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Helmut Hirt - One of the best experts on this subject based on the ideXlab platform.
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Enterococcus faecalis sex pheromone cCF10 enhances Conjugative Plasmid transfer in vivo
mBio, 2018Co-Authors: Helmut Hirt, Kerryl E. Greenwood-quaintance, Melissa J. Karau, Lisa M. Till, Purna C. Kashyap, Robin Patel, Gary M. DunnyAbstract:ABSTRACT Cell-cell communication mediated by peptide pheromones (cCF10 [CF]) is essential for high-frequency Plasmid transfer in vitro in Enterococcus faecalis. To examine the role of pheromone signaling in vivo, we established either a CF-producing (CF+) recipient or a recipient producing a biologically inactive variant of CF (CF− recipient) in a germfree mouse model 3 days before donor inoculation and determined transfer frequencies of the pheromone-inducible Plasmid pCF10. Plasmid transfer was detected in the upper and middle sections of the intestinal tract 5 h after donor inoculation and was highly efficient in the absence of antibiotic selection. The transconjugant/donor ratio reached a maximum level approaching 1 on day 4 in the upper intestinal tract. Plasmid transfer was significantly lower with the CF− recipient. While rescue of the CF− mating defect by coculture with CF+ recipients is easily accomplished in vitro, no extracellular complementation occurred in vivo. This suggests that most pheromone signaling in the gut occurs between recipient and donor cells in very close proximity. Plasmid-bearing cells (donors plus transconjugants) steadily increased in the population from 0.1% after donor inoculation to about 10% at the conclusion of the experiments. This suggests a selective advantage of pCF10 carriage distinct from antibiotic resistance or bacteriocin production. Our results demonstrate that pheromone signaling is required for efficient pCF10 transfer in vivo. In the absence of CF+ recipients, a low level of transfer to CF− recipients occurred in the gut. This may result from low-level host-mediated induction of the donors in the gastrointestinal (GI) tract, similar to that previously observed in serum. IMPORTANCE Horizontal gene transfer is a major factor in the biology of Enterococcus faecalis, an important nosocomial pathogen. Previous studies showing efficient Conjugative Plasmid transfer in the gastrointestinal (GI) tracts of experimental animals did not examine how the enterococcal sex pheromone response impacts the efficiency of transfer. Our study demonstrates for the first time pheromone-enhanced, high-frequency Plasmid transfer of E. faecalis Plasmid pCF10 in a mouse model in the absence of antibiotic or bacteriocin selection. Pheromone production by recipients dramatically increased Plasmid transfer in germfree mice colonized initially with recipients, followed by donors. The presence of a coresident community of common gut microbes did not significantly reduce in vivo Plasmid transfer between enterococcal donors and recipients. In mice colonized with enterococcal recipients, we detected Plasmid transfer in the intestinal tract within 5 h of addition of donors, before transconjugants could be cultured from feces. Surprisingly, pCF10 carriage provided a competitive fitness advantage unrelated to antibiotic resistance or bacteriocin production.
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Enterococcus faecalis Sex Pheromone cCF10 Enhances Conjugative Plasmid Transfer In Vivo
American Society for Microbiology, 2018Co-Authors: Helmut Hirt, Kerryl E. Greenwood-quaintance, Melissa J. Karau, Lisa M. Till, Purna C. Kashyap, Robin Patel, Gary M. Dunny, Peter E. GreenbergAbstract:Cell-cell communication mediated by peptide pheromones (cCF10 [CF]) is essential for high-frequency Plasmid transfer in vitro in Enterococcus faecalis. To examine the role of pheromone signaling in vivo, we established either a CF-producing (CF+) recipient or a recipient producing a biologically inactive variant of CF (CF− recipient) in a germfree mouse model 3 days before donor inoculation and determined transfer frequencies of the pheromone-inducible Plasmid pCF10. Plasmid transfer was detected in the upper and middle sections of the intestinal tract 5 h after donor inoculation and was highly efficient in the absence of antibiotic selection. The transconjugant/donor ratio reached a maximum level approaching 1 on day 4 in the upper intestinal tract. Plasmid transfer was significantly lower with the CF− recipient. While rescue of the CF− mating defect by coculture with CF+ recipients is easily accomplished in vitro, no extracellular complementation occurred in vivo. This suggests that most pheromone signaling in the gut occurs between recipient and donor cells in very close proximity. Plasmid-bearing cells (donors plus transconjugants) steadily increased in the population from 0.1% after donor inoculation to about 10% at the conclusion of the experiments. This suggests a selective advantage of pCF10 carriage distinct from antibiotic resistance or bacteriocin production. Our results demonstrate that pheromone signaling is required for efficient pCF10 transfer in vivo. In the absence of CF+ recipients, a low level of transfer to CF− recipients occurred in the gut. This may result from low-level host-mediated induction of the donors in the gastrointestinal (GI) tract, similar to that previously observed in serum
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characterization of the pheromone response of the enterococcus faecalis Conjugative Plasmid pcf10 complete sequence and comparative analysis of the transcriptional and phenotypic responses of pcf10 containing cells to pheromone induction
Journal of Bacteriology, 2005Co-Authors: Helmut Hirt, Dawn A Manias, Edward M Bryan, Joanna R Klein, Jesper K Marklund, Jack H Staddon, Michael L Paustian, Vivek Kapur, Gary M. DunnyAbstract:The sex pheromone Plasmids in Enterococcus faecalis are one of the most efficient Conjugative Plasmid transfer systems known in bacteria. Plasmid transfer rates can reach or exceed 10−1 transconjugants per donor in vivo and under laboratory conditions. We report the completion of the DNA sequence of Plasmid pCF10 and the analysis of the transcription profile of Plasmid genes, relative to Conjugative transfer ability following pheromone induction. These experiments employed a mini-microarray containing all 57 open reading frames of pCF10 and a set of selected chromosomal genes. A clear peak of transcription activity was observed 30 to 60 min after pheromone addition, with transcription subsiding 2 h after pheromone induction. The transcript activity correlated with the ability of donor cells to transfer pCF10 to recipient cells. Remarkably, aggregation substance (Asc10, encoded by the prgB gene) was present on the cell surface for a long period of time after pheromone-induced transcription of prgB and Plasmid transfer ability had ceased. This observation could have relevance for the virulence of E. faecalis.
Gary M. Dunny - One of the best experts on this subject based on the ideXlab platform.
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Enterococcus faecalis sex pheromone cCF10 enhances Conjugative Plasmid transfer in vivo
mBio, 2018Co-Authors: Helmut Hirt, Kerryl E. Greenwood-quaintance, Melissa J. Karau, Lisa M. Till, Purna C. Kashyap, Robin Patel, Gary M. DunnyAbstract:ABSTRACT Cell-cell communication mediated by peptide pheromones (cCF10 [CF]) is essential for high-frequency Plasmid transfer in vitro in Enterococcus faecalis. To examine the role of pheromone signaling in vivo, we established either a CF-producing (CF+) recipient or a recipient producing a biologically inactive variant of CF (CF− recipient) in a germfree mouse model 3 days before donor inoculation and determined transfer frequencies of the pheromone-inducible Plasmid pCF10. Plasmid transfer was detected in the upper and middle sections of the intestinal tract 5 h after donor inoculation and was highly efficient in the absence of antibiotic selection. The transconjugant/donor ratio reached a maximum level approaching 1 on day 4 in the upper intestinal tract. Plasmid transfer was significantly lower with the CF− recipient. While rescue of the CF− mating defect by coculture with CF+ recipients is easily accomplished in vitro, no extracellular complementation occurred in vivo. This suggests that most pheromone signaling in the gut occurs between recipient and donor cells in very close proximity. Plasmid-bearing cells (donors plus transconjugants) steadily increased in the population from 0.1% after donor inoculation to about 10% at the conclusion of the experiments. This suggests a selective advantage of pCF10 carriage distinct from antibiotic resistance or bacteriocin production. Our results demonstrate that pheromone signaling is required for efficient pCF10 transfer in vivo. In the absence of CF+ recipients, a low level of transfer to CF− recipients occurred in the gut. This may result from low-level host-mediated induction of the donors in the gastrointestinal (GI) tract, similar to that previously observed in serum. IMPORTANCE Horizontal gene transfer is a major factor in the biology of Enterococcus faecalis, an important nosocomial pathogen. Previous studies showing efficient Conjugative Plasmid transfer in the gastrointestinal (GI) tracts of experimental animals did not examine how the enterococcal sex pheromone response impacts the efficiency of transfer. Our study demonstrates for the first time pheromone-enhanced, high-frequency Plasmid transfer of E. faecalis Plasmid pCF10 in a mouse model in the absence of antibiotic or bacteriocin selection. Pheromone production by recipients dramatically increased Plasmid transfer in germfree mice colonized initially with recipients, followed by donors. The presence of a coresident community of common gut microbes did not significantly reduce in vivo Plasmid transfer between enterococcal donors and recipients. In mice colonized with enterococcal recipients, we detected Plasmid transfer in the intestinal tract within 5 h of addition of donors, before transconjugants could be cultured from feces. Surprisingly, pCF10 carriage provided a competitive fitness advantage unrelated to antibiotic resistance or bacteriocin production.
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Enterococcus faecalis Sex Pheromone cCF10 Enhances Conjugative Plasmid Transfer In Vivo
American Society for Microbiology, 2018Co-Authors: Helmut Hirt, Kerryl E. Greenwood-quaintance, Melissa J. Karau, Lisa M. Till, Purna C. Kashyap, Robin Patel, Gary M. Dunny, Peter E. GreenbergAbstract:Cell-cell communication mediated by peptide pheromones (cCF10 [CF]) is essential for high-frequency Plasmid transfer in vitro in Enterococcus faecalis. To examine the role of pheromone signaling in vivo, we established either a CF-producing (CF+) recipient or a recipient producing a biologically inactive variant of CF (CF− recipient) in a germfree mouse model 3 days before donor inoculation and determined transfer frequencies of the pheromone-inducible Plasmid pCF10. Plasmid transfer was detected in the upper and middle sections of the intestinal tract 5 h after donor inoculation and was highly efficient in the absence of antibiotic selection. The transconjugant/donor ratio reached a maximum level approaching 1 on day 4 in the upper intestinal tract. Plasmid transfer was significantly lower with the CF− recipient. While rescue of the CF− mating defect by coculture with CF+ recipients is easily accomplished in vitro, no extracellular complementation occurred in vivo. This suggests that most pheromone signaling in the gut occurs between recipient and donor cells in very close proximity. Plasmid-bearing cells (donors plus transconjugants) steadily increased in the population from 0.1% after donor inoculation to about 10% at the conclusion of the experiments. This suggests a selective advantage of pCF10 carriage distinct from antibiotic resistance or bacteriocin production. Our results demonstrate that pheromone signaling is required for efficient pCF10 transfer in vivo. In the absence of CF+ recipients, a low level of transfer to CF− recipients occurred in the gut. This may result from low-level host-mediated induction of the donors in the gastrointestinal (GI) tract, similar to that previously observed in serum
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Model of induction of the pCF10 Conjugative Plasmid and reporter systems.
2017Co-Authors: Rebecca J. Breuer, Arpan Bandyopadhyay, Sofie A. O’brien, Aaron M. T. Barnes, Ryan C. Hunter, Gary M. DunnyAbstract:(A) The QL transcript from the pCF10 Plasmid encodes the proteins that mediate conjugation and is induced upon signaling by C pheromone (green stars) from potential recipient cells. The I inhibitory peptide (red stars) counteracts C and is produced by Plasmid-containing cells from a short transcript from the PQ promoter (red). PrgX complexes (green circles) repress the PQ promoter whereby PrgX-C complexes allow induction of QL transcription and PrgX or PrgX-I complexes inhibit transcription of QL. (B) The pBK2 and pCIE-GFP reporter Plasmid constructs have the same PQ/ QL regulatory region as pCF10. However, either lacZ for pBK2 or gfp for pCIE-GFP have been inserted in place of the conjugation genes.
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characterization of the pheromone response of the enterococcus faecalis Conjugative Plasmid pcf10 complete sequence and comparative analysis of the transcriptional and phenotypic responses of pcf10 containing cells to pheromone induction
Journal of Bacteriology, 2005Co-Authors: Helmut Hirt, Dawn A Manias, Edward M Bryan, Joanna R Klein, Jesper K Marklund, Jack H Staddon, Michael L Paustian, Vivek Kapur, Gary M. DunnyAbstract:The sex pheromone Plasmids in Enterococcus faecalis are one of the most efficient Conjugative Plasmid transfer systems known in bacteria. Plasmid transfer rates can reach or exceed 10−1 transconjugants per donor in vivo and under laboratory conditions. We report the completion of the DNA sequence of Plasmid pCF10 and the analysis of the transcription profile of Plasmid genes, relative to Conjugative transfer ability following pheromone induction. These experiments employed a mini-microarray containing all 57 open reading frames of pCF10 and a set of selected chromosomal genes. A clear peak of transcription activity was observed 30 to 60 min after pheromone addition, with transcription subsiding 2 h after pheromone induction. The transcript activity correlated with the ability of donor cells to transfer pCF10 to recipient cells. Remarkably, aggregation substance (Asc10, encoded by the prgB gene) was present on the cell surface for a long period of time after pheromone-induced transcription of prgB and Plasmid transfer ability had ceased. This observation could have relevance for the virulence of E. faecalis.
Jacques Mahillon - One of the best experts on this subject based on the ideXlab platform.
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a novel t4ss mediated dna transfer used by pxo16 a Conjugative Plasmid from bacillus thuringiensis serovar israelensis
Environmental Microbiology, 2018Co-Authors: Lionel Makart, Annika Gillis, Pauline Hinnekens, Jacques MahillonAbstract:The entomopathogenic Bacillus thuringiensis serovar israelensis displays peculiar Conjugative transfer capabilities, accounted for by the large Conjugative Plasmid pXO16 (350 kb). The efficient and fast Conjugative transfers are accompanied by a macroscopic aggregation of bacterial partners. Moreover, pXO16 has proven capable of effective mobilization and the retro-transfer of both mobilizable and 'non-mobilizable' Plasmids. In this work, the aggregation phenomenon is shown to promote pXO16 transfer while not being mandatory for transfer. Transfer of pXO16 to B. thuringiensis recipient strains that do not display aggregation is observed as well, hence enlarging the previously defined host range. The use of variant calling analysis of transconjugants allowed for observation of up to 791 kb chromosomal regions mobilization. Previous analysis of pXO16 did not reveal any Type IV Secretion System (T4SS) homologs, which suggested the presence of an unusual Conjugative system. A FtsK/SpOIIIE ATPase gene proved here to be necessary for Conjugative transfer. Additionally, the analysis of natural restriction-modification systems in both Conjugative partners gave credit to a ssDNA transfer mechanism. A 'transfer israelensis Plasmid' (tip) region containing this ATPase gene was shown to code for other potential T4SS proteins, illustrating a Conjugative system distantly related to the other known Gram-positive T4SSs.
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Conjugative Plasmid paw63 brings new insights into the genesis of the bacillus anthracis virulence Plasmid pxo2 and of the bacillus thuringiensis Plasmid pbt9727
BMC Genomics, 2005Co-Authors: Geraldine A Van Der Auwera, Lars Andrup, Jacques MahillonAbstract:Bacillus cereus, Bacillus anthracis and Bacillus thuringiensis belong to the genetically close-knit Bacillus cereus sensu lato group, a family of rod-shaped Gram-positive bacteria. pAW63 is the first Conjugative Plasmid from the B. cereus group to be completely sequenced. The 71,777 bp nucleotide sequence of pAW63 reveals a modular structure, including a 42 kb tra region encoding homologs of the Type IV secretion systems components VirB11, VirB4 and VirD4, as well as homologs of Gram-positive conjugation genes from Enterococcus, Lactococcus, Listeria, Streptococcus and Staphylococcus species. It also firmly establishes the existence of a common backbone between pAW63, pXO2 from Bacillus anthracis and pBT9727 from the pathogenic Bacillus thuringiensis serovar konkukian strain 97-27. The alignment of these three Plasmids highlights the presence of well conserved segments, in contrast to distinct regions of high sequence plasticity. The study of their specific differences has provided a three-point reference framework that can be exploited to formulate solid hypotheses concerning the functionalities and the molecular evolution of these three closely related Plasmids. This has provided insight into the chronology of their divergence, and led to the discovery of two Type II introns on pAW63, matching copies of the mobile element IS231 L in different loci of pXO2 and pBT9727, and the identification on pXO2 of a 37 kb pathogenicity island (PAI) containing the anthrax capsule genes. The complete sequence determination of pAW63 has led to a functional map of the Plasmid yielding insights into its Conjugative apparatus, which includes T4SS-like components, as well as its resemblance to other large Plasmids of Gram-positive bacteria. Of particular interest is the extensive homology shared between pAW63 and pXO2, the second virulence Plasmid of B. anthracis, as well as pBT9727 from the pathogenic strain B. thuringiensis serovar konkukian strain 97-27.
Jean M. Whichard - One of the best experts on this subject based on the ideXlab platform.
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imipenem resistance in a salmonella clinical strain due to Plasmid mediated class a carbapenemase kpc 2
Antimicrobial Agents and Chemotherapy, 2003Co-Authors: Vivi Miriagou, Shannon Rossiter, Eva Tzelepi, Leonidas S. Tzouvelekis, Frederick J Angulo, Jean M. WhichardAbstract:A Salmonella enterica serotype Cubana isolate exhibiting resistance to most β-lactam antibiotics, including oxyimino-cephalosporins and imipenem, was isolated from a 4-year-old boy with gastroenteritis in Maryland. β-Lactam resistance was mediated by a Conjugative Plasmid that encoded KPC-2, a class A carbapenemase previously found in a Klebsiella pneumoniae isolate from the Maryland area as well. Sequence analysis of the flanking regions indicated a potential association of blaKPC-2 with mobile structures.
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Imipenem resistance in a Salmonella clinical strain due to Plasmid-mediated class A carbapenemase KPC-2
Antimicrobial Agents and Chemotherapy, 2003Co-Authors: Vivi Miriagou, Shannon Rossiter, Eva Tzelepi, Leonidas S. Tzouvelekis, Frederick J Angulo, Jean M. WhichardAbstract:A Salmonella enterica serotype Cubana isolate exhibiting resistance to most beta-lactam antibiotics, including oxyimino-cephalosporins and imipenem, was isolated from a 4-year-old boy with gastroenteritis in Maryland. beta-Lactam resistance was mediated by a Conjugative Plasmid that encoded KPC-2, a class A carbapenemase previously found in a Klebsiella pneumoniae isolate from the Maryland area as well. Sequence analysis of the flanking regions indicated a potential association of bla(KPC-2) with mobile structures.
Sheng Chen - One of the best experts on this subject based on the ideXlab platform.
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a Conjugative Plasmid that augments virulence in klebsiella pneumoniae
Nature microbiology, 2019Co-Authors: Xuemei Yang, Sheng Chen, Edward Waichi Chan, Rong ZhangAbstract:A virulence-encoding Plasmid, p15WZ-82_Vir, which formed as a result of the integration of a 100-kb fragment of the hypervirulence Plasmid pLVPK into a Conjugative IncFIB Plasmid, was recovered from a clinical Klebsiella variicola strain. Such a Plasmid could be conjugated to carbapenem-resistant Klebsiella strains, enabling them to simultaneously express the carbapenem resistance- and hypervirulence-associated phenotypes. Unlike the non-Conjugative pLVPK Plasmid, emergence of p15WZ-82_Vir may promote rapid dissemination of virulence-encoding elements among Gram-negative bacterial pathogens.
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identification and characterization of a Conjugative blavim 1 bearing Plasmid in vibrio alginolyticus of food origin
Journal of Antimicrobial Chemotherapy, 2019Co-Authors: Zhiwei Zheng, Edward Waichi Chan, Sheng ChenAbstract:Objectives To investigate the genetic features of the blaVIM-1 gene first detected in a cephalosporin-resistant Vibrio alginolyticus isolate, Vb1978. Methods The MICs of V. alginolyticus strain Vb1978 were determined, and the β-lactamases produced were screened and analysed using conjugation, S1-PFGE and Southern blotting. The complete sequence of the blaVIM-1-encoding Plasmid was also obtained using the Illumina and MinION sequencing platforms. Results V. alginolyticus strain Vb1978, isolated from a retail shrimp sample, was resistant to cephalosporins and exhibited reduced susceptibility to carbapenems. A novel blaVIM-1-carrying Conjugative Plasmid, designated pVb1978, was identified in this strain. Plasmid pVb1978 had 50 001 bp and comprised 59 predicted coding sequences (CDSs). The Plasmid backbone of pVb1978 was homologous to those of IncP-type Plasmids, while its replication region was structurally similar to non-IncP Plasmids. The blaVIM-1 gene was found to be carried by the class 1 integron In70 and associated with a defective Tn402-like transposon. Conclusions A novel blaVIM-1-carrying Conjugative Plasmid, pVb1978, was reported for the first time in V. alginolyticus, which warrants further investigation in view of its potential pathogenicity towards humans and widespread occurrence in the environment.
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complete nucleotide sequence of a Conjugative Plasmid carrying blaper 1
Antimicrobial Agents and Chemotherapy, 2015Co-Authors: Marcus Ho Yin Wong, Yuanjie Zhou, Edward Chan, Sheng ChenAbstract:The nucleotide sequence of a self-transmissible Plasmid pVPH1 harboring bla(PER-1) from Vibrio parahaemolyticus was determined. pVPH1 was 183,730 bp in size and shared a backbone similar to pAQU1 and pAQU2, differing mainly in an ∼40-kb multidrug resistance (MDR) region. A complex class 1 integron was identified together with ISCR1 and bla(PER-1) (ISCR1-bla(PER-1)-gst-abct-qacEΔ1-sul1), which was shown to form a circular intermediate playing an important role in the dissemination of bla(PER-1).
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molecular characterisation of a multidrug resistance Conjugative Plasmid from vibrio parahaemolyticus
International Journal of Antimicrobial Agents, 2013Co-Authors: Ming Liu, Marcus Ho Yin Wong, Sheng ChenAbstract:Vibrio parahaemolyticus is a major causative agent of gastroenteritis and is the leading cause of food-borne illness in Hong Kong. Recent studies of resistance to extended-spectrum β-lactams and fluoroquinolones in V. parahaemolyticus have caused huge concern. This work reports the characterisation of a multidrug resistance Conjugative Plasmid in V. parahaemolyticus isolated from shrimp samples from Hong Kong. The Plasmid is ca. 200 kb and carries multidrug resistance genes, including a novel Plasmid-mediated quinolone resistance gene qnrVC6 surrounded by several known and novel insertion sequence (IS) elements, an extended-spectrum β-lactamase gene bla(PER-1) mediated by ISCR1, and a ca. 3-kb four-gene cassette (aacA3, catB2, dfrA1 and aadA1) class 1 integron. Transmission of this multidrug resistance Conjugative Plasmid among Vibrio spp. would compromise the effectiveness of Vibrio infection control and pose a huge threat to public health.