Prevalence of the quinolone-modifying enzyme aac(6')-Ib-cr in extended-spectrum b-lactamase-producing enterobacterial isolates in Barcelona

Abstract number: P1523

Lavilla S., González-López J.J., Larrosa M.N., Bartolomé R.M., Prat G.

Background: Quinolone resistance usually results from mutations in chromosomal genes coding for type II topoisomerases, efflux pumps, and/or porins alterations. Recently, plasmid-mediated quinolone resistance mechanisms has been reported by the qnr and the aac(6')-Ib-cr genes.

Objective: To evaluate the presence of aac(6')-Ib-cr among enterobacterial isolates carrying ESBLs in Barcelona, determine the ESBL present in each isolate and establish if the aac(6')-Ib-cr and the ESBL genes were located in the same plasmid.

Methods: A total of 305 non-duplicate clinically relevant ESBL-producing enterobacterial isolates, obtained at Hospital Vall d'Hebron (Barcelona), collected between 2003 and 2004 were analysed for the presence of aac(6')-Ib. Antimicrobial susceptibility was tested by Etest and disc diffusion. The screening for the presence of aac(6')-Ib was studied by PCR using specific primers. Positive amplicons were digested with BstCI and sequenced to identify the aac(6')-Ib-cr variant. ESBLs of all aac(6')-Ib-cr-positive isolates were determined by IEF and PCR using specific primers. The quinolonone-resistance-determining region (QRDR) of gyrA and parC genes was amplified and sequenced. Plasmid number and location of aac(6')-Ib-cr and bla genes was performed on all aac(6')-Ib-cr-positive isolates by S1 nuclease digestion and hybridisation with specific probes.

Results: Nineteen isolates (6.2%) carried aac(6')-Ib (8 Escherichia coli, 7 Klebsiella pneumoniae, 3 Enterobacter cloacae and 1 Klebsiella oxytoca). Of these, 6 isolates (31.6%) carried the aac(6')-Ib-cr variant (5 E. coli and 1 K. pneumoniae). Among the 6 aac(6')-Ib-cr-positive isolates 4 were positive for CTX-M-1 group. The ESBLs of the remaining two isolates are currently been determined. The 5 aac(6')-Ib-cr-positive E. coli isolates were resistant to nalidixic acid and ciprofloxacin and the K. pneumoniae aac(6')-Ib-cr-positive isolate was susceptible. Sequencing of the QRDR of the gyrA and parC genes identified amino acid changes in gyrA (S83-L and D87-N) and parC (S80-I and E84-V) on the 5 E. coli isolates. In the K. pneumoniae isolate no aminoacid changes were detected. The 4 aac(6')-Ib-cr-positive isolates positive for CTX-M-1 group harboured the ESBL gene and aac(6')-Ib-cr located in the same plasmid.

Conclusion: The prevalence of aac(6')-Ib-cr, responsible for low-level quinolone resistance, among enterobacterial clinical isolates carrying ESBLs between 2003 and 2004 in Barcelona was 1.9%.