Biofilm infections between Scylla and Charybdis: interplay of host antimicrobial peptides and antibiotics

Figures & data

Table 1 Biofilm-related resistance and FLIP7–antibiotic interaction in biofilms (TTC assay)

Strain/antibiotic	BRR^1 quantification			FLIP7 and antibiotic interplay in biofilms
	Planktonic cells MIC90(µg/mL)	Biofilm MBIC90(µg/mL)	BRR^1	FICI	Interaction type	Antibiotic MBIC90 in FLIP7 presence (µg/mL)^2	Fold reduction^3
Staphylococcus aureus
Meropenem	0.02 ± 0.02	>50	>2000	0.168	Syn	<0.1	>500
Amikacin	1.88 ± 0.00	>500	>266	0.087	Syn	1.5 ± 0.0	>333
Kanamycin	3.51 ± 1.17	>500	>142	0.107	Syn	3.0 ± 0.0	>168
Ampicillin	0.03 ± 0.01	19.5 ± 3.94	650	<0.208	Syn	<1.0	>24
Vancomycin	0.41 ± 0.19	34.4 ± 15.6^4	83.9	0.165	Syn	1.5 ± 0.9	22.9
Benzalkonium Cl	0.30 ± 0.00	12.0 ± 0.00	40	0.749	Add	2.25 ± 0.75	5.3
Clindamycin	75.0 ± 0.0	>250	>3	0.355	Syn	10.0 ± 2.0	>25
Erythromycin	0.94 ± 0.00	9.4 ± 0.00	10	0.422	Syn	0.45 ± 0.15	20.9
Oxacillin	0.05 ± 0.00	0.12 ± 0.00	2.4	0.583	Add	0.01 ± 0.003	9.2
Tetracycline	0.12 ± 0.00	0.15 ± 0.00	1.25	<0.749	Add	<0.10	>1.5
Chloramphenicol	4.70 ± 0.00	5.0 ± 0.00	1.1	0.417	Syn	1.0 ± 0.5	5.0
Escherichia coli
Meropenem	0.03 ± 0.00	3.75 ± 0.00	125	0.297	Syn	<0.02	>188
Cefotaxime	0.09 ± 0.00	2.81 ± 0.94	31.2	0.179	Syn	0.07 ± 0.01	40
Polymyxin B	0.34 ± 0.11	7.03 ± 2.34	20.7	1.042	Ind	9.4 ± 0.0	0.75
Ciprofloxacin	0.03 ± 0.00	0.06 ± 0.00	2	0.542	Add	<0.02	>3
Tetracycline	0.46 ± 0.00	0.9 ± 0.30	1.9	0.708	Add	0.15 ± 0.0	6
Gentamicin	4.70 ± 0.00	6.25 ± 3.13	1.3	0.794	Add	0.6 ± 0.0	10.4
Chloramphenicol	4.69 ± 0.00	3.0 ± 0.00	0.6	0.667	Add	1.50	2
Pseudomonas aeruginosa
Meropenem	0.94 ± 0.31	10.9 ± 1.6	11.6	0.407	Syn	2.4 ± 0.0	4.5
Klebsiella pneumoniae
Meropenem	0.01 ± 0.00	7.03 ± 2.34	703	0.583	Add	0.25 ± 0.05	28.1
Acinetobacter baumannii
Meropenem	0.88 ± 0.13	9.4 ± 0.0	10.7	0.370	Syn	1.2 ± 0.0	7.8

Notes: ¹Biofilm-related resistance determined as antibiotic MBIC₉₀ to planktonic cells MIC₉₀ ratio. ²MBIC₉₀ of antibiotic in combination with FLIP7 concentration causing maximum fold reduction. ³Ratio of antibiotic MBIC₉₀ in the absence and presence of FLIP7 concentration causing maximum antibiotic fold reduction. ⁴The difference from the planktonic culture MIC₉₀ was statistically unreliable (P = 0.161). Data presented as mean ± SE.

Abbreviations: TTC, trimethyl tetrazolium chloride; BRR, biofilm-related resistance; MIC, minimum inhibiting concentration; MBIC, minimum biofilm-inhibiting concentration; FICI, fractional inhibitory concentration index; Syn, synergy; Add, additivity; Ind, independence.

Table 2 FLIP7 and antibiotics interaction on planktonic cells (TTC assay)

Strain/antibiotic	Antibiotic MIC90 (µg/mL)	Antibiotic MIC90 in FLIP7 presence (µg/mL)^1	FICI	Interaction type	Fold reduction^2
Staphylococcus aureus
Meropenem	0.02 ± 0.02	<0.002	0.667	Add	>10
Amikacin	1.88 ± 0.00	0.36 ± 0.12	0.833	Add	5.2
Kanamycin	3.51 ± 1.17	<0.1	0.527	add	>35.1
Ampicillin	0.03 ± 0.01	0.007 ± 0.001	0.542	add	4.3
Vancomycin	0.41 ± 0.19	0.12 ± 0.00	0.589	add	3.4
Benzalkonium Cl	0.30 ± 0.00	0.15 ± 0.00	0.750	Add	2.0
Clindamycin	75.0 ± 0.0	4.70 ± 0.00	0.729	add	15.9
Erythromycin	0.94 ± 0.00	0.03 ± 0.00	0.589	add	31.3
Oxacillin	0.05 ± 0.00	0.014 ± 0.002	1.042	Ind	3.6
Chloramphenicol	4.70 ± 0.00	0.60 ± 0.00	0.418	Syn	7.8
Escherichia coli
Meropenem	0.03 ± 0.00	0.03 ± 0.00	1.085	Ind	1.0
Cefotaxime	0.09 ± 0.00	0.02 ± 0.00	0.415	Syn	4.5
Polymyxin B	0.34 ± 0.11	<0.02	0.339	Syn	17.0
Ciprofloxacin	0.03 ± 0.00	0.015 ± 0.00	0.666	add	2.0
Tetracycline	0.46 ± 0.00	0.03 ± 0.00	0.729	add	15.3
Gentamicin	4.69 ± 0.00	3.53 ± 1.17	1.085	Ind	1.3
Chloramphenicol	4.69 ± 0.00	0.30 ± 0.00	0.670	add	15.6
Pseudomonas aeruginosa
Meropenem	0.94 ± 0.31	0.48 ± 0.00	0.516	add	2.0
Klebsiella pneumoniae
Meropenem	0.01 ± 0.00	0.01 ± 0.00	1.042	Ind	1.0
Acinetobacter baumannii
Meropenem	0.88 ± 0.13	0.03 ± 0.01	0.393	Syn	29.3

Notes: ¹MIC₉₀ of antibiotic in combination with FLIP7 concentration causing maximum antibiotic fold reduction. ²Ratio of antibiotic MIC₉₀ in the absence and presence of FLIP7 concentration causing maximum antibiotic fold reduction. Data presented as mean ± SE.

Abbreviations: TTC, trimethyl tetrazolium chloride; MIC, minimum inhibiting concentration; FICI, fractional inhibitory concentration index; Syn, synergy; Add, additivity; Ind, independence.

Figure 1 Doze–effect curves of antibiotics alone and in combination with FLIP7 against Staphylococcus aureus biofilm (TTC assay).

Notes: (A) Meropenem versus meropenem + FLIP7 125 µg/mL. (B) Amikacin versus amikacin + FLIP7 250 µg/mL. (C) Ampicillin versus ampicillin + FLIP7 125 µg/mL. (D) Kanamycin versus kanamycin + FLIP7 250 µg/mL. (E) Oxacillin versus oxacillin + FLIP7 250 µg/mL. (F) Chloramphenicol versus chloramphenicol + FLIP7 125 µg/mL. Open circles – antibiotic, closed circles – the antibiotic + FLIP7; Y-axis – optical density (mean ± standard error). Effective concentrations of FLIP7 were individually determined for each antibiotic based on the checkerboard experiments.
Abbreviations: TTC, trimethyl tetrazolium chloride; OD, optical density; BRR, biofilm-related resistance; FICI, fractional inhibitory concentration index.

Figure 2 Doze–effect curves of antibiotics alone and in combination with FLIP7 against Escherichia coli biofilm (TTC assay).

Notes: (A) Meropenem versus meropenem + FLIP7 500 µg/mL. (B) Cefotaxime versus cefotaxime + FLIP7 500 µg/mL. (C) Ciprofloxacin versus ciprofloxacin + FLIP7 500 µg/mL. (D) Chloramphenicol versus chloramphenicol + FLIP7 250 µg/mL.
Abbreviations: TTC, trimethyl tetrazolium chloride; OD, optical density; BRR, biofilm-related resistance; FICI, fractional inhibitory concentration index.

Table 3 FLIP7 and antibiotics interaction in crystal violet biofilm eradication assay

Strain/antibiotic	FICI	Interaction type	Antibiotic MBEC90 (Mg/mL)	Antibiotic MBEC90 in FLIP7 presence (Mg/mL)^1	Fold reduction^2
Staphylococcus aureus
Meropenem	0.169	Syn	>50	0.15 ± 0.0	>333
Amikacin	0.024	Syn	>500	1.5 ± 0.0	>333
Kanamycin	0.137	Syn	>500	3.5 ± 0.5	>143
Chloramphenicol	0.438	Syn	3.0 ± 1.0	<0.5	>6
Escherichia coli
Meropenem	0.349	Syn	2.19 ± 0.31	0.03 ± 0.0	73
Cefotaxime	0.397	Syn	1.56 ± 0.31	0.1 ± 0.02	16
Ciprofloxacin	0.583	Add	0.06 ± 0.0	<0.02	>3
Pseudomonas aeruginosa
Meropenem	0.282	Syn	9.4 ± 0.0	2.36 ± 0.0	4
Klebsiella pneumoniae
Meropenem	0.624	Add	6.25 ± 0.0	1.17 ± 0.0	5
Acinetobacter baumannii
Meropenem	0.291	Syn	9.4 ± 0.0	1.37 ± 0.19	7

Notes: ¹MBEC₉₀ of antibiotic in combination with FLIP7 concentration causing maximum antibiotic fold reduction. ²Ratio of antibiotic MBEC₉₀ in the absence and presence of FLIP7 concentration causing maximum antibiotic fold reduction. Data presented as mean ± SE.

Abbreviations: FICI, fractional inhibitory concentration index; MBEC, minimum biofilm eradication concentration; Syn, synergy; Add, additivity.

Figure 3 Doze–effect curves of antibiotics alone and in combination with FLIP7 against Staphylococcus aureus biofilm (crystal violet assay).

Notes: (A) Amikacin versus amikacin + FLIP7 500 µg/mL. (B) Kanamycin versus kanamycin + FLIP7 500 µg/mL. (C) Meropenem versus meropenem + FLIP7 250 µg/mL. (D) Chloramphenicol versus chloramphenicol + FLIP7 250 µg/mL. Open circles: antibiotic; closed circles: antibiotic + FLIP7.
Abbreviation: OD, optical density.

Figure 4 Doze–effect curves of antibiotics alone and in combination with FLIP7 against Escherichia coli biofilm (crystal violet assay).

Notes: (A) Cefotaxime versus cefotaxime + FLIP7 500 µg/mL. (B) Meropenem versus meropenem + FLIP7 500 µg/mL. (C) Ciprofloxacin versus ciprofloxacin + FLIP7 250 µg/mL. Open circles: antibiotic; closed circles: antibiotic + FLIP7.
Abbreviation: OD, optical density.

Figure 5 Staphylococcus aureus biofilm microscopic visualization (Nomarski optics ×1000).

Notes: Twenty-four-hour biofilms were formed on the coverslip and processed as follows: (A) control; (B) treated with meropenem 0.2 µg/mL; and (C) treated with meropenem 0.2 µg/mL + FLIP7 125 µg/mL. In control, bacteria formed a multilayered biofilm consisting of live cells of regular shape. Treatment with meropenem killed most of the bacteria but a portion of the cells (presumable persisters) survived (survivors are marked with arrows). After treatment with the combination, no signs of living cells were seen on the glass.

Table 4 Pharmacological windows of antibiotics used in combination with FLIP7

Antibiotic	Clinically achievable concentration^1		Pharmacological window^2
	µg/mL	Reference
			Staphylococcus aureus	>300
			Escherichia coli	>1500
Meropenem	40	35	Klebsiella pneumoniae	125
			Acinetobacter baumannii	25
			Pseudomonas aeruginosa	12.5
Ampicillin	281	36	S. aureus	>281
Cefotaxime	41	37	E. coli	586
Oxacillin	64	38	S. aureus	6400
Vancomycin	53	39	S. aureus	35
Amikacin	45	40	S. aureus	30
Kanamycin	16	41	S. aureus	5.5
Gentamicin	14	42	E. coli	23
Erythromycin	6.5	43^3	S. aureus	14
Ciprofloxacin	6.7	44	E. coli	>335
Tetracycline	5	45	S. aureus	>50
			E. coli	33
Chloramphenicol	20	46	S. aureus	20
			E. coli	13

Notes: ¹Plasma C_max at intravenous administration. ²The ratio of clinically achievable concentration and MBIC₉₀ of the antibiotic in the presence of FLIP7. The values of MBIC₉₀ are taken from Table 1. ³Plasma C_max at oral administration.

Abbreviation: MBIC, minimum biofilm-inhibiting concentration.