Novel Salmonella Phage, vB_Sen_STGO-35-1, Characterization and Evaluation in Chicken Meat.

Salmonellosis is one of the most frequently reported zoonotic foodborne diseases worldwide, and poultry is the most important reservoir of Salmonella enterica serovar Enteritidis. The use of lytic bacteriophages (phages) to reduce foodborne pathogens has emerged as a promising biocontrol intervention for Salmonella spp. Here, we describe and evaluate the newly isolated Salmonella phage STGO-35-1, including: (i) genomic and phenotypic characterization, (ii) an analysis of the reduction of Salmonella in chicken meat, and (iii) genome plasticity testing. Phage STGO-35-1 represents an unclassified siphovirus, with a length of 47,483 bp, a G + C content of 46.5%, a headful strategy of packaging, and a virulent lifestyle. Phage STGO-35-1 reduced S. Enteritidis counts in chicken meat by 2.5 orders of magnitude at 4 °C. We identified two receptor-binding proteins with affinity to LPS, and their encoding genes showed plasticity during an exposure assay. Phenotypic, proteomic, and genomic characteristics of STGO-35-1, as well as the Salmonella reduction in chicken meat, support the potential use of STGO-35-1 as a targeted biocontrol agent against S. Enteritidis in chicken meat. Additionally, computational analysis and a short exposure time assay allowed us to predict the plasticity of genes encoding putative receptor-binding proteins.

Comparative Analysis of Felixounavirus Genomes Including Two New Members of the Genus That Infect Salmonella Infantis.

Salmonella spp. is one of the most common foodborne pathogens worldwide; therefore, its control is highly relevant for the food industry. Phages of the Felixounavirus genus have the characteristic that one phage can infect a large number of different Salmonella serovars and, thus, are proposed as an alternative to antimicrobials in food production. Here, we describe two new members of the Felixounavirus genus named vB_Si_35FD and vB_Si_DR94, which can infect Salmonella Infantis. These new members were isolated and sequenced, and a subsequent comparative genomic analysis was conducted including 23 publicly available genomes of Felixounaviruses that infect Salmonella. The genomes of vB_Si_35FD and vB_Si_DR94 are 85,818 and 85,730 bp large and contain 129 and 125 coding sequences, respectively. The genomes did not show genes associated with virulence or antimicrobial resistance, which could be useful for candidates to use as biocontrol agents. Comparative genomics revealed that closely related Felixounavirus are found in distinct geographical locations and that this genus has a conserved genomic structure despite its worldwide distribution. Our study revealed a highly conserved structure of the phage genomes, and the two newly described phages could represent promising biocontrol candidates against Salmonella spp. from a genomic viewpoint.

Screening the Presence of Non-Typhoidal Salmonella in Different Animal Systems and the Assessment of Antimicrobial Resistance.

Salmonella is a major bacterial foodborne pathogen that causes the majority of worldwide food-related outbreaks and hospitalizations. Salmonellosis outbreaks can be caused by multidrug-resistant (MDR) strains, emphasizing the importance of maintaining public health and safer food production. Nevertheless, the drivers of MDR Salmonella serovars have remained poorly understood. In this study, we compare the resistance profiles of Salmonella strains isolated from 4047 samples from domestic and wild animals in Chile. A total of 106 Salmonella strains (2.61%) are isolated, and their serogroups are characterized and tested for susceptibility to 16 different antimicrobials. The association between antimicrobial resistance (AMR) and a subset of independent variables is evaluated using multivariate logistic models. Our results show that 47 antimicrobial-resistant strains were found (44.3% of the total strains). Of the 47, 28 correspond to single-drug resistance (SDR = 26.4%) and 19 are MDR (17.9%). S. Enteritidis is highly persistent in animal production systems; however, we report that serogroup D strains are 18 times less likely to be resistant to at least one antimicrobial agent than the most common serogroup (serogroup B). The antimicrobials presenting the greatest contributions to AMR are ampicillin, streptomycin and tetracycline. Additionally, equines and industrial swine are more likely to acquire Salmonella strains with AMR. This study reports antimicrobial-susceptible and resistant Salmonella in Chile by expanding the extant literature on the potential variables affecting antimicrobial-resistant Salmonella.

Salmonella in Raptors and Aquatic Wild Birds in Chile.

Salmonella enterica is one of the main causes of gastrointestinal disease worldwide. Wild birds are capable of harboring a variety of Salmonella serovars, which could have an important role in the epidemiology of salmonellosis in humans and production animals. We tested 519 fecal samples from raptors and aquatic birds from different regions of central (three rehabilitation centers for wildlife and the coastal area) and southern areas of Chile for Salmonella. All samples were obtained in 2015 and 2017, covering all four seasons. Salmonella was isolated from 12 of the 519 samples (2%) analyzed, from two carnivorous birds, four birds with generalist habits, and six waterfowl. Among the isolates obtained, one showed resistance to gentamicin, and one showed a multidrug-resistance phenotype, with resistance to ampicillin, ceftriaxone, ciprofloxacin, chloramphenicol, streptomycin, gentamicin, kanamycin, trimethoprim-sulfamethoxazole, and tetracycline. These results demonstrated the importance of characterizing Salmonella in wild birds because previous studies have shown genetic and phenotypic evidence suggesting interspecies transmission of Salmonella enterica that is resistant to antimicrobials between humans and wild and domestic birds.

Backyard Farms Represent a Source of Wide Host Range Salmonella Phages That Lysed the Most Common Salmonella Serovars.

The genus Salmonella has more than 2,600 serovars, and this trait is important when considering interventions for Salmonella control. Bacteriophages that are used for biocontrol must have an exclusively lytic cycle and the ability to lyse several Salmonella serovars under a wide range of environmental conditions. Salmonella phages were isolated and characterized from 34 backyard production systems (BPSs) with a history of Salmonella infections. BPSs were visited once, and cloacal or fecal samples were processed for phage isolation. Four hosts, Salmonella serovars Enteritidis, Heidelberg, Infantis, and Typhimurium, were used for phage isolation. The host range of the phages was later characterized with a panel of 23 Salmonella serovars (serovar diversity set) and 31 isolates obtained from the same farms (native set). Genetic relatedness for 10 phages with a wide host range was characterized by restriction fragment length polymorphism, and phages clustered based on the host range. We purified 63 phages, and 36 phage isolates were obtained on Salmonella Enteritidis, 16 on Salmonella Heidelberg, and 11 on Salmonella Infantis. Phages were classified in three clusters: (i) phages with a wide host range (cluster I), (ii) phages that lysed the most susceptible Salmonella serovars (serogroup D) and other isolates (cluster II), and (iii) phages that lysed only isolates of serogroup D (cluster III). The most susceptible Salmonella serovars were Enteritidis, Javiana, and Dublin. Seven of 34 farms yielded phages with a wide host range, and these phages had low levels of genetic relatedness. Our study showed an adaptation of the phages in the sampled BPSs to serogroup D Salmonella isolates and indicated that isolation of Salmonella phages with wide host range differs by farm. A better understanding of the factors driving the Salmonella phage host range could be useful when designing risk-based sampling strategies to obtain phages with a wide lytic host range for biocontrol purposes.

Short communication: Characterization of Salmonella phages from dairy calves on farms with history of diarrhea.

Salmonella enterica can cause disease and mortality in calves. This pathogen is also a zoonosis that can be transmitted by animal contact or by food. The prevalence of Salmonella in dairy farms has been reported to range from 0 to 64%, and, due to the diversity of Salmonella serovars that can be circulating, Salmonella is an important concern for dairy production. Bacteriophages that infect Salmonella have been documented to be abundant and widely distributed in the dairy environment. The current study investigated the diversity of Salmonella serovars and Salmonella phages in 8 dairy farms with a history of diarrhea in southern Chile. A total of 160 samples from sick calves, healthy calves, and the environment were analyzed for Salmonella and phage. Isolated phages were characterized and classified by their host range using a panel of 26 Salmonella isolates representing 23 serovars. Host ranges were classified according to lysis profiles (LP) and their spatial distribution was mapped. Salmonella-infecting phages were identified, but none of the 160 samples were positive for Salmonella. A total of 45 phage isolates were obtained from sick calves (11), healthy calves (16), or the environment (18). According to their host range, 19 LP were identified, with LP1 being the most common on all 8 farms; LP1 represents phages that only lyse serogroup D Salmonella. The identification of Salmonella phages but not Salmonella in the same samples could suggest that these phages are controlling Salmonella in these farms.

Hepatogenic and neurogenic differentiation of bone marrow mesenchymal stem cells from abattoir-derived bovine fetuses.

BACKGROUND: Mesenchymal stem cells (MSC) are multipotent progenitor cells characterized by their ability to both self-renew and differentiate into tissues of mesodermal origin. The plasticity or transdifferentiation potential of MSC is not limited to mesodermal derivatives, since under appropriate cell culture conditions and stimulation by bioactive factors, MSC have also been differentiated into endodermal (hepatocytes) and neuroectodermal (neurons) cells. The potential of MSC for hepatogenic and neurogenic differentiation has been well documented in different animal models; however, few reports are currently available on large animal models. In the present study we sought to characterize the hepatogenic and neurogenic differentiation and multipotent potential of bovine MSC (bMSC) isolated from bone marrow (BM) of abattoir-derived fetuses. RESULTS: Plastic-adherent bMSC isolated from fetal BM maintained a fibroblast-like morphology under monolayer culture conditions. Flow cytometric analysis demonstrated that bMSC populations were positive for MSC markers CD29 and CD73 and pluripotency markers OCT4 and NANOG; whereas, were negative for hematopoietic markers CD34 and CD45. Levels of mRNA of hepatic genes α-fetoprotein (AFP), albumin (ALB), alpha1 antitrypsin (α1AT), connexin 32 (CNX32), tyrosine aminotransferase (TAT) and cytochrome P450 (CYP3A4) were up-regulated in bMSC during a 28-Day period of hepatogenic differentiation. Functional analyses in differentiated bMSC cultures evidenced an increase (P < 0.05) in albumin and urea production and glycogen storage. bMSC cultured under neurogenic conditions expressed NESTIN and MAP2 proteins at 24 h of culture; whereas, at 144 h also expressed TRKA and PrPC. Levels of MAP2 and TRKA mRNA were up-regulated at the end of the differentiation period. Conversely, bMSC expressed lower levels of NANOG mRNA during both hepatogenic and neurogenic differentiation processes. CONCLUSION: The expression patterns of linage-specific markers and the production of functional metabolites support the potential for hepatogenic and neurogenic differentiation of bMSC isolated from BM of abattoir-derived fetuses. The simplicity of isolation and the potential to differentiate into a wide variety of cell lineages lays the foundation for bMSC as an interesting alternative for investigation in MSC biology and eventual applications for regenerative therapy in veterinary medicine.

Isolation and multilineage differentiation of bone marrow mesenchymal stem cells from abattoir-derived bovine fetuses.

BACKGROUND: Mesenchymal stem cells (MSC) are multipotent progenitor cells localized in the stromal compartment of the bone marrow (BM). The potential of MSC for mesenchymal differentiation has been well documented in different animal models predominantly on rodents. However, information regarding bovine MSC (bMSC) is limited, and the differentiation potential of bMSC derived from fetal BM remains unknown. In the present study we sought to isolate bMSC from abattoir-derived fetal BM and to characterize the multipotent and differentiation potential under osteogenic, chondrogenic and adipogenic conditions by quantitative and qualitative analyses. RESULTS: Plastic-adherent bMSC isolated from fetal BM maintained a fibroblast-like morphology under monolayer culture conditions. These cells expressed high levels of MSC surface markers (CD73, CD90, and CD105) and low levels of hematopoietic surface markers (CD34 and CD45). Culture of bMSC under osteogenic conditions during a 27-day period induced up-regulation of the osteocalcin (OC) gene expression and alkaline phosphatase (ALPL) activity, and promoted mineralization of the matrix. Increasing supplementation levels of ascorbic acid to culture media enhanced osteogenic differentiation of bMSC; whereas, reduction of FBS supplementation compromised osteogenesis. bMSC increased expression of cartilage-specific genes aggrecan (ACAN), collagen 2A1 (COL2A1) and SRY (sex-determining region Y) box 9 (SOX9) at Day 21 of chondrogenic differentiation. Treatment of bMSC with adipogenic factors increased levels of fatty acid-binding protein 2 (AP2) mRNA and accumulation of lipid vacuoles after 18 days of culture. NANOG mRNA levels in differentiating bMSC were not affected during adipogenic culture; however, osteogenic and chondrogenic conditions induced higher and lower levels, respectively. CONCLUSIONS: Our analyses revealed the potential multilineage differentiation of bMSC isolated from abattoir-derived fetal BM. NANOG mRNA pattern in differentiating bMSC varied according to differentiation culture conditions. The osteogenic differentiation of bMSC was affected by ascorbic acid and FBS concentrations in culture media. The simplicity of isolation and the differentiation potential suggest that bMSC from abattoir-derived fetal BM are appropriate candidate for investigating MSC biology and for eventual applications for regenerative therapy.