Efficacy of Octenidine Hydrochloride for Reducing Escherichia coli O157:H7, Salmonella spp., and Listeria monocytogenes on Cattle Hides

Abstract

The efficacy of octenidine hydrochloride (OH; 0.025, 0.15, and 0.25%) for inactivating Escherichia coli O157:H7, Salmonella spp., and Listeria monocytogenes on cattle hides was investigated at 23°C in the presence and absence of bovine feces. All tested concentrations of OH were effective in decreasing more than 5.0 log CFU of bacteria/cm² in 5 min (P < 0.01). The results suggest that OH could be used to decontaminate cattle hides; however, further studies under commercial settings are necessary to validate these results.

TEXT

Escherichia coli O157:H7 (19, 34), Salmonella spp. (11, 26), and Listeria monocytogenes (25, 32) are major food-borne pathogens that colonize the gastrointestinal tract of cattle. These pathogens are shed in the feces, thereby leading to pathogen contamination and persistence on hides for long periods of time (5, 10, 22, 37). The reported prevalence rate for E. coli O157:H7 on cattle hides ranges from 11% (24) to 76% (4), whereas Salmonella prevalence has been reported to be as high as 94% (17). The prevalence of Listeria spp. on cattle hides was found to be higher during cooler weather (28 to 92%) than warmer weather (6 to 77%) (27). Since E. coli O157:H7 may persist on cattle hides for extended periods of time, strategies that reduce fecal loads of the pathogen in animals may not be effective for preventing carcass contamination on a long-term basis (7). Moreover, the hide prevalence of E. coli O157:H7 has been reported to be a more accurate predictor for carcass contamination than the fecal prevalence of the pathogen (9).

Generally, carcass muscle surfaces are sterile, but bacterial contamination occurs as a result of pathogen transfer from hides onto the meat during slaughter and the hide removal processes. Previous studies revealed that carcass contamination with pathogens is strongly correlated to hide contamination (5, 6, 12, 15, 16). Thus, it is important to decrease pathogens on cattle hides to reduce the risk of human exposure to these pathogens from beef carcasses. Effective and practical treatments that eradicate or reduce pathogens on hides would also help in the successful implementation of Hazard Analysis Critical Control Points (HACCP) programs by the meat industry.

Octenidine hydrochloride (OH) is a positively charged bispyridinamine that exhibits antimicrobial activity against a wide range of microorganisms, including plaque-producing Streptococcus mutans and Streptococcus sanguis (8). Our laboratory previously observed that OH was effective in rapidly killing planktonic cells and biofilms of Listeria monocytogenes on different abiotic surfaces at 37, 21, 8, and 4°C in the presence and absence of organic matter (2). Octenidine hydrochloride exerts its antimicrobial activity by binding to the negatively charged bacterial cell envelope, thereby disrupting vital functions of the cell membrane and killing the bacterium (18). It has high affinity for cardiolipin, a prominent lipid in bacterial cell membranes, making it selectively lethal to bacterial cells without adversely affecting eukaryotic cells (18). Additionally, repeated exposure of Staphylococcus aureus to OH for up to 3 months did not induce resistance to the compound (1), suggesting a low potential of bacteria to develop resistance to OH. Octenidine chloride has a high degree of safety and has been found safe for skin disinfection in patients undergoing bone marrow transplantation (36). Toxicity studies in a variety of host species have revealed that OH is not absorbed through mucous membranes and the gastrointestinal tract, and there are no reports of carcinogenicity, genotoxicity, or mutagenicity (28, 29).

The objective of this study was to investigate the efficacy of OH for reducing E. coli O157:H7, Salmonella spp., and L. monocytogenes on cattle hides.

All bacteriological media were obtained from Difco (Sparks, MD). Five isolates each of E. coli O157:H7, Salmonella spp., and L. monocytogenes from our culture collection were used in the study. E. coli O157:H7 strains included E16 (meat isolate), E10 (meat isolate), E8 (meat isolate), E22 (calf feces isolate), and E6 (milk isolate); Salmonella spp. were S. enterica serovar Typhimurium DT104 43, S. Typhimurium DT104 H3380, S. Typhimurium Presque 3712, S. Typhimurium ATCC, and S. Newport. L. monocytogenes strains used for the study included ATCC 19115 (human isolate), ScottA (human isolate), 315 (pork isolate), 316 (pork isolate), and 24 (human isolate). All strains of the pathogens were induced for resistance to nalidixic acid (NA; 50 μg/ml; Sigma-Aldrich Chemical, St. Louis, MO), as described previously (38). For confirming resistance to the antibiotic, the cultures were streaked on tryptic soy agar (TSA) supplemented with 50 μg/ml of nalidixic acid, and growth was checked after incubation at 37°C for 24 h. Each bacterial isolate was cultured separately in 10 ml of sterile tryptic soy broth (TSB) supplemented with 50 μg/ml of NA at 37°C for 24 h with agitation (150 rpm). Following incubation, the cultures were sedimented by centrifugation (4°C, 8,000 × g, 10 min), washed twice, and resuspended in 10 ml of sterile phosphate-buffered saline (PBS; pH 7.3). The bacterial population in each culture was determined by plating 0.1-ml portions of serial dilutions (1:10 in PBS) on duplicate TSA plus NA plates, with incubation at 37°C for 24 h. For each pathogen, equal volumes containing approximately equal populations from each of the five strains were combined, and 500 μl of an appropriately diluted suspension was used as the inoculum (∼8 log CFU).

The antimicrobial efficacies of OH to reduce E. coli O157:H7, Salmonella spp., and L. monocytogenes on cattle hides were determined as described previously (3, 23), with slight modifications. Fresh cattle hides were collected from cows slaughtered at a local slaughterhouse. Hides were cut into square pieces (5 by 5 cm), cleaned with 70% ethanol, and air dried under a laminar flow hood prior to the experiment. Each bacterial inoculum (∼8.0 log CFU/0.5 ml) was applied in drops onto each hide piece and spread evenly using a sterile spreader. The inoculated hide pieces were dried in a laminar flow hood for 2 h. After drying, each hide piece was sprayed with 25 ml of sterile deionized water, 95% ethanol, or 0.05%, 0.15%, or 0.25% OH (dissolved in 95% ethanol) or not subjected to any spray (control). The control and treated hide samples were kept at room temperature (23°C) for 5 min, and the inoculated pathogen population was enumerated at 2 and 5 min. A sterile cellulose sponge premoistened with 10 ml of maximum recovery diluent (MRD) was used to swab each hide sample five times, and the sponge was placed in a sterile stomacher bag containing 40 ml of MRD and pummeled in a stomacher for 1 min. The diluent was serially diluted in PBS, and appropriate dilutions were plated on TSA plus NA plates and incubated at 37°C for 24 h. When the pathogens were not detected by direct plating, MRD samples were tested for surviving bacteria by enriching 1 ml of culture in 100 ml of TSB at 37°C for 24 h, followed by streaking on sorbitol MacConkey agar (SMA) containing 0.1% 4-methylumbelliferyl-β-d-glucuronide and 50 μg/ml NA (for E. coli O157:H7), xylose lysine deoxycholate (XLD) agar plus NA (for Salmonella), or Oxford agar plus NA (for L. monocytogenes) and incubated at 37°C for 24 h. Representative colonies of bacteria from SMA and XLD were confirmed for E. coli O157 and Salmonella, respectively, by an E. coli O157 latex agglutination test (Oxoid Division, Unipath Co.) and Salmonella latex agglutination test (Microgen, United Kingdom). Representative colonies from Oxford agar plates were confirmed for L. monocytogenes by using the API-20E bacterial identification kit (bioMérieux).

Since manure, a common contaminant present on cattle hides, could reduce the efficacy of OH for inactivating pathogens, the experiment was repeated in the presence of 5% autoclaved bovine feces added onto hide samples. Briefly, the inoculum of each bacterial pathogen prepared in PBS containing 5% (wt/vol) sterile feces was applied on each hide piece, allowed to dry, and subjected to the treatments as described above. Three replicate samples of the cattle hides were included for each treatment and control, and the entire experiment was repeated four times.

Data from the four independent replicate experiments were pooled. The bacterial counts of each pathogen were analyzed separately, and the effects of OH concentration, sampling time, and their interaction on bacterial counts were analyzed using the PROC MIXED program of SAS (SAS Institute, Cary, NC). Variation among replicates was used as the error term. Data were expressed as least-squares means; differences were considered significant at a P level of <0.01.

Since OH was found equally effective in killing all three pathogens on cattle hide in the presence and absence of bovine feces, only the results obtained from cattle hides containing 5% feces are presented in the manuscript. The efficacy of OH for inactivating E. coli O157:H7, Salmonella spp., and L. monocytogenes on cattle hides is depicted in Fig. 1A to C, respectively. The untreated hide samples (controls) yielded approximately 6.3 log CFU/cm² of E. coli O157:H7 after 2 and 5 min of incubation at 23°C. Similar counts of E. coli O157:H7 were also recovered from the hide samples sprayed with water. Hide treatment with 95% ethanol (control) decreased the bacterial pathogen count by approximately 1.5 log CFU/cm². However, all the concentrations of OH reduced substantial populations of the pathogen on hide samples after 2 and 5 min of treatment (P < 0.01). Octenidine hydrochloride decreased E. coli O157:H7 populations on hide samples by >5.0 log CFU at 5 min of exposure (Fig. 1A). The efficacies of OH for killing Salmonella (Fig. 1B) and L. monocytogenes (Fig. 1C) were similar to that observed for E. coli O157:H7. Compared to untreated controls and water-treated hide samples, all three concentrations of OH caused reductions in bacterial counts of ∼5.0 log CFU/cm² (Fig. 1B and C).

Fig 1.

Efficacies of octenidine hydrochloride for reducing E. coli O157:H7 (A), Salmonella spp. (B), and L. monocytogenes (C) on cattle hides in the presence of 5% bovine feces at 23°C. Values are expressed as means ± standard errors (bars). Bars with different lowercase letters denote significant differences between treatments (P < 0.01). Each treatment had three samples of hides, and the experiments were replicated four times.

Cattle hides constitute a primary source of pathogens that contaminate beef carcasses during slaughter and subsequent operations. Since washing of cattle hides with water has only a minimal effect in reducing pathogen load (31), there is an increasing interest in identifying effective decontamination treatments for cattle hides (30). A variety of antimicrobials used for inactivating E. coli O157:H7 and other bacteria on cattle hides have been reported in the literature, with various magnitudes of reduction in the bacterial populations. These antimicrobials include organic acids (20, 31), alcohols (31), mineral acids (15), cetylpyridinium chloride (13, 14), commercial detergents/disinfectants/sanitizers (35), ozonated and electrolyzed oxidizing water (16), a shellac (food-grade resin) and ethanol mixture (3), and bacteriophage application (33). Most of these treatments generally yielded about 3.0- to 4.0-log reductions in pathogen counts on hides under experimental conditions and 2.0- to 3.0-log reductions under commercial slaughter conditions (3). Another study (20) reported that treatment of cattle hides with 10% acetic acid and 10% lactic acid at 23°C reduced E. coli O157:H7 counts by 0.7 log and 2.9 log/cm², respectively. Spraying of cattle hides with 23% shellac-ethanol yielded about a 2.1-log reduction in E. coli O157:H7 counts on artificially inoculated cattle hides (3). Similarly, spraying of cattle hides with sodium hydroxide solution (3%) decreased E. coli O157:H7 (21) and Salmonella counts by 3.4 and 2.6 log/cm², respectively (21), whereas a combination of sodium hydroxide (1.6%) and chlorine (200 to 500 ppm) was more effective, reducing the pathogen counts by 4.5 to 5.0 log/cm² (21). In our study, we observed that OH was very effective in killing all three pathogens on cattle hides, decreasing the pathogen counts by >5.0 log CFU/cm² after 5 min of treatment at 23°C. Although all three tested concentrations of OH were effective in significantly decreasing pathogen populations on hides, in general, the efficacy of OH was greater with increased concentrations. It should be noted that most of the previously reported treatments were not investigated for their hide-decontaminating efficacies in the presence of bovine feces, which when present on cattle hides could potentially decrease antimicrobial effects. However, OH was found equally effective in killing the pathogens on cattle hides in the presence and absence of bovine feces.

In conclusion, the results of this study suggest the potential application for OH for decontaminating cattle hides and justify further investigations to validate its efficacy for use in slaughterhouses.