Adhesion Behavior of Escherichia coli Strains on Glass: Role of Cell Surface Qualitative and Quantitative Hydrophobicity in Their Attachment Ability

Microbial adhesion to surfaces is thought to involve physicochemical interactions between the substrate and microbial cells. Understanding the physicochemical aspects involved in the adhesion phenomenon, as a critical step in biofilm formation, is essential to finding ways to prevent their formation and control biocontamination risks. The aim of this study was to investigate the relation between the adhesion behavior of 12 Escherichia coli strains isolated from food and their surface hydrophobicities using qualitative (θw) and quantitative (ΔGiwi) approaches. The surface physicochemical properties of both bacterial cells and glass material were estimated through contact angle measurements. The adhesive behavior of E. coli strains on a glass surface was assessed. The results showed a good logarithmic relation between the percentage of the adhered cells and their surface hydrophobicity with the quantitative approach ΔGiwi; however, qualitative hydrophobicity (θw) appeared to demonstrate no effect regarding adhesion behavior. This work lays the foundation for future studies and opens an important debate on the mechanisms underlying the adhesion behavior of E. coli strains by using the thermodynamic approach (ΔGiwi) as an important model of hydrophobicity that could explain and predict better bacterial adhesion ability.


Introduction
In natural environments, as well as in the food industry, microorganisms are most often attached to solid surfaces and are generally provided with sufficient nutrients to ensure their viability and growth. is adhesion ability of bacteria to surfaces is cause for concern for many industries, particularly the food industry. In fact, microbial adhesion to surfaces can act as a source of contamination, which may compromise hygienic and food quality [1]. E. coli is an important foodborne pathogen that causes thousands of cases of foodborne diseases annually across the world [2,3]. Several studies have been conducted, therefore, to determine the factors and mechanisms leading to the microbial attachment to food contact surfaces [4][5][6][7][8][9][10].
Bacterial hydrophobicity generally refers to the tendency of a bacterial cell to interact with cells of similar hydrophobicity as opposed to water (θ w ) [19]. Most previous works have used the qualitative hydrophobicity (θ w ) measured directly by contact angle with water to explain the adhesion behavior of many bacterial species [15,[20][21][22]. However, several studies reported that the qualitative hydrophobicity expressed as wettability with water (θ w ) could not completely explain the adhesion behavior of many bacteria [4,7,20,21,23]. erefore, the free energy of interaction (ΔG iwi ) was proposed by Van Oss et al. [24] as a quantitative method to assess cell hydrophobicity. is parameter is directly related to the interfacial tension and considers not only the contact angle measured with water (θ w ) but also the Lifshitz-van der Waals and acid-base interactions (electron donor and electron acceptor properties). To our knowledge, no previous work has discussed which hydrophobicity method could better explain the microbial adhesion behavior of E. coli strains.
To control biocontamination in food environments, it is important to understand the physicochemical aspects involved in the initial deposition of bacteria onto surfaces. erefore, this study investigates the relation between the adhesion behavior of 12 E. coli strains isolated from food and their surface hydrophobicities using qualitative (θ w ) and quantitative (ΔG iwi ) approaches.

Bacterial Strain Selection.
Twelve Escherichia coli strains isolated from food and identified at the Laboratory of Microbiology and Hygiene of Food and Environment of the Pasteur Institute Morocco were used in this study. One hundred samples received for a month from different food sources (catering, food industries, hotels, and supermarkets) were analyzed. e procedures for isolation of E. coli from different samples given in this protocol follow the ISO-9308-1:2014 standards [25].
Among the examined samples, 12 out of 100 samples were tested positive for E. coli isolation. Positive samples showed typical pink colonies on MacConkey agar and showed characteristic green metallic sheen on EMB agar. Biochemical characters showed citrate utilization test negative, H2S production in TSI agar negative, indole production positive, urease activity negative, methyl red test positive, Voges-Proskauer negative, and lysine decarboxylase positive. e isolates were confirmed also using a biochemical test (Api 20E, BioMérieux, France).
Typing of E. coli isolates was performed in the Laboratory of Microbiology and Hygiene of Food and Environment of the Pasteur Institute Morocco according to Kauffmann and Das Kauffmann [26]. e E. coli strains were typed (13; 19; 38; 64; 76; 65; EI1; EI2; EI3; EI4; EM3; and EM4) as a function of the samples' codes and then stored at −20°C in glycerol stocks before analysis.

Bacterial Growth Conditions and Preparation of Bacterial
Suspensions. Each bacterial strain was incubated overnight at 37°C in Liquid Luria-Bertani (LLB) medium, which contains tryptone, yeast extract, and NaCl. After 24 h of incubation, bacterial cells were harvested by centrifugation at 8400 × g for 15 min, and washed twice with, and resuspended in, KNO 3 solution with ionic strength 0.1 M. e bacterial optical densities (ODs) were adjusted using a spectrophotometer to approximately 0.7 and 0.8 corresponding to 10 8 CFU/mL for subsequent experiments [9]. e bacterial suspension was filtered using a 0.45 μm cellulose acetate filter (Sartorius). A thick lawn of cells was obtained after filtration by means of negative pressure. e wet filters were placed carefully on a glass support with double-sided sticky tape and allowed to air-dry until the socalled stable plateau contact angles could be measured [9].

Cleaning and Preparation of Solid Surfaces.
e substrate used for the adhesion experiments was glass. e glass samples were microscope slides (RS, France). Before each experiment, substrates were immersed in 95% ethanol for 15 min and then rinsed six times with distilled water. Finally, the substrate was autoclaved for 15 min at 120 • C [9].

Cell Surface and Substratum Characterization.
e physicochemical properties of the bacterial surface and substratum were determined using a goniometer (GBX instruments) by the sessile drop method according to Busscher [27]. e surface energy of bacteria and substratum surfaces was determined by measuring the contact angle with three liquids: water, formamide 99%, and diiodomethane 99%. ree to six drops of liquid were placed on each solid material and bacterial filter (described earlier). e qualitative hydrophobicity expressed as θ w was directly analyzed by measuring the contact angle with water. e quantitative hydrophobicity was determined using the Van Oss approach [24], which explains hydrophobicity as the free energy of interaction between two materials when immersed in water, denoted as ∆G iwi . e surface is considered hydrophobic or hydrophilic if its free energy is negative (∆G iwi < 0) or positive (∆G iwi > 0), respectively. e free energy can be estimated from the surface tensions of interacting entities according to the following formula: Furthermore, the electron donor (c − ) and acceptor (c + ) characteristics and the Lifshitz-van der Waals (c LW ) interactions were estimated by the approach proposed by Van Oss [24]; the contact angle can be expressed as follows: where S and L denote solid surface and liquid phases, respectively. Lastly, the Lewis acid-base components can be identified as follows: e surface free energy components of water, formamide, and diiodomethane are known (Table 1), but the corresponding values for the entity i have to be determined.

Adhesion Assay.
e bacterial suspension was placed in a Petri dish containing the sterilized materials (glass). After 3 h of incubation at 25°C, nonadherent cells were eliminated by three consecutive rinses with sterile distilled water [28,29]. e glass samples were dried at room temperature, and then a Gram coloration was performed to assess bacterial adhesion. e adhesion on the glass surface was examined by using an optical microscope (× 400) (Olympus CH30). To estimate the percentage of the surface occupied by adherent cells, the obtained images after observation by microscopy were treated using an algorithm developed in the MATLAB software program [8].
2.6. Statistical Analysis. All physicochemical analyses and adhesion assays were conducted with three repetitions. ree to six contact angle measurements were performed on each bacterial filter and glass surface. e correlations studies were carried out between water contact angle measurement (θ w ), the free energy of interaction (ΔG iwi ), and the percentage of surface occupied by adhered cells using SPSS 20.0 software.

Cell Surface and Substratum Characterization.
e physicochemical characteristics of both microbial cells and material surfaces play an essential role in microbial adhesion phenomena. To control and inhibit adhesion, an understanding of the mechanisms involved in the interaction between microbial cells and substrates is required. ereby, the physicochemical surface properties of E. coli strains and glass surfaces were measured by the contact angle (Table 2).
Hydrophobicity is often expressed in terms of contact angle formed by a sessile drop of water θ w . In the present work, we used this parameter to express the qualitative hydrophobicity of both bacterial strains and substratum. According to Vogler [30], hydrophobic surfaces exhibit water contact angle values higher than 65°, whereas hydrophilic surfaces gave water contact angle values lower than 65°. Table 2 illustrates the bacterial physicochemical characterization by the contact angle method. Based on contact angle with the water value θ w , the results show that E. coli strains codified EI1, 38, EM3, 64, 19, EM4, and EI4 showed hydrophobic values ranging from 69.44°to 99.8°; however, the E. coli strains 13, 76, EI3, EI2, and 65 were hydrophilic (33.22-56.5°).
Referring to the results in Table 2, it appeared that the E. coli strains 13, 76, EI3, EI2, and 65 were hydrophilic according to both qualitative and quantitative approaches. Moreover, the glass surface was qualitatively (θ w � 36.14°) and quantitatively (ΔG iwi � 38.61 mJ/m 2 ) hydrophilic.

Adhesion Tests.
e adhesion ability of E. coli strains isolated from food origins on an inert surface (glass) was examined. Optical microscopic images of E. coli strains after 3 h of contact on the glass are provided in Figure 1. e percentages of the occupied surface by cells on the glass are presented in Figure 2.
A comprehensive understanding of E. coli adhesion relies on establishing how the physicochemical properties of bacterial surfaces are involved. erefore, we investigated the correlation between the adhesion behavior of E. coli strains and their surface hydrophobicities with qualitative (θ w ) and quantitative (ΔG iwi ) approaches.
Using SPSS software, we analyzed several correlation models (linear, logarithmic, and exponential) between the percentage of adhered cells of E. coli strains and their surface hydrophobicities with both approaches (θ w ; ΔG iwi ) to identify the best correlation model revealing the relationship between the adhesion capacity of our strains and their surface properties.
Correlation analysis showed no significant correlation between the cells' qualitative hydrophobicity (θ w ) and their adhesion intensity; however, correlation results revealed a highly significant logarithmic relation between the adhesion     (Figure 3). e coefficient of determination R 2 indicates a good correlation between the percentage of adhered cells of E. coli and their surface hydrophobicity for hydrophobic (R 2 � 0.9989) and hydrophilic (R 2 � 0.7914) characteristics separately. For a better understanding of this relationship between the adhesion behavior of E. coli strains and their hydrophobic nature, Figure 4 illustrates the distribution of the percentage of E. coli adhered cells as a function of their quantitative hydrophobicity (ΔG iwi ). e results indicated an increase and decrease in the percentage of adhered cells with negative (ΔG iwi < 0) and positive (ΔG iwi > 0) free energy of interaction values, respectively. In addition, the adhesion of E. coli strains onto the glass substrate was more pronounced when going from extreme negative and positive values of quantitative hydrophobicity (ΔG iwi ) to centric values (close to 0); however, this behavior was not observed with qualitative hydrophobicity (θ w ) (Figure 4).

Discussion
e adhesion of microorganisms to surfaces is the first step in biofilm formation. is adhesion stage is largely governed by physicochemical interactions, primarily Lifshitz-van der Waals, Lewis acid-base, and electrostatic ones.
ese interactions depend on the physicochemical properties that include hydrophobicity, electrostatic charge, and electron donor/electron acceptor characteristics.

International Journal of Microbiology
Surface hydrophobicity is generally thought to be the main factor in microbial adhesion. Several authors consider hydrophobicity to be the key parameter governing bacterial adhesion to surfaces [4,15,[31][32][33][34][35][36]. Contact angle with water expressed as wettability (θ w ) is generally used to assess surface hydrophobicity. However, with this approach, it is only possible to evaluate hydrophobicity qualitatively [9,10]. erefore, Van Oss and coworkers [24] proposed a quantitative determination of surface hydrophobicity ΔG iwi that takes into account several surface properties' parameters (the electron donor (c−) and acceptor (c + ) characteristics and the Lifshitz-van der Waals (c LW )).
Several research works have used either qualitative or quantitative hydrophobicity to explain the adhesive behavior of the studied microorganisms. However, to our knowledge, no previous studies have investigated which of these hydrophobicities' parameters could explain better the adhesive behavior of E. coli strains. erefore, the present study aimed to evaluate the adhesion ability of 12 E. coli strains isolated from food to adhere to an inert surface (glass) and then investigate the relation between their surface hydrophobicities and their adhesion behavior according to quantitative (ΔG iwi ) and qualitative (θ w ) approaches. Table 2 provides the contact angle measurement results and shows that the glass support had a hydrophilic surface as determined by both qualitative (θ w ) and quantitative (ΔG iwi ) approaches, which concurs with previous researchers [4,13,32,35,36]. e findings in Table 2 also indicate that E. coli strains had different hydrophobicities, depending on the two aforementioned approaches. In fact, only E. coli strains 13, 76, EI3, EI2, and 65 were hydrophilic according to these approaches.
ese results showed similarities with those of Hamadi [9,12], who reported that E. coli strains isolated from patients with urinary tract infections are hydrophilic with both approaches.
According to the literature, the hydrophobicity of a bacterial cell is largely influenced by the residues and structures on the cell surfaces, which can be hydrophilic or hydrophobic [10,19,37]. Hence, bacterial hydrophobicity varies among species and strains, even within the same strain, depending on the mode and stage of growth, growth medium composition, and even the analysis technique [7,9,38,39], potentially explaining the variation of hydrophilicities even within the same species in our work. e adhesion assay indicated that E. coli strains showed a different ability to adhere to glass materials (Figure 1). To investigate the relation between the adhesion behavior of E. coli strains and their physicochemical surface properties, we evaluated the correlation between E. coli adhesion behavior and their surface hydrophobicities using qualitative (θ w ) and quantitative (ΔG iwi ) strategies.
e results in Figure 3 demonstrated a strong logarithmic correlation between the free energy of the interaction (∆G iwi ) and the adhesion of E. coli strains onto glass supports; however, no significant correlation with the qualitative hydrophobicity was observed. Similarly, previous studies investigating the relationship between the hydrophobicity properties (θ w ) of E. coli and their attachment ability have not found a correlation between the degree of hydrophobicity of the supporting surfaces and the number of adhered cells [34,40,41]. Several works, including this one, have reported that qualitative hydrophobicity (θ w ) is a general concept that cannot explain systematically the microbial adhesion results for many supports [4,42,43]. According to Goulter [2], qualitative hydrophobicity is a general concept that cannot be directly measured but it can be only estimated by observing the bulk properties of numerous cells and interpreting these interactions as those of molecules. is could be a primary factor that makes many studies unable to find a significant correlation between qualitative hydrophobicity and adhesion behavior [4,7,23].
Few studies have used the thermodynamic approach in the interpretation of adhesion behavior. However, this strategy could represent an important tool in understanding microbial adhesion because it takes into account the Lifshitz-van der Waals interactions and acid-base interactions that are known to play an essential role in adhesion [9,44]. Based on the results obtained here, we suggest that the adhesion of E. coli strains on a glass substratum is mainly governed by surface quantitative hydrophobicity (ΔG iwi ). erefore, we propose that the free energy of interaction (ΔG iwi ) should be further investigated as an important parameter to understand and predict the adhesion behavior of bacterial strains.

Conclusion
e mechanism of the initial attachment of E. coli strains to surfaces is most likely a complex process involving several factors. Many aspects, such as hydrophobicity, have been shown to play an essential role in cell attachment. is study demonstrated that qualitative hydrophobicity (θ w ) could not explain systematically the adhesion behavior of 12 E. coli strains; however, the quantitative hydrophobicity (ΔG iwi ) showed a good logarithmic relation with the percentage of adhered cells. is work provides an important approach for understanding the mechanisms underlying different aspects of adhesion behavior by focusing on studying the thermodynamic approach (ΔG iwi ) as an important model of hydrophobicity that could predict E. coli adhesion behavior.
To end, a comparison of E. coli strains adhesion on other substrates and the study of their biofilm formation could better inform on their behavior according to their surface qualitative and quantitative hydrophobicities. is strategy could help in understanding and controlling the adhesive behavior of these strains in order to reduce biocontamination risks in food industries 6. Disclosure

Data Availability
e data used to support the findings of this study are included within the article.

Conflicts of Interest
e authors declare that there are no conflicts of interest.

Authors' Contributions
Hassan Latrache, Hafida Zahir, and Ellouali Mostafa designed the study and corrected the final manuscript. Elfazazi Kaoutar, Btissam Mayoussi, Safae Tankiouine, and Chorouk Zanane conducted the experimental work. Souad Lekchiri contributed to data analysis and article writing and correction. El Mostafa Mliji conducted the bacterial identification. Elfazazi Kaoutar performed the data analysis and wrote the article.