Association between childhood obesity and gut microbiota: 16S rRNA gene sequencing-based cohort study.

BACKGROUND: This study aimed to identify characteristic gut genera in obese and normal-weight children (8-12 years old) using 16S rDNA sequencing. The research aimed to provide insights for mechanistic studies and prevention strategies for childhood obesity. Thirty normal-weight and thirty age- and sex-matched obese children were included. Questionnaires and body measurements were collected, and fecal samples underwent 16S rDNA sequencing. Significant differences in body mass index (BMI) and body-fat percentage were observed between the groups. Analysis of gut microbiota diversity revealed lower α-diversity in obese children. Di-fferences in gut microbiota composition were found between the two groups. Prevotella and Firmicutes were more abundant in the obese group, while Bacteroides and Sanguibacteroides were more prevalent in the control group. AIM: To identify the characteristic gut genera in obese and normal-weight children (8-12-year-old) using 16S rDNA sequencing, and provide a basis for subsequent mechanistic studies and prevention strategies for childhood obesity. METHODS: Thirty each normal-weight, 1:1 matched for age and sex, and obese children, with an obese status from 2020 to 2022, were included in the control and obese groups, respectively. Basic information was collected through questionnaires and body measurements were obtained from both obese and normal-weight children. Fecal samples were collected from both groups and subjected to 16S rDNA sequencing using an Illumina MiSeq sequencing platform for gut microbiota diversity analysis. RESULTS: Significant differences in BMI and body-fat percentage were observed between the two groups. The Ace and Chao1 indices were significantly lower in the obese group than those in the control group, whereas differences were not significant in the Shannon and Simpson indices. Kruskal-Wallis tests indicated significant differences in unweighted and weighted UniFrac distances between the gut microbiota of normal-weight and obese children (P < 0.01), suggesting substantial disparities in both the species and quantity of gut microbiota between the two groups. Prevotella, Firmicutes, Bacteroides, and Sanguibacteroides were more abundant in the obese and control groups, respectively. Heatmap results demonstrated significant differences in the gut microbiota composition between obese and normal-weight children. CONCLUSION: Obese children exhibited lower α-diversity in their gut microbiota than did the normal-weight children. Significant differences were observed in the composition of gut microbiota between obese and normal-weight children.

Comparison of bacterial species and clinical outcomes in patients with diabetic hand infection in tropical and nontropical regions.

Hand infection is a rare complication in patients with diabetes. Its clinical outcomes depend on the severity of hand infection caused by bacteria, but the difference in bacterial species in the regional disparity is unknown. The purpose of this study was to explore the influence of tropical and nontropical regions on bacterial species and clinical outcomes for diabetic hand. A systematic literature review was conducted using PubMed, EMBASE, Web of Science, and Google Scholar. Moreover, the bacterial species and clinical outcomes were analyzed with respect to multicenter wound care in China (nontropical regions). Both mixed bacteria (31.2% vs. 16.6%, p = 0.014) and fungi (7.5% vs. 0.8%, p = 0.017) in the nontropical region were significantly more prevalent than those in the tropical region. Staphylococcus and Streptococcus spp. were dominant in gram-positive bacteria, and Klebsiella, Escherichia coli, Proteus and Pseudomonas in gram-negative bacteria occupied the next majority in the two regions. The rate of surgical treatment in the patients was 31.2% in the nontropical region, which was significantly higher than the 11.4% in the tropical region (p = 0.001). Although the overall mortality was not significantly different, there was a tendency to be increased in tropical regions (6.3%) compared with nontropical regions (0.9%). However, amputation (32.9% vs. 31.3%, p = 0.762) and disability (6.3% vs. 12.2%, p = 0.138) were not significantly different between the two regions. Similar numbers of cases were reported, and the most common bacteria were similar in tropical and nontropical regions in patients with diabetic hand. There were more species of bacteria in the nontropical region, and their distribution was basically similar, except for fungi, which had differences between the two regions. The present study also showed that surgical treatment and mortality were inversely correlated because delays in debridement and surgery can deteriorate deep infections, eventually leading to amputation and even death.

Analysis of Salt Stress on Soil Microbial Community Composition and Its Correlation with Active Components in the Rhizosphere of Acanthopanax senticosus.

Salt stress can adversely affect plant seed germination, growth and development, and eventually lead to slow growth and even death of plants. The purpose of this study was to investigate the effects of different concentrations of NaCl and Na2SO4 stress on the physicochemical properties, enzyme activities, rhizosphere microbial community and seven active components (L-phenylalanine, Protocatechuic acid, Eleutheroside B, Chlorogenic acid, Caffeic acid, Eleutheroside E, Isofraxidin) of Acanthopanax senticosus rhizosphere soil. Statistical analysis was used to explore the correlation between the rhizosphere ecological factors of Acanthopanax senticosus and its active components. Compared with Acanthopanax senticosus under NaCl stress, Na2SO4 generally had a greater effect on Acanthopanax senticosus, which reduced the richness of fungi in rhizosphere soil and adversely affected the content of multiple active components. Pearson analysis showed that pH, organic matter, ammonium nitrogen, available phosphorus, available potassium, catalase and urease were significantly correlated with active components such as Caffeic acid and Isofraxidin. There were 11 known bacterial genera, 12 unknown bacterial genera, 9 known fungal genera and 1 unknown fungal genus significantly associated with the active ingredient. Salt stress had great changes in the physicochemical properties, enzyme activities and microorganisms of the rhizosphere soil of Acanthopanax senticosus. In conclusion, different types and concentrations of salts had different effects on Acanthopanax senticosus, and the active components of Acanthopanax senticosus were regulated by rhizosphere soil ecological factors.

Changing patterns of bacterial profile and antimicrobial resistance in high-risk patients during the COVID-19 pandemic at a tertiary oncology hospital.

The widespread evolution of phenotypic resistance in clinical isolates over the years, coupled with the COVID-19 pandemic onset, has exacerbated the global challenge of antimicrobial resistance. This study aimed to explore changes in bacterial infection patterns and antimicrobial resistance during the COVID-19 pandemic. This study involved the periods before and during COVID-19: the pre-pandemic and pandemic eras. The surveillance results of bacterial isolates causing infections in cancer patients at an Egyptian tertiary oncology hospital were retrieved. The Vitek®2 or Phoenix systems were utilized for species identification and susceptibility testing. Statistical analyses were performed comparing microbiological trends before and during the pandemic. Out of 2856 bacterial isolates, Gram-negative bacteria (GNB) predominated (69.7%), and Gram-positive bacteria (GPB) comprised 30.3% of isolates. No significant change was found in GNB prevalence during the pandemic (P = 0.159). Elevated rates of Klebsiella and Pseudomonas species were demonstrated during the pandemic, as was a decrease in E. coli and Acinetobacter species (P < 0.001, 0.018, < 0.001, and 0.046, respectively) in hematological patients. In surgical patients, Enterobacteriaceae significantly increased (P = 0.012), while non-fermenters significantly decreased (P = 0.007). GPB species from either hematological or surgical wards exhibited no notable changes during the pandemic. GNB resistance increased in hematological patients to carbapenems, amikacin, and tigecycline and decreased in surgical patients to amikacin and cefoxitin (P < 0.001, 0.010, < 0.001, < 0.001, and 0.016, respectively). The study highlights notable shifts in the microbial landscape during the COVID-19 pandemic, particularly in the prevalence and resistance patterns of GNB in hematological and surgical wards.

Arbuscular mycorrhizal fungi and Streptomyces: brothers in arms to shape the structure and function of the hyphosphere microbiome in the early stage of interaction.

BACKGROUND: Fungi and bacteria coexist in a wide variety of environments, and their interactions are now recognized as the norm in most agroecosystems. These microbial communities harbor keystone taxa, which facilitate connectivity between fungal and bacterial communities, influencing their composition and functions. The roots of most plants are associated with arbuscular mycorrhizal (AM) fungi, which develop dense networks of hyphae in the soil. The surface of these hyphae (called the hyphosphere) is the region where multiple interactions with microbial communities can occur, e.g., exchanging or responding to each other's metabolites. However, the presence and importance of keystone taxa in the AM fungal hyphosphere remain largely unknown. RESULTS: Here, we used in vitro and pot cultivation systems of AM fungi to investigate whether certain keystone bacteria were able to shape the microbial communities growing in the hyphosphere and potentially improved the fitness of the AM fungal host. Based on various AM fungi, soil leachates, and synthetic microbial communities, we found that under organic phosphorus (P) conditions, AM fungi could selectively recruit bacteria that enhanced their P nutrition and competed with less P-mobilizing bacteria. Specifically, we observed a privileged interaction between the isolate Streptomyces sp. D1 and AM fungi of the genus Rhizophagus, where (1) the carbon compounds exuded by the fungus were acquired by the bacterium which could mineralize organic P and (2) the in vitro culturable bacterial community residing on the surface of hyphae was in part regulated by Streptomyces sp. D1, primarily by inhibiting the bacteria with weak P-mineralizing ability, thereby enhancing AM fungi to acquire P. CONCLUSIONS: This work highlights the multi-functionality of the keystone bacteria Streptomyces sp. D1 in fungal-bacteria and bacterial-bacterial interactions at the hyphal surface of AM fungi. Video Abstract.

Viruses contribute to microbial diversification in the rumen ecosystem and are associated with certain animal production traits.

BACKGROUND: The rumen microbiome enables ruminants to digest otherwise indigestible feedstuffs, thereby facilitating the production of high-quality protein, albeit with suboptimal efficiency and producing methane. Despite extensive research delineating associations between the rumen microbiome and ruminant production traits, the functional roles of the pervasive and diverse rumen virome remain to be determined. RESULTS: Leveraging a recent comprehensive rumen virome database, this study analyzes virus-microbe linkages, at both species and strain levels, across 551 rumen metagenomes, elucidating patterns of microbial and viral diversity, co-occurrence, and virus-microbe interactions. Additionally, this study assesses the potential role of rumen viruses in microbial diversification by analyzing prophages found in rumen metagenome-assembled genomes. Employing CRISPR-Cas spacer-based matching and virus-microbe co-occurrence network analysis, this study suggests that the viruses in the rumen may regulate microbes at strain and community levels through both antagonistic and mutualistic interactions. Moreover, this study establishes that the rumen virome demonstrates responsiveness to dietary shifts and associations with key animal production traits, including feed efficiency, lactation performance, weight gain, and methane emissions. CONCLUSIONS: These findings provide a substantive framework for further investigations to unravel the functional roles of the virome in the rumen in shaping the microbiome and influencing overall animal production performance. Video Abstract.

Sediment DNA Records the Critical Transition of Bacterial Communities in the Arid Lake.

It is necessary to predict the critical transition of lake ecosystems due to their abrupt, non-linear effects on social-economic systems. Given the promising application of paleolimnological archives to tracking the historical changes of lake ecosystems, it is speculated that they can also record the lake's critical transition. We studied Lake Dali-Nor in the arid region of Inner Mongolia because of the profound shrinking the lake experienced between the 1300 s and the 1600 s. We reconstructed the succession of bacterial communities from a 140-cm-long sediment core at 4-cm intervals and detected the critical transition. Our results showed that the historical trajectory of bacterial communities from the 1200 s to the 2010s was divided into two alternative states: state1 from 1200 to 1300 s and state2 from 1400 to 2010s. Furthermore, in the late 1300 s, the appearance of a tipping point and critical slowing down implied the existence of a critical transition. By using a multi-decadal time series from the sedimentary core, with general Lotka-Volterra model simulations, local stability analysis found that bacterial communities were the most unstable as they approached the critical transition, suggesting that the collapse of stability triggers the community shift from an equilibrium state to another state. Furthermore, the most unstable community harbored the strongest antagonistic and mutualistic interactions, which may imply the detrimental role of interaction strength on community stability. Collectively, our study showed that sediment DNA can be used to detect the critical transition of lake ecosystems.

Interactions between host and gut microbiota in gestational diabetes mellitus and their impacts on offspring.

Gestational diabetes mellitus (GDM) is characterized by insulin resistance and low-grade inflammation, and most studies have demonstrated gut dysbiosis in GDM pregnancies. Overall, they were manifested as a reduction in microbiome diversity and richness, depleted short chain fatty acid (SCFA)-producing genera and a dominant of Gram-negative pathogens releasing lipopolysaccharide (LPS). The SCFAs functioned as energy substance or signaling molecules to interact with host locally and beyond the gut. LPS contributed to pathophysiology of diseases through activating Toll-like receptor 4 (TLR4) and involved in inflammatory responses. The gut microbiome dysbiosis was not only closely related with GDM, it was also vital to fetal health through vertical transmission. In this review, we summarized gut microbiota signature in GDM pregnancies of each trimester, and presented a brief introduction of microbiome derived SCFAs. We then discussed mechanisms of microbiome-host interactions in the physiopathology of GDM and associated metabolic disorders. Finally, we compared offspring microbiota composition from GDM with that from normal pregnancies, and described the possible mechanism.

Intestinal Barrier Impairment Induced by Gut Microbiome and Its Metabolites in School-Age Children with Zinc Deficiency.

Zinc deficiency affects the physical and intellectual development of school-age children, while studies on the effects on intestinal microbes and metabolites in school-age children have not been reported. School-age children were enrolled to conduct anthropometric measurements and serum zinc and serum inflammatory factors detection, and children were divided into a zinc deficiency group (ZD) and control group (CK) based on the results of serum zinc. Stool samples were collected to conduct metagenome, metabolome, and diversity analysis, and species composition analysis, functional annotation, and correlation analysis were conducted to further explore the function and composition of the gut flora and metabolites of children with zinc deficiency. Beta-diversity analysis revealed a significantly different gut microbial community composition between ZD and CK groups. For instance, the relative abundances of Phocaeicola vulgatus, Alistipes putredinis, Bacteroides uniformis, Phocaeicola sp000434735, and Coprococcus eutactus were more enriched in the ZD group, while probiotic bacteria Bifidobacterium kashiwanohense showed the reverse trend. The functional profile of intestinal flora was also under the influence of zinc deficiency, as reflected by higher levels of various glycoside hydrolases in the ZD group. In addition, saccharin, the pro-inflammatory metabolites, and taurocholic acid, the potential factor inducing intestinal leakage, were higher in the ZD group. In conclusion, zinc deficiency may disturb the gut microbiome community and metabolic function profile of school-age children, potentially affecting human health.

ARGNet: using deep neural networks for robust identification and classification of antibiotic resistance genes from sequences.

BACKGROUND: Emergence of antibiotic resistance in bacteria is an important threat to global health. Antibiotic resistance genes (ARGs) are some of the key components to define bacterial resistance and their spread in different environments. Identification of ARGs, particularly from high-throughput sequencing data of the specimens, is the state-of-the-art method for comprehensively monitoring their spread and evolution. Current computational methods to identify ARGs mainly rely on alignment-based sequence similarities with known ARGs. Such approaches are limited by choice of reference databases and may potentially miss novel ARGs. The similarity thresholds are usually simple and could not accommodate variations across different gene families and regions. It is also difficult to scale up when sequence data are increasing. RESULTS: In this study, we developed ARGNet, a deep neural network that incorporates an unsupervised learning autoencoder model to identify ARGs and a multiclass classification convolutional neural network to classify ARGs that do not depend on sequence alignment. This approach enables a more efficient discovery of both known and novel ARGs. ARGNet accepts both amino acid and nucleotide sequences of variable lengths, from partial (30-50 aa; 100-150 nt) sequences to full-length protein or genes, allowing its application in both target sequencing and metagenomic sequencing. Our performance evaluation showed that ARGNet outperformed other deep learning models including DeepARG and HMD-ARG in most of the application scenarios especially quasi-negative test and the analysis of prediction consistency with phylogenetic tree. ARGNet has a reduced inference runtime by up to 57% relative to DeepARG. CONCLUSIONS: ARGNet is flexible, efficient, and accurate at predicting a broad range of ARGs from the sequencing data. ARGNet is freely available at https://github.com/id-bioinfo/ARGNet , with an online service provided at https://ARGNet.hku.hk . Video Abstract.

Application value of metagenomic next-generation sequencing in hematological patients with high-risk febrile neutropenia.

Background: Metagenomic next-generation sequencing (mNGS) is a novel non-invasive and comprehensive technique for etiological diagnosis of infectious diseases. However, its practical significance has been seldom reported in the context of hematological patients with high-risk febrile neutropenia, a unique patient group characterized by neutropenia and compromised immune responses. Methods: This retrospective study evaluated the results of plasma cfDNA sequencing in 164 hematological patients with high-risk febrile neutropenia. We assessed the diagnostic efficacy and clinical impact of mNGS, comparing it with conventional microbiological tests. Results: mNGS identified 68 different pathogens in 111 patients, whereas conventional methods detected only 17 pathogen types in 36 patients. mNGS exhibited a significantly higher positive detection rate than conventional methods (67.7% vs. 22.0%, P < 0.001). This improvement was consistent across bacterial (30.5% vs. 9.1%), fungal (19.5% vs. 4.3%), and viral (37.2% vs. 9.1%) infections (P < 0.001 for all comparisons). The anti-infective treatment strategies were adjusted for 51.2% (84/164) of the patients based on the mNGS results. Conclusions: mNGS of plasma cfDNA offers substantial promise for the early detection of pathogens and the timely optimization of anti-infective therapies in hematological patients with high-risk febrile neutropenia.

Microbiome mapping in beef processing reveals safety-relevant variations in microbial diversity and genomic features.

The microbiome of surfaces along the beef processing chain represents a critical nexus where microbial ecosystems play a pivotal role in meat quality and safety of end products. This study offers a comprehensive analysis of the microbiome along beef processing using whole metagenomics with a particular focus on antimicrobial resistance and virulence-associated genes distribution. Our findings highlighted that microbial communities change dynamically in the different steps along beef processing chain, influenced by the specific conditions of each micro-environment. Brochothrix thermosphacta, Carnobacterium maltaromaticum, Pseudomonas fragi, Psychrobacter cryohalolentis and Psychrobacter immobilis were identified as the key species that characterize beef processing environments. Carcass samples and slaughterhouse surfaces exhibited a high abundance of antibiotic resistance genes (ARGs), mainly belonging to aminoglycosides, ß-lactams, amphenicols, sulfonamides and tetracyclines antibiotic classes, also localized on mobile elements, suggesting the possibility to be transmitted to human pathogens. We also evaluated how the initial microbial contamination of raw beef changes in response to storage conditions, showing different species prevailing according to the type of packaging employed. We identified several genes leading to the production of spoilage-associated compounds, and highlighted the different genomic potential selected by the storage conditions. Our results suggested that surfaces in beef processing environments represent a hotspot for beef contamination and evidenced that mapping the resident microbiome in these environments may help in reducing meat microbial contamination, increasing shelf-life, and finally contributing to food waste restraint.

Chronic consumption of orange juice modifies urinary excretion of flavanone gut-derived metabolites through gut microbiota modulation.

The metabolism and absorption of citrus flavanones are intrinsically linked to the gut microbiota, creating a bidirectional relationship where these compounds influence the microbiome, and in turn, the microbiota affects their metabolism. This study evaluates the effect of acute and chronic consumption of orange juice (OJ) on the urinary excretion of gut-derived flavanone metabolites and the gut microbiota. Health volunteers ingested 500 mL of OJ for 60 days in a single-arm human intervention study. Blood and feces were collected at baseline and after 60 days, with an additional 24-hour urine collection after a single dose on day 1 and day 63. LC-MS/MS analyzed urinary flavanone metabolites, while 16S rRNA sequencing characterized gut microbiota. Total urinary hesperetin conjugates excretion significantly decreased over 60 days, while gut-derived total phenolic acids, particularly three hydroxybenzoic acids, increased. Moreover, the heterogeneity of the total amount of flavanone conjugates, initially categorizing individuals into high-, medium- and low- urinary excretor profiles, shifted towards medium-excretor, except for five individuals who remained as low-excretors. This alteration was accompanied by a decrease in intestinal ß-glucosidase activity and a shift in the relative abundance of specific genera, such as decreases in Blautia, Eubacterium hallii, Anaerostipes, and Fusicatenibacter, among which, Blautia was associated with higher urinary flavanone conjugates excretion. Conversely, an increase in Prevotella was observed. In summary, chronic OJ consumption induced transient changes in gut microbiota and altered the metabolism of citrus flavanones, leading to distinct urinary excretion profiles of flavanone metabolites.

Revealing the intrinsic relationship between microbial communities and physicochemical properties during ripening of Xuanwei ham.

To clarify the relationship between microorganisms and physicochemical indicators of Xuanwei ham. Six ham samples for the first, second and third year were selected, respectively. The changes of physicochemical properties, the free fatty acids and microbial communities of Xuanwei ham were investigated by GC-MS and high-throughput sequencing technology. Results showed that scores of colour, overall acceptability, texture, taste and aroma were the highest in the third year sample. With increasing ripening time, moisture content, water activity (Aw), lightness (L*), springiness, and resilience decreased continuously, and yellowness (b*) was the highest in the second year sample. 31 free fatty acids were detected, and unsaturated fatty acids such as palmitoleic acid, oleic acid, and linoleic acid were the major fatty acids. The content of palmitoleic acid, oleic acid and eicosenoic acid increased significantly during processing. At the phylum level, the dominant bacteria were Proteobacteria and Firmicutes, and fungi were Ascomycota. At the genus level, the dominant bacteria were Staphylococcus and Psychrobacter, and fungi were Aspergillus. Correlation analysis showed that water content and Aw were closely related to microorganisms, and most unsaturated fatty acids were significantly correlated with microorganisms. These findings showed that microorganisms played an important role in the quality of Xuanwei ham, and provided a scientific basis for the quality control of Xuanwei ham.

In vitro batch fermentation demonstrates variations in the regulation of gut microbiota and metabolic functions by ß-glucans of differing structures.

The gut microbiota is widely acknowledged as a crucial factor in regulating host health. The structure of dietary fibers determines changes in the gut microbiota and metabolic differences resulting from their fermentation, which in turn affect gut microbe-related health effects. ß-Glucan (BG) is a widely accessible dietary fiber to humans, and its structural characteristics vary depending on the source. However, the interactions between different structural BGs and gut microbiota remain unclear. This study used an in vitro fermentation model to investigate the effects of BG on gut microbiota, and microbiomics and metabolomics techniques to explore the relationship between the structure of BG, bacterial communities, and metabolic profiles. The four sources of BG (barley, yeast, algae, and microbial fermentation) contained different types and proportions of glycosidic bonds, which differentially altered the bacterial community. The BG from algal sources, which contained only ß(1 â†’ 4) glycosidic bonds, was the least metabolized by the gut microbiota and caused limited metabolic changes. The other three BGs contain more diverse glycosidic bonds and can be degraded by bacteria from multiple genera, causing a wider range of metabolic changes. This work also suggested potential synergistic degradation relationships between gut bacteria based on BG. Overall, this study deepens the structural characterization-microbial-functional understanding of BGs and provides theoretical support for the development of gut microbiota-targeted foods.

Metagenomic sequencing and reconstruction of 82 microbial genomes from barley seed communities.

Barley (Hordeum vulgare) is essential to global food systems and the brewing industry. Its physiological traits and microbial communities determine malt quality. Although microbes influence barley from seed health to fermentation, there is a gap in metagenomic insights during seed storage. Crucially, elucidating the changes in microbial composition associated with barley seeds is imperative for understanding how these fluctuations can impact seed health and ultimately, influence both agricultural yield and quality of barley-derived products. Whole metagenomes were sequenced from eight barley seed samples obtained at different storage time points from harvest to nine months. After binning, 82 metagenome-assembled genomes (MAGs) belonging to 26 distinct bacterial genera were assembled, with a substantial proportion of potential novel species. Most of our MAG dataset (61%) showed over 90% genome completeness. This pioneering barley seed microbial genome retrieval provides insights into species diversity and structure, laying the groundwork for understanding barley seed microbiome interactions at the genome level.

Investigation of a novel biofilm model close to the original oral microbiome.

A more optimized culture medium used in vitro to mimic the bacterial composition of original oral flora as similar as possible remains difficult at present, and the goal of this study is to develop a novel oral biofilm medium to restore the original oral microbiome. Firstly, we conducted a systematic literature review by searching PubMed and summarized the current reported culture media in vitro. Seven culture media were found. We used mixed saliva as the origin of oral species to compare the effects of the above media in culturing oral multispecies biofilms. Results indicated that among the seven media brain heart infusion containing 1% sucrose (BHIs) medium, PG medium, artificial saliva (AS) medium, and SHI medium could obviously gain large oral biofilm in vitro. The nutrients contained in different culture media may be suitable for the growth of different oral bacteria; therefore, we optimized several novel media accordingly. Notably, results of crystal violet staining showed that the biofilm cultured in our modified artificial saliva (MAS) medium had the highest amount of biofilm biomass. 16S rRNA gene sequencing showed that the operational taxonomic units (OTUs) and Shannon index of biofilm cultured in MAS medium were also the highest among all the tested media. More importantly, the 16S rRNA gene sequencing analysis indicated that the biofilm cultured in MAS medium was closer to the original saliva species. Besides, biofilm cultured by MAS was denser and produced more exopolysaccharides. MAS supported stable biofilm formation on different substrata. In conclusion, this study demonstrated a novel MAS medium that could culture oral biofilm in vitro closer to the original oral microbiome, showing a good application prospect. KEY POINTS: • We compare the effects of different media in culturing oral biofilms • A novel modified artificial saliva (MAS) medium was obtained in our study • The MAS medium could culture biofilm that was closer to oral microbiome.

Co-localization of antibiotic resistance genes is widespread in the infant gut microbiome and associates with an immature gut microbial composition.

BACKGROUND: In environmental bacteria, the selective advantage of antibiotic resistance genes (ARGs) can be increased through co-localization with genes such as other ARGs, biocide resistance genes, metal resistance genes, and virulence genes (VGs). The gut microbiome of infants has been shown to contain numerous ARGs, however, co-localization related to ARGs is unknown during early life despite frequent exposures to biocides and metals from an early age. RESULTS: We conducted a comprehensive analysis of genetic co-localization of resistance genes in a cohort of 662 Danish children and examined the association between such co-localization and environmental factors as well as gut microbial maturation. Our study showed that co-localization of ARGs with other resistance and virulence genes is common in the early gut microbiome and is associated with gut bacteria that are indicative of low maturity. Statistical models showed that co-localization occurred mainly in the phylum Proteobacteria independent of high ARG content and contig length. We evaluated the stochasticity of co-localization occurrence using enrichment scores. The most common forms of co-localization involved tetracycline and fluoroquinolone resistance genes, and, on plasmids, co-localization predominantly occurred in the form of class 1 integrons. Antibiotic use caused a short-term increase in mobile ARGs, while non-mobile ARGs showed no significant change. Finally, we found that a high abundance of VGs was associated with low gut microbial maturity and that VGs showed even higher potential for mobility than ARGs. CONCLUSIONS: We found that the phenomenon of co-localization between ARGs and other resistance and VGs was prevalent in the gut at the beginning of life. It reveals the diversity that sustains antibiotic resistance and therefore indirectly emphasizes the need to apply caution in the use of antimicrobial agents in clinical practice, animal husbandry, and daily life to mitigate the escalation of resistance. Video Abstract.