CrpP Is a Novel Ciprofloxacin-Modifying Enzyme Encoded by the Pseudomonas aeruginosa pUM505 Plasmid

Abstract

The pUM505 plasmid, isolated from a clinical isolate, confers resistance to ciprofloxacin (CIP) when transferred into the standard strain PAO1. CIP is an antibiotic of the quinolone family that is used to treat infections. analysis, performed to identify CIP resistance genes, revealed that the 65-amino-acid product encoded by the gene in pUM505 displays 40% amino acid identity to the aminoglycoside phosphotransferase (an enzyme that phosphorylates and inactivates aminoglycoside antibiotics). We cloned (renamed, for iprofloxacinesistance rotein,lasmid encoded) into the pUCP20 shuttle vector. The resulting recombinant plasmid, pUC-, conferred resistance to CIP on strain J53-3, suggesting that this gene encodes a protein involved in CIP resistance. Using coupled enzymatic analysis, we determined that the activity of CrpP on CIP is ATP dependent, while little activity against norfloxacin was detected, suggesting that CIP may undergo phosphorylation. Using a recombinant His-tagged CrpP protein and liquid chromatography-tandem mass spectrometry, we also showed that CIP was phosphorylated prior to its degradation. Thus, our findings demonstrate that CrpP, encoded on the pUM505 plasmid, represents a new mechanism of CIP resistance in, which involves phosphorylation of the antibiotic.

Changes to the resistome ofPseudomonas aeruginosaclone ST308 associated with corneal infection over time

“…The presence of the plasmid related fluoroquinolone resistance gene qnrVC1 [23] and the recently reported plasmid related gene crpP [24] are also novel findings in the current study related to clonal ST308 P. aeruginosa isolates. All isolates contained the fluoroquinolone r esistance gene crpP , but this had not been identified as a potential plasmid related fluoroquinolone resistance gene prior to the publication of resistance genes of the 1997 isolates [6].…”

Quantifying the contribution of four resistance mechanisms to ciprofloxacin minimum inhibitory concentration inEscherichia coli: a systematic review

“…The most recently discovered ciprofloxacin resistance determinant in E. coli is crpP , a plasmid-mediated gene coding for a protein with the putative ability to phosphorylate certain fluoroquinolones such as ciprofloxacin. 43 crpP was first detected in a clinical isolate of Pseudomonas aeruginosa , but was shown to confer a 7.5 fold-change increase in ciprofloxacin MIC when conjugated to E. coli J53.…”

“…The very recent discovery of crpP suggests that more resistance determinants or resistance mechanisms are still waiting to be discovered. 43 Additionally, complex mutation patterns influencing ciprofloxacin resistance through unknown pathways may exist, but current research methods do not usually detect these kinds of effects.…”

HypermutatorPseudomonas aeruginosaexploits multiple genetic pathways to develop multidrug resistance during long-term infections in the airways of cystic fibrosis patients

“…Nevertheless, the fact that many different mutations arose after fluoroquinolone treatments supports the previous observation that mutations involved in fluoroquinolone resistance can be highly variable (28). Importantly, with the use of the ResFinder tool we found the acquisition of a novel plasmid-encoded ciprofloxacin-modifying gene encoding the enzyme CrpP (65), which may explain the high-resistance profile observed in the two intermediate isolates from 1995 and 2002, and all isolates from 2011.…”