Inactivation of the virulence factors from 2,3-butanediol-producing Klebsiella pneumoniae.

The microbiological production of 2,3-butanediol (2,3-BDO) has attracted considerable attention as an alternative way to produce high-value chemicals from renewable sources. Among the number of 2,3-BDO-producing microorganisms, Klebsiella pneumoniae has been studied most extensively and is known to produce large quantity of 2,3-BDO from a range of substrates. On the other hand, the pathogenic characteristics of the bacteria have limited its industrial applications. In this study, two major virulence traits, outer core LPS and fimbriae, were removed through homologous recombination from 2,3-BDO-producing K. pneumoniae 2242 to expand its uses to the industrial scale. The K. pneumoniae 2242 ∆wabG mutant strain was found to have an impaired capsule, which significantly reduced its ability to bind to the mucous layer and evade the phagocytic activity of macrophage. The association with the human ileocecal epithelial cell, HCT-8, and the bladder epithelial cell, T-24, was also reduced dramatically in the K. pneumoniae 2242 ∆fimA mutant strain that was devoid of fimbriae. However, the growth rate and production yield for 2,3-BDO were unaffected. The K. pneumoniae strains developed in this study, which are devoid of the major virulence factors, have a high potential for the efficient and sustainable production of 2,3-BDO.

Paper-Based Radial Chromatographic Immunoassay for the Detection of Pathogenic Bacteria in Milk.

Here, a paper-based radial flow chromatographic immunoassay (RFCI) employing gold nanoparticles (AuNPs) as chromatic agents was developed for the detection of Escherichia coli O157:H7 in whole milk. A 4-repeated gold-binding peptide-tagged (4GBP) streptococcal protein G (SPG) fusion protein was constructed as a bifunctional linker to immobilize antibodies on the surface of AuNPs with a well-oriented form based on the specific affinity of GBP and SPG to the gold and Fc portion of the antibody, respectively. 4GS@AuNPs prepared with the bifunctional linker protein exhibited excellent colloidal stability even at high salt concentrations of up to 500 mM, which is a critical requirement for its application to a broad range of biological and food samples. The enhanced colloidal stability and excellent binding capability of the immuno-4GS@AuNPs toward target bacteria lowered the detection limit of RFCI for target pathogenic bacteria in whole milk as low as 103 CFU/mL, which is by an order of magnitude lower than that of conventional immuno-AuNPs prepared with physical adsorption of antibodies. The RFCI pattern could also be converted into a grayscale value by simple image processing for quantitative determination of target pathogenic bacteria. This paper-based detection system would provide an effective means of monitoring the presence of food-borne pathogens in real food samples with naked eyes.