 L7-0635 mprF 10773 L7-0653 mprF 85451 L7-0147 mprF 10813 L7-0854 mprF 17786 L7-0926 mprF 10815 L7-0640 mprF 10770 L7-0685 mprF 10771 L7-0214 mprF 10771 L7-0230 mprF 84484 L7-1401 mprF 17631 L7-0339 mprF 10823 L7-0862 mprF 3975 L7-0764 mprF 10770 L7-0218 mprF 85131 L7-1331 mprF 50211 L7-0648 mprF 10770 L7-0655 mprF 10770 L7-0627 mprF 85916 L7-0666 mprF 10842 L7-0157 mprF 10771 L7-0197 mprF 83859 L7-0629 mprF 10771 L7-0546 mprF 10770 L7-0551 mprF 86972 L7-1725 mprF 10888 L7-1723 mprF 10888 L7-0325 mprF 84791 L7-1765 mprF 89737 L7-1310 mprF 10770 L7-1309 mprF 10793 L7-1752 mprF 10770 L7-1731 mprF 10793 L7-1375 mprF 10771 L7-1755 mprF 89712 L7-1749 mprF 60764 L7-1317 mprF 10815 L7-0403 mprF 10771 L7-0829 mprF 10771 L7-0601 mprF 85132 L7-0408 mprF 10815 L7-0411 mprF 10815 L7-0421 mprF 86057 L7-0415 mprF 10827 L7-0399 mprF 10814 L7-0921 mprF 10792 L7-1035 mprF 85924 L7-0355 mprF 85777 L7-0437 mprF 85423 L7-0201 mprF 83857 L7-0139 mprF 83857 L7-0662 mprF 83854L7-0680 mprF 10792L7-0487 mprF 85627L7-0819 mprF 85628L7-1361 mprF 10761L7-0239 mprF 10763L7-0379 mprF 10761L7-0521 mprF 10763L7-0809 mprF 10760L7-0827 mprF 10760L7-0463 mprF 10814L7-0983 mprF 85624L7-0251 mprF 10814L7-1444 mprF 85623L7-0443 mprF 10761L7-0777 mprF 10764L7-0471 mprF 10760L7-1418 mprF 16192L7-1423 mprF 11137L7-0337 mprF 10759L7-0451 mprF 10762L7-1332 mprF 85630L7-0427 mprF 87577L7-0001 mprF 10810L7-0801 mprF 10762L7-0543 mprF 85623L7-1415 mprF 10760L7-0466 mprF 87571L7-0235 mprF 85629L7-1326 mprF 10760 L7-1432 mprF 85656L7-1431 mprF 85656L7-0261 mprF 10761L7-0479 mprF 10760L7-1336 mprF 10765L7-0609 mprF 10765L7-0602 mprF 87581L7-0327 mprF 85629L7-0347 mprF 10760L7-0495 mprF 85634L7-0957 mprF 86320L7-0769 mprF 86321L7-0846 mprF 86320L7-0745 mprF 86320L7-1775 mprF 85760L7-1802 mprF 84467L7-0375 mprF 84483L7-0594 mprF 10767L7-1856 mprF 10767L7-0163 mprF 10767L7-1173 mprF 86353L7-1783 mprF 10767L7-0013 mprF 10810L7-0503 mprF 10772L7-0369 mprF 10771L7-0523 mprF 10828L7-0329 mprF 10768L7-0331 mprF 84485L7-0350 mprF 10873L7-0349 mprF 10770L7-0529 mprF 10772L7-0515 mprF 10773 L7-0368 mprF 10772 L7-0504 mprF 10773 L7-0367 mprF 10774 L7-0373 mprF 10774 L7-0530 mprF 10772 L7-0233 mprF 10768 L7-0253 mprF 85438 L7-0229 mprF 86825 L7-1434 mprF 86180 L7-1425 mprF 86166 L7-1435 mprF 85425 L7-1447 mprF 10771 L7-1426 mprF 86178 L7-0478 mprF 84519 L7-0993 mprF 10771 L7-0123 mprF 84381 L7-0020 mprF 10817 L7-1193 mprF 89205 L7-1854 mprF 10816 L7-1073 mprF 10766 L7-0910 mprF 84951 L7-1187 mprF 10766 L7-0095 mprF 84643 L7-1787 mprF 84594 L7-1818 mprF 12496 L7-1269 mprF 84592 L7-1245 mprF 84596 L7-1215 mprF 10766 L7-1209 mprF 10766 L7-0699 mprF 10766 L7-1248 mprF 84959 L7-1620 mprF 10766 L7-0719 mprF 10766 L7-1868 mprF 10765 L7-1199 mprF 10766 L7-1247 mprF 84578 L7-1205 mprF 10816 L7-0693 mprF 85342 L7-1648 mprF 10746 L7-1616 mprF 88831 L7-0082 mprF 10816 L7-1251 mprF 10766 L7-0045 mprF 87778 L7-1220 mprF 10766 L7-1293 mprF 85338 L7-1860 mprF 10769 L7-0735 mprF 87509 L7-1070 mprF 51305 L7-1835 mprF 10767 L7-1845 mprF 3873 L7-1846 mprF 86855 L7-1832 mprF 10766 L7-1851 mprF 3872 L7-0905 mprF 10766 L7-0066 mprF 10915 L7-0717 mprF 84591 L7-1014 mprF 92128 L7-1320 mprF 10766 L7-1312 mprF 10766 L7-1303 mprF 10766 L7-1011 mprF 10806 L7-1198 mprF 10769 L7-1253 mprF 15785 L7-0489 mprF 90594 L7-0486 mprF 10766 L7-0493 mprF 10766 L7-0545 mprF 85372 L7-1013 mprF 85742 L7-0361 mprF 85372 L7-1646 mprF 87725 L7-1246 mprF 10766 L7-1862 mprF 10766 L7-1838 mprF 84597 L7-1657 mprF 84628 L7-1221 mprF 84632 L7-1295 mprF 84597 L7-1785 mprF 10766 L7-1263 mprF 84592 L7-1642 mprF 10867 L7-1257 mprF 84594 L7-0192 mprF 10766 L7-1287 mprF 86298 L7-1227 mprF 10766 L7-1197 mprF 84594 L7-1180 mprF 84593 L7-1201 mprF 84961 L7-1291 mprF 10766 L7-1275 mprF 10766 L7-1623 mprF 10766 L7-1837 mprF 55787 L7-1829 mprF 10816 L7-0481 mprF 10766 L7-1859 mprF 85736 L7-1606 mprF 10770 L7-1281 mprF 10767 L7-1612 mprF 10766 L7-0096 mprF 10816 L7-0106 mprF 85911 L7-1239 mprF 84598 L7-1203 mprF 10766 L7-1599 mprF 10836 L7-1233 mprF 84595 L7-0713 mprF 84578 L7-1041 mprF 10766 L7-0705 mprF 84604 L7-1255 mprF 10766 L7-1584 mprF 84561 L7-1590 mprF 84561 L7-0708 mprF 10766 L7-1204 mprF 84597 L7-1282 mprF 10766 L7-1210 mprF 84594 L7-1687 mprF 84594 L7-0381 mprF 8053 L7-0384 mprF 10766 L7-0371 mprF 10766 L7-1566 mprF 91338 L7-0031 mprF 10880 L7-1659 mprF 84618 L7-0078 mprF 10878 L7-1706 mprF 10766 L7-0036 mprF 84681 L7-0038 mprF 10816 L7-1710 mprF 84592 L7-0393 mprF 84579 L7-1600 mprF 10767 L7-1005 mprF 10766 L7-1191 mprF 10766 L7-0067 mprF 10816 L7-0060 mprF 91946 L7-1185 mprF 10766 L7-0480 mprF 10766 L7-0964 mprF 10766 L7-0215 mprF 11301 L7-1711 mprF 87513 L7-1717 mprF 10945 L7-1707 mprF 10765 L7-1705 mprF 84605 L7-0100 mprF 10815 L7-1718 mprF 84571 L7-1535 mprF 10766 L7-1601 mprF 10766 L7-1585 mprF 10766 L7-0387 mprF 10766 L7-1587 mprF 10829 L7-1591 mprF 10829 L7-1855 mprF 86292 L7-1866 mprF 10766 L7-1863 mprF 87522 L7-0711 mprF 10766 L7-1810 mprF 84562 L7-0737 mprF 10766 L7-1325 mprF 10766 L7-1192 mprF 87522 L7-1615 mprF 10766 L7-1542 mprF 10766 L7-1499 mprF 10766 L7-1107 mprF 80406 L7-1665 mprF 10816 L7-1661 mprF 92462 L7-1663 mprF 86962 L7-1675 mprF 91162 L7-1670 mprF 91161 L7-1726 mprF 10848 L7-1651 mprF 10818 L7-1743 mprF 90156 L7-0007 mprF 10811 L7-0796 mprF 10765 L7-0795 mprF 10790 L7-0789 mprF 70999 L7-0313 mprF 10762 L7-0794 mprF 10788 L7-0297 mprF 90211 L7-0311 mprF 10789 L7-0279 mprF 10762 L7-0285 mprF 10765 L7-1624 mprF 145644 L7-1625 mprF 91508 L7-0317 mprF 91493 L7-0312 mprF 10761 L7-0309 mprF 91497 L7-0288 mprF 90209 L7-0304 mprF 91498 L7-0280 mprF 10765 L7-0291 mprF 10763 L7-0303 mprF 91496 L7-0125 mprF 10788 L7-1067 mprF 10761L7-1059 mprF 92922L7-1062 mprF 62039L7-1693 mprF 88900L7-1699 mprF 88900L7-0091 mprF 10800L7-0072 mprF 10908L7-0083 mprF 90230L7-1113 mprF 10841L7-0724 mprF 86459L7-1467 mprF 10841L7-1117 mprF 10841L7-0896 mprF 10841L7-0949 mprF 10841L7-0912 mprF 10841L7-1081 mprF 10841L7-1054 mprF 10841L7-0946 mprF 86459L7-0937 mprF 10841L7-1087 mprF 10841L7-1021 mprF 10870L7-0835 mprF 10803L7-1134 mprF 56921L7-0925 mprF 86446L7-1000 mprF 10841L7-1123 mprF 56793L7-1548 mprF 73146L7-0900 mprF 87511L7-1179 mprF 87511L7-1580 mprF 57861L7-1639 mprF 10771L7-1459 mprF 57861 L7-1453 mprF 10821L7-0940 mprF 10771L7-0681 mprF 72086L7-0916 mprF 10771L7-0844 mprF 10771L7-0904 mprF 10770L7-1363 mprF 86424L7-0191 mprF 86423L7-0842 mprF 86425L7-1626 mprF 10770L7-1037 mprF 10770L7-1465 mprF 86424L7-0950 mprF 10770L7-1050 mprF 86424L7-0929 mprF 10770L7-1636 mprF 10770L7-0132 mprF 10770L7-0999 mprF 86455L7-1053 mprF 10806L7-0150 mprF 86455L7-0934 mprF 87693L7-0948 mprF 87693L7-1133 mprF 10870L7-0920 mprF 86424L7-0181 mprF 86424L7-1153 mprF 10771L7-0922 mprF 86424L7-1072 mprF 86424L7-0177 mprF 10771L7-0898 mprF 10771 L7-1581 mprF 10771 L7-0401 mprF 86421 L7-1051 mprF 10771 L7-0970 mprF 86421 L7-1574 mprF 86421 L7-1455 mprF 77L7-0723 mprF 61060 L7-1596 mprF 57594 L7-0875 mprF 10856 L7-0802 mprF 10908 L7-0976 mprF 10907 L7-0179 mprF 86424 L7-0198 mprF 10907 L7-1159 mprF 10856 L7-0679 mprF 10856 L7-1047 mprF 91320 L7-0952 mprF 10841 L7-1630 mprF 10841 L7-1068 mprF 10841 L7-1075 mprF 10841 L7-1357 mprF 10820 L7-0580 mprF 10869 L7-0783 mprF 91278 L7-0186 mprF 10820 L7-0207 mprF 92721 L7-0567 mprF 10820 L7-0586 mprF 10820 L7-0888 mprF 10531 L7-1560 mprF 2637 L7-0748 mprF 85714 L7-1348 mprF 85718 L7-0573 mprF 85718 L7-0559 mprF 85718 L7-1342 mprF 10820 L7-1356 mprF 10870 L7-0579 mprF 85718 L7-0185 mprF 10820 L7-0171 mprF 86702 L7-1358 mprF 10770 L7-1343 mprF 10870 L7-0199 mprF 10820 L7-0617 mprF 10530 L7-1175 mprF 10761 L7-1578 mprF 88737 L7-1509 mprF 88492 L7-1519 mprF 10860 L7-1515 mprF 88492 L7-1558 mprF 10860 L7-1510 mprF 91 L7-1554 mprF 84254 L7-1485 mprF 10860 L7-1517 mprF 10753 L7-0121 mprF 10738 L7-1049 mprF 90545 L7-1025 sul 3877 L7-1439 sul 3874 L7-1027 sul 623 L7-0213 sul 3877 L7-0817 sul 481601 L7-0535 sul 250 L7-0829 sul 3877 L7-0601 sul 3877 L7-0551 sul 3877 L7-0546 sul 3877 L7-0631 sul 398 L7-0626 sul 3877 L7-0973 sul 213 L7-1018 sul 3876 L7-1367 sul 3934 L7-0205 sul 3934 L7-0625 sul 742 L7-0222 sul 3934 L7-1145 sul 495616 L7-0627 sul 3877 L7-0323 sul 3877 L7-0115 sul 592 L7-0629 sul 3877 L7-0655 sul 3877 L7-0648 sul 453 L7-0678 sul 3877 L7-0669 sul 340 L7-0230 sul 214 L7-1750 sul 417 L7-0680 sul 264710 L7-0214 sul 3900 L7-0683 sul 511188 L7-0672 sul 4247 L7-0868 sul 3900 L7-0145 sul 334 L7-0147 sul 3877 L7-1393 sul 3974 L7-1401 sul 3907 L7-0144 sul 3928 L7-0763 sul 3928 L7-0764 sul 3928 L7-0218 sul 3928 L7-0830 sul 3877 L7-1129 sul 3877 L7-0960 sul 170 L7-0325 sul 513176 L7-1309 sul 3924 L7-0666 sul 3900 L7-0640 sul 3900 L7-0641 sul 3935 L7-0139 sul 511951 L7-0201 sul 3877 L7-0624 sul 424 L7-0355 sul 3877 L7-1383 sul 3877 L7-0880 sul 3900 L7-0635 sul 334 L7-0863 sul 334 L7-0866 sul 334 L7-0639 sul 3900 L7-0647 sul 546 L7-0677 sul 3877 L7-1026 sul 3877 L7-0221 sul 3878 L7-0755 sul 552792 L7-0661 sul 453 L7-0197 sul 504135 L7-0131 sul 3877 L7-0662 sul 515998 L7-1146 sul 3877 L7-0112 sul 116857 L7-0157 sul 620 L7-0874 sul 213 L7-1141 sul 213 L7-1165 sul 3877 L7-0408 sul 3877 L7-0921 sul 379 L7-1139 sul 3900 L7-0843 sul 689 L7-0854 sul 213 L7-1375 sul 213 L7-0862 sul 3900 L7-1315 sul 3877 L7-1331 sul 215 L7-0339 sul 3877 L7-0633 sul 3877 L7-0649 sul 3877 L7-0623 sul 3877 L7-0958 sul 3877 L7-1800 sul 3900 L7-0421 sul 3900 L7-0148 sul 235 L7-1035 sul 3900 L7-0593 sul 149303L7-1637 sul 3877 L7-0415 sul 3877 L7-0407 sul 3877 L7-0155 sul 437 L7-0653 sul 3900 L7-0685 sul 3877 L7-1635 sul 3877 L7-0411 sul 3877 L7-0926 sul 3900 L7-1310 sul 3877 L7-1808 sul 346 L7-1752 sul 3877L7-0845 sul 314 L7-1751 sul 417 L7-1735 sul 3877L7-1755 sul 298 L7-1749 sul 3878L7-1734 sul 405 L7-1723 sul 3877 L7-1758 sul 3877 L7-1765 sul 3877 L7-1737 sul 3877L7-1731 sul 337 L7-0419 sul 3877 L7-0403 sul 3877 L7-1725 sul 3877 L7-1317 sul 3877 L7-1093 sul 3877L7-1101 sul 3877 L7-0410 sul 3877L7-0399 sul 417 L7-0437 sul 3877L7-0745 sul 341 L7-1173 sul 3877L7-0594 sul 3877L7-1856 sul 3877L7-0769 sul 288 L7-1783 sul 3901L7-0163 sul 3901L7-0375 sul 214 L7-0846 sul 114235L7-0957 sul 3878L7-1802 sul 3948L7-1775 sul 3877L7-0463 sul 3880L7-0471 sul 289 L7-1431 sul 72231L7-1418 sul 4253L7-1423 sul 3879L7-0251 sul 3880L7-0479 sul 3879L7-0809 sul 3879L7-0827 sul 3879L7-0347 sul 3879L7-0495 sul 289L7-0327 sul 3879L7-0466 sul 4255L7-1336 sul 3879 L7-1415 sul 3879L7-1444 sul 616L7-0543 sul 3879L7-0239 sul 3880L7-0261 sul 3879L7-0487 sul 3879L7-0602 sul 3879L7-0337 sul 289L7-0983 sul 3878L7-0443 sul 290L7-0001 sul 3929L7-0451 sul 4253L7-0609 sul 3879L7-0379 sul 3879L7-1326 sul 3879L7-0427 sul 271L7-0235 sul 4255L7-1332 sul 3879L7-0013 sul 321L7-1361 sul 3830L7-0801 sul 3879L7-0819 sul 3879L7-1432 sul 3808L7-0521 sul 3933L7-0777 sul 4175L7-1699 sul 480955L7-1693 sul 3877L7-0072 sul 481133L7-0091 sul 144270L7-0083 sul 317225L7-1863 sul 3877 L7-0711 sul 3877 L7-1198 sul 271 L7-0095 sul 3927 L7-1787 sul 5604 L7-1818 sul 271 L7-1005 sul 3878 L7-1868 sul 3877 L7-1862 sul 228 L7-0361 sul 3877 L7-0717 sul 3933 L7-0705 sul 256 L7-1616 sul 228 L7-1185 sul 431 L7-1859 sul 3877 L7-0910 sul 339 L7-1325 sul 3877 L7-0708 sul 271 L7-1291 sul 3873 L7-1599 sul 3877 L7-1227 sul 3877 L7-1204 sul 271 L7-1210 sul 271 L7-1014 sul 3876 L7-0545 sul 320 L7-1215 sul 271 L7-0100 sul 330 L7-1295 sul 3877 L7-0393 sul 3877 L7-0031 sul 128204 L7-0078 sul 3927 L7-1011 sul 271 L7-1711 sul 813 L7-1717 sul 458 L7-1612 sul 314 L7-1718 sul 3877 L7-1203 sul 271 L7-1209 sul 3873 L7-1281 sul 3873 L7-1293 sul 340766 L7-1710 sul 344 L7-1248 sul 3877 L7-1205 sul 3926 L7-1199 sul 112569 L7-1620 sul 213 L7-0737 sul 3877 L7-1838 sul 123751 L7-0713 sul 333 L7-0719 sul 3874 L7-1251 sul 3878 L7-1646 sul 3877 L7-1197 sul 3877 L7-1180 sul 3877 L7-1070 sul 3877 L7-1187 sul 231 L7-1623 sul 3931 L7-1866 sul 3877 L7-0693 sul 3876 L7-1193 sul 3873 L7-1312 sul 19403 L7-0082 sul 62433 L7-1657 sul 546 L7-1707 sul 212 L7-1239 sul 3877 L7-1585 sul 3877 L7-1659 sul 352 L7-1303 sul 3877 L7-1705 sul 279 L7-0964 sul 3877 L7-0038 sul 3927 L7-1706 sul 3873 L7-1253 sul 3877 L7-1247 sul 65477 L7-0036 sul 329 L7-1835 sul 213 L7-1845 sul 3877 L7-1851 sul 3876 L7-1832 sul 3877 L7-1587 sul 213 L7-1591 sul 3877 L7-1810 sul 32070 L7-1542 sul 3877 L7-0387 sul 3934 L7-0106 sul 3984 L7-1041 sul 3877 L7-1499 sul 3934 L7-1263 sul 280 L7-1601 sul 308223 L7-1584 sul 109503 L7-1590 sul 3877 L7-1233 sul 64408 L7-0905 sul 275 L7-1855 sul 3950 L7-1642 sul 154798 L7-1221 sul 4024 L7-0066 sul 321 L7-0192 sul 273 L7-0045 sul 3927 L7-1860 sul 339275 L7-1287 sul 3877 L7-1220 sul 3873 L7-1282 sul 3876 L7-1275 sul 67247 L7-1846 sul 3828 L7-0060 sul 338344 L7-0067 sul 314 L7-1269 sul 122410 L7-1245 sul 3874 L7-1687 sul 3877 L7-1191 sul 3877 L7-0215 sul 415 L7-0486 sul 3878 L7-0480 sul 275 L7-1837 sul 3918 L7-1615 sul 3877 L7-1073 sul 3877 L7-0096 sul 435806 L7-0699 sul 3924 L7-1192 sul 271 L7-1854 sul 141531 L7-0735 sul 3877 L7-1013 sul 539 L7-1320 sul 170 L7-1201 sul 3877 L7-0489 sul 339 L7-0493 sul 3877 L7-1648 sul 213 L7-0371 sul 3877 L7-0381 sul 3877 L7-1785 sul 3876 L7-1257 sul 334 L7-1566 sul 516 L7-0384 sul 339 L7-1107 sul 238 L7-1246 sul 3877 L7-1255 sul 339 L7-0481 sul 488996 L7-1829 sul 31860 L7-1606 sul 3877 L7-1600 sul 213 L7-1535 sul 3934 L7-1661 sul 4270 L7-1675 sul 271 L7-1651 sul 478767 L7-1743 sul 171071 L7-1670 sul 3922 L7-1726 sul 3949 L7-1663 sul 3877 L7-1665 sul 3877 L7-1669 sul 3900 L7-1062 sul 3952 L7-1059 sul 3900 L7-1067 sul 3877 L7-0795 sul 3877 L7-0789 sul 281 L7-0007 sul 502883 L7-1625 sul 3877 L7-0291 sul 3877 L7-0303 sul 3877 L7-0279 sul 3877 L7-0794 sul 3915 L7-0312 sul 472641 L7-0288 sul 3923 L7-0304 sul 428 L7-0125 sul 75093 L7-0297 sul 3877 L7-0796 sul 251 L7-0311 sul 463279 L7-0280 sul 3900 L7-0309 sul 3900 L7-0313 sul 473042 L7-0317 sul 3900 L7-1624 sul 3900 L7-0285 sul 3900 L7-0020 sul 4025 L7-0530 sul 3876 L7-0529 sul 3932 L7-0504 sul 3900 L7-0368 sul 3877 L7-0515 sul 515 L7-0367 sul 3876 L7-0373 sul 3899 L7-0331 sul 3900 L7-0349 sul 3900 L7-0523 sul 3877 L7-0350 sul 3900 L7-0503 sul 3877L7-0369 sul 3900L7-0329 sul 3877L7-0478 sul 482690L7-1426 sul 489745L7-1425 sul 490484L7-1447 sul 3877L7-1435 sul 3900L7-1434 sul 3877L7-0229 sul 76898L7-0233 sul 3877L7-0253 sul 3877L7-0123 sul 257L7-0993 sul 213L7-0946 sul 3872L7-0999 sul 3976L7-0912 sul 133208L7-1081 sul 133206L7-0896 sul 315L7-1467 sul 133338L7-0949 sul 134793L7-0937 sul 133682L7-1053 sul 3976L7-1054 sul 3872L7-1117 sul 3872L7-1087 sul 315L7-0150 sul 226L7-1113 sul 3872L7-0724 sul 3872L7-1051 sul 3872L7-0970 sul 97101 L7-1581 sul 97100L7-1574 sul 3872L7-0401 sul 3872L7-1134 sul 3913L7-0723 sul 3873L7-1455 sul 149L7-0950 sul 4529L7-1636 sul 315L7-1021 sul 3872L7-0835 sul 3872 L7-0929 sul 315L7-0802 sul 315L7-1159 sul 315L7-1050 sul 315L7-0198 sul 315L7-0679 sul 3873L7-0976 sul 4529 L7-0875 sul 315L7-0179 sul 4529 L7-0904 sul 558L7-1037 sul 3873 L7-1363 sul 430L7-0842 sul 354L7-0191 sul 3873L7-1626 sul 3873 L7-1465 sul 315L7-0836 sul 3872L7-0920 sul 3872L7-0922 sul 97337 L7-0898 sul 3872 L7-0916 sul 171 L7-1072 sul 171 L7-0177 sul 171L7-1153 sul 3872 L7-0940 sul 3872 L7-0844 sul 248 L7-0681 sul 248 L7-0181 sul 453L7-0934 sul 3913 L7-0948 sul 3913 L7-1179 sul 3872 L7-1000 sul 3947 L7-0900 sul 3872 L7-0925 sul 3947 L7-1548 sul 3947 L7-1123 sul 3913L7-1047 sul 17897 L7-1639 sul 171L7-1596 sul 106697 L7-1459 sul 3872 L7-1453 sul 388 L7-1580 sul 234 L7-1133 sul 3976 L7-1068 sul 17897 L7-1075 sul 17937 L7-0952 sul 17897 L7-1630 sul 3872 L7-0207 sul 225 L7-0580 sul 3976 L7-0567 sul 225 L7-0783 sul 225 L7-1343 sul 3872 L7-0586 sul 225L7-0199 sul 397073 L7-0186 sul 3872 L7-1357 sul 225 L7-0888 sul 3872 L7-1560 sul 158421 L7-0559 sul 3873 L7-0185 sul 3873 L7-0573 sul 262087 L7-0579 sul 262474 L7-1342 sul 455572 L7-1356 sul 458041 L7-1348 sul 262088 L7-0748 sul 3872 L7-1175 sul 3872 L7-0617 sul 3872 L7-0171 sul 242695 L7-1358 sul 171 L7-0132 sul 3976 L7-1049 sul 3925 L7-0121 sul 3873 Listeria species color L. monocytogenes L. marthii L. cossartiae L. swaminathanii L. innocua L. farberi L. welshimeri L. seeligeri L. ivanovii L. immobilis sensu stricto L. booriae L. rocourtiae sensu lato .CC-BY-NC-ND 4.0 International licenseavailable under a (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 The copyright holder for this preprintthis version posted June 27, 2024. ; https://doi.org/10.1101/2024.06.27.600992doi: bioRxiv preprint of HGT was observed in a lin, b fosX, and c norB, while PS is observed in d mprF, and e sul. The 186 trees were constructed using sequences for lin, fosX, norB, mprF, and sul detected in 491, 361, 187 347, 489, and 482 genomes, respec4vely, with 1,000 bootstraps. The evolu4onary models used 188 for construc4ng the tree were TN+F+I+R4, HKY+F+I+R3, TVM+F+I+R4, GTR+F+I+R5, and 189 K3Pu+F+I+R3 for lin, fosX, norB, mprF, and sul, respec4vely. The trees were rooted by the 190 midpoint. Bootstrap values >80% are indicated by light blue circles. 191 192 To understand if maintaining ARGs may offer advantages in fitness to adapt to certain 193 environmental stressors, we performed a gene -wide test for PS for each of the five 194 functional ARGs. Results showed that mprF (Fig. 2d, LRT = 13.932, P = 0.0004718) and 195 sul (Fig. 2e, LRT =17.539, P = 7.77e -05) exhibited a better fit in the alternative 196 unconstrained model (allowing for PS) compared to a null model. This suggests the 197 presence of positive selection in mprF and sul, which were the only ARGs universally 198 present among all 594 Listeria strains, i.e., evidence that these genes confer an advantage 199 under environmental pressures. For the remaining ARGs – lin, fosX, and norB – there is 200 no evidence supporting the presence of PS acting on these three genes (P > 0.05 for all). 201 202 The probable mechanism of HGT of ARGs among soil-dwelling Listeria 203 204 To investigate potential mechanisms underlying the HGT in ARGs of Listeria in soil, we 205 employed a predictive approach focusing on MGEs, including prophages, IS, composite 206 transposons, and plasmids. The presence of ARGs within these MGEs could indicate their 207 role as carriers, informing specific mechanisms of HGT. Among the 594 genomes 208 examined, 1,338 prophages were identified (Fig. 3a). Out of these prophages, only 14.7% 209 (n = 197) were classified as ‘intact’. Of note, we found 12 ‘incomplete’ prophages in L. 210 booriae, 11 of which presented lin and one of which presented norB (Supplementary 211 Table 1). However, these ARGs appeared to be not functional (Fig. 3a). The presence of 212 remnants of ARGs in the prophages suggests a historical role of bacteriophages in 213 transferring ARGs from other species to L. booriae. Apart from these observations, the 214 only instance of recent HGT of a functional ARG potentially mediated by transduction was 215 the norB gene located within a prophage, PHAGE_Bacill_SPbeta_NC_001884, of L. 216 ivanovii L7-1049, a pathogenic species (Fig. 3b, Supplementary Table 1). The prophage 217 region, delimited by the left ( attL) and right ( attR) attachment sites, comprises the norB 218 gene and genes encoding other phage -related proteins and hypothetical proteins ( Fig. 219 3b). The norB gene identified in this prophage was positioned in the first split branch in 220 the norB tree (Fig. 2c), adjacent to the norB of another L. ivanovii strain L7-0121 rather 221 than strains of a different species. This proximity suggests the occurrence of transduction 222 events within this species, not across species. 223 .CC-BY-NC-ND 4.0 International licenseavailable under a (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 The copyright holder for this preprintthis version posted June 27, 2024. ; https://doi.org/10.1101/2024.06.27.600992doi: bioRxiv preprint 224 Fig. 3 | Overview of Listeria species phylogenies, mo:lity genes, competence genes, and MGEs. 225 a The maximum likelihood phylogene4c tree was adapted from a previously published 226 phylogene4c tree for Listeria, constructed from 594 genomes using core SNPs with 200 227 bootstraps29. The tree, rooted by the midpoint, features branches color-coded by Listeria species. 228 Annota4ons include the status (presence/absence) of mo4lity and competence genes, as well as 229 MGEs (plasmids, ISs, and prophages). In the annota4ons, a filled box indicates the presence of a 230 func4onal gene, an empty box indicates a non-func4onal gene (i.e., truncated or with premature 231 stop codons), and a white box indicates the absence of the gene. b Visualiza4on of a prophage 232 carrying a norB gene in L. ivanovii. norB, genes encoding phage-related proteins, and genes 233 encoding hypothe4cal proteins are shown in red, yellow, and purple, respec4vely. The lee and 234 right afachment sites for the phage are referred to as a8L and a8R, respec4vely. 235 236 A total of 4,023 ISs were identified (Fig. 3a), with only 18.3% (n = 735) being classified as 237 'complete'. IS3 and IS1182 families constituted 66.4% ( n = 488) and 15.5% ( n = 114) of 238 the complete IS, respectively. Using ISAbR_finder, we identified an IS -associated ARG 239 that matched with the functional fosX located on the negative strand in L. welshimeri L7-240 1846, showing 100% identity and coverage. The copy of the IS3 transposase was located 241 downstream of fosX on the positive strand, but IS3 transposition involves a copy -out-242 paste-in process that requires at least two copies of IS3 38. Thus, we expect that the IS3 243 transposase under the configuration that it was found would not be sufficient for gene 244 transfer. In addition, no composite transposons carrying ARGs were detected. For 245 plasmids, only 81 were identified across the 594 draft genomes ( Supplementary Table 246 Phage region (145981..173528) in L. ivanovii FSL L7-1049 that contains an ARG. Scale: 5kB attL norB PP_01023 PP_01024 PP_01025 PP_01026 PP_01027 PP_01028 PP_01029 PP_01030 PP_01031 PP_01032 PP_01033 PP_01034 PP_01035 PP_01036 PP_01037 attR PP_01038 b a monocytogenes welshimeri seeligeri booriae innocua marthii cossartiae immobilis farberi fleischmannii grandensis rocourtiae ivanoii weihenstephanensis portnoyi newyorkensis aquatica swaminathanii grayi L7-1507 L7-1629 L7-1645 L7-1641 L7-1523 L7-1479 L7-0741 L7-1572 L7-1582 L7-1614 L7-1621 L7-1319 L7-1681 L7-1698 L7-0435 L7-0058 L7-0033 L7-1299 L7-1658 L7-1769 L7-1063 L7-1071 L7-1250 L7-1773 L7-1069 L7-0729 L7-0035 L7-0039 L7-0054 L7-0051 L7-0094 L7-1594 L7-1588 L7-1529 L7-1586 L7-1541 L7-1547 L7-1497 L7-1491 L7-0859 L7-1017 L7-0149 L7-0153 L7-1387 L7-1385 L7-0335 L7-0767 L7-0217 L7-1844 L7-0259 L7-1831 L7-1761 L7-1843 L7-1850 L7-1834 L7-1848 L7-1816 L7-1826 L7-1824 L7-1830 L7-1827 L7-1836 L7-1825 L7-1842 L7-1833 L7-0256 L7-0269 L7-0273 L7-0459 L7-1427 L7-1430 L7-0255 L7-0236 L7-0278 L7-0245 L7-0232 L7-0231 L7-1409 L7-0994 L7-0978 L7-0979 L7-0990 L7-0984 L7-0029 L7-0360 L7-0030 L7-0025 L7-0815 L7-0811 L7-0816 L7-0587 L7-0527 L7-0605 L7-0818 L7-0510 L7-0370 L7-0385 L7-0519 L7-0514 L7-0084 L7-0509 L7-0520 L7-0751 L7-1517 L7-1510 L7-1554 L7-1485 L7-1578 L7-1558 L7-1515 L7-1519 L7-1509 L7-1049 L7-0121 L7-1175 L7-0185 L7-1356 L7-1348 L7-1342 L7-0573 L7-0579 L7-0559 L7-0748 L7-1560 L7-0888 L7-0617 L7-1358 L7-0171 L7-0567 L7-0586 L7-0186 L7-1357 L7-1343 L7-0580 L7-0199 L7-0783 L7-0207 L7-1596 L7-0723 L7-1455 L7-1580 L7-1459 L7-1453 L7-1123 L7-1134 L7-0925 L7-1000 L7-1548 L7-0900L7-1179L7-1133 L7-0842L7-0191L7-0904L7-1465L7-1363L7-1626L7-1037L7-0835L7-1021L7-0179L7-0802L7-0679L7-0198L7-0875L7-1159L7-0976L7-0934L7-0948L7-0999L7-1053L7-0150L7-0132L7-1050L7-0950L7-1636L7-0929L7-1051L7-1581L7-0970L7-0401L7-1574L7-0920L7-0922L7-1153L7-0898L7-1072 L7-0181L7-0177L7-0836L7-0681L7-0844L7-0916L7-0940L7-1639L7-1630L7-1047L7-0952L7-1068L7-1075L7-1117L7-1054L7-0912L7-0949L7-0946L7-1113L7-0937L7-1467L7-1087L7-0724L7-0896L7-1081L7-1710L7-1711L7-1717L7-0038L7-1623L7-1810L7-1707L7-1705L7-0036L7-1659L7-1706L7-1657L7-1718L7-0031 L7-0393 L7-0100 L7-0078 L7-1838 L7-1303 L7-1600 L7-1601 L7-1542 L7-1499 L7-0387 L7-0106 L7-1535 L7-1585 L7-1587 L7-1591 L7-1312 L7-0964 L7-1325 L7-1257 L7-1041 L7-1107 L7-1851 L7-1845 L7-1835 L7-1832 L7-1829 L7-1846 L7-1837 L7-0384 L7-1566 L7-0381 L7-0371 L7-0215 L7-0486 L7-0480 L7-0493 L7-0489 L7-0192 L7-0066 L7-1192 L7-1615 L7-0545 L7-0361 L7-1005 L7-1013 L7-0082 L7-1193 L7-1251 L7-1612 L7-1187 L7-1201 L7-1180 L7-0699 L7-0708 L7-1210 L7-1263 L7-1070 L7-1197 L7-1014 L7-1233 L7-1295 L7-0713 L7-1269 L7-1245 L7-1215 L7-1204 L7-1590 L7-1584 L7-1221 L7-1239 L7-1599 L7-1282 L7-1293 L7-1247 L7-0719 L7-1220 L7-1209 L7-1203 L7-1281 L7-1291 L7-1287 L7-1227 L7-1275 L7-1320 L7-1011 L7-1205 L7-1199 L7-1248 L7-1620 L7-1859 L7-1818 L7-0705 L7-0910 L7-1854 L7-1073 L7-1616 L7-1648 L7-1185 L7-0067 L7-0060 L7-1642 L7-1687 L7-1606 L7-1191 L7-1253 L7-1246 L7-1255 L7-1787 L7-1785 L7-0717 L7-1868 L7-0693 L7-0095 L7-0905 L7-1855 L7-0737 L7-1198 L7-1863 L7-0711 L7-1860 L7-0735 L7-0096 L7-1866 L7-0481 L7-1862 L7-1646 L7-0045 L7-0083 L7-0091 L7-0072 L7-1699 L7-1693 L7-1669 L7-1665 L7-1663 L7-1675 L7-1661 L7-1651 L7-1670 L7-1743 L7-1726 L7-1059 L7-1067 L7-1062 L7-1625 L7-0007 L7-0125 L7-0304 L7-0288 L7-0796 L7-0794 L7-1624 L7-0789 L7-0795 L7-0317 L7-0279 L7-0291 L7-0311 L7-0280 L7-0313 L7-0303 L7-0297 L7-0312 L7-0285 L7-0309 L7-0020 L7-0123 L7-0993 L7-0233 L7-0229 L7-0253 L7-1434 L7-1426 L7-1435 L7-0478 L7-1425 L7-1447 L7-0368 L7-0369 L7-0523 L7-0530 L7-0504 L7-0529 L7-0367 L7-0373 L7-0503 L7-0515 L7-0329 L7-0350 L7-0331 L7-0349 L7-0495 L7-1418 L7-0471 L7-1423 L7-1431 L7-1432 L7-0463 L7-0251 L7-0347 L7-0327 L7-0337 L7-0543 L7-0521 L7-0379 L7-0466 L7-0487 L7-0819 L7-0479 L7-0801 L7-1361 L7-0827 L7-0809 L7-0443 L7-1444 L7-1415 L7-0001 L7-0451 L7-0427 L7-1332 L7-1336 L7-0602 L7-0609 L7-0261 L7-0983 L7-0235 L7-0239 L7-0013L7-1326L7-0777L7-1802L7-1775L7-0375L7-0957L7-0769L7-0846L7-0163L7-1783L7-0594L7-1856L7-1173L7-0745L7-0230L7-1310L7-1309L7-1800L7-0845L7-1808L7-1315L7-1317L7-0403L7-1735L7-1750L7-1765L7-1752L7-1755L7-1749L7-0419L7-1725L7-1723L7-1734L7-1751L7-1731L7-1758L7-1737L7-0437 L7-0355L7-1439L7-1393L7-1401L7-1101L7-1093L7-0593L7-0546L7-0551L7-0601L7-0829L7-0535L7-0830L7-0817L7-0148L7-1331L7-0662L7-0139L7-0201L7-0661L7-0197L7-0631L7-0626L7-0157L7-0147L7-0629L7-0648L7-0655L7-0678L7-0131L7-0221L7-0669L7-0145L7-0155L7-0218L7-0764L7-0144L7-0763 L7-0755 L7-0921 L7-1635 L7-0112 L7-0421 L7-0415 L7-0410 L7-0407 L7-0325 L7-0115 L7-0926 L7-1637 L7-0399 L7-0323 L7-0408 L7-0411 L7-0627 L7-0339 L7-0214 L7-0685 L7-1375 L7-0683 L7-0653 L7-0677 L7-0672 L7-0623 L7-0649 L7-0633 L7-0680 L7-0213 L7-0868 L7-1141 L7-1165 L7-0635 L7-0666 L7-0624 L7-0639 L7-0647 L7-1027 L7-1025 L7-0863 L7-1383 L7-0880 L7-0866 L7-0874 L7-0640 L7-0625 L7-1367 L7-0641 L7-0205 L7-0222 L7-0862 L7-1139 L7-1145 L7-0843 L7-0854 L7-0958 L7-1129 L7-1035 L7-1026 L7-1146 L7-0960 L7-0973 L7-1018 fosXlmo0919 lmo1695 norB sul lmo0209 lmo0674 lmo0675 lmo0676 lmo0677 lmo0678 lmo0679 lmo0680 lmo0681 lmo0682 lmo0685 lmo0686 lmo0688 lmo0690 lmo0693 lmo0696 lmo0697 lmo0698 lmo0699 lmo0700 lmo0705 lmo0706 lmo0707 lmo0708 lmo0710 lmo0711 lmo0712 lmo0713 lmo0714 lmo0715 lmo0716 comKlmo0945 lmo1341 lmo1342 lmo1344 lmo1347 lmo1397 lmo1483 lmo1484 lmo2189 lmo2512 lmo2513 Tree scale: 10 Other details Antibiotic resistance genes (n) Motility genes (n) Competence genes (n) Plasmids (n) Insertion sequence (%) Prophage (%) Listeria species color sensu stricto L. monocytogenes L. marthii L. cossartiae L. swaminathanii L. innocua L. farberi L. welshimeri L. seeligeri L. ivanoii L. immobilis sensu lato L. grayi L. booriae L. rocourtiae L. newyorkensis L. portnoyi L. grandensis L. weihenstephanensis L. fleischmannii L. aquatica .CC-BY-NC-ND 4.0 International licenseavailable under a (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 The copyright holder for this preprintthis version posted June 27, 2024. ; https://doi.org/10.1101/2024.06.27.600992doi: bioRxiv preprint 2). The most dominant plasmid incompatibility (Inc) family and group were Inc18 (98.8%, 247 n = 80) and repUS25 (96.3%, n = 78), respectively. Still, none of the plasmids identified in 248 this study were found to carry any ARGs. 249 250 Given that conjugation and transduction did not appear to be substantial contributors to 251 the HGT of functional ARGs across Listeria species, we further explored whether natural 252 transformation might play a role. We predicted the presence of competence genes, which 253 signify a bacterium's capacity to uptake foreign DNA, or extracellular DNA (eDNA), from 254 its surroundings and integrate it into its genome39. Listeria sensu stricto species generally 255 possessed significantly more competence genes than Listeria sensu lato species 256 (adjusted Mann-Whitney U P < 0.05 for all; Fig. 3a, Supplementary Table 3). Among 257 Listeria sensu stricto species, competence genes were uniformly present, with over 90% 258 functionality observed for several key competence genes, including comK, coiA, cinA, 259 comEC, and comEB (Fig. 3a). On the contrary, among Listeria sensu lato species, specific 260 competence genes such as comGD, comG, and comGF were completely absent ( Fig. 261 3a). The sole functional competence gene among Listeria sensu lato species was comEC, 262 identified in three L. fleischmannii strains (L7-1629, L7-1641, and L7-1645). 263 264 As motility plays a crucial role in enabling bacteria to move, providing adaptive advantages 265 in new environments40 and facilitating DNA uptake41, we further predicted motility genes 266 across the Listeria genomes. Consistent with competence genes, Listeria sensu stricto 267 species possessed significantly more motility genes compared to sensu lato species 268 (adjusted Mann-Whitney U P < 0.05 for all; Fig. 3a, Supplementary Table 3), which may 269 increase their chance of being exposed to diverse DNA pools in the environment. 270 Specifically, all 31 motility genes were present in every Listeria sensu stricto species, with 271 90% of these genes being identified as functional in more than 99% of strains, except for 272 L. immobilis , the sole sensu stricto species identified thus far that lacks motility 42. In 273 contrast, almost none of the genomes among sensu lato species harbored any functional 274 motility genes. Since functional ARGs and their HGT events were predominately observed 275 among Listeria sensu stricto species, these collective observations regarding the 276 distribution of competence and motility genes suggest that natural transformation may play 277 a substantial role in the HGT of ARGs among Listeria sensu stricto species. 278 279 Environmental factors associated with the richness and genetic divergence of 280 ARGs among soil-dwelling Listeria 281 282 To assess the influence of the environment on ARG acquisition and evolution, we 283 performed correlation analyses between environmental variables and ARG richness and 284 sequence diversification. Considering the high correlation between ARG richness and 285 genetic similarity of isolates to L. monocytogenes (Spearman ρ = 0.88, P < 0.001; Fig. 1d, 286 Supplementary Fig. 1b) and a geographic signal likely driven by species ( Fig. 1e, 287 Supplementary Fig. 1c), genetic similarity could potentially confound identification of true 288 correlations among environmental variables and ARG richness. Thus, Spearman partial 289 correlation analysis, c ontrolling for the genetic similarity of Listeria species to L. 290 .CC-BY-NC-ND 4.0 International licenseavailable under a (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 The copyright holder for this preprintthis version posted June 27, 2024. ; https://doi.org/10.1101/2024.06.27.600992doi: bioRxiv preprint monocytogenes, was performed to investigate the relationship between ARG richness and 291 34 environmental variables. Thirteen environmental variables were significantly correlated 292 with ARG richness (adjusted Spearman P < 0.05 for all). Among these, seven variables, 293 including aluminum, forest, zinc, manganese, iron, longitude, and developed areas with < 294 20% impervious surface, exhibited a positive correlation, while the remaining six: copper, 295 wetland, molybdenum, magnesium, pH, and potassium, showed a negative correlation 296 (Fig. 4a). To further quantify the contributions of different environmental variable 297 categories to the observed variation in ARG richness, VPA was conducted. To exclude 298 potential confounding effects, genetic similarity to L. monocytogenes was also included in 299 this analysis. Of note, the climate category was excluded because no climatic variables 300 were found to be significant (Fig. 4a). As expected, genetic similarity alone accounted for 301 the majority of the variation (adjusted R2, 80.53%) ( Fig. 4b). Among the environmental 302 variables, soil properties explained 7.37% of the variation, similar to the 6.74% explained 303 by land use (Fig. 4b). Geolocation accounted for <1% of variation (Fig. 4b). MDS analysis 304 further demonstrated that isolates with and without functional ARGs formed significantly 305 different clusters based on environmental conditions ( Supplementary Fig. 2 ; 306 PERMANOVA P < 0.001). As functional ARGs were predominately detected in Listeria 307 sensu stricto species (Fig. 1c), we hypothesized that environmental variables significantly 308 differ between Listeria sensu stricto and sensu lato species. Indeed, latitude, minimum 309 temperature, coverage of pasture, cropland, and barren areas were found to be 310 significantly higher for Listeria sensu stricto species, while coverage of shrubland, 311 maximum temperature, precipitation, and several soil properties (aluminum, copper, iron, 312 molybdenum, and moisture) were found to be significantly higher for Listeria sensu lato 313 species (adjusted Mann-Whitney U P < 0.05 for all; Fig. 4c). These results collectively 314 suggest that environmental conditions, particularly soil properties, play a role in ARG 315 acquisition in Listeria. 316 .CC-BY-NC-ND 4.0 International licenseavailable under a (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 The copyright holder for this preprintthis version posted June 27, 2024. ; https://doi.org/10.1101/2024.06.27.600992doi: bioRxiv preprint 317 Fig. 4 | Environmental condi:ons associated with ARG richness and gene:c divergence among 318 the Listeria isolates. a Spearman's par4al correla4on between ARG richness in Listeria genomes 319 and environmental variables, controlled for gene4c similarity for L. monocytogenes. b Venn 320 diagram of varia4on par44oning analysis (VPA) showing the varia4on of the richness of func4onal 321 ARG being explained by the environmental variable groups with significant correla4ons detected 322 (i.e., geographic loca4ons, soil proper4es, and surrounding land use) and gene4c similarity for L. 323 monocytogenes. Residuals indicate unexplained varia4on. c Volcano plot illustra4ng the 324 significance (two-sided Mann-Whitney; y-axis) of the difference between environmental variables 325 (fold change; x-axis) among Listeria sensu stricto and sensu lato species). Variables above the red 326 dashed line have an adjusted P < 0.05. Dots are color-coded by environmental variable types. d 327 ARG predic4on accuracy of random forest model based on environmental variables (mean auROC 328 = 0.76). The light blue lines show ROC curves for models trained only on the training set. Each 329 curve reflects one evalua4on using holdout data, repeated 10 4mes. The dark blue line represents 330 the mean performance across these repe44ons. e The top ten most predic4ve features on overall 331 predic4on of ARG presence/absence in d (SHAP-based; X axis), sorted by descending importance. 332 f Mantel tests between ARG sequence dissimilarity and environmental variables. g Venn diagram 333 of VPA showing the varia4on of the gene4c divergence of norB explained by environmental 334 variable groups. For a and f, significance levels are denoted by “*”, “**”, “***”, and “****” for 335 adjusted P < 0.05, P < 0.01, P < 0.001, and P < 0.0001, respec4vely, and “ns” indicates not 336 significant. 337 .CC-BY-NC-ND 4.0 International licenseavailable under a (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 The copyright holder for this preprintthis version posted June 27, 2024. ; https://doi.org/10.1101/2024.06.27.600992doi: bioRxiv preprint Given the relationship between environmental variables and ARG richness that we 338 observed, we hypothesize that the status (presence/absence) of ARGs in Listeria is 339 predictable using environmental variables. To test our hypothesis, we compared different 340 machine learning algorithms (see Methods) and identified random forest as the best 341 algorithm for predicting the presence of ARGs with environmental variables as features. 342 The model utilized logistic loss to assess the quality of splits, had a maximum depth of 3, 343 considered the logarithm base 2 of the total number of features to determine the best split, 344 and comprised 800 trees in the forest. After hyperparameter tuning ( Supplementary 345 Table 4), the best model achieved a mean auROC of 0.76 (Fig. 4d) and a mean auPR of 346 0.88 (Supplementary Fig. 3). To interpret outputs from the best machine learning model, 347 we utilized SHAP 43 to assess the importance of each feature to the prediction. The ten 348 most influential environmental factors were barren land, precipitation, shrubland, cropland, 349 developed areas with < 20% impervious surface, pasture, latitude, aluminum, elevation, 350 and magnesium ( Fig. 4e). These findings underscore the predictive capability of 351 environmental factors for determining the presence of ARGs in Listeria species. 352 353 Lastly, to investigate the interplay between the genetic divergence of ARGs and 354 environmental factors, Mantel tests were conducted to assess correlations between the 355 sequence dissimilarity of each ARG and the distance of each environmental variable. We 356 identified seven universal variables – geographic distance, pH, potassium, precipitation, 357 maximum and minimum temperatures, and surrounding forest coverage – which exhibited 358 a consistent significant positive correlation with sequence dissimilarity across all five 359 functional ARGs (Mantel P < 0.05 for all; Fig. 4f). Among these ARGs, mprF exhibited 360 correlations with the greatest number of significant environmental variables ( n = 19). To 361 delineate the contribution of environmental variable groups to the observed variation in 362 ARG sequence dissimilarity, we further conducted VPA. Among the ARGs, norB (Fig. 4g) 363 sequence divergence was the most affected by environmental variables, accounting for 364 16.56% of the explained variation. For fosX, mprF, lin, and sul, environmental factors 365 contributed to 12.99%, 7.93%, 6.79%, and 6.26% of explained variation (Supplementary 366 Figs. 4a-d), respectively. Despite the varying contributions of environmental variables to 367 the sequence divergence of different ARGs, a consistent pattern emerged that 368 surrounding land use was the most influential factor across all five ARGs, independently 369 (and collectively) explaining 2.02% (11.13%), 3.04% (6.48%), 1.35% (4.77%), 1.57% 370 (4.42%), and 1.48% (3.45%) of the variation for norB, fosX, mprF, lin, and sul, respectively, 371 compared to other environmental variable groups. Collectively, these results suggest that, 372 like richness, the genetic diversification of ARGs might be influenced by environmental 373 conditions as well. However, instead of soil properties, surrounding land use tends to play 374 a more important role in the diversification of ARGs. 375 376

377 378 ARGs are predominantly present in Listeria sensu stricto species in soils, with 379 limited evidence of the impact of antibiotics used in clinical settings. 380 381 .CC-BY-NC-ND 4.0 International licenseavailable under a (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 The copyright holder for this preprintthis version posted June 27, 2024. ; https://doi.org/10.1101/2024.06.27.600992doi: bioRxiv preprint This study explored the dynamics of ARGs within soil-dwelling Listeria species, including 382 sensu stricto species , which are more closely related to L. monocytogenes, and sensu 383 lato species. We identified five functional ARGs, lin, mprF, sul, fosX, and norB, 384 predominantly in Listeria sensu stricto species. Most of these five ARGs are still present 385 in Listeria sensu lato species, but appear to be non -functional, suggesting that carrying 386 ARGs may cause metabolic costs in these species 44. In contrast, maintaining at least 387 some of these ARGs in the genomes might increase fitness in Listeria sensu stricto 388 species evidenced by the PS in two ARGs, mprF and sul, in these species. The large 389 discrepancy in the prevalence of ARGs between Listeria sensu stricto and sensu lato 390 species may also be partly attributed to the observed different conditions in the soil 391 environment they encounter. 392 393 The five ARGs, lin, mprF, sul, fosX, and norB, identified in soil-dwelling Listeria species in 394 this study are considered intrinsic because they are part of the core genome for L. 395 monocytogenes23,35. The current treatment protocol for listeriosis involves a combination 396 of penicillin and aminopenicillins (ampicillin or amoxicillin)9 or ampicillin and gentamicin10. 397 ARGs conferring resistance to these antibiotics used in clinical treatment, including ampR, 398 aacA4, and aadC, were not detected in Listeria soil isolates included in this study, which 399 suggests that in soil-dwelling Listeria examined in this study have not been influenced by 400 antibiotics used in clinical settings. Indeed, findings from ARG surveillance of L. 401 monocytogenes in food-related and clinical settings in France23, Denmark45, and Spain46 402 consistently report that acquired resistance is limited in L. monocytogenes and this 403 pathogen remains susceptible to antibiotics over time. Therefore, while emerging 404 resistance in L. monocytogenes is observed for certain clinical -use antibiotics, like 405 penicillin11 and rifampicin14, resistance to the antibiotics used in patients with listeriosis 406 (aminopenicillins and gentamicin) remains rare9,23. 407 408 ARGs in Listeria sensu stricto species show evidence of HGT, likely caused by 409 natural transformation. 410 411 HGT events were observed in lin, fosX, and norB among Listeria sensu stricto species, 412 including between pathogenic ( L. monocytogenes ) and non -pathogenic species ( L. 413 welshimeri, L. innocua, and L. seeligeri) in this study. This observation supports the notion 414 that HGT tends to display a bias toward individuals and species that are more closely 415 related47. HGT of ARGs has also been observed among both clinical and food isolates 416 from various L. monocytogenes clonal complexes, with tetracycline resistance identified 417 as the most prevalent acquired resistance phenotype48. This has been primarily attributed 418 to the presence of composite transposons like Tn916 -Tn1545 carrying tetracycline 419 resistance (Tn916-carrying tetM genes) in L. monocytogenes23,32,33. However, we did not 420 identify any tetracycline resistance genes (tetM and tetS) among the Listeria soil isolates 421 examined in this study. This is likely attributed to the widespread use of tetracycline in 422 clinical and food -related environments 49–51, whereas the baseline tetracycline 423 concentrations in less disturbed environments might be low52. 424 425 .CC-BY-NC-ND 4.0 International licenseavailable under a (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 The copyright holder for this preprintthis version posted June 27, 2024. ; https://doi.org/10.1101/2024.06.27.600992doi: bioRxiv preprint Given the limited instances of acquired resistance observed from transposons, prophages, 426 or plasmids in this study, we propose that transduction and conjugation may not be the 427 primary mechanisms for the HGT of ARGs observed in Listeria soil isolates. Instead, we 428 found that natural transformation is the most likely mechanism. Natural transformation 429 relies on the recipient bacterium that expresses the competence machinery53 and largely 430 depends on the uptake and incorporation of exogenous naked DNA from the environment 431 into the genomes of competent recipient organisms 54. We detected a complete set of 432 competence genes that enable the acquisition of foreign DNA in most of the genomes of 433 Listeria sensu stricto species, where HGT events of ARGs were observed. However, 434 despite the presence of genes associated with competence machinery, L. monocytogenes 435 is not recognized as naturally transformable in lab settings55. The absence of competence 436 in L. monocytogenes has been attributed to the truncation of the comK gene, which 437 cleaved into two parts by a 42-kb region containing several ORFs encoding phage-related 438 products56. The regulation of the Com system relies on the formation of a functional comK 439 gene via prophage excision57. In this study, based on the high coverage (> 80%) of comK 440 genes detected in the genomes, this gene does not appear to be truncated in Listeria 441 sensu stricto species. Nevertheless, even with strains containing an intact comK gene, 442 attempts at transforming L. monocytogenes have been unsuccessful under lab 443 conditions56. This suggests that Listeria may require unusual conditions (beyond 444 competence minimal medium, at 37°C, and selection on BHI agar supplemented with 445 chloramphenicol, the specifics of which are still unknown) for competence 56, which 446 complex soil environments may uniquely be able to provide, facilitating HGT of ARGs in 447 Listeria sensu stricto species via natural transformation. 448 449 Environmental selection plays a role in the acquisition and diversification of ARGs 450 among soil-dwelling Listeria. 451 452 Understanding the associations between environmental factors and ARGs is crucial for 453 unraveling the dynamics and evolution of antibiotic resistance under the context of climate 454 change. In this study, we observed that the richness of ARGs in soil -dwelling Listeria is 455 predominantly associated with soil physicochemical properties, such as pH, aluminum, 456 zinc, manganese, iron, copper, magnesium, and potassium. Consistent with this finding, 457 previous studies reported that nanoalumina can enhance the uptake of ARGs by aiding 458 the transfer of plasmid -mediated ARGs58 and zinc can increase ARG abundance by 459 promoting HGT59. However, contrary to the negative correlation identified in this study, 460 some studies found a significant positive correlation between potassium and the 461 coexistence of soil multidrug resistance genes 60. These conflicting findings suggest that 462 the relationship between potassium and ARG richness may be bacterium-dependent. 463 Another noteworthy environmental variable linked to ARG richness was soil pH. Studies 464 have consistently demonstrated that soil pH is a crucial determinant affecting the diversity 465 and composition of ARGs61. Our soil samples generally exhibited slightly acidic conditions 466 (mean pH ± SD 6.6 ± 1.1), and we observed a negative correlation between pH and ARG 467 richness, suggesting that lower soil pH levels are associated with higher ARG richness. 468 This negative correlation might be attributed to the relationship between pH and HGT, 469 .CC-BY-NC-ND 4.0 International licenseavailable under a (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 The copyright holder for this preprintthis version posted June 27, 2024. ; https://doi.org/10.1101/2024.06.27.600992doi: bioRxiv preprint where studies reported that acidic pH conditions promote the potential of HGT, while 470 alkaline pH conditions attenuate HGT 62,63, consequently leading to a change in ARG 471 richness. Given the complexity of natural environments and the co-correlations previously 472 detected between soil property variables used in this study (e.g., between aluminum with 473 calcium, magnesium, manganese, moisture, molybdenum, organic matter, total carbon, 474 total nitrogen, and zinc)29, further experiments are needed to better understand the role of 475 soil properties in influencing ARG richness. 476 477 Besides soil properties, surrounding land use, particularly forest coverage, was found to 478 be positively associated with ARG richness in soil-dwelling Listeria. This is consistent with 479 a previous global study which indicated that forests, irrespective of their location and type 480 (boreal, cold, temperate, or tropical), exhibit the highest richness of ARGs in their soils 4. 481 The elevated presence of ARGs among Listeria in soil environments where surrounding 482 areas have higher coverage of forest may be connected to the movement of wild animals, 483 such as deer 64, bird65, reptiles66, and rodents67 which serve as carriers of ARGs. These 484 genes are subsequently excreted into the soil through fecal matter, contributing to the 485 process of ARG acquisition. 486 487 Surrounding land use was also found to be a key driver of the diversification of ARG 488 sequences in soil-dwelling Listeria. Prior research has highlighted the contribution of land 489 use to the evolution of microbial antibiotic resistance 68. For example, in cropland areas, 490 the extensive use of antibiotics to enhance crop productivity 69 selects specific ARGs. 491 However, it is not clear how surrounding land use may affect the genetic diversification of 492 ARGs. A plausible explanation is that surrounding land use could influence the soil 493 properties of the natural environment, which indirectly impose selective pressures on the 494 genetic diversification of ARGs. For instance, surrounding cropland and pasture coverage, 495 in which we previously detected a positive correlation with soil magnesium29, were found 496 to be associated with the sequence diversification of mprF and norB in this study. 497 Moreover, we found evidence of PS in mprF and sul. There are four significant 498 environmental variables exclusively associated with their sequence divergence, all of 499 which are soil minerals (i.e., aluminum, copper, iron, and zinc). These minerals may induce 500 oxidative stress by generating mineral -induced reactive oxygen species (ROS), which 501 could stimulate genetic mutations 70 and/or represent a selection pressure driving gene 502 evolution70. Overall, the associations between surrounding land use and ARG richness 503 and diversification detected in this study reflect potential anthropogenic effects on the 504 dynamics of microbial antibiotic resistance in the natural environment. 505 506 In addition, we found the majority of environmental variables that were universally 507 significantly and positively correlated with genetic divergence for all five ARGs in this study 508 are climatic factors, including temperature and precipitation. It has been reported that 509 increased temperatures can trigger adaptive responses in organisms, resulting in 510 accelerated mutation rates, heightened genetic diversity, and enhanced genome 511 plasticity71. Elevated temperatures directly contribute to higher mutation rates by 512 promoting replication errors and inducing DNA damage 72, thus elevating the mean 513 .CC-BY-NC-ND 4.0 International licenseavailable under a (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 The copyright holder for this preprintthis version posted June 27, 2024. ; https://doi.org/10.1101/2024.06.27.600992doi: bioRxiv preprint strength of natural selection on genome-wide polymorphism73. Increased temperature also 514 affects various biochemical and biophysical processes within cells74, leading to increased 515 de novo genetic diversity. For instance, using enzyme kinetics theory, it has been shown 516 that higher temperatures are likely to strengthen natural selection throughout the genome 517 by amplifying the effects of DNA sequence variation on protein stability 73. Alternatively, 518 elevated temperatures might impact existing genetic variation by altering allele 519 frequencies in genes or linked genomic regions associated with climate adaptation75. 520 521

522 523 Through leveraging a national reconnaissance of Listeria isolates and further examination 524 of the genetic context of their ARGs, we demonstrated the intrinsic nature of genetic 525 antibiotic resistance traits predominately found in Listeria sensu stricto species in the soil 526 environment. Considering the limited occurrence of prophages and plasmids carrying 527 ARGs and the presence of a full set of functional competence genes in Listeria sensu 528 stricto species, we propose that natural transformation may be the more plausible route 529 for the HGT events observed in ARGs in soil-dwelling Listeria. In contrast, HGT of ARGs 530 appears to be often achieved via conjugation in food and clinical isolates, suggesting that 531 Listeria isolates from different environments may employ distinct HGT mechanisms for 532 ARG acquisition. We also identified evidence of environmental selection likely triggered 533 by soil properties, climate, and surrounding land use in the acquisition and diversification 534 of ARGs in Listeria, highlighting the importance of monitoring the impact of environmental 535 disturbance on the ecology and evolution of microbial antibiotic resistance. Overall, this 536 study provides a baseline understanding of ecological and evolutionary processes 537 governing ARG dynamics in soil environments and demonstrates Listeria as a model 538 organism for elucidating the environmental factors that drive ARG mobilization and 539 diversification across both Listeria pathogenic and non-pathogenic species. 540 541

542 543 Listeria isolates and environmental data 544 545 The genomic data of 594 Listeria strains collected from minimally disturbed natural 546 environments across the contiguous United States, which were described in a previous 547 study examining the mechanism underlying bacterial pangenome evolution29, was further 548 analyzed to consider their carriage of ARGs. These genomes represent 19 Listeria 549 species, predominantly L. monocytogenes (n = 177), followed by L. welshimeri (n = 141), 550 L. seeligeri (n = 98), and L. booriae (n = 90). Other Listeria genomes in our dataset 551 included L. innocua (n = 33), L. marthii (n = 14), L. cossartiae (n = 11), L. immobilis (n = 552 9), L. farberi (n = 5), L. grandensis (n = 3), L. ivanovii (n = 2), and L. rocourtiae (n = 2). 553 Single genomes were available for L. swaminathanii, L. grayi, L. aquatica, L. 554 weihenstephanensis, L. portnoyi, and L. newyorkensis. Among the species included in this 555 study, L. monocytogenes, L. seeligeri, L. marthii, L. ivanovii, L. welshimeri, L. innocua, L. 556 cossartiae, L. farberi, L. immobilis, and L. swaminathanii are classified as Listeria sensu 557 .CC-BY-NC-ND 4.0 International licenseavailable under a (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 The copyright holder for this preprintthis version posted June 27, 2024. ; https://doi.org/10.1101/2024.06.27.600992doi: bioRxiv preprint stricto species (491 genomes total), while others are sensu lato species (103 genomes 558 total)76. 559 560 Previously reported environmental metadata encompassing 34 variables29 paired with this 561 genomic dataset were also examined further in the present study. These environmental 562 variables include 3 spatial (latitude, longitude, and elevation), 17 soil properties (moisture, 563 total nitrogen, total carbon, pH, organic matter, aluminum, calcium, copper, iron, 564 potassium, magnesium, manganese, molybdenum, sodium, phosphorus, sulfur, and zinc), 565 4 climatic (precipitation, wind speed, maximum and minimum temperatures), and 10 566 surrounding land-use (open water, barren, forest, shrubland, grassland, cropland, pasture, 567 wetland, and developed open space categorized as > 20% and < 20% impervious cover)29 568 variables. 569 570 Detection of ARGs, competence genes, flagellar genes, and mobile genetic 571 elements (MGEs) 572 573 ARGs, competence genes, and flagellar genes were identified through BLASTN 574 searches77, using an E-value of 0.01 and without restrictions on percent identity, against 575 a reference database sourced from the BIGSdb -Lm platform35. BIGSdb-Lm is a curated 576 bacterial isolate genome sequence database specializing in L. monocytogenes35. A total 577 of 25 ARGs, 12 competence genes, and 31 flagellar genes were extracted from the 578 platform (Supplementary Table 5)35. The gene with the highest bit -score was chosen 579 for further analysis. Subsequently, the presence of premature stop codons and sequence 580 coverage (%) was assessed for each detected gene. Genes were categorized as 581 putatively functional if their sequence coverage exceeded 80% and no premature stop 582 codon was detected; non-functional if its sequence coverage ranged between 30% - 80% 583 or premature stop codon was detected; and absent if sequence coverage was < 30% or 584 no hits were observed in the BLASTN searches78. Based on this categorization, we further 585 simplified the classification as present but not necessarily functional (referred to as 586 “present gene” hereafter) and putatively functional (referred to as “functional gene” 587 hereafter). 588 589 To minimize the possibility of false negative predictions of ARGs in sensu lato species 590 potentially arising from dissimilarities in ARG sequences to those of L. monocytogenes 591 available in the BIGSdb -Lm database, we conducted cross -validation using two 592 approaches. First, we obtained the reference genome of L. rocourtiae FSL F6 -920 593 (accession number: AODK01) from the NCBI database and predicted its ARGs using our 594 approach described above. This strain was selected because it belongs to the Listeria 595 sensu lato group, and genotypic and phenotypic antibiotic resistance data for this strain 596 are available and published in literature79. Through this analysis, we identified three ARGs: 597 sul, mprF, and fosX. fosX appears to be functional, aligning with its known phenotypic 598 fosfomycin resistance79, while sul and mprF were categorized as “not functional” due to 599 the presence of a premature stop codon ( Supplementary Table 6). However, this strain 600 was found to be phenotypically resistant to sulfamethoxazole (conferred by sul)79, which 601 .CC-BY-NC-ND 4.0 International licenseavailable under a (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 The copyright holder for this preprintthis version posted June 27, 2024. ; https://doi.org/10.1101/2024.06.27.600992doi: bioRxiv preprint was inconsistent with prediction according to the above criteria in this study. The 602 functionality of sul might be attributed to its high coverage (94.6%) and the position of the 603 stop codon in a later part of the gene ( Supplementary Fig. 5). Secondly, we BLASTed 604 our genomes against the Comprehensive Antibiotic Resistance Database (CARD) 37, 605 which includes a more diverse set of reference ARGs than BIGSdb-Lm. A consistent trend 606 and no significant differences in prevalences for present ARGs (paired two-sided t-test P 607 = 0.81) and functional ARGs (paired two-sided t-test P = 0.11) were observed between the 608 predictions from BIGSdb -Lm and CARD ( Supplementary Fig. 6). In addition, BLAST 609 against CARD did not identify any sul, an ARG commonly found in L. monocytogenes23,35. 610 In contrast, it predicted fosXCC, an ARG with a similar function to fosX, and is commonly 611 identified in Campylobacter coli rather than Listeria species80. From this, we conclude that 612 our prediction of ARGs in Listeria species using BLAST against BIGSdb -Lm is robust, 613 sensitive, and conservative. 614 615 To predict MGEs, including plasmids, insertion sequences ( ISs), transposons, and 616 prophages, we employed specialized programs, including PlasmidFinder281 (0.6 cutoff) to 617 identify plasmids; ISEScan 82 and ISAbR_finder83 to detect ISs and ISs associated with 618 ARGs; TnFinder 83 to identify composite transposons; and PHASTER 84 for prophage 619 prediction. All programs were employed with default settings if not specified. 620 Subsequently, a comparison was made to determine if the functional ARGs are present in 621 (for plasmids and prophages) or near (for IS, within 2000 bp83) the predicted MGEs, based 622 on their genomic coordinates. Positive findings were visualized using Gene Graphics85. 623 624 Richness, diversity, and the spatial distribution of ARGs 625 626 The richness and Shannon-Wiener diversity index of present and functional ARGs were 627 computed for each genome using the skbio library in Python 3.6.8. Since a strong positive 628 correlation between ARG richness and diversity was observed (Spearman ρ = 1, P < 10e-629 30; Supplementary Fig. 7), subsequent analyses only focused on ARG richness. To 630 evaluate the association between the ARG richness of Listeria species and their genetic 631 similarity to L. monocytogenes, we averaged the previously reported pairwise average 632 nucleotide identity (ANI)29 for each genome based on their respective species compared 633 with one L. monocytogenes genome and correlated it with the average richness of ARGs 634 (both present and functional) using Spearman's rank correlation analysis. The distribution 635 of ARG richness for Listeria species was visualized using Mercator Projection and the 636 Basemap Matplotlib Toolkit v.1.2.1 in Python v.3.6.8. 637 638 Phylogenetic tree annotation, ARG tree construction, and PS detection 639 640 We annotated a previously published core single -nucleotide polymorphisms (SNPs) -641 based phylogenetic tree of 594 Listeria genomes29 with details about species, ARGs, 642 plasmids, competence genes, flagellar genes, and the proportions of ISs, transposons, 643 and prophages. The annotations were conducted using the Interactive Tree of Life (iTOL) 644 webserver86. 645 .CC-BY-NC-ND 4.0 International licenseavailable under a (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 The copyright holder for this preprintthis version posted June 27, 2024. ; https://doi.org/10.1101/2024.06.27.600992doi: bioRxiv preprint 646 To investigate the potential shared ancestry of ARGs within Listeria, we built a gene tree 647 for each ARG. First, we aligned the nucleotide sequences of each ARG using MUSCLE 648 v.3.8.3187. Then, we built the trees using a maximum likelihood method with 1,000 649 bootstraps in IQ -TREE88. IQ-TREE implements ModelFinder to select the best 650 evolutionary model for the phylogenetic estimates 89. The best-fit models, determined by 651 the Bayesian information criterion (BIC), were TN+F+I+R4, GTR+F+I+R5, K3Pu+F+I+R3, 652 HKY+F+I+R3, and TVM+F+I+R4 for lin, mprF, sul, fosX, and norB, respectively. All gene 653 trees were then visualized through the iTOL webserver86. 654 655 To assess whether PS occurs across the entire gene in the functional ARGs, we utilized 656 the BUSTED (branch-site unrestricted statistical test for episodic diversification) model in 657 HyPhy90. A likelihood ratio test (LRT) was performed, comparing two models: the 658 unconstrained model (allowing for PS) and the constrained model (disallowing PS). 659 Statistical significance was determined by approximating the test statistic to a χ2 660 distribution. ARGs with a P < 0.05 were considered to exhibit evidence of PS, at least at 661 one specific site in the ARG. 662 663 Assessment of the relationships between environmental variables and the richness 664 and diversification of ARGs 665 666 Two-sided Mann–Whitney U tests were employed to identify significant differences for 667 each of the environmental variables between samples positive for Listeria sensu stricto 668 and sensu lato species followed by a Benjamini-Hochberg (BH) false discovery rate (FDR) 669 adjustment to account for multiple testing. An FDR-adjusted Spearman’s partial correlation 670 analysis, controlling for genetic similarity, was performed to evaluate associations between 671 ARG richness and each environmental variable. This approach addresses potential 672 confounding effects of genetic similarity to L. monocytogenes. Environmental variables 673 with an FDR -adjusted P < 0.05 were considered significant. Following this, a variation 674 partitioning analysis (VPA) was conducted, controlling for genetic similarity to L. 675 monocytogenes, to evaluate the relative contribution of each group with significant 676 environmental variables (i.e., geolocation, soil property, climate, or land use) to ARG 677 richness. Both genetic similarity and environmental data were structured in matrix format, 678 enabling the use of adjusted R2 in redundancy analysis (RDA) ordination to partition the 679 variation. VPA was executed and the respective adjusted R2 value was visualized as a 680 Venn diagram using the 'varpart' function in the vegan package v.2.6 -4 within the R 681 environment. Furthermore, multidimensional scaling (MDS) along with a permutational 682 multivariate analysis of variance (PERMANOVA) test was used to compare overall 683 environmental conditions for isolates with and without functional ARGs. PERMANOVA P 684 < 0.05 indicates that there are significant differences in the environmental conditions 685 between groups of isolates with and without functional ARGs. 686 687 A series of Mantel tests were conducted to assess the relationships between the distance 688 matrices of environmental variables and ARG sequences. Briefly, genetic dissimilarity 689 .CC-BY-NC-ND 4.0 International licenseavailable under a (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 The copyright holder for this preprintthis version posted June 27, 2024. ; https://doi.org/10.1101/2024.06.27.600992doi: bioRxiv preprint between genomes for a given ARG was quantified using the Levenshtein distance91, while 690 dissimilarity between genomes for a given environmental variable (excluding longitude 691 and latitude) 29 was calculated using Euclidean distance. Geographic distance was 692 computed based on longitude and latitude using the haversine formula. FDR adjustment 693 was applied to correct multiple testing. VPA was then performed using the calculated 694 distance matrices to assess the relative contribution of each group with significant 695 environmental variables identified to the sequence diversification of ARG. 696 697 Machine learning models to predict the presence of ARGs using environmental 698 variables 699 700 To predict the presence of ARGs with environmental variables as features, we developed 701 an end-to-end machine learning -based framework that embodies a series of individual 702 software programs (e.g., scikit-learn and SHapley Additive exPlanations, SHAP) written in 703 Python for data preparation, hyperparameter tuning, model training, and testing, model 704 evaluation through cross-validation, and visualization. Samples were first cleaned and split 705 into the training set (80%) and the testing set (i.e., the holdout set; 20%) in a stratified 706 fashion. The training set was further split into 5 stratified folds for cross-validation, in which 707 a collection of predefined models (i.e., classifiers based on decision trees, random forest, 708 multilayer perceptron, support vector machines, and gradient boosting) were trained and 709 tested with a random set of hyperparameters ( Supplementary Table 4). The average 710 area under the receiver operating characteristic curve (auROC) score was used to 711 evaluate the performance of the models across the 5 rounds of cross -validation. To 712 account for stochasticity introduced by the random splitting of samples and division of 713 training data into 5 folds, we repeated these steps 10 times. We selected the best model 714 and its hyperparameter set with the highest interquartile mean of the auROC scores out 715 of the 10 repetitions among the predefined models. The interquartile means of the auROC 716 and area under the precision -recall curve (auPR) scores of the best model that was 717 exclusively trained on the training set were reported based on a single evaluation of the 718 holdout data from each of the 10 repetitions. The importance of the features was quantified 719 using SHAP43. 720 721 Data availability 722 723 The data used in this study, including Listeria genomic data and environmental metadata, 724 were previously published in Liao et al, 202129. 725 726 Code availability 727 728 Code to replicate all analyses is available at https://github.com/leaph -729 lab/Soil_Listeria_ARG_manuscript. 730 731 732 .CC-BY-NC-ND 4.0 International licenseavailable under a (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 The copyright holder for this preprintthis version posted June 27, 2024. ; https://doi.org/10.1101/2024.06.27.600992doi: bioRxiv preprint Acknowledgments 733 734 We thank all members of the Liao laboratory for their enriching discussions. This work was 735 funded by the Virginia Tech Center for Emerging, Zoonotic, and Arthropod -borne 736 Pathogens (CeZAP) Interdisciplinary Team -building Pilot Grant (JL) and the Infectious 737 Disease Interdisciplinary Graduate Education Program (ID IGEP) fellowship (YG). The 738 funder played no role in the study design, data collection, analysis and interpretation of 739 data, or the writing of this manuscript. 740 741 Author contributions 742 743 JL designed the study. YG, BA, AN, HZ, and JL analyzed the data. YG wrote the paper 744 with input from JL, MP, LK, and AP. 745 746 Competing interest 747 748 All authors declare no financial or non-financial competing interests. 749 750

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It is made The copyright holder for this preprintthis version posted June 27, 2024. ; https://doi.org/10.1101/2024.06.27.600992doi: bioRxiv preprint Supplementary Figures 992 993 Supplementary Fig. 1 | ARG profiles among soil-dwelling Listeria and their na:onal distribu:on. 994 a Propor4on of present ARGs among different Listeria species. b Correla4on between gene4c 995 similarity to L. monocytogenes and average richness of present ARGs. Gene4c similarity was 996 calculated based on pairwise ANI between different Listeria species and L. monocytogenes. ρ 997 represents the Spearman’s rank correla4on coefficient. c Richness of present ARGs among Listeria 998 genomes across the United States. Circles and crosses indicate genomes with and without ARGs, 999 respec4vely, and are color-coded by species. Circle size is propor4onal to the ARG richness 1000 calculated in each genome. 1001 .CC-BY-NC-ND 4.0 International licenseavailable under a (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 The copyright holder for this preprintthis version posted June 27, 2024. ; https://doi.org/10.1101/2024.06.27.600992doi: bioRxiv preprint 1002 Supplementary Fig. 2 | Mul4dimensional scaling (MDS) analysis for genomes with (red) and 1003 without (blue) ARGs based on environmental condi4ons. PERMONOVA P = 0.001 indicated that 1004 the environmental condi4ons were significantly different for genomes with and without ARGs. 1005 The size of the ellipse is determined by two 4mes the standard devia4on from the mean. 1006 1007 1008 1009 1010 1011 .CC-BY-NC-ND 4.0 International licenseavailable under a (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 The copyright holder for this preprintthis version posted June 27, 2024. ; https://doi.org/10.1101/2024.06.27.600992doi: bioRxiv preprint 1012 Supplementary Fig. 3 | ARG predic4on precision of random forest model based on environmental 1013 variables (mean auPR = 0.88). The light-colored lines show PR curves for models trained only on 1014 the training set. Each curve reflects one evalua4on using holdout data, repeated 10 4mes. The 1015 dark-colored line represents the mean performance across these repe44ons. This model has high 1016 precision and recall for iden4fying ARG instances based on environmental data. 1017 .CC-BY-NC-ND 4.0 International licenseavailable under a (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 The copyright holder for this preprintthis version posted June 27, 2024. ; https://doi.org/10.1101/2024.06.27.600992doi: bioRxiv preprint 1018 Supplementary Fig. 4 | Venn diagram of varia4on par44oning analysis ( VPA) showing the 1019 varia4on of the gene4c divergence of a fosX, b mprF, c lin, and d sul explained by environmental 1020 variable groups, including geographic loca4ons, soil proper4es, climate, and surrounding land 1021 use. Residuals indicate unexplained varia4on. 1022 .CC-BY-NC-ND 4.0 International licenseavailable under a (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 The copyright holder for this preprintthis version posted June 27, 2024. ; https://doi.org/10.1101/2024.06.27.600992doi: bioRxiv preprint 1023 1024 Supplementary Fig. 5 | Amino acid sequence alignment of Sul. DNA sequences for sul were 1025 retrieved from the BIGSdb-Lm database (seq_sul_197) and the BLASTN output (query_sul_197), 1026 respectively. These sequences were then translated into amino acids and aligned using MEGA11 1027 software92 to detect the position of the stop codons. The conserved region for amino acids was 1028 highlighted in yellow, and “*” indicates the identified stop codons. 1029 .CC-BY-NC-ND 4.0 International licenseavailable under a (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 The copyright holder for this preprintthis version posted June 27, 2024. ; https://doi.org/10.1101/2024.06.27.600992doi: bioRxiv preprint 1030 Supplementary Fig. 6 | Comparing the prevalence of a present and b functional ARGs between 1031 the predicted outputs from BIGSdb-Lm and CARD using paired two-sided t-test revealed a 1032 consistent trend. No significant differences in prevalences were observed between the 1033 predictions from BIGSdb-Lm and CARD. BLASTN against CARD did not identify any sul, and BLAST 1034 against BIGSdb-Lm did not predict fosXCC. It is noteworthy that fosXCC, a gene resistant to 1035 fosfomycin with a similar function to fosX, was commonly identified in Campylobacter coli rather 1036 than Listeria species80. 1037 mprF sul lin fosX norB fosXCCPresent ARGs 0.0 0.2 0.4 0.6 0.8 1.0Fraction among Listeria genomes Comparison on the prevalence of present ARGs predicted from BIGSdb-Lm and CARD (Paired two-sided t-test P = 0.8058) BIGSdb-Lm CARD a mprF sul lin fosX norB fosXCCFunctional ARGs 0.0 0.1 0.2 0.3 0.4 0.5 0.6 0.7 0.8Fraction among Listeria genomes Comparison on the prevalence of functional ARGs predicted from BIGSdb-Lm and CARD (Paired two-sided t-test P = 0.1115) BIGSdb-Lm CARD b .CC-BY-NC-ND 4.0 International licenseavailable under a (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 The copyright holder for this preprintthis version posted June 27, 2024. ; https://doi.org/10.1101/2024.06.27.600992doi: bioRxiv preprint 1038 Supplementary Fig. 7 | Spearman’s rank correlation between the richness and diversity of 1039 functional ARGs. ρ represents the Spearman’s rank correlation coefficient. This suggests a strong 1040 positive correlation between ARG richness and diversity. 1041 .CC-BY-NC-ND 4.0 International licenseavailable under a (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 The copyright holder for this preprintthis version posted June 27, 2024. ; https://doi.org/10.1101/2024.06.27.600992doi: bioRxiv preprint Supplementary Tables 1042 1043 Supplementary Table 1 List of incomplete prophages identified by PHASTER, along with 1044 the associated ARGs they contain. 1045 1046 Supplementary Table 2 List of plasmids predicted in the environmental Listeria isolates. 1047 1048 Supplementary Table 3 Statistical comparison for the abundance of motility and 1049 competence genes between Listeria sensu stricto and Listeria sensu lato species. 1050 1051 Supplementary Table 4 Hyperparameters for machine learning models. 1052 1053 Supplementary Table 5 List of ARGs, motility genes, and competence genes retrieved 1054 from BIGSdb-Lm for BLASTN searches. 1055 1056 Supplementary Table 6 BLASTN predicted output for L. rocourtiae FSL F6 -920 1057 (accession number: AODK01). 1058 .CC-BY-NC-ND 4.0 International licenseavailable under a (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 The copyright holder for this preprintthis version posted June 27, 2024. ; https://doi.org/10.1101/2024.06.27.600992doi: bioRxiv preprint