A case of 'blown pack' spoilage of vacuum-packaged pork likely associated with Clostridium estertheticum in Canada.

This study investigated the potential causative agents for vacuum-packaged pork that had shown gross package extension during a routine storage life study in a Canadian pork plant using both conventional and culture-independent methods. The spoilage-associated bacteria in purge samples from two packages were enumerated using selective media and profiled using 16S rDNA amplicon analysis. The presence of Clostridium estertheticum was detected using species-specific real-time PCR. An enrichment procedure was used to isolate C. estertheticum from one of the purge samples. The average population density in the two purge samples of total aerobes, lactic acid bacteria (LAB), coliforms and Brochothrix thermosphacta was 9·4, 9·1, 6·0 and 4·6 log CFU per ml respectively, as determined by plating. The estimated numbers of C. estertheticum were >7 log cells per ml. Clostridium estertheticum was recovered although the enrichment condition used for isolation favoured the growth of LAB more than that of Clostridium spp. Based on 16S rDNA amplicon analysis, the microbiota in the two purge samples had 64·7 and 20·7% of Clostridium spp., and 32·5 and 70·1% of LAB respectively. SIGNIFICANCE AND IMPACT OF THE STUDY: Blown pack spoilage of vacuum-packaged meat may lead to severe economic losses and is often associated with beef, venison and lamb. This study is the first to report vacuum-packaged chilled pork can also be subject to blown pack spoilage, and data support the conclusion that the causative agent is likely Clostridium estertheticum. The lysozyme-digestion step greatly improved the isolation efficiency for C. estertheticum, a spore-forming anaerobic organism that has been proven to be difficult to recover. This method can be used for isolating spore-forming organisms from food samples.

In situ determination of Clostridium endospore membrane fluidity during pressure-assisted thermal processing in combination with nisin or reutericyclin.

This study determined the membrane fluidity of clostridial endospores during treatment with heat and pressure with nisin or reutericyclin. Heating (90°C) reduced laurdan (6-dodecanoyl-2-dimethylaminonaphthalene) general polarization, corresponding to membrane fluidization. Pressure (200 MPa) stabilized membrane order. Reutericyclin and nisin exhibit divergent effects on heat- and pressure-induced spore inactivation and membrane fluidity.

Growth and filamentation of cold-adapted, log-phase Listeria monocytogenes exposed to salt, acid, or alkali stress at 3°C.

In Canada, there is a zero tolerance for Listeria in a 125-g sample of product in which growth of Listeria monocytogenes can occur, and a limit of ≤100 CFU/g in ready-to-eat (RTE) food products that support limited growth during the stated shelf life and/or RTE refrigerated foods with a shelf life of ≤5 days. L. monocytogenes can form filaments in response to pH and osmotic, atmospheric, and temperature stress, which can result in an underestimation of the risk of RTE foods as filaments form single colonies on plate count agars but can divide into individual cells once the stress is removed. The objective was to investigate the filamentation characteristics of three strains of L. monocytogenes exposed to saline, acidic, basic, and simultaneous acidic and saline environments at 3°C. After 4 days at 3°C, log-phase cells grown in tryptic soy broth (TSB) were longer than cells grown at 15°C, and 68% of cells were below the reference value of the 90th percentile of control cultures. When cultures growing at 3°C were exposed to additional stresses, increases in the proportion and length of filaments in the population were observed, while increases in log CFU per milliliter were reduced. After 4 days of incubation at 3°C, the log CFU per milliliter of L. monocytogenes increased by 1.1 U in TSB and 0.4 to 0.5 U in TSB with 4% NaCl, TSB with a pH of 6.0 with 4% NaCl, and TSB with a pH of 5.5. Moreover, the longest 10% of cells were 6.4 to 8.5 times longer than control cells, and only 20 to 30% of cells were below the reference value. Cultures grown in TSB at pH 6.0 with 4% NaCl experienced more sustained filamentation than cultures grown in TSB with 4% NaCl, but less than cultures grown in TSB at pH 6.0. The mechanism involved in filamentation could be different for cells exposed to NaCl than exposed to acid, and additional stress might not necessarily result in more extensive filament formation. These findings contribute to a better understanding of the widespread potential of filament formation and the potential implications for food safety.

Hatching egg and newly hatched chick yolk sac total IgY content at 3 broiler breeder flock ages.

During the first week of the posthatching period, before the immune system is mature enough to produce its own B lymphocytes, a chick's humoral immunity depends on maternal antibodies (IgY) received from the egg yolk. During incubation and after hatching, the yolk sac (YS) membrane transfers nutrients (including IgY) from the egg yolk to the developing embryo or newly hatched chick. The objective of this study was to determine the effects of breeder flock age on the total IgY content of egg yolks and chick YS from a commercial broiler breeder strain. Hatching eggs from the same broiler breeder flock were collected at 32, 40, and 55 wk of age. One group of eggs per flock age was used to determine the egg yolk total IgY content. Another group of eggs was incubated for 21.5 d, and upon hatching, the YS of newly hatched chicks were collected to determine the total IgY content. Egg and egg yolk weight increased with flock age, but YS weights did not reflect egg yolk weight. The total IgY content per gram of egg yolk increased with flock age; this fact plus the observed yolk weight increase with flock age notably increased the total IgY contained in yolks of eggs laid by 55-wk-old breeders. However, chicks hatching from 55-wk-old breeders had less IgY per gram of YS than chicks from 32- and 40-wk-old breeders. Whether there are differences in the rates of YS absorption between chicks of different breeder ages is unknown. This research provided total IgY values for broiler breeder egg yolk and chick YS of a commonly used meat-type chicken strain. Differences in egg yolk and YS total IgY contents due to flock age in this type of bird had not been previously reported. Research on the physiological consequences of YS absorption rates in chicks from different breeder ages is advised.

Characterization of an extremely heat-resistant Escherichia coli obtained from a beef processing facility.

AIMS: This study aimed to determine the survival of Escherichia coli strains during steam and lactic acid decontamination interventions currently used by the beef-processing industry, and to determine their heat resistance. METHODS AND RESULTS: Strains were grouped into cocktails of five strains each differing in their RAPD patterns for subsequent identification. Steam and lactic acid treatments on meat reduced cell counts of E. coli strain cocktails by 90-99%. The 20 slaughter plant isolates exhibited only minor variation in their resistance to steam and lactic acid treatments but were more resistant than reference strains (three strains) or isolates from live cattle (seven strains). D(60) values of strains from live cattle, and reference strains ranged from 0·1 to 0·5 min, in keeping with literature data. However, D(60) values of current slaughter plant isolates ranged between 15 for E. coli DM18.3 and 71 min AW 1.7. Cell counts of E. coli AW 1.7 were reduced by <5 log(10) CFU g(-1) in ground beef patties cooked to an internal temperature of 71°C. CONCLUSIONS: Strains of E. coli that survive cooking of ground beef to the recommended internal temperature of 71°C can be isolated from beef-processing facilities. SIGNIFICANCE AND IMPACT OF THE STUDY: Pathogen interventions in current commercial beef slaughter may select for extremely heat-resistant strains of E. coli.

Spraying hatching eggs with electrolyzed oxidizing water reduces eggshell microbial load without compromising broiler production parameters.

An experiment was conducted to determine whether spraying hatching eggs with electrolyzed oxidizing (EO) water would decrease eggshell microbial load and hence improve hatchability, chick quality, and broiler growth performance. Eggs were collected from a broiler breeder flock; half the eggs were sprayed with EO water and the other half were left untreated. Enterobacteriaceae and total aerobic bacteria present on the eggshells of eggs from both treatments were enumerated. The eggs were incubated, and the broiler chicks were grown out to 39 d. Eggshell microbial load was significantly decreased by spraying the eggs with acidic EO water before incubation, with no effect on cuticle structure [as measured by egg weight (moisture) loss], normal embryonic development, and hatchability. Chick quality, as determined by visual assessment and BW at the time of hatch, was also not affected. However, broiler mortality during the first 2 wk of the production period was significantly reduced in the chicks that hatched from eggs sprayed with EO water compared with chicks hatching from control eggs. The ability of EO water to reduce eggshell microbial load without negatively affecting hatchability or chick quality may make it a useful product for hatching egg sanitation.

Risk factors for ciprofloxacin resistance in reported Campylobacter infections in southern Alberta.

We conducted a case-control study examining risk factors for ciprofloxacin resistance in Campylobacter infections that were reported in 2004 and 2005 in two health regions in southern Alberta. The study questionnaire included questions about recent travel and antibiotic use, food consumption frequency, use of household and personal hygiene products with antibacterial agents, contact with animals, and potential misuse of antibiotics. Of the 210 patients who participated, 31.0% had ciprofloxacin-resistant Campylobacter infections. Foreign travel was the strongest predictor of resistance. Surprisingly, possession of antibiotics for future use was identified as a risk factor for resistance. We also examined the potential for participation bias and resistance misclassification to affect the resulting multivariable models. Participation bias appears to have had a substantial effect on the model results, but the estimated misclassification effect due to the use of different ciprofloxacin susceptibility testing methods was only slight.

Occurrence of pathogens in raw and ready-to-eat meat and poultry products collected from the retail marketplace in Edmonton, Alberta, Canada.

A total of 800 meat and poultry products were purchased from the retail marketplace in Edmonton, Alberta, Canada. The products consisted of raw ground beef, chicken legs, pork chops, and ready-to-eat fermented sausage, roast beef, processed turkey breast, chicken wieners, and beef wieners. The samples were analyzed to determine the prevalence of Shiga toxin-producing Escherichia coli, Salmonella, Campylobacter spp., and Listeria monocytogenes. Shiga toxin-producing E. coli 022: H8 was found in one raw ground beef sample. Salmonella and Campylobacter were found in 30 and 62% of raw chicken legs, respectively. L. monocytogenes was found in 52% of raw ground beef, 34% of raw chicken legs, 24% of raw pork chops, 4% of fermented sausages, 3% of processed turkey breast, 5% of beef wieners, and 3% of chicken wieners. The occurrence of pathogens in this study is similar to that in retail products in many other international locales.

Differential expression of proteins in cold-adapted log-phase cultures of Escherichia coli incubated at 8, 6 or 2 degrees C.

Temperature is used to control the growth of microorganisms in foods. The minimum temperature for sustained growth of Escherichia coli is 7 degrees C. E. coli cells in the logarithmic phase of growth at 15 degrees C were incubated at 8, 6 or 2 degrees C. The cells grew with the formation of filaments at the two higher temperatures, but did not grow at 2 degrees C. In order to investigate more thoroughly the nature of filament formation in E. coli at temperatures near the minimum temperature for sustained growth, cells were harvested after 1 day at 2 degrees C or at times up to 4 or 8 days at 8 or 6 degrees C, respectively. Proteins extracted from the cells were separated by two-dimensional polyacrylamide gel electrophoresis (2D-PAGE), and spots containing differentially expressed proteins were identified by quadropole time-of-flight tandem (Q-ToF-2) mass spectrometry. For most of the identified proteins, the amounts were not substantially different in cells grown at 15 degrees C or incubated at 2 degrees C. In cells incubated at 8 or 6 degrees C, proteins associated with stress responses, the tricarboxylic acid cycle and electron transport were present in substantially greater amounts, and proteins associated with protein synthesis were present in substantially smaller amounts than in cells grown at 15 degrees C. These findings suggest that the stringent response is induced in E. coli incubated at temperatures near the minimum for growth, so the formation of filaments at those temperatures may be a result of the stringent response.

Genetic diversity of Escherichia coli recovered from the oral cavity of beef cattle and their relatedness to faecal E. coli.

AIMS: To determine the genetic diversity of generic Escherichia coli recovered from the oral cavities of beef cattle and their relatedness to E. coli isolated from the faeces of cattle during pasture grazing and feedlot finishing. METHODS AND RESULTS: A total of 484 E. coli (248 oral and 236 faecal isolates) were obtained from eight beef cattle after 1 and 5 months of grazing on pasture and after 1 and 5 months in a feedlot. The random amplification of polymorphic DNA (RAPD) method was used to genetically characterize these isolates. The RAPD patterns showed that ca 60% of E. coli recovered from the oral cavities and faeces during pasture and feedlot shared a close genetic relatedness. A number of E. coli with unique RAPD types were also found either in the oral cavities or faeces. Most of the E. coli RAPD types recovered from the oral cavities were shared among animals, but there were also RAPD types which were unique to individual animals. The E. coli populations of the oral cavities were genetically diverse and changed over time. CONCLUSIONS: This study indicates that there are large numbers of E. coli carried in the oral cavities of beef cattle and those E. coli are closely related to strains found in the faeces. The oral cavities of cattle harbour a genetically diverse E. coli population. SIGNIFICANCE AND IMPACT OF THE STUDY: The oral cavity may be an important reservoir of enteric pathogens which may transfer to meat during carcass dressing. A better understanding of the molecular ecology of E. coli in cattle would assist the design of approaches to control pathogenic strains during beef production and processing.

The behaviour of log phase Escherichia coli at temperatures that fluctuate about the minimum for growth.

AIMS: To investigate the behaviour of cold-adapted, log phase Escherichia coli exposed to temperatures that fluctuate below and above the minimum for growth. METHODS AND RESULTS: Log phase E. coli cultures were incubated at a constant temperature of 2, 4 or 6 degrees C or with temperatures allowed to increase from those temperatures for 35 min, to 10 degrees C, at 6-, 12- or 24-h intervals, as commonly occurs during retail display of chilled foods. At suitable intervals for each culture, the optical absorbance value was determined using a spectrophotometer, the forward angle light scatter was determined using a flow cytometer, and portions were spread on plate count agar for enumeration of colony forming units (CFU). Numbers of CFU decreased by 3 log units or increased by 1 log unit for cultures incubated at 6 degrees C for 17 days without or with temperatures fluctuations at < or =12-h intervals, respectively. Cells elongated when cultures were incubated at 4 or 2 degrees C with temperatures fluctuating at 6-h intervals, and at 6 degrees C at constant or fluctuating temperatures, but cells did not elongate in cultures incubated at a constant temperature of 2 or 4 degrees C. SIGNIFICANCE AND IMPACT OF THE STUDY: The minimum growth temperature of E. coli is assumed to be > or =7 degrees C. Elongated cells were able to divide when temperatures rose from 6 degrees C to above 7 degrees C for <45 min at < or =12-h intervals. Such temperature fluctuations may be experienced by chilled foods during defrosting cycles of retail display cases. The finding that cells behave differently under fluctuating than at constant temperatures may significantly affect understanding of appropriate temperatures for the safe storage of chilled foods and for predictive modelling of bacterial growth in such foods.

Genotypic analysis of Escherichia coli recovered from product and equipment at a beef-packing plant.

AIMS: To identify sources of Escherichia coli on beef by characterizing strains of the organism on animals, equipment and product at beef-packing plant. METHODS AND RESULTS: Generic E. coli were recovered from hides, carcasses, beef trimmings, conveyers and ground beef during the summer of 2001 (750 isolates) and winter of 2002 (500 isolates). The isolates were characterized by Random Amplification of Polymorphic DNA (RAPD). The numbers of E. coli recovered from dressed carcasses were less than the numbers recovered from hides. The numbers recovered from chilled carcasses were too few for meaningful analysis of the strains present on them but the numbers recovered from trimmings and ground beef were larger. The RAPD patterns showed that the majority of isolates from hides, carcasses, beef trimmings, conveyers and ground beef were of similar RAPD types, but a few unique RAPD types were recovered from only one of those sources. The E. coli populations present on the hides of incoming animals and in the beef-processing environment were highly diverse. Randomly selected E. coli isolates from each of the five sources were further characterized by pulsed-field gel electrophoresis (PFGE). Most genotypes of E. coli defined by PFGE corresponded to the E. coli types defined by RAPD. CONCLUSIONS: The hides of the incoming animals appeared to be only one of the sources of the E. coli on trimmings and in ground beef, as additional sources were apparently present in equipment used for carcass breaking. SIGNIFICANCE AND IMPACT OF THE STUDY: This study indicates that hazardous microbiological contamination of meat may occur after the dressing of carcasses at commercial beef-packing plants, which suggests that attention should be given to the control of the contamination of meat during carcass breaking as well as during the dressing of carcasses.

Behaviour of log-phase Escherichia coli at temperatures near the minimum for growth.

The behaviour of cold-adapted, log-phase Escherichia coli in broth cultures incubated at temperatures between 7 and 15 degrees C was examined by determinations of numbers of colonies recovered on plate count agar (PCA); absorbance at 600 nm (A600); cell lengths from photomicrographs; and cell size distributions by flow cytometry. Cultures incubated between 7 and 10 degrees C were evaluated for 8 days or until A600 values approached 1.0. Cultures incubated at > or =12 degrees C were subcultured to maintain them in the log phase for up to 8 days. Numbers of colonies recovered declined when cultures were incubated at 7 degrees C, but increased when cultures were incubated at higher temperatures. However, A600 values increased during incubation at all temperatures. The mean lengths of cells doubled during incubation at 7 degrees C for 8 days, but remained constant during incubation at 10 degrees C for 1.25 days. Forward angle light scatter (FALS) measurements obtained by flow cytometry indicated that the mean length of cells increased at < or = 8 degrees C, but not at 10 degrees C. A reference value at the 90th percentile of FALS measurements on day 0 was used to determine changes in the distribution of the lengths of cells. About 80% or 17% of the cells were above the reference value after 5 days of incubation at 7 degrees C or 1.25 days of incubation at 10 degrees C, respectively. Cultures that were maintained in the log phase at 12 degrees C became increasingly heterogeneous in cell size after 2 days, but cultures that were maintained at 13 degrees C remained constant in cell size for 8 days. The observations have implications for the prediction of mesophile proliferation at temperatures that approach their minima for growth.

Genetic characterization and heterologous expression of brochocin-C, an antibotulinal, two-peptide bacteriocin produced by Brochothrix campestris ATCC 43754.

Brochocin-C, produced by Brochothrix campestris ATCC 43754, is active against many strains of the closely related meat spoilage organism Brochothrix thermosphacta and a wide range of other gram-positive bacteria, including spores of Clostridium botulinum. Purification of the active compound and genetic characterization of brochocin-C revealed that it is a chromosomally encoded, two-peptide nonlantibiotic bacteriocin. Both peptides of brochocin-C are ribosomally synthesized as prepeptides that are typical of class II bacteriocins. They are cleaved following Gly-Gly cleavage sites to yield the mature peptides, BrcA and BrcB, containing 59 and 43 amino acids, respectively. Fusion of the nucleotides encoding the signal peptide of the bacteriocin divergicin A in front of the structural genes for either BrcA or BrcB allowed independent expression of each component by the general protein secretion pathway. This revealed the two-component nature of brochocin-C and the necessity for both peptides for activity. A 53-amino-acid peptide encoded downstream of brcB functions as the immunity protein (BrcI) for brochocin-C. In addition, the cloned chromosomal fragment revealed open reading frames downstream of brcI, designated brcT and brcD, that encode proteins with homology to ATP-binding cassette translocator and accessory proteins, respectively, involved in the secretion of Gly-Gly-type bacteriocins.

Assessment of the hygienic performances of hamburger patty production processes.

The hygienic conditions of the hamburger patties collected from three patty manufacturing plants and six retail outlets were examined. At each manufacturing plant a sample from newly formed, chilled patties and one from frozen patties were collected from each of 25 batches of patties selected at random. At three, two or one retail outlet, respectively, 25 samples from frozen, chilled or both frozen and chilled patties were collected at random. Each sample consisted of 30 g of meat obtained from five or six patties. Total aerobic, coliform and Escherichia coli counts per gram were enumerated for each sample. The mean log (x) and standard deviation (s) were calculated for the log10 values for each set of 25 counts, on the assumption that the distribution of counts approximated the log normal. A value for the log10 of the arithmetic mean (log A) was calculated for each set from the values of x and s. A chi2 statistic was calculated for each set as a test of the assumption of the log normal distribution. The chi2 statistic was calculable for 32 of the 39 sets. Four of the sets gave chi2 values indicative of gross deviation from log normality. On inspection of those sets, distributions obviously differing from the log normal were apparent in two. Log A values for total, coliform and E. coli counts for chilled patties from manufacturing plants ranged from 4.4 to 5.1, 1.7 to 2.3 and 0.9 to 1.5, respectively. Log A values for frozen patties from manufacturing plants were between < 0.1 and 0.5 log10 units less than the equivalent values for chilled patties. Log A values for total, coliform and E. coli counts for frozen patties on retail sale ranged from 3.8 to 8.5, < 0.5 to 3.6 and < 0 to 1.9, respectively. The equivalent ranges for chilled patties on retail sale were 4.8 to 8.5, 1.8 to 3.7 and 1.4 to 2.7, respectively. The findings indicate that the general hygienic condition of hamburgers patties could be improved by their being manufactured from only manufacturing beef of superior hygienic quality, and by the better management of chilled patties at retail outlets.

Movement therapy with depressed inpatients: a randomized multiple single case design.

The relative lack of research on movement therapy in inpatient versus outpatient settings stems from the difficulty of conducting an interpretable study in clinical situations where multiple treatments exist. To control for the multiple treatment confound, this study used a randomized single-case experimental design with 12 replications. Results indicated that the movement therapy, which was designed to target the syndrome of a major depressive episode had a positive effect on mood across experiments (p < .001). From a clinical perspective, these results support the use of a movement program as adjunctive treatment, and challenge the view that movement is recreation but not therapy.

Quality of fresh retail pork cuts stored in modified atmosphere under temperature conditions simulating export to distant markets.

The effect of storage temperature on microbial and sensory quality of retail cuts of pork was determined on samples stored under temperature regimens designed to simulate conditions that could be encountered in accessing distant markets with retail-ready product. Samples were packaged in modified atmosphere with 100% CO(2) and <200 ppm O(2) in plastic film with extremely low gas transmission rates. All samples were stored at -1·5°C for three weeks. Reference samples were held at -1·5°C for the duration of the study; experimental samples were transferred to 4°C (-1·5 4° C ) or 7°C (-1· 517° C ) and analyzed for microbial content and sebsory attributes including appearance, confinement and meat odours. Storage life of reference samples at -1·5°C was seven weeks before rejection for loss of acceptable appearance. With transfer of samples to 4 and 7°C after three weeks at -1·5°C, samples remained acceptable for retail sale for two weeks and one week, restpectively. The microbial flora was dominated by lactic acid bacteria under all three storage conditions. Appearance of the cuts was the principal criterion limiting storage life. Discoloration of the meat was not a problem in this study, but purge and odour, including sour and sulphur notes, became a problem with time. The study indicated that export of retail-ready pork cuts to distant markets with a three-week time for delivery to market at -1·5°C can be achieved with one to two weeks of marketing time at retail market at 4 to 7°C.

Microbial ecology of fresh pork stored under modified atmosphere at -1, 4.4 and 10 degrees C.

The prevalent bacteria on fresh pork packaged in modified atmosphere with elevated CO2 were determined by selection of representative colonies from the greatest dilution of meat samples. The pork samples were stored in two packaging films of different oxygen permeability at three storage temperatures. Strains were classified and those identified as lactic acid bacteria were screened for production of inhibitory substances. The types of bacteria isolated from samples stored in the two packaging films were similar. Storage temperature influenced the type of bacteria that dominated the microbial population. At 10 degrees C the prevalent microflora consisted of aeromonads, Enterobacteriaceae and lactic acid bacteria but at 4.4 and -1 degrees C, aeromonads, Brochothrix thermosphacta and lactic acid bacteria dominated. Listeriae were detected as part of the prevalent microflora on samples stored at -1 degree C, but not on samples stored at 4.4 or 10 degrees C. Species of lactic acid bacteria dominating the microflora were influenced by growth medium. The majority of isolates taken from Plate Count agar were carnobacteria whereas those from Lactobacilli MRS agar were homofermentative lactic acid bacteria. Of the 538 lactic acid bacteria isolates screened for production of inhibitory substances, 162 strains showed deferred inhibition toward a range of lactic acid bacteria and nonlactic acid bacteria indicator strains.

WAIS characteristics of non-pathological obsessive and hysteric styles.

Tested traditional clinical hypotheses about the cognitive functioning of individuals (N = 16) with an obsessive or an hysteric style in a non-pathological population using selected subtests of the WAIS. Individuals identified as having an obsessive style displayed the predicted patterns (Information and Vocabulary greater than Comprehension), while their hysteric counterparts displayed only a trend toward certain predicted patterns (Comprehension greater than Information and Vocabulary). Predicted between-group differences were significant. Higher base rates of obsessive features in a college population may account for the weaker trends within the hysteric group. Generally, the results appear to support the likelihood that nonpathological forms of the two styles display patterns of cognitive functioning similar to those of their more pathological counterparts.