The gut microbiome is vital to the health and development of an organism, specifically in determining the host response to a chemical (drug) administration. To understand this, we investigated the effects of six antibiotic (AB) treatments (Streptomycin sulfate, Roxithromycin, Sparfloxacin, Vancomycin, Clindamycin and Lincomycin hydrochloride) and diet restriction (–20%) on the gut microbiota in 28-day oral toxicity studies on Wistar rats. The fecal microbiota was determined using 16S rDNA marker gene sequencing. AB-class specific alterations were observed in the bacterial composition, whereas restriction in diet caused no observable difference. These changes associated well with the changes in the LC–MS/MS- and GC–MS-based metabolome profiles, particularly of feces and to a lesser extent of plasma. Particularly strong and AB-specific metabolic alterations were observed for bile acids in both plasma and feces matrices. Although AB-group-specific plasma metabolome changes were observed, weaker associations between fecal and plasma metabolome suggest a profound barrier between them. Numerous correlations between the bacterial families and the fecal metabolites were established, providing a holistic overview of the gut microbial functionality. Strong correlations were observed between microbiota and bile acids, lipids and fatty acids, amino acids and related metabolites. These microbiome–metabolome correlations promote understanding of the functionality of the microbiome for its host.

The complex interaction between a higher organism and its resident gut flora is a subject of immense interest in the field of symbiosis. Many insects harbor a complex community of microorganisms in their gut. Larvae of Spodoptera littoralis, a lepidopteran pest which is prevalent in tropical and subtropical regions of the world, have a tube-like gut structure containing a simple bacterial community. This community varies both spatially (along the length of the gut) and temporally (during the life cycle of the insect).To monitor the dynamics and rapid adaptation of microbes to the gut conditions, a GFP-tagged reporter E. mundtii was constructed. After feeding to early instar S. littoralis larvae, the taggedmicrobes recovered from the fore and hind guts by flow cytometry. The fluorescent reporter confirmed the persistence of E. mundtii in the gut. RNA-sequencing of the sorted bacteria highlighted various strategies that the symbiont employs to survive, including upregulated pathways for tolerating alkaline stress, forming biofilms and two-component signaling systems, resisting oxidative stress and quorum sensing. Although these symbionts depend on the host for amino acid and fatty acids, differential regulation among various metabolic pathways points to an enriched lysine synthesis pathway in the hindgut of the larvae.

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Bile acid homeostasis plays an important role in many biological activities through the bile–liver–gut axis. In this study, two in vitro models were applied to further elucidate the mode of action underlying reported in vivo bile acid changes induced by antibiotics (colistin sulfate, tobramycin, meropenem trihydrate, and doripenem hydrate). 16S rRNA analysis of rat fecal samples anaerobically incubated with these antibiotics showed that especially tobramycin induced changes in the gut microbiota. Furthermore, tobramycin was shown to inhibit the microbial deconjugation of taurocholic acid (TCA) and the transport of TCA over an in vitro Caco-2 cell layer used as a model to mimic intestinal bile acid reuptake. The effects induced by the antibiotics in the in vitro model systems provide novel and complementary insight explaining the effects of the antibiotics on microbiota and fecal bile acid levels upon 28-day in vivo treatment of rats. In particular, our results provide insight in the mode(s) of action underlying the increased levels of TCA in the feces upon tobramycin exposure. Altogether, the results of the present study provide a proof-of-principle on how in vitro models can be used to elucidate in vivo effects on bile acid homeostasis, and to obtain insight in the mode(s) of action underlying the effect of an antibiotic, in this case tobramycin, on bile acid homeostasis via effects on intestinal bile acid metabolism and reuptake.

21The complex interaction between a higher organism and its resident gut flora is a subject of 22 immense interest in the field of symbiosis. Many insects harbor a complex community of 23 microorganisms in their gut. Larvae of Spodoptera littoralis, a lepidopteran pest which is 24 prevalent in tropical and subtropical regions of the world, have a tube-like gut structure 25 containing a simple bacterial community. This community varies both spatially (along the 26 length of the gut) and temporally (during the life cycle of the insect). 27To monitor the dynamics and rapid adaptation of microbes to the gut conditions, a GFP-tagged 28 reporter E. mundtii was constructed. After feeding to early instar S. littoralis larvae, the tagged-29 microbes recovered from the fore and hind guts by flow cytometry. The fluorescent reporter 30 confirmed the persistence of E. mundtii in the gut. RNA-sequencing of the sorted bacteria 31 highlighted various strategies that the symbiont employs to survive, including upregulated 32 pathways for tolerating alkaline stress, forming biofilms and two-component signaling systems, 33 resisting oxidative stress and quorum sensing. Although these symbionts depend on the host for 34 amino acid and fatty acids, differential regulation among various metabolic pathways points to 35 an enriched lysine synthesis pathway in the hindgut of the larvae. 36 37 animals, pests or pollinators of food crops, and as cyclers of carbon and nitrogen during the 43 decomposition of plant biomass (1) 44 There are several factors that determine the gut bacterial composition in insects. The gut can be 45 compartmentalized, resulting in structures that vary according to the complexity of the microbial 46 communities. Insects with a straight, tube-like gut usually possess a less diverse microbial 47 population compared to species with invaginations and deep pouches (1). Other factors that 48 shape the gut population include the following: oxygen level, gut pH, the presence of digestive 49 enzymes, antimicrobial compounds, and insect diet (2, 3). Although most bacteria have an 50 affinity for neutral pH, several acidophiles and alkalophiles have adapted to extreme pH 51 conditions. 52 Gut microbes can be either vertically or horizontally transmitted. Vertical transmission allows 53 bacterial transfer (from the ovaries to the egg shells) to the next generation (4), whereas 54 horizontal transmission occurs over the course of the life cycle, through diet and social behavior. 55 Regardless of how bacteria are transmitted, microbial populations may be unstable during early 56 developmental stages (5, 6). For example, in holometabolous insects, a complete metamorphosis 57 of the gut occurs in the larva, through pupal and adult stages, resulting in microbial turnover and 58 variable microbial counts (5).59 Insects are helped by their bacterial and fungal symbionts with functions relating to the digestion 60 of complex plant carbohydrates and amino acids, the assimilation of vitamins and the 61 development of defensive str...

Quinolinic carboxylic acids are known for their metal ion chelating properties in insects, plants and bacteria. The larval stages of the lepidopteran pest, Spodoptera littoralis, produce 8-hydroxyquinoline-2-carboxylic acid (8-HQA) in high concentrations from tryptophan in the diet. At the same time, the larval midgut is known to harbor a bacterial population. The motivation behind the work was to investigate whether 8-HQA is controlling the bacterial community in the gut by regulating the concentration of metal ions. Knocking out the gene for kynurenine 3-monooxygenase (KMO) in the insect using CRISPR/Cas9 eliminated production of 8-HQA and significantly increased bacterial numbers and diversity in the larval midgut. Adding 8-HQA to the diet of knockout larvae caused a dose-dependent reduction of bacterial numbers with minimal effects on diversity. Enterococcus mundtii dominates the community in all treatments, probably due to its highly efficient iron uptake system and production of the colicin, mundticin. Thus host factors and bacterial properties interact to determine patterns of diversity and abundance in the insect midgut.

An understanding of the changes in gut microbiome composition and its associated metabolic functions is important to assess the potential implications thereof on host health. Thus, to elucidate the connection between the gut microbiome and the fecal and plasma metabolomes, two poorly bioavailable carbapenem antibiotics (doripenem and meropenem), were administered in a 28-day oral study to male and female Wistar rats. Additionally, the recovery of the gut microbiome and metabolomes in doripenem-exposed rats were studied one and two weeks after antibiotic treatment (i.e., doripenem-recovery groups). The 16S bacterial community analysis revealed an altered microbial population in all antibiotic treatments and a recovery of bacterial diversity in the doripenem-recovery groups. A similar pattern was observed in the fecal metabolomes of treated animals. In the recovery group, particularly after one week, an over-compensation was observed in fecal metabolites, as they were significantly changed in the opposite direction compared to previously changed metabolites upon 28 days of antibiotic exposure. Key plasma metabolites known to be diagnostic of antibiotic-induced microbial shifts, including indole derivatives, hippuric acid, and bile acids were also affected by the two carbapenems. Moreover, a unique increase in the levels of indole-3-acetic acid in plasma following meropenem treatment was observed. As was observed for the fecal metabolome, an overcompensation of plasma metabolites was observed in the recovery group. The data from this study provides insights into the connectivity of the microbiome and fecal and plasma metabolomes and demonstrates restoration post-antibiotic treatment not only for the microbiome but also for the metabolomes. The importance of overcompensation reactions for health needs further studies.

The diversity of microbial species in the gut has a strong influence on health and development of the host. Further, there are indications that the variation in expression of gut bacterial metabolic enzymes is less diverse than the taxonomic profile, underlying the importance of microbiome functionality, particularly from a toxicological perspective. To address these relationships, the gut bacterial composition of Wistar rats was altered by a 28 day oral treatment with the antibiotics tobramycin or colistin sulfate. On the basis of 16S marker gene sequencing data, tobramycin was found to cause a strong reduction in the diversity and relative abundance of the microbiome, whereas colistin sulfate had only a marginal impact. Associated plasma and fecal metabolomes were characterized by targeted mass spectrometry-based profiling. The fecal metabolome of tobramycin-treated animals had a high number of significant alterations in metabolite levels compared to controls, particularly in amino acids, lipids, bile acids (BAs), carbohydrates, and energy metabolites. The accumulation of primary BAs and significant reduction of secondary BAs in the feces indicated that the microbial alterations induced by tobramycin inhibit bacterial deconjugation reactions. The plasma metabolome showed less, but still many alterations in the same metabolite groups, including reductions in indole derivatives and hippuric acid, and furthermore, despite marginal effects of colistin sulfate treatment, there were nonetheless systemic alterations also in BAs. Aside from these treatment-based differences, we also uncovered interindividual differences particularly centering on the loss of Verrucomicrobiaceae in the microbiome, but with no apparent associated metabolite alterations. Finally, by comparing the data set from this study with metabolome alterations in the MetaMapTox database, key metabolite alterations were identified as plasma biomarkers indicative of altered gut microbiomes resulting from a wide activity spectrum of antibiotics.