International Spread and Persistence of TEM-24 Is Caused by the Confluence of Highly Penetrating Enterobacteriaceae Clones and an IncA/C₂ Plasmid Containing Tn1696::Tn1 and IS5075-Tn21

Bacterial strains and epidemiological background.Twenty-eight TEM-24-producing isolates from four European countries collected between 1998 and 2004 were studied. They included 13 E. aerogenes isolates, 6 E. coli isolates, 6 K. pneumoniae isolates, 2 P. mirabilis isolates, and 1 Klebsiella oxytoca isolate recovered from Spain (E. aerogenes, n = 7; E. coli, n = 4), Portugal (K. pneumoniae, n = 6; E. aerogenes, n = 3; E. coli, n = 2; P. mirabilis, n = 2; K. oxytoca, n = 1), and France/Belgium (E. aerogenes, n = 3) in outbreak and nonoutbreak situations (Table 2 ). Representatives of the epidemic E. aerogenes clone associated with nosocomial outbreaks in France, Belgium, Spain, and Portugal were included (6, 18, 23, 31). Isolates were obtained from 26 patients located at medical wards (44%), intensive care units (32%), and surgical wards (12%), and 12% of patients were outpatients. Species identification, determination of patterns of susceptibility to 12 non-β-lactam antibiotics, and ESBL characterization were performed by standard procedures (15). Relationships among isolates were established by pulsed-field gel electrophoresis (PFGE) as previously described (6, 15, 43). The E. coli isolates were also characterized by multilocus sequence typing (MLST) and classified according to the phylogenetic groups identified by the multiplex PCR assay described by Clermont et al. (11; see also http://www.mlst.net ).

Plasmid analysis.Conjugative transfer was tested by broth and/or filter mating methods using E. coli K-12 strain BM21R (Nal^r, Rif^r, Lac⁺, plasmid free) as recipient at a 1:2 donor-to-recipient ratio and transconjugants were selected in plates supplemented with ceftazidime (2 μg/ml) and rifampin (100 μg/ml). Plasmid characterization was accomplished by determination of plasmid content and size, identification of the incompatibility group according to a PCR-based replicon typing scheme, sequencing, and further hybridization with specific probes and comparison of plasmid DNA patterns after digestion with EcoRI, PstI, and HpaI restriction enzymes (8, 43). Restriction fragment length polymorphism (RFLP) profiles corresponding to plasmids identified in this study were compared with those of other sequenced plasmids virtually generated by using the NEBcutter V2.0 tool (http://tools.neb.com/NEBcutter2/index.php ).

Genetic elements associated with antibiotic resistance.Screening for sequences of widely disseminated genetic elements generally associated with antibiotic resistance was achieved by PCR. They included sequences related to class 1, 2, and 3 integrons (intI1, intI2, and intI3), transposons (tnpR₃, tnpA₃, tnpR₂₁, tnpM₂₁, tni₄₀₂, and Tn5393), IS1326, IS1353, IS6100, merA, and other antibiotic resistance genes, such as sul2 (32, 43). Transposable elements were further characterized in selected isolates by a PCR mapping assay based on known sequences of Tn402, Tn3, Tn21, and Tn1696 (43). Oligonucleotide sequences used in this study appear in Table 3.

Molecular techniques.Different PCR mapping experiments were performed using primers described in Table 3 and the following amplification conditions. (i) The first set of conditions comprised 1.5 mM MgCl₂, 1× reaction buffer, 0.2 mM each deoxynucleoside triphosphate, 1 μM each primer, and 1.5 units of Taq DNA polymerase (AmpliTaq Gold; Applied Biosystems, Foster City, CA) with amplification programs of 12 min at 94°C and 35 cycles of 1 min at 94°C, 1 to 2 min at 55°C to 65°C, and 1 to 3 min at 72°C, with a final extension step of 10 min at 72°C for standard PCR assays (PCR products < 3 kb). (ii) The other set of conditions comprised 2.5 mM MgCl₂, 1× reaction buffer, 0.4 mM each deoxynucleoside triphosphate, 1 μM each primer, 5% dimethyl sulfoxide (when necessary), and 2.5 units of LA Taq polymerase (Takara Bio Inc., Shiga, Japan) with amplification conditions of 1 min at 94°C and 35 cycles of a denaturation step of 20 s at 96°C, annealing at 52°C to 68°C for 1 min, and an extension step at 72°C for 3 to 4 min, followed by a final step of 10 min at 72°C, for long PCRs (products > 3 kb). Southern blot DNA transfer and hybridization assays were performed by standard procedures (43). The bla_TEM-24 and repA/C probes used in the hybridization assays were generated by PCR using well-known positive controls as template DNA. Labeling and detection were carried out using the Gene Images Alkphos direct labeling system kit, following the manufacturer's instructions (Amersham Life Sciences, Uppsala, Sweden). PFGE was performed as described previously, using the following conditions: 14°C, 6 V/cm², 5- to 25-s pulses for 6 h followed by 30- to 45-s pulses for 18 h (S1 nuclease), and 10- to 40-s pulses for 24 h (XbaI) (43).

Sequence analysis.GenBank searches were performed using the NCBI BLASTN alignment tool. Open reading frame (ORF) identification and annotation were facilitated by the use of ARTEMIS (version 10.1) (http://www.sanger.ac.uk/Software ).

Nucleotide sequence accession numbers.The sequences shown in Fig. 1 have been submitted to the GenBank database and correspond to plasmid variants pRYC103T24.1 (GQ293498 and GQ293499) and pRYC103T24.2 (GQ293500 and GQ293501).

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FIG. 1.

Characterization of multidrug resistance regions identified in the IncA/C₂ plasmids carrying bla_TEM-24. (A) In4-Tn402 and In0-Tn402 variants identified in pRYC103T24.1. (B) In4-Tn402 and In0-Tn402 variants identified in pRYC103T24.2. (C) The pRYC103T24.3 variant is similar to pRYC103T24.2, differing in the gene cassette array of In0-Tn402. Horizontal dotted lines represent the PCR mapping strategy used to characterize Tn402 derivatives and mercury resistance transposon operons. The locations of the primers used are indicated by white (In4-like variant) and black (In0 variant) triangles. Numbers represent primers described in Table 3 or the size (kb) of the PCR products. Open reading frames (ORFs) correspond to arrow-shaped boxes. Dark gray boxes represent transposition and mercury resistance modules, light gray boxes represent Tn402/class 1 integron derivatives, and cross-hatched boxes represent gene cassettes. The P1/Pant promoter type (In2/In4), which directs the expression of the gene cassettes, is shown. White circles symbolize 59-base elements of the corresponding gene cassettes. Rectangles correspond to insertion sequences (IS). Boxes bordered by a dotted line indicate incomplete or truncated ORFs. Arrows inside rectangles express the direction of transcription of the corresponding ORFs. Vertical bars symbolize inverted repeats of the integrons (IRi and IRt) and transposons (TIR). Horizontal bold lines show identity with highly similar regions (>99% identity) available in the GenBank database.

Dissemination of the bla_TEM-24 gene is caused by clonal spread of Enterobacteriaceae.We identified similar PFGE patterns among all E. aerogenes, K. pneumoniae, and P. mirabilis TEM-24-producing isolates, in contrast to the clonal diversity found among E. coli strains (6 isolates/6 PFGE types), which belonged to phylogenetic groups B2 (n = 2), D (n = 2), and A (n = 2) (Table 2). The isolates exhibited resistance to β-lactams, chloramphenicol, sulfonamides, and trimethoprim, and most of them also exhibited resistance to certain aminoglycosides (amikacin, kanamycin, netilmicin, streptomycin, and tobramycin) except gentamicin (only 3.6%), while resistance to ciprofloxacin (71%) or tetracycline (61%) was variable (Table 2). This phenotype was variably expressed in the transconjugants obtained.

The E. aerogenes strain corresponded to the multidrug-resistant clone responsible for the dissemination of TEM-24 in hospitals in Belgium, France, Portugal, and Spain (6, 18, 23, 31). This clone was initially identified in the early 1990s and, since then, has been extensively detected in European hospitals and health care facilities associated either with the international spread of TEM-24 or with the local spread of TEM-3 in France (1, 6, 17, 18, 20, 23). A recent study has also identified isolates of this clone carrying plasmids encoding different ESBLs (bla_SHV-12, bla_SHV-5, and bla_TEM-20) and metallo-β-lactamases (bla_IMP-1 and bla_VIM-2), which highlights its role as a highly efficient vehicle of multiresistant conjugative plasmids (2). We cannot establish if the K. pneumoniae and P. mirabilis clones identified were related to other widespread clones, since MLST analysis was not performed for these species. However, some E. coli isolates corresponded to the B2-ST131, A-ST10, A-ST23, and D-ST405 clonal complexes, which are currently widespread in the community carrying plasmids encoding different ESBLs (14). These results indicate that the surveillance and identification of highly penetrating clones is becoming a critical issue for controlling antibiotic resistance (14). The term “penetration” is used here, by analogy with the term used in security engineering, for elements having a high attack profile regarding existing networks, in our case, the human microbiota (16).

bla _TEM-24 is located on an epidemic plasmid belonging to the IncA/C group.In all species analyzed, bla_TEM-24 was located on conjugative plasmids of approximately 180 kb, containing replication and relaxase proteins (data not shown) identical to those associated with incompatibility group A/C₂ (22). All studied plasmids showed similar restriction patterns, arbitrarily designated pRYC103T24 plasmids (variants differing in the regions encoding antibiotic multiresistance characterized in this study were represented by subtypes designated pRYC103T24.1-3 plasmids, with two of them selected for full characterization of genetic elements) (Fig. 1) . Comparison of RFLP profiles corresponding to bla_TEM-24-containing plasmids included in this study and known IncA/C plasmids (GenBank accession numbers CP000602, CP000603, CP000604, and FJ705807) virtually generated by using NEBcutter V2.0 tool (data not shown) showed common bands among them, suggesting recent evolution of this plasmid lineage (22, 38, 57; this study). The recovery of pRYC103T24 from different species (E. coli and E. aerogenes) from the same patient suggests the possibility of an efficient in vivo transfer, previously demonstrated for other large plasmids encoding TEM-24 (34-36, 41, 42), although environmental transfer cannot be excluded.

The broad-host-range IncA/C group of plasmids has been detected in multidrug-resistant human and animal pathogens of Enterobacteriaeceae species (E. coli, Klebsiella sp., Salmonella enterica, P. mirabilis, Providencia sp., S. marcescens, Yersinia pestis, Yersinia ruckeri, Edwarsiella ictaluri), Photobacterium damselae subsp. piscicida (Pasteurella piscicida), Vibrio cholerae El Tor 013, Aeromonas salmonicida, and Pseudomonas spp. on all continents since the first description of pRA1 in the late 1960s (22, 56, 57). Plasmids belonging to the IncA/C group have recently facilitated the spread of genes conferring antibiotic resistance to β-lactams such as bla_TEM-3, bla_TEM-21, bla_TEM-24, bla_SHV-2, bla_CTX-M-2, bla_CTX-M-3, bla_CTX-M-14, bla_CTX-M-15, bla_CMY-2, bla_CMY-4, bla_IMP-4, and bla_VIM-4 or conferring a high level of resistance to aminoglycosides such as those encoded by armA and rmtB (7, 13, 33, 38, 57; this study). Acquisition of different antibiotic resistance transposons and/or genetic islands seems to have occurred multiple times, as described for other particular plasmid groups (Table 1) (22, 46, 53, 57).

The multiresistance region is associated with defective Tn402 elements located within Tn21 and Tn1696.Two Tn402 variants belonging to the In0 and In4 lineages were found in each IncA/C₂ plasmid included in this study. They were associated with fully characterized mercury resistance transposons from two E. coli transconjugant strains (Fig. 1). The analyzed IncA/C₂ plasmids were designated pRYC103T24.1 and pRYC103T24.2.

(i) Tn402.The two In0-Tn402 constructs analyzed differed in their 5′ conserved sequence (CS) regions, containing an intI1 gene with a weak (TGGACA-17 bp-TAAGCT) or strong (TTGACA-17 bp-TAAACT) P1/Pant promoter, and in the identity and/or number of gene cassettes (aacA4, aacA4-aacC1-orfE-aadA2-cmlA1, or dfrA1-aadA1) (48). Nevertheless, they exhibited a common tni₄₀₂ deletion, which was identical to the case for the formerly described prototype in plasmid pVS1. The genetic platforms described here constitute one of the few complete In0-like integrons available in the GenBank sequence database (accession numbers U49101, DQ125241, and CP001232).

The two In4-Tn402 types identified also differed in the 5′ CS region (containing intI1 with either a weak or strong P1/Pant promoter) and the identity and/or number of gene cassettes (aacA4 or dfrA1-aadA1). In addition, a 19-bp duplication identical to that found in In4 in Tn1696 (GenBank accession number U12338) was detected within the attI integration site of the aacA4-containing integrons, creating an in-frame ATG codon which might direct the expression of the aacA4 cassette (Fig. 1) (48). Similar to what was observed for In0-Tn402, these two Tn402 types showed a common tni module. In this case, it represents a new In4-like configuration, differing from that of the prototype In4 (GenBank accession number U12338) by a 26-bp deletion of the 3′ CS region and the absence of the duplication of the last 321 bp of IS6100. Among the diverse In4 elements found in the GenBank database, In1 (GenBank accession number AY046276) has a 42-bp deletion of the same region, suggesting independent evolutionary events. In4-type integrons have been frequently detected among contemporary antibiotic-resistant bacteria and differ in several lengths of deletions of regions flanking IS6100 or in insertions of IS26 at different sites of the 5′ CS or 3′ CS regions, which seem to direct the evolution of members of this family (39, 49; this study). The high diversity of genetic platforms linked to In4 integrons further highlights their high degree of plasticity (29, 39, 49, 54, 55).

The gene cassettes of the integrons identified explain most of the multiresistance phenotypes observed, affecting in most cases trimethoprim (dfrA1), chloramphenicol (cmlA1), streptomycin and spectinomycin (aadA1), gentamicin (aacC1), and other different aminoglycosides, such as amikacin, kanamycin, tobramycin, and netilmicin (aacA4). Variations in the gene cassette content and expression level might explain slight differences in the resistance patterns observed in different strains and transconjugants (Table 2). The sul2 gene was part of a different module which has been identified within the common 100-kb backbone shared by all known IncA/C₂ plasmids (22, 57).

(ii) Tn21-like transposons.In0-Tn402 and In4-Tn402 were identified within Tn21 and Tn1696, respectively, in pRYC103T24.1. However, they were linked to mosaic transposons comprising tnp₂₁-mer₁₆₉₆ or tnp₁₆₉₆-mer₂₁ in pRYC103T24.2. IRtnp₂₁, IRmer₂₁, and IRtnp₁₆₉₆ were interrupted by IS5075 in different orientations, but we could not detect either IRmer₁₆₉₆ or an IS5075 copy adjacent to mer₁₆₉₆ (Fig. 1). These insertion sequences (ISs) belong to the IS1111 family, which typically shows target site specificity and has been found interrupting the terminal ends of members of the Tn21-like subgroup of transposons (GenBank accession numbers AY333434, AL513383, DQ310703, X64523, CP000971, CP000650, and FJ223605) (13, 21, 46, 47, 57). The presence of similar Tn21-like elements with IRtnp and IRmer interrupted by IS5075 in different plasmid types highlights the possible role of these ISs in the mobilization of these transposons to distinct genetic contexts. Although the precise locations of the transposons described in this work are not provided, we cannot exclude the possibility of insertion in a region of complex modular structure, as was already observed in other multiresistance plasmids (46). However, mercury resistance transposons identified in other IncA/C plasmids are flanked by direct repeats such as TTGTA in pCC416 and pSN254 and TACAA in pIP1202, indicating different acquisitions in distinct specific target sites (Fig. 2) (22, 57). Homologous and/or site-specific recombination events occurring within the plasmid or the transposon backbones seem to have played a key role in the evolution of this IncA/C plasmid group (45, 53, 58; this study). Although apparently diverse, the multiresistance regions described in this study and other studies consist of an assembly of a few highly modular genetic elements clustered in specific plasmid regions with potential for recombination (22, 44, 45, 46, 58; see below). They have also been observed in widely disseminated plasmids of the incompatibility groups FII, HI1, HI2, P1-α, P1-β, and W (GenBank accession numbers AP000342, AY458016, AJ851089, AF550679, AL513383, BX664015, NC_006352, and NC_004840) (44, 53).

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FIG. 2.

Comparison of mercury resistance transposon platforms identified in different IncA/C plasmids. Tn402 derivatives and mercury resistance transposon derivatives found in the IncA/C plasmids pIP1202 (GenBank accession number CP000603), pSN254 (GenBank accession number CP000604), pCC416 (GenBank accession number AJ704863), and pRYC103T24.1 (GenBank accession numbers GQ293498 and GQ293499) (this study) are represented. Vertical bars represent the inverted repeats of integrons or transposons. Arrows inside boxes show the direction of transcription corresponding to different ORFs. Plasmid pSN254 contains two integrons in a “tail-to-tail” configuration; plasmid pCC416 has an integron lacking the 3′ CS region. Gene cassettes from pIP1202, pSN254, and pCC416 were similar to others described in the GenBank database: the aadA2 gene cassette was 100% homologous to that identified in plasmid pSAL-1 from Salmonella enterica serovar Enteritidis (GenBank accession number AJ237702); the aadA1 and aac3-VI genes were identical to those from IncA/C plasmids pAR060302, pAM4528, and peH4H, which probably are pSN254 derivatives (GenBank accession numbers FJ621588, FJ621587, and FJ621586, respectively), or the IncHI2 plasmid pAPEC-O1-R (GenBank accession number DQ517526). The bla_VIM-4-aacA7-dfrA1-aadA1-smr array resembles that of the widespread In-e541 containing bla_VIM-1 (GenBank accession number AJ870988) (13).

bla _TEM-24 is located in a Tn1 construct.In all isolates analyzed (3 E. coli isolates, 2 K. pneumoniae isolates, and 1 E. aerogenes isolate), bla_TEM-24 was identified in a defective Tn1 containing the right inverted repeat (IR), tnpR, and a tnpA gene truncated at bp 1965. This transposon was inserted 465 nucleotides apart from the start codon of tnpA₁₆₉₆ (Fig. 1). A related configuration, IS5075-ΔTn1(bla_TEM-3)-ΔtnpA₁₆₉₆, showing a complete deletion of tnpA₁ was found in the IncL/M plasmid pCFF04 (GenBank accession number X64523) in which the Tn1 (bla_TEM-3) was identified in the same nucleotide position of tnpA of Tn3926 or Tn5036 from which Tn1696 was presumptively generated after the insertion of In4 (30, 48). This structure might have eventually arisen by insertion of Tn1 in a tnp₁₆₉₆ module followed by further independent deletions on tnpA₁. However, other hypotheses, like one involving a one-ended transposition event of an incomplete copy of Tn1 or a transposase complementation, have also been suggested (30). Interestingly, sequences associated with Tn1::Tn1696-like structures were also identified in other related plasmids from French clinical isolates producing TEM β-lactamases (TEM-7 and TEM-8) recovered during the 1980s (30). These and other variants (TEM-16, TEM-24, and TEM-66) are TEM-2 derivatives encoded by plasmids of similar sizes and RFLP patterns, conferring resistance to common antibiotics (kanamycin, tobramycin, amikacin, netilmicin, tetracyclines, trimethoprim, and sulfonamides) and showing identical genetic surroundings containing bla_TEM genes (Tn1 derivatives and/or presence of aac(6)′-Ib) in different species (Table 1) (3, 10, 30). The bla_TEM-24 gene identified in our study corresponded to the bla_TEM-24b variant which was 99% homologous (100% identity at amino acid level) to that first identified in 1992 by Chanal et al. and initially designated bla_CAZ-6 (GenBank accession number X65253) (9). It has been proposed that this gene arose by recombination between bla_TEM-5 and bla_TEM-8 variants (25), whereas the bla_TEM-24a variant (bla_CAZ-6) would have resulted from a recombination event between bla_TEM-5 and bla_TEM-3 (9). It is thus tempting to suggest a possible common origin for all these β-lactamases from a single insertion event of Tn1 containing bla_TEM-2 and/or bla_TEM-3 and further evolution by mutation and/or recombination of a variety of bla_TEM genes in a given clinical setting (30).

Tn3 elements are often identified in identical genetic surroundings such as tnpA (this case) or mer (as described in pRMH760, p1658/97, and TnSF1) in plasmids of the incompatibility groups A/C₂, L/M, and FII. The identical boundaries identified for Tn2-merT (pRMH760, p1658/97, and TnSF1) and Tn1-tnpA suggest single insertion events creating structures that have subsequently spread (22, 30, 46, 57; this study).

Concluding remarks.Our study indicates that the international dissemination of TEM-24 is driven by the confluence of highly penetrating clones of Enterobacteriaceae and a highly transmissible IncA/C₂ plasmid, confirming the contribution of IncA/C plasmids in the global spread of antibiotic resistance. It further demonstrates the evolution of this plasmid lineage by acquisition of new different mobile genetic elements as described for other particular incompatibility groups (22, 46, 53).

The variability of Tn402 and Tn21 within the pRYC103T24 group illustrates the plasticity of these genetic platforms, and it highlights the need for full multilevel characterization of the genetic environments linked to antibiotic resistance genes in order to understand how they spread. This paper contributes to extending the knowledge on the spread, distribution, and evolution of Tn3-like elements, helping to elucidate the origin and evolution of ESBLs derived from TEM-2 β-lactamase.