One hundred four bacterial strains mediating urinary tract infections in separate individuals from a Uruguayan community were isolated. Forty-six strains conferred a multidrug resistance phenotype. All 104 strains were examined for the presence of class 1, 2, and 3 integrons. Class 1 integrons were found in 21 isolates across four distinct bacterial genera. A large class 1 integron in a Klebsiella pneumoniae strain was fully sequenced and was 29,093 bp in length. This integron probably arose by homologous recombination since it was embedded in a hybrid Tn21-like transposon backbone which comprised a Tn5036-like tnp transposition module at the IRi integron end and a Tn21 mer module at the IRt integron end. The parent integron/transposon that contributed the Tn5036 module was not related to Tn1696 since the integron insertion points in the transposon backbones were 16 bases apart. Examination of the other 20 class 1 integron-containing strains revealed further evidence of genetic exchange. This included a strain that possessed a Tn5036 module at the IRt end but not at the IRi end and another that possessed a tnp module beyond IRi that was a hybrid of Tn21 and Tn5051 and that is presumed to have arisen by site-specific recombination. This study highlights the ability of different genetic elements to act cooperatively to spread and rearrange antibiotic resistance in a community.
Homologous recombination between closely related gene cassettes, such as aadA1 and aadA2, which are 89% identical, can create hybrid cassettes and hybrids of existing cassette arrays. A new cassette array, dfrA12-orfF-aadA8b, which was created by such a recombination event occurring within the aadA2 cassette in the dfrA12-orfF-aadA2 array, has been identified.Gene cassettes are a major source of the resistance genes found in clinical, commensal, and environmental isolates of bacteria that are resistant to antibiotics. Most commonly, they are found in association with class 1 or class 2 integrons (3, 13). One growing group of gene cassettes encodes aminoglycoside (3Ј) (9) adenylyltransferases that confer resistance to both streptomycin and spectinomycin (9). The genes and the cassettes, which are named after the genes, are designated aadA with an Arabic numeral to distinguish distinct genes, namely, those that differ by at least 2% in both the DNA and protein sequences. Two of these cassettes, aadA1 and aadA2, were present in two of the earliest-known plasmids that confer resistance to multiple antibiotics, namely, NR1, also called R100 (7), and pSa (1, 14), and remain very common in modern-day isolates. Though they have both been found in various contexts, they recur in a few specific cassette arrays, e.g., aadA1 or aadA2 alone, oxa1-aadA1, dfrA1-aadA1, and dfrA12-orfFaadA2, which appear to have become globally disseminated. These two cassettes have now been sequenced many times, leading to the identification of several variant sequences for each of them, each containing one or a few single base changes ( Fig. 1; also Fig. 3 in reference 9). The high level of similarity between the aadA1 and aadA2 cassette sequences (89.3% DNA identity; cassette length, 856 bp) means that they share many stretches of sequence identity that allow homologous recombination between them to occur. Several hybrids between aadA1 and aadA2 that presumably arose by homologous recombination have already been reported (Table 1). By combining existing knowledge of variant sequences and of cassette arrays, it is potentially possible to track the movement of specific sets of gene cassettes within bacterial populations and identify events that have been involved in their creation and dissemination.We used primers located in the 5Ј-and 3Ј-conserved segments (CS) of class 1 integrons (HS458, 5Ј-GTTTGATGT TATGGAGCAGCAACG-3Ј, and HS459, 5Ј-GCAAAAAGG CAGCAATTATGAGCC-3Ј [5]) to screen DNA isolated from mixed bacterial samples recovered from the feces of human volunteers with no recent exposure to antibiotics. Mixed cultures were grown in L broth at 37°C under aerobic conditions, and the organisms recovered were predominantly Escherichia coli. DNA was recovered using alkaline lysis, and PCR conditions were as follows: denaturation at 94°C for 3.0 min; 35 cycles at 94°C for 30 s, 65°C for 1.0 min, and 72°C for 1.5 min or 4.0 min in the final cycle. A significant proportion of the samples screened yielded an amplicon of 2.2 kb corresponding to a casset...
Integrons are genetic elements that can capture and express genes packaged as gene cassettes. Here we report new methods that allow integrons to be studied and manipulated in their native bacterial hosts. Synthetic gene cassettes encoding gentamicin resistance (aadB) and green fluorescence (gfp), or lactose metabolism (lacZY), were made by PCR and self-ligation, converted to large tandem arrays by multiple displacement amplification, and introduced into Escherichia coli or Pseudomonas stutzeri strains via electroporation or natural transformation. Recombinants (Gm R or Lac + ) were obtained at frequencies ranging from 10 1 to 10 6 c.f.u. (mg DNA) "1 .Cassettes were integrated by site-specific recombination at the integron attI site in nearly all cases examined (370/384), including both promoterless and promoter-containing cassettes. Fluorometric analysis of gfp-containing recombinants revealed that expression levels from the integron-associated promoter P C were five-to 10-fold higher in the plasmid-borne integron In3 compared with the P. stutzeri chromosomal integrons. Integration of lacZY cassettes into P. stutzeri integrons allowed the bacteria to grow on lactose, and the lacZY gene cassette was stably maintained in the absence of selection. This study is believed to be the first to show natural transformation by gene cassettes, and integron-mediated capture of catabolic gene cassettes. INTRODUCTIONMultiple antibiotic resistance in bacteria is a problem for human health (Davies & Davies, 2010), and is associated with the actions of mobile genetic elements (MGEs) such as plasmids, transposons and integrons (Frost et al., 2005). Integrons can assemble resistance genes from diverse sources at a single genetic locus (Hall & Collis, 1998;Mazel, 2006; Schlüter et al., 2007;Stokes & Hall, 1989; Gillings et al., 2008). The integron consists of the intI gene encoding integrase, an attachment site (attI), a promoter (P c ), and an array of mobile gene cassettes (Nunes-Düby et al., 1998;Recchia & Hall, 1997;Stokes & Hall, 1989; Lévesque et al., 1994;Jové et al., 2010). The cassettes typically consist of a single promoterless ORF and a recombination site (attC). The integron integrase recombines attC with attI, or attC with attC, enabling both integration and excision of gene cassettes, which shift between free circular and linear integrated forms. Integrons are common in bacteria, and diverse in their phylogeny and predicted functions (Boucher et al., 2007a; Elsaied et al., 2007;Gillings et al., 2005;Holmes et al., 2003a;Nield et al., 2001;Rowe-Magnus & Mazel, 2001;Rowe-Magnus et al., 2003). Integrons increasingly seem to have a role as general-purpose agents of adaptation, not just enablers of antibiotic resistance (Koenig et al., 2009(Koenig et al., , 2011Rosewarne et al., 2010;Wright et al., 2008).Previous studies have used excision assays (Collis & Hall, 1992;Drouin et al., 2002; Léon & Roy, 2003) or cointegrate assays (Biskri et al., 2005;Collis et al., 2002Collis et al., , 2001Holmes et al., 2003b;Martinez &...
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