Vesela I. Chalova

Foodborne Salmonella ecology in the avian gastrointestinal tract

Foodborne Salmonella continues to be a major cause of salmonellosis with Salmonella Enteritidis and S. Typhimurium considered to be responsible for most of the infections. Investigation of outbreaks and sporadic cases has indicated that food vehicles such as poultry and poultry by-products including raw and uncooked eggs are among the most common sources of Salmonella infections. The dissemination and infection of the avian intestinal tract remain somewhat unclear. In vitro incubation of Salmonella with mammalian tissue culture cells has shown that invasion into epithelial cells is complex and involves several genetic loci and host factors. Several genes are required for the intestinal phase of Salmonella invasion and are located on Salmonella pathogenicity island 1 (SPI 1). Salmonella pathogenesis in the gastrointestinal (GI) tract and the effects of environmental stimuli on gene expression influence bacterial colonization and invasion. Furthermore, significant parameters of Salmonella including growth physiology, nutrient availability, pH, and energy status are considered contributing factors in the GI tract ecology. Approaches for limiting Salmonella colonization have been primarily based on the microbial ecology of the intestinal tract. In vitro studies have shown that the toxic effects of short chain fatty acids (SCFA) to some Enterobacteriaceae, including Salmonella, have resulted in a reduction in population. In addition, it has been established that native intestinal microorganisms such as Lactobacilli provide protective mechanisms against Salmonella in the ceca. A clear understanding of the key factors involved in Salmonella colonization in the avian GI tract has the potential to lead to better approach for more effective control of this foodborne pathogen.

Campylobacter and Arcobacter species sensitivity to commercial orange oil fractions.

Seven orange oil fractions were screened for their ability to inhibit the growth of selected Campylobacter and Arcobacter spp. using the standard agar-disk diffusion assay. Cold pressed (CP) terpeneless Valencia orange oil was found to be the most inhibitory to both Campylobacter jejuni and Campylobacter coli, exhibiting maximum zones of inhibition up to 80+/-0.0 mm. Five-fold concentrated Valencia oil and distilled d-limonene resulted in Campylobacter inhibition zones ranging from 11.0+/-1.4 to 44+/-1.4 mm against both C. jejuni and C. coli. No inhibition of Arcobacter spp. was detected by 6 out of 7 orange fractions except CP terpeneless Valencia orange oil which produced inhibition zones varying from 9.5+/-0.7 to 29+/-1.4 mm. Naturally occurring C. jejuni UAF 244 was isolated from a whole retail chicken, confirmed by hippuricase gene PCR assay, and used to determine antimicrobial capacities of the CP terpeneless Valencia orange oil and limonene when applied on chicken legs and thighs. The two types of chicken parts did not influence the antimicrobial strength of both orange fractions. While the observed reduction of C. jejuni cells attached to the skin varied approximately 1.5 to 2 logarithms compared to the control, the growth inhibition of the bacterial cells by limonene in the rinse increased by 6-fold and complete inhibition without recovery of detectable viable cells occurred when CP Valencia orange oil was applied. The study demonstrated the potential of the selected commercial orange oil fractions to serve as natural antimicrobials against C. jejuni, C. coli, and Arcobacter spp.

The Influence of a Fructooligosaccharide Prebiotic Combined with Alfalfa Molt Diets on the Gastrointestinal Tract Fermentation, Salmonella Enteritidis Infection, and Intestinal Shedding in Laying Hens

Molting is a natural process, which birds undergo to rejuvenate their reproductive organs. The US poultry egg production industry has used feed withdrawal to effectively induce molt; however, susceptibility of Salmonella Enteritidis has encouraged the development of alternative methods. Previous research conducted in our laboratory showed that alfalfa is effective at molt induction and provides equivalent postmolt production numbers and quality when compared with feed withdrawal. In the attempt to further increase the efficacy of alfalfa molt diet and decrease the chicken susceptibility to Salmonella Enteritidis during molt, fructooligosaccharide (FOS) was added to a combination of 90% alfalfa and 10% layer ration in 2 levels (0.750 and 0.375%). Ovary and liver colonization by Salmonella Enteritidis in 3 and 2 of the 4 trials, respectively, were reduced (P <or= 0.05) in hens fed FOS-containing diets compared with hens subjected to feed withdrawal. Significant decreases in ce-cal Salmonella Enteritidis counts were also observed in 2 of the 4 trials. In 3 of the 4 trials, the same diets did not affect (P > 0.05) the production of cecal total volatile fatty acids when compared with hens undergoing feed withdrawal. However, in all 3 alfalfa molt diets, the concentrations of lactic acid were greater (P <or= 0.05) than hens with feed withdrawal, but no differences (P > 0.05) were observed among hens fed alfalfa combined with FOS and hens fed alfalfa/layer ration without FOS. Overall, given the similarities between hens fed 0.750% FOS (H) and 0.375% FOS (L), molt diets combined with the lower level of FOS should be sufficient.

Antimicrobial activity of commercial citrus-based natural extracts against Escherichia coli O157:H7 isolates and mutant strains.

Due to increasing concerns about the development of antimicrobial resistance amongst pathogenic bacteria, alternative strategies have been sought that do not use antibiotics to reduce pathogenic bacteria from foods and patients. A natural compound that has potent antimicrobial properties is citrus peel, which contains a variety of essential oils that inhibit the growth of or kill pathogenic bacteria. In the present study, seven citrus-based natural antimicrobials were evaluated for their ability to inhibit the growth of the pathogen Escherichia coli O157:H7. Zones of inhibition of E. coli O157:H7 by the citrus-derived fraction (10 microL/6 mm disk) were determined by a disk-diffusion assay on Sorbitol-MacConkey agar. Inhibition zones were observed after 48 h lawn growth of E. coli O157:H7 cells at 37 degrees C. Two citrus-based fractions, orange CP VAL terpeneless FAB 968611 and Limonene 1x Dist FAB 955430, inhibited E. coli O157:H7 with inhibition zones of approx. 11-24 mm dia. The remaining other five citrus-derived extracts (orange oil FL VAL 1121 ARR 974760, Orange 5x Conc VAL 4121 ARR 968374, orange terpenes ESS 1120 ARR 986259, orange terpenes CP 1100 ARR 986255, and orange terpenes OEO HP 1100 ARR 986257) were noninhibitory to E. coli O157:H7, yielding no clear inhibition zones. These studies show that citrus-derived natural compounds differ in their inhibitory activity against E. coli O157:H7 and some have potential applications as inhibitory agents against E. coli O157:H7 in various pathogen reduction strategies.

In Vitro Fermentation Response of Laying Hen Cecal Bacteria to Combinations of Fructooligosaccharide Prebiotics with Alfalfa or a Layer Ration

The objective of this in vitro study was to evaluate the effects of combining a prebiotic with alfalfa on fermentation by laying hen cecal bacteria. Cecal contents from laying hens were diluted to a 1:3,000 concentration with an anaerobic dilution solution and added to serum tubes filled with ground alfalfa or a layer ration with or without fructooligosaccharide (FOS) prebiotic. Samples were processed in an anaerobic hood, pressurized by using a pressure manifold, and incubated at 37 degrees C. Volatile fatty acid (VFA) and lactic acid concentrations were quantified at 6 and 24 h of substrate fermentation. In this study, fermentation of alfalfa resulted in greater production of acetate, VFA, and lactic acid compared with the layer ration. Although with a relative inconsistency in data between trials, the amendment of FOS to both alfalfa and the layer ration appeared to further increase fermentation as demonstrated by overall higher propionate, butyrate, VFA, and lactic acid concentrations. The effect was more pronounced after 24 h of fermentation, implying time constraints for the optimal production of fermentation products in the chicken gastrointestinal tract. These data indicate that in vitro cecal fermentation can be enhanced by the addition of FOS.

Extracellular antimutagenic activities of selected probiotic Bifidobacterium and Lactobacillus spp. as a function of growth phase.

The capabilities of selected strains from genera Lactobacillus and Bifidobacterium to produce extracellular bioactive compounds with antimutagenic properties against benzo[a]pyrene (BaP) and sodium azide (SA) were tested as a function of growth phase. The bacterial supernatants from exponential and stationary phases were characterized with different patterns of antimutagenic activity against the two mutagens. All lactobacilli exhibited either no effect or low antimutagenicity against BaP during exponential growth. Higher antimutagenic activities of lactobacilli supernatants were observed in the stationary phase against SA as well. An exception was Lactobacillus sakei 23K which expressed a relatively low percent of inhibition of mutagenesis (PI = 28.14 ± 7.41) in the exponential phase and no antimutagenic activity in the stationary phase. Of the bifidobacteria, only Bifidobacterium adoleascentis ATCC 15703 exhibited higher antimutagenecity against BaP in the exponential phase. The same bacterial supernatants however, did not possess any antimutagenicity against SA in either the exponential or stationary phases. B. bifidum ATCC 11863 did not express any significant differences in its activity against either BaP or SA in the exponential or stationary phases. Only B. breve ATCC 15700 expressed a high antimutagenic effect against SA in the stationary phase but exhibited no effect during exponential growth. Overall, bacterial antimutagenic responses were associated with growth phase and type of mutagen.

Quantification of total and bioavailable lysine in feed protein sources by a whole-cell green fluorescent protein growth-based Escherichia coli biosensor

Using a fluorescent whole-cell Escherichia coli biosensor previously developed in our laboratory, we determined total and bioavailable lysine in four feed ingredients (soybean, cottonseed, meat and bone meal, and sorghum) and three complete feeds (chick starter and finisher, and swine starter). The same feed sources were analyzed for total lysine by high performance liquid chromatography (HPLC) and bioavailable lysine by chick bioassay. No significant differences were found between bioavailable lysine estimates for soybean, cottonseed, meat and bone meal, chick starter and finisher, and swine starter obtained by the fluorescent E. coli biosensor and chick bioassay. Except for sorghum, the E. coli biosensor estimates for total lysine were highly comparable to those obtained by HPLC. Comparisons were also conducted between conventionally performed optical density-based and the newly developed fluorescence-based lysine assay. The lack of significant differences in data obtained for total and bioavailable lysine by both detection modes indicated reliance and accuracy of the fluorescent E. coli biosensor. Overall results suggest that the microbial assay based on green fluorescent protein fluorescence represents a promising alternative method for lysine quantification.

Microbial inhibitory and radical scavenging activities of cold-pressed terpeneless Valencia orange (Citrus sinensis) oil in different dispersing agents.

BACKGROUND Due to their low solubility in water, oil-based bioactive compounds require dispersion in a surface-active agent or appropriate solvents to ensure maximum contact with microorganisms. These combinations, however, may change their physical and/or chemical characteristics and consequently alter the desired functionality. The objective of this study was to determine the impact of selected dispersing agents, ethanol, dimethyl sulfoxide (DMSO), and Tween-80, on cold-pressed terpeneless (CPT) Valencia orange oil to function as a free radical scavenger and an antimicrobial food additive. RESULTS When dissolved in ethanol or DMSO, the orange oil fraction had similar minimum inhibitory concentrations (MIC) for Listeria monocytogenes ATCC 19 115 (0.3% and 0.25% v/v respectively), which were significantly lower (P <or= 0.5) than the MIC for Salmonella typhimurium ATCC 14 028 (1% v/v). Both ethanol and DMSO oil dispersion systems exhibited an intermediate MIC (0.75% v/v) for Lactobacillus plantarum WCFS1. The orange oil (up to 3%) in an aqueous solution of 0.1% Tween-80 yielded no inhibitory activities against any of the test bacteria. However, the 1% natural orange oil dispersed in Tween-80 exhibited 56.86% 2,2-diphenyl-1-picryl hydrazyl (DPPH) radical inhibition versus 18.37% and 16.60% when the same level of orange oil was dissolved in DMSO or ethanol, respectively. At the same orange oil concentration, the oil/Tween-80 suspension yielded 57.92% neutralization of hydroxyl radicals. This represents 71.37% of the mannitol antioxidant activity, which was used as a positive control. CONCLUSIONS These findings suggest that Tween-80 is an appropriate dispersing agent only if the antioxidant functionality is desired. If both antimicrobial and antioxidant properties are needed, the CPT Valencia orange oil should be dispersed in either DMSO or ethanol.

Escherichia coli, an Intestinal Microorganism, as a Biosensor for Quantification of Amino Acid Bioavailability

In animal diets optimal amino acid quantities and balance among amino acids is of great nutritional importance. Essential amino acid deficiencies have negative impacts on animal physiology, most often expressed in sub-optimal body weight gains. Over supplementation of diets with amino acids is costly and can increase the nitrogen emissions from animals. Although in vivo animal assays for quantification of amino acid bioavailability are well established, Escherichia coli-based bioassays are viable potential alternatives in terms of accuracy, cost, and time input. E. coli inhabits the gastrointestinal tract and although more abundant in colon, a relatively high titer of E. coli can also be isolated from the small intestine, where primary absorption of amino acids and peptides occur. After feed proteins are digested, liberated amino acids and small peptides are assimilated by both the small intestine and E. coli. The similar pattern of uptake is a necessary prerequisite to establish E. coli cells as accurate amino acid biosensors. In fact, amino acid transporters in both intestinal and E. coli cells are stereospecific, delivering only the respective biological l-forms. The presence of free amino- and carboxyl groups is critical for amino acid and dipeptide transport in both biological subjects. Di-, tri- and tetrapeptides can enter enterocytes; likewise only di-, tri- and tetrapeptides support E. coli growth. These similarities in addition to the well known bacterial genetics make E. coli an optimal bioassay microorganism for the assessment of nutritionally available amino acids in feeds.

Application of an Escherichia coli green fluorescent protein - based lysine biosensor under nonsterile conditions and autofluorescence background

Aims:  To examine the utility of an Escherichia coli green fluorescent protein (GFP) containing biosensor for quantification of bioavailable lysine in selected feed samples under nonsterile conditions and to estimate the background fluorescence of analyzed feed samples and evaluate the risk of confounding GFP emission from the lysine assay organism.