Literature-based safety assessment of an agriculture- and animal-associated microorganism: Weissella confusa.

Although Weissella confusa was established as a species over 25 years ago, it has been understudied until very recently. Several independent observations have driven the recent interest in this important microorganism. First, this Leuconostoc-like species of Lactic Acid Bacteria is associated with agricultural environments, many spontaneous food fermentations-especially carbohydrate-rich vegetable fermentations-and silage. Second, Weissella confusa are members of the autochthonous microbiota of healthy humans and livestock. Third, Weissella confusa-in a strain-specific fashion-are postulated to be good candidates for the development of novel direct-fed microbial products. Fourth, Weissella confusa-in a strain-specific fashion-have been described as opportunistic pathogens-especially in immunocompromised individuals. Last, a distantly related species (Weissella ceti) is the etiologic agent of weissellosis, a disease that affects farmed fish that are important for commercial aquaculture. The purpose of this literature-based safety assessment is to consolidate findings from primary research related to Weissella confusa and its natural associations with and effects on animals, humans, and their agricultural environments. Based on these assessments, it is reasonable to conclude that many Weissella confusa are safe for use in direct-fed microbial products for poultry.

From the Lab to the Farm: An Industrial Perspective of Plant Beneficial Microorganisms.

Any successful strategy aimed at enhancing crop productivity with microbial products ultimately relies on the ability to scale at regional to global levels. Microorganisms that show promise in the lab may lack key characteristics for widespread adoption in sustainable and productive agricultural systems. This paper provides an overview of critical considerations involved with taking a strain from discovery to the farmer's field. In addition, we review some of the most effective microbial products on the market today, explore the reasons for their success and outline some of the major challenges involved in industrial production and commercialization of beneficial strains for widespread agricultural application. General processes associated with commercializing viable microbial products are discussed in two broad categories, biofertility inoculants and biocontrol products. Specifically, we address what farmers desire in potential microbial products, how mode of action informs decisions on product applications, the influence of variation in laboratory and field study data, challenges with scaling for mass production, and the importance of consistent efficacy, product stability and quality. In order to make a significant impact on global sustainable agriculture, the implementation of plant beneficial microorganisms will require a more seamless transition between laboratory and farm application. Early attention to the challenges presented here will improve the likelihood of developing effective microbial products to improve crop yields, decrease disease severity, and help to feed an increasingly hungry planet.

Effective Sequestration of Clostridium difficile Protein Toxins by Calcium Aluminosilicate.

Clostridium difficile is a leading cause of antibiotic-associated diarrhea and the etiologic agent responsible for C. difficile infection. Toxin A (TcdA) and toxin B (TcdB) are nearly indispensable virulence factors for Clostridium difficile pathogenesis. Given the toxin-centric mechanism by which C. difficile pathogenesis occurs, the selective sequestration with neutralization of TcdA and TcdB by nonantibiotic agents represents a novel mode of action to prevent or treat C. difficile-associated disease. In this preclinical study, we used quantitative enzyme immunoassays to determine the extent by which a novel drug, calcium aluminosilicate uniform particle size nonswelling M-1 (CAS UPSN M-1), is capable of sequestering TcdA and TcdB in vitro. The following major findings were derived from the present study. First, we show that CAS UPSN M-1 efficiently sequestered both TcdA and TcdB to undetectable levels. Second, we show that CAS UPSN M-1's affinity for TcdA is greater than its affinity for TcdB. Last, we show that CAS UPSN M-1 exhibited limited binding affinity for nontarget proteins. Taken together, these results suggest that ingestion of calcium aluminosilicate might protect gastrointestinal tissues from antibiotic- or chemotherapy-induced C. difficile infection by neutralizing the cytotoxic and proinflammatory effects of luminal TcdA and TcdB.

Polyphenol-rich sorghum brans alter colon microbiota and impact species diversity and species richness after multiple bouts of dextran sodium sulfate-induced colitis.

The microbiota affects host health, and dysbiosis is involved in colitis. Sorghum bran influences butyrate concentrations during dextran sodium sulfate (DSS) colitis, suggesting microbiota changes. We aimed to characterize the microbiota during colitis, and ascertain if polyphenol-rich sorghum bran diets mitigate these effects. Rats (n = 80) were fed diets containing 6% fiber from cellulose, or Black (3-deoxyanthocyanins), Sumac (condensed tannins), or Hi Tannin black (both) sorghum bran. Inflammation was induced three times using 3% DSS for 48 h (40 rats, 2 week separation), and the microbiota characterized by pyrosequencing. The Firmicutes/Bacteroidetes ratio was higher in Cellulose DSS rats. Colonic injury negatively correlated with Firmicutes, Actinobacteria, Lactobacillales and Lactobacillus, and positively correlated with Unknown/Unclassified. Post DSS#2, richness was significantly lower in Sumac and Hi Tannin black. Post DSS#3 Bacteroidales, Bacteroides, Clostridiales, Lactobacillales and Lactobacillus were reduced, with no Clostridium identified. Diet significantly affected Bacteroidales, Bacteroides, Clostridiales and Lactobacillus post DSS#2 and #3. Post DSS#3 diet significantly affected all genus, including Bacteroides and Lactobacillus, and diversity and richness increased. Sumac and Hi Tannin black DSS had significantly higher richness compared to controls. Thus, these sorghum brans may protect against alterations observed during colitis including reduced microbial diversity and richness, and dysbiosis of Firmicutes/Bacteroidetes.

Draft Genome Sequence of Lactobacillus animalis 381-IL-28.

Lactobacillus animalis 381-IL-28 is an integral component of a multistrain commercial culture with food biopreservative and pathogen biocontrol functionality. A draft sequence of the L. animalis 381-IL-28 genome is described in this paper.

Inhibition of Escherichia coli O157:H7 and Salmonella enterica on spinach and identification of antimicrobial substances produced by a commercial Lactic Acid Bacteria food safety intervention.

The microbiological safety of fresh produce is of concern for the U.S. food supply. Members of the Lactic Acid Bacteria (LAB) have been reported to antagonize pathogens by competing for nutrients and by secretion of substances with antimicrobial activity, including organic acids, peroxides, and antimicrobial polypeptides. The objectives of this research were to: (i) determine the capacity of a commercial LAB food antimicrobial to inhibit Escherichia coli O157:H7 and Salmonella enterica on spinach leaf surfaces, and (ii) identify antimicrobial substances produced in vitro by the LAB comprising the food antimicrobial. Pathogens were inoculated on freshly harvested spinach, followed by application of the LAB antimicrobial. Treated spinach was aerobically incubated up to 12 days at 7 °C and surviving pathogens enumerated via selective/differential plating. l-Lactic acid and a bacteriocin-like inhibitory substance (BLIS) were detected and quantified from cell-free fermentates obtained from LAB-inoculated liquid microbiological medium. Application of 8.0 log10 CFU/g LAB produced significant (p < 0.05) reductions in E. coli O157:H7 and Salmonella populations on spinach of 1.6 and 1.9 log10 CFU/g, respectively. It was concluded the LAB antimicrobial inhibited foodborne pathogens on spinach during refrigerated storage, likely the result of the production of metabolites with antimicrobial activity.

Diet complexity and estrogen receptor ß status affect the composition of the murine intestinal microbiota.

Intestinal microbial dysbiosis contributes to the dysmetabolism of luminal factors, including steroid hormones (sterones) that affect the development of chronic gastrointestinal inflammation and the incidence of sterone-responsive cancers of the breast, prostate, and colon. Little is known, however, about the role of specific host sterone nucleoreceptors, including estrogen receptor ß (ERß), in microbiota maintenance. Herein, we test the hypothesis that ERß status affects microbiota composition and determine if such compositionally distinct microbiota respond differently to changes in diet complexity that favor Proteobacteria enrichment. To this end, conventionally raised female ERß(+/+) and ERß(-/-) C57BL/6J mice (mean age of 27 weeks) were initially reared on 8604, a complex diet containing estrogenic isoflavones, and then fed AIN-76, an isoflavone-free semisynthetic diet, for 2 weeks. 16S rRNA gene surveys revealed that the fecal microbiota of 8604-fed mice and AIN-76-fed mice differed, as expected. The relative diversity of Proteobacteria, especially the Alphaproteobacteria and Gammaproteobacteria, increased significantly following the transition to AIN-76. Distinct patterns for beneficial Lactobacillales were exclusive to and highly abundant among 8604-fed mice, whereas several Proteobacteria were exclusive to AIN-76-fed mice. Interestingly, representative orders of the phyla Proteobacteria, Bacteroidetes, and Firmicutes, including the Lactobacillales, also differed as a function of murine ERß status. Overall, these interactions suggest that sterone nucleoreceptor status and diet complexity may play important roles in microbiota maintenance. Furthermore, we envision that this model for gastrointestinal dysbiosis may be used to identify novel probiotics, prebiotics, nutritional strategies, and pharmaceuticals for the prevention and resolution of Proteobacteria-rich dysbiosis.

Draft Genome Sequence of the Pediocin-Encoding Biopreservative and Biocontrol Strain Pediococcus acidilactici D3.

We describe a draft genome sequence for Pediococcus acidilactici strain D3, a component of multistrain commercial cultures with biopreservative and biocontrol properties in food-based applications. Strain D3 encodes at least one antimicrobial peptide, pediocin AMPd3. The AMPd3-encoding operon exhibits high sequence similarity to the archetype pediocin, PA-1, encoded by P. acidilactici PAC 1.0.

A novel shift in estrogen receptor expression occurs as estradiol suppresses inflammation-associated colon tumor formation.

Postmenopausal women on estrogen replacement therapy (ERT) have a reduced risk of developing colon cancer compared with postmenopausal women not on ERT, suggesting a role for estradiol (E2) in protection against this disease. To determine whether E2 protects against inflammation-associated colon cancer when administered following the initiation of colonic DNA damage, in this study, we implanted E2-containing pellets into mice after co-treatment with azoxymethane and two rounds of dextran sulfate sodium (DSS). Wild-type (WT) E2-treated mice had reduced numbers and average area of adenocarcinomas compared with the control mice. These effects were lost in estrogen receptor-ß (Erß (Esr2)) knockout mice. Surprisingly, apoptosis was reduced and cell proliferation was increased in sections from tumors of the WT E2 mice compared with the WT control mice. These findings are probably due, in part, to a reduction in ERß expression in colonic epithelial cells as the cells progressed from a non-malignant to a cancerous state as enhanced apoptosis was observed in normal colonocytes expressing higher levels of ERß. Furthermore, epithelial cells within the tumors had dramatically increased ERα mRNA and protein expression compared with the non-diseased mice. We conclude that while E2 treatment resulted in an overall suppression of colonic adenocarcinoma formation, reduced ERß expression accompanied by enhanced ERα expression caused an altered colonocyte response to E2 treatment compared with the earlier stages of colon cancer development. These data are the first examples of decreased ERß expression concurrent with increased ERα expression as a disease develops and highlight the importance of understanding the timing of E2 exposure with regard to the prevention of inflammation-associated colon cancer.

Disruption of inducible 6-phosphofructo-2-kinase impairs the suppressive effect of PPARγ activation on diet-induced intestine inflammatory response.

PFKFB3 is a target gene of peroxisome proliferator-activated receptor gamma (PPARγ) and encodes for inducible 6-phosphofructo-2-kinase (iPFK2). As a key regulatory enzyme that stimulates glycolysis, PFKFB3/iPFK2 links adipocyte metabolic and inflammatory responses. Additionally, PFKFB3/iPFK2 is involved in the effect of active PPARγ on suppressing overnutrition-induced adipose tissue inflammatory response, which accounts for the insulin-sensitizing and antidiabetic effects of PPARγ activation. Using PFKFB3/iPFK2-disrupted mice, the present study investigated the role of PFKFB3/iPFK2 in regulating overnutrition-associated intestine inflammatory response and in mediating the effects of PPARγ activation. In wild-type mice, intestine PFKFB3/iPFK2 was increased in response to high-fat diet (HFD) feeding compared with that in mice fed a low-fat diet. However, intestine PFKFB3/iPFK2 was decreased in PFKFB3/iPFK2-disrupted mice and did not respond to HFD feeding. Furthermore, on an HFD, PFKFB3/iPFK2-disrupted mice displayed a significant increase in major intestine proinflammatory indicators such as toll-like receptor 4 expression, c-Jun N-terminal kinase 1 and nuclear factor kappa B phosphorylation, and proinflammatory cytokine expression compared with wild-type littermates. Upon treatment with rosiglitazone, an agonist of PPARγ, intestine proinflammatory indicators were markedly decreased in wild-type mice, but to a much lesser degree in PFKFB3/iPFK2-disrupted mice. Overall, the status of HFD-induced intestine inflammatory response in all treated mice correlated inversely with systemic insulin sensitivity, indicated by the homeostasis model assessment of insulin resistance data. Together, these results suggest that PFKFB3/iPFK2 is critically involved in the effect of PPARγ activation on suppressing diet-induced intestine inflammatory response.

Cryptic prophages help bacteria cope with adverse environments.

Phages are the most abundant entity in the biosphere and outnumber bacteria by a factor of 10. Phage DNA may also constitute 20% of bacterial genomes; however, its role is ill defined. Here, we explore the impact of cryptic prophages on cell physiology by precisely deleting all nine prophage elements (166 kbp) using Escherichia coli. We find that cryptic prophages contribute significantly to resistance to sub-lethal concentrations of quinolone and ß-lactam antibiotics primarily through proteins that inhibit cell division (for example, KilR of rac and DicB of Qin). Moreover, the prophages are beneficial for withstanding osmotic, oxidative and acid stresses, for increasing growth, and for influencing biofilm formation. Prophage CPS-53 proteins YfdK, YfdO and YfdS enhanced resistance to oxidative stress, prophages e14, CPS-53 and CP4-57 increased resistance to acid, and e14 and rac proteins increased early biofilm formation. Therefore, cryptic prophages provide multiple benefits to the host for surviving adverse environmental conditions.

Suppression of Listeria monocytogenes Scott A in Fluid Milk by Free and Liposome-Entrapped Nisin.

Nisin is an antimicrobial polypeptide inhibitory toward Gram-positive bacterial pathogens, including Listeria monocytogenes. Encapsulating nisin in lipid nanocapsules (i.e., liposomes) has been shown to protect antimicrobial functionality in complex food matrices. The capacity of liposomes to encapsulate a fluorescent reporter was determined via spectroscopy. Survival and growth of L. monocytogenes incubated in fluid milk containing 50 IU/ml free or liposome-entrapped nisin was assayed via periodic enumeration of survivors. Liposomes were formulated from phosphatidylcholine (PC) and phosphatidyl-DL-glycerol (PG) and prepared as PC, PC/PG 7/3 or PC/PG 6/4 (mol. fraction). Antilisterial activity of nisin-loaded liposomes was determined in ultra-high temperature processed fluid milk containing approximately 4.0 log10 CFU/ml L. monocytogenes Scott A plus liposomal or free nisin at 50 IU/mL. Samples were aerobically held at 5 or 20°C; L. monocytogenes were enumerated via plating after 0, 1, 3, 6, 12, 24, 48, and 72 incubation hours. Liposome entrapment did not enhance pathogen inhibition when compared to free nisin as a function of storage temperature or incubation duration.

Inhibition of bacteriophage replication in Streptococcus thermophilus by subunit poisoning of primase.

Invariant and highly conserved amino acids within a primase consensus sequence were targeted by site-specific mutations within the putative primase of Streptococcus thermophilus phage kappa3. PCR products containing the desired mutation(s) within putative ATPase/helicase and/or oligomerization domains of the kappa3-encoded primase gene were cloned into a high-copy-number vector and expressed in S. thermophilus NCK1125. The majority of the plasmid constructs failed to alter phage sensitivity; however, four of the constructs conferred strong phage resistance upon the host. Expression of the K238(A/T) and RR340-341AA mutant proteins in trans suppressed the function of the native phage primase protein in a dominant negative fashion via a proposed subunit poisoning mechanism. These constructs completely inhibited phage genome synthesis and reduced the efficiencies of plaquing and centre of infection formation by more than 9 and 3.5 logs, respectively. Amber mutations introduced upstream of the transdominant RR340-341AA and K238(A/T) mutations restored phage genome replication and sensitivity of the host, indicating that translation was required to confer phage resistance. Introduction of an E437A mutation in a putative oligomerization domain located downstream of the transdominant K238T mutation also completely suppressed phage resistance. This study appears to represent the first use of transdominant proteins to inhibit phages that are disruptive to cultures used in industrial fermentations.

Engineered bacteriophage-defence systems in bioprocessing.

Bacteriophages (phages) have the potential to interfere with any industry that produces bacteria as an end product or uses them as biocatalysts in the production of fermented products or bioactive molecules. Using microorganisms that drive food bioprocesses as an example, this review will describe a set of genetic tools that are useful in the engineering of customized phage-defence systems. Special focus will be given to the power of comparative genomics as a means of streamlining target selection, providing more widespread phage protection, and increasing the longevity of these industrially important bacteria in the bioprocessing environment.

Antisense RNA targeting of primase interferes with bacteriophage replication in Streptococcus thermophilus.

The putative primase gene and other genes associated with the Sfi21-prototype genome replication module are highly conserved in Streptococcus thermophilus bacteriophages. Expression of antisense RNAs complementary to the putative primase gene (pri3.1) from S. thermophilus phage kappa 3 provided significant protection from kappa 3 and two other Sfi21-type phages. Expression of pri3.10-AS, an antisense RNA that covered the entire primase gene, reduced the efficiency of plaquing (EOP) of kappa 3 to 3 x 10(-3) and reduced its burst size by 20%. Mutant phages capable of overcoming antisense inhibition were not recovered. Thirteen primase-specific antisense cassettes of different lengths (478 to 1,512 bp) were systematically designed to target various regions of the gene. Each cassette conferred some effect, reducing the EOP to between 0.8 and 3 x 10(-3). The largest antisense RNAs (1.5 kb) were generally found to confer the greatest reductions in EOP, but shorter (0.5 kb) antisense RNAs were also effective, especially when directed to the 5' region of the gene. The impacts of primase-targeted antisense RNAs on phage development were examined. The expression of pri3.10-AS resulted in reductions in target RNA abundance and the number of phage genomes synthesized. Targeting a key genome replication function with antisense RNA provided effective phage protection in S. thermophilus.

Expression of antisense RNA targeted against Streptococcus thermophilus bacteriophages.

Antisense RNA complementary to a putative helicase gene (hel3.1) of a cos-type Streptococcus thermophilus bacteriophage was used to impede the proliferation of a number of cos-type S. thermophilus bacteriophages and one pac-type bacteriophage. The putative helicase gene is a component of the Sfi21-type DNA replication module, which is found in a majority of the S. thermophilus bacteriophages of industrial importance. All bacteriophages that strongly hybridized a 689-bp internal hel3.1 probe were sensitive to the expression of antisense hel3.1 RNA. A 40 to 70% reduction in efficiency of plaquing (EOP) was consistently observed, with a concomitant decrease in plaque size relative to that of the S. thermophilus parental strain. When progeny were released, the burst size was reduced. Growth curves of S. thermophilus NCK1125, in the presence of variable levels of bacteriophage kappa3, showed that antisense hel3.1 conferred protection, even at a multiplicity of infection of approximately 1.0. When the hel3.1 antisense RNA cassette was expressed in cis from the kappa3-derived phage-encoded resistance (PER) plasmid pTRK690::ori3.1, the EOP for bacteriophages sensitive to PER and antisense targeting was reduced to between 10(-7) and 10(-8), beyond the resistance conferred by the PER element alone (less than 10(-6)). These results illustrate the first successful applications of antisense RNA and explosive delivery of antisense RNA to inhibit the proliferation of S. thermophilus bacteriophages.