Transfer of antimicrobial resistance plasmids from Klebsiella pneumoniae to Escherichia coli in the mouse intestine

Abstract

Objectives and methods: Klebsiella pneumoniae is a nosocomial pathogen and is considered the most common gram-negative bacterium that exhibits multiple antimicrobial resistances. In this study, the transfer of antimicrobial resistance genes from the clinical multiresistant K. pneumoniae MGH75875 isolate was assessed in vitro and in vivo in an intestinal colonization animal model. The ability to colonize and transfer was tested under different antimicrobial treatments. The frequency of the horizontal gene transfer was also examined in vitro.

Results: The clinical isolate of K. pneumoniae colonized the intestine of mice at levels up to 10(9) cfu/g faeces in antimicrobial-treated mice. In mice without antimicrobial treatment, the strain quickly decreased to below the detection limit due to competitive exclusion by the indigenous mouse flora. Onset of antimicrobial treatment gave immediate rise to detectable levels of the strain in the faeces of up to 10(9) cfu/g faeces. The experiment clearly shows that the treatment selects resistant strains and gives advantages to colonize the gastrointestinal tract. Furthermore, high transfer frequency of different plasmids was observed during colonization of the mouse intestine. The bla(SHV) and bla(TEM) genotypes were transferred to both an indigenous recipient in the in vivo setting and to an MG1655 Escherichia coli recipient strain in vitro.

Conclusions: K. pneumoniae is an excellent colonizer of the intestine and is extremely promiscuous with respect to the transferability of its numerous plasmids. Antimicrobial treatment enhances the selection of resistant strains and results in an increase in the resistance gene pool, which ultimately raises the risk of spreading resistance genes.

Figure 1

Plasmid profiles and PFGE profiles of donor, recipient and transconjugants. (a) Plasmid profiles where the marker is E. coli 39R861 (147, 63, 37 and 7 kb). (b) PFGE profiles using the restriction enzyme XbaI and Salmonella Branderup strain as a molecular weight marker. Lane 1, marker; lane; 2, recipient E. coli MG1655SR; lane 3, donor K. pneumoniae MGH75875; lanes 4–8, transconjugants (TCs) from different experiments (lane 4, TC MG vitro 1; lane 5, TC MG vitro 2; lane 6, TC indigenous E. coli vivo; lane 7, TC MG vitro 3; and lane 8, TC MG vivo).

Figure 2

cfu counts of K. pneumoniae from faecal samples of mice. Arrow indicates the shift in drinking water from 0 to 0.5 g/L ampicillin (AMP). The detection limit is illustrated by the dashed line. cfu of the inoculation suspension is shown at day 0 (2 × 10⁹ cfu/mouse).

Figure 3

cfu counts of K. pneumoniae from faecal samples of mice treated with 0.5 g/L ampicillin. The level of K. pneumoniae (filled squares and filled triangles) and indigenous E. coli transconjugants in Mouse 1 and 2 (open squares and open triangles). cfu of the inoculation suspension is shown at day 0 (1 × 10⁹ cfu/mouse).

Figure 4

cfu counts of donor and recipient inoculated in three mice treated with 0.5 g/L streptomycin. Recipient E. coli MG1655SR (filled circles) was inoculated first, and at day 7, the clinical K. pneumoniae strain (filled triangles) was introduced. Transconjugants (TCs) selected on different media: rifampicin and ampicillin (filled upside-down triangles), rifampicin and kanamycin (filled diamonds), and rifampicin and chloramphenicol (filled circles). cfu of the inoculation suspension of E. coli MG1655SR shown at day 0 (3 × 10⁸ cfu/mouse) and inoculation suspension of K. pneumoniae at day 7 (3 × 10⁸ cfu/mouse) (not depicted).