1 Cátedra de Microbiología, Facultad de Farmacia y Bioquímica, Universidad de Buenos Aires, Junín 956 (1113), Buenos Aires, Argentina; 2 Centre d'Ingéniérie des Protéines, Université de Liège, Institut de Chimie, B6 (B-4000), Sart Tilman, Belgium; 3 Centro de Biología Molecular Severo Ochoa, CSIC-UAM, Campus de Cantoblanco (28049), Madrid, Spain
Received 16 September 2004; returned 28 October 2004; revised 11 November 2004; accepted 27 November 2004
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Abstract |
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Methods: bla genes and class 1 integron elements were detected by PCR and DNADNA hybridization in a M. morganii strain isolated in 1996. PCR-mapping and sequencing of different fragments were carried out to determine the integron's architecture.
Results and conclusions: A class 1 integron (In116), strongly related to the In6/In7 family, was detected in a plasmid from an oxyimino-cephalosporin-resistant M. morganii strain, producing CTX-M-2 ß-lactamase. The variable region of In116 contains aacA4, blaOXA-2 and orfD cassettes. Downstream of the 3'-conserved-segment (3'-CS), an orf513-containing common region is followed by blaCTX-M-2 and flanking regions, having 9699% nucleotide identity with Kluyvera ascorbata's kluA-1 and neighbouring sequences. Some of the evidence supporting the incorporation of foreign DNA is as follows: a partial deletion in a second 3'-CS (3'-CS2), and the absence of 59-base element or IS-like structures upstream of blaCTX-M-2.
Keywords: Kluyvera ascorbata , In6/In7 , orf513 , aacA4 , blaOXA-2
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Introduction |
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Currently, nine integron classes are recognized, class 1 integrons being the most frequently reported and distributed among Gram-negative clinical isolates.2
The general organization of a class 1 integron includes: a 5'-conserved-segment (5'-CS), containing the divergently-transcribed intI1 gene, most of attI1, and the promoter region; the 3'-conserved-segment (3'-CS), including at least two genes, qacE1 (basal-level resistance to quaternary-ammonium compounds) and sul1 (sulphonamide resistance); and a variable region (between both conserved domains) harbouring the gene cassettes with the corresponding attC or 59-base element (59-be).1
Unusual class 1 integrons, having a partial duplication of 3'-CS, and a 2.1 kb region lying between both 3'-CSs, called the common region (CR), have been reported. Adjacent to CR resides a unique region containing an antibiotic resistance gene, not resembling a gene cassette. The first integrons of this family were In6 and In7,3,4 harbouring catA2 (chloramphenicol resistance) and dfrA10 (trimethoprim resistance), respectively. A growing number of these integrons have been reported in different regions and species,5,6 including In34 and InS21 (blaCTX-M-2) from Proteus mirabilis and Salmonella Infantis, respectively, isolated in Argentina.7,8
We have previously detected the presence of a plasmid-encoded CTX-M-2-type cefotaximase in six oxyimino-cephalosporin-resistant Morganella morganii isolates, collected during 19961997, being the first report of a cefotaximase-mediated resistance in the species.9 In this study, we establish the association of this cefotaximase with a complex class 1 integron, named In116, in a cefotaxime-resistant M. morganii strain isolated during 1996.
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Materials and methods |
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Morganella morganii PP16, isolated from a urinary tract infection during 1996 in Buenos Aires, was previously characterized as a producer of a pI 8.2 cefotaximase (cefotaxime MIC 64 mg/L) and TEM-1.9 Escherichia coli CAG12177(E. coli Genetic Stock Center, USA) and Escherichia coli Top10F' (Invitrogen, USA) were used as hosts for conjugation and transformation assays, respectively. Control strains: E. coli 1353 (blaTEM-1, blaCTX-M-2) kindly provided by M. Radice (Universidad de Buenos Aires); E. coli J53(RGK) (blaTEM-1) and E. coli J53.2 86-1 (blaSHV-2) kindly provided by A. Medeiros; Salmonella Infantis S21 (blaCTX-M-2, blaOXA-2, class 1 integron).8
Plasmid DNA from M. morganii PP16 (pM16), vectors and genomic DNA were purified by conventional methods,10 or by commercial systems (GFx Micro Plasmid Prep Kit; Amersham Biosciences, USA).
PCR screening
Screening for ß-lactamase-encoding genes (bla) and class 1-integron elements was carried out by PCR, using pM16 as template, 0.81 µM primers (Table 1), and 1.25 U of Taq DNA polymerase (Amersham Biosciences, USA) or 1.5 U of Pfu polymerase (Promega, USA), as required. PCR-amplicons were resolved in 11.3% agarose gels, using commercial standards (1 kb DNA Ladder; MBI-Fermentas, Lithuania).
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Overnight M. morganii colonies, transferred to nitrocellulose membranes, and DNA dot-blots were treated as described previously.10 Gene probes were generated by PCR from control strains and 32P-labelled by random priming.10 Hybridization was carried out overnight at 60°C, and detected by radioautography, using Kodak BioMax MS films and revealing solutions (Kodak, USA).
Conjugative transfer of pM16
The conjugative mobilization of pM16 was attempted by both liquid- and solid-medium mating, to recipient E. coli CAG1217710 Selection was carried out on LuriaBertani or MacConkey agar plates containing cefotaxime (10 mg/L) and tetracycline (20 mg/L).
Restriction endonuclease digestions
Standard reactions were carried out for 2 h at the corresponding temperature, and fragments resolved as above. Restriction endonucleases used: EcoRV, EcoRI, PstI, Eco47III, HindIII, HincII (New England Biolabs, USA), SphI, NotI, PvuII, AvaI and SacI (Pharmacia, USA).
PCR mapping of In116 and DNA sequencing
PCR amplifications, using different pairs of primers (Table 1 and Figure 1), were carried out to assess the architecture of the class 1 integron. PCR products were cloned into pGEM-T (Promega, USA) or pUC18 (Amersham Pharmacia, USA) vectors, according to manufacturers recommendations, and sequenced in both strands, from recombinant plasmids, by the automated Sanger method.11 Sequences were analysed with the NCBI (http://www.ncbi.nlm.nih.gov/) and EBI (http://www.ebi.ac.uk/) tools.
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The sequence of In116 has been submitted to the EMBL database under the accession no. AJ621187.
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Results and discussion |
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The presence of the following bla genes was detected in a 54 kb plasmid (pM16) from M. morganii PP16: blaTEM (0.85 kb), blaCTX-M-2-type (0.9 kb), and blaOXA (0.5 kb). Therefore, plasmid-borne ß-lactamases would be responsible for the acquired resistance to ß-lactams in this strain, oxyimino-cephalosporin resistance being mediated by the CTX-M-2-type enzyme. No blaSHV or blaPER-2 could be detected, indicating that M. morganii PP16 is not an SHV- or PER-producer.
Conjugative transfer of pM16
We failed to transfer pM16 plasmid to a recipient E. coli strain, both by solid- and liquid-medium reactions. This was probably due to a very low efficient conjugation, an incomplete mobilization because of the size of pM16, or even a host species not able to allow the conjugation. Though a Proteus mirabilis strain, phylogenetically closer to M. morganii, could have been used, the multiresistant Morganella strain made it extremely difficult to find the proper conditions for selection of transconjugant cells.
Sequencing and analysis of ESBL-encoding gene and upstream region
The sequence of a 1.4 kb fragment containing blaCTX-M and its upstream region showed a strict identity with the CTX-M-2-encoding gene. The deduced protein has a single amino acid shift (Arg-246Ser) compared with Toho-1 (99% identity). According to a theoretical 3D model of CTX-M-2 against Toho-1 (not shown), this shift would not influence the catalytic activity, and similar kinetic properties are expected. Moreover, these two cefotaximases are presumably derived from chromosome-encoded ß-lactamases from infrequent human pathogens, such as Kluyvera species.12
A putative promoter was predicted upstream of blaCTX-M-2 in pM16, in good agreement with the experimental evidence for a blaCTX-M-2 promoter in a Salmonella enterica strain (J. Di Conza, unpublished results). This 506 bp upstream region could be divided into a proximal 266 bp segment having 97% identity with an equivalent region upstream of Kluyvera ascorbata kluA-1 (AJ272538), reinforcing the hypothesis of their phylogenic relationship, and a distal 240 bp segment, showing strict identity with the CR, reported in In6/In7 integrons,4 including InS21 and In35, among others.7,8 The last 17 bp of this 240 bp region were postulated as a putative recombination site for insertion of resistance genes.13 It is worth noting the absence of ISEcp1 in M. morganii PP16, associated with blaCTX-M-9 and blaCTX-M-1 sub-families,14,15 including a chromosome-encoded blaCTX-M-3 from a K. ascorbata strain.16
These features suggest the acquisition of blaCTX-M-2 via non-IS-mediated mechanisms, although the presence of a hypothetical recombination site could indicate an earlier transposition, followed by the loss of the IS, probably by recombination events.
Detection and analysis of class 1 integron elements
Typical elements of class 1 integrons were detected by PCR and sequenced: intI1 from 5'-CS, sul1 and qacE1 from 3'-CS, and a 2 kb variable region (Figure 1a), including the following gene cassettes: aacA4-cassette, encoding an aminoglycoside acetyltransferase, which probably mediates the aminoglycoside resistance in this strain;9
blaOXA-2-cassette, encoding an OXA-2 ß-lactamase; and an open reading frame (orfD) of unknown function. Notably, this gene-cassette arrangement was also observed in four other M. morganii strains (data not shown), in InS21 and In35,7,8
and in a Klebsiella pneumoniae isolate from Uruguay (R. Vignoli, Universidad de la República, Montevideo, personal communication), suggesting a common origin of these integrons.
Description of In116 architecture
The map of In116, obtained by a PCR-based strategy and sequencing, is showed in Figure 1(a). In116 has 99.7% identity with InS21, and a similar structure to the In6/In7 family, reinforcing the idea of their common origin and probably a recent dissemination. Twenty nucleotide differences were detected in In116, compared with InS21, including the TGAACT 10 consensus sequence in the PANT promoter (TAAACT in InS21).
A 2.1 kb segment having 99% identity with the CR from In6/In7-type integrons was present downstream of 3'-CS.3,4,7,8 This extension includes an orf513, followed by a 28 bp conserved sequence containing a putative 17 bp signal for the hypothetical recombinase Orf513, which probably leads to the incorporation of resistance genes (Figure 1b). A resistance gene, depending on the integron, is located downstream of the 28 bp region: blaCTX-M-2 (In116), catA2 (In6),4 dfrA10 (In7),3 blaDHA-1 (pSAL-1 integron),5 blaCTX-M-9 (In60),6 etc. (Figure 1b). Notably, associated resistance genes and flanking sequences from In116 and the pSAL-1 integron have high homology with chromosomal DNA from other species: K. ascorbata and M. morganii, respectively. A 9699% identity between blaCTX-M-2 and flanking sequences with a homologous sequence including K. ascorbata kluA-1 suggests their incorporation was probably Orf513-mediated and used a 28 bp target sequence. This feature endorses the hypothesis of the chromosomal origin of plasmid-borne ß-lactamases, probably mobilized from different microorganisms to more promiscuous structures (integrons, transposons or plasmids).
As in In6/In7, In116 showed a partial duplication of 3'-CS (3'-CS2), lacking the first 70 bp. A 3' 28 bp deletion also occurred in orf3, probably allowing the continuity of orf3 and qacE1 from 3'-CS2, thus encoding a putative fusion protein, Orf3::QacE
1. These deletions (variable depending on the integron, Figure 1b) might have participated in recombination events implicated in the acquisition of foreign associated resistance genes, probably replacing less useful regions of DNA.
In summary, pieces of evidence supporting the hypothesis of the incorporation of blaCTX-M-2 and flanking sequences from K. ascorbata in In116 are: (i) the absence of attC-like structures, and the presence of a putative recombinase-encoding gene (orf513) suggesting the acquisition of the resistance gene by a non-site-specific recombination mechanism; (ii) a partial deletion in 3'-CS2, probably associated with the incorporation of foreign DNA (see above); and (iii) the absence of IS-like structures (ISEcp1 or related) upstream of blaCTX-M-2, suggesting a different mechanism, or even an earlier transposon-mediated incorporation followed by a loss of any trace from that event. If the latter had happened, In116 and related integrons could be more recent than the IS-associated ones.
So far, only blaCTX-M and blaDHA-1 genes have directly been associated with chromosome-borne genes. We could hypothesize that perhaps other In6/In7-associated resistance genes have been derived from a variety of chromosomal sources, probably using a similar mechanism: an initial collection from parental microorganisms by a still unknown strategy (probably transposon- or phage-mediated), followed by the acquisition of the whole fragment in the integron environment, presumably using a recombinase (Orf513) activity and not fully determined target sequences.
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Acknowledgements |
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