2 bacteria uc probiotic
TRANSCRIPT
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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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