Near Real Time Monitoring of <i>E. Coli</i> in Water

ABSTRACT Foodborne bacteria such as Escherichia coli O157:H7 can cause severe hemorrhagic colitis in humans following consumption of contaminated meat products. Contamination with pathogenic bacteria is frequently found in the food production environment, and adequate household storage conditions of purchased foods are vital for illness avoidance. Real-time monitoring was used to evaluate bacterial growth in ground horse, beef, and pork meats maintained under various storage conditions. Various levels of E. coli O157:H7 carrying the luxCDABE operon, which allows the cells to emit bioluminescence, were used to inoculate meat samples that were then stored at room temperature for 0.5 day, at 4°C (cold) for 7 or 9 days, or −20°C (frozen) for 9 days. Real-time bioluminescence imaging (BLI) of bacterial growth was used to assess bacterial survival or load. Ground horse meat BLI signals and E. coli levels were dose and time dependent, increasing during room temperature and −20°C storage, but stayed at low levels during 4°C storage. No bacteria survived in the lower level inoculum groups (101 and 103 CFU/g). With an inoculum of 107 CFU/g, pork meats had higher BLI signals than did their beef counterparts, displaying decreased BLI signals during 7 days storage at 4°C. Both meat types had higher BLI signals in the fat area, which was confirmed with isolated fat tissues in the beef meat. Beef lean and fat tissues contrasted with both pork fat and lean tissues, which had significantly higher BLI signals and bacterial levels. BLI appears to be a useful research tool for real-time monitoring of bacterial growth and survival in various stored livestock meats. The dependence of E. coli O157:H7 growth on meat substrate (fat or lean) and storage conditions may be used as part of an effective antibacterial approach for the production of safe ground horse, beef, and pork meats.

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Metabolic Flux Analysis of Catechin Biosynthesis Pathways Using Nanosensor

Antioxidants ◽

10.3390/antiox9040288 ◽

2020 ◽

Vol 9 (4) ◽

pp. 288

Author(s):

Habiba Kausar ◽

Ghazala Ambrin ◽

Mohammad K. Okla ◽

Walid Soufan ◽

Abdullah A. Al-Ghamdi ◽

...

Keyword(s):

Metabolic Engineering ◽

Real Time ◽

Metabolic Flux Analysis ◽

Metabolic Flux ◽

Regulatory Element ◽

Flux Analysis ◽

Bacterial Cells ◽

Biosynthesis Pathway ◽

Real Time Monitoring ◽

E Coli

(+)-Catechin is an important antioxidant of green tea (Camelia sinensis (L.) O. Kuntze). Catechin is known for its positive role in anticancerous activity, extracellular matrix degradation, cell death regulation, diabetes, and other related disorders. As a result of enormous interest in and great demand for catechin, its biosynthesis using metabolic engineering has become the subject of concentrated research with the aim of enhancing (+)-catechin production. Metabolic flux is an essential concept in the practice of metabolic engineering as it helps in the identification of the regulatory element of a biosynthetic pathway. In the present study, an attempt was made to analyze the metabolic flux of the (+)-catechin biosynthesis pathway in order to decipher the regulatory element of this pathway. Firstly, a genetically encoded fluorescence resonance energy transfer (FRET)-based nanosensor (FLIP-Cat, fluorescence indicator protein for (+)-catechin) was developed for real-time monitoring of (+)-catechin flux. In vitro characterization of the purified protein of the nanosensor showed that the nanosensor was pH stable and (+)-catechin specific. Its calculated Kd was 139 µM. The nanosensor also performed real-time monitoring of (+)-catechin in bacterial cells. In the second step of this study, an entire (+)-catechin biosynthesis pathway was constructed and expressed in E. coli in two sets of plasmid constructs: pET26b-PT7-rbs-PAL-PT7-rbs-4CL-PT7-rbs-CHS-PT7-rbs-CHI and pET26b-T7-rbs-F3H-PT7-rbs- DFR-PT7-rbs-LCR. The E. coli harboring the FLIP-Cat was transformed with these plasmid constructs. The metabolic flux analysis of (+)-catechin was carried out using the FLIP-Cat. The FLIP-Cat successfully monitored the flux of catechin after adding tyrosine, 4-coumaric acid, 4-coumaroyl CoA, naringenin chalcone, naringenin, dihydroquercetin, and leucocyanidin, individually, with the bacterial cells expressing the nanosensor as well as the genes of the (+)-catechin biosynthesis pathway. Dihydroflavonol reductase (DFR) was identified as the main regulatory element of the (+)-catechin biosynthesis pathway. Information about this regulatory element of the (+)-catechin biosynthesis pathway can be used for manipulating the (+)-catechin biosynthesis pathway using a metabolic engineering approach to enhance production of (+)-catechin.

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