Is E Coli Present in the Original Suspension of the Ground Beef
Appl Environ Microbiol. 2003 May; 69(5): 2794–2799.
Origin of Contamination and Genetic Diversity of Escherichia coli in Beef Cattle
Mueen Aslam
Lacombe Research Centre, Agriculture and Agri-Food Canada, Lacombe, Alberta, Canada T4L 1W1,1 Department of Agricultural, Food and Nutritional Science, University of Alberta, Edmonton, Alberta, Canada T6G 2P52
Frances Nattress
Lacombe Research Centre, Agriculture and Agri-Food Canada, Lacombe, Alberta, Canada T4L 1W1,1 Department of Agricultural, Food and Nutritional Science, University of Alberta, Edmonton, Alberta, Canada T6G 2P52
Gordon Greer
Lacombe Research Centre, Agriculture and Agri-Food Canada, Lacombe, Alberta, Canada T4L 1W1,1 Department of Agricultural, Food and Nutritional Science, University of Alberta, Edmonton, Alberta, Canada T6G 2P52
Chris Yost
Lacombe Research Centre, Agriculture and Agri-Food Canada, Lacombe, Alberta, Canada T4L 1W1,1 Department of Agricultural, Food and Nutritional Science, University of Alberta, Edmonton, Alberta, Canada T6G 2P52
Colin Gill
Lacombe Research Centre, Agriculture and Agri-Food Canada, Lacombe, Alberta, Canada T4L 1W1,1 Department of Agricultural, Food and Nutritional Science, University of Alberta, Edmonton, Alberta, Canada T6G 2P52
Lynn McMullen
Lacombe Research Centre, Agriculture and Agri-Food Canada, Lacombe, Alberta, Canada T4L 1W1,1 Department of Agricultural, Food and Nutritional Science, University of Alberta, Edmonton, Alberta, Canada T6G 2P52
Received 2002 Jul 29; Accepted 2003 Jan 25.
Abstract
The possible origin of beef contamination and genetic diversity of Escherichia coli populations in beef cattle, on carcasses and ground beef, was examined by using random amplification of polymorphic DNA (RAPD) and PCR-restriction fragment length polymorphism (PCR-RFLP) analysis of the fliC gene. E. coli was recovered from the feces of 10 beef cattle during pasture grazing and feedlot finishing and from hides, carcasses, and ground beef after slaughter. The 1,403 E. coli isolates (855 fecal, 320 hide, 153 carcass, and 75 ground beef) were grouped into 121 genetic subtypes by using the RAPD method. Some of the genetic subtypes in cattle feces were also recovered from hides, prechilled carcasses, chilled carcasses, and ground beef. E. coli genetic subtypes were shared among cattle at all sample times, but a number of transient types were unique to individual animals. The genetic diversity of the E. coli population changed over time within individual animals grazing on pasture and in the feedlot. Isolates from one animal (59 fecal, 30 hide, 19 carcass, and 12 ground beef) were characterized by the PCR-RFLP analysis of the fliC gene and were grouped into eight genotypes. There was good agreement between the results obtained with the RAPD and PCR-RFLP techniques. In conclusion, the E. coli contaminating meat can originate from cattle feces, and the E. coli population in beef cattle was highly diverse. Also, genetic subtypes can be shared among animals or can be unique to an animal, and they are constantly changing.
A large number of verotoxigenic Escherichia coli serotypes are associated with human intestinal infections (27), and some of these serotypes are recognized as important foodborne pathogens that may cause mild to severe bloody diarrhea and hemolytic uremic syndrome (33). Chilled raw beef is a major source of pathogenic E. coli and it has been assumed that such organisms in the feces of cattle are spread to meat during slaughter and processing (2, 7, 27). Cattle and their environment are among the most important sources of pathogenic E. coli, and they may be the origin of contamination of meat and meat products (11, 32). Cattle are also implicated in direct transmission of E. coli to humans (25, 38, 39).
Analyses of changes in bacterial numbers have led to a correlation between carcass contamination and E. coli originating from feces and from the hide (11), as well as the implication of the hide as a source of carcass contamination with E. coli during slaughter (4, 31). Identical genetic types of E. coli O157:H7 were isolated from feces of incoming cattle and carcasses by using pulsed-field gel electrophoresis (3), but the link from cattle through to meat was not made. Therefore, direct evidence that meat contamination originates from the fecal microflora of cattle is still lacking.
Although there has been considerable published research on the prevalence of E. coli O157:H7 in cattle and some evidence of its transfer to carcasses during beef processing (3, 13, 29), the significant variability in prevalence and the low numbers of this strain in meat make it a poor choice to track contamination during processing. The use of pathogens to study a meat production process has limitations, and generic E. coli has been used as an indicator of the microbiological status of the process and product (17, 18). The recovery and enumeration of generic E. coli are considered to be a more practical indicator of the hygienic efficiency of carcass processing (16, 17).
Considerable resources have been dedicated to reduce the introduction of bacteria from the animal to the processing plant, without conclusive evidence that the bacteria on the meat in fact originate from the live animal. With the development of genetic fingerprinting methods, the genetic diversity of strains in cattle and on meat can be studied and the sources of contamination can be more precisely determined. Random amplification of polymorphic DNA (RAPD) has been used extensively to study genetic diversity and epidemiological relationships of E. coli and other foodborne pathogens (10, 15, 21, 34, 37). It offers an efficient, sensitive, and relatively inexpensive method to characterize large numbers of E. coli isolates.
PCR-restriction fragment length polymorphism (PCR-RFLP) analysis of the fliC gene, encoding the H antigen, has also been used to characterize and to study genetic diversity of motile and nonmotile E. coli strains (14, 36). In the present study, a PCR-RFLP technique was used to supplement the results obtained with the RAPD method and to determine if the results were comparable with both methods for studying the genetic diversity of and tracking E. coli in ground beef.
There are some published data on the genetic diversity of E. coli in cattle (15, 26), but little is known about the ecology of E. coli in beef cattle on the farm. In this study, generic E. coli was characterized and used as an indicator of the potential for transfer of enteric pathogenic bacteria from cattle through processing to ground beef. This study was therefore designed to explore the genetic diversity in the E. coli population within and between beef cattle while they were grazing on pasture and during feedlot finishing. The genetic diversity of E. coli strains was monitored when these animals were slaughtered and processed in a research abattoir. The specific objectives of present study were (i) to identify and trace identical strains through the beef production process and (ii) to assess genetic diversity and the extent of distribution of genetic subtypes at each stage of the beef production process.
MATERIALS AND METHODS
Animals and slaughter.
Hereford × Angus cross cattle (five heifers and five steers) were placed on pasture after weaning. Following 138 days of pasture grazing, cattle were transferred to a feedlot (one pen of five steers and one pen of five heifers) and fed a ration of barley silage and barley grain supplemented with a feedlot ration. Steers were held for 146 days and heifers were held for 160 days to accommodate slaughter capabilities. At monthly intervals during pasture grazing and feedlot finishing, cattle were moved to a handling facility to collect routine performance data, and fecal samples were removed from the rectum by using a sterile glove and were transferred to a sterile specimen container (Fisher Scientific, Pittsburgh, Pa.). Fecal samples were stored on ice and transported to the laboratory within 2 h of sampling.
Cattle were slaughtered in groups of five on two slaughter days in a federally inspected research abattoir. At the time of slaughter, the age of the animals was approximately 13 months and the average weight was 564 kg.
Collection and processing of microbiological samples.
A 25-g sample of fecal material from each animal was homogenized in 225 ml of sterile 0.1% (wt/vol) peptone (Becton Dickinson Co., Sparks, Md.) for 2 min in a stomacher (Stomacher 400 Lab Blender; Seward Medical, London, United Kingdom) and diluted to 10−4 prior to plating.
Hides and carcasses were sampled as described by Gill and Jones (19). Briefly, 1,000-cm2 surface areas of the rump and brisket were swabbed with a sterile, 2- by 4-cm cellulose acetate sponge (Nasco Canada, Aurora, Ontario, Canada) moistened in 0.1% peptone. Within 4 h of sampling, each sponge sample was mixed with 10 ml of 0.1% peptone and blended for 2 min in a stomacher. Carcasses were sampled prior to chilling (prechilled carcass) and after being held for 18 h in a cooler operating at 2°C.
Ground beef with 20% fat was prepared from individual animals, using trim from the rump and brisket areas. Meat from each animal was processed separately by grinding (model 722; The Biro Manufacturing Co. Marblehead, Ohio) through a 10-mm-diameter plate and then through a 5-mm-diameter plate. A 200-g sample was obtained at the midpoint of the grinding operation for microbiological analysis. The grinder was dismantled between each sample, and all removable parts were washed with pressurized water at 85°C before processing of the sample from the next animal. Ground beef (200 g) from each animal was suspended in 200 ml of 0.1% peptone in a filter stomacher bag (model 400 bags; Seward Medical) and blended for 2 min. The homogenate was centrifuged at 4,000 × g (rotor SLA-1500, Sorvall RC-5B Refrigerated Superspeed centrifuge; Du Pont Instruments Co.) for 10 min. The supernatant was centrifuged at 24,000 × g for 10 min, and the resulting pellet was resuspended in 10 ml of 0.1% peptone containing 1% Tween 80 (BDH Inc., Toronto, Ontario, Canada). Samples were incubated with 1 ml of 0.5% papain (Oxoid Ltd., Hampshire, United Kingdom) for 30 min prior to microbiological analysis.
Bacterial isolation and enumeration.
Following sample preparation, samples were filtered through an ISO-GRID hydrophobic grid membrane (QA Life Sciences Inc., San Diego, Calif.), and filters were placed on SD-39 agar (Oxoid Ltd.). The plates were incubated at 42.5°C for 18 h, and green colonies were counted as presumptive E. coli as described by Entis and Lerner (12). Randomly selected green colonies were subcultured on SD-39 medium. Following 18 h of incubation at 42.5°C, individual colonies were suspended in PCR tubes containing 50 μl of sterile water. These samples were used for RAPD and PCR-RFLP analysis.
Confirmation of generic E. coli by PCR.
One hundred twenty-one E. coli isolates were randomly selected and were confirmed as generic E. coli by using a PCR assay targeting the universal stress protein A (uspA) gene as described previously (8).
RAPD analysis of E. coli isolates.
Template DNA was prepared from E. coli isolates that were suspended in 50 μl of sterile water. Samples were boiled for 15 min, followed by centrifugation at 1,700 × g (rotor F45-48-PCR, Eppendorf centrifuge 5417C; Brinkmann Instruments Inc., Mississauga, Ontario, Canada) for 2 min. RAPD analysis of E. coli isolates was performed according to procedures described previously (35) with a few modifications. Briefly, the 25-μl PCR mixture consisted of 5 μl of template DNA, 2.5 mM MgCl2, 0.8 nM 1254 decamer primer (5′-CCGCAGCCAA-3′) (Sigma-Aldrich Canada Ltd. Oakville, Ontario, Canada), a 300 μM concentration of each deoxynucleoside triphosphate (Invitrogen Corporation, Carlsbad, Calif.), and 1 U of Taq DNA polymerase (Sigma-Aldrich). PCR amplification was performed in a Robocycler Infinity thermocycler (Stratagene Inc., La Jolla, Calif.) according to the following protocol: 1 cycle of 5 min each at 94, 36, and 72°C; 10 cycles of 1 min at 94°C, 1 min at 36°C, and 2 min at 72°C; and 20 cycles of 1 min at 94°C, 1 min at 50°C, and 2 min at 72°C. Amplified DNA fragments were separated on 1.8% agarose gels (90 V for 2 h) and stained with ethidium bromide (0.25 μg/ml) for 45 min. A 1-kb DNA ladder (Invitrogen Corporation) was included as a size marker.
The stability of RAPD patterns over several bacterial generations was evaluated with five E. coli isolates recovered from feces. Frozen E. coli cells were inoculated into tryptic soy broth (Difco) and incubated at 37°C for 18 h. A loopful from this culture was streaked onto tryptic soy agar (Difco) and incubated overnight at 37°C. This was considered the generation 1 culture. An individual colony was picked from this culture and streaked onto a tryptic soy agar plate, followed by restreaking of individual colonies from three successive subcultures. DNA was isolated as described above from cultures at days 1, 2, 3, and 5 and used in RAPD analysis.
PCR-RFLP for analysis of the fliC gene.
The analysis of polymorphism associated with the fliC gene, encoding the H antigen of E. coli, was performed by a procedure described previously (14). Fragments (1.3 to 2.6 kb) of the fliC gene were amplified by PCR in a thermocycler (Robocyler Infinity; Stratagene Inc.) at 95°C for 30 s, 55°C for 1 min, and 72°C for 2 min for total of 35 cycles. The amplified fragments were digested with RsaI restriction enzyme at 37°C for 2 h, and the restriction DNA fragments were separated on 2% agarose gels (90 V, 2 h) and stained with ethidium bromide. A 1-kb DNA ladder (Invitrogen) was included as a size marker.
Analysis of DNA patterns.
Stained gels were scanned digitally with a Kodak EDAS290 system (Eastman-Kodak, Rochester, N.Y.), and images were stored as tagged image format files. DNA patterns obtained with the RAPD and PCR-RFLP techniques were analyzed with Molecular Analyst software, fingerprinting version 1.61 (Bio-Rad Laboratories, Hercules, Calif.). Similarities between the DNA patterns of E. coli isolates based on band positions were determined by using the Dice similarity coefficient, and a dendrogram was constructed by using the unweighted pair group method with arithmetic averages to reflect similarities in the matrix. E. coli isolates were grouped into genetic subtypes which were considered genetically related (≥80% similarity) based on DNA patterns.
RESULTS
Numbers of E. coli.
The numbers of E. coli isolated from the feces of 10 cattle while on pasture and in the feedlot were similar and ranged from log10 5.5 ± 0.5 to log10 5.9 ± 0.3 CFU g−1 (Fig. 1). At the time of slaughter, E. coli numbers were log10 5.0 ± 0.4 CFU/1,000 cm2 on the hides and log10 2.5 ± 1.0 CFU/1,000 cm2 on the carcasses. E. coli numbers declined to log10 0.3 ± 0.3 CFU/1,000 cm2 on the chilled carcasses and increased to log10 0.8 ± 0.6 CFU 200 g−1 in the ground beef (Fig. 1). A PCR assay using the primers derived from the uspA gene amplified a specific DNA fragment of 884 bp from all of the randomly selected E. coli isolates (data not shown).
Mean and standard error of the numbers of E. coli isolated at each stage of beef production. E. coli log10 counts are per gram of feces in the pasture and feedlot, per 1,000 cm2 on hides and carcasses, and per 200 g in ground beef. Data are for 10 animals. P, pasture; F, feedlot; H, hide; PC, prechilled carcasses; CC, chilled carcasses; GB, ground beef.
Stability of RAPD patterns.
The stability of RAPD patterns was evaluated with five different fecal E. coli isolates which were subcultured five times. DNA patterns of these isolates were compared by using the Dice similarity coefficient, and 100% similarity was demonstrated among four subcultures (data not shown).
Tracking of E. coli contamination.
A comparison of the RAPD patterns of E. coli isolates showed that 20 genetic subtypes (659 isolates) could be tracked through the continuum from pasture cattle to ground beef (Table 1). Out of 20 genetic subtypes, 16 E. coli types (639 isolates) were found among various sampling points from pasture to ground beef, whereas 4 types (20 isolates) were isolated only from hide, carcass, and ground beef samples during processing steps in a research abattoir (Table 1). Seven E. coli isolates representing one predominant genetic subtype (type 9) that was recovered through the entire continuum of meat production, from the pasture to the ground beef, were selected to demonstrate that genetically related strains were present at all stages (Fig. 2).
Ethidium bromide-stained agarose gel showing RAPD E. coli type 9 (≥80% similarity). Lanes: P, pasture; F, feedlot; H, hide; PC, prechilled carcasses; CC, chilled carcasses; GB, ground beef; M, DNA size marker in kilobases.
TABLE 1.
E. coli genotypes carried into chilled carcasses and ground beef that were also recovered at various stages of beef production
| RAPD type | No. of isolates a | ||||||||
|---|---|---|---|---|---|---|---|---|---|
| P | F | H | PC | CC | GB | Total | |||
| 1 mo | 5 mo | 1 mo | 5 mo | ||||||
| 2 | 10 | 1 | 11 | ||||||
| 9 | 42 | 3 | 80 | 47 | 32 | 3 | 20 | 227 | |
| 12 | 2 | 18 | 24 | 5 | 6 | 4 | 1 | 60 | |
| 14 | 1 | 4 | 4 | 2 | 11 | ||||
| 26 | 1 | 2 | 1 | 4 | |||||
| 29 | 27 | 9 | 1 | 2 | 1 | 1 | 1 | 42 | |
| 31 | 13 | 1 | 10 | 16 | 9 | 1 | 50 | ||
| 34 | 5 | 1 | 5 | 5 | 14 | 30 | |||
| 35 | 2 | 1 | 2 | 6 | 5 | 2 | 4 | 22 | |
| 36 | 1 | 2 | 3 | ||||||
| 39 | 5 | 1 | 21 | 17 | 4 | 3 | 51 | ||
| 41 | 1 | 1 | 2 | 5 | 1 | 14 | 24 | ||
| 51 | 1 | 1 | 2 | ||||||
| 53 | 1 | 10 | 4 | 3 | 2 | 20 | |||
| 62 | 3 | 2 | 5 | 1 | 1 | 12 | |||
| 63 | 3 | 2 | 1 | 1 | 1 | 8 | |||
| 67 | 26 | 26 | 1 | 53 | |||||
| 78 | 2 | 1 | 1 | 4 | |||||
| 107 | 1 | 1 | 1 | 2 | 5 | ||||
| 109 | 1 | 2 | 9 | 8 | 20 | ||||
Genetic variation among E. coli isolates.
A total of 1,403 E. coli isolates were examined from all sampling points and were grouped into 121 different genetic subtypes by using the RAPD technique. E. coli isolates recovered from feces after 1 month (218 isolates) and 5 months (195 isolates) on pasture were grouped into 45 and 40 genetic subtypes, respectively (Table 2). E. coli isolates recovered from feces after 1 and 5 months (221 isolates each) in the feedlot were grouped into 38 and 33 genetic subtypes, respectively. The majority of the E. coli types were shared among cattle between and within each sample time; however, unique types were also present at each sampling time (Table 2).
TABLE 2.
Total, shared and unique RAPD types of E. coli at each stage of beef production
| Sample source a | No. of types | ||||||||
|---|---|---|---|---|---|---|---|---|---|
| Total b | Shared c | Unique d | |||||||
| P (5 mo) | F | H | PC | CC | GB | ||||
| 1 mo | 5 mo | ||||||||
| P | |||||||||
| 1 mo | 45 | 14 | 14 | 18 | 27 | 20 | 3 | 10 | 9 |
| 5 mo | 40 | 17 | 12 | 23 | 11 | 3 | 9 | 9 | |
| F | |||||||||
| 1 mo | 38 | 17 | 22 | 12 | 3 | 4 | 5 | ||
| 5 mo | 33 | 21 | 16 | 3 | 9 | 3 | |||
| H | 72 | 29 | 3 | 15 | 17 | ||||
| PC | 37 | 1 | 12 | 3 | |||||
| CC | 5 | 1 | 1 | ||||||
| GB | 17 | 0 | |||||||
E. coli isolates recovered from the hides (320 isolates) at the time of slaughter were grouped into 72 genetic subtypes, and 17 types were found to be unique (Table 2). The majority of the types were shared with fecal E. coli types recovered from pasture and feedlot. E. coli isolates recovered from carcasses (141 isolates) prior to chilling were grouped into 37 genetic subtypes comprising only three unique types, whereas E. coli isolates recovered from chilled carcasses (12 isolates) were grouped into 5 genetic subtypes and only one type was found to be unique (Table 2). E. coli isolates recovered from ground beef (75 isolates) were grouped into 17 genetic subtypes, and no unique type was found in the ground beef (Table 2). All of the E. coli types recovered from the ground beef were shared with the types recovered from the fecal, hide, and carcass samples.
Analysis of DNA patterns obtained from PCR-RFLP, used to characterize 120 E. coli isolates, resulted in eight genetic subtypes of E. coli (Table 3). One predominant type (type A) was found in fecal samples collected after 5 months in the feedlot and also in the samples taken from the hides, the prechilled carcasses, the chilled carcasses, and the ground beef. A few unique genotypes were also isolated from the fecal and hide samples (Table 3). When the same E. coli isolates were characterized by using the RAPD technique, they were grouped into five RAPD types (data not shown). One predominant type (type A) was found at all sampling times, whereas four types were unique to fecal samples collected in the pasture, in the feedlot, and from the hides.
TABLE 3.
PCR-RFLP analysis of E. coli isolates recovered from one animal at each stage of beef production and characterized by PCR-RFLP analysis of the fliC gene
| PCR-RFLP type | No. of isolates a | |||||||
|---|---|---|---|---|---|---|---|---|
| P (5 mo) | F | H | PC | CC | GB | Total | ||
| 1 mo | 5 mo | |||||||
| A | 19 | 22 | 14 | 4 | 12 | 71 | ||
| B | 9 | 17 | 26 | |||||
| C | 7 | 1 | 8 | |||||
| D | 6 | 6 | ||||||
| E | 5 | 5 | ||||||
| F | 1 | 1 | ||||||
| G | 1 | 2 | ||||||
| H | 1 | 1 | ||||||
DISCUSSION
The total numbers of E. coli recovered from the feces of cattle (log10 5.0 to log10 6.0 CFU/g) were similar to those reported by others (32) and were not affected by time in pasture or in the feedlot. There was a decrease in the number of E. coli organisms recovered during carcass dressing, and very few were recovered from the chilled carcass, which is consistent with the results of a previous study (20). In the absence of spray chilling, it is conceivable that a reduction in bacterial numbers during chilling was due to desiccation at the carcass surface. Generic E. coli isolated by using the cultural method was further confirmed by PCR with uspA primers. The specificity of these primers for identification of generic E. coli has been previously reported (8).
In this study, RAPD patterns from subcultured E. coli isolates were stable over periods of subculturing, which has also been demonstrated in other studies (6, 35). The reproducibility of the RAPD technique has also been confirmed by previous researchers (30, 35). In the present study, consistent reproducibility of the RAPD technique allowed tracking of E. coli strains at various stages of the beef production system.
Tracking of E. coli contamination of meat.
Identical Campylobacter and Salmonella genotypes have been traced from live pigs through pork processing to pork cuts (9, 22). There have been no studies of a similar scope to determine the dissemination of E. coli from beef cattle through slaughter and processing to ground beef. Most published data have considered only the prevalence of E. coli O157:H7 in the preslaughter environment (11, 40), and more recent genotypic data have suggested that most of the E. coli O157 carcass contamination originates from live animals (3). It has been demonstrated, using generic E. coli, that the numbers of E. coli on decontaminated beef carcasses are lower than after carcass breaking and further processing, suggesting that the beef is recontaminated during these processes (16). The possible origin of these strains, therefore, becomes an important consideration.
Molecular fingerprinting methods such as RAPD and PFGE have been used to trace contamination with Listeria monocytogenes, Campylobacter spp., and Salmonella spp. during pork and shrimp production processes (9, 10, 21, 22). DNA profiling methods as well as standard bacteriological methods have also been used to establish a relationship between preslaughter E. coli O157 contamination in cattle and carcass contamination (3, 7, 11). Another study reported that genetically identical strains of E. coli O157:H7 carried in the feces of feedlot cattle appeared on the hides of animals and ultimately contaminated carcasses in the plant (3). However, samples were not necessarily taken from the same animals, and a limited numbers of isolates were analyzed. Additionally, only one serotype of E. coli O157:H7 was monitored, making it difficult to recover all of the O157:H7 isolates from competing microflora.
In the present study, generic E. coli was recovered from the entire production-processing continuum. The RAPD technique was used to monitor the genetically related E. coli strains as they appeared during various stages of the process. Multiple genetic subtypes of E. coli, found in the feces of pasture and feedlot cattle, were also found on the hides and carcasses at the time of slaughter, indicating that the carcasses were contaminated with E. coli of animal origin and that the strains were passed into the ground beef. This is the first presumptive evidence demonstrating that the E. coli types carried in the feces of cattle are the same as the ones isolated from ground beef.
There is a possibility that some of the E. coli genetic subtypes might have originated from the processing environment. However, due to efficient sanitizing procedures practiced in this federally inspected research facility, contamination from the abattoir environment should be minimal. It is, therefore, more likely that the majority of identical genetic subtypes of E. coli originated from the animals.
Genetic variation among E. coli isolates.
The RAPD technique was used to study the genetic diversity in E. coli isolates from 10 beef cattle at various stages of beef production. The population was diverse, with 121 E. coli genetic subtypes being recovered from beef cattle feces, hides, and carcasses and ground beef. A number of studies have reported observations of high genetic diversity in E. coli populations, particularly within strain O157:H7, by using various fingerprinting techniques (1, 13, 15, 24, 26, 34, 37), and diversity among E. coli serotypes O157:H7, O26, and other pathogenic serotypes isolated from a variety of sources, including beef cattle, has been described (15, 23, 36). Diverse strains of O157:H7 and other Shiga toxin-producing strains found in fecal samples of cattle and sheep have been shown to shift over time (1, 5, 28). These studies did not demonstrate whether the genetic shift in the E. coli population was due to divergence or succession. The high degree of genetic diversity in the generic E. coli population in cattle suggests that this species is comprised of numerous genetic subtypes. These subtypes may occupy different niches (26) or be optimized for survival and growth under various conditions in association with cattle or other animals.
Comparison of the RAPD patterns of E. coli isolates taken from individual animals for all sampling sources showed that within an individual animal, and within each sampling time, the majority of E. coli isolates shared close genetic relatedness (≥80% similarity). However unique genetic subtypes were also present at each sampling period, which is in agreement with other studies (1, 13, 26). Some genetic subtypes of E. coli were present at high frequency in feedlots, and they may constitute a resident population (15). In the present study predominant (resident) E. coli types were observed, and they were isolated at all samplings periods. However, some types were transient in nature, appearing at certain sampling times and not detected at others, suggesting that cattle could harbor transient as well as resident strains of E. coli throughout the beef production process.
Some genetic subtypes of E. coli isolated from hides and carcasses were similar to the fecal types found in the pasture, feedlot, or both, but a few unique types were also recovered from hides and carcasses, suggesting that hides and carcasses were exposed to more E. coli types than were recovered from the feces. During this study 20 additional cattle shared the pasture and feedlot with the 10 experimental cattle, so unique genetic subtypes of E. coli isolated from the hides and carcasses may have transferred horizontally from the feces and/or hides of other cattle that were not included in the analyses. The presence of unique genotypes on hides corroborates previous evidence that unique types of E. coli O157:H7 could be isolated from hides of feedlot cattle (3). In that study, however, the genetic variation was attributed to limitations in recovery of all of the types of E. coli from the hides and fecal samples and to the inability of all types to survive under various conditions. However, the significance of unique E. coli genotypes is limited in view of the objective of this study, which was to determine the predominant common types that appear at all stages of beef production.
To supplement and confirm the RAPD results, PCR-RFLP analysis of fliC gene, encoding the H antigen, was used to characterize 120 E. coli isolates recovered from one animal. The isolates were grouped into eight PCR-RFLP types, and the same isolates were grouped into five RAPD types. The molecular bases of the RAPD method and PCR-RFLP analysis of the fliC gene are different. The RAPD method explores genetic diversity within the whole genomic DNA, and PCR-RFLP provides information on a small portion of chromosomal DNA, in this case the single gene fliC (H antigen). The agreement between the two methods demonstrated that either could be used for strain differentiation. By using both types of analysis, it was possible to trace strains of E. coli through the entire beef production system from pasture to ground beef. Others have also shown that E. coli O157:H7 and O157:NM isolates could be grouped into distinct genotypes by PCR-RFLP analysis of the fliC gene (14), and results of RAPD and PCR-RFLP analysis of the fliC gene have been found to be comparable (36).
In conclusion, genetically diverse subpopulations of E. coli are present in beef cattle and appear to shift in their presence and abundance over time, the significance of which is yet to be ascertained. This research has, for the first time, monitored genetic subtypes of E. coli throughout the production of beef from the live animal, from their hides and carcasses, and also from the ground beef produced from them. It has established a link between fecal carriage of generic populations of E. coli by cattle, hide and carcass contamination, and contamination of ground beef. Since a research abattoir was used for this work and the system is not as complex as commercial facilities, future work will involve characterization of E. coli isolated at various stages from slaughter, processing, and finally ground beef at a commercial abattoir.
Acknowledgments
We acknowledge the technical support of Debbie Olsen, Bryan Dilts, and Madhu Badoni and the clerical support of Loree Verquin. We are also grateful for the assistance of the animal handling and beef processing staff at the Lacombe Research Centre.
This study was supported, in part, by the Alberta Agricultural Research Institute.
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