Detection of E. coli O157:H7 using its endogenous active membrane peroxidase

Shim, Kyu Hwan; Kang, Mino; Kim, Min-Gon; Chung, Bong Hyun; An, Seong Soo A.

doi:10.1007/s13530-011-0081-6

Cited by 5 publications

(4 citation statements)

References 27 publications

(20 reference statements)

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“…TMB (3, 3′, 5, 5′-tetramethylbenzidine) assay was used for determination of enzymatic activity of bacteria26. 80 μL of TMB, 100 μL of GO and 20 μL of bacteria (10 9 /mL) were mixed thoroughly.…”

Section: Methodsmentioning

confidence: 99%

Study of antibacterial mechanism of graphene oxide using Raman spectroscopy

Nanda

Kim

2016

Sci Rep

193

101

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Graphene oxide (GO) is extensively proposed as an effective antibacterial agent in commercial product packaging and for various biomedical applications. However, the antibacterial mode of action of GO is yet hypothetical and unclear. Here we developed a new and sensitive fingerprint approach to study the antibacterial activity of GO and underlying mechanism, using Raman spectroscopy. Spectroscopic signatures obtained from biomolecules such as Adenine and proteins from bacterial cultures with different concentrations of GO, allowed us to probe the antibacterial activity of GO with its mechanism at the molecular level. Escherichia coli (E. coli) and Enterococcus faecalis (E. faecalis) were used as model micro-organisms for all the experiments performed. The observation of higher intensity Raman peaks from Adenine and proteins in GO treated E. coli and E. faecalis; correlated with induced death, confirmed by Scanning electron Microscopy (SEM) and Biological Atomic Force Microscopy (Bio-AFM). Our findings open the way for future investigations of the antibacterial properties of different nanomaterial/GO composites using Raman spectroscopy.

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Section: Methodsmentioning

confidence: 99%

Study of antibacterial mechanism of graphene oxide using Raman spectroscopy

Nanda

Kim

2016

Sci Rep

193

101

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“…Among the four different bacteria, E. coli O157 demonstrated the highest level of membrane peroxidase activity which is inconsistent with previous report. 31 When we incorporated nanorods in the media, peroxidase activity greatly varied depending on the types of nanorods. Enzymatic activity was found quite low in case of AuNRs-treated bacteria.…”

Section: Discussionmentioning

confidence: 99%

“…31 Approximately 80 µL of the peroxidase substrate In each bacteria sample, continuous membrane peroxidase activity toward TMB substrate was further analyzed after repeated washing. The samples were washed five times and the OD was measured at 450 nm.…”

Section: Endogenous Membrane Peroxidase Activitymentioning

confidence: 99%

Enhanced detection sensitivity of Escherichia coli O157:H7 using surface-modified gold nanorods

Ramasamy¹,

Yi²,

An³

2015

IJN

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Escherichia coli O157:H7 (O157) is a Gram negative and highly virulent bacteria found in food and water sources, and is a leading cause of chronic diseases worldwide. Diagnosis and prevention from the infection require simple and rapid analysis methods for the detection of pathogens, including O157. Endogenous membrane peroxidase, an enzyme present on the surface of O157, was used for the colorimetric detection of bacteria by catalytic oxidation of the peroxidase substrate. In this study, we have analyzed the impact of the synthesized bare gold nanorods (AuNRs) and silica-coated AuNRs on the growth of E. coli O157. Along with the membrane peroxidase activity of O157, other bacteria strains were analyzed. Different concentrations of nanorods were used to analyze the growth responses, enzymatic changes, and morphological alterations of bacteria by measuring optical density, 3,3′,5,5′-tetramethylbenzidine assay, flow cytometry analysis, and microscopy studies. The results revealed that O157 showed higher and continuous membrane peroxidase activity than other bacteria. Furthermore, O157 treated with bare AuNRs showed a decreased growth rate in comparison with the bacteria with surface modified AuNRs. Interestingly, silica-coated AuNRs favored the growth of bacteria and also increased membrane peroxidase activity. This result can be particularly important for the enzymatic analysis of surface treated AuNRs in various microbiological applicants.

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“…Owing to the existence of endogenous peroxidases in E. coli O157:H7, peroxidase-based colorimetric reactions have generally been used in determining the presence of E. coli O157:H7 under standard buffer conditions at the lowest detectable concentration of ~10 5 cfu mL −1 [ 31 ]. However, most existing methods that use peroxidases for the visualization of the presence of Gram-negative bacteria still require overnight culture before peroxidase reaction tests can be carried out [ 32 , 33 , 34 , 35 , 36 ]. Moreover, Gram-positive bacteria, such as S. aureus , contain abundant catalases [ 37 ] that can catalyze the generation of oxygen in the presence of H 2 O 2 [ 37 ] ( SI Scheme S1 ).…”

Section: Introductionmentioning

confidence: 99%

Rapid Detection of Pathogenic Bacteria by the Naked Eye

2021

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Escherichia coli O157:H7 and Staphylococcus aureus are common pathogens. Gram-negative bacteria, such as E. coli, contain high concentrations of endogenous peroxidases, whereas Gram-positive bacteria, such as S. aureus, possess abundant endogenous catalases. Colorless 3,5,3′,5′-tetramethyl benzidine (TMB) changes to blue oxidized TMB in the presence of E. coli and a low concentration of H2O2 (e.g., ~11 mM) at pH of 3. Moreover, visible air bubbles containing oxygen are generated after S. aureus reacts with H2O2 at a high concentration (e.g., 180 mM) at pH of 3. A novel method for rapidly detecting the presence of bacteria on the surfaces of samples, on the basis of these two endogenous enzymatic reactions, was explored. Briefly, a cotton swab was used for collecting bacteria from the surfaces of samples, such as tomatoes and door handles, then two-step endogenous enzymatic reactions were carried out. In the first step, a cotton swab containing bacteria was immersed in a reagent comprising H2O2 (11.2 mM) and TMB for 25 min. In the second step, the swab was dipped further in H2O2 (180 mM) at pH 3 for 5 min. Results showed that the presence of Gram-negative bacteria, such as E. coli with a cell number of ≥ ~105, and Gram-positive bacteria, such as S. aureus with a cell number of ≥ ~106, can be visually confirmed according to the appearance of the blue color in the swab and the formation of air bubbles in the reagent solution, respectively, within ~30 min. To improve visual sensitivity, we dipped the swab carrying the bacteria in a vial containing a growth broth, incubated it for ~4 h, and carried out the two-stage reaction steps. Results showed that bluish swabs resulting from the presence of E. coli O157: H7 with initial cell numbers of ≥ ~34 were obtained, whereas air bubbles were visible in the samples containing S. aureus with initial cell numbers of ≥ ~8.5 × 103.

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Detection of E. coli O157:H7 using its endogenous active membrane peroxidase

Cited by 5 publications

References 27 publications

Study of antibacterial mechanism of graphene oxide using Raman spectroscopy

Study of antibacterial mechanism of graphene oxide using Raman spectroscopy

Enhanced detection sensitivity of Escherichia coli O157:H7 using surface-modified gold nanorods

Rapid Detection of Pathogenic Bacteria by the Naked Eye

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