RESUMEN
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.
Asunto(s)
Inmunoensayo/métodos , Listeria monocytogenes/aislamiento & purificación , Listeriosis/microbiología , Animales , Anticuerpos Inmovilizados/química , Microbiología de Alimentos , Humanos , Inmunoensayo/instrumentación , Listeriosis/diagnóstico , Ácidos Nucleicos/química , Sistemas de Atención de PuntoRESUMEN
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.
Asunto(s)
Listeria monocytogenes/aislamiento & purificación , Listeria monocytogenes/patogenicidad , Listeriosis/microbiología , Factores de Virulencia/análisis , Factores de Virulencia/inmunología , Animales , Anticuerpos Antibacterianos/inmunología , Antígenos Bacterianos/análisis , Antígenos Bacterianos/inmunología , Técnicas Biosensibles , Microbiología de Alimentos , Humanos , Inmunidad Innata , Listeria monocytogenes/crecimiento & desarrollo , Listeria monocytogenes/inmunología , Listeriosis/diagnóstico , Listeriosis/inmunología , Virulencia , Factores de Virulencia/metabolismoRESUMEN
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 in FBA allowed distinguishing Listeria spp. from closely-related bacteria, and was identified as part of the active site in the dimeric enzyme. However, its function in cell surface seems not to be host cell adhesion-related. Western and dot blot assays further demonstrated that mAb-3F8 together with anti-InlA mAb-2D12 could differentiate pathogenic from non-pathogenic Listeria isolated from artificially contaminated cheese. In summary, we report FBA as a novel immunogenic surface target useful for the detection of Listeria genus.
Asunto(s)
Antígenos de Superficie/inmunología , Proteínas Bacterianas/inmunología , Fructosa-Bifosfato Aldolasa/inmunología , Listeria/enzimología , Listeria/inmunología , Secuencia de Aminoácidos , Anticuerpos Monoclonales/inmunología , Antígenos de Superficie/metabolismo , Proteínas Bacterianas/genética , Proteínas Bacterianas/metabolismo , Western Blotting , Dominio Catalítico , Queso/microbiología , Clonación Molecular , Dimerización , Ensayo de Inmunoadsorción Enzimática , Mapeo Epitopo , Epítopos/inmunología , Microbiología de Alimentos , Fructosa-Bifosfato Aldolasa/genética , Fructosa-Bifosfato Aldolasa/metabolismo , Listeria/aislamiento & purificación , Espectrometría de Masas , Péptidos/análisis , Estructura Terciaria de Proteína , Proteínas Recombinantes/biosíntesis , Proteínas Recombinantes/inmunología , Proteínas Recombinantes/aislamiento & purificaciónRESUMEN
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 Dynabeads anti-Listeria antibody (9 %). Furthermore, capture efficiency for MyOne-2D12 was highly specific for L. monocytogenes and L. ivanovii. Subsequently, we captured L. monocytogenes by MyOne-2D12 and MyOne-3F8 from hotdogs inoculated with mono- or co-cultures of L. monocytogenes and L. innocua (10-40 CFU/g), enriched for 18 h and detected by fiber-optic sensor and confirmed by plating, light-scattering, and qPCR assays. The detection limit for L. monocytogenes and L. ivanovii by the fiber-optic immunosensor was 3 × 10² CFU/mL using MAb-2D12 as capture and reporter antibody. Selective media plating, light-scattering, and qPCR assays confirmed the IMS and fiber-optic results. CONCLUSIONS: IMS coupled with a fiber-optic sensor using anti-InlA MAb is highly specific for L. monocytogenes and L. ivanovii and enabled detection of these pathogens at low levels from buffer or food.
Asunto(s)
Técnicas Bacteriológicas/métodos , Tecnología de Fibra Óptica/métodos , Separación Inmunomagnética/métodos , Listeria/aislamiento & purificación , Animales , Anticuerpos Antibacterianos/aislamiento & purificación , Anticuerpos Monoclonales/aislamiento & purificación , Antígenos Bacterianos/inmunología , Femenino , Inmunoensayo/métodos , Ratones , Ratones Endogámicos BALB C , Sensibilidad y EspecificidadRESUMEN
The spreading of diseases through foods is a worldwide concern. Here, molecular and in vitro cell-culture assays were employed to characterize 63 Brazilian Listeria monocytogenes isolates (food, 47; clinical, 16). Serotype 4b was the most predominant (49%) followed by (1/2)b (30%), (1/2)a (10%), (1/2)c (6%), 3c (3%) and 3b (2%). Ribotyping yielded 17 ribopatterns, which were grouped into four phylogenetic clusters. Cluster A comprised of 39/63 isolates primarily of food origin, and clusters B, C and D contained both food and clinical isolates. Isolates were positive for virulence determinants prfA, hlyA and inlA: clinical isolates were more invasive to Caco-2 cells and expressed high levels of inlA transcripts than the food isolates. Highly invasive isolates also provoked more Ped-2E9 cells to die by apoptosis than the weakly-invasive strains. These data demonstrate a strong genetic relatedness among clinical and food isolates and suggest transmission of a subset of L. monocytogenes strains from food to humans.