Previous studies have shown that antibiotic treatment contributes to gut microbiota dysbiosis7,8,9. It has been reported that gut microbiota dysbiosis can be associated with many diseases, including colorectal cancer, obesity, and infection4,5,6. In this study, C57BL/6 mice were systematically administered ceftriaxone sodium with concomitant different treatment strategies for 7 days. Fecal bacterial diversity significantly decreased in the ceftriaxone treatment group compared to the control group. Although FMT is a promising therapy for treating recurrent C. difficile infection and antibiotic-induced dysbiosis16,17,28, FMT treatment in our study only showed a non-significant increase in diversity. The Ceftri FF AC group exhibited higher Chao1 and PD whole tree diversities compared to the Ceftri AC group. These results indicate a synergistic effect between FF and AC on the balance of microbial diversity in the mouse intestine during ceftriaxone administration.
Previous studies showed the impact of FMT after the withdrawal of antibiotics18,19. In contrast, this study demonstrated the effect of different treatments during the ceftriaxone administration, resembling real clinical settings where antibiotics cannot be discontinued at will. Theoretically, ceftriaxone administration may induce the production of AmpC beta-lactamase in Enterobacter29, promote horizontal transfer of AMR genes30, and favorably select bacteria with intrinsic resistance to ceftriaxone, such as Pseudomonas aeruginosa, Bacteroides, Lactobacillus, and Enterococcus31. Chakraborty et al. reported that short-term systemic ceftriaxone treatment induced expansion of the Enterococcus and Lactobacillus genera in the mouse intestine24. Consistently, our results showed that, on day 7, the Enterococcus and Lactobacillus genera increased in the gut of ceftriaxone-treated mice. However, Bacteroides and Enterobacter were comparable between groups in this study, while P. aeruginosa—not a normal flora for mice—could not be observed in any groups.
Previous studies have suggested that activated charcoal (AC) can adsorb antibiotics in the gut during treatment, potentially reducing the colonization and proliferation of resistant bacteria. A mouse model treated with cefotaxime and DAV131 (a charcoal-based adsorbent) for 3 days prior to infection with the Klebsiella pneumoniae strain (PUG-2) showed decreased colonization of K. pneumoniae in the fecal samples32. However, in this study, the Ceftri AC group showed a significantly higher abundance of Enterococcus genus compared to the Ceftri group. Although there is no definitive explanation, this may be due to desorption (the release of drugs, e.g., aspirin, from AC after adsorption33) of ceftriaxone from AC. Without AC, the ceftriaxone level in the gut would quickly rise due to biliary excretion and decline rapidly through normal defecation. However, with AC, the rapid increase in ceftriaxone levels in the gut might be blunted by AC adsorption, and the decline may be delayed due to desorption of ceftriaxone from AC and constipation caused by AC. In general, prolonged and low-level antibiotic exposure can promote overgrowth of drug-resistant bacteria more effectively than short, high-level exposure. We observed that mice in the CeftriAC group defecated more slowly than those in other groups, and our records indicated that collecting feces from this group was troublesome due to the relatively small amount produced. Unfortunately, we did not quantitatively record these data, so we cannot strongly confirm our claim. Unlike CeftriAC group, we found that the CeftriFFAC group did not exhibit a higher abundance of Enterococcus and Lactobacillus genera compared to the Ceftri group. FF may counteract the constipation caused by activated charcoal, as we did not encounter any issues with fecal collection in the CeftriFFAC group.
The balance between the residual fecal ceftriaxone and the dosage of AC might play an important role. An excess amount of AC should work as an adsorbent in the presence of a low amount of residual ceftriaxone. On the other hand, an excess amount of ceftriaxone might quickly saturate the adsorption ability of a low amount of AC, and the AC might then act as a reservoir instead. The outcomes could be better if the dosage of AC had been increased or used at the end of antibiotic treatment to adsorb a limited amount of residual antibiotic.
Previous studies have shown that FMT can restore the mucosal barrier functions in mice with ceftriaxone-induced dysbiosis18. However, the CeftriFMT and CeftriFMTAC groups in this study had a higher abundance of enterococci compared to the Ceftri group, although this increase was not statistically significant. FMT likely transferred a low number of enterococci already present in the fecal suspension, which were then selected and expanded by ceftriaxone in the gut. In contrast, FF did not contain any bacteria and could not introduce additional enterococci into the recipients. Ott et al. demonstrated that sterile fecal filtrate transfer restored normal stool habits and eliminated symptoms of C. difficile infection20.
The Muribaculaceae family is the most abundant in the gastrointestinal tract of mice34. Our results showed that the Muribaculaceae family significantly decreased in the Ceftri group compared to the control group. Some mice in the CeftriFFAC group retained comparable abundance to that of the control group. Wang et al. demonstrated that polyphenol rosemary acid treatment increased the abundance of Muribaculaceae and restored mucous secretion in mice with colitis35.
Li et al. reported that short-term oral ceftriaxone administration resulted in distorted tissue architecture, vascular congestion, and inflammatory cell infiltration18. Another study found histological lesions in the intestine following long-term oral ceftriaxone administration36. Unlike oral administration, our model with intraperitoneal administration showed that ceftriaxone-treated mice did not exhibit loss of mucosal architecture, crypt abscesses, or goblet cell depletion, except for a limited amount of inflammatory cell infiltration. Our results are consistent with previous reports indicating that short-term systemic ceftriaxone treatment does not affect the intestinal pathology of mice24. We suggest that the routes and doses of antibiotic administration may contribute to different pathologies in the colon.
Though oral antibiotics may better show the benefits of our treatments, they are impractical in clinical settings. AC can reduce the oral bioavailability of antibiotics, thus contraindicated for the oral route of antibiotics. The complexity of the FMT process and its potential risks (e.g., aspiration, infections from hidden or unknown pathogens) can limit its usage to carefully selected cases, typically inpatients who have prolonged intravenous broad-spectrum antibiotics and suffer recurrent severe infections caused by multidrug-resistant bacteria. On the other hand, relatively mild outpatient cases who can have oral antibiotics may not be worth receiving FMT due to its risks and hassle.
There are some limitations to our study. Only relative abundance was analyzed in this study. While absolute abundance can reflect the growth and decline of taxa directly, the increase or decrease of the relative abundance of any particular taxon may also be due to the decrease or increase of other taxa, respectively. Overall absolute abundance analysis requires bacterial cell count either by culture (colony-forming units) or by flow cytometry37. Multiplying each taxon relative abundance by the overall absolute abundace will give specific absolute abundance of that particular taxon. However, the culture method is limited to only culturable bacteria. For flow cytometry, host cell debris and small food particles may jeopardize the results of bacterial cell count. Real-time PCR or digital droplet PCR, utilizing a primer/probe set specific to a taxon of interest, may be more reliable for quantification of specific taxon absolute abundance.
We could not keep the same total volume of oral gavage for all groups. The CeftriFMTAC and CeftriFFAC groups received twice the volume compared to the other groups. Mixing FMT and AC into a single volume of 200 µl was too thick for oral gavage. Rather than preparing a 400 µl mix of FMT and AC, we decided to feed them sequentially, 200 µl each. Oral gavage with AC was performed first to adsorb intestinal ceftriaxone, keeping the residual antibiotic away from the FMT in the later gavage. However, the difference in volume fed could alter the chance of complications (e.g., aspiration, gut injuries) and the anatomical distribution of FMT components in the recipient’s gut38. Oral gavage with FMT to a mouse with a stomach full of AC suspension from the first gavage might also result in a different distribution of FMT components compared to a mouse with an empty stomach. These could become a confounder, affecting the observed characteristics of gut microbiota after the treatments.
In terms of microbiota diversity and compositions, the pretreatment heterogeneity across all seven groups was quite remarkable (Figs. 1B and 2A, and 3A). Two weeks of acclimation were probably not enough, because the Control group at day 7 was still quite different from the Control group at day 0 (Fig. 1C), meaning that the microbiota was still not stable at the time of rerandomization (day 0). Since we had many groups (7 in total), the rerandomization might not guarantee the balance of the rapidly changed and different microbiota in each mouse, resulting in baseline differences, which could then obscure the treatment effects.
Our experiments presented only a few phenotypic results, and we did not conduct any mechanistic studies. We have not yet explored the interaction mechanisms between the host gut microbiota and activated charcoal during ceftriaxone treatment in combination with fecal filtrate transplantation. This will be the focus of our future research.
