1. Introduction
The global spread of antimicrobial resistance within bacteria is a serious threat, causing many antibiotics to lose their effectiveness [
1,
2]. According to the United States (US) Center for Disease Control and Prevention’s (CDC) latest report, more than 2.8 million antibiotic-resistant infections occur in the US each year, and more than 35,000 people die as a result [
3]. According to this report, one of the most urgent threats is carbapenem-resistant
Enterobacterales: these bacteria account for more than 1100 deaths in the US annually, and cause more than
$130 million in financial losses every year. Multi-drug resistant
Pseudomonas aeruginosa has been designated a serious threat, causing more than 2700 annual deaths and resulting in a
$767 million financial burden on the US healthcare system. Overall, antibiotic-resistant infections cause more than 700,000 deaths worldwide every year, and it is projected that, by the middle of the 21st century, this figure will exceed the death rate of cancer and result in more than 10 million deaths a year [
2]. In this regard, there is an urgent need to discover new treatment options for infections caused by resistant microorganisms.
One of the strategies to combat antibiotic resistance is to use antimicrobial peptides (AMPs), which are an essential component of the innate immune system and protect against bacteria, viruses, fungi, parasites, and tumor cells [
4]. While conventional antibiotics act on one specific target, AMPs simultaneously affect membranes and intracellular targets of a bacterial cell, thereby providing high activity against resistant bacteria [
5,
6]. AMPs are mainly hydrophobic peptides with a positive charge. Their effectiveness is dependent on charge neutralization and penetration through bacterial membranes, producing bacterial cell death and reducing the likelihood for the development of bacterial resistance [
7]. AMPs are safer and less likely to have toxic side effects compared to conventional antibiotics [
8].
Previously, we investigated the in vitro activity of human AMPs from the defensins class against bacterial strains of
Staphylococcus aureus and
Escherichia coli, collected from hospitalized patients [
9]. Research has shown the most effective human defensins to be human beta-defensin-3 (hBD-3), which is produced by epithelial cells. We showed that human hBD-3 is highly active against methicillin-resistant
S. aureus (MRSA) and carbapenem-resistant
E. coli. In addition, hBD-3 showed a pronounced synergistic effect when used together with rifampicin (against MRSA) and amikacin (against
E. coli). Several studies have shown various AMPs to exhibit synergistic effects in vitro when combined with carbapenems [
10]. However, more research is needed to evaluate the effects of AMPs and carbapenems in experimental in vivo models.
Epinecidin-1 (Epi-1) is an AMP derived from the orange-spotted grouper (
Epinephelus coioides) and was discovered in 2005 [
11]. Epi-1 has a wide spectrum of antimicrobial activity against Gram-positive and Gram-negative bacteria and viruses, in addition to an antitumor effect, a wound healing effect, and an immune response regulator [
12,
13]. Despite numerous studies devoted to studying the properties of Epi-1, there is insufficient data documenting its effectiveness against resistant Gram-negative infections in vivo. The activity of Epi-1 in combination with any human AMPs has not been previously studied. In addition, the antibacterial effect of the combination of Epi-1 and vancomycin against MRSA has not been previously evaluated. In one study, the effectiveness of the combined use of Epi-1 + vancomycin was analyzed in experimental MRSA infection in pigs [
14]; however, the classical in vitro checkerboard assay for assessing the mutual antibacterial effect of two substances was not used here [
9,
15,
16,
17,
18].
The aim of this study was to evaluate the antimicrobial activity of: (i) Epi-1 + vancomycin against MRSA in vitro; (ii) Epi-1 + hBD-3 against carbapenem-resistant isolates of Klebsiella pneumoniae, Klebsiella aerogenes, Acinetobacter baumannii, and P. aeruginosa in vitro; (iii) Epi-1 + hBD-3 in the experimental model of K. pneumoniae-induced sepsis.
3. Discussion
In this study, the antimicrobial activity of the Epi-1 in combination with vancomycin against 22 clinical isolates of methicillin-resistant S. aureus was, for the first time, evaluated.
Different pharmacological effects of Epi-1 have been previously shown in vitro and in experimental animal models [
13]. We showed that Epi-1 is effective against a wide spectrum of MRSA clinical isolates, with an MIC range from 16 to 32 mg/L. In most previous studies, the range of MIC values for various
S. aureus strains (including MRSA) ranged from 6 to 50 mg/L. We also demonstrated the synergistic effect of Epi-1 + vancomycin against some MRSA isolates. In one study, the antistaphylococcal activity of Epi-1 was investigated when combined with conventional antibiotics (kanamycin or streptomycin): a synergistic effect was shown with FICI = 0.4 [
19]—the combined use of Epi-1 with these antibiotics resulted in a threefold decrease in MIC values. The authors suggested that the cause of synergy may be a different mechanism of action than that of aminoglycosides (namely, the effect on the translation of proteins in microbial cells) [
20] and AMPs (membrane permeabilization) [
7]. In addition, in this study, the authors obtained various modifications of truncated Epi-1, which also showed pronounced antibacterial activity. There are no other publications on the combined effect of Epi1-1 with other antimicrobial drugs/peptides.
Vancomycin is a glycopeptide antibiotic. It binds to D-alanyl D-alanine, which inhibits peptidoglycan synthase (glucosyltransferase), and the P-phospholipid carrier, thus preventing the synthesis and polymerization of N-acetylmuramic acid and N-acetylglucosamine within the peptidoglycan layer. This, in turn, weakens the bacterial cell wall, and leakage of intracellular components occurs, which causes the death of bacteria [
21]. Therefore, AMPs and vancomycin have different mechanisms of action, which can presumably lead to a synergistic effect. Previously, several studies have shown that vancomycin and various natural and newly designed AMPs can exhibit a synergistic effect against Gram-positive bacteria [
22,
23,
24,
25].
This study was the first investigation of the combined effect of Epi-1 with human AMP (namely, hBD-3) against broad spectrum of carbapenem-resistant strains of Gram-negative bacteria K. pneumoniae (n = 23), K. aerogenes (n = 17), P. aeruginosa (n = 13), and A. baumannii (n = 9).
Interestingly, the effectiveness of Epi-1 has not been previously studied in relation to these bacteria. In one of the first works on the Epi-1, it was shown that its MIC was 100 mg/L against
Klebsiella oxytoca [
26]. We showed that both peptides exhibited antibacterial effects in the concentration range of 2 to 32 mg/L. In more than half of the cases for
A. baumannii, in almost half of
P. aeruginosa isolates, and in almost one third of cases for
Klebsiella spp., the Epi-1 + hBD-3 combination showed synergy. We chose to study hBD-3, as we have previously investigated its activity in combination with amikacin against
E. coli, where classical synergism has also been demonstrated [
9]. These results provide new data that can be used in developing new treatments for multidrug-resistant bacterial infections.
It remains unclear why in some cases Epi-1 + hBD-3 enhanced each other’s effectiveness, and in some did not. Here it is necessary to conduct research on an even larger number of bacterial strains, as well as to study this effect at the molecular level. It seems that the phenotype of antibiotic resistance may somehow influence this, though many studies have shown that the antibiotic resistance phenotype of bacteria does not matter for the effectiveness of AMPs [
27]. Therefore, it can be speculated that this phenomenon (differences of FICI values for the same species of bacteria) can be explained by some surface characteristics of the bacterial cell wall, which is the main target for AMPs.
From a clinical point of view, it could seem that MIC values in the range of 2–32 mg/L is a hard-to-reach concentration at the site of infection. Therefore, the purpose of future research should be to find and develop any modifications and/or derivatives of Epi-1. However, it is worth noting that Epi-1 was found to be highly effective in combating
Pseudomonas aeruginosa-induced peritonitis infection in mouse models, without side effects or toxicity—Epi-1 increased survival in mice [
28]. In another study it was shown that, in the pyemia model in pigs, Epi-1 protects animals against MRSA-mediated mortality [
14].
In an experimental model of murine sepsis caused by K. pneumoniae or P. aeruginosa, we examined, for the first time, the efficacy of hBD-3 monotherapy in comparison with the following therapies: meropenem, hBD-3 + meropenem, and hBD-3 + Epi-1. For these animal studies, isolates were selected in which synergism was not shown during in vitro tests using the combination of hBD-3 and Epi-1.
In the
K. pneumoniae-sepsis model, the addition of Epi-1 to hBD-3 does not significantly reduce mortality compared to hBD-3 monotherapy. However, in the
P. aeruginosa-sepsis model, slight differences were found between the hBD-3 and hBD-3 + Epi-1 groups. We can cautiously assume that, in these mice, Epi-1 somewhat enhanced the effect of hBD-3. Although, at the same time, there was no difference between hBD-3 + meropenem compared to hBD-3 + Epi-1. One of the limitations of our work is that we did not investigate the effectiveness of Epi-1 monotherapy. In an earlier study, using a
P. aeruginosa-sepsis model, Epi-1 demonstrated reduced mortality in mice due to its antimicrobial and pronounced immunomodulatory effect [
28]. Epi-1 enhances the production of immunoglobulin G by activating the Th2-cell response [
29], and reduces the level of tumor necrosis factor-alpha by decreasing the levels of endotoxins [
28]. Based on these findings, Epi-1 and hBD-3 (and their derivatives) are the promising new therapeutic agents in the fight against antibiotic-resistant infections.
In the
K. pneumoniae-sepsis model, we found a weak statistically significant difference when comparing hBD-3 monotherapy to hBD-3 + meropenem. It can be speculated that hBD-3 overcomes the resistance of
K. pneumoniae to carbapenems in vivo, though additional studies are needed to confirm this hypothesis. Another limitation of this study is that we did not evaluate the in vitro combination of meropenem + hBD-3. Previously, several studies have shown that various AMPs demonstrate synergistic effects with beta-lactams [
10,
30,
31,
32]. However, we were unable to find studies on the combination of hBD-3 and carbapenems. We have previously reported that hBD-3 + amikacin has a synergistic effect against several
E. coli strains [
9]. In the same study, we demonstrated that the antibiotic resistance phenotype does not impact the effectiveness of AMPs.
The hBD-3 peptide reduced mortality, in both models of sepsis, when given as monotherapy. It can be assumed that this is directly associated with its antimicrobial effect, as well as some of the immunomodulatory effects of beta-defensins: chemoattraction of T-lymphocytes and dendritic cells, interaction with the CCR6 receptor, and increased production of IL-18. In addition, hBD-3 does not show cytotoxic or hemolytic effects at antimicrobial concentrations [
33].