2
The initial microbial adhesion of bacteria to different surfaces seems to be mediated by physico-chemical forces and this is the reason why the physico-chemical surface characterisation of bacteria has recently gained interest.
Type: Motivation |
Advantage: None |
Novelty: None |
ConceptID: Mot1
3
In this context, the adhesion of different microorganisms to biological substrata has been described from a physico-chemical point of view, aiming to simulate, as closely as possible, the conditions of interest.
Type: Goal |
Advantage: None |
Novelty: None |
ConceptID: Goa2
4
On this basis, the objective of this work is to characterise the surface of Enterococcus faecalis ATCC29212 through hydrophobicity, surface free energy and zeta potential at 37 °C, when cells grow in Trypticase Soy Broth (TSB) and TSB supplemented with serum or urine.
Type: Goal |
Advantage: None |
Novelty: None |
ConceptID: Goa1
5
These variations are used to provide a theoretical description of the bacterial adhesion to glass by interaction free energy, which is verified with experimental results employing a parallel plate flow chamber set at 37 °C, to simulate the conditions of flow inside the human body.
Type: Method |
Advantage: None |
Novelty: New |
ConceptID: Met1
6
The results show that the addition of serum to the growth medium increases the hydrophobicity and isoelectric point (i.e.p.) of the microorganisms, and this could indicate an increase in the protein content on the cell surface.
Type: Result |
Advantage: None |
Novelty: None |
ConceptID: Res1
7
However urine does not introduce a change in the above magnitudes.
Type: Observation |
Advantage: None |
Novelty: None |
ConceptID: Obs1
8
At short separation distances between the cells and the substratum, the interaction free energy predicts a favourable adhesion for serum-grown cells while non-favourable adhesion is expected for control (TSB-grown cells) and urine-grown cells.
Type: Result |
Advantage: None |
Novelty: None |
ConceptID: Res2
9
These results are in agreement with the experimental adhesion data, obtained with the flow chamber.
Type: Conclusion |
Advantage: None |
Novelty: None |
ConceptID: Con1
Introduction
10
Bacterial adhesion to different surfaces is a process that is of importance in many fields.1–3
Type: Motivation |
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ConceptID: Mot2
11
In particular, in the field of medicine, the adhesion of microorganisms to inert surfaces has gained importance in recent decades due to the increasing number of clinical practices which require the use of implants.
Type: Motivation |
Advantage: None |
Novelty: None |
ConceptID: Mot2
12
One of the microorganisms of current interest because of its adhesion to such biomaterials is Enterococcus faecalis.
Type: Motivation |
Advantage: None |
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ConceptID: Mot2
13
This bacterium is responsible for a great number of nosocomial infections including urinary tract and abdominal infections, wound infections, bacteremia and endocarditis.4,5
Type: Background |
Advantage: None |
Novelty: None |
ConceptID: Bac1
14
The initial adhesion of cells to biomaterials is followed by the formation of a biofilm which, because of its structure, is very difficult to eradicate.6
Type: Background |
Advantage: None |
Novelty: None |
ConceptID: Bac2
15
In these cases, the treatment with antimicrobial agents results in a non-effective therapy.7,8
Type: Background |
Advantage: None |
Novelty: None |
ConceptID: Bac3
16
Therefore, the prevention of the formation of biofilm at its initial stages, seems to be the most promising procedure.
Type: Background |
Advantage: None |
Novelty: None |
ConceptID: Bac4
17
For this reason many papers have studied the initial interaction between microorganisms and different substrata.9–11
Type: Background |
Advantage: None |
Novelty: None |
ConceptID: Bac5
18
It is widely accepted that the initial adhesion of bacteria to biomaterials is mediated by physico-chemical forces similar to those acting in the adhesion of colloidal particles.6,10
Type: Background |
Advantage: None |
Novelty: None |
ConceptID: Bac5
19
In this sense, theoretical predictions of the adhesion process based on interaction free energy calculations have been compared with experimental adhesion results in different investigations.10,12
Type: Method |
Advantage: None |
Novelty: Old |
ConceptID: Met2
20
The positive correlation between both magnitudes could help to predict and consequently control biofilm formation.
Type: Method |
Advantage: Yes |
Novelty: Old |
ConceptID: Met2
21
The interaction free energy between a particle (in this case bacterium) and a surface is described, from a theoretical point of view, as the sum of the Lifshitz–van der Waals, acid–base and electrical interaction free energies.13,14
Type: Model |
Advantage: None |
Novelty: None |
ConceptID: Mod1
22
This theory, known as the extended DLVO (X-DLVO: Derjaguin, Landau, Verwey and Overbeek), provides the dependence of the interaction free energy with the separation distance.
Type: Model |
Advantage: None |
Novelty: None |
ConceptID: Mod1
23
The long-range Lifshitz–van der Waals and the short-range acid–base forces are obtained through the surface free energy of both interacting surfaces, expressed as the sum of the Lifshitz–van der Waals and acid–base surface free energy components (γLW and γAB, respectively).
Type: Model |
Advantage: None |
Novelty: None |
ConceptID: Mod1
24
Both components are calculated from the contact angles that probe liquids form on lawns of bacteria deposited on filters.
Type: Method |
Advantage: None |
Novelty: Old |
ConceptID: Met3
25
The electrical interaction free energy is obtained from the zeta potential (ζ) of bacteria and substrata.
Type: Method |
Advantage: None |
Novelty: Old |
ConceptID: Met4
26
In relation to bacterial adhesion, there are different methods to quantify the adhesion of microorganisms to biomaterials.
Type: Background |
Advantage: None |
Novelty: None |
ConceptID: Bac6
27
Static and dynamic adhesion-based tests are the most commonly used.10
Type: Method |
Advantage: None |
Novelty: Old |
ConceptID: Met5
28
However, static tests have been criticised because they neglect the conditions of flow that bacteria find in different parts of the human body and because they usually need a biochemical method to permit the count of the adhered bacteria.
Type: Method |
Advantage: No |
Novelty: Old |
ConceptID: Met5
29
This work therefore utilises a flow chamber to carry out the adhesion of E. faecalis to glass, allowing a live observation of microorganisms adhered to solid substrata.
Type: Method |
Advantage: None |
Novelty: New |
ConceptID: Met6
30
In line with the above explanation, the goal of this work is the physico-chemical characterisation of E. faecalis ATCC29212 through measurements of water, formamide and diiodomethane contact angles and zeta potential determinations at 37 °C.
Type: Goal |
Advantage: None |
Novelty: None |
ConceptID: Goa3
31
The incubation of cells is carried out in a standard culture medium (TSB) and TSB supplemented with serum or urine, in order to simulate the physiological conditions in which bacteria cause infections.
Type: Experiment |
Advantage: None |
Novelty: None |
ConceptID: Exp1
32
The theoretical predictions of the adhesion process of such microorganisms to glass, defined by the X-DLVO, are verified with the adhesion experiments carried out in a parallel plate flow chamber at 37 °C.
Type: Experiment |
Advantage: None |
Novelty: None |
ConceptID: Exp2
Experimental
Bacteria preparation process
33
E. faecalis ATCC29212 bacteria were stored at −80 °C in porous beads (Microbank, Pro-Lab Diagnostics, Austin, Texas, USA).
Type: Experiment |
Advantage: None |
Novelty: None |
ConceptID: Exp3
34
From the frozen stock, blood agar plates were inoculated and incubated at 37 °C to obtain cultures.
Type: Experiment |
Advantage: None |
Novelty: None |
ConceptID: Exp4
35
Bacteria were incubated overnight in 100 ml of urine-free Trypticase Soy Broth (TSB) (BBL, Becton Dickinson, Cockeysville, Maryland, USA) or TSB containing 10% human serum or 50% human urine at 37 °C.
Type: Experiment |
Advantage: None |
Novelty: None |
ConceptID: Exp5
36
The cells were harvested by centrifugation at 37 °C for 5 min at 1000 g (Sorvall TC6, Dulont, Newtown, Pennsylvania, USA) and washed three times with phosphate buffer saline (PBS, 0.15 mol l−1) for flow experiments and with water (Millipore, Mosheim, France) for contact angle measurements.
Type: Experiment |
Advantage: None |
Novelty: None |
ConceptID: Exp6
Serum and urine collection
37
Serum and urine were collected from 5 healthy men, between the ages of 20 and 45.
Type: Experiment |
Advantage: None |
Novelty: None |
ConceptID: Exp7
38
They were pooled and sterilised by filtration employing 0.45 μm pore-size filters (Millipore, Mosheim, Francia) and volumes of approximately 10 ml and 50 ml of sterile serum and urine, respectively were stored at −20 °C in sterile flasks.
Type: Experiment |
Advantage: None |
Novelty: None |
ConceptID: Exp8
39
Prior to use, the flasks were warmed at room temperature.
Type: Experiment |
Advantage: None |
Novelty: None |
ConceptID: Exp9
Adhesion experiments—the flow chamber
40
Glass (Menzel-Glaser, Braunschweig, Germany) was employed as the substratum for the adhesion experiments in the parallel plate flow chamber.
Type: Experiment |
Advantage: None |
Novelty: None |
ConceptID: Exp10
41
It was cleaned by sonication for 5 min in 2% v/v commercially available surfactant solution (Vilenet, Vileda Iberica, Barcelona, Spain), rinsed thoroughly with water, sonicated in water for 5 min, and finally inserted in the flow chamber.
Type: Experiment |
Advantage: None |
Novelty: None |
ConceptID: Exp11
42
The parallel plate flow chamber (dimensions: l × w × h = 7.6 × 3.8 × 0.06 cm) has been described in detail before.15
Type: Experiment |
Advantage: None |
Novelty: None |
ConceptID: Exp10
43
For enumeration of the adhered bacteria, the flow chamber was placed on the stage of a microscope (Leitz Diaplan, Leica, Wetzlar, Germany) equipped with a 40x ultra-long working distance objective (Leica, Wetzlar, Germany) with a numerical aperture of 0.5.
Type: Experiment |
Advantage: None |
Novelty: None |
ConceptID: Exp12
44
The images obtained from the microscope focussed on microorganisms adhered to the bottom plate of the flow chamber were registered by a CCD camera (Hitachi, Tokyo, Japan).
Type: Experiment |
Advantage: None |
Novelty: None |
ConceptID: Exp13
45
Images were recorded in sets of three images, captured at intervals of a tenth of a second.
Type: Experiment |
Advantage: None |
Novelty: None |
ConceptID: Exp14
46
Adhered bacteria were identified from non-adhered ones by comparing the three images and counting the fixed bacteria.
Type: Method |
Advantage: None |
Novelty: New |
ConceptID: Met7
47
An image covered a surface area of 0.025 mm2.
Type: Experiment |
Advantage: None |
Novelty: None |
ConceptID: Exp15
48
Experimentally, E. faecalis ATCC29212 bacterium grown in TSB or in TSB with serum or urine, were suspended in PBS to a concentration of 3 × 108 cells ml−1 and the suspension flowed through the system at 37 °C for 4 h.
Type: Experiment |
Advantage: None |
Novelty: None |
ConceptID: Exp16
49
A pulse-free flow (0.034 ml s−1) was created by the hydrostatic pressure, and the suspension was recirculated by a Miniplus 2 peristaltic pump (Gilson, Villers le Bel, France).
Type: Experiment |
Advantage: None |
Novelty: None |
ConceptID: Exp17
50
During the first 30 min of adhesion, a large number of images were taken to determine the initial deposition rate, after that images were taken every 10 min approximately up to 4 h.
Type: Experiment |
Advantage: None |
Novelty: None |
ConceptID: Exp18
51
Experiments were repeated at least three times with separately cultured bacteria.
Type: Method |
Advantage: None |
Novelty: New |
ConceptID: Met8
52
The numbers of adhered bacteria were compared using an unpaired Students t-test.
Type: Method |
Advantage: None |
Novelty: New |
ConceptID: Met9
53
Changes were statistically significant if P < 0.05.
Type: Method |
Advantage: None |
Novelty: New |
ConceptID: Met9
54
P gives the probability of observing differences between samples means when the confidence interval selected is of 95%.
Type: Method |
Advantage: None |
Novelty: New |
ConceptID: Met9
Contact angle measurements. Surface energy calculations. Interaction free energy
55
Water (Milli-Q Plus), formamide (puriss > 99.0%, Fluka, Switzerland) and diiodomethane (purum > 98%, Fluka, Switzerland) contact angles on lawns of partially dried bacteria at 37 °C without the residual water were determined using the sessile drop technique.16
Type: Method |
Advantage: None |
Novelty: Old |
ConceptID: Met10
56
The values of the Lifshitz–van der Waals (LW) surface tension component (γLW), electron-donor (γ−) and electron-acceptor (γ+) parameters of the acid–base (AB) surface tension component (γAB) of these three liquids at 37 °C are the following: for water γLW = 20.3 mJ m−2, γ− = 25.0 mJ m−2, γ+ = 25.0 mJ m−2, γAB = 50.0 mJ m−2; for formamide γLW = 36.7 mJ m−2, γ− = 38.3 mJ m−2, γ+ = 2.2 mJ m−2, γAB = 18.4 mJ m−2 for diiodomethane γLW = 48.1 mJ m−2, γ− = 0.0 mJ m−2, γ+ = 0.7 mJ m−2, γAB = 0.0 mJ m−2 being calculated in terms of the constant relation for the γ+ and γ− parameters for water.17
Type: Experiment |
Advantage: None |
Novelty: None |
ConceptID: Exp19
57
Briefly, bacteria suspended in demineralised water were layered onto 0.45 μm pore size filters (Millipore, Molsheim, France) using a negative pressure.
Type: Experiment |
Advantage: None |
Novelty: None |
ConceptID: Exp20
58
Filters were left to air dry at 37 °C for 45 min.
Type: Experiment |
Advantage: None |
Novelty: None |
ConceptID: Exp21
59
The time of drying was the time at the “plateau contact angles” (θ) could be measured,17 and has been checked previously.
Type: Background |
Advantage: None |
Novelty: None |
ConceptID: Bac7
60
After that, the filters were introduced in an environmental chamber G211 (krüss, Hamburg, Germany), connected to a thermostat to maintain the temperature at 37 °C.
Type: Experiment |
Advantage: None |
Novelty: None |
ConceptID: Exp22
61
Before measuring the contact angle with a probe liquid, the chamber was allowed to saturate with vapour of the liquid employed.
Type: Experiment |
Advantage: None |
Novelty: None |
ConceptID: Exp23
62
Subsequently, the contact angles were obtained analysing the images captured with a computer.
Type: Method |
Advantage: None |
Novelty: New |
ConceptID: Met11
63
The surface free energy components, γLW, , were calculated from the three-equation system by applying the Young–Dupré equation to each probe liquid (L): where γL = γLWL + γABL is the surface tension of the probe liquid and the subscript B denotes bacteria.
Type: Method |
Advantage: None |
Novelty: Old |
ConceptID: Met12
64
The total interaction free energy, ΔGT, between microorganisms and substrata through water (W) as a function of the separation distance (d) is calculated by the sum of the Lifshitz–van der Waals (LW), the acid–base (AB) and the electrical interaction free energies (ΔGLW, ΔGAB and ΔGEL, respectively) as proposed by the extended DLVO theory (X-DLVO).13,14
Type: Method |
Advantage: None |
Novelty: Old |
ConceptID: Met13
65
These three components are calculated as follows.
Type: Method |
Advantage: None |
Novelty: Old |
ConceptID: Met13
66
Expression of ΔGLW: A is the Hamaker constant, A = −12πd20ΔGLWadh, a is the microorganism radius, d0 the distance of the closest approach between two surfaces and ΔGLWadh is obtained through the Lifshitz–van der Waals surface free energy as follows, subscripts B and S being bacterium and substratum, respectively, and W indicates that surface free energy of the suspending liquid is nearly equal to water.
Type: Method |
Advantage: None |
Novelty: Old |
ConceptID: Met13
67
Expression of ΔGAB: Expression of ΔGEL: ε0 is the dielectric constant of the vacuum and εr the relative dielectric constant of the suspending liquid.
Type: Method |
Advantage: None |
Novelty: Old |
ConceptID: Met13
68
ζB and ζS are the zeta potentials of bacteria and substrata and the subscript S denotes substratum.
Type: Method |
Advantage: None |
Novelty: Old |
ConceptID: Met13
69
Interaction free energies predict favourable adhesion when they are negative and no adhesion when they are positive.
Type: Method |
Advantage: None |
Novelty: Old |
ConceptID: Met13
Results and discussion
70
The addition of serum and urine to the growth medium of E. faecalis ATCC29212 involves a series of changes in its physico-chemical surface properties and adhesion behaviour as the following tables and figures indicate.
Type: Observation |
Advantage: None |
Novelty: None |
ConceptID: Obs2
Physico-chemical surface properties
71
The first columns of Table 1 contain the water, formamide and diiodomethane contact angles measured at 37 °C on lawns of bacteria grown in TSB (control), TSB with 10% serum and TSB plus 50% urine.
Type: Observation |
Advantage: None |
Novelty: None |
ConceptID: Obs3
72
Serum increases the water contact angle of microorganisms which, in terms of hydrophobicity, means that under such a condition E. faecalis becomes more hydrophobic.16,18
Type: Observation |
Advantage: None |
Novelty: None |
ConceptID: Obs3
73
On the other hand, urine has no effect on the surface hydrophobicity of the studied bacteria, which remains invariable compared to that of the control, within the experimental error.
Type: Observation |
Advantage: None |
Novelty: None |
ConceptID: Obs4
74
The formamide contact angle does not depend on the constituents of the growth medium, while the diiodomethane contact angle only changes for urine-grown cells.
Type: Result |
Advantage: None |
Novelty: None |
ConceptID: Res3
75
These contact angles of the probe liquids provide, through eqn. (1), the components and parameters of the surface free energy of the cells, which also appear in Table 1.
Type: Method |
Advantage: None |
Novelty: Old |
ConceptID: Met12
76
Serum-grown cells have the highest Lifshitz–van der Waals surface free energy component, but the lowest acid–base surface free energy component, making the total surface free energy in this case very similar to that of the control and urine-grown cells.
Type: Result |
Advantage: None |
Novelty: None |
ConceptID: Res4
77
The asymmetry found between the electron-donor and electron-acceptor parameters of γAB is very similar in the cases of the control and urine-grown cells and very much higher than that for serum-grown cells.
Type: Result |
Advantage: None |
Novelty: None |
ConceptID: Res5
78
According to Van Oss et al.,19 this result emphasises the higher hydrophobicity of serum-grown cells compared to the other two samples studied, taking into account that the degree of asymmetry between γ+ and γ− is an indicator of the level of hydrophilicity.
Type: Conclusion |
Advantage: None |
Novelty: None |
ConceptID: Con2
79
The change that serum introduces in the cellular surface hydrophobicity of cells is in agreement with the results obtained by microbial adhesion to hydrocarbons (MATH method) and recently published by this group.20
Type: Conclusion |
Advantage: None |
Novelty: None |
ConceptID: Con3
80
In relation to the electrical characterization of the cell surface, the last column of Table 1 presents the values of the zeta potential of bacteria when they are suspended in PBS, a liquid also employed in the adhesion experiments.
Type: Observation |
Advantage: None |
Novelty: None |
ConceptID: Obs5
81
The data show that, within the experimental error, the addition of urine to the culture medium produces a very small increase in the effective negative charge on the bacterial surface, while the addition of serum makes the net negative charge higher.
Type: Observation |
Advantage: None |
Novelty: None |
ConceptID: Obs5
82
It is interesting to point out that when we say that ζ is a measure of the effective charge, we mean that it measures the electrical potential which one particle manifests when it approaches another particle.
Type: Background |
Advantage: None |
Novelty: None |
ConceptID: Bac8
83
In this context, it is the quantity which characterises the electrical force between the approaching particles, as can be verified below.
Type: Background |
Advantage: None |
Novelty: None |
ConceptID: Bac8
84
Additional information on the surface charge of the microorganisms is provided by the isoelectric point (i.e.p.), which measures the value of the pH when the net surface charge is equal to zero.
Type: Background |
Advantage: None |
Novelty: None |
ConceptID: Bac9
85
Thereby, Fig. 1(a) shows the zeta potential versus pH for the three cases studied, when cells are suspended in KPi—buffer of low ionic strength, with physiological properties and widely used in determining the i.e.p. of biological samples.21,22
Type: Observation |
Advantage: None |
Novelty: None |
ConceptID: Obs6
86
When urine is added to the culture medium, the ζ–pH curve is in most cases similar to that of the control, with the exception of those points obtained around pH 3.3.
Type: Observation |
Advantage: None |
Novelty: None |
ConceptID: Obs6
87
Nevertheless, serum diminishes the net surface charge of E. faecalis over the entire pH range.
Type: Observation |
Advantage: None |
Novelty: None |
ConceptID: Obs7
88
These graphs indicate that the i.e.p.s of control and urine-grown cells are nearly equal (1.9 and 2.0, respectively), while the i.e.p. of serum-grown cells is higher and equal to 2.9.
Type: Result |
Advantage: None |
Novelty: None |
ConceptID: Res6
89
We also determined the i.e.p. when cells were suspended in the same liquid as that employed in the adhesion experiments, i.e. PBS, a liquid with a higher ionic strength.
Type: Object |
Advantage: None |
Novelty: New |
ConceptID: Obj1
90
The results are presented in Fig. 1(b).
Type: Observation |
Advantage: None |
Novelty: None |
ConceptID: Obs8
91
Firstly, it has to be mentioned that the amount of acid or base required to change the pH of PBS suspensions was very much higher than in the case of KPi, due to the higher stability of the buffer PBS.
Type: Result |
Advantage: None |
Novelty: None |
ConceptID: Res7
92
Despite the fact that PBS makes the curves less pronounced than those obtained with KPi, because of the reduction in the net surface charge of microorganisms (due to the compression of the electrical double layer), the effects of serum and urine on the zeta potential are similar to those observed in the case of KPi.
Type: Result |
Advantage: None |
Novelty: None |
ConceptID: Res8
93
This means that urine does not introduce important changes in ζ over the entire pH range selected, while serum makes the microbial surface less negatively charged, especially at low pHs.
Type: Conclusion |
Advantage: None |
Novelty: None |
ConceptID: Con4
94
Based on the curves (Fig. 1(b)), it can be seen that neither the control nor the urine-grown cells have their i.e.p.s in the pH range studied, while the i.e.p. of serum-grown cells is approximately 1.8, a unit less than with KPi, in agreement with the ionic characteristics of both suspending liquids.
Type: Result |
Advantage: None |
Novelty: None |
ConceptID: Res9
95
The increase in the i.e.p. of serum-grown cells with respect to the control and the invariability of the i.e.p. in the case of the urine-grown cells is positively correlated with the higher and equal hydrophobicity of serum-grown cells and urine-grown cells, respectively, compared to that of the control.
Type: Result |
Advantage: None |
Novelty: None |
ConceptID: Res10
96
In addition to the previous analysis of the i.e.p. as a magnitude to characterise the electrical behaviour of the cell surface, the i.e.p. value has been related by several authors to chemical components of the bacterial surface such as proteins.11,23,24
Type: Background |
Advantage: None |
Novelty: None |
ConceptID: Bac10
97
It is widely accepted that a direct relation between i.e.p.s and the ratio nitrogen–carbon (N/C, determined by X-ray electron spectroscopy) exists.11,23,24
Type: Background |
Advantage: None |
Novelty: None |
ConceptID: Bac10
98
The explanation is based on the excess of amino groups, NH4+, positively charged, against phosphate groups, PO43−, negatively charged on the bacterial surface.
Type: Background |
Advantage: None |
Novelty: None |
ConceptID: Bac10
99
This picture causes a reduction in the net value of ζ over the pHs, with the consequent increase in the i.e.p..
Type: Background |
Advantage: None |
Novelty: None |
ConceptID: Bac10
100
Some authors11,24 have related the increase in NH4+ groups (and also the i.e.p. increase) with a rise in the number of proteins exposed to the bacterial surface which, due to their hydrophobic character, contributes to an increase in the cellular surface hydrophobicity.
Type: Background |
Advantage: None |
Novelty: None |
ConceptID: Bac10
101
This result is in total agreement with the hydrophobic behaviour of E. faecalis as shown in the first column of Table 1, where serum-grown cells are presented as the most hydrophobic microorganisms.
Type: Conclusion |
Advantage: None |
Novelty: None |
ConceptID: Con5
102
The effect of serum on the hydrophobicity of the cell surface of enterococci is in agreement with the results obtained by Ljungh and Wadstrom25 when they determined the hydrophobicity of different microorganisms (among them E. faecalis) grown with serum.
Type: Conclusion |
Advantage: None |
Novelty: None |
ConceptID: Con6
103
Nevertheless, these authors could not attribute the phenomenon to a specific serum compound.
Type: Background |
Advantage: None |
Novelty: None |
ConceptID: Bac11
104
The direct relation between i.e.p. and hydrophobicity found in this work, in addition to the correlations between i.e.p., hydrophobicity and NH4+ obtained by our group and other researchers in previous studies, may indicate that the presence of proteins, contained in serum, on the bacterial surface is responsible for the increase in cellular surface hydrophobicity of serum-grown bacteria.
Type: Conclusion |
Advantage: None |
Novelty: None |
ConceptID: Con7
Theoretical predictions and experimental results of adhesion
105
The main objective when analysing the physico-chemical surface parameters of E. faecalis grown in TSB and TSB supplemented with serum or urine is to check whether they are able to predict the initial adhesion of the microorganisms.
Type: Goal |
Advantage: None |
Novelty: None |
ConceptID: Goa4
106
On this basis, interaction free energy, obtained through the above physico-chemical surface parameters, is related to the number of adhered cells discussed in this section.
Type: Background |
Advantage: None |
Novelty: None |
ConceptID: Bac12
107
When a bacterium approaches a surface, the value of the interaction free energy, ΔGT, is a function of the separation distance between both phases, as described by the extended DLVO theory (X-DLVO).
Type: Model |
Advantage: None |
Novelty: None |
ConceptID: Mod2
108
When representing ΔGTversus the separation distance (d) many systems present two minima, one called the secondary minimum (in most cases no higher than 100 kT, k and T being Boltman's constant and absolute temperature, respectively) and the other called the primary minimum, located at shorter distances, much deeper and separated from the secondary minimum by a potential barrier (in the order of some tens of kT).
Type: Model |
Advantage: None |
Novelty: None |
ConceptID: Mod2
109
The state of stable equilibrium corresponds to the primary minimum and the ‘jump’ from the secondary to the primary minimum is relatively easy for cells, which use in their metabolism an amount of energy in the range of 106–108kT.26
Type: Background |
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ConceptID: Bac13
110
When calculating the ΔGT in biological systems to predict non-specific bacterial adhesion, the minimal separation distance (1.57 Å) between cells and substrata is estimated in order to form a new interface.27
Type: Method |
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Novelty: Old |
ConceptID: Met14
111
This implies that bacteria falls into the primary minimum, an assumption that cannot be completely true if the primary minimum does not exist.
Type: Method |
Advantage: No |
Novelty: Old |
ConceptID: Met14
112
This reasoning led us in this work to take into account not only the value of ΔGT at 1.57 Å but also to draw the ΔGT curves as a function of the separation distances between the interacting surfaces.
Type: Method |
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Novelty: New |
ConceptID: Met15
113
The graphics are presented in Figs. 2–4, each relating to one of the samples studied.
Type: Observation |
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ConceptID: Obs9
114
At first glance, one can observe the similarity between Figs. 2 and 4, corresponding to control and urine-grown cells, while Fig. 3 appears very different from the others, not only at high separation distances but also when in close approximation (compare Figs. 2(a), 3(a) and 4(a) and Figs. 2(b), 3(b) and 4(b)).
Type: Result |
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ConceptID: Res11
115
This result is well correlated with all the data belonging to the previously mentioned physico-chemical characterisation of the cells, in which control and urine-grown cells behave in a similar way and differently from serum-grown cells.
Type: Conclusion |
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ConceptID: Con8
116
On analysing each type of force as a function of the separation distance, Lifshitz–van der Waals interaction free energy governs the interaction between E. faecalis and glass at high separation distances (this is clearly verified from 50 Å for all the cases studied).
Type: Result |
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Novelty: None |
ConceptID: Res12
117
At intermediate distances (from approximately 10 Å to 50 Å), the Lifshitz–van der Waals and mainly the electrical force control the sign and magnitude of the total interaction free energy, while at short distances (below 10 Å, see especially Figs denoted with (b)) the acid–base force is the dominant interaction.
Type: Result |
Advantage: None |
Novelty: None |
ConceptID: Res13
118
With respect to the shape of ΔGT, serum-grown cells exhibit a deep primary minimum (Fig. 2(b)), while control and urine-grown cells lack this potential well.
Type: Observation |
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ConceptID: Obs10
119
This fact implies, from a physico-chemical point of view, that serum-grown cells would have a far higher initial adhesion to glass than in the other cases.
Type: Result |
Advantage: None |
Novelty: None |
ConceptID: Res14
120
The high potential barrier that control and urine-grown cells have at the minimal separation distance means that the bacteria are not able to reach this state and so, from a theoretical point of view, the adhesion to glass of E. faecalis grown in TSB and in TSB supplemented with urine is not possible.
Type: Hypothesis |
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ConceptID: Hyp1
121
The cells in the last two cases would remain in the secondary minimum, which for the control is about −6 kT and for urine-grown cells −0.5 kT, at distances of 35 Å and 59 Å, respectively, far from the minimal interaction distance required to form a new interface.
Type: Hypothesis |
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ConceptID: Hyp1
122
In order to validate the theory, the interaction free energy of the adhesion process of E. faecalis to glass should be checked with the experimental adhesion data.
Type: Method |
Advantage: None |
Novelty: New |
ConceptID: Met16
123
In this sense, Fig. 5 represents the number of adhered cells per square centimetre throughout the adhesion period in a parallel plate flow chamber.
Type: Observation |
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ConceptID: Obs11
124
It can be clearly seen that the adhesion to glass of E. faecalis grown with serum is higher than that of the control and urine-grown cells (P < 0.05), the last two cases being mostly similar (P > 0.05).
Type: Result |
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Novelty: None |
ConceptID: Res15
125
These adhesion data are in good agreement with the theoretical predictions of adhesion dictated by the X-DLVO theory, in the sense that ΔG values anticipate favourable adhesion to glass of serum-grown cells against the control or urine-grown bacteria.
Type: Conclusion |
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ConceptID: Con9
126
In these last two cases, adhesion is expected to be similar between them and lower than serum-grown cells.
Type: Conclusion |
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ConceptID: Con10
Conclusion
127
The present study brings to light that components of serum modify the physico-chemical surface of E. faecalis by enhancing its hydrophobicity, isoelectric point and by changing the components and parameters of the surface free energy.
Type: Conclusion |
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ConceptID: Con11
128
Such changes predict a favourable adhesion to glass taking into account the X-DLVO theory, which is in agreement with the experimental adhesion data.
Type: Conclusion |
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Novelty: None |
ConceptID: Con12
129
On the other hand, the supplementation of the culture medium with urine does not produce any effect on the hydrophobicity and surface charge of microorganisms, as shown by the similar adhesion curves obtained for this sample and that of the control.
Type: Conclusion |
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ConceptID: Con13
130
This work can help to deepen in the knowledge of the infections by E. faecalis, which in the last decades has become an important nosocomial pathogen.
Type: Conclusion |
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ConceptID: Con14
131
Its ability to modify its physico-chemical properties in the presence of serum, favouring its adhesion to inert surfaces, could, for example, explain the importance of enterococcus in endocarditis.
Type: Conclusion |
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ConceptID: Con15
132
Moreover, enterococcal infections of the urinary tract could be related to other kinds of forces, different from physico-chemical interactions.
Type: Conclusion |
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ConceptID: Con16
133
This last point remains unclear and warrants further investigation.
Type: Conclusion |
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Novelty: None |
ConceptID: Con17