JAC Advance Access originally published online on February 16, 2007
Journal of Antimicrobial Chemotherapy 2007 59(4):746-750; doi:10.1093/jac/dkl549
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Occurrence and characteristics of class 1, 2 and 3 integrons in Escherichia coli, Salmonella and Campylobacter spp. in the Netherlands
1 Central Institute for Animal Disease Control Lelystad (CIDC-Lelystad), Wageningen UR, PO Box 2004, 8203 AA Lelystad, The Netherlands 2 Animal Science Group (ASG) – Wageningen UR, PO Box 65, 8200 AB Lelystad, The Netherlands
* Corresponding author. Tel: +31-320238800; Fax: +31-320239153; E-mail: dik.mevius{at}wur.nl
Received 2 November 2006; returned 28 November 2006; revised 11 December 2006; accepted 20 December 2006
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Abstract |
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Objectives: To determine the occurrence and transmission of class 1, 2 and 3 integrons in multidrug-resistant or sulfamethoxazole-resistant Salmonella from human and animal sources and in Campylobacter spp. and Escherichia coli from broilers isolated in the Netherlands in 2004.
Methods: PCR, restriction fragment length polymorphism (RFLP) and DNA sequencing were used to detect integrase genes and gene cassettes within 234 E. coli isolates, 40 Campylobacter isolates and 228 Salmonella isolates.
Results: Class 1 integrons were found in 76% of the E. coli and in 43% of the Salmonella isolates. Class 2 integrons were found in 11% of the E. coli and 1% of the Salmonella isolates. No class 1 or 2 integrons were detected in the Campylobacter isolates, and no class 3 integrons were detected in any of the bacterial species examined. The 22 different integrons detected harboured 20 different gene cassettes. The cassette arrays dfrA1-aadA1 and dfrA1-sat2-aadA1 were most frequently associated with class 1 and 2 integrons, respectively. For the first time linF was found to be associated with a class 2 integron as part of the linF-sat2-aadA1 cassette. The gene cassettes found within the integrons explain only a part of the resistance profile of the isolates. Conjugation experiments demonstrated transfer of class 1 and 2 integrons.
Conclusions: Our data demonstrate the importance of integrons for the occurrence and transmission of multidrug resistance. Identical predominant class 1 and 2 integrons in E. coli and Salmonella serovars indicate horizontal transfer between these species.
Keywords: Enterobacteriaceae , conjugation , multidrug resistant
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Introduction |
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Antimicrobial resistance and, in particular, multidrug resistance (MDR) is an increasing problem worldwide. MDR encoded by linked resistance genes occurs on integrons, which are potentially mobile genetic elements considered to be involved in the transfer of MDR.1 Exposure to MDR bacteria via the food chain is considered a potential risk to human health through food borne infections with resistant pathogens or because integrons can transfer horizontally from bacteria from food-producing animals to human pathogens.2
In Campylobacter spp., Salmonella and E. coli isolated from food-producing animals in the Netherlands the levels of resistance and MDR show a tendency to increase.3 So far however, little is known about the transmission of integrons from E. coli as part of the commensal intestinal flora of animals to pathogenic bacteria such as Campylobacter spp. and Salmonella.
The aim of this study was to determine the occurrence and the transmission of integrons of classes 1, 2 and 3 in a selection of MDR Salmonella from human and animal sources and in Campylobacter spp. and E. coli from broilers isolated in the Netherlands in 2004 and to characterize the gene cassettes associated with these integrons.
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Materials and methods |
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Bacterial isolates
From the strain collection obtained in the Dutch antimicrobial resistance monitoring programme in animals of 2004,3 Salmonella, E. coli and Campylobacter isolates resistant to three or more antimicrobial classes, excluding resistances encoded by chromosomal mutations were selected (MDR isolates). Also included in the selection were all isolates resistant to sulfamethoxazole as an indicator for the presence of class 1 integrons in Enterobacteriaceae. As a result 228 Salmonella, 234 E. coli and 40 Campylobacter isolates were selected. Excluded were Salmonella Paratyphi B var. Java (Salmonella Java) and Salmonella Typhimurium Definitive Type 104 (DT104). The Salmonella Java clone spreading in Dutch broilers harbours a chromosomal class 2 integron with the gene cassettes dfrA1-sat2-aadA1.4 Salmonella Typhimurium DT104 harbours a well-described chromosomal complex class 1 integron in the MDR region of Salmonella genomic island 1 (SGI1).
The E. coli, Campylobacter and Salmonella isolates were screened for the presence of integrons of classes 1, 2 and 3 and for the presence of conserved segments (CS) containing the gene cassettes of class 1 and 2 integrons by PCR as described before.5–9
As positive control E. coli 4H1 was used for the intI1 PCR; a Salmonella Paratyphi B var. Java isolated from Dutch broilers was used for the intI2 PCR and Serratia marcescens AK9373 was used for the intI3 PCR. As negative control E. coli ATCC 25922 was used.
Characterization of inserted gene cassettes by restriction fragment length polymorphism (RFLP) typing and DNA sequence analysis
RFLP analysis was performed on the conserved segments of the class 1 and 2 integrons. The amplification product was digested with a combination of restriction enzymes as described previously.2
The DNA sequence was determined for at least one of the variable region amplification products belonging to each of the individual RFLP patterns obtained using a 3100-Avant Genetic Analyzer (Applied Biosystems).
Integron-positive E. coli and Salmonella were used as donor isolates and a rifampicin-resistant, indole-negative E. coli K12 was used as recipient in conjugation experiments as described previously.5 Transconjugants were tested for susceptibility to a panel of antibiotics with the broth microdilution method.3 Transfer of antimicrobial resistance genes was confirmed by PCR as described above.
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Results |
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Selection of isolates
Most of the E. coli isolates selected were resistant to sulfamethoxazole (94%), trimethoprim (81%), tetracycline (78%) and amoxicillin (77%). In addition 30% were resistant to chloramphenicol, 15% to neomycin, 12% to cefotaxime and 5% to gentamicin.
In the selected Campylobacter spp. resistance to metronidazole (87%), doxycycline (87%) and amoxicillin (61%) was most commonly observed. Also 39% were resistant to sulfamethoxazole, 26% to streptomycin, 22% to neomycin and 4% to chloramphenicol.
In the selected Salmonella isolates resistance to sulfamethoxazole (99%), tetracycline (72%), amoxicillin (61%), or trimethoprim (52%) was most commonly observed. In addition 20% were resistant to chloramphenicol, 7% to neomycin and 5% to gentamicin.
Class 1 integrons were commonly found in the E. coli (76%) and Salmonella isolates (43%) tested (Table 1). Class 2 integrons were found in 11% of the E. coli isolates and 1% of the Salmonella isolates. No integrons of either class were detected in Campylobacter. Class 3 integrons were not detected in any of the bacterial species examined.
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In 222 of the 272 intI1-positive and in 24 of 26 intI2-positive Salmonella and E. coli isolates, the CS-PCR result was positive (Table 1). CS amplification products of 11 different sizes were found. Fragments varied in length between 600 and 2600 bp (Table 2).
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In two E. coli isolates and in 22 Salmonella isolates two CS1 amplicons were detected. One E. coli isolate harboured a 1000 and a 1550 bp CS1 amplicon, while the other E. coli harboured a 1450 and a 1550 bp CS1 amplicon. All 22 Salmonella isolates harboured a 1000 and 1200 bp CS1 amplicon. In five E. coli isolates and one Salmonella isolate a CS1 and a CS2 amplicon were detected. The E. coli isolates harboured a 1550 bp CS1 amplicon and a 1550 bp CS2 amplicon (three isolates); a 1000 bp CS1 amplicon and a 2300 bp CS2 amplicon and a 1550 bp CS1 amplicon and a 2600 bp CS2 amplicon. The Salmonella isolate harboured a 1000 bp CS1 amplicon and a 1300 bp CS2 amplicon.
Characterization of inserted gene cassettes by RFLP typing and DNA sequencing
Among the 11 different-sized CS amplification products found, 22 different RFLP patterns were detected (Table 2). Sequence analysis of the amplicons belonging to the 22 individual RFLP patterns showed that 21 different gene cassettes were found within the integrons (Table 2). The gene cassette array most frequently found associated with class 1 integrons, in E. coli as well as in Salmonella, was dfrA1-aadA1a. The cassette array most frequently found associated with class 2 integrons was dfrA1-sat2-aadA1.
Interestingly, two of the cassette arrays contained a linF gene. One of the linF genes was part of an aadA2-linF cassette and was found to be associated with a class 1 integron. The second linF gene was part of the linF-sat2-aadA1 cassette and was found to be associated with a class 2 integron.
Remarkably, only a small part of the resistance profile of the isolates could be explained by expression of the gene cassettes found within the integrons analysed. Apparently a considerable number of antibiotic resistance genes are located outside the integrons analysed here.
Transconjugants were obtained with 70% intI1-positive E. coli isolates, 66% intI2-positive E. coli isolates and 50% intI1-positive Salmonella isolates (Table 2). The antimicrobial resistance profiles of the transconjugants show that in addition to the transfer of resistance genes associated with the integron, other resistance genes present in the donor isolates were transferred as well. The number of additionally transferred genes differed by donor strain.
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Discussion |
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In E. coli and Salmonella isolates selected for this study class 1 integrons occurred frequently, while class 2 integrons were detected in a limited number of isolates. In Campylobacter no integrons of either class 1 or 2 were detected. Moreover, no class 3 integrons were detected in any of the bacterial species examined. Class 3 integrons are to date only described in Serratia marcescens and Klebsiella pneumoniae.10,11
The cassette array most frequently found associated with class 1 integrons, in E. coli as well as in Salmonella was dfrA1-aadA1a (Table 2). We confirmed the transferability of this class 1 integron in the conjugation experiment from E. coli and Salmonella to the E. coli K12 recipient.
The gene cassette array most frequently found associated with class 2 integrons was dfrA1-sat2-aadA1. This transposon is common in Salmonella Java isolated from broilers in the Netherlands and Germany4 and is recently found in Salmonella Typhimurium.12 Here, we also isolated one Salmonella Typhimurium from broilers containing a class 2 integron with the dfrA1-sat2-aadA1 gene cassette array. Moreover we demonstrated that in E. coli strains from broilers the dfrA1-sat2-aadA1 gene cassette array is the predominant class 2 integron, which indicates horizontal gene transfer between E. coli and Salmonella. We confirmed the transferability of this class 2 integron in the conjugation experiment from this E. coli isolate to the E. coli K12 recipient.
Two cassette arrays were found containing the linF gene. One of the linF genes was part of an aadA2-linF cassette associated with a class 1 integron, which was previously described in an E. coli strain isolated from bloodstream infections in human patients.13 Here this gene cassette array was found to be associated with a Salmonella Anatum strain isolated from poultry, which indicates the transfer of gene cassettes between E. coli and Salmonella or a common source for the gene. The second linF gene was part of the linF-sat2-aadA1 cassette and was found to be associated with a class 2 integron. This is the first time that the linF gene has been found to be associated with a class 2 integron. Both linF genes were identical except for the last two nucleotides of the 59 base element (be), the nucleotides AT in the class 1 integron are substituted by GC in the class 2 integron.
Eleven out of 18 gene cassette arrays associated with class 1 and two out of four class 2 integrons could efficiently be transferred from E. coli or Salmonella to the E. coli K12 recipient.
Although our study shows that integrons contribute to the occurrence and transmission of MDR in Enterobacteriaceae, further studies need to be conducted to determine the other factors involved in transmission of linked resistance genes.
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None to declare.
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Acknowledgements |
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The indole-negative E. coli K12 and the positive control strain for intI1 were kindly provided by Ad Fluit from the University Medical Centre of Utrecht, the Netherlands. The positive control for intI3 was kindly provided by Dr Arakawa, Nagoya University School of Medicine, Japan.
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References |
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1 Leverstein-van Hall MA, Blok HEM, Donders ART, et al. (2003) Multidrug resistance among Enterobacteriaceae is strongly associated with the presence of integrons and is independent of species or isolate origin. J Infect Dis 187:251–9.[CrossRef][ISI][Medline]
2 Box AT, Mevius DJ, Schellen P, et al. (2005) Integrons in Escherichia coli from food-producing animals in The Netherlands. Microb Drug Resist 11:53–7.[CrossRef][ISI][Medline]
3 MARAN. (2004) Monitoring of Antimicrobial Resistance and Antibiotic Usage in Animals in the Netherlands. http://www.cidc-lelystad.wur.nl/NL/publicaties/rapporten/maran-2004.pdf (30 November 2006, date last accessed).
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Miko A, Pries K, Schroeter A, et al. (2003) Multiple-drug resistance in D-tartrate-positive Salmonella enterica serovar Paratyphi B isolates from poultry is mediated by class 2 integrons inserted into the bacterial chromosome. Antimicrob Agents Chemother 47:3640–3.
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Leverstein-Van Hall MA, Paauw A, Box AT, et al. (2002) Presence of integron-associated resistance in the community is widespread and contributes to multidrug resistance in the hospital. J Clin Microbiol 40:3038–40.
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Martinez-Freijo P, Fluit AC, Schmitz FJ, et al. (1998) Class I integrons in Gram-negative isolates from different European hospitals and association with decreased susceptibility to multiple antibiotic compounds. J Antimicrob Chemother 42:689–96.
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Correia M, Boavida F, Grosso F, et al. (2003) Molecular characterization of a new class 3 integron in Klebsiella pneumoniae.. Antimicrob Agents Chemother 47:2838–43.
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Miko A, Pries K, Schroeter A, et al. (2005) Molecular mechanisms of resistance in multidrug-resistant serovars of Salmonella enterica isolated from foods in Germany. J Antimicrob Chemother 56:1025–33.
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