After two weeks of SHIME operation, the microbiota’s composition and diversity were stabilized, and most predominant phyla in the gut microbiome were Bacteriodetes, Proteobacteria, Synergistetes, and Firmicutes, which were also previously demonstrated by Yu’s group [33].{Yu, 2016 #1;Li, 2015 #3} These predominant phyla had also been observed in the fecal samples of humans and animals [34, 35]. As mentioned above, a large body of data showed that stabilized microbiota might be disturbed by antibiotics exposure, resulting in chronic and relapsing infections [9-11]. The effects of AMX or POL on the human intestinal microbiota have been extensively studied [18-24]. However, the combination effect of antibiotics cocktail on human intestinal microbiota dysbiosis and related health risk is not fully understood. Hence, this study for the first time evaluated and compared the in vitro ability of AMX and POL (used separately or combined) on ARGs as well as human disease-related pathways in the simulated human gut. As the bacteria-killing mechanism for AMX is interfering synthesis of the bacterial cell wall peptidoglycan [15, 16], whereas POL is binding to the anionic lipopolysaccharide of the gram-negative bacterial cell membrane [17]. Therefore, it is supposed that the combination of AMX and POL can cause a synergistic effect on human intestinal microbiota.
Combination treatment reduced occurrence of drug resistance development
As mentioned earlier, exposure to antibiotics may promote the spread of ARB and ARGs in the human gut, which may limit the treatment efficiency and resulting in chronic and relapsing infections [9-11]. For instance, ARGs from such classes, including aminoglycosides, beta-lactams, and tetracycline have been confirmed to enrich in human gut microbiota following administration of AMX-clavulanic acid for one week [36], while patients who have taken AMX lower the relative abundance of ARGs from 0.81% to 0.14% [37]. The controversial conclusions in their results may attribute to the subjects, antibiotics administration and, the statistical approach of ARGs varied widely between studies. The minor effect of AMX on ARGs found in our study was not totally in agreement with the results. Barraud and associates discovered that AMX exposure to women during labor significantly selected bla (TEM)-positive Enterobacteria in their gut microbiota as well as their children’s, indicating ARB would cumulate across generation [38]. Similarly, POL was suggested to cause the selection of antimicrobial-resistant bacteria and the exchange of ARGs through horizontal gene transfer (HGT) between bacterial species [23]. Buelow and associates also confirmed that cefotaxime, POL, tobramycin, and amphotericin B combination treatment would select the antibiotic-resistant bacteria and aminoglycoside resistance genes such as aph(2”)-Ib and the aadE-like gene [39]. Furthermore, the increment of relative log abundance of ARGs such as aac6ib (aminoglycoside) caused by POL treatment that showed in our research was also consistent with the previous results.
The extensive use and misuse of antibiotics are known as contributors to the development of antibiotic resistance, which is a recent threat to global health. Antibiotics combination therapy is one of the essential ways that can prevent the development of antimicrobial resistance [40-42]. For instance, the addition of an aminoglycoside to a beta-lactam therapy regimen has been suggested to have a beneficial effect in delaying or preventing the development of antimicrobial resistance [43]. Combination therapy also suppresses or minimizes the degree of resistance of daptomycin-resistant viridans group streptococci compared with daptomycin monotherapy [44]. This study, for the first time, revealed that the combination of AMX and POL treatment reduced the occurrence of drug resistance development, which was consistent with the above findings. However, the combination therapy with beta-lactam ceftazidime and the fluoroquinolone ciprofloxacin selected for mutants that displayed broad-spectrum resistance, leading to decreased susceptibility to the combination of drugs applied as well as to unrelated antibiotic classes [45]. Therefore, the increased risk of selecting for broad-spectrum resistance antibiotic combinations should also be considered before being implemented in the clinical settings.
Combination treatment promote the increase of the human disease-related genes
Antibiotics have been widely used for the prevention and treatment of diseases to humans. For instance, AMX is effective in resistant pneumococcal and Helicobacter pylori eradication therapy [19, 46]. Oral POL contributes to intestinal eradication of multidrug-resistant Enterobacteriaceae, ESBL-producing Escherichia coli, and Klebsiella pneumoniae in immunocompromised patients [23, 24]. However, the facilitating clearance of targeted infections, antibiotic-induced disruption of the microbiota and immune homeostasis can lead to disease [47]. AMX had been found to promote the selection of human disease category genes, including drug resistance categories in intestinal microbiota, and the increased gene numbers of human disease-related functional pathways shown in our results were in agreement with this [48]. Some studies demonstrated that AMX increased the beta-lactam resistance and antibiotic-resistant bacteria in the gut [19, 49]. Other papers reported AMX would activate innate immunity and cause infectious diseases such as urinary tract infections, diarrhea, and liver abscess [50-53]. AMX was also suggested to affect insulin sensitivity and increase succinate, monosaccharide, and oligosaccharide levels in the fecal samples, and prenatal exposure was also associated with the increase of childhood overweight risk [54-56]. AMX was even confirmed to increase the risk of colon cancer [57]. All the above might be explained by our findings that the human disease-related pathways, including cancer, drug resistance, endocrine, and metabolic diseases, infectious diseases were more abundant in the AMX treatment group. Similarly, POL suggested the effect of immune responses and induce disease incidence, including insulin-dependent diabetes, diarrhea, bacterial infections, and translocation using animal models [58-62].
A combination of antibiotic therapy is frequently used to treat severe infections, which would not only prevent the development of antimicrobial resistance but also improve the treatment efficacy [40-42]. For instance, a study confirmed that combination of colistin-polymyxin B or tigecycline with a carbapenem therapy is superior to monotherapy for carbapenemase-producing Klebsiella pneumoniae [63]. Besides, the combination of AMX and POL therapy would effectively attenuate bacterial endotoxin- and Shiga exotoxin-mediated cytotoxicity and reduce mortality from peritonitis [25, 64]. However, excessive use of combinations should be avoided because it might be associated with increased risk for the toxicity and super-infections [41]. Combination therapy was also reported to cause a rise in infections triggered by multidrug-resistant gram-negative organisms [65]. Another paper revealed that a combination of an aminoglycoside with beta-lactam is associated with an increased risk for nephrotoxicity [66]. This study, for the first time, revealed that the combination of AMX and POL treatment promote the increase of the human disease-related genes such as colorectal cancer, viral myocarditis, hepatitis B, and toxoplasmosis. Therefore, these results might indicate that the side effects of combination therapy should be carefully considered.
Combination effects on the genetic level attribute to microbiota shift
The phenomena of microbiota shift shown in our research are identical to several in vivo studies on intestinal microbiota; however, Proteobacteria phylum such as Enterobacter and Bacteroides were most predominantly detected following AMX administration [52, 67, 68]. Furthermore, the percentage of Escherichia-Shigella was decreased after POL treatment found in this study was further supported by earlier reports that confirmed the eradication ability of POL on Escherichia and its cytotoxicity [23, 64]. Some studies have also discovered a trend toward increasing taxonomic diversity after AMX treatment and a significant effect on the beta diversity pursued by POL exposure [23, 69]. Additionally, this study, for the first time, revealed that POL treatment caused a bloom of Pseudomonas and Bifidobacterium, while combination treatment (AMX and POL) caused the enrichment of Enterobacter and Pseudomonas. Bifidobacterium intrinsically carries ARGs such as ermx and tetw, and Pseudomonas possesses mexf, aada6, and blap1b, which make them inherently resistant to MLSB, tetracycline, aminoglycosides, and beta-lactam antibiotics, and consequently pose a significant therapeutic challenge [70-73]. All of the above findings supported the positive correlations between the abundances of Bifidobacterium and Pseudomonas with ARGs in our study. Significant Bifidobacterium shift discovered in this study also explained POL exposure caused more severe drug resistance development than combination treatment. Although combination treatment reduced the occurrence of drug resistance development produced by single POL treatment. However, the bloomed multidrug-resistant Pseudomonas was still an immense threat to human health because of its high virulence factor reported in many previous research papers [74-76].
Besides, Enterobacter was confirmed to increase after both AMX administration and combine exposure of antibiotics, and the combination treatment caused its more substantial enrichment. Although these bacteria are considered commensal in the human gut, fecal carriage of this opportunistic pathogen confirmed to be a severe risk factor for the infection [77-79]. The significant increase in opportunistic pathogen associated with human disease-related pathways of human intestinal microbiota following combination of AMX and POL treatment exhibited in this study have not been reported in details yet. More importantly, this study displayed a positive correlation between the opportunistic pathogen Enterobacter and human disease-related pathways, which suggested the bloom of this pathogen caused by antibiotics treatment that might contribute to human diseases. For instance, Enterobacter was reported to be related to human infectious diseases such as colitis, neonatal sepsis, and bloodstream infections [80-82]. Furthermore, some studies have also indicated that Enterobacter was enriched in cancer patients and related to cardiovascular diseases, endocrine and metabolic diseases, and neurosurgery diseases [83-87]. All of the findings, as mentioned above, were linked with positive correlations between the abundance of Enterobacter and human disease-related pathways. And this advocated that the enhanced effect of combination treatment on the increase of the pathways might attribute to apparent Enterobacter enrichment in our study. Therefore, the results of our research demonstrated a severe impact and a negative side-effect of combination antibiotics treatment that enriched pathogens related to health problems, which should be considered as a fundamental aspect of the cost-benefit equation for the antibiotics combination prescription.
Perspectives
The findings in this study suggested several numbers of opportunities for additional study. First, expansion of the analysis to incorporate multiple–omics approaches of the metagenome, metatranscriptome, and metabolome were not taken in this study, which attributed to the costs associated with sequencing and analysis. With multiple-omics data, the confirmed genome composition and expression of the gut microbiota would help us understand how antibiotics induced human intestinal microbiota dysbiosis and related disease. It is also of interest to inoculate the microbiota mixture from several patients to discover the different effects of antibiotics between healthy and sick individuals. However, it is difficult to obtain such samples, and the SHIME operation cost is very high. Further in vivo studies aimed to verify whether the findings in this in vitro study reflect the reality, which will provide new insights to measure how antibiotic affects the gut microbiota and the associated disease. Moreover, the combination antibiotics’ exposure was confirmed to cause the synergistic effect on increasing the human disease-related genes, and their prescription should be considered as an essential aspect of the risk assessment.