scholarly journals Cwp19 Is a Novel Lytic Transglycosylase Involved in Stationary-Phase Autolysis Resulting in Toxin Release inClostridium difficile

mBio ◽  
2018 ◽  
Vol 9 (3) ◽  
Author(s):  
Sandra Wydau-Dematteis ◽  
Imane El Meouche ◽  
Pascal Courtin ◽  
Audrey Hamiot ◽  
René Lai-Kuen ◽  
...  
Keyword(s):  
Health Care ◽  
Clostridium Difficile ◽  
Stationary Phase ◽  
Catalytic Domain ◽  
Brain Heart Infusion ◽  
Surface Protein ◽  
Wild Type ◽  
Content Type ◽  
Lytic Transglycosylase ◽  
Cell Autolysis

ABSTRACTClostridium difficileis the major etiologic agent of antibiotic-associated intestinal disease. Pathogenesis ofC. difficileis mainly attributed to the production and secretion of toxins A and B. Unlike most clostridial toxins, toxins A and B have no signal peptide, and they are therefore secreted by unusual mechanisms involving the holin-like TcdE protein and/or autolysis. In this study, we characterized the cell surface protein Cwp19, a newly identified peptidoglycan-degrading enzyme containing a novel catalytic domain. We purified a recombinant His6-tagged Cwp19 protein and showed that it has lytic transglycosylase activity. Moreover, we observed that Cwp19 is involved in cell autolysis and that aC. difficilecwp19mutant exhibited delayed autolysis in stationary phase compared to the wild type when bacteria were grown in brain heart infusion (BHI) medium. Wild-type cell autolysis is correlated to strong alterations of cell wall thickness and integrity and to release of cytoplasmic material. Furthermore, we demonstrated that toxins were released into the extracellular medium as a result of Cwp19-induced autolysis when cells were grown in BHI medium. In contrast, Cwp19 did not induce autolysis or toxin release when cells were grown in tryptone-yeast extract (TY) medium. These data provide evidence for the first time that TcdE and bacteriolysis are coexisting mechanisms for toxin release, with their relative contributionsin vitrodepending on growth conditions. Thus, Cwp19 is an important surface protein involved in autolysis of vegetative cells ofC. difficilethat mediates the release of the toxins from the cell cytosol in response to specific environment conditions.IMPORTANCEClostridium difficile-associated disease is mainly known as a health care-associated infection. It represents the most problematic hospital-acquired infection in North America and Europe and exerts significant economic pressure on health care systems. Virulent strains ofC. difficilegenerally produce two toxins that have been identified as the major virulence factors. The mechanism for release of these toxins from bacterial cells is not yet fully understood but is thought to be partly mediated by bacteriolysis. Here we identify a novel peptidoglycan hydrolase inC. difficile, Cwp19, exhibiting lytic transglycosylase activity. We show that Cwp19 contributes toC. difficilecell autolysis in the stationary phase and, consequently, to toxin release, most probably as a response to environmental conditions such as nutritional signals. These data highlight that Cwp19 constitutes a promising target for the development of new preventive and curative strategies.

scholarly journals Nisin Resistance of Listeria monocytogenes Is Increased by Exposure to Salt Stress and Is Mediated via LiaR

2013 ◽  
Vol 79 (18) ◽  
pp. 5682-5688 ◽  
Author(s):  
Teresa M. Bergholz ◽  
Silin Tang ◽  
Martin Wiedmann ◽  
Kathryn J. Boor
Keyword(s):  
Salt Stress ◽  
Listeria Monocytogenes ◽  
Safety Concern ◽  
Response Regulator ◽  
Brain Heart Infusion ◽  
Control Measures ◽  
Protective Effects ◽  
Wild Type ◽  
Content Type ◽  
Type Strains

ABSTRACTGrowth ofListeria monocytogeneson refrigerated, ready-to-eat food is a significant food safety concern. Natural antimicrobials, such as nisin, can be used to control this pathogen on food, but little is known about how other food-related stresses may impact how the pathogen responds to these compounds. Prior work demonstrated that exposure ofL. monocytogenesto salt stress at 7°C led to increased expression of genes involved in nisin resistance, including the response regulatorliaR. We hypothesized that exposure to salt stress would increase subsequent resistance to nisin and that LiaR would contribute to increased nisin resistance. Isogenic deletion mutations inliaRwere constructed in 7 strains ofL. monocytogenes, and strains were exposed to 6% NaCl in brain heart infusion broth and then tested for resistance to nisin (2 mg/ml Nisaplin) at 7°C. For the wild-type strains, exposure to salt significantly increased subsequent nisin resistance (P< 0.0001) over innate levels of resistance. Compared to the salt-induced nisin resistance of wild-type strains, ΔliaRstrains were significantly more sensitive to nisin (P< 0.001), indicating that induction of LiaFSR led to cross-protection ofL. monocytogenesagainst subsequent inactivation by nisin. Transcript levels of LiaR-regulated genes were induced by salt stress, and lmo1746 andtelAwere found to contribute to LiaR-mediated salt-induced nisin resistance. These data suggest that environmental stresses similar to those on foods can influence the resistance ofL. monocytogenesto antimicrobials such as nisin, and potential cross-protective effects should be considered when selecting and applying control measures for this pathogen on ready-to-eat foods.

scholarly journals Using a Novel Lysin To Help Control Clostridium difficile Infections

2015 ◽  
Vol 59 (12) ◽  
pp. 7447-7457 ◽  
Author(s):  
Qiong Wang ◽  
Chad W. Euler ◽  
Aurelia Delaune ◽  
Vincent A. Fischetti
Keyword(s):  
Clostridium Difficile ◽  
Catalytic Domain ◽  
Ex Vivo ◽  
Bacterial Species ◽  
Lytic Activity ◽  
Content Type ◽  
Intestinal Pathogen ◽  
Drug Treatments ◽  
Hospital Acquired

ABSTRACTAs a consequence of excessive antibiotic therapies in hospitalized patients,Clostridium difficile, a Gram-positive anaerobic spore-forming intestinal pathogen, is the leading cause of hospital-acquired diarrhea and colitis. Drug treatments for these diseases are often complicated by antibiotic-resistant strains and a high frequency of treatment failures and relapse; therefore, novel nonantibiotic approaches may prove to be more effective. In this study, we recombinantly expressed a prophage lysin identified from aC. difficilestrain, CD630, which we named PlyCD. PlyCD was found to have lytic activity against specificC. difficilestrains. However, the recombinantly expressed catalytic domain of this protein, PlyCD1–174, displayed significantly greater lytic activity (>4-log kill) and a broader lytic spectrum againstC. difficilestrains while still retaining a high degree of specificity towardC. difficileversus commensal clostridia and other bacterial species. Our data also indicated that noneffective doses of vancomycin and PlyCD1–174when combinedin vitrocould be significantly more bactericidal againstC. difficile. In anex vivotreatment model of mouse colon infection, we found that PlyCD1–174functioned in the presence of intestinal contents, significantly decreasing colonizingC. difficilecompared to controls. Together, these data suggest that PlyCD1–174has potential as a novel therapeutic for clinical application againstC. difficileinfection, either alone or in combination with other preexisting treatments to improve their efficacy.

scholarly journals A Nutrient-Regulated Cyclic Diguanylate Phosphodiesterase Controls Clostridium difficile Biofilm and Toxin Production during Stationary Phase

2017 ◽  
Vol 85 (9) ◽  
Author(s):  
Erin B. Purcell ◽  
Robert W. McKee ◽  
David S. Courson ◽  
Elizabeth M. Garrett ◽  
Shonna M. McBride ◽  
...  
Keyword(s):  
Clostridium Difficile ◽  
Stationary Phase ◽  
Nutrient Availability ◽  
Biofilm Formation ◽  
Biochemical Characterization ◽  
Toxin Production ◽  
Physiological Adaptation ◽  
Cyclic Diguanylate ◽  
Content Type ◽  
Wide Range

ABSTRACT The signaling molecule cyclic diguanylate (c-di-GMP) mediates physiological adaptation to extracellular stimuli in a wide range of bacteria. The complex metabolic pathways governing c-di-GMP synthesis and degradation are highly regulated, but the specific cues that impact c-di-GMP signaling are largely unknown. In the intestinal pathogen Clostridium difficile, c-di-GMP inhibits flagellar motility and toxin production and promotes pilus-dependent biofilm formation, but no specific biological functions have been ascribed to any of the individual c-di-GMP synthases or phosphodiesterases (PDEs). Here, we report the functional and biochemical characterization of a c-di-GMP PDE, PdcA, 1 of 37 confirmed or putative c-di-GMP metabolism proteins in C. difficile 630. Our studies reveal that pdcA transcription is controlled by the nutrient-regulated transcriptional regulator CodY and accordingly increases during stationary phase. In addition, PdcA PDE activity is allosterically regulated by GTP, further linking c-di-GMP levels to nutrient availability. Mutation of pdcA increased biofilm formation and reduced toxin biosynthesis without affecting swimming motility or global intracellular c-di-GMP. Analysis of the transcriptional response to pdcA mutation indicates that PdcA-dependent phenotypes manifest during stationary phase, consistent with regulation by CodY. These results demonstrate that inactivation of this single PDE gene is sufficient to impact multiple c-di-GMP-dependent phenotypes, including the production of major virulence factors, and suggest a link between c-di-GMP signaling and nutrient availability.

scholarly journals Phosphate Transporter PstSCAB of Campylobacter jejuni Is a Critical Determinant of Lactate-Dependent Growth and Colonization in Chickens

2020 ◽  
Vol 202 (7) ◽  
Author(s):  
Ritam Sinha ◽  
Rhiannon M. LeVeque ◽  
Marvin Q. Bowlin ◽  
Michael J. Gray ◽  
Victor J. DiRita
Keyword(s):  
Stationary Phase ◽  
Campylobacter Jejuni ◽  
Phosphate Transporter ◽  
Rna Seq ◽  
Wild Type ◽  
Content Type ◽  
High Affinity ◽  
Polyphosphate Metabolism

ABSTRACT Campylobacter jejuni causes acute gastroenteritis worldwide and is transmitted primarily through poultry, in which it is often a commensal member of the intestinal microbiota. Previous transcriptome sequencing (RNA-Seq) experiment showed that transcripts from an operon encoding a high-affinity phosphate transporter (PstSCAB) of C. jejuni were among the most abundant when the bacterium was grown in chickens. Elevated levels of the pstSCAB mRNA were also identified in an RNA-Seq experiment from human infection studies. In this study, we explore the role of PstSCAB in the biology and colonization potential of C. jejuni. Our results demonstrate that cells lacking PstSCAB survive poorly in stationary phase, in nutrient-limiting media, and under osmotic conditions reflective of those in the chicken. Polyphosphate levels in the mutant cells were elevated at stationary phase, consistent with alterations in expression of polyphosphate metabolism genes. The mutant strain was highly attenuated for colonization of newly hatched chicks, with levels of bacteria at several orders of magnitude below wild-type levels. Mutant and wild type grew similarly in complex media, but the pstS::kan mutant exhibited a significant growth defect in minimal medium supplemented with l-lactate, postulated as a carbon source in vivo. Poor growth in lactate correlated with diminished expression of acetogenesis pathway genes previously demonstrated as important for colonizing chickens. The phosphate transport system is thus essential for diverse aspects of C. jejuni physiology and in vivo fitness and survival. IMPORTANCE Campylobacter jejuni causes millions of human gastrointestinal infections annually, with poultry a major source of infection. Due to the emergence of multidrug resistance in C. jejuni, there is need to identify alternative ways to control this pathogen. Genes encoding the high-affinity phosphate transporter PstSCAB are highly expressed by C. jejuni in chickens and humans. In this study, we address the role of PstSCAB on chicken colonization and other C. jejuni phenotypes. PstSCAB is required for colonization in chicken, metabolism and survival under different stress responses, and during growth on lactate, a potential growth substrate in chickens. Our study highlights that PstSCAB may be an effective target to develop mechanisms for controlling bacterial burden in both chicken and human.

scholarly journals Point-Counterpoint: Active Surveillance for Carriers of Toxigenic Clostridium difficile Should Be Performed To Guide Prevention Efforts

2018 ◽  
Vol 56 (8) ◽  
Author(s):  
L. Clifford McDonald ◽  
Daniel J. Diekema
Keyword(s):  
Health Care ◽  
Clostridium Difficile ◽  
Health Care Systems ◽  
Laboratory Diagnosis ◽  
Value Based Purchasing ◽  
University Of Iowa ◽  
Content Type ◽  
Asymptomatic Patients ◽  
Care Systems ◽  
New Guidelines

INTRODUCTION In 2017, the Journal of Clinical Microbiology published a Point-Counterpoint on the laboratory diagnosis of Clostridium difficile infection (CDI). At that time, Ferric C. Fang, Christopher R. Polage, and Mark H. Wilcox discussed the strategies for diagnosing Clostridium difficile colitis in symptomatic patients (J Clin Microbiol 55:670–680, 2017, https://doi.org/10.1128/JCM.02463-16). Since that paper, new guidelines from the Infectious Diseases Society of America and the Society for Health Care Epidemiology have been published (L. C. McDonald, D. N. Gerding, S. Johnson, J. S. Bakken, K. C. Carroll, et al., Clin Infect Dis 66:987–994, 2018, https://doi.org/10.1093/cid/ciy149) and health care systems have begun to explore screening asymptomatic patients for C. difficile colonization. The theory behind screening selected patient populations for C. difficile colonization is that these patients represent a substantial reservoir of the bacteria and can transfer the bacteria to other patients. Hospital administrators are taking note of institutional CDI rates because they are publicly reported. They have become an important metric impacting hospital safety ratings and value-based purchasing, and hospitals may have millions of dollars of reimbursement at risk. In this Point-Counterpoint, Cliff McDonald of the U.S. Centers for Disease Control and Prevention discusses the value of asymptomatic C. difficile screening, while Dan Diekema of the University of Iowa discusses why caution should be used.

scholarly journals Transcriptional Analysis of an Ammonium-Excreting Strain of Azotobacter vinelandii Deregulated for Nitrogen Fixation

2017 ◽  
Vol 83 (20) ◽  
Author(s):  
Brett M. Barney ◽  
Mary H. Plunkett ◽  
Velmurugan Natarajan ◽  
Florence Mus ◽  
Carolann M. Knutson ◽  
...  
Keyword(s):  
Nitrogen Fixation ◽  
Stationary Phase ◽  
Gene Transcription ◽  
Biological Nitrogen Fixation ◽  
Azotobacter Vinelandii ◽  
Nitrogen Fixing ◽  
Wild Type ◽  
Content Type ◽  
Ammonium Production ◽  
Biological Nitrogen

ABSTRACT Biological nitrogen fixation is accomplished by a diverse group of organisms known as diazotrophs and requires the function of the complex metalloenzyme nitrogenase. Nitrogenase and many of the accessory proteins required for proper cofactor biosynthesis and incorporation into the enzyme have been characterized, but a complete picture of the reaction mechanism and key cellular changes that accompany biological nitrogen fixation remain to be fully elucidated. Studies have revealed that specific disruptions of the antiactivator-encoding gene nifL result in the deregulation of the nif transcriptional activator NifA in the nitrogen-fixing bacterium Azotobacter vinelandii, triggering the production of extracellular ammonium levels approaching 30 mM during the stationary phase of growth. In this work, we have characterized the global patterns of gene expression of this high-ammonium-releasing phenotype. The findings reported here indicated that cultures of this high-ammonium-accumulating strain may experience metal limitation when grown using standard Burk's medium, which could be amended by increasing the molybdenum levels to further increase the ammonium yield. In addition, elevated levels of nitrogenase gene transcription are not accompanied by a corresponding dramatic increase in hydrogenase gene transcription levels or hydrogen uptake rates. Of the three potential electron donor systems for nitrogenase, only the rnf1 gene cluster showed a transcriptional correlation to the increased yield of ammonium. Our results also highlight several additional genes that may play a role in supporting elevated ammonium production in this aerobic nitrogen-fixing model bacterium. IMPORTANCE The transcriptional differences found during stationary-phase ammonium accumulation show a strong contrast between the deregulated (nifL-disrupted) and wild-type strains and what was previously reported for the wild-type strain under exponential-phase growth conditions. These results demonstrate that further improvement of the ammonium yield in this nitrogenase-deregulated strain can be obtained by increasing the amount of available molybdenum in the medium. These results also indicate a potential preference for one of two ATP synthases present in A. vinelandii as well as a prominent role for the membrane-bound hydrogenase over the soluble hydrogenase in hydrogen gas recycling. These results should inform future studies aimed at elucidating the important features of this phenotype and at maximizing ammonium production by this strain.

scholarly journals High Prevalence of Clostridium difficile in Home Gardens in Western Australia

2020 ◽  
Vol 87 (1) ◽  
Author(s):  
Nirajmohan Shivaperumal ◽  
Barbara J. Chang ◽  
Thomas V. Riley
Keyword(s):  
Risk Factors ◽  
Clostridium Difficile ◽  
Western Australia ◽  
Brain Heart Infusion ◽  
Home Gardens ◽  
Home Garden ◽  
Toxin Gene ◽  
Content Type ◽  
High Prevalence ◽  
Shoe Soles

ABSTRACT In recent years, community-associated Clostridium difficile infection (CA-CDI) has emerged as a significant health problem, accounting for ∼50% of all CDI cases. We hypothesized that the home garden environment could contribute to the dissemination of C. difficile spores in the community and investigated 23 homes in 22 suburbs of Perth, Western Australia. We identified a high prevalence of toxigenic C. difficile in this environment. In total, 97 samples consisting of soil (n = 48), compost (n = 15), manure (n = 12), and shoe sole swabs (n = 22) were collected. All samples were cultured anaerobically on C. difficile ChromID agar and enriched in brain heart infusion broth, and isolates were characterized by toxin gene PCR and PCR ribotyping. Two-thirds (67%; 95% confidence interval [CI], 57 to 76%) of home garden samples, including 79% (95% CI, 68 to 91%) of soil, 67% (95% CI, 43 to 90%) of compost, 83% (95% CI, 62% to 100%) of manure, and 32% (95% CI, 12 to 51%) of shoe sole samples, contained C. difficile. Of 87 isolates, 38% (95% CI, 28 to 48%) were toxigenic, and 26 PCR ribotypes (RTs), 5 of which were novel, were identified. The toxigenic C. difficile strain RT014/020 was the most prevalent RT. Interestingly, 19 esculin hydrolysis-negative strains giving white colonies were identified on C. difficile ChromID agar, 5 of which were novel toxigenic RTs that produced only toxin A. Clearly, there is the potential for transmission of C. difficile in the community due to the contamination of home gardens. Our findings highlight the importance of a “One Health” approach to dealing with CDI. IMPORTANCE Recently, community-associated Clostridium difficile infection (CA-CDI) has emerged as a significant problem, accounting for ∼50% of all CDI cases and reported to affect a younger population without traditional risk factors. Possible sources of CA-CDI are soil, food, and water contaminated by animal feces, and recent reports show overlapping ribotypes of C. difficile in animals, humans, and the environment; however, the epidemiology of CA-CDI and related risk factors need to be better understood. Our research aimed to determine the prevalence of C. difficile in home gardens and on the shoe soles of homeowners in Perth, Western Australia. There were high rates of contamination with C. difficile in gardens, and some of the ribotypes identified had been isolated from human cases of CDI in Western Australia. This study shows that home gardens and shoes may be a source of C. difficile in CA-CDI.

scholarly journals AntisocialluxOMutants Provide a Stationary-Phase Survival Advantage in Vibrio fischeri ES114

2015 ◽  
Vol 198 (4) ◽  
pp. 673-687 ◽  
Author(s):  
John H. Kimbrough ◽  
Eric V. Stabb
Keyword(s):  
Stationary Phase ◽  
Vibrio Fischeri ◽  
Current Model ◽  
Homoserine Lactone ◽  
Survival Advantage ◽  
Mutant Library ◽  
Wild Type ◽  
Host Colonization ◽  
Content Type ◽  
Pheromone Signaling

ABSTRACTThe squid light organ symbiontVibrio fischericontrols bioluminescence using two acyl-homoserine lactone pheromone-signaling (PS) systems. The first of these systems to be activated during host colonization, AinS/AinR, produces and responds toN-octanoyl homoserine lactone (C8-AHL). We screened activity of a PainS-lacZtranscriptional reporter in a transposon mutant library and found three mutants with decreased reporter activity, low C8-AHL output, and other traits consistent with lowainSexpression. However, the transposon insertions were unrelated to these phenotypes, and genome resequencing revealed that each mutant had a distinct point mutation inluxO. In the wild type, LuxO is phosphorylated by LuxU and then activates transcription of the small RNA (sRNA) Qrr, which repressesainSindirectly by repressing its activator LitR. TheluxOmutants identified here encode LuxU-independent, constitutively active LuxO* proteins. The repeated appearance of theseluxOmutants suggested that they had some fitness advantage during construction and/or storage of the transposon mutant library, and we found thatluxO* mutants survived better and outcompeted the wild type in prolonged stationary-phase cultures. From such cultures we isolated additionalluxO* mutants. In all, we isolated LuxO* allelic variants with the mutations P41L, A91D, F94C, P98L, P98Q, V106A, V106G, T107R, V108G, R114P, L205F, H319R, H324R, and T335I. Based on the current model of theV. fischeriPS circuit,litRknockout mutants should resembleluxO* mutants; however,luxO* mutants outcompetedlitRmutants in prolonged culture and had much poorer host colonization competitiveness than is reported forlitRmutants, illustrating additional complexities in this regulatory circuit.IMPORTANCEOur results provide novel insight into the function of LuxO, which is a key component of pheromone signaling (PS) cascades in several members of theVibrionaceae. Our results also contribute to an increasingly appreciated aspect of bacterial behavior and evolution whereby mutants that do not respond to a signal from like cells have a selective advantage. In this case, although “antisocial” mutants locked in the PS signal-off mode can outcompete parents, their survival advantage does not require wild-type cells to exploit. Finally, this work strikes a note of caution for those conducting or interpreting experiments inV. fischeri, as it illustrates how pleiotropic mutants could easily and inadvertently be enriched in this bacterium during prolonged culturing.

scholarly journals A Phenotypically Silent vanB2 Operon Carried on a Tn1549-Like Element in Clostridium difficile

mSphere ◽  
2016 ◽  
Vol 1 (4) ◽  
Author(s):  
Daniel R. Knight ◽  
Grace O. Androga ◽  
Susan A. Ballard ◽  
Benjamin P. Howden ◽  
Thomas V. Riley
Keyword(s):  
Health Care ◽  
Clostridium Difficile ◽  
Resistance Genes ◽  
Vancomycin Resistance ◽  
Content Type ◽  
Vancomycin Resistant Enterococci ◽  
Vancomycin Resistant ◽  
Emergence Of Resistance ◽  
First Time

ABSTRACT In an era when the development of new antimicrobial drugs is slow, vancomycin remains the preferred antimicrobial therapy for Clostridium difficile infection (CDI), the most important health care-related infection in the world today. The emergence of resistance to vancomycin would have significant consequences in relation to treating patients with CDI. In this paper, we describe for the first time a complete set of vancomycin resistance genes in C. difficile. The genes were very similar to genes found in vancomycin-resistant enterococci (VRE) that were associated with the emergence and global dissemination of this organism. Fortunately, the C. difficile strain did not show any reduced susceptibility to vancomycin in vitro (MIC, 1 mg/liter), possibly because of a small difference in one gene. However, this observation signals that we may be very close to seeing a fully vancomycin-resistant strain of C. difficile. In the last decade, Clostridium difficile infection (CDI) has reached an epidemic state with increasing incidence and severity in both health care and community settings. Vancomycin is an important first-line therapy for CDI, and the emergence of resistance would have significant clinical consequences. In this study, we describe for the first time a vanB2 vancomycin resistance operon in C. difficile, isolated from an Australian veal calf at slaughter. The operon was carried on an ~42-kb element showing significant homology and synteny to Tn1549, a conjugative transposon linked with the emergence and global dissemination of vancomycin-resistant enterococci (VRE). Notably, the C. difficile strain did not show any reduced susceptibility to vancomycin in vitro (MIC, 1 mg/liter), possibly as a result of an aberrant vanRB gene. As observed for other anaerobic species of the animal gut microbiota, C. difficile may be a reservoir of clinically important vancomycin resistance genes. IMPORTANCE In an era when the development of new antimicrobial drugs is slow, vancomycin remains the preferred antimicrobial therapy for Clostridium difficile infection (CDI), the most important health care-related infection in the world today. The emergence of resistance to vancomycin would have significant consequences in relation to treating patients with CDI. In this paper, we describe for the first time a complete set of vancomycin resistance genes in C. difficile. The genes were very similar to genes found in vancomycin-resistant enterococci (VRE) that were associated with the emergence and global dissemination of this organism. Fortunately, the C. difficile strain did not show any reduced susceptibility to vancomycin in vitro (MIC, 1 mg/liter), possibly because of a small difference in one gene. However, this observation signals that we may be very close to seeing a fully vancomycin-resistant strain of C. difficile.

scholarly journals A Modified R-Type Bacteriocin Specifically Targeting Clostridium difficile Prevents Colonization of Mice without Affecting Gut Microbiota Diversity

mBio ◽  
2015 ◽  
Vol 6 (2) ◽  
Author(s):  
Dana Gebhart ◽  
Stephen Lok ◽  
Simon Clare ◽  
Myreen Tomas ◽  
Mark Stares ◽  
...  
Keyword(s):  
Health Care ◽  
Clostridium Difficile ◽  
Gut Microbiota ◽  
Genome Mining ◽  
Health Care Facilities ◽  
Enteric Infection ◽  
Colonization Resistance ◽  
Content Type ◽  
Strain Type ◽  
Type Strains

ABSTRACT Clostridium difficile is a leading cause of nosocomial infections worldwide and has become an urgent public health threat requiring immediate attention. Epidemic lineages of the BI/NAP1/027 strain type have emerged and spread through health care systems across the globe over the past decade. Limiting person-to-person transmission and eradicating C. difficile, especially the BI/NAP1/027 strain type, from health care facilities are difficult due to the abundant shedding of spores that are impervious to most interventions. Effective prophylaxis for C. difficile infection (CDI) is lacking. We have genetically modified a contractile R-type bacteriocin (“diffocin”) from C. difficile strain CD4 to kill BI/NAP1/027-type strains for this purpose. The natural receptor binding protein (RBP) responsible for diffocin targeting was replaced with a newly discovered RBP identified within a prophage of a BI/NAP1/027-type target strain by genome mining. The resulting modified diffocins (a.k.a. Avidocin-CDs), Av-CD291.1 and Av-CD291.2, were stable and killed all 16 tested BI/NAP1/027-type strains. Av-CD291.2 administered in drinking water survived passage through the mouse gastrointestinal (GI) tract, did not detectably alter the mouse gut microbiota or disrupt natural colonization resistance to C. difficile or the vancomycin-resistant Enterococcus faecium (VREF), and prevented antibiotic-induced colonization of mice inoculated with BI/NAP1/027-type spores. Given the high incidence and virulence of the pathogen, preventing colonization by BI/NAP1/027-type strains and limiting their transmission could significantly reduce the occurrence of the most severe CDIs. This modified diffocin represents a prototype of an Avidocin-CD platform capable of producing targetable, precision anti-C. difficile agents that can prevent and potentially treat CDIs without disrupting protective indigenous microbiota. IMPORTANCE Treatment and prevention strategies for bacterial diseases rely heavily on traditional antibiotics, which impose strong selection for resistance and disrupt protective microbiota. One consequence has been an upsurge of opportunistic pathogens, such as Clostridium difficile, that exploit antibiotic-induced disruptions in gut microbiota to proliferate and cause life-threatening diseases. We have developed alternative agents that utilize contractile bactericidal protein complexes (R-type bacteriocins) to kill specific C. difficile pathogens. Efficacy in a preclinical animal study indicates these molecules warrant further development as potential prophylactic agents to prevent C. difficile infections in humans. Since these agents do not detectably alter the indigenous gut microbiota or colonization resistance in mice, we believe they will be safe to administer as a prophylactic to block transmission in high-risk environments without rendering patients susceptible to enteric infection after cessation of treatment.

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