2'-Fucosyllactose inhibits proliferation of Clostridioides difficile ATCC 43599 in the CDi-screen, an in vitro model simulating Clostridioides difficile infection.

Background: Clostridioides difficile is a Gram-positive anaerobic bacterium that can produce the toxins TcdA and/or TcdB and is considered an opportunistic pathogen. C. difficile is mainly transmitted as endospores, which germinate to produce the pathogenic vegetative cells under suitable conditions in the gut. To efficiently screen novel therapeutic- interventions against the proliferation of C. difficile within a complex microbial community, platforms are needed that facilitate parallel experimentation. In order to allow for screening of novel interventions a medium-to-high throughput in vitro system is desirable. To this end, we have developed the 96-well CDi-screen platform that employs an adapted simulated ileal effluent medium (CDi-SIEM) and allows for culturing of pathogenic C. difficile. Methods: C. difficile strain ATCC 43599 was inoculated in the form of vegetative cells and spores into the CDi-screen in the presence and absence of a cultured fecal microbiota and incubated for 48h. To demonstrate its utility, we investigated the effect of the human milk oligosaccharide 2'-Fucosyllactose (2'-FL) at 4 and 8 mg/mL on C. difficile outgrowth and toxin production in the CDi-screen. The test conditions were sampled after 24 and 48 hours. C. difficile -specific primers were used to monitor C. difficile growth via qPCR and barcoded 16S rRNA gene amplicon sequencing facilitated the in-depth analysis of gut microbial community dynamics. Results: C. difficile ATCC 43599 proliferated in CDi-SIEM, both when inoculated as spores and as vegetative cells. The strain reached cell numbers expressed as C. difficile genome equivalents of up to 10 8 cells per mL after 24h of incubation. 2'-FL significantly inhibited the outgrowth of the ATTC 43599 strain within a complex human gut microbial community in the CDi-screen. In addition, a dose-dependent modulation of the gut microbial community composition by 2'-FL supplementation was detected, with a significant increase in the relative abundance of the genus Blautia in the presence of 2'-FL. Conclusion: The CDi-screen is suitable for studying C. difficile proliferation in a complex gut ecosystem and for screening for anti-pathogenic interventions that target C. difficile directly and/or indirectly through interactions with the gut microbiota. Different doses of compounds such as in this study the dose of the human milk oligosaccharide 2'-FL can be screened for efficacy in the inhibition of C. difficile proliferation.

The mechanistic overview of SARS-CoV-2 using angiotensin-converting enzyme 2 to enter the cell for replication: possible treatment options related to the renin-angiotensin system.

The SARS-CoV-2 pandemic is a healthcare crisis caused by insufficient knowledge applicable to effectively combat the virus. Therefore, different scientific discovery strategies need to be connected, to generate a rational treatment which can be made available as rapidly as possible. This relies on a solid theoretical understanding of the mechanisms of SARS-CoV-2 infection and host responses, which is coupled to the practical experience of clinicians that are treating patients. Because SARS-CoV-2 enters the cell by binding to angiotensin-converting enzyme 2 (ACE2), targeting ACE2 to prevent such binding seems an obvious strategy to combat infection. However, ACE2 performs its functions outside the cell and was found to enter the cell only by angiotensin II type 1 receptor (AT1R)-induced endocytosis, after which ACE2 is destroyed. This means that preventing uptake of ACE2 into the cell by blocking AT1R would be a more logical approach to limit entry of SARS-CoV-2 into the cell. Since ACE2 plays an important protective role in maintaining key biological processes, treatments should not disrupt the functional capacity of ACE2, to counterbalance the negative effects of the infection. Based on known mechanisms and knowledge of the characteristics of SARS-CoV we propose the hypothesis that the immune system facilitates SARS-CoV-2 replication which disrupts immune regulatory mechanisms. The proposed mechanism by which SARS-CoV-2 causes disease immediately suggests a possible treatment, since the AT1R is a key player in this whole process. AT1R antagonists appear to be the ideal candidate for the treatment of SARS-CoV-2 infection. AT1R antagonists counterbalance the negative consequences of angiotesnin II and, in addition, they might even be involved in preventing the cellular uptake of the virus without interfering with ACE2 function. AT1R antagonists are widely available, cheap, and safe. Therefore, we propose to consider using AT1R antagonists in the treatment of SARS-CoV-2.

Heat sterilisation dramatically reduces filter efficiency of the majority of FFP2 and KN-95 respirators.

BACKGROUND: Because of the enormous demand for personal protective equipment and especially respiratory protective devices (respirators) during the initial phase of the corona pandemic shortages arose. Sterilisation of used respirators can reduce these shortages. In our study, respirator testing was carried out after only one sterilisation cycle. AIM: To determine if steam sterilisation and reuse could be safely applied for used respirators. METHODS: In a cabinet an aqueous solution of NaCl (0.02% w/v) was nebulized and passed through a sample of the material of a respirator. Passing particle concentrations were measured directly from the cabinet and via the filter material of the respirator for particles ≥ 0.3 µm, ≥ 0.5 µm and ≥ 1.0 µm. FINDINGS: only three out of ten steam sterilised respirators met the requirements of 94% filtration efficiency. CONCLUSION: The results prove that heat sterilisation cannot be generically applied for reuse of respirators safely.

Dose-dependent impact of oxytetracycline on the veal calf microbiome and resistome.

BACKGROUND: Antibiotic therapy is commonly used in animal agriculture. Antibiotics excreted by the animals can contaminate farming environments, resulting in long term exposure of animals to sub-inhibitory levels of antibiotics. Little is known on the effect of this exposure on antibiotic resistance. In this study, we aimed to investigate the long term effects of sub-inhibitory levels of antibiotics on the gut microbiota composition and resistome of veal calves in vivo. Forty-two veal calves were randomly assigned to three groups. The first group (OTC-high) received therapeutic oral dosages of 1 g oxytetracycline (OTC), twice per day, during 5 days. The second group (OTC-low) received an oral dose of OTC of 100-200 µg per day during 7 weeks, mimicking animal exposure to environmental contamination. The third group (CTR) did not receive OTC, serving as unexposed control. Antibiotic residue levels were determined over time. The temporal effects on the gut microbiota and antibiotic resistance gene abundance was analysed by metagenomic sequencing. RESULTS: In the therapeutic group, OTC levels exceeded MIC values. The low group remained at sub-inhibitory levels. The control group did not reach any significant OTC levels. 16S rRNA gene-based analysis revealed significant changes in the calf gut microbiota. Time-related changes accounted for most of the variation in the sequence data. Therapeutic application of OTC had transient effect, significantly impacting gut microbiota composition between day 0 and day 2. By metagenomic sequence analysis we identified six antibiotic resistance genes representing three gene classes (tetM, floR and mel) that differed in relative abundance between any of the intervention groups and the control. qPCR was used to validate observations made by metagenomic sequencing, revealing a peak of tetM abundance at day 28-35 in the OTC-high group. No increase in resistance genes abundance was seen in the OTC-low group. CONCLUSIONS: Under the conditions tested, sub-therapeutic administration of OTC did not result in increased tetM resistance levels as observed in the therapeutic group.

Evaluating novel synthetic compounds active against Bacillus subtilis and Bacillus cereus spores using Live imaging with SporeTrackerX.

An empirical approach was taken to screen a novel synthetic compound library designed to be active against Gram-positive bacteria. We obtained five compounds that were active against spores from the model organism Bacillus subtilis and the food-borne pathogen Bacillus cereus during our population based experiments. Using single cell live imaging we were able to observe effects of the compounds on spore germination and outgrowth. Difference in sensitivity to the compounds could be observed between B. subtilis and B. cereus using live imaging, with minor difference in the minimal inhibitory and bactericidal concentrations of the compounds against the spores. The compounds all delayed the bursting time of germinated spores and affected the generation time of vegetative cells at sub-inhibitory concentrations. At inhibitory concentrations spore outgrowth was prevented. One compound showed an unexpected potential for preventing spore germination at inhibitory concentrations, which merits further investigation. Our study shows the valuable role single cell live imaging can play in the final selection process of antimicrobial compounds.

The Impact of Maltitol-Sweetened Chewing Gum on the Dental Plaque Biofilm Microbiota Composition.

Background: The oral cavity harbors a complex microbial ecosystem, intimately related to oral health and disease. The use of polyol-sweetened gum is believed to benefit oral health through stimulation of salivary flow and impacting oral pathogenic bacteria. Maltitol is often used as sweetener in food products. This study aimed to establish the in vivo effects of frequent consumption of maltitol-sweetened chewing gum on the dental plaque microbiota in healthy volunteers and to establish the cellular and molecular effects by in vitro cultivation and transcriptional analysis. Results: An intervention study was performed in 153 volunteers, randomly assigned to three groups (www.trialregister.nl; NTR4165). One group was requested to use maltitol gum five times daily, one group used gum-base, and the third group did not use chewing gum. At day 0 and day 28, 24 h-accumulated supragingival plaque was collected at the lingual sites of the lower jaw and the buccal sites of the upper jaw and analyzed by 16S ribosomal rRNA gene sequencing. At day 42, 2 weeks after completion of the study, lower-jaw samples were collected and analyzed. The upper buccal plaque microbiota composition had lower bacterial levels and higher relative abundances of (facultative) aerobic species compared to the lower lingual sites. There was no difference in bacterial community structure between any of the three study groups (PERMANOVA). Significant lower abundance of several bacterial phylotypes was found in maltitol gum group compared to the gum-base group, including Actinomyces massiliensis HOT 852 and Lautropia mirabilis HOT 022. Cultivation studies confirmed growth inhibition of A. massiliensis and A. johnsonii by maltitol at levels of 1% and higher. Transcriptome analysis of A. massiliensis revealed that exposure to maltitol resulted in changes in the expression of genes linked to osmoregulation, biofilm formation, and central carbon metabolism. Conclusion: The results showed that chewing itself only marginally impacted the plaque microbiota composition. Use of maltitol-sweetened gum lowered abundance of several bacterial species. Importantly, the species impacted play a key role in the early formation of dental biofilms. Further studies are required to establish if frequent use of maltitol gum impacts early dental-plaque biofilm development.

Amplicon sequencing for the quantification of spoilage microbiota in complex foods including bacterial spores.

BACKGROUND: Spoilage of food products is frequently caused by bacterial spores and lactic acid bacteria. Identification of these organisms by classic cultivation methods is limited by their ability to form colonies on nutrient agar plates. In this study, we adapted and optimized 16S rRNA amplicon sequencing for quantification of bacterial spores in a canned food matrix and for monitoring the outgrowth of spoilage microbiota in a ready-to-eat food matrix. RESULTS: The detection limit of bar-coded 16S rRNA amplicon sequencing was determined for the number of bacterial spores in a canned food matrix. Analysis of samples from a canned food matrix spiked with a mixture of equinumerous spores from the thermophiles, Geobacillus stearothermophilus and Geobacillus thermoglucosidans, and the mesophiles, Bacillus sporothermodurans, Bacillus cereus, and Bacillus subtilis, led to the detection of these spores with an average limit of 2 × 10(2) spores ml(-1). The data were normalized by setting the number of sequences resulting from DNA of an inactivated bacterial species, present in the matrix at the same concentration in all samples, to a fixed value for quantitative sample-to-sample comparisons. The 16S rRNA amplicon sequencing method was also employed to monitor population dynamics in a ready-to-eat rice meal, incubated over a period of 12 days at 7 °C. The most predominant outgrowth was observed by the genera Leuconostoc, Bacillus, and Paenibacillus. Analysis of meals pre-treated with weak acids showed inhibition of outgrowth of these three genera. The specificity of the amplicon synthesis was improved by the design of oligonucleotides that minimize the amplification of 16S rRNA genes from chloroplasts originating from plant-based material present in the food. CONCLUSION: This study shows that the composition of complex spoilage populations, including bacterial spores, can be monitored in complex food matrices by bar-coded amplicon sequencing in a quantitative manner. In order to allow sample-to-sample comparisons, normalizations based on background DNA are described. This method offers a solution for the identification and quantification of spoilage microbiota, which cannot be cultivated under standard laboratory conditions. The study indicates variable detection limits among species of bacterial spores resulting from differences in DNA extraction efficiencies.

Early respiratory microbiota composition determines bacterial succession patterns and respiratory health in children.

RATIONALE: Many bacterial pathogens causing respiratory infections in children are common residents of the respiratory tract. Insight into bacterial colonization patterns and microbiota stability at a young age might elucidate healthy or susceptible conditions for development of respiratory disease. OBJECTIVES: To study bacterial succession of the respiratory microbiota in the first 2 years of life and its relation to respiratory health characteristics. METHODS: Upper respiratory microbiota profiles of 60 healthy children at the ages of 1.5, 6, 12, and 24 months were characterized by 16S-based pyrosequencing. We determined consecutive microbiota profiles by machine-learning algorithms and validated the findings cross-sectionally in an additional cohort of 140 children per age group. MEASUREMENTS AND MAIN RESULTS: Overall, we identified eight distinct microbiota profiles in the upper respiratory tract of healthy infants. Profiles could already be identified at 1.5 months of age and were associated with microbiota stability and change over the first 2 years of life. More stable patterns were marked by early presence and high abundance of Moraxella and Corynebacterium/Dolosigranulum and were positively associated with breastfeeding in the first period of life and with lower rates of parental-reported respiratory infections in the consecutive periods. Less stable profiles were marked by high abundance of Haemophilus or Streptococcus. CONCLUSIONS: These findings provide novel insights into microbial succession in the respiratory tract in infancy and link early-life profiles to microbiota stability and respiratory health characteristics. New prospective studies should elucidate potential implications of our findings for early diagnosis and prevention of respiratory infections. Clinical trial registered with www.clinicaltrials.gov (NCT00189020).

Differential modulation by Akkermansia muciniphila and Faecalibacterium prausnitzii of host peripheral lipid metabolism and histone acetylation in mouse gut organoids.

UNLABELLED: The gut microbiota is essential for numerous aspects of human health. However, the underlying mechanisms of many host-microbiota interactions remain unclear. The aim of this study was to characterize effects of the microbiota on host epithelium using a novel ex vivo model based on mouse ileal organoids. We have explored the transcriptional response of organoids upon exposure to short-chain fatty acids (SCFAs) and products generated by two abundant microbiota constituents, Akkermansia muciniphila and Faecalibacterium prausnitzii. We observed that A. muciniphila metabolites affect various transcription factors and genes involved in cellular lipid metabolism and growth, supporting previous in vivo findings. Contrastingly, F. prausnitzii products exerted only weak effects on host transcription. Additionally, A. muciniphila and its metabolite propionate modulated expression of Fiaf, Gpr43, histone deacetylases (HDACs), and peroxisome proliferator-activated receptor gamma (Pparγ), important regulators of transcription factor regulation, cell cycle control, lipolysis, and satiety. This work illustrates that specific bacteria and their metabolites differentially modulate epithelial transcription in mouse organoids. We demonstrate that intestinal organoids provide a novel and powerful ex vivo model for host-microbiome interaction studies. IMPORTANCE: We investigated the influence of the gut microbiota and microbially produced short-chain fatty acids (SCFAs) on gut functioning. Many commensal bacteria in the gut produce SCFAs, particularly butyrate, acetate, and propionate, which have been demonstrated to reduce the risk of gastrointestinal disorders. Organoids-small crypt-villus structures grown from ileal intestinal stem cells-were exposed to SCFAs and two specific gut bacteria. Akkermansia muciniphila, found in the intestinal mucus, was recently shown to have a favorable effect on the disrupted metabolism associated with obesity. Faecalibacterium prausnitzii is a commensal gut bacterium, the absence of which may be associated with Crohn's disease. We showed that in our model, A. muciniphila induces stronger effects on the host than F. prausnitzii. We observed that A. muciniphila and propionate affect the expression of genes involved in host lipid metabolism and epigenetic activation or silencing of gene expression. We demonstrated that organoids provide a powerful tool for host-microbe interaction 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.

The human gastrointestinal microbiota--an unexplored frontier for pharmaceutical discovery.

The mammalian gastrointestinal tract (GIT) harbors microorganisms (the microbiota) of vast phylogentic, genomic, and metabolic diversity, and recent years have seen a rapid development in the techniques for studying these complex microbial ecosystems. It is increasingly apparent that the GIT microbiota plays an intricate role in host health and disease. Targeted strategies for modulating human health through the modification of the GIT microbiota, however, are developing and in their infancy. This perspective article discusses the rationale, benefits and limitations of using the GIT microbiota as a pharmacological and nutritional target in the treatment of various diseases and disorders linked to imbalances in our microbiota.

Effect of air drying on bacterial viability: A multiparameter viability assessment.

The effect of desiccation on the viability of microorganisms is a question of great interest for a variety of public health questions and industrial applications. Although viability is traditionally assessed by plate counts, cultivation-independent methods are increasingly applied with the aim to gain more insight into why cells might not form colonies and to optimize production processes. To evaluate their usefulness, we applied in this study a multiparameter viability assay to selected bacteria (Escherichia coli, Pseudomonas aeruginosa, Enterococcus hirae, and Staphylococcus aureus) subjected to air-drying in the absence or presence of supplements. Tests included growth on solid culture medium and the measurement of membrane integrity, membrane potential, esterase and respiratory activities using fluorescent dyes. All measured parameters were responsive to desiccation stress. Results suggested that extending plate count analysis with cultivation-independent methods can greatly enhance resolution especially for moderate stress conditions, which do not get reflected in plate counts due to cellular recovery. Whereas plate counts reflect the final effect on viability, immediate measurement of cellular functions provides a snapshot picture of the fitness status at a specific point in time. Special emphasis was given to MgCl(2) which in concentrations≥50mM dramatically increased the bacterial susceptibility to desiccation in the case of the gram-negative bacteria and to a lesser extent also for the gram-positive bacteria. The study in addition confirmed a good agreement of results obtained with the recently developed real-time viability (RTV) assay and the BacLight LIVE/DEAD method in combination with a fluorescence plate reader.

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.

Variability and diversity of nasopharyngeal microbiota in children: a metagenomic analysis.

The nasopharynx is the ecological niche for many commensal bacteria and for potential respiratory or invasive pathogens like Streptococcus pneumoniae, Haemophilus influenzae, and Neisseria meningitidis. Disturbance of a balanced nasopharyngeal (NP) microbiome might be involved in the onset of symptomatic infections with these pathogens, which occurs primarily in fall and winter. It is unknown whether seasonal infection patterns are associated with concomitant changes in NP microbiota. As young children are generally prone to respiratory and invasive infections, we characterized the NP microbiota of 96 healthy children by barcoded pyrosequencing of the V5-V6 hypervariable region of the 16S-rRNA gene, and compared microbiota composition between children sampled in winter/fall with children sampled in spring. The approximately 1,000,000 sequences generated represented 13 taxonomic phyla and approximately 250 species-level phyla types (OTUs). The 5 most predominant phyla were Proteobacteria (64%), Firmicutes (21%), Bacteroidetes (11%), Actinobacteria (3%) and Fusobacteria (1,4%) with Moraxella, Haemophilus, Streptococcus, Flavobacteria, Dolosigranulum, Corynebacterium and Neisseria as predominant genera. The inter-individual variability was that high that on OTU level a core microbiome could not be defined. Microbiota profiles varied strongly with season, with in fall/winter a predominance of Proteobacteria (relative abundance (% of all sequences): 75% versus 51% in spring) and Fusobacteria (absolute abundance (% of children): 14% versus 2% in spring), and in spring a predominance of Bacteroidetes (relative abundance: 19% versus 3% in fall/winter, absolute abundance: 91% versus 54% in fall/winter), and Firmicutes. The latter increase is mainly due to (Brevi)bacillus and Lactobacillus species (absolute abundance: 96% versus 10% in fall/winter) which are like Bacteroidetes species generally related to healthy ecosystems. The observed seasonal effects could not be attributed to recent antibiotics or viral co-infection.The NP microbiota of young children is highly diverse and appears different between seasons. These differences seem independent of antibiotic use or viral co-infection.

Exploring the oral microbiota of children at various developmental stages of their dentition in the relation to their oral health.

BACKGROUND: An understanding of the relation of commensal microbiota to health is essential in preventing disease. Here we studied the oral microbial composition of children (N = 74, aged 3 - 18 years) in natural transition from their deciduous to a permanent dentition and related the microbial profiles to their oral health status. The microbial composition of saliva was assessed by barcoded pyrosequencing of the V5-V6 hypervariable regions of the 16 S rRNA, as well as by using phylogenetic microarrays. RESULTS: Pyrosequencing reads (126174 reads, 1045 unique sequences) represented 8 phyla and 113 higher taxa in saliva samples. Four phyla--Firmicutes, Bacteriodetes, Proteobacteria and Actinobacteria--predominated in all groups. The deciduous dentition harboured a higher proportion of Proteobacteria (Gammaproteobacteria, Moraxellaceae) than Bacteroidetes, while in all other groups Bacteroidetes were at least as abundant as Proteobacteria. Bacteroidetes (mainly genus Prevotella), Veillonellaceae family, Spirochaetes and candidate division TM7 increased with increasing age, reflecting maturation of the microbiome driven by biological changes with age. Microarray analysis enabled further analysis of the individual salivary microbiota. Of 350 microarray probes, 156 gave a positive signal with, on average, 77 (range 48-93) probes per individual sample. A caries-free oral status significantly associated with the higher signal of the probes targeting Porphyromonas catoniae and Neisseria flavescens. CONCLUSIONS: The potential role of P. catoniae and N. flavescens as oral health markers should be assessed in large-scale clinical studies. The combination of both open-ended and targeted molecular approaches provides us with information that will increase our understanding of the interplay between the human host and its microbiome.

A mixed-species microarray for identification of food spoilage bacilli.

Failure of food preservation is frequently caused by thermostable spores of members of the Bacillaceae family, which show a wide spectrum of resistance to cleaning and preservation treatments. We constructed and validated a mixed-species genotyping array for 6 Bacillus species, including Bacillus subtilis, Bacillus licheniformis, Bacillus pumilus, Bacillus sporothermodurans, Bacillus cereus and Bacillus coagulans, and 4 Geobacillus species, including Geobacillus stearothermophilus, Geobacillus thermocatenulatus, Geobacillus toebii and Geobacillus sp., in order to track food spoilage isolates from ingredient to product. The discriminating power of the array was evaluated with sets of 42 reference and 20 test strains. Bacterial isolates contain a within-species-conserved core genome comprising 68-88% of the entire genome and a non-conserved accessory genome comprising 7-22%. The majority of the core genome markers do not hybridise between species, thus they allow for efficient discrimination at the species level. The accessory genome array markers provide high-resolution discrimination at the level of individual isolates from a single species. In conclusion, the reported mixed-species microarray contains discriminating markers that allow rapid and cost-effective typing of Bacillus food spoilage bacteria in a wide variety of food products.

Discrimination between live and dead cellsin bacterial communities from environmental water samples analyzed by 454 pyrosequencing.

SUMMARY: The preferential detection of cells with intact membranes by sample treatment with propidium monoazide (PMA) in combination with PCR amplification is gaining in popularity. This study evaluates the effect of PMA on 454 pyrosequencing profiles of environmental water samples from a canal in Amsterdam and seawater (with sediment) left untreated or exposed to elevated temperatures (50, 60, or 85 °C) for 10 min. Community analysis was based on the extraction of genomic DNA followed by PCR amplification of 16S rRNA genes using universal bacterial primers. Whereas the highest temperature in combination with PMA treatment completely suppressed PCR amplification, PCR products from the other samples were subjected to massively parallel tag sequencing. PMA treatment did not substantially affect the sequence profiles of non-heated samples, but heat exposure resulted in a clear difference in the relative proportions of certain groups. This difference was significantly more pronounced in heated seawater than in heated canal water. The effect of the chosen experimental conditions on the membrane integrity of cells was supported by BacLight LIVE/DEAD staining in combination with flow cytometry, which confirmed an increase in the uptake of propidium iodide in samples exposed to high temperatures.

Comparative genome analysis of a large Dutch Legionella pneumophila strain collection identifies five markers highly correlated with clinical strains.

BACKGROUND: Discrimination between clinical and environmental strains within many bacterial species is currently underexplored. Genomic analyses have clearly shown the enormous variability in genome composition between different strains of a bacterial species. In this study we have used Legionella pneumophila, the causative agent of Legionnaire's disease, to search for genomic markers related to pathogenicity. During a large surveillance study in The Netherlands well-characterized patient-derived strains and environmental strains were collected. We have used a mixed-genome microarray to perform comparative-genome analysis of 257 strains from this collection. RESULTS: Microarray analysis indicated that 480 DNA markers (out of in total 3360 markers) showed clear variation in presence between individual strains and these were therefore selected for further analysis. Unsupervised statistical analysis of these markers showed the enormous genomic variation within the species but did not show any correlation with a pathogenic phenotype. We therefore used supervised statistical analysis to identify discriminating markers. Genetic programming was used both to identify predictive markers and to define their interrelationships. A model consisting of five markers was developed that together correctly predicted 100% of the clinical strains and 69% of the environmental strains. CONCLUSIONS: A novel approach for identifying predictive markers enabling discrimination between clinical and environmental isolates of L. pneumophila is presented. Out of over 3000 possible markers, five were selected that together enabled correct prediction of all the clinical strains included in this study. This novel approach for identifying predictive markers can be applied to all bacterial species, allowing for better discrimination between strains well equipped to cause human disease and relatively harmless strains.

Real-time detection of viable microorganisms by intracellular phototautomerism.

BACKGROUND: To date, the detection of live microorganisms present in the environment or involved in infections is carried out by enumeration of colony forming units on agar plates, which is time consuming, laborious and limited to readily cultivable microorganisms. Although cultivation-independent methods are available, they involve multiple incubation steps and do mostly not discriminate between dead or live microorganisms. We present a novel generic method that is able to specifically monitor living microorganisms in a real-time manner. RESULTS: The developed method includes exposure of cells to a weak acid probe at low pH. The neutral probe rapidly permeates the membrane and enters the cytosol. In dead cells no signal is obtained, as the cytosolic pH reflects that of the acidic extracellular environment. In live cells with a neutral internal pH, the probe dissociates into a fluorescent phototautomeric anion. After reaching peak fluorescence, the population of live cells decays. This decay can be followed real-time as cell death coincides with intracellular acidification and return of the probe to its uncharged non-fluorescent state. The rise and decay of the fluorescence signal depends on the probe structure and appears discriminative for bacteria, fungi, and spores. We identified 13 unique probes, which can be applied in the real-time viability method described here. Under the experimental conditions used in a microplate reader, the reported method shows a detection limit of 10(6) bacteria ml(-1), while the frequently used LIVE/DEAD BacLight Syto9 and propidium iodide stains show detection down to 10(6) and 10(7) bacteria ml(-1), respectively. CONCLUSIONS: We present a novel fluorescence-based method for viability assessment, which is applicable to all bacteria and eukaryotic cell types tested so far. The RTV method will have a significant impact in many areas of applied microbiology including research on biocidal activity, improvement of preservation strategies and membrane permeation and stability. The assay allows for high-throughput applications and has great potential for rapid monitoring of microbial content in air, liquids or on surfaces.

Discrimination between live and dead cells in bacterial communities from environmental water samples analyzed by 454 pyrosequencing

The preferential detection of cells with intact membranes by sample treatment with propidium monoazide (PMA) in combination with PCR amplification is gaining in popularity. This study evaluates the effect of PMA on 454 pyrosequencing profiles of environmental water samples from a canal in Amsterdam and seawater (with sediment) left untreated or exposed to elevated temperatures (50, 60, or 85 ºC) for 10 min. Community analysis was based on the extraction of genomic DNA followed by PCR amplification of 16S rRNA genes using universal bacterial primers. Whereas the highest temperature in combination with PMA treatment completely suppressed PCR amplification, PCR products from the other samples were subjected to massively parallel tag sequencing. PMA treatment did not substantially affect the sequence profiles of non-heated samples, but heat exposure resulted in a clear difference in the relative proportions of certain groups. This difference was significantly more pronounced in heated seawater than in heated canal water. The effect of the chosen experimental conditions on the membrane integrity of cells was supported by BacLight LIVE/DEAD staining in combination with flow cytometry, which confirmed an increase in the uptake of propidium iodide in samples exposed to high temperatures (AU)

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