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Necrotic enteritis caused by Clostridium perfringens ( C. perfringens ) is characterized by poor performance and higher mortality rates in poultry farms. Novel dietary intervention involving bioactive molecules loaded into smart magnetized nano-system with a potent antioxidant function (quercetin-loaded Fe 3 O 4 -NPs), was evaluated for their impact on growth performance, intestinal immune and antioxidant defenses, and resistance against Clostridium perfringens in a necrotic enteritis challenge model. Four experimental groups comprising a total of 200 one-day-old Ross 308 broiler chickens were fed different diets: a control basal diet, a diet supplemented with quercetin (300 mg/kg), a diet with Fe 3 O 4 -NPs (60 mg/kg), and a diet with quercetin-loaded Fe 3 O 4 -NPs (300 mg/kg). These groups were then challenged with C. perfringens during the grower period. Dietary inclusion of quercetin-loaded Fe 3 O 4 -NPs prominently reduced C. perfringens colonization and its associated virulence genes expression, which subsequently restored the impaired growth performance and intestinal histopathological changes in challenged broilers. Quercetin-loaded Fe 3 O 4 -NPs supplemented group displayed higher Lactobacillus and Bifidobacterium counts, upregulation of intestinal host defense antimicrobial peptides related genes (avian β -defensin 6 and 12) and downregulation of intestinal inflammatory regulated genes (Interleukin-1 beta, C-X-C motif chemokine ligand 8, tumor necrosis factor- α , chemokine C-C motif ligand 20, inducible nitric oxide synthase and cycloox-ygenase-2). Intestinal redox balance was boosted via upregulation of catalase, superoxide dismutase, glutathione peroxidase and heme Oxygenase 1 genes along with simultaneous decrease in hydrogen peroxide , reactive oxygen species and malondialdehyde contents in groups fed quercetin-loaded Fe 3 O 4 -NPs. Overall, new nutritional intervention with quercetin-loaded Fe 3 O 4 -NPs impacted better immune and antioxidant defenses, attenuated C. perfringens induced necrotic enteritis and contributed to better performance in the challenged birds., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2024 Al-Nasser, El-Demerdash, Ibrahim, Abd El-Hamid, Al-Khalaifah, El-borady, Shukry, El-Azzouny, Ibrahim, Badr, Elshater, Ismail and El Sayed.)

Phytochemical nanoemulsions, such as thymoquinone nanoemulsions (TQN), are regarded as innovative alternatives to antimicrobials that significantly improve the performance, digestion, antioxidant potential and immunity of rabbits. Thus, the potential effects of TQN on growth, digestibility, antioxidant potential, immunity and resistance against Pasteurella multocida ( P. multocida ) in rabbits were assessed. Herein, 240 rabbits were offered either a basal diet or diets fortified with three TQN-graded concentrations. At 60 days of age, rabbits were challenged with multidrug-resistant (MDR) virulent P. multocida strain. Our outcomes described that dietary inclusion of TQN, especially at higher concentrations, significantly enhanced the growth performance of rabbits, which was supported by increasing the levels of jejunal lipase, amylase and trypsin enzymes. Of note, the levels of muscle and jejunal antioxidant enzymes [superoxide dismutase (SOD), glutathione peroxidase (GPX), catalase (CAT) and total antioxidant capacity (T-AOC)], serum immunological markers (IgG, IgG, IgM and total Igs) and blood phagocytic percentage were significantly provoked after TQN fortification; meanwhile, the levels of muscle and jejunal MDA, serum biochemical parameters (total cholesterol, TG and LDL), abdominal fat percentage, breast and thigh cholesterol were significantly decreased following TQN supplementations. Our findings showed that TQN protected rabbits against P. multocida experimental challenge as evidenced by reducing P. multocida counts in rabbits' lungs, downregulating the transcription levels of P. multocida virulence-related genes ( ptfA, toxA and nanB ) at 48 and 96 h post-infection and ameliorating the expression levels of cytokines-related genes ( IL-1β , IL-10, IL-8, IL-6 , DEFB1 , TNF-α , TLR-4 and TLR-2 ) at 96 h post-infection. Our findings suggest the utilization of TQN in rabbits' diets due to their stimulating effects on digestibility as well as their growth-promoting, anti-inflammatory, antioxidant, antibacterial, anti-virulence and immunostimulant properties, which enhance the rabbits' P. multocida resistance., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2024 Abd El-Hamid, El-Azzouny, El-Malt, Elkenawy, Abdelwarith, Younis, Youssef, Dawod, Elged, Habaka, El Oksh, Mekawy, Davies and Ibrahim.)

Introduction: As a growing direction, nano-based therapy has become a successful paradigm used to address the phytogenic delivery-related problems in overcoming multivirulent vancomycin-resistant Staphylococcus aureus (VRSA) infection., Methods: Hence, our aim was to develop and assess a novel nanocarrier system (mesoporous silica nanoparticles, MPS-NPs) for free berberine (Free-BR) as an antimicrobial alkaloid against strong biofilm-producing and multi-virulent VRSA strains using in vitro and in vivo mouse model., Results and Discussion: Our outcomes demonstrated vancomycin resistance in 13.7% of Staphylococcus aureus ( S. aureus ) strains categorized as VRSA. Notably, strong biofilm formation was observed in 69.2% of VRSA strains that were all positive for icaA gene. All strong biofilm-producing VRSA strains harbored a minimum of two virulence genes comprising clfA and icaA with 44.4% of them possessing all five virulence genes ( icaA , tst , clfA , hla , and pvl ), and 88.9% being multi-virulent. The study findings affirmed excellent in vitro antimicrobial and antibiofilm properties of BR-loaded MPS-NPs. Real-time quantitative reverse transcription PCR (qRT-PCR) assay displayed the downregulating role of BR-loaded MPS-NPs on strong biofilm-producing and multi-virulent VRSA strains virulence and agr genes in both in vitro and in vivo mice models. Additionally, BR-loaded MPS-NPs supplementation has a promising role in attenuating the upregulated expression of pro-inflammatory cytokines' genes in VRSA-infected mice with attenuation in pro-apoptotic genes expression resulting in reduced VRSA-induced apoptosis. In essence, the current study recommends the future scope of using BR-loaded MPS-NPs as auspicious alternatives for antimicrobials with tremendous antimicrobial, antibiofilm, anti-quorum sensing (QS), and anti-virulence effectiveness against problematic strong biofilm-producing and multi-virulent VRSA-associated infections., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2024 Abd El-Hamid, Ibrahim, Elazab, Gad, Shalaby, El-Neshwy, Alshahrani, Saif, Algendy, AlHarbi, Saleh, Alharthi and Mohamed.)

Locomotor disorders caused by multidrug-resistant (MDR) bacterial pathogens denote one of the most detrimental issues that collectively threaten the poultry industry leading to pronounced economic losses across the world. Hence, searching for effective alternatives, especially those extracted from plant origins became of great priority targeting a partial or complete replacement of chemical antimicrobials to tackle their developing resistance. Therefore, we aimed to determine the prevalence and antimicrobial resistance of Staphylococcus aureus (S. aureus), Salmonella species, Mycoplasma synoviae (M. synoviae), and Escherichia coli (E. coli) recovered from 500 broilers and ducks (250 each) with locomotor disorders in various farms in Dakahlia and Sharkia Governorates, Egypt. Additionally, we assessed, for the first time, the in vitro antimicrobial effectiveness of marjoram, garlic, ginger and cinnamon essential oils (EOs) against MDR and multivirulent bacterial isolates as well as the in vivo efficiency of the most effective antibiotics and EOs either separately or in combination in the treatment of experimentally induced poultry leg disorders. The overall prevalence rates of S. aureus, E. coli, Salmonella species, and M. synoviae were 54, 48, 36, and 2%, respectively. Salmonella species and S. aureus prevailed among ducks and broilers (36 and 76%, respectively). Notably, MDR was observed in 100, 91.7, 81.1, and 78.5% of M. synoviae, E. coli, Salmonella, and S. aureus isolates, respectively. Our in vitro results displayed that marjoram was the most forceful EO against MDR and multivirulent chicken vancomycin-resistant S. aureus (VRSA) and duck S. Typhimurium isolates. The current in vivo results declared that marjoram in combination with florfenicol or amoxicillin/clavulanic acid succeeded in relieving the induced duck and chicken leg disorders caused by S. Typhimurium and VRSA, respectively. This was evidenced by improvement in the clinical and histopathological pictures with a reduction of bacterial loads in the experimental birds. Our encountered successful in vitro and in vivo synergistic effectiveness of marjoram combined with florfenicol or amoxicillin/clavulanic acid recommends their therapeutic application for leg disorders and offers opportunities for reducing the antibiotics usage in the poultry industry., (Copyright © 2023 The Authors. Published by Elsevier Inc. All rights reserved.)

Introduction: There is an urgent need to develop therapeutic options for biofilm-producing Staphylococcus aureus (S. aureus). Therefore, the renewed interest in essential oils (EOs), especially carvacrol, linalool and eugenol, has attracted the attention of our research group., Methods: Multidrug resistance and multivirulence profiles in addition to biofilm production of S. aureus strains isolated from cows with mastitis were evaluated using both phenotypic and genotypic methods. The antimicrobial and antibiofilm activities of EOs were tested using both in vitro and molecular docking studies. Moreover, the interactions between commonly used antibiotics and the tested EOs were detected using the checkerboard method., Results: We found that all our isolates (n= 37) were biofilm methicillin resistant S. aureus (MRSA) producers and 40.5% were vancomycin resistant S. aureus (VRSA). Unfortunately, 73 and 43.2% of the recovered MRSA isolates showed multidrug resistant (MDR) and multivirulence patterns, respectively. The antimicrobial activities of the tested EOs matched with the phenotypic evaluation of the antibiofilm activities and molecular docking studies. Linalool showed the highest antimicrobial and antibiofilm activities, followed by carvacrol and eugenol EOs. Fortunately, synergistic interactions between the investigated EOs and methicillin or vancomycin were detected with fractional inhibitory concentration index (FICI) values ≤ 0.5. Moreover, the antimicrobial resistance patterns of 13 isolates changed to sensitive phenotypes after treatment with any of the investigated EOs. Treatment failure of bovine mastitis with resistant S. aureus can be avoided by combining the investigated EOs with available antimicrobial drugs., Conclusion: We hope that our findings can be translated into a formulation of new pharmaceutical dosage forms against biofilm-producing S. aureus pathogens., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2023 Abd El-Hamid, El-Tarabili, Bahnass, Alshahrani, Saif, Alwutayd, Safhi, Mansour, Alblwi, Ghoneim, Elmaaty, Al-harthi and Bendary.)

Control and management of life-threatening bacterial and fungal infections are a global health challenge. Despite advances in antimicrobial therapies, treatment failures for resistant bacterial and fungal infections continue to increase. We aimed to repurpose the anthelmintic drug rafoxanide for use with existing therapeutic drugs to increase the possibility of better managing infection and decrease treatment failures. For this purpose, we evaluated the antibacterial and antifungal potential of rafoxanide. Notably, 70% (70/100) of bacterial isolates showed multidrug resistance (MDR) patterns, with higher prevalence among human isolates (73.5% [50/68]) than animal ones (62.5% [20/32]). Moreover, 22 fungal isolates (88%) were MDR and were more prevalent among animal (88.9%) than human (87.5%) sources. We observed alarming MDR patterns among bacterial isolates, i.e., Klebsiella pneumoniae (75% [30/40; 8 animal and 22 human]) and Escherichia coli (66% [40/60; 12 animal and 28 human]), and fungal isolates, i.e., Candida albicans (86.7% [13/15; 4 animal and 9 human]) and Aspergillus fumigatus (90% [9/10; 4 animal and 5 human]), that were resistant to at least one agent in three or more different antimicrobial classes. Rafoxanide had antibacterial and antifungal activities, with minimal inhibitory concentration (MICs) ranging from 2 to 128 μg/mL. Rafoxanide at sub-MICs downregulated the mRNA expression of resistance genes, including E. coli and K. pneumoniae bla CTX-M-1 , bla TEM-1 , bla SHV , MOX , and DHA , C. albicans ERG11 , and A. fumigatus cyp51A . We noted the improvement in the activity of β-lactam and antifungal drugs upon combination with rafoxanide. This was apparent in the reduction in the MICs of cefotaxime and fluconazole when these drugs were combined with sub-MIC levels of rafoxanide. There was obvious synergism between rafoxanide and cefotaxime against all E. coli and K. pneumoniae isolates (fractional inhibitory concentration index [FICI] values ≤ 0.5). Accordingly, there was a shift in the patterns of resistance of 16.7% of E. coli and 22.5% of K. pneumoniae isolates to cefotaxime and those of 63.2% of C. albicans and A. fumigatus isolates to fluconazole when the isolates were treated with sub-MICs of rafoxanide. These results were confirmed by in silico and mouse protection assays. Based on the in silico study, one possible explanation for how rafoxanide reduced bacterial resistance is through its inhibitory effects on bacterial and fungal histidine kinase enzymes. In short, rafoxanide exhibited promising results in overcoming bacterial and fungal drug resistance. IMPORTANCE The drug repurposing strategy is an alternative approach to reducing drug development timelines with low cost, especially during outbreaks of disease caused by drug-resistant pathogens. Rafoxanide can disrupt the abilities of bacterial and fungal cells to adapt to stress conditions. The coadministration of antibiotics with rafoxanide can prevent the failure of treatment of both resistant bacteria and fungi, as the resistant pathogens could be made sensitive upon treatment with rafoxanide. From our findings, we anticipate that pharmaceutical companies will be able to utilize new combinations against resistant pathogens., Competing Interests: The authors declare no conflict of interest.

Background: Oxidative stress and its end products, such as malondialdehyde (MDA) play a leading role in the pathogenesis of hepatitis C. Melatonin is a hormone that helps regulate circadian rhythms, which likely play a role in infectious diseases in terms of susceptibility, clinical expression, and outcome. Objective: The present study was conducted to assess serum malondialdehyde and melatonin levels in patients with chronic hepatitis C infection before and after the intake of direct-acting antivirals. Method: Forty hepatitis C patients were the subjects of this study. While ten healthy volunteers who matched in age and socioeconomic status served as the control subjects. Malondialdehyde and melatonin were assayed in the serum of the three groups, and the results were statistically analyzed. Results: Hepatitis C patients had significantly higher malondialdehyde ( p < 0.001) but significantly lower melatonin ( p < 0.001) as compared to the healthy controls. After 12 weeks of treatment with direct-acting antivirals, the malondialdehyde level decreased significantly ( p < 0.001) and the melatonin level increased significantly ( p < 0.001). A significant negative correlation between malondialdehyde and melatonin was observed. Conclusion : The present findings suggest that treatment of hepatitis C patients with Direct-acting antivirals improves liver function parameters and antioxidant profiles., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2023 El-Mahdy, Abou-Saif, Abd EL hamid, Hashem, Hammad and Abu-Risha.)

Chronic respiratory disease (CRD) caused by Mycoplasma gallisepticum (MG) leads to impaired broiler growth performance and significant economic losses worldwide. The utilization of essential oils (EOs) as natural alternatives to antibiotics to control CRD outbreaks is not completely clarified yet. Thus, we investigated the effect of a commercial EOs mixture (toldin CRD), in comparison to tilmicosin antibiotic, on the clinical observations, growth performance, immunity, digestive enzymes, gut barrier functions, and bacterial loads in broilers experimentally infected with MG. A total of 400 one-day-old broiler chicks were assigned into four groups; negative control (NC), positive control (PC), tilmicosin, and toldin CRD treated groups. All groups except NC were experimentally infected with MG at 14 d of age. Our data showed that birds treated with toldin CRD showed significant enhancement in the body weight gain (BWG) and feed conversion ratio (FCR) (P = 0.001 each) over the whole experimental period. Likely, improved digestibility and intestinal barrier functions in the toldin CRD treated group was evidenced by the significant upregulation (P < 0.05) of cholecystokinin (CCK), alpha 2A amylase (AMY2A), pancreatic lipase (PNLIP), junctional adhesion molecule-2 (JAM-2), occludin, and mucin-2 (MUC-2) genes. Moreover, toldin CRD exhibited immunostimulant and ant-inflammatory activities via significant downregulation (P < 0.05) of tumor necrosis factor-alpha (TNF-α) and interleukin (IL)-6 genes, significant reduction of lysozyme (LYZ), myeloperoxidase (MPO), and nitric oxide (NO) levels (P = 0.03, 0.02, and 0.001, respectively) and significant increase in the immunoglobulin G (IgG) level (P = 0.03). Notably, immunohistochemistry and quantitative real-time polymerase chain reaction (qPCR) results showed prominent reductions (P < 0.05) in the levels of MG antigens and MG loads in the toldin CRD treated group, which were evidenced by relieving the clinical picture of MG experimental infection. In conclusion, we recommend the utilization of toldin CRD as a potential candidate for controlling MG infection in broiler chickens., (Copyright © 2022 The Authors. Published by Elsevier Inc. All rights reserved.)

Listeriosis is one of the most common foodborne diseases caused by Listeria monocytogenes ( L. monocytogenes ). A poor prognosis has been recorded for the invasive listeriosis, especially neurolisteriosis. In several countries throughout the world, foodborne infections with L. monocytogenes exceeded the legal safety limits in animal sourced foods. Therefore, we decided to investigate the variability, virulence and antimicrobial resistance profiles of this pathogen. Both phenotypic and genotypic methods were used for identifying L. monocytogenes isolates and confirming their virulence profiles. The antimicrobial resistances and their correlation analysis with the existence of virulence genes were detected. Additionally, sequencing and phylogenetic analysis based on L. monocytogenes inlA and inlB genes were undertaken. The prevalence rate (11.9%) and the resistance profiles of L. monocytogenes were shocking. The multi-drug resistance (MDR) phenotypes were common among our isolates (64.9%). Fortunately, the resistance phenotypes were always associated with low virulence arrays and the MDR strains possessed low virulence fitness. Herein, the high genotypic and phenotypic diversity of L. monocytogenes isolates and their weak clonality and adaptability highlighted the difficulty in controlling and managing this pathogen. Therefore, it is important to add more restriction guidelines from national authorities on the consumption of ready to eat foods.

Multi-strain probiotics (MSP) are considered innovative antibiotics' substitutes supporting superior gut health and immunity of farmed rabbits. The promising roles of MSP on performance, intestinal immunity, integrity and transporters, and resistance against Listeria monocytogenes ( L. monocytogenes ) were evaluated. In the feeding trial, 220 rabbits were fed a control diet or diet supplemented with three MSP graded levels. At 60 days of age, rabbits were experimentally infected with L. monocytogenes and the positive control, enrofloxacin, prophylactic MSP (MSPP), and prophylactic and therapeutic MSP (MSPTT) groups were included. During the growing period, MSP at the level of 1 × 10 8 CFU/kg diet (MSPIII) promoted the rabbits' growth, upregulated the nutrient transporters and tight-junction-related genes, and modified cytokines expression. Supplementing MSPTT for L. monocytogenes experimentally-infected rabbits restored the impaired growth and intestinal barriers, reduced clinical signs of severity and mortalities, and attenuated the excessive inflammatory reactions. Notably, enrofloxacin decreased L. monocytogenes and beneficial microbial loads; unlike MSPTT, which decreased pathogenic bacterial loads and sustained the beneficial ones. Histopathological changes were greatly reduced in MSPTT, confirming its promising role in restricting L. monocytogenes translocation to different organs. Therefore, our results suggest the use of MSPTT as an alternative to antibiotics, thereby conferring protection for rabbits against L. monocytogenes infection.

Among many bovine Mycoplasma species (spp.), Mycoplasma bovis is recognized as a significant causative agent of respiratory diseases in cattle. In recent years, resistant M. bovis isolates, especially to fluoroquinolones, have been reported globally as a result of the extensive usage of antimicrobials in the treatment of bovine pneumonia. Therefore, the aim of this study is to investigate the prevalence and antimicrobial susceptibility patterns of bovine Mycoplasma spp. isolated from the respiratory tracts of cattle in Egypt and to assess the fluoroquinolones resistance in the recovered mycoplasma isolates via broth microdilution and conventional PCR techniques. Conventional phenotypic methods identified 128 mycoplasma isolates (32%) from 400 different samples, with M. bovis being the predominant spp. (61%), followed by M. bovirhinis (15%). Of note, mycoplasma isolates were rarely isolated from total healthy lung tissues (7/55, 12.7%), but they were frequently isolated from pneumonic lungs (31/45, 68.9%). All the examined mycoplasma isolates ( n = 76) were sensitive to tilmicosin, tylosin, tulathromycin, spiramycin, and spectinomycin (100% each), while 60.5% and 43.4% of the examined isolates had high minimum inhibitory concentration (MIC) values to enrofloxacin and doxycycline, respectively. Three and two mycoplasma isolates with high enrofloxacin MICs were confirmed to be M. bovis and M. bovirhinis , respectively, by PCR assays. All molecularly confirmed mycoplasma isolates ( n = 5) were positive for the gyrA gene (100%); meanwhile, three isolates (60%) were positive for the parC gene. In conclusion, our findings revealed alarming resistance to enrofloxacin and doxycycline antibiotics; thus, antimicrobial usage must be restricted and molecular techniques can help in the rapid detection of the resistant strains.

The treatment failure recorded among patients and animals infected with diarrheagenic Escherichia coli (DEC) was increased due to the presence of specific virulence markers among these strains. These markers were used to classify DEC into several pathotypes. We analyzed the correlations between DEC pathotypes and antimicrobial resistances, the existence of virulence genes, serotypes, and hosts. The ETEC pathotype was detected with a high prevalence rate (25%). Moreover, the ETEC and EPEC pathotypes were highly associated with human infections in contrast to the EIEC and EAEC phenotypes, which were commonly recognized among animal isolates. Interestingly, the antimicrobial resistance was affected by E. coli pathotypes. With the exception of EIEC and STEC, imipenem represented the most effective antibiotic against the other pathotypes. There were fixed correlations between the DEC pathotypes and the presence of virulence markers and hosts; meanwhile, their correlation with serotypes was variable. Additionally, the vast majority of our isolates were highly diverse, based on both phenotypic and ERIC molecular typing techniques. Our promising results gave a clear indication for the heterogeneity and weak clonality of DEC pathotypes in Egypt, which can be utilized in the evaluation of the current therapeutic protocols and infection control guidelines.

Recently, the use of essential oils (EOs) or their bioactive compounds encapsulated by nanoparticles as alternative supplements for in-feed antimicrobials is gaining attention, especially in organic poultry production. Focusing on eugenol, its incorporation into the nanoformulation is a novel strategy to improve its stability and bioavailability and thus augment its growth-boosting and antimicrobial activities. Therefore, we explored eugenol nanoemulsion activities in modulating growth, digestive and gut barrier functions, immunity, cecal microbiota, and broilers response to avian pathogenic E. coli challenge (APEC) O78. A total of 1,000 one-day-old broiler chicks were allocated into five groups; negative control (NC, fed basal diet), positive control (PC), and 100, 250, and 400 mg/kg eugenol nanoemulsion supplemented groups. All groups except NC were challenged with APEC O78 at 14 days of age. The results showed that birds fed eugenol nanoemulsion displayed higher BWG, FI, and survivability and most improved FCR over the whole rearing period. Birds fed 400 mg/kg of eugenol nanoemulsion sustained a higher growth rate (24% vs. PC) after infection. Likely, the expression of digestive enzymes' genes ( AMY2A, CCK, CELA1 , and PNLIP ) was more prominently upregulated and unaffected by APEC O78 challenge in the group fed eugenol nanoemulsion at the level of 400 mg/kg. Enhanced gut barrier integrity was sustained post-challenge in the group supplemented with higher levels of eugenol nanoemulsion as evidenced by the overexpression of cathelicidins-2, β-defensin-1, MUC -2, JAM -2, occludin, CLDN -1, and FABP -2 genes. A distinct modulatory effect of dietary eugenol nanoemulsion was observed on cytokine genes (IL-1β, TNF-α, IL -6, IL -8, and IL -10) expression with a prominent reduction in the excessive inflammatory reactions post-challenge. Supplementing eugenol nanoemulsion increased the relative cecal abundance of Lactobacillus species and reduced Enterobacteriaceae and Bacteriods counts. Notably, a prominent reduction in APEC O78 loads with downregulation of papC, iroN, iutA , and iss virulence genes and detrimental modifications in E. coli morphological features were noticed in the 400 mg/kg eugenol nanoemulsion group at the 3rd-week post-challenge. Collectively, we recommend the use of eugenol nanoemulsion as a prospective targeted delivery approach for achieving maximum broilers growth and protection against APEC O78 infection., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2022 Ibrahim, Eldemery, Metwally, Abd-Allah, Mohamed, Ismail, Hamed, Al Sadik, Neamat-Allah and Abd El-Hamid.)

Several food-poisoning outbreaks have been attributed to Clostridium perfringens ( C. perfringens ) worldwide. Despite that, this crisis was discussed in a few studies, and additional studies are urgently needed in this field. Therefore, we sought to highlight the prevalence, antimicrobial resistance, toxin profiles, and toxinotypes of C. perfringens isolates. In this study, 50 C. perfringens isolates obtained from 450 different animal origin samples (beef, chicken meat, and raw milk) were identified by phenotypic and genotypic methods. The antimicrobial susceptibility results were surprising, as most of the isolates (74%) showed multidrug-resistant (MDR) patterns. The phenotypic resistance to tetracycline, lincomycin, enrofloxacin, cefoxitin/ampicillin, and erythromycin was confirmed by the PCR detections of tet , lnu , qnr , bla , and erm (B) genes, respectively. In contrast to the toxinotypes C and E, toxinotype A prevailed (54%) among our isolates. Additionally, we found that the genes for C. perfringens enterotoxin ( cpe ) and C. perfringens beta2 toxin ( cpb 2) were distributed among the tested isolates with high prevalence rates (70 and 64%, respectively). Our findings confirmed that the C. perfringens foodborne crisis has been worsened by the evolution of MDR strains, which became the prominent phenotypes. Furthermore, we were not able to obtain a fixed association between the toxinotypes and antimicrobial resistance patterns.

Campylobacter species (spp.) are one of the most important causes of human bacterial gastroenteritis in foods of animal origin. Recently, with the spread of multi-drug-resistant (MDR) and extensively drug-resistant (XDR) Campylobacter spp., natural alternative therapeutic methods are urgently required. Phytogenic active principles have gained considerable attention due to their proficiency to enhance gut health and, thereby, performance of broiler chickens. Thus, the current study aims to determine the prevalence and antimicrobial resistance of Campylobacter spp. of different chicken sources in Sharkia Governorate, Egypt, and to assess the growth-promoting, immunostimulant and antimicrobial effects of a mixture of eugenol and trans-cinnamaldehyde in an in vivo approach. A total of 101 (67.3%) campylobacter isolates was identified, according to both phenotypic and genotypic techniques. Moreover, all of the campylobacter isolates were resistant to erythromycin, trimethoprim/sulfamethoxazole, and ampicillin (100% each). Of note, a dietary supplementation of the mixture of eugenol and trans-cinnamaldehyde led to a significant improvement of the feed conversion ratio and body weight gain and a decrease in the cecal C. jejuni loads in the broilers challenged with XDR C. jejuni . Additionally, eugenol and the trans-cinnamaldehyde mixture had protective activities via the down-regulation of XDR C. jejuni ( flaA , virB11 and wlaN ) virulence genes and proinflammatory cytokines ( TNF-α , IL -2, IL-6 , and IL-8 ), and the up-regulation of anti-inflammatory cytokine IL-10 . Thus, we recommend the usage of a mixture of eugenol and trans-cinnamaldehyde as an alternative to antimicrobials for the control and treatment of campylobacter infections.

Methicillin-resistant Staphylococcus aureus (MRSA) is a major pathogen associated with severe morbidity and mortality and poses a significant threat to public health worldwide. The genetic diversity based on sequence types of MRSA strains was illustrated in previous studies; meanwhile, the diversity along with the predominant sequence type, especially in Egypt, remains unknown. The purpose of the current study was to determine the diversity of the predominant MRSA clone ST239-MRSA (n = 50) isolated from different hosts and clinical samples and to illustrate the correlation between the resistance patterns, toxin genes, and the genetic background in Port-said and El-Sharkia Governorates, Egypt. The ST239-MRSA clone was analyzed by phenotypic antibiotyping and various genotypic assays comprising SCC mec , agr , spa , coa , and coa -RFLP in addition to toxin gene profiles. Most of the analyzed strains (40/50, 80%) were multidrug resistant (MDR), belonged to SCC mec -III, agr -I, and coa genotype I, and harbored sea and pvl genes. A negative correlation between the toxin gene profiles and antimicrobial resistance was recorded. Meanwhile, the correlation between the toxin gene profiles and the genetic background was not observed in this study. Although ST239-MRSA strains belonged to a single sequence type, they exhibited a high degree of phenotypic and genotypic diversity, indicating weak clonality and adaptability. With such diversity, it is assumed that these strains may have undergone different evolutionary processes during transmission events among and/or within a single host or tissue niche., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2022 Abd El-Hamid, Sewid, Samir, Hegazy, Bahnass, Mosbah, Ghaith, Khalifa, Ramadan, Alshareef, Alshareef, Ghoneim, Al-Sanea and Bendary.)

In recent times, resistant foodborne pathogens, especially of the Campylobacter species, have created several global crises. These crises have been compounded due to the evolution of multidrug-resistant (MDR) bacterial pathogens and the emergence of extensively drug-resistant (XDR) and pan-drug-resistant (PDR) strains. Therefore, this study aimed to investigate the development of resistance and the existence of both XDR and PDR among Campylobacter isolates. Moreover, we explored the use of the polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) technique for the detection of fluoroquinolone (FQ)-resistant Campylobacter isolates. A total of 120 Campylobacter isolates were identified depending on both phenotypic and genotypic methods. Of note, cefoxitin and imipenem were the most effective drugs against the investigated Campylobacter isolates. Interestingly, the majority of our isolates (75%) were MDR. Unfortunately, both XDR and PDR isolates were detected in our study with prevalence rates of 20.8% and 4.2%, respectively. All FQ-resistant isolates with ciprofloxacin minimum inhibitory concentrations ≥4 µg/mL were confirmed by the genetic detection of gyrA chromosomal mutation via substitution of threonine at position 86 to isoleucine (Thr-86-to-Ile) using the PCR-RFLP technique. Herein, PCR-RFLP was a more practical and less expensive method used for the detection of FQ resistant isolates. In conclusion, we introduced a fast genetic method for the identification of FQ-resistant isolates to avoid treatment failure through the proper description of antimicrobials.

Necrotic enteritis (NE) caused by Clostridium perfringens ( C. perfringens ) results in impaired bird growth performance and increased production costs. Nanotechnology application in the poultry industry to control NE outbreaks is still not completely clarified. Therefore, the efficacy of dietary garlic nano-hydrogel (G-NHG) on broilers growth performance, intestinal integrity, economic returns and its potency to alleviate C. perfringens levels using NE challenge model were addressed. A total of 1200 male broiler chicks (Ross 308) were assigned into six groups; four supplemented with 100, 200, 300 or 400 mg of G-NHG/kg diet and co-challenged with C. perfringens at 21, 22 and 23 d of age and two control groups fed basal diet with or without C. perfringens challenge. Over the total growing period, the 400 mg/kg G-NHG group had the most improved body weight gain and feed conversion efficiency regardless of challenge. Parallel with these results, the mRNA expression of genes encoding digestive enzymes (alpha 2A amylase (AMY2A), pancreatic lipase (PNLIP) and cholecystokinin (CCK)) and intestinal barriers (junctional adhesion molecule-2 (JAM-2), occludin and mucin-2 (Muc-2)) were increased in groups fed G-NHG at higher levels to be nearly similar to those in the unchallenged group. At 14 d post challenge, real-time PCR results revealed that inclusion of G-NHG led to a dose-dependently decrease in the C. perfringens population, thereby decreasing the birds' intestinal lesion score and mortality rates. Using 400 mg/kg of G-NHG remarkably ameliorated the adverse effects of NE caused by C. perfringens challenge, which contributed to better growth performance of challenged birds with rational economic benefits.

Foodborne pathogens have been associated with severe and complicated diseases. Therefore, these types of infections are a concern for public health officials and food and dairy industries. Regarding the wide-spread multidrug resistant (MDR) and extensively drug resistant (XDR) foodborne pathogens such as Salmonella Enteritidis ( S. Enteritidis), new and alternative therapeutic approaches are urgently needed. Therefore, we investigated the antimicrobial, anti-virulence, and immunostimulant activities of a stable formulation of thymol as thymol nanoemulsion in an in vivo approach. Notably, treatment with 2.25% thymol nanoemulsion led to a pronounced improvement in the body weight gain and feed conversion ratio in addition to decreases in the severity of clinical findings and mortality percentages of challenged chickens with XDR S. Enteritidis confirming its pronounced antimicrobial activities. Moreover, thymol nanoemulsion, at this dose, had protective effects through up-regulation of the protective cytokines and down-regulation of XDR S. Enteritidis sopB virulence gene and interleukins ( IL ) -4 and IL-10 cytokines as those hinder the host defenses. Furthermore, it enhanced the growth of gut Bifidobacteria species, which increases the strength of the immune system. For that, we suggested the therapeutic use of thymol nanoemulsion against resistant foodborne pathogens. Finally, we recommended the use of 2.25% thymol nanoemulsion as a feed additive for immunocompromised individuals as well as in the veterinary fields.

Campylobacter jejuni is the leading cause of foodborne bacterial gastroenteritis in humans worldwide. Contaminated chickens and their products are the main sources of human campylobacteriosis. Therefore, this study aimed to detect the genotypic and virulence genes' profiles of multi-drug resistant (MDR) C. jejuni isolates and to assess the effects of sub-inhibitory concentrations (SICs) of eugenol and beta-resorcylic acid on the virulence of avian MDR C. jejuni isolates. These isolates were clustered together with the human isolates via enterobacterial repetitive intergenic consensus-PCR (ERIC-PCR) fingerprinting. A total of 345 samples were collected from human stool (100) and different chicken (245) samples in Sharkia Governorate, Egypt. Conventional phenotypic methods identified 113 isolates (32.8%) as C. jejuni, and all C. jejuni isolates were MDR and resistant to erythromycin and ampicillin. The genes vir B11, wla N, and fla A were detected in 52%, 36% and 100% strains, respectively. ERIC-PCR yielded 14 profiles and five main clusters. Interestingly, human and chicken C. jejuni isolates were clustered together in ERIC-PCR clusters II-V, which confirmed the genetic relatedness between the isolates from both origins. Beta-resorcylic acid and eugenol inhibited the invasion of C. jejuni isolates to chicken intestinal cells by 41.66-38.19% and 31.94-29.16%, respectively, and minimized the transcription of fla A, vir B11, and wla N genes in the tested isolates by real-time quantitative reverse transcription PCR (qRT-PCR). In essence, eugenol and beta-resorcylic acid are promising natural antimicrobials for minimizing the virulence of MDR C. jejuni in chickens, thereby managing human campylobacteriosis.

Multidrug resistant (MDR) methicillin-resistant Staphylococcus aureus (MRSA) is a superbug pathogen that causes serious diseases. One of the main reasons for the lack of the effectiveness of antibiotic therapy against infections caused by this resistant pathogen is the recalcitrant nature of MRSA biofilms, which results in an increasingly serious situation worldwide. Consequently, the development of innovative biofilm inhibitors is urgently needed to control the biofilm formation by this pathogen. In this work, we thus sought to evaluate the biofilm inhibiting ability of some promising antibiofilm agents such as zinc oxide nanoparticles (Zno NPs), proteinase K, and hamamelitannin (HAM) in managing the MRSA biofilms. Different phenotypic and genotypic methods were used to identify the biofilm producing MDR MRSA isolates and the antibiofilm/antimicrobial activities of the used promising agents. Our study demonstrated strong antibiofilm activities of ZnO NPs, proteinase K, and HAM against MRSA biofilms along with their transcriptional modulation of biofilm (intercellular adhesion A, ica A) and quorum sensing (QS) ( agr ) genes. Interestingly, only ZnO NPs showed a powerful antimicrobial activity against this pathogen. Collectively, we observed overall positive correlations between the biofilm production and the antimicrobial resistance/ agr genotypes II and IV. Meanwhile, there was no significant correlation between the toxin genes and the biofilm production. The ZnO NPs were recommended to be used alone as potent antimicrobial and antibiofilm agents against MDR MRSA and their biofilm-associated diseases. On the other hand, proteinase-K and HAM can be co-administrated with other antimicrobial agents to manage such types of infections.

Among many avian mycoplasmas, Mycoplasma gallisepticum (MG) and Mycoplasma synoviae (MS) are recognized as the main etiological agents of respiratory diseases and infectious synovitis in chickens and turkeys causing tremendous economic losses worldwide. Therefore, proper treatment is promoted for the control of these diseases. This study was the first in Egypt to evaluate the in vitro efficacy of various antimicrobials against field MG and MS isolates recovered from chicken and turkey flocks using both conventional broth microdilution and quantitative real-time polymerase chain reaction assays. Totally, 47 mycoplasma isolates were recovered from 160 collected tracheal samples (29.4%). Of these, 44 MG (27.5%) and 3 MS (1.9%) were identified using conventional and molecular assays. The in vitro susceptibilities of 4 representative mycoplasma field isolates (3 MG and one MS) to 8 antibiotics and 4 essential oils were investigated. The tested isolates showed various susceptibilities to tested antimicrobials. Toldin CRD, followed by clove, cumin, and cinnamon oils were effective against both MG and MS clinical isolates with minimum inhibitory concentration (MIC) values ranging from 0.49 to 15.63 µg/mL. Similarly, tylvalosin was the most active antibiotic against MG and MS isolates with the lowest MIC values (0.015 to 0.03 µg/mL). DNA loads of both MG mgc2 and MS vlhA genes were markedly decreased upon treatment with majority of the tested antimicrobials confirming their effectiveness as was also evaluated by conventional MIC results. In conclusion, Toldin CRD and tylvalosin were found to be the most effective antimicrobials in this study. This finding highlights the importance of using these antimicrobials in controlling mycoplasma infections in chickens and turkeys., (© 2019 Poultry Science Association Inc.)

Campylobacter jejuni ( C. jejuni ) are able to colonise and infect domestic poultry and also pose a risk for humans. The aim of this study was to determine the extent of genotypic diversity among C. jejuni isolates recovered from avian and human sources in Egypt. Furthermore, the short variable region (SVR) of flagellin A ( flaA ) gene was analysed for the presence of allelic variants. Our results showed that C. jejuni isolates differ in their capacity to harbour each of the virulence genes alone or when present in various combinations. The flaA gene was detected in all C. jejuni strains and none of the strains had all the studied virulence genes together. When considering C. jejuni strains from the investigated sources, the cdtC gene was the most similar, while the cdtB and iam genes were the most dissimilar. We could identify 13 novel alleles in the analysed strains. The analyses of virulence gene patterns, flaA gene sequences and allelic variants showed that C. jejuni strains from different sources overlapped largely suggesting potential involvement of poultry in transmitting C. jejuni to humans. We also found that the strains isolated from the same host were highly heterogeneous, with chicken strains exhibiting the highest diversity. Moreover, the human strains were clustered closer to chicken ones than to those from pigeon. The results of this study should be taken into consideration when assessing the epidemiology and risk potential of Egyptian C. jejuni not only in poultry, but also in humans., (Copyright © 2019 Abd El-Hamid, Abd El-Aziz, Samir, El-Naenaeey, Abo Remela, Mosbah and Bendary.)

Introduction: Salmonella serovars are one of the primary foodborne pathogens. Poultry consumption is responsible for the majority of disease cases worldwide. The prevalence of virulence determinants among Salmonella serovars appears to be lacking in Egypt. Therefore, this study investigated the occurrence, antibiotic resistance patterns, and virulence gene profiling of Salmonella serovars in broilers., Methodology: Three hundred samples from broiler chickens were examined for the presence of Salmonella by standard microbiological techniques. All Salmonella isolates were tested for their sensitivity against ten antibiotics and subjected to virulence genotyping by polymerase chain reaction (PCR)., Results: The overall isolation percentage of Salmonella was 17%. Seven different serovars were found, with the main one being Salmonella Typhimurium (52.94%). Salmonella isolates were sensitive to most of the tested antibiotics, but they exhibited absolute resistance against amoxicillin/clavulanic acid. Nine Salmonella strains (52.94%) were resistant to at least three antibiotics. Further PCR investigations into 17 Salmonella strains revealed different distribution patterns of eight virulence determinants among the isolates. The invA gene was the most prevalent one (100%), followed by hilA (88.24%), stn (58.82%), and fliC genes (52.94%), while each of sopB and pefA genes had a similar prevalence (41.18%), and sefC and spvC genes had the lowest prevalence (11.76 and 5.88%, respectively). PCR genotyping allowed grouping of Salmonella strains into ten genetic profiles., Conclusions: These results will help in understanding the spread of virulence genotypes and antibiotic resistance among Salmonella serovars in broilers.