A randomized double-blind placebo controlled pilot study of probiotics in adolescents with severe obesity.

PURPOSE: The purpose of the study is to assess the effect of probiotic supplementation on gut microbiota and insulin resistance in adolescents with severe obesity. METHODS: Through a randomized, double blind, placebo-controlled, 12-week pilot clinical trial, 15 adolescents with severe obesity received either an oral probiotic 'Visbiome®' (n = 8) or placebo (n = 7). Anthropometry, fasting glucose, insulin, hs-CRP and stool for microbiome and calprotectin were collected at baseline (week 0) and 12 weeks after intervention. RESULTS: Among completers (n = 4 in each of the two groups), mean change in fasting glucose was significantly lower in the probiotic group (0 ± 4 mg/dL) as compared to the placebo group (6.3 ± 1.7 mg/dL) (p = 0.028). Gut microbial Firmicutes to Bacteroidetes (F/B) ratio had a greater decline from week 0 to week 12 in the probiotic group (mean 17.7 ± 25.1 to 2.39 ± 2.0, respectively) but was not statistically significant (p = 0.06) as compared to in the placebo group (mean 12.8 ± 18.2 to 6.9 ± 5.61, respectively) (p = 0.89). Weight and BMI (mean ± SD) trended to remain stable in the treatment group (-1.07 ± 6.1 kg and -0.3 ± 2.2 kg/m2 respectively) as compared to the placebo group (3.9 ± 5.1 kg, 1.0 ± 1.6 kg/m2) but was not significant (p = 0.12 for weight and 0.38 for BMI). No significant change in the fasting insulin, HOMA-IR, or serum and stool inflammatory markers were noted between the two groups (p > 0.05). One participant in the treatment arm reported adverse effects of gastrointestinal intolerance. CONCLUSION: Probiotic therapy with Visbiome® may improve the fasting glucose and possibly decrease the gut microbial F/B ratio as compared to placebo in adolescents with severe obesity. Future larger studies are required to confirm these findings.U.S. Clinical Trial Registry number: NCT03109587. SUPPLEMENTARY INFORMATION: The online version contains supplementary material available at 10.1007/s40200-021-00855-7.

Interplay of Trypanosome Lytic Factor and innate immune cells in the resolution of cutaneous Leishmania infection.

Trypanosome Lytic Factor (TLF) is a primate-specific high-density lipoprotein (HDL) complex that, through the cation channel-forming protein apolipoprotein L-1 (APOL1), provides innate immunity to select kinetoplastid parasites. The immunoprotective effects of TLF have been extensively investigated in the context of its interaction with the extracellular protozoan Trypanosoma brucei brucei, to which it confers sterile immunity. We previously showed that TLF could act against an intracellular pathogen Leishmania, and here we dissected the role of TLF and its synergy with host-immune cells. Leishmania major is transmitted by Phlebotomine sand flies, which deposit the parasite intradermally into mammalian hosts, where neutrophils are the predominant phagocytes recruited to the site of infection. Once in the host, the parasites are phagocytosed and shed their surface glycoconjugates during differentiation to the mammalian-resident amastigote stage. Our data show that mice producing TLF have reduced parasite burdens when infected intradermally with metacyclic promastigotes of L. major, the infective, fly-transmitted stage. This TLF-mediated reduction in parasite burden was lost in neutrophil-depleted mice, suggesting that early recruitment of neutrophils is required for TLF-mediated killing of L. major. In vitro we find that only metacyclic promastigotes co-incubated with TLF in an acidic milieu were lysed. However, amastigotes were not killed by TLF at any pH. These findings correlated with binding experiments, revealing that labeled TLF binds specifically to the surface of metacyclic promastigotes, but not to amastigotes. Metacyclic promastigotes of L. major deficient in the synthesis of surface glycoconjugates LPG and/or PPG (lpg1- and lpg5A-/lpg5B- respectively) whose absence mimics the amastigote surface, were resistant to TLF-mediated lysis. We propose that TLF binds to the outer surface glycoconjugates of metacyclic promastigotes, whereupon it kills the parasite in the acidic phagosome of phagocytes. We hypothesize that resistance to TLF requires shedding of the surface glycoconjugates, which occurs several hours after phagocytosis by immune cells, creating a relatively short-lived but effective window for TLF to act against Leishmania.

Maintenance tobramycin primarily affects untargeted bacteria in the CF sputum microbiome.

RATIONALE: The most common antibiotic used to treat people with cystic fibrosis (PWCF) is inhaled tobramycin, administered as maintenance therapy for chronic Pseudomonas aeruginosa lung infections. While the effects of inhaled tobramycin on P. aeruginosa abundance and lung function diminish with continued therapy, this maintenance treatment is known to improve long-term outcomes, underscoring how little is known about why antibiotics work in CF infections, what their effects are on complex CF sputum microbiomes and how to improve these treatments. OBJECTIVES: To rigorously define the effect of maintenance tobramycin on CF sputum microbiome characteristics. METHODS AND MEASUREMENTS: We collected sputum from 30 PWCF at standardised times before, during and after a single month-long course of maintenance inhaled tobramycin. We used traditional culture, quantitative PCR and metagenomic sequencing to define the dynamic effects of this treatment on sputum microbiomes, including abundance changes in both clinically targeted and untargeted bacteria, as well as functional gene categories. MAIN RESULTS: CF sputum microbiota changed most markedly by 1 week of antibiotic therapy and plateaued thereafter, and this shift was largely driven by changes in non-dominant taxa. The genetically conferred functional capacities (ie, metagenomes) of subjects' sputum communities changed little with antibiotic perturbation, despite taxonomic shifts, suggesting functional redundancy within the CF sputum microbiome. CONCLUSIONS: Maintenance treatment with inhaled tobramycin, an antibiotic with demonstrated long-term mortality benefit, primarily impacted clinically untargeted bacteria in CF sputum, highlighting the importance of monitoring the non-canonical effects of antibiotics and other treatments to accurately define and improve their clinical impact.

Deciphering the Ecology of Cystic Fibrosis Bacterial Communities: Towards Systems-Level Integration.

Despite over a decade of cystic fibrosis (CF) microbiome research, much remains to be learned about the overall composition, metabolic activities, and pathogenicity of the microbes in CF airways, limiting our understanding of the respiratory microbiome's relation to disease. Systems-level integration and modeling of host-microbiome interactions may allow us to better define the relationships between microbiological characteristics, disease status, and treatment response. In this way, modeling could pave the way for microbiome-based development of predictive models, individualized treatment plans, and novel therapeutic approaches, potentially serving as a paradigm for approaching other chronic infections. In this review, we describe the challenges facing this effort and propose research priorities for a systems biology approach to CF lung disease.

Human and Extracellular DNA Depletion for Metagenomic Analysis of Complex Clinical Infection Samples Yields Optimized Viable Microbiome Profiles.

Metagenomic sequencing is a promising approach for identifying and characterizing organisms and their functional characteristics in complex, polymicrobial infections, such as airway infections in people with cystic fibrosis. These analyses are often hampered, however, by overwhelming quantities of human DNA, yielding only a small proportion of microbial reads for analysis. In addition, many abundant microbes in respiratory samples can produce large quantities of extracellular bacterial DNA originating either from biofilms or dead cells. We describe a method for simultaneously depleting DNA from intact human cells and extracellular DNA (human and bacterial) in sputum, using selective lysis of eukaryotic cells and endonuclease digestion. We show that this method increases microbial sequencing depth and, consequently, both the number of taxa detected and coverage of individual genes such as those involved in antibiotic resistance. This finding underscores the substantial impact of DNA from sources other than live bacteria in microbiological analyses of complex, chronic infection specimens.

How low can we go? The implications of low bacterial load in respiratory microbiota studies.

BACKGROUND: Culture-independent sequencing methods are increasingly used to investigate the microbiota associated with human mucosal surfaces, including sites that have low bacterial load in healthy individuals (e.g. the lungs). Standard microbiota methods developed for analysis of high bacterial load specimens (e.g. stool) may require modification when bacterial load is low, as background contamination derived from sterile laboratory reagents and kits can dominate sequence data when few bacteria are present. MAIN BODY: Bacterial load in respiratory specimens may vary depending on the specimen type, specimen volume, the anatomic site sampled and clinical parameters. This review discusses methodological issues inherent to analysis of low bacterial load specimens and recommends strategies for successful respiratory microbiota studies. The range of methods currently used to process DNA from low bacterial load specimens, and the strategies used to identify and exclude background contamination are also discussed. CONCLUSION: Microbiota studies that include low bacterial load specimens require additional tests to ensure that background contamination does not bias the results or interpretation. Several methods are currently used to analyse the microbiota in low bacterial load respiratory specimens; however, there is scant literature comparing the effectiveness and biases of different methods. Further research is needed to define optimal methods for analysing the microbiota in low bacterial load specimens.

Comparison of 3M Petrifilm Aerobic Count Plates to standard plating methodology for use with AOAC antimicrobial efficacy methods 955.14, 955.15, 964.02, and 966.04 as an alternative enumeration procedure: collaborative study.

A multilaboratory study was conducted to determine the equivalence of the 3M Petrifilm Aerobic Count Plate and standard plating methodology for measuring viable bacteria and spores recovered from hard-surface carriers (stainless steel and porcelain), also known as "control carrier counts," used in AOAC antimicrobial efficacy test methods. Six laboratories participated in the study in which carriers inoculated with Staphylococcus aureus, Pseudomonas aeruginosa, Salmonella enterica, and spores of Bacillus subtilis were evaluated using 3M Petrifilm Aerobic Count (AC) plates and standard plating side-by-side. The data were analyzed using a matched-pair t-test to determine the between-method effect with confidence intervals. For all test organisms pooled across all laboratories, the mean difference in log10 concentration between the standard plate count method and 3M Petrifilm AC Plates was -0.012, with a 95% confidence interval of (-0.090, +0.066), which was well within the -0.5, +0.5 interval established as the acceptance criterion. The between-carrier SD averaged 0.139; the between-replicate SD was 0.050. The carrier reproducibility, given that a single replicate per carrier is done, was estimated to be 0.148. Although differences were seen in the final concentrations of the test organisms among laboratories, there were no statistical differences between the enumeration methods. Based on the results from this study, 3M Petrifilm AC Plates are equivalent to standard plating methodology and can be used as an alternative procedure for the enumeration of test organisms used in AOAC Methods 955.14, 955.15, 964.02, and 966.04.

Probability of Detection (POD) as a statistical model for the validation of qualitative methods.

A statistical model is presented for use in validation of qualitative methods. This model, termed Probability of Detection (POD), harmonizes the statistical concepts and parameters between quantitative and qualitative method validation. POD characterizes method response with respect to concentration as a continuous variable. The POD model provides a tool for graphical representation of response curves for qualitative methods. In addition, the model allows comparisons between candidate and reference methods, and provides calculations of repeatability, reproducibility, and laboratory effects from collaborative study data. Single laboratory study and collaborative study examples are given.