Bacterial Culture Supernatants Research Articles

Inhibition of Aflatoxin Production in Aspergillus flavus by a Klebsiella sp. and Its Metabolite Cyclo(l-Ala-Gly).

During an experiment where we were cultivating aflatoxigenic Aspergillus flavus on peanuts, we accidentally discovered that a bacterium adhering to the peanut strongly inhibited aflatoxin (AF) production by A. flavus. The bacterium, isolated and identified as Klebsiella aerogenes, was found to produce an AF production inhibitor. Cyclo(l-Ala-Gly), isolated from the bacterial culture supernatant, was the main active component. The aflatoxin production-inhibitory activity of cyclo(l-Ala-Gly) has not been reported. Cyclo(l-Ala-Gly) inhibited AF production in A. flavus without affecting its fungal growth in a liquid medium with stronger potency than cyclo(l-Ala-l-Pro). Cyclo(l-Ala-Gly) has the strongest AF production-inhibitory activity among known AF production-inhibitory diketopiperazines. Related compounds in which the methyl moiety in cyclo(l-Ala-Gly) is replaced by ethyl, propyl, or isopropyl have shown much stronger activity than cyclo(l-Ala-Gly). Cyclo(l-Ala-Gly) did not inhibit recombinant glutathione-S-transferase (GST) in A. flavus, unlike (l-Ala-l-Pro), which showed that the inhibition of GST was not responsible for the AF production-inhibition of cyclo(l-Ala-Gly). When A. flavus was cultured on peanuts dipped for a short period of time in a dilution series bacterial culture broth, AF production in the peanuts was strongly inhibited, even at a 1 × 104-fold dilution. This strong inhibitory activity suggests that the bacterium is a candidate for an effective biocontrol agent for AF control.

Bioassay-Driven, Fractionation-Empowered, Focused Metabolomics for Discovering Bacterial Activators of Aryl Hydrocarbon Receptor.

Aryl hydrocarbon receptor (AhR) is a transcription factor that regulates gene expression upon ligand activation, enabling microbiota-dependent induction, training, and function of the host immune system. A spectrum of metabolites, encompassing indole and tryptophan derivatives, have been recognized as activators. This work introduces an integrated, mass spectrometry-centric workflow that employs a bioassay-guided, fractionation-based methodology for the identification of AhR activators derived from human bacterial isolates. By leveraging the workflow efficiency, the complexities inherent in metabolomics profiling are significantly reduced, paving the way for an in-depth and focused mass spectrometry analysis of bioactive fractions isolated from bacterial culture supernatants. Validation of AhR activator candidates used multiple criteria─MS/MS of the synthetic reference compound, bioassay of AhR activity, and elution time confirmation using a C-13 isotopic reference─and was demonstrated for N-formylkynurenine (NFK). The workflow reported provides a roadmap update for improved efficiency of identifying bioactive metabolites using mass spectrometry-based metabolomics. Mass spectrometry datasets are accessible at the National Metabolomics Data Repository (PR001479, Project DOI: 10.21228/M8JM7Q).

P135 Mixed probiotics containing Bifidobacterium longum and its metabolites inhibit the formation of NLRP3 inflammasomes by suppressing GBP5 expression in macrophages in ulcerative colitis

Abstract Background Ulcerative Colitis (UC) is a non-specific chronic inflammation of the intestine. As an intestinal disease with a gradually increasing incidence in recent years and increasingly valued by medical staff, UC has a close relationship with intestinal flora. At present, domestic and foreign guidelines point out that probiotics can be used to induce remission in ulcerative colitis, but there are no clear instructions on the selection of probiotics, the use of dosage forms, and the dosage of probiotics, and there is a lack of relevant clinical and basic research. This study aims to explore the distinct therapeutic effects of mixed probiotics and individual strains of probiotics (Bifidobacterium longum), as well as elucidate the underlying mechanisms responsible for these variations. Methods A total of 90 patients with ulcerative colitis (UC) were enrolled in our center, who were subsequently divided into three groups: control group, single strain probiotic group (Bifidobacterium longum), and mixed probiotic group. After three months of adjuvant probiotic therapy, serum and fecal samples were collected for comparative analysis of therapeutic effects using ELISA and fecal 16s rDNA and metabolome sequence analysis. Mixed probiotic and Bifidobacterium longum were cultured in BHIs in an anaerobic chamber. The supernatant obtained after bacterial cultivation was subjected to metabolome sequence analysis. Human colonic organoids and Caco-2 cells were co-cultured with mixed probiotic and Bifidobacterium longum subsequent to inflammation induction, and cytotoxicity, permeability, and inflammatory cytokine release were assessed. THP1 cells were also to co-cultured with mixed probiotic and Bifidobacterium longum, and expression of virulence genes was analyzed using RT-qPCR, transcriptome sequencing, and western blot. GBP5 gene manipulated cell and mouse model were used to evaluate inflammation and apoptosis. Results The utilization of mixed probiotic therapy in patients with UC demonstrated significantly superior therapeutic efficacy compared to those receiving a single strain probiotic (Bifidobacterium longum). Differential metabolite 8-Deoxylactucinwas identified among various groups through the metabolome sequence analysis of clinical patients’ feces, bacterial culture supernatant, and mouse feces. Through in vitro and in vivo experiments, it has been demonstrated that 8-Deoxylactucin exhibits inhibitory effects on the expression of GBP5 in THP-1 cells and ameliorates inflammation. Conclusion Collectively, these findings indicate that mixed probiotics and its metabolites suppress GBP5 expression in macrophages in ulcerative colitis.

Possible roles of short-chain fatty acids produced by oral bacteria in the development of alveolar osteitis.

Alveolar osteitis (dry sockets) is a painful condition characterized by a limited immune response. It is typically caused by the removal of blood clots from extracted tooth sockets, which leads to the fermentation of trapped food remnants by oral bacteria in the cavities, producing high concentrations of short-chain fatty acids (SCFAs). This study examined the effects of SCFAs on immunity and bone metabolism. Mouse macrophage Raw264.7 cells were treated with oral bacteria supernatants or SCFA mixtures, and inducible nitric oxide synthase (iNOS) levels were determined by western blot. The same cells were treated with SCFA mixtures in the presence of receptor activator of nuclear factor-kappa B ligand (RANKL), and osteoclast-like cells were counted. MC3T3-E1 cells were treated with SCFA mixtures and stained with alizarin red S. Raw264.7 cells treated with oral bacterial culture supernatants of Porphyromonas gingivalis and Fusobacterium nucleatum inhibited lipopolysaccharide (LPS)-induced iNOS production, likely due to SCFA content. SCFA mixtures mimicking these supernatants inhibited the number of RANKL-induced tartrate-resistant acid phosphatase (TRAP)-positive cells and MC3T3-E1 cell mineralization. These data suggest that SCFAs produced by P. gingivalis and F. nucleatum may reduce the inflammatory response and mildly induce mineralization of the alveolar walls. These results may contribute to the understanding of alveolar osteitis.

Differential proteome profiling of bacterial culture supernatants reveals candidates for the induction of oral immune priming in the red flour beetle.

Most organisms are host to symbionts and pathogens, which led to the evolution of immune strategies to prevent harm. Whilst the immune defences of vertebrates are classically divided into innate and adaptive, insects lack specialized cells involved in adaptive immunity, but have been shown to exhibit immune priming: the enhanced survival upon infection after a first exposure to the same pathogen or pathogen-derived components. An important piece of the puzzle are the pathogen-associated molecules that induce these immune priming responses. Here, we make use of the model system consisting of the red flour beetle (Tribolium castaneum) and its bacterial pathogen Bacillus thuringiensis, to compare the proteomes of culture supernatants of two closely related B. thuringiensis strains that either induce priming via the oral route, or not. Among the proteins that might be immunostimulatory to T. castaneum, we identify the Cry3Aa toxin, an important plasmid-encoded virulence factor of B. thuringiensis. In further priming-infection assays we test the relevance of Cry-carrying plasmids for immune priming. Our findings provide valuable insights for future studies to perform experiments on the mechanisms and evolution of immune priming.

Type I Lipoteichoic Acid (LTA) Detection by Western Blot.

Type I lipoteichoic acid (LTA) is a glycerolphosphate polymer found in the cell envelope of diverse Gram-positive bacteria including Staphylococcus aureus, Bacillus subtilis, and Listeria monocytogenes. The polymer is linked by a lipid anchor to the outer leaflet of the bacterial membrane and in some bacteria can also be shed and detected in the culture supernatant. Here, we describe a simple and rapid western blot method for the detection of Type I LTA in bacterial cell extracts and culture supernatant fractions using a polyglycerolphosphatespecific monoclonal LTA antibody.

Staphylococcus aureus-derived virulent phenol-soluble modulin α triggers alarmin release to drive IL-36-dependent corneal inflammation

Methicillin-resistant Staphylococcus aureus (MRSA) isolated from patients with keratitis produces substantial amounts of phenol-soluble modulin α (PSMα). However, the role of PSMα in S. aureus keratitis remains unclear. We observed that PSMα-producing and PSMα-deficient strains could infect the cornea in our experimental mouse keratitis model; however, only the PSMα-producing strain delayed epithelial wound healing and induced stromal inflammation. PSMα induced damage to the epithelium, the release of alarmins IL-1α and IL-36α, and the expression of inflammatory chemokines by resident corneal cells in the mouse corneal organ culture. The IL-36 (but not IL-1) receptor antagonist attenuated mouse keratitis induced by PSMα-containing bacterial culture supernatants, as well as by infection with PSMα-producing S. aureus, suggesting that the corneal inflammations were dependent on IL-36. Recombinant PSMα elicited IL-36-dependent corneal inflammation in mice. Thus, PSMα and the subsequently released IL-36 are critical factors triggering inflammation during S. aureus keratitis.

Bacterial mediated green synthesis of silver nanoparticles and their antibacterial and antifungal activities against drug-resistant pathogens.

In the healthcare sector, the production of bioactive silver nanoparticles (AgNPs) with antimicrobial properties is of great importance. In this study, a novel bacterial strain, Paenibacillus sp. MAHUQ-63, was identified as a potential candidate for facile and rapid biosynthesis of AgNPs. The synthesized AgNPs were used to control the growth of human pathogens, Salmonella Enteritidis and Candida albicans. The bacterial culture supernatant was used to synthesize the nanoparticles (NPs). Field emission transmission electron microscope examination showed spherical-shaped NPs with 15-55 nm in size. Fourier transform-infrared analysis identified various functional groups. The synthesized AgNPs demonstrated remarkable activity against S. Enteritidis and C. albicans. The zones of inhibition for 100 µl (0.5 mg ml-1) of AgNPs against S. Enteritidis and C. albicans were 18.0 ± 1.0 and 19.5 ± 1.3 mm, respectively. The minimum inhibitory concentrations were 25.0 and 12.5 µg ml-1 against S. Enteritidis and C. albicans, respectively. Additionally, the minimum bactericidal concentrations were 25.0 µg ml-1 against both pathogenic microbes. The field emission scanning electron microscopy analysis showed that the treatment of AgNPs caused morphological and structural damage to both S. Enteritidis and C. albicans. Therefore, these AgNPs can be used as a new and effective antimicrobial agent.

Biosolubilized zinc from ZnO in bacterial culture supernatant enhanced seed germination, early growth, and zinc accumulation in rice (Oryza sativa L.) under hydroponic system through seed priming method

Presently, zinc sulfate (ZnSO4) based fertilizers are reported inefficient in flooded paddy fields which necessitates to find a better alternative for sustainable rice cultivation. This study was conducted to find out the effectiveness of bacterially solubilized Zn (BSZn) on the early growth of rice plants and the concentration of Zn in the plant parts in comparison to conventional ZnSO4. BSZn preparations were obtained from culture supernatant following ZnO solubilization by bacterial isolates Burkholderia spp. and Achromobacter sp. The effectiveness of BSZn was assessed on rice seed germination on Petri plates and the growth of seedlings upon treatment in hydroponic culture under controlled conditions. In general, BSZn obtained from certain Burkholderia spp. increased seed germination percentage (SG%) (98–100%), mean germination rate (MGR), mean daily germination (MDG), decreased mean germination time (MGT) and impart stimulating effect on seed germination as indicated by increased vigor indices compared to ZnSO4 treatment regimes. The obtained data from the hydroponic culture of rice seedling growth indicated a positive influence of most of the BSZn preparation on dry and wet weight of roots and shoots, and seedling length compared to ZnSO4 at p < 0.05. Both chlorophyll and carotenoid content of the seedling increased in the presence of some BSZn preparations. In general, compared to shoot, the root of seedlings accumulated a higher amount of Zn (up to 888 µg Zn/g dry biomass). Zn concentration data indicated that BSZn16 obtained from Burkholderia sp. EIKU16 culture help to accumulate significantly higher amounts of Zn in rice seedlings under hydroponic culture than that while treated with ZnSO4.

Symbiosis of Carpenter Bees with Uncharacterized Lactic Acid Bacteria Showing NAD Auxotrophy.

Eusocial bees (such as honey bees and bumble bees) harbor core gut microbiomes that are transmitted through social interaction between nestmates. Carpenter bees are not eusocial; however, recent microbiome analyses found that Xylocopa species harbor distinctive core gut microbiomes. In this study, we analyzed the gut microbiomes of three Xylocopa species in Japan between 2016 and 2021 by V1 to V2 region-based 16S rDNA amplicon sequencing, and 14 candidate novel species were detected based on the full-length 16S rRNA gene sequences. All Xylocopa species harbor core gut microbiomes consisting of primarily lactic acid bacteria (LAB) that were phylogenetically distant from known species. Although they were difficult to cultivate, two LAB species from two different Xylocopa species were isolated by supplementing bacterial culture supernatants. Both genomes exhibited an average LAB genome size with a large set of genes for carbohydrate utilization but lacked genes to synthesize an essential coenzyme NAD, which is unique among known insect symbionts. Our findings of phylogenetically distinct core LAB of NAD auxotrophy reflected the evolution of Xylocopa-restricted bacteria retention and maintenance through vertical transmission of microbes during solitary life. We propose five candidate novel species belonging to the families Lactobacillaceae and Bifidobacteriaceae, including a novel genus, and their potential functions in carbohydrate utilization. IMPORTANCE Recent investigations found unique microbiomes in carpenter bees, but the description of individual microbes, including isolation and genomics, remains largely unknown. Here, we found that the Japanese Xylocopa species also harbor core gut microbiomes. Although most of them were difficult to isolate a pure colony, we successfully isolated several strains. We performed whole-genome sequencing of the isolated candidate novel species and found that the two Lactobacillaceae strains belonging to the Xylocopa-specific novel LAB clade lack the genes for synthesizing NAD, a coenzyme central to metabolism in all living organisms. Here, we propose a novel genus for the two LAB species based on very low 16S rRNA gene sequence similarities and genotypic characters.

Real-Time Volatile Metabolomics Analysis of Dendritic Cells.

Dendritic cells (DCs) actively sample and present antigen to cells of the adaptive immune system and are thus vital for successful immune control and memory formation. Immune cell metabolism and function are tightly interlinked, and a better understanding of this interaction offers potential to develop immunomodulatory strategies. However, current approaches for assessing the immune cell metabolome are often limited by end-point measurements, may involve laborious sample preparation, and may lack unbiased, temporal resolution of the metabolome. In this study, we present a novel setup coupled to a secondary electrospray ionization-high resolution mass spectrometric (SESI-HRMS) platform allowing headspace analysis of immature and activated DCs in real-time with minimal sample preparation and intervention, with high technical reproducibility and potential for automation. Distinct metabolic signatures of DCs treated with different supernatants (SNs) of bacterial cultures were detected during real-time analyses over 6 h compared to their respective controls (SN only). Furthermore, the technique allowed for the detection of 13C-incorporation into volatile metabolites, opening the possibility for real-time tracing of metabolic pathways in DCs. Moreover, differences in the metabolic profile of naı̈ve and activated DCs were discovered, and pathway-enrichment analysis revealed three significantly altered pathways, including the TCA cycle, α-linolenic acid metabolism, and valine, leucine, and isoleucine degradation.

Study of Copper Resistant Pseudomonas Strains Isolated from Soil in Sarcheshmeh Copper Mine, in Iran and Investigation of the Mechanisms Involved in Copper Resistance

Heavy metal contaminated sites are the potential source of metal resistance microorganisms. The major focus of this study is to isolate and identify copper ions resistant Pseudomonas from the soil samples collected from mining and refining sites of Sarcheshmeh copper mine in the Kerman Province of Iran and investigate on possible mechanisms for copper resistance. Isolates were selected based on high level of copper ions resistance. The screened isolate was identified as Pseudomonas putida Cu-2 by morphological, biochemical and 16S rDNA gene sequencing analysis. A high degree of copper ions resistance associated with multiple antibiotic resistances was also detected in the screened isolate. The growth pattern of screened isolate with all the studied Cu concentrations was similar to that of control (without Cu ions) indicating that Cu ions would not affect the growth of isolated strain. A decrease in the amount of exopolysaccharides (EPS) was detected after Cu ions – P. Putida Cu-2 culture supernatant interaction. The Fourier transform infrared spectroscopy (FT-IR) peaks for the EPS extracted from the bacterial culture supernatant and the interacted Cu ions were nearly similar. The EPS covering of the Cu ions was established by FT-IR. The study of bacterial EPS covered Cu ions with E. coli PTCC 1338 and S. aureus PTCC 1113 exhibited less toxicity compared to uncoated Cu ions. The presence of copA gene that encodes for the multi-copper oxidase was investigated in the selected strain. The present study suggests that the capping of Cu ions by bacterially produced EPS and the presence of copA gene serve as the probable mechanisms of copper resistance.

Shiga Toxin (Stx) Phage-Encoded Lytic Genes Are Not Required for the Mouse Virulence of O157:H7 Escherichia coli Stx2-Producing Clinical Isolates.

Shiga toxin (Stx)-producing Escherichia coli (STEC) is a major cause of foodborne diarrheal illness in the United States and globally, and serotype O157:H7 is frequently associated with STEC outbreaks and sporadic cases in the United States. Severe systemic diseases associated with STEC are mediated by Stx types, particularly subtype Stx2a, encoded on inducible bacteriophages. We previously identified two STEC O157:H7 clinical isolates, JH2010 and JH2012, that exhibit a large difference in virulence in a streptomycin (Str)-treated mouse model. In this study, we aimed to identify a genetic basis for the difference in virulence between those strains. Comparison of the stx2a phage sequences showed that JH2012 lacks the lytic genes S and R on the phage genome. We also demonstrated that compared to JH2012 cultures, cultures of JH2010 released more Stx2 into the supernatant and were more sensitive to bacterial lysis during growth with ciprofloxacin (Cip), an inducer of stx phages. We therefore generated an stx2a phage SR deletion mutant strain of JH2010 to determine if those genes were responsible for the high virulence of that strain. We found that deletion of the SR genes from the stx2a phage in JH2010, and another O157:H7 strain, JH2016, resulted in increased cellular retention of Stx2, but there was no difference in virulence compared to the wild-type strains. Our results indicate that the stx2a phage SR genes are involved in Stx2 localization and phage-mediated cell lysis in vitro but that they are not required in wild-type STEC strains for virulence in a mouse model. IMPORTANCE The release of Stx from STEC has been thought to be tied to phage-mediated lysis of the host bacterial cell. In this study, we found that the stx2a phage lytic genes are not required for the virulence of pathogenic O157:H7 clinical isolates in a murine model of STEC infection or for release of Stx2a into the supernatant of bacterial cultures. These results point to an alternate mechanism for Stx2a release from STEC strains.

Inhalable vaccine of bacterial culture supernatant extract mediates protection against fatal pulmonary anthrax

ABSTRACT Pulmonary anthrax is the most fatal clinical form of anthrax and currently available injectable vaccines do not provide adequate protection against it. Hence, next-generation vaccines that effectively induce immunity against pulmonary anthrax are urgently needed. In the present study, we prepared an attenuated and low protease activity Bacillus anthracis strain A16R-5.1 by deleting five of its extracellular protease activity-associated genes and its lef gene through the CRISPR-Cas9 genome editing system. This mutant strain was then used to formulate a lethal toxin (LeTx)-free culture supernatant extract (CSE) anthrax vaccine, of which half was protective antigen (PA). We generated liquid, powder, and powder reconstituted formulations that could be delivered by aerosolized intratracheal inoculation. All of them induced strong humoral, cellular, and mucosal immune responses. The vaccines also produced LeTx neutralizing antibodies and conferred full protection against the lethal aerosol challenges of B. anthracis Pasteur II spores in mice. Compared to the recombinant PA vaccine, the CSE anthrax vaccine with equal PA content provided superior immunoprotection against pulmonary anthrax. The preceding results suggest that the CSE anthrax vaccine developed herein is suitable and scalable for use in inhalational immunization against pulmonary anthrax.

Surface-enhanced Raman spectroscopy (SERS) for the characterization of supernatants of bacterial cultures of bacterial strains causing sinusitis.

Sinusitis is defined as inflammation of the paranasal sinus mucous membrane lining caused by bacteria which usually invade the sinus by upper respiratory tract viral infections (UTI). In the present study, Surface-enhanced Raman spectroscopy (SERS) has been applied to differentiate and characterize supernatant samples, in triplicate, of three different types of bacteria which are considered leading cause of sinusitis disease. For this purpose, supernatant samples of three different strains of bacteria namely Staphylococcus aureus, Klebsiella pneumoniae and Enterococcus faecalis. The SERS has identified significant changes as a result of secretions of biomolecules by these bacteria in their supernatants which can be helpful to explore the potential of this technique for the identification and characterization of different strains of bacteria causing same disease. These differentiating characteristic SERS spectral features including 552cm-1 (C-S-S-C bonds), 951cm-1 (CN stretching), 1008cm-1 (Phenylalanine), 1032cm-1 (In plane CH bending mode Phenylalanine), 1280cm-1, 1320cm-1, 1329cm-1 (Amide III band), 1368cm-1, 1400cm-1, 1420cm-1 (COO-sym. stretching and CH bending), 1583cm-1 (Tyrosine) correspond to Proteins and 1051cm-1 (C-C, C-O, -C-OH def.) correspond to carbohydrates contents of these three different types of bacterial secretions in their respective supernatants. Furthermore, multivariate data analysis techniques like principal component analysis (PCA) and a supervised method partial least squares-discriminant analysis (PLS-DA) were found to be useful for the identification and characterization of different bacterial supernatants. Surface-enhanced Raman spectroscopy is proven to be a helpful approach for the characterization and discrimination of three bacterial supernatants including S. aureus, K. pneumonia and E. faecalis.

Application of Digital Holographic Microscopy to Analyze Changes in T-Cell Morphology in Response to Bacterial Challenge

Quantitative phase imaging (QPI) is a non-invasive, label-free technique used to detect aberrant cell morphologies caused by disease, thus providing a useful diagnostic approach. Here, we evaluated the potential of QPI to differentiate specific morphological changes in human primary T-cells exposed to various bacterial species and strains. Cells were challenged with sterile bacterial determinants, i.e., membrane vesicles or culture supernatants, derived from different Gram-positive and Gram-negative bacteria. Timelapse QPI by digital holographic microscopy (DHM) was applied to capture changes in T-cell morphology over time. After numerical reconstruction and image segmentation, we calculated single cell area, circularity and mean phase contrast. Upon bacterial challenge, T-cells underwent rapid morphological changes such as cell shrinkage, alterations of mean phase contrast and loss of cell integrity. Time course and intensity of this response varied between both different species and strains. The strongest effect was observed for treatment with S. aureus-derived culture supernatants that led to complete lysis of the cells. Furthermore, cell shrinkage and loss of circular shape was stronger in Gram-negative than in Gram-positive bacteria. Additionally, T-cell response to bacterial virulence factors was concentration-dependent, as decreases in cellular area and circularity were enhanced with increasing concentrations of bacterial determinants. Our findings clearly indicate that T-cell response to bacterial stress depends on the causative pathogen, and specific morphological alterations can be detected using DHM.

Carbapenemase-loaded outer membrane vesicles protect Pseudomonas aeruginosa by degrading imipenem and promoting mutation of antimicrobial resistance gene

AimsTo investigate the effect of Klebsiella pneumoniae carbapenemase (KPC)-loaded outer membrane vesicles (OMVs) in protecting Pseudomonas aeruginosa against imipenem treatment and its mechanism. MethodsThe OMVs of carbapenem-resistant Klebsiella pneumonia (CRKP) were isolated and purified from the supernatant of bacterial culture by using ultracentrifugation and Optiprep density gradient ultracentrifugation. The transmission electron microscope, bicinchoninic acid, PCR and carbapenemase colloidal gold assays were applied to characterize the OMVs. Bacterial growth and larvae infection experiments were performed to explore the protective function of KPC-loaded OMVs for P. aeruginosa under imipenem treatment. Ultra-performance liquid chromatography, antimicrobial susceptibility testing, whole-genome sequencing and bioinformatics analysis were used to investigate the mechanism of P. aeruginosa resistance phenotype mediated by OMVs. ResultsCRKP secreted OMVs loaded with KPC, which protect P. aeruginosa from imipenem through hydrolysis of antibiotics in a dose- and time-dependent manner. Furthermore, carbapenem-resistant subpopulations were developed in P. aeruginosa by low concentrations of OMVs that were confirmed to inadequately hydrolyze imipenem. Interestingly, none of the carbapenem-resistant subpopulations obtained the exogenous antibiotic resistance genes, but all of them possessed OprD mutations, which was consistent with the mechanism of P. aeruginosa induced by sub-minimal inhibitory concentrations of imipenem. ConclusionsOMVs containing KPC provide a novel route for P. aeruginosa to acquire an antibiotic-resistant phenotype in vivo.

Protective role of colitis in inflammatory arthritis via propionate-producing Bacteroides in the gut.

To investigate whether and how inflammatory disease in the intestine influences the development of arthritis, considering that organ-to-organ communication is associated with many physiological and pathological events. First, mice were given drinking water containing dextran sodium sulfate (DSS) and then subjected to inflammatory arthritis. We compared the phenotypic symptoms between the cohoused and separately-housed mice. Next, donor mice were divided into DSS-treated and untreated groups and then cohoused with recipient mice. Arthritis was then induced in the recipients. The fecal microbiome was analyzed by 16S rRNA amplicon sequencing. We obtained type strains of the candidate bacteria and generated propionate-deficient mutant bacteria. Short-chain fatty acids were measured in the bacterial culture supernatant, serum, feces, and cecum contents using gas chromatography-mass spectrometry. Mice fed with candidate and mutant bacteria were subjected to inflammatory arthritis. Contrary to expectations, the mice treated with DSS exhibited fewer symptoms of inflammatory arthritis. Intriguingly, the gut microbiota contributes, at least in part, to the improvement of colitis-mediated arthritis. Among the altered microorganisms, Bacteroides vulgatus and its higher taxonomic ranks were enriched in the DSS-treated mice. B. vulgatus, B. caccae, and B. thetaiotaomicron exerted anti-arthritic effects. Propionate production deficiency further prevented the protective effect of B. thetaiotaomicron on arthritis. We suggest a novel relationship between the gut and joints and an important role of the gut microbiota as communicators. Moreover, the propionate-producing Bacteroides species examined in this study may be a potential candidate for developing effective treatments for inflammatory arthritis.

Selective purification of catecholate, hydroxamate and α-hydroxycarboxylate siderophores with titanium dioxide affinity chromatography

Siderophores, high affinity iron chelators, play a key role in the uptake of iron by microorganisms and regulate many biological functions. Siderophores are categorized by their chelating group, e.g., catecholates, hydroxamates, α-hydroxycarboxylates. Natural concentrations of siderophores are often either too low or sample matrices are too complex for direct analysis by, e.g., liquid chromatography – mass spectrometry. Therefore, both concentration and purification are prerequisite for reliable analyses. However, a chromatographic technique that is selective for all siderophore classes and affords high levels of purification is lacking. We developed a titanium dioxide affinity chromatography (TDAC) solid-phase extraction (SPE) that affords the selective purification of these siderophore classes from complex sample matrices with recoveries up to 82%. The one-step purification removed most non-ligand sample ‘contaminants’, therefore, affording the straightforward identification of siderophore peaks in base peak chromatograms. As a proof of concept, the bioinformatic processing, dereplication of known features and selection of significant features in the TDAC eluates afforded a fast identification of six novel siderophores (woodybactines) from bacterial supernatants. We propose TDAC SPE as a fast and cost-effective methodology to screen for known or discover novel siderophores in natural samples in combination with untargeted bioinformatic processing by, e.g., XCMS. The method is scalable and yielded large amounts of highly purified siderophores from bacterial culture supernatants, providing an effective quantitative sample clean-up for, e.g., NMR structure elucidation.

Brevibacillus laterosporus as a Natural Biological Control Agent of Soil-Dwelling Nematodes

The nematocidal potential of an entomopathogenic strain of Brevibacillus laterosporus previously known for its antimicrobial properties was assessed on the free-living nematode microworm Panagrellus redivivus and the root-knot nematode Meloidogyne incognita. Laboratory bioassays showed significant nematocidal properties of the culture supernatant of the bacterium, achieving over 90% mortality of both microworms and M. incognita juveniles when the supernatant was collected at the sporulation phase, which related to the progressive production and release of virulence factors and toxins in the culture medium at this stage of bacterial growth. A protein fraction obtained by precipitation from the bacterial culture supernatant was found to be very active against nematodes with a concentration-dependent effect and an LC50 value of 0.4 µg/µL on M. incognita. Bacterial preparations based on either spores or a culture supernatant proved to be effective in reducing M. incognita density in treated compared with untreated soil, which makes the use of B. laterosporus as a biological control agent of soil-dwelling nematode pests particularly promising.