Harsh Mathur

The potential for emerging therapeutic options for Clostridium difficile infection

Clostridium difficile is mainly a nosocomial pathogen and is a significant cause of antibiotic-associated diarrhea. It is also implicated in the majority of cases of pseudomembranous colitis. Recently, advancements in next generation sequencing technology (NGS) have highlighted the extent of damage to the gut microbiota caused by broad-spectrum antibiotics, often resulting in C. difficile infection (CDI). Currently the treatment of choice for CDI involves the use of metronidazole and vancomycin. However, recurrence and relapse of CDI, even after rounds of metronidazole/vancomycin administration is a problem that must be addressed. The efficacy of alternative antibiotics such as fidaxomicin, rifaximin, nitazoxanide, ramoplanin and tigecycline, as well as faecal microbiota transplantation has been assessed and some have yielded positive outcomes against C. difficile. Some bacteriocins have also shown promising effects against C. difficile in recent years. In light of this, the potential for emerging treatment options and efficacy of anti-C. difficile vaccines are discussed in this review.

Analysis of Anti-Clostridium difficile Activity of Thuricin CD, Vancomycin, Metronidazole, Ramoplanin, and Actagardine, both Singly and in Paired Combinations

ABSTRACT Due to the ongoing problem of recurrence of Clostridium difficile-associated diarrhea following antibiotic treatment, there is an urgent need for alternative treatment options. We assessed the MICs of five antimicrobials singly and in combinations against a range of C. difficile clinical isolates. Ramoplanin-actagardine combinations were particularly effective, with partial synergistic/additive effects observed against 61.5% of C. difficile strains tested.

Bacteriocin-Antimicrobial Synergy: A Medical and Food Perspective

The continuing emergence of multi-drug resistant pathogens has sparked an interest in seeking alternative therapeutic options. Antimicrobial combinatorial therapy is one such avenue. A number of studies have been conducted, involving combinations of bacteriocins with other antimicrobials, to circumvent the development of antimicrobial resistance and/or increase antimicrobial potency. Such bacteriocin-antimicrobial combinations could have tremendous value, in terms of reducing the likelihood of resistance development due to the involvement of two distinct mechanisms of antimicrobial action. Furthermore, antimicrobial synergistic interactions may also have potential financial implications in terms of decreasing the costs of treatment by reducing the concentration of an expensive antimicrobial and utilizing it in combination with an inexpensive one. In addition, combinatorial therapies with bacteriocins can broaden antimicrobial spectra and/or result in a reduction in the concentration of an antibiotic required for effective treatments to the extent that potentially toxic or adverse side effects can be reduced or eliminated. Here, we review studies in which bacteriocins were found to be effective in combination with other antimicrobials, with a view to targeting clinical and/or food-borne pathogens. Furthermore, we discuss some of the bottlenecks which are currently hindering the development of bacteriocins as viable therapeutic options, as well as addressing the need to exercise caution when attempting to predict clinical outcomes of bacteriocin-antimicrobial combinations.

The efficacy of thuricin CD, tigecycline, vancomycin, teicoplanin, rifampicin and nitazoxanide, independently and in paired combinations against Clostridium difficile biofilms and planktonic cells

BackgroundThuricin CD is a two-component antimicrobial, belonging to the recently designated sactibiotic subclass of bacteriocins. The aim of this study was to investigate the effects of thuricin CD, as well as the antibiotics, tigecycline, vancomycin, teicoplanin, rifampicin and nitazoxanide when used independently and when combined at low concentrations on the viability of Clostridium difficile 20291 R027, TL178 R002, Liv022 R106, DPC6350 and VPI10463 biofilms and planktonic cells.ResultsOn the basis of XTT (2,3-bis[2-methyloxy-4-nitro-5-sulphophenyl]-2H-tetrazolium-5-carboxanilide)-menadione biofilm viability assays, we found that thuricin CD was effective against biofilms of R027, Liv022 R106 and DPC6350 when used independently while nitazoxanide and rifampicin were also potent against biofilms of R027 and DPC6350, when applied on their own. Tigecycline was found to be effective against R027 and DPC6350 biofilms, whereas teicoplanin and vancomycin when used independently were only effective against DPC6350 biofilms. The efficacies of the antibiotics rifampicin, tigecycline, vancomycin and teicoplanin against C. difficile 20291 R027 biofilms were significantly potentiated when combined with thuricin CD, indicating effective antimicrobial combinations with this sactibiotic against R027 biofilms. However, the potency of nitazoxanide against R027 biofilms was significantly diminished when combined with thuricin CD, indicating an ineffective combination with this sactibiotic against R027 biofilms. Paired combinations of thuricin CD along with these five antibiotics were effective at diminishing the viability of DPC6350 biofilms. However, such combinations were largely ineffective against biofilms of TL178 R002, Liv022 R106 and VPI10463.ConclusionsTo the best of our knowledge, this is the first study to highlight the activity of a sactibiotic bacteriocin against biofilms and the first to reveal the potency of the antibiotics tigecycline, teicoplanin and nitazoxanide against C. difficile biofilms. On the basis of this study, it is apparent that different strains of C. difficile possess varying abilities to form biofilms and that the sensitivities of these biofilms to different antimicrobials and antimicrobial combinations are strain-dependent. Since the formation of relatively strong biofilms by certain C. difficile strains may contribute to increased cases of antibiotic resistance and recurrence and relapse of C. difficile infection, the findings presented in this study could provide alternative strategies to target this pathogen.

Insights into the Mode of Action of the Sactibiotic Thuricin CD

Thuricin CD is a two-component bacteriocin, consisting of the peptides Trnα and Trnβ, and belongs to the newly designated sactibiotic subclass of bacteriocins. While it is clear from studies conducted thus far that it is a narrow-spectrum bacteriocin, requiring the synergistic activity of the two peptides, the precise mechanism of action of thuricin CD has not been elucidated. This study used a combination of flow cytometry and traditional culture-dependent assays to ascertain the effects of the thuricin CD peptides on the morphology, physiology and viability of sensitive Bacillus firmus DPC6349 cells. We show that both Trnα and Trnβ are membrane-acting and cause a collapse of the membrane potential, which could not be reversed even under membrane-repolarizing conditions. Furthermore, the depolarizing action of thuricin CD is accompanied by reductions in cell size and granularity, producing a pattern of physiological alterations in DPC6349 cells similar to those triggered by the pore-forming single-component bacteriocin Nisin A, and two-component lacticin 3147. Taken together, these results lead us to postulate that the lytic activity of thuricin CD involves the insertion of thuricin CD peptides into the membrane of target cells leading to permeabilization due to pore formation and consequent flux of ions across the membrane, resulting in membrane depolarization and eventual cell death.

Heterologous expression of thuricin CD immunity genes in Listeria monocytogenes.

ABSTRACT Bacteriocins are ribosomally synthesized peptides that can have a narrow or broad spectrum of antimicrobial activity. Bacteriocin producers typically possess dedicated immunity systems that often consist of an ATP-binding cassette (ABC) transporter system and/or a dedicated immunity protein. Here we investigated the genes responsible for immunity to thuricin CD, a narrow-spectrum two-peptide sactibiotic produced by Bacillus thuringiensis DPC6431. Heterologous expression of putative thuricin CD immunity determinants allowed us to identify and investigate the relative importance of the individual genes and gene products that contribute to thuricin CD immunity. We established that TrnF and TrnG are the individual components of an ABC transporter system that provides immunity to thuricin CD. We also identified a hitherto overlooked open reading frame located upstream of trnF predicted to encode a 79-amino-acid transmembrane protein. We designated this newly discovered gene trnI and established that TrnI alone can provide protection against thuricin CD.

Flow Cytometry as a Tool to Study the Effects of Bacteriocins on Prokaryoticand Eukaryotic Cells

Researchers use a combination of techniques to study and contrast the impact of antimicrobials, such as bacteriocins, on sensitive and resistant variants. Flow cytometry is one such technique, which allows researchers to evaluate the activity of antimicrobials at a single-cell level in real-time. The generation of an increasing number of probes/dyes that can be used in flow cytometry studies has vastly expanded the potential applications of this technique. Furthermore, flow cytometry has the potential to replace, or at the very least be used as an adjunct to traditional growth-based techniques, including viable plate counts, growth curves, microscopic analysis and cell culture, many of which have limitations when used on their own. Here we review studies conducted using flow cytometry as a technique to assess the impact of antimicrobials from the bacteriocin family on individual cells, either prokaryotic or eukaryotic.

Fighting biofilms with lantibiotics and other groups of bacteriocins

Biofilms are sessile communities of bacteria typically embedded in an extracellular polymeric matrix. Bacterial cells embedded in biofilms are inherently recalcitrant to antimicrobials, compared to cells existing in a planktonic state, and are notoriously difficult to eradicate once formed. Avenues to tackle biofilms thus far have largely focussed on attempting to disrupt the initial stages of biofilm formation, including adhesion and maturation of the biofilm. Such an approach is advantageous as the concentrations required to inhibit formation of biofilms are generally much lower than removing a fully established biofilm. The crisis of antibiotic resistance in clinical settings worldwide has been further exacerbated by the ability of certain pathogenic bacteria to form biofilms. Perhaps the most notorious biofilm formers described from a clinical viewpoint have been methicillin-resistant Staphylococcus aureus (MRSA), Staphylococcus epidermidis, Pseudomonas aeruginosa, Gardnerella vaginalis and Streptococcus mutans, the latter of which is found in oral biofilms. Due to the dearth of novel antibiotics in recent decades, compounded by the increasing rate of emergence of resistance amongst pathogens with a propensity for biofilm formation, solutions are urgently required to mitigate these crises. Bacteriocins are a class of antimicrobial peptides, which are ribosomally synthesised and often are more potent than their antibiotic counterparts. Here, we review a selection of studies conducted with bacteriocins with the ultimate objective of inhibiting biofilms. Overall, a deeper understanding of the precise means by which a biofilm forms on a substrate as well as insights into the mechanisms by which bacteriocins inhibit biofilms is warranted.

Delivery of a Hydrophobic Drug into the Lower Gastrointestinal System via an Endogenous Enzyme-Mediated Carrier Mechanism: an in vitro study.

&NA; Clofazimine (CFZ) is a hydrophobic antibiotic agent which exhibits poor solubility. This poor solubility was overcome herein by the formulation of CFZ with the digestive enzyme pepsin. It is shown that pepsin can actively bind 11 CFZ molecules in the proteins native gastric environment, forming a CFZ‐pepsin complex. A dynamic dissolution system, representing both the gastric and intestinal system, was used to analyze this CFZ‐pepsin complex, revealing that only CFZ which binds to pepsin in the gastric environment remains in solution in the intestinal environment. The CFZ‐pepsin complex displays adequate solution stability for the delivery of CFZ into the lower intestinal system. In vitro bioactivity assays against Clostridium difficile demonstrated the effectiveness of this CFZ‐pepsin complex for the treatment of infectious diseases in the lower intestinal system. Graphical abstract Figure. No caption available.