I. INTRODUCTION

Section:

ChooseTop of pageABSTRACTI. INTRODUCTION <<II. MATERIALS AND METHODO...III. RESULTS AND DISCUSSI...IV. SUMMARY AND CONCLUSIO...REFERENCESThe World Health Organization (WHO) has stated Pseudomonas aeruginosa (P. aeruginosa) as one of the 12 types of notorious and fatal bacteria for which novel antibiotics and treatments are needed to treat related infections.11. WHO, 2017, see https://www.who.int/news/item/27-02-2017-who-publishes-list-of-bacteria-for-which-new-antibiotics-are-urgently-needed. Many severe infections such as pneumonia, septicemia, meningitis, and endocarditis can be related to P. aeruginosa, and it can be fatal for patients who have a weakened immune system.2,32. G. P. Bodey, R. Bolivar, V. Fainstein, and L. Jadeja, Rev. Infect. Dis. 5, 279 (1983). https://doi.org/10.1093/clinids/5.2.2793. CDC, 2019, see https://www.cdc.gov/hai/organisms/pseudomonas.html. The Centers for Disease Control and Prevention (CDC) cautions that patients who use medical devices, such as ventilators and catheters, or have burn and surgical wounds are at an increased risk of P. aeruginosa infection.33. CDC, 2019, see https://www.cdc.gov/hai/organisms/pseudomonas.html. The high nosocomial incidence rate (10%–15%) and mortality rate (18%–61%) make P. aeruginosa infections a global problem.4,54. Q. Shi, C. Huang, T. Xiao, Z. Wu, and Y. Xiao, Antimicrob. Resist. Infect. Control 8, 68 (2019). https://doi.org/10.1186/s13756-019-0520-85. C.-I. Kang, S.-H. Kim, H.-B. Kim, S.-W. Park, Y.-J. Choe, M.-D. Oh, E.-C. Kim, and K.-W. Choe, Clin. Infect. Dis. 37, 745 (2003). https://doi.org/10.1086/377200 Furthermore, many P. aeruginosa related infections are difficult to treat and can be resistant to the currently available antibiotics.As a Gram-negative bacterium, the structure of P. aeruginosa consists of inner and outer membranes, and antimicrobials need to penetrate both membranes to kill the bacterium. The outer membrane exhibits a barrier function to hinder antimicrobials from entering and can decrease the uptake of some antimicrobials and make treatments less effective.66. Q. Li, R. Cebrián, M. Montalbán-López, H. Ren, W. Wu, and O. P. Kuipers, Commun. Biol. 4, 31 (2021). https://doi.org/10.1038/s42003-020-01511-1 For example, vancomycin can penetrate the inner membrane but cannot penetrate the outer membrane; therefore, it is not effective against P. aeruginosa.66. Q. Li, R. Cebrián, M. Montalbán-López, H. Ren, W. Wu, and O. P. Kuipers, Commun. Biol. 4, 31 (2021). https://doi.org/10.1038/s42003-020-01511-1 Exploiting outer membrane permeabilization can be an effective strategy to help develop treatments.The outer membrane contains phospholipids and lipopolysaccharides (LPS), which are in the inner and outer leaflets, respectively.77. J. Ude et al., Proc. Natl. Acad. Sci. U.S.A. 118, e2107644118 (2021). https://doi.org/10.1073/pnas.2107644118 LPS has dense phosphate groups that form a negative charged electrostatic network and cause low outer membrane permeability.88. J. Li, J.-J. Koh, S. Liu, R. Lakshminarayanan, C. S. Verma, and R. W. Beuerman, Front. Neurosci. 11, 73 (2017). https://doi.org/10.3389/fnins.2017.00073 The low outer membrane permeability is a type of intrinsic resistance, which makes many antimicrobials inefficacious to treat P. aeruginosa related infections.99. D. Zhu, F. Chen, Y.-C. Chen, H. Peng, and K.-J. Wang, Front. Cell. Infect. Microbiol. 11, 11 (2021).The deficiency of efficacious antibiotics and the existence of extensively drug-resistant P. aeruginosa strains have prompted scientists to research new antibacterial therapy strategies such as combination strategies. For example, the antibacterial efficiency of single antibiotics can be improved in combination with other antimicrobials that have outer membrane-permeabilizing properties.66. Q. Li, R. Cebrián, M. Montalbán-López, H. Ren, W. Wu, and O. P. Kuipers, Commun. Biol. 4, 31 (2021). https://doi.org/10.1038/s42003-020-01511-1 A combination strategy can also expand the antibacterial spectrum of antibiotics, which is important for the absence of new effective treatments.1010. J. P. Horcajada, M. Montero, A. Oliver, L. Sorlí, S. Luque, S. Gómez-Zorrilla, N. Benito, and S. Grau, Clin. Microbiol. Rev. 32, e00031 (2019). https://doi.org/10.1128/CMR.00031-19 In 2019, the Food and Drug Administration (FDA) approved antibiotic combination treatments such as Zerbaxa (ceftolozane-tazobactam combination) and Zavicefta (ceftazidime-avibactam combination) to treat drug-resistant P. aeruginosa infections. However, there are concerns that the antibiotic combinations might worsen or accelerate existing drug resistance,1111. J. Liu, O. Gefen, I. Ronin, M. Bar-Meir, and N. Q. Balaban, Science 367, 200 (2020). https://doi.org/10.1126/science.aay3041 which means bacteria may develop more severe drug resistance against combination therapies.1111. J. Liu, O. Gefen, I. Ronin, M. Bar-Meir, and N. Q. Balaban, Science 367, 200 (2020). https://doi.org/10.1126/science.aay3041In the present study, we proposed a novel bacterial inactivation strategy by combining antimicrobial peptide (AMP) LL37 with the current commercially available antibiotics. AMPs are short peptides that can be potential and promising antimicrobials to replace the traditional antibiotics to treat bacterial infections and can even be used in implantable biomaterials to prevent bacterial colonization.12,1312. B. Domingues et al., Adv. Ther. 5, 2100158 (2022). https://doi.org/10.1002/adtp.20210015813. M. Xiao, J. Jasensky, J. Gerszberg, J. Chen, J. Tian, T. Lin, T. Lu, J. Lahann, and Z. Chen, Langmuir 34, 12889 (2018). https://doi.org/10.1021/acs.langmuir.8b02377 Currently, more AMPs have been found with LL37, which is a 37 amino acid cationic peptide that shows a crucial antimicrobial defense role in the human body.1414. J. M. Kahlenberg and M. J. Kaplan, J. Immunol. 191, 4895 (2013). https://doi.org/10.4049/jimmunol.1302005 LL37 exhibits therapeutic potential in clinical trials because it has antibacterial effects and is also less likely to promote drug resistance because LL37 damages the bacterial membrane through non-specific interactions rather than special targets.15–1715. U. H. N. Dürr, U. S. Sudheendra, and A. Ramamoorthy, Biochim. Biophys. Acta Biomembr. 1758, 1408 (2006). https://doi.org/10.1016/j.bbamem.2006.03.03016. A. Grönberg, M. Mahlapuu, M. Ståhle, C. Whately-Smith, and O. Rollman, Wound Repair Regener. 22, 613 (2014). https://doi.org/10.1111/wrr.1221117. L. D. Lozeau, S. Youssefian, N. Rahbar, T. A. Camesano, and M. W. Rolle, Biomacromolecules 19, 4513 (2018). https://doi.org/10.1021/acs.biomac.8b00802 As a cationic AMP, the net charge of LL37 is +6 at physiological pH and can interact with negatively charged bacterial membranes, and it further forms transmembrane pores to damage the integrity of the bacterium and achieve antibacterial effects.18,1918. K. E. Ridyard and J. Overhage, Antibiotics 10, 10 (2021).19. Z. Oren, J. C. Lerman, G. H. Gudmundsson, B. Agerberth, and Y. Shai, Biochem. J. 341, 501 (1999). https://doi.org/10.1042/bj3410501

In this research, we studied the minimum inhibitory concentrations (MICs) and minimum bactericidal concentrations (MBCs) of LL37 and different antibiotics against two P. aeruginosa strains in vitro. Furthermore, LL37 was combined with vancomycin, colistin, gentamicin, azithromycin, and polymyxin B to find synergistic inhibitory and bactericidal combinations against P. aeruginosa. To find possible mechanisms behind synergistic effects, we tested the outer membrane-permeabilizing properties of LL37 and also studied the charge neutralization among the interactions of LL37 and the outer membrane. Finally, we tested the uptake of antibiotics after LL37 permeabilization of the outer membrane for further elucidating the mechanism of interactions between antibiotic uptake and outer membrane permeabilization.

II. MATERIALS AND METHODOLOGY

Section:

ChooseTop of pageABSTRACTI. INTRODUCTIONII. MATERIALS AND METHODO... <<III. RESULTS AND DISCUSSI...IV. SUMMARY AND CONCLUSIO...REFERENCES

A. Materials

Two P. aeruginosa strains were studied in this research. Wild-type PAO1 strain (ATCC 15692) and wild-type PA103 strain (ATCC 29260) were kept in the freezer at −80 °C in our laboratory. PAO1 was originally isolated from an infected wound and is one of the most widely used P. aeruginosa strains in research. PA103, known as a cytotoxic strain, was originally isolated from the sputum of patients. Bacteria were cultured in the Mueller–Hinton broth (Sigma-Aldrich, St. Louis, MO) and Mueller–Hinton agar (MHA) (Hardy Diagnostics CRITERION™). LL37 (LLGDFFRKSKEKIGKEFKRIVQRIKDFLRNLVPRTES) was purchased from Anaspec, Inc. (Fremont, CA). Vancomycin, colistin, azithromycin, and polymyxin B were purchased from Sigma-Aldrich (St. Louis, MO). Gentamicin sulfate was purchased from Alexis Corporation. 4-(2-hydroxyethyl) piperazine-1-ethanesulfonic acid (HEPES) buffer solution (1M in H2O), fluorescent probe N-phenyl-1-naphthylamine (NPN), bovine serum albumin (BSA), acetic acid, acetone, magnesium chloride hexahydrate, and calcium chloride dihydrate were also procured from Sigma-Aldrich (St. Louis, MO).

B. Methods

1. Broth microdilution assay

The MICs of LL37 solutions and antibiotics were measured according to published methods based on the Clinical and Laboratory Standards Institute.2020. I. Wiegand, K. Hilpert, and R. E. W. Hancock, Nat. Protoc. 3, 163 (2008). https://doi.org/10.1038/nprot.2007.521 Briefly, twofold serial dilutions of antimicrobials were prepared using acidified water (0.01% acetic acid, 0.2% BSA) in a 96-well microtiter plate (polypropylene) with a final concentration of LL37 (256–0.5 μg/ml) or antibiotics [gentamicin (16–0.25 μg/ml), colistin (8–0.125 μg/ml), vancomycin (512–8 μg/ml), azithromycin (512–16 μg/ml), and polymyxin B (8–0.125 μg/ml)]. Colonies from MHA plates were incubated in 7 ml of the MHB medium in a tissue culture roller drum TC-7 (New Brunswick Scientific Co., Inc.) at 37 °C for 15 h at 40 rpm. Bacterial suspensions were diluted in a fresh MHB medium (1:20) and further incubated at 37 °C for 4 h in the tissue culture roller drum at 40 rpm and prepared for experiments. P. aeruginosa suspensions were diluted to reach 0.2 OD600 (approximately 1 × 108 CFU/ml). The bacteria suspensions were further diluted and 90 μl of the suspension was added to each well of the 96-well microtiter plate to reach the final concentration of 5 × 105 CFU/ml. Sterility control (MHB media) and negative control (bacterial solution only) were also prepared in a 96-well plate. The 96-well plate was incubated for 20 h with shaking at 37 °C, and the MICs were determined as the lowest concentrations of antimicrobials for which the visible growth of P. aeruginosa was inhibited. The experiments were repeated three times independently.

2. Minimum bactericidal concentration

MBCs were determined for LL37 and antibiotics, using a method adapted from the BSAC standardized susceptibility testing method.2121. J. M. Andrews, J. Antimicrob. Chemother. 60, 20 (2007). https://doi.org/10.1093/jac/dkm110 In short, after the MICs of LL37 and antibiotics were determined, 10 μl of the solution from the wells of the 96-well plate with no visible P. aeruginosa growth was seeded to the MHA plates. The seeded MHA plates were incubated for 20 h at 37 °C. MBC refers to the minimum concentration of an antimicrobial that kills 99.9% of bacteria with no visible colony growth on the MHA plate. The experiments were repeated three times independently.

3. Checkerboard assay

The combination effects (synergic, additive, and antagonistic) of LL37 with different antibiotics against P. aeruginosa strains were studied using a checkerboard assay via MIC and MBC determination.6,226. Q. Li, R. Cebrián, M. Montalbán-López, H. Ren, W. Wu, and O. P. Kuipers, Commun. Biol. 4, 31 (2021). https://doi.org/10.1038/s42003-020-01511-122. J. D. Cha, J. H. Lee, K. M. Choi, S. M. Choi, and J. H. Park, Evidence-Based Complementary Altern. Med. 2014, 450572. https://doi.org/10.1155/2014/450572 10 μl of different concentrations of LL37 and antibiotics were added to the 96-well plate together to reach a 1:1 mix of LL37 and antibiotics. Then, the P. aeruginosa suspension was diluted and 80 μl of the bacterial suspension was added to each well of the 96-well microtiter plate to reach the final concentration of 5 × 105 CFU/ml with a final concentration of LL37 (16–8 μg/ml) or antibiotics [gentamicin (2–0.0625 μg/ml), colistin (2–0.031 25 μg/ml), vancomycin (512–32 μg/ml), azithromycin (128–16 μg/ml), and polymyxin B (0.5–0.031 25 μg/ml)]. Sterility control (MHB media) and negative control (bacteria) were also prepared in a 96-well plate. The 96-well plate was further incubated for 20 h in a 37 °C room, and the visible growth of bacteria was then determined with the same method described for MIC. After the determination of the inhibition, 10 μl of solution from wells of the plate with no visible bacterial growth was seeded to the MHA plates to determine the bactericidal effects of LL37-antibiotic combinations. The fractional inhibitory concentration index (FICI) can be calculated as (MICofdrugAincombination)(MICofdrugAalone)+(MICofdrugBincombination)(MICofdrugBalone).The synergistic inhibitory effects can be defined when FICI ≤ 0.5.2323. V. Ng, S. A. Kuehne, and W. C. Chan, Chemistry 24, 9136 (2018). https://doi.org/10.1002/chem.201801423 The fractional bactericidal concentration index (FBCI) can be calculated as (MBCofdrugAincombination)(MBCofdrugAalone)+(MBCofdrugBincombination)(MBCofdrugBalone).The synergistic bactericidal effects can be defined when FBCI ≤ 0.5.2222. J. D. Cha, J. H. Lee, K. M. Choi, S. M. Choi, and J. H. Park, Evidence-Based Complementary Altern. Med. 2014, 450572. https://doi.org/10.1155/2014/450572 The experiments were repeated three times independently.

4. Outer membrane permeability test

The ability of LL37 to make the outer membrane more permeable was determined through measuring the uptake of the fluorescent dye NPN, which was adapted from a prior study.2424. J. Wang, S. Chou, L. Xu, X. Zhu, N. Dong, A. Shan, and Z. Chen, Sci. Rep. 5, 15963 (2015). https://doi.org/10.1038/srep15963 P. aeruginosa strains were incubated for 20 h at 37 °C and then centrifuged at 5000 rpm for 10 min, washed three times, and diluted to reach 5 × 105 CFU/ml by using the HEPES buffer (pH 7.25, 5 mM HEPES) with the addition of 5 mM glucose (referred to as the GHEPES buffer). 10 μM NPN was added to a quartz cuvette, which contained 2 ml of bacterial suspension, and fluorescence was measured and recorded as F0 by using an F-4500 fluorescence spectrophotometer (Hitachi, Japan) under the following parameters: excitation λ = 350 nm and emission λ = 420 nm. LL37 was further added to the cuvette to detect the fluorescence until the fluorescence stabilized. The increased outer membrane permeability was represented as the percentage of NPN uptake through the equation NPNuptake(%)=(FLL37–F0)/(FpolymyxinB–F0)×100%.FLL37 is the recorded fluorescence of P. aeruginosa strains with NPN at different concentrations of LL37, and F0 is the fluorescence of P. aeruginosa strains only with the existence of NPN. The fluorescence of P. aeruginosa strains with NPN after the addition of 20 μg/ml polymyxin B is named as Fpolymyxin B, which served as a positive control because polymyxin B is well known to permeabilize the outer membrane.2525. N. Dong, X. Zhu, S. Chou, A. Shan, W. Li, and J. Jiang, Biomaterials 35, 8028 (2014). https://doi.org/10.1016/j.biomaterials.2014.06.005 The experiments were repeated three times independently.

5. Zeta potential

Zeta potentials of P. aeruginosa strains with or without the addition of LL37 were measured by using a previously published method.2626. R. Kannan, P. Prabakaran, R. Basu, C. Pindi, S. Senapati, V. Muthuvijayan, and E. Prasad, ACS Appl. Bio Mater. 2, 3212 (2019). https://doi.org/10.1021/acsabm.9b00140 Briefly, P. aeruginosa strains were incubated for 20 h at 37 °C room and then centrifuged at 5000 rpm for 10 min, washed three times, and diluted to reach 5 × 105 CFU/ml by using the GHEPES buffer. Then, the bacterial suspension was added to disposable folded capillary cells and the zeta potential was measured by using Zetasizer Nano-ZS ZEN 3600 (Malvern Instruments, Inc.). After that, LL37 was added to the bacterial suspensions to reach the final peptide concentrations of 1–32 μg/ml for PAO1 and 0.25–2 μg/ml for PA103, and the change in zeta potential was measured for each solution. The experiments were repeated three times independently.

6. Antibiotic uptake test

The uptake of antibiotics was tested after the bacteria were treated with LL37. P. aeruginosa strains, cultured in MHB medium at 37 °C room for 20 h, and then diluted to 1 × 106 CFU/ml. An amount of LL37 equal to half the MIC was added to the bacterial suspensions and incubated for 2 h at 4 °C. Ca2+ and Mg2+ ions were added to bacterial solutions to reach a final concentration of 25 μg/ml of Ca2 and 12.5 μg/ml of Mg2+ after treatment to eliminate the antibacterial effect of LL37. The control groups were studied under the same conditions except for the treatment of LL37. Twofold serial dilutions of antibiotics were prepared in a 96-well plate. Treated or untreated bacteria suspensions were further diluted by an MHB2 medium (containing 25 μg/ml of Ca2 and 12.5 μg/ml of Mg2+), and 90 μl of treated or untreated bacteria suspensions were added to the 96-well plate to reach the bacterial concentration of 5 × 105 CFU/ml. The 96-well plate was incubated in a 37 °C room for 20 h, and the new MICs with different antibiotics were determined for both treated and untreated groups. The experiments were repeated three times independently.

III. RESULTS AND DISCUSSION

Section:

ChooseTop of pageABSTRACTI. INTRODUCTIONII. MATERIALS AND METHODO...III. RESULTS AND DISCUSSI... <<IV. SUMMARY AND CONCLUSIO...REFERENCES

A. Anti-Pseudomonas effect of LL37 and antibiotics

The antibacterial effects of LL37 and five different clinically used antibiotics were tested against P. aeruginosa strains PAO1 and PA103 (Table I). Among these antibiotics, colistin and polymyxin B are seen as last-resort treatments because of their strong clinical antibacterial effects.2727. M. A. E. El-Sayed Ahmed, L. L. Zhong, C. Shen, Y. Yang, Y. Doi, and G. B. Tian, Emerging Microbes Infect. 9, 868 (2020). https://doi.org/10.1080/22221751.2020.1754133 Gentamicin and azithromycin are useful for treating Gram-negative bacteria infections, but vancomycin is not used to treat P. aeruginosa infections because the low outer membrane permeability prevents vancomycin from entering the bacterium. As shown in Table I, LL37 showed better inhibitory effects to PA103 than PAO1, 32 vs 64 μg/ml, respectively, but exhibited the same bactericidal effects for each strain (64 μg/ml). The bacteriostatic and bactericidal effects of gentamicin were both better than vancomycin and azithromycin. As last-resort antibiotics, colistin and polymyxin B exhibited potent anti-Pseudomonas effects, although polymyxin B showed better bactericidal activity to both PAO1 and PA103.

TABLE I. MICs and MBCs of LL37 and different antibiotics against P. aeruginosa strains PAO1 and PA103.

AntimicrobialsMIC
(μg/ml)MBC
(μg/ml)PAO1PA103PAO1PA103LL3764326464Gentamicin2288Colistin0.50.584Vancomycin128512>2048>4096Azithromycin256>512>512>512Polymyxin B0.50.512

B. LL37 and antibiotic combination effects

After the MICs and MBCs of different antimicrobials were determined, LL37 was combined with gentamicin, colistin, vancomycin, azithromycin, and polymyxin B to find synergistic inhibitory combinations and synergistic bactericidal combinations against P. aeruginosa strains PAO1 and PA103 (Table II). Although vancomycin on its own is not an attractive treatment for PA infections, LL37-vancomycin combination showed a synergistic inhibitory effect to PAO1, as well as a synergistic bactericidal effect to both PAO1 and PA103. Vancomycin can target lipid II, which is embedded in the inner membrane and is also an important molecule needed for the synthesis of the bacterial cell wall.28,2928. B. D. Kruijff, V. V. Dam, and E. Breukink, Prostaglandins Leukotrienes Essent. Fatty Acids 79, 117 (2008). https://doi.org/10.1016/j.plefa.2008.09.02029. C. Harwood and G. Jensen, Imaging Bacterial Molecules, Structures and Cells (Academic, Cambridge, 2016). Typically, the outer membrane as a barrier decreases the uptake of vancomycin. However, the synergistic combination of LL37 and vancomycin extended the antibacterial spectrum of vancomycin. We hypothesized that LL37 has outer membrane-permeabilizing properties that allow vancomycin to enter P. aeruginosa.

TABLE II. Combined activity of LL37 and antibiotics against P. aeruginosa strains PAO1 and PA103.

LL37-Antibiotic combinationsFICIFBCIPAO1PA103PAO1PA103Gentamicin>0.5>0.5>0.5>0.5Colistin>0.50.380.50.31Vancomycin0.5>0.5<0.375<0.375Azithromycin0.27>0.5>0.5>0.5Polymyxin B0.38>0.50.38>0.5

We also found that the combination of LL37 and colistin was effective against Pseudomonas, showing a synergistic inhibitory effect with PA103 and also a synergistic bactericidal effect toward PAO1 and PA103. LL37 also exhibited synergistic inhibitory and bactericidal effects toward PAO1 when combined with polymyxin B, and LL37-azithromycin combination showed synergistic inhibitory effects toward PAO1. However, the combination of LL37 and gentamicin showed no synergistic effects toward either PAO1 or PA103.

C. Outer membrane-permeabilizing properties of LL37

To help uncover the mechanism of how LL37 promotes synergistic effects with some antibiotics, we studied the outer membrane-permeabilizing properties of LL37 through the NPN uptake assay. NPN can enter the outer membrane and show increased fluorescence at the permeabilized outer membrane. LL37 increased the outer membrane permeability of both P. aeruginosa strains PAO1 and PA103 at low concentrations, indicating strong outer membrane-permeabilizing properties (Fig. 1). Also, LL37 exhibited stronger outer membrane-permeabilizing properties to PA103 than PAO1, where the increased outer membrane permeability reached 100% permeabilization at a lower LL37 concentration.

D. Zeta potential

Zeta potentials of the bacteria were measured to characterize how the charge on the outer membrane is affected by various treatments. Maintaining the normal zeta potential is important for normal cellular function, and the charge neutralization process of the outer membrane is essential for antimicrobials to achieve an antibacterial effect.30–3230. S. Halder, K. K. Yadav, R. Sarkar, S. Mukherjee, P. Saha, S. Haldar, S. Karmakar, and T. Sen, SpringerPlus 4, 672 (2015). https://doi.org/10.1186/s40064-015-1476-731. F. Tokumasu, G. R. Ostera, C. Amaratunga, and R. M. Fairhurst, Exp. Parasitol. 131, 245 (2012). https://doi.org/10.1016/j.exppara.2012.03.00532. I. M. Torcato, Y. H. Huang, H. G. Franquelim, D. D. Gaspar, D. J. Craik, M. A. Castanho, and S. T. Henriques, Chembiochem 14, 2013 (2013). https://doi.org/10.1002/cbic.201300274 In our study, the zeta potential of P. aeruginosa strains PAO1 and PA103 strains were found to be −40.9 and −10.9 mV, respectively (Fig. 2), and our results were similar to the previous findings, which reported zeta potentials of −40 and −10.1 mV for PAO1 and PA103, respectively.33,3433. H. Zhang, H. Zeng, A. C. Ulrich, and Y. Liu, Water Resour. Res. 52, 1127, https://doi.org/10.1002/2015WR017821 (2016). https://doi.org/10.1002/2015WR01782134. I. E. Ivanov, E. N. Kintz, L. A. Porter, J. B. Goldberg, N. A. Burnham, and T. A. Camesano, J. Bacteriol. 193, 1259 (2011). https://doi.org/10.1128/JB.01308-10 The differences of zeta potential between two strains may be due to the differences among both composition and structure of the outer membranes, such as the differences in the amount of LPS in the outer membrane.3535. R. L. Soon, R. L. Nation, S. Cockram, J. H. Moffatt, M. Harper, B. Adler, J. D. Boyce, I. Larson, and J. Li, J. Antimicrob. Chemother. 66, 126 (2010). https://doi.org/10.1093/jac/dkq422Positively charged LL37 interacted with the negatively charged bacterial outer membrane through electrostatic forces, and the zeta potential was neutralized by an increasing concentration of LL37. PAO1 needed more LL37 to neutralize the negative charges compared to PA103 (16 vs 2 μg/ml), and this charge neutralization can cause destabilization of the outer membrane and, subsequently, increase the outer membrane permeability and cause antibacterial effects.30,3630. S. Halder, K. K. Yadav, R. Sarkar, S. Mukherjee, P. Saha, S. Haldar, S. Karmakar, and T. Sen, SpringerPlus 4, 672 (2015). https://doi.org/10.1186/s40064-015-1476-736. M. Arakha, M. Saleem, B. C. Mallick, and S. Jha, Sci. Rep. 5, 9578 (2015). https://doi.org/10.1038/srep09578 The outer membrane permeability is easier to increase for P. aeruginosa strains, which have lower surface charges because less LL37 is needed to neutralize the negative charge.

E. Increased uptake of antibiotics

The outer membrane-permeabilizing properties of LL37 were determined, and we hypothesized that the uptake of antibiotics would also increase due to the greater outer membrane permeability. To validate this hypothesis, we examined the uptake of antibiotics (Figs. 3 and 4). LL37 here served as the outer membrane-permeabilizing agent to increase the outer membrane permeability. However, LL37 itself also has an antibacterial effect on P. aeruginosa. To minimize the antibacterial effects of LL37, Ca2+ and Mg2+ ions were added, which inhibit LL37. We found that the antibacterial effect of LL37 can be substantially inhibited with the addition of Ca2+ and Mg2+, and the MICs of LL37 for both strains were >256 μg/ml when against P. aeruginosa strains PAO1 and PA103.For PAO1, the uptake of antibiotics increased among all tested antibiotics. The MIC of vancomycin decreased eight times compared with the control group, which provided further evidence that outer membrane permeability is the main barrier for vancomycin and also other antibiotics. For PA103, the MIC of polymyxin B, colistin, and gentamicin decreased twofold to fourfold, but the MIC of vancomycin and azithromycin was still >512 μg/ml, which was the highest concentration tested. Several factors may have contributed to this phenomenon. First, we used LL37 to increase the outer membrane permeability, but the increased outer membrane permeability might still not be enough for vancomycin or azithromycin to enter the outer membrane and achieve the antibacterial effect. Second, the existence of Ca2+ and Mg2+ can not only inhibit the antibacterial effect of LL37 but also stabilize and protect the outer membrane because Ca2+ and Mg2+ can build the salt bridge with LPS, and thus may also influence the permeability.37–3937. A. Z. Sahalan, A. H. Abdul, H. L. Hing, and M. K. A. Ghani, Sains Malays. 42, 301 (2013).38. L. A. Clifton, M. W. A. Skoda, A. P. Le Brun, F. Ciesielski, I. Kuzmenko, S. Holt, and J. H. Lakey, Langmuir 31, 404 (2015). https://doi.org/10.1021/la504407v39. H. Nikaido, Microbiol. Mol. Biol. Rev. 67, 593 (2003). https://doi.org/10.1128/MMBR.67.4.593-656.2003 The antibacterial inhibitive effects of Ca2+ and Mg2+ ions were also observed for colistin; our preliminary data showed that the MIC of colistin is 0.5 μg/ml when PAO1 was cultured in the MHB medium, but it increased to 1 μg/ml in a cation-adjusted MHB medium. Our results showed that LL37 increased the outer membrane permeability, which allowed the antibiotics to better penetrate the bacteria. The MICs of most antibiotics decreased in the presence of LL37 compared with the control groups.

F. Discussion

A strategy combining antimicrobial peptides with antibiotics can provide a new pathway for finding effective antibacterial therapies.4040. K. M. Papp-Wallace et al., J. Infect. Dis. 220, 666 (2019). https://doi.org/10.1093/infdis/jiz149 AMPs have great potential to be used in the clinic due to their antimicrobial effects and low resistance.4141. J. O'Neill, Tackling Drug-Resistant Infections Globally: Final Report and Recommendations (Government of the United Kingdom, 2016). In this research, we sought to find synergistic combinations among LL37 and five antibiotics, and we studied the mechanism behind these synergistic combinations.The combination of LL37 with antibiotics exhibited several synergistic inhibitory and bactericidal effects toward P. aeruginosa strains PAO1 and PA103. Additionally, the antibacterial spectrum of vancomycin was expanded when combined with LL37, showing antibacterial effects on both PAO1 and PA103. The latter expansion is important because it makes vancomycin possible to be used clinically and provides a potential solution to address the threat of antibiotic shortages. Antimicrobials with different antibacterial targets on P. aeruginosa may have a great potential to achieve the synergistic effect.42,4342. L. Ejim, M. A. Farha, S. B. Falconer, J. Wildenhain, B. K. Coombes, M. Tyers, E. D. Brown, and G. D. Wright, Nat. Chem. Biol. 7, 348 (2011). https://doi.org/10.1038/nchembio.55943. D. Field, N. Seisling, P. D. Cotter, R. P. Ross, and C. Hill, Front. Microbiol. 7, 1713 (2016). https://doi.org/10.3389/fmicb.2016.01713 Vancomycin can bind with d-alanyl-d-alanine, a component in the peptidoglycan layer, to prevent the synthesis of the bacterial cell wall to further achieve antibacterial effects,4444. R. Nagarajan, Antimicrob. Agents Chemother. 35, 605 (1991). https://doi.org/10.1128/AAC.35.4.605 and so we know that there is a cell membrane component involved in the action of vancomycin. Synergistic effects of vancomycin in combination with other outer membrane targeting antimicrobials against Gram-negative bacteria have been previously reported.6,456. Q. Li, R. Cebrián, M. Montalbán-López, H. Ren, W. Wu, and O. P. Kuipers, Commun. Biol. 4, 31 (2021). https://doi.org/10.1038/s42003-020-01511-145. A. Oliva, S. Garzoli, M. De Angelis, C. Marzuillo, V. Vullo, C. M. Mastroianni, and R. Ragno, Molecules 24, 886 (2019). https://doi.org/10.3390/molecules24050886 Prior research reported that LL37 combined with azithromycin could inhibit protein synthesis of P. aeruginosa,4646. K. Tateda, Y. Ishii, T. Matsumoto, T. Kobayashi, S. Miyazaki, and K. Yamaguchi, J. Infect. Chemother. 6, 1 (2000). https://doi.org/10.1007/s101560050042 and this combination is another example in which the peptide helps the antibiotic act against the bacteria.Furthermore, LL37 also exhibited synergistic effects toward PAO1 and/or PA103 when combined with colistin and polymyxin B, although these two antibiotics are already effective against PA strains. Colistin and polymyxin B both have strong outer membrane-permeabilizing properties and can act with the negatively charged LPS to influence and damage the integrity of the outer membrane, which further results in leakage of the intracellular components and also the death of bacteria.47,4847. A. P. Zavascki, L. Z. Goldani, J. Li, and R. L. Nation, J. Antimicrob. Chemother. 60, 1206 (2007). https://doi.org/10.1093/jac/dkm35748. S. Biswas, J. M. Brunel, J. C. Dubus, M. Reynaud-Gaubert, and J. M. Rolain, Expert Rev. Anti-Infect. Ther. 10, 917 (2012). https://doi.org/10.1586/eri.12.78 The synergistic combinations among LL37 with colistin and polymyxin B may be because they all have strong outer membrane-permeabilizing properties. Therefore, when combined together, these molecules can more easily disrupt PA membranes and the MICs of colistin and polymyxin B were much lower for each PA strain.LPS are essential parts of the outer membrane that cause it to be negatively charged and also control the outer membrane permeability.49,5049. B. W. Simpson and M. S. Trent, Nat. Rev. Microbiol. 17, 403 (2019). https://doi.org/10.1038/s41579-019-0201-x50. R. F. Maldonado, I. Sá-Correia, and M. A. Valvano, FEMS Microbiol. Rev. 40, 480 (2016). https://doi.org/10.1093/femsre/fuw007 The differences in the amount of LPS might cause the differences of zeta potential between these two PA strains. The neutralization of surface charges among LL37 and P. aeruginosa was observed, and this type of interaction mainly relies on electrostatic interactions, rather than targeting a specific molecule on the bacterial surfaces. Therefore, there is less concern of promoting drug resistance.5151. M. Seil, C. Nagant, J.-P. Dehaye, M. Vandenbranden, and M. F. Lensink, Pharmaceuticals 3, 3435 (2010). https://doi.org/10.3390/ph3113435 Also, we noted that more LL37 was needed to neutralize the negative charges of PAO1 compared to PA103 (16 vs 2 μg/ml), which may be because PAO1 has a stronger electrostatically negative charge than PA103. Furthermore, the neutralizing process may explain how LL37 increases the outer membrane permeability. As LL37 neutralizes the negative charges, this perturbs the normal condition of the outer membrane and increases the permeability. The increased permeability of PA103 can reach 100% with the addition of less LL37 compared with PAO1, which may be because PA103 has fewer negative charges to neutralize. The change of surface charge (zeta potential) can be related to an increased outer membrane permeability, which has been proved in other publications.30,52,5330. S. Halder, K. K. Yadav, R. Sarkar, S. Mukherjee, P. Saha, S. Haldar, S. Karmakar, and T. Sen, SpringerPlus 4, 672 (2015). https://doi.org/10.1186/s40064-015-1476-752. C. S. Alves et al., J. Biol. Chem. 285, 27536 (2010). https://doi.org/10.1074/jbc.M110.13095553. K. Madhongsa, S. Pasan, O. Phophetleb, S. Nasompag, S. Thammasirirak, S. Daduang, S. Taweechaisupapong, A. L. Lomize, and R. Patramanon, PLOS Negl. Trop. Dis. 7, e2267 (2013). https://doi.org/10.1371/journal.pntd.0002267

Another important finding in this study is the relationship between outer membrane permeability and the uptake of antibiotics. The uptake of most antibiotics increased after LL37 increased the outer membrane permeability, which was directly observed. Increased outer membrane permeability due to LL37 explains how some of the antibiotics were able to be more effective when applied in combination with the peptide.

The existence of Mg2+ and Ca2+ ions greatly affected the MIC of LL37 not only because of the salt sensitivity of peptide2020. I. Wiegand, K. Hilpert, and R. E. W. Hancock, Nat. Protoc. 3, 163 (2008). https://doi.org/10.1038/nprot.2007.521 but also because of the stabilization effect of Mg2+ and Ca2+ ions on the outer membrane through building the salt bridge with LPS.