CMY-16, a novel acquired AmpC-type beta-lactamase of the CMY/LAT lineage in multifocal monophyletic isolates of Proteus mirabilis from northern Italy

Abstract

We report multifocal detection (four different cities in northern Italy) of Proteus mirabilis isolates resistant to both oxyimino- and 7-alpha-methoxy-cephalosporins and producing a novel acquired AmpC-like beta-lactamase. The enzyme, named CMY-16, is a variant of the CMY/LAT lineage, which differs from the closest homologues, CMY-4 and CMY-12, by a single amino acid substitution (A171S or N363S, respectively) and from CMY-2 by two substitutions (A171S and W221R). Expression of the cloned bla(CMY-16) gene in Escherichia coli decreased susceptibility to penicillins, cephalosporins, and aztreonam. Tazobactam was more effective than clavulanate at antagonizing the enzyme activity. Genotyping, by random amplification of polymorphic DNA and pulsed-field gel electrophoresis of genomic DNA digested with SfiI, showed that isolates were clonally related to each other, although not identical. The bla(CMY-16) gene was not transferable to E. coli by conjugation or transformation. In all isolates, it was chromosomally located and inserted in a conserved genetic environment. PCR mapping experiments revealed that the bla(CMY-16) was flanked by ISEcp1 and the blc gene, similar to other genes of this lineage from plasmids of Salmonella enterica, Klebsiella spp., and E. coli. Overall, these results revealed multifocal spreading of a CMY-16-producing P. mirabilis clone in northern Italy. This finding represents the first report of an acquired AmpC-like beta-lactamase in Proteus mirabilis from Italy and underscores the emergence of similar resistance determinants in the European setting.

FIG. 1.

RAPD fingerprinting of isolates investigated in this work, carried out with primer 1254 (A) or AP12h (B). Lanes 1, BG-073/03; lanes 2, NO-080/03; lanes 3, VA-1017/03; lanes 4, NO-051/03; lanes 5, PM207RED; lanes 6, PM208RED; lanes 7, PM209RED; lanes 8, PM210RED. DNA size standards are indicated in bp on the left.

FIG. 2.

PFGE profiles of genomic DNAs of the eight P. mirabilis isolates producing CMY-16 after digestion with SfiI. Lane 1, PM207RED; lane 2, PM208RED; lane 3, PM209RED; lane 4, PM210RED; lane 5, NO-051/03; lane 6, VA-1017/03; lane 7, NO-080/03; lane 8, BG-073/03. DNA size standards, expressed in kbp, are indicated on the left.

FIG. 3.

Lanes 1 to 9, results of Southern blot hybridization of genomic DNAs of the eight P. mirabilis isolates producing CMY-16 digested with XbaI and hybridized to the bla_CMY-2 probe. Lane 1, PM207RED; lane 2, PM208RED; lane 3, PM209RED; lane 4, PM210RED; lane 5, BG-073/03; lane 6, NO-080/03; lane 7, NO-051/03; lane 8, VA-1017/03. DNA size standards, expressed in kbp, are indicated on the left. The hybridization signal obtained with undigested genomic DNA of one isolate (VA-1017/03) and the corresponding agarose gel electrophoresis images are shown in lanes 9 and 10.

FIG. 4.

PFGE profiles of genomic DNAs of the eight P. mirabilis isolates after digestion with I-CeuI (left) and Southern blot analysis of the same gel following hybridization with the bla_CMY-16 probe (right). All bands visible in the gel and an additional band of smaller size (indicated by the asterisk) were recognized by the 16S rRNA gene probe. Lanes 1, PM207RED; lanes 2, PM208RED; lanes 3, PM209RED; lanes 4, PM210RED; lanes 5, NO-051/03; lanes 6, NO-080/03; lanes 7, BG-073/03; lanes 8, VA-1017/03. DNA size standards, expressed in kbp, are indicated on the left.