The influence of manure amendment on the soil bacterial communities was still noticeable after one month, although the magnitude of changes varied between soils. Indeed, the bacterial community of soil A showed, compared to others, a stronger shift following the manure amendment, while that of soil D was the least affected. The most abundant OTUs in the soils remained stable but the rare ones were affected (i.e. the rarest one among those with relative abundance > 0.05%) and possibly undetected ones (i.e. those with relative abundance < 0.05% and/or those that were not present in the soil). Changes in the composition of microbial communities due to rare OTUs during coalescence processes is in line with the observation of the emergence of the rare-biosphere microorganisms following environmental disturbances . Indeed, the least abundant OTUs under stable conditions are often those favored by a disruption of the environmental conditions [32, 33]. They may benefit from the equilibrium disruption of the environment where they are normally outcompeted by the better-adapted to multiply and colonize newly available ecological niches .
The communities of soils amended with manure exhibited OTUs with a wider distribution along the phylogenetic tree. This increase in diversity within the soil bacterial community could either correspond to the emergence of endogenous rare OTUs with functional abilities better adapted to this new environment than the original one or to the invasion of soil community. While previous studies provided evidence suggesting soil invasion by manure-borne bacteria, they failed to distinguish surely allochthonous invaders from autochthonous OTUs that emerge because of environmental disturbance caused by the adjunction of exogenous matter [15, 20]. In the same line, we showed that some OTUs favored in response to manure amendment were not detected in the manure-free soils although they were almost all detected in the manure. However, even if these OTUs were not detected in control soils but detected in the manure, it is not possible to conclude definitively that there was an effective invasion of the soil with OTUs of manure origin since the lack of detection in the soil could be due to sequencing biases. Indeed, an absence of detection by 16S rDNA sequencing does not necessarily mean its effective absence in the community because of detection thresholds that ignore the endogenous rarest OTUs and make it impossible to perceive the entire bacterial community .
However, our results provided additional evidence in favor of the hypothesis of the invasion of manure-borne OTUs thanks to the use of different soils, including one already amended in situ 6 months earlier (i.e. soil D). First, in response to manure amendment some of these OTUs presented the same increase pattern in different soils. The extreme case involved the most abundant OTU in the manure, a Pseudomonodales, which was favored in each of the four soils and was not detected in control soils A, B and C not amended with manure. This response pattern suggests a common manure origin since all the soils presented different biotic and abiotic properties, limiting the chance that they hosted the same rarest OTUs. Secondly, all these OTUs were detected in the non-experimentally amended soil D, suggesting that they were successful in colonizing this soil following previous in situ manure amendment events (the last amendment having taken place six months before the experiment).
Considering this set of arguments, we assumed that all or almost all the OTUs detected in both manure and soil D and which increased in several soils were from manure origin. All of them were among the most abundant OTUs in manure. Their high abundance in manure bacterial community composition may explain the success of their invasion, since a high inoculation rate is more likely to be successful [20, 36]. One could argue that the detection of the most abundant bacteria of the manure in soil may be a bias due to DNA from dead cells that did not establish in the soil [37, 38]. However, this argument can be rejected since not all the most abundant OTUs from manure were detected in the amended soils.
Interestingly, when examining the abundance of the OTUs that are suspected to be invaders established in soil D during previous in situ amendment of manure (see above), two kinds of establishment patterns can be differentiated, depending on their taxonomic affiliation: the first one relates to the Firmicutes that are quite abundant in soil D, while the other relates to the Proteobacteria that are rare OTUs (Supplementary Tab.1). The ability of OTUs related to Firmicutes to have become well represented in the soil following previous manure amendment may be because of their physiological attributes. Firmicutes, contrary to Proteobacteria, have sporulation capacity that may confer upon them an ability to persist for a long period in a less hospitable environment until favorable conditions return . Indeed, the long-term survival of Firmicutes in soil after manure amendment has been already reported, with detection even after the winter season .
It is noticeable that the community of soil D was much less impacted by manure amendment than the other soils, with only one OTU being significantly increased. This is in contrast to the hypothesis formulated by Mallon et al. (2018), who suggested that previous invasions generate legacy effects in soil communities facilitating future invasion attempts. However, it is consistent with the study of Gravuer and Scow, (2021), who observed that soils’ communities after a first disturbance by manure amendment were less impacted by the following ones. They hypothesized that the endogenous soil bacteria may adapt to the occasional input of nutrients carried by manure, outcompeting manure-borne bacteria during subsequent amendments. Here, we rather hypothesized that it is the manure-borne OTUs already established in the soil that prevent the new ones from establishing since they are functionally similar and already adapted to soil.
Finally, the impact of the antibiotic SMZ on the structure of autochthonous soil bacterial communities was relatively low as it was significant in only one of the four soils (soil A). Previous results obtained from the same microcosms may explain this fact . After one month of incubation, the bioavailable fraction of SMZ in the SMZ-treated soils represented a low level of exposure for the community. Concordantly, the soil A in which the free-fraction of SMZ was the highest  is the one for which its bacterial community was differently impacted by manure amendment according the presence or not of SMZ. A first group of seven OTUs, most of them suspected of being manure-borne OTUs, had their abundance even more increased by manure amendment in the presence of SMZ. The observed effect of SMZ could be explained by the selection of antibiotic-resistant bacteria. Indeed, persistence of resistant bacteria and contamination by antibiotics could be positively linked . For instance, we showed in a previous study that the invasion of SMZ-resistant bacteria in soil can be favored in the presence of the antibiotic . The second group consisted of OTUs that were favored by the manure amendment in the absence of SMZ but that lost this advantage in the presence of the antibiotic. All were autochthonous soil bacteria. The lack of negative effects of SMZ on these OTUs in manure-free conditions (Supplementary Tab 1) suggests that they were not especially sensitive to SMZ.