The contribution of sampling uncertainty to total measurement uncertainty in the enumeration of microorganisms in foods.

Random samples of each of several food products were obtained from defined lots during processing or from retail outlets. The foods included raw milk (sampled on farm and from a bulk-milk tanker), sprouted seeds, raw minced meat, frozen de-shelled raw prawns, neck-flaps from raw chicken carcasses and ready-to-eat sandwiches. Duplicate sub-samples, generally of 100 g, were examined for aerobic colony counts; some were examined also for counts of presumptive Enterobacteriaceae and campylobacters. After log(10)-transformation, all sets of colony count data were evaluated for conformity with the normal distribution (ND) and analysed by standard ANOVA and a robust ANOVA to determine the relative contributions of the variance between and within samples to the overall variance. Sampling variance accounted for >50% of the reproducibility variance for the majority of foods examined; in many cases it exceeded 85%. We also used an iterative procedure of re-sampling without replacement to determine the effects of sample size (i.e. the number of samples) on the precision of the estimate of variance for one of the larger data sets. The variance of the repeatability and reproducibility variances depended on the number of replicate samples tested (n) in a manner that was characteristic of the underlying distribution. The results are discussed in relation to the use of measurement uncertainty in assessing compliance of results with microbiological criteria for foods.

Detection of cold-tolerant clostridia other than Clostridium estertheticum in raw vacuum-packed chill-stored meat.

Samples from raw chill-stored vacuum-packed beef, lamb and venison or the meat processing environment, associated with a spoilage problem, but negative for Clostridium estertheticum using a specific real-time PCR test, were examined for other Clostridium spp. using direct 16S rDNA PCR-RFLP and sequencing. Of 291 samples tested by PCR, presence of clostridia was indicated in 123 and there was sufficient PCR product in 35 to be further investigated. Presence of Clostridium spp. was confirmed by RFLP and sequencing in 25/35 samples (11 of 14 incidents). Species detected in spoiled meat were (incidents): Clostridium tagluense-like (4), Clostridium putrefaciens (2), Clostridium algidicarnis (3), Clostridium frigoris/estertheticum-like (3) and Clostridium. gasigenes (2). More than one species was detected in some incidents. All of the above species have previously been associated with spoiled meat apart from the Cl. tagluense-like species. Clostridia were also confirmed in 4/7 samples from the environment, with two Cl. frigoris/estertheticum-like and two mesophilic species of Clostridium. Our study showed that, cold-tolerant Clostridium species other than Cl. estertheticum are occasionally associated with spoiled vacuum-packed meat, particularly lamb. Further studies are required to confirm the exact identity of the Cl. tagluense-like species and its role in meat spoilage.

Minimising the between-sample variance in colony counts on foods.

The objective of this study was to assess whether it is possible to minimise the variance in colony counts on replicate target samples of foods by aseptic compositing of the samples or by increasing the quantity of sample examined. The results show that compositing reduces the overall variance, and hence the standard deviation, to very low levels, although in some cases the overall variance remains relatively high, reflecting the heterogeneous distribution of microorganisms in the foods. Increasing the weight of target sample examined (e.g. from 10 g to 100g) had a pronounced effect on the mean log(10) colony count and significantly reduced the variance of the mean. The results are discussed in relation to the quantity of sample that is recommended for examination in international and other standards.

Assessment of measurement uncertainty for quantitative methods of analysis: comparative assessment of the precision (uncertainty) of bacterial colony counts.

An interlaboratory trial was made, by three analysts in each of the 19 laboratories, of three International Standards Organisation (ISO) colony count methods for aerobic organisms (ACC), Enterobacteriaceae (ECC) and Escherichia coli (EcCC). All tests were done in duplicate and were further replicated by plating both on culture media supplied by the organisers and on each laboratory's own choice of culture media. In order to avoid any influence of food matrix on the results, the inoculum for each test was a freeze-dried ampoule of a standardised mixed culture. After collation of test results, individual data sets were examined for obvious non-consistency, and colony counts for each individual test were determined both as simple and 'weighted' mean values. The derived colony counts were then log(10)-transformed and examined statistically. Estimates of repeatability and reproducibility for each set of results were derived and used to calculate the parameters for the uncertainty of measurement. Estimated values of the uncertainty of reproducibility and repeatability for the ACC ranged, respectively, from 9.3 to 12.1% and 2.0 to 3.9% of the mean log(10) colony count, depending on the specific culture media, the method of deriving the mean count and the statistical procedure employed. Similarly, estimates of the uncertainty of reproducibility and repeatability for the ECC ranged, respectively, from 14.0 to 17.4% and 4.1 to 6.7%. The estimates of uncertainty of reproducibility and repeatability for the EcCC data ranged, respectively, from 21.1 to 30.9% and 6.7 to 15.4%. Whilst the uncertainty estimates for the ACC data conform to long-held views on the repeatability and reproducibility of microbial count data, the estimates for the ECC and EcCC are considerably greater. It was notable that no benefits were seen in the use of the weighted mean as compared to simple mean colony count. The importance of uncertainty estimates of colony count data in the assessment of the microbiological quality of foods is discussed.

Decontamination of poultry carcasses using steam or hot water in combination with rapid cooling, chilling or freezing of carcass surfaces.

The effects of the application of steam at atmospheric pressure for times up to 20 s on the numbers of inoculated Campylobacter jejuni and Escherichia coli on whole chicken carcasses were investigated in a pilot steam cabinet. Steam treatments reduced the numbers of C. jejuni AR6 by ca. 1.8, 2.6 and 3.3 log(10) cfu cm(-2) in 10, 12 and 20 s, respectively. Corresponding reductions in numbers of E. coli K12 were 1.7, 2.3 and 2.8 log(10) cfu cm(-2). However, such treatments caused the skin to shrink and change colour. The optimum treatment for maximum reductions of C. jejuni and E. coli, least skin shrinkage and change of colour was concluded to be <12 s. Further work was carried out to determine whether a modified air chilling system in combination with steam or hot water decontamination treatments could be used to reduce numbers of pathogens, particularly campylobacters, on the surface of poultry carcasses. Whole chicken carcasses inoculated with C. jejuni and E. coli were either not treated, treated with steam at atmospheric pressure for up to 10 s or treated with hot water at 80 degrees C for up to 20 s, then either chilled by crust freezing, chilled at 0 degrees C, or chilled at 15 degrees C, in a pilot chilling chamber. The optimum combination was treatment with water at 80 degrees C for 20 s followed by crust freezing, which reduced the numbers of C. jejuni and E. coli by ca. 2.9 and 3.2 log(10) cfu cm(-2), respectively, without extensive degradation of carcass appearance.

Humidification of unwrapped chilled meat on retail display using an ultrasonic fogging system.

The effects of an ultrasonic humidification system on unwrapped meat in a chilled retail display cabinet were assessed. Humidification raised the relative humidity of the cabinet air from a mean of 76.7% to just below saturation at 98.8%. This reduced the mean evaporative weight loss from whole samples of meat after 14h from 1.68% to 0.62% of their initial weight. The rate of deterioration in the appearance of the meat due to dehydration was reduced to the extent that while the unhumidified trial was terminated after 14h because all samples were judged to be unacceptable, the humidified trial was continued for 24h without any major changes in appearance. Levels of presumptive pseudomonas bacteria were relatively high in water samples taken from the humidification system and defrost water during the humidified trial, but Legionella spp. were not isolated. Significant increases in the numbers of bacteria on the meat during either trial were only found in one case, that of humidified minced beef. However, some of the samples had high counts even before display, and this may have masked any effect due to humidification. Differences in levels of air-borne contamination were small and inconsistent. Air temperatures were raised by humidification by between 1 and 2°C and this was reflected in similarly raised product temperatures. Temperatures of air leaving the evaporator indicated that this was due to icing of the evaporator in the periods leading up to defrosts.

Combined steam and ultrasound treatment of broilers at slaughter: a promising intervention to significantly reduce numbers of naturally occurring campylobacters on carcasses.

Steam or hot water decontamination treatment of broiler carcasses is hampered by process limitations due to prolonged treatment times and adverse changes to the epidermis. In this study, a combination of steam with ultrasound (SonoSteam®) was investigated on naturally contaminated broilers that were processed at conventional slaughter speeds of 8,500 birds per hour in a Danish broiler plant. Industrial-scale SonoSteam equipment was installed in the evisceration room, before the inside/outside carcass washer. The SonoSteam treatment was evaluated in two separate trials performed on two different dates. Numbers of naturally occurring Campylobacter spp. and TVC were determined from paired samples of skin excised from opposite sides of the breast of the same carcass, before and after treatments. Sampling was performed at two different points on the line: i) before and after the SonoSteam treatment and ii) before the SonoSteam treatment and after 80 min of air chilling. A total of 44 carcasses were examined in the two trials. Results from the first trial showed that the mean initial Campylobacter contamination level of 2.35 log10 CFU was significantly reduced (n=12, p<0.001) to 1.40 log10 CFU after treatment. A significant reduction (n=11, p<0.001) was also observed with samples analyzed before SonoSteam treatment (2.64 log10 CFU) and after air chilling (1.44 log10 CFU). In the second trial, significant reductions (n=10, p<0.05) were obtained for carcasses analyzed before (mean level of 2.23 log10 CFU) and after the treatment (mean level of 1.36 log10 CFU). Significant reductions (n=11, p<0.01) were also found for Campylobacter numbers analyzed before the SonoSteam treatment (2.02 log10 CFU) and after the air chilling treatment (1.37 log10 CFU). The effect of air chilling without SonoSteam treatment was determined using 12 carcasses pre- and postchill. Results showed insignificant reductions of 0.09 log10 from a mean initial level of 2.19 log10 CFU. Numbers of TVC before treatments ranged between 3.47 and 4.79 log10 CFU. In all cases, TVC was significantly (p<0.001, n=45 in each trial) reduced by approximately 0.7 log10 CFU. An authorized sensory panel at the Danish Veterinary and Food Administration concluded that broiler carcasses treated with SonoSteam were acceptable for purchase. These conclusions were based on organoleptic differences (smell, skin/meat consistency, texture and color) of treated vs. untreated carcasses. Results obtained from this study suggest that steam-ultrasound treatment of carcasses at broiler processing plants can significantly reduce numbers of Campylobacter on naturally contaminated broilers.

Wide variety of bioserotypes of enteropathogenic Yersinia in tonsils of English pigs at slaughter.

The tonsils of 630 pigs from 45 English farms using three different rearing methods (Assured British Pigs, Open Management and Organic) were examined between 2003 and 2005 in order to investigate if the low incidence of human yersiniosis could be attributed to a low prevalence of enteropathogenic Yersinia among English pigs. In addition, different isolation methods were compared, possible differences in prevalence among pigs were studied, as well as the prevalence of different bioserotypes of enteropathogenic Yersinia. A high prevalence and a wide diversity of bioserotypes of enteropathogenic Yersinia compared to other European countries were observed. The prevalence of pathogenic Yersinia enterocolitica was 44% and of Yersinia pseudotuberculosis 18%. Overall, 60% of pigs carried enteropathogenic Yersinia. Y. pseudotuberculosis was detected on 78% of farms and Y. enterocolitica on 69%. The most common bioserotypes of Y. enterocolitica were 2/O:9 (33%) and 2/O:5 (26%), and of Y. pseudotuberculosis 2/O:3 (34%), 1/O:1 (26%) and 1/O:4 (24%). Cold enrichment gave the highest isolation rate for both species. Y. enterocolitica was more prevalent (P<0.001) and Y. pseudotuberculosis less prevalent (P<0.05) in winter than in summer in Eastern England. Y. enterocolitica was more common in Eastern England and in assured British pigs, whereas Y. pseudotuberculosis was more common in Western England and in organic pigs. Y. pseudotuberculosis 1/O:1 was predominant (P<0.05) in Western England. Types 1/O:4 (P<0.05) and 2/O:3 (P<0.001) predominated in Eastern England. The high prevalence of Y. enterocolitica bioserotypes 2/O:9 and 2/O:5 found in this study suggests that English pigs are an important reservoir of these bioserotypes whereas in other European countries bioserotype 4/O:3 predominates.

Measurement uncertainty of the EU methods for microbiological examination of red meat.

Three parallel trials were made of EU methods proposed for the microbiological examination of red meat using two analysts in each of seven laboratories within the UK. The methods involved determination of aerobic colony count (ACC) and Enterobacteriaceae colony count (ECC) using simulated methods and a freeze-dried standardised culture preparation. Trial A was based on a simulated swab test, Trial B a simulated meat excision test and Trial C was a reference test on reconstituted inoculum. Statistical analysis (ANOVA) was carried out before and after rejection of outlying data. Expanded uncertainty values (relative standard deviation x2) for repeatability and reproducibility, based on the log10 cfu/ml, on the ACC ranged from +/-2.1% to +/-2.7% and from +/-5.5% to +/-10.5%, respectively, depending upon the test procedure. Similarly for the ECC, expanded uncertainty estimates for repeatability and reproducibility ranged from +/-4.6% to +/-16.9% and from +/-21.6% to +/-23.5%, respectively. The results are discussed in relation to the potential application of the methods.

A critical review of measurement uncertainty in the enumeration of food micro-organisms.

Derivation of uncertainty provides a way to standardize the expression of variability associated with any analytical procedure. The published information on uncertainty associated with data obtained using microbiological procedures is reviewed to highlight the causes and magnitude of such variability in food microbiology. We also suggest statistical procedures that can be used to assess variability (and hence, uncertainty), within and between laboratories, including procedures that can be used routinely by microbiologists examining foods, and the use of 'robust' methods which allow the retention of 'outlying' data. Although concerned primarily with variability associated with colony count procedures, we discuss also the causes of variability in presence/absence and indirect methods, such as limiting dilution, most probable number and modern instrumental methods of microbiological examination. Recommendations are also made concerning the most important precautions to be taken in order to minimize uncertainty in microbiology. These include strict internal controls at all stages of microbiological testing, as well as validation of methods, trend analysis, use of reference materials and participation in proficiency testing schemes. It is emphasized that the distribution of microbes in foods is inherently heterogeneous, and that this review only addresses uncertainty of measurement with respect to the sample taken, not the lot or consignment of food from which the sample was taken.

Heat resistance and mechanism of heat inactivation in thermophilic campylobacters.

The heat resistance of Campylobacter jejuni strains AR6 and L51 and the heat resistance of Campylobacter coli strains DR4 and L6 were measured over the temperature range from 50 to 60 degrees C by two methods. Isothermal measurements yielded D55 values in the range from 4.6 to 6.6 min and z values in the range from 5.5 to 6.3 degrees C. Dynamic measurements using differential scanning calorimetry (DSC) during heating at a rate of 10 degrees C/min yielded D55 values of 2.5 min and 3.4 min and z values of 6.3 degrees C and 6.5 degrees C for AR6 and DR4, respectively. Both dynamic and isothermal methods yielded mean D55 values that were substantially greater than those reported previously (0.75 to 0.95 min). DSC analysis of each strain during heating at a rate of 10 degrees C/min yielded a complex series of overlapping endothermic peaks, which were assigned to cell wall lipids, ribosomes, and DNA. Measurement of the decline in the numbers of CFU in calorimetric samples as they were heated showed that the maximum rate of cell death occurred at 56 to 57 degrees C, which is close to the value predicted mathematically from the isothermal measurements of D and z (61 degrees C). Both estimates were very close to the peak m1 values, 60 to 62 degrees C, which were tentatively identified with unfolding of the 30S ribosome subunit, showing that cell death in C. jejuni and C. coli coincided with unfolding of the most thermally labile regions of the ribosome. Other measurements indicated that several essential proteins, including the alpha and beta subunits of RNA polymerase, might also unfold at the same time and contribute to cell death.

Sources of Salmonella on broiler carcasses during transportation and processing: modes of contamination and methods of control.

AIMS: The prevalence and types of salmonella in broiler chickens during transportation and during slaughter and dressing were studied. This was part of a comprehensive investigation of salmonellas in two UK poultry companies, which aimed to find the origins and mechanisms of salmonella contamination. METHODS AND RESULTS: Salmonellas were isolated using cultural methods. Serovars of Salmonella detected during rearing were usually also found in a small proportion of birds on the day of slaughter and on the carcasses at various points during processing. There was little evidence of salmonellas spreading to large numbers of carcasses during processing. Many serovars found in the feedmills or hatcheries were also detected in the birds during rearing and/or slaughter. Transport crates were contaminated with salmonellas after washing and disinfection. CONCLUSIONS: Prevalence of salmonellas fell in the two companies during this survey. A small number of serovars predominated in the processing plants of each company. These serovars originated from the feed mills. Reasons for transport crate contamination were: (1) inadequate cleaning, resulting in residual faecal soiling; (2) disinfectant concentration and temperature of disinfectant too low; (3) contaminated recycled flume water used to soak the crates. SIGNIFICANCE AND IMPACT OF THE STUDY: Efforts to control salmonella infection in broilers need to concentrate on crate cleaning and disinfection and hygiene in the feed mills.