Rapid detection of Listeria monocytogenes in food using culture enrichment combined with real-time PCR

A rapid method for the detection of Listeria monocytogenes in foods combining culture enrichment and real-time PCR was compared to the ISO 11290-1 standard method. The culture enrichment component of the rapid method is based on the ISO standard and includes 24 h incubation in half-Fraser broth, 4 h incubation in Fraser broth followed by DNA extraction and real-time PCR detection of the ssrA gene of L. monocytogenes. An internal amplification control, which is co-amplified with the same primers as the L. monocytogenes DNA, was also included in the assay. The method has a limit of detection of 1–5 CFU/25 g food sample and can be performed in 2 working days compared to up to 7 days for the ISO standard. A variety of food samples from retail outlets and food processing plants (n = 175) and controls (n = 31) were tested using rapid and conventional methods. The rapid method was 99.44% specific, 96.15% sensitive and 99.03% accurate when compared to the standard method. This method has the potential to be used as an alternative to the standard method for food quality assurance providing rapid detection of L. monocytogenes in food.     

Rapid real-time PCR detection of Listeria monocytogenes in enriched food samples based on the ssrA gene, a novel diagnostic target

A real-time PCR assay was designed to detect a 162-bp fragment of the ssrA gene in Listeria monocytogenes. The specificity of the assay for L. monocytogenes was confirmed against a panel of 6 Listeria species and 26 other bacterial species. A detection limit of 1-10 genome equivalents was determined for the assay. Application of the assay in natural and artificially contaminated culture enriched foods, including soft cheese, meat, milk, vegetables and fish, enabled detection of 1-5 CFU L. monocytogenes per 25g/ml of food sample in 30h. The performance of the assay was compared with the Roche Diagnostics 'LightCycler foodproof Listeria monocytogenes Detection Kit'. Both methods detected L. monocytogenes in all artificially contaminated retail samples (n=27) and L. monocytogenes was not detected by either system in 27 natural retail food samples. The method developed in this study has the potential to enable the specific detection of L. monocytogenes in a variety of food types in a time-frame considerably faster than current standard methods. The potential of the ssrA gene as a nucleic acid diagnostic (NAD) target has been demonstrated in L. monocytogenes. We are currently developing NAD tests based on the ssrA gene for a range of common foodborne and clinically relevant bacterial pathogens.     

A stochastic approach for integrating strain variability in modeling Salmonella enterica growth as a function of pH and water activity

Strain variability of the growth behavior of foodborne pathogens has been acknowledged as an important issue in food safety management. A stochastic model providing predictions of the maximum specific growth rate (μ_max) of Salmonella enterica as a function of pH and water activity (a_w) and integrating intra-species variability data was developed. For this purpose, growth kinetic data of 60 S. enterica isolates, generated during monitoring of growth in tryptone soy broth of different pH (4.0-7.0) and a_w (0.964-0.992) values, were used. The effects of the environmental parameters on μ_max were modeled for each tested S. enterica strain using cardinal type and gamma concept models for pH and a_w, respectively. A multiplicative without interaction-type model, combining the models for pH and a_w, was used to describe the combined effect of these two environmental parameters on μ_max. The strain variability of the growth behavior of S. enterica was incorporated in the modeling procedure by using the cumulative probability distributions of the values of pH_min, pH_opt and a_wmin as inputs to the growth model. The cumulative probability distribution of the observed μ_max values corresponding to growth at pH 7.0-a_w 0.992 was introduced in the place of the model's parameter μ_opt. The introduction of the above distributions into the growth model resulted, using Monte Carlo simulation, in a stochastic model with its predictions being distributions of μ_max values characterizing the strain variability. The developed model was further validated using independent growth kinetic data (μ_max values) generated for the 60 strains of the pathogen at pH 5.0-a_w 0.977, and exhibited a satisfactory performance. The mean, standard deviation, and the 5th and 95th percentiles of the predicted μ_max distribution were 0.83, 0.08, and 0.69 and 0.96 h^− 1, respectively, while the corresponding values of the observed distribution were 0.73, 0.09, and 0.61 and 0.85 h^− 1. The stochastic modeling approach developed in this study can be useful in describing and integrating the strain variability of S. enterica growth kinetic behavior in quantitative microbiology and microbial risk assessment.     

Prevalence and characterization of Salmonella enterica serovar Weltevreden from imported seafood

During 2001-2005, 210 Salmonella enterica strains were isolated from seafood samples imported into US. Strains of S. enterica serovar Weltevreden were the most predominantly found among the 64 different serovars isolated. A total of 37 Salmonella Weltevreden isolates were characterized by pulsed-field gel electrophoresis (PFGE), plasmid profiles and antibiotic susceptibility to assess genetic diversity. Our results showed a low frequency of antibiotic resistance; 35 of the 37 isolates were sensitive to ampicillin, tetracycline, chloramphenicol, gentamicin, sulfisoxazole, streptomycin and kanamycin. Only two isolates, from samples originating in the Philippines and India, showed resistance to ampicillin and tetracycline and to streptomycin, sulfisoxazole and tetracycline, respectively. Of the 37 isolates, two isolates did not carry any plasmid and 35 isolates harbored several small and mega-plasmids. These isolates were differentiated into 10 distinct types based on plasmid profiles. Four different PFGE clusters were obtained with a genetic similarity of 66-76%. Four groups of isolates (formed by two or three isolates each) showed 100% similarity in the PFGE profiles. One of these groups included strains isolated in Vietnam in 2003, 2004 and 2005 from fish and shrimp. The other groups included strains isolated in Vietnam, Indonesia and Thailand in 2000, 2004 and 2005 from snail, shrimp and fish. Our findings show genetic diversity and temporal persistence of S. enterica serovar Weltevreden in recently monitored seafood imports.     

Effect of the growth environment on the strain variability of Salmonella enterica kinetic behavior

Intra-species variability of microbial growth kinetic behavior is an event with important implications for food safety research. Aiming at the evaluation of the growth variability among Salmonella enterica strains as affected by the growth environment, the kinetic behavior of 60 isolates of the pathogen was assessed at 37 °C in tryptone soy broth of different pH values (4.3-7.0) and NaCl concentrations (0.5-6.0%). Maximum specific growth rate (μ_max) values corresponding to each strain and growth condition were estimated by means of absorbance detection times of serially decimally diluted cultures using the automated turbidimetric system Bioscreen C. A total of 9600 optical density curves were generated for the strains and the growth conditions tested. The variability of μ_max among the S. enterica strains was important and greater than that observed within the strains (i.e. among replicates). Moreover, strain variability increased as the growth conditions became more stressful both in terms of pH and NaCl. The coefficient of variation of μ_max among the tested strains at pH 7.0-0.5% NaCl was 6.1%, while at pH 4.3-0.5% NaCl and pH 7.0-6.0% NaCl was 11.8% and 23.5%, respectively. Beyond the scientific interest in understanding strain variability, the findings of this study should be useful in strain selection for exploitation in food safety challenge studies as well as in incorporating strain variability in predictive microbiology and microbial risk assessment.     

Detection of changes in the cellular composition of Salmonella enterica serovar Typhimurium in the presence of antimicrobial compound(s) of Lactobacillus strains using Fourier transform infrared spectroscopy

It was previously established that Lactobacillus fermentum ACA-DC 179, Lactobacillus plantarum ACA-DC 287 and Lactobacillus plantarum ACA-DC 2350 exhibit antimicrobial activity against Salmonella enterica serovar Typhimurium. In order to further investigate the killing effect of these microorganisms against Salmonella cells, we employed Fourier transform infrared spectroscopy (FT-IR). Salmonella cells were incubated with different concentrated lactobacilli supernatants and their FT-IR spectra were recorded. The second derivative transformation of the original spectra revealed changes in spectral regions corresponding to absorptions of major cellular constituents (e.g. cell wall, cell membrane, and proteins of the cell) among the Salmonella cells treated with the supernatants and those treated with the control samples. Principal component analysis of the second derivative transformed spectra showed that the yet unidentified antimicrobial compound(s) produced by the lactobacilli tested clearly interfered with the fatty acids of the cell membrane, as well as the polysaccharides of the cell wall in Salmonella cells, pointing towards a dual killing mode. Our study shed light for the first time in the anti-Salmonella activity of the particular Lactobacillus strains.     

Reduction of Salmonella enterica on grape tomatoes using microwave heating

Grape tomatoes were surface inoculated with Salmonella enterica serovars Typhimurium, Senftenburg, Kentucky and Enteritidis and heated for 10, 20, 30, 40 and 50 s using a household microwave oven at two different power levels (medium and high). Following heating, viable counts, temperature measurements and quality measurements were performed on the tomatoes. At high power level, more than 2 log reduction of Salmonella enterica was detected on grape tomatoes after 50 s but the texture were damaged. Three heating treatments, 40 s heating at high power level, 40 and 50 s heating at medium power level, could achieve more than 1.45 log reduction of Salmonella enterica on grape tomatoes, and all the treatments except for 50 s at high power level did not affect the color, pH value and nutritional quality of grape tomato after heating (p > 0.05). However, 40 s heating at medium power was the only treatment among the three that did not affect the texture quality of grape tomato. Therefore, it might be a potential way for consumers to use microwave heating at medium power level (700 W) for 40 s to reduce Salmonella population on water immersed grape tomatoes.     

Development of multiplex real-time PCR with Internal amplification control for simultaneous detection of Salmonella and Cronobacter in powdered infant formula

Contamination of powdered infant formula (PIF) by the bacteria Cronobacter spp. and Salmonella enterica was deemed a matter of great concern by the World Health Organization and the Food and Agriculture Organization of the United Nations in 2004. Therefore, we developed a rapid and sensitive multiplex real-time PCR assay for the simultaneous detection of Cronobacter and Salmonella in PIF. In addition, an internal amplification control (IAC) was also included for exclusion of false negative results in this study. The quantitative detection range for pure cultures in this optimized multiplex real-time PCR assay was 10³ to 10⁸ CFU/ml for both Salmonella and Cronobacter. When our established multiplex real-time PCR system was applied to artificially contaminated PIF, the detection limit was 10³ CFU/ml for Salmonella and Cronobacter without enrichment. The commercial PIF was then inoculated with Salmonella and Cronobacter at 10, 1 and 0.1 CFU per gram of formula and the single enrichment broth samples were analyzed by multiplex real-time PCR after enrichment for 9, 12, and 24 h. At 12 h post-enrichment, we could detect Salmonella and Cronobacter at initial inoculation levels of approximately 0.1 CFU/g in PIF. Additionally, stable fluorescent IAC signals could be assessed between 29 and 34 cycles of PCR amplification. Results from this study showed that the multiplex real-time PCR assay is an effective method for the rapid and simultaneous detection and quantification of Cronobacter and Salmonella in PIF.     

Identification and molecular characterization of Salmonella spp. from unpasteurized orange juices and identification of new serotype Salmonella strain S. enterica serovar Tempe

Several Salmonella enterica serotypes were isolated from unpasteurized orange juice samples analysed as a follow-up to an outbreak in 1999 of S. enterica serotype Muenchen in the Pacific Northwest regions of United States. Eleven S. enterica strains were serotyped and identified as S. enterica serotype Muenchen (2), S. enterica serotype Hidalgo (2), S. enterica serotype Alamo (1), S. enterica serotype Gaminera (2), S. enterica serotype Javiana (2) and a new serotyped strain S. enterica serotype Tempe (2). The identity of the new serotype S. enterica serovar Tempe serotype 30:b:1,7:z33 was confirmed by the National Salmonella Reference Laboratory at NCID/CDC, Atlanta. These strains were sensitive to ampicillin, chloramphenicol, kanamycin, tetracycline, streptomycin and sulfisoxazole antibiotics. Isolates were screened for invasion (invA) and virulence (spvC) genes using specific primers for these two genes by polymerase chain reaction. All strains were positive for invA gene giving 321-bp fragment, however negative to virulence spvC gene. For pulsed-field gel electrophoresis (PFGE) analysis, Salmonella strain plugs were made and digested with XbaI and subjected to 18-h electrophoresis. The PFGE patterns were different for each S. enterica serotypes suggesting the several origins of contamination in outbreak. S. enterica serotype.     

Antimicrobial activity of different copper alloy surfaces against copper resistant and sensitive Salmonella enterica

Copper has shown antibacterial effects against foodborne pathogens. The objective of this study was to evaluate the antibacterial activity of copper surfaces on copper resistant and sensitive strains of Salmonella enterica. Six different copper alloy coupons (60–99.9% copper) were tested along with stainless steel as the control. The coupons were surface inoculated with either S. Enteritidis or one of the 3 copper resistant strains, S. Typhimurium S9, S19 and S20; stored under various incubation conditions at room temperature; and sampled at various times up to 2 h. The results showed that under dry incubation conditions, Salmonella only survived 10–15 min on high copper content alloys. Salmonella on low copper content alloys showed 3–4 log reductions. Under moist incubation conditions, no survivors were detected after 30 min–2 h on high copper content alloys, while the cell counts decreased 2–4 logs on low copper content coupons. Although the copper resistant strains survived better than S. Enteritidis, they were either completely inactivated or survival was decreased. Copper coupons showed better antimicrobial efficacy in the absence of organic compounds. These results clearly show the antibacterial effects of copper and its potential as an alternative to stainless steel for selected food contact surfaces.     

Development and validation of a novel real-time PCR method for the detection of celery (Apium graveolens) in food

The paper presents a novel real-time PCR method allowing the detection of traces of celery (Apium graveolens) in complex food matrices. The method is based on the amplification of a sequence of the gene coding for the Apium graveolens NADPH-dependent mannose-6-phosphate reductase. It allows the detection of three commonly used celery varieties, celery roots (Apium graveolens var. rapaceum), celery stalks (Apium graveolens var. dulce) and leaf celery (Apium graveolens var. secalinum) and does not show any cross-reactivity with 64 biological species, including ten members of the Apiaceae family. The limit of detection, determined by analysing serially diluted celery extracts, is 10 pg celery DNA for all three celery varieties. In spiked model sausages, the LOD is 0.005% celery. The real-time PCR method was applied to 26 commercial food products. Celery DNA was found in one out of ten samples without any information about the presence of celery.     

Development and validation of a real-time PCR assay specific for Clostridium estertheticum and C. estertheticum-like psychrotolerant bacteria

A new real-time PCR assay was developed targeted to the psychrotolerant spoilage bacteria, Clostridum estertheticum, a causative agent of 'blown-pack' spoilage of vacuum packaged meats during chilled storage. Further, a robust validation of the sensitivity and specificity in different meat processing related matrices was carried out. Results show that real-time PCR is a valid method for the detection of C. estertheticum spores as long as consideration is given to the matrix being tested and the sensitivity of detection required. For meat, hide, blood/drip and environmental swabs it was possible to detect low numbers of C. estertheticum spores (approx 3 spores per ml) by direct real-time PCR (without pre-enrichment of the samples). For faeces and soil matrices, a cold temperature enrichment step was required prior to DNA extraction and real-time PCR analysis to increase the ability to detect samples containing C. estertheticum spores; this was particularly important when the samples contained low numbers of spores (less than 3 spores per ml). For matrices with high levels of PCR inhibitors such as soil, it was necessary to dilute the extracted DNA sample 100 fold especially for detection of high levels of contamination (greater than 10(3)per ml) otherwise a pre-enrichment was required.     

Characterization of the quinolone resistance mechanism in foodborne Salmonella isolates with high nalidixic acid resistance

Sixteen Salmonella strains resistant to nalidixic acid isolated from kimbab, the most popular ready-to-eat (RTE) food in Korea, and chicken meat were selected for this study. The resistant strains were shown to have high minimal inhibitory concentrations (MICs) against nalidixic acid (512 ~ 4096 μg/mL). Among them, 4 Salmonella enterica serovar Haardt isolates showed multi-drug resistance (MDR) patterns with reduced susceptibility to fluoroquinolone (0.5 μg/mL of ciprofloxacin MICs). The mechanisms of quinolone resistance in the nalidixic acid resistant strains were characterized by PCR and sequence analysis. The presence of plasmid-mediated quinolone resistance (PMQR) genes and amino acid changes in the quinolone resistance determining region (QRDR) were investigated by PCR-based detection and sequencing, and the efflux pump inhibition test was also done using phe-arg-β-naphthylamide (PAβN). Although PMQR genes were not detected in any of the tested strains, the QRDR mutations were found in this study: single mutation in gyrA (Asp87Tyr, Asp87Gly, and Asp87Asn), double mutations in gyrA (Ser83Thr) and parC (Thr57Ser), and single mutation in parC (Thr57Ser). MICs of nalidixic acid were reduced by 2- to 32-folds by the efflux pump inhibitor, PAβN. Pulsed-field gel electrophoresis (PFGE) was carried out to confirm the epidemiological relationship between the nalidixic acid resistant strains. The PFGE patterns were classified into 6 groups at cutoff level of 70 ~ 100% correlation on the dendrogram. Some strains of serotype Haardt and Enteritidis showed several values of genomic identity in accordance with strains, sources, and isolation year. We suggest that point mutation on QRDR and efflux pump systems involved in antimicrobials had independent effects on drug-resistance regardless of bacterial genomic variation.     

Molecular characterization of Salmonella enterica serovar Saintpaul isolated from imported seafood, pepper, environmental and clinical samples

A total of 39 Salmonella enterica serovar Saintpaul strains from imported seafood, pepper and from environmental and clinical samples were analyzed for the presence of virulence genes, antibiotic resistance, plasmid and plasmid replicon types. Pulsed-field gel electrophoresis (PFGE) fingerprinting using the XbaI restriction enzyme and plasmid profiling were performed to assess genetic diversity. None of the isolates showed resistance to ampicillin, chloramphenicol, gentamicin, kanamycin, streptomycin, sulfisoxazole, and tetracycline. Seventeen virulence genes were screened for by PCR. All strains were positive for 14 genes (spiA, sifA, invA, spaN, sopE, sipB, iroN, msgA, pagC, orgA, prgH, lpfC, sitC, and tolC) and negative for three genes (spvB, pefA, and cdtB). Twelve strains, including six from clinical samples and six from seafood, carried one or more plasmids. Large plasmids, sized greater than 50 kb were detected in one clinical and three food isolates. One plasmid was able to be typed as IncI1 by PCR-based replicon typing. There were 25 distinct PFGE-XbaI patterns, clustered to two groups. Cluster A, with 68.5% similarity mainly consists of clinical isolates, while Cluster C, with 67.6% similarity, mainly consisted of shrimp isolates from India. Our findings indicated the genetic diversity of S. Saintpaul in clinical samples, imported seafood, and the environment and that this serotype possesses several virulent genes and plasmids which can cause salmonellosis.     

Development and evaluation of aptamer magnetic capture assay in conjunction with real-time PCR for detection of Campylobacter jejuni

A prototype method for the concentration and detection of Campylobacter jejuni was developed using a previously reported biotinylated DNA aptamer in conjunction with qPCR. The so-called aptamer-based magnetic capture-qPCR (AMC-qPCR) assay was compared to a similar immunomagnetic separation (IMS)-qPCR assay. In small volume experiments (300 μl) applied to serially diluted C. jejuni suspended in buffer containing a mixed culture of other common food borne pathogens, the lower detection limit of the AMC-qPCR method was 1.1 log₁₀/300 μl C. jejuni cells, one log₁₀ better (lower) than that of IMS-qPCR (2.1 log₁₀ CFU/300 μl). AMC-qPCR capture efficiency was 10–13% at assay detection limit. In 10 ml scale-up experiments, the lower detection limit of AMC-qPCR was 2.0 log₁₀ CFU/10 ml with corresponding capture efficiency of 4–7%. Nucleic acid aptamers are promising alternatives to antibodies for magnetic bead-based capture followed by qPCR detection.     

Prevalence and antimicrobial resistance of Salmonella serotypes isolated from poultry in Spain: comparison between 1993 and 2006

A total of 226 chicken samples (carcasses, legs, wings, necks and breasts) were obtained (73 in 1993 and 153 in 2006) from 10 retail outlets in North-Western Spain and screened for the presence of Salmonella. Isolates were subjected to serotyping, phage typing (Salmonella Enteritidis and Salmonella Typhimurium) and antimicrobial susceptibility testing (15 antimicrobials; disk diffusion method). Salmonella was detected in 40 (55%) samples in 1993 and 19 (12.4%) in 2006 (P < 0.001). The serotypes (S. Enteritidis, Salmonella Poona, Salmonella Infantis, Salmonella Newport and S. Typhimurium) and phage types (1, 4, 14b and 35 in the case of S. Enteritidis and 193 for S. Typhimurium) detected are among the main types responsible for human salmonellosis in Spain. All strains were multi-resistant (resistant to 3-13 antimicrobials). The average number of resistances per strain increased (P < 0.05) from 3.98 in 1993 to 5.00 in 2006. An increase in the incidence of resistance was observed between 1993 and 2006 for cephalothin (P < 0.01), enrofloxacin (P < 0.001) and tetracycline (P < 0.01). The decreases in the prevalence of Salmonella between 1993 and 2006 suggest that the mandatory measures introduced over the last decade in the European Union to reduce the incidence of Salmonella in poultry have apparently been successful. However, the increase in antibiotic resistance rates is of concern and constitutes a threat to public health. Because the data in this study demonstrated that chicken in North-Western Spain is a potential source of antibiotic-resistant Salmonella strains, the need of consumer education on good sanitary practices is highlighted.     

Development and validation of a duplex real-time PCR method for the simultaneous detection of celery and white mustard in food

The developed duplex real-time PCR method allows the simultaneous detection of traces of potentially allergenic white mustard (Sinapis alba) and celery roots (Apium graveolens var. rapaceum), celery stalks (A. g. var. dulce) and leaf celery (A. g. var. secalinum). The duplex assay does not show any cross-reactivity with 64 different biological species, including various members of the Brassicaceae and Apiaceae family. In raw model sausages spiked with white mustard and celery roots, the LOD was found to be 0.001% white mustard and 0.005% celery. In model sausages brewed at 75–78 °C for 15 min the LOD was found to be 0.005% white mustard and 0.005% celery. The duplex real-time PCR assay was applied to check if commercial food products are labelled in compliance with the legal regulations.     

Authentication of Atlantic salmon (Salmo salar) using real-time PCR

A real-time PCR assay based on LNA TaqMan probe technology was developed for the detection and identification of Atlantic salmon (Salmo salar). Among the advantages it is worth highlighting simplicity, rapidity, highest potential for automation and minor risk of contamination of this technique. The TaqMan real-time PCR is the currently most suitable method for screening, allowing the detection of fraudulent or unintentional mislabelling of this species. The method can be applied to all kind of products, fresh, frozen and processed products, including those undergoing intensive processes of transformation.     

Growth of Salmonella enteritidis and Salmonella typhimurium in the presence of quorum sensing signalling compounds produced by spoilage and pathogenic bacteria

The effect of acylated homoserine lactones (AHLs) and autoinducer-2 (AI-2) signalling compounds present in the cell-free culture supernatants (CFS), of Pseudomonas aeruginosa, Yersinia enterocolitica-like GTE 112, Serratia proteamaculans 00612, Y. enterocolitica CITY650 and Y. enterocolitica CITY844, on the growth of two Salmonella Enteritidis and two S. Typhimurium strains was assessed though monitoring of changes in conductance of the medium. Detection times (T_det), area and slope of conductance curves were recorded. Except for P. aeruginosa 108928, which was not found to produce AI-2, all other strains produced both AHLs and AI-2. Thereafter, aliquots (20% in the final volume) of these CFS were transferred into NZ Amine broth inoculated with ca. 10³ CFU/ml of stationary phase cultures of each Salmonella strain. While the CFS of P. aeruginosa induced a shorter detection time, i.e. acceleration of the metabolic activity, the CFS of the other microorganisms increased the detection time of Salmonella strains compared to control samples (i.e. without CFS). Results indicate that the growth of Salmonella may be affected by the presence of Quorum sensing (QS) signalling compounds and/or other novel signals existing in CFS, produced by other bacterial species and confirm the complexity of bacterial communication.