Helicobacter pylori-coccoid forms and biofilm formation

Abstract

Electron microscopic studies have shown that Helicobacter pylori occurs in three stages: spiral forms, coccoid forms and degenerative forms. The spiral forms are viable, culturable, virulent and can colonize experimental animals and induce inflammation. The coccoid forms may also be viable but are nonculturable, less virulent and are less likely to colonize and induce inflammation in experimental animals than the spiral forms. The degenerative forms are pyknotic, nonculturable, coccoid forms of dead H. pylori. These forms cannot be cultured and the cell membrane has disintegrated but gene material can be detected by PCR in water supplies. There is no substantial evidence for viable H. pylori persisting in water supplies. Epidemiological studies suggest that environmental water is a risk factor for H. pylori infection when compared with tap water, and formation of H. pylori biofilm cannot be excluded. Helicobacter pylori does not seem to take part in biofilm formation in the oral cavity even though the bacterium may be detected.

Helicobacter pylori, biofilm, coccoid forms, dental plaques

Introduction

Helicobacter pylori is a Gram-negative microaerobic curved rod that causes gastritis, peptic ulcers and plays an important role in gastric cancer and MALT lymphomas (Andersen & Wadström, 2001; Amieva & El-Omar, 2008; Kandulski et al., 2008). The prevalence of H. pylori is about 50% worldwide and up to 90% in developing countries. Helicobacter pylori primarily colonizes the antral part of the human stomach where some of the bacteria adhere to the gastric mucosa and a substantial proportion remain as floaters (nonadherent) in the gastric lumen and may continue to the intestine and be excreted with feces (Blaser & Kirschner, 1999; Blom et al., 2000). When patients have dyspeptic symptoms that include nausea and vomiting, the floating bacteria may be transported to the oral cavity where they may colonize gingival and dental plaques (Young et al., 2000, 2001). Thus, H. pylori has the opportunity to contaminate the environment by oral–oral or oral–fecal routes where it may survive as coccoid forms or in biofilm. The vitality of nonculturable coccid forms of H. pylori and biofilm formation by H. pylori has been controversial (Cellini et al., 1994, 2001; Kusters et al., 1997; Willén et al., 2000). In this review, the validity of these statements about coccoid forms and biofilm will be evaluated on the basis of the literature.

Coccoid forms of H. pylori

Formation of coccoid forms is a common feature in Gram-negative rods under conditions of stress, such as starvation (Roszak et al., 1984; Perez-Rosas & Hazen, 1989). Coccoid forms have been described as a nonculturable form of H. pylori (Nilius et al., 1993; Vijayakumari et al., 1995; Jones & Curry, 1998; Mizoguchi et al., 1999), but it has been questioned whether these coccoid forms are viable or degenerative forms of H. pylori (Cellini et al., 1994, 2001; Kusters et al., 1997; Willén et al., 2000). Electron microscopic studies have shown that H. pylori can exist in three forms: a viable spiral form, a viable coccoid form and a nonviable degenerative coccoid form (Nilius et al., 1993; Benaissa et al., 1996; Willén et al., 2000; Young et al., 2000; Saito et al., 2003). The viable spiral and coccoid forms have intact cytoplasm structures and a cell membrane that is characteristic for Gram-negative bacteria. The nonviable coccoid forms have degenerative organelles and a disintegrated cell membrane. The viable coccoid form is usually smaller and more condensed than the degenerative coccoid forms. It has been shown that H. pylori can survive as culturable forms in distilled water and saline for >14 days and in artificial seawater for >7 days (West et al., 1992). Viable culturable H. pylori can persist for >10 days and viable nonculturable coccoid H. pylori can survive for up to 1 year in fresh water (Shahamat et al., 1989). One H. pylori strain stored in deep ground water or in natural seawater at 4 °C survived significantly better for 7 days than the same strain stored in nutrient-rich media (Konishi et al., 2007). These studies indicate that H. pylori may survive as culturable forms for weeks in water and that they survive better in natural systems than in artificial nutrient-rich systems. The nonculturable coccoid form was able to persist for several months and it is unlikely that the fractions were contaminated with spiral forms after such a long period.

Studies have indicated that the protein content remains unchanged during the conversion from spiral to coccoid forms of H. pylori (Bumann et al., 2004) and that the bacterium remains genetically unchanged, which — the authors state — indicates that coccoid forms remain viable (Hua & Ho, 1996; Sisto et al., 2000). When H. pylori becomes degenerative with a disintegrated cell membrane, it is thought that enzyme systems and organelles are lost while major proteins and genetic material remain unchanged for a longer period. This is indicated by the decrease in urease activity and the ability to adhere to Hep2 cells of coccoid forms of H. pylori converted in sterile tap water compared with spiral forms of H. pylori (P<0.01) (She et al., 2003). It may also be indicated by a loss of virulence in coccoid forms of H. pylori.

Helicobacter pylori was cultured from 14/16 mice 28 days after inoculation with spiral forms and from 11/16 mice inoculated with coccoid forms of H. pylori, a difference that was not statistically significant. There was less gastric inflammation in the mice inoculated with coccoid forms (She et al., 2003). In one study, coccoid forms of H. pylori were unable to colonize gnotobiotic piglets (0/6), unlike spiral forms (6/6), and it was concluded that the coccoid forms were degenerative. The difference could also be due to differences in preparation of coccoid forms or differences in the gastric mucosa of pigs compared with mice and humans (Eaton et al., 1995). Some studies show that up to 95% of a culture consists of coccoid forms after 21 days of culture (Cole et al., 1997; Ren et al., 1999). This indicates that small amounts of spiral forms may occur in cultures after 3 weeks. This could also be the case in studies in which H. pylori was converted by different methods such as in water. Most studies that convert spiral bacteria to coccoid forms use time schedules of <4 weeks and it may be questioned whether small amounts of spiral bacteria may contaminate such preparations.

Thus, H. pylori occurs in three stages: viable spiral forms that are culturable, virulent and infectious and induce inflammation in experimental animals; viable coccoid forms that are nonculturable, less virulent and less likely to colonize and induce inflammation in experimental animals; and a third form that consists of nonviable degenerative forms of dying H. pylori.

Environmental and dental H. pylori

Helicobacter pylori is able to survive in sea and fresh water as viable and culturable forms for a few weeks and as nonculturable viable forms for up to a year (Shahamat et al., 1989; West et al., 1992; Konishi et al., 2007). Laboratory studies have shown that some strains of H. pylori have characteristics that make them able to produce biofilm in vitro. This formation of biofilm involves attachment of individual bacteria that form microcolonies, merging of microcolonies and growth in the third dimension (Cole et al., 2004; Cellini et al., 2008; Williams et al., 2008). The formation of biofilm and the cell morphology depends on the support material. One study showed that H. pylori only survived for a few days in biofilm in a water supply model (Azevedo et al., 2006). Helicobacter pylori could not be cultured and the cell membrane was disintegrated but gene material was still detected by PCR (Azevedo et al., 2006; Giao et al., 2008). It has only been possible to detect H. pylori by PCR in water supplies and seawater (Park et al., 2001; Cellini et al., 2004). Thus, there is no substantial evidence that viable H. pylori plays a role in biofilm formation in water supplies.

However, several epidemiological studies based on serology or the urea breath test have found from two to 13 times higher risk of H. pylori infection in people drinking river or well water compared with those drinking tap water (Klein et al., 1991; Goodman et al., 1996; Nurgalieva et al., 2002; Karita et al., 2003; Ahmed et al., 2007). One of the studies (Goodman et al., 1996) also showed that people swimming in rivers, streams or pools had a three times higher risk of H. pylori infection than people who did not swim in such environments. These studies were carried out in developing countries where water supplies may differ from those in developed countries and where river water may be more common as a source of drinking water, but they all agree that environmental water is a risk factor for H. pylori infection and that formation of H. pylori biofilm cannot be excluded.

Helicobacter pylori can be cultured from gastric juice from dyspeptic patients and these floating forms of H. pylori may be transmitted to the oral cavity by reflux or vomiting (Andersen et al., 1988; Young et al., 2000). Biofilm formations or dental plaque in the oral cavity of human beings have been found to harbor H. pylori. Electron microscopic studies have shown that both spiral and viable coccoid forms of H. pylori may be present in the oral cavity even though they were not cultured (Young et al., 2001). Different methods to detect H. pylori in the oral cavity have been evaluated and the conclusion was that PCR is most suited for the purpose (Teoman et al., 2007). Several studies using PCR as a detection method were able to detect H. pylori in the mouth both in patients with a history of gastric symptoms and in patients without any history of gastric symptoms (Teoman et al., 2007; Souto & Colombo, 2008). One study found H. pylori in the oral cavity of 60% (18/30) of patients 3 months after triple antibiotic therapy, but in the stomach in only 10% (3/30) of the same patients (Gebara et al., 2006). This indicates that the prevalence of H. pylori in the oral cavity may be independent of the prevalence of H. pylori in the stomach after treatment. Yet others believe that there may be a correlation between the prevalence of H. pylori found in dental plaque and the oral health status. One study found H. pylori in subgingival biofilm in 11% of periodontally healthy patients vs. 50% of patients suffering from periodontitis (Souto & Colombo, 2008).

It is possible that biofilm formations in the oral cavity can serve as a reservoir for H. pylori and may thereby cause gastric reinfection. The prevalence of H. pylori in the mouth may be affected by the health status of the oral cavity or the stomach. It is not clear whether or not H. pylori participates in biofilm formation in the oral cavity, and no studies have investigated the length of time that H. pylori can survive in the oral biofilm.

In conclusion, H. pylori normally exists as spiral forms that are usually culturable but may occasionally be difficult to culture. They can convert into viable but nonculturable coccoid forms under stress or they may die as degenerative coccoid forms. Certain environmental strains of H. pylori are able to form biofilm in laboratory experiments. However, several studies indicate, that H. pylori convert to coccoid forms rather quickly in water supply systems. Epidemiological studies agree that use of river or well water as drinking water is a risk factor for H. pylori infection compared with tap water. The oral cavity is another place where biofilm is common, and both spiral and coccoid forms of H. pylori can be found in dental plaques, but there are no indications that H. pylori participates in biofilm formation.

Author notes

Editor: Patrik Bavoil