Microevolution of Helicobacter pylori during Prolonged Infection of Single Hosts and within Families

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Abstract
Our understanding of basic evolutionary processes in bacteria is still very limited. For example, multiple recent dating estimates are based on a universal inter-species molecular clock rate, but that rate was calibrated using estimates of geological dates that are no longer accepted. We therefore estimated the short-term rates of mutation and recombination in Helicobacter pylori by sequencing an average of 39,300 bp in 78 gene fragments from 97 isolates. These isolates included 34 pairs of sequential samples, which were sampled at intervals of 0.25 to 10.2 years. They also included single isolates from 29 individuals (average age: 45 years) from 10 families. The accumulation of sequence diversity increased with time of separation in a clock-like manner in the sequential isolates. We used Approximate Bayesian Computation to estimate the rates of mutation, recombination, mean length of recombination tracts, and average diversity in those tracts. The estimates indicate that the short-term mutation rate is 1.4×10−6 (serial isolates) to 4.5×10−6 (family isolates) per nucleotide per year and that three times as many substitutions are introduced by recombination as by mutation. The long-term mutation rate over millennia is 5–17-fold lower, partly due to the removal of non-synonymous mutations due to purifying selection. Comparisons with the recent literature show that short-term mutation rates vary dramatically in different bacterial species and can span a range of several orders of magnitude. Mutation rates in bacteria have generally been considered to be much slower than in viruses. This is partly because estimates of long-term mutation rates for the evolution of distinct species have been inappropriately used for dating divergence within species. Furthermore, the most commonly used long-term mutation rate is based on geological dates that are no longer accepted. In addition, only few short-term mutation rates have been calculated within bacterial species, and these differ with the species by several orders of magnitude. Here, we provide robust estimates for short-term mutation and recombination rates within Helicobacter pylori, a bacterium that commonly infects the human gastric mucosa, based on serial isolates from long-term infections and on differences between isolates from multiple family members. These short-term mutation rates are 5–17-fold faster than long-term mutation rates in H. pylori that have been calibrated by parallel ancient migrations of humans. Short-term mutation rates in bacteria, including those for H. pylori, can be quite fast, partially overlapping with those for viruses. Future calculations of ages of bacterial species will need to account for dramatic differences in mutation rate between species and for dramatic differences between short- and long-term mutation rates.