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Using a Galleria mellonella animal model, we compared the virulence of two sequence type 258 (ST258) KPC-producing Klebsiella pneumoniae strains, which were representative of the two clades of this clonal lineage, with that of isogenic colistin-resistant mgrB mutants. With both strains, the mgrB mutants did not exhibit modification in virulence. In the G. mellonella model, the clade 1 strain (capsular type cps-1 [ wzi29 , producing KPC-2]) was significantly more virulent than the clade 2 strain (capsular type cps-2 [ wzi154 , producing KPC-3]).

Colistin is a key drug for the treatment of infections caused by extensively drug-resistant strains of Enterobacteriaceae producing carbapenemases. However, the emergence of colistin resistance is being increasingly reported, especially among Klebsiella pneumoniae strains producing KPC-type carbapenemases (KPC-KP). In this work, we investigated colistin-susceptible (KPB-1) and colistin-resistant (KPB-2) sequential isolates obtained from a patient with a KPC-KP infection before and after low-dosage colistin treatment, respectively. By using a next-generation sequencing approach and comparative genomic analysis of the two isolates, we detected in KPB-2 a nonsynonymous nucleotide substitution in the gene encoding the PmrB sensor kinase, resulting in a leucine-to-arginine substitution at amino acid position 82. Compared with KPB-1, KPB-2 exhibited upregulated transcription of pmrA and of pmrK , which is part of the pmrHFIJKLM operon responsible for modification of the colistin lipopolysaccharide target. Complementation with wild-type pmrB in KPB-2 restored colistin susceptibility and reduced the transcription of pmrA and pmrK to basal levels, while expression of PmrB L82R in KPB-1 did not alter colistin susceptibility or upregulate pmrA and pmrK expression, confirming the dominance of wild-type PmrB versus the PmrB L82R mutant. The present results indicated that PmrB mutations mediating colistin resistance may be selected during low-dosage colistin treatment. The colistin-resistant phenotype of KPB-2 was stable for up to 50 generations in the absence of selective pressure and was not associated with a significant fitness cost in a competition experiment.

Susceptibility to several β-lactams and β-lactamase production was investigated in a collection of 20 strains of Pseudomonas otitidis , a new Pseudomonas species that has been recently recognized in association with otic infections in humans. All strains appeared to be susceptible to piperacillin, cefotaxime, ceftazidime, and aztreonam, while resistance or decreased susceptibility to carbapenems was occasionally observed. All strains were found to express metallo-β-lactamase (MBL) activity and to carry a new subclass B3 MBL gene, named bla POM , that appeared to be highly conserved in this species. P. otitidis , therefore, is the first example of a pathogenic Pseudomonas species endowed with a resident MBL. The POM-1 protein from P. otitidis type strain MCC10330 exhibits the closest similarity (60 to 64%) to the L1 MBL of Stenotrophomonas maltophilia . Expression in Escherichia coli and Pseudomonas aeruginosa revealed that, similar to L1 and other subclass B3 MBLs, POM-1 confers decreased susceptibility or resistance to carbapenems, penicillins, and cephalosporins but not to aztreonam. Expression of the POM MBL in P. otitidis is apparently constitutive and, in most strains, does not confer a carbapenem-resistant phenotype. However, a strong inoculum size effect was observed for carbapenem MICs, and carbapenem-resistant mutants could be readily selected upon exposure to imipenem, suggesting that carbapenem-based regimens should be considered with caution for P. otitidis infections.

We report multifocal detection (four different cities in northern Italy) of Proteus mirabilis isolates resistant to both oxyimino- and 7-α-methoxy-cephalosporins and producing a novel acquired AmpC-like β-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 IS Ecp1 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 β-lactamase in Proteus mirabilis from Italy and underscores the emergence of similar resistance determinants in the European setting.

The sequenced chromosome ofCaulobacter crescentusCB15 encodes a hypothetical protein that exhibits significant similarity (30 to 35% identical residues) to metallo-β-lactamases of subclass B3. An allelic variant of this gene (divergent by 3% of its nucleotides) was cloned inEscherichia colifromC. crescentustype strain DSM4727. Expression studies confirmed the metallo-β-lactamase activity of its product, CAU-1. The enzyme produced inE. coliwas purified by two ion-exchange chromatography steps. CAU-1 contains a 29-kDa polypeptide with an alkaline isoelectric pH (>9), and unlike the L1 enzyme ofStenotrophomonas maltophilia, the native form is monomeric. Kinetic analysis revealed a preferential activity toward penicillins, carbapenems, and narrow-spectrum cephalosporins, while oxyimino cephalosporins were poorly or not hydrolyzed. Affinities for the various β-lactams were poor overall (Kmvalues were always >100 μM and often >400 μM). The interaction with divalent ion chelators appeared to occur by a mechanism similar to that prevailing in other members of subclass B3. InC. crescentus, the CAU-1 enzyme is produced independently of β-lactam exposure and, interestingly, theblaCAUdeterminant is bracketed by three other genes, including two genes encoding enzymes involved in methionine biosynthesis and a gene encoding a putative transcriptional regulator, in an operon-like structure. The CAU-1 enzyme is the first example of a metallo-β-lactamase in a member of the α subdivision of the classProteobacteria.