1 National Salmonella Reference Laboratory (Berlin) and 2 National Escherichia coli Reference Laboratory (Dessau), Federal Institute for Risk Assessment (BfR), P.O. Box 480447, D-12254 Berlin, Germany
Received 1 April 2003; returned 23 April 2003; revised 2 June 2003; accepted 9 June 2003
![]() |
Abstract |
---|
![]() ![]() ![]() ![]() ![]() ![]() |
---|
Materials and methods: Three hundred and seventeen isolates were tested for their resistance to 17 antimicrobial agents by broth microdilution. Resistant strains were screened by molecular methods for resistance genes, integrons and mutations in quinolone-resistance determining regions.
Results: Resistance was found in 40% and multiresistance in 32% of the strains. The resistance was significantly higher in isolates from poultry (61%) and swine (60%) than from cattle (25%) (P < 0.01). The most prevalent resistances were to sulfamethoxazole, tetracycline, streptomycin, ampicillin and spectinomycin (3015%). For each antibiotic, the predominant resistance genes were: ampicillin, blaTEM1-like (92%); chloramphenicol, catA (68%) and cmlA1-like (36%); gentamicin, aac(3)-IV (60%); kanamycin, aphA1 (100%); streptomycin, aadA1-like (61%) and strA/B (59%); sulfamethoxazole, sul2 (66%), sul1 (42%) and sul3 (14%); tetracycline, tet(A) (66%) and tet(B) (42%); and trimethoprim, dfrA1-like (77%), dfrA17 (13%) and dfrA12 (7%). Class 1 integrons were found in 30% of the strains. They carried dfrA1-aadA1a (40%), aadA1a (29%), sat1-aadA1a (16%), dfrA17-aadA5 (11%), oxa1-aadA1a (5%) and dfrA12-aadA2 (3%). Eleven percent of the strains were resistant to nalidixic acid. Of these, 61% presented a reduced susceptibility to ciprofloxacin (MIC = 0.122 mg/L) and single mutations in gyrA or gyrA and parC genes, and 39%, full resistance to ciprofloxacin (MIC 4 mg/L) and double and single mutations in gyrA and parC, respectively.
Conclusion: The study gives baseline information on the magnitude of the resistance problem and its genetic background in contemporary German E. coli from food-producing animals.
Keywords: resistance genes, quinolones, integrons
![]() |
Introduction |
---|
![]() ![]() ![]() ![]() ![]() ![]() |
---|
Integrons are gene expression systems that incorporate gene cassettes and make them functional. Class 1 integrons are the most frequently detected ones among Enterobacteriaceae. They comprise two conserved segments (CS) flanking a variable region (VR) where the gene cassettes can be located. The 5'CS consists of an integrase gene (intI) and specific insertion site (attI). The 3'CS usually contains a qacE1 and a sul1 gene (encoding quaternary ammonium compound and sulphonamide resistance).5
To generate baseline data to be used in future risk assessment of antimicrobial resistance, a number of surveillance systems on the local, continental or global scale have been initiated. Among the species proposed for surveillance is E. coli.6 Consequently, the prevalence and molecular basis of antimicrobial resistance in E. coli strains from humans, livestock and food have been investigated in several countries.3,79 This paper describes the antimicrobial drug resistance situation of German E. coli isolates obtained from cattle, poultry and swine (livestock and food) during 19992001.
![]() |
Materials and methods |
---|
![]() ![]() ![]() ![]() ![]() ![]() |
---|
The MICs of 17 antimicrobial agents were assessed by the NCCLS broth microdilution method. The NCCLS recommended breakpoints were used, except for ceftiofur (8 mg/L), colistin (
16 mg/L), florfenicol (
32 mg/L), neomycin (
16 mg/L), spectinomycin (
128 mg/L) and streptomycin (
32 mg/L) (DANMAP7 recommended breakpoints). In the phenotypic analysis, isolates with intermediate MICs were not considered as resistant.
The detection of antimicrobial resistance genes, class 1 integrons and mutations in the quinolone-resistance determining region (QRDR) was carried out by PCR and DNA sequencing. The identification of resistance genes followed a two-step screening approach. All isolates showing full or intermediate resistance to a certain antimicrobial were tested for the corresponding resistance genes shown in Table 1. Those strains that were PCR negative were screened for additional resistance determinants (legend of Table 1). Information about the primers used can be requested from the authors. Plasmid DNA was extracted by an alkaline denaturation method. The location of class 1 integrons was determined by Southern-hybridization with intI1 and sul1 probes.
|
![]() |
Results and discussion |
---|
![]() ![]() ![]() ![]() ![]() ![]() |
---|
The molecular investigations on the underlying resistance mechanisms showed that identical resistance phenotypes were based on different genes (Table 1): ampicillin (blaTEM1-like and blaOXA1-like genes), chloramphenicol (cmlA1-like and catA genes), streptomycin-spectinomycin (aadA1a, aadA2, aadA5 and strA/B genes), sulfamethoxazole (sul1, sul2 and sul3 genes), tetracycline [tet(A), tet(B) and tet(D) genes], and trimethoprim (dfrA1-like, dfrA7, dfrA12, dfrA14 and dfrA17 genes). Some of the genes (blaTEM1-like, catA, aac(3)-IV, aphA1, aadA1-like, strA/B, sul2, tet(A) and dfrA1-like) were widely spread (frequency >60%) among the resistant strains. In seven cases (three for ampicillin, two for chloramphenicol and two for gentamicin), the genes responsible for resistance could not be identified, indicating other possible resistance mechanisms. In 49 cases, more than one gene encoding the same resistance was present in one strain [the tandem strA/B and aadA1-like in 17 strains, sul1 and sul2 in 13, tet(A) and tet(B) in eight, sul3 and sul2 or sul1 in four and three, catA and cmlA in three and dfrA1-like and dfrA17 in one]. The newly described sul3 gene (accession number AJ459418) found in Swiss E. coli isolates from pigs was detected in 14% of the sulfamethoxazole-resistant isolates (eight strains from swine, four from poultry and one from cattle). No floR, blaCARB-2 (pse-1) or tet(G) genes, generally associated with a resistance island in Salmonella Typhimurium DT104-clone, were found, although floR has been found in plasmids of E. coli.4
About 30% (38 strains) of all resistant isolates investigated carried class 1 integrons. They were more prevalent in strains isolated from poultry (36% of resistant strains from poultry) and swine (32%) than from cattle (20%). These strains showed different resistance phenotypes, but all of them were resistant to streptomycin. Six types of integrons (five, four and three in swine, cattle and poultry, respectively) were detected. Gene cassettes which conferred resistance to streptomycin/spectinomycin (aadA1a, aadA2 and aadA7) were present in all integron-positive strains, and to trimethoprim (dfrA1, dfrA17 and dfrA12), in 53% of the cases. Fifteen strains (40% of integron positive) carried integrons (VR-amplicons of about 1600 bp) with dfrA1-aadA1a, and showed trimethoprim-resistance as well. However, two of the strains were sulfamethoxazole-susceptible, and their integrons lacked sul1 and qacE1/sul1. Additional resistances were also found. Eleven and six strains (29% and 16%) carried integrons (amplicons of 1000 and 1900 bp) with aadA1a and sat1 (streptothricin-resistance)-aadA1a, respectively. All these strains had the phenotype streptomycin-spectinomycin-sulfamethoxazole in common, but additional resistances were also found. Four strains (11%) carried integrons (amplicons of 1700 bp) with dfrA17-aadA5, and were of the trimethoprim-trimethoprimsulfamethoxazole-streptomycin-sulfamethoxazole-ampicillin-chloramphenicol-tetracycline phenotype. Again additional resistances were found as well. Two strains (5%) carried integrons (amplicons of 2000 bp) with the blaOXA1-aadA1a, and showed the phenotype ampicillin- coamoxiclav-streptomycin-spectinomycin-sulfamethoxazole-kanamycinneomycin in combination with additional resistances. One of these also carried the integron with dfrA1-aadA1a. The last strain (3%) carried an integron (amplicon of 1850 bp) with dfrA12-aadA2 and showed the trimethoprim-trimethoprimsulfamethoxazole-streptomycin-spectinomycin-sulfamethoxazole-ampicillin-tetracycline phenotype. The integron lacked the qacE
1 and sul1 genes. However, the strain carried a sul3 gene which probably encoded the sulfamethoxazole resistance. In 32 strains, the integron probes hybridized to plasmids of >50 kb. In two strains, more than one plasmid carried an integron.
The results presented show the wide distribution of integrons on resistance plasmids of E. coli and emphasize their high potential to contribute to the efficient spread of antibiotic resistance.5 The finding of strains which lacked qacE1/sul1 (two) or only sul1 (one), was interesting, and in fact, defective integrons have been described5 lacking some of these genes usually located in the 3'CS.
Table 2 shows the amino acid changes in the QRDRs of the 36 nalidixic acid-resistant strains. Fourteen of them (13 from poultry) exhibited resistance to ciprofloxacin (MIC 4 mg/L), and showed three-point mutations: two affecting gyrA and the third affecting parC. The other 22 strains (18 from poultry) exhibited reduced susceptibility to ciprofloxacin (MIC 0.122 mg/L). They showed mutations in the Ser-83 (18 strains) or Asp-87 (three strains) of the gyrA gene, or a double mutation affecting gyrA and parC (only one isolate). The fact that most of the nalidixic acid-resistant strains (31 of 36) are of poultry origin is remarkable. It points to the selective potential of fluoroquinolones, in commercial poultry (chicken and turkey) production.10,11
|
![]() |
Acknowledgements |
---|
![]() |
Footnotes |
---|
![]() |
References |
---|
![]() ![]() ![]() ![]() ![]() ![]() |
---|
2 . White, D. G., Zhao, S., Simjee, S. et al. (2002). Antimicrobial resistance of foodborne pathogens. Microbes and Infection 4, 40512.[CrossRef][ISI][Medline]
3 . Isaacson, R. E. & Torrence, M. E. (2002). The Role of Antibiotics in Agriculture. American Academy of Microbiology, Washington, DC, USA.
4 . Schwarz, S. & Chaslus-Dancla, E. (2001). Use of antimicrobials in veterinary medicine and mechanisms of resistance. Veterinary Research 32, 20125.[CrossRef][ISI][Medline]
5 . Carattoli, A. (2001). Importance of integrons in the diffusion of resistance. Veterinary Research 32, 24359.[CrossRef][ISI][Medline]
6 . Caprioli, A., Busani, L., Martel, J. L. et al. (2000). Monitoring of antibiotic resistance in bacteria of animal origin: epidemiological and microbiological methodologies. International Journal of Antimicrobial Agents 14, 295301.[CrossRef][ISI][Medline]
7 . DANMAP. (2001). Use of Antimicrobial Agents and Occurrence of Antimicrobial Resistance in Bacteria from Food Animals, Foods and Humans in Denmark (Bager, F. & Heuer, O. E., Eds). Danish Zoonosis Centre, Danish Veterinary Institute, Copenhagen, Denmark.
8 . Lanz, R., Kuhnert, P. & Boerlin, P. (2003). Antimicrobial resistance and resistance gene determinants in clinical Escherichia coli from different animal species in Switzerland. Veterinary Microbiology 91, 7384.[CrossRef][ISI][Medline]
9
.
Schroeder, C. M., Zhao, C., DebRoy, C. et al. (2002). Antimicrobial resistance of Escherichia coli O157 isolated from humans, cattle, swine, and food. Applied and Environmental Microbiology 68, 57681.
10
.
Van den Bogaard, A. E., London, N., Driessen, C. et al. (2001). Antibiotic resistance of faecal Escherichia coli in poultry, poultry farmers and poultry slaughterers. Journal of Antimicrobial Chemotherapy 47, 76371.
11 . Webber, M. & Piddock, L. J. V. (2001). Quinolone resistance in Escherichia coli. Veterinary Research 32, 27584.[CrossRef][ISI][Medline]