Alpha-sitosterol: a new antiviral agent produced by Streptomyces misakiensis and its potential activity against Newcastle disease virus.

BACKGROUND: Newcastle Disease Virus (NDV) causes severe economic losses in the poultry industry worldwide. Hence, this study aimed to discover a novel bioactive antiviral agent for controlling NDV. Streptomyces misakiensis was isolated from Egyptian soil and its secondary metabolites were identified using infrared spectroscopy (IR), gas chromatography-mass spectrometry (GC-MS), and nuclear magnetic resonance (NMR) spectroscopy. The inhibitory activity of bioactive metabolite against NDV were examined. Three experimental groups of 10-day-old specific pathogen-free embryonated chicken eggs (SPF-ECEs), including the bioactive metabolite control group, NDV control positive group, and α-sitosterol and NDV mixture-treated group were inoculated. RESULTS: α-sitosterol (Ethyl-6-methylheptan-2-yl]-10,13-dimethyl-dodecahydro-1H-cyclopenta[a]phenanthren-3-ol), a secondary metabolite of S. misakiensis, completely inhibited hemagglutination (HA) activity of the NDV strain. The HA activity of the NDV strain was 8 log2 and 9 log2 for 0.5 and 0.75% RBCs, respectively. The NDV HA activity for the two concentrations of RBCs was significantly (P < 0.0001) inhibited after α-sitosterol treatment. There was a significant (P < 0.0001) decrease in the log 2 of HA activity, with values of - 0.500 (75%, chicken RBCs) before inoculation in SPF-ECEs and - 1.161 (50%, RBCs) and - 1.403 (75%, RBCs) following SPF-ECE inoculation. Compared to ECEs inoculated with NDV alone, the α-sitosterol-treated group showed improvement in histological lesion ratings for chorioallantoic membranes (CAM) and hepatic tissues. The CAM of the α-sitosterol- inoculated SPF-ECEs was preserved. The epithelial and stromal layers were noticeably thicker with extensive hemorrhages, clogged vasculatures, and certain inflammatory cells in the stroma layer in the NDV group. However, mild edema and inflammatory cell infiltration were observed in the CAM of the treated group. ECEs inoculated with α-sitosterol alone showed normal histology of the hepatic acini, central veins, and portal triads. Severe degenerative alterations, including steatosis, clogged sinusoids, and central veins, were observed in ECEs inoculated with NDV. Mild hepatic degenerative alterations, with perivascular round cell infiltration, were observed in the treated group. CONCLUSION: To the best of our knowledge, this is the first study to highlight that the potentially bioactive secondary metabolite, α-sitosterol, belonging to the terpene family, has the potential to be a biological weapon against virulent NDV. It could be used for the development of innovative antiviral drugs to control NDV after further clinical investigation.

Streptomyces coeruleorubidus as a potential biocontrol agent for Newcastle disease virus.

BACKGROUND: Newcastle disease virus (NDV) is a severe disease that affects domestic and wild birds. Controlled antibiotics derived from probiotics have been examined as prospective solutions for preserving seroconversion in NDV-vaccinated fowl. In this study, the secondary metabolite "telomycin" was extracted from Streptomyces coeruleorubidus (S. coeruleorubidus) isolated from Egypt's cultivated soil. The structure of telomycin was determined by the elucidation of spectroscopic analysis, including nuclear magnetic resonance (NMR) and mass spectrometry (MS) spectra, and comparison with the literature. The antiviral activity of the secondary metabolite was tested by checking its effect on NDV hemagglutination activity (HA). Moreover, HA of NDV was tested after inoculation of NDV (control) and a combination of telomycin and NDV in 10- days- specific pathogen-free embryonated chicken eggs (SPF-ECE) daily candling. Histopathological examination was performed for chorioallantoic membranes and liver of SPF-ECE. RESULTS: S. coeruleorubidus secondary metabolite "telomycin" showed complete hemagglutination inhibition (HI) activity of NDV strain (MN635617) with log106 infectivity titers (EID50/mL). The HA of NDV strain was 8 log2 and 9 log2 with 0.5% and 0.75% of chicken RBCs, respectively. Preserved structures of chorioallantoic-membranes (CAM) with dilated capillary networks were observed in the treated group inoculated with telomycin and NDV. Histological changes in SPF-ECE liver were examined after inoculation in ova to further characterize the telomycin effect. Telomycin and NDV mixture inoculated group showed preserved cytoarchitecture of hepatocytes with the presence of perivascular foci of lymphocytes. The group that was inoculated with telomycin alone showed normal histology of hepatic acini, central veins, and portal triads. CONCLUSION: S. coeruleorubidus telomycin is a promising bioactive agent that might be a biological weapon against a deadly chicken NDV that costs farmers a lot of money.

Characterization and Antidermatophyte Activity of Henna Extracts: A Promising Therapy for Humans and Animals Dermatophytoses.

Dermatophytoses representing a major global health problem and dermatophyte species with reduced susceptibility to antifungals are increasingly reported. Therefore, we investigated for the first time the antidermatophyte activity and phytochemical properties of the sequential extracts of the Egyptian privet Henna (Lawsonia inermis) leaves. Total phenolic content (TPC), total flavonoids (TF), and antioxidant activity of chloroform, diethyl ether, acetone, ethanol 80%, and aqueous extracts were evaluated. The antifungal activity of henna leaves extracts (HLE) toward 30 clinical dermatophytes isolates, including Trichophyton mentagrophytes, Microsporum canis, and T. rubrum, was determined. Morphological changes in hyphae were investigated using scanning electron microscopy (SEM) analysis. Following the polarity of ethanol and acetone, they exhibited distinct efficiency for the solubility and extraction of polyphenolic polar antioxidants from henna leaves. Fraxetin, lawsone, and luteolin-3-O-glucoside were the major phenolic compounds of henna leaves, as assessed using high-performance liquid chromatography analysis. A high and significant positive correlation was found between TPC, TF, the antioxidants, and the antidermatophyte activities of HLE. Acetone and ethanol extracts exhibited the highest antifungal activity toward the tested dermatophyte species with minimum inhibitory concentration (MIC) ranges 12.5-37.5 and 25-62.5 µg/mL, respectively. Structural changes including collapsing, distortion, inflating, crushing of hyphae with corrugation of walls, and depressions on hyphal surfaces were observed in SEM analysis for dermatophyte species treated with MICs of griseofulvin, acetone, and ethanol extracts. In conclusion, acetone and ethanolic extracts of henna leaves with their major constituent fraxetin exhibited effective antifungal activity toward dermatophyte species and may be developed as an alternative for dermatophytosis treatment. These findings impart a useful insight into the development of an effective and safe antifungal agent for the treatment of superficial fungal infections caused by dermatophytes.

Ringworm in calves: risk factors, improved molecular diagnosis, and therapeutic efficacy of an Aloe vera gel extract.

BACKGROUND: Dermatophytosis in calves is a major public and veterinary health concern worldwide because of its zoonotic potential and associated economic losses in cattle farms. However, this condition has lacked adequate attention; thus, to develop effective control measures, we determined ringworm prevalence, risk factors, and the direct-sample nested PCR diagnostic indices compared with the conventional methods of dermatophytes identification. Moreover, the phenolic composition of an Aloe vera gel extract (AGE) and its in vitro and in vivo antidermatophytic activity were evaluated and compared with those of antifungal drugs. RESULTS: Of the 760 calves examined, 55.79% (424/760) showed ringworm lesions; 84.91% (360/424) were positive for fungal elements in direct-microscopy, and 79.72% (338/424) were positive in culture. Trichophyton verrucosum was the most frequently identified dermatophyte (90.24%). The risk of dermatophytosis was higher in 4-6-month-old vs. 1-month-old calves (60% vs. 41%), and in summer and winter compared with spring and autumn seasons (66 and 54% vs. 48%). Poor hygienic conditions, intensive breeding systems, animal raising for meat production, parasitic infestation, crossbreeding, and newly purchased animals were statistically significant risk factors for dermatophytosis. One-step PCR targeting the conserved regions of the 18S and 28S genes achieved unequivocal identification of T. verrucosum and T. mentagrophytes in hair samples. Nested-PCR exhibited an excellent performance in all tested diagnostic indices and increased the species-specific detection of dermatophytes by 20% compared with culture. Terbinafine and miconazole were the most active antifungal agents for dermatophytes. Gallic acid, caffeic acid, chlorogenic acid, cinnamic acid, aloe-Emodin, quercetin, and rutin were the major phenolic compounds of AGE, as assessed using high-performance liquid chromatography (HPLC). These compounds increased and synergized the antidermatophytic activity of AGE. The treated groups showed significantly lower clinical scores vs. the control group (P < 0.05). The calves were successfully treated with topical AGE (500 ppm), resulting in clinical and mycological cure within 14-28 days of the experiment; however, the recovery was achieved earlier in the topical miconazole 2% and AGE plus oral terbinafine groups. CONCLUSIONS: The nested PCR assay provided a rapid diagnostic tool for dermatophytosis and complemented the conventional methods for initiating targeted treatments for ringworm in calves. The recognized antidermatophytic potential of AGE is an advantageous addition to the therapeutic outcomes of commercial drugs.

Carvacrol essential oil stimulates growth performance, immune response, and tolerance of Nile tilapia to Cryptococcus uniguttulatus infection.

This study investigated the antifungal activity of 5 essential oils (EOs) towards yeasts recovered from diseased fishes; and focused on the efficacy of one EO (carvacrol) on growth performance, non-specific immunity, and disease resistance of Nile tilapia Oreochromis niloticus against Cryptococcus uniguttulatus challenge. Thymoquinone, thymol, carvacrol, eugenol, and cinnamon were first tested in vitro against 20 clinical yeast strains in comparison with antifungal drugs (fluconazole, ketoconazole, itraconazole, amphotericin B, nystatin, and clotrimazole) using disc diffusion and broth microdilution methods. For the in vivo challenge, fish (n = 150) were divided into 5 groups (carvacrol prophylaxis, carvacrol treatment, itraconazole treatment, unchallenged control, and positive control; 30 fish group-1) with 3 replicates. Phagocytic activity, reactive oxygen species production, reactive nitrogen species production, myeloperoxidase, lysozyme activity, and total immunoglobulins were tested before and after challenge. Relative percent survival (RPS) and mortality percent were determined as indicators for functional immunity. EOs displayed divergent degrees of antifungal activity, and carvacrol was the most effective against the tested yeasts. The dietary additive of carvacrol significantly enhanced growth performance, all immunological parameters, and the RPS values (90%) compared to other treatments. This unique experimental model indicates that carvacrol seems promising not only for enhancing immunity and promoting fish growth, but also for controlling emerging fungal infections. Future studies should investigate different concentrations of carvacrol as well as its antifungal activity in different fish species.

Equine pythiosis in Egypt: clinicopathological findings, detection, identification and genotyping of Pythium insidiosum.

BACKGROUND: Equine pythiosis is an emerging, devastating disease that is hard to treat. The tumour-like nodular skin masses grow rapidly and the outcome is generally fatal, and thus early diagnosis and intervention are important. OBJECTIVES: (i) To highlight the clinical, histological and haematological findings in pythiosis, and (ii) to evaluate the efficacy of direct sample multiplex-PCR targeting the single nucleotide polymorphisms within the ribosomal DNA region for detection and genotyping of Pythium insidiosum. ANIMALS: Two hundred and twenty horses including 204 Arabian and 16 draft horses were surveyed. METHODS: Case series study diagnosis was based on clinical, pathological and haematological findings typical of P. insidiosum infection, culture identification, immunohistochemical investigation and direct sample PCR. RESULTS: The affected horses (24 of 220, 10.91%) presented with unifocal or multiple lesions on the abdomen, limbs, chest, face and mammary gland. Cases commonly had a history of access to stagnant water, ponds and intentionally flooded rice fields. Most were pregnant mares (58.33%). Histopathology revealed granulomatous reaction, blood vessel endotheliosis, heavy infiltration of eosinophils in the dermal layer, multifocal necrosis and Splendore-Hoeppli phenomenon. Unlike direct microscopy (50%) and culture (91.6%), multiplex-PCR assay identified P. insidiosum (Clade II) in all tested samples. To the best of the authors knowledge, this is the first study determining a clade of P. insidiosum causing equine pythiosis in Egypt. CONCLUSIONS AND CLINICAL IMPORTANCE: Direct sample multiplex-PCR assay is a potential tool for the early and rapid diagnosis of equine pythiosis. It overcomes limitations associated with morphological identification and provides a definitive diagnosis.

Towards a rapid identification and a novel proteomic analysis for dermatophytes from human and animal dermatophytosis.

BACKGROUND: Since accurate identification of dermatophyte species is essential for epidemiological studies and implementing antifungal treatment, overcoming limitations of conventional diagnostics is a fruitful subject. OBJECTIVES AND METHODS: In this study, we investigated real-time polymerase chain reaction(q-PCR), matrix-assisted laser desorption/ionisation time of flight mass spectrometry (MALDI-TOF MS) and nano-electrospray ionisation mass spectrometry (nano-ESI-MS) to detect and identify the most frequently isolated dermatophytes from human and animal dermatophytosis in comparison with conventional methods. RESULTS: Among 200 samples, the identified species were Microsporum canis (78.22%), Trichophyton verrucosum (10.89%) and T. mentagrophytes (5.94%). Q-PCR assay displayed great execution attributes for dermatophytes detection and identification. Using MALDI-TOF MS, M. canis, but none of T. violacium, T. verrucosum or T. mentagrophytes, could be identified. Nano-ESI-MS accurately identified all species. The potential virulence attributes of secreted proteases were anticipated and compared between species. Secreted endoproteases belonging to families/subfamilies of metalloproteases, subtilisins and aspartic protease were detected. The analysed exoproteases are aminopeptidases, dipeptidyl peptidases and carboxypeptidases. Microsporum canis have three immunogenic proteins, siderophore iron transporter mirB, protease inhibitors, plasma membrane proteolipid 3 and annexin. CONCLUSION: In essence, q-PCR, MALDI-TOF MS and nano-ESI-MS assays are very nearly defeating difficulties of dermatophytes detection and identification, thereby, supplement or supplant conventional diagnosis of dermatophytosis.

Diagnostic performance of molecular and conventional methods for identification of dermatophyte species from clinically infected Arabian horses in Egypt.

BACKGROUND: Rapid and accurate identification of dermatophytes is crucial for the effective control of disease outbreaks. Current methods based on culture and microscopic characteristics may require weeks before positive identification is made. OBJECTIVES: To (i) identify the most common pathogenic dermatophytes affecting Arabian horses; (ii) compare the performance of direct microscopy (DM), culture, PCR using hair samples (PCRhair ) and PCR based on culture isolates (PCRculture ) for the diagnosis of dermatophytosis. METHODS: Samples of hair and crusts of skin lesions from Arabian horses were collected on a monthly basis by scraping skin of affected horses. Samples were divided into three portions: the first portion was used for microscopic examination, the second for culture and the third portion for PCR amplification of intergenic spacer (ITS) regions. RESULTS: Out of 200 horses examined, 70 (35%) showed cutaneous lesions characteristic of dermatophytosis. DM revealed that 70.4% were positive for fungal elements and 85.7% were culture positive. The identified species were Microsporum canis, Trichophyton verrucosum, T. mentagrophytes var. mentagrophytes and M. equinum. Among 25 selected samples, 64, 92, 91.3 and 52% were positive for dermatophytes, as determined by DM, culture, PCRculture and PCRhair , respectively. CONCLUSIONS: The dermatophytes M. canis, T. verrucosum, T. mentagrophytes var. mentagrophytes and M. equinum were the most common cause of dermatophytosis in Arabian horses. Although the number of samples was small, the ITS-based PCR may be a useful diagnostic tool when combined with culture.

Genetic diversity and antifungal susceptibilities of environmental Cryptococcus neoformans and Cryptococcus gattii species complexes.

Cryptococcosis is an opportunistic systemic mycosis caused by Cryptococcus neoformans and C. gattii species complexes and is of increasing global importance. Maintaining continued surveillance of the antifungal susceptibility of environmental C. neoformans and C. gattii isolates is desirable for better managing cryptococcosis by identifying resistant isolates and revealing the emergence of intrinsically resistant species. Relevant research data from Egypt are scarce. Thus, this study aimed to report the genetic diversity of C. neoformans and C. gattii species complexes originating from different environmental sources in Egypt, antifungal susceptibility profiles, antifungal combinations, and correlations of susceptibility with genotypes. A total of 400 environmental samples were collected, 220 from birds and 180 from trees. Cryptococcus spp. were found in 58 (14.5%) of the samples, 44 (75.9%) of the isolates were recovered from birds and 14 (24.1%) from trees. These isolates were genotyped using M13 polymerase chain reaction-fingerprinting and URA5 gene restriction fragment length polymorphism analysis. Of the 31 C. neoformans isolates, 24 (77.4%), 6 (19.4%) and one (4.4%) belonged to VNI, VNII, and VNIII genotypes, respectively. The 27 C. gattii isolates belonged to VGI (70.4%), VGII (18.5%), and VGIII (11.1%) genotypes. Non-wild type C. neoformans and C. gattii isolates that may have acquired resistance to azoles, amphotericin B (AMB), and terbinafine (TRB) were observed. C. gattii VGIII was less susceptible to fluconazole (FCZ) and itraconazole (ITZ) than VGI and VGII. C. neoformans isolates showed higher minimum inhibitory concentrations (MICs) to FCZ, ITZ, and voriconazole (VRZ) than those of C. gattii VGI and VGII. Significant (P < 0.001) correlations were found between the MICs of VRZ and ITZ (r = 0.64) in both C. neoformans and C. gattii isolates, FCZ and TRB in C. neoformans isolates, and FCZ and TRB (r = 0.52) in C. gattii isolates.There is no significant differences in the MICs of TRB in combination with FCZ (P = 0.064) or in combination with AMB (P = 0.543) and that of TRB alone against C. gattii genotypes. By calculating the fractional inhibitory concentration (FIC) index, the combination of FCZ + AMB was synergistic against all tested genotypes. These findings expand our knowledge of ecological niches, genetic diversity, and resistance traits of C. neoformans and C. gattii genotypes in Egypt. Further investigations into how they are related to clinical isolates in the region are warranted.

Fighting Emerging Caspofungin-Resistant Candida Species: Mitigating Fks1-Mediated Resistance and Enhancing Caspofungin Efficacy by Chitosan.

Invasive candidiasis poses a worldwide threat because of the rising prevalence of antifungal resistance, resulting in higher rates of morbidity and mortality. Additionally, Candida species, which are opportunistic infections, have significant medical and economic consequences for immunocompromised individuals. This study explores the antifungal potential of chitosan to mitigate caspofungin resistance in caspofungin-resistant Candida albicans, C. krusei, and C. tropicalis isolates originating from human and animal sources using agar well diffusion, broth microdilution tests, and transmission electron microscope (TEM) analysis of treated Candida cells. Reverse transcriptase quantitative polymerase chain reaction (RT-qPCR) was performed to assess the expression of SAGA complex genes (GCN5 and ADA2) and the caspofungin resistance gene (FKS) in Candida species isolates after chitosan treatment. The highest resistance rate was observed to ketoconazole (80%) followed by clotrimazole (62.7%), fluconazole (60%), terbinafine (58%), itraconazole (57%), miconazole (54.2%), amphotericin B (51.4%), voriconazole (34.28%), and caspofungin (25.7%). Nine unique FKS mutations were detected, including S645P (n = 3 isolates), S645F, L644F, S645Y, L688M, E663G, and F641S (one isolate in each). The caspofungin minimum inhibitory concentration (MIC) and minimum fungicidal concentration (MFC) values before chitosan treatment ranged from 2 to 8 µg/mL and 4 to 16 µg/mL, respectively. However, the MIC and MFC values were decreased after chitosan treatment (0.0625-1 µg/mL) and (0.125-2 µg/mL), respectively. Caspofungin MIC was significantly decreased (p = 0.0007) threefold following chitosan treatment compared with the MIC values before treatment. TEM analysis revealed that 0.5% chitosan disrupted the integrity of the cell surface, causing irregular morphologies and obvious aberrant changes in cell wall thickness in caspofungin-resistant and sensitive Candida isolates. The cell wall thickness of untreated isolates was 0.145 µm in caspofungin-resistant isolate and 0.125 µm in sensitive isolate, while it was significantly lower in chitosan-treated isolates, ranging from 0.05 to 0.08 µm when compared with the cell wall thickness of sensitive isolate (0.03 to 0.06 µm). Moreover, RT-qPCR demonstrated a significant (p < 0.05) decrease in the expression levels of histone acetyltransferase genes (GCN5 and ADA2) and FKS gene of caspofungin-resistant Candida species isolates treated with 0.5% chitosan when compared with before treatment (fold change values ranged from 0.001 to 0.0473 for GCN5, 1.028 to 4.856 for ADA2, and 2.713 to 12.38 for FKS gene). A comparison of the expression levels of cell wall-related genes (ADA2 and GCN5) between caspofungin-resistant and -sensitive isolates demonstrated a significant decrease following chitosan treatment (p < 0.001). The antifungal potential of chitosan enhances the efficacy of caspofungin against various caspofungin-resistant Candida species isolates and prevents the development of further antifungal resistance. The results of this study contribute to the progress in repurposing caspofungin and inform a development strategy to enhance its efficacy, appropriate antifungal activity against Candida species, and mitigate resistance. Consequently, chitosan could be used in combination with caspofungin for the treatment of candidiasis.

Ocimum basilicum and Lagenaria siceraria Loaded Lignin Nanoparticles as Versatile Antioxidant, Immune Modulatory, Anti-Efflux, and Antimicrobial Agents for Combating Multidrug-Resistant Bacteria and Fungi.

Lignin nanoparticles emerged as a promising alternative for drug delivery systems owing to their biodegradability and bioactive properties. This study investigated the antimicrobial activity of the ethanolic extract of Ocimum basilicum-loaded lignin nanoparticles (OB-LNPs) and Lagenaria siceraria seed oil-loaded lignin nanoparticles (LS-LNPs) to find a solution for antimicrobial resistance. OB-LNPs and LS-LNPs were tested for their antimicrobial potential against Escherichia coli, Enterococcus faecalis, Klebsiella pneumoniae, Staphylococcus aureus, Salmonella enterica, Trichophyton mentagrophytes, Trichophyton rubrum, and Microsporum canis. OB-LNPs and LS-LNPs were further tested for their anti-efflux activity against ciprofloxacin-resistant Salmonella enterica strains and for treating Salmonella infection in a rat model. We also investigated the antifungal efficacy of OB-LNPs and LS-LNPs for treating T. rubrum infection in a guinea pig model. Both OB-LNPs and LS-LNPs showed strong antimicrobial potential against S. Typhimurium and T. rubrum infections. LS-LNPs showed antibacterial activity against Salmonella enterica species with a MIC range of 0.5-4 µg/mL and antifungal activity against T. rubrum with a MIC range of 0.125-1 µg/mL. OB-LNPs showed antibacterial activity against Salmonella enterica species with a MIC range of 0.5-2 µg/mL and antifungal activity against T. rubrum with a MIC range of 0.25-2 µg/mL. OB-LNPs and LS-LNPs downregulated the expression of ramA and acrB efflux pump genes (fold change values ranged from 0.2989 to 0.5434; 0.4601 to 0.4730 for ramA and 0.3842-0.6199; 0.5035-0.8351 for acrB). Oral administration of OB-LNPs and LS-LNPs in combination with ciprofloxacin had a significant effect on all blood parameters, as well as on liver and kidney function parameters. Oxidative stress mediators, total antioxidant capacity, and malondialdehyde were abolished by oral administration of OB-LNPs and LS-LNPs (0.5 mL/rat once daily for 5 days). Interferon-γ and tumor necrosis factor-α were also reduced in comparison with the positive control group and the ciprofloxacin-treated group. Histopathological examination of the liver and intestine of OB-LNPs and LS-LNPs-treated rats revealed an elevation in Salmonella clearance. Treatment of T. rubrum-infected guinea pigs with OB-LNPs and LS-LNPs topically in combination with itraconazole resulted in a reduction in lesion scores, microscopy, and culture results. In conclusion, OB-LNPs and LS-LNPs possess immunomodulatory and antioxidant potential and can be used as naturally derived nanoparticles for drug delivery and treatment of Salmonellosis and dermatophytosis infections.

Emergence of pandrug-resistant carbapenemase-producing Enterobacterales in dogs and cats: a cross-sectional study in Egypt.

One of the most important emerging health problems is the increasing role of animals in the rapid global rise in resistance to last-resort antibiotics, such as carbapenems. However, there is limited information on the role of pet animals in harboring and spreading pandrug-resistant (PDR) carbapenemase-producing Enterobacterales (CPE), especially in Egypt. This cross-sectional study was conducted to screen for CPE in healthy and diseased pets using phenotypic and molecular methods and the NG-Test CARBA 5 immunochromatographic assay. Rectal swabs were collected from 62 dogs and 48 cats, incubated overnight in tryptic soy broth containing 10 µg of meropenem disc and subsequently cultured on MacConkey agar supplemented with meropenem (1 mg/L). Sixty-six isolates (60.6%), including 56 Klebsiella pneumoniae, seven Escherichia coli, and three K. oxytoca isolates, were confirmed to be carbapenem-resistant Enterobacterales (CRE) by the disc diffusion method, broth microdilution test, CNPt-direct, and PCR assay targeting carbapenemase genes. Forty-three (65.2%) dogs and 23 (34.8%) cats carried CPE. Of these, 35 (70.0%) were healthy (including 27 dogs and 8 cats) and 31 (52.5%) were diseased (including 16 dogs and 15 cats). bla OXA-181 was the most common gene detected (42/66, 63.6%), followed by bla IMP (40/66, 60.6%), bla OXA-48-like (29/66, 43.9%), bla KPC and bla VIM (20/66, 30.3% each), and bla NDM (17/66, 25.8%). The identified genotypes were bla KPC-2, bla IMP-1, bla VIM-1, bla NDM-1, and bla NDM-5. The CARBA 5 assay showed higher sensitivity and specificity for the detection of NDM, OXA and KPC than that for VIM and IMP genes. Antimicrobial resistance profiles of CRE isolates revealed 20 PDR, 30 extensively drug-resistant (XDR), and 16 multidrug-resistant (MDR) phenotypes. This study provides evidence of colonization with PDR CPE in dogs and cats. To manage the infection or colonization of pets in veterinary clinical settings, extended surveillance systems should be considered, and the use of critical antibiotics should be strictly controlled.

Development, Optimization, and In Vitro/In Vivo Evaluation of Azelaic Acid Transethosomal Gel for Antidermatophyte Activity.

Treatment of dermatophytosis is quite challenging. This work aims to investigate the antidermatophyte action of Azelaic acid (AzA) and evaluate its efficacy upon entrapment into transethosomes (TEs) and incorporation into a gel to enhance its application. Optimization of formulation variables of TEs was carried out after preparation using the thin film hydration technique. The antidermatophyte activity of AzA-TEs was first evaluated in vitro. In addition, two guinea pig infection models with Trichophyton (T.) mentagrophytes and Microsporum (M.) canis were established for the in vivo assessment. The optimized formula showed a mean particle size of 219.8 ± 4.7 nm and a zeta potential of -36.5 ± 0.73 mV, while the entrapment efficiency value was 81.9 ± 1.4%. Moreover, the ex vivo permeation study showed enhanced skin penetration for the AzA-TEs (3056 µg/cm2) compared to the free AzA (590 µg/cm2) after 48 h. AzA-TEs induced a greater inhibition in vitro on the tested dermatophyte species than free AzA (MIC90 was 0.01% vs. 0.32% for T. rubrum and 0.032% for T. mentagrophytes and M. canis vs. 0.56%). The mycological cure rate was improved in all treated groups, specially for our optimized AzA-TEs formula in the T. mentagrophytes model, in which it reached 83% in this treated group, while it was 66.76% in the itraconazole and free AzA treated groups. Significant (p < 0.05) lower scores of erythema, scales, and alopecia were observed in the treated groups in comparison with the untreated control and plain groups. In essence, the TEs could be a promising carrier for AzA delivery into deeper skin layers with enhanced antidermatophyte activity.

Proteomics and metabolomics analyses of Streptococcus agalactiae isolates from human and animal sources.

Streptococcus agalactiae (S. agalactiae), group B Streptococcus (GBS), a major cause of infection in a wide variety of diseases, have been compared in different human and animal sources. We aimed to compare the bacterial proteome and metabolome profiles of human and animal S. agalactiae strains to delineate biological interactions relevant to infection. With the innovative advancement in mass spectrometry, a comparative result between both strains provided a solid impression of different responses to the host. For instance, stress-related proteins (Asp23/Gls24 family envelope stress response protein and heat shock protein 70), which play a role in the survival of GBS under extreme environmental conditions or during treatment, are highly expressed in human and animal strains. One human strain contains ꞵ-lactamase (serine hydrolase) and biofilm regulatory protein (lytR), which are important virulence regulators and potential targets for the design of novel antimicrobials. Another human strain contains the aminoglycosides-resistance bifunctional AAC/APH (A0A0U2QMQ5) protein, which confers resistance to almost all clinically used aminoglycosides. Fifteen different metabolites were annotated between the two groups. L-aspartic acid, ureidopropionic acid, adenosine monophosphate, L-tryptophan, and guanosine monophosphate were annotated at higher levels in human strains. Butyric acid, fumaric acid, isoleucine, leucine, and hippuric acid have been found in both human and animal strains. Certain metabolites were uniquely expressed in animal strains, with fold changes greater than 2. For example, putrescine modulates biofilm formation. Overall, this study provides biological insights into the substantial possible bacterial response reflected in its macromolecular production, either at the proteomic or metabolomic level.

Bioactive metabolites of Streptomyces misakiensis display broad-spectrum antimicrobial activity against multidrug-resistant bacteria and fungi.

Background: Antimicrobial resistance is a serious threat to public health globally. It is a slower-moving pandemic than COVID-19, so we are fast running out of treatment options. Purpose: Thus, this study was designed to search for an alternative biomaterial with broad-spectrum activity for the treatment of multidrug-resistant (MDR) bacterial and fungal pathogen-related infections. Methods: We isolated Streptomyces species from soil samples and identified the most active strains with antimicrobial activity. The culture filtrates of active species were purified, and the bioactive metabolite extracts were identified by thin-layer chromatography (TLC), preparative high-performance liquid chromatography (HPLC), nuclear magnetic resonance (NMR) spectroscopy, and gas chromatography-mass spectrometry (GC-MS). The minimum inhibitory concentrations (MICs) of the bioactive metabolites against MDR bacteria and fungi were determined using the broth microdilution method. Results: Preliminary screening revealed that Streptomyces misakiensis and S. coeruleorubidus exhibited antimicrobial potential. The MIC50 and MIC90 of S. misakiensis antibacterial bioactive metabolite (ursolic acid methyl ester) and antifungal metabolite (tetradecamethylcycloheptasiloxane) against all tested bacteria and fungi were 0.5 µg/ml and 1 µg/mL, respectively, versus S. coeruleorubidus metabolites: thiocarbamic acid, N,N-dimethyl, S-1,3-diphenyl-2-butenyl ester against bacteria (MIC50: 2 µg/ml and MIC90: 4 µg/mL) and fungi (MIC50: 4 µg/ml and MIC90: 8 µg/mL). Ursolic acid methyl ester was active against ciprofloxacin-resistant strains of Streptococcus pyogenes, S. agalactiae, Escherichia coli, Klebsiella pneumoniae, and Salmonella enterica serovars, colistin-resistant Aeromonas hydrophila and K. pneumoniae, and vancomycin-resistant Staphylococcus aureus. Tetradecamethylcycloheptasiloxane was active against azole- and amphotericin B-resistant Candida albicans, Cryptococcus neoformans, C. gattii, Aspergillus flavus, A. niger, and A. fumigatus. Ursolic acid methyl ester was applied in vivo for treating S. aureus septicemia and K. pneumoniae pneumonia models in mice. In the septicemia model, the ursolic acid methyl ester-treated group had a significant 4.00 and 3.98 log CFU/g decrease (P < 0.05) in liver and spleen tissue compared to the infected, untreated control group. Lung tissue in the pneumonia model showed a 2.20 log CFU/g significant decrease in the ursolic acid methyl ester-treated group in comparison to the control group. The haematological and biochemical markers in the ursolic acid methyl ester-treated group did not change in a statistically significant way. Moreover, no abnormalities were found in the histopathology of the liver, kidneys, lungs, and spleen of ursolic acid methyl ester-treated mice in comparison with the control group. Conclusion: S. misakiensis metabolite extracts are broad-spectrum antimicrobial biomaterials that can be further investigated for the potential against MDR pathogen infections. Hence, it opens up new horizons for exploring alternative drugs for current and reemerging diseases.

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.

Virulence Determinants and Plasmid-Mediated Colistin Resistance mcr Genes in Gram-Negative Bacteria Isolated From Bovine Milk.

A major increase of bacterial resistance to colistin, a last-resort treatment for severe infections, was observed globally. Using colistin in livestock rearing is believed to be the ground of mobilized colistin resistance (mcr) gene circulation and is of crucial concern to public health. This study aimed to determine the frequency and virulence characteristics of colistin-resistant Gram-negative bacteria from the milk of mastitic cows and raw unpasteurized milk in Egypt. One hundred and seventeen strains belonging to Enterobacteriaceae (n = 90), Pseudomonas aeruginosa (n = 10), and Aeromonas hydrophila (n = 17) were screened for colistin resistance by antimicrobial susceptibility testing. The genetic characteristics of colistin-resistant strains were investigated for mcr-1-9 genes, phylogenetic groups, and virulence genes. Moreover, we evaluated four commonly used biocides in dairy farms for teat disinfection toward colistin-resistant strains. Multidrug-resistant (MDR) and extensive drug-resistant (XDR) phenotypes were detected in 82.91% (97/117) and 3.42% (4/117) of the isolates, respectively. Of the 117 tested isolates, 61 (52.14%) were colistin resistant (MIC >2 mg/L), distributed as 24/70 (34.29%) from clinical mastitis, 10/11 (90.91%) from subclinical mastitis, and 27/36 (75%) from raw milk. Of these 61 colistin-resistant isolates, 47 (19 from clinical mastitis, 8 from subclinical mastitis, and 20 from raw milk) harbored plasmid-borne mcr genes. The mcr-1 gene was identified in 31.91%, mcr-2 in 29.79%, mcr-3 in 34.04%, and each of mcr-4 and mcr-7 in 2.13% of the colistin-resistant isolates. Among these isolates, 42.55% (20/47) were E. coli, 21.28% (10/47) A. hydrophila, 19.12% (9/47) K. pneumoniae, and 17.02% (8/47) P. aeruginosa. This is the first report of mcr-3 and mcr-7 in P. aeruginosa. Conjugation experiments using the broth-mating technique showed successful transfer of colistin resistance to E. coli J53-recipient strain. Different combinations of virulence genes were observed among colistin-resistant isolates with almost all isolates harboring genes. Hydrogen peroxide has the best efficiency against all bacterial isolates even at a low concentration (10%). In conclusion, the dissemination of mobile colistin resistance mcr gene and its variants between MDR- and XDR-virulent Gram-negative isolates from dairy cattle confirms the spread of mcr genes at all levels; animals, humans, and environmental, and heralds the penetration of the last-resort antimicrobial against MDR bacteria. Consequently, a decision to ban colistin in food animals is urgently required to fight XDR and MDR bacteria.

Extensive Drug-Resistant Salmonella enterica Isolated From Poultry and Humans: Prevalence and Molecular Determinants Behind the Co-resistance to Ciprofloxacin and Tigecycline.

The emergence of extensive drug-resistant (XDR) Salmonella in livestock animals especially in poultry represents a serious public health and therapeutic challenge. Despite the wealth of information available on Salmonella resistance to various antimicrobials, there have been limited data on the genetic determinants of XDR Salmonella exhibiting co-resistance to ciprofloxacin (CIP) and tigecycline (TIG). This study aimed to determine the prevalence and serotype diversity of XDR Salmonella in poultry flocks and contact workers and to elucidate the genetic determinants involved in the co-resistance to CIP and TIG. Herein, 115 Salmonella enterica isolates of 35 serotypes were identified from sampled poultry (100/1210, 8.26%) and humans (15/375, 4.00%), with the most frequent serotype being Salmonella Typhimurium (26.96%). Twenty-nine (25.22%) Salmonella enterica isolates exhibited XDR patterns; 25 out of them (86.21%) showed CIP/TIG co-resistance. Exposure of CIP- and TIG-resistant isolates to the carbonyl cyanide 3-chlorophenylhydrazone (CCCP) efflux pump inhibitor resulted in an obvious reduction in their minimum inhibitory concentrations (MICs) values and restored the susceptibility to CIP and TIG in 17.24% (5/29) and 92% (23/25) of the isolates, respectively. Molecular analysis revealed that 89.66% of the isolates contained two to six plasmid-mediated quinolone resistance genes with the predominance of qepA gene (89.66%). Mutations in the gyrA gene were detected at codon S83 (34.62%) or D87 (30.77%) or both (34.62%) in 89.66% of XDR Salmonella. The tet(A) and tet(X4) genes were detected in 100% and 3.45% of the XDR isolates, respectively. Twelve TIG-resistant XDR Salmonella had point mutations at codons 120, 121, and 181 in the tet(A) interdomain loop region. All CIP and TIG co-resistant XDR Salmonella overexpressed ramA gene; 17 (68%) out of them harbored 4-bp deletion in the ramR binding region (T-288/A-285). However, four CIP/TIG co-resistant isolates overexpressed the oqxB gene. In conclusion, the emergence of XDR S. enterica exhibiting CIP/TIG co-resistance in poultry and humans with no previous exposure to TIG warrants an urgent need to reduce the unnecessary antimicrobial use in poultry farms in Egypt.

Methicillin- and Vancomycin-Resistant Staphylococcus aureus From Humans and Ready-To-Eat Meat: Characterization of Antimicrobial Resistance and Biofilm Formation Ability.

Methicillin-resistant and vancomycin-resistant Staphylococcus aureus (MRSA and VRSA) are zoonotic life-threatening pathogens, and their presence in food raises a public health concern. Yet, scarce data are available regarding MRSA and VRSA in both ready-to-eat (RTE) meat and food handlers. This study was undertaken to determine the frequency, antimicrobial resistance, and biofilm-forming ability of MRSA and VRSA isolated from RTE meat (shawarma and burger) and humans (food handlers, and hospitalized patients) in Zagazig city, Sharkia Governorate, Egypt. We analyzed 176 samples (112 human samples: 72 from hospitalized patients and 40 from food handlers, 64 RTE meat samples: 38 from shawarma and 26 from burger). Using phenotypic, PCR-based identification of nuc gene and matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF MS), 60 coagulase-positive S. aureus (COPS) isolates were identified in the samples as follow: RTE meat (15/64, 23.4%), hospitalized patients (33/72, 45.8%) and food handlers (12/40, 30%). All the COPS isolates were mecA positive (and thus were classified as MRSA) and multidrug resistant with multiple antibiotic resistance indices ranging from 0.25 to 0.92. Overall, resistance to cefepime (96.7%), penicillin (88.3%), were common, followed by ampicillin-sulbactam (65%), ciprofloxacin (55%), nitrofurontoin (51.7%), and gentamicin (43.3%). VRSA was detected in 30.3% of COPS hospitalized patient's isolates, 26.7% of COPS RTE meat isolates and 25% of COPS food handler's isolates. VanA, vanB, or both genes were detected in 64.7, 5.9, and 29.4% of all VAN-resistant isolates, respectively. The majority of the COPS isolates (50/60, 83.3%) have biofilm formation ability and harbored icaA (76%), icaD (74%), icaC (50%), and icaB (46%) biofilm-forming genes. The bap gene was not detected in any of the isolates. The ability of MRSA and VRSA isolates to produce biofilms in addition to being resistant to antimicrobials highlight the danger posed by these potentially virulent microorganisms persisting in RTE meat, food handlers, and patients. Taken together, good hygiene practices and antimicrobial surveillance plans should be strictly implemented along the food chain to reduce the risk of colonization and dissemination of MRSA and VRSA biofilm-producing strains.

Emergence of Colistin and Carbapenem Resistance in Extended-Spectrum ß-Lactamase Producing Klebsiella pneumoniae Isolated from Chickens and Humans in Egypt.

This study investigated the frequency of carbapenem and colistin resistance in ESBL-producing K. pneumoniae (ESBLK) isolates recovered from chickens and their environment, contact farm workers and hospitalized patients in Egypt. Further, the phenotypic and genotypic relationships between the community and hospital-acquired K. pneumoniae isolates in the same geographical area were investigated. From 272 total samples, 37 (13.6%) K. pneumoniae isolates were identified, of which 20 (54.1%) were hypervirulent. All isolates (100%) were multidrug-resistant (MDR) with multiple antibiotic resistance (MAR) indices ranging from 0.19 to 0.94. Colistin-resistant isolates (18.9%) displayed colistin MIC values >2 µg/mL, all harbored the mcr-1 gene. All isolates from patients (13/90, 14.4%), workers (5/22, 22.7%), chickens (9/100, 9%) and the environment (10/60, 16.7%) harbored a single or multiple ß-lactamase genes, blaSHV, blaTEM, blaCTX-M1 and blaOXA-1, often in combination with carbapenemase genes (blaVIM, blaNDM-1 or blaIMP; 45.9%), the mcr-1 gene (18.9%) or both (13.5%). Enterobacterial repetitive intergenic consensus (ERIC)-PCR genotyping revealed 24 distinct ERIC types (ETs) with a discrimination index of 0.961. Six ETs showed clusters of identical isolates from chicken and human sources. The increased frequency and genetic relatedness of ESBLK and carbapenemase-producing K. pneumoniae (CPK) from chickens and humans pose a public health threat that urge more prudent use of antimicrobials in chicken farms to avoid the propagation and expansion of both ESBLK and CPK from the chicken sources to humans.