Category Archives: C. diff. Research & Development

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

Confirmed Safety and Efficacy MGB-BP-3 and Highly Effective in the Target of Reducing C. difficile Recurrence In Promising Phase IIa Study

MGB’s antibacterial avoids infection recurrence cycle in promising phase IIa study

In the trial, 250-mg MGB-BP-3 enteric-coated tablets given twice a day for 10 days cured acute infections and prevented recurrence at eight weeks. That is in contrast to standard of care vancomycin where there is initial cure but the infection recurs in 20% to 30% of cases.

The company points to the ability to prevent recurrent infections as a key differentiator that gives the compound potential to become a first-line treatment.

MGB-BP-3 achieves that effect by rapidly killing vegetative C. difficile in the 10 to 15 hours before it sporulates and lies dormant in the gut.

“We are absolutely delighted to get that effect and to get a very clean safety profile,” said Chris Wardhaugh, chief business officer. “We’ve confirmed we have a compound that is both safe and highly effective in the target of reducing recurrence. There is a surprisingly high mortality from C. diff infections; it’s because the bugs aren’t killed,” he told BioWorld.

Glasgow, U.K.-based MGB Biopharma said the trial demonstrates the 250-mg dose is able to strike the right balance between maximal killing effects, whilst having a minimal effect on the normal gut flora.

That prevents MGB-BP-3 from sparking the usual vicious circle, in which broad-spectrum antibiotic treatment destroys the microbiota, giving resting C. difficile spores an opening to germinate and form vegetative cells that cause a further bout of diarrhea. Each recurrence is associated with increased risk of further recurrence.

The impact on the microbiome is one of the secondary endpoints, but that analysis is not completed yet. “We are still very much going through the data. We only got the safety and efficacy results last week,” Wardhaugh said. “But we’ve shown in previous studies that [MGB-BP-3] kills bacteria so they don’t form spores. We’ve been able to demonstrate in previous studies it is very rapidly bactericidal and wipes out bacteria in the vegetative state.”

It also has been demonstrated MGB-BP-3 is effective against a hypervirulent strain of
C. difficile that is largely resistant to current therapy. That strain accounts for 20% of cases in Europe and 50% in North America.

The cure rate is impressive, but the numbers are small, with only 10 patients in each of three dose cohorts, of which 250 mg was the middle dose. The company has drawn up plans to move straight to phase III, with an interim analysis on the way.

The next step will be to hold a phase II meeting with the FDA, which Wardhaugh suspects will be delayed because of the COVID-19 pandemic. “So we can’t confirm the size of the phase III yet. We did have a post phase I meeting and looked at a number of options. That gave us comfort a single phase III demonstrating superiority over vancomycin, in particular in hypervirulent strains of C. difficile, would be enough for approval,” Wardhaugh said.

“Phase III is really beyond us, so we are looking for partners or investors to take it on. We have it planned out so we can complete clinical development and have started to talk to companies large and small.”

Eligible for incentives

MGB Biopharma has had a hand to mouth existence since it was spun out of Strathclyde University in Glasgow in 2010, to commercialize research of chemistry professor Colin Suckling into compounds that are selective binders of the minor groove of pathogen DNA.

The main backers are Archangel Investors, a syndicate of business angels, and the government-backed Scottish co-investment fund, but the company has not disclosed the value of any rounds of investment. The phase IIa trial, which took place in North America, was funded through a £2.78 million (US$3.4 million) U.K. government grant.

C. difficile infection is recognized as an urgent threat pathogen by the U.S. CDC and MGB-BP-3 has FDA qualified infectious disease product status. That could lead to fast track submission and five years of extra marketing exclusivity.

The product also will be eligible for the prescribing incentives being put in place under the DISARM (Developing an innovative strategy for antimicrobial resistant micro-organism) Act. MGB Biopharma has also talked up the chances of MGB-BP-3 being recommended as first-line treatment in the Infectious Diseases Society of America guidelines, if it makes it through to approval.

The phase IIa dataset has even more resting on it, in that MGB-BP-3 also is active against seven of 21 priority pathogens listed in the GAIN (Generating antibiotic incentives now) Act. Those include the gram-positive infections vancomycin-resistant Staphylococcus aureus and Enterococcus, and gram-negative PseudomonasKlebsiella and Escherichia coli.

MGB Biopharma has developed other formulations of MGB-BP-3, including liquid-filled capsules and a freeze-dried product which is designed to be reconstituted for intravenous administration.

Final formulation optimization and scale up of the intravenous formulation have been completed, as have preclinical proof-of-concept studies against Staphylococcus aureusStreptococcus pyogenes and pneumoniae. There also is a topical formulation for treating MRSA-infected skin lesions in the works.

MGB Biopharma Ltd. is looking for new investors or a commercialization partner to take its lead program, MGB-BP-3, through to market, after reporting 100% initial and sustained cure of Clostridium difficile infections in a phase IIa dose-ranging study.

 

SOURCE:  https://www.bioworld.com/articles/435241-mgbs-antibacterial-avoids-infection-recurrence-cycle-in-promising-phase-iia-study