Impact of Phage CDHS-1 on the Transcription, Physiology and Pathogenicity of a Clostridioides difficile Ribotype 027 Strain, R20291

All known Clostridioides difficile phages encode integrases rendering them potentially able to lyse or lysogenise bacterial strains. Here, we observed the infection of the siphovirus, CDHS-1 on a ribotype 027 strain, R20291 and determined the phage and bacterial gene expression profiles, and impacts of phage infection on bacterial physiology and pathogenicity. Using RNA-seq and RT-qPCR we analysed transcriptomic changes during early, mid-log and late phases of phage replication at an MOI of 10. The phage has a 20 min latent period, takes 80 min to lyse cells and a burst size of ~37. All phage genes are highly expressed during at least one time point. The Cro/C1-transcriptional regulator, ssDNA binding protein and helicase are expressed early, the holin is expressed during the mid-log phase and structural proteins are expressed from mid-log to late phase. Most bacterial genes, particularly the metabolism and toxin production/regulatory genes, were downregulated from early phage replication. Phage-resistant strains and lysogens showed reduced virulence during Galleria mellonella colonization as ascertained by the larval survival and expression of growth (10), reproduction (2) and infection (2) marker genes. These data suggest that phage infection both reduces colonization and negatively impacts bacterial pathogenicity, providing encouraging data to support the development of this phage for therapy to treat C. difficile infection.

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Refining the Galleria mellonella Model by Using Stress Marker Genes to Assess Clostridioides difficile Infection and Recuperation during Phage Therapy

Microorganisms ◽

10.3390/microorganisms8091306 ◽

2020 ◽

Vol 8 (9) ◽

pp. 1306

Author(s):

Janet Y. Nale ◽

Mahananda Chutia ◽

Jeffrey K. J. Cheng ◽

Martha R. J. Clokie

Keyword(s):

Phage Therapy ◽

Galleria Mellonella ◽

Clinical Symptoms ◽

Expression Profiles ◽

Larval Survival ◽

Marker Genes ◽

Control Groups ◽

Stress Marker ◽

Clostridioides Difficile ◽

Bacterial Control

The Galleria mellonella is an effective model for probing Clostridioides difficile interactions with phages. Despite valuable insights from this model, the larvae are not easily amenable to assessing detailed clinical responses to either bacteria or phages. Here, larval survival, colonisation and toxin levels were compared to expression profiles of 17 G. mellonella stress genes to monitor Clostridiodes difficile infection (CDI), and recuperation during phage therapy. The larvae were infected with a ribotype 014/020 isolate and treated with an optimised phage cocktail. Larvae treated prophylactically with phages and the phage-control larval group were protected, showing the highest survival, and low C. difficile colonisation and toxin rates, compared to co-infection, remedial and bacterial-control larval groups. Expression of growth (9) and reproduction (2) genes were enhanced within prophylaxis and phage-control larval groups compared to the co-infection, remedial and bacterial control groups. In contrast, expression of infection (2), humoral (1) and cellular (3) immunity genes declined in the prophylactic and phage-control groups but increased in the co-infection, remedial and bacterial control larvae. The molecular markers augment the survival, colonisation and toxin data and allow detailed monitoring of CDI and recovery. This data support the use of stress marker genes as tools to analyse clinical symptoms in this model.

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Mechanisms by Which Small RNAs Affect Bacterial Activity

Journal of Dental Research ◽

10.1177/0022034519876898 ◽

2019 ◽

Vol 98 (12) ◽

pp. 1315-1323

Author(s):

L. Lei ◽

Y. Yang ◽

S. Wu ◽

X. Ma ◽

...

Keyword(s):

Stress Responses ◽

Small Rnas ◽

Environmental Changes ◽

Expression Profiles ◽

Bacterial Flora ◽

Oral Disease ◽

Bacterial Gene ◽

Bacterial Physiology ◽

Cellular Processes ◽

Regulatory Rnas

The oral cavity contains a distinct habitat that supports diverse bacterial flora. Recent observations have provided additional evidence that sRNAs are key regulators of bacterial physiology and pathogenesis. These sRNAs have been divided into 5 functional groups: cis-encoded RNAs, trans-encoded RNAs, RNA regulators of protein activity, bacterial CRISPR (clustered regularly interspaced short palindromic repeat) RNAs, and a novel category of miRNA-size small RNAs (msRNAs). In this review, we discuss a critical group of key commensal and opportunistic oral pathogens. In general, supragingival bacterial sRNAs function synergistically to fine-tune the regulation of cellular processes and stress responses in adaptation to environmental changes. Particularly in the cariogenic bacteria Streptococcus mutans, both the antisense vicR RNA and msRNA1657 can impede the metabolism of bacterial exopolysaccharides, prevent biofilm formation, and suppress its cariogenicity. In Enterococcus faecalis, selected sRNAs control the expression of proteins involved in diverse cellular processes and stress responses. In subgingival plaques, sRNAs from periodontal pathogens can function as novel bacterial signaling molecules that mediate bacterial-human interactions in periodontal homeostasis. In Porphyromonas gingivalis, the expression profiles of putative sRNA101 and sRNA42 were found to respond to hemin availability after hemin starvation. Regarding Aggregatibacter actinomycetemcomitans (previously Actinobacillus actinomycetemcomitans), a major periodontal pathogen associated with aggressive periodontitis, the predicted sRNAs interact with several virulence genes, including those encoding leukotoxin and cytolethal distending toxin. Furthermore, in clinical isolates, these associated RNAs could be explored not only as potential biomarkers for oral disease monitoring but also as alternative types of regulators for drug design. Thus, this emerging subspecialty of bacterial regulatory RNAs could reshape our understanding of bacterial gene regulation from their key roles of endogenous regulatory RNAs to their activities in pathologic processes.

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Parallel Genomics Uncover Novel Enterococcal-Bacteriophage Interactions

mBio ◽

10.1128/mbio.03120-19 ◽

2020 ◽

Vol 11 (2) ◽

Cited By ~ 13

Author(s):

Anushila Chatterjee ◽

Julia L. E. Willett ◽

Uyen Thy Nguyen ◽

Brendan Monogue ◽

Kelli L. Palmer ◽

...

Keyword(s):

Bacterial Infections ◽

Response Regulator ◽

Burst Size ◽

Bacterial Community Composition ◽

Model Organism ◽

Phage Infection ◽

Bacterial Cells ◽

Transcriptomic Profiling ◽

Bacterial Gene ◽

Content Type

ABSTRACT Bacteriophages (phages) have been proposed as alternative therapeutics for the treatment of multidrug-resistant bacterial infections. However, there are major gaps in our understanding of the molecular events in bacterial cells that control how bacteria respond to phage predation. Using the model organism Enterococcus faecalis, we used two distinct genomic approaches, namely, transposon library screening and RNA sequencing, to investigate the interaction of E. faecalis with a virulent phage. We discovered that a transcription factor encoding a LytR family response regulator controls the expression of enterococcal polysaccharide antigen (epa) genes that are involved in phage infection and bacterial fitness. In addition, we discovered that DNA mismatch repair mutants rapidly evolve phage adsorption deficiencies, underpinning a molecular basis for epa mutation during phage infection. Transcriptomic profiling of phage-infected E. faecalis revealed broad transcriptional changes influencing viral replication and progeny burst size. We also demonstrate that phage infection alters the expression of bacterial genes associated with intra- and interbacterial interactions, including genes involved in quorum sensing and polymicrobial competition. Together, our results suggest that phage predation has the potential to influence complex microbial behavior and may dictate how bacteria respond to external environmental stimuli. These responses could have collateral effects (positive or negative) on microbial communities, such as the host microbiota, during phage therapy. IMPORTANCE We lack fundamental understanding of how phage infection influences bacterial gene expression and, consequently, how bacterial responses to phage infection affect the assembly of polymicrobial communities. Using parallel genomic approaches, we have discovered novel transcriptional regulators and metabolic genes that influence phage infection. The integration of whole-genome transcriptomic profiling during phage infection has revealed the differential regulation of genes important for group behaviors and polymicrobial interactions. Our work suggests that therapeutic phages could more broadly influence bacterial community composition outside their intended host targets.

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Parallel genomics uncover novel enterococcal-bacteriophage interactions

10.1101/858506 ◽

2019 ◽

Cited By ~ 1

Author(s):

Anushila Chatterjee ◽

Julia L. E. Willett ◽

Uyen Thy Nguyen ◽

Brendan Monogue ◽

Kelli L. Palmer ◽

...

Keyword(s):

Bacterial Infections ◽

Response Regulator ◽

Burst Size ◽

Bacterial Community Composition ◽

Model Organism ◽

Multidrug Resistant ◽

Phage Infection ◽

Bacterial Cells ◽

Transcriptomic Profiling ◽

Bacterial Gene

AbstractBacteriophages (phages) have been proposed as alternative therapeutics for the treatment of multidrug resistant bacterial infections. However, there are major gaps in our understanding of the molecular events in bacterial cells that control how bacteria respond to phage predation. Using the model organism Enterococcus faecalis, we employed two distinct genomic approaches, transposon (Tn) library screening and RNA sequencing, to investigate the interaction of E. faecalis with a virulent phage. We discovered that a transcription factor encoding a LytR family response regulator controls the expression of enterococcal polysaccharide antigen (epa) genes that are involved in phage infection and bacterial fitness. In addition, we discovered that DNA mismatch repair mutants rapidly evolve phage adsorption deficiencies, underpinning a molecular basis for epa mutation during phage infection. Transcriptomic profiling of phage infected E. faecalis revealed broad transcriptional changes influencing viral replication and progeny burst size. We also demonstrate that phage infection alters the expression of bacterial genes associated with intra and inter-bacterial interactions, including genes involved in quorum sensing and polymicrobial competition. Together our results suggest that phage predation has the potential to influence complex microbial behavior and may dictate how bacteria respond to external environmental stimuli. These responses could have collateral effects (positive or negative) on microbial communities such as the host microbiota during phage therapy.ImportanceWe lack fundamental understanding of how phage infection influences bacterial gene expression and consequently how bacterial responses to phage infection affect the assembly of polymicrobial communities. Using parallel genomic approaches, we have discovered novel transcriptional regulators and metabolic genes that influence phage infection. The integration of whole genome transcriptomic profiling during phage infection has revealed the differential regulation of genes important for group behaviors and polymicrobial interactions. Our work suggests that therapeutic phages could more broadly influence bacterial community composition outside of their intended host targets.

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Molecular characterization of Clostridioides difficile ribotype 027 in a major Chinese hospital

Journal of Microbiology Immunology and Infection ◽

10.1016/j.jmii.2021.01.003 ◽

2021 ◽

Author(s):

Ren-feng Zhang ◽

Yu-xia Man ◽

Yuan-yuan Bai ◽

Chun-hong Shao ◽

Chun-mei Liu ◽

...

Keyword(s):

Molecular Characterization ◽

Ribotype 027 ◽

Clostridioides Difficile

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In VitroandIn VivoAntimicrobial Activities of Gallium Nitrate against Multidrug-Resistant Acinetobacter baumannii

Antimicrobial Agents and Chemotherapy ◽

10.1128/aac.01519-12 ◽

2012 ◽

Vol 56 (11) ◽

pp. 5961-5970 ◽

Cited By ~ 72

Author(s):

Luísa C. S. Antunes ◽

Francesco Imperi ◽

Fabrizia Minandri ◽

Paolo Visca

Keyword(s):

Human Serum ◽

Acinetobacter Baumannii ◽

Galleria Mellonella ◽

Therapeutic Potential ◽

Survival Rates ◽

Bloodstream Infections ◽

Multidrug Resistant ◽

Gallium Nitrate ◽

Content Type ◽

Bacterial Physiology

ABSTRACTMultidrug-resistantAcinetobacter baumanniiposes a tremendous challenge to traditional antibiotic therapy. Due to the crucial role of iron in bacterial physiology and pathogenicity, we investigated iron metabolism as a possible target for anti-A. baumanniichemotherapy using gallium as an iron mimetic. Due to chemical similarity, gallium competes with iron for binding to several redox enzymes, thereby interfering with a number of essential biological reactions. We found that Ga(NO3)3, the active component of an FDA-approved drug (Ganite), inhibits the growth of a collection of 58A. baumanniistrains in both chemically defined medium and human serum, at concentrations ranging from 2 to 80 μM and from 4 to 64 μM, respectively. Ga(NO3)3delayed the entry ofA. baumanniiinto the exponential phase and drastically reduced bacterial growth rates. Ga(NO3)3activity was strongly dependent on iron availability in the culture medium, though the mechanism of growth inhibition was independent of dysregulation of gene expression controlled by the ferric uptake regulator Fur. Ga(NO3)3also protectedGalleria mellonellalarvae from lethalA. baumanniiinfection, with survival rates of ≥75%. At therapeutic concentrations for humans (28 μM plasma levels), Ga(NO3)3inhibited the growth in human serum of 76% of the multidrug-resistantA. baumanniiisolates tested by ≥90%, raising expectations on the therapeutic potential of gallium for the treatment ofA. baumanniibloodstream infections. Ga(NO3)3also showed strong synergism with colistin, suggesting that a colistin-gallium combination holds promise as a last-resort therapy for infections caused by pan-resistantA. baumannii.

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Galleria mellonella Larvae as an Infection Model to Investigate sRNA-Mediated Pathogenesis in Staphylococcus aureus

Frontiers in Cellular and Infection Microbiology ◽

10.3389/fcimb.2021.631710 ◽

2021 ◽

Vol 11 ◽

Author(s):

Guillaume Ménard ◽

Astrid Rouillon ◽

Gevorg Ghukasyan ◽

Mathieu Emily ◽

Brice Felden ◽

...

Keyword(s):

Staphylococcus Aureus ◽

Galleria Mellonella ◽

Inflammatory Responses ◽

Expression Profiles ◽

Bacterial Pathogens ◽

Infection Model ◽

Animal Testing ◽

Easy Method ◽

Larval Infection ◽

Over Time

Small regulatory RNAs (sRNAs) are key players in bacterial regulatory networks. Monitoring their expression inside living colonized or infected organisms is essential for identifying sRNA functions, but few studies have looked at sRNA expression during host infection with bacterial pathogens. Insufficient in vivo studies monitoring sRNA expression attest to the difficulties in collecting such data, we therefore developed a non-mammalian infection model using larval Galleria mellonella to analyze the roles of Staphylococcus aureus sRNAs during larval infection and to quickly determine possible sRNA involvement in staphylococcal virulence before proceeding to more complicated animal testing. We began by using the model to test infected larvae for immunohistochemical evidence of infection as well as host inflammatory responses over time. To monitor sRNA expression during infection, total RNAs were extracted from the larvae and invading bacteria at different time points. The expression profiles of the tested sRNAs were distinct and they fluctuated over time, with expression of both sprD and sprC increased during infection and associated with mortality, while rnaIII expression remained barely detectable over time. A strong correlation was observed between sprD expression and the mortality. To confirm these results, we used sRNA-knockout mutants to investigate sRNA involvement in Staphylococcus aureus pathogenesis, finding that the decrease in death rates is delayed when either sprD or sprC was lacking. These results demonstrate the relevance of this G. mellonella model for investigating the role of sRNAs as transcriptional regulators involved in staphylococcal virulence. This insect model provides a fast and easy method for monitoring sRNA (and mRNA) participation in S. aureus pathogenesis, and can also be used for other human bacterial pathogens.

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Molecular Classification of Multiple Myeloma: A Distinct Transcriptional Profile Characterizes Patients Expressing CCND1 and Negative for 14q32 Translocations

Journal of Clinical Oncology ◽

10.1200/jco.2005.01.3870 ◽

2005 ◽

Vol 23 (29) ◽

pp. 7296-7306 ◽

Cited By ~ 90

Author(s):

Luca Agnelli ◽

Silvio Bicciato ◽

Michela Mattioli ◽

Sonia Fabris ◽

Daniela Intini ◽

...

Keyword(s):

Gene Expression ◽

Multiple Myeloma ◽

Expression Profiles ◽

Quantitative Polymerase Chain Reaction ◽

Gene Expression Pattern ◽

Marker Genes ◽

Published Data ◽

Cyclin D ◽

Chromosome 13Q ◽

13Q Deletion

Purpose The deregulation of CCND1, CCND2 and CCND3 genes represents a common event in multiple myeloma (MM). A recently proposed classification grouped MM patients into five classes on the basis of their cyclin D expression profiles and the presence of the main translocations involving the immunoglobulin heavy chain locus (IGH) at 14q32. In this study, we provide a molecular characterization of the identified translocations/cyclins (TC) groups. Materials and Methods The gene expression profiles of purified plasma cells from 50 MM cases were used to stratify the samples into the five TC classes and identify their transcriptional fingerprints. The cyclin D expression data were validated by means of real-time quantitative polymerase chain reaction analysis; fluorescence in situ hybridization was used to investigate the cyclin D loci arrangements, and to detect the main IGH translocations and the chromosome 13q deletion. Results Class-prediction analysis identified 112 probe sets as characterizing the TC1, TC2, TC4 and TC5 groups, whereas the TC3 samples showed heterogeneous phenotypes and no marker genes. The TC2 group, which showed extra copies of the CCND1 locus and no IGH translocations or the chromosome 13q deletion, was characterized by the overexpression of genes involved in protein biosynthesis at the translational level. A meta-analysis of published data sets validated the identified gene expression signatures. Conclusion Our data contribute to the understanding of the molecular and biologic features of distinct MM subtypes. The identification of a distinctive gene expression pattern in TC2 patients may improve risk stratification and indicate novel therapeutic targets.

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Antimicrobial susceptibility of Clostridioides difficile isolated from diarrhoeal stool specimens of Canadian patients: summary of results from the Canadian Clostridioides difficile (CAN-DIFF) surveillance study from 2013 to 2017

Journal of Antimicrobial Chemotherapy ◽

10.1093/jac/dkaa118 ◽

2020 ◽

Vol 75 (7) ◽

pp. 1824-1832

Author(s):

James A Karlowsky ◽

Heather J Adam ◽

Melanie R Baxter ◽

Christopher W Dutka ◽

Kim A Nichol ◽

...

Keyword(s):

Antimicrobial Susceptibility ◽

Susceptibility Testing ◽

Surveillance Study ◽

Dilution Method ◽

Agar Dilution Method ◽

In Vitro Susceptibility ◽

Ribotype 027 ◽

Clostridioides Difficile ◽

Vancomycin Mics ◽

Stool Specimens

Abstract Objectives To summarize data generated by the Canadian Clostridioides difficile (CAN-DIFF) surveillance study from 2013 to 2017. Methods Isolates of C. difficile (n = 2158) were cultured from toxin-positive diarrhoeal stool specimens submitted by eight hospital laboratories to a coordinating laboratory. Antimicrobial susceptibility testing was performed according to the CLSI agar dilution method (M11, 2018). Isolate ribotypes were determined using an international, standardized, high-resolution capillary gel-based electrophoresis protocol. Results Of the 2158 isolates of C. difficile, 2133 (98.8%) had vancomycin MICs ≤2 mg/L [i.e. were vancomycin susceptible (EUCAST breakpoint tables, v 9.0, 2019) or WT (CLSI M100, 29th edition, 2019)]. Fidaxomicin MICs were lower than those of all other agents tested (MIC90, 0.5 mg/L); however, one isolate with a fidaxomicin MIC of >8 mg/L was identified. Metronidazole MICs ranged from 0.12 to 4 mg/L; all isolates were metronidazole susceptible by the CLSI breakpoint (≤8 mg/L) compared with 96.8% susceptible by the EUCAST breakpoint (≤2 mg/L). In total, 182 different ribotypes were identified from 2013 to 2017. The most common ribotypes identified were 027 (19.3% of isolates) and 106 (8.2%). Ribotype 027 isolates were frequently moxifloxacin resistant (87.3% of isolates) and MDR (48.6%), associated with vancomycin (10/25, 40.0%) and metronidazole (58/69, 84.1%) resistance and from patients aged ≥80 years. The prevalence of ribotype 027 decreased significantly (P < 0.0001) from 2013 (27.5%) to 2017 (9.0%) and was replaced by increases in ribotype 106 (P = 0.0003) and multiple less common ribotypes. Conclusions Periodic surveillance is required to monitor clinical isolates of C. difficile for changes to in vitro susceptibility testing profiles and ribotype evolution.

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Spatial sampling approach to unravel the impact of soil texture and root genotype on maize root gene expression profiles

10.5194/egusphere-egu2020-9707 ◽

2020 ◽

Author(s):

Minh Ganther ◽

Marie-Lara Bouffaud ◽

Lucie Gebauer ◽

François Buscot ◽

Doris Vetterlein ◽

...

Keyword(s):

Gene Expression ◽

Soil Texture ◽

Expression Profiles ◽

Soil Column ◽

Column Experiment ◽

Spatial Sampling ◽

Marker Genes ◽

Sampling Depth ◽

Plant Growth Hormones ◽

The Impact

The complex interactions between plant roots and soil microbes enable a range of beneficial functions such as nutrient acquisition, defense against pathogens and production of plant growth hormones. The role of soil type and plant genotype in shaping rhizosphere communities has been explored in the past, but often without spatial context. The spatial resolution of rhizosphere processes enables us to observe pattern formation in the rhizosphere and investigate how spatial soil organization is shaped through soil&#8211;plant&#8211;microbiome interactions.We applied spatial sampling in a standardized soil column experiment with two maize genotypes (wildtype vs. roothairless3) and two different soil textures (loam vs. sand) in order to investigate how in particular functions of the maize roots relating to nutrient/water uptake, immunity/defense, stress and exudation are affected. RNA sequencing and differential gene expression analysis were used to dissect impact of soil texture, root genotype and sampling depth. Our results indicate that variance in gene expression is predominantly explained by soil texture as well as sampling depth, whereas genotype appears to play a less pronounced role at the analyzed depths. Gene Ontology enrichment analysis of differentially expressed genes between soil textures revealed several functional categories and pathways relating to phytohormone-mediated signaling, cell growth, secondary metabolism, and water homeostasis. Community analysis of rhizosphere derived ACC deaminase active (acdS gene including) plant beneficial bacteria, which suppress the phytohormone ethylene production, suggests that soil texture and column depth are the major factors that affect acdS community composition.From the comprehensive gene expression analyses we aim to identify maize marker genes from the relevant core functional groups. These marker genes will be potentially useful for future experiments; such as field plot experiments for investigation of later-emerging plant properties.This research was conducted within the research program &#8220;Rhizosphere Spatiotemporal Organisation &#8211; a Key to Rhizosphere Functions&#8221; of the German Science Foundation (TA 290/5-1).

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