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In vitro transcription studies reveal that phenylation and purified TyrR exhibit capacity to activate the in vitro transcription from the tyrP promoter of Escherichia coli by wild-type RNA polymerase. This activation involves a specific interaction between RNA polymerase and TyrR protein. A putative promoter is present in the strand opposite to tyrP and within the TyrR-binding region. Absence of TyrR-mediated activation in the mutant alpha 235 RNA polymerase reveals TyrR as a class I transcription activator.

Three TyrR boxes and one TrpR binding site constitute the promoter-operator region of the aroL gene of Escherichia coli K-12. TyrR-induced regulation of aroL expression requires the TyrR boxes/and 3 and TrpR protein-induced regulation requires TrpR and the TyrR boxes. The regulations indicate that TrpR and TrpR are capable of reacting with each other.

The Seneca Valley virus (SVV) is an oncolytic virus from the picornavirus family, characterized by a 7.3-kilobase RNA genome encoding for all the structural and functional viral proteins. Directed evolution by serial passaging has been employed for oncolytic virus adaptation to increase the killing efficacy towards certain types of tumors. We propagated the SVV in a small-cell lung cancer model under two culture conditions: conventional cell monolayer and tumorspheres, with the latter resembling more closely the cellular structure of the tumor of origin. We observed an increase of the virus-killing efficacy after ten passages in the tumorspheres. Deep sequencing analyses showed genomic changes in two SVV populations comprising 150 single nucleotides variants and 72 amino acid substitutions. Major differences observed in the tumorsphere-passaged virus population, compared to the cell monolayer, were identified in the conserved structural protein VP2 and in the highly variable P2 region, suggesting that the increase in the ability of the SVV to kill cells over time in the tumorspheres is acquired by capsid conservation and positively selecting mutations to counter the host innate immune responses.

Emerging SARS-CoV-2 variants pose a threat to human health worldwide. SARS-CoV-2 receptor binding domain (RBD)-based vaccines are suitable candidates for booster vaccines, eliciting a focused antibody response enriched for virus neutralizing activity. Although RBD proteins are manufactured easily, and have excellent stability and safety properties, they are poorly immunogenic compared to the full-length spike protein. We have overcome this limitation by engineering a subunit vaccine composed of an RBD tandem dimer fused to the N-terminal domain (NTD) of the spike protein. We found that inclusion of the NTD (1) improved the magnitude and breadth of the T cell and anti-RBD response, and (2) enhanced T follicular helper cell and memory B cell generation, antibody potency, and cross-reactive neutralization activity against multiple SARS-CoV-2 variants, including B.1.1.529 (Omicron BA.1). In summary, our uniquely engineered RBD-NTD-subunit protein vaccine provides a promising booster vaccination strategy capable of protecting against known SARS-CoV-2 variants of concern., Competing Interests: The authors declare no conflict of interests, other than the filing of patent application number AU2021902667 entitled “Fusion Polypeptide”. The funders had no role in the design of the study; in the collection, analyses, or interpretation of data; in the writing of the manuscript, or in the decision to publish the results., (© 2023 The Authors.)

A jeotgal-like product was processed from Chinook salmon (Oncorrhynchus tshawytscha) roe. Physicochemical, biochemical, and microbiological compositions were studied during 30 days of fermentation. Fermentation decreased water activity (a w ) and pH value. Total bacterial and LAB counts (log CFU/g) increased up to 12 days of processing and then no further changes occurred. Saturated fatty acids (SFA) decreased (p < 0.05), monounsaturated fatty acids (MUFA) did not change (p > 0.05), whereas fermentation time improved polyunsaturated fatty acids (PUFA) content significantly (p < 0.05). Astaxanthin, lutein and phospholipids (PC, LPC, PE, LPE and LPS) concentrations were found to increase, while cholesterol and tocopherol contents were decreased at the end of the fermentation (p < 0.05). This study indicates that the nutritional value of salmon roe can be enhanced by fermentation., Competing Interests: Declaration of Competing Interest The authors declare the following financial interests/personal relationships which may be considered as potential competing interests: Alaa El-Din Ahmed Bekhit reports financial support was provided by University of Otago. Alaa El-Din Ahmed Bekhit reports a relationship with University of Otago that includes: employment., (Copyright © 2022 Elsevier Ltd. All rights reserved.)

Obesity is a complex, multifactorial condition that is an important risk factor for noncommunicable diseases including cardiovascular disease and type 2 diabetes. While prevention and management require a healthy and energy balanced diet and adequate physical activity, the taxonomic composition and functional attributes of the colonic microbiota may have a supplementary role in the development of obesity. The taxonomic composition and metabolic capacity of the fecal microbiota of 286 women, resident in Auckland New Zealand, was determined by metagenomic analysis. Associations with BMI (obese, nonobese), body fat composition, and ethnicity (Pacific, n = 125; NZ European women [NZE], n = 161) were assessed using regression analyses. The fecal microbiotas were characterized by the presence of three distinctive enterotypes, with enterotype 1 represented in both Pacific and NZE women (39 and 61%, respectively), enterotype 2 mainly in Pacific women (84 and 16%) and enterotype 3 mainly in NZE women (13 and 87%). Enterotype 1 was characterized mainly by the relative abundances of butyrate producing species, Eubacterium rectale and Faecalibacterium prausnitzii , enterotype 2 by the relative abundances of lactic acid producing species, Bifidobacterium adolescentis, Bifidobacterium bifidum , and Lactobacillus ruminis , and enterotype 3 by the relative abundances of Subdoligranulum sp., Akkermansia muciniphila, Ruminococcus bromii , and Methanobrevibacter smithii . Enterotypes were also associated with BMI, visceral fat %, and blood cholesterol. Habitual food group intake was estimated using a 5 day nonconsecutive estimated food record and a 30 day, 220 item semi-quantitative Food Frequency Questionnaire. Higher intake of 'egg' and 'dairy' products was associated with enterotype 3, whereas 'non-starchy vegetables', 'nuts and seeds' and 'plant-based fats' were positively associated with enterotype 1. In contrast, these same food groups were inversely associated with enterotype 2. Fecal water content, as a proxy for stool consistency/colonic transit time, was associated with microbiota taxonomic composition and gene pools reflective of particular bacterial biochemical pathways. The fecal microbiotas of women of Pacific and New Zealand European ethnicities are characterized by distinctive enterotypes, most likely due to differential dietary intake and fecal consistency/colonic transit time. These parameters need to be considered in future analyses of human fecal microbiotas.

Evidence of gut microbiota involvement in regulating glucose metabolism and type 2 diabetes mellitus (T2DM) progression is accumulating. The understanding of microbial dysbiosis and specific alterations of gut microbiota composition that occur during the early stages of glucose intolerance, unperturbed by anti-diabetic medications, is especially essential. Hence, this systematic review was conducted to summarise the existing evidence related to microbiota composition and diversity in individuals with prediabetes (preDM) and individuals newly diagnosed with T2DM (newDM) in comparison to individuals with normal glucose tolerance (nonDM). A systematic search of the PubMed, MEDLINE and CINAHL databases were conducted from inception to February 2021 supplemented with manual searches of the list of references. The primary keywords of "type 2 diabetes", "prediabetes", "newly-diagnosed" and "gut microbiota" were used. Observational studies that conducted analysis of the gut microbiota of respondents with preDM and newDM were included. The quality of the studies was assessed using the modified Newcastle-Ottawa scale by independent reviewers. A total of 18 studies (5,489 participants) were included. Low gut microbial diversity was generally observed in preDM and newDM when compared to nonDM. Differences in gut microbiota composition between the disease groups and nonDM were inconsistent across the included studies. Four out of the 18 studies found increased abundance of phylum Firmicutes along with decreased abundance of Bacteroidetes in newDM. At the genus/species levels, decreased abundance of Faecalibacterium prausnitzii , Roseburia , Dialister , Flavonifractor, Alistipes, Haemophilus and Akkermansia muciniphila and increased abundance of Lactobacillus, Streptococcus , Escherichi a, Veillonella and Collinsella were observed in the disease groups in at least two studies. Lactobacillus was also found to positively correlate with fasting plasma glucose (FPG), HbA1c and/or homeostatic assessment of insulin resistance (HOMA-IR) in four studies. This renders a need for further investigations on the species/strain-specific role of endogenously present Lactobacillus in glucose regulation mechanism and T2DM disease progression. Differences in dietary intake caused significant variation in specific bacterial abundances. More studies are needed to establish more consistent associations, between clinical biomarkers or dietary intake and specific gut bacterial composition in prediabetes and early T2DM., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2022 Letchumanan, Abdullah, Marlini, Baharom, Lawley, Omar, Mohideen, Addnan, Nur Fariha, Ismail and Pathmanathan.)

SARS-CoV-2, the virus responsible for the COVID-19 pandemic, has wreaked havoc across the globe for the last two years. More than 300 million cases and over 5 million deaths later, we continue battling the first real pandemic of the 21st century. SARS-CoV-2 spread quickly, reaching most countries within the first half of 2020, and New Zealand was not an exception. Here, we describe the first isolation and characterization of SARS-CoV-2 variants during the initial virus outbreak in New Zealand. Patient-derived nasopharyngeal samples were used to inoculate Vero cells and, three to four days later, a cytopathic effect was observed in seven viral cultures. Viral growth kinetics was characterized using Vero and VeroE6/TMPRSS2 cells. The identity of the viruses was verified by RT-qPCR, Western blot, indirect immunofluorescence assays, and electron microscopy. Whole-genome sequences were analyzed using two different yet complementary deep sequencing platforms (MiSeq/Illumina and Ion PGM™/Ion Torrent™), classifying the viruses as SARS-CoV-2 B.55, B.31, B.1, or B.1.369 based on the Pango Lineage nomenclature. All seven SARS-CoV-2 isolates were susceptible to remdesivir (EC 50 values from 0.83 to 2.42 µM) and β-D-N 4 -hydroxycytidine (molnupiravir, EC 50 values from 0.96 to 1.15 µM) but not to favipiravir (>10 µM). Interestingly, four SARS-CoV-2 isolates, carrying the D614G substitution originally associated with increased transmissibility, were more susceptible (2.4-fold) to a commercial monoclonal antibody targeting the spike glycoprotein than the wild-type viruses. Altogether, this seminal work allowed for early access to SARS-CoV-2 isolates in New Zealand, paving the way for numerous clinical and scientific research projects in the country, including the development and validation of diagnostic assays, antiviral strategies, and a national COVID-19 vaccine development program.

The rapid global rise of COVID-19 from late 2019 caught major manufacturers of RT-qPCR reagents by surprise and threw into sharp focus the heavy reliance of molecular diagnostic providers on a handful of reagent suppliers. In addition, lockdown and transport bans, necessarily imposed to contain disease spread, put pressure on global supply lines with freight volumes severely restricted. These issues were acutely felt in New Zealand, an island nation located at the end of most supply lines. This led New Zealand scientists to pose the hypothetical question: in a doomsday scenario where access to COVID-19 RT-qPCR reagents became unavailable, would New Zealand possess the expertise and infrastructure to make its own reagents onshore? In this work we describe a review of New Zealand's COVID-19 test requirements, bring together local experts and resources to make all reagents for the RT-qPCR process, and create a COVID-19 diagnostic assay referred to as HomeBrew (HB) RT-qPCR from onshore synthesized components. This one-step RT-qPCR assay was evaluated using clinical samples and shown to be comparable to a commercial COVID-19 assay. Through this work we show New Zealand has both the expertise and, with sufficient lead time and forward planning, infrastructure capacity to meet reagent supply challenges if they were ever to emerge., Competing Interests: Authors JU and JG are employed by Southern Community Laboratories, Dunedin, New Zealand. Authors WR and LL are employed by South Pacific Sera, Washdyke, Timaru, New Zealand. Authors RO'B and PC are employed by MicroGEM NZ Ltd., 201 Princes Street, Dunedin, New Zealand. The remaining authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2022 Stanton, O'Brien, Hall, Chernyavtseva, Ha, Jelley, Mace, Klenov, Treece, Fraser, Clow, Clarke, Su, Kurup, Filichev, Rolleston, Law, Rendle, Harris, Wood, Scully, Ussher, Grant, Hore, Moser, Harfoot, Lawley, Quiñones-Mateu, Collins and Blaikie.)

Background: Our understanding of the peripheral human immunodeficiency virus type 1 (HIV-1) reservoir is strongly biased towards subtype B HIV-1 strains, with only limited information available from patients infected with non-B HIV-1 subtypes, which are the predominant viruses seen in low- and middle-income countries (LMIC) in Africa and Asia., Results: In this study, blood samples were obtained from well-suppressed ART-experienced HIV-1 patients monitored in Uganda (n = 62) or the U.S. (n = 50), with plasma HIV-1 loads < 50 copies/ml and CD4 + T-cell counts > 300 cells/ml. The peripheral HIV-1 reservoir, i.e., cell-associated HIV-1 RNA and proviral DNA, was characterized using our novel deep sequencing-based EDITS assay. Ugandan patients were slightly younger (median age 43 vs 49 years) and had slightly lower CD4 + counts (508 vs 772 cells/ml) than U.S. individuals. All Ugandan patients were infected with non-B HIV-1 subtypes (31% A1, 64% D, or 5% C), while all U.S. individuals were infected with subtype B viruses. Unexpectedly, we observed a significantly larger peripheral inducible HIV-1 reservoir in U.S. patients compared to Ugandan individuals (48 vs. 11 cell equivalents/million cells, p < 0.0001). This divergence in reservoir size was verified measuring proviral DNA (206 vs. 88 cell equivalents/million cells, p < 0.0001). However, the peripheral HIV-1 reservoir was more diverse in Ugandan than in U.S. individuals (8.6 vs. 4.7 p-distance, p < 0.0001)., Conclusions: The smaller, but more diverse, peripheral HIV-1 reservoir in Ugandan patients might be associated with viral (e.g., non-B subtype with higher cytopathicity) and/or host (e.g., higher incidence of co-infections or co-morbidities leading to less clonal expansion) factors. This highlights the need to understand reservoir dynamics in diverse populations as part of ongoing efforts to find a functional cure for HIV-1 infection in LMICs., (© 2022. The Author(s).)

The arrival of SARS-CoV-2 to Aotearoa/New Zealand in February 2020 triggered a massive response at multiple levels. Procurement and sustainability of medical supplies to hospitals and clinics during the then upcoming COVID-19 pandemic was one of the top priorities. Continuing access to new personal protective equipment (PPE) was not guaranteed; thus, disinfecting and reusing PPE was considered as a potential alternative. Here, we describe part of a local program intended to test and implement a system to disinfect PPE for potential reuse in New Zealand. We used filtering facepiece respirator (FFR) coupons inoculated with SARS-CoV-2 or clinically relevant multidrug-resistant pathogens ( Acinetobacter baumannii Ab5075, methicillin-resistant Staphylococcus aureus USA300 LAC and cystic-fibrosis isolate Pseudomonas aeruginosa LESB58), to evaluate the potential use of ultraviolet-C germicidal irradiation (UV-C) or dry heat treatment to disinfect PPE. An applied UV-C dose of 1000 mJ/cm 2 was sufficient to completely inactivate high doses of SARS-CoV-2; however, irregularities in the FFR coupons hindered the efficacy of UV-C to fully inactivate the virus, even at higher UV-C doses (2000 mJ/cm 2 ). Conversely, incubating contaminated FFR coupons at 65 °C for 30 min or 70 °C for 15 min, was sufficient to block SARS-CoV-2 replication, even in the presence of mucin or a soil load (mimicking salivary or respiratory secretions, respectively). Dry heat (90 min at 75 °C to 80 °C) effectively killed 10 6 planktonic bacteria; however, even extending the incubation time up to two hours at 80 °C did not completely kill bacteria when grown in colony biofilms. Importantly, we also showed that FFR material can harbor replication-competent SARS-CoV-2 for up to 35 days at room temperature in the presence of a soil load. We are currently using these findings to optimize and establish a robust process for decontaminating, reusing, and reducing wastage of PPE in New Zealand.

It has been 20 months since we first heard of SARS-CoV-2, the novel coronavirus detected in the Hubei province, China, in December 2019, responsible for the ongoing COVID-19 pandemic. Since then, a myriad of studies aimed at understanding and controlling SARS-CoV-2 have been published at a pace that has outshined the original effort to combat HIV during the beginning of the AIDS epidemic. This massive response started by developing strategies to not only diagnose individual SARS-CoV-2 infections but to monitor the transmission, evolution, and global spread of this new virus. We currently have hundreds of commercial diagnostic tests; however, that was not the case in early 2020, when just a handful of protocols were available, and few whole-genome SARS-CoV-2 sequences had been described. It was mid-January 2020 when several District Health Boards across New Zealand started planning the implementation of diagnostic testing for this emerging virus. Here, we describe our experience implementing a molecular test to detect SARS-CoV-2 infection, adapting the RT-qPCR assay to be used in a random-access platform (Hologic Panther Fusion ® System) in a clinical laboratory, and characterizing the first whole-genome SARS-CoV-2 sequences obtained in the South Island, right at the beginning of the SARS-CoV-2 outbreak in New Zealand. We expect that this work will help us and others prepare for the unequivocal risk of similar viral outbreaks in the future.

The human gut microbiota develops soon after birth and can acquire inter-individual variation upon exposure to intrinsic and environmental cues. However, inter-individual variation has not been comprehensively assessed in a multi-ethnic study. We studied a longitudinal birth cohort of 106 infants of three Asian ethnicities (Chinese, Malay, and Indian) that resided in the same geographical location (Singapore). Specific and temporal influences of ethnicity, mode of delivery, breastfeeding status, gestational age, birthweight, gender, and maternal education on the development of the gut microbiota in the first 2 years of life were studied. Mode of delivery, breastfeeding status, and ethnicity were identified as the main factors influencing the compositional development of the gut microbiota. Effects of delivery mode and breastfeeding status lasted until 6M and 3M, respectively, with the primary impact on the diversity and temporal colonization of the genera Bacteroides and Bifidobacterium . The effect of ethnicity was apparent at 3M post-birth, even before the introduction of weaning (complementary) foods, and remained significant after adjusting for delivery mode and breastfeeding status. Ethnic influences remained significant until 12M in the Indian and Chinese infants. The microbiota of Indian infants was characterized by higher abundances of Bifidobacterium and Lactobacillus , while Chinese infants had higher abundances of Bacteroides and Akkermansia . These findings provide a detailed insight into the specific and temporal influences of early life factors and ethnicity in the development of the human gut microbiota. Trial Registration: Clinicaltrials.gov registration no. NCT01174875.

The gut microbiota harvests energy from indigestible plant polysaccharides, forming short-chain fatty acids (SCFAs) that are absorbed from the bowel. SCFAs provide energy-presumably after easily digested food components have been absorbed from the small intestine. Infant night waking is believed by many parents to be due to hunger. Our objective was to determine whether faecal SCFAs are associated with longer uninterrupted sleep in infants. Infants (n = 57) provided faecal samples for determining SCFAs (7 months of age), and questionnaire data for determining infant sleep (7 and 8 months). Linear regression determined associations between SCFAs-faecal acetate, propionate and butyrate-and sleep. For each 1% higher propionate at 7 months of age, the longest night sleep was 6 (95% CI: 1, 10) minutes longer at both 7 and 8 months. A higher proportion of total faecal SCFA as propionate was associated with longer uninterrupted infant sleep.

Immuno-modulatory effects of infant gut bacteria were tested on poly(I:C) stimulated HT-29 intestinal epithelial cells. Blautia producta, Bacteroides vulgatus, Bacteroides fragilis and Bacteroides thetaiotaomicron decreased transcription of poly(I:C)-induced inflammatory genes. Modulation of basal level and poly(I:C)-induced IL-8 secretion varied between bacterial species, and between heat treated and non-heat treated bacterial cells., Competing Interests: Declaration of competing interest Authors declare no conflict of interest., (Copyright © 2019 Elsevier Ltd. All rights reserved.)

Dietary fiber provides growth substrates for bacterial species that belong to the colonic microbiota of humans. The microbiota degrades and ferments substrates, producing characteristic short-chain fatty acid profiles. Dietary fiber contains plant cell wall-associated polysaccharides (hemicelluloses and pectins) that are chemically diverse in composition and structure. Thus, depending on plant sources, dietary fiber daily presents the microbiota with mixtures of plant polysaccharides of various types and complexity. We studied the extent and preferential order in which mixtures of plant polysaccharides (arabinoxylan, xyloglucan, β-glucan, and pectin) were utilized by a coculture of five bacterial species ( Bacteroides ovatus , Bifidobacterium longum subspecies longum , Megasphaera elsdenii , Ruminococcus gnavus , and Veillonella parvula ). These species are members of the human gut microbiota and have the biochemical capacity, collectively, to degrade and ferment the polysaccharides and produce short-chain fatty acids (SCFAs). B. ovatus utilized glycans in the order β-glucan, pectin, xyloglucan, and arabinoxylan, whereas B. longum subsp. longum utilization was in the order arabinoxylan, arabinan, pectin, and β-glucan. Propionate, as a proportion of total SCFAs, was augmented when polysaccharide mixtures contained galactan, resulting in greater succinate production by B. ovatus and conversion of succinate to propionate by V. parvula Overall, we derived a synthetic ecological community that carries out SCFA production by the common pathways used by bacterial species for this purpose. Systems like this might be used to predict changes to the emergent properties of the gut ecosystem when diet is altered, with the aim of beneficially affecting human physiology. IMPORTANCE This study addresses the question as to how bacterial species, characteristic of the human gut microbiota, collectively utilize mixtures of plant polysaccharides such as are found in dietary fiber. Five bacterial species with the capacity to degrade polymers and/or produce acidic fermentation products detectable in human feces were used in the experiments. The bacteria showed preferential use of certain polysaccharides over others for growth, and this influenced their fermentation output qualitatively. These kinds of studies are essential in developing concepts of how the gut microbial community shares habitat resources, directly and indirectly, when presented with mixtures of polysaccharides that are found in human diets. The concepts are required in planning dietary interventions that might correct imbalances in the functioning of the human microbiota so as to support measures to reduce metabolic conditions such as obesity., (Copyright © 2020 American Society for Microbiology.)

Background: Gut microbiota data obtained by DNA sequencing are complex and compositional because of large numbers of detectable taxa, and because microbiota characteristics are described in relative terms. Nutrition researchers use principal component analysis (PCA) to derive dietary patterns from food data. Although compositional PCA methods are not commonly used to describe patterns from complex microbiota data, this approach would be useful for identifying gut microbiota patterns associated with diet and body composition., Objectives: To use compositional PCA to describe the principal components (PCs) of gut microbiota in 5-y-old children and explore associations between microbiota components, diet, and BMI z-score., Methods: A fecal sample was provided by 319 children aged 5 y. Their primary caregiver completed a validated 123-item quantitative FFQ. Body composition was determined using DXA, and a BMI z-score was calculated. Compositional PCA identified characterizing taxa and weightings for calculation of gut microbiota PC scores at the genus level, and was examined in relation to diet and body size., Results: Three gut microbiota PCs were found. PC1 (negative loadings on uncultured Christensenellaceae and Ruminococcaceae) was related to lower BMI z-scores and longer duration of breastfeeding (per month) (β = -0.14; 95% CI: -0.26, -0.02; and β = 0.02; 95% CI: 0.003, 0.34, respectively). PC2 (positive loadings on Fusicatenibacter and Bifidobacterium; negative loadings on Bacteroides) was associated with a lower intake of nuts, seeds, and legumes (β = -0.05 per gram; 95% CI: -0.09, -0.01). When adjusted for fiber intake, PC2 was also associated with higher BMI z-scores (β = 0.12; 95% CI: 0.01, 0.24). PC3 (positive loadings on Faecalibacterium, Eubacterium, and Roseburia) was associated with higher intakes of fiber (β = 0.02 per gram; 95% CI: 0.003, 0.04) and total nonstarch polysaccharides (β = 0.02 per gram; 95% CI: 0.003, 0.04)., Conclusions: Our results suggest that specific gut microbiota components determined using compositional PCA are associated with diet and BMI z-score.This trial was registered at clinicaltrials.gov as NCT00892983., (Copyright © The Author(s) 2019.)

Background and Aim: This study compared the oral bacteriome between HIV-1-infected and non-HIV-1-infected Brazilian children/teenagers., Methods: Whole saliva, biofilm from the dorsal surface of the tongue and biofilm from supragingival and subgingival sites were collected from 27 HIV-1-infected and 30 non-HIV-1-infected individuals. Bacterial genomic DNA was extracted and 16S rRNA genes were sequenced using next-generation sequencing technology (Ion Torrent)., Results: In the supragingival biofilm, the phylum Firmicutes and genus Streptococcus sp. were more frequent in HIV-1-infected (95% and 78%, respectively) than in non-HIV-1-infected individuals (40% and 24%, respectively). In the subgingival biofilm of HIV-infected participants, the relative abundance of the Veillonella sp. and Prevotella sp. genera were higher than in non-HIV-1-infected participants. On the tongue, the genera with greater relative abundance in HIV-1-infected individuals were Neisseria sp. (21%). In saliva, the difference of the genus Prevotella sp. between non-HIV-1-infected and HIV-1-infected individuals was 15% and 7%, respectively. The Chao index revealed an increase in the richness of both sub- and supragingival biofilms in the HIV-1-infected samples compared with non-HIV-1-infected samples., Conclusion: HIV-1-infected children/teenagers have a higher frequency of the phyla Firmicutes and genus Streptococcus, and their oral microbiome shows more complexity than that of non-HIV-1-infected children/teenagers., (© 2019 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.)

The biological succession that occurs during the first year of life in the gut of infants in Western countries is broadly predictable in terms of the increasing complexity of the composition of microbiotas. Less information is available about microbiotas in Asian countries, where environmental, nutritional, and cultural influences may differentially affect the composition and development of the microbial community. We compared the fecal microbiotas of Indonesian ( n  = 204) and New Zealand (NZ) ( n  = 74) infants 6 to 7 months and 12 months of age. Comparisons were made by analysis of 16S rRNA gene sequences and derivation of community diversity metrics, relative abundances of bacterial families, enterotypes, and cooccurrence correlation networks. Abundances of Bifidobacterium longum subsp. infantis and B. longum subsp. longum were determined by quantitative PCR. All observations supported the view that the Indonesian and NZ infant microbiotas developed in complexity over time, but the changes were much greater for NZ infants. B. longum subsp. infantis dominated the microbiotas of Indonesian children, whereas B. longum subsp. longum was dominant in NZ children. Network analysis showed that the niche model (in which trophic adaptation results in preferential colonization) of the assemblage of microbiotas was supported in Indonesian infants, whereas the neutral (stochastic) model was supported by the development of the microbiotas of NZ infants. The results of the study show that the development of the fecal microbiota is not the same for infants in all countries, and they point to the necessity of obtaining a better understanding of the factors that control the colonization of the gut in early life. IMPORTANCE This study addresses the microbiology of a natural ecosystem (the infant bowel) for children in a rural setting in Indonesia and in an urban environment in New Zealand. Analysis of DNA sequences generated from the microbial community (microbiota) in the feces of the infants during the first year of life showed marked differences in the composition and complexity of the bacterial collections. The differences were most likely due to differences in the prevalence and duration of breastfeeding of infants in the two countries. These kinds of studies are essential for developing concepts of microbial ecology related to the influence of nutrition and environment on the development of the gut microbiota and for determining the long-term effects of microbiological events in early life on human health and well-being., (Copyright © 2019 American Society for Microbiology.)

Background: The prevalence of obesity has increased substantially over recent decades and is associated with considerable health inequalities. Although the causes of obesity are complex, key drivers include overconsumption of highly palatable, energy-dense, and nutrient-poor foods, which have a profound impact on the composition and function of the gut microbiome. Alterations to the microbiome may play a critical role in obesity by affecting energy extraction from food and subsequent energy metabolism and fat storage., Objective: We report the study protocol and recruitment strategy of the PRedictors linking Obesity and the gut MIcrobiomE (PROMISE) study, which characterizes the gut microbiome in 2 populations with different metabolic disease risk (Pacific and European women) and different body fat profiles (normal and obese). It investigates (1) the role of gut microbiome composition and functionality in obesity and (2) the interactions between dietary intake; eating behavior; sweet, fat, and bitter taste perception; and sleep and physical activity; and their impact on the gut microbiome, metabolic and endocrine regulation, and body fat profiles., Methods: Healthy Pacific and New Zealand (NZ) European women aged between 18 and 45 years from the Auckland region were recruited for this cross-sectional study. Participants were recruited such that half in each group had either a normal weight (body mass index [BMI] 18.5-24.9 kg/m 2 ) or were obese (BMI ≥30.0 kg/m 2 ). In addition to anthropometric measurements and assessment of the body fat content using dual-energy x-ray absorptiometry, participants completed sweet, fat, and bitter taste perception tests; food records; and sleep diaries; and they wore accelerometers to assess physical activity and sleep. Fasting blood samples were analyzed for metabolic and endocrine biomarkers and DNA extracted from fecal samples was analyzed by shotgun sequencing. Participants completed questionnaires on dietary intake, eating behavior, sleep, and physical activity. Data were analyzed using descriptive and multivariate regression methods to assess the associations between dietary intake, taste perception, sleep, physical activity, gut microbiome complexity and functionality, and host metabolic and body fat profiles., Results: Of the initial 351 women enrolled, 142 Pacific women and 162 NZ European women completed the study protocol. A partnership with a Pacific primary health and social services provider facilitated the recruitment of Pacific women, involving direct contact methods and networking within the Pacific communities. NZ European women were primarily recruited through Web-based methods and special interest Facebook pages., Conclusions: This cross-sectional study will provide a wealth of data enabling the identification of distinct roles for diet, taste perception, sleep, and physical activity in women with different body fat profiles in modifying the gut microbiome and its impact on obesity and metabolic health. It will advance our understanding of the etiology of obesity and guide future intervention studies involving specific dietary approaches and microbiota-based therapies., Trial Registration: Australian New Zealand Clinical Trials Registry ACTRN12618000432213; https://www.anzctr.org.au/Trial/Registration/TrialReview.aspx?id=370874., International Registered Report Identifier (irrid): RR1-10.2196/14529., (©Sophie Kindleysides, Rozanne Kruger, Jeroen Douwes, Gerald W Tannock, Nikki Renall, Joanne Slater, Blair Lawley, Anne-Thea McGill, Niamh Brennan, Moana Manukia, Marilize Richter, Ridvan Tupai-Firestone, T Leigh Signal, Philippa Gander, Stephen R Stannard, Bernhard H Breier. Originally published in JMIR Research Protocols (http://www.researchprotocols.org), 26.08.2019.)

The introduction of "solids" (i.e., complementary foods) to the milk-only diet in early infancy affects the development of the gut microbiota. The aim of this study was to determine whether a "baby-led" approach to complementary feeding that encourages the early introduction of an adult-type diet results in alterations of the gut microbiota composition compared to traditional spoon-feeding. The Baby-Led Introduction to SolidS (BLISS) study randomized 206 infants to BLISS (a modified version of baby-led weaning [BLW], the introduction of solids at 6 months of age, followed by self-feeding of family foods) or control (traditional spoon-feeding of purées) groups. Fecal microbiotas and 3-day weighed-diet records were analyzed for a subset of 74 infants at 7 and 12 months of age. The composition of the microbiota was determined by sequencing of 16S rRNA genes amplified by PCR from bulk DNA extracted from feces. Diet records were used to estimate food and dietary fiber intake. Alpha diversity (number of operational taxonomic units [OTUs]) was significantly lower in BLISS infants at 12 months of age (difference [95% confidence interval {CI}] of 31 OTUs [3.4 to 58.5]; P = 0.028), and while there were no significant differences between control and BLISS infants in relative abundances of Bifidobacteriaceae , Enterobacteriaceae , Veillonellaceae , Bacteroidaceae , Erysipelotrichaceae , Lachnospiraceae , or Ruminococcaceae at 7 or 12 months of age, OTUs representing the genus Roseburia were less prevalent in BLISS microbiotas at 12 months. Mediation models demonstrated that the intake of "fruit and vegetables" and "dietary fiber" explained 29% and 25%, respectively, of the relationship between group (BLISS versus control) and alpha diversity. IMPORTANCE The introduction of solid foods (complementary feeding or weaning) to infants leads to more-complex compositions of microbial communities (microbiota or microbiome) in the gut. In baby-led weaning (BLW), infants are given only finger foods that they can pick up and feed themselves-there is no parental spoon-feeding of puréed baby foods-and infants are encouraged to eat family meals. BLW is a new approach to infant feeding that is increasing in popularity in the United States, New Zealand, the United Kingdom, and Canada. We used mediation modeling, commonly used in health research but not in microbiota studies until now, to identify particular dietary components that affected the development of the infant gut microbiota., (Copyright © 2018 American Society for Microbiology.)

Starches resistant to mammalian digestion are present in foods and pass to the large bowel, where they may be degraded and fermented by the microbiota. Increases in relative abundances of bifidobacteria (blooms) have been reported in rats whose diet was supplemented with Hi-Maize resistant starch. We determined that the bifidobacterial species present in the rat cecum under these circumstances mostly belonged to Bifidobacterium animalis However, cultures of B. animalis isolated from the rats failed to degrade Hi-Maize starch to any extent. In contrast, Bifidobacterium pseudolongum also detected in the rat microbiota had high starch-degrading ability. Transcriptional comparisons showed increased expression of a type 1 pullulanase, alpha-amylase, and glycogen debranching enzyme by B. pseudolongum when cultured in medium containing Hi-Maize starch. Maltose was released into the culture medium, and B. animalis cultures had shorter doubling times in maltose medium than did B. pseudolongum Thus, B. pseudolongum , which was present at a consistently low abundance in the microbiota, but which has extensive enzymatic capacity to degrade resistant starch, showed the attributes of a keystone species associated with the bifidobacterial bloom. IMPORTANCE This study addresses the microbiology and function of a natural ecosystem (the rat gut) using DNA-based observations and in vitro experimentation. The microbial community of the large bowel of animals, including humans, has been studied extensively through the use of high-throughput DNA sequencing methods and advanced bioinformatics analysis. These studies reveal the compositions and genetic capacities of microbiotas but not the intricacies of how microbial communities function. Our work, combining DNA sequence analysis and laboratory experiments with cultured strains of bacteria, revealed that the increased abundance of bifidobacteria in the rat gut, induced by feeding indigestible starch, involved a species that cannot itself degrade the starch ( Bifidobacterium animalis ) but cohabits with a species that can ( Bifidobacterium pseudolongum ). B. pseudolongum has the characteristics of a keystone species in the community because it had low abundance but high ability to perform a critical function, the hydrolysis of resistant starch., (Copyright © 2018 American Society for Microbiology.)

Kiwifruit are a nutrient dense food and an excellent source of vitamin C. Supplementation of the diet with kiwifruit enhances plasma vitamin C status and epidemiological studies have shown an association between vitamin C status and reduced insulin resistance and improved blood glucose control. In vitro experiments suggest that eating kiwifruit might induce changes to microbiota composition and function; however, human studies to confirm these findings are lacking. The aim of this study was to investigate the effect of consuming two SunGold kiwifruit per day over 12 weeks on vitamin C status, clinical and anthropometric measures and faecal microbiota composition in people with prediabetes. This pilot intervention trial compared baseline measurements with those following the intervention. Participants completed a physical activity questionnaire and a three-day estimated food diary at baseline and on completion of the trial. Venous blood samples were collected at each study visit (baseline, 6, 12 weeks) for determination of glycaemic indices, plasma vitamin C concentrations, hormones, lipid profiles and high-sensitivity C-reactive protein. Participants provided a faecal sample at each study visit. DNA was extracted from the faecal samples and a region of the 16S ribosomal RNA gene was amplified and sequenced to determine faecal microbiota composition. When week 12 measures were compared to baseline, results showed a significant increase in plasma vitamin C (14 µmol/L, p < 0.001). There was a significant reduction in both diastolic (4 mmHg, p = 0.029) and systolic (6 mmHg, p = 0.003) blood pressure and a significant reduction in waist circumference (3.1 cm, p = 0.001) and waist-to-hip ratio (0.01, p = 0.032). Results also showed a decrease in HbA1c (1 mmol/mol, p = 0.005) and an increase in fasting glucose (0.1 mmol/L, p = 0.046), however, these changes were small and were not clinically significant. Analysis of faecal microbiota composition showed an increase in the relative abundance of as yet uncultivated and therefore uncharacterised members of the bacterial family Coriobacteriaceae . Novel bacteriological investigations of Coriobacteriaceae are required to explain their functional relationship to kiwifruit polysaccharides and polyphenols.

Members of the bacterial genus Bifidobacterium generally dominate the fecal microbiota of infants. The species Bifidobacterium longum is prevalent, but the B. longum subsp. longum and B. longum subsp. infantis strains that are known to colonize the infant bowel are not usually differentiated in microbiota investigations. These subspecies differ in their capacities to metabolize human milk oligosaccharides (HMO) and may have different ecological and symbiotic roles in humans. Quantitative PCR provides a quick analytical method by which to accurately ascertain the abundances of target species in microbiotas and microcosms. However, amplification targets in DNA extracted from samples need to be dependably differential. We evaluated the tuf gene sequence as a molecular target for quantitative PCR measurements of the abundances of B. longum subsp. infantis and B. longum subsp. longum in fecal microbiotas. This approach resulted in the detection of a tuf gene variant (operational taxonomic unit 49 [OTU49]) in Chinese infants that has sequence similarities to both B. longum subsp. infantis and B. longum subsp. longum We compared the genome sequence and growth and transcriptional characteristics of an OTU49 isolate cultured in HMO medium to those of other B. longum subsp. infantis cultures. We concluded from these studies that OTU49 belongs to B. longum subsp. infantis , that dependable quantitative PCR (qPCR) differentiation between the B. longum subspecies cannot be achieved by targeting tuf gene sequences, and that functional genes involved in carbohydrate metabolism might be better targets because they delineate ecological functions. IMPORTANCE High-throughput DNA sequencing methods and advanced bioinformatics analysis have revealed the composition and biochemical capacities of microbial communities (microbiota and microbiome), including those that inhabit the gut of human infants. However, the microbiology and function of natural ecosystems have received little attention in recent decades, so an appreciation of the dynamics of gut microbiota interactions is lacking. With respect to infants, rapid methodologies, such as quantitative PCR, are needed to determine the prevalences and proportions of different bifidobacterial species in observational and microcosm studies in order to obtain a better understanding of the dynamics of bifidobacterial nutrition and syntrophy, knowledge that might be used to manipulate the microbiota and perhaps ensure the better health of infants., (Copyright © 2018 American Society for Microbiology.)

Premise of the Study: To study pollination networks in a changing environment, we need accurate, high-throughput methods. Previous studies have shown that more highly resolved networks can be constructed by studying pollen loads taken from bees, relative to field observations. DNA metabarcoding potentially allows for faster and finer-scale taxonomic resolution of pollen compared to traditional approaches (e.g., light microscopy), but has not been applied to pollination networks., Methods: We sampled pollen from 38 bee species collected in Florida from sites differing in forest management. We isolated DNA from pollen mixtures and sequenced rbcL and ITS2 gene regions from all mixtures in a single run on the Illumina MiSeq platform. We identified species from sequence data using comprehensive rbcL and ITS2 databases., Results: We successfully built a proof-of-concept quantitative pollination network using pollen metabarcoding., Discussion: Our work underscores that pollen metabarcoding is not quantitative but that quantitative networks can be constructed based on the number of interacting individuals. Due to the frequency of contamination and false positive reads, isolation and PCR negative controls should be used in every reaction. DNA metabarcoding has advantages in efficiency and resolution over microscopic identification of pollen, and we expect that it will have broad utility for future studies of plant-pollinator interactions.

Background: Members of the genus Bifidobacterium are abundant in the feces of babies during the exclusively-milk-diet period of life. Bifidobacterium longum is reported to be a common member of the infant fecal microbiota. However, B. longum is composed of three subspecies, two of which are represented in the bowel microbiota ( B. longum subsp. longum ; B. longum subsp. infantis ). B. longum subspecies are not differentiated in many studies, so that their prevalence and relative abundances are not accurately known. This may largely be due to difficulty in assigning subspecies identity using DNA sequences of 16S rRNA or tuf genes that are commonly used in bacterial taxonomy., Methods: We developed a qPCR method targeting the sialidase gene (subsp. infantis ) and sugar kinase gene (subsp. longum ) to differentiate the subspecies using specific primers and probes. Specificity of the primers/probes was tested by in silico, pangenomic search, and using DNA from standard cultures of bifidobacterial species. The utility of the method was further examined using DNA from feces that had been collected from infants inhabiting various geographical regions., Results: A pangenomic search of the NCBI genomic database showed that the PCR primers/probes targeted only the respective genes of the two subspecies. The primers/probes showed total specificity when tested against DNA extracted from the gold standard strains (type cultures) of bifidobacterial species detected in infant feces. Use of the qPCR method with DNA extracted from the feces of infants of different ages, delivery method and nutrition, showed that subsp. infantis was detectable (0-32.4% prevalence) in the feces of Australian ( n  = 90), South-East Asian ( n  = 24), and Chinese babies ( n  = 91), but in all cases at low abundance (<0.01-4.6%) compared to subsp. longum (0.1-33.7% abundance; 21.4-100% prevalence)., Discussion: Our qPCR method differentiates B. longum subspecies longum and infantis using characteristic functional genes. It can be used as an identification aid for isolates of bifidobacteria, as well as in determining prevalence and abundance of the subspecies in feces. The method should thus be useful in ecological studies of the infant gut microbiota during early life where an understanding of the ecology of bifidobacterial species may be important in developing interventions to promote infant health., Competing Interests: The authors declare there are no competing interests. Colin G. Prosser and Dianne Lowry are employees of Dairy Goat Cooperative (NZ) Ltd., Hamilton, New Zealand. Pheng Soon Lee and Khai Hong Wong are employees of Mead Johnson Nutrition, Marina Bay, Singapore.

The study of microbial ecology has undergone a paradigm shift in recent years, with rapid advances in molecular and bioinformatic tools allowing researchers with wide-ranging interests and backgrounds access to community profiling methods. While these advances have undoubtedly led to exciting new understanding of many systems, the array of protocols available and the idiosyncrasies of particular approaches can lead to confusion or, at worst, erroneous interpretation of results. Here, we describe a workflow from raw 16S rRNA gene amplicon sequence data, generated on an Illumina MiSeq instrument, to microbial community taxonomy profiles and basic diversity measures. The workflow can be adapted to input from major sequence platforms and uses freely available open source software that can be implemented on a range of operating systems.

Background: The Prevention of Overweight in Infancy (POI) study was a four-arm randomised controlled trial (RCT) in 802 families which assessed whether additional education and support on sleep (Sleep group); food, physical activity and breastfeeding (FAB group); or both (Combination group), reduced excessive weight gain from birth to 2 years of age, compared to usual care (Control group). The study had high uptake at recruitment (58 %) and retention at 2 years (86 %). Although the FAB intervention produced no significant effect on BMI or weight status at 2 years, the odds of obesity were halved in those who received the sleep intervention, despite no apparent effect on sleep duration. We speculate that enhanced self-regulatory behaviours may exist in the Sleep group. Self-regulation was not measured in our initial intervention, but extensive measures have been included in this follow-up study. Thus, the overall aim of the POI follow-up is to determine the extent to which augmented parental support and education on infant sleep, feeding, diet, and physical activity in the first 2 years of life reduces BMI at 3.5 and 5 years of age, and to determine the role of self-regulation in any such relationship., Methods/design: We will contact all 802 families and seek renewed consent to participate in the follow-up study. The families have received no POI intervention since the RCT finished at 2 years of age. Follow-up data collection will occur when the children are aged 3.5 and 5 years (i.e. up to 3 years post-intervention). Outcomes of interest include child anthropometry, body composition (DXA scan), diet (validated food frequency questionnaire), physical activity (accelerometry), sleep (questionnaire and accelerometry), and self-regulation (questionnaires and neuropsychological assessment)., Discussion: Our follow-up study has been designed primarily to enable us to determine whether the intriguing benefit of the sleep intervention suggested at 2 years of age remains as children approach school age. However, cohort analyses will also investigate how BMI, self-regulation, and sleep consolidation develop during the early years. This information will be valuable to researchers and policy makers progressing the field of early childhood obesity prevention., Trial Registration: ClinicalTrials.gov number NCT00892983 .

A feeding trial was performed with broilers receiving a diet of wheat-based feed (WBF), maize-based feed (MBF), or maize-based concentrates supplemented with 15% or 30% crimped kernel maize silage (CKMS-15 or CKMS-30, respectively). The aim of the study was to investigate the bacterial community compositions of the crop, gizzard, ileum, and cecum contents in relation to the feeding strategy and age (8, 15, 22, 25, 29, or 36 days). Among the four dietary treatments, bacterial diversity was analyzed for MBF and CKMS-30 by 454 pyrosequencing of the 16S rRNA gene. Since the diets had no significant influence on bacterial diversity, data were pooled for downstream analysis. With increasing age, a clear succession of bacterial communities and increased bacterial diversity were observed.Lactobacillaceae(belonging mainly to the genus Lactobacillus) represented most of the Firmicutesat all ages and in all segments of the gut except the cecum. The development of a "mature" microbiota in broilers occurred during the period from days 15 to 22. Striking increases in the relative abundances of Lactobacillus salivarius(17 to 36%) and clostridia (11 to 18%), and a concomitant decrease in the relative abundance of Lactobacillus reuteri, were found in the ileum after day 15. The concentration of deconjugated bile salts increased in association with the increased populations of L. salivarius and clostridia. Both L. salivarius and clostridia deconjugate bile acids, and increases in the abundances of these bacteria might be associated with growth reduction and gastrointestinal (GI) disorders occurring in the critical period of broiler life between days 20 and 30., (Copyright © 2016, American Society for Microbiology. All Rights Reserved.)