Sample collection, isolation, and species confirmation:
The present study was initiated with the screening of 545 E. coli isolates for colistin resistance. All isolates were collected from Microbiology laboratories of PIMS Islamabad (n= 260), HMC (n= 105), RMI (n=94), and KTH (n= 86) Peshawar - tertiary care hospitals in Pakistan from June 2018 to September 2019. The isolates obtained from human clinical samples included urine (n= 345), blood (n= 109), stool (n= 18), and pus (n= 73). The growth capacity of isolates was initially assessed on CLED and MacConkey agar. A single colony from each Petri plate was stored in LB media for maintaining strains. Species confirmation of E. coli was performed with 16S rDNA PCR using specific primer mentioned in Table S1. The PCR products were sequenced, and species confirmation was performed using EZ Biocloud online software (https://www.ezbiocloud.net/identify).
Phenotypic and molecular detection of colistin resistance:
Phenotypic detection of colistin resistance was performed applying the broth microdilution method. The Minimum inhibitory concentration results were interpreted according to CLSI guidelines [13]. The genomic DNA from resistant isolates was extracted with the conventional boiling method [14]. The DNA samples were subjected to PCR using mcr genes-specific primers mentioned in Table S1. The expected size amplicons were visualized on 1% Agarose gel, which was sequenced and analysed with BLAST (https://blast.ncbi.nlm.nih.gov/Blast.cgi)
Antimicrobial susceptibility testing:
The mcr-1 harboring isolates were subjected to antimicrobial susceptibility testing using the broth microdilution method. The antibiotics used were Ampicillin, Cefotaxime, Chloramphenicol, Ciprofloxacin, Fosfomycin, Cefoxitin, Gentamycin, Aztreonam, Amikacin, Meropenem, and Tetracycline. ESBL and MBL activity of resistant isolates were also determined by the Double Desk Synergy Test (DDST) and combined desk test (CDT), respectively. The results were interpreted according to CLSI guidelines [13].
PCR detection of ESBL genes:
ESBL genes blaTEM, blaSHV, blaCTX-M, and blaOXA variants were amplified from the genomic DNA of colistin-resistant isolates with ESBL genes-specific primers mentioned in Table S1. The expected sizes were visualized on 1% Agarose gel. For further confirmation, PCR products of ESBL genes were sequenced and blast via the NCBI blast tool (https://blast.ncbi.nlm.nih.gov/Blast.cgi).
Molecular typing:
The multilocus sequence typing of four mcr-1 harboring isolates was carried out. The sequence result of 7 alleles, adk, fumC, gyrB, icd, mdh, purA, and recA, were determined from the MLST database (http://enterobase.warwick.ac.uk/species/index/ecoli). To further determine the genetic link among mcr-1 harboring isolates, the Xba l PFGE was performed according to PulseNet PFGE protocol [15]. DNA fingerprint analysis and sketch drawing were performed by BioNumerics v.8.0 (Applied Maths, Sint-Martens-Latem, Belgium). Clusters were examined by the unweight pair-group method with arithmetic mean (UPGMA) analysis. The Dice similarity coefficient was calculated with a position tolerance of 1.5%, and a dendrogram was created based on the UPGMA.
Transconjugation and PCR based replicon typing:
In order to detect the transferability of four colistin-resistant isolates, trans-conjugation experiments were performed. These isolates were selected as the donor, and E. coli EC 600 (NalR, RifR) were taken as recipients. The experiment was performed, as described previously [16]. The transconjugants were analysed using antibiotic susceptibility testing and mcr-1 specific PCR, as described earlier. The conjugation transfer rates were calculated by dividing the number of transconjugants by the number of donors.
To further investigate the plasmid incompatibility type responsible for mcr-1, the plasmid DNA of transconjugants was extracted by the alkaline lysis method [17]. The plasmid type was determined by using the PBRT 2.0 kit (MBK0078, Diatheva, Italy). The PCR products of eight multiplex PCR were visualized on 2% Agarose gel, and the results were analysed according to the manufactured instructions.
S1 PFGE and southern blotting:
S1 PFGE and southern hybridization of the successfully trans-conjugant E. coli EC600 were performed to determine the plasmid location. For S1 PFGE, the bacterial isolates were embedded into 1% low melting agarose plugs. Following cell lysis and washing, the plugs were pre-incubated in 200µl of 1x S1 buffer at 37° c for 30 minutes. S1 digestion was performed by ten units of S1 enzymes (Thermo scientific) in 200 µl of 1x S1 buffer for 10 min at 37° c. The digestion was stopped by removing enzymes and adding 200 µl of 0.5M EDTA and left for 10 min at room temperature. Before running on the gel, the plug was incubated in 200 µl of TE buffer for 30 minutes. The products were electrophoresed on the CHEF mapper PFGE system (Bio-Rad USA) for 21 h at 6 V/cm with initial switch time 2.5 sec and final switch time 60 sec. The plasmids from the gel were transferred onto the nylon membrane via the capillary transfer technique [18]. Southern hybridization of plasmid DNA with a digoxin-labelled mcr-1-specific probe was performed according to kit instructions (Roche Diagnostics, Mannheim, Germany).
PCR mapping of mcr-1 genetic context:
To explore the genetic context of mcr-1, whether pHNSHP45[5] like key genetic component surrounds it, we designed seven pairs of primers targeting ParA, nikB, tnpA, mcr-1, hp, pilN, and vird4 genes. The plasmid DNA for PCR mapping was extracted by the alkaline lysis method and quantified by one drop [17]. The primers used for mapping are mentioned in Table S1. The PCR products were visualized on 1 % Agarose gel and subsequently sequenced and blasted via the NCBI blast tool.