Campylobacter spp. are among the most commonly reported bacterial causes of acute diarrheal disease in humans worldwide. Potential virulence factors include motility, chemotaxis, colonization ability, adhesion to intestinal cells, invasion and epithelial translocation, intracellular survival, and formation of toxins. Probiotic Lactobacillus and Bifidobacterium strains are known to have an inhibitory effect against the growth of various foodborne pathogens. The objective of this study was to investigate the effect of Lactobacillus acidophilus strain La-5 and Bifidobacterium longum strain NCC2705 cell-free spent media (CFSM) on the expression of virulence genes ( cadF, cdtB, flaA , and ciaB ) of Campylobacter jejuni strain 81-176 and a luxS mutant, using real-time PCR. Our results demonstrated that the CFSM of both probiotic strains were able to down-regulate the expression of ciaB (ratio of −2.80 and −5.51, respectively) and flaA (ratio of −7.00 and −5.13, respectively) in the wild-type Campylobacter strain. In the luxS mutant, where the activated methyl cycle is disrupted, only the ciaB gene (ratio −7.21) was repressed in the presence of La-5 CFSM. A supplementation of homocysteine to restore the disrupted cycle was able to partially reestablish the probiotic effect of both strains. luxS and the activated methyl cycle might play an active role in the modulation of virulence of C. jejuni by probiotic extracts.

Escherichia coli O157:H7 is responsible for a human toxico-infection that can lead to severe complications such as hemolytic uremic syndrome. Inside the intestine, E. coli O157:H7 forms typical attaching-effacing lesions and produces Shiga toxins. The genes that are responsible for these lesions are located in a pathogenicity island called the locus of enterocyte effacement (LEE). LEE gene expression is influenced by quorum sensing through the luxS system. In this study, the effect of glucose on the expression of several genes from LEE, on the expression of Shiga toxin genes, and on the expression of luxS was assessed with real-time, reverse transcription PCR. All concentrations of glucose (from 0.1 to 1%) were able to down-regulate genes from the LEE operon. A slight down-regulation of genes implicated in Shiga toxin expression was also observed but was significant for low doses of glucose (0.1 to 0.5%) only. A slight but significant increase in luxS expression was observed with 1% glucose. This confirms that in addition to quorum sensing, the presence or absence of nutrients such as glucose has an impact on the down- or upregulation of LEE-encoded virulence genes by the bacterium. The influence of glucose on the virulence of E. coli O157:H7 has received little attention, and these results suggest that glucose can have an important effect on the virulence of E. coli O157:H7.

A simple method was developed for the fast and inexpensive detection of Salmonella Typhimurium using a recombinant P22:: luxAB phage. All the steps from phage production to detection were considered. A strain of Salmonella Typhimurium harboring the prophage P22:: luxAB was grown in batch culture to produce spontaneously the recombinant bacteriophage. Batch production to stationary phase was better for propagation of the phage and led to a total population of 4.3 × 10 9 (±4.3 × 10 9 ) PFU/ml of P22, including only 1.4 × 10 6 (±1 × 10 6 ) PFU/ml harboring the luxAB genes. After preenrichment, a simple four-step bioassay was tested and optimized for several parameters. The detection limit of the luminometer was only 5 × 10 2 (±1.75 × 10 2 ) CFU Salmonella Typhimurium per ml, but increased to 1.5 × 10 4 (±1.17 × 10 4 ) CFU Salmonella Typhimurium per ml when the cells were in a complex matrix. The detection limit after the preenrichment was 6.5 × 10 3 (±1.5 × 10 3 ) CFU Salmonella Typhimurium per ml, but the detection limit after the preenrichment also increased markedly to 1.65 × 10 5 (±0.15 × 10 5 ) CFU Salmonella Typhimurium per ml when Salmonella Typhimurium was in a complex matrix. Finally, the bioassay was applied to the detection of Salmonella Typhimurium LT2 in 14 different feed and environmental samples (including duck feed, litters, and feces) spiked either before or after the preenrichment process. It was possible to detect Salmonella Typhimurium LT2 in all samples within 16 h.

The microaerophilic nature of Campylobacter jejuni has complicated its recovery from human and animal sources. In this study, enhancement of the growth and aerotolerance of C. jejuni ATCC 35921 in nutrient broth no. 2 (NB2) was investigated. The efficiency of recovery of C. jejuni in NB2 containing FBP (0.025% [each] ferrous sulfate, sodium metabisulfite, and sodium pyruvate), 5% laked horse blood, hemin, Oxyrase, or activated charcoal in an aerobic atmosphere was compared with that obtained under microaerophilic incubation. The shortest lag time (λ) for cells grown aerobically was observed with NB2 supplemented with FBP, 5% laked horse blood, 0.01 g/liter of hemin, or 0.15 U/ml of Oxyrase. The efficacy of these media to resuscitate C. jejuni cells in late exponential phase, as well as cells subjected to stress induced by cold, heat, starvation, or acid, was determined in aerobic or microaerobic atmospheres. The λ of cells grown aerobically in NB2 containing both FBP and blood was similar to that obtained in the same medium incubated in a microaerobic environment ( P > 0.05). However, the λ was longer during aerobic growth when low numbers of cells (approximately 1 log CFU/ml) in late exponential phase were used as the initial inoculum. The best recovery of stressed C. jejuni was observed in NB2 supplemented with FBP and blood and incubated aerobically. Enrichment in media incorporating FBP and 5% laked horse blood is a simple, convenient, and time-saving method to replace microaerophilic incubation methods for the resuscitation of C. jejuni .

Proteolytic pseudomonads dominate the spoilage flora of aerobically chill-stored proteinaceous raw foods. Proteolysis during spoilage of these food systems affects both food quality and the dynamics of the bacterial community because it increases the availability of nutrients to the community as a whole. Quorum sensing, or cell-cell signaling, is associated closely with ecological interactions among bacteria in mixed communities. The potential role of quorum sensing in proteolytic food spoilage was examined, based on the evaluation of N -acyl-homoserine lactone (AHL) signal molecules. The occurrence of proteolytic activity and AHL signals was studied during spoilage of aerobically chill-stored ground beef, fish, chicken, and raw milk. Pseudomonads dominated the psychrotrophic flora, followed distantly by members of the Enterobacteriaceae . The growth of pseudomonads was correlated with the occurrence of proteolytic activity in all food systems. AHL concentration began increasing significantly only after the onset of proteolytic activity. Widely divergent AHL profiles were revealed by thin-layer chromatography analysis of the different food samples, and these profiles were likely determined by the undefined bacterial flora in these systems and by the characterized pseudomonads and Enterobacteriaceae . Although Hafnia alvei was a major component of the Enterobacteriaceae flora in all foods tested and a strong AHL producer, the signal molecules produced by H. alvei strain EB1 did not influence protease production by Pseudomonas fluorescens strain 395 in vitro. These results do not indicate any clear correlation between the overall detectable AHL signal molecules accumulated in the food samples and proteolytic activity.

Groups of 25 skin-on thighs or skin-on, skinned, or tumbled breast portions of broiler chicken carcasses were sampled by excision of skin or muscle tissue, rinsing, or swabbing. Counts of total aerobic bacteria, coliforms, and Escherichia coli were recorded for each sample. For all types of carcass portions, the mean log counts and the total log counts obtained for each group of bacteria by excision or rinsing mostly differed by <0.5 log unit. However, the counts obtained by swabbing were generally >0.5 log unit lower than the smaller of the values obtained by the other two sampling methods.

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.

The ability of Salmonella Enteritidis to survive in the presence of phage, SJ2, during manufacture, ripening, and storage of Cheddar cheese produced from raw and pasteurized milk was investigated. Raw milk and pasteurized milk were inoculated to contain 10 4 CFU/ml of a luminescent strain of Salmonella Enteritidis ( lux ) and 10 8 PFU/ml SJ2 phage. The milks were processed into Cheddar cheese following standard procedures. Cheese samples were examined for Salmonella Enteritidis ( lux ), lactic acid bacteria, molds and yeasts, coliforms, and total counts, while moisture, fat, salt, and pH values were also measured. Salmonella Enteritidis ( lux ) was enumerated in duplicate samples by surface plating on MacConkey novobiocin agar. Bioluminescent colonies of Salmonella Enteritidis were identified in the NightOwl molecular imager. Samples were taken over a period of 99 days. Counts of Salmonella Enteritidis ( lux ) decreased by 1 to 2 log cycles in raw and pasteurized milk cheeses made from milk containing phage. In cheeses made from milks to which phage was not added, there was an increase in Salmonella counts of about 1 log cycle. Lower counts of Salmonella Enteritidis ( lux ) were observed after 24 h in pasteurized milk cheese containing phage compared to Salmonella counts in raw milk cheese with phage. Salmonella Enteritidis ( lux ) survived in raw milk and pasteurized milk cheese without phage, reaching a final concentration of 10 3 CFU/g after 99 days of storage at 8°C. Salmonella did not survive in pasteurized milk cheese after 89 days in the presence of phage. However, Salmonella counts of approximately 50 CFU/g were observed in raw milk cheese containing phage even after 99 days of storage. In conclusion, this study demonstrates that the addition of phage may be a useful adjunct to reduce the ability of Salmonella to survive in Cheddar cheese made from both raw and pasteurized milk.

To determine the long-term effects of a lactic acid rinse on viability and recovery of pathogens, Salmonella Hadar was isolated from poultry and bioluminescent constructs obtained by transformation with the lux (CDABE) gene cassette from Photobacterium phosphoreum . Results indicated that the transformed Salmonella Hadar lux was otherwise phenotypically similar to the wild-type strain. Viability studies were performed by measuring luminescence following lactic acid treatment of turkey breast and subsequent storage at −12, 0, 5, and 10°C. The ability of the S . Hadar lux strain to recover was determined by monitoring light output after incubation at 22°C for 10 h. The results showed that metabolic activity was significantly ( P < 0.05) affected by lactic acid and by storage temperatures of −12, 0, and 5°C. The lowest recovery rate was observed after rinsing with lactic acid and storing at 5°C. The study demonstrated that bacterial bioluminescence is an effective way of monitoring in “real time” the ability of bacteria to recover from stress.

Biofilms of luminescent Pseudomonas putida were developed on rubber surfaces by incubation in brain heart infusion (BHI) broth. Scanning electron microscopy (SEM) and epifluorescence microscopy (EFM) were used to examine biofilm formation. To test the efficacy of two sanitizers commonly employed in dairy plants for CIP (cleaning in place) procedures, a novel bioluminescence method and aerobic plating were used to enumerate cells. Immediately after the sanitizer treatments an apparent 5-log reduction of biofilm-associated cells was determined. However, when the samples were resuscitated for 18 h in BHI broth, high numbers of cells were detected which reached levels close to those of nontreated controls. The results demonstrated that neither sanitizer could completely eliminate biofilm-associated P. putida . The microbial bioluminescence method proved to be the best way for assessing effectiveness of sanitizers against microbial biofilms.

The Lactek test, marketed for antimicrobial residue detection in milk, was validated for the detection of antimicrobial residues in tissues. A previous study found that the LacTek test could confidently identify tissue samples spiked with antimicrobial residues. However, the test could not reliably distinguish violative from nonviolative spiked samples relative to Canadian maximum residue limits (MRLs). The objectives of this study were to assess and compare the performance of the LacTek tests for (β-lactams, tetracyclines, gentamicin, and sulfamethazine on samples containing naturally incurred residues by running the test in parallel with the standard microbial inhibition test (MIT) presently used for the routine testing of tissues at our facility and to assess the agreement with high pressure liquid chromatographic (HPLC) determinative methods. Parallel testing with the official MIT found that the Lactek tests could be confidently used for testing tissue samples containing incurred residues. Among 1,008 MIT-positive samples, the LacTek test found that 90% contained (β-lactams and/or tetracyclines. A further 7.3% of violative residues could not be identified to an antimicrobial class. In addition, 9%> of samples testing negative on the MIT were found to contain an antimicrobial residue by the LacTek tests. Comparative testing with HPLC methods found that there was very good agreement between the two tests and that most violations were due to penicillin G and oxytetracycline. Although the LacTek test cannot be used to distinguish violative from nonviolative residue levels, it does offer several advantages over the present MIT. These include speed, ease of use, the ability to identify residues to a specific class, and an improved sensitivity at the MRL level for the most commonly found antimicrobials in tissue.

The objective of this study was to evaluate the potential of Fourier transform infrared spectroscopy (FTIR) for rapid identification of Bacillus cereus isolates. Ten B. cereus group isolates (comprising B. cereus , Bacillus mycoides , and Bacillus thuringiensis strains), five other Bacillus spp., and five non- Bacillus spp. were used. Two types of media, brain heart infusion (BHI) and Trypticase soy agar (TSA), were tested. The results indicated that all B. cereus group isolates produced characteristic absorbance peaks at wave numbers between 1738 and 1740 cm −1 These peaks were not affected by the growth medium. None of the other bacteria tested showed a similar peak after growth on BHI or TSA. Absorbance peaks between 1800 and 1500 cm −1 of members of the B. cereus group had different shapes and sizes, suggesting that FTIR may be useful for rapid identification of species within the B. cereus group.

This paper presents a historical review of antimicrobial use in food animals, the causes of residues in meat and milk, the types of residues found, their regulation in Canada, tests used for their detection, and test performance parameters, with an emphasis on immunoassay techniques. The development of residue detection methods began shortly after the introduction of antimicrobials to food animal production in the late 1940s. From initial technical concerns expressed by the dairy industry to the present public health and international trade implications, there has been an ongoing need for reliable, sensitive, and economical methods for the detection of antimicrobial residues in food animal products such as milk and meat. Initially there were microbial growth inhibition tests, followed by more sensitive and specific methods based on receptor binding, immunochemical, and chromatographic principles. An understanding of basic test performance parameters and their implications is essential when choosing an analytical strategy for residue testing. While each test format has its own attributes, no one test will meet all the required analytical needs. Therefore the use of a tiered or integrated System employing assays designated for screening and confirmation is necessary to ensure that foods containing violative residues are not introduced into the food chain.

Outbreaks of enterohemorrhagic Escherichia coli O157:H7 have been commonly associated with products derived from ground beef, but recently the organism has been implicated as the causative agent in outbreaks involving yogurt and cheese. This finding has raised concern about the potential for its growth and survival in fermented dairy products. A bioluminescent strain of E. coli O157:H7 was used to determine postprocessing survival in yogurt with live cultures at pH 4.17, 4.39, and 4.47 stored at 4 and 10°C. In addition, survival of E. coli O157:H7 was monitored during the manufacture of Cottage, Colby, Romano, and Feta cheeses. Results indicated survival for 8 and 5 days at 4 and 10°C respectively in yogurt at pH 4.17, 17 and 15 days at 4 and 10°C respectively in yogurt at pH 4.39, and 17days at both 4 and 10°C in yogurt at pH 4.47. E. coli O157:H7 did not survive cooking procedures at 56°C in Cottage cheese. However, the pathogen survived for 27, 30, and 27 days in Colby, Romano, and Feta cheeses respectively. A high correlation of r 2 > 0.89 was obtained between counts of bioluminescenct colonies and standard plate count for all yogurt and cheese varieties, indicating that bioluminescence was a sensitive and rapid indicator of cellular viability for E. coli O157:H7. Survival of the pathogen, as indicated by this method, is possible in highly acidic environments even at refrigeration temperatures. This poses a potential hazard should postprocessing contamination occur.

Many marine microorganisms ( Vibrio , Photobacterium ) are capable of emitting light, that is, they are bioluminescent. The light-yielding reaction is catalyzed by a luciferase, and it involves the oxidation of reduced riboflavin phosphate and a long-chain aldehyde in the presence of oxygen to produce a blue green light. The genes responsible for the luciferase production, ( lux A and lux B), aldehyde synthesis ( lux C, D, and E), and regulation of luminescence ( lux I and lux R) have all been identified, and recent research has resulted in the discovery of three new genes ( lux F, G, and H). The ability to genetically engineer dark microorganisms to become light emitting by introducing the lux genes into them has opened up a wide range of applications of bioluminescence. Assays using bacterial bioluminescence for the detection and enumeration of microorganisms are rapid, sensitive, accurate, and can be made specific. It is these attributes that are making in vivo bioluminescent assays so attractive to the food industry.

Psychrotrophic bacteria have been recognized as a recurring problem in the refrigerated storage and distribution of fluid milk and cream and other perishable dairy products for several decades. Much emphasis has been focused on postpasteurization contaminants that are psychrotrophic, (e.g., Pseudomonas , Flavobacterium , and Alcaligenes spp.). Common sources of these gram-negative, non-sporeforming organisms are equipment surfaces and water supplies. Although these organisms are generally heat sensitive, many of their associated proteinases and lipases can withstand moderate to severe heat treatments and cause product deterioration. With the advance of improved control of postpasteurization contamination by nonheat-resistant psychrotrophs, more recent attention has been directed at psychrotrophic sporeformers and their potential impact on milk quality and shelf life properties. Heat-resistant psychrotrophs include members from the genera Clostridium , Arthrobacter , Microbacterium , Streptococcus , Corynebacterium , and Bacillus . However, the predominant microorganisms which comprise this category are Bacillus species. These bacteria can be introduced into milk supplies from water, udder and teat surfaces, or from soil and milkstone deposits on farm bulk tanks, pumps, pipelines, gaskets, and processing equipment. There is speculation that they can also be postpasteurization contaminants. When in the spore state, these microorganisms easily survive the typical range of pasteurization conditions with subsequent germination and outgrowth of vegetative cells. These organisms produce degradative enzymes (e.g., proteinases, lipases, and phospholipases) similar to those of non-sporeforming psychrotrophs. Enzymatic activity results in the development of objectionable flavor and quality defects in dairy products. The unique combination of both heat-resistant and psychrotrophic properties with the same microorganism represents substantial potential for causing spoilage of perishable milk products. Recent trends of higher pasteurization temperatures and extended refrigerated storage time of both raw and pasteurized milk and cream products exacerbates the significance of this group of microorganisms for the dairy foods industry.

Methods to rapidly assess the bacteriological quality of raw milk were investigated. Whereas direct microscopic count, modified psychrotrophic plate count, and direct epifluorescent filter technique (DEFT) did not correlate well with initial psychrotrophic bacterial count of raw milk, improvements were obtained after preincubation of the milk samples. The best preincubation conditions were identified as 30°C for 6 h, 21°C for 10 h, 13°C for 15 h, 13°C for 20 h, or 7°C for 37 h. The “square root” equation was applied to the data, and a model was produced for predicting growth of the native microflora of raw milk. Using this equation, a DEFT count after preincubation of the milk at 21°C for 10 h could accurately predict the initial psychrotroph count and the count after storage of the milk at 6°C for 48 h.

Using a reversed passive latex agglutination assay, about 85% of psychrotrophic Bacillus spp. tested were shown to produce diarrhoegenic toxin during growth on brain heart infusion broth at 25°C. The majority of these strains were identified as Bacillus cereus or cereus -related strains. However, a number of other species was capable of synthesizing the toxin. Further investigation of four psychrotrophic Bacilli showed that the toxin was produced during growth in milk at temperatures ranging from 6 to 21°C. Toxin production increased with increasing temperatures and was not synthesized in appreciable quantities until the bacterial count exceeded 1 × 10 7 cfu/ml.

A test involving preincubation of samples at 21°C for 25 h in the presence of a mixture of nisin:penicillin:crystal violet to prevent growth of gram-positive organisms was used to identify post-pasteurization contamination of milk and single cream. This test (P-INC test) could successfully predict the level of contamination after storage at 6°C for 7 din 85% of milk samples and 86.1% of cream samples studied. A Bacillus sp. which was resistant to the inhibitors used in the test was isolated from only cream samples. However, growth of this organism did not significantly affect accuracy of the test.