[Show abstract][Hide abstract] ABSTRACT: The spread of antibiotic-resistant pathogens requires new treatments. As the rate of development of new antibiotics has severely declined, alternatives to antibiotics must be considered in both animal agriculture and human medicine. Products for disease prevention are different from those for disease treatment, and examples of both are discussed here. For example, modulating the gut microbial community, either through feed additives or fecal transplantation, could be a promising way to prevent certain diseases; for disease treatment, non-antibiotic approaches include phage therapy, phage lysins, bacteriocins, and predatory bacteria. Interestingly, several of these methods augment antibiotic efficacy by improving bacterial killing and decreasing antibiotic resistance selection. Because bacteria can ultimately evolve resistance to almost any therapeutic agent, it is important to continue to use both antibiotics and their alternatives judiciously.
Annals of the New York Academy of Sciences 06/2014; · 4.38 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: In recent studies, we demonstrated that a deletion of hha caused increased secretion of locus of enterocyte encoded adherence proteins and reduced motility of enterohemorrhagic Escherichia coli (EHEC) O157:H7. In addition to the importance of hha in positive regulation of motility, a two-component quorum sensing pathway encoded by the qseBC genes has been shown to activate bacterial motility in response to mammalian stress hormones epinephrine and norepinephrine as well as bacterially produced autoinducer-3. In this study, we compared regulatory contribution and hierarchy of hha, a member of the Hha/YmoA family of nucleoid-associated proteins, to that of qseBC in the expression of EHEC O157:H7 motility. Since norepinephrine affects motility of EHEC O157:H7 through a qseBC-encoded two-component quorum sensing signaling, we also determined whether the hha-mediated regulation of motility is affected by norepinephrine and whether this effect is qseBC dependent. We used single (Δhha or ΔqseC) and double (Δhha ΔqseC) deletion mutants to show that hha exerts a greater positive regulatory effect in comparison to qseBC on the expression of motility by EHEC O157:H7. We also show that Hha is hierarchically superior in transcriptional regulation of motility than QseBC because transcription of qseC was significantly reduced in the hha deletion mutant compared to that in the parental and the hha-complemented mutant strains. These results suggest that hha regulates motility of EHEC O157:H7 directly as well as indirectly by controlling the transcription of qseBC.
PLoS ONE 01/2014; 9(1):e85866. · 3.53 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: The qseBC encoded quorum-sensing system regulates motility of Escherichia coli O157:H7 in response to bacterial autoinducer-3 (AI-3) and mammalian stress hormones epinephrine (E) and norepinephrine (NE). The qseC gene encodes a sensory kinase that auto-phosphorylates in response to AI-3, E or NE and subsequently phosphorylates its cognate response regulator QseB. In the absence of QseC, QseB down-regulates bacterial motility and virulence in animal models. In this study, we found that eight to ten months old calves orally inoculated with a mixture of the E. coli O157:H7 and its isogenic qseBC mutant showed significantly higher fecal shedding of the qseBC mutant. In vitro analysis revealed similar growth profiles and motilities of the qseBC mutant and the parental strain in the presence or absence of NE. The magnitude of the response to NE and expression of flagellar genes flhD and fliC were also similar between the qseBC mutant and the parental strain. The expression of ler, a positive regulator of locus of enterocyte effacement (LEE), the ler-regulated espA, and the csgA gene encoding curli fimbriae were increased in the qseBC mutant compared to the parental strain. On the other hand, growth, motility, and transcription of flhD, fliC, ler, espA, and csgA were significantly reduced in the qseBC mutant complemented with a plasmid-cloned copy of qseBC genes. Thus, in vitro motility and gene expression data indicate that the near-parental level of motility, ability to respond to NE, and enhanced expression of LEE and curli genes might in part be responsible for increased colonization and fecal shedding of the qseBC mutant in calves.
Applied and Environmental Microbiology 01/2014; · 3.95 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Antibiotics are used in livestock and poultry production to treat and prevent disease as well as to promote animal growth. Carbadox is an in-feed antibiotic that is widely used in swine production to prevent dysentery and to improve feed efficiency. The goal of this study was to characterize the effects of carbadox and its withdrawal on the swine gut microbiota. Six pigs (initially 3-weeks old) received feed containing carbadox and six received unamended feed. After 3-weeks of continuous carbadox administration, all pigs were switched to a maintenance diet without carbadox. DNA was extracted from feces (n = 142) taken before, during, and following (6-week withdrawal) carbadox treatment. Phylotype analysis using 16S rRNA sequences showed the gradual development of the non-medicated swine gut microbiota over the 8-week study, and that the carbadox-treated pigs had significant differences in bacterial membership relative to non-medicated pigs. Enumeration of fecal Escherichia coli showed that a diet change concurrent with carbadox withdrawal was associated with an increase in the E. coli in the non-medicated pigs, suggesting that carbadox pre-treatment prevented an increase of E. coli populations. In-feed carbadox caused striking effects within 4 days of administration, with significant alterations in both community structure and bacterial membership, notably a large relative increase in Prevotella populations in medicated pigs. Digital PCR was used to show that the absolute abundance of Prevotella was unchanged between the medicated and non-medicated pigs despite the relative increase shown in the phylotype analysis. Carbadox therefore caused a decrease in the abundance of other gut bacteria but did not affect the absolute abundance of Prevotella. The pending regulation on antibiotics used in animal production underscores the importance of understanding how they modulate the microbiota and impact animal health, which will inform the search for antibiotic alternatives.
[Show abstract][Hide abstract] ABSTRACT: Alternatives to antibiotics are urgently needed in animal agriculture. The form these alternatives should take presents a complex problem due to the various uses of antibiotics in animal agriculture, including disease treatment, disease prevention, and growth promotion, and to the relative contribution of these uses to the antibiotic resistance problem. Numerous antibiotic alternatives, such as pre- and probiotics, have been proposed but show variable success. This is because a fundamental understanding of how antibiotics improve feed efficiency is lacking, and because an individual alternative is unlikely to embody all of the performance-enhancing functions of antibiotics. High-throughput technologies need to be applied to better understand the problem, and informed combinations of alternatives, including vaccines, need to be considered.
Trends in Microbiology 03/2013; 21(3):114-9. · 8.43 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Type II heat-labile enterotoxins (LT-II) have been reported in Escherichia coli isolates from humans, animals, food and water samples. The goal here was to determine the specific roles of the antigenically distinguishable LT-IIa and LT-IIb subtypes in pathogenesis and virulence of enterotoxigenic E. coli (ETEC) which has not been previously reported. The prevalence of genes encoding for LT-II was determined by colony blot hybridization in a collection of 1648 E. coli isolates from calves and pigs with diarrhea or other diseases and from healthy animals. Only five isolates hybridized with the LT-II probe and none of these isolates contained genes for other enterotoxins or adhesins associated with porcine or bovine ETEC. Ligated intestinal loops in calves, pigs, and rabbits were used to determine the potential of purified LT-IIa and LT-IIb to cause intestinal secretion. LT-IIa and LT-IIb caused significant secretion in the intestinal loops in calves but not in the intestinal loops of rabbits or pigs. In contrast, neonatal pigs inoculated with isogenic adherent E. coli containing the cloned genes for LT-I, LT-IIa or LT-IIb developed severe watery diarrhea with weight loss that was significantly greater than pigs inoculated with the adherent, non-toxigenic parental or vector only control strains. The results demonstrate that the incidence of LT-II appeared to be very low in porcine and bovine E. coli. However, a potential role for these enterotoxins in E. coli-mediated diarrhea in animals was confirmed because purified LT-IIa and LT-IIb caused fluid secretion in bovine intestinal loops and adherent isogenic strains containing cloned genes encoding for LT-IIa or LT-IIb caused severe diarrhea in neonatal pigs.
[Show abstract][Hide abstract] ABSTRACT: Enterotoxigenic Escherichia coli (ETEC) strains expressing K88 (F4) or F18 fimbriae and heat-labile (LT) and/or heat-stable (ST) toxins are the major cause of diarrhea in young pigs. Effective vaccines inducing antiadhesin (anti-K88 and anti-F18) and antitoxin (anti-LT and anti-ST) immunity would provide broad protection to young pigs against ETEC. In this study, we genetically fused nucleotides coding for peptides from K88ac major subunit FaeG, F18 minor subunit FedF, and LT toxoid (LT(192)) A2 and B subunits for a tripartite adhesin-adhesin-toxoid fusion (FaeG-FedF-LT(192)A2:B). This fusion was used for immunizations in mice and pigs to assess the induction of antiadhesin and antitoxin antibodies. In addition, protection by the elicited antiadhesin and antitoxin antibodies against a porcine ETEC strain was evaluated in a gnotobiotic piglet challenge model. The data showed that this FaeG-FedF-LT(192)A2:B fusion elicited anti-K88, anti-F18, and anti-LT antibodies in immunized mice and pigs. In addition, the anti-porcine antibodies elicited neutralized cholera toxin and inhibited adherence against both K88 and F18 fimbriae. Moreover, immunized piglets were protected when challenged with ETEC strain 30302 (K88ac/LT/STb) and did not develop clinical disease. In contrast, all control nonvaccinated piglets developed severe diarrhea and dehydration after being challenged with the same ETEC strain. This study clearly demonstrated that this FaeG-FedF-LT(192)A2:B fusion antigen elicited antibodies that neutralized LT toxin and inhibited the adherence of K88 and F18 fimbrial E. coli strains and that this fusion could serve as an antigen for vaccines against porcine ETEC diarrhea. In addition, the adhesin-toxoid fusion approach used in this study may provide important information for developing effective vaccines against human ETEC diarrhea.
[Show abstract][Hide abstract] ABSTRACT: Escherichia coli O157:H7 colonizes cattle intestines by using the locus of enterocyte effacement (LEE)-encoded proteins. The induction of systemic immune response against LEE-encoded proteins, therefore, will prove effective in reducing E. coli O157:H7 colonization in cattle. The previous studies have demonstrated that a hha (encodes for a hemolysin expression modulating protein) deletion enhances expression of LEE-encoded proteins and a sepB (encodes an ATPase required for the secretion of LEE-encoded proteins) deletion results in intracellular accumulation of LEE proteins. In this study, we demonstrate the efficacy of the hha and hha sepB deletion mutants as bacterins for reducing fecal shedding of E. coli O157:H7 in experimentally inoculated weaned calves. The weaned calves were injected intramuscularly with the bacterins containing 10(9) heat-killed cells of the hha(+) wild-type or hha or hha sepB isogenic mutants, and boosted with the same doses 2- and 4-weeks later. The evaluation of the immune response two weeks after the last booster immunization revealed that the calves vaccinated with the hha mutant bacterin had higher antibody titers against LEE proteins compared to the titers for these antibodies in the calves vaccinated with the hha sepB mutant or hha(+) wild-type bacterins. Following oral inoculations with 10(10) CFU of the wild-type E. coli O157:H7, the greater numbers of calves in the group vaccinated with the hha or hha sepB mutant bacterins stopped shedding the inoculum strain within a few days after the inoculations compared to the group of calves vaccinated with the hha(+) wild-type bacterin or PBS sham vaccine. Thus, the use of bacterins prepared from the hha and hha sepB mutants for reducing colonization of E. coli O157:H7 in cattle could represent a potentially important pre-harvest strategy to enhance post-harvest safety of bovine food products, water and produce.
[Show abstract][Hide abstract] ABSTRACT: The feasibility of exploiting fluorescence spectra of the eye for diagnosis of transmissible spongiform encephalopathies (TSEs) was examined. Retinas from scrapie-positive sheep were compared with scrapie-negative sheep using fluorescence spectroscopy, and distinct differences in the fluorescence intensity and spectroscopic signatures were observed. The characteristic fluorescent signatures are thought to be the result of an accumulation of lipofuscin in the retina. It appears that the eye, in particular the retina, is a useful tissue for noninvasive examination of some neurological pathologies such as scrapie. The development of procedures based on examinations of the eye that permit the detection of neurological disorders in animals holds great promise.
[Show abstract][Hide abstract] ABSTRACT: Applications of fluorescence spectroscopy that enable the real-time or rapid detection of fecal contamination on beef carcasses and the presence of central nervous system tissue in meat products are discussed. The former is achieved by employing spectroscopic signatures of chlorophyll metabolites; the latter, by exploiting the characteristic structure and intensity of lipofuscin in central nervous system tissue. The success of these techniques has led us to investigate the possibility of diagnosing scrapie in sheep by obtaining fluorescence spectra of the retina. Crucial to this diagnosis is the ability to obtain baseline correlations of lipofuscin fluorescence with age. A murine model was employed as a proof of principle of this correlation.
[Show abstract][Hide abstract] ABSTRACT: We describe a comparison of the fluorescence spectra of bovine tissues with murine tissues in order to determine whether spectral features are conserved and whether an appropriate and practical laboratory small animal model system could be identified to be used for investigation of tissue- and age-related fluorescence signal patterns. Recently it has been shown that spectral signatures of lipofuscin have enabled the detection of bovine central nervous system (CNS) tissue in meat products with high sensitivity (Schönenbrücher, H., Adhikary, R., Mukherjee, P., Casey, T.A., Rasmussen, M.A., Maistrovich, F.D., Hamir, A.N., Kehrli, M.J., Richt, J., Petrich, J.W.  J Agric Food Chem56, 6220-6226). We report that brain and spinal cord of mice provide fluorescence spectra similar to those of bovine brain and spinal cord. It is concluded that murine CNS tissue is an appropriate model system for bovine CNS tissue for the development of fluorometric CNS detection assays.
Photochemistry and Photobiology 08/2009; 85(6):1322-6. · 2.29 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Micro-organisms may simultaneously encounter multiple stresses in their environment. To investigate the protection that several known Escherichia coli O157 : H7 acid-resistance systems might provide against both oxidative and acid stress, the addition of diamide, a membrane-permeable thiol-specific oxidizing agent, or hydrogen peroxide were used concurrent with acid challenge at pH 2.5 to determine bacterial survival. The addition of either diamide or hydrogen peroxide decreased bacterial survival in a dose-dependent manner for E. coli O157 : H7 during challenge at pH 2.5 following overnight growth in LB MES pH 5.5 (acid-resistance system 1, AR1). In contrast, the presence of either glutamate or arginine during challenge provided significant protection against diamide- and hydrogen peroxide-induced oxidative stress during pH 2.5 acid challenge. Oxidative stress protection during acid challenge required gadC and adiA for the glutamate- (AR2) and arginine- (AR3) dependent acid-resistance systems, respectively. In addition, maximal protection against oxidative stress in the presence of glutamate required a low external pH (pH 2.5), since pH 5.5 did not protect. This study demonstrates that the glutamate- and arginine-dependent acid-resistance systems of E. coli O157 : H7 can simultaneously protect against oxidative stress during extreme acid challenge.
[Show abstract][Hide abstract] ABSTRACT: A multiplex polymerase chain reaction (mPCR) assay was developed for detection and characterization of pathogenic Escherichia coli that cause diarrhea and edema disease in swine. The mPCR assay was designed as a single reaction for detecting 5 different adhesins (K88, K99, 987P, F41, and F18), 3 enterotoxins (LT, STaP, and STb), and the Shiga toxin (Stx2e) associated with porcine pathogenic E. coli. The specificity of the mPCR assay was evaluated by comparison with results from previous analysis of 100 porcine isolates characterized by colony blot hybridization with DNA probes for the 5 adhesins and 4 toxin genes. There was complete agreement between the 2 methods. The mPCR assay for E. coli pathogens isolated from swine was further evaluated by examination of strains containing virulence factors that are known to have different antigenic subtypes or DNA sequence variations. It was found that the mPCR assays targeting genes encoding for K88 and F18 amplified products with the appropriate sizes from strains containing genes for different K88 and F18 antigenic subtypes; mPCR assays targeting the gene encoding for STaP amplified product from only STaP-positive but not STaH-positive isolates; and mPCR assays targeting the gene encoding for the Stx2 amplified products from only Stx2-positive and not Stx1-positive isolates. Similarly, mPCR assays targeting the gene encoding for LTI did not produce the appropriate product from strains containing genes for LTII. The mPCR assays are simple to perform, and they should be useful for diagnosis of porcine colibacillosis, including the genotypic characterization of E. coli isolates from pigs with diarrhea or edema disease.
Journal of veterinary diagnostic investigation: official publication of the American Association of Veterinary Laboratory Diagnosticians, Inc 02/2009; 21(1):25-30. · 1.18 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: The integrated fluorescence of murine eyes is collected as a function of age. This fluorescence is attributed to pigments generally referred to as lipofuscin and is observed to increase with age. No difference in fluorescence intensity is observed between the eyes of males or females. This work provides a benchmark for further studies that are planned in order to use such signatures as markers of central nervous system (CNS) tissue or even of diseased CNS tissue and provides a basis for determining the age of a healthy animal.
Photochemistry and Photobiology 09/2008; 85(1):234-8. · 2.29 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: The removal of central nervous system (CNS) tissues as part of bovine spongiform encephalopathy (BSE) risk material is one of the highest priority tasks to avoid contamination of the human food chain with BSE. No currently available method enables the real-time detection of possible CNS tissue contamination on carcasses during slaughter. The fluorescent pigment lipofuscin is a heterogeneous, high-molecular weight material that has been shown to be enriched in high concentrations in neuronal tissues. In this study, lipofuscin fluorescence was investigated as a marker for real-time detection of CNS contamination. Front-faced fluorescence spectra of brain and spinal cord samples from 11 cattle gave identical, reproducible fluorescence signal patterns with high intensities. The specificity of these spectra was assessed by investigating 13 different non-CNS tissues enabling the differentiation of brain and spinal cord by signal intensity and structure of the spectra, respectively. Small quantities of bovine spinal cord were reliably detected in the presence of raw bovine skeletal muscle, fat, and vertebrae. The presented data are a fundamental basis for the development of a prototype device allowing real-time monitoring of CNS tissue contamination on bovine carcasses and meat cuts.
Journal of Agricultural and Food Chemistry 08/2008; 56(15):6220-6. · 3.11 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Although cattle develop humoral immune responses to Shiga-toxigenic (Stx+) Escherichia coli O157:H7, infections often result in long-term shedding of these human pathogenic bacteria. The objective of this study was to compare humoral and cellular immune responses to Stx+ and Stx- E. coli O157:H7. Three groups of calves were inoculated intrarumenally, twice in a 3-week interval, with different strains of E. coli: a Stx2-producing E. coli O157:H7 strain (Stx2+ O157), a Shiga toxin-negative E. coli O157:H7 strain (Stx- O157), or a nonpathogenic E. coli strain (control). Fecal shedding of Stx2+ O157 was significantly higher than that of Stx- O157 or the control. Three weeks after the second inoculation, all calves were challenged with Stx2+ O157. Following the challenge, levels of fecal shedding of Stx2+ O157 were similar in all three groups. Both groups inoculated with an O157 strain developed antibodies to O157 LPS. Calves initially inoculated with Stx- O157, but not those inoculated with Stx2+ O157, developed statistically significant lymphoproliferative responses to heat-killed Stx2+ O157. These results provide evidence that infections with STEC can suppress the development of specific cellular immune responses in cattle, a finding that will need to be addressed in designing vaccines against E. coli O157:H7 infections in cattle.
Clinical and Vaccine Immunology 01/2007; 13(12):1322-7. · 2.60 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Enterohemorrhagic Escherichia coli (EHEC) O157:H7 virulence factors, specifically those conferring intimate adherence to and formation of attaching and effacing lesions (A/E) on host cells, are encoded by a horizontally acquired locus of enterocyte effacement (LEE). Expression of several LEE-encoded genes, which are organized into operons LEE1 through LEE5, is under the positive regulation of ler, the first gene in the LEE1 operon. We have recently demonstrated that EHEC O157:H7 lacking hha exhibited greater than a 10-fold increase in ler expression and that the repression of ler results from the binding of Hha to the ler promoter. In this report, we show that an hha mutant of EHEC O157:H7 exhibited increased adherence to Hep-2 cells, had increased transcriptional activities of LEE1, LEE2, LEE3, and LEE5 as determined by reverse transcriptase-polymerase chain reaction assays, and expressed LEE5::lac transcriptional fusion at levels that were several-fold higher than that expressed by the parental hha+ strain. These results demonstrate that hha is an important regulatory component of the cascade that governs the expression of LEE operons and the resulting ability of EHEC O157:H7 to intimately adhere to host cells.
[Show abstract][Hide abstract] ABSTRACT: Lipofuscin is a yellow-brown, highly fluorescent pigment that undergoes an age-related progressive accumulation in animal cells, mainly in postmitotic cells. It is a heterogeneous, high-molecular weight material associated with proteins, lipids and nucleic acids. Lipofuscin is implicated in many aspects of human health, including aging, oxidative stress, macular degeneration, lipid peroxidation, atherosclerosis, dementia (Alzheimer's Disease) and diseases associated with prions. Although the fluorescent properties of lipofuscin have long been recognized, neither histologists nor chemists have yet isolated the pigments themselves or characterized their optical properties. We have prepared lipofuscinlike species by reacting malondialdehyde (MDA) with cysteine (Cys). MDA:Cys adducts 3:2 and 2:2 are two of those that have been identified among the many that were present in the reaction. Whereas previous attempts to synthesize lipofuscinlike species resulted in compounds that were either nonfluorescent or emitted principally in the blue, the MDA:Cys adducts reported in this study are not only fluorescent but also emit over a broader range.
Photochemistry and Photobiology 02/2004; 79(1):21-5. · 2.29 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Digestion of green plants in the gastrointestinal (GI) tract produces degradation products from chlorophyll that cause ingesta and feces to be highly fluorescent. This property was exploited for development and construction of instruments to noninvasively detect minute quantities of feces on meat samples in real time. The presence of feces on meat products is a primary source of foodborne pathogens, such as Escherichia coli O157:H7 and Salmonella. This new technology provides a rapid and accurate alternative to the practice of visual inspection and augments more time-consuming biological testing methods. This innovation can assist meat processors and government inspectors in their efforts to provide safe and wholesome food to consumers.
Journal of Agricultural and Food Chemistry 06/2003; 51(11):3502-7. · 3.11 Impact Factor