Transfer dynamics of multi-resistance plasmids in Escherichia coli isolated from meat.

Resistance plasmids are crucial for the transfer of antimicrobial resistance and thus form a matter of concern for veterinary and human healthcare. To study plasmid transfer, foodborne Escherichia coli isolates harboring one to five known plasmids were co-incubated with a general recipient strain. Plasmid transfer rates under standardized conditions varied by a factor of almost 106, depending on the recipient/donor strain combination. After 1 hour transconjugants never accounted for more than 3% of the total number of cells. Transconjugants were formed from 14 donors within 1 hour of co-incubation, but in the case of 3 donors 24 hours were needed. Transfer rates were also measured during longer co-incubation, between different species and during repeated back and forth transfer. Longer co-incubation resulted in the transfer of more types of resistance. Maximum growth rates of donor strains varied by a factor of 3. Donor strains often had higher growth rates than the corresponding transconjugants, which grew at the same rate as or slightly faster than the recipient. Hence, possessing one or more plasmids does not seem to burden the harboring strain metabolically. Transfer was species specific and repeated transfer of one plasmid did not result in different transfer rates over time. Transmission Electron microcopy was used to analyze the morphology of the connection between co-incubated strains. Connection by more pili between the cells resulted in better aggregate formation and corresponded with higher transfer rates.

Genetic editing of multi-resistance plasmids in Escherichia coli isolated from meat during transfer.

Resistance plasmids mediate the rapid spread of antimicrobial resistance, which poses a threat to veterinary and human healthcare. This study addresses the question whether resistance plasmids from Escherichia coli isolated from foodstuffs always transfer unchanged to recipient E. coli cells, or that genetic editing can occur. Strains containing between one and five different plasmids were co-incubated with a standard recipient strain. Plasmids isolated from transconjugant strains were sequenced using short and long read technologies and compared to the original plasmids from the donor strains. After one hour of co-incubation only a single plasmid was transferred from donor to recipient strains. If the donor possessed several plasmids, longer co-incubation resulted in multiple plasmids being transferred. Transferred plasmids showed mutations, mostly in mobile genetic elements, in the conjugative transfer gene pilV and in genes involved in plasmid maintenance. In one transconjugant, a resistance cluster encoding tetracycline resistance was acquired by the IncI1 plasmid from the IncX1 plasmid that was also present in the donor strain, but that was not transferred. A single plasmid transferred twelve times back and forth between E. coli strains resulted in a fully conserved plasmid with no mutations, apart from repetitive rearrangements of pilV from and back to its original conformation in the donor strain. The overall outcome suggests that some genetic mutations and rearrangements can occur during plasmid transfer. The possibility of such mutations should be taken into consideration in epidemiological research aimed at attribution of resistance to specific sources.

Multiplication of ampC upon Exposure to a Beta-Lactam Antibiotic Results in a Transferable Transposon in Escherichia coli.

Plasmids play a crucial role in spreading antimicrobial resistance genes. Plasmids have many ways to incorporate various genes. By inducing amoxicillin resistance in Escherichia coli, followed by horizontal gene transfer experiments and sequencing, we show that the chromosomal beta-lactamase gene ampC is multiplied and results in an 8-13 kb contig. This contig is comparable to a transposon, showing similarities to variable regions found in environmental plasmids, and can be transferred between E. coli cells. As in eight out of nine replicate strains an almost completely identical transposon was isolated, we conclude that this process is under strict control by the cell. The single transposon that differed was shortened at both ends, but otherwise identical. The outcome of this study indicates that as a result of exposure to beta-lactam antibiotics, E. coli can form a transposon containing ampC that can subsequently be integrated into plasmids or genomes. This observation offers an explanation for the large diversity of genes in plasmids found in nature and proposes mechanisms by which the dynamics of plasmids are maintained.

Antibiotic resistance plasmid composition and architecture in Escherichia coli isolates from meat.

Resistance plasmids play a crucial role in the transfer of antimicrobial resistance from the veterinary sector to human healthcare. In this study plasmids from foodborne Escherichia coli isolates with a known (ES)BL or tetracycline resistance were sequenced entirely with short- and long-read technologies to obtain insight into their composition and to identify driving factors for spreading. Resistant foodborne E. coli isolates often contained several plasmids coding for resistance to various antimicrobials. Most plasmids were large and contained multiple resistance genes in addition to the selected resistance gene. The majority of plasmids belonged to the IncI, IncF and IncX incompatibility groups. Conserved and variable regions could be distinguished in each of the plasmid groups. Clusters containing resistance genes were located in the variable regions. Tetracycline and (extended spectrum) beta-lactamase resistance genes were each situated in separate clusters, but sulphonamide, macrolide and aminoglycoside formed one cluster and lincosamide and aminoglycoside another. In most plasmids, addiction systems were found to maintain presence in the cell.