Absolute abundance calculation enhances the significance of microbiome data in antibiotic treatment studies

Stefanie Wagner ¹ Michael Weber ¹ Lena-Sophie Paul ¹ Angelika Grümpel-Schlüter ² Jeannette Kluess ² Klaus Neuhaus ³ Thilo Fuchs ^4*

• ¹ Institute of Molecular Pathogenesis, Friedrich Loeffler Institute, Jena, Thuringia, Germany
• ² Institute for Animal Nutrition, Federal Research Institute for Animal Health, Friedrich Loeffler Institute, Braunschweig, Berlin, Germany
• ³ Core Facility Microbiome Unit NGS, Institute for Food and Health, Technical University of Munich, Freising, Bavaria, Germany
• ⁴ Friedrich-Loeffler-Institute, Greifswald-Insel Riems, Germany

Background: The intestinal microbiota contributes to the colonization resistance of the gut towards bacterial pathogens. Antibiotic treatment often negatively affects the microbiome composition, rendering the host more susceptible for infections (1). However, a correct interpretation of such a perturbation requires quantitative microbiome profiling to reflect accurately the direction and magnitude of compositional changes within a microbiota. Standard 16S rRNA gene amplicon sequencing of microbiota samples offers compositional data in relative, but not absolute abundancies, and the presence of multiple copies of 16S rRNA genes in bacterial genomes introduces bias into compositional data. We explored whether improved sequencing data analysis influences the significance of the effect exerted by antibiotics on the faecal microbiota of young pigs using two veterinary antibiotics.Calculation of absolute abundances, either by flow cytometry-based bacterial cell counts or by spikein of synthetic 16S rRNA genes, was employed and 16S rRNA gene copy numbers (GCN) were corrected.Results: Cell number determination exhibited large interindividual variability in two pig studies, using either tylosin or tulathromycin. Following tylosin application, flow cytometry-based cell counting revealed decreased absolute abundances of five families and ten genera. These results were not detectable by standard 16S analysis based on relative abundances. Here, GCN correction additionally uncovered significant decreases of Lactobacillus and Faecalibacterium. In another experimental setting with tulathromycin treatment, bacterial abundance quantification by flow cytometry and by a spike-in method yielded similar results only on the phylum level. Even though the spike-in method identified the decrease of four genera, analysis by fluorescence-activated cell sorting (FACS) uncovered eight significantly reduced genera, such as Prevotella and Paraprevotella upon antibiotic treatment. In contrast, analysis of relative abundances only showed a decrease of Faecalibacterium and Rikenellaceae RC9 gut group and, thus, a much less detailed antibiotic effect.Flow cytometry is a laborious method, but identified a higher number of significant microbiome changes in comparison to common compositional data analysis and even revealed to be superior to a spike-in method. Calculation of absolute abundances and GCN correction are valuable methods that should be standards in microbiome analyses in veterinary as well as human medicine.