Abstract
Introduction
Antibiotics have been of paramount importance in the battle against infectious disease and have been used on an enormous scale.1 Concerns about the use of antibiotics have traditionally focused on bacterial resistance, but recent studies have found that antibiotic use is associated with permanent modification of the microorganisms that live in and on humans: the microbiota.2 An increasing number of studies have revealed that disturbance of the intestinal microbiota is linked to an increased prevalence of obesity, asthma and, paradoxically, susceptibility to infectious diseases.2-6 A number of studies have addressed the effect of a single antibiotic on the intestinal microbiota using sequencing technologies. A 5-10 day course of ciprofloxacin or clindamycin resulted in a drop in microbial diversity and shifts in community composition.7,8 The use of metronidazole and clarithromycin for Helicobacter pylori eradication had similar effects.9 However, although many hospitalized patients initially receive a combination of classes of antimicrobial agents, directed at both Gram-positive and Gram-negative bacteria, including anaerobes, limited studies have been published on the long-term consequences of combination antibiotic therapy on the microbiota. Here we report the effects of broad-spectrum antibiotic modulation of the intestinal microbiota of healthy human subjects for a period of up to 31 months.
Patients and methods
Study design
Healthy, non-smoking, Caucasian male subjects (aged 18-25 years) were recruited by advertising. Screening consisted of a questionnaire, physical examination and routine blood and urine investigation. Subjects had not been exposed to intravenous or oral antibiotics during the previous year. This study was part of the prospective MISSION-2 project (ClinicalTrials.gov identifier NCT02127749), in which the effect of microbiota modulation on systemic innate immune responses following LPS challenge was investigated (see Supplementary Methods, available as Supplementary data at JAC Online).10
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Ethics
Ethics approval was received from the Medical Ethics Committee of the Academic Medical Centre, The Netherlands. All subjects gave written informed consent and research was conducted in accordance with the Declaration of Helsinki. Ten healthy subjects received oral broad-spectrum antibiotics (ciprofloxacin 500 mg q12h, vancomycin 500 mg q8h and metronidazole 500 mg q8h) for 7 days. Subjects were asked to collect faecal samples before antibiotic treatment (day 0), 36 h after the 7 day course of antibiotics (day 9), and again 6 weeks (day 49) and 8-31 months after the end of antibiotic treatment [long term (LT), median time to collection: 12 months, IQR 10-30]. Three additional healthy subjects served as controls and did not receive any antibiotics. Faecal samples were collected in plastic containers, stored at −20°C at home and were transported to the study centre for storage at −80°C within 24 h.
Microbiota analysis and butyrate-producing bacteria selection
Bacterial 16S rRNA microbiome sequencing targeting the V1-V2 region was performed on the collected faecal samples (see Supplementary Methods). To investigate the effect of combination antibiotic therapy on alpha diversity and community richness, inverse Simpson’s (IS) and observed taxa (OT) indices were calculated for all samples. In addition, beta diversity of the microbiota communities at baseline and after antibiotics were portrayed by principal coordinate analysis (PCoA) of weighted and unweighted UniFrac distances. We also measured the abundance of bacterial taxa that are known to be drivers of butyrate production, in accordance with a study that analysed butyrate-producing pathways from 15 publicly available data sets (see Table S1).11
Results
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Baseline characteristics of study participants are provided in Table S2. Mean age was 21.5 years (SD 1.9) and mean BMI was 22.2 (SD 2.2). All subjects completed the study. No episodes of Clostridioides (Clostridium) difficile infection were recorded, nor any other disorders that are associated with dysbiosis.
Effects on microbiota community richness, alpha and beta diversity
We observed a drop in alpha diversity and richness at day 9, when compared with baseline (median IS: 40.9 at day 0 versus 5.3 at day 9, Wilcoxon P = 0.001; median OT: 163 at day 0 versus 85 at day 9, Wilcoxon P = 0.048), Figure 1(a and b). Community richness was restored at day 49 and at LT, but alpha diversity was incompletely recovered and remained different from baseline at day 49 (median IS index: 40.9 versus 18.3, Wilcoxon P = 0.011). Microbiota diversity was restored in samples collected 8-31 months following antibiotic administration. In addition, beta diversity of the microbiota communities at baseline and after antibiotics was affected by broad-spectrum antibiotics (Figure 1c and d) as samples that were collected at day 9 differed from baseline in both weighted and unweighted UniFrac analyses (PERMANOVA, P < 0.001 and P < 0.001, Table S3). Gut microbial composition returned to baseline at day 49 and remained comparable up to 31 months later in the weighted UniFrac analyses. However, differences compared with baseline were sustained on the unweighted UniFrac at day 49, and even 8-31 months after antibiotic exposure (PERMANOVA, P = 0.003, and P = 0.016, Table S3). The microbiota compositions of subjects that had not undergone antibiotic treatment remained stable across all timepoints and were comparable with baseline and LT microbiota compositions of the volunteers that received antibiotics (Figure 1). Finally, no differences were observed in either the weighted or unweighted UniFrac at baseline (day 9, PERMANOVA, P = 0.356 and P = 0.376, respectively) and at LT (PERMANOVA, P = 0.138 and P = 0.563, respectively) when comparing subjects that received LPS with those who did not receive LPS (Table S3).
Effects on microbiota community composition
As shown in both alpha- and beta-diversity metrics, the effect of broad-spectrum antibiotics on gut microbial communities was profound and rapid, with a loss of diversity and a shift in community composition at day 9. On a phylum level, a significant decrease in Bacteroidetes and an increase in Firmicutes was seen (day 0 versus day 9; median Firmicutes abundance 80% versus 98%), which returned to baseline at day 49 and at LT. (Figure 2a) On a genus level, antibiotic-treated subjects were shown to be significantly overgrown by Streptococcus and Lactobacillus genera at day 9, with a decrease in obligately anaerobic taxa, such as Bacteroides, Subdoligranulum and Faecalibacterium (Figure 2b and Figure S1A). Day 49 showed a partial reversal to baseline, with decreased abundance of Streptococcus and Lactobacillus genera and a restoration of the anaerobic communities (Figure S1B). LT samples were comparable with samples collected before antibiotic administration. However, taxa from the Lachnospiraceae family and other butyrate-producing taxa were present in LT samples at the expense of taxa of the closely related Ruminococcaceae family11 (Figure S1C). Finally, administration of antibiotics had a profound impact on butyrate-producing genera, with a strong drop from 27% to 0.3% median relative abundance (Wilcoxon P < 0.001) and a rapid recolonization to baseline levels at 49 days that was sustained in LT samples (Figure S2).
Discussion
In this study, we aimed to describe the effect of combined treatment with vancomycin, ciprofloxacin and metronidazole on the intestinal microbiota of healthy adults, for a period of up to 31 months. We observed profound changes shortly after the course of antibiotics, with a drop in microbial diversity, overgrowth of the genera Streptococcus and Lactobacillus, and an early loss of anaerobic bacterial taxa with important roles in short-chain fatty acid metabolism.11 Six weeks after antibiotic treatment, the overall composition of the microbiota showed an incomplete return to baseline levels. Microbiota composition subsequently normalized after 8-31 months, but was often altered from its initial state. The observation that only the unweighted UniFrac analysis was able to detect group separation at 8-31 months suggests that it is the presence or absence of particular bacteria that leads to the group distinctions. Specifically, it indicates that the taxa that drive the separation at 8-31 months are potentially of low abundance, as the weighted UniFrac (which considers not only the presence or absence of specific bacteria, but also the relative abundance) was unable to identify any differences. To the best of our knowledge, this is the first study that addresses the short- and long-term impact of broad-spectrum antibiotic combination therapy on the microbiota. These findings are in agreement with previous publications that reported profound short-term changes in microbiota composition shortly after antibiotic administration, with an altered and individualized long-term recovery to baseline values.8,9,12,13
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This study has a number of limitations. First, these results were obtained in healthy male adults and cannot be directly extrapolated to hospitalized patients. Secondly, seven subjects underwent an infusion with LPS, which induced a transient state of endotoxaemia. However, this intervention appears not to have impacted long-term microbiota recovery. Third, this study has not investigated changes in function of the microbiota. This could be of importance as it is known that despite alterations in gut communities, the genetic potential and function of these communities can remain the same.14 This is likely to be the case in our findings that Ruminococcaceae taxa were replaced by taxa of the Lachnospiraceae family, as these families have similar metabolic potential. Lastly, we did not address viruses and fungi in the present study.
Conclusions
The intestinal microbiota shows remarkable resilience upon broad-spectrum antibiotic modification, but changes can still be found months or even years after antibiotic perturbation. Therefore, further research is needed to provide insight into the extent of changes in the intestinal microbiota, as well as the clinical impact these changes cause in patients who receive combination antibiotic therapy, specifically those who are receiving long-term or recurrent treatments.
Acknowledgements
We thank all the subjects who participated in this study. We also thank Mark Davids for his aid in preprocessing the sequences and Steven Aalvink for his help with the workup of intestinal microbiota samples.
Funding
This work was financially supported by the Netherlands Organization for Scientific Research (VIDI grant 91716475 to W. J. W. and SIAM Gravity grant 024.002.002 to W. M. V.)
Transparency declarations
W. M. V. served on scientific advisory boards for or received funds from Johnson & Johnson, Shire, GSK, Merck, Valio, Winclove, Nestlé, NIHS, Danone, DSM, Chr Hansen, CSM, Corbion, APC, GI-Health, AAK Biotech, MicroDish and Caelus Pharmaceuticals. C. B. received funds from Danone Research. The remaining authors have none to declare.
References
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