Prevalence of the crpP gene conferring decreased ciprofloxacin susceptibility in enterobacterial clinical isolates from Mexican hospitals

Objectives: This study investigated the presence of the crpP gene, which encodes an enzymatic mechanism of antibiotic phosphorylation that decreases ciprofloxacin susceptibility, in ESBL-producing clinical isolates and its effect in transconjugants. Methods: A collection of 77 ESBL-producing clinical isolates of Enterobacteriaceae and 68 ESBL-producing transconjugants that had acquired plasmids from clinical isolates from hospitals in Mexico obtained from 1988 to 2012 was employed. The crpP homologue genes were identified by dot-blot and PCR assays; five of them were sequenced and an in silico analysis was conducted. Expression of CrpP proteins was determined by western blot assays using antibodies against CrpP from plasmid pUM505. Three crpP homologue genes were cloned and transferred to Escherichia coli J53-3 as recipient strain. Results: The crpP gene was identified in four (5.19%) ESBL-producing isolates and five (7.35%) ESBL-producing transconjugants with plasmids from clinical isolates. Analysis of the deduced amino acid (aa) sequence of the CrpP protein homologues revealed that they all corresponded to small proteins (63-70 aa) with an identity of 10.1%-43.7% with respect to the pUM505 CrpP sequence. In addition, all crpP-positive transconjugants expressed a CrpP protein. Finally, transfer of crpP homologues conferred lower ciprofloxacin susceptibility to E. coli. Conclusions: These findings indicate the presence of crpP genes among ESBL-producing isolates from Mexican hospitals and point to widespread crpP-type genes in old Enterobacteriaceae clinical isolates (from 1994). CrpP probably confers resistance by means of the phosphorylation of ciprofloxacin.