CC BY
17
CELL-FREE SUPERNATANT OF STAPHYLOCOCCUS AUREUS CULTURE INCREASES ANTIMICROBIALS SUSCEPTIBILITY OF PSEUDOMONAS AERUGINOSA
M.S. Fedorova, A.V. Mironova, A.R. Kayumov, E.Y. Trizna* Kazan Federal University, 18 Kremlyovskaya St., Kazan, 420008, Russia * Corresponding author: trizna91@mail.ru
Abstract. Along with the wide spread of bacterial antibiotic resistance over the world, the treatment efficiency of infectious disease is greatly affected by the mixed biofilm formation by pathogenic bacteria. Staphylococcus aureus and Pseudomonas aeruginosa, a frequent cause of nosocomial infections, exhibit both synergistic and antagonistic interactions in co-culture, leading to various changes in the metabolic profile of bacteria, which in turn affect their sensitivity to antimicrobials. Here we show that S. aureus cell-free culture liquid exhibits bacteriostatic properties and increases the efficacy of antimicrobials against P. aeruginosa. Thus, the MICs of amikacin, gentamicin, and ciprofloxacin decreased 2-4 fold in the presence of cell-free supernatant of S. aureus 24 h culture. Furthermore, the combination of the latter with antimicrobials increased the efficacy of amikacin up to 64-fold. Thus, the combined use of cell-free culture liquid of S. aureus with broad-spectrum antibiotics can be used to increase the effectiveness of antimicrobial therapy of P. aeruginosa.
Keywords: biofilm, Staphylococcus aureus, Pseudomonas aeruginosa, culture liquid, antibiotics, antibiotic resistance, synergy.
Introduction
Over the world, diseases caused by biofilms are difficult to treat since bacteria in biofilms exhibit increased resistance to antimicrobials (Ding et al., 2021; Beaudoin et al., 2017; Briaud et al., 2019). To date, it is believed that rather multispecies than monobacterial biofilms are formed during the development of infection. The multispecies communities are characterized by a different metabolic profile and properties in contrast to their monospecific counterparts (Harrison et al., 2020; Cendra et al., 2019). These changes may affect the sensitivity of bacteria to antibiotics (Hall et al., 2017; Uruén et al., 2020; Luo et al, 2021; Singh et al., 2021). Since very few antimicrobial agents effective against infections associated with the formation of biofilms are available, the discovery of new therapeutic strategies to combat biofilms is a modern challenge in medicine (Simöes et al., 2021; Xuan et al., 2021; Maka-benta et al., 2021). In patients with pneumonia, the most frequently isolated types of opportunistic pathogens are Staphylococcus aureus and Pseudomonas aeruginosa. These are the most common multidrug-resistant pathogens and exhibit both synergistic and antagonistic interac-
tions in mixed biofilms (Behzadi et al., 2021; Little et al., 2021; Cheung et al., 2021).
The antimicrobial peptides (AMPs), which can act individually and increase the activity of antibiotics, seem to be one of the alternatives to antimicrobial therapy (Grassi et al., 2017; Por-telinha et al., 2021). S. aureus is able to produce AMPs (aureocins), which have high bactericidal activity and increase the activity of known antibiotics against various microorganisms (Ceotto et al., 2012). We have shown previously that the sensitivity of P. aeruginosa to broad-spectrum antibiotics increases in the S. aureus - P. aeruginosa mixed community (Trizna et al., 2020). In the present study, we show that S. aureus cell-free culture liquid exhibits bacteriostatic properties and increases the efficacy of antimicrobials against P. aeru-ginosa.
Materials and Methods
Bacterial strains and growth conditions
S. aureus ATCC 29213 (Museum Strain of the American Collection of Microorganisms), P. aeruginosa ATCC 27853 (Museum Strain of the American Collection of Microorganisms) were used in this study. Bacteria were grown in
flasks with a medium: flask volumes ratio of 1:7.5 with a shaking intensity of 200 rpm at 37 °C. To obtain biofilms, bacteria were grown for 48 hours under static conditions at 37 °C in Basal medium broth (BM) (glucose 5g, peptone 7g, MgSO4*7№O 2.0g and CaChx2№O 0.05g in 1.0 liter tap water) in plates with an initial density of bacterial culture of 1*106 CFU/ml.
Determination of minimum inhibitory concentration (MIC) and minimum bactericidal concentration (MBC)
MICs of antimicrobials were determined by microdilution approach in BM broth according to EUCAST recommendations (Leclercq et al., 2013). Antibiotics were diluted with broth in a 96-well plastic plate (Eppendorf Cell Culture Plates) at concentrations of 0.25-512 pg/mL. The wells were inoculated with 200 pL of bacterial culture (2-9*105 CFU/mL) in BM and incubated at 37°C under static conditions. The minimum inhibitory concentration was defined as the lowest concentration of antibiotic at which no bacterial growth was observed after 24 hours of incubation. Next, the minimum bactericidal concentration (MBC) was determined. For that, a 1000* culture dilution was made from wells with no visible growth and incubated for 24 hours in broth without any antimicrobials. The MBC was taken as the lowest concentration of the substance at which bacterial growth was completely absent at 24 hours of incubation.
Determination of the permeability of the extracellular matrix of biofilms for antibacterial substances (Anderl et al., 2000)
The mono- and polymicrobial biofilms were obtained on sterile nitrocellulose discs. The bacterial suspension with density of 3 * 107 CFU/ml in BM broth were dropped on discs which were placed on plates with LB agar and incubated for 48 hours at 37°C. Then disks were transferred onto a new LB agar plate containing an antibiotic at a concentration corresponding to 1*MBC for corresponding bacterium. A smaller membrane disk moistened with BM broth was placed on the disks with biofilms. Finally, 6-mm Whatman disks were laid out on
the surface of upper membrane, allowing absorbing the antibiotic diffusing from the nutrient medium through biofilm. After 24 hours incubation at 37 °C, the Whatman disks were placed on new plates with bacterial culture spread on the surface of LB agar. After 24-hour incubation, the growth repression zones were measured. As a control, discs were placed on membranes with biofilms and kept in a medium without antibacterial substances. To assess the effect of antibiotics themselves on bacterial cultures, discs were incubated on sterile membranes without bacterial biofilms.
Obtaining cell-free culture liquid
S. aureus cells were grown for 24 hours in LB broth with a shaking intensity of 200 rpm at 37 °C. Next, cells were removed by centrifuga-tion for 15 minutes at 12000 rpm at 37 °C. The supernatant was filtered using sterile Minisart High Flow, 0.2 |im filters.
Alamar-blue test
To evaluate the bacterial viability, 100 pl of bacterial suspension was transferred into a 96-well plate. The biofilms were resuspended in 0.9% NaCl by mechanical scratching. The resazurin sodium salt (Sigma) solution was added to the culture liquid to a final concentration of 120 pM and incubated for 10 min. In the presence of a pink color, the bacteria were identified as viable. The blue color indicated death cells.
CFUs count
The CFUs count was assessed by drop plate assay (Herigstad et al., 2001) with modifications (Baidamshina et al., 2017). Briefly, a serial 10-fold dilutions of the bacterial suspension were prepared in 0.9% NaCl. For cells in biofilms, 0.9% NaCl was added to the wells and bacteria were suspended by scratching the well bottoms with subsequent treatment in an ultrasonic bath for 2 min to facilitate the disintegration of bacterial clumps. Then 5 pi from each dilution was dropped onto LB agar plate and incubated for 24 hours at 37 °C. CFUs were counted from dilutions containing 5-10 colonies.
Statistical analysis
Experiments were performed in three biological replicates with three technical repetitions in each. The statistical significance of differences in determining the number of colonies by counting CFU from a series of dilutions was evaluated by the formula 10 log10[c], where c is the number of cells obtained, using Pearson's Chi-square test for homogeneity. Differences were considered significant at p < 0.05.
Results
The permeability of the extracellular matrix of S. aureus and P. aeruginosa mono- and dual species biofilms for antimicrobials
The increased sensitivity of P. aeruginosa in a mixed S. aureus-P. aeruginosa community to broad-spectrum antibiotics has been shown previously (Trizna et al., 2020). We asked whether this is a consequence of the change in the biochemical composition of the extracellular matrix of a mixed biofilm and consequent alteration in its permeability for antimicrobials. To test that, Ampicillin, vancomycin, amikacin and ciprofloxacin were added to LB agar at a concentration corresponding to respective 1*MBC (Table 1) and antibiotics diffusion through the biofilm has been assessed as described in Materials and Methods.
For the S. aureus and P. aeruginosa biofilms, no significant changes were found compared to the control, indicating a low permeability of antibiotics through the extracellular matrix of biofilms (Fig. 1). Of note, a significant inhibition of S. aureus growth has been observed around the discs incubated on the mixed S. aureus-P. aeruginosa biofilm, apparently,
due to the synthesis of antimicrobial metabolites by P. aeruginosa in the mixed community. Thus, the change in the P. aeruginosa sensitivity in mixed S. aureus - P. aeruginosa biofilm is governed rather by the production of extracellular metabolites by S. aureus affecting P. aeruginosa than due to changes in the of the biofilm permeability.
Effect of S. aureus cell-free culture liquid on P. aeruginosa susceptibility to antibiotics
Some antibiotics are known to show synergy when combined with antimicrobial peptides (Grassi et al., 2017). We assumed that the S. aureus secrets AMPs exhibiting synergistic effect with various antibiotics against P. aeruginosa. To test this assumption, the S. aureus cell-free culture liquid was diluted 4-fold with fresh LB medium in combination with various antibiotics in the concentrations of 0.5-512 pg/mL and seeded by P. aeruginosa. After 24h the residual viability of P. aeruginosa cells was assessed. In presence of 25% S. aureus cell-free culture liquid, the efficacy of aminoglycosides (Amika-cin, Gentamicin) against P. aeruginosa increased 2-fold. Further, the effect of the S. au-reus cell-free culture liquid on biofilm-embed-ded P. aeruginosa susceptibility to antibiotics was evaluated. For that, the cell-free culture liquid of S. aureus was diluted with fresh BM broth to final concentrations of 6% and 12% in combination with various antibiotics at concentrations of 0.25-128 pg/mL. As a control of the nutrients depletion, NaCl solution was added in the same proportions (6% and 12%). After 24h of incubation the residual viability of P. aeru-ginosa in biofilm was assessed (Table 2).
Table 1
P. aeruginosa S. aureus
MIC, ^g/mL MBC, ^g/mL MIC, ^g/mL MBC, ^g/mL
Amikacin 1 64 8 32
Ciprofloxacin 4 64 0.25 16
Ampicillin ND ND 0.5 64
Vancomycin ND ND 2 64
Note: ND - not determined
Minimum inhibitory and bactericidal concentrations of antibiotics, ug/mL
Fig. 1. Evaluation of the permeability of mono- and dimicrobial S. aureus - P. aeruginosa biofilms for antimicrobials
Table 2
Synergism of cell-free culture liquid (CL) of S. aureus with antibiotics
Antibiotic 6% NaCl 6% CL The effect increase, fold 12% NaCl 12% CL The effect increase, fold
MIC, ug/mL MIC, ug/mL
Ciprofloxacin 32 32 0 64 16 4x
Gentamicin 64 16 4x 64 32 2x
Amikacin 128 2 64x 64 4 16x
A Biofilm
Amikacin, ng/mL
Ciprofloxaciri, ng/mL
b Detachted cells
Amikacin, ng/mL
Ciprofloxacin, ng/mL
CF S.a. 25% » CFS.a. 100%
* CF Sa.+ P.a. 25% ■ CFS.a + Pa 100%
♦ Control
Fig. 2. The number of viable detached cells and biofilm-embedded cells P. aeruginosa in the presence of the culture liquid of S. aureus, S. aureus - P. aeruginosa and antibiotics. Viability was assessed by counting CFU by the serial dilution. Antimicrobials were added to 48-hour biofilms (A - 24-hour incubation with culture liquid and amikacin, cells in the biofilm; B - 24-hour incubation with culture liquid and amikacin, detached cells; C - 24-hour incubation with culture liquid and ciprofloxacin, cells in the biofilm; D - 24-hour incubation with culture fluid and ciprofloxacin, cells in the biofilm)
The cell-free culture liquid of S. aureus significantly increased the efficiency of the antibiotics. Thus, ciprofloxacin together with 12% culture liquid led to the death of P. aeruginosa at a concentration 4 times lower than the combination with saline. The effectiveness of gen-tamicin in combination with 6% culture liquid also increased 4 times. The maximum effect was observed when using the culture liquid with amikacin, where the efficacy of the antibiotic increased up to 64 times.
The mutual action of the cell-free culture liquid of S. aureus with broad-spectrum antibiot-
ics against P. aeruginosa cells was also quantified by CFUs count. For that, 48 hours P. aeru-ginosa biofilms were established in 24-well plates, washed and filled with 25-100% culture liquid of S. aureus or a of S. aureus - P. aeru-ginosa mixed culture and antibiotics were added at concentrations equal to their 0.03 - 8 x MBC (Table 1). After a 24-hour incubation, the viability of detached cells and bio-film-embedded cells of P. aeruginosa was assessed by drop plate assay (Fig. 2).
The culture liquid itself of either S. aureus or S. aureus - P. aeruginosa did not decrease
CFUs count, suggesting that antimicrobial metabolites have rather bacteriostatic than bactericidal effect. The addition of amikacin with culture liquid increased the efficacy of antimicrobial. Thus, in presence of the latter, the decrease in CFUs count by 3 orders of magnitude was observed at concentrations of amikacin 4-16-fold lower compared to solely antimicrobial (Fig. 2).
When ciprofloxacin was added in 100% culture liquid of either S. aureus or S. aureus -P. aeruginosa, a decrease in the viability of detached P. aeruginosa cells by three orders of magnitude was observed at an antibiotic concentration of 2 pg/mL (which corresponds to 0.03xMBC of antibiotics), while the introduction of ciprofloxacin alone led to a similar effect only at an antibiotic concentration of 8*MBC In biofilm, the complete cell death of P. aeru-ginosa was observed when the culture liquid of S. aureus - P. aeruginosa was introduced together with ciprofloxacin at a concentration of 2*MBC, while solely antibiotic led to a decrease in CFUs by three orders of magnitude at 8xMBC (Fig. 2).
Discussion
Over the world, diseases caused by Pseudomonas aeruginosa in increased tolerance to antimicrobials raises significantly, suggesting that the development of novel approaches for treatment challenging. The antimicrobial peptides (AMPs), which can act individually and increase the activity of antibiotics, seem to be one of the alternatives to antimicrobial therapy (Grassi et al., 2017 Portelinha et al., 2021). S. aureus is able to produce AMPs (aureocins) with high bactericidal activity and increasing the activity of various antibiotics (Ceotto et al., 2012). Thus, the sensitivity of P. aeruginosa to
broad-spectrum antibiotics increases in the S. aureus - P. aeruginosa mixed community (Trizna et al., 2020). Here we show that S. au-reus produces extracellular metabolites which exhibit bacteriostatic properties and and can be used as enhancers of antimicrobials against P. aeruginosa.
The biofilm-diffusion test revealed that the change in the P. aeruginosa sensitivity in mixed S. aureus - P. aeruginosa biofilm is governed by the production of extracellular metabolites by S. aureus affecting P. aeruginosa. Indeed, in presence of 25% S. aureus cell-free culture liquid, the efficacy of aminoglycosides (Amikacin, Gentamicin) against P. aeruginosa increased 2 fold. In the case of biofilm-embed-ded P. aeruginosa, ciprofloxacin together with 12% culture liquid led to the death of P. aeru-ginosa at a concentration 4 times lower than the combination with saline. In combination with 6% culture liquid, the effectiveness of gentamicin increased 4 times and the MBC of amikacin decreased 64 times (Table 2, Fig. 2).
Taken together, these data confirm that S. aureus produces antimicrobial metabolites which increase the efficacy of antimicrobials against P. aeruginosa cells in both planktonic and biofilm-embedded form. These metabolites can serve as a promising approach to improve the antimicrobial therapy of infections associated with the formation of P. aeruginosa biofilms on various surfaces, while their identification remains challenging.
Acknowledgments
This research was funded by Russian Science Foundation (grant N 20-64-47014 to A.K.) and performed in frames of Kazan Federal University Strategic Academic Leadership Program (PRI0RITY-2030).
References
ANDERL J.N., FRANKLIN M.J. & STEWART P.S. (2000): Role of antibiotic penetration limitation in Klebsiella pneumoniae biofilm resistance to ampicillin and ciprofloxacin. Antimicrobial agents and chemotherapy 44(7), 1818-1824.
BAIDAMSHINA D R., TRIZNA E.Y., HOLYAVKA M.G., BOGACHEV M.I., ARTYUKHOV V.G., AKHATOVA F.S., ... & KAYUMOV A.R. (2017): Targeting microbial biofilms using Ficin, a nonspecific plant protease. Scientific reports 7(1), 1-12.
BEAUDOIN T., YAU Y.C.W., STAPLETON P.J., GONG Y., WANG P.W., GUTTMAN D.S. & WATERS V. (2017): Staphylococcus aureus interaction with Pseudomonas aeruginosa biofilm enhances tobramycin resistance. NPJbiofilms and microbiomes 3(1), 1-9.
BEHZADI P., BARÁTH Z. & GAJDÁCS M. (2021): It's not easy being green: a narrative review on the microbiology, virulence and therapeutic prospects of multidrug-resistant Pseudomonas aeruginosa. Antibiotics 10(1), 42.
BRIAUD P., CAMUS L., BASTIEN S., DOLÉANS-JORDHEIM A., VANDENESCH F. & MOREAU K. (2019): Coexistence with Pseudomonas aeruginosa alters Staphylococcus aureus transcriptome, antibiotic resistance and internalization into epithelial cells. Scientific reports 9(1), 1-14.
CHEUNG G.Y., BAE J.S. & OTTO M. (2021): Pathogenicity and virulence of Staphylococcus aureus. Virulence 12(1), 547-569.
CENDRA M.D.M., BLANCO-CABRA N., PEDRAZ L. & TORRENTS E. (2019): Optimal environmental and culture conditions allow the in vitro coexistence of Pseudomonas aeruginosa and Staphylococcus aureus in stable biofilms. Scientific reports 9(1), 1-17.
CEOTTO H., DA SILVA DIAS R.C., DOS SANTOS NASCIMENTO J., DE PAIVA BRITO M.A.V., GIAMBIAGI-DEMARVAL M. & DO CARMO DE FREIRE BASTOS M. (2012): Aureocin A70 production is disseminated amongst genetically unrelated Staphylococcus aureus involved in bovine mastitis. Letters in applied microbiology 54(5), 455-461.
DING L., WANG J., CAI S., SMYTH H. & CUI Z. (2021): Pulmonary biofilm-based chronic infections and inhaled treatment strategies. International Journal of Pharmaceutics 604, 120768.
GRASSI L., MAISETTA G., ESIN S. & BATONI G. (2017): Combination strategies to enhance the efficacy of antimicrobial peptides against bacterial biofilms. Frontiers in Microbiology 8, 2409.
HALL C.W. & MAH T.F. (2017): Molecular mechanisms ofbiofilm-based antibiotic resistance and tolerance in pathogenic bacteria. FEMS microbiology reviews 41(3), 276-301.
HARRISON F., ALLAN R.N. & MADDOCKS S.E. (2020): Polymicrobial Biofilms in Chronic Infectious Disease. Frontiers in Cellular and Infection Microbiology, 816.
HERIGSTAD B., HAMILTON M. & HEERSINK J. (2001): How to optimize the drop plate method for enumerating bacteria. Journal of microbiological methods 44(2), 121 -129.
LECLERCQ R., CANTÓN R., BROWN D.F., GISKE CG., HEISIG P., MACGOWAN A.P., ... & KAHLMETER G. (2013): EUCAST expert rules in antimicrobial susceptibility testing. Clinical Microbiology and Infection 19(2), 141-160.
LUO A., WANG F., SUN D., LIU X. & XIN B. (2021): Formation, development, and cross-species interactions in biofilms. Frontiers in Microbiology V. 12, 1-15.
LITTLE W., BLACK C. & SMITH A. C. (2021): Clinical Implications of Polymicrobial Synergism Effects on Antimicrobial Susceptibility. Pathogens 10(2), 144.
MAKABENTA J.M.V., NABAWY A., LI C.H., SCHMIDT-MALAN S., PATEL R. & ROTELLO V.M. (2021): Nanomaterial-based therapeutics for antibiotic-resistant bacterial infections. Nature Reviews Microbiology 19(1), 23-36.
NEWSTEAD L.L., VARJONEN K., NUTTALL T. & PATERSON G.K. (2020): Staphylococcal-produced bacteriocins and antimicrobial peptides: Their potential as alternative treatments for Staphylococcus aureus infections. Antibiotics 9(2), 40.
PORTELINHA J., DUAY S.S., YU S.I., HEILEMANN K., LIBARDO M.D.J., JULIANO S.A., ... & ANGELES-BOZA A.M. (2021): Antimicrobial peptides and copper (II) ions: Novel therapeutic opportunities. Chemical Reviews 121(4), 2648-2712.
SIMÖES M., PEREIRA A.R., SIMÖES L.C., CAGIDE F. & BORGES F. (2021): Biofilm control by ionic liquids. Drug Discovery Today 26(6), 1340-1346.
SINGH S., DATTA S., NARAYANAN K.B. & RAJNISH K.N. (2021): Bacterial exo-polysaccharides in biofilms: role in antimicrobial resistance and treatments. Journal of Genetic Engineering and Biotechnology 19(1), 1-19.
TRIZNA E.Y., YARULLINA M.N., BAIDAMSHINA D.R., MIRONOVA A V., AKHATOVA F.S., ROZHINA E.V., ... & KAYUMOV A.R. (2020): Bidirectional alterations in antibiotics susceptibility in Staphylococcus aureus — Pseudomonas aeruginosa dual-species biofilm. Scientific reports 10(1), 1-18.
URUEN C., CHOPO-ESCUIN G., TOMMASSEN J., MAINAR-JAIME R.C. & ARENAS J. (2020): Biofilms as promoters of bacterial antibiotic resistance and tolerance. Antibiotics 10(1), 3.
VO TD., SPAHN C., HEILEMANN M. & BODE H.B. (2021): Microbial Cationic Peptides as a Natural Defense Mechanism against Insect Antimicrobial Peptides. ACS Chemical Biology 16(3), 447-451.
XUAN T.F., WANG Z.Q., LIU J., YU H.T., LIN Q.W., CHEN W.M. & LIN J. (2021): Design and Synthesis of Novel c-di-GMP G-Quadruplex Inducers as Bacterial Biofilm Inhibitors. Journal of Medicinal Chemistry 64(15), 11074-11089.
YUAN L., HANSEN MF., R0DER H.L., WANG N., BURM0LLE M. & HE G. (2020): Mixed-species biofilms in the food industry: current knowledge and novel control strategies. Critical Reviews in Food Science and Nutrition 60(13), 2277-2293.
