The resistance of microorganisms to antibiotics has been developing for more than 2 billion years and is widely distributed among various representatives of the microbiological world. Bacterial enzymes play a key role in the emergence of resistance. Classification of these enzymes is based on their participation in various biochemical mechanisms: modification of the enzymes that act as antibiotic targets, enzymatic modification of intracellular targets, enzymatic transformation of antibiotics, and the implementation of cellular metabolism reactions. The main mechanisms of resistance development are associated with the evolution of superfamilies of bacterial enzymes due to the variability of the genes encoding them. The collection of all antibiotic resistance genes is known as the resistome. Tens of thousands of enzymes and their mutants that implement various mechanisms of resistance form a new community that is called the enzystome. Analysis of the structure and functional characteristics of enzymes, which are the targets for different classes of antibiotics, will allow us to develop new strategies for overcoming the resistance.

Extended-spectrum beta-lactamases (ESBL) of the TEM, SHV, or CTX-M type confer resistance to beta-lactam antibiotics in Gram-negative bacteria. The activity of these enzymes against beta-lactam antibiotics and their resistance against inhibitors can be influenced by genetic variation at the single-nucleotide level. Here, we describe the development and validation of an oligonucleotide microarray for the rapid identification of ESBLs in Gram-negative bacteria by simultaneously genotypingbla_TEM,bla_SHV, andbla_CTX-M. The array consists of 618 probes that cover mutations responsible for 156 amino acid substitutions. As this comprises unprecedented genotyping coverage, the ESBL array has a high potential for epidemiological studies and infection control. With an assay time of 5 h, the ESBL microarray also could be an attractive option for the development of rapid antimicrobial resistance tests in the future. The validity of the DNA microarray was demonstrated with 60 blinded clinical isolates, which were collected during clinical routines. Fifty-eight of them were characterized phenotypically as ESBL producers. The chip was characterized with regard to its resolution, phenotype-genotype correlation, and ability to resolve mixed genotypes. ESBL phenotypes could be correctly ascribed to ESBL variants ofbla_CTX-M(76%),bla_SHV(22%), or both (2%), whereas no ESBL variant ofbla_TEMwas found. The most prevalent ESBLs identified were CTX-M-15 (57%) and SHV-12 (18%).

Bacterial resistance to β-lactams, the most commonly used class of antibiotics, poses a global challenge. This resistance is caused by the production of bacterial enzymes that are termed β-lactamases (βLs). The evolution of serine-class A β-lactamases from penicillin-binding proteins (PBPs) is related to the formation of the Ω-loop at the entrance to the enzyme’s active site. In this loop, the Glu166 residue plays a key role in the two-step catalytic cycle of hydrolysis. This residue in TEM–type β-lactamases, together with Asn170, is involved in the formation of a hydrogen bonding network with a water molecule, leading to the deacylation of the acyl–enzyme complex and the hydrolysis of the β-lactam ring of the antibiotic. The activity exhibited by the Ω-loop is attributed to the positioning of its N-terminal residues near the catalytically important residues of the active site. The structure of the Ω-loop of TEM-type β-lactamases is characterized by low mutability, a stable topology, and structural flexibility. All of the revealed features of the Ω-loop, as well as the mechanisms related to its involvement in catalysis, make it a potential target for novel allosteric inhibitors of β-lactamases.