JAC Advance Access originally published online on September 7, 2007
Journal of Antimicrobial Chemotherapy 2007 60(6):1227-1234; doi:10.1093/jac/dkm336
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Detailed structure of integrons and transposons carried by large conjugative plasmids responsible for multidrug resistance in diverse genomic types of Salmonella enterica serovar Brandenburg
1 Departamento de Biología Funcional (Área de Microbiología) and Instituto Universitario de Biotecnología de Asturias (IUBA), Universidad de Oviedo, C/ Julián Clavería 6, 33006 Oviedo, Spain; 2 Laboratorio de Salud Pública, Consejería de Sanidad del Principado de Asturias, 33001 Oviedo, Spain
* Corresponding author. Tel: +34-985103562; Fax: +34-985103148; E-mail: rrodicio{at}fq.uniovi.es
Received 5 March 2007; returned 17 April 2007; revised 3 August 2007; accepted 8 August 2007
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Abstract |
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Objectives: To evaluate the incidence, molecular basis and distribution among genomic types of antimicrobial drug resistance in Salmonella enterica (S.) serovar Brandenburg isolates recorded in the Principality of Asturias, Spain.
Methods: Thirty-seven S. Brandenburg isolates were tested for susceptibility to antimicrobial agents and typed by random amplified polymorphic DNA (RAPD) and pulsed-field gel electrophoresis (PFGE). PCR amplifications, together with DNA cloning and sequencing, were used to identify resistance genes, integrons and transposons and to establish the structure and physical associations between them. Conjugation experiments were applied to establish the location of the identified elements.
Results: Twenty-one isolates were resistant to one or more unrelated drugs. Resistances to streptomycin, tetracycline, kanamycin, chloramphenicol, ampicillin and trimethoprim-sulfamethoxazole, encoded by aadA1, tet(A) or tet(B), aphA1, catA1, blaTEM and dfrA1-sul1-sul3, respectively, were most frequently observed. Multidrug resistance (32.4%) was mainly mediated by mobile genetic elements. These included: (i) class 1 integrons (with dfrA1-aadA1 gene cassettes in their variable region), which were part of Tn21-related transposons associated with Tn9; (ii) a Tn1721-derivative containing tet(A); (iii) a defective Tn10 that carried tet(B), and was linked to an integron; and (iv) large conjugative plasmids carrying a class 1 integron-Tn21-Tn9-like structure, together with the Tn1721- or the Tn10-related element. Two-way-RAPD and XbaI-PFGE discriminated the isolates into 15 and 12 profiles, respectively.
Conclusions: Complex genetic elements have apparently been responsible for the recruitment, assembly and dispersion of resistance genes among the highly diverse genomic types of S. Brandenburg, identified as causal agents of human salmonellosis in the Principality of Asturias, over recent years.
Keywords: conjugative plasmids , transposons , 1600 bp/dfrA1-aadA1 integrons , RAPD , PFGE
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Introduction |
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Non-typhoidal serovars of Salmonella enterica (S.) constitute an important cause of bacterial gastroenteritis in humans. For the last two decades, our laboratories [placed in the Principality of Asturias (PA) a region in the Northwest of Spain with more than one million inhabitants] have been investigating the molecular epidemiology and the genetic basis of antimicrobial drug resistance in such serovars.1–8 During the course of these studies, several facts regarding serovar Brandenburg came to our attention. First, 2 out of 32 cases of bacteraemia recorded from 1991 to 1996 could be attributed to S. Brandenburg.9 Second, 7 out of 15 clinical isolates recovered over 1995–97 were resistant to seven drugs (ampicillin, chloramphenicol, kanamycin, streptomycin, sulfadiazine, trimethoprim and tetracycline), in clear contrast to data reported for the same serovar by other groups.10–13 Third, although the incidence of S. Brandenburg in the PA is low, a sudden increase in the number of clinical cases was noted in 2000 by the Laboratorio de Salud Pública (LSP), acting as Salmonella Reference Centre. These facts prompted the present study, in which all S. Brandenburg isolates recorded by the LSP during 1993–2005 were characterized with regard to the incidence and molecular basis of resistance and multidrug resistance. Particular attention was paid to the involvement of mobile genetic elements in the recruitment and dispersion of resistance determinants, their structure, physical associations and distribution among genomic types.
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Bacterial strains
All S. Brandenburg isolates recorded at the LSP between 1993 and 2005 were used in this study (Table 1). Of a total of 37 isolates, 32 were collected from faeces of different patients with gastroenteritis, 2 from food samples, 1 from a rectal abscess, 1 from a urine sample and 1 from sewage water.
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Antimicrobial susceptibility testing and detection of resistance determinants
Susceptibility to antimicrobial agents was determined by the disc diffusion assay on Mueller–Hinton agar with commercially available discs (Oxoid, Madrid, Spain), according to CLSI (formerly NCCLS).14 The antimicrobials tested and the disc content (in µg) were as follows: ampicillin (10), ciprofloxacin (5), chloramphenicol (30), gentamicin (10), kanamycin (30), nalidixic acid (30), streptomycin (10), sulfadiazine (300), tetracycline (30), trimethoprim (5) and trimethoprim-sulfamethoxazole (SXT, 1.25/23.75). The MIC of ciprofloxacin was determined by a broth dilution method, with serial dilutions ranging from 0.01 to 4 mg/L. Drug resistance determinants [aadA1-like, aphA1, aac(3)-IV, blaTEM, catA1, cmlA1-like, dfrA1-like, dfrA12, strA, strB, sul1, sul2, sul3, tet(A), tet(B) and tet(G)] were screened for by PCR amplification using previously described primers and conditions.4,15 Amplification of gyrA from the only isolate resistant to nalidixic acid and sequencing of the PCR product (performed at Secugen, Madrid, Spain) were used to establish the genetic background of the resistance.5
Integron and transposon analysis
Integrons were screened by PCR amplifications performed with degenerate primers (hep35 and hep36) designed for conserved regions of classes 1, 2 and 3 integrase genes (intI1–intI3), followed by digestion of the generated products with HinfI.16 The variable regions of class 1 integrons were amplified with the 5'CS and 3'CS primers that anneal with sequences flanking the attI1 site.7 Gene cassettes in the variable region were identified by nested PCR with primer pairs selected according to the R-genotypes of the integron-containing isolates, followed by sequencing. Transposon-related sequences, including tnpA of Tn3 (which encodes the transposase);17 IS1, the insertion sequence flanking Tn9;18 tnpA, tnpR (encoding transposition functions), merA and merR (involved in mercury resistance) of Tn21;18 the tet(A) gene (coding for a tetracycline efflux pump) specific of Tn1721;19 and the tetR gene, encoding the tet(B) repressor of Tn10,20 were screened in isolates suspected to contain one or more transposons, according to their resistance phenotypes and/or genotypes. The presence and arrangement of genes outside the variable region of the integrons, including intI1, qacE
1, orf5, istA and istB (of IS1326), orfA and orfB (of IS1353), tniB1 and tniA,18 a more detailed structure of the transposons, as well as the physical associations between the latter, resistance genes and/or integrons, were investigated in relevant isolates (Table 1). In all cases, standard and nested PCR amplifications were performed using already described primers,4,6,21–24 and primers newly designed for the present work (Table 2), according to the schemes depicted in Figure 1.
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Plasmid analysis
To determine the number and molecular sizes of large plasmids, total DNA was digested with S1 nuclease (Amersham Biosciences) and subjected to PFGE (S1-PFGE).5 Lambda ladder PFG marker (New England BioLabs) was the size standard. Mating experiments were performed in Luria Broth25 at 37°C, using Escherichia coli K12J53 (resistant to rifampicin) as recipient.5 Transconjugants were selected on eosin methylene blue agar (Oxoid) containing rifampicin (50 mg/L) along with ampicillin (100 mg/L), chloramphenicol (50 mg/L) or tetracycline (30 mg/L). Transfer of resistance genes was phenotypically tested by the disc diffusion method, as before. The plasmid location of relevant genes, integron and/or transposons was confirmed by PCR.
Cloning and sequence analysis of the tet(A) and tet(B) regions
The tet(A) and tet(B) genes were cloned from relevant plasmids (pUO-SbR3 and pUO-SbR5, respectively) into pUC18, using standard techniques.25 Plasmids were extracted from E. coli transconjugants of S. Brandenburg LSP 145/01 and LSP 338/97, using the HiPure Plasmid Midiprep Kit (Invitrogen). In the successful strategies, pUO-SbR3 digested with BamHI and SphI, and pUO-SbR5 digested with XbaI and SphI were ligated to the vector previously cut with the same enzymes. The ligated DNA was transformed into chemically competent E. coli DH5
cells (Invitrogen), and direct selection was achieved in Luria Broth containing both ampicillin and tetracycline, at the above indicated concentrations. DNA insertions conferring resistance to tetracycline were sequenced, analysed by BLAST,26 and the generated sequences deposited in the EMBL database with accession numbers AM746674 and AM746675.
Molecular typing by RAPD and PFGE
Genomic variability of the isolates was assessed by two typing procedures that analyse the entire genome of the bacteria: RAPD and macrorestriction-PFGE. A two-way-RAPD procedure was performed with primers S and OPB-17 (also termed C), as reported.2 Lambda DNA digested with PstI was used as size standard. PFGE plugs were digested with the restriction enzyme XbaI (30 U, 4 h, 37°C; Takara Biomedicals). The obtained fragments were separated in a CHEF-DRIII system (Bio-Rad Laboratories), under standardized conditions.5 Lambda ladder PFG marker and DNA of S. enterica serovar Braenderup H9812 digested with XbaI were included as controls.
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Prevalence and genetic basis of antimicrobial drug resistance in Salmonella Brandenburg
The antimicrobial resistance profiles (R0–R12) of the 37 analysed isolates are shown in Table 1. Sixteen isolates (43.2%) were susceptible to all antimicrobials tested (R0 profile), whereas the remaining 21 (56.8%) were resistant to one (5; 13.5%), two (2; 5.4%), three (2; 5.4%) and six or more (12; 32.4%, considered as multidrug-resistant) unrelated agents. The most common individual resistances were streptomycin, tetracycline, kanamycin, chloramphenicol, ampicillin, trimethoprim-sulfamethoxazole, encoded by aadA1, either tet(A) or tet(B), aphA1, catA1, blaTEM, and dfrA1-sul1-sul3, displayed by 18, 15, 14, 13, 12 and 12 isolates, respectively. Two isolates (R11 profile) were resistant to gentamicin, due to acquisition of aac(3)-IV, and one (R4 profile) was resistant to nalidixic acid. The latter carried a mutation in the gyrA gene (GAC to GGC), changing Asp87 to Gly, and also exhibited an intermediate susceptibility to ciprofloxacin (MIC of 2 mg/L). Since the observed mutation does not fully explain this MIC value, additional changes in either gyrB, parC, or parE and/or other mechanisms such as overexpression of efflux pumps are likely to be involved.27 Apart from R0, the R12 profile was the most frequent, with seven isolates (18.9% of the total) recovered during 1995–97.
Integrons and transposons in resistant and mutlidrug-resistant isolates of Salmonella Brandenburg
Resistant isolates of S. Brandenburg were tested for the presence of mobile genetic elements. None contained integrons of classes 2 and 3. However, class 1 integrons with the dfrA1-aadA1 genes in the variable region (ca. 1600 bp) were found in all multidrug-resistant isolates (R9–R12 profiles), and its detailed structure was established for LSP 662/01, LSP 125/02, LSP 145/01, LSP 19/97 and LSP 338/97 (Table 1; Figure 1). Apart from dfrA1, aadA1 and sul1, all were positive for intI1, qacE1 and orf5, and negative for genes of IS1326 (istA and istB) and IS1353 (orfA and orfB), the two insertion sequences frequently found in class 1 integrons.18 In addition, tet(A)-positive isolates (R9–R11 profiles) yielded the amplicon expected for tniA but not for tniB1,18 and the linkage between orf5 and tniA could be demonstrated (Figure 1a). In contrast, tet(B)-positive isolates (R12 profile) lacked both tniA and tniB1, and the amplicons joining orf5 with either tniA or urf2 could not be obtained (Figure 1c). Association of the integron with Tn21- and Tn9-related elements was also investigated. In all selected isolates, the integron was part of a Tn21-like transposon that carried an apparently intact mer operon, and was in turn inserted into Tn9, where the catA1 gene was located (Figure 1a and c). Other integron-containing isolates proved to be positive for Tn21 (tnpA, tnpR, merA and merR)- and Tn9 (IS1 and catA1)-related sequences, although the actual linkage between the three elements was not tested.
With regard to other transposons, none of the S. Brandenburg isolates contained a Tn3-like element, where the blaTEM gene could have been located.17 However, all tet(A)-positive isolates, whether multiresistant (R9, R10 and R11 profiles) or not (R3, R6 and R8), yielded the expected amplicon with primers reported as specific for the tet(A) gene of Tn1721.23 The detailed structure of the Tn1721-like transposon was then investigated for LSP 662/01, LSP 125/02 and LSP 145/01, each displaying a different resistance profile (Table 1). The order and orientation of mcp (which encodes a polypeptide with features of a methyl-accepting chemotaxis protein), tnpR, tnpA, tetR, tet(A), pecM (putative regulatory component) and tnpA (which corresponds to a partial duplication of the transposase gene)19,28 was established by overlapping amplifications, followed by nested PCRs of individual genes (Figure 1b). However, we failed to associate tetR with tnpA, a result consistent with insertion of additional DNA between the two genes. This was in fact demonstrated by cloning and sequence analysis of a BamHI–SphI fragment (6.85 kb) containing the tet(A) region of LSP 145/01. Apart from the expected genes, IS186,29 an insertion sequence of the IS4 family, was detected upstream of tetR (Figure 1b).
On the other hand, the tnp gene that encodes the transposase of IS10 (the insertion sequence flanking Tn10)20 was identified in all isolates carrying tet(B) (R12 profile). In LSP 19/97 and LSP 338/97, the linkage between tet(B) and tetR (located in opposite directions), tetR and ybdA (which encodes a protein with homology to transcriptional repressors of metal resistance operons), ybfA (encodes a possible sodium/glutamate transporter) and tnpL was demonstrated (Figure 1c). However, tetC and tetD could not be amplified, and we also failed to associate tet(B) and tnpR. To avoid confusion, it is worth noting that the tetC and tetD genes of Tn10 (the latter encoding a transcriptional activator of genes that confer resistance to redox-cycling compounds and antibiotics, which is negatively regulated by the product of tetC)30 are different from the tet(C) and tet(D) tetracycline efflux genes. In LSP 338/97, cloning and sequencing of an SphI–XbaI fragment (ca. 6.5 kb) carrying the tet(B) gene, located the Tn10-related element next to the 1600 bp/dfrA1-aadA1 integron, together with one inverted repeat of IS1326, an additional copy of IS1, and a truncated tetC gene, between orf5 and tet(B). The Tn10 version of LSP 338/97 lacked IS10R and is defective in transposition.
Conjugative plasmids in multidrug-resistant isolates of Salmonella Brandenburg
Five types of resistance plasmids (termed pUO-SbR1 to pUO-SbR5) were detected in multiresistant isolates (R9–R12 profiles), by S1-PFGE and conjugation experiments, the latter only performed with representative strains of each profile (Figure 2a; Table 1). pUO-SbR1, pUO-SbR2 (of about the same size, ca. 300 kb) and pUO-SbR3 (ca. 280 kb) were found in R9, R10 and R11 isolates, respectively. The former two carried all the resistance genes detected in their hosts, whereas pUO-SbR3 failed to transfer aac(3)-IV, the gene responsible for gentamicin resistance in the R11 profile. Two other plasmids, pUO-SbR4 and pUO-SbR5 (of ca. 210 and 200 kb, respectively), found in two and five isolates of the prevalent R12 profile, transferred all the resistance determinants that defined the profile, but aadA1 failed to confer the expected phenotype (streptomycin resistance) in E. coli. At least in LSP 19/97 (pUO-SbR4) and LSP 338/97 (pUO-SbR5), the aadA1 gene was intact, as demonstrated by nucleotide sequencing, but host-dependent expression of integron-borne aadA genes has been previously reported.31 According to the resistance genotypes and phenotypes of the E. coli transconjugants, the presence of the class 1 integron, and the Tn21-, Tn9- and Tn1721-like transposons in pUO-SbR1, pUO-SbR2 and pUO-SbR3 was demonstrated by PCR amplification. In the same way, the integron, and Tn21-, Tn9-, and Tn10-related elements could be located in pUO-SbR4 and pUO-SbR5.
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Genomic typing of Salmonella Brandenburg isolates
RAPD analysis of the S. Brandenburg isolates was performed with primers S and C (Table 1; Figure 2b). Two distinct profiles (S1 and S2) were revealed by primer S, with most isolates (81.1%) belonging to S1, whereas 11 profiles (C1–C11) were generated by primer C. Considering results from both procedures, 15 combined profiles were found, with S1/C3 and S1/C1 (12 and 6 isolates, respectively) being the most frequent. Genomic macrorestriction using XbaI, followed by PFGE (Figure 2c), identified 12 profiles (X1–X12), hence confirming the high diversity of the S. Brandenburg isolates circulating in the PA during 1993–2005. In most cases, a clear correlation between year of isolation, resistance-, RAPD- and/or PFGE-profile could not be established (Table 1). However, 9 out of the 10 isolates recovered in 2000 were susceptible to all tested antimicrobials (R0-profile) and yielded S1/C3 RAPD- and X9 PFGE-profiles. Accordingly, they were probably associated with an outbreak, undetected at the time. On the other hand, the 12 integron-positive isolates, collected along 1995–2002, were highly polymorphic being discriminated into 8 C/S RAPD- and 7 X-profiles. Each of these isolates contained a large resistance and self-transferable plasmid and, again, a correlation between RAPD- and PFGE-profiles and plasmid could not be established.
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Increasing antimicrobial resistance in non-typhoid Salmonella serovars has become a global problem for public health. Studies on S. Brandenburg, performed in different countries, revealed this serovar as relatively susceptible to antimicrobial agents.10–13 In contrast, 56.8% of the isolates recovered during 1993–2005 in the Principality of Asturias (Spain), proved to be resistant to at least one antimicrobial agent, and 32.4% were multidrug-resistant. The latter contained class 1 integrons with the 1600 bp/dfrA1-aadA1 variable region. The same gene array has been previously found in many other serovars of S. enterica, collected in different countries from clinical samples, animals and/or foods of animal origin.1–3,23,32–35 However, the detailed organization of the integron and its association with transposons has been rarely addressed.32 Here, we demonstrated that the integrons of S. Brandenburg are inserted within transposons of the Tn21 subgroup. Members of the subgroup share the same transposition machinery and contain a class 1 integron.18 However, they differ in the identity and/or number of gene cassettes carried by the integron (aadA1 in the case of In2, the integron of Tn21), the presence or absence of an intact mer locus, and the associated IS elements and/or additional transposons. The oldest element of the subgroup, Tn2411, contains an integron with the aadA1 gene cassette, and the insertion sequence IS1326. Tn21 itself could have derived from an ancestral element of the Tn2411 type, through insertion of IS1353. In contrast, excision of IS1326 may have originated a separate branch of transposons lacking the two insertion sequences, and differing in the number and/or identity of the gene cassettes carried by the integron. The elements found in S. Brandenburg could be new members of this branch, in which loss of IS1326 was associated with a second deletion in the truncated tniB
1 gene in tet(A) isolates, and of both tniB1 and tniA in tet(B) isolates. In the latter, a defective Tn10 element, which provided the tet(B) gene, was linked to the integron through a vestige of IS1326 and a copy of IS1. Most tet(A) genes of S. enterica have been described within a deleted version of Tn1721, lacking a portion of the left arm, including mcp, tnpR and tnpA.23,36,37 The element of S. Brandenburg contained apparently intact left and right arms, but the central region was altered through insertion of additional DNA, including a copy of IS186 next to tetR. A similar organization has been described in plasmid pU302 of Typhimurium.38
S. Brandenburg is a minor serovar whose main reservoir is swine. In Spain, it is ranked at the 13th position, with only 54 clinical isolates recorded at the Spanish National Reference Laboratory for Salmonella and Shigella (LNRSS; Madrid) over 2002–03.39 Despite of this fact, it may be playing an important role in the dissemination of multidrug resistance in the animal reservoir. All multidrug-resistant isolates analysed in this work carried large conjugative plasmids, where all but one of the resistance genes, the integron and all transposon-related elements were located. Considering the high genomic heterogeneity of the isolates, horizontal transmission of the detected plasmids had probably contributed to the spread of such resistance. Interestingly, large self-transferable plasmids carrying integrons with the 1600 bp/dfrA1-aadA1 variable region were also found by our group in multidrug-resistant isolates of other serovars with swine as a host (including Wien, Ohio and Typhimurium; N. M. and I. R., unpublished results).3 Further experiments will be required to investigate a possible relationship between these plasmids and those found in S. Brandenburg.
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None to declare.
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Funding |
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This work has been supported by projects FIS PI020172 and P1052489 (Ministerio de Sanidad y Consumo, Spain). 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).
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