PorA specific primers for the identification of Campylobacter species in food and clinical samples

Campylobacteriosis is a public health problem with considerable socio-economic impact. As the European Food Safety Authority has emphasized the importance of a surveillance programme for campylobacteriosis, the aim of the present study was the optimization of a specific and sensitive PCR protocol able to detect Campylobacter species responsible for gastrointestinal infections. Raw poultry meat samples were analysed for the presence of Campylobacter sp., by plating onto mCCD (Modified Charcoal-Cefoperazone-Deoxycholate) Agar and Campylobacter Selective Preston Agar and using four sets of species-specific primers for Campylobacter jejuni, Campylobacter coli, Campylobacter upsaliensis and Campylobacter lari designed to bridge the porA gene. The resulting primers demonstrated a sensitivity of 0.01 ng/μl for the C. coli-specific, C. lari-specific, and C. upsaliensis-specific primer sets and 0.5 ng/μl for the C. jejuni-specific primer sets using DNA from pure cultures. Non-specific amplification of non-target DNA was not observed indicating excellent specificity. The primers were useful for the analyses of poultry meat samples both for direct plating onto mCCDA, and for DNA extracted directly from the cells grown for 48 h in Preston enrichment broth. The sets of primers were also useful when used for species identification of human isolates.     

Characterization of Escherichia coli from raw poultry in Belgium and impact on the detection of Campylobacter jejuni using Bolton broth

A comparative study examining Bolton broth and Preston broth for enrichment and reliable detection of Campylobacter jejuni (both healthy and freeze stressed cells) was performed. Tested as pure cultures, Bolton broth enabled faster resuscitation and growth of C. jejuni compared to Preston broth. When C. jejuni was co-incubated with extended-spectrum-beta-lactamase (ESBL) producing Escherichia coli isolated from Belgian poultry meat preparations, the latter dominated in the Bolton enrichment broth and crowded the mCCDA plates. This resulted in the inability to recover C. jejuni by ISO 10272-1:2006 standard method. Preston broth did not support the growth of the ESBL E. coli isolates, but showed longer detection time of C. jejuni compared to Bolton broth. The use of the same antibiotic (sodium cefoperazone) in Bolton broth and in mCCDA plates may explain the problems encountered for detection of C. jejuni, as high numbers of ESBL E. coli present after enrichment in Bolton broth, also caused overgrowth and masked the few C. jejuni colonies present on the mCCDA plates. The use of Campylobacter spp. specific real-time PCR circumvented these problems and enabled rapid detection of the pathogen after 24 h enrichment in both Bolton and Preston broth, for both healthy and freeze stressed cells.     

Performance characteristics and estimation of measurement uncertainty of three plating procedures for Campylobacter enumeration in chicken meat

In this work, we present an intra-laboratory study in order to estimate repeatability (r), reproducibility (R), and measurement uncertainty (U) associated with three media for Campylobacter enumeration, named, modified charcoal cefoperazone deoxycholate agar (mCCDA); Karmali agar; and CampyFood ID agar (CFA) a medium by Biomérieux SA. The study was performed at three levels: (1) pure bacterial cultures, using three Campylobacter strains; (2) artificially contaminated samples from three chicken meat matrixes (total n=30), whereby samples were spiked using two contamination levels; ca. 10(3)cfuCampylobacter/g, and ca. 10(4)cfuCampylobacter/g; and (3) pilot testing in naturally contaminated chicken meat samples (n=20). Results from pure culture experiment revealed that enumeration of Campylobacter colonies on Karmali and CFA media was more convenient in comparison with mCCDA using spread and spiral plating techniques. Based on artificially contaminated samples testing, values of repeatability (r) were comparable between the three media, and estimated as 0.15log(10)cfu/g for mCCDA, 0.14log(10)cfu/g for Karmali, and 0.18log(10)cfu/g for CFA. As well, reproducibility performance of the three plating media was comparable. General R values which can be used when testing chicken meat samples are; 0.28log(10), 0.32log(10), and 0.25log(10) for plating on mCCDA, Karmali agar, and CFA, respectively. Measurement uncertainty associated with mCCDA, Karmali agar, and CFA using spread plating, for combination of all meat matrixes, were +/-0.24log(10)cfu/g, +/-0.28log(10)cfu/g, and +/-0.22log(10)cfu/g, respectively. Higher uncertainty was associated with Karmali agar for Campylobacter enumeration in artificially inoculated minced meat (+/-0.48log(10)cfu/g). The general performance of CFA medium was comparable with mCCDA performance at the level of artificially contaminated samples. However, when tested at naturally contaminated samples, non-Campylobacter colonies gave similar deep red colour as that given by the typical Campylobacter growth on CFA. Such colonies were not easily distinguishable by naked eye. In general, the overall reproducibility, repeatability, and measurement uncertainty estimated by our study indicate that there are no major problems with the precision of the International Organization for Standardization (ISO) 10272-2:2006 protocol for Campylobacter enumeration using mCCDA medium.     

Yersinia enterocolitica in slaughter pig tonsils: Enumeration and detection by enrichment versus direct plating culture

Tonsil samples from 139 slaughter pigs were examined for the presence of pathogenic Yersinia enterocolitica by enrichment procedures based on the standard method ISO 10273:2003. In addition, samples were tested by direct plating method to evaluate its efficiency compared to the enrichment culture methods and to quantify the level of contamination in porcine tonsils. In total, 52 samples (37.4%) were positive for pathogenic Y. enterocolitica, all belonging to bioserotype 4/O:3. Fifty out of the 52 positive samples (96.2%) were detected by direct plating. Enumeration showed an average concentration of 4.5 log₁₀ CFU g⁻¹ and 4.4 log₁₀ CFU g⁻¹ tonsil on Salmonella-Shigella-desoxycholate-calcium chloride (SSDC) and cefsulodin-irgasan-novobiocin (CIN) agar plates, respectively. The enrichment procedures recommended by the ISO 10273:2003 method were not optimal for the isolation of pathogenic Y. enterocolitica from pig tonsils: two days enrichment in irgasan-ticarcillin-potassium chlorate (ITC) broth resulted in an isolation rate of 84.6%, while 5 days enrichment in peptone-sorbitol-bile (PSB) broth recovered only 59.6% of positive samples. Reducing the enrichment time in PSB from 5 to 2 days resulted in a significantly higher recovery rate (94.2%) and might serve as an appropriate enrichment protocol for the isolation of pathogenic Y. enterocolitica from pig tonsils. Compared to enrichment culture methods, results based on direct plating can be obtained in a shorter time course and provide quantitative data that might be needed for further risk assessment studies.     

Procedures for the isolation of Campylobacter jejuni and Campylobacter coli from poultry

The effect of different techniques for the isolation of Campylobacter from 198 samples of chicken giblets was studied. Four enrichment broths and two plating media were compared. A protocol of enrichment in Preston or Doyle and Roman's broth followed by plating on Preston agar is recommended for routine use. The duration of incubation of enrichment media was shown to have a significant effect on the number of Campylobacter isolates obtained, 48 h being statistically superior to either 24h or 72h for media incubated at 42°C. Of the 198 samples examined, 177 were found to contain Campylobacter by at least one of the procedures used.     

Comparison of three selective media and validation of the VIDAS Campylobacter assay for the detection of Campylobacter jejuni in ground beef and fresh-cut vegetables

In this study, three different selective media, modified cefoperazone charcoal deoxycholate agar (mCCDA), Karmali agar, and Preston agar, were compared for isolating Campylobacter jejuni from artificially contaminated ground beef and fresh-cut vegetables that have different levels of background microflora. Concurrently, an automated enzyme-linked immunosorbent assay method for detecting Campylobacter spp. (VIDAS Campylobacter) was evaluated by comparing it with the culture methods. Food samples inoculated with C. jejuni were enriched in Bolton broth at 42°C for 44 h and then streaked onto the three different selective media, followed by incubation under microaerobic conditions at 42°C for 48 h. The enriched Bolton broth (1 ml) was used in the VIDAS Campylobacter assay. No statistical differences in sensitivities were observed between the three selective media for ground beef and fresh-cut vegetables, but the selectivity of Preston agar was better (P < 0.05) than those of mCCDA and Karmali agar. The VIDAS Campylobacter assay showed a recovery rate similar (P > 0.05) to those of all of the medium combinations in ground beef. However, more positive samples (P < 0.05) were detected with the VIDAS Campylobacter than with the selective agars, except for the combinations of mCCDA plus Preston agar or mCCDA plus Karmali agar plus Preston agar in fresh-cut vegetables.     

A survey on the incidence of Campylobacter spp. and the development of a surface adhesion polymerase chain reaction (SA-PCR) assay for the detection of Campylobacter jejuni in retail meat products

A rapid and sensitive method for the detection of Campylobacter in meat products was developed.Campylobacter were isolated from a range of Irish retail meats (n=80) and poultry (n=100) samples by direct plating on Campylobacter Selective Agar (CCDA, Oxoid). A total of 30·5% of samples tested positive forCampylobacter and Campylobacter jejuni was the most commonly identified species. The pathogen was detected in 39 (65%) poultry, 12 (60%) offal and 4 (20%) minced beef samples examined. Estimates derived from direct plate counts ranged from log₁₀0·7 to log₁₀2·75 cfu g⁻¹. The data from this study was used in the development of a rapid and sensitive method for the detection of Campylobacter in poultry. A rapid method was developed based on an initial sample enrichment for 24 h in Campylobacter Enrichment Broth (CEB), recovery of the pathogen from the enriched sample by surface adhesion onto a polycarbonate membrane, phenol:chloroform extraction of DNA from the adherent bacteria, and PCR analysis using primers specific for the flagellin A gene (present in C. jejuni and C. coli). The developed surface adhesion PCR (SA-PCR) technique had a detection limit of log₁₀4–5 and could be completed within 29 h. Results from SA-PCR analysis of a number of retail samples (n=50) correlated favourably with traditional plate culture results, i.e. 34 samples were found to contain Campylobacter by both methods.     

Evaluation of Potassium‐Clavulanate‐Supplemented Modified Charcoal‐Cefoperazone‐Deoxycholate Agar for Enumeration of Campylobacter in Chicken Carcass Rinse

Potassium‐clavulanate‐supplemented modified charcoal‐cefoperazone‐deoxycholate agar (C‐mCCDA) that was described in our previous study was compared with original mCCDA for the enumeration of Campylobacter in pure culture and chicken carcass rinse. The quantitative detection of viable Campylobacter cells from a pure culture, plated on C‐mCCDA, is statistically similar (P > 0.05) to mCCDA. In total, 120 chickens were rinsed using 400 mL buffered peptone water. The rinses were inoculated onto C‐mCCDA and mCCDA followed by incubation at 42 °C for 48 h. There was no statistical difference between C‐mCCDA (45 of 120 plates; mean count, 145.5 CFU/mL) and normal mCCDA (46 of 120 plates; mean count, 160.8 CFU/mL) in the isolation rate and recovery of Campylobacter (P > 0.05) from chicken carcass rinse. The Pearson correlation coefficient value for the number of Campylobacter cells recovered in the 2 media was 0.942. However, the selectivity was much better on C‐mCCDA than on mCCDA plates (P < 0.05). Significantly fewer C‐mCCDA plates (33 out of 120 plates; mean count, 1.9 CFU/mL) were contaminated with non‐Campylobacter cells than the normal mCCDA plates (67 out of 120 plates; mean count, 27.1 CFU/mL). The C‐mCCDA may provide improved results for enumeration of Campylobacter in chicken meat alternative to mCCDA with its increased selectivity the modified agar possess.     

Addition of Rifampicin to Bolton Broth to Inhibit Extended‐Spectrum β‐Lactamase‐Producing Escherichia coli for the Detection of Campylobacter

Exponential growth of extended‐spectrum β‐lactamase (ESBL)‐producing Escherichia coli in Campylobacter media has become a common problem for the detection of Campylobacter in chicken meats. We investigated the minimum inhibitory concentration of 40 ESBL‐producing E. coli isolates from meats obtained from various countries against antibacterial agents in Bolton broth (cefoperazone, vancomycin, and trimethoprim). All ESBL‐producing E. coli strains were resistant to cefoperazone and vancomycin, whereas 50% of them were resistant to trimethoprim and grew in Bolton broth. We found that 20 μg/mL of rifampicin inhibited the growth of trimethoprim‐resistant E. coli strains. Hence, we added 20 μg/mL of rifampicin to Bolton broth to improve the isolation of Campylobacter from chicken carcass rinses. The isolation rate of Campylobacter was significantly higher in the modified broth (44 out of 58, 75.9%, P < 0.05) than in the normal broth (0 out of 58, 0%). Furthermore, the number of agar plates with non‐Campylobacter spp. was much lower after enrichment in the modified broth (4 out of 58, 6.9%, P < 0.05) than in the normal broth (58 out of 58, 100%).     

The physiologic state of Escherichia coli O157:H7 does not affect its detection in two commercial real-time PCR-based tests

Multiplex real-time PCR detection of Escherichia coli O157:H7 is an efficient molecular tool with high sensitivity and specificity for meat safety assurance. The Biocontrol GDS^® and DuPont Qualicon BAX^®-RT rapid detection systems are two commercial tests based on real-time PCR amplification with potential applications for quantification of specific E. coli O157:H7 gene targets in enriched meat samples. However, there are arguments surrounding the use of these tests to predict pre-enrichment concentrations of E. coli O157:H7, as well as arguments pertaining to the influence of non-viable cells causing false positive results. The present study attempts to illustrate the effects of different bacterial physiologic states and the presence of non-viable cells on the ability of these systems to accurately measure contamination levels of E. coli O157:H7 in ground beef. While the PCR threshold cycle (C_T) values of these assays showed a direct correlation with the number of bacteria present in pure cultures, this was not the case for ground beef samples spiked with various levels of injured or healthy cells. Furthermore, comparison of post-enrichment cell densities of bacteria did not correlate with injured or healthy cell numbers inoculated before enrichment process. Ground beef samples spiked with injured or healthy cells at different doses could not be distinguished by C_T values from either assay. In addition, the contribution of nonviable cells in generating positive real-time PCR signals was investigated using both assays on pre-enriched and post-enriched beef samples, but only if inoculated at levels of 10⁶ cells/sample or higher, which are levels not typically seen in ground beef.     

Evaluation of the ISO 10273:2003 method for the isolation of human pathogenic Yersinia enterocolitica from pig carcasses and minced meat

Pig carcass swabs (n = 254) and minced meat samples (n = 82) were examined for pathogenic Yersinia enterocolitica using different routinely used enrichment protocols. All samples were obtained in the context of the official Yersinia monitoring program in Belgium. In total, 28 carcasses (11.0%) were contaminated with Y. enterocolitica bioserotype 4/O:3 and one (0.4%) with bioserotype 2/O:9. Four minced meat samples (4.9%) tested positive for Y. enterocolitica bioserotype 4/O:3. Using the ISO 10273:2003 method, eight out of the 29 Yersinia-positive carcasses (27.6%) and none of the contaminated minced meat samples (0.0%) were detected. Reducing the enrichment time in PSB from 5 to 2 days increased the number of positive samples. Overall, enrichment in PSB at 25 °C recovered more positive carcasses and minced meat samples than selective enrichment and cold enrichment. As the exclusive use of the ISO 10273:2003 method results in a strong underestimation of Y. enterocolitica positive carcasses and minced meats, efforts are needed to optimize the current version of the ISO method. In addition, isolation of pathogenic Y. enterocolitica requires experience and the use of a stereomicroscope to avoid false negative results.     

Effect of mash maceration and ripening stage of apples on phenolic compounds and antioxidant power of cloudy juices: A study using chemometrics

The effects of different enzymatic preparations on total phenolic content, phenolic profile (HPLC), and ferric reducing antioxidant power (FRAP) of cloudy juices from Lis Gala and Fuji Suprema apples varieties, at three ripening stages (unripe, ripe and senescent) were investigated using Principal Component Analysis and Hierarchical Cluster Analysis. The commercial preparations enzymatic (Ultrazym^® AFPL; Pectinex^® Ultra Clear; Pectinex^® SMASH XXL; Panzym^® YieldMASH) increased the total phenolic compounds and ferric reducing capacity of the cloudy juice from unripe and ripe Lis Gala (respectively by 67 and 49% for unripe apples, and 28 and 33% for ripe apples) and unripe Fuji Suprema apples (23 and 55%), while for the ripe Fuji Suprema apples only Pectinex^® Ultra Clear and Panzym^® YieldMASH had this effect. No significant (p > 0.05) was observed on senescent stage, whatever the enzymatic preparation. Enzymatic preparations could increase phenolic compounds concentration and antioxidant capacity of cloudy apple juice, but this effect depended on the maturity of the apples.     

Comparison of three methods for detecting Campylobacter spp. in chilled or frozen meat

There is a demand from the meat industry as well as from public health authorities for a simple and rapid detection method for thermophilic Campylobacter spp. from food. Hence, we compared different isolation procedures for their usefulness for this purpose. Bolton enrichment medium without blood, incubated statically in stomacher bags in microaerophilic atmosphere, detected more samples positive for thermophilic Campylobacter spp. than did Preston enrichment broth in bottles with small headspace and tight caps, incubated in aerobic atmosphere. Use of an automated antigen detection system to identify enrichment cultures positive for Campylobacter spp. was as sensitive as selective agars, and reduced the detection time by 24 h. Campylobacter spp. were recovered from 18.4% of the 461 samples tested. The prevalence was highest in refrigerated poultry meat (52% of the 80 samples tested) and poultry offal (41% of the 44 samples tested).     

Development and evaluation of DNA and RNA real-time assays for food analysis using the hilA gene of Salmonella enterica subspecies enterica

The objective of this study was the development of DNA and RNA real-time PCR methods for detection of food-borne Salmonella sp. as rapid alternatives to the traditional cultural method (ISO 6579, 2004) in fresh meat carcasses and processed meat samples. These PCR methods were based on the hilA sequence, with primers and hybridisation probes designed against this gene target. The primers and probes were evaluated for their efficiency and dynamic range and subsequently the specificity of the assay was tested using 106 Salmonella enterica subspecies enterica strains and 30 non-salmonellae strains. An internal amplification control (IAC) was also developed for incorporation. The optimum copy number of IAC was determined to be 500 copies per reaction. A complementary enrichment protocol was adapted from the existing standard ISO 6579:2004 and consisted of enrichment in Buffered Peptone Water (BPW) 22 ± 2 h and a second selective enrichment for 6 h in Rappaport Vassiliadis with Soya (RVS). The DNA and RNA-based real-time PCR protocols, were applied to meat samples inoculated with Salmonella enterica subspecies enterica strains, including swabs from meat carcasses and minced beef samples which were heat treated or frozen. The developed methods have the potential as useful alternatives to the standard ISO 6579:2004 method for the detection of Salmonella enterica subspecies enterica on carcass swabs and raw meat using hilA as a target. The DNA assay is a useful tool for the screening of meat samples in the abattoir within 3 days of slaughter or in a food production process and the RNA-based assay has the potential to detect viable Salmonella enterica subspecies enterica in ready-to-eat products.     

Multiplex fiber optic biosensor for detection of Listeria monocytogenes, Escherichia coli O157:H7 and Salmonella enterica from ready-to-eat meat samples

Listeria monocytogenes, Escherichia coli O157:H7 and Salmonella enterica are the most common foodborne bacterial pathogens and are responsible for many outbreaks. Therefore, multiplex detection of these three using a single assay platform is highly desirable. The objective was to develop and optimize a fiber optic sensor for simultaneous detection of these three from food. The streptavidin coated optical waveguides were immobilized with biotinylated polyclonal antibodies and exposed to the bacterial suspensions or enriched food samples for 2 h. Pathogens were detected after reacting with Alexa-Fluor 647-labeled monoclonal antibodies. Ready-to-eat beef, chicken and turkey meats were inoculated with each pathogen (∼100 cfu/25 g), enriched in SEL (Salmonella, E. coli, Listeria), a multipathogen selective enrichment broth for 18 h and tested with the biosensor. The biosensor was able to detect each pathogen, individually or in a mixture with very little cross-reactivity. The limit of detection for the sensor was ∼10³ cfu/ml for all three pathogens. Furthermore, the biosensor successfully detected each pathogen, grown in a mixture from enriched meat samples under 24 h. The pathogen presence was further verified by PCR and immunofluorescence assay. The multiplex fiber optic sensor shows promise for detection of the three pathogens if present in the same sample eliminating the use of multiple single pathogen detection platforms.     

Campylobacter contamination of broiler caeca and carcasses at the slaughterhouse and correlation with Salmonella contamination

In order to estimate the prevalence of Campylobacter spp. and Salmonella spp. on broiler chicken carcasses and the prevalence of Campylobacter spp. in caeca, 58 French slaughterhouses were investigated in 2008. Enumeration of Campylobacter spp. was also performed in order to study the relation between caeca and carcass contamination. A pool of 10 caeca and one carcass were collected from 425 different batches over a 12-month period in 2008. Salmonella was isolated on 32 carcasses leading to a prevalence of 7.5% ([5.0-10.0]_95%CI). The prevalence of Campylobacter was 77.2% ([73.2-81.2]_95%CI) in caeca and 87.5% ([84.4-90.7]_95%CI) on carcasses. No significant correlation was found between Campylobacter and Salmonella. Positive values of Campylobacter were normally distributed and the average level was 8.05 log₁₀ cfu/g ([7.94-8.16]_95%CI) in caeca and 2.39 cfu/g ([2.30-2.48]_95%CI) on carcasses. A positive correlation (r = 0.59) was found between the mean of Campylobacter in caeca and on carcasses (p < 0.001). Thus, carcasses from batches with Campylobacter-positive caeca had significantly (p < 0.001) higher numbers of Campylobacter per gram than batches with negative caeca. These results show that Campylobacter can be present in both matrices and reduction in caeca could be a possible way to reduce the amount of bacteria on carcasses. Of the 2504 identifications performed, 3 species of Campylobacter (Campylobacter jejuni, Campylobacter coli and Campylobacter lari) were identified. The main species recovered were C. jejuni and C. coli, which were isolated in 55.3% and 44.5% of positive samples, respectively. These two species were equally represented in caeca but C. jejuni was the most frequently isolated on carcasses with 57.1% and 42.5% of positive carcasses for C. jejuni and C. coli, respectively. This study underlines that target a reduction of Campylobacter on final products requires a decrease of contamination in caeca.     

Effect of sample preparation and bacterial concentration on Salmonella enterica detection in poultry meat using culture methods and PCR assaying of preenrichment broths

We evaluated the sensitivity of a PCR assay in the detection of Salmonella enterica at the broth preenrichment step of poultry meat. A total of 162 retail poultry meat samples, which were prepared by manual massaging, stomacher or no homogenization were compared for Salmonella recovery. Using these homogenization methods, the PCR assay at the broth preenrichment step detected Salmonella in, respectively, 48.9%, 62.2% and 50.0% of meat and giblet samples detected as Salmonella-positive using the culture method. In ground chicken, however, Salmonella was detected in 21.7% of samples treated by stomacher homogenization, compared to 40.7% and 48% of untreated and hand-massaged samples, respectively. These results suggest that stomaching of ground chicken causes excessive effusion of food constituents, which affects PCR results. Using the most probable number (MPN) technique, Salmonella was detected at under 1.0 CFU/g in 12 ground chicken samples and under 10³ CFU/ml of broth in seven of the 12 broth-enriched samples, which considered the minimum concentration detectable by PCR assay. These results show that Salmonella detection using routine PCR assays is difficult in poultry meat, and in particular ground chicken, due to low amounts of Salmonella and the presence of inhibitors.     

Survival of Campylobacter jejuni on beef and pork under vacuum packaged and retail storage conditions: examination of the role of natural meat microflora on C. jejuni survival

The ability of Campylobacter jejuni ATCC 11168 to survive on beef and pork stored under chilled, vacuum packaged and retail display conditions were examined. In addition, the effect of natural microflora on commercial beef and pork on the survival of C. jejuni under these storage conditions was examined. When sterile cores of beef and pork were inoculated with ∼10⁵ to 10⁶ cfu cm⁻²C. jejuni, and were stored under aerobic or vacuum packaged conditions at −1.5 or 4 °C, its numbers dropped significantly and C. jejuni could not be enumerated by direct plating after 21 d of the 6 wks study. In contrast, survival of C. jejuni on commercial vacuum packaged beef and pork was significantly enhanced, resulting in only 1 log cfu cm⁻² reduction at the end of 6 wks. During 7 d of display in a retail case, numbers of C. jejuni dropped quickly, but could be enumerated by direct plating even after the 7 d. The presence of high numbers of inoculated C. jejuni on beef and pork had no significant effect on the natural microflora numbers compared to uninoculated controls when the meat was stored either in vacuum or in a retail display case. These results show that natural microflora on vacuum packaged meat afford enhanced survival of C. jejuni present on the surfaces of both beef and pork when stored at refrigeration temperatures. Hence, strict hygienic practices or the implementation of decontamination technologies are recommended to ensure safety of meat with respect to this pathogen.     

Comparison of methods for the isolation of Yersinia enterocolitica from porcine tonsils and pork

Three enrichment procedures and three plating media were evaluated for their efficiency in isolating Yersinia enterocolitica from porcine tonsils and pork. Cold enrichment in phosphate-sorbitol-bile medium (PBSSB) with alkali treatment before plating resulted in higher isolation rates than enrichment in modified Rappaport broth (MRB) and bile-oxalate-sorbose broth (BOS). Post-enrichment alkali treatment gave a considerable increase in isolation rate with all the enrichment media tested. A sample inoculum of 10 g showed a better recovery than 0.2 g. Cefsulodin-irgasan-novobiocin (CIN) agar was slightly better than MacConkey agar and MacConkey agar + Tween 80 as isolation medium for Yersinia spp. from porcine tonsils. Most of the serotype 0:3 and 0:9 strains were isolated after enrichment in MRB and PBSSB. Enrichment in PBSSB resulted in the isolation of some other potentially virulent Yersinia strains. For the isolation of Yersinia spp. from pork and porcine tonsils an inoculum of 10 g, parallel use of MRB and PBSSB, post-enrichment alkali treatment and isolation on CIN agar is recommended.     

Prevalence of Clostridium difficile in retailed meat in the Netherlands

Recent reports indicate that a large proportion of community-acquired Clostridium difficile infections (CA-CDI) are not linked to recent antibiotic therapy, older age, significant comorbidity or previous hospitalization. Possible community sources for CA-CDI include animals and food, and therefore a surveillance study on the prevalence of C. difficile in meat was performed. Samples of different meat species were collected from the retail trade and analyzed for the presence of C. difficile using a method that included selective enrichment in C. difficile broth, subsequent alcohol shock-treatment and plating onto C. difficile selective medium. C. difficile isolates were tested for the presence of toxin genes and were typed using PCR ribotyping. Of 500 samples tested, 8 (1.6%) were positive for the presence of C. difficile: 1 from lamb (6.3%) and 7 from chicken meat (2.7%). The isolated strains belonged to PCR ribotypes different from those that are currently most frequently found in patients with CDI in the Netherlands, except for C. difficile PCR ribotype 001 which was found in one chicken meat sample. This observation suggests that other matrices than meat may serve as a source for CA-CDI.