59-base Element Research Articles

Occurrence and Characteristics of Class 1 and 2 Integrons in Clinical Bacterial Isolates from Patients in South China

Recent studies have shown that integrons play a major role in spreading antibiotic resistance genes in clinical settings, as antibiotic resistance genes are frequently found located at gene cassettes. Polymerase chain reaction (PCR) analyses were carried out for the detection of the integrase genes of the three classes of integrons, and their gene cassettes were characterized in 118 clinical strains, including both gram-positive and gram-negative bacteria, isolated from a hospital in Guangzhou, China during 2004. Class 1 and 2 integrons were detected in 76.3% (90/118) and 0.8% (1/118) of the tested clinical isolates, respectively. Moreover four isolates were positive for both the integrons and no class 3 was detected. Some of them were first reported, such as Enterococcus faecalis, Enterococcus faecium, Alcaligenes sp., and Flavobacterium sp. Seven different arrays of gene cassettes of class 1 integron were found, and a high prevalence of dfrA12-orfF-aadA2 genes was observed. In addition, we identified a new none-open reading frame (ORF) cassette in a class 1 integron positive clinical coagulase-negative Staphylococcus strain GH69. The none-ORF cassette, a 700 bp sequence containing a 441 bp ORF and a 59-base element, is first characterized in this study. All class 2 integrons carried an array of gene cassettes dfrA1-sat2-aadA1. Moreover bacteria contain more than one integrons were also found in this study. Resistance to trimethoprim-sulfamethoxazole was frequently found to be encoded within integrons. Therefore, it is important that guidelines for the prudent use of antimicrobial agents are adopted and surveillance programs are established.

Molecular analysis of multiresistant porcine Salmonella enterica subsp. enterica serovar Bredeney isolates from Southern Brazil: identification of resistance genes, integrons and a group II intron

The relationships of 83 porcine Salmonella enterica subsp. enterica serovar Bredeney isolates obtained at two slaughterhouses in Southern Brazil were analysed by XbaI and BlnI macrorestriction analysis, plasmid profiling and determination of antimicrobial resistance patterns. Twenty-nine XbaI and 30 BlnI macrorestriction patterns were identified. The 72 plasmid-bearing isolates exhibited 20 different plasmid profiles. Multiresistance was detected in 49 isolates (59%), of which 39 isolates showed at least resistance to sulfonamides, tetracyclines, chloramphenicol, streptomycin, kanamycin and/or ampicillin. A representative subset of 12 isolates was chosen for identification of resistance genes, their localisation and transferability. The sulfonamide resistance genes sul1, sul2 and sul3, the tetracycline resistance genes tet(A) and tet(B), the phenicol resistance genes catA1 and floR, the streptomycin resistance gene strA, the kanamycin resistance gene aphA1 and the ampicillin resistance gene bla TEM were detected and found to be located most frequently on plasmids. In addition, class 1 and 2 integrons with the cassette arrangements dfrA21/ bla OXA-129/ aadA1 and dfrA1/ sat1/ aadA1, respectively, were detected. A group II intron was found to be inserted into the 59-base element of an aadA1 gene cassette in a class 1 integron. This study revealed a wide genomic variety among the S. Bredeney isolates, and the high number of multiresistant isolates may point towards the risks that these S. Bredeney isolates can represent to human health.

Genetic and biochemical characterization of GES-5, an extended-spectrum class A β-lactamase from Klebsiella pneumoniae

The present study was conducted to characterize a new class 1 integron containing the bla GES-5 gene cassette in Klebsiella pneumoniae clinical isolate CHAK36 and measure the kinetic parameters of GES-5 β-lactamase. Long-range polymerase chain reactions (PCRs) and sequence analysis were performed to identify and analyze the bla GES-5 gene cassette-containing integrons. Kinetic parameters were determined from purified GES-5. Sequencing of the 6190-bp PCR amplicon from K. pneumoniae CHAK36 isolate revealed the new structure of class 1 integron. The integron has 3 unique gene cassettes ( bla GES-5– aac(6′)-IIa– bla OXA-17/ orf4), but the 59-base element of the bla OXA-17 gene cassette was interrupted by a putative transposase gene, orf4. The kinetic parameters of GES-5 showed its broad-spectrum activity against most β-lactams, including benzylpenicillin, cefaloridine, cefotaxime, and imipenem. This work shows that the bla GES-5 gene was located on a new class 1 integron as a gene cassette. Our kinetic characterizations show that GES-5 was more active against impenem than GES-2 and GES-4.

LinG , a New Integron-Associated Gene Cassette Encoding a Lincosamide Nucleotidyltransferase

Lincosamide antibiotics include lincomycin, a compound produced by several actinomycetes, and its semisynthetic chlorinated derivative clindamycin. These antibiotics block the peptidyltransferase activity of the 50S subunit of the bacterial ribosome, inhibiting protein synthesis, and are active against most gram-positive cocci and anaerobes. However, they are not generally effective against gram-negative bacilli due to intrinsic resistance (3, 5). Resistance to lincosamides is most commonly due to N6 dimethylation of an adenine residue in the 23S rRNA, which usually confers broad-spectrum cross-resistance to macrolides, lincosamides, and streptogramin B antibiotics or to efflux (5). However, antibiotic inactivation by nucleotidylation (1, 2) has also been described as a mechanism of resistance. Despite the fact that lincosamides are not used to treat enterobacterial infections, a gene, linF, that confers low levels of resistance to both lincomycin (fourfold) and clindamycin (twofold) was recently found in a gene cassette in a class 1 integron recovered from an Escherichia coli blood isolate (4). The linF gene encodes a 273-amino-acid lincosamide nucleotidyltransferase. The LinF protein shares approximately 35% identity with the nucleotidyltransferases encoded by the linB gene from Enterococcus faecium (1) (GenBank accession no. {type:entrez-nucleotide,attrs:{text:AF110130,term_id:4633124}}AF110130) and linB′ from Enterococcus faecalis (GenBank accession no. {type:entrez-nucleotide,attrs:{text:AF408195,term_id:15667874}}AF408195). We have identified a second lin gene in a gene cassette. This cassette was recovered from a multiply antibiotic-resistant Salmonella enterica serovar Stanley strain (SRC54) isolated in 2001 from a traveler who had recently returned from Thailand. This strain was resistant to chloramphenicol, gentamicin, kanamycin, spectinomycin, streptomycin, sulfathiazole, and tetracycline but susceptible to ampicillin and nalidixic acid at levels described previously (6). It displayed intermediate resistance to ciprofloxacin. The gene cassette array was amplified by using standard primers (L2 and R1) in the 5′ conserved sequence and 3′ conserved sequence of class 1 integrons, and the 2.25-kb amplicon was cloned into pPCRscript and sequenced as previously described (see reference 6 for primer details). E. coli strain DH5α containing pPCR-Script with the cassette array was at least 10-fold more resistant to lincomycin (MIC, ≥2,000 μg/ml) than DH5α containing only pPCR-Script (MIC, 180 μg/ml). The first cassette in the array was identical to the aadA2 cassette in GenBank accession no. {type:entrez-nucleotide,attrs:{text:L06822,term_id:56786619}}L06822. The second cassette was 937 bp long and 93.4% identical to the linF gene cassette. It encoded a 273-amino-acid protein that is 93.1% identical to LinF (17 amino acid differences). An alignment of these proteins with LinB is shown in Fig. ​Fig.1A.1A. The sequence of the aadA2-linG cassette array was identical to a region found in GenBank accession no. {type:entrez-nucleotide,attrs:{text:AY522431,term_id:116294654}}AY522431. The 59-base elements (59-be; attC sites) of the linG and linF cassettes are 58 bp long (Fig. ​(Fig.1B)1B) and not closely related to any other known 59-be. They retain the critical features of 59-be, namely, complementary sites 1L-1R and 2L-2R (7). FIG. 1. Analysis of LinG sequences. (A) Alignment of Lin proteins. Amino acids that are completely conserved across all sequences are shown as white letters on a black background. The protein sequences of LinB and LinF were from GenBank accession numbers {type:entrez-nucleotide,attrs:{text:AF110130,term_id:4633124}} ...

Characterization of acquired -lactamases and their genetic support in multidrug-resistant Pseudomonas aeruginosa isolates in Taiwan: the prevalence of unusual integrons

The present study was conducted to investigate acquired beta-lactamases and their genetic support in 26 Pseudomonas aeruginosa isolates that were resistant to nearly all antipseudomonal drugs from six medical centres in Taiwan. Acquired beta-lactamases and their genetic support were determined by PCR-based strategies. Four and 16 of the 26 isolates were found to produce VIM-2 and VIM-3 metallo-beta-lactamases (MBLs), respectively, and 1, 1 and 2 isolates produced OXA-17, OXA-10 and PSE-1, respectively. These bla genes are all in class 1 integrons that are probably chromosomally located. The bla(VIM-3)-containing integron, with a deletion between int1 and the bla(VIM-3) structural gene, has six gene cassettes, bla(VIM-3), a probable fosfomycin resistance determinant, aacA4, aacA4, aadB and aacA4. The bla(VIM-2)-containing integron, without detectable 5'-conserved segment, contains four genes cassettes (aacA7-bla(VIM-2)-dhfr-aacA5) and is ended by tniC. The bla(OXA-10)-containing integron includes a catB3 cassette and a fused gene cassette, which is made up of bla(OXA-17) and a novel streptomycin-spectinomycin gene, designated aadA15. The bla(OXA-17)-containing integron has three gene cassettes (aacA4-catB2-bla(OXA-17)) but the 59-base element of the bla(OXA-17) cassette is interrupted by a putative transposase gene. The bla(PSE-1)-containing integron has three gene cassettes, aacA4, an aadA3-related gene designated aadA3b and bla(PSE-1). PFGE revealed genetic diversity among the multidrug-resistant isolates from different hospitals. This study demonstrated the high prevalence of VIM-type MBLs and the presence of unusual bla-encoding integrons in multidrug-resistant P. aeruginosa isolates in Taiwan. The spread of bla(VIM-2)-related genes by horizontal transfer might have occurred.

New Array of aacA4-catB3-dfrA1 Gene Cassettes and a Noncoding Cassette from a Class-1-Integron-Positive Clinical Strain of Pseudomonas aeruginosa

Pseudomonas aeruginosa is an opportunistic human pathogen that causes nosocomial infections. This bacterium is characterized by inherent resistance to a wide variety of antimicrobials; the inherent resistance is always mediated by antibiotic resistance genes (3, 10). A genetic element, the integron, is potentially a major agent in the dissemination of multidrug resistance among gram-negative bacteria, especially enteric bacteria and Pseudomonas (2, 6). Class 1 integrons are predominant among integrons that carry resistance cassettes (11, 12). The goal of our study was to analyze the gene cassettes and a noncoding cassette in a class-1-integron-positive clinical Pseudomonas aeruginosa strain isolated from a sputum specimen of a respiratory disease patient in Hebei Province, People's Republic of China. P. aeruginosa strain P11 was screened for antimicrobial susceptibility by using the disk agar dilution method (7). Results of antimicrobial susceptibility testing showed that strain P11 was resistant to several antibiotics, including cefaclor, cefazolin, cefuroxime, chloramphenicol, gentamicin, trimethoprim-sulfamethoxazole, tetracycline, and tobramycin. The intI1 gene, an integron-encoded integrase gene, was amplified by primers specific for intI1 (intM1-U [5′-ACGAGCGCAAGGTTTCGGT-3′] and intM1-D [5′-GAAAGGTCTGGTCATACATG-3′]). The variable region of the integron was determined to be 2,655 bp by PCR with primers specific for the variable region between the 5′ conserved sequence (5′-CS) (in-F [5′-GGCATCCAAGCAGCAAGC-3′]) and the 3′-CS (in-B [5′-AAGCAGACTTGACCTGAT-3′]) (8). The 3′-CS of the class 1 integron was also specifically amplified by primers (qacEΔ1-F [5′-ATCGCAATAGTTGGCGAAGT-3′] and sul1-B [5′-GCAAGGCGGAAACCCGCGCC-3′]) (5), indicating that the 3′-CS of the class 1 integron possessed the qacEΔ1 and sul1 genes. In order to identify the gene cassettes harbored in this integron, the 2,655-bp amplicon was purified by using the QIAquick PCR purification kit (QIAGEN, Hilden, Germany) and cloned into the pMD18-T vector (TaKaRa, Japan). The sequencing was done by an ABI PRISM 310 genetic analyzer. Analysis of the sequence by BLASTX and BLASTN revealed that the 2,655-bp amplicon (GenBank accession number {type:entrez-nucleotide,attrs:{text:AB195796,term_id:56342375}}AB195796) contained aacA4, catB3, and dfrA1 gene cassettes and the noncoding cassette, a cassette without any open reading frames (Fig. ​(Fig.1).1). It is noteworthy that aacA4-catB3-dfrA1 is a novel rearrangement in the class 1 integron. Also, this noncoding cassette had no significant BLASTN hits and had not been reported before. Noncoding cassettes were reported in vibrios only recently (1), but the details of their structures were not analyzed. Interestingly, this noncoding cassette has many adenines and thymines repeated in the sequence. In generic gene cassettes, 59-base elements (59-be) are typical and important components, because they can serve as integration sites for IntI1-catalyzed site-specific recombination (4, 9). Downstream, this noncoding cassette also contained a 59-be with an imperfectly inverted repeat sequence that was 71 bp long. Despite the fact that the 59-be had not been reported in other gene cassettes, it contained features common to 59-base elements, such as 1L, 2L, 2R, and 1R (13). As reported, a few scattered attC sites, apparently not associated with structural genes and not making up parts of integrons, were found in some partially sequenced genomes (3). Therefore, it is possible that the 59-be of this cassette could originally be derived from the scattered attC sites. However, the sequence of this noncoding cassette has not been reported before, which makes the mechanism of this cassette formation questionable. FIG. 1. (A) Schematic representation of the 2,655-bp fragment of class 1 integron in P. aeruginosa strain P11. Cassettes are represented by arrowheads, and their 59-base elements are represented by ellipses. (B) Sequence of the noncoding cassette (boldface type). ... Our study provides a new array of aacA4-catB3-dfrA1 gene cassettes in a class 1 integron and analyzes the structure of a new noncoding cassette which offers information for the formation and evolution of cassettes harbored by integrons.

Recovery and evolutionary analysis of complete integron gene cassette arrays from Vibrio

BackgroundIntegrons are genetic elements capable of the acquisition, rearrangement and expression of genes contained in gene cassettes. Gene cassettes generally consist of a promoterless gene associated with a recombination site known as a 59-base element (59-be). Multiple insertion events can lead to the assembly of large integron-associated cassette arrays. The most striking examples are found in Vibrio, where such cassette arrays are widespread and can range from 30 kb to 150 kb. Besides those found in completely sequenced genomes, no such array has yet been recovered in its entirety. We describe an approach to systematically isolate, sequence and annotate large integron gene cassette arrays from bacterial strains.ResultsThe complete Vibrio sp. DAT722 integron cassette array was determined through the streamlined approach described here. To place it in an evolutionary context, we compare the DAT722 array to known vibrio arrays and performed phylogenetic analyses for all of its components (integrase, 59-be sites, gene cassette encoded genes). It differs extensively in terms of genomic context as well as gene cassette content and organization. The phylogenetic tree of the 59-be sites collectively found in the Vibrio gene cassette pool suggests frequent transfer of cassettes within and between Vibrio species, with slower transfer rates between more phylogenetically distant relatives. We also identify multiple cases where non-integron chromosomal genes seem to have been assembled into gene cassettes and others where cassettes have been inserted into chromosomal locations outside integrons.ConclusionOur systematic approach greatly facilitates the isolation and annotation of large integrons gene cassette arrays. Comparative analysis of the Vibrio sp. DAT722 integron obtained through this approach to those found in other vibrios confirms the role of this genetic element in promoting lateral gene transfer and suggests a high rate of gene gain/loss relative to most other loci on vibrio chromosomes. We identify a relationship between the phylogenetic distance separating two species and the rate at which they exchange gene cassettes, interactions between the non-mobile portion of bacterial genomes and the vibrio gene cassette pool as well as intragenomic translocation events of integrons in vibrios.

Identification of an aadA2 Gene Cassette from Salmonella enterica subsp. enterica Serovar Derby

During a study on Salmonella enterica subsp. enterica serovar Derby from slaughter-age pigs in Brazil, two epidemiologically unrelated multi-resistant S. Derby isolates were found to carry a class 1 integron with a single gene cassette. Sequence analysis confirmed that this gene cassette harboured an aadA2 gene. The aadA2 gene codes for an aminoglycoside adenyltransferase, which mediates resistance to the aminoglycoside streptomycin and the aminocyclitol spectinomycin. Although aadA2 gene cassettes are widely distributed among Salmonella, database searches identified an AadA2 protein indistinguishable from that of S. Derby only in single isolates of S. enterica subsp. enterica Enteritidis from France and S. enterica subsp. enterica Typhimurium from Japan. Structural analysis of the 59-base element revealed at least one base pair difference between the 59-base element of the aadA2 cassette from S. Derby and any of the 59-base elements deposited in the databases.

VIM-12, a Novel Plasmid-Mediated Metallo-β-Lactamase from Klebsiella pneumoniae That Resembles a VIM-1/VIM-2 Hybrid

A transferable plasmid from Klebsiella pneumoniae carried a class 1 integron containing bla(VIM-12), a novel bla(VIM)-type gene, flanked by two copies of aacA7. bla(VIM-12) was clustered between bla(VIM-1) and bla(VIM-2) and differed from bla(VIM-1) by 18 nucleotides that were all located at the 3' end and matched the corresponding nucleotides in bla(VIM-2). The bla(VIM-12)-associated 59-base element was identical to that described in bla(VIM-2) alleles.

The ORF1 Gene Located on the Class-1-Integron-Associated Gene Cassette Actually Represents a Novel Fosfomycin Resistance Determinant

The class 1 integrons are genetic elements capable of integrating gene cassettes by a site-specific recombination mechanism ([1][1]). Gene cassettes are mobile units composed of a gene, most often an antibiotic resistance gene, and a recombination site, the 59-base element ([1][1]). We have

Description of In116, the first blaCTX-M-2-containing complex class 1 integron found in Morganella morganii isolates from Buenos Aires, Argentina

We analysed the architecture and probable origin of a class 1 integron from cefotaxime-resistant Morganella morganii isolates. bla genes and class 1 integron elements were detected by PCR and DNA-DNA 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. 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 beta-lactamase. The variable region of In116 contains aacA4, bla(OXA-2) and orfD cassettes. Downstream of the 3'-conserved-segment (3'-CS), an orf513-containing common region is followed by bla(CTX-M-2) and flanking regions, having 96-99% 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 bla(CTX-M-2).

Gene Cassettes Potentially Encoding Fosfomycin Resistance Determinants

Gene cassettes are small mobile genetic elements that are found in arrays of one or more cassettes within an integron (5, 9). In addition to a single open reading frame, or sometimes two, each cassette contains a unique recombination site that belongs to a family of sites known as 59-be (59-base elements). The genes found in cassettes recovered from class 1 integrons found in plasmids or chromosomally integrated mobile regions are extremely diverse but include mainly genes that confer resistance to antibiotics, including aminoglycosides, β-lactams, chloramphenicol, trimethoprim, streptothricin, rifampin, lincomycin, and erythromycin, or resistance to antiseptics and disinfectants (5, 7, 9). However, a number of cassettes that include open reading frames whose function is not known have also been recovered from the same source. Our recent searches using the predicted sequences of the proteins encoded by cassette-encoded open reading frames revealed two that are significantly related to fosfomycin resistance proteins. An open reading frame designated ORF1, found in a cassette in a class 1 integron from Pseudomonas aeruginosa (GenBank accession no. {type:entrez-nucleotide,attrs:{text:AY294333,term_id:31559084,term_text:AY294333}}AY294333) (11), encodes a protein that is 52.3% identical to FosA, which is the product of a fosfomycin resistance determinant in the composite transposon Tn2921 found in Serratia marcescens ({type:entrez-nucleotide,attrs:{text:M85195,term_id:154988,term_text:M85195}}M85195) (8) and 40.5% identical to the FosB fosfomycin resistance proteins encoded by plasmids from Staphylococcus epidermidis ({type:entrez-nucleotide,attrs:{text:X54227,term_id:46980,term_text:X54227}}X54227) (12) and Staphylococcus haemolyticus ({type:entrez-nucleotide,attrs:{text:X89875,term_id:927563,term_text:X89875}}X89875) (1). The ORF1 product also exhibits 54.5 and 38.6% identity to proteins encoded in the chromosomes of P. aeruginosa PAO1 (PA1129 in {type:entrez-nucleotide,attrs:{text:AE004543,term_id:9947047,term_text:AE004543}}AE004543) and Bacillus subtilis (yndH in {type:entrez-nucleotide,attrs:{text:Z99113,term_id:32468758,term_text:Z99113}}Z99113) that have recently been cloned and shown to confer resistance to fosfomycin (3, 10). It is also more than 50% identical to predicted products of uncharacterized open reading frames found in the genomes of Chromobacterium violaceum (fofB/CV0163 in {type:entrez-nucleotide,attrs:{text:AE016910,term_id:34101311,term_text:AE016910}}AE016910) and Nostoc species ({type:entrez-nucleotide,attrs:{text:AP003588,term_id:17131110,term_text:AP003588}}AP003588). An alignment of some of these proteins is shown in Fig. ​Fig.1.1. The level of identity between these proteins and the conservation of a number of key amino acids (2) suggest that the cassette gene is likely to confer resistance to fosfomycin. FIG. 1. Alignment of putative cassette-encoded fosfomycin resistance proteins with proteins known to confer resistance to fosfomycin. Completely conserved residues are shown as black on white, and the residues important in binding the divalent cations required ... A second cassette-encoded open reading frame, designated orf“i”, predicts a protein that is 37.9% identical to ORF1 (Fig. ​(Fig.1).1). This cassette lies within a class 1 integron from P. aeruginosa (cassette at positions 2193 to 2654 in GenBank accession no. {type:entrez-nucleotide,attrs:{text:AY029772,term_id:14017420,term_text:AY029772}}AY029772) (6), and the same open reading frame is also found in an integron in Pseudomonas putida ({type:entrez-nucleotide,attrs:{text:AY065966,term_id:17978819,term_text:AY065966}}AY065966). The orf“i” product is 34.6 and 39.1% identical to FosA and FosB, respectively. It is also colinear with and more than 50% identical to products of open reading frames found in the genomes of Mesorhizobium loti (mlr3345 in {type:entrez-nucleotide,attrs:{text:AP003001,term_id:14023393,term_text:AP003001}}AP003001), Listeria monocytogenes (mlo1702 in {type:entrez-nucleotide,attrs:{text:AL591981,term_id:16411141,term_text:AL591981}}AL591981), Listeria innocua (lin1810 in {type:entrez-nucleotide,attrs:{text:AL596170,term_id:16414292,term_text:AL596170}}AL596170), Brucella suis (BRA0578 in {type:entrez-nucleotide,attrs:{text:AE014554,term_id:23463930,term_text:AE014554}}AE014554), and Brucella melitensis (BMEII0689 in {type:entrez-nucleotide,attrs:{text:AE009704,term_id:17984870,term_text:AE009704}}AE009704). The genes from M. loti and L. monocytogenes have recently been cloned and designated fosX because they confer significant levels of resistance to fosfomycin (4). Thus, it is likely that orf“i” also determines resistance to fosfomycin. FosA, FosB, and FosX confer resistance to fosfomycin by adding glutathione, l-cysteine, or a hydroxyl group, respectively, to the oxirane ring of this antibiotic, rendering it inactive (see references 2, 3, 4, and 10 and references therein).

Recombination activity of a distinctive integron-gene cassette system associated with Pseudomonas stutzeri populations in soil.

Class 1 integrons have strongly influenced the evolution of multiple antibiotic resistance. Diverse integrons have recently been detected directly in a range of natural environments. In order to characterize the properties of these environmental integrons, we sought to isolate organisms containing integrons from soils, which resulted in the isolation of Pseudomonas stutzeri strain Q. Further isolation efforts targeted at this species resulted in recovery of two other strains (P and BAM). 16S rRNA sequences and chromosome mapping showed that these three strains are very closely related clonal variants in a single genomovar of P. stutzeri. Only strains Q and BAM were found to contain an integron and an associated gene cassette array. The intI and attI components of these strains showed 99 and 90% identity, respectively. The structure of these integrons and their associated gene cassettes was similar to that reported previously for other integron classes. The two integrons contained nonoverlapping sets of cassette-associated genes. In contrast, many of the cassette-associated recombination sites in the two integrons were similar and were considered to constitute a distinct subfamily consisting of 59-base element (59-be) recombination sites (the Pseudomonas subfamily). The recombination activity of P. stutzeri integron components was tested in cointegrate assays. IntIPstQ was shown to catalyze site-specific recombination between its cognate attI site and 59-be sites from antibiotic resistance gene cassettes. While IntIPstQ did not efficiently mediate recombination between members of the Pseudomonas 59-be subfamily and other 59-be types, the former sites were functional when they were tested with IntI1. We concluded that integrons present in P. stutzeri possess recombination activity and represent a hot spot for genomic diversity in this species.

Class 1 integron containing a new gene cassette, aadA10, associated with Tn1404 from R151.

The carbenicillin, gentamicin, kanamycin, streptomycin, spectinomycin, sulfonamide, and tobramycin resistance determinants found on Pseudomonas aeruginosa plasmid R151 have previously been shown to translocate to another plasmid, R388, and it was inferred that a transposon, Tn1404, carried the resistance determinants. Sequencing of the cassette array from the plasmid known as R388::Tn1404 revealed two known gene cassettes, oxa10 and aadB, and a previously unidentified cassette determining resistance to streptomycin and spectinomycin, here designated aadA10, in the order oxa10-aadB-aadA10. These cassettes replaced the dfrB2-orfA cassette array of R388, indicating that movement of the resistance determinants from R151 to R388 resulted from recombinational exchange between two class 1 integrons rather than transposition. The AadA10 protein is most closely related to AadA6 (85% identical) and AadA7 (80% identical). The aadA10 cassette found here has only a simple site containing a 7-bp spacer derived from attI1 in place of a 59-base element and is likely to represent a derivative of the complete cassette. IntI1-mediated deletion of the aadA10 cassette was not detected, indicating that this single simple site is either inactive or only weakly active.

Presence of a group II intron in a multiresistant Serratia marcescens strain that harbors three integrons and a novel gene fusion.

We analyzed the role of integrons in the dissemination of antibiotic resistance in a recent multiresistant clinical isolate, Serratia marcescens SCH88050909 (SCH909). This isolate harbors three integrons, all on a 60-kb conjugative plasmid. By PCR, hybridization, and sequencing analyses, we found that integron 1 has the dfrA1 and ant(3")-Ia cassettes. The first cassette in integron 2 contains the ant(2")-Ia gene, separated from its attC site (59-base element) by a 1,971-bp insert containing a group II intron; this intron codes for a putative maturase-reverse transcriptase on the complementary strand and is the first such intron to be found associated with an integron. The attC site is followed by a novel aminoglycoside resistance gene, ant(3")-Ii-aac(6')-IId, which has been characterized for its bifunctional ANT(3")-I and AAC(6')-II activities. DNA sequence analysis of this fused cassette suggests that insertion and excision due to the integrase activity could have an important role in the evolution of aminoglycoside resistance genes. This gene is followed by an unknown open reading frame with a typical attC site and a partial cassette composed of the beginning of the bla(OXA-10) cassette interrupted by IS1. The sequence downstream of IS1 revealed that the bla(OXA-10) cassette is incomplete and that the 3' conserved segment of this integron is absent. Integron 3 is in a Tn1696-like transposon with the aac(3)-Ia cassette followed by three unknown cassettes and ant(3")-Ia. The presence of the group II intron and the relationship of group II introns in eubacteria with mobile elements suggest a possible role of this element in events such as cassette formation and/or plasmid evolution.

Definition of the attI1 site of class 1 integrons.

Integron-encoded integrases recognize two distinct types of recombination site: attI sites, found in integrons, and members of the 59-base element (59-be) family, found in the integron-associated gene cassettes. The class 1 integron integrase, IntI1, catalyses recombination between attI1 and a 59-be, two 59-be, or two attI1 sites, but events involving two attI1 sites are less efficient than the reactions in which a 59-be participates. The full attI1 site is required for high-efficiency recombination with a 59-be site. It is 65 bp in length and includes a simple site, consisting of a pair of inversely oriented IntI1-binding domains, together with two further directly oriented IntI1-binding sites designated strong and weak. However, a smaller region that contains only the simple site is sufficient to support a lower level of recombination with a complete attI1 partner and the features that determine the orientation of attI1 reside within this region. An unusual reaction between the attI1 site and a 59-be appears to be responsible for the loss of the central region of a 59-be to create a potential fusion of two adjacent gene cassettes.

Antibiotic resistance in the ECOR collection: integrons and identification of a novel aad gene.

The 72 Escherichia coli strains of the ECOR collection were examined for resistance to 10 different antimicrobial agents including ampicillin, tetracycline, mercury, trimethoprim, and sulfonamides. Eighteen strains were resistant to at least one of the antibiotics tested, and nearly 20% (14 of 72) were resistant to two or more. Several of the resistance determinants were shown to be carried on conjugative elements. The collection was screened for the presence of the three classes of integrons and for the sul1 gene, which is generally associated with class 1 integrons. The four strains found to carry a class 1 integron also had Tn21-encoded mercury resistance. One of the integrons encoded a novel streptomycin resistance gene, aadA7, with an attC site (or 59-base element) nearly identical to the attC site associated with the qacF gene cassette found in In40 (M.-C. Ploy, P. Courvalin, and T. Lambert, Antimicrob. Agents Chemother. 42:2557-2563, 1998). The conservation of associated attC sites among unrelated resistance cassettes is similar to arrangements found in the Vibrio cholerae superintegrons (D. Mazel, B. Dychinco, V. A. Webb, and J. Davies, Science 280:605-608, 1998) and supports the hypothesis that resistance cassettes are picked up from superintegron pools and independently assembled from unrelated genes and related attC sites.

Characterization of the metallo-beta-lactamase determinant of Acinetobacter baumannii AC-54/97 reveals the existence of bla(IMP) allelic variants carried by gene cassettes of different phylogeny.

The metallo-beta-lactamase determinant of Acinetobacter baumannii AC-54/97, a clinical isolate from Italy that was previously shown to produce an enzyme related to IMP-1, was isolated by means of a PCR methodology which targets amplification of gene cassette arrays inserted into class 1 integrons. Sequencing revealed that this determinant was an allelic variant (named bla(IMP-2)) of bla(IMP) found in Japanese isolates and that it was divergent from the latter by 12% of its nucleotide sequence, which evidently had been acquired independently. Similar to bla(IMP), bla(IMP-2) was also carried by an integron-borne gene cassette. However, the 59-base element of the bla(IMP-2) cassette was unrelated to those of the bla(IMP) cassettes found in Japanese isolates, indicating a different phylogeny for the gene cassettes carrying the two allelic variants. Expression of the integron-borne bla(IMP-2) gene in Escherichia coli resulted in a significant decrease in susceptibility to a broad array of beta-lactams (ampicillin, carbenicillin, cephalothin, cefoxitin, ceftazidime, cefepime, and carbapenems). The IMP-2 enzyme was purified from an Escherichia coli strain carrying the cloned determinant, and kinetic parameters were determined with several beta-lactam substrates. Compared to IMP-1, the kinetic parameters of IMP-2 were similar overall with some beta-lactam substrates (cefoxitin, ceftazidime, cefepime, and imipenem) but remarkably different with others (ampicillin, carbenicillin, cephaloridine, and meropenem), revealing a functional significance of at least some of the mutations that differentiate the two IMP variants. Present findings suggest that the environmental reservoir of bla(IMP) alleles could be widespread and raise a question about the global risk of their transfer to clinically relevant species.

Biochemical sequence analyses of GES-1, a novel class A extended-spectrum beta-lactamase, and the class 1 integron In52 from Klebsiella pneumoniae.

Klebsiella pneumoniae ORI-1 was isolated in 1998 in France from a rectal swab of a 1-month-old girl who was previously hospitalized in Cayenne Hospital, Cayenne, French Guiana. This strain harbored a ca. 140-kb nontransferable plasmid, pTK1, that conferred an extended-spectrum cephalosporin resistance profile antagonized by the addition of clavulanic acid, tazobactam, or imipenem. The gene for GES-1 (Guiana extended-spectrum beta-lactamase) was cloned, and its protein was expressed in Escherichia coli DH10B, where this pI-5. 8 beta-lactamase of a ca. 31-kDa molecular mass conferred resistance to oxyimino cephalosporins (mostly to ceftazidime). GES-1 is weakly related to the other plasmid-located Ambler class A extended-spectrum beta-lactamases (ESBLs). The highest percentage of amino acid identity was obtained with the carbenicillinase GN79 from Proteus mirabilis; with YENT, a chromosome-borne penicillinase from Yersinia enterocolitica; and with L-2, a chromosome-borne class A cephalosporinase from Stenotrophomonas maltophilia (36% amino acid identity each). However, a dendrogram analysis showed that GES-1 clustered within a class A ESBL subgroup together with ESBLs VEB-1 and PER-1. Sequencing of a 7,098-bp DNA fragment from plasmid pTK1 revealed that the GES-1 gene was located on a novel class 1 integron named In52 that was characterized by (i) a 5' conserved segment containing an intI1 gene possessing two putative promoters, P(1) and P(2), for coordinated expression of the downstream antibiotic resistance genes and an attI1 recombination site; (ii) five antibiotic gene cassettes, bla(GES-1), aac(6')Ib' (gentamicin resistance and amikacin susceptibility), dfrXVb (trimethoprim resistance), a novel chloramphenicol resistance gene (cmlA4), and aadA2 (streptomycin-spectinomycin resistance); and (iii) a 3' conserved segment consisting of qacEDelta1 and sulI. The bla(GES-1) and aadA2 gene cassettes were peculiar, since they lacked a typical 59-base element. This work identified the second class A ESBL gene of a non-TEM, non-SHV series which was located in the plasmid and integron, thus providing it additional means for its spread and its expression.

Reconstruction of an active integron recombination site after integration of a gene cassette at a secondary site.

As the site of insertion of the aadB gene cassette on pRAY, from a clinical isolate of Acinetobacter, is almost identical to the preferred site on integrons, the composite 59-base element (59-BE) associated with this cassette is potentially recombinationally active. By using a conduction assay to quantitate site activity, the 59-BE was recognized by integrase with high frequency, indicating that the composite site is recombinationally active.