Supplementary appendix This appendix formed part of the original submission and has been peer reviewed. We post it as supplied by the authors. Supplement to: Quan J, Li X, Chen Y, et al. Prevalence of mcr-1 in Escherichia coli and Klebsiella pneumoniae recovered from bloodstream infections in China: a multicentre longitudinal study. Lancet Infect Dis 2017; published online Jan 27. http://dx.doi. org/10.1016/s147-099(16)0528-x.
Supplementary table 1. The primers of common chromosomal mutations used in the study. Genes primer name Sequence (5'-') Reference or source KP-mgrB KP-phoP KP-phoQ KP-pmrA KP-pmrB EC-mgrB EC-phoP EC-phoQ EC-pmrA EC-pmrB KPmgrB-F AAGGCGTTCATTCTACCACC KPmgrB-R TTAAGAAGGCCGTGCTATCC KPphoP-F GAGCTTCAGACTACTATCGA KPphoP-R GGGAAGATATGCCGCAACAG KPphoQ-F ATACCCACAGGACGTCATCA KPphoQ-R CAGGTGTCTGACAGGGATTA KPpmrA-F CATTTCCGCGCACTGTCTGC KPpmrA-R CAGGTTTCAGTTGCAAACAG KPpmrB-F ACCTACGCGAAAAGATTGGC KPpmrB-R GATGAGGATAGCGCCCATGC ECmgrB-F AAGGTAGGTGAAACGGAGATT This study ECmgrB-R CCGATACAACCAAAGACGC ECphoP-F ATGGCGATGCTGTCCG This study ECphoP-R TCCGTAGGCAAGCGAAA ECphoQ-F GCAAAGTGGTCAGCAAAGA This study ECphoQ-R AATCGGGCCAGTTAAGAGT ECpmrA-F TGCTGTGGCTGTCGGA This study ECpmrA-R AATCTGCTCGGTACTTTCATG ECpmrB-F CCAACACCCTGGAAGTGC This study ECpmrB-R TGATGAATAAGCTGAAACGGA 1 1 1 2 2
Supplementary table 2. The primers of ESBLs and carberpenemases used in the study. Genes Primers Primer sequence (5 ) Reference TEM-F TCGGGGAAATGTGCG bla TEM bla SHV bla CTX-M-1 bla CTX-M-2 bla CTX-M-8 bla CTX-M-9 bla VEB bla PER bla GES bla OXA-2 bla OXA-10 bla NDM TEM-R TGCTTAATCAGTGAGGCACC SHV-F GCCTTTATCGGCCTTCACTCAAG SHV-R TTAGCGTTGCCAGTGCTCGATCA CTX-M-1-F CAGCGCTTTTGCCGTCTAAG CTX-M-1-R GGCCCATGGTTAAAAAATCACTGC CTX-M-2-F GCATTCGCCGCTCAATGTTA CTX-M-2-R GGTTCGTTGCAAGACAAGAC CTX-M-8-F ACTTCAGCCACACGGATTCA CTX-M-8-R CGAGTACGTCACGACGACTT CTX-M-9-F GTTACAGCCCTTCGGCGATGATTC CTX-M-9-R GCGCATGGTGACAAAGAGAGTGCAA VEB-F GCGGTAATITAACCAGA VEB-R GCCTATGAGCCAGTGTT PER-F CCTGACGATCTGGAACCTTT PER-R TGGTCCTGTGGTGGTTTC GES-F CCGATCTTGAGAAGCTAGAG GES-R GACCGACAGAGGCAACTAAT OXA-2-F GGAGCAGCAACGATGTTACG OXA-2-R GGCGGTAACGCTTCAATAGA OXA-10-F CCACCAAGAAGGTGCCATGA OXA-10-R GCGACCTTGAGCGACTTGTT NDM-F GGCGGAATGGCTCATCACGA This study NDM-R CGCAACACAGCCTGACTTTC
Supplementary table. The chromosomal mutations identified in colistin-resistant or mcr-1 positive E. coli and K. pneumoniae isolates. Isolates MIC of colistin(mg/l) phop phoq pmra pmrb mgrb 04HAE12 0.06 WT a WT A64T WT WT 04HAE0 16 WT WT WT WT WT 05HAE09 8 I44L N46K WT H2R/D28G/A60V WT 05HAE16 4 I44L N46K WT H2R/D28G/A60V WT 05HAE0 4 I44L L477M WT H2R/D28G WT 06COE21 8 I44L WT G144S H2R/D28G WT 07HAE27 8 WT WT WT WT WT 10HAE1 8 WT E464D/A482T WT WT WT 18HAE25 4 I44L A449V WT D28G/Y58N WT 19COE11 8 WT WT WT WT WT 20COE1 8 WT WT WT H2R/D28G/A60V WT 20HAE28 4 I44L I175F WT D28G/Y58N WT 20HAE29 8 WT A482T WT H2R/D28G/A60V WT 2COE28 16 WT WT WT WT WT 2HAE4 8 I44L WT WT H2R/A242T/D28G/A60V WT 24COE0 8 WT WT WT WT L16V 24COE26 8 I44L WT G144S H2R/E12D/D28G/V51I WT 27COE18 8 I44L WT T1S/I128N/G144S H2R/D28G N41S 27COE19 8 I44L WT R18H D28G/Y58N WT 27HAE25 4 I44L WT R18H D28G/Y58N WT 28HAK10 2 WT WT WT WT WT 01COK02 2 WT WT WT R256G WT
0HAK08 64 WT WT WT R256G WT 06COE08 16 I44L R6H T1S/I128N/G144S H2R/E12D/D28G/V51I N8G\Y17H\N41S 10COE06 4 I44L WT T1S H2R/E12D/V161G/D28G/V51I Q1K\N41S 1COE09 8 I44L R6H T1S/I128N/G144S H2R/E12D/V161G/D28G/V51I N8G\Y17H\N41S 28COK16 128 WT WT WT R256G insersation inactivation a WT: wild type
Supplementary table 4. The antimicrobial susceptibility of index mcr-1-positive isolates and their transconjugants MIC (mg/l) Isolates name TZP FEP CTX CPS2/1 IPM MEM CST a AMK TGC MIN CIP ATM 05HAE09 4 8 2 2 0.125 0.25 8 2 0.125 4 2 4 05HAE09-EC600 4 0.25 0.06 1 0.5 0.25 4 2 0.06 1 0.25 0.25 05HAE16 8 8 256 2 0.125 0.25 4 8 0.25 8 2 2 05HAE16-EC600 4 0.25 0.06 1 0.5 0.25 4 1 0.06 4 0.25 0.25 06COE21 8 256 256 8 0.25 0.125 8 4 0.125 4 2 128 06COE21-EC600 4 128 256 8 0.5 0.125 4 2 0.06 2 0.25 128 10HAE1 4 256 256 256 0.125 0.125 8 4 0.125 2 0.25 2 10HAE1-EC600 2 2 0.25 64 0.5 0.125 4 2 0.06 1 0.25 0.5 18HAE25 2 4 256 2 0.06 0.25 4 4 0.125 4 2 16 18HAE25-EC600 2 0.25 0.125 1 1 0.25 2 2 0.06 1 0.25 0.25 19COE11 2 256 256 8 0.25 0.125 8 256 0.125 8 2 2 19COE11-EC600 2 0.25 0.125 1 0.5 0.125 2 1 0.06 1 0.25 0.25 27COE18 2 256 256 16 0.25 0.125 8 2 0.125 16 2 128 27COE18-EC600 2 64 256 8 0.5 0.125 4 2 0.06 1 0.25 128 27COE19 1 256 256 16 0.25 0.125 8 16 0.125 4 1 128 27COE19-EC600 2 64 256 16 0.5 0.125 4 2 0.06 1 0.25 128 ATCC25922 b 1 0.125 0.125 0.25 0.125 0.06 0.125 0.5 0.125 0.25 0.125 0.125 E. coli EC600 2 0.125 0.125 1 0.5 0.125 0.0 2 0.06 1 0.125 0.19 TZP, piperacillin/tazobactam;fep, cefepime;ctx, ceftriaxone;cps2/1, cefoperazone/sulbactam; IPM, imipenem; MEM, meropenem; CST, colistin; AMK, amikacin; TGC, tigecycline; MIN, minocycline; CIP, ciprofloxacin; ATM, aztreonam.
a Drug susceptibility was determined with broth microdilution method. All susceptibility tests were repeated at least three times according to CLSI method. The results of colistin susceptibility were interpreted according to EUCAST breakpoints. b quality control strain
Methods Patient and isolate data The E. coli 04HAE12 isolate was recovered from a 64-year-old female patient admitted to the hospital for biliary calculi in Beijing, China, April 2014. The strain was cultured from blood during the patient hospitalization. Initially, the patient only received cefuroxime (1.5g, q12h) for days. Following treatment with surgery, the therapeutic regimen was adjusted to cefoperazone/sulbactam (g, q12h) for 6 days and the patient was cured. Plasmid DNA extraction and analysis Plasmid extraction and analysis was performed as previously described. 4 Briefly, the plasmid DNA of strain E. coli 04HAE12 was extracted using a QIAamp DNA MiniKit (Qiagen, Valencia, CA, USA) following the manufacturer s recommendations. The plasmids were sequenced on an Illumina-Hiseq TM 2000 (Illumina Inc, San Diego, U.S.A) using a paired-end 2 100 bp protocol. Sequence reads were assembled using the CLC Genomics Workbench software package (CLC Bio 8.0). Gaps were closed by standard PCR and Sanger sequencing according to a protocol from a previous study. 5 The RAST annotation website server was then used to annotate the genomes of the plasmid. The CG viewer was used to compare the pec_04hae12 sequence with that of the reference plasmid phnshp45 (accession number KP47127). 6 Cloning experiments The mcr-1 gene and part of the flanking sequence were amplified with primers MCR-F/R from the strains E. coli 04HAE12 and E. coli 20COE1 (mcr-1-positive and colistin resistance strain, unpublished data). The purified PCR products were cloned into the pcr 2.1-TOPO vector (Invitrogen, Shanghai, China), giving recombinant plasmids pec_04hae12::mcr-1 and p20coe1::mcr-1. The recombinant plasmids were then introduced into the colistin-susceptible E. coli DH5α strain via electroporation. Transformants (Supplementary table 5) were selected on LB agar plates containing 2 mg/l colistin and were confirmed by sequencing. Nucleotide sequence accession number The nucleotide sequence reported in the present study has been deposited in the GenBank nucleotide database under accession no. KX592672. Results Isolate characteristics The mcr-1 gene was detected in E. coli 04HAE12 by PCR amplification and DNA sequencing revealed 100% identity compared with the originally reported gene sequence. Antimicrobial susceptibility testing showed that strain E. coli 04HAE12 was resistant to piperacillin/tazobactam, cefepime, ceftriaxone, ciprofloxacin, aztreonam, amikacin and tetracycline and sulfonamides, but susceptible to colistin (MIC 0.06 mg/l). PCR and sequence analyses showed various resistance genes, such as bla CTX-M-15, aada5, stra, aac()-iia, strb, mph(a), tet(a) and sul1, in isolate E. coli 04HAE12. The mcr-1 gene was detected in this strain with a colistin- susceptible phenotypic. Southern blot showed that the mcr-1 gene was located in a plasmid (ca. 60k). In addition, this strain belonged to a new ST clonal lineage (10-11-78-8-8-8-7). Plasmid analysis The mcr-1-harbouring plasmid from E. coli 04HAE12 belonged to IncI2 incompatibility group with a
G+C content of 42.5%. The BLASTn search results showed that pec_04hae12 exhibited only 92% query coverage and 97% identity to the originally described mcr-1-encoding plasmid phnshp45. The mcr-1-encoding pec_04hae12 plasmid sequence was 59,99 bp in size and was nearly identical to plasmid phnshp45, mainly differing by (i) the lack of a 2704 bp insertion of IS68 downstream of the repa gene; and (ii) the absence of ISApl1 transposable element in the plasmid pec_04hae12, however, insertion sequence IS1 was present. Notably, insertion sequence IS1 was identified among the -10 promoter sequence of mcr-1 and disrupted this box (TACAAT) (Supplementary figure 1). It is therefore plausible that this genetic event is responsible for the modified of expression of mcr-1. To confirm this hypothesis, cloning experiments were performed. As we expected, MIC of strain E. coli D04HAE12, transformed by pec_04hae12::mcr-1 plasmid, is 0.06 mg/l of colistin. In contrast, MIC of strain E. coli D20COE1, transformed by p20coe1::mcr-1 plasmid, displayed 4 mg/l of colistin (Supplementary table 6). In addition, wild type E. coli DH5α and plasmid control strain E. coli DT1 are both 0.06 mg/l of colistin. Overall,these results demonstrated that the insert sequence IS1 in the -10 promoter box breaks the functionality of the mcr-1 promoter region and conferred to colistin susceptibility, which might affect the expression of mcr-1.
Supplementary table 5. Bacterial isolates, plasmids and primers used in this study Isolate, plasmid, or primer Relevant description Source or reference Strains E. coli DH5α E. coli E. coli 04HAE12 mcr-1-positive and colistin susceptible strain This study E. coli 20COE1 mcr-1-positive and colistin resistance strain This study E. coli DT1 E. coli DH5α was transformed by expression plasmid pcr2.1-topo as a control This study E. coli D04HAE12 E. coli DH5α was transformed by pec_04hae12::mcr-1 plasmid This study E. coli D20COE1 E. coli DH5α was transformed by p20coe1::mcr-1 plasmid This study Plasmids pcr2.1-topo E. coli expression plasmid, Amp r, KM r pec_04hae12::mcr-1 IS1 and mcr-1 from E. coli 04HAE12 were together cloned into the plasmid pcr2.1-topo, Km r This study p20coe1::mcr-1 mcr-1 from E. coli 20COE1 was cloned into pcr2.1-topo plasmid, Km r This study Primers Nucleotide sequences (5 - ) mcr-ms_f AGACGGCAAGATTCTTGAGG; forward primer for amplification upstream of mcr-1 for cloning This study mcr-ms_r CCCCATACCCAGATAAAAC; reverse primer for amplification upstream of mcr-1 for cloning This study Km: kanamycin; Amp: ampicillin; r: resistance.
Supplementary table 6. MICs of colistin (mg/l) Strains colistin E. coli DH5α 0.06 E. coli DT1 0.06 E. coli D04HAE12 0.06 E. coli D20COE1 4 E. coli 20COE1 8 Supplementary figure 1. Circular genetic map and mechanism of pec_04hae12 from colistin-susceptible E. coli 04HAE12 carrying mcr-1. (A): Comparison of pec_04hae12 (KX592672) from E. coli 04HAE12 with complete plasmid sequence phnshp45 (KP47127) from E. coli SHP45. (B) The zoomed-in view of the mcr-1 mobile element in the plasmid pec_04hae12. The large blank arrow indicates ORF of mcr-1 (it is encoded on the forward strand). The blank block indicates mobile element of IS1. The -10 and -5 promoter sequences of the mcr-1 gene are indicated by the underline. IRL (Inverted Repeat Left) and IRR (Inverted Repeat Right) of IS1 are also indicated by the red body words. Reference 1 Cannatelli A, D'Andrea MM, Giani T, et al. In vivo emergence of colistin resistance in Klebsiella pneumoniae producing KPC-type carbapenemases mediated by insertional inactivation of the PhoQ/PhoP mgrb regulator. Antimicrob Agents Chemother 201; 57:
5521-6. 2 Jayol A, Poirel L, Brink A, Villegas MV, Yilmaz M, Nordmann P. Resistance to colistin associated with a single amino acid change in protein PmrB among Klebsiella pneumoniae isolates of worldwide origin. Antimicrob Agents Chemother 2014; 58: 4762-6. Quan J, Zhao D, Liu L, et al. High prevalence of ESBL-producing Escherichia coli and Klebsiella pneumoniae in community-onset bloodstream infections in China. J Antimicrob Chemother 2016. DOI: 10.109/jac/dkw72 4 Fu Y, Liu L, Li X, et al. Spread of a common blandm-1-carrying plasmid among diverse Acinetobacter species. Infect Genet Evol 2015; 2: 0-. 5 Fu Y, Du X, Ji J, Chen Y, Jiang Y, Yu Y. Epidemiological characteristics and genetic structure of blandm-1 in non-baumannii Acinetobacter spp. in China. J Antimicrob Chemother 2012; 67: 2114-22. 6 Liu YY, Wang Y, Walsh TR, et al. Emergence of plasmid-mediated colistin resistance mechanism MCR-1 in animals and human beings in China: a microbiological and molecular biological study. Lancet Infect Dis 2016; 16: 161-8.