Viable Microbiota Research Articles

Shifts and importance of viable bacteria in treatment of DSS-induced ulcerative colitis mice with FMT.

Ulcerative colitis (UC) has become a global public health concern, and is in urgent need of novel therapies. Fecal microbiota transplantation (FMT) targeting gut microbiota has recently been applied to the treatment of UC. Despite its recent successes, it is still largely unknown how FMT functionally modulates the gut microbiota and improves the disease. We prospectively collected fecal samples from the 40 mice (30 mice for dextran sulfate sodium (DSS)-induced, 10 for controls), followed by Propidium monoazide treatment for 16S rRNA gene sequencing. These 30 mice were divided equally into 3 groups, which were transplanted with original donor microbiota (DO), inactivated donor microbiota (DI) and saline, respectively. Subsequently, we used 16S rRNA gene sequencing to analyze the viable gut bacteria of ulcerative colitis (UC) mice and histological analysis to evaluate the effects of fecal microbiota transplantation (FMT) with viable microbiota. We demonstrated that the community structure of viable bacteria was significantly different from fecal bacteria based on total DNA. Furthermore, the intestinal viable microbiota and colonic mucosal structure of mice were significantly changed by DSS induction. The histological analysis showed that only the mice treated with original donor microbiota group (HF) achieved a significant improvement. Compared with inactivated donor microbiota group (IF) and saline (NF), Lactobacillus and Halomonas were significantly enriched in the HF group. We inferred that only live bacteria from human donor reversed the histopathology and symptoms of UC in mice and altered the gut microbiota. The activity of gut microbiota in donor samples should be considered in FMT and that detailed analysis of viable microbiota is essential to understand the mechanisms by which FMT produces therapeutic effects in the future.

Cryopreservation of stool samples altered the microbial viability quantitively and compositionally.

Stool is the most commonly used sample for gut microbiota analysis in humans and animals. Cryopreservation of stool at -80°C is a feasible and simple method in clinics and researches, especially in large-scale cohort studies. However, the viability of bacteria in stool after freezing has yet well-demonstrated quantitatively and compositionally. This study determined the viable microbiota of samples under cryopreservation at -80°C, relative to fresh samples and that stored at ambient. Stool samples were collected from three healthy adults. Propidium monoazide treatment combined with quantitative PCR and 16S rRNA gene sequencing was performed to target viable microbiota. After freezing, the number of viable bacteria decreased, though inter-individual difference existed. Notably, the alpha diversity of viable microbiota after freezing did not change significantly, while its composition changed. Freezing significantly reduced the viable bacteria in Gram-negative genera of Bacteroidetes and Firmicutes, and proportionally increased Gram-positive bacteria in genera of Actinobacteria and Firmicutes, including Bifidobacterium, Collinsella and Blautia, implying that the cell envelope structure associated with the bacterial sensitivity to freezing. On the contrary, the room temperature storage not only decreased the number of viable bacteria, but also decreased the microbial alpha diversity, and remarkably enriched facultative anaerobes of Escherichia-Shigella, Enterococcus and Lactococcus, some of which are opportunistic pathogens. Our findings suggested that changes in viable microbiota in stool samples caused by cryopreservation should be paid enough attention for downstream utilization.

Bacteria in the cavity-restoration interface after varying periods of clinical service — SEM description of distribution and 16S rRNA gene sequence identification of isolates

ObjectivesTo use extracted human teeth with amalgam (n = 26) or GIC (n = 3) restorations in service up to 20 years to evaluate microbiota at the cavity/restoration interface by SEM or culture.Materials and methodsExtracted teeth with intracoronal restorations (n = 20) of known history (2–20 years) were fixed, split, and prepared for SEM to ascertain the pattern and structure of bacterial aggregates on cavity and restoration surfaces. Another 9 teeth were anaerobically decontaminated, split and sampled (cavity/restorations), and cultured (anaerobically, aerobically); recovered isolates were identified by 16S rRNA gene sequencing.ResultsSEM showed rods, cocci, and filaments in 11/20 teeth (55%) on cavity and corresponding restoration surfaces; 4/20 (20%) on neither surface; 1/20 (5%) on just cavity; and 4/20 (20%) on just restoration. Microbial growth extended from marginal openings into the deeper interfacial microspace to varying extents but was not always evident. Restoration size or age did not predict bacterial presence. Bacteria-free surfaces (cavity/amalgam) showed possible calcification. Cultivation yielded 160 isolates, mainly Gram-positive (86%) and facultative (81%); and morphotypes of rods (43%), cocci (36%), and cocco-bacilli (18%) belonging to Actinobacteria (45%) and Firmicutes (50%). The most frequent genera were Staphylococcus, Streptococcus, Actinomyces, and Lactobacillus. Biofilms on cavity and restoration appeared independent of each other.ConclusionsCavity and amalgam surfaces were independently colonised and some not. The penetration of microbiota into marginal gaps varied; resembled root caries and was dominated by Gram-positive species.Clinical relevanceMarginal gaps around restorations are unavoidable but are not always colonised by bacteria after long-term clinical service. Calcification of biofilms in the restorative interface may prevent further colonisation. The viable microbiota in the restorative interface resembled root caries and may be subject to ecological fluxes of activity and arrest and therefore preventative management.

Does the Amniotic Fluid of Mice Contain a Viable Microbiota?

The existence of an amniotic fluid microbiota (i.e., a viable microbial community) in mammals is controversial. Its existence would require a fundamental reconsideration of fetal in utero exposure to and colonization by microorganisms and the role of intra-amniotic microorganisms in fetal immune development as well as in pregnancy outcomes. In this study, we determined whether the amniotic fluid of mice harbors a microbiota in late gestation. The profiles of the amniotic fluids of pups located proximally or distally to the cervix were characterized through quantitative real-time PCR, 16S rRNA gene sequencing, and culture (N = 21 dams). These profiles were compared to those of technical controls for bacterial and DNA contamination. The load of 16S rRNA genes in the amniotic fluid exceeded that in controls. Additionally, the 16S rRNA gene profiles of the amniotic fluid differed from those of controls, with Corynebacterium tuberculostearicum being differentially more abundant in amniotic fluid profiles; however, this bacterium was not cultured from amniotic fluid. Of the 42 attempted bacterial cultures of amniotic fluids, only one yielded bacterial growth – Lactobacillus murinus. The 16S rRNA gene of this common murine-associated bacterium was not detected in any amniotic fluid sample, suggesting it did not originate from the amniotic fluid. No differences in the 16S rRNA gene load, 16S rRNA gene profile, or bacterial culture were observed between the amniotic fluids located Proximally and distally to the cervix. Collectively, these data indicate that, although there is a modest DNA signal of bacteria in murine amniotic fluid, there is no evidence that this signal represents a viable microbiota. While this means that amniotic fluid is not a source of microorganisms for in utero colonization in mice, it may nevertheless contribute to fetal exposure to microbial components. The developmental consequences of this observation warrant further investigation.

Long-term population dynamics of viable microbes in a closed ecosystem of fermented vegetables

This study was performed to investigate the succession of various microorganisms naturally present in raw ingredients and the changes in metabolites following long-term fermentation of kimchi. Kimchi was stored at 4 °C for 500 days, and the composition of the microbial community and the nature of metabolites were analyzed using metataxonomics and metabolomics. We confirmed that the taxa belonging to Leuconostoc and Weissella were dominant in the early stages of fermentation, while Latilactobacillus and Levilactobacillus were dominant in the middle and late fermentation stages, respectively. In the eukaryotic community, Cladosporium was dominant in the early stages, while Pichia and Hanseniaspora tended to increase in the middle and late fermentation stages. The longitudinal metabolite profile demonstrated that about half (55.7%) of the metabolites present in kimchi after 500 days of fermentation were produced within 15 days of fermentation due to rapid fermentation in the initial stage. These results revealed that even in a closed environment, the viable microbiota in fermented vegetables are not static but dynamic, and the composition of metabolites evolves accordingly during long-term fermentation.

Stool preparation under anaerobic conditions contributes to retention of obligate anaerobes: potential improvement for fecal microbiota transplantation

BackgroundFecal microbiota transplantation (FMT) in patients with ulcerative colitis has shown variable efficacy depending on the protocol used. A previous randomized controlled trial reported that anaerobic preparation of donor stool contributes to improved efficacy. Despite the suggestion that viable obligate anaerobes would be decreased through aerobic handling, there have been only a limited number of reports on how these aerobic or anaerobic procedures affect the composition of viable microbiota in the fecal slurries used for FMT.MethodsWe adopted 16S and 23S rRNA-targeted reverse transcription-quantitative polymerase chain reaction to quantify viable bacteria in fecal slurries. This study utilized specific primers designed to detect obligate anaerobes (including Clostridium coccoides group, C. leptum subgroup, Bacteroides fragilis group, Bifidobacterium, Atopobium cluster, and Prevotella) and facultative anaerobes (including total lactobacilli, Enterobacteriaceae, Enterococcus, Streptococcus, and Staphylococcus). We then calculated the ratio change (RC) between before and after mixing, and compared the resulting values between anaerobic-prep and aerobic-prep in samples fixed immediately after blending (RCAn0 vs. RCAe0) and in samples maintained (under anaerobic or aerobic conditions) for 1 h after blending (RCAn1 vs. RCAe1).ResultsFor most obligate anaerobes, the median RC tended to be less than 1, indicating that the number of obligate anaerobes was decreased by the blending procedure. However, in samples maintained for 1 h after blending, anaerobic-prep counteracted the decrease otherwise seen for the C. coccoides group and B. fragilis groups (P < 0.01 for both). The C. leptum subgroup also tended to show higher RC by anaerobic-prep than by aerobic-prep, although this effect was not statistically significant. Among facultative anaerobes, Enterobacteriaceae, Enterococcus, and Staphylococcus showed median RC values of more than 1, indicating that these organisms survived and even grew after mixing. Moreover, oxygen exposure had no significant influence on the survival of the facultative anaerobes.ConclusionsThe conditions under which the blending procedure was performed affected the proportion of live anaerobes in fecal slurries. The obligate anaerobes tended to be decreased by blending processes, but anaerobic-prep significantly mitigated this effect. Anaerobic-prep may improve the efficacy of FMT by permitting the efficient transfer of obligate anaerobes to patients with ulcerative colitis.

Changes in physico-chemical characteristics and viable bacterial communities during fermentation of alfalfa silages inoculated with Lactobacillus plantarum.

This study investigated the effect of inoculating Lactobacillus (L.) plantarum PS-8 in fermentation of alfalfa silages. We monitored the fermentation characteristics and bacterial population dynamics during the ensiling process. PacBio single molecule real time sequencing was combined with propidium monoazide (PMA) treatment to monitor the viable microbiota dynamics. We found that inoculating L. plantarum PS-8 may improve the silage quality by accelerating acidification, reducing the amounts of clostridia, coliform bacteria, molds and yeasts, elevating the protein and organic acid contents (except butyrate), and enhancing lactic acid bacteria (LAB) while suppressing harmful microorganisms. Some significant differential abundant taxa were found between the PMA-treated and non-treated microbiota. For example, the relative abundances of L. brevis, L. plantarum, and Pediococcus pentosaceus were significantly higher in the PMA-treated group than the non-PMA-treated group, suggesting obvious differences between the viable and non-viable microbiota. It would thus be necessary to distinguish between the viable and non-viable microbial communities to further understand their physiological contribution in silage fermentation. By tracking the dynamics of viable microbiota in relation with changes in the physico-chemical parameters, our study provided novel insights into the beneficial effects of inoculating L. plantarum PS-8 in silage fermentation and the physiological function of the viable bacterial communities.

Long-Term Use of Oral Hygiene Products Containing Stannous and Fluoride Ions: Effect on Viable Salivary Bacteria.

The aim of this randomized, controlled clinical trial was to isolate and identify viable microorganisms in the saliva of study participants that continuously used a stannous and fluoride ion (F/Sn)-containing toothpaste and mouth rinse over a period of three years in comparison to a control group that used stannous ion free preparations (noF/Sn) over the same time period. Each group (F/Sn and noF/Sn) included 16 participants that used the respective oral hygiene products over a 36-month period. Stimulated saliva samples were collected at baseline (T0) and after 36 months (T1) from all participants for microbiological examination. The microbial composition of the samples was analyzed using culture technique, matrix-assisted laser desorption ionization–time of flight (MALDI–TOF) mass spectrometry, and 16S rDNA Polymerase Chain Reaction (PCR). There were only minor differences between both groups when comparing the absolute values of viable microbiota and bacterial composition. The treatment with F/Sn led to a slight decrease in disease-associated and a slight increase in health-associated bacteria. It was shown that the use of stannous ions had no negative effects on physiological oral microbiota even after prolonged use. In fact, a stabilizing effect of the oral hygiene products containing stannous ions on the health-associated oral microbiota could be expected.

Characterization of antibacterial activity of a N-acetylmuramoyl-L-alanine amidase produced by Latilactobacillus sakei isolated from salami.

Lactic acid bacteria are the predominant group within meat products, whose metabolites such as bacteriocins and peptidoglycan hydrolases inhibit pathogenic or spoilage bacteria. Fermented meat products, as a salami, is a good source to analyze the viable microbiota, due to these products present a low risk to consumer health. The aim of this work was to identify the lactic acid bacteria with broad antibacterial activity present in salami, purify the protein responsible for this activity, achieve antagonistic spectrum and perform the biochemical characterization. Five strains from salami were selected, isolated and identified by 16S rRNA gene sequencing. The antimicrobial activity was evaluated by antagonism assay and zymography, using spoilage microorganisms commonly found in meat products. The strain that showed a broad antibacterial activity was Latilactobacillus sakei and the antibacterial activity was given by a protein with 75-kDa of molecular mass, identified by LC/MALDI-TOF/TOF. The sequence analysis showed 67% of identity with a N-acetylmuramoyl-L-alanine amidase protein with five non-identical LysM domains. The purified protein showed an optimal pH of 8.0 and heat resistance at 80°C for 10min. L. sakei strain displayed antibacterial activity against Gram-negative and Gram-positive spoilage microorganisms. The results of this study provide the information to use Latilactobacillus sakei as a starter culture which will provide the necessary metabolites to combat undesirable microorganisms. Additionally, the conditions and properties for the best application and use of the antibacterial protein produced by this strain. This protein may have a potential use in the food industry as a new antibacterial agent.

Comparison of Blood Bacterial Communities in Periodontal Health and Periodontal Disease.

The use of Next Generation Sequencing (NGS) techniques has generated a wide variety of blood microbiome data. Due to the large variation in bacterial DNA profiles between studies and the likely high concentrations of cell-free bacterial DNA in the blood, it is still not clear how such microbiome data relates to viable microbiota. For these reasons much remains to be understood about the true nature of any possible healthy blood microbiota and of bacteraemic events associated with disease. The gut, reproductive tracts, skin, and oral cavity are all likely sources of blood-borne bacteria. Oral bacteria, especially those associated with periodontal diseases, are also commonly associated with cardiovascular diseases such as infective endocarditis, and also have been linked to rheumatoid arthritis and Alzheimer’s disease. Periodontal treatment, dental probing, and toothbrushing have been shown to cause transient bacteraemia and oral bacteria from the phyla Firmicutes (e.g. Streptococci) and Bacteroidetes (e.g. Porphyromonas) are found in cardiovascular lesions (CVD). Many studies of blood bacterial DNA content however, find Proteobacteria DNA to be the dominant microbiome component, suggesting a gut origin. Most studies of this type use total DNA extracted from either whole blood or blood fractions, such as buffy coat. Here, using a method that purifies DNA from intact bacterial cells only, we examined blood donated by those with active, severe periodontitis and periodontally healthy controls and show that 43–52% of bacterial species in blood are classified as oral. Firmicutes, consisting largely of members of the Streptococcus mitis group and Staphylococcus epidermidis, were predominant at 63.5% of all bacterial sequences detected in periodontal health and, little changed at 66.7% in periodontitis. Compared to studies using total DNA Proteobacteria were found here at relatively low levels in blood at 13.3% in periodontitis and 17.6% in health. This study reveals significant phylogenetic differences in blood bacterial population profiles when comparing periodontal health to periodontal disease cohorts.

Novel use of culturomics to identify the microbiota in hospital sink drains with and without persistent VIM-positive Pseudomonas aeruginosa

In hospitals, Verona Integron-encoded Metallo-beta-lactamase (VIM)-positive Pseudomonas aeruginosa may colonize sink drains, and from there, be transmitted to patients. These hidden reservoirs are difficult to eradicate since P. aeruginosa forms biofilms that resist disinfection. However, little is known on the composition of these biofilms. Therefore, culturomics was used for the first time to investigate the viable microbiota in four hospital sink drain samples with longstanding VIM-positive P. aeruginosa drain reservoirs (inhabited by high-risk clone, sequence type ST111), and four drain samples where VIM-positive P. aeruginosa was not present. Microbial load and composition varied between samples, yielding between 471–18,904 distinct colonies and 8–20 genera. In two VIM-positive drain samples, P. aeruginosa was the most abundantly-isolated microorganism, and found in combination with other Gram-negative bacteria, Citrobacter, Enterobacter, or Stenotrophomonas. P. aeruginosa was in low abundance in the other two VIM-positive samples, and found with Gram-positive cocci (Enterococcus and Staphylococcus) or Sphingomonas. In VIM-negative drain samples, high abundances of Gram-negative non-fermenting bacteria, including Acinetobacter, non-aeruginosa Pseudomonas spp., Acidovorax, Chryseobacterium, Flavobacterium, and Sphingobium, as well as Candida, were cultured. Although additional experiments are needed to draw more firm conclusions on which microorganisms enable or inhibit VIM-positive P. aeruginosa persistence, our data provide unique insights into the microbial compositions of sink drain inlets.

Impact of dietary ingredients on the interpretation of various fecal parameters in rats fed inulin

In the present study, the influences of diets (i.e. chow and AIN-93 diets) on the interpretation of various fecal parameters including viable microbiota, moisture, weight, and short-chain fatty acids in rats fed different amounts of inulin (0.5–2 g/kg). Eight groups of rats (n = 8/group) were fed, for 4 weeks, chow or AIN-93 diets with or without inulin supplementation. Fecal samples were analyzed for different fecal parameters. After a 2-week adaptation, apparent differences in some fecal parameters were observed between the chow and AIN-93 diet groups. Throughout the 4-week intervention period, significantly (p < 0.05) higher Lactobacillus spp. counts, fecal moisture (∼2.7-fold), and fecal weight (∼5.8-fold) were observed with chow diet over AIN-93 diet. More specifically, significant elevations in the levels of Bifidobacterium spp., Lactobacillus spp., fecal moisture, and fecal weight could be observed at low-dose (0.5 g/kg) of inulin in chow diet groups, while most of these changes could merely be seen at medium-dose (1 g/kg) in AIN-93 diet groups. These results demonstrated that the choice of experimental diets would affect the comparison of fecal parameters as well as the interpretation of effective dosage of prebiotic in intestinal health assessments.

Bacterial viability in faecal transplants: Which bacteria survive?

BackgroundThe therapeutic potential of faecal microbiota transplantation (FMT) is under investigation for a range of inflammatory conditions. While mechanisms of benefit are poorly understood, most models rely on the viability of transplanted microbes. We hypothesised that protocols commonly used in the preparation of faecal transplants will substantially reduce the number, diversity and functional potential of viable microbes.MethodsStools from eight screened donors were processed under strict anaerobic conditions, in ambient air, and freeze-thawed. Propidium monoazide (PMA) sample treatment was combined with quantitative PCR, 16S rRNA gene amplicon sequencing and short-chain fatty acid (SCFA) analysis to define the viable microbiota composition and functional potential.FindingsApproximately 50% of bacterial content of stool processed immediately under strict anaerobic conditions was non-viable. Homogenisation in ambient air or freeze-thaw reduced viability to 19% and 23% respectively. Processing of samples in ambient air resulted in up to 12-fold reductions in the abundance of important commensal taxa, including the highly butyrogenic species Faecalibacterium prausnitzii, Subdoligranulum variable, and Eubacterium hallii. The adverse impact of atmospheric oxygen exposure on the capacity of the transplanted microbiota to support SCFA biosynthesis was demonstrated by significantly reduced butyrate and acetate production by faecal slurries processed in ambient air. In contrast, while reducing overall levels of viable bacteria, freeze-thaw did not significantly alter viable microbiota composition.InterpretationThe practice of preparing material for faecal transplantation in ambient air profoundly affects viable microbial content, disproportionately reducing the abundance of anaerobic commensals and the capacity for biosynthesis of important anti-inflammatory metabolites.FundThis work was supported by the South Australian Health and Medical Research Institute. LP is supported by a scholarship from the Flinders Foundation. GR is supported by a Matthew Flinders Research Fellowship.

Quenching by sodium thiosulfate does not influence 16S rRNA gene sequencing profiles of reclaimed water from three sites in the Mid-Atlantic, United States

A quenching agent is commonly added to chlorinated, reclaimed water during sample collection to prevent chlorine-mediated die-off of viable microbiota. However, the effect of quenching on downstream 16S rRNA-based bacterial community analyses is unclear. We conducted a side-by-side comparison of 16S rRNA sequencing data from reclaimed water samples quenched with sodium thiosulfate and non-quenched samples. Our data showed that 16 S rRNA processing and sequencing methods, and resulting bacterial profiles, were not negatively impacted by quenching.

In Vivo Analysis of the Viable Microbiota and Helicobacter pylori Transcriptome in Gastric Infection and Early Stages of Carcinogenesis.

Emerging evidence shows that the human microbiota plays a larger role in disease progression and health than previously anticipated. Helicobacter pylori, the causative agent of gastric cancer and duodenal and gastric ulcers, was early associated with gastric disease, but it has also been proposed that the accompanying microbiota in Helicobacter pylori-infected individuals might affect disease progression and gastric cancer development. In this study, the composition of the transcriptionally active microbial community and H. pylori gene expression were determined using metatranscriptomic RNA sequencing of stomach biopsy specimens from individuals with different H. pylori infection statuses and premalignant tissue changes. The results show that H. pylori completely dominates the microbiota not only in infected individuals but also in most individuals classified as H. pylori uninfected using conventional methods. Furthermore, H. pylori abundance is positively correlated with the presence of Campylobacter, Deinococcus, and Sulfurospirillum Finally, we quantified the expression of a large number of Helicobacter pylori genes and found high expression of genes involved in pH regulation and nickel transport. Our study is the first to dissect the viable microbiota of the human stomach by metatranscriptomic analysis, and it shows that metatranscriptomic analysis of the gastric microbiota is feasible and can provide new insights into how bacteria respond in vivo to variations in the stomach microenvironment and at different stages of disease progression.

Metabolic gene-targeted monitoring of non-starter lactic acid bacteria during cheese ripening

Long ripened cheeses, such as Grana Padano (GP), a Protected Designation of Origin (PDO) Italian cheese, harbor a viable microbiota mainly composed of non-starter lactic acid bacteria (NSLAB), which contribute to the final characteristics of cheese. The NSLAB species Lactobacillus rhamnosus, Lb. casei and Lb. paracasei are frequently found in GP, and form a closely related taxonomic group (Lb. casei group), making it difficult to distinguish the three species through 16S rRNA sequencing. SpxB, a metabolic gene coding for pyruvate oxidase in Lb. casei group, was recently used to distinguish the species within this bacterial group, both in pure cultures and in cheese, where it could provide an alternative energy source through the conversion of pyruvate to acetate. The aim of this work was to study the evolution of the metabolically active microbiota during different stages of GP ripening, targeting 16S rRNA to describe the whole microbiota composition, and spxB gene to monitor the biodiversity within the Lb. casei group. Furthermore, activation of pyruvate oxidase pathway was measured directly in cheese by reverse transcription real time PCR (RT–qPCR). The results showed that Lb. casei group dominates throughout the ripening and high-throughput sequencing of spxB allowed to identify four clusters inside the Lb. casei group. The dynamics of the sequence types forming the clusters were followed during ripening. Pyruvate oxidase pathway was expressed in cheese, showing a decreasing trend over ripening time. This work highlights how the composition of the microbiota in the early manufacturing stages influences the microbial dynamics throughout ripening, and how targeting of a metabolic gene can provide an insight into the activity of strains relevant for dairy products.

Reducing Viability Bias in Analysis of Gut Microbiota in Preterm Infants at Risk of NEC and Sepsis.

Necrotising enterocolitis (NEC) and sepsis are serious diseases of preterm infants that can result in feeding intolerance, the need for bowel resection, impaired physiological and neurological development, and high mortality rates. Neonatal healthcare improvements have allowed greater survival rates in preterm infants leading to increased numbers at risk of developing NEC and sepsis. Gut bacteria play a role in protection from or propensity to these conditions and have therefore, been studied extensively using targeted 16S rRNA gene sequencing methods. However, exact epidemiology of these conditions remain unknown and the role of the gut microbiota in NEC remains enigmatic. Many studies have confounding variables such as differing clinical intervention strategies or major methodological issues such as the inability of 16S rRNA gene sequencing methods to determine viable from non-viable taxa. Identification of viable community members is important to identify links between the microbiota and disease in the highly unstable preterm infant gut. This is especially important as remnant DNA is robust and persists in the sampling environment following cell death. Chelation of such DNA prevents downstream amplification and inclusion in microbiota characterisation. This study validates use of propidium monoazide (PMA), a DNA chelating agent that is excluded by an undamaged bacterial membrane, to reduce bias associated with 16S rRNA gene analysis of clinical stool samples. We aim to improve identification of the viable microbiota in order to increase the accuracy of clinical inferences made regarding the impact of the preterm gut microbiota on health and disease. Gut microbiota analysis was completed on stools from matched twins (n = 16) that received probiotics. Samples were treated with PMA, prior to bacterial DNA extraction. Meta-analysis highlighted a significant reduction in bacterial diversity in 68.8% of PMA treated samples as well as significantly reduced overall rare taxa abundance. Importantly, overall abundances of genera associated with protection from and propensity to NEC and sepsis such as: Bifidobacterium; Clostridium, and Staphylococcus sp. were significantly different following PMA-treatment. These results suggest non-viable cell exclusion by PMA-treatment reduces bias in gut microbiota analysis from which clinical inferences regarding patient susceptibility to NEC and sepsis are made.

Viable cells differentiation improves microbial dynamics study of fermented milks

Understanding the changes that occur during storage of fermented milk products is of significant importance to dairy producers. Here, we investigated the microbiota dynamics of two fermented milk products, yoghurt and kefir, using culture-independent methods in combination with the use of a viability dye and quantitative-polymerase chain reaction. Propidium monoazide (PMA) was used to selectively detect viable cells in the food matrix. Samples treated with PMA showed results comparable with culturing methods and a lower copy number than samples not treated with PMA. The use of two culture-independent approaches combined with PMA treatment showed a more complete picture of the viable microbiota in the fermented milks during storage. Significant differences in microbiota were detected in kefir samples treated with and without PMA whereas no such significant differences were found in yoghurt. The microbiota in kefir also showed a high variation between batches at the species level.

Aerobiological investigations inside repositories of the National Archive of the Republic of Cuba

The objectives of this study were to evaluate the microbial prevalence inside six repositories of the National Archive of the Republic of Cuba in 2 months of the year and to examine some of the physiological features of fungi isolated in order to evaluate their potential for biodeterioration. The microbiological sampling was conducted in February and September using a slit impactor as air sampler. Appropriate selective culture media were used to isolate fungi and bacteria. Temperature and relative humidity were measured during the samplings. The cellulolytic activity and the production of acids and pigments of the fungi isolated were qualitatively determined. Total viable microbiota and bacteria concentrations were greater in February while the fungal concentration was higher in September. Aspergillus, Cladosporium, Penicillium, Curvularia and Alternaria were the predominant fungal genera in February while Cladosporium prevailed in September, although Fusarium, Mucor and Neurospora genera were also isolated in this month. The fungi isolated were capable of degrading cellulose and excreting pigments and acids. The Gram-positive bacteria group prevailed in the air and Corynebacterium, Streptomyces, Bacillus, Streptococcus, Staphylococcus, Enterobacter and Serratia were some of the genera identified.

The prevalence of opportunistic pathogens associated with intraoral implants

The aim of this study was to determine the prevalence and proportions of opportunistic pathogens harboured on orthodontic retainers. First, Staphylococcus spp. and Candida spp. were isolated from the retainer's inner surface and from other mucosal surfaces of the subject's mouth by routine bacterial culture. The prevalence and proportions of these micro-organisms on retainers was compared in different areas of the mouth within a group of retainer wearers, and mucosal carriage was compared to a group of nonretainer wearers. Staphylococcus spp. were isolated from 50% of the retainers and comprised on average 8·4% of the viable microbiota. Candida spp. comprised 0·13% of the viable microbiota and were recovered from 66·7% of the retainers. Neither genus was isolated from nonretainer wearers. Second, the two most commonly worn retainers manufactured from different materials were sampled; again Staphylococcus spp. and Candida spp. were recovered; however, no statistical differences were observed between the devices. Opportunistic, nonoral, pathogenic micro-organisms were recovered from orthodontic retainers. It is possible that an orthodontic retainer could be a reservoir for opportunistic pathogens and act as a source of cross-, self- and re-infection.