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Molecular characterization of a multidrug resistance IncF plasmid from the globally disseminated Escherichia coli ST131 clone.

ABSTRACT: Escherichia coli sequence type 131 (E. coli ST131) is a recently emerged and globally disseminated multidrug resistant clone associated with urinary tract and bloodstream infections. Plasmids represent a major vehicle for the carriage of antibiotic resistance genes in E. coli ST131. In this study, we determined the complete sequence and performed a comprehensive annotation of pEC958, an IncF plasmid from the E. coli ST131 reference strain EC958. Plasmid pEC958 is 135.6 kb in size, harbours two replicons (RepFIA and RepFII) and contains 12 antibiotic resistance genes (including the blaCTX-M-15 gene). We also carried out hyper-saturated transposon mutagenesis and multiplexed transposon directed insertion-site sequencing (TraDIS) to investigate the biology of pEC958. TraDIS data showed that while only the RepFII replicon was required for pEC958 replication, the RepFIA replicon contains genes essential for its partitioning. Thus, our data provides direct evidence that the RepFIA and RepFII replicons in pEC958 cooperate to ensure their stable inheritance. The gene encoding the antitoxin component (ccdA) of the post-segregational killing system CcdAB was also protected from mutagenesis, demonstrating this system is active. Sequence comparison with a global collection of ST131 strains suggest that IncF represents the most common type of plasmid in this clone, and underscores the need to understand its evolution and contribution to the spread of antibiotic resistance genes in E. coli ST131.

SUBMITTER: Phan MD

PROVIDER: S-EPMC4398462 | biostudies-literature | 2015

REPOSITORIES: biostudies-literature

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Molecular characterization of a multidrug resistance IncF plasmid from the globally disseminated Escherichia coli ST131 clone.

Phan Minh Duy MD Forde Brian M BM Peters Kate M KM Sarkar Sohinee S Hancock Steven S Stanton-Cook Mitchell M Ben Zakour Nouri L NL Upton Mathew M Beatson Scott A SA Schembri Mark A MA

PloS one 20150415 4

Escherichia coli sequence type 131 (E. coli ST131) is a recently emerged and globally disseminated multidrug resistant clone associated with urinary tract and bloodstream infections. Plasmids represent a major vehicle for the carriage of antibiotic resistance genes in E. coli ST131. In this study, we determined the complete sequence and performed a comprehensive annotation of pEC958, an IncF plasmid from the E. coli ST131 reference strain EC958. Plasmid pEC958 is 135.6 kb in size, harbours two r ...[more]

PMID: 25875675

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Project description:UnlabelledEscherichia coli sequence type 131 (ST131) is a clone of uropathogenic E. coli that has emerged rapidly and disseminated globally in both clinical and community settings. Members of the ST131 lineage from across the globe have been comprehensively characterized in terms of antibiotic resistance, virulence potential, and pathogenicity, but to date nothing is known about the methylome of these important human pathogens. Here we used single-molecule real-time (SMRT) PacBio sequencing to determine the methylome of E. coli EC958, the most-well-characterized completely sequenced ST131 strain. Our analysis of 52,081 methylated adenines in the genome of EC958 discovered three (m6)A methylation motifs that have not been described previously. Subsequent SMRT sequencing of isogenic knockout mutants identified the two type I methyltransferases (MTases) and one type IIG MTase responsible for (m6)A methylation of novel recognition sites. Although both type I sites were rare, the type IIG sites accounted for more than 12% of all methylated adenines in EC958. Analysis of the distribution of MTase genes across 95 ST131 genomes revealed their prevalence is highly conserved within the ST131 lineage, with most variation due to the presence or absence of mobile genetic elements on which individual MTase genes are located.ImportanceDNA modification plays a crucial role in bacterial regulation. Despite several examples demonstrating the role of methyltransferase (MTase) enzymes in bacterial virulence, investigation of this phenomenon on a whole-genome scale has remained elusive until now. Here we used single-molecule real-time (SMRT) sequencing to determine the first complete methylome of a strain from the multidrug-resistant E. coli sequence type 131 (ST131) lineage. By interrogating the methylome computationally and with further SMRT sequencing of isogenic mutants representing previously uncharacterized MTase genes, we defined the target sequences of three novel ST131-specific MTases and determined the genomic distribution of all MTase target sequences. Using a large collection of 95 previously sequenced ST131 genomes, we identified mobile genetic elements as a major factor driving diversity in DNA methylation patterns. Overall, our analysis highlights the potential for DNA methylation to dramatically influence gene regulation at the transcriptional level within a well-defined E. coli clone.

Project description:Escherichia coli ST131 is a globally disseminated, multidrug resistant clone responsible for a high proportion of urinary tract and bloodstream infections. The rapid emergence and successful spread of E. coli ST131 is strongly associated with antibiotic resistance; however, this phenotype alone is unlikely to explain its dominance amongst multidrug resistant uropathogens circulating worldwide in hospitals and the community. Thus, a greater understanding of the molecular mechanisms that underpin the fitness of E. coli ST131 is required. In this study, we employed hyper-saturated transposon mutagenesis in combination with multiplexed transposon directed insertion-site sequencing to define the essential genes required for in vitro growth and the serum resistome (i.e. genes required for resistance to human serum) of E. coli EC958, a representative of the predominant E. coli ST131 clonal lineage. We identified 315 essential genes in E. coli EC958, 231 (73%) of which were also essential in E. coli K-12. The serum resistome comprised 56 genes, the majority of which encode membrane proteins or factors involved in lipopolysaccharide (LPS) biosynthesis. Targeted mutagenesis confirmed a role in serum resistance for 46 (82%) of these genes. The murein lipoprotein Lpp, along with two lipid A-core biosynthesis enzymes WaaP and WaaG, were most strongly associated with serum resistance. While LPS was the main resistance mechanism defined for E. coli EC958 in serum, the enterobacterial common antigen and colanic acid also impacted on this phenotype. Our analysis also identified a novel function for two genes, hyxA and hyxR, as minor regulators of O-antigen chain length. This study offers novel insight into the genetic make-up of E. coli ST131, and provides a framework for future research on E. coli and other Gram-negative pathogens to define their essential gene repertoire and to dissect the molecular mechanisms that enable them to survive in the bloodstream and cause disease.

Dataset Information

Molecular characterization of a multidrug resistance IncF plasmid from the globally disseminated Escherichia coli ST131 clone.

Publications

Molecular characterization of a multidrug resistance IncF plasmid from the globally disseminated Escherichia coli ST131 clone.

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