Virulence and Pathogen Multiplication: A Serial Passage Experiment in the Hypervirulent Bacterial Insect-Pathogen Xenorhabdus nematophila

The trade-off hypothesis proposes that the evolution of pathogens' virulence is shaped by a link between virulence and contagiousness. This link is often assumed to come from the fact that pathogens are contagious only if they can reach high parasitic load in the infected host. In this paper we present an experimental test of the hypothesis that selection on fast replication can affect virulence. In a serial passage experiment, we selected 80 lines of the bacterial insect-pathogen Xenorhabdus nematophila to multiply fast in an artificial culture medium. This selection resulted in shortened lag phase in our selected bacteria. We then injected these bacteria into insects and observed an increase in virulence. This could be taken as a sign that virulence in Xenorhabdus is linked to fast multiplication. But we found, among the selected lineages, either no link or a positive correlation between lag duration and virulence: the most virulent bacteria were the last to start multiplying. We then surveyed phenotypes that are under the control of the flhDC super regulon, which has been shown to be involved in Xenorhabdus virulence. We found that, in one treatment, the flhDC regulon has evolved rapidly, but that the changes we observed were not connected to virulence. All together, these results indicate that virulence is, in Xenorhabdus as in many other pathogens, a multifactorial trait. Being able to grow fast is one way to be virulent. But other ways exist which renders the evolution of virulence hard to predict

Analysis of the time that elapses before absorbance starts increasing.

<p>This analysis was performed using a non-parametrical Cox model of survival. The total number of kinetics that are analyzed here is . The variation between the three replicate experiments is modeled as resulting from a random factor. The possible influence of initial bacterial density is controlled by considering the initial absorbance as a covariate. This analysis shows that selected lines, LDI or HDI, reach the threshold absorbance faster than the ancestral lineage.</p

Analysis of the time that elapses before absorbance starts increasing for a subset of 20 selected lineages and with dilution factors ranging from to (the total number of kinetics that are analyzed here is , see text for further details).

<p>The analysis is conducted similarly to that presented in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0015872#pone-0015872-t001" target="_blank">table 1</a>. The initial number of CFU present in the culture, which decreases with the dilution factor, is used here as a co-factor. The kinetics of selected lineages is contrasted to that of the ancestral lineage and the variance among replicate experiments is modeled as a random factor (with estimated variance ). This analysis demonstrates that the time that elapses before we first detect an increase in absorbance decreases with the initial number of bacteria present in the culture. Most importantly, the two significant interaction terms indicate that this decrease is sharper in selected bacteria than in the ancestral lineage. We found no difference between the LDI and HDI lineages.</p

Analysis of insect survival after they are injected with 2000 bacteria cells.

<p>In this analysis, only the 20 lineages we used in the dilution experiment and for phenotypic measurements are present. In addition to the insect weight and the number of CFU present in the injected solution, we considered the median lag, the phenotypic distance to the ancestral lineage, the proportion of non-motile clones and the proportion of clones with the total haemolysis phenotype as explanatory variables. The analysis of all lineages show that the factor that explains the best the highest virulence of selected lineages is the increase in the proportion of total haemolysis, although this effect is marginal. The analysis of HDI lineages demonstrated no significant correlation between the virulence and the phenotypes we have measured. Conversely, in LDI lineages, we found a highly significant correlation between the virulence and the median lag time, i.e. the time that elapses before the absorbance of 50% of the bacterial populations of a lineage overreach that of empty wells. Contrary to our expectations, though, the LDI bacteria that kill insects the fastest are those which take the longest to start growing.</p

Proportion of surviving insects as a function of the time that elapsed since they were injected with bacteria.

<p>PBS injected insects serve here as a negative control, as they were not injected with bacteria. 46 hours after injection, more than 95% of all insects injected with bacteria were dead.</p

Figure 1

<p><b>A.</b> The absorbance () of each culture as a function of time. For each curve, we analyzed separately the time that elapses before the absorbance starts increasing and the variation in absorbance after this first increase has occurred. <b>B.</b> In spite of the great variance observed in A, the shape of the growth curve each culture follows is fairly stereotyped. This is represented here by the humped relationship that exists between the increase in absorbance () and current absorbance (). The curves in this figure represent the prediction of a GAM adjusted for each treatment on all replicate experiments (see text). The maximum increase in absorbance is significantly lower and occurs for higher absorbance in selected bacteria than in the ancestral lineages. <b>C.</b> Proportion of cultures which absorbance has not yet increased above that of wells containing only LB, as a function of time, for the ancestral lineage, the HDI treatment and the LDI treatment lines. This time varies a lot from one culture to another, but always positively correlates to the number of cells present in the culture after 20 hours. Overall, we found that absorbance starts increasing sooner in selected bacteria than in the ancestral lineage, although this difference was not found in one of the three replicate experiments.</p

Median lethal time (i.e. the time at which 50% of the insects injected with a particular lineage are dead) as a function of median lag time (i.e. the time at which 50% of the populations of a particular lineage have overreached the absorbance of an empty well).

<p>The estimation of these times are performed using a parametric survival regression method (see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0015872#s2" target="_blank">method</a>). Horizontal and vertical bars correspond to the standard error of these estimates. In LDI lineages, the two quantities are negatively correlated, indicating that the fastest growing LDI lineages are the least virulent. No relation could be found between medium lag time and medium lethal time in HDI lineages. The size of symbols indicates the proportion of clones that display the total haemolysis phenotype. When analyzing both ancestral and selected lineages, we found a marginally significant link between this variable and virulence.</p

Distribution of motility halo diameter in the two selection treatments and the ancestral lineage.

<p>Distribution of motility halo diameter in the two selection treatments and the ancestral lineage.</p