Isolation and characterization of bacteriophages for combating multidrug-resistant Listeria monocytogenes from dairy cattle farms in conjugation with silver nanoparticles

This study aims to achieve biocontrol of multidrug-resistant Listeria monocytogenes in dairy cattle farms which poses a severe threat to our socio-economic balance and healthcare systems. Naturally occurring phages from dairy cattle environments were isolated and characterized, and the antimicrobial effect of isolated L. monocytogenes phages (LMPs) against multidrug-resistant L. monocytogenes strains were assessed alone and in conjugation with silver nanoparticles (AgNPs). Six different phenotypic LMPs (LMP1-LMP6) were isolated from silage (n = 4; one by direct phage isolation and three by enrichment method) and manure (n = 2; both by enrichment method) from dairy cattle farms. The isolated phages were categorized into three different families by transmission electron microscopy (TEM): Siphoviridae (LMP1 and LMP5), Myoviridae (LMP2, LMP4, and LMP6), and Podoviridae (LMP3). The host range of the isolated LMPs was determined by the spot method using 22 multidrug-resistant L. monocytogenes strains. All 22 (100%) strains were susceptible to phage infection; 50% (3 out of 6) of the isolated phages showed narrow host ranges, while the other 50% showed moderate host ranges. We found that LMP3 (the phage with the shortest tail) had the ability to infect the widest range of L. monocytogenes strains. Eclipse and latent periods of LMP3 were 5 and 45 min, respectively. The burst size of LMP3 was 25 PFU per infected cell. LMP3 was stable with wide range of pH and temperature. In addition, time-kill curves of LMP3 alone at MOI of 10, 1 and 0.1, AgNPs alone, and LMP3 in combination with AgNPs against the most phage-resistant L. monocytogenes strain (ERIC A) were constructed. Among the five treatments, AgNPs alone had the lowest inhibition activity compared to LMP3 at a multiplicity of infection (MOI) of 0.1, 1, and 10. LMP3 at MOI of 0.1 in conjugation with AgNPs (10 µg/mL) exhibited complete inhibition activity after just 2 h, and the inhibition activity lasted for 24 h treatment. In contrast, the inhibition activity of AgNPs alone and phages alone, even at MOI of 10, stopped. Therefore, the combination of LMP3 and AgNPs enhanced the antimicrobial action and its stability and reduced the required concentrations of LMP3 and AgNPs, which would minimize the development of future resistance. The results suggested that the combination of LMP3 and AgNPs could be used as a powerful and ecofriendly antibacterial agent in the dairy cattle farm environment to overcome multidrug-r