Cell-surface anchoring of Listeria adhesion protein on L. monocytogenes is fastened by internalin B for pathogenesis.

Listeria adhesion protein (LAP) is a secreted acetaldehyde alcohol dehydrogenase (AdhE) that anchors to an unknown molecule on the Listeria monocytogenes (Lm) surface, which is critical for its intestinal epithelium crossing. In the present work, immunoprecipitation and mass spectrometry identify internalin B (InlB) as the primary ligand of LAP (KD ∼ 42 nM). InlB-deleted and naturally InlB-deficient Lm strains show reduced LAP-InlB interaction and LAP-mediated pathology in the murine intestine and brain invasion. InlB-overexpressing non-pathogenic Listeria innocua also displays LAP-InlB interplay. In silico predictions reveal that a pocket region in the C-terminal domain of tetrameric LAP is the binding site for InlB. LAP variants containing mutations in negatively charged (E523S, E621S) amino acids in the C terminus confirm altered binding conformations and weaker affinity for InlB. InlB transforms the housekeeping enzyme, AdhE (LAP), into a moonlighting pathogenic factor by fastening on the cell surface.

Inactivation of Polymicrobial Biofilms of Foodborne Pathogens Using Epsilon Poly-L-Lysin Conjugated Chitosan Nanoparticles.

A mixed culture (polymicrobial) biofilm provides a favorable environment for pathogens to persist in the food processing environment and to contaminate food products. Inactivation and eradication of such biofilms from food processing environments are achieved by using harsh disinfectants, but their toxicity and environmentally hostile characteristics are unsustainable. This study aims to use food-grade natural nanoparticulated antimicrobials to control mixed-culture biofilms. Chitosan, a natural broad-spectrum antimicrobial biopolymer (polysaccharide) from crustaceans, was derivatized to produce chitosan nanoparticles (ChNP) as a carrier for another broad-spectrum antimicrobial agent, ε-poly-L-lysine (PL), to synthesize ChNP-PL conjugate. The antimicrobial activity of ChNP and ChNP-PL was tested against mixed-culture biofilms. ChNP-PL (~100 nm) exhibited a synergistic antimicrobial and anti-biofilm effect against mono or mixed-culture biofilms of five foodborne pathogens, including Listeria monocytogenes, Staphylococcus aureus, Salmonella enterica serovar Enteritidis, Escherichia coli O157:H7, and Pseudomonas aeruginosa. ChNP-PL treatment prevented biofilm formation by mono or mixed cultures of L. monocytogenes, P. aeruginosa, and E. coli O157:H7, and bacterial counts were either below the detection limit or caused 3.5-5 log reduction. ChNP-PL also inactivated preformed biofilms. In monoculture biofilm, ChNP-PL treatment reduced L. monocytogenes counts by 4.5 logs, S. Enteritidis by 2 logs, E. coli by 2 logs, and S. aureus by 0.5 logs, while ChNP-PL had no inhibitory effect on P. aeruginosa. In vitro mammalian cell-based cytotoxicity analysis confirmed ChNP-PL to have no deleterious effect on intestinal HCT-8 cell line. In conclusion, our results show ChNP-PL has strong potential to prevent the formation or inactivation of preformed polymicrobial biofilms of foodborne pathogens.

Bacterial Biofilms and Their Implications in Pathogenesis and Food Safety.

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.

A Low-Field Magnetic Resonance Imaging Aptasensor for the Rapid and Visual Sensing of Pseudomonas aeruginosa in Food, Juice, and Water.

In this work, we present a low-field magnetic resonance imaging (LF-MRI) aptasensor based on the difference in magnetic behavior of two magnetic nanoparticles with diameters of 10 (MN10) and 400 nm (MN400) for the rapid detection of Pseudomonas aeruginosa (P. aeruginosa). First, specific anti-P. aeruginosa aptamers were covalently immobilized onto magnetic nanoparticles via 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide/N-hydroxysuccinimide chemistry for the capture of the target bacteria. In the presence of P. aeruginosa, an MN10-bacteria-MN400 (MBM) complex was formed after binding between the aptamers on magnetic nanoparticles and P. aeruginosa cells. When a magnetic field was applied, the MBM complex and free MN400 were rapidly magnetically separated, and free MN10 left in the solution worked as a T2 (transverse relaxation time) single readout in MRI measurement. Under optimum conditions, the LF-MRI platform provides both image analysis and quantitative detection of P. aeruginosa, with a detection limit of 100 cfu/mL. The feasibility and specificity of the aptasensor were demonstrated in detecting real food, orange juice, and drinking water samples and validated using plate counting methods.

Current State of Development of Biosensors and Their Application in Foodborne Pathogen Detection.

ABSTRACT: Foodborne disease outbreaks continue to be a major public health and food safety concern. Testing products promptly can protect consumers from foodborne diseases by ensuring the safety of food before retail distribution. Fast, sensitive, and accurate detection tools are in great demand. Therefore, various approaches have been explored recently to find a more effective way to incorporate antibodies, oligonucleotides, phages, and mammalian cells as signal transducers and analyte recognition probes on biosensor platforms. The ultimate goal is to achieve high specificity and low detection limits (1 to 100 bacterial cells or piconanogram concentrations of toxins). Advancements in mammalian cell-based and bacteriophage-based sensors have produced sensors that detect low levels of pathogens and differentiate live from dead cells. Combinations of biotechnology platforms have increased the practical utility and application of biosensors for detection of foodborne pathogens. However, further rigorous testing of biosensors with complex food matrices is needed to ensure the utility of these sensors for point-of-care needs and outbreak investigations.

Effects of fulvic acid size on microcystin-LR photodegradation and detoxification in the chlorine/UV process.

Microcystin-LR (MC-LR), a polypeptide toxin generated by cyanobacteria, threatens the safety of drinking water supplies. In this study, fulvic acid (FA) was separated into two molecular weight (MW) ranges to evaluate the effects of FA size on MC-LR degradation in the chlorine/UV process. The rates of MC-LR degradation were significantly reduced in FA-containing water (3.7 × 10-3 s-1 for small MW FA; 4.3 × 10-3 s-1 for large MW FA) as compared with FA free water (4.9 × 10-3 s-1). The contributions of ClO• to MC-LR degradation were dramatically lower in small MW FA water (0.4%) than large MW FA (13.9%) and FA free water (17.4%), suggesting inhibition by lignin-like substances in FA in the transformation of Cl• to ClO• and scavenging ClO•. Monochlorination and hydroxylation occurred in the first step of the MC-LR degradation process. The accumulation of intermediate products in the chlorine/UV process indicated that small MW FA inhibited further degradation of MC-LR. Small MW FA, rather than MC-LR degradation, was the dominant factor in minimizing MC-LR cytotoxicity toward a human intestinal epithelial cell line.

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

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.

Mammalian Cell-Based Immunoassay for Detection of Viable Bacterial Pathogens.

Rapid detection of live pathogens is of paramount importance to ensure food safety. At present, nucleic acid-based polymerase chain reaction and antibody-based lateral flow assays are the primary methods of choice for rapid detection, but these are prone to interference from inhibitors, and resident microbes. Moreover, the positive results may neither assure virulence potential nor viability of the analyte. In contrast, the mammalian cell-based assay detects pathogen interaction with the host cells and is responsive to only live pathogens, but the short shelf-life of the mammalian cells is the major impediment for its widespread application. An innovative approach to prolong the shelf-life of mammalian cells by using formalin was undertaken. Formalin (4% formaldehyde)-fixed human ileocecal adenocarcinoma cell line, HCT-8 on 24-well tissue culture plates was used for the capture of viable pathogens while an antibody was used for specific detection. The specificity of the Mammalian Cell-based ImmunoAssay (MaCIA) was validated with Salmonella enterica serovar Enteritidis and Typhimurium as model pathogens and further confirmed against a panel of 15 S. Enteritidis strains, 8 S. Typhimurium, 11 other Salmonella serovars, and 14 non-Salmonella spp. The total detection time (sample-to-result) of MaCIA with artificially inoculated ground chicken, eggs, milk, and cake mix at 1-10 CFU/25 g was 16-21 h using a traditional enrichment set up but the detection time was shortened to 10-12 h using direct on-cell (MaCIA) enrichment. Formalin-fixed stable cell monolayers in MaCIA provide longer shelf-life (at least 14 weeks) for possible point-of-need deployment and multi-sample testing on a single plate.

Receptor-targeted engineered probiotics mitigate lethal Listeria infection.

Probiotic bacteria reduce the intestinal colonization of pathogens. Yet, their use in preventing fatal infection caused by foodborne Listeria monocytogenes (Lm), is inconsistent. Here, we bioengineered Lactobacillus probiotics (BLP) to express the Listeria adhesion protein (LAP) from a non-pathogenic Listeria (L. innocua) and a pathogenic Listeria (Lm) on the surface of Lactobacillus casei. The BLP strains colonize the intestine, reduce Lm mucosal colonization and systemic dissemination, and protect mice from lethal infection. The BLP competitively excludes Lm by occupying the surface presented LAP receptor, heat shock protein 60 and ameliorates the Lm-induced intestinal barrier dysfunction by blocking the nuclear factor-κB and myosin light chain kinase-mediated redistribution of the major epithelial junctional proteins. Additionally, the BLP increases intestinal immunomodulatory functions by recruiting FOXP3+T cells, CD11c+ dendritic cells and natural killer cells. Engineering a probiotic strain with an adhesion protein from a non-pathogenic bacterium provides a new paradigm to exclude pathogens and amplify their inherent health benefits.

Tunicamycin Mediated Inhibition of Wall Teichoic Acid Affects Staphylococcus aureus and Listeria monocytogenes Cell Morphology, Biofilm Formation and Virulence.

The emergence of bacterial resistance to therapeutic antibiotics limits options for treatment of common microbial diseases. Subinhibitory antibiotics dosing, often aid in the emergence of resistance, but its impact on pathogen's physiology and pathogenesis is not well understood. Here we investigated the effect of tunicamycin, a cell wall teichoic acid (WTA) biosynthesis inhibiting antibiotic at the subinhibitory dosage on Staphylococcus aureus and Listeria monocytogenes physiology, antibiotic cross-resistance, biofilm-formation, and virulence. Minimum inhibitory concentration (MIC) of tunicamycin to S. aureus and L. monocytogenes was 20-40 µg/ml and 2.5-5 µg/ml, respectively, and the subinhibitory concentration was 2.5-5 µg/ml and 0.31-0.62 µg/ml, respectively. Tunicamycin pre-exposure reduced cellular WTA levels by 18-20% and affected bacterial cell wall ultrastructure, cell membrane permeability, morphology, laser-induced colony scatter signature, and bacterial ability to form biofilms. It also induced a moderate level of cross-resistance to tetracycline, ampicillin, erythromycin, and meropenem for S. aureus, and ampicillin, erythromycin, vancomycin, and meropenem for L. monocytogenes. Pre-treatment of bacterial cells with subinhibitory concentrations of tunicamycin also significantly reduced bacterial adhesion to and invasion into an enterocyte-like Caco-2 cell line, which is supported by reduced expression of key virulence factors, Internalin B (InlB) and Listeria adhesion protein (LAP) in L. monocytogenes, and a S. aureus surface protein A (SasA) in S. aureus. Tunicamycin-treated bacteria or the bacterial WTA preparation suppressed NF-κB and inflammatory cytokine production (TNFα, and IL-6) from murine macrophage cell line (RAW 264.7) indicating the reduced WTA level possibly attenuates an inflammatory response. These results suggest that at the subinhibitory dosage, tunicamycin-mediated inhibition of WTA biosynthesis interferes with cell wall structure, pathogens infectivity and inflammatory response, and ability to form biofilms but promotes the development of antibiotic cross-resistance.

Erratum: Kim, K.-P.; Singh, A.K.; Bai, X.; Leprun, L.; Bhunia, A.K. Novel PCR Assays Complement Laser Biosensor-Based Method and Facilitate Listeria Species Detection from Food. Sensors 2015, 15, 22672-22691.

The authors wish to correct the oligonucleotide sequence of primer E-LAP-F1 and LIS-R1 in Table 1in their paper published in Sensors [1], doi:10.3390/s150922672, http://www.mdpi.com/1424-8220/15/9/22672. The following table should be used.[...].

Virulence Gene-Associated Mutant Bacterial Colonies Generate Differentiating Two-Dimensional Laser Scatter Fingerprints.

UNLABELLED: In this study, we investigated whether a laser scatterometer designated BARDOT (bacterial rapid detection using optical scattering technology) could be used to directly screen colonies of Listeria monocytogenes, a model pathogen, with mutations in several known virulence genes, including the genes encoding Listeria adhesion protein (LAP; lap mutant), internalin A (ΔinlA strain), and an accessory secretory protein (ΔsecA2 strain). Here we show that the scatter patterns of lap mutant, ΔinlA, and ΔsecA2 colonies were markedly different from that of the wild type (WT), with >95% positive predictive values (PPVs), whereas for the complemented mutant strains, scatter patterns were restored to that of the WT. The scatter image library successfully distinguished the lap mutant and ΔinlA mutant strains from the WT in mixed-culture experiments, including a coinfection study using the Caco-2 cell line. Among the biophysical parameters examined, the colony height and optical density did not reveal any discernible differences between the mutant and WT strains. We also found that differential LAP expression in L. monocytogenes serotype 4b strains also affected the scatter patterns of the colonies. The results from this study suggest that BARDOT can be used to screen and enumerate mutant strains separately from the WT based on differential colony scatter patterns. IMPORTANCE: In studies of microbial pathogenesis, virulence-encoding genes are routinely disrupted by deletion or insertion to create mutant strains. Screening of mutant strains is an arduous process involving plating on selective growth media, replica plating, colony hybridization, DNA isolation, and PCR or immunoassays. We applied a noninvasive laser scatterometer to differentiate mutant bacterial colonies from WT colonies based on forward optical scatter patterns. This study demonstrates that BARDOT can be used as a novel, label-free, real-time tool to aid researchers in screening virulence gene-associated mutant colonies during microbial pathogenesis, coinfection, and genetic manipulation studies.

Novel PCR Assays Complement Laser Biosensor-Based Method and Facilitate Listeria Species Detection from Food.

The goal of this study was to develop the Listeria species-specific PCR assays based on a house-keeping gene (lmo1634) encoding alcohol acetaldehyde dehydrogenase (Aad), previously designated as Listeria adhesion protein (LAP), and compare results with a label-free light scattering sensor, BARDOT (bacterial rapid detection using optical scattering technology). PCR primer sets targeting the lap genes from the species of Listeria sensu stricto were designed and tested with 47 Listeria and 8 non-Listeria strains. The resulting PCR primer sets detected either all species of Listeria sensu stricto or individual L. innocua, L. ivanovii and L. seeligeri, L. welshimeri, and L. marthii without producing any amplified products from other bacteria tested. The PCR assays with Listeria sensu stricto-specific primers also successfully detected all species of Listeria sensu stricto and/or Listeria innocua from mixed culture-inoculated food samples, and each bacterium in food was verified by using the light scattering sensor that generated unique scatter signature for each species of Listeria tested. The PCR assays based on the house-keeping gene aad (lap) can be used for detection of either all species of Listeria sensu stricto or certain individual Listeria species in a mixture from food with a detection limit of about 104 CFU/mL.

Streptomycin Induced Stress Response in Salmonella enterica Serovar Typhimurium Shows Distinct Colony Scatter Signature.

We investigated the streptomycin-induced stress response in Salmonella enterica serovars with a laser optical sensor, BARDOT (bacterial rapid detection using optical scattering technology). Initially, the top 20 S. enterica serovars were screened for their response to streptomycin at 100 µg/mL. All, but four S. enterica serovars were resistant to streptomycin. The MIC of streptomycin-sensitive serovars (Enteritidis, Muenchen, Mississippi, and Schwarzengrund) varied from 12.5 to 50 µg/mL, while streptomycin-resistant serovar (Typhimurium) from 125-250 µg/mL. Two streptomycin-sensitive serovars (Enteritidis and Mississippi) were grown on brain heart infusion (BHI) agar plates containing sub-inhibitory concentration of streptomycin (1.25-5 µg/mL) and a streptomycin-resistant serovar (Typhimurium) was grown on BHI containing 25-50 µg/mL of streptomycin and the colonies (1.2 ± 0.1 mm diameter) were scanned using BARDOT. Data show substantial qualitative and quantitative differences in the colony scatter patterns of Salmonella grown in the presence of streptomycin than the colonies grown in absence of antibiotic. Mass-spectrometry identified overexpression of chaperonin GroEL, which possibly contributed to the observed differences in the colony scatter patterns. Quantitative RT-PCR and immunoassay confirmed streptomycin-induced GroEL expression while, aminoglycoside adenylyltransferase (aadA), aminoglycoside efflux pump (aep), multidrug resistance subunit acrA, and ribosomal protein S12 (rpsL), involved in streptomycin resistance, were unaltered. The study highlights suitability of the BARDOT as a non-invasive, label-free tool for investigating stress response in Salmonella in conjunction with the molecular and immunoassay methods.

Label-free, non-invasive light scattering sensor for rapid screening of Bacillus colonies.

Bacillus species are widely distributed in nature and have great significance both as industrially beneficial microbes and as public health burdens. We employed a novel light-scattering sensor, BARDOT (bacterial rapid detection using optical scattering technology) for instant screening of colonies of Bacillus species on agar plates. A total of 265 Bacillus and non-Bacillus isolates from our collection were used to develop and verify scatter image libraries including isolates from food, environmental and clinical samples. All Bacillus species (n=118) were detected with a high positive predictive value, PPV (≥90%) while non-Bacillus spp. had very low PPV (<5%) when compared with scatter images from the library. Among all media tested for culturing, Bacillus colonies on phenol red mannitol (PRM) generated the highest differential scatter patterns and were used in subsequent studies. Surface plot analysis of scatter patterns confirmed differences for Bacillus and non-Bacillus isolates. BARDOT successfully detected Bacillus from inoculated baby formula, cheese, and naturally contaminated bovine unpasteurized milk in 7-16h. Ten of 129 colonies (isolates) from seven milk samples were Bacillus and remainders were non-Bacillus spp. BARDOT results were confirmed by PCR and 16S rDNA sequencing. This study demonstrates that BARDOT could be used as a screening tool to identify relevant Bacillus colonies from a community prior to genome sequencing.