2 Bacteria UC Probiotic

8/2/2019 2 Bacteria UC Probiotic

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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 andLactobacillus 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.


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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 host-

microbiota 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

Clostridiumdifficile (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


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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


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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 (Croucheret 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


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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 usingan 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


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0

1

2

3

4

5

Log10bacteria(cm

2biopsymaterial)-1

U Healthy

Strict anaerobes

Facultative species

Figure 2 Comparison of strictly anaerobic (dark bars) bacterial populations andfacultative anaerobes (light bars) on the rectal mucosa in healthy subjects and UCpatients. Results show means (n=3-5) SEM.

surface is in broad agreement with data obtained from colonic tissue at autopsy

(Croucheret al. 1983), but differs from the results of Poxton etal. (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


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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.


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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 rangesand means (vertical bars). Values in parentheses indicate the number of species andstrains in each bacterial group or genus, those in italics show the number ofindividuals 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


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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 statusquo 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 asrgenes (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


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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 forE.

coli, Staphylococcusaureus 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 ofB. 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 ofin 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 (Guarneret 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 ofStreptococcus 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


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baseline levels during feeding (P


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prevalence in biofilms formed on silicone tubes in voice prostheses in vitro

(Busscheret 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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2 Bacteria UC Probiotic