Zearalenone (ZEN) is an estrogenic mycotoxin produced by the Fusarium species and induces severe ... more Zearalenone (ZEN) is an estrogenic mycotoxin produced by the Fusarium species and induces severe reproductive disorders in animals thus a major concern in the livestock industry. Probiotic bacteria treatments have been shown to inactivate mycotoxins, therefore, in this study, we investigated the effect of two commercial probiotic feed additives on the sequestration of ZEN. Commercial probiotic blends containing clay-based binder with Aspergillus niger, Bacillus licheniformis, Bacillus pumilus, and Bacillus subtilis at various proportions from BioMatrix International were incubated with ZEN in a time-dependent manner and then analyzed by Enzyme-Linked Immunosorbent Assay (ELISA) to quantify unbound ZEN. Sequestration of ZEN was further verified by using MCF-7 cell-based cytotoxicity and/or cell proliferation assays. ZEN, or probiotic mix, was nontoxic to MCF-7 cells. Probiotic blends decreased ZEN concentration by 45% (∼100 μg L−1) and prevented ZEN from inducing MCF-7 cell prolifera...
Classical microbiology has paved the path forward for the development of modern biotechnology and... more Classical microbiology has paved the path forward for the development of modern biotechnology and microbial biosensing platforms. Microbial culturing and isolation using the Petri plate revolutionized the field of microbiology. In 1887, Julius Richard Petri invented possibly the most important tool in microbiology, the Petri plate, which continues to have a profound impact not only on reliably isolating, identifying, and studying microorganisms but also manipulating a microbe to study gene expression, virulence properties, antibiotic resistance, and production of drugs, enzymes, and foods. Before the recent advances in gene sequencing, microbial identification for diagnosis relied upon the hierarchal testing of a pure culture isolate. Direct detection and identification of isolated bacterial colonies on a Petri plate with a sensing device has the potential for revolutionizing further development in microbiology including gene sequencing, pathogenicity study, antibiotic susceptibilit...
<p>(a) Plasmid map (10.6 kb) of LAP expression vector pLP401T (9.8 kb)-LAP (2.6 kb) <a h... more <p>(a) Plasmid map (10.6 kb) of LAP expression vector pLP401T (9.8 kb)-LAP (2.6 kb) <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0029277#pone.0029277-Pouwels1" target="_blank">[50]</a>. <b>Ery</b>, erythromycin resistance gene; <b>Amp</b>, ampicillin resistance gene; Ori+ = origin of replication of <i>E. coli</i>, Ori- = origin of replication of <i>Lactobacillus</i>; <b>LAP</b>, <i>Listeria</i> adhesion protein; <b>Pamy</b>, α-amylase promoter gene; <b>ssAmy</b>, secretion signal (36 aa) and the N-terminus (26 aa) of α-amylase gene; <b>Anchor</b>, anchor peptide (117 aa) gene of <i>Lb. casei</i>; <b>Tcbh</b>, transcription terminator of the cbh (conjugated bile acid hydrolase) gene; <b>Rep</b>, <i>repA</i> gene. (b) Western blot showing LAP expression in the supernatant, cell wall, and intracellular fractions of <i>Listeria monocytogenes</i> (Lm) and Lbp<sup>LAP</sup> but absent in wild type <i>Lb. paracasei</i> (Lbp<sup>WT</sup>). Molecular weight of LAP from Lm and the recombinant Lbp<sup>LAP</sup> was similar. (c) Immunofluorescence staining of bacteria (magnification 1000×) with anti-LAP MAb-H7 and fluorescein isothiocyanate-conjugated second antibody (left panel) and Hoechst dye (blue; right panel). Lbp<sup>LAP</sup> and Lm (control) cells indicated the presence of surface-expressed LAP (green) that was absent in Lbp<sup>WT</sup>. (d) Binding (capture) of recombinant <i>Lb. paracasei</i> cells to paramagnetic beads coated with Hsp60 relative to <i>L. monocytogenes</i> (considered 100%). The data are average (SD) of two independent experiments performed in duplicate. Letters (a, b) indicate significant difference at <i>P</i><0.05.</p
Listeria monocytogenes strain HPB5415—isolated from deli meat—was found in 2008 to have the same ... more Listeria monocytogenes strain HPB5415—isolated from deli meat—was found in 2008 to have the same pulsed-field gel electrophoresis patterns as a clinical strain (08-5923). However, whether nucleotide differences (single nucleotide polymorphisms [SNPs]) exist between their genomes was not determined. We sequenced the L. monocytogenes strain HPB5415 genome and identified 52 SNPs relative to strain 08-5923.
Applied and environmental microbiology, Jan 18, 2016
In this study, we investigated whether a laser scatterometer designated BARDOT (bacterial rapid d... more 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 Listeria adhesion protein (lap (-)), Internalin A (ΔinlA), and an accessory secretory protein (ΔsecA2). Here, we show that the scatter patterns of the lap(-), ΔinlA and ΔsecA2 colonies are markedly different from that of the wild type (WT) with >95% positive predictive values (PPV), whereas in the complemented mutant strains, scatter patterns were restored to the WT. The scatter image library successfully distinguished the lap(-) and the ΔinlA mutant strains from the WT in a mixed culture experiment including in a co-infection 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 mut...
Listeria monocytogenes is a ubiquitous food-borne pathogen, and its presence in food or productio... more Listeria monocytogenes is a ubiquitous food-borne pathogen, and its presence in food or production facilities highlights the importance of surveillance. Increased understanding of the surface exposed antigens on Listeria would provide potential diagnostic and therapeutic targets. In the present work, using mass spectrometry and genetic cloning, we show that fructose-1,6-bisphosphate aldolase (FBA) class II in Listeria species is the antigen target of the previously described mAb-3F8. Western and dot blot assays confirmed that the mAb-3F8 could distinguish all tested Listeria species from close-related bacteria. Localization studies indicated that FBA is present in every fraction of Listeria cells, including supernatant and the cell wall, setting Listeria spp. as one of the few bacteria described to have this protein on their cell surface. Epitope mapping using ORFeome display and a peptide membrane revealed a 14-amino acid peptide as the potential mAb-3F8 epitope. The target epitope...
Environmental cues promote microbial biofilm formation and physiological and genetic heterogeneit... more 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- ...
A mixed culture (polymicrobial) biofilm provides a favorable environment for pathogens to persist... more 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 ...
Background Immunomagnetic separation (IMS) and immunoassays are widely used for pathogen detectio... more Background Immunomagnetic separation (IMS) and immunoassays are widely used for pathogen detection. However, novel technology platforms with highly selective antibodies are essential to improve detection sensitivity, specificity and performance. In this study, monoclonal antibodies (MAbs) against Internalin A (InlA) and p30 were generated and used on paramagnetic beads of varying diameters for concentration, as well as on fiber-optic sensor for detection. Results Anti-InlA MAb-2D12 (IgG2a subclass) was specific for Listeria monocytogenes and L. ivanovii, and p30-specific MAb-3F8 (IgM) was specific for the genus Listeria. At all bacterial concentrations (103–108 CFU/mL) tested in the IMS assay; the 1-μm diameter MyOne beads had significantly higher capture efficiency (P < 0.05) than the 2.8-μm diameter M-280 beads with both antibodies. The highest capture efficiency for MyOne-2D12 (49.2% for 105 CFU/mL) was significantly higher (P < 0.05) than that of MyOne-3F8 (16.6 %) and Dyn...
Foodborne microorganisms are an important cause of human illness worldwide. Two-thirds of human f... more Foodborne microorganisms are an important cause of human illness worldwide. Two-thirds of human foodborne diseases are caused by bacterial pathogens throughout the globe, especially in developing nations. Despite enormous developments in conventional foodborne pathogen detection methods, progress is limited by the assay complexity and a prolonged time-to-result. The specificity and sensitivity of assays for live pathogen detection may also depend on the nature of the samples being analyzed and the immunological or molecular reagents used. Bacteriophage-based biosensors offer several benefits, including specificity to their host organism, the detection of only live pathogens, and resistance to extreme environmental factors such as organic solvents, high temperatures, and a wide pH range. Phage-based biosensors are receiving increasing attention owing to their high degree of accuracy, specificity, and reduced assay times. These characteristics, coupled with their abundant supply, make...
As the world population has grown, new demands on the production of foods have been met by increa... more As the world population has grown, new demands on the production of foods have been met by increased efficiencies in production, from planting and harvesting to processing, packaging and distribution to retail locations. These efficiencies enable rapid intranational and global dissemination of foods, providing longer “face time” for products on retail shelves and allowing consumers to make healthy dietary choices year-round. However, our food production capabilities have outpaced the capacity of traditional detection methods to ensure our foods are safe. Traditional methods for culture-based detection and characterization of microorganisms are time-, labor- and, in some instances, space- and infrastructure-intensive, and are therefore not compatible with current (or future) production and processing realities. New and versatile detection methods requiring fewer overall resources (time, labor, space, equipment, cost, etc.) are needed to transform the throughput and safety dimensions ...
Foodborne disease outbreaks continue to be a major public health and food safety concern. Testing... more 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 ...
Zearalenone (ZEN) is an estrogenic mycotoxin produced by the Fusarium species and induces severe ... more Zearalenone (ZEN) is an estrogenic mycotoxin produced by the Fusarium species and induces severe reproductive disorders in animals thus a major concern in the livestock industry. Probiotic bacteria treatments have been shown to inactivate mycotoxins, therefore, in this study, we investigated the effect of two commercial probiotic feed additives on the sequestration of ZEN. Commercial probiotic blends containing clay-based binder with Aspergillus niger, Bacillus licheniformis, Bacillus pumilus, and Bacillus subtilis at various proportions from BioMatrix International were incubated with ZEN in a time-dependent manner and then analyzed by Enzyme-Linked Immunosorbent Assay (ELISA) to quantify unbound ZEN. Sequestration of ZEN was further verified by using MCF-7 cell-based cytotoxicity and/or cell proliferation assays. ZEN, or probiotic mix, was nontoxic to MCF-7 cells. Probiotic blends decreased ZEN concentration by 45% (∼100 μg L−1) and prevented ZEN from inducing MCF-7 cell prolifera...
Classical microbiology has paved the path forward for the development of modern biotechnology and... more Classical microbiology has paved the path forward for the development of modern biotechnology and microbial biosensing platforms. Microbial culturing and isolation using the Petri plate revolutionized the field of microbiology. In 1887, Julius Richard Petri invented possibly the most important tool in microbiology, the Petri plate, which continues to have a profound impact not only on reliably isolating, identifying, and studying microorganisms but also manipulating a microbe to study gene expression, virulence properties, antibiotic resistance, and production of drugs, enzymes, and foods. Before the recent advances in gene sequencing, microbial identification for diagnosis relied upon the hierarchal testing of a pure culture isolate. Direct detection and identification of isolated bacterial colonies on a Petri plate with a sensing device has the potential for revolutionizing further development in microbiology including gene sequencing, pathogenicity study, antibiotic susceptibilit...
<p>(a) Plasmid map (10.6 kb) of LAP expression vector pLP401T (9.8 kb)-LAP (2.6 kb) <a h... more <p>(a) Plasmid map (10.6 kb) of LAP expression vector pLP401T (9.8 kb)-LAP (2.6 kb) <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0029277#pone.0029277-Pouwels1" target="_blank">[50]</a>. <b>Ery</b>, erythromycin resistance gene; <b>Amp</b>, ampicillin resistance gene; Ori+ = origin of replication of <i>E. coli</i>, Ori- = origin of replication of <i>Lactobacillus</i>; <b>LAP</b>, <i>Listeria</i> adhesion protein; <b>Pamy</b>, α-amylase promoter gene; <b>ssAmy</b>, secretion signal (36 aa) and the N-terminus (26 aa) of α-amylase gene; <b>Anchor</b>, anchor peptide (117 aa) gene of <i>Lb. casei</i>; <b>Tcbh</b>, transcription terminator of the cbh (conjugated bile acid hydrolase) gene; <b>Rep</b>, <i>repA</i> gene. (b) Western blot showing LAP expression in the supernatant, cell wall, and intracellular fractions of <i>Listeria monocytogenes</i> (Lm) and Lbp<sup>LAP</sup> but absent in wild type <i>Lb. paracasei</i> (Lbp<sup>WT</sup>). Molecular weight of LAP from Lm and the recombinant Lbp<sup>LAP</sup> was similar. (c) Immunofluorescence staining of bacteria (magnification 1000×) with anti-LAP MAb-H7 and fluorescein isothiocyanate-conjugated second antibody (left panel) and Hoechst dye (blue; right panel). Lbp<sup>LAP</sup> and Lm (control) cells indicated the presence of surface-expressed LAP (green) that was absent in Lbp<sup>WT</sup>. (d) Binding (capture) of recombinant <i>Lb. paracasei</i> cells to paramagnetic beads coated with Hsp60 relative to <i>L. monocytogenes</i> (considered 100%). The data are average (SD) of two independent experiments performed in duplicate. Letters (a, b) indicate significant difference at <i>P</i><0.05.</p
Listeria monocytogenes strain HPB5415—isolated from deli meat—was found in 2008 to have the same ... more Listeria monocytogenes strain HPB5415—isolated from deli meat—was found in 2008 to have the same pulsed-field gel electrophoresis patterns as a clinical strain (08-5923). However, whether nucleotide differences (single nucleotide polymorphisms [SNPs]) exist between their genomes was not determined. We sequenced the L. monocytogenes strain HPB5415 genome and identified 52 SNPs relative to strain 08-5923.
Applied and environmental microbiology, Jan 18, 2016
In this study, we investigated whether a laser scatterometer designated BARDOT (bacterial rapid d... more 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 Listeria adhesion protein (lap (-)), Internalin A (ΔinlA), and an accessory secretory protein (ΔsecA2). Here, we show that the scatter patterns of the lap(-), ΔinlA and ΔsecA2 colonies are markedly different from that of the wild type (WT) with >95% positive predictive values (PPV), whereas in the complemented mutant strains, scatter patterns were restored to the WT. The scatter image library successfully distinguished the lap(-) and the ΔinlA mutant strains from the WT in a mixed culture experiment including in a co-infection 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 mut...
Listeria monocytogenes is a ubiquitous food-borne pathogen, and its presence in food or productio... more Listeria monocytogenes is a ubiquitous food-borne pathogen, and its presence in food or production facilities highlights the importance of surveillance. Increased understanding of the surface exposed antigens on Listeria would provide potential diagnostic and therapeutic targets. In the present work, using mass spectrometry and genetic cloning, we show that fructose-1,6-bisphosphate aldolase (FBA) class II in Listeria species is the antigen target of the previously described mAb-3F8. Western and dot blot assays confirmed that the mAb-3F8 could distinguish all tested Listeria species from close-related bacteria. Localization studies indicated that FBA is present in every fraction of Listeria cells, including supernatant and the cell wall, setting Listeria spp. as one of the few bacteria described to have this protein on their cell surface. Epitope mapping using ORFeome display and a peptide membrane revealed a 14-amino acid peptide as the potential mAb-3F8 epitope. The target epitope...
Environmental cues promote microbial biofilm formation and physiological and genetic heterogeneit... more 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- ...
A mixed culture (polymicrobial) biofilm provides a favorable environment for pathogens to persist... more 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 ...
Background Immunomagnetic separation (IMS) and immunoassays are widely used for pathogen detectio... more Background Immunomagnetic separation (IMS) and immunoassays are widely used for pathogen detection. However, novel technology platforms with highly selective antibodies are essential to improve detection sensitivity, specificity and performance. In this study, monoclonal antibodies (MAbs) against Internalin A (InlA) and p30 were generated and used on paramagnetic beads of varying diameters for concentration, as well as on fiber-optic sensor for detection. Results Anti-InlA MAb-2D12 (IgG2a subclass) was specific for Listeria monocytogenes and L. ivanovii, and p30-specific MAb-3F8 (IgM) was specific for the genus Listeria. At all bacterial concentrations (103–108 CFU/mL) tested in the IMS assay; the 1-μm diameter MyOne beads had significantly higher capture efficiency (P < 0.05) than the 2.8-μm diameter M-280 beads with both antibodies. The highest capture efficiency for MyOne-2D12 (49.2% for 105 CFU/mL) was significantly higher (P < 0.05) than that of MyOne-3F8 (16.6 %) and Dyn...
Foodborne microorganisms are an important cause of human illness worldwide. Two-thirds of human f... more Foodborne microorganisms are an important cause of human illness worldwide. Two-thirds of human foodborne diseases are caused by bacterial pathogens throughout the globe, especially in developing nations. Despite enormous developments in conventional foodborne pathogen detection methods, progress is limited by the assay complexity and a prolonged time-to-result. The specificity and sensitivity of assays for live pathogen detection may also depend on the nature of the samples being analyzed and the immunological or molecular reagents used. Bacteriophage-based biosensors offer several benefits, including specificity to their host organism, the detection of only live pathogens, and resistance to extreme environmental factors such as organic solvents, high temperatures, and a wide pH range. Phage-based biosensors are receiving increasing attention owing to their high degree of accuracy, specificity, and reduced assay times. These characteristics, coupled with their abundant supply, make...
As the world population has grown, new demands on the production of foods have been met by increa... more As the world population has grown, new demands on the production of foods have been met by increased efficiencies in production, from planting and harvesting to processing, packaging and distribution to retail locations. These efficiencies enable rapid intranational and global dissemination of foods, providing longer “face time” for products on retail shelves and allowing consumers to make healthy dietary choices year-round. However, our food production capabilities have outpaced the capacity of traditional detection methods to ensure our foods are safe. Traditional methods for culture-based detection and characterization of microorganisms are time-, labor- and, in some instances, space- and infrastructure-intensive, and are therefore not compatible with current (or future) production and processing realities. New and versatile detection methods requiring fewer overall resources (time, labor, space, equipment, cost, etc.) are needed to transform the throughput and safety dimensions ...
Foodborne disease outbreaks continue to be a major public health and food safety concern. Testing... more 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 ...
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