Ureaplasma parvum undergoes selection in utero resulting in genetically diverse isolates colonising the chorioamnion of fetal sheep Running Title: In utero selection of U. parvum variants Summary Sentence: The chorioamnion selects for different ureaplasma sub-types within a non-clonal population during chronic intrauterine infection; ureaplasmas isolated from chorioamnion tissue contained highly polymorphic 23S rRNA gene sequences. Abstract 1 Ureaplasmas are the microorganisms most frequently

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... olated from the amniotic fluid of 2 pregnant women and can cause chronic intrauterine infections. These tiny bacteria are 3 thought to undergo rapid evolution and exhibit a hypermutatable phenotype; however, little is 4 known about how ureaplasmas respond to selective pressures in utero. Using an ovine model 5 of chronic intra-amniotic infection, we investigated if exposure of ureaplasmas to sub-6 inhibitory concentrations of erythromycin could induce phenotypic or genetic indicators of 7 macrolide resistance. At 55 days gestation, 12 pregnant ewes received an intra-amniotic 8 injection of a non-clonal, clinical U. parvum strain, followed by: (i) erythromycin treatment 9 (IM, 30 mg/kg/day, n=6); or (ii) saline (IM, n=6) at 100 days gestation. Fetuses were then 10 delivered surgically at 125 days gestation. Despite injecting the same inoculum into all ewes, 11 significant differences between amniotic fluid and chorioamnion ureaplasmas were detected 12 following chronic intra-amniotic infection. Numerous polymorphisms were observed in 13 domain V of the 23S rRNA gene of ureaplasmas isolated from the chorioamnion (but not the 14 amniotic fluid), resulting in a mosaic-like sequence. Chorioamnion isolates also harboured 15 the macrolide resistance genes erm(B) and msr(D) and were associated with variable 16 roxithromycin minimum inhibitory concentrations. Remarkably, this variability occurred 17 independently of exposure of ureaplasmas to erythromycin, suggesting that low-level 18 erythromycin exposure does not induce ureaplasmal macrolide resistance in utero. Rather, the 19 significant differences observed between amniotic fluid and chorioamnion ureaplasmas 20 suggest that different anatomical sites may select for ureaplasma sub-types within non-clonal, 21 clinical strains. This may have implications for the treatment of intrauterine ureaplasma 22 infections. 23