Penicillin-binding Protein Research Articles

Modified Carba PBP test for rapid detection and differentiation between different classes of carbapenemases in Enterobacterales.

An advanced biochemical assay named modified Carba PBP test was innovated to identify and differentiate distinct categories of clinically significant carbapenemases (Ambler classes A, B, and D) within the Enterobacterales. The mechanism of mCarba PBP hinges on two core attributes: (i) the hydrolysis of the meropenem substrate by various carbapenemases, (ii) the immobilized penicillin and free meropenem in their affinity to interact with a limited quantity of penicillin-binding protein (PBP). Specific inhibitors for class A (phenylboronic acid, PBA) and class B (ethylenediaminetetraacetic acid, EDTA) were employed to inhibit the hydrolysis activity of carbapenemase and facilitate the classification of carbapenemase classes within 25min. A comprehensive evaluation was undertaken using 94 clinical Enterobacterales isolates, comprising 75 carbapenemase-producing strains and 19 non-carbapenemase-producing strains. Its overall specificity and sensitivity were 100% and 97.3%, respectively, including detection of all types of OXA-48-like carbapenemases. For precise carbapenemase type identification, the assay exhibited remarkable sensitivities for class A, class B, and class D detection at 94.7%, 100%, and 100%, respectively. This user-friendly test presents a promising tool for carbapenemase identification, refining the selection of β-lactam/β-endoenzyme inhibitor combinations for effectively treating infections due to carbapenemase-producing organisms.

Kinact/KI Value Determination for Penicillin-Binding Proteins in Live Cells.

Penicillin-binding proteins (PBPs) are an essential family of bacterial enzymes that are covalently inhibited by the β-lactam class of antibiotics. PBP inhibition disrupts peptidoglycan biosynthesis, which results in deficient growth and proliferation, and ultimately leads to lysis. IC50 values are often employed as descriptors of enzyme inhibition and inhibitor selectivity, but can be misleading in the study of time-dependent, covalent inhibitors. Due to this disconnect, the second-order rate constant, kinact/KI, is a more appropriate metric of covalent-inhibitor potency. Despite being the gold standard measurement of potency, kinact/KI values are typically obtained from in vitro assays, which limits assay throughput if investigating an enzyme family with multiple homologues (such as the PBPs). Therefore, we developed a whole-cell kinact/KI assay to define inhibitor potency for the PBPs in Streptococcus pneumoniae using the fluorescent, activity-based probe, Bocillin-FL. Our results align with in vitro kinact/KI data and show a comparable relationship to previously established IC50 values. These results support the validity of our in vivo kinact/KI method as a means of obtaining β-lactam potency for a suite of PBPs to enable structure-activity relationship studies.

Revisiting mutational resistance to ampicillin and cefotaxime in Haemophilus influenzae

BackgroundHaemophilus influenzae is an opportunistic bacterial pathogen that can cause severe respiratory tract and invasive infections. The emergence of β-lactamase-negative ampicillin-resistant (BLNAR) strains and unclear correlations between genotypic (i.e., gBLNAR) and phenotypic resistance are challenging empirical treatments and patient management. Thus, we sought to revisit molecular resistance mechanisms and to identify new resistance determinants of H. influenzae.MethodsWe performed a systematic meta-analysis of H. influenzae isolates (n = 291) to quantify the association of phenotypic ampicillin and cefotaxime resistance with previously defined resistance groups, i.e., specific substitution patterns of the penicillin binding protein PBP3, encoded by ftsI. Using phylogenomics and a genome-wide association study (GWAS), we investigated evolutionary trajectories and novel resistance determinants in a public global cohort (n = 555) and a new clinical cohort from three European centers (n = 298), respectively.ResultsOur meta-analysis confirmed that PBP3 group II- and group III-related isolates were significantly associated with phenotypic resistance to ampicillin (p < 0.001), while only group III-related isolates were associated with resistance to cefotaxime (p = 0.02). The vast majority of H. influenzae isolates not classified into a PBP3 resistance group were ampicillin and cefotaxime susceptible. However, particularly group II isolates had low specificities (< 16%) to rule in ampicillin resistance due to clinical breakpoints classifying many of them as phenotypically susceptible. We found indications for positive selection of multiple PBP3 substitutions, which evolved independently and often step-wise in different phylogenetic clades. Beyond ftsI, other possible candidate genes (e.g., oppA, ridA, and ompP2) were moderately associated with ampicillin resistance in the GWAS. The PBP3 substitutions M377I, A502V, N526K, V547I, and N569S were most strongly related to ampicillin resistance and occurred in combination in the most prevalent resistant haplotype H1 in our clinical cohort.ConclusionsGradient agar diffusion strips and broth microdilution assays do not consistently classify isolates from PBP3 groups as phenotypically resistant. Consequently, when the minimum inhibitory concentration is close to the clinical breakpoints, and genotypic data is available, PBP3 resistance groups should be prioritized over susceptible phenotypic results for ampicillin. The implications on treatment outcome and bacterial fitness of other extended PBP3 substitution patterns and novel candidate genes need to be determined.

Production of Natural Penicillin-Binding Protein 2 and Development of a Signal-Amplified Fluorescence Polarization Assay for the Determination of 28 Beta-Lactam Antibiotics in Milk

Production of Natural Penicillin-Binding Protein 2 and Development of a Signal-Amplified Fluorescence Polarization Assay for the Determination of 28 Beta-Lactam Antibiotics in Milk

Green Fabrication of Silver Nanoparticles, Statistical Process Optimization, Characterization, and Molecular Docking Analysis of Their Antimicrobial Activities onto Cotton Fabrics

Nanotechnological methods for creating multifunctional fabrics are attracting global interest. The incorporation of nanoparticles in the field of textiles enables the creation of multifunctional textiles exhibiting UV irradiation protection, antimicrobial properties, self-cleaning properties and photocatalytic. Nanomaterials-loaded textiles have many innovative applications in pharmaceuticals, sports, military the textile industry etc. This study details the biosynthesis and characterization of silver nanoparticles (AgNPs) using the aqueous mycelial-free filtrate of Aspergillus flavus. The formation of AgNPs was indicated by a brown color in the extracellular filtrate and confirmed by UV-Vis spectroscopy with a peak at 426 nm. The Box-Behnken design (BBD) is used to optimize the physicochemical parameters affecting AgNPs biosynthesis. The desirability function was employed to theoretically predict the optimal conditions for the biosynthesis of AgNPs, which were subsequently experimentally validated. Through the desirability function, the optimal conditions for the maximum predicted value for the biosynthesized AgNPs (235.72 µg/mL) have been identified as follows: incubation time (58.12 h), initial pH (7.99), AgNO3 concentration (4.84 mM/mL), and temperature (34.84 °C). Under these conditions, the highest experimental value of AgNPs biosynthesis was 247.53 µg/mL. Model validation confirmed the great accuracy of the model predictions. Scanning electron microscopy (SEM) revealed spherical AgNPs measuring 8.93–19.11 nm, which was confirmed by transmission electron microscopy (TEM). Zeta potential analysis indicated a positive surface charge (+1.69 mV), implying good stability. X-ray diffraction (XRD) confirmed the crystalline nature, while energy-dispersive X-ray spectroscopy (EDX) verified elemental silver (49.61%). FTIR findings indicate the presence of phenols, proteins, alkanes, alkenes, aliphatic and aromatic amines, and alkyl groups which play significant roles in the reduction, capping, and stabilization of AgNPs. Cotton fabrics embedded with AgNPs biosynthesized using the aqueous mycelial-free filtrate of Aspergillus flavus showed strong antimicrobial activity. The disc diffusion method revealed inhibition zones of 15, 12, and 17 mm against E. coli (Gram-negative), S. aureus (Gram-positive), and C. albicans (yeast), respectively. These fabrics have potential applications in protective clothing, packaging, and medical care. In silico modeling suggested that the predicted compound derived from AgNPs on cotton fabric could inhibit Penicillin-binding proteins (PBPs) and Lanosterol 14-alpha-demethylase (L-14α-DM), with binding energies of −4.7 and −5.2 Kcal/mol, respectively. Pharmacokinetic analysis and sensitizer prediction indicated that this compound merits further investigation.

Affinity of cefditoren for penicillin-binding proteins in bacteria and its relationship with antibiotic sensitivity.

Penicillin-binding proteins (PBPs) are the targets of β-lactam antibiotics; however, changes in the affinity of PBPs for beta-lactam antibiotics often affect the susceptibility of bacteria to antibiotics. The purpose of this study was to elucidate the mechanism by which cefditoren, an oral third-generation cephalosporin, binds PBPs. The minimal inhibitory concentration (MIC), bactericidal curves, and inhibition zone comparisons were assessed to evaluate the antibacterial activity of cefditoren. PBP1A and PBP2X proteins from Streptococcus pneumoniae were purified, and their ability to bind to cefditoren was investigated via microscale thermophoresis. The Kd of cefditoren toward PBP1A was 0.005±0.004 µM, which was lower than those of other cephalosporins (cefcapene, cefixime and cefdinir). In contrast, the Kd of cefditoren toward PBP2X of S. pneumoniae was 9.70±8.24 µM, which was lower than that of cefixime but higher than those of cefcapene and cefdinir. Additionally, the biotinylated ampicillin (BIO-AMP) method was employed to evaluate the affinity of cefditoren toward PBPs of Haemophilus influenzae, and the results demonstrated that cefditoren and PBP3A/B had the lowest IC50 values (0.060±0.002 µM). These findings indicate that cefditoren has a strong affinity for PBP1A of H. influenzae. Cefditoren has a high affinity toward the PBP1As of S. pneumoniae and PBP1A and PBP3A/B of H. influenzae, which may contribute to the effective antibacterial effects of cefditoren against clinical strains and its low propensity for inducing resistance. The data presented in this article help elucidate the mechanism by which cefditoren, an oral third-generation cephalosporin, binds to PBPs and provide theoretical support for the wider use of cefditoren as an antibiotic therapy.

Biosynthesis; Characterization; and Antibacterial, Antioxidant, and Docking Potentials of Doped Silver Nanoparticles Synthesized from Pine Needle Leaf Extract

The current study focused on the synthesis of doped silver nanoparticles (doped AgNPs) with yttrium (Y), gadolinium (Gd), and chromium (Cr) from pine needle leaf extract (PNLE). X-ray diffraction (XRD) was performed to assess the phase formation, detecting 61.83% from Ag and 38.17% for secondary phases of AgCl, AgO, Y, Cr-, and Gd phases. The size and shape of the NPs were determined by transmission electron microscopy (TEM), showing a spherical shape with an average particle size of 26.43 nm. X-ray photoelectron spectroscopy (XPS) detected the oxidation state of the presented elements. The scanning electron microscope (SEM) and the energy-dispersive X-ray analysis (EDX) determined the morphology and elemental composition of the NPs, respectively. Fourier transform infrared spectroscopy (FTIR) determined the different functional groups indicating the presence of Ag, Y, Gd, Cr, and other groups. Photoluminescence (PL) spectroscopy showed the optical properties of the NPs. A vibrating sample magnetometer (VSM) revealed the ferromagnetic behavior of the doped AgNPs. The antibacterial activity of the doped AgNPs was tested against six uro-pathogenic bacteria (Staphylococcus aureus, Staphylococcus haemolyticus, Enterococcus faecalis, Escherichia coli, Klebsiella pneumonia, and Pseudomonas aeruginosa) using the minimum inhibitory concentration (MIC) and minimum bactericidal concentration (MBC) microdilution assays, agar well diffusion assay, time–kill test, and antibiofilm screening assays, revealing significant activity, with MICs ranging between 0.0625 and 0.5 mg/mL and antibiofilm activity between 40 and 85%. The antioxidant activity was determined by the 1,1, diphenyl 1-2 picrylhydrazyl (DPPH) radical scavenging assay with a potential of 61.3%. The docking studies showed that the doped AgNPs had the potential to predict the inhibition of crucial enzymes such as penicillin-binding proteins, LasR-binding proteins, carbapenemase, DNA gyrase, and dihydropteroate synthase. The results suggest that the doped AgNPs can be applied in different medical domains.

CRISPR-Cas9-Mediated Targeting of Multidrug Resistance Genes in Methicillin-Resistant Staphylococcus aureus.

Antibiotic resistance poses a global health crisis limiting the efficacy of available therapeutic agents. We explored CRISPR-Cas-based antimicrobials to combat multidrug resistance in methicillin-resistant Staphylococcus aureus (MRSA), targeting methicillin (mecA), gentamicin (aacA), and ciprofloxacin (grlA, grlB) resistance genes. Engineered CRISPR plasmids with specific single-guide RNAs were electroporated into MRSA strains. Real-time polymerase chain reaction assessed gene expression changes, while antibiotic susceptibility tests (ASTs) evaluated resistance status. Results showed a 1.5-fold decrease in mecA, a 5.5-fold decrease in grlA, a 6-fold decrease in grlB, and a 4-fold decrease in aacA expression. ASTs demonstrated the reversal of resistance to beta-lactam, quinolone, and aminoglycoside antibiotics. Western blot analysis revealed a 70% decrease in penicillin-binding protein 2a expression. Sanger sequencing confirmed point mutations in the grlB and aacA genes. Our findings highlight the potential of CRISPR-Cas9 technology to restore antibiotic efficacy against multidrug-resistant pathogens.

Cefiderocol in Combating Carbapenem-Resistant Acinetobacter baumannii: Action and Resistance.

Acinetobacter baumannii (A. baumannii) has emerged as a prominent multidrug-resistant (MDR) pathogen, significantly complicating treatment strategies due to its formidable resistance mechanisms, particularly against carbapenems. Reduced membrane permeability, active antibiotic efflux, and enzymatic hydrolysis via different β-lactamases are the main resistance mechanisms displayed by A. baumannii, and they are all effective against successful treatment approaches. This means that alternate treatment approaches, such as combination therapy that incorporates beta-lactams, β-lactamase inhibitors, and novel antibiotics like cefiderocol, must be investigated immediately. Cefiderocol, a new catechol-substituted siderophore cephalosporin, improves antibacterial activity by allowing for better bacterial membrane penetration. Due to its unique structure, cefiderocol can more efficiently target and destroy resistant bacteria by using iron transport systems. Through its inhibition of peptidoglycan formation through binding to penicillin-binding proteins (PBPs), cefiderocol avoids conventional resistance pathways and induces bacterial cell lysis. The possibility of resistance development due to β-lactamase synthesis and mutations in PBPs, however, emphasizes the need for continued investigation into cefiderocol's efficacy in combination treatment regimes. Cefiderocol's siderophore mimic mechanism is especially important in iron-limited conditions because it can use ferric-siderophore transporters to enter cells. Additionally, its passive diffusion through bacterial porins increases its intracellular concentrations, making it a good option for treating carbapenem-resistant A. baumannii, especially in cases of severe infections and ventilator-associated diseases (IVACs). Cefiderocol may reduce MDR infection morbidity and mortality when combined with customized antimicrobial treatments, but further investigation is needed to improve patient outcomes and address A. baumannii resistance issues.

Conjugation of Polycationic Peptides Extends the Efficacy Spectrum of β-Lactam Antibiotics.

Antibiotic-resistant enterococci represent a significant global health challenge. Unfortunately, most β-lactam antibiotics are not applicable for enterococcal infections due to intrinsic resistance. To extend their antimicrobial spectrum, polycationic peptides are conjugated to examples from each of the four classes of β-lactam antibiotics. Remarkably, the β-lactam-peptide conjugates gained an up to 1000-fold increase in antimicrobial activity against vancomycin-susceptible and vancomycin-resistant enterococci. Even against β-lactam-resistant Gram-negative strains, the conjugates are found to be effective despite their size exceeding the exclusion volume of porins. The extraordinary gain of activity can be explained by an altered mode of killing. Of note, the conjugates showed a concentration-dependent activity in contrast to the parent β-lactam antibiotics that exhibited a time-dependent mode of action. In comparison to the parent β-lactams, the conjugates showed altered affinities to the penicillin-binding proteins. Furthermore, it is found that peptide conjugation also resulted in a different elimination route of the compounds when administered to rodents. In mice systemically infected with vancomycin-resistant enterococci, treatment with a β-lactam-peptide conjugate reduced bacterial burden in the liver compared to its originator. Therefore, peptide modification of β-lactam antibiotics represents a promising platform strategy to broaden their efficacy spectrum, particularly against enterococci.

Solution structure, oxidative DNA damage, biological activity and molecular docking of ternary copper(II) L−argininato complexes

Continuing our search for metal drugs with markedly higher toxicity to cancer cells than to normal cells, we evaluated the effect of 2,2′-bipyridine (bpy) as a co-ligand in the compounds [Cu(μ−O,O’−NO3)(L−Arg)(bpy)]NO3}n (1) and [CuCl(L−Arg)(bpy)]Cl·3H2O (2) (L−Arg = L−arginine), on DNA interaction, cytotoxic and antiproliferative activity, compared to the effects induced by other co-ligands i.e. 1,10-phenanthroline (phen) and SCN− ions, in similar Cu(II) compounds we have studied previously. Potentiometric, X-band EPR and UV–Vis experiments were first used to structurally characterise the complexes formed in solutions 1 and 2 and in model Cu(II)/bpy/L−Arg systems. Gel electrophoresis in the presence of H2O2 was used to identify DNA damage by 1 and 2. In addition, cyclic voltammetry of both compounds was performed to confirm the existence of Cu(II)/Cu(I) redox pairs involved in the free radical mechanism of this DNA damage. The DNA binding constants of 1 and 2 were determined spectrophotometrically. The selectivity of the cytotoxic and antiproliferative activity of compounds 1 and 2 was tested in vitro against human lung adenocarcinoma (A549), liver cancer (HepG2) and normal cells in comparison with those previously observed by us for compounds consisting of phen and SCN− ligands. Molecular docking calculations were performed for [Cu(L−Arg)(bpy)]2+ species (arraised in solutions of 1 and 2) interacting with B-DNA (aureolin), metalloproteinase (S. aureus) and penicillin-binding protein (E. coli) to determine the nature of the complex-receptor interaction, potential binding modes and energies.

Generation and characterization of a monoclonal antibody Fab fragment targeting PBP2a in methicillin-resistant Staphylococcus aureus

Methicillin-resistant Staphylococcus aureus (MRSA) is one of the main pathogens associated with nosocomial and community infections that are difficult to treat owing to its resistance to all β-lactams and other classes of antibiotics. Reports of MRSA demonstrate the pathogen relevance and urgency for developing innovative diagnostic and treatment strategies against this microorganism. In this context, monoclonal antibodies (mAbs) represent a powerful tool for such purposes. Beta-lactam resistance in MRSA is caused by penicillin-binding protein 2a (PBP2a). The characteristics of PBP2a make this protein a potential target for immunobiologicals to combat this pathogen. This study describes the development of a recombinant Fab fragment from a mAb directed against the PBP2a protein, designed to identify and treat MRSA infections. The Fd and light chain coding sequences for Fab expression were amplified and ligated into the mammalian cell expression vector. Recombinant DNA constructs were used to transfect Expi293F cells expressing anti-PBP2a Fab. A purification based on ion-exchange chromatography was used for Fab separation, followed by analysis of antigen target recognition and interaction, either with the isolated antigen or with the antigen on the MRSA cell surface. The experimental approach allowed us to obtain significant Fab expression levels in the Expi293F system when transfecting the cells with the genetic constructs developed in pCDNA3.4 vector. Antigen target interaction assays revealed the capacity of Fab to recognize and interact with the PBP2a protein. Biodistribution analysis indicated serum Fab presence, in the serum, kidneys, lungs, and spleen, and a plasma half-life averaging 6–8 h.

Two codependent routes lead to high-level MRSA.

Methicillin-resistant Staphylococcus aureus (MRSA), in which acquisition of mecA [which encodes the cell wall peptidoglycan biosynthesis component penicillin-binding protein 2a (PBP2a)] confers resistance to β-lactam antibiotics, is of major clinical concern. We show that, in the presence of antibiotics, MRSA adopts an alternative mode of cell division and shows an altered peptidoglycan architecture at the division septum. PBP2a can replace the transpeptidase activity of the endogenous and essential PBP2 but not that of PBP1, which is responsible for the distinctive native septal peptidoglycan architecture. Successful division without PBP1 activity requires the alternative division mode and is enabled by several possible chromosomal potentiator (pot) mutations. MRSA resensitizing agents differentially interfere with the two codependent mechanisms required for high-level antibiotic resistance, which provides opportunities for new interventions.

Global emergence of Escherichia coli with PBP3 insertions.

Escherichia coli producing metallo-β-lactamases with penicillin-binding protein 3 (PBP3) insertions have reduced susceptibility to aztreonam-avibactam and cefiderocol. Here, we analysed high-quality E. coli genomes for PBP3 insertions. E. coli genomes (n = 167 518) were retrieved from EnteroBase with CheckM2 for quality control, fastANI for species confirmation, multi-locus sequencing typing for sequence type (ST) determination and AMRFinderPlus for resistance gene identification. For PBP3 insertion analysis, we used Prokka for predicting coding sequences, BLAST+ for comparing resulted protein sequences and SnpEff for annotating variants. Among the included 159 341 genomes, PBP3 insertions with 11 variants were found in 2.01% (n = 3198). The predominant variant is a duplication of 334-337 amino acids (aa) (94.75%, n = 3030), comprising YRIN (65.92%, n = 2108) and its single-aa-variant YRIK (28.83%, n = 922), followed by a similar PYRI duplication of 333-336 (4.16%, n = 133). The less common ones are a TIPY duplication of 331-334 (n = 24) and its single-aa-variant TVPY's duplication: TVVPY (n = 1), TVPYTVPY (n = 1) and TVPYPVPY (n = 1). Rare duplications include VGDR of 106-109 (n = 3), ANALNIPL of 114-121 (n = 3), AL of 567-568 (n = 1) and TG of 584-585 (n = 1). Insertion variants were detected across 62 countries on six continents, primarily in human samples, and associated with 85 STs, concentrated in high-risk clones ST410 (29.18%, n = 1923), ST167 (23.40%, n = 1740) and ST405 (10.56%, n = 1334), with 83.32% (n = 2218) encoding metallo-β-lactamase NDM. Global spread of E. coli harbouring PBP3 insertion, often with NDM β-lactamase, high-risk ST410, ST167 and ST405 clones and various hosts, underscores the escalating antimicrobial resistance crisis and the urgency for a 'One Health' strategy.

In-vitro antibacterial and antibiofilm activities and in-silico analysis of a potent cyclic peptide from a novel Streptomyces sp. strain RG-5 against antibiotic-resistant and biofilm-forming pathogenic bacteria.

The proliferation of multidrug-resistant and biofilm-forming pathogenic bacteria poses a serious threat to public health. The limited effectiveness of current antibiotics motivates the search for new antibacterial compounds. In this study, a novel strain, RG-5, was isolated from desert soil. This strain exhibited potent antibacterial and antibiofilm properties against multidrug-resistant and biofilm-forming pathogenic bacteria. Through phenotypical characterizations, 16S rRNA gene sequence and phylogenetic analysis, the strain was identified as Streptomyces pratensis with 99.8% similarity. The active compound, RG5-1, was extracted, purified by reverse phase silica column HPLC, identified by ESI-MS spectrometry, and confirmed by 1H and 13C NMR analysis as 2,5-Piperazinedione, 3,6-bis(2-methylpropyl), belonging to cyclic peptides. This compound showed interesting minimum inhibitory concentrations (MICs) of 04 to 15µg/mL and minimum biofilm inhibitory concentrations (MBICs 50%) of ½ MIC against the tested bacteria. Its molecular mechanism of action was elucidated through a molecular docking study against five drug-protein targets. The results demonstrated that the compound RG5-1 has a strong affinity and interaction patterns with glucosamine-6-phosphate synthase at - 6.0kcal/mol compared to reference inhibitor (- 5.4kcal/mol), medium with penicillin-binding protein 1a (- 6.1kcal/mol), and LasR regulator protein of quorum sensing (- 5.4kcal/mol), confirming its antibacterial and antibiofilm activities. The compound exhibited minimal toxicity and favorable physicochemical and pharmacological properties. This is the first report that describes its production from Streptomyces, its activities against biofilm-forming and multidrug-resistant bacteria, and its mechanism of action. These findings indicate that 2,5-piperazinedione, 3,6-bis(2-methylpropyl) has the potential to be a promising lead compound in the treatment of antibiotic-resistant and biofilm-forming pathogens.

Unveiling the antibacterial potential of latex-derived phytocomponents from Calotropis gigantea targeting bacterial Penicillin-binding proteins: chemical profiling, in silico docking, and in vitro lab validation

Uncontrolled use of currently accessible medicines has caused antibiotic resistance in bacteria worldwide. This work aimed to undertake molecular docking and chemical profiling of bioactive components extracted from Calotropis gigantea latex, with wet lab confirmation to follow. Gas chromatography was used to analyze the phytochemical profile. The docking tool, Cb-dock2, was used for in-silico analysis. G+ and G- bacterial strains were utilized to confirm the wet lab results. Chemical profiling revealed 18 peaks, and some major bioactive compounds were like phytol, 4-methyl-2- phenylindole and α-tocospiro A. Docking analysis revealed strong interaction against bacterial Penicillin-binding Protein (PBP1a) with docking scores ranging from -5.2 to -7.9 (Vina score). The PBP-Ligand complex has Van der Waals, hydrogen bond, pi-cation, and alkyl bonding interactions, according to the results of the interaction investigations. Calotropis latex successfully suppressed the development of bacterial strains (G+ and G-) with inhibition zones ranging from 1.1 cm to 2.2 cm, as demonstrated by in-vitro tests, indicating their potential as antibacterial medications. The study found that latex-based bioactive compounds have the potential to be used in the pharmaceutical and biotechnology sectors by facilitating the creation of structural alterations that lead to stronger medications. Therefore, this latex-derived compound may open a new pipeline into the development of multi-target antibiotics against a broad-spectrum multidrug-resistant G- bacterium.

A potent antimicrobial glycolipopeptide GLIP and its promising combined antimicrobial effect

Here, the glycolipopeptide GLIP was obtained by coupling IL-C8 and the monosaccharide molecule D-(+)-glucosamine to the N-terminal and C-terminal of the peptide P, which was designed on the basis of the biological characteristics of the antimicrobial peptides. In vitro bioactivity and physicochemical properties assays confirmed that GLIP had excellent antimicrobial activity against Gram-negative E. coli ATCC 25922 and Gram-positive S. aureus ATCC 29213, as well as good stability in serum and trypsin, low hemolysis, and good bacterial membrane-disrupting ability. In addition, the glycolipopeptide GLIP could self-assembly in aqueous solution to form spherical nano-aggregates, which could encapsulate the small molecule antibiotic TC to form the nanomedicine GLIP@TC and release the TC continuously and slowly in a sustained-release manner, exerting the combined antimicrobial effect of both. The results of animal experiments demonstrated the excellent in vivo antimicrobial activities of GLIP and nanomedicine GLIP@TC. Finally, molecular docking experiment showed that the GLIP could effectively bind to penicillin-binding protein 5 (PBP5) of E. coli and possibly inhibit its D-Ala carboxypeptidase (CPase) activity. All these results may imply the great potential of GLIP for clinical application against bacterial drug resistance.

Antibacterial activity of green synthesized copper oxide nanoparticles against multidrug-resistant bacteria

Using plant extracts in the green synthesis of nanoparticles has become an environmentally acceptable approach. In our study, copper oxide nanoparticles (CuO NPs) were synthesized using ethanolic extracts of Azadirachta indica and Simmondsia chinensis. CuO NP formation was confirmed by the change in color and by UV‒visible spectroscopy (CuO NPs peaked at a wavelength of 344 nm). TEM images confirmed the semispherical shape of the CuO NPs, with particle sizes ranging from 30.9 to 10.7 nm. The antibacterial activity of these NPs was evaluated by using the agar diffusion method against clinical isolates, including methicillin-resistant Staphylococcus aureus (MRSA), Escherichia coli, Pseudomonas aeruginosa, Acinetobacter spp., Klebsiella pneumoniae, and Stenotrophomonas maltophilia. The minimum inhibitory concentration (MIC) of CuO NPs ranged from 62.5 to 125 µg/ml. In contrast, the antioxidant activity and antibiofilm activity of CuO NPs ranged from 31.1 to 92.2% at 125–500 µg/ml and 62.2–95%, respectively, at 125 –62.5 µg/ml. Our results confirmed that CuO NPs had IC50s of 383.41 ± 3.4 and 402.73 ± 1.86 at 250 µg/mL against the HBF4 cell line. Molecular docking studies with CuO NPs suggested that penicillin-binding protein 4 (PBP4) and beta-lactamase proteins (OXA-48) strongly bind to S. aureus and K. pneumoniae, respectively, with CuO NPs. Our study confirms the promising use of CuO NPs in treating pathogenic bacteria and that CuO NPs could be possible alternative antibiotics. This study supports the pharmaceutical and healthcare sectors in Egypt and worldwide.

Frameshift Mutations in Genes Encoding PBP3 and PBP4 Trigger an Unusual, Extreme β-Lactam Resistance Phenotype in Burkholderia multivorans.

In our curated panel of Burkholderia cepacia complex isolates, Burkholderia multivorans strain AU28442 was unusually highly β-lactam resistant. To explore the molecular mechanisms leading to this phenotype, we performed whole genome sequencing (WGS) and microbiological and biochemical assays. WGS analysis revealed that strain AU28442 produced two β-lactamases, AmpC22 and a novel PenA-like β-lactamase denominated PenA39. Additionally, the strain presented frame-shift mutations in the genes encoding penicillin binding proteins 3 (PBP3) and 4 (PBP4). The antibiotic susceptibilities of the parent AU28442 strain carrying blaPenA39 vs the isogenic E. colistrain producing blaPenA39 were discrepant with ceftazidime MICs of >512 and 1 μg/mL, respectively. Accordingly, PenA39 was found to poorly hydrolyze β-lactams with kcat values of ≤8.8 s-1. An overlay of the crystal structure of PenA39 with PenA1 revealed a shift in the SDN loop in the variant, which may affect the catalytic efficiency of PenA39 toward substrates and inhibitors. Moreover, microscopic examination of AU28442 revealed shortened rod-shaped cells compared to B. multivoransATCC 17616, which carries a full complement of intact PBPs. Further complementation assays confirmed that the loss of PBP3 and PBP4 was the main factor contributing to the high-level β-lactam resistance observed in B. multivoransAU28442. This information allowed us to revert susceptibility by pairing a potent β-lactamase inhibitor with a β-lactam with promiscuous PBP binding. This detailed characterization of B. multivoransprovides an illustration of the myriad ways in which bacteria under antibiotic selection can develop resistance and demonstrates a mechanism to overcome it.

Probing the interaction mechanisms between three β-lactam antibiotics and penicillin-binding proteins of Escherichia coli by molecular dynamics simulations

The presence of antibiotic residues in the aquatic environments poses great potential risks to the aquatic organisms, and even human health. Elucidating the interaction mechanisms between antibiotics and biomacromolecules is crucial for accurately assessing and preventing their potential risks. Therefore, the toxicity of three beta-lactam antibiotics on Escherichia coli (E. coli) was investigated by using the time-dependent toxicity microplate analysis method in this study. Then, molecular docking and molecular dynamics simulation technologies were used to elucidate the potential molecular interactions between β-lactam antibiotics and penicillin-binding proteins of E. coli, and their correlation with the physical and chemical behaviors observed in the physiological and biochemical experiments. The results show that three antibiotics exert inhibitory effects on E. coli cells by modifying their membrane permeability, and even more severe cell damage including rupture, wrinkling, adhesion, indentation, elongation and size alterations. But, toxic effect of the three antibiotics on E. coli varies, and toxicity order is followed by meropenem > cefoperazone > amoxicillin. Van der Waals forces play a vital role in the molecular interactions between the three antibiotics penicillin binding protein of E. coli and the sequence of binding free energy is consistent with the observed toxicity order. Shape compensation is the principal determinant for the binding of antibiotics to penicillin binding proteins, which pertains to the drug-induced alteration in the three-dimensional conformation of penicillin binding proteins.