Dr. Jon Kabara was a professor at Michigan State University (20 years)
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Author Jon Kabara begins by revealing the surprisingly varied roles played by fats and cholesterol in the body
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Book Review
Lipids and MRSA
Antibiotics when first discovered changed the course of treatment of infectious disease. Their successful application became wide spread and their misuse quickly followed. Due to their inappropriate use and over use resistant organisms quickly appeared soon after discovery of an antibiotic. Although these newly resistant organisms could be controlled by newer and even more effective agents, soon afterwards resistant organisms again reappeared. This cycle of antibiotic discovery and inevitable resistance continues to frustrate their continued clinical benefits. What is needed are agents that have biocidal activity but low frequency of forming resistant organisms. Fatty acids and their corresponding monoesters may be the answer. It is shown here that both nonresistant and formerly antibiotic resistant organisms are equally inactivated by such lipids. Both in vitro and in vivo data are presented which support the new role of lipids as antimicrobial agent.
Antibiotic Resistant Organisms
When first discovered antibiotics like penicillin were hailed as medical miracles against infectious disease (1). The first bacterium to be successfully inactivated by penicillin was Staphylococcus aureus (SA). This bacterium, often a harmless passenger in the human body, can cause illness, such as pneumonia or toxic shock syndrome, when it overgrows or produces a toxin. Staphylococcus aureus is one of the major human pathogens, causing a variety of infections from suppurative disease to food poisoning.
Shortly after mass-producing of penicillin in 1943, microbes began appearing in 1947 that could resist it. Alexander Fleming, the name most associated with penicillin, had commented that penicillin given in small doses could produce resistant organisms. This warning was not heeded since increasingly effective antibiotics were being discovered at a rapid pace.
The increase and inappropriate uses of antibiotics however has caused problems, more than could ever have been fully anticipated. Overuse of antibiotics results in bacterial resistance not only to the antibiotic prescribed, but also often to other antibiotics. The abuse or misuse of antibiotics, especially in animal feed, is costly because it leads to the emergence of antibiotic resistance among microorganisms.
Penicillin-resistant pneumonia (or pneumococcus, caused by Streptococcus pneumoniae) was first detected in 1967, as was penicillin-resistant gonorrhea. Resistance to penicillin substitutes is also known beyond S. aureus. By 1993 Escherichia coli was resistant to five fluoroquinolone variants. Mycobacterium tuberculosis is commonly resistant to isoniazid and rifampin and sometimes universally resistant to present available common treatments. Other pathogens showing some resistance include Salmonella, Campylobacter, and Streptococci.(2)
However, bacteria were not only becoming resistant to penicillin but also to other important alternatives. Methicillin was introduced in 1959 and was then the antibiotic of choice for penicillin resistant organisms. Soon afterward its introduction however Methicillin-resistant Staphylococcus aureus (MRSA) were detected in 1961. Methicillin resistance is due to the formation by the bacteria of beta-lactamase an enzyme that inactivates this and other lactam antibiotic. Half of all Staphylococcus aureus infections in the US are resistant to penicillin, methicillin, tetracycline, and erythromycin.
Today Methicillin-resistant S. aureus (MRSA) is a huge clinical problem. Many strains of MRSA are resistant to all antimicrobial agents except glycopeptides (vancomycin and teicoplanin). Even this has changed. The emergence of multidrug-resistant isolates of Methicillin-resistant S. aureus (MRSA) exhibited decreased susceptibilities to even glycopeptides (glycopeptide-intermediate Staph. aureus, GISA). Staphylococcus aureus resistance to the glycopeptide antibiotic vancomycin is of noteworthy concern since vancomycin had been the only antibiotic to which there has been uniform susceptibility to multidrug-resistant Methicillin-resistant S. aureus (MRSA). Vancomycin-resistant Staphylococcus aureus (VRSA) were identified in Japan in 1997, and has since been found in hospitals in England, France and the US. VRSA is also termed GISA (glycopeptide intermediate Staphylococcus aureus) or VISA (vancomycin insensitive Staphylococcus aureus), indicating resistance to all glycopeptide antibiotics. (3).
To overcome these problems a new class of antibiotics, oxazolidinones, became commercially available in the 1990s, Oxazolidinone was comparable to the earlier in effectiveness of vancomycin against MRSA. The resistant story continues however since oxazolidinone resistance in Enterococcus faecium (ORE) was reported in the late 1990s.
The situation became worse when in a report by B. E. Murray in the April 28, 1994, New England Journal of Medicine; researchers had identified bacteria in patient samples that resist all currently available antibiotic drugs. The problem continues to expand (4)
In November, 2004, the Centers for Disease Control and Prevention (CDC) reported an increasing number of Acinetobacter baumannii bloodstream infections in patients at military medical facilities in which service members injured in the Iraq/Kuwait region during military operations in Iraq and Afghanistan were treated.
Total antibiotic production today is more like 35 million pounds and 70% of total antibiotic production is devoted to non-therapeutic use in three livestock sectors. The bottom line is that current information suggests that the medical increase use and agricultural abuse of antibiotics is likely to be a larger part of an antibiotic-resistance problem than was currently thought (5).
A different approach to medical problems associated with microorganisms is desperately needed. The answer is to discover products that create fewer complications and negative side effects than present conventional drug-based pharmaceutical.
Lipids as Antimicrobial Agents
Rather than producing new and stronger antibiotics, a different approach is needed. An approach that will reduce the formation of resistant organisms is critically needed. Initial work by Kabara et al on lipids suggested that the self-disinfecting properties of fatty acids and monoglycerides may show promise (6-8) Their disinfecting power have long been recognized. It is a contention of the present review that, unlike drug antibiotics antimicrobial lipids are also part of an innate immune system.
Natural endogenous antimicrobial lipids would have advantages over other exogenous materials. These endogenous antimicrobials would have been arrived at through the process of natural selection to provide protection against the most common potential pathogens. It could be anticipated that their mechanism of action is such that they do not readily give rise to resistant strains otherwise they would no longer be effective. This has been supported by recent studies detailed later in this paper. Since lipids are normal constituents, it would be expected that they would not be irritating, sensitizing, or toxic.
Our earlier identification of the most effective antimicrobial lipids could lead to new strategies for treatment of or prophylaxis against infections. One such group for consideration is free fatty acids and monoglycerides. These lipids have been shown to have antimicrobial affects against a wide spectrum of microorganisms (6-8).
In Vitro Activity of Skin Fatty acids
In specific support of this hypothesis skin fatty acids have been selected as examples. It is generally thought that the normal bacterial flora on the skin surface have been selected through a combination of limited water availability and differential sensitivity to antimicrobial lipids at the skin surface. Lipids at the skin surface have been earlier shown to have antibacterial activity against S. aureus. Burtenshaw (9) showed in the 1940s that lipid extracts from the skin surface had the ability to kill Staphylococcus aureus in vitro, and it was thought that free fatty acids were the active agent however, this proposition was not extensively tested with fatty acids actually found at the human skin surface until much later.
As shown by Kabara et al (6-8) it was not surprising that fatty acids from epidermal stratum corneum would have antibacterial activity against a range of Gram positive bacteria, but not against Candida albicans or a number of gram negative bacteria.. Our seminal studies demonstrated that antimicrobial action was directly related to structure of the fatty acid.
Fractionation of the sebum lipids confirmed, as predicted by Kabara et al (6), that both saturated and unsaturated fatty acids contained the bulk of the antimicrobial activity (10). The conclusions from these studies supported Kabara’s earlier conclusions which were: a) a) The most active saturated fatty acid was C12 (lauric acid): b) The most active monounsaturated acid was palmitoleic acid ((16:1, D9). c) Monoesters were more active than their free fatty acids
The monounsaturated fatty acid found in sebum, sapienic acid (C16-1,D6), was both the most predominant and active monoene. Purified sapienic acid (>99%) yielded typical minimal inhibitory concentration (MIC) values of 10-20 µg/ml against gram-positive bacteria. A second prominent fatty acid, lauric acid (C12:0) was the most active saturated fatty acid.(11)
Sapienic acid was found to be the most active sebum lipid fraction. Since these natural antimicrobials have been selected by evolutionary forces, it seems relatively unlikely that resistant bacterial strains would arise. This is supported by a study in which incubation of Helicobacter pylori cells of three different strains overnight with two times the minimum inhibitory concentration of lauric acid resulted in no resistant colonies (12). This raises the possibility that these lipids could be incorporated into topical formulations for prophylaxis in individuals at risk of infection as well as to treat skin infections, including those caused by antibiotic resistant organisms.
The antimicrobial activities of free fatty acids and their more active monoglyceride form until recently have not been investigated systematically against MRSA.
Extraordinarily (sapienic acid, C161,D6) in combination with a low concentration of ethanol exerts a synergistic bactericidal activity against several methacillin-resistant strains of S. aureus (MRSA) and even gram-negative pathogenic bacteria, including Pseudomonas aeruginosa, Propionibacterium acnes, Escherichia coli, In fact, this combination was far more effective than Mupirocin with or without ethanol. Mupirocin is the “gold standard” antibiotic for antimicrobial activity against MRSA.
Similar results were obtained with methicillin antibiotic-resistant clinical isolates of S. aureus (MRSA) strains Figure 1
Figure 1 illustrates that the relatively rapid killing of a methacillin-resistant strain of Staphylococcus aureus (MRSA) by sapienic acid in combination with ethanol.
Synergy of the antimicrobial activity between sapienic acid and ethanol was expected. Since fatty acids must partition into the cell, ethanol increase the cell membrane fluidity, or alternatively this could enhance alter partitioning of fatty acid into the membrane. Ethanol is a known permeability enhancer. It could also facilitate diffusion of the fatty acid into the cytoplasm of bacteria.
This finding could have important implications for dealing with antibiotic resistance. In addition to ethanol, there are a number of compounds known to increase fluidity of membranes. This permits rapid diffusion of compounds thru cell membranes. Known collectively as penetration enhancers, this group includes some fatty acids (C12, C18), monolaurin, terpenes, and azones, among others. Interesting sapienic acid (16:1,D6) has not been identified in any other human tissues or in the sebaceous gland secretions of other animals (13)
In early studies (circa 1966) on structure-function of lipids on microorganisms, Kabara et al noted that some 26 different clinical isolates of Staphylococcus aureus had similar MIC values to active fatty acids and monolaurin (Lauricidin®). The problem of resistance to antibiotics was known but did not seem to be a pressing problem since new antibiotics were constantly being discovered Recent studies on MRSA have indicated however that newer antibiotics were not the long-term solution. At this juncture the role of antimicrobial lipids needed to be explored because of recent findings Table 1 .(14)
TABLE 1 MIC (mg/ml) of Biocides against 6 Staphylococcus aureus
Antimicrobial MSSA MRSA
agents ATCC
29213 4952 6849 3818 352 5914
____________________________________________________________
Oxacillin < 0.5 16 >16 16 >16 >16
Ampicillin 2 16 >16 >16 >16 >16
Cefpirone <0.5 4 4 4 >16 >16
Vancomicin 1 2 1 1 1 2
Caprylic acid (C8)>1600 >1600 >1600 >1600 >1600 >1600
Capric acid (10) 800 800 800 800 800 800
Lauric acid (12) 400 400 400 400 400 400
Myristic acid (14) 1600 >1600 800 1600 >1600 >1600
Palmitic acid (16) >1600 >1600 >1600 >1600 >1600 >1600
Stearic acid (18) >1600 >1600 >1600 >1600 >1600 >1600
MSSA= Methicillin-Susceptible Staphylococcus aureus
MRSA= Methicillin-Resistant Staphylococcus aureusDepending on the antibiotic the MIC values against any particular organism changes with their individual susceptibility. Contrary to this are the MIC values for the various fatty acids that have the same values for MSSA and MRSA Lauric acid, as an example, has the same low MIC value for the susceptible strain MSSA as it does for the resistant MRSA strain. From other studies, it is known that antimicrobial lipids, in contrast to other antibiotics, do not form resistant organisms.
What is true for lauric acid has also been shown to be true for its monoester, monolaurin (LauricidinÆ) as seen in table 2 (15) Sensitivity rates of Gram-positive Staphylococcus aureus, Streptococcus spp., and coagulase-negative Staphylococcus. to 20 mg/ml LauricidinÆ was 100% and of Klebsiella rhinoscleromatis was 92.31%.. Staphylococcus aureus, coagulase-negative Staphylococcus, and Streptococcus sp. did not exhibit any resistance to monolaurin and had statistically significant (P .05) differences in resistance rates to the antibiotics.
Table 2 Comparison of Resistance Rates (%) of skin bacterial isolates to LauricidinÆ and six antibiotics
--------------------------------------------------------------
Antibiotic Staph. Coagulase(-) Streptococcuss sp. Klebsiellasp.
aureus Staphylococcus
--------------------------------------------------------------
LauricidinÆ 0 0 0 7.69
Penicillin 92.75 85.00 37.50 100
Oxacillin 36.23 60.00 6.25 100
Erythromycin 2.89 15.00 0 92.32
Mupirocin 0 10.00 0 92.30
Fusidic Acid 15.94 55.00 81.25 100
Vancomycin 5.79 15.00 12.50 92.30The problems of antibiotic resistance become more acute when MRSA organisms that were susceptible to vancomycin at one time are now becoming resistant to vancomycin. (Methicillin-resistant Staphylococcus aureus (MRSA) strains with reduced susceptibility to vancomycin (so called VISA strains) have been detected among clinical isolates in several countries (17)
Ploy et al raised serious concern about the impact of such a resistance mechanism on the chemotherapy of multidrug-resistant staphylococci. MRSA isolates with gradually increasing vancomycin MICs (18).
Fortunately, an ester of lauric acid (monolaurin, glycerol monolaurate (GML, Lauricidin®) has been found to inhibit the induction of beta -lactamase in Staphylococcus aureus. In addition LauricidinÆ suppresses growth of vancomycin-resistant Enterococcus faecalis on plates with vancomycin and blocks the induction of vancomycin resistance, which involves a membrane-associated signal transduction mechanism, either at or before initiation of transcription (19,20). Monolaurin at sub inhibitory concentrations prevents the synthesis of staphylococcal exoproteins and does so at the level of transcription (21) Since monolaurin is likely to act at the cell membrane and therefore inhibit either of the two membrane-related processes involved in exoprotein production, namely signal transduction and secretion. It has been shown previously that monolaurin blocks the induction but not the secretion of beta -lactamase or the constitutive synthesis of the enzyme, suggesting that signal transduction is the primary target .
IN VIVO EFFECTS OF LAURIC ACID ESTERS
Nasal carriage of Methicillin-resistant Staphylococcus aureus (MRSA) by hospitalized patients has been associated with nosocomial transmission of MRSA (22).
Mupirocin resistance in S. aureus was first reported in 1987, 2 years after Mupirocin was introduced into clinical practice (3). Mupirocin resistance in staphylococci has been classified as low-level (MIC, 8 to 256 μg/ml) and high-level (MIC, >256 μg/ml) (13).. Because of emerging Mupirocin resistance in S. aureus, alternatives to Mupirocin are needed for S. aureus nasal decolonization. The antistaphylococcal activity and minimal toxicity of fatty acids make lauric acid formulations potential alternatives to Mupirocin for S. aureus nasal decolonization (23).
As an example Table 3, administration of two lauric acid monoesters (LAM) was associated with greater eradication of MRSA carriage (24/34 [71%] or 33/40 [83%]) of animals, respectively) than bland ointment (12/38 [32%]) . Lauric monoester administration resulted in greater MRSA carriage eradication than even the present standard Mupirocin (19/38 [50%]) in this animal model.
TABLE 3
Intranasal infection with Methicillin-resistant Staphylococcus aureus
Treatment animals animals No. (%)of animals
challenged decolonized colonized
Bland ointment 53 38 12 (32)
Mupirocin 56 38 19 (50)
128774-49D 60 39 18 (46)
128774-49E 50 34 24 (71)
128774-53A 60 40 33 (83)These in vivo experiments with lauric acid esters indicate activity against nasal MRSA comparable or greater than standard drug treatment with Mupirocin. The enormous advantage is that lipid antimicrobials as compared to drug antibiotics do not readily form resistant organisms (23).
The LAM formulations studied were lipophilic surfactant/emulsifiers. Their exact mechanism of action is unknown but likely involves effects on the bacterial cell envelope and/or induction of autolysin activity and inhibition of protein synthesis. For example, Bergsson et al. demonstrated that S. aureus is killed by fatty acids, and especially by monocaprin, through disintegration of the cell membrane, leaving the cell wall intact (24).
Ved et al. showed that the ester form of monolaurin ,dodecylglycerol, inhibits peptidoglycan synthesis and stimulates a proteinase that activates peptidoglycan-degrading enzyme autolysin (25,26).
Several investigators have reported effects on toxin synthesis. For example, Schlievert et al. demonstrated that S. aureus elaboration of hemolysin, toxic shock syndrome toxin 1, and exfoliative toxin A was inhibited at glycerol monolaurate concentrations below those necessary to inhibit growth (21). Mechanistic studies performed by Projan et al. showed that glycerol monolaurate inhibits synthesis of staphylococcal toxins (and other exoproteins) at the level of transcription by interfering with signal transduction. Interference with signal transduction has also been shown in other genera;(20). Rusin and Novick demonstrated that glycerol monolaurate suppresses growth of vancomycin-resistant Enterococcus faecalis (19) in the presence of vancomycin and blocks the induction of vancomycin resistance, which involves a membrane-associated signal transduction mechanism, either at or before initiation of transcription (27).
IN VIVO EFFECTS OF MONOLAURIN (LAURICIDIN®)
In two separate experiments, groups of mice (6 and 8 respectively) were infected with Staphylococcus aureus (28) In the first in vivo experiment the daily dose of LauricidinÆ was either 2.0 ul (1.6 mg) or 4.0 ul (3.2 mg). In the second in vivo study, the daily dose of LauricidinÆ was given at the higher dose (3.2 mg).
Animals were gavaged daily with 0.2 ml of olive oil for 30 days. Control mice also received daily gavages of either olive oil alone (negative control) or olive oil orally plus vancomycin (400 mcg) i.p. (positive control). Experiments were terminated at the end of 30 days.
In examining the comparative effects of various antibiotics at the selected concentrations, penicillin had essentially no effect, and streptomycin did not completely kill the bacteria. However, vancomycin destroyed the bacteria.
Groups of 6 mice infected with Staphylococcus aureus (ATCC#14775) (5 X LD50) were treated i.p. with vancomycin (400 ug) daily for 30 days.
Fifty percent of those receiving vancomycin (400 ug) survived for 30 days, while all in the Control group died within a three-day period. Although prolonged survival was noted in mice, in all 30-days survivors, no internal abscesses were noted at post mortem, and renal cultures showed no bacterial growth. In contrast, numerous abscesses were noted and renal cultures were positive in all dying mice.
Groups of 8 mice were infected with five times the LD50 of Staphylococcus aureus (ATTC #14775). Fifty percent of the mice (4/8) injected daily with vancomycin (400 ug i.p.) survived for the thirty days of study in contrast to the control group where all eight mice died within a week’s time. In the LauricidinÆ group (3.2 mg) 5/8 survived. no abscesses were seen on inspection, and no bacterial growth was found in the kidney tissue of the 30-day survivors.
While Lauricidin® was known to kill Staphylococcus aureus effectively in vitro (6-9 ), in these experiments, it was found that Lauricidin® was also protective in vivo . In the 14 animals used as control, Staphylococcus aureus (ATCC #14775) killed all the mice within 7 days. In contrast, 50% of the mice survived for thirty day after receiving daily gavages of vancomycin (7/14) or Lauricidin® (4/8).
Conclusions
The current way that antibiotics are used has limitations. A different approach to the problem includes natural non-toxic lipids. Of the lipids examined pure monolaurin (Lauricidin® ) has properties that best fit a wide spectrum antimicrobial agent. Lauricidin® has been designated by the FDA (Federal Drug Administration (FDA) to be non-toxic.
First discovered by Kabara, et all (6) Lauricidin®, a saturated monoglyceride, is now known to affect microorganisms in several ways. That several mechanisms are involved may make it more difficult for the affected organisms to develop resistance.
Lauricidin®, like any fatty acid ester, is a lipophilic compound and hence its inhibitory activity is probably through interactions with the cytoplasmic membrane. The mechanism of antibacterial action of fatty acids and their derivatives is while not clearly defined, seems to involve disruption of the cell membrane permeability barrier and inhibition of amino acid uptake (29).
Lee and Shafer (30) studied the resistance of gonococci to long-chain fatty acids and discovered that resistance was mediated by an efflux pump encoded by farAB (fatty acid resistance).
The first effect seen after the use of monoglycerides against microorganisms is destabilization of the bacterial/virus membrane which allow constituents to “leak” out.( ) Biocides that have this mechanism for inactivation do not readily form resistant organisms.
An effect noted in viral inactivation is another mechanism. This involves a fatty acid interfering with viral assembly. In other words viral constituents in the cell are made but these cannot form an infective particle (32). The most active inhibitor was lauric acid (C12), which reduced virus yields of several attenuated and pathogenic strains of JUNV in a dose dependent manner, without affecting cell viability. Fatty acids with shorter or longer chain length had a reduced or negligible anti-JUNV activity. From mechanistic studies, it was concluded that lauric acid inhibited a late maturation stage in the replicative cycle of JUNV. Viral protein synthesis was not affected by the compound, but the expression of glycoproteins in the plasma membrane was diminished.
Lauricidin® has been found to inhibit the post-exponential phase activation of virulence factor production and the induction of beta-lactamase in Staphylococcus aureus. It has been suggested that signal transduction is the most probable target for GML (4).
It was found that Lauricidin® suppresses growth of vancomycin-resistant Enterococcus faecalis on plates with vancomycin and blocks the induction of vancomycin resistance, which involves a membrane-associated signal transduction mechanism, either at or before initiation of transcription. It is suggested that Lauricidin® blocks signal transduction. In contrast, GML has no effect on the induction of erythromycin-inducible macrolide resistance in S. aureus, which does not involve signal transduction. These results are consistent with the possibility that monolaurin acts at the level of signal transduction (31).
Monolaurin, a food grade glycerol monoester of lauric acid, has been reported to have the greatest antimicrobial activity of all of the monoglycerides (50). Monolaurin, like any fatty acid ester, is a lipophilic compound and hence its inhibitory activity is probably through interactions with the cytoplasmic membrane. Although the mechanism of antibacterial action of fatty acids and their derivatives is not defined, it has been suggested to involve disruption of the cell membrane permeability barrier and inhibition of amino acid uptake (6-9), Glycerol monolaurate has been shown to inhibit the production of exoenzymes and virulence factors in Staphylococcus aureus (41), to block the induction of vancomycin resistance in Enterococcus faecalis (46), and to modulate T-cell proliferation (62), all of which involve membrane-bound signal transduction systems. Dodecylglycerol (corresponding ether of monolaurin) has been shown to activate the proteolytic enzyme responsible for the activation of autolysin in the cell wall of E. faecium (40, 56, 57) and to inhibit glycerolipid and lipoteichoic acid biosynthesis in Streptococcus mutans (6).
Lauricidin®is effective in blocking or delaying the production of exotoxins by pathogenic gram-positive bacteria (31) and inhibits the synthesis of most staphylococcal infections and other exoproteins--it does so at the level of transcription. (32) Monolaurin also inhibits the expression of virulence factors in Staphylococcus aureus and the induction of vancomycin resistance in Enterococcus faecalis. (33,34)
As a simple food supplement, Lauricidin® could be given before or in conjunction with antibiotic therapy. This would help reduce the number of bacteria becoming resistant to drugs as well as overcoming those bacteria like MRSA that are already resistant. In view of all these positive effects LauricidinÆ should be more widely used for clinical problems involving microorganisms either alone or in conjunction with classical antibiotics.
References
1. Podolsky,ML: in Cures out of Chaos. Hardwood Academic Press, 1997, pp 179-194.
2. . Tenover FC, Lancaster MV, Hill BC, Steward CD, Stocker SA, Hancock GA, O'Hara CM, McAllister SK, Clark NC, Hiramatsu K. Characterization of staphylococci with reduced susceptibilities to vancomycin and other glycopeptides. J Clin Microbiol 1998; 36::1020-1027.
3. Weigel LM, Clewell DB, Gill SR, Clark NC, McDougal LK, Flannagan SE, Kolonay JF, Shetty J, Killgore GE, Tenover FC. Genetic analysis of a high-level vancomycin-resistant isolate of Staphylococcus aureus. Science 2003; 302:1569-1571.
4. Murray,BE. Can Antibiotics Resistance be Controlled. New Eng. J Med. 1994; 330:1229-1230.
5. Chambers HF. The Changing Epidemiology of Staphylococcus aureus. Emerg Infect Dis. 2001; 7:178-182.
6. Kabara, J. J., Swieczkowski D.M,. Conley, A. J and Truant, J. P.: Fatty acids and derivatives as antimicrobial agents. Antimicrob. Agents Chemother. 1972; 2:23-28.
7. Conley, A. J., and. Kabara J. J. Antimicrobial action of esters of polyhydric alcohols. Antimicrob. Agents Chemother. 1973; 4:501-506.
8. Kabara, J. J. Lipids as host-resistance factors of human milk. Nutr Rev. 1980; 38:65-73.
9. Burtenshaw, J.M.: The mechanisms disinfection of the human skin and its appendages. J Hyg, 1942; 42:184-209.
10. Willea,J, A. Kydonieus: Palmitoleic Acid Isomer (C16-1,D6) in Human Skin Sebum Is Effective Against Gram-Positive Bacteria, Skin Pharmacology and Applied Skin Physiology. 2003; 16:176-187
11. Drake,DR, Brogden,KA, Dawson, DV and Wertz,PW: Skin Lipids. Antimicrobial lipids at the skin surface. Journal of Lipids Research, 2008; 49: 4-11
12. Petschow,BW, Batema,RP,and Ford,LL. Susceptibility of Helicobacter pylori to bactericidal properties of medium-chain monoglycerides and free fatty acids. Antimicrob. Agents & Chemother. 1996; 40:302-306.
13. Nicolaides, N. Lipids: their biochemical uniqueness. Science 1974; 186: 19–26.
14. Kitahara T, Koyama N, Matsuda J, Aoyama,Y,Hirata Y, et al. Antimicrobial Activity of Saturated Fatty Acis and Fatty Amines against Methicillin-Resistant Staphylococcus aureus. Biol. Pharm. Bull. 2004; 27: 1321—1326.
15. Carpo BG, Verallo-Rowell VM, Kabara J. Novel antibacterial activity of monolaurin compared with conventional antibiotics against organisms from skin infections: an in vitro study. J Drugs Dermatol. 2007; Oct;6 (10):991-8.
16. Hiramatsu, K., Hanaki H, Ino,T. Yabuta,K. Oguri,T. and Tenover. F. C. Methicillin-resistant Staphylococcus aureus clinical strain with reduced vancomycin susceptibility. J. Antimicrob. Chemother. 1997;40:135-136.
17. Ploy MC, Grelaud C, Martin C, Lumley,L de and Denis,F: First clinical isolate of vancomycin-intermediate Staphylococcus aureus in a French hospital [letter]. Lancet 1998;351: 1212.
18. Ruzin,A and Novick,R.P.: Glycerol Monolaurate Inhibits Induction of Vancomycin Resistance in Enterococcus faecalis. J Bacteriol, 1998; 180: 182-185.
19. Projan, S. J., Brown-Skrobot,S. Schlievert,P.M. Vandenesch, F and. Novick. R. P. Glycerol monolaurate inhibits the production of beta -lactamase, toxic shock syndrome toxin-1, and other staphylococcal exoproteins by interfering with signal transduction. J. Bacteriol. 1994;176: 4204-4209 .
20. Schlievert, P. M., J. R. Deringer ,J.R. Kim M. H., Projan, S. J. and Novick. R. P.. Effect of glycerol monolaurate on bacterial growth and toxin function. Antimicrob. Agents Chemother. 1992; 36: 626-631
21. Boyce, J. M., MRSA patients: proven methods to treat colonization and infection. J. Hosp. Infect. 2001; 48 (Suppl. A): S9-S14.
22. Rouse MS. Rotger,M, Piper,KE, Steckelberg,MJ. Scholz,M. Andrews J. and Robin Patell In Vitro and In Vivo Evaluations of the Activities of Lauric Acid Monoester Formulations against Staphylococcus aureus Antimicrob Agents Chemother. 2005 August; 49(8): 3187–3191
23. Bergsson, G., J. Arnfinnsson, O. Steingrimsson, and H. Thormar. Killing of gram-positive cocci by fatty acids and monoglycerides. APMIS. 2001; 109: 670-678.
24. Ved, H. S. Gustow E, Mahadevan V, and Pieringer, R. A. Dodecylglycerol. A new type of antibacterial agent which stimulates autolysin activity in Streptococcus faecium ATCC 9790. J. Biol. Chem. 1984; 259: 8115-8121
25. Ved, H. S., Gustow E, and Pieringer, R. A. Inhibition of peptidoglycan synthesis of Streptococcus faecium ATCC 9790 and Streptococcus mutans BHT by the antibacterial agent dodecyl glycerol. Biosci. Rep. 1984; 4:659-664.
26. Holland, K. T., Taylor D, and Farrell A. M.. The effect of glycerol monolaurate on growth of, and production of toxic shock syndrome toxin-1 and lipase by, Staphylococcus aureus. J. Antimicrob. Chemother. 1994; 33:41-55
27. Preuss, H. Echard,G, Dadgar,B. Talpur,A. Manohar N., Enig V, Bagchi M, Ingram D., C. Effects of Essential Oils and Monolaurin on Staphylococcus aureus: In Vitro and In Vivo Studies Toxicology Mechanisms and Methods 2005;15: pages 279-286
28. Kato, N., and Shibasaki,I.. Combined effect of citric and polyphosphoric acid on the antibacterial activity of monoglycerides. J. Antibact. Antifung. Agents 1976; 4:254-261.
29. Lee, E.H., and W.M. Shafer. The farAB-encoded efflux pump mediates resistance of gonococci to long-chained antibacterial fatty acids. Mol. Microbiol. 1999;33:839-845.).
30. Bartolotta S, GarcÌa CC, Candurra NA, Damonte EB. Effect of fatty acids on arenavirus replication: inhibition of virus production by lauric acid. Arch Virol. 2001;146(4):777-90
