BMC Microbiology

IntroductionIndigenous groups across the world have been underrepresented in the ongoing efforts to map human microbiome diversity. This study investigates the gut microbiome and resistome diversities of three indigenous Malaysian (Orang Asli, OA) communities with different lifestyles and degree of urbanisation. We included an urban Malay group as a comparison. MethodsHealthy participants over 18 years old gave were recruited from three indigenous communities, namely Temuan (urban, n=12), Temiar (semi-urban, n=9) and Jahai (rural, hunter-gatherer, n=12), and Malay (urban, non-indigenous, n=9). Stools were collected on dry ice and sequenced using shotgun metagenomics. ResultsApproximately 65% of the reads were classified across the dataset. Microbial alpha diversity (Shannon) decreased but antibiotic resistance genes (ARGs) diversity increased as the degree of urbanisation in the groups increased (P < 0.05). The groups contributed to 13% of the variation observed in the microbial composition (PERMANOVA, P = 0.001), and 14.5% in resistome composition (PERMANOVA, P = 0.001). Romboutsia timonensis was significantly depleted in Jahai compared to Malay (FDR = 0.04). Shared ARGs conferring resistance to beta-lactams (cfxA), tetracyclines (tet), and macrolides (erm) were observed across all groups, irrespective of geographical location, ethnicity and lifestyle. ConclusionThis study provides initial characterisation of the gut microbiome and resistome of three underrepresented indigenous OA communities in Malaysia. Our findings offer foundational evidence of antimicrobial resistance patterns and underscores the need for broader inclusion of underrepresented populations in national surveillance and stewardship efforts.

Disruptions to gut microbial communities in early life can have lasting effects on metabolism, immune function, and resistance to infections. Antibiotics, including levofloxacin, can alter gut microbiota composition, potentially leading to long-term dysbiosis. The long-term impact of levofloxacin on the gut microbiota, especially in young children, remains poorly understood. This study investigated the effects of prolonged levofloxacin therapy over 6 months on gut microbiota in children and the stability of these changes after treatment cessation. This work used samples that were collected as part of a cluster-randomized, double-blind, placebo-controlled trial that investigated the efficacy and safety of levofloxacin for multidrug-resistant (MDR) tuberculosis (TB) preventive treatment in healthy children under the age of five years exposed to MDR-TB in the home. Levofloxacin or placebo were administered daily for 24 weeks following randomization, and stool samples were taken at baseline, and at 24- and 48-week follow-up visits. Bacterial 16S rRNA sequencing was performed on the Illumina MiSeq platform and the changes in bacterial gut microbiota composition and diversity were assessed at different time points and compared between the levofloxacin and placebo arms for different age group. Changes in the functional potential of the gut microbiome were predicted based on the observed taxonomy. Gut microbiota analysis was stratified into three age groups: 0 to <1 year, 1 to <2 years, and 2 to <5 years. The richness and evenness of microbiota were not significantly reduced following 24 weeks of levofloxacin therapy in any group. However, in infants (<1 year), the expected natural microbial diversification was significantly stunted at the end of treatment and remained impaired 24 weeks after treatment completion (48-week visit). Differential abundance testing supported this finding, revealing that a greater number of taxa were negatively impacted in the levofloxacin-treated group. Despite these shifts, beta-diversity analysis indicated no significant differences in overall microbial composition between baseline and follow-up visits after antibiotic treatment. This study showed that the natural diversification of the gut microbiota is stunted in infants and does not recover even at 24 weeks following cessation of treatment. The gut microbiota of 2 to <5-year-old children demonstrated more resilience to the influence of antibiotics.

The gut microbiota plays an essential role in the conversion of anthocyanins and (epi-)catechins into smaller phenolic acids, which are then absorbed into the blood stream. The phenolic composition of a commercial bilberry extract and grape seed extract was assessed, as well as a formulation extract containing a combination of both extracts. The extracts were subjected to an in vitro salivary, gastric and intestinal digestion environment, based on the INFOGEST Model. The solid fraction end-product of the combined extract from the in vitro digestion was further fermented with faecal samples from six healthy donors, for 72 hours, to assess phenolic acid metabolism, short-chain fatty acid formation and changes in microbial composition. During the in vitro digestion, flavonoid content in all three extract samples (bilberry, grape seed and the formulation extracts) decreased significantly. In the process of anthocyanin and flavonoid digestion, smaller phenolic acid compounds such as benzoic acid, cinnamic acid and mandelic acid increased in bilberry, grape seed and formulation extract samples. All faecal donors harboured unique microbiota compositions, however all faecal microbiota were able to fully convert catechin/epicatechin, the most prominent flavonoids in the formulation extract sample, into smaller phenolic metabolites (phenylacetic, phenylpropionic and benzoic acids) within 24 hours. Using 16S rRNA gene amplicon sequencing, Anaerobutyricum and Enterocloster spp. were correlated with catechin/epicatechin metabolism in the fermentation procedure, however, in single bacterial strain fermentation experiments with the formulation extract or catechin standard, these bacteria were not capable of metabolising flavonoids. HighlightsO_LIFaecal microbiota converted (epi-)catechin to phenolic metabolites within 24 h. C_LIO_LI(Epi-)catechin correlated negatively with Anaerobutyricum and Enterocloster spp. C_LIO_LIFaecal bacterial cultures did not show (epi-)catechin metabolism capacity. C_LI

Marine Aspergillus terreus has been explored as an important chitinase-producing fungal strain for-N-Acetyl-D-Glucosamine (GlcNAc) production from chitin substrates. Here, a purified extracellular 45 kDa chitinase of marine Aspergillus terreus (accession number JQ248076) was characterized in terms of substrate specificity. Conventionally, endochitinase cleaves the chitin substrate randomly to produce GlcNAc and its different multimers. So, it requires at least tetramer to characterize the endochitinases; whereas, exochitinases cleaves the chitin substrate from its reducing end and produce either GlcNAc or chitobiose (GlcNAc dimer). In present chitinase characterization, the HPLC followed by HRMS analyses revealed differential product formation from the chitin substrates of varied chain length. With swollen chitin polymer, the enzyme produced GlcNAc as a sole product; whereas with chitohexaose substrate, a mixture of GlcNAc and its oligomers were obtained. Although, mass spectrometry-based proteomics analysis identified the isolated chitinase as an endochitinase 1 precursor (Accession XP_001217186). However, the enzyme kinetic study exhibited higher catalytic efficiency for exochitinase specific dimeric chromogenic substrate in comparison to endochitinase specific tetrameric fluorogenic substrate, which indicated predominantly exochitinase behavior of the enzyme. Further, the in-silico study predicted the differential cleavage pattern of the enzyme, which could be due to different mode of substrate binding and processive mechanism through the tunnel shaped binding cleft of the enzyme. The dual mode of catalytic activity of the present chitinase was further confirmed by a molecular docking study with different lengths of substrates. With the unique dual mode of action, the chitinase of marine Aspergillus terreus offers a great promise towards its utility in the production of GlcNAc.

The role of gut microbiome in regulating vertebrate metabolism has been well-recognized. However, the effects of gut bacteria on growth have been less studied. Bacillus is a prevalent genus in the gut microbiota of human and animals. In this study, the effect of gut-derived Bacillus velezensis T23 on growth was investigated in zebrafish. B. velezensis T23 improved the growth of zebrafish and promoted IGF1 production in the liver and muscle, with a concomitant activation of the AKT/mTOR signaling pathway. The growth-promoting effect of B. velezensis T23 was not dependent on lipopeptides and polyketides. Cell wall peptidoglycan isolated from B. velezensis T23, as well as muramyl dipeptide (MDP), was sufficient to stimulate IGF1 signaling and growth. Further, the effect of B. velezensis T23 on growth and IGF1 production was abrogated in nod2-/- zebrafish, confirming that B. velezensis T23 promoted growth via MDP-NOD2 signaling. Gut transcriptomic analysis indicated that B. velezensis T23 promoted renewal and differentiation of intestinal cells, suggesting an involvement of gut-liver axis in the effect of B. velezensis T23 on systemic IGF1 production. Together, our results revealed an effect of gut Bacillus-derived muropeptide on growth via NOD2-IGF1 signaling, and provided novel mechanistic insights in the beneficial effect of Bacillus spp. as probiotics.

The genus Gardnerella comprises a group of fastidious bacteria associated with the female urogenital tract and has undergone extensive taxonomic revision in recent years. In this study, a bacterial strain, designated CCPDSM, was isolated from the female urinary microbiome and subjected to a comprehensive polyphasic taxonomic characterization. The 16S rRNA gene sequence confirmed that this strain is a member of the genus Gardnerella, and phylogenetic analyses based on cpn60 sequences, together with phylogenomic reconstruction placed strain CCPDSM within the genus Gardnerella as a distinct and well-supported lineage. Genome-based relatedness indices (ANIb, ANIm, TETRA and dDDH), demonstrated clear separation of CCPDSM from all validly published Gardnerella species. In contrast, comparisons with two publicly available closely related genomes yielded values above accepted species delineation thresholds, supporting their assignment to the same taxon. Phenotypic characterization, together with genome-based functional predictions, revealed a fastidious, fermentative metabolic profile that further differentiated CCPDSM from its closest relatives, while remaining consistent with traits characteristic of the genus. On the basis of combined phylogenetic, genomic and phenotypic evidence, strain CCPDSM is proposed as representing a novel species within the genus Gardnerella, for which the name Gardnerella fastidiominuta sp. nov. is proposed, with strain CCPDSM (=CECT 31324=CCP 588) designated as the type strain. This study expands the recognized diversity of Gardnerella and highlights the female urinary tract as a reservoir of previously uncharacterized species within this genus.

The human gut mycobiome, though less diverse than the bacterial microbiome, plays a significant role in health and disease. This study investigates the culturable fungal communities in fecal samples from hospitalized patients with diarrhea in Mexico City. We isolated and characterized 26 fungal strains using culture-dependent methods, including 20 yeasts and six filamentous fungi. The most prevalent organisms were Candida albicans, Rhodotorula mucilaginosa, Penicillium spp., and Paecilomyces spp. Fungal isolates were tested for their ability to withstand gut-like conditions, including temperature, pH, oxidative stress, and bile salts. Notably, Paecilomyces variotii demonstrated thermotolerance, surviving at 42{degrees}C, and exhibited competitive growth against other fungi. Co-occurrence analysis revealed associations between fungal isolates and bacterial pathogens such as Salmonella and Clostridioides difficile, suggesting potential interkingdom interactions. Cytotoxicity assays on Caco-2 cells showed that cell-free supernatants from Candida inospicua and filamentous fungi reduced cell viability by up to 40%. Finally, dereplication and untargeted metabolomic analyses of P. variotii, Penicillium crustosum, and Penicillium chrysogenum revealed the presence of several bioactive metabolites, including mycotoxins and antimicrobial compounds, highlighting their potential roles in gut ecology and disease. Overall, this study underscores the importance of the gut mycobiome in dysbiosis and its interactions with bacterial pathogens. The findings suggest that fungi, particularly thermotolerant species such as P. variotii, may contribute to gut dysbiosis and disease progression, particularly in immunocompromised patients. Further research is needed to elucidate the functional roles of these fungi and their metabolites in gut health and disease.

BackgroundAcinetobacter baumannii is a common bacterial pathogen in nosocomial infections. It has become one of the greatest threats to human health for its growing resistance to last resort antibiotics, which has led to a revival of phage therapy as a potential treatment. However, conventional methods for isolating A. baumannii-infecting phages are labor-intensive and often unsuccessful. MethodsOur approach involves a computational pipeline to identify temperate phages (prophages) integrated into A. baumannii genomes, followed by mitomycin C (MMC) induction of those strains to screen for active prophages. ResultsHere we show a prophage analysis for nearly 900 A. baumannii genomes. We observed MMC-triggered excision of nine prophages from eight A. baumannii strains by PCR and sequencing. Further we show four prophage form virions detectable by transmission electron microscopy, and two which can plaque on other A. baumannii isolates. ConclusionThis work demonstrates the utility and diversity of prophages for further development as therapeutics for antibiotic resistant A. baumannii.

IntroductionAcinetobacter is a highly diverse genus which includes a range of common pathogenic species such as A. baumannii, A. lwoffii etc. Acinetobacter species causes bacteremia, pneumonia, wound infections, Urinary tract infections in community as well as hospital settings. A. baumannii is one of the ESKAPE pathogen which makes it even more lethal as antibiotics cannot action on this. AimTo isolate Acinetobacter species from various clinical samples and to check their antimicrobial susceptibility pattern by VITEK {square} Compact in SGT Hospital, Gururam, Haryana. ResultsOut of total 6673 samples 595 were the positive isolates from which 35 were Acinetobacter isolates which were received from various wards of the hospital. Occurrence of Acinetobacter was seen more in males(57.14%) as compare to females (46.8%). A total of 31 strains were A. baumannii, 3 were A. lwoffi and 1 strain was of A. haemolyticus. Prominent presence of Acinetobacter was seen in Blood (48.57%) specimen along with pus(22.85%), endotracheal (22.85%), tracheal (2.85%) and eye swabs (2.85%). All the isolates were resistant to piperacillin/tazobactam (100%), ceftriazone (100%), amikacin (100%), gentamicin (100%) ciprofloxacin (91.42%), ceftazidime (91.42%), cefepime (88.57%), levofloxacin (88.57%) and trimethoprim/sulfamethoxazole (80%). Colistin susceptibility was observed in 88.57% of the isolates. ConclusionAcinetobacter is a common pathogen in hospital acquired as well as in community acquired infections as it is a opportunistic pathogen hence to identify the Acinetobacter species and to understand their antimicrobial resistance pattern this study was conducted.

The strain LEGE 10371, isolated from the surface of a marine sponge at Praia da Memoria, Portugal, was characterized as a new Thalassoporum species (Pseudanabaenales) using a polyphasic approach that included 16S rRNA gene phylogenetic analysis (Maximum Likelihood and Bayesian Inference), 16S-23S ITS secondary structures, p-distance calculations, MALDI-TOF MS profiling, and morphological analysis by optical and scanning electron microscopy, as well as ecological and biochemical characterization. Phylogenetically, LEGE 10371 clustered within the Thalassoporum clade, however distant from the other existent species of the genus. The p-distance analysis revealed low sequence identity with other Thalassoporum species, with a maximum value of 97.2% to Th. komareki. The MALDI-TOF profile displayed high-intensity peaks at approximately 3,000, 4,000, 6,000 and 8,000 m/z, representing strong candidates for diagnostic markers of the new species. Morphologically, the new species differ from the other species of the genus by presenting trichomes with more than 10 cells and lack of aerotopes. Biocompatibility of the fractions was evaluated in HaCaT keratinocytes, showing no cytotoxic effects at most tested concentrations. PCR screening targeting mcyE, sxtG, anaC, and cyrA confirmed the absence of the genetic potential for the production of major cyanotoxins. Chemical characterization revealed a pigment-rich profile dominated by chlorophyll-a and carotenoids, including {beta}-carotene, zeaxanthin, lutein, and mixoxanthophyll. Bioactivity assays showed superoxide anion radical scavenging by the aqueous fraction (IC2 {approx} 0.042-0.045 mg mL-{superscript 1}), strong nitric oxide radical scavenging by the acetonic fraction (IC = 0.045 mg mL-{superscript 1}), and lipoxygenase inhibition ([~]41%, for a fraction concentration of 0.25 mg mL-), suggesting a potential contribution of these fractions to modulate inflammation-related pathways. Additionally to this results, the polyphasic analysis permitted to confirm previous data that Pseudanabaena and Limnothrix represent the same generic entity. Both genera clustered together, presented high 16S rRNA gene identity (up to 99.9%) and share the same morphological and ecological features. Consequently, we formally proposed the synonimization of Limnothrix into Pseudanabaena.

BackgroundThe role of the gut microbiome and specific enteric bacteria in influencing the development of colorectal cancer (CRC) remains incompletely understood. Recently, it was shown that human CRC-derived strains of Clostridioides difficile were capable of inducing colonic tumorigenesis in a susceptible mouse model. We hypothesized that C. difficile contributes to the pathogenesis of human CRC and would be enriched in CRC tumors compared to paired normal tissues from the same individual. MethodsWe analyzed matched tumor/normal tissue samples from a cohort of 108 individuals presenting to a tertiary care hospital in Kuala Lumpur, Malaysia for CRC resection between 2013-2014. We assessed the prevalence of C. difficile detection using 16S rRNA amplicon sequencing with high-resolution taxonomic assignment as well as culture and PCR. ResultsWe found that detection of C. difficile was prevalent (38% of individuals), but of low abundance (tumor median relative abundance 0.01%, paired normal 0.006% [p=0.4]). Detection of C. difficile was more prevalent in individuals with biofilm-positive tumor tissues than biofilm-negative (i.e., 81% of C. difficile-positive individuals were biofilm-positive vs. 63% of C. difficile-negative individuals [p=0.04]). Additionally, in exploratory analyses, we describe patterns of taxonomic and inferred functional pathway differences between C. difficile-positive and C. difficile-negative groups. ConclusionThese findings suggest that C. difficile is frequently present in low abundance in the tumor microbiome with a potentially significant impact on community composition and function.

During weaning period, the intake of dietary fibres changes and increases dramatically. Given the considerable structural differences, we hypothesized that different fibres may vary in their function. The objective of the study was to explore the impact of specific dietary fibres (arabinoxylan, cellulose, pectin and xyloglucan) on the gut microbiome of children below 3 years. By using ex vivo fecal fermentation experiments, cellular models and cohort data analysis, we assessed how these fibres and their combinations influence infants gut microbiota composition, diversity, metabolite production and possible actions on the gut epithelial barrier function. We found that the fermentation with arabinoxylan, xyloglucan and pectin resulted in an increased production of short-chain fatty acids. These fibres also promoted the generation of metabolites with potential health benefits, such as indole-3-lactic acid. By combining the ex vivo fermentation and cellular co-culture experiments, arabinoxylan and xyloglucan were found to be able to maintain gut epithelial barrier integrity upon lipopolysaccharide challenge, and a blend of cellulose, pectin, and xyloglucan dampened different LPS induced cytokines. Moreover, pectin was found supporting the growth of a wide range of microbial species ex vitro and correlated positively with -diversity in young children in an observational cohort. Our findings provided insights into the potential benefits of diverse fibre intakes during early life. Further studies are needed to understand the mechanisms and the effectiveness of specific fibres on the gut microbiome development in young children.

We report the isolation and characterization of Streptomyces albidoflavus MD102, a strain that can be used as a microbial chassis for the heterologous production of secondary metabolites. This strain, closely related to the widely used S. albidoflavus J1074, exhibits a compact genome, exceptional genetic tractability, rapid growth, and susceptibility to antibiotics. Whole-genome sequencing revealed the metabolic capabilities of S. albidoflavus MD102, highlighting its versatility in supporting the production of diverse secondary metabolites. Employing CRISPR/Cas9-assisted genome editing tools, we created mutant strains with reduced genome and cleaner chromatographic background. In addition to the deletion of several biosynthetic gene clusters (BGC), we inserted the global regulator bldA gene and geranyl diphosphate synthase (gpps) genes and an additional {Phi}BT1-attB attachment site into the chromosome to enhance the strains capability in producing secondary metabolites. S. albidoflavus MD102 will be a new addition to the repertoire of existing Streptomyces chassis, contributing to the advancement of secondary metabolite discovery and synthetic microbiology. IMPORTANCEThe pursuit of a universal Streptomyces microbial chassis for the heterologous production of secondary metabolites has proven elusive, prompting a more pragmatic approach to develop a suite of Streptomyces chassis. The current study introduces Streptomyces albidoflavus MD102 as a promising heterologous chassis with rapid growth, susceptibility to common antibiotics, and genetic tractability. Its close phylogenetic relation with the widely used versatile S. albidoflavus J1074 chassis and the traits gained from strain improvement place the engineered S. albidoflavus MD102 strains as useful chassis for the heterologous production of microbial secondary metabolites. A notable feature of S. albidoflavus MD102 that distinguishes it from J1074 and other Streptomyces chassis is the presence of metabolic genes in its genome putatively responsible for the degradation of aromatic compounds. This characteristic may endow the strain with the capability to convert petrogenic polycyclic aromatic hydrocarbons (PAHs) and substituted aromatics into valuable secondary metabolites.

The human gut microbiome is a complex community that plays an important role in health, where perturbations can result in dysbiosis and disease. Bacteriophages (phages) can provide treatment for bacterial gastrointestinal disease, and commercial preparations such as the Intesti bacteriophage cocktail can be taken orally to target bacterial pathogens. However, interactions between these phages and the native gut microbiota are understudied. To investigate the impact of phage treatment, we used simulated gut models seeded with healthy donor microbiota from three individuals, sequenced the DNA, and analysed the bacterial and viral portion from samples obtained over time. Each donor had a unique bacterial composition which diverged with time. When comparing phage treated to control samples, we observed that Escherichia coli abundance accounted for the largest portion of bacterial community variance and was more associated with the controls. The lower abundance in phage treated samples may have resulted from the lytic action of phages from the cocktail. Additionally, our analyses of the viral portion revealed a phage bloom exclusive to phage treated samples. A highly abundant phage in this bloom was matched with the Intesti bacteriophage cocktail, showed similarity to Enterobacteria phage phi92, and provided evidence of productive infection within the model. While we did observe fluctuations in relative abundance of additional viral sequences in the presence of the phage cocktail, these changes were often transient. Furthermore, we detected only slight differences to typical members of the virome, and low numbers of active prophages. Our experiments suggest that the phage cocktail had minimal interruption to the native gut microbiota within the model. Impact statementBacteriophages are increasingly investigated and tested for their efficacy in treating infections and are a key component in fight against antimicrobial resistant bacterial infections. Because of their specificity, it has become almost a dogma to state that they do not alter the gut microbiome. We have now tested this in an in vitro study using a commercially available cocktail and real human faecal microbiota. We show minimal effects on the composition of the healthy microbiota with an individual-specific effect on Escherichia coli caused by productive infection of one phage in the cocktail.

Accurate identification of microbial species in complex populations and communities relies on the isolation of representative marker 16S, ITS, and 18S sequences through the use of DNA extraction, PCR, and sequencing. Aviti sequencing has brought an improvement in the read quality and depth of marker gene sequencing technology. Quality scores exceeding Q40 representing highly accurate sequencing allows researchers to ask more questions of their marker gene data. However, this improvement in quality and throughput also brings with it a surprising increase in diversity of amplicon sequencing variants (ASVs) making further analysis and comparisons to previous studies on Illumina platforms challenging. This increased diversity causes downstream processing issues, including an over-reporting of chimeric ASVs. Here we identify this problem and put forward straightforward solutions to retain counts and reduce technically introduced diversity, as well as tying chimeric read identification to minimum parent distance. Through the use of synthetic mock samples, we discovered that erroneous ASVs are systematically substitution errors introduced by the upstream PCR methods. This error can be reduced significantly bioinformatically through clustering of ASVs within 99% similarity. Further we highlight technically introduced variation as a result of variable region length, sample misassignment, and sample biomass. Collectively, these results improve the similarity of Aviti and Illumina datasets for better comparisons of microbial studies from different platforms.

Functional oligosaccharides, such as galacto-oligosaccharides (GOS), are valued for modulating gut microbiota and promoting health. This study aimed to produce a high-purity GOS ingredient (ALPINA GOS) via nanofiltration/diafiltration and assess its prebiotic efficacy using an in vitro fermentation model. GOS-rich syrup was obtained from transgalactosylation of lactose in concentrated whey permeate (30% lactose) and processed by diafiltration/nanofiltration to reduce monosaccharides and enrich oligosaccharide content. Carbohydrate composition was analyzed by HPAEC-PAD. Prebiotic activity was evaluated using a MicroColon model with fecal inocula from healthy adults, measuring pH, short-chain fatty acids (SCFAs), and microbiota shifts. Membrane processing increased oligosaccharides from 55.5% to 70.2% (dry basis) and reduced monosaccharides from 25.2% to 5.1%. ALPINA GOS induced a dose-dependent pH reduction and significantly enhanced lactate and acetate production, with stronger effects at 10 mg/mL. Microbiota profiling showed increased abundance of beneficial bacteria, especially Bifidobacterium, versus control. The findings confirm that GOS can be sustainably produced from whey permeate and exhibits potent prebiotic activity, supporting its application in functional foods aimed at gut health.

The carbohydrate-binding proteins (LecA and LecB) present within the extracellular polymeric substance (EPS) matrix of Pseudomonas aeruginosa play an essential role in maintaining the structural integrity of the biofilms through interactions with the EPS polysaccharides. Therefore, targeting the above lectins can turn out to be one of the promising strategies for disrupting P. aeruginosa biofilms. In the current study, we investigated the potency of antimicrobial peptides (AMPs) produced by the human gut microbiome in targeting LecA and LecB proteins of P. aeruginosa. Initially, a comprehensive in-silico pipeline was developed to identify and characterize putative antibacterial and antibiofilm AMPs produced by the human gut microbiome. These AMPs were then subsequently studied for their interaction with the lectin proteins through molecular docking, MM-GBSA, residue analysis, and molecular dynamics (MD) simulation. Among the studied peptides, amp21 and amp24 exhibited the strongest interactions with the lectin protein, occupying binding sites overlapping with key active-site residues previously reported for raffinose binding. amp6, amp21, and amp24 were selected for in vitro validation based on the MD simulation results of both LecA and LecB proteins. The above selected peptides exhibited minimal hemolytic activity across the tested concentration range. amp21 and amp6 were non-toxic to mammalian cells while amp24 demonstrated cytotoxicity only at higher doses. amp21 was found to be the most potent AMP and inhibited the growth of P. aeruginosa by [~]60% at 50 {micro}g mL{square}{superscript 1}. amp6 and amp21 resulted in a significant disruption of P. aeruginosa biofilms. Membrane permeabilization assays and scanning electron microscopy revealed that amp6, amp21, and amp24 damaged the bacterial cell membranes apart from compromising the integrity of the biofilm EPS matrix. Lastly, through in-silico studies, we designed ultrashort peptides (USPs) from the lead AMPs. The USPs (amp21.4 and amp24.2) exhibit superior antibiofilm efficacy compared to their parent AMPs. These findings establish human-gut microbiome-derived AMPs as promising candidates to target P. aeruginosa biofilms via inhibition of lectin proteins.

ObjectivesBiofilms are the dominant mode of bacterial life. The gut microbiota itself has characteristics of a biofilm that grows on the intestinal mucosa. C. difficile and VRE are commonly co-isolated from patients but biofilm formation has not been studied in a multi-species context. Here we study the interactions between C. difficile and VRE in surface adherent community. ResultsWe found that VRE inhibits C. difficile biofilm formation in dual-species culture in the presence of excess glucose. Robust dual-species biofilms were produced when the carbon source was changed to a non-fermentable sugar such as fucose and xylose. We observed a high level of vancomycin tolerance in C. difficile biofilms that was not affected by the presence of VRE. Finally we also found that a nutrient step-change is sufficient to induce dispersion of single and dual-species biofilms. ConclusionsVRE can inhibit the development of C. difficile biofilms in the presence of a fermentable carbon source. VRE does not appear to affect vancomycin tolerance or nutrient-induced dispersion of C. difficile biofilms. Highlights- VRE inhibits C. difficile biofilm formation in the presence of fermentable glucose. - Stable VRE - C. difficile biofilms are formed by managing the available carbon source. - VRE does not affect C. difficile vancomycin tolerance in this model. - A 10-fold increase in available nutrients is sufficient to induce biofilm dispersion in C. difficile and VRE.

Exopolysaccharides (EPS) produced by lactic acid bacteria (LAB) and other microorganisms have attracted considerable interest due to their structural diversity and physicochemical properties, which makes them valuable across various industrial applications. To achieve high cell densities and maximize EPS yields, microorganisms are typically cultivated in nutrient-rich media containing yeast extract. However, yeast extract may contain high molecular weight polysaccharides that are not metabolized by the bacteria. This can lead to an overestimation of EPS yields and contamination of the bacterial EPS, potentially resulting in misinterpretation of their structure and biological activity. In this study, we demonstrate the presence of high molecular weight -mannan and {beta}-glucan in yeast extract in EPS isolates using both ultrafiltration and the commonly used trichloroacetic acid/ethanol (TCA/EtOH) precipitation method. These polysaccharides were characterized by size-exclusion chromatography, high-performance anion-exchange chromatography, and nuclear magnetic resonance spectroscopy. Their abundances were estimated to range from 10 to 50 mg/L in MRS medium, depending on the supplier of the yeast extract. The main contaminant identified was yeast -mannan. By cultivating L. rhamnosus GG (ATCC 53103) and L. pentosus KW1 and isolating their respective EPS, we illustrate how these yeast extract contaminants affect the structural interpretation of the EPS and that the contaminants can be completely removed by ultrafiltration of the growth medium prior to bacterial cultivation. In conclusion, we emphasize the necessity of stringent controls in the production and purification of microbial EPS, with particular attention to the chemical purity of medium constituents.

The spread and rise of antimicrobial resistance poses a risk to public health due to limited effective treatment options. Alternative antimicrobials that are effective against gram-negative multi-drug resistant pathogens. The increasing rate of carbapenem resistance observed in Klebsiella pneumoniae, indicates the need for alternative antimicrobial options. Bacteriophages that target Klebsiella pneumoniae are promising alternative antimicrobial option, with successful treatments being reported. Here we characterized 30 lytic bacteriophages from various environmental sources and tested their effectiveness against nine clinically relevant carbapenem-resistant K. pneumoniae isolates. These phages were characterized through genomic sequencing, bioinformatic analysis, virulence in liquid medium, and host range on different mediums. Bioinformatic analysis revealed a diverse collection of phages that span 9 ICTV recognized families and 13 genera with genome sizes ranging from 39-349 kbp. The phages were able to inhibit bacterial growth, and no virulence or antibiotic resistance genes were detected within the phage genomes. Host range testing demonstrated phages with broad host range have varying infectivity when plated on different common growth mediums. This study includes candidate phages for further potential development as potential antimicrobial agents against CR-KP, and the complexity in understanding phage-host dynamics of non-capsule phages that target against K. pneumoniae.