Timothy R. Walsh

Metallo-β-Lactamases: the Quiet before the Storm?

SUMMARY The ascendancy of metallo-β-lactamases within the clinical sector, while not ubiquitous, has nonetheless been dramatic; some reports indicate that nearly 30% of imipenem-resistant Pseudomonas aeruginosa strains possess a metallo-β-lactamase. Acquisition of a metallo-β-lactamase gene will invariably mediate broad-spectrum β-lactam resistance in P. aeruginosa, but the level of in vitro resistance in Acinetobacter spp. and Enterobacteriaceae is less dependable. Their clinical significance is further embellished by their ability to hydrolyze all β-lactams and by the fact that there is currently no clinical inhibitor, nor is there likely to be for the foreseeable future. The genes encoding metallo-β-lactamases are often procured by class 1 (sometimes class 3) integrons, which, in turn, are embedded in transposons, resulting in a highly transmissible genetic apparatus. Moreover, other gene cassettes within the integrons often confer resistance to aminoglycosides, precluding their use as an alternative treatment. Thus far, the metallo-β-lactamases encoded on transferable genes include IMP, VIM, SPM, and GIM and have been reported from 28 countries. Their rapid dissemination is worrisome and necessitates the implementation of not just surveillance studies but also metallo-β-lactamase inhibitor studies securing the longevity of important anti-infectives.

Use of antimicrobial agents in veterinary medicine and food animal production.

Antimicrobial resistance is a growing area of concern in both human and veterinary medicine. This review presents an overview of the use of antimicrobial agents in animals for therapeutic, metaphylactic, prophylactic and growth promotion purposes. In addition, factors favouring resistance development and transfer of resistance genes between different bacteria, as well as transfer of resistant bacteria between different hosts, are described with particular reference to the role of animals as a reservoir of resistance genes or resistant bacterial pathogens which may cause diseases in humans.

blaVIM-7, an Evolutionarily Distinct Metallo-β-Lactamase Gene in a Pseudomonas aeruginosa Isolate from the United States

ABSTRACT As part of the CANCER Antimicrobial Surveillance Program in North America, a Pseudomonas aeruginosa isolate, strain 07-406, was shown to possess a metallo-β-lactamase, designated VIM-7. blaVIM-7 is located on a 24-kb plasmid which can be readily transferred into Enterobacteriaceae and other pseudomonads. This is the first report of a mobile metallo-β-lactamase gene, blaVIM-7, being detected within the United States.

Sequence analysis of the L1 metallo-β-lactamase from Xanthomonas maltophilia

The amino acid sequence deduced from the L1 β-lactamase gene of Xanthomonas maltophilia shows a significant variation from that of the CphA and Blm metallo-β-lactamases of Aeromonas hydrophila and Bacillus cereus, respectively. Whilst the N-terminus of the L1 protein shows some similarity, particularly at the histidine residues previously suggested as a zinc-binding motif, the C-terminus of the protein demonstrates very little similarity. Such differences amongst this group of enzymes would argue for at least three subclasses within the Group 3 β-lactamases. However, in order to predict their phylogenetic ancestry more sequence data are required from other possible metallo-β-lactamases.

Overexpression, Purification, and Characterization of the Cloned Metallo-β-Lactamase L1 from Stenotrophomonas maltophilia

ABSTRACT The metallo-β-lactamase L1 from Stenotrophomonas maltophilia was cloned, overexpressed, and characterized by spectrometric and biochemical techniques. Results of metal analyses were consistent with the cloned enzyme having 2 mol of tightly bound Zn(II) per monomer. Gel filtration chromatography demonstrated that the cloned enzyme exists as a tightly held tetramer with a molecular mass of ca. 115 kDa, and matrix-assisted laser desorption ionization and time-of-flight mass spectrometry indicated a monomeric molecular mass of 28.8 kDa. Steady-state kinetic studies with a number of diverse penicillin and cephalosporin antibiotics demonstrated that L1 effectively hydrolyzes all tested compounds, withkcat/Km values ranging between 0.002 and 5.5 μM−1 s−1. These characteristics of the recombinant enzyme are contrasted to those previously reported for metallo-β-lactamases isolated directly fromS. maltophilia.

Emerging Metallo-β-Lactamase-Mediated Resistances: A Summary Report from the Worldwide SENTRY Antimicrobial Surveillance Program

The rates of occurrence of metallo-beta-lactamase-mediated resistances in Pseudomonas aeruginosa, Acinetobacter species, and Serratia marcescens, among other gram-negative bacilli, have escalated since 2000, severely limiting treatment options in Asia, Europe, and Latin America to non-beta-lactam antimicrobial classes. Clinical isolates harboring metallo-beta-lactamases have also recently been reported in western Canada and in Texas, signaling the need for development of accurate diagnostic tests by clinical laboratories to detect their presence and for new, and more potent, antimicrobial agents.

Molecular and Biochemical Characterization of OXA-45, an Extended-Spectrum Class 2d′ β-Lactamase in Pseudomonas aeruginosa

ABSTRACT As part of the CANCER Antimicrobial Surveillance Program in North America, a clinical strain of Pseudomonas aeruginosa, strain 07-406, isolated in Texas was found to be resistant to all antimicrobials except polymyxin B. Genetic analysis of this isolate identified two unique extended-spectrum β-lactamase genes. One, blaVIM-7, encoded a metallo-β-lactamase (unpublished data), and the other, blaOXA-45, described here, encoded a class D extended-spectrum β-lactamase. blaOXA-45 was isolated on a Sau3A1 genomic fragment of 1.8 kb and encodes a protein of 264 amino acids with the highest identities to OXA-18 (65.9%), OXA-9 (42.8%), OXA-22 (40.2%), OXA-12 (38.6%), and OXA-29 (35.2%) but weak identities with other class D β-lactamases. blaOXA-45 was found to be harbored on a 24-kb plasmid in a region that displays high identities with a section of the 43-kb genomic island of Salmonella enterica serovar Typhimurium DT104. Biochemically OXA-45 is most similar to OXA-18 in its substrate profile and inhibition by clavulanic acid and is a member of the 2d′ class of β-lactamases.

Nosocomial outbreak of CTX-M-15-producing E. coli in Norway.

Seven E. coli isolates expressing resistance to 3rd generation cephalosporins were recovered from blood (n=2), kidney and lung tissue (n=1), and urinary tract (n=4) samples from seven patients hospitalised or recently discharged from the Divisions of Geriatrics and Pulmonary Medicine, Central Hospital of Rogaland, between July and September 2004. All isolates expressed a typical ESBL‐cefotaximase profile (cefotaxime MIC>ceftazidime MIC) with clavulanic acid synergy. A blaCTX‐M‐15 genotype was confirmed in six strains that were coresistant to gentamicin, nitrofurantoin, trimethoprim‐sulfamethoxazole and ciprofloxacin. A blaCTX‐M‐3 genotype was detected in the last strain. XbaI‐PFGE patterns of the six blaCTX‐M‐15 isolates revealed a clonal relationship. BlaCTX‐M‐15 strains were also positive for the ISEcp1‐like insertion sequences that have been shown to be involved in the mobilization of blaCTX‐M. Further analyses revealed two blaCTX‐M‐15‐positive E. coli urinary isolates clonally related to the outbreak strain from two different patients at the same divisions in January and February 2004. These patients were later re‐hospitalised and one had E. coli with an ESBL‐cefotaximase profile in sputum and nasopharyngeal specimen during the outbreak period. Clinical evaluation suggests that the CTX‐M‐producing E. coli strains contributed to death in three patients due to delayed efficient antimicrobial therapy. The outbreak emphasises the epidemic potential of multiple‐antibiotic‐resistant CTX‐M‐15‐producing E. coli also in a country with low antibiotic usage and low prevalence of antimicrobial resistance.

A novel metallo-β-lactamase, Mbl1b, produced by the environmental bacterium Caulobacter crescentus1

Caulobacter crescentus 101123 possesses a gene (Mbl1b) encoding a metallo‐β‐lactamase with 32% amino acid identity to the L1 metallo‐β‐lactamase from Stenotrophomonas maltophilia. The gene was cloned into an expression vector and the enzyme, Mbl1b, was expressed in Escherichia coli. Mbl1b was purified. Catalytic properties for several antibiotics were determined. The enzyme exhibits Michaelis–Menten kinetics for imipenem, meropenem and nitrocefin but substrate inhibition kinetics with cefoxitin, cefaloridine, penicillin G and ampicillin. A homology model predicts Mbl1b has the same structural fold as other metallo‐β‐lactamases with a detailed structure very similar to L1 but whereas L1 is a homotetramer, Mbl1b is monomeric. The main differences between Mbl1 and L1 are in the N‐terminal region.

CorrespondenceGlobal spread of New Delhi metallo-β-lactamase 1

Karthikeyan Kumarasamy and colleagues express concern about the emergence of New Delhi metallo-βlactamase (NDM-1) in enterobacteria in India, Pakistan, and the UK. One of the fi rst acquired metallo-βlactamase-producing enterobacteria reported was an IMP-1-producing Klebsiella pneumoniae isolated in Singapore in 1996. Since then we have undertaken hospital surveillance for carbapenem-resistant enterobacteria. Until 2009, the only other metallo-β-lactamase enterobacteria we isolated was another unrelated IMP-1-producing K pneumoniae in 2004. In early 2010, we isolated two K pneumoniae with a high level of resistance (determined by Etest; bioMerieux, Marcy l’Etoile, France) to many antibiotics including to carbapenems (table). The fi rst—DU1301/10—was from the urine of a patient who had just returned from a 5-month stay in India where he had an indwelling catheter inserted. The second isolate, DU7433/10, was from THK, CTK, and TYK did the experimental work to identify the β-lactamase genes, and did the typing studies. LW, YLL, HNL, and LCL managed and provided background information about the two cases. All authors were involved in writing the letter. We declare that we have no confl icts of interest.