Prevalence and characterization of class 1 and class 2 integrons in Escherichia coli isolated from meat and meat products of Norwegian origin

Marianne Sunde*

Section of Bacteriology, National Veterinary Institute, 0033 Oslo, Norway


* Tel: +47-23-21-63-81; Fax: +47-23-21-63-01; E-mail: marianne.sunde{at}vetinst.no

Received 1 July 2005; returned 8 September 2005; revised 18 September 2005; accepted 21 September 2005


    Abstract
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Objectives: The aim of the study was to investigate the prevalence of integrons and to characterize inserted gene cassettes in Escherichia coli isolated from meat and meat products of Norwegian origin.

Methods: The strains investigated (n = 241 resistant out of 944 investigated) were collected within the frame of the Norwegian monitoring programme for antimicrobial resistance in bacteria from feed, food and animals (NORM-VET) during the years 2000–2003. PCR and DNA sequencing were used for detection of the integrase genes and gene cassettes within the integrons.

Results: Integrons were detected in 43 (18%) of the 241 resistant isolates. Class 1 integrons were detected in 29 (12%) strains and class 2 integrons were detected in 14 (6%) strains. Ten different gene cassettes were detected: dfrA1, dfr2a, dfrA12, aadA1, aadA2, catB2, oxa-30, sat, sat1 and orfF. The dfrA1 + aadA1 combination was the most prevalent cassette combination, detected in 12 of 29 class 1 integrons. Twelve (of 14) class 2 integrons contained a cassette area consistent with that on Tn7, the remaining two contained the cassettes sat + sat1 + aadA1. Nearly one-third of the class 1 integrons (9 of 29) lacked the sul1 gene. Ten gene cassettes (one dfr2a, two catB2 and seven aadA1) were expressed at levels below breakpoint values normally used to classify strains as resistant.

Conclusions: Integrons of class 1 or 2 were present in ~18% of the resistant E. coli strains investigated. Certain cassette combinations in class 1 integrons seem to be more widespread than others, like the dfrA1 + aadA1. Low-level expression of antimicrobial resistance, caused by the expression of certain gene cassettes in some integrons represents an obstacle in classifying strains as susceptible or resistant.

Keywords: gene cassettes , antibiotic resistance monitoring programme , resistant bacteria in food


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The awareness of antimicrobial resistance as an emerging problem worldwide is increasing. Exposure to resistant bacteria via the food-chain has gained increased attention because the presence of resistant bacteria in food and water might have an impact on the development and dissemination of antibiotic resistance among human bacterial pathogens.

The EU member states have to implement monitoring programmes that provide comparable data on the occurrence of antimicrobial resistance in zoonotic agents, and insofar as they present a threat to public health, other agents.1 The Norwegian monitoring programmes for antimicrobial resistance, NORM (for humans) and NORM-VET (for food, feed and animals), have been running since the year 2000. Indicator bacteria (Escherichia coli and Enterococcus spp.) isolated from meat and meat products of Norwegian origin have been included in the NORM-VET programme every year.

The sampling of meat samples in the NORM-VET programme is performed in order to obtain a representative random sample of meat from poultry, swine, cattle and sheep. A detailed description of the sampling procedures can be found in the annual NORM-VET reports.25 One E. coli isolate from each meat sample is included in the monitoring programme and subjected to further analysis. The isolates may be regarded as representing a stratified random sample of the respective populations and products. The occurrence of resistance provides an estimate of the true occurrence in the populations. Investigations of the genetic background of antimicrobial resistance in isolates collected within the frame of monitoring programmes can provide an estimate of the true occurrence of the various genetic elements responsible for resistance in bacteria occurring in the products of concern.

Multiresistance integrons are considered to be important contributors to the development of antibiotic resistance among Gram-negative bacteria.6,7 Integrons are capable of incorporating, collecting, and expressing mobile gene cassettes by site-specific recombination. The backbone structure of an integron contains a conserved region encoding an integrase (intI) and a variable region with integrated gene cassettes.8 To date, ~70 different cassette genes have been discovered in multiresistance integrons, the majority of which encode resistance to antimicrobial agents.9,10

Multiresistance integrons harboured by human clinical isolates are disseminated worldwide, and the class 1 integron is probably the most prevalent type of integron harboured by such isolates. Several studies have also shown that Tn7,11 carrying a class 2 integron with the gene cassettes dfrA1, sat1 and aadA1a, can be present in clinical pathogens.12

The aim of this study was to investigate the prevalence of integrons of classes 1 and 2, and further to characterize inserted gene cassettes in E. coli isolates from meat and meat products included in the NORM-VET programme during the years 2000–2003.


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Bacterial strains

A total of 944 E. coli isolates from meat and meat products of poultry (359), swine (295), cattle (190) and sheep (100) were included in the NORM-VET programme during the years 2000 to 2003. Meat from swine and poultry was investigated every second year, alternating with meat of cattle (also investigated every second year). Meat from sheep was only investigated in year 2001. The 944 E. coli isolates from meat have been subjected to susceptibility testing, and the minimum inhibitory concentrations to various antimicrobials are known.25

The breakpoints used by NORM-VET have varied somewhat between the years. The following breakpoint values have been used in this study for classification of the strains as resistant (also used by NORM-VET 2004): sulphonamides >258 mg/L, streptomycin >8 mg/L, trimethoprim >4 mg/L, ampicillin >8 mg/L, chloramphenicol >16 mg/L, florfenicol >16 mg/L, tetracycline >8 mg/L, kanamycin >32 mg/L, gentamicin >4 mg/L, nalidixic acid >16 mg/L, enrofloxacin >0.25 mg/L, ceftiofur >2 mg/L. Based on these breakpoints, a total of 241 isolates were classified as resistant to one or more of the tested antimicrobials and included in this study. Most commonly, resistance was to streptomycin (15%), sulphonamides (14%), tetracyclines (7%), ampicillin (6%) and trimethoprim (4%). Resistance to quinolones (including fluoroquinolones), cephalosporins, kanamycin and gentamicin was rare (>1%). Kanamycin resistance was only investigated in year 2000, and ~1% of the strains investigated (n = 362) were resistant to kanamycin. Of the 241 isolates classified as resistant and included in this study, 117 were investigated for kanamycin resistance.

Polymerase chain reaction

The 241 resistant isolates were screened for integron-associated structures in the following way: all isolates were screened for the presence of the integrase genes of class 1 and class 2 integrons, intI1 and intI2.8,11 Strains containing the intI1 or intI2 genes were subsequently subjected to PCR for amplification of the variable regions of class 1 and class 2 integrons, respectively. Integron-positive strains expressing resistance to trimethoprim (n = 35) were investigated for the presence of a dfrA1 cassette encoding resistance to trimethoprim.13 All strains containing class 1 integrons were investigated for the presence of the sul1 gene located on the 3'-conserved segment of the class 1 integron.

Primers and annealing temperatures used are listed in Table 1. The template was prepared by boiling a bacterial pellet in sterile distilled water and a 5 µL suspension was added to a 45 µL of a mix of PCR reagents containing 1x PCR buffer (Qiagen PCR Buffer, Qiagen GmbH, Hilden, Germany) with 1 U of Taq DNA polymerase (Qiagen), 10 pmol of each primer and 200 µM of (each) dNTP. Positive and negative controls were included in each run. The following positive control strains were used: E. coli Se 131 (accession no. AJ238350) for detection of intI1, dfrA1, sul1 and the variable part of class 1 integrons, and E. coli U56 (containing Tn7) for detection of intI2 and the variable part of the class 2 integron on Tn7.


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Table 1. Primers and annealing temperatures used in the PCRs carried out in this study

 
In an earlier study, most of the streptomycin-resistant strains (MIC of ≥16 mg/L, n = 136) have been screened for the presence of the integron-associated aadA gene cassettes conferring streptomycin and spectinomycin resistance.14

Characterization of inserted gene cassettes

Amplicons generated with primers for amplification of the variable regions of class 1 and class 2 integrons were sequenced in order to determine the content and order of inserted cassettes, with the exception of the following amplicons: amplicons of 1 kb generated with primers for the variable part of class 1 integrons and DNA from strains harbouring an aadA gene cassette. These strains were suspected to harbour an integron with an aadA gene cassette as the sole cassette. The amplicons were restricted with PvuI in order to confirm the presence of an aadA1a or aadA12 cassette within the integron (yielding a 525 bp fragment). The recently characterized aadA12 cassette originating from the faecal microflora of a pig (accession no. AY665771) is closely related to the aadA1a cassette and contains the same PvuI site. The aadA1a/aadA12 cassettes are hereafter termed aadA1-like cassettes.

Strains that contained both a dfrA1 cassette and an aadA gene cassette, and which generated 1.6 kb amplicons with primers for the variable part of class 1 integrons, were suspected to contain integrons with a dfrA1 cassette and an aadA cassette. These amplicons were restricted with PvuI for confirmation of the aadA1-like gene cassette closest to the 3'-conserved segment (yielding a 525 bp fragment).

Strains that contained a dfrA1 cassette and an aadA cassette, and generated amplicons of ~2.2 kb with primers for the variable part of class 2 integrons, were suspected to contain a class 2 integron with dfrA1, sat1 and aadA1a inserted (Tn7). The 2.2 kb amplicons were restricted with PvuI in order to confirm the insertion of an aadA1-like cassette as the third cassette within the integron (yielding a 635 bp fragment).

Several of the class 1 integrons were negative for the sul1 gene. The cassette area in three of these strains was amplified with an aadA specific primer in combination with an integrase-specific primer (5'-CS and aadaI, annealing temperature 48°C). The amplicons generated were subsequently sequenced.

DNA sequencing

Prior to the sequencing reaction, the PCR products that were sequenced were purified using the QIAquick PCR purification kit (Qiagen GmbH, Hilden, Germany) following the manufacturer's instructions, and the nucleotide sequence was determined using the BigDye Terminator v3.1 Cycle Sequencing kit (Applied Biosystems, Foster City, CA, USA) with oligonucleotide primers (primer walking). The sequencing reactions were run on a capillary sequencer 3100-Avant Genetic Analyzer (Applied Biosystems). Sequences were analysed using BioEdit (http://www.mbio.ncsu.edu/BioEdit/page2.html—14 September 2005, date last accessed), NCBI GeneBlast2 (http://www.ebi.ac.uk/blastall/index.html—14 September 2005, date last accessed) and ClustalW (http://www.ebi.ac.uk/clustalw/index.html—14 September 2005, date last accessed) programs via the internet.

Nucleotide accession number

The sequence of a dfr2a-like cassette has been submitted to GenBank under accession number DQ104737.


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Prevalence of class 1 and class 2 integrons

Integrons were detected in 43 of the 241 E. coli isolates included in the study (18%). Class 1 integrons were detected in 29 strains (12%) and class 2 integrons were detected in 14 strains (6%). Some differences in the presence of integrons in relation to type of meat were observed: 31% of the resistant strains from meat of swine contained integrons, 16% of the resistant strains from meat of cattle were integron-positive, 15% of the resistant strains from meat of poultry carried integrons, whereas none of the resistant strains from meat of sheep were integron-positive (only three strains from meat of sheep were classified as resistant and included in this investigation).

Ten different gene cassettes and seven different cassette combinations were detected within the integrons. Table 2 shows an overview of the various integrons and cassette arrays detected.


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Table 2. Characterization of integrons and cassette arrays in the 42 integron-positive E. coli isolates detected

 
Amplicons were produced with DNA from 19 of the 29 intI1-positive strains when subjected to PCR for amplification of the variable region of class 1 integrons. No PCR products were generated with DNA from the remaining 10 intI-positive strains. Nine of these strains lacked the sul1 gene. The hybridization site for the 3'-CS primer may be lacking, and this could explain the negative PCR results. Gene cassettes that might be present within seven of these integrons have not been determined in this study. The remaining three integrons contained an aadA-like cassette and the cassette areas were amplified with an aadA specific primer in combination with an integrase-specific primer. The three integrons contained the cassettes dfrA12, orfF and aadA2 (Table 2).

One isolate contained both intI1 and sul1 but no amplicons were generated with DNA from this strain when PCR was repeatedly preformed for amplification of the variable region. Rearrangements or absence of primer hybridization site(s) may explain this.

Amplicons were produced with DNA from all 14 intI2-positive strains when subjected to PCR for amplification of the variable region of the class 2 integron (Table 2). The content and order of gene cassettes are shown in Table 2. Twelve of the class 2 integrons contained the cassette array usually associated with the integron on Tn7: dfrA1, sat1 and aadA1. The remaining two integrons contained the unusual cassette combination sat, sat1 and aadA1. This cassette array was recently described from class 2 integrons in Salmonella serovars isolated in Japan.15

The aadA1-like cassette within seven of the class 1 integrons was expressed with streptomycin MICs of 8 mg/L (n = 4) and 4 mg/L (n = 3). These values are below the breakpoint values normally used to classify E. coli strains as resistant to streptomycin. The aadA-like cassette was inserted as the second cassette after a dfrA1 cassette in six of the integrons, and after an oxa-30 cassette in one integron. The two catB2 gene cassettes detected were also associated with low-level expression. Their chloramphenicol MICs were 8 mg/L and 16 mg/L. The breakpoint used by NORM-VET and by the CLSI is >16 mg/L. The catB2 cassettes were inserted as the third cassette in an array of three in both class 1 integrons containing them.

The remaining gene cassettes associated with antibiotic resistance were expressed with MICs above breakpoint values normally used, with the exception of one dfr2a cassette located within a class 1 integron. The trimethoprim MIC of this strain was 0.75 mg/L. The nucleotide sequence of the gene cassette revealed an intact reading frame when compared with another dfr2a gene cassette (accession no. U36276). However, a 72 bp region located upstream of the reading frame in the cassette investigated was lacking when compared with the sequence available under accession no. U36276. This 72 bp stretch is repeated twice in the latter dfr2a cassette. The lack of one of these 72 bp regions in the gene cassette investigated alters the predicted reading frame of the dfr2a gene, extending it with 19 amino acids. This may explain the loss of action of the dihydrofolate reductase encoded by this cassette. The sequence of the dfr2a cassette investigated is available under GenBank accession number DQ104737.


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The bacterial isolates collected within the frame of the monitoring programmes for antibiotic resistance are of great value as they reflect, under the premisses that the most optimal sampling procedure has been chosen, the prevalence of resistance for the area under concern. Most investigations related to antibiotic resistance have so far been carried out with bacterial isolates chosen in a non-random way. The data reported here provide an estimate of the prevalence of integrons in E. coli from meat and meat products of Norwegian origin.

Integrons of class 1 or 2 were detected in 18% of the resistant E. coli isolates. The majority of the isolates must be equipped with resistance elements other than integrons responsible for expression of antimicrobial resistance. Such resistance determinants could be the sul2 or sul3 genes (responsible for sulphonamide resistance), the linked strA–strB genes (mediating streptomycin resistance), the blaTEM genes (responsible for ampicillin resistance), or dfr3, 4, 8, 9 and 10 (mediating resistance to trimethoprim). Resistance to tetracycline has so far not been associated with integrons.9

In other investigations, when screening for integrons in resistant E. coli has been performed, a considerably higher portion of the E. coli isolates was shown to contain integrons.12,1620 These studies have mainly investigated clinical isolates, many of them of human origin. Such isolates have probably been subjected to a considerably higher antibiotic selection pressure or to more broad-spectrum antimicrobials. This may in turn explain the differences in dissemination of integrons.

The portion of class 2 integrons was surprisingly high as approximately one-third of the detected integrons were of class 2. In previous studies, class 2 integrons have been shown to have a more limited distribution than reported here.12,18,19

A total of 10 different gene cassettes and six different combinations of gene cassettes were detected. Of the 29 class 1 integrons detected, as many as 12 had the same cassette content, consisting of the dfrA1 + aadA1-like cassettes. This cassette array has been described in many other investigations as well, indicating a wide distribution of class 1 integrons containing these two cassettes.

Three class 1 integrons contained the cassette combination dfrA12, orfF and aadA2. All three integrons lacked the sul1 gene on the 3'-conserved segment. The isolates containing these integrons were recovered from meat samples obtained within geographically distinct areas. An equal cassette combination has also been detected in class 1 integrons from E. coli isolated in Spain, Germany, Sweden and the USA.12,16,19,21 Lack of the sul1 gene was reported in association with this cassette combination in one of the strains.16 The reason for the wide distribution of some integrons with a specific cassette combination is so far unknown. One explanation could be that such integrons could be a part of successful plasmids or transposons with a wide, perhaps global dissemination. Further investigations should be carried out in order to determine why some cassette combinations are much more prevalent than others.

A surprisingly high portion (nearly one-third) of the class 1 integrons lacked the sul1 gene. No PCR products were amplified with DNA from these strains and primers hybridizing to the conserved segments of class 1 integrons. This phenomenon has been reported previously.16,22 Such integrons represent an obstacle when inserted gene cassettes are to be detected. When screening for integrons is to be carried out, it is preferable to do an initial screening for the integrase genes. Use of primers specific for the conserved segments can produce false-negative results, when class 1 integrons with antibiotic resistance gene cassettes might be present.

Other investigations have shown that streptomycin-susceptible E. coli and Salmonella enterica Serotype Newport isolates can harbour aadA gene cassettes within integrons.20,21 This was also demonstrated in this study. It was also shown that the catB2 cassettes were expressed at a level below the breakpoint value normally used to classify a strain as resistant to chloramphenicol. The catB2 cassettes were inserted as the third cassette in an array consisting of three cassettes in both integrons containing this cassette. This may explain the weak expression of the catB2 cassettes detected. Gene cassettes in integrons can have a variable expression and this is caused by several factors, for example, if the integron is located on a high-copy-number plasmid or not. Several versions of the integron promoter located in the 5'-conserved segment of the integron exist causing differences in the strength of the promoter.23 The expression of a cassette in an integron containing more than one inserted cassette is also influenced by the position of the cassette. The expression weakens as the cassette is situated nearer the 3'-conserved segment.23 All these factors may lead to considerable variations of MIC values when gene cassettes in integrons are responsible for antimicrobial resistance. Low-level expression of antimicrobial resistance, caused by the presence of certain gene cassettes within integrons represents an obstacle in classifying strains as susceptible or resistant. Furthermore, the determination of an epidemiological cut-off value for surveillance purposes24 can be complicated by dissemination of integrons containing cassettes involved in conferring low-level resistance to antimicrobials.14

The findings presented in this study show that E. coli in meat can contain integrons responsible for multiresistance. Bacteria in meat may therefore be a source for exposure of human bacteria to integrons. This reflects the importance of proper hygiene during slaughter and food-processing routines. However, it was recently demonstrated that meat can also be contaminated with pathogenic extraintestinal E. coli.25 Meat may therefore be an important vehicle for dissemination of both antibiotic-resistant E. coli and pathogenic E. coli.

It is not known if the presence of resistant commensals, like non-pathogenic E. coli, in food might be a potential health problem. Further investigations should be carried out to evaluate to what extent this is a risk factor for dissemination of antibiotic resistance among human bacterial pathogens. Important zoonotic bacteria such as Salmonella spp., Yersinia spp. and shiga toxin-producing E. coli (STEC) are often associated with meat.26 The potential transfer of integrons from the indicator bacteria to such pathogens might contribute to a further dissemination of antibiotic-resistant zoonotic bacteria.


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    Acknowledgements
 
The control strain E. coli U56 containing Tn7 was kindly donated by Malin Grape, Karolinska Institute, Sweden. Madelaine Norström (National Veterinary Institute) and Henning Sørum (The Norwegian School of Veterinary Science) are acknowledged for helpful discussions and comments on this paper. This work was supported by a grant (153080/120) from the Norwegian Research Council.


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1. Directive 2003/99/EC of the European Parliament and of the Council of 17 November 2003 on the Monitoring of Zoonoses and Zoonotic Agents, amending Council Decision 90/424/EEC and repealing Council Directive 92/117/EEC. Official Journal L 325, 12/12/2003, pp. 0031–0040.

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