Trends in Antimicrobial Susceptibility in Relation to Antimicrobial Usage

Veterinary Microbiology 89 (2002) 83–94

Trends in antimicrobial susceptibility in relation to antimicrobial usage and presence of resistance genes in Staphylococcus hyicus isolated from exudative epidermitis in pigs Frank Møller Aarestrup*, Lars Bogø Jensen Danish Veterinary Institute, 27 Bu¨lowsvej, DK-1790 Copenhagen V, Denmark Received 11 March 2002; received in revised form 11 July 2002; accepted 16 July 2002

Abstract

From 1996 to 2001 a total of 467 Staphylococcus hyicus isolates from exudative epidermitis (EE) in pigs in Denmark were examined for susceptibility to 13 different antimicrobial agents. The presence of selected genes encoding macrolide (erm(A), erm(B) and erm(C)), penicillin (blaZ ), streptogramin (vat, vga, vga(B), vat(B), vat(D) and vat(E)), streptomycin (aadE ) and tetracycline resistance (tet(K), tet(L), tet(M) and tet(O)) were determined in selected isolates. The occurrence of erythromycin resistance increased from 33% in 1996 to a maximum of 62% in 1997 and decreased to 26% in 2001. Resistance to sulphametazole increased from 17% in 1996 to 30% in 1998 but has since decreased to 4% in 2001. Resistance to trimethoprim increased to 51% in 1997 and decreased to 21% in 2001. Resistance to tetracycline (21–31%) remained relatively constant during 1996–2000, but increased to 47% in 2001. Resistance to penicillin (54–75%) streptomycin (33–53%) and tetracycline (21–47%) remained relatively constant over the time investigated. All 48 penicillin resistant isolates examined contained the blaZ gene and 40 (85%) of the streptomycin resistant isolates the aadE gene. It was not possible to detect any streptogramin resistance gene in four streptogramin resistant isolates. Of the 55 erythromycin resistant isolates examined, five contained erm(A), 13 erm(B), 35 erm(C) and two both erm(A) and erm(C). The presence of erm(B) was confirmed by hybridization to plasmid profiles in all 13 PCR-positive isolates. Of 52 tetracycline resistant isolates examined, two contained tet(L), 38 tet(K) and 12 both tet(K) and tet(L). # 2002 Published by Elsevier Science B.V.

Keywords: Staphylococcus hyicus; Pig-bacteria; Antimicrobial agents; Genetics

* Corresponding author. Tel.: þ45-35-30-01-00; fax: þ45-35-30-01-20. E-mail addresses: [email protected], [email protected] (F.M. Aarestrup).

0378-1135/02/$ – see front matter # 2002 Published by Elsevier Science B.V. PII: S 0378-1135(02)00177-3 84 F.M. Aarestrup, L.B. Jensen / Veterinary Microbiology 89 (2002) 83–94

1. Introduction

Staphylococcus hyicus is the causative agent of exudative epidermitis (EE) in pigs, a generalized infection of the skin characterized by greasy exudation, exfoliation, and vesicle formation (Sompolinsky, 1953; Jones, 1961; L’Ecuyer, 1966). The disease is frequently treated with antimicrobial agents, but treatment is problematic because of the limited number of antimicrobial drugs available for this purpose. A frequent occurrence of antimicrobial resistance has previously been reported among S. hyicus in different countries (Devriese, 1977; Teranishi et al., 1987; Schwarz and Blobel, 1989; Aarestrup et al., 1998a,b). A large number of different antimicrobial agents has been used for therapy and until 1998 also for growth promotion in the production of pigs in Denmark and a frequent occurrence of resistance to these compounds has been observed among several different bacterial species (Aarestrup et al., 1998a,b). However, major changes in the usage of antimicrobial agents especially those used for growth promotion has occurred in the period 1995–1999. This has been for pig production especially, the case for tylosin that belongs to the macrolides. Among enterococci from pigs the decreased usage of tylosin has been followed by a major decrease in the occurrence of macrolide resistance (Aarestrup et al., 2001). Several studies have determined the occurrence of different genes encoding antimicrobial resistance in staphylococci of human origin. In contrast there is only limited information about the distribution of different resistance genes in staphylococci of animal origin. This study describes the trends in antimicrobial susceptibility and presence of selected resistance genes among S. hyicus isolated from EE in Denmark. In addition, the occurrence of different genes encoding resistance to macrolides, penicillins, streptomycin, and tetracycline among selected isolates is reported.

2. Materials and methods

2.1. Sampling of bacterial species

As part of the Danish monitoring for antimicrobial resistance S. hyicus isolates from diagnostic submissions are continuously isolated from cases of EE, tested for susceptibility to antimicrobial agents and stored (Aarestrup et al., 1998a). All isolates were from different herds all over Denmark. In the period of January 1996 to December 2001 a total of 467 isolates were included in the monitoring of resistance at the Danish Veterinary Institute.

2.2. Susceptibility testing

Isolates collected during 1996 were, with the exception of avilamycin, tested for their susceptibility using tablet diffusion as previously described (Aarestrup et al., 1998a,b). Isolates collected during 1997–2001 were tested for susceptibility to antimicrobial agents using a commercial dehydrated MIC panel and a semiautomatic inoculation (SensiTitre, F.M. Aarestrup, L.B. Jensen / Veterinary Microbiology 89 (2002) 83–94 85

Trek Diagnostic, UK) following NCCLS guidelines (NCCLS, 2000). Susceptibility of the following antimicrobial agents was determined during the entire time-period: avilamycin, bacitracin, chloramphenicol, erythromycin, gentamicin, oxacillin, penicillin, streptomycin, sulphametoxazole, tetracycline, trimethoprim, and vancomycin. From 1996 to 1997 susceptibility was determined for the fluoroquinolone, enrofloxacin whereas susceptibility was determined for ciprofloxacin from 1998 to 2001. Susceptibility to quinupristin/dalfopristin (Q/D) was only determined from 1998 to 2001.

Table 1 Primers used for detection of genes encoding resistance to erythromycin, penicillin, sreptogramin, streptomycin, and tetracycline among S. hyicus from pigs

Primer Sequence (50 ! 30) Reference erm(A)-1 50-TCAAAGCCTGTCGGAATTGG-30 Jensen et al. (1998) erm(A)-2 50-AAGCGGTAAACCCCTCTGAG-30 erm(B)-1 50-CATTTAACGACGAAACTGGC-30 Jensen et al. (1998) erm(B)-2 50-GGAACATCTGTGGTATGGCG-30 erm(C)-1 50-ATCTTTGAAATCGGCTCAGG-30 Jensen et al. (1998) erm(C)-2 50-CAAACCCGTATTCCACGATT-30 vat-1 50-TGGAGTGTGACAAGATAGGC-30 Hammerum et al. (1998) vat-2 50-GTGACAACAGCTTCTGCAGC-30 vga-1 50-AGTGGTGGTGAAGTAACACG-30 Hammerum et al. (1998) vga-2 50-CTTGTCTCCTCCGCGAATAC-30 vga(B)-1 50-TGACAATATGAGTGGTGGTG-30 Hammerum et al. (1998) vga(B)-2 50-GCGACCATGAAATTGCTCTC-30 vat(B)-1 50-GGCCCTGATCCAAATAGCAT-30 Hammerum et al. (1998) vat(B)-2 50-GTGCTGACCAATCCCACCAT-30 vat(D) 50-GCTCAATAGGACCAGGTGTA-30 Hammerum et al. (1998) vat(D) 50-TCCAGCTAACATGTATGGCG-30 vat(E) 50-ACTATACCTGACGCAAATGC-30 Jensen et al. (2000) vat(E) 50-GGTTCAAATCTTGGTCCG-30 blaZ-1 50-AAGAGATTTGCCTATGCTTC-30 Vesterholm-Nielsen et al. (1999) blaZ-2 50-GCTTGACCACTTTTATCAGC-30 tet(K)-1 50-TTAGGTGAAGGGTTAGGTCC-30 Aarestrup et al. (2000) tet(K)-2 50-GCAAACTCATTCCAGAAGCA-30 tet(L)-1 50-GTTGCGCGCTATATTCCAAA-30 Aarestrup et al. (2000) tet(L)-2 50-TTAAGCAAACTCATTCCAGC-30 tet(M)-1 50-GTTAAATAGTGTTCTTGGAG-30 Aarestrup et al. (2000) tet(M)-2 50-CTAAGATATGGCTCTAACAA-30 tet(O)-1 50-GATGGCATACAGGCACAGAC-30 Aarestrup et al. (2000) tet(O)-2 50-CAATATCACCAGAGCAGGCT-30 aadE-1 50ATGGAATTATTCCCACCTGA-30 This study aadE-2 50-TCAAAACCCCTATTAAAGCC-30 86 F.M. Aarestrup, L.B. Jensen / Veterinary Microbiology 89 (2002) 83–94

2.3. Detection of genes encoding macrolide, streptogramin, streptomycin, and tetracycline resistance

The presence of antimicrobial resistance genes was examined in selected resistant isolates using PCR as previously described (Hammerum et al., 1998; Jensen et al., 1999; Vesterholm-Nielsen et al., 1999; Aarestrup et al., 2000). The presence of the erm(A), erm(B) and erm(C) genes encoding macrolide resistance were examined in 55 erythromycin resistant isolates, the blaZ gene encoding penicillin resistance in 48 penicillin resistant isolates, the presence of the tet(K), tet(L), tet(M) and tet(O) genes encoding tetracycline resistance in 52 tetracycline resistant isolates, the vat, vga, vga(B), vat(B), vat(D) and vat(E) genes encoding streptogramin resistance in four Q/D resistant isolates and the presence of the aadE gene encoding streptomycin resistance in 47 streptomycin resistant isolates. The primers used are given in Table 1. DNA sequencing (Sears et al., 1992) veriﬁed the identity of the gene products in selected isolates.

2.4. Plasmid profiling

Plasmid DNA was purified from all erythromycin resistant isolates, that gave amplicons for the erm(B) gene, using a modified protocol for the Qiagen Plasmid Midi kit (Qiagen, Cat. no. 12243). Cells from a 10 ml overnight culture were harvested in a Beckman centrifuge at 6000 Â g for 10 min and resuspended in 10 ml P1 buffer (50 mM Tris–Cl, pH 8.0; 10 mM EDTA) containing RNase (100 mg/ml) and lysozyme (20 mg/ml). The suspension was placed on a rotary shaker (250 rpm) at 37 8C for 15 min and then 10 ml P2 buffer (200 mM NaOH, 1% SDS) and 10 ml P3 buffer (3.0 M potassium acetate, pH 5.5) was added as described in the Qiagen protocol. The DNA was then purified on a Qiagen Midi column as described by the Qiagen Plasmid Midi Protocol.

2.5. Hybridization

Digoxigenin-labeled DNA-probes were prepared by PCR ampliﬁcation using primers for erm(B) and labeled with the Boehringer Mannheim DNA labeling kit. Southern transfer and hybridization of all plasmid proﬁles were performed as previously described (Jensen et al., 1998), using the erm(B) probe.

3. Results

3.1. Distribution of antimicrobial resistance using MIC-determination

The susceptibility of the different S. hyicus isolates examined with MIC-determination is shown in Table 2. All isolates examined were susceptible to chloramphenicol, gentamicin, oxacillin, and vancomycin with MICs below the NCCLS break points. For avilamycin, the isolates gave one large distribution with most isolates having MICs around 2–4 mg/ml. Two isolates had a MIC above the break point chosen at 16 mg/ml. Similarly for bacitracin the isolates gave one large population with most isolates having MICs from 16 to 64 mg/ml. Table 2 Distribution of MICs for S. hyicus isolates from pigs with EE Denmarka

Antimicrobial agentb Number of Number of isolates with a MIC (mg/ml) of ..Arsrp ..Jne eeiayMcoilg 9(02 83 (2002) 89 Microbiology Veterinary / Jensen L.B. Aarestrup, F.M. isolates tested 0.125 0.25 0.5 1 2 4 8 16 32 64 128 256 512 >512

Avilamycin 467 Bacitracin 377 Chloramphenicol 377 Ciprofloxacinc 288 Enrofloxacind 89 Erythromycin 377 Gentamicin 377 Oxacillin 377 Penicillin 377 Streptomycin 377 Sulphametazole 377 Q/De 288 Tetracycline 377 Trimethoprim 377 Vancomycin 377 a The shaded areas indicate the concentrations of the different antimicrobial agents for which the bacterial isolates were not tested. Dark lines indicate the break points for resistance according to NCCLS (2000) or Aarestrup et al. (1998a). b MIC-determinations only performed on isolates collected from 1997 to 2001, except avilamycin where it was performed on all isolates. c 1998–2001. d – 1997. 94 e MICs for quinupristrin/dalfopristin only performed on isolates from 1998 to 2001. 87 88 F.M. Aarestrup, L.B. Jensen / Veterinary Microbiology 89 (2002) 83–94

A few isolates had MIC above or close to the NCCLS break points for the ﬂuoroquinolones. For erythromycin, two large populations clearly separated by the NCCLS break point were observed. Most isolates had either a MIC at 0.5 mg/ml or below or 32 mg/ml. Most penicillin resistant isolates had MICs 4 mg/ml. However, a few isolates had MICs closer to the break point at 0.25 mg/ml. For streptomycin the isolates were not as clearly separated using the break point of 32 mg/ml. Most susceptible isolates had MICs 4 mg/ml and most resistant isolates MICs 128 mg/ml. However, 16 isolates had a MIC of 16 mg/ml and 19 isolates had a MIC of 32 mg/ml. Thus, 9% of the isolates had MICs just below or above the break point. For sulphametazole, the susceptible isolates gave MICs around 16–32 mg/ ml, while most resistant isolates gave MICs >512 mg/ml. A total of 24 (6%) isolates had MICs just around the break point. For Q/D most isolates were clearly susceptible, but six isolates had MICs of 4 mg/ml. These isolates had also decreased MICs to another streptogramin antibiotic, and virginiamycin (data not shown). For tetracycline, most susceptible isolates were highly susceptible with MICs 1 mg/ml, while the resistant isolates had MICs around 32 mg/ml. Thus, most isolates were clearly separated with the NCCLS break points, but a few isolates had MICs just around the break point at 16 mg/ ml. For trimethoprim, the susceptible isolates were distributed around 4 mg/ml and most resistant at 64 mg/ml. A large number of isolates were just around the break point of 16 mg/ml.

3.2. Trends in resistance over time

The trends over time in antimicrobial resistance are given in Table 3 for seven antimicrobial agents where more than 5% of the isolates were resistant. All isolates were susceptible to chloramphenicol, gentamicin, oxacillin and vancomycin, while a limited number of isolates tested resistant to avilamycin, bacitracin and Q/D. The occurrence of erythromycin resistance increased from 33% in 1996 to a maximum of 62% in 1997 followed by a decrease to 17% in 2000 and 26% in 2001. Resistance to ﬂuoroquino- lones increased from 1996 to 1997 and has increased to 8% in 2001. Resistance to penicillin increased from 59% in 1996 to 75% in 1999, but then decreased to 62% in 2001. Resistance to streptomycin increased from 33% in 1996 to 53% in 1998 and

Table 3 Trends in resistance of S. hyicus from EE in pigs in Denmark to seven antimicrobial agents

Antimicrobial Rate of resistance of S. hyicus (%) agent 1996 1997 1998 1999 2000 2001 ðn ¼ 90Þ ðn ¼ 89Þ ðn ¼ 95Þ ðn ¼ 59Þ ðn ¼ 81Þ ðn ¼ 53Þ

Erythromycin 33 62 26 15 17 26 Fluoroquinolones 1 64458 Penicillin 59 62 64 75 54 62 Streptomycin 33 46 53 36 40 43 Sulphametazole 17 26 30 5 0 4 Tetracycline 31 29 28 24 21 47 Trimethoprim 43 51 40 22 15 21 F.M. Aarestrup, L.B. Jensen / Veterinary Microbiology 89 (2002) 83–94 89

Table 4 Occurrence of resistance genes among selected erythromycin and tetracycline resistant S. hyicus isolated from EE in pigs in Denmark

Antimicrobial No. of isolates Occurrence of resistance genes among the isolates examined resistance examined erm(A) erm(B) erm(C) ermðAÞþ tet(K) tet(L) tetðKÞþ ermðCÞ tetðLÞ Erythromycin 55 5 13 35 2 –– – Tetracycline 52 –––– 38 2 12

decreased to 43% in 2001. Resistance to sulphametazole increased from 17% in 1996 to 30% in 1998 but have since decreased to around 0% in 2000 and 2001. Resistance to tetracycline has slowly decreased from 31% in 1996 to 21% in 2000, but increased again in 2001 to 47%. Resistance to trimethoprim increased to 51% in 1997 and decreased to 21% in 2001.

3.3. Occurrence of resistance genes

All 48 penicillin resistant isolates examined contained the blaZ gene, while 40 (85%) of the streptomycin resistant isolates contained aadE. It was not possible to detect any of the resistance genes tested for in the four Q/D resistant isolates. All 55 erythromycin resistant isolates contained one or more of the erm genes. Five contained erm(A), 13 erm(B), 35 erm(C) and two both erm(A) and erm(C) (Table 4). The presence of erm(B) was conﬁrmed by hybridization to plasmid proﬁles in all 13 PCR-positive isolates. All 52 tetracycline resistant isolates examined gave positive results for tet genes. Two contained tet(L), 38 tet(K) and 12 both tet(K) and tet(L) (Table 4).

4. Discussion

In the present study a frequent occurrence of resistance to erythromycin, penicillin, streptomycin, sulfonamides, tetracycline and trimethoprim was observed among S. hyicus from pigs with EE in Denmark. Among S. hyicus isolates from Germany, Schwarz and Blobel (1989) reported higher levels of resistance to tetracycline (66%) and sulfonamides (100%), similar levels of resistance to streptomycin (43%), but lower level of resistance to penicillin (25%) and especially erythromycin (3%). In Belgium, Devriese (1977) reported 74, 60 and 60% resistance to erythromycin, penicillin and tetracycline, respectively, whereas in Japan, Teranishi et al. (1987) found very low levels of resistance to penicillin (4%) and streptomycin (2%) and more moderate levels of resistance to tetracycline (22%) and erythromycin (40%) among isolates from diseased pigs. Thus, in general the occurrence of resistance to erythromycin, but also to other antimicrobial agents is relatively high in Denmark compared to most other reports. However, the isolates from Denmark were collected in the period 1996–2001 and thus, more recent than the isolates from other countries. As can be seen from Table 3 major changes in the occurrence of 90 F.M. Aarestrup, L.B. Jensen / Veterinary Microbiology 89 (2002) 83–94 resistance can take place over a short time making comparison between different countries very difficult. The occurrence of resistance to penicillin (54–75%), streptomycin (33–53%) and tetracycline (21–47%) has been relatively constant over the period 1996–2001. In contrast major changes have taken place with respect to resistance to erythromycin, sulfonamides and trimethoprim. The occurrence of erythromycin resistance increased from 33% in 1996 to 62% in 1997 followed by a decrease to 17% in 2000 and 26% in 2001. For several years the macrolide tylosin were the most commonly used antimicrobial agent for growth promotion in Denmark. The usage increased from 52 t in 1995 to 68 t in 1996 and decreased to 62 t in 1997, 13 t in 1998, and 2 t in 1999, until its total ban as a growth promoter in 1999 (DANMAP, 2002). Approximately 10 t of macrolides, primarily tylosin are used every year for treatment of infections in Danish pigherds (DANMAP, 2002). Thus, the changes in occurrence of erythromycin resistance among S. hyicus seem to have followed the changes in usage of tylosin. This is in agreement with the observations among enterococci from pigs in Denmark, where major changes in the occurrence of resistance to erythromycin have followed the usage of tylosin for growth promotion (Aarestrup et al., 2001). In contrast, there is no similar explanation for the changes in the occurrence of resistance to sulfonamides and trimethoprim that have occurrence during 1999–2001. An increase in the usage of tetracycline for pigs have been observed from 1999 and during 2000 and 2001 and this could explain the increased occurrence of tetracycline resistance observed during 2001. Among staphylococci, the most commonly found tetracycline resistance gene is tet(K) (Bismuth et al., 1990; Warsa et al., 1996; Schwarz et al., 1998; Aarestrup et al., 2000). This was also the case in the present study where 50 of 52 isolates contained tet(K). The tet(L) gene has only been detected at a low frequency among Staphylococcus aureus and CNS isolated from humans (Bismuth et al., 1990; Warsa et al., 1996; Trzcinski et al., 2000). Schwarz and Noble (1994) detected the tet(L) gene among 10 (21%) of 47 tetracycline resistant staphylococci isolated from the skin of pigs. In contrast, Schwarz et al. (1998) only detected the tet(L) gene among a limited number of tetracycline resistant staphylococci of different animal origin. It the latter study only one of 24 tetracycline resistant S. hyicus examined contained tet(L). In a previous study on avian staphylococci from Denmark the tet(L) gene was not detected among 50 tetracycline resistant isolates (Aarestrup et al., 2000). In the present study the tet(L) gene was detected in 14 (27%) of the tetracycline resistant isolates and was the only tetracycline resistance gene detected in two isolates. Thus, in contrast to most other reports on the occurrence of tet genes in staphylococci, the tet(L) gene seems to have established itself among S. hyicus from pigs in Denmark. In staphylococci of human origin, the erm(A) and erm(C) genes are responsible for macrolide resistance in almost all isolates (Thakker-Varia et al., 1987; Eady et al., 1993; Westh et al., 1995; Jensen et al., 1999; Nicola et al., 1998; Martineau et al., 2000; Nawaz et al., 2000; Schmitz et al., 2000). Among S. aureus, erm(A) was responsible for the resistance among historical isolates (Westh et al., 1995; Nicola et al., 1998), whereas, in more recent years there has been a change towards erm(C) (Westh et al., 1995). The erm(A) has been commonly found in staphylococci of avian origin (Nawaz et al., 1999, 2000; Aarestrup et al., 2000) and in milk samples from cows (Khan et al., 2000), but only among a F.M. Aarestrup, L.B. Jensen / Veterinary Microbiology 89 (2002) 83–94 91 limited number of S. hyicus (Eady et al., 1993; Jensen et al., 1999). This is in agreement with the present study, where the erm(C) gene was the most commonly found macrolide resistance gene and the erm(A) gene only detected in seven of the 52 erythromycin resistant isolates examined. The erm(B) gene is the predominant gene among Staphylococcus intermedius (Eady et al., 1993; Boerlin et al., 2001), but has only been detected in a limited number of other staphylococcal species (Werckenthin et al., 1996; Martineau et al., 2000) and was not found in a previous study on S. hyicus in Denmark (Jensen et al., 1999). This is in contrast to the present study where the erm(B) gene was detected by PCR in 13 (25%) of the isolates and the presence of this gene verified by hybridization to plasmid profiles. The erm(B) gene is the most commonly observed gene encoding resistance in streptococci and enterococci (Weisblum, 1995), but seems now also to have emerged among S. hyicus in Denmark. The aadE (also called ant(6)-Ia) gene encoding streptomycin resistance was detected in most of the streptomycin resistant S. hyicus isolates. There is no other report on the occurrence of genes encoding streptomycin resistance in S. hyicus, but this gene seems to be the most common gene encoding streptomycin resistance in staphylococci of human origin (Shaw et al., 1993). Penicillin resistant resistant S. aureus emerged already in the 1940s (North and Christie, 1946; Barber, 1947). When beta-lactamase producing strains of S. aureus were first described (Kirby, 1944), resistance was uncommon, but have since emerged worldwide and today from 70 to 100% of staphylococci of human origin can be resistant (Lacey et al., 1984; Thornsberry, 1988; Renneberg and Rosdahl, 1992; Cormican and Jones, 1996). A more limited occurrence of resistance has been observed among staphylococci of animal origin, but resistance is still very common. The most common mechanisms mediating resistance to penicillin in staphylococci of human origin is the production of beta- lactamase (Lyon and Skurray, 1987). There have only been a limited number of studies determining the genetic background for resistance among animal isolates. However, among S. aureus from bovine mastitis a blaZ gene similar to those detected in isolates of human origin has been found (Vesterholm-Nielsen et al., 1999; Yazdankhah et al., 2000). In the present study all the penicillin resistant S. hyicus examined harboured the blaZ gene confirming that this is the main mechanism of penicillin resistance in all staphylococci. A number of different genes encoding resistance to streptogramin antimicrobials have been detected in staphylococci (Allignet et al., 1996; Allignet and El Solh, 1997) and enterococci (Hammerum et al., 1998; Jensen et al., 2000. It was, however, not possible to detect any of these mechanisms in any of the four Q/D resistant S. hyicus examined in this study. The present study showed a frequent occurrence of resistance to erythromycin, penicillin, streptomycin, sulphonamides, tetracycline and trimethoprim among S. hyicus isolated from EE in pigs in Denmark. The occurrence of resistance to erythromycin, sulphonamides and trimethoprim has decreased from 1996 to 2001 and for erythromycin the most likely explanation is the ban of the use of tylosin for growth promotion. Similar genes encoding resistance to erythromycin, penicillin, streptomycin and tetracycline to those previously observed were found. However, the erm(B) and tet(L) genes were more frequently found than have previously been observed. 92 F.M. Aarestrup, L.B. Jensen / Veterinary Microbiology 89 (2002) 83–94

Acknowledgements

We are grateful to Rene´ Hendriksen, Christina Svendsen, and Anette Nielsen for technical assistance. This study is a part of the Danish Integrated Antimicrobial Resistance Monitoring and Research Programme (DANMAP) conducted in collaboration between Statens Serum Institut, the National Food Agency of Denmark and the Danish Veterinary Institute and funded jointly by the Danish Ministry of Health and the Danish Ministry of Food, Agriculture and Fisheries.

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