Antimicrobial resistance in pig faecal samples from The Netherlands (five abattoirs) and Sweden

A. E. J. M. van den Bogaard*, N. London and E. E. Stobberingh

Department of Medical Microbiology, University of Maastricht, PO Box 616, NL-6200 MD Maastricht, The Netherlands


    Abstract
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
The prevalence and degree of antibiotic resistance of faecal indicator bacteria, Escherichia coli and enteroccoci, were determined in 1321 faecal samples collected from pigs at five abattoirs in The Netherlands and in 100 samples from Swedish pigs. In the Dutch samples a high prevalence of resistance was observed in E. coli for three commonly used antibiotics in pig medicine, amoxycillin (70–94%), oxytetracycline (78–98%) and trimethoprim (62–96%). Also, the prevalence for chloramphenicol (55–67%) and neomycin (38–67%) was relatively high. For the other compounds tested the prevalence was less than 10%. The percentage of samples with a high degree of resistant E. coli showed the same tendency in all Dutch abattoirs although significant differences between the abattoirs were observed. The percentage of Swedish samples with a high degree of resistant E. coli was significantly lower for all antibiotics except nitrofurantoin, gentamicin, flumequin and ciprofloxacin. All enterococci were susceptible to amoxycillin and high-level resistance to gentamicin was observed in 4–6% of the Dutch samples. A high prevalence of resistance and a high degree of resistance was found for erythromycin and oxytetracycline. The prevalence of resistance to dalfopristin–quinupristin ranged from 6 to 8% and for vancomycin from 24 to 46%. Significant differences between the abattoirs were found for all compounds tested except amoxycillin. In the Swedish population both the prevalence and degree of resistance in enterococci were significantly lower except for amoxycillin and gentamicin. This point prevalence study showed that the prevalence and degree of antibiotic resistance in indicator bacteria, E. coli and enterococci, in faecal samples from pigs differed between two countries and reflected differences in antibiotic usage in pigs. To analyse the differences observed between the slaughterhouses, additional information about the farms of origin and antibiotic consumption is necessary.


    Introduction
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
The prevalence and extent of antibiotic resistance in a population are strongly correlated with antibiotic usage, as selection and dissemination of resistant bacteria are heavily augmented under selective pressure caused by antibiotics. As a consequence, resistance is most common where there is heavy use of antibiotics and appreciable host-to-host contact, as in hospitals, day-care centres for children and nursing homes.1,2 Intensive farming units also represent a large reservoir of antibiotic-resistant bacteria.2 Here resistance is selected and perpetuated not only by the regular veterinary use of antibiotics, but also by the continuous feeding of antibiotics to enhance growth of animals. Moreover, resistant microorganisms are very easily disseminated within units via faecal contact. Many studies on bacterial resistance have been published, but mainly of hospital and animal pathogens and based on data generated to support therapy. Therefore non-therapeutic antibiotics such as antimicrobial growth promoters (AGP) have seldom been studied. Moreover, the selection of clinical cases from which samples are tested represents a worst-case scenario. Knowledge of the transfer of resistance via plasmids, phages, transposition and ‘free’ DNA3 has increased considerably since the 1970s and 1980s, when transfer was considered to be uncommon, it was thought that only zoonotic bacteria from animals could infect humans and transfer of resistance genes from animal bacteria to human bacteria was thought to be rare.48 The fear now is that antibiotic-resistant bacteria, pathogenic or non-pathogenic to humans, are selected in the intestinal flora of animals, contaminate foods of animal origin and transfer their resistance to other bacteria in the human gut.9,10

Humans and animals live in close association with large numbers of bacteria, of which the majority are found in the large intestine, where they are exposed to antimicrobial agents, exchange genetic material with other bacteria and, on excretion, contaminate the environment or colonize other animals and humans. Hence, the intestinal flora of healthy animals and humans is considered to be the most important reservoir of resistant bacteria and resistance genes.1113 As contamination of carcasses with faecal flora during slaughtering inevitably occurs, food of animal origin may serve as a vehicle to transport resistant bacteria and resistance genes between animals and humans. The prevalence and degree of antibiotic resistance found in indicator bacteria in the faecal flora of humans and animals are considered to be a good indicator of the selective pressure of antibiotic usage.1418 They correlate with the amounts and types of antimicrobial agents consumed by these populations1921 and changes in resistance can be considered as an early warning system for resistance to be expected in potentially pathogenic bacteria.20,21 Therefore it has been suggested that a low level of carriage of resistant intestinal (indicator) bacteria in humans should become a public health goal, as have normal blood pressure and low serum cholesterol levels.21 In food animals a low prevalence and degree of antibiotic resistance in the intestinal flora should be considered a distinguishing quality and safety mark.22,23 Indicator bacteria such as Escherichia coli and enterococci are organisms that constitute a natural part of the intestinal flora of humans and animals. Using these species makes it feasible to compare levels of resistance between populations.

The aim of the present study was to determine and compare the prevalence and degree of resistance to antibiotics commonly used in pig medicine and AGP in indicator bacteria, E. coli and enterococci, in faecal samples from pigs slaughtered in five regions in The Netherlands and in Sweden. In Sweden the use of AGP was banned in 1986.


    Materials and methods
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Collection of faecal samples

Fresh faecal samples were collected in November and December 1995 at the slaughterhouse in Weert and in March and April 1996 at the other four slaughterhouses (Table IGo) by inspectors from the RVV (Rijkskeuringsdienst voor Vee en Vlees: Dutch National Inspection Service for Livestock and Meat). The slaughterhouses were widely located in The Netherlands. The Swedish samples were collected during September 1997. To prevent multiple sampling from pigs from the same farm, faeces (approximately 20 g in a plastic vial) were collected from every 300th or more pig after evisceration at the slaughtering line. The samples were stored at 4°C and sent weekly to the bacteriological laboratory. On the day of arrival at the laboratory they were diluted (10–1) in 0.9% saline containing 20% (v/v) glycerol and stored at –20°C until examined. The faecal samples collected in the slaughterhouse in Weert and in Sweden were frozen immediately after collection and stored at –20°C until examined. All faecal samples were analysed within 61 days of collection.


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Table I. Prevalence of resistant Escherichia coli and percentages of samples with a high degree of resistance in pig faecal samples collected in The Netherlands (five abattoirs) and Sweden
 
Bacteriological analysis

After thawing, the samples were inoculated on selective media with and without antibiotics.

For E. coli, 40 µL of 10–2 and 10–4 dilutions in 0.9% saline were inoculated on Levine agar plates (BBL 11221; Becton Dickenson BV, Etten-Leur, The Netherlands) with a spiral plater (Salm en Kip, Utrecht, The Netherlands). For enterococci, KF-Streptococcus agar plates (Oxoid CM701; Basingstoke, UK) were inoculated with 40 µL of the 10–1 and 10–3 dilutions with a spiral plater. If no vancomycin-resistant enterococci (VRE) were detected on the vancomycin agar plate a new vancomycin agar plate was inoculated by swabbing 0.5 mL of the 10–1 dilution. For trimethoprim testing 5% lysed horse blood was added to the agar. The antibiotics tested and concentrations used are given in Tables I and IIGoGo. E. coli isolates from plates with chloramphenicol were tested for resistance to florfenicol (MIC > 8 mg/L) by broth microdilution. The antibiotics were selected because they, or related antibiotics, are or have been until recently regularly used in pigs, either on veterinary prescription or as AGP. E. coli grows on Levine agar as purple colonies with a black centre and metallic shine. Only these colonies were counted after 18–24 h incubation at 37°C. The minimum detection level by this method was 300 cfu/g faeces and it has been shown that more than 95% of the presumptively identified colonies are E. coli.24 Previous studies also showed that all isolates from antibiotic-containing plates had MICs identical to or higher than the concentration of the antibiotic in the selective plate from which they had been isolated.2527


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Table II. Prevalence of antibiotic-resistant enterococci and percentage of samples with a high degree of resistance in pig faecal samples collected in The Netherlands (five abattoirs) and Sweden
 
Enterococci appear as typical red or pink colonies on KF-Streptococcus agar. After 48 h incubation at 42°C only the typical pink colonies were counted. At random, typical colonies collected from 100 KF-Streptococcus agar plates with and without antibiotics (five colonies per plate) were subcultured on blood agar (Oxoid CM854 with 5% sheep blood) and identified on the basis of tolerance for bile, aesculin hydrolysis, growth on 6.5% NaCl (w/v) and a positive pyrrolidonyl-arylamidase reaction (pyrrolidonyl-arylamidase tablets, Rosco, Taastrup, Denmark). In total 500 colonies were identified, and all belonged to the genus Enterococcus. The minimum detection level was 102 cfu/g faeces. For the prevalence of resistance to dalfopristin– quinupristin (Synercid) a specimen was considered positive only if at least one Enterococcus faecium was isolated and identified from the dalfopristin–quinupristin agar plate. For this reason the degree of resistance could not be calculated. For identification to species level, acid production from different sugars was determined.28 Isolates from a previous study had all MICs identical to or higher than the concentration of the antibiotic in the selective plate from which they had been isolated.29

The samples from the abattoir in Weert were processed first and susceptibility was tested against a smaller number of antibiotics than the other samples.

Statistical analysis

A one-way analysis of variance was used to estimate overall differences between the group means. Group means were compared pairwise using t-tests controlled for overall error rate (Bonferroni test) where P < 0.05 was regarded as statistically significant.30

Definitions

Prevalence of antibiotic resistance.
The prevalence of resistance (%) for either E. coli or enterococci in the population for an antibiotic is given by the number of samples showing growth on the plates containing that antibiotic, divided by the total numbers of samples tested x 100.

Degree of antibiotic resistance.
The degree (%) of antibiotic resistance in each faecal sample to each antimicrobial agent tested was calculated as the number of cfu growing on the plate containing that agent divided by the total number of typical colonies on the antibiotic-free control plate x 100.

Two degrees of antibiotic resistance to a particular antibiotic could be distinguished: low degree, i.e. less than 50% of the indicator microorganisms resistant; and high degree, 50% or more (thus the majority) of the indicator microorganisms being resistant to that agent.

The prevalence of high degree of resistance (%) is the number of samples with a high degree of resistance to a particular agent divided by the total number of samples tested x 100.


    Results
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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
E. coli

No significant differences in the population of samples from which E. coli were isolated and the total numbers of E. coli per positive sample were observed between the slaughterhouses or between the Swedish and Dutch samples (data not shown). The mean ± S.D. number of E. coli (log10 cfu/g faeces) was 6.4 ± 1.1 for the Dutch samples and 6.2 ± 0.9 for the Swedish samples. The prevalence and percentages of samples with a high degree of antibiotic-resistant E. coli in samples collected at the slaughterhouses in The Netherlands and from Sweden are depicted in Table IGo. A high prevalence of resistance in the samples from all slaughterhouses was observed for amoxycillin, oxytetracycline and trimethoprim, and a relatively high prevalence for chloramphenicol and neomycin. For the other compounds the prevalence was less than 10%. Florfenicol resistance was present in only two samples from Meppel. The percentage of samples with a high degree of resistance showed the same trend in all abattoir populations and was always highest for oxytetracycline followed by trimethoprim and amoxycillin. Significant differences (P < 0.05) in prevalence of resistance and prevalence of a high degree of resistance between samples received from the various slaughterhouses were observed for all compounds tested except gentamicin and ciprofloxacin. The prevalence of resistance to trimethoprim and nitrofurantoin in the samples from Apeldoorn was significantly higher than the mean of all Dutch samples tested. In contrast, the prevalence of resistance and/or a high degree of resistance to amoxycillin, trimethoprim and neomycin in the samples from Olst and Weert were significantly lower than the mean. Compared with the total Dutch population, and also with each of the abattoir populations, both the prevalence and degree of resistance in the Swedish population were significantly lower (P <= 0.005) for amoxycillin, oxytetracycline and trimethoprim. For chloramphenicol and neomycin, only the prevalence was significantly lower in the Swedish population. No differences were found for nitrofurantoin, gentamicin, flumequin and ciprofloxacin. For all tested antibiotics combined, the proportion of resistant E. coli in faecal samples with a high degree of resistance was 76% (range 74–79%) and for low-degree samples 7% (range 0.2–22%).

Enterococci

No significant differences in the percentage of samples (91–100%) from which enterococci were isolated and the total numbers of enterococci per positive sample were observed between the slaughterhouses or between the Swedish and the Dutch samples (data not shown). The mean ± S.D. number of enterococci (log10 cfu/g faeces) was 5.1 ± 1.0 for the Dutch samples and 5.1 ± 1.3 for the Swedish samples. No amoxycillin-resistant enterococci were found. The prevalence of antibiotic-resistant enterococci and the prevalence of high-degree antibiotic resistance in enterococci in the various populations of pig faecal samples is presented in Table IIGo. In the abattoir groups the prevalence of high-level gentamicin resistance ranged from 1 to 6%. A high prevalence of resistance was found for erythromycin and oxytetracycline. The prevalence of resistance to dalfopristin–quinupristin ranged from 62 to 82%, and for vancomycin from 24 to 46%. Overall, a high degree of resistance was found for erythromycin and oxytetracycline: 70% and 46%, respectively. Significant differences in prevalence and high degree of resistance between the slaughterhouses were found for all compounds tested except amoxycillin (no resistance). As shown in Table IIGo, significantly lower prevalences of resistance and high degree of resistance were found in the Swedish samples for oxytetracycline and erythromycin, and of resistance for dalfopristin–quinupristin either compared with the total population or with the various slaughterhouse populations separately (P <= 0.001). No VRE were found in the Swedish samples. The percentages of resistant enterococci in faecal specimens with low and high degree of resistance showed a bimodal distribution. In samples with a high degree of resistance approximately 80% (range 73–86%) of all the enterococci were resistant, whereas in samples with a low degree of resistance this varied from 2% to 28% (mean 17%).


    Discussion
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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Given the large number of samples tested and their geographical distribution of origin, the resistance percentages found in the present study can be considered to be representative of resistance in the faecal flora of pigs in The Netherlands. As the population of positive samples and the total numbers of bacteria isolated did not significantly differ between the populations, the different methods of storage of samples before testing did not seem to have had any influence on the results. All the antibiotics for which the prevalence of resistance in E. coli was determined are used only for therapy on veterinary prescription. The overall high prevalence of E. coli resistant to oxytetracycline and trimethoprim is in accordance with the amounts of these compounds used in pig medicine. Approximately 50% of all antimicrobial agents used in pigs are tetracyclines and 30% a combination of trimethoprim with a sulphonamide.31 In the Dutch veterinary antibiotic guidelines both agents are recommended as first choice antibiotics for many indications for therapy in fattening pigs.32 Only 4% of the antibiotics used on veterinary prescription are penicillins, of which more than 90% are ampicillin or amoxycillin for oral medication.31 The observed high prevalence of resistance to amoxycillin can therefore not be explained by a high usage. It has been recognized, however, that these broad-spectrum penicillins disturb the colonization resistance of the intestinal tract profoundly,3335 facilitating overgrowth by and increasing excretion of resistant bacteria. An additional explanation is that the genes encoding ß-lactamase production are often located on the same plasmid as the genes for tetracycline and chloramphenicol resistance.36 However, it is most likely that, as a result of co-selection, the prevalence of resistance to broad-spectrum penicillins (only limited) and chloramphenicol (use prohibited for more than 5 years) is higher than would be expected in comparison with the amounts of these drugs used in the pig population. In contrast, the observed resistance to nitrofurantoin was much lower. The use of nitrofurans in food animals has also been prohibited for more than 5 years and resistance to this class of agents seems to be disappearing from the population. This process might be faster than with other antibiotics because no transferable resistance to nitrofurans exists. Aminoglycosides represent only 2% of the veterinary use of antibiotics and neomycin is the most widely used agent of this group. For gentamicin, florfenicol and fluoroquinolones such as ciprofloxacin no oral formulations for mass medication of pigs are available in The Netherlands and therefore these agents have only limited use in pigs. In contrast, enrofloxacin accounts in The Netherlands for 14% of all veterinary antibiotic use in poultry and the prevalence of fluoroquinolone resistance in faecal E. coli from poultry is 50%.19,37 Florfenicol is a structural analogue of chloramphenicol that is not inactivated by chloramphenicol acetyl transferase.38 In The Netherlands it is used only for parenteral therapy of bacterial infections in individual animals.

In the present study no differentiation was made between Enterococcus spp., except for resistance to dalfopristin– quinupristin, because Enterococcus faecalis is considered intrinsically resistant to pristinamycins such as dalfopristin– quinupristin. The highest prevalence and degree of resistance was found for erythromycin and oxytetracycline. The resistance to tetracyclines was in line with the extensive usage of these drugs in pigs. Despite the relatively low susceptibility of enterococci to amoxycillin, with MICs of most E. faecalis of 1–8 mg/L and even higher for E. faecium,39 no amoxycillin-resistant enterococci (MIC > 25 mg/L) were found. In recent clinical studies the majority of E. faecium isolates had MICs lower than 25 mg/L and resistant isolates with higher MICs were related to hospital stay.4042 The samples collected at the various slaughterhouses showed significant differences in resistance for most antibiotics tested. As most slaughterhouses tend to collect pigs from a particular region the differences observed are probably due to differences in antibiotic usage, qualitatively and quantitatively, between these regions. However, the influence of differences in husbandry systems, stocking densities and density of animal populations between regions should also be considered.

In enterococci tylosin use is probably responsible for the high prevalence and degree of resistance to erythromycin, and virginiamycin use as AGP for the observed prevalence of resistance to dalfopristin–quinupristin in E. faecium. In this study the prevalence of VRE ranged from 24% in Boxtel to 46% in Apeldoorn, with a mean prevalence of 39%. Similar43 or lower44 prevalences of VRE in pigs have been reported from other European countries that used avoparcin as AGP. In the USA, where avoparcin has never been licensed for use in animals, no VRE have been isolated from food animals.45,46 A high degree of vancomycin resistance was observed in only 2% of the samples, which might reflect the fact that use of avoparcin, a glycopeptide like vancomycin, as AGP has been banned in the EU recently, because a relationship between avoparcin use and the prevalence of VRE in the faecal flora of food animals and humans was suggested.47,48

The observed differences in resistance to antimicrobial growth promoters between abattoirs is probably a regional difference, because one or two feedmills provide pig feeds to the majority of the farms in a region and feedmills tend to use the same AGP for a long time. Most striking was the difference in prevalence of VRE between the Dutch (39%) and Swedish (0%) population. Avoparcin, tylosin (a macrolide like erythromycin) and virginiamycin (a mixture of two pristinamycins like dalfopristin–quinupristin) were at the time of the study extensively used as AGP in The Netherlands, but not in Sweden. The observed differences are probably a result of the Swedish ban on AGP in 1986, as before that year AGP were also commonly used in pig feeds in Sweden. Unfortunately, no data on prevalence of resistance to AGP from before 1986 are available.

Prevalence and degree of resistance to antibiotics in veterinary use was also significantly lower in Sweden than in The Netherlands. This contradicts the common opinion that a prohibition of the use of AGP inevitably must lead to an increase in veterinary use of antibiotics. As the prevalence and degree of resistance in indicator bacteria is strongly correlated with the amounts of antibiotics used in a population, the present data show clearly that the prohibition of AGP in Sweden has not increased the therapeutic usage of antibiotics in pigs in Sweden to such an extent that the selection pressure on the intestinal flora was higher than in a country using AGP. The lower prevalence and degree of resistance to most antibiotics in E. coli and enterococci strongly suggests that veterinary use in Sweden is even lower. The observed prevalence of resistance to dalfopristin–quinupristin in E. faecium in the Swedish population is probably due to veterinary usage in Sweden of virginiamycin or macrolides such as tylosin, which show cross resistance and select for the same erm genes.

The marked bimodal distribution of the proportion of resistant bacteria in the indicator populations studied for all antibiotics was unexpected. There was a clear difference between low- and high-degree resistance in the faecal samples studied. In the majority of positive samples only a low degree of resistance was found. For example, the mean ± s.e.m. of the proportion of VRE in the total enterococcal population in samples (n = 492) with a low degree of resistance was 6% ± 0.4%, but in samples (n = 24) with a high degree of resistance the proportion of VRE was 73% ± 4%. Therefore susceptibility testing at random of one to three isolates from faecal specimens as is done in most studies inevitably results in a serious underestimation of the prevalence of resistance of indicator bacteria in a population.

In this study, current frequent usage of a particular antibiotic in a pig population seemed to correlate not only with a high prevalence of resistance to that antibiotic, but also with a higher prevalence of animals with a high degree of resistance in the population. Frequent usage of an antibiotic in the past was also associated with a relatively high prevalence of resistance in the population, but with reduced numbers of resistant bacteria in individual animals, and hence a decrease in the prevalence of animals with a high degree of resistance. Increasing usage of an already commonly used antibiotic, against which there was a high prevalence of resistance in the population, was reflected in an increase in the prevalence of faecal samples with a high degree of resistance to that antibiotic.

In conclusion, because of the increasing problem of bacterial resistance, a rational usage of antibiotics including the implementation of a veterinary antibiotic policy is of utmost importance to safeguard the efficacy of veterinary antibiotic therapy for the future and to minimize public health risks from veterinary use. Surveillance is an essential part of an antibiotic policy and should consist of the regular or continuous monitoring not only of antimicrobial resistance, but also of antibiotic usage in the population under study or at risk, followed by analysis and dissemination of the results. Differences between countries, regions, farms and veterinary practices can be observed and investigated, and therapeutic recommendations adapted when resistance exceeds threshold levels. Moreover, emergence and spread of new resistance genes, resistant clones or new resistance profiles can be detected early and action taken to prevent further dissemination.


    Acknowledgments
 
We would like to thank the inspectors of The Dutch National Inspection Service for Meat and Livestock (RVV) for collecting the Dutch faecal samples, Dr Christina Greko for providing the Swedish specimen, and Ms C. Driessen and Mr P. Terporten for technical assistance. This study has been made possible by a grant of The Dutch Veterinary Public Health Inspectorate and The Dutch Ministry for Agriculture, Nature Management and Fisheries.


    Notes
 
1 Corresponding author. Tel: +31-43-3881015; Fax: +31-43-3670996; E-mail: A.vandenBogaard{at}CPV.Unimaas.NL Back


    References
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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
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Received 2 September 1999; accepted 4 January 2000