Non-pathogenic Strains Research Articles

Genomic analysis, culturing optimization, and characterization of Escherichia bacteriophage OSYSP, previously studied as effective pathogen control on fresh produce

Advances in bacteriophage genome sequencing and regulatory approvals of some bacteriophages in various applications have renewed interest in these antibacterial viruses as a potential solution to persistent food safety challenges. Here, we analyzed in depth the genome of the previously studied Escherichia bacteriophage OSYSP (phage OSYSP), revealed its application-related characteristics, and optimized its enumeration techniques for facilitating industrial implementation. We previously sequenced phage OSYSP genome completely by combining results from Illumina Miseq and Ion Torrent sequencing platforms and completing the remaining sequence gaps using PCR. Based on the genomics analysis completed herein, phage OSYSP was confirmed as an obligate lytic phage of the Caudoviricetes class. The genome encodes 81 proteins of identifiable functions, including two endolysins and 45 proteins that support host-independent DNA replication, transcription, and repair. Despite its similarities to T5-like phages, unique genome arrangements confirm phage OSYSP’s novelty. The genomic analysis also confirmed the absence of DNA sequences encoding virulence or antibiotic resistance factors. For optimizing phage detection and quantification in the conventional plaque assay, it was observed that decreasing the concentration of agar or agarose, when used as a medium gelling agent, increased phage recovery (p < 0.05), but using agarose resulted in smaller plaque diameters (p < 0.05). Phage OSYSP inactivated pathogenic and non-pathogenic strains of E. coli and some Salmonella enterica serovars, with more pronounced effect against E. coli O157:H7. Phage titers remained fairly unchanged throughout a 24-month storage at 4°C. Incubation for 30 min at 4°C−47°C or pH 4–11 had no significant detrimental effect (p > 0.05) on phage infectivity. In vitro application of phage OSYSP against E. coli O157:H7 EDL933 decreased the pathogen’s viable population by >5.7-log CFU/mL within 80 min, at a multiplicity of infection as low as 0.01. The favorable genome characteristics, combined with improved enumeration methodology, and the proven infectivity stability, make phage OSYSP a promising biocontrol agent against pathogenic E. coli for food or therapeutic applications.

Differential TLR2 and TLR4 mediated inflammatory and apoptotic responses in asymptomatic and symptomatic Leptospira interrogans infections in canine uterine tissue

Leptospirosis is major zoonotic disease with global implications, affecting both domestic animals and humans. It is caused by Leptospira interrogans (L. interrogans), which can damage multiple organs, including the kidneys, liver, testes, and uterus. Despite this, L. interrogans can also persist asymptomatically in tissues, akin to nonpathogenic strains. The mechanisms driving asymptomatic infections remain poorly understood. This study investigated the role of L. interrogans in asymptomatic infection within the uterine tissue of canines, focusing on the differential expression of Toll-like receptors (TLRs)2 and 4 and their roles in inflammatory and apoptotic pathways. We hypothesized that TLR2 and TLR4 coexpression is crucial for eliciting inflammation and apoptosis, whereas TLR4 alone might be insufficient. Our findings revealed that in symptomatic infections, both TLR2 and TLR4 are coexpressed, leading to markedly elevated levels of the proinflammatory cytokines IL-10, IL-1β, TNF-α, and IL-6. This enhanced inflammatory response is further evidenced by increased CD4 expression, indicating robust T helper cell activation. In contrast, asymptomatic infections are characterized by exclusive TLR4 expression, with inflammatory markers remaining at baseline levels. Additionally, we observed that L. interrogans induces apoptosis in symptomatic animals through TLR2 and TLR4 mediated activation of Caspase 8 and Caspase 3. These findings illustrate that L. interrogans drives both inflammation and apoptosis via the combination of TLR2 and TLR4 actions. When only TLR4 is activated, the immune response is insufficient, resulting in an asymptomatic disease course. This study provides novel insights into the differential roles of TLR receptors in leptospirosis, offering potential directions for targeted therapeutic strategies.

Extensive genomic characterization, pre-clinical probiotic evaluation, and safety analysis of Bifidobacterium longum subsp. longum BL21 isolated from infant feces

ObjectiveThis study aimed to evaluate the safety and probiotic properties of Bifidobacterium longum subsp. longum BL21 isolated from infant feces for use as a commercial probiotic strain. MethodsWhole-genome sequencing; physiological and biochemical assessments; enzymatic assays; metabolite, antibiotic sensitivity, cell adhesion and cytotoxicity, and tolerance tests; and a 14-day oral toxicity study were conducted. ResultsBL21 exhibited genetic integrity, and its genome lacked genes related to antibiotic resistance or virulence. It was found to be non-pathogenic, had efficient carbohydrate metabolism and mucin degradation ability, and was free from biogenic amines. It also showed susceptibility to antibiotics, strong cell adhesion, and resilience to adverse conditions. The aforementioned results confirm that BL21 is a functional probiotic strain with genetic stability, enzymatic capabilities, and non-pathogenic properties that mean it is safe for oral consumption, demonstrating that it is a promising candidate for probiotic applications. ConclusionThe study demonstrates that BL21 is a genetically stable, non-pathogenic probiotic strain with metabolic potential. The strain lacks virulence and antibiotic resistance genes, and its resilience to gastrointestinal conditions, as well as the results of the 14-day oral toxicity study, suggest that BL21 is safe for oral consumption. However, further long-term studies and clinical trials are needed to confirm its safety and efficacy for therapeutic use.

From cactus to crop: genomic insights of a beneficial and non-pathogenic Curtobacterium flaccumfaciens strain and the evolution of its pathosystem.

With the advent of advanced sequencing technologies, new insights into the genomes of pathogens, including those in the genus Curtobacterium, have emerged. This research investigates a newly isolated C. flaccumfaciens strain 208 (Cf208) from Arthrocereus glaziovii, and endemic plant from Iron Quadrangle. Previous results show that Cf208 exhibits the potential to remediate soils, facilitating the growth of tomato plants. Furthermore, Cf208 showed no virulence towards bean plants, thus, confounding its phytopathogenic origins. Using a comprehensive comparative genomics approach, we analyzed the Cf208 genome against 34 other Curtobacterium strains, aiming to discern the genomic landmarks associated with its adaptation as an endophyte and its avirulence in bean crops. This revealed a predominant core genome comprising about 2426 genes (68%). Notably, Cf208 possesses a unique plasmid, pCF208-73, which contains 84 unique genes (2.5%). However, unlike the plasmids previously described for pathogenic strains, pCF208-73 does not feature genes associated with virulence induction. In contrast, while several genes traditionally linked to virulence, like pectate lyases and proteases were identified, but the T4P apparatus emerged as new crucial factor for understanding virulence in the Curtobacterium genus. The presence or absence of this apparatus, especially in strains from different clades, may determine their virulence towards leguminous plants. In conclusion, this work highlights the significance of comparative genomics in unraveling the complexities of pathogenicity within the Curtobacterium genus. Our findings suggest that, although the limited genetic variations, specific genes, particularly those linked to the T4P apparatus, play a fundamental role in their interactions with host plants.

Use of stable isotope combined with intact cell lipidomic by routine MALDI mass spectrometry analysis for rapid drug susceptibility assay in mycobacteria.

Rapid, accurate, and easy-to-perform diagnostic assays are required to address the current need for the diagnosis of resistant pathogens. That is particularly the case for mycobacteria, such as the human pathogen Mycobacterium tuberculosis, which requires up to 2weeks for the determination of the drug susceptibility profile using the conventional broth microdilution method. To address this challenge, we investigated the incorporation of deuterium, the stable isotope of hydrogen, into lipids as a read out of the drug susceptibility profile. Deuterium is incorporated into newly synthesized proteins or lipids in place of hydrogen as bacterial cells grow, increasing the mass of the macromolecules, which can then be observed via matrix-assisted laser desorption ionization time of flight mass spectrometry (MALDI-TOF MS). As proof-of-concept, we used the non-pathogenic Mycobacterium smegmatis mc2155 strain, which is susceptible to the aminoglycoside antibiotic kanamycin, and M.smegmatis mc2155 containing the empty vector pVV16, which is kanamycin-resistant. Bacteria were incubated in a culture medium containing 50% of deuterium oxide (D2O) and either 1 or 2 times the minimal inhibitory concentration (MIC50) of kanamycin. Lipids were then analyzed using the MBT lipid Xtract matrix combined with routine MALDI mass spectrometry in the positive ion mode to evaluate the changes in the lipid profile. Using this approach, we were able to distinguish susceptible from resistant bacteria in less than 5h, a process that would take 72 h using the conventional broth microdilution method. We therefore propose a solution for the rapid determination of drug susceptibility profiles using a phenotypic assay combining D2O stable isotope labelling and lipid analysis by routine MALDI mass spectrometry.

Biosorption and transformation of cadmium and lead by Staphylococcus epidermidis AS-1 isolated from industrial effluent.

Rapid utilization of natural resources and other anthropogenic activities intruded heavy metals into the food chain and raised alarming concern for all life forms. The available methods proved insufficient in handling waste and pollutants due to the high cost and generation of toxic residues. Bioremediation strategies have offered sustainable solutions for toxic pollutants. In the current study, cadmium and lead (Cd and Pb respectively) tolerant strains have been isolated from industrial effluent and characterized for tolerance towards target pollutants. The strain was identified by 16s rRNA gene and further used for metal removal from the industrial effluents. Bacterial isolates were obtained from industrial discharge and evaluated for their tolerance towards Cd and Pb. AS-1 bacterial isolate exhibited maximum tolerance towards both the metals and hence was selected for further study. The isolate was identified as Staphylococcus epidermidis. ICP-MS and energy dispersive X-ray (EDX) analysis of biomass revealed that a significant proportion of cadmium (90.89%) and lead (94.87%) available in effluent were sequestered within bacterial biomass. Characteristic peaks at 2Ɵ (31.8637 and 45.6247 for cadmium) and (21.0397, 27.0127, 46.0537, 54.2707 and 75.6547 for lead) confirmed the crystalline nature of the sequestered metals. The selected strain was characterized on biochemical and molecular basis and was found to be Staphylococcus epidermidis. Based on 16S rDNA sequence analysis, a phylogenetic dendrogram was created for the maximum likelihood of the bacterial strain. The sequence was deposited in the NCBI repository (accession number PP587422). The work has shown the possible way out of heavy metal pollution sustainably. To the best of the author's knowledge, this is the first report on the sequestration and reduction of cadmium and lead by a nonpathogenic strain of Staphylococcus epidermidis AS-1 that may be useful for alleviating heavy metal contamination.

Bovine Lactoferrin Impairs in-vitro Growth of Neonatal Sepsis-causingEscherichia Coli Clinical Isolates

Abstract Background E. coli is a dominant cause of neonatal sepsis with no prevention currently available. Lactoferrin (LF) is an iron-sequestering antibacterial protein that has been shown to decrease all-cause neonatal late-onset sepsis. However, the impact of LF on impairing growth of neonatal E. coli septicemia isolates carrying various iron acquisition virulence genes has not been investigated. We aimed to determine the effects of bovine lactoferrin (bLF) on in vitro growth of neonatal E. coli septicemia strains and to determine the presence of iron-acquisition virulence genes in the isolates. Methods We tested six clinical E. coli strains obtained from blood of septic neonates, including the archetypal RS218 strain. Isolates represent the most frequent phylogroups (B2 and D) and multi-locus sequence types (ST95, ST69, and ST131) among neonatal E. coli septicemia strains. The nonpathogenic strain DH5α was used as control. Strains were grown in media containing 0, 0.1, 1.0, and 10 mg/mL bLF. Optical density (OD) readings were obtained every 30 min. for 20 h. Experiments were repeated 3 times. OD area under the curve (AUC) values were compared with ANOVA. Whole genome sequencing (WGS) was obtained to determine the presence of iron-acquisition virulence genes using VirulenceFinder v. 2.0.3 (J Clin Micro 2020. 58 (10):e01269-20). Results For all strains, the growth decrease with 10 mg/mL bLF compared to zero bLF was highly significant (p < 0.001) (Fig.1). Growth was also significantly reduced at 1 mg/mL for most strains (p ≤ 0.02). Growth decrease at 0.1 mg/mL was only significant for isolate SCB31. Growth suppression at 10 mg/mL was greatest in DH5α. WGS analyses showed that all pathogenic strains carried several iron acquisition virulence genes, with chuA (hemin receptor), fyuA (siderophore receptor) in irp2 (protein 2 peptide synthetase) and sitA (iron transport protein) found in all (Fig. 2). DH5α had no iron acquisition virulence genes. Conclusion Bovine LF is effective in impairing growth of neonatal sepsis-causing E. coli regardless of the presence of iron acquisition genes known to contribute to virulence. Future studies will examine additional isolates and the effect of greater bLF concentrations. These data have the potential to inform dosing regimens for additional in vivo studies to further assess bLF’s protective effects against neonatal sepsis. Figure 1. Comparison of growth curve data among E. coli strains. Optical density AUC values of six neonatal septicemia E. coli isolates growth with increasing concentrations of bovine lactoferrin. DH5α is a nonpathogenic control strain. Bars depict means and standard deviations for each condition tested. Asterisks indicate statistically significant differences compared to the 0 mg/mL bLF condition (*p < 0.001, **p < 0.05). Figure 2. Presence of iron acquisition virulence genes in E. coli isolates. WGS analyses detected several genes, which are indicated in black. White indicates absence of gene. ST, Sequence type; chuA, Outer membrane hemin receptor; fyuA, Siderophore receptor (yersiniabactin); iroN, Siderophore receptor; irp2, High-molecular-weight protein 2 nonribosomal peptide synthetase; iucC, Aerobactin siderophore synthetase; iutA, Ferric aerobactin siderophore receptor; sitA, Iron transport protein. *Multi-drug resistant isolate.

Modulating effects of fodder grasses extracts on antibiotic sensitivity and biofilm production in avian pathogenic Escherichia coli strains

Extracts of certain fodder grasses may be viewed as powerful agents against infections induced by avian pathogenic Escherichia coli strains. Here we demonstrated ability of Galega orientalis and Rhaponticum carthamoides extracts, alone or in combination with antibiotics, to inhibit growth, viability and biofilm formation in avian pathogenic Escherichia coli strains with different sensitivity to antibiotics and non-pathogenic laboratory strain E. coli BW25113 as well as its mutant derivatives. Modulation of motility and production of extracellular structures in the presence of the extracts correlated with their anti-biofilm effects. Interestingly, an increase in antibacterial action of kanamycin, streptomycin, ciprofloxacin, and cefotaxime on both biofilms and planktonic cultures of the studied strains was observed in the presence of the extracts, including antibiotic resistant APEC strain #45. The extracts alone showed weak prooxidant activity which could contribute to modification of redox-sensitive sites of various regulatory circuits, resulting to synergetic effects in combination with antibiotics.

Оценка степени высвобождения и токсичности соединений цинка, используемых в фармацевтических и косметических средствах

The degree of release from ointments and the toxicity of three zinc compounds were assessed to establish their minimum sufficient concentration in antibacterial pharmaceutical and cosmetic products. The properties of zinc oxide (ZnO), zinc pyrrolidone carboxylate (ZnРСА) and zinc salicylate (Zn(Sal)2·2H2О) were compared. The release rate of active substances from petrolatum-based ointments (the mass fraction of zinc compounds is 1%) was determined by the Kruvchinsky method. A cellophane film for dialysis served as a semipermeable membrane, which was fixed on a glass tube (length 15 cm, cross section 20 mm2). A sample of ointment (2.00 g) was applied to the inner surface of the membrane. The dialysis tube was inserted into a beaker with dialysis medium and immersed to a depth of no more than 3 mm. The toxicity of aqueous solutions (distilled water) of zinc compounds was determined in a bioluminescent biotest using “Ecolume” bacterial preparation based on a non-pathogenic strain of Escherichia coli. It was found that a high level of toxicity of is achieved at 10-5 mol/dm3 zinc salts in a solution, zinc oxide – 10-4 mol/dm3. A similar amount of Zn2+ diffuses from ointments through a semipermeable cellophane membrane used for the study of dialysis into a 50% aqueous alcohol solution. Zinc ion release from ointment with ZnO was only 0.31%, as well as from ointments with ZnPCA and Zn(Sal)2·2H2О – 1.59% and 1.87% respectively. Thus, the tested approach is economically and environmentally effective, as it allows you to determine the lowest effective concentration of active substances, reducing the burden on the human microbiome and the environment.

Therapeutic potential of Zanha africana (Radlk.) Exell: antimycobacterial activity and safety evaluation

BackgroundZanha africana (Radlk.) Exell extracts are commonly utilized in Tanzania and other countries for treating conditions associated with HIV and AIDS, including tuberculosis (TB). In Tanzania, it is listed among the most frequently traded plant species for medicinal use in different marketplaces. However, limited literature exists regarding its antimycobacterial properties and safety profile. ObjectiveThis study aimed to evaluate Z. africana ethanolic extract for safety and antimycobacterial activity. MethodsThe antimycobacterial activity of the crude extracts was assessed using the broth microdilution method against various non-pathogenic mycobacteria strains including Mycobacterium aurum (MA), Mycobacterium madagascariense (MM), and Mycobacterium indicus pranii (MIP). Bioassay-guided fractionation was employed to isolate the active compounds. The safety profile of the crude extract was investigated through cytotoxicity assay on peripheral blood mononuclear cells (PBMCs) and the brine shrimp lethality test. ResultsThe 80% ethanolic extracts of Z. africana demonstrated activity against all tested non-pathogenic mycobacteria. The antimycobacterial activity of ethanolic extract of the stem bark was 625 µg/mL, 313 µg/mL and 125 µg/mL against MA, MM, and MIP, respectively. The most potent fraction was the ethyl acetate fraction, from which two active compounds were isolated: proanthocyanidin A2; Compound (1) and its monomer epicatechin, Compound (2). Compound (1) exhibited higher antimycobacterial activity compared to compound (2) with minimum inhibitory concentrations (MICs) of 19.50 µg/mL and 12.98 µg/mL against MM and MIP respectively. The ethanolic extract did not demonstrate toxicity on PBMCs and brine shrimps. ConclusionThis study demonstrates that Z. africana extracts and the isolated compounds possess antimycobacterial activity. This provides evidence supporting the traditional use of Z. africana extracts for TB management. Further research is recommended to comprehensively evaluate the efficacy and safety of the plant constituents using different models.

Unveiling Microbial Diversity: Raman Spectroscopy's Discrimination of Clostridium and Related Genera.

In the clinical environment, the identification of phylogenetic closely related genera and species like Clostridium and Bacillus spp. is challenging. Both genera contain representatives of pathogenic and nonpathogenic species that need to be distinguished for a proper diagnostic read-out. Therefore, reliable and accurate detection methods must be employed for the correct identification of these genera and species. Despite their high pathogenicity, clostridial infections and food contaminations present significant challenges due to their unique cultivation conditions and developmental needs. Therefore, in many diagnostic protocols, the toxins are used for microbiological documentation. However, the applied laboratory methods suffer in accuracy, sometimes require large bacterial loads to provide reliable results, and cannot differentiate pathogenic from nonpathogenic strains. Here, Raman spectroscopy was employed to create an extensive Raman database consisting of pathogenic and nonpathogenic Bacillus and Clostridium species. These genera, as well as representatives of Paraclostridium and Clostridioides were specifically selected for their phylogenetic relation, cultivation conditions, and metabolic activity. A chemometric evaluation of the Raman spectra of single vegetative cells revealed a high discriminating power at the genus level. However, bacilli are considerably easier to classify at the species level than clostridia. The discrimination between the genera and species was based on their phylogeny and not their aerobic and anaerobic cultivation conditions. These encouraging results demonstrated that Raman spectroscopy coupled with chemometrics is a robust and helpful method for differentiating Clostridium species from Bacillus, Clostridioides, and Paraclostridium species. This approach has the potential to be a valuable tool in clinical diagnostics.

Harnessing microbial potential: Exploiting heavy oil-laden soil microbiota for sustainable production of high-yield rhamnolipids from waste cooking oil

Oil polluted soil microbiota plays an important role in the production of biosurfactants. In comparison to synthetic surfactants, biosurfactants offer unique advantages, such as lower toxicity, biodegradability, selectivity, and effectiveness under unpleasant conditions. Despite these benefits, the widespread use of biosurfactants is limited by enormous production costs. To address this challenge, this study aimed to explore the adoption of waste cooking for rhamnolipids production. Two prominent bacterial strains: Kosakonia cowanni and Acinetobacter colcoaceticus, were obtained from heavy oil-laden soil samples, and further demonstrated their capability for rhamnolipids production from waste cooking oil (Acinetobacter colcoaceticus: 0.51 g/L, Kosakonia cowanii: 0.39 g/L). The biosurfactants obtained were characterized through TLC, FTIR, and H NMR to confirm their rhamnolipid identities as mono-rhamnolipids. The findings in our study emphasizes the potential of cost-effective production of rhamnolipids that possess interesting biotechnological features through the synergy of oil-polluted environments and waste cooking oil. This study contributes significantly to the development of sustainable rhamnolipid production using non-pathogenic strains. By harnessing these microorganisms, we advance towards addressing critical environmental challenges, such as heavy metal contamination in water. This research aligns with broader sustainability goals, including clean water and sanitation.

High-resolution 2D solid-state NMR provides insights into nontuberculous mycobacteria

We present a high-resolution magic-angle spinning (MAS) solid-state NMR (ssNMR) study to characterize nontuberculous mycobacteria (NTM). We studied two different NTM strains, Mycobacterium smegmatis, a model, non-pathogenic strain, and Mycobacterium abscessus, an emerging and important human pathogen. Hydrated NTM samples were studied at natural abundance without isotope-labelling, as whole-cells versus cell envelope isolates, and native versus fixed sample preparations. We utilized 1D13C and 2D 1H-13C ssNMR spectra and peak deconvolution to identify NTM cell-wall chemical sites. More than ∼100 distinct 13C signals were identified in the ssNMR spectra. We provide tentative assignments for ∼30 polysaccharides by using well resolved 1H/13C chemical shifts from the 2D INEPT-based 1H-13C ssNMR spectrum. The signals originating from both the flexible and rigid fractions of the whole-cell bacteria samples were selectively analyzed by utilizing either CP or INEPT based 13C ssNMR spectra. CP buildup curves provide insights into the dynamical similarity of the cell-wall components for NTM strains. Signals from peptidoglycan, arabinogalactan and mycolic acid were identified. The majority of the 13C signals were not affected by fixation of the whole cell samples. The isolated cell envelope NMR spectrum overlap with the whole-cell spectrum to a large extent, where the latter has more signals. As an orthogonal way of characterizing these bacteria, electron microscopy (EM) was used to provide spatial information. ssNMR and EM data suggest that the M. abscessus cell-wall is composed of a smaller peptidoglycan layer which is more flexible compared to M. smegmatis, which may be related to its higher pathogenicity. Here in this work, we used high-resolution 2D ssNMR first time to characterize NTM strains and identify chemical sites. These results will aid the development of structure-based approaches to combat NTM infections.

Hydrolysis probe assays for the detection of pathogenic Enterohemorrhagic Escherichia coli: Multi-Country validation study

Enterohemorrhagic Escherichia coli (EHEC) strains are foodborne pathogens frequently associated with outbreaks linked to red meat, posing significant global food safety concerns. While several commercial assays exist for detecting EHEC strains belonging to serogroups O26, O103, O111, and O121, they often fail to distinguish pathogenic strains from non-pathogenic strains lacking virulence genes. These non-pathogenic E. coli interfere with the accuracy of existing assays, resulting in false-positives rates between 81 – 100 % for beef products. This study aimed to standardize and validate four hydrolysis probe assays for the specific detection of pathogenic strains of E. coli O26, O103, O111, and O121. Primers and probes were designed to target single nucleotide polymorphisms conserved among pathogenic strains of each target serogroup. The standardized assays were validated using a comprehensive collection of pure culture and DNA samples (n = 248) from Canada, France, Switzerland, and the United States; laboratory-inoculated beef and spinach (n = 132), fractionally inoculated ground beef (n = 390), and samples from the federal red meat surveillance program (n = 166). The results demonstrated an overall accuracy of 94.8 % in predicting the virulence profile of pure culture strains. These four assays are a single-step PCR screening tool that can determine the presence of virulent strains. These assays provide food testing laboratories worldwide with a set of reliable methods for identifying pathogenic O26, O103, O111, and O121 strains. Thus, this assay will help to reduce product waste and financial losses caused by misleading positive results caused by presence of non-pathogenic strains.

The Impact of a Non-Pathogenic Strain of Fusarium Oxysporum on Structural and Biochemical Properties of Flax Suspension Cultures.

Flax (Linum usitatissimum L.) is an important crop plant with pharmaceutical significance. It is described in pharmacopoeias (the United States Pharmacopeia and the European Pharmacopoeia), which confirms that it (especially the seeds) is a valuable medicinal product. Similar to flax seeds, which accumulate bioactive compounds, flax in vitro cultures are also a rich source of flavonoids, phenolics, lignans and neolignans. In the present study, flax suspension cultures after treatment of the non-pathogenic Fusarium oxysporum strain Fo47 were established and analyzed. The study examined the suitability of Fo47 as an elicitor in flax suspension cultures and provided interesting data on the impact of these endophytic fungi on plant metabolism and physiology. Two flax cultivars (Bukoz and Nike) and two compositions of media for flax callus liquid cultures were tested. Biochemical analysis revealed enhanced levels of secondary metabolites (total flavonoid and total phenolic content) and photosynthetically active pigments in the flax callus cultures after treatment with the non-pathogenic fungal strain F. oxysporum Fo47 when compared to control, untreated cultures. In cultures with the selected, optimized conditions, FTIR analysis was performed and revealed changes in the structural properties of cell wall polymers after elicitation of cultures with F. oxysporum Fo47. The plant cell wall polymers were more strongly bound, and the crystallinity index (Icr) of cellulose was higher than in control, untreated samples. However, lignin and pectin levels were lower in the flax callus liquid cultures treated with the non-pathogenic strain of Fusarium when compared to the untreated control. The potential application of the non-pathogenic strain of F. oxysporum for enhancing the synthesis of desired secondary metabolites in plant tissue cultures is discussed.

Polyphenolic extracts from Diospyros kaki and Vitis vinifera by-products stimulate cytoprotective effects in bacteria-cell host interactions by mediation of transcription factor Nrf2

BackgroundThe intestinal and skin epithelium play a strong role against bacterial stimuli which leads to inflammation and oxidative stress when overwhelmed. Polyphenols from fruit-rich diets and by-products show promise against bacterial deleterious effects; however, their antibacterial and health-promoting effects remain understudied. PurposeThis study aimed to assess the impact of polyphenolic extracts of grape (GrPE), persimmon (PePE) and pomegranate (PoPE) by-products on bacterial pathogen-host interactions, focusing beyond growth inhibition to explore their effects on bacterial adhesion, invasion, and modulation of host responses. MethodsThe microdilution method, as well as the tetrazolium based MTT cell proliferation and cytotoxicity assay with crystal violet staining were used to identify extracts sub-inhibitory concentrations that interfere with bacterial adhesion, invasion or lipopolysaccharides (LPS) effect on cell hosts without compromising host viability. The cytoprotective effects of extracts were assessed in a knock-down model of nuclear factor erythroid 2-related factor 2 (Nrf2). ResultsAll extracts demonstrated significant reductions in pathogen adhesion to Caco-2 and HaCaT cells while preserving cellular integrity. Notably, PePE exhibited specific efficacy against Salmonella enterica adhesion, attributed mostly to its gallic acid content, whereas PoPE reduced S. enterica invasion in Caco-2 cells. The extracts supported the prevalence of non-pathogenic and commensal strains of intestinal and skin surfaces, selectively reducing pathogenic adhesion. The extracts mitigated the oxidative stress, enhanced the barrier function, and modulated the pro-inflammatory cytokines in LPS-challenged cells. GrPE, rich in anthocyanins, and PePE were found to mediate their protective effects through Nrf2 activation, while PoPE exerted multifaceted actions independent of Nrf2. ConclusionOur results highlight the therapeutic potential of GrPE, PePE, and PoPE in shaping bacterial-host interactions, endorsing their utility as novel nutraceuticals for both oral and topical applications to prevent potential bacterial infections through innovative mechanisms.

Daily and Weekly Urine Variations in Bacterial Growth Susceptibility in Postmenopausal Women With no History of Urinary Tract Infection: A Pilot Study

ObjectivesTo evaluate bacterial growth in the bladder as a major virulence factor during urinary tract infections (UTIs), we assessed the variability of uropathogenic bacterial growth in urine samples over multiple timepoints from three postmenopausal women with no history of UTI. MethodsFollowing IRB approval, postmenopausal women who never had a UTI provided mid-stream urine samples three times daily (8 a.m., 12 p.m. and 4 p.m.), twice a week over two weeks, as well as dietary intake logs. Each sample was studied for pH and bacterial growth using three uropathogenic Escherichia coli (E.coli) strains (LRPF007, KE40, UTI89), one uropathogenic Enterococcus faecalis strain (HRH40), and one non-pathogenic E. coli strain (W3110). ResultsSimilar mean growth yields were observed for the uropathogenic strains (mean ∆OD600 = 0.1 – 0.13) with high variability (standard deviation (SD) = 0.13 – 0.28) and lower mean growth for the non-pathogenic strain (mean ∆OD600 = 0.05). Urine from each individual at different collection times never had the same bacterial growth potential for any bacterial strain. There were significant associations between urine pH decrease with higher fat (p=0.017), vitamin D (p=0.02), magnesium (p=0.049), fluid (p=0.013), and ash (p=0.01) intake. ConclusionsMajor variations were observed in the bacterial growth response in the urine of the same individuals. These variations imply a requirement for a normalization procedure, e.g., growth in synthetic urine, for analysis of bacterial growth in urine. There were significant associations between decreased urine pH and several nutrients found in foods that are classified as highly acidic.

Cryo-EM-based discovery of a pathogenic parvovirus causing epidemic mortality by black wasting disease in farmed beetles

We use cryoelectron microscopy (cryo-EM) as a sequence- and culture-independent diagnostic tool to identify the etiological agent of an agricultural pandemic. For the past 4 years, American insect-rearing facilities have experienced a distinctive larval pathology and colony collapse of farmed Zophobas morio (superworm). By means of cryo-EM, we discovered the causative agent: a densovirus that we named Zophobas morio black wasting virus (ZmBWV). We confirmed the etiology of disease by fulfilling Koch's postulates and characterizing strains from across the United States. ZmBWV is a member of the family Parvoviridae with a 5,542 nt genome, and we describe intersubunit interactions explaining its expanded internal volume relative to human parvoviruses. Cryo-EM structures at resolutions up to 2.1Å revealed single-strand DNA (ssDNA) ordering at the capsid inner surface pinned by base-binding pockets in the capsid inner surface. Also, we demonstrated the prophylactic potential of non-pathogenic strains to provide cross-protection invivo.

Insights into the genomic and phenotypic characteristics of Bacillus spp. strains isolated from biofilms in broiler farms.

The characterization of surface microbiota living in biofilms within livestock buildings has been relatively unexplored, despite its potential impact on animal health. To enhance our understanding of these microbial communities, we characterized 11 spore-forming strains isolated from two commercial broiler chicken farms. Sequencing of the strains revealed them to belong to three species Bacillus velezensis, Bacillus subtilis, and Bacillus licheniformis. Genomic analysis revealed the presence of antimicrobial resistance genes and genes associated with antimicrobial secretion specific to each species. We conducted a comprehensive characterization of the biofilm formed by these strains under various conditions, and we revealed significant structural heterogeneity across the different strains. A macro-colony interaction model was employed to assess the compatibility of these strains to coexist in mixed biofilms. We identified highly competitive B. velezensis strains, which cannot coexist with other Bacillus spp. Using confocal laser scanning microscopy along with a specific dye for extracellular DNA, we uncovered the importance of extracellular DNA for the formation of B. licheniformis biofilms. Altogether, the results highlight the heterogeneity in both genome and biofilm structure among Bacillus spp. isolated from biofilms present within livestock buildings.IMPORTANCELittle is known about the microbial communities that develop on farms in direct contact with animals. Nonpathogenic strains of Bacillus velezensis, Bacillus subtilis, and Bacillus licheniformis were found in biofilm samples collected from surfaces in contact with animals. Significant genetic and phenotypic diversity was described among these Bacillus strains. The strains do not possess mobile antibiotic resistance genes in their genomes and have a strong capacity to form structured biofilms. Among these species, B. velezensis was noted for its high competitiveness compared with the other Bacillus spp. Additionally, the importance of extracellular DNA in the formation of B. licheniformis biofilms was observed. These findings provide insights for the management of these surface microbiota that can influence animal health, such as the use of competitive strains to minimize the establishment of undesirable bacteria or enzymes capable of specifically deconstructing biofilms.

Production of unsulfated chondroitin and associated chondro-oligosaccharides in recombinant Escherichia coli

We designed metabolically engineered non-pathogenic strains of Escherichia coli to produce unsulfated chondroitin with and without chondroitin lyase to produce the chondroitin polymer or its related oligosaccharides. Chondroitin was synthesized using chondroitin synthase KfoC and chondroitin was degraded using Pl35, a chondroitin lyase from Pedobacter heparinus. Pl35 behaved as a true endo-enzyme generating a large panel of oligosaccharides ranging from trimers to 18-mers instead of the di- and tetramers obtained with most chondroitin lyases. Two series of oligosaccharides were characterized, sharing an unsaturated uronic acid (4-deoxy-α-L-threo-hex-4-enepyranosyluronic acid, △UA) residue at their non-reducing end. The major “even-numbered” series was characterized by a terminal reducing N-acetylgalactosaminyl residue. The minor “odd-numbered” series oligosaccharides carried a terminal reducing glucuronic acid residue instead.Cultures were conducted in fed-batch conditions, and led to the production of up to 10 g L−1 chondroitin or chondroitin oligosaccharides.All products were purified and fully characterized using NMR and mass spectrometry analyses.This is the first report of the microbial production of large chondro-oligosaccharides.