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Sir, Metallo-b-lactamase (MBL) is an important resistance determinant among Gram-negative bacteria, and its clinical relevance is increasing. Some MBL genes are carried on mobile gene elements that have spread among various clinically important bacterial species. Here we report a case of a novel MBL-positive Pseudomonas alcaligenes strain, MRY13-0052, that caused a bloodstream infection in a medical institution in Japan. P. alcaligenes is a Gram-negative aerobic bacillus belonging to the bacterial family Pseudomonadaceae, the members of which are common inhabitants of soil and water, and it is a rare opportunistic human pathogen. However, little is known about the clinical importance of P. alcaligenes, mainly because of the difficulties in identifying and distinguishing this bacterium from closely related Pseudomonas species, such as Pseudomonas aeruginosa, Pseudomonas mendocina and Pseudomonas pseudoalcaligenes, in medical settings. We report here our investigation of the draft genome sequence of P. alcaligenes strain MRY13-0052 and our finding that this strain contains a subclass B3 MBL, PAM-1 (P. alcaligenes MBL-1), that can hydrolyse cephalosporins and carbapenems. In 2012, Pseudomonas strain MRY13-0052 was recovered from a blood sample of a patient who was receiving therapy for Guillain–Barre syndrome. The patient had no recent history of travel abroad. Although the primary site of infection was unknown, the patient became afebrile soon after combination therapy with ceftazidime and clindamycin. The MRY13-0052 strain was identified as P. mendocina by the VITEK2 system (bioMerieux; 96% probability), but subsequently as P. alcaligenes based on 16S rRNA gene sequence analysis. MRY13-0052 was resistant to penicillins, cephalosporins, aztreonam and fosfomycin, but susceptible to imipenem, meropenem, amikacin, fluoroquinolones, minocycline and trimethoprim/sulfamethoxazole, according to MICs determined using the VITEK2 system and the Etest (bioMerieux), applying the recommended breakpoints described by CLSI (2013). The production of MBL was screened for using a disc containing sodium mercaptoacetic acid (SMA) (Eiken). Apparent expansion of the growth inhibitory zone around the ceftazidime and meropenem discs was observed around the SMA disc following overnight incubation at 378C, strongly suggesting that MRY13-0052 produces MBL. PCR tests to detect the blaNDM, blaIMP, blaVIM and blaTMB genes in MRY13-0052 were all negative; therefore, we analysed the whole-genome shotgun (WGS) sequence of MRY13-0052, obtained using the GS Junior system (Roche), to identify the responsible MBL gene (DDBJ/EMBL/GenBank accession number of the WGS project: BATO01). BLAST-based similarity searches revealed that MRY13-0052 carries three class C b-lactamase genes and a novel subclass B3 MBL gene [which we named blaPAM-1 (DDBJ/EMBL/GenBank accession number of the gene: AB858498)] that might confer resistance to b-lactams. The PCR product of the blaPAM-1 gene was ligated into pUCP19 (ATCC), a Pseudomonas–Escherichia shuttle vector, resulting in the PAM-1 expression vector pUCP19-blaPAM-1. P. aeruginosa strain PAO1 and Escherichia coli strain MC1061 were transformed with this vector, and transformants were selected on agar plates containing 20 mg/L piperacillin. Expression of the blaPAM-1 gene was driven by the tac promoter regardless of IPTG induction and confirmed by SMA disc-mediated expansion of the growth inhibitory zone around ceftazidime and meropenem discs. As shown in Table 1, blaPAM-1-producing P. aeruginosa bacteria were more resistant to ceftazidime, imipenem, meropenem and doripenem than control bacteria harbouring the empty vector (MICs increased 32-fold, 2-fold, 6-fold and 21-fold, respectively), but were still as susceptible as control bacteria to aztreonam. Although blaPAM-1producing E. coli bacteria were slightly more resistant to ceftazidime and meropenem than control bacteria (MICs increased 4-fold and 1.4-fold), there was no apparent change in the susceptibility to aztreonam and other carbapenems. The differences in the contribution of the PAM-1 enzyme to cephalosporin and carbapenem resistance among P. aeruginosa and E. coli could reflect differences in expression levels, outer-membrane permeability and/or efflux systems in these hosts. The blaPAM-1 gene in P. alcaligenes strain MRY13-0052 is encoded in contig 73, which is part of the chromosome, and there is no transposable element, such as a transposon or integron, around the gene, suggesting that blaPAM-1 is an intrinsic species-specific MBL gene of P. alcaligenes. Pseudomonas otitidis, a Pseudomonas species that is associated with otic infections in humans, also produces a resident MBL named POM-1 (P. otitidis MBL-1), which is active against carbapenems. The PAM-1 protein exhibits close similarity to POM-1 (72.4% amino acid identity), suggesting that these enzymes have a common ancestor (Figure 1). PAM-1 and POM-1 are homologous with the L1 MBL of Stenotrophomonas maltophilia (63.3% and 62.1% identity,

A subclass B3 metallo- -lactamase found in Pseudomonas alcaligenes