Microorganisms as Therapy

MICROORGANISMS AS THERAPY      

A)                   Bacteriophages

In the early 1900s, before the discovery of penicillin, Felix D’Herelles observed that patients with high titres of bacteriophages in their faeces recovered from dysentery and typhoid fever more rapidly. This paved the way for the commercialization of bacteriophage preparations for a variety of bacterial infections by, for example, the Société Française de Teintures Inoffensives pour Cheveux (The French Society for Safe Hair Colouring) or L’Oréal as it is known today. Following the advent of modern antibiotic therapy in the 1930s the science of phage therapy was all but bankrupted, but the emergence of antibiotic resistance has led to a resurgent interest in research and development of phage therapeutics and at least eight commercial enterprises are involved in the development of clinically relevant phage medicines. Most phages have a specific affinity for only a small group of bacteria, predicated by the interaction of phage components with bacterial surface receptors. Upon interaction, the viral DNA is translocated into the bacterial cell for transcription where lytic or lysogenic replication may occur. Lytic phages replicate and assemble and then ‘burst’ from the host cell, resulting in cell death. In the lysogenic lifecycle, bacteriophage DNA becomes integrated in the host bacterium’s genome. This newly generated material termed a prophage is replicated during cell division. The lysogenic lifecycle is shifted to a lytic one when exposed to some external trigger, such as UV radiation.

Lytic phage are in many ways an ideal antibacterial agent. They are target specific and the existence of more than 1 × 108 species of phage suggests there may be a phage therapeutic for every bacterial species; they kill bacteria rapidly and amplify at the site of infection, and are relatively inexpensive to produce. In addition, the FDA recognizes that humans ingest vast quantities of phages on a daily basis and tacitly accept that they are safe for oral administration. It is perhaps topical administration that has seen the most interest, however, with the application of cocktails of phage to chronic wounds either as simple suspensions or incorporated into some form of dressing system such as a biodegradable polymer infused with phage and antibiotics. The systemic administration of phage therapeutics is complicated by an inadequate knowledge of the pharmacokinetic and pharmacodynamic properties of most phage species, with many studies indicating that the timing of administration is critical for infection control. Recent reports suggest, however, that phages, which are naturally immunostimulatory, may be useful as vaccine delivery vehicles either by vaccinating with phages displaying the antigen or by utilizing phages to deliver a DNA expression cassette integrated into the phage genome.

B)                        Probiotics

The bacterial microflora that colonizes the gastrointestinal (GI) tract is an essential feature of normal human physiology and represents a symbiotic relationship where the bacteria both protect the host against pathogenic microbes and aid in the digestion of food, contributing to the production of essential host nutrients. Under certain conditions (e.g. illness, infection, antibiotic therapy), the bacterial population in the GI tract may be diminished, contributing to disease states. Probiotics are live cultures of ‘good’ bacteria that are purported to survive transit through the stomach, subsequently colonizing the intestinal mucosa and replacing the diminished natural microflora or displacing pathogenic microorganisms. Bifidobacteria and Lactobacillus spp. are the most commonly encountered probiotic bacteria, primarily because they are reported to survive the harsh environment of the upper GI tract more readily than other species. Of note, Bifidobacteria spp. are amongst the first colonizers of the neonate intestine, as a consequence of both Bifidobacteria and prebiotic content in breast milk, and contribute to defence against pathogenic invaders and maturation of the immune system. Probiotics are generally formulated as capsules or as food supplements particularly as dairy products such as yoghurts. Often probiotic formulations are combined with prebiotics— indigestible oligosaccharides that are fermented by anaerobic bacteria in the gut, yielding metabolic substrates that promote probiotic growth.

Probiotics have been investigated for efficacy in a range of conditions that may be associated with diminished bacterial microflora. Several probiotic species including Lactobacillus spp. have shown utility in both the prevention and treatment of nosocomial, antibiotic and traveller’s diarrhoea. Long-term treatment with E.coli Nissle 1917 (>12 months) is reported to be at least equivalent to mesalazine therapy in preventing relapse in ulcerative colitis. In irritable bowel syndrome (IBS) the results are equivocal but hopeful, with some studies reporting a reduction in symptoms such as abdominal discomfort.

A cautionary note, however: probiotics are increasingly marketed as a ‘lifestyle’ nutrient to healthy individuals to promote general GI and immune health, despite limited evidence of any significant effect. Nevertheless, research into the benefits of probiotics in both healthy and diseased individuals is ongoing, using recognized and novel probiotic species, and may in the future reap significant reward.

C)              Toxins 

i)  Botilinum Toxin

In the late 19th century, a Belgian professor of microbiology, van Ermengem, conducted a serious of experiments to identify the cause of a fatal outbreak of food poisoning, the clinical symptoms of which had been described over a century before by the German physician Justinus Kerner. van Ermengem’s endeavours resulted in the identification of botulinum toxin, a potent exotoxin produced by the Gram-positive anaerobic bacterium Clostridium botulinum. It is this toxin that is responsible for what is now widely recognized as botulism food poisoning. The symptoms of botulism, which remains a common cause of fatal food poisoning, include GI disturbances, dysphagia, facial paralysis and, depending on the ingested dose, more widespread muscle weakness resulting in possible respiratory paralysis and subsequent death.

In the mid-20th century the work of Burgen and colleagues established that the basic mechanism of action for botulinum toxin is neuromuscular blockade. Now, some 60 years later, we understand that the toxic component of the protein complex is a 150 kDa single-chain polypeptide, consisting of a 100 kDa heavy chain linked to a 50 kDa light chain by a disulphide bridge, which temporarily inhibits acetylcholine release from the presynaptic membrane of cholinergic nerve terminals. Seven different serotypes of the botulinum neurotoxin (A–G), have been identified. The 150 kDa toxic component of these macromolecular protein complexes is relatively homologous, conferring only subtle differences between the mechanisms of action of the serotypes. However the non-toxic proteins within the bacterium-derived botulinum toxin protein complex also differ, depending on the strain of Cl. botulinum. Botulinum toxin serotypes therefore possess molecular weights between 300 and 900 kDa.

In the late 1970s and 1980s nanogram quantities of the botulinum toxin were being locally injected, by clinical researchers, into the muscles of human volunteers to induce local paralysis in an attempt to treat various movement disorders. In 1989, after more than a decade of clinical development, the FDA approved the first botulinum toxin therapy. This commercial product contained the botulinum toxin A serotype and was used for the treatment of strabismus, blepharospasm and hemifacial spasm. In 1991 Allergan obtained both the license and the manufacturing facilities to become the sole supplier of botulinum toxin A for clinical therapy and they branded their product Botox.

There are currently five licensed pharmaceutical forms of the botulinum toxin A: Botox, Vistabel, Dysport, Xeomin and Azzalure. These pharmaceutical preparations contain different forms of the toxin, are formulated differently and/or are licensed for different therapeutic indications. For example, Xeomin contains only the 150 kDa light chain region of the toxin and contains human albumin and sucrose as excipients, whereas Botox contains the 900 kDa macromolecular protein complex and contains human albumin and sodium chloride as excipients. Doses of commercial botulinum toxin A preparations are therefore not interchangeable and specific brands should be prescribed for specific clinical indications. Doses are significantly less than the lethal dose for a human, but systemic side effects of the toxin have been observed, albeit rarely. Clinical administration of the toxin relies on multiple localized injections, often in the secondary care setting, directly into the target tissue. However, the therapeutic effect of botulinum toxin is transient, typically 6–12 months, and patients therefore return for treatment at regular intervals.

In the past two decades the use of botulinum toxin A in clinical practice has increased almost exponentially and it is now used to treat a diversity of medical conditions. Specific licensed indications include blepharospasm, cervical dystonia, hemifacial spasm, glabellar lines and hyperhidrosis. However, it has been used more widely, often unlicensed, for a range of clinical indications related to movement disorders, spasticity, ophthalmic disorders, GI disorders, genitourinary disorders, surgical interventions, tendon release in the Ponseti treatment of talipes, and more recently pain. The toxin is even better known for its widespread use in the cosmetic industry. A significant population of patients and cosmetic clients have therefore now been treated with the neurotoxin, and in general it appears to be a safe and effective addition to the therapeutic armoury. Botulinum toxin type B is also available commercially, as Neurobloc, but at present this serotype is used less widely in therapy. The clinical use of botulinum toxin is primarily restricted to conditions that are associated with superficial/ accessible tissues, to minimize the risk of systemic uptake, but there is no doubt that the toxin is emerging as a useful therapeutic entity and has already revolutionized the treatment of some conditions including hyperhidrosis.

ii)                Cholera toxin

Vibrio cholerae is a bacterial pathogen that colonizes the small intestine leading to cholera, an infection characterized by life-threatening acute diarrhoea. Cholera is endemic in developing countries and in areas where hygiene and sanitary conditions are poor, and even with supportive therapy that includes rehydration and restoration of electrolytes, morbidity and mortality rates remain high. An oligomeric protein (87 kDa) secreted by V. cholerae was confirmed as the causative agent of cholera in 1963 by Finkelstein and colleagues and has been termed cholera toxin (CT). CT is a member of the superfamily of AB toxins comprised of a catalytic heterodimeric A subunit (A1 and A2 chains) and a glycolipid receptor binding homopentameric B-subunit connected by a disulphide bond. When V. cholerae colonizes the small intestine it secretes CT which subsequently interacts, via the B-subunit, with an enterocyte membrane receptor GM1 (monosialotetrahexosylganglioside) localized in lipid rafts. The CT is then internalized into early endosomes and trafficked to the trans-Golgi network, eventually ending up in the endoplasmic reticulum where protein disulphide isomerase dissociates and unfolds the A1 chain from CT. The A1 chain is then translocated by the Sec61 channel into the cytosol where it interacts with proteins that regulate adenylate cyclase (AC) leading to the constitutive activation of AC. This is accompanied by an increase in intracellular cAMP concentration resulting in phosphorylation of the cystic fibrosis transmembrane conductance regulator (CFTR). The net consequence is extracellular secretion of chloride ions into the small intestine, producing an osmotic gradient that draws water into the lumen, resulting in diarrhoea.

CT, despite its pathogenicity, has significant immunological properties and has been proposed as a mucosal adjuvant for subunit vaccines where the toxin is coadministered or complexed with the antigen. Mucosally administered vaccines, i.e. oral, nasal, rectal or vaginal, have a number of advantages over the more traditional intravenous vaccines, not least the ability to stimulate mucosal and systemic protection and may perhaps enhance vaccine uptake rates given no needles are involved. The mechanism of adjuvanticity of cholera toxin is controversial and remains to be fully elucidated but evidence suggests the A-subunit, when administered orally, enhances antigen presentation following complex interactions with mucosal cells and cells of the immune system. The role of the B-subunit in adjuvanticity is interesting; CT-B does not appear to survive transit through the GI tract but there is growing evidence of autoimmune stimulation following nasal or intravenous administration and therefore the B-subunit may offer utility in the treatment of autoimmune diseases. A number of preclinical in vivo studies have been undertaken to ascertain the effectiveness of CT as a mucosal adjuvant for antigens derived from (for example) Helicobacter pylori, influenza, tetanus, HIV and Streptococcus pneumoniae, and for the treatment of diabetes mellitus. To date, however, it appears that oral doses of CT that elicit adjuvanticity are similar to those that induce diarrhoea amongst other adverse reactions, although efforts to ‘detoxify’ CT through genetic engineering are under way.