IncI shufflons: Assembly issues in the next-generation sequencing era

Brouwer, Michael S.M.; Tagg, Kaitlin A.; Mevius, Dik; Iredell, Jonathan R.; Bossers, Alex; Smith, Hilde E.; Partridge, Sally R.

doi:10.1016/j.plasmid.2015.04.009

Cited by 32 publications

(37 citation statements)

References 35 publications

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“…The shufflon of R64 consists of four invertible DNA fragments embedded with seven 19-bp repeat sequences. These 19-bp repeats can be subdivided into a 7-bp core-site region where the DNA crossover occurs and the highly conserved 12-bp right arm region (Komano, 1999; Brouwer et al, 2015). …”

Section: Resultsmentioning

confidence: 99%

Plasmids Carrying blaCMY -2/4 in Escherichia coli from Poultry, Poultry Meat, and Humans Belong to a Novel IncK Subgroup Designated IncK2

et al. 2017

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The blaCMY -2/4-carrying IncB/O/K-like plasmids of seven Escherichia coli strains from poultry, poultry meat and human urine samples were examined using comparative analysis of whole plasmid sequences. The incompatibility group was determined by analysis of the incRNAI region and conjugation assays with strains containing the IncK and IncB/O reference plasmids. Strains were additionally characterized using MLST and MIC determination. The complete DNA sequences of all plasmids showed an average nucleotide identity of 91.3%. Plasmids were detected in E. coli sequence type (ST) 131, ST38, ST420, ST1431, ST1564 and belonged to a new plasmid variant (IncK2) within the IncK and IncB/O groups. Notably, one E. coli from poultry meat and one from human contained the same plasmid. The presence of a common recently recognized IncK2 plasmid in diverse E. coli from human urine isolates and poultry meat production suggests that the IncK2 plasmids originated from a common progenitor and have the capability to spread to genetically diverse E. coli in different reservoirs. This discovery is alarming and stresses the need of rapidly introducing strict hygiene measures throughout the food chain, limiting the spread of such plasmids in the human settings.

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Section: Resultsmentioning

confidence: 99%

Plasmids Carrying blaCMY -2/4 in Escherichia coli from Poultry, Poultry Meat, and Humans Belong to a Novel IncK Subgroup Designated IncK2

et al. 2017

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“…also identified five IncI2 plasmids carrying mcr-1 in animal isolates 12, 18, 20, 21 . This gene has been reported in various plasmid types (namely IncF 13, 14, 22–25 , P 15, 26–28 , I2 11, 16–18, 29 , HI2 19, 20, 30 , repB (pO111) 31 , HI1 20 and X4 18, 20, 21, 32 ) of the Enterobacteriaceae with diverse origins worldwide, suggesting horizontal gene transfer and plasmid-mediated conjugal transfer of mcr-1 among the Enterobacteriaceae . Therefore, analysis of plasmids carrying mcr-1 has become important for the surveillance and control of drug-resistant bacteria.…”

Section: Introductionmentioning

confidence: 93%

Elucidation of quantitative structural diversity of remarkable rearrangement regions, shufflons, in IncI2 plasmids

Sekizuka

Kawanishi

Ohnishi

et al. 2017

Sci Rep

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A multiple DNA inversion system, the shufflon, exists in incompatibility (Inc) I1 and I2 plasmids. The shufflon generates variants of the PilV protein, a minor component of the thin pilus. The shufflon is one of the most difficult regions for de novo genome assembly because of its structural diversity even in an isolated bacterial clone. We determined complete genome sequences, including those of IncI2 plasmids carrying mcr-1, of three Escherichia coli strains using single-molecule, real-time (SMRT) sequencing and Illumina sequencing. The sequences assembled using only SMRT sequencing contained misassembled regions in the shufflon. A hybrid analysis using SMRT and Illumina sequencing resolved the misassembled region and revealed that the three IncI2 plasmids, excluding the shufflon region, were highly conserved. Moreover, the abundance ratio of whole-shufflon structures could be determined by quantitative structural variation analysis of the SMRT data, suggesting that a remarkable heterogeneity of whole-shufflon structural variations exists in IncI2 plasmids. These findings indicate that remarkable rearrangement regions should be validated using both long-read and short-read sequencing data and that the structural variation of PilV in the shufflon might be closely related to phenotypic heterogeneity of plasmid-mediated transconjugation involved in horizontal gene transfer even in bacterial clonal populations.

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“…Another significant difference between pHeN867 and pHNSHP45 is the shufflon segments located between the rci and pilV genes. pHeN867 contains segment A (574 bp), segment C (499 bp), and a fusion of segments B and D (568 bp), the same components but with a different order than that of another IncI2-type plasmid, R721 (20). In contrast, the shufflon region in pHNSHP45 showed a 507-bp difference from pHeN867, which results from the lack of segment C (499 bp) and an 8-bp flanking sequence (GGAGGCCA) between segment C and the fusion region of segments D and B.…”

Section: Plasmid-encoding Colistin Resistance Gene Mcr-13mentioning

confidence: 99%

Colistin Resistance Gene mcr-1 and Its Variant in Escherichia coli Isolates from Chickens in China

Yang

Song

et al. 2017

Antimicrob Agents Chemother

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The mcr-1 gene was detected in 5.11% (58/1136) of Escherichia coli isolates of chicken origin from 13 provinces in China. A novel mcr-1 variant, named mcr-1.3, encoding an Ile-to-Val functional variant of MCR-1 was identified in a sequence type 155 (ST155) strain. An mcr-1.3-containing IncI2 plasmid, pHeN867 (60,757 bp), was identified. The transfer of pHeN867 led to a 32-fold increase in the MIC of colistin in the recipient, exhibiting an effect on colistin resistance that was similar to that of mcr-1. KEYWORDS E. coli, colistin resistance, mcr-1.3, plasmid P olymyxins (polymyxin B and colistin) are a last-resort treatment for infections caused by multidrug-resistant (MDR) Gram-negative bacteria (1). In veterinary use, colistin is administered with food in pig and poultry farming to prevent infections caused by pathogens (2). The mcr-1 gene, which confers plasmid-mediated colistin resistance to Enterobacteriaceae, was identified in an IncI2 plasmid from Escherichia coli and Klebsiella pneumoniae in China in 2016 (3). The mcr-1 gene found in E. coli (4), K. pneumoniae (5), and Salmonella spp. (6) has been proven to disseminate ubiquitously. The transmission of mcr-1-mediated colistin resistance between animals and human has been a threat to human health. It has also been demonstrated that the mcr-1 gene can coexist with bla CTX-M (5), bla NDM (7), and other resistance genes (4), which threatens a return of untreatable infections worldwide. Previous reports described the unique mcr-1 gene sequence compared with that of the originally published sequence (3), which indicates that mcr-1 is relatively conserved. Recently, a point mutation of A¡T at position 8 in mcr-1 was identified in K. pneumoniae (8). To investigate the epidemiology of mcr-1 and its variant, E. coli isolates collected from chickens nationwide in China were assessed.In total, 1,136 nonduplicate E. coli isolates were collected between 2010 and 2015 from sick chickens in 20 provinces and municipalities in China. All of these isolates were preliminarily screened on Mueller-Hinton agar medium with 2 g/ml colistin. Because the cooccurrence of mcr-1 with bla CTX-M may accelerate the transmission of resistance to colistin and cephalosporins, the mcr-1 (3) and bla CTX-M (9) genes were detected by PCR amplification of the isolates with resistance to colistin. The corresponding primers used to amplify the whole mcr-1 gene and parts of the ISApl1 element are listed in Table S1 in the supplemental material. For all of the positive PCR products of mcr-1, Sanger sequencing was performed (Tsingke Biological Technology, Chengdu, China) by using a DNA analyzer (Applied Biosystems, Life Technologies, Carlsbad, CA). We found a total of 58 (5.11%) mcr-1-positive isolates, including one isolate harboring the mcr-1 gene with mutations not found in the originally published gene sequence (3). MICs of colistin

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IncI shufflons: Assembly issues in the next-generation sequencing era

Cited by 32 publications

References 35 publications

Plasmids Carrying blaCMY -2/4 in Escherichia coli from Poultry, Poultry Meat, and Humans Belong to a Novel IncK Subgroup Designated IncK2

Plasmids Carrying blaCMY -2/4 in Escherichia coli from Poultry, Poultry Meat, and Humans Belong to a Novel IncK Subgroup Designated IncK2

Elucidation of quantitative structural diversity of remarkable rearrangement regions, shufflons, in IncI2 plasmids

Colistin Resistance Gene mcr-1 and Its Variant in Escherichia coli Isolates from Chickens in China

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