Rapid proteomic characterization of bacteriocin-producing Enterococcus faecium strains from foodstuffs

Enterococcus belongs to a group of microorganisms known as lactic acid bacteria (LAB), which constitute a broad heterogeneous group of generally food-grade microorganisms historically used in food preservation. Enterococci live as commensals of the gastrointestinal tract of warm-blooded animals, although they also are present in food of animal origin (milk, cheese, fermented sausages), vegetables, and plant materials because of their ability to survive heat treatments and adverse environmental conditions. The biotechnological traits of enterococci can be applied in the food industry; however, the emergence of enterococci as a cause of nosocomial infections makes their food status uncertain. Recent advances in high-throughput sequencing allow the subtyping of bacterial pathogens, but it cannot reflect the temporal dynamics and functional activities of microbiomes or bacterial isolates. Moreover, genetic analysis is based on sequence homologies, inferring functions from databases. Here, we used an end-to-end proteomic workflow to rapidly characterize two bacteriocin-producing Enterococcus faecium (Efm) strains. The proteome analysis was performed with liquid chromatography coupled to a trapped ion mobility spectrometry-time-of-flight mass spectrometry instrument (TimsTOF) for high-throughput and high-resolution characterization of bacterial proteins. Thus, we identified almost half of the proteins predicted in the bacterial genomes (>1100 unique proteins per isolate), including quantifying proteins conferring resistance to antibiotics, heavy metals, virulence factors, and bacteriocins. The obtained proteomes were annotated according to function, resulting in 22 complete KEGG metabolic pathway modules for both strains. The workflow used here successfully characterized these bacterial isolates and showed great promise for determining and optimizing the bioengineering and biotechnology properties of other LAB strains in the food industry

Potential immunosuppressive effects of Escherichia coli O157:H7 experimental infection on the bovine host

Background: Enterohaemorrhagic Escherichia coli (EHEC), like E. coli O157:H7 are frequently detected in bovine faecal samples at slaughter. Cattle do not show clinical symptoms upon infection, but for humans the consequences after consuming contaminated beef can be severe. The immune response against EHEC in cattle cannot always clear the infection as persistent colonization and shedding in infected animals over a period of months often occurs. In previous infection trials, we observed a primary immune response after infection which was unable to protect cattle from reinfection. These results may reflect a suppression of certain immune pathways, making cattle more prone to persistent colonization after re-infection. To test this, RNA-Seq was used for transcriptome analysis of recto-anal junction tissue and ileal Peyer's patches in nine Holstein-Friesian calves in response to a primary and secondary Escherichia coli O157: H7 infection with the Shiga toxin (Stx) negative NCTC12900 strain. Non-infected calves served as controls. Results: In tissue of the recto-anal junction, only 15 genes were found to be significantly affected by a first infection compared to 1159 genes in the ileal Peyer's patches. Whereas, re-infection significantly changed the expression of 10 and 17 genes in the recto-anal junction tissue and the Peyer's patches, respectively. A significant downregulation of 69 immunostimulatory genes and a significant upregulation of seven immune suppressing genes was observed. Conclusions: Although the recto-anal junction is a major site of colonization, this area does not seem to be modulated upon infection to the same extent as ileal Peyer's patches as the changes in gene expression were remarkably higher in the ileal Peyer's patches than in the recto-anal junction during a primary but not a secondary infection. We can conclude that the main effect on the transcriptome was immunosuppression by E. coli O157: H7 (Stx(-)) due to an upregulation of immune suppressive effects (7/12 genes) or a downregulation of immunostimulatory effects (69/94 genes) in the ileal Peyer's patches. These data might indicate that a primary infection promotes a re-infection with EHEC by suppressing the immune function

Mold contamination and total aflatoxin content in marketed raw milk in Zagazig city, Egypt

Milk contains a lot of bioactive peptides, vitamins, and trace minerals including calcium and magnesium. Mold contamination of milk and aflatoxin formation are major concerns in the food industry. One of the primary tasks of the food safety and public health sectors is to ensure that the population receives safe animal products. Given these considerations, the current investigation attempted to examine into mold contamination of retailed raw milk from cattle, buffaloes, and sheep. Furthermore, the total aflatoxins in the analyzed samples were estimated, and their potential health risks were explored further. The obtained results revealed that cattle milk had the highest mold contamination, followed by buffalo and sheep milk, with 60%, 40%, and 35%, respectively. In the current study, the identification of distinct mold species indicated four mold genera recovered from the milk samples, namely Aspergillus spp., Penicillium spp., Cladosporium spp., and Fusarium spp. Aspergillus spp. was the most prevalent mold genera isolated from the milk samples of cattle, sheep, and buffaloes, with 34%, 13.2%, and 11.3%, respectively. The mean total aflatoxins (ppb) levels in the milk samples tested were 5.05±0.25 (cattle), 4.22±0.18 (buffaloes), and 3.1±0.11 (sheep), respectively. In conclusion, mold contamination was found in retailed raw milk from cattle, buffaloes, and sheep in Zagazig, Egypt. Aflatoxin was found in several samples. As a result, efficient heat treatment of milk to pasteurization temperatures and avoidance of raw milk consumption are strongly advised

Mold contamination and total aflatoxin content in marketed raw milk in Zagazig city, Egypt

Milk contains a lot of bioactive peptides, vitamins, and trace minerals including calcium and magnesium. Mold contamination of milk and aflatoxin formation are major concerns in the food industry. One of the primary tasks of the food safety and public health sectors is to ensure that the population receives safe animal products. Given these considerations, the current investigation attempted to examine into mold contamination of retailed raw milk from cattle, buffaloes, and sheep. Furthermore, the total aflatoxins in the analyzed samples were estimated, and their potential health risks were explored further. The obtained results revealed that cattle milk had the highest mold contamination, followed by buffalo and sheep milk, with 60%, 40%, and 35%, respectively. In the current study, the identification of distinct mold species indicated four mold genera recovered from the milk samples, namely Aspergillus spp., Penicillium spp., Cladosporium spp., and Fusarium spp. Aspergillus spp. was the most prevalent mold genera isolated from the milk samples of cattle, sheep, and buffaloes, with 34%, 13.2%, and 11.3%, respectively. The mean total aflatoxins (ppb) levels in the milk samples tested were 5.05±0.25 (cattle), 4.22±0.18 (buffaloes), and 3.1±0.11 (sheep), respectively. In conclusion, mold contamination was found in retailed raw milk from cattle, buffaloes, and sheep in Zagazig, Egypt. Aflatoxin was found in several samples. As a result, efficient heat treatment of milk to pasteurization temperatures and avoidance of raw milk consumption are strongly advised

Selection, Molecular Identification and Testing of Potentially Probiotic Bacteria Recovered from Popular Artisanal Egyptian Cheeses

The present study was aimed to select potential probiotic and functional strains among lactic acid bacteria (LAB) isolated from some artisanal Egyptian dairy products. For this, 75 samples comprising karish (fresh skimmed), mish (aged salted skimmed) and Domiati (brined ripened) cheese varieties were surveyed in this study. Approximately, 300 lactic acid bacterial (LAB) strains were isolated and initially screened for their antibacterial activity against the two common food-borne pathogens; Staphylococcus aureus and Escherichia coli O157:H7. Using cell free extract of isolated LAB strains, various antimicrobial inhibition patterns have been noted against the tested pathogens. Upon further proteolytic and neutralization treatments, only 16 strains were proved to have potent antimicrobial attribute. Based on 16S rRNA gene sequencing identification of those 16 strains, 8 different species were identified (Lactobacillus plantarum, Lb. fermentum, Pediococcus acidilacticii and Enterococcus faecium). Additionally, those 16 strains were tested for different probiotic, functional and safety criteria (acid and bile resistance, salt tolerance, milk acidification ability, heamolytic activity and antibiotic sensitivity).  The present study showed that artisanal Egyptian artisanal chesses were proven to be sources of safe potentially probiotic LAB with interesting physiological properties, thus it could be further incorporated in manufacture of various dairy products as starter and non-starter cultures