Fast and Accurate Large-Scale Detection of ß-Lactamase Genes Conferring Antibiotic Resistance.

Fast detection of ß-lactamase (bla) genes allows improved surveillance studies and infection control measures, which can minimize the spread of antibiotic resistance. Although several molecular diagnostic methods have been developed to detect limited bla gene types, these methods have significant limitations, such as their failure to detect almost all clinically available bla genes. We developed a fast and accurate molecular method to overcome these limitations using 62 primer pairs, which were designed through elaborate optimization processes. To verify the ability of this large-scale bla detection method (large-scaleblaFinder), assays were performed on previously reported bacterial control isolates/strains. To confirm the applicability of the large-scaleblaFinder, the assays were performed on unreported clinical isolates. With perfect specificity and sensitivity in 189 control isolates/strains and 403 clinical isolates, the large-scaleblaFinder detected almost all clinically available bla genes. Notably, the large-scaleblaFinder detected 24 additional unreported bla genes in the isolates/strains that were previously studied, suggesting that previous methods detecting only limited types of bla genes can miss unexpected bla genes existing in pathogenic bacteria, and our method has the ability to detect almost all bla genes existing in a clinical isolate. The ability of large-scaleblaFinder to detect bla genes on a large scale enables prompt application to the detection of almost all bla genes present in bacterial pathogens. The widespread use of the large-scaleblaFinder in the future will provide an important aid for monitoring the emergence and dissemination of bla genes and minimizing the spread of resistant bacteria.

Expression, crystallization and preliminary X-ray crystallographic analysis of alanine racemase from Acinetobacter baumannii OXA-23.

Acinetobacter baumannii has received much attention owing to its exceptional ability to develop resistance to currently available antibiotics. Alanine racemase (ALR) catalyzes the racemization of L-alanine to D-alanine with pyridoxal 5'-phosphate (PLP) as a cofactor. The D-alanine product is an essential component of the bacterial cell wall and ALR is a potential target for the development of novel antibacterial drugs. The alr gene from A. baumannii was cloned and the protein (AbALR) was expressed, purified and crystallized. The AbALR crystal diffracted to 2.3 Šresolution and belonged to the primitive orthorhombic space group P2(1)2(1)2(1), with unit-cell parameters a = 55.1, b = 85.0, c = 167.7 Å. Two protomers were present in the asymmetric unit, with a corresponding V(M) value of 2.3 Å(3) Da(-1) and a solvent content of 47.5%.

Crystallization and preliminary diffraction studies of GIM-1, a class B carbapenem-hydrolyzing ß-lactamase.

GIM-1 is a member of the class B carbapenemases (metallo-ß-lactamases; MBLs) and has a wide spectrum of activity against carbapenems, penicillins and extended-spectrum cephalosporins, but not aztreonam. GIM-1 presents an enormous challenge to infection control, particularly in the eradication of Gram-negative pathogens including Enterobacteriaceae, Pseudomonas aeruginosa, Acinetobacter baumannii and nonfermenters. There are presently few or no drugs in late-stage development for these pathogens and GIM-1 is a potential target for the development of antimicrobial agents against pathogens producing MBLs. In this study, GIM-1 was cloned, overexpressed and crystallized. The GIM-1 crystals diffracted to 1.4 Šresolution and belonged to the orthorhombic space group P2(1)2(1)2(1), with unit-cell parameters a = 38.5, b = 67.6, c = 72.8 Å. One molecule is present in the asymmetric unit, with a corresponding V(M) of 1.69 Å(3) Da(-1) and a solvent content of 27.1%.