Isolation and biochemical identification of bacteria
Two hundred clinical specimens were collected from two teaching hospitals (Sayed Jalal and Al Hussein hospitals) and two private clinical laboratories in Cairo, Egypt. These were isolated from urine samples, sputum, pleural fluid, endotracheal secretion and swaps. The samples were collected at November 2016- October 2017. Isolates were collected from patients admitted in ICU,PICU as well as outpatients. Their ages ranged from 6 – 50 years. They suffered from respiratory infection, UTI or Meningitis. Gram negative isolates were identified using standard microbiological tests; Gram stain, motility test, catalase test, triple sugar iron, citrate agar test, oxidase test and witek test.

Antimicrobial susceptibility testing
The antibiotic susceptibility testing was performed by Kirby Bauer disk diffusion method⁷. Gram negative isolates were screened for susceptibility to six b-lactam antibiotics; meropenem, imipenem, amoxicillin-clavulinic, cefotaxime, amoxicillin and ceftazidime. Resistant isolates were screened for susceptibility to polymyxinB, colistin, amikacin and levofloxacin.

DNA extraction and manipulation
DNA extraction for double resistant (Colistin/Carbapenem) isolates consisted of boiling lysates prepared from the strains; a loopful of culture was suspended in 1 mL of sterile PBS buffer, centrifuged 5 min at 20000 g and the supernatant rejected. Then, the pellet was re-suspended in 100 µL TE buffer (10:1) and boiled for 10 min at 100÷C. For use as template in the polymerase chain reaction, this DNA was further diluted at 1:10 in TE buffer. The DNA extract was checked by running on 0.8 % agarose gel ,stained with ethidium bromide (Thermo Fischer Scientific, USA) using gel electrophoresis (Labnet, USA). The gel was  illuminated under Whitman U.V Trans-illuminator (Biometra, Germany) according to Sambrook et al. ²⁰.

PCR amplification of specific sequences of some genes encoding for colistin and polymyxin resistance
Three genes contributing to colistin and polymyxin resistance were selected in this study and amplified by PCR; mcr-1 primers {( CLRr/CLRf (5’CGGTCAGTCCGTTTGTTC’3) CLR/r (5’CTTGGTCGGTCTGTAGGG’3) 94÷C 3min+ 25X(94÷C 30 sec+ 58÷C 90 sec+ 72÷C 60 sec) + 72÷C 10min}¹⁰, pmrA primers LT₂PMRA/REV{( F (5’CAT TTC CGC GCA CTG TCT GC’3)R (5’CAG CTT TCA GTT GCA AAC AG’3) 95°C for 10s,52°Cfor 5s, 72°C for 5s} and pmrB primers 1519/1520{( F (5’ACC TAC GCG AAA AGA TTG GC’3)R (5’GAT GAG GAT AGC GCC CAT GC’3) 95°C for 30s ,62°C for 30s,72°C for 180s)} ²¹. Amplification was performed using thermocycler in total volume of 25µL containing 1 µL DNA extract, 1µL of forward primer (10 µM), 1µL reverse primer (10 µM), 12.5µL tag master mix (Qiagen, Germany) and 9.5µL of nuclease-free water.

Amplification using Gene-AMP-PCR-system-9700 thermocycler (Applied bio system). After amplification, 10µL of the PCR mixture was analyzed by gel electrophoresis.(1.5% agarose in tris-EDTA stained with ethidium bromide). . As mcr-1 mostly carried on plasmid and pmr-A, pmr-B are chromosomal genes so mcr-1 dissemination is highly predicted that lead to increase occurrence of colistin resistance globally, so SYBR Green-based real-time PCR assay was designed for specific detection of the mcr-1 gene. Cell lysates (3 µL), prepared as described above, were used as DNA template, MCR -F: 5’-ACGGCGTATTCTGTGCCGTGTAT-3’ and MCR -R 5’- GCTGTTCTTTTGGTGCAAAGGCATTT-3’ were used as primers for PCR analysis of mcr-1real-time PCR were performed using QuantiNova SYBR Green mixture and Qiagen Rotor Gene system (Qiagen, Hilden, Germany). A typical 20 µL of PCR reaction includes 0.7 µM of each primer, 3 µL of lysate and SYBR Green mixture (1x). The PCR conditions used are: 95°C, 10 minute; 30 cycles including 95°C, 15 seconds, 60°C, 1 min; and 72°C, 30 seconds.

Confirmation of the identity of isolate harboring mcr-1 by 16S rRNA gene sequence analysis
Identification was confirmed by sequencing of partially amplified 16S rRNA gene of the bacterial isolate that harbored mcr-1 gene. Molecular characterization of the selected strain was carried out according to modified Sambrook et al. ²⁰. The 16S rRNA gene was amplified using universal primers pair: the forward primer sequence was 5-AGAGTTTGATCATGGCTCAG-3 and the reverse primer sequence was 5-ACGAGCTGACGACAGCCATG-3 (IDT, USA). PCR was carried out in a total volume of 50 µL using 25 µL of My Taq PCR Master Mix (2X) (Bio line, Germany), containing 1 µL (10 µM) of each forward and reverse primers together with equivalent micro liters containing 50 µg of the genomic DNA and finally completed to 50 µL by nuclease free water.  Thermo cycling was carried out using thermo cycler (Primus 25 Advanced, Germany). It was carried out as follow: first step was denaturation for 3 minutes at 94°C, followed by 40 cycles of denaturation at 94°C for 30 sec, annealing at 50°C C for 30 sec and extension at 72°C for 2 min. Finally, extension was done at 72°C for 10 min. The amplified sequence was verified by gel electrophoresis. PCR products were purified by Gene Jet Gel Extraction Kit (Thermo Fisher Scientific, USA). The purified products were subjected to  sequencing. Nucleotide sequence similarities were determined using other known sequences found in the GenBank database using BLAST program of National Center for Biotechnology Information (NCBI) databases.