2 

Evaluation of non-pathogenic surrogate bacteria as process validation indicators 

for Salmonella enteric for selected antimicrobial treatments, cold storage and 

fermentation in meat 

S. E. Niebuhr1

, A. Laury1, G. R. Acuff2, and J. S. Dickson1* 

* Corresponding Author: 515.294.4733; 515.294.5066(FAX); 

jdickson@iastate.edu 

1. Department of Animal Science, Food Safety Research Laboratory, 215F 

Meat Lab, Iowa State University, Ames, IA 50011 

Department of Animal Science, 2471 TAMU, Texas A & M University, 

College Station, TX 77843-2471 

Running Title: Surrogates for salmonella in meat 

Key words: salmonella; E coli, indicators; surrogate; meat processing; process 

validation 

1 
ABSTRACT 

Pre-rigor lean and adipose beef carcass tissue was artificially inoculated 

individually with stationary phase cultures of five non-pathogenic Escherichia coli 

cultures which had been previously identified as surrogates for E. coli O157:H7 

or a mixture of five Salmonella strains in a fecal inoculum. Each tissue sample 

was processed with microbial interventions comparable to those used in the meat 

industry. The log10 reductions of the E. coli isolates were generally not 

statistically different from the salmonellae inoculum within a specific treatment. 

Inoculation experiments were also conducted with ground beef stored at either 

4C or -20C. When compared to the Salmonella inoculum, at least three of the 

five E. coli strains survived in a manner which was not statistically different from 

the salmonellae. The E. coli strains and the Salmonella mixed culture were also 

inoculated into summer sausage batter, and the population enumerated both 

before and after fermentation. Four of the E. coli strains showed a lower 

population reduction (higher survival) than the Salmonella mixed culture. The 

five non-pathogenic E. coli strains may be used as individually or collectively for 

specific process validation indicators for Salmonella. 

2 
Introduction 

Salmonella enterica is one of the major foodborne bacterial pathogens in 

the United States food supply (4). The significance of this bacterium in meat and 

poultry products was emphasized by the U.S. Department of Agriculture, Food 

Safety and Inspection Service (USDA-FSIS), by incorporating Salmonella 

performance standards in the Pathogen Reduction/HACCP final rule (6). In 2006, 

the USDA-FSIS began a new initiative to reduce the levels of Salmonella in 

meat and poultry products (7). 

One of the practical limitations of process validation is that the actual 

pathogens cannot be taken in to a food processing establishment to verify a 

specific process. While laboratory research can be used as a reference point, it is 

not a true substitute for actual, in-plant process validation. The availability of nonpathogenic bacteria, which have similar responses to specific food processes as 

the pathogenic bacteria, offers the food processor the ability to validate a process 

in-plant, without the use of the actual pathogens. For example, coliforms have 

been used as process indicators for pasteurization in the dairy industry. 

Recently, several non-pathogenic Escherichia coli strains were isolated 

which responded to meat process interventions in a manner similar to E. coli 

O157:H7 (3). Since the bacteria were shown to be not statistically different from 

E. coli O157:H7 in their responses to various antimicrobial interventions, it was 

reasonable to determine if they could also be used as surrogates for 

salmonellae. The objective of this research was to compare the responses of 

3 
these specific non-pathogenic surrogate bacteria to the response of a mixed 

culture of Salmonella to various meat processes. The intent was to determine 

which, if any, of the non-pathogenic surrogates could be used for process 

validation for the reduction of Salmonella on meat. 

Materials and Methods 

Culture Preparation: Five generic Escherichia coli beef cattle isolates (P1, P3, 

P8, P14, P68), previously characterized and selected based upon their heat 

resistance (3) and five Salmonella enterica strains (Table 1) were used in this 

study. The E. coli isolates were deposited with the American Type Culture 

Collection (www.ATCC.org), and the accession numbers are given in Table 1. 

All strains were maintained on Tryptic Soy Agar (TSA, Difco) slants at 4o

C until 

use. 

Preparation of Tissue Surface Inoculum: The preparation of the inoculum 

procedure was adapted from Hardin et al. (2). Feces collected from randomly 

selected beef cattle which had not been treated with or fed antimicrobials from 

the Iowa State University Beef Research Farm, were brought back to the 

laboratory and stored at 4o

C. The cultures were grown to late logarithmic phase 

at 37C in 45 ml Tryptic Soy Broth (TSB, Difco), and then centrifuged at 3,000 x g 

for 15 minutes at 4oC (Sorvall Super T21, Kendro Lab Products, Newtown, CT). 

The supernatant was discarded and the strains were re-suspended with 9.0 ml of 

0.1% sterile peptone water (Difco, Detroit, MI). The prepared inoculum of each 

isolate (ca. 108

 CFU/ml) was added to individual bags containing 10 g of feces 

4 
-

and hand kneaded for 1 minute. All five S. enterica strains were grown and 

harvested as described above, and each strain was re-suspended in 2.0 ml of 

0.1% sterile peptone. The individual S. enterica strains were combined, and the 

inoculum (ca. 109 CFU/ml) was pooled into one bag containing 10 g of feces and 

hand kneaded for 1 minute. A control inoculum was prepared by adding 9.0 ml 

of 0.1% sterile peptone water to 10 g of feces and hand kneaded for 1 minute. 

Carcass tissue inoculation: The cutaneous trunci (lean tissue) and adipose 

carcass trim was excised from randomly selected carcasses of cows immediately 

after slaughter (pre-rigor) at a Federally inspected beef slaughter establishment. 

The tissue samples were placed in individual Whirl-Pak® bags, packed in an 

insulated cooler and delivered to the Food Safety Research Lab (FSRL; Iowa 

State University, Ames, IA) within two hours after collection. 

The tissue samples were separated into lean and adipose tissue and each 

tissue type was cut into strips 2 cm wide by 6 cm long by 1 cm thick. A sample 

was taken of each tissue type prior to inoculation with feces to determine the 

background aerobic bacterial population of the tissues (typically <102 CFU/cm2; 

data not shown). Tissue samples of lean and adipose were inoculated by 

applying the fecal suspensions with a foam paint brush, and then allowed to 

stand 20-30 minutes at 10o

C prior to treatment. 

5 
Application of wash treatments: Spray washes were conducted in a 

BioSafety Level II Pilot Plant of the FSRL maintained at 10oC. The treatments, 

designed to simulate commercial processes included (a) 90 oC water wash (b) 90 

o

C water wash followed by a 55 oC, 2% (wt/vol, pH 2.0) lactic acid rinse (c) 90 oC 

water wash followed by a 20 oC, 2% lactic acid rinse (d) 20 oC water wash (e) 20 

o

C water wash followed by a 20 oC, 2% lactic acid rinse (f) 20 oC water wash 

followed by 20 oC, 20 ppm chlorine rinse (g) 20 oC water wash followed by a 20 

o

C, 10% (wt/vol, pH 11) trisodium phosphate (TSP) wash. Inoculated tissues 

were placed on sterile metal hooks and were hung in the same orientation as the 

tissue would hang on a carcass. Each set of hanging inoculated tissues was 

separated by a square piece of high density polyethylene (HDPE) plastic to 

prevent aerosol contamination between samples. The wash and rinse 

treatments were applied using a low pressure, Shur flow diaphragm pump 

sprayer with a flat fan nozzle (ME Anderson Engineering Inc., Model No. MEA10-1, Columbia MO) at a pressure of 20 psi for 3 seconds (flow rate 77 ml/s) at a 

distance of 10 – 15 cm. All of the treatments were conducted within a chamber 

constructed of PVC pipe and polyethylene plastic sheeting to provide protection 

from the biological agents. 

6 
Microbiological Analysis: A 1 cm x 2 cm sample of each tissue type was 

excised both prior to inoculation, after inoculation with the fecal suspension, and 

then after treatment. Each tissue sample was placed in a sterile filter stomacher 

bag and homogenized for 1 min in 25 ml buffered peptone water (BPW; Difco). 

Previous research had shown that the BPW neutralized the residual chemicals 

from the treatments, based on the presence of free available chlorine or pH 

measurements. Serial dilutions were performed in BPW, and total aerobic 

populations were determined by spread plating 0.1 ml of the sample from the 

appropriate dilution onto duplicate plates of TSA. Populations of each indicator 

organism were determined by spread plating 0.1 ml of the sample from the 

appropriate dilution onto duplicate plates of Violet Red Bile Glucose (VRBG; 

Difco) agar. All plates were incubated at 37o

C for 24 h. The minimum detection 

limit for the assay was log10 1.8 colony forming units/cm2. 

Survival During Refrigerated and Frozen Storage: Frozen ground beef was 

obtained from local retail establishments. The ground beef was irradiated to a 

minimum dose of 3 kGy at the Iowa State University Linear Accelerator Facility to 

eliminate the background gram negative microflora. Twenty five grams of the 

thawed meat was placed into sterile Whirl-Pak bags (Ft. Atkinson, WI) and 

inoculated with either the mixed culture of S. enterica or individually with the five 

surrogate E. coli bacteria, to an approximate population of ca. 107

 colony forming 

units/gram (cfu/g). The samples were stored at either 4o

C or -20oC, and sampled 

at 0, 1,3,5,7,10,14 and 21 days (4o

C) or 0,1,5,10,15,30,60 and 90 days (-20oC). 

7 
-

The populations of the bacteria were enumerated on VRBG after incubation at 

37oC for 24 h. 

Summer Sausage Fermentation: The survival of surrogate bacteria during 

summer sausage were compared to the mixed culture of S. enterica using the 

“beaker sausage” method (1) Briefly, 100 g of prepared summer sausage batter 

(50:50 beef : pork; 72:28 lean:fat; 2% NaCl) obtained from the Iowa State 

University Meat Laboratory and inoculated with Bactiferm HP starter culture, Chr. 

Hansen, Milwaukee WI. The summer sausage batter was subsequently 

inoculated with ca. 107 cfu/g of either the mixed S. enterica culture or the 

individual surrogate E. coli bacteria, and fermented at 37.8o C until the pH < 4.8 

(approximately 12 h). Samples were collected at the beginning and end of the 

fermentation, and the bacterial populations were enumerated on VRBG after 

incubation at 37o

C for 24 h. 

Statistical Analysis: All experiments were independently replicated three 

times. The microbial populations were converted from colony forming units (CFU) 

per cm2

 or g to log10 CFU/cm2 or g. The mean log10 reduction was calculated by 

subtracting the log10 (final) count of each organism after each microbial 

intervention from the log10 (initial) count obtained of each organism before the 

microbial interventions. The mean log reductions of each E. coli isolate and for 

the S. enterica composite culture for each treatment were compared by the 

general linear model procedure (GLM). Significant differences among means 

8 
(P<0.05) were reported using GLM procedures. All statistical analyses were 

conducted using SAS procedures (5). 

Results and Discussion 

The population reductions in the indicator bacteria were compared to the 

population reductions in the mixed salmonellae culture. Using this method of 

calculation, a lesser population reduction of the indicator bacteria, as compared 

to the salmonellae population, would indicate that the treatment would reduce the 

population of salmonellae more than that of the indicator. Conversely, a larger 

population reduction in the indicator bacteria, when compared to the salmonellae 

population, indicated that the treatment would reduce population of the indicator 

more than that of the salmonellae. A lesser population reduction in the indicator 

bacteria suggests that the indicator would under-predict the actual reduction in 

salmonellae, and this under-prediction could be considered a margin of safety for 

validating the process. 

The appropriateness of the E. coli isolates as potential Salmonella 

indicators was found to be dependent upon the microbial intervention utilized. 

On pre-rigor lean tissue (Table 2), the indicator organisms were not significantly 

different from the mixed culture of salmonellae for five of the treatments. For the 

20 oC water rinse only, two of the indicators (BAA-1430 and BAA-1431) resulted 

in a statistically (P<0.05) lower reduction in population than the mixed 

salmonellae culture. For the 20 oC water/20 oC acid and 20 oC water/20 oC 

9 
chlorine treatments, indicator culture BAA-1430 showed a statistically (P<0.05) 

lower reduction in population than the mixed salmonellae culture. 

On adipose tissue (Table 3), the indicator organisms were not significantly 

different from the mixed culture of salmonellae for four of the treatments. Two of 

the indicators (BAA-1427 and BAA-1428) resulted in a statistically (P<0.05) lower 

reduction in population than the mixed salmonellae culture for the 90o

 C water/ 

55 o C acid. Indicator BAA-1429 showed a statistically (P<0.05) greater reduction 

in population than the mixed salmonellae culture for the 90 o C water/20 o C acid 

and 20 o C water/20 o C acid. 

The surrogate E. coli bacteria were also evaluated in comparison to the 

survival of Salmonella enterica during storage. During both frozen (Figure 1) and 

refrigerated (Figure 2) storage, the surrogate bacteria survived in a manner either 

comparable to the mixed culture of S enterica, or in some cases to a greater 

extent than the mixed culture. That is, the surrogate bacteria would survive not 

less than the mixed culture of S enterica. This would provide a margin of safety, 

suggesting that the surrogate bacteria are in fact adequate predictors of the 

survival of S. enterica under normal storage conditions. 

The surrogate E. coli bacteria were also evaluated for their survival during 

summer sausage fermentation. Figure 3 shows the log10 reductions, calculated 

as log10 initial population – log10 final population, of both the mixed culture of S. 

enterica and the individual surrogate cultures. As with the storage conditions, the 

surrogate bacteria had population reductions generally less than those of the 

mixed salmonellae culture. Cultures BAA-1427, 1428, 1429 and 1430 had 

10 
population reductions from fermentation which were significantly less than the 

population reduction of the mixed S. enterica culture. The population reductions 

for the salmonellae inoculum were approximately 5 log10, while those for the four 

surrogate bacteria were approximately 3 to 3.5 log10. Only one isolate, BAA1431, was not statistically different (P>0.05) from the reduction in the 

salmonellae population. Again, this provides a margin of safety, as the reduction 

in the population of the surrogates is either equal to, or less than those of 

salmonellae. 

In conclusion, at least some of the non-pathogenic surrogate E. coli were 

equivalent to a mixed culture of salmonellae for all of the processes studied in 

these experiments. This study demonstrates that these bacteria, either 

individually or collectively, have the potential to be used to validate meat 

processes for the reduction of salmonellae. There are clearly limitations to these 

experiments, with the most significant being the use of a larger initial population 

than would be expected to be encountered in meat, as well as the potential for 

variation in response among strains of salmonellae not evaluated. These 

limitations are common with most experiments, in which laboratory data is used 

to predict performance in a processing establishment. However, indicator 

cultures evaluated in this research may allow a meat processing establishment to 

internally validate their own processes for salmonellae reduction. 

11 
ACKNOWLEDEGEMENTS 

The authors gratefully acknowledge the support of the staff of the Food 

Safety Research Laboratory. This research was supported by a grant from 

USDA-FSIS, FSIS-C-7-2005, and the Tri- State Food Safety Consortium. 

12 
References 

1. Glass, K.A., J. M. Loeffelholz, J. P. Ford and M. P. Doyle. 1992. Fate of 

Escherichia coli O157:H7 as affected by pH or sodium chloride and in fermented, 

dry sausage. Appl. Environ. Microbiol. 58:2513-2516. 

2. Hardin, M. D., G. R. Acuff, L. M. Lucia, J. S. Oman, and J. W. Savell. 

1995. Comparison of methods for contamination removal from beef carcass 

surfaces. J. Food Prot. 58:368-374. 

3. Marshall, K.M., S. E. Niebuhr, G. R. Acuff, L. M. Lucia and J. S. Dickson. 

2005. The identification of Escherichia coli O157:H7 meat processing indicators 

for fresh meat through the comparison of the effects of selected anti-microbial 

interventions. J. Food Protect. 68: 2580–2586. 

4. Mead, P.S., L. S., V. Dietz, L. F. McCaig, J. S. Bresee, C. Shapiro, P. M. 

Griffin, and R. V. Tauxe. 1999. Food-Related Illness and Death in the United 

States. Emerging Infectious Dis. 5:607-625. 

5. SAS Institute, Inc. 1985. SAS© User’s Guide: Statistics, Version 5 

Edition, SAS Institute Inc., Statistical Analysis System, Cary, NC. 

13 
6. USDA-FSIS. 1996. Pathogen Reduction; Hazard Analysis and Critical 

Control Point (HACCP) Systems; Final Rule. Federal Register 61(144):38806 – 

38989. 

7. USDA-FSIS. 2006. FSIS Announces Initiative to Reduce Salmonella in 

Meat and Poultry 

www.fsis.usda.gov/News_&_Events/NR_022306_01/index.asp (accessed 19 

Feb. 2007) 

14 
 

Table 1. Sources and identification of Salmonella enterica serovars and 

Escherichia coli cultures used as a reference cultures. 

Bacterium Strain Designator1 Source 

S. enterica var. 

Typhimurium 

G-24 Meat product isolate 

S. enterica var. 

Typhimurium 

G-25 Meat product isolate 

S. enterica var. 

Typhimurium 

G-26 ATCC 14028 

S. enterica var. 

Typhimurium G-5 

Poultry isolate; USDAARS, Russell Research 

Laboratory, Athens GA 

S. enterica var. 

Heidelberg 

G-1 Dairy cattle isolate; Iowa 

State University 

Veterinary Diagnostic 

Laboratory 

Bacterium Strain Designator1 ATCC accession number 

Escherichia coli P1 BAA-1427 

Escherichia coli P3 BAA-1428 

Escherichia coli P8 BAA-1429 

Escherichia coli P14 BAA-1430 

Escherichia coli P68 BAA-1431 

1 Iowa State University Food Safety Research Laboratories. Salmonella 

strain designators (G series) are from the internal culture collection. E. coli strain 

designators (P series) are as referenced on Marshall et al., 2005. 

15 
 

Table 2. Mean log10 reduction in populations of a five strain mixture of 

salmonellae and five individual non-pathogenic indicators on pre-rigor lean 

tissue. 

Micro 

organism 

90oC 

water 

90 oC 

water 

55 oC 

acid1 

90 oC 

water 

20 oC 

acid 

20 oC 

water 

20 oC 

water 

20 oC 

acid 

20 oC 

water 

20 oC 

chlorine2 

20 oC 

water 

20 oC 

TSP3 

salmonellae 1.40 2.72 1.55 1.614b 2.07 b 1.24 b 1.57 

BAA-1427 1.77 2.55 1.80 1.02b 1.82 b 0.91 b 1.21 

BAA-1428 1.43 2.45 1.55 0.76b 1.4 b 1.18 b 1.45 

BAA-1429 1.09 2.55 1.29 0.78b 1.59 b 0.87 b 1.19 

BAA-1430 1.19 2.03 1.32 0.41a 1.12a 0.81a 0.99 

BAA-1431 1.30 2.04 1.58 0.69a 1.24 b 1.04 b 0.94 

1 The acid rinse was 2% (wt/vol). 

2 The chlorine rinse was 20 ppm. 

3 The TSP rinse was 10% trisodium phosphate. 

4 Means within columns with different superscripts are significantly (P<0.05) 

different. If no superscripts are present, there was no significant difference 

between the means. 

16 
 

Table 3. Mean log10 reduction in populations of a five strain mixture of 

salmonellae and five individual non-pathogenic indicators on pre-rigor adipose 

tissue. 

Micro 

organism 

90oC 

water 

90 oC 

water 

55 oC 

acid1 

90 oC 

water 

20 oC 

acid 

20 oC 

water 

20 oC 

water 

20 oC 

acid 

20 oC 

water 

20 oC 

chlorine2 

20 oC 

water 

20 oC 

TSP3 

salmonellae 1.10 2.764b 1.93 b 1.29 2.31 b 1.30 1.56 

BAA-1427 0.96 1.20a 1.52 b 0.61 1.92 b 0.95 1.37 

BAA-1428 0.92 1.63a 2.01 b 0.78 2.25 b 1.25 2.14 

BAA-1429 1.13 2.97 b 3.02a 0.90 3.33a 1.03 1.45 

BAA-1430 0.77 2.68 b 1.74 b 0.99 2.10 b 1.05 2.21 

BAA-1431 0.96 2.63 b 2.06 b 0.69 2.67 b 1.28 1.59 

1 The acid rinse was 2% (wt/vol). 

2 The chlorine rinse was 20 ppm. 

3 The TSP rinse was 10% trisodium phosphate. 

4 Means within columns with different superscripts are significantly (P<0.05) 

different. If no superscripts are present, there was no significant difference 

between the means. 

17 
• 

----l:,.--

....... 111···'"· 

-·-0·-· 

······O······ 

--◊--

Figure 1. Survival of Salmonella enterica and surrogate E. coli bacteria in ground 

beef at -20 oC. 

Log10 Colony Forming Units/gram 

5 

6 

7 

8 

5 

6 

7 

8 

S. enterica 

BAA-1427 

BAA-1428 

BAA-1429 

BAA-1430 

BAA-1431 

0 20 40 60 80 100 

Time (days) 

18 
....... 111 ...... 

-·-0·-· 

········l .. 

Figure 2. Survival of Salmonella enterica and surrogate E. coli bacteria in ground 

beef at 4oC. 

4.5 

5.0 

5.5 

6.0 

6.5 

7.0 

7.5 

4.5 

5.0 

5.5 

6.0 

6.5 

7.0 

7.5 

Log10 Colony Forming Units/gram 

S. enterica 

BAA-1427 

BAA-1428 

BAA-1429 

BAA-1430 

BAA-1431 

0 5 10 15 20 25 

Time (days) 

19 
I c::J I 

Figure 3. Reduction of Salmonella enterica and surrogate E. coli bacteria during 

summer sausage fermentation. Reduction in log10 Colony Forming Units/g 

0 

1 

2 

3 

4 

5 

6 

Log10 initial - log10 final a a 

b 

b 

b 

b 

S. enterica BAA-1427 BAA-1428 BAA-1429 BAA-1430 BAA-1431 

Microorganism 

20