In-situ study of early stages of biofilm formation under different environmental stresses by ATR-FTIR spectroscopy

F. Humbert and F. Quilès

Laboratoire de Chimie Physique et Microbiologie pour l’Environnement (LCPME), UMR 7564 CNRS - Nancy Université, 405 rue de Vandoeuvre - 54600 Villers-lès-Nancy, France

The Attenuated Total Reflectance - Fourier Transform InfraRed (ATR-FTIR) spectroscopy has been very early applied for studying biofilms, in situ, nondestructively, in real time, and under fully hydrated conditions. Because its analysis depth is very thin, typically of the order of 1-2 µm, the ATR-FTIR technique is an interesting tool for monitoring in situ, via the ATR-FTIR fingerprints of biomolecules, not only initial stages of biofilm formation but also subsequently response of the base sessile bacteria monolayer to environmental condition changes and for studying the influence of environmental conditions on bacterial adhesion, biofilm growth or detachment processes. For example, the time evolution of the bands assigned mainly to proteins, nucleic acids, and polysaccharides allows for conveniently monitoring biomass accumulation on the ATR crystal, whereas the detailed analysis of the whole spectral profile may provide information about biochemical and structural changes accompanying bacterial adhesion, biofilm development, detachment processes and environmental condition changes (temperature, hydration rate, nutrient quality and quantity, biocide addition, …). Consequently, ATR-FTIR technique is a valuable tool for gaining more insight, at the molecular level, into bioadhesion mechanisms and biochemical and physiological changes during the early stages of biofilm development. This chapter presents an overview of the uses of ATR-FTIR spectroscopy in this research field and illustrates this approach with some of our recent findings. The basis, the potential, the artefacts, and the current limitations of this approach as well as future directions are discussed.

Keywords: nascent biofilm; early stage; FTIR-ATR spectroscopy; in situ monitoring; Pseudomonas fluorescens

1. Introduction

Bioadhesion and biofilm research as well as biofilm control and prevention require tools capable of probing and monitoring at a molecular level, in situ and in real time, physicochemical, physiological and biochemical properties of biofilms especially during the initial stages of their formation. Throughout the past two decades, a great variety of in situ biofilm monitoring methods and devices have been suggested and investigated. They have been reviewed in detail in several papers [1-5]. However, among these numerous techniques, only a few spectroscopies are capable to in situ explore solid-liquid interfaces and to provide this type of information [6], namely, the fluorescence and Attenuated Total Reflectance - Fourier Transform InfraRed (ATR-FTIR) spectroscopies. This chapter addresses the ATR-FTIR technique which, as noted by Nivens et al. [1], provides a unique “inside-out” view of biofilm. This technique is a extremely valuable tool for studying in situ, at the molecular level, any process (adsorption, desorption, speciation, (bio)adhesion, (bio)corrosion,…) occurring at the solid-liquid interface [7]. That is why it is used in various areas other than the microbiology. However, to take fully advantage of this technique in bioadhesion studies and biofilm control it is necessary to have in mind some of its pitfalls and limitations.

2. Attenuated total reflection infrared spectroscopy for studying in situ bioadhesion

2.1 Principle of attenuated total reflection infrared spectroscopy

The infrared (IR) spectroscopy is a well-established technique to identify, in non-destructive way and within a few seconds, functional groups in molecules. It belongs to the class of vibrational spectroscopies because it exploits molecular vibrations. An infrared spectrum is commonly obtained by radiating the sample with IR light and determining what fraction of the incident radiation is absorbed at a particular energy. Conveniently, the frequency is given in wavenumber defined as the reciprocal of the wavelength. Each peak, or vibrational band, in the spectrum corresponds to a vibration of a part of a sample molecule. Molecules experience a wide variety of vibrational motions, characteristic of their component atoms and chemical bonds. For example, vibrations can involve either a change in bond length (stretching usually denoted ) or bond angle (bending denoted ). Some bonds can stretch in-phase (symmetrical stretching, s) or out-of-phase (asymmetric stretching, as). Furthermore, the spectral features of vibrational bands such as the frequency, the full width at half maximum, and the intensity can be highly sensitive to the overall structure and configuration of molecules, as well as their environment. Consequently, an IR spectrum provides a highly specific vibrational fingerprint of the sample under investigation. The drawback is the complexity of IR spectra. However, several methods can aid in spectral peak assignments and extract the principal spectral components for example in using

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chemometric analysis [8]. Infrared spectra are usually collected in transmission mode, that is, the IR beam is passed through the sample and the transmitted IR intensity is measured. However, for the studies of bioadhesion and early stages of biofilm formation the most appropriate mode is the Attenuated Total Reflectance (ATR) mode mainly because of its surface sensitivity. In ATR mode, the sample is placed in contact with a crystal of high refractive index, superior to the sample refractive index (Fig. 1). Typical materials for ATR crystals include germanium, zinc selenide, diamond and silicon. The IR beam is focused onto the edge of the ATR crystal, multiply reflected on the inner surface of the crystal, and then directed to a suitable detector. At each reflection at the sample – ATR crystal interface, an infrared evanescent wave is created into the sample where it can be absorbed, allowing obtaining the infrared fingerprint of the sample. An important feature is that the evanescent wave decays exponentially with distance from the crystal surface. It protrudes only a few microns (0.5 - 5 μm) beyond the crystal surface and into the sample. This is usually characterized by the penetration depth (dp) defined as the distance required for the electric field amplitude of the evanescent wave to fall to e-1 of its value at the surface. It is noteworthy that the depth actually probed by the evanescent wave is greater than dp. However, the more distant molecules are from the crystal surface, the weaker their contribution in the resulting ATR spectrum. dp varies according to the following equation: dp = /[2 (nc

2 sin2 – ns2)1/2] where is the wavelength of the incident radiation,

nc is the refractive index of the crystal, ns is the refractive index of the sample in contact with the crystal, and is the angle of incidence [9]. Therefore the penetration depth is proportional to the wavelength. The longer the wavelength is, the greater the depth of penetration. For example, the probed sample thickness is four smaller at 4000 cm-1 than at 1000 cm-1. This point must be well borne in mind in interpreting an ATR spectrum. The thin penetration depth makes that the ATR-FTIR technique is also ideal for highly absorbing samples such as water suspensions or even bulk water and biological samples which contain very much water. This set of features led as early as the eighties to use the ATR-FTIR spectroscopy for studying bioadhesion in situ, in real time and under fully hydrated conditions [10-12]. Given the very thin analysis depth, only bacteria that are in the immediate vicinity of the crystal surface are detected in ATR mode. That is why Nivens wrote in 1995 [1] that “ATR-FTIR provides a unique “inside-out” view of biofilms”. This feature can be a drawback for studying biofilm development since only the base layer of biofilms is analyzed, except if an optical fiber can be used. Nevertheless, it is also a strength of the method for studying in situ the initial stages of biofilm formation as shown hereafter. Further details about the ATR technique are given in reference 9.

Fig. 1 Principle of the ATR-FTIR technique and some values of evanescent wave penetration depth dp for a sample refractive index of 1.5. is the wavelength of the incident radiation, nc is the refractive index of the crystal, ns is the refractive index of the sample in contact with the crystal, and is the angle of incidence. E0 and Ez are the evanescent electric field amplitudes at the surface of the crystal and at a distance z from the surface, respectively. ATR crystal: Germanium or zinc selenide, =45°; 6 internal reflections; size: 10x80x6mm.

2.2 Bacterial attachment and colonization onto the ATR crystal: ATR-IR fingerprints of planktonic and sessile bacteria

Figure 2 presents the ATR-FTIR spectrum of a partially air-dried pellet of planktonic Pseudomonas fluorescens (Pf), a gram-negative bacterium, harvested at the end of exponential growth phase. This typical bacterial spectrum is mainly the superposition of the ATR-IR fingerprints of five biomolecules:

- water with the symmetric and asymmetric stretching modes of the OH bond in the 3800-3000 cm-1 range, the bending mode at 1640 cm-1, and in the low wavenumber range a broad band which arises from so-called librations, a collective normal mode involving many water molecules, water molecule rotations restricted by hydrogen bonding;

- proteins mainly identified by the N-H stretching and the vibrations of the peptide linkage in particular the amide I band arising mainly from backbone amide C=O stretching vibrations and the amide II band arising mainly of the out-of-phase combination of the NH in-plane bend and the CN stretching vibrations;

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- lipids and phospholipids with mainly asymmetric and symmetric methylene stretching vibrations, and bending (scissoring and rocking) vibrations and, for phospholipids, the asymmetric and symmetric stretching vibrations of phosphate groups;

- polysaccharides mainly localized in the region 1200 – 900 cm-1; - and, finally, nucleic acids associated mainly to the asymmetric and symmetric stretching vibrations of

phosphate groups.

Fig. 2 ATR-FTIR spectra of a) a partially dehydrated pellet of planktonic P. fluorescens harvested at the end of exponential growth phase and b) a nascent Pf biofilm after 1, 1.5, 2, 2.5 and 3h of colonization onto a Ge ATR crystal by flowing a bacterial suspension (~108 CFU/ml) in sterile Luria-Bertani medium (0.5 g/liter) into the ATR flow cell. The spectra are normalized to the amide II band. See ref. 16 for more details. See references 13 and 14 for a more complete assignment. These IR bacterial fingerprints contain a wealth of information which can advantageous be used for example for rapid microbial detection and identification [13, 15]. Concerning bioadhesion studies, a first approach, relatively simple, is to consider only the intensity or area of the amide II band. This band is indeed of particular interest because it is known as a good marker of biomass and is relatively little overlapped with others bands. Thus, a common way for in-situ studying biofilm formation consists in monitoring its intensity or area. In practice, monitoring can be easily performed using a flow system built around an ATR-IR flow cell as described in detail in reference 16. Figure 3a shows a typical spectral evolution when the ATR flow cell is inoculated for 3h with a suspension of bacteria containing ~ 108 CFU/ml in sterile Luria-Bertani (LB) medium (0.5 g/liter). Over the inoculation period the intensity of all bands increases except for the water bands, especially the OH stretching band around 3400 cm-1, which in contrast decrease because the water molecules on the crystal surface are progressively replaced by bacteria that contain a lower water concentration than the bacterial suspension. Note in the OH large band a small positive peak at 3300 cm-1 associated with the NH stretch vibration in proteins.

Fig. 3 Evolution of a) ATR-FTIR spectra over P. fluorescens attachment and colonization onto Ge ATR crystal and b) the amide II band intensity over bacterial colonization on Ge and ZnSe ATR crystals. The solid lines represent the non linear regression fit using the equation A = Amax (1-e-kt) (see the text). c) Another possible kinetic. Same experimental conditions that in Fig 2. This evolution is characteristic of bacterial colonization onto the crystal surface. In the case of Pf, bacteria obey monolayer attachment kinetic as confirmed by epifluorescence microscopy [17]. For this period, mainly of attachment, the rate of increase is reduced as the surface coverage growth. The plot of the amide II band area, A, versus time can be fitted using the following equation: A = Amax (1-e-kt) where Amax is the amide II band area corresponding to a complete bacteria single layer and k is an absorption rate constant [16, 18]. It should be noted that this type of kinetic is an ideal case. Small changes in culture conditions, temperature, growth phase, number of bacteria inoculated in the flow system, hydrodynamic conditions, crystal surface state, and so on can lead to different adhesion kinetics as shown in fig 3c. This

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simple monitoring of the amide II band is an excellent way to control bacterial adhesion quality and to study influence of various parameters on bacterial attachment. However, the intensity of the amide II band is only one element in the wealth of information provided by an IR spectrum. To gain more insight, at the molecular level, into biochemical and physiological changes it is necessary to exploit the whole ATR-IR fingerprints of biomolecules. To illustrate this point, consider again the period of colonization of ATR crystal by P. fluorescens bacteria. During 3-h bacterial inoculation period, as seen previously, the process is essentially an attachment process but not only. After 1h, an increase of the region of the asymmetric and symmetric stretching vibrations of phosphate groups is indeed observed (Fig 2b) reflecting a more important synthesis of nucleic acids associated with cell division. Subsequently, if this 3-h monolayer young biofilm is fed with sterile culture medium to promote biofilm formation, the intensity of all bands continues to increase during 3 to 4 hours but the polysaccharide band increases at a higher rate than the amide II band, reflecting not only cell division but also an overproduction of extracellular polysaccharides [17]. At this stage, the biofilm still consists of a single layer of bacteria monolayer as confirmed by epifluorescence microscopy. Subsequently, the band intensity still continues to increase during about 8 hours but significant changes occur in the spectral profile reflecting structural and compositional changes in the biofilm such the formation of a second layer and secretion of glycogen [17].

2.3 What is actually probed in ATR experiments? Pitfalls and tips

As illustrated above and hereafter, ATR-FTIR has undeniably a great potential to gain more insight, at the molecular level, into the physiological and structural changes accompanying bioadhesion processes and biofilm development. However, it is necessary to be extremely cautious in interpreting ATR spectra which are more complex than transmission spectra. Indeed, what part of biofilm does contribute to the recorded ATR spectrum? One, two, three bacterial layers? Only the envelope of the cells in the first monolayer? Or even only the slime layer? Furthermore, do the spectral changes result from physiological changes of bacteria, changes in environmental physicochemical conditions or from artefacts of the ATR technique? It is essential to bear in mind that an ATR –FTIR spectrum has a spatial component. This feature is especially exploited in adhesion science to obtain depth profiles by recording ATR spectra as a function of the incidence angle. However, it is very difficult to apply this method for biofilm study. Considering the mean size of bacteria and the dp values given in Fig. 1, the evanescent wave can penetrate the interior of bacterial cells, even two or three bacterial monolayers, provided that the bacteria are in direct contact with the ATR crystal surface. In this case, the bacterial ATR-IR spectrum reflects all of the components of the cells, including the cell walls, cell membrane, internal structures and the cytoplasm but in varying degrees depending on the distance between the component and the crystal surface. An additional difficulty is that over biofilm formation these bacterial components are not static. But any change in distance between the ATR crystal surface and a given molecule modifies the contribution of this molecule in the resulting ATR spectrum. Various factors such as pH, ionic strength, surface charge, hydration rate, can influence the distance between the bacteria and the crystal [18-20]. Changes in these factors or in the structure and spatial organization of biomolecules can significantly modify ATR-IR spectra. This issue is illustrated in Fig. 4a with the effect of drastic dehydration on the ATR-FTIR fingerprint of a 7-h-nascent biofilm. The shrinkage of the bacterial monolayer results in increasing all bands but not in the same proportion. The band shapes are in particular affected by conformational changes from an open to condensed molecular structure over dehydration as confirmed by a detailed analysis of the amide I and II bands. Another pitfall concerns with the changes in the refraction index of bacterial film over biofilm formation because of biochemical composition and structural changes. In this case not only the penetration depth can be affected but band shapes can be distorted [21]. This last point is illustrated in a simple manner in Fig. 4b. This problem can also occur with the subtraction of a background spectrum from the ATR spectrum of biofilm at a given time in order to eliminate the contribution of solvent or nutritive medium or highlight the spectral variations between two times. A difference between the optic characteristics of the two samples corresponding respectively to two spectra to subtract can cause significant distortions in the band shapes of the resulting spectrum [22]. As shown hereafter these limitations do not hamper development of interesting applications. However, the interpretation at the molecular level of spectral changes measured is sometimes tricky and non-trivial, requiring good knowledge and experience of ATR-IR bacterial fingerprints in order to be able to identify and to differentiate the spectral changes induced by such or such metabolic change or simply by changes in environmental physicochemical conditions. This explains why the ATR-FTIR technique is relatively little exploited, even underexploited, in biofilm studies despite of its great potential.

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Fig. 4 a) Effect of nitrogen drying on the ATR-FTIR fingerprint of a 7-hour old nascent Pf biofilm formed on a germanium ATR crystal. b) Effect of refractive index of the ATR crystal on band shapes of the water ATR-FTIR spectrum.

3. A few current applications

Bearing in mind the pitfalls previously mentioned, various applications can be considered. We will address only three of them which well exemplify the potential of the ATR-FTIR technique concerning the knowledge, detection, control and prevention of biofilm formation. In line with the attachment and colonization monitoring described above, the fate of attached pioneer bacteria can be monitored in the same manner and, interestingly, their response to changes in various environmental parameters: temperature, hydration rate, nutrient quality and quantity, biocide addition, hydrodynamic, and so on. Thus, we have applied this approach to monitor, over several days, the response of 6-h nascent Pseudomonas fluorescens biofilms to variations in dissolved organic carbon (DOC) level in tap water [16]. We reported that any variation in DOC level modified the kinetic of biofilm development within 1-2 hours. Biofilm detachment and regrowth kinetics determined from changes in the area of infrared bands associated with proteins and polysaccharides were directly dependent upon DOC level. In addition, they were consistent with bacterial adhesion or growth kinetic models and the EPS overproduction or starvation-dependent detachment mechanisms. These findings are very interesting for the research and development of new decision making tools for managing finished water quality because DOC level is often a key factor of the bacterial regrowth in drinking water. They allow us to possibly consider monitoring, in situ and in real time, drinking water biostability in using a biofilm-type biosensor consisting of a monolayer reference biofilm, as biological recognition element, in contact with an ATR crystal which should be the transduction element [17]. Such a device would be a decision support tool for water suppliers which could take in time appropriate counter-measures against drinking water quality degradation and optimize the amount of disinfectant used. Another issue of importance concerning the control and prevention of biofilm formation is the improvement of the antimicrobial agent activity against sessile bacteria. As early as 1993, Sucy et al. [23] reported the use of the ATR-FTIR technique for providing in situ and in real time information on both transport of an antimicrobial agent, namely ciprofloxacin, to the biofilm-substratum interface, and its interaction with biofilm components. Significant changes in IR bands of the biofilm in the regions of the spectrum associated with RNA and DNA vibrational modes appeared following exposure to the antibiotic, indicating chemical modification of biofilm components. Astonishingly, to date, the Suci’s studies [23-25] are nearly the only ones in this area using the ATR-FTIR spectroscopy. Yet the latter could be a valuable help to understand mechanisms of action of antimicrobial agents and design new ones. Thus, we currently use ATR-FTIR spectroscopy to probe in situ the changes induced by antimicrobial peptides (AMP) in sessile bacterial cells over the early stages of biofilm formation. AMPs play a central role in the innate immunity of system of many organisms and are active towards a wide range of microorganisms. In addition, due to their membranolytic activity, development of microbial resistance to AMPs should be rarer. Several studies have already investigated by ATR-FTIR the mode of action of AMPs against planktonic bacteria [26-27]. But resistance of bacteria inside biofilms being much greater than that of their planktonic counterparts, it is essential to probe “in live” the effects of AMPs against sessile bacteria in order to gain more insight into their precise molecular mechanisms of action which are still largely in dispute, and to ultimately improve their antimicrobial activity and toxicity profile in optimizing their structure. Our current ATR-FTIR investigations on the activity of analogues of dermaseptine S4 against 6-h-old Pseudomonas fluorescens biofilms reveal important and rapid changes in ATR-FTIR bacterial fingerprints when AMPs are injected. Depending on concentration, peptide type, injection time, these spectral changes reflect adsorption of peptides on the cell walls, inhibition of biofilm growth, major biochemical and physiological changes, bacterial detachment but also, at some time, bacterial regrowth [28]. Another recent nice application is in situ monitoring chemical and structural changes induced by proton uptake and heavy metal cation biosorption onto microbial cells [18, 29]. The binding of metal ions to bacteria can be detected from

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changes in bacterial absorbances allowing identification of major bacterial components involved in proton uptake and metal binding. This approach is particularly interesting in bioremediation research as well as in the studies of bio-physicochemical processes occurring in the environment.

4. Conclusion and future trends

The ATR-FTIR technique offers the unique advantage to probe and monitor at the molecular scale, in situ, non-destructively, in real-time, and under fully hydrated conditions, the biochemical composition of the first two or three bacteria layers attached onto the crystal surface and the interphase between the crystal and bacteria and between bacteria. Consequently, it is undeniably a tool of choice to study the early stages of biofilm formation and the physiological and biochemical changes of the biofilm base layer in response to various environmental changes. The pitfalls associated with the ATR-FTIR technique are not hampering but they require a particular attention to avoid misinterpreting spectral changes. The main limitation of the ATR-FTIR technique is its poor spatial resolution which prevents probing heterogeneity of biofilm and localizing, at the cellular scale, the biochemical changes revealed in spectra. Indeed, the diameter of IR beam in ATR-FTIR spectrometers being of the order of millimeter, the ATR-FTIR spectrum recorded do not correspond to a single cell but bacteria colonies which can be highly heterogeneous. Chemometric or multivariate statistical techniques can be employed to extract the spectral fingerprints of these different populations from the whole spectrum [8,13]. However, this approach is not always straightforward and successful. Thanks to an IR beam diameter of the order of a few tenths of microns, the ATR-FTIR microspectroscopy, which combines the ATR-FTIR spectroscopy with microscopy, can help to better probe heterogeneity in biofilms, even to map it, but without the possibility of in situ monitoring like in the experiments described above [30, 31]. Another approach which should bring significant advances in this field is the combination, in varying degrees, of vibrational spectroscopies (ATR-FTIR but also Raman) with the atomic force microscopy (AFM) and, especially, AFM force spectroscopy. AFM provides imaging with nanometer resolution under wet or physiological conditions and is also able to measure, in the so-called single-molecule force spectroscopy mode, the nanoscale chemical and physical properties of cells. Force curve analyses by means of various models can indeed provide information on the unfolding pathways of membrane proteins, the conformational properties of cell-surface polymers, the localization of surface receptors, and so on [32, 33]. The ideal would be to combine directly the instruments in order to analyze exactly the same area of the sample. Some attempts in this direction have been recently reported [34, 35] but some difficulties still must be overcome. Finally, efforts must still be worn in two others directions: i) functionalization of ATR crystal surfaces with a thin organic [36] or metal film [37] in order to study bioadhesion on surfaces less specific than those of the conventional ATR crystals; ii) development of devices using optical fibers operating in mid-infrared region, such as chalcogenide glass fibers [38], for remote monitoring bioadhesion in various systems and not only in laboratory.

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Science against microbial pathogens: communicating current research and technological advances A. Méndez-Vilas (Ed.)_______________________________________________________________________________