Class 1 integron-associated gene cassettes in Salmonella enterica subsp. enterica serovar Agona isolated from pig carcasses in Brazil

Geovana Brenner Michael1,2, Marisa Cardoso2 and Stefan Schwarz1,*

1 Institut für Tierzucht, Bundesforschungsanstalt für Landwirtschaft (FAL), Höltystr. 10, 31535 Neustadt-Mariensee, Germany; 2 Departamento de Medicina Veterinária Preventiva, Universidade Federal do Rio Grande do Sul (UFRGS), Porto Alegre, Brazil


* Corresponding author. Tel: +49-5034-871-241; Fax: +49-5034-871-246; Email: stefan.schwarz{at}fal.de

Received 5 January 2005; returned 26 January 2005; revised 31 January 2005; accepted 2 February 2005


    Abstract
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 Abstract
 Introduction
 Materials and methods
 Results and discussion
 References
 
Objectives: Two multiresistant Salmonella enterica subsp. enterica serovar Agona isolates from pig carcasses were investigated for antimicrobial resistance genes and their location with particular reference to the detection of class 1 integrons.

Methods: The two S. Agona isolates were investigated for their in vitro susceptibility to antimicrobial agents and their plasmid content. The resistance genes and class 1 amplicons were identified by PCR assays. Amplicons of class 1 integrons were cloned and sequenced. Transferability of resistance plasmids was confirmed by conjugation.

Results: Both S. Agona isolates carried conjugative plasmids of approximately 150 kb which harboured all resistance genes detected in the respective isolates. S. Agona 231 was resistant to chloramphenicol by catA1, to tetracycline and minocycline by tet(B), and to sulphonamides by sul1. In addition, it harboured a streptomycin resistance gene strA and a class 1 integron with a new aadA variant designated aadA23, which mediates resistance to streptomycin and spectinomycin. S. Agona 242 also carried the genes catA1, tet(B), and sul1. Moreover, it harboured a second sulphonamide resistance gene, sul2, and a class 1 integron with intact gene cassettes carrying new variants of the trimethoprim resistance gene dfrA15b or the chloramphenicol resistance gene cmlA4. The third gene cassette consisted of a truncated aadA2 gene.

Conclusions: The results of this study show that large conjugative multiresistance plasmids are present in S. Agona from pigs. Analysis of the class 1 integrons revealed the presence of new variants of resistance genes so far not detected in Salmonella isolates.

Keywords: 59-base element , trimethoprim , chloramphenicol , streptomycin , spectinomycin , resistance


    Introduction
 Top
 Abstract
 Introduction
 Materials and methods
 Results and discussion
 References
 
Isolates of Salmonella enterica subsp. enterica serovar Agona play a role as food-borne pathogens worldwide.15 During recent years, analysis of multiresistant S. Agona strains led to the identification of several variants of the Salmonella genomic island 1 multidrug resistance gene cluster,1 but also the gene blaTEM-52 coding for an extended-spectrum ß-lactamase in S. Agona.2 An S. Agona strain from imported food was found to possess a class 1 integron with aadA and blaPSE-1 gene cassettes for resistance to streptomycin and ampicillin, respectively.3 Moreover, a 105 kb multiresistance plasmid detected in an S. Agona strain from a hospitalized patient in Brazil was shown to carry the genes catA1 for chloramphenicol resistance, blaTEM-1 for ampicillin resistance as well as a class 1 integron with the novel oxacillinase gene blaOXA-53 and a novel aminoglycoside resistance gene aac(6')-I30.4 Multiresistant S. Agona strains have also been detected in slaughter-age pigs and environmental samples collected at modern swine raising facilities in Brazil.5

In this study, we analysed two non-related multiresistant S. Agona strains obtained from pig carcasses in Southern Brazil for their antimicrobial resistance genes with particular reference to the detection of class 1 integrons and their gene cassettes.


    Materials and methods
 Top
 Abstract
 Introduction
 Materials and methods
 Results and discussion
 References
 
The two S. Agona strains 231 and 242 were identified during a survey on the prevalence of Salmonella serovars in healthy pigs in Southern Brazil at two different slaughterhouses. In vitro susceptibility testing was carried out by disc diffusion according to the NCCLS document M31-A2.6 Plasmids were prepared by alkaline lysis.7 Conjugation into the rifampicin-resistant Escherichia coli strain HK225 was conducted by filter mating.2 Plasmid DNA obtained from the original strains and their transconjugants was screened for the presence of class 1 integrons as well as chloramphenicol, streptomycin, spectinomycin, tetracycline, sulphonamides, or trimethoprim resistance genes by previously described PCR assays.810 The amplicons specific for class 1 integrons were cloned into either pCR-Blunt II-TOPO (Invitrogen, Groningen, The Netherlands) or pGEM-T Easy (Promega, Mannheim, Germany) and transformed into E. coli recipient strains TOP10 or JM109, respectively. Initial sequence analyses were conducted with the M13 reverse and forward primers. For determination of the complete sequence of the amplicon from S. Agona 242, primer walking was carried out with oligonucleotide primers (MWG, Ebersberg, Germany) designed from sequences obtained with the previously mentioned standard primers. Sequence analysis was carried out with the BLAST programs blastn and blastp (http://www.ncbi.nlm.nih.gov/BLAST/; last accessed 15 December 2004) and with the ORF Finder program (http://www.ncbi.nlm.nih.gov/gorf/gorf.html; last accessed 15 December 2004). To confirm the linkage between aadA23 and sul1 as well as {Delta}aadA2 and sul1, a specific PCR assay [common forward primer from aadA23 and {Delta}aadA2 (5'-GTGGATGGCGGCCTGAAGCC-3') and reverse primer from sul1 (5'-CTAGGCATGATCTAACCCTCGGTCT-3'), annealing temperature 63 °C] was used. Based on the sequences obtained from the two S. Agona integrons and the 3' conserved segments of class 1 integrons, an ~2 kb amplicon was indicative for the linkage of sul1 to aadA23 and {Delta}aadA2, respectively. The nucleotide sequences of the two amplicons have been deposited in the European Molecular Biology Laboratory (EMBL) database under accession numbers AJ809407 (S. Agona 231) and AJ867237 (S. Agona 242).


    Results and discussion
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 Abstract
 Introduction
 Materials and methods
 Results and discussion
 References
 
S. Agona 231 was resistant to chloramphenicol, streptomycin, spectinomycin, sulphonamides, and tetracycline/minocycline whereas S. Agona 242 exhibited resistance to chloramphenicol, sulphonamides, trimethoprim, and tetracycline/minocycline. All these resistance properties were associated with conjugative plasmids of ~150 kb in both S. Agona isolates (data not shown).

The multiresistance plasmid from S. Agona strain 231 carried the chloramphenicol resistance gene catA1 coding for a type A chloramphenicol acetyltransferase and the resistance gene tet(B) coding for a tetracycline/minocycline exporter. This plasmid also harboured the streptomycin resistance gene strA coding for an aminoglycoside phosphotransferase and the sulphonamide resistance gene sul1 which represents part of the 3' conserved segment of class 1 integrons. PCR analysis for class 1 integrons revealed the presence of an amplicon of 1009 bp which contained a single gene cassette carrying a new aadA gene variant, designated aadA23 (Figure 1a). The aadA23 gene has a GTG translational start codon and codes for an aminoglycoside 3'-(9)-O-adenyltransferase of 259 amino acids (aa) that mediates resistance to streptomycin and spectinomycin. The AadA23 protein from S. Agona was most closely related to the 259-aa AadA23b protein of Escherichia coli (accession no. AB189176) which was deposited in the databases soon after the aadA23 sequence was released. Two amino acid exchanges, N170 and V257 in AadA23 versus D170 and I257 in AadA23b, were detectable. The next best matches were the 263-aa proteins AadA21 from S. Typhimurium (accession no. AY171244), AadA22 from S. Choleraesuis (accession no. AY550883), and an AadA1-like protein from Pasteurella multocida (accession no. AY232671). In addition to the differences at the N terminus—these three genes were reported to start with an ATG start codon which is located 12 bp upstream of the GTG start codon of aadA23—only single amino acid exchanges were seen between the AadA23 sequence and those of the aforementioned three AadA proteins. It is questionable whether the annotation of the ATG start codon is correct since there is no ribosome binding site detectable for any of these genes coding for 263-aa AadA proteins, whereas the ATG start codon and the adjacent three nucleotides (ATGAGG) might represent a suitable ribosome binding site for an aadA gene that starts with a GTG (Figure 1a). The 59-base element of the aadA23 cassette is 60 bp (Figure 1a). The observation that E. coli JM109 carrying the cloned aadA23 gene cassette was resistant to streptomycin and spectinomycin confirmed that the aadA23 gene is functionally active.



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Figure 1. Schematic presentation of (a) the aadA23 gene cassette (accession no. AJ809407) as well as (b) the dfrA15b, cmlA4, and {Delta}aadA2 gene cassettes (accession no. AJ867237) described in this study. The aadA23, dfrA15b, cmlA4, and {Delta}aadA2 reading frames are shown as arrows while the 5' and 3' conserved segments (5' CS, 3' CS) of the class 1 integron are shown as boxes. In the case of the {Delta}aadA2 reading frame, the dotted arrow shows the aadA2 reading frame without the loss of a single ‘C’ after codon 37. The beginning and the end of the gene cassettes are shown in detail below. The translational start (GTG) and stop (TAA, TGA) codons are underlined. The 59-base elements are depicted as stem–loop structures and the integrase 1 binding domains 1L, 2L, 2R, and 1R indicated by arrows. The 59-base elements of the gene cassettes are shown in bold type. The lower-case letters in the 59-base element of the {Delta}aadA2 gene cassette indicate the bases that are commonly present in aadA2 cassettes, but are absent in the truncated 59-base element identified in this study. Numbers indicating important positions of bases in the 59-base elements refer to the corresponding database entries. The dotted line in scheme (b) indicates the connection between the three gene cassettes.

 
The multiresistance plasmid from S. Agona 242 also carried the aforementioned resistance genes catA1, tet(B), and sul1 as well as a second sulphonamide resistance gene, sul2. The amplicon specific for class 1 integrons was 3159 bp and sequence analysis revealed the presence of three different gene cassettes (Figure 1b). The first gene cassette contained a trimethoprim resistance gene dfrA15b which codes for a trimethoprim-resistant dihydrofolate reductase of 157 aa. This protein differed from the DfrA15b protein of the Klebsiella pneumoniae integron In52 (accession no. AF156486) by a single amino acid exchange, K141 in S. Agona versus Q141 in K. pneumoniae. The 59-base element of the dfrA15b gene cassette was 104 bp in size (Figure 1b). The second gene cassette in this integron carried a cmlA4 gene which codes for a 418-aa chloramphenicol exporter composed of 12 transmembrane segments. As seen previously in other cmlA gene cassettes, a regulatory region consisting of two pairs of inverted repeated sequences of 12 bp and a reading frame for a 9-aa peptide were found upstream of the cmlA4 gene. This region is believed to play a role in the chloramphenicol-inducible expression of the cmlA genes.11 The deduced CmlA4 amino acid sequence differed from that of the 419-aa CmlA4 protein from K. pneumoniae (accession no. AF156486) by 10 aa exchanges. In addition to the exchange of F234 in K. pneumoniae by S234 in S. Agona, the amino acids located between positions 264 and 272 in CmlA4 from K. pneumoniae (264-FTARFMGRV-272) were changed completely in CmlA4 from S. Agona (264-LRLVLWAL-271). The loss of three single base pairs caused consecutive frame-shift mutations which are responsible for the loss of one amino acid and the alteration of the codons 264–271 in CmlA4 from S. Agona (Figure 2). A similar observation has previously been made for a tet(A) gene from E. coli.7 An analysis of transmembrane domains (http://www.ch.embnet.org/software/TMPRED_form.html) revealed that these altered amino acids were located in the terminal part of the transmembrane segment 8 and did not change significantly the hydrophobicity of this transmembrane segment. Analysis of the third gene cassette revealed a truncated aadA2-like gene whose N-terminal 37 amino acids corresponded exactly to those found at the N terminus of several AadA2 proteins deposited in the databases. The loss of a single ‘C’ after codon 37 in the aadA2 sequence caused a frame-shift mutation that turned the next codon into a translational stop codon. Susceptibility testing showed that E. coli JM109 that carried this cloned amplicon was resistant to trimethoprim and chloramphenicol, but susceptible to streptomycin and spectinomycin. This observation confirms that the truncated aadA2 gene is in fact functionally inactive. Moreover, analysis of this {Delta}aadA2 gene cassette showed that most of the 59-base element, including the integrase binding sites 2L and 2R, was lost. Binding of the IntI1 integrase, which also catalyses the excision of the gene cassettes, to the 59-base element is essential for the mobility of the gene cassettes. Thus, impaired mobility resulting from a deletion of most of the 59-base element might explain why such an inactive gene cassette persists in a class 1 integron. The same deletion has also been detected in the 59-base element of a functionally active aadA2 gene cassette from K. pneumoniae.12 In both integrons, the linkage between sul1 and the aadA23 or {Delta}aadA2 gene cassettes was confirmed by PCR amplification of the expected 2 kb segment.



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Figure 2. Comparative analysis of the nucleotide sequences of codons 261–274 of the cmlA4 reading frame of S. Agona 242 and its counterpart in cmlA4 from K. pneumoniae. Amino acids are shown in the single-letter code. The variable part is displayed as a box. Deleted bases in the S. Agona sequence are indicated as horizontal bars.

 
In conclusion, the results of this study showed that conjugative multiresistance plasmids, which also carried class 1 integrons, are present in S. Agona from carcasses of apparently healthy pigs. Such asymptomatic carriers may further the dissemination of S. Agona not only to other animals, but also to humans when they enter the food chain. In Brazil, S. Agona has been reported to be the fourth most frequently detected Salmonella serovar from non-human sources and to be among the 10 most frequently seen serovars associated with diseases in humans.13 These observations underline the role of S. Agona as food-borne pathogens and support the requirement for the establishment of monitoring and control programmes to reduce the prevalence of S. Agona and other Salmonella serovars in pigs and other food-producing animals.


    Acknowledgements
 
We thank Marjo Cado Bessa and Sandra Maria Ferraz Castagna for providing S. Agona strains, Roswitha Becker and Vera Nöding for excellent technical assistance, and Kristina Kadlec for helpful discussions. G. Brenner Michael received a scholarship from the German Academic Exchange Service (DAAD).


    References
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 Abstract
 Introduction
 Materials and methods
 Results and discussion
 References
 
1 . Doublet, B., Butaye, P., Imberechts, H. et al. (2004). Salmonella genomic island 1 multidrug resistance gene clusters in Salmonella enterica serovar Agona isolated in Belgium in 1992 to 2002. Antimicrobial Agents and Chemotherapy 48, 2510–7.[Abstract/Free Full Text]

2 . Lee, K., Yong, D., Yum, J. H. et al. (2003). Diversity of TEM-52 extended-spectrum ß-lactamase-producing non-typhoidal Salmonella isolates in Korea. Journal of Antimicrobial Chemotherapy 52, 493–6.[Abstract/Free Full Text]

3 . Zhao, S., Datta, A. R., Ayers, S. et al. (2003). Antimicrobial-resistant Salmonella serovars isolated from imported foods. International Journal of Food Microbiology 84, 87–92.[ISI][Medline]

4 . Mulvey, M. R., Boyd, D. A., Baker, L. et al. (2004). Characterization of a Salmonella enterica serovar Agona strain harbouring a class 1 integron containing novel OXA-type ß-lactamase (blaOXA-53) and 6'-N-aminoglycoside acetyltransferase genes [aac(6')-I30]. Journal of Antimicrobial Chemotherapy 54, 354–9.[Abstract/Free Full Text]

5 . Oliveira, C. J., Carvalho, L. F., Fernandes, S. A. et al. (2002). Antimicrobial resistance of Salmonella serotypes isolated from slaughter-age pigs and environmental samples. Microbial Drug Resistance 8, 407–11.[CrossRef][ISI][Medline]

6 . National Committee for Clinical Laboratory Standards. (2002). Performance Standards for Antimicrobial Disk and Dilution Susceptibility Tests for Bacteria Isolated From Animals—Second Edition: Approved Standard M31-A2. NCCLS, Wayne, PA, USA.

7 . Ojo, K. K., Kehrenberg, C., Odelola, H. A. et al. (2003). Structural analysis of the tetracycline resistance gene region of a small multiresistance plasmid from uropathogenic Escherichia coli isolated in Nigeria. Journal of Antimicrobial Chemotherapy 52, 1043–4.[Free Full Text]

8 . Frech, G., Kehrenberg, C. & Schwarz, S. (2003). Resistance phenotypes and genotypes of multiresistant Salmonella enterica subsp. enterica serovar Typhimurium var. Copenhagen isolates from animal sources. Journal of Antimicrobial Chemotherapy 51, 180–2.[Free Full Text]

9 . Waturangi, D. E., Suwanto, A., Schwarz, S. et al. (2003). Identification of class 1 integron-associated gene cassettes in Escherichia coli isolated from Varanus spp. in Indonesia. Journal of Antimicrobial Chemotherapy 51, 175–7.[Free Full Text]

10 . Schwarz, S., Kehrenberg, C., Salmon, S. A. et al. (2004). In vitro activities of spectinomycin and comparator agents against Pasteurella multocida and Mannheimia haemolytica from respiratory tract infections of cattle. Journal of Antimicrobial Chemotherapy 53, 379–82.[Abstract/Free Full Text]

11 . Stokes, H. W. & Hall, R. M. (1991). Sequence analysis of the inducible chloramphenicol resistance determinant in the Tn1696 integron suggests regulation by translational attenuation. Plasmid 26, 10–19.[CrossRef][ISI][Medline]

12 . Poirel, L., Le Thomas, I., Naas, T. et al. (2000). Biochemical sequence analyses of GES-1, a novel class A extended-spectrum ß-lactamase, and the class 1 integron In52 from Klebsiella pneumoniae. Antimicrobial Agents and Chemotherapy 44, 622–32.[Abstract/Free Full Text]

13 . Tavechio, A., Ghilardi, A., Peresi, J. et al. (2002). Salmonella serotypes isolated from nonhuman sources in Sao Paulo Brazil, from 1996 through 2000. Journal of Food Protection 65, 1041–4.[ISI][Medline]