有抗菌作用的游离脂肪酸和单甘油酯：生物活性，实验检测和治疗应用

Antibacterial Free Fatty Acids and Monoglycerides： Biological Activities,Experimental Testing and Therapeutic Applications

Int J Mol Sci. 2018 Apr; 19(4): 1114.

Published online 2018 Apr 8. doi:  10.3390/ijms19041114

PMCID: PMC5979495

PMID: 29642500

antimicrobial lipids—single-chain lipid amphiphiles that destabilize bacterial cell membranes—are attractive candidates to become next-generation antibacterial agents for treating bacterial infections. Curiously, the antibacterial properties of antimicrobial lipids have been known since early reports by Dr. Robert Koch and colleagues in the late 1880s, when it was shown that fatty acids, a prominent class of antimicrobial lipid, inhibited growth of the Bacillus anthracis pathogen that causes anthrax [9]. A few decades later, Burtenshaw and colleagues showed that antimicrobial lipids are an important component of human skin’s innate immune system [10,11], lending credence to the possibility that exogenous addition of antimicrobial lipids would be medically opportune. Despite strong promise and demonstrated results, the prospects for antimicrobial lipids faded away by the late 1940s due to the emergence of antibiotics, but have received renewed attention amidst the growing impact of antibiotic-resistant bacteria. Indeed, one attractive feature of antimicrobial lipids is that it is difficult for bacteria to mutate to become resistant to them. As such, bacterial cell cultures can be grown in the presence of antimicrobial lipids (at sub-lethal concentrations) for at least one year, without signs of drug-resistant strains emerging [12].

 

Particular attention is drawn to two classes of antimicrobial lipid, namely fatty acids (hydrocarbon chains with a carboxylic acid functional group [13]) and monoglycerides (esterified adducts of a fatty acid and glycerol molecule). The motivation for studying these two classes of antimicrobial lipid arose from pioneering studies by Kabara and colleagues in the 1970s, which systematically investigated the antibacterial potency of medium-chain saturated fatty acids and monoglycerides with different chain lengths [14,15,16,17]. It was discovered that lauric acid (LA), which possesses a 12 carbon-long chain, had the most potent activity to inhibit growth of Gram-positive bacteria, and its monoglyceride derivative, glycerol monolaurate (GML), exhibited even stronger inhibitory activity than LA. Importantly, both LA and GML are Generally Recognized As Safe (GRAS) by the United States Food and Drug Administration [18] and abundant in nature. These factors have led to wide exploration of LA and GML for anti-infective applications [19,20,21], including application topics such as agriculture [22] and cosmetics [13,23,24]. Other antimicrobial lipids such as capric acid, which possesses a 10 carbon-long chain, and its monoglyceride derivative, monocaprin, have also received attention. While numerous studies have been conducted to empirically investigate the inhibitory properties of antimicrobial lipids, clarifying how the physicochemical properties of antimicrobial lipids influence biological activities remains an outstanding goal in many respects.

 

Spectrum of Antibacterial Activity

 

Broad-spectrum inhibitory activity of antimicrobial lipids against algae, bacteria, fungi, protozoa, and virus has been reported for several decades [13,14,15,23,57,58,59]. The antibacterial potency of fatty acids and monoglyceride derivatives has been investigated extensively against a wide range of bacteria, including pathogenic strains such as methicillin-resistant Staphylococcus aureus (MRSA), as presented in Table 1. Extensive screening of the antibacterial activities of fatty acids was conducted by Kabara and colleagues in the 1970s, and helped to establish the modern-day field of antimicrobial lipids from a chemical perspective. Fatty acids with hydrocarbon chains ranging from 6 to 18 carbons long and selected derivatives with different functionalized headgroups were evaluated, resulting in the comprehensive identification of LA (C12:0) as the most potent antimicrobial lipid to inhibit growth of Gram-positive bacteria, as mentioned above. As a general principle, the esterification of a fatty acid to its corresponding monoglyceride derivative increases antibacterial activity [14,15,17]. Another supportive study for the high antibacterial potency of LA was conducted with different types of Gram-positive bacteria, and further demonstrated that unsaturated fatty acids with 18 carbon long chains—oleic acid (C18:1), linoleic (C18:2), and linolenic acid (C18:3)—have potent antibacterial activities as well [50].

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Of the tested fatty acids and monoglycerides, LA, linolenic acid, and GML had the strongest bactericidal activity at 10–20 µg/mL in brain heart infusion broth at pH 5. Interestingly, the bactericidal activity of the fatty acids depended on solution pH, showing increased activity at pH 5 as compared to pH 6, while the activity of the monoglyceride was not influenced by solution pH across this range [28]. This is consistent with the ionizable headgroup of fatty acids while monoglycerides are nonionic molecules.

 

Another target bacterium that is susceptible to fatty acids is Helicobacter pylori, which is a Gram-negative bacterium that can be pathogenic in the stomach or duodenum leading to diseases such as chronic gastritis, gastric ulcers, and stomach cancer. Petschow et al. tested a wide range of saturated fatty acids and corresponding monoglycerides with chain lengths from 4 to 17 carbons against H. pylori and observed the following trend in bactericidal activities. Monoglycerides with 10–14 carbon long chains showed appreciable bactericidal efficacy at 1 mM concentration and had a lower tendency of spontaneous resistance development, while LA was the only saturated fatty acid that had bactericidal potency under the tested conditions [62]. Similarly, the anti-H. pylori activity of LA and GML was demonstrated by Sun et al., in which case selected unsaturated fatty acids were evaluated along with a range of saturated fatty acids (4–16 carbon chains) and monoglycerides (12–16 carbon chains) [54]. LA completely killed H. pylori at minimum bactericidal concentration (MBC) of 1 mM, and GML showed even more potent activity than LA with a lower MBC value around 0.5 mM. Additionally, it was identified that, among unsaturated fatty acids, myristoleic (C14:1) and linolenic acid have the most potent activity against this bacterium [54]. Bergsson and colleagues also assessed inhibitory activity against Gram-negative bacteria, including H. pylori, for a wide range of fatty acids and corresponding monoglycerides [64]. Interestingly, the tested compounds that had hydrocarbon chains between 10–16 carbons long exhibited inactivation of H. pylori at 10 mM concentration, however, there was no significant activity observed against Salmonella species and Escherichia coli. Bergsson et al. conducted additional studies in order to characterize the antibacterial potency of selected antimicrobial lipids against different bacteria, and it was demonstrated that monocaprin (MG C10:0) is the most potent bactericidal agent against Chlamydia trachomatis and Neisseria gonorrhoeae [29,63]. Additional studies have been reported investigating the effects of fatty acids (with chains of 2 to 18 carbon length) on Gram-negative bacteria, including E. coli [65] and Salmonella species [66], and demonstrated that caprylic acid (C8:0) has particularly high antibacterial activity against these bacteria. Antibacterial activity of caprylic acid and its monoglyceride, monocaprylate, were also identified against fish pathogens including Edwardsiella species by Kollanoor and colleagues [69]. Of particular note, caprylic acid and monocaprylate have showed potent inhibitory activity against important foodborne pathogens, including E. coli O157:H7 [73,74,75,76,77], along with Salmonella species [78,79], L. monocytogenes [80,81], and mastitis pathogens [82].

 

2.3. Mechanisms of Antibacterial Activity

 

The mechanism(s) of antibacterial activity of fatty acids and monoglycerides have been explored using diverse experimental methods to identify that they mainly target bacterial cell membranes and interrupt crucial processes involved in cellular protection and function. The membrane-lytic behavior of fatty acids and monoglycerides stem from their amphipathic properties, leading to overlapping sets of biophysical phenomena including membrane destabilization and pore formation. In particular, membrane-destabilizing activity causes increased cell permeability and cell lysis, leading to inhibition of bacterial cell growth (bacteriostatic action) or cell death (bactericidal action).

 

Among vital processes involving bacterial cell membranes, two of the most important ones involve the electron transport chain and oxidative phosphorylation, which are essential for energy production in bacterial cells. The two processes are interconnected, and fatty acids have the potential to disrupt the electron transport chain process by binding to electron carriers or altering membrane integrity as well as interfering with oxidative phosphorylation by decreasing the membrane potential and proton gradient. Moreover, fatty acids can directly inhibit membrane enzymes such as glucosyltransferase, presumably due to similar molecular structures of fatty acids with known small molecule inhibitors [90,91], and also target other membrane-associated proteins as well [92]. Figure 2 presents a schematic illustration describing key antibacterial activities of fatty acids and monoglycerides that relate to targeting bacterial cell membranes. The details of the mechanistic processes can be classified based on the relationship between the following three aspects: (i) increased membrane permeability and cell lysis, (ii) disruption of electron transport chain and uncoupling oxidative phosphorylation, and (iii) inhibition of membrane enzymatic activities and nutrient uptake.

 

4.1. Systemic Treatment of Stomach Infection

 

Helicobacter pylori is a pathogenic Gram-negative bacterium that colonizes the stomach of over half of the world’s population, and is a leading cause of stomach infections, chronic gastritis, gastric ulcers, and stomach cancer [134,135]. Indeed, H. pylori infection is a serious health problem because it is estimated that ~75% of all stomach cancer cases worldwide are caused by H. pylori infection [136]. As the standard first-line treatment for H. pylori infection involves a combination of three of four compounds in total (typically a proton-pump inhibitor and/or bismuth along with two of three antibiotics), however, there are several key challenges faced, including poor patient compliance, history of past antibiotic usage, drug side effects, uncertain eradication rate, and most importantly the presence of antibiotic-resistant H. pylori strains [137,138,139]. As such, there is strong motivation to identify new antibacterial agents that work against H. pylori, particulary ones that are effective with high potency and have a low risk of drug-resistant strains emerging.

 

In this regard, fatty acids and monoglycerides are promising agents that have demonstrated antibacterial efficacy against H. pylori and there is a high barrier for drug-resistant strains to emerge. The antibacterial activity of fatty acids and monoglycerides was identified through extensive in vitro screening to detremine the antibacterial spectrum of various fatty acids and derivatives thereof. Petschow et al. demonstrated that LA and monoglycerides containing 10–14 carbon long chains effectively killed H. pylori at 1 mM treatment concentrations, with a low tendency for resistance development [62]. Similarly, in vitro bacterial susceptibility tests showed that saturated fatty acids and corresponding monoglycerides with 10–14 carbon long chains as well as two additional unsaturated fatty acids and monoglycerides—palmitoleic acid and monopalmitolein—showed an appreciable killing effect against H. pylori [64]. Among the findings, monocaprin and GML were the most active against H. pylori. Subsequently, Sun et al. reported in vitro inhibitory activity of fatty acids and monoglycerides against H. pylori as well [54]. According to the study, LA completely killed H. pylori at 1 mM concentration and its monoglyceride derivative, GML, showed similar bactericidal action at lower concentrations around 0.5 mM. Additionally, another unsaturated fatty acid, linolenic acid, also exhibited bactericidal activity at 0.5 mM concentration, in line with the GML potency. Polyunsaturated fatty acids such as docosahexaenoic acid (DHA) also have anti-H.pylori activity and been reported to reduce H.pylori-associated disease pathogenesis in mouse models [140,141,142,143].

 

To improve delivery of antimicrobial lipids in highly concentated forms (i.e., to avoid dilution effect), liposomal formulations have been developed that encapsulate the active agents within the liposomal bilayer. The development of liposomal formulations containing intercalated fatty acids are first characterized—usually entailing size, zeta potential, and loading characterization—followed by in vitro efficacy and safety studies, and then in vivo testing. Obonyo et al. developed linolenic acid-loaded liposomes (LipoLLA) and identified that LipoLLA effectively killed both spiral and coccoid forms of H. pylori by disrupting the bacterial cell membrane [144]. Compared to free linolenic acid, LipoLLA provided greater killing effect and there was a high barrier to resistance emerging. Jung et al. extended this line of investigation to better understand how LipoLLA interacts with and disrupts the membranes surrounding H. pylori cells [145]. Linolenic acid and 18-carbon long analogues were prepared in liposomal formulations, and, out of the tested species, it was confirmed that LipoLLA had the most potent antibacterial activity against H. pylori and showed complete killing of the bacterium at 200 μg/mL based on causing an increase in bacterial membrane permeability.

 

Motivated by these in vitro findings, Thamphiwatana et al. further investigated the therapeutic efficacy of LipoLLA to treat H. pylori infection in an in vivo mouse model [146]. After systemic administration of LipoLLA, H. pylori burden in the mouse stomach was measured in comparison to treatment with free linolenic acid or conventional triple-therapy involving antibiotics. Treatment with LipoLLA showed superior activity to reduce bacterial burden and the level of H. pylori-induced proinflammatory cytokines, indicating that LipoLLA is promising therapeutic agent to treat H. pylori infection with high biocompatibility as well. The latter feature was further confirmed recently by Zhong and colleagues by demonstrating that LipoLLA administration induced only minor changes in the gastrointestinal microbiota while conventional triple therapy with three antibiotics caused more dramatic changes in the microbiotica [147].

 

Following this approach, Seabra et al. reported another promising formulation that involves loading DHA into solid lipid nanoparticles [148]. The nanoparticle enhanced bactericidal activity of DHA against H. pylori by enabling delivery of poorly water-soluble DHA and the DHA-loaded nanoparticles were not cytotoxic against human adenocarcinoma cells. Taken together, the results highlight that antimicrobial lipids can be developed into therapeutically viable formulations that can be used for in vivo applications.

https://www.ncbi.nlm.nih.gov/pmc/articles/PMC5979495/