High-resolution X-ray structure of an acyl-enzyme species for the class D OXA-10 beta-lactamase

Beta-lactamases are resistance enzymes for beta-lactam antibiotics. These enzymes hydrolyze the beta-lactam moieties of these antibiotics, rendering them inactive. Of the four classes of known beta-lactamases, the enzymes of class D are the least understood. We report herein the high-resolution (1.9 A) crystal structure of the class D OXA-10 beta-lactamase inhibited by a penicillanate derivative. The structure provides evidence that the carboxylated Lys-70 (a carbamate) is intimately involved in the mechanism of the enzyme.     

An in silico approach to elucidate structure based functional evolution of oxacillinase

Bacterial Oxacillinases (OXAs), genetically being extremely diverse and highly versatile in hydrolyzing antibiotics of different classes, holds utmost significant clinical importance. Hence, to analyze functional evolution of this enzyme, plausible changes in drug profile, affinity and binding stability of different subclasses of OXA with their preferred drugs, viz. penicillin, ceftazidime, imipenem/meropenem were investigated. Maximum-Likelihood dendrogram was constructed and based on tree topology, the least and most divergent variants of each clade were selected. Pocket characterization, enzyme structural stability and mutational effect were analyzed in silico. Modes of interaction of selected OXA variants with respective antibiotics were analyzed by Autodock4.0 and LIGPLOT. Comparative mobility profiling and subsequent ΔG° and Km calculations of representative OXA variants revealed that after RSBL evolution, perhaps, two competitive strategies evolved among the OXA variants. Either loops flanking helix5 gets stabilized or it becomes more flexible. Therefore, while OXA variants (e.g. OXA-2, OXA-32, OXA-23, OXA-133, OXA-24, OXA-25, OXA-51 and OXA-75) with highly stabilized loops flanking helix5 exhibited improved binding stability and affinity towards carbapenems, especially meropenem, OXA variants (e.g. OXA-10, OXA-251, OXA-48 and OXA-247) possessing highly flexibile loops flanking helix5 revealed their catalytic proficiency towards ceftazidime. Moreover, LIGPLOT and PROMALS3D jointly identified ten consensuses/conserved residues, viz. P68, A69, F72, K73, W105, V120, W164, L169, K216 and G218 to be critical for drug hydrolysis. Hence, novel inhibitors could be designed to target these sites.     

Rational design of inhibitors against LpxA protein of Acinetobacter baumannii using a virtual screening method

BackgroundAcinetobacter baumannii is a highly antimicrobial resistant nosocomial pathogen. Resistance to currently used antibiotics has limited effective drugs against this bacterium. This study aimed to propose a rational inhibitor design against the LpxA protein of A. baumannii using a virtual screening method based on a similar structure of ligands.MethodsIn this study, we targeted LpxA protein, which is involved in the early stage of LPS biosynthesis. In the next step, we used Peptide920 and 1,2- Ethanediol as templates to find similar compounds using Drugbank and Zinc15 webservers, respectively. Subsequently, molecular dynamics (MD) simulations were carried out for LpxA protein and two complexes of ZINC895081 and Macrolactam-1 which represented the highest binding affinity and best conformation. Finally, ADMET properties, water solubility and drug-likeness of the desired compounds were evaluated using SwissADME and DruLiTo softwares.ResultsAccording to considered criteria, Drugbank suggested 5 compunds including Ilomastat, Macrolactam-1, Macrolactam-2, Macimorelin, and Oglufanide. On the other hand, Zinc15 webserver suggested 4 compunds including ZINC895048, ZINC895081, ZINC901061 and ZINC1531008. The result of the HDOCK server and Molegro virtual docker (MVD) showed that Macrolactam-1 and ZINC895081 (Citrate) had the highest docking score. In addition, MD simulations showed that ZINC895081 and Macrolactam-1 ligands have the stable binding to the LpxA protein. According to Lipinski's rule, these two compounds are non-carcinogenic, non-toxic and promising inhibitors against LpxA of A. baumannii.ConclusionIt seems that Macrolactam-1 and ZINC895081 (Citrate) are two valuable promising inhibitors against the LpxA protein of A. baumannii. Further in vitro and in vivo experiments are needed to confirm the capabilities of these proposed compounds against A. baumannii.     

Antimicrobial Activity of Dihydroisocoumarin Isolated from Wadi Lajab Sediment-Derived Fungus Penicillium chrysogenum: In Vitro and In Silico Study

Antibiotic resistance is considered a major health concern globally. It is a fact that the clinical need for new antibiotics was not achieved until now. One of the most commonly prescribed classes of antibiotics is β-Lactam antibiotics. However, most bacteria have developed resistance against β-Lactams by producing enzymes β-Lactamase or penicillinase. The discovery of new β-Lactamase inhibitors as new antibiotics or antibiotic adjuvants is essential to avoid future catastrophic pandemics. In this study, five dihydroisocoumarin: 6-methoxy mellein (1); 5,6-dihydroxymellein (2); 6-hydroxymellein (3); 4-chloro-6-hydroxymellein (4) and 4-chloro-5,6-di-hydroxymellein (5) were isolated from Wadi Lajab sediment-derived fungus Penicillium chrysogenum, located 15 km northwest of Jazan, KSA. The elucidation of the chemical structures of the isolated compounds was performed by analysis of their NMR, MS. Compounds 1–5 were tested for antibacterial activities against Gram-positive and Gram-negative bacteria. All of the compounds exhibited selective antibacterial activity against Gram-positive bacteria Staphylococcus aureus and Bacillus licheniformis except compound 3. The chloro-dihydroisocoumarin derivative, compound 4, showed potential antimicrobial activities against all of the tested strains with the MIC value between 0.8–5.3 μg/mL followed by compound 5, which exhibited a moderate inhibitory effect. Molecular docking data showed good affinity with the isolated compounds to β-Lactamase enzymes of bacteria; NDM-1, CTX-M, OXA-48. This work provides an effective strategy for compounds to inhibit bacterial growth or overcome bacterial resistance.     

Profiling of Potential Antibacterial Compounds of Lactic Acid Bacteria against Extremely Drug Resistant (XDR) Acinetobacter baumannii

A total of 20 of isolates of lactic acid bacteria (LAB) were selected and screened for antagonistic activity against clinical strains of 30 clinical isolates of extremely drug-resistant (XDR) Acinetobacter baumannii using the well diffusion assay method. Results showed that 50% of the highly LAB strains possessed inhibitory activity against (up to 66%) of the XDR A. baumannii strains tested. The supernatant of the twenty LAB strains was subjected to gas chromatography mass spectrometry (GCMS) revealed that the common compound found in the active isolates against XDR A. baumannii was 3-Isobutyl-2,3,6,7,8,8a-hexahydropyrrolo[1,2-a]pyrazine-1,4-dione, a known potential diketopiperazine group. The molecular docking study against potential antibacterial targets with selected ligands was performed to predict the binding mode of interactions, which is responsible for antibacterial activity. The docking analysis of the potent compounds supported the potential antibacterial activity exhibiting high inhibition constant and binding affinity in silico.     

Structure-based approach for identification of novel phenylboronic acids as serine-β-lactamase inhibitors

β-Lactamases are bacterial enzymes conferring resistance to β-lactam antibiotics in clinically-relevant pathogens, and represent relevant drug targets. Recently, the identification of new boronic acids (i.e. RPX7009) paved the way to the clinical application of these molecules as potential drugs. Here, we screened in silico a library of ~1400 boronic acids as potential AmpC β-lactamase inhibitors. Six of the most promising candidates were evaluated in biochemical assays leading to the identification of potent inhibitors of clinically-relevant β-lactamases like AmpC, KPC-2 and CTX-M-15. One of the selected compounds showed nanomolar K _i value with the clinically-relevant KPC-2 carbapenemase, while another one exhibited broad spectrum inhibition, being also active on Enterobacter AmpC and the OXA-48 class D carbapenemase.     

大肠埃希菌高耐药率回顾性分析

目的了解和分析天津市泰达医院大肠埃希菌耐药情况,指导临床合理使用抗菌药物。方法对2010年至2012年间从临床标本中分离的859株大肠埃希菌的耐药情况进行回顾性总结分析。结果 859株大肠埃希菌对多种抗菌药物均产生不同程度的耐药性,其中对氨苄西林的耐药率达到85.0%,对碳青霉烯类抗菌药物如亚胺培南及四代头孢菌素的耐药率均低于5%。结论天津市泰达医院分离的大肠埃希菌存在严重的耐药性,耐药率达到50%的有11种,原因是滥用抗生素,应加强其耐药性监测,合理规范使用抗生素。     

Total synthesis of monocyclic β-lactam antibiotics,nocardicin A and D

The total synthesis of monocyclic β-lactam antibiotics, nocardicins A (1a) and D (1d), is described. 3-Aminonocardicinic acid (3-ANA, 2) was synthesized from p-hydroxyphenylglycine via an acid chloride-imine cyloaddition reaction. The side chain amino acid 13 was prepared via a key step of condensation of p-hydroxyacetophenone and α-phthalimidobutyrolactone. Acylation of 3-ANA with 13 gave nocardicin D, from which nocardicin A was obtained by oximation.     

A photo-sensitizable phage for multidrug-resistant Acinetobacter baumannii therapy and biofilm ablation

Antibiotic abuse causes the emergence of bacterial resistance. Photodynamic antibacterial chemotherapy (PACT) has great potential to solve serious bacterial resistance, but it suffers from the inefficient generation of ROS and the lack of bacterial targeting ability. Herein, a unique cationic photosensitizer (NB) and bacteriophage (ABP)-based photodynamic antimicrobial agent (APNB) is developed for precise bacterial eradication and efficient biofilm ablation. Thanks to the structural modification of the NB photosensitizer with a sulfur atom, it displays excellent reactive oxygen species (ROS)-production ability. Moreover, specific binding to pathogenic microorganisms can be provided by bacteriophages. The developed APNB has multiple functions, including bacteria targeting, near-infrared fluorescence imaging and combination therapy (PACT and phage therapy). Both in vitro and in vivo experiments prove that APNB can efficiently treat A. baumannii infection. Particularly, the recovery from A. baumannii infection after APNB treatment is faster than that with ampicillin and polymyxin B in vivo. Furthermore, the strategy of combining bacteriophages and photosensitizers is employed to eradicate bacterial biofilms for the first time, and it shows the excellent biofilm ablation effect as expected. Thus, APNB has huge potential in fighting against multidrug-resistant bacteria and biofilm ablation in practice.

APNB for multidrug-resistant A. Baumannii therapy and biofilms ablation.     

Modulatory Effects of Caffeine and Pentoxifylline on Aromatic Antibiotics: A Role for Hetero-Complex Formation

Antimicrobial resistance is a major healthcare threat globally. Xanthines, including caffeine and pentoxifylline, are attractive candidates for drug repurposing, given their well-established safety and pharmacological profiles. This study aimed to analyze potential interactions between xanthines and aromatic antibiotics (i.e., tetracycline and ciprofloxacin), and their impact on antibiotic antibacterial activity. UV-vis spectroscopy, statistical-thermodynamical modeling, and isothermal titration calorimetry were used to quantitatively evaluate xanthine-antibiotic interactions. The antibacterial profiles of xanthines, and xanthine-antibiotic mixtures, towards important human pathogens Staphylococcus aureus, Enterococcus faecium, Escherichia coli, Acinetobacter baumannii, Klebsiella pneumoniae, and Enterobacter cloacae were examined. Caffeine and pentoxifylline directly interact with ciprofloxacin and tetracycline, with neighborhood association constant values of 15.8–45.6 M⁻¹ and enthalpy change values up to −4 kJ·M⁻¹. Caffeine, used in mixtures with tested antibiotics, enhanced their antibacterial activity in most pathogens tested. However, antagonistic effects of caffeine were also observed, but only with ciprofloxacin toward Gram-positive pathogens. Xanthines interact with aromatic antibiotics at the molecular and in vitro antibacterial activity level. Given considerable exposure to caffeine and pentoxifylline, these interactions might be relevant for the effectiveness of antibacterial pharmacotherapy, and may help to identify optimal treatment regimens in the era of multidrug resistance.     

Purification and Analytical Application of <Emphasis Type="Italic">Vigna mungo</Emphasis> Chitinase for Determination of Total Fungal Load of Stored Cereals

Chitinases are glycosyl hydrolases that catalyze the hydrolysis of β-(1,4)-glycosidic bonds in chitin, the major structural polysaccharide presented in the cuticle and gut peritrophic matrix of insects. Two aspartate residues (D143, D145) and one tryptophan (W146) in the Lymantria dispar chitinase are highly conserved residues observed within the second conserved motif of the family 18 chitinase catalytic region. In this study, a chitinase cDNA, LdCht5, was cloned from L. dispar, and the roles of the three residues were investigated using site-directed mutagenesis and substituting them with three other amino acids. Seven mutant proteins, D143E, D145E, W146G, D143E/D145E, D143E/W146G, D145E/W146G, and D143E/D145E/W146G, as well as the wild-type enzyme, were produced using the baculovirus-insect cell line expression system. The enzymatic and kinetic properties of these mutant enzymes were measured using the oligosaccharide substrate MU-(GlcNAc)₃. Among the seven mutants, the D145E, D143E/D145E, and D145E/W146G mutations kept some extant catalytic activity toward MU-(GlcNAc)₃, while the D143E, W146G, D143E/W146G, and D143E/D145E/W146G mutant enzymes were inactivated. Compared with the mutant enzymes, the wild-type enzyme had higher values of k _cat and k _cat / K _m . A study of the multiple point mutations in the second conserved catalytic region would help to elucidate the role of the critical residues and their relationships.     

Cu-OMS-2-catalyzed decarboxylation–acetoxylation of (3R,4R)-4-acid-3-[(R)-1-((t-butyl-dimethylsilyl)oxy)-ethyl]-1-methoxyphenyl-2-azetidinone with NaBrO3/NaOAc

(3R,4R)-4-Acetoxy-3-[(R)-1((t-butyldimethylsilyl)oxy)-ethyl]-1-methoxyphenyl-2-azetidinone (an important precursor for the synthesis of carbapenem antibiotics) was synthesized under mild and green conditions catalyzed by Cu-OMS-2. Normal to excellent isolated yields were obtained under given conditions.     

Co-culture of tumor spheroids and monocytes in a collagen matrix-embedded microfluidic device to study the migration of breast cancer cells

A 3D co-culture microfluidic device was developed to study the effects of ECM stiffness and TAMs on tumor cells migration.     

Biosynthesis of Zinc Oxide Nanoparticles from Acacia nilotica (L.) Extract to Overcome Carbapenem-Resistant Klebsiella Pneumoniae

Recently, concerns have been raised globally about antimicrobial resistance, the prevalence of which has increased significantly. Carbapenem-resistant Klebsiella pneumoniae (KPC) is considered one of the most common resistant bacteria, which has spread to ICUs in Saudi Arabia. This study was established to investigate the antibacterial activity of biosynthesized zinc oxide nanoparticles (ZnO-NPs) against KPC in vitro and in vivo. In this study, we used the aqueous extract of Acacia nilotica (L.) fruits to mediate the synthesis of ZnO-NPs. The nanoparticles produced were characterized by UV-vis spectroscopy, zetasizer and zeta potential analyses, X-ray diffraction (XRD) spectroscopy, Fourier transform infrared spectroscopy (FTIR), scanning electron microscopy (SEM), energy-dispersive X-ray spectroscopy (EDX), and transmission electron microscopy (TEM). The antimicrobial activity of ZnO-NPs against KPC was determined via the well diffusion method, and determining minimum inhibitory concentration (MIC) and minimum bactericidal concentration (MBC), the results showed low MIC and MBC when compared with the MIC and MBC of Imipenem and Meropenem antibiotics. The results of in vitro analysis were supported by the results upon applying ZnO-NP ointment to promote wound closure of rats, which showed better wound healing than the results with imipenem ointment. The biosynthesized ZnO-NPs showed good potential for use against bacteria due to their small size, applicability, and low toxicity to human cells.     

Carboxylation and decarboxylation of active site Lys 84 controls the activity of OXA-24 β-lactamase of Acinetobacter baumannii: Raman crystallographic and solution evidence

The class D β-lactamases are characterized by the presence of a carboxylated lysine in the active site that participates in catalysis. Found in Acinetobacter baumannii, OXA-24 is a class D carbapenem hydrolyzing enzyme that exhibits resistance to most available β-lactamase inhibitors. In this study, the reaction between a 6-alkylidiene penam sulfone inhibitor, SA-1-204, in single crystals of OXA-24 is followed by Raman microscopy. Details of its reaction with SA-1-204 provide insight into the enzyme's mode of action and help define the mechanism of inhibition. When the crystal is maintained in HEPES buffer, the reaction is fast, shorter than the time scale of the Raman experiment. However, when the crystal holding solution contains 28% PEG 2000, the reaction is slower and can be recorded by Raman microscopy in real time; the inhibitor's Raman bands quickly disappear, transient features are seen due to an early intermediate, and, at approximately 2-11 min, new bands appear that are assigned to the late intermediate species. At about 50 min, bands due to all intermediates are replaced by Raman signals of the unreacted inhibitor. The new population remains unchanged indicating (i) that the OXA-24 is no longer active and (ii) that the decarboxylation of Lys84 occurred during the first reaction cycle. Using absorbance spectroscopy, a one-cycle reaction could be carried out in aqueous solution producing inactive OXA-24 as assayed by the chromogenic substrate nitrocefin. However, activity could be restored by reacting aqueous OXA-24 with a large excess of NaHCO(3), which recarboxylates Lys84. In contrast, the addition of NaHCO(3) was not successful in reactivating OXA-24 in the crystalline state; this is ascribed to the inability to create a concentration of NaHCO(3) in large excess over the OXA-24 that is present in the crystal. The finding that inhibitor compounds can inactivate a class D enzyme by promoting decarboxylation of an active site lysine suggests a novel function that could be exploited in inhibitor design.     

Polybenzimidazole-based thiol-ene photosensitive composition for DLP 3D printing

Aphotosensitive composition based on N-allylated poly[2,2'-(p-oxydiphenylene)-5,5'-dibenzimidazole] and pentaerythritol tetrakis(3-mercaptopropionate) has been developed. This photosensitive composition is capable of forming cross-linked 3D-structures due to UV-initiated thiol-ene polymerization. Using digital light processing 3D printing, 3D-objects with high resolution, mechanical strength and thermal resistance up to 397 °C are formed.     

Identification of Druggable Targets for Acinetobacter baumannii Via Subtractive Genomics and Plausible Inhibitors for MurA and MurB

Emergence of the multidrug-resistant pathogens has rendered the current therapies ineffective thereby, resulting in the need for new drugs and drug targets. The accumulating protein sequence data has initiated a drift from classical drug discovery protocols to structure-based drug designing. In the present study, in silico subtractive genomics approach was implemented to find a set of potential drug targets present in an opportunist bacterial pathogen, Acinetobacter baumannii (A. baumannii). Out of the 43 targets identified, further studies for protein model building and lead-inhibitor identification were carried out on two cell-essential targets, MurA and MurB enzymes (of A. baumannii designated as MurA_Ab and MurB_Ab) involved in the peptidoglycan biosynthesis pathway of bacteria. The homology model built for each of them was further refined and validated using various available programs like PROCHECK, Errat, ProSA energy plots, etc. Compounds showing activity against MurA and MurB enzymes of other organisms were collected from the literature and were docked into the active site of MurA_Ab and MurB_Ab enzymes. Three inhibitors namely, T6361, carbidopa, and aesculin, showed maximum Glide score, hydrogen bonding interactions with the key amino acid residues of both the enzymes and acceptable ADME properties. Furthermore, molecular dynamics simulation studies on MurA_Ab–T6361 and MurB_Ab–T6361 complexes suggested that the ligand has a high binding affinity with both the enzymes and the hydrogen bonding with the key residues were stable in the dynamic condition also. Therefore, these ligands have been propsed as dual inhibitors and promising lead compounds for the drug design against MurA_Ab and MurB_Ab enzymes.     

Chemical Composition and Synergistic Potential of Mentha pulegium L. and Artemisia herba alba Asso. Essential Oils and Antibiotic against Multi-Drug Resistant Bacteria

The essential oils were obtained by hydrodistillation from aerial parts of Mentha pulegium L. (M. pulegium L.) and Artemisia herba alba (A. herba alba) Asso. and analyzed by gas chromatography–flame ionization detector chromatograpy (GC–FID) and gaz chromatography–mass spectrometry (GC–MS). The antibacterial activities of the oils were determined by the disk diffusion method and a microdilution broth assay against six bacteria stains. The combinations of these essential oils with antibiotics were evaluated against two multi-drug-resistant bacteria strains: imipenem-resistant Acinetobacter baumannii (IRAB S3310) and methicillin-resistant Staphylococcus aureus (MRSA S19). The chemical analysis of M. pulegium essential oil revealed the presence of pulegone (74.8%) and neoisomenthol (10.0%). A. herba alba essential oil was characterized by camphor (32.0%), α-thujone (13.7%), 1,8-cineole (9.8%), β-thujone (5.0%), bornéol (3.8%), camphene (3.6%), and p-cymene (2.1%). All strains tested except Pseudomonas aeruginosa were susceptible to these oils. The combinations of essential oils with antibiotics exerted synergism, antagonism, or indifferent effects. The best effect was observed with A. herba alba essential oil in association with cefoxitin (CX) against MRSA S19. However, for IRAB S3310, the strongest synergistic effect was observed with M. pulegium in association with amikacin (AK). This study demonstrated that M. pulegium and A. herba alba essential oils have antibacterial activities which could be potentiated by antibiotics especially in the case of IRAB S3310.     

Identification and biotyping of clinical isolates of Acinetobacter

A total of 343 Acinetobacter strains, most isolated from hospital patients, were identified using a 16-test system (acid production from glucose, gelatin hydrolysis and utilization of 14 carbon sources) associated with tests for growth at 37, 41 and 44°C. Of 299 nosocomial isolates, 253 were identified as A. baumannii, 20 as Acinetobacter genospcies 3, 8 as A. haemolyticus, 8 as A. lwoffii, 4 as A. johnsonii and 6 as other (presently) unnamed species. A biotyping system based on the utilization of levulinate, citraconate, L-phenylanine, phenylacetate, 4-hydroxybenzoate and L-tartrate allowed recognition of 17 biotypes among 247 A. baumannii isolates. This biotyping system should be useful in epidemiological studies of Acinetobacter strains.     

Occurrence and Characteristics of Staphylococcus aureus Isolated from Dairy Products

Food, particularly milk and cheese, may be a reservoir of multi-drug resistant Staphylococcus aureus strains, which can be considered an important issue in terms of food safety. Furthermore, foods of animal origin can be a cause of staphylococcal food poisoning via the production of heat-stable enterotoxins (SE). For this reason, we investigated the prevalence of and characterized Staphylococcus aureus strains isolated from milk and fresh soft cheese obtained from farms located in Wielkopolskie and Zachodniopomorskie Provinces in Poland. Overall, 92% of S. aureus isolates were positive for at least one of the 18 enterotoxin genes identified, and 26% of the strains harbored 5 to 8 enterotoxin genes. Moreover, the S. aureus strains contained genes conferring resistance to antibiotics that are critically important in both human and veterinary medicine, i.e., β-lactams (mecA), aminoglycosides (aac(6′)/aph(2″), aph(3′)-IIIa, ant(4′)-Ia) and MLS_B (erm(A), msr(A), lun(A)). The antimicrobial susceptibility of S. aureus to 16 antibiotics representing 11 different categories showed that 74% of the strains were resistant to at least 1 antibiotic. Moreover, 28% of the strains showed multidrug resistance; in particular, two methicillin-resistant S. aureus strains (MRSA) exhibited significant antibiotic resistance. In summary, our results show that dairy products are contaminated by S. aureus strains carrying genes encoding a variety of enterotoxins as well genes conferring resistance to antibiotics. Both MRSA strains and MSSA isolates showing multidrug resistance were present in foods of animal origin.