scholarly journals Function of the CRISPR-Cas System of the Human Pathogen Clostridium difficile

mBio ◽  
2015 ◽  
Vol 6 (5) ◽  
Author(s):  
Pierre Boudry ◽  
Ekaterina Semenova ◽  
Marc Monot ◽  
Kirill A. Datsenko ◽  
Anna Lopatina ◽  
...  
Keyword(s):  
Clostridium Difficile ◽  
Type I ◽  
Human Pathogen ◽  
Range Analysis ◽  
Data Set ◽  
Content Type ◽  
Crispr System ◽  
Nosocomial Diarrhea ◽  
Active Expression ◽  
Foreign Dna

ABSTRACT Clostridium difficile is the cause of most frequently occurring nosocomial diarrhea worldwide. As an enteropathogen, C. difficile must be exposed to multiple exogenous genetic elements in bacteriophage-rich gut communities. CRISPR (clustered regularly interspaced short palindromic repeats)-Cas (CRISPR-associated) systems allow bacteria to adapt to foreign genetic invaders. Our recent data revealed active expression and processing of CRISPR RNAs from multiple type I-B CRISPR arrays in C. difficile reference strain 630. Here, we demonstrate active expression of CRISPR arrays in strain R20291, an epidemic C. difficile strain. Through genome sequencing and host range analysis of several new C. difficile phages and plasmid conjugation experiments, we provide evidence of defensive function of the CRISPR-Cas system in both C. difficile strains. We further demonstrate that C. difficile Cas proteins are capable of interference in a heterologous host, Escherichia coli. These data set the stage for mechanistic and physiological analyses of CRISPR-Cas-mediated interactions of important global human pathogen with its genetic parasites. IMPORTANCE Clostridium difficile is the major cause of nosocomial infections associated with antibiotic therapy worldwide. To survive in bacteriophage-rich gut communities, enteropathogens must develop efficient systems for defense against foreign DNA elements. CRISPR-Cas systems have recently taken center stage among various anti-invader bacterial defense systems. We provide experimental evidence for the function of the C. difficile CRISPR system against plasmid DNA and bacteriophages. These data demonstrate the original features of active C. difficile CRISPR system and bring important insights into the interactions of this major enteropathogen with foreign DNA invaders during its infection cycle.

scholarly journals A Role for Tetracycline Selection in Recent Evolution of Agriculture-Associated Clostridium difficile PCR Ribotype 078

mBio ◽  
2019 ◽  
Vol 10 (2) ◽  
Author(s):  
Kate E. Dingle ◽  
Xavier Didelot ◽  
T. Phuong Quan ◽  
David W. Eyre ◽  
Nicole Stoesser ◽  
...  
Keyword(s):  
Clostridium Difficile ◽  
Evolutionary History ◽  
Selective Pressure ◽  
Tetracycline Resistance ◽  
Clonal Expansion ◽  
Human Pathogen ◽  
Content Type ◽  
Resistance Determinants ◽  
Ribotype 078 ◽  
Human Infections

ABSTRACT The increasing clinical importance of human infections (frequently severe) caused by Clostridium difficile PCR ribotype 078 (RT078) was first reported in 2008. The severity of symptoms (mortality of ≤30%) and the higher proportion of infections among community and younger patients raised concerns. Farm animals, especially pigs, have been identified as RT078 reservoirs. We aimed to understand the recent changes in RT078 epidemiology by investigating a possible role for antimicrobial selection in its recent evolutionary history. Phylogenetic analysis of international RT078 genomes (isolates from 2006 to 2014, n = 400), using time-scaled, recombination-corrected, maximum likelihood phylogenies, revealed several recent clonal expansions. A common ancestor of each expansion had independently acquired a different allele of the tetracycline resistance gene tetM. Consequently, an unusually high proportion (76.5%) of RT078 genomes were tetM positive. Multiple additional tetracycline resistance determinants were also identified (including efflux pump tet40), frequently sharing a high level of nucleotide sequence identity (up to 100%) with sequences found in the pig pathogen Streptococcus suis and in other zoonotic pathogens such as Campylobacter jejuni and Campylobacter coli. Each RT078 tetM clonal expansion lacked geographic structure, indicating rapid, recent international spread. Resistance determinants for C. difficile infection-triggering antimicrobials, including fluoroquinolones and clindamycin, were comparatively rare in RT078. Tetracyclines are used intensively in agriculture; this selective pressure, plus rapid, international spread via the food chain, may explain the increased RT078 prevalence in humans. Our work indicates that the use of antimicrobials outside the health care environment has selected for resistant organisms, and in the case of RT078, has contributed to the emergence of a human pathogen. IMPORTANCE Clostridium difficile PCR ribotype 078 (RT078) has multiple reservoirs; many are agricultural. Since 2005, this genotype has been increasingly associated with human infections in both clinical settings and the community. Investigations of RT078 whole-genome sequences revealed that tetracycline resistance had been acquired on multiple independent occasions. Phylogenetic analysis revealed a rapid, recent increase in numbers of closely related tetracycline-resistant RT078 (clonal expansions), suggesting that tetracycline selection has strongly influenced its recent evolutionary history. We demonstrate recent international spread of emergent, tetracycline-resistant RT078. A similar tetracycline-positive clonal expansion was also identified in unrelated nontoxigenic C. difficile, suggesting that this process may be widespread and may be independent of disease-causing ability. Resistance to typical C. difficile infection-associated antimicrobials (e.g., fluoroquinolones, clindamycin) occurred only sporadically within RT078. Selective pressure from tetracycline appears to be a key factor in the emergence of this human pathogen and the rapid international dissemination that followed, plausibly via the food chain.

scholarly journals Genomic Epidemiology of a Protracted Hospital Outbreak Caused by a Toxin A-Negative Clostridium difficile Sublineage PCR Ribotype 017 Strain in London, England

2015 ◽  
Vol 53 (10) ◽  
pp. 3141-3147 ◽  
Author(s):  
M. D. Cairns ◽  
M. D. Preston ◽  
T. D. Lawley ◽  
T. G. Clark ◽  
R. A. Stabler ◽  
...  
Keyword(s):  
Clostridium Difficile ◽  
De Novo ◽  
University Hospital ◽  
Nucleotide Polymorphisms ◽  
Single Nucleotide ◽  
Conjugative Transposon ◽  
Content Type ◽  
Genomic Epidemiology ◽  
Hospital Outbreak ◽  
Nosocomial Diarrhea

Clostridium difficileremains the leading cause of nosocomial diarrhea worldwide, which is largely considered to be due to the production of two potent toxins: TcdA and TcdB. However, PCR ribotype (RT) 017, one of five clonal lineages of human virulentC. difficile, lacks TcdA expression but causes widespread disease. Whole-genome sequencing was applied to 35 isolates from hospitalized patients withC. difficileinfection (CDI) and two environmental ward isolates in London, England. The phylogenetic analysis of single nucleotide polymorphisms (SNPs) revealed a clonal cluster of temporally variable isolates from a single hospital ward at University Hospital Lewisham (UHL) that were distinct from other London hospital isolates.De novoassembled genomes revealed a 49-kbp putative conjugative transposon exclusive to this hospital clonal cluster which would not be revealed by current typing methodologies. This study identified three sublineages ofC. difficileRT017 that are circulating in London. Similar to the notorious RT027 lineage, which has caused global outbreaks of CDI since 2001, the lineage of toxin-defective RT017 strains appears to be continually evolving. By utilization of WGS technologies to identify SNPs and the evolution of clonal strains, the transmission of outbreaks caused by near-identical isolates can be retraced and identified.

scholarly journals Bacteriophage Combinations Significantly Reduce Clostridium difficile GrowthIn Vitroand ProliferationIn Vivo

2015 ◽  
Vol 60 (2) ◽  
pp. 968-981 ◽  
Author(s):  
Janet Y. Nale ◽  
Janice Spencer ◽  
Katherine R. Hargreaves ◽  
Anthony M. Buckley ◽  
Przemysław Trzepiński ◽  
...  
Keyword(s):  
Clostridium Difficile ◽  
Oral Delivery ◽  
Therapeutic Potential ◽  
Bacterial Load ◽  
Range Analysis ◽  
Hamster Model ◽  
Content Type ◽  
Phage Types ◽  
Specific Phage

ABSTRACTThe microbiome dysbiosis caused by antibiotic treatment has been associated with both susceptibility to and relapse ofClostridium difficileinfection (CDI). Bacteriophage (phage) therapy offers target specificity and dose amplificationin situ, but few studies have focused on its use in CDI treatment. This mainly reflects the lack of strictly virulent phages that target this pathogen. While it is widely accepted that temperate phages are unsuitable for therapeutic purposes due to their transduction potential, analysis of sevenC. difficilephages confirmed that this impact could be curtailed by the application of multiple phage types. Here, host range analysis of six myoviruses and one siphovirus was conducted on 80 strains representing 21 major epidemic and clinically severe ribotypes. The phages had complementary coverage, lysing 18 and 62 of the ribotypes and strains tested, respectively. Single-phage treatments of ribotype 076, 014/020, and 027 strains showed an initial reduction in the bacterial load followed by the emergence of phage-resistant colonies. However, these colonies remained susceptible to infection with an unrelated phage. In contrast, specific phage combinations caused the complete lysis ofC. difficilein vitroand prevented the appearance of resistant/lysogenic clones. Using a hamster model, the oral delivery of optimized phage combinations resulted in reducedC. difficilecolonization at 36 h postinfection. Interestingly, free phages were recovered from the bowel at this time. In a challenge model of the disease, phage treatment delayed the onset of symptoms by 33 h compared to the time of onset of symptoms in untreated animals. These data demonstrate the therapeutic potential of phage combinations to treat CDI.

scholarly journals Phage ϕC2 Mediates Transduction of Tn6215, Encoding Erythromycin Resistance, between Clostridium difficile Strains

mBio ◽  
2013 ◽  
Vol 4 (6) ◽  
Author(s):  
Shan Goh ◽  
Haitham Hussain ◽  
Barbara J. Chang ◽  
Warren Emmett ◽  
Thomas V. Riley ◽  
...  
Keyword(s):  
Clostridium Difficile ◽  
Genetic Manipulation ◽  
Recipient Strain ◽  
Open Reading Frames ◽  
Human Pathogen ◽  
Erythromycin Resistance ◽  
Content Type ◽  
Filter Mating ◽  
First Time ◽  
Reading Frames

ABSTRACTIn this work, we show thatClostridium difficilephage ϕC2 transduceserm(B), which confers erythromycin resistance, from a donor to a recipient strain at a frequency of 10−6per PFU. The transductants were lysogenic for ϕC2 and contained theerm(B) gene in a novel transposon, Tn6215. This element is 13,008 bp in length and contains 17 putative open reading frames (ORFs). It could also be transferred at a lower frequency by filter mating.IMPORTANCEClostridium difficileis a major human pathogen that causes diarrhea that can be persistent and difficult to resolve using antibiotics.C. difficileis potentially zoonotic and has been detected in animals, food, and environmental samples.C. difficilegenomes contain large portions of horizontally acquired genetic elements. The conjugative elements have been reasonably well studied, but transduction has not yet been demonstrated. Here, we show for the first time transduction as a mechanism for the transfer of a novel genetic element inC. difficile. Transduction may also be a useful tool for the genetic manipulation ofC. difficile.

scholarly journals Mining the Methylome Reveals Extensive Diversity in Staphylococcus epidermidis Restriction Modification

mBio ◽  
2019 ◽  
Vol 10 (6) ◽  
Author(s):  
Jean Y. H. Lee ◽  
Glen P. Carter ◽  
Sacha J. Pidot ◽  
Romain Guérillot ◽  
Torsten Seemann ◽  
...  
Keyword(s):  
Staphylococcus Epidermidis ◽  
Genetic Manipulation ◽  
Gene Arrangement ◽  
Type I ◽  
Content Type ◽  
Efficient Approach ◽  
Molecular Studies ◽  
Foreign Dna ◽  
Clinically Significant ◽  
Restriction Modification

ABSTRACT Staphylococcus epidermidis is a significant opportunistic pathogen of humans. Molecular studies in this species have been hampered by the presence of restriction-modification (RM) systems that limit introduction of foreign DNA. Here, we establish the complete genomes and methylomes for seven clinically significant, genetically diverse S. epidermidis isolates and perform the first systematic genomic analyses of the type I RM systems within both S. epidermidis and Staphylococcus aureus. Our analyses revealed marked differences in the gene arrangement, chromosomal location, and movement of type I RM systems between the two species. Unlike S. aureus, S. epidermidis type I RM systems demonstrate extensive diversity even within a single genetic lineage. This is contrary to current assumptions and has important implications for approaching the genetic manipulation of S. epidermidis. Using Escherichia coli plasmid artificial modification (PAM) to express S. epidermidis hsdMS, we readily overcame restriction barriers in S. epidermidis and achieved electroporation efficiencies equivalent to those of modification-deficient mutants. With these functional experiments, we demonstrated how genomic data can be used to predict both the functionality of type I RM systems and the potential for a strain to be electroporation proficient. We outline an efficient approach for the genetic manipulation of S. epidermidis strains from diverse genetic backgrounds, including those that have hitherto been intractable. Additionally, we identified S. epidermidis BPH0736, a naturally restriction-defective, clinically significant, multidrug-resistant ST2 isolate, as an ideal candidate for molecular studies. IMPORTANCE Staphylococcus epidermidis is a major cause of hospital-acquired infections, especially those related to implanted medical devices. Understanding how S. epidermidis causes disease and devising ways to combat these infections have been hindered by an inability to genetically manipulate clinically significant hospital-adapted strains. Here, we provide the first comprehensive analyses of the barriers to the uptake of foreign DNA in S. epidermidis and demonstrate that these are distinct from those described for S. aureus. Using these insights, we demonstrate an efficient approach for the genetic manipulation of S. epidermidis to enable the study of clinical isolates for the first time.

scholarly journals Avoidance of Trinucleotide Corresponding to Consensus Protospacer Adjacent Motif Controls the Efficiency of Prespacer Selection during Primed Adaptation

mBio ◽  
2018 ◽  
Vol 9 (6) ◽  
Author(s):  
Olga Musharova ◽  
Danylo Vyhovskyi ◽  
Sofia Medvedeva ◽  
Jelena Guzina ◽  
Yulia Zhitnyuk ◽  
...  
Keyword(s):  
Selection Process ◽  
Natural Populations ◽  
Type I ◽  
Dna Fragments ◽  
Dna Arrays ◽  
Content Type ◽  
Factors Affecting ◽  
Crispr Array ◽  
Protospacer Adjacent Motif ◽  
Foreign Dna

ABSTRACT CRISPR DNA arrays of unique spacers separated by identical repeats ensure prokaryotic immunity through specific targeting of foreign nucleic acids complementary to spacers. New spacers are acquired into a CRISPR array in a process of CRISPR adaptation. Selection of foreign DNA fragments to be integrated into CRISPR arrays relies on PAM (protospacer adjacent motif) recognition, as only those spacers will be functional against invaders. However, acquisition of different PAM-associated spacers proceeds with markedly different efficiency from the same DNA. Here, we used a combination of bioinformatics and experimental approaches to understand factors affecting the efficiency of acquisition of spacers by the Escherichia coli type I-E CRISPR-Cas system, for which two modes of CRISPR adaptation have been described: naive and primed. We found that during primed adaptation, efficiency of spacer acquisition is strongly negatively affected by the presence of an AAG trinucleotide—a consensus PAM—within the sequence being selected. No such trend is observed during naive adaptation. The results are consistent with a unidirectional spacer selection process during primed adaptation and provide a specific signature for identification of spacers acquired through primed adaptation in natural populations. IMPORTANCE Adaptive immunity of prokaryotes depends on acquisition of foreign DNA fragments into CRISPR arrays as spacers followed by destruction of foreign DNA by CRISPR interference machinery. Different fragments are acquired into CRISPR arrays with widely different efficiencies, but the factors responsible are not known. We analyzed the frequency of spacers acquired during primed adaptation in an E. coli CRISPR array and found that AAG motif was depleted from highly acquired spacers. AAG is also a consensus protospacer adjacent motif (PAM) that must be present upstream from the target of the CRISPR spacer for its efficient destruction by the interference machinery. These results are important because they provide new information on the mechanism of primed spacer acquisition. They add to other previous evidence in the field that pointed out to a “directionality” in the capture of new spacers. Our data strongly suggest that the recognition of an AAG PAM by the interference machinery components prior to spacer capture occludes downstream AAG sequences, thus preventing their recognition by the adaptation machinery.

Group acceptance sampling plans based on time truncated life tests for compound Weibull-exponential distribution

2021 ◽  
Vol ahead-of-print (ahead-of-print) ◽  
Author(s):  
Ayten Yiğiter ◽  
Canan Hamurkaroğlu ◽  
Nazan Danacıoğlu
Keyword(s):  
Acceptance Sampling ◽  
Type I ◽  
Consumer’S Risk ◽  
Producer’S Risk ◽  
Data Set ◽  
Sampling Plans ◽  
Content Type ◽  
Acceptance Sampling Plans ◽  
Life Tests ◽  
Group Acceptance

PurposeAcceptance sampling plans are a decision-making process on the basis of a randomly selected sampling from a party, where it is not possible to completely scan the products for reasons such as time and cost being limited or the formation of damaged products during the inspection. For some products, the life span (time from beginning to failure) may be an important quality characteristic. In this case, the quality control adequacy of the products can be checked with an acceptance sampling plan based on the truncated life test with a censored scheme for the lifetime of the products. In this study, group acceptance sampling plans (GASPs) based on life tests are studied under the Type-I censored scheme for the compound Weibull-exponential (CWE) distribution.Design/methodology/approachGASPs based on life tests under the Type-I censored scheme for the CWE distribution are developed by using both the producer's risk and the consumer's risk.FindingsIn this study, optimum sample size, optimum number of groups and acceptance number are obtained under the Type-I censored scheme for the CWE distribution. Real data set illustration is given to show GASPs how to be used for the industry applications.Originality/valueDifferent from acceptance sampling plans with just considering the producer's risk, GASPs are constructed by using two-point approach included both the producer's risk and the consumer's risk for CWE distribution.

scholarly journals Usefulness of Adjunctive Fecal Calprotectin and Serum Procalcitonin in Individuals Positive for Clostridium difficile Toxin Gene by PCR Assay

2015 ◽  
Vol 53 (11) ◽  
pp. 3667-3669 ◽  
Author(s):  
Kristin Y. Popiel ◽  
Romina Gheorghe ◽  
Jennifer Eastmond ◽  
Mark A. Miller
Keyword(s):  
Clostridium Difficile ◽  
Fecal Calprotectin ◽  
Toxin Gene ◽  
Pcr Assay ◽  
Content Type ◽  
Nosocomial Diarrhea ◽  
Clostridium Difficile Toxin ◽  
Stool Samples

In 54/64 subjects with nosocomial diarrhea, fecal calprotectin levels correlated with the results of stool samples tested forClostridium difficiletoxin gene by PCR. Fecal calprotectin levels can be used as an adjunctive measure to PCR to support the diagnosis ofC. difficileinfection.

Financial distress determinants among SMEs: empirical evidence from Sweden

2020 ◽  
Vol 47 (3) ◽  
pp. 547-560 ◽  
Author(s):  
Darush Yazdanfar ◽  
Peter Öhman
Keyword(s):  
Financial Crisis ◽  
Financial Distress ◽  
Large Scale ◽  
Global Financial Crisis ◽  
Binary Logistic Regression ◽  
Data Availability ◽  
Cross Sectional ◽  
Data Set ◽  
Content Type ◽  
The Global Financial Crisis

PurposeThe purpose of this study is to empirically investigate determinants of financial distress among small and medium-sized enterprises (SMEs) during the global financial crisis and post-crisis periods.Design/methodology/approachSeveral statistical methods, including multiple binary logistic regression, were used to analyse a longitudinal cross-sectional panel data set of 3,865 Swedish SMEs operating in five industries over the 2008–2015 period.FindingsThe results suggest that financial distress is influenced by macroeconomic conditions (i.e. the global financial crisis) and, in particular, by various firm-specific characteristics (i.e. performance, financial leverage and financial distress in previous year). However, firm size and industry affiliation have no significant relationship with financial distress.Research limitationsDue to data availability, this study is limited to a sample of Swedish SMEs in five industries covering eight years. Further research could examine the generalizability of these findings by investigating other firms operating in other industries and other countries.Originality/valueThis study is the first to examine determinants of financial distress among SMEs operating in Sweden using data from a large-scale longitudinal cross-sectional database.

scholarly journals Modulation of Chicken Intestinal Immune Gene Expression by Small Cationic Peptides as Feed Additives during the First Week Posthatch

2013 ◽  
Vol 20 (9) ◽  
pp. 1440-1448 ◽  
Author(s):  
Michael H. Kogut ◽  
Kenneth J. Genovese ◽  
Haiqi He ◽  
Christina L. Swaggerty ◽  
Yiwei Jiang
Keyword(s):  
Gene Expression ◽  
Proinflammatory Cytokines ◽  
Proinflammatory Cytokine ◽  
Feed Additives ◽  
Feed Additive ◽  
Cationic Peptides ◽  
Type I ◽  
Immune Gene ◽  
Content Type ◽  
Functional Efficiency

ABSTRACTWe have been investigating modulation strategies tailored around the selective stimulation of the host's immune system as an alternative to direct targeting of microbial pathogens by antibiotics. One such approach is the use of a group of small cationic peptides (BT) produced by a Gram-positive soil bacterium,Brevibacillus texasporus. These peptides have immune modulatory properties that enhance both leukocyte functional efficiency and leukocyte proinflammatory cytokine and chemokine mRNA transcription activitiesin vitro. In addition, when provided as a feed additive for just 4 days posthatch, BT peptides significantly induce a concentration-dependent protection against cecal and extraintestinal colonization bySalmonella entericaserovar Enteritidis. In the present studies, we assessed the effects of feeding BT peptides on transcriptional changes on proinflammatory cytokines, inflammatory chemokines, and Toll-like receptors (TLR) in the ceca of broiler chickens with and withoutS. Enteritidis infection. After feeding a BT peptide-supplemented diet for the first 4 days posthatch, chickens were then challenged withS. Enteritidis, and intestinal gene expression was measured at 1 or 7 days postinfection (p.i.) (5 or 11 days of age). Intestinal expression of innate immune mRNA transcripts was analyzed by quantitative real-time PCR (qRT-PCR). Analysis of relative mRNA expression showed that a BT peptide-supplemented diet did not directly induce the transcription of proinflammatory cytokine, inflammatory chemokine, type I/II interferon (IFN), or TLR mRNA in chicken cecum. However, feeding the BT peptide-supplemented diet primed cecal tissue for increased (P≤ 0.05) transcription of TLR4, TLR15, and TLR21 upon infection withS. Enteritidis on days 1 and 7 p.i. Likewise, feeding the BT peptides primed the cecal tissue for increased transcription of proinflammatory cytokines (interleukin 1β [IL-1β], IL-6, IL-18, type I and II IFNs) and inflammatory chemokine (CxCLi2) in response toS. Enteritidis infection 1 and 7 days p.i. compared to the chickens fed the basal diet. These small cationic peptides may prove useful as alternatives to antibiotics as local immune modulators in neonatal poultry by providing prophylactic protection againstSalmonellainfections.

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