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1.
Int J Antimicrob Agents ; 52(5): 724-729, 2018 Nov.
Article in English | MEDLINE | ID: mdl-30145248

ABSTRACT

The increasing incidence and severity of diarrhoea and colitis caused by Clostridium difficile, together with a high rate of relapse following treatment with currently recommended antimicrobials, calls for novel interventions for C. difficile infection (CDI). Rhodomyrtone, a bioactive compound derived from the leaves of the rose myrtle (Rhodomyrtus tomentosa) has demonstrated antibacterial activity against several Gram-positive bacteria. This study compared the in vitro antimicrobial activity of rhodomyrtone on C. difficile with that of vancomycin, a recommended agent for the treatment of CDI. Determination of the minimum inhibitory concentrations (MICs) and minimum bactericidal concentrations (MBCs) of rhodomyrtone and vancomycin for ten C. difficile isolates showed that the MICs of rhodomyrtone for C. difficile vegetative cells (0.625-2.5 mg/L) were comparable with that of vancomycin (1.25 mg/L), but the MBCs of rhodomyrtone (1.25-5 mg/L) were significantly lower than those for vancomycin (5 mg/L to ˃40 mg/L; P < 0.001). Time-kill assays showed rapid bactericidal activity for rhodomyrtone, with ≥99% killing within 4 h. Rhodomyrtone was also four-fold more potent than vancomycin in inhibiting C. difficile spore outgrowth. Transmission electron microscopy of rhodomyrtone-treated C. difficile revealed cell lysis and evidence of defective cell division and spore formation. These studies indicate that rhodomyrtone should be further investigated as a potential treatment for CDI.


Subject(s)
Anti-Bacterial Agents/pharmacology , Clostridioides difficile/drug effects , Spores, Bacterial/drug effects , Xanthones/pharmacology , Bacteriolysis/drug effects , Cell Division/drug effects , Clostridioides difficile/isolation & purification , Clostridioides difficile/ultrastructure , Clostridium Infections/microbiology , Humans , Microbial Sensitivity Tests , Microbial Viability/drug effects , Microscopy, Electron, Transmission , Spores, Bacterial/ultrastructure , Vancomycin/pharmacology
2.
Sci Rep ; 7(1): 3665, 2017 06 16.
Article in English | MEDLINE | ID: mdl-28623367

ABSTRACT

The increased incidence of antibiotic resistant 'superbugs' has amplified the use of broad spectrum antibiotics worldwide. An unintended consequence of antimicrobial treatment is disruption of the gastrointestinal microbiota, resulting in susceptibility to opportunistic pathogens, such as Clostridium difficile. Paradoxically, treatment of C. difficile infections (CDI) also involves antibiotic use, leaving patients susceptible to re-infection. This serious health threat has led to an urgent call for the development of new therapeutics to reduce or replace the use of antibiotics to treat bacterial infections. To address this need, we have developed colostrum-derived antibodies for the prevention and treatment of CDI. Pregnant cows were immunised to generate hyperimmune bovine colostrum (HBC) containing antibodies that target essential C. difficile virulence components, specifically, spores, vegetative cells and toxin B (TcdB). Mouse infection and relapse models were used to compare the capacity of HBC to prevent or treat primary CDI as well as prevent recurrence. Administration of TcdB-specific colostrum alone, or in combination with spore or vegetative cell-targeted colostrum, prevents and treats C. difficile disease in mice and reduces disease recurrence by 67%. C. difficile-specific colostrum should be re-considered as an immunotherapeutic for the prevention or treatment of primary or recurrent CDI.


Subject(s)
Antibodies, Bacterial/immunology , Cattle Diseases/immunology , Cattle Diseases/microbiology , Clostridioides difficile/immunology , Clostridium Infections/veterinary , Animals , Anti-Bacterial Agents/pharmacology , Anti-Bacterial Agents/therapeutic use , Antibiotic Prophylaxis , Antibodies, Bacterial/therapeutic use , Antibodies, Neutralizing/immunology , Antibodies, Neutralizing/therapeutic use , Antibody Specificity/immunology , Bacterial Proteins/immunology , Cattle , Cattle Diseases/drug therapy , Cattle Diseases/pathology , Clostridioides difficile/drug effects , Cross Reactions/immunology , Mice , Neutralization Tests , Recurrence , Repressor Proteins/immunology
3.
Nat Med ; 22(11): 1277-1284, 2016 11.
Article in English | MEDLINE | ID: mdl-27694934

ABSTRACT

Bacterial infection is highly prevalent in patients who have had a stroke. Despite the potential contribution of micro-aspiration in post-stroke pneumonia, we found that the majority of the microorganisms detected in the patients who developed infections after having a stroke were common commensal bacteria that normally reside in the intestinal tracts. In a mouse model of ischemic stroke, post-stroke infection was only observed in mice that were born and raised in specific-pathogen-free facilities; this was not seen in mice that were born and raised in germ-free facilities. Using high-throughput 16S rRNA gene amplicon sequencing and bioinformatics analyses, we provide evidence demonstrating that the source of the bacteria forming the microbial community in the lungs of post-stroke mice was indeed the host small intestine. Additionally, stroke-induced gut barrier permeability and dysfunction preceded the dissemination of orally inoculated bacteria to peripheral tissues. This study identifies a novel pathway in which stroke promotes the translocation and dissemination of selective strains of bacteria that originated from the host gut microbiota.


Subject(s)
Bacterial Infections/immunology , Bacterial Translocation/immunology , Gastrointestinal Microbiome/genetics , Gram-Positive Bacterial Infections/immunology , Intestine, Small/metabolism , RNA, Ribosomal, 16S/genetics , Stroke/immunology , Adrenergic beta-Antagonists/pharmacology , Aged , Aged, 80 and over , Animals , Bacteremia/immunology , Bacteremia/metabolism , Bacteremia/microbiology , Bacterial Infections/metabolism , Bacterial Infections/microbiology , Blood Culture , Computational Biology , Disease Models, Animal , Enterococcus faecalis , Female , Goblet Cells/cytology , Goblet Cells/metabolism , Gram-Positive Bacterial Infections/metabolism , Gram-Positive Bacterial Infections/microbiology , High-Throughput Nucleotide Sequencing , Humans , Infarction, Middle Cerebral Artery/immunology , Intestine, Small/cytology , Intestine, Small/drug effects , Intestine, Small/microbiology , Male , Mice , Microbiota/genetics , Middle Aged , Permeability/drug effects , Pneumonia, Bacterial/immunology , Pneumonia, Bacterial/metabolism , Pneumonia, Bacterial/microbiology , Receptors, Adrenergic, beta/metabolism , Sequence Analysis, RNA , Specific Pathogen-Free Organisms , Urinary Tract Infections/immunology , Urinary Tract Infections/metabolism , Urinary Tract Infections/microbiology , Zonula Occludens-1 Protein/metabolism
4.
Genes (Basel) ; 6(4): 1347-60, 2015 Dec 21.
Article in English | MEDLINE | ID: mdl-26703737

ABSTRACT

Clostridium difficile is well recognized as the leading cause of antibiotic-associated diarrhea, having a significant impact in both health-care and community settings. Central to predisposition to C. difficile infection is disruption of the gut microbiome by antibiotics. Being a Gram-positive anaerobe, C. difficile is intrinsically resistant to a number of antibiotics. Mobile elements encoding antibiotic resistance determinants have also been characterized in this pathogen. While resistance to antibiotics currently used to treat C. difficile infection has not yet been detected, it may be only a matter of time before this occurs, as has been seen with other bacterial pathogens. This review will discuss C. difficile disease pathogenesis, the impact of antibiotic use on inducing disease susceptibility, and the role of antibiotic resistance and mobile elements in C. difficile epidemiology.

5.
Anaerobe ; 34: 80-3, 2015 Aug.
Article in English | MEDLINE | ID: mdl-25944720

ABSTRACT

Some Australian strain types of Clostridium difficile appear unique, highlighting the global diversity of this bacterium. We examined recent and historic local isolates, finding predominantly toxinotype 0 strains, but also toxinotypes V and VIII. All isolates tested were susceptible to vancomycin and metronidazole, while moxifloxacin resistance was only detected in recent strains.


Subject(s)
Anti-Bacterial Agents/pharmacology , Clostridioides difficile/isolation & purification , Clostridium Infections/microbiology , Genetic Variation , Bacterial Toxins/genetics , Clostridioides difficile/classification , Clostridioides difficile/drug effects , Clostridioides difficile/genetics , Drug Resistance, Bacterial , Fluoroquinolones/pharmacology , Humans , Metronidazole/pharmacology , Microbial Sensitivity Tests , Moxifloxacin , Vancomycin/pharmacology , Victoria
7.
Appl Environ Microbiol ; 80(12): 3597-3603, 2014 Jun.
Article in English | MEDLINE | ID: mdl-24682304

ABSTRACT

TnpX is a site-specific recombinase responsible for the excision and insertion of the transposons Tn4451 and Tn4453 in Clostridium perfringens and Clostridium difficile, respectively. Here, we exploit phenotypic features of TnpX to facilitate genetic mutagenesis and complementation studies. Genetic manipulation of bacteria often relies on the use of antibiotic resistance genes; however, a limited number are available for use in the clostridia. The ability of TnpX to recognize and excise specific DNA fragments was exploited here as the basis of an antibiotic resistance marker recycling system, specifically to remove antibiotic resistance genes from plasmids in Escherichia coli and from marked chromosomal C. perfringens mutants. This methodology enabled the construction of a C. perfringens plc virR double mutant by allowing the removal and subsequent reuse of the same resistance gene to construct a second mutation. Genetic complementation can be challenging when the gene of interest encodes a product toxic to E. coli. We show that TnpX represses expression from its own promoter, PattCI, which can be exploited to facilitate the cloning of recalcitrant genes in E. coli for subsequent expression in the heterologous host C. perfringens. Importantly, this technology expands the repertoire of tools available for the genetic manipulation of the clostridia.


Subject(s)
Bacterial Proteins/metabolism , Cloning, Molecular/methods , Clostridium perfringens/genetics , DNA, Bacterial/genetics , Escherichia coli/genetics , Genome, Bacterial , Recombinases/metabolism , Bacterial Proteins/genetics , Clostridium perfringens/enzymology , DNA Nucleotidyltransferases , Escherichia coli/metabolism , Genetic Complementation Test , Recombinases/genetics , Recombination, Genetic
8.
Clin Infect Dis ; 58(12): 1723-30, 2014 Jun.
Article in English | MEDLINE | ID: mdl-24704722

ABSTRACT

BACKGROUND: We identified 12 patients with Clostridium difficile infection between July 2011 and March 2012 from whom an unusual C. difficile strain was isolated. This strain had a single-nucleotide deletion of the tcdC gene at position 117 and binary toxin genes, which are characteristic of the hypervirulent ribotype (RT) 027 strain. METHODS: A retrospective cohort study of 12 patients infected with C. difficile RT244 and 24 patients infected with non-RT244/non-RT027 strains matched for place of diagnosis and time of collection of specimen was performed. We performed whole-genome sequencing to understand the relationship of the RT244 strain to other C. difficile strains and further understand its virulence potential. RESULTS: Clostridium difficile RT244 was associated with more severe disease and a higher mortality rate. Phylogenomic analysis using core genome single-nucleotide polymorphisms showed that RT244 is in the same genetic clade (clade 2) as RT027 but is distinct from all RT027 strains. The pathogenicity locus of the RT244 strain encodes a variant toxin B, and this was confirmed by demonstration of Clostridium sordellii-like cytopathic effect on Vero cells. Toxin B production in culture supernatants was lower than that seen with a RT027 strain. CONCLUSIONS: Our findings demonstrate the pathogenic potential of this RT244 C. difficile strain and emphasize the importance of ongoing surveillance for emergent strains.


Subject(s)
Clostridioides difficile/genetics , Communicable Diseases, Emerging/epidemiology , Communicable Diseases, Emerging/microbiology , Disease Outbreaks , Enterocolitis, Pseudomembranous/epidemiology , Enterocolitis, Pseudomembranous/microbiology , Adult , Aged , Aged, 80 and over , Bacterial Proteins/genetics , Bacterial Toxins/genetics , Female , Frameshift Mutation , Genome, Bacterial , Humans , Male , Middle Aged , Phylogeny , Polymorphism, Single Nucleotide , Repressor Proteins/genetics , Retrospective Studies , Ribotyping , Severity of Illness Index
9.
FEMS Microbiol Lett ; 352(2): 140-9, 2014 Mar.
Article in English | MEDLINE | ID: mdl-24372713

ABSTRACT

Clostridium difficile is the leading cause of bacterial antibiotic-associated diarrhoea in hospitals in the developed world. Despite this notoriety, the complex mechanisms employed by this pathogen to overcome innate host defences and induce fulminant disease are poorly understood. Various animal models have been used extensively for C. difficile research to study disease pathogenesis. Until recently, the most commonly used C. difficile disease model has utilised hamsters; however, mouse and pig models have now been developed that unravel different aspects of C. difficile pathology. This review summarises key aspects of the small animal models currently used in C. difficile studies with a specific focus on major differences between them. Furthermore, this review highlights the advantages and disadvantages of each model and illustrates that careful consideration is required when selecting models for use in C. difficile research.


Subject(s)
Clostridioides difficile/physiology , Clostridium Infections/microbiology , Clostridium Infections/pathology , Diarrhea/microbiology , Diarrhea/pathology , Disease Models, Animal , Animals , Anti-Bacterial Agents/adverse effects , Clostridioides difficile/pathogenicity , Cricetinae , Diarrhea/chemically induced , Humans , Mice , Swine
10.
PLoS One ; 8(11): e79666, 2013.
Article in English | MEDLINE | ID: mdl-24236153

ABSTRACT

Clostridium difficile is an important pathogen of humans and animals, representing a significant global healthcare problem. The last decade has seen the emergence of epidemic BI/NAP1/027 and ribotype 078 isolates, associated with the onset of more severe disease and higher rates of morbidity and mortality. However, little is known about these isolates at the molecular level, partly due to difficulties in the genetic manipulation of these strains. Here we report the development of an optimised Tn916-mediated plasmid transfer system, and the use of this system to construct and complement spo0A mutants in a number of different C. difficile strain backgrounds. Spo0A is a global regulator known to control sporulation, but may also be involved in the regulation of potential virulence factors and other phenotypes. Recent studies have failed to elucidate the role of Spo0A in toxin A and toxin B production by C. difficile, with conflicting data published to date. In this study, we aimed to clarify the role of Spo0A in production of the major toxins by C. difficile. Through the construction and complementation of spo0A mutants in two ribotype 027 isolates, we demonstrate that Spo0A acts as a negative regulator of toxin A and toxin B production in this strain background. In addition, spo0A was disrupted and subsequently complemented in strain 630Δerm and, for the first time, in a ribotype 078 isolate, JGS6133. In contrast to the ribotype 027 strains, Spo0A does not appear to regulate toxin production in strain 630Δerm. In strain JGS6133, Spo0A appears to negatively regulate toxin production during early stationary phase, but has little effect on toxin expression during late stationary phase. These data suggest that Spo0A may differentially regulate toxin production in phylogenetically distinct C. difficile strain types. In addition, Spo0A may be involved in regulating some aspects of C. difficile motility.


Subject(s)
Bacterial Proteins/metabolism , Bacterial Toxins/biosynthesis , Clostridioides difficile/genetics , Clostridioides difficile/metabolism , Animals , Bacterial Proteins/biosynthesis , Bacterial Proteins/genetics , Clostridioides difficile/growth & development , Clostridium/genetics , Clostridium/metabolism , Conjugation, Genetic , Enterotoxins/biosynthesis , Flagella/metabolism , Gene Order , Mutation , Plasmids/genetics , Plasmids/metabolism , Ribotyping
12.
PLoS Pathog ; 7(10): e1002317, 2011 Oct.
Article in English | MEDLINE | ID: mdl-22022270

ABSTRACT

Nosocomial infections are increasingly being recognised as a major patient safety issue. The modern hospital environment and associated health care practices have provided a niche for the rapid evolution of microbial pathogens that are well adapted to surviving and proliferating in this setting, after which they can infect susceptible patients. This is clearly the case for bacterial pathogens such as Methicillin Resistant Staphylococcus aureus (MRSA) and Vancomycin Resistant Enterococcus (VRE) species, both of which have acquired resistance to antimicrobial agents as well as enhanced survival and virulence properties that present serious therapeutic dilemmas for treating physicians. It has recently become apparent that the spore-forming bacterium Clostridium difficile also falls within this category. Since 2000, there has been a striking increase in C. difficile nosocomial infections worldwide, predominantly due to the emergence of epidemic or hypervirulent isolates that appear to possess extended antibiotic resistance and virulence properties. Various hypotheses have been proposed for the emergence of these strains, and for their persistence and increased virulence, but supportive experimental data are lacking. Here we describe a genetic approach using isogenic strains to identify a factor linked to the development of hypervirulence in C. difficile. This study provides evidence that a naturally occurring mutation in a negative regulator of toxin production, the anti-sigma factor TcdC, is an important factor in the development of hypervirulence in epidemic C. difficile isolates, presumably because the mutation leads to significantly increased toxin production, a contentious hypothesis until now. These results have important implications for C. difficile pathogenesis and virulence since they suggest that strains carrying a similar mutation have the inherent potential to develop a hypervirulent phenotype.


Subject(s)
Bacterial Proteins/biosynthesis , Bacterial Proteins/genetics , Bacterial Proteins/metabolism , Bacterial Toxins/biosynthesis , Clostridioides difficile/pathogenicity , Enterocolitis, Pseudomembranous/microbiology , Enterotoxins/biosynthesis , Repressor Proteins/genetics , Repressor Proteins/metabolism , Animals , Bacterial Toxins/genetics , Chlorocebus aethiops , Cloning, Molecular , Clostridioides difficile/genetics , Clostridioides difficile/isolation & purification , Cricetinae , Cross Infection/microbiology , Enterotoxins/genetics , Mesocricetus , Mutation , Plasmids , Repressor Proteins/biosynthesis , Vero Cells , Virulence Factors/metabolism
13.
J Bacteriol ; 189(20): 7290-301, 2007 Oct.
Article in English | MEDLINE | ID: mdl-17693517

ABSTRACT

Clostridium difficile binary toxin (CDT) is an actin-specific ADP-ribosyltransferase that is produced by various C. difficile isolates, including the "hypervirulent" NAP1/027 epidemic strains. In contrast to the two major toxins from C. difficile, toxin A and toxin B, little is known about the role of CDT in virulence or how C. difficile regulates its production. In this study we have shown that in addition to the cdtA and cdtB toxin structural genes, a functional cdt locus contains a third gene, here designated cdtR, which is predicted to encode a response regulator. By introducing functional binary toxin genes into cdtR(+) and cdtR-negative strains of C. difficile, it was established that the CdtR protein was required for optimal expression of binary toxin. Significantly increased expression of functional binary toxin was observed in the presence of a functional cdtR gene; an internal deletion within cdtR resulted in a reduction in binary toxin production to basal levels. Strains that did not carry intact cdtAB genes or cdtAB pseudogenes also did not have cdtR, with the entire cdt locus, or CdtLoc, being replaced by a conserved 68-bp sequence. These studies have shown for the first time that binary toxin production is subject to strict regulatory control by the response regulator CdtR, which is a member of the LytTR family of response regulators and is related to the AgrA protein from Staphylococcus aureus.


Subject(s)
ADP Ribose Transferases/biosynthesis , Bacterial Proteins/biosynthesis , Clostridioides difficile/physiology , Gene Expression Regulation, Bacterial/physiology , Transcription Factors/physiology , ADP Ribose Transferases/genetics , Amino Acid Sequence , Bacterial Proteins/genetics , DNA, Bacterial/chemistry , DNA, Bacterial/genetics , Genes, Bacterial , Genes, Regulator , Genetic Complementation Test , Molecular Sequence Data , Sequence Analysis, DNA , Sequence Deletion , Sequence Homology, Amino Acid , Transcription Factors/genetics
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