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606. Identification and Characterization of HMB-2, a Novel Metallo-β-Lactamase in a Pseudomonas aeruginosa Isolate
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October 23 2019
Source: Open Forum Infect Dis. 2019; 6(Suppl 2):S283-S284
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Alternative Title:Open Forum Infect Dis
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Description:Background
Carbapenemases, a global health threat, are a diverse group of β-lactamases active against cephalosporins and carbapenems, which are often last resort treatments for multidrug-resistant gram-negative infections. The most common carbapenemases reported among Pseudomonas aeruginosa are metallo-β-lactamase (MBLs). We describe a novel MBL (designated HMB-2) identified in a P. aeruginosa isolate from a urine specimen collected in 2015 as part of CDC’s Emerging Infections Program.
Methods
We performed antimicrobial susceptibility testing (AST) by broth microdilution, real-time PCR to screen for common carbapenemases (IMP, KPC, NDM, VIM, and OXA-48), and modified carbapenem inactivation method (mCIM) to test for carbapenemase production. The isolate underwent whole-genome sequencing (WGS) using Illumina MiSeq and PacBio RS II (Pacific Biosciences) platforms. Long read sequences were polished using Quiver and corrected by Pilon utilizing Illumina reads. We further characterized a putative novel MBL identified in WGS data by amplifying and cloning the gene into the pCR2.1-TOPO II vector (Invitrogen), which was then sub-cloned into a pET21 expression vector (Sigma–Aldrich). The resulting hmb2+ pET21 plasmid was transformed into a susceptible Escherichia coli for AST, including the imipenem-EDTA method to confirm MBL activity.
Results
The isolate displayed resistance to carbapenems and demonstrated phenotypic carbapenemase activity (mCIM positive), but was negative for carbapenemase genes by PCR. WGS analyses identified a putative MBL gene located on the chromosome. The gene shared 98% DNA and protein sequence identity with an MBL reported in 2016 in a P. aeruginosa isolate from Germany (HMB-1) and thus was named hmb-2. The cloned hmb-2 gene conferred resistance to carbapenems (meropenem and ertapenem) and third-generation cephalosporins (cefotaxime and ceftazidime) in transformed E. coli. The Minimum Inhibitory Concentrationratio for the imipenem-EDTA method was ≥4.
Conclusion
A putative, novel β-lactamase gene, blaHMB-2, was identified and cloned. The imipenem-EDTA results indicated that HMB-2 is an MBL. This discovery underscores the important role WGS plays in identifying new mechanisms of antimicrobial resistance.
Disclosures
All authors: No reported disclosures.
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Pubmed Central ID:PMC6811285
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