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

Abstract

Background 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. Methods 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). Results 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. Conclusions 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-resistant L. monocytogenes.

An effective antibiofilm strategy based on bacteriophages armed with silver nanoparticles

“…This effect included not only the dissipation of the biofilm but also lethality toward the biofilm-forming cells. Similarly, phages ZCSE2 and ZCSE6 (of Myoviridae and Siphoviridae family, respectively) exhibited a synergistic effect with AgNPs against free Salmonella enterica cultures 56 , 57 , as did the mixture of phage LMP3 (of Podoviridae family) and AgNPs against liquid cultures of Listeria monocytogenes 58 . Additionally, mixing of phage ZCEC5 with AgNPs improved the effectiveness against free E. coli O157:H7 59 .…”

Comprehensive Review on the Biocontrol of Listeria monocytogenes in Food Products

“…The former combination reduced the bacterial counts below the detection limits after 2 h, while the latter combination allowed the re-growth of Listeria cells [ 117 ]. In a recent article, Elsayed et al [ 118 ] isolated and characterized six different phages from dairy cattle environments and assessed their antimicrobial effect against 22 different multidrug-resistant L. monocytogenes strains alone and in conjugation with silver nanoparticles (AgNPs). The authors showed that the bacteriophages attached to silver nanoparticles were protected from the harsh environmental conditions, enhancing their effectiveness in reducing L. monocytogenes growth (without regrowth) and excluding the possibility of phage to induce bacterial resistance.…”

Bacteriophage Therapy in Companion and Farm Animals

“…Still, in the absence of clinical trials, to pose the basis for developing bacteriophage therapy against multidrug-resistant strains of L. monocytogenes, six (LMP1-LMP6) new phages were recently isolated and characterized for the host range and stability at wide ranges of pH and temperature. This led to the selection of one phage (LMP3) being more suitable for killing multidrug-resistant L. monocytogenes [60]. The in vitro conjugation of this phage with silver nanoparticles further demonstrated enhanced anti-Listeria monocytogenes activity and more stability [60].…”

“…This led to the selection of one phage (LMP3) being more suitable for killing multidrug-resistant L. monocytogenes [60]. The in vitro conjugation of this phage with silver nanoparticles further demonstrated enhanced anti-Listeria monocytogenes activity and more stability [60]. New technologies, such as nanotechnology, may therefore be used to develop carriers of bacteriophages that are able to inexpensively improve stability and help to combat multidrug-resistant L. monocytogenes.…”