Showing papers by "Frank Møller Aarestrup published in 2006"

Antimicrobial resistance in bacteria of animal origin

Abstract: Table of Contents 1. Modes of Antimicrobial Action and Mechanisms of Bacterial Resistance, Luca Guardabassi and Patrice Courvalin 2. History of Antimicrobial Usages in Agriculture: an Overview, John F. Prescott 3. Antimicrobial Susceptibility Testing of Bacteria of Veterinary Origin, Jeffrey L. Watts and Cynthia J. Lindeman 4. Molecular Methods for Detection of Antibiotic Resistance, Henk J. M. Aarts, Beatriz Guerra, and Burkhard Malorny 5. Drug Selection and Optimization of Dosage Schedules to Minimize Antimicrobial Resistance, Peter Lees, Didier Concordet, Fariborz Shojaee Aliabadi, and Pierre-Louis Toutain 6. Mechanisms and Spread of Bacterial Resistance to Antimicrobial Agents, Stefan Schwarz, Axel Cloeckaert, and Marilyn C. Roberts 7. Resistance to Metals Used in Agricultural Production, Henrik Hasman, Sylvia Franke, and Christopher Rensing 8. Disinfectant Resistance in Bacteria, Mark A. Webber, Martin J. Woodward, and Laura J. V. Piddock 9. Antimicrobial Resistance in Clostridium and Brachyspira spp. and Other Anaerobes, Anders Franklin, Marit Pringle, and David J. Hampson 10. Antimicrobial Resistance in Pathogenic Escherichia coli from Animals, David G. White 11. Antimicrobial Resistance in Members of the Family Pasteurellaceae, Corinna Kehrenberg, Robert D. Walker, Ching Ching Wu, and Stefan Schwarz 12. Antimicrobial Resistance in Staphylococci and Steptococci of Animal Origin, Frank M. Aarestrup and Stefan Schwarz 13. Antimicrobial Drug Resistance in Fish Pathogens, Henning Sorum 14. Mycoplasma, Frank M. Aarestrup and Isabelle Kempf 15. Other Pathogens, Frank M. Aarestrup 16. Antimicrobial Resistance in Campylobacter Species, Jorgen Engberg, Monika Keelan, Peter Gerner-Smidt, and Diane E. Taylor 17. Antimicrobial Resistance in Nontyphoidal Salmonellae, Patrick F. McDermott 18. Enterococcus, Shabbir Simjee, Lars B. Jensen, Susan M. Donabedian, and Marcus J. Zervos 19. The Clinical Importance of Animal-Related Resistance, Kare Molbak 20. The Origin, Evolution, and Local and Global Dissemination of Antimicrobial Resistance, Frank M. Aarestrup 21. Licensing and Approval of Antimicrobials for Use in Animals, Linda Tollefson, Deborah Morris, Christopher Boland, and Jack Kay 22. Monitoring of Antimicrobial Drug Usage in Animals: Methods and Applications, Kari Grave, Vibeke Frokjaer Jensen, Scott McEwen, and Hilde Kruse 23. Monitoring of Antimicrobial Resistance in Animals: Principles and Practices, Scott A. McEwen, Frank M. Aarestrup, and David Jordan 24. Risk Assessment and Its Use in Approval, Licensing, and Prudent Use Guidelines, David Vose 25. Concluding Remarks and Future Aspects: Some Personal Views, Frank M. Aarestrup

Diversity and evolution of blaZ from Staphylococcus aureus and coagulase-negative staphylococci

Abstract: Objectives To elucidate the diversity and evolutionary history of plasmid- and chromosomally-located blaZ, to detect indications of frequent exchange of blaZ between human and bovine staphylococci and to estimate the frequency of transfer of blaZ between coagulase-negative staphylococci (CoNS) and Staphylococcus aureus of bovine origin. Methods blaZ was detected in 143 strains of penicillin-resistant S. aureus and CoNS from five Danish cattle herds (n = 25/23), random CoNS isolates from Denmark (n = 37), a collection of S. aureus from six different countries (n = 52), humans in Denmark (n = 3) and beta-lactamase control strains (n = 3). The sequence was determined in 105 strains and compared to published sequences by pairwise and multiple alignments. Maximum likelihood analysis was performed including bootstrap analysis. Parsimony, neighbour joining and consensus comparisons were performed for recombination. The localization of blaZ was determined by Southern blotting in 108 isolates. Results All penicillin-resistant strains carried blaZ and showed a similar organization of blaR1 and blaZ. The blaZ gene was localized to a plasmid in only 16 of the resistant strains. Sixty-nine sequences representing 105 isolates and sequences retrieved from public databases were compared. A phylogenetic tree showed that blaZ exists in three evolutionary lines: one group was of plasmid origin, one group was of chromosomal origin and one intermediate group. Sixty-nine sequence types were demonstrated. They translated into 11 BlaZ protein types. The major types all contained strains of both human and bovine origin, and more than one Staphylococcus species, demonstrating a shared gene pool. In a comparison of S. aureus and CoNS obtained from five Danish cattle herds, the same type of blaZ was only detected in one case. Conclusions Results indicated a separate evolution for plasmid- and chromosomally-encoded blaZ. Although a common gene pool seems to exist among staphylococci, exchange of blaZ between strains and species is judged to be an extremely rare event.

Copper resistance in Enterococcus faecium, mediated by the tcrB gene, is selected by supplementation of pig feed with copper sulfate.

Abstract: The tcr gene cluster mediates in vitro copper resistance in Enterococcus faecium. Here we describe the selection of tcr-mediated copper resistance in E. faecium in an animal feeding experiment with young pigs fed 175 mg copper/kg feed (ppm), which is the concentration commonly used for piglets in European pig production. tcr-mediated copper resistance was not selected for in a control group fed low levels of copper (6 ppm). We also show coselection of macrolide- and glycopeptide-resistant E. faecium in the animal group fed the high level of copper. Finally, we identify the tcr genes in the enterococcal species E. mundtii, E. casseliflavus, and E. gallinarum for the first time.

Molecular characterization and occurrence of extended-spectrum beta-lactamase resistance genes among Salmonella enterica serovar Corvallis from Thailand, Bulgaria, and Denmark.

Abstract: Fifty nine Salmonella Corvallis isolates from humans and food products in Bulgaria, Denmark, and Thailand were examined for antimicrobial susceptibility and characterized by pulsed-field gel electrophoresis (PFGE). Cephalosporin-resistant isolates were examined for the presence of genes encoding beta-lactamases by PCR and sequencing. Ten different PFGE types were observed. One type (30 isolates) was recovered in all three countries; three types were found only in Bulgaria, two only in Denmark, two only in Thailand, and two both in Denmark and Thailand. Ten isolates were susceptible to all antimicrobial agents tested, whereas 41 were resistant to three or more antimicrobials. Most resistance was observed among the isolates from Bulgaria. Of the 25 isolates from Bulgaria, 20 displayed resistance to ampicillin and the cephalosporins ceftiofur and cephalothin. All 20 isolates tested negative for bla (CMY-1), bla (CMY-2), and bla (ACC), but positive for bla (SHV), of which five were sequenced to bla (SHV-2). Plasmid profiling and hybridization revealed that the bla (SHV) gene was located on plasmids of approximately 70 kb. Five plasmid profiles were found among these 20 isolates. The plasmid profiling confirmed the PFGE-type and was able to further subdivide the strains. Seventeen of these 20 isolates contained also bla (TEM), of which nine representatives were sequenced to bla (TEM-1B), or bla (TEM-1H). One isolate contained bla (CTX-M-15), bla (SHV-2), and bla (TEM-1H), with the bla (CTX-M-15), and bla (TEM-1H) genes located on a 63-kb transferable plasmid. This study showed a high frequency of resistance among S. Corvallis isolated from humans and food products in Bulgaria, with a lower frequency in Thailand and Denmark. The clonal relatedness among the isolates from three countries could indicate a recent spread of this serovar.

Identification of Tn5397-like and Tn916-like transposons and diversity of the tetracycline resistance gene tet(M) in enterococci from humans, pigs and poultry

Abstract: Objectives To analyse the sequence diversity of the tetracycline resistance gene tet(M) and its location on mobile elements in Enterococcus faecium and Enterococcus faecalis from humans, pigs and poultry in Denmark. Methods A total of 76 isolates were screened for Tn916/Tn1545-like and Tn5397-like transposons using PCR. tet(M) was sequenced in 15 of the isolates and compared with tet(M) sequences submitted to GenBank (phylogenetic analysis and signs of recombination). Plasmids were extracted, filter-mating experiments were performed and Tn5397-like transposons were further characterized in selected isolates. Results In 8 of 13 isolates of E. faecium from broilers, tet(M) was present on Tn5397-like transposons, whereas tet(M) was predominantly associated with Tn916/Tn1545-like transposons in E. faecium from pigs and humans, as well as in E. faecalis from humans, pigs and broilers (50 of 63 isolates). The tet(M) genes were divided into three major subgroups according to the phylogenetic analysis. Subgroup I consisted of tet(M) from Clostridium difficile and E. faecium associated with Tn5397-like elements, subgroup II consisted of tet(M) located on Tn916/Tn1545 family transposons and subgroup III consisted of tet(M) associated with composite elements containing several resistance genes. We found evidence of recombination both within and between these groups. Moreover, we identified an E. faecium isolate with both Tn916/Tn1545-like and Tn5397-like elements. Conclusions This study showed that enterococci contain diverse tet(M) genes present on different mobile elements, which may suggest that enterococci play an important role in the evolution and horizontal spread of mobile elements carrying tet(M). This is the first report of Tn5397-like elements in enterococci.

Occurrence of integrons and antimicrobial resistance genes among Salmonella enterica from Brazil

Abstract: Objectives: To determine the occurrence of antimicrobial resistance genes and role of integrons among 135 antimicrobial-resistant Salmonella enterica from Brazil. Methods: The presence of antimicrobial resistance genes, class 1 and 2 integrons and gene cassettes was analysed by PCR and sequencing. The genetic location of class 1 integrons was determined in 25 isolates by hybridization and plasmid transfer experiments. Results: Fifty-five of the isolates were positive for class 1 integrons. Integron-positive isolates represented 17 different serovars and were mainly from human (n = 28) and animal (n = 13) sources. The gene cassette arrangements could be determined in 51 of the positive isolates, which harboured one [dfrA22, aadA1 or orf3 (putative trimethoprim resistance)], two [aadA1-dfrA1, aac(6 0 )-Ib-orf1 (unknown function) or aacA4-aadA1], three [dfrA15b-cmlA4-aadA2, orf2 (unknown function)-dfrA5-orfD] or four [orf4-aacA4blaOXA-30 (interrupted by an IS1 element)-aadA1] cassettes in their variable region. Only one isolate harboured a class 2 integron with the gene cassette array dfrA1-sat-aadA1. Several integron unrelated resistance genes were also detected in the isolates. Sulphonamide resistance was primarily mediated by sul2 and sul3, tetracycline resistance by tet(B) and tet(A), chloramphenicol resistance by catA1, streptomycin resistance by strA and ampicillin resistance by blaTEM. blaCTX and blaCMY-2 were found in cephalosporin-resistant isolates. Mating and hybridization experiments demonstrated that a high-molecular-weight plasmid mediated the gene transfer of integrons and additional resistance determinants. Conclusions: The present study revealed that integron-mediated resistance genes contributed to the multiresistance phenotype observed in the isolates, but most resistance genes were located outside the integron structure, as independent genes. However, they might be located on the same conjugative plasmid.

First description of an oxyimino-cephalosporin-resistant, ESBL-carrying Escherichia coli isolated from meat sold in Denmark

Abstract: Sir, Gram-negative bacteria expressing extended-spectrum blactamases (ESBLs) have emerged globally and this has limited the treatment strategies available for bacterial infections. Very few studies have demonstrated the presence of resistant ESBL-expressing Escherichia coli in foods and food animals. In a Spanish study from 2003, the prevalence of resistant ESBLexpressing E. coli was 2.8% in isolates tested from sick animals and the genes were identified as CTX-M-, SHVand TEM-type. In a Danish study from 2000 to 2002 on E. coli and Salmonella isolates no ESBLs were detected. In a study of 34 resistant ESBL-expressing Salmonella from the Netherlands from 2001 to 2002 from poultry, poultry products and patients, a high prevalence of blaTEM-52, CTX-M-type ESBLs and the Ambler class C ACC-1 was found among the tested isolates of animal origin. Nine unrelated isolates of animal origin, one Salmonella and eight E. coli, from the UK tested as ESBL-resistant. All E. coli isolates contained ampC promoter mutations and the Ambler class C CMY-2 was detected in the Salmonella isolate tested. As part of the continuous surveillance of antimicrobial resistance in food animals, foods and humans in Denmark (DANMAP), E. coli isolates are obtained from meat sold at retail in Denmark. A total of 732 samples were positive for isolation of E. coli; only one isolate per sample was investigated. MICs of 17 antimicrobials were determined according to CLSI guidelines. The b-lactams tested included ampicillin (MIC >32 mg/L); cefalotin (MIC >64 mg/L), a first-generation cephalosporin; and ceftiofur (MIC >8 mg/L), a third-generation cephalosporin that is used only in veterinary practice. Furthermore, sensitivity to b-lactamase inhibitors was tested using amoxicillin in combination with clavulanic acid (MIC = 8/4 mg/L). Among these 732 E. coli isolates obtained from poultry, beef and pork from 2004 (Table 1) the first resistant ESBL-expressing E. coli isolated from food products sold in Denmark was identified (7633094-7). This resistant ESBL-expressing E. coli was isolated from sliced beef (Goulash) imported from Germany, and was also resistant to quinolones [nalidixic acid (MIC >128 mg/L) and ciprofloxacin (MIC >4 mg/L)], sulphonamides (MIC > 1024 mg/L), tetracycline (MIC >32 mg/L) and trimethoprim (MIC >32 mg/L). Using primers previously designed for TEM-type genes, a 957 bp amplicon was obtained from E. coli 7633094-7 and by sequencing was identified as blaTEM-52. 2 This was the first E. coli isolated in Denmark from meat containing blaTEM-52. Previously, blaTEM-52 has been found in high prevalence among E. coli from patients in Canadian hospitals. Recently, bacterial isolates containing blaTEM-52 have been detected in Salmonella enterica serovars Blockley, Thomsen, London, Enteritidis, Paratyphi B, Virchow and Typhimurium from poultry and patients in the Netherlands and from patients in Scotland. Using E. coli MT102 and S. enterica serovar Typhimurium JEO3817 and serovar Dublin JEO66 as recipients, cephalosporin resistance was transferred by conjugation, indicating that blaTEM-52 was located on a mobile DNA element. Only resistance to b-lactams was transferred in these experiments. These results show the importance of monitoring the prevalence of antimicrobial resistance not only in food animals but also in meat sold at retail. Both locally produced and imported meat should be examined, especially since global trade is expected to increase in the future. National programmes to limit the selection for resistance will have to be combined with international measures and regulations to ensure that consumers are not exposed to unnecessary hazards from antimicrobial-resistant bacteria in food bought at retail. Furthermore, since resistance genes, as identified in this study, are located on mobile DNA elements, resistance may be transferred in the human gut to virulent human adapted strains, resulting in reduced antimicrobial treatment options and prolonged hospitalization.

First description of blaCTX-M-1-carrying Escherichia coli isolates in Danish primary food production

Abstract: was prevented completely by DW-224a at concentrations of 1·, 2· and 4· MIC by 24 h, although regrowth occurred in the presence of ciprofloxacin at 1· MIC concentration. DW-224a also showed a dose-dependent bactericidal activity against all test organisms up to 4· the MIC. The MBCs of DW-224a in Mueller–Hinton broth (MHB) were identical to 1·MIC or at most twice the MIC for all strains tested. Because the bactericidal activities of DW-224a increased with increasing concentrations (up to a concentration of 2 mg/L) for all test strains, and because the slope of the lines (rate of activity) in the time–kill curve varied with concentration, we concluded that DW-224a had concentration-dependent bactericidal activities regardless of the tested strains. Mutations within QRDR of the genes encoding DNA gyrase in S. pneumoniae strains did not affect the bactericidal activities of DW-224a, ciprofloxacin and gemifloxacin. PAE of DW-224a was determined using a broth technique in MHB. The PAE was calculated by the following equation: PAE = T – C, where T is the time to achieve 1 log10 cfu/mL growth for the antibiotic-exposed sample and C is the time to achieve 1 log10 cfu/mL growth for the untreated control sample (Table 1). In summary, DW-224a showed very potent and dosedependent bactericidal effect against all strains tested. The clinical usefulness of DW-224a should be established by further studies.

Resistance to penicillin of Staphylococcus aureus isolates from cows with high somatic cell counts in organic and conventional dairy herds in Denmark

Abstract: Background Quarter milk samples from cows with high risk of intramammary infection were examined to determine the prevalence of Staphylococcus aureus (SA) and penicillin resistant SA (SAr) in conventional and organic dairy herds and herds converting to organic farming in a combined longitudinal and cross-sectional study.

The Origin, Evolution, and Local and Global Dissemination of Antimicrobial Resistance

Abstract: The origin of acquired antimicrobial resistance is in general believed to be adoption of resistance genes from antimicrobial-producing organisms and/or nucleotide changes in housekeeping genes. This chapter provides a brief overview of the putative origins of some of the most important antimicrobial resistance determinants and some of the genetic mechanisms. The actual origin of antimicrobial resistance genes is unknown, but environmental microbes, including the natural producers of antimicrobial substances, are believed to be important primary sources. The s-lactamases are believed to have evolved from enzymes involved in cell wall biosynthesis. The main mechanism of resistance to aminoglycosides is enzymatic inactivation. Comparison of the DNA and deduced amino acid sequence of the erm genes from macrolide producing organisms and macrolide-resistant bacteria strongly suggests that the origin of macrolide resistance is macrolide-synthesizing organisms. Resistance to tetracyclines is mediated either by genes encoding ribosomal protection proteins (RPPs) or efflux pumps. Food animals and pets produce large amounts of waste products, which may contain many pathogenic bacteria, antimicrobial-resistant bacteria, and resistance genes. Human waste contains large amounts of antimicrobial-resistant bacteria and resistance genes. Such waste is normally treated to avoid transmission of pathogenic bacteria.

Quality-control ranges for antimicrobial susceptibility testing by broth dilution of the Brachyspira hyodysenteriae type strain (ATCC 27164(T))

Abstract: There are no approved standards for antimicrobial susceptibility testing of the fastidious spirochete Brachyspira hyodysenteriae. An interlaboratory study was performed to establish MIC quality control ranges for six antimicrobial agents for the type strain of B. hyodysenteriae using broth dilution. The results showed that B. hyodysenteriae B78T ATCC 27164T is a suitable quality control strain. This is a first step toward standardization of methods regarding this anaerobe.

Monitoring of Antimicrobial Resistance in Animals: Principles and Practices

Abstract: This chapter reviews information relevant to the design and scope of antimicrobial resistance monitoring and surveillance programs for animals and food, with emphasis on program purposes and methods. The chapter describes some of the essential features of existing monitoring and surveillance programs in various countries around the world. It shows how these programs have been useful in improving understanding of resistance and its relation to antimicrobial use and other factors, guiding public policy, and measuring the impact of interventions on antimicrobial resistance in bacteria from animals, food, and humans. The major methodological considerations for the monitoring program include the types of samples to be collected, sampling strategies, species of bacteria, antimicrobials for susceptibility testing, data collection and analysis, and reporting of results. Comprehensive monitoring of antimicrobial resistance in animals in the context of animal and human health covers the entire farm-to-fork continuum. The Food and Drug Administration (FDA) Center for Veterinary Medicine (CVM) has been active in developing new approaches for the preapproval assessment of antimicrobial resistance risks from antimicrobials used in animals. The Japanese Veterinary Antimicrobial Resistance Monitoring (JVARM) program examines the susceptibility of bacteria from food-producing animals to antimicrobial agents. Most programs focus on pathogenic bacteria or Salmonella, but some also report data on resistance in indicator bacteria isolated from healthy animals. Knowledge about antimicrobial resistance should be combined with knowledge regarding the usage of antimicrobial agents for different food animal species, which also should be performed on an internationally comparable basis.

Heterologous expression of glycopeptide resistance vanHAX gene clusters from soil bacteria in Enterococcus faecalis

Abstract: OBJECTIVES The aim of the study was to determine whether glycopeptide resistance gene clusters from soil bacteria could be heterologously expressed in Enterococcus faecalis and adapt to the new host following exposure to vancomycin. METHODS The vanHAX clusters from Paenibacillus thiaminolyticus PT-2B1, Paenibacillus apiarius PA-B2B and Amycolatopsis coloradensis DSM 44225 were separately cloned in an appropriately constructed shuttle vector containing the two-component regulatory system (vanRS) of Tn1546. The complete vanA(PT) operon (vanRSHAXY) from P. thiaminolyticus PT-2B1 was cloned in the same shuttle vector lacking enterococcal vanRS. All plasmid constructs were electroporated into E. faecalis JH2-2 and the MICs of vancomycin and teicoplanin were determined for each recombinant strain before and following exposure to sublethal concentrations of vancomycin. RESULTS The vanHAX clusters from P. thiaminolyticus and P. apiarius conferred high-level vancomycin resistance (MIC > or = 125 mg/L) in E. faecalis JH2-2. In contrast, cloning of the vanHAX cluster from A. coloradensis did not result in a significant increase of vancomycin resistance (MIC = 0.7 mg/L). Resistance to vancomycin was not observed after cloning the complete vanA(PT) operon from P. thiaminolyticus (MIC = 2 mg/L), but this recombinant rapidly adapted to high concentrations of vancomycin (MIC = 500 mg/L) following exposure to sub-lethal concentrations of this antibiotic. CONCLUSION The results showed that vanA(PT) in P. thiaminolyticus is a possible ancestor of vanA-mediated glycopeptide resistance in enterococci. Experimental evidence supported the hypothesis that enterococci did not acquire glycopeptide resistance directly from glycopeptide-producing organisms such as A. coloradensis.

dfrA25, a novel trimethoprim resistance gene from Salmonella Agona isolated from a human urine sample in Brazil

Abstract: OBJECTIVES To describe a novel trimethoprim resistance gene, designated dfrA25, which was detected as a gene cassette within a class 1 integron in Salmonella Agona. METHODS The gene was cloned into Escherichia coli MT102 and resistance to 10 different antimicrobial drugs was measured. A phylogenetic tree was constructed based on representative trimethoprim-resistance-mediating DfrA proteins retrieved from GenBank. Filter-mating experiments and Southern blots of plasmid preparations were performed with the donor and selected transconjugants. RESULTS AND CONCLUSIONS dfrA25 encodes a dihydrofolate reductase of 157 amino acids with closest identity (85%) to dfrA5 dihydrofolate reductase. dfrA25 was located on a transferable plasmid (approximately 150 kb) that also harboured the tetracycline resistance gene tet(A).

Concluding Remarks and Future Aspects: Some Personal Views

Abstract: The antimicrobial agents are among the most important medical discoveries of the 20th century and are unique in the respect that their usage might itself lead them to become useless. It is very difficult to estimate the true human health risk associated with the use of antimicrobial agents for animals, since it involves estimation of selection for resistance to different compounds; the spread of clones, plasmids, and genes through the food chain; and the consequences of human infections with different bacterial species that are resistant to different antimicrobial agents. Several international bodies and conferences have addressed the need to have concrete and common approaches in order to control the global emergence of antimicrobial resistance. In 2000, the World Health Organization issued global principles for the containment of antimicrobial resistance in animals intended for food. In most countries it is now required that pharmaceutical companies perform postmarketing monitoring of antimicrobial resistance. International meetings and conferences have pointed to the need for more data through monitoring of the occurrence of resistance in different reservoirs. Antimicrobials are essential for the sake of both human and animal health. Antimicrobial-free production of food animals, as is currently attempted in organic production, is seemingly associated with so many welfare problems that it will become a viable production method only for a minor market.

Salmonella lamphun: first isolation of a new Salmonella serovar in Thailand.

Abstract: Two isolates of a new Salmonella serovar, Salmonella Lamphun were discovered from animal feeds in Thailand, in 2003, which belongs to group C, with antigenic formula 6,8:y: 1,2. Both isolates were susceptible to all antimicrobials tested. The pulsed field gel electrophoresis pattern of both isolates comprises 11 DNA fractions sized 48,65,77, 105, 110, 170, 244, 330, 337, 453 and 1,135 kbp. Up to April 2005, no human or animal infection by this new Salmonella serovar was reported.