Effective reduction of Salmonella Enteritidis in broiler chickens using the UPWr_S134 phage cocktail.

Salmonella is a poultry-associated pathogen that is considered one of the most important zoonotic bacterial agents of contaminated food of animal origin including poultry products. Many efforts are taken to eliminate it from the food chain, and phages are one of the most promising tools to control Salmonella in poultry production. We investigated the usefulness of the UPWr_S134 phage cocktail in reducing Salmonella in broiler chickens. For this purpose, we analyzed the survivability of phages in the harsh environment encountered in the chicken gastrointestinal tract, which has low pH, high temperatures, and digestive activity. Phages in the cocktail UPWr_S134 showed the ability to remain active after storage at temperatures ranging from 4 to 42°C, reflecting temperatures of storage conditions, broiler handling, and the chicken body, and exhibited robust pH stability. We found that although simulated gastric fluids (SGF) caused phage inactivation, the addition of feed to gastric juice allows maintenance of UPWr_S134 phage cocktail activity. Further, we analyzed UPWr_S134 phage cocktail anti-Salmonella activity in live animals such as mice and broilers. In an acute infection model in mice, the application of doses of 107 and 1014 PFU/ml UPWr_S134 phage cocktail resulted in delaying symptoms of intrinsic infection in all analyzed treatment schedules. In Salmonella-infected chickens orally treated with the UPWr_S134 phage cocktail the number of pathogens in internal organs in comparison to untreated birds was significantly lower. Therefore we concluded that the UPWr_S134 phage cocktail could be an effective tool against this pathogen in the poultry industry.

Characterization and Comparative Genomic Analysis of Three Virulent E. coli Bacteriophages with the Potential to Reduce Antibiotic-Resistant Bacteria in the Environment.

The emerging global crisis of antibiotic resistance demands new alternative antibacterial solutions. Although bacteriophages have been used to combat bacterial infections for over a century, a dramatic boost in phage studies has recently been observed. In the development of modern phage applications, a scientific rationale is strongly required and newly isolated phages need to be examined in detail. In this study, we present the full characterization of bacteriophages BF9, BF15, and BF17, with lytic activity against extended-spectrum ß-lactamases (ESBLs)- and AmpC ß-lactamases (AmpC)-producing Escherichia coli, the prevalence of which has increased significantly in livestock in recent decades, representing a great hazard to food safety and a public health risk. Comparative genomic and phylogenetic analysis indicated that BF9, BF15, and BF17 represent the genera Dhillonvirus, Tequatrovirus, and Asteriusvirus, respectively. All three phages significantly reduced in vitro growth of their bacterial host and retained the ability to lyse bacteria after preincubation at wide ranges of temperature (-20-40 °C) and pH (5-9). The results described herein indicate the lytic nature of BF9, BF15, and BF17, which, along with the absence of genes encoding toxins and bacterial virulence factors, represents an undoubted asset in terms of future phage application.

Bacteriophage Cocktail Can Effectively Control Salmonella Biofilm in Poultry Housing.

Salmonella enterica serovar Enteritidis (S. Enteritidis) is the major contaminant of poultry products, and its ability to form biofilms on produced food and poultry farm processing surfaces contributes to Salmonella transmission to humans. Bacteriophages have come under increasing interest for anti-Salmonella biofilm control. In this study, we used the three previously sequenced and described phages UPWr_S1, UPWr_S3, and UPWr_S4 and a phage cocktail, UPWr_S134, containing these three phages to degrade biofilms formed by two S. Enteritidis strains, 327 lux and ATCC 13076, in vitro. It was found that treatment with bacteriophages significantly reduced biofilm on a 96-well microplate (32-69%) and a stainless steel surface (52-98%) formed by S. Enteritidis 327 lux. The reduction of biofilm formed by S. Enteritidis ATCC 13076 in the 96-well microplate and on a stainless steel surface for bacteriophage treatment was in the range of 73-87% and 60-97%, respectively. Under laboratory conditions, an experimental model utilizing poultry drinkers artificially contaminated with S. Enteritidis 327 lux and treated with UPWr_S134 phage cocktail was applied. In in vitro trials, the phage cocktail significantly decreased the number of Salmonella on the surface of poultry drinkers. Moreover, the phage cocktail completely eradicated Salmonella from the abundant bacterial load on poultry drinkers in an experimentally infected chickens. Therefore, the UPWr_S134 phage cocktail is a promising candidate for Salmonella biocontrol at the farm level.

Genomic and functional characterization of five novel Salmonella-targeting bacteriophages.

BACKGROUND: The host-unrestricted, non-typhoidal Salmonella enterica serovar Enteritidis (S. Enteritidis) and the serovar Typhimurium (S. Typhimurium) are major causative agents of food-borne gastroenteritis, and the host-restricted Salmonella enterica serovar Gallinarum (S. Gallinarum) is responsible for fowl typhoid. Increasing drug resistance in Salmonella contributes to the reduction of effective therapeutic and/or preventive options. Bacteriophages appear to be promising antibacterial tools, able to combat infectious diseases caused by a wide range of Salmonella strains belonging to both host-unrestricted and host-restricted Salmonella serovars. METHODS: In this study, five novel lytic Salmonella phages, named UPWr_S1-5, were isolated and characterized, including host range determination by plaque formation, morphology visualization with transmission electron microscopy, and establishment of physiological parameters. Moreover, phage genomes were sequenced, annotated and analyzed, and their genomes were compared with reference Salmonella phages by use of average nucleotide identity, phylogeny, dot plot, single nucleotide variation and protein function analysis. RESULTS: It was found that UPWr_S1-5 phages belong to the genus Jerseyvirus within the Siphoviridae family. All UPWr_S phages were found to efficiently infect various Salmonella serovars. Host range determination revealed differences in host infection profiles and exhibited ability to infect Salmonella enterica serovars such as Enteritidis, Gallinarum, Senftenberg, Stanley and Chester. The lytic life cycle of UPWr_S phages was confirmed using the mitomycin C test assay. Genomic analysis revealed that genomes of UPWr_S phages are composed of 51 core and 19 accessory genes, with 33 of all predicted genes having assigned functions. UPWr_S genome organization comparison revealed 3 kinds of genomes and mosaic structure. UPWr_S phages showed very high sequence similarity to each other, with more than 95% average nucleotide identity. CONCLUSIONS: Five novel UPWr_S1-5 bacteriophages were isolated and characterized. They exhibit host lysis range within 5 different serovars and are efficient in lysis of both host-unrestricted and host-restricted Salmonella serovars. Therefore, because of their ability to infect various Salmonella serovars and lytic life cycle, UPWr_S1-5 phages can be considered as useful tools in biological control of salmonellosis.

Application of a New Engineered Strain of Yarrowia lipolytica for Effective Production of Calcium Ketoglutarate Dietary Supplements.

The present study aimed to develop a technology for the production of dietary supplements based on yeast biomass and α-ketoglutaric acid (KGA), produced by a new transformant of Yarrowia lipolytica with improved KGA biosynthesis ability, as well to verify the usefulness of the obtained products for food and feed purposes. Transformants of Y. lipolytica were constructed to overexpress genes encoding glycerol kinase, methylcitrate synthase and mitochondrial organic acid transporter. The strains were compared in terms of growth ability in glycerol- and oil-based media as well as their suitability for KGA biosynthesis in mixed glycerol-oil medium. The impact of different C:N:P ratios on KGA production by selected strain was also evaluated. Application of the strain that overexpressed all three genes in the culture with a C:N:P ratio of 87:5:1 allowed us to obtain 53.1 g/L of KGA with productivity of 0.35 g/Lh and yield of 0.53 g/g. Finally, the possibility of obtaining three different products with desired nutritional and health-beneficial characteristics was demonstrated: (1) calcium α-ketoglutarate (CaKGA) with purity of 89.9% obtained by precipitation of KGA with CaCO3, (2) yeast biomass with very good nutritional properties, (3) fixed biomass-CaKGA preparation containing 87.2 µg/g of kynurenic acid, which increases the health-promoting value of the product.

Bacteriophage amplification - A comparison of selected methods.

The bactericidal properties of bacteriophages have been used almost since the moment of the discovery of bacterial viruses. In the light of the rapidly growing number of antibiotic-resistant bacteria, phage therapy is considered one of the most promising alternatives to classical treatment. Phage amplification is one of the most common procedures of working with phages, and high-titer preparations are beneficial at the experimental stage of studies as well as in practice. The objective of this study was to compare five commonly applied methods of phage amplification: (i) pooled plaques method, (ii) the plate wash method, (iii) the agar culture method, (iv) the two-stage culture method, and (v) in liquid culture. All methods were tested for fifteen different phages. The results described herein indicate that there is no optimal, universal method for phage amplification, and the most effective method has to be established individually for each phage.

The Efficacy of Isolated Bacteriophages from Pig Farms against ESBL/AmpC-Producing Escherichia coli from Pig and Turkey Farms.

Extended-spectrum ß-lactamases (ESBLs) and AmpC ß-lactamases are plasmid (but also chromosomally) encoded enzymes found in Enterobacteriaceae, determining resistance to a variety of important antibiotics including penicillins, cephalosporins, and monobactams. In recent decades, the prevalence of ESBL/AmpC-producing bacteria has increased rapidly across the world. Here, we evaluate the potential use of bacteriophages in terms of a reduction of antibiotic-resistant bacteria in healthy animals. The aim of our studies was to isolate bacteriophages capable of destroying ESBL/AmpC-producing Escherichia coli isolated from livestock habitats. The efficacy of isolated phages against ESBL/AmpC E. coli strains varies, but creation of a phage cocktail with broad activity spectrum is possible. This may indicate that the role of phages may not be limited to phage therapy, but bacterial viruses may also be applied against spread of bacteria with antibiotic resistance genes in the environment. We also addressed the hypothesis, that phages, effective for therapeutic purposes may be isolated from distant places and even from different environments other than the actual location of the targeted bacteria. This may be beneficial for practical purposes, as the construction of effective phage preparations does not require access to disease outbreaks.

Type 1 fimbriae are important factors limiting the dissemination and colonization of mice by Salmonella Enteritidis and contribute to the induction of intestinal inflammation during Salmonella invasion.

We have recently shown that Salmonella Gallinarum type 1 fimbriae with endogenous mannose-resistant (MR) variant of the FimH protein increase systemic dissemination of S. Gallinarum and colonization of internal organs in comparison to the S. Gallinarum fimH knockout strain or the mutant expressing mannose-sensitive (MS) FimH variant from S. Enteritidis. Elaborating from these studies, we proposed that MS variants of FimH are advantageous in gastrointestinal infections, in contrast to MR FimH variants which decrease intestinal colonization and promote their systemic spreading. To support our hypothesis, we carried out in vivo studies using mice infected with wild-type S. Enteritidis and its fimH knockout strain (S. Enteritidis), which was characterized by significantly lower adhesion and invasiveness of murine ICE-1 intestinal cells. Using bioluminescence imaging, we observed that the loss of MS FimH adhesin correlates well with the highly increased colonization of mice by these bacteria. The appearance of the mutant strain was observed much earlier than wild-type Salmonella, and mice infected with 10(4)-10(7) S. Enteritidis fimH::kan CFUs had significantly (P < 0.05) shorter infection-free time than animals inoculated with wild-type S. Enteritidis. Infections caused by non-typhoid Salmonella, such as S. Enteritidis, are associated with massive inflammation of the lamina propria and lymph nodes in the intestinal tract. Therefore, we evaluated the role of MS type 1 fimbriae in the induction of cytokine expression and secretion, using murine ICE-1 intestinal cells. We showed that the expression, as well as secretion, of Il-1b, Il-6, Il-10, and Il-12b was significantly higher in cells infected with wild-type S. Enteritidis compared to cells infected with the mutant strain. Based on our results, we propose that type 1 fimbriae may play an important role in the pathogenicity of S. Enteritidis and may contribute to an intestinal inflammatory response.

FimH adhesin from host unrestricted Salmonella Enteritidis binds to different glycoprotein ligands expressed by enterocytes from sheep, pig and cattle than FimH adhesins from host restricted Salmonella Abortus-ovis, Salmonella Choleraesuis and Salmonella Dublin.

Adhesion to gut tissues and colonization of the alimentary tract, two important stages in the pathogenesis of Salmonella, are mediated by FimH adhesin of type 1 fimbriae. It was suggested that minor differences in the structure of FimH are most likely associated with differences in adhesion specificities, and may determine the tropism of various Salmonella serovars to different species and tissues. We investigated this hypothesis by comparing the binding properties of FimH proteins from three Salmonella enterica serovars with limited (Choleraesuis, Dublin) or restricted (Abortusovis) host ranges to FimH from broad host range S. Enteritidis and mannose inactive FimH from S. Gallinarum. Although all active variants of FimH protein were able to bind mannose-rich glycoproteins (RNase B, HRP and Man-BSA) with comparable affinity measured by surface plasmon resonance, there were significant differences in the binding profiles of the FimH proteins from host restricted serovars and host unrestricted serovar Enteritidis, to glycoproteins from enterocyte cell lines established in vitro and derived from sheep, pig and cattle. When low-binding FimH adhesin from S. Enteritidis was subjected to Western blot analysis, it bound to surface membrane protein of about 130 kDa, and high-binding FimH adhesins from S. Abortusovis, S. Choleraesuis and S. Dublin bound to surface membrane protein of about 55 kDa present in each cell line. Differential binding of FimH proteins from host-restricted and broad-host-range Salmonella to intestinal receptors was confirmed using mutant FimH adhesins obtained by site-directed mutagenesis. It was found that the low-binding variant of FimH from S. Choleraesuis with mutation Leu57Pro lost the ability to bind protein band of 55 kDa, but instead interacted with glycoprotein of about 130 kDa. On the other hand, the high-binding variant of FimH adhesin from S. Enteritids with mutation Asn101Ser did not bind to its receptor of 130 kDa, but instead it interacted with glycoprotein ligand of 55 kDa. These results suggest that FimH adhesins of type 1 fimbriae are one of the factors responsible for different host-specificities of these Salmonella serovars.

Decreased colonization of chicks by Salmonella enterica serovar Gallinarum expressing mannose-sensitive FimH adhesin from Salmonella enterica serovar Enteritidis.

To investigate the role of non-hemagglutinating type 1 fimbriae in the pathogenesis of Salmonella Gallinarum, the isogenic mutant elaborating type 1 fimbriae with mannose-sensitive (MS) variant of the FimH adhesin from Salmonella Enteritidis and the mutant strain with no FimH expression were constructed. Their binding to chicken leukocytes in vitro and invasiveness in 1-day-old chicks were studied. Our results demonstrated that S. Gallinarum type 1 fimbriae with an endogenous variant of the FimH adhesin mediated mannose-resistant (MR) binding to avian leukocytes and did not bind to human epithelial cells. However, after allelic replacement of the FimH, mutated fimbriae with S. Enteritidis variant of the FimH adhesin bound to both cell types in a mannose-dependent manner. In chick model, S. Gallinarum expressing wild-type FimH variant colonized cecal tonsils and bursa of Fabricius more effectively and invaded the spleen and liver in greater numbers than S. Gallinarum fimH knockout strain or mutant expressing MS FimH variant from S. Enteritidis. The invasive potential of the latter was greatly reduced in chicks since no viable bacteria expressing MS variant of the adhesin could be recovered from intestinal lymphoid tissues or liver over a 6 days course of infection. Together, these results demonstrate that the S. Gallinarum type 1 fimbriae with the endogenous MR variant of the FimH protein increase systemic dissemination of S. Gallinarum and colonization of internal organs in chicks indicating the importance of these adhesive structures in the virulence of S. Gallinarum.

The high-adhesive properties of the FimH adhesin of Salmonella enterica serovar Enteritidis are determined by a single F118S substitution.

The binding properties of low- and high-adhesive forms of FimH adhesins from Salmonella enterica serovars Enteritidis and Typhimurium (S. Enteritidis and S. Typhimurium) were studied using chimeric proteins containing an additional peptide that represents an N-terminal extension of the FimF protein. This modification, by taking advantage of a donor strand exchange mechanism, closes the hydrophobic groove in the fimbrial domain of the FimH adhesin. Such self-complemented adhesins (scFimH) did not form aggregates and were more stable (resistant to proteolytic cleavage) than native FimH. High-adhesive variants of scFimH proteins, with alanine at position 61 and serine at position 118, were obtained by site-directed mutagenesis of fimH genes from low-adhesive variants of S. Enteritidis and S. Typhimurium, with glycine at position 61 and phenylalanine at position 118. Direct kinetic analysis using surface plasmon resonance (SPR) and glycoproteins carrying high-mannose carbohydrate chains (RNase B, horseradish peroxidase and mannan-BSA) revealed the existence of high- and low-adhesive allelic variants, not only in S. Typhimurium but also in S. Enteritidis. Using two additional mutants of low-adhesive FimH protein from S. Enteritidis (Gly61Ala and Phe118Ser), SPR analysis pointed to Ser118 as the major determinant of the high-adhesive phenotype of type 1 fimbriae from S. Enteritidis. These studies demonstrated for the first time that the functional differences observed with whole fimbriated bacteria could be reproduced at the level of purified adhesin. They strongly suggest that the adhesive properties of type 1 fimbriae are determined only by structural differences in the FimH proteins and are not influenced by the fimbrial shaft on which the adhesin is located.