Genome-based studies indicate that the Enterococcus faecium Clade B strains belong to Enterococcus lactis species and lack of the hospital infection associated markers.

Enterococcus lactis and the heterotypic synonym Enterococcus xinjiangensis from dairy origin have recently been identified as a novel species based on 16S rRNA gene sequence analysis. Enterococcus faecium type strain NCTC 7171T was used as the reference genome for determining E. lactis and E. faecium to be separate species. However, this taxonomic classification did not consider the diverse lineages of E. faecium, and the double nature of hospital-associated (clade A) and community-associated (clade B) isolates. Here, we investigated the taxonomic relationship among isolates of E. faecium of different origins and E. lactis, using a genome-based approach. Additional to 16S rRNA gene sequence analysis, we estimated the relatedness among strains and species using phylogenomics based on the core pangenome, multilocus sequence typing, the average nucleotide identity and digital DNA-DNA hybridization. Moreover, following the available safety assessment schemes, we evaluated the virulence profile and the ampicillin resistance of E. lactis and E. faecium clade B strains. Our results confirmed the genetic and evolutionary differences between clade A and the intertwined clade B and E. lactis group. We also confirmed the absence in these strains of virulence gene markers IS16, hylEfm and esp and the lack of the PBP5 allelic profile associated with ampicillin resistance. Taken together, our findings support the reassignment of the strains of E. faecium clade B as E. lactis.

Fermentation as a tool for increasing food security and nutritional quality of indigenous African leafy vegetables: the case of Cucurbita sp.

Sub-Saharan region is often characterized by food and nutrition insecurity especially "hidden hunger" which results from inadequate micronutrients in diets. African indigenous leafy vegetables (AILVs) can represent a valid food source of micronutrients, but they often go to waste resulting in post-harvest losses. In an attempt to prolong AILVs shelf-life while enhancing their nutritional quality, fermentation was studied from a microbiological and nutritional point of view. Pumpkin leaves (Cucurbita sp.) were spontaneously fermented using the submerged method with 3% NaCl and 3% sucrose. Controls were set up, consisting of leaves with no additions. During fermentation, samples of both treatments were taken at 0, 24, 48, 72 and 168 h to monitor pH and characterize the microbial population through culture-based and molecular-based analyses. Variations between fresh and treated leaves in B-group vitamins, carotenoids, polyphenols, and phytic acid were evaluated. Data revealed that the treatment with addition of NaCl and sucrose hindered the growth of undesired microorganisms; in controls, unwanted microorganisms dominated the bacterial community until 168 h, while in treated samples Lactobacillaceae predominated. Furthermore, the content in folate, ß-carotene and lutein increased in treated leaves compared to the fresh ones, while phytic acid diminished indicating an amelioration in the nutritional value of the final product. Thus, fermentation could help in preserving Cucurbita sp. leaves, avoiding contamination of spoilage microorganisms and enhancing the nutritional values.

Transcriptome analysis of Bacillus thuringiensis spore life, germination and cell outgrowth in a vegetable-based food model.

Toxigenic species belonging to Bacillus cereus sensu lato, including Bacillus thuringiensis, cause foodborne outbreaks thanks to their capacity to survive as spores and to grow in food matrixes. The goal of this work was to assess by means of a genome-wide transcriptional assay, in the food isolate B. thuringiensis UC10070, the gene expression behind the process of spore germination and consequent outgrowth in a vegetable-based food model. Scanning electron microscopy and Energy Dispersive X-ray microanalysis were applied to select the key steps of B. thuringiensis UC10070 cell cycle to be analyzed with DNA-microarrays. At only 40 min from heat activation, germination started rapidly and in less than two hours spores transformed in active growing cells. A total of 1646 genes were found to be differentially expressed and modulated during the entire B. cereus life cycle in the food model, with most of the significant genes belonging to transport, transcriptional regulation and protein synthesis, cell wall and motility and DNA repair groups. Gene expression studies revealed that toxin-coding genes nheC, cytK and hblC were found to be expressed in vegetative cells growing in the food model.

Adhesive behaviour and virulence of coagulase negative staphylococci isolated from Italian cheeses.

Coagulase-negative staphylococci (CoNS) belong to saprophytic microbiota on the skin and mucous membranes of warm-blooded animals and humans, but are also isolated from foodstuffs such as meat, cheese, and milk. In other circumstances, some CoNS can act as pathogens. Thus the presence of CoNS may not be an immediate danger to public health, but can become a risk factor. In particular antibiotic-resistant genes could be transferred to other potentially pathogenic microorganisms. Furthermore, CoNS are known to be strong biofilm producers and this is also a risk factor for public health. The aim of the present work was to determine the genotypic and phenotypic profiles of 106 CoNS belonging to four different species isolated from five different Italian cheeses for the presence of some adhesion and virulence features. In order to verify a possible correlation between the formation of biofilm and staphylococcal virulence factors, we checked the presence of adhesin genes by PCR and we investigated the ability of these strains to make biofilm at different temperatures. Furthermore, in some conditions, we analyzed surface proteins and autolytic pattern of selected strains. In conclusion, we checked the presence of norA and mecA genes responsible for fluoroquinolones and methicillin resistance, respectively. We found resistant genes in a proportion of the food isolates in amounts of 9.4% (mecA) and 5.7% (norA). These data support the importance to continuously examine the microbiota not only for the creation of a database but also to safeguard public health.

Understanding the bacterial communities of hard cheese with blowing defect.

The environment of hard cheese encourages bacterial synergies and competitions along the ripening process, which might lead in defects such as clostridial blowing. In this study, Denaturing Gradient Gel Electrophoresis (DGGE), a quantitative Clostridium tyrobutyricum PCR and next-generation Illumina-based sequencing of 16S rRNA gene were applied to study 83 Grana Padano spoiled samples. The aim was to investigate the community of clostridia involved in spoilage, the ecological relationships with the other members of the cheese microbiota, and the effect of lysozyme. Three main genera were dominant in the analysed cheeses, Lactobacillus, Streptococcus and Clostridium, and the assignment at the species level was of 94.3% of 4,477,326 high quality sequences. C. tyrobutyricum and C. butyricum were the most prevalent clostridia. Hierarchical clustering based on the abundance of bacterial genera, revealed three main clusters: one characterized by the highest proportion of Clostridium, a second where Lactobacillus was predominant and the last, dominated by Streptococcus thermophilus. Ecological relationships among species were found: cheeses characterized by an high abundance of S. thermophilus and L. rhamnosus were spoiled by C. tyrobutyricum while, when L. delbrueckii was the most abundant Lactobacillus, C. butyricum was the dominant spoiling species. Lysozyme also shaped the bacterial community, reducing C. tyrobutyricum in favour of C. butyricum. Moreover, this preservative increased the proportion of L. delbrueckii and obligate heterofermentative lactobacilli and lowered L. helveticus and non-starter species, such as L. rhamnosus and L. casei.

Enterococcus faecium PBP5-S/R, the missing link between PBP5-S and PBP5-R.

During a study to investigate the evolution of ampicillin resistance in Enterococcus faecium, we observed that a number of E. faecium strains, mainly from the recently described subclade A2, showed PBP5 sequences in between PBP5-S and PBP5-R. These hybrid PBP5-S/R patterns reveal a progression of amino acid changes from the S form to the R form of this protein; however, these changes do not strictly correlate with changes in ampicillin MICs.

Microbiological, physico-chemical, nutritional and sensory characterization of traditional Matsoni: selection and use of autochthonous multiple strain cultures to extend its shelf-life.

Matsoni, a traditional Georgian fermented milk, has variable quality and stability besides a short shelf-life (72-120 h at 6 °C) due to inadequate production and storage conditions. To individuate its typical traits as well as select and exploit autochthonous starter cultures to standardize its overall quality without altering its typicality, we carried out a thorough physico-chemical, sensorial and microbial characterization of traditional Matsoni. A polyphasic approach, including a culture-independent (PCR-DGGE) and two PCR culture-dependent methods, was employed to study the ecology of Matsoni. Overall, the microbial ecosystem of Matsoni resulted largely dominated by Streptococcus (S.) thermophilus and Lactobacillus (Lb.) delbrueckii subsp. bulgaricus. High loads of other lactic acid bacteria species, including Lb. helveticus, Lb. paracasei and Leuconostoc (Leuc.) lactis were found to occur as well. A selected autochthonous multiple strain culture (AMSC) composed of one Lb. bulgaricus, one Lb. paracasei and one S. thermophilus strain, applied for the pilot-scale production of traditional Matsoni, resulted the best in terms of enhanced shelf-life (one month), sensorial and nutritional quality without altering its overall typical quality. This AMSC is at disposal of SMEs who need to exploit and standardize the overall quality of this traditional fermented milk, preserving its typicality.

Evaluating the Genome-Based Average Nucleotide Identity Calculation for Identification of Twelve Yeast Species.

Classifying a yeast strain into a recognized species is not always straightforward. Currently, the taxonomic delineation of yeast strains involves multiple approaches covering phenotypic characteristics and molecular methodologies, including genome-based analysis. The aim of this study was to evaluate the suitability of the Average Nucleotide Identity (ANI) calculation through FastANI, a tool created for bacterial species identification, for the assignment of strains to some yeast species. FastANI, the alignment of in silico-extracted D1/D2 sequences of LSU rRNA, and multiple alignments of orthologous genes (MAOG) were employed to analyze 644 assemblies from 12 yeast genera, encompassing various species, and on a dataset of hybrid Saccharomyces species. Overall, the analysis showed high consistency between results obtained with FastANI and MAOG, although, FastANI proved to be more discriminating than the other two methods applied to genomic sequences. In particular, FastANI was effective in distinguishing between strains belonging to different species, defining clear boundaries between them (cutoff: 94-96%). Our results show that FastANI is a reliable method for attributing a known yeast species to a particular strain. Moreover, although hybridization events make species discrimination more complex, it was revealed to be useful in the identification of these cases. We suggest its inclusion as a key component in a comprehensive approach to species delineation. Using this approach with a larger number of yeasts would validate it as a rapid technique to identify yeasts based on whole genome sequences.

Microplastic-Mediated Transfer of Tetracycline Resistance: Unveiling the Role of Mussels in Marine Ecosystems.

The global threat of antimicrobial resistance (AMR) is exacerbated by the mobilization of antimicrobial resistance genes (ARGs) occurring in different environmental niches, including seawater. Marine environments serve as reservoirs for resistant bacteria and ARGs, further complicated by the ubiquity of microplastics (MPs). MPs can adsorb pollutants and promote bacterial biofilm formation, creating conditions favorable to the dissemination of ARGs. This study explores the dynamics of ARG transfer in the marine bivalve Mytilus galloprovincialis within a seawater model, focusing on the influence of polyethylene MPs on the mobilization of the Tn916-carrying tetM gene and plasmid-encoded ermB. Experiments revealed that biofilm formation on MPs by Enterococcus faecium and Listeria monocytogenes facilitated the transfer of the tetM resistance gene, but not the ermB gene. Furthermore, the presence of MPs significantly increased the conjugation frequency of tetM within mussels, indicating that MPs enhance the potential for ARG mobilization in marine environments. These findings highlight the role of MPs and marine organisms in ARG spread, underscoring the ecological and public health implications.

Safety evaluation of an extension of use of the food enzyme triacylglycerol lipase from the non-genetically modified Mucor circinelloides strain AE-LMH.

The food enzyme triacylglycerol lipase (triacylglycerol acylhydrolase, EC 3.1.1.3) is produced with the non-genetically modified Mucor circinelloides strain AE-LMH by Amano Enzyme Inc. A safety evaluation of this food enzyme was made previously, in which EFSA concluded that this food enzyme did not give rise to safety concerns when used in three food manufacturing processes. Subsequently, the applicant requested to extend its use to include two additional processes. In this assessment, EFSA updated the safety evaluation of this food enzyme when used in a total of five food manufacturing processes. The dietary exposure to the food enzyme-total organic solids (TOS) was estimated to be up to 0.845 mg TOS/kg body weight (bw) per day in European populations. When combined with the no observed adverse effect level previously reported (784 mg TOS/kg bw per day, the highest dose tested), the Panel derived a margin of exposure of at least 928. Based on the data provided for the previous evaluation and the revised margin of exposure, the Panel concluded that this food enzyme does not give rise to safety concerns under the revised intended conditions of use.

Safety evaluation of an extension of use of the food enzyme peroxidase from the genetically modified Aspergillus niger strain MOX.

The food enzyme peroxidase (phenolic donor: hydrogen-peroxide oxidoreductase, EC 1.11.1.7) is produced with the genetically modified Aspergillus niger strain MOX by DSM Food Specialties B.V. A safety evaluation of this food enzyme was made previously, in which EFSA concluded that this food enzyme did not give rise to safety concerns when used in one food manufacturing process. Subsequently, the applicant requested to extend its use to include an additional process. In this assessment, EFSA updated the safety evaluation of this food enzyme when used in a total of two food manufacturing processes: processing of dairy products for the production of modified milk proteins and the production of plant-based analogues of milk and milk products. The dietary exposure to the food enzyme-total organic solids (TOS) was estimated to be up to 0.091 mg TOS/kg body weight (bw) per day in European populations. Using the no observed adverse effect level previously reported (2162 mg TOS/kg bw per day), the Panel derived a margin of exposure (MoE) of at least 23,758. Based on the data provided for the previous evaluation and the revised MoE, the Panel concluded that this food enzyme does not give rise to safety concerns under the revised intended conditions of use.

Safety evaluation of an extension of use of the food enzyme α-glucosidase from the non-genetically modified Aspergillus niger strain AE-TGU.

The food enzyme α-glucosidase (α-d-glucoside glucohydrolase; EC 3.2.1.20) is produced with the non-genetically modified Aspergillus niger strain AE-TGU by Amano Enzyme Inc. A safety evaluation of this food enzyme was made previously, in which EFSA concluded that this food enzyme did not give rise to safety concerns when used in four food manufacturing processes. Subsequently, the applicant requested to extend its use to include three additional processes. In this assessment, EFSA updated the safety evaluation of this food enzyme when used in a total of seven food manufacturing processes. The dietary exposure to the food enzyme-total organic solids (TOS) was estimated to be up to 0.693 mg TOS/kg body weight (bw) per day in European populations. When combined with the no observed adverse effect level previously reported (1062 mg TOS/kg bw per day, the highest dose tested), the Panel derived a margin of exposure of at least 1532. Based on the data provided for the previous evaluation and the revised margin of exposure, the Panel concluded that this food enzyme does not give rise to safety concerns under the revised intended conditions of use.

Safety evaluation of a food enzyme with phospholipase A1 and lysophospholipase activities from the genetically modified Aspergillus niger strain PLN.

The food enzyme with phospholipase A1 (phosphatidycholine 1-acylhydrolase, EC 3.1.1.32) and lysophospholipase (2-lysophosphatidylcholine acylhydrolase, EC 3.1.1.5) activities is produced with the genetically modified Aspergillus niger strain PLN by DSM. The genetic modifications do not give rise to safety concerns. The food enzyme is free from viable cells of the production organism and its DNA. It is intended to be used for the production of refined edible fats and oils by degumming. Since residual amounts of total organic solids are removed during this process, dietary exposure was not calculated and toxicological studies were considered unnecessary for the assessment of this food enzyme. A search for the similarity of the amino acid sequence of the food enzyme to known allergens was made and no matches were found. The Panel considered that the risk of allergic reactions upon dietary exposure cannot be excluded, but the likelihood is low. Based on the data provided, the Panel concluded that this food enzyme does not give rise to safety concerns, under the intended conditions of use.

Safety evaluation of the food enzyme asparaginase from the genetically modified Aspergillus niger strain AGN.

The food enzyme asparaginase (l-asparagine amidohydrolase; EC 3.5.1.1) is produced with the genetically modified Aspergillus niger strain AGN by DSM Food Specialties B.V. The genetic modifications do not give rise to safety concerns. The food enzyme is free from viable cells of the production organism and its DNA. It is intended to be used to prevent acrylamide formation in food processing. The dietary exposure to the food enzyme-total organic solids (TOS) was estimated to be up to 1.434 mg TOS/kg body weight (bw) per day in European populations. The toxicity studies were carried out with an asparaginase from A. niger (strain ASP). The Panel considered this food enzyme as a suitable substitute for the asparaginase to be used in the toxicological studies, because the genetic differences between the production strains are not expected to result in a different toxigenic potential, and the raw materials and manufacturing processes of both food enzymes are comparable. Genotoxicity tests did not indicate a safety concern. The systemic toxicity was assessed by means of a repeated dose 90-day oral toxicity study in rats. The Panel identified a no observed adverse effect level of 1038 mg TOS/kg bw per day, which, when compared with the estimated dietary exposure, resulted in a margin of exposure of at least 724. A search for the similarity of the amino acid sequence of the food enzyme to known allergens was made and no match was found. The Panel considered that the risk of allergic reactions by dietary exposure cannot be excluded, but the likelihood is low. Based on the data provided, the Panel concluded that this food enzyme does not give rise to safety concerns under the intended conditions of use.

Safety evaluation of an extension of use of the food enzyme glucan 1,4-α-glucosidase from the non-genetically modified Rhizopus arrhizus strain AE-G.

The food enzyme glucan 1,4-α-glucosidase (4-α-d-glucan glucohydrolase; EC 3.2.1.3) is produced with the non-genetically modified Rhizopus arrhizus strain AE-G by Amano Enzyme Inc. A safety evaluation of this food enzyme was made previously, in which EFSA concluded that this food enzyme did not give rise to safety concerns when used in one food manufacturing process. Subsequently, the applicant requested to extend its use to nine additional processes and revised the use levels. In this assessment, EFSA updated the safety evaluation of this food enzyme for uses in a total of 10 food manufacturing processes. As the food enzyme-total organic solids (TOS) is removed from the final foods in two food manufacturing processes, the dietary exposure to the food enzyme-TOS was estimated only for the remaining eight processes. Dietary exposure was up to 0.424 mg TOS/kg body weight (bw) per day in European populations. When combined with the no observed adverse effect level previously reported (1868 mg TOS/kg bw per day, the highest dose tested), the Panel derived a margin of exposure of at least 4406. Based on the data provided for the previous evaluation and the margin of exposure revised in the present evaluation, the Panel concluded that this food enzyme does not give rise to safety concerns under the revised intended conditions of use.

Safety evaluation of an extension of use of the food enzyme ß-amylase from the non-genetically modified Bacillus flexus strain AE-BAF.

The food enzyme ß-amylase (4-α-d-glucan maltohydrolase, EC 3.2.1.2) is produced with the non-genetically modified Bacillus flexus strain AE-BAF by Amano Enzyme Inc. A safety evaluation of this food enzyme was made previously, in which EFSA concluded that this food enzyme did not give rise to safety concerns when used in three food manufacturing processes. Subsequently, the applicant requested to extend its use to four additional processes and revised the use levels. In this assessment, EFSA updated the safety evaluation of this food enzyme for use in a total of seven food manufacturing processes. As the food enzyme-total organic solids (TOS) are removed from the final foods in one food manufacturing process, the dietary exposure to the food enzyme-TOS was estimated only for the remaining six processes. The dietary exposure was estimated to be up to 0.247 mg TOS/kg body weight per day in European populations. Based on the data provided for the previous evaluation and the dietary exposure revised in the present evaluation, the Panel concluded that this food enzyme does not give rise to safety concerns under the revised intended conditions of use.

Safety evaluation of the food enzyme endo-1,4-ß-xylanase from the genetically modified Bacillus velezensis strain AR-112.

The food enzyme endo-1,4-ß-xylanase (4-ß-d-xylan xylanohydrolase, EC 3.2.1.8) is produced with the genetically modified Bacillus velezensis strain AR-112 by AB Enzymes GmbH. The genetic modifications do not give rise to safety concerns. The food enzyme is free from viable cells of the production organism and its DNA. It is intended to be used in baking processes. Dietary exposure to the food enzyme-total organic solids (TOS) was estimated to be up to 0.024 mg TOS/kg body weight (bw) per day in European populations. As the production strain B. velezensis strain AR-112 meets the requirements for the qualified presumption of safety (QPS) approach to safety assessment and no issue of concern arose from the production process, no toxicological data are required. A search for the similarity of the amino acid sequence of the food enzyme to known allergens was made and no match was found. The Panel considered that, under the intended conditions of use, the risk of allergic reactions upon dietary exposure cannot be excluded, but the likelihood is low. Based on the data provided, the Panel concluded that this food enzyme does not give rise to safety concerns under the intended conditions of use.

Safety evaluation of the food enzyme glucan 1,4-α-maltohydrolase from the genetically modified Bacillus subtilis strain BABSC.

The food enzyme glucan 1,4-α-maltohydrolase (4-α-d-glucan α-maltohydrolase, EC 3.2.1.133) is produced with the genetically modified Bacillus subtilis strain BABSC by Advanced Enzyme Technologies Ltd. The requirements for the qualified presumption of safety (QPS) approach have not been met. The food enzyme is free from viable cells of the production organism and its DNA. It is intended to be used in baking processes and starch processing for the production of glucose syrups and other starch hydrolysates. Since residual amounts of total organic solids (TOS) are removed, dietary exposure was not calculated for starch processing for the production of glucose syrups and other starch hydrolysates. For baking processes, the dietary exposure was estimated to be up to 0.101 mg TOS/kg body weight per day in European populations. No toxicological studies were provided by the applicant. A search for the similarity of the amino acid sequence of the food enzyme to known allergens was made and one match with a respiratory allergen was found. The Panel considered that the risk of allergic reactions by dietary exposure cannot be excluded, but the likelihood is low. In the absence of appropriate data to fully characterise the production strain, the Panel was unable to conclude on the safety of the food enzyme under the intended conditions of use.

Safety evaluation of the food enzyme rennet containing chymosin and pepsin A from the abomasum of suckling calves, goats, lambs and buffaloes.

The food enzyme containing chymosin (EC 3.4.23.4) and pepsin (EC 3.4.23.1) is prepared from the abomasum of suckling calves, goats, lambs and buffaloes by Caglificio Clerici S.p.A. It is intended to be used in the production of cheese. As no concerns arise from the source of the food enzyme, from its manufacture and based on the history of safe use and consumption, the Panel considered that toxicological data were not required and no exposure assessment was necessary. The similarity of the amino acid sequences of the two proteins (chymosin and pepsin A) to those of known allergens was searched and two matches were found with respiratory allergens. The Panel considered that the risk of allergic reactions by dietary exposure cannot be excluded, but the likelihood is low. Based on the data provided, the Panel concluded that this food enzyme does not give rise to safety concerns under the intended conditions of use.

Safety evaluation of an extension of use of the food enzyme 4-α-glucanotransferase from the non-genetically modified Aeribacillus pallidus strain AE-SAS.

The food enzyme 4-α-glucanotransferase (1,4-α-d-glucan:1,4-α-d-glucan 4-α-d-glycosyltransferase, EC 2.4.1.25) is produced with the non-genetically modified Aeribacillus pallidus strain AE-SAS by Amano Enzyme Inc. A safety evaluation of this food enzyme was made previously, in which EFSA concluded that this food enzyme did not give rise to safety concerns when used in two food manufacturing processes. Subsequently, the applicant requested to extend its use to two additional processes. In this assessment, EFSA updated the safety evaluation of this food enzyme for use in a total of four food manufacturing processes. As the food enzyme-total organic solids (TOS) is removed from the final foods in one food manufacturing process, the dietary exposure to the food enzyme-TOS was estimated only for the remaining three processes. Dietary exposure was up to 0.040 mg TOS/kg body weight (bw) per day in European populations. When combined with the no observed adverse effect level reported in the previous opinion (900 mg TOS/kg bw per day, the highest dose tested), the Panel derived a margin of exposure of at least 22,500. Based on the data provided for the previous evaluation and the revised margin of exposure, the Panel concluded that this food enzyme does not give rise to safety concerns under the revised intended conditions of use.