Rare Spontaneous Loss of Multiresistance Gene Carrying IncI/ST12 Plasmid in Escherichia coli in Pig Microbiota

Abstract

Resistance to extended-spectrum cephalosporins (ESCs) is a matter of considerable concern for public health. Here, we studied the spontaneous loss of an extended-spectrum beta-lactamase (ESBL)-encoding plasmid from a rifampin-resistant Escherichia coli isolate orally inoculated into pigs under controlled conditions. Fecal samples were collected and cultured on rifampin-supplemented medium, and the resistance of the E. coli isolates to ESCs was studied by phenotypic tests, PCR detection of plasmid genes, and complete sequencing. The results showed that only 3 out of 353 rifampin-resistant E. coli isolates were ESC susceptible, and PCR and bioinformatics analysis confirmed the loss of the plasmid. These in vivo experiments indicate that the loss of an ESBL-encoding plasmid seems a rare event in gut microbiota. Resistance to extended-spectrum cephalosporins (ESCs) is a considerable public health concern. Food and companion animals may also harbor ESC-resistant Enterobacteriaceae in their intestinal microbiota. ESC resistance in animals in Europe is most commonly due to conjugative plasmids carrying extended-spectrum beta-lactamase (ESBL)-encoding genes. It is sometimes hypothesized that, for bacteria, the presence of a conjugative plasmid may have a biological cost so that plasmid-free bacteria would outcompete their plasmid-harboring homologs in an antimicrobial-free environment. This assumption relies on the hypothesis that either both plasmid-bearing and plasmid-free strains are present in this environment or bacteria can lose their plasmid. However, plasmids have developed sophisticated mechanisms to ensure their transmission to daughter cells during cell division (1). Although several studies have addressed the in vitro stability of plasmids (2-4), the in vivo maintenance of a resistance plasmid has never been reported, to our knowledge. Thus, we took advantage of a series of experiments including pigs inoculated with ESCresistant Escherichia coli to evaluate the maintenance of an ESBLencoding plasmid in its E. coli host in pig gut. MATERIALS AND METHODSFour different trials were conducted in 2012 to 2013, using the previously described ESBL-producing E. coli M63 strain (5). The experiments were performed in accordance with French animal welfare regulations, and the protocols were approved by the ANSES/ENVA/UPEC ethics committee (ComEth approval no. 12/032 and authorization no. 12-003). Briefly, E. coli M63 was obtained by conjugation between a randomly chosen pansusceptible E. coli isolate, UB12/059-3, obtained from a specific-pathogen-free pig of our herd, made resistant to 250 mg/liter rifampin, and an ESC-resistant E. coli isolate, 05-M63-1, from our slaughter pig strain collection. Thus, E. coli M63 is resistant to rifampin because of chromosomal mutations, and sequencing of the ESC resistance plasmid showed that it was an IncI1/ST12 plasmid harboring the bla CTX-M-1 , bla CMY-2 , sul2, dfrA17, and aadA5 genes (5). The E. coli M63 cultures used for pig inoculation were prepared in cefotaxime (CTX)-supplem...

A large self-transmissible plasmid from Nigeria confers resistance to multiple antibacterials without a carrying cost

“…Antimicrobial use is the principal driving force behind the current epidemic of antimicrobial resistance. However, research has shown that, once evolved, multidrug resistance plasmids can be maintained in the absence of selection (67). There is a dearth of fitness defects reported for resistance plasmids in the literature and the reasons why some of them are evolutionarily successful are not well understood (58,68).…”

A large self-transmissible resistance plasmid from Nigeria contains genes that ameliorate a carrying cost

“…Antimicrobial use is the principal driving force behind the current epidemic of antimicrobial resistance. However, research has shown that, once evolved, multidrug resistance plasmids can be maintained in the absence of selection 44 . There is a dearth of fitness defects reported for resistance plasmids in the literature and the reasons why some of them are evolutionarily successful are not well understood 41,45 .…”

Dissemination of IncI plasmid encodingblaCTX-M-1is not hampered by its fitness cost in the pig’s gut

“…Animals and experimental design. As previously described in Mourand et al (10,12), the experiments were conducted in the ANSES, Ploufragan, France, animal facilities. The trials were conducted with 6-to 8-week-old specific pathogen-free and initially ESC-resistant E. colifree Large White piglets.…”

“…Previous studies using a series of experiments that included pigs inoculated with E. coli harboring bla CTX-M-1 -encoding IncI plasmid showed the maintenance of the plasmid in its E. coli host, in the pig’s gut. Moreover, the loss of the plasmid was a rare event in gut microbiota during these longitudinal studies of in vivo carriage in pigs (10). In these models, the transfer of the bla CTX-M-1 -encoding IncI plasmid between different E. coli strains occurred and underlines the dissemination capacity of the ESC resistance, independently of the exposure to antibiotic drugs (11, 12).…”