Alternative therapies for Helicobacter pylori: probiotics and phytomedicine

Abstract

Helicobacter pylori is a common human pathogen infecting about 30% of children and 60% of adults worldwide and is responsible for diseases such as gastritis, peptic ulcer and gastric cancer. Treatment against H. pylori is based on the use of antibiotics, but therapy failure can be higher than 20% and is essentially due to an increase in the prevalence of antibiotic-resistant bacteria, which has led to the search for alternative therapies. In this review, we discuss alternative therapies for H. pylori, mainly phytotherapy and probiotics. Probiotics are live organisms or produced substances that are orally administrated, usually in addition to conventional antibiotic therapy. They may modulate the human microbiota and promote health, prevent antibiotic side effects, stimulate the immune response and directly compete with pathogenic bacteria. Phytomedicine consists of the use of plant extracts as medicines or health-promoting agents, but in most cases the molecular mode of action of the active ingredients of these herbal extracts is unknown. Possible mechanisms include inhibition of H. pylori urease enzyme, disruption of bacterial cell membrane, and modulation of the host immune system. Other alternative therapies are also reviewed.

Helicobacter pylori, alternative therapy, probiotics, phytomedicine, phage therapy, bacteriocin

Introduction

Human stomach colonization with Helicobacter pylori is asymptomatic in about 70% of the population. Although approximately half of the human population is colonized by H. pylori, only about 10–20% are likely to develop peptic ulcer, and only 1–2% are at risk for either gastric cancer or mucosa-associated lymphoid tissue lymphoma (International Agency for Research on Cancer, 1994, Dunn et al., 1997; Kusters et al., 2006; Marshall, 2006). Triple therapy, which consists of two antibiotics and a proton pump inhibitor (PPI), or ranitidine bismuth, administered for 7 days (Megraud, 2004; Kusters et al., 2006), is commonly used to eradicate H. pylori (Megraud & Lehours, 2007). The most commonly used antibiotics are tetracycline, amoxicillin, imidazole (metronidazole or tinidazol) and macrolids (clarithromycin or azithromycin). A recent phase 3 trial in Europe showed that quadruple therapy — two antibiotics, a PPI and bismuth — should be considered for first-line treatment in view of the rising prevalence of clarithromycin-resistant H. pylori (Malfertheiner et al., 2011). Antibiotic therapy fails in about 20% of patients (Parente et al., 2003; Kusters et al., 2006; Vale & Vitor, 2010), mainly due to antibiotic-resistant bacteria and patient non-compliance (Megraud, 2004), but also because the bacteria may be present in a protective environment such as the stomach mucus or even intracellularly (Dubois & Boren, 2007). The problem of antibiotic resistance is common to all bacteria. Regardless of the current need to develop new effective antibiotics, some studies have started looking into new treatment approaches, sometimes in addition to antibiotics. In this brief review, we summarize the experience with non-antibiotic therapies for H. pylori, considering mainly probiotics and phytotherapy. Other alternatives therapies, such as the potential use of bacteriophages against H. pylori and the use of photodynamic therapy, are also discussed.

The human gut microbiota

The human organism is colonized by a large number of microorganisms that play an important role in several biochemical reactions. The microorganisms that colonize the human gastrointestinal tract are collectively described as microbiota, and a typical human may carry over 40 000 bacterial species in the intestinal microbiome (McFarland, 2010). The microbiota of the human stomach and its influence on H. pylori colonization has been characterized (Bik et al., 2006). Most phylotypes belong to the phyla Proteobacteria, Firmicutes, Actinobacteria, Bacteroidetes and Fusobacteria. Lactobacillus species are acid-resistant and commensal and their concentrations in the normal human stomach vary between 0 and 10³ mL⁻¹. They are able to survive in the stomach for periods of up to 2 h and some strains adhere to gastric epithelial cells in vitro, probably through lipoteichoic acid (Gotteland et al., 2006).

Considering the close relationship between prokaryotic and eukaryotic cells, the human body may be considered a multiorganism or complex ecosystem in which any change may affect human physiology and health (Dethlefsen et al., 2006; Salminen et al., 2006; Palmer et al., 2007). Moreover, changes in cultural habits leading to microbiota variations may affect human health. A good example is the introduction of agriculture 10 000 years ago, followed by demographic expansion and a switch from a nomad to a sedentary society. This alteration of cultural habits shortened the proximity between human beings and animals (both domestic and opportunistic, for example rats) and brought new infectious diseases such as plague, tuberculosis and other zoonoses. During the 19th and 20th centuries, the improvement of sanitary conditions, the implementation of vaccine programs, and the introduction of antibiotics led us to believe that infectious diseases could be eradicated. The increase of antibiotic-resistant bacteria has proved otherwise.

The human microbiome co-evolved with mankind, is part of human physiology and contributes to homeostasis. Microbiota–host interactions through metabolic exchange and co-metabolism of substrates, or metabolome–metabolome interactions, are poorly understood, but may be implicated in the aetiology of many human diseases (O'Hara & Shanahan, 2007). Diseases whose pathogenicity is caused by a biochemical, physiological or immunological imbalance may be explained by autochthonous or indigenous microorganisms that colonize the human body. Also, perturbations within this dynamic ecosystem may be associated with the host's health (Blaser, 2005). The administration of antibiotics to treat H. pylori infection also perturb the microbiota, with potential effects on human health (Jakobsson et al., 2010). The regeneration of the microbiota after infection or antibiotic therapy appears to be important for health recovery, which may be facilitated by the administration of live organisms, i.e. probiotics (Wolvers et al., 2010).

Antibiotic therapy failure and alternative therapies

Routinely applied triple therapy consists of two antibiotics and a PPI or ranitidine bismuth (Megraud, 2004). In the last two decades, several multiresistant bacteria were identified (Krause, 2002). Therapy failure is observed in 10–23% of patients (Parente et al., 2003; Kusters et al., 2006) and is mainly due to loss of antibiotic efficacy. Helicobacter pylori acquires resistance mainly via point mutations (Megraud, 2004) and resistance to clarithromycin and metronidazole (Krause, 2002), in particular, has high clinical significance. As clarithromycin is the first-choice antibacterial, resistance to this antibiotic, as well as to metronidazole, has led to the utilization of other drugs, such as amoxicillin-rifabutin, amoxicillin-levofloxacin or amoxicillin-furazolidone. Although it is possible to eradicate resistant bacteria, there are practical limitations on drug availability in some countries (Megraud, 2004). A recent phase 3 trial in Europe showed that 10 days of treatment with quadruple therapy was superior in eradicating H. pylori compared with 7 days of standard therapy in patients with or without presence or history of peptic ulcer disease. Therefore, the authors of this study recommend that in regions with high levels of clarithromycin resistance, quadruple therapy should be considered the first-line therapy for eradication of H. pylori (Malfertheiner et al., 2011).

Considering drug resistance mechanisms, increased side effects, low compliance and the high cost of antibiotic therapy, treatment failure rates are increasing and second-line treatment strategies need to be developed (Bytzer & O'Morain, 2005). A vaccine against H. pylori would solve the problem of pathogenic strains and work as a prophylactic (Velin & Michetti, 2006). There is still no vaccine available but several are being developed (Permin & Andersen, 2005; Kabir, 2007; Svennerholm & Lundgren, 2007). Stomach colonization by H. pylori induces a strong and un-protective immune response, mainly effected by Th1 cytokines (Mohammadi et al., 1996; Romagnani, 1999; Luzza et al., 2001). Th1 cells produce interferon-gamma, interleukin (IL)-2 and tumour necrosis factor (TNF)-beta, which activate macrophages and are responsible for cell-mediated immunity and phagocyte-dependent protective responses. By contrast, Th2 cells produce IL-4, IL-5, IL-10 and IL-13, which stimulate antibody production and eosinophil activation and inhibit several macrophage functions, providing phagocyte-independent protective responses. It is therefore believed that the efficiency of a potential H. pylori vaccine will depend on the induction of humoral immune response (antibody production) and of Th2 cells (Bergman et al., 2006; Kusters et al., 2006).

Antibiotics and vaccines are not the only weapons that can be used against H. pylori. The elimination of risk factors such as poverty and poor hygiene, and the improvement in living conditions could also decrease the prevalence of infection (Feldman, 1995; Vale, 2011).

Probiotics

The use of probiotics as monotherapy or in combination with antibiotics for the treatment of H. pylori is an active research field. Probiotics are live organisms or produced substances that are orally administered to promote health. PubMed displays more than 170 references when a search for ‘H. pylori and probiotics’ is submitted. Probiotics usage may prevent pathogenic bacteria infection, either by stimulating the immune response, or by directly competing with pathogenic bacteria. Probiotics may also prevent antibiotic side effects, as well as improve eradication rates (Lionetti et al., 2006, 2010; Lesbros-Pantoflickova et al., 2007; Miki et al., 2007). In view of the antagonism from the production of inhibitory substances, competition for adhesion or nutrients, host immune modulation or inhibition of toxins, probiotics are potentially important in preventing pathogen infection (Drouin, 1999; Broekaert & Walker, 2006; Lesbros-Pantoflickova et al., 2007). Several randomized controlled trials showed a decrease in the frequency and severity of antibiotic side effects (Table 1), but an improvement in eradication rate was not always observed (Lionetti et al., 2010).

Table 1

Examples of probiotics tested in blind, placebo-controlled, randomized trials

Probiotic	>No. of patients enrolled	Improved eradication	Improved side effects	Other comments	References
Saccharomyces boulardii	991	Yes	Yes	Helped completing therapy	Song et al. (2010)
	90	No	Yes		Hurduc et al. (2009)
	124	No	Yes		Cindoruk et al. (2007)
	389	No	Yes	Prevented antibiotic-associated diarrhoea	Duman et al. (2005)
Lactobacillus GG	83	No	No		Szajewska et al. (2009)
Lactobacillus reuteri	40	No	Yes	Decreased the occurrence of dyspeptic symptoms	Francavilla et al. (2008)
	40	No	Yes		Lionetti et al. (2006)
Lactobacillus casei DN-114 001	86	Yes	−		Sykora et al. (2005)
Lb. casei ssp. casei DG	70	Yes	Yes		Tursi et al. (2004)
Lactobacillus johnsonii La1	326	Yes	−		Cruchet et al. (2003)
Lactobacillus gasseri OLL2716	31	Yes	−		Sakamoto et al. (2001)
Bacillus subtilis and Streptococcus faecium	352	Yes	Yes		Park et al. (2007)
Bacillus clausii	120	No	Yes		Nista et al. (2004)
Bacillus animalis and Lb. casei	65	No	No		Goldman et al. (2006)
Lactobacillus and Bifidobacterium	138	Yes	Yes		Sheu et al. (2006)
Lactobacillus acidophilus LB and Streptococcus boulardii	254	Yes	−	S. boulardii eradicated H. pylori in 12% of the colonized children, and Lb. acidophilus in 6%	Gotteland et al. (2005)
Lb. acidophilus La5 or B. lactis Bb12	70	Yes	−		Wang et al. (2004)
Lactobacillus and Bifidobacterium	160	Yes	Yes	Restored the depletion of Bifidobacterium in stools after triple therapy	Sheu et al. (2002)
Lb. acidophilus HY2177, Lb. casei HY2743, Bifidobacterium longum HY8001, and Streptococcus thermophilus B-1 (Will yogurt)	337	No	No		Yoon et al. (2011)
347	Yes	No		Kim et al. (2008)
Lactobacillus rhamnosus GG, Lb. rhamnosus LC705, Propionibacterium freudenreichii ssp. shermanii JS, and Biphidobacterium breve Bb99	58	−	Yes	Probiotic combination resulted in only minor changes in the microbiota	Myllyluoma et al. (2007)
Lactobacillus GG, S. boulardii, Lactobacillus ssp. and Biphidobacterium	85	No	Yes		Cremonini et al. (2002)

Probiotics administered alone (without antibiotic therapy).

Two recent meta-analysis studies showed that standard triple therapy supplementation with the yeast Saccharomyces boulardii increases eradication rates and decreases overall therapy-related side effects, particularly diarrhoea (McFarland, 2010; Szajewska et al., 2010). Another meta-analysis concluded that Lactobacillus has a similar effect in H. pylori eradication (Zou et al., 2009), which favours the co-administration of probiotics with antibiotics in the treatment of H. pylori. Saccharomyces boulardii and Lactobacillus strains are the most common probiotics used in clinical trials, even though others have been tested, especially preparations of multiple strains of different species (Table 1). Indeed, Lactobacillus strains are known to be part of the transient gastric flora (Gotteland et al., 2006).

The mode of action of probiotics is not completely understood but they may act as surrogate normal microflora following antibiotic therapy until recovery is achieved (McFarland, 2010). However, probiotic combinations appeared to induce only minor changes in the microbiota (Myllyluoma et al., 2007). For instance, the mechanisms of action of S. boulardii include luminal action (anti-toxic effect, antimicrobial activity), trophic action (enzymatic activity, increased IgA) and mucosal-anti-inflammatory signalling effects (decreased synthesis of inflammatory cytokines) (McFarland, 2010).

Short-chain fatty acids (SCFAs) and bacteriocin proteins have been implicated in the inhibition of H. pylori by lactic acid bacteria. SCFAs such as formic, acetic, propionic, butyric and lactic acids are produced as a result of the metabolism of carbohydrates by probiotics and play an important role in decreasing the pH in vitro. Their antimicrobial activity could be due to the inhibition of urease activity by high lactic acid producers, such as Lactobacillus salivarius and Lactobacillus casei Shirota (Gotteland et al., 2006). Lactobacillus salivarius significantly decreased IL-8 production [IL-8 is induced after injection of virulence factor CagA (cytotoxin-associated gene A) into epithelial cells] upon exposure to H. pylori and led to CagA accumulation in H. pylori cells, presumably as a result of loss of functionality of the Cag secretion system (Ryan et al., 2009). Alterations in gastrointestinal permeability are an initial step in the development of lesions such as ulcers. Probiotics may stabilize the intestinal barrier by stimulating the expression of gastric mucins, decreasing bacterial overgrowth and stimulating local immune responses and the release of antioxidant substances (Gotteland et al., 2006).

Bacteriocins are proteins lethal to bacteria of the same species other than the producing strain. They are a heterogeneous group of proteins, ranging from small peptides of 19–37 amino acid residues to large peptides with molecular weights of 90 000 Da (Joerger, 2003). Bacteriocins are considered safer for humans than antibiotics as they have been present in food ever since the origin of humankind. The antimicrobial activity of some of the probiotics may even be based on the production of bacteriocins. In fact, the release of heat-stable, proteinaceous bacteriocins with anti-H. pylori activity has been identified in probiotic strains of Lactobacillus, Enterococcus faecium, Bacillus subtilis and Bifidobacterium (Gotteland et al., 2006). Bacteriocins produced by lactic acid bacteria include nisin A, pediocin PO2, leucocin K and various types of lacticins (Gotteland et al., 2006). Another study demonstrated that bacteriocin action is dependent on H. pylori strain and load (Kim et al., 2003). This may explain the diversity of results obtained in randomized trials (Table 1). Also, the gut microbiota may act through the production of bacteriocins. For instance, Fusobacterium mortiferum, a phylotype isolated from chicken described in the human stomach (Bik et al., 2006), synthesizes bacteriocins that inhibit Salmonella typhimurium (Portrait et al., 2000). Moreover, the mouth microbiota seems to inhibit H. pylori growth through the synthesis of bacteriocins (Ishihara et al., 1997). Therefore, in vivo bacteriocin production may be important for the dynamic stability of the gastrointestinal microbiota (Joerger, 2003) and, consequently, for human health. The administration of bacteriocin-producing bacteria, instead of purified products, may be a simpler strategy. Further research is needed as the mechanism of action of bacteriocins is not known, and as for other antimicrobial drugs, the resistance issue should be addressed.

Phytotherapy

Phytotherapy can be defined as the use of plant extracts as medicines or health- promoting agents and is as old as human civilization. Its origins are based on empirical knowledge and scientific validation of these products is still very limited. For example, tea tree oil has broad anti-bacterial activity, including activity against methicillin-resistant Staphylococcus aureus or Candida albicans (Carson & Riley, 2003) and green tea catechins also possess antimicrobial activity (Song & Seong, 2007). However, the study of herbal medicines to treat H. pylori infection is in its early days and few reports are available. Some authors describe H. pylori as susceptible to garlic extract at moderate concentrations in vitro (Sivam, 2001), whereas others report no effect (Martin & Ernst, 2003).

The extract of Pelargonium sidoides roots (EPs) 7630, a South African herbal remedy currently used to treat acute bronchitis, prevents bacteria from attaching to cell membranes. EPs 7630 inhibit H. pylori growth and high-potency adhesion to gastric AGS cells and to intact human stomach tissue from patient resections in situ. This suggests that their mode of action is mainly related to their anti-adhesive activity (Beil & Kilian, 2007; Wittschier et al., 2007). Similarly, the use of a high molecular mass constituent of cranberry juice inhibits H. pylori adhesion to human gastric mucus, suggesting that a combination of antibiotics and a cranberry preparation may improve H. pylori eradication (Burger et al., 2000; Shmuely et al., 2004). A synergistic combination of oregano and cranberry phenolics has been suggested to inhibit H. pylori in a laboratory medium. The likely mode of action may be through urease inhibition and disruption of energy production by inhibition of proline dehydrogenase at the plasma membrane (Lin et al., 2005). The inhibition of urease, which catalyzes the hydrolysis of urea to carbon dioxide and ammonia, hence increasing the pH, is a potential target for H. pylori eradication (Kosikowska & Berlicki, 2011). For instance, ethanol extracts of Magnolia officinalis Rehd. et Wils. (Magnoliaceae) and Cassia obtusifolia L. (Leguminosae) inhibit urease (Shi et al., 2011). Hydroxamic acids, phosphoramidates, urea derivatives, quinones and heterocyclic compounds constitute the main classes of substances exhibiting such activity (Kosikowska & Berlicki, 2011). Other phytomedicines implicated in urease inhibition are extracts of Camellia sinensis (Hassani et al., 2009) and apple peel polyphenols (Pastene et al., 2009).

The bark of Calophyllum brasiliense Camb. (Clusiaceae), which grows in Brazil and is also known as ‘guanandi’, and its hydroethanolic extract and dichloromethanic fraction have demonstrated anti-H. pylori activity in humans (Souza et al., 2009). Extracts of the Brazilian Mouriri elliptica Martius (Melastomataceae) (Moleiro et al., 2009), Hancornia speciosa Gomez (Mangaba) (Moraes et al., 2008), Byrsonima fagifolia Nied. (Malpighiaceae) (Lima et al., 2008b) and Alchornea triplinervia (Lima et al., 2008a) also appear to have anti-H. pylori action. Mexican Amphipterygium adstringens (Schltdl.) Standl. (Anacardiaceae), an anacardic acids mixture, exhibits potent dose-dependent anti-H. pylori activity (Castillo-Juarez et al., 2007). Extract of Japanese rice also demonstrated anti-H. pylori activity (Ishizone et al., 2007) and African São Tomé plants are also used in traditional therapies for several gastric disorders. These include Leonotis nepetifolia (L.) W. T. Ainton var. nepetifolia (gastric indisposition), Solenostenom monastachyus (P. Beauv.) Briq. ssp. monostachyus (stomach pain), Piper umbellatum L. (stomach problems), Bertiera racemosa (G. Don) K. Shum var. elephantina N. Hallé (stomach pain), Allophyllus grandifolius (Baker) Radlk (gastric affection) and Solanum gilo Raddi (stomach pain) (Madureira et al., 2008). The anti-Helicobacter action of these compounds, either in vitro or in vivo, has not been tested yet.

Isothiocyanate sulforaphane, abundant in broccoli sprouts, may have both a direct antibacterial effect on H. pylori, leading to reduced gastritis and an indirect (systemic) effect by increasing the mammalian cytoprotective response (Yanaka et al., 2009).

The cold extract, infusion, decoction and simulated digestion of Larrea divaricata Cav (jarilla) inhibit clarithromycin- and metronidazole-susceptible and -resistant H. pylori strains. This plant, usually applied in the treatment of gastric disturbances, may also be useful in peptic ulcer and gastric cancer therapy (Stege et al., 2006).

Bacopa monniera is currently used in Indian medicine to improve intellectual function. A standardized extract of B. monniera has anti-H. pylori activity in vitro. The antimicrobial effect is due to augmentation of defensive mucosal responses such as mucin secretion, life span of mucosal cells and gastric antioxidant effects, rather than offensive acid-pepsin secretion (Goel et al., 2003).

Propolis is a resinous hive product collected by honeybees from various plant sources usually used in food, beverages and folk medicine for treating various diseases. It shows a broad spectrum biological activity and offers antibacterial activity against H. pylori (Banskota et al., 2001). Other kinds of honey also have anti-H. pylori activity (Manyi-Loh et al., 2010).

Alkyl methyl quinolone alkaloids (AM quinolones) from gosyuyu (Wu-Chu-Yu) and psoralen (extract from Psoralea corylifolia) (Zhang et al., 2010), a Chinese herbal medicine (Hamasaki et al., 2000), and amu-ru 7, a Mongolian folk medicine (Bai et al., 2010), show antibacterial activity against H. pylori in vitro. Luteolin, a component in herbal medicine, can also inhibit H. pylori (Chung et al., 2001). However, the molecular mechanism of action of these herbal extracts is unknown and further investigation is needed.

A recent study identified new herbal extracts with antimicrobial activity against H. pylori. Agrimonia eupatoria, Hydrastis canadensis, Filipendula ulmaria and Salvia officinalis were the most active herbal extracts (Cwikla et al., 2010). The same study also tested essential oils and the most effective against H. pylori, Syzygium aromaticum oil, showed a half maximal inhibitory concentration value 10 times lower than the standard antibiotic ampicillin. Cytotoxic studies are needed to prove the selective toxicity of these essential oils and to establish the concentrations at which anti-Helicobacter activities are not harmful for epithelial cells in the gastrointestinal tract. Finally, clinical trials are necessary to explore the possibility of using herbal medicines as an efficient and low-cost remedy for eradicating H. pylori.

Curcumin (diferuloylmethane), the major yellow pigment present in the rhizome of turmeric (Curcuma longa), has a healing effect in H. pylori infection (De et al., 2009), probably by suppressing secretion of metalloproteinases 3 and 9 by gastric cells, believed to be involved in the development of gastric ulcer and gastric cancer (Kundu et al., 2011).

Nigella sativa (Ranunculaceae) grows in the Middle East, Eastern Europe and Eastern and Middle Asia and its oil is used as a food additive because of its anti-inflammatory, anti-cancer and antimicrobial activity. Nigella sativa seeds contain essential oils such as thymoquinone, dihydrothymoquinone and terpenes, that may exercise their antimicrobial activity by disrupting the lipid structure of the cell membrane (Salem et al., 2010). Other herbal extracts also have potential H. pylori inhibitory properties, but their mode of action is unknown. These include crude methanol extract of the leaf of Allium ascalonicum (Adeniyi & Anyiam, 2004); flavonoid constituents of herbal medicines (Shin et al., 2005), such as the ones isolated from the leaves of Piper carpunya Ruiz & Pav. (syn. Piper lenticellosum C.D.C.) (Piperaceae), widely used in folk medicine in tropical and subtropical South American countries and known for their anti-inflammatory and anti-ulcer action (Quilez et al., 2010); ‘compound with anti-Helicobacter activity’, extracted from celery (Apium graveolens) seeds (Zhou et al., 2009); and phenolic acid derivatives, acylglycoflavonoids and condensed tannins from Davilla elliptica and Davilla nitida (Kushima et al., 2009). Resveratrol, a polyphenol highly abundant in red grapes (Daroch et al., 2001) (Zaidi et al., 2009), exhibits anti-inflammatory and anti-cancer activity and has cardioprotective and neuroprotective properties (Mahady & Pendland, 2000). This protective activity may be due to modulation of inflammatory cytokines such as IL-6, transcription factors such as nuclear factor-kB and regulatory enzymes such as mitogen-activated protein kinases, or to multiple modulatory effects on H. pylori-induced IL-8 secretion, reactive oxygen species production and morphological changes (Goswami & Das, 2009; Zaidi et al., 2009).

Although these substances show promising anti-H. pylori properties, a systematic review of Chinese herbal medicines does not support a role as stand-alone therapy (Lin & Huang, 2009). The active ingredients of phytomedicines should be identified to confirm their selective toxicity and determine their mode of action and the dose required to treat infections without harming the patients.

Other alternative therapies

Bacteriophage therapy consists of the use of bacteriophages to treat infectious diseases and was developed and applied in the pre-antibiotic era by Felix d'Herelle, in France, Eastern Europe (ex-Soviet Union), Vietnam and India (D'Herelle, 1921, 1929). Although the development of antibiotics put the research field on hold, this old idea is being revived, given the present emergence of antibiotic resistance (Hanlon, 2007; Gill & Hyman, 2010; Kutateladze & Adamia, 2010). The discovery of antibiotics led to the assumption that infectious diseases could be controlled, but bacteria are sophisticated cells that have existed for 3.5 billion years (Mojzsis et al., 1996; Holland, 1997) and rapidly (in a brief period of 50 years) developed several antibiotic resistance mechanisms, bringing back the classic problem of treating infectious diseases. But not all phages are harmless to humans and animals. Several known toxins encoded by phages played an important role in epidemics of diphtheria (Corynebacterium diphteriae), cholera (Vibrio cholerae), scarlet fever (Streptococcus pyogenes) (Aziz et al., 2005) and the recent haemolytic–uremic syndrome in children (Escherichia coli O157:H7) (Miao & Miller, 1999). The complete sequencing of the phage genome should be the first step of a rigorous characterization of bacteriophages to be introduced in therapy in order to guarantee the absence of virulence factors.

Nonetheless, bacteriophages have already been applied in environmental decontamination (Walter, 2003; Withey et al., 2005), food industry (Huff et al., 2005; Kim et al., 2007) and plants (Goodridge, 2004) and have been administered orally or intravenously in animals (Doyle & Erickson, 2006; Sheng et al., 2006; Wang et al., 2006) and humans (Sulakvelidze et al., 2001; Bruttin & Brussow, 2005; Clark & March, 2006). Phage therapy has been used successfully to reduce colonization by Campylobacter jejuni, a closely related species of H. pylori, in poultry (Connerton et al., 2004; Loc et al., 2005; Wagenaar et al., 2005; Carvalho et al., 2010).

In addition, genetically manipulated bacteriophages may be used as vaccine delivery vehicles, either directly by vaccination with phages carrying vaccine antigens on the surface, or indirectly by using the phage particles to deliver a DNA vaccine expression cassette incorporated in the phage genome. Due to their viral nature, these vectors may be rapidly delivered to antigen-presenting cells, effecting a stronger antibody response (Clark & March, 2006; Ranallo et al., 2006; Lankes et al., 2007).

Helicobacter pylori phage screening is a rare topic in the literature, despite the possibility of using phage therapy to treat antibiotic multiresistant strains. To our knowledge, there are only five references to H. pylori bacteriophages in the literature (Marshall et al., 1987; Goodwin et al., 1989; Schmid et al., 1990; Heintschel von et al., 1993; Vale et al., 2008). Marshall et al. (1987) and Goodwin et al. (1989) initially described intracellular phage-like particles in human gastric biopsies. Schmid et al. (1990) reported that the strain SchReck (also designated IMMi290/89), isolated from a patient with chronic antral gastritis, intestinal metaplasia and atrophy, spontaneously produced bacteriophages in vitro. Three years later, the same group published another study of the same phage (designated HP1). HP1 characterization revealed a head of 50 nm, a flexible tail of 170 nm in length by 9.5 nm in diameter and a DNA genome of 22 000 bp (Heintschel von et al., 1993). An H. pylori phage screening procedure revealed clear lysis plaques on a cell lawn, whereas transmission electron microscopy showed bacteriophage-like structures with a diameter of approximately 100 nm without a tail in one strain, and 15-nm filaments in another strain. Both can represent phage particles (Vale et al., 2008). Helicobacter pylori strain B38 contains prophage remnant sequences (Thiberge et al., 2010) and sequencing of the H. pylori strain B45 revealed the presence of a complete inducible prophage (Vale et al., 2010). These recent data make testing phage therapy against H. pylori possible.

Finally, photodynamic therapy, in which harmless visible light is used to excite non-toxic photosensitive molecules to highly reactive triplet states that can then react with molecules in their immediate vicinity to originate free radicals and reactive singlet oxygen, has been suggested to treat H. pylori (Wilder-Smith et al., 2002; Lembo et al., 2009).

Conclusions

The mode of action of the human gastrointestinal microbiota is not fully understood, but there is some evidence that it plays an important role in human health and that any disturbance may be responsible for emergent diseases. As some H. pylori strains are antibiotic-resistant, leading to therapeutic failure, finding alternative non-antibiotic approaches to treat H. pylori and other pathogenic bacteria is imperative. Probiotics diminish side effects and improve the efficacy of antibiotics, probably because they mimic the function of the human microbiota. Phytotherapy is a very promising therapeutic approach that has been used for centuries in traditional medicine. The herbal species used across the globe are highly diverse and only a few studies have identified the nature of the active ingredient or its mechanism of action. Safety must be assessed and each active substance must be characterized. Resistance against these new phyto-therapeutic agents should also be addressed. Bacteriophage therapy shows potential as an alternative to antibiotics, but it will take some time to verify whether it is feasible as it requires phage isolation and characterization. In summary, the mode of action underlying the described alternative therapies is largely unknown and future research in the field is greatly needed.

Acknowledgements

We thank Elsa Anes for critical reading of the manuscript and Patrícia Fonseca for editorial support.