Enterococcal colonization of the gastro-intestinal tract: role of biofilm and environmental oligosaccharides
- Roberta Creti†1, 2,
- Stefanie Koch†1,
- Francesca Fabretti2, 3,
- Lucilla Baldassarri2 and
- Johannes Huebner1, 3Email author
© Creti et al; licensee BioMed Central Ltd. 2006
Received: 10 May 2006
Accepted: 11 July 2006
Published: 11 July 2006
Biofilm formation in E. faecalis is presumed to play an important role in a number of enterococcal infections. We have previously identified a genetic locus provisionally named bop that is involved in maltose metabolism and biofilm formation. A transposon insertion into the second gene of the locus (bopB) resulted in loss of biofilm formation, while the non-polar deletion of this gene, together with parts of the flanking genes (bopA and bopC) resulted in increased biofilm formation. A polar effect of the transposon insertion on a transcriptional regulator (bopD) was responsible for the reduced biofilm formation of the transposon mutant.
The amount of biofilm formed is related to the presence of maltose or glucose in the growth medium. While the wild-type strain was able to produce biofilm in medium containing either glucose or maltose, two mutants of this locus showed opposite effects. When grown in medium containing 1% glucose, the transposon mutant showed reduced biofilm formation (9%), while the deletion mutant produced more biofilm (110%) than the wild-type. When grown in medium containing 1% maltose, the transposon mutant was able to produce more biofilm than the wild-type strain (111%), while the deletion mutant did not produce biofilm (4%). Biofilm formation was not affected by the presence of several other sugar sources. In a gastrointestinal colonization model, the biofilm-negative mutant was delayed in colonization of the mouse intestinal tract.
The biofilm-positive phenotype of the wild-type strain seems to be associated with colonization of enterococci in the gut and the presence of oligosaccharides in food may influence biofilm formation and therefore colonization of enterococci in the gastrointestinal system.
Biofilm formation of the E. faecalis strains grown in glucose or maltose medium
Scanning electron microscopy
To obtain a comparable view of the biofilm composition, micrographs were taken from randomly chosen fields at the same magnification for each sample.
Biofilm formed in glucose and maltose appears to differ slightly: T9 cells grown in glucose presented small globular aggregates on the cell surface (Fig. 5a) that became larger and less regular when bacteria were grown in maltose (Fig. 5b). Larger aggregates where also visible on the surface of 10D5 grown in maltose (Fig. 5d) and on the deletion mutant grown in glucose (Fig 5e).
Extracellular material visible on scanning electron micrographs represents polysaccharides partially collapsed due to dehydration caused by processing for SEM. However, we always examined samples processed in a single run, to avoid biased observation caused by artifacts; the differential "collapse" of the extracellular material when bacteria were grown in glucose or maltose, suggests that the material does in fact differ.
Mouse colonization model
Enterococci are important inhabitants of the gastrointestinal tract of humans and many animals . While a number of colonization and adhesion factors have been studied, the ability of enterococci to effectively colonize the gut is not well understood. Several studies have investigated biofilm formation of enterococci, which is thought to be a multifactorial event [6, 8, 12–16].
Le Breton and colleagues identified the locus described by us previously  as being responsible for the uptake and metabolism of maltose . From these data and from our results it seems clear that bop D is likely to be a Maltose-sensitive negative regulatory protein that may repress both bopABC, and the divergently transcribed malT operon. Two insertional mutants were studied by Le Breton et al., one in the malT and one in bopA (named malP by Breton et al.). These results confirm the role of this locus in the utilization of maltose and corroborate our observations with regard to the growth curves in our mutants. E. faecalis TDM shows significantly decreased growth in medium containing maltose as the single sugar source compared with the other strains (T9 and 10D5). However, the ability to form biofilm seems to be independently reduced under these conditions because all the optical densities measured for the biofilm formation were normalized to take into account the different growth rates.
Trehalose and maltose are abundant disaccharides in nature and serve as important carbon and energy sources to lactic acid bacteria. Maltose is generated by enzyme-catalyzed hydrolysis of starch by amylases present in the gastrointestinal tract. In L. lactis the genes encoding maltose phosphorylase (MP), trehalose-6-phosphate phosphorylase (TrePP), and β-phosphoglucomutase are induced by maltose and trehalose, indicating that the trehalose and maltose catabolic pathways are closely connected [17, 18]. However, when our wild-type and mutants were grown in medium containing 1% trehalose, the effect on growth was different with respect to growth in maltose and no biofilm was formed (see Figure 4).
It has been observed that, in the hyperthermophilic bacterium T. maritima, at high growth rates, maltose consumption increased significantly, although it appeared that carbon was used in the formation of extracellular polysaccharide (EPS) rather than accumulation of biomass . The authors speculated that EPS formation could reflect the processing of excess carbon or, alternatively, could be coupled to a specific ecological strategy, such as biofilm formation.
It has been hypothesized that whenever bacteria are in non-optimal growth conditions (such as excess of carbon sources in the environment and/or altered sugar-metabolizing gene expression), the accumulation of reducing equivalents can be disposed of through the production of biofilm to transport these molecules out of the cell. In fact, it has been proposed that bacteria form exopolysaccharide matrices as a by-product to release reducing equivalents that could otherwise function as a bottleneck in the metabolism of an excess of the carbon source [19, 20].
The observed effects of reduced biofilm formation in the transposon mutant grown in glucose could be attributable to the lower expression of the BopD protein and its subsequent minor efficiency in carbon catabolite regulation.
On the other hand, lower expression of bopD results in derepression of the bopA gene (a maltose phosphorylase), and to a minor extent also of the upstream sugar transport gene malT, as confirmed by real-time PCR (data not shown), which in turn may lead to a higher efficiency in transport and maltose utilization and enhanced biofilm formation ability when bacteria are grown in maltose.
In the deletion mutant TDM, the transcription of the bopD gene seems to be somewhat enhanced with respect to the wild-type. This probably results in increased repression of the transcription of bopA compared to the wild-type and the transposon mutant 10D5, as confirmed by real-time PCR (data not shown). However, E. faecalis TDM lacks functional BopA, BopB (a phosphoglucomutase), and BopC (an aldose-1-epimerase). Consequently, when this strain is grown in BFM-M, it may not be able to use maltose to produce the extracellular macromolecules necessary for biofilm formation, while its ability to metabolize glucose may not be affected.
Moreover, when E. faecalis TDM is grown in glucose, the overexpression of bopD could increase alternative pathways for glucose metabolism and in turn lead to enhanced biofilm formation.
In the gastrointestinal colonization model, we could demonstrate that the biofilm-negative mutant was delayed in colonizing the mouse intestinal tract, although the levels achieved after 9 days were eventually as high as for the wild-type strain. However, since the transposon mutation also leads to different expression of other genes putatively involved in biofilm formation , these experiments cannot completely rule out pleiotropic effects that may be responsible for the observed differences. Using stringent decolonizing methods in this experiment that provide a very artificial "mono-organism" colonization, interactions of these strains with other organisms cannot be ruled out.
A mechanism that integrates the availability of certain carbohydrates into the signal transduction pathway regulating biofilm expression could be important for the ability of enterococci to colonize the gastro-intestinal system of many animals and humans. Biofilm formation could help the bacteria adhere to the gut wall and may represent an advantage for certain strains. The formation of a biofilm seems to be related to a multicellular architecture and copious amounts of extracellular macromolecules, as shown in the scanning electron micrographs. This organization depends on the presence of specific genes and specific carbohydrate pathways as well as specific oligosaccharides in the environment.
Although the functional role of the above-mentioned mechanism needs to be further elucidated, we speculate that the availability of starch and maltose in food and gastrointestinal contents may influence the expression of biofilm by enterococci and that this biofilm formation may enable these bacteria to colonize and persist in the gut.
Strains of Enterococcus faecalis used in the present study
strong BF in glucose or maltose
Tn917 insertion into bop B
no BF in glucose-containing medium
deletion of bop A, bop B, and bop C
strong BF in glucose-containing medium
The primary culture was diluted 1:10 into polystyrene tissue culture-treated microtiter plates (Corning, Corning, NY) and grown at 37°C for 18 h. Growth was measured spectrophotometrically (OD595 nm); the plates were emptied, washed three times with PBS, and dried at 60°C for 1 h. The biofilm was subsequently stained for 2 min with Hucker's crystal violet . The plates were washed thoroughly with tap water and dried, and the OD was measured with an ELISA reader at 595 nm. Biofilm formation was normalized to growth with the biofilm index (BFI), which was calculated as ODbiofilm × (0.5/ODgrowth) . Multigroup comparisons were made by ANOVA with Tukey's multiple comparison test using the Prism3 software package.
Scanning electron microscopy
For biofilm formation, inocula were prepared in BFM-M and BFM-G, which were used to inoculate 24-well tissue culture plates (Costar, Corning Inc., Corning, NY) containing segments of polystyrene and BFM-M or BFM-G. Biofilm formed on polystyrene pieces was fixed according to Fassell et al.  for best preservation of polysaccharides. Briefly, samples were pre-fixed for 20' with Na-cacodylate-1% glutaraldehyde, supplemented with lysine 75 mM and ruthenium red 0.075% (w/v). Samples were then treated with Na-cacodylate-1% glutaraldehyde supplemented with ruthenium red 0.075% (w/v) for 1 hour at room temperature, and OsO4 1% for 1 hour. Dehydration with graded series of ethanol solutions was followed by critical point drying, gold sputtering, and observation with a Cambridge SE 360 scanning electron microscope.
Mouse colonization with E. faecalis wild-type and mutant strains
Female BALB/c mice (Harlan-Sprague Dawley, Inc.) were kept in groups of 4 mice in cages with microisolator tops. The bedding (alpha chip, Northeastern products corporation, Warrensburg, NY), cages, and drinking bottles were autoclaved and changed every other day. Mice were fed irradiated mouse chow (PicoLab mouse diet 20, #5038, from PMI Nutrition International, Inc., Brentwood, MO). The drinking water, regular tap water, was autoclaved and supplemented with 1 g/l vancomycin hydrochloride (for intravenous use, Novaplus, Abbott Laboratories, North Chicago, IL), 100 mg/L metronidazole (Sigma), 1 g/L gentamicin (Sigma), and 1 g/L cefoxitin (Baxter Healthcare Corp., Syracuse NY). Mice were kept on this decolonizing antibiotic regimen for 10 days. Fecal pellets (1 per mouse) were collected every other day, weighed, homogenized in 750 μl Todd-Hewitt-Broth with 0.05% Tween, diluted, and plated on tryptic soy agar, bile esculin azide (PML microbiologicals, Wilsonville, OR), and McConkey plates to monitor decolonization of the intestinal flora.
After 10 days, mice were switched to drinking water supplemented with 125 mg/L cefoxitin and 100 mg/L metronidazole, as well as 5 × 107 cfu/ml bacteria from a fresh overnight culture. One group of eight mice received the wild-type strain T9; the other group of eight mice received the biofilm-negative transposon mutant 10D5. Both strains showed equal viability in water with the antibiotics mentioned above, which was tested by plating dilutions at days 0, 1, 2, and 3. The cages, water, and food were again changed every other day, and fecal pellets were collected, homogenized, diluted, and plated every or every other day. The colonization was documented as the number of bacteria per gram of stool per mouse.
We thank C. Theilacker and A. Koh for helpful discussions and D. Lawrie-Blum for editorial assistance. Financial support: National Institute of Allergy and Infectious Diseases (AI50667) to J.H.
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