Acquired fluoroquinolone resistance genes in corneal isolates of Pseudomonas aeruginosa
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.
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.
Summary (2 min read)
- Pseudomonas aeruginosa is a highly adaptable opportunistic pathogen which is ubiquitously present in the environment.
- This bacterium is naturally resistant to many antimicrobials and can acquire antibiotic resistance through mutations in chromosomal genes and lateral gene transfer [1, 2].
- Fluoroquinolone resistance in various clinical isolates is on the rise .
- These studies were based on a single strain and the prevalence of acquired fluoroquinolone resistance genes in P. aeruginosa remains unclear.
Materials and Methods
- Pseudomonas aeruginosa strains Isolates in this study were collected from the cornea of microbial keratitis patients in India and Australia between 1992 and 2018.
- Information on antibiotic susceptibility and the whole genome data of 13 isolates was obtained from their previous study  (Supplementary Table 1).
- DNA was sequenced on MiSeq (Illumina, San Diego, CA, USA) generating 300 bp paired end reads.
- The core genome SNPs were used to construct a maximum likelihood phylogenetic tree.
- Long reads were basecalled using Guppy v3.3.0 and adapters removed using Porechop v0.2.4.
- Population structure, phylogeny and fluoroquinolone resistance of P. aeruginosa strains Draft genomes were mapped against the reference genome P. aeruginosa PAO1 and a total of 202,232 SNPs were observed among the 33 isolates, which were used to construct core genome phylogeny using Parsnp v1.2 .
- The detection rate of exoU was 68% in Indian isolates and 36% in Australian isolates.
- In addition, four strains from the latter cohort of 11 strains carried another fluoroquinolone resistance gene, qnrVC1, in combination with mutations in gyrA and parC, and this was associated with a very high MIC (>128 µg/mL) to all three fluoroquinolones (Fig 1).
- Only one of the exoU, and none of the qnrVC1 carrying strains, were positive for CRISPR-Cas genes.
Bayesian phylogenetic reconstruction
- To examine the evolutionary trends of isolates, a Bayesian analysis was performed based on MLST sequences.
- This study also investigated the presence of the crpP homologues in NCBI databases using local BLAST with a cut off of 80% coverage and 90% sequence identity to examine the distribution of crpP gene in the bacterial database including plasmids as of 2019-06-15.
- Protein-coding regions are represented by the orange arrows and common key features/associated genes among all strains are shown in various coloured arrows.
- This suggests that the qnrVC1 gene is prevalent in various bacterial families.
- Integration of VOC with other antibiotic resistance genes leads us to hypothesise that VOC can be associated with antibiotic resistance.
- While fluoroquinolones are the preferred empirical therapy for corneal ulcers, which are often caused by P. aeruginosa, increasing resistance in this bacterium raises the concern about the efficacy of these antibiotics [4-11, 59, 60].
- This result, together with finding of high divergence in crpP orthologues between strains and lower CAI compared to a highly expressed chromosomal gene (rlpS)  suggests that acquisition of the crpP gene was a relatively old evolutionary event in P. aeruginosa.
- The acquired fluoroquinolone resistance gene qnrVC1 was observed in isolates sampled in 2017 and 2018, indicating that transferable fluoroquinolone resistance genes may be recently acquired or passed unnoticed in ocular isolates of P. aeruginosa.
- Integration of several other antibiotic resistance genes in these mobile genetic elements suggests these elements may concentrate antibiotic resistance genes.
- Given that qnrVC1 has been reported as being responsible for lowlevel fluoroquinolone resistance , the high MICs observed for these isolates is not clear but may indicate some synergistic activity between the QRDR mutations and qnrVC1.
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