Competitive Inhibition of E. coli O157:H7 and Salmonella spp. in Ground Beef Products Dr. Mindy Brashears Dr. Mark Miller Department of Animal and Food Sciences Texas Tech University Final Report Submitted to the Texas Beef Council National Cattleman’s Beef Association October 10, 2002 Competitive Inhibition of E. coli O157:H7 and Salmonella spp. in Ground Beef Products -FINAL REPORT- Principle Investigator Mindy Brashears, Ph.D. Mark Miller, Ph.D. Institution: Department Animal and Food Sciences Texas Tech University Box 42141 Lubbock, TX 79409 (806)742-2469 (806)742-2427 – fax Project Title:
Competitive Inhibition of E. coli O157:H7 and Salmonella spp. in Ground Beef Products
Stated Project Objectives:
The overall objective of this study is to eliminate E. coli O157:H7 and Salmonella in
ground beef products by competitive inhibition by lactic acid bacteria and thus reduce the
number of outbreaks associated with these products.
Background Information about the Need for This Research
According to the Centers for Disease Control (CDC), E. coli O157:H7causes an
estimated 73,000 cases of illness each year and 61 deaths. Salmonella causes 40,000
reported cases with the estimated actual number of cases being 20 times more than the
reported amount. More than 1000 deaths occur each year due to Salmonella infection
making it the deadliest food-borne pathogen.
Ground beef products are commonly implicated in outbreaks of Salmonella and
E. coli O157:H7. While there are many intervention technologies applied to beef
carcasses, very few interventions exist that have been validated to be effective in
Lactic acid bacteria (LAB) are inhibitory towards various pathogenic bacteria and
spoilage organisms during growth and refrigerated storage in associative cultures
(Brashears et al. 1998; Brashears and Gilliland, 1997; Brashears et al. 1996; Brashears
and Durre; Dahiya and Speck, 1967; Gilliland and Speck, 1975; Gilliland and Speck,
1977; Martin and Gilliland, 1980; Price and Lee, 1969; Kao and Frazier, 1966; Shahani
et al., 1976). The inhibitory actions were due to the production of acid, hydrogen
peroxide, or bacteriocins. Recent reviews of the inhibitory actions of LAB towards food-
borne pathogens were done by Barefoot and Nettles (1993), Klaenhammer (1988) and
Hopzafel et. al (1995). These reviews suggest that pathogens and spoilage organisms
in fermented foods such as yogurt and summer sausage may be inhibited during growth
of LAB. Additionally, inhibition can occur during refrigerated storage. Growth of lactic
acid bacteria in a fresh meat product would not be desirable, but adding the cells to the
meat held at refrigeration temperature could still give an inhibitory effect due to the
production of inhibitory substances by the cells. It is possible to select strains of lactic
acid bacteria that do not grow at refrigeration temperatures, but produce inhibitory
substances. The production of inhibitory compounds by the LAB can continue during
storage of the product so there is continuous inhibition of the pathogen during the
storage period instead of a one-time reduction that occurs with other antimicrobial
interventions (Gilliland and Villegas, 1998; Jaroni and Brashears, 2000).
In a previous study in our laboratory, several strains of lactic acid bacteria were
isolated. This strain was selected for its ability to completely eliminate E. coli O157;H7,
Salmonella, and Listeria monocytogenes in laboratory media. The unique properties of
these organisms is that they eliminate the pathogens at refrigeration temperatures, but it
do not grow during refrigerated storage. The organisms are GRAS and can be added to
A comprehensive study was conducted in our laboratory with this LAB to
determine if it inhibited Listeria monocytogenes in ready-to-eat meat products. All
pathogen growth was completely eliminated for the entire 60-day duration of the study
in cold cuts and in frankfurters. A second study was conducted with non-pathogen
inoculated products to determine if the LAB had a detrimental effect on the sensory
properties of the products. For the entire 60-day duration of the sensory study, there
were no differences in the control samples and the inoculated samples. Plate counts of
LAB in both studies indicated that there was no growth of the LAB during storage, but
there was significant inhibition. Further studies have indicated that a protein-based
product, most likely a bacteriocin, is produced by this organism at refrigeration
Reduction of E. coli O157:H7 is an important concern in the beef industry.
Ground beef processors currently do not have effective intervention steps in ground
beef processes. Intervention steps need to be investigated to ensure the safety of the
ground beef supply. This research indicates that the use of competitive inhibition in
ground beef products could be an important hurdle to reduce E. coli O157 and
Salmonella in the ground beef supply.
Achievement of the Specific Objectives Stated in the Proposal
All objectives in the original proposal have been achieved.
Materials and Methods
The treatments in this study were, 1) ground beef containing both the pathogen and the
LAB, 2) ground beef containing only the pathogen, and 3) a background control containing
Frozen concentrated cultures of the lactic acid bacteria culture were prepared as
described by Brashears et al. (1998) and combined into a “cocktail” mixture and added to the
food products. Products were inoculated with the pathogen by adding a cocktail mixture of
Salmonella spp. or E. coli O157:H7 (two separate studies) containing approximately 1 x 104
cfu/ml. Streptomycin resistant (1000ug/mg) strains of the pathogens were used to facilitate
recovery of injured cells. The use of these organisms has been validated to be equivalent to
non-resistant cells and recovery rates are similar to recovery on non-selective media
(Brashears et. al., 2001). Fresh ground beef samples were obtained from the Texas Tech
University Meat Laboratory. It is important to use ground beef containing the natural flora to
determine if the added LAB competes well in that particular environment. Cells of the LAB
were added to the ground beef by pouring a designated amount of a diluted frozen
concentrated culture to the products to yield a population of 1 x 107 cfu/g and thoroughly
mixing. Background controls received no treatment. All samples were vacuum packaged.
Samples were stored for 10 days at 5oC. Samples were taken on days 4, 8 and 12 and
the total number of pathogens present were determined by plating on non-selective media
(Trypticase Soy Agar) containing 1000 ug/ml streptymycin on a Spiral Biotech Spiral Plating
System. The numbers were compared to the control that contained only added pathogen, no
LAB, to determine the amount of inhibition.
Results and Discussion
Selection of Antibiotic Resistant Organisms
Because we proposed the use of nalidixic acid resistant strains of the pathogens to
facilitate recovery of injured cells in nonselective media in the original proposal, we plated
uninoculated ground beef on TSA plus 50ug nalidixic acid to ensure that no background flora
in the meat would grow on the media. Because a recent project using pork indicated that there
were significant background flora in raw pork products that grew in the presence of nalidixic
acid, we also tested the beef in media containing streptomycin. We had to identify an antibiotic
that suppressed background flora in the raw product.
The raw ground beef samples obtained from several sources were plated on TSA
with the following antibiotic concentrations:
The plates were incubated at 37 C for 48 hrs. There was evident growth for all
dilutions plated on both the 50 ug and 100 ug nalidixic acid plates. Therefore this
antibiotic was not used because it allowed the growth of the background flora.
There was no observed growth for any of the plates containing streptomycin for
either the 1000 ug or 2000 ug concentrations. Therefore the lower concentration of
streptomycin was used in the study to inhibit background flora in the meat while allowing
streptomycin resistant pathogens to grow and recover.
To validate this observation, more ground beef samples were obtained and
plated on plates containing streptomycin. The results from this beef sample were the
same as the results observed before. There was growth on the 100 ug nalidixic acid
plates but there was no growth on either the 1000 ug or 2000 ug streptomycin plates.
In addition to confirming that the background flora would not grow on the media,
we also had to be sure that the LAB would not grow and that the pathogens would grow.
Four different cultures of E. coli O157 and 2 different lactic acid bacteria cultures on
pre-poured TSA plates. One set of plates contained 1000 ug streptomycin and the
other set contained 2000 ug of streptomycin.
After 48 hrs. of incubation at 37C all of the E. coli cultures grew confirming their
antibiotic resistance. Neither of the lactic acid bacteria cultures grew. Therefore the
media containing 1000 ug of streptomycin was used to suppress the background flora in
We stored samples at 5 C for 12 days to determine the impact of Lactic Acid
Bacteria (LAB) on the growth and inhibition of E. coli O157:H7. After 4 days of storage,
one of the LAB cultures, M35 resulted in significantly lower populations of E. coli
O157:H7 compared to the control samples (Figure 1). After 8 days of storage, the
other 3 LAB cultures resulted in significant differences between control samples and
treated samples with more than a 1.5 log difference (>90% reduction) between the
control samples and the treated samples.
A more inhibitory effect was observed against Salmonella spp. After 4 days of
storage, M35, LA51 and D3 resulted in significant reductions of Salmonella compared to
the control samples (Figure 2). Each of these resulted in a 1.5 log reduction after 4
days of storage at refrigeration temperatures. After 8 days of storage, M35 resulted in a
3.5 log difference compared to the control and even more inhibition was observed on
day 12 with more than a 4 log reduction. All LAB cultures except for L7 resulted in
significantly lower populations of Salmonella compared to the control on days 8 and 12.
While we did observe significant reductions in this process, a higher amount of
reduction is desirable. Currently we are continuing with this study to examine the
behavior of E. coli and Salmonella in the presence of a combined cocktail containing all
4 cultures at a higher inoculation level. Preliminary evidence indicates that we can
achieve more than a 3 log reduction of E. coli and a 4 log of Salmonella using this
Additionally, we plan to study the effects on the sensory properties of the product
in non-inoculated samples to determine if the use of the LAB is feasible from a quality
Publications
• Manuscript will be submitted before the end of the year to Journal of Food
• Presentation/abstract will be presented at the 2003 meeting of the
International Association of Food Protection
Lay Interpretation
Lactic acid bacteria (LAB), similar to cultures used to produce cheese and yogurt,
were added to ground beef to determine if the cultures inhibited E. coli O157:H7 and
Salmonella during refrigerated storage. Some of the cultures reduced E. coli O157:H7
by more than 90% and Salmonella by more than 99.9%. Adding the cultures to ground
beef may be an effective strategy to control food-borne pathogens in ground beef
Figure 1. Competitive inhibition of E. coli O157 at 5 C in during a 12 day storage period Days of Storage Figure 2. Competitive inhibition of Salmonella spp. at 5 C in ground beef during a 12 day storage period Days of Storage
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