Investigation of Formation of Bacterial Biofilm upon Dead Siblings

Subscriber access provided by Karolinska Institutet, University Library

Biological and Environmental Phenomena at the Interface

Investigation of Formation of Bacterial Biofilm upon Dead Siblings Zhi Wang, Xiangjun Gong, Jinhong Xie, Zhenbo Xu, Guangming Liu, and Guangzhao Zhang Langmuir, Just Accepted Manuscript • DOI: 10.1021/acs.langmuir.8b01962 • Publication Date (Web): 07 Aug 2018 Downloaded from http://pubs.acs.org on August 18, 2018

Just Accepted “Just Accepted” manuscripts have been peer-reviewed and accepted for publication. They are posted online prior to technical editing, formatting for publication and author proofing. The American Chemical Society provides “Just Accepted” as a service to the research community to expedite the dissemination of scientific material as soon as possible after acceptance. “Just Accepted” manuscripts appear in full in PDF format accompanied by an HTML abstract. “Just Accepted” manuscripts have been fully peer reviewed, but should not be considered the official version of record. They are citable by the Digital Object Identifier (DOI®). “Just Accepted” is an optional service offered to authors. Therefore, the “Just Accepted” Web site may not include all articles that will be published in the journal. After a manuscript is technically edited and formatted, it will be removed from the “Just Accepted” Web site and published as an ASAP article. Note that technical editing may introduce minor changes to the manuscript text and/or graphics which could affect content, and all legal disclaimers and ethical guidelines that apply to the journal pertain. ACS cannot be held responsible for errors or consequences arising from the use of information contained in these “Just Accepted” manuscripts.

is published by the American Chemical Society. 1155 Sixteenth Street N.W., Washington, DC 20036 Published by American Chemical Society. Copyright © American Chemical Society. However, no copyright claim is made to original U.S. Government works, or works produced by employees of any Commonwealth realm Crown government in the course of their duties.

Page 1 of 33 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Langmuir

Investigation of Formation of Bacterial Biofilm upon Dead Siblings Zhi Wang1, Xiangjun Gong1,*, Jinhong Xie2, Zhenbo Xu2,3, Guangming Liu4,*, Guangzhao Zhang1 1

Faculty of Materials Science and Engineering, South China University of Technology,

Guangzhou 510640, P. R. China. 2

School of Food Science and Engineering, South China University of Technology, Guangzhou

510640, P. R. China. 3

Department of Microbial Pathogenesis, School of Dentistry, University of Maryland, Baltimore,

Maryland, MD 21201, USA. 4

Hefei National Laboratory for Physical Sciences at the Microscale, Department of Chemical

Physics, University of Science and Technology of China, Hefei 230026, P. R. China.

*Correspondence should be addressed. E-mail: [email protected] (X. J. G.); [email protected] (G. M. L.) . 1

ACS Paragon Plus Environment

Langmuir 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Abstract Biocides can effectively kill bacteria. However, whether the dead bacterial cells left on the surface influence the later growth of biofilm is unknown. In this study, we have cultured Pseudomonas aeruginosa (PAO1) biofilm on their dead siblings and have investigated their evolution by using magnetic force modulation atomic force microscopy (MF-AFM). The time dependence of the biofilm thickness indicates that the deposited dead siblings can slow down the growth of PAO1 biofilm. The biofilm growing on dead bacteria layers is softer in comparison with those upon alive siblings, as reflected by the static elastic modulus (E) and dynamic stiffness (kd) scaled to the 

disturbing frequency (f) as kd  kd ,0 f , where kd,0 is the scaling factor and γ is the power-law exponent. We reveal that the smaller population instead of the variation of extracellular polymeric substances (EPS) within the biofilm upon the dead siblings is responsible for the softer biofilm. The present study provides a better understanding of the biofilm formation, thus making significance for designing antimicrobial medical materials and antifouling coatings.

2


Page 2 of 33

Page 3 of 33 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Langmuir

Introduction Bacteria attaching to a surface usually form biofilm that consists of microbial communities embedded into a protective layer or extracellular polymeric substances (EPS), causing problems in healthcare and industries.1 To inhibit or prevent the formation of such biofilm, antibiotics and disinfectants are often used.2,3 For example, ethanol (normally 75% v/v), a widely-used antimicrobial that is effective to clean microbial contaminated surfaces.4 Alternatively, antimicrobial surface can also inhibit the formation of biofilm by either contact killing or biocide leaching to biochemically induce bacterial death once the bacteria come into contact with the surface.5,6 Construction of a contact-killing surface involves the conjugation with antibiotic functional groups such as antimicrobial peptides7 and polyelectrolytes.8 On the other hand, biocide leaching surface consists of biodegradable polymer carrying cytotoxic compounds, where the latter are released from the surfaces in a controlled manner to degrade the former.9,10 For either antimicrobial agents or antibacterial surfaces, the killed bacteria or disrupted biofilm are not removed but left on the surfaces for hours, days or even longer, which gradually lay over and inactivate the initial surface. Since surface properties can significantly alter the biofilm architecture,11 many antimicrobial surfaces lack long-term resistance to biofilms. Previous studies have shown that the surviving bacteria (Legionella pneumophila, Bacillus subtilis, Prevotella intermedia and Porphyromonas gingivalis) have a necrotrophic growth in the dispersion by using the dead bacteria as a nutritional source after antimicrobial treatment.12-14 Virulence 3


Langmuir 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

genes of oral species P. intermedia and P.gingivalis have been reported to be up-regulated when they are cocultured with dead oral bacteria.13 Clearly, the surface-deposited bacteria casualties have profound effects on the long-term performances of antimicrobial surfaces and surface treatment with antimicrobial agents. Yet, the mechanism still remains unclear. Pseudomonas aeruginosa (P. aeruginosa) is one of the major pathogens with serious threats to human due to its ability to form biofilm and drug resistance.15,16 In the present work, we have examined the evolution of P. aeruginosa developed upon the corresponding ethanol-killed siblings by using magnetic force modulation atomic force microscopy (MF-AFM). A magnetic force driven cantilever penetrates into the bacterial colonies and oscillates around an indentation depth (δ) at a certain frequency (f),17 such that the viscoelasticity of growing biofilm as a function of indentation depth and disturbing frequency (f) can be measured by MF-AFM.18 Our aim is to understand the mechanism about the formation of bacterial biofilm on dead siblings. Experimental Section Culture of P. aeruginosa biofilm. PAO1 (wild-type strain) and WFPA800 (∆psl, the psl promoter deletion mutant19) were streaked onto a tryptic soy agar (TSA, Becton, Dickinson and Company) plate and incubated at 37 °C for 24 h. Psl is one of the polysaccharides in P. aeruginosa that plays a vital role in structure maintenance and antibiotic resistance of the biofilm. PAO1 colonies from the plate were inoculated into tryptic soy broth (TSB, Becton, Dickinson and Company), while WFPA800 colonies were grown in liquid cultures in lysogeny broth (LB, Becton, Dickinson and 4


Page 4 of 33

Page 5 of 33 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Langmuir

Company) lacking sodium chloride. An orbital shaker (Model ZWY-200D, Zhicheng Instruments) was used for incubation at 37 °C for 16 h. After culturing overnight, 1 mL P. aeruginosa suspension was mixed with 1 mL 75% ethanol (Nanjing Chem. Reagent Co.) for 10 min. To remove the residual ethanol, the dead bacterial cells were centrifuged for 10 min at 1,800 rpm, washed twice in the phosphate-buffered saline (PBS) buffer and suspended in PBS. The suspension was diluted to a final concentration of OD600 = 0.4 (approximately 4 × 108 CFU/mL). The culture with alive P. aeruginosa bacterial cells was diluted to OD600 = 0.4 as a control. The suspension consisting of either dead or alive cells was added to a sterile 24-well plate containing poly(ethylenimine) (PEI, Mw = 70,000 g/mol, Sigma-Aldrich) coated silicon wafers (1.5 × 1.5 cm2 for each piece, oblique in the well plate). The silicon wafers were firstly cleaned and hydroxylated by immersing in a piranha solution (a mixture of 3:1 (v/v) (98% H2SO4 and 30% H2O2) at 90 °C for 30 min. The cleaned wafers were then placed into a dilute aqueous solution of PEI (0.2 wt.%) and held for 12 h. After rinsed by ultra-pure water and flushed by nitrogen gas, a thin layer of PEI was adsorbed on the silicon surface. We recorded 0 h as the time that the surface was completely covered by dead or alive bacteria. Afterwards, the surfaces were washed with PBS, transferred into the container with fresh suspension of alive PAO1 (OD600 = 0.04), and incubated at 37 °C for biofilm formation. Fluorescence

imaging of

bacterial

attachment

and biofilm formation.

Fluorescence microscopy was used to assess the initial adhesion of dead/alive PAO1 and WFPA800 cells as well as the formation process of the biofilm. The surfaces 5


Langmuir 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

covered with the attached cells and the biofilm with different incubation times were rinsed with PBS to remove planktonic bacteria, and treated with 1 mL of a staining solution containing 5 × 10-3 mM SYTO 9 and propidium iodide (Molecular Probes) in the dark for 30 min. The surfaces were then rinsed with PBS and examined using a microscope (Axio Scope A1, Zeiss) operating under the epi-fluorescence mode. Three different areas were randomly chosen representative of each surface, and the percentage of bacterial coverage was calculated using the ImageJ software (NIH Image). Biofilm thickness determination and morphology of individual cells. For the determination of the biofilm thickness, the sample was firstly scraped with a sterile blade to expose the substrate. Afterwards, the probe was allowed to approach and contact with the biofilm and the substrate using AFM (AFM, Park Systems, XE-100). The thickness of biofilm was obtained from the relative height between the biofilm skeleton and the substrate.20 For each biofilm, the measurements were taken at least three different locations and repeated for three times at the same location. Furthermore, the silicon substrates covered with individual PAO1/WFPA800 cells were taken out from the well-plate and rinsed with PBS to remove the planktonic cells, and dried in ambient air. AFM performed in non-contact mode was used to characterize the morphology of individual P. aeruginosa cells in air. Silicon cantilever (PPP-NCHR, Nanosensors) with spring constant of 42 N/m and resonance frequency of 330 kHz was used. The scan rate was 0.5 Hz and the image resolution was 256 pixels × 256 pixels. 6


Page 6 of 33

Page 7 of 33 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Langmuir

Viscoelasticity measurements. Viscoelasticity of P. aeruginosa biofilm was measured with MF-AFM, (AFM, Park Systems, XE-100) where a magnetic cantilever was used to penetrate into samples and oscillate in the surface normal direction around an initial indentation at a certain f, and the amplitude and phase shift of the cantilever deflection were used to extract the viscoelasticity of the samples. The magnetic colloidal probe was prepared by attaching a small permanent SmCo magnet piece onto the backside and by attaching a 20-μm diameter silica bead (Suzhou Nano-Micro Technology Co., Ltd, China) on the underside of the same cantilever (DNP-S10 D, Bruker) with hot melt adhesives (purified Epikote 1004 resin, Shell). The procedure was detailed elsewhere.18 The spring constant of this probe (kc) was calibrated by the thermal tune method. Briefly, viscoelasticity measurements of P. aeruginosa biofilm were performed with MF-AFM equipped with an open liquid cell at 25 °C. The silicon substrates covered with biofilm were taken out from the well-plate and rinsed with PBS. Afterwards, the substrate was placed at the bottom of a liquid cell which was filled with PBS to prevent biofilms from desiccation.21 The tests were completed within 3 h after taking the biofilm out from the well-plate to exclude any variation of the rheological properties of biofilms due to ageing.22 In this case, force-indentation curves and viscoelasticity measurements were directly performed. For each biofilm, the measurements were taken three times at different locations, and usually two to three locations were selected. Since the adhesive force between the probe and biofilms could be neglected (see Figures S1 and S2 in Supporting Information), the Hertzian contact model was used to 7


Langmuir 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 8 of 33

analyze the force-indentation curves of biofilms.18 From the fitting of the force-indentation curves at the linear elasticity region with the Hertzian contact model,23 the static elastic modulus (E) of P. aeruginosa biofilm could be obtained from Eq. 1: F

4 E 3 (1  2 )

R 3/ 2

(1)

where R, δ and υ were the radius of the colloidal probe, indentation depth and Poisson’s ratio, respectively. υ was assumed to be 0.5 for gel-like and biological samples.24,25 The force was exerted on the magnetic colloidal probe at an arbitrary height far from the biofilm samples, resulting in an oscillation with amplitude of about 10 nm (A0) of the probe (a free oscillation). The probe was then moved to contact with biofilm with initial contact forces (Fc). Here, we used Fc = 1, 2, 3, and 4 nN. The indentation depth of the probe into the biofilm increased with increasing Fc. Afterwards, the same oscillated magnetic force was applied to further oscillate the probe. Consequently, the oscillation resulted in small amplitude A1 and a phase shift φ with respect to the free oscillation. The amplitude (A0 and A1) and φ were obtained by using a lock-in amplifier (SR830, Stanford Research Systems) from the deflection signal of the magnetic colloidal probe. The sinusoidal current generated by the function generator was used as reference. The dynamic stiffness (kd) of the sample was determined by Eq. 217

k d  kc (

A0  1) A1

(2)

where kc was the spring constant of the probe. Viable-cell enumeration. The silicon substrates with biofilm were rinsed with PBS to 8


Page 9 of 33 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Langmuir

remove the planktonic cells. Biofilm cells were carefully scraped out with a pipette and resuspended in PBS. The suspension was sonicated using an ultrasound bath for 10 min. This sonication step was pre-optimized to ensure that biofilm aggregates were dispersed into single bacteria. This suspension was diluted with PBS, plated on TSA plates and incubated at 37 °C for 24 h. The biofilm viable cell density was then counted as colony-forming units (CFU) per mL. The mean density was obtained from at least three repeated experiments.

Results and discussion

Figure 1. (a) Schematic illustration of PAO1 biofilm growing upon a deposited layer of dead or alive bacteria siblings as a function of time. Fluorescent images of bacteria adhered to the PEI-coated silicon surface, PAO1 with (b, c) and without (d, e) the application of 75% v/v ethanol. The bacteria were stained with SYTO9 and PI. The deposited bacteria whether they are alive or dead are stained by green, whereas the dead ones with damaged cell membranes are red.

9


Langmuir 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Figure 1a schematically illustrates that the evolution of PAO1 colonies from individual cells to biofilm deposited upon their alive or dead siblings. A layer of dead PAO1 cells deposited on a silicon substrate is prepared as described in the experimental section. It is noteworthy that a positively-charged polymeric adhesive-PEI layer is adsorbed on the silicon surface before depositing the dead cells. As a result, the dead PAO1 are firmly glued to the substrate during the measurements. After that, PEI-coated silicon substrates are immersed in PAO1 suspensions with or without the application of ethanol. Figure 1b to 1e depict a saturated layer of PAO1 settled on the silicon substrate with and without the application of 75% v/v ethanol. Figure 1b and 1c show the deposition of all the PAO1 cells (Figure 1b, green color) and the dead cells (Figure1c, red color) in the sight of view after treating with ethanol. The deposited cells disperse in a homogeneous manner on the substrate. From image analysis, the surface coverage of stained green cells is 45.3% ± 0.6% whereas the coverage of dead cells (stained as red color) is 44.9% ± 0.5%. Clearly, the application with 75% v/v ethanol is highly efficient with the dead rate as 99.1%. From Figure 1e, without the application of ethanol, the adsorbed PEI molecules are not lethal for the contacting PAO1 cells since no dead cells (stained in red color) are observed. Besides, the coverage of alive PAO1 in Figure 1d on PEI-coated substrate is 42.2% ± 0.3%, quite close to that of the dead cells. The standard deviation of the surface coverage is smaller than 1%, indicating that a similar surface coverage for dead and alive PAO1 cells can be achieved and reproduced by this surface preparation method.

10


Page 10 of 33

Page 11 of 33 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Langmuir

Figure 2. Fluorescence microscopy images of the PAO1 biofilm growing upon dead siblings with incubation time of 0 h (a), 1 h (b), 3 h (c), 5 h (d) and 7 h (e), as well as that upon alive bacteria with incubation time of 0 h (f), 1 h (g), 3 h (h), 5 h (i) and 7 h (j). The scale bar is 20 μm.

Figure 2 illustrates the evolution of PAO1 colonies from individual cells to biofilm deposited upon their alive or dead siblings by fluorescence microscope imaging. For 11


Langmuir 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

both cases, individual bacteria cells can be distinguished at the incubation time of 1 h and 3 h. In contrast, the bacteria profiles become blurred after 3 h, indicating that the amount of EPS increases and the biofilm gradually forms. Besides, the cell number upon the dead siblings seems to be slightly smaller than that upon the alive cells at the same incubation time from 1 h. We will discuss detailedly this later. Meanwhile, the morphology of the deposited biofilm after 3 h shows PAO1 tend to gather in the form of small clusters upon dead siblings, whereas they grow in a random way upon alive siblings.

Figure 3. The thickness (d) of biofilm upon a dead/alive PAO1 layer as a function of incubation time.

Figure 3 shows the incubation time dependence of the average thickness (d) of PAO1 biofilm. To confirm the statistical significance, the P-value (p) is obtained to be smaller than 0.05 for most of the cases (Table S1, Supporting Information). As the incubation time increases, the thickness increases during the growth of biofilm. For either alive or dead PAO1 cell, the thickness of the initial deposited layer is ~1.5 μm, i.e., one to two layers of the PAO1 cells (see Figure S3 for the morphology of single 12


Page 12 of 33

Page 13 of 33 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Langmuir

PAO1 cells).26,27 The slope reflects the growth rate of the biofilm. Within the first 2 h, the PAO1 colonies upon dead bacteria grow a little faster than those upon alive bacteria. However, 3 h later, the latter grow more rapidly than the former. We will further discuss this below.

Figure 4. Static elastic modulus (E) of PAO1 biofilm as a function of incubation time with dead or alive bacteria as the bottom layer. (*) denotes statistically significant at p < 0.05.

Force spectroscopy and the static elastic modulus of the growing PAO1 biofilms are measured with MF-AFM in PBS buffer, where the force-indentation curves are obtained by measuring the contact force (Fc) at the corresponding indentation depth into the bacteria colonies (δ) (see Figure S4). For PAO1 colonies upon either alive or dead bacteria layer, δ non-linearly varies with the incubation time. At the initial phase, the dead layer is stiffer than the alive one. This is because the former’s cells are more dehydrated and have more disrupted membranes after ethanol application (Figure S3). In terms of Hertzian contact model, the static elastic modulus (E) of biofilm is 13


Langmuir 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

obtained by fitting the linear elasticity region of the force-indentation curves with Eq. 1 (Figure S4). It should be noted that due to the heterogeneity nature of biofilms, the standard deviation of E is large in some cases. To confirm the statistical significance, dynamic modulus measured by MF-AFM is also introduced and compared in the later section. A good accordance between the static and dynamic modulus of the biofilms developed at different incubation times can be obtained. As the incubation time increases from 1 to 3 h, E increases from 780 ± 350 to 1000 ± 300 Pa for PAO1 upon alive siblings, but significantly increases from 360 ± 220 to 820 ± 320 Pa for PAO1 upon dead siblings. In Figure 3, during the early 3 h, the thickness of PAO1 upon the dead siblings increases for 0.3 μm, and the thickness of PAO1 upon alive cells increases for 0.5 μm. As a result, the increments in thickness are comparable to the thickness of a single bacteria (0.6-0.8 μm). This fact indicates that the elastic modulus at 1 and 3 h represent the average stiffness of the deposited cells which contact with the initial dead/alive bacterial layer. This can also be confirmed from the morphology of the deposited PAO1 cells in Figure 2. It was known that bacterial cells can change their shapes significantly to squeeze into crevices, this change may also exist in our case.28,29 In Figure 4, E is 780 Pa for PAO1 cells upon alive siblings and 360 Pa for those upon dead siblings at 1 h. At 3 h, E of PAO1 upon alive bacteria is still slightly larger than those upon dead siblings. As a result, the bacterial cells become slightly softer when they come into contact with the dead siblings compared with those contact with alive cells. As incubation time increases from 3 to 5 h, E decreases to 550 ± 280 for PAO1 14


Page 14 of 33

Page 15 of 33 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Langmuir

upon alive cells and 100 ± 60 Pa for PAO1 upon dead cells. It is reported that E of biofilm decreases with the amount of produced EPS,30 which is consistent with the result in Figure 2. After 3 h, the profiles of bacteria are difficult to distinguish since the bacteria are embedded into the self-produced EPS. Anyhow, the facts indicate that PAO1 cells attach to the surface individually from 1 to 3 h and form biofilm after 3 h. From 5 to 7 h, for biofilm growing upon the dead siblings, E changes from 100 ± 60 to 340 ± 180 Pa. In contrast, for biofilm growing upon alive cells, E keeps decreasing from 550 ± 280 to 300 ± 130 Pa. Previous study has shown the elastic modulus (E) to be 17 to 240 Pa for P. aeruginosa biofilm measured by the flow-cell method,31 and 0.5 to 200 Pa for E. coli biofilm measured by magnetic tweezers.32 Clearly, the E of P. aeruginosa biofilm measured here is consistent with those measured by other methods. Besides, the thickness of the biofilm upon dead siblings increases more slowly after incubation for 3 h, indicating that the biofilm turns softer with a slower growth rate.

15


Langmuir 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Figure 5. Frequency (f) dependence of dynamic stiffness (kd) of PAO1 biofilm growing upon dead siblings with incubation time of 0 h (a), 1 h (b), 3 h (c) and 5 h (d), as well as that upon alive bacteria with incubation time of 0 h (e), 1 h (f), 3 h (g) and 5 h (h), where initial forces Fc = 1 nN (□), 2 nN (○), 3 nN (∆), and 4 nN (◊) are applied. The dashed lines are the fitting curves of kd with Eq. 3.

Figure 5 shows the disturbing frequency (f) dependence of dynamic stiffness (kd) of the biofilm formed on the deposited alive and dead bacterial layer as a function of incubation time. Here, Fc is the initial contact force between probe and biofilm, and

16


Page 16 of 33

Page 17 of 33 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Langmuir

the indentation depth of the probe into the biofilm increases as Fc increases. kd measured under higher Fc reflects the mechanics inside the biofilm. At 0 h, kd of the dead bacteria layer does not vary with f, but it increases as Fc increases from 1 to 4 nN. In contrast, kd of the alive bacteria layer shows a power law relation with f, and it also increases as Fc increases. Such a power law is also observed for biofilm evolved either on the dead or alive bacterial layer. From 1 h to 7 h, kd increases as f increases, indicating that the upper biofilm gradually develops on the initial dead and alive cells (see Figure S5 for the results at 7 h). For Fc = 1 nN corresponding to a depth inside the surface layer of the biofilm, kd sharply decreases when the incubation time increases from 3 to 5 h for biofilm upon either dead or alive cells. This suggests that PAO1 cells in the biofilm are now covered with a layer of self-produced EPS, leading to a softer mechanics. In Figure 5, we find that kd is scaled to the disturbing frequency (f) as follows:

kd  kd ,0 f 

(3)

where kd,0 is the scaling factor which is independent of f, and γ is the power index. Clearly, the scaling relation indicates that the biofilm behaves like a soft glass. The phenomenon is also observed in E. coli biofilm.18 A material behaves like a solid without responding to the mechanical perturbation at different frequencies when γ = 0. It turns fluid-like when γ = 1, namely, where the material may deform, flow, and reorganize under the influence of perturbation.33-35 Figure 6 show γ and kd,0 which are obtained by fitting kd measured at Fc = 1, 2, 3, and 4 nN with Eq. 3. kd,0 and γ measured under higher Fc reflect the mechanics deeper inside the biofilm. At 0 h and 17


Langmuir 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Fc = 1 nN, kd

f

0.08

and kd  f 0.22 for the dead and alive bacteria layer, respectively,

suggesting that the layer composed by the dead PAO1 cells killed by ethanol is closer to the solid due to the cell dehydration. In contrast, as the incubation time increases from 1 to 5 h, the upper biofilm upon the dead and alive cells becomes more fluid-like due to the secretion of EPS.

Figure 6. Initial contact force (Fc) dependence of the power-law exponent (γ) for PAO1 biofilm growing upon dead/alive siblings with incubation time of 0 h (a), 1 h 18


Page 18 of 33

Page 19 of 33 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Langmuir

(b), 3 h (c) and 5 h (d), as well as the scaling factor (kd,0) for PAO1 biofilm growing upon dead/alive siblings with incubation time of 0 h (e), 1 h (f), 3 h (g) and 5 h (h), obtained by fitting the dynamic stiffness (kd) of PAO1 biofilm in Figure 5 with Eq. 3.

On the other hand, at a certain Fc, γ of biofilm upon the dead cells is always slightly higher than that upon the alive cells from 1 to 5 h in Figure 6, indicating that the growing biofilm upon the dead cells is more fluid-like than that upon the alive cells at the moment. Meanwhile, the larger kd,0 at start indicates the dead PAO1 layer is stiffer. However, the biofilm growing on dead siblings becomes softer than that growing on the alive bacteria layer from 1 to 5 h. In particular, similar to a smaller E for bacterial cells upon dead siblings compared with those upon alive cells at 1 and 3 h, kd,0 in Figure 6 shows similar trends. This result confirms that PAO1 cells that contact with dead siblings are softer than those contact with alive cells. Meanwhile, a sharper increase in kd,0 is observed for the latter as Fc increases from 1 to 4 nN. This elucidates the former is more homogeneous than the latter at different depths. When the incubation time is 7 h, kd,0 for the former is slightly higher than that for the latter, but γ makes little difference in both cases (see Figure S6 for the results at 7 h). In short, the PAO1 biofilm growing upon dead siblings is softer and more fluid-like than that upon alive siblings, and the impact of the dead siblings on the upper biofilms becomes slight after 7 h. To confirm whether the softer PAO1 biofilm upon dead siblings is associated with the bacterial population within biofilm, we have studied the viable cells amount of the biofilm with dead or without bacteria as the bottom layer by CFU counting. The 19


Langmuir 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

viable cells amount of biofilm growing on a blank silicon substrate is counted instead of that growing upon an alive bacteria layer to exclude the contribution from an initial alive bacteria layer. Figure 7 shows that the viable cells of PAO1 biofilm growing upon dead siblings are 58.5%, 66.8%, and 89.2% of those growing on a blank substrate with the same incubation time from 1 to 5 h. These results present significant difference since p < 0.05. However, at the time of 7 h, the viable cells upon dead siblings are 96.9% of those on the blank substrate and p > 0.05, indicating that the cell number of biofilm upon dead siblings shows no significant difference from that upon blank substrate (Table S2, Supporting Information). This suggests that the presence of a dead bacterial layer slows down the growth of the bacteria colonies, which contributes to the formation of softer biofilm with the same incubation time. Such an impact of the dead siblings on the upper biofilms becomes slight after 7 h. As stated above, the thickness of the biofilm increases more rapidly for those upon dead siblings in the period from 1 to 2 h (Figure 3). Therefore, the initial dead layer gives rise to a looser packing structures of the sessile bacteria at the beginning, leading to a smaller bacterial population of the above alive siblings within the whole incubation time. Previous studies have shown that some neurotrophic bacteria can produce toxins to kill siblings for nutrition.13,36 This phenomenon might also exist in our case, so that the bacterial population is smaller in the case upon dead siblings. In addition to the viable cell amount, the dead bacterial layer might also influence the self-produced EPS of the upper biofilm, which can result in a softer structure. We have measured the viscoelasticity of WFPA800 biofilm, a mutant that cannot produce 20


Page 20 of 33

Page 21 of 33 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Langmuir

Psl polysaccharide. Psl is one of the dominant polysaccharides in P. aeruginosa that functions as a scaffold for biofilm development,37 thus plays a vital role in structure maintenance of the biofilm.

Figure 7. Enumeration of viable bacteria within PAO1 biofilm as a function of incubation time with dead/without bacteria as the bottom. (*) denotes statistically significant at p < 0.05.

A layer of dead or alive WFPA800 cells deposited on a silicon substrate is prepared. At the beginning, the coverage rate of ethanol-killed WFPA800 is 32.0% ± 0.2%, and the surface coverage of alive WFPA800 is 32.9% ± 0.6%. Note that without the protection of Psl, 24.2% of the deposited WFPA800 is disrupted by the adsorbed PEI molecules. Moreover, the morphology of WFPA800 biofilm with different incubation times shows that the amount of EPS increases and the biofilm gradually forms as the incubation time increases from 3 to 5 h (Figure S7). Furthermore, we have measured E of the growing WFPA800 biofilm (Figure S8). At 0 h, WFPA800 after the application of ethanol is stiffer than the alive cells, which is caused by dehydration

21


Langmuir 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

(Figure S9). The biofilms growing on dead siblings are always softer than those on the alive bacteria layer before 7 h. When the incubation time is 7 h, E of biofilm growing on dead bacteria is slightly larger than that on alive siblings.

Figure 8. Initial contact force (Fc) dependence of the power-law exponent (γ) for WFPA800 biofilm growing upon dead/alive siblings with incubation time of 0 h (a), 1 h (b), 3 h (c) and 5 h (d), as well as the scaling factor (kd,0) upon dead/alive bacteria with incubation time of 0 h (e), 1 h (f), 3 h (g) and 5 h (h), obtained by fitting the dynamic stiffness (kd) of WFPA800 biofilm in Figure. S10 with Eq. 3. 22


Page 22 of 33

Page 23 of 33 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Langmuir

Figure 8 shows γ and kd,0 by fitting kd (see Figure S10) with Eq. 3. At 0 h, the layer of initially deposited dead WFPA800 cells is stiffer and more solid-like. However, the WFPA800 biofilm growing on dead bacteria is always softer than the one growing on the alive bacteria layer within 5 h. In contrast, kd,0 of biofilm growing on dead bacteria is slightly larger than that on alive siblings, and there is no significant difference in γ within 7 h (Figure S11). The above results are in accordance with those in Figure 6 and Figure S6 for PAO1 biofilm, where the presence of dead siblings leads to a softer biofilm within 7 h. The impact of the dead siblings becomes weak after 7 h. Particularly, similar to a smaller E and kd,0 for PAO1 cells upon dead siblings compared with those upon alive cells at 1 and 3 h, kd,0 of WFPA800 cells shows the same trend. This result reveals that WFPA800 cells that contact with dead siblings are also softer than those contact with alive cells.

Figure 9. Incubation time dependence of thickness (d) (a) of WFPA800 biofilm upon a dead or alive bacterial layer, and enumeration of viable bacteria (b) within WFPA800 biofilm with dead/without bacteria as the bottom. (*) denotes statistically significant at p < 0.05.

23


Langmuir 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Figure 9a shows the average thickness (d) of WFPA800 biofilm. Here, the slope reflects the growth rate of biofilm. In the early 5 h, WFPA800 colonies upon dead bacteria grow comparably with those upon alive bacteria. However, the former grow slower 5 h later. Concerning that the PAO1 biofilm upon dead bacteria grows slower than those upon alive cells after 3 h, we have found that the development of WFPA800 biofilm without the Psl is more difficult than the PAO1 biofilm. This is not surprising since Psl is one of the dominant polysaccharides in P. aeruginosa that functions as a scaffold for biofilm development. Another reason might be that Psl in the path of chemical signaling. Figure 9b illustrates that the WFPA800 biofilm developed upon dead siblings presents similar behaviors with the PAO1 biofilm. From 1 to 5 h, the viable cells of WFPA800 biofilm growing upon dead siblings are 62.1%, 72.3%, and 85.1% of those growing upon blank substrates. These values are always less than those growing on a blank substrate with statistical significance (p < 0.05, Table S2). In contrast, at the time of 7 h, p > 0.05, indicating that the cell number of biofilm upon dead siblings or blank substrate shows no significant difference. In short, the smaller bacterial population in the biofilm growing upon dead siblings rather than the EPS compositions leads to a softer biofilm. Finally, the variation in binding strength between bacterial cells and EPS may also contribute to the mechanical change. Therefore, the approach-retraction force separation curves between the probe and the PAO1/WFPA800 biofilms at different incubation times are mapped, as shown in Figures S1 and S2. The retraction gives the information of adhesion between the probe and the biofilms. Both of the two figures 24


Page 24 of 33

Page 25 of 33 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Langmuir

reveal that the forces between the probe and either the individual cells or the biofilms are always repulsive and the adhesion between the probe and the biofilms is negligible at different incubation times. The change in binding strength at different incubation times is represented as the area between the approach and retraction curves, as shown in Figure S12. No significant difference or trend of the binding strength of PAO1/WFPA800 biofilm upon a dead or alive bacterial layer is observed. As a result, the binding strength between bacterial cells and EPS has no obvious influences on the softening of the biofilm.

Conclusion We have investigated the growth of bacterial biofilm upon deposited layer of dead siblings by using MF-AFM. The initially deposited dead P. aeruginosa can suppress the growth of upper biofilm. The individual bacteria cells that contact with the dead siblings as well as the biofilm growing upon dead siblings are softer than those grow upon alive siblings, as reflected by the static modulus and dynamic stiffness (kd) of 

MF-AFM. Moreover, kd is scaled to the disturbing frequency (f) as kd  kd ,0 f , as a consequence, the biofilm is glassy. Such a relation together with the incubation time and penetration depth dependences of γ and kd,0 indicate that the biofilms upon the dead bacteria become softer and more fluid-like than those growing upon the alive cells, but the impact of the dead siblings becomes weak for the upper biofilms as the incubation time increases. Our study reveals that the smaller population instead of the

25


Langmuir 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

variation in EPS compositions inside the biofilms upon the dead siblings is mainly responsible for the softer biofilm.

Associated Content Supporting Information Force-separation curves of the PAO1/WFPA800; Morphology of individual PAO1 cells measured by AFM; Force-indentation curves of PAO1 biofilm; Dynamic stiffness (kd) of PAO1 biofilm at 7 h; the power-law exponent γ, or scaling factor kd,0 of PAO1 biofilm at 7 h; Fluorescence microscopy images of the WFPA800 biofilm; Force-indentation curves and static elastic modulus of WFPA800 biofilm; Morphology of the individual WFPA800 cells measured by AFM; Dynamic stiffness (kd) of the WFPA800 biofilm; the power-law exponent γ, and scaling factor kd,0 of WFPA800 biofilm at 7 h; the binding energy of PAO1/ WFPA800 biofilm; Probability-value (p) of thickness and enumeration of viable bacteria of PAO1/WFPA800 biofilm, after student’s t test. This material is available free of charge via the Internet at http://pubs.acs.org.

Author Information Corresponding Authors *Email: [email protected] (X. J. G.). *Email: [email protected] (G. M. L.). 26


Page 26 of 33

Page 27 of 33 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Langmuir

Notes The authors declare no competing financial interests.

Acknowledgements The financial support of National Natural Science Foundation of China (21574046, 51573061) and the Fundamental Research Funds for the Central Universities (WK2340000066) is acknowledged.

References (1) Flemming, H. C.; Wingender, J., The biofilm matrix. Nat. Rev. Microbiol. 2010, 8, 623-633. (2) Høiby, N.; Bjarnsholt, T.; Givskov, M.; Molin, S.; Ciofu, O., Antibiotic resistance of bacterial biofilms. Int. J. Antimicrob. Agents 2010, 35, 322-332. (3) Hallstoodley, L.; Costerton, J. W.; Stoodley, P., Bacterial biofilms: from the natural environment to infectious diseases. Nat. Rev. Microbiol. 2004, 2, 95-108. (4) Graziano, M. U.; Graziano, K. U.; Pinto, F. M.; Bruna, C. Q.; de Souza, R. Q.; Lascala, C. A., Effectiveness of disinfection with alcohol 70% (w/v) of contaminated surfaces not previously cleaned. REV LAT-AM ENFERM 2013, 21, 618-623. (5) Hasan, J.; Crawford, R. J.; Ivanova, E. P., Antibacterial surfaces: the quest for a new generation of biomaterials. Trends Biotechnol. 2013, 31, 295-304. (6) Lichter, J. A.; Van Vliet, K. J.; Rubner, M. F., Design of Antibacterial Surfaces and Interfaces: Polyelectrolyte Multilayers as a Multifunctional Platform. 27


Langmuir 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Macromolecules 2009, 42, 8573-8586. (7) Statz, A. R.; Park, J. P.; Chongsiriwatana, N. P.; Barron, A. E.; Messersmith, P. B., Surface-immobilized antimicrobial peptoids. Biofouling 2008, 24, 439-448. (8) Buffet-Bataillon, S.; Tattevin, P.; Bonnaure-Mallet, M.; Jolivet-Gougeon, A., Emergence of resistance to antibacterial agents: the role of quaternary ammonium compounds--a critical review. Int. J. Antimicrob. Agents 2012, 39, 381-389. (9) Chen, S.; Ma, C.; Zhang, G., Biodegradable polymer as controlled release system of organic antifoulant to prevent marine biofouling. Prog. Org. Coat. 2017, 104, 58-63. (10) Ma, C.; Zhang, W.; Zhang, G.; Qian, P. Y., Environmentally friendly anti-fouling coatings based on biodegradable polymer and natural antifoulant. ACS Sustainable Chem. Eng. 2017, 5, 6304-6309. (11) Rzhepishevska, O.; Hakobyan, S.; Ruhal, R.; Gautrot, J.; Barbero, D.; Ramstedt, M., The surface charge of anti-bacterial coatings alters motility and biofilm architecture. Biomaterialsence 2013, 1, 589-602. (12) Temmerman, R.; Vervaeren, H.; Noseda, B.; Boon, N.; Verstraete, W., Necrotrophic growth of Legionella pneumophila. Appl. Environ. Microbiol. 2006, 72, 4323-4328. (13) Herrero, E. R.; Boon, N.; Pauwels, M.; Bernaerts, K.; Slomka, V.; Quirynen, M.; Teughels, W., Necrotrophic growth of periodontopathogens is a novel virulence factor in oral biofilms. Sci. Rep. 2017, 7, 1107-1117. (14) Ma, W.; Peng, D.; Walker, S. L.; Cao, B.; Gao, C. H.; Huang, Q.; Cai, P., 28


Page 28 of 33

Page 29 of 33 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Langmuir

Bacillus subtilisbiofilm development in the presence of soil clay minerals and iron oxides. Npj Biofilms & Microbiomes 2017, 3, 4-13. (15) Blackledge, M. S.; Worthington, R. J.; Melander, C., Biologically-Inspired Strategies for Combating Bacterial Biofilms. Curr. Opin. Pharmacol. 2013, 13, 699-706. (16) Bhagirath, A. Y.; Li, Y.; Somayajula, D.; Dadashi, M.; Badr, S.; Duan, K., Cystic fibrosis lung environment and Pseudomonas aeruginosa infection. Bmc Pulmonary Medicine 2016, 16, 174-196. (17) Rebêlo, L. M.; de Sousa, J. S.; Mendes, F. J.; Schäpe, J.; Doschke, H.; Radmacher, M., Microrheology of cells with magnetic force modulation atomic force microscopy. Soft Matter 2014, 10, 2141-2149. (18) Gan, T.; Gong, X.; Schönherr, H.; Zhang, G., Microrheology of growing Escherichia coli biofilms investigated by using magnetic force modulation atomic force microscopy. Biointerphases 2016, 11, 041005. (19) Ma, L.; Jackson, K. D.; Landry, R. M.; Parsek, M. R.; Wozniak, D. J., Analysis of Pseudomonas aeruginosa Conditional Psl Variants Reveals Roles for the Psl Polysaccharide in Adhesion and Maintaining Biofilm Structure Postattachment. J. Bacteriol. 2006, 188, 8213-8221. (20) Pellizzoni, E., Ravalico, F., Scaini, D., Delneri, A., Rizzo, R., & Cescutti, P. Biofilms produced by burkholderia cenocepacia: influence of media and solid supports on composition of matrix exopolysaccharides. Microbiol. 2016, 162, 283-294. 29


Langmuir 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

(21) Lau, P. C.; Dutcher, J. R.; Beveridge, T. J.; Lam, J. S., Absolute quantitation of bacterial biofilm adhesion and viscoelasticity by microbead force spectroscopy. Biophys. J. 2009, 96, 2935-2948. (22) Aggarwal, S.; Poppele, E. H.; Hozalski, R. M., Development and testing of a novel microcantilever technique for measuring the cohesive strength of intact biofilms. Biotechnology & Bioengineering 2010, 105, 924-934. (23) Dimitriadis; Emilios, K.; Horkay; Ferenc; Maresca; Julia; Kachar; Bechara; Chadwick; Richard, S., Determination of Elastic Moduli of Thin Layers of Soft Material Using the Atomic Force Microscope. Biophys. J. 2002, 82, 2798-2810. (24) Alcaraz, J.; Buscemi, L.; Grabulosa, M.; Trepat, X.; Fabry, B.; Farre, R.; Navajas, D., Microrheology of Human Lung Epithelial Cells Measured by Atomic Force Microscopy: Biophysical Journal. Biophys. J. 2003, 84, 2071-2079. (25) And, J. D., & Radmacher, M. Measuring the elastic properties of thin polymer films with the atomic force microscope. Langmuir 1998, 14, 3320-3325. (26) Medilanski, E., Kaufmann, K., Wick, L. Y., Wanner, O., & Harms, H. Influence of the surface topography of stainless steel on bacterial adhesion. Biofouling 2002, 18, 193-203. (27) Waszczuk, K., Gula, G., Swiatkowski, M., Olszewski, J., Drulis-Kawa, Z., & Gutowicz, J. Evaluation of pseudomonas aeruginosa, biofilm formation using piezoelectric tuning forks mass sensors. Procedia Eng. 2012, 170, 7-12. (28) Männik, J., Driessen, R., Galajda, P., Keymer, J. E., & Dekker, C. Bacterial growth and motility in sub-micron constrictions. PNAS 2009, 106, 14861-14866. 30


Page 30 of 33

Page 31 of 33 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Langmuir

(29) Lai, C. Bacterial attachment, aggregation and alignment on subcellular nanogratings. Langmuir 2018, 34, 4059-4070. (30) Safari, A.; Habimana, O.; Allen, A.; Casey, E., The significance of calcium ions on Pseudomonas fluorescens biofilms - a structural and mechanical study. Biofouling 2014, 30, 859-869. (31) Stoodley, P.; Lewandowski, Z.; Boyle, J. D.; Lappin-Scott, H. M., Structural deformation of bacterial biofilms caused by short-term fluctuations in fluid shear: an in situ investigation of biofilm rheology. Biotechnology & Bioengineering 1999, 65, 83-92. (32) Galy, O.; Latourlambert, P.; Zrelli, K.; Ghigo, J. M.; Beloin, C.; Henry, N., Mapping of Bacterial Biofilm Local Mechanics by Magnetic Microparticle Actuation. Biophys. J. 2012, 103, 1400-1408. (33) Chen, Y. Y.; Wu, C. C.; Hsu, J. L.; Peng, H. L.; Chang, H. Y.; Yew, T. R., Surface Rigidity Change of Escherichia coli after Filamentous Bacteriophage Infection. Langmuir 2009, 25, 4607-4614. (34) Fabry, B.; Maksym, G. N.; Butler, J. P.; Glogauer, M.; Navajas, D.; Fredberg, J. J., Scaling the microrheology of living cells. Phys. Rev. Lett. 2001, 87, 148102. (35) Moreno, J.; Vargas, M. A.; Madiedo, J. M.; Muñoz, J.; Rivas, J.; Guerrero, M. G., Chemical and rheological properties of an extracellular polysaccharide produced by the cyanobacterium Anabaena sp. ATCC 33047. Biotechnology & Bioengineering 2000, 67, 283-290. (36) González-Pastor, J. E., Hobbs, E. C., & Losick, R. Cannibalism by sporulating 31


Langmuir 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

bacteria. Science 2003, 301, 510-513. (37) Wei, Q.; Ma, L., Biofilm Matrix and Its Regulation in Pseudomonas aeruginosa. Int. J. Mol. Sci. 2013, 14, 20983-21005.

32


Page 32 of 33

Page 33 of 33 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Langmuir

Table of Contents

33


Investigation of Formation of Bacterial Biofilm upon Dead Siblings

Recommend Documents