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PubMed2003

Polymerase chain reaction screening for integrons can be used to complement resistance surveillance programs.

Integrons have been recognised as important contributors to the acquisition and dissemination of antibiotic resistance in Gram-negative bacteria. In a collection of 19 multi-antibiotic resistant Gram-negative clinical isolates, 47 per cent (9/19) of strains were found to contain one or more integron, using a polymerase chain reaction (PCR) based screening method. Resistance gene cassettes within the integrons were amplified, sequenced and characterised. Antibiotic susceptibility testing demonstrated that resistance phenotypes correlated with the resistance conferred by gene cassettes identified. PCR-screening for integrons and gene cassettes provides a rapid technique for the identification of genetic determinants of resistance in Gram-negative bacteria. Such screening could assist in guiding treatment regimens and complement existing antibiotic resistance surveillance programs by providing information on molecular mechanisms of both resistance and resistance dissemination.

Anti-Bacterial Agents↗
PubMed2005

Prevalence of class 1 integrons and antimicrobial resistance gene cassettes among enteric bacteria found in multisite group-level cohorts of humans and swine.

The prevalence of antimicrobial resistance genotypic characteristics (class 1 integrons and antimicrobial resistance gene cassettes) among commensal Escherichia coli isolated from humans and swine in a semiclosed, integrated farrow-to-fork population was evaluated in a cross-sectional study. The objective of this study was to establish baseline antimicrobial resistance patterns of enteric bacteria from animals and humans within the study population; specifically, genotypic traits both unique and common to commensal E. coli derived from the different sources were evaluated. There were significant differences between host species; swine isolates were more likely to harbor integrons (odds ratio = 2.33, P = 0.0487). No significant differences were found for facility location, facility type, human housing cohort, or time of day (P > 0.05). There were significant differences (P = 0.006) among swine production groups (fecal samples from boars, dry sows, finishers, growers, intake boars, lactating sows, the lagoon, nursery piglets, influent, and piglets); the grower group was less likely than the nursery group to harbor a class 1 integron (nursery as referent: odds ratio = 0.22, P = 0.04). Among all isolates with an integron present, human isolates were more likely to harbor an antimicrobial resistance gene cassette (odds ratio = 6.36, P = 0.003). When isolates that possessed gene cassettes coding for resistance to specific antimicrobials were compared, no significant differences between host species (P > 0.05) were observed.

Animals↗
PubMed1999

The cassettes and 3' conserved segment of an integron from Klebsiella oxytoca plasmid pACM1.

pACM1 is a conjugative multiresistance plasmid from Klebsiella oxytoca that encodes SHV-5 extended-spectrum beta-lactamase (ESBL) and has two integrons. The first is a type I (sul type); the second, detected by hybridization with an intI gene probe, has been putatively identified as a defective type I integron. The cassette region of the first integron has now been fully sequenced and contains three aminoglycoside resistance determinants (aac(6')-Ib, aac(3)-Ia, and ant(3")-Ia) and two open reading frames of unknown function. In addition, sequencing of a region downstream from the qacEDelta1-sulI-ORF 5 gene cluster of the first integron revealed a copy of insertion sequence IS6100 flanked by inverted copies of sequence from the 11.2-kb insert (In2) of Tn21. This arrangement is similar to that found in In4 of Tn1696. The coincidence of an ESBL gene and mobile elements on a conjugative plasmid has potential implications for the spread of ESBL-mediated drug resistance, though evidence of bla((SHV-5)) movement mediated by these elements has not been found.

Amino Acid Sequence↗
PubMed1991

'Integron'-bearing vectors: a method suitable for stable chromosomal integration in highly restrictive corynebacteria.

A pBR322-derived plasmid (pCGL107) that carries the Corynebacterium melassecola ATCC17965 analogue of Escherichia coli gdhA gene (encoding glutamate dehydrogenase), was introduced into the related strain, Brevibacterium lactofermentum CGL2002, by electroporation and integrated into its chromosome by homologous recombination. However, pCGL107 cannot integrate into C. melassecola, since the host restriction prevents successful electroporation by E. coli-modified DNA. Nevertheless, B. lactofermentum-modified replicative plasmid DNA can be transformed by electroporation into C. melassecola; thus pCGL519-2, a shuttle plasmid that carries the C. melassecola analogue of E. coli gltA (encoding citrate synthase), was extracted from the former host and electroporated into the latter. Rare restriction sites conveniently placed in pCGL519-2 were used to recover a replicon-less cartridge called 'integron', that contains a selectable marker and gtlA within a single fragment. Integron prepared from pCGL519-2 DNA which had been extracted from C. melassecola, and thus, was capable of eluding the C. melassecola restriction barrier(s), was successfully electroporated into this host. The molecular analysis of the resulting transformants suggests that they result from the integration of a single circular integron molecule by homologous recombination between the gltA regions of the host genome and the integron. These transformants were stable for 30 generations in the absence of selection.

Base Sequence↗
PubMed2002

Epidemiological typing and prevalence of integrons in multiresistant Acinetobacter strains.

Seventy-seven Acinetobacter isolates were recovered from patients in a Korean hospital during the period from November to December 1998. The isolates were genotyped using randomly amplified polymorphic DNA (RAPD) analysis for epidemiological relationship, and investigated for antibiotic susceptibility and presence of integrons. Sixty-nine Acinetobacter baumannii isolates were distributed into five groups by RAPD profiles, with 5, 1, 60, 2 and 1 in each group. The major RAPD group of 60 isolates was further divided into six subgroups by antibiograms. Eight isolates belonging to Acinetobacter DNA group 13TU were distributed among six RAPD groups. Seventy-three of the Acinetobacter isolates were resistant to eight or more of the antibiotics tested. Integrase genes were detected in 66 of the 69 A. baumannii (96%) and in 5 of the 8 Acinetobacter DNA group 13TU isolates (63%). The intI1 and intI2 genes were found in 63 and 8 isolates, respectively. The intI3 gene was not detected. All integron-carrying isolates were resistant to multiple antibiotics. All strains isolated from more than one patient carried integrons. According to the results, the presence of integrons was significantly (p<0.01) associated with multiple antibiotic resistance and nosocomial spread in Acinetobacter strains.

Acinetobacter↗
PubMed2026

High-throughput recovery of integron cassettes for gene discovery screens.

Integrons capture functional genes in mobile genetic elements called integron cassettes, which represent an untapped source of genes of biotechnological interest. Here we present two tools, cassette gatherer and cassette hunter, that enable high-throughput establishment of gene libraries either from genetically tractable strains or directly from DNA. We re-engineered a class 1 integron into counterselection markers on a plasmid or on the chromosome of a naturally competent Vibrio cholerae, which enabled capture of single cassettes in a sequence- and function-independent manner. When applied to Vibrio strains and genomic libraries, our tools recovered hundreds of single cassettes per assay with more than 99% specificity. We further subjected the library of cassettes generated by the hunter and gatherer tools to screens against phages ICP2 and T4, and identified nine phage-defence systems, including five previously undescribed. These tools enable rapid and large-scale recovery of integron cassettes that could be leveraged for functional gene discovery.

Journal Article↗
PubMed2001

Characterization of class 1 integrons associated with R-plasmids in clinical Aeromonas salmonicida isolates from various geographical areas.

Class 1 integrons were found in 26 of 40 antibiotic-resistant isolates of the fish pathogen Aeromonas salmonicida from Northern Europe and North America. Three different dhfr genes, conferring trimethoprim resistance, and one ant(3")1a aminoglycoside resistance gene were identified as gene inserts. The gene cassettes tended to be conserved among isolates from a particular geographical area. Nineteen isolates transferred R-plasmids carrying different tet determinants to Escherichia coli in filter mating assays, and in 15 cases, the class 1 integrons were co-transferred. Transferable sulphadiazine, trimethoprim and streptomycin resistances were invariably encoded by integrons. It thus appears that integron-encoded antibiotic resistance genes contribute substantially to the horizontal spread of antimicrobial resistance within this species, being associated with conjugative plasmids.

Aeromonas↗
PubMed2004

Contribution of integrons, and SmeABC and SmeDEF efflux pumps to multidrug resistance in clinical isolates of Stenotrophomonas maltophilia.

OBJECTIVES: The contribution of integrons and efflux pumps to multidrug resistance in Stenotrophomonas maltophilia was evaluated. MATERIALS AND METHODS: Ninety-three S. maltophilia clinical isolates were studied. PCR and direct sequencing were used to detect the presence of integrons. Real-time PCR was performed to assess and quantify the expression of the Sme efflux pumps of S. maltophilia. RESULTS: Class 1 integrons were detected in 22% of clinical isolates and carried cassettes conferring resistance mainly to aminoglycosides and trimethoprim. The small multidrug resistance gene, smr, was found on class 1 integrons in six isolates. Thirty-one percent of the isolates overexpressed the smeDEF gene, as compared with a control strain, and 59% overexpressed the smeABC gene. Extrusion of ciprofloxacin and meropenem was specific to the SmeABC and SmeDEF pumps, respectively. CONCLUSION: SmeABC and SmeDEF efflux pumps play important roles in resistance of S. maltophilia to ciprofloxacin and meropenem.

Anti-Bacterial Agents↗
PubMed2000

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.

Attachment Sites, Microbiological↗
PubMed1991

Site-specific insertion of genes into integrons: role of the 59-base element and determination of the recombination cross-over point.

From examination of published DNA sequences of genes found inserted at a specific site in integrons, all genes are shown to be associated, at their 3' ends, with a short imperfect inverted repeat sequence, a 59-base element or relative of this element. The similarity of the arrangement of gene inserts in the integron and in the Tn7 transposon family is described. A refined consensus for the 59-base element is reported. Members of this family are highly diverged and the relationship of a group of longer elements to the 59-base elements is demonstrated. The ability of 59-base elements of different length and sequence to act as sites for recombination catalysed by the integron-encoded DNA integrase is demonstrated, confirming that elements of this family have a common function. The ability of elements located between gene pairs to act as recombination sites has also been demonstrated. The recombination cross-over point has been localized to the GTT triplet which is conserved in the core sites, GTTRRRY, found at the 3' end of 59-base elements. Recombination at the core site found in inverse orientation at the 5' end of the 59-base elements was not detected, and the sequences responsible for orientation of the recombination event appear to reside within the 59-base element. A model for site-specific insertion of genes into integrons and Tn7-like transposons is proposed. Circular units consisting of a gene associated with a 59-base element are inserted into an ancestral element which contains neither a gene nor a 59-base element.(ABSTRACT TRUNCATED AT 250 WORDS)

Base Sequence↗
PubMed1992

Gene cassettes from the insert region of integrons are excised as covalently closed circles.

Integrons are DNA elements which generally include one or more discrete gene cassettes inserted at a specific site. We have recently proposed a model for the acquisition and dissemination of genes found in the insert region of integrons, which requires the existence of circularized gene cassettes. Evidence for the existence of covalently closed circular molecules consisting of one or more gene cassettes has now been obtained. Low levels of small molecules which hybridize to probes specific for individual gene cassettes were detected in plasmid DNA isolated from cells containing a plasmid which includes an integron fragment with three gene cassettes aacC1, orfE and aadA2. These molecules were only detected when the gene encoding the integron DNA integrase was also present and are thus products of site-specific cassette excision. The excised cassettes have been shown to be in the form of covalently closed supercoiled circles, by digestion with restriction enzymes exonuclease III and DNase I. The circular excision products detected included either one cassette, aadA2 or orfE, two cassettes, aacC1 and orfE or all three cassettes. The predicted sequence of the recombinant junction in the excised aadA2 cassette confirmed that excision was precise. The predicted unique sequences of the 59-base elements associated with individual genes in the circular cassette form were compiled, and the sequences of the seven-base core sites which flank 59-base elements are now, with few exceptions, exact inverted repeats.

Base Sequence↗
PubMed1993

Site-specific insertion of gene cassettes into integrons.

Site-specific insertion of gene cassettes into the insert region of integrons has been demonstrated. Insertion was only observed if the integron DNA integrase was expressed in the recipient cell and if the cassette DNA was ligated prior to transformation. The essential ligation products were resistant to treatment with exonuclease III, indicating that they were closed circular molecules. Insertion of cassettes into integron fragments containing either no insert (one recombination site), or one gene cassette (two recombination sites), was demonstrated. In the latter case, insertion occurred predominantly at the core site located 5' to the resident cassette, which corresponds to the only site available when no insert is present in the recipient. When DNA molecules including two gene cassettes were used, insertion of only one of the gene cassettes was generally observed, suggesting that resolution of the circular molecule to generate two independent circular cassettes occurred more rapidly than insertion into the recipient integron.

Base Sequence↗
PubMed1995

Isolation and structure of a new integron that includes a streptomycin resistance gene from the R plasmid of Pseudomonas aeruginosa.

A new integron, located on the R plasmid of Pseudomonas aeruginosa, was isolated in Japan. This integron was made up of two conserved segments (5'-and 3'-conserved segments) and a single streptomycin resistance gene as a gene cassette. The structure of this integron resembles that of integron InC, the existence of which was postulated by Bissonnette and Roy (J. Bacteriol. 174, 1248-1257, 1992).

Biological Evolution↗
PubMed1998

Presence of integrons in isolates of different biotypes of Acinetobacter baumannii from Chilean hospitals.

Class 1 and class 2 integrons were investigated by hybridisation in 100 isolates of multiresistant biotypes of Acinetobacter baumannii from Chilean hospitals. Most isolates of A. baumannii biotype 9, the prevalent biotype, harboured integrons of class 2 (Tn7-like) whereas no integrons were detected in infrequent biotypes. Integron-carrying isolates exhibited broader antibiotic resistance patterns as well as higher resistance levels to various antimicrobials.

Acinetobacter↗
PubMed2000

Class I integrons containing a dhfrI trimethoprim resistance gene cassette in aquatic Acinetobacter spp.

The presence of antibiotic resistance gene cassettes in class I integrons was investigated in 24 sulfamethoxazole-resistant and -sensitive Acinetobacter isolates derived from two Danish freshwater trout farms. Integrons were detected in five isolates from one of the fish farms, and their inserts were characterised by DNA sequencing. Each isolate contained a dhfrI gene cassette encoding resistance to trimethoprim and an open reading frame orfC of unknown function identical to the content of an integron previously found in a clinical enterobacterial isolate. Among the five isolates, at least two different strains were differentiated based on phenotypic tests and randomly amplified polymorphic DNA analysis. To our knowledge, this is the first report and characterisation of an integron in environmental bacteria.

Acinetobacter↗
PubMed2000

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.

Amino Acid Sequence↗
PubMed2000

Molecular characterization of Irish Salmonella enterica serotype typhimurium: detection of class I integrons and assessment of genetic relationships by DNA amplification fingerprinting.

Salmonella enterica is among the principal etiological agents of food-borne illness in humans. Increasing antimicrobial resistance in S. enterica is a cause for worldwide concern. There is concern at present in relation to the increasing incidence of human infection with antimicrobial agent-resistant strains of S. enterica serotype Typhimurium, in particular of phage type DT104. Integrons appear to play an important role in the dissemination of antimicrobial resistance genes in many Enterobacteriaceae including S. enterica. In this study the antimicrobial susceptibilities and phage types of 74 randomly collected strains of S. enterica serotype Typhimurium from the Cork region of southern Ireland, obtained from human, animal (clinical), and food sources, were determined. Each strain was examined for integrons and typed by DNA amplification fingerprinting (DAF). Phage type DT104 predominated (n = 48). Phage types DT104b (n = 3), -193 (n = 9), -195 (n = 6), -208 (n = 3), -204a (n = 2), PT U302 (n = 1), and two nontypeable strains accounted for the remainder. All S. enterica serotype Typhimurium DT104 strains were resistant to ampicillin, chloramphenicol, streptomycin, Sulfonamide Duplex, and tetracycline, and one strain was additionally resistant to trimethoprim. All DT104 strains but one were of a uniform DAF type (designated DAF-I) and showed a uniform pattern of integrons (designated IP-I). The DT104b and PT U302 strains also exhibited the same resistance phenotype, and both had the DAF-I and IP-I patterns. The DAF-I pattern was also observed in a single DT193 strain in which no integrons were detectable. Greater diversity of antibiograms and DAF and IP patterns among non-DT104 phage types was observed. These data indicate a remarkable degree of homogeneity at a molecular level among contemporary isolates of S. enterica serotype Typhimurium DT104 from animal, human, and food sources in this region.

Animals↗
PubMed1991

Characterization of the nonenzymatic chloramphenicol resistance (cmlA) gene of the In4 integron of Tn1696: similarity of the product to transmembrane transport proteins.

Integrons constitute a novel family of DNA elements which evolved by site-specific integration of discrete units between two conserved segments. On the In4 integron of Tn1696, a precisely inserted gene cassette of 1,549 bp conferring nonenzymatic chloramphenicol resistance (cmlA) is present between the streptomycin-spectinomycin resistance (aadA2) gene cassette and the 3'-conserved segment of the integron. In this study, we present the nucleotide sequence of the cmlA gene cassette of Tn1696, show its similarity to bacterial efflux systems and other transport proteins, and present evidence for alterations that its expression exerts on bacterial membranes. The cmlA gene cassette apparently carries its own promoter(s), a situation that has not heretofore been observed in the integrons of multiresistance plasmids and transposons of gram-negative bacteria. One or more of these promoters were shown to be functionally active in expressing a cat marker gene from promoter-probe vectors. The putative CmlA polypeptide appears to provoke a reduction of the content of the major porins OmpA and OmpC.

Amino Acid Sequence↗