Molecular characterisation of acquired and overproduced chromosomal blaAmpC in Escherichia coli clinical isolates.

Abstract Escherichia coli recovered from three hospitals in Barcelona (Spain) were studied to determine the prevalence of isolates with acquired AmpC (ac-AmpC) and/or overproduced chromosomal AmpC (c-AmpC). Mechanisms involved in bla c-AmpC overexpression, bla ac-AmpC and the plasmids associated with their distribution as well as the prevalence of plasmid-mediated quinolone resistance (PMQR) in AmpC-producing isolates were also determined. Isolates were selected according to their resistance phenotype. bla ac-AmpC , alterations in the bla c-AmpC promoter/attenuator, and PMQR genes [ qnrA , qnrB , qnrS , aac(6′)-Ib -cr and qepA ] were characterised by PCR and sequencing. bla c-AmpC expression was determined by qRT-PCR. Population structure analysis was performed using PFGE, MLST and phylogenetic group PCR. Plasmids carrying bla ac-AmpC were characterised by PCR-based replicon typing and S1-PFGE. IncI1 and IncF plasmids were also analysed by plasmid MLST and replicon sequence typing, respectively. Among 21563 E. coli isolates, 240 (1.1%) overproduced AmpC β-lactamases, including 180 (75.0%) harbouring ac-AmpC (132 CMY-2 variants and 48 DHA-1) and 60 (25.0%) c-AmpC enzymes. Three mutation profiles in the bla c-AmpC promoter/attenuator were associated with a 72.5-, 19.9- and 5.8-fold increased expression, respectively. Moreover, 63.3% of ac-AmpC and 43.3% of c-AmpC isolates belonged to B2, D, E or F phylogenetic groups. PMQR was found in 31% of ac-AmpC isolates [38 qnrB4 , 8 aac(6′)-Ib -cr, 6 qnrS1 and 3 qnrB19 ] and in 10% of c-AmpC isolates [5 aac(6′)-Ib -cr and 1 qnrS1 ]. IncI1-ST12 and IncF were associated with bla CMY-2 and bla DHA-1 , respectively. These results suggest that ac-AmpC β-lactamases were the main mechanism of AmpC production. Isolates and plasmids both showed high genetic diversity.