Beta-Lactamase Database (BLDB) is a comprehensive, manually curated public resource providing up-to- date structural and functional information focused on this superfamily of enzymes with a great impact on antibiotic resistance. All the enzymes reported and characterised in the literature are presented according to the class (A, B, C and D), family and subfamily to which they belong. All three-dimensional structures of b-lactamases present in the Protein Data Bank are also shown. The characterisation of representative mutants and hydrolytic profiles (kinetics) completes the picture and altogether these four elements consti- tute the essential foundation for a better understanding of the structure-function relationship within this enzymes family. BLDB can be queried using different protein- and nucleotide-based BLAST searches, which represents a key feature of particular importance in the context of the surveillance of the evolution of the antibiotic resistance. BLDB is available online at http://bldb.eu without any registration and supports all modern browsers.
The production of extended-spectrum beta-lactamase in Enterobacteriaceae has been associated with increased treatment failure and higher management costs.
The prevalence of extended-spectrum beta-lactamase producing Escherichia coli in urinary samples from outpatients has increased significantly in France to reach 3.3%.
EMERGENCE DE SOUCHES DE SALMONELLA VIRCHOW MULTIRESITANTES ET
PRODUCTRICES D’UNE BETA-LACTAMASE A SPECTRE ETENDU EN BELGIQUE EN 2003
L’émergence des souches de salmonelles multirésitantes représente un problème important tant en médecine humaine que pour la médecine vétérinaire (1). Bien qu’une antibiothérapie ne soit pas recommandée pour traiter les salmonelloses non-typhoïdes chez l’humain, un traitement devient essentiel en cas d’infection invasive extra-intestinale chez les patients à risque ou chez les patients présentant des symptômes sévères ou prolongés (2).
5. Loucif L, Gacemi-Kirane D, Cherak Z, Chamlal N, Grainat N, Rolain J-M. 2016. First report of German cockroaches (Blattella germanica) as reser- voirs of CTX-M-15 extended-spectrum-beta-lactamase- and OXA-48 carbapenemase-producing Enterobacteriaceae in Batna University Hos- pital, Algeria. Antimicrob Agents Chemother 60:6377– 6380. https://doi .org/10.1128/AAC.00871-16 .
Presence of extended-spectrum beta-lactamase-producing Enterobacteriaceae (ESBL-E) in the fecal flora of patients from general practice
18 th ECCMID Abstract 631
Cécile Meex 1 , Pierrette Melin 1 , Jean Denis Docquier 2 , Tukumbane Kabasele 1 , Pascale Huynen 1 , Paul M. Tulkens 3 , Patrick De Mol 1
 Bonten MJ, Slaughter S, Ambergen AW, Hayden MK, van Voorhis J, Nathan C, et al. The role of “colonization pressure” in the spread of vancomycin-resistant enterococci: an important infection control variable. Arch Intern Med 1998;158:1127–32.
 Harris AD, Perencevich EN, Johnson JK, Paterson DL, Morris JG, Strauss SM, et al. Patient-to-patient transmission is important in extended-spectrum beta-lactamase- producing Klebsiella pneumoniae acquisition. Clin Infect Dis 2007;45:1347–50. doi:10.1086/522657.
on the synthesis and the biochemical evaluation of potential inhib- itors of b-lactamases. Numerous b-lactamase inhibitors have been reported in the literature. 2 However, most of these drugs are b-lac-
tam derivatives, and when exposed to such molecules, bacteria ac- quire resistance. To disrupt this vicious circle, non b-lactam inhibitors may be an alternative. The search of novel structures considered as ‘hits’ in medicinal chemistry proceeds from different strategies, such as the mechanism-based design and the screening of various chemical libraries, but also from serendipity. Our pres- ent work is precisely based on a fortuitous observation related to protein crystallography, when using 100 mM sodium citrate buffer in the crystallization protocols.
manual, 3rd ed. Cold Spring Harbor Laboratory Press, Cold Spring Har- bor, NY.
16. Senda, K., Y. Arakawa, S. Ichiyama, K. Nakashima, H. Ito, S. Ohsuka, K.
Shimokata, N. Kato, and M. Ohta. 1996. PCR detection of metallo- ␤- lactamase gene (blaIMP) in gram-negative rods resistant to broad-spectrum ␤-lactams. J. Clin. Microbiol. 34:2909–2913.
From a B2 to a B3 Metallo-β-lactamase
The Arg 121 residue is conserved in the BcII and CphA enzymes. Dal Peraro et al. (23) have shown by quantum chemistry calculations that, in the mono-zinc form of BcII, Arg 121 anchors the Asp 120 side chain by forming a strong ionic bond, ultimately orienting the Zn(II)-bound hydroxide for nucleophilic attack of the antibiotic β- lactam ring. Moreover, Rasia et al. (24) have shown that the R121H mutation in BcII leads to poor positioning of Asp 120 , and thus the k cat values of this mutant are lower than those of WT BcII. Our kinetic results are consistent with a similar role for the Arg 121 residue in CphA, because the k cat value for imipenem is strongly decreased with R121H.
It is important to emphasize that in the BS3-cefoxitin structure, the R-methoxy group does not directly cause the conformational change but that it does so by influencing the 7β side chain orientation. In agreement with that, and as observed by NMR experiments, the 7β-formamidoyl-7R- methoxycephalosporanic acid does not induce structural modifications in the B. licheniformis 749/C β-lactamase. 2 Indeed, in this case, there is no steric incompatibility between the short 7β side chain and the Ω-loop. Nevertheless, for all compounds bearing a 7R-methoxy group, all steps of the hydrolysis pathway are severely impeded. To both accom- modate the 7R and eventually the 7β side chains into the catalytic pocket and bring the substrate in a suitable position for the nucleophilic attack, the β-lactamase must necessarily undergo some conformational changes. The catalytic mech- anism leading to formation of the acyl-enzyme complex is thus greatly impaired. Comparatively, the k cat /K m value determined for BS3 and cephalothin is more than 10 5 -fold larger than with a similar substrate bearing a 7R-methoxy group (Table 1). Similarly, the deacylation process is also impaired by the introduction of a new hydrogen bonding scheme, which directly involves essential amino acid residues such as Ser70, Lys73, and Glu166, and by the disappearance of the hydrolytic water molecule.
C (P99 26 ), class D (OXA-10 27 ), and class B (BcII 28 , VIM-4 29 ). Class A enzymes are primarily
penicillinases, susceptible to inhibition by the presently marketed -lactamase inhibitors, namely potassium clavulanate, sulbactam and tazobactam which are also efficient against some class D -lactamases. Class C enzymes are primarily cephalosporinases, resistant to inhibition by the previous agents. There is no clinically useful inhibitor for class B enzymes that emerge now as worldwide source of carbapenem resistance.
coding a mutant of the chromosomal ␤-lactamase of Kluyvera cryocrescens, was also identified at a plasmid location in E. cloacae 7506, suggesting the ISEcp1-assisted escape of bla KLUC from the chromosome. Determi-
nation of the KPC-2 structure at 1.6 Å revealed that the binding site was occupied by the C-terminal (C-ter) residues coming from a symmetric KPC-2 monomer, with the ultimate C-ter Glu interacting with Ser130, Lys234, Thr235, and Thr237 in the active site. This mode of binding can be paralleled to the inhibition of the TEM-1 ␤-lactamase by the inhibitory protein BLIP. Determination of the 1.23-Å structure of a KPC-2 mutant in which the five C-ter residues were deleted revealed that the catalytic site was filled by a citrate molecule. Structure analysis and docking simulations with cefotaxime and imipenem provided further insights into the molecular basis of the extremely broad spectrum of KPC-2, which behaves as a cefotaximase with significant activity against carbapenems. In particular, residues 104, 105, 132, and 167 draw a binding cavity capable of accommodating both the aminothiazole moiety of cefotaxime and the 6 ␣-hydroxyethyl group of imipenem, with the binding of the former drug being also favored by a significant degree of freedom at the level of the loop at positions 96 to 105 and by an enlargement of the binding site at the end of strand ␤3.
* Measured as Ki.
Measurements were done at 30 °C in 15 mM sodium cacodylate, pH 6.5. N.D., not determined.
Mutation of Cys221
Cys221 was replaced by a Ser or an Ala residue. The purified proteins were analyzed by MS. For the C221S mutant, the expected mass of 25,177 Da was found. By contrast, analysis of the C221A mutant indicated the presence of three majors peaks of 24,346, 24,276 and 23,503 Da (for an expected mass of 25,156 Da), indicating that the mutant protein had lost 8, 9 and 15 residues at the C terminus. There was no degradation at the N terminus, as confirmed by N-terminal sequencing (not shown). Surprisingly, we found that cefoxitin treatment of the C221S preparation resulted in a significant decrease in the hydrolytic activity against imipenem. This was completely unexpected because cefoxitin inactivates the CphA β-lactamase via a covalent modification of the Cys221 side chain , and we anticipated that the C221S mutant would be insensitive to this compound. We therefore concluded that the mutant preparation was contaminated by traces of wild-type protein. Similar findings have been reported before, and were suggested to occur through a 'misreading' at the transcriptional and/or trans-lational level [19, and references therein]. In the present case, however, the wild-type enzyme could be completely inactivated by repeated treatments with cefoxitin (see Materials and methods). This resulted in a ~200-fold decrease in the hydrolytic activity of the preparation against imipenem (not shown). All the data reported here (e.g. in table 3) were obtained after wild-type-enzyme inactiva-tion.
with aryl boronic acids were investigated. From the sta- tistical search with Relibase, apart from the structure of cocaine esterase in complex with phenyl boronic acid (PDB code 1JU3 ), aryl boronic acid ligands are ap- parently not found in other structures deposited at the PDB. This does not mean that the aryl boronic fragment is specific for class C b-lactamase but probably reflects
Since 2012, a decrease of β-lactam resistance of pathogenic Escherichia (E.) coli from calves has been observed at ARSIA, the Regional Veterinary Diagnostics laboratory in Wallonia, Belgium. This may be a consequence of the recommendation for a prior antibiotic sensitivity test of bovine pathogenic E. coli for human critical antibiotics, like 3 rd and 4 th generation cefalosporins. The most frequent -lactam resistance mechanism is the production of a -lactamase enzyme that inactivates the antibiotic. Actual classification(s) of β-lactamases (BLA) is highly complex, but four groups can be summarily described: classical BLA (BLAC), extended cefalosporinases (BLAAmpC), extended spectrum BLA (BLAESBL) and carbapenemases (BLACPE). The aim of this study was to identify the resistance genes coding for β-lactamases in pathogenic E. coli from calves with a resistance phenotype at the disk diffusion assay.
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