Contaminated Food Samples Research Articles

Simplified detection of food-borne pathogens: An in situ high affinity capture and staining concept

Food industries need simple, rapid and cost-effective solutions for pathogen detection in food and environmental samples. In this paper, we describe a simple but novel detection concept combining an affinity capture surface and intracellular metabolic marker to visualize the bacterial presence on the affinity surface. The surface of a Solid Phase Support (SPS) is functionalized with specific phage tail proteins targeted to the bacterial pathogen of interest. The SPS is placed directly into the primary food enrichment bag after stomaching. Following incubation, the captured bacteria are visually detected in situ as a result of the bacterial reduction of the colorless soluble substrate triphenyltetrazolium chloride (TTC) (present in the primary culture medium) to an intracellular red insoluble formazan product. Detection on the SPS is observed as an intense red color after 22 to 40hours of enrichment. This is not impaired by the presence of food particles and the natural background microflora. The in situ method significantly simplifies pathogen detection by eliminating any post-enrichment intervention that is necessary in the traditional methods of analysis. We have demonstrated the application of this new approach for the detection of Escherichia coli O157: H7, Listeria spp. and Salmonella spp. in artificially contaminated food samples.

Development of an Improved Selective and Differential Medium for Isolation of Salmonella spp

We describe an improved selective, differential, and cost-effective medium, XA medium, which contains d-arabinose, to facilitate the selective isolation of Salmonella spp. The sensitivity and the specificity of XA medium were compared to those of xylose lysine desoxycholate agar (XLD) using stock cultures and naturally contaminated food samples. XA medium and XLD were evaluated with a total of 82 Salmonella and 69 non-Salmonella stock cultures. Of 82 strains of Salmonella spp. tested, 76 produced a characteristic black colony on XA medium and XLD. The remaining 6 strains belonged to Salmonella enterica serovars Berta (n = 1), Paratyphi A (n = 1), Gallinarum (n = 2), and Pullorum (n = 2). The sensitivities of XA medium and XLD were identical (92.7%). Citrobacter freundii (n = 21) and Proteus mirabilis (n = 21) stock cultures produced black colonies on XLD, whereas only 4 strains of P. mirabilis appeared as black colonies on XA medium. In the second phase of the study, a total of 180 food samples were cultured onto XA medium and XLD after selective enrichment. The sensitivities of XA medium and XLD were equal (100%), and a total of 6 Salmonella strains were isolated from the 180 food samples. The specificity of XA medium (92.0%) was superior to that of XLD (73.0%), with a total of 14 and 47 false-positive results found on XA medium and XLD, respectively. On the basis of its good specificity, XA medium is useful for the isolation of Salmonella spp. from food samples.

A real-time reverse-transcriptase PCR technique for detection and quantification of viable Alternaria spp. in foodstuffs

A real-time reverse-transcriptase PCR (RT-PCR) technique was developed for the rapid and specific detection and enumeration of viable Alternaria spp. in foodstuffs. The method uses Alernaria-specific primers and probe targeting the internal transcribed spacer regions ITS1 and ITS2 of the rRNA gene. The detection limit of the real-time RT-PCR assay to detect viable Alternaria spp. in food samples was 1 CFU/g. The estimated Alternaria counts obtained by real-time RT-PCR showed a good correlation (R2 = 0.9881, P < 0.01) in the range of 1–105 CFU/mL with the Alternaria counts obtained by culture methods. The applicability of the real-time RT-PCR protocol was assessed through analysis of 110 commercial food samples, including 60 fresh fruit and vegetable samples and 50 processed foodstuffs. The assay developed provides a useful tool for early detection of low concentrations of viable Alternaria spp. in naturally contaminated food samples, and could be applied as a quality and biosecurity marker of raw materials and final products in the fruits and vegetables processing industries.

Rapid and simple DNA extraction method for the detection of enterotoxigenic Staphylococcus aureus directly from food samples: comparison of PCR and LAMP methods

The study describes the development of simple and rapid DNA extraction method in combination with loop-mediated isothermal amplification (LAMP) to detect enterotoxigenic Staphylococcus aureus in food samples. In this study, isolation of genomic DNA of enterotoxigenic Staph. aureus from spiked milk, milk burfi, khoa, sugarcane juice and boiled rice was carried out by boiling the isolated sample pellets for 10 min with 1% Triton X-100. The isolated DNA was evaluated by polymerase chain reaction (PCR) and LAMP method. The LAMP was found to be 100 times more sensitive than PCR. The LAMP assay was very specific for Staph. aureus, and the presence of other contaminating bacterial DNAs and food matrix did not interfere or inhibit the LAMP assay. The template DNA extraction method developed in this study for food samples is simple, rapid and cost-effective. LAMP was found to be less sensitive to matrix effect of food, compared to PCR. The method is suitable for direct detection of Staph. aureus without any enrichment in contaminated food samples and hence finds its application in food safety analysis, in permutation with LAMP.

Potential Function of a Novel Protective Culture Enterococcus faecium-MC13 Isolated From the Gut of Mughil cephalus: Safety Assessment and its Custom as Biopreservative

The effects of protective culture Enterococcus faecium MC-13 on the shelf life of beheaded, scaled and gutted oil sardines (Sardinella longiceps) and tiger shrimp (Penaeus monodon) during refrigerated storage were examined. The control and the treated samples were analyzed periodically for chemical (PV, TVB-N), microbiological (total viable count), and sensory characteristics. The in vivo safety following oral administration to wistar rats was investigated before its usage as a biopreservative. By the end of the experiment, the lowest total volatile base microbial load was observed in E. faecium inoculated seafood samples and the highest in controls. Addition of protective culture to the artificially contaminated food sample in the assayed storage condition inhibited Listeria monocytogenes by 1.3 and 1.17 log CFU/g in Penaeus monodon and Sardinella longiceps, respectively. Our preliminary results validate the feasibility of using Enterococcus faecium as a possible candidate for use as a starter culture in increasing the quality and nutritive value in seafood.

Development of a PCR Protocol To Detect Aflatoxigenic Molds in Food Products

Aflatoxins are secondary metabolites produced mainly by Aspergillus species growing in foodstuffs. Because aflatoxins have important health effects, the detection of early contamination of foods by aflatoxigenic molds should be useful. In the present work, a reliable conventional PCR method for detecting aflatoxigenic molds of various species was developed. Fifty-six aflatoxigenic and nonaflatoxigenic strains commonly reported in foodstuffs were tested. Aflatoxin production was first confirmed by micellar electrokinetic capillary electrophoresis or/and high-pressure liquid chromatography-mass spectrometry. Based on the conserved regions of the O-methyltransferase gene (omt-1) involved in the aflatoxin biosynthetic pathway, six primer pairs were designed. With only the designed primer pair AFF1-AFR3, the expected PCR product (381 bp) was obtained in all of the tested aflatoxigenic strains of various species and genera. Amplification products were not obtained with this primer pair for any of the nonaflatoxigenic reference molds. However, an amplicon of 453 bp was obtained for all aflatoxigenic and nonaflatoxigenic mold reference strains with a PCR protocol based on the constitutive fungal β-tubulin gene, which was used as a positive fungal control. The PCR protocol based on omt-1 detected as little as 15 pg of DNA from aflatoxigenic molds and 10(2) to 10(3) CFU/g in contaminated food samples. This PCR protocol should be used as a routine technique to detect aflatoxigenic molds in foods.

Real-time PCR with internal amplification control for the detection of Cronobacter spp. (Enterobacter sakazakii) in food samples

Cronobacter spp. (Enterobacter sakazakii) is an opportunistic pathogen and is linked with life-threatening infections in neonates. The organism has been isolated from a wide variety of foods and environments. In this study, a Taqman real-time PCR assay incorporating an internal amplification control (IAC) was developed and evaluated for specific detection of Cronobacter spp. in foods. Previously reported macromolecular synthesis (MMS) operon sequence was selected for specificity, and 67 bacterial strains, including four strains of Cronobacter spp., were evaluated. All Cronobacter strains were successfully identified, however no cross-reactivity was observed with non-Cronobacter strains. Detection limit of the assay in pure culture and formula infant without enrichment was 1.2 × 103 CFU/ml (1.2 × 101 CFU/assay). After 24 h enrichment in broth, as few as 100 CFU/ml or g of Cronobacter could be detected in artificially contaminated food samples (infant formula, sterilized milk and chicken meat). The detection limit of the real-time PCR assay however remained unaffected in the presence of 108 CFU/ml Salmonella typhimurium in another analysis. A total of 92 food samples were analyzed for the presence of Cronobacter, out of which two pork samples were found as positive by real-time PCR, whereas only one was detected by the ISO standard method. The adjusted real-time PCR assay can be adopted to rapidly detect Cronobacter spp. in food samples with high specificity and sensitivity, and can prevent false negative results by using the IAC.

Zearalenone removal in synthetic media and aqueous part of canned corn by montmorillonite K10 and pillared montmorillonite K10.

The capacities of montmorillonite K10 (K10), aluminum pillared K10 (Al-K10), and iron pillared K10 (Fe-K10) to eliminate zearalenone (ZEN) from synthetic media and the aqueous part of canned corn were studied. Original clay and pillared clays were characterized in terms of X-ray powder diffraction analysis and N(2) adsorption-desorption isotherms. The maximum amounts of adsorption of ZEN by K10, Al-K10, and Fe-K10 at 25°C and pH 7 were 0.202, 1.305, and 1.028 mg/g and 0.264, 0.096, and 0.255 mg/g, calculated from Langmuir and Freundlich isotherms, respectively. The adsorption of ZEN was also studied as a function of adsorbent amount (1 to 30 mg), ZEN concentration (2 to 20 mg/liter), pH of solution (pH 4 to 10), and contact time. Pillared clays could be an excellent alternative for removing ZEN in contaminated food samples and are potentially low-cost adsorbents with a promising future as an alternative to more costly materials.

Rapid and sensitive detection of Vibrio vulnificus by loop-mediated isothermal amplification combined with lateral flow dipstick targeted to rpoS gene

A novel loop-mediated isothermal amplification (LAMP) combined with amplicon detection by chromatographic lateral flow dipstick (LFD) assay was developed and evaluated for the detection of Vibrio vulnificus. Biotinylated LAMP amplicons were produced by a set of six designed primers that recognized the V. vulnificus RNA polymerase subunit sigma factor S ( rpoS) gene followed by hybridization with an FITC-labeled probe and LFD detection. The optimized time and temperature conditions for the LAMP assay were 90 min at 65 °C. The LAMP–LFD method accurately identified 14 isolates of V. vulnificus but did not detect 25 non- vulnificus Vibrio isolates and 37 non -Vibrio isolates. The sensitivity of LAMP–LFD for V. vulnificus detection in pure culture was 1.5 × 10 3 CFU ml −1 or equivalent to 2.8 CFU per reaction. In the case of spiked oyster samples without enrichment, the detection limit for V. vulnificus was 1.2 × 10 4 CFU g −1 or equivalent to 11 CFU per reaction. The results show that this method appears to be accurate, precise and valuable tool for identification of V. vulnificus and can be used efficiently for detection of V. vulnificus in contaminated food sample.

The development of loop-mediated isothermal amplification combined with lateral flow dipstick for detection of Vibrio parahaemolyticus

The current study was aimed to develop a loop-mediated isothermal amplification (LAMP) combined with amplicon detection by chromatographic lateral flow dipstick (LFD) assay for rapid and specific detection of Vibrio parahaemolyticus. Biotinylated LAMP amplicons were produced by a set of four designed primers that recognized specifically the V. parahaemolyticus thermolabile haemolysin (tlh) gene followed by hybridization with an FITC-labelled probe and LFD detection. The optimized time and temperature conditions for the LAMP assay were 90 min at 65 °C. The LAMP-LFD method accurately identified 28 isolates of V. parahaemolyticus but did not detect 24 non-parahaemolyticus Vibrio isolates and 35 non-Vibrio bacterial isolates. The sensitivity of LAMP-LFD for V. parahaemolyticus detection in pure cultures was 120 CFU ml⁻¹. In the case of spiked shrimp samples without enrichment, the detection limit for V. parahaemolyticus was 1·8 x 10³ CFU g⁻¹ or equivalent to 3 CFU per reaction while that of conventional PCR was 30 CFU per reaction. The established LAMP-LFD assay targeting tlh gene was specific, rapid and sensitive for identification of V. parahaemolyticus. The developed LAMP-LFD assay provided a valuable tool for detection of V. parahaemolyticus and can be used effectively for identification of V. parahaemolyticus in contaminated food sample.

Flower-shaped PdI2nanomaterials with remarkable surface-enhanced Raman scattering activity

Uniform dispersed flower-shaped palladium iodide (PdI2) nanoparticles were prepared successfully by a simple wet chemical method. The as-synthesized products were structurally and morphologically characterized by X-ray diffraction (XRD), scanning electron microscopy (SEM), transmission electron microscopy (TEM), as well as high resolution transmission electron microscopy (HRTEM). Large numbers of twin and stacking faults were observed at the tip of the petals of flower-shaped morphology. At the same time, a series of palladium iodide structures with different morphology and size were obtained through the manipulation of the concentration of KI and the reaction temperature. The formation of typical flower-shaped palladium iodide was correlated with their reaction conditions. The as-produced PdI2 nanoparticles exhibit high surface-enhanced Raman scattering (SERS) activities for malachite green (MG) probe molecules with 1.0 × 10−7 M concentration. As a result, the flower-shaped PdI2 nanostructures coupled with SERS hold a great potential as a rapid and ultra-sensitive agent for detecting trace amounts of prohibited substances in contaminated food samples.

Listeria spp. in Street-Vended Ready-to-Eat Foods.

Street-vended ready-to-eat food sold in Egypt, including sandwiches and dishes of traditional food, was examined for the presence of Listeria species. Out of 576 samples, 24% were found to contain Listeria species. L. monocytogenes and L. innocua were isolated from 57% and 39% of the contaminated samples, respectively. Other Listeria spp. were detected with lower frequency. L. monocytogenes of ≥103 CFU/g were detected in 7% of the total examined samples, which represent 49% of the contaminated food samples (meat, poultry, seafood, dairy products, and products of plant origin). Most of the samples contaminated by L. monocytogenes had high levels of total viable bacterial counts. The results obtained may help to clarify the epidemiology of listeriosis in the country and draw the attention of the decision makers to issue hygienic regulations for food processing industries as well as street vendors in order to ensure safe street-vended ready-to-eat food.

Anti-listerial activity of coatings entrapping living bacteria

Polyvinyl alcohol (PVOH) based coatings entrapping either living bacteriocin-producer Enterococcus casseliflavusIM 416K1 bacteria or Enterocin 416K1 have been prepared and applied to poly(ethylene terephthalate) (PET) films. The antimicrobial activity of coated PET films was evaluated against Listeria monocytogenes NCTC 10888 by qualitative agar diffusion assays and by direct contact with artificially contaminated food samples (wurstel and seasoned cheese) stored at 4 °C and 22 °C. Anti-listerial activity of both coatings was observed for both tests. However, the live-enterococcus doped coatings showed a much more remarkable anti-listerial activity than enterocin doped ones. Interestingly, live-enterococcus doped coatings lead to a strong decrease of L. monocytogenes viable counts even at 22 °C, indicating that they are able to contrast efficiently the fast L. monocytogenesgrowth occurring at this temperature in wurstel samples. In this respect, they can be considered smart coatings, being able to be responsive towards an accidental rise of temperature during food storage. The capability of bacteria to survive for a long time can also assure a long lasting antibacterial activity.

Compact DryR X-BC for the Enumeration of Bacillus cereus in Food Samples

We evaluated the effectiveness of using Compact Dry(R) X-BC (CD-XBC), a ready-to-use and self-diffusing dry medium sheet culture system based on a novel detection principle, for the detection and enumeration of Bacillus cereus. All 13 B. cereus strains, which were studied for the inclusivity study, grew as blue/green colonies on the CD-XBC. When 3 yeast strains and 103 bacterial strains other than B. cereus were tested for the exclusivity study, 5 strains formed white colonies, and 4 strains formed blue/green colonies, while 94 other strains failed to grow. The 4 strains that formed blue/green colonies were B. thuringiensis, which is known to have the same biochemical features as B. cereus. The CD-XBC method was compared with the MYP agar method (MYP) and the NGKG agar method (NGKG) in 130 artificially contaminated food samples. The correlation coefficients between CD-XBC and MYP, and CD-XBC and NGKG were 0.972 and 0.971, respectively.

Design of an imprinted clean-up method for mycophenolic acid in maize

In the present work, the development of imprinted polymers selective towards mycophenolic acid and their application in food analysis are reported for the first time. To synthesize the molecularly imprinted polymer (MIP) 4-vinylpyridine and ethyleneglycol dimethacrylate were applied as functional monomer and cross-linker, respectively. Besides the toxin itself, the implementation of structural analogues as templates was evaluated. A molecularly imprinted solid-phase extraction (MISPE) procedure was designed for the selective clean-up of maize extracts. Binding experiments and Scatchard analysis indicated the presence of specific binding sites in the imprinted polymers. The imprinting effect varied along with the selected template. The dissociation constant ( K D) of the higher affinity binding sites ranged from 0.8 μmol/l to 15.6 μmol/l, while the K D of the lower affinity binding sites was in the range of 138.5–519.3 μmol/l. The performance of the MIPs throughout the clean-up of spiked maize sample extracts was evaluated and compared with the results obtained when applying a non-imprinted polymer. Depending on the polymers and the spiked concentration, recoveries after MISPE and non-imprinted solid-phase extraction varied respectively from 49% to 84% and from 28% to 31%. The imprinted polymers were superior regarding matrix effect, limit of detection (LOD) and limit of quantification (LOQ). LOD ranged from 0.17 μg/kg to 0.25 μg/kg and LOQ varied from 0.57 μg/kg to 0.82 μg/kg. Analysis of 15 maize samples by liquid chromatography tandem mass spectrometry revealed that the MIPs could be excellent sorbents for clean-up of contaminated food samples.

Performance characteristics of the Duopath® Cereus Enterotoxins assay for rapid detection of enterotoxinogenic Bacillus cereus strains

The Duopath® Cereus Enterotoxins test (Merck KGaA) is a newly developed gold-labeled lateral flow immunoassay for the detection of Bacillus cereus enterotoxins. The test uses monoclonal antibodies (MAbs) against the L 2 component of hemolysin BL (Hbl) and NheB of the non-hemolytic enterotoxin (Nhe), respectively. The inclusivity and exclusivity of the assay was tested using 44 B. cereus, B. cereus group and Bacillus spp. strains. Apart from the B. mycoides type strain the results were in full agreement with those obtained by other immunological and molecular biological methods. The detection limit of the assay was 6 ng/ml for NheB and 20 ng/ml for the Hbl-L 2-component, respectively. Using artificially and naturally contaminated food samples (n = 76) the assay was positive after 18–24 h enrichment if at least 10 2 enterotoxin producing B. cereus/g were present. After 30 h enrichment samples contaminated with as low as 1 enterotoxin producing B. cereus/g gave positive results. In addition, testing of suspected colonies for enterotoxin production is possible. The assay is easy to perform and results can be clearly read without instrumentation.

Rapid separation and immunoassay for low levels of Salmonella in foods using magnetosome–antibody complex and real‐time fluorescence quantitative PCR

A rapid and economical method for detecting Salmonella was developed, based on a novel complex for immunomagnetic separation, which was composed of anti-Salmonella polyclonal antibody (Ab) and magnetosome (bacterial magnetic particle, BMP) produced by the bacterium Magnetospirillum gryphiswaldense MSR-1. BMP-Ab complex was used to capture Salmonella from pure suspensions of S. dublin, S. enteritidis, S. aesch, S. agona, S. abony and S. bareily, from mixed suspensions of S. dublin and Vibrio parahaemolyticus, and from artificially contaminated food samples. Captured Salmonella were then detected by plate count, or real-time fluorescence quantitative PCR. Capture efficiencies, calculated from plate count, were >80% for the pure Salmonella suspensions of all six strains, and >70% for the mixed suspension. Samples of six food products, with artificial contamination by 6000, 600, 60, or 0.6 cfu/mL S. dublin, were captured by complex and detected by real-time fluorescence quantitative PCR. Threshold cycle values varied depending on type of food. The lower limit of detectability was 60 cfu/mL without pre-enrichment, and <0.6 cfu/mL after 3-h pre-enrichment. The method described here, based on capture pathogens by BMP-Ab complex, is sensitive, rapid, and considerably simpler than traditional methods for Salmonella detection. It can be extended to other pathogens by the use of appropriate antibodies.

A multiparametric PCR-based tool for fast detection and identification of spore-forming bacteria in food

The presence of psychrotrophic or highly thermoresistant spore-forming bacteria in food and feedstuff responsible for food poisoning and spoilage raises major safety and economical issues. The aim of this study was to evaluate the performances of a ready-to-use PCR assay (alternative method) in comparison with the standard microbiological plating method regarding spore-forming bacteria detection in food samples. An overnight sample enrichment was selected to increase sporeformer diversity recovery, spore germination, bacterial growth and favour DNA extraction. A total of 180 sporeformer isolates representing 38 different species and 8 genera were tested in the PCR assays. Inclusivity and exclusivity results ensured specific detection and identification of the majority of targeted genera and species. Validation studies carried on artificially contaminated food samples showed detection of the inoculated contaminants in most cases, with increased detection limit for the alternative method which enabled detection with up 1 spore of B. cereus in 25 g food sample. Using naturally contaminated food samples, standard method comforted the alternative method. In a number of cases, the alternative method was able to identify species not detected with the standard method. In addition, identification and discrimination between the B. cereus group members was possible. Thus, associated to a key element, i.e., the enrichment step, the developed multiparametric PCR-based assays reported in this study provide a fast, sensitive and reliable detection and identification tool for mostly encountered spore-forming food contaminants.

Synthesis and application of a T-2 toxin imprinted polymer

The synthesis of a T-2 toxin imprinted polymer and its application in food analysis are reported for the first time. A molecularly imprinted polymer (MIP) for the selective recognition of T-2 toxin (T-2) was synthesized by bulk polymerization. Methacrylamide and ethyleneglycol dimethacrylate were applied as functional monomer and cross-linker, respectively. Molecularly imprinted solid-phase extraction (MISPE) procedures were optimized for further application in the analysis of T-2. Scatchard plot analysis revealed that two classes of imprinted binding sites were formed in the imprinted polymer. The dissociation constant ( K D) of the higher affinity binding sites was 7.0 μmol/l, while the K D of the lower affinity binding sites was 54.7 μmol/l. The performance of the MIP throughout the clean-up of spiked maize, barley and oat sample extracts was compared with the results obtained when using non-imprinted polymer, OASIS HLB ® and immunoaffinity columns (IAC). Depending on the food matrix and the spiked concentration, recoveries after MISPE and non-imprinted solid-phase extraction varied respectively from 60% to 73% and from 21% to 57%. Recoveries obtained after clean-up using OASIS HLB ® and IAC were in the range of 74–104% and 60–85%, respectively. Although highest recoveries were obtained with OASIS HLB ® sorbents, the designed MIP and the IAC were superior regarding selectivity, cross-reactivity, matrix effect, limits of detection (LOD) and limits of quantification (LOQ). Depending on the matrix, LOD after MISPE ranged from 0.4 μg/kg to 0.6 μg/kg and LOQ from 1.4 μg/kg to 1.9 μg/kg. LOD and LOQ after OASIS HLB ® clean-up varied from 0.9 μg/kg to 3.5 μg/kg and from 3.1 μg/kg to 11.7 μg/kg, respectively. The LOD and LOQ values obtained with IAC were in the range of 0.3–2.3 μg/kg and 1.0–7.7 μg/kg, respectively. Analysis of 39 naturally contaminated samples (maize, barley and oat) by liquid chromatography tandem mass spectrometry revealed that the MIP could be an excellent alternative for clean-up of contaminated food samples.

Evaluation of the Compact Dry X-SA Method for Enumerating Staphylococcus aureus in Artificially Contaminated Food Samples

Compact Dry X-SA (CD-XSA), a ready-to-use and self-diffusible dry medium sheet culture system for the detection and enumeration of Staphylococcus aureus, was evaluated. A total of 50 S. aureus strains, which were studied for the inclusivity study, grew as blue-colored colonies on the CD-XSA. When 114 bacteria other than S. aureus and 3 yeasts were inoculated for the exclusivity study, 37 strains produced white colonies, and 4 strains produced blue colonies, and 3 strains produced magenta colonies, while 73 other strains failed to grow. The CD-XSA method was compared with the mannitol salt agar with egg yolk (MSEY) method, the Baird-Parker agar (BP) method and the 3M Petrifilm(TM) STX (3M-STX) method in 105 artificially contaminated food samples. The correlation coefficients between CD-XSA and MSEY, CD-XSA and BP, and CD-XSA and 3M-STX were 0.945, 0.960 and 0.977, respectively.