Non-Hydrolytic β-Lactam Antibiotic Fragmentation by l,d-Transpeptidases and Serine β-Lactamase Cysteine Variants.
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ABSTRACT:
SUBMITTER: Lohans CT
PROVIDER: S-EPMC6391942 | biostudies-literature |
REPOSITORIES: biostudies-literature
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Project description:B. licheniformis exo-small beta-lactamase (ESBL) has two nonsequential domains and a complex architecture. We replaced ESBL serine residues 126 and 265 with cysteine to probe the conformation of buried regions in each domain. Spectroscopic, hydrodynamic, and chemical methods revealed that the mutations do not alter the native fold but distinctly change stability (S-126C > wild-type > S-126/265C > S-265C ESBL) and the features of partially folded states. The observed wild-type ESBL equilibrium intermediate has decreased fluorescence but full secondary structure. S-126C ESBL intermediate has the fluorescence of the unfolded state, no thiol reactivity, and partial secondary structure. S-265C and S-126/265C ESBL populate intermediate states unfolded by fluorescence and thiol reactivity but with full secondary structure. Mass analysis of S-126/265C ESBL in the partially folded state proved that both thiol groups become exposed simultaneously. None of the intermediates is compatible with sequential domain unfolding. Molecular dynamics simulation suggests that the stabilizing effect of the S-126C substitution is due to optimization of van der Waals interactions and packing. On the other hand, destabilization induced by the S-265C mutation results from alteration of the hydrogen-bond network. The results illustrate the large impact that seemingly conservative serine-to-cysteine changes can have on the energy landscape of proteins.
| S-EPMC1948053 | biostudies-literature
Project description:β-Lactamase positive bacteria represent a growing threat to human health because of their resistance to commonly used antibiotics. Therefore, development of new diagnostic methods for identification of β-lactamase positive bacteria is of high importance for monitoring the spread of antibiotic-resistant bacteria. Here, we report the discovery of a new biodegradation metabolite (H2S), generated through β-lactamase-catalyzed hydrolysis of β-lactam antibiotics. This discovery directed us to develop a distinct molecular technique for monitoring bacterial antibiotic resistance. The technique is based on a highly efficient chemiluminescence probe, designed for detection of the metabolite, hydrogen sulfide, that is released upon biodegradation of β-lactam by β-lactamases. Such an assay can directly indicate if antibiotic bacterial resistance exists for a certain examined β-lactam. The assay was successfully demonstrated for five different β-lactam antibiotics and eight β-lactam resistant bacterial strains. Importantly, in a functional bacterial assay, our chemiluminescence probe was able to clearly distinguish between a β-lactam resistant bacterial strain and a sensitive one. As far as we know, there is no previous documentation for such a biodegradation pathway of β-lactam antibiotics. Bearing in mind the data obtained in this study, we propose that hydrogen sulfide should be considered as an emerging β-lactam metabolite for detection of bacterial resistance.
| S-EPMC8382227 | biostudies-literature
Project description:Yersiniosis caused by Yersinia enterocolitica has been reported from all continents. The bacterial species is divided into more than fifty serovars and six biovars viz. 1A, 1B, 2, 3, 4 and 5 which differ in geographical distribution, ecological niches and pathogenicity. Most Y.enterocolitica strains harbor chromosomal genes for two β-lactamases, blaA an Ambler class A penicillinase and blaB an Ambler class C inducible cephalosporinase. In the present study, susceptibility to b-lactam antibiotics and β-lactamase inhibitor was studied for Y. enterocolitica strains of biovars 1A, 1B, 2 and 4. We observed that β-lactamases were expressed differentially among strains of different biovars. To understand the molecular mechanisms underlying such differential expression, the sequences of genes and promoters of blaA were compared. Also, the variants of blaA present in different biovars were modeled and docked with amoxicillin and clavulanic acid. The mRNA secondary structures of blaA variants were also predicted in-silico. Our findings indicated that neither variations in the promoter regions, nor the secondary structures of mRNA contributed to higher/lower expression of blaA in different biovars. Analysis of H-bonding residues of blaA variants with amoxicillin and clavulanic acid revealed that if amino acid residues of a β-lactamase interacting with amoxicillin and the clavulanic acid were similar, clavulanic acid was effective in engaging the enzyme, accounting for a significant reduction in MIC of amoxicillin-clavulanate. This finding might aid in designing better β-lactamase inhibitors with improved efficiencies in future.
| S-EPMC4414059 | biostudies-literature
Project description:ObjectiveAntibiotic resistance by beta lactamase expression is a serious and growing threat. We aimed to determine whether beta-lactamase activity is detectable in urine specimens to enable faster identification of resistance.MethodsUrine specimens from patients with extended spectrum beta lactamase (ESBL)-expressing urinary infections were incubated with beta lactam antibiotics. Beta lactam hydrolysis was determined by mass spectrometry methods.ResultsCeftriaxone hydrolysis was observed in 45 of 45 ESBL-containing specimens from patients not treated with a beta lactamase inhibitor before specimen collection. Ceftriaxone hydrolysis was not observed in 108 of 108 non-ESBL-containing specimens. Spiking studies show that beta lactam hydrolysis can be observed within 30 minutes. Beta lactam hydrolysis is evidenced by mass spectrometry preceded by either liquid chromatography or matrix-assisted laser desorption ionization specimen processing methods.ConclusionClinically significant beta lactamase activity is detectable directly from urine specimens. The described methods would enable the detection of beta lactam resistance 24 to 48 hours sooner than culture based methods.
| S-EPMC8900932 | biostudies-literature
Project description:Antibiotic resistance caused by β-lactamase production continues to present a growing challenge to the efficacy of β-lactams and their role as the most important class of clinically used antibiotics. In response to this threat however, only a handful of β-lactamase inhibitors have been introduced to the market over the past thirty years. The first-generation β-lactamase inhibitors (clavulanic acid, sulbactam and tazobactam) are all β-lactam derivatives and work primarily by inactivating class A and some class C serine β-lactamases. The newer generations of β-lactamase inhibitors including avibactam and vaborbactam are based on non-β-lactam structures and their spectrum of inhibition is extended to KPC as an important class A carbapenemase. Despite these advances several class D and virtually all important class B β-lactamases are resistant to existing inhibitors. The present review provides an overview of recent FDA-approved β-lactam/β-lactamase inhibitor combinations as well as an update on research efforts aimed at the discovery and development of novel β-lactamase inhibitors.
| S-EPMC6151480 | biostudies-literature
Project description:By use of a new computer-assisted u.v.-spectrophotometric assay method, the kinetic parameters of the reaction catalysed by Bacillus licheniformis 749/C beta-lactamase were re-examined and the mode of inhibition of the enzyme by compound PS-5, a novel beta-lactam antibiotic, was studied with benzylpenicillin as substrate. (1) The fundamental assay conditions for the determination of Km and V were examined in detail with benzylpenicillin as substrate. In 0.1 M-sodium/potassium phosphate buffer, pH 6.8, at 30 degrees C, initial substrate concentrations of benzylpenicillin above 0.7 mM were very likely to lead to substrate inhibition. The Km value of the enzyme for benzylpenicillin at initial concentrations from 1.96 to 0.07 mM was calculated to be 97-108 microM. (2) The Km values of the enzyme for 6-aminopenicillanic acid, ampicillin and cephaloridine were found to be 25, 154-161 and 144-161 microM respectively. (3) Compound PS-5 was virtually unattacked by Bacillus licheniformis 749/C beta-lactamase. (4) The activity of the enzyme was diminished by compound PS-5, to extents depending on the duration of incubation and the concentration of the inhibitor. The rate of inactivation of the enzyme by compound PS-5 followed first-order kinetics. (5) In an Appendix, a new computer-assisted u.v.-spectrophotometric enzyme assay method, in which a single reaction progress curve of time-absorbance was analysed by the integrated Michaelis-Menten equation, was devised for the accurate and precise determination of the kinetic constants of beta-lactamase. For conversion of absorbance readings into molar substrate concentrations, the initial or final absorbance reading that was independent of the reaction time was used as the basis of calculation. In calculation of Km and V three systematic methods of data combination were employed for finer analysis of the reaction progress curve. A list of the computer program named YF6TAIM is obtainable from the author on request or as Supplementary Publication SUP 50100 (12 pages) from the British Library Lending Division, Boston Spa, Wetherby, West Yorkshire LS23 7BQ, U.K., on the terms indicated in Biochem. J. (1978) 169, 5.
| S-EPMC1161281 | biostudies-other
Project description:Our current understanding of how low antibiotic concentrations shape the evolution of contemporary β-lactamases is limited. Using the widespread carbapenemase OXA-48, we tested the long-standing hypothesis that selective compartments with low antibiotic concentrations cause standing genetic diversity that could act as a gateway to developing clinical resistance. Here, we subjected Escherichia coli expressing blaOXA-48, on a clinical plasmid, to experimental evolution at sub-MICs of ceftazidime. We identified and characterized seven single variants of OXA-48. Susceptibility profiles and dose-response curves showed that they increased resistance only marginally. However, in competition experiments at sub-MICs of ceftazidime, they demonstrated strong selectable fitness benefits. Increased resistance was also reflected in elevated catalytic efficiencies toward ceftazidime. These changes are likely caused by enhanced flexibility of the Ω- and β5-β6 loops and fine-tuning of preexisting active site residues. In conclusion, low-level concentrations of β-lactams can drive the evolution of β-lactamases through cryptic phenotypes which may act as stepping-stones toward clinical resistance.IMPORTANCE Very low antibiotic concentrations have been shown to drive the evolution of antimicrobial resistance. While substantial progress has been made to understand the driving role of low concentrations during resistance development for different antimicrobial classes, the importance of β-lactams, the most commonly used antibiotics, is still poorly studied. Here, we shed light on the evolutionary impact of low β-lactam concentrations on the widespread β-lactamase OXA-48. Our data indicate that the exposure to β-lactams at very low concentrations enhances β-lactamase diversity and drives the evolution of β-lactamases by significantly influencing their substrate specificity. Thus, in contrast to high concentrations, low levels of these drugs may substantially contribute to the diversification and divergent evolution of these enzymes, providing a standing genetic diversity that can be selected and mobilized when antibiotic pressure increases.
| S-EPMC8092134 | biostudies-literature
Project description:Pseudomonas aeruginosa can cause infections in the blood, lungs (pneumonia), or other parts of the body after surgery. To investigate the molecular characteristics of β-lactam antibiotic resistance of P. aeruginosa isolated from a hospital population between 2015 and 2017, in this study, the antimicrobial susceptibility and the resistance gene profile of the bacteria were determined. The Pulsed-field gel electrophoresis (PFGE) was used to characterize the clonal relatedness and sequencing and comparative genomic analysis were performed to analyze the structure of the resistance gene-related sequences. As a result, of the 260 P. aeruginosa strains analyzed, the resistance rates for 6 β-lactam antibiotics ranged from 4.6 to 9.6%. A total of 7 genotypes of 44 β-lactamase genes were identified in 23 isolates (8.9%, 23/260). Four transconjugants from different donors carrying bla CARB-3 exhibited a phenotype of reduced susceptibility to piperacillin-tazobactam, ceftazidime, and cefepime, and 2 transconjugants harboring bla IMP-45 exhibited a phenotype of reduced susceptibility to carbapenems. bla CARB positive isolates (n = 12) presented six PFGE patterns, designated groups A to F. Two bla genes (bla IMP-45 and bla OXA-1) in PA1609 related to a class 1 integron (intI1-bla IMP-45- bla OXA-1-aac(6')-Ib7-catB3-qacE∆1-sul1) were encoded on a plasmid (pPA1609-475), while the bla CARB-3 gene of PA1616 also related to a class 1 integron was located on the chromosome. The results suggest that β-lactam antibiotic resistance and clonal dissemination exist in this hospital population. It indicates the necessity for molecular surveillance in tracking β-lactamase-producing strains and emphasizes the need for epidemiological monitoring.
| S-EPMC9096163 | biostudies-literature
Project description:An equation is derived from first principles for describing the change in concentration with time of a beta-lactam antibiotic in the presence of intact Gram-negative bacteria possessing a beta-lactamase located in the periplasmic space. The equation predicts a first-order decline in beta-lactam concentration of the form [S] = [Si]e lambda t, where [S] is the exogenous concentration of beta-lactam, [Si] is the value of [S] at time zero, t is the time from mixing of cells and antibiotic and lambda (less than 0) is the decay constant. The value of lambda is exactly described by the theory in terms of experimentally measurable quantities. Quantitative data concerning cephaloridine hydrolysis by intact cells of Haemophilus influenzae agreed well with the theory, as did data concerning the uptake of 2-nitrophenyl galactoside by intact cells of Escherichia coli. Cephalosporin C hydrolysis by intact cells of Pseudomonas aeruginosa did not progress as predicted by the theory. The theory is applicable to any substrate which is acted on by an enzyme that is located solely in the periplasmic space and that obeys the Michaelis-Menten equation of enzyme kinetics.
| S-EPMC1148025 | biostudies-other
Project description:Significant advances were made in antibiotic development during the past 5 years. Novel agents were added to the arsenal that target critical priority pathogens, including multidrug-resistant Pseudomonas aeruginosa and carbapenem-resistant Enterobacterales. Of these, 4 novel β-lactam-β-lactamase inhibitor combinations (ceftolozane-tazobactam, ceftazidime-avibactam, meropenem-vaborbactam, and imipenem-cilastatin-relebactam) reached clinical approval in the United States. With these additions comes a significant responsibility to reduce the possibility of emergence of resistance. Reports in the rise of resistance toward ceftolozane-tazobactam and ceftazidime-avibactam are alarming. Clinicians and scientists must make every attempt to reverse or halt these setbacks.
| S-EPMC7609624 | biostudies-literature