Development of Molecularly Imprinted Polymers To Block Quorum Sensing and Inhibit Bacterial Biofilm Formation

Bacterial biofilms are responsible for most clinical infections and show increased antimicrobial resistance. In this study, molecularly imprinted polymers (MIPs) were developed to specifically capture prototypical quorum sensing autoinducers [i.e., <i>N</i>-(3-oxododecanoyl)-l-homoserine lactone (3-oxo-C₁₂AHL)], interrupt quorum sensing, and subsequently inhibit biofilm formation of Pseudomonas aeruginosa, an important human nosocomial pathogen. The synthesis of MIPs was optimized by considering the amount and type of the functional monomers itaconic acid (IA) and 2-hydroxyethyl methacrylate (HEMA). IA-based MIPs showed high adsorption affinity toward 3-oxo-C₁₂AHL with an imprinting factor of 1.68. Compared to IA-based MIPs, the adsorption capacity of HEMA-based MIPs was improved fivefold. HEMA-based MIPs significantly reduced biofilm formation (by ∼65%), whereas biofilm suppression by IA-based MIPs was neutralized because of increased bacterial attachment. The developed MIPs represent promising alternative biofilm intervention agents that can be applied to surfaces relevant to clinical settings and food processing equipment

Determination of α‑Tocopherol in Vegetable Oils Using a Molecularly Imprinted Polymers–Surface-Enhanced Raman Spectroscopic Biosensor

We report the development of a novel hybrid “capture–detection” molecularly imprinted polymers–surface-enhanced Raman spectroscopic (MIPs-SERS) biosensor for the detection and quantification of α-tocopherol (α-Toc) in vegetable oils. α-Toc served as the template for MIPs synthesis. Methacrylic acid formed as the functional monomer. Ethylene glycol dimethacrylate was the cross-linking agent, and 2,2′-azobisisobutyronitrile was used as the initiator. The synthesized MIPs functioned to rapidly and selectively adsorb and separate α-Toc from oil components. We validated a dendritic silver nanostructure synthesized by a displacement reaction to be a suitable SERS substrate for the enhancement of Raman signals. Second-derivative transformations and chemometric models based upon SERS spectral features confirmed the possibility of a rapid and precise detection and quantification of different spiking levels of α-Toc in four different sources of vegetable oils (Mahalanobis distance from 15.93 to 34.01 for PCA model; <i>R</i> > 0.92, RMSE < 0.41 for PLSR model). The MIPs-SERS biosensor had a high sensitivity as well as a good recovery for α-Toc analysis in vegetable oils. The entire analysis required 15 min or less to complete with limited sample preparation

Detecting and Tracking Nosocomial Methicillin-Resistant <i>Staphylococcus aureus</i> Using a Microfluidic SERS Biosensor

Rapid detection and differentiation of methicillin-resistant <i>Staphylococcus aureus</i> (MRSA) are critical for the early diagnosis of difficult-to-treat nosocomial and community acquired clinical infections and improved epidemiological surveillance. We developed a microfluidics chip coupled with surface enhanced Raman scattering (SERS) spectroscopy (532 nm) “lab-on-a-chip” system to rapidly detect and differentiate methicillin-sensitive <i>S. aureus</i> (MSSA) and MRSA using clinical isolates from China and the United States. A total of 21 MSSA isolates and 37 MRSA isolates recovered from infected humans were first analyzed by using polymerase chain reaction (PCR) and multilocus sequence typing (MLST). The <i>mecA</i> gene, which refers resistant to methicillin, was detected in all the MRSA isolates, and different allelic profiles were identified assigning isolates as either previously identified or novel clones. A total of 17 400 SERS spectra of the 58 <i>S. aureus</i> isolates were collected within 3.5 h using this optofluidic platform. Intra- and interlaboratory spectral reproducibility yielded a differentiation index value of 3.43–4.06 and demonstrated the feasibility of using this optofluidic system at different laboratories for bacterial identification. A global SERS-based dendrogram model for MRSA and MSSA identification and differentiation to the strain level was established and cross-validated (Simpson index of diversity of 0.989) and had an average recognition rate of 95% for <i>S. aureus</i> isolates associated with a recent outbreak in China. SERS typing correlated well with MLST indicating that it has high sensitivity and selectivity and would be suitable for determining the origin and possible spread of MRSA. A SERS-based partial least-squares regression model could quantify the actual concentration of a specific MRSA isolate in a bacterial mixture at levels from 5% to 100% (regression coefficient, >0.98; residual prediction deviation, >10.05). This optofluidic platform has advantages over traditional genotyping for ultrafast, automated, and reliable detection and epidemiological surveillance of bacterial infections

Lactic acid kills <i>C. jejuni in vitro</i>.

<p>An overnight MH broth culture of <i>C. jejuni</i> was inoculated into fresh media to an O.D.₆₀₀ of 0.1. Each broth culture was either untreated, supplemented with 10 mM, 25 mM, or 100 mM lactic acid, or supplemented with hydrochloric acid to achieve a pH equivalent to the lactic acid treated cultures (pH 5.12, 4.32, and 3.46, respectiveley). Samples were serially diluted and plated onto MHB agar for enumeration at 1, 2, 4, and 8 h after incubation. No viable <i>C. jejuni</i> were detected after 1 h when the media was supplemented with 100 mM lactic acid or acidified to pH 3.46 using HCl, therefore these data are not displayed.</p

<i>Lactobacillus</i> reduces <i>C. jejuni</i> colonization of broiler chicks.

<p>Broiler chicks were administered <i>Lactobacillus</i> by oral gavage (∼10⁸ CFU) at day of hatch and 4 days post-hatch. Chicks receiving <i>C. jejuni</i> challenge were administered <i>C. jejuni</i> F38011 by oral gavage (∼10⁸ CFU) at day 14 post-hatch. Chickens were euthanized and necropsied at seven days post-challenge. Cecal contents were serially diluted and plated onto Campy Cefex agar for enumeration of <i>C. jejuni</i>. No <i>C. jejuni</i> were detected in non-challenged chickens.</p

Computer-Aided Molecular Modeling Study on Antibody Recognition of Small Molecules: An Immunoassay for Triazine Herbicides

Most immunoassays for determination of small molecules are still designed on the basis of the “trial and error” method, due to the lack of understanding of antibody recognition. In the present study, we developed a heterologous indirect competitive enzyme-linked immunosorbent assay for determination of triazine herbicides, with limits of detection for 11 triazines ranging from 0.05 to 29.4 μg/L. Mechanisms of the antigen–antibody interaction were studied by computer-aided molecular modeling (CAMM)-based quantitative structure–activity relationship analyses. Co-effects of the analytes’ substructural hydrophobic, electrostatic, and steric fields on antibody recognition were further revealed. Hydrophobicity of the antigens was demonstrated to have the most important impact. Even less exposed substituents provided hydrophobic force to the antigen–antibody interaction. Dislocated orientation of analyte functional groups could lead to steric hindrance and hydrophobic misleading of antibody recognition. This may happen even when the antigens contained the same substituent as the hapten. Frontier orbital energies also affect the reaction significantly. This study highlights of the power of CAMM-based analyses, providing insights into antibody recognition of small molecules

Inhibition of <i>C. jejuni</i> by lactobacilli.

<p>Overnight cultures of <i>C. jejuni</i> were inoculated into 10 ml MH soft agar, overlaid on MH agar, and incubated 24 h at 37°C. Supernatants from overnight cultures of <i>Lactobacillus</i> were left untreated, neutralized with 6.25 N NaOH, or heat treated (boiled). Treated supernatants were filter sterilized and spotted onto the <i>C. jejuni</i> inoculated soft agar. <i>Lactobacillus</i> strains used are indicated as follows: (A) <i>L. acidophilus</i>, (B) <i>L. crispatus</i>, (C) <i>L. gallinarum</i> and (D) <i>L. helveticus</i>.</p

Production of lactic acid^a.

a<p>±represent one standard deviation from the mean of triplicate assays.</p

<i>L. crispatus</i> dominates the metabolic activity in co-culture.

<p>Partial least squares 2-component (PSL2) models were calibrated with pure monocultures of <i>C. jejuni</i> (circles) and <i>L. crispatus</i> (squares), and co-culture samples (triangles) were projected into the PLS2 model. Color spectrum represents log₁₀ CFU/mL (cell count).</p

Both regions 9 and 4N modulate NiV membrane fusion.

<p>CSE (HA) and cell-cell fusion levels of region 4N (<b>A</b>) and 9 (<b>B</b>) mutants. CSE levels were measured in 293T cells as in <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1003770#ppat-1003770-g004" target="_blank">Fig. 4E</a>. 293T cell-cell fusion levels induced by wt NiV-F and wt or mutant NiV-G, normalized to values of wt NiV-F/G. n = 3–8. <b>C) & D)</b> Depictions of regions 4N (C), or 9 (D), from the crystalized NiV-G head structure in <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1003770#ppat-1003770-g004" target="_blank">Fig. 4G</a>. Blue and red colored residues mark hypo- or hyper-fusogenic mutants, respectively.</p