Abstract
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.
Highlights
Foodborne illnesses caused by pathogenic bacteria can result in diarrhea, abdominal pain, nausea, fever, and even death [1,2,3,4,5], and these pathogens cannot be identified on the basis of these symptoms [3]
Given that the goal of this study was to use endogenous enzymatic reactions derived from bacteria for distinguishing Gram-negative bacteria from Gram-positive bacteria, two common pathogenic bacteria, S. aureus and E. coli O157: H7, were initially selected as the models for investigation
The cotton swab at the bottom of the photograph was tainted with Gram-negative bacteria (E. coli O157:H7), whereas the cotton swab at the top of the photograph was shown after sampling Gram-positive bacteria (S. aureus) and remained colorless
Summary
Foodborne illnesses caused by pathogenic bacteria can result in diarrhea, abdominal pain, nausea, fever, and even death [1,2,3,4,5], and these pathogens cannot be identified on the basis of these symptoms [3]. S. aureus, a Gram-positive bacterium, is another common pathogen that can cause foodborne illnesses [15]. Meaningful information necessary to medical treatment can be obtained by determining whether infections or contaminations are caused by either Gram-positive or Gram-negative bacteria. 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]. We developed a rapid sensing method for detecting Gram-negative and Gram-positive, catalase-positive bacteria in samples by using cotton swabs as the tool In this method, a swab is used as a sampling and sensing probe, and endogenous enzymatic reactions derived from target bacteria are used for distinguishing the presence of bacteria. The optimal experimental conditions were examined, and cherry tomatoes and door handles contaminated by bacteria were used as real samples
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