Metallo-beta-lactamase production by Pseudomonas otitidis: a species-related trait

Abstract

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.

FIG. 1.

Tree showing the similarity of the POM-1 protein (GenBank/EMBL accession no. EU315252) with other known subclass B3 MBLs. The tree has been constructed using the Clustal2 program (http://www.ebi.ac.uk/tools/clustalw2). Other MBLs (Swiss-Prot accession nos.) are FEZ-1 from Fluoribacter gormanii (Q9K578), GOB-1 from Elizabethkingia meningoseptica (Q9RB00), CAR-1 from Erwinia carotovora (ECA2849), L1 from S. maltophilia (Q9RBQ3), THIN-B from Janthinobacterium lividum (Q9AEF9), CAU-1 from Caulobacter crescentus (Q8KKG1), and BJP-1 from Bradyrhizobium japonicum (Q89GW5).

FIG. 2.

(A) Genetic context of bla_POM-1 and (B) homologies of flanking regions with the genomes of other Pseudomonas species. The location of primers used for PCR amplification (Table 2) is shown by numbered arrows: 1, Pom-seq/F; 2, Pom-seq/R; 3, POTSEQ/F; 4, POM948/R; 5, CT1ft/F; 6, CT1Prom/R. The NCBI accession numbers for the Pseudomonas genome sequences are as follows: NC_002516.2 for P. aeruginosa PAO1, NC_007005.1 for P. syringae pv. syringae B728a, NC_008027.1 for P. entomophila L48, NC_002947.3 for P. putida KT22440, NC_009439.1 for P. mendocina ymp, NC_007492.2 for P. fluorescens Pf-01, and NC_009434.1 for P. stutzeri A1501. Colinearity of genes/operons is indicated by thick black lines; where no such lines are drawn, the homologues are present but located apart from each other on the chromosome.

FIG. 3.

Sequence alignment of the POM-1-like proteins from different P. otitidis strains. The conserved residues known to be involved in metal binding are indicated by gray shading. Identical residues (*), strongly similar residues (:), and weakly similar residues (.) are indicated below the sequences. Gaps introduced to optimize alignment are indicated by dashes.