Beta-lactam Antibiotics Research Articles

Private benefit of β-lactamase dictates selection dynamics of combination antibiotic treatment

β-lactam antibiotics have been prescribed for most bacterial infections since their discovery. However, resistance to β-lactams, mediated by β-lactamase (Bla) enzymes such as extended spectrum β-lactamases (ESBLs), has become widespread. Bla inhibitors can restore the efficacy of β-lactams against resistant bacteria, an approach which preserves existing antibiotics despite declining industry investment. However, the effects of combination treatment on selection for β-lactam resistance are not well understood. Bla production confers both private benefits for resistant cells and public benefits which faster-growing sensitive cells can also exploit. These benefits may be differentially impacted by Bla inhibitors, leading to non-intuitive selection dynamics. In this study, we demonstrate strain-to-strain variation in effective combination doses, with complex growth dynamics in mixed populations. Using modeling, we derive a criterion for the selection outcome of combination treatment, dependent on the burden and effective private benefit of Bla production. We then use engineered strains and natural isolates to show that strong private benefits of Bla are associated with increased selection for resistance. Finally, we demonstrate that this parameter can be coarsely estimated using high-throughput phenotyping of clonal populations. Our analysis shows that quantifying the phenotypic responses of bacteria to combination treatment can facilitate resistance-minimizing optimization of treatment.

Validation of An HPLC Method for the Determination of Some Β-Lactams Antibiotics Using A Green Analytical Technique

Green analytical chemistry (GAC) primary goal is to develop environmental and health-friendly analytical methods by reduction or replacement of hazardous substances from analytical procedures. In this research, the application of GAC to quantify five β-lactams antibiotics (cefuroxime sodium, cefaclor, ertapenem sodium, ampicillin sodium and sulbactam sodium) by high-performance liquid chromatography (HPLC) using photodiode arrays (PDA) detector was developed. To achieve this, ethanol was used as a green replacement for methanol and acetonitrile, common solvents in chromatographic procedures. Several chromatographic conditions were investigated to accomplish the optimal conditions. The method validation followed the ICH Q2 (R1) guideline, and the developed method was successfully applied for quantifying some pharmaceutical products.

Machine Learning and Deep Learning Models for Predicting Noncovalent Inhibitors of AmpC β-Lactamase.

Continuous use of antibiotics leads to the ability of bacteria to adapt by developing complex antibiotic resistance (AR) mechanisms. The synthesis of β-lactamases is a widely observed AR mechanism. The class C β-lactamase (AmpC) causes significant resistance toward β-lactam antibiotics, and new treatments are urgently needed. Noncovalent inhibitors have been developed against a broad spectrum of β-lactamases. In this study, we developed robust and accurate models for predicting noncovalent inhibitors of AmpC using large compound data sets and machine/deep learning modeling. We created support vector machine (SVM), random forest (RF), and feed-forward neural network (FFNN) classification models. The cross-validation (CV) accuracies varied between 80 and 82%, as combined models reached an accuracy of 83%. We analyzed the physicochemical characteristics of the noncovalent inhibitors and predicted the binding modes for some of them. Such models are helpful for identifying new noncovalent inhibitors in order to establish novel solutions against the growing resistance to standard β-lactam inhibitors. The best RF, SVM, and FFNN models for predicting noncovalent inhibitors of AmpC β-lactamase are available in the GitHub repository, https://github.com/UPCmctr/ML-DL-AmpC-B-lactamase.

Effect of Ceftaroline, Ceftazidime/Avibactam, Ceftolozane/Tazobactam, and Meropenem/Vaborbactam on Establishment of Colonization by Vancomycin-Resistant Enterococci and Klebsiella pneumoniae in Mice.

The potential for promotion of intestinal colonization with healthcare-associated pathogens by new antibiotics used to treat infections due to multidrug-resistant Gram-negative bacilli is unclear. Mice treated for 3 days with daily subcutaneous phosphate-buffered saline (control), ceftazidime/avibactam, ceftolozane/tazobactam, ceftaroline, and meropenem/vaborbactam were challenged with 10,000 colony-forming units (CFU) of vancomycin-resistant Enterococcus (VRE) resistant to each of the antibioics or carbapenemase-producing Klebsiella pneumoniae 1 day after the final treatment dose. The concentrations of VRE or K. pneumoniae in stool were measured on days 1, 3, 6, and 15 after challenge. Control mice had transient low levels of VRE or K. pneumoniae (<3 log10 CFU/g) detected in stool with negative cultures on days 6 and 15 after challenge. In comparison to control mice, each of the antibiotics promoted establishment of high-density colonization with VRE (mean concentration, >8 log10 CFU/g of stool on day 1 after challenge) that persisted at >4 log10 CFU/g of stool through day 15 (P<0.01). In comparison to control mice, meropenem/vaborbactam and ceftaroline promoted high-density colonization with K. pneumoniae (peak concentration, >8 log10 CFU/g of stool) (P<0.01), ceftolozane/tazobactam promoted colonization to a lesser degree (peak concentration, >5 log10 CFU/g of stool), and ceftazidime/avibactam did not promote colonization (P>0.05). Our results suggest that several beta-lactam antibiotics recently developed for treatment of infections with resistant Gram-negative bacilli have the potential to promote colonization by healthcare-associated pathogens. Additional studies are needed to examine the impact of these agents in patients.

Continuous infusion of beta-lactam antibiotics in critically ill patients with sepsis: implementation considerations

Continuous infusion of beta-lactam antibiotics in critically ill patients with sepsis: implementation considerations

Antimicrobial Resistance and Public Health Risks Associated with Staphylococci Isolated from Raw and Processed Meat Products.

This study aimed at evaluating the occurrence, antibiotic resistance, and β-lactamase production in Staphylococcus isolates recovered from meat and meat products, as well as the incidence of antimicrobial resistance (AMR) genes in these bacterial isolates. The prevalence of Staphylococcus was very high (75% and 50%) in street kebab and raw buffalo meat, respectively. The antibiotic resistance and susceptibility behavior showed that 82% of the Staphylococcus isolates were resistant to β-lactam antibiotics such as aztreonam, followed by methicillin (68%), oxacillin (54%), cefepime (36%), ceftazidime (34%), cefaclor (24%), cefotaxime (22%), ertapenem (4%), meropenem and imipenem (2%). Among non-β-lactam antibiotics, the most widespread resistance was observed against nalidixic acid (80%), sulfadiazine (76%), vancomycin (24%), erythromycin (10%), chloramphenicol (6%), and kanamycin and gatifloxacin (4%). One hundred percent of the isolates were susceptible to ciprofloxacin, tetracycline, gemifloxacin, and cefotaxime/clavulanic acid. In vitro drug-resistant characteristics revealed 36 distinct resistance patterns of Staphylococcus isolates, with 82% of them being multidrug resistant (MDR). Iodometric assay showed that 48% of the Staphylococcus isolates produced β-lactamase and 24% of the isolates were capable of producing extended-spectrum beta-lactamases phenotypically. The most commonly detected AMR gene was mecA (29.2%), followed by Sul 1 (25%) and qnrS and qnrB (20.8%), in Staphylococcus isolates. Current findings show widespread occurrence of MDR Staphylococcus strains in raw meat and street meat products, which is a potential risk to public health. Therefore, the study suggests strict monitoring of hygiene through the whole food chain and judicious use of antibiotics.

Antibiotic resistance plasmids in Enterobacteriaceae isolated from fresh produce in northern Germany.

This study showed that Enterobacteriaceae from raw vegetables carried plasmids ranging in size from 2,715 to 326,286 bp, of which about less than one-third carried antibiotic resistance genes encoding resistance toward antibiotics such as tetracyclines, aminoglycosides, fosfomycins, sulfonamides, quinolones, and β-lactam antibiotics. Some strains encoded multiple resistances, and some encoded extended-spectrum β-lactamases. The study highlights the potential of produce, which may be eaten raw, as a potential vehicle for the transfer of antibiotic-resistant bacteria.

Identification of novel genetic factors that regulate c-di-AMP production in Staphylococcus aureus using a riboswitch-based biosensor.

Nucleotide secondary messengers regulate various processes in bacteria allowing them to rapidly respond to changes in environmental conditions. c-di-AMP is an essential second messenger required for the growth of the human pathogen Staphylococcus aureus, regulating potassium, osmolyte uptake, and beta-lactam resistance. Cellular concentrations of c-di-AMP are regulated by the activities of two enzymes, DacA and GdpP, which synthesize and hydrolyze c-di-AMP, respectively. Besides these, only a limited number of other factors are known to regulate c-di-AMP levels. Using a c-di-AMP biosensor consisting of the Bacillus subtilis c-di-AMP-binding kimA riboswitch and yfp, we were able to efficiently detect differences in cellular c-di-AMP levels in S. aureus. To identify novel factors that regulate c-di-AMP levels, we introduced the biosensor into a library of S. aureus transposon mutants. In this manner, we obtained mutants with increased c-di-AMP levels that contained insertions in gdpP coding for the c-di-AMP hydrolase and ybbR (cdaR) coding for a c-di-AMP cyclase regulator, thus validating our screen. We also identified two high c-di-AMP mutants with insertions upstream of the nrdIEF operon coding for the ribonucleotide reductase enzyme. Further analysis revealed that the insertion down-regulated nrdIEF expression, indicating that the enzyme is a negative regulator of c-di-AMP production. This negative regulation was dependent on rsh, encoding for the synthase of the endogenous GdpP inhibitor (p)ppGpp. The methods established in this work can be readily adapted for use in other bacteria to uncover genetic or environmental factors regulating c-di-AMP levels.IMPORTANCEc-di-AMP is an important secondary messenger, produced by many bacterial species including the opportunistic pathogen Staphylococcus aureus. In this bacterium, c-di-AMP controls cell wall homeostasis, cell size, and osmotic balance. In addition, it has been shown that strains with high c-di-AMP levels exhibit increased resistance to beta-lactam antibiotics. Here, we developed a biosensor-based method for the rapid detection of c-di-AMP levels in S. aureus. We utilized the biosensor in a genetic screen for the identification of novel factors that impact cellular c-di-AMP. In this manner, we identified the ribonucleotide reductase as a novel factor altering cellular c-di-AMP levels and showed that reducing its expression leads to increased cellular c-di-AMP levels. As methicillin-resistant S. aureus strains are considered as a global health threat, it is important to study processes that dictate cellular c-di-AMP levels, which are associated with antibiotic resistance.

Heterologous Expression of Ketoreductase ChKRED20 Mutant in Pichia pastoris and Bioreductive Production of (R)-1, 3-Butanediol.

(R)-1, 3-Butanediol (1, 3-BDO) is an important intermediate in the synthesis of aromatics, pheromones, insecticides, and beta-lactam antibiotics. The ChKRED20 is a robust NADH-dependent ketoreductase identified from Chryseobacterium sp. CA49. We obtained a ChKRED20 mutant (M12) through directed evolutionary screening of ChKRED20, the mutant with significantly improved activity to asymmetrically reduce 4-hydroxy-2-butanone (4H2B) to (R)-1, 3-BDO. So far, both ChKRED20 and its mutants have been expressed in intracellular in E. coli, the process of purification after intracellular expression is complicated, which leads to high cost. Here, we expressed M12 by constructing multicopy expression strains in P. pastoris, and the target protein yield was 302 mg/L in shake-flask fermentation and approximately 3.5 g/L in high-density fermentation. The recombinant M12 showed optimal enzyme activity at 30 °C and had high activity within a broad pH range of 6.0-8.0, and also showed high thermal stability. The recombinant M12 was further used for the reduction of 4H2B to (R)-1, 3-BDO, and 98.9% yield was achieved at 4540 mM 4H2B. The crude M12 enzyme extract was found to catalyze the bioreductive production of (R)-1, 3-BDO with excellent stereoselectivity (ee > 99%) and meet the production requirements. Our research shows that the M12 mutant can be used for the synthesis of (R)-1, 3-BDO, and the P. pastoris expression system is an ideal platform for the large-scale, low-cost preparation of ChKRED20 or its mutants, which may have applications in industrial settings.

489 Antibiotic Resistance of cecal Escherichia coli isolates from Lohmann LSL lite pullets and laying hens fed omega-3 fatty acids or yeast bioactives, reared at different spacing allowance

Abstract Poultry diets provide components that alter gut microbiota composition. We investigated antimicrobial resistance (AMR) phenotypes of E. coli isolates from ceca digesta of pullets and hens reared at different spacing allowance (SA) and dietary source of omega-3 fatty acids (N-3 FA) and yeast bioactives (YB) from placement to 17 wk of age (woa). A total of 2,632-day old chicks were placed in cages at two SA (24 cages each): High SA (HSA, 348 cm2/bird; ~50 birds/cage) and Low SA (LSA, 284 cm2/bird; ~68 birds/cage). Birds were fed corn and soybean-based diets (C), C + 3% co-extruded full fat flaxseed and pulse mixture (N-3 FA) and C + 0.05% YB processed with β-1,3-glucan hydrolase. At 4, 16 and 35 woa, ceca samples were collected to isolate E. coli using Chromocult agar. Their susceptibility to 14 antibiotics were determined by automated Sensititre system according to CLSI and CIPARS guidelines to determine their antimicrobial susceptibility profile. Number of antibiotics resistant E. coli isolates were analyzed using general linear mixed model procedure of SAS with diets, SA, sampling age and associated interactions as fixed factors. Cochran-Mantel-Haenszel test was used to determine the relationship between these treatments, and differences between treatments were considered significant at P &amp;lt; 0.05. Out of 428 presumptive E. coli isolated, 153 of 428 isolates (35.7%) were resistant to one antimicrobial class but no resistance against ciprofloxacin, azithromycin and nalidixic acid were detected. The prevalence of antimicrobial resistant E. coli decreased as the birds aged from wk 4–35 (P &amp;lt; 0.05). At wk 4 (P = 0.002) and 16 (P = 0.011), the least prevalence of ampicillin (AMP) resistance was in ceca digesta from N-3 FA-fed birds. At 35 woa, no AMP and cefoxitin (FOX) resistance were detected in birds fed YB (Table 2). At 16 woa, the prevalence of streptomycin resistance was greater in N-3 FA-fed than in YB-fed birds (P = 0.02), which was less than control-fed birds. Of the 428 isolates, 59 (13.8%) were resistant to more than two antimicrobial classes, with most from TIO-FOX-AMP-TET and STR-SULF-TET. At wk-4, SA had a significant effect on multiple resistance spectrum with LSA having the lowest MDR E. coli (P = 0.02) mainly from N-3 FA-fed pullets. This study showed an overall low AMR prevalence which decreases with age of the bird. This study is the first to report that feed additives (N-3 FA or YB) influence the AMR profile of cecal E. coli in pullets and layers. Prevalence of AMP resistance was least in N-3 FA-fed birds, whereas least STEP- and SULF resistance was observed in YB-fed birds. Our next study would help understand the molecular and evolutionary aspect of this resistant E. coli and their potential to cause colibacillosis.

Molecular detection of OXA-48 and NDM-1 carbapenemase genes among clinical isolates of Klebsiella pneumoniae recovered from patients attending a private tertiary hospital in Southwestern Nigeria

There have been increasing reports of Klebsiella pneumoniae resistant to β-lactam antibiotics. This study aimed to determine the prevalence of some selected carbapenemase genes among clinical isolates of Klebsiella pneumoniae recovered from patients attending a private tertiary hospital in Southwestern Nigeria. The study was conducted over two months (February-March 2024). A total of 50 clinical isolates of Klebsiella pneumoniae from different clinical specimens were obtained from the Medical Microbiology Department, Babcock University Teaching Hospital (BUTH). The clinical isolates were then characterized using standard microbiological procedures and were tested for susceptibility to meropenem and other classes of antibiotics according to Clinical and Laboratory Standards Institute (CLSI) guidelines. Polymerase Chain Reaction (PCR) detection for OXA-48 and NDM-1 carbapenemase genes was performed on the 50 clinical isolates. PCR analysis showed that 9 (18%) clinical isolates were positive for the OXA-48 gene, 22 (44%) were positive for the NDM-1 gene, 4 (8%) possessed both the OXA-48 and NDM-1 genes, and 23 (46%) possessed neither the OXA-48 nor NDM-1 genes. Antibiotic Susceptibility Testing (AST) revealed that all the clinical isolates were resistant to meropenem. In conclusion, this study demonstrates the presence of OXA-48 and NDM-1 genes in clinical isolates of Klebsiella pneumoniae recovered from patients attending a private tertiary hospital in Southwestern Nigeria, highlighting the role of ESBL (extended-spectrum beta-lactamase) as a major resistance mechanism alongside other mechanisms. Population-based surveillance programs should be implemented to monitor the prevalence and epidemiology of Klebsiella pneumoniae infections at the community level, facilitating early detection of outbreaks and identification of emerging antimicrobial resistance patterns.Core TipThis study highlights the significant prevalence of NDM-1 and OXA-48 carbapenemase genes among Klebsiella pneumoniae clinical isolates in a private tertiary hospital in Southwestern Nigeria, with 44% and 18% of isolates harboring these genes, respectively. Notably, 46% of isolates were resistant to carbapenems despite lacking these genes, suggesting alternative resistance mechanisms. The findings underscore the urgent need for enhanced surveillance, infection control measures, and antibiotic stewardship programs to combat the spread of multidrug-resistant Klebsiella pneumoniae in healthcare settings.

Predictors of mortality and severe illness from Escherichia coli sepsis in neonates.

Neonatal Escherichia coli (E. coli) sepsis is increasing. There is limited data on the factors contributing to increased mortality and severity of illness in neonatal E. coli sepsis. A retrospective review of neonates (<30 days) admitted to a Level IV NICU in the United States from 2008 to 2022 diagnosed with E. coli bloodstream or cerebrospinal fluid infection was conducted. Primary outcome was defined as mortality from or severe illness during E. coli infection (defined as a need for inotropic support or metabolic acidosis). E. coli neonatal sepsis rate increased from 2008 to 2022 (average of 1.12 per 1000 live births). The primary outcome, which occurred in 57.4% of cases, was independently associated with prematurity, neutropenia, and thrombocytopenia. Ampicillin resistance was not associated with the primary outcome. GA, neutropenia, and thrombocytopenia but not ampicillin resistance, are associated with mortality or severe illness from E. coli sepsis.

Synthetic Strategies towards the Generation of Penicillin-Containing Hybrids in the Search for Anticancer Activity.

An extended library of hybrids that combined a penicillin derivative with a peptoid moiety was designed and synthetized using either a solid-phase or a mixed solid-phase/solution-phase strategy. The library was further evaluated for antiproliferative activity. While none of the different synthesized compounds showed significant cytotoxicity against a normal cell line, tumor cell results drew several conclusions, when comparing with our reference, the highly active triazolylpeptidyl penicillin derivative, TAF7f. Thus, when the 1,2,3-triazole group was exchanged by its "retro-inverse" analogue, no change was noted in the activity of the hybrids; however, better performance was generally obtained if the triazole is replaced by a glycine moiety. Additionally, the absence of hydrogen bond donor groups decreased the compounds activity, which could explain that, in general, this set of derivatives were less active than their peptide-containing analogues. From this study, is indisputable that, regardless of the type of chain (peptide, peptoid or mixture) attached to penicillin, an isobutyl side chain placed in the position closest to penicillin and a benzyl in the next position are determinant for the activity.

Characterization of acquired β-lactamases in Pseudomonas aeruginosa and quantification of their contributions to resistance.

Pseudomonas aeruginosa is a highly problematic opportunistic pathogen that causes a range of different infections. Infections are commonly treated with β-lactam antibiotics, including cephalosporins, monobactams, penicillins, and carbapenems, with carbapenems regarded as antibiotics of last resort. Isolates of P. aeruginosa can contain horizontally acquired bla genes encoding β-lactamase enzymes, but the extent to which these contribute to β-lactam resistance in this species has not been systematically quantified. The overall aim of this research was to address this knowledge gap by quantifying the frequency of β-lactamase-encoding genes in P. aeruginosa and by determining the effects of β-lactamases on susceptibility of P. aeruginosa to β-lactams. Genome analysis showed that β-lactamase-encoding genes are present in 3% of P. aeruginosa but are enriched in carbapenem-resistant isolates (35%). To determine the substrate antibiotics, 10 β-lactamases were expressed from an integrative plasmid in the chromosome of P. aeruginosa reference strain PAO1. The β-lactamases reduced susceptibility to a variety of clinically used antibiotics, including carbapenems (meropenem, imipenem), penicillins (ticarcillin, piperacillin), cephalosporins (ceftazidime, cefepime), and a monobactam (aztreonam). Different enzymes acted on different β-lactams. β-lactamases encoded by the genomes of P. aeruginosa clinical isolates had similar effects to the enzymes expressed in strain PAO1. Genome engineering was used to delete β-lactamase-encoding genes from three carbapenem-resistant clinical isolates and increased susceptibility to substrate β-lactams. Our findings demonstrate that acquired β-lactamases play an important role in β-lactam resistance in P. aeruginosa, identifying substrate antibiotics for a range of enzymes and quantifying their contributions to resistance.IMPORTANCEPseudomonas aeruginosa is an extremely problematic pathogen, with isolates that are resistant to the carbapenem class of β-lactam antibiotics being in critical need of new therapies. Genes encoding β-lactamase enzymes that degrade β-lactam antibiotics can be present in P. aeruginosa, including carbapenem-resistant isolates. Here, we show that β-lactamase genes are over-represented in carbapenem-resistant isolates, indicating their key role in resistance. We also show that different β-lactamases alter susceptibility of P. aeruginosa to different β-lactam antibiotics and quantify the effects of selected enzymes on β-lactam susceptibility. This research significantly advances the understanding of the contributions of acquired β-lactamases to antibiotic resistance, including carbapenem resistance, in P. aeruginosa and by implication in other species. It has potential to expedite development of methods that use whole genome sequencing of infecting bacteria to inform antibiotic treatment, allowing more effective use of antibiotics, and facilitate the development of new antibiotics.

Effect of cephalexin on chlorophyll and carotenoid content of Chlorella pyrenoidosa-2378 and its biodegradation in BG-11 medium

Cephalexin (CFX), a β-lactam antibiotic which is identified for the treatment of different disease infections, has been reported as a micropollutant in wastewater released from sewage, hospital, or pharmaceutical industries. Chlorella pyrenoidosa-2378, a green microalgal strain, is recognized for its degradation ability against wastewater pollutants and the potential of the biomass it produces. In this present study, the cultivation process of Chlorella pyrenoidosa-2378 strain with CFX concentration of 50 mg/L, 100 mg/L, 150 mg/L, and 200 mg/L added to its culture medium labeled as test I, test II, test III, and test IV, respectively, is the process being observed for evaluation of its degradation ability and as sustainable approach for antibiotic degradation. chlorophyll a in test IV, chlorophyll b in test III, and carotenoid content in test I were observed to be higher in amount than control by 0.775 mg/L, .069 mg/L, and 0.356 mg/L, respectively. Using the HPLC method, the total removal efficiency was observed to be 79.09%, 86.26%, 89.62%, and 88.03% against 50, 100, 150, and 200 mg/L concentrations of CFX, respectively. The observations that C. pyrenoidosa-2378 remained stable while being used as an alternative bioremediation method, provide an alibi for its novelty and potentiality to be used commercially for biomass production and at industrial scale for degradation.Graphical

Antibiotics at End of Life: Where Are We Now and Where Are We Going? A Narrative Review.

Antibiotics are frequently prescribed at the end of life, though the benefits and harms are not well understood. We abstracted relevant findings from articles published in English in the past 25years to answer questions generated by discussion among the authors and with stakeholders in Palliative Care and Infectious Diseases. Prescribing practices vary based on individual situation and geographic location. Patients with cancer and those hospitalized receive more antibiotics than those enrolled in outpatient hospice. Urinary tract infections and pulmonary infections are the most common conditions treated with antibiotics at the end of life -most often with penicillin derivatives and vancomycin in the hospital, fluoroquinolones in outpatient, and cephalosporins in both settings. When asked, patients most often prefer limiting antibiotics to symptom management at the end of life. Physicians' over-estimation of patient preference for antibiotics and the increased probability of misdiagnosis increases antibiotic prescription rates. Antibiotics can improve symptoms when used for specific diseases at the cost of drug reactions, resistant organisms, and delayed discharge. Antibiotic use has variable results on survival duration. Antimicrobial stewardship exists in hospital and long-term care facilities, but not outpatient hospice groups. Stewardship interventions could increase proper use of antibiotics, but more information is needed to apply these interventions to hospice groups. Antibiotics at the end of life are impactful and efforts to educate patients and providers will be invaluable in optimizing care.

Pharmacophore-Based Study: An In Silico Perspective for the Identification of Potential New Delhi Metallo-β-lactamase-1 (NDM-1) Inhibitors.

In the ongoing battle against antibiotic-resistant bacteria, New Delhi metallo-β-lactamase-1 (NDM-1) has emerged as a significant therapeutic challenge due to its ability to confer resistance to a broad range of β-lactam antibiotics. This study presents a pharmacophore-based virtual screening, docking, and molecular dynamics simulation approach for the identification of potential inhibitors targeting NDM-1, a critical enzyme associated with antibiotic resistance. Through the generation of a pharmacophore model and subsequent virtual screening of compound libraries, candidate molecules (ZINC29142850 (Z1), ZINC78607001 (Z2), and ZINC94303138 (Z3)) were prioritized based on their similarity to known NDM-1 binder (hydrolyzed oxacillin (0WO)). Molecular docking studies further elucidated the binding modes and affinities of the selected compounds towards the active site of NDM-1. These compounds demonstrated superior binding affinities to the enzyme compared to a control compound (-7.30 kcal/mol), with binding scores of -7.13, -7.92, and -8.10 kcal/mol, respectively. Binding interactions within NDM-1's active site showed significant interactions with critical residues such as His250, Asn220, and Trp93 for these compounds. Subsequent molecular dynamics simulations were conducted to assess the stability of the ligand-enzyme complexes, showing low root mean square deviation (RMSD) values between 0.5 and 0.7 nm for Z1, Z2, which indicate high stability. Z2's compactness in principal component analysis (PCA) suggests that it can stabilize particular protein conformations more efficiently. Z2 displays a very cohesive landscape with a notable deep basin, suggesting a very persistent conformational state induced by the ligand, indicating robust binding and perhaps efficient inhibition. Z2 demonstrates the highest binding affinity among the examined compounds with a binding free energy of -25.68 kcal/mol, suggesting that it could offer effective inhibition of NDM-1. This study highlights the efficacy of computational tools in identifying novel antimicrobial agents against resistant bacteria, accelerating drug discovery processes.

Effects of ZinT-Mediated Zinc Sequestration on Metallo-β-Lactamase Function in Escherichia coli

β-lactams are the most prescribed antibiotic class, but their clinical utility is threatened by the production of metallo-β-lactamase (MBL) enzymes by resistant bacteria. MBLs such as NDM-1 employ zinc ions as cofactors, allowing them to degrade β-lactams. The zinc chaperone ZinT transports zinc into the cytoplasm for intracellular functions, potentially decreasing the availability of zinc in the periplasm for MBLs. We hypothesize that when zinc is limited, ZinT could impair NDM-1 activity and make bacteria more susceptible to β-lactams. To determine whether ZinT and NDM-1 compete for periplasmic zinc, bacterial growth and NDM-1 activity were tested in zinc-poor M9 minimal media for NDM-1-producing Escherichia coli strains with different levels of zinT expression. In addition to wild-type and ΔzinT knockout strains, a recombinant zinT-containing plasmid was transformed into ΔzinT knockout cells to produce a complementation strain with increased zinT expression. Growth assays were conducted in M9 with various concentrations of the β-lactam antibiotic, meropenem. Under these conditions, the ΔzinT strain grew in significantly higher meropenem concentrations than the other strains tested. NDM-1 activity assays revealed that the ΔzinT strain hydrolyzed meropenem notably faster than wild-type cells, whereas the complementation strain showed almost no hydrolysis. This suggests that the increased ZinT production in the complementation strain effectively sequestered zinc from NDM-1. These differences in meropenem hydrolysis and bacterial growth were absent in zinc-sufficient Cation-Adjusted Mueller-Hinton Broth, providing evidence that ZinT and NDM-1 compete for periplasmic zinc under nutrient deficiency. Overall, our results elucidate the effects of competing zinc requirements under zinc-deficient conditions resembling the host environment during a bacterial infection. In these zinc-deficient environments, ZinT can deprive NDM-1 of zinc, causing impaired enzymatic activity and bacterial survival. By better understanding how MBLs function under physiological conditions, future investigations can more reliably explore countermeasures to the growing threat of antibiotic resistance.

Negative Predictive Value of Single-Day Drug Provocation Test for Immediate and Non-Immediate Beta-Lactam Hypersensitivity Reactions in Children

Introduction: Beta-lactam antibiotics (BLAs) commonly cause hypersensitivity reactions in children. These reactions are categorized into immediate reactions, which include urticaria, angioedema, bronchospasm, and anaphylaxis, and non-immediate reactions, such as maculopapular rashes and delayed-onset urticaria/angioedema. Rashes in children, often caused by infections, may be misdiagnosed as BLA allergy. However, over 90% tolerate the medication following an allergic evaluation. Methods: We aimed to evaluate patients with negative single-day drug provocation test (sdDPT) results for subsequent reactions and to determine the negative predictive value (NPV) of sdDPT for immediate (less than 1 h) and non-immediate (more than 1 h) suspected BLA allergy. In addition, non-immediate reactions were assessed by classifying them as occurring within 1–6 h or after 6 h. Patients who underwent sdDPT for suspected BLA allergy and tested negative between 2019 and 2023 were included in the study. They were questioned via telephone interviews about their reuse of the tested drug. Results: 404 patients who underwent sdDPT for suspected BLA allergy were evaluated. The NPV of BLA sdDPT was determined to be 97.3%. When patients were categorized based on the time interval between the last dose and the reaction, the NPV was 97% for those experiencing a reaction within the first hour of drug use and 96.7% for reactions occurring after more than 1 h. Non-immediate reactions were further evaluated, revealing an NPV of 98.7% for reactions occurring between 1 and 6 h, and 92.5% for reactions occurring after 6 h. Conclusion: Our findings demonstrate that sdDPT has a high NPV for both immediate and non-immediate reactions. However, the NPV of sdDPT was lower for reactions occurring more than 6 h after the last dose.

RcsF-independent mechanisms of signaling within the Rcs Phosphorelay.

The Rcs (regulator of capsule synthesis) phosphorelay is a conserved cell envelope stress response mechanism in enterobacteria. It responds to perturbations at the cell surface and the peptidoglycan layer from a variety of sources, including antimicrobial peptides, beta-lactams, and changes in osmolarity. RcsF, an outer membrane lipoprotein, is the sensor for this pathway and activates the phosphorelay by interacting with an inner membrane protein IgaA. IgaA is essential; it negatively regulates the signaling by interacting with the phosphotransferase RcsD. We previously showed that RcsF-dependent signaling does not require the periplasmic domain of the histidine kinase RcsC and identified a dominant negative mutant of RcsD that can block signaling via increased interactions with IgaA. However, how the inducting signals are sensed and how signal is transduced to activate the transcription of the Rcs regulon remains unclear. In this study, we investigated how the Rcs cascade functions without its only known sensor, RcsF and characterized the underlying regulatory mechanisms for three distinct RcsF-independent inducers. Previous reports showed that Rcs signaling can be induced in the absence of RcsF by a loss of function mutation in the periplasmic oxidoreductase DsbA or by overexpression of the DnaK cochaperone DjlA. We identified an inner membrane protein, DrpB, as a multicopy RcsF-independent Rcs activator in E. coli. The loss of the periplasmic oxidoreductase DsbA and the overexpression of the DnaK cochaperone DjlA each trigger the Rcs cascade in the absence of RcsF by weakening IgaA-RcsD interactions in different ways. In contrast, the cell-division associated protein DrpB uniquely requires the RcsC periplasmic domain for signaling; this domain is not needed for RcsF-dependent signaling. This suggests the possibility that RcsC acts as a sensor for some Rcs signals. Overall, the results add new understanding to how this complex phosphorelay can be activated by diverse mechanisms.