Molecular Epidemiology of mcr-1, bla_KPC-2, and bla_NDM-1 Harboring Clinically Isolated Escherichia coli from Pakistan

Figures & data

Figure 1 Antibiotic resistance profile of all tested E. coli isolates.

Table 1 Antimicrobial Susceptibility Profile of mcr-1 Harbored Isolates

Isolate ID			MIC of Antibiotics in mg/L											ESBL	ESBL Genes	CRE
	AMP		FOX	CAP	CIP	COL	FOM	AZT	GEN	AN	MEM	CTX	TET	DDST		MHT
PKE211	256 (R)		16 (R)	8 (I)	4 (R)	4 (R)	32 (S)	128 (R)	4 (S)	64 (R)	0.064 (S)	256 (R)	1 (S)	+ve	CTXM-15/TEM-1	-ve
PKE141	256 (R)		32 (R)	2 (S)	0.032 (S)	4 (R)	4 (S)	128 (R)	16 (R)	128 (R)	0.032 (S)	64 (R)	256 (R)	+ve	CTXM-15	-ve
PKE196	>256 (R)		32 (R)	2 (S)	1 (S)	16 (R)	128 (I)	128 (R)	64 (R)	128 (R)	0.032 (S)	64 (R)	128 (R)	+ve	CTXM-15 TEM-1	-ve
PKE051	>256 (R)		64 (R)	1 (S)	0.064 (S)	8 (R)	8 (S)	256 (R)	32 (R)	256 (R)	0.032 (S)	128 (R)	128 (R)	+ve	CTXM-15 TEM-1	-ve

Abbreviations: ID, identification; ESBL, extended-spectrum β lactamases; MIC, minimum inhibitory concentration; MBL, metallo β lactamases; AMP, ampicillin; COL, colistin; CTX, cefotaxime; CAP, chloramphenicol; CIP, ciprofloxacin; fox, cefoxitin; FOM, fosfomycin; GEN, gentamycin;, An, amikacin; TET, tetracycline; AZT, aztreonam; DDST, double desk synergy test; CDT, combined desk test; CTX-M, cefotaximase Munich β-lactamase of ESBL-A type; TEM, temoneira, β-lactamase of ESBL-A type; CR, carbapenem resistant; (R), resistant; (S), susceptible; (I), moderate resistant.

Table 2 Prevalence of Solo or in Co-Existence of Antibiotic Resistance Genes Detected in This Study

Colistin Resistance Gene	Carbapenem Resistance Genes Among 120 MHT Positive Isolates					ESBL Producing Genes Among 139 ESBL Positive Strains				Non-ESBL SHV	No. of Isolates Having Multiples Genes (Total n=545)
mcr-1	blaKPC-2	blaOXA-48	blaNDM-1	blaIMP	blaVIM	blaCTX-M	blaTEM	blaSHV-75	blaOXA	blaSHV-11
+						+	+				3 (0.55%)
+						+					1 (0.18%)
	+					+	+				7 (1.28%)
	+						+	+			4 (0.73%)
	+					+					5 (0.92%)
					+					+	6 (1.1%)
	+				+		+				2 (0.37%)
	+		+								2 (0.37%)
	+				+						4 (0.73%)
	+										6 (1.1%)
		+	+				+				2 (0.37%)
		+						+			4 (0.73%)
		+	+		+						1 (0.18%)
		+			+						1 (0.18%)
		+									2 (0.37%)
			+			+			+		12 (21.8%)
			+			+					11 (2.02%)
			+		+						3 (0.55%)
			+	+	+						1 (0.18%)
			+		+				+		1 (0.18%)
			+	+							3 (0.55%)
			+								7 (1.28%)
				+		+	+				8 (1.47%)
				+					+		8 (1.47%)
				+	+						2 (0.37%)
				+							3 (0.55%)
					+	+					2 (0.37%)
					+						7 (1.28%)
						+					24 (4.40%)
						+	+				9 (1.65%)
						+	+		+		3 (0.55%)
						+			+		6 (1.1%)
						+			+	+	5 (0.92%)
						+				+	8 (1.47%)
							+		+		3 (0.55%)
							+			+	2 (0.37%)
							+				5 (0.92%)
										+	8 (1.47%)
									+	+	2 (0.37%)
4 (0.73%)	32 (26.6%)	10 (8.3%)	43 (35.8%)	25 (20.8%)	30 (25%)	104 (74.8%)	48 (34.5%)	8 (5.75%)	40 (28.7%)	31 (5.69%)	< ˂ No. of Isolates having Particular gene

Notes: Cell having (+) represent presence of particular gene in each cell. Empty cell represent the absence of particular gene. The underneath row represent the percentage of each gene.

Figure 2 Minimum spinning tree of mcr-1, bla_KPC-2, and bla_NDM-1 harboring E. coli by MLST type and alleles. Each node represent a sequence type, size of node represent the number of isolates, length of branches represent number of different alleles out of seven MLST alleles. Nodes are labeled with corresponding sequence type.

Table 3 Number of mcr-1, bla_KPC-2, and bla_NDM-1 Harboring Plasmids Incompatibility Type Prevalent on Various Sequence Types

Plasmid Type	Plasmid Sizes	ST	ST Cplx	Resistant Gene	No. of Isolates
Incl2	~ 60-kb	ST405	ST405 Cplx	mcr-1	¼ (25%)
Incl2	~ 60-kb	ST117	ST23Cplx	mcr-1	¼ (25%)
Incl2	~ 60-kb	ST156	ST156Cplx	mcr-1	2/4 (50%)
IncH12	~ 140-kb	ST 131	ST131 Cplx	blaKPC-2	11/32 (34.38%)
IncN	~ 100-kb	ST 1196	None	blaKPC-2	5/32 (15.63%)
Incl1α	~ 90-kb	ST 7584	None	blaKPC-2	4/32 (12.5%)
IncN	~ 100-kb	ST 405	ST405 Cplx	blaKPC-2	3/32 (9.37%)
IncH12	~ 140-kb	ST 1196	None	blaKPC-2	2/32 (6.25%)
IncH12	~ 140-kb	ST 405	ST405 Cplx	blaKPC-2	2/32 (6.25%)
IncFIIK	~ 70-kb	ST 1196	None	blaNDM-1	17/43 (39.53%)
IncH12	~ 140-kb	ST 131	ST131 Cplx	blaNDM-1	9/43 (20.93%)
IncH12	~ 140-kb	ST 10,184	ST69 Cplx	blaNDM-1	5/43 (11.63%)
IncN	~ 100-kb	ST 1196	None	blaNDM-1	4/43 (9.30%)
IncN	~ 100-kb	ST 8671	None	blaNDM-1	1/43 (2.32%)
IncA/C	~ 60-kb	ST 7584	None	blaNDM-1	4/43 (9.3%)
IncN	~ 100-kb	ST 405	ST405 Cplx	blaNDM-1	2/43 (4.65%)
IncFII	~ 45-kb	ST 10,184	ST69 Cplx	blaNDM-1	1/43 (2.33%)

Note: 5 ST131 bla_KPC-2 strains plasmid were not detected.

Abbreviations: kb, kilo base; ST, sequence type; ST Cplx, sequence type complex.

Figure 3 S1 PFGE and Southern blot of mcr-1 harboring strains. H9812 is a molecular size marker (1135 to 20.5-kb). PKE211, PKE196, and PKE14, PKE051 are the isolates; digested with S1 enzymes. All isolates show 60-kb plasmid. The red arrows represent plasmids on PFGE gel. Black arrows represent plasmids on nylon membrane.

Figure 4 S1 PFGE and Southern blot of bla_KPC-2 harboring strains. H9812 is a molecular size marker. PKE003 and PKE091 isolates have 90-kb plasmid, PKE201 isolate has 140-kb plasmid, and PKE470 and PKE482 isolates have 100-kb plasmid. The red arrows represent plasmids on PFGE gel. Black arrows represent plasmids on nylon membrane.

Figure 5 S1 PFGE and Southern blot of bla_NDM-1 harboring strains. H9812 is a molecular size marker. PKE470 isolate have 100-kb plasmid, PKE131 isolate have 70-kb plasmid, PKE134 isolate have 45-kb plasmid, PKE263 isolate have 140-kb plasmid, and PKE303 isolate have 60-kb plasmid. The red arrows represent plasmids on PFGE gel. Black arrows represent plasmids on nylon membrane.

Figure 6 PCR mapping of mcr-1 genetic background. (A) PCR amplified vird4, pilN, hp, ParA, mcr-1, tnpA, and nikB, and tnpA genes, red circle show the shortened band of tnpA. (B) pHNSPH45 reference map, indicating Pap2 on downstream of mcr-1 and IsApl1 on upstream and. (C) mcr-1 genetic setting in our strains, IsApl1 is missing due to truncated tnpA. The black, red, and grey arrows represent particular genes having a name written underneath of each arrow. (*) on tnpA indicates truncation in tnpA gene.