Category Archives: C. diff. Research Community

Vanderbilt University Medical Center Scientists Demonstrated C. diff. Exposed To Heme Increases Expression of a Protein System Not Previously Studied

 

Vanderbilt University Medical Center scientists have identified a C. diff protein system that senses and captures heme (part of hemoglobin) to build a protective shield that fends off threats from our immune system and antibiotics. The findings, reported in the journal Cell Host & Microbe, reveal a unique mechanism for C. diff survival in the human gut and suggest novel strategies for weakening its defenses.

In a cruel twist, the bacterium Clostridioides difficile (C. diff) makes us bleed and then uses our blood to defend itself against us.

C. diff the most common cause of health care-associated infections (HAI’s) in the United States causes diarrhea and inflammation of the colon (colitis). Individuals taking antibiotics, which disturb the protective gut microbiota, have increased risk for acquiring a C. diff infection, and 20% of patients suffer recurrent C. diff infections despite treatment.

When C. diff colonizes the gut, it produces toxins that cause tissue damage and inflammation. Blood cells burst, releasing heme, the part of hemoglobin that binds iron and oxygen.

Eric Skaar, Ph.D., MPH, Ernest W. Goodpasture Professor of Pathology, Microbiology and Immunology, and colleagues have studied how bacteria respond to heme, which is both a source of the nutrient iron and a reactive, toxic compound.

“Organisms that experience large amounts of heme have to have ways to deal with heme toxicity,” said Skaar, director of the Vanderbilt Institute for Infection, Immunology and Inflammation (VI4). “We wanted to understand how C. diff deals with heme exposure.”

The investigators demonstrated that C. diff exposed to heme increases expression of a protein system that had not been previously studied. They named the system HsmRA (heme sensing membrane proteins R and A) and showed that HsmR senses heme and deploys HsmA to capture it. They also found that the HsmRA system is genetically conserved in many bacterial species.

The binding of heme in the bacterial membrane by HsmA serves a protective purpose first by simply reducing the concentration of free heme, Skaar explained. The researchers also discovered that HsmA uses heme binding to protect C. diff from oxidative stress, including that produced by neutrophils and macrophages from our immune system to kill bacteria.

“C. diff is using cofactors from our own cells as a shield to protect against our innate immune response,” Skaar said.

Oxidative stress also plays a role in antibiotic action.

“Antibiotics have different molecular targets—they may prevent cell wall synthesis; they may prevent protein translation—but the net result of that stress on the cell is often the massive accumulation of oxidative stress that many believe to be a major contributor to why antibiotics kill bacteria,” Skaar said.

The investigators studied whether the HsmRA system protected C. diff against antibiotics.

“We found a really impressive phenotype with vancomycin and metronidazole, two of the front-line antibiotics used to treat C. diff,” Skaar said. “C. diff that expresses HsmA, when HsmA is bound to heme, is much more resistant to vancomycin and metronidazole.”

They also showed that C. diff strains with inactivated HsmR or HsmA had reduced colonization in a mouse model of relapse C. diff infection.

Skaar said it has not been clear why C. diff produces toxins that cause so much tissue damage.

“It’s interesting to speculate that a benefit of toxin-related damage is that C. diff can capture liberated heme and use it as a shield to protect itself against various insults that cause oxidative stress—that would be immune cells, antibiotics and potentially other bacteria.”

The findings suggest that targeting the HsmA-heme shield might increase the sensitivity of C. diff to antibiotics such as vancomycin and metronidazole. It’s not clear that HsmA, a membrane protein, will be a druggable target, Skaar said.

It might be possible, however, to deprive C. diff of heme building blocks by reducing tissue damage or by administering proteins that bind heme, he said. The researchers will explore whether they can increase the sensitivity of C. diff to antibiotics by co-administering a heme-binding protein during infection in an animal model.

“We’re excited about this as a potentially powerful strategy for treating C. diff,” Skaar said.

In other studies, the researchers will explore if the HsmRA system that is genetically conserved in many different organisms has the same functional role to protect against reactive oxygen species. They are also trying to understand the exact mechanism that HsmA-heme uses to detoxify oxidative stress.

To read article in its entirety, please click on the following link to be redirected:

https://phys.org/news/2020-06-clostridioides-difficile-captures-blood-cell.html

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

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

Facile Therapeutics of Belmont, California, To Develop a New Oral Drug for Recurrent Clostridioides difficile Infections

CARB-X today announced an award of up to $1.26 million to  Facile Therapeutics of Belmont, California, to develop a new oral drug for recurrent Clostridioides difficile infections.Facile Therapeutics of Belmont, California, to develop a new oral drug for recurrent Clostridioides difficile infections.

The money will help fund preclinical development of Ebselen, a small-molecule anti-toxin that inhibits a key biochemical function of C difficile toxins A and B, which attack the lining of the intestine. Previous studies showed Ebselen provided protection against severe intestinal damage in mice after they were exposed to virulent C difficile infections. The drug has also been tested in humans in clinical trials for stroke, and although it was not approved for that indication, it was shown to be safe.

“This is a terrific example of an attempt to repurpose a compound for use in the infectious-disease arena,” CARB-X chief of research and development Erin Duffy, PhD, said in a press release. “If successful and ultimately approved for use in patients, Facile’s project could represent tremendous progress in the prevention of recurrent C. difficile infections, and save many lives.”

C difficile infections are traditionally treated with antibiotics, which can cure the infection but also further disrupt the microbiome and clear a path for C difficile bacteria to spread, leading to recurrent infections. At least 20% of patients who get an initial C difficile infection have a recurrent infection.

Facile could receive an additional $17 million if the project achieves certain milestones.

Since its launch in 2016, CARB-X (the Combating Antibiotic Resistant Bacteria Biopharmaceutical Accelerator) has awarded more than $222 million to companies developing new treatments and diagnostics for drug-resistant pathogens.
May 18 CARB-X press release