An Enterobacteriaceae bloom in aging animals is restrained by the gut microbiome

The gut microbiome plays important roles in host function and health. Core microbiomes have been described for different species, and imbalances in their composition, known as dysbiosis, are associated with pathology. Changes in the gut microbiome and dysbiosis are common in aging, possibly due to multi-tissue deterioration, which includes metabolic shifts, dysregulated immunity, and disrupted epithelial barriers. However, the characteristics of these changes, as reported in different studies, are varied and sometimes conflicting. Using clonal populations of C. elegans to highlight trends shared among individuals, and employing NextGen sequencing, CFU counts and fluorescent imaging to characterize age-dependent changes in worms raised in different microbial environments, we identified an Enterobacteriaceae bloom as a common denominator in aging animals. Experiments using Enterobacter hormachei, a representative commensal, suggested that the Enterobacteriaceae bloom was facilitated by a decline in Sma/BMP immune signaling in aging animals and demonstrated its detrimental potential for increasing susceptibility to infection. However, such detrimental effects were context-dependent, mitigated by competition with commensal communities, highlighting the latter as determinants of healthy versus unhealthy aging, depending on their ability to restrain opportunistic pathobionts.


Enterobacteriaceae-specific primers and normalized to worm DNA (represented by actin genes). 124
This evaluation showed a much steeper increase in Enterobacteriaceae strains compared to the 125 increase in the total bacterial load (Fig. 2D). These results support the notion that an 126 Enterobacteriaceae bloom is a hallmark of aging, regardless of the initial conditions such as high 127 or low environmental diversity, or high or low initial proportion of Enterobacteriaceae. This 128 bloom involves a large increase in total bacterial load, but a proportionally larger increase in the 129 Enterobacteriaceae load per worm. in worms disrupted for DBL-1/BMP immune signaling, gut abundance of CEent1 increased and 135 the otherwise beneficial commensal became an exacerbating factor in host health outcomes 25 . We 136 thus used CEent1 to examine the functional significance of the age-dependent Enterobacteriaceae 137 bloom. Worms raised on CEent1 and shifted to E. faecalis at the end of larval development showed 138 higher pathogen resistance compared to worms raised on the E. coli control, as previously shown. 139 In contrast, worms raised on CEent1 to middle age before shifting to E. faecalis (day four of 140 adulthood, at which stage worms are well colonized), showed significantly lower pathogen 141 resistance compared to worms raised on E. coli controls (Fig. 3A, B). Thus, the Enterobacteriaceae 142 bloom has the potential to have detrimental consequences in aging worms. What may be the cause for the Enterobacteriaceae bloom? Experiments in microcosm 147 environments and with defined communities showed that environmental availability was not a 148 likely cause (Fig. 1B, 2A-D). Ecological succession, driven by accumulating effects of interactions 149 over time, also did not appear to contribute to the expansion (Fig. 1C). This was further supported 150 by a comparison of CEent1-dsRed colonization in worms raised continuously on CEent1-dsRed 151 monocultures versus worms shifted to CEent1-dsRed in different ages for a fixed duration of two 152 days, which showed comparable age-dependent increases in Enterobacteriaceae abundance (Fig.  153 . CC-BY 4.0 International license available under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprint this version posted June 14, 2023. ; https://doi.org/10.1101/2023.06.13.544815 doi: bioRxiv preprint causing the bloom. To this end, we compared CEent1-dsRed colonization, as part of the SC20 155 community, in wildtype worms, which show an age-dependent decline in pharyngeal pumping 156 (and thus bacterial uptake), and eat-2 mutants, which lack a pharyngeal receptor for the 157 neurotransmitter acetylcholine resulting in slow pumping rate, which does not change considerably 158 during aging (Fig. 4B inset). In both strains we observed a similar course of age-dependent 159 increases in CEent1-dsRed colonization ( Figure 4B), indicating that age-dependent changes in 160 bacterial uptake likely did not contribute to the Enterobacteriaceae bloom. 161 DBL-1/BMP signaling is a conserved regulator of development, body size and immunity 31 . 162 Previous work at the lab identified a role for DBL-1-dependent immune regulation in shaping the 163 worm gut microbiome, particularly affecting Enterobacter strains, which bloomed when genes 164 encoding different components of this pathway were disrupted 25 . Gene expression data in 165 Wormbase (https://wormbase.org) suggested that expression of the pathway's components may 166 decline at the end of larval development. To examine whether this indicated an age-dependent 167 decline in DBL-1 signaling and downstream gene expression, which might affect the gut 168 microbiome, we used a transgenic worm strain expressing GFP from the spp-9 promoter, 169 previously shown to be negatively regulated by DBL-1 signaling 32 . Fluorescent imaging 170 demonstrated age-dependent increase in the expression of the GFP reporter, indicating a decline 171 in DBL-1 signaling in aging worms (Fig. 5A). In line with this, the effects of either disruption or 172 over-expression of the dbl-1 ligand gene diminished with age. Reduced effects of dbl-1 disruption 173 were also observed in worm colonization with CEent1, which in middle-aged mutants was 174 comparable to that seen in wildtype animals, indicating a decline in DBL-1 signaling and in its 175 involvement in controlling gut Enterobacteriaceae abundance during early aging (Fig. 5B). 176 Further support for a decline in DBL-1 control of gut bacteria was provided by experiments with 177 sma-4(syb2546) mutants, which carry a gain-of-function (gof) mutation in DBL-1's transcriptional 178 mediator, exhibiting a 20% longer body length compared to wildtype animals (Fig. 5C inset). 179 These mutants showed lower CEent1-dsRed colonization compared to wildtype animals in early 180 days of adulthood, up to day five (blue box compared to dotted line in Fig. 5C), signifying a delay 181 in the CEent1 bloom. The ability to delay the CEent1 bloom had beneficial consequences, as sma-182 4(gof) mutants were partially protected from CEent1's detrimental effects on infection resistance 183 in day four adults (Fig. 5D). Together, these results suggest that an age-dependent decline in DBL-184 . CC-BY 4.0 International license available under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The ability of the sma-4(gof) mutation to partially mitigate the detrimental effects of CEent1 191 expansion suggested that protection from an Enterobacteriaceae bloom is possible. Considering 192 that the decline in DBL-1 signaling also increased abundance of non-Enterobacteriaceae bacteria 193 ( Fig. 2), we examined whether other bacteria could compete with CEent1 and help prevent its 194 detrimental effects. Recent work identified members of the genus Pantoea as common worm 195 commensals effectively colonizing the gut and capable of competing with an invading pathogen 196 23 . Wildtype worms raised on a community consisting of three such Pantoea commensals in 197 addition to CEent1-dsRed (in equal parts) and shifted to E. faecalis in middle-age were as resistant 198 as worms raised on E. coli alone, and significantly more resistant than worms raised on a similar 199 inoculum of CEent1-dsRed mixed with E. coli (Fig. 6A). While mortality on E. faecalis plates was 200 attributed to the pathogen, 96.7% of the worms raised on the CEent1-dsRed/E. coli mix, which 201 died in any of the days of the infection assay, were heavily colonized with CEent1-dsRed ( Fig. 6A  202 inset), indicating proliferation alongside E. faecalis. In contrast, only 62.5% of the worms who 203 were initially raised on the CEent1-dsRed/Pantoea mix were colonized, together indicating that 204 the Pantoea community was able to mitigate CEent1 proliferation in some of the worms and to 205 reduce mortality in the population. To examine whether mitigating the detrimental effects of 206 CEent1 proliferation was unique to Pantoea, worms were raised on a subset of seven members of 207 CeMBio (see Methods), with or without BIGb393 (one of the protective Pantoea strains, which is 208 also a member of CeMBio) and with an excess of CEent1-dsRed (50% of total) and shifted at 209 middle age to E. faecalis. Raising worms on the CeMBio subsets, with or without BIGb0303, 210 conferred significantly higher resistance to infection than in worms raised on CEent1-dsRed alone 211 (Fig. 6B). Again, fewer of the dead worms were colonized with CEent1-dsRed among those raised 212 on CeMBio/CEent1-dsRed (51.4%), compared to those raised on CEent1-dsRed alone (84.6%). 213 These experiments demonstrate that over proliferation of Enterobacteriaceae and its detrimental 214 consequences in aging worms can be mitigated with more than one combination of gut 215 . CC-BY 4.0 International license available under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprint this version posted June 14, 2023. ; https://doi.org/10.1101/2023.06.13.544815 doi: bioRxiv preprint did not compromise the lifespan of their host, as worms grown on CeMBio with or without its 217 Enterobacteriaceae members (CEent1 and JUb66) had a comparable lifespan (Fig. 6C). 218

Discussion 219
Our experiments identify an Enterobacteriaceae bloom as a hallmark of the gut microbiome in 220 aging C. elegans. This bloom was observed in worms raised in natural-like microcosm 221 environments with varying initial microbial diversity, as well as in worms raised on defined 222 bacterial communities differing in the environmental availability of Enterobacteriaceae, 223 indicating that it is independent of initial conditions. The Enterobacteriaceae bloom is not due to 224 bacteria-driven ecological succession or to age-dependent changes in bacterial uptake. Rather, it 225 is due to intrinsic age-dependent changes in the intestinal niche, suggesting that the bloom is a 226 signature of chronological age. Our results demonstrate that increased gut abundance of 227 Enterobacteriaceae strains may have detrimental consequences for aging animals, at least for 228 infection resistance. However, in the context of a community, even such with restricted diversity, 229 the detrimental consequences of this bloom can be mitigated. Our results highlight the 230 Enterobacteriaceae bloom as a hallmark of chronological aging but suggest that the consequences 231 of this bloom are context-dependent, with microbiome composition representing the context that 232 can differentiate between healthy or unhealthy aging. 233 It is accepted that aging is accompanied by gut dysbiosis 7-9,14,33 . Human studies have documented 234 diverse changes in gut microbiome composition. Among those, increased abundance of 235 Proteobacteria/Pseudomonadota, and specifically of Enterobacteriaceae, is a recurring theme 236 17,34 . In line with this, our results show a replicable age-associated expansion of 237 Enterobacteriaceae, suggesting that it may be an evolutionarily conserved signature of aging. aging. Using this model, we identified what seems to be an evolutionary conserved signature of 269 dysbiosis in aging animals and have begun to dissect its causes as well as its consequences. As 270 often seen in different scenarios of gut dysbiosis, the Enterobacteriaceae bloom that we identified 271 is associated with pathology. However, this pathology can be circumvented by manipulating the 272 gut microbiome using various commensal communities. Thus, while an Enterobacteriaceae bloom 273 seems to be an inevitable consequence of aging, its extent and outcomes can be restrained by other 274 . CC-BY 4.0 International license available under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprint this version posted June 14, 2023. ; https://doi.org/10.1101/2023.06.13.544815 doi: bioRxiv preprint achieve this remains to be seen. 276

Bacterial strains and communities 285
Escherichia coli strain OP50 and the Gram-positive pathogen, Enterococcus faecalis strain V583 286 were obtained from the CGC. Two defined communities of worm gut commensals were used: 287 CeMBio, with twelve strains, represents the worm core gut microbiome 29 and SC20, a subset of 288 twenty strains of the previously described SC1 25 , with eight species out of the 20 of the 289 Enterobacteriaceae family (Table 1). In addition, a subset of of CeMBio strains was used in 290 experiments testing effects on susceptibility to Enterococcus faecalis infection, including only 291 strains that are sensitive to gentamycin, which is used in E. faecalis plates, to prevent enrichment 292 of gut commensals through the environment. Additional commensals, of the genus Pantoea, family 293 Erwiniaceae (a recent splinter off Enterobacteriaceae), included BIGB0393 (also in CeMBio) and 294 the recently characterized Pantoea cypripedii strains V8 and T16 23 . 295 Bacterial communities were prepared for experiments by growing individual strains in LB at 28 C 296 for two days, adjusting cultures to 1 OD, concentrating 10-fold and mixing equal volumes from 297 each culture. 100-200 L aliquots of the mix were plated on either minimal nematode growth 298 medium (NGM) or on peptone-free medium (PFM), which further limits bacterial growth 29 , as 299 described, and air-dried for 2 to 12 hours prior to the addition of worms. 300

Construction of fluorescently-tagged Enterobacter hormaechei CEent1-dsRed 301
E. hormaechei CEent1, previously misidentified as E. cloacae 22 , is a member in both the CeMBio 302 and SC20 communities. The construction of its dsRed-expressing derivative was achieved by 303 integrating the dsRed gene into the functionally neutral attTn7 site in the CEent1 genome using 304 . CC-BY 4.0 International license available under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprint this version posted June 14, 2023.

Microcosm experiments 328
Compost microcosms harboring diverse microbial communities were prepared from local soil 329 composted with produce for up to two weeks essentially as previously described 19,45 . Briefly, local 330 soils were supplemented with banana peels or chopped apple, composted soils were split into two 331 parts: one part (6 gr in a glass vial) autoclaved to eliminate native nematodes and the other (10 gr 332 soil) suspended in M9 buffer to obtain a microbial extract which was concentrated and added to 333 the autoclaved samples to reconstitute the original microbial community. 334 . CC-BY 4.0 International license available under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprint this version posted June 14, 2023. ; https://doi.org/10.1101/2023.06.13.544815 doi: bioRxiv preprint 20 C in separate vials containing the same compost and harvested at advancing ages up to day five 336 of adulthood (D5) (Fig. 1A). The final time point was determined by the need to distinguish 337 between the original cohort (post-gravid at D5) and progeny (mid-stage gravids), which could not 338 be achieved in subsequent time points. In experiments with fixed time colonization, worms were 339 raised on live E. coli until the L4 stage to ensure proper development, then transferred to 340 kanamycin-killed E. coli 46 from which worms were further transferred at advancing ages to 341 microcosm environments for two days before harvesting for analysis. For the earliest time point 342 (gravids, day zero of adulthood), worms were raised on live E. coli from L1 to the L4, then shifted 343 to dead E. coli for 4 hours to minimize carry over of live E. coli, before transferring to microcosms. 344 Worm harvesting was carried out using a Baermann funnel as described 45 . 345 Soil samples (1g) were taken from microcosms of the same compost batch used to grow worms 346 ("soil"), or from the same microcosm from which worms were harvested ("grazed soil"). 347

Experiments with defined bacterial communities 348
Aging experiments on bacterial communities/strains were carried out similarly to the description 349 for microcosm experiments, with bacteria seeded on NGM or PFM plates as described. In

Survival assays 415
For infection resistance experiments, synchronized worm populations were raised from L1 on PFM 416 plates with E. coli OP50, CEent1, or designated communities, and shifted, at L4, or at day 4 of 417 adulthood, to E. faecalis plates prepared with Brain Heart Infusion Agar containing 25 g/ L 418 gentamicin and seeded with bacteria a day before the transfer of worms. Assays were carried out 419 at 25 C and dead or live worms were counted every day 30 . For lifespan assays, worms were raised, 420 on designated strains or communities in PFM plates at 20 C and scored daily for survival 421 beginning at L4 (t0). 422  (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprint this version posted June 14, 2023. ; https://doi.org/10.1101/2023.06.13.544815 doi: bioRxiv preprint results and wrote the manuscript. 441

Ethics declarations 442
The authors declare that the research was conducted in the absence of any commercial or financial 443 relationships that could be construed as a potential conflict of interest. (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made   (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprint this version posted June 14, 2023. ; Figure 2: An Enterobacteriaceae bloom is a common denominator of aging worms raised in different microbial environments. A. Colonization of individual aging worms raised on CeMBio with CEent1dsRed, n=21-23/group; p < 0.01, pairwise t-tests; B. Bacterial load in aging worms raised on CeMBio, based on CFU counts of Enterobacteriaceae on VRBG plates and of total bacteria on LB plates. Shown are averages ± SDs for 3 plates per time point (n=4-12 worms/time point). C. CEent1 colonization in aging worms raised on SC20 with CEent1dsRed (n=9-18 worms/time point); p <0.001, pairwise t-tests. D. Fold change in bacterial load in worms aging on SC20, assessing bacterial load with qPCR using primers specific for Enterobacteriaceae 16S or Eubacterial 16s, normalized to worm DNA assessed by qPCR with primers specific for C. elegans actin (shapes represent replicate plates, each evaluated by qPCR in duplicate or triplicate).

A B
Shifted at L4 Shifted at D4 . CC-BY 4.0 International license available under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprint this version posted June 14, 2023. ; CEent1dsRed colonization in wildtype and eat-2 worms raised on the SC20 community (n = 10-36 worms/group/time point); inset demonstrates age-dependent declines in pumping rates; n = 4-10 worms/time point).

A B
Days post L4  CEent1dsRed colonization in worms of designated strains at designated days of adulthood. C. dbl-1 null mutants, and sma-4 gof mutants aging on CeMBio containing CEent1-dsRed (n = 39-40/group/time point); fold over wt median. Inset. Body length of designated strains (n=9-12, p < 0.0001) D. Survival of worms of designated strains raised on E. coli or CEent1 and shifted to E. faecalis at D4 of adulthood (n = 106-112/group); ***, p < 0.001, logrank test. wt . CC-BY 4.0 International license available under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprint this version posted June 14, 2023. ; https://doi.org/10.1101/2023.06.13.544815 doi: bioRxiv preprint Figure 6: Commensal communities mitigate age-dependent susceptibility to infection. A. Survival of worms raised on the designated strains/communities and shifted to E. faecalis at D4; Pan, a community of three Pantoea strains; **, p < 0.0001, log rank test (n = 82-90/group); averages ± SDs for three plate replicates. Inset. CEent1colonized dead worms, one day after shift to E. faecalis. B. BIGb393, a Pantoea strain in CeMBio.(CB) **, p < 0.0001, n = 95-105/group). Shown are results of one representative experiment out of two with similar results. C. Lifespan of wildtype worms raised on designated communities. Entero stands for Lelliotia Jub66 and Enterobacter CEent1. Averages ± SDs for three plate replicates (n = 67-79/group).   . CC-BY 4.0 International license available under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprint this version posted June 14, 2023. ; https://doi.org/10.1101/2023.06.13.544815 doi: bioRxiv preprint