scholarly journals Biofilm regulation in Clostridioides difficile: Novel systems linked to hypervirulence

2021 ◽  
Vol 17 (9) ◽  
pp. e1009817
Author(s):  
Megan G. Taggart ◽  
William J. Snelling ◽  
Patrick J. Naughton ◽  
Roberto M. La Ragione ◽  
James S. G. Dooley ◽  
...  
Keyword(s):  
Biofilm Formation ◽  
Gastrointestinal Disease ◽  
Regulatory System ◽  
Disease Recurrence ◽  
The United States ◽  
Extracellular Dna ◽  
Published Data ◽  
Bacterial Cells ◽  
Healthcare Associated Infection ◽  
Clostridioides Difficile

Clostridiodes difficile (C. difficile) was ranked an “urgent threat” by the Centers for Disease Control and Prevention (CDC) in 2019. C. difficile infection (CDI) is the most common healthcare-associated infection (HAI) in the United States of America as well as the leading cause of antibiotic-associated gastrointestinal disease. C. difficile is a gram-positive, rod-shaped, spore-forming, anaerobic bacterium that causes infection of the epithelial lining of the gut. CDI occurs most commonly after disruption of the human gut microflora following the prolonged use of broad-spectrum antibiotics. However, the recurrent nature of this disease has led to the hypothesis that biofilm formation may play a role in its pathogenesis. Biofilms are sessile communities of bacteria protected from extracellular stresses by a matrix of self-produced proteins, polysaccharides, and extracellular DNA. Biofilm regulation in C. difficile is still incompletely understood, and its role in disease recurrence has yet to be fully elucidated. However, many factors have been found to influence biofilm formation in C. difficile, including motility, adhesion, and hydrophobicity of the bacterial cells. Small changes in one of these systems can greatly influence biofilm formation. Therefore, the biofilm regulatory system would need to coordinate all these systems to create optimal biofilm-forming physiology under appropriate environmental conditions. The coordination of these systems is complex and multifactorial, and any analysis must take into consideration the influences of the stress response, quorum sensing (QS), and gene regulation by second messenger molecule cyclic diguanosine monophosphate (c-di-GMP). However, the differences in biofilm-forming ability between C. difficile strains such as 630 and the “hypervirulent” strain, R20291, make it difficult to assign a “one size fits all” mechanism to biofilm regulation in C. difficile. This review seeks to consolidate published data regarding the regulation of C. difficile biofilms in order to identify gaps in knowledge and propose directions for future study.

scholarly journals Phylogenomic analysis of Clostridioides difficile ribotype 106 strains reveals novel genetic islands and emergent phenotypes

2020 ◽  
Vol 10 (1) ◽  
Author(s):  
Bryan Angelo P. Roxas ◽  
Jennifer Lising Roxas ◽  
Rachel Claus-Walker ◽  
Anusha Harishankar ◽  
Asad Mansoor ◽  
...  
Keyword(s):  
Biofilm Formation ◽  
Diarrheal Disease ◽  
The United States ◽  
Genomic Islands ◽  
Rapid Expansion ◽  
Phylogenomic Analysis ◽  
Community Settings ◽  
Hamster Model ◽  
Clostridioides Difficile ◽  
Healthcare Associated

AbstractClostridioides difficile infection (CDI) is a major healthcare-associated diarrheal disease. Consistent with trends across the United States, C. difficile RT106 was the second-most prevalent molecular type in our surveillance in Arizona from 2015 to 2018. A representative RT106 strain displayed robust virulence and 100% lethality in the hamster model of acute CDI. We identified a unique 46 KB genomic island (GI1) in all RT106 strains sequenced to date, including those in public databases. GI1 was not found in its entirety in any other C. difficile clade, or indeed, in any other microbial genome; however, smaller segments were detected in Enterococcus faecium strains. Molecular clock analyses suggested that GI1 was horizontally acquired and sequentially assembled over time. GI1 encodes homologs of VanZ and a SrtB-anchored collagen-binding adhesin, and correspondingly, all tested RT106 strains had increased teicoplanin resistance, and a majority displayed collagen-dependent biofilm formation. Two additional genomic islands (GI2 and GI3) were also present in a subset of RT106 strains. All three islands are predicted to encode mobile genetic elements as well as virulence factors. Emergent phenotypes associated with these genetic islands may have contributed to the relatively rapid expansion of RT106 in US healthcare and community settings.

scholarly journals Spo0A Suppresses sin Locus Expression in Clostridioides difficile

mSphere ◽  
2020 ◽  
Vol 5 (6) ◽  
Author(s):  
Babita Adhikari Dhungel ◽  
Revathi Govind
Keyword(s):  
Diarrheal Disease ◽  
Toxin Production ◽  
The United States ◽  
Upstream Region ◽  
Pcr Analysis ◽  
Bacterial Cells ◽  
Master Regulator ◽  
Content Type ◽  
Clostridioides Difficile

ABSTRACT Clostridioides difficile is the leading cause of nosocomial infection and is the causative agent of antibiotic-associated diarrhea. The severity of the disease is directly associated with toxin production, and spores are responsible for the transmission and persistence of the organism. Previously, we characterized sin locus regulators SinR and SinR′ (we renamed it SinI), where SinR is the regulator of toxin production and sporulation. The SinI regulator acts as its antagonist. In Bacillus subtilis, Spo0A, the master regulator of sporulation, controls SinR by regulating the expression of its antagonist, sinI. However, the role of Spo0A in the expression of sinR and sinI in C. difficile had not yet been reported. In this study, we tested spo0A mutants in three different C. difficile strains, R20291, UK1, and JIR8094, to understand the role of Spo0A in sin locus expression. Western blot analysis revealed that spo0A mutants had increased SinR levels. Quantitative reverse transcription-PCR (qRT-PCR) analysis of its expression further supported these data. By carrying out genetic and biochemical assays, we show that Spo0A can bind to the upstream region of this locus to regulates its expression. This study provides vital information that Spo0A regulates the sin locus, which controls critical pathogenic traits such as sporulation, toxin production, and motility in C. difficile. IMPORTANCE Clostridioides difficile is the leading cause of antibiotic-associated diarrheal disease in the United States. During infection, C. difficile spores germinate, and the vegetative bacterial cells produce toxins that damage host tissue. In C. difficile, the sin locus is known to regulate both sporulation and toxin production. In this study, we show that Spo0A, the master regulator of sporulation, controls sin locus expression. Results from our study suggest that Spo0A directly regulates the expression of this locus by binding to its upstream DNA region. This observation adds new detail to the gene regulatory network that connects sporulation and toxin production in this pathogen.

Simulation of bacterial biofilms and estimation of the sensitivity of healthcare-associated infection pathogens to bactericide Sekusept active

2020 ◽  
Vol 65 (10) ◽  
pp. 652-658
Author(s):  
U. M. Nemchenko ◽  
E. A. Kungurtseva ◽  
E. V. Grigorova ◽  
N. L. Belkova ◽  
Y. A. Markova ◽  
...  
Keyword(s):  
Biofilm Formation ◽  
Bacterial Cells ◽  
Healthcare Associated Infection ◽  
Planktonic Cells ◽  
Biocidal Activity ◽  
Fermenting Bacteria ◽  
Planktonic Form ◽  
Biochemical Identification ◽  
Microbial Strains ◽  
Healthcare Associated

The effect of bactericide Sekusept active (B SA), a peracetic acid-based preparation, on microbial strains, isolated from patients with severe infectious diseases who were treated in a regional children’s multi-specialty hospital, was studied. Based on the biochemical identification, the strains were classified as gram-negative non-fermenting bacteria (22 strains), Enterobacteriaceae family (18 strains), and bacilli - 3 strains. The biocidal activity of B SA was evaluated by the degree of inhibition of the growth of bacterial cells, existing in the planktonic form and in the form of biofilm (on a flat-bottomed plastic immunological tablet). It was shown that all the studied strains had the ability to biofilm formation, most of them (67,4%) formed moderately pronounced biofilms, and non-fermenting bacteria had a particularly pronounced coefficient of biofilm formation. The selected concentrations of B CA inhibited the growth of planktonic cells, and the ability of bactericide to prevent the formation of biofilms depended on the concentration (the most effective concentrations were 0,8 and 3,0%). Sensitivity of the strains existed in the aged biofilm to the bactericide was significantly lower, especially resistant to this effect were biofilms formed by non-fermenting bacteria and representatives of fam. Enterobacteriaceae. Our results confirm the importance of testing the effectiveness of biocides not only in accordance with standard methods developed for microorganisms in planktonic form, but also for biofilms.

scholarly journals Defining the black box: a narrative review of factors associated with adverse outcomes from severe Clostridioides difficile infection

2021 ◽  
Vol 14 ◽  
pp. 175628482110481
Author(s):  
Adam Ressler ◽  
Joyce Wang ◽  
Krishna Rao
Keyword(s):  
The United States ◽  
Adverse Outcomes ◽  
Chronic Illnesses ◽  
Narrative Review ◽  
Healthcare Associated Infection ◽  
Clostridioides Difficile ◽  
Clostridioides Difficile Infection ◽  
Patients At Risk ◽  
Hospital Deaths ◽  
Healthcare Associated

In the United States, Clostridioides difficile infection (CDI) is the leading cause of healthcare-associated infection, affecting nearly half a million people and resulting in more than 20,000 in-hospital deaths every year. It is therefore imperative to better characterize the intricate interplay between C. difficile microbial factors, host immunologic signatures, and clinical features that are associated with adverse outcomes of severe CDI. In this narrative review, we discuss the implications of C. difficile genetics and virulence factors in the molecular epidemiology of CDI, and the utility of early biomarkers in predicting the clinical trajectory of patients at risk of developing severe CDI. Furthermore, we identify associations between host immune factors and CDI outcomes in both animal models and human studies. Next, we highlight clinical factors including renal dysfunction, aging, blood biomarkers, level of care, and chronic illnesses that can affect severe CDI diagnosis and outcome. Finally, we present our perspectives on two specific treatments pertinent to patient outcomes: metronidazole administration and surgery. Together, this review explores the various venues of CDI research and highlights the importance of integrating microbial, host, and clinical data to help clinicians make optimal treatment decisions based on accurate prediction of disease progression.

scholarly journals High Adhesion and Increased Cell Death Contribute to Strong Biofilm Formation in Klebsiella pneumoniae

Pathogens ◽  
2019 ◽  
Vol 8 (4) ◽  
pp. 277
Author(s):  
Siddhi Desai ◽  
Kinjal Sanghrajka ◽  
Devarshi Gajjar
Keyword(s):  
Cell Death ◽  
Klebsiella Pneumoniae ◽  
Biofilm Formation ◽  
Laser Scanning ◽  
Laser Scanning Microscopy ◽  
Extracellular Dna ◽  
Confocal Laser ◽  
Bacterial Cells ◽  
High Adhesion ◽  
Biofilm Matrix

Klebsiella pneumoniae (Kp), is a frequent cause of hospital and community-acquired infections and WHO had declared it as a “priority pathogen”. Biofilm is a major virulence factor of Kp and yet the mechanism of strong biofilm formation in Kp is unclear. A key objective of the present study is to investigate the differences between strong and weak biofilms formed by clinical isolates of Kp on various catheters and in different media conditions and to identify constituents contributing to strong biofilm formation. Quantification of matrix components (extracellular DNA (eDNA), protein, exopolysaccharides (EPS), and bacterial cells), confocal laser scanning microscopy (CLSM), field emission gun scanning electron microscopy (FEG-SEM) and flow-cytometry analysis were performed to compare strong and weak biofilm matrix. Our results suggest increased biofilm formation on latex catheters compared to silicone and silicone-coated latex catheters. Higher amounts of eDNA, protein, EPS, and dead cells were observed in the strong biofilm of Kp. High adhesion capacity and cell death seem to play a major role in formation of strong Kp biofilms. The enhanced eDNA, EPS, and protein in the biofilm matrix appear as a consequence of increased cell death.

scholarly journals Environmental Stress-Induced Bacterial Lysis and Extracellular DNA Release Contribute toCampylobacter jejuniBiofilm Formation

2017 ◽  
Vol 84 (5) ◽  
Author(s):  
Jinsong Feng ◽  
Lina Ma ◽  
Jiatong Nie ◽  
Michael E. Konkel ◽  
Xiaonan Lu
Keyword(s):  
Environmental Stress ◽  
Campylobacter Jejuni ◽  
Biofilm Formation ◽  
Extracellular Dna ◽  
Bacterial Cells ◽  
Starvation Stress ◽  
Content Type ◽  
Bacterial Lysis ◽  
Major Form ◽  
Spatial Locations

ABSTRACTCampylobacter jejuniis a microaerophilic bacterium and is believed to persist in a biofilm to antagonize environmental stress. This study investigated the influence of environmental conditions on the formation ofC. jejunibiofilm. We report an extracellular DNA (eDNA)-mediated mechanism of biofilm formation in response to aerobic and starvation stress. The eDNA was determined to represent a major form of constitutional material ofC. jejunibiofilms and to be closely associated with bacterial lysis. Deletion mutation of the stress response genesspoTandrecAenhanced the aerobic influence by stimulating lysis and increasing eDNA release. Flagella were also involved in biofilm formation but mainly contributed to attachment rather than induction of lysis. The addition of genomic DNA from eitherCampylobacterorSalmonellaresulted in a concentration-dependent stimulation effect on biofilm formation, but the effect was not due to forming a precoating DNA layer. Enzymatic degradation of DNA by DNase I disruptedC. jejunibiofilm. In a dual-species biofilm, eDNA allocatedCampylobacterandSalmonellaat distinct spatial locations that protectCampylobacterfrom oxygen stress. Our findings demonstrated an essential role and multiple functions of eDNA in biofilm formation ofC. jejuni, including facilitating initial attachment, establishing and maintaining biofilm, and allocating bacterial cells.IMPORTANCECampylobacter jejuniis a major cause of foodborne illness worldwide. In the natural environment, the growth ofC. jejuniis greatly inhibited by various forms of environmental stress, such as aerobic stress and starvation stress. Biofilm formation can facilitate the distribution ofC. jejuniby enabling the survival of this fragile microorganism under unfavorable conditions. However, the mechanism ofC. jejunibiofilm formation in response to environmental stress has been investigated only partially. The significance of our research is in identifying extracellular DNA released by bacterial lysis as a major form of constitution material that mediates the formation ofC. jejunibiofilm in response to environmental stress, which enhances our understanding of the formation mechanism ofC. jejunibiofilm. This knowledge can aid the development of intervention strategies to limit the distribution ofC. jejuni.

scholarly journals Extracellular DNA, cell surface proteins and c-di-GMP promote biofilm formation in Clostridioides difficile

2021 ◽  
Vol 11 (1) ◽  
Author(s):  
Lisa F. Dawson ◽  
Johann Peltier ◽  
Catherine L. Hall ◽  
Mark A. Harrison ◽  
Maria Derakhshan ◽  
...  
Keyword(s):  
Cell Surface ◽  
Biofilm Formation ◽  
Bacterial Species ◽  
Surface Proteins ◽  
Extracellular Dna ◽  
Cell Surface Proteins ◽  
Secondary Messenger ◽  
Clostridioides Difficile ◽  
Antibiotic Efficacy ◽  
Biofilm Matrix

AbstractClostridioides difficile is the leading cause of nosocomial antibiotic-associated diarrhoea worldwide, yet there is little insight into intestinal tract colonisation and relapse. In many bacterial species, the secondary messenger cyclic-di-GMP mediates switching between planktonic phase, sessile growth and biofilm formation. We demonstrate that c-di-GMP promotes early biofilm formation in C. difficile and that four cell surface proteins contribute to biofilm formation, including two c-di-GMP regulated; CD2831 and CD3246, and two c-di-GMP-independent; CD3392 and CD0183. We demonstrate that C. difficile biofilms are composed of extracellular DNA (eDNA), cell surface and intracellular proteins, which form a protective matrix around C. difficile vegetative cells and spores, as shown by a protective effect against the antibiotic vancomycin. We demonstrate a positive correlation between biofilm biomass, sporulation frequency and eDNA abundance in all five C. difficile lineages. Strains 630 (RT012), CD305 (RT023) and M120 (RT078) contain significantly more eDNA in their biofilm matrix than strains R20291 (RT027) and M68 (RT017). DNase has a profound effect on biofilm integrity, resulting in complete disassembly of the biofilm matrix, inhibition of biofilm formation and reduced spore germination. The addition of exogenous DNase could be exploited in treatment of C. difficile infection and relapse, to improve antibiotic efficacy.

scholarly journals Exploring the Diversity of Listeria monocytogenes Biofilm Architecture by High-Throughput Confocal Laser Scanning Microscopy and the Predominance of the Honeycomb-Like Morphotype

2014 ◽  
Vol 81 (5) ◽  
pp. 1813-1819 ◽  
Author(s):  
Morgan Guilbaud ◽  
Pascal Piveteau ◽  
Mickaël Desvaux ◽  
Sylvain Brisse ◽  
Romain Briandet
Keyword(s):  
Listeria Monocytogenes ◽  
High Throughput ◽  
Biofilm Formation ◽  
Spatial Organization ◽  
Confocal Imaging ◽  
Laser Scanning Microscopy ◽  
Extracellular Dna ◽  
Confocal Laser ◽  
Published Data ◽  
Content Type

ABSTRACTListeria monocytogenesis involved in food-borne illness with a high mortality rate. The persistence of the pathogen along the food chain can be associated with its ability to form biofilms on inert surfaces. While most of the phenotypes associated with biofilms are related to their spatial organization, most published data comparing biofilm formation byL. monocytogenesisolates are based on the quantitative crystal violet assay, which does not give access to structural information. Using a high-throughput confocal-imaging approach, the aim of this work was to decipher the structural diversity of biofilms formed by 96L. monocytogenesstrains isolated from various environments. Prior to large-scale analysis, an experimental design was created to improveL. monocytogenesbiofilm formation in microscopic-grade microplates, with special emphasis on the growth medium composition. Microscopic analysis of biofilms formed under the selected conditions by the 96 isolates revealed only weak correlation between the genetic lineages of the isolates and the structural properties of the biofilms. However, a gradient in their geometric descriptors (biovolume, mean thickness, and roughness), ranging from flat multilayers to complex honeycomb-like structures, was shown. The dominant honeycomb-like morphotype was characterized by hollow voids hosting free-swimming cells and localized pockets containing mixtures of dead cells and extracellular DNA (eDNA).

scholarly journals Spo0A suppresses sin locus expression in Clostridioides difficile

2020 ◽  
Author(s):  
Babita Adhikari Dhungel ◽  
Revathi Govind
Keyword(s):  
Diarrheal Disease ◽  
Toxin Production ◽  
The United States ◽  
Upstream Region ◽  
Pcr Analysis ◽  
Bacterial Cells ◽  
Master Regulator ◽  
Clostridioides Difficile ◽  
Severity Of The Disease

AbstractClostridioides difficile is the leading cause of nosocomial infection and is the causative agent of antibiotic-associated diarrhea. The severity of the disease is directly associated with the production of toxins, and spores are responsible for the transmission and persistence of the organism. Previously we characterized sin locus regulators SinR and SinR’, where SinR is the regulator of toxin production and sporulation, while the SinR’ acting as its antagonist. In Bacillus subtilis, Spo0A, the master regulator of sporulation, regulates SinR, by regulating the expression of its antagonist sinI. However, the role of Spo0A in the expression of sinR and sinR’ in C. difficile is not yet reported. In this study, we tested spo0A mutants in three different C. difficile strains R20291, UK1, and JIR8094, to understand the role of Spo0A in sin locus expression. Western blot analysis revealed that spo0A mutants had increased SinR levels. The qRT-PCR analysis for its expression further supported this data. By carrying out genetic and biochemical assays, we have shown that Spo0A can bind to the upstream region of this locus to regulates its expression. This study provides vital information that Spo0A regulates sin locus, which controls critical pathogenic traits such as sporulation, toxin production, and motility in C. difficile.IMPORTANCEClostridioides difficile is the leading cause of antibiotic-associated diarrheal disease in the United States. During infection, C. difficile spores germinate, and the vegetative bacterial cells produce toxins that damage host tissue. In C. difficile, sin locus is known to regulate both sporulation and toxin production. In this study, we have shown that Spo0A, the master regulator of sporulation to control the sin locus expression. We performed various genetic and biochemical experiments to show that Spo0A directly regulates the expression of this locus by binding to its upstream DNA region. This observation adds new detail to the gene regulatory network that connects sporulation and toxin production in this pathogen.

scholarly journals Clostridioides difficile Spores: Bile Acid Sensors and Trojan Horses of Transmission

2020 ◽  
Vol 33 (02) ◽  
pp. 058-066
Author(s):  
Aimee Shen
Keyword(s):  
Disease Transmission ◽  
Disease Recurrence ◽  
The United States ◽  
Life Forms ◽  
Obligate Anaerobe ◽  
Infectious Particle ◽  
Clostridioides Difficile ◽  
Healthcare Associated ◽  
High Treatment ◽  
Annual Treatment Cost

AbstractThe Gram-positive, spore-forming bacterium, Clostridioides difficile is the leading cause of healthcare-associated infections in the United States, although it also causes a significant number of community-acquired infections. C. difficile infections, which range in severity from mild diarrhea to toxic megacolon, cost more to treat than matched infections, with an annual treatment cost of approximately $6 billion for almost half-a-million infections. These high–treatment costs are due to the high rates of C. difficile disease recurrence (>20%) and necessity for special disinfection measures. These complications arise in part because C. difficile makes metabolically dormant spores, which are the major infectious particle of this obligate anaerobe. These seemingly inanimate life forms are inert to antibiotics, resistant to commonly used disinfectants, readily disseminated, and capable of surviving in the environment for a long period of time. However, upon sensing specific bile salts in the vertebrate gut, C. difficile spores transform back into the vegetative cells that are responsible for causing disease. This review discusses how spores are ideal vectors for disease transmission and how antibiotics modulate this process. We also describe the resistance properties of spores and how they create challenges eradicating spores, as well as promote their spread. Lastly, environmental reservoirs of C. difficile spores and strategies for destroying them particularly in health care environments will be discussed.

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