dh

and Salmonella enterica serovar Typhimurium to intestinal mucus. Moreover, selected probiotic strains were used to study

An often necessary step in the infection process is

the adhesion of pathogenic bacteria to host tissues

ourable event mediated through the adhesion of

bacteria to various surfaces. A considerable amount

of research has been done to understand how bacteria

Journal of Microbiological Methods 60 (20whether probiotics or the adhesion method used affected the results. As a result, we show that the best reproducibility and

sensitivity were obtained using radioactive labelling. With other methods, the sensitivity was too low due to poorly adhering

bacteria and low signal-to-background ratio.

D 2004 Elsevier B.V. All rights reserved.

Keywords: Adhesion; Crystal violet; DAPI; EYFP; Fluorescence; GFP; Probiotic; Radioactively labelled

1. Introduction (Finlay and Falkow, 1997). Also the formation of

biofilms in industrial processes is mostly an unfav-of different methods

Satu Vesterlunda,*, Johanna Palttab, Matti Karpb, Arthur C. Ouwehanda

aDepartment of Biochemistry and Food Chemistry, University of Turku, Itainen Pitkakatu 4A, 20014 Turku, FinlandbDepartment of Biotechnology, University of Turku, Tykistokatu 6, 20014 Turku, Finland

Received 27 September 2004; accepted 27 September 2004

Abstract

The adhesion of bacteria to host tissue is the first step in pathogenesis. Similarly, bacterial adhesion to inanimate surfaces is

the first step in formation of biofilmsa real problem in industrial processes and medical devices. Various agents capable of

blocking the adhesion of bacteria to surfaces have been identified, such as probiotics, which are supposed to prevent the

adhesion of pathogenic bacteria to the intestinal mucosa. Although measurement of bacterial adhesion is important itself,

especially when agents used to prevent adhesion are developed, a relative small number of techniques can be used in the

measurement of adhesion. These techniques are not well validated and there is lack of studies where those methods are

compared to each other. Here we have compared different commonly used methods to measure adhesion of bacteria; radioactive

labelling, fluorescence tagging, and staining of bacteria. The methods were used to measure the adhesion of Escherichia coliMeasurement of bacterial a0167-7012/$ - see front matter D 2004 Elsevier B.V. All rights reserved.

doi:10.1016/j.mimet.2004.09.013

* Correspon

333 6860.

E-mail address: satu.vesterlund@utu.fi (S. Vesterlund).esionin vitro evaluation

05) 225233

www.elsevier.com/locate/jmicmethilarly different agents capableadhere to surfaces. Simding author. Tel.: +358 2 333 6823; fax: +358 2of blocking the adhesion of bacteria to certain

surfaces have been developed. Oral probiotics are

plates supplemented with 100 Ag ml ampicillin.Plates were grown for 16 h at 37 8C and stored for

robiolsuch agents that can interfere with the adhesion of

other microbes and have been defined as blivingmicroorganisms, which, upon ingestion in certain

numbers, exert health benefits beyond inherent basic

nutritionQ (Guarner and Schaafsma, 1998). Althoughadhesion is the cornerstone of pathogenesis, a relative

small number of techniques can be used to measure

the adhesion. Conventional methods used to enumer-

ate the adherent bacteria are based on plating or

microscopic counting. Plating is often used when the

amount of bacteria adhered to eukaryotic cells

(Bermudez et al., 1994) or inanimate surfaces

(Gristina et al., 1989; Sheehan et al., 2004) is

detected. However, this method is laborious and

requires that bacteria are released first and that

bacteria remain culturable after the release process.

Microscopic evaluation can be used after fixation and

Gram staining of the bacteria (Tuomola and Salminen,

1998), but the method is equally laborious as plating.

When the amount of released bacteria is sufficient for

turbidometric analysis, spectrophotometry can be

used (Styriak et al., 1999), but the sensitivity and

accuracy are maybe less compared to plating or

microscopic enumeration. These methods cannot be

used when adhesion of one bacterial strain is studied

in an environment where other bacteria are present. In

order to distinguish bacteria in a mixed population,

radiolabels (Ahearn et al., 2000; Jin et al., 1998;

Tuomola et al., 1999), fluorochromes (Bosch et al.,

2003; Drudy et al., 2001), or bacteria-specific anti-

bodies (Sanchez et al., 1993) can be used. Radiolabels

are regarded as undesirable due to safety and cost

concerns. Fluorochromes are used to replace radio-

labels, but they may alter the surface properties of

bacteria or affect the viability of bacteria (Fuller et al.,

2000). Bacteria-specific antibodies can be used, but

the availability can cause problems as well as cross-

reactions.

There is a lack of studies where different adhesion

methods are compared to each other. Since detection

of bacterial adhesion is important (e.g., in the

development of probiotics), much more studies

should be done where different methods are eval-

uated. In this study, we have compared commonly

used methods to measure the adhesion of bacteria.

The methods were based on radiolabelling, fluores-

S. Vesterlund et al. / Journal of Mic226cent tagging (measured by microscopy or fluore-

scence), and staining (with crystal violet and DAPI).not more than 2 weeks in 4 8C. For culturing, onecolony was inoculated into 5 ml of LB broth

supplemented with 100 Ag ml1 ampicillin, and theculture was grown for 16 h without agitation at 30 8Cto reach stationary growth phase. LcS and LGG were

inoculated directly from glycerol stocks as a 0.5%

inoculum into de Man, Rogosa, and Sharpe (MRS)

broth (Oxoid, Basingstoke, UK). Bacteria were

grown for 1820 h without agitation at 37 8C underThese methods are used to detect the adhesion of

Escherichia coli and Salmonella enterica serovar

Typhimurium bacteria to intestinal mucus. Moreover,

we studied whether the selected commercial pro-

biotic strains, Lactobacillus casei Shirota (hereafter

LcS) and Lactobacillus rhamnosus GG (hereafter

LGG) affected the adhesion of pathogenic bacteria,

and, more importantly, whether the adhesion method

used affected the results.

2. Materials and methods

2.1. Plasmid constructs of transformed strains

Plasmids containing genes for fluorescent proteins

(GFPmut2 and EYFP; Clontech, Palo Alto, CA) were

transformed into E. coli and S. enterica serovar

Typhimurium, respectively, by electroporation (Dower

et al., 1988) and selection for ampicillin (100 Ag ml1)resistance.

2.2. Bacterial strains and culture conditions

Bacterial strains used were E. coli MC1061,

GFPmut2-tagged variant from E. coli MC1061, S.

enterica serovar Typhimurium ATCC 14028 and

EYFP-tagged variant of this strain. Probiotic strains

used were lactic acid bacteria (LAB): L. casei

Shirota (isolated from a YakultR product; LcS) andL. rhamnosus GG (ATCC 53103; LGG). All strains

were stored at 86 8C in 40% glycerol. E. coli andS. enterica serovar Typhimurium were plated first

onto LuriaBertani (LB; yeast extract and tryptone

were purchased from Pronadisa, Madrid, Spain)1

ogical Methods 60 (2005) 225233anaerobic conditions in order to reach the late

logarithmic growth phase. Bacteria were harvested

robiolby centrifugation (1500g, 7 min) and washed twicewith phosphate-buffered saline (PBS; pH 7.2). The

optical density of bacterial suspensions at 600 nm

was adjusted with PBS to 0.5F0.02, giving approx-imately 4108 CFU ml1 for E. coli and S. entericaserovar Typhimurium strains and 12108 CFUml1 for LcS and LGG.

2.3. Human intestinal mucus

Human intestinal tissue was used as a source of

mucus. The use of resected human intestinal tissue

was approved by the joint ethical committee of the

University of Turku and Turku University Central

Hospital and informed written consent was obtained

from the patients. The mucus was isolated from the

healthy part of tissue obtained from a patient with

diverticulitis. In short, resected material was col-

lected on ice within 20 min and processed immedi-

ately by washing gently with PBS containing 0.01%

gelatin. Mucus was collected into a small amount of

HEPESHanks buffer (10 mmol l1 HEPES; pH7.4) by gently scraping with a rubber spatula and

centrifuged (13,000g, 10 min) in order to removecell debris and bacteria. After measurement of the

protein content, mucus was stored at 20 8C. Thesame stock of mucus was used in all experiments in

order to avoid the possible effect of variations in the

mucus on adhesion. In adhesion assays, mucus was

diluted to a protein concentration of 0.5 mg ml1

with HEPESHanks and 100 or 50 Al of thissolution was immobilized passively into microtiter

plate wells (Maxisorp; Nunc, Denmark) or into

microscope slides, respectively, by overnight incu-

bation at 4 8C.

2.4. In vitro adhesion assays

2.4.1. Adhesion of radioactively labelled bacteria

When the original and fluorescent strains of E.

coli and S. enterica serovar Typhimurium were

inoculated as described above, 10 Al ml1 [5V-3H]thymidine (16.7 Ci mmol1) was added to thecultures to metabolically radiolabel the bacteria.

After growth, washing, and adjustment of optical

density at 600 nm to 0.5, bacteria were added as a

S. Vesterlund et al. / Journal of Micvolume of 100 Al into microtiter plate wells coatedwith human intestinal mucus. Bacteria were allowedto adhere at 37 8C for 1 h and the wells werewashed three times with 250 Al of HEPESHanksbuffer to remove the nonadherent bacteria. The

bacteria bound to mucus were released and lysed

with 1% SDS0.1 M NaOH by incubation at 60 8C.The radioactivity of the suspension was measured by

liquid scintillation. Three parallel wells were used in

each experiment. The adhesion ratio (%) of bacteria

was calculated by comparing the radioactivity of the

adhered bacteria to the radioactivity of the added

bacteria.

2.4.2. Adhesion of fluorescent-tagged bacteria

measurement by fluorometer

After adjustment of optical density at 600 nm to

0.5, GFPmut2/E. coli and EYFP/S. enterica serovar

Typhimurium were added as a volume of 100 Al intomicrotiter plate wells coated with mucus. After incu-

bation and washing as described in Section 2.4.1, the

wells were covered with 100 Al of HEPESHanksbuffer to prevent drying of the bacteria. Fluorescence

of the bacteria was measured by using a Victor2 1420

Multilabel counter (PerkinElmer, Turku, Finland).

The filters used were 485 nm filter for excitation

and 535 nm filter for emission. The sensitivity of the

measurement was increased by measuring 12 different

points (the diameter of one point was 4 mm) from one

well. Fluorescence of immobilized mucus covered

with 100 Al of HEPESHanks buffer was used as abackground in measurements, and it was reduced

from the fluorescence of the samples. Three parallel

wells were used in each experiment. The adhesion

ratio (%) of bacteria was calculated by comparing the

fluorescence of the adhered bacteria to the fluores-

cence of the added bacteria.

2.4.3. Adhesion of fluorescent-tagged bacteria

measurement by microscopic counting

In microscopic analysis, the fluorescent E. coli

MC1061 and S. enterica serovar Typhimurium were

added as a volume of 50 Al into microscope slidescovered with mucus (Krovacek et al., 1987). Micro-

scope slides were incubated in humidified chamber

at 37 8C for 1 h and the nonadherent bacteria werewashed away by dipping the slides into 0.9% NaCl

solution. The slides were covered with a coverslip

ogical Methods 60 (2005) 225233 227and stored in humidified chamber until enumeration

of adherent bacteria with epifluorescence microscope

ously), and displacement (pathogenic bacteria were

incubated first with the mucus, washed away, and

robiol(Olympus BX51, Japan). Adhered bacteria in 20

randomly selected fields were enumerated.

2.4.4. Detection of bacterial adhesion with crystal

violet

The crystal violet method was modified after

Styriak et al. (1999). In short, E. coli and S.

enterica serovar Typhimurium (nonfluorescent and

fluorescent) bacteria were added as a volume of 100

Al into microtiter plate wells coated with 150 Al ofhuman intestinal mucus. The bigger volume of

mucus compared to the volume of added bacteria

was used to avoid contact of the stain with the

polystyrene. Bacteria were adhered at 37 8C for 1 hand the nonadherent bacteria were removed by

washing the wells three times with 250 Al ofPBS. The adherent bacteria were fixed at 60 8Cfor 20 min and stained with crystal violet (100 Alwell1, 0.1% solution) for 45 min. Wells weresubsequently washed five times with PBS to remove

excess stain. The stain bound to bacteria was

released by adding 100 Al of citrate buffer (20mmol l1; pH 4.3). After 45-min incubation at roomtemperature, the absorbance values at 570 nm were

determined by using Victor2 1420 Multilabel coun-

ter (PerkinElmer). Stained mucus without added

bacteria was used as negative control and the

absorbance value of this negative control was

subtracted from the absorbance value of the

samples. Four parallel wells were used in two

independent experiments.

2.4.5. Detection of bacterial adhesion with 4V,6-diamidino-2-phenylindole (DAPI)

PBS-washed E. coli and S. enterica serovar

Typhimurium bacteria were stained with DAPI by

adding the stain as a final concentration of 0.2 Agml1. Bacteria were incubated for 30 min with mildshaking at room temperature and washed three times

with PBS. Adhesion assay was done as described in

Sections 2.4.1 and 2.4.2. Then, wells were washed

with 250 Al of HEPESHanks buffer and thefluorescence from the wells was determined by using

Victor2 1420 Multilabel counter (PerkinElmer). The

filters used were 355 nm filter for excitation and 460

nm filter for emission. Sensitivity of the measurement

S. Vesterlund et al. / Journal of Mic228was increased, as was done in the fluorescence study,

by measuring 12 different points (the diameter of onefollowed by incubation with LAB) assays. The effect

of LAB on the adhesion of E. coli and S. enterica

serovar Typhimurium was assessed with different

methods (Sections 2.4.1, 2.4.2, and 2.4.3) in order to

study whether the method used had effects on the

results.

2.6. Statistical analysis

Results shown from Sections 2.4.1, 2.4.2, and 2.4.3

are the averageFstandard deviation (S.D.) of fourindependent experiments. Students t test was used to

determine the significant difference (Pb0.05) betweenthe samples.

3. Results

3.1. Sensitivity of the methods

Sensitivity of the methods was determined by

measuring the signal obtained from a single bacte-

rium. In radiolabelling, the lowest amount of

detectable bacteria after background subtraction was

2.7103 CFU for both GFPmut2/E.coli MC1061 andEYFP/S. enterica serovar Typhimurium (Fig. 1). In

the fluorescence method, the lowest detectable signal

after background subtraction was obtained from

6.4104 CFU of GFPmut2/E. coli and 1.1105CFU of EYFP/S. enterica serovar Typhimuriumpoint was 4 mm) from one well. The staining

efficiency of the bacteria was determined microscopi-

cally (Section 2.4.3). Four parallel wells were used in

three independent experiments. The adhesion ratio

was calculated as described in Section 2.4.2.

2.5. Effect of LAB on adhesion ability of pathogens

The effect of LcS and LGG on the adhesion

ability of E. coli MC1061 and S. enterica serovar

Typhimurium was assessed in exclusion (LAB was

incubated first with the mucus, washed away, and

followed by incubation with pathogenic bacteria),

competition (bacteria were incubated simultane-

ogical Methods 60 (2005) 225233bacteria (Fig. 2). Staining with crystal violet (Section

2.4.4) was not a sensitive-enough method to detect

Fig. 1. Linear relationship between radioactivity and CFU of GFPmut2/E. coli and EYFP/S. typhimurium bacteria. Results shown are

S. Vesterlund et al. / Journal of Microbiological Methods 60 (2005) 225233 229low levels of adherent bacteria as the signal was not

different from the background; absorbance values at

570 nm were between 0 and 0.05 in two independent

experiments. Similarly with DAPI staining (Section

2.4.5), the fluorescence obtained from the sample was

the same or close to the background. This was the

reason for high S.D. values in the experiments;

adhesion of E. coli was 2.80F3.04% and S. entericaserovar Typhimurium was 0.41F0.72%.

3.2. Effect of LAB on adhesion ability of pathogens

When radiolabelling (Section 2.4.1) was used, the

averageFS.D. of three samples.adhesion percentages of E. coli, GFPmut2/E. coli, S.

Fig. 2. Linear relationship between fluorescence and CFU of GFPmut

averageFS.D. of three samples.enterica serovar Typhimurium, and EYFP/S. enterica

serovar Typhimurium bacteria were 0.70, 0.60, 0.50,

and 0.85, respectively (Table 1). Displacement with

LcS significantly reduced the binding (%) of E. coli

and S. enterica serovar Typhimurium to 0.41

(P=0.006) and 0.34 (P=0.013), respectively. Simi-

larly, displacement with LGG significantly reduced

the binding (%) of E. coli, S. enterica serovar

Typhimurium, and EYFP/S. enterica serovar Typhi-

murium to 0.49 (P=0.041), 0.27 (P=0.020), and 0.27

(P=0.032), respectively. Also with other bacteria, LcS

and LGG caused a trend for lowered adhesion in

displacement, but this did not reach statisticalsignificance: LcS and GFPmut2/E. coli (P=0.310),

2/E. coli and EYFP/S. typhimurium bacteria. Results shown are

number of adherent bacteria was almost always

significantly lower when compared to the radiolabel-

ling method (Tables 3 and 4). In the sample where

LcS and GFPmut2/E. coli were incubated together

(competition), a trend for a reduction in adhesion was

Table 1

Adhesion assay using radioactively labelled bacteriaeffect of L. casei Shirota (LcS) and L. rhamnosus GG (LGG) on adhesion of pathogens

Assay E. coli GFPmut2/

E. coli

S. typhimurium EYFP/

S. typhimurium

Alone 0.70F0.19 0.60F0.17 0.50F0.18 0.85F0.32Exclusion by LcS 0.47F0.09 0.45F0.05 0.71F0.48 0.68F0.09Exclusion by LGG 0.78F0.52 0.62F0.12 0.44F0.11 0.64F0.29Competition by LcS 0.44F0.12 0.70F0.45 0.74F0.32 0.67F0.26Competition by LGG 0.57F0.13 0.50F0.16 1.69F1.42 0.95F0.22Displacement by LcS 0.41F0.15a 0.47F0.06 0.34F0.18a 0.62F0.37Displacement by LGG 0.49F0.17a 0.40F0.07 0.27F0.10a 0.27F0.09a

Adhesion (%); meanFS.D. of four independent experiments.a Significantly lower than bacteria incubated alone with the mucus ( Pb0.05).

S. Vesterlund et al. / Journal of Microbiological Methods 60 (2005) 225233230LGG and GFPmut2/E. coli (P=0.064), as well as LcS

and EYFP/S. enterica serovar Typhimurium (P=

0.398). It was also important for the use of fluorescent

bacteria that the fluorescent phenotype did not affect

the adhesion of bacteria (Table 1).

Use of fluorescent bacteria and fluorometer (Sec-

tion 2.4.2) gave lower binding (%) for GFPmut2/E.

coli and EYFP/S. enterica serovar Typhimurium (0.14

and 0.07, respectively) when compared to radio-

labelling (Table 2). The S.D. values were high due

to relative low reproducibility, use of poorly adherent

bacteria, and probably also light scattering and

autofluorescence of the mucus. Thus, LcS and LGG

did not statistically affect the adhesion ability of the

pathogens.

In order to compare the microscopic method

(Section 2.4.3) to other methods, the results from

Sections 2.4.1, 2.4.2, and 2.4.3 were represented as a

number of adherent bacteria per area of the mucus

(mm2). With fluorescent bacteria and fluorometer, theTable 2

Adhesion assay using fluorescent-tagged bacteriameasurement by

fluorometer and effect of L. casei Shirota (LcS) and L. rhamnosus

GG (LGG) on adhesion of pathogens

Assay GFPmut2/

E. coli

EYFP/

S. typhimurium

Alone 0.14F0.11 0.07F0.02Exclusion by LcS 0.09F0.10 0.07F0.07Exclusion by LGG 0.12F0.12 0.16F0.12Competition by LcS 0.21F0.06 0.06F0.05Competition by LGG 0.20F0.07 0.07F0.03Displacement by LcS 0.14F0.03 0.05F0.04Displacement by LGG 0.19F0.05 0.07F0.03

Adhesion (%); meanFS.D. of four independent experiments.observed (P=0.076). Using fluorescent bacteria and

microscopy, the number of bacteria observed was

often higher when compared to radiolabelling,

although statistical significance was obtained only in

competition of YFP/S. enterica serovar Typhimurium

by LGG (P=0.007; Tables 3 and 4). Similarly, when

microscopy was compared to fluorometry, signifi-

cantly higher numbers of bacteria were obtained with

microscopy: exclusion of GFPmut2/E. coli by LGG

Table 3

Adhesion of GFPmut2/E. coli per surface area (mm2)

Assay Radiolabelled Fluorescent

fluorometry

Fluorescent

microscopyAlone 6.26F1.72 1.46F1.13a 22.00F24.03Exclusion by LcS 4.65F0.57 0.89F1.05b 9.63F10.93Exclusion by LGG 6.48F1.26 1.22F1.29a 4.75F2.26c

Competition by LcS 7.23F4.69 2.16F0.65 3.50F2.16Competition by LGG 5.24F1.70 2.08F0.74a 39.88F24.03Displacement by LcS 4.88F0.57 1.47F0.27b 10.50F6.72c

Displacement by LGG 4.11F0.68 2.01F0.55a 8.38F7.94

Measurement with different methods and effect of L. casei Shirota

(LcS) and L. rhamnosus GG (LGG) on adhesion of pathogens.

Results shown are 103 of adherent bacteria per square millimeter;meanFS.D. of four independent experiments.

a Significantly lower than the result obtained with radiolabel-

ling ( Pb0.05).b Significantly lower than the result obtained with radiolabel-

ling ( Pb0.001).c Significantly higher than the result obtained with fluorometry

( Pb0.05).

c Significantly higher than the result obtained with radio-

robiollabelling ( Pb0.05).d Significantly lower than the result obtained with radiolabel-

ling ( Pb0.001).e Significantly higher than the result obtained with fluorometry

( Pb0.001).Table 4

Adhesion of EYFP/S. typhimurium per surface area (mm2)

Assay Radiolabelled Fluorescent

fluorometry

Fluorescent

microscopy

Alone 8.78F3.36 0.71F0.25a 23.88F10.21b,c

Exclusion by LcS 7.09F0.96 0.73F0.71d 15.00F6.65b

Exclusion by LGG 6.62F2.98 1.61F1.27a 19.25F11.87b

Competition by LcS 7.00F2.74 0.66F0.51a 20.63F16.19b

Competition by LGG 9.89F2.34 0.68F0.31d 30.13F9.78c,e

Displacement by LcS 6.41F3.80 0.51F0.40a 14.75F12.84Displacement by LGG 2.81F0.90 0.68F0.30a 26.88F29.71

Measurement with different methods and effect of L. casei Shirota

(LcS) and L. rhamnosus GG (LGG) on adhesion of pathogens.

Results shown are 103 of adherent bacteria per square millimeter;meanFS.D. of four independent experiments.

a Significantly lower than the result obtained with radiolabel-

ling ( Pb0.05).b Significantly higher than the result obtained with fluorometry

( Pb0.05).

S. Vesterlund et al. / Journal of Mic(P=0.035), displacement of GFPmut2/E. coli by LcS

(P=0.036), exclusion of EYFP/S. typhimurium by

LcS (P=0.005) and LGG (P=0.025), as well as

competition of YFP/S. typhimurium by LcS

(P=0.049) and LGG (P=0.001) (Tables 3 and 4).

4. Discussion

Bacterial adhesion is one of the main concerns in

the areas of medicine, industry and environment. In

many cases, bacterial adhesion is unwanted as it can

lead to infection or interruption of the industrial

processes. However, bacterial adhesion can also be an

advantage as in the case when probiotics are used to

promote intestinal health (Mattila-Sandholm et al.,

1999). As bacterial adhesion is involved in many

sectors of life and health, the development of methods

to measure adhesion is an important area.

The adhesion method used is often selected on the

basis of what people are accustomed to use. As

adhesion of bacteria is thought to be a complex

interplay between bacteria and surface, the adhesion

method used could affect the results. The initial step in

the adhesion process is mainly a physicochemicalprocess, based on nonspecific interactions (i.e.,

repulsive electrostatic and attractive van der Waals

interactions) (van Loosdrecht et al., 1990). This kind

of adhesion can be reversible and, therefore, the

number of washings used in adhesion assays should

remain constant between experiments. Initial adhesion

is followed by firm attachment where adhesins on the

bacterial cell surface recognize receptors on the target

surface (Miron et al., 2001). As the stain used could

change the surface properties of the bacterial cell, for

example, by affecting to the hydrophobicity of the cell

(Olofsson et al., 1998), the stain may affect the

adhesion of bacteria. Adhesion of microbes has also

been found to be increased during exponential growth,

possibly as a result of increased cell wall hydro-

phobicity (van Loosdrecht et al., 1990). Thus, when

adhesion properties of different strains are compared,

the growth phase of microbes should be the same in

order to reach comparable results (Blum et al., 1999).

A limited number of methods used to measure

bacterial adhesion are commonly used. The conven-

tional methods used are enumeration by plating or

microscopy. However, plating is laborious and

insensitive as it requires selective media, and the

method does not detect cells that are viable but not

culturable (Rahman et al., 1994; Steinert et al., 1997)

or which died during the release process. Similarly

enumeration by microscopy after staining of the

sample is laborious as it requires counting of many

fields and may also be prone to observer error.

Furthermore, these methods cannot be used when

adhesion of bacteria is studied in a mixed popula-

tion. In these kind of applications, fluorochromes can

be used. Although these are usually developed to

stain eukaryotic cells, many fluorochromes are also

suitable for prokaryotic use. Use of genetically

modified, fluorescent-tagged bacteria is preferred

over fluorescent stains as they make the experiments

shorter, do not stain other bacteria when mixed

populations are studied, and, above all, the signal

propagates with the dividing bacteria, avoiding the

dilution of the signal. Green fluorescent protein

(GFP) is the most widely used fluorescent marker.

The popularity of the GFP is due to its properties:

heat stability (up to 65 8C), pH stability (pH 7 to11), resistance to denaturants and proteases, and no

ogical Methods 60 (2005) 225233 231requirement of added cofactors for fluorescence

(Aspiras et al., 2000), as well as its expression in a

Ahearn, D.G., Grace, D.T., Jennings, M.J., Borazjani, R.N., Boles,

a species-specific marker in coadhesion with Streptococcus

oralis 34 in saliva-conditioned biofilms in vitro. Appl. Environ.

robiolwide variety of bacterial species (Valdivia et al.,

1998). However, certain difficulties can occur when

wild-type GFP is used. The fluorescence intensity or

folding can be too low, or protein is found in

nonfluorescent inclusion bodies. Thus, the properties

of GFP have been improved by mutagenesis in order

to overcome these problems. Here we used GFPmut2

variant, which has been shown to fluoresce at

approximately 100-fold higher intensity and to be

more soluble compared to wild type (Cormack et al.,

1996). Similarly, for Salmonella, another improved

GFP variant, EYFP, was used. Both fluorochromes

were bright under microscope and showed 100%

labelling, and no photobleaching was observed

during experiments. The fluorescent phenotype was

not observed to affect the growth of the bacteria,

indicating that the expression levels did not pose any

metabolic burden on bacteria (results not shown).

However, the sensitivity of the method where

fluorescence was detected by fluorometry did not

reach the sensitivity that was obtained with radio-

labelled bacteria. The signal obtained from radio-

labelled bacteria was approximately two to four log

units higher than the signal obtained with fluoro-

metery (Figs. 1 and 2). Although the fluorescence

intensity of bacteria could be improved by increasing

the gene copy number or using a stronger promoter,

the higher level of fluorescent protein may interfere

with the adhesion properties of bacteria (Wendland

and Bumann, 2002). Longer maturation of the

protein was not a solution for low fluorescence

either; when bacteria were kept overnight at 4 8C,the fluorescence was at the same level, indicating

proper maturation of the protein. When the fluores-

cent bacteria were enumerated by microscope, the

S.D. was high due to low adhesion of bacteria (010

bacteria per field). Thus, the use of fluorescent-

tagged bacteria is probably not a suitable method

when poorly adherent bacteria (b1%) are studied.Similarly, the use of common stains, fluorescent

DAPI, and crystal violet did not work with poorly

adherent bacteria. In these methods, also the use of

mixed bacterial population (i.e., here the intact

bacterial microbiota present in the mucus) made

the methods more insensitive as the microbiota was

probably also stained.

S. Vesterlund et al. / Journal of Mic232In summary, the use of radioactive labels in

bacterial adhesion assays offers the best reproduci-Microbiol. 66, 40744083.

Bermudez, L.E., Young, L.S., Inderlied, C.B., 1994. Rifabutin and

sparfloxacin but not azithromycin inhibit binding of Mycobac-

terium avium complex to HT-29 intestinal mucosal cells.

Antimicrob. Agents Chemother. 38, 12001202.

Blum, S., Reniero, R., Schiffrin, E.J., Crittenden, R., Mattila-

Sandholm, T., von Wright, A., Saarela, M., Saxelin, M.,

Collins, K., Morelli, L., 1999. Adhesion studies for probiotics:

need for validation and refinement. Trends Food Sci. Technol.

10, 405410.

Bosch, J.A., Veerman, E.C., Turkenburg, M., Hartog, K.,

Bolscher, J.G., Nieuw Amerongen, A.V., 2003. A rapid

solid-phase fluorimetric assay for measuring bacterial adher-

ence, using DNA-binding stains. J. Microbiol. Methods 53,

5156.

Cormack, B.P., Valdivia, R.H., Falkow, S., 1996. FACS-optimizedK.J., Rose, L.J., Simmons, R.B., Ahanotu, E.N., 2000. Effects

of hydrogel/silver coatings on in vitro adhesion to catheters of

bacteria associated with urinary tract infections. Curr. Microbiol.

41, 120125.

Aspiras, M.B., Kazmerzak, K.M., Kolenbrander, P.E., McNab, R.,

Hardegen, N., Jenkinson, H.F., 2000. Expression of green

fluorescent protein in Streptococcus gordonii DL1 and its use asbility and sensitivity when poorly adherent bacteria

(b1%) are studied. The use of fluorescent-taggedbacteria enables also easy and reproducible enumer-

ation of adherent bacteria, but the sensitivity is low

for poorly adherent bacteria. This is due to high

signal-to-background noise especially when the

adhesion surface is autofluorescent. Because with

radioactive labels the safety issues are the main

disadvantage, and considering the advantages and

potential of fluorescent-tagged bacteria, more studies

are needed to increase the sensitivity of bacterial

adhesion methods based on the use of such tagged

bacteria.

Acknowledgements

Financial support was obtained from the Academy

of Finland (grant no. 53758), the Danisco Foundation,

and the Paulo Foundation.

References

ogical Methods 60 (2005) 225233mutants of the green fluorescent protein (GFP). Gene 173,

3338.

Dower, W.J., Miller, J.F., Ragsdale, C.W., 1988. High efficiency

transformation of E. coli by high voltage electroporation.

Nucleic Acids Res. 16, 61276145.

Drudy, D., ODonoghue, D.P., Baird, A., Fenelon, L., OFarrelly,

C., 2001. Flow cytometric analysis of Clostridium difficile

adherence to human intestinal epithelial cells. J. Med. Micro-

biol. 50, 526534.

Finlay, B.B., Falkow, S., 1997. Common themes in microbial patho-

genicity revisited. Microbiol. Mol. Biol. Rev. 61, 136169.

Fuller, M.E., Streger, S.H., Rothmel, R.K., Mailloux, B.J., Hall,

J.A., Onstott, T.C., Fredrickson, J.K., Balkwill, D.L., DeFlaun,

M.F., 2000. Development of a vital fluorescent staining method

for monitoring bacterial transport in subsurface environments.

Appl. Environ. Microbiol. 66, 44864496.

Gristina, A.G., Jennings, R.A., Naylor, P.T., Myrvik, Q.N., Webb,

L.X., 1989. Comparative in vitro antibiotic resistance of surface-

colonizing coagulase-negative staphylococci. Antimicrob.

Rahman, I., Shahamat, M., Kirchman, P.A., Russek-Cohen, E.,

Colwell, R.R., 1994. Methionine uptake and cytopathogenicity

of viable but nonculturable Shigella dysenteriae type 1. Appl.

Environ. Microbiol. 60, 35733578.

Sanchez, R., Kanarek, L., Koninkx, J., Hendriks, H., Lintermans, P.,

Bertels, A., Charlier, G., Van Driessche, E., 1993. Inhibition of

adhesion of enterotoxigenic Escherichia coli cells expressing

F17 fimbriae to small intestinal mucus and brush-border

membranes of young calves. Microb. Pathog. 15, 207219.

Sheehan, E., McKenna, J., Mulhall, K.J., Marks, P., McCormack,

D., 2004. Adhesion of Staphylococcus to orthopaedic metals, an

in vivo study. J. Orthop. Res. 22, 3943.

Steinert, M., Emody, L., Amann, R., Hacker, J., 1997. Resuscitation

of viable but nonculturable Legionella pneumophila Philadel-

phia JR32 by Acanthamoeba castellanii. Appl. Environ. Micro-

biol. 63, 20472053.

Styriak, I., Demeckova, V., Nemcova, R., 1999. Collagen (Cn-I)

binding by gut lactobacilli. Berl. Mqnch. Tier7rztl. Wochenschr.

S. Vesterlund et al. / Journal of Microbiological Methods 60 (2005) 225233 233Guarner, F., Schaafsma, G.J., 1998. Probiotics. Int. J. Food

Microbiol. 39, 237238.

Jin, L.Z., Baidoo, S.K., Marquardt, R.R., Frohlich, A.A., 1998. In

vitro inhibition of adhesion of enterotoxigenic Escherichia coli

K88 to piglet intestinal mucus by egg-yolk antibodies. FEMS

Immunol. Med. Microbiol. 21, 313321.

Krovacek, K., Ahmed, F., Ahne, W., M3nsson, I., 1987. Adhesionof Aeromonas hydrophila and Vibrium angillarum to fish cells

and to mucus-coated glass slides. FEMS Microbiol. Lett. 42,

8589.

Mattila-Sandholm, T., M7ttf, J., Saarela, M., 1999. Lactic acidbacteria with health claimsinteractions and interference with

gastrointestinal flora. Int. Dairy J. 9, 2535.

Miron, J., Ben-Ghedalia, D., Morrison, M., 2001. Invited review:

adhesion mechanisms of rumen cellulolytic bacteria. J. Dairy

Sci. 84, 12941309.

Olofsson, A.C., Zita, A., Hermansson, M., 1998. Floc stability and

adhesion of green-fluorescent-protein-marked bacteria to flocs

in activated sludge. Microbiology 144 (Part 2), 519528.112, 301304.

Tuomola, E.M., Salminen, S.J., 1998. Adhesion of some probiotic

and dairy Lactobacillus strains to Caco-2 cell cultures. Int. J.

Food Microbiol. 41, 4551.

Tuomola, E.M., Ouwehand, A.C., Salminen, S.J., 1999. The

effect of probiotic bacteria on the adhesion of pathogens to

human intestinal mucus. FEMS Immunol. Med. Microbiol. 26,

137142.

Valdivia, R.H., Cormack, B.P., Falkow, S., 1998. The uses of

green fluorescent protein in prokaryotes. In: Chalfie, M., Kain,

S. (Eds.), Green Fluorescent Protein: Properties, Applications,

and Protocols. John Wiley & Sons, Ltd., Chichester, England,

pp. 121138.

van Loosdrecht, M.C., Lyklema, J., Norde, W., Zehnder, A.J., 1990.

Influence of interfaces on microbial activity. Microbiol. Rev. 54,

7587.

Wendland, M., Bumann, D., 2002. Optimization of GFP levels for

analyzing Salmonella gene expression during an infection.

FEBS Lett. 521, 105108.Agents Chemother. 33, 813816.

Measurement of bacterial adhesion-in vitro evaluation of different methodsIntroductionMaterials and methodsPlasmid constructs of transformed strainsBacterial strains and culture conditionsHuman intestinal mucusIn vitro adhesion assaysAdhesion of radioactively labelled bacteriaAdhesion of fluorescent-tagged bacteria-measurement by fluorometerAdhesion of fluorescent-tagged bacteria-measurement by microscopic countingDetection of bacterial adhesion with crystal violetDetection of bacterial adhesion with 4,6-diamidino-2-phenylindole (DAPI)

Effect of LAB on adhesion ability of pathogensStatistical analysis

ResultsSensitivity of the methodsEffect of LAB on adhesion ability of pathogens

DiscussionAcknowledgementsReferences