Appendix 2: Figures and Tables
100
% resistant E. coli
80
60
40
20
0
0
20
40
60
80
100
120
time (days)
Fig 1: Percentage E. coli resistant to tetracycline (), to sulphadiazine () and to both
tetracycline and sulphadiazine () in pigs treated with chlortetracycline (black bar below x-axis)
followed by sulfadiazine (grey bar below x-axis) (group 3); and the percentage of E. coli resistant
to tetracycline (), to sulphadiazine () and to both tetracycline and sulphadiazine () in
untreated pigs (group 5).
1
100
% resistant enterococci
80
60
40
20
0
0
20
40
60
80
100
120
time (days)
Fig 2: Percentage of tetracycline-resistant enterococci in pigs treated () with chlortetracycline
(black bar below x-axis) followed by sulphadiazine (grey bar below x-axis) and in untreated pigs
120
% resistant enterococci
100
80
60
40
20
0
0
20
40
60
80
100
120
time (days)
pigs ().
Fig 3: Percentage of enterococci resistant to tetracycline (), to erythromycin () and to both
tetracycline and erythromycin () in pigs treated with chlortetracycline (black bar below x-axis)
followed by tylosin (grey bar below x-axis) (group 4); and the percentage of enterococci resistant
to tetracycline (), to erythromycin () and to both tetracycline and erythromycin () in untreated
pigs (group 5).
2
100
% resistant E. coli
80
60
40
20
0
0
20
40
60
80
100
120
time (days)
Fig 4: Percentage of tetracycline-resistant E. coli in pigs treated () with chlortetracycline (black
bar below x-axis) followed by tylosin (grey bar below x-axis) and in untreated pigs ().
1.00E+04
mar derivatives
parent strain
CFU/100cm2
1.00E+03
1.00E+02
1.00E+01
1.00E+00
Leg
Belly
Fig. 5 Number of viable E. coli re-isolated from carcasses pre-chill.
3
Back
4
Fig. 6a
% tetracycline resistant
enterococci
100
80
60
40
20
0
0
2
4
6
8
10
12
14
8
10
12
14
time (weeks)
Fig. 6b
% erythromycin resistant
enterococci
100
80
60
40
20
0
0
2
4
6
time (weeks)
Figs. 6a and b Percentage of tetracycline- (Fig 6a) and erythromycin- (Fig 6b) resistant
enterococci in pigs from group 1 (), which were treated with copper sulphate only and in pigs
from group 4 (), which remained untreated. Copper sulphate was added to the feed at 175
ppm from weeks 1 to 6 (inclusive) and at 25 ppm from weeks 7 to 14 (inclusive). The arrow
indicates the point at which the copper sulphate concentration was reduced from 175ppm to 25
ppm.
5
Fig. 7a
% tetracycline resistant
enterococci
120
100
80
60
40
20
0
0
2
4
6
8
10
12
14
10
12
14
time (weeks)
Fig. 7b
% erythromycin resistant
enterococci
120
100
80
60
40
20
0
0
2
4
6
8
time (weeks)
Fig. 7a and b Percentage of tetracycline- (Fig7a) and erythromycin- (Fig 7b) resistant
enterococci in pigs from group 2 (), which were treated with both copper sulphate and tylosin,
in pigs from group 3 (), which were treated with tylosin only and in untreated pigs (). Copper
sulphate was added to the feed at 175 ppm from weeks 1 to 6 (inclusive) and at 25 ppm from
weeks 7 to 14. Tylosin (black bar below x-axis) was added to the feed from weeks 4 to 6. The
arrow indicates the point at which the copper sulphate concentration was decreased from
175ppm to 25 ppm.
6
% tcrB-positive enterococci
100
80
60
40
20
0
0
2
4
6
8
10
12
14
time (weeks)
Fig. 8 Percentage of enterococci carrying the tcrB gene in group 1 pigs (), which were treated
with copper alone, in group 2 (), which were treated with both copper sulphate and tylosin, in
group 3 (), which were treated with tylosin only and in untreated pigs (). Copper sulphate
was added to the feed at 175 ppm from weeks 1 to 6 (inclusive) and at 25 ppm from weeks 7 to
14 (inclusive). Tylosin (black bar below x-axis) was added to the feed from weeks 4 to 6. The
arrow represents the point at which the copper sulphate concentration was decreased from
175ppm to 25 ppm.
7
7
MIC geomeans (mg/L)
6
5
4
3
2
1
0
group 1
group 2
group 3
group 4
Fig. 9 Geomean of copper sulphate MIC values of tcrB-positive enterococci. Data is generated
from enterococci (n=204) isolated prior to the start of the treatments (white); in weeks 1, 2 and 3
(light grey) while groups 1 and 2 were receiving copper sulphate (175ppm); in weeks 4, 5 and 6
(dark grey) while groups 2 and 3 were receiving tylosin and groups 1 and 2 continued to be
treated with copper sulphate (175ppm); and in weeks 8, 10 and 12 after the end of tylosin
treatment, and while groups 1 and 2 were receiving copper sulphate (25ppm).
8
120
% resistance E. coli
100
80
60
40
20
0
0
2
4
6
8
10
12
14
time (weeks)
Fig. 10 Percentage of tetracycline-resistant E. coli in pigs from group 1 (O), group 2 () and
group 4 (). Group 2 was treated with tylosin treatment (black bar below x-axis) from week 4 to
6. Groups 1 and 2 were treated with 175 ppm copper sulphate weeks 1 to 6 (inclusive) and with
25 ppm copper sulphate weeks 7 to 14 (the change in concentration is indicated by the arrow).
100
% E. coli
80
60
40
20
0
group 1
group 2
group 3
group 4
Fig. 11 Percentage of E. coli with a triclosan MIC≥0.125 mg/L. Measurements were done on E.
coli (n=288) isolated prior to the start of the treatments (white); in week6 (light grey) on the last
day of 175ppm copper sulphate for groups 1 and 2 and the last day of tylosin treatment for
groups 2 and 3, in week 8 (dark grey) 2 weeks post tylosin treatment, and 2 weeks into 25ppm
copper sulphate for groups 1 and 2, and in week 14 (black), 8 weeks post tylosin treatment and
8 weeks into copper sulphate treatment at the lower dose.
9
Table 1. Primers used to produce PCR amplicon for sequencing and for subsequent
sequencing
Forward
primer
Sequence (5’ to 3’)
Reverse
primer
Sequence (5’ to 3’)
Amplified
bp's
Accession number U00734
acrROF
GAACCTGAAGAACGACC
TGA
acrROR
CATCAGAACGACCGCCAG
AG
AcrRO1F
GATTACGTTGTGCCTGTT
GC
AcrRO1R
ACATCCTCGATGTGGCTCT
A
marROR
CGCTTGTCATTCGGGTTC
G
GGATAGAGCAGAGCACTT
A
CTGGACATCGTCATACCTC
T
GACGTAGAGCCACATCG
AGG
Accession number M96235
marROF CCAGCCCCAGGCCAATT
GC
marRO1F CGTGGCATCGGTCAATT
CAT
marRO2F TGTCTCCGCTGGATATTA
CC
197-1140
AcrRO2F
marRO1R
marRO2R
Accession number X59593
soxROF TAAGCGGCTGGTCAATAT
soxROR
AATGAGGTGTGTTGACGT
GC
CG
soxRO1F GGCAATCAGCGGCGATA
SoxRO1R CGGAATGCCAATACGCTG
TAA
AG
soxRO2F CAATGGCGAGAAGAGTT
SoxRO2R AATGCGCCGATCCAACTCT
GGA
T
Primers underlined are primers to amplify the entire region.
1200-1726
614-1329
Table 2: Antibiotic treatments received by the pigs. Each group was composed of 6 pigs.
TREATMENT 1
(from day 0)
Active ingredient
TREATMENT 2
(from day 21)
Duration
Active
Duration
ingredient
Group 1
Chlortetracycline*
7 days
None
Group 2
Chlortetracycline
7 days
Chlortetracycline
7 days
Group 3
Chlortetracycline
7 days
Sulfadiazine^
5 days
Group 4
Chlortetracycline
7 days
Tylosin0
21 days
Group 5
None
None
*Product name: AureofacTM from Alpharma; ^Product name: UniprimTM 150 powder from Pfizer
ltd; 0Product name: Tylan G50 PremixTM from Elanco Animal Health.
10
Table 3 Percentage of enterococci and E. coli isolates carrying resistance genes investigated in pigs before, during and post treatments (n=504).
Resistance genotype
tet(M)
Before treatment
Tetracycline
10 days post treatment
4 wks post treatment
12 wks post treatment
11
72 (5)*1
39
72 (16)*1
28 (17)*1
Enterococci
tet(M)tet(L)ermB
E. coli
tet(A)
tet(B)
tet(B)sul2
11
82 (58)*3
5.5
33
5.5
16
0
0
0
27
0
0
27
11
5.5
11
82 (58)*3
67 (33)*3
83
5.5
16
0
17
0
22
0
0
10
0
11
0
72 (5)*3
5 (5)*3
0
0
5
17
0
17
0
0
11
83
72
0
0
72 (5)*3
0
11
22
5
17
61
11
5.5
0
11
0
0
0
0
5
5
0
11
0
0
0
5
0
0
0
0
0
0
Group 1
0
0
0
0
0
Group 2
Before treatment
Tetracycline
Tetracycline
10-days post treatment
10 wks post treatment
11
72 (5)*1
44
62 (6)*1
34 (6)*1
0
0
17
0
0
Group 3
Before treatment
Tetracycline
Sulfadiazine
5-days post treatment
10 wks post treatment
16
65
50
44
44 (11)*1
Before treatment
Tetracycline
Tylosin
2 wks post-treatment
10 wks post treatment
16
67
0
0
23
Before treatment
Treatment 1
Treatment 2
2 wks post-treatment
10 wks post treatment
5
27
39 (6)*1
33
0
0
0
6
0
0
Group 4
0
0
89
45 (17)*2
38 (5)*2
Group 5 (Control*4)
0
0
0
0
0
*1 Percentage of isolates also carrying the tet(L) resistance gene.
*2 Percentage of isolates not carrying the tet(M) resistance gene.
*3 Percentage isolates also carrying the sul 2 resistance gene
*4Group 5 was not exposed to any treatment, measurements were taken at same time points as the other groups.
11
Table 4: Percentage and phenotype of multi-resistant E. coli isolated from pigs during
treatment.
Drug treatment
Tetracycline (1)
% Multi-resistance
22%
Resistance phenotype
AMP-TET-SUL
Tetracycline (2)
5%
AMP-TET-SUL *
Sulphonamide
16%
AMP-TET-SUL
NAL-TET-SUL *
Tylosin
16%
CHL-TET-SUL-CYCLO
TET-SUL-TRI
NAL-TET-SUL-TRI
Control
11%
AMP-TET-SUL
AMP, ampicillin; CHL, chloramphenicol; CYCLO, cyclohexane; NAL, nalidixic acid; SUL,
sulphonamide; TET, tetracycline; TRI: triclosan
* isolates with this phenotype were identified at the end of the withdrawal period in 5% E. coli
tested.
Table 5. MIC values of antibiotics and cyclohexane tolerance for E. coli 345-2RifC and for MAR
mutants derived from this strain.
E. coli strains
Number
tested
MIC (mg/l)
345-2RifC
1
AMP
4
CHLOR
8
NAL
4
TET
2
CYCLO
sensitive
MAR mutants
20
8
32
8
4
resistant
AMP, ampicillin; CHLOR, chloramphenicol; NAL, nalidixic acid; TET, tetracycline; CYCLO,
cyclohexane.
12
Table 6. Numbers of parent and MAR derivative E. coli re-isolated from sample sites in the
abattoir during processing.
Parent
MAR
CFU/ item
Scald tank
Bleeding knives
Scraper
Dehairing knives
Worker hand
Tools (degutting)
Saw (split carcass)
Knives to trim fat
< 2 per 10mL
< 2 x 100
< 2 x 100
2.50 x 100
7.30 x 102
< 2 x 100
< 2 x 100
< 2 x 100
< 2 per 10mL
8.40 x 101
3.20 x 101
2.50 x 100
4.18 x 102
6.50 x 101
1.92 x 102
2.50 x 100
CFU/ 100 cm2 of surface
Gambrelling table
3.80 x 101
4.76 x 102
Dehairer
1.13 x 101
7.15 x 101
4
Lairage
6.00 x 10
3.58 x 102
0
Apron washer
< 2 x 10
< 2 x 100
1
Apron
3.00 x 10
4.45 x 102
The detection limit was 2 CFU/item or 2 CFU/100 cm2. Anything below the detection limit was
scored as < 2 x 100 CFU.
Table 7 Percentage and phenotype of multi-resistant E. coli isolated in pigs.
Treatment
Copper
Percentage multi-resistance
4%
Phenotype
AMP-TET-SUL
CHL-TET-SUL
AMP-CHL-TET-SUL
Copper and tylosin
21%
AMP-CHL-TET-SUL
CHL-TET-SUL
AMP-CHL-TET-SUL-NAL
Tylosin
14%
AMP-CHL-TET-SUL
CHL-TET-SUL
AMP-CHL-TET-SUL-NAL
Control
1%
AMP-TET-SUL
AMP, ampicillin; TET, tetracycline; SUL, sulphonamide; CHL, chloramphenicol; NAL, naladixic;
13