Novel Ciprofloxacin-Modifying Enzyme (CrpP) Encoded by the Pseudomonas aeruginosa pUM505 Plasmid [PublishAheadOfPrint]

The pUM505 plasmid, isolated from a clinical Pseudomonas aeruginosa isolate, confers resistance to ciprofloxacin (CIP) when transferred into the standard P. aeruginosa PAO1 strain. CIP is an antibiotic of the quinolone family that is used to treat P. aeruginosa infections. In silico analysis, performed to identify CIP-resistance genes, revealed that the 65-amino acid product encoded by the orf131 gene in pUM505 displays 40% amino acid identity to the Mycobacterium smegmatis aminoglycoside phosphotransferase (an enzyme that phosphorylates and inactivates aminoglycoside antibiotics). We cloned orf131 (renamed as crpP for ciprofloxacin-resistance protein plasmid-encoded) into the pUCP20 shuttle vector. The resulting recombinant plasmid, pUC-crpP, conferred resistance to CIP in the Escherichia coli J53-3 strain, suggesting that the product of this gene encodes a protein involved in CIP resistance. Using a coupled enzymatic analysis, we determined that the activity of CrpP on CIP is ATP-dependent, while little activity was detected towards norfloxacin, 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 P. aeruginosa, which involves phosphorylation of the antibiotic.

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