Preclinical class 1 integron with a complete Tn402-like transposition module.

The presence of integrons was assessed in gut bacteria isolated from wild-caught prawns. A pseudomonad was recovered that contained a Tn402-like class 1 integron with a complete transposition module and two gene cassettes. One cassette was identical to a previously described cassette from a chromosomal class 3 integron in Delftia tsuruhatensis.

Mobilization of a Tn402-like class 1 integron with a novel cassette array via flanking miniature inverted-repeat transposable element-like structures.

A Tn402-like class 1 integron was recovered from a prawn-associated bacterium. One of its cassettes included methionine sulfoxide reductase genes, the first example of such genes being captured by an integron. The integron was flanked by direct repeats that resemble miniature inverted-repeat transposable element sequences. Excision of the integron by homologous recombination through these sequences was demonstrated.

Evidence for dynamic exchange of qac gene cassettes between class 1 integrons and other integrons in freshwater biofilms.

Class 1 integrons carried by pathogens have acquired over 100 different gene cassettes encoding resistance to antimicrobial compounds, helping to generate a crisis in the management of infectious disease. It is presumed that these cassettes originated from environmental bacteria, but exchange of gene cassettes has surprisingly never been demonstrated outside laboratory or clinical contexts. We aimed to identify a natural environment where such exchanges might occur, and determine the phylogenetic range of participating integrons. Here we examine freshwater biofilms and show that families of cassettes conferring resistance to quaternary ammonium compounds (qac) are found on class 1 integrons identical to those from clinical contexts, on sequence variants of class 1 integrons only known from natural environments, and on other diverse classes of integrons only known from the chromosomes of soil and freshwater Proteobacteria. We conclude that gene cassettes might be readily shared between different integron classes found in environmental, commensal and pathogenic bacteria. This suggests that class 1 integrons in pathogens have access to a vast pool of gene cassettes, any of which could confer a phenotype of clinical relevance. Exploration of this resource might allow identification of resistance or virulence genes before they become part of multi-drug-resistant human pathogens.

Gene cassettes encoding resistance to quaternary ammonium compounds: a role in the origin of clinical class 1 integrons?

DNA sequencing, phylogenetic and mapping studies suggest that the class 1 integron found in pathogens arose when one member of the diverse family of environmental class 1 integrons became embedded into a Tn402 transposon. However, the timing of this event and the selective forces that first fixed the newly formed element in a bacterial lineage are still unknown. Biocides have a longer use in clinical practice than antibiotics, and a qac (quaternary ammonium compound) resistance gene, or remnant thereof, is a normal feature of class 1 integrons recovered from clinical isolates. Consequently, it is possible that the initial selective advantage was conferred by resistance to biocides, mediated by qac. Here, we show that diverse qac gene cassettes are a dominant feature of cassette arrays from environmental class 1 integrons, and that they occur in the absence of any antibiotic resistance gene cassettes. They are present in arrays that are dynamic, acquiring and rearranging gene cassettes within the arrays. The abundance of qac gene cassettes makes them a likely candidate for participation in the original insertion into Tn402, and as a source of a readily selectable phenotype. More broadly, the increasing use of qac and other biocides at the present time seems likely to promote the fixation of further novel genetic elements, with unpredictable and potentially adverse consequences for human health and agriculture.

An enhanced miniaturized assay for antimicrobial prospecting.

Bioprospecting for novel antimicrobials increasingly relies on extremely small samples unsuitable for conventional bulk extraction and assay. We developed a microtitre plate assay for minimal amounts of test materials which is rapid, extremely sensitive, allows time-course analysis and reduces false negatives. Developed for the analyses of antimicrobial sensitivity and resistance, the technique is appropriate for assays where source materials are scarce.

Integrons in Xanthomonas: a source of species genome diversity.

Integrons are best known for assembling antibiotic resistance genes in clinical bacteria. They capture genes by using integrase-mediated site-specific recombination of mobile gene cassettes. Integrons also occur in the chromosomes of many bacteria, notably beta- and gamma-Proteobacteria. In a survey of Xanthomonas, integrons were found in all 32 strains representing 12 pathovars of two species. Their chromosomal location was downstream from the acid dehydratase gene, ilvD, suggesting that an integron was present at this site in the ancestral xanthomonad. There was considerable sequence and structural diversity among the extant integrons. The majority of integrase genes were predicted to be inactivated by frameshifts, stop codons, or large deletions, suggesting that the associated gene cassettes can no longer be mobilized. In support, groups of strains with the same deletions or stop codons/frameshifts in their integrase gene usually contained identical arrays of gene cassettes. In general, strains within individual pathovars had identical cassettes, and these exhibited no similarity to cassettes detected in other pathovars. The variety and characteristics of contemporary gene cassettes suggests that the ancestral integron had access to a diverse pool of these mobile elements, and that their genes originated outside the Xanthomonas genome. Subsequent inactivation of the integrase gene in particular lineages has largely fixed the gene cassette arrays in particular pathovars during their differentiation and specialization into ecological niches. The acquisition of diverse gene cassettes by different lineages within Xanthomonas has contributed to the species-genome diversity of the genus. The role of gene cassettes in survival on plant surfaces is currently unknown.

Mobile gene cassettes: a fundamental resource for bacterial evolution.

Horizontal gene transfer increases genetic diversity in prokaryotes to a degree not allowed by the limitations of reproduction by binary fission. The integron/gene cassette system is one of the most recently characterized examples of a system that facilitates horizontal gene transfer. This system, discovered in the context of multidrug resistance, is recognized in a clinical context for its role in allowing pathogens to adapt to the widespread use of antibiotics. Recent studies suggest that gene cassettes are common and encode functions relevant to many adaptive traits. To estimate the diversity of mobile cassettes in a natural environment, a molecular technique was developed to provide representative distributions of cassette populations at points within a sampling area. Subsequently, statistical methods analogous to those used for calculating species diversity were employed to assess the diversity of gene cassettes within the sample area in addition to gaining an estimate of cassette pool size. Results indicated that the number of cassettes within a 5x10-m sample area was large and that the overall mobile cassette metagenome was likely to be orders of magnitude larger again. Accordingly, gene cassettes appear to be capable of mobilizing a significant genetic resource and consequently have a substantial impact on bacterial adaptability.

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.