A facile fluorescence method for the effective detection of ampicillin using antioxidant carbon dots with specific fluorescent response to ˙OH.

Monitoring methods for beta-lactam (ß-lactam) antibiotics, especially for ampicillin (AMP), with simple operation and sensitivity for realtime applications are highly required. To address this need, antioxidant carbon dots (E-CDs) with excellent fluorescence properties were synthesized using citric acid and ethylenediamine as raw materials. With a quantum yield of 81.97%, E-CDs exhibited a specific and sensitive response to ˙OH. The quenched fluorescence of E-CDs by the formed ˙OH could be restored through a competition reaction with AMP. Leveraging the signal-quenching strategy of E-CDs, H2O2, and Fe2+, a fluorescence signal-on strategy was developed using AMP as the fluorescence recovery agent for the sensitive detection of AMP. The mechanism of the quenching of E-CDs by ˙OH was attributed to the damaging effect of ˙OH on E-CDs. Under optimal conditions, the detection limit of this method for AMP was determined to be 0.38 µg mL-1. This method was successful in drug quality control and the spiked detection of AMP in lake water, milk, and sea cucumber, presenting a viable option for convenient and rapid antibiotic monitoring methods.

Gold nanoparticles-doped MXene heterostructure for ultrasensitive electrochemical detection of fumonisin B1 and ampicillin.

Early transition metal carbides (MXene) hybridized by precious metals open a door for innovative electrochemical biosensing device design. Herein, we present a facile one-pot synthesis of gold nanoparticles (AuNPs)-doped two-dimensional (2D) titanium carbide MXene nanoflakes (Ti3C2Tx/Au). Ti3C2Tx MXene exhibits high electrical conductivity and yields synergistic signal amplification in conjunction with AuNPs leading to excellent electrochemical performance. Thus Ti3C2Tx/Au hybrid nanostructure can be used as an electrode platform for the electrochemical analysis of various targets. We used screen-printed electrodes modified with the Ti3C2Tx/Au electrode and functionalized with different biorecognition elements to detect and quantify an antibiotic, ampicillin (AMP), and a mycotoxin, fumonisin B1 (FB1). The ultralow limits of detection of 2.284 pM and 1.617 pg.mL-1, which we achieved respectively for AMP and FB1 are far lower than their corresponding maximum residue limits of 2.8 nM in milk and 2 to 4 mg kg-1 in corn products for human consumption set by the United States Food and Drug Administration. Additionally, the linear range of detection and quantification of AMP and FB1 were, respectively, 10 pM to 500 nM and 10 pg mL-1 to 1 µg mL-1. The unique structure and excellent electrochemical performance of Ti3C2Tx/Au nanocomposite suggest that it is highly suitable for anchoring biorecognition entities such as antibodies and oligonucleotides for monitoring various deleterious contaminants in agri-food products.

Antimicrobial Functionalization of Silicone-graft-poly(N-vinylimidazole) Catheters.

In this work, we present the modification of a medical-grade silicone catheter with the N-vinylimidazole monomer using the grafting-from method at room temperature and induced by gamma rays. The catheters were modified by varying the monomer concentration (20-100 vol%) and the irradiation dose (20-100 kGy). Unlike the pristine material, the grafted poly(N-vinylimidazole) chains provided the catheter with hydrophilicity and pH response. This change allowed for the functionalization of the catheters to endow it with antimicrobial features. Thus, the quaternization of amines with iodomethane and bromoethane was performed, as well as the immobilization of silver and ampicillin. The inhibitory capacity of these materials, functionalized with antimicrobial agents, was challenged against Escherichia coli and Staphylococcus aureus strains, showing variable results, where loaded ampicillin was amply better at eliminating bacteria.

Aggregation-Induced Near-Infrared Emission and Electrochemiluminescence of an Iridium(III) Complex for Ampicillin Sodium Sensing.

A new iridium(III) complex was synthesized and characterized. Its photophysical properties and aggregation-induced emission and electrochemiluminescence in the near-infrared range were studied. The large conjugated cyclometallic ligand 1,2-phenylbenzoquinoline (pbq) was selected to form the Ir-C bond with the metal iridium(III) center and provide near-infrared emission of the complex. The auxiliary ligand 4,4'-diamino-2,2'-bipyridine (dabpy) can form hydrogen bonds, which was beneficial for the generation of aggregation-induced emission. The complex was aggregated into small spherical nanoparticles in 80% water and fascinating nanorings in 90% water. The sensing of ampicillin sodium (AMP) antibiotic by the iridium(III) complex were also investigated by photoluminescent and electrochemiluminescent methods. The complex showed a good selectivity toward AMP antibiotic compared to sodium phenylacetate and other eight antibiotics. The detection limits for AMP antibiotic was 0.76 µg/mL. This work provided a new strategy for the design of iridium(III) complex-based aggregation-induced emission and electrochemiluminescence probes for the sensing application.

An Isotope-Labeled Single-Cell Raman Spectroscopy Approach for Tracking the Physiological Evolution Trajectory of Bacteria toward Antibiotic Resistance.

Understanding evolution of antibiotic resistance is vital for containing its global spread. Yet our ability to in situ track highly heterogeneous and dynamic evolution is very limited. Here, we present a new single-cell approach integrating D2 O-labeled Raman spectroscopy, advanced multivariate analysis, and genotypic profiling to in situ track physiological evolution trajectory toward resistance. Physiological diversification of individual cells from isogenic population with cyclic ampicillin treatment is captured. Advanced multivariate analysis of spectral changes classifies all individual cells into four subsets of sensitive, intrinsic tolerant, evolved tolerant and resistant. Remarkably, their dynamic shifts with evolution are depicted and spectral markers of each state are identified. Genotypic analysis validates the phenotypic shift and provides insights into the underlying genetic basis. The new platform advances rapid phenotyping resistance evolution and guides evolution control.

KPC-2 ß-lactamase enables carbapenem antibiotic resistance through fast deacylation of the covalent intermediate.

Serine active-site ß-lactamases hydrolyze ß-lactam antibiotics through the formation of a covalent acyl-enzyme intermediate followed by deacylation via an activated water molecule. Carbapenem antibiotics are poorly hydrolyzed by most ß-lactamases owing to slow hydrolysis of the acyl-enzyme intermediate. However, the emergence of the KPC-2 carbapenemase has resulted in widespread resistance to these drugs, suggesting it operates more efficiently. Here, we investigated the unusual features of KPC-2 that enable this resistance. We show that KPC-2 has a 20,000-fold increased deacylation rate compared with the common TEM-1 ß-lactamase. Furthermore, kinetic analysis of active site alanine mutants indicates that carbapenem hydrolysis is a concerted effort involving multiple residues. Substitution of Asn170 greatly decreases the deacylation rate, but this residue is conserved in both KPC-2 and non-carbapenemase ß-lactamases, suggesting it promotes carbapenem hydrolysis only in the context of KPC-2. X-ray structure determination of the N170A enzyme in complex with hydrolyzed imipenem suggests Asn170 may prevent the inactivation of the deacylating water by the 6α-hydroxyethyl substituent of carbapenems. In addition, the Thr235 residue, which interacts with the C3 carboxylate of carbapenems, also contributes strongly to the deacylation reaction. In contrast, mutation of the Arg220 and Thr237 residues decreases the acylation rate and, paradoxically, improves binding affinity for carbapenems. Thus, the role of these residues may be ground state destabilization of the enzyme-substrate complex or, alternatively, to ensure proper alignment of the substrate with key catalytic residues to facilitate acylation. These findings suggest modifications of the carbapenem scaffold to avoid hydrolysis by KPC-2 ß-lactamase.

Multiplex Detection of Antibiotic Residues in Milk: Application of MCR-ALS on Excitation-Emission Matrix Fluorescence (EEMF) Data Sets.

The presence of antibiotics and their metabolites in milk and dairy products is a serious concern because of their harmful effects on human health. In the current study, a novel synergistic bimetallic nanocluster with gold and silver as an emission fluorescence probe was investigated for the simultaneous determination of tetracycline (TC), ampicillin (AMP), and sulfacetamide (SAC) antibiotics in the milk samples using excitation-emission matrix fluorescence (EEMF) spectroscopy. The multivariate curve resolution-alternating least squares (MCR-ALS) method was implemented to analyze augmented EEMF data sets to quantify the multicomponent systems in the presence of interferences with considerable spectral overlap. A pseudo-univariate calibration curve of the resolved emission spectra intensity against the concentration of the mentioned antibiotics was linear in the range of 5-5000 ng mL-1 for AMP and 50-5000 ng mL-1 for TC and SAC. The calculated values of the limit of detection ranged between 1.4 and 14.6 ng mL-1 with a relative standard deviation (RSD) of less than 4.9%. The obtained results show that the EEMF/MCR-ALS methodology using an emission fluorescence probe is a powerful tool for the simultaneous quantification of TC, AMP, and SAC in complex matrices with highly overlapped spectra.

Dual Activity BLEG-1 from Bacillus lehensis G1 Revealed Structural Resemblance to B3 Metallo-ß-Lactamase and Glyoxalase II: An Insight into Its Enzyme Promiscuity and Evolutionary Divergence.

Metallo-ß-lactamases (MBLs) are class B ß-lactamases from the metallo-hydrolase-like MBL-fold superfamily which act on a broad range of ß-lactam antibiotics. A previous study on BLEG-1 (formerly called Bleg1_2437), a hypothetical protein from Bacillus lehensis G1, revealed sequence similarity and activity to B3 subclass MBLs, despite its evolutionary divergence from these enzymes. Its relatedness to glyoxalase II (GLXII) raises the possibility of its enzymatic promiscuity and unique structural features compared to other MBLs and GLXIIs. This present study highlights that BLEG-1 possessed both MBL and GLXII activities with similar catalytic efficiencies. Its crystal structure revealed highly similar active site configuration to YcbL and GloB GLXIIs from Salmonella enterica, and L1 B3 MBL from Stenotrophomonas maltophilia. However, different from GLXIIs, BLEG-1 has an insertion of an active-site loop, forming a binding cavity similar to B3 MBL at the N-terminal region. We propose that BLEG-1 could possibly have evolved from GLXII and adopted MBL activity through this insertion.

Apigenin and Ampicillin as Combined Strategy to Treat Severe Streptococcus suis Infection.

As an important zoonotic pathogen, Streptococcus suis (S. suis) can cause a variety of diseases both in human and animals, especially Streptococcal toxic shock-like syndrome (STSLS), which commonly appears in severe S. suis infection. STSLS is often accompanied by excessive production of inflammatory cytokines, which is the main cause of host death. Therefore, it is urgent to find a new strategy to relieve the damage caused by STSLS. In this study, we found, for the first time, that apigenin, as a flavonoid compound, could combine with ampicillin to treat severe S. suis infection. Studies found that apigenin did not affect the growth of S. suis and the secretion of suilysin (SLY), but it could significantly inhibit the hemolytic activity of SLY by directly binding to SLY and destroying its secondary structure. In cell assays, apigenin was found to have no significant toxic effects on effective concentrations, and have a good protective effect on S. suis-infected cells. More importantly, compared with the survival rate of S. suis-infected mice treated with only ampicillin, the survival rate of apigenin combined with an ampicillin-treated group significantly increased to 80%. In conclusion, all results indicate that apigenin in combination with conventional antibiotics can be a potential strategy for treating severe S. suis infection.

Trajectory Taken by Dimeric Cu/Zn Superoxide Dismutase through the Protein Unfolding and Dissociation Landscape Is Modulated by Salt Bridge Formation.

Native mass spectrometry (MS) is a powerful means for studying macromolecular protein assemblies, including accessing activated states. However, much remains to be understood about what governs which regions of the protein (un)folding funnel, which can be explored by activation of protein ions in a vacuum. Here, we examine the trajectory that Cu/Zn superoxide dismutase (SOD1) dimers take over the unfolding and dissociation free energy landscape in a vacuum. We examined wild-type SOD1 and six disease-related point mutants by using tandem MS and ion-mobility MS as a function of collisional activation. For six of the seven SOD1 variants, increasing activation prompted dimers to transition through two unfolding events and dissociate symmetrically into monomers with (as near as possible) equal charges. The exception was G37R, which proceeded only through the first unfolding transition and displayed a much higher abundance of asymmetric products. Supported by the observation that ejected asymmetric G37R monomers were more compact than symmetric G37R ones, we localized this effect to the formation of a gas-phase salt bridge in the first activated conformation. To examine the data quantitatively, we applied Arrhenius-type analysis to estimate the barriers on the corresponding free energy landscape. This reveals a heightening of the barrier to unfolding in G37R by >5 kJ/mol-1 over the other variants, consistent with expectations for the strength of a salt bridge. Our work demonstrates weaknesses in the simple general framework for understanding protein complex dissociation in a vacuum and highlights the importance of individual residues, their local environment, and specific interactions in governing product formation.

Ampicillin-mediated functionalized gold nanoparticles against ampicillin-resistant bacteria: strategy, preparation and interaction studies.

Antibiotic resistance is a highly challenging concern of infectious diseases, and it requires a rational approach to overcome. Through this work, we have synthesized ampicillin-capped gold nanoparticles (Amp-Au NPs) and studied its interaction with bacterial cells. In this process of synthesis, the primary amine group of ampicillin acts as both reducing as well as capping agent. In addition to synthesized gold nanoparticles, the ß-lactam ring remains free to interact with bacteria. This approach not only utilizes the maximum efficiency of nanoparticles and antibiotics towards ampicillin sensitive bacterial cells but also proves to be effective against ampicillin resistance bacteria. Our results illustrate that the optimized system of Amp-Au NPs was formulated by taking 1.25 mM ampicillin and 10-2 of gold ions concentration. UV-vis spectrum of gold nanoparticles and the presence of ampicillin were recorded at around 540 nm and 259 nm, respectively. Microscopic images indicate that particles are nearly spherical and are in size range between 25 and 50 nm. Moreover, formulated Amp-Au NPs show successful accumulation onto the surface of the bacterial cell as a result of which pores were formed into the bacterial membrane. The entry of nanoparticles into bacterial cells was validated through both atomic force microscopy and fluorescent microscopy. The adhesive properties of this coating material and its stability in various pH, i.e. pH 3, pH 7 and pH 10 conditions, could make them a good candidate in the prevention of biofilm formation. Amp-Au NPs show promising antimicrobial activity against ampicillin resistance Escherichia coli bacteria. Furthermore, antimicrobial studies indicate that the efficacy of Amp-Au NPs increased against both ampicillin sensitive and ampicillin resistance bacteria up to sixteen folds and four folds respectively.

Modification of titanium implants using biofunctional nanodiamonds for enhanced antimicrobial properties.

The present study describes a novel antimicrobial surface using anodic oxidation of titanium and biofunctional detonation nanodiamonds (ND). ND have been loaded with antibiotics (amoxicillin or ampicillin) using poly(diallyldimethylammonium chloride) (PDDA). Successful conjugation with PDDA was determined by dynamic light scattering, which showed increase in the hydrodynamic diameter of ND agglomerates and shift of zeta potential towards positive values. The surface loading of amoxicillin was determined using UV-vis spectroscopy and the maximum of 44% surface loading was obtained. Biofunctional ND were immobilized by anodic oxidation within a titanium oxide layer, which was confirmed by scanning electron microscopy. The in vitro antimicrobial properties of ND suspensions were examined using Kirby-Bauer test with E. coli. Modified titanium surfaces comprising biofunctional ND were evaluated with E. coli inoculum by live/dead assay staining. Both biofunctional ND suspensions and modified titanium surfaces presented inhibition of bacteria growth and increase in bacteria lethality.

Ultrasonication-facilitated synthesis of functionalized graphene oxide for ultrasound-assisted magnetic dispersive solid-phase extraction of amoxicillin, ampicillin, and penicillin G.

A simplistic approach is presented for the synthesis of ultrasonically fabricated graphene oxide functionalized with polyaniline and N-[3-(Trimethoxysilyl)propyl]ethylenediamine. The synthesized nanocomposite was then employed for the facile, green, ultrasound-assisted, magnetic dispersive solid-phase extraction of amoxicillin, ampicillin, and penicillin G in milk samples and infant formula prior to high-performance liquid chromatography-ultraviolet determination. The designed nanocomposites were comprehensively characterized using field emission scanning electron microscopy, energy-dispersive X-ray spectroscopy, transmission electron microscopy, X-ray powder diffraction, and Fourier transform infrared spectroscopy. In order to achieve the best extraction efficiencies, the influential parameters including pH, amount of magnetic sorbent, type and volume of elution solvent, extraction time, sample volume, and desorption time were assessed. At the optimum conditions, linear ranges of 2.5-1000 (µg L-1) for ampicillin and penicillin G and a linear range of 2.5-750 (µg L-1) were obtained for amoxicillin at optimum conditions. Moreover, the limits of detection (S/N = 3) of 0.5, 0.8, and 0.9 (µg L-1) were obtained for amoxicillin, ampicillin, and penicillin G, respectively. The precision (relative standard deviations (%)) values of 3.1, 2.6, and 2.5 at the concentration of 50 µg L-1 for seven replicates were obtained for ampicillin, amoxicillin, and penicillin G, respectively. The efficiencies of ≤ 96% and relative standard deviations of less than 3.1% were also obtained thereby confirming the high potential of the synthesized nanocomposites for simultaneous preconcentration and separation of the ß-lactam antibiotics in complex matrixes. Graphical Abstract.

The Impact of [C16Pyr][Amp] on the Aggressiveness in Breast and Prostate Cancer Cell Lines.

Breast (BrCa) and prostate (PCa) cancers are the most common malignancies in women and men, respectively. The available therapeutic options for these tumors are still not curative and have severe side effects. Therefore, there is an urgent need for more effective antineoplastic agents. Herein, BrCa, PCa, and benign cell lines were treated with two ionic liquids and two quinoxalines and functional experiments were performed-namely cell viability, apoptosis, cytotoxicity, and colony formation assays. At the molecular level, an array of gene expressions encompassing several molecular pathways were used to explore the impact of treatment on gene expression. Although both quinoxalines and the ionic liquid [C2OHMIM][Amp] did not show any effect on the BrCa and PCa cell lines, [C16Pyr][Amp] significantly decreased cell viability and colony formation ability, while it increased the apoptosis levels of all cell lines. Importantly, [C16Pyr][Amp] was found to be more selective for cancer cells and less toxic than cisplatin. At the molecular level, this ionic liquid was also associated with reduced expression levels of CPT2, LDHA, MCM2, and SKP2, in both BrCa and PCa cell lines. Hence, [C16Pyr][Amp] was shown to be a promising anticancer therapeutic agent for BrCa and PCa cell lines.

Interaction of Ampicillin and Amoxicillin with Mn2+: A Speciation Study in Aqueous Solution.

A potentiometric and UV spectrophotometric investigation on Mn2+-ampicillin and Mn2+-amoxicillin systems in NaCl aqueous solution is reported. The potentiometric measurements were carried out under different conditions of temperature (15 ≤ t/°C ≤ 37). The obtained speciation pattern includes two species for both the investigated systems. More in detail, for system containing ampicillin MLH and ML species, for that containing amoxicillin, MLH2 and MLH ones. The spectrophotometric findings have fully confirmed the results obtained by potentiometry for both the systems, in terms of speciation models as well as the stability constants of the formed species. Enthalpy change values were calculated via the dependence of formation constants of the species on temperature. The sequestering ability of ampicillin and amoxicillin towards Mn2+ was also evaluated under different conditions of pH and temperature via pL0.5 empirical parameter (i.e., cologarithm of the ligand concentration required to sequester 50% of the metal ion present in traces).

Design and Characterisation of Inhibitory Peptides against Bleg1_2478, an Evolutionary Divergent B3 Metallo-ß-lactamase.

Previously, a hypothetical protein (HP) termed Bleg1_2437 (currently named Bleg1_2478) from Bacillus lehensis G1 was discovered to be an evolutionary divergent B3 subclass metallo-ß-lactamase (MBL). Due to the scarcity of clinical inhibitors for B3 MBLs and the divergent nature of Bleg1_2478, this study aimed to design and characterise peptides as inhibitors against Bleg1_2478. Through in silico docking, RSWPWH and SSWWDR peptides with comparable binding energy to ampicillin were obtained. In vitro assay results showed RSWPWH and SSWWDR inhibited the activity of Bleg1_2478 by 50% at concentrations as low as 0.90 µM and 0.50 µM, respectively. At 10 µM of RSWPWH and 20 µM of SSWWDR, the activity of Bleg1_2478 was almost completely inhibited. Isothermal titration calorimetry (ITC) analyses showed slightly improved binding properties of the peptides compared to ampicillin. Docked peptide-protein complexes revealed that RSWPWH bound near the vicinity of the Bleg1_2478 active site while SSWWDR bound at the center of the active site itself. We postulate that the peptides caused the inhibition of Bleg1_2478 by reducing or blocking the accessibility of its active site from ampicillin, thus hampering its catalytic function.

Efficiency of Ampicillin-Associated Biogenic Ferrihydrite Nanoparticles in Combination with a Magnetic Field for Local Treatment of Burns.

We studied the effectiveness of using magnetic ferrihydrite nanoparticles of bacterial origin carrying ampicillin for local treatment of burn wounds in rats using a magnetic field. It was found that the use of these nanoparticles in combination with a magnetic field accelerated wound healing and reduced the titer of microorganisms in comparison with the corresponding parameters in the untreated animals and animals treated with nanoparticles or ampicillin alone.

Tuning the mechanical and physicochemical properties of cross-linked protein films.

Films derived from natural sources such as proteins provide an advantage over synthetic films due to their noncytotoxicity, biodegradability, and vast functionality. A new protein source gained from the cataractous eye protein isolate (CEPI) obtained after surgery has been investigated for this purpose. Glycerol has been employed as the plasticizer and glutaraldehyde (GD) as a cross-linker. Fourier transform infrared spectroscopy was employed to characterize the films. Nanoindentation and thermogravimetric analyses reveal improved mechanical and thermal properties of the cross-linked films. The films with 20% (w/w) GD exhibited properties such as the highest modulus and low water solubility. It is possible to tune the properties based on the extent of cross-linking. All the films were completely degraded by the enzyme trypsin. The similarity of these films was checked by using the prepared films as a delivery vehicle for a model compound, ampicillin sodium. The encapsulation efficiency was found to be 74%, and in vitro release studies showed significant amounts of drug release at physiological pH. This study will help us understand how the properties of protein films can be tuned to obtain the desired physicochemical properties. These biodegradable protein films could find use in pharmaceutical industries as delivery carriers.

Effects of boric acid-linked ampicillin on the rat intra-abdominal sepsis model.

The aim of this study was to determine efficiency of a new molecule that was obtained by linking boric acid with ampicillin in treating intra-abdominal infection.Following intraperitoneal E. coli injection totwenty-one female Wistar albino rats, group 1 was administered boron-linked ampicillin, group 2 was administered only ampicillin and group 3 was injected intraperitoneally with physiological serum. IL-6, and a white blood cell analysis was performed from the blood before and on the seventh day of treatment.No statistically significant difference in blood WBC levels after treatment was found among the groups. There was no statistically significant difference in the IL-6 values of group 2 and group 3 before and after the treatment (p=0.195 and 0.193, respectively); however, the reduction in the serum IL-6 values of group 1 was statistically significant (p=0.003).Boric acid-linked ampicillin is a more effective intra-abdominal infection treatment compared with ampicillin alone.

Stability of benzylpenicillin potassium and ampicillin in an elastomeric infusion pump.

Some infectious diseases, such as infective endocarditis, osteomyelitis, and abscesses, require treatment with long-term intravenous antimicrobial treatment. Therefore, the patient is required to stay in the hospital to receive therapy, which lowers their quality of life. Establishing an outpatient parenteral antimicrobial therapy (OPAT) by continuous infusion pump is desired in Japan to overcome these issues. However, the 24-h stability of antimicrobial agents dissolved in infusion solutions is unclear. Thus, we investigated the stability of antimicrobial agents in five different infusion solutions in a clinical setting. Benzylpenicillin potassium (PCG) and ampicillin (ABPC) were dissolved separately in five different infusion solutions and kept at 25 or 31.1 °C for 24 h. The residual ratios were determined by high-performance liquid chromatography (HPLC). Dissolved PCG in acetate ringer solution remained stable for 24 h at temperatures of 25 and 31.1 °C (101.7 ± 1.4% and 92.9 ± 1.3%, respectively). In addition, the PCG solution did not adsorb onto the elastomeric infusion pump after 24 h at 31.1 °C. PCG dissolved in acetate ringer solution was also stable for 10 days after being kept in an elastomeric infusion pump at 4 °C (99.7 ± 0.5%). ABPC was unstable in all of the tested infusion solutions and temperatures. Based on our results, PCG in acetate ringer solution can be used in OPAT with continuous infusion pumps.