Miconazole and phenothiazine hinder the quorum sensing regulated virulence in Pseudomonas aeruginosa.

Antibiotic resistance is a major health problem worldwide. Pseudomonas aeruginosa is a Gram-negative pathogen with an arsenal of virulence factors and elevated antimicrobial resistance. It is a leading cause of nosocomial infections with high morbidity and mortality. The significant time and effort required to develop new antibiotics can be circumvented using alternative therapeutic strategies, including anti-virulence targets. This study aimed to investigate the anti-virulence activity of the FDA-approved drugs miconazole and phenothiazine against P. aeruginosa. The phenotypic effect of sub-inhibitory concentrations of miconazole and phenothiazine on biofilm, pyocyanin, protease, rhamnolipid and hemolysin activities in PAO1 strain was examined. qRT-PCR was used to assess the effect of drugs on quorum-sensing genes that regulate virulence. Further, the anti-virulence potential of miconazole and phenothiazine was evaluated in silico and in vivo. Miconazole showed significant inhibition of Pseudomonas virulence by reducing biofilm-formation approximately 45-48%, hemolytic-activity by 59%, pyocyanin-production by 47-49%, rhamnolipid-activity by approximately 42-47% and protease activity by 36-40%. While, phenothiazine showed lower anti-virulence activity, it inhibited biofilm (31-35%), pyocyanin (37-39%), protease (32-40%), rhamnolipid (35-40%) and hemolytic activity (47-56%). Similarly, there was significantly reduced expression of RhlR, PqsR, LasI and LasR following treatment with miconazole, but less so with phenothiazine. In-silico analysis revealed that miconazole had higher binding affinity than phenothiazine to LasR, RhlR, and PqsR QS-proteins. Furthermore, there was 100% survival in mice injected with PAO1 treated with miconazole. In conclusion, miconazole and phenothiazine are promising anti-virulence agents for P. aeruginosa.

Phenotypic, molecular, and in silico characterization of coumarin as carbapenemase inhibitor to fight carbapenem-resistant Klebsiella pneumoniae.

BACKGROUND: Carbapenems represent the first line treatment of serious infections caused by drug-resistant Klebsiella pneumoniae. Carbapenem-resistant K. pneumoniae (CRKP) is one of the urgent threats to human health worldwide. The current study aims to evaluate the carbapenemase inhibitory potential of coumarin and to test its ability to restore meropenem activity against CRKP. Disk diffusion method was used to test the antimicrobial susceptibility of K. pneumoniae clinical isolates to various antibiotics. Carbapenemase genes (NDM-1, VIM-2, and OXA-9) were detected using PCR. The effect of sub-MIC of coumarin on CRKP isolates was performed using combined disk assay, enzyme inhibition assay, and checkerboard assay. In addition, qRT-PCR was used to estimate the coumarin effect on expression of carbapenemase genes. Molecular docking was used to confirm the interaction between coumarin and binding sites within three carbapenemases. RESULTS: K. pneumoniae clinical isolates were found to be multi-drug resistant and showed high resistance to meropenem. All bacterial isolates harbor at least one carbapenemase-encoding gene. Coumarin significantly inhibited carbapenemases in the crude periplasmic extract of CRKP. The checkerboard assay indicated that coumarin-meropenem combination was synergistic exhibiting a fractional inhibitory concentration index ≤ 0.5. In addition, qRT-PCR results revealed that coumarin significantly decreased carbapenemase-genes expression. Molecular docking revealed that the binding energies of coumarin to NDM1, VIM-2, OXA-48 and OXA-9 showed a free binding energy of -7.8757, -7.1532, -6.2064 and - 7.4331 Kcal/mol, respectively. CONCLUSION: Coumarin rendered CRKP sensitive to meropenem as evidenced by its inhibitory action on hydrolytic activity and expression of carbapenemases. The current findings suggest that coumarin could be a possible solution to overcome carbapenems resistance in CRKP.

Evaluation of polymethyl-methacrylate and acetal denture base resins processed by two different techniques before and after nano-chlorohexidine surface treatment.

BACKGROUND: Flexible denture base polymers have gained popularity in modern dentistry however, their biofilm formation tendency, adversely affecting the oral tissue heath, remains a concern. Consequently, this study aimed to evaluate surface roughness and biofilm formation tendency of two types of denture base resins manufactured with two techniques before and after surface coating with chlorohexidine (CHX) NPs. MATERIALS AND METHODS: Acetal (AC) and Polymethyl-methacrylate (PMMA) resins manufactured by conventional and CAD/CAM methods were shaped into disk (10 X 10 X 1 mm). They were dipped for 8 h and 24 h in colloidal suspension prepared by mixing aqueous solution of CHX digluconate and hexa-metaphosphate (0.01 M). Surface roughness, optical density (OD) of microbial growth media and biofilm formation tendency were evaluated directly after coating. Elutes concentrations of released CHX were evaluated for 19 days using spectrophotometer. Three-way ANOVA and Tukey's post-hoc statistical analysis were used to assess the outcomes. RESULTS: AC CAD/CAM groups showed statistically significant higher roughness before and after coating (54.703 ± 4.32 and 77.58 ± 6.07 nm, respectively). All groups showed significant reduction in OD and biofilm formation tendency after surface coating even after 19 days of CHX NPs release. CONCLUSIONS: Biofilm formation tendency was highly relevant to surface roughness of tested resins before coating. After CHX NPs coating all tested groups showed significant impact on microbial growth and reduction in biofilm formation tendency with no relation to surface roughness. Significant antimicrobial effect remained even after 19 days of NPs release and specimens storage.

Optimizing Eco-Friendly Degradation of Polyvinyl Chloride (PVC) Plastic Using Environmental Strains of Malassezia Species and Aspergillus fumigatus.

Worldwide, huge amounts of plastics are being introduced into the ecosystem, causing environmental pollution. Generally, plastic biodegradation in the ecosystem takes hundreds of years. Hence, the isolation of plastic-biodegrading microorganisms and finding optimum conditions for their action is crucial. The aim of the current study is to isolate plastic-biodegrading fungi and explore optimum conditions for their action. Soil samples were gathered from landfill sites; 18 isolates were able to grow on SDA. Only 10 isolates were able to the degrade polyvinyl chloride (PVC) polymer. Four isolates displayed promising depolymerase activity. Molecular identification revealed that three isolates belong to genus Aspergillus, and one isolate was Malassezia sp. Three isolates showed superior PVC-biodegrading activity (Aspergillus-2, Aspergillus-3 and Malassezia) using weight reduction analysis and SEM. Two Aspergillus strains and Malassezia showed optimum growth at 40 °C, while the last strain grew better at 30 °C. Two Aspergillus isolates grew better at pH 8-9, and the other two isolates grow better at pH 4. Maximal depolymerase activity was monitored at 50 °C, and at slightly acidic pH in most isolates, FeCl3 significantly enhanced depolymerase activity in two Aspergillus isolates. In conclusion, the isolated fungi have promising potential to degrade PVC and can contribute to the reduction of environmental pollution in eco-friendly way.

Valuable effects of lactobacillus and citicoline on steatohepatitis: role of Nrf2/HO-1 and gut microbiota.

Non-alcoholic steatohepatitis (NASH) is a more dangerous form of chronic non-alcoholic fatty liver disease (NAFLD). In the current investigation, the influence of citicoline on high-fat diet (HFD)-induced NASH was examined, both alone and in combination with Lactobacillus (probiotic). NASH was induced by feeding HFD (10% sugar, 10% lard stearin, 2% cholesterol, and 0.5% cholic acid) to rats for 13 weeks and received single i.p. injection of streptozotocin (STZ, 30 mg/kg) after 4 weeks. Citicoline was given at two dose levels (250 mg and 500 mg, i.p.) at the beginning of the sixth week, and in combination with an oral suspension of Lactobacillus every day for eight weeks until the study's conclusion. HFD/STZ induced steatohepatitis as shown by histopathological changes, elevated serum liver enzymes, serum hyperlipidemia and hepatic fat accumulation. Moreover, HFD convinced oxidative stress by increased lipid peroxidation marker (MDA) and decreased antioxidant enzymes (GSH and TAC). Upregulation of TLR4/NF-kB and the downstream inflammatory cascade (TNF-α, and IL-6) as well as Pentaraxin, fetuin-B and apoptotic markers (caspase-3 and Bax) were observed. NASH rats also had massive increase in Bacteroides spp., Fusobacterium spp., E. coli, Clostridium spp., Providencia spp., Prevotella interrmedia, and P. gingivalis while remarkable drop in Bifidobacteria spp. and Lactobacillus spp. Co-treatment with citicoline alone and with Lactobacillus improve histopathological NASH outcomes and reversed all of these molecular pathological alterations linked to NASH via upregulating the expression of Nrf2/HO-1 and downregulating TLR4/NF-kB signaling pathways. These results suggest that citicoline and lactobacillus may represent new hepatoprotective strategies against NASH progression.

Genome Analysis of Pseudomonas aeruginosa Strains from Chronically Infected Patients with High Levels of Persister Formation.

The appearance of persister cells with low metabolic rates are key factors leading to antibiotic treatment failure. Such persisters are multidrug tolerant and play a key role in the recalcitrance of biofilm-based chronic infections. Here, we present the genomic analyses of three distinct Pseudomonas aeruginosa Egyptian persister-isolates recovered from chronic human infections. To calculate the persister frequencies, viable counts were determined before and after treatment with levofloxacin. The susceptibilities of isolates to different antibiotics were determined using the agar-dilution method. To determine their recalcitrance, the levofloxacin persisters were further challenged with lethal concentrations of meropenem, tobramycin, or colistin. Furthermore, the biofilm formation of the persister strains was estimated phenotypically, and they were reported to be strong biofilm-forming strains. The genotypic characterization of the persisters was performed using whole genome sequencing (WGS) followed by phylogenetic analysis and resistome profiling. Interestingly, out of the thirty-eight clinical isolates, three isolates (8%) demonstrated a persister phenotype. The three levofloxacin-persister isolates were tested for their susceptibility to selected antibiotics; all of the tested isolates were multidrug resistant (MDR). Additionally, the P. aeruginosa persisters were capable of surviving over 24 h and were not eradicated after exposure to 100X-MIC of levofloxacin. WGS for the three persisters revealed a smaller genome size compared to PAO1-genome. Resistome profiling indicated the presence of a broad collection of antibiotic-resistance genes, including genes encoding for antibiotic-modifying enzymes and efflux pump. Phylogenetic analysis indicated that the persister isolates belong to a distinct clade rather than the deposited P. aeruginosa strains in the GenBank. Conclusively, the persister isolates in our study are MDR and form a highly strong biofilm. WGS revealed a smaller genome that belongs to a distinct clade.

Impact of short chain fatty acids (SCFAs) on antimicrobial activity of new ß-lactam/ß-lactamase inhibitor combinations and on virulence of Escherichia coli isolates.

In a healthy gut microbiota, short chain fatty acids (SCFAs) are produced. The antibacterial action of SCFAs against intestinal pathogens makes them useful for ensuring the safety of food and human health. In this study, we aimed to assess the in vitro inhibitory activity of SCFAs, and to report, for the first time, their impact on the activity of new ß-lactam/ß-lactamase inhibitor combinations. The minimum inhibitory concentrations of acetic, propionic, and butyric acids were determined against E. coli clinical isolates recovered from gastrointestinal infections. Cefoperazone/sulbactam, ceftazidime/avibactam and cefepime/enmetazobactam are new ß-lactam/ß-lactamase inhibitor combinations that were studied for their combined therapeutic effects. Also, the effects of pH and concentration of SCFAs were evaluated on in vitro bacterial growth and expression of genes encoding for motility, adhesion, invasion, and biofilm formation. SCFAs were tested at concentrations of 12 mM at pH 7.4 (ileum-conditions), in addition to 60 mM and 123 mM, at pH 6.5 (colon-conditions). The tested SCFAs showed the same MIC (3750 µg ml-1 ≃ 60 mM) against all isolates. Furthermore, the addition of SCFAs to the tested ß-lactam/ß-lactamase inhibitor combinations greatly restored the susceptibility of the isolates. SCFAs had significant effect on bacterial growth and virulence in a pH and concentration-dependent manner; low ileal concentration potentiated E. coli growth, while higher colonic concentration significantly suppressed growth and down-regulated the expression of virulence genes (fliC, ipaH, FimH, BssS). Therefore, the significant inhibitory effect of colonic SCFAs on ß-lactam/ß-lactamase inhibitor combinations might lead to the development of promising treatment strategies.

In vitro activity of celastrol in combination with thymol against carbapenem-resistant Klebsiella pneumoniae isolates.

Klebsiella pneumoniae is an opportunistic pathogen causing nosocomial and community-acquired infections. Klebsiella has developed resistance against antimicrobials including the last resort class; carbapenem. Currently, treatment options for carbapenem-resistant-Klebsiella (CRK) are very limited. This study aims to restore carbapenem effectiveness against CRK using celastrol and thymol. Clinical Klebsiella isolates were identified using biochemical and molecular methods. Antimicrobial susceptibility was determined using disk-diffusion method. Carbapenemase-production was tested phenotypically and genotypically. Celastrol and thymol-MICs were determined and the carbapenemase-inhibitory effect of sub-MICs was investigated. Among 85 clinical Klebsiella isolates, 72 were multi-drug-resistant and 43 were meropenem-resistant. Phenotypically, 39 isolates were carbapenemase-producer. Genotypically, blaNDM1 was detected in 35 isolates, blaVIM in 17 isolates, blaOXA in 18 isolates, and blaKPC was detected only in 6 isolates. Celastrol showed significant inhibitory effect against carbapenemase-hydrolytic activity. Meropenem-MIC did not decrease in presence of celastrol, only 2-fold decrease was observed with thymol, while 4-64 fold decrease was observed when meropenem was combined with both celastrol and thymol. Furthermore, thymol increased CRK cell wall-permeability. Molecular docking revealed that celastrol is superior to thymol for binding to KPC and VIM-carbapenemase. Our study showed that celastrol is a promising inhibitor of multiple carbapenemases. While meropenem-MIC were not affected by celastrol alone and decreased by only 2-folds with thymol, it decreased by 4-64 folds in presence of both celastrol and thymol. Thymol increases the permeability of CRK-envelope to celastrol. The triple combination (meropenem/celastrol/thymol) could be useful for developing more safe and effective analogues to restore the activity of meropenem and other ß-lactams.

Repurposing pantoprazole and haloperidol as efflux pump inhibitors in azole resistant clinical Candida albicans and non-albicans isolates.

Candida species have a major role in nosocomial infections leading to high morbidity and mortality. Increased resistance to various antifungals, especially azoles is a significant problem. One of the main mechanisms for azole resistance is the up-regulation of efflux pump genes including CDR1 and MDR1. In the current study, clinical Candida isolates were identified to the species level and the antifungal susceptibility (AFS) of different Candida species was determined by disk diffusion method. Furthermore, the main mechanisms of azole resistance were investigated. Finally, haloperidol and pantoprazole were tested for their potential synergistic effect against fluconazole-resistant isolates. One hundred and twenty-two Candida clinical isolates were used in this study. 70 isolates were Candida albicans (57.4%), the non-albicans Candida species include: C. krusei (20.5%), C. tropicalis (6.6%), C. parapsilosis (5.7%), C. dubliniensis (4.9%) and C. glabrata (4.9%). The AFS testing showed that resistance to fluconazole and voriconazole were 13.1% (n = 16) and 9.8% (n = 12), respectively. Among the 16 resistant isolates, eight isolates (50%) were strong biofilm producers, seven (43.8 %) formed intermediate biofilm and one had no biofilm. All resistant strains overexpressed efflux pumps. Using RT-PCR, the efflux genes CDR1, MDR1 and ABC2 were over-expressed in azole resistant isolates. Haloperidol-fluconazole and pantoprazole-fluconazole combinations reduced the MIC of fluconazole in resistant isolates. The current study showed an increase in azole resistance of Candida species. The majority of resistant isolates form biofilm, and overexpress efflux pumps. Pantoprazole and Haloperidol showed a noteworthy effect as efflux pump inhibitors which oppose the fluconazole resistance in different Candida species.

The promising anti-virulence activity of candesartan, domperidone, and miconazole on Staphylococcus aureus.

Staphylococcus aureus is a primary cause of hospital and community-acquired infections. With the emergence of multidrug-resistant S. aureus strains, there is a need for new drugs discovery. Due to the poor supply of new antimicrobials, targeting virulence of S. aureus may generate weaker selection for resistant strains, anti-virulence agents disarm the pathogen instead of killing it. In this study, the ability of the FDA-approved drugs domperidone, candesartan, and miconazole as inhibitors of S. aureus virulence was investigated. The effect of tested drugs was evaluated against biofilm formation, lipase, protease, hemolysin, and staphyloxanthin production by using phenotypic and genotypic methods. At sub-inhibitory concentrations, candesartan, domperidone, and miconazole showed a significant inhibition of hemolysin (75.8-96%), staphyloxanthin (81.2-85%), lipase (50-65%), protease (40-64%), and biofilm formation (71.4-90%). Domperidone and candesartan have similar activity and were more powerful than miconazole against S. aureus virulence. The hemolysins and lipase inhibition were the greatest under the domperidone effect. Candesartan showed a remarkable reduction in staphyloxanthin production. The highest inhibitory effect of proteolytic activity was obtained with domperidone and candesartan. Biofilm was significantly reduced by miconazole. Expression levels of crtM, sigB, sarA, agrA, hla, fnbA, and icaA genes were significantly reduced under candesartan (68.98-82.7%), domperidone (62.6-77.2%), and miconazole (32.96-52.6%) at sub-MIC concentrations. Candesartan showed the highest inhibition activity against crtM, sigB, sarA, agrA, hla, and icaA expression followed by domperidone then miconazole. Domperidone showed the highest downregulation activity against fnbA gene. In conclusion, candesartan, domperidone, and miconazole could serve as anti-virulence agents for attenuation of S. aureus pathogenicity.

Impeding Virulence of Candida albicans by Candesartan and Domperidone.

Candida albicans is the most common human fungal pathogen that has developed extensive virulence factors which allows successful colonization and infection of the host. Anti-virulence agents can alleviate the pathogenesis of fungi and help the immune system to eradicate them easily. This study aimed to explore the anti-virulence effect of domperidone and candesartan against C. albicans standard strain. Sub-inhibitory concentrations (1/4 and 1/8 of minimum inhibitory concentration) of domperidone and candesartan significantly inhibited the virulence factors hemolysin, lipase, protease, phospholipase, and bioflim formation. It was found that candesartan inhibited biofilm formation by 60.48-67.91%, hemolysin activity (61.21-74.14%), phospholipase activity (40-49.67%), lipase activity (58.97-73%), and protease activity (52.63%), while domperidone was found to inhibit biofilm formation by 70.54-77.49%, hemolysin activity (64.84-69.84%), phospholipase activity (49.67-60%), lipase activity (50-54.87%), and protease activity (52.63-57.9%). Quantitative real time-PCR confirmed the anti-virulence activity of domperidone and candesartan as both drugs significantly reduce the expression of the virulence genes SAP2, SAP6, PLB1, PLB2, LIP4, LIP5. In conclusion, domperidone and candesartan could serve as anti-virulence agents for treatment of C. albicans infections.

Could the analgesic drugs, paracetamol and indomethacin, function as quorum sensing inhibitors?

The current failure of antimicrobials in treating life-threatening diseases, the high rate of multidrug resistant pathogens and the slow progress in the development of new antibiotics directed scientists to develop antivirulence drugs that targets quorum sensing (QS). In many microbes, QS acts as a communication system which control pathogenicity of microbes. Analgesics can be beneficial in controlling virulence traits of microbes and hence they may augment the efficacy of antimicrobials. In this study, two analgesics were screened for the inhibition of QS in Chromobacterium violaceum CV026 and their effects on virulence production in Pseudomonas aeruginosa PAO1 strain and clinical isolates of Acinetobacter baumannii were evaluated. The traits investigated were biofilm formation, pyocyanin and rhamnolipid production, twitching, swarming or surface associated motilities, production of protease, phospholipase and gelatinase enzymes and sensitivity to oxidative stress. Relative expression of abaI gene was calculated by performing qRT-PCR. Docking analysis of paracetamol as QSI (quorum sensing inhibitor) of AbaI and AbaR proteins was performed. Paracetamol inhibited QS in CV026, but indomethacin devoids anti-QS activity. Paracetamol inhibited virulence factors of PAO1. It strongly inhibited biofilm formation, and swarming by 66.4% and 57.1%, respectively. While, it moderately to slightly inhibited rhamnolipid, pyocyanin, gelatinase, resistance to oxidative stress, protease and twitching motility by 33.3%, 33.1% 17.5%, 9.1%, 8.7% and 7.7%, respectively. For A. baumannii, paracetamol strongly inhibited biofilm by 39.7-93% and phospholipase enzyme by 8.7-100%, reduced twitching and surface motility by 6.7-82.5% and 7.7-29.4%, respectively, And slightly reduced sensitivity to oxidative stress by 3.3-36.4%. Paracetamol at sub-MIC suppressed the expression of abaI gene by 32% in A. baumannii. Docking studies suggested that paracetamol can bind to AbaR and AbaI proteins and bind more to AbaR, hence it may act by inhibiting AHL signal reception. As a conclusion, paracetamol, beside its analgesic activity, has anti-QS activity and could be used in the eradication of P. aeruginosa and A. baumannii infections in combination with antibiotics.

The Link between Occurrence of Class I Integron and Acquired Aminoglycoside Resistance in Clinical MRSA Isolates.

Methicillin-resistant Staphylococcus aureus (MRSA) is a major cause of nosocomial infections because of its high resistance. Here, we study the antibiotic resistance in MRSA clinical isolates and their relation to integron I occurrence. A total of 88 clinical Staphylococcusaureus isolates were collected. MRSA were identified by the disk diffusion method (DDM) and confirmed by PCR, and antibiogram was determined by DDM. Integron I, II and the aacA4 gene were investigated by PCR. Integrase-positive strains were analyzed for the presence of resistance gene cassettes by sequencing. All isolates were identified as MRSA by DDM and confirmed by PCR. All isolates were resistant to ampicillin and cefoxitin. Concerning aminoglycosides, the frequency of resistance was reported for streptomycin (60.7%), tobramycin (37.1%) gentamicin (36%), and for amikacin (15.9%). Integron I was detected in 41 isolates (46.6%), while integron II was detected in three isolates (3.4%). Sequencing of the integron I-cassette indicated the exclusive prevalence of addA gene variants mediating aminoglycoside resistance. The aacA4 gene was found in DNA of 31 isolates (35.22%). This study revealed the high existence of MRSA. Furthermore, the AacA4 gene and class I integron harboring aadA gene were predominant in MRSA isolates.

The alarming association between antibiotic resistance and reduced susceptibility to biocides in nosocomial MRSA isolates from two regional hospitals in Egypt.

Methicillin-resistant Staphylococcus aureus is one of the major clinical problems in hospitals because of its resistance to many antimicrobials. Biocides are used in hospitals to control nosocomial infections. This work aimed to investigate the relationship between the presence of integrons and reduced susceptibility to both biocides and antimicrobials in nosocomial multidrug-resistant (MDR)-MRSA isolates. A total of 114 clinical and eight environmental MRSA isolates were collected from Zagazig University Hospitals and El-Ahrar Educational Hospital, Egypt. These isolates were identified as MRSA by disk diffusion method (DDM) and confirmed by PCR. Susceptibility profile against 12 antibiotics and five biocides was determined by DDM and agar dilution method, respectively. Presence of integrons was investigated by PCR in MDR isolates. Seventy-five clinical and six environmental isolates were MDR and had reduced susceptibility to biocides. Class I integron was detected in plasmid DNA of 34 isolates and genomic DNA of 14 isolates. Meanwhile, class II integron was only detected in plasmid DNA of 10 clinical isolates. This study revealed a high prevalence of MDR-MRSA clinical and environmental isolates, both had reduced susceptibility to investigated biocides. Class I integron was more predominant in plasmid DNA of isolates, indicating that plasmid is a major carrier for integrons that transfer resistance genes. In conclusion, the association between antibiotic resistance and biocides reduced susceptibility is alarming. The selection of curative antibiotic should depend on the antimicrobial susceptibility profile. Furthermore, biocides should always be used at appropriate concentrations to prevent the evolution of resistance and to control the hospital-transmission of MRSA.

Back to Nature: Combating Candida albicans Biofilm, Phospholipase and Hemolysin Using Plant Essential Oils.

Candida albicans is the causative agent of fatal systemic candidiasis. Due to limitations of antifungals, new drugs are needed. The anti-virulence effect of plant essential oils (EOs) was evaluated against clinical C. albicans isolates including cinnamon, clove, jasmine and rosemary oils. Biofilm, phospholipase and hemolysin were assessed phenotypically. EOs were evaluated for their anti-virulence activity using phenotypic methods as well as scanning electron microscopy (SEM) and atomic force microscopy (AFM). Among the C. albicans isolates, biofilm, phospholipase and hemolysins were detected in 40.4, 86.5 and 78.8% of isolates, respectively. Jasmine oil showed the highest anti-biofilm activity followed by cinnamon, clove and rosemary oils. SEM and AFM analysis showed reduced adherence and roughness in the presence of EOs. For phospholipase, rosemary oil was the most inhibitory, followed by jasmine, cinnamon and clove oils, and for hemolysins, cinnamon had the highest inhibition followed by jasmine, rosemary and clove oils. A molecular docking study revealed major EO constituents as promising inhibitors of the Als3 adhesive protein, with the highest binding for eugenol, followed by 1,8-cineole, 2-phenylthiolane and cinnamaldehyde. In conclusion, EOs have a promising inhibitory impact on Candida biofilm, phospholipase and hemolysin production, hence EOs could be used as potential antifungals that impact virulence factors.

Diclofenac mitigates virulence of multidrug-resistant Staphylococcus aureus.

Staphylococcus aureus is an opportunistic pathogen that has the ability to cause a wide range of diseases including superficial infection and severe invasive life threatening infections. The pathogenicity of S. aureus is mediated by a group of virulence factors that mediate the colonization and penetration. The antibiotic resistance of S. aureus has evolved due to the abuse of antibiotics rendering the cure of infection very difficult especially with the shortage in new antibiotic production. To combat this shortage, repurposing of FDA-approved drugs against the virulence factors is a new strategy. The analgesic drug Diclofenac was found to have anti-virulence activity against Pseudomonas aeruginosa and Proteus mirabilis. This study aimed to demonstrate the anti-virulence effect of diclofenac against clinical MRSA isolates phenotypically and genotypically using qRT-PCR. In this study, diclofenac showed significant reduction in biofilm formation when compared to controls, the inhibition ranged between 22.67% and 70%. Also, remarkable inhibition of hemolysin activity was found (5.4-66.34%). Additionally, diclofenac has inhibitory activity against the staphyloxanthin production (8-57.2%). The results were confirmed by qRT-PCR that showed significant down-regulation of tested virulence genes. The down-regulation ranged from 43 to 64.05% for SarA, 36.85-64.75% for AgrA, 50-63.2% for hla, 38.55-60.35% for FnbA, 46.75-61.05% for IcaA, 27.55-64% for SigB and 51.05-72.8% for CrtM. In conclusion, diclofenac can be used in combination with antibiotics as anti-virulence agent against MDR-MRSA which will enhance the ability of immune system to eradicate infection.

Distribution of genes encoding adhesins and biofilm formation capacity among Uropathogenic Escherichia coli isolates in relation to the antimicrobial resistance.

BACKGROUND: Escherichia coli is the most predominant pathogen involved in UTIs. Mainly, fimbrial surface appendages are implicated in adherence to urothelium besides non-fimbrial proteins. OBJECTIVES: to determine prevalence of genes encoding fimbrial and non-fimbrial proteins among Uropathogenic Escherichia coli (UPEC). Furthermore, distribution of these genes and biofilm formation capacity were investigated in relation to antimicrobial resistance. METHODS: Antimicrobial susceptibility of 112 UPEC isolates was performed using disc diffusion method. ESBL production was confirmed by double disc synergy test. Genes encoding fimbrial and non-fimbrial proteins were detected using PCR and biofilm formation was investigated using microtitre plate assay. RESULTS: UPEC isolates exhibited high resistance against doxycyclines (88.39 %), ß-lactams (7.14-86.6%), sulphamethoxazole-trimethoprim (53.75%) and fluoro-quinolones (50%). Fifty percent of tested isolates were ESBL producers. PapGII gene was statistically more prevalent among pyelonephritis isolates. SfaS, focG and picU genes were statistically associated with fluoroquinolone (FQs) sensitive isolates and Dr/afaBC gene was statistically associated with ESBL production. Moreover, non-MDR isolates produced sturdier biofilm. CONCLUSION: PapGII adhesin variant seems to have a critical role in colonization of upper urinary tract. There is a possible link between antimicrobial resistance and virulence being capable of affecting the distribution of some genes besides its negative impact on biofilm formation.

Phenotypic and genotypic detection of antibiotic resistance of Pseudomonas aeruginosa isolated from urinary tract infections.

BAKGROUND: Pseudomonas aeruginosa is a major nosocomial uropathogen. It can tolerate a wide variety of physical conditions and many antibiotics by different resistance mechanisms. OBJECTIVES: This study aimed to investigate the mechanisms of antibiotics resistance in uropathogenic P. aeruginosa clinical isolates. METHODS: Two hundred sixty six urine samples were collected from Zagazig University Hospitals, Zagazig, Egypt. P. aeruginosa isolates were identified using standard microbiological tests. The sensitivity to different antibiotics was determined by disc diffusion method. Anti-microbial resistance mechanisms were investigated using phenotypic methods and confirmed by PCR. RESULTS: Fifty P. aeruginosa isolates were recovered. All isolates were MDR and were resistant to amoxicillin/clavulinic, sulphamethaxzole/trimethoprim, doxycycline and ceftazidime. Phenotypic detection of resistance mechanisms revealed that all strains have efflux mechanism, outer membrane porins, and AmpC ß-lactamase; none of the strains showed ESBL activity and two of the imipenem resistant strains showed MßL activity. PCR analysis showed that all strains have MexAB-R, OprD and AmpC genes, 42 strains had PSE gene, while VEB and VIM genes were not detected. CONCLUSION: The resistance rates in P. aeruginosa were higher than global values; this resistance was attributed to several mechanisms. This high resistance is alarming and necessitates applying strict antibiotic prescription policies.

Deletion of Aspergillus nidulans GDP-mannose transporters affects hyphal morphometry, cell wall architecture, spore surface character, cell adhesion, and biofilm formation.

Systemic human fungal infections are increasingly common. Aspergillus species cause most of the airborne fungal infections. Life-threatening invasive aspergillosis was formerly found only in immune-suppressed patients, but recently some strains of A. fumigatus have become primary pathogens. Many fungal cell wall components are absent from mammalian systems, so they are potential drug targets. Cell-wall-targeting drugs such as echinocandins are used clinically, although echinocandin-resistant strains were discovered shortly after their introduction. Currently there are no fully effective anti-fungal drugs. Fungal cell wall glycoconjugates modulate human immune responses, as well as fungal cell adhesion, biofilm formation, and drug resistance. Guanosine diphosphate (GDP) mannose transporters (GMTs) transfer GDP-mannose from the cytosol to the Golgi lumen prior to mannosylation. Aspergillus nidulans GMTs are encoded by gmtA and gmtB. Here we elucidate the roles of A. nidulans GMTs. Strains engineered to lack either or both GMTs were assessed for hyphal and colonial morphology, cell wall ultrastructure, antifungal susceptibility, spore hydrophobicity, adherence and biofilm formation. The gmt-deleted strains had smaller colonies with reduced sporulation and with thicker hyphal walls. The gmtA deficient spores had reduced hydrophobicity and were less adherent and less able to form biofilms in vitro. Thus, gmtA not only participates in maintaining the cell wall integrity but also plays an important role in biofilm establishment and adherence of A. nidulans. These findings suggested that GMTs have roles in A. nidulans growth and cell-cell interaction and could be a potential target for new antifungals that target virulence determinants.

Cinnamomum zeylanicum bark essential oil induces cell wall remodelling and spindle defects in Candida albicans.

BACKGROUND: Cinnamon (Cinnamomum zeylanicum) bark extract exhibits potent inhibitory activity against Candida albicans but the antifungal mechanisms of this essential oil remain largely unexplored. RESULTS: We analyzed the impact of cinnamon bark oil on C. albicans RSY150, and clinical strains isolated from patients with candidemia and candidiasis. The viability of RSY150 was significantly compromised in a dose dependent manner when exposed to cinnamon bark oil, with extensive cell surface remodelling at sub inhibitory levels (62.5 µg/mL). Atomic force microscopy revealed cell surface exfoliation, altered ultrastructure and reduced cell wall integrity for both RSY150 and clinical isolates exposed to cinnamon bark oil. Cell wall damage induced by cinnamon bark oil was confirmed by exposure to stressors and the sensitivity of cell wall mutants involved in cell wall organization, biogenesis, and morphogenesis. The essential oil triggered cell cycle arrest by disrupting beta tubulin distribution, which led to mitotic spindle defects, ultimately compromising the cell membrane and allowing leakage of cellular components. The multiple targets of cinnamon bark oil can be attributed to its components, including cinnamaldehyde (74%), and minor components (< 6%) such as linalool (3.9%), cinamyl acetate (3.8%), α-caryophyllene (5.3%) and limonene (2%). Complete inhibition of the mitotic spindle assembly was observed in C. albicans treated with cinnamaldehyde at MIC (112 µg/mL). CONCLUSIONS: Since cinnamaldehyde disrupts both the cell wall and tubulin polymerization, it may serve as an effective antifungal, either by chemical modification to improve its specificity and efficacy or in combination with other antifungal drugs.