Effect of Sanitizers Applied by Electrostatic Spraying on Pathogenic and Indicator Bacteria Attached to the Surface of Eggs
Russell, S M
Primary Audience: Quality Assurance Personnel, Directors of Research, Microbiologists, Hatchery Managers, Table Egg Producers
Research was conducted to compare the effectiveness of BioSentry 904 and Biox H applied by an electrostatic spraying system for killing populations of bacteria that are of concern to the poultry industry. Populations of pathogenic bacteria Salmonella enteridis, Staphylococcus aureus, and Listeria monocytogenes and the indicator bacterium Escherichia coli were applied to eggs and allowed to attach for 1 h. BioSentry 904 completely eliminated all S. enteritidis on 100, 93, and 60% of eggs in experiments 1, 2, and 3, respectively. BioSentry Biox H completely eliminated all S. enteritidis on 100, 93, and 93% of eggs in experiments 1, 2, and 3, respectively. BioSentry 904 completely eliminated all S. aureus on 100, 87, and 100% of eggs in experiments 1, 2, and 3, respectively. BioSentry Biox H completely eliminated all S. aureus on 100, 100, and 80% of eggs in experiments 1, 2, and 3, respectively. BioSentry 904 completely eliminated all L. monocytogenes on 100% of eggs in experiments 1, 2, and 3. BioSentry Biox H completely eliminated all L. monocytogenes on 93, 87, and 73% of eggs in experiments 1, 2, and 3, respectively. BioSentry 904 completely eliminated all E. coli on 93, 93, and 60% of eggs in experiments 1, 2, and 3, respectively. BioSentry 904 and Biox H completely eliminated all E. coli on 93% of eggs in experiments 1, 2, and 3, respectively. BioSentry 904 and Biox H were extremely effective when used in conjunction with electrostatic spraying for eliminating pathogenic and indicator populations of bacteria from eggshell surfaces.
Key words: electrostatic spraying, disinfection, egg 2003 J. Appl. Poult. Res. 12:183-189
DESCRIPTION OF PROBLEM
The National Advisory Committee on Microbiological Criteria for Foods (NACMCF)  has reported that contaminated chicks are a potential source of Salmonella contamination within the growout house. Delivery of Salmonella-free chicks to the growout house is considered to be critical for the control of Salmonella in flocks . This recommendation is supported by the observed relationship between cross-contamination in the hatchery from Salmonella-infected eggs or surfaces to uninfected baby chicks during the hatching process and contamination of raw poultry products with Salmonella spp. Goren et al.  isolated salmonellae from three different commercial hatcheries in Europe and reported that the same serotypes found in the hatcheries could also be found on processed broiler chicken carcass skin. According to the NACMCF , it is important that chickens with as few pathogens as feasible be delivered to the processing plant because during poultry processing, there is no lethal heat process to reduce populations of Salmonella.
Studies have demonstrated that 71 % of egg-shell fragments, 80% of chick conveyor belt swabs, and 74% of pad samples placed under newly hatched chicks contained Salmonella spp. in a modern broiler hatchery . Cason et al.  reported that during the hatching process, Salmonella spp. is readily spread throughout the hatching cabinet due to rapid air movement by circulation fans. When eggs were inoculated with a marker strain of Salmonella during hatching, greater than 80% of the chicks in the trays above and below the inoculated eggs were contaminated . In an earlier study, Cason et al. [51 demonstrated that salmonellae on the exterior of eggs or in eggshell membranes could be transmitted to baby chicks during pipping.
Because of temperature, humidity, and the presence of high concentrations of organic material, Salmonella may survive in hatchery environments for extended periods. Salmonella is able to survive in chick fluff for 4 yr at room temperature, and as many as 1,000,000 Salmonella cells/g have been recovered from these samples .
Research has been conducted to evaluate the efficacy of various sanitizers for disinfecting eggshell surfaces and membranes. A 2.5% solution of H^sub 2^O^sub 2^, administered at 100 or 500 mL/h, was able to reduce Salmonella Typhimurium (ST)-positive eggshells by 55% and the number of chicks that are positive for ST by 53% . Others have reported that higher concentrations (5%) of H^sub 2^O^sub 2^ were able to reduce aerobic plate counts (APC) on hatching eggs by 2.99 log^sub 10^ cfu/ egg . A 3.0% quaternary ammonium sanitizer solution reduced APC on hatching eggs by 3 log^sub 10^ within 30 min of application . These studies evaluated spraying in addition to gaseous exposure and dipping.
The method of application may have a dramatic impact on efficacy of the sanitizers. Law  developed an electrostatic means of depositing liquids onto surfaces. This system electrically charges and atomizes the liquid as it passes through a nozzle. Electrostatic spraying (ESS) has been used to achieve a sevenfold increase in spray deposition over conventional application methods. Other researchers have reported up to a 24-fold increase in spray deposition using ESS . Thus, the ESS method may be an appropriate means of applying sanitizers in the hatchery environment by distributing the sanitizer more effectively over the surface of eggs and equipment.
The purpose of this research was to determine the effect of BioSentry 904 (904) and Biox H (BioxH) applied using electrostatic spraying on Salmonella enteritidis (SE), Listeria monocytogenes (LM), Staphylococcus aureus (SA), and Escherichia coli (EC). The hypothesis was that treating the eggs by ESS would enhance disinfection of hatching eggs while using less sanitizer.
MATERIALS AND METHODS
Pathogenic Bacterial Isolates
Salmonella enteritidis, LM, SA, and EC were obtained from the USDA, Agricultural Research Service (ARS) Poultry Microbiological Safety Unit laboratory. These isolates were originally collected from commercial broiler car-casses. Each isolate was assayed for Gram reaction, cytochrome oxidase activity, and production of catalase and was identified by the Vitek , Biolog , or Micro-ID  rapid identification method.
Stock Solution Preparation
A stock solution of 904  was prepared by adding 50 mL to 950 mL of tap water to achieve the manufacturer’s recommended concentration (5%). A stock solution of BioxH was prepared by adding 30 mL to 970 mL of tap water to achieve the manufacturer’s recommended concentration (3%).
Eggs were collected from Single Comb White Leghorn chickens housed at University of Georgia, Poultry Research Center. After collection, the eggs were washed using a commercially available chlorine-based sanitizer and were allowed to dry. Each egg was then rinsed thoroughly three times using sterile deionized water to remove any residual sanitizer that might have remained from the washing process.
An inoculation solution was prepared by placing 2 mL of an actively multiplying pure bacterial culture, incubated 24 h in brain heart infusion broth (BHI)  at 35°C, into 200 mL of sterile 1 % peptone broth. The bacterial cultures used were SE, LM, SA, and EC. Eggs were individually dipped into the inoculum and allowed to dry under a laminar flow hood for 1 h. This procedure provided time for the bacteria to attach to the surface of the egg. A total of 42 eggs were inoculated per experiment (two control eggs to determine the number that attached to the egg surface, 10 tap-water-treated to determine the effect of spraying with tap water, 15 eggs treated with 904, and 15 treated with BioxH). Inoculation levels were defined as follows: low (10,000 cfu/ mL).
Electrostatic Spraying of Eggs
After inoculation and drying, each egg was placed onto a clean egg flat and positioned in an electrostatic spraying chamber. Tap water or stock solution of sanitizer was sprayed onto the eggs using two electrostatic spray nozzles for 20 s each hour for 6 h. After treatment, the eggs were allowed to dry under a laminar flow hood for 1 h. In addition, two eggs were dipped in each bacterial isolate, allowed to dry, and stored for 6 h under a laminar flow hood as controls.
Neutralization of Sanitizers
Each control and treated egg was cracked using a sterile blade, and the contents were removed. Eggshells and membranes were placed into 25 mL of sterile 1% peptone broth  containing 3% Tween 80 , 0.3% lecithin , and 0.1% histidine  for at least 10 min to neutralize the sanitizers according to Opitz .
One milliliter of this mixture was placed into 9 mL of sterile BHI broth , which was used as a growth medium for conducting impedance or conductance assays, and vortexed. One milliliter of the sample and BHI mixture was placed into a Bactometer module well in duplicate. Samples were monitored using the Bactometer Microbial Monitoring System M128 . All of the bacterial isolates tested were monitored at 350C. All samples were monitored for 48 h using impedance except for EC, which was monitored using conductance.
The experimental design was a 3 × 4 × 3 of experiment (rep), bacterial type, and treatment (water, 904, or BioxH). All microbiological analyses were conducted in duplicate. Data were analyzed after averaging the duplicates. Results were analyzed using logistic regression and SAS software . All values reported as significant were analyzed at [alpha] = 0.05.
RESULTS AND DISCUSSION
As bacteria multiply in microbiological growth media, they produce metabolites that begin to accumulate. Production of these metabolites may be detected by monitoring the electrical characteristics of the medium. Once an electrical shift in the medium occurs, the time required for it to take place is recorded and is inversely proportional to the number of bacteria in the original sample.
Impedance and conductance have been demonstrated to be effective means of determining the minimum inhibitory concentration of sanitizing agents for inhibiting growth of psychrotrophic bacteria . If impedance or conductance detection times are significantly increased when bacterial populations are exposed to a chemical sanitizer, then the sanitizer has an inhibitory effect on the microorganism that is being measured. Moreover, if no detection time is recorded in 48 h, then it is assumed that the bacteria were deactivated or injured beyond repair by the sanitizer, as they were unable to multiply under optimal growth conditions.
In this study, significant differences in bacterial inhibition by each sanitizer were observed among experiments. For each experiment, different types and concentrations of bacteria were used. Thus, the differences observed among experiments may be attributed to the individual resistance of each bacterial isolate to 904 or BioxH, or it may be related to application of high numbers of bacteria in some instances.
Log^sub 10^ colony-forming units of bacteria per milliliter of inoculum exposed to 904 or BioxH are presented in Table 1. It should be noted that, in some cases, very high concentrations of bacteria were challenged in this study to determine the effect of the sanitizer on high numbers of actively growing pathogens and indicator populations of bacteria.
Impedance detection times (hours) for pure cultures of SE, LM, SA, and EC on eggs that were treated with 904 or BioxH using electrostatic spraying are presented in Tables 2, 3, 4, and 5, respectively. BioSentry 904 and BioxH completely eliminated all SE on all eggs in experiment 1 and all but one egg in experiment 2 (Table 2). In experiment 3, 904 was only able to completely eliminate all SE on nine of 15 eggs, with six eggs remaining positive. The detection times were significantly increased, indicating that it lowered the number of SE on eggs that remained positive as compared to controls. This result may be explained by the fact that a much higher inoculate of SE (12,880 cfu/mL) was used in this experiment compared to other experiments.
BioSentry 904 and BioxH completely eliminated all SA in all experiments, except in experiment 2 (904; two of 15 eggs remained positive) and experiment 3 (BioxH; three of 15 eggs remained positive) (Table 3). Again, this effect may be attributed to high levels of inoculate (Table 1).
With regard to LM, there was a distinct difference between the two sanitizers. The 904 eliminated all LM from all eggs in all experiments (Table 4), even when high levels of inoculate were used (4,450 cfu/mL). BioxH was unable to eliminate LM from all eggs in any experiment (Table 4), even though fairly low levels were used in experiments 2 and 3 (Table 1). Thus, 904 may be more appropriate to use in environments where LM is a concern.
Both sanitizers were able to eliminate EC from all but one egg in all experiments, except 904 in experiment 3 (Table 5). Only nine of 15 eggs were completely sanitized in this experiment, even though low concentrations of inoculate (395 cfu/mL) were used.
These data are promising in that 904 and BioxH are approved for use as a means of sanitizing equipment surfaces. Because Salmonella testing is part of the USDA-FSIS Pathogen Reduction Final Rule , these sanitizers may prove to be useful as a means of eliminating Salmonella and other pathogens on equipment surfaces within the processing plant during cleanup. Moreover, new regulations regarding the presence of Listeria on fully cooked poultry have been implemented, and these sanitizers may assist processors who must ensure that no Listeria remain in or on processing equipment from day to day.
In addition, bacteria that are often resistant to environmental extremes, such as SA and LM were susceptible to these sanitizers. Antimicrobial substances that inhibit gram-negative bacteria such as tellurite, neomycin, sodium azide, polymyxin, and acriflavine, do not adversely affect Staphylococcus [23, 24]. Other researchers have reported that SA may survive inside food processing equipment for months or years |25, 26]. Some SA may become resident in poultry processing plants because they are more resistant to chlorine and tend to clump together and form protective biofilms .
Other research has demonstrated that some SA are resistant to quaternary ammonium sanitizers. Thirteen percent of Staphylococcus strains isolated from meat and poultry processing plants were resistant to benzalkonium chloride (quaternary ammonium compound) . Therefore, 904 and BioxH may be useful for inactivating SA, which are resistant to commonly used compounds such as quaternary ammonium.
The LM has been shown to be resistant to the effects of sanitizing agents that are commonly used in the poultry industry. LM was reported to be resistant to the effects of trisodium phosphate (TSP) . In another study  5% Tween 80 and 1% TSP enhanced removal of psychrotrophic bacterial populations and Salmonella but had little effect on LM. Franco et al.  found Listeria spp. on processing plant surfaces after washing and disinfection had taken place. The authors concluded that commonly used disinfection procedures were not effective for controlling Listeria spp. These findings are promising in that 904 completely eliminated LM from all eggs tested, and BioxH eliminated it from most of the eggshell surfaces.
CONCLUSIONS AND APPLICATIONS
1. BioSentry 904 and BioxH were effective for eliminating SE from eggshell surfaces.
2. BioSentry 904 and BioxH were extremely effective for eliminating SA from eggshells, except when very high levels of inoculate were present; however, this is rarely observed in an industrial setting.
3. BioSentry 904 was excellent for eliminating LM from eggshells even when high levels of inoculate were used; however, BioxH was unable to eliminate LM from all eggs in any experiment, although low levels were used in experiments 2 and 3.
4. BioSentry 904 may be more appropriate to use in environments where LM is a concern.
5. BioSentry 904 and BioxH were effective for eliminating EC from eggshells, except 904 in experiment 3. Only nine of 15 eggs were completely sanitized in this experiment, even though low concentrations of inoculate were used.
6. BioSentry 904 and BioxH are excellent sanitizers and are effective when used in combination with electrostatic spraying for disinfection of pathogenic and indicator bacteria that are firmly attached to eggshells.
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This study was supported in part by state and Hatch funds allocated to the Georgia Agricultural Experiment Station. Appreciation is extended to bioMerieux Vitek, Inc. for its generous support, which included the Bactometer Microbial Monitoring System, technical assistance, and supplies.
S. M. Russell1
Department of Poultry Science, University of Georgia, Athens, Georgia 30602-2772
1 To whom correspondence should be addressed: email@example.com.
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