This study proposes a novel chemiluminescent assay of bacterial activity. Luminol chemiluminescence (LC) was amplified on addition of menadione to Escherichia coli suspension, and it was effectively inhibited by addition of superoxide dismutase rather than catalase. This fact suggests that H2O2 produced from O2 by superoxide dismutase is decomposed by catalase of E. coli. NAD(P)H:menadione reductase activities in periplasm and cytosol corresponded to the amplification of menadione-catalyzed LC, and outer and cytoplasmic membranes were only slightly involved in the LC. The total activity and Vmax of NAD(P)H:menadione reductase in the cytoplasm were greater than those in the periplasm. A transient increase in menadione-catalyzed LC was observed in the exponential phase and the LC decreased in the stationary phase during growth of E. coli. Menadione-catalyzed LC was sensitive to antibiotic action. A decrease in menadione-catalyzed LC by the impairment of membrane functions and by the inhibition of protein synthesis was observed at 5 min and 3 hr, respectively. These findings suggest the possibility that menadione-catalyzed luminol chemiluminescent assay is applicable to rapid antimicrobial assay because LC is sensitive to the change in growth and cytotoxic events caused by antimicrobial agents.

https://onlinelibrary.wiley.com/doi/pdf/10.1111/j.1348-0421.2001.tb02628.x

Menadione-catalyzed O2- production by Escherichia coli cells: application of rapid chemiluminescent assay to antimicrobial susceptibility testing.

An amphiphilic, cationic peptide composed of eight leucines and six lysines was synthesized by solid phase peptide synthesis (SPPS). The synthetic peptide was bactericidal within 10 min at concentrations as low as 3 μg ml−1 against mid-exponential Escherichia coli O157:H7 suspended in buffer. Concentrations of 25 μg ml−1 caused up to 7 log10 cfu ml−1 reductions. When tested against E. coli O157:H7 grown in TSB, the peptide was bactericidal and bacteriostatic at concentrations of 50 and 25 μg ml−1, respectively. An inhibitory effect was also observed against stationary phase cells. The synthetic peptide caused the release of u.v.-absorbing materials from the E. coli O157:H7 as well as an increase in its O.D.600 nm. Intracellular K+ and ATP depletion were also observed. These results suggest that the peptide increased the cell membrane permeability but it did not lyse the cells.

Antimicrobial activity of a 14-residue peptide against Escherichia coli O157:H7.

Escherichia coli ATCC 25922 produced O2- in the presence of menadione, and O2- -dependent luminol chemiluminescence intensity was proportional to colony-forming unit (CFU) in the exponential phase. CFU was determined by using a 96-well plate at a range of 3 X 10(3) to 8 x 10(7) CFU /well (0.1 ml) after a 10-min incubation with menadione, followed by chemiluminescent assay for 5 s. After a 4-hr incubation of E. coli (10(5) CFU/0.1 ml) with menadione and an antimicrobial agent inhibiting the synthesis of peptidoglycan, protein, and DNA, the inhibitory concentration (IC) of the antimicrobial agent determined by menadione-catalyzed luminol chemiluminescent assay was in good agreement with minimal inhibitory concentration (MIC) of the NCCLS (National Committee for Clinical Laboratory Standard) method requiring 18 hr. Menadione-catalyzed luminol chemiluminescent assay is expected to be useful for the rapid determination of cell viability under the conditions of various cell growths and stresses.

Menadione-catalyzed luminol chemiluminescent assay for the viability of Escherichia coli ATCC 25922

The current study focused on treatment of phenolic wastewater using an integrated process – dosing of ozone directly to activated sludge. The main goal was to analyze the effect of ozonation on viability of activated sludge in different systems – activated sludge in distilled water and activated sludge in wastewater. Two viability detection methods, oxygen uptake (OUR) rate and adenosine-5'-triphosphate measurement (ATP), were compared. The linear correlation between ATP and OUR measurements in studied range was found to be good (r2 = 0.90). In case of ozonation of activated sludge in wastewater, ozone doses up to 42 mgO3·gMLVSS−1 did not influence the viability of sludge. In addition, contrary to ozonation of sludge in distilled water, soluble COD was reduced by 15.6% (at ozone dose of 42 mgO3·gMLVSS−1).

Effect of Ozone on Viability of Activated Sludge Detected by Oxygen Uptake Rate (OUR) and Adenosine-5'-triphosphate (ATP) Measurement

The relative constancy of ATP content per unit of dry biomass in different microorganisms enables to use the specific, sensitive and quick luciferin-luciferase method for ATP assay for indirect quantitation of microbial biomass by measuring the ATP content in biomass samples.The aim of this work was to investigate the feasibility of using the ATP concentration for the monitoring of the growth of Escherichia coli K 12 W 3350 in different cultivation conditions. Statistically significant high correlation coefficients (r > 0.9) between the ATP content (ATP/ml) and the other growth characteristics examined showed that the ATP content of the culture is as suitable characteristic as the optical density of the culture, total cell number per ml or total cell volume per ml for the monitoring of the growth of E.coliin batch culture in mineral medium with glucose at different growth temperatures (27–42 °C) as well as in fed-batch culture.

R. Vilu

Papers

Use of the luciferin‐luciferase assay of ATP for measuring the bacterial growth: Application to Escherichia coli