Thymoquinone' potent impairment of multidrug-resistant Staphylococcus aureus NorA efflux pump activity.

The drug efflux pump is a crucial mechanism implicated in resistance to multiple antimicrobials. Thymoquinone (TQ) has evidently demonstrated multiple activities, antibacterial being the most effective. Knowledge about TQ activity against multidrug-resistant Staphylococcus aureus is very scarce. Therefore, the present study was conducted to investigate TQ resistance modulation in ciprofloxacin (CIP) and doxycycline (DO) multidrug-resistant S. aureus. Forty-seven samples were collected from different sources, and S. aureus was isolated and identified. Then, S. aureus resistance profiles to antimicrobials, N. sativa essential oil, and TQ; the correlation between TQ-MIC readings and disc diffusion; cartwheel and ethidium bromide (EtBr) accumulation assays; and norA gene expression were all described within silico molecular docking for TQ interactions with norA efflux pump protein. TQ-MICs ranged from 5-320 µg/ml. TQ down-regulated norA gene expression, resulting in a drop in efflux pump activity of 77.5-90.6% in the examined strains, comparable to that observed with verapamil. Exposure of S. aureus strains to CIP and DO raises the initial basal efflux pumping expression to 34.2 and 22.9 times, respectively. This induced efflux pumping overexpression was substantially reduced by 97.7% when TQ was combined with CIP or DO. There was a significant reduction of MICs of CIP and DO MICs by 2-15 and 2-4 folds, respectively, after treatment with 0.5XMIC-TQ in resistance modulation assays. These results refer to TQ ligand inhibitory interactions with NorA protein in molecular docking. Interpretations of inhibition zone diameters (IZDs) of disc diffusion and TQ-MICs exhibit independence of MICs from IZDs, as indicated by invalid linear regression analysis. TQ significantly reduced efflux pumping S. aureus induced by CIP and DO, but further investigations are needed to improve TQ-pharmacokinetics to restore CIP and DO activity and suppress fluoroquinolone and doxycycline-resistant S. aureus selection in clinical and animal settings.

Immunomodulatory, antioxidant, and growth-promoting activities of dietary fermented Moringa oleifera in Nile tilapia (Oreochromus niloticus) with in-vivo protection against Aeromonas hydrophila.

BACKGROUND: Moringa oleifera, a well-known medicinal plant, has been used in aquafeed as a dietary supplement. Based on previous studies, insufficient research is available on the dietary supplementation of Nile tilapia with M. oleifera leaf and seed mixtures, specifically the fermented form. Therefore, this study aimed to investigate the efficacy of fermented (FMO) versus non-fermented M. oleifera (MO) leaf and seed mixtures on immunological parameters, antioxidant activity, growth performance, and resistance to A. hydrophila infection after a 30-day feeding trial on Nile tilapia. METHODS: A total of 180 fingerlings were randomly divided into four groups in addition to the control group (36 fish each, in triplicate). Fish in the tested groups were fed on basal diet supplemented with MO5%, MO10%, FMO5%, and FMO10%, while those in control were fed on basal diet only. After the feeding trial, fish were challenged with A. hydrophila. The immunomodulatory activity of M. oleifera was evaluated in terms of phagocytic and lysozyme activities, immune-related cytokines and IgM gene expression. Antioxidants, and growth-promoting activities were also assessed. RESULTS: The results revealed that fish supplemented FMO markedly in FMO10% group followed by FMO5%, exhibited significant (P < 0.05) improvement in the tested immunological, hepatic antioxidants, and growth performance parameters. Furthermore, the highest survival rate post-challenge with mild clinical symptoms, and the lowest A. hydrophila bacterial count were reported in these groups. Meanwhile, MO10%-supplementation exhibited the opposite trend. CONCLUSIONS: The study' conclusion suggests that fermented M. oleifera leaf and seed mixture is a promising growth-promoting and immunostimulatory feed-additive candidate for Nile tilapia and could reduce the losses caused by A. hydrophila infection.

Nigella sativa-chitosan nanoparticles: Novel intestinal mucosal immunomodulator controls and protects against Salmonella enterica serovar Enteritidis infection in broilers.

BACKGROUND: Salmonella Enteritidis (SE) propagates in chickens' gastrointestinal surfaces and is transmitted to humans, causing food poisoning. Oral supplementation with natural nanoparticles can overcome the harsh gastrointestinal conditions facing oral vaccines and requires no antibiotic administration to protect against microbial infection. This study was designed to study Nigella sativa-chitosan nanoparticles (CNP-NS) prophylactic immunomodulatory efficacy against SE infection in broiler chicks. The CNP-NS was prepared and characterized, and its in vivo immunomodulatory activities against an avian virulent-MDR SE-induced challenge in chicks were investigated. RESULT: To verify the immune-protective activities of the CNP-NS, colony forming units (CFU) in the liver and fecal droppings; intestinal histopathological alterations and immune cell recruitment; MUC-2, TLR-4, cecal cytokines, and specific IgA gene expression levels were assessed. On the 7th and 12th days after the SE challenge, the CNP-NS supplemented chicks showed complete clearance of SE CFU in livers and fecal droppings, as well as an improvement in food conversion rate compared to non-supplemented CNP-NS that revealed the presence of the challenge SE CFU on the same days. A prominent influx of antigen presenting cells and lymphoid aggregates into the intestinal wall, spleen, and liver was detected with improvements in the intestinal villi morphometry of the CNP-NS-supplemented chicks. The changes of INF-γ, IL-1ß, and IL-4 cecal cytokines, as well as TLR-4, MUC-2, and IgA mRNA expression levels, confirm CNP-NS immunomodulatory activities and provide a mechanism(s) for its protective actions against the induced SE challenge of the tested chickens. CONCLUSION: These findings suggest promising useful insights into CNP-NS supplementation as a safe food additive for poultry meat consumers' and a protective immunomodulator of the chickens' mucosal immune systems. It could be recommended for epidemiological purposes to reduce the risk of SE food poisoning and transmission to humans.

ß-1,3-glucan improved the health and immunity of juvenile African catfish (Clarias gariepinus) and neutralized the histological changes caused by lead and fipronil pollutants.

BACKGROUND: Water pollutants cause adverse effects in aquatic ecosystems. The immunomodulatory and mitigating effects of dietary 1,3-glucan on fipronil and lead-induced intoxication in African catfish (Clarias gariepinus) were investigated. Two hundred forty catfish were randomly divided into four equal groups: those in the first group were fed basic diet and served as controls; those in the second group were supplemented with ß-1,3-glucan (0.1%); those in the third group were exposed to combination of lead nitrate at 0.041 mg/L (1/10 96 h LC50) and fipronil at 2.8 mg/l (1/10 96 h LC50); and those in the fourth group were exposed to combination of fipronil, lead, and ß-1,3-glucan. The health status, haematological, immunological, and histological changes were all evaluated. RESULT: Swelling on the dorsolateral side, spinal column deviation, sluggish movement, skin bleaching, excessive mucus secretion, significant variations in blood indices-related measures, and a 45% death rate were observed in the third group. There was a significant reduction in interleukin-1 (IL-1) and interleukin-6 (IL-6) and immunoglobulin M (IgM) concentrations, as well as decrease in their corresponding gene expression, indicating that fipronil and lead had immunosuppressive activity. Severe catarrhal enteritis and mucinous degeneration of the lining epithelium, and notable depletion of white pulp, congested red pulp and hemosiderosis were common pathological findings in the spleen. ß-1,3-glucan alone or in combination with fipronil and lead provoked physical activity, blood indices, with elevations in IL-1ß, IL-2, IL-6, and IgM concentrations, as well as up-regulation in their genes' expression in splenic tissues, when compared to the third group. The spleen and intestine had normal histological architecture with 5% mortalities. There were no fish deaths in the ß-1,3-glucan-alone or control groups. CONCLUSION: The use of ß-1,3-glucan (0.1%) as dietary supplement could be implemented to protect against the toxic effects of fipronil and lead toxicity by improving the health and immunological parameters of intoxicated catfish.

Promising Anti-Biofilm Agents and Phagocytes Enhancers for the Treatment of Candida albicans Biofilm-Associated Infections.

Little is known about the interactions among phagocytes and antifungal agents and the antifungal immunomodulatory activities on Candida species biofilms. Here, inhibition of C. albicans biofilms and the interactions among biofilms and phagocytes alone or in combination with essential oils, biological, and chemical agents, or fluconazole were investigated. Biofilm formation by a panel of 28 C. albicans clinical isolates from hospitalized patients, birds, and cattle was tested. The anti-biofilm activities of cinnamon and clove oils, sodium dodecyl sulfate (SDS), cetyltrimethylammonium bromide (CTAB), and Enterococcus faecalis cell-free supernatant (CFS) in comparison with fluconazole were investigated using crystal violet and XTT reduction assays, expression of hypha-specific and hyphal regulator genes, and scanning electron microscopy (SEM) analysis. Of the tested C. albicans isolates, 15 of 28 (53.6%) were biofilm producers. Cinnamon followed by E. faecalis-CFS, SDS, and CTAB was the most effective inhibitors of planktonic C. albicans and biofilms. Fluconazole was an ineffective inhibitor of C. albicans biofilms. Sessile minimal inhibitory concentration (SMIC50) of cinnamon, SDS, CTAB, and E. faecalis-CFS downregulated the hypha-specific and regulator genes, albeit to various extents, when compared with untreated biofilms (P < 0.001). SEM analysis revealed disruption and deformity of three-dimensional structures in cinnamon oil-treated biofilms. C. albicans sessile cells within biofilm were less susceptible to phagocytosis than planktonic cells. The additive effects of phagocytes and the tested antifungals enabled phagocytes to engulf C. albicans cells rapidly in cinnamon, E. faecalis-CFS, or SDS-treated biofilms. No differences in anti-Candida or anti-biofilm eradication activities were detected among the tested isolates. Our findings reinforce the substantial anti-biofilm activity of cinnamon oil, SDS, and E. faecalis-CFS and provide new avenues for the development of novel anti-biofilm immunotherapies or antifungals that could be used prior to or during the management of cases with biofilm-associated infections.

Nigella sativa oil extract: A natural novel specific conjugal transfer inhibitor of vancomycin resistance from vanA/B-resistant Enterococcus faecium to Staphylococcus aureus.

AIM: The emergence of vancomycin-resistant Staphylococcus aureus (VRSA) has been identified as one of the most challenging problems in healthcare settings worldwide. Specific conjugation inhibitors' development is critical in the fight against the spread of emerging VRSA. The impact of Nigella sativa oil on VR genes conjugal transfer from Enterococcus faecium (VREtfm) to vancomycin-sensitive S. aureus (VSSA) was investigated in this study. METHODS AND RESULTS: Enterococciwere isolated from retail broilers, fish, cows' milk, and human urine. VR E. faecalis and VREtfm VanA phenotypes were prevalent in retail broiler samples. The VREtfm isolates were dominant, exhibiting high levels of resistance to gentamycin and ciprofloxacin antibiotics, as well as the existence of both vanA and vanB genes and virulence traits (ESP+ , asa1+ ) as determined by PCR. Transconjugant VREtfm strains containing vanA/vabB and 20 kb plasmids (transfer frequency around 103 ) and carrying the Tn1546 transposon were identified. Tn1546 transposon transfer with its VR markers to VSSA was effectively inhibited in treated VREtfm donor strains with a sub-minimum inhibitory concentration of N. sativa oil. THE SIGNIFICANCE AND IMPACT OF THE STUDY: This work offers new insights for overcoming VR conjugal transfer utilizing natural N. sativa oil, as well as a suggestion for a novel specialized conjugation inhibitor that could effectively facilitate the difficulty of eliminating VR bacteria from healthcare settings.

CCR2 promotes monocyte recruitment and intestinal inflammation in mice lacking the interleukin-10 receptor.

Macrophages are a heterogeneous population of mononuclear phagocytes abundantly distributed throughout the intestinal compartments that adapt to microenvironmental specific cues. In adult mice, the majority of intestinal macrophages exhibit a mature phenotype and are derived from blood monocytes. In the steady-state, replenishment of these cells is reduced in the absence of the chemokine receptor CCR2. Within the intestine of mice with colitis, there is a marked increase in the accumulation of immature macrophages that demonstrate an inflammatory phenotype. Here, we asked whether CCR2 is necessary for the development of colitis in mice lacking the receptor for IL10. We compared the development of intestinal inflammation in mice lacking IL10RA or both IL10RA and CCR2. The absence of CCR2 interfered with the accumulation of immature macrophages in IL10R-deficient mice, including a novel population of rounded submucosal Iba1+ cells, and reduced the severity of colitis in these mice. In contrast, the absence of CCR2 did not reduce the augmented inflammatory gene expression observed in mature intestinal macrophages isolated from mice lacking IL10RA. These data suggest that both newly recruited CCR2-dependent immature macrophages and CCR2-independent residual mature macrophages contribute to the development of intestinal inflammation observed in IL10R-deficient mice.

The role of sulpiride in attenuating the cardiac, renal, and immune disruptions in rats receiving clozapine: mRNA expression pattern of the genes encoding Kim-1, TIMP-1, and CYP isoforms.

The present study was aimed to explore the cardio-, immuno-, and nephrotoxic effects of the antipsychotic agent clozapine (CLZ) and the alleviative potency of sulpiride (SPD) on these impairments in rats. For this purpose, 40 male rats were divided into four groups and were orally treated with saline (control), CLZ (0.5 mg/kg bw), SPD (28 mg/kg bw), or a combination of CLZ and SPD (CLZ+SPD), daily for 30 consecutive days. At necropsy, blood samples and specimens from the heart, kidneys, and spleen were collected for biochemical, molecular, and histopathological investigations. The results showed that CLZ administration was associated with significantly lower immune status indices and increased serum levels of pro-inflammatory cytokines, lactate dehydrogenase, malondialdehyde, cardiac, and renal tissues injury markers. Moreover, the mRNA expression levels of Kidney Injury Molecule-1 (Kim-1), tissue inhibitor of metalloproteinase-1 (TIMP-1), and cytochrome P450 (CYP) isoforms were markedly upregulated in CLZ-treated rats, compared to the control group. On the other hand, rats treated with SPD alone showed non-significant differences in terms of immune response indices, tissue injury markers, and mRNA expression levels of Kim-1, TIMP-1, and CYP isoforms. Finally, CLZ+SPD co-treatment significantly modulated almost all biochemical indices. Besides, Kim-1, TIMP-1, and CYP2C19 mRNA expression levels were significantly downregulated, while other CYP isoforms showed no modulation, compared with CLZ-treated group. Histopathologically, CLZ-treated rats showed severe lesions in renal, splenic, and cardiac tissues, compared with control rats, which were restored in CLZ+SPD-co-treated rats. Overall, these findings demonstrate that CLZ treatment induces significant cardiac, immune, and nephropathic alterations, which were reduced with CLZ+SPD co-treatment.

Garlic and ginger extracts modulated broiler chicks innate immune responses and enhanced multidrug resistant Escherichia coli O78 clearance.

Administration of antibiotics as feed additives in broilers resulted in prompting of some undesirable effects such as the rising emergence of multi-drug resistant (MDR) bacteria, so scrutinizing for new alternatives like herbs is the up to date task for global health. This study was designed to determine the in-vitro antibacterial and ex-vivo immunomodulatory efficacy of garlic (Allium sativum) and ginger (Zingiber officinale) extracts post dietary supplementation for 900-one-day-old Sasso broiler chicks. The in-vivo protective actions of these extracts against avian pathogenic MDR Escherichia coli (E. coli) O78 challenge was evaluated after 21 days of extracts supplementation. Garlic extract exhibited broader antimicrobial spectra against MDR E. coli O78 and S. aureus isolates. Through the 21 days of garlic or ginger dietary supplementation, the chicks' innate immune response was modulated via various mechanisms including phagocytosis augmentation, bactericidal activity enhancement and nitric oxide (NO) production reduction, together with triggering the IL-1ß, IL-6 and IFN-γ cytokines expression levels in comparison with the non-supplemented chicks. It is tempting to speculate that protection against pathogenic E. coli O78 challenge was high in chicks supplemented with each individual extract with severe reduction in the bacterial colony forming units in chicks' vital organs that confirm the extracts immunomodulatory activity and provide a mechanism(s) of their protective actions. Our data suggest promising useful insights to garlic and ginger dietary supplementation in broilers that may be safe for consumers from antibiotic toxic metabolites' residues and protective against the risk of infection with bacterial pathogens.

Prevalence of plasmid-mediated AmpC in Enterobacteriaceae isolated from humans and from retail meat in Zagazig, Egypt.

Background: The objective of this study was to determine the prevalence of plasmid-mediated AmpC (pAmpC) among Enterobacteriaceae isolated from humans and from retail meat in Egypt. Methods: Enterobacteriaceae were isolated from patients with suspected bloodstream infection, human fecal samples, retail chicken meat samples and retail sheep meat samples. All group I Enterobacteriaceae were analyzed for presence of pAmpC genes by PCR. Antibiotic susceptibility testing was performed in all pAmpC positive isolates, followed by phenotypic and genotypic ESBL and carbapenemase testing on indication. Results: The prevalence of pAmpC among group I Enterobacteriaceae isolated from 225 patients with bloodstream infection was 5.6% [95%CI 2.2-13.4]. Among 100 patients with community-onset gastroenteritis the prevalence in fecal samples was 4.8% [95%CI 2.1-10.7]. The prevalence among 112 chicken carcasses and 100 sheep meat samples was 2.4% [95%CI 0.7-8.4] and 1.1% [95%CI 0.2-5.7], respectively. In half of the AmpC positive isolates we detected an ESBL gene and 2 isolates harbored a carbapenemase gene. In five isolates there was resistance to at least three important alternative antibiotic drugs. Conclusions: We consider the prevalence of pAmpC in Egypt, as found in our study, moderately low. To follow future trends in prevalence of pAmpC worldwide, a standardized screening algorithm for the detection of pAmpC is needed.

Molecular Detection of New SHV ß-lactamase Variants in Clinical Escherichia coli and Klebsiella pneumoniae Isolates from Egypt.

The emergence of multidrug-resistant (MDR) pathogens was reported worldwide. Herein, SHV extended-spectrum ß-lactamase (SHV-ESBL) variants detection was investigated in MDR E. coli and K. pneumoniae isolates recovered from human subjects (n = 144), one day-old chicks (n = 36) and broiler clinical samples (n = 90). All examined samples were positive for E. coli (n = 246/270; 91.11%) and Klebsiella pneumoniae (n = 24/270; 8.89%). Antimicrobial susceptibility testing was performed on E. coli and K. pneumoniae. SHV-ESBL producing isolates were defined followed by SHV-ESBL amino acids sequence and proteins structure-function analyses. Phylogenetic analysis of 11 MDR isolates resistant to at least 6 ß-lactams was designed to determine their genetic relationship with those previously identified in Egypt. SHV-ESBL variants were detected in 28% and 16% of E. coli and K. pneumoniae isolates, respectively. Among the 11 SHV-ESBL producing isolates, one isolate displayed 100% blaSHV-12 similarity with three point mutations, while the other 10 isolates displayed amino acid substitutions at previously non-reported sites. Amino acid sequence analyses of these 10 isolates displayed 96-100% identity to blaSHV-10 (2 isolates with 3-6 point mutations), blaSHV-18 (one isolate with 4 point mutations), blaSHV-58 (4 isolates with 4-5 point mutations), and blaSHV-91 (3 isolates with 3-7 point mutations). These mutations altered SHV-enzyme pocket dimensions and its binding sites chargeability. The blaSHV phylogeny analysis revealed occurrence of variants in closely related lineages with blaSHV-5 and blaSHV-12 with possibility of blaSHV gene transfer between human and birds. The occurrence of these variants in Egypt could help in epidemiological studies and could explain the emergent resistance to ß-lactams.

Immunization with hepatitis C virus-like particles results in control of hepatitis C virus infection in chimpanzees.

Recombinant hepatitis C virus (HCV)-like particles (HCV-LPs) containing HCV structural proteins (core, E1, and E2) produced in insect cells resemble the putative HCV virions and are capable of inducing strong and broad humoral and cellular immune responses in mice and baboons. Here, we present evidence on the immunogenicity and induction of protective immunity by HCV-LPs in chimpanzees. Chimpanzees (two in each group), were immunized with HCV-LPs or HCV-LPs plus AS01B adjuvant. After immunizations, all animals developed an HCV-specific immune response including IFN-gamma(+), IL-2(+), CD4(+), and CD8(+) T cell and proliferative lymphocyte responses against core, E1, and E2. Upon challenge with an infectious HCV inoculum, one chimpanzee developed transient viremia with low HCV RNA titers (10(3) to 10(4) copies per ml) in the third and fourth weeks after the challenge. The three other chimpanzees became infected with higher levels of viremia (10(4) to 10(5) copies per ml), but their viral levels became unquantifiable (<10(3) copies per ml) 10 weeks after the challenge. After the HCV challenge, all four chimpanzees demonstrated a significant increase in peripheral and intrahepatic T cell and proliferative responses against the HCV structural proteins. These T cell responses coincided with the fall in HCV RNA levels. Four naïve chimpanzees were infected with the same HCV inoculum, and three developed persistent infection with higher viremia in the range of 10(5) to 10(6) copies per ml. Our study suggests that HCV-LP immunization induces HCV-specific cellular immune responses that can control HCV challenge in the chimpanzee model.