Antibiotic resistance is a serious threat to human and animal health and new ways to combat it are urgently needed. Broad-host range plasmids, such as those belonging to the IncN and IncP1 groups are important to the dissemination of antibiotic resistance due to their ability to replicate in a variety clinically relevant bacterial species and environments [1
]. Indeed, both IncN and IncP1 group plasmids have been shown to encode clinically important resistance determinants such as blaCTX-M
], whilst IncN plasmids have also been strongly implicated in the recent spread of blaKPC
encoded carbapenemases [9
Antimicrobial resistance can sometimes be accompanied by a reduction in biological fitness in the absence of antibiotic selection. Hence, less fit resistant bacteria may be outcompeted and displaced by fitter, susceptible bacteria in the absence of antibiotic use, leading to the suggestion that it may be possible to reduce the prevalence of antibiotic resistance by temporarily restricting prescribing. In practice, however, such approaches have enjoyed mixed success [10
A fitness cost of antibiotic resistance has often been demonstrated in the case of chromosomal mutations conferring resistance, for example in the case of fusA
mutations conferring resistance to fusidic acid [15
] and gyrA
mutations conferring resistance to fluoroquinolones [16
]. However, compensatory mutations can arise at secondary sites that reduce or eliminate this cost [17
]. In the case of acquired antibiotic resistance genes encoded on mobile genetic elements such as plasmids and transposons, the existence of a fitness cost is less clear. While early studies which often investigated cloning plasmids and/or laboratory strains demonstrated a cost to plasmid carriage [18
], some more recent data using naturally-occurring plasmids and/or wild-type bacteria have failed to demonstrate significant costs and have sometimes shown a benefit. For example, the small sulphonamide and streptomycin resistance plasmid p9123 confers a 4% per generation fitness benefit in E. coli
], and a benefit has also been demonstrated for some apramycin resistance plasmids isolated from bovine E. coli
]. A number of antibiotic resistance encoding plasmids and transposons conferred only a low fitness cost or were cost-neutral in the wild-type E. coli
strain 345-2RifC in vitro
and in the pig gut [24
], whilst the resistance plasmid R751 and variants of it enhanced fitness under some growth conditions in E. coli
]. It is likely that the fitness cost a particular plasmid exerts on its host is variable depending on the plasmid as well as on the host itself. However, few studies have examined the fitness cost of a single plasmid on different strains of bacteria. The genetic factors, be they plasmid or host-encoded, that influence fitness are poorly understood, and it is not known whether related plasmids influence fitness in similar ways.
There are theoretically three ways in which a bacterial host can counteract the potential fitness cost exerted by antibiotic resistance genes carried on mobile genetic elements; the first is to acquire compensatory mutations, while the second is outright loss of the mobile genetic element. A third possibility is that bacteria could switch off the expression of resistance genes when they are not required whilst retaining the genes themselves in order to lower costs. We have previously demonstrated silencing of antibiotic resistance genes carried on the broad-host range plasmids pVE46 and RP1 by the wild-type E. coli
strain 345-2RifC [26
]. Following passage through the pig gut, a small proportion (0.5%) of 345-2RifC(pVE46) colonies recovered lost expression of one or more of the four resistance genes encoded on the plasmid. Such isolates had retained the pVE46 plasmid and in most cases, intact, wild-type resistance genes and promoters were present, but no resistance gene mRNA was expressed. Similar results were found for three colonies of 345-2RifC(RP1) that also lost resistance following passage through the pig gut. Antibiotic resistance gene silencing appears to be restricted to only the plasmid with minimal effect on the remainder of the genome and is thought to be due to a mutation on the chromosome of E. coli
]. Its precise mechanism is yet to be elucidated.
Here, we examine several unexplored questions regarding the fitness impact of broad host range IncP and IncN plasmids on their hosts; namely, the effect of the host background on fitness, whether related plasmids have similar fitness impacts and the fitness impact of antimicrobial resistance gene. To facilitate this task we also report the complete nucleotide sequence of the IncN plasmid N3.