Abstract
The emergence of antibiotic resistant microorganisms is a great public health concern and has triggered an urgent need to develop alternative antibiotics. Chitosan microparticles (CM), derived from chitosan, have been shown to reduce E. coli O157:H7 shedding in a cattle model, indicating potential use as an alternative antimicrobial agent. However, the underlying mechanism of CM on reducing the shedding of this pathogen remains unclear. To understand the mode of action, we studied molecular mechanisms of antimicrobial activity of CM using in vitro and in vivo methods. We report that CM are an effective bactericidal agent with capability to disrupt cell membranes. Binding assays and genetic studies with an ompA mutant strain demonstrated that outer membrane protein OmpA of E. coli O157:H7 is critical for CM binding, and this binding activity is coupled with a bactericidal effect of CM. This activity was also demonstrated in an animal model using cows with uterine diseases. CM treatment effectively reduced shedding of intrauterine pathogenic E. coli (IUPEC) in the uterus compared to antibiotic treatment. Since Shiga-toxins encoded in the genome of bacteriophage is often overexpressed during antibiotic treatment, antibiotic therapy is generally not recommended because of high risk of hemolytic uremic syndrome. However, CM treatment did not induce bacteriophage or Shiga-toxins in E. coli O157:H7; suggesting that CM can be a potential candidate to treat infections caused by this pathogen. This work establishes an underlying mechanism whereby CM exert antimicrobial activity in vitro and in vivo, providing significant insight for the treatment of diseases caused by a broad spectrum of pathogens including antibiotic resistant microorganisms.
Highlights
Chitosan has been highlighted as a potential candidate for targeting antibiotic resistant microorganisms due to a broad spectrum of antimicrobial activity and biocompatibility [1,2,3,4,5,6]
We suggested that reduction of E. coli O157:H7 by oral Chitosan microparticles (CM) administration might be a result of the pathogen binding activity of CM, the previous study failed to differentiate whether the reduction of E
To understand if the E. coli O157:H7 reduction was caused by either scrubbing or antimicrobial action, antimicrobial activity was determined by a standard plating method after incubation of E. coli O157:H7 with CM at various concentrations, ranging from 0% to 0.2%
Summary
Chitosan has been highlighted as a potential candidate for targeting antibiotic resistant microorganisms due to a broad spectrum of antimicrobial activity and biocompatibility [1,2,3,4,5,6]. Though the exact mechanism has yet to be elucidated, the intracellular leakage hypothesis is widely accepted [1,10,11,12] In this mechanism, positively charged chitosan binds to the negatively charged bacterial surface leading to altered membrane permeability, which results in leakage of intracellular constituents causing cell death [3,5,11]. It has been reported that antimicrobial activity of chitosan is limited to acidic conditions due to the loss of positive charges on the amino group at neutral pH [3,5]. This restricts the use of chitosan as an antimicrobial agent at neutral pH
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