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.