The Protected Designation of Origin (PDO) indication for foods intends to guarantee the conditions of production and the geographical origin of regional products within the European Union. Honey products are widely consumed due to their health-promoting properties and there is a general interest in tracing their authenticity. In this regard, metagenomics sequencing and machine learning (ML) have been proposed as complementary technologies to improve the traceability methods of foods. Therefore, the aim of this study was to analyze the metagenomic profiles of Spanish honeys from three different PDOs (Granada, Tenerife and Villuercas-Ibores), and compare them with non-PDO honeys using ML models (PLS, RF, LOGITBOOST, and NNET). According to the results obtained, non-PDO honeys and Granada PDO showed higher beta diversity values than Tenerife and Villuercas-Ibores PDOs. ML classification of honey products allowed the identification of different microbial biomarkers of the geographical origin of honeys: Lactobacillus kunkeei, Parasaccharibacter apium and Lactobacillus helsingborgensis for PDO honeys and Paenibacillus larvae, Lactobacillus apinorum and Klebsiella pneumoniae for non-PDO honeys. In addition, potential microbial biomarkers of some honey varieties including L. kunkeei for Albaida and Retama del Teide varieties, and P. apium for Tajinaste variety, were identified. ML models were validated on an independent set of samples leading to high accuracy rates (above 90 %). This work demonstrates the potential of ML to differentiate different types of honey using metagenome-based methods, leading to high performance metrics. In addition, ML models discriminate both the geographical origin and variety of products corresponding to different PDOs and non-PDO products. Results here presented may contribute to develop enhanced traceability and authenticity methods that could be applied to a wide range of foods.

Salmonella enterica serovar Derby (S. Derby) ranks fifth among nontyphoidal Salmonella serovars causing human infections in the European Union. S. Derby isolates (36) collected between 2006 and 2018 in a Spanish region (Asturias) from human clinical samples (20) as well as from pig carcasses, pork- or pork and beef-derived products, or wild boar (16) were phenotypically characterized with regard to resistance, and 22 (12 derived from humans and 10 from food-related samples) were also subjected to whole genome sequence analysis. The sequenced isolates belonged to ST40, a common S. Derby sequence type, and were positive for SPI-23, a Salmonella pathogenicity island involved in adherence and invasion of the porcine jejune enterocytes. Isolates were either susceptible (30.6%), or resistant to one or more of the 19 antibiotics tested for (69.4%). Resistances to tetracycline [tet(A), tet(B) and tet(C)], streptomycin (aadA2), sulfonamides (sul1), nalidixic acid [gyrA (Asp87 to Asn)] and ampicillin (bla_TEM-1-like) were detected, with frequencies ranging from 8.3% to 66.7%, and were higher in clinical than in food-borne isolates. The fosA7.3 gene was present in all sequenced isolates. The most common phenotype was that conferred by the tet(A), aadA2 and sul1 genes, located within identical or closely related variants of Salmonella Genomic Island 1 (SGI1), where mercury resistance genes were also present. Diverse IncI1-I(α) plasmids belonging to distinct STs provided antibiotic [bla_TEM-1, tet(A) and/or tet(B)] and heavy metal resistance genes (copper and silver), while small pSC101-like plasmids carried tet(C). Regardless of their location, most resistance genes were associated with genetic elements involved in DNA mobility, including a class one integron, multiple insertion sequences and several intact or truncated transposons. By phylogenetic analysis, the isolates were distributed into two distinct clades, both including food-borne and clinical isolates. One of these clades included all SGI1-like positive isolates, which were found in both kinds of samples throughout the entire period of study. Although the frequency of S. Derby in Asturias was very low (0.5% and 3.1% of the total clinical and food isolates of S. enterica recovered along the period of study), it still represents a burden to human health linked to transmission across the food chain. The information generated in the present study can support further epidemiological surveillance aimed to control this zoonotic pathogen.

Faecalibacterium is among the most abundant bacterial genera in the healthy human colon, comprising approximately 10–15% of the total gut microbiota. Species within this genus ferment complex carbohydrates, including pectin, to produce butyrate, a short-chain fatty acid with anti-inflammatory and anti-carcinogenic properties. Butyrate is the primary energy source for colonocytes and in Faecalibacterium is synthesized via the butyryl-CoA:acetate CoA transferase pathway. Reduced levels of Faecalibacterium are often associated with increased abundance of Escherichia coli and may be linked to early-onset colorectal cancer. Here, genomic analysis of Faecalibacterium strains revealed that several lack antibiotic resistance genes, suggesting a favorable safety profile. Additional genome mining revealed multiple biosynthetic gene clusters (BGCs) involved in the synthesis of secondary metabolites, including ranthipeptides, which may exhibit antimicrobial activity. Understanding the functional roles of these BGCs, particularly their potential to inhibit E. coli , is critical for advancing microbiome-based therapies. Moreover, developing effective delivery strategies to maintain Faecalibacterium populations in the colon is essential for promoting gut health and preventing disease.

Background/Objective: The present study focused on the analysis of the Spanish clone belonging to the successful 4,[5],12:i:- monophasic variant of Salmonella enterica serovar Typhimurium. Methods: All isolates of the clone recovered in a Spanish region from human clinical samples between 2008 and 2018 (N = 14) were investigated using microbiological approaches and genome sequence analysis. In addition, they were compared with isolates from the years 2000 to 2003 (N = 21), which were previously characterized but had not yet been sequenced. Results: Phylogenetic analyses indicate that all isolates are closely related (differing by 1 to 103 SNPs) but belong to two clades termed A and B. With few exceptions, clade A comprised isolates of the first period, also including two “older” control strains, LSP 389/97 and LSP 272/98. Clade B only contained isolates from the second period. Isolates from both periods were resistant to antibiotics and biocides, with almost all resistance genes located on large IncC plasmids, additionally carrying pSLT-derived virulence genes. The number of resistance genes was highly variable, resulting in a total of 22 ABR (antibiotic biocide resistance) profiles. The number of antibiotic resistance genes, but not that of biocide resistance genes, was considerably lower in isolates from the second than from the first period (with averages of 5.5 versus 9.6 genes). Importantly, IS26, which resides in multiple copies within these plasmids, appears to be playing a crucial role in the evolution of resistance, and it was also responsible for the monophasic phenotype, which was associated with four different deletions eliminating the fljAB region. Conclusions: the observed reduction in the number of antibiotic resistance genes could correlate with the loss of adaptive advantage originating from the ban on the use of antibiotics as feed additives implemented in the European Union since 2006, facilitated by the intrinsic instability of the IncC plasmids. Two consecutive IS26 transposition events, which can explain both the clonal relationship of the isolates and their variability, may account for the observed fljAB deletions.

Salmonella enterica subspecies enterica serovar 4,[5],12:i:- is a monophasic variant of S. Typhimurium which has emerged as a world-wide distributed pathogen in the last decades. Several clones have been identified within this variant, the European clone, the Spanish clone, the Southern European clone and the U.S./American clone. The present study focused on isolates of the Southern European clone that were obtained from clinical samples at Spanish hospitals. The selected isolates were multidrug resistant, with most resistance genes residing on IncR plasmids that also carried virulence genes. These plasmids had a mosaic structure, comprising a highly reduced IncR backbone, which has acquired a large amount of exogenous DNA mostly derived from pSLT and IncI1-I(alfa) plasmids. Although composed of approximately the same elements, the investigated plasmids displayed a high diversity, consistent with active evolution driven by a wealth of mobile genetic elements. They comprise multiple intact or truncated insertion sequences, transposons, pseudo-compound transposons and integrons. Particularly relevant was the role of IS26 (with six to nine copies per plasmid) in generating insertions, deletions and inversions, with many of the rearrangements uncovered by tracking the patterns of eight bp target site duplications. Most of the resistance genes detected in the analyzed isolates have been previously associated with the Southern European clone. However, erm(B), lnu(G) and bla_TEM-1B are novel, with the last two carried by a second resistance plasmid found in one of the IncR-positive isolates. Thus, evolution of resistance in the Southern European clone is not only mediated by diversification of the IncR plasmids, but also through acquisition of additional plasmids. All isolates investigated in the present study have the large deletion affecting the fljBA region previously found to justify the monophasic phenotype in the Southern European and U.S./American clones. An SNP-based phylogenetic analysis revealed the close relationship amongst our isolates, and support that those sharing the large fljBA deletion could be more heterogeneous than previously anticipated.