Cellulose Nanocrystal/Zinc Oxide Bio-Nanocomposite Activity on Planktonic and Biofilm Producing Pan Drug-Resistant Clostridium perfringens Isolated from Chickens and Turkeys

<b>Background/Objectives:</b><i>Clostridium perfringens</i> 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 <i>C. perfringens</i> strains. <b>Methods:</b> The antimicrobial and antibiofilm activities of cellulose nanocrystals/zinc oxide nanocomposite (CNCs/ZnO) were assesses against pan drug-resistant (PDR) <i>C. perfringens</i> isolated from chickens and turkeys using phenotypic and molecular assays. <b>Results:</b> The overall prevalence rate of <i>C. perfringens</i> was 44.8% (43.75% in chickens and 58.33% in turkeys). Interestingly, the antimicrobial susceptibility testing of <i>C. perfringens</i> 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 <i>C. perfringens</i> isolates could synthesize biofilms; among them, 21.7% were strong biofilm producers. The antimicrobial potentials of CNCs/ZnO against PDR <i>C. perfringens</i> 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 <i>C. perfringens</i> 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 <i>agrB</i> quorum sensing gene and <i>pilA2</i> 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. <b>Conclusions:</b> To the best of our knowledge, this is the first report of CNCs/ZnO nanocomposite's antimicrobial and antibiofilm activities against PDR <i>C. perfringens</i> isolated from chickens and turkeys.