Search

Horizontal gene transfer (HGT) is a key driver of bacterial evolution via transmission of genetic materials across taxa. Class 1 integrons are genetic elements that correlate strongly with anthropogenic pollution and contribute to the spread of antimicrobial resistance (AMR) genes via HGT. Despite their significance to human health, there is a shortage of robust, culture-free surveillance technologies for identifying uncultivated environmental taxa that harbour class 1 integrons. We developed a modified version of epicPCR (emulsion,pairedisolation andconcatenationpolymerasechainreaction) that links class 1 integrons amplified from single bacterial cells to taxonomic markers from the same cells in emulsified aqueous droplets. Using this single-cell genomic approach and Nanopore sequencing, we successfully assigned class 1 integron gene cassette arrays containing mostly AMR genes to their hosts in coastal water samples that were affected by pollution. Our work presents the first application of epicPCR for targeting variable, multi-gene loci of interest. We also identified theRhizobactergenus as novel hosts of class 1 integrons. These findings establish epicPCR as a powerful tool for linking taxa to class 1 integrons in environmental bacterial communities and offer the potential to direct mitigation efforts towards hotspots of class 1 integron-mediated dissemination of AMR.SynopsisWe present a novel single-cell genomic surveillance technology for identifying environmental bacterial hosts of a class of mobile genetic elements that are linked to anthropogenic pollution and contribute to the dissemination of antimicrobial resistance.

SummaryHorizontal gene transfer between different domains of life is increasingly being recognised as an important driver of evolution, with the potential to provide the recipient with new gene functionality and assist niche adaptation1-3. However, the molecular mechanisms underlying the integration of exogenous genes from foreign domains are mostly unknown. Integrons are a family of genetic elements that facilitate this process within Bacteria via site-specific DNA recombination4-7. Integrons, however, have not been reported outside Bacteria, and thus their potential role in cross-domain gene transfer has not been investigated. Here we show that integrons are also present among diverse phyla within the domain Archaea. Further, we provide experimental evidence that integron-mediated recombination can facilitate the recruitment of archaeal genes by bacteria. Our findings establish a new mechanism that can facilitate horizontal gene transfer between the two domains of prokaryotes, which has important implications for prokaryotic evolution in both clinical and environmental contexts.

Integrons were first identified because of their central role in assembling and disseminating antibiotic resistance genes in commensal and pathogenic bacteria. However, these clinically relevant integrons represent only a small proportion of integron diversity. Integrons are now known to be ancient genetic elements that are hotspots for genomic diversity, helping to generate adaptive phenotypes. This perspective examines the diversity, functions, and activities of integrons within both natural and clinical environments. We show how the fundamental properties of integrons exquisitely pre-adapted them to respond to the selection pressures imposed by the human use of antimicrobial compounds. We then follow the extraordinary increase in abundance of one class of integrons (class 1) that has resulted from its acquisition by multiple mobile genetic elements, and subsequent colonisation of diverse bacterial species, and a wide range of animal hosts. Consequently, this class of integrons has become a significant pollutant in its own right, to the extent that it can now be detected in most ecosystems. As human activities continue to drive environmental instability, integrons will likely continue to play key roles in bacterial adaptation in both natural and clinical settings. Understanding the ecological and evolutionary dynamics of integrons can help us predict and shape these outcomes that have direct relevance to human and ecosystem health.

Integrons are bacterial genetic elements that can integrate mobile gene cassettes. They are mostly known for spreading antibiotic resistance cassettes among human pathogens. However, beyond clinical settings, gene cassettes encode an extraordinarily diverse range of functions important for bacterial adaptation. The recovery and sequencing of cassettes has promising applications, including: surveillance of clinically important genes, particularly antibiotic resistance determinants; investigating the functional diversity of integron-carrying bacteria; and novel enzyme discovery. Although gene cassettes can be directly recovered using PCR, there are no standardised methods for their amplification and, importantly, for validating sequences as genuine integron gene cassettes. Here, we present reproducible methods for the PCR amplification, sequence processing, and validation of gene cassette amplicons from complex communities. We describe two different PCR assays that either amplify cassettes together with integron integrases, or gene cassettes together within cassette arrays. We compare the use of Nanopore and Illumina sequencing, and present bioinformatic pipelines that filter sequences to ensure that they represent amplicons from genuine integrons. Using a diverse set of environmental DNAs, we show that our approach can consistently recover thousands of unique cassettes per sample and up to hundreds of different integron integrases. Recovered cassettes confer a wide range of functions, including antibiotic resistance, with as many as 300 resistance cassettes found in a single sample. In particular, we show that class 1 integrons appear to be collecting and concentrating antibiotic resistance genes out of the broader diversity of cassette functions. The methods described here can be applied to any environmental or clinical microbiome sample.

Objectives Antibiotic therapy is increasingly becoming difficult to treat Pseudomonas infections. Two multi-drug resistant, clinically derived Pseudomonas strains contained related transposons that are responsible for giving rise to resistance determinants. In this study we characterise these transposons. Methods Two clinical, multidrug resistant Pseudomonas isolates were obtained from a medical facility in Cyprus. The strains were identified as Pseudomonas putida C54 and Pseudomonas aeruginosa C69. DNA was extracted from both strains and sequenced. Transposons were identified, annotated and compared to DNA sequences in GenBank. Results Two related nested transposons, here, named Tn6608 (from P. putida C54) and Tn6609 (from P. aeruginosa C69) were characterised. The transposons are built on an ancestral Tn1403 base element (here named Tn1403A) that contains only the transposition module (tnpA and tnpR) and the associated cargo gene module (orfA, orfB, orfC and orfD) flanked by 38 bp IR. The nested transposons identified in this study have evolved via the acquisition of multiple transposons, adding multiple resistance genes to an ancestral transposon that originally lacked any resistance determinants. Conclusion Transposons related to Tn6608 and Tn6609 have evolved and are globally disseminated. Of particular interest is that most of these nested transposons are located within the same site in a genomic island providing alternative avenues for dissemination.

Transposons of the Pseudomonasaeruginosa accessory gene pool contribute to phenotype and to genome plasticity. We studied local P. aeruginosa strains to ascertain the encroachment of mer-type res site hunter transposons into clinical settings and their associations with other functional modules. Five different Tn5053 family transposons were detected, all chromosomal. Some were solitary elements, one was in res of Tn1013#, a relative of a reported carrier of int-type res site hunters (class 1 integrons), but most were in res of Tn6603, a new Tn501-related transposon of unknown phenotype. Most of the Tn6603::Tn elements, and some Tn6603 and Tn6603::Tn elements found in GenBank sequences, were at identical sites in an hypothetical gene of P. aeruginosa genomic island PAGI-5v. The island in clonally differing strains was at either of two tRNALys loci, suggesting lateral transfer to these sites. This observation is consistent with the membership of the prototype PAGI-5 island to the ICE family of mobile genetic elements. Additionally, the res site hunters in the nested transposons occupied different positions in the Tn6603 carrier. This suggested independent insertion events on five occasions at least. Tn5053 family members that were mer-/tni-defective were found in Tn6603- and Tn501-like carriers in GenBank sequences of non-clinical Pseudomonas spp. The transposition events in these cases presumably utilized tni functions in trans, as can occur with class 1 integrons. We suggest that in the clinical context, P. aeruginosa strains that carry Tn6603 alone or in PAGI-5v can serve to disseminate functional res site hunters, these in turn can provide the requisite trans-acting tni functions to assist in the dissemination of class 1 integrons, and hence of their associated antibiotic resistance determinants.

Transposons of the Pseudomonas aeruginosa accessory gene pool contribute to phenotype and to genome plasticity. We studied local P. aeruginosa strains to ascertain the encroachment of mer-type res site hunter transposons into clinical settings and their associations with other functional modules. Five different Tn5053 family transposons were detected, all chromosomal. Some were solitary elements; one was in res of Tn1013#, a relative of a reported carrier of int-type res site hunters (class 1 integrons), but most were in res of Tn6603, a new Tn501-related transposon of unknown phenotype. Most of the Tn6603::Tn elements, and some Tn6603 and Tn6603::Tn elements found in GenBank sequences, were at identical sites in an hypothetical gene of P. aeruginosa genomic island PAGI-5v. The island in clonally differing strains was at either of two tRNALys loci, suggesting lateral transfer to these sites. This observation is consistent with the membership of the prototype PAGI-5 island to the ICE family of mobile genetic elements. Additionally, the res site hunters in the nested transposons occupied different positions in the Tn6603 carrier. This suggested independent insertion events on five occasions at least. Tn5053 family members that were mer-/tni-defective were found in Tn6603- and Tn501-like carriers in GenBank sequences of non-clinical Pseudomonas spp. The transposition events in these cases presumably utilized tni functions in trans, as can occur with class 1 integrons. We suggest that in the clinical context, P. aeruginosa strains that carry Tn6603 alone or in PAGI-5v can serve to disseminate functional res site hunters; these in turn can provide the requisite trans-acting tni functions to assist in the dissemination of class 1 integrons, and hence of their associated antibiotic resistance determinants.

The taxonomic positions of Microterricola viridarii JCM 15926T, Phycicola gilvus DSM 18319T and Leifsonia pindariensis JCM 15132T were re-examined. Phylogenetic analysis and 16S rRNA gene sequence similarities revealed that all three strains are closely related with each other and form a monophyletic cluster with high sequence similarity (99.2 -99.9 %). A dendrogram constructed based on the protein spectra generated by matrix-assisted laser desorption/ionization time-of-flight mass spectroscopy also displayed close clustering of these three strains. The fatty acid profiles of three strains were very similar to each other and contained branched fatty acids (anteiso-C15 : 0, anteiso-C17 : 0 and iso-C16 : 0) as the predominant cellular fatty acids. The polar lipid profiles of the three stains were similar and consisted of diphosphatidylglycerol, phosphatidylglycerol and phosphatidylcholine as major polar lipids and an unknown lipid. Comparisons of morphological, chemotaxonomic and physiological data of Microterricola viridarii JCM 15926T, Leifsonia pindariensis JCM 15132T and Phycicola gilvus DSM 18319T are in agreement with the features of a common genus. DNA-DNA hybridization data generated during this study showed less than 70 % reassociation value with each other indicating that they are different at species level. Based on the present study, we conclude that Phycicola gilvus DSM 18319T and Leifsonia pindariensis JCM 15132T should be reclassified under the genus Microterricola, since this genus has the nomenclatural priority, and reclassified as Microterricolagilva comb. nov. (type strain SSWW-21T=DSM 18319T=KCTC 19185T=JCM 30550T) and Microterricolapindariensis comb. nov. (type strain PON10T=LMG 24222T=JCM 15132T=MTCC9128T). An emended description of the genus Microterricola is also presented.