The Evaluation of a Lateral Flow Strip Based on the Covalently Fixed "End-On" Orientation of an Antibody for Listeria monocytogenes Detection.

In this study, the covalently fixed "end-on" orientation of a monoclonal Listeria monocytogenes antibody (mAb-Lis) to amino terminated oligo (ethylene glycol)-capped gold nanoparticles (NH2-TEG-AuNPs) was used to fabricate an in-house lateral flow strip (LFS), namely, the fixed "end-on" Lis-mAb-NH-TEG-AuNPs LFS. The aim was to evaluate the performance of the fixed "end-on" Lis-mAb-NH-TEG-AuNPs LFS in detecting L. monocytogenes. The proposed LFS enabled the sensitive detection of L. monocytogenes in 15 min with a visual limit of detection of 102 CFU/mL. Quantitative analysis indicated an LOD at 10 CFU/mL. The fixed "end-on" Lis-mAb-NH-TEG-AuNPs LFS showed no cross-reactivity with other pathogenic bacteria and practical performance across different food matrices, including human blood, milk, and mushroom samples. Furthermore, the clinical performance of the fixed "end-on" Lis-mAb-NH-TEG-AuNPs LFS for detecting L. monocytogenes was evaluated by using 12 clinical samples validated by the hemoculture method. It demonstrated excellent concordance with the reference methods, with no false-positive or false-negative results observed. Therefore, the fixed "end-on" Lis-mAb-NH-TEG-AuNPs LFS serves as a promising candidate for a point-of-care test (POCT), enabling the rapid, precise, and highly sensitive detection of L. monocytogenes in clinical samples and contaminated food.

Tannic Acid-Stabilized Silver Nanoparticles Used in Biomedical Application as an Effective Antimelioidosis and Prolonged Efflux Pump Inhibitor against Melioidosis Causative Pathogen.

Burkholderia pseudomallei is the causative pathogen of melioidosis and this bacterium is resistant to several antibiotics. Silver nanoparticles (AgNPs) are an interesting agent to develop to solve this bacterial resistance. Here, we characterize and assess the antimelioidosis activity of AgNPs against these pathogenic bacteria. AgNPs were characterized and displayed a maximum absorption band at 420 nm with a spherical shape, being well-monodispersed and having high stability in solution. The average size of AgNPs is 7.99 ± 1.46 nm. The antibacterial efficacy of AgNPs was evaluated by broth microdilution. The bactericidal effect of AgNPs was further assessed by time-kill kinetics assay. Moreover, the effect of AgNPs on the inhibition of the established biofilm was investigated by the crystal violet method. In parallel, a study of the resistance induction development of B. pseudomallei towards AgNPs with efflux pump inhibiting effect was performed. We first found that AgNPs had strong antibacterial activity against both susceptible and ceftazidime-resistant (CAZ-resistant) strains, as well as being efficiently active against B. pseudomallei CAZ-resistant strains with a fast-killing mode via a bactericidal effect within 30 min. These AgNPs did not only kill planktonic bacteria in broth conditions, but also in established biofilm. Our findings first documented that the resistance development was not induced in B. pseudomallei toward AgNPs in the 30th passage. We found that AgNPs still showed an effective efflux pump inhibiting effect against these bacteria after prolonged exposure to AgNPs at sublethal concentrations. Thus, AgNPs have valuable properties for being a potent antimicrobial agent to solve the antibiotic resistance problem in pathogens.

Detection of aflatoxin B1 in chili powder using attenuated total reflectance-Fourier transform infrared spectroscopy.

Aflatoxin B1, a major global food safety concern, is produced by toxigenic fungi during crop growing, drying, and storage, and shows increasing annual prevalence. This study aimed to detect aflatoxin B1 in chili samples using ATR-FTIR coupled with machine learning algorithms. We found that 83.6% of the chili powder samples were contaminated with Aspergillus and Penicillium species, with aflatoxin B1 levels ranging from 7.63 to 44.32 µg/kg. ATR-FTIR spectroscopy in the fingerprint region (1800-400 cm-1) showed peak intensity variation in the bands at 1587, 1393, and 1038 cm-1, which are mostly related to aflatoxin B1 structure. The PCA plots from samples with different trace amounts of aflatoxin B1 could not be separated. Vibrational spectroscopy combined with machine learning was applied to address this issue. The logistic regression model had the best F1 score with the highest %accuracy (73%), %sensitivity (73%), and %specificity (71%), followed by random forest and support vector machine models. Although the logistic regression model contributed significant findings, this study represents a laboratory research project. Because of the peculiarities of the ATR-FTIR spectral measurements, the spectra measured for several batches may differ, necessitating running the model on multiple spectral ranges and using increased sample sizes in subsequent applications. This proposed method has the potential to provide rapid and accurate results and may be valuable in future applications regarding toxin detection in foods when simple onsite testing is required.

Development in competitive immunoassay of a point-of-care testing for cotinine (COT) detection in urine.

We present a sensitive and selective lateral flow immunoassay (LFIA) for cotinine (COT), the primary metabolite of nicotine. COT is widely recognized as a superior biomarker to evaluate tobacco smoke exposure. The LFIA uses a competitive assay format where the COT-BSA capture competes with the target COT in urine samples for binding to the monoclonal antibody against COT (mAb-COT) conjugated with gold nanoparticles (mAb-COT-AuNPs). To improve the sensitivity and selectivity of the LFIA-COT, we focused on optimizing the diameter of AuNPs, the conjugation of mAb-COT, and the concentration of the COT-BSA capture. Our findings reveal that the utilization of 40 nm AuNPs in conjugation with a concentration of 4 mg mL-1 of mAb-COT demonstrated significantly greater efficacy compared to LFAs utilizing 20 nm AuNPs. Under the optimal conditions, the LFIA-COT demonstrated sensitive detection of COT at a level of 150 ng mL-1 within 15 min, as observed by the naked eye. It possesses a linear range of 25 to 200 ng mL-1 of COT, with the limit of detection (LOD) of 11.94 ng mL-1 in human urine samples when the color intensity is analyzed using ImageJ software. Our LFIA described here is simple and requires less time for COT detection. It can be used for the rapid and quantitative detection of COT in urine samples in clinical settings.

Andrographolide stabilized-silver nanoparticles overcome ceftazidime-resistant Burkholderia pseudomallei: study of antimicrobial activity and mode of action.

Burkholderia pseudomallei (B. pseudomallei) is a Gram-negative pathogen that causes melioidosis, a deadly but neglected tropical disease. B. pseudomallei is intrinsically resistant to a growing list of antibiotics, and alternative antimicrobial agents are being sought with urgency. In this study, we synthesize andrographolide-stabilized silver nanoparticles (andro-AgNPs, spherically shaped with 16 nm average diameter) that show excellent antimicrobial activity against B. pseudomallei, including ceftazidime-resistant strains, being 1-3 orders of magnitude more effective than ceftazidime and 1-2 orders of magnitude more effective than other green-synthesized AgNPs. The andro-AgNPs are meanwhile non-toxic to mammalian cell lines. The mode of action of Andro-AgNPs toward B. pseudomallei is unraveled by killing kinetics, membrane neutralization, silver ions (Ag+) release, reactive oxygen species (ROS) induction, membrane integrity, and cell morphology change studies. The antimicrobial activity and mode of action of andro-AgNPs against B. pseudomallei reported here may pave the way to alternative treatments for melioidosis.

TA-AgNPs/Alginate Hydrogel and Its Potential Application as a Promising Antibiofilm Material against Polymicrobial Wound Biofilms Using a Unique Biofilm Flow Model.

The presence of biofilm within a chronic wound may delay the healing process. Thus, control of biofilm formation and providing bactericidal effect are crucial factors for wound healing management. Alginate-based nanocomposite hydrogels have been suggested as dressing materials for wound treatment, which are employed as a biocompatible matrix. Therefore, in this study, we aimed to develop a biocompatible antimicrobial wound dressing containing AgNPs and demonstrate its efficacy against polymicrobial wound biofilms by using a biofilm flow device to simulate a chronic infected, exuding wound and specific wound environment. The results from agar well diffusion, the Minimum Inhibitory Concentration (MIC) and Minimum Bactericidal Concentration (MBC) assays showed that TA-AgNPs exhibited antibacterial activity against wound pathogens. Additionally, the Minimum Biofilm Eradication Concentration assay (MBEC) demonstrated it could impair biofilm formation. Importantly, our TA-AgNPs/Alginate hydrogel clearly showed antibacterial activities against Streptococcus pyogenes, Staphylococcus aureus and Pseudomonas aeruginosa. Furthermore, we used the biofilm flow device to test the topical antimicrobial hydrogel against a three-species biofilm. We found that TA-AgNPs/Alginate hydrogel significantly showed a 3-4 log reduction in bacterial numbers when applied with multiple doses at 24 h intervals, and was especially effective against the chronic wound pathogen P. aeruginosa. This work highlighted that the TA-AgNPs/Alginate hydrogel is a promising material for treating complex wound biofilms.

Colorimetric detection of Hg(II) by γ-aminobutyric acid-silver nanoparticles in water and the assessment of antibacterial activities.

In this work, we report the synthesis of silver nanoparticles (AgNPs) via a wet-chemical reduction procedure using citrate (Cit) and γ-aminobutyric acid (GABA) as stabilizers. The formation of GABA-Cit@AgNPs was confirmed by UV-vis spectroscopy with a surface plasmon resonance band at 393 nm clearly confirming the formation of silver nanoparticles. AgNPs were characterized using UV-vis spectroscopy, attenuated total reflectance-fourier transform infrared spectroscopy (ATR-FTIR), transmission electron microscope (TEM), energy-dispersive X-ray spectroscopy (EDX), X-ray diffraction (XRD), dynamic light scattering (DLS), and zeta potential. The as-prepared AgNPs can be used for the detection of hazardous mercury ions (Hg2+) in water by colorimetric method with a limit of detection (LOD) and limit of quantitation (LOQ) of 2.37 µM and 3.99 µM, respectively. The linear working range for Hg2+ detection is 5-35 µM and the sensor probe was applied to investigate Hg2+ in real drinking water samples with satisfied results. Rapid response to Hg2+ is also observed when the nanoparticles are composited within hydrogels. Moreover, GABA-Cit@AgNPs shows antibacterial activity against Escherichia coli (E. coli) and Staphylococcus aureus (S. aureus). The fast and sensitive response of the proposed Hg2+ sensor, together with its antibacterial activities, makes GABA-Cit@AgNPs potentially applicable for the development of cheap, portable, colorimetric sensors in fieldwork.

Anisotropic Silver Nanoparticles Gel Exhibits Antibacterial Action and Reduced Scar Formation on Wounds Contaminated with Methicillin-Resistant Staphylococcus pseudintermedius (MRSP) in a Mice Model.

Staphylococcus pseudintermedius (S. pseudintermedius) infected wounds can cause seriously delayed wound healing processes in animals. Antimicrobial agents that have antimicrobial and wound healing efficacy have become an essential tool for overcoming this problem. In our previous study, anisotropic AgNPs have been reported to have antimicrobial efficiency against animal and human pathogens, and could be suitable as antimicrobial agents for infected wounds. Here, antimicrobial and wound healing activities of anisotropic AgNPs gels were assessed in vivo. BALB/cAJcl mice wounds were infected by Methicillin-resistant Staphylococcus pseudintermedius (MRSP). Then, antibacterial and wound healing activities were evaluated by bacterial cell count, wound contraction, digital capture, and histology. The results show that anisotropic AgNPs gels could eliminate all bacterial cell infected wounds within 7 days, the same as povidone iodine. Wound healing activity was evaluated by wound contraction (%). The results showed 100% wound contraction in groups treated with anisotropic AgNPs gels within 14 days that was not significantly different from povidone iodine and control gel without AgNPs. However, the digital capture of wounds on day 4 showed that anisotropic AgNPs gel prevented pus formation and reduced scar appearance within 21 days. The histology results exhibit improved collagen fiber alignment that supports scar disappearance. In conclusion, these results indicate that anisotropic AgNPs gels are suitable for treating infected wounds. The gel is effective in eliminating bacteria that supports the natural process of wound repair and also causes reduced scar formation.

AFM Study of Nanoscale Membrane Perturbation Induced by Antimicrobial Lipopeptide C14 KYR.

Lipopeptides have been extensively studied as potential antimicrobial agents. In this study, we focused on the C14-KYR lipopeptide, a modified version of the KYR tripeptide with myristic acid at the N-terminus. Here, membrane perturbation of live E. coli treated with the parent KYR and C14-KYR peptides was compared at the nanoscale level using AFM imaging. AFM analyses, including average cellular roughness and force spectroscopy, revealed the severe surface disruption mechanism of C14-KYR. A loss of surface roughness and changes in topographic features included membrane shrinkage, periplasmic membrane separation from the cell wall, and cytosolic leakage. Additional evidence from synchrotron radiation FTIR microspectroscopy (SR-FTIR) revealed a marked structural change in the membrane component after lipopeptide attack. The average roughness of the E. coli cell before and after treatment with C14-KYR was 129.2 ± 51.4 and 223.5 ± 14.1 nm, respectively. The average rupture force of the cell treated with C14-KYR was 0.16 nN, four times higher than that of the untreated cell. Our study demonstrates that the mechanistic effect of the lipopeptide against bacterial cells can be quantified through surface imaging and adhesion force using AFM.

The Decoration of ZnO Nanoparticles by Gamma Aminobutyric Acid, Curcumin Derivative and Silver Nanoparticles: Synthesis, Characterization and Antibacterial Evaluation.

Zinc oxide nanoparticles (ZnO NPs) are applied in various applications in catalysis, biosensing, imaging, and as antibacterial agents. Here we to prepare ZnO nanomaterials decorated by γ-amino butyric acid (GABA), curcumin derivatives (CurBF2) and silver nanoparticles (CurBF2-AgNPs). The structures of all ZnO nanostructures were characterized using Fourier transform infrared (FT-IR) spectroscopy, X-ray diffraction (XRD), UV-VIS spectrophotometry, fluorescence spectrophotometry, scanning electron microscopy (SEM), transmission electron microscopy (TEM), and high-resolution transmission electron microscopy (HR-TEM). Further, their antibacterial activities against Gram-negative (Escherichia coli) and Gram-positive (Staphylococcus aureus) bacteria were investigated through analysis of minimum inhibitory concentration (MIC) method. Among the prepared nanostructures, the ZnO NPs-GABA/CurBF2-AgNPs showed excellent antibacterial activity against both Gram-positive and -negative bacteria. ZnO NPs fabricated here may have potential use in future anti-bacterial compositions and coatings technologies.