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Laboratory of Ecology of Pathogenic Bacteria, Gamaleya Research Center of Epidemiology and Microbiology, 123098 Moscow, RussiaInstitutue of Biochemical Technology and Nanotechnology, Peoples' Friendship University of Russia (RUDN University), 117198 Moscow, Russia
The gram-positive bacterium Listeria monocytogenes is an important foodborne pathogen contaminating dairy products. Closely related to L. monocytogenes saprophytic Listeria spp. are also frequent contaminators of food and, particularly, dairy products. To distinguish L. monocytogenes from nonpathogenic Listeria spp. and other bacteria, a dot-immunoassay was developed. The immunoassay is based on the polyclonal antibody to the secreted form of the surface virulence-associated L. monocytogenes-specific InlB protein. To increase InlB production, bacteria were grown on the brain-heart infusion agar supplemented with 0.2% activated charcoal (BHIC agar). Direct plating of artificially contaminated raw milk samples on the BHIC agar followed by the dot-immunoassay allowed a rapid identification of L. monocytogenes in concentrations as little as 10 cfu/mL. Using the developed approach, preliminary results were obtained within 14 h, and the final results were obtained after 26 h. The dot-immunoassay was tested on L. monocytogenes strains belonging to different clonal complexes and phylogenetic lineages, Listeria spp., and other bacterial species. Results showed the exceptional specificity of the developed dot-immunoassay for the rapid identification of L. monocytogenes.
). The incidence of listeriosis ranges from 0.4 to 1.8 per 100,000 population, while the highest number of fatal cases among foodborne infections in developed countries is due to high mortality rates reaching up to 30%, especially among groups of risk, such as immunocompromised and elderly persons, newborns, and pregnant women (
The high L. monocytogenes incidence in dairy products caused several serious outbreaks associated with ready-to-eat products including raw milk, soft and creamery cheeses, and ice cream (
). In the last years, the prevalence of L. monocytogenes in raw milk was reported at 7.5–9%, which is higher than for Salmonella or Escherichia coli O157:H7 (
Prevalence, molecular characterization, and antimicrobial resistance profiles of Listeria monocytogenes, Salmonella enterica, and Escherichia coli O157:H7 on dairy cattle farms in Jordan.
). Listeria monocytogenes multiplies in a wide range of temperatures, salt concentrations, and pH, which contributes to its long-term preservation and reproduction in food products and provides conditions for pathogen proliferation in fresh cheese and other dairy products (
The genus Listeria comprises 17 species including 6 species that share common phenotypic characteristics with L. monocytogenes (Listeria sensu strictu species;
). Four of 6 L. sensu strictu species are nonpathogenic, excluding L. monocytogenes and Listeria ivanovii, an animal pathogen, and rare contaminants of food products (
). Apart from L. monocytogenes, other predominant food contaminants from L. sensu strictu species are nonpathogenic, such as Listeria innocua, Listeria seeligeri, and Listeria welshimeri (
). Listeria innocua is the closest to L. monocytogenes, as they share 90% of the genome including 2,523 genes that encode housekeeping proteins, transporters, transcriptional regulators, and so on. (
The multiple similarities with L. innocua and other nonpathogenic Listeria species make the detection of L. monocytogenes from food samples a very challenging task. Selective media such as polymyxin acriflavine lithium-chloride ceftazidime esculin mannitol (PALCAM) or Oxford agars are not specific to L. monocytogenes, as they also support the growth of other Listeria spp. and some other bacterial genera (
). Competitive flora of food samples might overgrow low numbers of L. monocytogenes putting a limitation on chromogenic media such as listeria agar Ottaviani and Agosti (ALOA), which allows for direct differentiation of L. monocytogenes from other Listeriae (
). However, these methods are expensive and require specialized hardware. Immunological methods are cheap and easy, but L. monocytogenes shares many cell surface antigens with other Listeria spp., which makes it difficult to differentiate L. monocytogenes from nonpathogenic species frequent in food samples and restricts application of immunological methods for L. monocytogenes detection. Therefore, the development of reliable immunological tests based on antigenic markers specific to L. monocytogenes is required.
Listeria monocytogenes-specific surface proteins, which are absent in other Listeria spp., are mainly represented by virulence factors (
). Among them, the protein InlB, a specific surface protein of L. monocytogenes, is a virulence factor mediating bacterial invasion into the host cell and is absent from the other Listeria spp. (
). Internalin B is a secreted protein that can be anchored on the cell surface via noncovalent interactions of its carboxyterminal GW-domains with cell wall teichoic acids (
). Positive regulatory factor A activity is downregulated when L. monocytogenes is cultivated on the rich media, unless the medium is supplemented with activated charcoal or another hydrophobic adsorbent. Hydrophobic adsorbents activate PrfA and induce expression of PrfA-controlled virulence factors including InlB (
Thus, the aim of this work was to assess the potential use of the detection of the InlB protein in conditions of PrfA regulon upregulation to reliably differentiate L. monocytogenes from nonpathogenic Listeria spp. and other bacteria contaminating raw milk.
MATERIALS AND METHODS
No human or animal subjects were used, so this analysis did not require approval by an Institutional Animal Care and Use Committee or Institutional Review Board.
Bacterial Strains and Growth Conditions
The strains used are listed in the Table 1. Strains were kept frozen in the collection of the Laboratory of Ecology of Pathogenic Bacteria, Gamaleya Research Centre of Epidemiology and Microbiology, until the work started. Thawed bacteria were plated on the Brain-Heart Infusion (BHI, BD) agar and grown at 37°C for 18–24 h.
For the PrfA regulon and InlB upregulation, the BHI agar medium was supplemented with 0.2% (wt/vol) charcoal (BHIC) as described in our previous work (
). To activate PrfA when bacteria were grown in the liquid medium, BHI broth (BD) was supplemented with 0.2% (wt/vol) charcoal or 1% (wt/vol) AmberlitXAD4 (Sigma-Aldrich;
Recombinant InlB321 Protein Purification and Development of Polyclonal Antibodies
The inlB gene fragment encoding the secreted amino-terminal InlB domain (amino acids 29–321, InlB321) lacking the signaling peptide and the cell wall bounding GW-domain was obtained by PCR on DNA from the strain EGDe as previously described in the work of
. Primers InlBF 5′-aca-agc-gga-tcc-tat-cac-tgtgcc-3′ and InlBR 5′-tgt-aaa-gct-ttt-tca-gtg-gtt-ggg-3′ (Syntol) were used to obtained the PCR product, which was cloned into the auxiliary vector pGEM-T Easy (Promega), and then subcloned into the vector pET28b(+) (Novagene) by BamHI and HindIII restriction sites. The recombinant plasmid encoding the InlB321 with the C-terminal His-Tag was transformed into the E. coli strain BL21.
The recombinant strain was grown in the LB broth supplemented with 100 μg/mL kanamycin and 1 mM IPTG. Bacteria were pelleted by centrifugation at 4,200 × g for 10 min at 4°C, resuspended in the Buffer A (30 mM imidazole, 150 mM NaCl, 10 mM Na2PO4; pH 7.4), and disrupted using ultrasound. Cell debris was sedimented by centrifugation at 11,000 × g for 10 min at 4°C. The supernatant was applied to the HisTrapTM FF Crude (Sigma-Aldrich) column. The column was washed with 5 volumes of the Buffer A, and then the bound InlB-HisTag protein was eluted with the eluting buffer (500 mM imidazole, 150 mM NaCl, 10 mM Na2HPO4; рН 7.4). Eluted fractions were checked with SDS-PAGE. The purified protein was dialyzed against PBS using Slide-A-Lyzer Dialysis Cassettes (ThermoFisher Scientific Inc.). The final protein concentration was determined according to the Bradford method. The purified protein was kept at −20°C.
Polyclonal antibodies were developed in a rabbit after immunization that was carried out in 3 stages using 1 mg of purified InlB321. The hyperimmune serum was taken from the rabbit 3 times at 2-wk intervals starting 1-mo after immunization was finished. All serum samples were combined to be used in further experiments.
Western Blotting
Listeria monocytogenes was grown in the BHI broth supplemented with 0.2% charcoal overnight. Surface proteins were prepared as described in previous work (
). Surface proteins were separated on 10% SDS-PAGE, transferred onto a nitrocellulose membrane, and Western blotting was performed in the TTBS (0.05% Tween 20, 10 mM Tris HCl, pH 7.5-0.15 M NaCl) buffer with the rabbit antiserum diluted 1:5,000. Internalin B was visualized with horseradish peroxidase (HRP)-conjugated anti-rabbit antibodies (Bio-Rad) and the TMB (3,3′,5,5′-tetramethylbenzidine) substrate (Thermo Fisher Scientific).
Purification of InlB-Specific Polyclonal Antibodies
To get specific immunosorbent, purified InlB321 was immobilized on the Cyanogen bromide-activated-Sepharose 4B (Sigma-Aldrich) according to manufacturer instructions. The hyperimmune serum was loaded to a handmade immunosorbent column. The column was washed with a phosphate-salt buffer with 0.3 M NaCl to remove nonbound antibodies. Internalin B-specific antibodies were eluted with 4.5 M MgCl2 and dialyzed against PBS, pH 7.4. Purified InlB-specific IgG antibodies were dissolved up to concentration of 5 mg/mL and stored in 50% glycerol at −20°C.
Preparation of the a-InlB-IgG–Horseradish Peroxidase Conjugate
Internalin B-specific IgG antibodies were labeled with horseradish peroxidase using a 2-stage method and glutaraldehyde as a crosslinking agent. Ten mg of horseradish peroxidase was dissolved in 0.2 mL of 0.1 M phosphate buffer containing 0.15 M NaCl and 1.25% glutaraldehyde, pH 6.8, by overnight mixing at room temperature. Excessed glutaraldehyde was removed by dialysis against 0.15 M NaCl. The horseradish peroxidase solution was diluted up to 1 mL using 0.15 M NaCl. Then, 1 mL of IgG solution (5 mg/mL) containing 0.15 M NaCl and 0.1 mL of 1 M carbonate buffer, pH 9.5 was added. The mixture was incubated at 4°C for 24 h, then 0.1 mL of 0.2 M lysine solution was introduced to stop the reaction. The mixture was incubated for 2 h at room temperature and dialyzed overnight against a PBS (0.01 M phosphate buffer supplemented with 0.15 M NaCl, pH 7.2). The a-InlB-IgG–horseradish peroxidase (a-InlB-IgG-HRP) conjugate was precipitated with an equivalent volume of saturated NH4(SO4)2 solution and dissolved in 1 mL of phosphate buffer. The solution was dialyzed against PBS. The solution was centrifuged at 10,000 × g for 30 min at 4°C, the precipitate was removed, BSA was added to 1%, and the solution was filtered through a filter with a pore diameter of 0.2 μm. The obtained a-InlB-IgG–HRP conjugate was stored at −20°C.
Direct ELISA
For direct ELISA, bacteria were grown in the BHI supplemented with 1% (wt/vol) Amberlite XAD4 resin (Sigma-Aldrich) overnight. Bacteria were harvested, washed, and resuspended in PBS at the concentration of 1010 cfu/mL. Decimal dilutions of the initial bacterial suspension were prepared in PBS. Corner Notch Pierce 96-Well Polystyrene Plates (Thermo Scientific) were coated with 100 µL each of bacterial suspensions and incubated at +4°C overnight. After 3 times washing with the TTBS solution, a-InlB-IgG–HRP conjugate diluted 1:10,000 was added. Plates were incubated at room temperature for 1 h, washed 3 times with TTBS, and the HRP substrate 1-Step Ultra TMB-ELISA Substrate Solution (Thermo Fisher Scientific) was added. Results of the colorimetric reaction was read with the iMark (Bio-Rad) plate reader at 450 nm.
Dot-immunoassay
Colonies were grown on 55 mm BHIC agar plates for 18–24 h unless otherwise stated and then imprinted on a 55 mm nitrocellulose membrane (Bio-Rad). The membrane was dried before it was immersed into chloroform for 15 min for fixation. Then the membrane was dried from chloroform residues and incubated for 30 min in a blocking buffer (2% bovine serum albumin in PBS) at room temperature. The developed a-InlB-IgG–HRP conjugate was diluted at 1:4,000 in TTBS. The dried membrane was incubated with the diluted conjugate at room temperature for an hour using a 150 rpm shaker. The membranes were washed 3 times, each for 5 min with TTBS. The colonies were revealed with the 1-Step Ultra TMB-Blotting Solution (Thermo Fisher Scientific). The reaction was stopped by washing the membranes with distilled water. The development from light to the dark purple color indicated a positive reaction and the presence of colonies of the species L. monocytogenes.
PCR Assay
The PCR assay was performed to confirm the belonging of the colonies positive in the dot-assay to the species L. monocytogenes. Briefly, colonies imprinted onto the membrane were sampled with sterile toothpicks. Samples were transferred to the 50 μL 1× PCR buffer and suspended by vortexing. Lysozyme was added up to 40 µg/mL and samples were incubated at 37°C for 1 h, then Proteinase K was added up to 40 µg/mL, and samples were incubated at 56°C for 1 h more. Then samples were boiled for 5 min. PCR was performed with 1 μL of lysates and the following primers: InlBF 5′-aca-agc-gga-tcc-tat-cac-tgtgcc-3′ and InlBR 5′-tgt-aaa-gct-ttt-tca-gtg-gtt-ggg-3′. The PCR with these primers gave an 872-bp inlB gene fragment. Amplification was performed for 35 cycles at the annealing temperature of 58°C using Tertsik amplificator (DNA-Technology).
To confirm that the colonies belonged to the genus Listeria, PCR was performed with genus-specific primers that recognized the dat gene datF 5′-gaaagagaagatgccacagttga-3′ and datR 5′-gtccataatacaccatcttt-3′. Amplification was performed for 35 cycles at the annealing temperature of 55°C using Tertsik amplificator (DNA-Technology). The PCR products were separated in 1% agarose gel.
Real-Time PCR
To confirm that the colonies belonged to L. monocytogenes, real-time PCR analysis was performed with a commercial kit (DNA-Technology) according to the manufacturer's instructions on thermocycler QuantStudio 5 (Thermo Scientific). The thermocycler program steps were: first at 80°C for 1 min, 94°C for 1 min, second (repeated 5 times) at 94°C for 30 s, 64°C for 15 s, and third (repeated 45 times) at 94°C for 10 s, 64°C for 15 s.
Raw Milk Inoculation and Analysis
Overnight the L. monocytogenes strain EGDe culture was concentrated by centrifugation, washed, and resuspended in PBS. The suspension taken at the concentration of 109 cfu/mL according to the McFarland standard was diluted up to 1, 10, 102, and 103 cfu/mL in PBS. Raw milk was taken from the farm market (Moscow, Russia) on the day of the experiment. Bacterial suspensions were mixed with raw milk samples in the ratio 1:9 (bacterial suspension: milk). 100 μL samples were plated on the BHIC agar and grown for 18–24 h. Then the dot-immunoassay was performed with colonies grown on BHIC as described above. The intact milk samples plated on BHIC and treated in the same way were used as a negative control. As a positive control, the same samples were pated on the PALCAM agar. The colonies grown on the PALCAM agar were tested in PCR as described above.
Statistics
All experiments were repeated at least 3 times. When it was applicable, one-way ANOVA was perfomed, and P-values of less than 0.05 were considered statistically significant.
RESULTS
InlB-Specific Polyclonal Antibody Development
The recombinant InlB321-His-tag protein, which included amino acids 29–321 of the InlB protein and C-end His-tag, was purified from the E. coli strain BL21:pET28b(+):inlB321 as described in Materials and Methods. The recombinant protein was used to develop an InlB-specific antiserum in the rabbit. To check the ability of the antiserum developed against InlB321-His-tag to recognize full-length InlB, it was applied to lysates of 3 wild type L. monocytogenes strains belonging to different clonal complexes of I and II phylogenetic lineages, and the strain EGDeΔinlB lacking the inlB gene in Western blotting. For all strains except EGDeΔinlB, antibodies reacted with a protein with a molecular weight of about 66 kDa, correspondent to the molecular mass of full-length InlB (Figure 1).
Figure 1SDS-PAGE and Western blot analysis of Listeria monocytogenes lysates using polyclonal antibodies developed against InlB321 [secreted amino-terminal InlB domain (amino acids 29–321)]. Bacterial lysates were obtained from 1 mL of the overnight culture grown in the brain heart infusion (BHI) broth supplemented with 0.2% charcoal, separated on 10% SDS-PAGE, and transferred onto the nitrocellulose membrane. The InlB protein was visualized with the developed rabbit antiserum used in the dilution 1:5,000 and the 3,3′,5,5′-tetramethylbenzidine (TMB) substrate. The figure shows the results of the Western blot for the following strains of L. monocytogenes: (1) EGDeΔinlB, EGDe derivative lacking the inlB gene; (2) EGDe, type strain belonging to the clonal complex CC9, phylogenetic lineage II; (3) VIMHA004 clinic isolate, CC2, phylogenetic lineage I (
Next, InlB-specific IgG antibodies were purified from the serum using a handmade immunosorbent column and conjugated with HRP to get a-InlB-IgG–HRP conjugate (see Materials and Methods). To verify that the conjugated antibodies retain the ability to bind specifically to the antigen, a direct ELISA was performed using the L. monocytogenes strains EGDe and EGDeΔinlB, the L. innocua strain SLCC3379, and the L. ivanovii strain ATCC 19119. Conjugated a-InlB-IgG antibodies bound EGDe cells in a dose dependent manner, but did not bind EGDeΔinlB, L. innocua, and L. ivanovii (Supplemental Figure S1; https://data.mendeley.com/datasets/bhr63fh9rr;
). Thus, obtained results demonstrated that the developed a-InlB-IgG–HRP conjugate specifically reacted with L. monocytogenes carrying the InlB protein on its surface.
Development of the a-InlB-IgG-HRP Conjugate Based L. monocytogenes-Specific Dot-Immunoassay
The developed a-InlB-IgG-HRP conjugate was used in the dot-immunoassay. We grew bacteria on the BHIC agar to upregulate the inlB gene and improve InlB production, and made imprints of the colonies on the nitrocellulose membrane. As an alternative approach, we tried to grow bacteria directly on the membrane placed over the BHIC agar overnight. The first approach allowed better bacterial growth and gave better results, so, colony imprints were used in all further experiments. The membrane was stained with the a-InlB-IgG-HRP conjugate, and results were visualized with the TMB substrate as described in Materials and Methods. The positive reaction with L. monocytogenes looked similar to a solid dot, at the place where the colony was imprinted (Figure 2).
Figure 2The dot-immunoassay with the a-InlB-IgG-HRP conjugate (purified InlB-specific IgG antibodies-HRP conjugate). (A) Membrane imprints of L. monocytogenes EGDe colonies grown on brain-heart infusion agar supplemented with 0.2% activated charcoal (BHIC); (B) the dot-immunoassay of the membrane with the a-InlB-IgG-HRP conjugate. HRP = horseradish peroxidase.
To check that the secreted InlB does not affect the results of the dot-blot, we conducted a similar experiment, but with the addition of 2.5 µg/mL of the soluble form of InlB. The resulting mixture was plated on BHIC agar, and a dot-blot was carried out (Supplemental Figure S2; https://data.mendeley.com/datasets/bhr63fh9rr;
). We observed no additional background when adding a soluble form of InlB. Further, the wild type strain EGDe and the isogenic strain EGDeΔinlB were premixed in different ratios, and the mixture was used in the dot-blot assay. Results supported the view that the strain lacking InlB gives a negative result independently on the presence of the InlB-positive bacteria on the culture (Supplemental Figure S3; https://data.mendeley.com/datasets/bhr63fh9rr;
To check its specificity, the dot-immunoassay was performed with 9 L. monocytogenes strains belonging to different clonal complexes and serovars, other Listeria spp., and non-Listeria species. All L. monocytogenes strains tested gave a positive signal, despite the presence of certain AA substitutions that distinct InlB isoforms from strains belonging to different phyla (Figure 3, Figure 4). Meantime, no signal was obtained when other bacterial species were tested, including L. innocua, L. seeligeri, L. welshimeri, L. ivanovii, Staphylococcus aureus, and E. coli (Figure 3) Particularly, the assay did not recognize L. ivanovii, even though this species carries the homologous protein as well, designated as InlB (
A spontaneous genomic deletion in Listeria ivanovii identifies LIPI-2, a species-specific pathogenicity island encoding sphingomyelinase and numerous internalins.
). Multiple substitutions differentiate L. monocytogenes and L. ivanovii InlB (Figure 4), which seem to be important for binding specific antibodies.
Figure 3The dot-immunoassay with the a-InlB-IgG-HRP conjugate (purified InlB-specific IgG antibodies-HRP conjugate). (A) The dot-immunoassay with Listeria monocytogenes strains, strain designations, and clonal complexes the strain belongs to shown in the picture; (B) the dot-immunoassay with Listeria spp. and non-Listeria species. HRP = horseradish peroxidase.
Figure 4Alignment of the Listeria monocytogenes EGDe InlB domain used to develop a polyclonal antibody with the domains of L. monocytogenes strains used in the study, including VIMHA004 (CC2), VIMHA034 (CC7), 1300 (CC1), 56-T (CC315), 87014 (CC2), 8712 (CC8), 129/3 (CC9), 114/26 (CC37) VIMPR134 (CC7), VIMHA036 (CC20), and the homologous domain of the InlB protein from the Listeria ivanovii strain ATCC19119. The Unipro UGENE software version 41.0 was used.
L. monocytogenes Detection with the Dot-Immunoassay in the Mixed Culture
Listeria innocua is the closest to L. monocytogenes species and is one of the most often Listeria contaminators of dairy products. To evaluate the potential of InlB as a marker to differentiate L. monocytogenes from L. innocua in a mixed culture, we took overnight cultures of L. monocytogenes EGDe and L. innocua SLCC3379 in the ratios 1:1, 1:5, 1:10, and plated the mixed cultures onto BHIC agar. The colonies of the 2 species were morphologically indistinguishable, but dot-blotting with the a-InlB-IgG-HRP conjugate applied to colony imprints allowed unambiguous identification of colonies as positive (Figure 5). The proportion of immune-positive colonies corresponded to the percentage of L. monocytogenes in the mixed culture. To confirm the results of the dot-immunoassay, the positive and negative colonies were tested in PCR and real-time PCR (Figure 6). The commercially available L. monocytogenes-specific real-time PCR test-system (DNA-Technology) gave a signal with InlB-positive colonies only, proving that the dot-blot assay distinguished L. monocytogenes in the mixed culture. Only InlB-positive colonies gave a signal in the PCR assay with the inlB-specific primers, supporting their belonging to the species L. monocytogenes. When the Listeria genus-specific primers were used, both InlB-positive and InlB-negative colonies gave a signal making sure that all colonies belonged to bacteria of the genus Listeria (Figure 6E).
Figure 5Testing mixed samples of Listeria monocytogenes and Listeria innocua. Listeria monocytogenes EGDe and L. innocua SLCC3379 were taken in ratios 1:10, 1:5, and 1:1, plated onto the membrane overlying the brain-heart infusion agar supplemented with 0.2% activated charcoal (BHIC), and grown for 24 h. Then colonies were imprinted on the membrane, and membranes were processed with the a-InlB-IgG-HRP conjugate (purified InlB-specific IgG antibodies-HRP conjugate). Imprints of colonies and results of the dot-immunoassay are shown. Arrowheads show imprints of L. monocytogenes colonies and corresponding dot-blots. HRP = horseradish peroxidase.
Figure 6Verification of the dot-blot results. Listeria monocytogenes EGDe and Listeria innocua SLCC 3379 were taken in the ratio 1:1 and grown on brain-heart infusion agar supplemented with 0.2% activated charcoal (BHIC) for 24 h. The imprinted colonies were sampled before washing out, then the membrane was processed with the a-InlB-IgG-HRP conjugate (purified InlB-specific IgG antibodies-HRP conjugate) as described above. To identify L. monocytogenes, PCR with the inlB gene-specific primers was performed. (A) Imprints of colonies grown on the BHIC agar; (B) results of the dot-immunoassay before imprinting on the membrane; (C) PCR was performed with inlB-specific primers, PCR products were separated on the 1% agarose gel, and the inlB gene fragment has a size of 872 bp. Numbers designate individual colonies. Marker fragment sizes are shown; (D) real-time PCR using a commercially available kit; numbers 1–9 correspond to colony numbers (see A); C+ = Listeria monocytogenes EGDe; C = Listeria innocua SLCC 3379; (E) PCR was performed with genus-specific primers that recognized the dat gene. HRP = horseradish peroxidase.
To determine the minimum time required to reveal colonies using the suggested dot-blotting method, the analysis was performed with L. monocytogenes strain EGDe colonies grown on BHIC agar for 8, 12, 16, 24 h. Eight h postplating, the colonies were indistinguishable, and dot-blotting showed negative results. Starting from 12 h postplating, when small colonies appeared, the dot-immunoassay with the a-InlB-IgG-HRP conjugate gave a positive result (Figure 7). Therefore, the appearance of visible colonies, even small ones, was enough to get a positive result in the dot-immunoassay with antibodies to InlB. The dot-assay took 2 h, so, together with the dot-assay, the minimal time required to reveal L. monocytogenes colonies was 14 h. Still, the best readable results were obtained with colonies grown for 24 h and a total analysis duration of 26 h.
Figure 7Testing of the minimal time to detect Listeria monocytogenes using brain-heart infusion agar supplemented with 0.2% activated charcoal (BHIC) growth and a-InlB-IgG-HRP conjugate (purified InlB-specific IgG antibodies-HRP conjugate) based dot-blot analysis. Bacteria were grown on the BHIC agar for 8, 12, 16, or 24 h. The immunoassay with the a-InlB-IgG-HRP conjugate was performed as described above. The results of the immunoassay, including enlarged colony imprints, are shown. HRP = horseradish peroxidase.
Application of the Developed Dot-Immunoassay to Detect L. monocytogenes in Raw Milk
Raw milk samples acquired at the farm market were inoculated with 1, 10, 102, or 103 cfu/mL of the L. monocytogenes strain EGDe. Contaminated milk samples were plated on the BHIC agar, and grown colonies were analyzed in the dot-immunoassay as described above (Figure 8). In parallel, the same samples were plated on the selective PALCAM agar to analyze grown colonies in PCR. Detection of as little L. monocytogenes concentration as 1 cfu/mL was successful in 1 experiment using the dot-immunoassay and in 2 experiments using PALCAM plating supplemented with PCR colony checking. Results of all 3 experiments were successful from both methods, with higher bacterial concentrations starting from 10 cfu/mL. According to colony counting, the sensitivity of the PALCAM or PCR method was slightly higher than the sensitivity of the dot-immunoassay (Supplemental Figure S4; https://data.mendeley.com/datasets/bhr63fh9rr;
; Table 2; P < 0.05). This discrepancy might be caused by competition and overgrowth of L. monocytogenes by milk microflora. Using a selective medium is not appropriate for the method based on InlB detection, as InlB is poorly expressed in selective media (
). Still, obtained results demonstrated that sensitivity of the developed dot-blot immunoassay ranged from 1 to 10 cfu/mL. The same range was observed for the PALCAM/PCR method.
Figure 8Listeria monocytogenes detection in artificially contaminated milk samples. Raw milk samples were inoculated with L. monocytogenes EGDe up to concentrations shown. One hundred-microliter samples were plated on selective polymyxin acriflavine lithium-chloride ceftazidime esculin mannitol (PALCAM) and nonselective brain-heart infusion agar supplemented with 0.2% activated charcoal (BHIC). Belonging to the species L. monocytogenes was proven by PCR for bacteria grown on PALCAM agar and by the developed dot-blot immunoassay for bacteria grown on BHIC agar. A, B, C: milk samples plated on PALCAM agar; D, E, F: milk samples plated on BHIC agar, note the growth of intrinsic milk microflora; G, H, I: results of the dot-immunoassay with BHIC-grown samples. A, D, G: intact milk; B, E, H: 1-mL milk sample was inoculated with 10 cfu/mL; C, F, I: 1-mL milk sample was inoculated with 100 cfu/mL. Arrowheads show dots corresponding to L. monocytogenes colonies.
Table 2Listeria monocytogenes detection in milk samples artificially contaminated with 1 and 10 cfu/mL of L. monocytogenes EGDe1 Belonging to the species L. monocytogenes was proven by PCR for bacteria grown on PALCAM agar (PALCAM/PCR)
Belonging to the species L. monocytogenes was proven by PCR for bacteria grown on polymyxin acriflavine lithium-chloride ceftazidime esculin mannitol (PALCAM) agar (PALCAM/PCR).
Experiment
Negative control (intact milk)
1 cfu/mL
10 cfu/mL
PALCAM/PCR
Dot-assay
PALCAM/PCR
Dot-assay
PALCAM/PCR
Dot-assay
1
0
0
1
1
11
8
2
0
0
1
0
8
6
3
0
0
0
0
9
8
1 Belonging to the species L. monocytogenes was proven by PCR for bacteria grown on polymyxin acriflavine lithium-chloride ceftazidime esculin mannitol (PALCAM) agar (PALCAM/PCR).
In this work, we developed a simple method that combined bacterial growth on the nonselective medium and a dot-immunoassay to differentiate L. monocytogenes, which is an important foodborne pathogen, from Listeria spp. and other bacterial genera. The method allowed the detection of L. monocytogenes in artificially contaminated milk with a sensitivity as low as 10 cfu/mL within 26 h. Listeria monocytogenes is widely spread in the farm and industrial environment and is an often contaminant of dairy products, which underscores the significance of its surveillance. Up-to-date cultural methods that use various enrichment and selective media supplemented with biochemical assays are of major importance for the detection of L. monocytogenes in food and environmental samples (
Detection of Listeria monocytogenes in foods and environmental samples, and enumeration of Listeria monocytogenes in foods. Chapter 10 in Bacteriological Analytical Manual (BAM).
). These methods are reliable but time consuming. In the last years, biochemical assays were replaced by MALDI-TOF MS. The MALDI-TOF MS provides reliable identification and simultaneous subtyping of L. monocytogenes for an isolated colony grown on selective media (
). The need for expensive equipment and infrastructure limits the applicability of this method. The PCR-based methods are highly sensitive, but their application directly to contaminated samples is restricted by food-specific substances that inhibit the reaction, which require additional steps including pre-enrichment, immunomagnetic separation, or DNA purification (
Biotin exposure-based immunomagnetic separation coupled with sodium dodecyl sulfate, propidium monoazide, and multiplex real-time PCR for rapid detection of viable Salmonella Typhimurium, Staphylococcus aureus, and Listeria monocytogenes in milk.
The pathogen-specific immunoassays are specific, do not require expensive equipment, and allow to substantially shorten the time required for the identification. Using monoclonal antibodies developed against peptides derived from the surface protein p60,
described an ELISA system that provides efficient identification of L. monocytogenes using bacterial lysates. Using monoclonal antibodies against the p60-derived L. monocytogenes-specific peptide PepD,
developed a fluorescence sandwich immunoassay that allowed analysis for 12 h with a limit of detection of as little as 102 cfu/mL.
Here we demonstrated that the first results can be obtained within 14 h with a system based on growth on the nonselective BHIC medium and InlB-specific antibodies. Within 26 h, reliable results can be obtained for raw milk samples contaminated with 10 cfu/mL. The BHIC supports an effective bacterial growth and improves the expression of the L. monocytogenes-specific virulence factor InlB, whereas antibodies developed against InlB reliably differentiated L. monocytogenes from other bacteria, including closely related L. sensu strictu species.
Internalin B belongs to the family of Internalin-like proteins found in Listeria spp. (
A spontaneous genomic deletion in Listeria ivanovii identifies LIPI-2, a species-specific pathogenicity island encoding sphingomyelinase and numerous internalins.
). Another representative of the internalin family, InlA was shown to be useful for L. monocytogenes identification and differentiation of highly virulent and low-virulent strains (
The development of rapid fluorescence-based immunoassays, using quantum dot-labeled antibodies for the detection of Listeria monocytogenes cell surface proteins.
Int. J. Biol. Macromol.2006; 39 (16600361): 127-134
used monoclonal an InlB-specific antibody in the quantum dot-assay. Anti-IgG developed against the secreted InlB form was applied to develop a highly sensitive immunoassay based on electrochemical impedance spectroscopy (
). The method detected L. monocytogenes at a concentration of 1.7 × 105 cfu/mL and was successfully tested on food samples.
The method suggested in this work allowed the detection of L. monocytogenes in artificially contaminated milk at a concentration of 10 cfu/mL with 100% effectiveness and at a concentration of 1 cfu/mL with 30% effectiveness. The developed inexpensive and fast assay might be applied in monitoring dairy products for the presence of L. monocytogenes to ensure their microbiological quality and safety.
ACKNOWLEDGMENTS
This study was funded by the Russian Foundation for Basic Research (grant no. 20-38-90260; Moscow, Russia). The authors thank J. Vazquez-Boland, University of Edinburgh, for the gift of the strain EGDeΔinlB. The authors have not stated any conflicts of interest.
REFERENCES
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A spontaneous genomic deletion in Listeria ivanovii identifies LIPI-2, a species-specific pathogenicity island encoding sphingomyelinase and numerous internalins.
Detection of Listeria monocytogenes in foods and environmental samples, and enumeration of Listeria monocytogenes in foods. Chapter 10 in Bacteriological Analytical Manual (BAM).
Prevalence, molecular characterization, and antimicrobial resistance profiles of Listeria monocytogenes, Salmonella enterica, and Escherichia coli O157:H7 on dairy cattle farms in Jordan.
Biotin exposure-based immunomagnetic separation coupled with sodium dodecyl sulfate, propidium monoazide, and multiplex real-time PCR for rapid detection of viable Salmonella Typhimurium, Staphylococcus aureus, and Listeria monocytogenes in milk.
The development of rapid fluorescence-based immunoassays, using quantum dot-labeled antibodies for the detection of Listeria monocytogenes cell surface proteins.
Int. J. Biol. Macromol.2006; 39 (16600361): 127-134
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