Genetic Elements Orchestrating Lactobacillus crispatus Glycogen Metabolism in the Vagina.

Glycogen in the female lower reproductive tract is a major carbon source for colonization and acidification by common vaginal Lactobacillus species, such as Lactobacillus crispatus. Previously, we identified the amylopullulanase encoding gene pulA of Lactobacillus crispatus to correlate with the ability to autonomously utilize glycogen for growth. Here, we further characterize genetic variation and differential regulation of pulA affecting the presence of its gene product on the outer surface layer. We show that alpha-glucan degrading activity dissipates when Lactobacillus crispatus is grown on glucose, maltose and maltotriose, in agreement with carbon catabolite repression elements flanking the pulA gene. Proteome analysis of the S-layer confirmed that the amylopullulanase protein is highly abundant in an S-layer enriched fraction, but not in a strain with a defective amylopullulanase variant or in an amylopullulanase-sufficient strain grown on glucose. In addition, we provide evidence that Lactobacillus crispatus pulA mutants are relevant in vivo, as they are commonly observed in metagenome datasets of human vaginal microbial communities. Analysis of the largest publicly available dataset of 1507 human vaginal metagenomes indicates that among the 270 samples that contain a Lactobacillus crispatuspulA gene, 62 samples (23%) had a defective variant of this gene. Taken together, these results demonstrate that both environmental, as well as genetic factors explain the variation of Lactobacillus crispatus alpha-glucosidases in the vaginal environment.

Different Resource Allocation in a Bacillus subtilis Population Displaying Bimodal Motility.

To cope with sudden changes in their environment, bacteria can use a bet-hedging strategy by dividing the population into cells with different properties. This so-called bimodal or bistable cellular differentiation is generally controlled by positive feedback regulation of transcriptional activators. Due to the continuous increase in cell volume, it is difficult for these activators to reach an activation threshold concentration when cells are growing exponentially. This is one reason why bimodal differentiation is primarily observed from the onset of the stationary phase, when exponential growth ceases. An exception is the bimodal induction of motility in Bacillus subtilis, which occurs early during exponential growth. Several mechanisms have been put forward to explain this, including double-negative feedback regulation and the stability of the mRNA molecules involved. In this study, we used fluorescence-assisted cell sorting (FACS) to compare the transcriptomes of motile and nonmotile cells and noted that expression of ribosomal genes is lower in motile cells. This was confirmed using an unstable green fluorescent protein (GFP) reporter fused to the strong ribosomal rpsD promoter. We propose that the reduction in ribosomal gene expression in motile cells is the result of a diversion of cellular resources to the synthesis of the chemotaxis and motility systems. In agreement with this, single-cell microscopic analysis showed that motile cells are slightly shorter than nonmotile cells, an indication of slower growth. We speculate that this growth rate reduction can contribute to the bimodal induction of motility during exponential growth. IMPORTANCE To cope with sudden environmental changes, bacteria can use a bet-hedging strategy and generate different types of cells within a population-so-called bimodal differentiation. For example, a Bacillus subtilis culture can contain both motile and nonmotile cells. In this study, we compared the gene expression between motile and nonmotile cells. It appeared that motile cells express fewer ribosomes. To confirm this, we constructed a ribosomal promoter fusion that enabled us to measure expression of this promoter in individual cells. This reporter fusion confirmed our initial finding. The reallocation of cellular resources from ribosome synthesis toward synthesis of the motility apparatus results in a reduction in growth. Interestingly, this growth reduction has been shown to stimulate bimodal differentiation.

The Two-Way Interaction between the Molecules That Cause Vaginal Malodour and Lactobacilli: An Opportunity for Probiotics.

Vaginal malodour is a sign of dysbiosis. The biogenic amines (BAs) cadaverine, putrescine and tyramine are known to be causative compounds. Recent reports suggest these compounds produced by pathogens might have a role beyond causing malodour; namely inhibiting the growth of lactobacilli bacteria that are crucial in the maintenance of vaginal homeostasis. The aim of this study was to identify whether certain lactobacilli strains could reduce BAs and to evaluate how Lactobacillus species were affected by these compounds. Using LC-MS and HPLC-UV, five Lactobacillus crispatus strains were identified as being capable of significantly reducing BAs from the media under in vitro conditions. Through 16S rRNA gene sequencing of vaginal swabs exposed to Bas, cadaverine was found to reduce the relative abundance of lactobacilli. When L. crispatus was exposed to media supplemented with BAs with an HCl adjusted lower pH, its growth was enhanced, demonstrating the relevance of the maintenance of an acidic vaginal environment. If strains are to be developed for probiotic application to alleviate bacterial vaginosis and other conditions affecting large numbers of women worldwide, their ability to adapt to Bas and regulate pH should be part of the experimentation.

Interstrain Variability of Human Vaginal Lactobacillus crispatus for Metabolism of Biogenic Amines and Antimicrobial Activity against Urogenital Pathogens.

Lactobacillus crispatus is the dominant species in the vagina of many women. With the potential for strains of this species to be used as a probiotic to help prevent and treat dysbiosis, we investigated isolates from vaginal swabs with Lactobacillus-dominated and a dysbiotic microbiota. A comparative genome analysis led to the identification of metabolic pathways for synthesis and degradation of three major biogenic amines in most strains. However, targeted metabolomic analysis of the production and degradation of biogenic amines showed that certain strains have either the ability to produce or to degrade these compounds. Notably, six strains produced cadaverine, one produced putrescine, and two produced tyramine. These biogenic amines are known to raise vaginal pH, cause malodour, and make the environment more favourable to vaginal pathogens. In vitro experiments confirmed that strains isolated from women with a dysbiotic vaginal microbiota have higher antimicrobial effects against the common urogenital pathogens Escherichia coli and Enterococcus faecium. The results indicate that not all L. crispatus vaginal strains appear suitable for probiotic application and the basis for selection should not be only the overall composition of the vaginal microbiota of the host from which they came, but specific biochemical and genetic traits.

Fermented foods in a global age: East meets West.

Fermented foods and alcoholic beverages have long been an important part of the human diet in nearly every culture on every continent. These foods are often well-preserved and serve as stable and significant sources of proteins, vitamins, minerals, and other nutrients. Despite these common features, however, many differences exist with respect to substrates and products and the types of microbes involved in the manufacture of fermented foods and beverages produced globally. In this review, we describe these differences and consider the influence of geography and industrialization on fermented foods manufacture. Whereas fermented foods produced in Europe, North America, Australia, and New Zealand usually depend on defined starter cultures, those made in Asia and Africa often rely on spontaneous fermentation. Likewise, in developing countries, fermented foods are not often commercially produced on an industrial scale. Although many fermented products rely on autochthonous microbes present in the raw material, for other products, the introduction of starter culture technology has led to greater consistency, safety, and quality. The diversity and function of microbes present in a wide range of fermented foods can now be examined in detail using molecular and other omic approaches. The nutritional value of fermented foods is now well-appreciated, especially in resource-poor regions where yoghurt and other fermented foods can improve public health and provide opportunities for economic development. Manufacturers of fermented foods, whether small or large, should follow Good Manufacturing Practices and have sustainable development goals. Ultimately, preferences for fermented foods and beverages depend on dietary habits of consumers, as well as regional agricultural conditions and availability of resources.

Promising Prebiotic Candidate Established by Evaluation of Lactitol, Lactulose, Raffinose, and Oligofructose for Maintenance of a Lactobacillus-Dominated Vaginal Microbiota.

Perturbations to the vaginal microbiota can lead to dysbiosis, including bacterial vaginosis (BV), which affects a large portion of the female population. In a healthy state, the vaginal microbiota is characterized by low diversity and colonization by Lactobacillus spp., whereas in BV, these species are displaced by a highly diverse population of bacteria associated with adverse vaginal health outcomes. Since prebiotic ingestion has been a highly effective approach to invigorate lactobacilli for improved intestinal health, we hypothesized that these compounds could stimulate lactobacilli at the expense of BV organisms to maintain vaginal health. Monocultures of commensal Lactobacillus crispatus, Lactobacillus vaginalis, Lactobacillus gasseri, Lactobacillus johnsonii, Lactobacillus jensenii, and Lactobacillus iners, in addition to BV-associated organisms and Candida albicans, were tested for their ability to utilize a representative group of prebiotics consisting of lactitol, lactulose, raffinose, and oligofructose. The disaccharide lactulose was found to most broadly and specifically stimulate vaginal lactobacilli, including the strongly health-associated species L. crispatus, and importantly, not to stimulate BV organisms or C. albicans Using freshly collected vaginal samples, we showed that exposure to lactulose promoted commensal Lactobacillus growth and dominance and resulted in healthy acidity partially through lactic acid production. This provides support for further testing of lactulose to prevent dysbiosis and potentially to reduce the need for antimicrobial agents in managing vaginal health.IMPORTANCE Bacterial vaginosis (BV) and other dysbioses of the vaginal microbiota significantly affect the quality of life of millions of women. Antimicrobial therapy is often poorly effective, causes side effects, and does not prevent recurrences. We report one of very few studies that have evaluated how prebiotics-compounds that are selectively fermented by beneficial bacteria such as Lactobacillus spp.-can modulate the vaginal microbiota. We also report use of a novel in vitro polymicrobial model to study the impact of prebiotics on the vaginal microbiota. The identification of prebiotic lactulose as enhancing Lactobacillus growth but not that of BV organisms or Candida albicans has direct application for retention of homeostasis and prevention of vaginal dysbiosis and infection.

A practical guide for probiotics applied to the case of antibiotic-associated diarrhea in The Netherlands.

BACKGROUND: Antibiotic-associated diarrhea (AAD) is a side-effect frequently associated with the use of broad spectrum antibiotics. Although a number of clinical studies show that co-administration of specific probiotics reduces the risk for AAD, there is still unclarity among healthcare professionals on the recommendation of probiotic products. This paper aims at a practical guide to inform healthcare professionals, patients and consumers about the exact product characteristics of available probiotics with a proven efficacy to prevent AAD. METHODS: The workflow in this paper includes three consecutive steps: 1) systematic review of relevant clinical studies for effective probiotics by a meta-analysis, 2) compilation of a list of available probiotic products, and 3) recommendation of probiotic products that match effective formulations. Our systematic review on the efficacy of probiotics for the prevention of AAD included only studies with randomized, double blind placebo-controlled trials, a clear definition of antibiotic associated diarrhea, and a probiotic administration regime for at least the duration of the antibiotic therapy. RESULTS: Using our inclusion criteria, we selected 32 out of 128 identified trials and pooled the results of these studies for each specific dairy product and food supplement. The results indicate a total of seven single or multiple-strain formulations favoring the probiotic treatment group, with the strain Lactobacillus rhamnosus GG being the most effective [relative risk ratio of probiotic versus placebo 0.30 (95% CI 0.16-0.5)]. We selected products for recommendation from a compiled list of all probiotic dairy products and food supplements available in The Netherlands and categorized them into groups of products showing effects against the incidence of AAD in at least one, two or three independent clinical studies. We excluded all products which did not unambiguously declare on the label the specific probiotic strain(s) and the number of colony forming units. CONCLUSION: Here we present a practical guide that informs healthcare professionals and patients on the availability of probiotic products with a proven efficacy for the prevention of AAD.

Molecular assessment of bacterial vaginosis by Lactobacillus abundance and species diversity.

BACKGROUND: To date, women are most often diagnosed with bacterial vaginosis (BV) using microscopy based Nugent scoring or Amsel criteria. However, the accuracy is less than optimal. The aim of the present study was to confirm the identity of known BV-associated composition profiles and evaluate indicators for BV using three molecular methods. METHODS: Evaluation of indicators for BV was carried out by 16S rRNA amplicon sequencing of the V5-V7 region, a tailor-made 16S rRNA oligonucleotide-based microarray, and a PCR-based profiling technique termed IS-profiling, which is based on fragment variability of the 16S-23S rRNA intergenic spacer region. An inventory of vaginal bacterial species was obtained from 40 females attending a Dutch sexually transmitted infection outpatient clinic, of which 20 diagnosed with BV (Nugent score 7-10), and 20 BV negative (Nugent score 0-3). RESULTS: Analysis of the bacterial communities by 16S rRNA amplicon sequencing revealed two clusters in the BV negative women, dominated by either Lactobacillus iners or Lactobacillus crispatus and three distinct clusters in the BV positive women. In the former, there was a virtually complete, negative correlation between L. crispatus and L. iners. BV positive subjects showed cluster profiles that were relatively high in bacterial species diversity and dominated by anaerobic species, including Gardnerella vaginalis, and those belonging to the Families of Lachnospiraceae and Leptotrichiaceae. Accordingly, the Gini-Simpson index of species diversity, and the relative abundance Lactobacillus species appeared consistent indicators for BV. Under the conditions used, only the 16S rRNA amplicon sequencing method was suitable to assess species diversity, while all three molecular composition profiling methods were able to indicate Lactobacillus abundance in the vaginal microbiota. CONCLUSION: An affordable and simple molecular test showing a depletion of the genus Lactobacillus in combination with an increased species diversity of vaginal microbiota could serve as an alternative and practical diagnostic method for the assessment of BV.

A novel consortium of Lactobacillus rhamnosus and Streptococcus thermophilus for increased access to functional fermented foods.

BACKGROUND: The lactic acid bacterium Lactobacillus rhamnosus GG is the most studied probiotic bacterium with proven health benefits upon oral intake, including the alleviation of diarrhea. The mission of the Yoba for Life foundation is to provide impoverished communities in Africa increased access to Lactobacillus rhamnosus GG under the name Lactobacillus rhamnosus yoba 2012, world's first generic probiotic strain. We have been able to overcome the strain's limitations to grow in food matrices like milk, by formulating a dried starter consortium with Streptococcus thermophilus that enables the propagation of both strains in milk and other food matrices. The affordable seed culture is used by people in resource-poor communities. RESULTS: We used S. thermophilus C106 as an adjuvant culture for the propagation of L. rhamnosus yoba 2012 in a variety of fermented foods up to concentrations, because of its endogenous proteolytic activity, ability to degrade lactose and other synergistic effects. Subsequently, L. rhamnosus could reach final titers of 1E+09 CFU ml(-1), which is sufficient to comply with the recommended daily dose for probiotics. The specific metabolic interactions between the two strains were derived from the full genome sequences of L. rhamnosus GG and S. thermophilus C106. The piliation of the L. rhamnosus yoba 2012, required for epithelial adhesion and inflammatory signaling in the human host, was stable during growth in milk for two rounds of fermentation. Sachets prepared with the two strains, yoba 2012 and C106, retained viability for at least 2 years. CONCLUSIONS: A stable dried seed culture has been developed which facilitates local and low-cost production of a wide range of fermented foods that subsequently act as delivery vehicles for beneficial bacteria to communities in east Africa.

Development of a locally sustainable functional food based on mutandabota, a traditional food in southern Africa.

A probiotic dairy product was developed on the basis of a traditional dish called mutandabota to enable resource-poor populations in southern Africa to benefit from a functional food. Mutandabota is widely consumed in rural southern Africa, making it an ideal food matrix to carry probiotics. First, a process to produce probiotic mutandabota was designed. Raw cow milk was boiled and subsequently cooled to ambient temperature (25°C). Next, dry pulp from the fruit of the baobab tree (Adansonia digitata L.) was added to the milk at a concentration of 4% (wt/vol). This mixture was inoculated with the probiotic Lactobacillus rhamnosus yoba and left to ferment for 24h, while the growth of the bacterial culture was monitored. Final ingredients were then added to produce probiotic mutandabota that had 14% (wt/vol) baobab fruit pulp and 7% (wt/vol) sugar in cow milk. The pH of probiotic mutandabota was pH 3.5, which ensures that the product is microbiologically safe. The viable plate count of L. rhamnosus yoba increased from 5.8 ± 0.3 log cfu/mL at the point of inoculation to 8.8 ± 0.4 log cfu/mL at the moment of consumption, thereby meeting the criterion to have a viable count of the probiotic bacterium in excess of 6 log cfu/mL of a product. Baobab fruit pulp at 4% promoted growth of L. rhamnosus yoba with a maximal specific growth rate (µmax) of 0.6 ± 0.2/h at 30°C. The developed technology, though specific for this particular product, has potential to be applied for the delivery of probiotics through a variety of indigenous foods in different regions of the world. Upon consumption, probiotic mutandabota is expected to improve the population's intestinal health, which is especially relevant for vulnerable target groups such as children and elderly people.

Genome instability in Lactobacillus rhamnosus GG.

We describe here a comparative genome analysis of three dairy product isolates of Lactobacillus rhamnosus GG (LGG) and the ATCC 53103 reference strain to the published genome sequence of L. rhamnosus GG. The analysis showed that in two of three isolates, major DNA segments were missing from the genomic islands LGGISL1,2. The deleted DNA segments consist of 34 genes in one isolate and 84 genes in the other and are flanked by identical insertion elements. Among the missing genes are the spaCBA genes, which encode pilin subunits involved in adhesion to mucus and persistence of the strains in the human intestinal tract. Subsequent quantitative PCR analyses of six commercial probiotic products confirmed that two more products contain a heterogeneous population of L. rhamnosus GG variants, including genotypes with or without spaC. These results underline the relevance for quality assurance and control measures targeting genome stability in probiotic strains and justify research assessing the effect of genetic rearrangements in probiotics on the outcome of in vitro and in vivo efficacy studies.

Abiotic and microbiotic factors controlling biofilm formation by thermophilic sporeformers.

One of the major concerns in the production of dairy concentrates is the risk of contamination by heat-resistant spores from thermophilic bacteria. In order to acquire more insight in the composition of microbial communities occurring in the dairy concentrate industry, a bar-coded 16S amplicon sequencing analysis was carried out on milk, final products, and fouling samples taken from dairy concentrate production lines. The analysis of these samples revealed the presence of DNA from a broad range of bacterial taxa, including a majority of mesophiles and a minority of (thermophilic) spore-forming bacteria. Enrichments of fouling samples at 55°C showed the accumulation of predominantly Brevibacillus and Bacillus, whereas enrichments at 65°C led to the accumulation of Anoxybacillus and Geobacillus species. Bacterial population analysis of biofilms grown using fouling samples as an inoculum indicated that both Anoxybacillus and Geobacillus preferentially form biofilms on surfaces at air-liquid interfaces rather than on submerged surfaces. Three of the most potent biofilm-forming strains isolated from the dairy factory industrial samples, including Geobacillus thermoglucosidans, Geobacillus stearothermophilus, and Anoxybacillus flavithermus, have been characterized in detail with respect to their growth conditions and spore resistance. Strikingly, Geobacillus thermoglucosidans, which forms the most thermostable spores of these three species, is not able to grow in dairy intermediates as a pure culture but appears to be dependent for growth on other spoilage organisms present, probably as a result of their proteolytic activity. These results underscore the importance of abiotic and microbiotic factors in niche colonization in dairy factories, where the presence of thermophilic sporeformers can affect the quality of end products.

Raw milk kefir: microbiota, bioactive peptides, and immune modulation.

This study aims to characterize the microbiota and peptidomic composition of raw milk kefir, and to address the potential anti-allergic effects of raw milk kefir using validated research models for food allergy. Raw milk kefir was produced after incubation with a defined freeze-dried starter culture. Kefir was sampled during fermentation at seven time intervals. For comparison, kefir was also prepared from heat-treated milk. Peptide compositions were determined for the raw and heated milk, and kefir end products made from these milks. In a murine food allergy model, the two kefir end products were investigated for their allergy modulating effects. In both kefirs, we identified amplicon sequence variants identical to those in the starter culture, matching the bacteria Lactococcus lactis, Streptococcus thermophilus, Leuconostoc and the yeast Debaryomyces. In raw milk kefir, additional sequence variants of Lactococcus lactis and the yeasts Pichia and Galactomyces could be identified, which were absent in heated milk kefir. Analysis of peptide compositions in both kefirs indicated that the number and intensity of peptides drastically increased after fermentation. Heating of the milk negatively affected the diversity of the peptide composition in kefir. Only raw milk kefir suppressed the acute allergic skin response to the food allergen ovalbumin in sensitised mice. These effects coincided with differences in the T-cell compartment, with lower percentages of activated Th1 cells and IFNg production after treatment with kefir made from heated milk. The results of this study indicate specific properties of raw milk kefir that may contribute to its additional health benefits.

Complete genome sequence of Geobacillus thermoglucosidans TNO-09.020, a thermophilic sporeformer associated with a dairy-processing environment.

Thermophilic spore-forming bacteria are a common cause of contamination in dairy products. We isolated the thermophilic strain Geobacillus thermoglucosidans TNO-09.020 from a milk processing plant and report the complete genome of a dairy plant isolate consisting of a single chromosome of 3.75 Mb.

Normative cognition and the effects of a probiotic food intervention in first grade children in Côte d'Ivoire.

The cognitive skills critical for success have largely been studied in Western populations, despite the fact that children in low- and middle-income countries are at risk to not reach their full developmental potential. Moreover, scientists should leverage recent discovery to explore means of boosting cognition in at-risk populations. This semi-randomized controlled trial examined normative cognitive development and whether it could be enhanced by consumption of a probiotic food in a sample of 251 4- to 7-year-old children in urban schools in Côte d'Ivoire. Participants completed executive functioning measures at baseline (T1) and 5 months later (T2). After T1, children in one school received a probiotic (N = 74) or placebo (N = 79) fermented dairy food every day they were in school for one semester; children in the other school (N = 98) continued their diet as usual. Children improved on all tests across time (Cohen's d = 0.08-0.30). The effects of probiotic ingestion were inconclusive and are interpreted with caution due to socio-political factors affecting daily administration. Given the general feasibility of the study, we hope that it will serve as an inspiration for future research into child development and sustainable (health-promoting) interventions for school children in developing nations.

Gel-free proteomic identification of the Bacillus subtilis insoluble spore coat protein fraction.

Species from the genus Bacillus have the ability to form endospores, dormant cellular forms that are able to survive heat and acid preservation techniques commonly used in the food industry. Resistance characteristics of spores towards various environmental stresses are in part attributed to their coat proteins. Previously, 70 proteins have been assigned to the spore coat of Bacillus subtilis using SDS-PAGE, 2-DE gel approaches, protein localization studies and genome-wide transcriptome studies. Here, we present a "gel-free" protocol that is capable of comprehensive B. subtilis spore coat protein extraction and addresses the insoluble coat fraction. Using LC-MS/MS we identified 55 proteins from the insoluble B. subtilis spore coat protein fraction, of which 21 are putative novel spore coat proteins not assigned to the spore coat until now. Identification of spore coat proteins from a B. subtilis food-spoilage isolate corroborated a generic and "applied" use of our protocol. To develop specific and sensitive spore detection and/or purification systems from food stuff or patient material, suitable protein targets can be derived from our proteomic approach. Finally, the protocol can be extended to study cross-linking among the spore coat proteins as well as for their quantification.

Multiparameter viability assay for stress profiling applied to the food pathogen Listeria monocytogenes F2365.

A novel generic approach for stress profiling was applied to Listeria monocytogenes strain F2365. This food-borne pathogen was exposed to gradients of five different stresses of increasing intensity, typically ranging from moderate to lethal conditions. The stress factors included heat, acidic pH, a detergent disinfectant, an oxidant, and hyperosmotic conditions. In addition to CFU counts and lag time, five different molecular viability parameters were measured by fluorescence-based assays, including membrane integrity, membrane potential, esterase activity, redox activity, and intracellular pH stability. The last was measured by our recently invented real-time viability assay. Exposure to all stresses resulted in clear dose-response relationships for all viability parameters with the exception of hyperosmotic conditions. A statistical analysis showed strong correlations for (i) the growth parameters plate counts and lag times, (ii) the enzyme-associated functions redox and esterase activity, and (iii) the membrane-associated pH stability and membrane integrity. Results indicated a pronounced difference in the susceptibilities of the measured parameters depending on the stress factor applied. However, at relatively high stress intensities, all of the viability parameters became affected independent of the stress factor. Applications of the approach presented here include studies on the mechanism of action of unknown compounds with biocidal activity and a comparative analysis of the severities of the impact of stress conditions of interest. It appears that a meaningful evaluation of the impact of mild stress conditions can be obtained only through measurement of multiple viability parameters.

Microarray-based identification of clinically relevant vaginal bacteria in relation to bacterial vaginosis.

OBJECTIVE: The objective was to examine the use of a tailor-made DNA microarray containing probes representing the vaginal microbiota to examine bacterial vaginosis. STUDY DESIGN: One hundred one women attending a health center for HIV testing in South Africa were enrolled. Stained, liquid-based cytology slides were scored for bacterial vaginosis. An inventory of organisms was obtained using microarray technology, probing genera associated with bacterial vaginosis in more detail, namely Gardnerella, Atopobium, Dialister, Leptotrichia, Megasphaera, Mobiluncus, Peptostreptococcus, Prevotella, and Sneathia. RESULTS: Of 101 women, 34 were diagnosed positive for bacterial vaginosis. This condition was associated with an increased microbial diversity. It is no longer useful to base the diagnosis of bacterial vaginosis on Gardnerella alone. Rather, its presence with Leptotrichia and Prevotella species, and especially Atopobium was more indicative of an aberrant state of the vaginal flora. CONCLUSION: To understand the vaginal microbiota in more detail, microarray-based identification can be used after microscopic scoring.

Challenges and advances in systems biology analysis of Bacillus spore physiology; molecular differences between an extreme heat resistant spore forming Bacillus subtilis food isolate and a laboratory strain.

Bacterial spore formers are prime organisms of concern in the food industry. Spores from the genus Bacillus are extremely stress resistant, most notably exemplified by high thermotolerance. This sometimes allows surviving spores to germinate and grow out to vegetative cells causing food spoilage and possible intoxication. Similar issues though more pending toward spore toxigenicity are observed for the anaerobic Clostridia. The paper indicates the nature of stress resistance and highlights contemporary molecular approaches to analyze the mechanistic basis of it in Bacilli. A molecular comparison between a laboratory strain and a food borne isolate, very similar at the genomic level to the laboratory strain but generating extremely heat resistant spores, is discussed. The approaches cover genome-wide genotyping, proteomics and genome-wide expression analyses studies. The analyses aim at gathering sufficient molecular information to be able to put together an initial framework for dynamic modelling of spore germination and outgrowth behaviour. Such emerging models should be developed both at the population and at the single spore level. Tools and challenges in achieving the latter are succinctly discussed.