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