2.4 Conjugation Experiments and evaluation of plasmid stability
To investigate the transferability of plasmid inKlebsiella pneumoniae M297-l isolate, we performed conjugation assays with sodium-azide resistanceE.coli J53 as a recipient strain. Briefly, overnight cultures of MDRKlebsiella pneumoniaeM297-l as a donor and the recipient E. coli J53 strain were 1:10 mix and conducted on nitrocellulose membranes on a MacConkey Agar plate by incubation at 35oC for 16-20 hours. After incubation, we subsequently ten-fold serial diluted the mixed culture in sterile saline and aliquoted 100μl of diluted culture onto MacConkey Agar plates supplemented with 20mg/L of cefotaxime and 200mg/L of sodium azide. E.coli transconjugants were screened by drug susceptibility testing. To further evaluate the stability of the plasmid of E.coli transconjugants,E.coli transconjugants were passaged continuously in MHB without antibiotics and detected in McConkey Agar plate containing 20mg/L cefotaxime according to previous reports(Di Luca et al., 2017; Walsh, Weeks, Livermore, & Toleman, 2011; Wein, Hulter, Mizrahi, & Dagan, 2019).
Accession numbers: the sequence data and details of the sequenced samples, including the date and location of collection and source, were submitted to the GenBank. Accession numbers for Chr-M297-1, plasmid pM297-1.1and pM297-1.2 from Klebsiella pneumoniae M297-1, respectively. Klebsiella pneumoniae M297-1 assembly contigs are deposited under study accession number CP051490, CP051491, and CP051492.