A comprehensive survey of integron-associated genes present in metagenomes.

BACKGROUND: Integrons are genomic elements that mediate horizontal gene transfer by inserting and removing genetic material using site-specific recombination. Integrons are commonly found in bacterial genomes, where they maintain a large and diverse set of genes that plays an important role in adaptation and evolution. Previous studies have started to characterize the wide range of biological functions present in integrons. However, the efforts have so far mainly been limited to genomes from cultivable bacteria and amplicons generated by PCR, thus targeting only a small part of the total integron diversity. Metagenomic data, generated by direct sequencing of environmental and clinical samples, provides a more holistic and unbiased analysis of integron-associated genes. However, the fragmented nature of metagenomic data has previously made such analysis highly challenging. RESULTS: Here, we present a systematic survey of integron-associated genes in metagenomic data. The analysis was based on a newly developed computational method where integron-associated genes were identified by detecting their associated recombination sites. By processing contiguous sequences assembled from more than 10 terabases of metagenomic data, we were able to identify 13,397 unique integron-associated genes. Metagenomes from marine microbial communities had the highest occurrence of integron-associated genes with levels more than 100-fold higher than in the human microbiome. The identified genes had a large functional diversity spanning over several functional classes. Genes associated with defense mechanisms and mobility facilitators were most overrepresented and more than five times as common in integrons compared to other bacterial genes. As many as two thirds of the genes were found to encode proteins of unknown function. Less than 1% of the genes were associated with antibiotic resistance, of which several were novel, previously undescribed, resistance gene variants. CONCLUSIONS: Our results highlight the large functional diversity maintained by integrons present in unculturable bacteria and significantly expands the number of described integron-associated genes.

The resistomes of six carbapenem-resistant pathogens - a critical genotype-phenotype analysis.

Carbapenem resistance is a rapidly growing threat to our ability to treat refractory bacterial infections. To understand how carbapenem resistance is mobilized and spread between pathogens, it is important to study the genetic context of the underlying resistance mechanisms. In this study, the resistomes of six clinical carbapenem-resistant isolates of five different species - Acinetobacter baumannii, Escherichia coli, two Klebsiella pneumoniae, Proteus mirabilis and Pseudomonas aeruginosa - were characterized using whole genome sequencing. All Enterobacteriaceae isolates and the A. baumannii isolate had acquired a large number of antimicrobial resistance genes (7-18 different genes per isolate), including the following encoding carbapenemases: blaKPC-2, blaOXA-48, blaOXA-72, blaNDM-1, blaNDM-7 and blaVIM-1. In addition, a novel version of blaSHV was discovered. Four new resistance plasmids were identified and their fully assembled sequences were verified using optical DNA mapping. Most of the resistance genes were co-localized on these and other plasmids, suggesting a risk for co-selection. In contrast, five out of six carbapenemase genes were present on plasmids with no or few other resistance genes. The expected level of resistance - based on acquired resistance determinants - was concordant with measured levels in most cases. There were, however, several important discrepancies for four of the six isolates concerning multiple classes of antibiotics. In conclusion, our results further elucidate the diversity of carbapenemases, their mechanisms of horizontal transfer and possible patterns of co-selection. The study also emphasizes the difficulty of using whole genome sequencing for antimicrobial susceptibility testing of pathogens with complex genotypes.