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Supplementary datas

Supplementary data 1

Fig S1. A total of 459 K. pneumoniae clinical isolates collected from six provinces of

China with different carbapenem resistance rates.

Fig S2. The R-M units profiles (excluding 14 major clades) identified in K. pneumoniae.

Table S1.

Oligonucleotides for PCR

Table S2. The distribution of type I R-M systems in 459 K. pneumoniae clinical isolates

Table S3. Example of type I R-M systems matches on clinical plasmid p187-2

Supplementary data 2

Maximum Likelihood-based phylogenetic tree (Jukes–Cantor model) based on aligned

amino acid sequences of HdsM of RM positive K. pneumoniae genomes

Supplementary data 3

Supplementary data 3a. 217 completely sequenced K. pneumoniae strains used in this

study

Supplementary data 3b. Type I R-M systems linked with plasmids

Supplementary data 3c. Type I R-M information of 217 completely sequenced K.

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Fig S1. A total of 459 K. pneumoniae clinical isolates collected from six provinces

of China with different carbapenem resistance rates. Carbapenem resistance rates

of K. pneumoniae among different provinces of China in 2017 was taken from data of

China Antimicrobial Resistance Surveillance System (CARSS)*. The six red bars

characterize the six provinces, from which our 459 strains collected and the resistance

rate in these provinces typically represent high, medium and low levels. The variance

distribution of resistance levels in the six provinces ensures the universality of our

research results.

(3)

Fig S2 The R-M units profiles (excluding 14 major clades) identified in K.

pneumoniae. Genes in R-M systems are depicted as arrows in different colors.

* N, any

nucleotide; R, either purine; Y, either pyrimidine. Red bold type identifies either the

adenine that is the target for methylation or the thymine complementary to the target

adenine. # No. base pairs between target adenines.

(4)

Table S1 Oligonucleotides for PCR

Name Sequence (5'-3')

For R-M systems screening

RM clade1-F TTGTGGTCAGACAGGCGTTC RM clade1-R GTTGCAGGAACAGATCCGCC RM clade2-F TGCTGGTACTTGGATTCCACC RM clade2-R AGAGCGGCAGTCACAGAAG RM clade3-F GCGGCGTCTCCTATCAAAAC RM clade3-R GTGGTCGAGGTTGCCTTCAA RM clade4-F (plasmid-borne) TCAAGCATACGGACTTTGGC RM clade4-R (plasmid-borne) GTTTCCGGCTCCAACTCCTG RM clade5-F CTGAGCCTCGTGTTCCTCAA RM clade5-R GTTCGAGCATTTCGGTCAGC RM clade6-F (plasmid-borne) CACCAAACTGAAAGACGCCG RM clade6-R (plasmid-borne) GCCCGGAGACAATTTTACGC RM clade7-F (plasmid-borne) CTGATTTCTAACTATGCGGC RM clade7-R (plasmid-borne) CATGTTCATACGCGCCAGG

RM clade8-F AAGATACGCTGGAGCATCCG RM clade8-R TGGTTTTATCGATCTCCGCC RM clade9-F GATTGACGGTTCGGTCTCCA RM clade9-R GGCATCTTAGCCGGATTGGT RM clade10-F CCGAGCATGAATTCGCCAAG RM clade10-R ACGACGTAGTTTTCGTGGCT RM clade11-F TGATGACCGTCTACGCGATG RM clade11-R ATTCGATCAGCCACAGGCAA RM clade12-F CTTGCTTCCTGGGATGAGCA RM clade12-R TAGCTGGTTACGAAGCTCGC RM clade13-F ACAACCATCCCTTGGCGAAT RM clade13-R ACCAGTTGGTAAGCGCAAGA RM clade14-F AGGTCCGCAATACCAGCAAA RM clade14-R

For blaKPC screening

CGGCGGGTTGGCAAAAATTA

blaKPC-F TCGCTAAACTCGAACAGG

(5)

Table S2 The distribution of Type I R-M systems in 459 K. pneumoniae clinical

isolates

247 KPC-positive clinical isolates

ID MLST blaKPC I-RM in chromosome(clades) I-RM in plasmids (clades)

220 11 + clade6,7 221 11 + clade6,7 223 11 + 224 11 + 225 11 + 226 11 + 227 11 + 228 11 + 229 11 + 230 11 + 231 11 + 232 11 + 233 11 + 234 11 + 235 11 + 236 11 + 237 11 + 238 11 + 239 11 + 240 11 + clade6 241 11 + 242 11 + 243 11 + 244 11 + 245 11 + 246 11 + 247 11 + 248 11 + 249 11 + 250 11 + 251 11 + 252 11 + 253 11 + 254 11 + 255 11 +

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257 11 + 258 11 + 259 11 + 260 11 + 261 11 + 262 11 + 263 11 + 264 11 + 265 11 + 266 11 + 267 11 + 268 11 + 269 11 + 270 11 + 271 11 + 272 11 + 273 11 + 274 11 + 275 11 + 276 11 + 277 11 + 278 11 + 279 11 + 280 11 + 281 11 + 282 11 + 283 11 + 284 11 + 285 11 + 286 11 + 287 11 + 288 11 + 289 11 + 290 11 + 291 11 + 292 11 + 293 11 + 294 11 + 295 11 + 296 11 + 297 11 + 298 11 + 299 11 +

(7)

300 11 + 301 11 + 302 11 + 303 11 + 304 11 + 305 11 + 306 11 + 307 11 + 308 11 + 309 11 + 310 11 + 311 11 + 312 11 + 313 11 + 314 11 + 315 11 + 316 11 + 317 11 + 318 11 + 319 11 + 320 11 + 321 11 + 322 11 + 323 11 + 324 11 + 325 11 + 326 11 + clade6 327 11 + 328 11 + 329 11 + clade6 330 11 + 331 11 + 333 11 + 334 11 + 335 11 + 336 11 + 337 11 + 338 11 + 339 11 + clade6 340 11 + clade6 341 11 + clade6 344 11 +

(8)

347 11 + clade8 clade6 349 11 + 350 11 + 351 11 + 352 11 + clade6 353 11 + clade6 354 11 + clade6 355 11 + 356 11 + clade6 357 11 + 358 11 + clade6 359 11 + clade4 360 11 + 361 11 + 362 11 + 363 11 + 364 11 + 365 11 + 366 11 + 367 11 + 371 11 + 372 11 + 373 11 + 376 11 + 377 11 + 379 11 + 380 11 + clade6 383 11 + 384 11 + 385 11 + 386 11 + 387 11 + 388 11 + clade3 390 11 + 391 11 + 393 11 + 394 11 + 395 11 + 396 11 + clade6 397 11 + 398 11 + clade6 399 11 + 400 11 +

(9)

401 11 + 402 11 + 405 11 + 406 11 + 407 11 + clade6 408 11 + 409 11 + 410 11 + clade6 411 11 + 412 11 + 413 11 + 414 11 + 415 11 + clade6 416 11 + 417 11 + clade6 418 11 + clade6 419 11 + 420 11 + 421 11 + 422 11 + 423 11 + 424 11 + 425 11 + 426 11 + 427 11 + 428 11 + 429 11 + clade6 430 11 + 431 11 + 432 11 + 433 11 + 434 11 + 435 11 + 436 11 + 437 11 + 438 11 + 439 11 + 440 11 + 441 11 + 442 11 + 443 11 + 444 11 +

(10)

446 11 + 447 11 + 448 11 + 449 11 + 450 11 + 451 11 + 452 11 + 453 11 + 454 11 + 455 11 + 456 11 + 457 11 + 458 11 + 459 11 + 348 15 + clade2 368 15 + clade6 369 15 + clade3 clade6 370 15 + clade3,8 374 15 + clade8 375 15 + clade3 clade6 378 15 + clade3 clade6 381 15 + clade3 clade6 382 15 + clade3 389 15 + 392 15 + clade3 403 15 + clade3 404 23 + 218 65 + clade10 222 65 + clade10 342 81 + 343 81 + 345 81 + 332 290 + 213 423 + clade12 clade4,7 214 423 + clade12 clade4,7 215 423 + clade12 clade4,7 216 423 + clade12 clade4,7 217 423 + clade12 clade4,7 219 977 +

212 KPC-negative clinical isolates

ID MLST blaKPC I-RM in chromosome(clades) I-RM in plasmids (clades)

(11)

118 1 - clade12 183 6 - 5 11 - 15 11 - clade4 66 11 - clade6 67 11 - 158 11 - 19 14 - clade3 21 14 - clade3 22 14 - clade3 116 14 - clade3 52 15 - clade8 68 15 - clade8 clade7 166 15 - clade3 clade7 170 15 - clade3 clade7 182 15 - clade2,8 10 17 - 11 17 - clade6 55 17 - clade6 16 20 - 37 20 - clade5 65 20 - clade6 139 20 - 140 20 - 142 20 - clade12 147 20 - 148 20 - 150 20 - 152 20 - 153 20 - 154 20 - 155 20 - clade1 156 20 - 168 20 - 4 23 - clade2 6 23 - clade4 12 23 - 30 23 - 34 23 - 36 23 - 39 23 - 42 23 -

(12)

54 23 - 72 23 - 105 23 - clade12 109 23 - clade12 113 23 - clade12 clade7 185 23 - 186 23 - 191 23 - 193 23 - clade3 203 25 - clade3,10 clade4 13 29 - 32 29 - clade3,12 38 29 - 53 29 - clade6 92 29 - clade13 93 29 - clade13 97 29 - clade13 98 29 - clade13 125 29 - clade12 114 34 - 61 35 - 88 35 - clade6,7 96 35 - clade14 clade4,6 169 35 - clade14 190 35 - 59 36 - 76 36 - 77 36 - 78 36 - 31 37 - 51 37 - 95 37 - clade2,9 119 37 - clade12 14 39 - 62 39 - 86 39 - 84 43 - 110 48 - clade12 clade6 112 48 - clade12 clade6 133 54 - clade1 clade6,7 135 54 - clade1,12 clade6,7 136 54 - clade1,12 clade6,7 137 54 - clade1,12 clade6,7

(13)

138 54 - clade1 clade6,7 144 54 - clade6 145 54 - clade1 clade7 146 54 - clade1 clade7 149 54 - clade1 clade7 151 54 - clade1 clade7 157 64 - clade1 clade6,7 3 65 - clade10 clade4 8 65 - clade10 clade4 18 65 - clade10 clade4 174 65 - clade4 197 65 - clade10 clade4 202 65 - clade3 178 76 - clade1 clade6 184 81 - clade1 7 86 - clade11 73 86 - clade7 94 86 - clade11 121 86 - clade11,12 189 86 - clade11 131 91 - clade3 28 101 - 70 101 - 80 101 - 129 107 - 194 111 - 111 133 - 74 134 - clade11 17 147 - clade1,3,5 35 152 - 101 152 - clade5 87 188 - clade13 clade7 1 218 - clade1,14 45 218 - clade1,10 171 254 - clade6 63 261 - 175 268 - 134 290 - 43 309 - 44 309 - 160 334 - clade8 clade6,7 181 347 -

(14)

187 412 - 188 412 - clade11 192 412 - 195 412 - 196 412 - 117 420 - clade3 103 455 - 49 461 - clade8,10 clade6 127 490 - clade1,5 41 494 - clade4,7 57 494 - clade6 58 494 - clade6 69 494 - clade4 71 494 - clade4,7 100 494 - clade5 clade6 115 502 - clade7 122 502 - clade7 173 580 - clade6 108 584 - clade12 48 592 - 120 592 - 204 612 - 47 626 - clade10 clade6 64 655 - clade2,8 26 660 - clade1,3 123 661 - clade3,12 128 661 - clade3,12 23 686 - 2 690 - clade7 46 690 - 167 692 - clade3 clade7 161 697 - 162 697 - 141 705 - clade12 clade6,7 143 705 - clade1,12 clade6,7 132 785 - 20 793 - 172 814 - clade10 clade4,7 29 872 - clade2 clade7 176 876 - clade7 90 881 - clade10 clade4 91 881 - clade10 clade4 126 893 - clade11,12

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200 893 - clade10 clade4 107 896 - clade7 89 1031 - clade7 177 1265 - 180 1265 - clade14 106 1326 - clade2 clade6 27 1419 - 102 1419 - 104 1419 - 164 1446 - clade3 163 1536 - 201 1536 - 179 1653 - clade5 83 1764 - 85 1770 - clade7 124 1783 - 99 1914 - 25 1933 - clade1 33 1934 - 75 1935 - 79 1936 - 82 1937 - 24 1938 - clade2 40 1938 - 56 1939 - clade1 clade7 60 1940 - clade7 198 2158 - 165 2464 - 159 2695 - clade5 81 ND - 199 ND - 205 ND - clade3 206 ND - clade3,14 207 ND - 208 ND - clade10 clade4 209 ND - 210 ND - clade10 clade4 211 ND - clade3,10 clade4 212 ND - clade3

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Table S3. Example of type I R-M systems matches on clinical plasmid p187-2

plasmid p187-2, an IncFII plasmid harboring blaKPC, CP025468.1

Recognition Sequence Matches on plasmid(start to end) Matches elements on plasmid Type Numbers

I A/B(calde1) 12 11424-11438 ISKpn24 GAAYNNNNNNNCTGG 20832-20846 DUF262 domain-containing protein

23032-23046 DUF262 domain-containing protein

28958-28972 YecA family protein

60344-60358 TraC 70553-70567 conjugal transfer protein TrbJ

71992-72006 RelE/ParE 73030..73044 RelE/ParE 80820-80834 RelE/ParE 84208-84222 RelE/ParE 89032-89046 DNA methylase 122617-122631 ISKpn6 IC (clade4) > 23 35198-35209 traX ACGNNNNNNCTG 35446-35457 traI 37477-37488 traI 38155-38166 traI 38362-38373 traI 39073-39084 traI 39368-39379 traI 40100-40111 traI 40471-40482 traI

(17)

42456-42467 traD 46787-46798 traG 49543-49554 traH 51635-51646 traF 53612-53623 traN 57225-57236 traW 58124-58135 traC 58312-58323 traC 59461-59472 traC 60259-60270 traC 62737-62748 traV 62911-62922 traV 64060-64071 traB 65422-65433 traE ID (clade8) 9 2060-2073 bacteriocin immunity protein CATCNNNNNNTTYG 11248-11261 bacteriocin immunity protein

15029-15042 DUF4011 domain-containing protein 20863-20876 DUF262 domain-containing protein

26504-26517 HsdR 67022-67035 conjugal transfer protein TrbJ

92846-92859 ltrA 118675-118688 adenine-specific DNA-methyltransferase

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102 M1 GAAYNNNNNNNCTGG 105 M1 GAAYNNNNNNNCTGG 98 M GAAYNNNNNNNCTGG 96 M3 GAAYNNNNNNNCTGG 89 M1 GAAYNNNNNNNCTGG 84 M1 GAAYNNNNNNNCTGG 54 M2 GAAYNNNNNNNCTGG 19 M GAAYNNNNNNNCTGG 121 M GAAYNNNNNNNRTDCC 119 M1 GAAYNNNNNNNCTGG 136 M1 GAAYNNNNNNNCTGG 135 M1 GAAYNNNNNNNCT 134 M1 GAAYNNNNNNNCTGG 133 M1 GAAYNNNNNNNCTGG 132 M1 GAAYNNNNNNNCTGG 130 M1 GAAYNNNNNNNCTGG 209 M1 GAAYNNNNNNNCTGG 198 M2 GAAYNNNNNNNCTGG 197 M1 GAAYNNNNNNNCTGG 178 M1 GAAYNNNNNNNCTGG 176 M1 GAAYNNNNNNNCTGG 165 M1 GAAYNNNNNNNCTGG 164 M2 GAAYNNNNNNNCTGG 161 M1 GAAYNNNNNNNCTGG 195 M1 unknown 196 M1 unknown 97 M unknow 5 M2 unknown 124 M CAAYNNNNNGTRC 195 M2 AGCNNNNNCTTC 196 M2 AGCNNNNNCTTC 182 M AGCNNNNNCTTC 185 M AGCNNNNNCTTC 159 M AGCNNNNNCTTC 91 M1 unknown 65 M unknown 94 M AGCNNNNNCTTC 197 M2 AGCNNNNNCTTC 198 M1 161 M2 AGCNNNNNCTTC 105 M2 AGCNNNNNCTTC 93 M2 AGCNNNNNCTTC 8 M2 unknown 93 M1 CCANNNNNNNRTRTC 8 M1 unknown 156 M GAACNNNNNNGTG 157 M1 ACANNNNNNNNTGAC 215 M1 ACANNNNNNNNTGAC 151 M2 ACANNNNNNNNTGAC 145 M1 ACANNNNNNNNTGAC 129 M1 115 M2 unknown 108 M1 unknown 3 M2 unknown 2 M1 unknown 1 M1 ACANNNNNNNNTGAC 21 M unknown 22 M unknown 54 M1 unknown 84 M2 CGANNNNNNNNTGCC 89 M2 CGANNNNNNNNTGCC 96 M2 CGANNNNNNNNTGCC 102 M2 CGANNNNNNNNTGCC 119 M2 CGANNNNNNNNTGCC 130 M2 CGANNNNNNNNTGCC 132 M2 CGANNNNNNNNTGCC 133 M2 CGANNNNNNNNTGCC 134 M2 CGANNNNNNNNTGCC 135 M2 CGANNNNNNNNTGCC 136 M2 CGANNNNNNNNTGCC 164 M1 CGANNNNNNNNTGCC 165 M2 CGANNNNNNNNTGCC 176 M2 CGANNNNNNNNTGCC 209 M2 CGANNNNNNNNTGCC 11 M1 unknown 23 M unknown 79 M unknown 142 M unknown 203 M1 unknown 16 M1 unknown 214 M unknown 99 M unknown 14 M1 unknown 155 Mp1186 unknown 17 M1 unknown 150 M1 unknown 68 M unknown 118 M unknown 97 MpKpQIL-9b8 unknow 10 MpKPN4 CAGNNNNNNCGT 67 MpAUSMDU8079-2 ACGNNNNNNCTG 66 M pACGNNNNNNCTG 58 Mplasmid ACGNNNNNNCTG 58 M pACGNNNNNNCTG 57 Mp2MR0617ctx ACGNNNNNNCTG 49 MpUHKPC07-113.639 ACGNNNNNN 47 MpUHKPC33-113.638 ACGNNNNNN 46 Mp500 1420-130.552 ACGNNNNN 40 MpKpQIL-531 ACGNNNNNNCTG 37 MpKpQIL-6e6 ACGNNNNNNCTG 36 MpKpQIL-6e6 ACGNNNNNNCTG 34 MpKpQIL-531 ACGNNNNNNCTG 10 M1 p CAGNNNNNNCGT 151 Mp3 ACGNNNNNNCTG 165 Mp200 ACGNNNNNNCTG 164 Mp217 GAAYNNNNNNNCTGG 200 Mp8119 ACGNNNNNNCTG 181 Mpd1 ACGNNNNNNCTG 181 M pACGNNNNNNCTG 178 Mpd1 ACGNNNNNNCTG 177 Mpd2 ACGNNNNNNCTG 176 Mp2 2 ACGNNNNNNCTG 95 MpKpQIL-6e6 107 Mp2 ACGNNNNNNCTG 104 MpCR14 p3 unknown 96 Mp2NDM-1fa ACGNNNNNNCTG 96 Mp1KPN-04f ACGNNNNNNCTG 96 M4 p ACGNNNNNNCTG 140 Mp1 ACGNNNNNNCTG 139 Mp ACGNNNNNNCTG 115 Mp2 ACGNNNNNNCTG 115 Mp1 ACGNNNNNNCTG 115 M3 ACGNNNNNNCTG 110 Mp2IncFIB ACGNNNNNNCTG 70 Mp2 ACGNNNNNNCTG 4 Mp2PMK1-C GACNNNNNNGTC 101 M TTACNNNNNTAG 190 Mpd4 unknown 27 M unknown 83 M unknown 173 M1 unknown 173 M2 unknown 87 M unknown 88 M unknown 57 M unknown 85 M unknown 177 M unknown 15 M1 unknown 26 M CCAYNNNNNTCC 5 M1 unknown 204 M2 unknown 205 M2 unknown 203 M2 unknown 204 M1 unknown 205 M1 unknown 176 Mp2 1 unknown 185 Mp617 CAANNNNNNCATC 174 Mp01 CAANNNNNNCATC 157 Mp853 CAANNNNNNCATC 74 Mp2187-2 CAANNNNNNCATC 33 MpKPN-262 GATGNNNNNNTGC 179 Mpd1 GTGANNNNNNNTGG 151 Mp2 nuknown 86 MpUCLAOXA232-6.X unknown 155 Mp1160 TGGANNNNNNNTTG 166 Mp168 TGGANNNNNNNTTG 91 M2 unknown 9 M1 unknown 149 M2 unknown 168 M2 GGCANNNNNNNTNNCG 202 M unknown 18 M1 unknown 168 Mp8 CACNNNNNRTAT 30 MpKPHS1 unknown 74 Mp187-1 unknown 98 MpKPN-065 GAGNNNNNNTAYG 84 MpSg1-1 unknown 156 Mp161 CTANNNNNNCTG 4 Mp1PMK1-B unknown 140 Mp2 unknown 57 Mp1MR0617ndm-1 unknown 177 Mpd1 CAAYNNNNNCTGG 129 M2 CATCNNNNNNTTYG 169 M CATCNNNNNNTTYG 115 M1 unknown 108 M2 unknown 4 M1 unknown 211 M TGAYNNNNNCCT 168 M1 ACGNNNNNGTTG 215 M2 ACGNNNNNGTTG 157 M2 ACGNNNNNGTTG 157 Mp1 ACGNNNNNGTTG 145 M2 ACGNNNNNGTTG 149 M1 ACGNNNNNGTTG 151 M1 ACGNNNNNGTTG 96 M1 ACGNNNNNGTTG 78 M 1 M2 ACGNNNNNGTTG 2 M2 unknown 3 M1 unknown 110 Mp1IncFIB unknown 99 74 99 99 99 86 95 99 99 99 99 99 99 99 75 99 68 95 55 52 52 8 55 99 99 99 99 58 99 99 99 66 64 99 35 99 99 69 97 89 98 86 99 44 86 32 86 86 99 56 99 71 70 35 17 23 15 17 43 55 95 99 0.1

1

2

3 10 14 11 13 12 4 5 6 8 9 7 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 Recognize sites ID* HsdM#

Note:*All the numbers are consistent with the strain ID in Supplementary data 3.

Figure

Fig S1. A total of 459 K. pneumoniae clinical isolates collected from six provinces  of China with different carbapenem resistance rates
Fig  S2  The  R-M  units  profiles  (excluding  14  major  clades)  identified  in  K
Table S1 Oligonucleotides for PCR
Table S2 The distribution of Type I R-M systems in 459 K. pneumoniae clinical  isolates
+2

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