Fitness costs resulting in a lower bacterial growth rate or a lower maximum density due to the presence of the plasmid IncI1 carrying the bla
CTX-M-1 gene were not observed here. No differences were found between donor D, recipient R and transconjugant T in growth rate ψ, maximum density K or lag-phase λ in single population experiments 1a-j. Fitness costs might have arisen in a competition setting with mixed populations of D and R due to competition for resources or inhibition by the competitor. However, also in the mixed populations of the conjugation experiments 2a-b, we could not find a difference in growth parameters between the recipient R and donor D.
San Millan et al. neither found a difference in percentage of plasmid free and plasmid carrying bacteria for their pB1000 plasmid in the first 12 hours. However, starting at day 2 they observed a clear decrease in the fraction of plasmid carrying bacteria. Also in our experiments, the fitness costs of the plasmid carrying bacteria were not evident in the early phase. Small fitness costs may not be observable at all in experiments with a short duration, but when the experiments are maintained longer, fitness costs other than costs related to the growth rate can play a role. In 12 or 24 hours experiments, these differences might be too small to measure. This is why we conducted the long term experiment 3 both with intervals of 24 and 48 hours, as the duration of our experiments 1 and 2 (up to 24 hours) may have been too short to observe fitness costs. We showed by simulation (illustrated in Figure 3) that only for large fitness costs resulting in a 20% smaller maximum density K by carrying the IncI1 plasmid, a distinct decrease in population size would have been observed within the time-frame of experiment 3. This was, however, not observed in experiment 3, underlining the conclusion that this plasmid does not infer sufficient fitness costs to its host bacterium to let it go extinct in the absence of antimicrobials. Thus, our results suggest that reduction of the use of antimicrobials might not result in a decrease, let alone extinction, of such a plasmid. This is in accordance with the conclusions of Poole et al..
The extrapolation of in vitro experiments to in vivo dynamics might show to be invalid, due to the presence of other bacterial species and a different environment. Furthermore, our study focussed on only one plasmid and host (E. coli) combination. Although this combination is relevant, because of its high prevalence in Dutch broilers, other plasmid – host combination might exhibit different behaviour.
Plasmid loss was not observed as expected because of the presence of two addiction systems, which account for stable inheritance of the plasmid to daughter cells . The presence of these addiction systems is common in IncI1 plasmids . The reduction of the ESBL-gene carrying plasmid shall thus depend on fitness costs involving reduced growth or maximum density of its host.
Conjugation was modelled as a mass action process, which is often used to describe the spread of infectious diseases among host individuals . This mass action assumption is commonly used for modelling the conjugation process, as it explains mechanistically that at higher concentrations of bacteria, conjugation is more efficient because cells make more frequent contacts [12, 24]. With mass action we assume that the time taken by the actual conjugation process is much smaller than the time between contacts of bacteria, which seems a valid assumption, because much higher conjugation coefficients are found with similar conjugation systems . Furthermore, assuming mass action means that we assume homogeneous mixing, this is thought to occur in our in vitro experiments, but might not be the case under natural conditions. When under natural conditions in the gut mixing is not homogeneous, the conjugation will be less efficient because fewer contacts are made. This might lead to a decrease of bacteria carrying the plasmid when small fitness costs exist, which cannot be measured in our in vitro experiments.
For our analyses, we used a logistic growth model by Barany and Roberts  for which we separated the population into three subpopulations (D, R and T) and added conjugation and plasmid loss dynamics. The model does not describe a death phase in which the bacterial population dies out. A death phase occurs when the medium in which the populations are grown is depleted of nutrients. Such a death phase was not observed in the experiments. Therefore, the model was appropriate to describe the population dynamics in our experiments.
The conjugation coefficient γ
of the transconjugant was found to be much higher than that of the donor. This might be due to repression of conjugation [9, 26]. By such a mechanism conjugation becomes repressed after a certain period since acquiring the plasmid. Newly formed transconjugants have a transient period in which conjugation is de-repressed and the conjugation coefficient is higher. The population of donors might be in a repressed state such that the increase of transconjugants is slower in the beginning of the experiment, and the accumulation of new transconjugants increases the overall conjugation coefficient. Such a repression-depression system is, however, to our knowledge not described for IncI1 plasmids.
The results of this study, although obtained in vitro, indicate that the IncI1 plasmid carrying the bla
CTX-M-1 gene does not impose or only imposes small fitness costs in the absence of antimicrobials. Apart from abandoning the use of antimicrobials, additional measures might be required to reduce the occurrence of this plasmid, such as competitive exclusion with other bacteria carrying incompatible plasmids [6, 16]. If the IncI1 plasmid shows the same absence of fitness costs in vivo as in our in vitro experiments and additional control measures cannot be found, it is expected that this plasmid remains present in poultry even without the use of antimicrobials.