Showing papers by "Arun K. Bhunia published in 2021"

Bacterial Biofilms and Their Implications in Pathogenesis and Food Safety.

Abstract: Biofilm formation is an integral part of the microbial life cycle in nature. In food processing environments, bacterial transmissions occur primarily through raw or undercooked foods and by cross-contamination during unsanitary food preparation practices. Foodborne pathogens form biofilms as a survival strategy in various unfavorable environments, which also become a frequent source of recurrent contamination and outbreaks of foodborne illness. Instead of focusing on bacterial biofilm formation and their pathogenicity individually, this review discusses on a molecular level how these two physiological processes are connected in several common foodborne pathogens such as Listeria monocytogenes, Staphylococcus aureus, Salmonella enterica and Escherichia coli. In addition, biofilm formation by Pseudomonas aeruginosa is discussed because it aids the persistence of many foodborne pathogens forming polymicrobial biofilms on food contact surfaces, thus significantly elevating food safety and public health concerns. Furthermore, in-depth analyses of several bacterial molecules with dual functions in biofilm formation and pathogenicity are highlighted.

Biofilm-isolated Listeria monocytogenes exhibits reduced systemic dissemination at the early (12-24 h) stage of infection in a mouse model

Abstract: Environmental cues promote microbial biofilm formation and physiological and genetic heterogeneity. In food production facilities, biofilms produced by pathogens are a major source for food contamination; however, the pathogenesis of biofilm-isolated sessile cells is not well understood. We investigated the pathogenesis of sessile Listeria monocytogenes (Lm) using cell culture and mouse models. Lm sessile cells express reduced levels of the lap, inlA, hly, prfA, and sigB and show reduced adhesion, invasion, translocation, and cytotoxicity in the cell culture model than the planktonic cells. Oral challenge of C57BL/6 mice with food, clinical, or murinized-InlA (InlAm) strains reveals that at 12 and 24 h post-infection (hpi), Lm burdens are lower in tissues of mice infected with sessile cells than those infected with planktonic cells. However, these differences are negligible at 48 hpi. Besides, the expressions of inlA and lap mRNA in sessile Lm from intestinal content are about 6.0- and 280-fold higher than the sessle inoculum, respectively, suggesting sessile Lm can still upregulate virulence genes shortly after ingestion (12 h). Similarly, exposure to simulated gastric fluid (SGF, pH 3) and intestinal fluid (SIF, pH 7) for 13 h shows equal reduction in sessile and planktonic cell counts, but induces LAP and InlA expression and pathogenic phenotypes. Our data show that the virulence of biofilm-isolated Lm is temporarily attenuated and can be upregulated in mice during the early stage (12–24 hpi) but fully restored at a later stage (48 hpi) of infection. Our study further demonstrates that in vitro cell culture assay is unreliable; therefore, an animal model is essential for studying the pathogenesis of biofilm-isolated bacteria.

Listeria monocytogenes: review of pathogenesis and virulence determinants-targeted immunological assays

Abstract: Listeria monocytogenes is one of the most invasive foodborne pathogens and is responsible for numerous outbreaks worldwide. Most of the methods to detect this bacterium in food require selective enrichment using traditional bacterial culture techniques that can be time-consuming and labour-intensive. Moreover, molecular methods are expensive and need specific technical knowledge. In contrast, immunological approaches are faster, simpler, and user-friendly alternatives and have been developed for the detection of L. monocytogenes in food, environmental, and clinical samples. These techniques are dependent on the constitutive expression of L. monocytogenes antigens and the specificity of the antibodies used. Here, updated knowledge on pathogenesis and the key immunogenic virulence determinants of L. monocytogenes that are used for the generation of monoclonal and polyclonal antibodies for the serological assay development are summarised. In addition, immunological approaches based on enzyme-linked immunosorbent assay, immunofluorescence, lateral flow immunochromatographic assays, and immunosensors with relevant improvements are highlighted. Though the sensitivity and specificity of the assays were improved significantly, methods still face many challenges that require further validation before use.

Validation of Bioinformatic Modeling for the Zeta Potential of Vicilin, Legumin, and Commercial Pea Protein Isolate

Abstract: The zeta potential of a molecule is an important property in the food industry, since the electrostatic potential of a material governs its ability to interact through ionic forces with other molecules in solution. In particular, emulsions that are kept in solutions with a high magnitude of the zeta potential are shown to be more stable over time, as the electrostatic repulsive forces of protein far from its isoelectric point can help prevent oil globule coalescence over time. However, modeling the zeta potential of protein is difficult given the anisotropy of protein molecules, the shifts in amino acid side chain ionization across pH, and understanding at what distance to measure the zeta potential from the molecular surface to accurately capture the shear plane between the particle and solvent under flow. In this work, we use the Poisson-Boltzmann Equation to model the net electrostatic surface potential of pea vicilin and legumin. We then use a weighted average of these globular proteins to predict the measured zeta potential in pea protein. The R2 between the bioinformatically modeled net surface charge and the measured commercial isolate zeta potential is 0.987 between pH 2.50 and 9.50, and this equation predicted the zeta potential of a different commercial pea protein isolate with a standard error of 0.040. This shows that using the Poisson-Boltzmann Equation to solve for the net electrostatic surface potential, it is possible to accurately estimate the zeta potential of pea protein.

Cold Denaturation of Proteins: Where Bioinformatics Meets Thermodynamics to Offer a Mechanistic Understanding: Pea Protein As a Case Study.

Abstract: Protein structure can be altered with heat, but models which predict denaturation show that globular proteins also spontaneously unfold at low temperatures through cold denaturation. By an analysis of the primary structure of pea protein using bioinformatic modeling, a mechanism of pea protein cold denaturation is proposed. Pea protein is then fractionated into partially purified legumin and vicilin components, suspended in ethanol, and subjected to low temperatures (-10 to -20 °C). The structural characterizations of the purified fractions are conducted through FTIR, ζ potential, dynamic light scattering, and oil binding, and these are compared to the results of commercial protein isolates. The observed structural changes suggest that pea protein undergoes changes in structure as the result of low-temperature treatments, which could lead to innovative industrial processing techniques for functionalization by low-temperature processing.

Antibody- and nucleic acid-based lateral flow immunoassay for Listeria monocytogenes detection

Abstract: Listeria monocytogenes is an invasive opportunistic foodborne pathogen and its routine surveillance is critical for protecting the food supply and public health. The traditional detection methods are time-consuming and require trained personnel. Lateral flow immunoassay (LFIA), on the other hand, is an easy-to-perform, rapid point-of-care test and has been widely used as an inexpensive surveillance tool. In recent times, nucleic acid–based lateral flow immunoassays (NALFIA) are also developed to improve sensitivity and specificity. A significant improvement in lateral flow–based assays has been reported in recent years, especially the ligands (antibodies, nucleic acids, aptamers, bacteriophage), labeling molecules, and overall assay configurations to improve detection sensitivity, specificity, and automated interpretation of results. In most commercial applications, LFIA has been used with enriched food/environmental samples to ensure detection of live cells thus prolonging the assay time to 24–48 h; however, with the recent improvement in LFIA sensitivity, results can be obtained in less than 8 h with shortened and improved enrichment practices. Incorporation of surface-enhanced Raman spectroscopy and/or immunomagnetic separation could significantly improve LFIA sensitivity for near-real-time point-of-care detection of L. monocytogenes for food safety and public health applications.