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.

Various cold storage-backslopping cycles show the robustness of Limosilactobacillus fermentum IMDO 130101 as starter culture for Type 3 sourdough production.

Type 3 sourdoughs, which are starter culture-initiated and subsequently backslopped, are less studied than other sourdough types. Yet, they can serve as a model to assess how competitive starter culture strains for sourdough production are and how the microbial composition of such sourdoughs may evolve over time. In the present study, Limosilactobacillus fermentum IMDO 130101 was used to produce Type 3 sourdoughs, prepared from wheat and wholemeal wheat flours. Therefore, an initial fermentation of the flour-water mixture was performed at 30 °C for 48 h. This was followed by cold storage-backslopping cycles, consisting of refreshments (50 %, v/v), fermentation steps of 16 h, and storage at 4 °C each week, every three weeks, and every six weeks. The microbial dynamics (culture-dependent and -independent approaches) and metabolite dynamics were measured. In all sourdoughs produced, starter culture strain monitoring, following an amplicon sequence variant approach, showed that Liml. fermentum IMDO 130101 prevailed during one month when the sourdoughs were refreshed each week, during 24 weeks when the sourdoughs were refreshed every three weeks, and during 12 weeks when the sourdoughs were refreshed every six weeks. This suggested the competitiveness and robustness of Liml. fermentum IMDO 130101 for a considerable duration but also showed that the strain is prone to microbial interference. For instance, Levilactobacillus brevis and Pediococcus spp. prevailed upon further cold storage and backslopping. Also, although no yeasts were inoculated into the flour-water mixtures, Kazachstania unispora, Torulaspora delbrueckii, and Wickerhamomyces anomalus were the main yeast species found. They appeared after several weeks of storage and backslopping, which however indicated the importance of an interplay between LAB and yeast species in sourdoughs. The main differences among the mature sourdoughs obtained could be explained by the different flours used, the refreshment conditions applied, and the sampling time (before and after backslopping). Finally, the metabolite quantifications revealed continued metabolite production during the cold storage periods, which may impact the sourdough properties and those of the breads made thereof.

Impact of process parameters on the specific volume of wholemeal wheat bread made using sourdough- and baker's yeast-based leavening strategies.

The final quality of wholemeal wheat bread is determined by the process parameter settings and leavening strategy. We hypothesise that the used leavening strategy may influence the optimal process parameter settings and, as such, the specific volume of the bread loaf. To analyse this interaction, bread was leavened with (i) a type 1 sourdough (SB), (ii) a type 1 sourdough combined with baker's yeast (YSB), or (iii) baker's yeast (YB). For each leavening strategy, the specific volume of bread, in response to variations in mixing time (4-10/4-14 min), water absorption (60-85 %), and proofing time (1-7/1-3 h), was analysed using an I-optimal response surface experimental design. Data modelling identified a substantially lower maximal specific volume of SB (2.13 mL/g), compared to YSB (3.30 mL/g) and YB (3.26 mL/g). The proofing time and water absorption mostly influenced the specific volume of the SB and YSB, respectively. However, the mixing and proofing times mainly affected the specific volume of YB. The type 1 sourdough reduced the mixing time and water absorption required for an optimal specific volume of bread compared to baker's yeast. These results challenge the idea of yielding higher volumes upon using sourdough compared to baker's yeast and highlight the importance of optimisation of bread dough formulations and breadmaking processes.

Enterococcus faecium Bacteriophage vB_EfaH_163, a New Member of the Herelleviridae Family, Reduces the Mortality Associated with an E. faecium vanR Clinical Isolate in a Galleria mellonella Animal Model.

The rise of antimicrobial resistant (AMR) bacteria is a major health concern, especially with regard to members of the ESKAPE group, to which vancomycin-resistant (VRE) Enterococcus faecium belongs. Phage therapy has emerged as a novel alternative for the treatment of AMR infections. This, however, relies on the isolation and characterisation of a large collection of phages. This work describes the exploration of human faeces as a source of new E. faecium-infecting phages. Phage vB_EfaH_163 was isolated and characterised at the microbiological, genomic, and functional levels. vB_EfaH_163 phage, a new member of Herelleviridae, subfamily Brockvirinae, has a dsDNA genome of 150,836 bp that does not harbour any virulence factors or antibiotic resistance genes. It infects a wide range of E. faecium strains of different origins, including VRE strains. Interestingly, it can also infect Enterococcus faecalis strains, even some that are linezolid-resistant. Its capacity to control the growth of a clinical VRE isolate was shown in broth culture and in a Galleria mellonella animal model. The discovery and characterisation of vB_EfaH_163 increases the number of phages that might be used therapeutically against AMR bacteria.

Contact Effect of a Methylobacterium sp. Extract on Biofilm of a Mycobacterium chimaera Strain Isolated from a 3T Heater-Cooler System.

Mycobacterium chimaera is an opportunistic slowly growing non-tuberculous mycobacteriumof increasing importance due to the outbreak of cases associated with contaminated 3T heater-cooler device (HCD) extracorporeal membrane oxygenator (ECMO). The aim of this study was to evaluate the effect of pre-treating a surface with a Methylobacterium sp. CECT 7180 extract to inhibit the M. chimaera ECMO biofilm as well as of the treatment after different dehydration times. Surface adherence, biofilm formation and treatment effect were evaluated by estimating colony-forming units (CFU) per square centimeter and characterizing the amount of covered surface area, thickness, cell viability, and presence of intrinsic autofluorescence at different times using confocal laser scanning microscopy and image analysis. We found that exposing a surface to the Methylobacterium sp. CECT 7180 extract inhibited M. chimaera ECMO biofilm development. This effect could be result of the effect of Methylobacterium proteins, such as DNaK, trigger factor, and xanthine oxidase. In conclusion, exposing a surface to the Methylobacteriumsp. extract inhibits M. chimaera ECMO biofilm development. Furthermore, this extract could be used as a pre-treatment prior to disinfection protocols for equipment contaminated with mycobacteria after dehydration for at least 96 h.

Mycoredoxins Are Required for Redox Homeostasis and Intracellular Survival in the Actinobacterial Pathogen Rhodococcus equi.

Rhodococcus equi is a facultative intracellular pathogen that can survive within macrophages of a wide variety of hosts, including immunosuppressed humans. Current antibiotherapy is often ineffective, and novel therapeutic strategies are urgently needed to tackle infections caused by this pathogen. In this study, we identified three mycoredoxin-encoding genes (mrx) in the genome of R. equi, and we investigated their role in virulence. Importantly, the intracellular survival of a triple mrx-null mutant (Δmrx1Δmrx2Δmrx3) in murine macrophages was fully impaired. However, each mycoredoxin alone could restore the intracellular proliferation rate of R. equi Δmrx1Δmrx2Δmrx3 to wild type levels, suggesting that these proteins could have overlapping functions during host cell infection. Experiments with the reduction-oxidation sensitive green fluorescent protein 2 (roGFP2) biosensor confirmed that R. equi was exposed to redox stress during phagocytosis, and mycoredoxins were involved in preserving the redox homeostasis of the pathogen. Thus, we studied the importance of each mycoredoxin for the resistance of R. equi to different oxidative stressors. Interestingly, all mrx genes did have overlapping roles in the resistance to sodium hypochlorite. In contrast, only mrx1 was essential for the survival against high concentrations of nitric oxide, while mrx3 was not required for the resistance to hydrogen peroxide. Our results suggest that all mycoredoxins have important roles in redox homeostasis, contributing to the pathogenesis of R. equi and, therefore, these proteins may be considered interesting targets for the development of new anti-infectives.