Tag Archives: C. difficile research

Study Hypothesized Commensal Clostridia are Important for Providing Colonization Resistance Against C. difficile Due to Their Ability to Produce Secondary Bile Acids

ABSTRACT

Clostridioides difficile is one of the leading causes of antibiotic-associated diarrhea.

Gut microbiota-derived secondary bile acids and commensal Clostridia that carry the bile acid-inducible (bai) operon are associated with protection from C. difficile infection (CDI), although the mechanism is not known.

In this study, we hypothesized that commensal Clostridia are important for providing colonization resistance against C. difficile due to their ability to produce secondary bile acids, as well as potentially competing against C. difficile for similar nutrients.

To test this hypothesis, we examined the abilities of four commensal Clostridia carrying the bai operon (Clostridium scindens VPI 12708, C. scindens ATCC 35704, Clostridium hiranonis, and Clostridium hylemonae) to convert cholate (CA) to deoxycholate (DCA) in vitro, and we determined whether the amount of DCA produced was sufficient to inhibit the growth of a clinically relevant C. difficile strain.

We also investigated the competitive relationships between these commensals and
C. difficile using an in vitro coculture system.

We found that inhibition of C. difficile growth by commensal Clostridia supplemented with CA was strain dependent, correlated with the production of ∼2 mM  DCA, and increased the expression of bai operon genes.

We also found that C. difficile was able to outcompete all four commensal Clostridia in an in vitro coculture system. These studies are instrumental in understanding the relationship between commensal Clostridia and C. difficile in the gut, which is vital for designing targeted bacterial therapeutics. Future studies dissecting the regulation of the bai operon in vitro and in vivo and how this affects CDI will be important.

IMPORTANCE : Commensal Clostridia carrying the bai operon, such as C. scindens, have been associated with protection against CDI; however, the mechanism for this protection is unknown. Herein, we show four commensal Clostridia that carry the bai operon and affect                                C. difficile growth in a strain-dependent manner, with and without the addition of cholate. Inhibition of C. difficile by commensals correlated with the efficient conversion of cholate to deoxycholate, a secondary bile acid that inhibits C. difficile germination, growth, and toxin production.

Competition studies also revealed that C. difficile was able to outcompete the commensals in an in vitro coculture system.

These studies are instrumental in understanding the relationship between commensal Clostridia and C. difficile in the gut, which is vital for designing targeted bacterial therapeutics.

 

 

 

 

 

 

SOURCE: https://jb.asm.org/content/202/11/e00039-20

Study of Hospital-Associated Infection – Clostridioides difficile Identified NpmA In the Genome of a Clinical Isolate

Clostridioides difficile: a potential source of NpmA in the clinical environment

Aminoglycosides are widely used to treat MDR Gram-negative bacterial infections with bactericidal activity mediated by binding the 16S rRNA aminoacyl-tRNA recognition site to prevent protein synthesis. Multiple aminoglycoside resistance mechanisms have been documented including 16S rRNA modification. NpmA, an uncommon 16S rRNA methyltransferase originally identified in a clinical Escherichia coli isolate confers pan-aminoglycoside resistance.

In this study, routine WGS of hospital-acquired Clostridioides (Clostridiumdifficile identified npmA in the genome of a clinical isolate (CD7814).

C. difficile is a Gram-positive, spore-forming enteric pathogen and the cause of most hospital-acquired, antibiotic-associated diarrhoea. The epidemiology of C. difficile has changed in the past several decades with infections increasingly being reported outside of acute care settings.

The discovery of npmA in the genome of a clinical C. difficile isolate has implications for the spread of aminoglycoside resistance.

Clinical C. difficile isolates are routinely sequenced using Illumina NextSeq500 and Nextera libraries as part of an infection prevention initiative at our hospital. Genomes are assembled using SPAdes, annotated with Prokka and characterized by searches of ResFinder and pubMLST databases (https://pubmlst.org/cdifficile/).5–7 This analysis identified NpmA-coding sequences in C. difficile isolate CD7814 belonging to ST11. CD7814 carries two additional aminoglycoside resistance determinants: (i) aph(3′)-III, which encodes an aminoglycoside phosphotransferase; and (ii) ant(6)-Ia, which encodes an aminoglycoside nucleotidyltransferase.

Aminoglycoside susceptibility testing was performed by Etest on CD7814 and two additional ST11 isolates from our hospital that lack npmA (CD7861 and CD7786). Cell suspensions corresponding to 0.5 McFarland were prepared and plated onto Brucella blood agar plates supplemented with vitamin K1 and haemin (Anaerobe Systems, Morgan Hill, CA, USA). Etest strips (bioMérieux, Durham, NC, USA) were applied and the plates were incubated anaerobically at 37°C for 48 h as previously described. Etests were read according to the manufacturer’s instructions. CD7814 demonstrated high-level resistance to gentamicin (>256 mg/L) relative to CD7861 (64 mg/L) and CD7786 (24 mg/L). High-level resistance to tobramycin and amikacin was observed in all ST11 C. difficile isolates tested. Although CLSI breakpoints for C. difficile are not defined for aminoglycosides, these data suggest that NpmA is expressed and associated with increased gentamicin resistance in CD7814.

Genomic analysis of the CD7814 assembly identified a large, presumably chromosomal contig of ∼150 kb containing the npmA gene. The majority of predicted ORFs surrounding npmA in CD7814 are hypothetical proteins whereas others encode proteins involved in recombination suggesting that npmA was acquired via horizontal gene transfer, which is consistent with the mosaic structure of the C. difficile chromosome (Figure 1a). Five additional C. difficile genomes bearing npmA gene sequences that show 99% nucleotide identity to npmA from CD7814 and pARS3 were identified through BLAST and PubMed literature searches (Figure 1a). The DNA sequence flanking npmA in CD7814 shows little or no nucleotide identity to either E. coli pARS3 or the five C. difficile npmA flanking regions. The five C. difficile genomes are of human and animal origin and were collected from three different continents over a period of at least 10 years. Interestingly, these C. difficile isolates belong to three different STs but their genomes share 99% nucleotide identity across ∼3kb of the npmA region (Figure 1a).

None of the five C. difficile genomes share any other sequence similarity to pARS3 outside of npmA. Together, these genomic data suggest that npmA is carried on a conserved element in the five C. difficile genomes and that the mechanism of npmA acquisition in CD7814 is different. In addition, all five C. difficile genomes encode a missense mutation in npmA resulting in a K131N substitution in NpmA relative to CD7814 and pARS3 sequences (Figure 1b). To maintain the established nomenclature for 16S rRNA methyltransferase genes, the CD7814 and, by default, the E. coli pARS3 npmA genes can be re-designated as npmA1 while npmA sequences containing the K131N mutation can be designated npmA2. CD7814 npmA1 has been deposited in GenBank under accession number MH249957.

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(a) Predicted ORFs surrounding the npmA gene (dark grey) in CD7814 (light grey), the E. coli pARS3 plasmid (black) and five C. difficile genomes of human and animal origin (white). Shaded areas highlight regions of sequence homology. Chequered arrows indicate ORFs encoding recombinases potentially associated with horizontal transfer of npmA into CD7814. hu, human; sw, swine; bv, bovine; US, USA, JP, Japan; AU, Australia; CA, Canada. (b) NpmA protein alignment depicting the K131N substitution in CD7814 and E. coli pARS3.

To the best of our knowledge, this is the first description of npmA and high-level aminoglycoside resistance in a hospital-acquired C. difficile isolate. Because of strict anaerobic growth conditions, Etest is the most practical method to measure antibiotic susceptibility in C. difficile. As Etests for aramycin and neomycin are unavailable, a limitation of this study is our inability to demonstrate the specificity of NpmA methyltransferase activity for the N1-A1408 16S rRNA. However, the nucleotide identity between CD7814 npmA and the original E. coli sequence and the high-level aminoglycoside resistance observed support NpmA-mediated resistance in this clinical C. difficile isolate. To the best of our knowledge, no evidence to support high-level gentamicin resistance in the presence of ant(6)-Ia and aph(3′)-III has been reported.

In conclusion, this study demonstrates the presence of npmA and high-level aminoglycoside resistance in a clinical C. difficile isolate. The ability of C. difficile to persist in the environment as a spore former may facilitate acquisition of novel antibiotic resistance determinants. These data suggest that hospital-acquired C. difficile may be a reservoir for uncommon antibiotic resistance determinants such as npmA.

Funding

This work was supported by a research grant from the National Institute of Allergy and Infectious Diseases (R01AI127472 to L. H. H.).

Transparency declarations

None to declare.

References

1. Doi Y, Wachino JI, Arakawa Y.. Aminoglycoside resistance: the emergence of acquired 16S ribosomal RNA methyltransferasesInfect Dis Clin North Am 2016; 30: 523–37. [PMC free article] [PubMed[]
2. Wachino J, Shibayama K, Kurokawa H. et al. Novel plasmid-mediated 16S rRNA m1A1408 methyltransferase, NpmA, found in a clinically isolated Escherichia coli strain resistant to structurally diverse aminoglycosidesAntimicrob Agents Chemother 2007; 51: 4401–9. [PMC free article] [PubMed[]
3. Lessa FC, Winston LG, McDonald LC. et al. Burden of Clostridium difficile infection in the United StatesN Engl J Med 2015; 372: 2369–70. [PubMed[]
4. Baym M, Kryazhimskiy S, Lieberman TD. et al. Inexpensive multiplexed library preparation for megabase-sized genomesPLoS One 2015; 10: e0128036.. [PMC free article] [PubMed[]
5. Bankevich A, Nurk S, Antipov D. et al. SPAdes: a new genome assembly algorithm and its applications to single-cell sequencingJ Comput Biol 2012; 19: 455–77. [PMC free article] [PubMed[]
6. Seemann T. Prokka: rapid prokaryotic genome annotationBioinformatics 2014; 30: 2068–9. [PubMed[]
7. Zankari E, Hasman H, Cosentino S. et al. Identification of acquired antimicrobial resistance genesJ Antimicrob Chemother 2012; 67: 2640–4. [PMC free article] [PubMed[]
8. Citron DM, Ostovari MI, Karlsson A. et al. Evaluation of the E test for susceptibility testing of anaerobic bacteriaJ Clin Microbiol 1991; 29: 2197–203. [PMC free article] [PubMed[]
9. Amy J, Johanesen P, Lyras D.. Extrachromosomal and integrated genetic elements in Clostridium difficilePlasmid 2015; 80: 97–110. [PubMed[]
10. Doi Y, Wachino J, Arakawa Y.. Nomenclature of plasmid-mediated 16S rRNA methylases responsible for panaminoglycoside resistanceAntimicrob Agents Chemother 2008; 52: 2287–8. [PMC free article] [PubMed[]

Research Aimed To Identify Bacterial Signatures Associated with Resistance and Susceptibility to C. difficile Colonization (CDC) and C. difficile Infection (CDI)

Biologists Develop Models to Aid Development of Novel Therapies to Fight Clostridioides difficile (C. diff.) Pathogen

The Clostridium difficile pathogen takes its name from the French word for “difficult.” A bacterium that is known to cause symptoms ranging from diarrhea to life-threatening colon damage,

 

 

 

C. difficile is part of a growing epidemic of concern for the elderly and patients on antibiotics.

Outbreaks of C. difficile-infected cases have progressively increased in Western countries, with 29,000 reported deaths per year in the United States alone.

Now, biologists at the University of California San Diego are drawing parallels from newly developed models of the common fruit fly to help lay the foundation for novel therapies to fight the pathogen’s spread. Their report is published in the journal iScience.

C. difficile infections pose a serious risk to hospitalized patients,” said Ethan Bier, a distinguished professor in the Division of Biological Sciences and science director of the UC San Diego unit of the Tata Institute for Genetics and Society (TIGS). “This research opens a new avenue for understanding how this pathogen gains an advantage over other beneficial bacteria in the human microbiome through its production of toxic factors. Such knowledge could aid in devising strategies to contain this pathogen and reduce the great suffering it causes.”

As with most bacterial pathogens, C. difficile secretes toxins that enter host cells, disrupt key signaling pathways and weaken the host’s normal defense mechanisms. The most potent strains of C. difficile unleash a two-component toxin that triggers a string of complex cellular responses, culminating in the formation of long membrane protrusions that allow the bacteria to attach more effectively to host cells.

UC San Diego scientists in Bier’s lab-created strains of fruit flies that are capable of expressing the active component of this toxin, known as “CDTa.” The strains allowed them to study the elaborate mechanisms underlying CDTa toxicity in a live model system focused on the gut, which is key since the digestive system of these small flies is surprisingly similar to that of humans.

“The fly gut provides a rapid and surprisingly accurate model for the human intestine, which is the site of infection by C. difficile,” said Bier. “The vast array of sophisticated genetic tools in flies can identify new mechanisms for how toxic factors produced by bacteria disrupt cellular processes and molecular pathways. Such discoveries, once validated in a mammalian system or human cells, can lead to novel treatments for preventing or reducing the severity of C. difficile infections.”

The fruit fly model gave the researchers a clear path to examine genetic interactions disrupted at the hands of CDTa. They ultimately found that the toxin induces a collapse of networks that are essential for nutrient absorption. As a result, the model flies’ body weight, fecal output and overall lifespan were severely reduced, mimicking symptoms in human C. difficile-infected patients.

In addition to Bier, study coauthors include first-author Ruth Schwartz, Annabel Guichard, Nathalie Franc, and Sitara Roy.

The National Institutes of Health (R01 AI110713) funded the research.


Story Source:

Materials provided by the University of California – San Diego. Original written by Mario Aguilera. Note: Content may be edited for style and length.


Journal Reference:

  1. Ruth Schwartz, Annabel Guichard, Nathalie C. Franc, Sitara Roy, Ethan Bier. A Drosophila Model for Clostridium difficile Toxin CDT Reveals Interactions with Multiple Effector Pathways. iScience, 2020; 100865 DOI: 10.1016/j.isci.2020.100865

Transgenic fruit flies help scientists trace the cascade of symptoms caused by toxic infection

Date: February 7, 2020

Source: University of California – San Diego
Summary: Clostridium difficile, a bacterium is known to cause symptoms from diarrhea to life-threatening colon damage, is part of a growing epidemic for the elderly and hospitalized patients. Biologists have now developed models of the common fruit fly to help develop novel therapies to fight the pathogen

DEINOVE Announced Enrollment of First Patient in Phase II Trial Testing DNV3837 in Clostridioides difficile infections

On January 27, 2020, DEINOVE announced the inclusion of the first patient in the Phase II trial testing DNV3837.

 

  • The Phase II clinical trial aims to evaluate the efficacy, safety, and pharmacokinetics of DNV3837 in patients with Clostridioides difficile gastrointestinal infection (CDI).
  • The trial will be conducted mainly in 15 centers in the United States, in two successive stages:
    • a cohort of 10 patients with moderate to severe CDI treated with DNV3837,
    • a randomized cohort study testing DNV3837 against the standard of care in 30 patients with severe CDI.
  • The final results of this trial are expected by the end of 2020.
  • DEINOVE is the only French player to conduct a clinical trial with an antibiotic.
  • On 17 January, the WHO warned about the extreme lack of new antibiotics and the threat posed by antibiotic resistance.

DEINOVE (Euronext Growth Paris: ALDEI), a French biotech company that uses a disruptive approach to develop innovative antibiotics and bio-based active ingredients for cosmetics, announced the inclusion of the first patient in the Phase II trial testing DNV3837.

DNV3837 targets the treatment of Clostridioides difficile infections (CDI), a disease classified as a priority by the WHO and one of the global leading causes of healthcare-related infections*.

DNV3837 is an intravenous antibiotic that, when converted to its active form DNV3681, crosses the gastrointestinal barrier and accumulates in the intestinal lumen, allowing it to precisely target the infection site. DNV3837 has demonstrated a promising efficacy profile and acceptable tolerance in Phase I trials (on healthy volunteers). It has also demonstrated its ability to eliminate Clostridioides bacteria without affecting the gut microbiota. It has been granted Fast Track status and QIDP designation**.

The Phase II trial aims to evaluate the efficacy of DNV3837 in pathological conditions (through monitoring of symptoms, stool analysis, etc.), as well as to consolidate the safety and pharmacokinetic data of the antibiotic candidate.

This trial is concentrated in the United States. It will take place in two stages:

  • In the first phase, involving 5 centers, a cohort of 10 patients with moderate to severe CDI will be treated with DNV3837. At the end of this phase, the DSMB*** will review the interim results.
  • The second phase will involve 30 patients with severe CDI and will be carried out in 15 investigation centers. This will be an open-label randomized trial testing DNV3837 (in 2/3 of patients) against an approved standard of care**** (1/3 of patients) for comparison purposes.

The results of this clinical trial should be available by the end of 2020.

 “The start of this Phase II clinical trial is a significant step forward for DEINOVE and a great hope for patients. We are very proud to provide a potential solution to this unmet medical need and, to this end, work with the best American specialists in this area. The investigation centers are very committed to conducting this trial which, in the event of positive results, will be an important milestone towards the registration of DNV3837,” said Dr. Georges Gaudriault, Scientific Director of DEINOVE.

This announcement echoes warnings issued by the WHO about the lack of antibiotics renewal.

Dr. Tedros Adhanom Ghebreyesus, Director-General of WHO, declared last January 17 « Never has the threat of antimicrobial resistance been more immediate and the need for solutions more urgent ».

https://www.who.int/news-room/detail/17-01-2020-lack-of-new-antibiotics-threatens-global-efforts-to-contain-drug-resistant-infections

 

* Source: CDC (US Centers for Disease Control and Prevention)

** ‘Fast Track’ status facilitates the development of the molecule through a faster and more flexible regulatory review of the application. The QIDP designation gives the drug exclusive access to the market for an additional five-year period. These designations are granted by the FDA to drugs under development that meet critical and unmet therapeutic needs.

*** DSMB – Data Safety Monitoring Board: a group of independent experts tasked to review the data generated during the trial and make recommendations on patient safety as well as trial relevance and validity.

**** Standard treatments approved in the United States for the treatment of CDIs include vancomycin, fidaxomicin and metronidazole (all three antibiotics). The choice will be at the discretion of the clinicians.