I am better than I look: genome based safety assessment of the probiotic Lactiplantibacillus plantarum IS-10506.

BACKGROUND: Safety of probiotic strains that are used in human and animal trials is a prerequisite. Genome based safety assessment of probiotics has gained popularity due its cost efficiency and speed, and even became a part of national regulation on foods containing probiotics in Indonesia. However, reliability of the safety assessment based only on a full genome sequence is not clear. Here, for the first time, we sequenced, assembled, and analysed the genome of the probiotic strain Lactiplantibacillus plantarum IS-10506, that was isolated from dadih, a traditional fermented buffalo milk. The strain has already been used as a probiotic for more than a decade, and in several clinical trials proven to be completely safe. METHODS: The genome of the probiotic strain L. plantarum IS-10506 was sequenced using Nanopore sequencing technology, assembled, annotated and screened for potential harmful (PH) and beneficial genomic features. The presence of the PH features was assessed from general annotation, as well as with the use of specialised tools. In addition, PH regions in the genome were compared to all other probiotic and non-probiotic L. plantarum strains available in the NCBI RefSeq database. RESULTS: For the first time, a high-quality complete genome of L. plantarum IS-10506 was obtained, and an extensive search for PH and a beneficial signature was performed. We discovered a number of PH features within the genome of L. plantarum IS-10506 based on the general annotation, including various antibiotic resistant genes (AMR); however, with a few exceptions, bioinformatics tools specifically developed for AMR detection did not confirm their presence. We further demonstrated the presence of the detected PH genes across multiple L. plantarum strains, including probiotics, and overall high genetic similarities between strains. CONCLUSION: The genome of L. plantarum IS-10506 is predicted to have several PH features. However, the strain has been utilized as a probiotic for over a decade in several clinical trials without any adverse effects, even in immunocompromised children with HIV infection and undernourished children. This implies the presence of PH feature signatures within the probiotic genome does not necessarily indicate their manifestation during administration. Importantly, specialized tools for the search of PH features were found more robust and should be preferred over manual searches in a general annotation.

Promoting vegetable acceptance in toddlers using a contingency management program: A cluster randomised trial.

Children learn to accept novel foods by repeated exposure to these foods. In the current study, we investigated in toddlers whether a contingency management program (The Vegetable Box), comprising repeated vegetable taste exposure with contingent non-food rewards, is particularly potent at increasing recognition of and willingness to try vegetables. A total of 598 children (1-4 years old) recruited at 26 different day-care centres in the Netherlands took part. The day-care centres were randomly assigned to one of three conditions ('exposure/reward', 'exposure/no reward', or 'no exposure/no reward'). At the start and directly after the 3-month intervention period, all children were asked to identify various vegetables (recognition test; max score = 14) and whether they would like to taste and consume 1 or 2 bite-size pieces of tomato, cucumber, carrot, bell pepper, radish, and cauliflower (willingness to try test). Data were analysed with linear mixed-effects regression analyses (for recognition and willingness to try separately) with condition and time as independent variables, and adjusting for day-care centre clustering. Vegetable recognition significantly increased in both the 'exposure/reward' and the 'exposure/no reward' group, relative to the 'no exposure/no reward' control group. The willingness to try vegetables only increased significantly in the 'exposure/reward' group. Offering vegetables to children at day-care centres significantly increased toddlers' ability to identify various vegetables, but rewards contingent upon tasting vegetables appear particularly effective to also increase children's willingness to try (i.e., taste and consume) different vegetables. This result corroborates and strengthens previous findings demonstrating the efficacy of similar reward-based programs.

Do apes smell like humans? The role of skin bacteria and volatiles of primates in mosquito host selection.

Anthropophilic mosquitoes are effective vectors of human disease because of their biting preferences. To find their host, these mosquitoes are guided by human odours, primarily produced by human skin bacteria. By analysing the skin bacterial and skin volatile profiles of humans, bonobos, chimpanzees, gorillas, lemurs and cows, we investigated whether primates that are more closely related to humans have a skin bacterial community and odour profile that is similar to that of humans. We then investigated whether this affected discrimination between humans and closely related primates by anthropophilic and zoophilic mosquitoes that search for hosts. Humans had a lower skin bacterial diversity than the other animals and their skin bacterial composition was more similar to that in other primates than it was to the skin bacteria of cows. Like the skin bacterial profiles, the volatile profiles of the animal groups were clearly different from each other. The volatile profiles of cows and lemurs were more closely related to the human profiles than expected. Human volatiles were indeed preferred above cow volatiles by anthropophilic mosquitoes and no preference was observed when tested against non-human primate odour, except for bonobo volatiles, which were preferred over human volatiles. Unravelling the differences between mosquito hosts and their effect on host selection is important for a better understanding of cross-species transmission of vector-borne diseases.

Romboutsia hominis sp. nov., the first human gut-derived representative of the genus Romboutsia, isolated from ileostoma effluent.

A Gram-stain-positive, motile, rod-shaped, obligately anaerobic bacterium, designated FRIFIT, was isolated from human ileostoma effluent and characterized. On the basis of 16S rRNA gene sequence similarity, strain FRIFIT was most closely related to the species Romboutsia ilealis CRIBT (97.7 %), Romboutsia lituseburensis DSM 797T (97.6 %) and Romboutsia sedimentorum LAM201T (96.6 %). The level of DNA-DNA relatedness between strain FRIFIT and R. ilealis CRIBT was 13.9±3.3 % based on DNA-DNA hybridization. Whole genome sequence-based average nucleotide identity between strain FRIFIT and closely related Romboutsia strains ranged from 78.4-79.1 %. The genomic DNA G+C content of strain FRIFIT was 27.8 mol%. The major cellular fatty acids of strain FRIFIT were saturated and unsaturated straight-chain C12-C19 fatty acids as well as cyclopropane fatty acids, with C16 : 0 being the predominant fatty acid. The polar lipid profile comprised five phospholipids and six glycolipids. These results, together with differences in phenotypic features, support the proposal that strain FRIFIT represents a novel species within the genus Romboutsia, for which the name Romboutsiahominis sp. nov. is proposed. The type strain is FRIFIT (=DSM 28814T=KCTC 15553T).

Detection of coronaviruses in insectivorous bats of Fore-Caucasus, 2021.

Coronaviruses (CoVs) pose a huge threat to public health as emerging viruses. Bat-borne CoVs are especially unpredictable in their evolution due to some unique features of bat physiology boosting the rate of mutations in CoVs, which is already high by itself compared to other viruses. Among bats, a meta-analysis of overall CoVs epizootiology identified a nucleic acid observed prevalence of 9.8% (95% CI 8.7-10.9%). The main objectives of our study were to conduct a qPCR screening of CoVs' prevalence in the insectivorous bat population of Fore-Caucasus and perform their characterization based on the metagenomic NGS of samples with detected CoV RNA. According to the qPCR screening, CoV RNA was detected in 5 samples, resulting in a 3.33% (95% CI 1.1-7.6%) prevalence of CoVs in bats from these studied locations. BetaCoVs reads were identified in raw metagenomic NGS data, however, detailed characterization was not possible due to relatively low RNA concentration in samples. Our results correspond to other studies, although a lower prevalence in qPCR studies was observed compared to other regions and countries. Further studies should require deeper metagenomic NGS investigation, as a supplementary method, which will allow detailed CoV characterization.

Classification and prediction of Mycobacterium Avium subsp. Paratuberculosis (MAP) shedding severity in cattle based on young stock heifer faecal microbiota composition using random forest algorithms.

BACKGROUND: Bovine paratuberculosis is a devastating infectious disease caused by Mycobacterium avium subsp. paratuberculosis (MAP). The development of the paratuberculosis in cattle can take up to a few years and vastly differs between individuals in severity of the clinical symptoms and shedding of the pathogen. Timely identification of high shedding animals is essential for paratuberculosis control and minimization of economic losses. Widely used methods for detection and quantification of MAP, such as culturing and PCR based techniques rely on direct presence of the pathogen in a sample and have little to no predictive value concerning the disease development. In the current study, we investigated the possibility of predicting MAP shedding severity in cattle based on the faecal microbiota composition. Twenty calves were experimentally infected with MAP and faecal samples were collected biweekly up to four years of age. All collected samples were subjected to culturing on selective media to obtain data about shedding severity. Faecal microbiota was profiled in a subset of samples (n = 264). Using faecal microbiota composition and shedding intensity data a random forest classifier was built for prediction of the shedding status of the individual animals. RESULTS: The results indicate that machine learning approaches applied to microbial composition can be used to classify cows into groups by severity of MAP shedding. The classification accuracy correlates with the age of the animals and use of samples from older individuals resulted in a higher classification precision. The classification model based on samples from the first 12 months of life showed an AUC between 0.78 and 0.79 (95% CI), while the model based on samples from animals older than 24 months showed an AUC between 0.91 and 0.92 (95% CI). Prediction for samples from animals between 12 and 24 month of age showed intermediate accuracy [AUC between 0.86 and 0.87 (95% CI)]. In addition, the results indicate that a limited number of microbial taxa were important for classification and could be considered as biomarkers. CONCLUSIONS: The study provides evidence for the link between microbiota composition and severity of MAP infection and shedding, as well as lays ground for the development of predictive diagnostic tools based on the faecal microbiota composition.

Genetic and phenotypic analysis of the pathogenic potential of two novel Chlamydia gallinacea strains compared to Chlamydia psittaci.

Chlamydia gallinacea is an obligate intracellular bacterium that has recently been added to the family of Chlamydiaceae. C. gallinacea is genetically diverse, widespread in poultry and a suspected cause of pneumonia in slaughterhouse workers. In poultry, C. gallinacea infections appear asymptomatic, but studies about the pathogenic potential are limited. In this study two novel sequence types of C. gallinacea were isolated from apparently healthy chickens. Both isolates (NL_G47 and NL_F725) were closely related to each other and have at least 99.5% DNA sequence identity to C. gallinacea Type strain 08-1274/3. To gain further insight into the pathogenic potential, infection experiments in embryonated chicken eggs and comparative genomics with Chlamydia psittaci were performed. C. psittaci is a ubiquitous zoonotic pathogen of birds and mammals, and infection in poultry can result in severe systemic illness. In experiments with embryonated chicken eggs, C. gallinacea induced mortality was observed, potentially strain dependent, but lower compared to C. psittaci induced mortality. Comparative analyses confirmed all currently available C. gallinacea genomes possess the hallmark genes coding for known and potential virulence factors as found in C. psittaci albeit to a reduced number of orthologues or paralogs. The presence of potential virulence factors and the observed mortality in embryonated eggs indicates C. gallinacea should rather be considered as an opportunistic pathogen than an innocuous commensal.

Metagenomic- and Cultivation-Based Exploration of Anaerobic Chloroform Biotransformation in Hypersaline Sediments as Natural Source of Chloromethanes.

Chloroform (CF) is an environmental contaminant that can be naturally formed in various environments ranging from forest soils to salt lakes. Here we investigated CF removal potential in sediments obtained from hypersaline lakes in Western Australia. Reductive dechlorination of CF to dichloromethane (DCM) was observed in enrichment cultures derived from sediments of Lake Strawbridge, which has been reported as a natural source of CF. No CF removal was observed in abiotic control cultures without artificial electron donors, indicating biotic CF dechlorination in the enrichment cultures. Increasing vitamin B12 concentration from 0.04 to 4 µM in enrichment cultures enhanced CF removal and reduced DCM formation. In cultures amended with 4 µM vitamin B12 and 13C labelled CF, formation of 13CO2 was detected. Known organohalide-respiring bacteria and reductive dehalogenase genes were neither detected using quantitative PCR nor metagenomic analysis of the enrichment cultures. Rather, members of the order Clostridiales, known to co-metabolically transform CF to DCM and CO2, were detected. Accordingly, metagenome-assembled genomes of Clostridiales encoded enzymatic repertoires for the Wood-Ljungdahl pathway and cobalamin biosynthesis, which are known to be involved in fortuitous and nonspecific CF transformation. This study indicates that hypersaline lake microbiomes may act as a filter to reduce CF emission to the atmosphere.

Occupancy strongly influences faecal microbial composition of wild lemurs.

The microbiota of the mammalian gut is a complex ecosystem, the composition of which is greatly influenced by host genetics and environmental factors. In this study, we aim to investigate the influence of occupancy (a geographical area of habitation), species, age and sex on intestinal microbiota composition of the three lemur species: Eulemur fulvus, E. rubriventer and E. rufifrons. Faecal samples were collected from a total of 138 wild lemurs across Madagascar, and microbial composition was determined using next-generation sequencing of PCR-amplified 16S rRNA gene fragments. Consistent with reports from other primate species, the predominant phyla were Firmicutes (43 ± 6.4% [s.d.]) and Bacteroidetes (30.3 ± 5.3%). The microbial composition was strongly associated with occupancy in the E. fulvus population, with up to 19.9% of the total variation in microbial composition being explained by this factor. In turn, geographical differences observed in faecal microbiota of sympatric lemur species were less pronounced, as was the impact of the factors sex and age. Our findings showed that among the studied factors occupancy had the strongest influence on intestinal microbiota of congeneric lemur species. This suggests adaptation of microbiota to differences in forest composition, climate variations and correspondingly available diet in different geographical locations of Madagascar.