Visit 3
Impact of Antibiotic and Probiotic Intervention on the Gut Microbiome and Resistome of Healthy Adults Chad MacPherson1, Olivier Mathieu1, Julien Tremblay2, André Nantel2, Thomas A. Tompkins1, Stéphanie-Anne Girard1 and Pierre Burguière1 1
Lallemand Health Solutions Inc., 6100 Royalmount avenue, Montreal, Quebec, H4P 2R2, Canada. 2 National Research Council Canada, Montreal-Royalmount building, 6100 Royalmount Avenue, Montreal, Quebec, H4P 2R2, Canada.
Introduction
The aim of this study was to evaluate the impact of Lacidofil® STRONG following antibiotic administration on the gut microbiome and resistome of 70 healthy participants (a subgroup of the clinical study cohort) using 16S rRNA targeted metagenomic sequencing, as well shotgun sequencing and a custom-designed ABR microarray.
Placebo
1 week Visit 2 (Baseline)
Visit 3 (Antibiotic + Treatment)
1 week Visit 4 (Treatment-only)
Visit 5 (Wash Out)
Figure 1. Clinical study design.
Experimental Procedures Community DNA
A
Metagenomics Sequencing
16S Microbiome Sequencing (n=280 Samples)
DNA Extraction
B
Placebo Visit 3
Shotgun Sequencing Microbiome & ABR (Pooled Visit 3 Samples Placebo n= 35 & Lacidofil® STRONG n= 35)
PCR of 16S rRNA gene (V4 region)
MiSeq: DNA Sequencing
Shear DNA & Add Adaptors (red)
Pooled DNA
Bind ssDNA to Flow Cell Channels Shotgun Sequencing
Taxonomy: Kingdom Phylum Class Order Family Genus
Reference Database to Determine Relative Operational Taxonomic Units (OTUs)
Species Level & ABR Genes
Lacidofil® STRONG Visit 3
HiSeq: Massively Parallel Sequencing
Reference Database to Determine Relative OTUs and ABR Genes at the Species Level
C Gut Resistome ABR Microarray
+
V2 vs. V3
V2 vs. V5
V2 vs. V3
V2 vs. V3
V2 vs. V5
Visit 2
Visit 4
Visit 5
Visit 2
Visit 3
Visit 4
Visit 5
Visit 3
Placebo Lacidofil® STRONG
Lacidofil® STRONG
Figure 5. Top 15 OTUs from shotgun sequencing of pooled samples of Visit 3 for Placebo and Lacidofil® STRONG for the detection of species in the Enterobacteriaceae family. 0.0
900
Aminoglycosides 157/39
800
141/39
17.0
Placebo Lacidofil® STRONG
Beta-lactams Lincosamides
700
Macrolides
78/49
84/49
Miscellaneous
600 86/39
500
21/49
92/39
91/39
30/49
26/49
400
104/39 107/39 103/39
28/49
Mobile Elements Multidrug resistance
25/49
Aminoglycosides Distinct ABR Genes
Phenicols
16/49
Quaternary ammonium
300
Quinolones
200
Tetracyclines
100
138/36
194/36
128/36
131/36
102/36
197/36
132/36
Visit 3 Placebo Visit 3 Lacidofil® STRONG Aminoglycoside Beta-lactam Tetracycline
141/36
Trimethoprim Vancomycins
Lacidofil® STRONG
Figure 6. Total number of ABR genes whose transcript abundance increased by 2-fold or more after median normalization per participant. One-Way ANOVA with Dunn’s multiple comparisons test was used to evaluate the statistical differences of antibiotic + treatment (V3) compared to the other visits in both the Placebo and Lacidofil® STRONG groups.
Beta-lactams Distinct ABR Genes
Sulfonamides
0
Placebo
Visit 3
Figure 4. Microbiome community composition of top 20 OTUs down to the family level detected by 16S rRNA gene (V4 region) targeted metagenomic sequencing.
Cy5 dCTP
Placebo
Tetracycline Distinct ABR Genes
Lacidofil® STRONG
Figure 7. Total ABR genes of all participants for each visit categorized into antibiotic classes or types. Major increases of specific ABR classes are highlighted for Aminoglycosides, Betalactams and Tetracycline. The ratio depicts the total number of ABR gene hits over the number of ABR genes on the custom-designed ABR microarray.
Figure 8. Hierarchical clustering for each visit for both placebo and Lacidofil® STRONG groups for classes of Aminoglycosides, Betalactams and Tetracycline showing distinct and redundant ABR genes.
Discussion & Conclusion Results showed significant changes of the gut resistome and microbiome composition profiles after one week of antibiotic administration (V3). However, there was no major impact of Lacidofil® STRONG compared to the placebo on these antibiotic-induced changes as both gut resistome and microbiome compositions were very similar. Relative enrichment of specific ABR genes for the classes of Aminoglycosides, Beta-lactams and Tetracycline in Visit 3 was correlated back to the relative enrichment of the Enterobacteriaceae family by shotgun sequencing revealing the antibiotic-induced enrichment of bacterial genera (e.g. Klebsiella, Enterobacter, Salmonella and possible Escherichia) known to include pathogenic strains. Lastly, gut microbiome and resistome reversion to baseline levels one week after the end of antibiotic administration (V4) implied a static rebalancing after short-term antibiotic perturbation that may be explained by the hypothesis of community resilience2 and functional redundancy3.
References
(n=280 Samples)
267 ABR Genes DNA Extraction Cy5 Dye Labeling
Visit 3
Placebo
Lacidofil® STRONG
Figure 3. Weighted UniFrac phylogenetic distance analysis comparing how alike the microbiome profiles are between the visits based on the frequency of OTUs and the phylogenetic distance of the DNA sequences.
No Treatment
1 week
n = 35 Participants x 2 Groups (Placebo & Lacidofil® STRONG) x 4 visits = 280 Fecal Samples
V2 vs..V3
Placebo
Amoxicillin 875 mg / Clavulanic Acid 125 mg BID Placebo
1 week
No Treatment
Total Number of ABR Genes per Visit
Screening Visit 1
Lacidofil® STRONG
***p
Lallemand Health Solutions Inc., 6100 Royalmount avenue, Montreal, Quebec, H4P 2R2, Canada. 2 National Research Council Canada, Montreal-Royalmount building, 6100 Royalmount Avenue, Montreal, Quebec, H4P 2R2, Canada.
Introduction
The aim of this study was to evaluate the impact of Lacidofil® STRONG following antibiotic administration on the gut microbiome and resistome of 70 healthy participants (a subgroup of the clinical study cohort) using 16S rRNA targeted metagenomic sequencing, as well shotgun sequencing and a custom-designed ABR microarray.
Placebo
1 week Visit 2 (Baseline)
Visit 3 (Antibiotic + Treatment)
1 week Visit 4 (Treatment-only)
Visit 5 (Wash Out)
Figure 1. Clinical study design.
Experimental Procedures Community DNA
A
Metagenomics Sequencing
16S Microbiome Sequencing (n=280 Samples)
DNA Extraction
B
Placebo Visit 3
Shotgun Sequencing Microbiome & ABR (Pooled Visit 3 Samples Placebo n= 35 & Lacidofil® STRONG n= 35)
PCR of 16S rRNA gene (V4 region)
MiSeq: DNA Sequencing
Shear DNA & Add Adaptors (red)
Pooled DNA
Bind ssDNA to Flow Cell Channels Shotgun Sequencing
Taxonomy: Kingdom Phylum Class Order Family Genus
Reference Database to Determine Relative Operational Taxonomic Units (OTUs)
Species Level & ABR Genes
Lacidofil® STRONG Visit 3
HiSeq: Massively Parallel Sequencing
Reference Database to Determine Relative OTUs and ABR Genes at the Species Level
C Gut Resistome ABR Microarray
+
V2 vs. V3
V2 vs. V5
V2 vs. V3
V2 vs. V3
V2 vs. V5
Visit 2
Visit 4
Visit 5
Visit 2
Visit 3
Visit 4
Visit 5
Visit 3
Placebo Lacidofil® STRONG
Lacidofil® STRONG
Figure 5. Top 15 OTUs from shotgun sequencing of pooled samples of Visit 3 for Placebo and Lacidofil® STRONG for the detection of species in the Enterobacteriaceae family. 0.0
900
Aminoglycosides 157/39
800
141/39
17.0
Placebo Lacidofil® STRONG
Beta-lactams Lincosamides
700
Macrolides
78/49
84/49
Miscellaneous
600 86/39
500
21/49
92/39
91/39
30/49
26/49
400
104/39 107/39 103/39
28/49
Mobile Elements Multidrug resistance
25/49
Aminoglycosides Distinct ABR Genes
Phenicols
16/49
Quaternary ammonium
300
Quinolones
200
Tetracyclines
100
138/36
194/36
128/36
131/36
102/36
197/36
132/36
Visit 3 Placebo Visit 3 Lacidofil® STRONG Aminoglycoside Beta-lactam Tetracycline
141/36
Trimethoprim Vancomycins
Lacidofil® STRONG
Figure 6. Total number of ABR genes whose transcript abundance increased by 2-fold or more after median normalization per participant. One-Way ANOVA with Dunn’s multiple comparisons test was used to evaluate the statistical differences of antibiotic + treatment (V3) compared to the other visits in both the Placebo and Lacidofil® STRONG groups.
Beta-lactams Distinct ABR Genes
Sulfonamides
0
Placebo
Visit 3
Figure 4. Microbiome community composition of top 20 OTUs down to the family level detected by 16S rRNA gene (V4 region) targeted metagenomic sequencing.
Cy5 dCTP
Placebo
Tetracycline Distinct ABR Genes
Lacidofil® STRONG
Figure 7. Total ABR genes of all participants for each visit categorized into antibiotic classes or types. Major increases of specific ABR classes are highlighted for Aminoglycosides, Betalactams and Tetracycline. The ratio depicts the total number of ABR gene hits over the number of ABR genes on the custom-designed ABR microarray.
Figure 8. Hierarchical clustering for each visit for both placebo and Lacidofil® STRONG groups for classes of Aminoglycosides, Betalactams and Tetracycline showing distinct and redundant ABR genes.
Discussion & Conclusion Results showed significant changes of the gut resistome and microbiome composition profiles after one week of antibiotic administration (V3). However, there was no major impact of Lacidofil® STRONG compared to the placebo on these antibiotic-induced changes as both gut resistome and microbiome compositions were very similar. Relative enrichment of specific ABR genes for the classes of Aminoglycosides, Beta-lactams and Tetracycline in Visit 3 was correlated back to the relative enrichment of the Enterobacteriaceae family by shotgun sequencing revealing the antibiotic-induced enrichment of bacterial genera (e.g. Klebsiella, Enterobacter, Salmonella and possible Escherichia) known to include pathogenic strains. Lastly, gut microbiome and resistome reversion to baseline levels one week after the end of antibiotic administration (V4) implied a static rebalancing after short-term antibiotic perturbation that may be explained by the hypothesis of community resilience2 and functional redundancy3.
References
(n=280 Samples)
267 ABR Genes DNA Extraction Cy5 Dye Labeling
Visit 3
Placebo
Lacidofil® STRONG
Figure 3. Weighted UniFrac phylogenetic distance analysis comparing how alike the microbiome profiles are between the visits based on the frequency of OTUs and the phylogenetic distance of the DNA sequences.
No Treatment
1 week
n = 35 Participants x 2 Groups (Placebo & Lacidofil® STRONG) x 4 visits = 280 Fecal Samples
V2 vs..V3
Placebo
Amoxicillin 875 mg / Clavulanic Acid 125 mg BID Placebo
1 week
No Treatment
Total Number of ABR Genes per Visit
Screening Visit 1
Lacidofil® STRONG
***p
