EVALUATION OF ADENOSINE TRIPHOSPHATE (ATP) BIOLUMINESCENCE FOR ESTIMATING BACTERIA

ON SURFACES OF BEEF CARCASSES

D.A. BAUTISTA’, G . KOZUB’, K.W.F. JERICH03 and M.W. GRIFFITHS1*4

‘Department of Food Science University of Guelph

Guelph, Ontario, Canada NIG 2Wl

2Agriculture and Agri-Food Canada Research Branch, Research Station Statistics Unit, P. 0. Box 3000 Main

Lethbridge, Alberta, Canada TIJ 4812

’Agriculture and Agri-Food Canada Food Production and Inspection Branch

Animal Diseases Research Institute P.O. Box 640, Lethbridge Alberta, Canada TIJ 324

Accepted for Publication September 4, 1996

ABSTRACT

A rapid (< 15 min), inexpensive and simple method has been developed to estimate the concentration of bacteria on surfaces of beef carcasses using adenosine triphosphate (ATP) bioluminescence. Surfaces (5 x 5 cm’) of beef carcasses (n = 159) were collected by excision. An ATP assay and aerobic plate count were performed on each sample. A signipcant < 0.001) positive linear relationship (r = 0.83) between plate count and ATP assay was obtained for 159 beef carcass samples. When thresholds levels were set at I x lo‘, 1 x 16 and 1 x lo6 CFU/cm2, there was moderate to good agreement between the ATP bioluminescence assay and the aerobic plate count as determined by the K-

statistic. The application of this ATP bioluminescence test to HACCP systems for -beef slaughter processes is discussed.

Correspondent: Dr. Manse1 W. Griffiths, Tel: 519-824-4120 x2269, Fax: 519-824-6631, E-mail: MGriffiths@APS.UoGuelph.CA

Journal of Rapid Methods and Automation in Microbiology 5 (1997) 37-45. All Righfs Reserved. 37 Copyright 1997 by Food & Nutrition Press, Inc., Trumbull, Connecticut.

INTRODUCTION

Agriculture and Agri-food Canada has implemented the Food Safety Enhancement Program (FSEP). It is designed to encourage plant inspection systems based on the Hazard Analysis Critical Control Point (HACCP) principle (FSEP 1991). One important criterion that the HACCP system requires is the identification and measurement of potential risks within a food processing system. For this purpose, the enumeration of total bacterial counts and/or specific pathogens is needed; preferably based on methods which are rapid, inexpensive and easy to use. The need for such an approach has been highlight- ed by the outbreak of foodborne illness due to the consumption of inadequately cooked hamburgers contaminated with E. culi 0157:H7 associated with the Jack- in-the-Box chain of restaurants. This resulted in a call for revisions in methods used for assessing the quality of meat (Powell 1993).

One method that may be applicable for rapidly monitoring total microbial load in foods is adenosine triphosphate (ATP) bioluminescence. It is an indirect assessment of contamination based upon the estimation of microbial ATP. This can be assayed by addition of firefly luciferase/luciferin to generate a light output that is proportional to the concentration of bacteria present in the sample (Bautista ef al. 1994a; Bautista ef al. 1994b; Griffiths 1991). The entire assay takes less than 15 min. A previous report has indicated that ATP biolumines- cence compares favorably to plate counting procedures for estimating the microbial load in milk and meat (Bautista ef al. 1994a; Bautista ef al. 1994b; Griffiths 1991; Olson 1991; Siragusa and Cutter 1995; Siragusa ef al. 1995; Van Crombrugge ef al. 1989). The ATP method has been also successfully employed to estimate microbial populations in chicken carcass rinses and process waters (Bautista ef al. 1994c,d).

In the search for a faster method, the use of ATP bioluminescence for the estimation of microbial numbers on surfaces of beef carcasses was investigated.

MATERIALS AND METHODS

Beef Carcass Sampling

Beef carcass samples were obtained from 3 beef processing facilities; Plant 1 (n=79); Plant 2 (n=40); Plant 3 (n=40). Samples from plant 1 were maintained at 4C during transportation (24 h) from Lethbridge, Alberta to the laboratory in insulated Styrofoam containers. Samples from Plants 2 and 3 were obtained and analyzed directly at the plant.

Samples were prepared by excision (approximately 5 x 5 cm) from several areas on the beef carcass. To increase the range of microbial counts in

RAPID ESTIMATION BACTERIA ON BEEF CARCASSES 39

the samples examined, some samples were pressed against hide at the abattoir before shipment and some were stored at 4C, 1OC and 37C for an additional 24 h before analysis. Carcass sample rinses were obtained by adding sterile water (1OOmL) to the samples contained in stomacher bags which were then manually shaken 30 times in an arc of approximately 50 cm. The rinse water samples were collected and analyzed by both aerobic plate count and by the ATP bioluminescence assay.

Microbial Analysis

Aerobic Plate Counts. Aerobic plate counts were performed by either using PetrifilmTM (3M PetrifilmTM, St. Paul, MN) (n=80) or a Spiral Plater Model D (Spiral Biotech, Inc., Bethesda, MD) to inoculate Standard Plate Count Agar (Oxoid: CM463) (n = 79). Plates and petrifilms were incubated at 37C for approximately 48 h and then counted.

ATP Bioluminescence Assay. ATP bioluminescence assays were performed on all samples. The assay involved the use of a modified Raw Milk Microbial ATP kit (Biotrace, Inc., Bridgend, UK: Raw Milk Quality Kit/ Product Code MMKAOO). Digestion of the sample with lipase, together with a pre-filtration step, was incorporated into the procedure recommended by the manufacturer to facilitate extraction of microbial ATP from the rinses. Lipase (Sigma L-1754, St. Louis, MO) was dissolved in Somex A (at 37C) to a final concentration of 10 mg/mL. This lipase solution (200-pL) was combined with a solution of Somex A (800-pL), also prewarmed to 37C. One mL of rinse was added to the lipase/Somex A reagent and placed in a water bath at 37C for 5 min. The mixture was loaded in a 5-mL sterile, disposable syringe and was filtered simultaneously through two sterile filter units. The first filter unit contained a Whatman No. 114 filter cut to a diameter of 13 mm and placed into a 13 mm filter holder (Millipore Swinnex filter unit, SX0001300). The second filter unit contained the bacteria-retaining filter supplied with the Raw Milk Microbial ATP kit. Microbial ATP was extracted from the microorganisms retained on the filter with Bactex (microbial ATP releasing agent; Biotrace, Inc.)(SOO-pL) and was assayed using the Enzyme-HM (luciferase/luciferh; Biotrace, Inc.) (100-pL) prepared as directed in the manufacturer’s instructions. Light emission was measured using a Biotrace Multilite luminometer (Biotrace, Inc., Bridgend, U.K.).

Statistical Analysis

Log,,, transformations of the result from both the ATP assay and plate counts were obtained and subjected to linear and a nonlinear regression analysis (Steel and Torrie 1980). In addition, least square means analysis was carried out

40 D.A. BAUTISTA ETAL.

to compare processing facilities and method used for aerobic plate count. The linear regressions of log,, aerobic plate count on log,, ATP based on the type of aerobic plate count or processing facility were compared by the General Linear Models program of SAS (1988).

As a further measure of the relation between plate counts and ATP assay, the kappa statistic (Martin el al. 1987) was used to measure the agreement of log,, aerobic plate count and log,, ATP readings for samples based on threshold levels of 1 x lo4, 1 x lo5 and 1 x lo6 CFU/cm*. For the log,, transformation of each of these threshold levels, a corresponding threshold level for log,, ATP count was estimated from the linear regression equation of log,, plate count on log,, ATP count. The kappa statistic ( K ) was calculated for each threshold value. A K-value between 0.50-0.69 and 2 0.70 is considered to be indicative of moderate agreement and very good to excellent agreement beyond chance, respectively (Martin et al. 1987). A K-value of 1.00 indicates complete agreement.

Samples (n=20) were analyzed in duplicate by the ATP bioluminescence assay and aerobic plate count and the repeatability calculated by the general linear model program.

RESULTS

Duplicate samples (n=20) analyzed by both the ATP bioluminescence assay and by aerobic plate count were not significantly (p > 0.10) different. Therefore, duplicates were consistent and single assays were carried out on subsequent samples. Comparison of least square means indicated that they were not significantly different (p > 0.10) for both results obtained at the different processing facilities and using the different methods used to determine plate count. The linear regressions of log,, aerobic plate count on log,, ATP using the different microbiological tests and on the samples obtained at different processing facilities were also not significantly different (p > 0.10). Therefore, all results from the three facilities and the corresponding aerobic plate count analysis were homogeneous and could be pooled into one data set.

Microbial levels for all beef carcass rinses determined by both ATP and aerobic plate count are compared in Fig. 1. There was a strong linear and nonlinear relation between results obtained by both methods. The relations are given by the equations:

(1) nonlinear relationship

log,, (aerobic plate count) = 7.58 - 5.29 x (ln(log,,(ATP count)))

(n = 159, r = 0.83, p < 0.001)

RAPID ESTIMATION BACTERIA ON BEEF CARCASSES 41

(2) linear relationship

log,, (aerobic plate count) = 1.83 - 2.28 x log,,(ATP count)

(n = 159, r = 0.83, p < 0.001)

10

9

8

7

6

5

4

3

2

1 2 3 4 5 6 7

Log ATP count (RLU/cmZ)

FIG. 1 . LINEAR AND NONLINEAR RELATION BETWEEN log,, ATP COUNT (RLU) AND log,, AEROBIC PLATE COUNT (CFU/ML) FOR 159 BEEF CARCASS SAMPLES;

PLANT 1 (m) (n = 79), PLANT 2 ( 0 ) (n = 40), PLANT 3 (*) (n = 40) Predictive quartiles determined from the regression equation obtained by analysis

of group means are shown as follows: lines numbered (1 ) log,, aerobic plate count of 4 and log,, ATP count of 3.43: lines numbered (2) log,, aerobic plate count

of 5 and log,, ATP count of 3.98; lines numbered (3) log,, aerobic plate count of 6 and log,, ATP count of 4.52.

When threshold levels were set at log,, aerobic plate count 4, 5 and 6 CFU/cm2, the corresponding log,, ATP count levels were 3.43, 3.98 and 4.52 RLU/cm2, respectively. These were calculated using the linear regression equation (2). The observed agreement, agreement beyond chance and the K-

statistic between log,, ATP count based on the frequency of samples that were above or below these threshold values are shown in Table 1. For all threshold levels, the observed agreement was moderate for levels set at 1 x lo4 and very good for levels set at 1 x lo5 and 1 x lo6 CFU/cm2.

42 D.A. BAUTISTA ETAL.

TABLE 1 . MEASURE OF AGREEMENT BETWEEN THE ATP BIOLUMINESCENCE ASSAY

AND SPIRAL PLATE COUNTS (SPC) FOR DETERMINING MICROBIAL LEVELS ON SAMPLES OF SURFACES OF BEEF CARCASSES

Threshold levels % agreement

log SPC log ATP2 Observed' Expected value^ (CFU/ml) (RLU) agreement agreement

4.0 5.0 6.0

3.43 74.1 3.98 90.0 4.52 82.3

49.0 0.50 44.0 0.82 39.3 0.71

' Percentage of samples with log,, SPC and log,, ATP counts above or below SPC and ATP threshold levels, respectively. Determined from regression of log,, ATP on log,, SPC. Kappa statistics

DISCUSSION

HACCP procedures, as part of a Food Quality Management system, are generally recognized as one of the best process control systems for food safety (Powell et al. 1993). To properly monitor, verify, and/or characterize critical control points (CCPs) in a meat processing facility, a testing program is required that quantifies the microbiological risks that may be conveyed to, or associated with, the food product. Estimates based on standard bacterial plate counts are accepted as useful indirect measures of hygienic risk during food processing (Mackey and Roberts 1993). To be useful for monitoring CCPs, results should be generated as soon as possible to allow enough reaction time for corrective action to be taken (Mackey and Roberts 1993). The ATP assay may serve this purpose since it is relatively fast, inexpensive and very easy to use. Results can be obtained within 15-20 min of sampling and the test is also relatively inexpensive. The ATP assay can be easily learned. An inexperienced laboratory assistant would require a training period of no more than 2 h to produce reliable results.

Both the linear and nonlinear regression analysis showed a strong correlation between plate count values and ATP bioluminescence readings. Microbial levels below 1 x lo3 CFU/cm2 showed an apparent lack of fit. At this level, populations of microorganisms cannot be accurately determined by the bioluminescence assay due to the background levels of ATP from nonmicrobial sources. Similar results have been reported for other foods (Bautista et al. 1994a; Griffiths 1991; Olson 1991). However, Siragusa et al. (1995a) have recently developed an ATP bioluminescence swab test for beef carcasses which

RAPID ESTIMATION BACTERIA ON BEEF CARCASSES 43

claimed a sensitivity of 1 X lo2 CFU/cm2 and a good correlation (n = 108, r = 0.92) with aerobic plate count. However, the sensitivity of their test was determined from a segmented nonlinear model whereas a single linear model may be more appropriate. Using their method of analysis, the sensitivity of the ATP bioluminescence assay developed in this study was calculated to be about 2 x lo2 CFU/cm2.

In an in-plant study by Siragusa ef al. (1995b), high correlations (r > 0.90) between aerobic plate count and their ATP bioluminescence swab tests were also generated for beef carcass samples and the ATP assay again was shown to have high sensitivity (1 x lo2 CFU/cm2). However, a sample area of 500 cm2 was used instead of the 25 cm2 sample area in the present study. The sensitivity per unit area of the ATP bioluminescence assay of this study could be increased by treating more than one 5 x 5 cm2 excised piece per sample. Assuming a homogeneous distribution of microbial load, it was calculated that if ten pieces were bulked together, the sensitivity would be reduced to about 1 x 102CFU/cm2. This method would also provide a more representative sample of the carcass.

The observed agreement and %-statistic showed good agreement between both the ATP assay and aerobic plate count at all threshold values (Table 1). These results are comparable to those found with chicken rinses (Bautista ef al. 1994~). Therefore, this study shows that the ATP assay would be useful as a monitoring test indicating breach of certain threshold microbial levels on surfaces of beef carcasses and may have application in attribute plans for cuts of processed carcasses (Hildebrant ef al. 1994). For example, the brisket site of some 220 carcasses sampled (5 x 5 cm2) at one high-line speed beef abattoir over a twelve month period had a maximum aerobic bacterial count 5.52 log,, CFU/mL (Jericho ef al. 1994). This corresponds to a standard pour plate count of 5.61 log,, CFU/cm2 which is well above the sensitivity of the ATP bioluminescence method (1 x lo3 CFU/cm2). According to that only published “Advisory Scale” of beef carcass hygiene, this level of contamination would signal corrective action (Mackey and Roberts 1993).

This test may also be useful for the development of HACCP programs. Where the HACCP program may be early in its development, the ATP bioluminescence assay could be used to verify that the CCP points are eliminating microbial contamination. This wouldallow faster implementation and less “down time” to evaluate problems with the program. Perhaps to further the progress of this technology, automation, combined with other speedier sampling methods, could be used to enhance the microbiological sampling regimes required for quality control (Hildebrant ef al. 1994). The method may also replace some of the indirect methods used to monitor product safety associated with CCPs.

44 D.A. BAUTISTA ETAL

In conclusion, this report indicates that the ATP assay can reliably determine upper levels of bacterial contamination in beef carcass rinses. However, the test should be used to give an estimate of bacterial population rather than exact counts on beef carcasses. The ATP bioluminescence assay can provide fast and reliable results for determining hygienic risks associated with beef carcasses on a near “real-time” basis.

ACKNOWLEDGMENTS

We thank Ms. J. Dixon-MacDougall for assistance in sampling and shipment of samples and Mr. B.J. Nishiyama for assistance with the statistical processing. This work was supported by Food Production and Inspection Branch of Agriculture and Agri-foods Canada, Dairy Farmers of Ontario, Natural Science and Engineering Research Council of Canada and the Ontario Cattlemen’s Association.

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