Biology subjects

Stapleton, F.

Publications and source records attributed to Stapleton, F..

4 recordsLinked to original sources

bioRxiv2021

Human Tear Metabolomics Using Liquid Chromatography-Q Exactive-HF Mass Spectrometry

AimTo putatively identify and characterise human tear metabolites in a normal subject on an untargeted platform of liquid chromatography-Q exactive-HF mass spectrometry. MethodsFour samples of unstimulated tears were collected from both eyes on four consecutive days between 1 - 2 pm using a microcapillary tube and pooled from both eyes each day. Untargeted analysis of the tears was performed by chromatographic separation of constituent metabolites in both CSH-C18RP (Charged Surface Hybrid-C18 Reversed Phase) and SeQuant ZIC-pHILIC (Zwitterionic-polymeric Hydrophilic Interaction Liquid Chromatography) columns, followed by heated electrospray ionization (HESI) and the acquisition of mass spectra using QExactive-HF mass spectrometer. Compound Discoverer software (v2.0) was used for data analysis. ResultEighty-two metabolites were tentatively identified. Seventy compounds (85.4 %) were observed in all four samples with a coefficient of variation (CV) less than 25 %. Fifty-nine metabolites (71.9 %) were novel in the healthy tears. Amino acids were the most frequently detected metabolites in the tears (28 %), followed by carbohydrates (12.2 %), carboxylic acids (8.5 %), carnitines (6.1 %) and glycerophospholipids (4.9 %), respectively. ConclusionThe current untargeted platform is capable of detecting a range of tear metabolites across several biological categories. This study provides a baseline for further ocular surface studies.

molecular biology↗

bioRxiv2020

Evaluation of optically tailored fluorescent silicon quantum dots for bioimaging of the tear film

This experimental study aimed to investigate the feasibility of using silicon quantum dots doped with transition metals: scandium, copper and zinc as contrast agents for eventual application for the study of the tear film in eyes. Si-QDs were synthesized and characterized by transmission electron microscopy, photoluminescence, absorbance and transient absorption measurements. The fluorescence of Si-QDs was investigated when combined with TheraTears(R) (a balanced electrolyte formula for dry eye therapy). An optical imaging system composed of a modified slit lamp biomicroscope combined with a high-resolution Zyla sCMOS camera, SOLIS software, custom-made optical mounts and emission filters (460 nm, 510 nm and 530 nm) were used for in vitro imaging of Si-QDs with TheraTears(R). The average size of Si-QDs was 2.65 nm. In vitro imaging of Sc-Si-QDs and Cu-Si-QDs indicated their stable and bright fluorescence with TheraTears(R). Sc-Si-QDs were significantly brighter compared to Cu-Si-QDs and Zn-Si-QDs, and the Zn-Si-QDs showed a tendency to clump in TheraTears(R). The fluorescence of the Si-QDs was detected down to a concentration of 0.01 {micro}g/mL within a total volume of 5 {micro}L. Cu-Si-QDs and Sc-Si-QDs showed brighter fluorescence than Zn-Si-QDs. However, Zn-Si-QDs and to a lesser extent, Cu-Si-QDs showed some aggregation at specific concentrations. Sc-Si-QDs are proposed as a better option for further development as an in vivo bioimaging agent to study the tear film dynamics.

biochemistry↗

bioRxiv2020

Acquired fluoroquinolone resistance genes in corneal isolates of Pseudomonas aeruginosa

Fluroquinolones are widely used as an empirical therapy for pseudomonal ocular infections. Based on increasing reports on acquired fluroquinolone resistance genes in clinical isolates of Pseudomonas aeruginosa, we investigated 33 strains of P. aeruginosa isolated from the cornea of microbial keratitis patients in India and Australia between 1992 and 2018 to understand the prevalence of acquired fluroquinolone resistance genes in ocular isolates and to assess whether the possession of those genes was associated with fluoroquinolone susceptibility. We obtained the whole genome sequence of 33 isolates using Illumina MiSeq platform and investigated the prevalence of two fluoroquinolone resistance genes crpP and qnrVC1. To examine the associated mobile genetic elements of qnrVC1 positive strains, we obtained long read sequences using Oxford Nanopore MinION and performed hybrid assembly to combine long reads with Illumina short sequence reads. We further assessed mutations in QRDRs and antibiotic susceptibilities to ciprofloxacin, levofloxacin and moxifloxacin to examine the association between resistance genes and phenotype. Twenty strains possessed crpP in genetic islands characterised by possession of integrative conjugative elements. The qnrVC1 gene was carried by four isolates on class I integrons and Tn3 transposons along with aminoglycoside and beta-lactam resistance genes. We did not observe any evidence of plasmids carrying fluroquinolone resistance genes. Resistance to fluroquinolones was observed in those strains which possessed crpP, qnrVC1 and that had QRDRs mutations. The presence of crpP was not a sole cause of fluroquinolone resistance.

microbiology↗

bioRxiv2020

Changes to the resistome of Pseudomonas aeruginosa clone ST308 associated with corneal infection over time

ObjectivesThis study compared the resistomes of isolates of Pseudomonas aeruginosa clone ST308 from 2018 and 1997 from India. MethodsTwo ocular clonal type ST308 isolates of Pseudomonas aeruginosa (198 and 219) isolated in 2018 and five historical isolates (31, 32, 33, 35 and 37) isolated in 1997 at the LV Prasad Eye Institute in India were analysed for their susceptibilities to ciprofloxacin, levofloxacin, gentamicin, tobramycin, piperacillin, imipenem, ceftazidime and polymyxin B. DNA was extracted using the DNeasy(R) Blood and Tissue. Paired-end library was prepared using Nextera XT DNA library preparation kit. Libraries were sequenced on Illumina(R) MiSeq bench top sequencer generating 300 bp paired-end reads. Spades v3.12.0 was used for assembly, Resfinder v3.1. for acquired resistance genes and Snippy V2 for variants calling. Integron finder v1.5.1 was used to identify the integrons present in the genomes. ResultsThe recent isolate 219 was resistant to all tested antibiotics except polymyxin while isolate 198 was resistant to ciprofloxacin, levofloxacin, gentamicin and tobramycin. Among historical isolates five were resistant to gentamicin, tobramycin and ciprofloxacin, four were resistant to levofloxacin while two were resistant to polymyxin. Twenty-four acquired resistance genes were present in the 2018 isolates compared to 11 in the historical isolates. All isolates contained the following genes encoding for aminoglycoside aph(6)-Id, aph(3')-lIb, aph(3'')-Ib), beta-lactam (blaPAO), tetracycline (tet(G)), fosfomycin (fosA), chloramphenicol (catB7), sulphonamide (sul1), quaternary ammonium (qacEdelta1) and fluoroquinolone (crpP) resistance. Isolate 198 possessed aph(3')-VI, rmtD2, qnrVC1, blaOXA-488, blaPME-1, while 219 possessed aadA1, rmtB, aac(6')-Ib-cr, blaTEM-1B, blaVIM-2, mph(E), mph(A), msr(E). In the isolate 219 genes blaTEM-1b, blaVIM-2, sul1, qnrvc1, rmtB and aadA1 were carried on class 1 integron. While an incomplete class 1 integron was also found in isolate 198 which was located on the genome where gene rmtB, blaPME-1, qnrVC1 and sul1 genes were positioned. There were no notable differences in the number of single nucleotide polymorphisms, but recent isolates carried more insertions and deletions in their genes. ConclusionP. aeruginosa ocular clonal isolates have changed over time, with strains acquiring genes and having more insertions and deletions in their chromosomal genes that confirm resistance to antibiotics. HighlightsO_LIRecent clonal ocular isolates of Pseudomonas aeruginosa from India have acquired a number of resistance genes compared to historical clones C_LIO_LIConsequently, resistance to antibiotics particularly fluoroquinolones in recent clones of P. aeruginosa appears to have increased. C_LIO_LIThe acquired resistance genes found in the recent P. aeruginosa isolates were related to mobile genetic elements. C_LI

microbiology↗