Development and in vitro evaluation of an Escherichia coli probiotic able to inhibit the growth of pathogenic E. coli K88

Department of Animal Science, University of Manitoba, Winnipeg, Manitoba R3T 2N2, Canada.
Journal of Animal Science (Impact Factor: 2.11). 04/2009; 87(6):2005-12. DOI: 10.2527/jas.2008-1400
Source: PubMed


Weaned piglets commonly suffer from gastroenteritis caused by enterotoxigenic Escherichia coli K88. Our aim was to produce E. coli strains that inhibited the growth of E. coli K88 and could be used as a probiotic against postweaning diarrhea. The inclusion criteria for the probiotics were that in addition to being able to inhibit E. coli K88, they also needed to be negative for virulence genes commonly associated with E. coli. A total of 463 E. coli isolates from the cattle rumen, cattle feces, swine feces, and soil were screened against 18 E. coli K88 clinical isolates using an agar diffusion technique. Growth inhibition of the most sensitive K88 indicator strain 2-12 occurred for 121/463 isolates: 96/358 from cattle feces, 0/33 from rumen fluid, 9/35 from swine feces, and 16/37 from soil. Of the 121 positive strains, 71/121 were negative for toxin genes (LT, STa, STb, VT1, and VT2). The 14 most inhibitory strains were screened against a range of substrates to assess the ability to utilize carbohydrates that could be included in the diet to enhance their ability to compete in the gut. Two strains, UM-2 and UM-7, were weak utilizers of starch and inulin. In vitro competition assays between the probiotic strains and E. coli K88 strain 2-12 were conducted with glucose as the only carbon source (minimal medium; MM), MM + 2% starch, or MM + 2% inulin. The UM-2 and UM-7 strains were able to outcompete strain 2-12 when glucose was the only carbon source, indicating that inhibitory activity was produced against 2-12 independent of carbon source. The UM-2 strain outcompeted strain 2-12 in assays in which potato starch or inulin was the only carbon source; the ability of 2-12 to maintain its concentrations in the culture were probably the result of cross feeding of breakdown sugars of starch and inulin that could be utilized by 2-12. In contrast, UM-7 did not grow as well as UM-2 on starch and inulin and 2-12 declined rapidly in successive cultures likely because of the lack of breakdown products of starch and inulin produced by UM-7. We conclude that probiotic E. coli without known toxins and that produce inhibitory activity against E. coli that cause postweaning diarrhea can be produced. In addition, the ability to utilize starch or inulin is an important phenotype because it likely gives the probiotic a competitive advantage in the gut.

Download full-text


Available from: James Duncan House,
  • Source
    • "Probiotics can also compete with other micro-organisms for binding to specific receptors on host epithelial cells, thereby preventing potential pathogen invasion (Mukai et al., 2002; Setia et al., 2009). Transient colonization of the GIT by Bifidobacteria have been previously demonstrated as early as 1 week after supplementation (Langhendries et al., 1995) indicating that these effects can be Table 1 | Characteristics of an ideal probiotic. "
    [Show abstract] [Hide abstract]
    ABSTRACT: The prevalence of allergic disease has increased dramatically in Western countries over the past few decades. The hygiene hypothesis, whereby reduced exposure to microbial stimuli in early life programs the immune system toward a Th2-type allergic response, is suggested to be a major mechanism to explain this phenomenon in developed populations. Such microbial exposures are recognized to be critical regulators of intestinal microbiota development. Furthermore, intestinal microbiota has an important role in signaling to the developing mucosal immune system. Intestinal dysbiosis has been shown to precede the onset of clinical allergy, possibly through altered immune regulation. Existing treatments for allergic diseases such as eczema, asthma, and food allergy are limited and so the focus has been to identify alternative treatment or preventive strategies. Over the past 10 years, a number of clinical studies have investigated the potential of probiotic bacteria to ameliorate the pathological features of allergic disease. This novel approach has stemmed from numerous data reporting the pleiotropic effects of probiotics that include immunomodulation, restoration of intestinal dysbiosis as well as maintaining epithelial barrier integrity. In this mini-review, the emerging role of probiotics in the prevention and/or treatment of allergic disease are discussed with a focus on the evidence from animal and human studies.
    Frontiers in Pharmacology 09/2012; 3:171. DOI:10.3389/fphar.2012.00171 · 3.80 Impact Factor
  • Source
    • "Beneficial effects of probiotics are also mediated via production of microbicidal substances such as bacteriosins and organic acids that act against pathogens (Juven et al., 1991; Gibson and Wang, 1994b; Li et al., 2003). Setia et al. (2009) reported that colicin-producing E. coli exhibited inhibitory activities against ETEC in an in vitro assay. Probiotic bacteria increased the production of short chain fatty acids in an in vitro study (Sakata et al., 2003), and these may help to reduce digesta pH and subsequently depress the growth of pathogenic bacteria (Gibson, 1999). "
    [Show abstract] [Hide abstract]
    ABSTRACT: For the last several decades, antimicrobial compounds have been used to promote piglet growth at weaning through the prevention of subclinical and clinical disease. There are, however, increasing concerns in relation to the development of antibiotic-resistant bacterial strains and the potential of these and associated resistance genes to impact on human health. As a consequence, European Union (EU) banned the use of antibiotics as growth promoters in swine and livestock production on 1 January 2006. Furthermore, minerals such as zinc (Zn) and copper (Cu) are not feasible alternatives/replacements to antibiotics because their excretion is a possible threat to the environment. Consequently, there is a need to develop feeding programs to serve as a means for controlling problems associated with the weaning transition without using antimicrobial compounds. This review, therefore, is focused on some of nutritional strategies that are known to improve structure and function of gastrointestinal tract and (or) promote post-weaning growth with special emphasis on probiotics, prebiotics, organic acids, trace minerals and dietary protein source and level.
    J Anim Physiol a Anim Nutr 03/2012; 97(2). DOI:10.1111/j.1439-0396.2012.01284.x · 1.41 Impact Factor
  • Source
    • "To evaluate the proliferation of ETEC and to differentiate the inoculum from the indigenous strains, the pure ETEC strain was made resistant to ciprofloxacin by exposing it to increasing doses of ciprofloxacin in Müller-Hinton broth (Becton, Dickinson and Company, Sparks, MD) as described by Opapeju et al. (2009). Before being used to challenge pigs, the ciprofloxacin-resistant ETEC was confirmed to be positive for K88 fimbrial antigen, heatlabile enterotoxin, and heat-stable enterotoxin genes by PCR genotyping using published primers (Kotlowski et al., 2007; Setia et al., 2009). Each pig was orally challenged with 6 mL (2 × 10 9 cfu/mL) of the freshly grown ETEC inoculants via a polyethylene tube attached to a syringe placed in the back of the oral cavity. "
    [Show abstract] [Hide abstract]
    ABSTRACT: Lysozyme is a low-molecular-weight protein with antimicrobial properties. An experiment was conducted to investigate the response of piglets receiving a water-soluble lysozyme supplement [Entegard (EG), Neova Technologies Inc., Abbotsford, British Columbia, Canada; 4,000 lysozyme units/mg] after oral challenge with enterotoxigenic Escherichia coli (ETEC). A total of 36 individually housed weanling pigs were randomly allotted to 1 of the 4 treatments, with 9 replicates per treatment. Treatments were a control (CONT, no additive), antibiotic (AB; 2.5 g/kg of feed of antibiotic with chlortetracycline, sulfamethazine, and penicillin), and EG delivered in the drinking water at concentrations of 0.1% (EG1) and 0.2% (EG2). All pigs received a basal diet similar in composition and nutrients, except for pigs receiving the AB diet, which had an added antibiotic. Pigs were acclimated to treatments for a 7-d period to monitor growth performance. On d 8, blood samples were collected from each pig to obtain serum, and each pig was gavaged with 6 mL (2 × 10(9) cfu/mL) of ETEC solution. Pigs were monitored for another 7 d to assess incidences of diarrhea and growth performance, and then all pigs were killed to obtain intestinal tissue and digesta samples. Treatments did not influence growth performance throughout the study. Greater ETEC counts were observed in the ileal mucosal scrapings (P = 0.001) and colonic digesta (P = 0.025) of pigs in the CONT group compared with pigs in the AB and EG1 groups. Pigs receiving AB and EG1 had greater (P < 0.05) small intestinal weights and ileal villus heights than pigs receiving CONT; however, the ileal villus height-to-crypt depth ratio was greater in pigs fed the AB diet (1.69) compared with those fed the CONT diet (1.34), whereas pigs receiving EG1 were intermediate. Pigs in the EG1 group showed greater (P < 0.001) serum tumor necrosis factor α and IL-6 concentrations before ETEC challenge; however, at 7 d postchallenge, pigs receiving EG2 showed the least (P < 0.05) circulating tumor necrosis factor α and IL-6 concentrations. Overall, better intestinal growth and development, as well as decreased ETEC counts on the intestinal mucosa and serum proinflammatory cytokines, suggest that EG can maintain gut health and function in piglets commensurate with antibiotics. However, it is noteworthy that at the largest dose tested, EG seemed to have a dramatic effect on proinflammatory cytokines but had a minimal or no effect on the other response criteria.
    Journal of Animal Science 09/2011; 90(1):252-60. DOI:10.2527/jas.2010-3596 · 2.11 Impact Factor
Show more