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. 2020 Jun 25;15(6):e0235093.
doi: 10.1371/journal.pone.0235093. eCollection 2020.

Combinatorial effects of antibiotics and enzymes against dual-species Staphylococcus aureus and Pseudomonas aeruginosa biofilms in the wound-like medium

Rima Fanaei Pirlar  1 , Mohammad Emaneini  1 , Reza Beigverdi  1 , Maryam Banar  1 , Willem B van Leeuwen  2 , Fereshteh Jabalameli  1
Affiliations

Combinatorial effects of antibiotics and enzymes against dual-species Staphylococcus aureus and Pseudomonas aeruginosa biofilms in the wound-like medium

Rima Fanaei Pirlar et al. PLoS One. .

Abstract

Bacterial biofilms are one of the major issues in the treatment of chronic infections such as chronic wounds, where biofilms are typically polymicrobial. The synergy between species can occur during most polymicrobial infections, where antimicrobial resistance enhances as a result. Furthermore, self-produced extracellular polymeric substance (EPS) in biofilms results in a high tolerance to antibiotics that complicates wound healing. Since most antibiotics fail to remove biofilms in chronic infections, new therapeutic modalities may be required. Disruption of EPS is one of the effective approaches for biofilm eradication. Therefore, degradation of EPS using enzymes may result in improved chronic wounds healing. In the current study, we investigated the efficacy of trypsin, β-glucosidase, and DNase I enzymes on the degradation of dual-species biofilms of Pseudomonas aeruginosa and Staphylococcus aureus in a wound-like medium. These species are the two most common bacteria associated with biofilm formation in chronic wounds. Moreover, the reduction of minimum biofilm eradication concentration (MBEC) of meropenem and amikacin was evaluated when combined with enzymes. The minimum effective concentrations of trypsin, β-glucosidase, and DNase I enzymes to degrade biofilms were 1 μg/ml, 8 U/ml, and 150 U/ml, respectively. Combination of 0.15 μg/ml trypsin and 50 U/ml DNase I had a significant effect on S. aureus-P. aeruginosa biofilms which resulted in the dispersal and dissolution of all biofilms. In the presence of the enzymatic mixture, MBECs of antibiotics showed a significant decrease (p < 0.05), at least 2.5 fold. We found that trypsin/DNase I mixture can be used as an anti-biofilm agent against dual-species biofilms of S. aureus-P. aeruginosa.

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Conflict of interest statement

The authors declare that they have no competing interests.

Figures

Fig 1
Fig 1
Coagulated media in a plate (left) and in a microtube (right). Wound-Like Medium containing heparinized human plasma, 45% Bolton broth base, 1% gelatin, and 5% laked sheep red blood cells, was inoculated with 10 μl of the combined and normalized culture (1×106 CFU/ml) of S. aureus-P. aeruginosa bacteria. After 24 h incubation at 37° C, media was coagulated.
Fig 2
Fig 2. Scanning electron micrographs of S. aureus-P. aeruginosa biofilms.
Rod (solid arrow) and cocci bacteria (dashed arrow) are shown. (6000x magnification).
Fig 3
Fig 3. Dispersion of S. aureus-P. aeruginosa biofilms with 18 h enzymatic treatments.
Biofilms developed in WLM and then treated with minimum effective concentrations of tested enzymes containing 1 μg/ml trypsin, 8 U/ml β-glucosidase, and 150 U/ml DNase I (T) or enzyme buffer (NT). After treatment, the number of dispersed bacterial cells were estimated by CFU enumeration on staphylococcus/pseudomonas isolation media. Graphs were drawn based on the logarithm of CFU/ml. Values represent the mean of three independent experiments with three replicates per condition. Asterisks indicate the statistically significant difference (P< 0.05) in the number of dispersed bacteria from treated biofilm compared to non-treated (control) biofilm. Error bars represent the standard errors of the means (SEM). Abbreviation: NT, Non-treated; T, Treated; PA0, PA ATCC 27853; PA1, PA1185; PA2, PA1179; PA3, PA1162; PA4, PA1326; PA5, PA1329; SA0, SA ATCC 29213; SA1, SA639.
Fig 4
Fig 4. Effect of the trypsin/DNase I mixture on S. aureus-P. aeruginosa biofilms.
Combination of 0.15 μg/ml trypsin and 50 U/ml DNase I enzymes was treated on established S. aureus-P. aeruginosa biofilms in WLM. The mixture degraded all biofilms completely and dispersed bacterial cells. Data show the logarithm of CFU/ml of biofilm-released cells. Asterisks indicate the statistically significant difference (p < 0.05) in the number of released bacteria from treated biofilms compared to non-treated (control) biofilms. The data shown are the mean (±standard error of the mean) of at least three replicates (three independent experiments). Abbreviation: NT, Non-treated; T, Treated; PA0, PA ATCC 27853; PA1, PA1185; PA2, PA1179; PA3, PA1162; PA4, PA1326; PA5, PA1329; SA0, SA ATCC 29213; SA1, SA639.
Fig 5
Fig 5. MBCs in different culture conditions.
Data show the MBCs of meropenem and amikacin against monoculture strains, co-culture strains, and MBECs of the antibiotics against S. aureus-P. aeruginosa biofilms. A shows MBC values obtained against monocultures of SA0, PA0, SA1, PA1, and PA2 isolates. B indicates MBCs of the antibiotics against SA0, PA0, SA1, PA1, and PA2 strains when they were co-cultured (co-cultures: SA0-PA0, PA1-SA1, and PA2-SA1). C shows MBECs of the antibiotics against dual-species biofilms of SA0-PA0, PA1-SA1, and PA2-SA1. With the exception of SA1, all other strains showed a significant increase in MBC values in co-culture mode (B) over that of MBC values against monoculture strains (A). A significant increase in MBECs of meropenem and amikacin (C) compared to MBCs of antibiotics against co-culture (B) and monoculture (A) strains was observed. Values represent the antibiotic concentration in μg/ml. Asterisks indicate the statistically significant difference (p < 0.05) between MBC values in each chart (5B and 5C) compared to the previous chart/charts (5A and 5B). One-Way ANOVA was used to compare the MBC values obtained against monocultures, co-cultures, and MBECs (comparison among data of A, B, and C). The data shown are the mean of at least three replicates (three independent experiments).
Fig 6
Fig 6. Comparison of MBEC results.
Data shown represent MBECs of meropenem and amikacin alone (Antibiotic alone) and in combination with trypsin/DNase I mixture (Antibiotic with enzyme). MBECs of both antibiotics decreased significantly (p < 0.05) when combined with trypsin/DNase I mixture. Error bars represent the standard errors of the means.
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This work was supported by Tehran University of Medical Sciences under 97-01-30-38032.