in april 2008 a carbapenem - nonsusceptible e. coli strain , marked as eco2 , was recovered from the gall bladder drainage of a 91-year - old man with dialysis - dependent end - stage renal disease , congestive heart failure , anemia , and peptic ulcer disease . a month earlier , the patient had been hospitalized with sepsis that developed after an infected heel wound had required amputation of the left leg below the knee . the patient was treated with ertapenem , metronidazole , colistin , and vancomycin . acute cholecystitis developed , and the patient underwent cholecystostomy . during his hospital stay , the patient underwent screening for carriage of carbapenem - resistant enterobacteriacae ( cre ) as part of a routine infection control program aimed at limiting the spread of cre . the first swab specimen was negative for cre by culture , and the second swab specimen showed a carbapenem - resistant k. pneumoniae strain ( marked kpn1 ) , which was pcr positive for blakpc . one month after the patient s admission , a carbapenem - nonsusceptible e. coli ( eco2 ) was isolated from drainage at the cholecystectomy site , which prompted this study . microbiologic and molecular investigations ( pulsed - field gel electrophoresis [ pfge ] , dna isolation , isoelectric focusing analysis [ ief ] , pcr detection of resistance genes , plasmid isolation , transformation , and southern analysis ) were performed as described ( 2,4,7 ) . the carbapenem - nonsusceptible e. coli isolated from the clinical specimen ( eco2 ) was initially identified by vitek-2 ( biomrieux , marcy - letoile , france ) as resistant to imipenem ( mic>16 mg / l ) . further antimicrobial - drug susceptibility testing of the strain by using agar dilution and etest ( ab biodisk , solna , sweden ) showed mics in the resistant range for ceftriaxone and aztreonam ; in the intermediate range for ceftazidime and piperacillin / tazobactam ; and in the susceptible range for cefepime , ertapenem , meropenem , imipenem , aminoglycosides , quinolones , tigecycline , and colistin ( appendix table ) . ief identified 2 -lactamases with isoelectric ph values of 5.4 and 6.7 , corresponding to those of tem - type and kpc . transformation of plasmids purified from eco2 into an e. coli dh10b recipient strain ( eco2-t ) indicated transfer of a single plasmid that encoded these blatem-1 and blakpc-3 ( figure 1 , panel a ) , and increased the mics of the recipient strains to broad - spectrum cephalosporins and carbapenems ( appendix table ) . pfge identified the genetic similarity between the colonizing klebsiella ( kpn1 ) and a representative kpnst258 ( isolate kpn557 described previously ) ( figure 2 ) . susceptibility testing of kpn1 reflected the extremely drug - resistant phenotype characteristic of isolates belonging to this clone ( 12 ) ( appendix table ) . a ) analysis of klebsiella pneumoniae carbapenemase ( kpc)encoding plasmids in isolates kpn1 ( 1 ) , eco2 ( 3 ) , kpn1-t ( 2 ) , and eco2-t ( 4 ) , israel , 2008 . plasmid size estimation was performed by digestion of dna with s1 nuclease ( 20 u ; promega , madison , wi , usa ) followed by pulsed - field gel electrophoresis ( pfge ) with the chef - dr iii apparatus ( bio - rad laboratories , inc . , lambda ladder pfg marker ( new england biolabs , beverly , ma , usa ) was used as a molecular size marker ( lane m ) . b ) restriction fragment length polymorphism of the kpc-3encoding plasmid from kpn1-t ( k ) and eco2-t ( e ) . plasmid dna was digested with bamhi , bglii , ecori , and saci endonucleases ( new england biolabs ) and underwent pfge on a 1% agarose gel . the level of similarity between restriction patterns was calculated by using gelcomparii software version 5 ( applied maths , kortrigk , belgium ) . plasmid restriction products were transferred to a hybond n membrane ( amersham biosciences , little chalfont , united kingdom ) , cross - linked with uv light , and hybridized with a blakpc-3-labeled probe ( 892-bp product of blakpc-3 ) . pulsed - field gel electrophoresis demonstrating genetic relatedness of study isolates eco2 , eco1 , and kpn1 , and a representative klebsiella pneumoniae isolate of the epidemic clone , kpn st258 , israel , 2008 . bacterial dna was prepared and cleaved with 20u spei endonuclease ( new england biolabs , beverly , ma , usa ) , followed by electrophoresis in a chef - dr iii apparatus ( bio - rad laboratories , inc . , hercules , ca , usa ) , as described ( 4 ) . the macrorestriction patterns of the isolates were compared according to the dice similarity index ( 1.5% tolerance interval ) by using gelcomparii software ( applied maths , kortrigk , belgium ) . kpn1 carried 4 different plasmids , whereas eco2 carried 1 plasmid that correlated with the 105-kb plasmid of kpn1 . experiments to transform eco2 and kpn1 plasmids into an e. coli dh10b recipient , followed by selection on plates containing 100 g / ml ampicillin and screening for blakpc - positive colonies , showed that dh10b was transformed with the105-kb kpc-3encoding plasmid ( figure 1 , panel a ) . this plasmid correlated in size with that of pkpqil , the kpc-3-encoding plasmid of klebsiella st258 in israel ( 13 ) . plasmid dna restriction fragment length polymorphism showed that band patterns of the 2 kpc-3encoding plasmids of kpn1 and eco2 were highly similar ( 98% similarity ) ( figure 1 , panel b ) , and southern analysis with a blakpc-3 probe showed the same hybridization pattern ( figure 1 , panel c ) . we aimed to determine whether the patient s gut was colonized with a carbapenem - susceptible e. coli strain , which would ultimately serve as the in vivo recipient of the blakpc-3-encoding plasmid . thus , the first broth culture prepared ( obtained before the patient was colonized with kpc-3producing e. coli ) was processed . aliquots ( 0.1 ml ) were directly plated onto a macconkey agar plate ( hy - labs , rehovot , israel ) . e. coli colonies isolated from the plate were restreaked onto a macconkey agar plate , yielding an e. coli strain 7364 ( eco1 ) that was susceptible to all antimicrobial drugs tested ( appendix table ) . pfge dna fingerprinting showed that eco1 was 100% identical to the kpc-3-producing clinical strain eco2 , isolated from the clinical specimen ( figure 2 ) . plasmid analysis of this strain , however , proved that it lacked plasmid pkpqil ( results not shown ) . elements belonging to kpc transposon tn4401 , including tnpa , tnpr , iskpn6 , and iskpn7 ( 14 ) , were identified by pcr and sequencing on both kpc-3encoding plasmids originating from kpn1 and eco2 . these data suggest that eco1 has acquired pkpqil from kpn1 in the patient s gut , leading to the formation of eco2 . although acquisition of the plasmid increased mics for imipenem , meropenem , and ertapenem considerably , it did not confer full resistance ( appendix table ) presumably due to copy number of the plasmid or the expression level of blakpc-3 in e. coli . curing of pkpqil from eco2 was performed by sequential transfers at an elevated temperature ( 42c ) . the cured strain , which lacked the kpc - encoding plasmid , showed full susceptibility to all antimicrobial drugs tested , similar to the eco1 strain isolated from the patient s gut flora . the patient received a combination of 4 antimicrobial agents concomitantly ( ertapenem , metronidazole , colistin , and vancomycin ) during the period in which eco1 acquired in vivo the plasmid pkpqil , thereby becoming eco2 . we believe that the selection pressure imposed by these antimicrobial agents contributed to the sequence of events that led to plasmid transfer . we hypothesize that interspecies conjugation and antimicrobial pressure led to the preferential selection of eco2 , rather than eco1 , as a determinant of infection in this patient . interspecies kpc transfer can presumably occur through the dissemination of mobile genetic elements as has been described for transfer of the meca gene between strains of staphylococcus aureus ( 15 ) . blakpc may spread through transfer by virtue of its location on the tn4401 transposon ( 14 ) , or by dissemination of the intact kpc - encoding plasmid , likely through natural conjugation . multiple attempts to mimic the natural transfer of pkpqil , the kpc-3-encoding plasmid from kpn1 into eco1 by using conjugation experiments were not successful . the isolation of an isogenic , antimicrobial drug  susceptible e. coli clone enabled us to decipher the natural order of the interspecies genetic transfer event with increasing global spread of kpc - producing k. pneumoniae st258 , the likelihood increases of interspecies transfer of drug - resistance determinants into a highly fit e. coli clone . such an event may have severe public health consequences , leading to elimination of any effective antimicrobial drug treatment against the most common human bacterial pathogens . patient 's bacterial isolates and escherichia coli dh10b transformed with their kpc-3-encoding plasmids , israel , 2008 *
klebsiella pneumoniae carbapenemase ( kpc ) 3producing escherichia coli was isolated from a carrier of kpc-3producing k. pneumoniae . 
 the kpc-3 plasmid was identical in isolates of both species . 
 the patient 's gut flora contained a carbapenem - susceptible e. coli strain isogenic with the kpc-3producing isolate , which suggests horizontal interspecies plasmid transfer .