Discovery of a novel integron-borne aminoglycoside resistance gene present in clinical pathogens by screening environmental bacterial communities

Abstract

Background: New antibiotic resistance determinants are generally discovered too late, long after they have irreversibly emerged in pathogens and spread widely. Early discovery of resistance genes, before or soon after their transfer to pathogens could allow more effective measures to monitor and reduce spread, and facilitate geneticsbased diagnostics. Results: We modified a functional metagenomics approach followed by in silico filtering of known resistance genes to discover novel, mobilised resistance genes in class 1 integrons in wastewater-impacted environments. We identified an integron-borne gene cassette encoding a protein that conveys high-level resistance against aminoglycosides with a garosamine moiety when expressed in E. coli. The gene is named gar (garosamine-specific aminoglycoside resistance) after its specificity. It contains none of the functional domains of known aminoglycoside modifying enzymes, but bears characteristics of a kinase. By searching public databases, we found that the gene occurs in three sequenced, multiresistant clinical isolates (two Pseudomonas aeruginosa and one Luteimonas sp.) from Italy and China, respectively, as well as in two food-borne Salmonella enterica isolates from the USA. In all cases, gar has escaped discovery until now. Conclusion: To the best of our knowledge, this is the first time a novel resistance gene, present in clinical isolates, has been discovered by exploring the environmental microbiome. The gar gene has spread horizontally to different species on at least three continents, further limiting treatment options for bacterial infections. Its specificity to garosamine-containing aminoglycosides may reduce the usefulness of the newest semisynthetic aminoglycoside plazomicin, which is designed to avoid common aminoglycoside resistance mechanisms. Since the gene appears to be not yet common in the clinics, the data presented here enables early surveillance and maybe even mitigation of its spread.

A Novel, Integron-Regulated, Class C β-Lactamase

“…Since no known ampC gene was discovered as a gene cassette in sequenced isolates, we chose to search the functional metagenomics amplicons containing only integron gene cassettes that were selected on cefotaxime [48] for ampC genes with the same method. Preparation of the functional metagenomic amplicons is summarized briefly as follows: DNA was isolated from river sediments contaminated with municipal and hospital sewage.…”

“…Preparation of the functional metagenomic amplicons is summarized briefly as follows: DNA was isolated from river sediments contaminated with municipal and hospital sewage. Gene cassettes were amplified from these samples, with primer pairs targeting the gene cassette array of class 1 integrons [48,51]. The amplification products were cloned into the vector pZE21-P bla and screened for resistance against different antibiotics in E. coli DH10β.…”

“…Functional screening of the metagenomic libraries included four β-lactam antibiotics (cefotaxime, ertapenem, imipenem, and meropenem [48]). The two identified novel ampC genes were both clearly selected on cefotaxime, but not on carbapenems (Table S1 in the Supplementary Materials), and were assumed to be AmpCs on the basis of sequence similarities.…”

“…The overexpression of blaIDC-1 caused a four-fold increase in the ertapenem MIC (Table 2). However, the strain remains susceptible (clinical breakpoint of Enterobacterales = 0.5 µg/mL; EUCAST breakpoint [48,56]. Attachment sites: attI marked as blue rhomb, attC marked as green rectangle; bla IDC-1 and bla IDC-2 : novel integron-derived cephalosporinases; bla OXA-10 : class D β-lactamase OXA-10; bla GES-5 : class A β-lactamase GES-5 (carbapenemase); qacE∆: quarternary ammonium compound resistance protein (truncated).…”

“…The ability of integrons to accumulate resistances also favors co-selection, and would enhance the spread of cassette-encoded ampC. To answer the question of whether ampC genes appear as gene cassettes, we searched all sequences encoding potential ampC genes from NCBI's non-redundant protein database and the metagenomic datasets from a previous study, which have focused specifically on the identification of antibiotic resistance genes in class 1 integrons [48]. We discovered two novel AmpC proteins that are encoded as gene cassettes and verified their functionality in E. coli.…”