JAC Advance Access originally published online on May 7, 2008
Journal of Antimicrobial Chemotherapy 2008 62(2):360-363; doi:10.1093/jac/dkn196
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Original research |
Ten years of antimicrobial susceptibility testing of Salmonella from Danish pig farms
National Food Institute, Technical University of Denmark, Mørkhøj Bygade 19, DK-2860 Søborg, Denmark
* Corresponding author. Tel: +45-72-34-70-68; Fax: +45-72-34-70-01; E-mail: hde{at}food.dtu.dk
Received 31 January 2008; returned 4 March 2008; revised 9 April 2008; accepted 9 April 2008
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Abstract |
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Objectives: This study analysed the trends in antimicrobial resistance in Salmonella serovars and phage types from pigs in Denmark from 1997 to 2006.
Methods: Salmonella isolates collected through the Salmonella surveillance programme in pigs were serotyped and phage-typed, and susceptibilities to the following antimicrobials were determined: ampicillin, chloramphenicol, gentamicin, nalidixic acid, colistin, streptomycin, sulphonamide, tetracycline and trimethoprim.
Results: No significant development of resistance occurred within the most important serovars, except Salmonella Typhimurium. A major decrease in Salmonella Typhimurium DT12 occurred from 46.5% in 1998 to 16.8% in 2006 while DT120, DT170 and DT104 increased. Throughout the study period, 80.9% of the DT12 isolates remained susceptible to the antimicrobials tested despite an increase in antimicrobial consumption in pigs during the period. In DT120, DT170 and DT104, only 20.1%, 33.1% and 23.0%, respectively, remained fully susceptible.
Conclusions: The results support that the use of antimicrobial agents might select for multiple resistant clones and that this might be the driver of changes in antimicrobial resistance within a serovar, rather than an emergence of resistance within clones. The results of this study also support that susceptible serovars only slowly become resistant to the antimicrobials tested.
Keywords: serovars , phage types , resistance profiles , trend analyses
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Introduction |
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Salmonella enterica is a common cause of human gastroenteritis and bacteraemia worldwide, and a wide variety of animals, particularly food animals, have been identified as reservoirs for non-typhoid Salmonella infections. In Denmark, for many years, pigs have been an important source of human Salmonella infections.1–4
In recent years, an increase in the occurrence of antimicrobial resistance among Salmonella isolated from animals and humans has been observed in several countries.5 The emergence of antimicrobial resistance is a matter of concern, since humans with infections caused by antimicrobial-resistant Salmonella, particularly nalidixic-acid-resistant Salmonella, have a higher fatality rate, are more likely to be hospitalized and are hospitalized for a longer time period than patients with infections caused by susceptible strains.6 This emergence of resistance is either driven by the emergence of multiresistant clones or by already established clones acquiring resistance genes. The importance of these possibilities has, to our knowledge, not previously been analysed in a large food animal reservoir.
The use of antimicrobial agents is normally considered an important factor in the emergence and spread of antimicrobial resistance. In a previous study, we showed that the emergence of tetracycline resistance in Salmonella Typhimurium from pigs was associated with the consumption of tetracycline in pig farms and that the emergence of resistance was caused by an emergence of resistant clones rather than the development of resistance in already established susceptible clones.7
Here we present an analysis of trends in resistance to several antimicrobial classes in Salmonella serovars and Salmonella Typhimurium phage types most commonly isolated from pigs in Denmark during the 10 year period 1997–2006. The changes in the occurrence of resistances were analysed in relation to the consumption of antimicrobial agents for pigs in Denmark over the same time period.
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Materials and methods |
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Collection of Salmonella isolates
In 1995, the Danish serological Salmonella surveillance programme in pigs was initiated, which identifies farms with medium-to-high-level Salmonella infection.2 In order to further characterize the Salmonella infection, veterinarians are required to collect 20 pen faecal samples in each farm. A pen faecal sample is a composite sample from the pigs in the pen. Before 2003, one Salmonella isolate per positive faecal sample was serotyped, and a random subsample of all Salmonella Typhimurium isolates was phage-typed; the majority of these isolates were also tested for antimicrobial susceptibility. From 2003 and onwards, 20 pen faecal samples are still collected but at the laboratory samples are pooled into five batches, each batch comprising 4 samples. All Salmonella suspect colonies are tested for chloramphenicol resistance to screen for DT104. If the Salmonella suspect colonies are resistant to chloramphenicol, one isolate from each positive pooled sample is serotyped, phage-typed and tested for antimicrobial susceptibility. If none of the colonies are resistant to chloramphenicol, only one Salmonella suspect colony from the five pooled samples is serotyped. If Salmonella Typhimurium is detected, the isolate is also phage-typed and tested for antimicrobial susceptibility. From 1996 to mid-2005, a random subsample of all non-Typhimurium serovars was tested for antimicrobial susceptibility.
Isolation, identification and antimicrobial susceptibility testing
Identification, serotyping, phage typing and antimicrobial susceptibility testing were performed by conventional microbiological methods as described previously.8 During the entire 10 year time period, all Salmonella isolates were tested for antimicrobial susceptibility by determining MICs using a commercially prepared, dehydrated panel (Sensititre®) according to the CLSI standards.9 From 1997 and onwards, the Salmonella isolates were tested for susceptibility to the following nine antimicrobials: ampicillin, chloramphenicol, gentamicin, nalidixic acid, colistin, streptomycin, sulphonamide, tetracycline and trimethoprim, representing different antimicrobial classes. In categorizing the results, CLSI breakpoints were used.
Salmonella isolates collected through the Salmonella surveillance programme in pigs from 1996 to 2006 were included to describe the changes in serovar and phage type distributions over time. Only one isolate per serovar/Salmonella Typhimurium phage type per farm per year was included. Information on Salmonella Typhimurium phage types was not maintained in the database for isolates collected in 1996 and 1997.
The trends over time for the most prevalent serovars and phage types were analysed for each serovar and phage type using logistic regression in generalized linear mixed models with farm included as a random effect (SAS Enterprise Guide® Version 3.0). The criteria for keeping fixed effects in the model were P < 0.05.
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Results and discussion |
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The handling of pen faecal samples was changed in 2003; however, this change did not decrease the sensitivity of detecting Salmonella in pen faecal samples from pigs. Since 1995, chloramphenicol resistance occurred rarely among Salmonella isolates from Danish pigs; the only exception was Salmonella Typhimurium DT104 and the occurrence of this phage type did not change significantly after 2003 (Figure 1). This shows that the change in method had little or no impact on the probability of detecting susceptible Salmonella isolates.
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A total of 12 788 Salmonella isolates were available for the analysis. Salmonella Typhimurium accounted for 8501 isolates (66.5%), and of these, 7199 Salmonella Typhimurium isolates were characterized by phage typing; between 529 and 1075 isolates per year. The results of the logistic regressions showed that the occurrence of Salmonella Typhimurium in Danish pigs has decreased significantly (P < 0.0001) from 74.9% in 1996 to 62.6% in 2006. In addition, the occurrence of Salmonella Typhimurium DT12 (P < 0.0001) and DT66 (P < 0.0001) decreased significantly, whereas DT120 (P < 0.0001), DT170 (P < 0.0001) and DT104 (P < 0.0001) increased significantly and DT17 (P = 0.054) remained unchanged from 1998 to 2006. The largest change over time was observed for Salmonella Typhimurium DT12 (Figure 1).
Table 1 shows the resistance profiles for the most prevalent serovars and phage types based on the nine antimicrobial agents tested. From 1997 to 2006, between 80.9% and 83.4% of all DT12, DT17 and DT66 isolates remained fully susceptible to all nine antimicrobials.
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DT120 was rarely detected from 1998 to 2001 (Figure 1); however, from 2002 to 2006, this phage type became more prevalent and in 2006, DT120 was for the first time the most common phage type in Danish pigs. The predominant resistance profile in DT120 was ampicillin-streptomycin-sulphonamide-tetracycline. DT170 occurrence was similar to that of DT120. However, among DT170, the dominant resistance profile was streptomycin-sulphonamide-tetracycline. Concomitant with the significant decrease in DT12 and the emergence of phage types where only 20.1% to 33.1% of the isolates remained fully susceptible, the antimicrobial consumption, and in particular tetracycline consumption, increased in Danish animal production from 12 900 kg of active tetracycline in 1996 to 32 650 kg in 2006. This increase was particularly pronounced from 1999 to 2006, where tetracycline consumption doubled. Approximately 80% of this tetracycline consumption was used for pigs.8
Among the resistant phage types (DT104, DT120 and DT170), one resistance profile in each phage type was dominant (Table 1). A previous study7 indicated that an increase in tetracycline consumption in Danish pigs selected for Salmonella Typhimurium phage types that were resistant to tetracycline, resulting in a change in the phage type distribution over time. The results of the present study support that the use of antimicrobial agents might select for multiresistant clones and that this might be the driver of changes in antimicrobial resistance within a serovar.
Based on the experiences in the UK in particular, where DT104 has spread rapidly, it was expected that DT104 would have become more prevalent in Danish pig production. Since DT104 was first detected in Danish pigs, a trace-back strategy based on trade relations was initiated to reduce the spread of DT104; these precautions were not taken against any other phage types. Results from this study and results from a recent study by Skov et al.10 indicated that the occurrence of DT104 in Danish pig production would have been higher if no precautions were taken.
Salmonella Derby was the second most common Salmonella serovar in pigs and the occurrence increased significantly (P < 0.0001) from 4.5% in 1996 to 25.1% in 2006. Tetracycline resistance in Salmonella Derby increased from 2000 to 2006 (Table 1), and during the same period, tetracycline consumption increased. The prevalence of Salmonella Infantis and Salmonella Livingstone remained unchanged during the study period and the majority of the isolates were susceptible to all nine antimicrobials tested (Table 1). We indicated in a previous study7 that antimicrobial-susceptible Salmonella Typhimurium phage types only slowly become resistant, although tetracycline consumption increased. The results of this study further support that susceptible serovars only slowly become resistant to the antimicrobials tested.
To our knowledge, this is the first time that the Salmonella serovar distributions over time have been analysed in an animal population. In addition, it is to our knowledge also the first time that changes in resistance profiles for different Salmonella serovars and phage types have been analysed over time. The results support that the use of antimicrobial agents might select for multiresistant clones and that this might be the driver of changes in antimicrobial resistance within a serovar, rather than an emergence of resistance within clones.
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Funding |
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The Ministry of Family and Consumer Affairs is funding the DANMAP and VetStat programmes while The Ministry of the Interior and Health is funding the DANMAP programme.
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None to declare.
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Acknowledgements |
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We would like to thank the technicians at the National Food Institute for characterization of bacterial specimens and antimicrobial susceptibility testing.
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References |
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1 Anon. Annual Report on Zoonoses in Denmark 2006 (2007) Technical University of Denmark.
2 Christensen J, Baggesen DL, Soerensen V, et al. Salmonella level of Danish swine herds based on serological examination of meat-juice samples and Salmonella occurrence measured by bacteriological follow-up. Prev Vet Med (1999) 40:277–92.[CrossRef][Web of Science][Medline]
3 Christensen J, Baggesen DL, Nielsen B, et al. Herd prevalence of Salmonella spp. in Danish pig herds after implementation of the Danish Salmonella Control Program with reference to a pre-implementation study. Vet Microbiol (2002) 88:175–88.[CrossRef][Web of Science][Medline]
4 Stege H, Christensen J, Nielsen JP, et al. Prevalence of subclinical Salmonella enterica infection in Danish finishing pig herds. Prev Vet Med (2000) 44:175–88.[CrossRef][Web of Science][Medline]
5 McDermott PF. Antimicrobial resistance in nontyphoidal salmonellae. In: Antimicrobial Resistance in Bacteria of Animal Origin—Aarestrup FM, ed. (2006) Washington, DC: ASM Press. 293–314.
6 Mølbak K. The clinical importance of animal-related resistance. In: Antimicrobial Resistance in Bacteria of Animal Origin—Aarestrup FM, ed. (2006) Washington, DC: ASM Press. 329–37.
7 Emborg H-D, Vigre H, Jensen VF, et al. Tetracycline consumption and occurrence of tetracycline resistance in Salmonella Typhimurium phage types from Danish pigs. Microb Drug Resist (2007) 13:289–94.[CrossRef][Web of Science][Medline]
8 DANMAP. Use of Antimicrobial Agents and Occurrence of Antimicrobial Resistance in Bacteria From Food Animals, Foods and Humans in Denmark. A combined reference for 11 DANMAP reports, 1996–2006.
9 Clinical and Laboratory Standards Institute. Methods for Dilution Antimicrobial Susceptibility Tests for Bacteria That Grow Aerobically—Second Edition: Approved Standard M7-A2 (2003) Wayne, PA, USA: CLSI.
10 Skov MN, Andersen JS, Baggesen DL. Occurrence and spread of multiresistant Salmonella Typhimurium DT104 in Danish animal herds investigated by the use of DNA typing and spatio-temporal analysis. Epidemiol Infect (2007) 1–7.
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