Fungal volatiles mediate cheese rind microbiome assembly

In vitro studies in plant, soil, and human systems have shown that microbial volatiles can mediate microbe–microbe or microbe–host interactions. These previous studies have often used artificially high concentrations of volatiles compared to in situ systems and have not demonstrated the roles volatiles play in mediating community-level dynamics. We used the notoriously volatile cheese rind microbiome to identify bacteria responsive to volatiles produced by five widespread cheese fungi. Vibrio casei had the strongest growth stimulation when exposed to all fungi. In multispecies community experiments, fungal volatiles caused a shift to a Vibrio-dominated community, potentially explaining the widespread occurrence of Vibrio in surface-ripened cheeses. RNA sequencing identified activation of the glyoxylate shunt as a possible mechanism underlying volatile-mediated growth promotion and community assembly. Our study demonstrates how airborne chemicals could be used to control the composition of microbiomes and illustrates how volatiles may impact the development of cheese rinds.     

Temporal and Spatial Differences in Microbial Composition during the Manufacture of a Continental-Type Cheese

We sought to determine if the time, within a production day, that a cheese is manufactured has an influence on the microbial community present within that cheese. To facilitate this, 16S rRNA amplicon sequencing was used to elucidate the microbial community dynamics of brine-salted continental-type cheese in cheeses produced early and late in the production day. Differences in the microbial composition of the core and rind of the cheese were also investigated. Throughout ripening, it was apparent that cheeses produced late in the day had a more diverse microbial population than their early equivalents. Spatial variation between the cheese core and rind was also noted in that cheese rinds were initially found to have a more diverse microbial population but thereafter the opposite was the case. Interestingly, the genera Thermus, Pseudoalteromonas, and Bifidobacterium, not routinely associated with a continental-type cheese produced from pasteurized milk, were detected. The significance, if any, of the presence of these genera will require further attention. Ultimately, the use of high-throughput sequencing has facilitated a novel and detailed analysis of the temporal and spatial distribution of microbes in this complex cheese system and established that the period during a production cycle at which a cheese is manufactured can influence its microbial composition.     

Effects of Proteus vulgaris growth on the establishment of a cheese microbial community and on the production of volatile aroma compounds in a model cheese

Aims:  To investigate the impact of Proteus vulgaris growth on a multispecies ecosystem and on volatile aroma compound production during cheese ripening.
Methods and Results:  The microbial community dynamics and the production of volatile aroma compounds of a nine-species cheese ecosystem were compared with or without the presence of P. vulgaris in the initial inoculum. Proteus vulgaris was able to colonize the cheese surface and it was one of the dominant species, representing 37% of total isolates at the end of ripening with counts of 9·2 log₁₀ CFU g⁻¹. In the presence of P. vulgaris , counts of Arthrobacter arilaitensis , Brevibacterium aurantiacum and Hafnia alvei significantly decreased. Proteus vulgaris influenced the production of total volatile aroma compounds with branched-chain aldehydes and their corresponding alcohols being most abundant.
Conclusions:  Proteus vulgaris was able to successfully implant itself in a complex cheese ecosystem and significantly contributed to the organoleptic properties of cheese during ripening. This bacterium also interacted negatively with other bacteria in the ecosystem studied.
Significance and Impact of the Study:  This is the first time that the impact of a Gram-negative bacterium on cheese microbial ecology and functionality has been described.     

Altering environmental conditions induce shifts in simulated deep terrestrial subsurface bacterial communities—Secretion of primary and secondary metabolites

The deep terrestrial subsurface (DTS) harbours a striking diversity of microorganisms. However, systematic research on microbial metabolism, and how varying groundwater composition affects the bacterial communities and metabolites in these environments is lacking. In this study, DTS groundwater bacterial consortia from two Fennoscandian Shield sites were enriched and studied. We found that the enriched communities from the two sites consisted of distinct bacterial taxa, and alterations in the growth medium composition induced changes in cell counts. The lack of an exogenous organic carbon source (ECS) caused a notable increase in lipid metabolism in one community, while in the other, carbon starvation resulted in low overall metabolism, suggesting a dormant state. ECS supplementation increased CO₂ production and SO₄²⁻ utilisation, suggesting activation of a dissimilatory sulphate reduction pathway and sulphate-reducer-dominated total metabolism. However, both communities shared common universal metabolic features, most probably involving pathways needed for the maintenance of cell homeostasis (e.g., mevalonic acid pathway). Collectively, our findings indicate that the most important metabolites related to microbial reactions under varying growth conditions in enriched DTS communities include, but are not limited to, those linked to cell homeostasis, osmoregulation, lipid biosynthesis and degradation, dissimilatory sulphate reduction and isoprenoid production.     

The spatial distribution of bacteria in Grana-cheese during ripening

The microbial composition and its spatial distribution of Grana Trentino, a hard Parmesan-like cheese, was determined, from vat milk to cheese. After cutting along the vertical axis of the cheese wheels, three layers were sampled diagonally across the cheese: under the cheese rind, an intermediate section and the cheese core. After two different ripening periods (9 and 18 months), the cheese samples were analysed using traditional culture dependent and culture independent methods. Milk samples were dominated by mesophilic and psychrophilic bacterial counts. Thermophilic bacteria (Lactobacillus helveticus) were found in high amounts in cooked whey and natural whey starter cultures. After 9 months of ripening, lactic acid bacteria (LAB) counts were higher than those after 18 months. Furthermore, the LAB numbers in the cheese core was lower than those under the rind or in the intermediate section. The main LAB species isolated from milk (Lactococcus lactis, Pediococcus pentosaceus, Streptococcus uberis and Lactococcus garvieae) were not found in the corresponding cheeses. Some differences were observed in the species composition among the three cheese sections. Microbiota under the rind and in the intermediate section was similar and dominated by Lactobacillus paracasei and Lactobacillus rhamnosus. The core, after 18 months of ripening, was characterized by a total absence of LAB. In each sample, all LAB were genotypically grouped and the different biotypes were subjected to several technological tests indicating that some non-starter LAB (NSLAB) displayed technological features that are favorable for the production of Grana Trentino cheese.     

Spatial Distribution of the Metabolically Active Microbiota within Italian PDO Ewes' Milk Cheeses

Italian PDO (Protected Designation of Origin) Fiore Sardo (FS), Pecorino Siciliano (PS) and Pecorino Toscano (PT) ewes’ milk cheeses were chosen as hard cheese model systems to investigate the spatial distribution of the metabolically active microbiota and the related effects on proteolysis and synthesis of volatile components (VOC). Cheese slices were divided in nine sub-blocks, each one separately subjected to analysis and compared to whole cheese slice (control). Gradients for moisture, and concentrations of salt, fat and protein distinguished sub-blocks, while the cell density of the main microbial groups did not differ. Secondary proteolysis differed between sub-blocks of each cheese, especially when the number and area of hydrophilic and hydrophobic peptide peaks were assessed. The concentration of free amino acids (FAA) agreed with these data. As determined through Purge and Trap (PT) coupled with Gas Chromatography-Mass Spectrometry (PT-GC/MS), and regardless of the cheese variety, the profile with the lowest level of VOC was restricted to the region identified by the letter E defined as core. As shown through pyrosequencing of the 16S rRNA targeting RNA, the spatial distribution of the metabolically active microbiota agreed with the VOC distribution. Differences were highlighted between core and the rest of the cheese. Top and bottom under rind sub-blocks of all three cheeses harbored the widest biodiversity.The cheese sub-block analysis revealed the presence of a microbiota statistically correlated with secondary proteolysis events and/or synthesis of VOC.     

Microbial ecology of chlorinated solvent biodegradation

This study focused on the microbial ecology of tetrachloroethene (PCE) degradation to trichloroethene, cis‐1,2‐dichloroethene and vinyl chloride to evaluate the relationship between the microbial community and the potential accumulation or degradation of these toxic metabolites. Multiple soil microcosms supplied with different organic substrates were artificially contaminated with PCE. A thymidine analogue, bromodeoxyuridine (BrdU), was added to the microcosms and incorporated into the DNA of actively replicating cells. We compared the total and active bacterial communities during the 50‐day incubations by using phylogenic microarrays and 454 pyrosequencing to identify microorganisms and functional genes associated with PCE degradation to ethene. By use of this integrative approach, both the key community members and the ecological functions concomitant with complete PCE degradation could be determined, including the presence and activity of microbial community members responsible for producing hydrogen and acetate, which are critical for Dehalococcoides‐mediated PCE degradation. In addition, by correlation of chemical data and phylogenic microarray data, we identified several bacteria that could potentially oxidize hydrogen. These results demonstrate that PCE degradation is dependent on some microbial community members for production of appropriate metabolites, while other members of the community compete for hydrogen in soil at low redox potentials.     

Global dynamics of microbial communities emerge from local interaction rules

Most microbes live in spatially structured communities (e.g., biofilms) in which they interact with their neighbors through the local exchange of diffusible molecules. To understand the functioning of these communities, it is essential to uncover how these local interactions shape community-level properties, such as the community composition, spatial arrangement, and growth rate. Here, we present a mathematical framework to derive community-level properties from the molecular mechanisms underlying the cell-cell interactions for systems consisting of two cell types. Our framework consists of two parts: a biophysical model to derive the local interaction rules (i.e. interaction range and strength) from the molecular parameters underlying the cell-cell interactions and a graph based model to derive the equilibrium properties of the community (i.e. composition, spatial arrangement, and growth rate) from these local interaction rules. Our framework shows that key molecular parameters underlying the cell-cell interactions (e.g., the uptake and leakage rates of molecules) determine community-level properties. We apply our model to mutualistic cross-feeding communities and show that spatial structure can be detrimental for these communities. Moreover, our model can qualitatively recapitulate the properties of an experimental microbial community. Our framework can be extended to a variety of systems of two interacting cell types, within and beyond the microbial world, and contributes to our understanding of how community-level properties emerge from microscopic interactions between cells.     

The effect of the intra- and extracellular metabolites of microorganisms isolated from various ecotopes on the catalase activity of Staphylococcus aureus 6538P

The cell extracts (i.e., intracellular metabolites) and culture liquids (i.e., extracellular metabolites) of microorganisms isolated from various ecotopes were found to inhibit the catalase activity of Staphylococcus aureus ATCC 6538P, which resulted in a considerable inhibition of the growth of metabolite-treated S. aureus cells by hydrogen peroxide. The inhibitory effect of microbial metabolites on S. aureus catalase can be considered as a mechanism of intercellular interactions responsible for the formation of microbiocenoses.     

Succession of sulfur-oxidizing bacteria in the microbial community on corroding concrete in sewer systems

Microbially induced concrete corrosion (MICC) in sewer systems has been a serious problem for a long time. A better understanding of the succession of microbial community members responsible for the production of sulfuric acid is essential for the efficient control of MICC. In this study, the succession of sulfur-oxidizing bacteria (SOB) in the bacterial community on corroding concrete in a sewer system in situ was investigated over 1 year by culture-independent 16S rRNA gene-based molecular techniques. Results revealed that at least six phylotypes of SOB species were involved in the MICC process, and the predominant SOB species shifted in the following order: Thiothrix sp., Thiobacillus plumbophilus, Thiomonas intermedia, Halothiobacillus neapolitanus, Acidiphilium acidophilum, and Acidithiobacillus thiooxidans. A. thiooxidans, a hyperacidophilic SOB, was the most dominant (accounting for 70% of EUB338-mixed probe-hybridized cells) in the heavily corroded concrete after 1 year. This succession of SOB species could be dependent on the pH of the concrete surface as well as on trophic properties (e.g., autotrophic or mixotrophic) and on the ability of the SOB to utilize different sulfur compounds (e.g., H2S, S0, and S2O3(2-)). In addition, diverse heterotrophic bacterial species (e.g., halo-tolerant, neutrophilic, and acidophilic bacteria) were associated with these SOB. The microbial succession of these microorganisms was involved in the colonization of the concrete and the production of sulfuric acid. Furthermore, the vertical distribution of microbial community members revealed that A. thiooxidans was the most dominant throughout the heavily corroded concrete (gypsum) layer and that A. thiooxidans was most abundant at the highest surface (1.5-mm) layer and decreased logarithmically with depth because of oxygen and H2S transport limitations. This suggested that the production of sulfuric acid by A. thiooxidans occurred mainly on the concrete surface and the sulfuric acid produced penetrated through the corroded concrete layer and reacted with the sound concrete below.     

The Effect of the Intra- and Extracellular Metabolites of Microorganisms Isolated from Various Ecotopes on the Catalase Activity of Staphylococcus aureus ATCC 6538 P

The cell extracts (i.e., intracellular metabolites) and culture liquids (i.e., extracellular metabolites) of microorganisms isolated from various ecotopes were found to inhibit the catalase activity of Staphylococcus aureus ATCC 6538 P, which resulted in a considerable inhibition of the growth of metabolite-treated S. aureus cells by hydrogen peroxide. The inhibitory effect of microbial metabolites on S. aureus catalase can be considered as a mechanism of intercellular interactions responsible for the formation of microbiocenoses.     

An analysis of the volatile flavour compounds in a soft raw goat milk cheese

The major components of the musty cow rind cheeses were identified in a soft raw goat milk cheese as heptan-2-one, nonan-2-one, their corresponding secondary alcohols, some esters and sulfur compounds. Their production was associated with the manufacturing process and its influence on the microbial activity. However a specificity in goat cheese compounds was displayed concerning in particular limonene and some ketones, alcohols and aldehydes.     

Diffusion of lactate and ammonium in relation to growth of Geotrichum candidum at the surface of solid media

Geotrichum candidum was cultivated at the surface of solid model media containing peptone to simulate the composition of Camembert cheese. The surface growth of G. candidum induced the diffusion of substrates from the core to the rind and the diffusion of produced metabolites from the rind to the core. In the range of pH measured during G. candidum growth, constant diffusion coefficients were found for lactate and ammonium, 0.4 and 0.8 cm(2) day(-1), respectively, determined in sterile culture medium. Growth kinetics are described using the Verlhust model and both lactate consumption and ammonium production are considered as partially linked to growth. The experimental diffusion gradients of lactate and ammonium recorded during G. candidum growth have been fitted. The diffusion/reaction model was found to match with experimental data until the end of growth, except with regard to ammonium concentration gradients in the presence of lactate in the medium. Indeed, G. candidum preferentially assimilated peptone over lactate as a carbon source, resulting in an almost cessation of ammonium release before the end of growth. On peptone, it was found that the proton transfer did not account for the ammonium concentration gradients. Indeed, amino acids, being positively charged, are involved in the proton transfer at the beginning of growth. This effect can be neglected in the presence of lactate within the medium, and the sum of both lactate consumption and ammonium release gradients corresponded well to the proton transfer gradients, confirming that both components are responsible for the pH increase observed during the ripening of soft Camembert cheese.     

Linking cell structure to gene regulation: Signaling events and expression controls on the model genes PAI-1 and CTGF

The microtubule and microfilament cytoskeletal systems as well as cell-to-cell contacts and cell–matrix interactions are critical regulators of cell structure and function. Alterations in cell shape profoundly influence signaling events and gene expression programs that impact a spectrum of biological responses including cell growth, migration and apoptosis. These same pathways also contribute to the progression of several important pathologic conditions (e.g., arteriosclerosis, vascular fibrosis, and endothelial dysfunction). Indeed, hemodynamic forces in the vascular compartment are established modifiers of endothelial and smooth muscle cell cytoarchitecture and orchestrate complex genetic and biological responses in concert with contributions from the extracellular matrix (ECM), growth factors (e.g., EGF, and TGF-β) and cell adhesion receptors (e.g., integrins, and cadherins). The profibrotic matricellular proteins plasminogen activator inhibitor-1 (PAI-1) and connective tissue growth factor (CTGF) are prominent members of a subset of genes the expression of which is highly responsive to cell shape-altering stimuli (i.e., disruption of the actin-based and microtubule networks, shear strain and cyclic stretch). Since both PAI-1 and CTGF are major mediators of cardiovascular fibrotic disease, understanding cell structure-linked signaling cascades provides potential avenues for focused therapy. It is increasingly evident that growth factor receptors (EGFR) are activated by changes in cytoarchitecture and that the “repressive state” of certain signaling proteins (e.g., SMAD, and Rho-GEFs) is maintained by sequestration on cell structural networks. Functional repression can be relieved by cytoskeletal perturbations (e.g., in response to treatment with network-specific drugs) resulting in activation of signaling cascades (e.g., Rho, and MAPK) with associated changes in gene reprogramming. Recent studies document a complex network of both similar and unique signaling control elements leading to the induction of PAI-1 and CTGF in response to modifications in cell shape. The purpose of this review is to highlight our current understanding of “cell deformation”-responsive signaling cascades focusing on the potential value of targeting such pathways, and their model response genes (e.g., PAI-1, and CTGF), as a therapeutic option for the treatment of fibrotic diseases.     

Intertwined interspecies relationships: approaches to untangle the microbial network

In nature, microorganisms live by interacting with each other. Microbiological studies that only consider pure cultures are not sufficient to adequately describe the natural behaviour of microbes. Several microbial interactions have been recognized to affect the growth or metabolism of others; e.g. syntrophic cometabolism, competition, production of inhibitors or activators, and predation. It is believed that third‐party organisms easily affect the two‐species relationships and these relationships form the basis of interspecies networks within microbial communities. A microbial network contributes to ‘functional redundancy’ or ‘structural diversity’ and the microbial communities effectively act as a multicellular organism. It is necessary to understand not only the physiological activity of members within microbial communities but also their roles to regulate the activity or population of others. To access the microbial network, we require (i) comprehensive determination of all possible interspecies relationships among microbes, (ii) knock‐out experiments by which certain members can be removed or suppressed, and (iii) supplemental addition of microbes or activation of certain members. Microbial network studies have started using defined microbial communities, i.e. a mixed culture that is composed of three or four species. In order to expand these studies to microflora in nature, microbial ecology requires the help of mathematical biology.     

Microbial Interactions within a Cheese Microbial Community

The interactions that occur during the ripening of smear cheeses are not well understood. Yeast-yeast interactions and yeast-bacterium interactions were investigated within a microbial community composed of three yeasts and six bacteria found in cheese. The growth dynamics of this community was precisely described during the ripening of a model cheese, and the Lotka-Volterra model was used to evaluate species interactions. Subsequently, the effects on ecosystem functioning of yeast omissions in the microbial community were evaluated. It was found both in the Lotka-Volterra model and in the omission study that negative interactions occurred between yeasts. Yarrowia lipolytica inhibited mycelial expansion of Geotrichum candidum, whereas Y. lipolytica and G. candidum inhibited Debaryomyces hansenii cell viability during the stationary phase. However, the mechanisms involved in these interactions remain unclear. It was also shown that yeast-bacterium interactions played a significant role in the establishment of this multispecies ecosystem on the cheese surface. Yeasts were key species in bacterial development, but their influences on the bacteria differed. It appeared that the growth of Arthrobacter arilaitensis or Hafnia alvei relied less on a specific yeast function because these species dominated the bacterial flora, regardless of which yeasts were present in the ecosystem. For other bacteria, such as Leucobacter sp. or Brevibacterium aurantiacum, growth relied on a specific yeast, i.e., G. candidum. Furthermore, B. aurantiacum, Corynebacterium casei, and Staphylococcus xylosus showed reduced colonization capacities in comparison with the other bacteria in this model cheese. Bacterium-bacterium interactions could not be clearly identified.     

Adaptive Evolution of Synthetic Cooperating Communities Improves Growth Performance

Symbiotic interactions between organisms are important for human health and biotechnological applications. Microbial mutualism is a widespread phenomenon and is important in maintaining natural microbial communities. Although cooperative interactions are prevalent in nature, little is known about the processes that allow their initial establishment, govern population dynamics and affect evolutionary processes. To investigate cooperative interactions between bacteria, we constructed, characterized, and adaptively evolved a synthetic community comprised of leucine and lysine Escherichia coli auxotrophs. The co-culture can grow in glucose minimal medium only if the two auxotrophs exchange essential metabolites — lysine and leucine (or its precursors). Our experiments showed that a viable co-culture using these two auxotrophs could be established and adaptively evolved to increase growth rates (by ∼3 fold) and optical densities. While independently evolved co-cultures achieved similar improvements in growth, they took different evolutionary trajectories leading to different community compositions. Experiments with individual isolates from these evolved co-cultures showed that changes in both the leucine and lysine auxotrophs improved growth of the co-culture. Interestingly, while evolved isolates increased growth of co-cultures, they exhibited decreased growth in mono-culture (in the presence of leucine or lysine). A genome-scale metabolic model of the co-culture was also constructed and used to investigate the effects of amino acid (leucine or lysine) release and uptake rates on growth and composition of the co-culture. When the metabolic model was constrained by the estimated leucine and lysine release rates, the model predictions agreed well with experimental growth rates and composition measurements. While this study and others have focused on cooperative interactions amongst community members, the adaptive evolution of communities with other types of interactions (e.g., commensalism, ammensalism or parasitism) would also be of interest.     

Scanning electron and light microscopic study of microbial succession on bethlehem st. Nectaire cheese

St. Nectaire cheese is a semisoft cheese of French origin that, along with Brie and Camembert cheeses, belongs to the class of surface mold-ripened cheese. The surface microorganisms that develop on the cheese rind during ripening impart a distinctive aroma and flavor to this class of cheese. We have documented the sequential appearance of microorganisms on the cheese rind and in the curd over a 60-day ripening period. Scanning electron microscopy was used to visualize the development of surface fungi and bacteria. Light microscopy of stained paraffin sections was used to study cross sections through the rind. We also monitored the development of bacterial and yeast populations in and the pH of the curd and rind. The earliest stage of ripening (0 to 2 days) is dominated by the lactic acid bacterium Streptococcus cremoris and multilateral budding yeasts, primarily Debaryomyces and Torulopsis species. Geotrichum candidum follows closely, and then zygomycetes of the genus Mucor develop at day 4 of ripening. At day 20, the deuteromycete Trichothecium roseum appears. From day 20 until the end of the ripening process, coryneforms of the genera Brevibacterium and Arthrobacter can be seen near the surface of the cheese rind among fungal hyphae and yeast cells.     

Biodiversity in Oscypek, a traditional Polish cheese, determined by culture-dependent and -independent approaches

Oscypek is a traditional Polish scalded-smoked cheese, with a protected-designation-of-origin (PDO) status, manufactured from raw sheep's milk without starter cultures in the Tatra Mountains region of Poland. This study was undertaken in order to gain insight into the microbiota that develops and evolves during the manufacture and ripening stages of Oscypek. To this end, we made use of both culturing and the culture-independent methods of PCR followed by denaturing gradient gel electrophoresis (PCR-DGGE) and pyrosequencing of 16S rRNA gene amplicons. The culture-dependent technique and PCR-DGGE fingerprinting detected the predominant microorganisms in traditional Oscypek, whereas the next-generation sequencing technique (454 pyrosequencing) revealed greater bacterial diversity. Besides members of the most abundant bacterial genera in dairy products, e.g., Lactococcus, Lactobacillus, Leuconostoc, Streptococcus, and Enterococcus, identified by all three methods, other, subdominant bacteria belonging to the families Bifidobacteriaceae and Moraxellaceae (mostly Enhydrobacter), as well as various minor bacteria, were identified by pyrosequencing. The presence of bifidobacterial sequences in a cheese system is reported for the first time. In addition to bacteria, a great diversity of yeast species was demonstrated in Oscypek by the PCR-DGGE method. Culturing methods enabled the determination of a number of viable microorganisms from different microbial groups and their isolation for potential future applications in specific cheese starter cultures.     

Growth and aroma contribution of <Emphasis Type="Italic">Microbacterium foliorum</Emphasis>, <Emphasis Type="Italic">Proteus vulgaris</Emphasis> and <Emphasis Type="Italic">Psychrobacter</Emphasis> sp. during ripening in a cheese model medium

The growth and aroma contribution of Microbacterium foliorum, Proteus vulgaris and Psychrobacter sp., some common but rarely mentioned cheese bacteria, were investigated in a cheese model deacidified by Debaryomyces hansenii during the ripening process. Our results show that these bacteria had distinct growth and cheese flavour production patterns during the ripening process. P. vulgaris had the greatest capacity to produce not only the widest variety but also the highest quantities of volatile compounds with low olfactive thresholds, e.g. volatile sulphur compounds and branched-chain alcohols. Such compounds produced by P. vulgaris increased after 21 days of ripening and reached a maximum at 41 days. The three bacteria studied exhibited various degrees of caseinolytic, aminopeptidase and deaminase activities. Moreover, P. vulgaris had a greater capacity for hydrolysing casein and higher deaminase activity. Our results show that P. vulgaris, a Gram-negative bacterium naturally present on the surface of ripened cheeses, could produce high concentrations of flavour compounds from amino acid degradation during the ripening process. Its flavouring role in cheese cannot be neglected. Moreover, it could be a useful organism for producing natural flavours as dairy ingredients.