Synergistic combination of ultrasound and bacteriophages therapy for effective control of biofilm formation by multidrug-resistant Salmonella Typhimurium

Abstract

Salmonella Typhimurium is a foodborne pathogen threatening public health by forming resilient biofilms on food-processing surfaces. Bacteriophage-based intervention has gained significant recognition as an eco-friendly and sustainable approach for combating pathogenic bacteria and addressing antimicrobial resistance dissemination. However, the effectiveness of phage treatment is limited by the physical barrier of biofilms and the formation of phage-resistant strains. This study aimed to develop an ultrasound-assisted phage therapy to control multidrug-resistant Salmonella biofilms in the food industry. Specifically, using multidrug-resistant S. Typhimurium N20 as the host, three novel virulent phages, including two short-tailed phages (ST-YZU-14 and ST-YZU-19) and one Myoviridae phage (ST-YZU-21) were identified. These phages showed high efficiency, with large burst sizes (50, 158, and 138 PFU/host cell, respectively), and short latency periods (10, 5, and 15 min, respectively). Their optimal multiplicity of infection (MOI) values were 0.1, 0.1, and 1, respectively. The complete genomic sequences of the phages ranged from 40,083 to 44,328 bp and GC content of 48.25 %–49.56 %, which consisted of 43, 47, and 275 open reading frames for ST-YZU-14, ST-YZU-19, and ST-YZU-21, respectively. Genomes without drug-resistant genes and virulence determinants were confirmed. Phages exhibited strong inhibitory effects against S. Typhimurium in pork at 4 °C (0.32–0.58 Log CFU/g) and 37 °C (1.14–1.54 Log CFU/g). Finally, ultrasound-phage combination therapy effectively eradicated S. Typhimurium biofilms on polyethylene and stainless steel substrates, with synergistic treatments reducing biofilm formation to undetectable levels in some cases, and achieving up to 79.9 % reduction in thickness and 99.15 % reduction in coverage via confocal laser scanning microscopy (CLSM) analysis. This study established a phage-ultrasound combination therapy as a powerful strategy against biofilms of multidrug-resistant Salmonella strains.