Siamese crocodile plasma synergizes with ceftazidime against ceftazidime-resistant Enterobacter cloacae

Purpose: To evaluate whether Siamese crocodile plasma exhibits antibacterial properties and if it synergizes with ceftazidime against ceftazidime-resistant Enterobacter cloacae (CREnC).Methods: Protein fractions were from crocodile plasma and tested them on CREnC strains. Multiplex polymerase chain reaction (PCR) screening test was performed for extended-spectrum β-lactamase (ESBL) phenotype and AmpC gene. The effects of the antibacterial agents were analyzed using a bacterial suspension standard curve, minimum inhibitory concentration (MIC), Checkerboard assays, viability curves, membrane permeability assays, enzyme assays, and transmission electron microscopy.Results: CREnC strains expressed ESBL-AmpC gene combinations. The MICs of resuspended protein 1 (P1), protein 5 (P5), ceftazidime, cefotaxime, and benzylpenicillin against all tested CREnC and E. coli strains were in the range of > 1024 μg/mL, indicating resistance. However, P1 and P5 exhibited a synergistic effect against test CREnC and E. coli strains when used in combination with ceftazidime and cefotaxime, with fraction inhibitory concentration indices of < 0.062 and 0.28, respectively. A kill curve demonstrated that the combination treatments had synergistic activity and inhibited β-lactamase.Conclusion: The synergistic activity of P1 and P5 in combination with ceftazidime is achieved in multiple ways, including increased cytoplasmic and outer membrane permeability, β-lactamase inhibition, and peptidoglycan damage. Therefore, the combination therapy of Siamese crocodile plasma and ceftazidime may be a novel therapeutic approach for treating recalcitrant E. cloacae infection.Keywords: Crocodylus siamensis, ceftazidime-resistant Enterobacter cloacae, synergistic activity, β-lactamase activit

Phenotypic detection of AmpC β-lactamases, extended-spectrum- β-lactamases and metallo-β-lactamases in Enterobacteriaceae using a resazurin microtitre assay with inhibitor-based methods.

Dissemination of antibiotic resistance in Enterobacteriaceae mediated by AmpC, ESBL and MBL β-lactamases is clinically significant. A simple, relatively quick method for the detection of these resistance phenotypes would greatly improve chemotherapeutic recommendation. This technology would provide valuable input in our surveillance of resistance on a global stage, particularly if the methodology could be applicable to resource poor settings. A resazurin microtitre plate (RMP) assay incorporating cloxacillin, clavulanic acid, and EDTA for the rapid phenotypic identification of AmpC, Extended-spectrum-β-lactmase (ESBL), metallo-β-lactamase (MBL) and the co-existence of β-lactamases has been developed. A total of 47 molecularly characterised Enterobacteriaceae clinical isolates producing AmpCs, ESBLs, co-producers of ESBL and AmpC, MBLs, and co-producers of ESBL and MBL were phenotypically examined using the RMP assay. The ceftazidime (CAZ)-based and cefotaxime (CTX)-based RMP assay successfully detected all 16 AmpC, 14 ESBL, 9 MBL producers, 6 ESBL-AmpC co-producers, and 2 ESBL-MBL co-producers without false positive results. The CAZ-based assay was more reliable in detecting AmpC alone, while the CTX-based assay performed better in identifying co-producers of ESBL and AmpC. There was no difference in detection of ESBL and MBL producers. The findings of the present study suggest that use of the RMP assay with particular β-lactamase inhibitors explicitly detects three different β-lactamases, as well as co-existence of β-lactamases within 6 h after initial isolation of the pathogen. This assay is applicable to carry out in any laboratory, is cost-effective and easy to interpret. It could be implemented in screening patients, controlling infection and for surveillance purposes

Oroxylum indicum (L.) Kurz extract inhibits adipogenesis and lipase activity in vitro

Background: Oroxylum indicum (L.) Kurz (O. indicum) is found in Thailand. It has been used for the treatment of obesity. This study aimed to investigate the effects of an O. indicum extract (OIE) on the adipogenic and biomolecular change in 3T3-L1 adipocytes. Methods: Initial studies examined the chemical components of OIE. The cell line 3T3-L1 was used to establish potential toxic effects of OIE during the differentiation of pre-adipocytes to adipocytes. The inhibitory effect of OIE on lipid accumulation in 3T3-L1 cells was investigated. Moreover, the impact of OIE on pancreatic lipase activity was determined. In further experiments, Fourier Transform Infrared (FTIR) was used to monitor and discriminate biomolecular changes caused by the potential anti-adipogenic effect of OIE on 3T3-L1 cells. Results: Chemical screening methods indicated that OIE was composed of flavonoids, alkaloids, steroids, glycosides, and tannins. The percentage viability of 3T3-L1 cells was not significantly decreased after exposure to either 200 or 150 μg/mL of OIE for 2 and 10 days, respectively compared to control cells. The OIE exhibited a dose-dependent reduction of lipid accumulation compared to the control (p < 0.05). The extract also demonstrated a dosedependent inhibitory effect upon lipase activity compared to the control. The inhibitory effect of the OIE on lipid accumulation in 3T3-L1 cells was also confirmed using FTIR microspectroscopy. The signal intensity and the integrated areas relating to lipids, lipid esters, nucleic acids, glycogen and carbohydrates of the OIE-treated 3T3-L1 adipocytes were significantly lower than the non-treated 3T3-L1 adipocytes (p < 0.05). Principal component analysis (PCA) indicated four distinct clusters for the FTIR spectra of 3T3-L1 adipocytes based on biomolecular changes (lipids, proteins, nucleic acids, and carbohydrates). This observation was confirmed using Unsupervised hierarchical cluster analysis (UHCA). Conclusions: These novel findings provide evidence that the OIE derived from the fruit pods of the plant is capable of inhibiting lipid and carbohydrate accumulation in adipocytes and also has the potential to inhibit an enzyme associated with fat absorption. The initial observations indicate that OIE may have important properties which in the future may be exploited for the management of the overweight or obese

Reversing B-lactam antibiotic resistance with some flavonoids in gram-positive bacteria

ผลงานวิชาการคณาจารย์มหาวิทยาลัยเทคโนโลยีสุรนาร

Free radical scavenging and anti-isolated human LDL oxidation activities of Butea superba Roxb. extract

Abstract Background Butea superba Roxb. (B. superba), is an herbal plant traditionally used for rejuvenation. Additionally, there have been reports on its antioxidant properties. Low-density lipoproteins (LDL) oxidation is the leading cause of cardiovascular diseases (CVDs). Natural products with antioxidant properties have the potential to inhibit LDL oxidation. However, no work has been done about the anti-isolated human LDL oxidation of B. superba extract (BSE). This study aimed to investigate the antioxidant potential of BSE and its ability to prevent isolated human (LDL) oxidation induced by free radical agents. Methods The antioxidant properties were investigated by antioxidant assays, including 2,2-diphenyl-1-picrylhydrazyl (DPPH), 2,2-azinobis-(3-ethylbenzothiazoline)-6-sulfonic acid (ABTS), ferric reducing ability power (FRAP), nitric oxide (NO) and peroxynitrite scavenging assay. More so, anti-isolated human LDL oxidation activities were evaluated by 2,2'-azobis (2-amidinopropane) dihydrochloride (AAPH) and 3-morpholinosydnonimine hydrochloride (SIN-1) induced LDL oxidation assay. Results BSE exhibited a significant (p  0.05). Identifying the main constituents of BSE revealed the presence of genistein, daidzein, and biochanin A through Liquid Chromatography-Mass Spectrometer/Mass Spectrometer (LC–MS/MS) analysis. Conclusion This is the first report that the presence of isoflavones in BSE could play an important role in its antioxidation and isolated human LDL oxidation scavenging properties. These findings suggest the potential for developing antioxidant herbal supplements. However, further studies must be investigated, including efficacious and safe human dosages

The Synergy and Mode of Action of Cyperus rotundus L. Extract Plus Ampicillin against Ampicillin-Resistant Staphylococcus aureus

Cyperus rotundus L. has been used for pharmaceutical applications including antibacterial infections. Nevertheless, there is still no data regarding the mode of actions. This study aimed to determine the antibacterial activity and mode of actions of Cyperus rotundus extract (CRE) against ampicillin-resistant Staphylococcus aureus (ARSA) which poses a serious problem for hospitalized patients. The majority of chemical compounds of CRE were flavonoids and alkaloids. The minimum inhibitory concentrations (MICs) for ampicillin and CRE against all ARSA strains were 64 μg/ml and 0.5 mg/ml, respectively. Checkerboard assay revealed synergistic activity in the combination of ampicillin and CRE at the lowest fractional inhibitory concentration index (FICI) at 0.27. The killing curve assay had confirmed the synergistic and bactericidal activity of the combination against ARSA. Electron microscopic results showed that these ARSA cells treated with this combination caused peptidoglycan and cytoplasmic membrane (CM) damage and average cell areas significantly smaller than control. Also, this combination caused an increase in CM permeability of ARSA. CRE revealed the inhibitory activity against β-lactamase. It is normally known that some drugs are derived from flavonoids or alkaloids. So, this CRE proposes the potential to develop a novel adjunct phytopharmaceutical to ampicillin for the remedy of ARSA

Synergy and Mode of Action of Ceftazidime plus Quercetin or Luteolin on Streptococcus pyogenes

Streptococcus pyogenes causes streptococcal toxic shock syndrome. The recommended therapy has been often failure through the interfering of beta-lactamase-producing bacteria (BLPB). The present study was to investigate antibacterial activity, synergy, and modes of action of luteolin and quercetin using alone and plus ceftazidime against S. pyogenes. The MICs of ceftazidime, luteolin, and quercetin against all S. pyogenes were 0.50, 128, and 128 µg mL−1, respectively. A synergistic effect was exhibited on luteolin and quercetin plus ceftazidime against these strains at fractional inhibitory concentration indices 0.37 and 0.27, respectively, and was confirmed by the viable count. These combinations increased cytoplasmic membrane (CM) permeability, caused irregular cell shape, peptidoglycan, and CM damage, and decreased nucleic acid but increased proteins in bacterial cells. Enzyme assay demonstrated that these flavonoids had an inhibitory activity against β-lactamase. In summary, this study provides evidence that the inhibitory mode of action of luteolin and quercetin may be mediated via three mechanisms: (1) inhibiting of peptidoglycan synthesis, (2) increasing CM permeability, and (3) decreasing nucleic acid but increasing the protein contents of bacterial cells. So, luteolin and quercetin propose the high potential to develop adjunct to ceftazidime for the treatment of coexistence of the BLPB and S. pyogenes infections

Antiadipogenesis of Oroxylum indicum (L.) Kurz Extract via PPARγ2 in 3T3-L1 Adipocytes

Oroxylum indicum is regarded as a traditional food with medicinal properties and is used widely throughout Asia. It has previously been demonstrated that O. indicum extract (OIE) was able to suppress the differentiation of 3T3-L1 preadipocytes to adipocytes. However, the mechanism underlying the antiadipogenesis of this plant has not been fully investigated. The present study aimed to explore the impact of OIE at 50 to 200 μg mL−1 on the molecular mechanism involved in the antiadipogenic activity in 3T3-L1 cells at day 0 of their differentiation to adipocytes. The morphology and biochemistry of the cells on day 12 were investigated and compared to the relevant controls. Adiponectin was measured using enzyme-linked immunosorbent assay (ELISA). The mRNA expression of peroxisome proliferator-activated receptor-gamma 2 (PPARγ2), sterol regulatory element-binding proteins 1c (SREBP-1c), fatty acid synthetase (FAS), glucose transporter (GLUT4), and leptin in adipocytes was determined by real-time PCR. The results demonstrated that the OIE at 200 μg mL−1 exhibited strongest suppression on intracellular lipid accumulation. The levels of adiponectin were dramatically increased in the untreated adipocytes, whereas significantly decreased in the 200 μg mL−1 OIE-treated adipocytes (P<0.05). Expression of the mRNAs revealed that OIE-treated adipocytes at 200 μg mL−1 significantly inhibited the expression of PPARγ2 and SREBP-1c and lowered the level of expression of GLUT4, FAS, and leptin compared to the control (P<0.05). These findings suggest that OIE inhibits adipocyte differentiation along with the downregulation of PPARγ2, SREBP-1c, and GLUT4, leading to the decrease in the expression of FAS and adipokine (leptin and adiponectin). Thus, OIE might be developed for hyperlipidemia and obesity prevention

Gymnema inodorum (Lour.) Decne. Extract Alleviates Oxidative Stress and Inflammatory Mediators Produced by RAW264.7 Macrophages

Gymnema inodorum (Lour.) Decne. (G. inodorum) is widely used in Northern Thai cuisine as local vegetables and commercial herb tea products. In the present study, G. inodorum extract (GIE) was evaluated for its antioxidant and anti-inflammatory effects in LPS plus IFN-γ-induced RAW264.7 cells. Major compounds in GIE were evaluated using GC-MS and found 16 volatile compounds presenting in the extract. GIE exhibited antioxidant activity by scavenging the intracellular reactive oxygen species (ROS) production and increasing superoxide dismutase 2 (SOD2) mRNA expression in LPS plus IFN-γ-induced RAW264.7 cells. GIE showed anti-inflammatory activity through suppressing nitric oxide (NO), proinflammatory cytokine production interleukin 6 (IL-6) and also downregulation of the expression of cyclooxygenase-2 (COX-2), inducible nitric oxide synthase (iNOS), and IL-6 mRNA levels in LPS plus IFN-γ-induced RAW264.7 cells. Mechanism studies showed that GIE suppressed the NF-κB p65 nuclear translocation and slightly decreased the phosphorylation of NF-κB p65 (p-NF-κB p65) protein. Our studies applied the synchrotron radiation-based FTIR microspectroscopy (SR-FTIR), supported by multivariate analysis, to identify the FTIR spectral changes based on macromolecule alterations occurring in RAW264.7 cells. SR-FTIR results demonstrated that the presence of LPS plus IFN-γ in RAW264.7 cells associated with the increase of amide I/amide II ratio (contributing to the alteration of secondary protein structure) and lipid content, whereas glycogen and other carbohydrate content were decreased. These findings lead us to believe that GIE may prevent oxidative damage by scavenging intracellular ROS production and activating the antioxidant gene, SOD2, expression. Therefore, it is possible that the antioxidant properties of GIE could modulate the inflammation process by regulating the ROS levels, which lead to the suppression of proinflammatory cytokines and genes. Therefore, GIE could be developed into a novel antioxidant and anti-inflammatory agent to treat and prevent diseases related to oxidative stress and inflammation

The Effects of Cordyceps sinensis (Berk.) Sacc. and Gymnema inodorum (Lour.) Decne. Extracts on Adipogenesis and Lipase Activity In Vitro

This study aimed to investigate the effects of Cordyceps sinensis extract (CSE) and Gymnema inodorum extract (GIE), used alone and combined, on antiadipogenesis in 3T3-L1 cells. Oil Red O staining was used to examine the effects of these extracts on inhibition of intracellular lipid accumulation in 3T3-L1 adipocytes and on lipid droplet morphology. Fourier transform-infrared (FTIR) microspectroscopy was used to examine biomolecular changes in 3T3-L1 adipocytes. The pancreatic lipase assay was used to evaluate the inhibitory effects of CSE and GIE on pancreatic lipase activity. Taken together, the results indicated that CSE, GIE, and their combination suppressed lipid accumulation. The FTIR microspectroscopy results indicated that CSE, GIE, and their combination had inhibitory effects on lipid accumulation in the adipocytes. Compared with the untreated adipocytes, the signal intensity and integrated areas of glycogen and other carbohydrates, the acyl chain of phospholipids, and the lipid/protein ratios of the CSE, GIE, alone, and combined treated adipocytes were significantly lower (p < 0.05). Combination treatment resulted in a synergistic effect on lipid accumulation reduction in the adipocytes. Principal component analysis of the biomolecular changes revealed six distinct clusters in the FTIR spectra of the sample cells. The pancreatic lipase assay results indicated that CSE and GIE inhibited the pancreatic lipase activity in a dose-dependent manner (mean ± standard error of the mean IC50 values, 2312.44 ± 176.55 μg mL−1 and 982.24 ± 44.40 μg mL−1, resp.). Our findings indicated that FTIR microspectroscopy has potential application for evaluation of the effectiveness of medicinal plants and for the development of infrared biochemical obesity markers useful for treating patients with obesity. These results suggested that use of CSE and GIE alone and in combination may be efficacious as a complementary therapy for hyperlipidemia and obesity management. However, clinical trials in animals and humans must first be completed