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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

Majda Fetahagić
Majda Fetahagić
, 
Amir Ibrahimagić
Amir Ibrahimagić
, 
Selma Uzunović
Selma Uzunović
, 
Nataša Beader
Nataša Beader
, 
Vesna Elveđi-Gašparović
Vesna Elveđi-Gašparović
, 
Josefa Luxner
Josefa Luxner
, 
Muhamed Gladan
Muhamed Gladan
 et 
Branka Bedenić
Branka Bedenić
   | 30 déc. 2021
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Article Category: Original article
Publié en ligne: 30 déc. 2021
Pages: 305 - 314
Reçu: 01 mai 2021
Accepté: 01 oct. 2021
DOI: https://doi.org/10.2478/aiht-2021-72-3560
© 2021 Heinz-Elmar Tenorth, published by Sciendo
This work is licensed under the Creative Commons Attribution-NonCommercial-NoDerivatives 3.0 License.

Figure 1

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 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

Figure 2

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
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

Figure 3

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
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

Figure 4

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)
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
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
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
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