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Metagenomic strategies identify diverse integron‐integrase and antibiotic resistance genes in the Antarctic environment

ABSTRACT: Abstract The objective of this study is to identify and analyze integrons and antibiotic resistance genes (ARGs) in samples collected from diverse sites in terrestrial Antarctica. Integrons were studied using two independent methods. One involved the construction and analysis of intI gene amplicon libraries. In addition, we sequenced 17 metagenomes of microbial mats and soil by high‐throughput sequencing and analyzed these data using the IntegronFinder program. As expected, the metagenomic analysis allowed for the identification of novel predicted intI integrases and gene cassettes (GCs), which mostly encode unknown functions. However, some intI genes are similar to sequences previously identified by amplicon library analysis in soil samples collected from non‐Antarctic sites. ARGs were analyzed in the metagenomes using ABRIcate with CARD database and verified if these genes could be classified as GCs by IntegronFinder. We identified 53 ARGs in 15 metagenomes, but only four were classified as GCs, one in MTG12 metagenome (Continental Antarctica), encoding an aminoglycoside‐modifying enzyme (AAC(6´)acetyltransferase) and the other three in CS1 metagenome (Maritime Antarctica). One of these genes encodes a class D β‐lactamase (blaOXA‐205) and the other two are located in the same contig. One is part of a gene encoding the first 76 amino acids of aminoglycoside adenyltransferase (aadA6), and the other is a qacG2 gene. The objective of this study is to identify and analyze integrons and antibiotic resistance genes (ARGs) in samples collected from diverse sites in terrestrial Antarctica. The metagenomic analysis allowed us to identify novel predicted intI integrases and gene cassettes (GCs), which mostly encode unknown functions.

SUBMITTER: Antelo V

PROVIDER: S-EPMC8435808 | biostudies-literature |

REPOSITORIES: biostudies-literature

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Project description:UnlabelledClass 1 integrons are genetic systems that enable bacteria to capture and express gene cassettes. These integrons, when isolated in clinical contexts, most often carry antibiotic resistance gene cassettes. They play a major role in the dissemination of antibiotic resistance among Gram-negative bacteria. The key element of integrons is the integrase, which allows gene cassettes to be acquired and shuffled. Planktonic culture experiments have shown that integrase expression is regulated by the bacterial SOS response. In natural settings, however, bacteria generally live in biofilms, which are characterized by strong antibiotic resilience and by increased expression of stress-related genes. Here, we report that under biofilm conditions, the stringent response, which is induced upon starvation, (i) increases basal integrase and SOS regulon gene expression via induction of the SOS response and (ii) exerts biofilm-specific regulation of the integrase via the Lon protease. This indicates that biofilm environments favor integron-mediated acquisition of antibiotic resistance and other adaptive functions encoded by gene cassettes.ImportanceMultidrug-resistant bacteria are becoming a worldwide health problem. Integrons are bacterial genetic platforms that allow the bacteria to capture and express gene cassettes. In clinical settings, integrons play a major role in the dissemination of antibiotic resistance gene cassettes among Gram-negative bacteria. Cassette capture is catalyzed by the integron integrase, whose expression is induced by DNA damage and controlled by the bacterial SOS response in laboratory planktonic cultures. In natural settings, bacteria usually grow in heterogeneous environments known as biofilms, which have very different conditions than planktonic cultures. Integrase regulation has not been investigated in biofilms. Our results showed that in addition to the SOS response, the stringent response (induced upon starvation) is specifically involved in the regulation of class 1 integron integrases in biofilms. This study shows that biofilms are favorable environments for integron-mediated acquisition/exchange of antibiotic resistance genes by bacteria and for the emergence of multidrug-resistant bacteria.

Project description:A collection of 313 motile aeromonads isolated at Danish rainbow trout farms was analyzed to identify some of the genes involved in high levels of antimicrobial resistance found in a previous field trial (A. S. Schmidt, M. S. Bruun, I. Dalsgaard, K. Pedersen, and J. L. Larsen, Appl. Environ. Microbiol. 66:4908-4915, 2000), the predominant resistance phenotype (37%) being a combined oxytetracycline (OTC) and sulphadiazine/trimethoprim resistance. Combined sulphonamide/trimethoprim resistance (135 isolates) appeared closely related to the presence of a class 1 integron (141 strains). Among the isolates containing integrons, four different combinations of integrated resistance gene cassettes occurred, in all cases including a dihydrofolate reductase gene and a downstream aminoglycoside resistance insert (87 isolates) and occasionally an additional chloramphenicol resistance gene cassette (31 isolates). In addition, 23 isolates had "empty" integrons without inserted gene cassettes. As far as OTC resistance was concerned, only 66 (30%) out of 216 resistant aeromonads could be assigned to resistance determinant class A (19 isolates), D (n = 6), or E (n = 39); three isolates contained two tetracycline resistance determinants (AD, AE, and DE). Forty OTC-resistant isolates containing large plasmids were selected as donors in a conjugation assay, 27 of which also contained a class 1 integron. Out of 17 successful R-plasmid transfers to Escherichia coli recipients, the respective integrons were cotransferred along with the tetracycline resistance determinants in 15 matings. Transconjugants were predominantly tetA positive (10 of 17) and contained class 1 integrons with two or more inserted antibiotic resistance genes. While there appeared to be a positive correlation between conjugative R-plasmids and tetA among the OTC-resistant aeromonads, tetE and the unclassified OTC resistance genes as well as class 1 integrons were equally distributed among isolates with and without plasmids. These findings indicate the implication of other mechanisms of gene transfer besides plasmid transfer in the dissemination of antibiotic resistance among environmental motile aeromonads.

Project description:Evolved resistance to one antibiotic may be associated with "collateral" sensitivity to other drugs. Here, we provide an extensive quantitative characterization of collateral effects in Enterococcus faecalis, a gram-positive opportunistic pathogen. By combining parallel experimental evolution with high-throughput dose-response measurements, we measure phenotypic profiles of collateral sensitivity and resistance for a total of 900 mutant-drug combinations. We find that collateral effects are pervasive but difficult to predict because independent populations selected by the same drug can exhibit qualitatively different profiles of collateral sensitivity as well as markedly different fitness costs. Using whole-genome sequencing of evolved populations, we identified mutations in a number of known resistance determinants, including mutations in several genes previously linked with collateral sensitivity in other species. Although phenotypic drug sensitivity profiles show significant diversity, they cluster into statistically similar groups characterized by selecting drugs with similar mechanisms. To exploit the statistical structure in these resistance profiles, we develop a simple mathematical model based on a stochastic control process and use it to design optimal drug policies that assign a unique drug to every possible resistance profile. Stochastic simulations reveal that these optimal drug policies outperform intuitive cycling protocols by maintaining long-term sensitivity at the expense of short-term periods of high resistance. The approach reveals a new conceptual strategy for mitigating resistance by balancing short-term inhibition of pathogen growth with infrequent use of drugs intended to steer pathogen populations to a more vulnerable future state. Experiments in laboratory populations confirm that model-inspired sequences of four drugs reduce growth and slow adaptation relative to naive protocols involving the drugs alone, in pairwise cycles, or in a four-drug uniform cycle.

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Metagenomic strategies identify diverse integron‐integrase and antibiotic resistance genes in the Antarctic environment

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