Susceptibility of Pasteurella multocida isolated from cattle in Egypt to antibiotics, silver, chitosan and curcumin nanoparticles

Abstract

Pasteurella multocida is a Gram-negative bacterium causing economically significant diseases in cattle. This study aimed to determine P. multocida susceptibility to different antibiotics and antibiotic alternatives. In this study, 246 samples (180 nasal swabs and 66 lung tissue specimens) were collected from cattle showing respiratory manifestations in Egypt. Suspected P. multocida colonies following culture were subjected to polymerase chain reaction (PCR) for molecular confirmation of the isolates. A multiplex PCR was employed to identify P. multocida capsular groups. Susceptibility of the isolated P. multocida to different antibiotics and nanoparticles as antibiotic alternatives including silver (AgNPs), chitosan (CNPs) and curcumin (CurNPs) were tested using broth microdilution method. Thirty-two P. multocida isolates were obtained, kmt1 gene was detected in these isolates, and molecular capsular types classification revealed that all isolates were belonged to the capsular type A. Based on broth microdilution method findings, 20 (62.50%) isolates were considered as multi-drug resistant (MDR); the isolates were most sensitive to danofloxacin and kanamycin, whereas they were most resistant to doxycycline and tilmicosin. Antibiotic alternatives showed high anti-microbial activity against tested isolates with minimum inhibitory concentrations ranging from 1.56 - 6.25 μg mL^-1, 156 - 625 μg mL^-1, and 128 - 512 μg mL^-1 for AgNPs, CNPs and CurNPs, respectively. Our finding demonstrated that MDR P. multocida was evident in cattle in Egypt. Although antibiotic alternatives showed promising in vitro anti-microbial effects against MDR isolates, additional studies are required to be actually applicable in veterinary practices.

Keywords: Antibiotic susceptibility; Multi-drug resistance; Nanoparticles antibiotic alternatives; Pasteurella multocida.

Fig. 1

Transmission electron microscopy of prepared nanoparticles. A) Silver nanoparticles (bar = 200 µm); B) Chitosan nanoparticles (bar = 100 µm); and C) Curcumin nanoparticles (bar = 200 µm).

Fig. 2. A)

Molecular confirmation of Pasteurella multocida isolates. Polymerase chain reaction for detection of kmt1 gene in P. multocida isolates at amplicon size of 460 bp. Lane L: Molecular weight marker (100 - 600 bp, 100 bp plus DNA ladder; Vivantis, Shah Alam, Malaysia); Lane Pos: Positive control; Lanes 5, 8, and 9: Positive samples; Lanes 1 - 4, 6 - 7, and 10 - 11: Negative samples; Lane Neg: Negative control; B) Multiplex PCR showing Cap A (hyaD-hyaC gene) in Pasteurella multocida isolates at amplicon size of 1,044 bp. Lane L: Molecular weight marker (100 – 1,500 bp); Lanes 1, 2, and 3: Positive samples; Lane Pos: Positive control; Lane Neg: Negative control.

Fig. 3

Sensitive, intermediate and resistant isolates percentages for each antibiotics according to the minimum inhibitory concentration (MIC) results. DAN; Danofloxacin, DOX: Doxycycline, KAN: Kanamycin, FLO: Florfenicol, SPT: Spectinomycin, and TIL: Tilmicosin.