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The ability of phages to specifically interact with and lyse their host bacteria makes them ideal antibacterial agents. The range of applications of bacteriophage can be extended by their immobilization on inert surfaces. A novel method for the oriented immobilization of bacteriophage has been developed. The method was based on charge differences between the bacteriophage head, which exhibits an overall net negative charge, and the tail fibers, which possess an overall net positive charge. Hence, the head would be more likely to attach to positively charged surfaces, leaving the tails free to capture and lyse bacteria. Cellulose membranes modified so that they had a positive surface charge were used as the support for phage immobilization. It was established that the number of infective phages immobilized on the positively charged cellulose membranes was significantly higher than that on unmodified membranes. Cocktails of phages active against Listeria or Escherichia coli immobilized on these membranes were shown to effectively control the growth of L. monocytogenes and E. coli O157:H7 in ready-to-eat and raw meat, respectively, under different storage temperatures and packaging conditions. The phage storage stability was investigated to further extend their industrial applications. It was shown that lyophilization can be used as a phage-drying method to maintain their infectivity on the newly developed bioactive materials. In conclusion, utilizing the charge difference between phage heads and tails provided a simple technique for oriented immobilization applicable to a wide range of phages and allowed the retention of infectivity.

Quorum sensing (QS) is a signalling mechanism through which bacteria cellular functions are modified to promote access to nutrients and more favorable environmental niches. The frequent occurrence of Pseudomonas spp. in fresh and spoiled meat may involve enhanced gene expression regulated by QS. Several Pseudomonas spp. produce different N-acyl homoserine lactone (AHL) signal molecules. Meat spoilage during aerobic, refrigerated storage is often associated with the presence of Pseudomonas fragi. As with other Pseudomonas species in natural habitats, the dominance and activities of P. fragi in meat may be regulated by QS. In this study, five biosensor strains were used to detect AHL production on three different media by 72 different P. fragi strains isolated from fresh and spoiled meat. Positive and negative AHL-producing strains were used to verify the assays. None of the strains produced detectable quantities of AHLs, even when concentrated cell-free culture supernatants were assayed, nor did exogenous lactones increase biofilm formation in P. fragi strains. However, all isolates produced furanosyl borate diesters (type II autoinducers; AI-2) when tested using the bioluminescent biosensor strain of Vibrio harveyi (BB170). The production of AI-2 was presumed to be of metabolic origin even though Pseudomonas spp. have not been shown to harbor the luxS gene. Thus, the efficient development of P. fragi in fresh meat is not regulated by an AHL-mediated QS system. The mechanism of AI-2 production and its possible role in spoilage dynamics needs further study.

In this work, the feasibility of using a large-volume immunocapture system as a sample pretreatment before detection of Campylobacter was studied. Real-time PCR was used for detection of captured cells after immunocapture. This immunocapture system is able to process high-volume samples by recirculation, increasing the possibility of capturing cells in low numbers. After 30 min of recirculation, the sample is concentrated from 250 ml to 200 microl. In this study, different parameters were compared in order to improve cell capture. The analysis of inoculated chicken skin showed that detection of Campylobacter at levels of 10(3) CFU/25 g was possible after 8 h of enrichment. The low recovery of Campylobacter cells (< 1%) makes this separation method qualitative rather than quantitative. The detection limit of the entire protocol was increased due to the low cell recovery of the sample pretreatment. Therefore, this immunoseparation is able to recover cells present in high concentration after enrichment but not cells present in low concentration. Isolation of Campylobacter cells is achievable using this separation method rather than rapid detection.

A new two-step filtration protocol followed by a real-time PCR assay based on SYBR green I detection was developed to directly quantitate salmonellae in two types of biological samples: i.e., chicken rinse and spent irrigation water. Four prefiltration filters, one type of final filter, and six protocols for recovery of salmonellae from the final filter were evaluated to identify an effective filtration protocol. This method was then combined with a real-time PCR assay based on detection of the invA gene. The best results were obtained by subsequent filtration of 100 ml of chicken rinse or 100 ml of spent irrigation water through filters with pore diameters of >40 mum to remove large particles and of 0.22 microm to recover the Salmonella cells. After this, the Salmonella cells were removed from the filter by vortexing in 1 ml of physiological saline, and this sample was then subjected to real-time quantitative PCR. The whole procedure could be completed within 3 h from sampling to quantitation, and cell numbers as low as 7.5 x 10(2) CFU per 100-ml sample could be quantified. Below this limit, qualitative detection of concentrations as low as 2.2 CFU/100 ml sample was possible on occasion. This study has contributed to the development of a simple, rapid, and reliable method for quantitation of salmonellae in food without the need for sample enrichment or DNA extraction.

Primers designed to amplify a Campylobacter jejuni cadF gene sequence were used in an SYBR Green I real-time PCR assay as an alternative to conventional bacteriological methods for the rapid detection of C. jejuni in foods. Twenty-five grams of chicken skin (breast and thigh) was contaminated by adding approximately 1, 10, or 50 CFU of C. jejuni ATCC 35560. Twenty-five grams of pork and 25-ml aliquots of milk were also inoculated with 1 and 10 CFU of the pathogen. The samples were incubated in Bolton broth for different periods at 37 and 42 degrees C under microaerophilic conditions. Using a commercial robotic DNA purification system, DNA was extracted and purified from 1-ml aliquots of the enrichment cultures before and after centrifugation of the 250-ml enrichment broth at 15,900 x g for 10 min at 4 degrees C. The DNA was used as the template in a real-time PCR assay. C. jejuni was detected after 12 h of enrichment from samples inoculated with about 50 CFU/25-g sample. After centrifugation, an enrichment step of 8 h was sufficient to allow detection of pathogen in samples inoculated with 10 CFU/25 g. However, 24 h of enrichment was necessary to detect pathogen in samples inoculated with approximately 1 CFU/25 g. The real-time PCR protocol developed in this study significantly reduced the detection time of C. jejuni in foods.

Contamination of poultry by Campylobacter spp. is a significant source of human diarrheal diseases. Traditional methods currently used to detect Campylobacter in foods are time-consuming and labor-intensive. In this study, primers designed for the Campylobacter jejuni cadF gene sequence were used in a SYBR Green I real-time PCR assay as an alternative to a conventional bacteriological method for the rapid detection of C. jejuni from poultry. Twelve portions of chicken purchased from two local grocery stores and 39 portions obtained from a commercial processing plant were examined. Samples of the skin were enriched in Bolton broth at 37 degrees C for 3 h and then at 42 degrees C for 9, 21, or 45 h under microaerobic conditions. DNA was extracted from 1-ml aliquots of the enrichment cultures using 1% Triton X-100. The DNA was used as the template in a real-time polymerase chain reaction (PCR) assay. After 24 h of enrichment, C. jejuni was isolated from 13 samples and all of the positive cultures were also detected by the real-time PCR procedure. C. jejuni was detected by both methods from samples artificially contaminated with 1 or 10 CFU of C. jejuni per 10 g, after 24 h of enrichment. The real-time PCR method was found to be sensitive and specific. It significantly reduced the time required for the detection of C. jejuni in poultry following enrichment of samples.

Human campylobacteriosis has become the major cause of foodborne gastroenteritis in industrialized countries. Although there have been numerous studies investigating the prevalence of Campylobacter in animals and raw meat, sensitive and low-cost detection methods are needed to implement effective control measures during primary production and to use as tools in risk assessment studies. Thermophilic Campylobacter spp. in naturally contaminated (n = 64) and inoculated (n = 16) broiler samples were detected using two International Organization for Standardization (ISO)-approved methods. Both enrichment broths (those of Preston and of Park and Sanders) were inoculated with (i) Campylobacter jejuni ATCC35921, (ii) boneless breast from broilers, (iii) boneless breast rinse solution, (iv) boneless breast rinse solution inoculated with C. jejuni ATCC35921 before centrifugation, and (v) boneless breast rinse solution inoculated with C. jejuni ATCC35921 after centrifugation. The results indicated that the Park and Sanders broth was superior to the Preston broth for recovery of Campylobacter spp., and no significant differences (P > 0.05) were found between ISO (meat pieces) and modified ISO (centrifuged chicken rinse solution) methods for the detection of Campylobacter spp.

Nineteen Listeria monocytogenes strains were characterized by automated ribotyping, pulsed-field gel electrophoresis, and plasmid profiling to determine the relationship between genotype and sanitizer resistance. Isolates within a ribogroup had a consistent sensitivity or resistance phenotype except for ribogroup C isolates. All isolates with resistance phenotypes harbored two plasmids. The sensitivity of L. monocytogenes strains to quaternary ammonium compounds (QACs) was correlated with sensitivity to sanitizers and antibiotics with other modes of action. All isolates tested contained the mdrL gene, which encodes an efflux pump that confers resistance to QACs and is both chromosome and plasmid borne.

The microbiota of commercially produced, cooked and modified atmosphere packaged poultry meat was followed during storage at 3.5 degrees C for up to 7 weeks. The dominant microbiota consisted of Lactococcus raffinolactis (117 isolates), Carnobacterium divergens (61 isolates), Carnobacterium piscicola (11 isolates), Lactococcus garvieae (four isolates), Lactococcus lactis (one isolate) and Enterococcus faecalis (three isolates). All isolates were screened for production of bacteriocins. Only C. piscicola isolates produced an inhibitory substance active against other lactic acid bacteria and against several Listeria spp. Species-specific polymerase chain reaction (PCR) primers were used for the differentiation of Carnobacterium, L. raffinolactis, L. lactis, and L. garvieae strains associated with the modified atmosphere packaged poultry products. No false PCR products were observed with other closely related bacterial species.

The Reveal (Neogen Corp., Lansing, Mich.) and SafePath (SafePath Laboratories LLC, St. Paul, Minn.) tests were evaluated for their performance as beef fecal and beef carcass Escherichia coli O157:H7 monitoring tests. Agreement between these tests and a reference test system was determined using naturally contaminated bovine feces and beef carcasses. The reference system utilized immunomagnetic separation with plating onto cefixime, tellurite, sorbitol MacConkey agar, followed by colony testing using a serum agglutination test for the O157 antigen. Relative to this reference method, the Reveal test showed a sensitivity of 46% and a specificity of 82% on bovine feces and a specificity of 99% on carcass samples. The SafePath test, demonstrated a significantly higher sensitivity at 79% and a similar specificity of 79%. On carcass samples the SafePath test performed similarly to the Reveal test, demonstrating a specificity of 100% relative to the reference system. There was an insufficient number of E. coli O157-positive carcass samples to estimate precisely the sensitivity of these two methods. Both methods show promise as rapid carcass monitoring tests, but further field testing to estimate sensitivity is needed to complete their evaluation. The proportion of positive fecal samples for E. coli O157:H7 by the reference method ranged from 10.2% to 36% in 10 lots of cattle with an overall mean of 17.3% (39/225). Quarter carcass sponging of 125 carcasses revealed 1.6% positive for the pathogen (2/125).

LacTek tests are competitive enzyme-linked immunosorbent assays intended for rapid detection of antimicrobial residues in bovine milk. In this study, the LacTek test protocol was modified for use with extracts of bovine tissue to detect beta-lactam, tetracycline, and sulfamethazine residues. Test performance characteristics--precision, accuracy, ruggedness, practicability, and analytical specificity and sensitivity--were investigated. Results suggest that LacTek tests can be easily adapted to detect antimicrobial residues in extracts of lean ground beef. However, positive samples may not contain residues at violative concentrations (i.e., Canadian maximum residue limits), and therefore, additional analysis would be required for final confirmation and quantitation (e.g., chromatography).

The Petrifilm HEC test (3M Canada Inc., London, Ontario), a quantitative microbiological test for Escherichia coli O157:H7, was evaluated for its performance as a beef-carcass monitoring test. Test repeatability and agreement and agreement with an E. coli O157:H7 detection method using a hydrophobic grid membrane filter (HGMF) overlaid onto cefixime-tellurite-sorbitol MacConkey agar (CT-SMAC) followed by a latex agglutination test for the O157 antigen were determined by using pure cultures of E. coli O157:H7, beef samples experimentally contaminated with bovine feces containing E. coli O157:H7, and naturally contaminated beef carcasses of unknown E. coli O157:H7 status from a local abattoir. The Petrifilm HEC test showed excellent repeatability and excellent agreement with the HGMF-CT-SMAC method when test samples were obtained from pure cultures and experimentally contaminated meat. All 125 naturally contaminated beef carcasses surveyed were negative for E. coli O157:H7 with both microbial methods. The Petrifilm HEC test, however, demonstrated a significantly lower proportion of cross-reactive organisms (false-positive reactions) than the HGMF-CT-SMAC method. Given the performance of this test coupled with its ease of use and compact size, it shows considerable promise for carcass testing where abattoir laboratory facilities are limited and as a substitute for more complex laboratory testing methods used in established laboratories.

A PCR-amplified, digoxigenin (DIG)-labelled yst probe was evaluated in naturally and artificially contaminated pork and milk samples for its ability to detect and differentiate between pathogenic and nonpathogenic strains of Yersinia enterocolitica following enrichment in irgasan-ticarcillin-chlorate (ITC) enrichment broth. A total of 44 raw pork samples were obtained from one meat-processing factory and two local retail stores. Y. enterocolitica serogroup O:3/biovar 4 was isolated from 6 of the 44 (14%) pork samples tested. The hybridization results were in good agreement when compared with those from standard biochemical and serological tests. The lowest concentration detectable by a colony hybridization technique in the milk samples inoculated with Y. enterocolitica O:8 PA-A corresponded to 10 cfu/ml in the original sample. No pathogenic serovars were detected in 6 raw milk and 3 pasteurized milks tested. The results of this investigation demonstrate that the DIG-labelled yst probe is a reliable tool for rapid detection of pathogenic Y. enterocolitica in naturally and artificially contaminated food samples.