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A study of pH effects on the bacterial surface physicochemical properties of Acinetobacter baumannii
Colloids and Surfaces B: Biointerfaces 102 (2013) 540–545
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Colloids and Surfaces B: Biointerfaces journal homepage: www.elsevier.com/locate/colsurfb
A study of pH effects on the bacterial surface physicochemical properties of Acinetobacter baumannii Ryad Djeribi a,∗ , Zahia Boucherit b , Warda Bouchloukh a , Wafa Zouaoui a , Hassan Latrache c , Fatima Hamadi c , Bouzid Menaa a,d a
Laboratoire des Biofilms et Biocontamination des Matériaux, Faculté des Sciences, Université Badji Mokhtar, Annaba, Algeria Laboratoire Antibiotiques, Antifongiques: Physico-Chimie, Synthèse et Activité Biologique, Université Abou Bekr Belkaïd, Tlemcen, Algeria c Laboratoire de Valorisation et Sécurité des Produits Alimentaires, Département des Sciences et Techniques, Béni Mellal, B.P 523, Morocco d Fluorotronics, Inc., Department of Nano-Biotechnology, 2453 Cades Way, Vista, CA 92081, USA b
a r t i c l e
i n f o
Article history: Received 7 May 2012 Received in revised form 16 August 2012 Accepted 28 August 2012 Available online 17 September 2012 Keywords: A. baumannii adhesion pH Cell surface physicochemical properties Microbial adhesion to solvents method Contact angle measurement
a b s t r a c t The first step in the biofilm formation is the bacterial attachment to solid surfaces, which is dependent on the bacteria cell surface physico-chemical properties. The purpose of this work was to analyze the effect of pH on the physicochemical cell surface properties of Acinetobacter baumannii by two different methods. The cell surface properties were evaluated using the microbial adhesion to solvents method (MATS) and contact angle measurements (CAM). MATS technique allowed us to enlighten that A. baumannii was hydrophilic at the different values of pH. It was found that at a desired pH of 6.5, the strain presents a maximum and stable value of electron-donor characteristic, while the electron acceptor character increased as the pH increased. Regardless of the methods employed, the obtained results using MATS and CAM confirmed the influence of the pH on the surface physicochemical properties of A. baumannii. The cell surface electron-donor and electron-acceptor character at pH 6.5 was found to be quite similar using both methods. © 2012 Elsevier B.V. All rights reserved.
1. Introduction Acinetobacter baumannii has emerged as an important nosocomial pathogen with clinical implications including respiratory [1] and urinary tract infections, meningitis, endocarditis, wound infections and bacteremia, especially in intensive care unit patients [2–7]. Bacteria adhering as a biofilm are usually treated with antibiotics, although sometimes the available antibiotic therapies are ineffective because of the antibiotic resistance of the strains and the protection offered by the biofilm formation in contrast with the planktonic growth which induces less antibiotic resistance [8,9]. The interest in A. baumannii has been growing rapidly because of the emergence of multidrug-resistant (MDR) strains of this species, some of which are pan-resistant to antimicrobial agents [5–7]. Moreover, the ability of these strains to adhere to surfaces constitutes an important impact on the pathogenicity of these bacteria [3–12]. Indeed, A. baumannii survives for several days on inanimate objects and surfaces found normally in medical environments, even
∗ Corresponding author. Tel.: +213 5 59 46 97 23. E-mail address: [email protected] (R. Djeribi). 0927-7765/$ – see front matter © 2012 Elsevier B.V. All rights reserved. http://dx.doi.org/10.1016/j.colsurfb.2012.08.047
in dry conditions on dust particles. These survival properties play a significant role in the outbreaks caused by this pathogen [3–14]. Bacterial adhesion in relation to urinary-tract infections has gained importance in the last few years because of the increasing catheterization in hospitals to assist post-surgery flow of urine [15,16]. Bacterial attachment to an inert surface results from complex physicochemical interactions among the cell, the surface, and the liquid phase, which are caused by the cell surface charge, the hydrophobicity, and electron acceptor and donor properties [17]. The expression of these physicochemical interactions depends on the bacterial cell surface properties, which depend on their culture and experimental conditions [17,18] but also on the methods used to determine them. For this reason, it is difficult to discuss specifically about an absolute hydrophobicity value; therefore, a good estimation of the hydrophobic cell properties should take into account different methods [19]. In such context, the aim of this work was to study the pH effect on A. baumannii cell surface physicochemical properties to determinate the hydrophobic and hydrophilic character of the cell surface but also its electron donor and acceptor characters to have an evaluation and to further control the biofilm formation and adhesion owing to these properties and the environmental conditions (acidic or basic environment). These properties have been evaluated via
R. Djeribi et al. / Colloids and Surfaces B: Biointerfaces 102 (2013) 540–545
two different methods using the microbial adhesion to solvents method (MATS) and contact angle measurements (CAM).
Table 1 Affinities of A. baumannii cells to the four solvents used in the MATS analysis at the different pH values. pH
2. Materials and methods
% of adhesion (±SD)a to: Hexadecane
2.1. Bacterial strain, culture conditions and preparation of microbial suspension A. baumannii was isolated from a patient having an urinary tract infection. The strain was identified by morphological and chemical tests as belonging to the genus Acinetobacter. The identification of the strain at the species level was confirmed by the simplified test of growth at 37, 41, and 44 ◦ C. The strain was sub-cultured overnight and grown in Liquid Luria–Bertani medium (LLB) at 37 ◦ C for 18 h. After the culture, the cells were harvested by centrifugation for 15 min at 5000 × g, washed twice and suspended in phosphate buffered saline solution (PBS, 0, 1 M) at the desired pH used in the experimental hydrophobicity assay.
541
pH 4 pH 5.5 pH 6.5 pH 7.2 pH 8 a
13.26 11.03 11.30 5.88 9.32
± ± ± ± ±
2.04 1.52 1.04 0.66 0.52
Chloroform 24.39 22.85 37.02 17.95 20.70
± ± ± ± ±
Hexane
2.51 1.69 2.37 1.28 0.78
17.44 8.42 13.11 8.24 13.90
Diethylether
± ± ± ± ±
0.76 0.87 0.91 0.94 0.62
15.15 29.79 42.55 49.78 52.41
± ± ± ± ±
1.72 1.46 1.59 7.19 2.04
±Standard deviations of three measures of at least three separate experiments.
(S) were estimated from the approach proposed by van Oss et al. In this approach, which neglects spreading pressure, the pure liquid (L) contact angles () can be expressed as: Cos = −1 +
2(SLW LLW ) L
1/2
+2
(S+ L− ) L
1/2
+
2(S− L+ )
1/2
L
2.2. Microbial adhesion to solvents (MATS) 2.4. Biofilm observation by scanning electron microscopy (SEM) Acid–base interactions can be assessed from microbial adhesion to solvents MATS. The partitioning method previously described by Bellon-Fontaine et al. is based on the comparison between the microbial cell affinity to a monopolar solvent and an apolar solvent [20]. The monopolar solvent can be acidic (electron accepting) or basic (electron donating) but both solvents must have similar Lifshitz–van der Waals surface tension components. On this basis, the following pairs of solvents were selected: (a) chloroform, an acidic solvent (electron acceptor) with hexadecane, an apolar solvent; (b) diethyl ether, a strong basic solvent (electron donor) and hexane, an apolar solvent. Chemical products (chloroform, hexadecane, diethyl ether and hexane) having a highest purity grade ≥ 99.8% were obtained commercially (Aldrich Chemical, Co., Inc. USA). Experimentally, the bacteria were suspended at an optical density (A0 ) at 600 nm of 0.8–0.9 in PBS 0.1 M, with the pH adjusted to 4, 5.5, 6.5, 7.2, 8 by the addition of HCl or NaOH. Subsequently, 0.3 ml of the solvent was added to 1.8 ml of the bacterial suspension, after which the two phase system was vortexed for 2 min and allowed for 20 min to ensure complete separation of the two-phases (organic and aqueous phase). The optical density (A) of the aqueous phase was measured. The percentage of bound cells was subsequently calculated by the formula: Adherence (%) =
(A − A) 0 A0
× 100
where A0 is the optical density of the bacterial suspension measured at 600 nm before mixing. Experiments were repeated three times with separate cultured bacteria. 2.3. Contact angle measurements and estimation of bacterial surface tension components Cell surface hydrophobicity is an expression of the Lifshitz–Van der Waals and acid–base surface tensions, LW and AB of the cell surface [9] and is usually assessed from contact angles of water, formamide and diiodomethane (purity ≥ 99%) on lawns of partially dehydrated bacteria using the sessile drop technique [21]. Briefly, the bacteria suspended in 10 ml of PBS (with the appropriate pH) were layered onto 0.45 m pore size filters (Sartorius) using a negative pressure. The filters were left during about 30 min to air dry at room temperature until the so-called ‘plateau contact angles’ could be measured. Experiments were carried out in triplicate with separately cultured bacteria. The Lifshitz–van der Waals ( LW ), electrondonor ( − ) and electron-acceptor ( + ) components of the bacteria surface tension
Scanning electron microscope analysis was used to observe A. baumannii biofilm colonizing the intraluminal surface of urinary catheters. Sterile silicone Foley catheters were cut into 2 mm-thick discs. The discs were aseptically introduced into tubes containing nutrient broth, which was previously inoculated with A. baumannii culture. After homogenization, the culture was incubated at 37 ◦ C. After one week, the tubes were cleaned of bacterial cultures, and the discs extensively washed with sterile distilled water in order to analyze them by SEM. Support samples were then dried at 37 ◦ C for 24 h. Once coated with gold–palladium (Sputtering Device), samples were examined under a SEM (Cambridge S200). 3. Results 3.1. Influence of pH on cell surface physicochemical properties using the MATS method Different solvents were employed to evaluate the hydrophobic/hydrophilic cell surface properties of A. baumannii and their Lewis acid–base characteristics. Both apolar solvents hexadecane and hexane were used to estimate the hydrophobicity properties of A. baumannii, while the two monopolar solvents, chloroform and diethyl ether, were selected for the estimation of the Lewis acid/base (i.e., electron donor/acceptor) character. The MATS results obtained for A. baumannii at different pH are shown in Table 1. Assays were performed in high ionic strength solution (PBS, 0.1 mol/l) to minimize electrostatic interactions between A. baumannii and solvents. First, direct measurements of the cell surface hydrophobicity and hydrophilicity were carried out by partitioning the cells between aqueous and hexadecane phases. The percentages of bacteria adhering to this apolar solvent, range from 5.88 to 13.26%. This result demonstrates the hydrophilic surface of A. baumannii at the different values of the pH tested. Regardless the pH, the affinity of A. baumannii was always higher with chloroform (an electron acceptor solvent) than with hexadecane (a nonpolar solvent). The differences in bacterial affinity between these two solvents were due to Lewis acid–base interactions (i.e., electron donor–electron acceptor interactions resulting from the electron donor nature of the bacteria) [20]. As shown in Fig. 1, the electron donor character was stable and relatively low at the pH values (pH 4, pH 5.5, pH 7.2 and basic pH 8). Nevertheless, at pH 6.5, A. baumannii strain presents a clear maximum value of electron donor.
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Fig. 1. Variations in the surface electron donor character of A. baumannii at the different pH, estimated with the MATS method.
Fig. 3. SEM picture of Acinetobacter baumannii biofilm formed on an urinary catheter (magnifications: ×3000).
Fig. 2. Variations of the surface electron-acceptor character of A. baumannii at the different pH estimated with the MATS method.
Similarly, the acidic character of A. baumannii was evaluated by comparing the adhesion of A. baumannii to the monopolar basic diethyl ether (a strong electron-donor solvent) and to the apolar hexane solvent [20]. Fig. 2 showed the electron acceptor character of A. baumannii at the different pH. The affinity to diethyl ether increased with the pH and A. baumannii showed the maximum electron acceptor character at pH 7.2 and a non-negligible value at pH 8. However, this electron-acceptor character was null or negligible at the most acidic pH (pH 4). 3.2. Influence of the pH on the cell surface physicochemical properties using the contact angle measurements The results of the contact angle measurements are shown in Table 2. The measured contact angle with water indicates the qualitative change in the hydrophobicity of the bacterial surface as a
function of pH. With the exception of pH 6.5, the results show that A. baumannii has a hydrophilic character for the different pH values. At pH 6.5, the strain is hydrophobic as shown by the contact angle, E = 75.60◦ ± 5.54. Moreover, the value of Giwi allowed us to obtain a quantitative measure of the hydrophobicity [22]. The bacterium surface is very hydrophobic at pH 6.5, relatively hydrophobic at pH 4 while and it is hydrophilic at pH 5.5, 7.2 and 8. The results showed also the increasing electron-donor character with increasing pH, except for pH 6.5, at which a significant drop in the value of the electron-donor character can be noticed. The electron-acceptor character decreases with the increasing pH, with an exception at pH 5.5, for which we observe that the value of the electron-acceptor character is almost negligible (Table 2). 3.3. Scanning electron microscopy analysis of the biofilm A. baumannii biofilm colonizing the intraluminal surfaces of the urinary catheters was examined by SEM analysis (Fig. 3). The biofilm is characterized by a multitude of intercellular pores and interconnected channels, which allow not only the exchange of information and the flow of water and ions, but also the transport of nutrients and the efflux of waste products. SEM analysis suggests heterogeneous mosaic architecture of biofilm containing micro-colonies of bacterial cells encased in an extracellular polymeric substances (EPS) matrix and separated by interstitial voids. It is worth noting that the biofilm is observed at the urinary pH of about 6.5, which is consistent with the hydrophobicity of A. baumannii at this pH.
Table 2 Lifshitz–Van der Waals surface tension components ( LW ), electron-donor ( − ), and electron-acceptor ( + ) values of A. baumannii at the different pH. pH values
pH 4 pH 5.5 pH 6.5 pH 7 pH 8
Contact angles (◦ )
Surface tension components (mJ m−2 )
E
F
D
LW
+
−
AB
T
Giwi
51.70 (±2.95) 55.10 (±5.94) 75.60 (±5.54) 27.40 (±3.53) 43.30 (±2.82)
42.60 (±4.52) 62.40 (±2.79) 60.60 (±2.80) 41.00 (±4.27) 66.00 (±4.75)
92.20 (±5.78) 67.10 (±0.27) 93.00 (±4.89) 70.80 (±3.90) 59.60 (±4.81)
11.8 24.5 11.4 22.4 28.8
11.3 0.1 7.7 2.4 0.7
26.4 42.0 9.6 59.1 69.4
34.6 3.6 17.2 23.7 14.0
46.4 28.1 28.6 46.1 42.8
−2.45 26.94 −21.10 28.78 54.32
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4. Discussion Several studies have reported the effect of pH on the physicochemical properties of the bacterial surfaces [17–24]. The results using MATS method revealed that A. baumannii has a hydrophilic character at the different pH tested. According to several studies, it was admitted from the chemical point of view that the hydrophobic cells tend to adhere to a hydrophobic substrate, while the hydrophilic cell surface tend to adhere onto a hydrophilic substrate [10–25]. However, our results from the MATS method do not reflect this assumption. It is worth noting that the strain was isolated from a hydrophobic silicone-based urinary catheter surface and that A. baumannii has a hydrophilic character. At the present time, such contradictions for this hydrophobic/hydrophilic aspect [22] exist because the adhesion phenomenon cannot be treated only by taking in account the sole hydrophobicity effect, but the other parameters such as physical and chemical properties of bacterial cells and surface colonization have to be investigated and be taken in account. On the other hand, the results of the MATS method showed that the hydrophilic nature of this strain is associated with a more important Lewis acid–base character. The electron-donor character may be an indication of the nature of chemical groups exposed on the surface of the bacteria. The predominance of electron-donor character is attributed to the chemical groups generally negatively charged or neutral, such as carboxylate groups (COO ), amino groups (NH2 ) and phosphate (PO4 ) groups from phospholipids, lipoproteins and lipopolysaccharides as well as SO3 groups from sulfur clusters [17–23]. The donor character would normally be stronger if such carboxylic or phosphate groups are deprotonated, giving rise to the corresponding anions. It will be then expected to observe an increase in the donor character when the pH increases. On the contrary, our results show a decrease in the basic character of the bacterial surface at pH 7.2 and pH 8. It can be then be assumed that increasing the pH causes the deprotonation of chemical groups which increases the electron donor character. This deprotonation causes, in parallel, a negatively charged bacterial surface. Based on similar studies which reported that the deprotonated carboxylate groups and phosphate play a predominant role in determining the negative charge on the bacterial surface and that the contribution of these groups in the negative charge is pH dependent [24], we can assume that the increase of negative charges on the surface of the bacteria will act in a repulsive way with negatively charged solvent [17]. This could then explain the reduction of the affinity of the isolated strain to solvents. The results showed also that the electron-acceptor character for A. baumannii increases when we increase the pH of the buffer. The importance of the electron-acceptor character can be attributed to the presence of acidic groups (such as NH3 + ) groups and hydroxyl (OH) groups exposed on the cell surface [24]. However, the results obtained by Hamadi et al. showed that the electron-acceptor character of the two strains studied, Escherichia coli and Staphylococcus aureus, is maximal at acidic pH values (pH 2 and pH 3), and low at the other pH values studied (pH 5, 6.2, 9 and 11) [23]. Based on the results of the MATS method, we can deduce that the adherence of A. baumannii is due to the surfaces, at least in part, to Lewis acid–base interactions of the surface which is in agreement with the studies of several authors who have demonstrated the importance of the acid–base character in bacterial adhesion [17]. The method that consists of measuring the contact angle was aimed to characterize, by a second method, the changes in the physicochemical properties of the A. baumannii surface as function of the pH and to correlate these results with the results obtained previously by the MATS method. To our knowledge, this is the first study that used CAM method to evaluate the effect of pH on the physicochemical properties of a microbial surface. Unlike the
Fig. 4. Correlation between the variation of electron-donor character and the hydrophobicity of A. baumannii.
results obtained with the MATS method, for which the bacteria was found to be hydrophilic at the different values of pH tested, the CAM method results showed a very significant effect of pH on the hydrophobicity of the surface of the A. baumannii bacterium. Indeed, the bacterium is very hydrophobic at pH 6.5, relatively hydrophobic at pH 4 and hydrophilic at pH 5.5, 7.2 and 8. This paradox in the determination of hydrophobicity/hydrophilicity between the two methods was reported in some studies [26]. The results in the acid–base character as function of pH suggest that the electron-donor character increases with the pH. This increase is explained by the protonation of chemical groups, particularly groups of the type COO− and PO4 , which play an important role in the determination of electron-donor estimated by the CAM method. However, the decrease in the electron-donor character at pH 6.5 cannot be explained in terms of protonation/deprotonation of chemical groups. It is assumed that the hydrophobicity at this pH masked the chemical groups. This suggestion is reinforced when comparing the change in the electron-donor character of the strain with the change in hydrophobicity obtained at different pH (Fig. 4). The results showed a negative correlation (r = −0.84) between the variation of the electron-donor character and the hydrophobicity of the surface, indicating that the increase in hydrophobicity induced a decrease in the electron-donor character, which is observed at pH 6.5 and pH 4. The study of the electron-acceptor character revealed its increase along with a pH decrease. The importance of the electronacceptor character can be attributed to the protonation of groups such as NH3 + groups, and OH groups exposed on the surface of the bacteria [24]. Our results are consistent with the results obtained by Hamadi et al. who reported that the electron-acceptor character is more pronounced at acidic pH. No explanation has yet been found for the reduction of electron-acceptor character at pH 5.5 [23]. A limited number of publications dealing with the comparison between the results from both methods MATS and CAM were found. The comparison between the two methods was made for the first time by Bellon-Fontaine et al. [20]. The authors have shown that the results from both methods are comparable. In this present study, the results obtained from both methods are compared in order to properly estimate the effect of pH on the physicochemical properties of the surface of A. baumannii. According to the results obtained by the method MATS, we note that whatever the pH studied, A. baumannii has a hydrophilic character. By contrast, the results based on the contact angle measurements method showed a significant influence of the pH on the hydrophobic/hydrophilic nature of the A. baumannii surface (Table 3).
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Table 3 Changes in hydrophobicity of A. baumannii estimated by both methods as function of the pH. pH values
Hydrophobicity/ hydrophilicity estimated by (MATS)
Hydrophobicity/ hydrophilicity estimated by (CAM)
4 5.5 6.5 7.2 8
Hydrophilic Hydrophilic Hydrophilic Hydrophilic Hydrophilic
Relatively hydrophobic Hydrophilic Hydrophobic Hydrophilic Hydrophilic
Fig. 5. Correlation between the results of the hydrophobicity of A. baumannii measured by both methods MATS and CAM at different pH values.
Although the findings on the hydrophilic/hydrophobic surface behavior of the strain are different at certain pH, both methods show identical trends. For instance, Fig. 5 shows a correlation between both methods (r = 0.72) in estimating the change in hydrophobic/hydrophilic surface A. baumannii. Some differences exist at pH 6.5 and pH 4. The strain surface is hydrophilic by the MATS method and hydrophobic by the CAM method. In a similar study [26], using seven different strains and different culture conditions, the authors showed that the results of both methods MATS and MAC do not give the same conclusions for the different strains studied. In Fig. 6, we can also notice that the results concerning with the acidic/base character study of the bacterial surface as a function of the pH differ from both methods. Indeed, at pH 5.5, 7.2 and 8, the bacterium has a larger electron-donor character using the CAM method (Fig. 6b). By contrast, A. baumannii presents an electronacceptor character by the MATS method (Fig. 6a). However, a fairly good correlation on the acid–base character as measured by both methods is verified in the case of pH 6.5. Indeed, the results from the MATS method and the results from the CAM method show a balance between characters, donor and electron-acceptor. This suggests that at pH 6.5 the bacterium is in acid–base equilibrium. The contradiction in the measurements on the surface physicochemical properties of the bacterium using both methods can be attributed to the characteristics of each method. The measurements by the MATS method are performed on fully hydrated cells and two types of interactions are considered: one interaction between bacterial cells and the solvent and the other between the bacterial cells and the buffer. In contrast, for the contact angle measurements method, the estimation of hydrophobicity and the acid/base character of the bacterial surface are carried out on semi-hydrated bacterial cells. Therefore, the interactions evaluated by this method concern only with interactions between bacterial cells and solvent molecules (water, formamide or diodomethane). According to Rosenberg, the different methods for estimating the hydrophobicity (BATH, SAT, CAM, etc. . .) can differ depending on
Fig. 6. Variation of the acid–base character for A. baumannii surface as function of the pH. (a) Results from the MATS method; (b) results from the CAM method.
the nature of the hydrophobicity, and the way to measure it [27]. Hamadi and Latrache showed that the phosphate and amine groups play an important role in the electron-donor/acceptor character determination estimated by CAM [26]. The protein/polysaccharide interactions may be the source of the electron-donor character estimated by MATS [26]. It should be also noted that the MATS method provides a qualitative estimation of the hydrophobic/hydrophilic and electron-donor/acceptor characteristics. In the other hand, the CAM method combined with the equation of van Oss can determinate quantitatively the acid–base character and the hydrophobic/hydrophilic surface properties more accurately in terms of surface free energy [19]. 5. Conclusions In conclusion, the combination of the results from both techniques, taking into account the limitations of each, confirms, on one hand, the effect of the pH on the physicochemical properties of the A. baumannii surface, and on the other hand, it shows a reasonable correlation on the acid–base character measured by both methods at pH 6.5. The acid–base behavior of this strain at pH 6.5 may suggest a particular effect of this pH on the physicochemical properties of the A. baumannii surface that will provide us with a better understanding and information on the ability of this bacterium to adhere to urinary catheters. Acknowledgement This work was supported by the Grants-in-Aid from the Algerian Ministry of Education and Scientific Research (Direction of Scientific Research and Technological Development) to which the authors are very thankful. References [1] J. Baraibar, H. Correa, D. Mariscal, M. Gallego, J. Valles, J. Rello, Chest 112 (1997) 1050–1954. [2] E. Bergogne-Berezin, K.J. Towner, Clin. Microbiol. Rev. 9 (1996) 148–165.
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Colloids and Surfaces B: Biointerfaces journal homepage: www.elsevier.com/locate/colsurfb
A study of pH effects on the bacterial surface physicochemical properties of Acinetobacter baumannii Ryad Djeribi a,∗ , Zahia Boucherit b , Warda Bouchloukh a , Wafa Zouaoui a , Hassan Latrache c , Fatima Hamadi c , Bouzid Menaa a,d a
Laboratoire des Biofilms et Biocontamination des Matériaux, Faculté des Sciences, Université Badji Mokhtar, Annaba, Algeria Laboratoire Antibiotiques, Antifongiques: Physico-Chimie, Synthèse et Activité Biologique, Université Abou Bekr Belkaïd, Tlemcen, Algeria c Laboratoire de Valorisation et Sécurité des Produits Alimentaires, Département des Sciences et Techniques, Béni Mellal, B.P 523, Morocco d Fluorotronics, Inc., Department of Nano-Biotechnology, 2453 Cades Way, Vista, CA 92081, USA b
a r t i c l e
i n f o
Article history: Received 7 May 2012 Received in revised form 16 August 2012 Accepted 28 August 2012 Available online 17 September 2012 Keywords: A. baumannii adhesion pH Cell surface physicochemical properties Microbial adhesion to solvents method Contact angle measurement
a b s t r a c t The first step in the biofilm formation is the bacterial attachment to solid surfaces, which is dependent on the bacteria cell surface physico-chemical properties. The purpose of this work was to analyze the effect of pH on the physicochemical cell surface properties of Acinetobacter baumannii by two different methods. The cell surface properties were evaluated using the microbial adhesion to solvents method (MATS) and contact angle measurements (CAM). MATS technique allowed us to enlighten that A. baumannii was hydrophilic at the different values of pH. It was found that at a desired pH of 6.5, the strain presents a maximum and stable value of electron-donor characteristic, while the electron acceptor character increased as the pH increased. Regardless of the methods employed, the obtained results using MATS and CAM confirmed the influence of the pH on the surface physicochemical properties of A. baumannii. The cell surface electron-donor and electron-acceptor character at pH 6.5 was found to be quite similar using both methods. © 2012 Elsevier B.V. All rights reserved.
1. Introduction Acinetobacter baumannii has emerged as an important nosocomial pathogen with clinical implications including respiratory [1] and urinary tract infections, meningitis, endocarditis, wound infections and bacteremia, especially in intensive care unit patients [2–7]. Bacteria adhering as a biofilm are usually treated with antibiotics, although sometimes the available antibiotic therapies are ineffective because of the antibiotic resistance of the strains and the protection offered by the biofilm formation in contrast with the planktonic growth which induces less antibiotic resistance [8,9]. The interest in A. baumannii has been growing rapidly because of the emergence of multidrug-resistant (MDR) strains of this species, some of which are pan-resistant to antimicrobial agents [5–7]. Moreover, the ability of these strains to adhere to surfaces constitutes an important impact on the pathogenicity of these bacteria [3–12]. Indeed, A. baumannii survives for several days on inanimate objects and surfaces found normally in medical environments, even
∗ Corresponding author. Tel.: +213 5 59 46 97 23. E-mail address: [email protected] (R. Djeribi). 0927-7765/$ – see front matter © 2012 Elsevier B.V. All rights reserved. http://dx.doi.org/10.1016/j.colsurfb.2012.08.047
in dry conditions on dust particles. These survival properties play a significant role in the outbreaks caused by this pathogen [3–14]. Bacterial adhesion in relation to urinary-tract infections has gained importance in the last few years because of the increasing catheterization in hospitals to assist post-surgery flow of urine [15,16]. Bacterial attachment to an inert surface results from complex physicochemical interactions among the cell, the surface, and the liquid phase, which are caused by the cell surface charge, the hydrophobicity, and electron acceptor and donor properties [17]. The expression of these physicochemical interactions depends on the bacterial cell surface properties, which depend on their culture and experimental conditions [17,18] but also on the methods used to determine them. For this reason, it is difficult to discuss specifically about an absolute hydrophobicity value; therefore, a good estimation of the hydrophobic cell properties should take into account different methods [19]. In such context, the aim of this work was to study the pH effect on A. baumannii cell surface physicochemical properties to determinate the hydrophobic and hydrophilic character of the cell surface but also its electron donor and acceptor characters to have an evaluation and to further control the biofilm formation and adhesion owing to these properties and the environmental conditions (acidic or basic environment). These properties have been evaluated via
R. Djeribi et al. / Colloids and Surfaces B: Biointerfaces 102 (2013) 540–545
two different methods using the microbial adhesion to solvents method (MATS) and contact angle measurements (CAM).
Table 1 Affinities of A. baumannii cells to the four solvents used in the MATS analysis at the different pH values. pH
2. Materials and methods
% of adhesion (±SD)a to: Hexadecane
2.1. Bacterial strain, culture conditions and preparation of microbial suspension A. baumannii was isolated from a patient having an urinary tract infection. The strain was identified by morphological and chemical tests as belonging to the genus Acinetobacter. The identification of the strain at the species level was confirmed by the simplified test of growth at 37, 41, and 44 ◦ C. The strain was sub-cultured overnight and grown in Liquid Luria–Bertani medium (LLB) at 37 ◦ C for 18 h. After the culture, the cells were harvested by centrifugation for 15 min at 5000 × g, washed twice and suspended in phosphate buffered saline solution (PBS, 0, 1 M) at the desired pH used in the experimental hydrophobicity assay.
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pH 4 pH 5.5 pH 6.5 pH 7.2 pH 8 a
13.26 11.03 11.30 5.88 9.32
± ± ± ± ±
2.04 1.52 1.04 0.66 0.52
Chloroform 24.39 22.85 37.02 17.95 20.70
± ± ± ± ±
Hexane
2.51 1.69 2.37 1.28 0.78
17.44 8.42 13.11 8.24 13.90
Diethylether
± ± ± ± ±
0.76 0.87 0.91 0.94 0.62
15.15 29.79 42.55 49.78 52.41
± ± ± ± ±
1.72 1.46 1.59 7.19 2.04
±Standard deviations of three measures of at least three separate experiments.
(S) were estimated from the approach proposed by van Oss et al. In this approach, which neglects spreading pressure, the pure liquid (L) contact angles () can be expressed as: Cos = −1 +
2(SLW LLW ) L
1/2
+2
(S+ L− ) L
1/2
+
2(S− L+ )
1/2
L
2.2. Microbial adhesion to solvents (MATS) 2.4. Biofilm observation by scanning electron microscopy (SEM) Acid–base interactions can be assessed from microbial adhesion to solvents MATS. The partitioning method previously described by Bellon-Fontaine et al. is based on the comparison between the microbial cell affinity to a monopolar solvent and an apolar solvent [20]. The monopolar solvent can be acidic (electron accepting) or basic (electron donating) but both solvents must have similar Lifshitz–van der Waals surface tension components. On this basis, the following pairs of solvents were selected: (a) chloroform, an acidic solvent (electron acceptor) with hexadecane, an apolar solvent; (b) diethyl ether, a strong basic solvent (electron donor) and hexane, an apolar solvent. Chemical products (chloroform, hexadecane, diethyl ether and hexane) having a highest purity grade ≥ 99.8% were obtained commercially (Aldrich Chemical, Co., Inc. USA). Experimentally, the bacteria were suspended at an optical density (A0 ) at 600 nm of 0.8–0.9 in PBS 0.1 M, with the pH adjusted to 4, 5.5, 6.5, 7.2, 8 by the addition of HCl or NaOH. Subsequently, 0.3 ml of the solvent was added to 1.8 ml of the bacterial suspension, after which the two phase system was vortexed for 2 min and allowed for 20 min to ensure complete separation of the two-phases (organic and aqueous phase). The optical density (A) of the aqueous phase was measured. The percentage of bound cells was subsequently calculated by the formula: Adherence (%) =
(A − A) 0 A0
× 100
where A0 is the optical density of the bacterial suspension measured at 600 nm before mixing. Experiments were repeated three times with separate cultured bacteria. 2.3. Contact angle measurements and estimation of bacterial surface tension components Cell surface hydrophobicity is an expression of the Lifshitz–Van der Waals and acid–base surface tensions, LW and AB of the cell surface [9] and is usually assessed from contact angles of water, formamide and diiodomethane (purity ≥ 99%) on lawns of partially dehydrated bacteria using the sessile drop technique [21]. Briefly, the bacteria suspended in 10 ml of PBS (with the appropriate pH) were layered onto 0.45 m pore size filters (Sartorius) using a negative pressure. The filters were left during about 30 min to air dry at room temperature until the so-called ‘plateau contact angles’ could be measured. Experiments were carried out in triplicate with separately cultured bacteria. The Lifshitz–van der Waals ( LW ), electrondonor ( − ) and electron-acceptor ( + ) components of the bacteria surface tension
Scanning electron microscope analysis was used to observe A. baumannii biofilm colonizing the intraluminal surface of urinary catheters. Sterile silicone Foley catheters were cut into 2 mm-thick discs. The discs were aseptically introduced into tubes containing nutrient broth, which was previously inoculated with A. baumannii culture. After homogenization, the culture was incubated at 37 ◦ C. After one week, the tubes were cleaned of bacterial cultures, and the discs extensively washed with sterile distilled water in order to analyze them by SEM. Support samples were then dried at 37 ◦ C for 24 h. Once coated with gold–palladium (Sputtering Device), samples were examined under a SEM (Cambridge S200). 3. Results 3.1. Influence of pH on cell surface physicochemical properties using the MATS method Different solvents were employed to evaluate the hydrophobic/hydrophilic cell surface properties of A. baumannii and their Lewis acid–base characteristics. Both apolar solvents hexadecane and hexane were used to estimate the hydrophobicity properties of A. baumannii, while the two monopolar solvents, chloroform and diethyl ether, were selected for the estimation of the Lewis acid/base (i.e., electron donor/acceptor) character. The MATS results obtained for A. baumannii at different pH are shown in Table 1. Assays were performed in high ionic strength solution (PBS, 0.1 mol/l) to minimize electrostatic interactions between A. baumannii and solvents. First, direct measurements of the cell surface hydrophobicity and hydrophilicity were carried out by partitioning the cells between aqueous and hexadecane phases. The percentages of bacteria adhering to this apolar solvent, range from 5.88 to 13.26%. This result demonstrates the hydrophilic surface of A. baumannii at the different values of the pH tested. Regardless the pH, the affinity of A. baumannii was always higher with chloroform (an electron acceptor solvent) than with hexadecane (a nonpolar solvent). The differences in bacterial affinity between these two solvents were due to Lewis acid–base interactions (i.e., electron donor–electron acceptor interactions resulting from the electron donor nature of the bacteria) [20]. As shown in Fig. 1, the electron donor character was stable and relatively low at the pH values (pH 4, pH 5.5, pH 7.2 and basic pH 8). Nevertheless, at pH 6.5, A. baumannii strain presents a clear maximum value of electron donor.
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Fig. 1. Variations in the surface electron donor character of A. baumannii at the different pH, estimated with the MATS method.
Fig. 3. SEM picture of Acinetobacter baumannii biofilm formed on an urinary catheter (magnifications: ×3000).
Fig. 2. Variations of the surface electron-acceptor character of A. baumannii at the different pH estimated with the MATS method.
Similarly, the acidic character of A. baumannii was evaluated by comparing the adhesion of A. baumannii to the monopolar basic diethyl ether (a strong electron-donor solvent) and to the apolar hexane solvent [20]. Fig. 2 showed the electron acceptor character of A. baumannii at the different pH. The affinity to diethyl ether increased with the pH and A. baumannii showed the maximum electron acceptor character at pH 7.2 and a non-negligible value at pH 8. However, this electron-acceptor character was null or negligible at the most acidic pH (pH 4). 3.2. Influence of the pH on the cell surface physicochemical properties using the contact angle measurements The results of the contact angle measurements are shown in Table 2. The measured contact angle with water indicates the qualitative change in the hydrophobicity of the bacterial surface as a
function of pH. With the exception of pH 6.5, the results show that A. baumannii has a hydrophilic character for the different pH values. At pH 6.5, the strain is hydrophobic as shown by the contact angle, E = 75.60◦ ± 5.54. Moreover, the value of Giwi allowed us to obtain a quantitative measure of the hydrophobicity [22]. The bacterium surface is very hydrophobic at pH 6.5, relatively hydrophobic at pH 4 while and it is hydrophilic at pH 5.5, 7.2 and 8. The results showed also the increasing electron-donor character with increasing pH, except for pH 6.5, at which a significant drop in the value of the electron-donor character can be noticed. The electron-acceptor character decreases with the increasing pH, with an exception at pH 5.5, for which we observe that the value of the electron-acceptor character is almost negligible (Table 2). 3.3. Scanning electron microscopy analysis of the biofilm A. baumannii biofilm colonizing the intraluminal surfaces of the urinary catheters was examined by SEM analysis (Fig. 3). The biofilm is characterized by a multitude of intercellular pores and interconnected channels, which allow not only the exchange of information and the flow of water and ions, but also the transport of nutrients and the efflux of waste products. SEM analysis suggests heterogeneous mosaic architecture of biofilm containing micro-colonies of bacterial cells encased in an extracellular polymeric substances (EPS) matrix and separated by interstitial voids. It is worth noting that the biofilm is observed at the urinary pH of about 6.5, which is consistent with the hydrophobicity of A. baumannii at this pH.
Table 2 Lifshitz–Van der Waals surface tension components ( LW ), electron-donor ( − ), and electron-acceptor ( + ) values of A. baumannii at the different pH. pH values
pH 4 pH 5.5 pH 6.5 pH 7 pH 8
Contact angles (◦ )
Surface tension components (mJ m−2 )
E
F
D
LW
+
−
AB
T
Giwi
51.70 (±2.95) 55.10 (±5.94) 75.60 (±5.54) 27.40 (±3.53) 43.30 (±2.82)
42.60 (±4.52) 62.40 (±2.79) 60.60 (±2.80) 41.00 (±4.27) 66.00 (±4.75)
92.20 (±5.78) 67.10 (±0.27) 93.00 (±4.89) 70.80 (±3.90) 59.60 (±4.81)
11.8 24.5 11.4 22.4 28.8
11.3 0.1 7.7 2.4 0.7
26.4 42.0 9.6 59.1 69.4
34.6 3.6 17.2 23.7 14.0
46.4 28.1 28.6 46.1 42.8
−2.45 26.94 −21.10 28.78 54.32
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4. Discussion Several studies have reported the effect of pH on the physicochemical properties of the bacterial surfaces [17–24]. The results using MATS method revealed that A. baumannii has a hydrophilic character at the different pH tested. According to several studies, it was admitted from the chemical point of view that the hydrophobic cells tend to adhere to a hydrophobic substrate, while the hydrophilic cell surface tend to adhere onto a hydrophilic substrate [10–25]. However, our results from the MATS method do not reflect this assumption. It is worth noting that the strain was isolated from a hydrophobic silicone-based urinary catheter surface and that A. baumannii has a hydrophilic character. At the present time, such contradictions for this hydrophobic/hydrophilic aspect [22] exist because the adhesion phenomenon cannot be treated only by taking in account the sole hydrophobicity effect, but the other parameters such as physical and chemical properties of bacterial cells and surface colonization have to be investigated and be taken in account. On the other hand, the results of the MATS method showed that the hydrophilic nature of this strain is associated with a more important Lewis acid–base character. The electron-donor character may be an indication of the nature of chemical groups exposed on the surface of the bacteria. The predominance of electron-donor character is attributed to the chemical groups generally negatively charged or neutral, such as carboxylate groups (COO ), amino groups (NH2 ) and phosphate (PO4 ) groups from phospholipids, lipoproteins and lipopolysaccharides as well as SO3 groups from sulfur clusters [17–23]. The donor character would normally be stronger if such carboxylic or phosphate groups are deprotonated, giving rise to the corresponding anions. It will be then expected to observe an increase in the donor character when the pH increases. On the contrary, our results show a decrease in the basic character of the bacterial surface at pH 7.2 and pH 8. It can be then be assumed that increasing the pH causes the deprotonation of chemical groups which increases the electron donor character. This deprotonation causes, in parallel, a negatively charged bacterial surface. Based on similar studies which reported that the deprotonated carboxylate groups and phosphate play a predominant role in determining the negative charge on the bacterial surface and that the contribution of these groups in the negative charge is pH dependent [24], we can assume that the increase of negative charges on the surface of the bacteria will act in a repulsive way with negatively charged solvent [17]. This could then explain the reduction of the affinity of the isolated strain to solvents. The results showed also that the electron-acceptor character for A. baumannii increases when we increase the pH of the buffer. The importance of the electron-acceptor character can be attributed to the presence of acidic groups (such as NH3 + ) groups and hydroxyl (OH) groups exposed on the cell surface [24]. However, the results obtained by Hamadi et al. showed that the electron-acceptor character of the two strains studied, Escherichia coli and Staphylococcus aureus, is maximal at acidic pH values (pH 2 and pH 3), and low at the other pH values studied (pH 5, 6.2, 9 and 11) [23]. Based on the results of the MATS method, we can deduce that the adherence of A. baumannii is due to the surfaces, at least in part, to Lewis acid–base interactions of the surface which is in agreement with the studies of several authors who have demonstrated the importance of the acid–base character in bacterial adhesion [17]. The method that consists of measuring the contact angle was aimed to characterize, by a second method, the changes in the physicochemical properties of the A. baumannii surface as function of the pH and to correlate these results with the results obtained previously by the MATS method. To our knowledge, this is the first study that used CAM method to evaluate the effect of pH on the physicochemical properties of a microbial surface. Unlike the
Fig. 4. Correlation between the variation of electron-donor character and the hydrophobicity of A. baumannii.
results obtained with the MATS method, for which the bacteria was found to be hydrophilic at the different values of pH tested, the CAM method results showed a very significant effect of pH on the hydrophobicity of the surface of the A. baumannii bacterium. Indeed, the bacterium is very hydrophobic at pH 6.5, relatively hydrophobic at pH 4 and hydrophilic at pH 5.5, 7.2 and 8. This paradox in the determination of hydrophobicity/hydrophilicity between the two methods was reported in some studies [26]. The results in the acid–base character as function of pH suggest that the electron-donor character increases with the pH. This increase is explained by the protonation of chemical groups, particularly groups of the type COO− and PO4 , which play an important role in the determination of electron-donor estimated by the CAM method. However, the decrease in the electron-donor character at pH 6.5 cannot be explained in terms of protonation/deprotonation of chemical groups. It is assumed that the hydrophobicity at this pH masked the chemical groups. This suggestion is reinforced when comparing the change in the electron-donor character of the strain with the change in hydrophobicity obtained at different pH (Fig. 4). The results showed a negative correlation (r = −0.84) between the variation of the electron-donor character and the hydrophobicity of the surface, indicating that the increase in hydrophobicity induced a decrease in the electron-donor character, which is observed at pH 6.5 and pH 4. The study of the electron-acceptor character revealed its increase along with a pH decrease. The importance of the electronacceptor character can be attributed to the protonation of groups such as NH3 + groups, and OH groups exposed on the surface of the bacteria [24]. Our results are consistent with the results obtained by Hamadi et al. who reported that the electron-acceptor character is more pronounced at acidic pH. No explanation has yet been found for the reduction of electron-acceptor character at pH 5.5 [23]. A limited number of publications dealing with the comparison between the results from both methods MATS and CAM were found. The comparison between the two methods was made for the first time by Bellon-Fontaine et al. [20]. The authors have shown that the results from both methods are comparable. In this present study, the results obtained from both methods are compared in order to properly estimate the effect of pH on the physicochemical properties of the surface of A. baumannii. According to the results obtained by the method MATS, we note that whatever the pH studied, A. baumannii has a hydrophilic character. By contrast, the results based on the contact angle measurements method showed a significant influence of the pH on the hydrophobic/hydrophilic nature of the A. baumannii surface (Table 3).
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Table 3 Changes in hydrophobicity of A. baumannii estimated by both methods as function of the pH. pH values
Hydrophobicity/ hydrophilicity estimated by (MATS)
Hydrophobicity/ hydrophilicity estimated by (CAM)
4 5.5 6.5 7.2 8
Hydrophilic Hydrophilic Hydrophilic Hydrophilic Hydrophilic
Relatively hydrophobic Hydrophilic Hydrophobic Hydrophilic Hydrophilic
Fig. 5. Correlation between the results of the hydrophobicity of A. baumannii measured by both methods MATS and CAM at different pH values.
Although the findings on the hydrophilic/hydrophobic surface behavior of the strain are different at certain pH, both methods show identical trends. For instance, Fig. 5 shows a correlation between both methods (r = 0.72) in estimating the change in hydrophobic/hydrophilic surface A. baumannii. Some differences exist at pH 6.5 and pH 4. The strain surface is hydrophilic by the MATS method and hydrophobic by the CAM method. In a similar study [26], using seven different strains and different culture conditions, the authors showed that the results of both methods MATS and MAC do not give the same conclusions for the different strains studied. In Fig. 6, we can also notice that the results concerning with the acidic/base character study of the bacterial surface as a function of the pH differ from both methods. Indeed, at pH 5.5, 7.2 and 8, the bacterium has a larger electron-donor character using the CAM method (Fig. 6b). By contrast, A. baumannii presents an electronacceptor character by the MATS method (Fig. 6a). However, a fairly good correlation on the acid–base character as measured by both methods is verified in the case of pH 6.5. Indeed, the results from the MATS method and the results from the CAM method show a balance between characters, donor and electron-acceptor. This suggests that at pH 6.5 the bacterium is in acid–base equilibrium. The contradiction in the measurements on the surface physicochemical properties of the bacterium using both methods can be attributed to the characteristics of each method. The measurements by the MATS method are performed on fully hydrated cells and two types of interactions are considered: one interaction between bacterial cells and the solvent and the other between the bacterial cells and the buffer. In contrast, for the contact angle measurements method, the estimation of hydrophobicity and the acid/base character of the bacterial surface are carried out on semi-hydrated bacterial cells. Therefore, the interactions evaluated by this method concern only with interactions between bacterial cells and solvent molecules (water, formamide or diodomethane). According to Rosenberg, the different methods for estimating the hydrophobicity (BATH, SAT, CAM, etc. . .) can differ depending on
Fig. 6. Variation of the acid–base character for A. baumannii surface as function of the pH. (a) Results from the MATS method; (b) results from the CAM method.
the nature of the hydrophobicity, and the way to measure it [27]. Hamadi and Latrache showed that the phosphate and amine groups play an important role in the electron-donor/acceptor character determination estimated by CAM [26]. The protein/polysaccharide interactions may be the source of the electron-donor character estimated by MATS [26]. It should be also noted that the MATS method provides a qualitative estimation of the hydrophobic/hydrophilic and electron-donor/acceptor characteristics. In the other hand, the CAM method combined with the equation of van Oss can determinate quantitatively the acid–base character and the hydrophobic/hydrophilic surface properties more accurately in terms of surface free energy [19]. 5. Conclusions In conclusion, the combination of the results from both techniques, taking into account the limitations of each, confirms, on one hand, the effect of the pH on the physicochemical properties of the A. baumannii surface, and on the other hand, it shows a reasonable correlation on the acid–base character measured by both methods at pH 6.5. The acid–base behavior of this strain at pH 6.5 may suggest a particular effect of this pH on the physicochemical properties of the A. baumannii surface that will provide us with a better understanding and information on the ability of this bacterium to adhere to urinary catheters. Acknowledgement This work was supported by the Grants-in-Aid from the Algerian Ministry of Education and Scientific Research (Direction of Scientific Research and Technological Development) to which the authors are very thankful. References [1] J. Baraibar, H. Correa, D. Mariscal, M. Gallego, J. Valles, J. Rello, Chest 112 (1997) 1050–1954. [2] E. Bergogne-Berezin, K.J. Towner, Clin. Microbiol. Rev. 9 (1996) 148–165.
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