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Identification of Enterobacteriaceae on Vacuum Loaders in Shell Egg Processing

USDA, Agricultural Research Service, Egg Safety and Quality Research Unit, Russell Research Center, Athens, GA 30605

¹ Corresponding author: Deana.Jones{at}ars.usda.gov

	ABSTRACT

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Cleaning and sanitation are paramount in food processing. Gaining an understanding of the microbial populations present in a processing facility can help in the development of effective and efficient cleaning. The current study was undertaken to gain a better understanding of the Enterobacteriaceae present on vacuum loader cups used in shell egg processing to transfer nest run eggs to the processing line. Twenty cups were rinsed on each of 3 visits to both an off-line operation and a mixed operation. A total of 442 Enterobacteriaceae isolates were biochemically identified from vacuum loader cup rinses. The predominant genera isolated from the 2 facilities were Enterobacter, Klebsiella, Escherichia, Citrobacter, and Serratia. The primary organisms from the off-line facility were Klebsiella oxytoca, Enterobacter amnigenus 2, and Klebsiella pneumoniae. The isolates found in the greatest proportion from the mixed operation were Enterobacter cloacae and Klebsiella oxytoca. A total of 18 genera were recovered from the 2 facilities, with 9 being present in both processing facilities. The findings of this study can be used in assessing the sources of bacterial contamination in egg processing and in developing more effective, targeted cleaning programs for processing equipment and facilities.

Key Words: Enterobacteriaceae • vacuum loader cup • shell egg • processing • sanitation

	INTRODUCTION

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Since as early as the 1940s, it has been known that disinfecting shell egg processing equipment reduces egg spoilage (Sayers, 1943). In a more comprehensive examination of current shell egg processing methods, Moats (1981) reported that the bacterial counts on the shells of washed eggs were closely related to the counts on equipment surfaces and in wash water. A general assessment of food processing noted that the main sources of product contamination come from direct and indirect contact surfaces, water, air, and personnel (Slade, 2002). Previous studies have examined the bacterial levels on both direct and indirect contact surfaces as well as in the air of shell egg processing facilities (Jones et al., 2003; Musgrove et al., 2004b; Northcutt et al., 2004).

When processing plant sanitation practices were examined in 9 US southern region shell egg processing plants, there were no significant differences in bacterial levels before and after sanitation procedures for both contact and noncontact surfaces (Jones et al., 2003; Musgrove et al., 2004b). Davies and Breslin (2003) discussed the need for better cleaning and disinfection of equipment in egg-packing facilities in the United Kingdom. They observed poor cleaning practices and residual contamination of equipment. Salmonella was detected in 16.7% of the egg transfer units tested. Northcutt et al. (2004) monitored the microbial levels present in the air of shell egg processing facilities. They found average aerobic organism levels of 4.0 log cfu/mL of air, yeast and molds of 3.5 log cfu/ mL of air, pseudomonads of 2.7 log cfu/mL of air, and coliform levels of 1.5 log cfu/mL of air.

Although the US egg industry has made a shift to inline production and processing facilities (hens on site with the processing facility), there are still operations that require eggs to be brought in from off-site production. When nest run eggs, as they are called, enter a processing facility, they must be loaded onto the processing line. Vacuum loaders are used for this transfer. Rubber corrugated cups are attached to the vacuum loaders to form a seal with the egg, allowing the vacuum to be drawn and the egg to be transferred from the egg flat to the processing line (Figure 1).

View larger version (154K): [in this window] [in a new window]   Figure 1. Vacuum loader in a shell egg processing facility.

	MATERIALS AND METHODS

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Vacuum loaders were sampled on 3 occasions each at 2 shell egg processing facilities (one off-line and one mixed operation) according to the methods described by Jones and Musgrove (2008). An off-line operation requires nest run eggs to be transported to the processing facility and transferred to the processing line. A mixed operation has laying hens on site but also supplements processing volume with nest run eggs that are transported to the facility. Briefly, 20 vacuum loader cups were randomly chosen at each visit, removed from the processing line, and rinsed for 1 min with 50 mL of sterile PBS. Rinsates were transported to the laboratory on ice and immediately analyzed for microbial levels. This work has been summarized in Jones and Musgrove (2008).

Enterobacteriaceae were enumerated by plating 1 mL of rinsate into violet red bile glucose agar with overlay (Becton Dickinson, Sparks, MD). The duplicate plates per sample were incubated at 37°C for 18 to 24 h before assessing microbial growth. A random sampling of colonies from positive plates was conducted according to the methods of Musgrove et al. (2004a). Briefly, up to 5 colonies were selected from samples exhibiting presumptive positive growth on violet red bile glucose agar. The isolates were streaked for purity by passing 3 consecutive times onto plate count agar and incubating overnight. After the third pass, a culture solution was prepared in 5 mL of sterile PBS. This culture was used to inoculate API20E strips for identification (bioMérieux Inc., Marcy l’Etoile, France). Results were analyzed in accordance with the manufacturer’s recommendations.

	RESULTS AND DISCUSSION

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A total of 442 Enterobacteriaceae isolates were identified in the current study (Table 1). There were 225 (51%) from the off-line operation and 217 (49%) from the mixed operation. Eighteen different genera were identified. Enterobacter spp. was the most frequently detected (184 isolates, 42%). Forty-one percent of the Enterobacter spp. were found in the off-line operation, with 59% coming from the mixed operation. When both processing facilities were combined, the most frequently identified genera were Enterobacter spp. (42%), Klebsiella spp. (21%), Escherichia spp. (8%), Serratia spp. (7%), and Citrobacter spp. (7%). For the 225 off-line operation isolates identified, the predominant genera or species were Enterobacter spp. (33%), Klebsiella spp. (27%), Serratia liquefaciens (12%), Citrobacter spp. (9%), and Pantoea spp. (8%). Comparatively, in the mixed operation the predominant genera or species were Enterobacter spp. (50%), Klebsiella spp. (15%), Escherichia spp. (12%), Citrobacter spp. (5%), Leclercia adecarboxylata (3%), and S. liquefaciens (3%).

There were 9 occurrences of organisms being isolated from only one of the facilities. In the off-line operation, Cedecea davisae and Yersinia enterocolitica were each isolated once in the second replicate. Four isolates were identified as Aeromonas spp. over 2 replicates. Futher-more, Proteus spp. were found on 3 occasions in 2 replicates. In the mixed operation, 7 isolates were identified as L. adecarboxylata over all 3 replicates. Acinetobacter baumanii (7 isolates), Kluyvera spp. (1 isolate), Moellerella wisconsensis (1 isolate), and Shigella sonnei (1 isolate) were found in single replicates in the mixed operation.

Several of the species isolated in this study have been associated with egg spoilage (Jay et al., 2005). Acinetobacter and Pseudomonas are aerobic gram-negative rods and not members of the family Enterobacteriaceae. However, both genera may cause egg spoilage. Pseudomonas spp. have been linked as a cause for green and pink rots in eggs. Pseudomonas and Acinetobacter are associated with clear rots. Black rots have been associated with the genera Proteus, Pseudomonas, and Aeromonas. Red rots are often caused by Serratia spp. and "custard" rots by Proteus vulgaris. Citrobacter has also been linked to rotting in shell eggs (Banwart, 1989). All of these organisms were isolated from the vacuum loader cups sampled in the current study. The vacuum loaders transfer each nest run egg onto the processing line, therefore serving as a potential contamination point. According to processing plant personnel, the vacuum loader cups are replaced as they begin to wear and do not effectively hold a suction for egg transfer (approximately 2 wk).

Previous work conducted in our laboratories examined the Enterobacteriaceae populations present on the surfaces of washed and unwashed eggs during cold storage (Musgrove et al., 2004a). Many of the same predominant genera were detected in the current study. Musgrove and colleagues (2004a) have isolated Escherichia and Enterobacter most frequently. Ibeh and Izuagbe (1986) isolated Escherichia coli, Pseudomonas, Proteus, Klebsiella, Alcaligenes, Bacillus, Staphylococcus aureus, and Streptococcus faecalis from unwashed, cracked eggs being used by the confectionary industry in Nigeria. Klebsiella, Staph. aureus, and Alcaligenes were the predominant organisms isolated. A survey conducted in Korea found E. coli, Escherichia hermanii, and Citrobacter freundii on the shells of retail eggs (Chang, 2000). In the current study, the samples were collected from rubber vacuum loader cup rinses. These vacuum cups transfer the unwashed, nest run eggs onto the processing line. Although Escherichia spp. were identified during the current study, they represented only 8% of the identified organisms, compared with Klebsiella spp. (21%) and Enterobacter spp. (42%).

Many of the genera found in this study are opportunistic pathogens. Escherichia, Klebsiella, Shigella, Yersinia, Aeromonas, Enterobacter, and Leclercia have all previously been implicated in human illnesses (Farmer et al., 1985; Sheldon and Schuman, 1996; Stock et al., 2004). When considering the thermal resistance of Aeromonas hydrophila in liquid whole egg during pasteurization, Schuman et al. (1997) determined that the 2 strains isolated from egg processing plants were more heat resistant than the laboratory culture. A study conducted in Trinidad and Tobago determined that 88% of E. coli isolated from retail shell eggs was resistant to one or more antimicrobials tested (Adesiyun et al., 2007). High levels of Enterobacteriaceae have been detected on the contact and noncontact surfaces in shell egg processing facilities (Jones et al., 2003; Musgrove et al., 2004b). Further work (Jones et al., 2004) found 1 washed egg out of more than 240 to have Enterobacteriaceae present on the shell surface during a 6-wk period of cold storage. Although the presence of these organisms in the egg processing environment does not necessarily correlate to a direct food safety hazard, their presence does draw attention to the need to enhance cleaning and sanitation practices to reduce the potential of pathogen transfer to the processed egg.

Although washed eggs in the United States have not shown a high incidence in Enterobacteriaceae on the shell or in the egg contents, elevated levels of these organisms on equipment surfaces have been found (Jones et al., 2003; Musgrove et al., 2004b; Jones and Musgrove, 2008). A greater understanding of the particular organisms present on a processing surface can afford a more targeted and comprehensive approach to cleaning and sanitation processes. Furthermore, realizing what is in the processing environment can aid in discovering the sources of equipment contamination, which also enhances plant sanitation programs.

	ACKNOWLEDGMENTS

 
The authors appreciate the technical assistance of Patsy Mason, Jordan Shaw, Tod Stewart, Susan Akins, and Otis Freeman over the course of this project.

Received for publication December 17, 2007. Accepted for publication April 3, 2008.

	REFERENCES

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS AND DISCUSSION REFERENCES

 
Adesiyun, A., N. Offiah, N. Seepersadsingh, S. Rodrigo, V. Lashley, and L. Musai. 2007. Antimicrobial resistance of Salmonella spp. and Escherichia coli isolated from table eggs. Food Contr. 18:306–311.[CrossRef]

Banwart, G. J. 1989. Basic Food Microbiology. 2nd ed. Van Nostrand Reinhold, New York, NY.

Chang, Y. H. 2000. Prevalence of Salmonella spp. in poultry broilers and shell eggs in Korea. J. Food Prot. 63:655–658.[Web of Science][Medline]

Davies, R. H., and M. Breslin. 2003. Investigation of Salmonella contamination and disinfection in farm egg-packing plants. J. Appl. Microbiol. 94:191–196.[CrossRef][Medline]

Farmer, J. J., B. R. Davis, F. W. Hickman-Brenner, A. McWhorter, G. P. Huntley-Carter, M. A. Ashbury, C. Riddle, H. G. Wathen-Grady, C. Elias, G. R. Fanning, A. G. Steigerwalt, C. M. O’Hara, G. K. Morris, P. B. Smith, and D. J. Brenner. 1985. Biochemical identification of new species and biogroups of Enterobacteriaceae isolated from clinical specimens. J. Clin. Microbiol. 21:46–76.[Abstract/Free Full Text]

Ibeh, I. N., and Y. S. Izuagbe. 1986. An analysis of the microflora of broken eggs used in confectionary products in Nigeria and the occurrence of enterotoxigenic gram-negative bacteria. Int. J. Food Microbiol. 3:71–77.[CrossRef][Web of Science]

Jay, J. M., M. J. Loessner, and D. A. Golden. 2005. Miscellaneous food products. Pages 197–213 in Modern Food Microbiology. 7th ed. Springer Science and Business Media Inc., New York, NY.

Jones, D. R., and M. T. Musgrove. 2008. Assessment of microbial contaminants present on vacuum loaders in shell egg processing facilities. J. Food Saf. (In press).

Jones, D. R., M. T. Musgrove, and J. K. Northcutt. 2004. Variations in external and internal microbial populations in shell eggs during extended storage. J. Food Prot. 67:2657–2660.[Web of Science][Medline]

Jones, D. R., J. K. Northcutt, M. T. Musgrove, P. A. Curtis, K. E. Anderson, D. L. Fletcher, and N. A. Cox. 2003. Survey of shell egg processing plant sanitation programs: Effects on egg contact surfaces. J. Food Prot. 66:1486–1489.[Web of Science][Medline]

Moats, W. A. 1981. Factors affecting bacterial loads on shells of commercially washed eggs. Poult. Sci. 60:2084–2090.[Web of Science]

Musgrove, M. T., D. R. Jones, J. K. Northcutt, N. A. Cox, and M.A. Harrison. 2004a. Identification of Enterobacteriaceae from washed and unwashed commercial shell eggs. J. Food Prot. 67:2613–2616.[Web of Science][Medline]

Musgrove, M. T., D. R. Jones, J. K. Northcutt, P. A. Curtis, K. E. Anderson, D. L. Fletcher, and N. A. Cox. 2004b. Survey of shell egg processing plant sanitation programs: Effects on non-egg contact surfaces. J. Food Prot. 67:2801–2804.[Web of Science][Medline]

Northcutt, J. K., D. R. Jones, K. D. Ingram, A. Hinton Jr., and M. T. Musgrove. 2004. Airborne microorganisms in commercial shell egg processing facilities. Int. J. Poult. Sci. 3:195–200.

Sayers, C. W. 1943. Rotting in eggs. Agric. Gaz. 64:292–296.

Schuman, J. D., B. W. Sheldon, and P. M. Foegeding. 1997. Thermal resistance of Aeromonas hydrophila in liquid whole egg. J. Food Prot. 60:231–236.[Web of Science]

Sheldon, B. W., and J. D. Schuman. 1996. Thermal and biological treatments to control psychrotrophic pathogens. Poult. Sci. 75:1126–1132.[Web of Science][Medline]

Slade, P. J. 2002. Verification of effective sanitation control strategies. Food Saf. Mag. 8:24–29, 42–43.

Stock, I., S. Burak, and B. Wiedemann. 2004. Natural and antimicrobial susceptibility patterns and biochemical profiles of Leclercia adecarboxylata strains. Clin. Microbiol. Infect. 10:724–733.[CrossRef][Web of Science][Medline]

This Article Abstract Full Text (PDF) Alert me when this article is cited Alert me if a correction is posted Services Similar articles in this journal Similar articles in Web of Science Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Citing Articles Citing Articles via Google Scholar Google Scholar Articles by Jones, D. R. Articles by Musgrove, M. T. Search for Related Content PubMed PubMed Citation Articles by Jones, D. R. Articles by Musgrove, M. T.