Selective Enrichment Media Affect the Antibody-Based Detection of Stress-Exposed Listeria monocytogenes due to Differential Expression of Antibody-Reactive Antigens Identified by Protein Sequencing

2006 ◽  
Vol 69 (8) ◽  
pp. 1879-1886 ◽  
Author(s):  
TAO GENG ◽  
BYOUNG-KWON HAHM ◽  
ARUN K. BHUNIA
Keyword(s):  
Listeria Monocytogenes ◽  
Differential Expression ◽  
Brain Heart Infusion ◽  
Antigen Expression ◽  
Surface Proteins ◽  
Protein Sequencing ◽  
Enzyme Linked Immunosorbent Assay ◽  
Selective Enrichment ◽  
Elevated Expression ◽  
Detection Of Stress

Selective enrichment broths are frequently used to recover stressed Listeria cells to detectable levels, but the ability of antibodies to detect these cells from various commonly used enrichment media is unknown. In this study, a polyclonal (PAb) and monoclonal (MAb) antibody were used to examine the variation in antigen expression on healthy or stress-recovered Listeria monocytogenes cells grown in brain heart infusion broth, buffered Listeria enrichment broth (BLEB), Listeria repair broth (LRB), University of Vermont medium (UVM), and Fraser broth (FB) for immunodetection. Indirect enzyme-linked immunosorbent assay (ELISA) data showed that L. monocytogenes subjected to stresses (acid, cold, heat, and salt) and then grown in BLEB gave the highest reaction with the anti-Listeria PAb while those grown in LRB gave the highest reaction with the MAb C11E9. Cells grown in UVM and FB gave poor ELISA values with both antibodies. Western blotting with PAb revealed differential expression of surface proteins of 62, 58, 50, 43, and 30 kDa on L. monocytogenes cells, with most proteins displaying elevated expression in BLEB and LRB but reduced or no expression in UVM or FB. Similar differential expressions were noticed for C11E9. PAb-reactive proteins were identified as putative LPXTG-motif cell-wall anchor-domain protein (62 kDa; lmo0610), flavocytochrome C fumarate reductase chain A homolog protein (58 kDa; lmo0355), enolase (50 kDa; lmo2455), glyceraldehyde 3-phosphate dehydrogenase (43 kDa; lmo2459), and hypothetical phospho-sugar binding protein (30 kDa; lmo0041), respectively, and the MAb-reactive 66-kDa protein was confirmed to be N-acetylmuramidase (lmo2691). In conclusion, BLEB and LRB favorably supported increased expression of antigens and proved to be superior to UVM and FB for immunodetection of stressed L. monocytogenes cells.

scholarly journals Bias in the Listeria monocytogenes Enrichment Procedure: Lineage 2 Strains Outcompete Lineage 1 Strains in University of Vermont Selective Enrichments

2005 ◽  
Vol 71 (2) ◽  
pp. 961-967 ◽  
Author(s):  
Jesper Bartholin Bruhn ◽  
Birte Fonnesbech Vogel ◽  
Lone Gram
Keyword(s):  
Growth Rate ◽  
Listeria Monocytogenes ◽  
Brain Heart Infusion ◽  
Selective Medium ◽  
Individual Growth ◽  
Selective Enrichment ◽  
Enrichment Procedure ◽  
True Representation ◽  
Lineage 2 ◽  
University Of Vermont

ABSTRACT Listeria monocytogenes can be isolated from a range of food products and may cause food-borne outbreaks or sporadic cases of listeriosis. L. monocytogenes is divided into three genetic lineages and 13 serotypes. Strains of three serotypes (1/2a, 1/2b, and 4b) are associated with most human cases of listeriosis. Of these, strains of serotypes 1/2b and 4b belong to lineage 1, whereas strains of serotype 1/2a and many other strains isolated from foods belong to lineage 2. L. monocytogenes is isolated from foods by selective enrichment procedures and from patients by nonselective methods. The aim of the present study was to investigate if the selective enrichment procedure results in a true representation of the subtypes of L. monocytogenes present in a sample. Eight L. monocytogenes strains (four lineage 1 strains and four lineage 2 strains) and one Listeria innocua strain grew with identical growth rates in the nonselective medium brain heart infusion (BHI), but differed in their growth rate in the selective medium University of Vermont medium I (UVM I). When coinoculated in UVM I, some strains completely outgrew other strains. This outcome was dependent on the lineage of L. monocytogenes rather than the individual growth rate of the strains. When inoculated at identical cell densities in UVM I, L. innocua outcompeted L. monocytogenes lineage 1 strains but not lineage 2 strains. In addition, lineage 2 L. monocytogenes strains outcompeted lineage 1 L. monocytogenes strains in all combinations tested, indicating a bias in strains selected by the enrichment procedures. Bias also occurred when coinoculating two lineage 2 or lineage 1 strains; however, it did not appear to correlate with origin (clinical versus food). Identical coinoculation experiments in BHI suggested that the selective compounds in UVM I and II influenced this bias. The results of the present study demonstrate that the selective procedures used for isolation of L. monocytogenes may not allow a true representation of the types present in foods. Our results could have a significant impact on epidemiological studies, as lineage 1 strains, which are often isolated from clinical cases of listeriosis, may be suppressed during enrichment by other L. monocytogenes lineages present in a food sample.

scholarly journals Identification of Surface Protein Biomarkers of Listeria monocytogenes via Bioinformatics and Antibody-Based Protein Detection Tools

2016 ◽  
Vol 82 (17) ◽  
pp. 5465-5476 ◽  
Author(s):  
Cathy X. Y. Zhang ◽  
Brian W. Brooks ◽  
Hongsheng Huang ◽  
Franco Pagotto ◽  
Min Lin
Keyword(s):  
Monoclonal Antibodies ◽  
Listeria Monocytogenes ◽  
Surface Protein ◽  
Protein Detection ◽  
Food Samples ◽  
Surface Proteins ◽  
Content Type ◽  
Selective Enrichment ◽  
Wide Range ◽  
Serotype 4B

ABSTRACTThe Gram-positive bacteriumListeria monocytogenescauses a significant percentage of the fatalities among foodborne illnesses in humans. Surface proteins specifically expressed in a wide range ofL. monocytogenesserotypes under selective enrichment culture conditions could serve as potential biomarkers for detection and isolation of this pathogen via antibody-based methods. Our study aimed to identify such biomarkers. Interrogation of theL. monocytogenesserotype 4b strain F2365 genome identified 130 putative or known surface proteins. The homologues of four surface proteins, LMOf2365_0578, LMOf2365_0581, LMOf2365_0639, and LMOf2365_2117, were assessed as biomarkers due to the presence of conserved regions among strains ofL. monocytogeneswhich are variable among otherListeriaspecies. Rabbit polyclonal antibodies against the four recombinant proteins revealed the expression of only LMOf2365_0639 on the surface of serotype 4b strain LI0521 cells despite PCR detection of mRNA transcripts for all four proteins in the organism. Three of 35 monoclonal antibodies (MAbs) to LMOf2365_0639, MAbs M3643, M3644, and M3651, specifically recognized 42 (91.3%) of 46L. monocytogeneslineage I and II isolates grown in nonselective brain heart infusion medium. While M3644 and M3651 reacted with 14 to 15 (82.4 to 88.2%) of 17L. monocytogeneslineage I and II isolates, M3643 reacted with 22 (91.7%) of 24 lineage I, II, and III isolates grown in selective enrichment media (UVM1, modified Fraser, Palcam, and UVM2 media). The three MAbs exhibited only weak reactivities (the optical densities at 414 nm were close to the cutoff value) to some otherListeriaspecies grown in selective enrichment media. Collectively, the data indicate the potential of LMOf2365_0639 as a surface biomarker ofL. monocytogenes, with the aid of specific MAbs, for pathogen detection, identification, and isolation in clinical, environmental, and food samples.IMPORTANCEL. monocytogenesis traditionally divided into at least 12 serotypes. Currently, there are no monoclonal antibodies (MAbs) available that are capable of binding to the surface ofL. monocytogenesstrains representing all 12 serotypes. Such antibodies would be useful and are needed for the development of methods to detect and isolateL. monocytogenesfrom food samples. In our study, we aimed to identify surface proteins that possess regions of well-conserved amino acid sequences among various serotypes and then to employ them as antigen targets (biomarkers) for the development of MAbs. Through bioinformatics and protein expression analysis, we identified one of the four putative surface protein candidates, LMOf2365_0639, encoded by the genome of theL. monocytogenesserotype 4b strain F2365, as a useful surface biomarker. Extensive assessment of 35 MAbs raised against LMOf2365_0639 in our study revealed three MAbs (M3643, M3644, and M3651) that recognized a wide range ofL. monocytogenesisolates.

Unstable Expression and Thermal Instability of a Species-Specific Cell Surface Epitope Associated with a 66-Kilodalton Antigen Recognized by Monoclonal Antibody EM-7G1 within Serotypes of Listeria monocytogenes Grown in Nonselective and Selective Broths

1998 ◽  
Vol 64 (8) ◽  
pp. 3070-3074 ◽  
Author(s):  
Ramakrishna Nannapaneni ◽  
Robert Story ◽  
Arun K. Bhunia ◽  
Michael G. Johnson
Keyword(s):  
Monoclonal Antibody ◽  
Listeria Monocytogenes ◽  
Cell Surface ◽  
Brain Heart Infusion ◽  
Live Cells ◽  
Enzyme Linked Immunosorbent Assay ◽  
Specific Cell ◽  
Enrichment Broth ◽  
Species Specific ◽  
Heat Instability

ABSTRACT Conditions that resulted in unstable expression and heat instability of a cell surface epitope associated with a 66-kDa antigen in Listeria monocytogenes serotypes were identified with the probe monoclonal antibody (MAb) EM-7G1 in an enzyme-linked immunosorbent assay. This epitope appeared to be absent in three serotypes (serotypes 3b, 4a, and 4c), which did not react with MAb EM-7G1 irrespective of the enrichment broth tested. The remaining 10 serotypes were detected by MAb EM-7G1 only when cells were grown in nonselective brain heart infusion broth (BHI) or selectiveListeria enrichment broth (LEB). When cells were grown inListeria repair broth (LRB), only 6 of the 13 serotypes were detected by MAb EM-7G1, and recognition of serogroup 4 was completely lost. None of the 13 serotypes was detected by MAb EM-7G1 when cells were grown in two other commonly usedListeria-selective media, UVM1 broth and Fraser broth (FRB), indicating that possible loss of epitope expression occurred under these conditions. MAb EM-7G1 maintained species specificity without cross-reacting with live or heat-killed cells of six otherListeria spp. (Listeria ivanovii,Listeria innocua, Listeria seeligeri,Listeria welshimeri, Listeria grayi, andListeria murrayi) irrespective of the enrichment conditions tested. Due to heat instability of the cell surface epitope when it was exposed to 80 or 100°C for 20 min, MAb EM-7G1 is suitable for detection of live cells of L. monocytogenes in BHI or LEB but not in LRB, UVM1, or FRB enrichment medium.

scholarly journals Glucose and Nutrient Concentrations Affect the Expression of a 104-Kilodalton Listeria Adhesion Protein in Listeria monocytogenes

2002 ◽  
Vol 68 (10) ◽  
pp. 4876-4883 ◽  
Author(s):  
Ziad W. Jaradat ◽  
Arun K. Bhunia
Keyword(s):  
Listeria Monocytogenes ◽  
Brain Heart Infusion ◽  
Luria Bertani ◽  
Nutrient Concentrations ◽  
Enzyme Linked Immunosorbent Assay ◽  
Growth Media ◽  
Adhesion Protein ◽  
Sugar Fermentation ◽  
Rich Media ◽  
High Concentrations

ABSTRACT 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 K2HPO4 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.

Identification of Listeria monocytogenes Based on the Detection of a 68-Kilodalton Cell-Surface Antigen

1996 ◽  
Vol 59 (11) ◽  
pp. 1176-1181 ◽  
Author(s):  
SHU C. CHEN ◽  
TSUNG C. CHANG
Keyword(s):  
Listeria Monocytogenes ◽  
Cell Surface ◽  
Surface Antigen ◽  
Hemolytic Activity ◽  
Polyclonal Antibodies ◽  
Cell Surface Antigen ◽  
Enzyme Linked Immunosorbent Assay ◽  
Elisa Method ◽  
Selective Enrichment ◽  
Positive Results

Conventional procedures for the detection of Listeria monocytogenes require selective enrichment and isolation, followed by several potentially lengthy identification steps. To facilitate the identification of the bacterium, polyclonal antibodies against a 68-kDa cell-surface antigen of L. monocytogenes were used to develop a sandwich enzyme-linked immunosorbent assay (ELISA). Of 51 strains of L. monocytogenes tested, 50 (98%) produced positive results using the ELISA method, whereas 17 non-Listeria strains yielded negative reactions. However, the polyclonal antibodies were found to cross-react with L. innocua and some strains of L. welshimeri. Further purification of the antibodies by affinity chromatography was impractical, since the increase in sensitivity was accompanied with decreased specificity. It is proposed that a combination of the ELISA with a single biochemical test, such as phosphoinositol-specific phospholipase C (PI-PLC), arylamidase, or hemolytic activity could differentiate L. monocytogenes (PI-PLC [+], hemolysin [+], arylamidase [−]) from L. innocua and L. welshimeri (PI-PLC [−], hemolysin [−], arylamidase [+]) , thus considerably reducing the number of steps required and the length of time necessary for the identification of L. monocytogenes.

scholarly journals PSIX-18 Laurate and its glycerol monoester, monolaurin, as potential additives to control Listeria monocytogenes and tetracycline resistant Enterococcus faecalis in air-exposed silage

2020 ◽  
Vol 98 (Supplement_4) ◽  
pp. 414-415
Author(s):  
Yamicela Castillo-Castillo ◽  
Marina Ontiveros ◽  
Eric J Scholljegerdes ◽  
Robin Anderson ◽  
Claudio Arzola-Alvarez ◽  
...  
Keyword(s):  
Listeria Monocytogenes ◽  
Enterococcus Faecalis ◽  
Brain Heart Infusion ◽  
Bactericidal Effect ◽  
Complete Elimination ◽  
Gram Positive Bacteria ◽  
Viable Cells ◽  
Colony Forming Units ◽  
Feed Value ◽  
Risk Infection

Abstract Silages can harbor pathogenic and antimicrobial resistant microbes which risk infection of food-producing animals. Livestock producers need effective yet environmentally friendly interventions to preserve the feed value of these fermented materials. Medium chain fatty acids such as laurate and its glycerol monoester, monolaurin, are potent inhibitors of many Gram-positive bacteria and when tested at 5 mg/mL in anaerobic cultures (n = 3/treatment) inoculated with 105 colony forming units (CFU) of Listeria monocytogenes and grown at 37oC in ½ strength Brain Heart infusion broth achieved near complete elimination of viable cells after 6 h compared to a 2.2 ± 0.1 log10 CFU/mL increase observed in controls. Culture of a tetracycline-resistant Enterococcus faecalis with 5 mg laurate/mL likewise achieved near complete elimination of viable cells (5 log10 CFU/mL) by 6 h incubation. The bactericidal effect of 5 mg monolaurin was less against E. faecalis, achieving a decrease of 1.8 ± 0.2 log10 CFU/mL and not decreased further after 24 h. When tested against air-exposed silage, pH 7.53 (4 g), mixed with 4 mL water, 5 mg laurate or monolaurin decreased viability of experimentally-inoculated L. monocytogenes (105 CFU/g silage) more (P < 0.05) than untreated controls after 24 h aerobic incubation (22oC), with viable counts being decreased 6.3 ± 0.1, 5.9 ± 0.8 and 4.5 ± 0.1 log10 CFU/g, respectively. In contrast, viable recovery of the experimentally-inoculated (105 CFU/g) tetracycline-resistant E. faecalis was reduced more (P < 0.05) than controls (decreased 0.7 ± 0.1 log10 CFU/g) after 6 h incubation when similarly tested with laurate and monolaurin (1.7 ± 0.5 and 3.0 ± 0.9 log10 CFU/g, respectively) but counts after 24 h were similar, decreasing on average 2.0 ± 0.5 log10 CFU/g). Results indicate laurate and monolaurin may be useful in killing L. monocytogenes and tetracycline-resistant E. faecalis during silage feed-out.

scholarly journals Identification of Listeria monocytogenes Genes Contributing to Intracellular Replication by Expression Profiling and Mutant Screening

2006 ◽  
Vol 188 (2) ◽  
pp. 556-568 ◽  
Author(s):  
Biju Joseph ◽  
Karin Przybilla ◽  
Claudia Stühler ◽  
Kristina Schauer ◽  
Jörg Slaghuis ◽  
...  
Keyword(s):  
Listeria Monocytogenes ◽  
Stress Proteins ◽  
Carbon Sources ◽  
Brain Heart Infusion ◽  
Nitrogen Sources ◽  
Pentose Phosphate ◽  
Intracellular Growth ◽  
Cell Infection ◽  
Phosphotransferase System ◽  
Isoleucine Leucine

ABSTRACT A successful transition of Listeria monocytogenes from the extracellular to the intracellular environment requires a precise adaptation response to conditions encountered in the host milieu. Although many key steps in the intracellular lifestyle of this gram-positive pathogen are well characterized, our knowledge about the factors required for cytosolic proliferation is still rather limited. We used DNA microarray and real-time reverse transcriptase PCR analyses to investigate the transcriptional profile of intracellular L. monocytogenes following epithelial cell infection. Approximately 19% of the genes were differentially expressed by at least 1.6-fold relative to their level of transcription when grown in brain heart infusion medium, including genes encoding transporter proteins essential for the uptake of carbon and nitrogen sources, factors involved in anabolic pathways, stress proteins, transcriptional regulators, and proteins of unknown function. To validate the biological relevance of the intracellular gene expression profile, a random mutant library of L. monocytogenes was constructed by insertion-duplication mutagenesis and screened for intracellular-growth-deficient strains. By interfacing the results of both approaches, we provide evidence that L. monocytogenes can use alternative carbon sources like phosphorylated glucose and glycerol and nitrogen sources like ethanolamine during replication in epithelial cells and that the pentose phosphate cycle, but not glycolysis, is the predominant pathway of sugar metabolism in the host environment. Additionally, we show that the synthesis of arginine, isoleucine, leucine, and valine, as well as a species-specific phosphoenolpyruvate-dependent phosphotransferase system, play a major role in the intracellular growth of L. monocytogenes.

scholarly journals Visualizing the dynamics of exported bacterial proteins with the chemogenetic fluorescent reporter FAST

2020 ◽  
Author(s):  
Yankel Chekli ◽  
Caroline Peron-Cane ◽  
Dario Dell’Arciprete ◽  
Jean-François Allemand ◽  
Chenge Li ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Listeria Monocytogenes ◽  
Cell Surface ◽  
Bacterial Cell ◽  
Surface Proteins ◽  
Cellular Functions ◽  
Reporter Protein ◽  
Cell Surface Proteins ◽  
Fluorescent Reporter ◽  
Bacterial Proteins

AbstractBacterial proteins exported to the cell surface play key cellular functions. However, despite the interest to study the localization of surface proteins such as adhesins, transporters or hydrolases, monitoring their dynamics in live imaging remains challenging, due to the limited availability of fluorescent probes remaining functional after secretion. In this work, we used the Escherichia coli intimin and the Listeria monocytogenes InlB invasin as surface exposed scaffolds fused with the recently developed chemogenetic fluorescent reporter protein FAST. Using both membrane permeant (HBR-3,5DM) and non-permeant (HBRAA-3E) fluorogens that fluoresce upon binding to FAST, we demonstrated that fully functional FAST can be exposed at the cell surface and specifically tagged on the external side of the bacterial envelop in both diderm and monoderm bacteria. Our work opens new avenues to study of the organization and dynamics of the bacterial cell surface proteins.

scholarly journals Bioaffinity-based Surface Immobilization of Antibodies to Capture Endothelial Colony-forming Cells

2021 ◽  
Author(s):  
Mariève D Boulanger ◽  
Mohamed A Elkhodiry ◽  
Omar Bashth ◽  
Gaétan Laroche ◽  
Corinne A Hoesli
Keyword(s):  
Surface Modification ◽  
Surface Proteins ◽  
Enzyme Linked Immunosorbent Assay ◽  
Protein G ◽  
Cell Capture ◽  
Igg Antibodies ◽  
Immobilized Antibodies ◽  
Endothelial Colony Forming Cells ◽  
Covalent Grafting

Maximizing the re-endothelialization of vascular implants such as prostheses or stents has the potential to significantly improve their long-term performance. Endothelial progenitor cell capture stents with surface-immobilized antibodies show significantly improved endothelialization in the clinic. However, most current antibody-based stent surface modification strategies rely on antibody adsorption or direct conjugation via amino or carboxyl groups which leads to poor control over antibody surface concentration and/or molecular orientation, and ultimately bioavailability for cell capture. Here, we assess the utility of a bioaffinity-based surface modification strategy consisting of a surface-conjugated cysteine-tagged protein G molecules that immobilize Immunoglobulin G (IgG) antibodies via the Fc domain to capture circulating endothelial colony-forming cells (ECFCs). The cysteine-tagged protein G was grafted onto aminated substrates at different concentrations as detected by an enzyme-linked immunosorbent assay and fluorescence imaging. Different IgG antibodies were successfully immobilized on the protein G-modified surfaces and higher antibody surface concentrations were achieved compared to passive adsorption methods. Surfaces with immobilized antibodies targeting endothelial surface proteins, such as CD144, significantly enhanced the capture of circulating ECFCs in vitro compared to surfaces with non-endothelial specific antibodies such as anti-CD14. This work presents a potential avenue for enhancing the clinical performance of vascular implants by using covalent grafting of protein G to immobilize IgG antibodies more effectively.

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