Amphiphilic cyclic peptide [W₄KR₅]-Antibiotics combinations as broad-spectrum antimicrobial agents

Highlights

• [W₄KR₅] and [W₄KR₅]-meropenem covalent conjugate were synthesized.

• MICs were evaluated against non-resistant and multi-drug resistant strains.

• Time-kill kinetics assay indicated the time-dependent synergistic effect.

• Combination therapy enhanced the efficacy of commercial antibiotics.

• Flow cytometry analysis indicated the membrane disruption mechanism.

Abstract

Linear and cyclic amphiphilic peptides, (W₄KR₅) and [W₄KR₅], were evaluated as antibacterial agents against Gram-positive and Gram-negative bacteria, including four multi-drug resistant strains and the corresponding four non-resistant strains. Cyclic peptide [W₄KR₅] showed higher antibacterial activity than the linear (W₄KR₅) counterpart. Cyclic [W₄KR₅] was subjected to combination (physical mixture or covalent conjugation) with meropenem as a model antibiotic to study the impact of the combination on antimicrobial activity. A physical mixture of meropenem and [W₄KR₅] showed synergistic antibacterial activity against Gram-negative P. aeruginosa (ATCC BAA-1744) and P. aeruginosa (ATCC 27883) strains. [W₄KR₅] was further subjected to extensive antibacterial studies against additional 10 bacteria strains, showing significant antibacterial efficacy against Gram-positive bacteria strains. Combinations studies of [W₄KR₅] with an additional 9 commercially available antibiotics showed significant enhancement in antibacterial activity for all tested combinations, especially with tetracycline, tobramycin, levofloxacin, clindamycin, daptomycin, polymyxin, kanamycin, and vancomycin. Time-kill kinetics assay and flow cytometry results exhibited that [W₄KR₅] had a time-dependent synergistic effect and membrane disruption property. These data indicate that [W₄KR₅] improves the antibacterial activity, presumably by facilitating the internalization of antibiotics and their interaction with the intracellular targets. This study introduces a potential strategy for treating multidrug-resistant pathogens by combining [W₄KR₅] and a variety of classical antibiotics to improve the antibacterial effectiveness.

Introduction

The rise and spread of bacterial resistance have become one of the most serious public health crises and a major challenge for the scientific community. Over-prescribing antibiotics, unnecessary and inappropriate use of antibiotics and poor infection control in hospitals and clinics are the leading cause of bacterial resistance that result in the generation of super bacteria [[1], [2], [3]]. According to the Centers for Disease Control and Prevention (CDC), antibiotic resistance causes millions of illnesses worldwide every year, the number estimated to reach tens of millions by 2050 [4]. Among the major threats is the Gram-positive methicillin-resistant Staphylococcus aureus (MRSA), which causes around 19,000 deaths by year in the U.S.A with a health care cost of $3–4 billion. In recent years, the cases for multidrug-resistant Gram-negative bacteria, such as Escherichia coli (E. coli), Klebsiella pneumonia (K. pneumonia), and Pseudomonas aeruginosa (P. aeruginosa) have increased constantly [5]. The discovery of novel therapeutics alternative to commercially available antibiotics is urgently required to fight against or delay bacterial resistance. Several strategies have been applied to address this problem, such as developing new antimicrobial agents [6] and the revival of traditional antibiotics [7], but none of them are ideal.

Antimicrobial peptides (AMPs) are considered promising candidates to fight multi-drug resistant bacterial pathogens due to their excellent broad-spectrum antibacterial activity and unique non-specific bacterial membrane rupture mechanism, which does not allow the bacterial pathogens to develop resistance [[8], [9], [10]]. However, some AMPs cross the membrane without destroying it and act on intracellular targets, such as nucleic acids, protein biosynthesis, or protease inhibition [11]. AMPs are direct-acting antibacterial agent that directly inhibiting or killing bacteria without requiring any additional therapy [12]. Positively charged residues of the AMPs help them to accumulate at bacterial membrane surfaces through electrostatic interactions with the anionic phosphate head groups of membrane lipids, leading to membrane damage, cytoplasmic leakage, membrane depolarization and lysis, and cell death [8,13,14]. Modulation of the efficacy of AMPs via covalent conjugation with antibiotics is one of the strategies that is expected to increase antibacterial activity and decrease administration dose, leading to lowering the risk for adverse side effects [[15], [16], [17], [18]]. They were also found to be effective in combination therapy to fight against resistant bacteria [[19], [20], [21], [22]].

Meropenem (1) (Fig. 1), one of the carbapenem class antibiotics, is effective against most Gram-positive, Gram-negative, anaerobic, and even extended beta-lactamase–producing bacteria, and has good cerebrospinal fluid penetration [23,24]. However, meropenem is known to be less effective against New Delhi metallo-β-lactamases (NDMs) positive resistant bacterial strain and other metallo-β-lactamses producing bacteria. We have previously reported cyclic peptide [R₄W₄] (Fig. 1) containing arginine (Arg, R) and tryptophan (Trp, W) residues in a sequential manner to have antibacterial activity against Gram-positive and Gram-negative bacteria [[25], [26], [27]]. Moreover, we have previously investigated the conjugation between antibacterial cyclic peptides and antibiotics as an effective approach against bacterial resistance [28].

To improve the utility of [R₄W₄] as a promising antimicrobial agent, linear and cyclic peptides, (W₄KR₅) and [W₄KR₅] (Fig. 1), were successfully synthesized. Parentheses () and brackets [] represent the linear and cyclic peptides, respectively. We hypothesized that with the combination of peptides with antibiotics, the antibacterial activity could improve. Meropenem was used as the model antibiotic in the physical mixture with the peptides. Our goal here was to improve the activity of current antibiotics, including those that have low susceptibility to metallo-β-lactamses producing bacteria, through combination with the antibacterial peptide that has a different mechanism of action, such as targeting bacterial membrane. Furthermore, covalent conjugation of meropenem and [W₄KR₅] was accomplished to study the impact of conjugation on antimicrobial activity in comparison. Minimum inhibitory concentration (MIC) of the synthesized compounds along with the physical mixture (meropenem + [W₄KR₅]) were evaluated using multi-drug resistant strains as well as the corresponding non-resistant strains. Based on these data, combinations studies of [W₄KR₅] with an additional 9 commercially available antibiotics was accomplished. Then the synergistic and time-killing studies were performed for further understanding of the antimicrobial activity in combination and bactericidal action of the compounds, respectively. Flow cytometry analysis was conducted to study the effect of [W₄KR₅] on the bacterial cell membrane.