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This study investigates the microbiological and immunological basis underlying the efficacy of electron beam-inactivated immune modulators. The underlying hypothesis is that exposure to eBeam-based ionization reactions inactivate microorganisms without modifying their antigenic properties and thereby creating immune modulators. The immunological correlates of protection induced by such eBeam based Salmonella Typhimurium (EBST) immune modulators in dendritic cell (DC) (in vitro) and mice (in vivo) models were assessed. The EBST stimulated innate pro inflammatory response (TNFα) and maturation (MHC-II, CD40, CD80 and CD86) of DC. Immuno-stimulatory potential of EBST was on par with both a commercial Salmonella vaccine, and live Salmonella cells. The EBST cells did not multiply under permissive in vitro and in vivo conditions. However, EBST cells remained metabolically active. EBST immunized mice developed Salmonella-specific CD4+ T-cells that produced the Th1 cytokine IFNγ at a level similar to that induced by the live attenuated vaccine (AroA- ST) formulation. The EBST retained stable immunogenic properties for several months at room temperature, 4°C, and -20°C as well as after lyophilization. Therefore, such eBeam-based immune modulators have potential as vaccine candidates since they offer the safety of a “killed” vaccine, while retaining the immunogenicity of an “attenuated” vaccine. The ability to store eBeam based immune modulators at room temperature without loss of potency is also noteworthy.

Clostridium perfringens (Cp) is a Gram-positive anaerobe that is one of the causative agents of necrotic enteritis (NE) in chickens, which leads to high mortality. Owing to the ban of administering antibiotics in feed to chickens, there has been an increase in the number of NE outbreaks all over the world, and the estimated loss is approximately 6 billion U.S. dollars. The best alternative method to control NE without antibiotics could be vaccination. In this study, we exposed three different strains of Cp to electron beam (eBeam) irradiation to inactivate them and then used them as a killed vaccine to control the colonization of Cp in broiler chickens. The vaccine was delivered to 18-day old embryos in ovo and the chickens were challenged with the respective vaccine strain at two different time points (early and late) to test the protective efficacy of the vaccine. The results indicate that an effective eBeam dose of 10 kGy inactivated all three strains of Cp, did not affect the cell membrane or epitopes, induced significant levels of IgY in the vaccinated birds, and further reduced the colonization of Cp strains significantly (p &lt, 0.0001) in late challenge (JGS4064: 4 out of 10, JGS1473: 0 out of 10, JGS4104: 3 out of 10). Further studies are necessary to enhance the efficacy of the vaccine and to understand the mechanism of vaccine protection.

Harmful cyanobacterial blooms (cyanoHABs) pose threats to human and animal health due to the production of harmful cyanotoxins. Microcystis aeruginosa is a common cyanobacterium associated with these blooms and is responsible for producing the potent cyclic hepatotoxin microcystin-LR (MC-LR). Concerns over the public health implications of these toxins in water supplies have increased due to rising occurrence of these blooms. High energy electron beam (eBeam) irradiation technology presents a promising strategy for the mitigation of both cyanobacterial cells and cyanotoxins within the water treatment process. However, it is imperative that both cellular and chemical responses to eBeam irradiation are understood to ensure efficient treatment. We sought to investigate the effect of eBeam irradiation on M. aeruginosa cells and MC-LR degradation. Results indicate that doses as low as 2 kGy are lethal to M. aeruginosa cells and induce cell lysis. Even lower doses are required for degradation of the parent MC-LR toxin. However, it was observed that there is a delay in cell lysis after irradiation where M. aeruginosa cells may still be metabolically active and able to synthesize microcystin. These results suggest that eBeam may be suitable for cyanoHAB mitigation in water treatment if employed following cell lysis.

Ionizing radiation such as Electron beam (EB) and gamma irradiation inactivate microbial cells preventing their multiplication. These cells, however, are structurally intact and appear to have residual metabolic activity. We were interested in understanding the metabolic pathways that were still functional in EB-inactivated cells. Therefore, the primary objective of this study was to compare the metabolites accumulating in EB-inactivated pathogens E. coli 026:H11 and S. Typhimurium immediately after EB inactivation and 24 h post inactivation. Defined aliquots (109 CFU/mL) of E. coli O26-H11 (TW 1597) and S. Typhimurium (ATCC 13311) suspended in phosphate-buffered saline were exposed to lethal EB doses of 3 kGy and 2 kGy, respectively. Complete inactivation (inability of cells to multiply) was confirmed by traditional plating methods. An untargeted analysis of the primary metabolites accumulating in un-irradiated (control) cells, EB-inactivated cells immediately after irradiation, and EB-inactivated cells that were incubated at room temperature for 24 h post EB inactivation was performed using gas chromatography/mass spectrometry. A total of 349 different metabolites were detected in the EB-inactivated S. Typhimurium and E. coli O26:H11 cells, out of which, only 50% were identifiable. In S. Typhimurium, 98 metabolites were expressed at statistically different concentrations (P < 0.05) between the three treatment groups. In E. coli O26:H11, 63 metabolites were expressed at statistically different concentrations (P < 0.05) between the three treatment groups. In both these pathogens, the β-alanine, alanine, aspartate, and glutamate metabolic pathways were significantly impacted (P < 0.01). Furthermore, the metabolomic changes in EB-inactivated cells were amplified significantly after 24 h storage at room temperature in phosphate-buffered saline. These results suggest that EB-inactivated cells are very metabolically active and, therefore, the term Metabolically Active yet Non-culturable is an apt term describing EB-inactivated bacterial cells.

Bacterial cells exposed to lethal doses of ionizing radiation, i.e. electron beam, are unable to replicate yet they remain metabolically active. To investigate this phenomenon further, we electron beam irradiated Escherichia coli cells to a lethal dose and measured their membrane integrity, metabolic activity, ATP levels and overall cellular functionality in terms of ability to propagate bacteriophages. We also visualized the DNA double-strand breaks in the cells. We used non-irradiated (live) and heat-killed cells as positive and negative controls, respectively. Our results show that the membrane integrity of E. coli cells is maintained and that the cells remain metabolically active up to 9 days post-irradiation when stored at 4°C. The ATP levels in lethally irradiated cells are similar to non-irradiated control cells. We also visualized extensive DNA damage within the cells and confirmed their cellular functionality based on their ability to propagate bacteriophages for up to 9 days post-irradiation. Overall, these findings confirm that lethally irradiated E. coli cells which have extensively damaged DNA are unable to replicate. However, they are metabolically active based on multiple indicators. The ability of bacterial cells to be metabolically active and capable of propagating phages in spite of physically damaged DNA is intriguing and deserves further studies.

AIMS Acid exposure induces accumulation of certain metabolites in bacteria. The experimental objective was to identify the primary metabolites accumulating in Escherichia coli O26:H11 as a function of acid (pH 3·6) exposure. METHODS AND RESULTS Different buffers of pH 7·5 and 3·6 were used to study the metabolites accumulating in E. coli O26:H11 cells during such pH exposure. After 24 h of acid exposure, there was a 7-log decline in E. coli populations on trypticase soy agar plates. Untargeted metabolomic analysis identified 293 primary metabolites of which 145 metabolites were differentially (P

The response of the antimicrobial compounds sulfamethoxazole (SMX) and trimethoprim (TMP) - individually and in mixtures - to ionizing radiation was investigated using laboratory prepared mixtures and a commercial pharmaceutical formulation. The residual antibacterial activity of the solutions was monitored using Staphylococcus aureus and Escherichia coli test strains. Based on antibacterial activity, SMX was more susceptible to ionizing radiation as compared to TMP. The antibacterial activity of SMX and TMP was completely eliminated at 0.2 kGy and 0.8 kGy, respectively. However, when SMX and TMP were in a mixture, the dose required to eliminate the antibacterial activity was 10 kGy, implying a synergistic antibacterial activity when these are present in mixtures. Only when the antibiotic concentration was below the Minimum Inhibitory Concentration of TMP (i.e., 2 µmol dm

The effect of attenuating the energy of a 10 MeV electron beam on Salmonella inactivation kinetics was investigated. No statistically significant differences were observed between the D 10 values of either Salmonella 4,[5],12:i:- or a Salmonella cocktail ( S . 4,[5],12:i:-, Salmonella Heidelberg, Salmonella Newport, Salmonella Typhimurium, Salmonella ) when irradiated with either a non-attenuated 10 MeV eBeam or an attenuated 10 MeV eBeam (~2.9±0.22 MeV). The results show that attenuating the energy of a 10 MeV eBeam to achieve low doses does not affect the inactivation kinetics of Salmonella spp. when compared to direct 10 MeV eBeam irradiation.

Infection with porcine epidemic diarrhea virus (PEDV) causes diarrhea, vomiting, and high mortality in suckling pigs. Contaminated feed has been suggested as a vehicle of transmission for PEDV. The objective of this study was to compare thermal and electron beam processing, and the inclusion of feed additives on the inactivation of PEDV in feed. Feed samples were spiked with PEDV and then heated to 120-145°C for up to 30 min or irradiated at 0-50 kGy. Another set of feed samples spiked with PEDV and mixed with Ultracid P (Nutriad), Activate DA (Novus International), KEM-GEST (Kemin Agrifood), Acid Booster (Agri-Nutrition), sugar or salt was incubated at room temperature (~25°C) for up to 21 days. At the end of incubation, the virus titers were determined by inoculation of Vero-81 cells and the virus inactivation kinetics were modeled using the Weibull distribution model. The Weibull kinetic parameter delta represented the time or eBeam dose required to reduce virus concentration by 1 log. For thermal processing, delta values ranged from 16.52 min at 120°C to 1.30 min at 145°C. For eBeam processing, a target dose of 50 kGy reduced PEDV concentration by 3 log. All additives tested were effective in reducing the survival of PEDV when compared with the control sample (delta = 17.23 days). Activate DA (0.81) and KEM-GEST (3.28) produced the fastest inactivation. In conclusion, heating swine feed at temperatures over 130°C or eBeam processing of feed with a dose over 50 kGy are effective processing steps to reduce PEDV survival. Additionally, the inclusion of selected additives can decrease PEDV survivability.

Citrus limonoids were reported to inhibit autoinducer-mediated bacterial cell–cell signaling, Escherichia coli O157:H7 biofilms and the Type III secretion system. The goal of the present study was to understand the effect of specific modifications in limonin molecule on cell–cell signaling inhibitory activity. The limonin was modified into de-furan limonin, limonin 7-oxime, limonin 7-methoxime. The modified limonoids were examined for their cell–cell signaling and biofilm inhibitory properties. The results of the study suggest that the furan ring and C-7 substitutions were important structural features in the limonin molecule to inhibit cell–cell signaling and biofilm formation in Vibrio harveyi. The C-7 modifications of limonin showed increased potency to inhibit the E. coli biofilm. Furthermore, the data indicates that limonin 7-methoxime reduced the attachment of E. coli to plastic surface by interacting with type 1 pili and Ag43. In addition, limonin 7-methoxime seems to reduce the adhesion of E. coli to spinach leaves and colon epithelial cells.

Our laboratories are investigating the use of High Energy (10 MeV) Electron-Beam (E-beam) irradiation for is potential use in vaccine development. Ionizing radiation inactivates microorganisms by “direct and indirect” effects on nucleic acids and other cellular components. Though the cells are inactivated, the surface antigenic properties of the microorganisms remain unaltered. We hypothesized that electron-beam (E-beam) inactivated Salmonella enterica serovars could be used as a potential immune modulator to activate the innate immune response and thus reduce Salmonella intestinal colonization and shedding in neonatal chickens. Three replicate experiments were designed to evaluate the efficacy of a high energy E-beam irradiated Salmonella enterica serovar Typhimurium (ST) administered in ovo to: (a) induce a functional innate immune response and (b) reduce ST colonization in the ceca of chicks three-weeks post-hatch. We have previously shown that unmethylated CpG motifs of bacteria DNA oligodeoxynucleotides (CpG-ODN) given in ovo stimulates innate immune responsiveness of chicken heterophils and increases resistance of young chickens to SE colonization; thus were used as positive controls in these experiments Eighteen-day-old chicken embryos were equally divided into four independent treatment groups: (1) a negative control (sham injected, no challenge) group, (2) an infected control (sham injected, challenged) group, (3) a CpG-ODN injected, challenged positive control, and (4) an E-beam ST-injected, challenged group. All treatment groups contained 100 birds, half of the animals from each treatment group were euthanized on day 4 post-hatch so that peripheral blood granulocytes (heterophils) could be collected to evaluate the functional innate immune response. The remaining birds where reared under normal housing conditions for the remainder of the experiment. On day 18 post-hatch the birds were challenged with the homologous ST strain and five days later (day 23 post-hatch), the experiment was terminated to evaluate the colonization of ST in the ceca of the birds. Differences in the leukocyte function and in the log10 cfu of ST counts among treatment groups were determined by analysis of variance. Significant differences were further separated using Duncan's multiple range tests. Here, heterophil function was measured using in vitro assays for (1) oxidative burst and (2) degranulation. Heterophils from the CpG-ODN and E-beam ST-treated birds exhibited a significant increase (P < 0.05) in both the oxidative response and degranulation when compared to all other treatment groups with no differences in heterophil functions between the CpG-ODN and e-beam-treated groups. ST colonization of the ceca was significantly reduced (P < 0.05) in both the CpG-ODN and the E-beam ST-treated birds when compared to the non-vaccinated control birds. These results demonstrate that in ovo administration of E-beam irradiated Salmonella induced a primed heterophil-mediated innate immune response and provide a protective intestinal colonization-inhibition effect against a homologous Salmonella challenge.

Salmonellosis is one of the leading health problems worldwide. With the rise of drug resistance strains, it has become imperative to identify alternative strategies to counter bacterial infection. Natural products were used historically to identify novel compounds with various bioactivities. Citrus species is a rich source of flavonoids. Naringenin, a flavonone, is present predominantly in grapefruit. Previously we have demonstrated that naringenin is potent inhibitor of cell-cell signaling. The current study was undertaken to understand the effect of naringenin on Salmonella Typhimurium LT2. The cDNA microarrays were employed to study the response of S. Typhimurium to naringenin treatment. Naringenin specifically repressed 24 genes in the Salmonella pathogenicity island 1 and down-regulated 17 genes involved in flagellar and motility. Furthermore, phenotypic assays support the result of microarray analysis. In addition, naringenin seems to repress SPI-1 in pstS/hilD-dependent manner. Altogether the data suggest that naringenin attenuated S. Typhimurium virulence and cell motility. This is the first molecular evidence to demonstrate effect of naringenin on bacterial virulence and cell motility.

The food industry utilizes a variety of stressors such as heat treatment to inactivate or prevent the multiplication of L. monocytogenes in foods. The aim of this study was to identify the extent to which pre-exposure to sub-lethal temperatures would induce temperature tolerance to 60 °C and result in increased D values.The D value of an avirulent L. monocytogenes strain (4ab No 10) was initially determined in Tryptic Soy Broth at 55 °C, 60 °C and 65 °C. The cells were pre-exposed to sub-lethal heat stress of 46 °C (for 30 and 60 min), 48 °C (for 30 and 60 min) and 50 °C (for 30 and 60 min). Then they were exposed to 60 °C and the D 60 values were calculated. The pre-treatment at sub-lethal temperatures enhanced the D 60 value of the strain from 3.03 min to longer times. The D 60 values increased as a function of pre-treatment time at 46 °C: after 30 min exposure it was 5.24 min, and 16.18 min after exposure to 60 min. Similarly, the D 60 value after 30 min and 60 min exposure at 48 °C was 6.72 min and 14...

The enteric pathogen Salmonella enterica serovar Typhimurium uses autoinducer-2 (AI-2) as a signaling molecule. AI-2 requires the luxS gene for its synthesis. The regulation of global gene expression in Salmonella Typhimurium by luxS/AI-2 is currently not known; therefore, the focus of this study was to elucidate the global gene expression patterns in Salmonella Typhimurium as regulated by luxS/AI-2. The genes controlled by luxS/AI-2 were identified using microarrays with RNA samples from wild-type (WT) Salmonella Typhimurium and its isogenic DeltaluxS mutant, in two growth conditions (presence and absence of glucose) at mid-log and early stationary phases. The results indicate that luxS/AI-2 has very different effects in Salmonella Typhimurium depending on the stage of cell growth and the levels of glucose. Genes with p < or = 0.05 were considered to be significantly expressed differentially between WT and DeltaluxS mutant. In the mid-log phase of growth, AI-2 activity was higher (1500-fold) in the presence of glucose than in its absence (450-fold). There was differential gene expression of 13 genes between the WT and its isogenic DeltaluxS mutant in the presence of glucose and 547 genes in its absence. In early stationary phase, AI-2 activity was higher (650-fold) in the presence of glucose than in its absence (1.5-fold). In the presence of glucose, 16 genes were differentially expressed, and in its absence, 60 genes were differentially expressed. Our microarray study indicates that both luxS and AI-2 could play a vital role in several cellular processes including metabolism, biofilm formation, transcription, translation, transport, and binding proteins, signal transduction, and regulatory functions in addition to previously identified functions. Phenotypic analysis of DeltaluxS mutant confirmed the microarray results and revealed that luxS did not influence growth but played a role in the biofilm formation and motility.

Bacterial cell-to-cell communication is mediated by autoinducer (AI) molecules such as AI-2 and has been reported to regulate gene expression in Escherichia coli O157:H7. We have previously shown that ground beef contains compounds that can inhibit sensing of AI-2 like activity. The hypothesis of this study was that AI-2 activ- ity observed in conditioned medium (CM) will enhance E. coli O157:H7 survival and expression of virulence genes, whereas compounds inhibitory (such as those present in ground beef extracts) to AI-2 activity will negate these ef- fects. E. coli O157:H7 luxS mutant strain VS 94 (incapable of synthesizing AI-2) was employed in these studies. The survival of this enteric bacterial pathogen as a function of AI-2 activity and the presence of AI-2 inhibitory com- pounds was studied at 4 ◦ C. The number of survivors in the presence of AI-2 was significantly higher compared to the absence of AI-2, and the addition of ground beef extracts to conditioned medium negated the influence of AI- 2 activity. Autoinducer AI-2 upregulated selected genes virulence genes (yadK ,a ndhha), whereas the ground beef extract reversed the effect of AI-2 on the expression of the selected genes.

Enteric viruses are key foodborne pathogens. The objective of this study was to compare the relative contributions of electrostatic and hydrophobic forces with the nonspecific attachment of virus to butterhead lettuce. The attachment of four viruses (echovirus 11, feline calicivirus [FCV], MS2, and phiX174) was studied. Three different conditions, namely (i) 1% Tween 80, (ii) 1 M NaCl, and (iii) 1% Tween 80 with 1 M NaCl, were investigated to determine the role of hydrophobic, electrostatic, and combined hydrophobic and electrostatic forces, respectively. Attachment above the pI of FCV and echovirus 11 was reduced or eliminated in the presence of NaCl, indicating an electrostatic interaction between the animal viruses and lettuce. The bacteriophage phiX174 was not significantly affected by any treatment, indicating a lack of electrostatic or hydrophobic interactions between the lettuce and phage phiX174. Overall, 1 M NaCl was the most effective treatment in desorbing viruses from the surface of lettuce at pH 7 and 8. The results imply that electrostatic forces play a major role in controlling virus adsorption to lettuce. The results indicate that 1 M NaCl solution would improve the recovery or elution of unenveloped viruses from lettuce.

Microorganisms employ autoinducer molecules to modulate various bacterial processes including virulence expression, biofilm development, and bioluminescence. The universal autoinducer molecule AI-2 is hypothesized to mediate cell signaling in Escherichia coli O157:H7. We investigated the role of AI-2 on the E. coli O157:H7 cellular proteins using a two-dimensional (2D) gel electrophoresis-based proteomic approach. The protein expression patterns between two experimental comparisons were studied namely, 1) a wild type E. coli O157:H7 and its isogenic luxS mutant, and 2) the luxS mutant and the luxS mutant supplemented with AI-2 molecules. Eleven proteins were differentially expressed between the wild type and the luxS mutant strain, whereas 18 proteins were differentially expressed in the luxS mutant strain when supplemented with AI-2. The tryptophan repressor binding protein (WrbA), phosphoglycerate mutase (GpmA), and a putative protein YbbN were found to be differentially expressed under both experimental comparisons. The FliC protein which is involved in flagellar synthesis and motility was up-regulated in the wild type strain but was not influenced by the addition of synthetic AI-2 molecules to the luxS mutant suggesting the involvement of signaling molecules other than AI-2 on flagellar synthesis and motility.

Most library-dependent bacterial source tracking studies using Escherichia coli (E. coli) have focused on strain diversity of isolates obtained from known human and animal faecal sources for library development. In contrast, this study evaluated the genotype variation of E. coli isolated from natural surface water using pulsed field gel electrophoresis (PFGE) and enterobacterial repetitive intergenic consensus sequence polymerase chain reaction (ERIC-PCR) to better understand these naturally occurring populations. A total of 650 water samples were collected over a nine month period from eleven sampling stations from Lake Waco and Belton Lake in Central Texas. Of the 650 water samples collected, 412 were positive for E. coli, yielding a total of 631 E. coli isolates (1-12 isolates collected per sample). PFGE and ERIC-PCR patterns were successfully generated for 555 isolates and were compared using the curve-based Pearson's product-moment correlation coefficient. The 555 E. coli isolates represented 461 PFGE genotypes, with 84% (386/461) of the genotypes being represented by individual isolates. The remaining 75 genotypes were represented by 2-5 isolates each. Using ERIC-PCR, the 555 E. coli isolates represented 175 genotypes, with 63% (109/175) of the genotypes being represented by individual isolates. In contrast to the PFGE results, two ERIC-PCR genotypes represented 37% of the E. coli isolates, (83 and 124 isolates, respectively), and were found throughout the watersheds both spatially and temporally. Based on the PFGE genotype diversity of water isolates, there is little evidence that a small number of environmentally-adapted E. coli represent dominant populations in the studied waterbodies. However, with the lower discriminatory power technique ERIC-PCR, an opposing conclusion might have been drawn. These results emphasize the importance of considering the resolving power of the source tracking technique being used when assessing strain diversity and geographical stability.

Dried fruits and nuts make up a significant portion of the commodities traded globally, and the presence of yeasts and molds on dried fruits and nuts can be a public health risk because of the potential for exposure to toxigenic fungi. Since current postharvest treatment technologies are rather limited for dried fruits and nuts, electron beam (E-beam) radiation experiments were performed to determine the doses required to reduce the yeast and mold bioburden of raisins, walnuts, and dates. The indigenous yeast and mold bioburden on a select number of commodities sold at retail ranged from 10(2) to 10(3) CFU/g. E-beam inactivation kinetics based on the linear model suggest that the decimal reduction dose required to eliminate 90% of the microbial population (D10-value) of these indigenous fungal populations ranges from 1.09 to 1.59 kGy. Some samples, however, exhibited inactivation kinetics that were better modeled by a quadratic model. The results indicate that different commodities can contain molds and yeasts of varying resistance to ionizing radiation. It is thus essential for the dried fruit and nut industry to determine empirically the minimum E-beam dose that is capable of reducing or eliminating the bioburden of yeasts and molds in their specific commodities.

Bacterial cells communicate with each other and respond to external stimuli using signal molecules called autoinducers (AIs). Poultry meat contains inhibitors that apparently interfere with AI-2 signaling. Our objective was to understand the expression of Salmonella Typhimurium genes (using spotted microarrays) in response to AI-2 in the presence, and absence, of poultry meat (PM) derived AI-2 inhibitors. Expression of 1136 virulence-related genes in Salmonella Typhimurium wildtype and its isogenic luxS mutant strain (unable to produce AI-2) was monitored when the cells were exposed for 3 hours to different treatments containing in vitro synthesized AI-2 and a PM inhibitor (AI-2, AI-2 + PM, or PM alone). The responses of the genes were unique in the presence of in vitro synthesized AI-2. Of 1136 genes on the array, 23 genes were differentially expressed (either upregulated or downregulated) at least 1.5-fold (P < 0.05) in the presence of AI-2. Exposure to the PM inhibitor resulted in 36 genes being differentially expressed, while the combined AI-2 + PM treatment resulted in 22 genes being differentially expressed, of which only 7 genes showed overlap with the PM treatment, suggesting a unique response when AI-2 interacts with the inhibitor molecules. The results suggest that Salmonella gene expression can vary depending on the presence or absence of the poultry meat matrix and/or AI-2 molecules. Understanding the interaction of AI-2 and inhibitors of AI-2 activity found in poultry meat can help explain Salmonella survival and virulence on poultry products.

Food-borne salmonellosis continues to be a major public health concern, and contamination with Salmonella spp. in pre-harvest animal production is considered a primary contributor to this problem. Animal feeds can easily become contaminated during primary production, feed mixing and processing as well as during feeding. Consequently, monitoring and surveillance of feeds and feed ingredients for Salmonella spp. contamination may be useful or necessary in the prevention and control of this organism. Cultural and immunological detection methods for salmonellae have been used or suggested as possible approaches for use in animal feeds. Cultural methods remain advantageous owing to their ability to detect viable bacterial cells, while immunological methods have the capability of detecting nonculturable bacterial cells. Advancements and improvements in both methodologies offer opportunities for eventual routine use of these detection technologies in animal feed assays.

Enteric viruses account for most foodborne illness in the United States. The objective of this study was to determine whether the isoelectric point (pI) of viruses such as feline calicivirus (FCV), echovirus 11, and bacteriophages phiX174 and MS2 had any effect on their attachment to butterhead lettuce. The adsorption of virus particles to the lettuce was variable. Bacteriophage MS2 was the only virus that fit the current Derjaguin-Landau-Verway-Overbeek model of virus attachment. Echovirus 11 had the highest affinity to lettuce surface. Echovirus 11 appeared to exhibit reversible attachment above its pI, whereas below its pI strong adsorption was observed. Adsorption of FCV was at its maximum above its pI. Bacteriophage phiX174 exhibited the most complex adsorption pattern, with attachment occurring only at the pH extremes (pH 3.0 and 8.0). These results suggest the current model for virus adsorption to sediment does not adequately explain the attachment of virus to lettuce. Importantly, the results indirectly suggest that current sample processing methods to recover viruses from lettuce may differentially select for the recovery of only certain virus types.

The autoinducer-2 molecule, AI-2, is considered to be a universal signal for regulating a wide variety of physiological processes in bacteria by modulating gene expression. Studies were conducted to observe how Escherichia coli cells would respond to subtherapeutic tetracycline concentrations under continuous culture (chemostat) conditions, to observe AI-2 activity within the probiotic chicken microbial consortium (Preempt CF3; MS Bioscience, Dundee, IL) under in vitro conditions simulating a chicken cecum, and to observe the AI-2 activity in vivo within a chicken cecum as a function of exposure to subtherapeutic levels of chlortetracydine, tylosin, and vancomycin. The AI-2 activity in the E. coli continuous culture showed a 20-fold increase over baseline conditions for up to 24 hours. When the E. coli culture was subsequently exposed to pulses of chlortetracydine additions at subtherapeutic concentrations (2 microg/ml), AI-2 activity increased with increasing levels of tetracycline additions. The probiotic Preempt CF3 culture, however, did not exhibit any AI-2 activity in Viande Levure (VL) medium in the presence or absence of subtherapeutic levels of tetracycline. In vivo studies in the cecum of poultry chicks demonstrate that though AI-2 activity increased initially in the presence of vancomycin, there was no significant increase in AI-2 activity in the presence of tetracydine or tylosin. These results indicate that detectable levels of AI-2 activity are not evident within the probiotic culture (CF3) or within the chicken cecum. Understanding the relationships between AI-2 activity and microbial consortia characteristics could provide dues regarding the vulnerability of poultry chicks to enteric bacterial pathogen colonization.

Foodborne salmonellosis continues to be a public health issue of considerable concern. Animal feed has been a major link in pre-harvest food animal production. Although monitoring systems and control measures are available to limit Salmonella spp. contamination on animal feeds detection methodology is relatively time consuming in the context of time inputs for feed processing and mixing. Current cultural methods of Salmonella spp. detection in feeds require several days for confirmation. This amount of time represents significant problems if control measures are to be effectively implemented in a fashion that keeps feed processing costs low. Molecular methods offer improved sensitivity and potential reduction in assay time. In particular, several commercial polymerase chain reaction (PCR) assays, and combined PCR-hybridization assays have been suggested as possible means to implement more rapid detection of Salmonella spp. extracted from animal feeds. It has now become possible to rapidly detect and confirm the presence of foodborne Salmonella spp. in feed matrices by commercial amplification detection systems. The primary challenges remaining are to develop more reliable recovery and extraction procedures for routine processing of samples from a wide variety of feed matrices and apply molecular techniques for assessing physiological status of Salmonella spp. contaminants in animal feeds.

Fresh produce has been repeatedly implicated as a vehicle in the transmission of foodborne gastroenteritis. In an effort to assess the risk factors involved in the contamination of fresh produce with pathogenic bacteria, a total of 1,257 samples were collected from cantaloupe, oranges, and parsley (both in the field and after processing) and from the environment (i.e., irrigation water, soil, equipment, etc.). Samples were collected twice per season from two production farms per commodity and analyzed for the presence of Salmonella and Escherichia coli. E. coli was detected on all types of commodities (cantaloupe, oranges, and parsley), in irrigation water, and on equipment surfaces. A total of 25 Salmonella isolates were found: 16 from irrigation water, 6 from packing shed equipment, and 3 from washed cantaloupes. Salmonella was not detected on oranges or parsley. Serotyping, pulsed-field gel electrophoresis (PFGE), and repetitive element sequence-based PCR (rep-PCR) assays were applied to all Salmonella isolates to evaluate the genetic diversity of the isolates and to determine relationships between sources of contamination. Using PFGE, Salmonella isolates obtained from irrigation water and equipment were determined to be different from cantaloupe isolates; however, DNA fingerprinting did not conclusively define relationships between contamination sources. All Salmonella isolates were subjected to antimicrobial susceptibility testing using the disk diffusion method, and 20% (5 of 25) of the isolates had intermediate sensitivity to streptomycin. One Salmonella isolate from cantaloupe was resistant to streptomycin.

Cryptosporidium parvum and Giardia lamblia are protozoa capable of causing gastrointestinal diseases. Currently, these organisms are identified using immunofluorescent antibody (IFA)-based microscopy, and identification requires trained individuals for final confirmation. Since artificial neural networks (ANN) can provide an automated means of identification, thereby reducing human errors related to misidentification, ANN were developed to identify Cryptosporidium oocyst and Giardia cyst images. Digitized images of C. parvum oocysts and G. lamblia cysts stained with various commercial IFA reagents were used as positive controls. The images were captured using a color digital camera at 400× (total magnification), processed, and converted into a binary numerical array. A variety of “negative” images were also captured and processed. The ANN were developed using these images and a rigorous training and testing protocol. The Cryptosporidium oocyst ANN were trained with 1,586 images, while Giardia cyst ANN were trained with 2,431 images. After training, the best-performing ANN were selected based on an initial testing performance against 100 images (50 positive and 50 negative images). The networks were validated against previously “unseen” images of 500 Cryptosporidium oocysts (250 positive, 250 negative) and 282 Giardia cysts (232 positive, 50 negative). The selected ANNs correctly identified 91.8 and 99.6% of the Cryptosporidium oocyst and Giardia cyst images, respectively. These results indicate that ANN technology can be an alternate to having trained personnel for detecting these pathogens and can be a boon to underdeveloped regions of the world where there is a chronic shortage of adequately skilled individuals to detect these pathogens.

Transmission of food-poisoning salmonellae in the poultry industry is often associated with a contaminated feed supply. Occurrence of food-borne Salmonella spp. continues to be documented on poultr...

Many fruits and vegetables are irrigated with water from rivers, lakes and even wastewater systems. Irrigation may be a route for the introduction of Salmonella. Our objectives in this study were to determine survivability and virulence expression in a strain of Salmonella typhimurium when exposed to environmental water sources. Virulence expression was measured using a beta-galactosidase assay on a hilA:lacZY fusion strain of S. typhimurium. Water samples for environmental impact studies were taken from a local pond and specific sites along the Rio Grande River, which serves as a source of irrigation water in southern Texas. There was a significant difference (p0.05) of virulence expression among the water sites. Certain regions along the Rio Grande River yielded greater amounts of beta-galactosidase activity than others. All sites yielded at least a two-fold greater virulence response than S. typhimurium grown in brain heart infusion. Salmonella survivors were enumerated as colony forming units (CFU)/ml as plated on a selective medium for the duration of 1 week and beta-galactosidase assays were performed to determine a possible correlation between culturable cells and virulence gene expression. Bacterial cells remained viable but decreased after 7 days incubation. In conclusion, water sampled at specific locations and at different times water samples exhibited differences in virulence expression in S. typhimurium.

Municipal and agricultural pollution affects the Rio Grande, a river that separates the United States from Mexico. Three hundred and twenty-two Escherichia coli isolates were examined for multiple antibiotic resistance phenotypes and the prevalence of class 1 and class 2 integron sequences. Thirty-two (10%) of the isolates were resistant to multiple antibiotics. Four (13%) of these isolates contained class 1-specific integron sequences; one isolate contained class 2 integron-specific sequences. Sequencing showed that the class 1 integron-bearing strain contained two distinct gene cassettes, sat-1 and aadA. Although three of the four class 1 integron-bearing strains harbored the aadA sequence, none of the strains was phenotypically resistant to streptomycin. These results suggest that integron-bearing E. coli strains can be present in contaminated irrigation canals and that these isolates may not express these resistance markers.

Salmonella is regarded as a primary foodborne pathogen commonly found in the gastrointestinal tract and internal organs of poultry. However, Salmonella is also capable of surviving in fresh water supplies. In this study, our efforts were to determine which media controls of varying nutrient availability would allow for accurately assessing the potential of a poultry house water source to influence virulence gene expression in Salmonella. The cages of the sampled layer house were divided into four separate quadrants. Virulence expression was measured using a β-galactosidase assay on a hilA:lacZY fusion strain of S. Typhimurium and calculated as Miller units to adjust for bacterial cell concentration measured as optical density. Virulence assays were performed with brain heart infusion broth (BHI) serving as the primary overall control which yielded values ranging from approximately 60–100 Miller units. Results showed that standing water obtained from quadrant 2 yielded a significantly different (P

The objectives of this study were to determine if indigenous male specific and somatic bacteriophages could be detected in animal feeds and if isolated phages contained RNA or DNA. Seven fresh feeds, 2 fresh feed ingredients, 7 stored feeds, 2 stored feed ingredients, and 8 samples of poultry diets suspected to contain Salmonella spp. were enriched and spot plated for indigenous phages using Escherichia coli Famp and CN-13 as hosts. Bacteriophage numbers were below detection without enrichment, but both male specific and somatic coliphages were detected in all animal feeds, feed ingredients, and poultry diets after 16 h of enrichment, even after the samples had been stored for 14 months of storage at -20 C. Five out of 9 fresh feeds and 7 out of 8 stored feeds contained RNA somatic phages.

The ability to rapidly screen environmental samples for specific pathogens such as Salmonella spp., is of particular importance in molecular epidemiology. Although gene amplification reactions allow the rapid detection of microorganisms, the use of appropriate oligonucleotide primers targeted against specific microbial genes is critical for accurate detection specificity and sensitivity. Primers such as fimA and hns have been previously shown to be specific for pure cultures of Salmonella. However, the analysis of environmental samples requires post-amplification hybridizations to detect amplicons, since the presence of inhibitory environmental components reduces amplification efficiency of the target organism. These sensitive post-amplification approaches also enable the detection of spurious amplification from non-target sequences. Bioaerosols associated with animal facilities and municipal wastes contain a diverse array of pathogens including Clostridium spp. In our studies, hybridization sensor data revealed spurious amplification of clostridial species with Salmonella hns primers. Specificity checks using type cultures of Clostridium spp. revealed non-specific amplification by hns primers. These results suggest that fimA primers may be better suited to screen Salmonella-specific sequences in environmental samples, especially those obtained from animal and municipal waste facilities.

Salmonellosis is a cyclic problem in the food industry, to which animal feed has been contributory. Current conventional methods of Salmonella spp. detection require 96 h for detection and confirmation. With modern and just-in-time production schedules, a 96-h hold represents a significant expense in storage and decontamination. The commercially available assay, 'BAX for Screening/Salmonella' (BAX), is based on the principle of the polymerase chain reaction and may represent a significant decrease in assay time. Seven fresh feed formulations, two fresh feed ingredients, seven stored feeds and two stored feed ingredients were artificially contaminated with a primary poultry isolate of Salmonella typhimurium and analysed by conventional and BAX methodology. The results of BAX agreed with conventional plating results for 16 of 18 samples spiked with 1200 cfu 10 g(-1) of feed and 13 of 18 samples spiked with 40 cfu 10 g(-1) of feed. Indigenous Salmonella spp. were detected in five of eight samples of poultry diets by conventional methods. With BAX, Salmonella spp. could not be detected in any of the samples after only 7 h of enrichment but could be detected in two dietary samples after 13 h of enrichment and four dietary samples after 24 h of enrichment. Specific sequences of salmonella DNA that were extracted from poultry diets could be detected with BAX.

Rapid screening of poultry houses for contamination is critical for Salmonella control. Use of air filter sampling has great potential for efficient and reliable monitoring of Salmonella spp., as it could represent an entire poultry house and solve sample-size problems. Two sampling methods (litter and air filter) were compared for detection in four chicken pens inoculated with a S. typhimurium antibiotic resistant strain. Salmonella levels in both litter and air filter samples were determined by PCR amplification and by conventional enrichment. Although amplified DNA was not directly detected, amplified DNA could be detected using a dual probe hybridization sensor. The ratio of the positive samples to total samples determined by gene amplification was much lower than that obtained by conventional enrichments (29/128 versus 102/128 samples). However, the ratio obtained by gene amplification with air filter samples was greater than that with litter samples (26/64 versus 3/64). These results demonstrate that the air filter sampling method is an alternative method of Salmonella detection in poultry house using PCR gene amplification protocol. Journal of Industrial Microbiology & Biotechnology (2000) 24, 379–382.

An integrated effort to reduce the occurrence of Salmonella in poultry products should include the control of Salmonella in poultry production in early stages, which requires an efficient monitoring of Salmonella spp. in poultry houses. Studies were conducted to evaluate the potential advantages of using air samples as an alternative to litter samples for PCR detection of Salmonella spp. from poultry houses. The air filter and litter samples from a poultry house were spiked with a culture of Salmonella typhimurium. The intact samples and the DNA extracted from the samples were used for amplification of Salmonella-specific DNA using hns gene specific primers. The ***hns gene was more effectively amplified with filter samples than litter samples when the intact sample was used as template. When extracted DNA was used, however, there was no difference between the two samples in their intensities of PCR products as visualized on an agarose gel. The dilution of the matrices from both sampling methods greatly increased the amplification efficiency, indicating that it was the background matrices that hindered the PCR amplification. The results in this study suggest that air filter sampling may be a labor-saving alternative to litter sampling, which can be used to increase the sensitivity of PCR detection of Salmonella spp. without prior sample processing.

The rapid detection of pathogenic microbial species in aerosols is of paramount importance considering its public health and animal husbandry implications. Oligonucleotide primers targeted against the DNA binding protein genes (hns) under appropriate amplification conditions is capable of specifically detecting Salmonella spp. and Clostridium spp. To demonstrate a methodology for rapidly detecting specific bacterial gene sequences, aerosol samples were collected from an outdoor biosolid application site and from indoor poultry houses for use in the protocol. Aliquots of these aerosol samples were used as templates in a gene amplification‐hybridization sensor protocol to rapidly detect the DNA binding protein (hns) gene sequences. Using the combination of gene amplification and the hybridization sensor, the presence of the hns gene sequences was confirmed in the aerosol samples. The primary advantage of employing the hybridization sensor is that it confirms the sequence specificity of the amplifie...

The rapid detection of pathogenic microbial species in feed is of paramount importance considering its implications for animal production and food safety. More sensitive and rapid detection of contaminated feedstuffs may lead to more selective and therefore less expensive treatment of feeds, reduced rates of transmission to a poultry host and reduced carcass contamination. In order to interrupt the cycle of Salmonella spp. transmission from feed to poultry to the consumer, more rapid detection methods to monitor these sources are needed that provide conclusive results within the time frame of feed mixing or broiler processing. Within the last decade, new variations of selective media have been investigated to increase selectivity without reducing Salmonella spp. recovery. Immunological assay methods may also decrease assay time from 96 h to within 24–30 h. But all commercially available methods still require 16 to 57 h for preenrichment, enrichment, and in some cases, postenrichment to recover sublethally injured cells before the assay can be performed. Among the molecular methods that are currently available, the polymerase chain reaction (PCR) represents a tremendous potential for the detection of low levels of pathogenic bacteria. Once optimized, rapid methods may be used to quickly, reliably and inexpensively screen a variety of feedstuffs and feed components for the presence of Salmonella spp., with the goal of minimizing both the cost of feed treatment and the horizontal transmission of Salmonella spp. from feed to poultry.

The rapid detection of pathogenic microbial species in feed is of paramount importance considering its implications for animal production and food safety. To demonstrate the feasibility of rapidly detecting Salmonella spp. and fecal pollution microbial indicators in feed using gene amplification protocols, commercial and mixed feed samples were inoculated with two levels of a marker strain of S. typhimurium. Liquid extracts of the feed samples were used as templates in gene amplification reactions to amplify sequences associated with fecal contamination indicators. The sequence specificity of the amplification products (amplicons) were confirmed using biotin and fluorescein labeled probes in a navel dual probe based hybridization sensor. Using the combination of gene amplification and the hybridization sensor, the presence of sequences associated with fecal contamination were detected in 15 different feed matrices without employing preenrichment steps. Using this detection methodology, fecal pollution can be confirmed in feed at naturally occurring concentrations. The study demonstrates that it is possible to rapidly detect and confirm the presence of pathogenic bacterial genera in feed matrices by combining robust gene amplification reactions with appropriate post amplification detection systems.

Microbial contamination of groundwater is a serious threat to public health along the US‐Mexico border. The survival and transport of two pathogenic bacteria (Salmonella typhimurium and Klebsiella sp.) and two indicator viruses (bacteriophages PRD‐1 and MS‐2), were studied using microcosms to determine the behavior of pathogenic microorganisms in the Rio Grande alluvium, which underlies the border between the United States and Mexico. Culturable populations of Salmonella typhimurium declined rapidly (>5 log units) to below detection limits within 12 days, while as much as 103 CFU/mL of Klebsiella sp. was viable even after 30 days. Less than 1% of the surviving Salmonella sp. population was viable based on microscopic viability assays. The population of both MS‐2 and PRD‐1 also declined rapidly (>6 log units) to below detection limits within 10 days. Salmonella sp. exhibited a relatively greater “straining”; or adsorbtion than the Klebsiella sp. under saturated conditions in a 0.2 m column. Even a...

Sludge and waste water samples from a variety of locations (in the United States and from one location in Mexico) were evaluated to determine whether multiple antibiotic resistant resistance patterns correlated with location and type of waste. The occurrence of antibiotic resistant strains of Escherichia coli was dependent upon site among the eleven locations sampled (P < 0.001). The E. coli strains from digested municipal sewage sludge from El Paso, Texas and those from an El Paso army hospital were resistant to the widest range of antibiotics and were resistant to a greater number of antibiotics than any other site (P < 0.01). When isolates from undigested waste water samples were analyzed, there was no significant difference (P ≥ 0.05) between those obtained from two neighboring cities located on either side of the US‐Mexico border. There was no significant difference in the number of antibiotics to which isolates were resistant when comparing digested sewage sludge samples from rural and urba...

Consumption of raw oysters is an exposure route for human norovirus (NoV) and hepatitis A virus (HAV). Therefore, efficient postharvest oyster treatment technology is needed to reduce public health risks. This study evaluated the inactivation of HAV and the NoV research surrogate, murine norovirus-1 (MNV-1), in oysters ( Crassostrea virginica ) by electron beam (E-beam) irradiation. The reduction of potential infection risks was quantified for E-beam irradiation technology employed on raw oysters at various virus contamination levels. The E-beam dose required to reduce the MNV and HAV titer by 90% (D 10 value) in whole oysters was 4.05 (standard deviations [SD], �0.63) and 4.83 (SD, �0.08) kGy, respectively. Microbial risk assessment suggests that if a typical serving of 12 raw oysters was contaminated with 10 5 PFU, a 5-kGy treatment would achieve a 12% reduction (from 4.49 out of 10 persons to 3.95 out of 10 persons) in NoV infection and a 16% reduction (from 9.21 out of 10 persons to 7.76 out of 10 persons) in HAV infections. If the serving size contained only 10 2 PFU of viruses, a 5-kGy treatment would achieve a 26% reduction (2.74 out of 10 persons to 2.03 out of 10 persons) of NoV and 91% reduction (2.1 out of 10 persons to 1.93 out of 100 persons) of HAV infection risks. This study shows that although E-beam processing cannot completely eliminate the risk of viral illness, infection risks can be reduced.

The prevalence of fluoroquinolone-resistant enteric bacteria in agricultural and municipal waste streams was investigated by surveying for resistant Escherichia coli in samples collected from nine locations in the United States and one location in Mexico. None of the 102 isolates that were tested were resistant to either ciprofloxacin or ofloxacin. Microcosm studies using a clinical (ciprofloxacin resistant) Pseudomonas aeruginosa isolate suggest that the resistance phenotype is stable over multiple generations in the absence of selective antibiotic pressure.

Background E. coli O157:H7 (EHEC) is an important human pathogen. The antibiotic treatment of EHEC reportedly results in release of Shiga toxin and is therefore discouraged. Consequently, alternative preventive or therapeutic strategies for EHEC are required. The objective of the current study was to investigate the effect of citrus limonoids on cell-cell signaling, biofilm formation and type III secretion system in EHEC. Results Isolimonic acid and ichangin were the most potent inhibitors of EHEC biofilm (IC25=19.7 and 28.3 μM, respectively) and adhesion to Caco-2 cells. The qPCR analysis revealed that isolimonic acid and ichangin repressed LEE encoded genes by ≈3 to 12 fold. In addition, flhDC was repressed by the two limonoids (≈3 to 7 fold). Further studies suggested that isolimonic acid interferes with AI-3/epinephrine activated cell-cell signaling pathway. Loss of biofilm inhibitory activity of isolimonic acid in ΔqseBC mutant, which could be restored upon complementation, suggested a dependence on functional QseBC. Additionally, overexpression of qseBC in wild type EHEC abated the inhibitory effect of isolimonic acid. Furthermore, the isolimonic acid failed to differentially regulate ler in ΔqseA mutant, while plasmid borne expression of qseA in ΔqseA background restored the repressive effect of isolimonic acid. Conclusions Altogether, results of study seem to suggest that isolimonic acid and ichangin are potent inhibitors of EHEC biofilm and TTSS. Furthermore, isolimonic acid appears to interfere with AI-3/epinephrine pathway in QseBC and QseA dependent fashion.

The present study was designed to evaluate cycloheximide as a potential media amendment for differential bacterial and fungal enumeration of animal feeds. The objectives of this study were to determine the effect of cycloheximide on bacterial growth rates and to evaluate its efficacy for the reduction of indigenous spreading fungi when enumerating bacterial populations in three types of feeds and after short or long-term storage. Escherichia coli, Bacillus cereus, Staphylococcus aureus, Listeria monocytogenes and Pseudomonas fluorescens were grown in tryptic soy broth containing cycloheximide to determine its effect on bacterial specific growth rates. Growth rates of B. cereus and S. aureus were significantly decreased by the addition of 600 and 1000 mg/L cycloheximide respectively, but other pure cultures were not significantly influenced by cycloheximide addition. Intrinsic bacterial populations from feed were not significantly affected by cycloheximide additions at concentrations from 10 to 300 mg/L, but the indigenous spreading molds from feeds were significantly decreased by these cycloheximide concentrations and were decreased below detection levels by 300 mg/L of cycloheximide. The addition of 300 mg/L of cycloheximide effectively eliminates fungal growth for accurate enumeration of bacterial populations in feeds.

The present study was designed to evaluate cycloheximide as a potential media amendment to prevent fungal overgrowth on selective media for salmonellae enumeration. The objectives were to determine the effect of cycloheximide on Salmonella spp growth rates and to determine the effect of cycloheximide addition on Salmonella enumeration in selective media. The bacteria tested included two strains of Salmonella typhimurium (NO/NA and LT2) and one strain of Salmonella arizonae. All strains were grown in tryptic soy broth containing cycloheximide to determine the effect of cycloheximide on bacterial specific growth rates. The growth rate of all strains grown in tryptic soy broth were not significantly influenced by addition of cycloheximide at concentrations up to 1,000 mg/L. Growth rates of S. typhimurium NO/NA in minimal media were significantly decreased by addition of cycloheximide aerobically (300 mg/L) and anaerobically (600 mg/L). However, S. typhimurium NO/NA populations on brilliant green agar, MacConkey agar, and from selenite cysteine broth and tetrathionate broth were not affected by cycloheximide additions at concentrations up to 1,000 mg/L. Cycloheximide has potential as a fungistat additive for salmonellae selective media.

Traditionally, microbiological testing of meat products has involved isolating microorganisms and performing specific biochemical, and in some cases serological, tests to confirm the presence or absence of suspected food-borne pathogens. Given the public attention meat products have received as sources of food-borne disease, there has been considerable interest in the application of rapid detection techniques that require hours rather than days for completion. Theoretically, rapid detection methods could reduce the time from the initial sampling to confirmation so that conclusive results would be available by the time to process the meat product. Both direct gene probe hybridization as well as gene amplification methods show promise as rapid detection techniques. At present, direct gene probe hybridization are being commercially utilized to confirm the presence of a suspected pathogen. A number of gene amplification protocols for detecting food-borne bacterial pathogens have been published. However, many of these studies have utilized spiked samples rather than naturally contaminated samples and many of them have involved extended template extraction/purification methodologies. There is still only a very limited amount of information on the efficacies of the various protocols in detecting bacterial pathogens, especially toxigenic Escherichia coli, Salmonella spp., Campylobacter spp., and Listeria spp., in naturally contaminated food samples. In order to develop gene amplification protocols that have relevance to the meat industry, there must be a concerted effort to utilize naturally contaminated samples in the development and evaluation of protocols, as well as to initiate multilaboratory round robin evaluations of select protocols. Availability of multilaboratory tested methodologies would provide a means to design pathogen detection strategies at the quality control level rather than an end product confirmatory response to an already documented outbreak.

The objectives of this study were to screen the effect of different clinoptilolite compounds on S. typhimurium survival and to compare the most effective compounds as amendments using agricultural soil and aqueous microcosm conditions. These microcosms were inoculated with the bacterial culture and at periodic intervals (over 12–30 days) viable Salmonella populations were enumerated using selective media. In screening studies Salmonella populations were found to be lower from the smaller mesh clinoptilolite compounds than from wood-chip sources or larger mesh clinoptilolite compounds at the end of 14 days. However, the response was highly correlated with the change in moisture content. In soil microcosm studies where moisture content was kept constant, there was an overall reduction of about 4 log units but there was no significant difference between the unamended control and the treatments. When smaller mesh clinoptilolites were evaluated in phosphate saline buffer there was a significantly lower (P < 0·05) number of viable S. typhimurium than in the unamended treatments. However, when the clinoptilolite compounds were sterilized prior to use, it was observed that the bacterial populations were protected from viability loss when compared with the unamended control.

Obacunone belongs to a class of unique triterpenoids called limonoids, present in Citrus species. Previous studies from our laboratory suggested that obacunone possesses antivirulence activity and demonstrates inhibition of cell-cell signaling in Vibrio harveyi and Escherichia coli O157:H7. The present work sought to determine the effect of obacunone on the food-borne pathogen Salmonella enterica serovar Typhimurium LT2 by using a cDNA microarray. Transcriptomic studies indicated that obacunone represses Salmonella pathogenicity island 1 (SPI1), the maltose transporter, and the hydrogenase operon. Furthermore, phenotypic data for the Caco-2 infection assay and maltose utilization were in agreement with microarray data suggesting repression of SPI1 and maltose transport. Further studies demonstrated that repression of SPI1 was plausibly mediated through hilA . Additionally, obacunone seems to repress SPI2 under SPI2-inducing conditions as well as in Caco-2 infection models. Furthermore, obacunone seems to repress hilA in an EnvZ-dependent fashion. Altogether, the results of the study seems to suggest that obacunone exerts an antivirulence effect on S. Typhimurium and may serve as a lead compound for development of antivirulence strategies for S. Typhimurium.

Planetary and sample return missions require surface cleaning procedures using biocides. However, a deep understanding of how microorganisms respond to biocides is needed. The response of different Salmonella serovars to defined concentrations of chlorine, chlorine dioxide, and cetylpyridinium chloride (CPC) was studied. Microbial responses in terms of culturability, and virulence gene expression were also evaluated. Inactivation when exposed to chlorine and CPC was extremely rapid. Inactivation due to chlorine dioxide was, however, gradual. Exposure to these biocides caused a number of physiological states in Salmonella cells including dormancy, injury, and a viable but non-culturable state. Based on real-time PCR assays, all virulence-related genes were down-regulated in the presence of chlorine, chlorine dioxide and CPC. These studies highlight the importance of understanding the possible responses of organisms to biocides before they are used in mission critical applications such as pre-flight cleaning and laboratory protocols involved in sample return missions.

Colloids on the nanometer size scale are beginning to find increased applications in drinks, foods, food-contact surfaces, and food packaging. While these particles add intrinsic value to the food industry, their potential toxicities warrant careful studies. The physicochemical changes and possible perturbations to microbial communities within the gastrointestinal tract have not been adequately studied. The purpose of this study was to design and perform a simulated digestion protocol to evaluate the effect of colloidal silver in an orange juice suspension when exposed to planktonic bacterial cultures and biofilms. The model system includes four precursor steps in which the silver is exposed to varying pH conditions and incubation times. The gastrointestinally “digested” samples were then incubated with Escherichia coli strains for up to 4 h, the average residence time of foods in the GI tract. The physicochemical changes of the colloids and their corresponding biological effects were characterized at every step. The results showed differences between (1) bacterial cultures versus bacterial biofilms, (2) “digested” versus “undigested” silver on bacteria, and (3) differences between “digested” silver nitrate versus silver colloids on bacteria. We conclude that simulated digestion, as well as manner in which bacterial cells are grown, influences the results of toxicity.