Teaming up for animal health
Monitoring of Antimicrobial Susceptibility of E. coli and Salmonella from pigs in The Netherlands, April 2013 – December 2015 Heuvelink AE, Holstege MMC, Hout AJ van GD Animal Health, Deventer, The Netherlands
Introduction GD Animal Health (AH), a leading organisation in animal health, conducts the monitoring of antimicrobial susceptibility (AMS) of isolates of animal pathogens from different animal species. Isolates included in this monitoring originate from routine bacteriological examinations of clinical submissions or post-mortem examinations. In 2012, GD AH replaced the agar diffusion method with tablets by a broth microdilution system and minimal inhibitory concentrations (MICs) are being determined since.
Aim
Table 2. MIC distribution (%) for Escherichia coli (ECO) pig isolates from April 2013 – December 2015
Analysis and evaluation of the in vitro antimicrobial susceptibility of Escherichia coli (ECO), Salmonella group B (SGB) and Salmonella Typhimurium (STY) isolates from pigs.
Antimicrobial agent
Materials and methods Isolates originated from post-mortem samples from pigs routinely submitted to the Pathology Department of GD AH from April 2013 up to (including) December 2015.
Escherichia coli (n = 774)# MIC values (μg/mL) 0.25
0.5
1
AMCa
0.4
0.4
7.2
AMP
0.1
0.1
5.8
2
4
64
18.2 13.5 25.2 28.3
5.9
0.9
8
16
18.6
69.2
64
64
8
8
1
1
0.5
0.5
1
1
0.25
0.25
4
8
2
2
2
2
4
4
512
512
64
256
64
128
32
32
APR CEP
Antimicrobial susceptibility testing: MICs of in total 20 antimicrobials were assessed using a broth microdilution system with commercially manufactured plates (Merlin Diagnostics, Bornheim-Hersel, Germany). Isolates were incubated for 21 ± 3 hour at 35 ± 1°C in Mueller-Hinton broth. MIC50 and MIC90 values (lowest MIC at which at least 50% and 90% of the isolates are inhibited, respectively) were determined. In addition, MICs were interpreted as susceptible, intermediate and resistant (based on CLSI breakpoints, or, if not available, other standards).
COL
92.3
CTX ENR
96.3
2.6
16
0.1
0.5
0.3
98.0
1.2
0.8
97.4
1.1
0.4
0.4
0.1
0.5
2.5
0.5
1.5
1.5
0.7
1.0
98.0
0.4
0
1.6
0.6
0.1
0.4
FLL
13.3 47.3 31.3
8.1
FLM
93.8
2.5
1.8
0.9
GEN
98.3
0.9
0.4
0.4
94.5
1.2
0.6
NEO SPT
MIC distributions (%) for ECO (n=774) are shown in a separate table. SGB (n=58) and STY (n=44) showed AMS patterns similar to ECO. However, it should be noted that AMS for colistin was 100% for STY. Additionally, lower AMS of Salmonella isolates for trimethoprim-sulfamethoxazole (66 to 69%) was observed. Due to the relatively limited numbers of SGB and STY isolates, effect of age on AMS could only be analysed for ECO isolates. In total, from 518 ECO isolates the age of the animals was known (< 4 weeks (n=94); ≥ 4 weeks and ≤10 weeks (n=362); > 10 weeks (n=62)). ECO from pigs > 10 weeks were significantly more often sensitive for amoxicillin/ clavulanic acid, compared to ECO from pigs ≤ 10 weeks (p < 0.05). ECO from pigs between 4 and 10 weeks of age were significantly more often resistant to sulfamethoxazole, spectinomycin, streptomycin and/or tetracycline, compared to ECO from pigs < 4 weeks (and, for sulfamethoxazole, from pigs > 10 weeks). Table 1. MIC50 and MIC90 values for Salmonella group B (SGB)and Salmonella Typhimurium (STY) Antimicrobial agent
SGB (n=58) MIC50
MIC90
(μg/mL) AMC
STY (n=44) MIC50
MIC90
Antimicrobial agent
(μg/mL)
SGB (n=58) MIC50
MIC90
3.7
TET
0.4
3.1
14.3
TLS
0
TMP T/Sb
26.4
1.7
1.7
9.7
30.4
82.4
0.6
1.6
27.1 30.6
11.7 12.3
6.0
6.5
6.5
6.5
0.9
4.4
70.3
0
0.4
1.8
97.8
64
64
0
0.8
99.2
64
64
4
4
32
32
8
8
0.1
TIA TIL
14.2
(μg/mL)
0
0
0
0
0
0
0
100
27.3
0.5
0.3
0.1
0
1.9
1.0
0
0.1
70.6
0.4
10.4 46.6
71.4
The white areas indicate the dilution range tested for each antimicrobial agent. Values above this range indicate MIC values higher than the highest concentration within the range. Values corresponding to the lowest concentration tested indicate MIC values lower than or equal to the lowest concentration within the range. When available, susceptible and resistance breakpoints are indicated in vertical green and red lines. AMC = amoxicillin/clavulanic acid; AMP = ampicillin; APR = apramycin; CEP = cefepime; COL = colistin; CTX = cefotaxime; ENR = enrofloxacin; FFL = florfenicol; FLM = flumequine; GEN = gentamicin; NEO = neomycin; SMO = sulfamethoxazole; SPT = spectinomycin; STR = streptomycin; TET = tetracycline; TIA = tiamulin; TIL = tilmicosin; TLS = tylosin; TMP = trimethoprim; T/S = trimethoprim/sulphamethoxazole. For some antimicrobial agents, 768 isolates instead of 774 isolates were tested. a Concentration for amoxicillin given, tested with clavulanic acid in a concentration ratio 2:1. b Concentration for trimethoprim given, tested with sulfamethoxazole in a concentration ratio 1:19. #
STY (n=44) MIC50
MIC90
(μg/mL)
(μg/mL)
8
32
16
32
NEO
4
4
4
4
AMP
64
64
64
64
SMO
512
512
512
512
APR
8
8
8
8
SPT
64
256
64
256
CEP
1
1
1
1
STR
128
128
8
128
COL
0.5
1
0.5
0.5
TET
32
32
32
32
CTX
1
1
1
1
TIA
64
64
64
64
ENR
0.25
0.25
0.25
0.25
TIL
64
64
64
64
FLL
4
16
4
16
TLS
4
4
4
4
FLM
2
2
2
2
TMP
0.5
32
0.5
32
GEN
2
2
2
4
T/Sb
0.25
8
0.25
8
a
STR
0.1
1.0
SMO
Results
128 256 512
MIC90
32
5.3
8
MIC50
Figuur 1. MIC distribution of colistin for ECO (n = 774), SGB (n = 58) and STY (n = 44) from April 2013 - December 2015
Explanation of abbreviations for antimicrobial agents: see MIC distribution for ECO a Concentration for amoxicillin given, tested with clavulanic acid in a concentration ratio 2:1. b Concentration for trimethoprim given, tested with sulfamethoxazole in a concentration ratio 1:19.
Conclusions Emergence of resistance among ECO and Salmonella derived from post-mortem samples from pigs appears to be limited. Specifically very low levels of resistance to colistin, cefotaxime and cefepim were observed, a very important observation in relation to human health care. It has to be kept in mind that these results represent only part of the Dutch pig population and a certain bias might result from the fact that by submission of animals to the Pathology Department, it is unknown whether animals had already been treated with antimicrobials. To conduct a clinically reliable monitoring of AMS of veterinary pathogens, more clinical veterinary breakpoints are urgently needed.
Dashed red line indicates the breakpoint value.
References: Clinical and Laboratory Standards Institute (CLSI), 2015. Performance Standards for Antimicrobial Disk and Dilution Susceptibility Tests for Bacteria Isolated from Animals; Second Informational Supplement. Clinical and Laboratory Standards Institute, Wayne, PA, USA. 3rd ed. CLSI Document VET01S.
GD, P.O. Box 9, 7400 AA Deventer, the Netherlands, T. +31 (0)570-63 33 91, F. +31 (0)570-63 41 04 www.gdanimalhealth.com, info@gdanimalhealth.com
GDOV0876/09-16
contact: a.heuvelink@gdanimalhealth.com or j.v.hout@gdanimalhealth.com