Short Proline-Rich Lipopeptide Potentiates Minocycline and Rifampin against Multidrug- and Extensively Drug-Resistant Pseudomonas aeruginosa

Ronald Domalaon; Yaroslav Sanchak; Linet Cherono Koskei; Yinfeng Lyu; George G. Zhanel; Gilbert Arthur; Frank Schweizer

doi:10.1128/AAC.02374-17

DOI: 10.1128/AAC.02374-17

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FIG 1
Evaluation of cytotoxicity of amphiphilic C₁₂-PRP (red circles) via hemolytic activity against erythrocytes (A), inhibition of cellular proliferation against human liver carcinoma HepG2 cells (B), inhibition of cellular proliferation against human embryonic kidney HEK-293 cells (C), cytotoxic effects against human liver carcinoma HepG2 cells (D), and cytotoxic effects against human embryonic kidney HEK-293 cells (E). All experiments were performed in three or more replicates. Colistin (blue squares) and adriamycin (green triangles) were used to represent clinically used peptide antibiotics and anticancer drugs, respectively. Error bars indicate standard deviations of results from three independent experiments (n = 3).

TABLE 1

SPRLP sequences under consideration

Compound	Sequence	Molecular mass (g/mol of TFA salt)^a
C₈-PRP	CH₃(CH₂)₆CO-PRPRPRP-NH₂	1,342.33
C₁₂-PRP	CH₃(CH₂)₁₀CO-PRPRPRP-NH₂	1,398.44
C₁₆-PRP	CH₃(CH₂)₁₄CO-PRPRPRP-NH₂	1,454.55
Ad-PRP	Adamantyl-CH₂CO-PRPRPRP-NH₂	1,392.39
C₈-PGP	CH₃(CH₂)₆CO-PRPGPRP-NH₂	1,129.17
C₁₂-PGP	CH₃(CH₂)₁₀CO-PRPGPRP-NH₂	1,185.28
C₁₆-PGP	CH₃(CH₂)₁₄CO-PRPGPRP-NH₂	1,241.39
Ad-PGP	Adamantyl-CH₂CO-PRPGPRP-NH₂	1,179.23
C₈-PLP	CH₃(CH₂)₆CO-PRPLPRP-NH₂	1,185.28
C₁₂-PLP	CH₃(CH₂)₁₀CO-PRPLPRP-NH₂	1,241.39
C₁₆-PLP	CH₃(CH₂)₁₄CO-PRPLPRP-NH₂	1,297.50
Ad-PLP	Adamantyl-CH₂CO-PRPLPRP-NH₂	1,235.34
C₈-PWP	CH₃(CH₂)₆CO-PRPWPRP-NH₂	1,258.34
C₁₂-PWP	CH₃(CH₂)₁₀CO-PRPWPRP-NH₂	1,314.44
C₁₆-PWP	CH₃(CH₂)₁₄CO-PRPWPRP-NH₂	1,370.55
Ad-PWP	Adamantyl-CH₂CO-PRPWPRP-NH₂	1,308.40
C₁₂-prp	CH₃(CH₂)₁₀CO-prprprp-NH₂ (all d-peptide)	1,398.44

↵a TFA, trifluoroacetic acid.

TABLE 2

Biological activity of SPRLPs belonging to PRP and PGP sequence subsets

Organism	MIC (μg/ml)
Organism	C₈-PRP	C₁₂-PRP	C₁₆-PRP	Ad-PRP	C₈-PGP	C₁₂-PGP	C₁₆-PGP	Ad-PGP
Staphylococcus aureus ATCC 29213	>128	128	8	>128	>128	>128	32	>128
MRSA^a ATCC 33592	>128	>128	16	>128	>128	>128	32	>128
MSSE^b CANWARD-2008 81388	>128	32	4	>128	>128	128	8	>128
MRSE^c CAN-ICU 61589	>128	128	8	>128	>128	>128	16	>128
Enterococcus faecalis ATCC 29212	>128	>128	16	>128	>128	>128	16	>128
Enterococcus faecium ATCC 27270	>128	128	8	>128	>128	>128	16	>128
Escherichia coli ATCC 25922	>128	>128	16	>128	>128	>128	32	>128
E. coli CAN-ICU 61714^d	>128	>128	8	>128	>128	>128	32	>128
E. coli CAN-ICU 63074^e	>128	>128	8	>128	>128	>128	32	>128
E. coli CANWARD-2011 97615^f	>128	>128	8	>128	>128	>128	32	>128
Pseudomonas aeruginosa ATCC 27853	>128	>128	32	>128	>128	>128	128	>128
P. aeruginosa CAN-ICU 62308^g	>128	>128	32	>128	>128	>128	128	>128
P. aeruginosa CANWARD-2011 96846^h	>128	>128	64	>128	>128	>128	128	>128
P. aeruginosa PAO1	>512	128	32	>512	>512	>512	64	>512
Stenotrophomonas maltophilia CAN-ICU 62584	>128	>128	64	>128	>128	>128	128	>128
Acinetobacter baumannii CAN-ICU 63169	>128	>128	16	>128	>128	>128	32	>128
Klebsiella pneumoniae ATCC 13883	>128	>128	64	>128	>128	>128	64	>128
MHCⁱ	>512	>512	16	>512	>512	>512	16	>512

↵a MRSA, methicillin-resistant S. aureus.
↵b MSSE, methicillin-susceptible Staphylococcus epidermidis.
↵c MRSE, methicillin-resistant S. epidermidis. Ceftazidime resistant.
↵d Gentamicin resistant.
↵e Amikacin intermediate resistant.
↵f Gentamicin, tobramycin, and ciprofloxacin resistant; aac(3′)iia.
↵g Gentamicin resistant.
↵h Gentamicin and tobramycin resistant.
↵i Minimum concentration (μg/ml) that resulted in 5% red blood cell hemolysis.

TABLE 3

Biological activity of SPRLPs belonging to PLP and PWP sequence subsets

Organism	MIC (μg/ml)
Organism	C₈-PLP	C₁₂-PLP	C₁₆-PLP	Ad-PLP	C₈-PWP	C₁₂-PWP	C₁₆-PWP	Ad-PWP
Staphylococcus aureus ATCC 29213	>128	128	64	>128	>128	32	8	>128
MRSA^a ATCC 33592	>128	128	64	>128	>128	32	8	>128
MSSE^b CANWARD-2008 81388	>128	64	32	>128	>128	16	4	>128
MRSE^c CAN-ICU 61589	>128	64	64	>128	>128	16	8	>128
Enterococcus faecalis ATCC 29212	>128	128	64	>128	>128	32	8	>128
Enterococcus faecium ATCC 27270	>128	128	64	>128	>128	32	8	>128
Escherichia coli ATCC 25922	>128	>128	128	>128	>128	128	32	>128
E. coli CAN-ICU 61714^d	>128	>128	64	>128	>128	128	32	>128
E. coli CAN-ICU 63074^e	>128	>128	64	>128	>128	128	16	>128
E. coli CANWARD-2011 97615^f	>128	>128	128	>128	>128	128	64	>128
Pseudomonas aeruginosa ATCC 27853	>128	>128	>128	>128	>128	>128	64	>128
P. aeruginosa CAN-ICU 62308^g	>128	>128	>128	>128	>128	>128	64	>128
P. aeruginosa CANWARD-2011 96846^h	>128	>128	>128	>128	>128	>128	64	>128
P. aeruginosa PAO1	>512	512	32	>512	>512	64	32	>512
Stenotrophomonas maltophilia CAN-ICU 62584	>128	>128	>128	>128	>128	>128	64	>128
Acinetobacter baumannii CAN-ICU 63169	>128	>128	128	>128	>128	128	64	>128
Klebsiella pneumoniae ATCC 13883	>128	>128	128	>128	>128	>128	64	>128
MHCⁱ	>512	>512	16	>512	>512	64	16	>512

↵a MRSA, methicillin-resistant S. aureus.
↵b MSSE, methicillin-susceptible Staphylococcus epidermidis.
↵c MRSE, methicillin-resistant S. epidermidis. Ceftazidime resistant.
↵d Gentamicin resistant.
↵e Amikacin intermediate resistant.
↵f Gentamicin, tobramycin, and ciprofloxacin resistant; aac(3′)iia.
↵g Gentamicin resistant.
↵h Gentamicin and tobramycin resistant.
↵i Minimum concentration (μg/ml) that resulted in 5% red blood cell hemolysis.

TABLE 4

Adjuvant potency of amphiphilic C₁₂-PRP in combination with minocycline against wild-type and MDR/XDR P. aeruginosa^a

P. aeruginosa strain	MIC_MIN (μg/ml)	MIC_C12-PRP (μg/ml)	FIC index	Absolute MIC_MIN^b (μg/ml)	Potentiation (fold)^c
PAO1	8	128	0.19	1	8
259-96918	16	>128	0.12 < x < 0.19	2	8
260-97103	16	128	0.12	1	16
262-101856	64	>128	0.12 < x < 0.25	16	4
264-104354	32	>128	0.06 < x < 0.12	2	16
91433^d	32	>128	0.12 < x < 0.25	8	4
100036	16	>128	0.12 < x < 0.25	4	4
101243^d	2	128	0.31	0.5	4
101885	16	64	0.37	4	4

↵a MIN, minocycline; MDR, multidrug resistant; XDR, extensively drug resistant.
↵b MIC of minocycline in the presence of 8 μg/ml (5 μM) C₁₂-PRP.
↵c Degree of antibiotic potentiation in the presence of 8 μg/ml (5 μM) C₁₂-PRP.
↵d Colistin resistant.

TABLE 5

Adjuvant potency of amphiphilic C₁₂-PRP in combination with rifampin against wild-type and MDR/XDR P. aeruginosa^a

P. aeruginosa strain	MIC_RMP (μg/ml)	MIC_C12-PRP (μg/ml)	FIC index	Absolute MIC_RMP^b (μg/ml)	Potentiation (fold)^c
PAO1	8	128	0.14	1	8
259-96918	16	>128	0.01 < x < 0.14	2	8
260-97103	16	128	0.16	2	8
262-101856	512	>128	0.25 < x < 0.37	512	None
264-104354	8	>128	0.06 < x < 0.12	0.5	16
91433^d	16	>128	0.06 < x < 0.09	1	16
100036	16	>128	0.01 < x < 0.14	1	16
101243^d	4	128	0.09	0.125	32
101885	16	64	0.25	2	8

↵a RMP, rifampin; MDR, multidrug resistant; XDR, extensively drug resistant.
↵b MIC of rifampin in the presence of 8 μg/ml (5 μM) C₁₂-PRP.
↵c Degree of antibiotic potentiation in the presence of 8 μg/ml (5 μM) C₁₂-PRP.
↵d Colistin resistant.

TABLE 6

Adjuvant potency of amphiphilic C₁₂-prp in combination with minocycline against wild-type and MDR/XDR P. aeruginosa^a

P. aeruginosa strain	MIC_MIN (μg/ml)	MIC_C12-prp (μg/ml)	FIC index	Absolute MIC_MIN^b (μg/ml)	Potentiation (fold)^c
PAO1	8	>128	0.25 < x < 0.31	2	4
259-96918	16	>128	0.12 < x < 0.25	4	4
260-97103	16	>128	0.12 < x < 0.19	2	8
262-101856	64	>128	0.12 < x < 0.25	16	4
264-104354	32	>128	0.12 < x < 0.19	4	8
91433^d	32	>128	0.12 < x < 0.19	4	8
100036	16	>128	0.25 < x < 0.37	8	2
101243^d	4	>128	0.12 < x < 0.37	2	2
101885	16	>128	0.25 < x < 0.37	8	2

↵a MIN, minocycline; MDR, multidrug resistant; XDR, extensively drug resistant.
↵b MIC of minocycline in the presence of 8 μg/ml (5 μM) C₁₂-prp.
↵c Degree of antibiotic potentiation in the presence of 8 μg/ml (5 μM) of all d-lipopeptide C₁₂-prp.
↵d Colistin resistant.

TABLE 7

Adjuvant potency of amphiphilic C₁₂-prp in combination with rifampin against wild-type and MDR/XDR P. aeruginosa^a

P. aeruginosa strain	MIC_RMP (μg/ml)	MIC_C12-prp (μg/ml)	FIC index	Absolute MIC_RMP^b (μg/ml)	Potentiation (fold)^c
PAO1	8	>128	0.12 < x < 0.19	1	8
259-96918	16	>128	0.03 < x < 0.16	2	8
260-97103	16	>128	0.06 < x < 0.19	4	4
262-101856	512	>128	0.12 < x < 0.25	256	2
264-104354	8	>128	0.12 < x < 0.25	2	4
91433^d	16	>128	0.12 < x < 0.14	2	8
100036	16	>128	0.06 < x < 0.19	4	4
101243^d	4	>128	0.06 < x < 0.19	0.5	8
101885	16	>128	0.12 < x < 0.25	4	4

↵a RMP, rifampin; MDR, multidrug resistant; XDR, extensively drug resistant.
↵b MIC of rifampin in the presence of 8 μg/ml (5 μM) C₁₂-prp.
↵c Degree of antibiotic potentiation in the presence of 8 μg/ml (5 μM) of all d-lipopeptide C₁₂-prp.
↵d Colistin resistant.