scholarly journals Functional Analysis of a Bacitracin Resistance Determinant Located on ICECp1, a Novel Tn916-Like Element from a Conjugative Plasmid in Clostridium perfringens

2015 ◽  
Vol 59 (11) ◽  
pp. 6855-6865 ◽  
Author(s):  
Xiaoyan Han ◽  
Xiang-Dang Du ◽  
Luke Southey ◽  
Dieter M. Bulach ◽  
Torsten Seemann ◽  
...  
Keyword(s):  
Clostridium Perfringens ◽  
Wild Type Strain ◽  
Regulatory Gene ◽  
Conjugative Plasmid ◽  
Resistance Determinant ◽  
Toxin Gene ◽  
Resistance Locus ◽  
Necrotic Enteritis ◽  
Content Type ◽  
Integrative Conjugative Elements

ABSTRACTBacitracins are mixtures of structurally related cyclic polypeptides with antibiotic properties. They act by interfering with the biosynthesis of the bacterial cell wall. In this study, we analyzed an avian necrotic enteritis strain ofClostridium perfringensthat was resistant to bacitracin and produced NetB toxin. We identified a bacitracin resistance locus that resembled a bacitracin resistance determinant fromEnterococcus faecalis. It contained the structural genesbcrABDand a putative regulatory gene,bcrR. Mutagenesis studies provided evidence that bothbcrAandbcrBare essential for bacitracin resistance, and that evidence was supported by the results of experiments in which the introduction of both thebcrAandbcrBgenes into a bacitracin-susceptibleC. perfringensstrain was required to confer bacitracin resistance. The wild-type strain was shown to contain at least three large, putatively conjugative plasmids, and thebcrRABDlocus was localized to an 89.7-kb plasmid, pJIR4150. This plasmid was experimentally shown to be conjugative and was sequenced. The sequence revealed that it also carries atpeLtoxin gene and is related to the pCW3 family of conjugative antibiotic resistance and toxin plasmids fromC. perfringens. Thebcrgenes were located on a genetic element, ICECp1, which is related to the Tn916family of integrative conjugative elements (ICEs). ICECp1appears to be the first Tn916-like element shown to confer bacitracin resistance. In summary, we identified in a toxin-producingC. perfringensstrain a novel mobile bacitracin resistance element which was experimentally shown to be essential for bacitracin resistance and is carried by a putative ICE located on a conjugative plasmid.

scholarly journals Conjugation-Mediated Horizontal Gene Transfer of Clostridium perfringens Plasmids in the Chicken Gastrointestinal Tract Results in the Formation of New Virulent Strains

2017 ◽  
Vol 83 (24) ◽  
Author(s):  
Jake A. Lacey ◽  
Anthony L. Keyburn ◽  
Mark E. Ford ◽  
Ricardo W. Portela ◽  
Priscilla A. Johanesen ◽  
...  
Keyword(s):  
Antibiotic Resistance ◽  
Clostridium Perfringens ◽  
Plasmid Transfer ◽  
Conjugative Plasmid ◽  
Conjugative Transfer ◽  
Necrotic Enteritis ◽  
Content Type ◽  
Gastrointestinal Pathogen

ABSTRACT Clostridium perfringens is a gastrointestinal pathogen capable of causing disease in a variety of hosts. Necrotic enteritis in chickens is caused by C. perfringens strains that produce the pore-forming toxin NetB, the major virulence factor for this disease. Like many other C. perfringens toxins and antibiotic resistance genes, NetB is encoded on a conjugative plasmid. Conjugative transfer of the netB-containing plasmid pJIR3535 has been demonstrated in vitro with a netB-null mutant. This study has investigated the effect of plasmid transfer on disease pathogenesis, with two genetically distinct transconjugants constructed under in vitro conditions, within the intestinal tract of chickens. This study also demonstrates that plasmid transfer can occur naturally in the host gut environment without the need for antibiotic selective pressure to be applied. The demonstration of plasmid transfer within the chicken host may have implications for the progression and pathogenesis of C. perfringens-mediated disease. Such horizontal gene transfer events are likely to be common in the clostridia and may be a key factor in strain evolution, both within animals and in the wider environment. IMPORTANCE Clostridium perfringens is a major gastrointestinal pathogen of poultry. C. perfringens strains that express the NetB pore-forming toxin, which is encoded on a conjugative plasmid, cause necrotic enteritis. This study demonstrated that the conjugative transfer of the netB-containing plasmid to two different nonpathogenic strains converted them into disease-causing strains with disease-causing capability similar to that of the donor strain. Plasmid transfer of netB and antibiotic resistance was also demonstrated to occur within the gastrointestinal tract of chickens, with approximately 14% of the isolates recovered comprising three distinct, in vivo-derived, transconjugant types. The demonstration of in vivo plasmid transfer indicates the potential importance of strain plasticity and the contribution of plasmids to strain virulence.

scholarly journals Transposase-Mediated Excision, Conjugative Transfer, and Diversity of ICE6013 Elements in Staphylococcus aureus

2017 ◽  
Vol 199 (8) ◽  
Author(s):  
Emily A. Sansevere ◽  
Xiao Luo ◽  
Joo Youn Park ◽  
Sunghyun Yoon ◽  
Keun Seok Seo ◽  
...  
Keyword(s):  
Staphylococcus Aureus ◽  
Sequence Analysis ◽  
Integration Site ◽  
Consensus Sequence ◽  
Open Reading Frames ◽  
Conjugative Plasmid ◽  
Site Preference ◽  
Conjugative Transfer ◽  
Content Type ◽  
Integrative Conjugative Elements

ABSTRACT ICE6013 represents one of two families of integrative conjugative elements (ICEs) identified in the pan-genome of the human and animal pathogen Staphylococcus aureus. Here we investigated the excision and conjugation functions of ICE6013 and further characterized the diversity of this element. ICE6013 excision was not significantly affected by growth, temperature, pH, or UV exposure and did not depend on recA. The IS30-like DDE transposase (Tpase; encoded by orf1 and orf2) of ICE6013 must be uninterrupted for excision to occur, whereas disrupting three of the other open reading frames (ORFs) on the element significantly affects the level of excision. We demonstrate that ICE6013 conjugatively transfers to different S. aureus backgrounds at frequencies approaching that of the conjugative plasmid pGO1. We found that excision is required for conjugation, that not all S. aureus backgrounds are successful recipients, and that transconjugants acquire the ability to transfer ICE6013. Sequencing of chromosomal integration sites in serially passaged transconjugants revealed a significant integration site preference for a 15-bp AT-rich palindromic consensus sequence, which surrounds the 3-bp target site that is duplicated upon integration. A sequence analysis of ICE6013 from different host strains of S. aureus and from eight other species of staphylococci identified seven divergent subfamilies of ICE6013 that include sequences previously classified as a transposon, a plasmid, and various ICEs. In summary, these results indicate that the IS30-like Tpase functions as the ICE6013 recombinase and that ICE6013 represents a diverse family of mobile genetic elements that mediate conjugation in staphylococci. IMPORTANCE Integrative conjugative elements (ICEs) encode the abilities to integrate into and excise from bacterial chromosomes and plasmids and mediate conjugation between bacteria. As agents of horizontal gene transfer, ICEs may affect bacterial evolution. ICE6013 represents one of two known families of ICEs in the pathogen Staphylococcus aureus, but its core functions of excision and conjugation are not well studied. Here, we show that ICE6013 depends on its IS30-like DDE transposase for excision, which is unique among ICEs, and we demonstrate the conjugative transfer and integration site preference of ICE6013. A sequence analysis revealed that ICE6013 has diverged into seven subfamilies that are dispersed among staphylococci.

scholarly journals Draft Genome Sequence of Clostridium perfringens Strain TAMU, Which Causes Necrotic Enteritis in Broiler Chickens

2020 ◽  
Vol 9 (4) ◽  
Author(s):  
Catherine Ausland ◽  
Adil Sabr Al-Ogaili ◽  
Juan D. Latorre ◽  
Guillermo Tellez-Isaias ◽  
Billy M. Hargis ◽  
...  
Keyword(s):  
Clostridium Perfringens ◽  
Genome Sequence ◽  
Broiler Chickens ◽  
Draft Genome ◽  
Gastrointestinal Diseases ◽  
Intervention Strategies ◽  
Draft Genome Sequence ◽  
Necrotic Enteritis ◽  
Content Type ◽  
Deadly Disease

Clostridium perfringens causes severe gastrointestinal diseases, which include necrotic enteritis (NE) in chickens, a deadly disease worldwide. We report here the draft genome sequence of Clostridium perfringens strain TAMU, which was used in developing an NE chicken challenge model. This C. perfringens TAMU genome sequence will aid in advancing potential intervention strategies to reduce NE pathogenesis.

scholarly journals Necrotic Enteritis-Derived Clostridium perfringens Strain with Three Closely Related Independently Conjugative Toxin and Antibiotic Resistance Plasmids

mBio ◽  
2011 ◽  
Vol 2 (5) ◽  
Author(s):  
Trudi L. Bannam ◽  
Xu-Xia Yan ◽  
Paul F. Harrison ◽  
Torsten Seemann ◽  
Anthony L. Keyburn ◽  
...  
Keyword(s):  
Antibiotic Resistance ◽  
Clostridium Perfringens ◽  
Tetracycline Resistance ◽  
Necrotic Enteritis ◽  
Sequencing Analysis ◽  
Content Type ◽  
Conjugative Plasmids ◽  
Resistance Plasmid ◽  
Large Plasmids ◽  
Beta2 Toxin

ABSTRACTThe pathogenesis of avian necrotic enteritis involves NetB, a pore-forming toxin produced by virulent avian isolates ofClostridium perfringenstype A. To determine the location and mobility of thenetBstructural gene, we examined a derivative of the tetracycline-resistant necrotic enteritis strain EHE-NE18, in whichnetBwas insertionally inactivated by the chloramphenicol and thiamphenicol resistance genecatP. Both tetracycline and thiamphenicol resistance could be transferred either together or separately to a recipient strain in plate matings. The separate transconjugants could act as donors in subsequent matings, which demonstrated that the tetracycline resistance determinant and thenetBgene were present on different conjugative elements. Large plasmids were isolated from the transconjugants and analyzed by high-throughput sequencing. Analysis of the resultant data indicated that there were actually three large conjugative plasmids present in the original strain, each with its own toxin or antibiotic resistance locus. Each plasmid contained a highly conserved 40-kb region that included plasmid replication and transfer regions that were closely related to the 47-kb conjugative tetracycline resistance plasmid pCW3 fromC. perfringens. The plasmids were as follows: (i) a conjugative 49-kb tetracycline resistance plasmid that was very similar to pCW3, (ii) a conjugative 82-kb plasmid that contained thenetBgene and other potential virulence genes, and (iii) a 70-kb plasmid that carried thecpb2gene, which encodes a different pore-forming toxin, beta2 toxin.IMPORTANCEThe anaerobic bacteriumClostridium perfringenscan cause an avian gastrointestinal disease known as necrotic enteritis. Disease pathogenesis is not well understood, although the plasmid-encoded pore-forming toxin NetB, is an important virulence factor. In this work, we have shown that the plasmid that carries thenetBgene is conjugative and has a 40-kb region that is very similar to replication and transfer regions found within each of the sequenced conjugative plasmids fromC. perfringens. We also showed that this strain contained two additional large plasmids that were also conjugative and carried a similar 40-kb region. One of these plasmids encoded beta2 toxin, and the other encoded tetracycline resistance. To our knowledge, this is the first report of a bacterial strain that carries three closely related but different independently conjugative plasmids. These results have significant implications for our understanding of the transmission of virulence and antibiotic resistance genes in pathogenic bacteria.

scholarly journals Quantification of Cell Proliferation and Alpha-Toxin Gene Expression of Clostridium perfringens in the Development of Necrotic Enteritis in Broiler Chickens

2007 ◽  
Vol 73 (21) ◽  
pp. 7110-7113 ◽  
Author(s):  
Weiduo Si ◽  
Joshua Gong ◽  
Yanming Han ◽  
Hai Yu ◽  
John Brennan ◽  
...  
Keyword(s):  
Gene Expression ◽  
Cell Proliferation ◽  
Regression Model ◽  
Clostridium Perfringens ◽  
Broiler Chickens ◽  
Toxin Gene ◽  
Necrotic Enteritis ◽  
Lesion Score ◽  
Alpha Toxin ◽  
Toxin Gene Expression

ABSTRACT Cell proliferation and alpha-toxin gene expression of Clostridium perfringens in relation to the development of necrotic enteritis (NE) were investigated. Unlike bacitracin-treated chickens, non-bacitracin-treated birds exhibited typical NE symptoms and reduced growth performance. They also demonstrated increased C. perfringens proliferation and alpha-toxin gene expression that were positively correlated and progressed according to the regression model y = b 0 + b 1 X − b 2 X 2. The average C. perfringens count of 5 log10 CFU/g in the ileal digesta appears to be a threshold for developing NE with a lesion score of 2.

scholarly journals Clostridium perfringens–Associated Necrotic Enteritis-Like Disease in Coconut Lorikeets (Trichoglossus haematodus)

2020 ◽  
pp. 030098582097178
Author(s):  
Llorenç Grau-Roma ◽  
Mauricio Navarro ◽  
Sohvi Blatter ◽  
Christian Wenker ◽  
Sonja Kittl ◽  
...  
Keyword(s):  
Clostridium Perfringens ◽  
Toxin Gene ◽  
Necrotic Enteritis ◽  
Gene Encoding ◽  
Formalin Fixed Paraffin ◽  
Formalin Fixed Paraffin Embedded ◽  
Ffpe Tissues ◽  
Polymerase Chain ◽  
Beta Toxin ◽  
Formalin Fixed

Several outbreaks of necrotic enteritis-like disease in lorikeets, from which Clostridium perfringens was consistently isolated, are described. All lorikeets had acute, segmental, or multifocal fibrinonecrotizing inflammatory lesions in the small and/or the large intestine, with intralesional gram-positive rods. The gene encoding C. perfringens alpha toxin was detected by PCR (polymerase chain reaction) on formalin-fixed, paraffin-embedded (FFPE) tissues in 20 out of 24 affected lorikeets (83%), but it was not amplified from samples of any of 10 control lorikeets ( P < .0001). The second most prevalent C. perfringens toxin gene detected was the beta toxin gene, which was found in FFPE from 7 out of 24 affected lorikeets (29%). The other toxin genes were detected inconsistently and in a relatively low number of samples. These cases seem to be associated with C. perfringens, although the specific type involved could not be determined.

scholarly journals Prevalence of netB-positive Clostridium perfringens in Italian poultry flocks by environmental sampling

2019 ◽  
Vol 32 (2) ◽  
pp. 252-258 ◽  
Author(s):  
Francesca Profeta ◽  
Cristina E. Di Francesco ◽  
Andrea Di Provvido ◽  
Massimo Scacchia ◽  
Alessandra Alessiani ◽  
...  
Keyword(s):  
Clostridium Perfringens ◽  
Toxin Gene ◽  
Environmental Sampling ◽  
Necrotic Enteritis ◽  
Enteric Diseases ◽  
Enteric Disorders ◽  
The Impact ◽  
Encoding Genes ◽  
Very High ◽  
Turkey Flock

Clostridium perfringens type G is one of the pathogens involved in enteric diseases in poultry. NetB, a pore-forming toxin, is considered the main virulence factor responsible for necrotic enteritis during C. perfringens infection. We carried out a field study involving 14 farms to evaluate the occurrence of netB-positive C. perfringens and the impact of infection in Italian poultry flocks. Environmental samples ( n = 117) and 50 carcasses were screened by microbiologic and molecular methods. Microbiologic investigations yielded 82 C. perfringens isolates. DNA was extracted from all samples and screened for α-toxin and NetB encoding genes by real-time PCR. The C. perfringens α-toxin gene was detected in 151 of 167 extracts (90.4%), and 31 of 151 (20.5%) were netB gene positive also. Sixteen isolates from a turkey flock with mild enteric disorders were also netB positive, demonstrating their occurrence not only in broiler but also in turkey flocks. A pulsed-field gel electrophoresis protocol was optimized to evaluate the diversity among isolates and revealed high genetic heterogeneity. The complete NetB toxin-coding gene of 2 C. perfringens isolates from turkey and broiler flocks were analyzed and showed very high relatedness with analogous sequences worldwide.

scholarly journals The VirS/VirR Two-Component System Regulates the Anaerobic Cytotoxicity, Intestinal Pathogenicity, and Enterotoxemic Lethality of Clostridium perfringens Type C Isolate CN3685

mBio ◽  
2011 ◽  
Vol 2 (1) ◽  
Author(s):  
Menglin Ma ◽  
Jorge Vidal ◽  
Juliann Saputo ◽  
Bruce A. McClane ◽  
Francisco Uzal
Keyword(s):  
Clostridium Perfringens ◽  
Regulatory System ◽  
Gas Gangrene ◽  
Necrotic Enteritis ◽  
Vegetative Cells ◽  
Content Type ◽  
Two Component ◽  
Type C ◽  
Beta Toxin

ABSTRACT Clostridium perfringens vegetative cells cause both histotoxic infections (e.g., gas gangrene) and diseases originating in the intestines (e.g., hemorrhagic necrotizing enteritis or lethal enterotoxemia). Despite their medical and veterinary importance, the molecular pathogenicity of C. perfringens vegetative cells causing diseases of intestinal origin remains poorly understood. However, C. perfringens beta toxin (CPB) was recently shown to be important when vegetative cells of C. perfringens type C strain CN3685 induce hemorrhagic necrotizing enteritis and lethal enterotoxemia. Additionally, the VirS/VirR two-component regulatory system was found to control CPB production by CN3685 vegetative cells during aerobic infection of cultured enterocyte-like Caco-2 cells. Using an isogenic virR null mutant, the current study now reports that the VirS/VirR system also regulates CN3685 cytotoxicity during infection of Caco-2 cells under anaerobic conditions, as found in the intestines. More importantly, the virR mutant lost the ability to cause hemorrhagic necrotic enteritis in rabbit small intestinal loops. Western blot analyses demonstrated that the VirS/VirR system mediates necrotizing enteritis, at least in part, by controlling in vivo CPB production. In addition, vegetative cells of the isogenic virR null mutant were, relative to wild-type vegetative cells, strongly attenuated in their lethality in a mouse enterotoxemia model. Collectively, these results identify the first regulator of in vivo pathogenicity for C. perfringens vegetative cells causing disease originating in the complex intestinal environment. Since VirS/VirR also mediates histotoxic infections, this two-component regulatory system now assumes a global role in regulating a spectrum of infections caused by C. perfringens vegetative cells. IMPORTANCE Clostridium perfringens is an important human and veterinary pathogen. C. perfringens vegetative cells cause both histotoxic infections, e.g., traumatic gas gangrene, and infections originating when this bacterium grows in the intestines. The VirS/VirR two-component regulatory system has been shown to control the pathogenicity of C. perfringens type A strains in a mouse gas gangrene model, but there is no understanding of pathogenicity regulation when C. perfringens vegetative cells cause disease originating in the complex intestinal environment. The current study establishes that VirS/VirR controls vegetative cell pathogenicity when C. perfringens type C isolates cause hemorrhagic necrotic enteritis and lethal enterotoxemia (i.e., toxin absorption from the intestines into the circulation, allowing targeting of internal organs). This effect involves VirS/VirR-mediated regulation of beta toxin production in vivo. Therefore, VirS/VirR is the first identified global in vivo regulator controlling the ability of C. perfringens vegetative cells to cause gas gangrene and, at least some, intestinal infections.

scholarly journals The Agr-Like Quorum Sensing System Is Required for Pathogenesis of Necrotic Enteritis Caused by Clostridium perfringens in Poultry

2017 ◽  
Vol 85 (6) ◽  
Author(s):  
Qiang Yu ◽  
Dion Lepp ◽  
Iman Mehdizadeh Gohari ◽  
Tao Wu ◽  
Hongzhuan Zhou ◽  
...  
Keyword(s):  
Quorum Sensing ◽  
Clostridium Perfringens ◽  
Global Gene Expression ◽  
Toxin Production ◽  
Phospholipid Metabolism ◽  
Necrotic Enteritis ◽  
Content Type ◽  
Protein Levels ◽  
Global Gene Expression Analysis

ABSTRACT Clostridium perfringens encodes at least two different quorum sensing (QS) systems, the Agr-like and LuxS, and recent studies have highlighted their importance in the regulation of toxin production and virulence. The role of QS in the pathogenesis of necrotic enteritis (NE) in poultry and the regulation of NetB, the key toxin involved, has not yet been investigated. We have generated isogenic agrB-null and complemented strains from parent strain CP1 and demonstrated that the virulence of the agrB-null mutant was strongly attenuated in a chicken NE model system and restored by complementation. The production of NetB, a key NE-associated toxin, was dramatically reduced in the agrB mutant at both the transcriptional and protein levels, though not in a luxS mutant. Transwell assays confirmed that the Agr-like QS system controls NetB production through a diffusible signal. Global gene expression analysis of the agrB mutant identified additional genes modulated by Agr-like QS, including operons related to phospholipid metabolism and adherence, which may also play a role in NE pathogenesis. This study provides the first evidence that the Agr-like QS system is critical for NE pathogenesis and identifies a number of Agr-regulated genes, most notably netB, that are potentially involved in mediating its effects. The Agr-like QS system thus may serve as a target for developing novel interventions to prevent NE in chickens.

Sign in / Sign up

Export Citation Format

Share Document