1. Introduction
Antimicrobial resistance is a worldwide public health concern. The administration of antimicrobials leads to the selection of antimicrobial-resistant bacteria, and food-producing animals are one of several potential sources of antimicrobial resistance [
1]. Although antimicrobial use is thought to be low in sheep and beef cattle [
2,
3], the large numbers of sheep and cattle in the UK may potentially contribute to the dissemination of antimicrobial-resistant bacteria [
4,
5]. National surveillance of antimicrobial resistance from bacteria isolated from sheep and beef cattle only uses samples that are submitted for clinical diagnostics [
6]. The use of clinical isolates suggests that antimicrobial resistance to commonly used antimicrobials, such as tetracycline and ampicillin, is relatively high in sheep and cattle [
7]. However, clinical samples are potentially biased as they usually come from sick animals which may have been treated with antimicrobials. At present in the UK, active national surveillance of healthy sheep or cattle does not exist.
There are few studies investigating antimicrobial susceptibility of organisms isolated from healthy sheep and beef cattle in the UK. These studies suggest that antimicrobial resistance on sheep and beef farms is relatively uncommon [
8,
9,
10], although extended spectrum beta-lactamase (ESBL)-positive beef farms may be increasing [
11,
12]. Examining the presence of ESBL
E. coli has been the focus of more recent studies on beef farms [
11,
12]. Therefore, other resistance types may have been missed in these studies. Other studies have investigated a larger range of antimicrobial resistances, but only investigated a few farms [
8,
10]. Hence, variance between farms with respect to antimicrobial resistance patterns was not investigated. More information regarding antimicrobial resistance on sheep and beef farms in the UK is required. Indeed, a systematic review of antimicrobial resistance on British sheep and cattle farms called for additional efforts in collecting farm-level antimicrobial resistance data [
7].
Previously identified factors associated with antimicrobial resistance in pigs and veal calves in countries other than the UK include the use of antimicrobials, either as therapeutics to treat sick animals or as growth promoters [
13,
14,
15]. Antimicrobial growth promoters are not used in the UK. The number of animals on the farm, region of the farm and type of animals sampled have also been reported as factors associated with antimicrobial resistance in bacteria isolated from animals [
16,
17]. It has been shown that bacterial isolates of animal origin may present with resistance even when the animals have not been exposed to antimicrobials [
18,
19]. Markland et al. [
20] illustrated that when cefotaxime-resistant bacteria were present in samples from beef cattle, resistant bacteria were more abundant in soil samples. This indicated that the environment, such as soils and forage, may be a natural source of antimicrobial-resistant bacteria for food-producing animals [
20]. However, the factors that affect the prevalence of antimicrobial-resistant bacteria in soils are unclear [
21]. One potential explanation is that heavy metals such as copper and zinc may co-select for antimicrobial resistance in soil. The effect of metal concentrations in soil on the prevalence of antimicrobial resistance in farm animals requires further investigation.
Disk diffusion testing is a commonly used phenotypic method for determining antimicrobial susceptibility. Scientists typically interpret the results of such tests using clinical breakpoints and will mainly adhere to guidelines set by the European Committee on Antimicrobial Susceptibility Testing (EUCAST) or Clinical and Laboratory Standards Institute (CLSI) [
22,
23]. However, these clinical breakpoints are only relevant for human medicine [
24]. CLSI has set very few veterinary clinical breakpoints, and at present, European veterinary breakpoints do not exist. Research suggests that using human clinical breakpoints to interpret veterinary data may lead to calculating a higher antimicrobial resistance prevalence than actually occurs [
10].
An alternative method to interpret antimicrobial susceptibility data is to use epidemiological cut-off (ECOFF) values to determine fully susceptible isolates (wild type, WT) from non-fully susceptible isolates (non-wild type, NWT). EUCAST defines a WT organism as one with the absence of acquired and mutational resistance mechanisms to the drug in question [
25]. Thus, the ECOFFs determine microbiological resistance, whereas clinical breakpoints determine clinical resistance. The ECOFF values are established by EUCAST through analysis of the distribution of their inhibitory zone diameters [
26]. However, the distributions of inhibitory zone diameters for isolates of animal origin may differ from the distributions of inhibitory zone diameters for EUCAST isolates [
10,
27]. Therefore, ECOFF values may not reflect WT organisms isolated from livestock.
Instead, the normalised resistance interpretation (NRI) method can be used to calculate tailor-made cut-off values. The method was originally developed to calibrate the disk diffusion test to compare results between laboratories [
28]. It has also been used to investigate the susceptibility of organisms of animal origin when EUCAST or CLSI breakpoints do not exist [
29,
30]. Furthermore, the NRI method has been used when clinical breakpoints or ECOFF values do not appear appropriate [
10,
27]. An inappropriate cut-off value occurs when the cut-off splits the normal distribution of inhibition zone diameters. Therefore, it may be useful to interpret the inhibitory zone diameters of isolates of animal origin using clinical breakpoints, ECOFF values and the NRI method so that comparisons can be made and the appropriate cut-off value can be chosen. A previous study based on isolates from four sheep farms compared these three interpretation methods and suggested that sheep-specific cut-off values were most fitting [
10]. There needs to be additional studies with a larger number of participating farms to confirm these results, and similar studies have not been carried out for other livestock species. Additionally, the implications in terms of interpretation of antimicrobial resistance patterns requires further investigation. Therefore, the aim of this study was to investigate and compare antimicrobial resistance patterns on thirty-five sheep and beef farms in England and Wales using multiple interpretation methods, based on bacteria isolated from feces. Further objectives were to identify clusters of antimicrobial resistance and to identify factors that were associated with antimicrobial resistance on sheep and beef farms.
3. Discussion
In this study, antimicrobial resistance patterns in
E. coli isolated from sheep and beef farms were assessed using different interpretation methods. The results show that antimicrobial resistance in feces on sheep and beef farms in England and Wales is generally low regardless of interpretation method, compared with samples from other livestock species in the UK [
6]. However, interpretation method can have important implications on the understanding of resistance patterns. Different clusters of resistance patterns were determined when using clinical breakpoints compared with bespoke cut-off values using the NRI method.
The comparison of susceptibility interpretation methods showed that differences in the proportion of susceptible isolates may occur depending on the method used. There was little correlation between interpretations of susceptibility using clinical breakpoints, ECOFFs and CO
WT values for amoxicillin/clavulanic acid in particular. For the beef isolates, there was also little correlation between the interpretations for ciprofloxacin and sulfamethoxazole-trimethoprim. This is possibly because clinical breakpoints detect human clinical resistance, whereas ECOFFs and CO
WT values detect microbiological resistance. The results suggest that clinical breakpoints and ECOFFs are not appropriate for interpreting antimicrobial resistance in bacteria isolated from sheep or cattle feces as they do not fit the wild type distribution of isolates. Silva et al. [
10] also showed that clinical breakpoints and ECOFFs may be inappropriate for the classification of ovine isolates as resistant and that these interpretation methods may over-report antimicrobial resistance in sheep populations [
10]. Our results also indicate that clinical breakpoints and/or ECOFFs may slightly under-report, as well as over-report, antimicrobial resistance in sheep and beef isolates, particularly for sulfamethoxazole-trimethoprim, tetracycline and cefotaxime.
Silva et al. [
10] further suggest that the sheep industry could establish sheep-specific cut-offs to avoid over-interpretation of resistance in ovine isolates. It is important to highlight that some of the sheep-specific cut-offs determined in the study by Silva et al. [
10] were vastly different to those determined in our study. For example, Silva et al. [
10] calculated the sheep CO
WT for tetracycline to be 14 mm, whereas we calculated a sheep CO
WT of 25 mm. The difference could be because of differences in the study population. The present study investigated sheep samples from 27 farms in England and Wales, whereas Silva et al. [
10] used samples from just three farms in Scotland and one farm in Norway. Additionally, some isolates collected by Silva et al. [
10] were from diseased animals and not from fecal samples. Nevertheless, this demonstrates the need for large-scale data collection from a variety of different farms before industry-wide cut-off values can be developed. These results also demonstrate the need to use the NRI method to calibrate the disk diffusion test to compare results between laboratories.
The differences in interpretation methods led to disparities in the groupings of antimicrobial resistances through cluster analysis. There was a cluster of isolates that were non-susceptible to ampicillin and amoxicillin/clavulanic acid for both the sheep and beef samples when the clinical breakpoints were used, but were not clustered as non-wild type when CO
WT values were used. A cluster of sheep isolates that were non-wild type for cefotaxime and amoxicillin/clavulanic acid was only determined with the CO
WT values. This may have implications for elucidating resistance mechanisms. Non-susceptibility to amoxicillin/clavulanic acid and ampicillin or cefotaxime suggests mechanisms of beta-lactam resistance, which requires further investigation through genotypic analysis [
32]. These non-susceptibility and non-wild type patterns may have been missed if only one interpretation method was used.
A few cases of cefotaxime non-susceptibility in sheep and beef fecal isolates were reported in this study. Reduced susceptibility to cefotaxime has been reported in British beef cattle [
12]. Although cefotaxime resistance in isolates from sheep has previously been reported in England and Wales, this was from clinical diagnostic samples [
11], and to the authors’ knowledge, it has not been reported for apparently healthy sheep in England and Wales before. This may be because third-generation cephalosporins are very rarely used on sheep farms. The use of highest-priority critically important antimicrobials should only be used as a last resort, when susceptibility testing has been conducted and no other antimicrobial would be effective. As resistance to other lower priority antimicrobials is uncommon in sheep and beef cattle, the use of third-generation cephalosporins is usually not required. It has previously been shown that cefotaxime-resistant bacteria may be present on beef farms without any antimicrobial use and that the environment may be a source of cefotaxime resistance [
19,
20]. The presence of cefotaxime non-susceptibility raises concerns around the existence of extended spectrum beta-lactamases (ESBLs) in healthy sheep and beef cattle [
32], especially as a group of sheep isolates resistant to both cefotaxime and amoxicillin/clavulanic acid was identified from the cluster analysis. Further screening of the cefotaxime non-susceptible isolates is required to determine the presence of ESBL resistance mechanisms [
33].
The results suggest that antimicrobial resistance to commonly used antimicrobials (such as tetracycline and penicillin) in apparently healthy sheep and beef cattle in England and Wales is much lower than that reported from national clinical surveillance [
6,
34]. This is probably because clinical samples are more likely to come from sick animals that have already been treated with antimicrobials before submission. Additionally, beef and dairy cattle samples are often not separated when reporting antimicrobial resistance [
6]. In apparently healthy dairy cattle, the proportion of resistant isolates is much higher than the beef cattle reported here [
35]. Although antimicrobial resistance appears to be low on sheep and beef farms in England and Wales, these figures were higher than what was reported historically. In 1999, 3% of isolates from sheep, and 6% of isolates from cattle were resistant to one or more antimicrobials [
9]. In contrast, in our study, 10% of isolates from sheep, and 15% of isolates from cattle were non-wild type for one or more antimicrobials. Similarly, less than 4% of
E. coli isolates from Scottish beef farms between 2001 and 2004 were resistant to tetracycline [
8], compared with 12% of beef isolates in our study. This difference in susceptibility may be due to differences in study design, for example, differences in the antimicrobials studied or sampling technique. Additionally, the use of different interpretation methods may play a role in the varying antimicrobial susceptibilities as an organism that is classed as non-wild type does not necessarily display clinical resistance. Alternatively, differences in susceptibilities between the studies may be due to changes in farm practices over the last twenty years. This highlights the need for regular and consistent surveillance of antimicrobial resistance on sheep and beef farms in the UK so that longitudinal comparisons can be made.
The majority of the variance in antimicrobial susceptibility between isolates was due to between-sample differences, whereas only a small proportion of variance was due to between-farm differences. Around half of the between-farm variance could be explained by the use of tetracycline on the farm and the concentration of copper in the soils; however, there was still a large proportion of between-sample variation that was unexplained. This suggests that differences in antimicrobial resistance patterns are due to the variability in management of individual animals rather than any whole flock or herd management practices. The results probably reflect that antimicrobials are not usually used as routine prophylactic (preventative) treatments on sheep and beef farms in the UK, and in most cases farmers only use antimicrobials for the treatment of sick individual animals [
3,
36]. This is encouraging as there has been a large push in the UK agriculture industry to voluntarily reduce antimicrobial use over the last five years, particularly targeted at whole flock and herd treatments [
36,
37]. The large sample-level variance may also be due to the individual characteristics of the animals. For example, cattle over the age of 25 months have been shown to carry significantly less antimicrobial-resistant
E. coli compared with younger cattle [
17]. It was not possible to gather much sample-level information in this study as fecal samples were collected from the ground rather than directly from the animals. To understand the sources of variance at the sample level, further investigation using samples obtained directly from individual animals is recommended. The high variability between samples suggests that future studies that aim to understand the drivers of antimicrobial resistance on farms should consider taking individual animal samples rather than pooled samples.
The use of phenicols and tetracyclines was significantly associated with tetracycline non-wild type isolates in the univariable analysis. The association between tetracycline use and tetracycline non-wild type isolates is not surprising and has previously been reported for other livestock species [
13,
15]. The use of a particular antimicrobial will result in the direct selection of the corresponding resistance [
38]. Phenicol use might indirectly select for tetracycline resistance via cross-resistance mechanisms. Alternatively, phenicol use may be associated with tetracycline non-wild type isolates if farmers change their antimicrobial drug of choice from tetracycline to phenicol when they find tetracyclines are no longer as effective for them. Tetracyclines are used as first-line treatments for livestock in the UK, whereas phenicols are in a higher category of antimicrobial which should only be used when first-line treatments are unavailable, for example, in cases of clinical tetracycline resistance [
39]. Other factors that were identified as significant influences on antimicrobial resistance in previous studies, such as weather [
35] and farm size [
17], were not significant in our study. One possible reason for this is that for the dependent variable, there was only a small proportion of non-wild type isolates for tetracycline.
The concentration of copper in the soils in the farm area and the use of tetracyclines were significantly associated with tetracycline non-wild type isolates in the multivariable analysis. Previous research indicates that the geochemical conditions of soils, particularly copper concentrations, are correlated with the abundance of antimicrobial resistance genes [
40,
41]. Furthermore, copper has been shown to co-select for tetracycline resistance in experimental conditions [
42], and mathematical models suggest that this co-selection may occur at copper concentrations as low as 5.5 mg/mL [
43]. Our results suggest that tetracycline non-susceptibility may be more prevalent on some farms due to the environmental exposure to copper in soils. There needs to be further investigation into the presence of copper resistance genes in the tetracycline non-wild type isolates obtained in this study.
Limitations
The study sample was small; however, the number of farms was comparable to that of similar studies investigating antimicrobial resistance at the farm level and the farms represented a range of farm types and sizes [
44,
45]. Additionally, the farms were mainly located in Wales and the West of England, where sheep and cattle are more densely populated [
46,
47]. This was a cross-sectional study and so changes in antimicrobial resistance over time could not be measured. Additionally, information was mainly only collected at the farm level, but analysis indicated that most of the variation in resistance was at the sample level. Further investigation using a longitudinal study design and collecting sample-level information from individual animals is required to understand additional variation in resistance levels on farms. The β-glucuronidase-positive
E. coli isolates were the focus here, although other species not studied are likely to also be environmentally important.