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Anaerobe 13 (2007) 208–214 www.elsevier.com/locate/anaerobe
Food microbiology
In vitro anaerobic incubation of Salmonella enterica serotype Typhimurium and laying hen cecal bacteria in poultry feed substrates and a fructooligosaccharide prebiotic L.M. Donalsona,c,1, Woo-Kyun Kima,d,2, V.I. Chalovaa,e, P. Herreraa,e, C.L. Woodwarda, J.L. McReynoldsb, L.F. Kubenab, D.J. Nisbetb, S.C. Rickea,e, a Department of Poultry Science, Texas A&M University, College Station, TX 77843-2472, USA USDA-ARS, Southern Plains Agricultural Research Center, Food and Feed Safety Research Unit, College Station, TX 77845, USA c Southwest Foundation for Biomedical Research, San Antonio, TX 78245, USA d Department of Cardiology, David Geffen School of Medicine, Los Angeles, CA 90095, USA e Center for Food Safety and Microbiology, IFSE, and Department of Food Science, University of Arkansas, Fayetteville, AR 72704, USA b
Received 19 March 2007; received in revised form 3 May 2007; accepted 10 May 2007 Available online 21 May 2007
Abstract The objective of this study was to investigate the effect of combining a prebiotic with poultry feeds on the growth of Salmonella enterica serotype Typhimurium (ST) in an in vitro cecal fermentation system. Cecal contents from three laying hens were pooled and diluted to a 1:3000 concentration in an anaerobic dilution solution. The cecal dilution was added to sterile test tubes filled with alfalfa and layer ration with and without fructooligosaccharide (FOS). Two controls containing cecal dilutions and anaerobic dilution solution were used. The samples were processed in the anaerobic hood and incubated at 37 1C. Samples were inoculated with Salmonella at 0 and 24 h after in vitro cecal fermentation and plated at 0 and 24 h after inoculation with ST. Plates were incubated for 24 h and colony forming units (CFU) enumerated. The samples immediately inoculated with ST without prior cecal fermentation did not significantly lower ST counts 24 h later. However, samples pre-incubated for 24 h with cecal microflora prior to ST inoculation exhibited reduced ST CFU by approximately 2 logarithms, with the most dramatic decreases seen in alfalfa and layer ration combined with FOS. The addition of FOS to feed substrate diets in combination with cecal contents acted in a synergistic manner to decrease ST growth only after ST was introduced to 24 h cecal incubations. r 2007 Elsevier Ltd. All rights reserved. Keywords: Alfalfa; Prebiotics; Salmonella Typhimurium; Fructooligosaccharide
1. Introduction Nontyphoidal Salmonella serotypes are a significant problem for the layer industry in the United States and Corresponding author. Department of Food Science, University of Arkansas, 2650 North Young Ave., Fayetteville, AR 72704, USA. Tel.: +479 575 4678; fax: +479 575 6936. E-mail address:
[email protected] (S.C. Ricke). 1 Current address: Department of Cardiology, David Geffen School of Medicine, Los Angeles, CA 90095, USA. 2 Current address: Center for Food Safety and Microbiology, IFSE, and Department of Food Science, University of Arkansas, Fayetteville, AR 72704, USA.
1075-9964/$ - see front matter r 2007 Elsevier Ltd. All rights reserved. doi:10.1016/j.anaerobe.2007.05.001
Europe [1,2] causing 1.4 million cases of illness and 550 deaths annually in the United States. Salmonella colonizes the intestinal epithelium and is able to spread to a variety of organs such as the ovaries and oviducts without physical symptoms of illness being exhibited by the infected hens [3,4]. The cecum, however, is the main site for pathogen colonization including Salmonella [5]. This is especially a concern during feed deprivation induction of layer hen molt because it is known to increase the susceptibility to Salmonella enteritidis in laying hens [6–8]. Alternative molting approaches and diets have been examined and high fiber diets such as wheat middlings and alfalfa meal have been demonstrated to reduce S. enteritidis colonization
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[9–12]. Alfalfa meal diets fed during molt retained similar egg production and quality when compared to hens molted by feed deprivation [12–15]. In vitro evidence suggests that alfalfa is fermentable by cecal bacteria cultured from laying hens [16]. Although high fiber substrates are fermentable by cecal bacteria it would be beneficial to provide specific substrates that were more selective to indigenous cecal bacteria to ensure a more consistent microbial intestinal tract barrier. Prebiotics were first defined by Gibson and Roberfroid [17] as indigestible food ingredients which benefit the host by selectively stimulating the growth and/or activity of bacteria in the colon. In order to be considered a prebiotic, a food ingredient must be neither hydrolyzed nor absorbed in the upper gastrointestinal tract, serve as a selective substrate, cause shifts to beneficial microflora populations, and induce luminal or systemic effects which are also beneficial to the host [17]. The addition of feed additives such as prebiotics is known to increase fermentation both in vitro [18] and in vivo [19]. The majority of fermentation in laying hens occurs in the ceca, which provides a stable environment for indigenous microflora such as Bifidobacterium, Eubacterium, and Propioniobacterium [20,21]. There has been considerable interest in the synergistic effect of prebiotics and the indigenous microflora for potential protection of the avian host against Salmonella spp. Minimal effects of fructooligosaccharide (FOS) combined with broiler starter crumble feed on Salmonella reduction were observed when compared to control [22]. The same study reported higher protection of birds from Salmonella with competitive exclusion culture and an increase of this effect upon the addition of FOS to the latter. Likewise, lactose did not exert any effect on Salmonella population in chickens unless combined with undefined bacterial suspension obtained from adult chicken cecal content [23]. The objective of this study was to examine the in vitro influence of FOS combined with alternative feed substrates (alfalfa high fiber or corn-based) in conjunction with undefined layer hen cecal microflora.
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Rockford, IL, USA). Butterfield’s buffer was prepared according to manufacturer’s instructions.’ 2.2. Animals and cecal preparation Laying hens aged between 100 and 105 weeks old were obtained from a commercial laying facility and the handling procedures were approved by the Texas A&M University Laboratory Animal Care Committee (College Station, TX, USA). Birds were fed a complete layer ration (Table 1) ad libitum and allowed full access to water. Three hens were chosen at random and euthanized by CO2 asphyxiation. The ceca were collected aseptically and squeezed out into sterile 50 ml conical tubes (Becton Dickinson, Franklin Lakes, NJ, USA). Approximately 0.1 g of each cecal content was weighed, placed into the anaerobic chamber and diluted to a 1:3000 concentration (w/vol) with anaerobic phosphate buffer. The anaerobic phosphate buffer was prepared as previously described [24] with the addition of cysteine–HCL prior to autoclaving [25]. 2.3. In vitro incubation procedure Alfalfa meal was obtained from a local cooperative while layer ration (Table 1) was obtained from the Texas A&M University Poultry Science Center feed mill. Approximately 0.25 g of each substrate was added to presterilized 20 ml serum tubes and approximately 0.02 75% FOS (Encore Technologies, Plymouth, MN, USA) was added to FOS treatment tubes. Two groups of experiments differing in the length of incubation were performed. The experimental approach is schematically presented in Fig. 1. In group I, the test tubes were inoculated with ST and samples were collected either immediately (0 h cecal incubation; 0 h Table 1 Composition of Texas A&M University (TAMU) layer ration Ingredient
Amount (g/kg of mash)a
2. Materials and methods 2.1. Bacterial strain A chicken isolate of Salmonella enterica serotype Typhimurium (ST; ATCC 14028) resistant to novobiocin (NO) and nalidixic acid (NA) was used in this study. The double antibiotic resistance of ST was used to selectively enumerate the pathogen from a mixed microbial background. Luria–Bertani broth (LB; Difco Laboratories, Sparks, MD, USA) was used for the maintenance and the growth of the strain. The bacterial strain was grown overnight in a water bath with agitation at 37 1C, washed with sterile Butterfields buffer (Difco Laboratories, Sparks, MD, USA), resuspended in sterile Butterfield’s buffer and diluted to an optical density of 0.300 (600 nm) measured on a spectrophotometer (Milton Roy Spectronic 20D,
Corn, yellow Soybean meal Vegetable oil Mono calcium phosphate Calcium carbonate Methionine, 98% Vitamin premixb NaCl Trace mineral premixc Total a
567.18 316.33 76.82 16.86 15.62 1.69 2.50 2.50 0.50 1000.00
For diet formulation, crude fat concentrations were fixed at 100 g/kg. Provides mg/kg of diet unless otherwise noted: vitamin A, 8.818 IU; vitamin D, 2.205 IU; vitamin E, 5.86 IU; vitamin K, 2.2 IU; thiamine, 1.1 IU; riboflavin, 4.4 IU; niacin, 22 IU; pantothenic acid; choline, 500 IU; vitamin B12, 0.013 IU; biotin, 0.055 IU. c Trace mineral premix (Nutrius Premix Division, Bioproducts Inc., Cleveland, OH, USA), provided as milligrams per kilogram of diet unless otherwise noted: Mn, 68.2; Zn, 55; Cu, 4.4; I, 1.1; Se, 0.1. b
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Group I: Without cecal pre-incubation Cecal samples, substrates, +/- FOS Inoculated with Salmonella Incubation with Salmonella (24 h) Plating of baseline (0;0)
Plating of treatment groups (0;24)
Group II: With cecal pre-incubation Cecal samples, substrates, +/- FOS
Inoculated with Salmonella
Cecal fermentation (24 h)
Plating of baseline (24;0)
Incubation with Salmonella (24 h)
Plating of treatment groups (24;24)
Fig. 1. Diluted cecal samples, substrates, and FOS were added to sterile tubes. In group I, the tubes were inoculated with S. Typhimurium at time 0. Samples were collected and plated as baselines for this group (0:0 h). The tubes were incubated at 37 1C for 24 h and subsequently plated (0:24 h). In group II, the test tubes were incubated at 37 1C for 24 h to allow for cecal microbial activity to occur prior to the inoculation with S. Typhimurium. At the time of inoculation, samples were taken and plated as baselines for this group (24:0 h). The tubes were then incubated for an additional 24 h at 37 1C and plated (24:24 h).
time of sampling) when no microbial incubation was allowed to occur, or after 24 h of incubation (0:24 h) for ST enumeration. In group II, the test tubes containing cecal inocula and substrates were first subjected to 24 h incubation allowing cecal activity to occur before being inoculated with ST and sampled accordingly (24:0 h and 24:24 h). All tubes were placed and incubated at 37 1C in an anaerobic chamber (10% CO2, 5% H2, and 85% N2 gas phase; Coy Laboratory Products, Ann Arbor, MI, USA). Subsequently, tube contents were spread plated on Brilliant Green Agar (Difco Laboratories, Sparks, MD, USA) with the addition of 25 mg/ml each of NO and NA to enumerate ST.
an average of 2 log higher ST colony forming units (CFU) than the treatments without a substrate (CS, S). A similar trend was seen in Trial 2 (Fig. 3). In both trials, the addition of FOS to either alfalfa or layer ration did not significantly reduce (p40.05) ST counts 24 h after the initial inoculation (0:24 h). Instead, an increase in Salmonella population was detected potentially due to the presence of nutritive compounds in cecal contents [26] and the initial low concentration of the cecal fermentation byproducts [23,27–29]. A similar increase in Salmonella cell number 24 h after initial incubation with Veillonella sp. and E. avium versus pure cultures was obtained by Durant et al. [30] in a study of the probiotic effect of these two microorganisms on Salmonella growth.
2.4. Statistical analysis Differences among treatment groups were analyzed using a one-way analysis of variance (ANOVA) of general linear model procedures and Duncan’s multiple test (SAS Institute Inc., Cary, NC, 2000) with a 0.05 significance level. 3. Results and discussion 3.1. S. Typhimurium response to FOS without cecal preincubation Upon inoculation of the first group with ST, samples were immediately collected without incubation to determine a baseline (Figs. 2 and 3; time point 0:0 h). Twentyfour hours later, samples were again collected and evaluated for ST growth (Figs. 2 and 3; time point 0:24 h). In Trial 1 (Fig. 2), all substrates (ACS and LRCS) and FOS-based treatments (ACFS and LRCFS) exhibited
3.2. S. Typhimurium response to FOS after 24 h cecal preincubation The second group of treatments was allowed to ferment by cecal microflora for 24 h before being inoculated with ST (Figs. 4 and 5). Samples were subsequently collected immediately after inoculation (24:0 h) and 24 h after inoculation (24:24 h). This allowed the natural microflora present in the cecal contents to continue to metabolize substrates [31]. ST growth in Trial 1 (Fig. 4) was inhibited by all treatments with the highest inhibition observed in both alfalfa and layer ration combined with FOS. The effects of the extended incubation on ST inhibition were more pronounced in Trial 2 (Fig. 5) than in Trial 1 (Fig. 4) with the most dramatic decreases seen in the ACFS treatment. However, the decreases seen in this treatment were significantly different only from AFS. Therefore, optimal ST reduction required cecal contents and alfalfa in addition to the FOS.
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S. Typhimurium log counts
12 A
10 8
A
A
A
a
a
a
ACFS
ACS
LRCS
B
a
B
b
b
6 4 2 0 LRCFS
0;0 h
CS
S
0;24 h
Fig. 2. Effects of in vitro fermentation with hen cecal contents on S. Typhimurium growth after 0 and 24 h post-inoculation fermentation. Data are generated in Trial 1 at the following time points: 0:0 h (inoculated with S. Typhimurium at time 0, collected samples at time 0) and 0:24 h (inoculated with S. Typhimurium at time 0, fermented for 24 h then collected). Abbreviation key: ACFS ¼ alfalfa+cecal contents+FOS+ S. Typhimurium; ACS ¼ alfalfa+cecal contents+ S. Typhimurium; LRCS ¼ layer ration+cecal contents+ S. Typhimurium; LRCFS ¼ layer ration+cecal contents+FOS+ S. Typhimurium; CS ¼ cecal contents+ S. Typhimurium; S ¼ S. Typhimurium only. a–b Means with same letter and case do not differ significantly. A–B Means with same letter and case do not differ significantly.
S. Typhimurium log counts
10 A
9 8
a,b B
a,b
B
a
a,b C
7
b
A
A a,b
a,b
6 5 4 3 2 D*
1 0 ACFS
ACS
CS
AFS
S 0;0 h
AS
CFS
0;24 h
Fig. 3. Effects of in vitro fermentation with hen cecal contents on S. Typhimurium growth after 0 and 24 h post-inoculation fermentation. Data are generated in Trial 2 at the following time points: 0:0 h (inoculated with S. Typhimurium at time 0, collected samples at time 0) and 0:24 h (inoculated with S. Typhimurium at time 0, fermented for 24 h then collected). Abbreviation key: ACFS ¼ alfalfa+cecal contents+FOS+ S. Typhimurium; ACS ¼ alfalfa+cecal contents+ S. Typhimurium; CS ¼ cecal contents+ S. Typhimurium; S ¼ S. Typhimurium only AFS ¼ alfalfa+FOS+ S. Typhimurium; AS ¼ alfalfa+ S. Typhimurium; CFS ¼ cecal contents+FOS+ S. Typhimurium. a–b Means with same letter and case do not differ significantly. A–D Means with same letter and case do not differ significantly. * Corresponds to S. Typhimurium log counts of 0.0 7 0.0.
A possible explanation might be the decreases in nutrient availability in the test tubes exposed to 24 h cecal incubation prior to ST incubation. The diverse growth requirements of undefined cecal microbial population could lead to a depletion of growth limiting substances making the experimental environment upon inoculation less favorable for ST compared to the microenvironmental conditions generated in group (I) experiments. Indirect evidence indicates that limiting nutrients can influence the competition between pathogens and gastrointestinal microflora. It has been reported that in a competition for limited
serine availability, the chicken cecal bacterium Escherichia fergusonii had outgrown S. Typhimurium [32]. In a coculture of mixed bacteria isolated from human feces and S. Typhimurium, Ushyima and Seto [33] observed considerable reduction of S. Typhimurium due to a bacterial competition for growth limiting amino acids including arginine, serine, threonine, and aspartate. This ecological relationship was substantiated by Coleman et al. [34] using a computer simulation of the microbial competition. In conclusion, it appears that the addition of FOS to alfalfa and layer ration may inhibit ST growth but only
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10 S. Typhimurium log counts
9
a
a,b
a,b,c
8
a
A
b,c
c A
A,B
A
7 B
B
6 5 4 3 2 1 0
ACFS
ACS
LRCS
LRCFS
24;0 h
CS
S
24;24 h
Fig. 4. Effects of 24 h in vitro fermentation with hen cecal contents on S. Typhimurium growth after 0 and 24 h post-inoculation. Data are generated in Trial 1 at the following time points: 24:0 h (fermented for 24 h then inoculated with S. Typhimurium, samples collected with no further fermentation) and 24:24 h (fermented for 24 h then inoculated with S. Typhimurium, fermented another 24 h then samples were collected). Abbreviation key: ACFS ¼ alfalfa+cecal contents+FOS+ S. Typhimurium; ACS ¼ alfalfa+cecal contents+ S. Typhimurium; LRCS ¼ layer ration+cecal contents+ S. Typhimurium; LRCFS ¼ layer ration+cecal contents+FOS+ S. Typhimurium; CS ¼ cecal contents+ S. Typhimurium; S ¼ S. Typhimurium only. a–c Means with same letter and case do not differ significantly. A–B Means with same letter and case do not differ significantly.
S. Typhimurium log counts
8 A
7
a
a
6 a,b
a,b A,B
5 b
A,B
4
A,B
a,b A,B
b
3 2 1
B*
B*
0 ACFS
ACS
CS
S 24;0 h
AFS
AS
CFS
24;24 h
Fig. 5. Effects of 24 h in vitro fermentation with hen cecal contents on S. Typhimurium growth after 0 and 24 h post-inoculation. Data are generated in Trial 2 at the following time points: 24:0 h (fermented for 24 h then inoculated with S. Typhimurium, samples collected with no further fermentation) and 24:24 h (fermented for 24 h then inoculated with S. Typhimurium, fermented another 24 h then samples collected). Abbreviation key: ACFS ¼ alfalfa+cecal contents+FOS+ S. Typhimurium; ACS ¼ alfalfa+cecal contents+ S. Typhimurium; CS ¼ cecal contents+ S. Typhimurium; S ¼ S. Typhimurium only; AFS ¼ alfalfa+FOS+ S. Typhimurium; AS ¼ alfalfa+ S. Typhimurium; CFS ¼ cecal contents+FOS+ S. Typhimurium. a–b Means with same letter and case do not differ significantly. A–B Means with same letter and case do not differ significantly. * Corresponds to S. Typhimurium log counts of 0.0 7 0.0.
after cecal microbial metabolism has been extended. Most of the bacteria in the cecum are considered being capable of growing anaerobically and include species belonging to groups such as Lactobacilli, Bifidobacterium, Propioniobacterium, and Methanogens [20,35–39]. Indigenous cecal microflora are capable of fermenting FOS which is nondigestible for enteric pathogens and cannot be used as a sole carbon source by them [40,41]. The microflora in the ceca is in symbiosis and maintains a stable ecosystem thus
developing a natural resistance to infections produced by enteric pathogens [38]. The microenvironment created by cecal bacteria after FOS fermentation is thought to be highly unfavorable to pathogens such as Salmonella [23]. This is partially accomplished by forming a physical barrier which prevents the penetration of the intestinal epithelium by pathogens [39]. FOS are naturally occurring oligosaccharides of plant origin and are the only product recognized and used as a colonic food ingredient and probiotic [17,42].
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The delayed effectiveness of fermentation responses seen in the in vitro experiments reported here may also help to explain some of the variable responses reported from in vivo studies involving FOS and Salmonella infection in chickens. Bailey et al. [22] did not observe reduction in Salmonella colonization in vivo by FOS alone until combined with a competitive exclusion culture prepared from the ceca of 25-week-old, Salmonella-free laying chickens. Chambers et al. [43] reported little or no reduction of S. Typhimurium scores to refined FOS in chicken 3–5 weeks old. However, they observed considerable decreases of S. Typhimurium population in 6-week-old chicks when fed refined FOS. Therefore, the effect of FOS in a cecal fermentation system on the Salmonella reduction is highly related to either the diversity and the quality of indigenous cecal microflora [44], or the amount of the cecal byproducts released during fermentation [28], or both. These results imply a complexity of the beneficial factors involved in the establishment of gastrointestinal conditions promoting host health. Achievement of consistent in vivo responses to dietary FOS may require feeding for more sustained periods of time to allow the indigenous microflora to adapt to it as a substrate. Acknowledgments This research was supported by Hatch grant H8311 administered by the Texas Agricultural Experiment Station, USDA-NRI Grant number 2002-02614 and US Poultry and Egg Association Grant # 485. We would also like to thank Encore Technologies, Plymouth, MN, USA, for donating the FOS. L.M.D. was partially supported by the Maurice Stein Fellowship. P.H. is supported by a postdoctoral fellowship (USDA-NRI Grant # 2004-04571). References [1] Holt PS, Macri NP, Porter Jr RE. Microbiological analysis of the early Salmonella enteritidis infection in molted and unmolted hens. Avian Dis 1995;39:55–63. [2] Ba¨umler AJ. Foodborne Salmonella infections. In: Beier RC, Pillai SD, Phillips TD, editors. Preharvest and postharvest food safety, contemporary issues and future directions. Ames, IA: Blackwell Publishing Professional; 2004. [3] Gast RK. Understanding Salmonella enteritidis in laying chickens: the contributions of experimental infections. Int J Food Microbiol 1994;21:107–16. [4] Guard-Petter J. The chicken, the egg and Salmonella enteritidis. Environ Microbiol 2001;3:421–30. [5] Hudault S, Bewa H, Bridonneau C, Raibaud P. Efficiency of various bacterial suspensions derived from cecal floras of conventional chickens in reducing the population level of Salmonella Typhimurium in gnotobiotic mice and chicken intestines. Can J Microbiol 1985;31:832–8. [6] Durant JA, Corrier DE, Byrd JA, Stanker LH, Ricke SC. Feed deprivation affects crop environment and modulates Salmonella enteritidis colonization and invasion of Leghorn hens. Appl Environ Microbiol 1999;65:1919–23. [7] Ricke SC. The gastrointestinal tract ecology of Salmonella enteritidis colonization in molting hens. Poult Sci 2003;82:1003–7.
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