Emergence of Metallo-β-Lactamase GIM-1 in a Clinical Isolate of Serratia marcescens

Metallo-␤-lactamases (MBLs) have rapidly disseminated worldwide among clinically important Gram-negative bacteria and have challenged the therapeutic use of ␤-lactam antibiotics, particularly carbapenems. The bla GIM-1 gene, encoding one such enzyme, was first discovered in a Pseudomonas aeruginosa isolate from 2002 and has more recently been reported in Enterobacteriaceae. Here, we present crystal structures of GIM-1 in the apo-zinc (metal-free), mono-zinc (where Cys221 was found to be oxidized), and di-zinc forms, providing nine independently refined views of the enzyme. GIM-1 is distinguished from related MBLs in possessing a narrower active-site groove defined by aromatic side chains (Trp228 and Tyr233) at positions normally occupied by hydrophilic residues in other MBLs. Our structures reveal considerable flexibility in two loops (loop 1, residues 60 to 66; loop 2, residues 223 to 242) adjacent to the active site, with open and closed conformations defined by alternative hydrogen-bonding patterns involving Trp228. We suggest that this capacity for rearrangement permits GIM-1 to hydrolyze a wide range of ␤-lactams in spite of possessing a more constrained active site. Our results highlight the structural diversity within the MBL enzyme family.

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confidence: 99%

Crystal Structures of Pseudomonas aeruginosa GIM-1: Active-Site Plasticity in Metallo-β-Lactamases

Borra

Samuelsen

Spencer

et al. 2013

“…Recently, GIM-1 has been identified in other bacterial species, such as Serratia marcescens (7), Enterobacter cloacae (8), and Acinetobacter pittii (9), indicating that it is transmitted on mobile genetic elements (6,7). A new variant of GIM-1 (GIM-2) with a single mutation, A290G, was recently discovered (10).…”

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confidence: 99%

Role of Residues W228 and Y233 in the Structure and Activity of Metallo-β-Lactamase GIM-1

Skagseth

Carlsen

Bjerga

et al. 2016

e Metallo-␤-lactamases (MBLs) hydrolyze virtually all ␤-lactam antibiotics, including penicillins, cephalosporins, and carbapenems. The worldwide emergence of antibiotic-resistant bacteria harboring MBLs poses an increasing clinical threat. The MBL German imipenemase-1 (GIM-1) possesses an active site that is narrower and more hydrophobic than the active sites of other MBLs. The GIM-1 active-site groove is shaped by the presence of the aromatic side chains of tryptophan at residue 228 and tyrosine at residue 233, positions where other MBLs harbor hydrophilic residues. To investigate the importance of these two residues, eight site-directed mutants of GIM-1, W228R/A/Y/S and Y233N/A/I/S, were generated and characterized using enzyme kinetics, thermostability assays, and determination of the MICs of representative ␤-lactams. The structures of selected mutants were obtained by X-ray crystallography, and their interactions with ␤-lactam substrates were modeled in silico. Steady-state kinetics revealed that both positions are important to GIM-1 activity but that the effects of individual mutations vary depending on the ␤-lactam substrate. Activity against type 1 substrates bearing electron-donating C-3/C-4 substituents (cefoxitin, meropenem) could be enhanced by mutations at position 228, whereas hydrolysis of type 2 substrates (benzylpenicillin, ampicillin, ceftazidime, imipenem) with methyl or positively charged substituents was favored by mutations at position 233. The crystal structures showed that mutations at position 228 or the Y233A variant alters the conformation of GIM-1 loop L1 rather than that of loop L3, on which the mutations are located. Taken together, these data show that point mutations at both positions 228 and 233 can influence the catalytic properties and the structure of GIM-1. Members of the carbapenem class of ␤-lactams are among the most important antimicrobial agents available for the treatment of serious bacterial infections (1). However, their use against Gram-negative bacteria is now threatened by the dissemination of carbapenemases, ␤-lactamases that hydrolyze the amide bond of the ␤-lactam ring, thereby inactivating carbapenems and other ␤-lactams (2). To date, only a few effective carbapenemase inhibitors have been available for clinical use. Enzymes with carbapenemase activity have been identified in multiple ␤-lactamase classes. ␤-Lactamases are divided into four classes, of which classes A, C, and D are serine enzymes that catalyze hydrolysis of the ␤-lactam through a serine-bound acyl intermediate, whereas class B ␤-lactamases, or metallo-␤-lactamases (MBLs), coordinate one or two zinc ions in the active site, which are essential for their enzymatic activity (1-3). The class B MBLs can be divided into four subclasses, according to their primary structure: B1a (e.g., VIM, IMP, DIM, and SPM), B1b (NDM), B2 (e.g., CphA), and B3 (e.g., L1 and AIM) (3). Currently, no clinically effective MBL inhibitor has been approved for use (4). Avibactam, the only clinically approved effective carbap...

“…mid-mediated ampC genes (bla ACC , bla CMY , bla DHA , bla FOX , bla MOX ) was performed for all isolates with reduced susceptibility or resistance to cefoxitin (zone diameter of Ͻ19 mm), and isolates with reduced susceptibility or resistance to ertapenem (zone diameter of Ͻ25 mm) or imipenem or meropenem (zone diameter of Ͻ22 mm) were tested for the presence of carbapenemase genes bla KPC-type , bla OXA-48-type , bla NDM-type , bla GIM-type , bla IMP-type , and bla VIM-type , as described recently (12,13). Additionally, isolates with reduced susceptibility or resistance to nalidixic acid (zone diameter of Ͻ19 mm) and/or ciprofloxacin (zone diameter of Ͻ22 mm) were screened for the presence of the plasmid-mediated quinolone resistance (PMQR) genes aac(6=)-Ib-cr, qnrA/B/C/D/S, and qepA (14).…”

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confidence: 99%

Extended-Spectrum-β-Lactamase-Producing Escherichia coli as Intestinal Colonizers in the German Community

Valenza¹,

Nickel²,

Pfeifer

et al. 2014

Self Cite

134

We determined the presence of extended-spectrum-␤-lactamase (ESBL)-producing Escherichia coli among 3,344 study participants from the German community. Intestinal colonization was detected in 211 persons (6.3%), without significant differences among the different age groups. The majority (95.2%) of isolates harbored CTX-M-type ESBL, with CTX-M-15 (46%) and CTX-M-1 (24.2%) as the most common types. The finding of ESBL producers and one isolate additionally producing carbapenemase OXA-244 indicates a risk of dissemination of resistant bacteria outside the hospitals. Escherichia coli strains producing extended-spectrum ␤-lactamases (ESBLs) represent a major threat among the increasing number of drug-resistant Gram-negative bacteria (1). Fecal carriage of ESBL-producing E. coli has been described worldwide in hospitals and the ambulatory care setting (2-6) as well as in the community (7-9). A recent study investigating healthy infection control personnel (n ϭ 231) in Germany revealed that 3.5% are carriers of ESBL-producing E. coli (10). In the present study, we determined the fecal carriage rate of ESBL-producing E. coli in a large sample of persons from the German community, including molecular analysis of the isolates.From October 2009 to November 2012, we collected 3,344 nonreplicate fecal samples from individuals living in seven different areas of Bavaria (Upper Bavaria, 29.2%; Central Franconia, 20.8%; Upper Franconia, 15.0%; Upper Palatinate, 12.0%; Swabia, 10.8%; Lower Franconia, 9.0%; Lower Bavaria, 3.1%), Germany. The median age of the study participants was 32.0 years (range, 0 to 98 years), with a male/female ratio of 0.96. All probands had at the time of investigation a close contact to patients with bacterial gastroenteritis and were subsequently screened for fecal carriage of intestinal bacterial pathogens by 74 local health authorities. However, they did not show any symptoms related to gastroenteritis, and intestinal bacterial pathogens were not detected in any of these study participants. All 3,344 fecal samples were investigated for the presence of ESBL-producing E. coli by inoculation on MacConkey agar supplemented with cefotaxime (1 mg/liter). Identification to the species level was performed using API E strips (bioMérieux, Nürtingen, Germany), and ESBL production was confirmed by the combined disc method (Mast Diagnostica, Rheinfeld, Germany) using cefotaxime and ceftazidime with and without clavulanic acid. Susceptibility testing for 18 antimicrobial substances (ampicillin, cefotaxime, ceftriaxone, ceftazidime, cefepime, cefpodoxime, cefoxitin, ertapenem, imipenem, meropenem, aztreonam, amikacin, gentamicin, tetracycline, chloramphenicol, ciprofloxacin, nalidixic acid, and trimethoprim-sulfamethoxazole) was performed by disc diffusion (Oxoid Ltd., Basingstoke, United Kingdom), and interpretation was done according to EUCAST (http://www.eucast.org/clinical _breakpoints) and CLSI criteria (nalidixic acid, tetracycline) (11). E. coli isolates with the ESBL phenotype were investigated for the pres...