The Antibacterial Effect of Silver and Zinc Oxide Nanoparticles against Intracellular Brucella melitensis
Authors: ALzubaidy Zainab , Amin Narin , Sabah Mahabad 
Published: July 2023

Background: Brucellosis is a zoonotic disease of worldwide importance among animal and humans. The most important virulence factor of Brucella melitensis is related to intra-macrophage survival. On the other hand, the side effects of the current brucellosis treatment regime, it is necessary to find out new antimicrobial agents to treat the disease.Objective: To Isolate Brucella melitensis from local white cheese and raw milk by culture method with identification using PCR and determination of their susceptibility to classical antibiotics and silver and zinc oxide nanoparticles.Materials and methods: A total of 150 local soft cheese and raw milk samples collected from different local markets in Erbil city and were examined for the presence of Brucella melitensis during a period of (6) months (November 2015 to April 2016).Selective media; such as, Brucella agar were used for the isolation, and identified according to standard biochemical tests and confirmed by PCR.Antibiotic susceptibility test determined by Kirby-Bauer disk diffusion method and the antimicrobial activity of silver and zinc oxide nanoparticles were carried out in Muller–Hinton agar with 1% sheep’s blood by well diffusion method. Five mm diameter wells were prepared and loaded with Ag(20 nm size) and ZnO (20 nm size) nanoparticles dilutions, finally the combination of ampicillin/ cloxacillin with the nanoparticles were tested against isolates of Brucella melitensis.Results: Out of the 150 food samples, 53(35.33%) were contaminated; 21(28%) of the total samples of local soft cheese and 32(42.6%) of raw milk was contaminated with Brucella melitensis. Twenty isolates were identified according to standard biochemical tests, and confirmed by PCR for detection of pure Brucella genomic DNA in PCR. Brucella-specific primer BgF/BgR, were evaluated for detection of pure Brucella genomic DNA, provided bands on agarose gel corresponding to a 208 base pair product when compared to molecular ladder, the specific primer IS711were detected for Brucella melitensis, and achieved by given bands on agarose electrophoresis corresponding to 731 base pair from the total isolates. Seventeen from 20 isolates were found positive for Brucella with BgR/BgF primers and 15 isolates were positive for IS711 primer. The sensitivity to antibioticsshowed that the resistance to ampicillin and ceftazidime were (85%), for aampicillin/cloxacillin was (45%), and for imipenem and piperacillin were 80% and 90% respectively. The inhibition zone of ZnO-NP against Brucella melitenisis ranged between 10-20 mm when the concentration was 10-1000 ppm. While the inhibition zone of Ag NP was 11.5-24.5 mm. Combination of amoxicillin/ cloxacillin with nanoparticles show a synergistic effect as demonstrated by the increase of inhibition zone to 20 mm fir ZnO-NP and 23 mm for AgNP nanoparticles.Conclusion: Brucella melitensis contaminate local white soft cheese and raw milk and showed variable sensitivity to antibiotics and some isolates were resistant to ampicillin/ cloxacillin, ceftazidime, imipenem, piperacillin, and trimethoprim-sulphamethoxazole. The present study data show that silver and zinc oxide nanoparticles inhibit the growth of Brucella melitensis. The combination of silver nanoparticles and ampicillin/ cloxacillin showed a synergistic effect.