Saponin treatment for eukaryotic DNA depletion alters the microbial DNA profiles by reducing the abundance of Gram-negative bacteria in metagenomics analyses

Abstract

Background: Recent advances in microbiome sequencing techniques have provided new insights into the role of the microbiome on human health with potential diagnostic implications. However, these developments are often hampered by the presence of a large amount of human DNA interfering with the analysis of the bacterial content. Nowadays, extensive scientific literature focuses on eukaryotic DNA depletion methods, which successfully remove host DNA in microbiome studies, even if a precise assessment of the impact on bacterial DNA is often missing.

Methods: Here, we have investigated a saponin-based DNA isolation protocol commonly applied to different biological matrices to deplete the released host DNA.

Results: The bacterial DNA obtained was used to assess the relative abundance of bacterial and human DNA, revealing that the inclusion of 2.5% wt/vol saponin allowed the depletion of most of the host’s DNA in favor of bacterial DNA enrichment. However, shotgun metagenomic sequencing showed inaccurate microbial profiles of the DNA samples, highlighting an erroneous increase in Gram-positive DNA. Even the application of 0.0125% wt/vol saponin altered the bacterial profile by depleting Gram-negative bacteria, resulting in an overall increase of Gram-positive bacterial DNA.

Conclusion: The application of the saponin-based protocol drastically changes the detection of the microbial composition of human-related biological specimens. In this context, we revealed that saponin targets not only host cells but also specific bacterial cells, thus inducing a drastic reduction in the profiling of Gram-negative bacterial DNA.

Keywords

Host DNA depletion / saponin / bacterial DNA enrichment / microbiome profiling

References

[1] Procop GW.Molecular diagnostics for the detection and characterization of microbial pathogens.Clin Infect Dis2007;45:S99-111

[2] Nikkari S,Lepp PW.Broad-range bacterial detection and the analysis of unexplained death and critical illness.Emerg Infect Dis2002;8:188-94 PMCID:PMC2732447

[3] Clarridge JE III.Impact of 16S rRNA gene sequence analysis for identification of bacteria on clinical microbiology and infectious diseases.Clin Microbiol Rev2004;17:840-62 PMCID:PMC523561

[4] Cummings LA,Hoogestraat DR.Clinical next generation sequencing outperforms standard microbiological culture for characterizing polymicrobial samples.Clin Chem2016;62:1465-73

[5] Ranjan R,Metwally A,Perkins DL.Analysis of the microbiome: advantages of whole genome shotgun versus 16S amplicon sequencing.Biochem Biophys Res Commun2016;469:967-77 PMCID:PMC4830092

[6] Wensel CR,Salzberg SL.Next-generation sequencing: insights to advance clinical investigations of the microbiome.J Clin Invest2022;132:e154944 PMCID:PMC8970668

[7] Feigelman R,Baty F.Sputum DNA sequencing in cystic fibrosis: non-invasive access to the lung microbiome and to pathogen details.Microbiome2017;5:20 PMCID:PMC5303297

[8] Marotz CA,Zuniga C,Knight R.Improving saliva shotgun metagenomics by chemical host DNA depletion.Microbiome2018;6:42 PMCID:PMC5827986

[9] Horz HP,Vianna ME.New methods for selective isolation of bacterial DNA from human clinical specimens.Anaerobe2010;16:47-53

[10] Ahannach S,Spacova I.Microbial enrichment and storage for metagenomics of vaginal, skin, and saliva samples.iScience2021;24:103306 PMCID:PMC8571498

[11] Bruggeling CE,Achouiti S,Dutilh BE.Optimized bacterial DNA isolation method for microbiome analysis of human tissues.Microbiologyopen2021;10:e1191 PMCID:PMC8208965

[12] Goltsman DSA,Proctor DM.Metagenomic analysis with strain-level resolution reveals fine-scale variation in the human pregnancy microbiome.Genome Res2018;28:1467-80 PMCID:PMC6169887

[13] Byrd AL,Segre JA.The human skin microbiome.Nat Rev Microbiol2018;16:143-55

[14] Malone M,Jensen SO.Next generation DNA sequencing of tissues from infected diabetic foot ulcers.EBioMedicine2017;21:142-9 PMCID:PMC5514496

[15] Thoendel M,Greenwood-Quaintance KE.Comparison of microbial DNA enrichment tools for metagenomic whole genome sequencing.J Microbiol Methods2016;127:141-5 PMCID:PMC5752108

[16] Hasan MR,Tang P.Depletion of human DNA in spiked clinical specimens for improvement of sensitivity of pathogen detection by next-generation sequencing.J Clin Microbiol2016;54:919-27 PMCID:PMC4809942

[17] Castelino M,Moat J.Optimisation of methods for bacterial skin microbiome investigation: primer selection and comparison of the 454 versus MiSeq platform.BMC Microbiol2017;17:23 PMCID:PMC5251215

[18] Heravi FS,Vickery K.Host DNA depletion efficiency of microbiome DNA enrichment methods in infected tissue samples.J Microbiol Methods2020;170:105856

[19] Amar Y,Silva RL.Pre-digest of unprotected DNA by Benzonase improves the representation of living skin bacteria and efficiently depletes host DNA.Microbiome2021;9:123 PMCID:PMC8157445

[20] Charalampous T,Richardson H.Nanopore metagenomics enables rapid clinical diagnosis of bacterial lower respiratory infection.Nat Biotechnol2019;37:783-92

[21] Masud T.Corticosteroids in treatment of disseminated tuberculosis in patient with HIV infection.Br Med J1988;296:464-5 PMCID:PMC2545044

[22] He Y,Shi X.Enhanced DNA and RNA pathogen detection via metagenomic sequencing in patients with pneumonia.J Transl Med2022;20:195 PMCID:PMC9066823

[23] Khan MI,Shin JH,Kim MY.Green tea seed isolated saponins exerts antibacterial effects against various strains of gram positive and gram negative bacteria, a comprehensive study in vitro and in vivo.Evid Based Complement Alternat Med2018;2018:3486106 PMCID:PMC6287149

[24] Milani C,Foroni E.Assessing the fecal microbiota: an optimized ion torrent 16S rRNA gene-based analysis protocol.PLoS One2013;8:e68739 PMCID:PMC3711900

[25] Handschur M,Hertel C,Haslberger AG.Preanalytic removal of human DNA eliminates false signals in general 16S rDNA PCR monitoring of bacterial pathogens in blood.Comp Immunol Microbiol Infect Dis2009;32:207-19

[26] Milani C,Duranti S.Bifidobacteria exhibit social behavior through carbohydrate resource sharing in the gut.Sci Rep2015;5:15782 PMCID:PMC4623478

[27] Li H.Fast and accurate short read alignment with Burrows-Wheeler transform.Bioinformatics2009;25:1754-60 PMCID:PMC2705234

[28] Milani C,Lugli GA.Tracing mother-infant transmission of bacteriophages by means of a novel analytical tool for shotgun metagenomic datasets: METAnnotatorX.Microbiome2018;6:145 PMCID:PMC6102903

[29] Milani C,Fontana F.METAnnotatorX2: a comprehensive tool for deep and shallow metagenomic data set analyses.mSystems2021;6:e0058321 PMCID:PMC8269244

[30] Hillmann B,Shields-Cutler RR.Evaluating the information content of shallow shotgun metagenomics.mSystems2018;3:e00069-18 PMCID:PMC6234283

[31] Lupetti A,Morici P,Nibbering PH.Saponin promotes rapid identification and antimicrobial susceptibility profiling of Gram-positive and Gram-negative bacteria in blood cultures with the Vitek 2 system.Eur J Clin Microbiol Infect Dis2013;32:493-502

[32] Lee JE,Park KG.Evaluation of modified saponin preparation method for the direct identification and antimicrobial susceptibility testing from positive blood culture.J Microbiol Methods2018;154:118-23

[33] Mai-Prochnow A,Hong J.Gram positive and Gram negative bacteria differ in their sensitivity to cold plasma.Sci Rep2016;6:38610 PMCID:PMC5146927

[34] Tagousop CN,Kengne IC,Voutquenne-Nazabadioko L.Antimicrobial activities of saponins from Melanthera elliptica and their synergistic effects with antibiotics against pathogenic phenotypes.Chem Cent J2018;12:97 PMCID:PMC6768134

[35] Patra AK.The effect and mode of action of saponins on the microbial populations and fermentation in the rumen and ruminant production.Nutr Res Rev2009;22:204-19

[36] Marquet M,Pastuschek J.Evaluation of microbiome enrichment and host DNA depletion in human vaginal samples using Oxford Nanopore’s adaptive sequencing.Sci Rep2022;12:4000 PMCID:PMC8901746

[37] Ganda E,Haiminen N.DNA extraction and host depletion methods significantly impact and potentially bias bacterial detection in a biological fluid.mSystems2021;6:e0061921 PMCID:PMC8574158