Genomic exploration of the fermented meat isolate Staphylococcus shinii IMDO-S216 with a focus on competitiveness-enhancing secondary metabolites.

BACKGROUND: Staphylococcus shinii appears as an umbrella species encompassing several strains of Staphylococcus pseudoxylosus and Staphylococcus xylosus. Given its phylogenetic closeness to S. xylosus, S. shinii can be found in similar ecological niches, including the microbiota of fermented meats where the species may contribute to colour and flavour development. In addition to these conventional functionalities, a biopreservation potential based on the production of antagonistic compounds may be available. Such potential, however, remains largely unexplored in contrast to the large body of research that is available on the biopreservative properties of lactic acid bacteria. The present study outlines the exploration of the genetic basis of competitiveness and antimicrobial activity of a fermented meat isolate, S. shinii IMDO-S216. To this end, its genome was sequenced, de novo assembled, and annotated. RESULTS: The genome contained a single circular chromosome and eight plasmid replicons. Focus of the genomic exploration was on secondary metabolite biosynthetic gene clusters coding for ribosomally synthesized and posttranslationally modified peptides. One complete cluster was coding for a bacteriocin, namely lactococcin 972; the genes coding for the pre-bacteriocin, the ATP-binding cassette transporter, and the immunity protein were also identified. Five other complete clusters were identified, possibly functioning as competitiveness factors. These clusters were found to be involved in various responses such as membrane fluidity, iron intake from the medium, a quorum sensing system, and decreased sensitivity to antimicrobial peptides and competing microorganisms. The presence of these clusters was equally studied among a selection of multiple Staphylococcus species to assess their prevalence in closely-related organisms. CONCLUSIONS: Such factors possibly translate in an improved adaptation and competitiveness of S. shinii IMDO-S216 which are, in turn, likely to improve its fitness in a fermented meat matrix.

Genome-Based Characterization of a Plasmid-Associated Micrococcin P1 Biosynthetic Gene Cluster and Virulence Factors in Mammaliicoccus sciuri IMDO-S72.

Analysis of the de novo assembled genome of Mammaliicoccus sciuri IMDO-S72 revealed the genetically encoded machinery behind its earlier reported antibacterial phenotype and gave further insight into the repertoire of putative virulence factors of this recently reclassified species. A plasmid-encoded biosynthetic gene cluster was held responsible for the antimicrobial activity of M. sciuri IMDO-S72, comprising genes involved in thiopeptide production. The compound encoded by this gene cluster was structurally identified as micrococcin P1. Further examination of its genome highlighted the ubiquitous presence of innate virulence factors mainly involved in surface colonization. Determinants contributing to aggressive virulence were generally absent, with the exception of a plasmid-associated ica cluster. The native antibiotic resistance genes sal(A) and mecA were detected within the genome, among others, but were not consistently linked with a resistance phenotype. While mobile genetic elements were identified within the genome, such as an untypeable staphylococcal cassette chromosome (SCC) element, they proved to be generally free of virulence- and antibiotic-related genes. These results further suggest a commensal lifestyle of M. sciuri and indicate the association of antibiotic resistance determinants with mobile genetic elements as an important factor in conferring antibiotic resistance, in addition to their unilateral annotation. IMPORTANCEMammaliicoccus sciuri has been put forward as an important carrier of virulence and antibiotic resistance genes, which can be transmitted to clinically important staphylococcal species such as Staphylococcus aureus. As a common inhabitant of mammal skin, this species is believed to have a predominant commensal lifestyle, although it has been reported as an opportunistic pathogen in some cases. This study provides an extensive genome-wide description of its putative virulence potential taking into consideration the genomic context in which these genes appear, an aspect that is often overlooked during virulence analysis. Additional genome and biochemical analysis linked M. sciuri with the production of micrococcin P1, gaining further insight into the extent to which these biosynthetic gene clusters are distributed among different related species. The frequent plasmid-associated character hints that these traits can be horizontally transferred and might confer a competitive advantage to its recipient within its ecological niche.

Raw meat quality and salt levels affect the bacterial species diversity and community dynamics during the fermentation of pork mince.

Acidification level and temperature modulate the beneficial consortia of lactic acid bacteria (LAB) and coagulase-negative staphylococci (CNS) during meat fermentation. Less is known about the impact of other factors, such as raw meat quality and salting. These could for instance affect the growth of the pathogen Staphylococcus aureus or of Enterobacterales species, potentially indicative of poor fermentation practice. Therefore, pork batters from either normal or borderline quality (dark-firm-dry, DFD) were compared at various salt concentrations (0-4%) in meat fermentation models. Microbial ecology of the samples was investigated with culture-dependent techniques and (GTG)5-PCR fingerprinting of genomic DNA. Whilst Lactobacillus sakei governed the fermentation of normal meat, Lactobacillus curvatus was more prominent in the fermentation of the DFD meat variant. CNS were favoured during fermentation at rising salt concentrations without much effects on species diversity, consisting mostly of Staphylococcus equorum, Staphylococcus saprophyticus, and Staphylococcus xylosus. During fermentation of DFD meat, S. saprophyticus was less manifest than during that of normal meat. Enterobacterales mainly emerged in DFD meat during fermentation at low salt concentrations. The salt hurdle was insufficient to prevent Enterobacterales when acidification and initial pH were favourable for their growth.

Challenge tests reveal limited outgrowth of proteolytic Clostridium botulinum during the production of nitrate- and nitrite-free fermented sausages.

Nitrate and nitrite salts perform a versatile role in fermented meats, including the inhibition of food pathogens (in particular proteolytic group I Clostridium botulinum). Despite the increasing interest in clean-label products, little is known about the behaviour of this pathogen in response to the removal of chemical preservatives from fermented meat formulations. Therefore, challenge tests with a cocktail of nontoxigenic group I C. botulinum strains were performed to produce nitrate/nitrite-free fermented sausages under different acidification conditions and starter culture formulations, including the use of an anticlostridial Mammaliicoccus sciuri strain. Results showed limited outgrowth of C. botulinum, even in the absence of acidification. The anticlostridial starter culture did not lead to an additional inhibitory effect. The selective plating procedure adopted within this study proofed robust to follow germination and growth of C. botulinum, inhibiting common fermentative meat microbiota. The challenge tests constitute a suitable tool to assess the behaviour of this food pathogen within fermented meats upon nitrate- and nitrite omission.

Bacterial species diversity of traditionally ripened sheep legs from the Faroe Islands (skerpikjøt).

Skerpikjøt is a traditionally ripened sheep leg product from the Faroe Islands, constituting a relatively underexplored microbial ecosystem. The objective of this study is to achieve a deeper understanding of the microbial composition of this artisanal product. Nine ripened hind legs, obtained from three different producers, were assessed regarding their bacterial communities and contents of biogenic amines, including both surface and core samples. Biogenic amine concentrations were generally low, although one sample had a somewhat elevated concentration of cadaverine. Bacterial diversity was investigated by culture-dependent and culture-independent techniques. Gram-positive catalase-positive cocci (GCC) constituted the most abundant group. Within this group, Staphylococcus equorum was the most prevailing species, followed by Kocuria sp., Mammaliicoccus vitulinus, and Staphylococcus saprophyticus. Lactic acid bacteria prevailed in only one sample and were mainly represented by Latilactobacillus curvatus. Enterobacterial communities were characterised by the prevalence of Serratia proteamaculans. Despite the majority of GCC, Clostridium putrefaciens was the most abundant bacterial species in some core samples. Taken together, the culture-dependent and culture-independent identification methods gave complementary results.

Assessing levels of traditionality and naturalness depicted on labels of fermented meat products in the retail: Exploring relations with price, quality and branding strategy.

Food labelling is a tool to inform consumers about the specifications and characteristics of a product. Additionally, labels display information about traditionality and naturalness, of which the meaning is highly subjective. There is a paucity of research examining attributes both of tradition and naturalness. In this study, traditionality was assessed by a model that included temporal, geographical, know-how, and cultural components. Naturalness was evaluated based on bio/organic elements, 'free-from' claims, and natural ingredients. Therefore, a content analysis tool was developed to analyze and score labels of fermented meat products, which generated insights in the key label characteristics of tradition and naturalness. The degree of tradition and naturalness was the average of their subdimensions which were scored based on the displayed elements. A higher degree of tradition and naturalness was linked to higher prices. Fermented meat labels were found to be strongly embedded in 'authenticity', and less in naturalness, an element more attractive for private labels than for branded products.

High-throughput amplicon sequencing to assess the impact of processing factors on the development of microbial communities during spontaneous meat fermentation.

During spontaneous meat fermentation, diverse microbial communities develop over time. These communities consist mainly of lactic acid bacteria (LAB) and coagulase-negative staphylococci (CNS), of which the species composition is influenced by the fermentation temperature and the level of acidification. Recent development and application of amplicon-based high-throughput sequencing (HTS) methods have allowed to gain deeper insights into the microbial communities of fermented meats. The aim of the present study was to investigate the effect of different fermentation temperatures and acidification profiles on the CNS communities during spontaneous fermentation, using a previously developed amplicon-based HTS method targeting both the 16S rRNA and tuf genes. Spontaneous fermentations were performed with five different lots of meat to assess inter-lot variability. The process influence was investigated by fermenting the meat batters for seven days at different fermentation temperatures (23 °C, 30 °C, and 37 °C) and in the absence or presence of added glucose to simulate different acidification levels. Additionally, the results were compared with a starter culture-initiated fermentation process. The data revealed that the fermentation temperature was the most influential processing condition in shaping the microbial communities during spontaneous meat fermentation processes, whereas differences in pH were only responsible for minor shifts in the microbial profiles. Furthermore, the CNS communities showed a great level of variability, which depended on the initial microbial communities present and their competitiveness.

Application of a High-Throughput Amplicon Sequencing Method to Chart the Bacterial Communities that Are Associated with European Fermented Meats from Different Origins.

Insight into the microbial species diversity of fermented meats is not only paramount to gain control over quality development, but also to better understand the link with processing technology and geographical origin. To study the composition of the microbial communities, the use of culture-independent methods is increasingly popular but often still suffers from drawbacks, such as a limited taxonomic resolution. This study aimed to apply a previously developed high-throughput amplicon sequencing (HTS) method targeting the 16S rRNA and tuf genes to characterize the bacterial communities in European fermented meats in greater detail. The data obtained broadened the view on the microbial communities that were associated with the various products examined, revealing the presence of previously underreported subdominant species. Moreover, the composition of these communities could be linked to the specificities of individual products, in particular pH, salt content, and geographical origin. In contrast, no clear links were found between the volatile organic compound profiles of the different products and the country of origin, distinct processing conditions, or microbial communities. Future application of the HTS method offers the potential to further unravel complex microbial communities in fermented meats, as well as to assess the impact of different processing conditions on microbial consortia.

The Use of Less Conventional Meats or Meat with High pH Can Lead to the Growth of Undesirable Microorganisms during Natural Meat Fermentation.

The bacterial communities that are established during natural meat fermentation depend on the processing conditions and the type of meat substrate used. Six pork samples of variable quality (reflected in pH values) and six less conventional meats (beef, horse, hare, wild deer, wild duck, and wild boar) were naturally fermented under controlled conditions in model systems. The development of lactic acid bacteria (LAB), coagulase-negative staphylococci (CNS), and enterobacteria was followed using culture-dependent techniques and (GTG)5-PCR fingerprinting of genomic DNA from the isolates obtained. Taken together, Latilactobacillus sakei was the most abundant LAB species, although Latilactobacillus curvatus was more manifest in high-pH pork. Within staphylococci, common species were encountered (i.e., Staphylococcus equorum, Staphylococcus saprophyticus, and Staphylococcus xylosus), although some atypical ones (i.e., Staphylococcus succinus) were also recovered. Within enterobacteria, Serratia spp. prevailed in more acidic pork batches and in beef, whereas Hafnia spp. prevailed in game meat fermentations. Enterobacterial counts were particularly high in fermentations with low acidity, namely for some pork batches, hare, wild duck, and wild boar. These findings should be considered when naturally fermented meat products are manufactured, as the use of game meat or meat with high pH can give rise to safety concerns.

Exploring the Ambiguous Status of Coagulase-Negative Staphylococci in the Biosafety of Fermented Meats: The Case of Antibacterial Activity Versus Biogenic Amine Formation.

A total of 332 staphylococcal strains, mainly isolated from meat, were screened for antibacterial activity. Eighteen strains exhibited antibacterial activity towards species within the same genus. These antibacterial strains were further screened against Clostridium botulinum, to assess their potential as anticlostridial starter cultures for the development of fermented meat products without added nitrate or nitrite. Only Staphylococcus sciuri IMDO-S72 had the ability to inhibit all clostridial strains tested, whilst displaying additional activity against Bacillus cereus, Listeria monocytogenes and Staphylococcus aureus. Apart from their potential as bioprotective cultures, the staphylococcal collection was also screened for biogenic amine production, as these compounds may compromise food quality. To this end, ultra-high-performance liquid chromatography coupled to tandem mass spectrometry (UPLC-MS/MS) was applied. A low incidence of biogenic amine production was found, with tyramine and ß-phenylethylamine being the most prevalent ones. Concentrations remained relatively low (< 52 mg/L) after a prolonged incubation period, posing no or little threat towards food safety. Taken together, S. sciuri IMDO-S72 could serve as an interesting candidate for the bioprotection of fermented meats as it showed promising antibacterial activity as well as absence of biogenic amine production.

Exploring the Link Between the Geographical Origin of European Fermented Foods and the Diversity of Their Bacterial Communities: The Case of Fermented Meats.

European fermented meat products are prepared according to a wide variety of different recipes and processing conditions, which can influence their fermentative microbiota. However, due to the diverse processing conditions applied across Europe, it remained unclear to which degree bacterial heterogeneity can be encountered in commercially available fermented meat products and whether this is linked to their geographical origin. Therefore, the bacterial species diversity of 80 fermented meat products available in the Belgian retail, coming from five different countries, was investigated. It was also assessed how this related to the country of origin and the key processing parameters pH and salt concentration. The samples originated from Belgium, France, Germany, Italy, and Spain. In general, Southern European fermented meat products commonly had a higher pH, with their lactic acid bacteria (LAB) communities being represented by Lactobacillus sakei and with mostly Staphylococcus xylosus and Staphylococcus equorum governing over the coagulase-negative staphylococci (CNS) communities. Among these products, the Spanish variants showed a higher prevalence of S. equorum, whereas S. xylosus was the prevailing CNS species in most French and Italian fermented meat products. In contrast, Northern European fermented meat products were generally more acidified and showed a higher prevalence of Pediococcus pentosaceus in their LAB communities, whereas Staphylococcus carnosus represented the CNS communities. Non-parametric statistical tests indicated the impact of the geographical origin on the prevalence of the LAB and CNS species. The latter was likely due to the combination of differences in process technology as well as starter culture use.