JAC Advance Access originally published online on October 20, 2006
Journal of Antimicrobial Chemotherapy 2006 58(6):1124-1132; doi:10.1093/jac/dkl400
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Class 1 and class 2 integrons in non-prevalent serovars of Salmonella enterica: structure and association with transposons and plasmids
1 Departamento de Biología Funcional-Microbiología, Universidad de Oviedo and Instituto Universitario de Biotecnología de Asturias (IUBA) 33006-Oviedo, Spain 2 Instituto de Productos Lácteos de Asturias (CSIC) 33300-Villaviciosa, Asturias, Spain
*Correspondence address. Área de Microbiología, Facultad de Medicina, Universidad de Oviedo, Julián Clavería 6, 33006-Oviedo, Spain. Tel: +34-985103562; Fax: +34-985103148; E-mail: rrodicio{at}fq.uniovi.es
Received 26 July 2006; returned 30 August 2006; revised 7 September 2006; accepted 11 September 2006
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Abstract |
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Objectives: To characterize class 1 and class 2 integrons which were simultaneously detected in non-typhoid Salmonella enterica strains of non-prevalent serovars, and to investigate their possible association with transposons and/or plasmids.
Methods: Eight multidrug-resistant S. enterica strains belonging to serovars Virchow (4), Panama (2), Grumpensis (1) and Worthington (1), each containing a class 1 and a class 2 integron, were analysed. Nested PCR amplification was used to determine the gene-cassette configuration of the integrons. Overlapping PCR amplifications were applied in integron–transposon linkage experiments. Conjugation and hybridization experiments were used to localize integrons and transposons in the bacterial genome (plasmid and chromosome associated).
Results: One of two different class 1 integrons (with variable regions of 1000 bp/aadA1 and 2300 bp/sat-smr-aadA1) inserted into Tn21-like transposons, were found to coexist with the class 2 integron (2300 bp/dfrA1-sat1-aadA1) of Tn7 in the analysed strains. Class 1 integrons were always found in large conjugative plasmids whereas apparently intact or defective copies of the Tn7 integron could be located on the same plasmid and/or the bacterial chromosome.
Conclusions: This report describes different associations between mobile genetic elements that play a crucial role in the capture and spread of antimicrobial drug resistance. As far as we are aware, this is the first description of class 2 integrons in serovars Panama, Grumpensis and Worthington.
Keywords: multidrug resistance , mobile genetic elements , gene mapping , PFGE
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Introduction |
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Nowadays, the prevalence of multidrug-resistant (MDR) bacterial strains in different environments has become a significant public health concern. This situation is mostly due to the mobility of the antimicrobial-drug resistance (R) determinants, mediated by genetic elements such as integrons, transposons and plasmids.1–3 Integrons have the potential to capture gene cassettes via site-specific recombination events catalysed by the integron-encoded integrase (IntI). On the basis of differences in sequence of the integrase genes (intI), several classes of integrons have been described.3,4 Class 1 integrons are widely distributed among Gram-negative bacteria including different zoonotic serovars of Salmonella enterica.5–11 Class 1 integrons consist of two conserved segments (5'-CS and 3'-CS) separated by a variable region (VR) that usually comprises one or more gene cassettes. The 5'-CS region contains the integrase gene (intI1), the integration site (attI1) and a promoter region that allows expression of any number of gene cassettes inserted at the attI1 site in a suitable orientation. The 3'-CS region usually comprises qacE
1 (encoding resistance to quaternary ammonium compounds), sul1 (resistance to sulphonamides), followed by ORF5 (of unknown function) and/or tni genes (transposition functions). Integrons belonging to class 1 are usually associated with Tn21-related transposons.12 Tn21 itself includes In2, a class 1 integron that carries the aadA1 gene cassette (resistance to streptomycin and spectinomycin), and two insertion sequences, IS1326 and IS1353. The latter, which is inserted within IS1326, was probably acquired after In2 moved into Tn21, because close relatives of Tn21, such as Tn2411, do not contain IS1353.12 Tn21-like transposons are typically carried by large self-transferable plasmids, for which the archetype is NR1 (R100), originally isolated from Shigella flexneri. In this plasmid, Tn21 is inserted within Tn9, between catA1 and IS1b forming the composite Tn2670, which is itself transposable.13 Class 2 integrons share a common structure with class 1 integrons in the 5' region, but possess a distinct integrase gene (intI2) that includes an internal stop codon (TAA). They occur on the Tn7 family of transposons and, like class 1 integrons, contribute to dissemination of resistance genes in different bacteria.7,8,14–16 The integron of Tn7 carries the dfrA1-sat1-aadA1 array of gene cassettes, which mediate resistance to trimethoprim, streptothricin and streptomycin/spectinomycin, respectively17 and a fourth excisable unit (ORFX) that encodes a 165-amino-acid protein of unknown function.18 Nevertheless, variations on this configuration have been observed among Tn7-related elements (such as Tn1825, Tn1826 and Tn4132).19,20 In comparison with class 1 integrons, information about the distribution of class 2 integrons is still scarce, although they were shown to predominate in Shigella sonnei,14,15 and have been also found in serovars Paratyphi B dT+ (formerly known as Salmonella Java), Typhimurium, Enteritidis and Virchow of S. enterica.16,21–23
Previously, we have reported the detailed characterization of two MDR strains of Salmonella Virchow (LSP 231/90 and LSP 205/98), each containing a class 2 and a class 1 integron in a large conjugative plasmid.23 Here we extended the study to eight other MDR strains belonging to four non-typhoid and non-prevalent serovars of S. enterica, to broaden the knowledge on the involvement of mobile genetic elements in the capture and dissemination of resistance to antimicrobial agents.
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Bacterial strains
Eight Salmonella strains of serovars Virchow (4), Panama (2), Grumpensis (1) and Worthington (1), positive for intI1 and intI2 integrase genes, were characterized in this study. They were selected from among a number of Salmonella isolates recovered in the Principality of Asturias (Spain) that were screened for the presence of classes 1–3 integrase genes7 in our laboratory (I. Rodríguez, unpublished data). All were isolated from patients with acute gastroenteritis, except Salmonella Panama MUO 72/93, which was collected from sewage.
Antimicrobial susceptibility testing and integron analysis
The strains were tested for susceptibility to the following antimicrobial drugs: ampicillin, chloramphenicol, gentamicin, kanamycin, nalidixic acid, spectinomycin, streptomycin, sulfadiazine, tetracycline and trimethoprim, by the disc diffusion method, according to CLSI (formerly NCCLS).24 The following drug resistance determinants: aacC2 (encoding gentamicin resistance), aadA1, aphA1 (kanamycin resistance), blaTEM (ß-lactam resistance), catA1 (chloramphenicol resistance), dfrA1, strA, strB (streptomycin resistance) and tet(A) (tetracycline resistance), were screened by PCR amplification using previously described primers and conditions.25 VRs were amplified with the 5'-CS and 3'-CS primers that anneal with sequences flanking the attI1 site of class 1 integrons,26 and with the hep74 and hep51 primers that bind to attI2 and ORFX within class 2 integrons, respectively.7 Gene cassettes within the VRs, as well as the sul1 and qacE1 genes characteristic of class 1 integrons, were screened by PCR amplification, as described previously.25
Transposon detection and integron–transposon associations
The presence of transposon-related sequences and the physical association between transposons and integrons, was investigated by standard and nested PCR amplifications, as described previously.23,25 The screened genes were tnpA, tnpR (encoding transposition functions) and merEDACPTR (encoding mercury resistance) of Tn21-like transposons; ybfA, ybfB, ybgA (of unknown function), tnsE, tnsD, tnsC, tnsB and tnsA (encoding transposition functions) of Tn7; and catA1 and IS1 of Tn9.
Conjugation experiments
Transfer of antimicrobial resistance genes was studied by conjugation experiments using the S. enterica strains as donors and the rifampicin-resistant Escherichia coli K12 J53 as recipient. Conjugations were performed in Luria–Bertani broth27 for 4 h at 37°C, and transconjugants were selected on Eosin Methylene Blue agar (Oxoid, Madrid, Spain) containing rifampicin (50 mg/L) plus chloramphenicol (30 mg/L) or trimethoprim (5 mg/L). Purified colonies were further tested for additional antimicrobial resistances by the disc diffusion method (see above).
Plasmid analysis, PFGE-macrorestriction analysis and hybridization procedures
Plasmid DNA was extracted from S. enterica strains, their transconjugants and E. coli K12 J53, using the S1-PFGE method.28 Total DNA from the indicated strains was also subjected to PFGE-macrorestriction analysis with XbaI. Agarose plug preparation and PFGE running conditions used a standard protocol,29 and the CHEF-DRIII SYS220/240 system (Bio-Rad laboratories, S.A., Madrid, Spain). S1 and XbaI fragments were transferred onto membranes by Southern blotting,27 and hybridized to selected probes for antimicrobial resistance determinants, and for class 1 integron-, class 2 integron-, Tn21- Tn9- and Tn7-related genes. The probes were prepared with the commercial ‘PCR DIG labelling mix’ (Roche Diagnostics, Barcelona, Spain), using Salmonella Virchow LSP 231/90 and LSP 205/9823 as the source of template DNA.
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Characterization of integrons
Eight S. enterica strains belonging to four serovars were selected for further characterization on the basis of the simultaneous presence of a class 1 and a class 2 integron (Table 1). The class 1 integrons contained two different VRs: 2300 bp/sat-smr-aadA1 (in Salmonella Virchow LSP 244/90 and Salmonella Panama MUO 72/93; with the two former genes encoding streptothricin resistance and a small drug resistance protein, respectively), and 1000 bp/aadA1 (in the remaining strains). In contrast, all class 2 integrons were apparently identical, and carried the dfrA1-sat1-aadA1 gene array characteristic of Tn7, in their ca. 2300 bp VRs. The eight strains displayed additional resistance properties, apart from those conferred by the integrons. In total, they were resistant to 4–6 unrelated antimicrobial drugs, and could hence be considered as MDR. The resistance phenotypes and genotypes are shown in Table 1.
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Large conjugative plasmids and MDR
In order to determine the location of integrons and non-integron-associated resistance determinants, conjugation experiments were performed, using the S. enterica strains as donors and E. coli K12 J53 as the recipient. For Salmonella Virchow LSP 244/90, LSP 69/92, LSP 87/92 and LSP 142/92, Salmonella Grumpensis LSP 199/93, and Salmonella Worthington LSP 7/93, transconjugants (designated Tc-S followed by the initial of the serovar and the number of the strain; Table 1) were obtained that showed identical resistance phenotypes as the parental strains. Transconjugants could also be recovered in crosses involving Salmonella Panama MUO 72/93, though they differed from the parental strain by being susceptible to trimethoprim. In contrast, transconjugants of Salmonella Panama LSP 580/95 could not be obtained under the applied conditions.
Using S1-PFGE, plasmids of ca. 240–275 kb (termed pUO-S followed by the initial of the serovar and a serial number), were detected in the obtained transconjugants and in all S. enterica strains, including Salmonella Panama LSP 580/95 (Table 1; Figure 1a). Smaller plasmids were also detected in some of the strains (Figure 1). PCR amplifications using the transconjugants as the source of template DNA confirmed the presence of the expected genes and integrons in the conjugative plasmids. Thus, the two integrons and all non-integron associated resistance genes were of plasmid location in all strains except Salmonella Panama MUO 72/93, from which transconjugants could be obtained. In agreement with the resistance phenotype and genotype of Tc-Sp72/93, the class 2 integron (2300 bp/dfrA1-sat1-aadA1) was not carried by the conjugative plasmid of this strain. Finally, the failure to obtain transconjugants from Salmonella Panama LSP 580/95 made it impossible to establish the location of resistance genes and integrons at this stage. None of the screened genes could be amplified from E. coli K12 J53, as expected.
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ladder PFG marker (New England BioLabs). Sizes in kb are shown to the left of panel (a). The tnsD probe is expected to hybridize with 2.7 and 0.8 kb fragments in strains carrying an intact Tn7 (the latter cannot be detected under the conditions applied in the PFGE). Hybridization of the dfrA1 probe with a second XbaI fragment on the macrorestriction profile of Salmonella Worthington LSP 7/93 indicates the presence of a second dfrA gene within the chromosome of the strain. Linkage of this gene to integrons and/or transposons could not be established. Hybridizations with 2.7 and 48 kb fragments in strains carrying a truncated transposon can be explained by loss of XbaI sites at the right of the 0.8 kb fragment.Integron–transposon associations
To investigate a possible linkage between integrons and transposons, the presence of Tn21-like-, Tn9- and Tn7-related sequences in the S. enterica strains and their transconjugants was first investigated. All these bacteria yielded right-sized amplicons with primers specific for the tnpA, tnpR and merA genes of Tn21-related transposons. The same result was obtained for the catA1 gene, used as an indicator of Tn9, in the chloramphenicol-resistant strains (all except Salmonella Panama LSP 580/95). In addition, tnsE- and tnsD-derived amplicons could be generated from Tn7 in all S. enterica strains, and also in all transconjugants except Tc-Sv244/90 and Tc-Sp72/93. Results were negative for E. coli K12 J53, as expected.
The physical linkage between integrons and transposons (Figure 2) was established by PCR amplification of overlapping fragments using different combinations of primers.23 In all S. enterica strains and the obtained transconjugants, the class 1 integron was carried by a Tn21-related transposon that lacked IS1353, and was therefore of the Tn2411 type. Moreover, in all except Salmonella Panama LSP 580/95, this transposon was in turn inserted within Tn9. The element found in Salmonella Panama LSP 580/95 (susceptible to chloramphenicol) lacked IS1a and the catA1 gene of Tn9, although IS1b was still present. Similarly, we have also established the presence of: (i) an apparently complete Tn7, including the class 2 integron, in the conjugative plasmid of Salmonella Worthington LSP 7/93 and the chromosome of Salmonella Panama MUO 72/93; (ii) two copies of the class 2 integron, one in the chromosome and another in the plasmid of Salmonella Virchow LSP 244/90, the latter associated to a truncated Tn7; and (iii) a unique copy of the class 2 integron linked to a defective Tn7 within the conjugative plasmids of the remaining strains. A truncated Tn7 also exists in Salmonella Panama LSP 580/95, although its precise location could not be still determined (see below).
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Mapping of classes 1 and 2 integrons on the genome of the S. enterica strains
The S. enterica strains and the obtained transconjugants were also analysed by macrorestriction using XbaI-PFGE (Figures 1a and 3a). As expected, strains belonging to the same serovar generated closely related profiles that were clearly different from those yielded by strains belonging to other serovars.
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Hybridizations of selected probes for class 1 (qacE
1-sul1) and class 2 (dfrA1) integrons, and for Tn21 (merA), Tn9 (catA1) and Tn7 (tnsD and/or tnsA) transposons with the XbaI- and S1-PFGE profiles of the parental strains and E. coli transconjugants (Figures 1 and 3) confirmed the mapping results inferred by PCR amplifications, as well as the physical state (intact or deleted) of the transposons. Thus, probes for the class 1 integron and the merA gene of Tn21 mapped on the large extrachromosomal fragments detected by S1-PFGE in all Salmonella strains and the obtained transconjugants (Figures 1b and 3b). The same result was observed with the catA1 probe, for all S. enterica strains except Salmonella Panama LSP 580/95 (the isolate susceptible to chloramphenicol that carries a defective Tn9, see above), which failed to hybridize with catA1 (Figure 1c). Moreover, hybridization of the probes with XbaI fragments of the same size on the macrorestriction profiles of each Salmonella strain and the corresponding transconjugant is consistent with the presence of a single extrachromosomal copy of the class 1 integron and the associated transposons.
With regard to the Tn7 integron (Figure 1d–f and Figure 3c and d), hybridization experiments confirmed the existence of a single and apparently intact copy of the element on the chromosome of Salmonella Panama MUO 72/93. In fact, the relevant probes (dfrA1, tnsD and/or tnsA) failed to hybridize with the S1 profiles of the strain and its transconjugant, and with the XbaI profile of the latter. In contrast, positive signals were obtained with the XbaI profile of the strain. Similarly, hybridization of the same probes with the S1 profiles of Salmonella Worthington LSP 7/93 and Tc-Sw7/93, and with XbaI fragments of the same size in both strains, are consistent with an intact class 2-Tn7 structure of plasmid location. Hybridization experiments also corroborated the presence of a single extrachromosomal copy of the class 2 integron associated with a deleted Tn7, in Salmonella Virchow LSP 69/92, LSP 87/92 and LSP 142/92, Salmonella Grumpensis LSP 199/93 and Salmonella Panama LSP 580/95. For all except the latter, dfrA1 and tnsD, but not tnsA mapped on the S1 profiles of the strains and transconjugants, demonstrating both the extrachromosomal location and the deletion affecting Tn7. As before, hybridization of dfrA1 and tnsD on the same XbaI fragment of each strain and the corresponding transconjugant supports the existence of a single copy of the class 2-Tn7 structure. In the case of Salmonella Panama LSP 580/95, hybridizations with the S1 and XbaI profiles led to identical conclusions, although transconjugants carrying pUO-SpR1 could not be obtained. Finally, in Salmonella Virchow LSP 244/90, hybridization of dfrA1 with the S1 profiles of the strain and its transconjugant, and with the same XbaI fragment in the two strains, together with hybridization of tnsD and tnsA with the XbaI profile of the strain but not with the S1 fragments, confirm that intact and defective copies of the class 2-Tn7 structure are placed on the chromosome and pUO-SvR3, respectively.
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Discussion |
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In this study we have characterized class 2 integrons that coexist with class 1 integrons in clinical isolates of S. enterica recovered in Asturias (Spain), which belonged to four different serovars (Worthington, Panama, Grumpensis and Virchow). In all isolates, the class 1 integron was carried by Tn21-related transposons, which share the same transposition machinery, as well as an integron in the position of In2 (see the Introduction section). However, they differ in the identity and/or number of gene cassettes carried by the integron, the associated IS elements and/or additional transposons, and the presence or absence of an intact mer locus.12 The elements described here differed by the cassette array (aadA1 and sat-smr-aadA1 in six and two isolates, respectively), but all contained IS1326, lacked IS1353 and carried an apparently intact mer operon. Accordingly, they coincided with the ‘European Tn21-like elements’ (see Grinsted et al.30) from which Tn21, first identified in Japan, could have derived.31 These elements, represented by Tn2411, have probably spread globally before acquisition of IS1353 by the ancestor of Tn21.12 Tn2411 was discovered as part of R1767, a large self-transferable plasmid from a clinical strain of Salmonella Typhimurium recovered in Germany in the 1980s.32,33 The Tn2411-like transposons of the Spanish isolates were also carried by large plasmids whose conjugative transfer could be demonstrated in all but one case. Carriage on conjugative plasmids is regarded as the main reason for successful dissemination of Tn2411-like transposons in different bacteria (including the various Salmonella serovars) and countries (including Spain) while having the opportunity of further evolution.
The class 1 integrons characterized in this work were accompanied by a class 2 integron associated with Tn7. This transposon is unusual in that it inserts into a unique site in the bacterial chromosome (attTn7), and also within self-transmissible plasmids that can conjugate between cells.34,35 The chromosomal attTn7 site was only occupied in two out of the eight strains characterized in this work (Salmonella Virchow LSP 244/90 and Salmonella Panama MUO 72/93). In both cases, the chromosomal copy of the transposon was apparently intact. A class 2 integron with the same gene array and associated to a complete Tn7 was also detected in the chromosome of two other Salmonella Virchow strains of clinical origin (LSP 231/90 and LSP 205/98), which were previously characterized in our laboratory.23 In addition, plasmids from all clinical isolates contained the Tn7 integron, but only in pUO-SwR1 (from Salmonella Worthington LSP 7/93) Tn7 appeared to be intact. In the remaining strains the transposon contained deletions that removed genes essential for transposition (Figure 2). Since Tn7 transposes by a non-replicative mechanism36 such deletions could stabilize the resistance determinants within the conjugative plasmids, favouring their dissemination to new bacterial populations. This would be especially true for gene cassettes carried by class 2 integrons, which contain an inactive integrase.
The actual incidence of class 2 integrons in Asturias was not accurately determined. However, the available information supports that they are by far less common than those of class 1 (I. Rodríguez, M. C. Martín, M. R. Rodicio and M. C. Mendoza, unpublished data). Moreover, they were only detected in isolates recovered during or before 1998, in serovars with relatively low incidence (but not in the predominant Enteritidis and Typhimurium). At that time, the group of plasmids carrying the variety of structures depicted in Figure 2 could have played an important role in dissemination of resistance. It would be interesting to determine whether these plasmids are related (as it is probably true for those found in Salmonella Virchow), and if similar elements are still circulating in our region, despite of the absence of the class 2 integron.
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We declare no conflict of interest in connection with this article.
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Acknowledgements |
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We thank M. A. González-Hevia (Laboratorio de Salud Pública del PA, Oviedo) for clinical strains. I. R. is the recipient of a grant from the ‘Fundación para el Fomento en Asturias de la Investigación Científica Aplicada y la Tecnología’ (FICYT-BP04-086). This work has been supported by projects from ‘Fondo de Investigación Sanitaria’ (PI02-0172) and ‘Ministerio de Educación y Ciencia’ (SAF-2005-04212) of Spain.
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  N. Martinez, M. C. Mendoza, I. Rodriguez, S. Soto, M. Bances, and M. R. Rodicio Detailed structure of integrons and transposons carried by large conjugative plasmids responsible for multidrug resistance in diverse genomic types of Salmonella enterica serovar Brandenburg J. Antimicrob. Chemother., December 1, 2007; 60(6): 1227 - 1234. [Abstract] [Full Text] [PDF]
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