We recently investigated the applicability of antibiotic-treated recipient mice for transfer of different gut microbiota profiles. With this addendum we elaborate on perspectives and limitations of using antibiotics as an alternative to germ-free (GF) technology in microbial transplantation studies, and we speculate on the housing effect. It is possible to transfer host phenotypes via fecal transplantation to antibiotic-treated animals, but problems with reproducibility, baseline values, and antibiotic resistance genes should be considered. GF animals maintained in isolators still seem to be the best controlled models for long-term microbial transplantation, but antibiotic-treated recipients are also commonly utilized. We identify a need for systematic experiments investigating the stability of microbial transplantations by addressing 1) the recipient status as either GF, antibiotic-treated or specific pathogen free and 2) different levels of protected housing systems. In addition, the developmental effect of microbes on host physiological functions should be evaluated in the different scenarios.

Germ-free rodents colonized with microbiotas of interest are used for host-microbiota investigations and for testing microbiota-targeted therapeutic candidates. Traditionally, isolators are used for housing such gnotobiotic rodents due to optimal protection from the environment, but research groups focused on the microbiome are increasingly combining or substituting isolator housing with individually ventilated cage (IVC) systems. We compared the effect of housing systems on the gut microbiota composition of germ-free mice colonized with a complex microbiota and housed in either multiple IVC cages in an IVC facility or in multiple open-top cages in an isolator during three generations and five months. No increase in bacterial diversity as assessed by 16S rRNA gene sequencing was observed in the IVC cages, despite not applying completely aseptic cage changes. The donor bacterial community was equally represented in both housing systems. Time-dependent clustering between generations was observed in both systems, but was strongest in the IVC cages. Different relative abundance of a Rikenellaceae genus contributed to separate clustering of the isolator and IVC communities. Our data suggest that complex microbiotas are protected in IVC systems, but challenges related to temporal dynamics should be addressed.

Transplantation of germ-free (GF) mice with microbiota from mice or humans stimulates the intestinal immune system in disparate ways. We transplanted a human microbiota into GF C57BL/6 mice and a murine C57BL/6 microbiota into GF C57BL/6 mice and Swiss-Webster (SW) mice. Mice were bred to produce an offspring generation. 56% of the Operational Taxonomic Units (OTUs) present in the human donor microbiota established in the recipient mice, whereas 81% of the C57BL/6 OTUs established in the recipient C57BL/6 and SW mice. Anti-inflammatory bacteria such as Faecalibacterium and Bifidobacterium from humans were not transferred to mice. Expression of immune-related intestinal genes was lower in human microbiota-mice and not different between parent and offspring generation. Expression of intestinal barrier-related genes was slightly higher in human microbiota-mice. Cytokines and chemokines measured in plasma were differentially present in human and mouse microbiota-mice. Minor differences in microbiota and gene expression were found between transplanted mice of different genetics. It is concluded that important immune-regulating bacteria are lost when transplanting microbiota from humans to C57BL/6 mice, and that the established human microbiota is a weak stimulator of the murine immune system. The results are important for study design considerations in microbiota transplantation studies involving immunological parameters. The gut microbiota is an important component of human health. For studying its role in health and disease, aiming for the development of microbiota-targeting therapeutics, food products and ingredients, mice transplanted with human microbiotas (HMs) have been described and applied for several decades 1-6 , although concerns pertaining to limitations of this model system have been raised 7-10. Often such models are only studied on their phenotypic expression and the microbiota is not characterized, or the opposite is the case 8. Laboratory rodents are routinely raised in specific pathogen free (SPF) barrier facilities strictly protected from their wild conspecifics. For many years the microbial starting point for many rodent breeding colonies has been the Altered Schaedler Flora (ASF) or variants thereof. ASF consists of eight specific bacterial strains originating from conventional laboratory mice from the 1960's and 1970's, and it is therefore considered mouse-specific 11,12. In addition to this, laboratory rodents are exposed to microbes deriving from human staff in the facility, at least if bedding, food, and other materials are sterilized before introduced to the facility. Evolutionary adaptation to the host environment may drive formation of mouse-specific species and strains originally derived from humans, as

Background Despite low genetic variation of broilers and deployment of considerate management practices, there still exists considerable body weight (BW) heterogeneity within broiler flocks which adversely affects the commercial value. The purpose of this study was to investigate the role of the cecal microbiome in weight differences between animals. Understanding how the gut microbiome may contribute to flock heterogeneity helps to pave the road for identifying methods to improve flock uniformity and performance. Results Two hundred eighteen male broiler chicks were housed in the same pen, reared for 37 days, and at study end the 25 birds with highest BW (Big) and the 25 birds with lowest BW (Small) were selected for microbiome analysis. Cecal contents were analyzed by a hybrid metagenomic sequencing approach combining long and short read sequencing. We found that Big birds displayed higher microbial alpha diversity, higher microbiome uniformity (i.e. lower beta diversity within the group of Big birds), higher levels of SCFA-producing and health-associated bacterial taxa such as Lachnospiraceae, Faecalibacterium, Butyricicoccus and Christensenellales, and lower levels of Akkermansia muciniphila and Escherichia coli as compared to Small birds. Conclusion Cecal microbiome characteristics could be linked to the size of broiler chickens. Differences in alpha diversity, beta diversity and taxa abundances all seem to be directly associated with growth differences observed in an otherwise similar broiler flock.