Abstract
Background:
Mastitis poses a significant economic impact in the dairy sector and serves as a potential source of infection for healthy cows, impeding attempts to become self-sufficient in milk production. Treatment failure due to the development of multidrug-resistant pathogens raises concern about the need to investigate antimicrobial alternatives. Nanoparticles are attracting attention as a therapeutic approach for various infectious disorders. Despite the importance of this issue, limited research has been conducted on resistant bacteria that cause mastitis.

Aim:
This study aimed to investigate the occurrence and antimicrobial susceptibility profile of Escherichia coli and Salmonella spp. as environmental pathogens causing mastitis and the antibacterial activity of chitosan nanoparticles (ChNPs) against multidrug-resistant pathogens.

Methods:
A total of 100 milk samples were collected from dairy cattle and buffaloes with clinical and subclinical mastitis using a convenience sampling approach, with no randomization. As such, the sample may not fully represent all dairy sources in the study area. Mastitis was initially detected using the California mastitis test. Following standard biochemical tests and bacteriological culture, suspected positive samples were molecularly identified using polymerase chain reaction and phylogenetic analysis of 16S rRNA gene sequences. Along with antimicrobial susceptibility testing. The qualitative antimicrobial activity of ChNPs was assessed using the disc diffusion method along with quantitative determination methods of minimum inhibitory concentration (MIC) and minimum bactericidal concentration (MBC). Additionally, the effect of ChNPs on the bacterial cells was evaluated through TEM imaging.

Results:
Results revealed that E. coli was recovered with percentages of 56%, 32%, 22%, and 24%, and Salmonella spp. was recovered with percentages of 10.5%, 8.1%, 2.1%, and 0% from clinical and subclinical cases of cattle and buffaloes, respectively. The tested E. coli and Salmonella spp. isolates displayed variable sensitivity and resistance patterns toward the evaluated antimicrobials. The ChNPs exhibited potent MIC and MBC effects at 2.5 and 10 mg/ml, respectively, against both multidrug-resistant E. coli and Salmonella spp. TEM imaging revealed reduced bacterial cell viability by disrupting the membrane integrity.

Conclusion:
The outcomes of this study collectively provide a crucial starting point for developing a novel treatment plan for bovine mastitis.

Key words: Antimicrobial resistance; Bovine mastitis; E. coli; Nanoparticles; Salmonella.