Small Molecule Adjuvants Potentiate Colistin Activity and Attenuate Resistance Development in Escherichia coli by Affecting pmrAB System

Figures & data

Table 1 Minimum Bactericidal Concentration (MBC) of Antibiotics When Tested in Combination with SMs

AB	MBC (μg/mL)
	AB Alone	AB + SM
		SM1	SM2	SM3	SM4	SM5	SM6	SM7	SM8	SM9	SM10	SM11
Col	2	1.6^I	0.2^S	0.2^S	0.2^S	0.2^S	0.2^S	0.2^S	0.8^S	0.2^S	0.8^I	0.2^S
Tet	128	76.8^I	12.8^S	12.8^S	128^I	12.8^S	76.8^I	12.8^S	128^I	51.2^I	128^I	25.6^I
Cip	1	1^I	0.1^I	0.1^I	0.1^S	0.1^S	0.1^I	0.1^I	0.2^I	0.1^I	1^I	0.1^S

Notes: ^SSynergistic, ^IIndifferent.

Abbreviations: AB, antibiotic; Col, colistin; Tet, tetracycline; Cip, ciprofloxacin.

Table 2 Minimum Bactericidal Concentration (MBC) of SMs When Tested with Antibiotics

	MBC (µM)
	SM Alone	SM + Col	SM + Tet	SM + Cip
SM1	150	15	60	150
SM2	100	10	40	60
SM3	100	10	10	100
SM4	50	5	50	5
SM5	15	1.5	6	6
SM6	15	1.5	9	12
SM7	50	5	5	30
SM8	15	1.5	15	15
SM9	30	3	12	24
SM10	50	20	50	50
SM11	100	10	40	40

Abbreviations: Col, colistin; Tet, tetracycline; Cip, ciprofloxacin.

Table 3 Minimum Bactericidal Concentration (MBC) of Colistin Against APEC O1 and O2 When Tested with Selected SMs

	MBC (μg/mL)
	APEC O1			APEC O2
	Col Alone	Col + SM	FIC	Col alone	Col + SM	FIC
SM2	4	0.4	0.2^S	4	0.4	0.2^S
SM3	4	0.4	0.2^S	4	0.4	0.2^S

Note: ^SSynergistic.

Abbreviation: Col, colistin.

Figure 1 (A) Survival curve of wax moth larvae treated with colistin alone or in combination with SMs. Larvae (n=15 larva/group) were infected with 6.4×10⁴ CFU of Rif^r APEC O78 and then treated with colistin (0.3125 mg/kg) and combination of colistin and SMs (0.3125 mg/kg+12.5 µg) within 30 min of infection. Larval survival was monitored every 12 h for 3 days. (B) APEC load inside wax moth larvae treated with colistin alone or in combination with SMs. APEC load in larvae was quantified by plating the homogenized larval suspension on MacConkey agar plates supplemented with 50 µg/mL rifampicin. PC-infected and buffer mix (DMSO+PBS) treated larvae, ***P<0.0001.

Figure 2 (A) Minimum inhibitory concentration (MIC) of APEC cultures passaged only in presence of colistin or in combination with SMs. Six parallel independent APEC cultures (Col 1–6, Col + SM2 1–6, and Col+ SM3 1–6) were serially passaged (8 passages) in M63 media starting with the sub-inhibitory concentration of colistin (0.0625 µg/mL) to up to 8 µg/mL (beyond resistance breakpoint) in presence and absence of 0.5× MIC of SMs. APEC cultures containing no colistin and no SM (1% DMSO; DMSO 1–6) were used as controls and passaged similarly to cultures containing colistin or colistin and SMs. The black dotted line indicates the MIC breakpoint for colistin resistance. (B) Chemical structure and ChemBridge ID of selected SMs.

Table 4 Mutations Observed in Col^R APEC Isolates Analyzed Using PointFinder Database

Isolate	Mutation	Nucleotide Change	Amino Acid Change
Col-1	pmrA p.A80V	GCT -> GTT	A -> V
Col-3	pmrB p.T92P	ACC -> CCC	T -> P
Col-5	pmrB p.L14Q	CTG -> CAG	L -> Q
Col-6	pmrB p.L14Q	CTG -> CAG	L -> Q
Col+SM2-2	pmrB p.L14Q	CTG -> CAG	L -> Q
Col+SM3-5	pmrB p.L14Q	CTG -> CAG	L -> Q

Table 5 Non-Synonymous Mutations Observed in Col^R APEC Isolates

Product (Protein Id)	Type of Mutations	Amino Acid Changes
Sensor protein BasS/PmrB [AGC85036.1]	SNVs (A to T, T to G)	L14Q, T92P
Two-component response regulator BasR/PmrA [AGC85037.1]	SNV (G to A)	A80V
Hypothetical proteins [AGC85973.1, AGC85984.1, AGC85542.1, AGC85972.1]	SNVs, MNV, deletions, insertion	R15L, Q92R, D27N, A496_Q527del, A55_D59dup, S40fs
Putative transposases [AGC87419.1]	SNVs, MNVs	C22R, G33P, H219Y, E179D, V177I, N76K, R18L, K201R, L190T, A157V
Phage replication protein O [AGC84960.1]	SNVs, insertion	H152Q, S101A, A55_D59dup
Antitermination protein Q from phage origin [AGC86222.1]	SNVs, MNVs	D9E, A15V, I20V, V21G, S27A, G30R, C33G, C33_C34delinsWR, L41_R42delinsRL, I49V, I50V, G37C, Y38_I39delinsWV, A56M, E51D
Increased serum survival (Iss) [AGC85979.1]	deletion	*98fs
Rz endopeptidase from lambdoid prophage DLP12 [AGC86226.1]	SNVs, deletion, insertion	*154fs, E142*, T144A, R125Q, T129fs, T129S
Lambdoid prophage DLP12 Bor-like protein [AGC86227.1]	SNV, MNV, deletion	*98fs, L83F, K49E
Bacteriophage lysis protein [AGC85980.1]	Deletion	*154fs
Putative portal protein [AGC87035.1]	SNV	R237S
Terminase large subunit [AGC87037.1]	SNV, insertion, replacement	W318fs, M308V
YD repeat protein [AGC86138.1]	SNV	A68G
Uroporphyrinogen III C-methyltransferase [AGC89286.1]	Insertion	P389_Q390insAPAP

Note: *Stop codon.

Abbreviations: del, deletion; ins, insertion; dup, duplication; fs, frame shift.

Figure 3 Heatmap displaying the non-synonymous mutational comparison between Col^R isolates (Col-1, Col-3, Col-5, Col-6, Col + SM2-2 and Col + SM3-5) as compared to Col^S (DMSO-1, DMSO-3, Col + SM2-4, Col + SM2-6, Col + SM3-2 and Col + SM3-6) isolates. Phenotype “R” indicates resistant APEC isolate and “S” indicates susceptible APEC isolate.

Abbreviation: NA, not available.

Figure 4 (A) Expression (fold change) of pmrC and pmrH in APEC isolates passaged with colistin alone (Col-1, Col-3, Col-5 and Col-6), colistin and SM2 (Col + SM2-2, Col + SM2-4 and Col + SM2-6), and colistin and SM3 (Col + SM3-2, Col + SM3-5 and Col + SM3-6) as compared to control (DMSO-1 and DMSO-3) isolates. (B) Expression (fold change) of pmrA and pmrB in APEC isolates passaged with colistin alone (Col-1, Col-3, Col-5 and Col-6), colistin and SM2 (Col + SM2-2, Col + SM2-4 and Col + SM2-6), and colistin and SM3 (Col + SM3-2, Col + SM3-5 and Col + SM3-6) as compared to control (DMSO-1 and DMSO-3) isolates. Phenotype “R” indicates resistant APEC isolate and “S” indicates susceptible APEC isolate.

Table 6 Summary of Colistin Adjuvants Tested Against Different Pathogenic Bacteria

Bacteria	Adjuvant Compound	Increase in Efficacy in vitro	Increase in Efficacy in vivo	Reference
Col^S A. baumannii	Urea containing class of 2-aminoimidazoles	MIC reduction by 1000-fold	Increased survival (36–50%) of G. mellonella larva	[^Citation13]
Col^R E. coli	Eugenol	MIC reduction up to 8-fold	NA	[^Citation21]
Col^R A. baumannii	Pentamidine	NA	Rescued (10 of 11) mice infected with Col^R A. baumannii	[^Citation19]
Col^S and Col^R A. baumannii	Protegrin-1	Synergistic (FIC<0.5)	NA	[^Citation25]
Methicillin-resistant S. aureus and MDR P. aeruginosa	LL-37, cecropin	MIC reduction by at least eight-fold	NA	[^Citation26]
MDR A. baumannii	Daptomycin	Synergistic (≥2 log reduction compared to colistin alone)	NA	[^Citation24]
P. aeruginosa	Clomiphene citrate, mitoxantrone dihydrochloride, methyl benzethonium chloride, benzethonium chloride, and auranofin	Synergistic (FIC<0.5)	NA	[^Citation27]
Col^S and Col^R K. pneumoniae, A. baumannii	Compound 1, 2, 3 and 4	MIC reduction by >32-fold	NA	[^Citation22]
Col^R P. aeruginosa, K. pneumoniae, A. baumannii, E. coli, and Enterobacter cloacae	Niclosamide	MIC reduction by 64-fold	NA	[^Citation18]
Col^S and Col^R K. pneumoniae, A. baumannii, E. coli, E. cloacae, Citrobacter braakii, Stenotrophomonas maltophilia, Serratia marcescens, and Proteus mirabilis	Resveratrol	Synergistic (FIC<0.5)	NA	[^Citation17]
Col^S and Col^R A. baumannii	N-acetylcysteine	Synergistic (FIC<0.5)	NA	[^Citation16]
Col^S and potentially Col^R E. coli	Pyrrolidinyl SMs (SM2 and SM3)	MIC reduction by at least 10-fold	Twofold (>4.5 logs reduction compared to colistin alone), increased survival (>45%) of G. mellonella larva	This study

Abbreviation: NA, not available.

Table 7 Summary of pmrB and pmrA Mutations Reported in Col^R E. coli

Bacteria/Strain/Source	Observed Mutations	Reference
E. coli O25 (human clinical specimens)	Deletion ∆27-45 LISVFWLWHESTEQIQLFE; pmrB	[^Citation53]
E. coli O25 and O18 (human clinical specimens)	G206D; pmrB	[^Citation53]
E. coli O25 (human clinical specimens)	L105P; pmrA	[^Citation53]
E. coli LC711/14 (urinary tract infected human patient)	L10P; pmrB	[^Citation48]
E. coli (human clinical specimens)	A118T, E123D, Y315F, and Y358N; pmrB	[^Citation54]
E. coli (Parisian fecal samples)	L10P, L10R, C84R, C84Y, P94A, P94L, P94Q, E121K, E121Q, A159P, and A159V; pmrB G53A, G53C, G53E, G53R, G53S, G53V, G53W, R81L, and R81S; pmrA	[^Citation57]
E. coli (swine)	S138N, E123D, T156A, G160E, V161G, and V351I; pmrB G144S, S39I, and R81S; pmrA	[^Citation55,Citation56]
E. coli (calf cecum samples)	D283G and Y358N; pmrB	[^Citation58]
E. coli (poultry farms)	P94L and D283G; pmrB S29G; pmrA	[^Citation59]

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