The continuous evolutionary race between antibacterial innovation and antimicrobial resistance (AMR) has become one of the greatest challenges in the management of Gram-negative bacterial infections. Although numerous antibiotics and β-lactam–β-lactamase inhibitor combinations have been introduced since 2000, their clinical utility has been progressively eroded by the rapid emergence and global dissemination of resistance mechanisms, including carbapenemase production, reduced outer membrane permeability, multidrug efflux, target modification, and antibiotic-inactivating enzymes. This review provides a chronological and mechanistic overview of anti-Gram-negative antibiotic development over the past two decades while examining the parallel evolution of bacterial resistance that has limited the therapeutic lifespan of both established and newly approved agents. We critically discuss the mechanisms of action, antibacterial spectrum, pharmacological characteristics, clinical utility, and resistance determinants of recently approved antibiotics, with particular emphasis on therapeutic innovations that overcome conventional resistance barriers, including advanced β-lactam–β-lactamase inhibitor combinations, the siderophore cephalosporin cefiderocol, next-generation tetracyclines, aminoglycosides, and novel bacterial topoisomerase inhibitors. Emerging therapeutic strategies, including investigational antibacterial agents, artificial intelligence-assisted antibiotic discovery, bacteriophage therapy, and CRISPR-based antimicrobial approaches, are also reviewed as potential solutions to the growing challenge of multidrug-resistant Gram-negative pathogens. Collectively, these advances illustrate that successful antibiotic development increasingly depends on innovative target engagement, improved bacterial uptake, and resistance-informed drug design rather than incremental modification of existing scaffolds alone. Ultimately, preserving the clinical effectiveness of new antibiotics will require the integration of antimicrobial stewardship, rapid diagnostics, genomic surveillance, and mechanism-guided drug development to slow the continuing evolutionary arms race between bacterial adaptation and therapeutic innovation.

Background and Objectives: Chronic kidney disease (CKD) poses a worldwide health issue that greatly heightens the risk of infections, especially urinary tract infections (UTIs), which are often caused by antibiotic-resistant pathogens. The purpose of this research was to examine the occurrence, and responsiveness of bacterial strains from UTI patients with chronic kidney disease in Jordan.
Materials and Methods: A cross-sectional study was carried out with 150 participants, comprising 100 individuals with CKD and 50 as control. Urine samples were collected, cultured, and bacterial isolates were examined through biochemical assessments, and tests for antimicrobial susceptibility.
Results: Among 107 bacterial isolates, a significant number (85%) were identified as Gram-negative, with the most common being Escherichia coli (30%) and Klebsiella pneumoniae (18.7%). Gram-positive bacteria made up 15% of the isolates, including Enterococcus faecalis and Staphylococcus saprophyticus. There was a notable resistance to norfloxacin (59.4%) and ciprofloxacin (56.1%), whereas colistin was the most effective, showing 94.5% susceptibility. Multidrug resistance (MDR) was widespread, particularly in E. coli, which had the highest MDR rate at 82.2%.
Conclusion: The study emphasizes the predominance of Gram-negative bacteria, along with alarming rates of antibiotic resistance in CKD patients with UTIs. The findings of the study highlight the importance of customized antibiotic treatments and strong antimicrobial stewardship initiatives to effectively address UTIs in this vulnerable group.

Background and Objectives: Urinary tract infections (UTIs) are among the most prevalent bacterial infections in women, with Escherichia coli and Klebsiella pneumoniae as leading causative pathogens. The emergence of multidrug-resistant (MDR) and carbapenem-resistant strains poses a significant therapeutic challenge. Ceftazidime–avibactam (CZA) has shown activity against β-lactamase-producing Gram-negative bacteria; however, data on its activity against urinary carbapenem-resistant isolates remain limited. This study aimed to determine CZA activity against urinary carbapenem-resistant E. coli and K. pneumoniae isolates.
Materials and Methods: In this cross-sectional study, antimicrobial susceptibility testing was performed using the disk diffusion method according to CLSI guidelines. The tested antibiotics included carbapenems, third-generation cephalosporins, aminoglycosides, levofloxacin, fosfomycin, and CZA. Extended-spectrum β-lactamase (ESBL) production was assessed using the combined-disk method.
Results: A total of 76 previously collected urinary bacterial isolates, including 44 (57.9%) E. coli and 32 (42.1%) K. pneumoniae isolates were included in this study. Carbapenem resistance was detected in 25.0% (11/44) of E. coli and 28.1% (9/32) of K. pneumoniae isolates. ESBL production was observed in 36.4% and 37.5%, respectively. Overall, CZA susceptibility was 68.2% for E. coli and 68.7% for K. pneumoniae, decreasing to 27.3% and 22.2%, respectively, among carbapenem-resistant isolates. Overall, 25.0% of carbapenem-resistant isolates remained susceptible to CZA. Fosfomycin susceptibility was 81.8% and 46.9% among E. coli and K. pneumoniae, respectively.
Conclusion: CZA demonstrated substantial in vitro activity against urinary E. coli and K. pneumoniae isolates; however, its activity was markedly reduced among carbapenem-resistant strains. These findings highlight the importance of susceptibility-guided use of CZA and continued surveillance of antimicrobial resistance.

Background and Objectives: Surgical site infection (SSI) is an important complication following hysterectomy. Given the rising antimicrobial resistance in Iran, this multi-year cohort study aimed to identify independent predictors of SSI after hysterectomy.
Materials and Methods: This retrospective cohort study included patients undergoing total abdominal hysterectomy (2019-2024). Independent risk factors were identified via multivariable binary logistic regression. Model performance was validated using receiver operating characteristic (ROC) curve analysis. Continuous variable cut-off points were determined using the Youden index.
Results: Among 690 patients, the SSI rate was 5.2%. Multivariable analysis identified obesity (BMI ≥ 30 kg/m²) (Adjusted Odds Ratio [aOR]: 4.11; 95% CI: 1.81–9.32; p < 0.001) and operative duration (aOR: 1.02 per minute; p < 0.001) as the strongest independent predictors. ROC analysis demonstrated excellent discrimination (AUC = 0.88). Infection risk increased critically when surgery exceeded 180 minutes or in obese patients. Preoperative non-standard antibiotic prophylaxis consisting solely of clindamycin was associated with a 7.5-fold increased SSI risk (P=.006). Microbiological analysis revealed a 30.6% prevalence of multidrug-resistant and ESBL-producing bacteria.
Conclusion: Obesity and prolonged operative time are independent predictors of SSI. We recommend weight-based dosing, strict intraoperative re-dosing during prolonged procedures, and mandatory antimicrobial stewardship to mitigate risks associated with non-standard prophylaxis.

Background and Objectives: Surgical site infection (SSI) is an important complication among liver transplant (LT) recipients. This study aimed to determine the prevalence, risk factors and microbial etiologies of SSI in LT recipients.
Materials and Methods: This retrospective observational study was conducted at a tertiary care hospital, Imam Khomeini Hospital complex, Tehran, Iran, between 2015 and 2020. Clinical data were collected from password-protected database.
Results: A total of 200 patients were included, of whom 121 (60.5%) were men. The mean age was 43.85 ± 14.59 years. Ninety-six patients (48%) developed SSI. broad-spectrum antibiotics use (p=.005), preoperative corticosteroids and immunosuppressive therapy (P = .000), Prolonged ICU stay (P=.000), and higher MELD score before transplantation (P = .002) were significantly associated with SSI. Graft rejection and one-year mortality rates were higher among patients with SSI; however, these differences were not statistically significant. SSI was also more common among patient with diabetes mellitus, other non-hepatic comorbidities, moderate-to-severe ascites, and those receiving piperacillin-tazobactam prophylaxis after surgery. The most frequently isolated microorganisms from SSI culture were Klebsiella pneumoniae (25%), Enterococcus spp. (22.9%) and Escherichia coli (16.6%).
Conclusion: SSI remains a common complication among LT in our canter and should be suspected in patients presenting with infection during the first postoperative month. Recipients with advanced liver disease, prolonged ICU stay, preoperative immunosuppressive therapy, and post-transplant hemodialysis may require monitoring.

Background and Objectives: Postoperative wound infection is a serious complication in presence of prosthetic implantation (infectivity rate 10.4%). Implant infection is particularly high in developing countries. Hence there is a need to know the clinical presentation, common organisms and their susceptibility pattern in a particular region.
Materials and Methods: The patients who had undergone orthopaedic prosthesis or implant surgeries and presented with signs and symptoms of infection were included in this study. Pus, tissue, fluid samples obtained were streaked in Blood agar and MacConkey agar plates and incubated at 37°C, two Sabouraud Dextrose agar slants incubated at 28°C and 37°C and Gram staining and Acid-fast staining performed. Antimicrobial susceptibility testing was performed for all bacterial isolates. Data collection was done using a proforma and analysis of data was done and expressed as frequency and percentage.
Results: A total of 149 samples obtained from patients with Orthopaedic implant associated infections were processed. Of these 131 (87.9%) were culture positive. Staphylococcus aureus was the most common pathogen, followed by Pseudomonas spp. Escherichia coli and Staphylococcus other than S. aureus. Ten (7.6%) cases had multiple organisms and 2 fungi were also isolated (1.5%). Among the S. aureus, MSSA was the commonest and 42 (30.2%) isolates were multidrug resistant organisms.
Conclusion: Infection is the major cause of delayed healing in patients with Orthopaedic implants. Early identification and aggressive treatment with the appropriate antibiotics are necessary to decrease infection rate, reduce hospital stay, prevent biofilm formation, decrease the need for Implant removal or replacement, and prevent disability in the patients.

Background and Objectives: Bloodstream infections (BSIs) are an important cause of morbidity and mortality among neonates and children. Increasing antimicrobial resistance (AMR), particularly among Gram-negative organisms, complicates empirical antimicrobial therapy. Local antimicrobial susceptibility data are essential for guiding empirical treatment and antimicrobial stewardship.
Materials and Methods: A retrospective descriptive study was conducted at Children Hospital, Sukkur, Pakistan, from July 2024 to June 2025. Blood culture data from pediatric patients from birth to 14 years of age were reviewed. Only the first positive blood culture from each patient during the study period was included in the primary analysis, while subsequent positive cultures from the same patient were excluded. Organism distribution and antimicrobial susceptibility patterns were analyzed. Antimicrobial susceptibility testing (AST) had been performed in the hospital microbiology laboratory according to applicable Clinical and Laboratory Standards Institute (CLSI) recommendations. Because the retrospective records did not contain sufficient information to reliably confirm the exact AST technique used, the specific susceptibility-testing methodology could not be retrospectively established.
Results: A total of 11,962 blood cultures were processed during the study period, of which 4,151 (34.7%) were positive. Gram-positive bacteria accounted for 2,276 (54.8%) isolates and Gram-negative bacteria for 1,856 (44.7%). Nineteen (0.5%) positive cultures yielded fungal organisms and were excluded from the bacterial analysis. Coagulase-negative staphylococci (CoNS) were the most frequently isolated bacterial organisms, accounting for 2,125 isolates (51.2% of all positive cultures), followed by Klebsiella spp. (526, 12.7%), Serratia spp. (490, 11.8%), and Acinetobacter spp. (259, 6.2%). Gram-negative organisms demonstrated marked resistance to several commonly used antimicrobial agents. Salmonella Typhi demonstrated high susceptibility to azithromycin (93.6%) and meropenem (97.6%), whereas susceptibility to ceftriaxone (43.6%), cefixime (9%), ciprofloxacin (0%), and trimethoprim-sulfamethoxazole (15.7%) was low. Vancomycin and linezolid demonstrated 100% susceptibility among the tested CoNS, Staphylococcus aureus, and Enterococcus spp. isolates.
Conclusion: This one-year analysis demonstrates a substantial burden of antimicrobial resistance among pediatric bloodstream isolates at Children Hospital, Sukkur. CoNS were the most frequently isolated organisms; however, this finding should be interpreted cautiously because the clinical and microbiological information required to distinguish true CoNS bacteremia from blood culture contamination was not consistently available. Regular institution-specific antibiogram surveillance, antimicrobial stewardship, and strengthened infection prevention and control measures are warranted.

Background and Objectives: Candidemia is an important cause of healthcare-associated bloodstream infection, and changes in the distribution of Candida species may influence antifungal treatment strategies. This study aimed to determine the species distribution of yeasts isolated from blood cultures and evaluate their antifungal susceptibility profiles.
Materials and Methods: This retrospective study included 269 yeast isolates recovered from blood cultures between December 2017 and December 2021. Species identification was performed using the VITEK 2 YST system. Antifungal susceptibility testing was performed using Sensititre YeastOne for 215 (79.9%) isolates and VITEK 2 AST-YS08 for 54 (20.1%). Minimum inhibitory concentration results were interpreted according to applicable CLSI species-specific clinical breakpoints.
Results: Of the 269 isolates, 112 (41.6%) were Candida albicans and 157 (58.4%) were non-albicans Candida. The most frequent non-albicans species were C. parapsilosis (n=73, 27.1%), Nakaseomyces glabrata (C. glabrata) (n=37, 13.8%), and C. tropicalis (n=34, 12.6%). Fluconazole MIC₅₀/MIC₉₀ values were 0.5/256 mg/L for C. albicans, 1/16 mg/L for C. parapsilosis, 8/32 mg/L for C. glabrata, and 1/4 mg/L for C. tropicalis.
Conclusion: The predominance of non-albicans Candida, particularly C. parapsilosis, highlights the importance of continuous local surveillance. Methodological differences between commercial susceptibility testing systems should be considered when interpreting antifungal susceptibility results.

Background and Objectives: Inappropriate antibiotic use can lead to resistance, particularly in Gram-negative bacteria. The aimd of this study was to identify the distribution of Gram-negative bacteria responsible for lower respiratory tract infections and to assess their susceptibility profiles to beta-lactam antibiotics in the periods before and during the COVID-19 pandemic.
Materials and Methods: This retrospective cross-sectional study utilized secondary data from WHONET 2022, drawing on a database of Gram-negative bacteria isolated from lower respiratory tract specimens processed at the Clinical Microbiology Laboratory, Faculty of Medicine, Universitas Indonesia, during the period of 2018-2021. Susceptibility to ampicillin-sulbactam, ceftazidime, cefepime, meropenem, and piperacillin-tazobactam was assessed. Comparisons between the pre-pandemic and pandemic periods were conducted using the chi-square test.
Results: A total of 569 samples were analyzed, comprising 383 samples from the pre-pandemic period (2018-2019) and 186 samples from the COVID-19 pandemic period (2020-2021). Twenty-nine Gram-negative bacterial species were identified before the pandemic, compared with 19 species during the COVID-19 pandemic. Klebsiella pneumoniae emerged as the most common pathogen, with a prevalance of 30.29% before and 27.96% during the COVID-19 pandemic. Statistical analysis revealed significant differences in the resistance patterns of Acinetobacter baumannii to cefepime (p= 0.036), ceftazidime (p= 0.018), and piperacillin-tazobactam (p= 0.002). Conversely, no significant differences were observed for K. pneumoniae and Pseudomonas aeruginosa (p > 0.05).
Conclusion: K. Pneumoniae remains the predominant Gram-negative pathogen causing lower respiratory tract infections, whereas A. baumannii shows an increased disease burden and resistance during the COVID-19 pandemic. This underscores the importance of continuous surveillance of antimicrobial resistance.

Background and Objectives: Staphylococcus aureus poses clinical challenges through its virulence and antibiotic resistance, including SCCmec-mediated methicillin resistance. Integrated data on virulence profiling, molecular typing, and resistance are limited in Southwest Iran. We characterized virulence genes, SCCmec types, integrons, and antimicrobial resistance in S. aureus from diverse clinical sources in Yasuj, Iran.
Materials and Methods: We analyzed 214 S. aureus isolates recovered from tonsil, wound, sinus, and blood specimens (2020–2023). Species confirmation was by nuc PCR. Antimicrobial susceptibility testing followed CLSI disk diffusion guidelines; MRSA was confirmed by mecA PCR. PCR assays detected hemolysin genes (hla, hlb, hld), toxin genes (tst, pvl, eta, etb, etd), MSCRAMM genes (sdrC, sdrD, sdrE), sak, integrase genes (intI, intII), and SCCmec types I, III, and IVa.
Results: Isolates were from tonsils (59.8%), wounds (29.9%), sinusitis (6.1%), and blood (4.2%). MRSA prevalence was 29.0%. Highest resistance was to penicillin (98.6%), erythromycin (59.3%), and tetracycline (54.7%), with MDR in 32.7%. Prevalent virulence genes were hld (87.9%), hla (68.2%), sak (60.3%), sdrC (44.4%), eta (37.9%), sdrD (36.9%), tst (32.2%), sdrE (29.0%), etd (18.2%), and pvl (3.7%). Among 104 MDR/MRSA isolates, intI and intII were found in 14.4% and 10.6%. SCCmec type III dominated (80.5%), followed by I (14.5%) and IVa (5.0%); types I and III were significantly linked to MDR (P < 0.05). Four isolates co-harbored pvl, tst, and hla.
Conclusion: Considerable virulence gene carriage and SCCmec type III dominance were observed. Gentamicin, ciprofloxacin, cotrimoxazole, and clindamycin remain effective. Coexistence of life-threatening toxin genes and high resistance rates necessitates enhanced infection control.

Background and Objectives: The global rise of antimicrobial resistance threatens effective treatment of bacterial infections. Methicillin-resistant Staphylococcus aureus (MRSA) is a major pathogen whose resistance to β-lactam antibiotics is primarily mediated by the mecA gene, encoding the low-affinity penicillin-binding protein PBP2a. This study aimed to investigate whether CRISPR-dCas9-mediated targeting of mecA could suppress resistance-associated gene expression and restore β-lactam susceptibility in MRSA.
Materials and Methods: Seven target sites within mecA, comprising two non-coding and five coding regions, were selected for CRISPR-dCas9 targeting. The effects of gene interference were evaluated using antibiotic disk diffusion assays, minimum inhibitory concentration (MIC) testing with gradient E-strips, RT-PCR, and Sanger sequencing.
Results: Targeting non-coding regions suppressed mecA transcription and reduced PBP2a production, whereas targeting coding regions generated mutations associated with dysfunctional PBP2a. CRISPR-dCas9 treatment significantly reduced mecA expression and increased MRSA susceptibility to β-lactam antibiotics, particularly ampicillin and cefixime. MIC values decreased by up to 12-fold and 6-fold, respectively (p<0.05).
Conclusion: CRISPR-dCas9-mediated targeting of mecA effectively reduced β-lactam resistance in MRSA. This approach demonstrates potential for precision-based antimicrobial resistance management and warrants further investigation as a strategy for combating drug-resistant bacterial infections.

Background and Objectives: Enterococcus faecium is a major nosocomial pathogen frequently associated with catheter-related infections and biofilm-mediated antimicrobial resistance. This study aimed to investigate the genomic determinants and structural characteristics underlying biofilm formation in a clinical E. faecium isolate recovered from a catheter-associated urinary tract infection (CAUTI).
Materials and Methods: A clinical isolate, E. faecium MFKCC-BF.UR33, was subjected to whole-genome sequencing and comprehensive genome annotation to identify biofilm-associated and antimicrobial resistance genes. Phylogenetic analysis was performed to determine its relatedness to reference clinical strains. Structural modeling of selected biofilm-associated proteins was conducted using AlphaFold2 to assess their three-dimensional conformations and potential as therapeutic targets.
Results: Whole-genome sequencing revealed a 3.32 Mb genome with a G+C content of 37.35%, comprising 2,321 predicted coding sequences and no detectable plasmids. Genome annotation identified several biofilm-associated genes, including sgrA, acm, fss3, scm, and efbA, as well as antimicrobial resistance genes (antA/B, tetM, tetL, efmA, and liaFSR). Phylogenetic analysis placed the isolate within the E. faecium clade, closely related to other clinical reference strains. Structural modeling demonstrated high-confidence 3D structures for Fss3 and EfbA proteins.
Conclusion: The integration of genomic and structural analyses provides insights into the genetic and structural basis of biofilm formation in E. faecium and establishes a foundation for future functional and structure-based investigations.

Background and Objectives: Gastric cancer remains a leading cause of cancer mortality and exhibits significant geographic variation. Although Helicobacter pylori (H. pylori) is a well-established carcinogen, the contributions of Epstein–Barr virus (EBV) and human papillomavirus (HPV), individually or in conjunction with H. pylori, are not fully understood. This study assessed the prevalence and co-detection of these pathogens in gastric tissues from Yazd, Iran.
Materials and Methods: An archive-based cross-sectional study was conducted using 130 formalin-fixed, paraffin-embedded (FFPE) gastric specimens collected between 2019 and 2022, comprising 83 gastric adenocarcinomas and 47 non-malignant tissues. H. pylori was detected by conventional PCR targeting 16S rRNA, while EBV and HPV were identified by nested PCR targeting EBNA-1 and L1, respectively. PCR products were confirmed by agarose gel electrophoresis, and selected amplicons were verified by Sanger sequencing.
Results: H. pylori was detected in 74 of 130 specimens (56.9%), with a prevalence of 62.7% in malignant tissues and 46.8% in non-malignant tissues (p=0.080). EBV was identified in three malignant cases (3.6%) and HPV in one malignant case (1.2%), with no positive findings in non-malignant samples (p=0.187 and p=0.450, respectively). Three co-detections were observed in malignant tissues, including two cases of H. pylori with EBV and one case of H. pylori with HPV. Due to the limited number of EBV- and HPV-positive cases, association analyses lacked statistical power.
Conclusion: H. pylori was the predominant microorganism detected, while EBV and HPV were rare and co-detections were infrequent. Larger prospective studies employing complementary molecular techniques and H. pylori virulence profiling are required to elucidate the clinical significance of these infections in gastric carcinogenesis.

Background and Objectives: Helicobacter pylori infection primarily contributes to gastric carcinogenesis. This study examined the expression of cyclin E1, β-catenin, PPAR-δ, and miR-9 in different gastric regions of gastritis patients with and without H. pylori infection.
Materials and Methods: Gastric biopsies were obtained from 108 participants divided into three groups, including healthy (n = 15), gastritis (n = 53), and H. pylori-positive gastritis (n = 40). Bacterial culture and molecular characterization were performed to confirm H. pylori presence. Gene expression was assessed using SYBR Green-based RT-qPCR. Statistical analyses revealed correlations between the expression of cyclin E1, β-catenin, PPAR-δ, miR-9, and the presence of H. pylori in patients with gastritis compared to healthy controls.
Results: The cagA-positive strains were significantly associated with the expression of cyclin E1, β-catenin, PPAR-δ (P < 0.001), and miR-9 (P < 0.01). Cyclin E1 and β-catenin expression were upregulated in gastritis and H. pylori-positive gastritis patients. The PPAR-δ expression was highest in H. pylori-positive gastritis patients, specifically in the antrum tissues. miR-9 showed significant downregulation in H. pylori-positive gastritis samples.
Conclusion: cagA-positive H. pylori infection was linked to distinct gene expression changes that varied by gastric region. Further studies are needed to explore the mechanisms involved.

Background and Objectives: Apis dorsata (A. dorsata), the giant honeybee, is an important pollinator of Asia. Gut-microbial assemblages contribute to nutrient utilization and metabolism, yet their diversity in the gut of A. dorsata remains underexplored. The main objective of the current study was to explore the impact of local geographical conditions on the gut microbial diversity and the functional potential of A. dorsata gut-associated bacteria.
Materials and Methods: Bacterial community profiling and characterization were performed using high-throughput sequencing on the DNA Nanoball Sequencing (DNBseq) platform.
Results: Fifty-four bacterial species were detected from the gut of A. dorsata, expanding the known microbial diversity of this host species. The comprehensive analysis of pooled samples from two regions revealed that higher microbial diversity in district Bhimber than in Jhelum. Nevertheless, site-specific putative differences were observed in the pathways related to carbohydrate metabolism, amino acid synthesis, and other cellular activities.
Conclusion: This exploratory pilot investigation revealed site-wise preliminary differences in taxonomy and putative metabolic functions of bacterial species. These results provide a baseline for future studies on the diversity and dynamics of functioning of gut-associated microbial communities of A. dorsata.

Background and Objectives: In mammals, milk provides the nutrients required for growth and development, including proteins, fats, carbohydrates, vitamins, and essential minerals. Donkey milk has recently attracted increasing interest as an alternative to cow’s milk, particularly for infants with cow’s milk protein allergy, due to its favorable nutritional composition. However, limited information is available on the microbiological quality of donkey milk based on molecular confirmation of Staphylococcus aureus and the detection of antimicrobial resistance genes, particularly in Iran. The present study assessed the microbiological quality of raw donkey milk through conventional bacteriological culture, PCR confirmation of Staphylococcus aureus, antimicrobial susceptibility testing, and molecular detection of the mecA and blaZ resistance genes.
Materials and Methods: Between January and April 2023, a total of 50 raw donkey milk samples were collected and subjected to conventional bacteriological examination. Presumptive Staphylococcus aureus isolates were identified by culture-based methods and confirmed by PCR targeting the 23S rRNA gene. PCR assays were also performed to detect the coa, blaZ, and mecA genes. Antimicrobial susceptibility testing was conducted using the CLSI disk diffusion method with ten antimicrobial agents.
Results: Of 12 bacterial isolates, 8 (16%) were confirmed as S. aureus. The blaZ gene, was present in all isolates (100%), while mecA, was absent. Antibiotic susceptibility testing showed variable resistance among isolates, with notable resistance against penicillin, with all isolates showing resistance (100%). The second highest resistance rate was observed for trimethoprim- sulfamethoxazole, where 25% of the isolates showed resistance.
Conclusion: These findings indicate the presence of β-lactam resistance determinants despite the absence of molecular evidence of methicillin resistance. The study provides baseline data on the occurrence and antimicrobial resistance characteristics of S. aureus in raw donkey milk in Iran and highlights the value of combining phenotypic methods with PCR for microbiological surveillance of dairy products.

Background and Objectives: Pulmonary inflammation is central to the pathogenesis of various respiratory diseases, driving the need for novel therapeutics. Lipopolysaccharide (LPS) is a potent inflammatory trigger that induces proinflammatory cytokine synthesis via distinct signaling cascades. The objectives of this study were to evaluate the cytotoxic and anti-inflammatory effects of recombinant TGF-Arazyme fusion protein on LPS-stimulated human lung A549 and MRC-5 cell lines.
Materials and Methods: A gene construct fusing Arazyme with a non-mitogenic TGFα fragment was expressed, purified via Ni-NTA chromatography, and validated enzymatically. Cytotoxicity was assessed by MTT assay. Inflammatory cytokine levels (TNF-α, IL-1β, IL-6, IL-10) were quantified by RT-PCR and ELISA. Anti-inflammatory efficacy was further validated in a murine (BALB/c) air pouch model infected with E. coli.
Results: At 20 μg/mL, the chimeric protein showed no significant toxicity in MRC-5 cells but dose-dependently reduced A549 viability (p<0.01). Co-treatment with LPS and the chimeric protein significantly downregulated proinflammatory cytokines (TNF-α, IL-1β, IL-6) at both mRNA and protein levels (p<0.01, p<0.001), while increasing IL-10 (p<0.05). In the air pouch model, TGF-Arazyme reduced leukocyte counts by 48.3% (p<0.01), comparable to dexamethasone (51.2%).
Conclusion: TGF-Arazyme exhibits both anti-inflammatory and selective cytotoxic properties, positioning it as a promising candidate for inflammatory lung diseases and lung cancer.

Background and Objectives: Candida albicans (C. albicans) is the most common cause of candidiasis in immunocompromised patients. Biofilm-associated Candida can lead to treatment complications and systemic infections.
Materials and Methods: In this study, the antifungal activity of the cell-free supernatant (CFS) derived from Lactobacillus casei was assessed against C. albicans strains. To differentiate between pH- and H₂O₂-mediated effects and specific metabolite activity, all antifungal and antibiofilm assays were performed using CFS that had been neutralized to pH 7.0 and treated with catalase.
Results: The ranges of minimum inhibitory concentration (MIC) and Half-maximal inhibitory concentration (IC50) for CFS were 7.81-62.5 and 95.1-159.6 µl/ml, respectively. Using corrected FICI criteria (synergy ≤ 0.5; additive > 0.5-1.0; indifference > 1.0-4.0), additive effects were observed in 50% of strains, synergy in 20% (including the standard strain PTCC 5027), and indifference in 30%.
Conclusion: The results demonstrate that the CFS of L. casei exhibits promising in vitro antifungal and antibiofilm activity against C. albicans. When used in combination with nystatin, L. casei CFS showed additive or synergistic effects, suggesting its potential as an adjunctive agent for further investigation. However, these findings are preliminary, and in vivo studies and clinical assessments are necessary to validate the therapeutic potential of L. casei CFS in the management of oral candidiasis.

Background and Objectives: Pseudomonas aeruginosa is an opportunistic Gram-negative pathogen responsible for severe infections and increasing therapeutic difficulties due to multidrug resistance and virulence traits such as motility and biofilm formation. This study aimed to evaluate the in vitro antibacterial, antibiotic-synergistic, and anti-motility effects of eugenol in combination with cefixime and gentamicin against nine clinical P. aeruginosa isolates obtained from hospitalized animals.
Materials and Methods: This preliminary in vitro laboratory investigation was conducted on a convenience sample of nine independent non-duplicate clinical P. aeruginosa isolates obtained from wound swabs and urinary tract samples of hospitalized animals at the Small Animal Hospital, Faculty of Veterinary Medicine, University of Tehran. Minimum inhibitory concentrations (MICs) of eugenol alone were determined across all nine isolates using the broth microdilution method in accordance with CLSI guidelines (M07-A11). Synergistic interactions between eugenol and the antibiotics cefixime and gentamicin were evaluated using the checkerboard assay and calculation of the fractional inhibitory concentration index (FICI) on a subset of three selected representative isolates (P3, P6, and P9). Furthermore, the effects of sub-inhibitory concentrations of eugenol ("MIC/2" ) on bacterial motility were assessed using standardized media containing 0.3%, 0.7%, and 1.5% agar to evaluate swimming, swarming, and twitching motilities, respectively, across this same subset of isolates (P3, P6, and P9) in biological triplicate (n=3).
Results: Antimicrobial analysis across all nine isolates demonstrated intrinsic antibacterial activity of eugenol, with MIC values ranging from 2.5 to 10 µg/mL. Checkerboard synergy testing on the selected representative subset (P3, P6, and P9) revealed synergistic interactions with cefixime ("FICI"=0.5 for isolate P6, P3 and P9) as well as interactions with gentamicin yielding FICI values of 0.256 (for isolates P6 and P9) and 0.384 (for isolate P3), classifying these combinations within synergistic interaction ranges ("FICI"≤0.5). Additionally, sub-inhibitory eugenol treatment of the tested subset significantly reduced bacterial motility compared to untreated controls, decreasing swimming by 44-57%, swarming by 43-46%, and twitching by 39-64% (P<0.05).
Conclusion: Eugenol demonstrated notable in vitro antibacterial and anti-motility activities against tested clinical P. aeruginosa isolates from veterinary sources, alongside favorable FICI interactions with gentamicin and cefixime. Because these findings are strictly restricted to limited in vitro laboratory observations without dosage optimization, toxicity, or in vivo efficacy evaluations, further comprehensive investigations are required before establishing any potential clinical relevance.

Background and Objectives: Influenza A virus remains a major global health concern because antigenic drift reduces the long-term efficacy of current vaccines. This study evaluated a multi-epitope DNA vaccine targeting conserved regions of hemagglutinin (HA) and polymerase acidic (PA) proteins from influenza A/H1N1 and A/H3N2 viruses.
Materials and Methods: Conserved B-cell, CD4⁺ T-cell, and CD8⁺ T-cell epitopes were identified using immunoinformatic tools and linked to β-defensin-2 in the pcDNA3.1(+) vector. Protein expression was confirmed in HEK293 cells. BALB/c mice were immunized intramuscularly and challenged with lethal doses of H1N1 or H3N2 viruses. Humoral and cellular immune responses, lung viral loads, histopathology, and clinical outcomes were assessed.
Results: Vaccinated mice exhibited undetectable or markedly reduced lung viral loads (1,000-10,000-fold; p<0.01), significantly decreased pulmonary lesions, and milder clinical manifestations than controls. Vaccination induced significant CD4⁺ (p=0.041) and CD8⁺ (p=0.038) T-cell responses in bronchoalveolar lavage fluid, accompanied by increased interferon-γ and interleukin-4 production, indicating balanced cellular and humoral immunity. Complete protection against mortality was observed following viral challenge.
Conclusion: This multi-epitope DNA vaccine elicited cross-protective immunity against influenza A/H1N1 and A/H3N2 by targeting conserved HA and PA epitopes, supporting its potential as a candidate for universal influenza vaccine development.

Background and Objectives: West Nile virus (WNV) is a mosquito-borne flavivirus that represents a significant public health concern, and the development of effective vaccine candidates remains a priority. This study aimed to design a chimeric multi-epitope protein against WNV using immunoinformatics approaches and evaluate its immunogenicity in mice.
Materials and Methods: Selected regions of the WNV envelope (E) and non-structural 1 (NS1) proteins were analyzed to identify B- and T-cell epitopes using immunoinformatics tools. The selected epitopes were incorporated into a chimeric construct fused with cholera toxin B subunit, and its antigenicity, allergenicity, structural properties, and TLR3 interaction were evaluated in silico. The optimized construct was expressed in Escherichia coli, purified by Ni-NTA affinity chromatography, and confirmed by SDS-PAGE and western blotting. BALB/c mice were immunized with the recombinant protein plus adjuvant, and immune responses were assessed using ELISA, cytokine assays, and flow cytometry.
Results: Immunized mice developed high IgG antibody titers and showed detectable IFN-γ and IL-5 production, suggesting induction of Th1-associated responses with limited Th2-related cytokine production. The chimeric protein also stimulated CD4+ and CD8+ T-cell responses, indicating activation of both humoral and cellular immunity.
Conclusion: The designed chimeric protein showed immunogenic potential and induced humoral and cellular immune responses in mice, supporting further evaluation as a candidate for WNV vaccine development.