Mirela Ahmadi

Necrotic enteritis (NE), caused by the Gram-positive, anaerobic bacterium Clostridium perfringens (C. perfringens; CP), is one of the most economically important diseases affecting the poultry industry worldwide. The pathogenicity of C. perfringens is attributed to the production of more than twenty extracellular toxins and enzymes, with strains classified into seven toxinotypes (A–G) based on their major toxin genes. While the alpha-toxin gene (cpa) is present across all C. perfringens toxinotypes, netB is recognized as the primary virulence determinant of avian NE. However, toxin-gene carriage alone does not establish toxin expression or disease causation. Cpb2 and tpeL have also been associated with virulence in some C. perfringens isolates, although their independent contributions to NE pathogenesis remain less firmly established. The increasing prevalence of antimicrobial-resistant C. perfringens strains, including multidrug-resistant (MDR), extensively drug-resistant (XDR), and pandrug-resistant (PDR) isolates, together with the organism’s ability to form biofilms, has complicated disease prevention and treatment by enhancing environmental persistence and tolerance to antimicrobial agents. Distinct from reviews that primarily address NE pathogenesis or antibiotic alternatives as broad terms, this review integrates antimicrobial resistance with planktonic and biofilm-associated C. perfringens. Furthermore, it critically evaluates alternative interventions based on their activity against these bacterial states and the strength of supporting in vitro, in vivo, and field evidence. In conclusion, this review highlights the urgent need for sustainable, antibiotic-free strategies to combat NE. Advancing the development and field validation of these alternative approaches may contribute to reducing antimicrobial resistance, limiting biofilm-associated persistence, and improving poultry health and productivity.

Chronic high-fat diet (HFD) feeding in male rats causes significant metabolic as well as inflammatory disturbances, including obesity, insulin resistance, dyslipidemia, liver and kidney dysfunction, oxidative stress, and hypothalamic dysregulation. This study assessed the therapeutic effects of chlorogenic acid (CGA), a natural polyphenol, administered at 10 mg and 100 mg/kg/day for the last 4 weeks of a 12-week HFD protocol. Both CGA doses reduced body weight gain, abdominal circumference, and visceral fat accumulation, with the higher dose showing greater efficacy. CGA improved metabolic parameters by lowering fasting glucose and insulin and enhancing lipid profiles. CGA suppressed orexigenic genes (Agrp, NPY) and upregulated anorexigenic genes (POMC, CARTPT), suggesting appetite regulation in the hypothalamus. In abdominal white adipose tissue (WAT), CGA boosted antioxidant defenses (SOD, CAT, GPx, HO-1), reduced lipid peroxidation (MDA), and suppressed pro-inflammatory cytokines including TNF-α, IFN-γ, and IL-1β, while increasing the anti-inflammatory cytokine IL-10. CGA modulated inflammatory signaling via upregulation of miR-146a and inhibition of IRAK1, TRAF6, and NF-κB. It also reduced apoptosis by downregulating p53, Bax, and Caspase-3, and restoring Bcl-2. These findings demonstrate that short-term CGA administration effectively reverses multiple HFD-induced impairments, highlighting its potential as an effective therapeutic for obesity-related metabolic disorders.

Background/Objectives: Clostridium perfringens is a normal inhabitant of the intestinal tract of poultry, and it has the potential to induce cholangiohepatitis and necrotic enteritis (NE). The poultry industry suffers significant financial losses because of NE, and treatment becomes more challenging due to resistant C. perfringens strains. Methods: The antimicrobial and antibiofilm activities of cellulose nanocrystals/zinc oxide nanocomposite (CNCs/ZnO) were assesses against pan drug-resistant (PDR) C. perfringens isolated from chickens and turkeys using phenotypic and molecular assays. Results: The overall prevalence rate of C. perfringens was 44.8% (43.75% in chickens and 58.33% in turkeys). Interestingly, the antimicrobial susceptibility testing of C. perfringens isolates revealed the alarming PDR (29.9%), extensively drug-resistant (XDR, 54.5%), and multidrug-resistant (MDR, 15.6%) isolates, with multiple antimicrobial resistance (MAR) indices ranging from 0.84 to 1. All PDR C. perfringens isolates could synthesize biofilms; among them, 21.7% were strong biofilm producers. The antimicrobial potentials of CNCs/ZnO against PDR C. perfringens isolates were evaluated by the agar well diffusion and broth microdilution techniques, and the results showed strong antimicrobial activity of the green nanocomposite with inhibition zones’ diameters of 20–40 mm and MIC value of 0.125 µg/mL. Moreover, the nanocomposite exhibited a great antibiofilm effect against the pre-existent biofilms of PDR C. perfringens isolates in a dose-dependent manner [MBIC50 up to 83.43 ± 1.98 for the CNCs/ZnO MBC concentration (0.25 μg/mL)]. The transcript levels of agrB quorum sensing gene and pilA2 type IV pili gene responsible for biofilm formation were determined by the quantitative real time-PCR technique, pre- and post-treatment with the CNCs/ZnO nanocomposite. The expression of both genes downregulated (0.099 ± 0.012–0.454 ± 0.031 and 0.104 ± 0.006–0.403 ± 0.035, respectively) when compared to the non-treated isolates. Conclusions: To the best of our knowledge, this is the first report of CNCs/ZnO nanocomposite’s antimicrobial and antibiofilm activities against PDR C. perfringens isolated from chickens and turkeys.

Background: Thermophilic Campylobacter species are among the main culprits behind bacterial gastroenteritis globally and have grown progressively resistant to clinically important antimicrobials. Many studies have been carried out to explore innovative and alternative strategies to control antibiotic-resistant campylobacters in animal reservoirs and human hosts; however, limited studies have been performed to develop efficient control schemes against Campylobacter biofilms. Methods: This study investigated the antimicrobial and antibiofilm activities of some herbal extracts against multidrug-resistant (MDR) Campylobacter species recovered from different sources using phenotypic and molecular techniques. Results: The overall Campylobacter species prevalence was 21.5%, representing 15.25% and 6.25% for C. jejuni and C. coli, respectively. Regarding C. jejuni, the highest resistance rate was observed for amoxicillin–clavulanic acid and colistin (85.25% each), followed by cefotaxime (83.61%) and tetracycline (81.97%), whereas C. coli isolates showed absolute resistance to cefotaxime followed by erythromycin (92%) and colistin (88%). Remarkably, all Campylobacter isolates were MDR with elevated multiple antimicrobial resistance (MAR) indices (0.54–1). The antimicrobial potentials of green tea (Camellia sinensis), rosemary (Rosmarinus officinalis) and ginger (Zingiber officinale) extracts against MDR Campylobacter isolates were assessed by the disk diffusion assay and broth microdilution technique. Green tea extract showed a marked inhibitory effect against tested isolates, exhibiting growth inhibition zone diameters of 8 to 38 mm and a minimum inhibitory concentration (MIC) range of 1.56–3.12 mg/mL, unlike the rosemary and ginger extracts. Our findings reveal a respectable antibiofilm activity (>50% biofilm formation inhibition) of green tea against the preformed biofilms of Campylobacter isolates. Furthermore, real-time quantitative polymerase chain reaction (RT-qPCR) results showed a significant decrease (p < 0.05) in the expression levels of biofilm biosynthesis gene and its regulator (FlaA and LuxS, respectively) in Campylobacter isolates treated with the green tea extract in comparison with untreated ones. Conclusion: This is the first in vitro approach that has documented the inhibitory activity of green tea extract against MDR-biofilm-producing Campylobacter species isolated from different sources. Further in vivo studies in animals’ models should be performed to provide evidence of concept for the implementation of this alternative candidate for the mitigation of MDR Campylobacter infections in the future.

Antimicrobial resistance (AMR) poses a significant global health challenge affecting food safety and development. Residues of antibiotics in food from animal sources, particularly milk, contribute to the development and spread of AMR, alter intestinal microbiota, and potentially lead to allergies, serious health conditions, and environmental and technological problems within the dairy industry. Therefore, this study investigated the residue levels of veterinary drugs from β-lactam antibiotics and tetracyclines in milk and milk products and assessed human health risks. Two hundred milk and milk product samples (pasteurized milk, sterile milk, soft white cheese, and processed cheese, 50 each) were collected from different hospitals in the State of Kuwait and screened for antibiotic residues using a microbial inhibition assay (Delvotest SP-NT) and high-performance liquid chromatography (HPLC). Delvotest SP-NT and HPLC analyses showed that 30, 28, 26, and 24% of the pasteurized milk, sterilized milk, white soft cheese, and processed cheese samples tested positive for antibiotic residues. Forty-eight milk and cheese samples were confirmed as positive by both methods, and six samples initially found to be negative by Delvotest SP-NT were confirmed as positive by HPLC. Multi-antibiotic residues were detected in five samples by using HPLC. The kappa coefficient (0.921; p < 0.0001) revealed complete concordance between the HPLC and Delvotest SP-NT results. Ampicillin was the most abundant residue in the positive samples (31.48%), ranging from 2.44 to 3.89 μg/L, with an overall mean concentration of 3.492 ± 0.094 μg/L, followed by tetracycline and oxytetracycline (27.78% each), ranging from 54.13 to 220.3 μg/L and from 41.55 to 160.7 μg/L, with mean concentrations of 129.477 ± 14.22 and 91.86 ± 9.92 μg/L, respectively. The amoxicillin levels in the samples (12/54; 22.22%) ranged from 3.11 to 5.5 μg/L, with an overall mean concentration of 3.685 ± 0.186 μg/L. The maximum concentrations of ampicillin, amoxicillin, and tetracycline were detected in processed cheese with mean concentrations of 3.89 ± 0.28 µg/L, 3.95 ± 0.15 µg/L, and 170.3 ± 0.27 µg/L, respectively. Pasteurized milk contained the maximum concentrations of oxytetracycline, with a mean concentration of 120.45 ± 0.25 µg/L. The tetracycline residues exceeded the standard maximum residue limits (MRLs; 100 µg/L) in 6% of both pasteurized and sterilized milk samples, and in 4% of processed cheese. Additionally, the oxytetracycline levels in pasteurized milk (6%) and amoxicillin levels in processed cheese (2%) were higher than the permitted MRLs (100 µg/L and 4 µg/L, respectively). Furthermore, the antibiotic residues detected in 12.5% (25/200) of the samples were close to standard permissible MRL limits for ampicillin (5%), amoxicillin and oxytetracycline (3% each), and tetracycline (1.5%). Hazard quotients, which compare the standard acceptable daily intake (ADI) to the estimated daily exposure (EDI), indicated that the overall risk associated with antibiotic residues in these dairy products is low. The EDI was lower than the ADI for the tested antibiotics, indicating an elevated safety margin. While the overall hazard quotients are low, the potential for the development of antibiotic resistance due to long-term exposure to low levels of antibiotics should be considered. Hence, strict regulations and enforcement are necessary to prevent excessive residue levels and to promote responsible antibiotic use in dairy production. Regular monitoring of antibiotic residues in dairy products is essential for ensuring consumer safety.

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.

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.

Antimicrobial resistance poses considerable issues for current clinical care, so the modified use of antimicrobial agents and public health initiatives, coupled with new antimicrobial approaches, may help to minimize the impact of multidrug-resistant (MDR) bacteria in the future. This study aimed to evaluate the antimicrobial, antioxidant, and immunomodulatory activities of Lagenaria siceraria, Thymus vulgaris, and their chitosan nanocomposites against extensive drug-resistant (XDR) Pseudomonas aeruginosa and vancomycin-resistant Staphylococcus aureus (VRSA) using both in vitro and in vivo assays. The in vitro antimicrobial susceptibilities of P. aeruginosa and VRSA strains revealed 100% sensitivity to imipenem (100%). All P. aeruginosa strains were resistant to cefoxitin, cefepime, trimethoprim + sulfamethoxazole, and fosfomycin. However, S. aureus strains showed a full resistance to cefoxitin, amoxicillin, ampicillin, erythromycin, chloramphenicol, and fosfomycin (100% each). Interestingly, all S. aureus strains were vancomycin-resistant (MIC = 32–512 μg/mL), and 90% of P. aeruginosa and S. aureus strains were XDR. The antimicrobial potential of Lagenaria siceraria and Thymus vulgaris nanocomposites with chitosan nanoparticles demonstrated marked inhibitory activities against XDR P. aeruginosa and VRSA strains with inhibition zones’ diameters up to 50 mm and MIC values ranging from 0.125 to 1 μg/mL and 1 to 8 μg/mL, respectively. The results of the in vivo approach in male Sprague Dawley rats revealed that infection with P. aeruginosa and S. aureus displayed significant changes in biochemical, hematological, and histopathological findings compared to the negative control group. These values returned to the normal range after treatment by chitosan nanoparticles, either loaded with Lagenaria siceraria or Thymus vulgaris. Real-time quantitative polymerase chain reaction (RT-qPCR) findings presented significant upregulation of the relative expression of the IL10 gene and downregulation of the IFNG gene throughout the experimental period, especially after treatment with chitosan nanoparticles loaded either with Lagenaria siceraria or Thymus vulgaris in comparison to the positive control groups. In conclusion, this is the first report suggesting the use of Lagenaria siceraria and Thymus vulgaris nanocomposites with chitosan nanoparticles as a promising contender for combating XDR P. aeruginosa and VRSA infections as well as a manager for inflammatory situations and oxidative stress-related disorders.