Detection of Low Levels of Listeria monocytogenes Cells by Using a Fiber-Optic Immunosensor

Biosensor technology has a great potential to meet the need for sensitive and nearly real-time microbial detection from foods. An antibody-based fiber-optic biosensor to detect low levels of Listeria monocytogenes cells following an enrichment step was developed. The principle of the sensor is a sandwich immunoassay where a rabbit polyclonal antibody was first immobilized on polystyrene fiber waveguides through a biotin-streptavidin reaction to capture Listeria cells on the fiber. Capture of cells on the fibers was confirmed by scanning electron microscopy. A cyanine 5-labeled murine monoclonal antibody, C11E9, was used to generate a specific fluorescent signal, which was acquired by launching a 635-nm laser light from an Analyte 2000 and collected by a photodetector at 670 to 710 nm. This immunosensor was specific for L. monocytogenes and showed a significantly higher signal strength than for other Listeria species or other microorganisms, including Escherichia coli, Enterococcus faecalis, Salmonella enterica, Lactobacillus plantarum, Carnobacterium gallinarum, Hafnia alvei, Corynebacterium glutamicum, Enterobacter aerogenes, Pseudomonas aeruginosa, and Serratia marcescens, in pure or in mixed-culture setup. Fiber-optic results could be obtained within 2.5 h of sampling. The sensitivity threshold was about 4.3 × 10³ CFU/ml for a pure culture of L. monocytogenes grown at 37°C. When L. monocytogenes was mixed with lactic acid bacteria or grown at 10°C with 3.5% NaCl, the detection threshold was 4.1 × 10⁴ or 2.8 × 10⁷ CFU/ml, respectively. In less than 24 h, this method could detect L. monocytogenes in hot dog or bologna naturally contaminated or artificially inoculated with 10 to 1,000 CFU/g after enrichment in buffered Listeria enrichment broth.     

Development of a rapid 1-h fluorescence-based cytotoxicity assay for Listeria species

Listeria monocytogenes is cytotoxic to the lymphocyte-origin hybridoma Ped-2E9 cell line. The relative cytotoxicity can be calculated by assaying the release of alkaline phosphatase (ALP) from the infected cell line. In this study, a fluorogenic substrate (4-methylumbelliferyl phosphate, MUP) was used to quantify the ALP activity. The assay is 3.5-fold more sensitive than the colorimetric-based assay and requires only 1 h to differentiate virulent from avirulent strains. In addition to various Listeria species, 27 different common foodborne or clinical microorganisms were tested with the fluorescence-based cytotoxicity assay and only six cultures (Bacillus cereus, Citrobacter freundii, Serratia marcescens, Pseudomonas putida, Corynebacterium glutamicum and Micrococcus luteus) showed cytotoxic effects similar to L. monocytogenes. To use this assay as a confirmatory test for virulent L. monocytogenes suspect strains, pure cultures must be isolated from the sample prior to testing.     

PKD1 induces p21(waf1) and regulation of the cell cycle via direct activation of the JAK-STAT signaling pathway in a process requiring PKD2

Autosomal dominant polycystic kidney disease is characterized by cyst formation in the kidney and other organs and results from mutations of PKD1 or PKD2. Previous studies suggest that their gene products have an important role in growth regulation. We now show that expression of polycystin-1 activates the JAK-STAT pathway, thereby upregulating p21(waf1) and inducing cell cycle arrest in G0/G1. This process requires polycystin-2, a channel protein, as an essential cofactor. Mutations that disrupt polycystin-1/2 binding prevent activation of the pathway. Mouse embryos lacking Pkd1 have defective STAT1 phosphorylation and p21(waf1) induction. These results suggest that one function of the polycystin-1/2 complex is to regulate the JAK/STAT pathway and explain how mutations of either gene can result in dysregulated growth.     

Glucose and nutrient concentrations affect the expression of a 104-kilodalton Listeria adhesion protein in Listeria monocytogenes

Growth media and environmental conditions influence the expression of adhesion and invasion proteins in Listeria monocytogenes. Here, the expression of the 104-kDa Listeria adhesion protein (LAP) was studied in nutrient-rich media (Trypticase soy broth [TSB] and brain heart infusion [BHI]), minimal medium (Luria-Bertani [LB]), or nutrient-deficient medium (peptone water [PW]) by immunoblotting, enzyme-linked immunosorbent assay (ELISA), and immunoelectron microscopy. Also, the effect of incorporating different concentrations of glucose on LAP expression was studied. Immunoblotting showed that LAP expression was at least twofold higher in LB medium than in TSB or BHI, while PW supported very poor cell growth and LAP expression. ELISA and immunoblotting results showed that higher concentrations of glucose (>1.6 g/liter) lowered the culture pH and suppressed LAP expression by more than 75%; however, the addition of K(2)HPO(4) reduced this effect. L. monocytogenes cells grown in LB media with lower concentrations of glucose showed higher adhesion to Caco-2 cells (3,716 and 4,186 cpm of attached bacteria for 0 and 0.2 g of glucose/liter, respectively), while L. monocytogenes cells grown in LB with higher glucose concentrations exhibited lower adhesion (2,126 and 2,221 cpm for 1.6 and 3.2 g of glucose/liter, respectively). A LAP-negative L. monocytogenes strain (A572) showed low adhesion profiles regardless of the amount of glucose added. Transmission electron microscopy revealed that LAP is localized mainly in the cytoplasm, with only a few molecules located on the cell surface. Growth in LB with high glucose (3.2 g/liter) showed the presence of only a few molecules in the cells, corroborating the results observed with ELISA or immunoblotting. In summary, nutrient-rich media and high concentrations of glucose suppressed LAP expression, which possibly is due to the changes in the pH of the media during growth from the accumulation of sugar fermentation by-products.     

High-resolution solution structure of a designed peptide bound to lipopolysaccharide: transferred nuclear Overhauser effects, micelle selectivity, and anti-endotoxic activity

Designing peptides that would interact with lipopolysaccharides (LPS) and acquire a specific folded conformation can generate useful structural insights toward the development of anti-sepsis agents. In this work, we have constructed a 12-residue linear peptide, YW12, rich in aromatic and aliphatic amino acid residues with a centrally located stretch of four consecutive positively charged (KRKR) residues. In absence of LPS, YW12 is predominantly unstructured in aqueous solution. Using transferred nuclear Overhauser effect (Tr-NOE) spectroscopy, we demonstrate that YW12 adopts a well-folded structure as a complex with LPS. Structure calculations reveal that YW12 assumes an extended conformation at the N-terminus followed by two consecutive beta-turns at its C-terminus. A hydrophobic core is formed by extensive packing between number of aromatic and nonpolar residues, whereas the positively charged residues are segregated out to a separate region essentially stabilizing an amphipathic structure. In an in vitro LPS neutralization assay using NF-kappaB induction as the readout, YW12 shows moderate activity with an IC50 value of approximately 10 microM. As would be expected, tryptophan fluorescence studies demonstrate that YW12 shows selective interactions only with the negatively charged lipid micelles including sodium dodecyl sulfate (SDS), 1-palmitoyl-2-oleoylphosphatidyl-dl-glycerol (POPG), and LPS, and no significant interactions are detected with zwitterionic lipid micelles such as dodecyl-phosphocholine (DPC). Far-UV CD studies indicate the presence of beta-turns or beta-sheet-like conformations of the peptide in negatively charged micelles, whereas no structural transitions are apparent in DPC micelles. These results suggest that structural features of YW12 could be utilized to develop nontoxic antisepsis compounds.     

Influence of temperature and growth phase on expression of a 104-kilodalton Listeria adhesion protein in Listeria monocytogenes.

Interaction of Listeria monocytogenes with mammalian intestinal cells is believed to be an important first step in Listeria pathogenesis. Transposon (Tn916) mutagenesis provided strong evidence that a 104-kDa surface protein, designated the Listeria adhesion protein (LAP), was involved in adherence of L. monocytogenes to a human enterocyte-like Caco-2 cell line (V. Pandiripally, D. Westbrook, G. Sunki, and A. Bhunia, J. Med. Microbiol. 48:117-124, 1999). In this study, expression of LAP in L. monocytogenes at various growth temperatures (25, 37, and 42 degrees C) and in various growth phases was determined by performing an enzyme-linked immunoassay (ELISA) and Western blotting with a specific monoclonal antibody (monoclonal antibody H7). The ELISA and Western blot results indicated that there was a significant increase in LAP expression over time only at 37 and 42 degrees C and that the level of LAP expression was low during the exponential phase and high during the stationary phase. In contrast, there were not significant differences in LAP expression between the exponential and stationary phases at 25 degrees C. Examination of the adhesion of L. monocytogenes cells from exponential-phase (12-h) or stationary-phase (24-h) cultures grown at 37 degrees C to Caco-2 cells revealed that there were not significant differences in adhesion. Although expression of L. monocytogenes LAP was different at different growth temperatures and in different growth phases, enhanced expression did not result in increased adhesion, possibly because only a few LAP molecules were sufficient to initiate binding to Caco-2 cells.     

Antigenic property of pediocin AcH produced by Pediococcus acidilactici H

Pediocin AcH, a bacteriocin of Pediococcus acidilactici H, inhibits the growth of several food spoilage and pathogenic bacteria. The antigenic property of partially purified pediocin AcH was tested by immunizing mice and a rabbit. Pediocin AcH was not immunogenic in these animals as determined by immunoblotting even after conjugation to bovine serum albumin. The non-immunogenic nature of pediocin AcH, its non-toxicity to laboratory animals and its hydrolysis by gastric proteolytic enzymes may be considered favourably in its possible use as a food preservative.     

Delimitation of kala-azar risk areas in the district of Vaishali in Bihar (India) using a geo-environmental approach

Remote sensing and geographical information technologies were used to discriminate areas of high and low risk for contracting kala-azar or visceral leishmaniasis. Satellite data were digitally processed to generate maps of land cover and spectral indices, such as the normalised difference vegetation index and wetness index. To map estimated vector abundance and indoor climate data, local polynomial interpolations were used based on the weightage values. Attribute layers were prepared based on illiteracy and the unemployed proportion of the population and associated with village boundaries. Pearson's correlation coefficient was used to estimate the relationship between environmental variables and disease incidence across the study area. The cell values for each input raster in the analysis were assigned values from the evaluation scale. Simple weighting/ratings based on the degree of favourable conditions for kala-azar transmission were used for all the variables, leading to geo-environmental risk model. Variables such as, land use/land cover, vegetation conditions, surface dampness, the indoor climate, illiteracy rates and the size of the unemployed population were considered for inclusion in the geo-environmental kala-azar risk model. The risk model was stratified into areas of "risk"and "non-risk"for the disease, based on calculation of risk indices. The described approach constitutes a promising tool for microlevel kala-azar surveillance and aids in directing control efforts.     

Simultaneous Carbon and Nitrogen Removal from Domestic Wastewater using High Rate Vermifilter

Being a cost-effective and environmentally benign technology, vermifiltration has significantly replaced the available conventional wastewater remediation methods in many cases over the last few decades. The present work emphasizes on the investigation of the nitrogen transformation dynamics, in addition to the organic carbon abatement in the designed high rate hybrid vermifilter. Moreover, the economical sustainability of the vermifiltration technology has also been enlightened by creating a bridge with the concept of circular bio-economy. The designed high rate macrophyte-assisted vermifilter (MAVF) ascertained significant high nitrogen and organic carbon removal efficiencies from the real domestic sewage, considering the chemical oxygen demand (COD) of the influent and hydraulic loading rate (HLR) as the input variables. The designed MAVF facilitated the maximum ammonium nitrogen (NH₄⁺-N), organic nitrogen, and total kjeldahl nitrogen removal efficiencies up to 98.2 ± 0.70%, 100%, and 99 ± 0.47%, respectively when COD of the influent and HLR were 200 ± 25 mg/L and 3 ± 0.1 m³/m²-d, respectively. On the other hand, substantial enhancement in the nitrate nitrogen (NO₃⁻-N) in the effluent (73 ± 10.55 times its influent concentration) was observed with influent COD of 200 ± 25 mg/L and HLR of 7 ± 0.2 m³/m²-d. When the influent COD and HLR were maintained at 700 ± 45 mg/L and 3 ± 0.1 m³/m²-d, respectively, the highest total nitrogen removal of 87 ± 2.25% was obtained. Alternatively, the influent COD of 200 ± 25 mg/L and HLR of 3 ± 0.1 m³/m²-d yielded the highest COD removal efficiency of 77 ± 1.59%. Hence, the outcome of the present research work strengthens the suitability of the vermifiltration technology as an economically and ecologically sound natural wastewater bio-remediation technology for the treatment of domestic wastewater.     

NMR structures and interactions of temporin-1Tl and temporin-1Tb with lipopolysaccharide micelles: mechanistic insights into outer membrane permeabilization and synergistic activity

Temporins are a group of closely related short antimicrobial peptides from frog skin. Lipopolysaccharide (LPS), the major constituent of the outer membrane of gram-negative bacteria, plays important roles in the activity of temporins. Earlier studies have found that LPS induces oligomerization of temporin-1Tb (TB) thus preventing its translocation across the outer membrane and, as a result, reduces its activity on gram-negative bacteria. On the other hand, temporin-1Tl (TL) exhibits higher activity, presumably because of lack of such oligomerization. A synergistic mechanism was proposed, involving TL and TB in overcoming the LPS-mediated barrier. Here, to gain insights into interactions of TL and TB within LPS, we investigated the structures and interactions of TL, TB, and TL+TB in LPS micelles, using NMR and fluorescence spectroscopy. In the context of LPS, TL assumes a novel antiparallel dimeric helical structure sustained by intimate packing between aromatic-aromatic and aromatic-aliphatic residues. By contrast, independent TB has populations of helical and aggregated conformations in LPS. The LPS-induced aggregated states of TB are largely destabilized in the presence of TL. Saturation transfer difference NMR studies have delineated residues of TL and TB in close contact with LPS and enhanced interactions of these two peptides with LPS, when combined together. Fluorescence resonance energy transfer and (31)P NMR have pointed out the proximity of TL and TB in LPS and conformational changes of LPS, respectively. Importantly, these results provide the first structural insights into the mode of action and synergism of antimicrobial peptides at the level of the LPS-outer membrane.