In vitro analysis of probiotic strain combinations to inhibit pathogen adhesion to human intestinal mucus

Competition with pathogens for adhesion and colonization of the mucosal surfaces are possible protective mechanisms of probiotics. In this study we evaluated the ability of commercial strains (Lactobacillus rhamnosus GG, L. rhamnosus LC705, B. breve 99 and Propionibacterium freudenreichii ssp. shermanii JS) each strain alone and in different combinations to inhibit, displace and compete with selected pathogens in order to test the influence of the presence of the other probiotic in the adhesion pathogens to immobilized mucus. The ability to inhibit the adhesion or to displace adhered pathogens was variable depending on both the probiotic combination and the pathogen tested. Our results demonstrate that different probiotic combinations were able to enhance the inhibition percentages to pathogen adhesion to intestinal mucus. All probiotic combinations tested in this study showed inhibition abilities against pathogen infection with values which were over 40% for some pathogens tested. Combinations had specific properties and inhibition abilities against some or all of the tested pathogens. These results suggest that combinations of probiotics strains could be useful and more effective in inhibition of pathogen adhesion. The inhibition and displacement profiles were very different suggesting that different mechanisms are implied in both processes. Selection of new probiotic combinations should be conducted for specific target pathogens or pathogen associated microbiota aberrancies.     

Probiotics/direct fed microbials for Salmonella control in poultry

Bacterial antimicrobial resistance in both the medical and agricultural fields has become a serious problem worldwide. During the last 15 years, our laboratories have worked toward the identification of probiotic candidates for poultry which can actually displace Salmonella and other enteric pathogens which have colonized the gastrointestinal tract of chickens and turkeys, indicating that selection of therapeutically efficacious probiotic cultures with marked performance benefits in poultry is possible, and that defined cultures can sometimes provide an attractive alternative to conventional antimicrobial therapy. Our studies have been focused on specific pathogen reduction, performance under commercial conditions, and effects on both idiopathic and defined enteritis. We have also confirmed that selected heat-resistant spore-forming Bacillus species can markedly reduce Salmonella and Clostridium when administered in very high numbers, and we have developed a novel and simple technique for obtaining cultured Bacillus spore counts, providing a cost-effective feed-stable inclusion in commercial poultry diets. In order to select even more effective isolates, we are still currently focused on the mechanistic action of the Lactobacillus probiotic previously developed as well as new Bacillus candidates. Current indications are that mechanism of action involves rapid activation of innate host immune responses, providing an exciting possibility for identification of vastly superior and more potent probiotics. In this review, we summarize the safety and efficacy of individual monocultures for prophylactic and/or therapeutic efficacy against Salmonella infections under both laboratory and field conditions as well as the development of a novel, cost-effective, feed-stable direct-fed microbials (DFM) with potential for widespread utilization and improved production, delivery and clinical efficacy for animal use.     

Lactobacillus salivarius: Bacteriocin and probiotic activity

Lactic acid bacteria (LAB) antimicrobial peptides typically exhibit antibacterial activity against food-borne pathogens, as well as spoilage bacteria. Therefore, they have attracted the greatest attention as tools for food biopreservation. In some countries LAB are already extensively used as probiotics in food processing and preservation. LAB derived bacteriocins have been utilized as oral, topical antibiotics or disinfectants. Lactobacillus salivarius is a promising probiotic candidate commonly isolated from human, porcine, and avian gastrointestinal tracts (GIT), many of which are producers of unmodified bacteriocins of sub-classes IIa, IIb and IId. It is a well-characterized bacteriocin producer and probiotic organism. Bacteriocins may facilitate the introduction of a producer into an established niche, directly inhibit the invasion of competing strains or pathogens, or modulate the composition of the microbiota and influence the host immune system. This review gives an up-to-date overview of all L. salivarius strains, isolated from different origins, known as bacteriocin producing and/or potential probiotic.     

Monostrain, multistrain and multispecies probiotics--A comparison of functionality and efficacy

This literature review was carried out to make a comparison of functionality and efficacy between monostrain, multistrain and multispecies probiotics. A monostrain probiotic is defined as containing one strain of a certain species and consequently multistrain probiotics contain more than one strain of the same species or, at least of the same genus. Arbitrarily, the term multispecies probiotics is used for preparations containing strains that belong to one or preferentially more genera. Multispecies probiotics were superior in treating antibiotic-associated diarrhea in children. Growth performance and particularly mortality in broilers could be improved with multistrain probiotics. Mice were better protected against S. Typhimurium infection with a multistrain probiotic. A multispecies probiotic provided the best clearance of E. coli O157:H7 from lambs. Rats challenged with S. Enteritidis showed best post-challenge weight gains when treated with a multispecies probiotic. Possible mechanisms underlying the enhanced effects of probiotic mixtures are discussed. It is also emphasized that strains used in multistrain and multispecies probiotics should be compatible or, preferably, synergistic. The design and use of multistrain and multispecies probiotics should be encouraged.     

Probiotic bacteria induced improvement of the mucosal integrity of enterocyte-like Caco-2 cells after exposure to Salmonella enteritidis 857

Probiotics protect the intestinal epithelium. Whether their protection against Salmonella is mediated via stabilizing the barrier integrity was examined here. The intestinal barrier integrity was assessed by measuring the trans-epithelial electrical resistance (TER) of Caco-2 cell monolayer. Cells were exposed for 1 h to Lactobacillus casei W56, Lactobacillus salivarius W24, Lactobacillus acidophilus W70, Lactococcus lactis W58, Bifidobacterium infantis W52 or Bifidobacterium bifidum W23 at 200 bacteria/cell or to Salmonella enteritidis 857 at 1–200 bacteria/cell or to a combination of either of the probiotic and Salmonella both at 200 bacteria/cell. Heat shock protein (Hsp) 70 was assessed by Western blotting. Probiotics induced a significant increase while Salmonella decreased the TER. The latter was attenuated by probiotics but without full recovery. In addition, probiotics induced over-expression of Hsp70. It may be concluded that probiotics stabilise the intestinal integrity and impede Salmonella infection via, at least in part, production of Hsp70.     

Anticarcinogenecity of microbiota and probiotics in breast cancer

Breast cancer is one of the most important causes of cancer related morbidity and mortality in the world. Along with genetic, environmental factors also play a multifaceted role in the development of disease. Breast contains several bacterial species performing specialized functions. Probiotics, as functional food, play pivotal role against breast cancer development in vivo and in vitro. Current review summarized all the available data related to diet, probiotics, and their association with breast cancer risk along with underlying mechanisms. Presently, it was believed that many of the commercially available probiotic products were safe to use and had some beneficial health effects for the host. Probiotics had a potential to act against breast cancer progression evidenced by many animal model and cell-based experiments. Some probiotics strains may be useful as an adjuvant therapy for breast cancer prevention or treatment, by modulating immune response or breast microbial community. However, large-scale clinical trials and intense research are mandatory to explore probiotics-related metabolic and molecular mechanisms in breast cancer.     

Microbiological,immunological, and histological changes in the gut of Salmonella Enteritidis-challenged rats fed goat cheese containing Lactobacillus rhamnosus EM1107

Cheeses are able to serve as suitable matrices for supplying probiotics to consumers, enabling appropriate conditions for bacteria to survive gastric transit and reach the gut, where they are assumed to promote beneficial processes. The present study aimed to evaluate the microbiological, immunological, and histological changes in the gut of Salmonella Enteritidis-challenged rats fed goat cheese supplemented with the probiotic strain Lactobacillus rhamnosus EM1107. Thirty male albino Wistar rats were randomly distributed into 5 experimental groups with 6 animals each: negative (NC) and positive (PtC) control groups, control goat cheese (CCh), goat cheese added with L. rhamnosus EM1107 (LrCh), and L. rhamnosus EM1107 only (EM1107). All animals, except NC group were challenged with Salmonella Enteritidis (10⁹ cfu in 1 mL of saline through oral gavage). Microbial composition was assessed with high-throughput 16S rRNA sequencing by means of Illumina MiSeq (Illumina, San Diego, CA). Nuclear factor kappa B (NF-κB) from the animal cecum tissue was determined by real-time PCR and interleukins (TNF-α, IL-1β, IL-10, and IFN-γ) by means of ELISA. Myeloperoxidase and malondialdehyde levels were determined biochemically. The administration of the L. rhamnosus EM1107 probiotic strain, either as a pure culture or added to a cheese matrix, was able to reduce Salmonella colonization in the intestinal lumen and lessen tissue damage compared with rats from PtC group. In addition, the use of cheese for the probiotic strain delivery (LrCh) was associated with a marked shift in the gut microbiota composition toward the increase of beneficial organisms such as Blautia and Lactobacillus and a reduction in NF-κB expression. These findings support our hypothesis that cheeses might be explored as functional matrices for the efficacious delivery of probiotic strains to consumers.     

Probiotics interaction with foodborne pathogens: a potential alternative to antibiotics and future challenges

Abstract

Antibiotics are a key tool used nowadays in health care industry to fight against bacterial infections; however, repeated antibiotic use or misuses, have led to bacterial resistance, causing significant threats for many people with common bacterial infections. The use of probiotics to enhance gastrointestinal health has been proposed for many years. In recent years, there has been an increasing interest in the use of probiotic bacteria as alternatives for antibiotics for preventing or treating various intestinal infections. Several important underlying mechanisms responsible for the antagonistic effects of probiotics on different microorganisms include: (1) competitive exclusion for adhesion sites and nutritional sources; (2) secretion of antimicrobial substances; (3) enhancement of intestinal barrier function; and (4) immunomodulation. However, their mode of action is not very well understood and therefore a clearer understanding of these mechanisms is necessitated. This will enable appropriate probiotic strains to be selected for particular applications and may reveal new probiotic functions. The goal of this review was to highlight some studies from literature describing the probiotic interaction with several major foodborne pathogens, as well as explore the mechanisms for such probiotic-pathogen interaction. The review will conclude by presenting future perspective and challenges of probiotic application in food products.     

Short communication: In vitro and in vivo probiotic potential of Lactobacillus plantarum B7 and Lactobacillus rhamnosus D1 isolated from Minas artisanal cheese

Some Lactobacillus strains may contribute to the health of the host when administered in adequate concentrations, demonstrating their probiotic potential. In contrast, Listeria monocytogenes is a foodborne pathogen that can cause enteropathy, meningoencephalitis, abortion, and septicemia. The aim of this survey was to evaluate the in vitro and in vivo probiotic potential of Lactobacillus plantarum B7 and Lactobacillus rhamnosus D1, isolated from Minas artisanal cheese of the Serra da Canastra (Minas Gerais, Brazil), against Lis. monocytogenes. We submitted B7 and D1 to in vitro testing (antibiogram, tolerance to bile salts and artificial gastric fluid, and spot-on-lawn) and in vivo testing (relative weight gain in mice). Both Lactobacillus strains demonstrated in vitro inhibitory activity against Lis. monocytogenes, as well as sensitivity to antimicrobials and resistance to gastric acids and bile salts. In the in vivo assays, mice treated with D1 gained more weight than mice in the other groups. These results indicate that D1 could have higher probiotic potential than B7 because improvements in feed conversion may help animals fight infection.     

Probiotics in the new millennium

Beneficial effects on human health by specific probiotic microorganisms such as prevention of gastrointestinal tract infections, immune stimulation, and balancing of the intestinal microflora have been established in numerous clinical trials. The successful probiotic strains, which are mainly members of Lactobacillus and more recently Bifidobacterium naturally found in the human intestinal tract, have been traditionally incorporated into fermented milk products but have excellent potential for further inclusion in functional foods and health-related products. While the health claims are generally accepted by both scientists and consumers, often the molecular mechanisms underlying the probiotic properties remain controversial. Further progress concerning the molecular basis of probiotic traits will give vital reinforcement to the probiotic concept and is a prerequisite for rational development into further applications. In this review, current research on the genetics of properties of the intestinal lactobacilli that may contribute to the activity and effectiveness of probiotics is described. The potential of molecular biology for future probiotic applications is addressed and a probiotic strain developed by modern biotechnology with advantages for specific consumers is presented.     

Salmonella Typhimurium general virulence factors: A battle of David against Goliath?

The genus Salmonella is the most common agent causative of foodborne diseases. Although genus Salmonella members are genetically close microorganisms, they show wide variations in host-specificity, virulence and disease manifestations. Salmonellosis caused by contaminated water or food is usually present as two clinical forms: typhoid fever and nontyphoidal diseases. The latter producing gastroenteritis is frequently caused by Salmonella Typhimurium and Salmonella Enteritidis. The nontyphoidal S. Typhimurium infection involves the following steps: bacterial adhesion, invasion, SCV maturation and replication. During these steps there is a strong interaction between host and pathogen, in which the pathogen must resist different host defense mechanisms. In each of these stages the bacteria modulate the expression of diverse groups of genes, most of which are encoded in Salmonella pathogenicity islands (SPI). This modulation is in general under control of the so-called two-component systems (TCS). The TCSs are capable of sensing different environmental conditions and trigger a physiological response. Currently, the risk of contracting salmonellosis disease has been considerably increased due to the emergence of new serovars showing multiple-drug resistance that presents a high risk to human health. In this work, we summarize the new advances in the study of host–pathogen interactions during the Salmonella infection that leads to the establishment of the disease. This finding highlights the role of the S. Typhimurium secretion systems and effectors during infection. In addition, we mentioned some strategies that could be explored in order to take control of Salmonella infections.     

Ecological and functional implications of the acid-adaptation ability of Bifidobacterium: A way of selecting improved probiotic strains

Probiotics are live micro-organisms that exert health benefits on the host when ingested in appropriate numbers. Bifidobacterium strains are some of the most important probiotics used for human consumption. The high sensitivity of the strains of this genus to acidic conditions can, however, impair their survival in adverse environments such as the stomach and in fermented foods, and, therefore, limits their use as live probiotic cultures. The optimization of strategies based on stress adaptation and cross-protection mechanisms of bifidobacteria constitutes an attractive option to improve their technological performance and functionality. The ability of Bifidobacterium strains from different ecosystems to survive to acidic conditions as well as the success and limitations of the application of acid stress adaptation treatments to improve their stress tolerance and biological properties are reported. The potential use of these strategies to expand the spectra of probiotic strains and products currently marketed is discussed.     

Evaluation of probiotic characteristics of newly isolated Lactobacillus spp.: immune modulation and longevity

In the current study, the probiotic potential of approximately 350 strains of lactic acid bacteria isolated from Korean infant feces and Kimchi was investigated. Common probiotic properties of the bacterial strains, such as acid tolerance, bile tolerance and adhesion to human intestinal epithelial cells (HT-29 cells), were examined. Some strains were found to have immune modulatory and antimicrobial properties. Antagonistic activity against a panel of pathogenic bacteria was found to be strain dependent. To evaluate the immune modulatory activity of the strains, lymphocyte interferon (IFN)-γ secretion was determined in conjunction with cell proliferation. Some strains of Lactobacillus gasseri, L. fermentum and L. plantarum exhibited increased IFN-γ levels and lymphocyte proliferation. To evaluate the effects of these immune modulating lactobacilli on host life span, Caenorhabditis elegans was used as an in vivo model. Nematodes that were supplied heat-killed lactobacilli as a food source exhibited obvious differences in life span compared with those fed Escherichia coli OP50. The mean life span (determined as mean percent survival) of worms fed L. plantarum CJLP133 and L. fermentum LA12 was 13.89% and 13.69% greater, respectively, than that of control nematodes after 21 days (P = 0.036 and 0.043, respectively). In addition, some of safety profiles, including hemolytic type, gelatin hydration and degradation of urea, were found to be positive. These newly identified lactobacilli hold promise for use as probiotic agents, feed additives and/or in food applications.     

Economic production of probiotics from kitchen waste

In the present work, a novel treatment method of kitchen waste for economic production of probiotics was investigated. This required the selection of suitable probiotic microorganisms. Based on the pure cultures of probiotic strains for the fermentation kitchen waste, 5 strains of microorganisms including 1 strain of Lactobacillus, 2 strains of Bacillus, and 2 strains of yeast were selected, respectively. These probiotic microorganisms were mixed at the same ratio and cultured using the kitchen waste as culture medium at pH of 7.2 and temperature of 37°C. After 24 h, the total count of the viable cells reached 2.24 ×10¹⁰ CFU/g, which was higher than that obtained in any single probiotic strain pure culture. It was found that the presence of yeasts and Bacillus species enhanced the growth of Lactobacillus strain. Bench scale experiments were also done in a self-designed rotating drum type bioreactor. The experimental results indicate that there is a good possibility of utilizing kitchen waste for the economic production of probiotics.     

Influence of whey-based fruit juice containing Lactobacillus rhamnosus on intestinal well-being and humoral immune response in healthy adults

The ability of probiotics to balance intestinal microbes and to modulate gut immune system has been reported for several probiotic strains. In the present study, Lactobacillus rhamnosus strain VTT E-97800 (E800) or L. rhamnosus Lc705 (the latter in combination with Propionibacterium freudenreichii ssp. shermanii JS) were separately administered to healthy adult volunteers (n=11 in both study groups) in a whey-based fruit juice. Survival of the potentially probiotic L. rhamnosus strains in the GI-tract and their effect on intestinal well-being and peripheral immune parameters were assessed. Both L. rhamnosus strains were recovered in high numbers in faecal samples during the consumption period and with few exceptions they were not detected after the 2 weeks follow-up period. Consumption of the juice supplemented with either of the L. rhamnosus strains did not have significant effect on faecal consistency or defecation frequency. No changes were observed in the immunological parameters (number or immunoglobulin A-, G- and M-secreting cells, expression of Fcα-receptor or complement receptors 1 and 3), except for a slight decrease in the number of IgA secreting cells during ingestion of the juice supplemented with L. rhamnosus Lc705 and P. freudenreichii ssp. shermanii JS. The results indicate that L. rhamnosus strains E800 and Lc705 had good survival ability in the GI-tract when administered in a whey-based fruit juice matrix. No adverse effects on intestinal function or on studied immunological parameters were observed during consumption of the whey based juice drink supplemented with the potential probiotic strains.     

罗非鱼源肠道益生菌的分离鉴定及其体外益生特性分析

为了筛选出优良的鱼源益生菌,作者以吉富罗非鱼(Oreochromis niloticus)为实验材料,进行肠道芽孢杆菌和乳酸菌的分离、鉴定,并对分离得到的菌株进行抑菌活性、耐酸性、耐胆盐、人工胃肠液耐受、溶血活性和抗生素敏感性等体外益生特性分析。结果表明,从罗非鱼肠道中共分离得到109株菌,其中包括79株乳酸菌和30株芽孢杆菌,经过一系列的实验最终筛选出3株乳酸乳球菌（Lactococcus lactis）。生物学特性研究表明,3株菌均无溶血活性,对抗生素具有一定的敏感性,对强酸、胆盐和人工胃肠液具有一定的耐受性,对水产病原菌具有不同的抑菌活性。筛选得到的3株菌丰富了鱼源益生菌资源,它们均可作为优良的鱼源益生菌候选菌株。其中,菌株R1对强酸和人工胃肠液耐受性最强,菌株R29耐胆盐能力最强,可进行进一步的益生特性研究,挖掘其作为食品源益生菌的益生效果。     

Fermented whey dairy beverage offers protection against Salmonella enterica ssp. enterica serovar Typhimurium infection in mice

Fermented whey dairy beverages are dairy products obtained by fermentation from a mixture of milk and whey. These beverages have important health benefits, which could be improved with the addition of probiotic cultures. This study assessed the protective effect of the cosupplementation of a probiotic culture (Lactobacillus casei 01) with a fermented whey dairy beverage against infection by Salmonella enterica ssp. enterica serovar Typhimurium in a murine model. Two fermented whey dairy beverages were prepared: conventional (FWB; starter culture) and probiotic (PFWB; starter and probiotic cultures). In the first set of experiments, Balb/C female mice were treated with FWB or PFWB, challenged with Salmonella Typhimurium, and analyzed for clinical signs, weight loss, and mortality for 20 d postinfection. In the second set of experiments, mice were treated with FWB or PFWB, challenged with Salmonella Typhimurium, and killed on d 10 postinfection. The liver, colon, and ileum were used for myeloperoxidase, eosinophil peroxidase, and histological analysis and translocation to the liver. The contents from the small intestine were used for secretory IgA determination. The FWB treatment showed a better effect on animal survival (70%), translocation of the pathogen to the liver (2 out of 10), histopathology (fewer lesions), and inflammation than PFWB, which presented 50% animal survival, translocation in 5 out of 10 animals, and higher lesions. The control group presented 40% animal survival, translocation in 6 out of 10 animals, and severe lesions. Therefore, FWB was deemed to have a greater protective effect against Salmonella Typhimurium infection in the murine model compared with PFWB.     

The intestinal microbiota and its modulation for Salmonella control in chickens

Salmonella control has been one of the major tasks in poultry production to ensure poultry food safety. Effective control is difficult due to the numerous potential sources of Salmonella infection and product contamination in integrated poultry enterprises. In addition, the lack of clinical symptoms of infection in poultry further increases the challenge of achieving proper control. The chicken intestinal microbiota plays an important role in the host health and its modulation has achieved considerable success in the past to reduce Salmonella contamination at the farm level. The use of pro/prebiotics has been the major approach in modulating the microbiota. There is increased consumer pressure demanding the reduction of antibiotics in feed and elimination of Salmonella from poultry and their products. Modulation of the chicken intestinal microbiota continues to offer an attractive option for natural control and sustainable chicken production. In this article, the role of intestinal microbiota in chicken health and the progress in microbiota modulation for Salmonella control have been reviewed. Major technologies for modulating intestinal microbiota, such as the use of probiotics, prebiotics, or phytobiotics (essential oils) and phage therapy that have either received much attention in research and application or demonstrated promise in the development of novel techniques for Salmonella control, are discussed. The potential and drawbacks of each of these are also critically reviewed.