Identifying novel β-lactamase substrate activity through in silico prediction of antimicrobial resistance.
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ABSTRACT:
SUBMITTER: Tsang KK
PROVIDER: S-EPMC8115898 | biostudies-literature |
REPOSITORIES: biostudies-literature
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Project description:AmpC is a group I, class C -lactamase present in most Enterobacteriaceae and in Pseudomonas aeruginosa and other nonfermenting gram-negative bacilli. The β-lactam class of antibiotics is one of the most important structural classes of antibacterial compounds and act by inhibiting the bacterial D ,D - transpeptidases that are responsible for the final step of peptidoglycan cross-linking. Our main aim in the study is to screen possible inhibitors against AmpC / β - lactamase (an enzyme responsible for antimicrobial activity in Pseudomonas aeruginosa), through virtual screening of 1364 NCI (National Cancer Institute) diversity set II compounds. Homology Model of AmpC / β - lactamase was constructed using MODELLER and the Model was validated using PROCHECK and Verify 3D programs to obtain a stable structure, which was further used for virtual screening of NCI (National Cancer Institute) diversity set II compounds through molecular Docking studies using Autodock. The amino acid sequence of the β - lactamase was also subjected to ScanProsite web server to find any pattern present in the sequence. After the prediction of 3-dimensional model of AmpC/ β-lactamase, the possible Active sites ofβ - lactamase were determined using LIGSITE(csc) and CastP web servers simultaneously. The Docked complexes were validated and Enumerated based on the Autodock Scoring function to pick out the best inhibitor based on Autodock energy score. Thus from the entire 1364 NCI diversity set II compounds which were Docked, the best four docking solutions were selected (ZINC12670903, ZINC17465965, ZINC11681166 and ZINC13099024). Further the Complexes were analyzed through LIGPLOT for their interaction for the 4 best docked NCI diversity set II compounds. Thus from the Complex scoring and binding ability it is deciphered that these NCI diversity set II compounds could be promising inhibitors for Pseudomonas aeruginosa using AmpC /β - lactamase as Drug target yet pharmacological studies have to confirm it.
| S-EPMC2957765 | biostudies-literature
Project description:Penicillin (PEN) is a low-cost option for anthrax treatment, but naturally occurring resistance has been reported. β-Lactamase expression (bla1, bla2) in Bacillus anthracis is regulated by a sigma factor (SigP) and its cognate anti-sigma factor (RsiP). Mutations leading to truncation of RsiP were previously described as a basis for PEN resistance. Here, we analyze whole-genome sequencing (WGS) data and compare the chromosomal sigP-bla1 regions from 374 B. anthracis strains to determine the frequency of mutations, identify mutations associated with PEN resistance, and evaluate the usefulness of WGS for predicting PEN resistance. Few (3.5%) strains contained at least 1 of 11 different mutations in sigP, rsiP, or bla1. Nine of these mutations have not been previously associated with PEN resistance. Four strains showed PEN resistance (PEN-R) by conventional broth microdilution, including 1 strain with a novel frameshift in rsiP. One strain that carries the same rsiP frameshift mutation as that found previously in a PEN-R strain showed a PEN-susceptible (PEN-S) phenotype and exhibited decreased bla1 and bla2 transcription. An unexpectedly small colony size, a reduced growth rate, and undetectable β-lactamase activity levels (culture supernatant and cell lysate) were observed in this PEN-S strain. Sequence analysis revealed mutations in genes associated with growth defects that may contribute to this phenotype. While B. anthracis rsiP mutations cannot be exclusively used to predict resistance, four of the five strains with rsiP mutations were PEN-R. Therefore, the B. anthracis sigP-bla1 region is a useful locus for WGS-based PEN resistance prediction, but phenotypic testing remains essential. IMPORTANCE Determination of antimicrobial susceptibility of B. anthracis is essential for the appropriate distribution of antimicrobial agents for postexposure prophylaxis (PEP) and treatment of anthrax. Analysis of WGS data allows for the rapid detection of mutations in antimicrobial resistance (AMR) genes in an isolate, but the presence of a mutation in an AMR gene does not always accurately predict resistance. As mutations in the anti-sigma factor RsiP have been previously associated with high-level penicillin resistance in a limited number of strains, we investigated WGS assemblies from 374 strains to determine the frequency of mutations and performed functional antimicrobial susceptibility testing. Of the five strains that contained mutations in rsiP, only four were PEN-R by functional antimicrobial susceptibility testing. We conclude that while sequence analysis of this region is useful for AMR prediction in B. anthracis, genetic analysis should not be used exclusively and phenotypic susceptibility testing remains essential.
| S-EPMC6290263 | biostudies-literature
Project description:The decreasing effectiveness of antimicrobial agents is a global public health threat, yet risk factors for community-acquired antimicrobial resistance (CA-AMR) in low-income settings have not been clearly elucidated. Our aim was to identify risk factors for CA-AMR with extended-spectrum β-lactamase (ESBL)-producing organisms among urban-dwelling women in India. We collected microbiological and survey data in an observational study of primigravidae women in a public hospital in Hyderabad, India. We analyzed the data using multivariate logistic and linear regression and found that 7% of 1,836 women had bacteriuria; 48% of isolates were ESBL-producing organisms. Women in the bottom 50th percentile of income distribution were more likely to have bacteriuria (adjusted odds ratio 1.44, 95% CI 0.99-2.10) and significantly more likely to have bacteriuria with ESBL-producing organisms (adjusted odds ratio 2.04, 95% CI 1.17-3.54). Nonparametric analyses demonstrated a negative relationship between the prevalence of ESBL and income.
| S-EPMC6056104 | biostudies-literature
Project description:Resistance to β-lactams is constantly increasing due to the emergence of totally new enzymes but also to the evolution of preexisting β-lactamases. GES-1 is a clinically relevant extended-spectrum β-lactamase (ESBL) that hydrolyzes penicillins and broad-spectrum cephalosporins but spares monobactams and carbapenems. However, several GES-1 variants (i.e., GES-2 and GES-5) previously identified among clinical isolates display an extended spectrum of activity toward carbapenems. To study the evolution potential of the GES-1 β-lactamase, this enzyme was submitted to in vitro-directed evolution, with selection on increasing concentrations of the cephalosporin cefotaxime, the monobactam aztreonam, or the carbapenem imipenem. The highest resistance levels were conferred by a combination of up to four substitutions. The A6T-E104K-G243A variant selected on cefotaxime and the A6T-E104K-T237A-G243A variant selected on aztreonam conferred high resistance to cefotaxime, ceftazidime, and aztreonam. Conversely, the A6T-G170S variant selected on imipenem conferred high resistance to imipenem and cefoxitin. Of note, the A6T substitution involved in higher MICs for all β-lactams is located in the leader peptide of the GES enzyme and therefore is not present in the mature protein. Acquired cross-resistance was not observed, since selection with cefotaxime or aztreonam did not select for resistance to imipenem, and vice versa. Here, we demonstrate that the β-lactamase GES-1 exhibits peculiar properties, with a significant potential to gain activity against broad-spectrum cephalosporins, monobactams, and carbapenems.
| S-EPMC4325776 | biostudies-literature
Project description:Beta-lactamases are the major resistance mechanism to beta-lactam antibiotics and pose a growing threat to public health. Recently, bacteria have become resistant to beta-lactamase inhibitors, making this problem pressing. In an effort to overcome this resistance, non-beta-lactam inhibitors of beta-lactamases were investigated for complementarity to the structure of AmpC beta-lactamase from Escherichia coli. This led to the discovery of an inhibitor, benzo(b)thiophene-2-boronic acid (BZBTH2B), which inhibited AmpC with a Ki of 27 nM. This inhibitor is chemically dissimilar to beta-lactams, raising the question of what specific interactions are responsible for its activity. To answer this question, the X-ray crystallographic structure of BZBTH2B in complex with AmpC was determined to 2.25 A resolution. The structure reveals several unexpected interactions. The inhibitor appears to complement the conserved, R1-amide binding region of AmpC, despite lacking an amide group. Interactions between one of the boronic acid oxygen atoms, Tyr150, and an ordered water molecule suggest a mechanism for acid/base catalysis and a direction for hydrolytic attack in the enzyme catalyzed reaction. To investigate how a non-beta-lactam inhibitor would perform against resistant bacteria, BZBTH2B was tested in antimicrobial assays. BZBTH2B significantly potentiated the activity of a third-generation cephalosporin against AmpC-producing resistant bacteria. This inhibitor was unaffected by two common resistance mechanisms that often arise against beta-lactams in conjunction with beta-lactamases. Porin channel mutations did not decrease the efficacy of BZBTH2B against cells expressing AmpC. Also, this inhibitor did not induce expression of AmpC, a problem with many beta-lactams. The structure of the BZBTH2B/AmpC complex provides a starting point for the structure-based elaboration of this class of non-beta-lactam inhibitors.
| S-EPMC2144209 | biostudies-other
Project description:The antibiotic resistance crisis continues to threaten human health. Better predictions of the evolution of antibiotic resistance genes could contribute to the design of more sustainable treatment strategies. However, comprehensive prediction of antibiotic resistance gene evolution via laboratory approaches remains challenging. By combining site-specific integration and high-throughput sequencing, we quantified relative growth under the respective selection of cefotaxime or ceftazidime selection in ∼23,000 Escherichia coli MG1655 strains that each carried a unique, single-copy variant of the extended-spectrum β-lactamase gene blaCTX-M-14 at the chromosomal att HK022 site. Significant synergistic pleiotropy was observed within four subgenic regions, suggesting key regions for the evolution of resistance to both antibiotics. Moreover, we propose PEARP and PEARR, two deep-learning models with strong clinical correlations, for the prospective and retrospective prediction of blaCTX-M-14 evolution, respectively. Single to quintuple mutations of blaCTX-M-14 predicted to confer resistance by PEARP were significantly enriched among the clinical isolates harboring blaCTX-M-14 variants, and the PEARR scores matched the minimal inhibitory concentrations obtained for the 31 intermediates in all hypothetical trajectories. Altogether, we conclude that the measurement of local fitness landscape enables prediction of the evolutionary trajectories of antibiotic resistance genes, which could be useful for a broad range of clinical applications, from resistance prediction to designing novel treatment strategies.
| S-EPMC9087888 | biostudies-literature
Project description:CTX-M β-lactamases are prevalent antibiotic resistance enzymes and are notable for their ability to rapidly hydrolyze the extended-spectrum cephalosporin, cefotaxime. We hypothesized that the active site sequence requirements of CTX-M-mediated hydrolysis differ between classes of β-lactam antibiotics. Accordingly, we use codon randomization, antibiotic selection, and deep sequencing to determine the CTX-M active-site residues required for hydrolysis of cefotaxime and the penicillin, ampicillin. The study reveals positions required for hydrolysis of all β-lactams, as well as residues controlling substrate specificity. Further, CTX-M enzymes poorly hydrolyze the extended-spectrum cephalosporin, ceftazidime. We further show that the sequence requirements for ceftazidime hydrolysis follow those of cefotaxime, with the exception that key active-site omega loop residues are not required, and may be detrimental, for ceftazidime hydrolysis. These results provide insights into cephalosporin hydrolysis and demonstrate that changes to the active-site omega loop are likely required for the evolution of CTX-M-mediated ceftazidime resistance.
| S-EPMC9837174 | biostudies-literature
Project description:BackgroundCefiderocol is a novel siderophore β-lactam with improved hydrolytic stability toward β-lactamases, including carbapenemases, achieved by combining structural moieties of two clinically efficient cephalosporins, ceftazidime and cefepime. Consequently, cefiderocol represents a treatment alternative for infections caused by MDR Gram-negatives.ObjectivesTo study the role of cefiderocol on resistance development and on the evolution of β-lactamases from all Ambler classes, including KPC-2, CTX-M-15, NDM-1, CMY-2 and OXA-48.MethodsDirected evolution, using error-prone PCR followed by selective plating, was utilized to investigate how the production and the evolution of different β-lactamases cause changes in cefiderocol susceptibility determined using microbroth dilution assays (MIC and IC50).ResultsWe found that the expression of blaOXA-48 did not affect cefiderocol susceptibility. On the contrary, the expression of blaKPC-2, blaCMY-2, blaCTX-M-15 and blaNDM-1 substantially reduced cefiderocol susceptibility by 4-, 16-, 8- and 32-fold, respectively. Further, directed evolution on these enzymes showed that, with the acquisition of only 1-2 non-synonymous mutations, all β-lactamases were evolvable to further cefiderocol resistance by 2- (NDM-1, CTX-M-15), 4- (CMY-2), 8- (OXA-48) and 16-fold (KPC-2). Cefiderocol resistance development was often associated with collateral susceptibility changes including increased resistance to ceftazidime and ceftazidime/avibactam as well as functional trade-offs against different β-lactam drugs.ConclusionsThe expression of contemporary β-lactamase genes can potentially contribute to cefiderocol resistance development and the acquisition of mutations in these genes results in enzymes adapting to increasing cefiderocol concentrations. Resistance development caused clinically important cross-resistance, especially against ceftazidime and ceftazidime/avibactam.
| S-EPMC9410664 | biostudies-literature
Project description:We developed and tested a glass-based microarray suitable for detecting multiple tetracycline (tet) resistance genes. Microarray probes for 17 tet genes, the beta-lactamase bla(TEM-1) gene, and a 16S ribosomal DNA gene (Escherichia coli) were generated from known controls by PCR. The resulting products (ca. 550 bp) were applied as spots onto epoxy-silane-derivatized, Teflon-masked slides by using a robotic spotter. DNA was extracted from test strains, biotinylated, hybridized overnight to individual microarrays at 65 degrees C, and detected with Tyramide Signal Amplification, Alexa Fluor 546, and a microarray scanner. Using a detection threshold of 3x the standard deviation, we correctly identified tet genes carried by 39 test strains. Nine additional strains were not known to harbor any genes represented on the microarray, and these strains were negative for all 17 tet probes as expected. We verified that R741a, which was originally thought to carry a novel tet gene, tet(I), actually harbored a tet(G) gene. Microarray technology has the potential for screening a large number of different antibiotic resistance genes by the relatively low-cost methods outlined in this paper.
| S-EPMC201141 | biostudies-literature
Project description: Not available
| S-EPMC10795760 | biostudies-literature