Assessment of a food microbiology senior undergraduate course as a potential food safety distance education course for poultry science majors.

Distance education courses have become popular due to the increased number of commuter students as well as people already in the workforce who need further education for advancement within their careers. A graduate-level Web-based course entitled Special Topics-Poultry Food Safety Microbiology was developed from an existing senior undergraduate advanced food microbiology course in the Poultry Science Department at Texas A&M University. Conversion of standard lecture material into a distance education course can provide unique challenges to maintain comparable course content in an asynchronous manner. The overall objective for this course was to examine bacterial activities including ecology in food, animals, raw and processed meat, eggs, and human pathogenesis. Students were surveyed at the end of the class and the majority agreed that they would be willing to take the course as an online course, although they were not willing to pay an extra fee for an online course. The majority of students used the online version of the course as a supplement to the classroom rather than as a substitute.

Dilution rates influence ammonia-assimilating enzyme activities and cell parameters of Selenomonas ruminantium strain D in continuous (glucose-limited) culture.

AIMS: The objective of this study was to examine the effect of dilution rates (Ds, varying from 0.05 to 0.42 h(-1)) in glucose-limited continuous culture on cell yield, cell composition, fermentation pattern and ammonia assimilation enzymes of Selenomonas ruminantium strain D. METHODS AND RESULTS: All glucose-limited continuous culture experiments were conducted under anaerobic conditions. Except for protein, all cell constituents including carbohydrates, RNA and DNA yielded significant cubic responses to Ds with the highest values at Ds of either 0.10 or 0.20 h(-1). At Ds higher than 0.2 h(-1), fermentation acid pattern shifted primarily from propionate and acetate to lactate production. Succinate also accumulated at the higher Ds (0.30 and 0.42 h(-1)). Glucose was most efficiently utilized by S. ruminantium D at 0.20 h(-1) after which decreases in glucose and ATP yields were observed. Under energy limiting conditions, glutamine synthetase (GS) and glutamate dehydrogenase (GDH) appeared to be the major enzymes involved in nitrogen assimilation suggesting that other potential ammonia incorporating enzymes were of little importance in ammonia assimilation in S. ruminantium D. GS exhibited lower activities than GDH at all Ds, which indicates that the bacterial growth rate is not a primary regulator of their activities. CONCLUSIONS: Studied dilution rates influenced cell composition, fermentation pattern and nitrogen assimilation of S. ruminantium strain D grown in glucose-limited continuous culture. SIGNIFICANCE AND IMPACT OF THE STUDY: Selenomonas ruminantium D is an ecologically and evolutionary important bacterium in ruminants and is present under most rumen dietary conditions. Characterizing the growth physiology and ammonia assimilation enzymes of S. ruminantium D during glucose limitation at Ds, which simulate the liquid turnover rates in rumen, will provide a better understanding of how this micro-organism responds to differing growth conditions.

In vitro antilisterial effects of citrus oil fractions in combination with organic acids.

The objectives of this study were to screen activity of citrus essential oil fractions (EOs) alone and in combination with organic acids against 2 species of Listeria. Five citrus EOs were initially screened by disc diffusion assay for antibacterial activity. Cold pressed terpeneless Valencia orange oil (CP terpeneless oil) had the strongest bacteriostatic (MIC) and bactericidal (MBC) properties at 0.55% and 1.67%, respectively. Four organic acids were tested for effectiveness against Listeria. Citric and malic acids proved to be the most effective with MBC of 1.1% alone. Assays were conducted to determine synergistic effects of EOs and citric or malic acids. There was a significant decrease in MIC and MBC to 0.04% EO plus 0.12% malic or citric acid. EOs from citrus paired with organic acids offer the potential as an all-natural antimicrobial for improving the safety of all-natural foods.

Induction of cadBA in an Escherichia coli lysine auxotroph transformed with a cad-gfp transcriptional fusion.

CadBA functions as a part of overall Escherichia coli response to low extracellular pH. A gfpmut3 structural gene transcriptionally fused to the cadBA promoter (Pcad) was used as a reporter to monitor changes in intracellular lysine as a potential factor influencing cadBA induction. Different patterns of cadBA induction were observed in two E. coli strains with different lysine biosynthetic capabilities. In E. coli ZK126 (pJBA25-Pcad), a lysine prototroph, maximum levels of induction were detected 3 h after the transfer of bacterial cells under inducing conditions (pH 5.8; 3.4 microM extracellular lysine). The induction subsequently decreased until hour 7 after which no further change in expression was observed. However, in the lysine depleted strain E. coli ATCC 23812 (pJBA25-Pcad) which is an auxotroph for lysine, no decrease in cadBA expression was observed over time under the same induction conditions. Although no time dependent statistical differences in intracellular lysine were observed, bacterial cells depleted for no longer than 4 h (1.38 +/- 0.25 micromol lysine/g cell dry weight) exhibited more rapid induction of cadBA (after 3 h) and a lower maximum level of induction compared to cells with relatively lower intracellular lysine (approximately 1.08 micromol/g cell dry weight). For the latter, the detectable level of induction was delayed for 1 h but the maximum level of induction response was higher.

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.

Ciprofloxacin-sensitive and ciprofloxacin-resistant Campylobacter jejuni are equally susceptible to natural orange oil-based antimicrobials.

A total of 10 ciprofloxacin-sensitive (ciprofloxacin minimum inhibitory concentration, MIC < 0.5 micro g/ml) and 10 ciprofloxacin-resistant (MIC 16 to 32 micro g/ml) presumptive C. jejuni were further characterized and evaluated for their inhibition by natural orange oil fractions. Partial species identification was performed by using a hippuricase gene-based polymerase chain reaction (PCR) assay. One of the isolates appeared to be atypical and failed to hydrolyze hippurate. Of the ciprofloxacin-resistant C. jejuni isolates tested, six were found to have their quinolone resistance determined by a C --> T mutation in codon 86 of gyrA. Both groups of ciprofloxacin-sensitive and -resistant C. jejuni isolates were most susceptible to cold-pressed terpeneless Valencia orange oil (C4) which yielded inhibition zones from 44.0 +/- 1.4 to 80 +/- 0.0 mm. Less inhibitory responses were recorded for 5-fold concentrated Valencia orange oil (C3) and distilled d-limonene (C7) which exerted similar effects on both ciprofloxacin-sensitive and -resistant C. jejuni isolates. In general, ciprofloxacin-resistant and -sensitive C. jejuni isolates were equally susceptible to the respective orange oil fractions.

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 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 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.

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.

Effects of Maillard reaction products on hilA expression in Salmonella typhimurium.

Salmonella is a foodborne pathogen causing severe gastroenteritis. Three types of Maillard reaction products (MRP) generated by heat sterilization of D-glucose and L-lysine, L-histidine, and L-arginine were studied at 2 different levels of supplementation (0.5% and 1.0%) for their influence on growth and virulence of Salmonella. Two methods, namely, real-time polymerase chain reaction (RT-PCR) and a beta-galactosidase gene fusion assay, were used to determine the expression of hilA, a regulatory gene for Salmonella pathogenicity. Neither the type of MRP nor their quantities up to 1.0% affected the growth rates of S. Typhimurium EE658 (P > 0.05). When determined by beta-galactosidase assay, lysine MRP in both levels of supplementation were not found to have any effect on the hilA expression compared to the control. The addition of histidine and arginine MRP to M9 media (0.5%) increased by 2-fold hilA induction and up to 6-fold at the higher level (1%) supplementation of these compounds. Although somewhat inconsistent, RT-PCR analyses of hilA expression confirmed the greater induction effect of arginine MRP on hilA compared to lysine MRP. In contrast to beta-galactosidase assay results, however, lysine MRP were found to increase hilA expression compared to the control in both supplementation levels in all trials. The potential of MRP serving as a bacterial virulence modulator may be a factor to be considered in food thermal processing when assessing Salmonella risk for causing foodborne disease.

A cad-gfpmut3 plasmid construct in Escherichia coli for gene induction-based quantification of lysine in acid hydrolysates of feedstuffs.

AIMS: To generate an inducible plasmid-borne cad-gfpmut3 transcriptional fusion and develop a method for quantification of total lysine. METHODS AND RESULTS: The cad-gfpmut3 transcriptional fusion was constructed by cloning the cad promoter (Pcad) upstream of a promotorless gfpmut3 located on a high-copy plasmid. The construct was electroporated into Escherichia coli ZK126 and the transformed strain was subsequently used to quantify lysine in feed ingredients. Lysine standard curves based on gene induction of the bacterial cells were used for estimating acid hydrolysate lysine concentrations in four feed ingredients. Except for sorghum, no substantial differences were observed when the data for lysine in soybean (2 x 49 +/- 0 x 37%), cottonseed (1 x 82 +/- 0 x 15%), and meat and bone meal (2 x 31 +/- 0 x 24%) generated by the newly developed construct were compared with previously published data. CONCLUSIONS: Using the cad-gfpmut3 fusion, feed derived lysine induction was measured easily and accurately, and could be a useful tool for the estimation of lysine in acid hydrolysates of feed ingredients. SIGNIFICANCE AND IMPACT OF THE STUDY: The described approach for lysine quantification in feed ingredients represents a cost- and time-efficient method offering rapid and accurate lysine quantification of multiple samples.

Growth and genetic responses of Salmonella Typhimurium to pH-shifts in an anaerobic continuous culture.

Salmonella infection of chickens that leads to potential human foodborne salmonellosis continues to be a concern. Changes in the pH of poultry gastrointestinal tract could influence Salmonella growth and virulence response. In the current study, growth responses of a chicken isolate Salmonella enterica serovar Typhimurium (ST) to three incremental pH-shifts (6.17-7.35) in continuous cultures (CC) were evaluated. The expression of rpoS and hilA was determined by real time-polymerase chain reaction (RT-PCR) as well. Increases in pH resulted in higher cell protein concentrations, glucose disappearance, and glucose and ATP yields. Although with some inconsistency between the two trials, the data indicated that the ammonia release into media was favored by low pH. The pH shifts did not significantly affect acetate biosynthesis. No consistent trends of pH influence on propionate and butyrate production could be detected. In all three pH shifts, relative expression of hilA was dominant at 0h which represented CC steady state. In pH shift 7.35-6.86 (Trial 1), the relative expression of rpoS at time 0 and 1h were over five-fold higher than after 3 and 6h of growth. Overall, the results suggest that ST physiology is altered by changes in pH, which could be determinant factors for ST survival in the poultry gastrointestinal ecosystems.

Orange essential oils antimicrobial activities against Salmonella spp.

Seven citrus essential oils (EOs) were screened by disc diffusion assay for their antibacterial act against 11 serotypes/strains of Salmonella. The 3 most active oils were selected to determine the minimal inhibitory concentration (MIC) against the same Salmonella. Orange terpenes, single-folded d-limonene, and orange essence terpenes all exhibited inhibitory activity against the Salmonella spp. on the disc diffusion assay. EOs were stabilized in broth by the addition of 0.15% (w/v) agar for performance of the MIC tests. Orange terpenes and d-limonene both had MICs of 1%. The most active compound, terpenes from orange essence, produced an MIC that ranged from 0.125% to 0.5% against the 11 Salmonella tested. Gas chromatography-mass spectrometry (GC/MS) analysis revealed that this orange essence oil was composed principally of d-limonene, 94%, and myrcene at about 3%. EOs from citrus offer the potential for all natural antimicrobials for use in improving the safety of organic or all natural foods.

In vitro anaerobic incubation of Salmonella enterica serotype Typhimurium and laying hen cecal bacteria in poultry feed substrates and a fructooligosaccharide prebiotic.

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 degrees C. Samples were inoculated with Salmonella at 0 and 24h after in vitro cecal fermentation and plated at 0 and 24h after inoculation with ST. Plates were incubated for 24h and colony forming units (CFU) enumerated. The samples immediately inoculated with ST without prior cecal fermentation did not significantly lower ST counts 24h later. However, samples pre-incubated for 24h 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 24h cecal incubations.

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.