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

The Effect of Pluchea indica (L.) Less. Tea on Adipogenesis in 3T3-L1 Adipocytes and Lipase Activity

Obesity and hyperlipidemia are a major problem in the world. Pluchea indica (L.) Less. tea (PIT) is a beverage that has various indications. This study focused on the effect of the PIT on inhibiting adipogenesis of 3T3-L1 cells and pancreatic lipase enzyme activity. The viability of 3T3-L1 cells was not significantly decreased after exposure to 200 to 1000 μg mL−1 PIT compared to controls (p < 0.05). The PIT at 750 to 1000 μg mL−1 exhibited a significantly reduced lipid accumulation compared to the control (p < 0.05). The inhibitory effects of the PIT at 250 to 1000 μg mL−1 on lipase activity were significantly increased compared to control (p < 0.05). The FTIR results showed that the integrated areas of lipids, proteins, nucleic acids, glycogen, and carbohydrates of the PIT-treated 3T3-L1 adipocytes were significantly lower than the untreated 3T3-L1 adipocytes (p < 0.05). These findings may indicate that the PIT is not only capable of inhibiting lipids and carbohydrate accumulation in adipocytes but also has a potential to inhibit pancreatic lipase activity. So, the PIT may be further developed to the novel lipid-lowering herbal supplement for the management of overweight or obesity

Mode of Action and Synergy of Ceftazidime and Baicalein against Streptococcus pyogenes

Purpose: To investigate the antibacterial activity of baicalein used alone, or in combination with ceftazidime, against Streptococcus pyogenes.Methods: Minimum inhibitory concentration (MIC), checkerboard assay parameters, and viability curves were determined for S. pyogenes DMST 30653, 30654, and 30655. Cytoplasmic membrane (CM) permeability technique, enzyme assays, transmission electron microscopy and Fourier transforminfrared microspectroscopy were used to investigate the changes in the bacterial biomolecules.Results: The MIC of ceftazidime and baicalein against all the S. pyogenes strains were 0.50 and &gt; 256.0 μg/ml, respectively. A synergistic effect against these strains was exhibited by the ceftazidime/baicalein combination (fractional inhibitory concentration index, &lt; 0.37). The results for the viable counts indicate that this synergistic activity was present. Baicalein exerted inhibitory activity against β-lactamase. Compared with the controls, combining baicalein with ceftazidime caused peptidoglycan and morphological damage, significantly increased CM permeability and protein concentrations, and decreased cellular fatty acid and nucleic acid concentrations.Conclusion: Baicalein is a potential synergistic adjunct to ceftazidime for the treatment of S. pyogenes infections.Keywords: Streptococcus pyogenes, Cytoplasmic membrane permeability, Baicalein, Ceftazidime, Synergistic activity, Fourier Transform-infrared microspectroscopy, Transmission electron microscop

Pluchea indica (L.) Less. Tea Ameliorates Hyperglycemia, Dyslipidemia, and Obesity in High Fat Diet-Fed Mice.

Pluchea indica (L.) Less. (P. indica) tea has been used for a health-promoting drink, especially in Southeast Asia. The effect of P. indica tea (PIT) on amelioration of hyperglycemia; dyslipidemia that was total cholesterol (TC), LDL-cholesterol (LDL-C), HDL-cholesterol (HDL-C), and triglyceride (TG); and obesity in high fat diet-induced (HFD) mice was investigated. Oral glucose tolerance test (OGTT) displayed that PIT at 400 and 600 mg/kg orally ameliorated hyperglycemia with a dose-dependent manner compared to the untreated group. Moreover, PIT at these dosages exhibited significantly lower TC, LDL-C, TG, and perigonadal fat weight in HFD treated mice compared to HFD mice (P 0.05). The PIT chemical analysis results demonstrated that PIT contained total phenolic content (TPC), total flavonoid content (TFC), 4-O-caffeoylquinic acid (4-CQ), 5-O-caffeoylquinic acid (5-CQ), 3,4-O-dicaffeoylquinic acid (3,4-CQ), 3,5-O-dicaffeoylquinic acid (3,5-CQ), 4,5-O-dicaffeoylquinic acid (4,5-CQ), beta-caryophyllene, and gamma-gurjunene that may play an important role in inhibiting hyperlipidemia and hyperglycemia. Also, histological analysis expressed that the mean area and amount of perigonadal fat adipocytes of PIT treated groups were significantly lower and higher than the HFD group (P 0.05). These results suggest that PIT does not become toxic to the kidney, liver, and blood. In conclusion, PIT has the potential to develop into healthy food supplement or medicine for the prevention and treatment of hyperglycemic, hyperlipidemic, and obese patients

Nitro-Carba test, a novel and simple chromogenic phenotypic method for rapid screening of carbapeneamase-producing Enterobacteriaceae.

OBJECTIVES: The present study developed Nitro-Carba Test (NCT), a rapid and simple chromogenic method for screening of Carbapenemase-producing Enterobacteriaceae (CPE). METHODS: The NCT was validated with a total of 31 carbapenemase-producing isolates (9 KPCs, 11 MBLs and 11 OXA-48s) and with 56 non-carbapenemase-producing strains. The assay relies on the hydrolysis of nitrocefin in the presence of carbapenems. The carbapenemases were extracted with lysis buffer prior to addition to wells with and without imipenem (IPM), meropenem (MEM) and ertapenem (ETP). Following the addition of nitrocefin, a change in colour from yellow to red, indicating carbapenemase production, was observed within 20min. The susceptibility profiles of each bacterial strain were also investigated. RESULTS: A NCT detected all 31 CPEs within a timeframe of only 10seconds to 12min. All carbapenemase producers hydrolyzed nitrocefin in all wells. No colour change in the wells with carbapenems was observed in non-carbapenemase producers. The sensitivity for all three carbapenems was 100%, while specificity of IPM, MEM and ETP were 64.29%, 91.07% and 100%, respectively. IPM, MEM and ETP against all carbapenemase-producing strains had MIC values ranging from 0.5 to ≥256μg/mL, 0.25 to ≥256μg/mL and 1 to ≥256μg/mL, respectively. OXA-48-producing isolates showed lower MIC values compared with producers of MBL and KPC. CONCLUSION: This assay is a promising method detecting CPE rapidly. The NCT is a simple and reliable method, capable of detecting CPE in even carbapenem-susceptible strains

The Free Radical Scavenging and Anti-Isolated Human LDL Oxidation Activities of Pluchea indica (L.) Less. Tea Compared to Green Tea (Camellia sinensis).

Tea is one of the most popular beverages in the world. Camellia sinensis tea (CST) or green tea is widely regarded as a potent antioxidant. In Thailand, Pluchea indica (L.) Less. tea (PIT) has been commercially available as a health-promoting drink. This study focused on free radical scavenging activities of PIT, and its ability to protect isolated human low-density lipoproteins (LDL) from oxidation by chemical agents. A preliminary study to investigate the antioxidant nature of PIT was undertaken. These included common antioxidant assays involving 2,2-Diphenyl-1-picrylhydrazyl (DPPH), 2,2-azinobis-(3-ethylbenzothiazoline)-6-sulfonic acid (ABTS), hypochlorous acid (HOCl), and its potential to scavenge peroxynitrite. In separated experiments, isolated human LDL was challenged with either 2,2'-azobis(2-amidinopropane) dihydrochloride (AAPH), copper (Cu2+), or 3-Morpholinosydnonimine hydrochloride (SIN-1) to induce LDL oxidation. PIT exhibited antioxidant activity in all test systems and performed significantly better than CST in both DPPH (P < 0.05; IC50PIT = 245.85 ± 15.83 and CST = 315.41 ± 24.18 μg/ml) and peroxynitrite scavenging assays. PIT at 75 μg/ml almost fully prevented the peroxynitrite over a 5 h period. Moreover, it displayed similar properties to CST during the antioxidation of isolated human LDL using AAPH, Cu2+, SIN-1, and hypochlorous acid scavenging assays. However, it revealed a significantly lower ABTS scavenging activity than CST (P < 0.05; IC50PIT = 30.47 ± 2.20 and CST = 21.59 ± 0.67 μg/ml). The main constituents of the PIT were identified using LC-MS/MS. It contained 4-O-caffeoylquinic acid (4-CQ), 5-O-caffeoylquinic acid (5-CQ), 3,4-O-dicaffeoylquinic acid (3,4-CQ), 3,5-O-dicaffeoylquinic acid (3,5-CQ), and 4,5-O-dicaffeoylquinic acid (4,5-CQ). In conclusion, caffeoyl derivatives in PIT could play an important role in potent antioxidant properties. So, it may be further developed to be antioxidant beverages for preventing atherosclerosis and cardiovascular diseases associated with oxidative stress

Intracellular ROS Scavenging and Anti-Inflammatory Activities of Oroxylum indicum Kurz (L.) Extract in LPS plus IFN-gamma-Activated RAW264.7 Macrophages

Oroxylum indicum (L.) Kurz has been used as plant-based food and herbal medicine in many Asian countries. The aim of the present study was to examine the antioxidant and anti-inflammatory activities of O. indicum extract (O. indicum) in RAW264.7 cells activated by LPS plus IFN-γ. The phytochemical compounds in O. indicum were identified by GC-MS and LC-MS/MS. Five flavonoids (luteolin, apigenin, baicalein, oroxylin A, and quercetin) and 27 volatile compounds were found in O. indicum. O. indicum presented antioxidant activities, including reducing ability by FRAP assay and free radical scavenging activity by DPPH assay. Moreover, O. indicum also suppressed LPS plus IFN-γ-activated reactive oxygen species generation in RAW264.7 macrophages. It possessed the potent anti-inflammatory action through suppressing nitric oxide (NO) and IL-6 secretion, possibly due to its ability to scavenge intracellular ROS. The synchrotron radiation-based Fourier transform infrared (SR-FTIR) spectroscopy results showed the alteration of signal intensity and integrated areas relating to lipid and protein of the activated RAW264.7 macrophages compared to unactivated cells. This is the first report of an application of the SR-FTIR technique to evaluate biomolecular changes in activated RAW264.7 cells. Our results indicate that O. indicum may be used as a potential source of nutraceutical for the development of health food supplement or a novel anti-inflammatory herbal medicine

The performance of a resazurin chromogenic agar plate with a combined disc method for rapid screening of extended-spectrum-β-lactamases, AmpC β-lactamases and co-β-lactamases in Enterobacteriaceae.

A resazurin chromogenic agar (RCA) along with combined disc method has been developed as a promising method for rapid screening of extended-spectrum-β-lactamase (ESBL), AmpC β-lactamase, and co-production of ESBL and AmpC. Cefpodoxime (CPD) discs supplemented with and without clavulanic acid (CA), cloxacillin (CX), or CA+CX were evaluated against 86-molecularly confirmed β-lactamase-producing Enterobacteriaceae, including 15 ESBLs, 32 AmpCs, 9 co-producers of ESBL and AmpC, and 30 carbapenemase producers. The CA and CX synergy test successfully detected all ESBL producers (100% sensitivity and 98.6% specificity) and all AmpC producers (100% sensitivity and 96.36% specificity). This assay also exhibited a good performance in the screening for the co-existence of ESBL and AmpC (88.89% sensitivity and 100% specificity). The RCA assay is a simple and inexpensive method that allows observation of results within 7 h. It can be applicable in any microbiological laboratory, especially in the endemic areas of ESBL, AmpC, or co-β-lactamase-producing Enterobacteriaceae

A nitrocefin disc supplemented with ertapenem for rapid screening of carbapenemase-producing Enterobacteriaceae

Reliable, simple and rapid methods for laboratory detection of carbapenemases are important for an appropriate antibiotic administration. A nitrocefin disc containing ertapenem for rapid screening of carbapenemase production among Enterobacteriaceae is developed in the present study. A total of 87 molecularly-confirmed Enterobacteriaceae including 31 carbapenemase producers and 56 non-carbapenemase producers, were tested with nitrocefin discs supplemented with and without ertapenem (20 μg/disc). Nitrocefin discs with ertapenem successfully discriminated all 31 carbapenemase and all non-carbapenemase producers within 30 minutes. The sensitivity and specificity of the method were 100%. The minimum inhibitory concentrations (MICs) of ertapenem against all carbapenemase-producing isolates ranged from 1 to ≥ 256 μg/mL. This simple test could help to minimize the treatment failure and control the dissemination of infections caused by carbapenemase-associated resistant bacteria. It is a promising approach that could be performed routinely in any laboratory

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