Gut Microbiota Disturbance During Antibiotic Therapy
Gut Microbiota Disturbance During Antibiotic Therapy
The effects of ABs and the mechanisms underlying the connection between AB treatment and microbial gut metabolism require clarification, which can only be achieved through an integrated approach that goes well beyond the 16S DNA analysis that has been the cornerstone of previous research in this area. Thus, the aims of this study were as follows: to provide a proof of concept for an integrated workflow to assess the nature of such changes in the intestines of patients undergoing AB treatment at the structural and functional levels; and to evaluate whether there is a relationship between these types of changes. For instance, changes in the total composition at a given time point may also be associated with changes in the AB-resistant bacteria, although not necessarily at the same moment or during the same interval. Two major active factors that modulate changes in the microbiota should be considered. First, it is important to understand how AB treatment determines the emergence of bacterial species that are resistant to β-lactams (such as ampicillin and cephalosporins) and how the microbiota recovers once ABs have been removed. Second, the speed at which such changes are observed according to the different levels under consideration should be carefully evaluated.
In our study, we demonstrated that the greatest change in the active microbial fraction occurred later (day 14) than that in the total microbial fraction, which reached a minimum biodiversity and richness on the 11th day of AB treatment (Figure 1). Further, oscillatory population dynamics were observed (at both the DNA and RNA levels). An early reduction in Gram-negative bacteria at day 6 and an overall collapse in diversity was followed by possible colonisation of the upper gut by naturally resistant Bacteroidetes by day 11, a consequent increase in the colonisation of the lower gut with dominance at 11–14 days and eventual re-growth of the Gram-positive bacteria at day 14. Various studies based on 16S rDNA analysis revealed important variability in the recovery of the baseline bacterial composition after AB therapy depending on the individual and the AB used (type and dose). The large fluctuations in the relative abundances of the various bacterial taxa for the total and active microbiota throughout the follow-up study were most likely associated with an additive effect of ampicillin/sulbactam and the first-generation cephalosporin cefazolin as well as the widespread development of β-lactamases (for details, see the Discussion in the online supplement).
The apparent oscillations in the population dynamics were shown to further influence the biodiversity and richness of metabolites and active proteins; these changes, some of which may play essential roles in protection against ABs (for details, see the Discussion in the online supplement), may also have important ecological implications. In our study, we observed a drastic shift 6 days after the onset of AB treatment, at which time the predominantly active taxa were mainly members of the Streptococcaceae, Clostridiaceae and Bacteroidaceae, and at 40 days after the end of AB treatment, when the most abundant active bacteria were members of the Burkholderiaceae (Proteobacteria phylum). Thus, these bacteria may have contributed to the distinct functional profiles and metabolic statuses of colonic bacteria during the follow-up therapy.
A notable finding in this study was that protein expression appeared to decrease as a consequence of AB treatment; furthermore, the production of proteins needed for glycolysis, pyruvate decarboxylation, the tricarboxylic acid cycle, glutamate metabolism, iron uptake, GTP hydrolysis and translation termination were enhanced at the initial stages of AB treatment (day 6), most likely to cope with an intermittent nutrient supply and the stress caused by the ABs, but decreased at later stages and after treatment cessation. Together, these results suggest for first time that AB treatment may 'presumptively' negatively affect the overall metabolic status of the colonic space (for an example, see the Discussion in the online supplement), although further studies may be required to further confirm this hypothesis. Additionally, the expression levels of all the genes belonging to the 'mobile and extrachromosomal element functions' category were decreased during treatment, and all of them were associated with clustered regulatory interspaced short palindromic repeats (CRISPRs). These genes encode a system that functions as a type of bacterial adaptive 'immune' response. Specifically, the genes that belong to the CRISPR/Cas system are involved in protecting cells from invasion by foreign DNA (viruses and plasmids) through an RNA-interference-like process. Thus, the decreases in the expression of these genes may render the bacteria more susceptible to the acquisition of foreign DNA. This could provide an advantage in an AB-containing environment because it increases the likelihood of obtaining resistance genes by horizontal gene transfer.
Of major metabolic significance was the observation that the production of metabolites that are known to be produced by the host and further metabolised by colonic bacteria, such as derivatives of bile acids, cholesterol and hormones, was altered during the AB treatment and was significantly improved after treatment cessation. In essence, this finding suggests that AB treatment altered the continual interplay between the liver/pancreas and bacterial enzymes operating in the colonic space and that AB therapy may have a positive long-term effect in human biology. This result is consistent with the finding that the biological production of host-beneficial molecules such as vitamin B12 and the uptake of key metals such as Co by colonic bacteria were affected by AB therapy because the expression of genes and proteins associated with those functions was restored after treatment cessation. This is particularly important because it has been demonstrated that the microbiota from the distal guts of different individuals exhibit partial functional redundancy in addition to clear differences in community structure in the absence of ABs. By contrast, although further experimental evidence is required, our results suggest that the presence of ABs per se may have additional 'presumptive' collapse effects in key metabolic pathways independent of the community structure and that functional replacement events may be affected under AB stress.
Although, the investigation reported here was for a single patient and should generally be considered qualitative, it constitutes a proof of concept for an integrated, multi-omics approach towards unravelling the dynamics and mechanisms underlying the response of intestinal microbiota to AB treatment. A 'presumptive' model related to the follow-up effects of ABs on bacterial and metabolic composition is summarised in Figure 8. These data may help to identify specific strains of gut microbiota with potential benefits in human health or to design specific therapies to decrease intestinal inflammation or normalise dysfunctions of the gut mucosa; for example, minor bacterial taxa such as Proteobacteria have been shown to play a significant, active role in overall gut metabolism and host interaction despite their low number. Further studies investigating different ABs and (un)related individuals are required to better ascertain the link between bacterial producers and the presence of particular proteins and molecules, and the metabolic consequences of AB treatment; this may serve as a promising focus for therapeutic interventions or the treatment of pathogenic infections and diseases.
(Enlarge Image)
Figure 8.
The 'presumptive' model related to the follow-up effect of antibiotics (ABs) on the microbial and metabolic composition of the human gut. The model is based on the combination of experimental multi-omics data. The biliary excretion of ABs triggers a cascade of metabolic events. At the earlier stages of AB therapy, the bacteria respond by promoting systems to avoid the antimicrobial effects of the drugs (expressing beta-lactamases, antimicrobial peptide transporters and multidrug efflux pumps and producing glycero(lyso)phospholipids—G(L)PL) and to cope with an intermittent nutrient supply while decreasing polysaccharides and lipopolysaccharide (LPS) production. Genes involved in cell envelope biosynthesis and the degradation of peptidoglycan-like components are increasingly expressed until the end of AB treatment but with a time delay compared with other drug-detoxifying mechanisms. Finally, the bacterial metabolism of the bile acid, hormones and cholesterol synthesised in the liver and pancreas is attenuated by AB therapy, thus possibly affecting entero-hepatic recirculation and systemic lipid metabolism, that is, the emulsification, absorption and transport of dietary fats; however, after treatment cessation, the metabolism of these factors improved significantly. Similarly, the pool of vitamins that are directly synthesised by gut bacteria was significantly improved after treatment cessation. The nutrient supply mechanisms, such as glycolysis, pyruvate decarboxylation, tricarboxylic acid (TCA) cycle, glutamate metabolism, and iron uptake, that are induced at earlier stages (day 6) become attenuated during the late stages of the therapy and become significantly attenuated after treatment cessation, suggesting that the entero-hepatic recirculation system may contain a lower amount of iron, sugars, branched amino acids, short organic acids and pyruvate produced or transported by colonic bacteria. At the active bacterial structure level, an apparently oscillatory population dynamic was further observed, with the initially predominant active Bacteroidaceae becoming replaced by Burkholderiaceae after treatment cessation. The broken line indicates the overall trend in each of the gut bacteria components during the follow-up treatment.
Discussion
The effects of ABs and the mechanisms underlying the connection between AB treatment and microbial gut metabolism require clarification, which can only be achieved through an integrated approach that goes well beyond the 16S DNA analysis that has been the cornerstone of previous research in this area. Thus, the aims of this study were as follows: to provide a proof of concept for an integrated workflow to assess the nature of such changes in the intestines of patients undergoing AB treatment at the structural and functional levels; and to evaluate whether there is a relationship between these types of changes. For instance, changes in the total composition at a given time point may also be associated with changes in the AB-resistant bacteria, although not necessarily at the same moment or during the same interval. Two major active factors that modulate changes in the microbiota should be considered. First, it is important to understand how AB treatment determines the emergence of bacterial species that are resistant to β-lactams (such as ampicillin and cephalosporins) and how the microbiota recovers once ABs have been removed. Second, the speed at which such changes are observed according to the different levels under consideration should be carefully evaluated.
In our study, we demonstrated that the greatest change in the active microbial fraction occurred later (day 14) than that in the total microbial fraction, which reached a minimum biodiversity and richness on the 11th day of AB treatment (Figure 1). Further, oscillatory population dynamics were observed (at both the DNA and RNA levels). An early reduction in Gram-negative bacteria at day 6 and an overall collapse in diversity was followed by possible colonisation of the upper gut by naturally resistant Bacteroidetes by day 11, a consequent increase in the colonisation of the lower gut with dominance at 11–14 days and eventual re-growth of the Gram-positive bacteria at day 14. Various studies based on 16S rDNA analysis revealed important variability in the recovery of the baseline bacterial composition after AB therapy depending on the individual and the AB used (type and dose). The large fluctuations in the relative abundances of the various bacterial taxa for the total and active microbiota throughout the follow-up study were most likely associated with an additive effect of ampicillin/sulbactam and the first-generation cephalosporin cefazolin as well as the widespread development of β-lactamases (for details, see the Discussion in the online supplement).
The apparent oscillations in the population dynamics were shown to further influence the biodiversity and richness of metabolites and active proteins; these changes, some of which may play essential roles in protection against ABs (for details, see the Discussion in the online supplement), may also have important ecological implications. In our study, we observed a drastic shift 6 days after the onset of AB treatment, at which time the predominantly active taxa were mainly members of the Streptococcaceae, Clostridiaceae and Bacteroidaceae, and at 40 days after the end of AB treatment, when the most abundant active bacteria were members of the Burkholderiaceae (Proteobacteria phylum). Thus, these bacteria may have contributed to the distinct functional profiles and metabolic statuses of colonic bacteria during the follow-up therapy.
A notable finding in this study was that protein expression appeared to decrease as a consequence of AB treatment; furthermore, the production of proteins needed for glycolysis, pyruvate decarboxylation, the tricarboxylic acid cycle, glutamate metabolism, iron uptake, GTP hydrolysis and translation termination were enhanced at the initial stages of AB treatment (day 6), most likely to cope with an intermittent nutrient supply and the stress caused by the ABs, but decreased at later stages and after treatment cessation. Together, these results suggest for first time that AB treatment may 'presumptively' negatively affect the overall metabolic status of the colonic space (for an example, see the Discussion in the online supplement), although further studies may be required to further confirm this hypothesis. Additionally, the expression levels of all the genes belonging to the 'mobile and extrachromosomal element functions' category were decreased during treatment, and all of them were associated with clustered regulatory interspaced short palindromic repeats (CRISPRs). These genes encode a system that functions as a type of bacterial adaptive 'immune' response. Specifically, the genes that belong to the CRISPR/Cas system are involved in protecting cells from invasion by foreign DNA (viruses and plasmids) through an RNA-interference-like process. Thus, the decreases in the expression of these genes may render the bacteria more susceptible to the acquisition of foreign DNA. This could provide an advantage in an AB-containing environment because it increases the likelihood of obtaining resistance genes by horizontal gene transfer.
Of major metabolic significance was the observation that the production of metabolites that are known to be produced by the host and further metabolised by colonic bacteria, such as derivatives of bile acids, cholesterol and hormones, was altered during the AB treatment and was significantly improved after treatment cessation. In essence, this finding suggests that AB treatment altered the continual interplay between the liver/pancreas and bacterial enzymes operating in the colonic space and that AB therapy may have a positive long-term effect in human biology. This result is consistent with the finding that the biological production of host-beneficial molecules such as vitamin B12 and the uptake of key metals such as Co by colonic bacteria were affected by AB therapy because the expression of genes and proteins associated with those functions was restored after treatment cessation. This is particularly important because it has been demonstrated that the microbiota from the distal guts of different individuals exhibit partial functional redundancy in addition to clear differences in community structure in the absence of ABs. By contrast, although further experimental evidence is required, our results suggest that the presence of ABs per se may have additional 'presumptive' collapse effects in key metabolic pathways independent of the community structure and that functional replacement events may be affected under AB stress.
Although, the investigation reported here was for a single patient and should generally be considered qualitative, it constitutes a proof of concept for an integrated, multi-omics approach towards unravelling the dynamics and mechanisms underlying the response of intestinal microbiota to AB treatment. A 'presumptive' model related to the follow-up effects of ABs on bacterial and metabolic composition is summarised in Figure 8. These data may help to identify specific strains of gut microbiota with potential benefits in human health or to design specific therapies to decrease intestinal inflammation or normalise dysfunctions of the gut mucosa; for example, minor bacterial taxa such as Proteobacteria have been shown to play a significant, active role in overall gut metabolism and host interaction despite their low number. Further studies investigating different ABs and (un)related individuals are required to better ascertain the link between bacterial producers and the presence of particular proteins and molecules, and the metabolic consequences of AB treatment; this may serve as a promising focus for therapeutic interventions or the treatment of pathogenic infections and diseases.
(Enlarge Image)
Figure 8.
The 'presumptive' model related to the follow-up effect of antibiotics (ABs) on the microbial and metabolic composition of the human gut. The model is based on the combination of experimental multi-omics data. The biliary excretion of ABs triggers a cascade of metabolic events. At the earlier stages of AB therapy, the bacteria respond by promoting systems to avoid the antimicrobial effects of the drugs (expressing beta-lactamases, antimicrobial peptide transporters and multidrug efflux pumps and producing glycero(lyso)phospholipids—G(L)PL) and to cope with an intermittent nutrient supply while decreasing polysaccharides and lipopolysaccharide (LPS) production. Genes involved in cell envelope biosynthesis and the degradation of peptidoglycan-like components are increasingly expressed until the end of AB treatment but with a time delay compared with other drug-detoxifying mechanisms. Finally, the bacterial metabolism of the bile acid, hormones and cholesterol synthesised in the liver and pancreas is attenuated by AB therapy, thus possibly affecting entero-hepatic recirculation and systemic lipid metabolism, that is, the emulsification, absorption and transport of dietary fats; however, after treatment cessation, the metabolism of these factors improved significantly. Similarly, the pool of vitamins that are directly synthesised by gut bacteria was significantly improved after treatment cessation. The nutrient supply mechanisms, such as glycolysis, pyruvate decarboxylation, tricarboxylic acid (TCA) cycle, glutamate metabolism, and iron uptake, that are induced at earlier stages (day 6) become attenuated during the late stages of the therapy and become significantly attenuated after treatment cessation, suggesting that the entero-hepatic recirculation system may contain a lower amount of iron, sugars, branched amino acids, short organic acids and pyruvate produced or transported by colonic bacteria. At the active bacterial structure level, an apparently oscillatory population dynamic was further observed, with the initially predominant active Bacteroidaceae becoming replaced by Burkholderiaceae after treatment cessation. The broken line indicates the overall trend in each of the gut bacteria components during the follow-up treatment.