Development of a real-time PCR assay with an internal amplification control for the screening of Shiga toxin-producing Escherichia coli in foods.

AIMS: To develop and evaluate a real-time PCR assay incorporating an internal amplification control (IAC) suitable for the screening of Shiga toxin (Stx)-producing Escherichia coli (STEC) in foods. METHODS AND RESULTS: A competitive IAC was constructed and included in an stx-specific real-time PCR assay. Coupled to 18-h enrichment and automated DNA extraction, the assay could reliably detect the presence of STEC in minced meats inoculated at 10 CFU per 25 g. Its performance was evaluated on 415 minced beef and 112 raw milk cheese samples and compared with that of a PCR-ELISA method. Fifty-three minced meats and 31 cheeses were found stx-positive, giving 98.3% and 93.75% concordance, respectively, with the PCR-ELISA reference method. CONCLUSIONS: A highly sensitive stx-specific real-time PCR method including an IAC was developed, facilitating monitoring of false-negative results due to PCR inhibitors. SIGNIFICANCE AND IMPACT OF THE STUDY: Combined with automated DNA extraction, the stx-IAC real-time PCR assay represents a suitable method for rapid screening of STEC in foods.

Screening of food raw materials for the presence of Shiga toxin-producing Escherichia coli O91:H21.

AIMS: To provide information on the prevalence and detection, in foods, of Shiga toxin-producing Escherichia coli (STEC) O91:H21. METHODS AND RESULTS: Seven hundred fifteen minced beef meats and 205 raw milk samples were analysed by stx-specific PCR-ELISA. Samples positive for stx were subsequently tested for the presence of wzy-O91, fliC-H21 and the adhesin-encoding gene saa. For minced meat, 16 (2.2%) and 11 (1.5%) samples were found positive for (stx, wzy-O91, fliC-H21) and (stx, wzy-O91, fliC-H21, saa) combinations, respectively. For raw milk, seven (3.4%) samples were found positive for the (stx, wzy-O91, fliC-H21) combination but none of these contained saa. Two STEC O91:H21 saa-positive strains and three STEC O91 H21- and saa-negative strains were isolated by colony hybridization. CONCLUSIONS: A low prevalence of potentially pathogenic STEC O91:H21 in food products was found using a combination of PCR assays targeting stx, wzy-O91, fliC-H21 and saa. SIGNIFICANCE AND IMPACT OF THE STUDY: The PCR-based approach described here represents a valuable method for rapid screening of food samples contaminated by STEC O91:H21.

Use of specific oligonucleotides for direct enumeration of Listeria monocytogenes in food samples by colony hybridization and rapid detection by PCR.

Two 18-mer oligonucleotides derived from the sequence of hly, the gene coding for listeriolysin O, were shown to be specific for Listeria monocytogenes in the genus Listeria in colony hybridization tests. The oligonucleotides did not hybridize with any of the bacterial species found in food and co-isolated with Listeria on selective media. They were used in colony hybridization tests for enumeration of L. monocytogenes present in food samples after direct plating on selective media plates. In addition, two 24-mer oligonucleotides, each including the sequence of one of the 18-mers, were successfully used for the PCR-based detection of L. monocytogenes bacilli present in food samples after 48-h enrichment period. Using this technique, as little as 10(2) bacteria per ml of enrichment broth can be detected.

Comparison of a cultural method with ListerScreen plus Rapid'L.mono or PCR-ELISA methods for the enumeration of L. monocytogenes in naturally contaminated sewage sludge.

Cultural methods used to count Listeria monocytogenes in sewage sludge are laborious and time consuming, and alternative methods are needed to reduce analysis time and improve detection limits. In this study, a survey of L. monocytogenes in sewage sludge is presented with a comparative study between a cultural method and immunomagnetic separation using a ListerScreen test followed by identification of L. monocytogenes with Rapid'L.mono agar or PCR-ELISA. These two alternative methods improved the detection of L. monocytogenes in different types of sludge, irrespective of their physical and chemical characteristics. The ListerScreen method coupled with detection of L. monocytogenes on Rapid'L.mono offers the advantage of being less sophisticated than the molecular method and allows isolation of the organism, which may be useful in epidemiological studies. However, ListerScreen coupled with PCR-ELISA proved best for high-sensitivity detection of L. monocytogenes in sewage samples.

Comparison of five typing methods for the epidemiological study of Listeria monocytogenes.

Five typing methods were compared in a study designed to adapt a strategy for epidemiologically typing large numbers of Listeria monocytogenes strains. The methods studied were serotyping, electrophoretic typing of esterases (zymotyping), restriction fragment length polymorphism of ribosomal DNA (ribotyping), random amplified polymorphic DNA (RAPD) and pulsed-field gel electrophoresis (PFGE). Data were analysed by computer-assisted statistical analysis. Included in the analysis were 35 strains of L. monocytogenes, including 14 epidemic strains isolated during outbreaks in France in 1992 and 1993, and 21 strains isolated from food and the environment. Five serotypes, eight zymotypes, ten ribotypes, 13 RAPD patterns and 12 PFGE patterns were identified among the 35 strains. The most discriminating combination of typing methods was ribotyping and PFGE typing [27 types, discriminatory index (D.I.) = 0.978]. A factorial analysis of correspondence for each method differentiated the epidemic strains from the environmental strains. This study shows that computer-assisted statistical treatment of the data, combined with the use of discriminating typing methods, is a powerful tool for the epidemiological analysis of Listeria monocytogenes.

Evaluation of a polymerase chain reaction-based test for detecting Salmonella spp. in food samples: Probelia Salmonella spp.

A commercially available polymerase chain reaction (PCR) kit was evaluated for the detection of Salmonella spp. in food samples. The test combines PCR amplification and sandwich hybridization of the amplified DNA in microtiter plates. The sensitivity and specificity was evaluated with 52 Salmonella strains and 51 non-Salmonella strains and showed that the test was entirely reliable. The threshold sensitivity was 10(2) CFU/ml. The limit of detection of dead cells that determines the minimum detection level of dead cells in food samples was superior to 10(6) CFU/25 g, a level rarely achieved in naturally contaminated samples. After an 18-h pre-enrichment step, the test could detect viable Salmonella in artificially contaminated food samples, even for the lower contamination level (3 CFU/25 g). There was complete agreement between the PCR test and the ISO 6579 bacteriological reference method with artificially contaminated samples. Regarding the accuracy of the results obtained from 253 naturally or noncontaminated foods and from 32 artificially contaminated foods, the agreement percentage was 99.6%. The fidelity of the technique was evaluated in a collaborative study with eight European laboratories and showed a correlation of 98.4%.

A quantitative PCR assay for the detection and quantification of Shiga toxin-producing Escherichia coli (STEC) in minced beef and dairy products.

Shiga toxin (Stx)-producing Escherichia coli (STEC) are amongst major causes of food-borne infectious diseases and outbreaks. A new quantitative PCR (qPCR) assay was designed to detect all known stx gene subtypes in a single reaction, including the most distant variant stx2f. Performance of this assay was evaluated in combination with two different internal amplification controls (IAC), a competitive one specific for the assay and a universal IAC based on plasmid pUC19. The qPCR assay was 100% specific and showed analytical sensitivity of two STEC genome copies per reaction. The diagnostic approach proposed, combining enrichment, automated DNA extraction and qPCR detection, could reliably detect the presence of STEC in minced beef and cheese inoculated before enrichment at <4 CFU per 25 g. A comparative study performed on 240 minced beef and 113 raw milk cheese samples demonstrated that the method developed was as effective as two PCR screening assays used routinely in our laboratory to detect STEC. The new assay also proved to be appropriate for the direct quantification of STEC in milk and minced meat. It was found to be quantitative over a five log dynamic range, from 4 × 106 to 40 CFU/mL for milk and from 107 to 10² CFU/g for minced beef. In conclusion, the qPCR assay developed here represents a valuable tool for rapid detection and quantification of STEC in foods such as minced beef and dairy products as it ensures a high sensitivity and an optimal STEC diagnostic spectrum, taking into account the genetic stx variability observed in STEC population.

Detection of Escherichia coli serogroup O103 by real-time polymerase chain reaction.

AIMS: The aims of the study were to identify the specific genes of O-antigen gene cluster from Shiga toxin-producing Escherichia coli (STEC) O103 and to provide the basis for a specific real-time PCR test for rapid detection of E. coli O103. METHODS AND RESULTS: The published primers complementary to JUMPstart and gnd gene, the conserved flanking sequences of O-antigen genes clusters in E. coli and related species, were used to amplify the 12-kbp O103 O-antigen biosynthesis locus of STEC O103. A DNA library representative of this cluster allowed two O103-specific probes to be identified in the flippase (wzx) and UDP-galactose-4-epimerase (galE) genes. Two specific O103 serotyping real-time PCR tests based on these two genes were successfully developed. CONCLUSIONS: These results confirm that the O-antigen gene cluster sequences of E. coli allow rapidly a specific O-antigen real-time PCR assay to be designed. SIGNIFICANCE AND IMPACT OF THE STUDY: These findings increase the number of real-time PCR-assays available to replace the classical O-serotyping among E. coli O-antigen.

Polymerase chain reaction for Salmonella virulence-associated plasmid genes detection: a new tool in Salmonella epidemiology.

The important role of plasmid genes in assessing virulence for BALB/c mice in salmonella, and the difficulty of using standard techniques to detect them, led us to develop a detection method by gene amplification. One hundred and forty-three strains (71 serovars) of salmonella and 35 strains of other species were tested using specific oligonucleotide primers. The amplification products were identified by a specific oligonucleotide probe. Forty-nine salmonella strains from ten serovars (S. abortus ovis, S. choleraesuis, S. dublin, S. enteritidis, S. gallinarum/pullorum, S. hessarek, S. typhimurium, S. IIIa 48:z4, z23, S. IV 43:z4, z23:-, S. V 28:a:-) produced a positive and specific response. Because of various origins of the strains possessing the gene sought and the diversity of the responses, both from one serovar to another and in the same serovar, this search has its place among the epidemiological markers in general use. This method appears well suited to the research and detection of plasmid genes associated with mouse virulence in salmonella.

Identification of the O-antigen biosynthesis genes of Escherichia coli O91 and development of a O91 PCR serotyping test.

AIMS: The aims of the study were to characterize the O91 O-antigen gene cluster from Shiga toxin-producing Escherichia coli (STEC) O91 and to provide the basis for a specific PCR test for rapid detection of E. coli O91. METHODS AND RESULTS: The published primers complementary to JUMPstart and gnd gene, the conserved flanking sequences of O-antigen genes clusters in E. coli and related species were used to amplify the 10-kbp O91 O-antigen biosynthesis locus of STEC O91. A DNA library representative of this cluster allowed two O91 specific probes to be identified, and two specific PCR O91 serotyping tests to be successfully developed. CONCLUSIONS: These results confirm that the O-antigen gene cluster sequences of E. coli allow rapidly a specific O-antigen PCR assay to be designed. SIGNIFICANCE AND IMPACT OF THE STUDY: These findings increase the number of PCR-assays available to replace the classical O-serotyping among E. coli O-antigen.

Development of a 5'-nuclease PCR assay for detecting Shiga toxin-producing Escherichia coli O145 based on the identification of an 'O-island 29' homologue.

AIMS: A DNA sequence, from Escherichia coli STEC O145, homologous to O-island 29 from STEC O157 is described, together with a real-time PCR assay for detecting it. METHODS AND RESULTS: PCR and sequencing were used to identify the 'O-island 29' homologous DNA sequence from STEC O145 (strain VTH34). The sequence divergence between the STEC O145 and O157 'O-island 29' allowed a STEC O145 5'-nuclease PCR assay to be developed. CONCLUSIONS: The characterization of a novel locus in STEC O145 has allowed a specific O145 serogroup 5'-nuclease PCR assay to be designed. SIGNIFICANCE AND IMPACT OF THE STUDY: These findings increase the number of serogroup PCR assays available as alternatives to classical O-serotyping of E. coli.

Identification of a new putative enterotoxin SEU encoded by the egc cluster of Staphylococcus aureus.

AIMS: This paper reports on a new putative enterotoxin SEU encoded by the enterotoxin gene cluster egc from Staphylococcus aureus and on a real-time polymerase chain reaction (PCR) assay for detecting the seu gene. METHODS AND RESULTS: PCR and sequencing revealed a new putative enterotoxin SEU encoded by some egc clusters. The seu gene resulted from sequence divergence in the psient1 and psient2 pseudogenes previously described in the egc cluster (Jarraud et al. [2001] Journal of Immunology166, 669). The presence of the seu gene was investigated in a collection of S. aureus strains by conventional PCR and by a specific 5'-nuclease PCR assay. Among the 24 strains harbouring the egc cluster, four tested positive for the seu gene. CONCLUSIONS: The existence of the seu gene adds to the number of newly described se genes and underlines the need for a better understanding of their role in the pathogenesis of S. aureus. SIGNIFICANCE AND IMPACT OF THE STUDY: A thorough study of the seu gene should provide further insight into the phylogenetics of the staphylococcal enterotoxins.

Comparison between a PCR-ELISA test and the vero cell assay for detecting Shiga toxin-producing Escherichia coli in dairy products and characterization of virulence traits of the isolated strains.

AIMS: This paper provides information on a PCR-ELISA method for detecting Shiga toxin-producing Escherichia coli (STEC), and on their prevalence in dairy products. METHODS AND RESULTS: The sensitivity and specificity of the test was evaluated using pure cultures, spiked and naturally-contaminated samples. A comparative study with vero cytotoxicity testing was conducted, and STEC isolated from naturally-contaminated samples were characterized. The PCR-ELISA test was highly specific and sensitive, and detected 14% more positive samples than the vero cell assay. The prevalence of STEC in raw milk and unpasteurized cheese was 21.5% and 30.5%, respectively, while samples from the 'dairy environment' and from pasteurized cheese were less contaminated. The 34 strains of STEC isolated from natural samples showed that some of them carried virulence genes. CONCLUSION: No conclusion can be drawn at the moment concerning the potential risk to consumers. SIGNIFICANCE AND IMPACT OF THE STUDY: These data show the necessity of valuable screening methods to appreciate the virulence of STEC.

A PCR test for detecting Escherichia coli O157:H7 based on the identification of the small inserted locus (SILO157).

AIMS: This paper provides identification of a DNA sequence derived from Shiga toxin-producing Escherichia coli (STEC) O157:H7 and information on its utilization for detecting STEC O157 by PCR. METHODS AND RESULTS: Random Amplified Polymorphic DNA and DNA library were used to identify in STEC O157:H7 (strain EDL 933) a 2634-bp Small Inserted Locus, designated SILO157. Analysis of 211 bacterial strains showed that the PCR assays amplifying the SILO157 region could be used to detect STEC O157 with a good specificity. CONCLUSIONS: Characterization of a novel locus in STEC O157 is attractive since the serotype O157:H7 of STEC is still by far the most important serotype associated with more serious diseases. This island encodes putative proteins and especially one that is predicted to be an outer membrane protein designated IHP1. SIGNIFICANCE AND IMPACT OF THE STUDY: Further investigations could now be developed to appreciate the role of the SILO157 in pathogenicity.

Screening of staphylococci for the toxic shock syndrome toxin-1 (TSST-1) gene.

Dot blot hybridization was used to screen 820 staphylococci for the presence of the gene coding for TSST-1. The DNA of 33 strains among 70 Staph. aureus strains isolated from suspected toxic shock syndrome (TSS) cases hybridized with the probe. These results agreed perfectly with those obtained with a phenotypic method (immunodiffusion). Among 608 Staph. aureus strains isolated over a period of one month from hospitalized patients, 66 (11%) hybridized with the probe; of these strains, 64 (97%) were found to produce TSST-1 in vitro. None of 145 coagulase-negative staphylococcal strains harboured DNA hybridizing with the probe. The data indicate that this genotypic assay is suitable for epidemiological studies.

Evaluation of a ribosomal RNA gene probe for the identification of species and subspecies within the genus Staphylococcus.

To evaluate a 16S rRNA gene probe for the identification of staphylococcal species and subspecies, we have augmented previous studies involving 12 staphylococcal species by analysing the remaining 16 species currently classified in the genus Staphylococcus. HindIII- and EcoRI-restricted DNA of isolates from validly described species of Staphylococcus was probed with radiolabelled plasmid pBA2 containing 16S rDNA from Bacillus subtilis. The Dice coefficient was used to assess similarity between the 74 HindIII- and the 81 EcoRI-hybridization patterns obtained from a total of 271 isolates belonging to 31 staphylococcal taxa (28 species, of which three include two subspecies). The use of HindIII yielded a better discrimination of the staphylococci than the use of EcoRI. All of the isolates belonging to the same species or subspecies, except S. hyicus isolates, were recovered as homogeneous clusters using their HindIII hybridization patterns. The phenotypically close taxa were clearly distinguished. Thus, the method presented in this study constitutes a powerful tool for the identification of taxa within the genus Staphylococcus.