Incidence of Acinetobacter spp. and other gram-negative, oxidase-negative bacteria in fresh and spoiled ground beef.

A total of 1,409 gram-negative bacterial colonies were randomly selected from 19 samples of fresh and spoiled ground beef plated on six media. Only 137 (9.7%) were oxidase negative, and 20 (14.6%) of these were Acinetobacter spp., all of which were recovered from fresh meat samples. The importance of this group in both fresh and spoiled beef is less than is generally believed.     

Aerobic and Facultative Microflora of Fresh and Spoiled Refrigerated Dough Products

The microbial flora of fresh, unsterile, dough products held at refrigeration temperatures was compared with the microbial flora of the same products that had spoiled spontaneously. Various methods based on selective media were used to determine molds, yeasts, and bacteria present. Except for two special cases in which a yeast and Penicillium roqueforti induced spoilage, all of the samples deteriorated because of bacterial growth. A total of 1,132 bacterial isolates was subjected to further classification. In the spoiled products, 92% of the isolates belonged to the Lactobacillaceae. More than one-half of these (53%) belonged to the genus Lactobacillus, and an additional 36% were in the genus Leuconostoc. In the genus Leuconostoc almost all of the strains (94%) were L. mesenteroides. The third most common genus present was Streptococcus, represented by 3% of the total isolates. A preliminary taxonomic study of the microflora of refrigerated dough products revealed none of the isolates to be indicators of fecal contamination and none to be forms known to produce toxins. The highest counts encountered in the moist, fresh products were up to 200 million lactic acid bacteria per g in buttermilk biscuits, with a psychrophilic count as high as 4.8 million. In the spoiled samples, the highest total counts were 820 million in buttermilk biscuits. Mold counts were no higher than 1,800, except in the sample ruined by P. roqueforti where the count was 130,000 mold colonies.     

Isolation of Escherichia coli O157:H7 from retail fresh meats and poultry.

A total of 896 samples of retail fresh meats and poultry was assayed for Escherichia coli serogroup O157:H7 by a hydrophobic grid membrane filter-immunoblot procedure developed specifically to isolate the organism from foods. The procedure involves several steps, including selective enrichment, filtration of enrichment culture through hydrophobic grid membrane filters, incubation of each filter on nitrocellulose paper on selective agar, preparation of an immunoblot (by using antiserum to E. coli O157:H7 culture filtrate) of each nitrocellulose paper, selection from the filters of colonies which corresponded to immunopositive sites on blots, screening of isolates by a Biken test for precipitin lines from metabolites and antiserum to E. coli O157:H7 culture filtrate, and confirmation of isolates as Vero cell cytotoxic E. coli O157:H7 by biochemical, serological, and Vero cell cytotoxicity tests. E. coli O157:H7 was isolated from 6 (3.7%) of 164 beef, 4 (1.5%) of 264 pork, 4 (1.5%) of 263 poultry, and 4 (2.0%) of 205 lamb samples. One of 14 pork samples and 5 of 17 beef samples contaminated with the organism were from Calgary, Alberta, Canada, grocery stores, whereas all other contaminated samples were from Madison, Wis., retail outlets. This is the first report of the isolation of E. coli O157:H7 from food other than ground beef, and results indicate that the organism is not a rare contaminant of fresh meats and poultry.     

Origin and ecological selection of core and food-specific bacterial communities associated with meat and seafood spoilage

The microbial spoilage of meat and seafood products with short shelf lives is responsible for a significant amount of food waste. Food spoilage is a very heterogeneous process, involving the growth of various, poorly characterized bacterial communities. In this study, we conducted 16S ribosomal RNA gene pyrosequencing on 160 samples of fresh and spoiled foods to comparatively explore the bacterial communities associated with four meat products and four seafood products that are among the most consumed food items in Europe. We show that fresh products are contaminated in part by a microbiota similar to that found on the skin and in the gut of animals. However, this animal-derived microbiota was less prevalent and less abundant than a core microbiota, psychrotrophic in nature, mainly originated from the environment (water reservoirs). We clearly show that this core community found on meat and seafood products is the main reservoir of spoilage bacteria. We also show that storage conditions exert strong selective pressure on the initial microbiota: alpha diversity in fresh samples was 189±58 operational taxonomic units (OTUs) but dropped to 27±12 OTUs in spoiled samples. The OTU assemblage associated with spoilage was shaped by low storage temperatures, packaging and the nutritional value of the food matrix itself. These factors presumably act in tandem without any hierarchical pattern. Most notably, we were also able to identify putative new clades of dominant, previously undescribed bacteria occurring on spoiled seafood, a finding that emphasizes the importance of using culture-independent methods when studying food microbiota.     

Evaluation of the Extract-Release Volume Phenomenon as a Rapid Test for Detecting Spoilage in Beef

Ground beef of several grades, obtained fresh and held refrigerated until spoiled, was presented to a test panel for scoring on color, odor, and tactile response (tackiness) as to degree of acceptance. Panel scores were correlated with total bacterial counts, ninhydrin-reactive substances, and ERV (extract-release volume) on the same meat. ERV correlated highest with bacterial counts the largest number of times; tackiness, odor, ninhydrin, and color followed in that order. Correlation between bacterial numbers and organoleptic qualities was best, with tackiness followed closely by odor, and then by color. Correlation between tackiness and odor was high. The degree of correlation between bacterial numbers, tackiness, and ERV was high enough to warrant the use of the ERV phenomenon as a rapid test of microbial quality of beef. An ERV value of 25 under the conditions of the test was supported as a divider between acceptable and unacceptable ground beef.     

Changes in the spoilage-related microbiota of beef during refrigerated storage under different packaging conditions

The microbial spoilage of beef was monitored during storage at 5 degrees C under three different conditions of modified-atmosphere packaging (MAP): (i) air (MAP1), (ii) 60% O2 and 40% CO2 (MAP2), and (iii) 20% O2 and 40% CO2 (MAP3). Pseudomonas, Enterobacteriaceae, Brochothrix thermosphacta, and lactic acid bacteria were monitored by viable counts and PCR-denaturing gradient gel electrophoresis (DGGE) analysis during 14 days of storage. Moreover, headspace gas composition, weight loss, and beef color change were also determined at each sampling time. Overall, MAP2 was shown to have the best protective effect, keeping the microbial loads and color change to acceptable levels in the first 7 days of refrigerated storage. The microbial colonies from the plate counts of each microbial group were identified by PCR-DGGE of the variable V6-V8 region of the 16S rRNA gene. Thirteen different genera and at least 17 different species were identified after sequencing of DGGE fragments that showed a wide diversity of spoilage-related bacteria taking turns during beef storage in the function of the packaging conditions. The countable species for each spoilage-related microbial group were different according to packaging conditions and times of storage. In fact, the DGGE profiles displayed significant changes during time and depending on the initial atmosphere used. The spoilage occurred between 7 and 14 days of storage, and the microbial species found in the spoiled meat varied according to the packaging conditions. Rahnella aquatilis, Rahnella spp., Pseudomonas spp., and Carnobacterium divergens were identified as acting during beef storage in air (MAP1). Pseudomonas spp. and Lactobacillus sakei were found in beef stored under MAP conditions with high oxygen content (MAP2), while Rahnella spp. and L. sakei were the main species found during storage using MAP3. The identification of the spoilage-related microbiota by molecular methods can help in the effective establishment of storage conditions for fresh meat.     

The effects of feeding broiler litter on microbial contamination of beef carcasses

Two experiments were conducted to test the effects of feeding broiler litter, either directly in the diet or indirectly through pasture-fertilization, to beef cattle on the incidence of Salmonella typhimurium (S) and Escherichia coli O157:H7 (EC) contamination of carcasses and ground beef. In Experiment 1, beef cows (n = 32) were allotted either ad libitum access to grass hay or a formulated diet (80% deep-stacked broiler litter and 20% corn). In Experiment 2, beef cows (n = 32) were assigned to graze on pastures fertilized with a commercial fertilizer or fresh broiler litter. Cows in Experiment 1 were harvested following a 56-d feeding period; whereas, cows in Experiment 2 were harvested after 5, 10, 20, and 40 d of grazing pastures. All samples of muscle, purge, and ground beef were culture-negative for S and EC, suggesting that beef cattle may consume properly handled deep-stacked broiler litter, or pastures fertilized with fresh litter, without increasing the likelihood of carcass/meat contamination with S and (or) EC.     

Aflatoxin Production in Meats. I. Stored Meats

Aflatoxins were produced on fresh beef (in which bacterial spoilage was delayed with antibiotics), ham, and bacon inoculated with toxinogenic fungi and stored at 15, 20 and 30 C. Meats stored at 10 C were spoiled by bacteria and yeast before detectable levels of aflatoxins were produced. High levels of aflatoxins were formed in meats stored at 20 C; one sample supported the production of 630 mug of aflatoxins per g of meat, the major portion (580 mug) of which was aflatoxin G(1). Meats stored below 30 C developed higher levels of aflatoxin G(1) than B(1), but at 30 C Aspergillus flavus produced equal amounts of B(1) and G(1), whereas A. parasiticus continued to produce more G(1) than B(1).     

Culture medium for selective isolation and enumeration of Gram-negative bacteria from ground meats.

We developed a new medium, designated peptone bile amphotericin cycloheximide (PBAC) agar, which contains (per liter) 10 g of peptone, 300 mg of bile salts, 1 mg of amphotericin B, 1 g of cycloheximide, and 15 g of agar. When 21 samples of fresh ground beef were studied and plate count agar counts were used as references, we obtained a mean recovery of 28% of total counts with violet red bile agar overlay, whereas we obtained 48% recovery with PBAC agar. With 12 samples of frozen ground beef, recovery on violet red bile agar overlay was 29% of the recovery on plate count agar, whereas the corresponding value on PBAC agar was 45%. PBAC agar allowed the enumeration of 1.4 times as many gram-negative bacteria as violet red bile agar overlay. None of eight strains of gram-positive bacteria and none of eight strains of yeasts grew on PBAC agar. Of 158 colonies randomly selected from pour plates of eight fresh ground meat samples, 95% stained gram negative. In comparison, only 70% of 151 colonies selected from corresponding plate count agar plates were gram negative. The lack of background color, turbidity, and ease of use make PBAC agar easier to handle than other media used for gram-negative bacteria, such as violet red bile agar, violet red bile agar overlay, and crystal violet tetrazolium agar. In the preparation PBAC agar, all ingredients are autoclaved together except amphotericin B, which is filter sterilized and added before the plates are poured.     

Detection and recovery rates achieved using direct plate and enrichment/immunomagnetic separation methods for Escherichia coli O157:H7 in minced beef and on bovine hide

AIMS: To assess the detection and recovery rates achieved with commonly used cultural methods for the enumeration and recovery of Escherichia coli O157:H7 from minced beef and bovine hide. METHODS AND RESULTS: Minced beef and bovine hide were inoculated with varying concentrations (log(10) 1.58-2.58 CFU g(-1) and log(10) 2.42-4.49 CFU 100 cm(2) respectively) of E. coli O157:H7 and recovered using a direct plate method or an enrichment/immunomagnetic separation (IMS) method and then plated onto SMAC or SMAC-CT in both cases. The direct plate method detected the pathogen consistently from minced beef samples with an average recovery of 69.2-91.2%. From faecal material on the bovine hide the recovery of the pathogen ranged from 1.80 to 64.5% with fresh faeces depending on the inocula while from dried faeces on hide the results ranged from no recovery at all to 25.1%. Enrichment/IMS recovered E. coli O157:H7 at all inocula levels tested in minced beef while the pathogen was only detected consistently at an average inocula level of log(10) 2.73 CFU 100 cm(2) from fresh faeces and log(10) 4.49 CFU 100 cm(2) from dried faeces on bovine hide. CONCLUSIONS: The direct count enumeration method for E. coli O157:H7 underestimated the numbers of pathogens present. The enrichment/IMS procedure consistently detected the pathogen from minced beef but did not always detect E. coli O157:H7 from faeces on bovine hide. SIGNIFICANCE AND IMPACT OF THE STUDY: Overall this study highlights that any microbial data, used in either predictive microbiology or risk assessment, must take account of the sensitivity and associated performance of the methods employed, in order to make an accurate reflection of the true microbiology of the examined sample.     

Use of a Titrimetric Method to Assess the Bacterial Spoilage of Fresh Beef

A new method of determining bacterial spoilage in fresh beef is presented. The technique is based upon the fact that as beef undergoes refrigerator spoilage, there is a gradual increase in the production of alkaline substances by the spoilage flora. The level of these substances was measured by titrating meat homogenates to a pH 5.00 end point, employing 0.02 n HCl and an autotitrator. When 23 samples of ground beef from retail stores were tested, an average of 1.32 ml of acid was required for titration of 1 g of fresh beef to pH 5.00, whereas 2.58 ml was required for the same meat at the onset of spoilage. Preliminary data indicate that beef which requires more than 2 ml of 0.02 n HCl/g to lower its pH to 5.00 under the conditions of the test is in some state of incipient spoilage. The statistical correlation between titration values, log bacterial numbers, and extract-release volume was high (P < 0.001). The technique is simple to execute and is highly reproducible, and duplicate samples can be run within 15 min.     

A numerical taxonomic study of Pseudomonas strains from spoiled meat

A numerical taxonomic study using 160 unit characters has been performed on 110 isolates of pseudomonads from aerobically spoiled beef and pork and on 13 named strains from the genus Pseudomonas. Four clusters of meat strains were detected at 87% S. Non-fluorescent strains were contained in two closely related clusters (1 and 2) which were identified with P. fragi. Clusters 3 and 4 contained fluorescent strains which were distinct from P. fluorescens, P. putida and the other named strains examined. An identification scheme based on 11 carbon source tests is presented for the recognition of cluster members.     

Effect of microbial cell‐free meat extract on the growth of spoilage bacteria

Aims: This study examined the effect of microbial cell‐free meat extract (CFME) derived from spoiled meat, in which quorum sensing (QS) compounds were present, on the growth kinetics (lag phase, and growth rate) of two spoilage bacteria, Pseudomonas fluorescens and Serratia marcescens. Methods and Results: Aliquots of CFME from spoiled meat were transferred to Brain Heart Infusion broth inoculated with 10³ CFU ml^?1 of 18 h cultures of Ps. fluorescens or Ser. marcescens, both fresh meat isolates; CFME derived from unspoiled fresh meat (‘clean’ meat) served as a control. Changes in impedance measurements were monitored for 48 h, and the detection time (Tdet) was recorded. It was found that in the absence of CFME containing QS compounds the Tdet was shorter (P < 0·05) than that in broth samples with added CFME from spoiled meat. The rate of growth of Ps. fluorescens, recorded as the maximum slope rate of conductance changes (MSrCC), after Tdet, was higher (P < 0·05) in samples with CFME containing QS compounds compared to samples without CFME or CFME derived from ‘clean’ meat. Similar results in MSrCC of impedance changes were obtained for Ser. marcescens. Conclusions: The study indicated that the growth rate (expressed in MSrCC units) of meat spoilage bacteria in vitro was enhanced in samples supplemented with CFME containing QS compounds compared to control samples (i.e., without CFME or with CFME from ‘clean’ meat). This behaviour may explain the dominant role of these two bacteria in the spoilage of meat. Significance and Impact of the Study: These results illustrate the potential effect of signalling compounds released during storage of meat on the behaviour of meat spoilage bacteria. Understanding such interactions may assist in the control of fresh meat quality and the extension of its shelf life.     

ESTIMATION OF MICROBIOLOGICAL QUALITY OF CHILLED BEEF USING AUTOMATED TURBIDIMETRY

Total bacterial counts on chilled beef samples were estimated by the standard plate count method and by an automated turbidimetric system. The latter method is based on product-specific calibration curves constructed by correlating growth curve parameters calculated for the turbidimeter to the log CFU values obtained by plate counts. A total of 74 beef samples was used to construct the calibration curves. Correlation analysis between turbidimetric parameters and plate count values showed that detection time was the best predictor to estimate microbial loads on fresh (r=0.91) and aged beef (r=0.94). Microbial loads for a different set of aged beef samples (n = 37) refrigerated for 7, 9, 10, 17 and 45 days were compared by turbidimetric measurements and plate counts. Mean total viable counts were log 5.92 ± 1.17 and log 5.54 ± 1.28 CFU/mL, respectively. Results showed that total bacterial counts on chilled beef could be estimated accurately from turbidimetric parameters. Furthermore, setting a cut-off value of log 6 CFU/mL allowed to accepting/rejecting samples according to their microbial condition in shorter periods of time compared to the traditional plate count method.     

Incidence of Salmonellae in Meat and Meat Products

Standard enrichment, plating, and biochemical techniques were used to assess the incidence of Salmonella species on beef and pork carcasses and processed meat products. The incidence of Salmonella in pork carcasses was 56% and in beef carcasses, 74%. These figures are about the same as previously reported for pork but much higher than previously reported for beef carcasses; however, they represent only three to five abattoirs in Georgia and do not necessarily represent contamination levels throughout the country. Examination of carcasses by area did not indicate greater incidence of Salmonella in any one area. Two areas suggested for representative sampling are the cervical and anal areas of the carcass. Of the sausage samples examined, 38% of the fresh pork sausage, 9% of the smoked pork sausage, and 1 sample (souse) of 16 samples of miscellaneous sausage products were contaminated. Examination of subsamples indicated that Salmonella, when present in sausage products, could be found in any section of the entire sample.     

Growth hormone gene polymorphism and reproductive performance of AI bulls

Relationships between the growth hormone gene RFLP polymorphism and bull sperm characteristics were the objects of the present study. DNA was extracted from blood or sperm samples collected from 113 AI bulls and submitted for polymerase chain reaction (PCR) followed by digestion with Alu I restriction enzyme. The bGH genotypes were visualized on 10% polyacrylamide gel. The analyzed population of AI bulls consisted of dairy (Holstein Fresian [HF] crossbred [HF x Polish Black and White]) and beef breeds (Limousine, Charolaise, Piemontese, Angus and Hereford). The frequency of the Leu allele was 0.86 among dairy bulls and 0.38 in beef bulls (0.14 and 0.62 for the Val allele, respectively). Eight sperm characteristics and Day 60 non-return rates (NRR) were analyzed. The 3 genotype groups (LL, VV and LV) and the effect of production type (dairy or beef) on sperm characteristics were considered. None of the traits showed significant variability in relation to the bGH genotype, although a tendency was observed for LL bulls to have a lower ejaculate volume and VV bulls higher NRR. Moreover some statistically significant associations with production type were noticed: beef bulls were superior in sperm concentration and non-return rate, whereas dairy bulls excelled in individual fresh sperm motility.     

Rapid procedure for detecting enterohemorrhagic Escherichia coli O157:H7 in food.

A sensitive, specific procedure was developed for detecting Escherichia coli O157:H7 in food in less than 20 h. The procedure involves enrichment of 25 g of food in 225 ml of a selective enrichment medium for 16 to 18 h at 37 degrees C with agitation (150 rpm). The enrichment culture is applied to a sandwich enzyme-linked immunosorbent assay (ELISA) with a polyclonal antibody specific for E. coli O157 antigen as the capture antibody and a monoclonal antibody specific for enterohemorrhagic E. coli of serotypes O157:H7 and O26:H11 as the detection antibody. The ELISA can be completed within 3 h. The sensitivity of the procedure, determined by using E. coli O157:H7-inoculated ground beef and dairy products, including different varieties of cheese, was 0.2 to 0.9 cell per g of food. A survey of retail fresh ground beef and farm raw milk samples with this procedure revealed that 3 (2.8%) of 107 ground beef samples and 11 (10%) of 115 raw milk samples were positive for E. coli O157:H7. Most-probable-number determinations revealed E. coli O157:H7 populations of 0.4 to 1.5 cells per g in the three ground beef samples. In addition to being highly specific, sensitive, and rapid, this procedure is easy to perform and is amenable to use by laboratories performing routine microbiological testing.     

Factors affecting frozen and fresh embryo transfer pregnancy rates in cattle.

The effects of a large number of factors on the pregnancy rates of fresh and frozen cattle embryos were examined over a period of years at several different locations. For fresh embryos, overall pregnancy rates were 68.3% (n=9023) and 77.1% (n=2650) at different locations and time periods. Frozen-thawed embryo pregnancy rates were 56.1% (n=3616) in The Netherlands and 58.4% (n=5297) and 68.7% (n=774) for two studies in the United States. Pregnancy rates of surgical versus nonsurgical transfers were very similar. There were no differences in the pregnancy rates of beef versus dairy embryos, but the pregnancy rate was higher in dairy and beef heifers and beef cows than in dairy cows. Although on-farm pregnancy rates in California were higher than in the northeast United States, there was no influence of season on pregnancy rate. Estrous asynchrony between plus and minus 24 h did not affect pregnancy rate for frozen-thawed or fresh embryos. Neither breed nor parity of recipients affected the influence of asynchrony on pregnancy rates. Embryo grade was a significant factor in pregnancy rate for both fresh and frozen-thawed embryos, but neither embryo stage nor age was a significant factor. Pregnancy rate was not affected by holding embryos after flushing for up to 3 h prior to freezing.     

A Note on the Microflora of Beef Muscle Stored in Nitrogen at 0°

Fresh beef stored in nitrogen at 0° was spoiled by Gram positive rods. Almost all the organisms isolated were Microbacterium spp., although in one experiment about 10% of the organisms isolated were Lactobacillus spp. The numbers and types of organisms isolated were similar on each of two media incubated under aerobic and anaerobic conditions.     

Rapid procedure for detecting enterohemorrhagic Escherichia coli O157:H7 in food.

A sensitive, specific procedure was developed for detecting Escherichia coli O157:H7 in food in less than 20 h. The procedure involves enrichment of 25 g of food in 225 ml of a selective enrichment medium for 16 to 18 h at 37 degrees C with agitation (150 rpm). The enrichment culture is applied to a sandwich enzyme-linked immunosorbent assay (ELISA) with a polyclonal antibody specific for E. coli O157 antigen as the capture antibody and a monoclonal antibody specific for enterohemorrhagic E. coli of serotypes O157:H7 and O26:H11 as the detection antibody. The ELISA can be completed within 3 h. The sensitivity of the procedure, determined by using E. coli O157:H7-inoculated ground beef and dairy products, including different varieties of cheese, was 0.2 to 0.9 cell per g of food. A survey of retail fresh ground beef and farm raw milk samples with this procedure revealed that 3 (2.8%) of 107 ground beef samples and 11 (10%) of 115 raw milk samples were positive for E. coli O157:H7. Most-probable-number determinations revealed E. coli O157:H7 populations of 0.4 to 1.5 cells per g in the three ground beef samples. In addition to being highly specific, sensitive, and rapid, this procedure is easy to perform and is amenable to use by laboratories performing routine microbiological testing.