Undecanoic Acid, Lauric Acid, and N-Tridecanoic Acid Inhibit Escherichia coli Persistence and Biofilm Formation
Department of Chemical and Biological Engineering, Illinois Institute of Technology, Chicago, IL 60616, USACorrespondence to:
J. Microbiol. Biotechnol. 2021; 31(1): 130-136
Published January 28, 2021
Copyright © The Korean Society for Microbiology and Biotechnology.
Persister cells are tolerant to conventional antibiotic treatment because they are metabolically dormant or grow slowly without acquiring inherent antibiotic resistance via genetic modifications , whereas resistant cells undergo genetic changes that block antibiotic activity . Persister pathogens become active after the level of antibiotics decreases and thus cause chronic recalcitrant infections . Persister cells are also highly tolerant to environmental stresses, such as low pH , nutrient starvation , hyperosmolarity , or heat shock . Because of their high tolerance, persister cells are enriched in biofilms , sessile multi-microbial communities that are formed in response to environmental stresses  through secreted self-synthesized polymeric matrices . Many bacteria, including pathogens, can form biofilms, but inhibiting biofilms is challenging because of poor antibiotic penetration, biofilm-specific gene regulation, and persister cell formation . Hence, identifying new antimicrobial agents other than antibiotics is critical to decrease persister cell formation.
Fatty acid molecules, composed of a chain of carbon atoms with attached hydrogen atoms, are widely used in therapeutics, food preservation, and agriculture . Some fatty acids inhibit or kill bacteria directly , whereas others affect virulence factors or prevent bacterial adhesion . Fatty acids are produced in natural sources, such as plants that are inexpensive and non-toxic . The antimicrobial activities of fatty acids depend on the structure, chain length, and degree of saturation . As a result of these properties, fatty acids are considered as an alternative to conventional antibiotics for the treatment of infectious diseases. Furthermore, fatty acids exhibit antibiofilm properties in relation to antivirulence . However, the roles of fatty acid molecules in altering persister cells have not been extensively studied.
In this study, we screened 65 fatty acids with chain lengths of 10 to 24 carbons, including saturated and unsaturated forms, to determine whether combined antibiotic and fatty acid treatment might decrease persister cell formation. We identified antipersister and/or antibiofilm fatty acid molecules with the potential to be used with antibiotics to treat bacterial infections efficiently.
Materials and Methods
Bacterial Strains and Fatty Acids
Persister Cell Formation Assays
Persister assays were conducted according to previously reported procedures . Briefly, overnight
Biofilm Formation Assays
Biofilm formation assays were conducted according to a previously described method  with modifications. Briefly, overnight cultures were adjusted to an OD600nm of 0.01 (approximately 5 × 106 CFU/ml) with LB medium, and then 1.5 ml of adjusted culture was added to a polypropylene culture tube (Falcon PN352006, Corning, USA) with or without fatty acids. After incubation at 37°C for 24 h without shaking, the planktonic cultures were discarded, and the culture tubes containing biofilm were rinsed three times with 3 ml of 0.85% NaCl solution. Biofilm cells were carefully collected with a cotton swab  and resuspended in 3 ml of 0.85% NaCl solution via vortexing for 30 s. Biofilm cells were diluted serially (101–106 dilution), and 10 μl was applied on LB agar plates, which were incubated at 37°C. Biofilm cells were quantified as colony forming units (CFU) per surface area (cm2).
Overnight cultures were adjusted to OD600nm 0.05 with fresh LB medium, and then 200 μl cell suspensions were added to a 96-well plate. After the addition of different concentrations of fatty acids, cell growth was measured at OD600nm every 20 min at 37°C on a Synergy HTX plate reader in fast shaking mode (Biotek, USA). Each data point was obtained from three replicate wells with two independent cultures.
Statistical analysis was performed with two-tailed Student’s
Results and Discussion
Identification of Undecanoic Acid, Lauric Acid, and N-Tridecanoic Acid as Inhibitors of
E. coli BW25113 Persister Cell Formation
To explore whether fatty acids can inhibit persister cell formation, we examined the ability of 65 fatty acids to alter persister cell formation. These fatty acids ranged in length from 10 to 24 carbons and were saturated or unsaturated. First, we treated exponential phase
Fatty acid induced alterations in( E. colipersister formation. A) Exponentially grown E. coliBW25113 cells were exposed to 5 μg/ml of ciprofloxacin (Cipro) and 1 mM of nine fatty acids for 6 h in LB medium. ( B) Exponentially grown BW25113 and ( C) EDL933 (Enterohemorrhagic E. coli) cells were exposed to 5 μg/ml of ciprofloxacin and 1 mM of undecanoic acid, lauric acid, and N-tridecanoic acid for 3, 6, 24, 48, and 72 h in LB medium. Error bars indicate the standard deviation of two independent cultures with three replicates. *Indicates a significant difference relative to ciprofloxacin treatment only, with a p-value < 0.001.
Interestingly, the three fatty acids obtained from the persister inhibition screening were all saturated fatty acids of medium-chain length (undecanoic acid, 11 carbon atoms (C11); lauric acid, C12; and N-tridecanoic acid, C13)(Table 1), whereas a collection of various-length (C10 to C24) fatty acids of saturated and unsaturated states were studied (Table S1).
Undecanoic Acid, Lauric Acid, and N-Tridecanoic Acid Inhibit Enterohemorrhagic
E. coli (EHEC) Persister Formation
To investigate the effects of these fatty acids on other
Typically, antibiotics repress bacterial growth through different inhibition mechanisms. For example, ciprofloxacin (fluoroquinolone) inhibits DNA replication and repair, ampicillin (β-lactam) blocks cell-wall synthesis, and kanamycin (aminoglycosides) represses protein synthesis . Therefore, we investigated the effects of the medium-chain saturated fatty acids during EHEC persister formation in the presence of different classes of antibiotics in addition to ciprofloxacin. Specifically, we examined EHEC persister formation with 100 μg/ml of ampicillin (Fig. 2A) or kanamycin (Fig. 2B) in the presence of 1 mM of each fatty acid for 6 and 48 h. Similarly to the results with ciprofloxacin and each fatty acid cotreatment (Fig. 1B), the addition of undecanoic acid, lauric acid, and N-tridecanoic acid repressed persister cell formation caused by ampicillin treatment (6-, 14-, and 10-fold lower, respectively, than that after ampicillin-only treatment after 6 h; Fig. 2A). However, we did not observe a noticeable difference during kanamycin treatment with or without fatty acids (Fig. 2B). Cell survival after 48 h of kanamycin exposure (10 CFU/ml) was considered as negligible as it was close to the complete cell killing with the cotreatment (Fig. 2B). These results suggest that the fatty acid molecules that we tested behave differently when persister cells are formed through various bacterial inactivation mechanisms in the presence of different antibiotics.
EHEC persister cell formation in the presence of different antibiotics.EHEC persister formation with 100 μg/ml of ( A) ampicillin (Amp) and ( B) kanamycin (Kan) together with 1 mM of fatty acids for 6 and 48 h in LB. Error bars indicate the standard deviation of two independent cultures with three replicates. *Indicates a significant difference relative to Cipro treatment only, with a p-value < 0.001.
Persister cell formation is known to be an antibiotic-specific response rather than a global metabolic dormancy . The SOS response after DNA damage by ciprofloxacin induces toxin-antitoxin (TA) transcripts in
We determined that fatty acids did not repress persister formation induced by kanamycin (Fig. 2B). Kanamycin binds the 30S ribosomal subunit and inhibits protein synthesis ; as a result, ribosome dimerization induced by the presence of aminoglycosides, including kanamycin, results in the ribosome hibernation and thereby increases bacterial tolerance . Recently, the alarmone guanosine pentaphosphate/tetraphosphate (ppGpp) ribosome dimerization persister model has been proposed to understand how cells enter and exit the persister state [46, 48]. Persister cells are directly formed by inactivating ribosomes via ppGpp with the evidence that persister cells contain a large fraction of 100S ribosomes . Therefore, it is possible that the fatty acids might interact with ribosomes to interrupt the pathway of persistence formation, whereas kanamycin-induced persister cells containing inactivated ribosomes might not be affected by the fatty acids.
Persister Formation in Stationary Phase Cells Is Not Affected by Fatty Acids
To investigate the effect of fatty acids on stationary phase persister cell formation, we directly harvested the overnight culture (18 h after inoculation) and adjusted its optical density to 1.0 with fresh LB media in the presence of 1 mM of each fatty acid and 5 μg/ml of ciprofloxacin. The population of persister cells in the stationary phase with ciprofloxacin-only treatment was increased by more than 10-fold in comparison to that in the exponential phase. However, unlike the exponential phase of persister cell formation, the effect of fatty acid inhibition on persister cell formation was diminished in the stationary phase cells of BW25113 and EHEC (Fig. S2). Stationary phase cells have more robust membranes with a rigid cell envelope, a highly cross-linked cell wall, and a reduced membrane fluidity in comparison to exponential phase cells, which exhibit a high membrane fluidity . Bacteria can incorporate free exogenous fatty acids from its environment to their cellular membrane , such that the incorporation of fatty acids into the membrane may increase membrane fluidity and thereby enhance antimicrobial susceptibility [11, 28]. Our results that fatty acids are effective toward decreasing the persister formation of exponential phase cells but are less effective in regard to stationary phase cells, implying that undecanoic acid, lauric acid, and N-tridecanoic acid might increase membrane fluidity and improve the ciprofloxacin efficacy for inhibiting persister formation. It remains necessary to elucidate the relation between the cell membrane fluidity, persister formation, and the effect of fatty acids on membrane fluidity.
Undecanoic Acid, Lauric Acid, and N-Tridecanoic Acid Do Not Exhibit Antimicrobial Activity against EHEC Cells
Some fatty acids, such as linolenic acid, myristic acid, and lauric acid, have antimicrobial activity against microorganisms such as
EHEC growth with undecanoic acid, lauric acid, and N-tridecanoic acid.The growth of EHEC culture was monitored until 18 h in the presence of 1 mM of each fatty acid at 37°C in LB. Cell growth at OD600nm was measured every 20 min. Error bars indicate standard deviations of two independent cultures with three replicates.
Fatty Acids Repress EHEC Biofilm Formation
High persister populations in biofilms enhance the tolerance of biofilm cells [8, 39]. Because the examined fatty acid molecules inhibited persister cell formation, we reasoned that these fatty acids might be applied for repressing biofilm formation. We exposed EHEC cells to undecanoic acid, lauric acid, and N-tridecanoic acid during 24 h of biofilm formation. As expected, all fatty acids tested repressed EHEC biofilms, and lauric acid exhibited the highest efficacy (8-fold; Fig. 4). Lauric acid inhibits
EHEC biofilm formation.EHEC biofilms were formed in the presence of 1 mM of undecanoic acid, lauric acid, and N-tridecanoic acid for 24 h at 37°C without shaking. Error bars indicate standard deviations of two independent cultures with three replicates. *Indicates a significant difference relative to the DMSO control, with a p-value < 0.001.
This study demonstrates that the three fatty acid molecules undecanoic acid, lauric acid, and N-tridecanoic acid effectively inhibit
Supplementary data for this paper are available on-line only at http://jmb.or.kr.
This study was supported by Illinois Institute of Technology, the Research Experiences for Undergraduates (REU) program of the National Science Foundation (1757989), and the National Institute of Allergy and Infectious Diseases of the National Institutes of Health (R15AI130988).
Conflict of Interest
The authors have no financial conflicts of interest to declare.
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