An expectation–maximization algorithm for estimating proportions of deletions among bacterial populations with application to study antibiotic resistance gene transfer in Enterococcus faecalis

Abstract

The emergence of antibiotic resistance in bacteria limits the availability of antibiotic choices for treatment and infection control, thereby representing a major threat to human health. The de novo mutation of bacterial genomes is an essential mechanism by which bacteria acquire antibiotic resistance. Previously, deletion mutations within bacterial immune systems, ranging from dozens to thousands of base pairs (bps) in length, have been associated with the spread of antibiotic resistance. Most current methods for evaluating genomic structural variations (SVs) have concentrated on detecting them, rather than estimating the proportions of populations that carry distinct SVs. A better understanding of the distribution of mutations and subpopulations dynamics in bacterial populations is needed to appreciate antibiotic resistance evolution and movement of resistance genes through populations. Here, we propose a statistical model to estimate the proportions of genomic deletions in a mixed population based on Expectation–Maximization (EM) algorithms and next-generation sequencing (NGS) data. The method integrates both insert size and split-read mapping information to iteratively update estimated distributions. The proposed method was evaluated with three simulations that demonstrated the production of accurate estimations. The proposed method was then applied to investigate the horizontal transfers of antibiotic resistance genes in concert with changes in the CRISPR-Cas system of E. faecalis.

Keywords

Bacterial genomes / CRISPR-Cas system / Antibiotic resistance / EM algorithm / Proportion estimation / Biological Sciences / Genetics / Microbiology

References

[1] AriasCA, MurrayBE. The rise of the Enterococcus: beyond vancomycin resistance. Nat Rev Microbiol, 2012, 10: 266-278.

[2] BaoZ, StodghillPV, MyersCR, LamH, WeiHL, ChakravarthyS, KvitkoBH, CollmerA, CartinhourSW, SchweitzerP, SwingleB. Genomic plasticity enables phenotypic variation of Pseudomonas syringae pv. tomato dc3000. PLoS ONE, 2014, 9: e86628.

[3] BarrangouR, FremauxC, DeveauH, RichardsM, BoyavalP, MoineauS, RomeroDA, HorvathP. Crispr provides acquired resistance against viruses in prokaryotes. Science, 2007, 315: 1709-1712.

[4] Centers for Disease Control and Prevention (2019) Antibiotic resistance threats in the United States. Atlanta, GA: U.S. Department of health and human services, Centers for disease control and prevention. https://www.cdc.gov/drugresistance/biggest-threats.html

[5] Centers for Disease Control and Prevention (2021) COVID-19 & Antibiotic resistance. Atlanta, GA: U.S. Department of health and human services, Centers for disease control and prevention. https://www.cdc.gov/drugresistance/covid19.html

[6] ChenYC, LiuT, YuCH, ChiangTY, HwangCC. Effects of GC bias in next-generation-sequencing data on de novo genome assembly. PLoS ONE, 2013, 8: 1-20

[7] DeatherageDE, BarrickJE. Identification of mutations in laboratory-evolved microbes from next-generation sequencing data using breseq. Methods Mol Biol, 2014, 1151: 165-188.

[8] DempsterAP, LairdNM, RubinDB. Maximum likelihood from incomplete data via the EM algorithm. J R Stat Soc B, 1977, 39: 1-38

[9] DijkEL, JaszczyszynY, ThermesC. Library preparation methods for next-generation sequencing: Tone down the bias. Exp Cell Res, 2014, 322: 12-20.

[10] GuanP, SungWK. Structural variation detection using next generation sequencing data: a comparative technical review. Methods, 2016, 102: 36-49.

[11] Hatoum-AslanA, MarraffiniLA. Impact of CRISPR immunity on the emergence and virulence of bacterial pathogens. Curr Opin Microbiol, 2014, 17: 82-90.

[12] HershbergR. Mutation–the engine of evolution: studying mutation and its role in the evolution of bacteria. Cold Spring Harb Perspect Biol, 2015, 7. a018077

[13] HiguitaNI, HuyckeMMGilmoreMS, ClewellDB, IkeY, ShankarN. Enterococcal disease, epidemiology, and implications for treatment. Enterococci from commensals to leading causes of drug resistant infection, 2014BostonMassachusetts Eye and Ear Infirmary

[14] HullahalliK, RodriguesM, PalmerKL. Exploiting CRISPR-Cas to manipulate Enterococcus faecalis populations. Elife, 2017, 6: e26664.

[15] Huo W, Price VJ, Sharifi A, Zhang MQ, Palmer KL (2018) Deep sequencing analysis of CRISPR-escaping plasmid transconjugants in Enterococcus faecalis. bioRxiv1:220467

[16] JiangW, BikardD, CoxD, ZhangF, MarraffiniLA. RNA-guided editing of bacterial genomes using CRISPR-Cas systems. Nat Biotechnol, 2013, 31: 233-239.

[17] Kaiser G (2020) Horizontal gene transfer in bacteria. Biology LibreTexts. https://bio.libretexts.org/Bookshelves/Microbiology/Book:_Microbiology_(Kaiser)/Unit_2:_Bacterial_Genetics_and_the_Chemical_Control_of_Bacteria/3:_Bacterial_Genetics/3.1:_Horizontal_Gene_Transfer_in_Bacteria

[18] KeelingPJ, PalmerJD. Horizontal gene transfer in eukaryotic evolution. Nat Rev Genet, 2008, 9: 605-618.

[19] KorbelJO, UrbanAE, AffourtitJP, GodwinB, GrubertF, SimonsJF, KimPM, PalejevD, CarrieroNJ, DuL, TaillonBE, ChenZ, TanzerA, SaundersAC, ChiJ, YangF, CarterNP, HurlesME, WeissmanSM, HarkinsTT, et al. . Paired-end mapping reveals extensive structural variation in the human genome. Science, 2007, 318: 420-426.

[20] KosalaiS, SenP, NookaewI. Evaluation and assessment of read-mapping by multiple next-generation sequencing aligners based on genome-wide characteristics. Genomics, 2017, 109: 186-191.

[21] LebretonF, WillemsRJL, GilmoreMSGilmoreMS, ClewellDB, IkeY, ShankarN. Enterococcus diversity, origins in nature, and gut golonization. Enterococci from commensals to leading causes of drug resistant infection, 2014BostonMassachusetts Eye and Ear Infirmary

[22] LiB, WebsterTJ. Bacteria antibiotic resistance: new challenges and opportunities for implant-associated orthopedic infections. J Orthop Res, 2018, 36: 22-32

[23] Lopez-SanchezMJ, SauvageE, Da CunhaV, ClermontD, Ratsima HariniainaE, Gonzalez-ZornB, PoyartC, Rosinski-ChupinI, GlaserP. The highly dynamic CRISPR1 system of Streptococcus agalactiae controls the diversity of its mobilome. Mol Microbiol, 2012, 85: 1057-1071.

[24] LourensS, ZhangY, LongJD, PaulsenJS. Bias in estimation of a mixture of normal distributions. J Biom Biostat, 2013, 4: 1000179

[25] LuckeyTD. Introduction to intestinal microecology. Am J Clin Nutr, 1972, 25: 1292-1294.

[26] MarraffiniLA, SontheimerEJ. CRISPR interference limits horizontal gene transfer in staphylococci by targeting DNA. Science, 2008, 322: 1843-1845.

[27] MartinezJL, BaqueroF. Mutation frequencies and antibiotic resistance. Antimicrob Agents Chemother, 2000, 44: 1771-1777.

[28] PriceVJ, HuoW, SharifiA, PalmerK. CRISPR-Cas and restriction-modification act additively against conjugative antibiotic resistance plasmid transfer in Enterococcus faecalis. mSphere, 2016, 1: e00064-e116.

[29] PriceVJ, McBrideSW, HullahalliK, ChatterjeeA, DuerkopBA, PalmerKL. Enterococcus faecalis CRISPR-Cas is a robust barrier to conjugative antibiotic resistance dissemination in the murine intestine. mSphere, 2019, 4: e00464-e519.

[30] PuninaNV, MakridakisNM, RemnevMA, TopunovAF. Whole-genome sequencing targets drug-resistant bacterial infections. Hum Genomics, 2015, 9: 19.

[31] RauschT, ZichnerT, SchlattlA, StützAM, BenesV, KorbelJO. DELLY: structural variant discovery by integrated paired-end and split-read analysis. Bioinformatics, 2012, 28: i333-i339.

[32] ReAct (2005). Antibiotic resistance. https://www.reactgroup.org/toolbox/understand/antibiotic-resistance/

[33] RileyDR, SieberKB, RobinsonKM, WhiteJR, GanesanA, NourbakhshS, Dunning HotoppJC. Bacteria-human somatic cell lateral gene transfer is enriched in cancer samples. PLoS Comput Biol, 2013, 9: e1003107.

[34] Sage Science (2016) Why DNA size selection matters in NGS pipelines. https: //bitesizebio.com/27694/why-dna-size-selection-matters-in-ngs-pipelines/

[35] TreangenTJ, SalzbergSL. Repetitive DNA and next-generation sequencing: computational challenges and solutions. Nat Rev Genet, 2011, 13: 36-46.

[36] TreangenTJ, AbrahamAL, TouchonM, RochaEP. Genesis, effects and fates of repeats in prokaryotic genomes. FEMS Microbiol Rev, 2009, 33: 539-571.

[37] Von WintersdorffCJ, PendersJ, van NiekerkJM, MillsND, MajumderS, van AlphenLB, SavelkoulPH, WolffsPF. Dissemination of antimicrobial resistance in microbial ecosystems through horizontal gene transfer. Front Microbiol, 2016, 7: 173

[38] WoodfordN, EllingtonMJ. The emergence of antibiotic resistance by mutation. Clin Microbiol Infect, 2007, 13: 5-18.

[39] YeK, SchulzMH, LongQ, ApweilerR, NingZ. Pindel: a pattern growth approach to detect break points of large deletions and medium sized insertions from paired-end short reads. Bioinformatics, 2009, 25: 2865-2871.

RIGHTS & PERMISSIONS

Ocean University of China