To evaluate the microbiota of culture negative Corneal Impression Membrane (CIM) microbial keratitis samples with the use of shotgun metagenomics analysis.
Methods
DNA of microbial keratitis samples were collected with CIM and extracted using the MasterPure™ Complete DNA and RNA Purification Kit (Epicentre). DNA was fragmented by sonication into fragments of 300 to 400 base pairs (bp) using Bioruptor® (Diagenode, Belgium) and then used as a template for library preparation. DNA libraries were sequenced on Illumina® HiSeq2500. The resulting reads were quality controlled, trimmed and mapped against the human reference genome. The unmapped reads were taxonomically classified using the Kraken software.
Results
18 microbial keratitis samples were included in the study. Brevundimonas diminuta was found in 5 samples while 6 samples showed the presence of viral infections. Cutibacterium acnes, Staphylococcus aureus, Moraxella lacunata and Pseudomonas alcaligenes were also identified as the presumed putative cause of the infection in 7 samples.
Conclusions
Shotgun sequencing can be used as a diagnostic tool in microbial keratitis samples. This diagnostic method expands the available tests to diagnose eye infections and could be clinically significant in culture negative samples.
UrwinLOkurowskaKCrowtherG, et al.Corneal infection models: tools to investigate the role of biofilms in bacterial keratitis. Cells2020; 9: 2450.
2.
GhoshAKaurHGuptaA, et al.Emerging dematiaceous and hyaline fungi causing keratitis in a tertiary care centre from north India. Cornea2020; 39: 868–876.
SahayPGoelSNagpalR, et al.Infectious keratitis caused by rare and emerging micro-organisms. Curr Eye Res2020; 45: 761–773.
5.
UngLChodoshJ. Urgent unmet needs in the care of bacterial keratitis: An evidence-based synthesis. Ocul Surf2021.: S1542-0124(21)00095-1. DOI: 10.1016/j.jtos.2021.08.013. Epub ahead of print
6.
UngLBispoPJMShanbhagSS,et al.The persistent dilemma of microbial keratitis: global burden, diagnosis, and antimicrobial resistance. Surv Ophthalmol2019; 64: 255–271.
7.
LowLFuentes-UtrillaPHodsonJ, et al.Evaluation of full-length nanopore 16S sequencing for detection of pathogens in microbial keratitis. PeerJ2021; 9: e10778.
8.
BorroniDde LossadaCR. Microbial keratitis: the clinical impact of metagenomic next-generation sequencing (mNGS). Arch Soc Esp Oftalmol (Engl Ed2020; 95: 621–623.
9.
BorroniDRomanoVKayeSB, et al.Metagenomics in ophthalmology: current findings and future prospectives. BMJ Open Ophthalmol2019; 4: e000248.
10.
Su MunLWye LumSKong Yuiin SzeG, et al. Association of Microbiome with Oral Squamous Cell Carcinoma: A Systematic Review of the Metagenomic Studies. Int J Environ Res Public Health2021; 18: 7224.
11.
GallonPParekhMFerrariS,et al.Metagenomics in ophthalmology: hypothesis or real prospective?Biotechnol Rep (Amst2019; 23: e00355.
12.
WilsonMRShanbhagNMReidMJ, et al.Diagnosing balamuthia mandrillaris encephalitis with metagenomic deep sequencing. Ann Neurol2015; 78: 722–730.
13.
QuinceCNurkSRaguideauS, et al.STRONG: metagenomics strain resolution on assembly graphs. Genome Biol2021; 22: 214.
14.
DoanTWilsonMRCrawfordED, et al.Illuminating uveitis: metagenomic deep sequencing identifies common and rare pathogens. Genome Med2016; 8: 90.
15.
ParekhMBorroniDRomanoV, et al.Next-generation sequencing for the detection of microorganisms present in human donor corneal preservation medium. BMJ Open Ophthalmol2019; 4: e000246.
16.
NormanJMHandleySAVirginHW. Kingdom-agnostic metagenomics and the importance of complete characterization of enteric microbial communities. Gastroenterology2014; 146: 1459–1469.
17.
ParekhMRomanoVFranchA, et al.Shotgun sequencing to determine corneal infection. Am J Ophthalmol Case Rep2020; 19: 100737.
18.
DelbekeHYounasSCasteelsI,et al.Current knowledge on the human eye microbiome: a systematic review of available amplicon and metagenomic sequencing data. Acta Ophthalmol2021; 99: 16–25.
UngLWangYVangelM, et al.Validation of a comprehensive clinical algorithm for the assessment and treatment of microbial keratitis. Am J Ophthalmol2020; 214: 97–109.
21.
MiaoQMaYWangQ, et al.Microbiological diagnostic performance of metagenomic next-generation sequencing when applied to clinical practice. Clin Infect Dis2018; 67: S231–Ss40.
22.
TingDSJHoCSCairnsJ, et al.12-year Analysis of incidence, microbiological profiles and in vitro antimicrobial susceptibility of infectious keratitis: the nottingham infectious keratitis study. Br J Ophthalmol2021; 105: 328–333.
23.
PaliouraSGibbonsAMillerD,et al.Clinical features, antibiotic susceptibility profile, and outcomes of infectious keratitis caused by Stenotrophomonas maltophilia. Cornea2018; 37:326–330.
24.
GuWDengXLeeM, et al.Rapid pathogen detection by metagenomic next-generation sequencing of infected body fluids. Nat Med2021; 27: 115–124.
25.
LiuHYHoppingGCVaidyanathanU,et al.Polymerase chain reaction and its application in the diagnosis of infectious keratitis. Med Hypothesis Discov Innov Ophthalmol2019; 8: 152–155.
26.
SomervilleTFCorlessCESuekeH,et al.16S Ribosomal RNA PCR versus conventional diagnostic culture in the investigation of suspected bacterial keratitis. Transl Vis Sci Technol2020; 9: 2.
27.
SungJYLeeYJChoYJ, et al.A large-scale metagenomic study for enzyme profiles using the focused identification of the NGS-based definitive enzyme research (FINDER) strategy. Biotechnol Bioeng2021; 118: 4360–4374.
28.
ZhangYCuiPZhangHC, et al.Clinical application and evaluation of metagenomic next-generation sequencing in suspected adult central nervous system infection. J Transl Med2020; 18: 199.
29.
SharmaAImSH. Special issue on the human microbiome: from symbiosis to therapy. Exp Mol Med2020; 52: 1361–1363.
XingXWZhangJTMaYB, et al.Metagenomic next-generation sequencing for diagnosis of infectious encephalitis and meningitis: a large, prospective case series of 2020; 213, patients. Front Cell Infect Microbiol2020; 10: 88.
32.
ZhangPChenYLiS, et al.Metagenomic next-generation sequencing for the clinical diagnosis and prognosis of acute respiratory distress syndrome caused by severe pneumonia: a retrospective study. PeerJ2020; 8: e9623.
33.
MostafaHHFisselJAFanelliB, et al.Metagenomic next-generation sequencing of nasopharyngeal specimens collected from confirmed and suspect COVID-19 patients. mBio2020: 11.
34.
UngLBispoPJMDoanT, et al.Clinical metagenomics for infectious corneal ulcers: rags to riches?Ocul Surf2020; 18: 1–12.
35.
LiZBreitwieserFPLuJ, et al.Identifying corneal infections in formalin-fixed specimens using next generation sequencing. Invest Ophthalmol Vis Sci2018; 59: 280–288.
36.
BorroniD. Clinical metagenomic next-generation sequencing has the potential to reduce costs in the management of microbial keratitis patients. Cornea2021; 40: e14.
37.
SinghASahuSKSharmaS,et al.Acanthamoeba keratitis versus mixed acanthamoeba and bacterial keratitis: comparison of clinical and microbiological profiles. Cornea2020; 39: 1112–1116.
38.
RaghavanABaidwalSVenkatapathyN,et al.The acanthamoeba-fungal keratitis study. Am J Ophthalmol2019; 201: 31–36.
39.
PelletierJSIdeTYooSH. Brevundimonas vesicularis keratitis after laser in situ keratomileusis. J Cataract Refract Surg2010; 36: 340–343.
