Silver nanoparticle with potential antimicrobial and antibiofilm efficiency against multiple drug resistant, extensive drug resistant Pseudomonas aeruginosa clinical isolates.

BACKGROUND: The study aims to investigate the effect of combining silver nanoparticles (AGNPs) with different antibiotics on multi-drug resistant (MDR) and extensively drug resistant (XDR) isolates of Pseudomonas aeruginosa (P. aeruginosa) and to investigate the mechanism of action of AGNPs. METHODS: AGNPs were prepared by reduction of silver nitrate using trisodium citrate and were characterized by transmission electron microscope (TEM) in addition to an assessment of cytotoxicity. Clinical isolates of P. aeruginosa were collected, and antimicrobial susceptibility was conducted. Multiple Antibiotic Resistance (MAR) index was calculated, and bacteria were categorized as MDR or XDR. Minimum inhibitory concentration (MIC) of gentamicin, ciprofloxacin, ceftazidime, and AGNPs were determined. The mechanism of action of AGNPs was researched by evaluating their effect on biofilm formation, swarming motility, protease, gelatinase, and pyocyanin production. Real-time PCR was performed to investigate the effect on the expression of genes encoding various virulence factors. RESULTS: TEM revealed the spherical shape of AGNPs with an average particle size of 10.84 ± 4.64 nm. AGNPS were safe, as indicated by IC50 (42.5 µg /ml). The greatest incidence of resistance was shown against ciprofloxacin which accounted for 43% of the bacterial isolates. Heterogonous resistance patterns were shown in 63 isolates out of the tested 107. The MAR indices ranged from 0.077 to 0.84. Out of 63 P. aeruginosa isolates, 12 and 13 were MDR and XDR, respectively. The MIC values of AGNPs ranged from 2.65 to 21.25 µg /ml. Combination of AGNPs with antibiotics reduced their MIC by 5-9, 2-9, and 3-10Fold in the case of gentamicin, ceftazidime, and ciprofloxacin, respectively, with synergism being evident. AGNPs produced significant inhibition of biofilm formation and decreased swarming motility, protease, gelatinase and pyocyanin production. PCR confirmed the finding, as shown by decreased expression of genes encoding various virulence factors. CONCLUSION: AGNPs augment gentamicin, ceftazidime, and ciprofloxacin against MDR and XDR Pseudomonas isolates. The efficacy of AGNPs can be attributed to their effect on the virulence factors of P. aeruginosa. The combination of AGNPs with antibiotics is a promising strategy to attack resistant isolates of P. aeruginosa.

Microbiota-derived short chain fatty acids in fermented Kidachi Aloe promote antimicrobial, anticancer, and immunomodulatory activities.

BACKGROUND: Fermented Aloe leaf juice is a commonly used food supplement in Japan. In a previous study, fermentation of A. arborescence juice was performed and the presence of short-chain fatty acids (SCFAs) was confirmed and quantified. Samples were collected before and after the fermentation process to be subjected, in the present study, to DNA extraction, 16S rRNA gene (V3-V4 regions) amplification, and sequencing by the next-generation Illumina MiSeq sequencer. Our work aims to analyze the sequences to assess the bacterial diversity in the juice before and after fermentation, identify the beneficial microbes responsible for the production of SCFAs, and evaluate some of the biological activities of the fermented juice. RESULTS: Data revealed the richness and diversity of the bacterial community in the fermented juice compared to the unfermented control. Relative abundance of bacterial phyla showed that the majority of the microbial community in the test samples corresponded to Pseudomonadota (unfermented; 10.4%, fermented; 76.36%), followed by Bacillota (unfermented; 4.71%, fermented; 17.13%) and then Bacteroidota (unfermented; 0.57%, fermented; 1.64%). For the fermented sample, 84% of Bacillota were lactobacilli. A hierarchically clustered heatmap revealed that Lactobacillus was the most abundant genus in both samples suggesting its involvement in the production of SCFAs. To assess potential health benefits, the anticancer efficacy of the fermented product of A. arborescens was investigated against colorectal cancer (IC50 = 3.5 µg/ml) and liver cancer (IC50 = 6.367 µg/ml) compared to the normal peripheral blood mononuclear cells (PBMCs). Flow cytometric analysis of the cell cycle pattern revealed remarkable population arrest in G0 and G1, however, the highest percentages were mainly in the G1 phase for Hep-G2 (40.1%) and HCT-116 (53.2%) cell lines. This effect was accompanied by early apoptotic profiles of HCT-116 (36.9%) and late apoptosis for Hep-G2 (17.3%). Furthermore, immunomodulatory properties demonstrated a significantly (p < 0.001) reduced percentage of induced TNF-α while enhancing IFN-γ dramatically. For antimicrobial activities, marked broad-spectrum activities were recorded against some bacterial and fungal pathogens (17-37 mm inhibition zone diameter range). CONCLUSION: Therefore, this study affords the basis of bacterial community composition in fermented A. arborescens juice as well as its potential biological benefits.

Antibacterial, antibiofilm, and anti-quorum sensing activities of pyocyanin against methicillin-resistant Staphylococcus aureus: in vitro and in vivo study.

BACKGROUND: Methicillin-resistant Staphylococcus aureus (MRSA) infections are considered a major public health problem, as the treatment options are restricted. Biofilm formation and the quorum sensing (QS) system play a pivotal role in S. aureus pathogenicity. Hence, this study was performed to explore the antibacterial effect of pyocyanin (PCN) on MRSA as well as its effect on MRSA biofilm and QS. RESULTS: Data revealed that PCN exhibited strong antibacterial activity against all test MRSA isolates (n = 30) with a MIC value equal to 8 µg/ml. About 88% of MRSA biofilms were eradicated by PCN treatment using the crystal violet assay. The disruption of MRSA biofilm was confirmed using confocal laser scanning microscopy, which showed a reduction in bacterial viability (approximately equal to 82%) and biofilm thickness (approximately equal to 60%). Additionally, the disruption of the formation of microcolonies and the disturbance of the connection between bacterial cells in the MRSA biofilm after PCN treatment were examined by scanning electron microscopy. The 1/2 and 1/4 MICs of PCN exerted promising anti-QS activity without affecting bacterial viability; Agr QS-dependent virulence factors (hemolysin, protease, and motility), and the expression of agrA gene, decreased after PCN treatment. The in silico analysis confirmed the binding of PCN to the AgrA protein active site, which blocked its action. The in vivo study using the rat wound infection model confirmed the ability of PCN to modulate the biofilm and QS of MRSA isolates. CONCLUSION: The extracted PCN seems to be a good candidate for treating MRSA infection through biofilm eradication and Agr QS inhibition.

Improvement and enhancement of oligosaccharide production from Lactobacillus acidophilus using statistical experimental designs and its inhibitory effect on colon cancer.

Colorectal cancer (CRC) is the third cause of death by cancers worldwide and is one of the most common cancer types reported in both Egypt and the United States. The use of probiotics as a dietary therapy is increasing either as a prevention or as a treatment for many diseases, particularly, in the case of CRC. The increasing acceptance of lactic acid bacterial (LAB) oligosaccharides as bioactive agents has led to an increase in the demand for the large-scale production of LAB-oligosaccharides using fermentation technology. Therefore, in the current study, we are using the Plackett- Burman design (PBD) approach, where sixteen experimental trials were applied to optimize the production of the target oligosaccharide LA-EPS-20079 from Lactobacillus acidophilus. Glucose, yeast extract and sodium acetate trihydrate were the top three significant variables influencing LA-EPS production. The maximum concentration of LA-EPS-20079 achieved by L. acidophilus was 526.79 µg/ml. Furthermore, Box-Behnken design (BBD) as response surface methodology (RSM) was used to complete the optimization procedure. The optimal levels of the chosen variables which were 30.0 g/l, glucose; 5 g/l, yeast extract and 10.0 g/l sodium acetate trihydrate with the predicted LA-EPS-20079 concentration of 794.82 µg/ml. Model validity reached 99.93% when the results were verified. Both optimized trials showed great cytotoxic effects against colon cancer line (CaCo-2) with inhibition percentages ranging from 64.6 to 81.9%. Moreover, downregulation in the expression level of BCL2 and Survivin genes was found with a fold change of 3.377 and 21.38, respectively. Finally, we concluded that the optimized LA-EPS-20079 has maintained its anticancer effect against the CaCo-2 cell line that was previously reported by our research group.

Pseudomonas aeruginosa's greenish-blue pigment pyocyanin: its production and biological activities.

A subject of great interest is the bioprospecting of microorganisms and their bioactive byproducts, such as pigments. Microbial pigments have various benefits, including being safe to use due to their natural makeup, having therapeutic effects, and being produced all year round, regardless of the weather or location. Pseudomonas aeruginosa produces phenazine pigments that are crucial for interactions between Pseudomonas species and other living things. Pyocyanin pigment, which is synthesized by 90-95% of P. aeruginosa, has potent antibacterial, antioxidant, and anticancer properties. Herein, we will concentrate on the production and extraction of pyocyanin pigment and its biological use in different areas of biotechnology, engineering, and biology.

A promising microbial α-amylase production, and purification from Bacillus cereus and its assessment as antibiofilm agent against Pseudomonas aeruginosa pathogen.

BACKGROUND AND AIM: The purpose of the current study is to isolate a heavily amylase-producing bacteria of the genus Bacillus from soil samples, optimize the production of the enzyme, purify it, and evaluate its activity against biofilm-producing bacteria. A total of 12 soil samples were collected and screened for promising Bacillus species with good amylolytic activity. Isolation was done by serial dilution and plating technique and amylolytic activity was determined by starch agar plate method. Among the 12 Bacillus isolates recovered from soil samples, 7 showed positive α-amylase production. The best isolate that recorded the greatest amylolytic activity was selected for further studies. This isolate was identified by 16S rRNA sequencing as Bacillus cereus and registered under gene bank accession number OP811897. Furthermore, the α-amylase enzyme was produced by a submerged fermentation technique using best production media and partially purified by ammonium sulfate and chilled ethanol and molecular weight had been determined by SDS-PAGE gel electrophoresis. The production of α-amylase was optimized experimentally by one-factor at a time protocol and statistically by Plackett-Burman design as well as RSM CCD design. Data obtained from OFAT and CCD revealed that α-amylase activities were 1.5- and twofold respectively higher as compared to un-optimized conditions. The most significant factors had been identified and optimized by CCD design. RESULTS: Among the eleven independent variables tested by PBD, glucose, peptone, (NH4)2SO4, and Mg SO4 were the most significant parameters for α-amylase production with an actual yield of 250U/ml. The best physical parameters affecting the enzyme production were incubation time at 35 °C, and pH 5.5 for 48 h. The partially purified enzyme with 60% ammonium sulphate saturation with 1.38- fold purification showed good stability characteristics at a storage temperature of 4 °C and pH up to 8.5 for 21 days. Antibiofilm activity of purified α-amylase was determined against Pseudomonas aeruginosa (ATCC 35659) by spectrophotometric analysis and CLSM microscopic analysis. Results demonstrated biofilm inhibition by 84% of the formed Pseudomonas biofilm using a microtiter plate assay and thickness inhibition activity by 83% with live/Dead cells percentage of 17%/83% using CLSM protocol. CONCLUSIONS: A highly stable purified α-amylase from B. cereus showed promising antibiofilm activity against one of the clinically important biofilm-forming MDR organisms that could be used as a cost-effective tool in pharmaceutical industries.

A purified and lyophilized Pseudomonas aeruginosa derived pyocyanin induces promising apoptotic and necrotic activities against MCF-7 human breast adenocarcinoma.

BACKGROUND: Pyocyanin, a specific extracellular secondary metabolite pigment produced by Pseudomonas aeruginosa, exhibits redox activity and has toxic effects on mammalian cells, making it a new and potent alternative for treating cancer. Breast cancer (BC) treatment is now defied by acquired and de novo resistance to chemotherapy, radiation, or targeted therapies. Therefore, the anticancer activity of purified and characterized pyocyanin was examined against BC in our study. RESULTS: The maximum production of pyocyanin (53 µg/ml) was achieved by incubation of the highest pyocyanin-producing P. aeruginosa strain (P32) in pH-adjusted peptone water supplemented with 3% cetrimide under shaking conditions at 37 °C for 3 days. The high purity of the extracted pyocyanin was proven by HPLC against standard pyocyanin. The stability of pyocyanin was affected by the solvent in which it was stored. Therefore, the purified pyocyanin extract was lyophilized to increase its shelf-life up to one year. Using the MTT assay, we reported, for the first time, the cytotoxic effect of pyocyanin against human breast adenocarcinoma (MCF-7) with IC50 = 15 µg/ml while it recorded a safe concentration against human peripheral blood mononuclear cells (PBMCs). The anticancer potential of pyocyanin against MCF-7 was associated with its apoptotic and necrotic activities which were confirmed qualitatively and quantitively using confocal laser scanning microscopy, inverted microscopy, and flow cytometry. Caspase-3 measurements, using real-time PCR and western blot, revealed that pyocyanin exerted its apoptotic activity against MCF-7 through caspase-3 activation. CONCLUSION: Our work demonstrated that pyocyanin may be an ideal anticancer candidate, specific to cancer cells, for treating MCF-7 by its necrotic and caspase-3-dependent apoptotic activities.

In vitro and in silico ß-lactamase inhibitory properties and phytochemical profile of Ocimum basilicum cultivated in central delta of Egypt.

CONTEXT: Some studies reported the chemical content and antimicrobial properties of Ocimum basilicum L. (Lamiaceae), relevant to the ecological variations in some areas of Egypt and other countries, yet no research was conducted on the plant cultivated in the central delta region of Egypt. Also, no previous data reported on inhibition of ß-lactamases by O. basilicum. OBJECTIVE: To assess ß-lactamases inhibition by O. basilicum extracts and the individual constituents. MATERIALS AND METHODS: Dried aerial parts of O. basilicum were extracted by hydrodistillation for preparation of essential oil and by methanol for non-volatile constituents. Essential oil content and the methanol extract were analysed by GC-MS and UPLC-PDA-MS/MS, respectively. Methyl cinnamate was isolated and analysed by NMR. Broth microdilution method was used to investigate the antimicrobial against resistant clinical isolates of Escherichia coli identified by double disc synergy, combination disc tests and PCR. The most active oil content was further tested with a nitrocefin kit for ß-lactamase inhibition and investigated by docking. RESULTS: O. basilicum was found to contain methyl cinnamate as the major content of the essential oil. More interestingly, methyl cinnamate inhibited ESBL ß-lactamases of the type CTX-M. The in vitro IC50 using nitrocefin kit was 11.6 µg/mL vs. 8.1 µg/mL for clavulanic acid as a standard ß-lactamase inhibitor. DISCUSSION AND CONCLUSIONS: This is the first study to report the inhibitory activity of O. basilicum oil and methyl cinnamate against ß-lactamase-producing bacteria. The results indicate that methyl cinnamate could be a potential alternative for ß-lactamase inhibition.

A novel purified Lactobacillus acidophilus 20079 exopolysaccharide, LA-EPS-20079, molecularly regulates both apoptotic and NF-κB inflammatory pathways in human colon cancer.

BACKGROUND: The direct link between inflammatory bowel diseases and colorectal cancer is well documented. Previous studies have reported that some lactic acid bacterial strains could inhibit colon cancer progression however; the exact molecules involved have not yet been identified. So, in the current study, we illustrated the tumor suppressive effects of the newly identified Lactobacillus acidophilus DSMZ 20079 cell-free pentasaccharide against colon cancer cells. The chemical structure of the purified pentasaccharide was investigated by MALDI-TOF mass spectrum, 1D and 2D Nuclear Magnetic Resonance (NMR). The anticancer potentiality of the purified pentasaccharide against both Human colon cancer (CaCo-2) and Human breast cancer (MCF7) cell lines with its safety usage pattern were evaluated using cytotoxicity, annexin V quantification and BrdU incorporation assays. Also, the immunomodulatory effects of the identified compound were quantified on both LPS-induced PBMC cell model and cancer cells with monitoring the immunophenotyping of T and dendritic cell surface marker. At molecular level, the alteration in gene expression of both inflammatory and apoptotic pathways were quantified upon pentasaccharide-cellular treatment by RTqPCR. RESULTS: The obtained data of the spectroscopic analysis, confirmed the structure of the newly extracted pentasaccharide; (LA-EPS-20079) to be: α-D-Glc (1→2)][α-L-Fuc(1→4)] α-D-GlcA(1→2) α-D-GlcA(1→2) α-D-GlcA. This pentasaccharide, recorded safe dose on normal mammalian cells ranged from 2 to 5 mg/ml with cancer cells selectivity index, ranged of 1.96-51.3. Upon CaCo-2 cell treatment with the non-toxic dose of LA-EPS-20079, the inhibition percentage in CaCo-2 cellular viability, reached 80.65 with an increase in the ratio of the apoptotic cells in sub-G0/G1 cell cycle phase. Also, this pentasaccharide showed potentialities to up-regulate the expression of IKbα, P53 and TGF genes. CONCLUSION: The anticancer potentialities of LA-EPS-20079 oligosaccharides against human colon cancer represented through its regulatory effects on both apoptotic and NF-κB inflammatory pathways.

Exploring the potential efficacy of phage therapy for biocontrol of foodborne pathogenic extensively drug-resistant Escherichia coli in gastrointestinal tract of rat model.

AIM: The emergence of extensively drug-resistant (XDR) Escherichia coli leaves little or no therapeutic options for the control of these foodborne pathogens. The goal is to isolate, characterize, and assess the potential efficacy of a bacteriophage in the treatment of an induced gastrointestinal tract infection. MAIN METHODS: Sewage water was used to isolate phage phPE42. Transmission electron microscope was used for the visualization of phage morphology. Lysis profile, growth kinetics, and stability studies were determined. The ability of phage to eradicate biofilms was assessed by crystal violet staining, resazurin assay, compound bright field microscope, and confocal laser scanning microscope (CLSM). Moreover, the efficacy of phage phPE42 as a potential therapy was evaluated in a rat model. KEY FINDINGS: A newly lytic Myoviridae phage phPE42 was isolated and exhibited broad coverage activity (48.6 %) against E. coli clinical isolates. It demonstrated favorable growth kinetics and relative stability under a variety of challenging conditions. The resazurin colorimetric assay and CLSM provided evidence of phage potential's ability to significantly (P < 0.05) decrease the viability of biofilm-embedded cells. The bacterial burden in animal faeces was effectively eradicated (P < 0.05) by oral administration of phage phPE42. Phage-treated rats exhibited a significant decrease in tissue damage with no signs of inflammation, necrosis, or erosion. Furthermore, phage therapy significantly (P < 0.05) reduced the expression level of the apoptotic marker caspase-3 and the inflammatory cytokine TNF-α. SIGNIFICANCE: Treatment with phage phPE42 is considered a promising alternative therapy for the control of severe foodborne infections spurred by pathogenic XDR E. coli.

Characterization and genomic analysis of novel bacteriophage NK20 to revert colistin resistance and combat pandrug-resistant Klebsiella pneumoniae in a rat respiratory infection model.

AIM: We investigated the therapeutic capacity of the isolated Klebsiella bacteriophage NK20 against pandrug-resistant strains. Moreover, we assessed the impact of resistance development on the overall therapeutic outcome both in vitro and in vivo. MAIN METHODS: The pandrug-resistant K. pneumoniae Kp20 is used as a host strain for the isolation of bacteriophages using sewage samples. Spot assay was then used to compare the spectra of the isolated phages, while kinetic and genomic analysis of the phage with the broadest spectrum was assessed. Antibacterial potential of the phage was assessed using turbidimetric assay and MIC with and without colistin. Finally, the therapeutic efficacy was evaluated in vivo using a rat respiratory infection model. KEY FINDINGS: The isolated lytic bacteriophage (NK20) showed a relatively broad spectrum and an acceptable genomic profile. In vitro antibacterial assay revealed bacterial resistance development after 12 h. Colistin inhibited bacterial regrowth and reduced pandrug-resistant strains' colistin MICs. Despite the isolation of resistant clones, intranasal administration of NK20 significantly (p < 0.05) reduced the bacterial load in both the pulmonary and blood compartments and rescued 100 % of challenged rats. Histological and immunological analysis of treated animals' lung tissue revealed less inflammation and lower TNF-α and caspase-3 expression. SIGNIFICANCE: NK20 is a promising candidate that rescued rats from untreatable, pan-drug-resistant K. pneumoniae Kp20. Moreover, it steers the evolution of resistant mutants with higher sensitivity to colistin and less virulence, opening the door for using phages as sensitizing and anti-virulence entities rather than direct killer.

A Novel Triple Combination To Combat Serious Infections with Carbapenem-Resistant Acinetobacter baumannii in a Mouse Pneumonia Model.

The ongoing crisis of antimicrobial resistance demands novel combinations between antimicrobials and nonantimicrobials to manage infections caused by highly resistant pathogens. This study aimed to evaluate the effect of combining sodium ascorbate and/or apo-transferrin with imipenem, forming double and triple combinations, against 20 multiple-carbapenemase-producing Acinetobacter baumannii strains using the checkerboard test, time-kill assay, and disc diffusion test. The results of the checkerboard assay revealed that all double combinations showed indifference, while only triple combination recorded a synergistic effect (fractional inhibitory concentration index [FICI] < 0.8) in 95% the test isolates. Moreover, the MIC of imipenem (MICimp) was strongly reduced (up to 128-fold reduction) after treatment with the triple combination against highly resistant isolates and reached the susceptible range. The time-kill assay revealed that the triple combination led to a 4-log10 reduction in the CFU at 8 h compared with the initial bacterial count, and no viable count was recorded at 10 h. The mouse pneumonia model showed restoration of lung function and structure, with mild to moderate residual inflammation and moderately congested vessels observed 8 h following administration of the triple rescue therapy. Additionally, normal lungs with normal patent alveoli were detected 72 h following treatment. Accordingly, sodium ascorbate and apo-transferrin are promising adjunct biological agents with the potential to restore the effectiveness of critically essential antibiotics like imipenem, commonly used for the treatment of A. baumannii infections. IMPORTANCE Combination therapy provides a perspective to threat multidrug-resistant (MDR) strains. The present study sheds light on a novel and effective triple combination against carbapenem-resistant A. baumannii. Our in vitro results showed that combining imipenem with apo-transferrin and sodium ascorbate yielded synergism in 95% of test isolates, and this was associated with a marked reduction in imipenem MIC, shifting it below the breakpoint. Furthermore, a bactericidal effect was recorded, with no viable count detected at 10 h. An in vivo murine model of pneumonia was induced to mimic human disease. The triple combination therapy restored lung function and structure, with mild to moderate residual inflammation and moderately congested vessels observed 8 h following the initiation of therapy. Therefore, our findings suggest novel insights about a promising new combination therapy against highly resistant carbapenemase-producing A. baumannii to restore the effectiveness of imipenem.

Characterization of newly isolated bacteriophage to control multi-drug resistant Pseudomonas aeruginosa colonizing incision wounds in a rat model: in vitro and in vivo approach.

AIMS: Pseudomonas aeruginosa is one of the most common causes of opportunistic and hospital-acquired infections in the world, which is repeatedly associated with treatment challenges. The evolution of new approaches such as phage therapy may be a novel alternative strategy for the treatment of these life-threatening infections. This paper aims to characterize the isolated bacteriophage and evaluate its potential therapy for the treatment of induced skin infection. MAIN METHODS: Enrichment method and double-layer overlay agar were used for isolation and purification of bacteriophages. The lysis profiles of isolated phages were evaluated using spot method. The phage morphology was visualized by transmission electron microscope. The growth kinetics such as adsorption rate, latent period, burst size, and in vitro challenging activity were determined. Biofilm eradication was analyzed using confocal laser scanning microscope (CLSM). Furthermore, the potential activity of phage therapy was evaluated in a rat model. KEY FINDINGS: Eight phages were isolated while phage phPS127 displayed the strongest lytic spectra. This phage is a member of Siphoviridae family that showed good growth kinetics. Our in vitro results showed that phage phPS127 significantly decreased the bacterial density (P < 0.05). CLSM revealed the significant reduction in the viability of the biofilm-adhered cells (P < 0.05). Phage therapy provided a significant level of treatment and promoted wound healing. Moreover, phage therapy significantly decreased bacterial burden (P < 0.05), inflammatory cytokine (TNF-α) and apoptosis (caspase-3) expression level. SIGNIFICANCE: Phage phPS127 can be considered as a promising candidate for treatment of clinical P. aeruginosa infections.

Exploring the Therapeutic Potentials of Exopolysaccharides Derived From Lactic Acid Bacteria and Bifidobacteria: Antioxidant, Antitumor, and Periodontal Regeneration.

The metabolites of lactic acid bacteria (LAB) and bifidobacteria (Bb) have recently received a lot of attention due to their ability to protect interactions in blood and tissues, as well as their biodegradability and biocompatibility in human tissue. Exopolysaccharides (EPS) derived from bacteria have a long history of use in therapeutic and other industrial applications with no adverse effects. In this regard, EPSs were isolated and characterized from LAB and Bb culture supernatants to determine their antioxidant, antitumor, and periodontal regeneration properties. The antioxidant capacity of the EPSs varied with concentration (0.625-20 mg/ml). The highest antioxidant activity was found in LAB: Streptococcus thermophiles DSM 24731-EPS1, Lactobacillus delbrueckii ssp. bulgaricus DSM 20081T-EPS5, Limosilactobacillus fermentum DSM 20049-EPS6, and Bb; Bifidobacterium longum ssp. longum DSM 200707-EPS10. Human breast cancer cells (MCF7), human colon cancer cells (CaCo2), human liver cancer cells (HepG2), and human embryonic kidney 293 (HEK 293) cells were used as controls to assess the antitumor properties of the selected EPSs. According to the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium-bromide (MTT) assay, EPS5 had the highest cytotoxicity against MCF7, CaCo2, and HepG2, with IC50 values of 7.91, 10.69, and 9.12 mg/ml, respectively. Lactate dehydrogenase (LDH) activity was significantly higher in cell lines treated with EPS5-IC50 values compared to other EPSs-IC50 values (p < 0.05). Real time (RT)-PCR results showed that EPS5 treatment increased Bax, Caspase 8, Caspase 3, and p53 gene expression. The expression of the BCL2, MCL1, and Vimentin genes, on the other hand, was reduced. The MTT test was used to examine the effect of EPS5 on the viability of human periodontal ligament fibroblast cells (hPDLFCs), and it was discovered that EPS5 increased hPDLFC viability. According to high-performance liquid chromatography (HPLC) analysis, galactose made up 12.5% of EPS5. The findings of this study pave the way for the use of EPS, which hold great promise for a variety of therapeutic purposes such as antioxidant, antitumor, and periodontal regeneration.

Novel Preclinical Study of Galloylquinic Acid Compounds from Copaifera lucens with Potent Antifungal Activity against Vaginal Candidiasis Induced in a Murine Model via Multitarget Modes of Action.

Vaginal candidiasis is a medical condition characterized by the overgrowth of Candida spp. in the vaginal cavity with complex recurrent pathogenicity as well as tolerance to antifungal therapy and hence is awaiting more safe and effective treatments. This work aimed to assess the potential antifungal activity of galloylquinic acid compounds (GQAs) from Copaifera lucens leaves against vaginal Candida albicans. The antifungal susceptibility test was performed against 20 isolates of multidrug-resistant (MDR) C. albicans using agar diffusion and broth microdilution assays. The results showed that GQAs exhibited strong antagonistic activity against the test isolates, with inhibition zone diameters ranging from 26 to 38 mm and low MICs (1 to 16 µg/mL) as well as minimum fungicidal concentrations (2 to 32 µg/mL). The MTT [3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2H-tetrazolium bromide] assay confirmed the safety of GQAs against the Vero cell line, showing a 50% inhibitory concentration (IC50) of 168.17 mg/mL. A marked difference in the growth pattern of the treated and untreated pathogens was also observed, where a concentration-dependent reduction in the growth rate occurred. Moreover, a pronounced fungicidal effect was demonstrated 6 h after treatment with 1× the minimum fungicidal concentration (MFC), as evidenced by time-kill assays, where the number of survivors was decreased a 6-fold. GQAs effectively inhibited and eradicated about 80% of C. albicans biofilm at 6 µg/mL and 32 µg/mL, respectively. Interestingly, GQAs disturbed the fungal membrane integrity, induced cell lysis, and reduced the virulence factors (proteinase and phospholipase) as well as the catalase activity. Moreover, the ergosterol content in the plasma membrane decreased in a concentration-dependent manner. Additionally, the altered mitochondrial membrane potential was associated with an increased release of cytochrome c from mitochondria to the cytosol, suggesting the initiation of early apoptosis in GQA-treated cells. Transcriptional analysis revealed that all test genes encoding virulence traits, including SAP1, PLB1, LIP1, HWP1, and ALS1, were markedly downregulated in GQA-treated cells compared to the control. The in vivo murine model of vaginal candidiasis further confirmed the therapeutic activity of GQAs (4 mg/kg of body weight) against C. albicans. This work comprehensively evaluated the antifungal, antivirulence, and antibiofilm activities of GQAs against C. albicans isolates using in vitro and in vivo models, providing molecular-level insights into the antifungal mechanism of action and experimental evidence that supports the potential use of GQAs for the treatment of vaginal candidiasis. IMPORTANCE Our work presents a new perspective on the potential use of GQAs as safe and highly effective phytochemicals against MDR C. albicans. This microorganism colonizes the human vaginal epithelium, causing vaginal candidiasis, a condition characterized by recurrent pathogenicity and tolerance to traditional antifungal therapy. Based on the results of in vitro tests, our study reports GQAs antifungal modes of action. These compounds exhibited an anticandidal effect by deactivating the fungal hydrolytic enzymes, reducing ergosterol content in the plasma membrane, altering the potential of the mitochondrial membrane, and inducing apoptosis. Additionally, GQAs showed high activity in eradicating the biofilm formed by the fungus via the downregulation of HWP1, ALS, SAP, PLB, and LIP genes, which are constitutively expressed in the biofilm. In an in vivo murine model of vaginal candidiasis, GQAs further demonstrated strong evidence of their effectiveness as an antifungal therapy. In this regard, our findings provide novel insights into the potential therapeutic use of these phytoactive molecules for vaginal candidiasis treatment.

Unravelling the In Vitro and In Vivo Anti-Helicobacter pylori Effect of Delphinidin-3-O-Glucoside Rich Extract from Pomegranate Exocarp: Enhancing Autophagy and Downregulating TNF-α and COX2.

Fruits containing antioxidants, e.g., anthocyanins, exhibit antimicrobial activities. The emergence of drug resistance represents a major challenge in eradicating H. pylori. The current study aims to explore the effect of pomegranate exocarp anthocyanin methanol extract (PEAME) against H. pylori isolates recovered from antral gastric biopsies. The UPLC-PDA-MS/MS and 1H NMR analyses indicated delphinidin-3-O-glucoside as the major anthocyanin in the extract. The PEAME showed activity against all tested resistant isolates in vitro recording minimum inhibitory concentration (MIC) and minimum bactericidal concentration (MBC) values of 128 and 256 µg/mL, respectively. In vivo investigation included evaluation of the rat gastric mucosa for malondialdehyde (MDA), catalase activity, COX2, TNF-α, and key autophagy gene expression. The combination of pomegranate with metronidazole markedly reduced the viable count of H. pylori and the level of COX2, with alleviation of H. pylori-induced inflammation and oxidative stress (reduction of MDA, p-value < 0.001; and increase in catalase activity, p-value < 0.001). Autophagy gene expression was significantly upregulated upon treatment, whereas TNF-α was downregulated. In conclusion, we comprehensively assessed the effect of PEAME against H. pylori isolates, suggesting its potential in combination with metronidazole for eradication of this pathogen. The beneficial effect of PEAME may be attributed to its ability to enhance autophagy.

Molecular characterisation of an Acinetobacter baumannii outbreak.

BACKGROUND: Acinetobacter baumannii are problematic hospital pathogens, and the increased incidence of multi drug resistance has significantly limited treatment options. The global epidemiology is not fully characterised due to large data gaps from low- and middle-income countries. This study characterised the molecular epidemiology of an A. baumanniii outbreak in Egypt. METHODS: Fifty-four A. baumannii isolates were recovered from a 4-month-outbreak at Tanta University Hospitals (TUH). Associated clinical and demographic data, and the antibiograms were analysed, and Carbapenem resistant isolates were screened for acquired carbapenemase genes by PCR and sequencing. Epidemiological typing was performed by single-locus sequencing of bla OXA-51-like and Multi Locus Sequence Typing (MLST), and sequence types (STs) were analysed based on maximum-likelihood phylogeny (PhyML) to identify relatedness. FINDINGS: Immune suppression and ICU admission were the most common co-morbidity and risk factor. Carbapenem resistance accounted for 81%, and correlated with the presence of OXA-23, NDM-1 and -2, and VIM-1 and -2 carbapenemases. Nine different bla OXA-51-like genes were identified which corresponded to 22 different Sequence Types (STs), including 10 novel. International clone (IC2) was the predominant clone. PhyML analysis revealed the presence of 2 distinct clones with multiple sub-lineages. CONCLUSION: Given the short duration of the study, there was a rare heterogeneous population in the hospital. Carbapenem resistance is mediated by acquired carbapenemases in diverse lineages indicating the possibility of horizontal gene transfer. The diversity indicates the influx of multiple lineages of IC2 into TUH from unknown sources. Molecular epidemiological studies are essential for infection prevention and control measures.

Preparation, and Assessment of Antidermatophyte Activity of Miconazole-Urea Water-Soluble Film.

Cutaneous mycoses, particularly tinea pedis caused by Trichophyton rubrum, are commonly known infections in humans. They are still considered as a major public health problem worldwide affecting the quality of life due to prolonged period of treatment and development of drug resistance, which leads to recurrence of infections. The objective of our study was to assess the effectiveness of miconazole in the presence and absence of urea, as a penetration enhancer, against T. rubrum and to formulate both of them in a water-soluble film to be applied topically for the purpose of treating tinea pedis caused by this fungus. Drug combination revealed synergism where miconazole minimum inhibitory concentration (MIC) and minimum fungicidal concentration (MFC) (0.5 and 1 mg/L) were considerably declined to 0.001 and 0.004 mg/L, respectively, when combined with 20% urea. This enhanced drug interaction activity against the test strain was explained by the alterations raised on the morphology and ultrastructures observed microscopically. Minimal fungicidal dose of miconazole/urea combination displayed plasmolysis and shrink cytoplasm; however, necrotic cells with punctured walls and degraded cytoplasmic content were observed at high fungicidal dose. Water-soluble films, prepared using increasing values of miconazole MFC and urea, were transparent, smooth, uniform, and flexible. Their physicochemical characters showed homogeneity in weight, thickness, drug content, and folding endurances with normal surface pH values, indicating the reproducibility of the preparation method. The novel simulation model for the film mechanism of action supported the idea and the suggested application method of the new dosage form. Evaluation of these films was carried in vitro using disk diffusion assay as well as in vivo using guinea pig dermatophytosis model. The in vitro assessment revealed an increase in the inhibition zone diameters in a concentration-dependent manner upon using 10 or 20% of urea combined with miconazole. In vivo test showed that combination of 0.004 mg/L miconazole with 20% urea (M + U20) showed the highest efficacy percentage (95.83%), which was statistically superior to the infected untreated control (p < 0.001) in fungal burden reduction as well as improvement in clinical scores (p < 0.001). This work supports the hypothesis and suggests a new promising dosage form for the treatment of T. rubrum infections.

Purification, Characterization, Identification, and Anticancer Activity of a Circular Bacteriocin From Enterococcus thailandicus.

New anticancer agents are continually needed because cancerous cells continue to evolve resistance to the currently available chemotherapeutic agents. The aim of the present study was to screen, purify and characterize a hepatotoxic bacteriocin from Enterococcus species. The production of bacteriocin from the Enterococcus isolates was achieved based on their antibacterial activity against indicator reference strains. Enterococcus isolates showed a broad spectrum of antibacterial activity by forming inhibition zones with diameters ranged between 12 and 29 mm. The most potent bacteriocin producing strain was molecularly identified as Enterococcus thailandicus. The crude extracted bacteriocin was purified by cation exchange and size exclusion chromatography that resulted in 83 fractions. Among them, 18 factions were considered as bacteriocins based on their positive antibacterial effects. The anticancer effects of the purified bacteriocins were tested against HepG2 cell line. The most promising enterocin (LNS18) showed the highest anticancer effects against HepG2 cells (with 75.24% cellular inhibition percentages), with IC50 value 15.643 µM and without any significant cytotoxic effects on normal fibroblast cells (BJ ATCC® CRL-2522™). The mode of anticancer action of enterocin LNS18 against HepG2 cells could be explained by its efficacy to induce cellular ROS, decrease HepG2 CD markers and arrest cells in G0 phase. Amino acid sequence of enterocin LNS18 was determined and the deduced peptide of the structural gene showed 86 amino acids that shared 94.7% identity with enterocin NKR-5-3B from E. faecium. Enterocin LNS18 consisted of 6 α-helices; 5 circular and one linear. Model-template alignment constructed between enterocin LNS18 and NKR-5-3B revealed 95.31% identity. The predicted 3D homology model of LNS18, after circularization and release of 22 amino acids, showed the formation of a bond between Leu23 and Trp86 amino acid residues at the site of circularization. Furthermore, areas of positive charges were due to the presence of 6 lysine residues resulting in a net positive charge of +4 on the bacteriocin surface. Based on the above mentioned results, our characterized bacteriocin is a promising agent to target liver cancer without any significant toxic effects on normal cell lines.

Stress-Based Production, and Characterization of Glutathione Peroxidase and Glutathione S-Transferase Enzymes From Lactobacillus plantarum.

More attention has been recently directed toward glutathione peroxidase and s-transferase enzymes because of the great importance they hold with respect to their applications in the pharmaceutical field. This work was conducted to optimize the production and characterize glutathione peroxidase and glutathione s-transferase produced by Lactobacillus plantarum KU720558 using Plackett-Burman and Box-Behnken statistical designs. To assess the impact of the culture conditions on the microbial production of the enzymes, colorimetric methods were used. Following data analysis, the optimum conditions that enhanced the s-transferase yield were the De Man-Rogosa-Sharp (MRS) broth as a basal medium supplemented with 0.1% urea, 0.075% H2O2, 0.5% 1-butanol, 0.0125% amino acids, and 0.05% SDS at pH 6.0 and anaerobically incubated for 24 h at 40°C. The optimum s-transferase specific activity was 1789.5 U/mg of protein, which was ~12 times the activity of the basal medium. For peroxidase, the best medium composition was 0.17% urea, 0.025% bile salt, 7.5% Na Cl, 0.05% H2O2, 0.05% SDS, and 2% ethanol added to the MRS broth at pH 6.0 and anaerobically incubated for 24 h at 40°C. Furthermore, the optimum peroxidase specific activity was 612.5 U/mg of protein, indicating that its activity was 22 times higher than the activity recorded in the basal medium. After SDS-PAGE analysis, GST and GPx showed a single protein band of 25 and 18 kDa, respectively. They were able to retain their activities at an optimal temperature of 40°C for an hour and pH range 4-7. The 3D model of both enzymes was constructed showing helical structures, sheet and loops. Protein cavities were also detected to define druggable sites. GST model had two large pockets; 185Å3 and 71 Å3 with druggability score 0.5-0.8. For GPx, the pockets were relatively smaller, 71 Å3 and 32 Å3 with druggability score (0.65-0.66). Therefore, the present study showed that the consortium components as well as the stress-based conditions used could express both enzymes with enhanced productivity, recommending their application based on the obtained results.