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 Conjugative Delivery of CRISPR-Cas9 for the Selective Depletion of Antibiotic-Resistant Enterococci

2019 ◽  
Vol 63 (11) ◽  
Author(s):  
Marinelle Rodrigues ◽  
Sara W. McBride ◽  
Karthik Hullahalli ◽  
Kelli L. Palmer ◽  
Breck A. Duerkop
Keyword(s):  
Antibiotic Resistance ◽  
Bacterial Infections ◽  
Antibiotic Resistance Genes ◽  
Multidrug Resistant ◽  
Conjugative Plasmid ◽  
Antibiotic Resistant ◽  
Content Type ◽  
Resistance Determinants

ABSTRACT The innovation of new therapies to combat multidrug-resistant (MDR) bacteria is being outpaced by the continued rise of MDR bacterial infections. Of particular concern are hospital-acquired infections (HAIs) that are recalcitrant to antibiotic therapies. The Gram-positive intestinal pathobiont Enterococcus faecalis is associated with HAIs, and some strains are MDR. Therefore, novel strategies to control E. faecalis populations are needed. We previously characterized an E. faecalis type II CRISPR-Cas system and demonstrated its utility in the sequence-specific removal of antibiotic resistance determinants. Here, we present work describing the adaption of this CRISPR-Cas system into a constitutively expressed module encoded on a pheromone-responsive conjugative plasmid that efficiently transfers to E. faecalis for the selective removal of antibiotic resistance genes. Using in vitro competition assays, we show that these CRISPR-Cas-encoding delivery plasmids, or CRISPR-Cas antimicrobials, can reduce the occurrence of antibiotic resistance in enterococcal populations in a sequence-specific manner. Furthermore, we demonstrate that deployment of CRISPR-Cas antimicrobials in the murine intestine reduces the occurrence of antibiotic-resistant E. faecalis by several orders of magnitude. Finally, we show that E. faecalis donor strains harboring CRISPR-Cas antimicrobials are immune to uptake of antibiotic resistance determinants in vivo. Our results demonstrate that conjugative delivery of CRISPR-Cas antimicrobials may be adaptable for future deployment from probiotic bacteria for exact targeting of defined MDR bacteria or for precision engineering of polymicrobial communities in the mammalian intestine.

scholarly journals Clinical extended-spectrum beta-lactamase antibiotic resistance plasmids have diverse transfer rates and can spread in the absence of antibiotic selection

2019 ◽  
Author(s):  
Fabienne Benz ◽  
Jana S. Huisman ◽  
Erik Bakkeren ◽  
Joana A. Herter ◽  
Tanja Stadler ◽  
...  
Keyword(s):  
Antibiotic Resistance ◽  
Conjugative Transfer ◽  
Beta Lactamase ◽  
Extended Spectrum Beta Lactamase ◽  
Extended Spectrum ◽  
Resistance Plasmids ◽  
Serovar Typhimurium ◽  
Spreading Potential

AbstractHorizontal gene transfer, mediated by conjugative plasmids, is a major driver of the global spread of antibiotic resistance. However, the relative contributions of factors that underlie the spread of clinically relevant plasmids are unclear. Here, we quantified conjugative transfer dynamics of Extended Spectrum Beta-Lactamase (ESBL) producing plasmids in the absence of antibiotics. We showed that clinical Escherichia coli strains natively associated with ESBL-plasmids conjugate efficiently with three distinct E. coli strains and one Salmonella enterica serovar Typhimurium strain, reaching final transconjugant frequencies of up to 1% within 24 hours in vitro. The variation of final transconjugant frequencies varied among plasmids, donors and recipients and was better explained by variation in conjugative transfer efficiency than by variable clonal expansion. We identified plasmid-specific genetic factors, specifically the presence/absence of transfer genes, that influenced final transconjugant frequencies. Finally, we investigated plasmid spread within the mouse intestine, demonstrating qualitative agreement between plasmid spread in vitro and in vivo. This suggests a potential for the prediction of plasmid spread in the gut of animals and humans, based on in vitro testing. Altogether, this may allow the identification of resistance plasmids with high spreading potential and help to devise appropriate measures to restrict their spread.

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 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 Strong Environment-Genotype Interactions Determine the Fitness Costs of Antibiotic Resistance In Vitro and in an Insect Model of Infection

2020 ◽  
Vol 64 (10) ◽  
Author(s):  
C. James Manktelow ◽  
Elitsa Penkova ◽  
Lucy Scott ◽  
Andrew C. Matthews ◽  
Ben Raymond
Keyword(s):  
Antibiotic Resistance ◽  
Resistance Mutations ◽  
Fitness Costs ◽  
Mechanisms Of Resistance ◽  
Content Type ◽  
Poor Predictor ◽  
Fitness Consequences ◽  
Rifampin Resistance

ABSTRACT The acquisition of antibiotic resistance commonly imposes fitness costs, a reduction in the fitness of bacteria in the absence of drugs. These costs have been quantified primarily using in vitro experiments and a small number of in vivo studies in mice, and it is commonly assumed that these diverse methods are consistent. Here, we used an insect model of infection to compare the fitness costs of antibiotic resistance in vivo to those in vitro. Experiments explored diverse mechanisms of resistance in a Gram-positive pathogen, Bacillus thuringiensis, and a Gram-negative intestinal symbiont, Enterobacter cloacae. Rifampin resistance in B. thuringiensis showed fitness costs that were typically elevated in vivo, although these were modulated by genotype-environment interactions. In contrast, resistance to cefotaxime via derepression of AmpC β-lactamase in E. cloacae resulted in no detectable costs in vivo or in vitro, while spontaneous resistance to nalidixic acid, and carriage of the IncP plasmid RP4, imposed costs that increased in vivo. Overall, fitness costs in vitro were a poor predictor of fitness costs in vivo because of strong genotype-environment interactions throughout this study. Insect infections provide a cheap and accessible means of assessing the fitness consequences of resistance mutations, data that are important for understanding the evolution and spread of resistance. This study emphasizes that the fitness costs imposed by particular mutations or different modes of resistance are extremely variable and that only a subset of these mutations is likely to be prevalent outside the laboratory.

scholarly journals Antibody-Based Immunotherapy To Treat and Prevent Infection with Hypervirulent Klebsiella pneumoniae

2016 ◽  
Vol 24 (1) ◽  
Author(s):  
Elizabeth Diago-Navarro ◽  
Isabel Calatayud-Baselga ◽  
Donglei Sun ◽  
Camille Khairallah ◽  
Inderjit Mann ◽  
...  
Keyword(s):  
Antibiotic Resistance ◽  
Klebsiella Pneumoniae ◽  
Intravital Microscopy ◽  
Protective Antigen ◽  
Protective Efficacy ◽  
Mesenteric Lymph Nodes ◽  
Content Type ◽  
Anthrax Protective Antigen

ABSTRACT Hypervirulent Klebsiella pneumoniae (hvKp) strains are predicted to become a major threat in Asia if antibiotic resistance continues to spread. Anticapsular antibodies (Abs) were developed because disseminated infections caused by hvKp are associated with significant morbidity and mortality, even with antibiotic-sensitive strains. K1-serotype polysaccharide capsules (K1-CPS) are expressed by the majority of hvKp strains. In this study, K1-CPS-specific IgG Abs were generated by conjugation of K1-CPS to immunogenic anthrax protective antigen (PA) protein. Opsonophagocytic efficacy was measured in vitro and in vivo by intravital microscopy in murine livers. In vivo protection was tested in murine models, including a novel model for dissemination in hvKp-colonized mice. Protective efficacy of monoclonal antibodies (MAbs) 4C5 (IgG1) and 19A10 (IgG3) was demonstrated both in murine sepsis and pulmonary infection. In hvKp-colonized mice, MAb treatment significantly decreased dissemination of hvKp from the gut to mesenteric lymph nodes and organs. Intravital microscopy confirmed efficient opsonophagocytosis and clearance of bacteria from the liver. In vitro studies demonstrate that MAbs work predominantly by promoting FcR-mediated phagocytosis but also indicate that MAbs enhance the release of neutrophil extracellular traps (NETs). In anticipation of increasing antibiotic resistance, we propose further development of these and other Klebsiella-specific MAbs for therapeutic use.

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 Enterococcus faecalis Sex Pheromone cCF10 Enhances Conjugative Plasmid Transfer In Vivo

mBio ◽  
2018 ◽  
Vol 9 (1) ◽  
Author(s):  
Helmut Hirt ◽  
Kerryl E. Greenwood-Quaintance ◽  
Melissa J. Karau ◽  
Lisa M. Till ◽  
Purna C. Kashyap ◽  
...  
Keyword(s):  
Enterococcus Faecalis ◽  
High Frequency ◽  
Intestinal Tract ◽  
Bacteriocin Production ◽  
Plasmid Transfer ◽  
Conjugative Plasmid ◽  
Conjugative Plasmid Transfer ◽  
Pheromone Signaling

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.

scholarly journals Assessment of Immune Response and Efficacy of Essential Oils Application on Controlling Necrotic Enteritis Induced by Clostridium perfringens in Broiler Chickens

Molecules ◽  
2021 ◽  
Vol 26 (15) ◽  
pp. 4527
Author(s):  
Mohammad Gharaibeh ◽  
Mohammad Khalifeh ◽  
Adi Nawasreh ◽  
Wael Hananeh ◽  
Mofleh Awawdeh
Keyword(s):  
Antibacterial Activity ◽  
Clostridium Perfringens ◽  
In Vitro Study ◽  
Vitro Study ◽  
In Vivo Study ◽  
Necrotic Enteritis ◽  
Bacterial Counts ◽  
Small Intestines

Necrotic enteritis (NE) caused by Clostridium perfringens is one of the most important enteric diseases in poultry. The antibacterial activity of two different essential oil (EO) blends against C. perfringens was investigated both in vitro and in vivo. Additionally, the immunological response to EO treatment was assessed. In the in vitro study, the antibacterial activity of EO formulas and commonly used antibiotics was evaluated against C. perfringens using disk diffusion assay, minimum inhibitory concentration (MIC) assay, and minimum bactericidal concentration (MBC) assay. In the in vivo study, NE experimental infection was performed on 440 Ross broiler chicks at 19 days of age for 4 continuous days. The chicks were treated with either EOs or amoxicillin at 22 days of age for 5 continuous days. One day after the end of treatment, the birds’ performance was evaluated by calculating the feed conversion ratio. Serum samples from 120 birds were collected to measure the levels of IL-1β, IFN-γ, IL-8, IL-10, and IL-17. After that, all birds were slaughtered, and their small intestines were subjected to gross and histopathological evaluation. In addition, bacterial counts in the small intestines were evaluated. In the in vitro study, EOs showed higher antimicrobial activities in comparison with antibiotics against C. perfringens. In the in vivo study, birds treated with EOs showed a significant decrease in bacterial counts, a significant decrease in intestinal lesions, and a significant improvement in performance compared with untreated birds (p < 0.05). Moreover, treating birds with EOs directed the immune system toward an anti-inflammatory pathway. None of the treated birds died due to NE compared with the 10% mortality rate in untreated birds. In conclusion, EOs might be an effective and safe alternative to antibiotics in the treatment of chicken NE.

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