RESEARCH NOTE
José Di Conza1 Juan A. Ayala2* Ayelén Porto1 Marta Mollerach3 Gabriel Gutkind3 1Fac. of Biochem. & Biological Sciences (UNL), Santa Fe, Argentina 2Center for Molecular Biology "Severo Ochoa" (CSIC-UAM) Cantoblanco, Madrid, Spain 3Fac. of Pharmacy and Biochemistry (UBA), Buenos Aires, Argentina	Molecular characterization of InJR06, a class 1 integron located in a conjugative plasmid of Salmonella enterica ser. Typhimurium   Summary. The presence of class 1, 2, and 3 integrons was investigated in four pediatric isolates of Salmonella enterica ser. Typhimurium (S. Typhimurium). A class 1 integron was detected in one S. Typhimurium strain, the only one that also showed resistance to various aminoglycoside antibiotics. This integron, called InJR06, and the aminoglycoside resistance determinants were located in pS06, a large (≥ 55 kb) conjugative plasmid. A single mobile cassette (encoding the aminoglycoside adenylyltransferase ANT(3´´)-Ia) was detected in the variable region of InJR06, while the architecture of the attI1 and attC sites was conserved. [Int Microbiol 2005; 8(4):287-290] Key words: Salmonella Typhimurium · class 1 integron · aminoglycoside · ant(3´´)-Ia · gene cassette
Received 14 April 2005 Accepted 15 October 2005   *Corresponding author: Tel. +34-914978083. Fax +34-914978087 E-mail: jayala@cbm.uam.es

 

Caracterización molecular de InJR06, un integron de clase 1 situado en un plásmido conjugativo de Salmonella enterica ser. Typhimurium Resumen. Se investigó la presencia de integrones de clase 1, 2 y 3 en cuatro aislamientos pediátricos de Salmonella enterica ser. Typhimurium (S. Typhimurium). Un integrón de clase 1 se detectó en una cepa de S. Typhimurium, la única que además presentaba resistencia a varios antibióticos aminoglucósidos. Este integrón, llamado InJR06, y los determinantes de resistencia a aminoglucósidos se localizaron en pS06, un plásmido conjugativo de tamaño grande (≥ 55 kb). El análisis de la región variable de InJR06 mostró que un casete génico codifica la aminoglucósido adeniltransferasa ANT(3´´)-Ia y que la arquitectura de los sitios attI1 y attC está conservada. [Int Microbiol 2005; 8(4):287-290] Palabras clave: Salmonella Typhimurium · integrón de clase 1 · aminoglucósidos · ant(3´´)-Ia · casete génico

Caracterização molecular de InJR06, um integron de classe 1 situado em um plásmido conjugativo de Salmonela enterica ser. Typhimurium Resumo. Se averiguou a presença de integrones de classe 1, 2 e 3 em quatro isolamentos pediátricos de Salmonela enterica ser. Typhimurium (S. Typhimurium). Um integrón de classe 1 se pôde detectar em uma cepa de S. Typhimurium, a única que além disso apresentava resistência a vários antibióticos aminoglucósidos. Este integrón, chamado InJR06, e os determinantes de resistência a aminoglucósidos se localizaram em pS06, um plásmido conjugativo de tamanho grande (≥ 55 kb). A análise da região variável de InJR06 mostrou que um cassete genético codifica a aminoglucósido adeniltransferasa ANT(3´´)-Ia e que a arquitetura dos lugares attI1 e attC está conservada. [Int Microbiol 2005; 8(4):287-290] Palavras chiave: Salmonella Typhimurium · integrón de classe 1 · aminoglucósidos · ant(3´´)-Ia · cassete genético

 

Introduction

Integrons are genetic elements that can integrate gene cassettes, usually antibiotic resistance genes, by site-specifc recombination [28]. To date, ten integron classes have been identified and, among them, class 1, 2, and 3 integrons have been implicated in antibiotic resistance [5,23]. All integrons have a 5´ conserved segment (5´-CS), including an intI gene encoding an integrase and an attI recombination site, but they have distinct 3´ conserved segments (3´-CS). However, integrons most frequently found in resistant clinical isolates of members of the Enterobacteriaceae belong to class 1 [2,8,31]. In class 1 integrons, which is the only well-characterized group, a specific recombination site, attI1, is located next to the intI1 gene and is recognized by the IntI1 integrase, and a promoter, Pc, which directs transcription of the cassette-borne genes, lies within the intI1 gene [4,28].

The 3´ conserved segment of class 1 integrons includes qacEΔ1, a deletion derivate of the antiseptic resistance gene qacE, and the sul1 gene, which encodes sulfonamide resistance [11,21]. The attC site (59-base element) is usually found associated with a single open reading frame in a structure termed a gene cassette. More than 70 different gene cassettes encoding proteins that confer resistance to many antibiotic families have been characterized within integrons so far; i.e., enzymes that inactivate antibiotics (β-lactams, aminoglycosides, chloramphenicol), metabolic by-passes (trimetoprim), and efflux systems (chloramphenicol) [12,22]. Most of the attC sites of integron-associated resistance gene cassettes identified to date share only slight homologies. Their lengths and sequences vary considerably (from 51 bp to 141 bp) and their sequence similarities are primarily restricted to their 7-bp boundaries, which correspond to the inverse core site (RYYYAAC) and the core site (G↓TTRRRY, where R is a purine, Y is a pyrimidine, and the arrow shows the recombination point) [3,29].

Integrons can be considered as natural cloning and expression systems; since their genetic flexibility allows numerous cassette rearrangements under selective pressure. The study of these assortments can lead to a better understanding of multidrug-resistant strain evolution [24]. Nontyphoid Salmonella infections are increasingly common, appear with variable geographical incidence, and are often resistant to multiple antibiotics [25]. The aim of our study was to investigate the presence of the three classes of integrons in S. Typhimurium clinical isolates to characterize their gene cassette assortment.

Material and methods

Bacterial strains. Four Salmonella enterica serovar Typhimurium strains were isolated in 1997 from the stool and blood of children at the Ricardo Gutiérrez Hospital, Santa Fe, Argentina.

Susceptibility testing. Antibiotic susceptibility (of both the clinical isolates and the Escherichia coli transconjugants) was calculated by the disk diffusion method on Mueller-Hinton (MH) agar according to NCCLS (National Committee for Clinical Laboratory Standards, USA) recommendations [18]. Antimicrobial agents tested were ampicillin, cephalotin, cefotaxime, sulfisoxazole, tetracycline, chloramphenicol, nalidixic acid, gentamicin, amikacin, kanamycin, netilmicin, tobramycin, and streptomycin (Britania, Argentina). The minimum inhibitory concentration (MIC) of selected aminoglycosides was then determined by the agar dilution technique on MH agar plates with an inoculum of 10⁴ CFU per spot according to NCCLS recommendations [17].

Plasmid content and conjugation. Plasmid DNA from S. Typhimurium S06 (pS06) was extracted and analyzed as described by Sambrook et al. [26]. Plasmid size was estimated using Escherichia coli V517 as a reference [15]. Conjugative mobilization of pS06 was attempted by liquid-medium mating [26] to E. coli CAG12177 (M. Berlyn, E. coli Genetic Stock Center). Selection was carried out on Luria-Bertani (LB) agar plates containing gentamicin (30 mg/l) and tetracycline (20 mg/l).

PCR amplification. Architecture of the class 1 integron was determined by PCR mapping using different primer combinations (see Fig. 1) [7]. PCR amplification of intI2 and intI3 was done employing specific primers [9].

Cloning and DNA sequencing. The PCR product was ligated into pGEM-T vector (pGEM-T easy vector system I, Promega, USA) and transformed into E. coli TOP10F" (Invitrogen, Holland). Bacteria harboring recombinant plasmids were selected on LB agar plate containing ampicillin (100 mg/l), IPTG (1 mM), and X-Gal (40 mg/l). Two recombinant plasmids were sequenced in both strands by the automated Sanger method [27]. Sequences were analyzed with the NCBI [http://www.ncbi.nlm.nih.gov/] tools.

Nucleotide sequence accession number. The nucleotide sequence data reported in this work are available from the EMBL database (EBI, European Bioinformatics Institute) under the accession number AJ496285.

Results and Discussion

One of the four S. Typhimurium strains (S06) showed decreased susceptibility to sulfisoxazole and the aminoglycosides gentamicin, kanamycin, tobramycin, and streptomycin, but not to β-lactams (Table 1). Gentamicin and amikacin MICs were 128 mg/l and 1 mg/l, respectively.

The intI2 and intI3 genes were not detected in any of these isolates. Typical elements of class 1 integron were detected by PCR only in S06: intI1 from 5´-CS, qacEΔ1 and sul1 from 3´-CS, and only one fragment of 1 kb corresponding to the variable region. The map of this class 1 integron (called InJR06), obtained by a PCR-based strategy and sequencing, is shown in Fig. 1A.

A larger, 55-kb plasmid (pS06) was detected in S06 and transferred to a recipient E. coli strain by liquid-medium conjugation. The transconjugant (called CAG06) strain had reduced susceptibility to aminoglycosides and sulfisoxazole. As expected, fragments corresponding to intI1, qacEΔ1, and sul1 genes were amplified by PCR in the CAG06 strain (Fig. 1B). These findings clearly show that InJR06-class 1 integron and the aminoglycoside-resistant determinants are associated and are located in pS06 conjugative plasmid.

The 1-kb variable region PCR product was cloned and sequenced. Sequence analysis of the 1009-bp fragment revealed two different regions: (i) a 5´ segment, of approximately 100 bp, where the attI1 site included into the 5´-CS of class 1 integrons is identified (Fig. 2A), and (ii) an open reading frame that is 100% identical to the ant(3´´)-Ia gene (also called aadA1a gene) followed by an attC site (Fig. 2B). This aadA1a-integrated cassette encodes a 3´´ aminoglycoside adenylyltransferase (AadA1a protein) associated with streptomycin and spectinomycin resistance. Sequence analysis of the variable region showed that a recombination site (attI1) is present between nucleotides 46 and 103 (Fig. 2A). As described by Partridge et al. [20], the last 38 nucleotides correspond to the minimal size for the insertion of a new gene cassette. The core sites of the directed repeat regions (DR1 and DR2), the IntI1 binding site (simple site), and the recombination crossover point are shown in Fig. 2A

The 60-bp attC site of aadA1a cassette has the typical configuration of two simple sites (LH and RH) separated by a central region, as shown in Fig. 2B [29]. Each simple site contains a pair of inversely oriented core sites whose consensus sequences are RYYYAAC and GTTRRRY. These core sites are designated 1L and 2L at the left-hand (LH) end and 2R and 1R at the right-hand (RH) end. The IntI1 integrase binds in these simple sites and recombination crossover occurs between the G and the first T in the 1R core site [29].

It is already known that pS06 is a conjugative plasmid carrying a class 1 integron containing a single aadA1a cassette. Although this cassette encodes a 263-amino-acid adenylyltransferase associated with both streptomycin and spectinomycin resistance, we found no data relating AadA enzymes with the resistance profile to other aminoglycosides. Therefore, according to the results described in this work, we assumed that pS06 harbors another aminoglycoside resistance mechanism. The aadA1a gene has been described previously as part of Tn1331 or as a gene cassette of unusual class 1 integrons (bearing orf513 and a partial duplication of 3´-CS), both present in different Enterobacteriaceae [1,16,19]. However, these unusual class 1 integrons harbor the bla_CTX-M-2-β-lactamase gene in their architecture; therefore, InJR06 could be a precursor of them (bla_CTX-M-2 gene was not detected in S06; Di Conza J, PhD thesis).

The detection of typical attI1 and attC sequences adjacent to the aadA1a gene suggests that a new gene cassette can be integrated by site-specific recombination into the variable region of these genetic elements. Thus, the accumulation of resistance genes by integrons is a plausible explanation for the emergence of multiple resistant strains, and their location in a conjugative plasmid can contribute to the widespread dissemination of antibiotic resistance. An increasing number of cases of uptake of resistant genes by integrons has been reported in the last few years [6,10,13,14,30]. In addition, PCR mapping of integrons can be a useful epidemiological tool to study the evolution of multiresistance plasmids and transposons.

Acknowledgements. This work was supported in part by grants from the University of Buenos Aires, Argentina (TB 039), CONICET (PID 4413), and SEPCYT (PICT 0693) to GG, grants from the Fundación A. J. Roemmers to JDC and grants from the Ministerio de Ciencia y Tecnología, Spain (BMC-2001-2346), and the European Union (LSHM-CT-2003-503335) to JAA. GG is a member of Carrera de Investigador Científico, CONICET, Argentina.

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