Recombination between the dfrA12 -orfF- aadA2 Cassette Array and an aadA1 Gene Cassette Creates a Hybrid Cassette, aadA8b

Abstract

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...

Dissemination and Persistence of bla CTX-M-9 Are Linked to Class 1 Integrons Containing CR1 Associated with Defective Transposon Derivatives from Tn 402 Located in Early Antibiotic Resistance Plasmids of IncHI2, IncP1-α, and IncFI Groups

“…Plasticity of gene cassettes was also observed. The dfrA12-orfX-aadA8b array from our E79 isolate seems to have arisen by recombination events involving the aadA2 gene from the globally disseminated dfrA12-orfX-aadA2 array first described in Finland in 1969 (15). Our results are in agreement with other studies and show that the propensity for recombination and genetic exchange in nature seems to be more frequent than previously believed, playing a relevant role in the adaptation of specific plasmids to different cellular environments (9,29).…”

“…1. A high degree of homology was found among sequences from all of the identified gene cassette arrays: dfrA16-aadA2 (indistinguishable among all our isolates and those carrying qnr from China and North America, GenBank accession number AY259085), aadA1 (indistinguishable from that of the worldwide disseminated Tn21, GenBank accession number AF071413), and dfrA12-orfX-aadA8b from our E79 isolate (identical to that described in fecal isolates from Australia and clinical isolates from the United Kingdom) (15,21,45) (GenBank accession number AM040708). Analysis of the integrase intI1 of one representative isolate of each integron type revealed the presence of P c promoter sequences that correspond to the weak and intermediate versions of the P c promoter (33).…”

SGI2, a Relative ofSalmonellaGenomic Island SGI1 with an Independent Origin

“…However, the new segment is not derived from cmlA9 (not shown). Hence, it is clear that recombination is at least partly responsible for the generation of diversity in the cmlA (floR) genes, as previously found for the aadA1 and aadA2 genes (19).…”

Associations between Antimicrobial Resistance Genes in Fecal Generic Escherichia coli Isolates from Cow-Calf Herds in Western Canada

“…Although an integron probe was not included, the sul1 gene is an integral part of the conserved region of class I integrons. In addition, the ant(3Љ)-Ia (aadA1) gene and several different dhfr genes have been shown to be present frequently in the variable regions of integrons (6,11,14,24). Despite the lack of testing specifically for integrons, the various patterns consisting of two or more of these genes together with sul1 suggested the presence of integrons in this sample population.…”