Spatial and Temporal Distribution of Escherichia coli on Beef Trimmings Obtained from a Beef Packing Plant.

The objective of this study was to determine the immediate source of Escherichia coli on beef trimmings produced at a large packing plant by analyzing the E. coli on trimmings at various locations of a combo bin filled on the same day and of bins filled on different days. Ten 2,000-lb (907-kg) combo bins (B1 through B10) of trimmings were obtained from a large plant on 6 days over a period of 5 weeks. Thin slices of beef with a total area of approximately 100 cm(2) were excised from five locations (four corners and the center) at each of four levels of the bins: the top surface and 30, 60, and 90 cm below the top. The samples were enriched for E. coli in modified tryptone soya broth supplemented with 20 mg/liter novobiocin. The positive enrichment cultures, as determined by PCR, were plated on E. coli/coliform count plates for recovery of E. coli. Selected E. coli isolates were genotyped using multiple-locus variable-number tandem repeat analysis (MLVA). Of the 200 enrichment cultures, 43 were positive by PCR for E. coli, and 32 of these cultures yielded E. coli isolates. Two bins did not yield any positive enrichment cultures, and three PCR-positive bins did not yield any E. coli isolates. MLVA of 165 E. coli isolates (30, 62, 56, 5, and 12 from B6 through B10, respectively) revealed nine distinct genotypes. MLVA types 263 and 89 were most prevalent overall and on individual days, accounting for 49.1 and 37.6% of the total isolates, respectively. These two genotypes were also found at multiple locations within a bin. All nine genotypes belonged to the phylogenetic group A0 of E. coli, suggesting an animal origin. The finding that the trimmings carried very few E. coli indicates an overall effective control over contamination of beef with E. coli at this processing plant. The lack of strain diversity of the E. coli on trimmings suggests that most E. coli isolates may have come from common sources, most likely equipment used in the fabrication process.

Characterization of germination of spores of Clostridium estertheticum, the primary causative agent of blown pack spoilage of vacuum packaged beef.

The aim of this study was to investigate the effect of various factors on the germination of Clostridium estertheticum endospores (spores) in relation to beef. The effect of heat on germination was determined by recovering C. estertheticum on Columbia agar from spore suspensions not heated or heated at 63, 70 or 80°C for various times. The effects of pH, temperature and oxygen were determined, by enumeration of remaining ungerminated spores during incubation in Meat Juice medium (MJM). Amino acids and lactate were tested for their ability to trigger germination of C. estertheticum spores by monitoring dipicolinic acid (DPA) release. Heat treatment of spores at 80°C for ≤20min significantly (p<0.05) increased the numbers of spores recovered on blood agar. Neither incubation temperature nor oxygen affected germination in MJM. The optimal pH for germination was 7.0 to 7.5. Incubation with leucine or aspartic acid caused a 1.3% release of DPA, the highest among all amino acids tested. Incubation with lactate resulted in a 4.1% release of DPA, which was significantly (p<0.05) higher than those from incubation with amino acids. The DPA release from incubation with lactate, lactate with amino acids, or MJM was similar (p>0.05).

Determination of Sources of Escherichia coli on Beef by Multiple-Locus Variable-Number Tandem Repeat Analysis.

The possible origin of Escherichia coli found on cuts and trimmings in the breaking facility of a beef packing plant was examined using multiple-locus variable-number tandem repeat analysis. Coliforms and E. coli were enumerated in samples obtained from 160 carcasses that would enter the breaking facility when work commenced and after each of the three production breaks throughout the day, from the conveyor belt before work and after each break, and from cuts and trimmings when work commenced and after each break. Most samples yielded no E. coli, irrespective of the surface types. E. coli was recovered from 7 (<5%) carcasses, at numbers mostly ≤1.0 log CFU/160,000 cm(2). The log total numbers of E. coli recovered from the conveyor belt, cuts, and trimmings were mostly between 1 and 2 log CFU/80,000 cm(2). A total of 554 E. coli isolates were recovered. Multiple-locus variable-number tandem repeat analysis of 327 selected isolates identified 80 distinct genotypes, with 37 (46%) each containing one isolate. However, 28% of the isolates were of genotypes that were recovered from more than one sampling day. Of the 80 genotypes, 65 and 2% were found in one or all four sampling periods throughout the day. However, they represented 23 and 14% of the isolates, respectively. Of the genotypes identified for each surface type, at least one contained ≥9 isolates. No unique genotypes were associated with carcasses, but 10, 17, and 19 were uniquely associated with cuts, trimmings, and the belt, respectively. Of the isolates recovered from cuts, 49, 3, and 19% were of genotypes that were found among isolates recovered from the belt, carcasses, or both the belt and carcasses, respectively. A similar composition was found for isolates recovered from trimmings. These findings show that the E. coli found on cuts and trimmings at this beef packing plant mainly originated from the conveyor belt and that small number of E. coli strains survived the daily cleaning and sanitation process, thus persisting in the plant.

Microbiological effects of a routine treatment for decontaminating hide-on carcasses at a large beef packing plant.

To investigate the microbiological effects of a hide-on carcass decontaminating treatment recently implemented at a beef packing plant, carcasses undergoing routine processing at the plant were sampled during successive periods in January/February, April/May, and September/October. During each period, samples were collected from carcasses before and after the decontamination of hide-on carcasses, after skinning, before decontamination of the skinned carcasses, and at the end of the carcass dressing process. At each stage of processing during each period, samples were obtained by swabbing an area of 1,000 cm(2) on each of 25 carcasses. Aerobes, coliforms, and Escherichia coli were enumerated. In most samples, coliforms were predominantly E. coli. In all three periods, the log mean numbers of aerobes and E. coli recovered from hides before decontamination were between 6.6 and 6.8 and between 5.3 and 5.9 log CFU/1,000 cm(2), respectively. The log mean numbers of aerobes recovered from decontaminated hides were 6.6 log CFU/1,000 cm(2) in January/February and April/May but 5.4 log CFU/1,000 cm(2) in September/October. The log total numbers of E. coli recovered from decontaminated hides in January/February and April/May were 2.4 and 3.8 log CFU/25,000 cm(2), respectively, but no E. coli was recovered from such carcasses in September/October. Log total numbers of aerobes and E. coli recovered from skinned or dressed carcasses were mostly >4 and between 1 and 2 log CFU/25,000 cm(2), respectively. Typing of 480 E. coli isolates by multiple-locus variable-number tandem repeat analysis (MLVA) identified 218 MLVA types. Most isolates recovered from carcasses in different periods or at different stages of processing were of different MLVA types. However, small numbers of MLVA types were recovered in more than one period or from both hides before and after decontamination and skinned or dressed carcasses. The findings show that the hide-decontaminating treatment disrupted the usual transfer of E. coli from hides to meat surfaces during carcass skinning.

Storage life at 2 °C or -1.5 °C of vacuum-packaged boneless and bone-in cuts from decontaminated beef carcasses.

BACKGROUND: The microbiological condition of beef produced at North American plants has been improved as a result of the use of effective carcass-decontaminating treatments. The effect of these treatments on the storage life of beef has not been established. In this study, beef primal cuts in vacuum packs stored at -1.5 or 2 °C for up to 160 days were assessed for their microbiological and organoleptic properties. RESULTS: The odours of boneless cuts were acceptable after storage at either temperature for ≤160 days; and the flavours of steaks from boneless cuts stored at 2 or -1.5 °C for ≤70 or ≤120 days, respectively, were acceptable. The storage life of bone-in cuts stored at 2 or -1.5 °C was, respectively, shorter or the same as that of boneless cuts stored at the same temperature. More than 20 microbial species that were mostly obligate aerobes were present on both types of cuts before storage. After storage for ≥30 days, the microflora was dominated by carnobacteria and Enterobacteriaceae were present in the flora from early storage times. CONCLUSIONS: A storage life of 120-140 days was attained by vacuum-packaged beef primals from decontaminated carcasses stored at -1.5 °C. The bone-in cuts stored at 2 °C were spoiled at earlier times, probably by Enterobacteriaceae.

Effects of meat pH on growth of 11 species of psychrotolerant clostridia on vacuum packaged beef and blown pack spoilage of the product.

The aim of the study was to determine the effects of meat pH on the abilities of 11 psychrotolerant Clostridium spp. to grow on, and to possibly cause blown pack spoilage of vacuum packaged beef. Beef steaks of pH 5.4-5.6, 5.7-5.9 or ≥6.0, i.e. of normal, intermediate or high pH were prepared and vacuum packaged. Groups of 3 steaks of the same pH range were inoculated with log phase cultures of Clostridium algoriphilum, Clostridium algidixylanolyticum, Clostridium bowmanii, Clostridium estertheticum, Clostridium frigoris, Clostridium frigidicarnis, Clostridium gasigenes, Clostridium lacusfryxellense, Clostridium psychrophilum, Clostridium tagluense or Clostridium vincentii. Each pack was resealed immediately after the steak was inoculated, and pack volumes were determined by water displacement, immediately after resealing and at intervals during storage at 2 °C for 56 days. All of the clostridia grew in packs of high pH beef but none caused pack swelling. Packs of intermediate pH beef inoculated with C. estertheticum began to swell after 14 days, with a mean rate of increase of pack volumes of 6.80 ml/day. One pack of intermediate pH beef inoculated with C. frigoris was swollen after 37 days. Packs of normal pH beef that had been inoculated with C. estertheticum began swelling after 14 days with a mean rate of increase of pack volumes of 7.70 ml/day. Packs of normal or intermediate pH beef inoculated with other clostridia did not swell. After storage, the numbers of most Clostridium spp., as determined by real-time PCR were greater on beef of high pH than of lower pH values, but the numbers of C. frigidicarnis and C. lacusfryxellense were highest on intermediate pH meat, the numbers of C. estertheticum were higher on meat of lower than of high pH, and the numbers of C. tagluense were the same on meat of all pH values. With high pH meat, glucose was reduced to very low level in rinse fluids from packs that had been inoculated with any Clostridium sp. With intermediate and normal pH meat, glucose was reduced to very low concentrations in only rinse fluids from beef that had been inoculated with C. estertheticum.

Use of sodium lauroyl sarcosinate (sarkosyl) in viable real-time PCR for enumeration of Escherichia coli.

The cell membranes of inactivated Escherichia coli are not always permeable to propidium monoazide (PMA). This limits the use of PMA real-time PCR (PMA-qPCR) for quantification of DNA from only viable cells for enumeration of E. coli. The aim of this study was to develop PMA-qPCR procedures for E. coli with improved selectivity for viable cells. E. coli inactivated by incubation at 52°C were treated with 12 detergents before PMA treatment, and DNA was quantified by real-time PCR. Treatment with each of the 12 detergents and PMA increased the cycle threshold (Ct) values for heat inactivated E. coli suspensions. The greatest increase, of 10.68 Ct was obtained with sarkosyl. Treatment with sodium deoxycholate (NaDC) increased the Ct value by 8.99 Ct. Treatment with sarkosyl or NaDC of 16 heat treated 5-strain cocktails of verotoxigenic E. coli (VTEC) increased the mean Ct values by 8.15 or 6.82 Ct, respectively. Those mean values were significantly (p<0.05) different. When used to enumerate viable E. coli in suspensions treated with lactic acid or in mixtures of viable E. coli and E. coli inactivated by peroxyacetic acid, the slopes relating the Ct values from sarkosyl treated samples to the numbers of viable E. coli were 2.24 and 2.47, respectively, with regression coefficient values ≥0.85. The findings show that sarkosyl was more effective than NaDC for dissipation of PMA-barrier properties of membranes of inactivated E. coli cells. Viable E. coli in mixtures of viable E. coli and E. coli inactivated by heat, lactic acid or peroxyacetic acid could be reliably enumerated by sarkosyl PMA-qPCR.

Morphological and viability changes in Escherichia coli and E. coli O157:H7 cells upon rapid shift from 6 °C to 37 °C.

Cells in log phase cultures of Escherichia coli ATCC 23739 and E. coli O157:H7 02:0627 incubated at 6 °C for 8 days grew by elongation and the formation of filaments. When suspensions of cells from the cultures were incubated at 37 °C for 4 h, there was little or no change in mean cell lengths during the first hour of incubation; but subsequently the fractions of elongated (>4 ≤ 10 µm) or filamentous (>10 µm) cells declined with the most cells being of normal size (≤4 µm) after 3 h. LIVE/DEAD BacLight staining indicated that ≥94% of cells were alive after all times at 37 °C. Direct observation of cells on slides incubated at 37 °C, from culture incubated at 6 °C for 5 days, showed that few or no cells of normal size divided. Elongated cells of both strains, and filamentous cells of E. coli ATCC 23739 divided to multiple daughter cells; but filamentous cells of E. coli O157:H7 lysed. The results indicate that abrupt shifts of log phase E. coli from refrigeration to warm temperatures lead to inactivation of some cells and division of others to multiple daughter cells, and suggest that the extents of these opposing responses may vary widely among strains.

The viabilities of cells in cultures of Escherichia coli growing with formation of filaments at 6 °C.

Cold adapted, exponential phase cells of three strains of Escherichia coli were incubated at 6 °C for 10 days. Cells in all cultures grew by elongation, with formation of filaments to different extents, but with generally only small changes in the numbers of colony forming units (cfu). At later incubation times some cells of all lengths were identified as dead by LIVE/DEAD staining, with cell wall damage being apparent in some cells stained as dead but not in cells stained as live. When samples of cultures were incubated at 37 °C for 2h, the numbers of cfu of two strains (ATCC 11775 and ATCC 23739) increased similarly and were not affected by the time of prior exposure to 6 °C. When incubated at 37 °C, the numbers of cfu of the other strain (8WT) increased less after incubation at 6 °C for 1, 2 or 3 days than when transferred to 37 °C from 15 °C. After>3 days at 6 °C numbers of strain 8WT were reduced after incubation at 37 °C for 2h. The findings show that in cultures of E. coli elongating at 6 °C, elongating cells and filaments do not divide; and cells of all lengths lose viability at similar rates. Also, the findings indicate that substantial fractions of cells in cultures elongating at refrigerator temperatures are inactivated by an abrupt change of temperature to 37°C.

Use of propidium monoazide and quantitative PCR for differentiation of viable Escherichia coli from E. coli killed by mild or pasteurizing heat treatments.

Suspensions of Escherichia coli in peptone water were heated at temperatures between 52 and 90 °C, inclusive. Samples withdrawn at suitable times were not or were treated with propidium monoazide (PMA) or deoxycholate then PMA before extraction of DNA. DNA was quantified by real-time PCR for estimation of the numbers of E. coli from which template DNA for the PCR was obtained. Numbers of viable E. coli in suspensions at the times of sampling were determined from plate counts. For samples from suspensions heated at temperatures ≥ 52 ≤ 72 °C, PCR cycle threshold (Ct) values were little or no different for DNA from corresponding samples that were or were not treated with PMA. PMA treatment of samples heated to ≥ 80 °C largely inactivated E. coli DNA for PCR. When samples heated to ≤ 72 °C were treated with deoxycholate before treatment with PMA, Ct values for treated samples were greater than the Ct values for the corresponding untreated samples. Similar results were obtained with E. coli suspended in milk or fluid from ground beef pummeled with diluent. The results indicate that cells killed by heating to ≥ 80 °C are permeable to PMA, but most cells killed by heating to ≤ 72 °C are not. However, treatment with deoxycholate renders a substantial fraction of the latter cells permeable to PMA. Numbers of viable or dead E. coli can then be estimated from Ct values for samples not treated or treated with deoxycholate and PMA, provided viable cells are ≥ 1% of the total.

Non-O157 verotoxigenic Escherichia coli and beef: a Canadian perspective.

Verotoxigenic Escherichia coli (VTEC) are important foodborne pathogens in Canada. VTEC of the O157:H7 serogroup have been the focus of regulatory action and surveillance in both Canada and the USA, due to their role in a number of high profile outbreaks. However, there is increasing evidence that other VTEC serogroups cause a substantial proportion of human illness. This issue is of particular importance to the cattle industry due to the role of beef as a vehicle for VTEC transmission. In this review, the evidence for non-O157 VTEC as cause of human illness in Canada and the potential for Canadian beef and cattle to serve as a source of VTEC are presented. In addition, the available strategies for the control of VTEC in cattle and beef are discussed.

Products of glucose and lactate fermentation, and utilization of amino acids by Clostridium estertheticum subspp. laramiense and estertheticum growing in meat juice medium.

The type strains of Clostridium estertheticum subsp. laramiense and C. estertheticum subsp. estertheticum both utilized glucose and glycogen when growing in meat juice medium and fermented lactate, but ceased growth when glucose was exhausted. The fermentation products from glucose were butyrate, acetate, and formate; those from lactate were 1-butanol, ethanol, butyrate, and formate. Both organisms utilized several amino acids (not containing sulfur) during their cultivation in meat juice medium and did not produce H(2)S. The optimum and maximum temperatures for growth of C. estertheticum subsp. laramiense were 10 degrees C, and 20 to 22 degrees C, respectively. Those same optimum and maximum temperatures have previously been determined for C. estertheticum subsp. estertheticum. The pH range for growth of the two organisms, 5.5 to 7.5, was also the same. Both organisms were beta-hemolytic and formed subterminal spores. Thus, the organisms did not display the difference in fermentation products, optimum and maximum temperatures, hemolysis, and spore position that were reported to be the differentiating characteristics of the subspecies. The findings indicate that vacuum-packaged meat should be spoiled similarly by the two type strains.

Effects of temperature and pH on the growth of bacteria isolated from blown packs of vacuum-packaged beef.

Bacteria recovered from the microflora of blown packs of vacuum-packaged beef were identified as Leuconostoc mesenteroides, Lactococcus lactis, Carnobacterium maltaromaticum, and Clostridium estertheticum, with L. mesenteroides predominant. Isolates of these lactic acid bacteria all grew in peptone yeast extract glucose starch broth (PYGSB) at temperatures between -2 and 30 degrees C but generally grew more slowly and over a more restricted temperature range in meat juice medium (MJM). A C. estertheticum isolate and the type strain of C. estertheticum subsp. estertheticum (ATCC 51377) both grew in PYGSB and MJM at similar rates at temperatures between -2 and 17 degrees C and grew at 20 degrees C in MJM but not in PYGSB. Square root models of the variation of the growth rate with temperature indicated that the C. maltaromaticum isolate and the C. estertheticum strains grew at similar rates that were faster than those of the other isolates at temperatures between -2 and 0 degrees C. The L. mesenteroides and L. lactis isolates grew in PYGSB at pH 5.0, but the C. maltaromaticum isolate and both strains of C. estertheticum did not grow in PYGSB at pH

Substrate utilization by Clostridium estertheticum cultivated in meat juice medium.

Blown pack spoilage of vacuum packaged beef, which results in packs being grossly distended with gas, is caused by the psychrophile Clostridium estertheticum. To determine what substrates are utilized by C. estertheticum during growth on beef, C. estertheticum subsp. estertheticum ATCC 51377, the type strain for that organism, and two isolates from blown pack spoiled beef that were identified as C. estertheticum by 16 S rRNA gene sequencing were grown in meat juice medium at 10 degrees C for up to 14 days. Analysis of the growth media showed that all three organisms grew exponentially on glucose with simultaneous hydrolysis of glycogen. Growth ceased when glucose in the media was depleted; but hydrolysis of glycogen continued at a reduced rate, and lactate was consumed rapidly. The pH values of media fell during growth of the organisms, but rose as the concentrations of lactate subsequently decreased. The major products of fermentation during utilization of glucose were butyrate and acetate, with butyrate greatly predominating. During fermentation of lactate the major products were butyrate and butanol, which were produced in similar amounts. The findings suggest that growth of C. estertheticum on vacuum packaged beef may be limited by the availability of glucose, as is the growth of other organisms that usually predominate in the flora of vacuum packaged meat. However, production of gas by fermentation of lactate will likely continue after growth ceases.

Implementation of a validated HACCP system for the control of microbiological contamination of pig carcasses at a small abattoir.

To guide the implementation of a Hazard Analysis Critical Control Point (HACCP) system at a small abattoir, the microbiological conditions of pig carcasses at various stages of processing were assessed by enumerating total aerobes, coliforms, and Escherichia coli in samples collected from randomly selected sites on the carcasses. Those data indicated that carcasses were contaminated with bacteria mainly during dehairing and operations on the head. When carcasses were pasteurized after head removal, the numbers of total aerobes on dressed carcasses were reduced by about 1 order and the numbers of coliforms and E. coli were reduced by more than 2 orders of magnitude. Implementation of an HACCP system on the basis of the microbiological data gave cooled carcasses with mean numbers of total aerobes < 100/cm2, and mean numbers of coliforms and E. coli about 1/1000 cm2.