- Email: [email protected]
In Vitro Inactivation of Salmonella Enteritidis in Autoclaved Chicken Cecal Contents by Caprylic Acid
2005 Poultry Science Association, Inc.
In Vitro Inactivation of Salmonella Enteritidis in Autoclaved Chicken Cecal Contents by Caprylic Acid P. Vasudevan,* P. Marek,* M. K. M. Nair,* T. Annamalai,* M. Darre,* M. Khan,† and K. Venkitanarayanan*,1
Primary Audience: Researchers, Farm managers, Extension agents SUMMARY Contamination of poultry and poultry products by Salmonella Enteritidis is a serious problem worldwide. The effects of caprylic acid (50 and 100 mM) on S. Enteritidis in autoclaved chicken cecal contents and tryptic soy broth at 40°C was studied. Both concentrations of caprylic acid reduced the population of S. Enteritidis by ∼5.0 log10 cfu/mL within 1 min, with complete inactivation of the pathogen at 24 h. Results of the study revealed that caprylic acid was highly effective in killing S. Enteritidis in autoclaved chicken cecal contents in a rapid time frame. Key words: caprylic acid, Salmonella Enteritidis, cecal content, chicken 2005 J. Appl. Poult. Res. 14:122–125
DESCRIPTION OF PROBLEM Salmonella enterica serovar Enteritidis is an important pathogen causing foodborne infections in humans in the United States and elsewhere [1]. Poultry meat and eggs have been reported to be the major sources of S. Enteritidis infection to humans. Numerous surveys conducted in various parts of the world indicate that S. Enteritidis is the most common serotype of Salmonella isolated from poultry products [2, 3, 4, 5, 6, 7]. Efforts to reduce the incidence of S. Enteritidis in chickens will reduce the likelihood of foodborne outbreaks of this pathogen and decrease economic losses to the poultry industry. A primary colonization site of S. Enteritidis in chickens is the cecum [8]. Chicken cecal carriage of Salmonella can lead to horizontal transmission of the infection, contamination of egg1
shell with feces, and carcass contamination during slaughter [9]. Transovarian transmission of S. Enteritidis has also been reported [10]. Reducing the population of S. Enteritidis in the chicken intestinal tract would potentially lead to a decreased excretion of the pathogen in the droppings, which in turn could reduce contamination of poultry meat and eggs. Fatty acids and monoglycerides are reported to have bacteriostatic and bactericidal properties against a diverse range of microorganisms [11]. Chickens have been fed organic acids such as propionic acid as feed supplements to reduce or eliminate pathogenic bacterial and fungal loads in the birds [12]. Treating feed with such organic acids can reduce existing salmonellae contamination and prevent recontamination of feed [13, 14]. Caprylic acid (octanoic acid) is a natural, 8carbon fatty acid present in human milk, bovine
To whom correspondence should be addressed: [email protected].
Downloaded from http://japr.oxfordjournals.org/ at Wayne State University on April 16, 2015
*Department of Animal Science, Unit 4040 and †Department of Pathobiology, Unit 4089, University of Connecticut, Storrs, Connecticut 06269
6.2 0 0 0 6.1 0 0 0 Positive after enrichment but not detected by direct plating.
1
0 1 8 24 100 mM
min min h h
6.2 0 0 0
6.2 0 0 0
6.1 0 0 0
6.0 0 0 0
6.1 0 0 0
6.1 0 0 0
6.0 0 0 0
6.2 0 0 0
5.9 0 0 0 6.1 0 0 0 6.0 0 0 0 0 1 8 24 50 mM
min min h h
6.0 <11 0 0
6.4 <11 0 <11
6.1 0 0 0
6.1 0 0 0
6.0 <11 0 <11
6.0 0 0 0
6.1 0 0 0
6.3 8.5 8.9 6.1 8.6 9.0 6.0 8.2 9.0 6.2 8.5 8.8 6.0 8.4 8.8 6.3 8.6 9.0 6.1 8.7 8.9 0 min 8h 24 h Control
Treatment
6.2 8.5 8.9
6.2 8.6 8.9
Sample 10 Sample 9 Sample 8 Sample 7 Sample 6 Sample 5 Sample 4 Sample 1
Sample 2
Sample 3
Surviving population of S. Enteritidis (Mean log10 cfu/mL)
Sampling time
Five isolates of S. Enteritidis [SE12 (chicken liver, phage type 14b), SE22 (chicken intestine, phage type 8), SE 28 (chicken ovary, phage type 13a), SE31 (chicken gut, phage type 13a), and SE90 (human, phage type 8)] were obtained from the Department of Pathobiology at the University of Connecticut. Each strain was cultured separately in 10 mL of sterile TSB [19] at 37°C for 24 h with agitation (100 rpm). Equal portions from each of the 5 strains were combined to make a 5-strain mixture of the pathogen, and appropriate dilutions of the 5-strain mixture were used to obtain the desired level of inoculum. Cecal contents were obtained from 60, 6-wkold, male, Arbor Acres broiler chickens slaughtered at the University of Connecticut meat laboratory. To obtain a sufficient amount of cecal material for sampling during the whole experiment, cecal contents from 6 birds were combined into 1 composite sample, resulting in a total of 10 samples for the study. The 10 pooled cecal samples were immediately transported to the laboratory, mixed with an equal amount of sterile PBS, and autoclaved at 121°C for 15 min to eliminate background cecal bacteria. A volume of 10 mL each of the autoclaved cecal contents from each pooled sample was dispensed aseptically into separate 30-mL sterile tubes and placed in a waterbath set at 40°C to bring the temperature of the autoclaved cecal contents to 40°C (normal body temperature of chickens). Appropriate quantities of caprylic acid [20] were added to each 10-mL sample of autoclaved
TABLE 1. Effect of caprylic acid on Salmonella Enteritidis in autoclaved chicken cecal contents
MATERIALS AND METHODS
123
Downloaded from http://japr.oxfordjournals.org/ at Wayne State University on April 16, 2015
milk [15], and coconut oil [16, 17]. Caprylic acid is a foodgrade chemical recognized by the FDA as Generally Regarded as Safe (GRAS 184.1025). Isaacs and coworkers [18] observed that caprylic acid is effective in killing human infant pathogens such as herpes simplex virus (HSV-1), respiratory syncytial virus (RSV), Haemophilus influenza, and group B streptococci. Preliminary research conducted in our laboratory indicated that caprylic acid was very effective in killing S. Enteritidis in chicken droppings (data not shown). Therefore, the objective of this study was to determine the antibacterial effect of caprylic acid on S. Enteritidis in autoclaved cecal contents of broiler chickens and in a nutrient-rich medium (tryptic soy broth; TSB).
6.1 8.2 8.8
VASUDEVAN ET AL.: CAPRYLIC ACID AND SALMONELLA ENTERITIDIS
JAPR: Research Report
124
RESULTS AND DISCUSSION The average pH of the cecal contents was approximately 6.77 ± 0.01. At the end of 24 h, the pH of cecal samples containing 50 and 100 mM caprylic acid were 6.09 ± 0.08 and 5.75 ± 0.17, respectively.
The natural cecal bacterial flora of chickens can exert an inhibitory effect on Salmonella numbers in the ceca [22, 23]. Further, preliminary experiments on the viability of S. Enteritidis cultures (preinduced for nalidixic acid resistance) in nonautoclaved cecal contents revealed that the antibiotic-resistant strains rapidly declined even in control cecal samples devoid of caprylic acid, when incubated anaerobically at 40°C. Therefore, to alleviate any potential antagonistic effect of the background bacteria on S. Enteritidis in the cecal samples, and to obtain accurate counts of the pathogen throughout the experiment, we used autoclaved cecal contents. Caprylic acid at 50 mM completely inactivated S. Enteritidis (enrichment negative) by 1 min of incubation in 7 of 10 samples, whereas in samples 1, 2, and 5 the pathogen was reduced by approximately 5.0 log10 cfu/mL (Table 1). At 24 h of incubation, sample 1 containing 50 mM caprylic acid was devoid of S. Enteritidis, whereas samples 2 and 5 tested positive for the pathogen by enrichment. Caprylic acid at 100 mM eliminated S. Enteritidis in all the samples at 1 min of incubation (Table 1). During incubation at 40°C, the population of S. Enteritidis in the control samples increased to approximately 8.4 log10 and 8.9 log10 cfu/mL by 8 h and 24 h, respectively (Table 1). As observed in autoclaved cecal contents, caprylic acid was also highly effective in killing S. Enteritidis in the defined, nutrient-rich menstruum, TSB. Caprylic acid at 50 and 100 mM concentrations reduced the population of S. Enteritidis by approximately 5.0 log10 cfu/mL in 1 min, with complete inactivation of the pathogen at 24 h (data not shown). Results of this study revealed that caprylic acid was highly effective in inactivating S. Enteritidis in autoclaved cecal contents and tryptic soy broth. Substantial populations of the pathogen were killed by caprylic acid in a rapid time frame. However, further experiments are needed to determine if the tested concentrations of caprylic acid can be achieved in vivo. In addition, follow-up experiments to determine the in vivo effects of caprylic acid on S. Enteritidis in chickens are necessary, and the effect of feeding caprylic acid on chicken health needs to be investigated.
Downloaded from http://japr.oxfordjournals.org/ at Wayne State University on April 16, 2015
cecal contents to obtain a final concentration of 50 or 100 mM. Control samples were prepared by adding sterile distilled water instead of caprylic acid to the autoclaved cecal contents. A volume of 100 µL of the appropriately diluted 5-strain mixture of S. Enteritidis was added to each of the tubes to get an inoculation level of approximately 106 cfu/mL. The tubes were then incubated anaerobically at 40°C in a CO2-water jacketed incubator flushed with 85% nitrogen and 15% carbon dioxide. The population of surviving S. Enteritidis was determined at 0 min, 1 min (only in caprylic acid containing samples), 8 h, and 24 h of incubation by plating 0.1-mL portions of the samples directly or after serial dilutions (1:10 in PBS) on duplicate TSA plates. The plates were incubated at 37°C for 24 h before the colonies were counted. At each sampling time, 1 mL of the cecal sample was also transferred to separate 250-mL Erlenmeyer flasks containing 100 mL of sterile TSB for enrichment at 37°C for 24 h. When growth was observed in TSB, the culture was streaked on xylose lysine deoxycholate agar (XLD) [21] plates and observed for typical colonies of S. Enteritidis. Representative colonies from the XLD plates were confirmed as S. Enteritidis by Salmonella latex agglutination kit test [19]. In addition to bacterial counts, the pH of treatment and control samples of cecal contents was determined with an Accumet pH meter at 0 and 24 h of incubation after mixing equal amounts of cecal contents and PBS. The effect of caprylic acid on S. Enteritidis in TSB was determined under the same experimental conditions that were followed for the cecal samples, except that cecal contents were replaced with sterile TSB. In the experiments with TSB, triplicate samples were assayed for each treatment and control at each specified sampling time, and the study was replicated twice.
VASUDEVAN ET AL.: CAPRYLIC ACID AND SALMONELLA ENTERITIDIS
125
CONCLUSIONS AND APPLICATIONS 1. Caprylic acid is highly effective in killing S. Enteritidis in autoclaved chicken cecal contents and tryptic soy broth. 2. Caprylic acid kills S. Enteritidis rapidly, reducing the pathogen by 5.0 log cfu/mL in 1 min.
REFERENCES AND NOTES 1. Allen-Vercoe, E., M. Dibb-Fuller, C. Thorns, and M. Woodward. 1997. SEF17 fimbriae are essential for convoluted colonial morphology of Salmonella enteritidis. FEMS Microbiol. Lett. 153:33–42.
3. Arvanitidou, M., A. Tsakris, D. Sofianou, and V. Katsouyannopoulos. 1998. Antimicrobial resistance and R-factor transfer of salmonellae isolated from chicken carcasses in Greek hospitals. Int. J. Food Microbiol. 40:197–201. 4. Carramin˜ana, J. J., J. Yanguela, D. Blanco, C. Rota, A. I. Agustin, A. Arin˜o, and A. Herrera. 1997. Salmonella incidence and distribution of serotypes throughout processing in a Spanish poultry slaughterhouse. J. Food Prot. 60:1312–1317.
11. Kabara, J. J., D. M. Swieczkowski, A. J. Conley, and J. P. Truant. 1972. Fatty acids and derivatives as antimicrobial agents. Antimicrob. Agents Chemother. 2:23–28. 12. Hume, M. E., D. E. Corrier, S. Ambrus, A. Hinton, Jr., and J. R. DeLoach. 1993. Effectiveness of dietary propionic acid in controlling Salmonella typhimurium colonization in broiler chicks. Avian Dis. 37:1051–1056. 13. Hinton, M., and A. H. Linton. 1988. Control of salmonella infections in broiler chickens by the acid treatment of their feed. Vet. Rec. 123:416–421. 14. Humphrey, T. J., and D. G. Lanning. 1988. The vertical transmission of salmonellas and formic acid treatment of chicken feed. A possible strategy for control. Epidemiol. Infect. 100:43–49.
5. Machado, J., and F. Bernardo. 1990. Prevalence of Salmonella in chicken carcasses in Portugal. J. Appl. Bacteriol. 69:477–480.
15. Jensen, R. G. 2002. The composition of bovine milk lipids: January 1995 to December 2000. J. Dairy Sci. 85:295–350.
6. Plummer, R. A. S., S. J. Blissett, and C. E. R. Dodd. 1995. Salmonella contamination of retail chicken products sold in the UK. J. Food Prot. 58:843–846.
16. Jensen, R. G., A. M. Ferris, C. J. Lammi-Keefe, and R. A. Henderson. 1990. Lipids of bovine and human milks: a comparison. J. Dairy Sci. 73:223–240.
7. Uyttendaele, M., P. De Troy, and J. Debevere. 1999. Incidence of Salmonella, Campylobacter jejuni, Campylobacter coli, and Listeria monocytogenes in poultry carcasses and different types of poultry products for sale on the Belgian retail market. J. Food Prot. 62:735–740.
17. Sprong, R. C., M. F. Hulstein, and R. Van der Meer. 2001. Bactericidal activities of milk lipids. Antimicrob. Agents Chemother. 45:1298–1301.
8. Allen-Vercoe, E., and M. J. Woodward. 1999. Colonisation of the chicken caecum by afimbriate and aflagellate derivatives of Salmonella enterica serotype Enteritidis. Vet. Microbiol. 69:265– 275.
18. Isaacs, C. E., R. E. Litov, and H. Thormar. 1995. Antimicrobial activity of lipids added to human milk, infant formula, and bovine milk. J. Nutr. Biochem. 6:362–366. 19. Difco, Division of Becton-Dickinson, Sparks, MD. 20. Sigma-Aldrich, St. Louis, MO. 21. Oxoid, Ogdensburg, NY.
9. Keller, L. H., C. E. Benson, K. Krotec, and R. J. Eckroade. 1995. Salmonella enteritidis colonization of the reproductive tract and forming and freshly laid eggs of chickens. Infect. Immun. 63:2443–2449.
22. Corrier, D. E., A. Hinton, Jr., R. L. Ziprin, and J. R. DeLoach. 1990. Effect of dietary lactose on Salmonella colonization of market age broiler chickens. Avian Dis. 34:668–676.
10. Okamura, M., Y. Kamijima, T. Miyamoto, H. Tani, K. Sasai, and E. Baba. 2001. Differences among six Salmonella serovars in
23. Stavric, S. 1992. Defined cultures and prospects. Int. J. Food Microbiol. 15:245–263.
Downloaded from http://japr.oxfordjournals.org/ at Wayne State University on April 16, 2015
2. Antunes, P., C. Reu, J. C. Sousa, L. Peixe, and N. Pestana. 2003. Incidence of Salmonella from poultry products and their susceptibility to antimicrobial agents. Int. J. Food Microbiol. 82:97–103.
abilities to colonize reproductive organs and to contaminate eggs in laying hens. Avian Dis. 45:61–69.
In Vitro Inactivation of Salmonella Enteritidis in Autoclaved Chicken Cecal Contents by Caprylic Acid P. Vasudevan,* P. Marek,* M. K. M. Nair,* T. Annamalai,* M. Darre,* M. Khan,† and K. Venkitanarayanan*,1
Primary Audience: Researchers, Farm managers, Extension agents SUMMARY Contamination of poultry and poultry products by Salmonella Enteritidis is a serious problem worldwide. The effects of caprylic acid (50 and 100 mM) on S. Enteritidis in autoclaved chicken cecal contents and tryptic soy broth at 40°C was studied. Both concentrations of caprylic acid reduced the population of S. Enteritidis by ∼5.0 log10 cfu/mL within 1 min, with complete inactivation of the pathogen at 24 h. Results of the study revealed that caprylic acid was highly effective in killing S. Enteritidis in autoclaved chicken cecal contents in a rapid time frame. Key words: caprylic acid, Salmonella Enteritidis, cecal content, chicken 2005 J. Appl. Poult. Res. 14:122–125
DESCRIPTION OF PROBLEM Salmonella enterica serovar Enteritidis is an important pathogen causing foodborne infections in humans in the United States and elsewhere [1]. Poultry meat and eggs have been reported to be the major sources of S. Enteritidis infection to humans. Numerous surveys conducted in various parts of the world indicate that S. Enteritidis is the most common serotype of Salmonella isolated from poultry products [2, 3, 4, 5, 6, 7]. Efforts to reduce the incidence of S. Enteritidis in chickens will reduce the likelihood of foodborne outbreaks of this pathogen and decrease economic losses to the poultry industry. A primary colonization site of S. Enteritidis in chickens is the cecum [8]. Chicken cecal carriage of Salmonella can lead to horizontal transmission of the infection, contamination of egg1
shell with feces, and carcass contamination during slaughter [9]. Transovarian transmission of S. Enteritidis has also been reported [10]. Reducing the population of S. Enteritidis in the chicken intestinal tract would potentially lead to a decreased excretion of the pathogen in the droppings, which in turn could reduce contamination of poultry meat and eggs. Fatty acids and monoglycerides are reported to have bacteriostatic and bactericidal properties against a diverse range of microorganisms [11]. Chickens have been fed organic acids such as propionic acid as feed supplements to reduce or eliminate pathogenic bacterial and fungal loads in the birds [12]. Treating feed with such organic acids can reduce existing salmonellae contamination and prevent recontamination of feed [13, 14]. Caprylic acid (octanoic acid) is a natural, 8carbon fatty acid present in human milk, bovine
To whom correspondence should be addressed: [email protected].
Downloaded from http://japr.oxfordjournals.org/ at Wayne State University on April 16, 2015
*Department of Animal Science, Unit 4040 and †Department of Pathobiology, Unit 4089, University of Connecticut, Storrs, Connecticut 06269
6.2 0 0 0 6.1 0 0 0 Positive after enrichment but not detected by direct plating.
1
0 1 8 24 100 mM
min min h h
6.2 0 0 0
6.2 0 0 0
6.1 0 0 0
6.0 0 0 0
6.1 0 0 0
6.1 0 0 0
6.0 0 0 0
6.2 0 0 0
5.9 0 0 0 6.1 0 0 0 6.0 0 0 0 0 1 8 24 50 mM
min min h h
6.0 <11 0 0
6.4 <11 0 <11
6.1 0 0 0
6.1 0 0 0
6.0 <11 0 <11
6.0 0 0 0
6.1 0 0 0
6.3 8.5 8.9 6.1 8.6 9.0 6.0 8.2 9.0 6.2 8.5 8.8 6.0 8.4 8.8 6.3 8.6 9.0 6.1 8.7 8.9 0 min 8h 24 h Control
Treatment
6.2 8.5 8.9
6.2 8.6 8.9
Sample 10 Sample 9 Sample 8 Sample 7 Sample 6 Sample 5 Sample 4 Sample 1
Sample 2
Sample 3
Surviving population of S. Enteritidis (Mean log10 cfu/mL)
Sampling time
Five isolates of S. Enteritidis [SE12 (chicken liver, phage type 14b), SE22 (chicken intestine, phage type 8), SE 28 (chicken ovary, phage type 13a), SE31 (chicken gut, phage type 13a), and SE90 (human, phage type 8)] were obtained from the Department of Pathobiology at the University of Connecticut. Each strain was cultured separately in 10 mL of sterile TSB [19] at 37°C for 24 h with agitation (100 rpm). Equal portions from each of the 5 strains were combined to make a 5-strain mixture of the pathogen, and appropriate dilutions of the 5-strain mixture were used to obtain the desired level of inoculum. Cecal contents were obtained from 60, 6-wkold, male, Arbor Acres broiler chickens slaughtered at the University of Connecticut meat laboratory. To obtain a sufficient amount of cecal material for sampling during the whole experiment, cecal contents from 6 birds were combined into 1 composite sample, resulting in a total of 10 samples for the study. The 10 pooled cecal samples were immediately transported to the laboratory, mixed with an equal amount of sterile PBS, and autoclaved at 121°C for 15 min to eliminate background cecal bacteria. A volume of 10 mL each of the autoclaved cecal contents from each pooled sample was dispensed aseptically into separate 30-mL sterile tubes and placed in a waterbath set at 40°C to bring the temperature of the autoclaved cecal contents to 40°C (normal body temperature of chickens). Appropriate quantities of caprylic acid [20] were added to each 10-mL sample of autoclaved
TABLE 1. Effect of caprylic acid on Salmonella Enteritidis in autoclaved chicken cecal contents
MATERIALS AND METHODS
123
Downloaded from http://japr.oxfordjournals.org/ at Wayne State University on April 16, 2015
milk [15], and coconut oil [16, 17]. Caprylic acid is a foodgrade chemical recognized by the FDA as Generally Regarded as Safe (GRAS 184.1025). Isaacs and coworkers [18] observed that caprylic acid is effective in killing human infant pathogens such as herpes simplex virus (HSV-1), respiratory syncytial virus (RSV), Haemophilus influenza, and group B streptococci. Preliminary research conducted in our laboratory indicated that caprylic acid was very effective in killing S. Enteritidis in chicken droppings (data not shown). Therefore, the objective of this study was to determine the antibacterial effect of caprylic acid on S. Enteritidis in autoclaved cecal contents of broiler chickens and in a nutrient-rich medium (tryptic soy broth; TSB).
6.1 8.2 8.8
VASUDEVAN ET AL.: CAPRYLIC ACID AND SALMONELLA ENTERITIDIS
JAPR: Research Report
124
RESULTS AND DISCUSSION The average pH of the cecal contents was approximately 6.77 ± 0.01. At the end of 24 h, the pH of cecal samples containing 50 and 100 mM caprylic acid were 6.09 ± 0.08 and 5.75 ± 0.17, respectively.
The natural cecal bacterial flora of chickens can exert an inhibitory effect on Salmonella numbers in the ceca [22, 23]. Further, preliminary experiments on the viability of S. Enteritidis cultures (preinduced for nalidixic acid resistance) in nonautoclaved cecal contents revealed that the antibiotic-resistant strains rapidly declined even in control cecal samples devoid of caprylic acid, when incubated anaerobically at 40°C. Therefore, to alleviate any potential antagonistic effect of the background bacteria on S. Enteritidis in the cecal samples, and to obtain accurate counts of the pathogen throughout the experiment, we used autoclaved cecal contents. Caprylic acid at 50 mM completely inactivated S. Enteritidis (enrichment negative) by 1 min of incubation in 7 of 10 samples, whereas in samples 1, 2, and 5 the pathogen was reduced by approximately 5.0 log10 cfu/mL (Table 1). At 24 h of incubation, sample 1 containing 50 mM caprylic acid was devoid of S. Enteritidis, whereas samples 2 and 5 tested positive for the pathogen by enrichment. Caprylic acid at 100 mM eliminated S. Enteritidis in all the samples at 1 min of incubation (Table 1). During incubation at 40°C, the population of S. Enteritidis in the control samples increased to approximately 8.4 log10 and 8.9 log10 cfu/mL by 8 h and 24 h, respectively (Table 1). As observed in autoclaved cecal contents, caprylic acid was also highly effective in killing S. Enteritidis in the defined, nutrient-rich menstruum, TSB. Caprylic acid at 50 and 100 mM concentrations reduced the population of S. Enteritidis by approximately 5.0 log10 cfu/mL in 1 min, with complete inactivation of the pathogen at 24 h (data not shown). Results of this study revealed that caprylic acid was highly effective in inactivating S. Enteritidis in autoclaved cecal contents and tryptic soy broth. Substantial populations of the pathogen were killed by caprylic acid in a rapid time frame. However, further experiments are needed to determine if the tested concentrations of caprylic acid can be achieved in vivo. In addition, follow-up experiments to determine the in vivo effects of caprylic acid on S. Enteritidis in chickens are necessary, and the effect of feeding caprylic acid on chicken health needs to be investigated.
Downloaded from http://japr.oxfordjournals.org/ at Wayne State University on April 16, 2015
cecal contents to obtain a final concentration of 50 or 100 mM. Control samples were prepared by adding sterile distilled water instead of caprylic acid to the autoclaved cecal contents. A volume of 100 µL of the appropriately diluted 5-strain mixture of S. Enteritidis was added to each of the tubes to get an inoculation level of approximately 106 cfu/mL. The tubes were then incubated anaerobically at 40°C in a CO2-water jacketed incubator flushed with 85% nitrogen and 15% carbon dioxide. The population of surviving S. Enteritidis was determined at 0 min, 1 min (only in caprylic acid containing samples), 8 h, and 24 h of incubation by plating 0.1-mL portions of the samples directly or after serial dilutions (1:10 in PBS) on duplicate TSA plates. The plates were incubated at 37°C for 24 h before the colonies were counted. At each sampling time, 1 mL of the cecal sample was also transferred to separate 250-mL Erlenmeyer flasks containing 100 mL of sterile TSB for enrichment at 37°C for 24 h. When growth was observed in TSB, the culture was streaked on xylose lysine deoxycholate agar (XLD) [21] plates and observed for typical colonies of S. Enteritidis. Representative colonies from the XLD plates were confirmed as S. Enteritidis by Salmonella latex agglutination kit test [19]. In addition to bacterial counts, the pH of treatment and control samples of cecal contents was determined with an Accumet pH meter at 0 and 24 h of incubation after mixing equal amounts of cecal contents and PBS. The effect of caprylic acid on S. Enteritidis in TSB was determined under the same experimental conditions that were followed for the cecal samples, except that cecal contents were replaced with sterile TSB. In the experiments with TSB, triplicate samples were assayed for each treatment and control at each specified sampling time, and the study was replicated twice.
VASUDEVAN ET AL.: CAPRYLIC ACID AND SALMONELLA ENTERITIDIS
125
CONCLUSIONS AND APPLICATIONS 1. Caprylic acid is highly effective in killing S. Enteritidis in autoclaved chicken cecal contents and tryptic soy broth. 2. Caprylic acid kills S. Enteritidis rapidly, reducing the pathogen by 5.0 log cfu/mL in 1 min.
REFERENCES AND NOTES 1. Allen-Vercoe, E., M. Dibb-Fuller, C. Thorns, and M. Woodward. 1997. SEF17 fimbriae are essential for convoluted colonial morphology of Salmonella enteritidis. FEMS Microbiol. Lett. 153:33–42.
3. Arvanitidou, M., A. Tsakris, D. Sofianou, and V. Katsouyannopoulos. 1998. Antimicrobial resistance and R-factor transfer of salmonellae isolated from chicken carcasses in Greek hospitals. Int. J. Food Microbiol. 40:197–201. 4. Carramin˜ana, J. J., J. Yanguela, D. Blanco, C. Rota, A. I. Agustin, A. Arin˜o, and A. Herrera. 1997. Salmonella incidence and distribution of serotypes throughout processing in a Spanish poultry slaughterhouse. J. Food Prot. 60:1312–1317.
11. Kabara, J. J., D. M. Swieczkowski, A. J. Conley, and J. P. Truant. 1972. Fatty acids and derivatives as antimicrobial agents. Antimicrob. Agents Chemother. 2:23–28. 12. Hume, M. E., D. E. Corrier, S. Ambrus, A. Hinton, Jr., and J. R. DeLoach. 1993. Effectiveness of dietary propionic acid in controlling Salmonella typhimurium colonization in broiler chicks. Avian Dis. 37:1051–1056. 13. Hinton, M., and A. H. Linton. 1988. Control of salmonella infections in broiler chickens by the acid treatment of their feed. Vet. Rec. 123:416–421. 14. Humphrey, T. J., and D. G. Lanning. 1988. The vertical transmission of salmonellas and formic acid treatment of chicken feed. A possible strategy for control. Epidemiol. Infect. 100:43–49.
5. Machado, J., and F. Bernardo. 1990. Prevalence of Salmonella in chicken carcasses in Portugal. J. Appl. Bacteriol. 69:477–480.
15. Jensen, R. G. 2002. The composition of bovine milk lipids: January 1995 to December 2000. J. Dairy Sci. 85:295–350.
6. Plummer, R. A. S., S. J. Blissett, and C. E. R. Dodd. 1995. Salmonella contamination of retail chicken products sold in the UK. J. Food Prot. 58:843–846.
16. Jensen, R. G., A. M. Ferris, C. J. Lammi-Keefe, and R. A. Henderson. 1990. Lipids of bovine and human milks: a comparison. J. Dairy Sci. 73:223–240.
7. Uyttendaele, M., P. De Troy, and J. Debevere. 1999. Incidence of Salmonella, Campylobacter jejuni, Campylobacter coli, and Listeria monocytogenes in poultry carcasses and different types of poultry products for sale on the Belgian retail market. J. Food Prot. 62:735–740.
17. Sprong, R. C., M. F. Hulstein, and R. Van der Meer. 2001. Bactericidal activities of milk lipids. Antimicrob. Agents Chemother. 45:1298–1301.
8. Allen-Vercoe, E., and M. J. Woodward. 1999. Colonisation of the chicken caecum by afimbriate and aflagellate derivatives of Salmonella enterica serotype Enteritidis. Vet. Microbiol. 69:265– 275.
18. Isaacs, C. E., R. E. Litov, and H. Thormar. 1995. Antimicrobial activity of lipids added to human milk, infant formula, and bovine milk. J. Nutr. Biochem. 6:362–366. 19. Difco, Division of Becton-Dickinson, Sparks, MD. 20. Sigma-Aldrich, St. Louis, MO. 21. Oxoid, Ogdensburg, NY.
9. Keller, L. H., C. E. Benson, K. Krotec, and R. J. Eckroade. 1995. Salmonella enteritidis colonization of the reproductive tract and forming and freshly laid eggs of chickens. Infect. Immun. 63:2443–2449.
22. Corrier, D. E., A. Hinton, Jr., R. L. Ziprin, and J. R. DeLoach. 1990. Effect of dietary lactose on Salmonella colonization of market age broiler chickens. Avian Dis. 34:668–676.
10. Okamura, M., Y. Kamijima, T. Miyamoto, H. Tani, K. Sasai, and E. Baba. 2001. Differences among six Salmonella serovars in
23. Stavric, S. 1992. Defined cultures and prospects. Int. J. Food Microbiol. 15:245–263.
Downloaded from http://japr.oxfordjournals.org/ at Wayne State University on April 16, 2015
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