MEDICAL ASPECTS OF GASTROINTESTINAL BIOFILMS Graeme A. OMay*, Jennifer A.J.

Madden, Aileen Kennedy & Sandra Macfarlane

University of Dundee, MRC Microbiology and Gut Biology Group, Department of Molecular and Cellular Pathology, Level 6, Ninewells Hospital and Medical School, Dundee, UK. Abstract The human gastrointestinal tract has a large surface area available for biofilm formation. Biofilm communities are in direct contact with the host and are thus prime candidates for involvement in host-microbe interaction. In this article, we focus on the role of these communities in patients with inflammatory bowel disease and those undergoing percutaneous endoscopic gastrostomy tube feeding. Rectal mucosal populations in both healthy and ulcerative colitis patients are outlined. Anaerobic bacteria outnumbered facultative anaerobes in both patient groups. However, healthy people had more bifidobacteria and prevotella and fewer Gram-positive cocci, lactobacilli and clostridia than UC patients. Biofilms dominated by Yeasts, Enterococcus, Staphylococcus, Bacillus and Lactobacillus spp. were detected on percutaneous endoscopic gastronomy (PEG) tubes. PEG tube biofilms contribute to tube deterioration and may provide reservoirs for potential pathogens, making them difficult to eradicate using chemotherapeutic methods. Treatment with probiotics offers an alternative to chemotherapy in some instances, although the mechanisms by which probiotic microorganisms interact with gastrointestinal (GI) biofilms remain poorly understood.

Introduction The human gastrointestinal (GI) tract extends from the oesophagus to the rectum and harbours a diversity of microhabitats which are colonised by microorganisms to varying degrees, depending on local environmental conditions. A gradient of colonisation exists from the sparsely-populated oesophagus and stomach to the descending colon and rectum, which may contain up to 1012 culturable bacteria per gram contents (Hopkins et al. 2002). Evolution has dictated that these organs possess a large surface area to facilitate efficient nutrient uptake. This, together with high nutrient availability and a constant influx of microorganisms as well as stable autochthonous populations, makes the GI tract an ideal site for the development of sessile microbial communities. Those microorganisms in closest proximity to host tissues have the most opportunity for interaction with host physiology and metabolism; thus mucosal populations are an important component of any hostmicrobiota interaction, whether it be beneficial or detrimental. The human GI microbiota performs a number of beneficial functions. These include vitamin synthesis (Conly et al. 1994), absorption of calcium, magnesium and iron (Miyazawa et al. 1996, Younes et al. 2001), production of colonic enterocyte nutrients (Cummings et al. 1987) and immune stimulation/regulation (Tannock 2001). Additionally, in colonisation resistance the normal microbiota is known to assist in preventing colonisation of the GI tract by opportunistic invaders such as Clostridium difficile (van der Waaij 1989). Conversely, the GI tract microbiota has also been implicated in disease states such as inflammatory bowel disease (Macpherson et al. 1996), colonic (Horie et al. 1999) and gastric (Bjorkholm et al. 2003) carcinoma and irritable bowel syndrome (Wyatt et al. 1988). Additionally, the microbiota has an important role in almost any

medical situation involving the GI tract; for example, abdominal surgery and enteral nutrition. In these situations, although the GI tract microbiota may not be the original cause of the required intervention, it often influences the outcome. The research outlined in this article concentrates on the effect of the GI microbiota, particularly sessile populations, on patients receiving enteral nutrition through a percutaneous endoscopic gastrostomy (PEG) tube and in those suffering from inflammatory bowel disease (IBD).

Mucosal populations in ulcerative colitis Ulcerative colitis (UC) is a chronic relapsing form of IBD of unknown aetiology. The inflammatory response in UC is primarily located in the colonic mucosa and submucosa. The distal colon is always affected and the disease may progress towards the proximal bowel with crypt abscesses causing severe tissue damage. Bacterial involvement has been proposed in both the initiation and maintenance stages of UC (Hill et al. 1971). Antimicrobial agents specifically active against obligate anaerobes have been shown to prevent ulceration in guinea pigs (Onderdonk & Bartlett 1979) and experiments using germ-free animals show that they only develop colitis when repopulated with bacteria (Sadlack et al. 1993). A variety of species including fusobacteria, Shigella (Onderdonk 1983) and adhesive E. coli (Chadwick 1991) isolated from the colitic bowel have been implicated; however, no specific organism has been found in all patients. The luminal microbiota of UC patients has been examined in many studies (van der Wiel-Korstanje & Winkler 1975, von Wufflen et al. 1989) and there is good evidence for postulating that bacteria growing on the gut wall play a major role in UC, since they exist in close juxtaposition to host tissues and can interact with the host immune and

neuroendocrine systems. Electron microscope studies of human colonic biopsy tissue have suggested that mucosal bacteria are associated more closely with the mucus layer than the epithelial surface (Hartley et al. 1979). Distinct populations are known to exist on the mucosal surface and in the mucus layer in the large gut, where bacteroides and fusobacteria appear to predominate, but other groups such as eubacteria, clostridia and anaerobic Gram-positive cocci are also present as either heterogeneous populations or microcolonies (Croucher et al. 1983, Edmiston et al. 1982). There have been few studies on bacteria that inhabit the colonic mucosa because faeces and material from the gut lumen are more readily available for investigation and in most studies, the patients have been pre-treated with antibiotics and drugs, or the bowel has been purged before colonoscopy. As a consequence the metabolic and health-related significance of bacteria growing on the colonic mucosa is largely unknown. The objectives of the study were to enumerate and characterise mucosal bacterial communities in healthy people and in patients with UC. Rectal biopsies were chosen because the rectum is usually devoid of faecal material and patients did not need to be treated before the tissues were removed. Samples (four UC, five normal) were obtained from patients attending the Gastroenterology Out-patients Clinic at Ninewells Hospital, Dundee. None of the patients were taking antibiotics or any other drugs. Tissue samples were immediately placed in anaerobic transport medium, brought to the laboratory and measured, homogenised and plated out onto a range of selective and non-selective agars. The bacteria were then characterised on the basis of their Gram staining characteristics, cellular morphology and cellular fatty acid methyl ester (FAME) profiles using the MIDI system. Tissue samples were also placed in fixative for

analysis by fluorescent in situ hybridisation (FISH) with 16S rRNA oligonucleotide probes. Complex bacterial communities colonised the rectal mucosa in both healthy and UC patients. Bacteria were found to occur as microcolonies on the biopsies showing that they were actively growing and that their presence was not simply due to passive transfer from faecal material (Fig. 1).

Figure 1 Bacterial microcolonies on the rectal mucosa visualised by FISH using an enterococcal probe labelled with FITC. Total bacterial counts ranged from 104 to 106 cells per cm2 which differs from other studies, where only low numbers of bacteria were found in healthy patients compared with controls (Shultsz et al. 1999, Swidinski et al. 2002). Anaerobic bacteria outnumbered facultative anaerobes in both UC and healthy subjects (Fig. 2). Total anaerobic counts were 3-20 times higher than facultative anaerobes. This occurrence of a relatively high number of facultative anaerobes on the epithelial 5

Strict anaerobes Facultative species

5
Log10 bacteria (cm2 biopsy material)-1

0 UC Healthy

Figure 2 Comparison of strictlyfacultative anaerobes on the rectal populations and populations and anaerobic (dark bars) bacterial facultative anaerobes (light bars)subjects and UCmucosa Results mucosa in healthy on the rectal patients. in healthy subjects and UC patients. Resultsshow means (n =(n=3-5) SEM. show means 3-5) SEM surface is in broad agreement with data obtained from colonic tissue at autopsy (Croucher et al. 1983), but differs from the results of Poxton et al. (1997), where strict anaerobes on the mucosal surface were 10- to 100-fold higher than facultative anaerobes. However, in this study the patients were prepared for colonscopy and several were taking antibiotics. Enterobacteria, bacteroides, Gram positive cocci and bifidobacteria had the highest prevalance in both healthy and UC subjects with bacteroides having the greatest species diversity (Fig. 3). Other studies using colonic and rectal biopsies have also indicated that bacteroides and bifidobacteria 6

Figure 3 Comparison of strictly anaerobic bacterial

are the major anaerobes associated with the mucosal surface (Poxton et al. 1997). Gram-positive cocci, lactobacilli and clostridia were present in higher numbers in the UC patients, whereas the reverse was found with bifidobacteria and prevotella. Bacteroides fragilis was the main bacteroides found in both UC and healthy subjects. Bifidobacterium adolescentis and Bif. angulatum were predominant in healthy people whereas Bif. angulatum was the principal species in UC. Peptostreptococci and Enterococcus faecalis were not found on the rectal mucosa in healthy people, but did occur in UC patients. These results suggest that bacteria occur in broadly similar numbers on the rectal mucosa of UC and healthy patients, each having their own distinct subpopulations. Whether these bacteria on the rectal mucosa have a role in UC or those on the normal healthy mucosa are protective is currently under investigation.

Figure 3 Comparison of bacterial populations on the rectal mucosa in UC patients (closed bars, N=4) and healthy subjects (open bars, N=5). Results represent ranges and means (vertical bars). Values in parentheses indicate the number of species and strains in each bacterial group or genus, those in italics show the number of individuals that harboured these microorganisms in each subject group. Percutaneous endoscopic gastrostomy (PEG) tube biofilms Enteral tube feeding (ETF) through a PEG tube is sometimes advised when a clinical condition (most commonly cerebrovascular disease or head and neck trauma) results in impairment of a patients ability to ingest food normally. PEG tubes are placed during upper GI endoscopy and pass from the gastric lumen to the exterior of the abdomen. Feeding fluid is passed through the tube into the gastrum at predetermined intervals. ETF alters drastically the mechanisms by which a normal upper gastrointestinal microbiota is maintained. In normal individuals the upper GI tract is colonised sparsely. The gastrum is thought to be devoid of any significant resident microbiota; other than Helicobacter pylori and some lactobacilli (ca. 101 - 103 CFU ml-1) (Gustaffson 1982) any 8

microorganisms present are transients originating from food or the oral cavity. Lactobacilli, streptococci and Bifidobacterium spp. (102 - 104 CFU ml-1) are present in the duodenum (Berg 1996). This status quo is maintained by multiple mechanisms including peristalsis, a low pH (ca. one to four in normal individuals) and the enterosalivary circulation of nitrate and thiocyanate. A proportion of ingested nitrate is converted to nitrite by facultatively anaerobic bacteria on the tongue (Duncan et al. 1995, Xu et al. 2001b). The remainder is absorbed in the duodenum, enters the bloodstream and is concentrated in the salivary glands from where it is secreted back into the GI tract. Nitrite which reaches the gastrum is acidified to form nitric oxide along with other nitrogenous compounds which exert a strong antimicrobial effect in the low pH environment (Allaker et al. 2001, Dykhuizen et al. 1996, Xu et al. 2001a). Thiocyanate is also concentrated in saliva and enhances the antimicrobial effect of nitrite (Xu et al. 2001a). Recent studies have suggested that Enterobacteriaceae can survive exposure to extremely low pH environments (ca. pH 2) through expression of the asr genes (Seputiene et al. 2003). Perhaps, therefore, the acid environment of the stomach may not be sufficient to kill invading microorganisms under some circumstances. If this were true then the nitrite/thiocyanate system might play a central role in the defence of the gastrum. Each of these three protective mechanisms is degraded in ETF patients. Absence of any food-related sensory stimuli (smell, taste, sound) inhibits the production of saliva. Lack of normal mastication results in both reduced volumes of saliva reaching the gastrum and lower acid secretion. Additionally, lack of solid food inhibits peristaltic motion. The end result of this is that the antimicrobial defences of the stomach are compromised and it is thus open to colonisation. Invading

microorganisms may originate from one (or more) of three sources; (i) the lower gut, (ii) the oral cavity or (iii) from the external environment via the PEG tube and/or the nutrient fluid. Bacterial overgrowth in the upper GI tract has a number of potential sequelae. The most common is diarrhoea although other more serious complications occur; for example, malabsorption and sepsis (Cabre & Gassull 1993). Biofilm formation on PEG tubes is likely to be an unavoidable consequence of gastral bacterial overgrowth and will itself have consequences. PEG tube biofilms may act as a reservoir of microorganisms which will be difficult to eradicate with antimicrobial chemotherapy. Although replacement of the PEG tube would provide an answer to such colonisation this would consume more valuable medical resources. Thus a greater understanding of PEG tube biofilm composition, formation and physiology would be beneficial to both patients and clinicians. A number of studies have been conducted relating to bacterial colonisation of PEG tubes. Several detailed colonisation of PEG tubes by fungi, a phenomenon associated with deterioration of tube integrity. Several genera of fungi were isolated, including Candida albicans (Gottlieb et al. 1992, Gottlieb et al. 1993). Other authors have conducted a more comprehensive microbiological assessment of PEG tubes. Enterococcus, Escherichia, Bacillus, Lactobacillus and Staphylococcus spp. were isolated from 15 paediatric patients in one study (Dautle et al. 2002). The authors also used randomly amplified polymorphic DNA amplification (RAPD) to type microorganisms cultured from PEG tubes. Isolates cultured from different parts of a PEG tube were identical by RAPD profiling, suggesting that the biofilm spread from the initial attachment point. Three pairs of patients had identical RAPD profiles for E. coli, Staphylococcus aureus and E. faecalis. Thus PEG tube biofilm-associated

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microorganisms can be spread from patient to patient, raising concerns of transfer of detrimental attributes such as antibiotic resistance. The culture methods in this study involved extensive use of an antifungal (cycloheximide) at all stages of isolation. Given the evidence that a variety of fungi are to be found within PEG tube biofilms (Gottlieb et al. 1992; Gottlieb et al. 1993) the authors reasons for deliberately excluding such an important element of the biofilm community are difficult to understand. Another study by the same group used cultural methods in conjunction with scanning electron microscopy (SEM) and confocal scanning laser microscopy (CSLM) to visualise biofilms on different areas of PEG tubes taken from paediatric patients (Dautle et al. 2003). The majority of isolates were of the genera Bacillus, Enterococcus and Staphylococcus. SEM showed that control PEG tube surfaces were punctuated by cracks and crevices. Microcolonies were observed in PEG tubes removed from patients; these were often found in association with aberrations in the surface, leading to the suggestion that improved manufacturing methods might be of use in limiting biofilm formation on PEG tubes. Biofilm thickness was assessed using CSLM and ranged from 28.4 mm to 128.4 mm. Depth varied between patients and was not related to location on the PEG tube. Additionally, bacteria were surrounded by a protective layer of fungi; bacterial cell mass was lowest at the silicone surface and highest adjacent to the fungal layer. As before, an antifungal was used during culture again with the result that the fungi in these communities remained uncharacterised. Resistance to antibacterials was also investigated. Forty three percent of isolates of the genera Staphylococcus, Enterococcus and the family Enterobacteriaceae

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possessed multi-drug resistance, as determined by RAPD profiling (Dautle et al. 2003). The available literature suggests that biofilms can form on PEG tubes in vivo. Such communities comprise a range of microorganisms including both fungi (primarily Candida spp.) and prokaryotes. Bacteria isolated from PEG tube biofilms were primarily facultative anaerobes of the family Enterobacteriaceae and the genera Enterococcus, Lactobacillus and Staphylococcus together with Bacillus and Pseudomonas spp. Almost half of the isolates were multi-drug resistant. However, much work remains to be done: spatial organisation of PEG tube biofilms is unknown as is the sequence of colonisation. Additionally, the effect of immune defence mechanisms of the gastrum (acid, nitrite) on PEG tube biofilm formation is unclear. Knowledge of all of these factors will be vital to the elucidation of appropriate interventions and/or preventions.

Probiotics and gastrointestinal biofilms Fermented milks and milk products have been in use since antiquity, though it was the Russian Nobel laureate Eli Metchkinoff who proposed in 1907 that the longevity of the Balkan people could be attributed to their ingestion of fermented milks. Probiotics are live microbial food supplements that change either the composition or metabolic activities of the microbiota or modulate immune system reactivity in a way that benefits health (Macfarlane & Cummings 2002). They are commercially available in the form of yoghurts, drinks and as capsule, powder or tablet supplements. The role of probiotic bacteria in intestinal biofilms is poorly understood. The indigenous microbiota of gastric and intestinal surfaces certainly contributes to the

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stability of GI ecosystem (Savage 1987). However, while the role of potential pathogens in the aetiology of infection has been extensively studied, there is a lack of information regarding mechanisms by which the indigenous microbiota establishes and maintains colonisation, probably due to the innate complexity of the intestinal ecosystem (Greene & Klaenhammer 1994). Nonetheless, it is generally recognised that for probiotic bacteria to exert an effect in the intestine they must be able to adhere, at least temporarily, to the intestinal mucosa. Several in vitro studies have shown that Lactobacillus strains can adhere to either HT-29 or CaCo-2 epithelial cell lines (Chauviere et al. 1992) while bifidobacteria adhere competently to intestinal mucus (He et al. 2001, Ouwehand et al. 1999). The adherence mechanisms of lactobacilli and bifidobacteria are unclear and the amount of adhesion also differs greatly between species (Tuomola & Salminen 1998). In earlier animal studies it has been suggested that concanavalin A receptors on some Lactobacillus spp. influenced their ability to attach to epithelial cells (Fuller 1975). In a more recent study the authors showed that L. acidophilus LA1 exhibited a strong calcium-independent adherence property and that adhesion of this strain to Caco-2 cells required a strong proteinaceous adhesion-promoting property (Bernet et al. 1994). When 13 strains of B. longum were tested for adhesion to both gastric and colonic cell lines, adhesion was found to be strongly related to autoaggregation ability and the authors classified the adherence capabilities of the strains according to this ability (Del Re et al. 2000). The adherence of bifidobacteria is thought to be species-specific and possibly mediated by a proteinaceous adhesion-promoting factor, rather than a calcium-dependent one (Bernet et al. 1993). Research on adherence of probiotic bacteria to in vitro colonic and gastric cell lines has provided useful data on the mechanisms of adherence, but are probably

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not indicative of in vivo situations; the beneficial effects of probiotics may result from competitive interactions with pathogenic and non-pathogenic organisms in the intestine and with the immune system.

Probiotics in inflammatory bowel disease Crohn's disease (CD) is a chronic IBD where inflammation involves full thickness of the intestinal wall and may affect any point along the GI tract (Guarner et al. 2002). Increasing research in the area of probiotics in either UC or CD indicates that there may be some therapeutic benefits of bacterial supplementation in these patients. Although most research on probiotics in IBD has been on the maintenance and remission of UC, Lactobacillus strain GG was shown to help promote the barrier function in children with CD and also improve symptoms (Gupta et al. 2000, Malin et al. 1996), though it had no effect in adult CD patients after colonic resection (Prantera et al. 2002). L. salivarius strain UCC118 was also shown to transit the GI tract of patients with CD (Dunne 2001) though actual adherence to the mucosa of these patients was not demonstrated. Probiotics have had success in the achievement and maintenance of remission in UC. Pouchitis is a frequent chronic complication which occurs after pouch surgery for UC and manifests itself as a non-specific inflammation of the ileal reservoir (Gionchetti et al. 2000). When 40 patients were randomised to receive either VSL#3 (a probiotic containing four strains of lactobacilli, three strains of bifidobacteria and one strain of Streptococcus salivarius subsp. thermophilus) or a placebo, 15% of patients in the probiotic group experienced relapses during the nine month follow-up period, compared to 100% in the placebo group (P<0.001). In the probiotic group only lactobacilli, bifidobacteria and S. thermophilus increased significantly from 14

baseline levels during feeding (P<0.01), indicating transition through the GI tract (Gionchetti et al. 2000). The same probiotic was also successful in the maintenance of remission of UC patients allergic to standard drug therapy (Venturi et al. 1999). Other probiotics, such as non-pathogenic E. coli strain Nissle 1917 and Saccharomyces boulardii, a yeast, have proved as successful as conventional drug treatment for UC and CD (Guslandi et al. 2000, Kruis et al. 1997). As described earlier levels of bifidobacteria and lactobacilli are decreased in CD and UC, so there is clearly a potential for their use in these chronic conditions. However, the mechanistic role of probiotic bacteria in competing for cell receptor sites and their integration in the indigenous luminal and adherent communities of these patients, is an area that requires further research in order to fully understand their beneficial effects. Probiotics in enteral feeding Enteral feeds are an excellent nutrient source for bacteria; such patients are nine times more likely to develop Clostridium difficile-related diarrhoea than matched nontube fed patient (Bliss et al. 1998). The role of probiotics in enteral feeding is, as yet, undefined though they have had success in the prevention of diarrhoea associated with enteral feeding (Bleichner et al. 1997, Whelan et al. 2001). These results have been verified by other studies where probiotics have had good success in the prevention or treatment of antibiotic-associated diarrhoea, as summarised in two recent meta-analyses (Cremonini et al. 2002, D'Souza et al. 2002). An area where probiotics may have an significant role in enteral feeding-related complications is in the formation of biofilms on the feeding tubes, as described earlier. To date, no studies involving probiotics have been published in this area, though lactobacilli have had some success in reducing yeast and bacteria

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prevalence in biofilms formed on silicone tubes in voice prostheses in vitro (Busscher et al. 1997, Free et al. 2001, van der Mei et al. 2000). It may be that the use of probiotics in biofilm formation on voice prostheses tubing could be applied to those on ETF tubes.

Concluding remarks Despite increasing interest in complex gastrointestinal biofilms the health significance of these complex communities is still largely unknown. Microbiological analysis of the rectal mucosa has demonstrated marked differences in UC patients when compared with healthy subjects indicating a possible role for specific genera, or groups of genera, in disease aetiology. Of several novel treatments for the management of UC and the complications of PEG tube feeding probiotics appear the most promising. The existing clinical data supports a role for probiotics in maintaining quiescent disease and pouchitis in remission; it is therefore likely that such microorganisms can indeed colonise the GI mucosa.

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