Recent methods for discovering novel bioactive metabolites, specifically antimicrobial agents, from marine-associated micro-organisms.

Marine micro-organisms are a promising source for novel natural compounds with many medical and biotechnological applications. Here, we demonstrate limitations and recent strategies for investigating the marine microbial community for novel bioactive metabolites, specifically those of antimicrobial potential. These strategies include culture-dependent methods such as modifying the standard culture media, including changing the gelling agent, dissolving vehicle, media supplementation and preparation to access a broader range of bacterial diversity from marine samples. Furthermore, we discuss strategies such as in situ cultivation, dilution-to-extinction cultivation and long-term incubation. We are presenting recent applications of culture-independent methods such as genome mining, proteomics profiling and the application of metagenomics as a novel strategy for structure confirmation in the discovery of the marine micro-organism for novel antimicrobial metabolites. We present this review as a simple guide and a helpful resource for those who seek to enter the challenging field of applied marine microbiology.

Quantitative profiling of built environment bacterial and fungal communities reveals dynamic material dependent growth patterns and microbial interactions.

Indoor microbial communities vary in composition and diversity depending on material type, moisture levels, and occupancy. In this study, we integrated bacterial cell counting, fungal biomass estimation, and fluorescence-assisted cell sorting (FACS) with amplicon sequencing of bacterial (16S rRNA) and fungal (ITS) communities to investigate the influence of wetting on medium density fiberboard (MDF) and gypsum wallboard. Surface samples were collected longitudinally from wetted materials maintained at high relative humidity (~95%). Bacterial and fungal growth patterns were strongly time-dependent and material-specific. Fungal growth phenotypes differed between materials: spores dominated MDF surfaces while fungi transitioned from spores to hyphae on gypsum. FACS confirmed that most of the bacterial cells were intact (viable) on both materials over the course of the study. Integrated cell count and biomass data (quantitative profiling) revealed that small changes in relative abundance often resulted from large changes in absolute abundance, while negative correlations in relative abundances were explained by rapid growth of only one group of bacteria or fungi. Comparisons of bacterial-bacterial and fungal-bacterial networks suggested a top-down control of fungi on bacterial growth, possibly via antibiotic production. In conclusion, quantitative profiling provides novel insights into microbial growth dynamics on building materials with potential implications for human health.

Listeria monocytogenes infectious periaortitis: a case report from the infectious disease standpoint.

BACKGROUND: Endograft infection is a rare but extremely dangerous complication of aortic repair (25-100% of mortality). We describe here the first case of Listeria monocytogenes abdominal periaortitis associated with a vascular graft. We also discuss the differential diagnosis of periaortitis and provide a literature review of L. monocytogenes infectious aortitis. CASE PRESENTATION: Nine months after endovascular treatment of an abdominal aortic aneurysm (abdominal stent graft), a 76-year-old man was admitted for severe abdominal pain radiating to the back. Laboratory tests were normal apart from elevated C-reactive protein (CRP). Injected abdominal computed tomography (CT) showed infiltration of the fat tissues around the aortic endoprosthesis and aneurysmal sac expansion; positron emission tomography with 2-deoxy-2-[fluorine-18]fluoro- D-glucose integrated with computed tomography (18F-FDG PET/CT) showed a hypermetabolic mass in contact with the endoprosthesis. Blood cultures were negative. At surgical revision, an infra-renal peri-aortic abscess was evident; post-operative antibiotic therapy with ciprofloxacin and doxycycline was started. Cultures of intraoperative samples were positive for L. monocytogenes. Results were further confirmed by a broad-range polymerase chain reaction (PCR) and next-generation sequencing. Antibiotic treatment was switched to intravenous amoxicillin for 6 weeks. Evolution was uneventful with decrease of inflammatory parameters and regression of the abscess. CONCLUSION: An etiologic bacterial diagnosis before starting antibiotic therapy is paramount; nevertheless, culture-independent methods may provide a microbiological diagnosis in those cases where antimicrobials are empirically used and when cultures remain negative.

Assessment of the bacterial viability of chlorine- and quaternary ammonium compounds-treated Lactobacillus cells via a multi-method approach.

AIMS: The assessment of the bacterial viability of chlorine- and quaternary ammonium compounds (QACs)-treated Lactobacillus cells by culture-dependent and -independent methods. METHODS AND RESULTS: Lactobacillus isolates (Lactobacillus plantarum G1, Lactobacillus plantarum B1, Lactobacillus brevis S1 and Lactobacillus paracasei W1) in biofilm and planktonic cell suspensions were treated with chlorine-based (0·018 and 0·18%) and QACs-based (0·2 and 2·0%) disinfectants for 5 min and then analysed by plate counting, flow cytometry (FCM) and fluorescence activated cell sorting (FACS). The reaction of sessile cells to disinfectants was assessed with the confocal laser scanning microscopy (CLSM). Plate counts revealed L. paracasei W1 to be substantially inactivated by both disinfectants, while counts of the other isolates to be significantly reduced only by QACs, with L. plantarum B1 and L. brevis S1 showing a greater difference between QACs concentrations and cell types. In several cases, the disinfectants caused slightly higher inactivation of planktonic than biofilm cells, with L. plantarum B1 being significantly less sensitive to QACs in biofilm cells (P < 0·05). Following FCM with a Syto® 9/PI assay, which addresses cell membrane integrity, the emergence of damaged (Syto® 9- PI+ ) and injured (Syto® 9+ PI+ ) subpopulations was often observed in cells when they were treated with QACs, whereas intact (Syto® 9+ PI- ) and unstained (Syto® 9- PI- ) subpopulations were mostly encountered in chlorine-treated cells. Except Syto® 9- PI+ , all subpopulations were recovered on agar plates following FACS, with biofilm cells showing higher culturability irrespective of conditions, probably because of the residues of the biofilm matrix which serve as a protective cover for the bacteria. The CLSM revealed a substantial cell membrane damage within the QACs-treated biofilms, however, some cells deep in the biofilm were still intact and thus remained protected against this disinfectant. CONCLUSION: We found that FCM/FACS proved useful in the analysis of lactobacilli membrane integrity in disinfection experiments as well as in recovery evaluation of planktonic-biofilm cell subpopulations. In turn, CLSM was particularly useful in investigating the resistance mechanism when Lactobacillus cells were embedded in biofilms. SIGNIFICANCE AND IMPACT OF THE STUDY: This study highlights the need for treatment optimization on a case-by-case basis to avoid the emergence of cells in intermediate states with recovery potential and to reach and, thus, kill all bacteria in already developed lactobacilli biofilms.

Veterinary ocular microbiome: Lessons learned beyond the culture.

Ocular pathogens cause many painful and vision-threatening diseases such as infectious keratitis, uveitis, and endophthalmitis. While virulent pathogens and pathobionts play important roles in disease pathogenesis, the scientific community has long assumed disruption of the ocular surface occurs prior to microbial colonization and subsequent infection. While nonpathogenic bacteria are often detected in corneal and conjunctival cultures from healthy eyes, cultures also frequently fail to yield growth of common ocular pathogens or nonpathogenic bacteria. This prompts the following question: Is the ocular surface populated by a stable microbial population that cannot be detected using standard culture techniques? The study of the microbiome has recently become a widespread focus in physician and veterinary medicine. Research suggests a pivotal symbiotic relationship with these microbes to maintain healthy host tissues, and when altered is associated with various disease states ("dysbiosis"). The microbiota that lives within and on mammalian bodies have long been known to influence health and susceptibility to infection. However, limitations of traditional culture methods have resulted in an incomplete understanding of what many now call the "forgotten organ," that is, the microbiome. With the introduction of high-throughput sequencing, physician ophthalmology has recognized an ocular surface with much more diverse microbial communities than suspected based on traditional culture. This article reviews the salient features of the ocular surface microbiome and highlights important future applications following the advent of molecular techniques for microbial identification, including characterizing ocular surface microbiomes in our veterinary species and their potential role in management of infectious and inflammatory ocular diseases.

Advancement in LH-PCR methodology for multiple microbial species detections in fermented foods.

The length-heterogeneity PCR is a low throughput molecular biology methods explored to monitor bacteria populations in different environments. It could be more used in food microbiology analysis, not only for fingerprinting analysis, but it has been hampered until now by a limiting factor which relates to the high percentage of secondary peaks. With the aim to overcome this problem, different experiments were performed focusing on changing PCR parameters in order to obtain more specific amplicon patterns and also to reduce the complexity of community patterns. With this purpose, different annealing temperatures were tested on complex fermented food matrices taken from both animal and vegetable origin and also on the bacteria isolated from the same food source. In particular, the optimal annealing temperature identified for the fermented food samples is 59 °C and the optimal for bacterial strains varied between 63 °C and 65 °C. The approach allowed the modification of the LH-PCR protocol increasing the amplification efficiency and therefore the bacteria species discrimination. These temperatures also allowed the implementation of the previous LH-PCR published database. The modification in the level of accuracy of the LH-PCR technique could also allow an improvement in the relative species quantification by the peak area evaluation.

Rapid assemblage of diverse environmental fungal communities on public restroom floors.

An increasing proportion of humanity lives in urban environments where they spend most of their lives indoors. Recent molecular studies have shown that bacterial assemblages in built environments (BEs) are extremely diverse, but BE fungal diversity remains poorly understood. We applied culture-independent methods based on next-generation sequencing (NGS) of the fungal internal transcribed spacer to investigate the diversity and temporal dynamics of fungi in restrooms. Swab samples were collected weekly from three different surfaces in two public restrooms (male and female) in San Diego, CA, USA, over an 8-week period. DNA amplification and culturing methods both found that the floor samples had significantly higher fungal loads than other surfaces. NGS sequencing of floor fungal assemblages identified a total of 2550 unique phylotypes (~800 per sample), less than half of which were identifiable. Of the known fungi, the majority came from environmental sources and we found little evidence of known human skin fungi. Fungal assemblages reformed rapidly in a highly consistent manner, and the variance in the species diversity among samples was low. Overall, our study contributes to a better understanding of public restroom floor fungal communities.

Bacterial diversity of baechu-kimchi with seafood based on culture-independent investigations.

Baechu-kimchi is a traditional Korean dish of fermented vegetables, in which kimchi cabbage is the major ingredient. Seafood is added to baechu-kimchi in coastal areas, giving this dish regional diversity. However, little is known about how the addition of seafood affects the bacterial diversity of kimchi. Therefore, in this study, the bacterial diversity of five varieties of baechu-kimchi with seafood and one variety of baechu-kimchi without seafood was analyzed using culture-independent techniques. In 81.7% of all kimchi analyzed, the predominant species were members of the phylum Firmicutes and the lactic acid bacteria, Latilactobacillus sakei, Leuconostoc mesenteroides, Pediococcus inopinatus, and Weissella koreensis. These organisms were similar to those identified in baechu-kimchi without the addition of seafood, which was used as a control group, and bacterial community of previously reported kimchi. Therefore, the results of this study confirmed that the addition of seafood did not significantly affect the bacterial community in baechu-kimchi.

The Skin Microbiome: Current Techniques, Challenges, and Future Directions.

Skin acts as a barrier that promotes the colonization of bacteria, fungi, archaea, and viruses whose membership and function may differ depending on the various specialized niches or micro-environments of the skin. The group of microorganisms inhabiting the skin, also known as the skin microbiome, offers protection against pathogens while actively interacting with the host's immune system. Some members of the skin microbiome can also act as opportunistic pathogens. The skin microbiome is influenced by factors such as skin site, birth mode, genetics, environment, skin products, and skin conditions. The association(s) of the skin microbiome with health and disease has (have) been identified and characterized via culture-dependent and culture-independent methods. Culture-independent methods (such as high-throughput sequencing), in particular, have expanded our understanding of the skin microbiome's role in maintaining health or promoting disease. However, the intrinsic challenges associated with the low microbial biomass and high host content of skin microbiome samples have hindered advancements in the field. In addition, the limitations of current collection and extraction methods and biases derived from sample preparation and analysis have significantly influenced the results and conclusions of many skin microbiome studies. Therefore, the present review discusses the technical challenges associated with the collection and processing of skin microbiome samples, the advantages and disadvantages of current sequencing approaches, and potential future areas of focus for the field.

Fermented fish and fermented fish-based products, an ever-growing source of microbial diversity: A literature review.

Fermented fish and fermented fish-based products are part of the diet of many countries all over the world. Their popularity is not only due to the unique flavor, the distinct texture, and the good nutritional quality, but also to the easiness of the production process, that is commonly based on empirical traditional methods. Fish fermentation techniques ususally rely on the combination of some key steps, including salting, addition of spices or additives, and maintenance of anaerobic conditions, thus selecting for the multiplication of some pro-technological microorganisms. The objective of the present review was to provide an overview of the current knowledge of the microbial communities occurring in fermented fish and fish-based products. Specific information was collected from scientific publications published from 2000 to 2022 with the aim of generating a comprehensive database. The production of fermented fish and fish-based foods was mostly localized in West African countries, Northern European countries, and Southeast Asian countries. Based on the available literature, the microbial composition of fermented fish and fish-based products was delineated by using viable counting combined with identification of isolates, and culture-independent techniques. The data obtained from viable counting highlighted the occurrence of microbial groups usually associated with food fermentation, namely lactic acid bacteria, staphylococci, Bacillus spp., and yeasts. The identification of isolates combined with culture-independent methods showed that the fermentative process of fish-based products was generally guided by lactobacilli (Lactiplantibacillus plantarum, Latilactobacillus sakei, and Latilactobacillus curvatus) or Tetragenococcus spp. depending on the salt concentration. Among lactic acid bacteria populations, Lactococcus spp., Pediococcus spp., Leuconostoc spp., Weissella spp., Enterococcus spp., Streptococcus spp., and Vagococcus spp. were frequently identified. Staphylococcus spp. and Bacillus spp. confirmed a great adaptation to fermented fish-based products. Other noteworthy bacterial taxa included Micrococcus spp., Pseudomonas spp., Psychrobacter spp., Halanaerobium spp., and Halomonas spp. Among human pathogenic bacteria, the occurrence of Clostridium spp. and Vibrio spp. was documented. As for yeast populations, the predominance of Candida spp., Debaryomyces spp., and Saccharomyces spp. was evidenced. The present literature review could serve as comprehensive database for the scientific community, and as a reference for the food industry in order to formulate tailored starter or adjunctive cultures for product improvement.

Ecological and Oceanographic Perspectives in Future Marine Fungal Taxonomy.

Marine fungi are an ecological rather than a taxonomic group that has been widely researched. Significant progress has been made in documenting their phylogeny, biodiversity, ultrastructure, ecology, physiology, and capacity for degradation of lignocellulosic compounds. This review (concept paper) summarizes the current knowledge of marine fungal diversity and provides an integrated and comprehensive view of their ecological roles in the world's oceans. Novel terms for 'semi marine fungi' and 'marine fungi' are proposed based on the existence of fungi in various oceanic environments. The major maritime currents and upwelling that affect species diversity are discussed. This paper also forecasts under-explored regions with a greater diversity of marine taxa based on oceanic currents. The prospects for marine and semi-marine mycology are highlighted, notably, technological developments in culture-independent sequencing approaches for strengthening our present understanding of marine fungi's ecological roles.

Bioprospecting of Novel Extremozymes From Prokaryotes-The Advent of Culture-Independent Methods.

Extremophiles are remarkable organisms that thrive in the harshest environments on Earth, such as hydrothermal vents, hypersaline lakes and pools, alkaline soda lakes, deserts, cold oceans, and volcanic areas. These organisms have developed several strategies to overcome environmental stress and nutrient limitations. Thus, they are among the best model organisms to study adaptive mechanisms that lead to stress tolerance. Genetic and structural information derived from extremophiles and extremozymes can be used for bioengineering other nontolerant enzymes. Furthermore, extremophiles can be a valuable resource for novel biotechnological and biomedical products due to their biosynthetic properties. However, understanding life under extreme conditions is challenging due to the difficulties of in vitro cultivation and observation since > 99% of organisms cannot be cultivated. Consequently, only a minor percentage of the potential extremophiles on Earth have been discovered and characterized. Herein, we present a review of culture-independent methods, sequence-based metagenomics (SBM), and single amplified genomes (SAGs) for studying enzymes from extremophiles, with a focus on prokaryotic (archaea and bacteria) microorganisms. Additionally, we provide a comprehensive list of extremozymes discovered via metagenomics and SAGs.

Current knowledge on the microbiota of edible insects intended for human consumption: A state-of-the-art review.

Because of their positive nutritional characteristics and low environmental impact, edible insects might be considered a 'food of the future'. However, there are safety concerns associated with the consumption of insects, such as contaminating chemical and biological agents. The possible presence of pathogenic and toxigenic microorganisms is one of the main biological hazards associated with edible insects. This review presents an overview of the microbiota of edible insects, highlighting the potential risks for human health. Detailed information on the microbiota of edible insects from literature published in 2000-2019 is presented. These data show complex ecosystems, with marked variations in microbial load and diversity, among edible insects as well as stable and species-specific microbiota for some of the most popular edible insect species, such as mealworm larvae (Tenebrio molitor) and grasshoppers (Locusta migratoria). Raw edible insects generally contain high numbers of mesophilic aerobes, bacterial endospores or spore-forming bacteria, Enterobacteriaceae, lactic acid bacteria, psychrotrophic aerobes, and fungi, and potentially harmful species (i.e. pathogenic, mycotoxigenic, and spoilage microbes) may be present. Several studies have focused on reducing the microbial contamination of edible insects by applying treatments such as starvation, rinsing, thermal treatments, chilling, drying, fermentation, and marination, both alone and, sometimes, in combination. Although these studies show that various heat treatments were the most efficient methods for reducing microbial numbers, they also highlight the need for species-specific mitigation strategies. The feasibility of using edible insects as ingredients in the food industry in the development of innovative insect-based products has been explored; although, in some cases, the presence of spore-forming bacteria and other food-borne pathogens is a concern. Recent studies have shown that a risk assessment of edible insects should also include an evaluation of the incidence of antibiotic-resistance (AR) genes and antibiotic-resistant microorganisms in the production chain. Finally, as proposed in the literature, microbial hazards should be limited through the implementation of good hygienic practices during rearing, handling, processing, and storage, as well as the implementation of an appropriate HACCP system for edible insect supply chains. Another issue frequently reported in the literature is the need for a legislative framework for edible insect production, commercialisation, and trading, as well as the need for microbiological criteria specifically tailored for edible insects. Microbiological criteria like those already been established for the food safety and hygiene (e.g. those in the European Union food law) of different food categories (e.g. ready-to-eat products) could be applied to edible insect-based products.

A Functional Metagenomic Analysis of Tetracycline Resistance in Cheese Bacteria.

Metagenomic techniques have been successfully used to monitor antibiotic resistance genes in environmental, animal and human ecosystems. However, despite the claim that the food chain plays a key role in the spread of antibiotic resistance, metagenomic analysis has scarcely been used to investigate food systems. The present work reports a functional metagenomic analysis of the prevalence and evolution of tetracycline resistance determinants in a raw-milk, blue-veined cheese during manufacturing and ripening. For this, the same cheese batch was sampled and analyzed on days 3 and 60 of manufacture. Samples were diluted and grown in the presence of tetracycline on plate count milk agar (PCMA) (non-selective) and de Man Rogosa and Sharpe (MRS) agar (selective for lactic acid bacteria, LAB). DNA from the cultured bacteria was then isolated and used to construct four fosmid libraries, named after the medium and sampling time: PCMA-3D, PCMA-60D, MRS-3D, and MRS-60D. Clones in the libraries were subjected to restriction enzyme analysis, PCR amplification, and sequencing. Among the 300 fosmid clones analyzed, 268 different EcoRI restriction profiles were encountered. Sequence homology of their extremes clustered the clones into 47 groups. Representative clones of all groups were then screened for the presence of tetracycline resistance genes by PCR, targeting well-recognized genes coding for ribosomal protection proteins and efflux pumps. A single tetracycline resistance gene was detected in each of the clones, with four such resistance genes identified in total: tet(A), tet(L), tet(M), and tet(S). tet(A) was the only gene identified in the PCMA-3D library, and tet(L) the only one identified in the PCMA-60D and MRS-60D libraries. tet(M) and tet(S) were both detected in the MRS-3D library and in similar numbers. Six representative clones of the libraries were sequenced and analyzed. Long segments of all clones but one showed extensive homology to plasmids from Gram-positive and Gram-negative bacteria. tet(A) was found within a sequence showing strong similarity to plasmids pMAK2 and pO26-Vir from Salmonella enterica and Escherichia coli, respectively. All other genes were embedded in, or near to, sequences homologous to those of LAB species. These findings strongly suggest an evolution of tetracycline resistance gene types during cheese ripening, which might reflect the succession of the microbial populations. The location of the tetracycline resistance genes in plasmids, surrounded or directly flanked by open reading frames encoding transposases, invertases or mobilization proteins, suggests they might have a strong capacity for transference. Raw-milk cheeses should therefore be considered reservoirs of tetracycline resistance genes that might be horizontally transferred.

Polyphasic insights into the microbiomes of the Takamatsuzuka Tumulus and Kitora Tumulus.

Microbial outbreaks and related biodeterioration problems have affected the 1300-year-old multicolor (polychrome) mural paintings of the special historic sites Takamatsuzuka Tumulus (TT) and Kitora Tumulus (KT). Those of TT are designated as a national treasure. The microbiomes of these tumuli, both located in Asuka village, Nara, Japan, are critically reviewed as the central subject of this report. Using culture-dependent methods (conventional isolation and cultivation), we conducted polyphasic studies of the these microbial communities and identified the major microbial colonizers (Fusarium spp., Trichoderma spp., Penicillium spp., dark Acremonium spp., novel Candida yeast spp., Bacillus spp., Ochrobactrum spp., Stenotrophomonas tumulicola, and a few actinobacterial genera) and noteworthy microbial members (Kendrickiella phycomyces, Cephalotrichum verrucisporum (≡Doratomyces verrucisporus), Sagenomella striatispora, Sagenomella griseoviridis, two novel Cladophialophora spp., Burgoa anomala, one novel species Prototheca tumulicola, five novel Gluconacetobacter spp., three novel Bordetella spp., and one novel genus and species Krasilnikoviella muralis) involved in the biodeterioration of mural paintings, plaster walls, and stone chamber interiors. In addition, we generated microbial community data from TT and KT samples using culture-independent methods (molecular biological methods, including PCR-DGGE, clone libraries, and pyrosequence analysis). These data are comprehensively presented, in contrast to those derived from culture-dependent methods. Furthermore, the microbial communities detected using both methods are analytically compared, and, as a result, the complementary roles of these methods and approaches are highlighted. In related contexts, knowledge of similar biodeterioration problems affecting other prehistoric cave paintings, mainly at Lascaux in France and Altamira in Spain, are referred to and commented upon. Based on substrate preferences (or ecological grouping) and mapping (plotting detection sites of isolates), we speculate on the possible origins and invasion routes whereby the major microbial colonizers invaded the TT stone chamber interior. Finally, concluding remarks, lessons, and future perspectives based on our microbiological surveys of these ancient tumuli, and similar treasures outside of Japan, are briefly presented. A list of the microbial taxa that have been identified and fully or briefly described by us as known and novel taxa for TT and KT isolates since 2008 is presented in Supplementary Materials.

Marine-derived fungi: diversity of enzymes and biotechnological applications.

The ocean is considered to be a great reservoir of biodiversity. Microbial communities in marine environments are ecologically relevant as intermediaries of energy, and play an important role in nutrient regeneration cycles as decomposers of dead and decaying organic matter. In this sense, marine-derived fungi can be considered as a source of enzymes of industrial and/or environmental interest. Fungal strains isolated from different substrates, such as invertebrates, decaying wood, seawater, sediments, and mangrove detritus, have been reported to be producers of hydrolytic and/or oxidative enzymes, with alginate lyase, amylase, cellulase, chitinase, glucosidase, inulinase, keratinase, ligninase, lipase, nuclease, phytase, protease, and xylanase being among the enzymes produced by fungi of marine origin. These enzymes present temperature and pH optima ranging from 35 to 70(∘)C, and 3.0 to 11.0, respectively. High-level production in bioreactors is mainly performed using submerged-state fermentation. Certain marine-derived fungal strains present enzymes with alkaline and cold-activity characteristics, and salinity is considered an important condition in screening and production processes. The adaptability of marine-derived fungi to oceanic conditions can be considered an attractive point in the field of fungal marine biotechnology. In this review, we focus on the advances in discovering enzymes from marine-derived fungi and their biotechnological relevance.

New developments in the study of the microbiota of raw-milk, long-ripened cheeses by molecular methods: the case of Grana Padano and Parmigiano Reggiano.

Microorganisms are an essential component of cheeses and play important roles during both cheese manufacture and ripening. Both starter and secondary flora modify the physical and chemical properties of cheese, contributing and reacting to changes that occur during the manufacture and ripening of cheese. As the composition of microbial population changes under the influence of continuous shifts in environmental conditions and microorganisms interactions during manufacturing and ripening, the characteristics of a given cheese depend also on microflora dynamics. The microbiota present in cheese is complex and its growth and activity represent the most important, but the least controllable steps. In the past, research in this area was dependent on classical microbiological techniques. However, culture-dependent methods are time-consuming and approaches that include a culturing step can lead to inaccuracies due to species present in low numbers or simply uncultivable. Therefore, they cannot be used as a unique tool to monitor community dynamics. For these reasons approaches to cheese microbiology had to change dramatically. To address this, in recent years the focus on the use of culture-independent methods based on the direct analysis of DNA (or RNA) has rapidly increased. Application of such techniques to the study of cheese microbiology represents a rapid, sound, reliable, and effective way for the detection and identification of the microorganisms present in dairy products, leading to major advances in understanding this complex microbial ecosystem and its impact on cheese ripening and quality. In this article, an overview on the recent advances in the use of molecular methods for thorough analysis of microbial communities in cheeses is given. Furthermore, applications of culture-independent approaches to study the microbiology of two important raw-milk, long-ripened cheeses such as Grana Padano and Parmigiano Reggiano, are presented.

Culture independent methods to assess the diversity and dynamics of microbiota during food fermentation.

Culture independent methods first appeared in the food microbiology field at the end of the 90s and since then they have been applied extensively. These methods do not rely on cultivation and target nucleic acids (DNA and RNA) to identify and follow the changes that occur in the main populations present in a specific ecosystem. The method that has most often been used as a culture independent method in food microbiology is denaturing gradient gel electrophoresis (DGGE). The number of papers dealing with DGGE grew exponentially in the late nineties and, by analysing the studies available in the literature, it is possible to describe a trend in the subjects that have been investigated. DGGE was first used as a tool to monitor the ecology of fermented food, such as fermented sausage, cheese and sourdough, and later it also showed its potential in microbial spoilage process. In the last few years, the main application of DGGE has been to study fermented food from Asia, Africa and South America. The information collected using DGGE has made it possible to confirm the existing knowledge on food fermentation and spoilage. However, in some cases, new evidence that helps scientists to fully comprehend a specific microbial ecosystem has emerged. In this review, the roadmap of culture independent methods in food microbiology will be summarized, focusing on the DGGE technique. Examples of how this approach is useful to obtain a better understanding of microbial diversity are reported for several kinds of fermented food, such as fermented sausage, cheese and wine. The future of culture independent methods in food microbiology, with the increasing availability of next generation sequencing techniques, is also discussed.