The antagonistic interactions between a polyvalent phage SaP7 and β-lactam antibiotics on combined therapies

Highlights

• Seoulvirus phage SaP7 is a polyvalent phage that can control Salmonella and Escherichia coli.

• Phage SaP7 and three kinds of β-lactam antibiotics combined treatment had a negative interference on Salmonella growth in broth.

• Phage SaP7 combined with two kinds of β-lactam antibiotics showed antagonistic interactions on piglets and BALB/c mice infected with Salmonella.

Abstract

Phage therapy is a promising alternative antibiotic strategy to combat multidrug-resistant bacteria infections. Most studies focus on the synergistic effects, while the antagonistic interactions between phage and antibiotics is rarely studied. Here, we isolated and identified a novel polyvalent phage SaP7, which is capable of infecting multidrug-resistant Salmonella S7 and several E. coli strains. Morphology via electron microscopy showed that SaP7 belonged to the Myoviridae family. Genomic analysis revealed that the genome of SaP7 lacked any genes associated with antibiotic resistance, toxins, lysogeny, and virulence factors. We discovered the antagonism efficacy of SaP7 combined amoxicillin/potassium clavulanate (AMC) in counteracting Salmonella S7 in piglet-models by bacterial loads in feces and tissues. The consistent result as above between SaP7 and amoxicillin (AMX) was further verified in BALB/c mice-models. Furthermore, in vitro, plaque assay and minimum inhibitory concentration (MIC) determinations showed that AMX or AMC or cefepime (FEP) inhibited SaP7 plaque formation respectively and SaP7 decreased bacterial susceptibility of Salmonella S7 to AMX, AMC and FEP. And the negative interference of SaP7 with the bacteriostasis to Salmonella S7 of these three β-lactam antibiotics was observed in planktonic cultures via microtiter plates, but could not prevent the bacteriostasis of high titer of phage or high concentration of antibiotics. Finally, our research suggested that a polyvalent phage SaP7 existed antagonism with several β-lactam antibiotics. It is therefore crucial to fully and cautiously evaluate phage/antibiotic interactions and probable outcomes to avoid antagonistic impacts and failure of antibiotic and phage combination therapy.

Introduction

Antimicrobial resistance, especially multidrug resistance (MDR), is a growing global challenge. MDR bacteria are frequently detected in humans and animals and pose a serious threaten to human health (Doyle, 2015). In the case of Salmonella, the frequency of isolation of Salmonella serotypes resistant to one or more antibiotics has increased worldwide since antibiotic resistance was first reported in the early 1960s (Jajere, 2019). In fact, Salmonella remains one of the leading food-borne bacteria responsible for death, especially in low- and middle-income countries (Jajere, 2019; Gilchrist and MacLennan, 2019). There is an urgent need for more effective treatments for multidrug resistant Salmonella other than antibiotics.

Phage therapy is considered as an alternative strategy for the treatment of multidrug resistant bacterial infections (Danis-Wlodarczyk et al., 2021). Yet bacteria tend to develop resistance to phages easily just like they do to antibiotics, which greatly limits the practical effectiveness of phage therapy. It is argued that bacterial mutations which confer phage resistance might result in fitness costs such as reversal of drug resistance in the resistant bacteria (Edgar et al., 2012). Accordingly, phage combined with antibiotic therapy probably improve the therapeutic effect compared with phage therapy and antibiotic therapy alone. For instance, it’s considered by Rotem Edgar that phage-mediated transformation of plasmids carrying rpsL-wt and pRpsl-wt reduced the MIC of resistant mutants Sm6 and Sm13 to streptomycin from 100 μg/mL to 12.5 μg/mL and 200 μg/mL to 3.125 μg/mL, respectively (Edgar et al., 2012). In addition, a study reported that non-active antibiotic sulfamethoxazole-trimethoprim united with bacteriophage cocktail could constrain the phage-resistant mutation (Bao et al., 2020). Improper incompatibility of antibiotics will produce antagonistic effect and reduce the treatment effect (Gu Liu et al., 2020). Does antagonism exist between phage and antibiotics? It was reported that nalidixic acid could nearly or fully prevent the production of virions both by double-stranded phages (T2 and T5) and by single-stranded DNA phages (φR and φX174), but did not prevent the production of virions by RNA phages (β or 10) (Taketo and HW, 1967). Complete inhibition of E. coli phage T7 also using 50 μg/mL nalidixic acid and over 95 % burst-size reduction with 10 μg/mL was observed (Baird et al., 1972). Nevertheless, the research of the negative effect of nalidixic acid on phage was reported decades ago, antagonism between phages and other antibiotics are rarely reported.

Here we isolated and characterized a polyvalent phage named SaP7. Our study aimed to detect the interaction effect between SaP7 and several antibiotics in vitro, as well as the therapeutic effect of SaP7 combined with antibiotics on Salmonella infection in piglets and mice models. This information would provide an experimental basis to prove antagonism exists between phage and several β-lactam antibiotics.