Biocontrol of multidrug resistant pathogens isolated from fish farms using silver nanoparticles combined with hydrogen peroxide insight to its modulatory effect.

This study was divided into two parts. The first part involved the isolation, and detection of the prevalence and antimicrobial resistance profile of Aeromonas hydrophila, Pseudomonas aeruginosa, and Vibrio species from Nile tilapia fish and marine aquatic water. One hundred freshly dead Nile tilapia fish were collected from freshwater aquaculture fish farms located in Al-Abbassah district, Sharkia Governorate, and 100 samples of marine aquatic water were collected from fish farms in Port Said. The second part of the study focused on determining the in vitro inhibitory effect of dual-combination of AgNPs-H2O2 on bacterial growth and its down regulatory effect on crucial virulence factors using RT-PCR. The highest levels of A. hydrophila and P. aeruginosa were detected in 43%, and 34% of Nile tilapia fish samples, respectively. Meanwhile, the highest level of Vibrio species was found in 37% of marine water samples. Additionally, most of the isolated A. hydrophila, P. aeruginosa and Vibrio species exhibited a multi-drug resistance profile. The MIC and MBC results indicated a bactericidal effect of AgNPs-H2O2. Furthermore, a transcriptional modulation effect of AgNPs-H2O2 on the virulence-associated genes resulted in a significant down-regulation of aerA, exoU, and trh genes in A. hydrophila, P. aeruginosa, and Vibrio spp., respectively. The findings of this study suggest the effectiveness of AgNPs-H2O2 against drug resistant pathogens related to aquaculture.

Bacteriological evaluation and advanced SYBR-green multiplex real-time PCR assay for detection of minced meat adulteration.

Background: Minced meat is a valuable source of nutrients, but it is vulnerable to contamination by microorganisms commonly present in the environment. In addition, there is a risk of adulteration with cheaper meat sources, which can be harmful to consumers. Aim: It is crucial to identify meat adulteration with distinct microbiological analysis for legal, economic, religious, and public health purposes. Methods: A total of 100 minced meat samples were collected from several markets in Sharkia Governorate, Egypt. These samples were then subjected to bacteriological testing and an advanced multiplex PCR method. This method enables the detection of bovine, equine, porcine, and dog species in meat samples with just one step. Results: The adulterated samples had a higher total bacterial count and pH values compared to pure bovine meat. These differences in bacterial count and pH values were statistically significant, with p-values of 0.843 (log10) and 0.233, respectively. The frequency of Escherichia coli occurrence was 13%, and the O111 serotype was predominant in the adulterated samples. Listeria monocytogenes and Staphylococcus aureus were isolated with prevalence rates of 3% and 29%, respectively. Besides, the SYBR-green multiplex real-time PCR assay used in this study detected adulteration with dog, equine, and porcine meats in the examined samples at rates of 9%, 5%, and 4%, respectively. Conclusion: This method provides a sensitive and specific approach to detect issues related to well-being and safety.

The ameliorative role of Aloe vera-loaded chitosan nanoparticles on Staphylococcus aureus induced acute lung injury: Targeting TLR/NF-κB signaling pathways.

Background: Acute lung injury (ALI) is a severe condition distinguished by inflammation and impaired gas exchange in the lungs. Staphylococcus aureus, a common bacterium, can cause ALI through its virulence factors. Aloe vera is a medicinal plant that has been traditionally used to treat a variety of illnesses due to its anti-inflammatory properties. Chitosan nanoparticles are biocompatible and totally biodegradable materials that have shown potential in drug delivery systems. Aim: To explore the antibacterial activity of Aloe vera-loaded chitosan nanoparticles (AV-CS-NPs) against S. aureus in vitro and in vivo with advanced techniques. Methods: The antibacterial efficacy of AV-CS-NPs was evaluated through a broth microdilution assay. In addition, the impact of AV-CS-NPs on S. aureus-induced ALI in rats was examined by analyzing the expression of genes linked to inflammation, oxidative stress, and apoptosis. Furthermore, rat lung tissue was scanned histologically. The rats were divided into three groups: control, ALI, and treatment with AV-CS-NPs. Results: The AV-CS-NPs that were prepared exhibited clustered semispherical and spherical forms, having an average particle size of approximately 60 nm. These nanoparticles displayed a diverse structure with an uneven distribution of particle sizes. The maximum entrapment efficiency of 95.5% ± 1.25% was achieved. The obtained findings revealed that The minimum inhibitory concentration and minimum bactericidal concentration values were determined to be 5 and 10 ug/ml, respectively, indicating the potent bactericidal effect of the NPs. Also, S. aureus infected rats explored upregulation in the mRNA expression of TLR2 and TLR4 compared to healthy control groups. AV-CS-NP treatment reverses the case where there was repression in mRNA expression of TLR2 and TLR4 compared to S. aureus-treated rats. Conclusion: These NPs can serve as potential candidates for the development of alternative antimicrobial agents.

An Alternative Approach Using Nano-garlic Emulsion and its Synergy with Antibiotics for Controlling Biofilm-Producing Multidrug-Resistant Salmonella in Chicken.

Surface-growing antibiotic-resistant pathogenic Salmonella is emerging as a global health challenge due to its high economic loss in the poultry industry. Their pathogenesis, increasing antimicrobial resistance, and biofilm formation make them challenging to treat with traditional therapy. The identification of antimicrobial herbal ingredients may provide valuable solutions to solve this problem. Therefore, our aim is to evaluate the potency of nano garlic as the  alternative of choice against multidrug-resistant (MDR) Salmonella isolates using disc diffusion and microdilution assays. Then, checkerboard titration in trays was applied, and FIC was measured to identify the type of interaction between the two antimicrobials. A disc diffusion assay revealed that neomycin was the drug of choice. The range of nano garlic MIC was 12.5-25 µg/ml, while the neomycin MIC range was 32-64 µg/ml. The FIC index established a synergistic association between the two tested drugs in 85% of isolates. An experimental model was used including nano garlic and neomycin alone and in combination against Salmonella infection. The combination therapy significantly improved body productivity and inhibited biofilm formation by more than 50% down regulating the CsgBAD, motB, and sipA operons, which are responsible for curli fimbriae production and biofilm formation in Salmonella serotypes.

Pathognomonic features of Pasteurella multocida isolates among various avian species in Sharkia Governorate, Egypt.

The present study aimed to isolate Pasteurella multocida (P. multocida) from pulmonary cases in several avian species and then investigate the histopathological features, antimicrobial resistance determinants, virulence characteristics, and risk factors analysis of the isolates in each species in correlation with epidemiological mapping of pasteurellosis in Sharkia Governorate, Egypt. The obtained data revealed a total occurrence of 9.4% (30/317) of P. multocida among the examined birds (chickens, ducks, quails, and turkeys). The incidence rate was influenced by avian species, climate, breed, age, clinical signs, and sample type. Antimicrobial susceptibility testing revealed that all isolates were sensitive to florfenicol and enrofloxacin, while 86.6 and 73.3% of the isolates displayed resistance to amoxicillin-clavulanic acid and erythromycin, respectively. All of the P. multocida isolates showed a multiple-drug resistant pattern with an average index of 0.43. Molecular characterization revealed that the oma87, sodA, and ptfA virulence genes were detected in the all examined P. multocida isolates. The ermX (erythromycin), blaROB-1 (ß-lactam), and mcr-1(colistin) resistance genes were present in 60, 46.6, and 40% of the isolates, respectively. Ducks and quails were the most virulent and harbored species of antimicrobial-resistant genes. These results were in parallel with postmortem and histopathological examinations which detected more severe interstitial pneumonia lesions in the trachea and lung, congestion, and cellular infiltration especially in ducks. Epidemiological mapping revealed that the Fakous district was the most susceptible to pasteurellosis infection. Thus, farmers are recommended to monitor their flocks for signs of respiratory disease, seek veterinary care promptly if any birds are sick, and avoid the random usage of antibiotics. In conclusion, this study presents a comprehensive picture of the risk factors in correlation to the pathognomonic characteristics of P. multocida infection in poultry sectors to help in developing more effective strategies for prevention and control.

The Potential Effects of Quercetin-Loaded Nanoliposomes on Amoxicillin/Clavulanate-Induced Hepatic Damage: Targeting the SIRT1/Nrf2/NF-κB Signaling Pathway and Microbiota Modulation.

Amoxicillin/clavulanate (Co-Amox), a commonly used antibiotic for the treatment of bacterial infections, has been associated with drug-induced liver damage. Quercetin (QR), a naturally occurring flavonoid with pleiotropic biological activities, has poor water solubility and low bioavailability. The objective of this work was to produce a more bioavailable formulation of QR (liposomes) and to determine the effect of its intraperitoneal pretreatment on the amelioration of Co-Amox-induced liver damage in male rats. Four groups of rats were defined: control, QR liposomes (QR-lipo), Co-Amox, and Co-Amox and QR-lipo. Liver injury severity in rats was evaluated for all groups through measurement of serum liver enzymes, liver antioxidant status, proinflammatory mediators, and microbiota modulation. The results revealed that QR-lipo reduced the severity of Co-Amox-induced hepatic damage in rats, as indicated by a reduction in serum liver enzymes and total liver antioxidant capacity. In addition, QR-lipo upregulated antioxidant transcription factors SIRT1 and Nrf2 and downregulated liver proinflammatory signatures, including IL-6, IL-1ß, TNF-α, NF-κB, and iNOS, with upregulation in the anti-inflammatory one, IL10. QR-lipo also prevented Co-Amox-induced gut dysbiosis by favoring the colonization of Lactobacillus, Bifidobacterium, and Bacteroides over Clostridium and Enterobacteriaceae. These results suggested that QR-lipo ameliorates Co-Amox-induced liver damage by targeting SIRT1/Nrf2/NF-κB and modulating the microbiota.

Evaluation and development of diagnostic tools for rapid detection of Riemerella anatipestifer and Pasteurella multocida in ducks.

Objectives: Ducks suffer a huge economic loss as a result of infections with Pasteurella multocida and Riemerella anatipestifer, which cause high morbidity and mortality. Because these pathogens induce similar clinical symptoms when coinfections occur, it is very difficult to differentiate between them based just on clinical signs. Hence, these major pathogens must be quickly and accurately detected. Materials and Methods: A total of 104 birds ranging from 2 days to 4 weeks old were collected from Egyptian farms, and the outcomes were compared statistically. Conventional cultural identification procedures and a direct multiplex polymerase chain reaction assay were utilized to recognize both pathogens in a single tube reaction simultaneously. Then, the obtained isolates were characterized phenotypically and genotypically. Results: Clinical signs appear at 2-4 weeks of age with respiratory distress (dyspnea), white fluid feces, and stunting. The scrutinized data demonstrated a significantly higher detection rate by PCR directly compared to classical culture procedures. Pasteurella multocida was detected only by PCR. The disc diffusion technique against ten antibiotics showed absolute susceptibilities to amikacin, doxycycline, and florfenicol. High levels of beta-lactam resistance were observed. Riemerella anatipestifer isolates were screened for pathogenicity and plasmid-borne blaTEM genes. All six isolates harbored five virulence genes: aspC, RA46, m28, pstS, and Nlp/P60. Moreover, blaTEM was identified into four isolates and deposited to GenBank with accession numbers OP347083, OP347084, OP347085, and OP347086. Conclusion: These results suggest advanced PCR assays can be applied to the field for rapid and valuable diagnosis of two significant pathogens and focus on the worth of ducks in the propagation of transferable antibiotic resistance genes into the environment.

Molecular Characterization and Antimicrobial Susceptibilities of Corynebacterium pseudotuberculosis Isolated from Caseous Lymphadenitis of Smallholder Sheep and Goats.

Caseous lymphadenitis (CLA) is a bacterial infection caused by Corynebacterium pseudotuberculosis (C. pseudotuberculosis) that affects sheep and goats, leading to abscess formation in their lymph nodes. The present study aimed to isolate and identify C. pseudotuberculosis from CLA in smallholder sheep and goats, and determine the resistance patterns, virulence, and resistance genes of the isolates. Additionally, genotypic and phylogenetic analysis of the isolates was conducted using ERIC-PCR and DNA sequencing techniques. A cross-sectional study examined 220 animals (130 sheep and 90 goats) from 39 smallholder flocks for clinical signs of CLA. Fifty-four (24.54%) animals showed CLA-compatible lesions, confirmed by C. pseudotuberculosis isolation and PCR identification. Sheep had a lower infection rate of CLA (18.46%) compared with goats (33.3%). Antimicrobial susceptibility testing of 54 C. pseudotuberculosis isolates to 24 antimicrobial drugs revealed that they were 100% resistant to bacitracin and florfenicol, while none of the isolates were resistant to norfloxacin. A high resistance rate was observed for penicillin and erythromycin (92.6% each). Interestingly, 16.7% of C. pseudotuberculosis isolates recovered from sheep showed vancomycin resistance. Molecular characterization of C. pseudotuberculosis isolates revealed that PLD, PIP, and FagA virulence genes were present in all examined isolates. However, the FagB, FagC, and FagD genes were detected in 24 (100%), 20 (83%), and 18 (75%) of the sheep isolates, and 26 (87%), 26 (87%), and 18 (60%) of the goat isolates, respectively. The ß-lactam resistance gene was present in all isolates. Furthermore, 83% of the sheep isolates carried the aminoglycoside (aph(3″)-lb), chloramphenicol (cat1), and bacitracin (bcrA) resistance genes. Among the isolates recovered from goats, 73% were found to contain macrolides (ermX), sulfonamide (sul1), and bacitracin (bcrA) resistance genes. It is worrisome that the glycopeptide (vanA) resistance gene was detected in 8% of the sheep isolates as a first report. ERIC-PCR genotyping of 10 multi-drug-resistant C. pseudotuberculosis isolates showed a high similarity index of 83.6% between isolates from sheep and goats. Nucleotide sequence analysis of partial 16S rRNA sequences of C. pseudotuberculosis revealed 98.83% similarity with biovar Ovis of globally available reference sequences on the Genbank database. Overall, our findings might indicate that C. pseudotuberculosis infection in smallholders in Egypt might be underestimated despite the significant financial impact on animal husbandry and potential health hazards it poses. Moreover, this study highlights the importance of implementing a sustainable control strategy and increasing knowledge and awareness among smallholder breeders to mitigate the economic impact of CLA.

Modulatory Effect of Competitive Exclusion on the Transmission of ESBL E. coli in Chickens.

The extensive use of antimicrobial agents in broiler farms causes the emergence of antimicrobial resistance of E. coli producing severe economic losses to the poultry industry; therefore, monitoring the transmission of ESBL E. coli is of great significance throughout broiler farms. For this reason, we investigated the efficiency of competitive exclusion (CE) products to control the excretion and transmission of ESBL-producing E. coli in broiler chickens. Three hundred samples from 100 broiler chickens were screened for the incidence of E. coli by standard microbiological techniques. The overall isolation percentage was 39% and differentiated serologically into ten different serotypes: O158, O128, O125, O124, O91, O78, O55, O44, O2, and O1. The isolates represented absolute resistance to ampicillin, cefotaxime, and cephalexin. The effectiveness of CE (commercial probiotic product; Gro2MAX) on ESBL-producing E. coli (O78) isolate transmission and excretion was studied in vivo. The results showed that the CE product has interesting properties, making it an excellent candidate for targeted drug delivery by inhibiting bacterial growth and downregulating biofilm, adhesins, and toxin-associated genes loci. The histopathological findings demonstrated the capability of CE in repairing internal organ tissues. Our outcomes suggested that the administration of CE (probiotic products) in broiler farms could be a safe and alternative approach to control the transmission of ESBL-producing virulent E. coli in broiler chickens.

Evaluation of gene expression related to immunity, apoptosis, and gut integrity that underlies Artemisia's therapeutic effects in necrotic enteritis-challenged broilers.

The experiment was designed to validate the effect of Artemisia annua and its novel commercial product (Navy Cox) on the control of necrotic enteritis (NE). A total of one hundred forty broiler chicks were randomly distributed into seven equal groups: G1, control negative; G2, infected with Eimeria (day 15) and C. perfringens (day 19); G3, treated with Navy Cox before challenge; G4, treated with Artemisia before challenge; G5, infected and then treated with Navy Cox; G6, infected and then treated with Artemisia; and G7, infected and treated with amoxicillin. Chicken response and immune organ indicants were recorded during the observation period (4 weeks). Whole blood and serum samples were collected for immunological evaluation, and tissue samples were collected for bacterial counts and estimation of mRNA expression of genes encoding apoptosis, tight junctions, and immunity. Chickens in the infected group revealed a significant decrease in RBCS, HB, PCV% total protein, Lysozyme, and nitric oxide activity in addition to leukocytosis, heterophilia, monocytosis, increase in cortisol, interleukins, and malondialdehyde. Treated groups displayed lower lesions, colony-forming units, and no mortality. Concurrently, a complete blood profile, antioxidants, and immune markers showed significant improvements. The mRNA expression levels of CASP, CLDN-1, OCLN, TJPI, MUC2, and cell-mediated immune response genes (p < 0.0001) were significantly alleviated in the treated groups compared with the challenged counterpart. This is the first-ever report on the efficacy valuation of Navy Cox compared to standard antibiotic treatment of clostridial NE. Navy Cox proved remarkable capability to minimize C. perfringens colonization in broiler intestines, modulation of mucus production, gut health integrity, immune organs, and immune response when used as a prophylactic agent in this form or naturally as Artemisia.

Green synthesis of cellulose nanocrystal/ZnO bio-nanocomposites exerting antibacterial activity and downregulating virulence toxigenic genes of food-poisoning bacteria.

Recently, cellulose nanocrystals (CNs) have attracted wide attention owing to their superior properties compared to their bulk materials. For example, they represent an outstanding model for fabricating green metallic/metal oxide nanoparticles (NPs). In this study, two CNs (carboxylated CNs and sulfated CNs) extracted from agro-wastes of palm sheath fibers were used as templates for the facile and green synthesis of ZnO NPs by employing the sono-co-precipitation method. The obtained nanomaterials were characterized using TEM, EDX, UV-visible, DLS, FT-IR, and XRD analysis. As a result, the size and concentration of synthesized ZnO NPs were inversely proportional to one another and were affected by the CNs utilized and the reaction temperature used. Contagious diseases incited by multifarious toxigenic bacteria present severe threats to human health. The fabricated bio-nanocomposites were evaluated in terms of their antimicrobial efficacy by agar well diffusion method and broth microdilution assay, showing that CN-ZnO bio-nanocomposites were effective against the tested Gram-negative (Escherichia coli and Salmonella) and Gram-positive (Listeria monocytogenes and Staphylococcus aureus) bacteria. The influence of the subinhibitory concentrations of these suspensions on the expression of the most critical virulence toxin genes of the tested strains was effective. Significant downregulation levels were observed through toxigenic operons to both fabricated CN-ZnO bio-nanocomposites with a fold change ranging from 0.004 to 0.510, revealing a decline in the capacity and virulence of microorganisms to pose infections. Therefore, these newly fabricated CNS-ZnO bio-nanocomposites could be employed rationally in food systems as a novel preservative to inhibit microbial growth and repress the synthesis of exotoxins.

Rhamnolipid-Coated Iron Oxide Nanoparticles as a Novel Multitarget Candidate against Major Foodborne E. coli Serotypes and Methicillin-Resistant S. aureus.

Surface-growing antibiotic-resistant pathogenic bacteria such as Escherichia coli and Staphylococcus aureus are emerging as a global health challenge due to dilemmas in clinical treatment. Furthermore, their pathogenesis, including increasingly serious antimicrobial resistance and biofilm formation, makes them challenging to treat by conventional therapy. Therefore, the development of novel antivirulence strategies will undoubtedly provide a path forward in combatting these resistant bacterial infections. In this regard, we developed novel biosurfactant-coated nanoparticles to combine the antiadhesive/antibiofilm properties of rhamnolipid (RHL)-coated Fe3O4 nanoparticles (NPs) with each of the p-coumaric acid (p-CoA) and gallic acid (GA) antimicrobial drugs by using the most available polymer common coatings (PVA) to expand the range of effective antibacterial drugs, as well as a mechanism for their synergistic effect via a simple method of preparation. Mechanistically, the average size of bare Fe3O4 NPs was ~15 nm, while RHL-coated Fe3O4@PVA@p-CoA/GA was about ~254 nm, with a drop in zeta potential from -18.7 mV to -34.3 mV, which helped increase stability. Our data show that RHL-Fe3O4@PVA@p-CoA/GA biosurfactant NPs can remarkably interfere with bacterial growth and significantly inhibited biofilm formation to more than 50% via downregulating IcaABCD and CsgBAC operons, which are responsible for slime layer formation and curli fimbriae production in S. aureus and E. coli, respectively. The novelty regarding the activity of RHL-Fe3O4@PVA@p-CoA/GA biosurfactant NPs reveals their potential effect as an alternative multitarget antivirulence candidate to minimize infection severity by inhibiting biofilm development. Therefore, they could be used in antibacterial coatings and wound dressings in the future. IMPORTANCE Antimicrobial resistance poses a great threat and challenge to humanity. Therefore, the search for alternative ways to target and eliminate microbes from plant, animal, and marine microorganisms is one of the world's concerns today. Furthermore, the extraordinary capacity of S. aureus and E. coli to resist standard antibacterial drugs is the dilemma of all currently used remedies. Methicillin-resistant S. aureus (MRSA) and vancomycin-resistant S. aureus (VRSA) have become widespread, leading to no remedies being able to treat these threatening pathogens. The most widely recognized serotypes that cause severe foodborne illness are E. coli O157:H7, O26:H11, and O78:H10, and they display increasing antimicrobial resistance rates. Therefore, there is an urgent need for an effective therapy that has dual action to inhibit biofilm formation and decrease bacterial growth. In this study, the synthesized RHL-Fe3O4@PVA@p-CoA/GA biosurfactant NPs have interesting properties, making them excellent candidates for targeted drug delivery by inhibiting bacterial growth and downregulating biofilm-associated IcaABCD and CsgBAC gene loci.

Virulence attitude estimation of Pasteurella multocida isolates in embryonated chicken eggs.

A total of 220 birds of age ranging from 3 to 14 weeks old were collected from several backyards and different farms in Sharkia Governorate, Egypt, and surveyed for the presence of fowl cholera. Twenty Pasteurella multocida from chickens (15/145, 10%) and ducks (5/75, 6%) were bacteriologically isolated, and it was shown that the infection was significantly related to age and breed. Capsular typing, using multiplex polymerase chain reaction (PCR), demonstrated that all strains were type A (100%). Disk diffusion assay towards ten antimicrobials revealed high susceptibilities to amikacin, doxycycline, chloramphenicol, and neomycin with varying degrees. Doxycycline was effective at the lowest concentration (MIC 0.125-1 µg/ml). Multidrug resistance was detected with a percentage of 25%. Multidrug-resistant isolates (five isolates) were subjected to study their pathogenicity in embryonated chicken eggs (ECE). The results showed a variation in indices between different dilutions of the tested strains. The resulting pathogenicity indices showed significant differences (P < 0.05) according to the origin and dilution of the isolate. From the original inoculum to 10-4 dilutions, the mortality of inoculated embryos occurred within 1-2 days with pathological findings, including maceration and lesions on chorioallantoic membrane (CAM). From dilutions ranging from 10-5 to 10-9, no death occurred until 7 days post-inoculation, but a variation in the lesions on CAM was observed. In conclusion, P. multocida serogroup A could be intensely pathogenic for mature chickens thus causing considerable economic losses, and PCR provides a suitable technique for early and rapid diagnosis of fowl cholera.

Validation of Camel's Fetal Fluids as Antimicrobial Agents.

The present work investigated the effect of camel's fetal fluids on a variety of bacterial and fungal pathogens. Ten samples of camel's amniotic and allantoic fluids were collected aseptically during parturition and their antimicrobial activities were evaluated by disc diffusion method and minimum inhibitory concentration (MIC) assay. The majority of tested pathogens were inhibited by both fluids up to 25% concentration. The fluids showed zones of inhibition ranging from 8 to 30 mm. The most pronounced inhibition was detected for Staphylococcus aureus, Listeria monocytogenes, Klebsiella pneumonia, and Aspergillus niger but the weak inhibition was obtained for Enterococcus faecalis, Bacillus subtilis, and Candida albicans. Also, the MIC values of amniotic fluids (0.25-2 µg/ml) against Gram-positive bacteria and yeast were lower than the values of allantoic fluids (2-8 µg/ml). But in Gram-negative bacteria and molds, the MIC values of allantoic fluids (0.5-2 µg/ml) were the lowermost. Mucor circinelloides was the only pathogen that resisted both fluids. Analysis of fluid samples revealed the presence of several factors that are known to act as antimicrobial. All tested camel's fetal fluids harbored immunoglobulins, complements, and transferrin. Lysozyme was present in only one of 10 examined samples. We firstly report the prevalence of a profound in-vitro antimicrobial activity of camel's fetal fluids. This activity encourages their use as therapeutic alternative agents to overcome multidrug resistance problems.

Antimicrobial Effect of Garlic (Allium sativum) and Thyme (Zataria multiflora Boiss) Extracts on Some Food Borne Pathogens and Their Effect on Virulence Gene Expression.

The incrementing scope of pathogenic resistance to antibiotics has encouraged the search for antivirulence natural extracts. Therefore, our study designed to demonstrate the antimicrobial activity of an aqueous-garlic and thyme oil extracts against Gram-positive (Staphylococcus aureus) and Gram-negative (Salmonella spp.) bacteria by evaluating the influence of sub-inhibitory concentrations on the expression of the most critical virulence genes of the tested isolates. The antibacterial potential of both herbs was checked by the agar well diffusion method and minimum inhibitory concentration (MIC) assay. Interestingly, all isolates were inhibited by both extracts up to 50% concentration. Also, the MIC values of garlic extract (0.125-1µg/ml) against Salmonella isolates were lower than the values of thyme extract (0.5- 8µg/ml). But in S. aureus isolates, the MIC values of thyme extract (0.25- 2µg/ml) were the lowermost. Conventional PCR investigated that all S. aureus isolates carried the hlg (hemolysin) and icaA (intracellular adhesion) genes, but only six Salmonella isolates (three S. typhimurium and one each of S. kentucky, S. anatum, and S. lagos) had both the sopB (Salmonella outer protein B) and mgtC (membrane protein) genes. Real-time RT-PCR assays were performed to evaluate the extract's effect on the virulence genes. The thyme-oil extract has significantly repressed S. aureus virulence genes expression more than aqueous-garlic extract, which later one has effectively more than thyme-oil extract in downregulating the Salmonella virulence genes. In conclusion, garlic and thyme extracts can be used not only as a flavor, but also as potential antimicrobial agents against Gram-positive and negative bacteria.

Molecular analysis of integron gene cassette arrays associated multi-drug resistant Enterobacteriaceae isolates from poultry.

The study investigated 110 Enterobacteriaceae isolates from broiler chickens isolated from Sharkia poultry farms and analyzed the isolates antimicrobial resistance and the presence of integrons as a potential basis for this resistance. Antibiotic susceptibilities against 12 different antibiotics were determined by the disk diffusion method. Prevalences and classes of integrons were then detected in multi-drug resistant (MDR) strains using polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) followed by sequencing of the variable parts. Fifty-three isolates were MDR (resistant to three or more antimicrobial agents). High resistance was detected for rifamycin (82.7%), erythromycin (67.2%), and amoxicillin-clavulanic acid (63%). Classes 1 and 2 integrons were detected in 38 of 53 MDR Enterobacteriaceae isolates of which the most common were Salmonella species (n=19), followed by Escherichia coli (12), Klebsiella pneumoniae (3), Proteus species (3), and Citrobacter freundii (1). Three isolates only harbored class 1 integrons while the remaining 35 isolates carried class 2.  All class 1 integron positive isolates exhibited the same gene cassettes arrangements: 1.) dfrA12-orfF-aadA27 (1.6 kbp); 2.) aadA23 (1.0 kbp); and 3.) dfrA15 (0.8 kbp). Moreover, four different gene cassettes were identified within class 2 integrons: 1.) dfrA1-sat2-aadA30 (2 kbp) in all isolates; 2.) sat2-aadA1 (1.7 kbp) in only one isolate; 3.) catB2 (0.9 kbp) in four isolates; and 4.) a new variant of sat2 (0.65 kbp) in three isolates. Efforts should be made to introduce surveillance programs for monitoring antimicrobial resistance that could potentially be transmitted from broiler chickens to human via integrons.