Synergistic action of phages and lytic proteins with antibiotics: a combination strategy to target bacteria and biofilms

Abstract

Background: Multidrug-resistant bacteria continue to emerge owing to the abuse of antibiotics and have a considerable negative impact on people and the environment. Bacteria can easily form biofilms to improve their survival, which reduces the efficacy of antibacterial drugs. Proteins such as endolysins and holins have been shown to have good antibacterial activity and effectively removal bacterial biofilms and reduce the production of drug-resistant bacteria. Recently, phages and their encoded lytic proteins have attracted attention as potential alternative antimicrobial agents. The aim of the present study was to investigate the sterilising efficacy of phages (SSE1, SGF2, and SGF3) and their encoded lytic proteins (lysozyme and holin), and to further explore their potential in combination with antibiotics. To the ultimate aim is to reduce or replace the use of antibiotics and provide more materials and options for sterilisation.

Results: Phages and their encoded lytic proteins were confirmed to have great advantages in sterilisation, and all exhibited significant potential for reducing bacterial resistance. Previous studies on the host spectrum demonstrated the bactericidal efficacy of three Shigella phages (SSE1, SGF2, and SGF3) and two lytic proteins (LysSSE1 and HolSSE1). In this study, we investigated the bactericidal effects on planktonic bacteria and bacterial biofilms. A combined sterilisation application of antibiotics, phages, and lytic proteins was performed. The results showed that phages and lytic proteins had better sterilisation effects than antibiotics with 1/2 minimum inhibitory concentrations (MIC) and their effect was further improved when used together with antibiotics. The best synergy was shown when combined with β- lactam antibiotics, which might be related to their mechanism of sterilising action. This approach ensures a bactericidal effect at low antibiotic concentrations.

Conclusions: This study strengthens the idea that phages and lytic proteins can significantly sterilise bacteria in vitro and achieve synergistic sterilisation effects with specific antibiotics. Therefore, a suitable combination strategy may decrease the risk of drug resistance.

Keywords: Antibiotic; Phages and lytic proteins; Synergistic sterilization.

Fig. 1

Phages were combined with antibiotics to remove different planktonic Shigella. a Shigella 1.1869 were removed by phage SSE1 and antibiotics, b Shigella 1.1868 were removed by phage SGF2 and antibiotics, c Shigella 1.10599 were removed by phage SGF3 and antibiotics. Gray represents negative control without phage and antibiotics, green represents phage SSE1 and its antibiotic combinations, yellow represents phage SGF2 and its antibiotic combinations, orange represents phage SGF3 and its antibiotic combinations, and blue represents antibiotic alone. Groups 1, 2, 3, 4, 5, 6, 7, 8, and 9 correspond to erythromycin, gentamicin sulphate, chloramphenicol, cefotaxime, cefoxitin, cephalothin, cardelmycin, tetracycline hydrochloride, and polymyxin B sulphate, respectively. The concentration of all the antibiotics used was 1/2 MIC. Error bars represent standard deviation of three biological replicates. The letters a, b, c, d, e, f, g, and h indicate significant results; the significance level was set at p < 0.05

Fig. 2

Removal effect of different bacterial biofilms by phages and cocktails. a S. dysentery 1.1869 biofilm was treated with phage SSE1 and its cocktails. b S. baumannii 1.10618 biofilm was treated with phage SSE1 and its cocktails. c S. flexneri 1.1868 biofilm was treated with phage SGF2 and its cocktails. d S. flexneri 1.10599 biofilm was treated with phage SSE1, SGF3, and their cocktails. Gray indicates control groups without phage addition, orange indicates only phage SSE1, yellow indicates only phage SGF2, green indicates phage SGF3 alone, and blue indicates phage cocktails of several different combinations. Error bars represent standard deviation of three biological replicates. Statistical analysis was performed using a paired sample t-test, and double asterisks indicate p < 0.01

Fig. 3

LysSSE1, HolSSE1 and antibiotics were combined to remove planktonic a S. dysentery 1.1869, b S. flexneri 1.1868, and c S. flexneri 1.10599. Gray represents negative control without lytic proteins and antibiotics, orange represents LysSSE1 and antibiotics combination, green represents HolSSE1 and antibiotic combination, and blue represents antibiotic alone. Groups 1, 2, 3, 4, 5, 6, 7, 8, and 9 correspond to erythromycin, gentamicin sulphate, chloramphenicol, cefotaxime, cefoxitin, cephalothin, cardelmycin, tetracycline hydrochloride, and polymyxin b sulphate, respectively. The concentration of the antibiotics used was 1/2 MIC. Error bars represent standard deviation of three biological replicates. The letters a, b, c, d, e, and α, β, γ, δ indicate significant results; the significance level was set at p < 0.05

Fig. 4

LysSSE1, HolSSE1 and antibiotics were combined to remove the biofilms of a S. dysentery 1.1869, b S. flexneri 1.1868, and c S. flexneri 1.10599. Gray represents negative control without lytic proteins and antibiotics, orange represents LysSSE1 and antibiotics combination, green represents HolSSE1 and antibiotic combination, and blue represents antibiotic alone. Groups 1, 2, 3, 4, 5, 6, 7, 8, and 9 correspond to erythromycin, gentamicin sulphate, chloramphenicol, cefotaxime, cefoxitin, cephalothin, cardelmycin, tetracycline hydrochloride, and polymyxin b sulphate, respectively. The concentration of all the antibiotics used was 1/2 MIC. Error bars represent standard deviation of three biological replicates. The letters a, b, c, d, and α, β, γ, δ indicate significant results; the significance level was set at p < 0.05

Fig. 5

LysSSE1 and HolSSE1 were used to remove the biofilms of S. aureus. Gray represented negative controls without lytic proteins, orange for the LysSSE1 added group and green for the HolSSE1 added group. Error bars represented standard deviation of three biological replicates. Statistical analysis was performed using a paired-matched sample t-test, and the double asterisks indicated p < 0.01

Fig. 6

The removal effects of multiple bacterial biofilms. The concentration of isorhamnetin and luteolin was 20 mg/L. Error bars represent standard deviation of three biological replicates. Statistical analysis was performed using a paired sample t-test, and double asterisks indicate p < 0.01