Characterization of antimicrobial resistance mechanisms in carbapenem-resistant Pseudomonas aeruginosa carrying IMP variants recovered from a Mexican Hospital

Figures & data

Table 1 Phenotype and genotype of resistance of P. aeruginosa clinical isolates carrying bla_IMP gene

Strain	PFGE	Phenotype of resistance	^aMIC (μg/mL)							Genotype	Amino acid changes
	type		CAZ	FEP	MEM (+ ^bEPI)	IMP	CIP (+ ^bEPI)	AN	GM		GyrA	ParC
PE06	15	PIP, TIC, TZP, CAZ, FEP, ATM, IPM, MEM, GM, NN, AN, CIP, NOR	>512	>512	>512 (>512)	>512	>512 (512)	>512	32	blaIMP-18, blaIMP-56, blaOXA-2, blaOXA-50, aadA1, aacA6	T831	S87L
PE09	6a	TIC, CAZ, FEP, IPM, MEM, GM, NN, AN, CIP, NOR	>512	512	>512 (32)	64	64 (32)	256	>512	blaIMP-18, blaOXA-2, blaOXA-50, aacA4	T831	S87L
PE10	8	PIP, TIC, CAZ, FEP, ATM, IPM, MEM, GM, NN, AN, CIP, NOR	512	256	64 (64)	64	32 (16)	64	>512	blaIMP-18, blaOXA-2, blaOXA-50, aacA4	T831	S87L
PE15	7	TIC, CAZ, FEP, IPM, MEM, GM, NN, AN, CIP, NOR	512	256	64 (64)	>512	32 (16)	64	>512	blaIMP-18, blaOXA-2, blaOXA-50, aacA4	T831	S87L
PE16	6b	TIC, CAZ, FEP, IPM, MEM, GM, NN, AN, CIP, NOR	512	256	64 (64)	>512	32 (16)	64	>512	blaIMP-18, blaOXA-2, blaOXA-50, aacA4	T831	S87L
PE20	1	PIP, TIC, CAZ, FEP, IPM, MEM, GM, NN, AN, CIP, NOR	>512	>512	>512 (128)	>512	128 (64)	>512	>512	blaIMP-18, blaOXA-2, blaOXA-50, aacA4	T831	S87L
PE21	28	TIC, CAZ, FEP, IPM, MEM, GM, NN, AN, CIP, NOR	>512	512	>512 (>512)	128	128 (64)	>512	>512	blaIMP-62, blaGES-32, blaOXA-2, blaOXA-50, aacA33, aadA1, aadA6, aacA4, aacA7	T831	S87L
PE22	12	TIC, CAZ, FEP, IPM, MEM, GM, NN, AN, CIP, NOR	512	256	16 (16)	64	128 (64)	128	>512	blaIMP-18, blaOXA-2, blaOXA-50, aac(3′)-IIa, aacA4	T831	S87L
PE23	10	TIC, FEP, IPM, MEM, GM, NN, AN, CIP, NOR	16	32	32 (32)	64	32 (16)	64	>512	blaIMP-62, blaGES-2, blaOXA-2, blaOXA-50, aacA4, aacA29b	T831	S87L
PE27	25	TIC, CAZ, FEP, IPM, MEM, GM, NN, AN, CIP, NOR	512	256	64 (64)	>512	64 (32)	128	512	blaIMP-62, blaOXA-2, blaOXA-50, aacA4	T831	S87L
PE46	32	TIC, CAZ, FEP, IPM, MEM, GM, NN, AN, CIP, NOR	>512	512	>512 (32)	64	64 (32)	512	>512	blaIMP-18, blaOXA-2, blaOXA-50, aacA4	T831	S87L
PE52	16	PIP, TIC, TZP, CAZ, FEP, ATM, IPM, MEM, GM, AN, CIP, NOR	>512	>512	>512 (>512)	>512	>512 (512)	>512	32	blaIMP-18, blaIMP-56, blaOXA-2, blaOXA-50, aadA1, aacA6	T831	wild type
PE62	20	PIP, TIC, CAZ, FEP, ATM, IPM, MEM, GM, NN, AN, CIP, NOR	>512	>512	>512 (>512)	>512	>512 (512)	>512	16	blaIMP-18, blaOXA-2, blaOXA-50	T831	S87L
PE63	38	PIP, TIC, TZP, CAZ, FEP, ATM, IPM, MEM, GM, NN, AN, CIP, NOR	256	232	512 (512)	512	128 (64)	512	>512	blaIMP-18, blaGES-1, blaOXA-2, blaOXA-50, aacA4, aadAI	wild type	P105T
PE64	21	PIP, TIC, CAZ, FEP, ATM, IPM, MEM, GM, NN, AN, CIP, NOR	256	512	>512 (512)	>512	512 (256)	>512	32	blaIMP-18, blaOXA-2, blaOXA-50	T831	S87L
PE73	26	PIP, TIC, CAZ, FEP, ATM, IPM, MEM, GM, NN, AN, CIP, NOR	>512	>512	>512 (>512)	>512	>512 (512)	>512	32	blaIMP-56, blaOXA-2, blaOXA-50	T831	S87L
PE83	13	TIC, CAZ, FEP, IPM, MEM, GM, NN, AN, CIP, NOR	>512	>512	>512 (>512)	64	256 (128)	>512	>512	blaIMP-62, blaGES-26, blaOXA-2, blaOXA-50, aac(3′)-IIa, aacA4, aadA6, aacA7	T831	S87L

Notes: ^aMinimum Inhibitory Concentration;

b PAβN (L-phenylalanine-arginine-β-naphthylamide).

Abbreviations: PFGE, pulsed-field gel electrophoresis; PIP, piperacillin; TIC, ticarcillin; TZP, tazobactam; CAZ, ceftazidime; FEP, cefepime; IMP, imipenem; MER, meropenem; GM, gentamicin; NN, tobramycin; AN, amikacin; CIP, ciprofloxacin; NOR, norfloxacin; ATM, aztreonam.

Figure 1 Diversity of class 1 integron arrangements detected in bla_IMP-carrying P. aeruginosa clinical isolates.

Notes: The In1215 containing bla_IMP-18 with the accession number KY646160 and the In1343 containing bla_IMP-56 with the accession number KY646161. Arrows indicate the open reading frames.

Figure 2 Detection of the OprD protein in 17 clinical bla_IMP-carrying P. aeruginosa. (A, C, E) PAGE-SDS of extract total protein, (B, D, F) Western blot of extract total protein with polyclonal antisera anti-oprD.

Notes: For each lane, 30 μg of proteins were loaded. P. aeruginosa PAO1 and OprD protein recombinant were used as positive controls. (A, B) Line 1: Molecular weight marker (Spectra^tm); Line 2: recombinant OprD protein; Line 3: P. aeruginosa PAO1; Line 4: PE06 (stop codon); Line 5: PE09 (L7-short); Line 6: PE15 (not amplified); Line 7: PE16 (stop codon), Line 8: PE20 (IS1394); Line 9: PE21 (stop codon). (C, D) Line 1: Size marker; Line 2: recombinant OprD protein; Line 3: P. aeruginosa PAO1; Line 4: PE22 (L7-short); Line 5: PE23 (stop codon); Line 6: PE27 (partially amplified), Line 7: PE46 (L7-short); Line 8: PE52 (stop codon); Line 9: PE62 (stop codon); Line 10: PE63 (wild type). (E, F) Line 1: Size marker; Line 2: recombinant OprD protein; Line 3 P. aeruginosa PAO1; Line 4: PE64 (stop codon); Line 5: PE73 (stop codon); Line 6: PE83 (stop codon) and Line 7: PE10 (L7-short). The experiment was made in duplicate.

Table 2 Changes in OprD porin in clinical isolates of P. aeruginosa carrying bla_IMP

Strain	^aMIC (μg/mL)			OprD size	Amino acid changes in OprD sequence	Insertion/deletion
	IMP		MEM	(Number of amino acids)
PE06	>512	>512		426	D43N, S57E, S59R, E202Q, I210A, E230K, S240T, N262T, A267S, A281G, K296Q, Q301E, R310G, V359L, M372V, S373D, D374S, N375S, N376S, V377S, Δ378, Δ379, K380Y, N381A, Y382G, G383L E426STOP	Stop codon
PE09	64	>512		441	D43N, S57E, S59R, E202Q, 1210a, E230K, S240T, A267S, K296Q, Q301E, R310G, V359L, M372V, S373D, D374S, N375S, N376S, V377S, Δ378, Δ379, K380Y, N381A, Y382G, G383L	Loop L7-short
PE10	64	64		441	D43N, S57E, S59R, E202Q, I210A, E230K, S240T, N262T, A267S, A281G, K296Q, Q301E, R310G, V359L, M372V, S373D, D374S, N375S, N376S, V377S, Δ378, Δ379, K380Y, N38IA, Y382G, G383L	Loop L7-short
PE 15	>512	64		^bNA	^bNA	^bNA
PE 16	>512	64		422	D43N, S57E, S59R, E202Q, I210A, E230K, S240T, N262T, A267S, A281G, K296Q, Q301E, R310G, V359L, M372V, S373D, D374S, N375S, N376S, V377S, Δ378, Δ379, K380Y, N381A, Y382G, G383L A422STOP	Stop codon
PE20	>512	>512		232	D43N, S57E, S59R, E202Q, I210A, E230K, I232M, Y233A, R234A, IS/394	Truncated by IS/394
PE21	128	>512		417	D43N, S57E, S59R, E202Q, I210A, E230K, S240T, N262T, A267S A281G, K296Q, Q30IE R310G V359L, M372V, S373D, D374S, N375S, N376S, V377S, Δ378, Δ379, K380Y, N381A, Y382G, G383L W417STOP	Stop codon
PE22	64	16		441	D43N, S57E, S59R, E202Q, I210A, L224F, E230K, S240T, N262T, A267S A281G, K296Q, Q301E, R310G, V359L, M372V, S373D, D374S, N375S, N376S, V377S, Δ378, Δ379, K380Y, N381A, Y382G, G383L	Loop L7-short
PE23	64	32		417	D43N, S57E, S59R, E202Q, I210A, E230K, S240T, N262T, A267S A281G, K296Q, Q30IE, L307M, R310G, A316G, M372V, S373D, D374S, N375S, N376S, V377S, Δ378, Δ379, K380Y, N381A, Y382G, G383L W417STOP	Stop codon
PE27	>512	64		^bNA	D43N, S57E, S59R, E202Q, I210A, E230K, S240T, N262T, A267S	^bNA
PE46	64	>512		441	D43N, S57E, S59R, E202Q, I210A, E230K, S240T, N262T, A267S, W277R, A281G, A282G, Y283S, T284G, K296Q, Q30IE, R310G, V359L, M372V, S373D, D374S, N375S, N376S, V377S, Δ378, Δ379, K380Y, N38IA, Y382G, G383L	Loop L7-short
PE52	>512	>512		426	D43N, S57E, S59R, E202Q, I210A, E230K, S240T, N262T, A267S, A281G, K296Q, Q301E, R310G, V359L, M372V, S373D, D374S, N375S, N376S, V377S, Δ378, Δ379, K380Y, N381A, Y382G, G383L, E426STOP	Stop codon
PE62	>512	>512		426	D43N, S57E, S59R, E202Q, I210A, E230K, S240T, N262T, A267S, A280G, A281G, Y283S, T284A, K296Q, Q301E, R310G, V359L, M372V, S373D, D374S, N375S, N376S, V377S, Δ378, Δ379 K380Y, N381A, Y382G, G383L, E426STOP	Stop codon
PE63	512	512		443	Wild type	-
PE64	>512	>512		295	D43N, S57E, S59R E202Q, I210A, E230K, S240T, N262T, A267S, A281G, K295STOP	Stop codon
PE73	>512	>512		426	D43N, S57E, S59R, E202Q, I210A, E230K, S240T, N262T, A267S, A281G, K296Q, Q30IE, R310G, V359L, M372V, S373D, D374S, N375S, N376S, V377S, Δ378, Δ379, K380Y, N381A, Y382G, G383L, E426STOP	Stop codon
PE83	64	>512		417	D43N, S57E, S59R, E202Q, I210A, E230K, S240T, N262T, A267S A281G, K296Q, Q301E, R310G, V359L, M372V, S373D, D374S, N375S, N376S, V377S Δ378, Δ379, K380Y, N381A, Y382G, G383L W417STOP	Stop codon

Notes: ^aMinimum Inhibitory Concentration;

b Not amplified or partially amplified; IMP, imipenem; MEM, meropenem.

Figure 3 PFGE-S1 plasmids of 10 clinical isolates of P. aeruginosa carrying bla_IMP.

Notes: The arrows indicate the plasmids. Lane 1: PE06, Lane 2: PE09, Lane 3: PE20, Lane 4: PE21, Lane 5: PE46, Lane 6: PE52, Lane 7: PE62, Lane 8: PE63, Lane 9: PE73, Lane 10: PE83, Lane 11: Control strain Rhizobium etli CFN42 Δe, Lane 12: Negative control PAO1, Lane 13: Control strain E. coli NCTC 50192 with four plasmids: 154 kb, 66 kb, 48 kb and 7 kb. The experiment was performed twice.
Abbreviation: PFGE-S1, Pulsed-field gel electrophoresis with nuclease S1.

Table 3 Plasmid characterization of 17 clinical bla_IMP-carrying P. aeruginosa isolates

Strain	β-lactamase gene	Plasmids size (kb)	^aRelaxase MOB subfamily
PE06	blaIMP-18, blaIMP-56	2(approx 224, approx 188)	MOBP11
PE09	blaIMP-18	Absent	Absent
PE 10	blaIMP-18	Absent	Absent
PE 15	blaIMP-18	Absent	Absent
PE 16	blaIMP-18	Absent	Absent
PE20	blaIMP-18	Absent	Absent
PE21	blaIMP-62, blaGES-32	1(approx 368)	MOBP11 MOBH2
PE22	blaIMP-18	Absent	Absent
PE23	blaIMP-62, blaGES-2	Absent	Absent
PE27	blaIMP-62	Absent	Absent
PE46	blaIMP-18	Absent	Absent
PE52	blaIMP-18, blaIMP-56	2(approx 242, approx 194)	MOBP11
PE62	blaIMP-18	2(approx 248, approx 200)	MOBP11
PE63	blaIMP-18, blaGES-1	3(approx 230, approx 200, approx 182)	MOBP14
PE64	blaIMP-18	1(approx 146)	MOBP11
PE73	blaIMP-56	1(approx 266)	MOBP11
PE83	blaIMP-62, blaGES-26	1(approx 350)	MOBP11 MOBH2

Notes: ^aDetection of relaxases was performed by Degenerate Primer MOB Typing (DPMT).¹⁷

Figure 4 Detection of bla_IMP in plasmids of eight P. aeruginosa strains by Southern blot.

Notes: (A) PFGE after digestion with nuclease S1 (180 U/μL, Takara Bio Inc.), (B) Southern hybridization of plasmids (DIG HIGH Prime DNA Labeling and Detection Starter Kit II) using bla_IMP as a probe. Line 1: Control strain E. coli NCTC 50192 with four plasmids: 154 Kb, 66 Kb, 48 Kb and 7 Kb, Line 2: PE06; Line 3: PE21; Line 4: PE52; Line 5: PE62; Line 6: PE63; Line 7: PE64; Line 8: PE73 and Line 9: PE83. The black arrows indicate the positive signal corresponding to the hybridization with bla_IMP in plasmids. The experiment was done three times.
Abbreviation: PFGE-S1, Pulsed-field gel electrophoresis with nuclease S1.

Figure S1 Dendrogram of 41 multidrug-resistant P. aeruginosa clinical isolates obtained in ISSSTE Regional Hospital of Puebla, Mexico.

Notes: The isolates showing a Dice coefficient ≥80% were considered as genetically related for this study. PFGE type (PT), PFGE clusters (A, B and C).
Abbreviation: PFGE, pulsed-field gel electrophoresis.

Figure S2 Plasmid extraction by the Quick Prep method of 17 clinical bla_IMP-carrying P. aeruginosa isolates.

Notes: The position of chromosomal DNA is indicated by an arrow at the right. Line 1: Size marker; Line 2: PE06; Line 3: PE09; Line 4: PE15; Line 5: PE16; Line 6: PE20; Line 7: PE21; Line 8: PE22; Line 9: PE23; Line 10: PE27; Line 11: PE46; Line 12: PE52; Line 13: PE62, Line 14: PE63; Line 15: PE64; Line 16: PE73; Line 17: PE10; Line 18: PE83; Line 19: Control E. coli NCTC 50192; Line 20: P. aeruginosa PAO1.