Spatiotemporal analyses of the interaction between E. coli
O157:H7 and beef carcass tissue surfaces were conducted by using transformed bacteria which expressed the complete lux
operon of P. luminescens
) as an optical tag. The interaction of the tagged bacterium with tissues was monitored by using bioluminescence as a real-time indicator. The complete lux
operon includes genes that encode the biosynthetic enzymes for the substrate, and thus, exogenous substrate addition is not required. Although addition of an exogenous aldehyde, such as decanal, may increase the output of the luxAB
genes compared to the output when the substrate is supplied through expression of all of the lux
), a system that does not require exogenous substrate addition removes any variability due to substrate availability and streamlines the assay. Moreover, any potential effects of exogenous substrate addition on the physical or temporal location of bacterial cells on the surface tissues are eliminated. The P. luminescens
luciferase genes have the advantage of functioning at a higher temperature (up to 45°C) than other luciferases that have been characterized (2
), a trait which may be useful in future in vivo applications of the bioluminescent strain of E. coli
The utility and validity of using luminescent bacterial strains to study biological processes have been reviewed previously (33
). Data from this study and elsewhere indicate that there is a strong correlation between the viable counts of bioluminescent bacteria and light emission (3
We observed that as few as 50 cells of E. coli
O157:H7 strain L-2 could be detected by bioluminescence and that there was an approximately 10-fold decrease in sensitivity when bacteria were suspended in bovine fecal slurries compared with bacteria suspended buffer or broth. The slopes of the curves in plots of intensity versus cell number indicated that the sensitivity of detection was probably affected more by the optical properties of the fecal slurry than by direct effects of feces components on light output per cell. Bioluminescence from mouse fecal suspensions inoculated with Yersinia enterocolitica
) and luxCDABE
-bearing Salmonella typhimurium
) and bioluminescence from bovine feces from cows fed luxAB
-bearing Y. enterocolitica
have been reported previously (16
Initial inoculation of E. coli
O157:H7 strain L-2 onto tissues revealed that bacteria could be visualized directly on a large area of beef carcass surface tissue. The luminescence appeared to be especially concentrated along muscle striations on the surface of the tissue, which differed in appearance, composition, and topography (Fig. ). In a separate experiment, increases in bioluminescence were observed after incubation on a section of beef tissue (Fig. ). A similar observation was reported by Chen and Griffiths for luxAB
-bearing Salmonella enteritidis
inoculated onto chicken meat following incubation (3
Water washing of beef tissues inoculated with bioluminescent E. coli
O157:H7 strain L-2 by the immersion-shaking method or by using a bottle to squirt water onto the inoculated surface revealed that the effects of both decontamination methods could be visualized in real time, whether the surface tissue was lean or adipose. Although these washing procedures were not entirely comparable in terms of pressure and volume to the spray washing procedures used by the meat industry or during pilot-scale research (7
), our results suggest that imaging bioluminescent bacterial reporter strains can be used to assess carcass decontamination processes.
Differences in the attachment of food-borne bacterial pathogens to various tissue surfaces are an important issue in food microbiology and food processing. Attachment to tissue-specific molecules or polymers (30
) and attachment to or detachment from carcass surface tissues and excised sections of meat are typically determined by sampling and by performing plate count experiments (9
). The ability to monitor the process of attachment of pathogens to or association of pathogens with carcass surfaces in real time without exogenous sampling should accelerate such studies and has specific advantages. Unlike using physical biochemical indicators, using luciferase as the reporter ensures that the signal observed is from viable, metabolically active cells which can be detected as they exist in or on a food matrix.
Data presented in this paper indicate that bacterial association with the beef carcass surface may be influenced by the physical topography and structure of the surface. We observed that following a water wash, tissues that were rougher retained more bioluminescence (which correlated with the viable counts) than tissues that were relatively smooth and homogeneous. It has been reported that bacterial removal from or decontamination of beef carcass surface tissue is influenced by the level of lean or adipose tissue in the underlying tissue menstruum (7
). In these previous studies, excised samples of treated tissues were more likely to retain bacterial inocula if the surface tissue was lean than if it was adipose. Our bioluminescence data are in agreement with these observations; however, our bioluminescence method generated both spatial and quantitative information in real time. Although bioluminescence monitoring will not entirely replace culture methods in the study of attachment and decontamination, obviating the need for excision sampling, it has distinct advantages as a screening tool for selecting decontamination protocols for validation.
The observation that increased bioluminescence indicative of bacterial growth occurs on tissue surfaces after incubation has broad implications for studying decontamination processes. Since small numbers of tagged organisms (approximately 50 cells) can be detected on tissue surfaces and since luciferase-based photon counting imaging has a broad dynamic range of many logs, low levels of microbial contamination can be monitored over time. Moreover, spatially resolved analyses of bacterial survival and growth patterns after antimicrobial treatments could be rapidly performed by using the molecular biophotonic method described here, which would eliminate the need for exogenous sampling and culturing.
Eliminating back-extrapolation of sample plate count data would greatly improve the validity of carcass surface microbial count data. Biophotonics eliminates the need for the assumption that bacterial loads are homogeneously distributed across the surfaces of carcasses. Extrinsic factors that influence attachment of microorganisms to fascia-covered animal carcasses, such as the degree of hydration and composition, as well as putative intrinsic attachment factors, such as cell surface receptors, host tissue components (e.g., hyaluronan, collagen, and chondroitin sulfate [36
]), could be systematically examined in situ over significantly larger areas of carcass tissue surface than the areas previously examined to determine their roles in attachment by using the biophotonic system described in this study.
Cloning promoters of interest upstream of a complete lux
operon could provide indicator strains that could report metabolic, regulatory, or gene expression activity under different environmental conditions found in food processing. A similar approach has been described by Dodd et al., who used a spvA-lux
fusion in S. enteritidis
to monitor levels of the RpoS product in cells in the presence and absence of a competitive microflora (10
Since bioluminescence has been used as a real-time genetic reporter in live animal models (4
), the growth and survival of E. coli
O157:H7 and other enterohemorrhagic E. coli
organisms could be evaluated in animal models involving human infection and carcass processing. Specifically, expression of genotypic traits that influence survival in animals, such as the factors which determine the minimum infectious dose in human hosts, and transfer from the animal or gastrointestinal tract to the carcass during processing could be studied by this biophotonic procedure.
Our findings demonstrate that real-time macroscopic imaging of bacteria on beef carcass tissue surfaces is possible if bacterial strains with a lux
gene reporter are used. We present evidence showing that different tissue surfaces, especially lean and adipose surfaces, retain different levels of contaminating bacteria. The magnitude of the differences is consistently less than 1 log10
unit of inoculated population per unit of area. Therefore, any practical implications of this observation are yet to be determined. Previously, however, Thomas and McMeekin (36a
) documented the role of the water microlayer on poultry skin, which has a very significant effect on microbial attachment. It is not unlikely that in our experiments adipose tissues retained less water on their surfaces (due to their inherent hydrophobicity) and thus retained fewer bacteria after simulated spray washing. The clear differences between the prewashed and postwashed tissues indicate that a molecular biophotonic approach to studying microbial association with carcass tissue surfaces offers a truly in situ strategy for understanding a problem previously approached only retrospectively (i.e., through sampling, culturing, and subsequent back-calculation of microbial densities on tissue surfaces).