1. Introduction
Species of members of the genus
Staphylococcus are divided into two major groups based upon their ability to coagulate plasma. By far the best-known coagulase-positive species is
Staphylococcus aureus, which is a virulent pathogen that is often resistant to multiple antibiotics, including β-lactam antibiotics, often through presence of the
mecA determinant. In contrast the coagulase-negative staphylococci (CONS) are a large heterogeneous group of fifty-three validly described species, as of 2019 [
1], from animal host-associated to environmental species varying in pathogenic potential from considerable down to non-pathogenic. Antibiotic resistance rates continue to increase in CONS [
1,
2].
Novel antimicrobial agents are needed for the therapy of
S. aureus infections given resistance to multiple anti-staphylococcal agents exhibited by the organism [
3]. The
S. aureus cytoplasmic membrane plays an important role in the susceptibility and resistance to various antimicrobial agents of the bacterium. The cytoplasmic membrane of
S. aureus is composed of the phospholipids phosphatidyl glycerol, lysyl-phosphatidyl glycerol and cardiolipin, and the glycolipids monoglucosyldiglyceride and diglucosyldiglyceride [
4,
5]. Decrease in phosphatidyl glycerol, and increases in lysyl-phosphatidyl glycerol and cardiolipin contents have been associated with decreased susceptibility to the membrane active antimicrobial daptomycin [
6]. The fatty acids esterified to the glycerol moiety of these glycerolipids are a mixture of branched-chain fatty acids (BCFAs) and straight-chain fatty acids (SCFAs) biosynthesized by the organism when grown in conventional laboratory media [
7,
8]. There are two types of BCFA in
S. aureus, namely iso fatty acids where the methyl branch is on the penultimate carbon of the fatty acid chain, and anteiso fatty acids where the methyl branch is on the antepenultimate carbon. Even-numbered iso fatty acids, odd-numbered iso fatty acids, and odd-numbered anteiso fatty acids are biosynthesized from the branched-chain amino acids valine, leucine, and isoleucine, respectively [
9]. BCFAs increase membrane fluidity, whereas SCFAs decrease it [
10]. Anteiso fatty acids increase membrane fluidity more than iso fatty acids because they pack into the membrane more loosely [
10]. The growth medium has significant effects on the balance of BCFAs and SCFAs, with Mueller–Hinton broth (MHB) resulting in a much higher proportion of BCFAs than a medium such as tryptic soy broth (TSB) [
7]. Interestingly, MHB is the recommended medium for routine determination of antibiotic susceptibilities in the clinical microbiology laboratory [
11]. The other major
S. aureus membrane lipid component is the golden yellow carotenoid known as staphyloxanthin [
12]. Staphyloxanthin is generally thought to decrease membrane fluidity and impacts
S. aureus susceptibility to various antimicrobial peptides and fatty acids [
13,
14]. For the most part, this pigment is absent from coagulase-negative species. Staphyloxanthin production varies with different culture conditions and different
S. aureus strains [
15]. In a carotenoid-deficient mutant, growth in MHB resulted in a more fluid membrane [
15].
In laboratory media, the
S. aureus BCFAs and SCFAs are biosynthesized by the fatty acid biosynthesis system II (FASII) [
16]. This pathway is unique to bacteria and considerable effort has been devoted to developing novel antimicrobials that target this pathway [
16]. However, the typical fatty acid composition of
S. aureus grown in laboratory media, characterized by a mixture of BCFAs and SCFAs, is unlikely to mimic that of the organism growing in vivo in an infection.
S. aureus has the ability, like many pathogenic bacteria, to utilize preformed host fatty acids, typically straight-chain unsaturated fatty acids (SCUFAs) and SCFAs, in what is thought to be a means of saving energy and carbon that would otherwise be devoted to fatty acid biosynthesis [
17,
18]. Thus, when
S. aureus is grown in the presence of complex host biological materials such as serum [
7], or low-density lipoprotein [
19], BCFAs decrease and SCFAs and SCUFAs increase. Fatty acids are incorporated into cellular polar lipids through the activities of the fatty acid kinase system, FakAB, and PlsX and PlsY [
20]. It has been proposed that there is a requirement for fatty acid anteiso C15:0 on the
sn-2 portion of
S. aureus glycerolipids, and hence a requirement for operation of the FASII system when
S. aureus is growing in the host. However, demonstration of phosphatidyl glycerol species with no biosynthesized fatty acids on either the
sn-1 or
sn-2 position of the glycerol moiety seriously undermines the viability of agents targeting the FASII system [
21,
22].
Although
S. aureus can incorporate free fatty acids into lipids from media supplemented with them [
20,
23], the bacterium has a complex relationship with the variety of SCUFAs it might encounter. Very closely related fatty acid structures can either be inhibitory to growth at low concentrations or can have little effect on growth at relatively high concentrations [
24,
25,
26]. For example, sapienic acid (C16:1Δ6) and palmitoleic acid (C16:1Δ9) are highly inhibitory, whereas oleic acid (C18:1Δ9) and vaccenic acid (C18:1Δ11) are not inhibitory and are actually incorporated into the phospholipids by the pathogen [
26]. Sapienic acid (C16:1Δ6) is a major antimicrobial fatty acid of human skin, whereas palmitoleic acid (C16:1Δ9) performs this function in other mammalian species [
27,
28]. Little information is available on how CONS respond to antimicrobial fatty acids to which they are constantly exposed given that they are prominent members of the skin bacterial flora.
The CONS are a heterogeneous group of organisms colonizing the skin and mucus membranes of human and other animal hosts. Based upon sequences of four loci, Lamers et al. [
29] placed species into 15 phylogenetic cluster groups [
29]. Different species may have characteristic body ecological niches [
30].
S. epidermidis is prevalent on moist areas and is the most prevalent staphylococcal species on skin. It was cultured from the nose of 97% of people in a recent study [
31].
S. haemolyticus and
S. hominis are associated with areas high in apocrine glands.
S. capitis is isolated from the forehead and scalp.
S. saprophyticus colonizes the genitourinary tract and rectum, and is a common cause of urinary tract infection in women [
32].
S. auricularis is uniquely found in the external ear canal [
30]. As a group, the CONS tend to cause infections in immunocompromised patients and those with indwelling biomedical devices given the propensity for these bacteria to form biofilms [
1,
30].
The well documented plasticity of
S. aureus membrane lipid composition clearly is an important consideration in antibiotic response and resistance, lack of susceptibility to FASII inhibitors, and susceptibility to antimicrobial skin SCUFAs. In contrast, there is little information on the impact of different media on the fatty acid composition and membrane fluidity of CONS. Fatty acids in the CONS are a mixture of BCFAs and SCFAs with differences in the proportions of individual fatty acids such as anteiso C15:0, anteiso C17:0, iso C15:0, C18:0 and C20:0 amongst individual species [
33,
34].
In this study, we investigate the effect of growth medium on the fatty acid composition and membrane fluidity of CONS. We also investigated the response of the different species to host-derived antimicrobial SCUFAs, sapienic acid, palmitoleic acid, and linoleic acid. As in S. aureus, growth in MHB increased the proportion of BCFAs and membrane fluidity in CONS. CONS lack staphyloxanthin, which removes a complication from the determination of membrane fluidity. The different species had the ability to incorporate SCFAs and SCUFAs from serum.
3. Materials and Methods
Staphylococcus auricularis ATCC 33757
T,
Staphylococcus capitis ATCC 27840
T,
Staphylococcus epidermidis ATCC 12228
T,
Staphylococcus haemolyticus ATCC 27836
T,
Staphylococcus hominis ATCC 27844
T,
Staphylococcus saprophyticus ATCC 15305
T, and
Staphylococcus aureus RN450 were grown in various culture media and analyzed for growth characteristics through the determination of culture turbidity [
7], fatty acid composition and membrane fluidity. The growth media used were BactoTM tryptic soy broth (TSB; Becton, Dickinson and Company, Sparks Glencoe, MD, USA), BactoTM Mueller–Hinton broth (MHB; Becton, Dickinson and Company, Sparks Glencoe, MD, USA) and 100% human serum (BioreclammationIVT, Westbury, NY, USA). MHB was supplemented with 25 mg/liter Ca
2+ and 12.5 mg/liter Mg
2+. Human serum was heated to 56 °C for 30 min to inactivate complement before using. Staphylococci were grown in 50 mL of medium in a 250 mL Erlenmeyer flask with shaking at 200 rpm at 37 °C unless otherwise specified.
The different species were grown to an optical density (OD
600) of about 0.8 in TSB, MHB and 100% human serum, harvested by centrifugation at 2000×
g for 5 min, followed by washing two times with cold 1x phosphate buffer saline (PBS; 8 g/L NaCl, 0.2 g/L KCl, 1.44 g/L Na
2HPO
4 and 0.24 g/L KH
2PO
4, pH 7.4). The samples were sent for fatty acid methyl ester analysis by gas-liquid chromatography at Microbial ID, Inc. (Newark, DE, USA) and identified by the MIDI microbial identification system (Sherlock 4.5 microbial identification system, Microbial ID Inc., Newark, DE, USA), as described previously [
7]. The value of the percent composition of an individual fatty acid typically has a reproducibility of ±0.02 to ±1.44 standard error of the mean (SEM) in extensive experience in our laboratory [
7,
15].
The different strains were grown as described for the fatty acid analysis and membrane fluidities of the cells were determined using DPH (Sigma-Aldrich, Cleveland, OH, USA) as described previously [
37]. Briefly, DPH stock solution (10 mM) was prepared in tetrahydrofuran (Sigma-Aldrich, Cleveland, OH, USA), vortexed for 10 min and working solution (10 µM) was prepared in PBS followed by vortexing for 10 min. The cell pellets were washed twice with cold 1x PBS (pH 7.4) and resuspended to an OD600 of ~0.75, DPH dye was added at 5 µM concentration, the tube was wrapped with aluminum foil and incubated at 30 °C in a water bath. All steps involving DPH were carried out in the dark. Fluorescence polarization emitted by the fluorophore was measured using a PTIModel Quanta Master-4 Scanning Spectrofluorometer at an excitation wavelength of 360 nm and an emission wavelength of 430 nm. The experiments were performed with three separate fresh batches of the cultures.
MICs of known antimicrobial fatty acids, viz., palmitoleic acid (C16:1Δ9, Larodan Fine Chemicals, Solna, Sweden), sapienic acid (C16:1Δ6, Larodan Fine Chemicals, Solna, Sweden), and linoleic acid (C18:2Δ9,12; Larodan Fine Chemicals, Solna, Sweden), dissolved in absolute ethanol, were determined against the staphylococcal species by the broth microdilution method as recommended by the Clinical Laboratory Standards Institute [
10] guidelines. Briefly, serial dilutions of each antimicrobial fatty acid were prepared in a microtiter plate with 90 µL of TSB containing defined concentrations of the fatty acids for 100 µL total volume. Each strain was grown to OD600 of 1.0 at 37 °C with shaking at 200 rpm, diluted 20 times in sterile TSB, and 10 µL of the cell suspension was inoculated in each well in triplicate on the microtiter plate. The plates were incubated at 37 °C for 16 h and the MIC was determined as the concentration of the fatty acid that inhibited growth of the given species. At least three independent microtiter plate assays were carried out for each species.