Detection and characterisation of extended-spectrum and plasmid-mediated AmpC β-lactamase produced by Escherichia coli isolates found at poultry farms in Bosnia and Herzegovina

Fetahagić, Majda; Ibrahimagić, Amir; Uzunović, Selma; Beader, Nataša; Elveđi-Gašparović, Vesna; Luxner, Josefa; Gladan, Muhamed; Bedenić, Branka

doi:10.2478/aiht-2021-72-3560

Figures & Tables

Phenotypic detection of ESBL producers with DDST. The elliptical inhibitory zone between cephalosporins and clavulanic acid (augmentin) indicates an ESBL-producing organism. AUG – augmentin; CAZ – ceftazidime; CPM – cefepime; CRO – ceftriaxone; CTX – cefotaxime

Phenotypic detection of ESBL-producing organisms with the combined disk test. Positive results are those with visible inhibition zones (>5 mm in diameter) around CAZ, CTX, and CRO disks in combination with clavulanate vs those without clavulanate. CAZ – ceftazidime; CRO – ceftriaxone; CTX – cefotaxime

Cephalosporin inactivation method (CIM) with the control E. coli ATCC 25922 strain. Left: cefotaxime disk (10 mg) was placed in a heavy suspension of test strains. Right: cefotaxime disk on the surface of the plate as negative control

Combined disk test for the detection of plasmid-mediated AmpC β-lactamases with the cefotaxime disk alone (left) and in combination with 3-aminophenylboronic acid (right)

Conjugation frequency and cotransferred resistance markers

Isolate No.	Protocol number	Frequency	Cotransferred resistance markers
1	31	8×10^-5	Gm, Smx
2	32	5.5×10^-4
3	35	3.5×10^-5
4	36	1.2×10^-4
5	37	1.4×10^-5
6	39	8.4×10^-5
7	40	4.5×10^-5
8	41	0
9	43	10^-4	Tet
10	44	5×10^-4	Tet
11	45	1.6×10^-6	Tet
12	46	4.5×10^-6	Tet
13	47	1.6×10^-4	Tet
14	49	0
15	50	0
16	51	7.2×10^-6	Smx
17	53	1.57×10^-5
18	54	0
19	55	5.6×10^-6
20	56	0
21	58	0
22	59	0
23	60	0
24	64	1.5×10^-4
25	65	1.2×10^-4
26	66	3.1×10^-5	Tet
27	69	0

Antibiotic minimum inhibitory concentrations, genes, plasmids, and genotyping of E_ coli isolates from poultry farms in Bosnia and Herzegovina

No.	Protocol number	Minimum inhibitory concentrations (mg/L) and resistance breakpoint (≥) of antibiotics													Genes, plasmids, and genotypes
No.	Protocol number	AMX >32	AMC 128/16	TZP >128/4	CZ >4	CXM >32	CAZ >32	CTX >4	CRO >4	FEP >32	IMI >4	MEM >4	GM >16	CIP >4	ESBL	bla genes	PBI and ST
1	31	>128	8	32	>128	>128	64	>128	>128	64	0.5	0.25	>128	>128	+	TEM, CTX-M-15	FIA
2	32	>128	32	8	>128	32	0.5	32	32	0.25	0.12	0.06	2	0.25	+	ND	ND
3	35	>128	1	2	>128	>128	2	>128	>128	0.5	0.06	0.06	0.5	2	+	CTX-M-1 cluster	FIA
4	36	>128	4	8	>128	>128	2	64	64	1	0.06	0.06	1	0.25	+	CTX-M-1 cluster	FIB, ST 117
5	37	>128	8	16	>128	>128	1	>128	64	1	0.12	0.06	1	0.5	+	ND	FIB
6	39	>128	4	8	>128	>128	2	64	64	0.5	0.12	0.12	2	0.12	+	TEM, CTX-M-15	ND
7	40	>128	8	4	>128	>128	0.5	>128	>128	0.5	0.06	0.06	1	0.25	+	ND	ND
8	41	>128	1	4	>128	32	1	64	32	1	0.25	0.12	2	0.25	+	CTX-M-1 cluster	ND
9	43	>128	16	16	>128	>128	8	>128	>128	1	0.25	0.06	4	0.06	+	ND	ND
10	44	>128	32	16	>128	>128	32	>128	>128	2	0.06	0.06	0.5	0.12	+	ND	FIB
11	45	>128	16	16	>128	>128	1	>128	>128	0.5	0.5	0.06	2	0.12	+	CTX-M-1 cluster	ND
12	46	>128	8	4	>128	>128	2	>128	64	1	0.12	0.12	4	0.06	+	TEM, CTX-M-1 cluster	ND
13	47	>128	8	8	>128	>128	16	64	64	1	0.06	0.06	1	0.25	+	TEM, CTX-M-1 cluster	I1
14	49	>128	4	16	>128	>128	4	>128	>128	16	0.25	0.06	0,5	16	+	TEM, CTX-M-1 cluster	I1
15	50	>128	8	16	>128	>128	8	>128	>128	16	0.25	0.12	2	64	+	TEM, CTX-M-15	I1, FIB
16	51	>128	2	4	>128	>128	0.5	>128	>128	0.5	0.12	0.12	2	0.25	+	ND	FIB
17	53	>128	2	4	>128	>128	8	>128	>128	8	0.25	0.06	1	0.25	+	TEM, CTX-M-15	ND
18	54	>128	4	2	>128	>128	1	>128	64	0.5	0.5	0.06	0.5	0.12	+	TEM, CTX-M-15	I1, FIB
19	55	>128	1	2	>128	>128	1	>128	>128	2	0.06	0.06	0.5	0.5	+	TEM, CTX-M-1 cluster	I1
20	56	>128	4	32	>128	>128	16	>128	>128	16	0.5	0.25	2	4	+	TEM, CTX-M-1 cluster	I1, HI1
21	58	>128	2	2	>128	>128	2	>128	>128	8	0.25	0.25	1	0.25	+	CTX-M-1 cluster	ND
22	59	>128	32	4	>128	64	0.5	>128	64	0.5	0.12	0.12	16	32	+	TEM, CTX-M-1 cluster	I1
23	60	>128	32	16	>128	>128	16	>128	32	2	0.5	0.25	1	32	+	CTX-M-1 cluster	I1, HI1, ST155
24	64	>128	32	64	>128	16	>128	>128	32	4	1	0.5	2	>128	+	TEM, CTX-M,-1 cluster	I1, FIB
25	65	>128	4	8	>128	>128	2	64	32	2	0.06	0.06	0.25	0.12	+	TEM, CTX-M-1 cluster	I1
26	66	>128	32	4	>128	32	>128	>128	16	1	0.06	0.06	0.5	64	+	TEM, CTX-M-1 cluster	FIB
27	69	>128	8	2	>128	>128	2	>128	>128	2	0.25	0.12	0.25	0.12	+	CTX-M-15	FIA

Primers for ESBL detection with PCR

Primer designation	Target gene	Sequence	Amplicon size	Ref.
OT 3	TEM	5’-ATG-AGT-ATT- CAA-CAT-TTC-CG-3’	850	25
OT 4	TEM	5’-CCA-ATG-CTT-AAT-CAG-TGA-GG-3’	850	25
SHV-F	SHV	5’-TTC-GCC-TGT-GTA-TTA-TCT-CCC-3	1000	26
SHV-R	SHV	5’-TTA-GCG-TTG-CCA-GTG-YTC-GAT-3’	1000	26
MA-1	CTX-M	5’-SCS-ATG-TGC-AGY-ACC-AGT-AA-3’	550	27
MA-2	CTX-M	5’-CGC-CRA-TAT-GRT-TGG-TGG-TG-3’	550	27
M1-F	CTX-M-1 cluster	5’-AAA-AAT-CAC-TGC-GCC-AGT--TC-3’	415	30
M1-R	CTX-M-1 cluster	5’-TTG-GTG-ACG-ATT-TTA-GCC-GC-3’	415	30
M2-F	CTX-M-2 cluster	5’-CGA-CGC-TAC-CCC-TGC-TAT-T--3’	552	30
M2-R	CTX-M-2 cluster	5’-CCA-GCG-TCA-GAT-TTT-TCA-GG-3’	552	30
M8-F	CTX-M-8 cluster	5-TCG-CGT-TAA-GCG-GAT-GAT-GC	666	30
M9-F	CTX-M-9 cluster	5’-CAA-AGA-GAG-TGC-AAC-GGA-TG	205	30
M9-R	CTX-M-9 cluster	5’ATT-GGA-AAG-CGT-TCA-TCA-CC	205	30
M25-F	CTX-M-25 cluster	5’GCA-CGA-TGA-CAT-TCG-GG	327	30
M9/M25-R	CTX-M-8/25 clusters	5’AAC-CCA-CGA-TGT-GGG-TAG-C		30
IS26-F	IS26	5’-AAA-AAT-GAT-TGA-AAG-GTG-GT-3’		31
IS26-R	IS26	5’-ATT-CGG-CAA-GTT-TTT-GCT-GT-3		31
ISEcp-F	ISEcp	5’-AAA-AAT-GAT-TGA-AAG-GTG-GT-3’		31
ISEcp-R	ISEcp	5’-AAT-ACT-ACC-TTG-CTT-TCT-GA-3’		31
QNR A-F	QNR A	5’-ATT-TCT-CAC-GCC-AGG-ATT-TG-3’		29
QNR A-R	QNR A	5’-GAT-CGG-CAA-AGG-TTA-GGT-CA-3’		29
QNR B-F	QNR B	5’-GAT-CGT-GAA-AGC-CAG-AAA-GG		29
QNR B-R	QNR B	5’-ACG-ATG-CCT-GGT-AGT-TGT-CC		29
QNR S-F	QNR S	5’-ACG-ACA-TTC-GTC-AAC-TGC-AA		29
QNR S-R	QNR S	5’-TAA-ATT-GGC-ACC-CTG-TAG-GC		29

Antibiotic susceptibility of E_ coli isolates established with the disk-diffusion test

Isolate No.	Protocol number	Disk-diffusion method
Isolate No.	Protocol number	FOX	AMI	ERT	SXT
1	31	S	S	S	R
2	32	S	S	S	S
3	35	S	S	S	S
4	36	S	S	S	S
5	37	S	S	S	S
6	39	S	S	S	S
7	40	S	S	S	S
8	41	S	S	S	S
9	43	S	S	S	S
10	44	S	S	S	S
11	45	S	S	S	S
12	46	S	S	S	S
13	47	S	S	S	S
14	49	S	S	S	S
15	50	S	S	S	S
16	51	S	S	S	S
17	53	S	S	S	S
18	54	S	S	S	S
19	55	S	S	S	S
20	56	S	S	S	R
21	58	R	S	S	S
22	59	S	S	S	S
23	60	R	S	S	S
24	64	R	S	S	S
25	65	S	S	S	S
26	66	R	S	S	S
27	69	S	S	S	S

Detection and characterisation of extended-spectrum and plasmid-mediated AmpC β-lactamase produced by Escherichia coli isolates found at poultry farms in Bosnia and Herzegovina

Figures & Tables

Figure 1

Figure 2

Figure 3

Figure 4

Conjugation frequency and cotransferred resistance markers

Antibiotic minimum inhibitory concentrations, genes, plasmids, and genotyping of E_ coli isolates from poultry farms in Bosnia and Herzegovina

Primers for ESBL detection with PCR

Antibiotic susceptibility of E_ coli isolates established with the disk-diffusion test

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