Acquired fluoroquinolone resistance genes in corneal isolates of Pseudomonas aeruginosa
Mahjabeen Khan,Stephen Summers,Scott A. Rice,Scott A. Rice,Fiona Stapleton,Mark D. P. Willcox,Dinesh Subedi,Dinesh Subedi +7 more
TL;DR: Investigation of 33 strains of P. aeruginosa isolated from the cornea of microbial keratitis patients in India and Australia between 1992 and 2018 found the prevalence of acquired fluroquinolone resistance genes in ocular isolates was unclear and mutations in QRDRs and antibiotic susceptibilities to ciprofloxacin, levofloxacins and moxifloxACin were assessed to examine the association between resistance genes and phenotype.
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Abstract: 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.
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Figures
![Figure 5. Comparison of qnrVC1-associated genomic islands of P. aeruginosa strains. Protein-coding regions are represented by the arrows and common key features/associated genes among all strains are shown in various coloured arrows. The gradient blue and red shading represent regions of nucleotide sequence identity (100% to 65%) in forward and reverse directions, respectively, determined by BLASTn analysis. The sequences are from strains top to bottom; PA198, PA219, PA202 and PA221. Figures are drawn to scale using Easyfig [34]. (Tn3-tnp = Tn3-transposase HP= hypothetical protein, ydhC= Inner membrane transport protein, tet(R)-tet(g) = tetracycline resistance genes, qnrVC1=quinolone resistance gene, VOC = VOC family protein, aph(6)-Id = aminoglycoside resistance protein, DNA-inv = DNA invertase, aadA10= aminoglycoside resistance protein, IS110= IS110 family transposase, qacEdealta1= quaternary ammonium compound-resistance protein, sul1= Dihydropteroate synthase, blaLCR-1=betalactamase gene, attC = recombination sites of gene cassette, attI = integron recombination site, IR=invert repeat).](/figures/figure-5-comparison-of-qnrvc1-associated-genomic-islands-of-1uogchkk.png)
Figure 5. Comparison of qnrVC1-associated genomic islands of P. aeruginosa strains. Protein-coding regions are represented by the arrows and common key features/associated genes among all strains are shown in various coloured arrows. The gradient blue and red shading represent regions of nucleotide sequence identity (100% to 65%) in forward and reverse directions, respectively, determined by BLASTn analysis. The sequences are from strains top to bottom; PA198, PA219, PA202 and PA221. Figures are drawn to scale using Easyfig [34]. (Tn3-tnp = Tn3-transposase HP= hypothetical protein, ydhC= Inner membrane transport protein, tet(R)-tet(g) = tetracycline resistance genes, qnrVC1=quinolone resistance gene, VOC = VOC family protein, aph(6)-Id = aminoglycoside resistance protein, DNA-inv = DNA invertase, aadA10= aminoglycoside resistance protein, IS110= IS110 family transposase, qacEdealta1= quaternary ammonium compound-resistance protein, sul1= Dihydropteroate synthase, blaLCR-1=betalactamase gene, attC = recombination sites of gene cassette, attI = integron recombination site, IR=invert repeat). 
Table 1: BLAST matrix showing nucleotide identity (in percentage) of crpP sequences of each isolate. Colour intensity corresponds to percentage identity. 
Figure 3. Codon adaptation index of crpP orthologues of P. aeruginosa strains. PA3742_rplS denotes 50s ribosomal protein L19 (rplS) of P. aeruginosa PAO1. ![Figure 1. Maximum likelihood phylogenetic tree based on core genome SNPs analysis using Pseudomonas aeruginosa PAO1 as the reference, excluding SNPs identified in regions that had arisen by recombination, using the default parameters of Parsnp v1.2 [43]. Isolates from India are labelled red and Australian isolates are labelled blue. Numbers given at the nodes represent bootstrap values. The presence of crpP, exoU, qnrVC1, and CRISPR cas are represented by red squares. Orange squares represent the presence of mutations in the quinolone resistance determining regions (QRDRs) and fluoroquinolone (CIP = Ciprofloxacin; LEVO = Levofloxacin; and MOX = Moxifloxacin) susceptibilities are shown as a heat map with the ranges indicated in the figure. The figure was drawn using iTol v4 [41].](/figures/figure-1-maximum-likelihood-phylogenetic-tree-based-on-core-twtxvxs9.png)
Figure 1. Maximum likelihood phylogenetic tree based on core genome SNPs analysis using Pseudomonas aeruginosa PAO1 as the reference, excluding SNPs identified in regions that had arisen by recombination, using the default parameters of Parsnp v1.2 [43]. Isolates from India are labelled red and Australian isolates are labelled blue. Numbers given at the nodes represent bootstrap values. The presence of crpP, exoU, qnrVC1, and CRISPR cas are represented by red squares. Orange squares represent the presence of mutations in the quinolone resistance determining regions (QRDRs) and fluoroquinolone (CIP = Ciprofloxacin; LEVO = Levofloxacin; and MOX = Moxifloxacin) susceptibilities are shown as a heat map with the ranges indicated in the figure. The figure was drawn using iTol v4 [41]. ![Figure 2. Consensus tree of 33 P. aeruginosa isolates, based on Bayesian evolutionary analysis by sampling trees (BEAST) of concatenated multi-locus sequence type (MLST) under strict clock analysis [40]. The tip of the tree was constrained by date of isolation. The time scale is shown in years at the top and each internal node is labelled with posterior probability limit. Isolates from India are labelled red and Australian isolates are labelled blue. The presence of genes crpP, exoU, qnrVC1, and CRISPRcas are represented by red squares. Orange square represents presence of mutations in quinolone resistance determining region (QRDRs), Fluoroquinolone (CIP = Ciprofloxacin; LEVO = Levofloxacin; and MOX = Moxifloxacin) susceptibilities are shown as heat maps in the grey scale indicated in the figure. The figure was drawn using iTol v4 [41].](/figures/figure-2-consensus-tree-of-33-p-aeruginosa-isolates-based-on-3rwqoqu2.png)
Figure 2. Consensus tree of 33 P. aeruginosa isolates, based on Bayesian evolutionary analysis by sampling trees (BEAST) of concatenated multi-locus sequence type (MLST) under strict clock analysis [40]. The tip of the tree was constrained by date of isolation. The time scale is shown in years at the top and each internal node is labelled with posterior probability limit. Isolates from India are labelled red and Australian isolates are labelled blue. The presence of genes crpP, exoU, qnrVC1, and CRISPRcas are represented by red squares. Orange square represents presence of mutations in quinolone resistance determining region (QRDRs), Fluoroquinolone (CIP = Ciprofloxacin; LEVO = Levofloxacin; and MOX = Moxifloxacin) susceptibilities are shown as heat maps in the grey scale indicated in the figure. The figure was drawn using iTol v4 [41].
![Figure 5. Comparison of qnrVC1-associated genomic islands of P. aeruginosa strains. Protein-coding regions are represented by the arrows and common key features/associated genes among all strains are shown in various coloured arrows. The gradient blue and red shading represent regions of nucleotide sequence identity (100% to 65%) in forward and reverse directions, respectively, determined by BLASTn analysis. The sequences are from strains top to bottom; PA198, PA219, PA202 and PA221. Figures are drawn to scale using Easyfig [34]. (Tn3-tnp = Tn3-transposase HP= hypothetical protein, ydhC= Inner membrane transport protein, tet(R)-tet(g) = tetracycline resistance genes, qnrVC1=quinolone resistance gene, VOC = VOC family protein, aph(6)-Id = aminoglycoside resistance protein, DNA-inv = DNA invertase, aadA10= aminoglycoside resistance protein, IS110= IS110 family transposase, qacEdealta1= quaternary ammonium compound-resistance protein, sul1= Dihydropteroate synthase, blaLCR-1=betalactamase gene, attC = recombination sites of gene cassette, attI = integron recombination site, IR=invert repeat).](/figures/figure-5-comparison-of-qnrvc1-associated-genomic-islands-of-1uogchkk.webp)
![Figure 1. Maximum likelihood phylogenetic tree based on core genome SNPs analysis using Pseudomonas aeruginosa PAO1 as the reference, excluding SNPs identified in regions that had arisen by recombination, using the default parameters of Parsnp v1.2 [43]. Isolates from India are labelled red and Australian isolates are labelled blue. Numbers given at the nodes represent bootstrap values. The presence of crpP, exoU, qnrVC1, and CRISPR cas are represented by red squares. Orange squares represent the presence of mutations in the quinolone resistance determining regions (QRDRs) and fluoroquinolone (CIP = Ciprofloxacin; LEVO = Levofloxacin; and MOX = Moxifloxacin) susceptibilities are shown as a heat map with the ranges indicated in the figure. The figure was drawn using iTol v4 [41].](/figures/figure-1-maximum-likelihood-phylogenetic-tree-based-on-core-twtxvxs9.webp)
![Figure 2. Consensus tree of 33 P. aeruginosa isolates, based on Bayesian evolutionary analysis by sampling trees (BEAST) of concatenated multi-locus sequence type (MLST) under strict clock analysis [40]. The tip of the tree was constrained by date of isolation. The time scale is shown in years at the top and each internal node is labelled with posterior probability limit. Isolates from India are labelled red and Australian isolates are labelled blue. The presence of genes crpP, exoU, qnrVC1, and CRISPRcas are represented by red squares. Orange square represents presence of mutations in quinolone resistance determining region (QRDRs), Fluoroquinolone (CIP = Ciprofloxacin; LEVO = Levofloxacin; and MOX = Moxifloxacin) susceptibilities are shown as heat maps in the grey scale indicated in the figure. The figure was drawn using iTol v4 [41].](/figures/figure-2-consensus-tree-of-33-p-aeruginosa-isolates-based-on-3rwqoqu2.webp)
Citations
SPAdes, a new genome assembly algorithm and its applications to single-cell sequencing ( 7th Annual SFAF Meeting, 2012)
Glenn Tesler
- 01 Jun 2012
TL;DR: SPAdes as mentioned in this paper is a new assembler for both single-cell and standard (multicell) assembly, and demonstrate that it improves on the recently released E+V-SC assembler and on popular assemblers Velvet and SoapDeNovo (for multicell data).
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Understanding clinical and immunological features associated with Pseudomonas and Staphylococcus keratitis.
TL;DR: The role of macrophage inflammatory protein-2 (MIP-2) and intercellular adhesion molecule (ICAM-1) in neutrophil recruitment based on both in-vivo scratch models of bacterial keratitis and bacterial challenged in cell culture models is discussed.
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Guaiacol augments quorum quenching potential of ciprofloxacin against Pseudomonas aeruginosa
TL;DR: The present study aims to investigate the antimicrobial as well as antivirulence potential and the principle mechanism of action of guaiacol against Pseudomonas aeruginosa.
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Antibiotic Recommendations for Treatment of Canine Stromal Corneal Ulcers
Milan Joksimovic,Bradley Ford,Tatjana Lazic,Ivan Soldatovic,Sergey Luzetsky,Sinisa D. Grozdanic +5 more
TL;DR: In this article , the authors identify the aerobic bacterial isolates and determine corresponding antibiotic susceptibility profiles in vitro in canine clinical specimens with stromal corneal ulcers, with the goal of providing recommendations for first-line treatment with antibiotics.
Patterns of antimicrobial resistance and metal tolerance in environmental Pseudomonas aeruginosa isolates and the genomic characterization of the rare O6/ST900 clone
Micaela Santana Ramos,João Pedro Rueda Furlan,Lucas David Rodrigues dos Santos,Rafael da Silva Rosa,Eduardo Angelino Savazzi,Eliana Guedes Stehling +5 more
TL;DR: Describing potentially pathogenic, antimicrobial-resistant, and/or metal-tolerant P. aeruginosa isolates from different environmental samples and performing a whole-genome sequence-based analysis of a rare clone from residual water revealed a classification of O6/ST900 clone as rare, potentially pathogenic, and predisposed to acquire multidrug resistance genes.
4
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