Sensitive Detection of Deliquescent Bacterial Capsules through

Subscriber access provided by University of Otago Library

Article

Sensitive detection of deliquescent bacterial capsules through nanomechanical analysis Song Ha Nguyen, and Hayden K. Webb Langmuir, Just Accepted Manuscript • DOI: 10.1021/acs.langmuir.5b02546 • Publication Date (Web): 01 Oct 2015 Downloaded from http://pubs.acs.org on October 5, 2015

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 free 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 accessible to all readers and 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.

Langmuir 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 24

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

1

Sensitive detection of deliquescent bacterial

2

capsules through nanomechanical analysis

3

Song Ha Nguyen and Hayden K. Webb*

4

Faculty of Science, Engineering and Technology, Swinburne University of Technology, P.O.

5

Box 218, Hawthorn 3122, Victoria, Australia

6

*

7

Email: [email protected]

Corresponding author

8

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

1

ABSTRACT:

2

Encapsulated bacteria usually exhibit strong resistance to a wide-range of sterilisation

3

methods, and are often virulent. Early detection of encapsulation can be crucial in microbial

4

pathology. This work demonstrates a fast and sensitive method for the detection of

5

encapsulated bacterial cells. Nanoindentation force measurements were used to confirm the

6

presence of deliquescent bacterial capsules surrounding bacterial cells. Force/distance

7

approach curves contained characteristic linear – non-linear – linear domains, indicating co-

8

compression of the capsular layer and cell, indentation of the capsule and compression of the

9

cell alone. This is a sensitive method for the detection and verification of the encapsulation

10

status of bacterial cells. Given that this method was successful in detecting the

11

nanomechanical properties of two different layers of cell material, i.e. distinguishing between

12

the capsule and the remainder of the cell, further development may potentially lead to the

13

ability to analyse even thinner cellular layers, e.g. lipid bilayers.

14


Page 2 of 24

Page 3 of 24

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

1

INTRODUCTION

2

Deliquescence is the property of a material or substance that enables it to absorb moisture

3

from the surrounding environment and subsequently dissolve in it.1-3 As a concept, it is

4

similar to hygroscopy in that water is adsorbed by the substance, however while a

5

hygroscopic material remains in the solid phase, a deliquescent material transits into the

6

aqueous phase.4-5 Deliquescence is thought to obfuscate the detection of bacterial capsules

7

when performed by AFM imaging.6-8 It was reported that while an apparent capsule is often

8

observable surrounding bacterial cells when imaged in air, the same cells appear to lack any

9

capsules once imaged in an aqueous medium.6 Even some abiotic particles, e.g. Al2O3

10

nanoparticles appear to behave in the same way.6

11

Bacterial capsules vary in composition, however they are often exist as long chains of

12

sugar molecules. Pseudomonas aeruginosa, the object of this study, is known to produce

13

capsules consisting of long chains of alginate.9-12 The capsule forms the outermost layer of

14

bacteria, and is covalently attached to the cell;13-16 therefore it is extremely difficult to

15

remove mechanically.17-20 Encapsulated bacteria possess greater resilience against external

16

stresses such as desiccation or antibiotics, and the capsule often provides virulence factors to

17

pathogenic strains.21-26

18

It is well known that encapsulation of a cell aids it in resisting desiccation.19-20 Here, it is

19

hypothesised that the mechanism by which the capsule does so is by absorbing water from

20

the environment and dissolving in it, i.e. by deliquescence. Covalent bonding between the

21

capsule and the cell wall would ensure that the deliquescent water/capsule remain in

22

association with the cell. This would explain why the capsule is no longer detected in an

23

aqueous medium, as it is highly soluble and therefore indistinguishable from the surrounding

24

medium. This would also suggest that the detection of a bacterial cell surrounded by

25

deliquescent water in ambient scanning environments may be a good indication of


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

1

encapsulation. To truly distinguish between an encapsulated cell in deliquescent water and a

2

cell that has adsorbed water hygroscopically, simple imaging without nanomechanical

3

analysis is insufficient. For this reason, atomic force microscopy was employed to

4

characterise and detect encapsulation of P. aeruginosa cells in two steps: first, tapping mode

5

image scans were performed to locate candidate cells that may be encapsulated, followed by

6

confirmation of the encapsulation status via nanoindentation measurements.

7

EXPERIMENTAL SECTION

8

Bacterial strain and culture conditions

9

The bacterial strain used in all experiments was Pseudomonas aeruginosa ATCC 10145T.

10

Prior to immobilisation for AFM experiments, bacteria were refreshed from stock cultures on

11

nutrient agar (Difco, BD) and incubated for 24 hours at 37 °C, before streaking and

12

incubation under the same conditions to verify purity.

13

Cell immobilisation for AFM experiments

14

Suspensions of OD600 = 0.1 were prepared from plate cultures in 3 mL of phosphate buffered

15

saline. A volume of 100 µL of cell suspension was spread on a glass coverslip over an area of

16

approximately 1 cm2, and incubated at 37 °C for 30 minutes to allow the bacterial cells to

17

adhere. Subsequently, the coverslips were gently, but copiously, rinsed with MilliQ H2O and

18

semi-dried with sterile N2, before mounting on steel discs for AFM measurements.

19

Atomic force microscopy

20

AFM experiments were performed in air at ambient conditions, using an Innova (Veeco)

21

scanning probe microscope in tapping mode. Tapping mode tips (Cont20A, Bruker Probes)

22

with nominal spring constants of 0.9 N m-1, and resonance frequencies of approximately 21

23

kHz were used for all experiments. Spring constants were determined using the thermal noise

24

method.27-29 Image scans were typically performed at 1 Hz scanning perpendicular to the axis


Page 4 of 24

Page 5 of 24

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

1

of the cantilever and collecting 512 × 512 data points. Cells were predicted to either possess

2

or lack capsular material by combined assessment of topographical and phase-difference

3

images, which was later confirmed via nanoindentation measurements.

4

Once a cell of interest was located by image scanning, nanoindentation force curves were

5

collected on the cell in question. Force/distance curves were collected beginning from 200-

6

400 nm above the point of contact, finishing with the piezo at a position no more than 600 nm

7

beyond the contact point. Cells were then re-scanned to confirm indentation. This process

8

was repeated on more than 20 different cells so as to enable selection of representative data.

9

RESULTS AND DISCUSSION

10

Pseudomonas aeruginosa cells in three different states of encapsulation/hydration were

11

visually detected in AFM scans (Figure 1). Cells with apparently thick capsules were

12

identifiable by examination of phase difference images, appearing bright and surrounded by a

13

thick dark halo, indicative of the relative softness of the capsule (Figure 1a). The apparent

14

thickness of the capsule appeared to correlate well with equivalent cell capsules

15

biochemically stained according to the method of Anthony (Figure S1).30 Cells in the

16

remaining two states both lacked this dark halo, however their hydration status was apparent

17

from the cellular morphology; hydrated cells were relatively smooth in appearance (Figure

18

1b), whilst dehydrated cells appeared shrivelled and deflated, and slightly smaller than the

19

hydrated cells (Figure 1c). This would suggest that the hydrated cells possess capsules that

20

are thinner than those that can be seen in Figure 1a. However without understanding the

21

nanomechanical properties the question still remains as to whether or not the observed

22

structures are truly capsular material or simply hygroscopic water.


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

1 2

Figure 1. Encapsulation/hydration states of P. aeruginosa cells immobilised on glass.

3

Topography (left) and phase difference (right) data from AFM scans of each cell state are

4

presented, together with three dimensional representations correlating the two. (a) Thick

5

capsules surrounding the cells were apparent through examination of phase difference images

6

as dark halos. (b) Thin layers of encapsulating material surrounding cells were inferred from

7

AFM scans in which no halos were observed but cells appeared to remain smooth and

8

hydrated, whilst (c) dehydrated cells had a characteristic shrivelled appearance.

9

Nanoindentation force curves performed on cells with thick capsules provided clear

10

confirmation of the capsular presence (Figure 2). Force curves recorded both directly on top

11

of encapsulated cells and in positions peripheral to the cell but still within the capsular

12

material both demonstrated non-linear deformations that were absent from empty control

13

regions of the surface. These non-linear deformations were further highlighted by calculation

14

of the first derivative of each force curve, the result demonstrating the compression of the

15

capsular material as a ‘step’. As the piezo moves the sample toward the tip, the capsule, or

16

the capsule and the cell compress linearly at first, and then there is a brief non-linear

17

deformation as the capsule is indented, before the force curve transitions into a second linear

18

region corresponding to either compression of the cell alone or deflection against the


Page 6 of 24

Page 7 of 24

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

1

underlying substratum. It should be noted that the relatively large attraction force experienced

2

by the AFM tip on approach to position B likely arises from traces of water adhering to the

3

tip from the previous measurement. This however does not affect the compressibility of the

4

material located at position B.

5 6

Figure 2. Nanomechanical analysis of P. aeruginosa cells with thick capsules. Force-distance

7

curves were recorded either on top of encapsulated cells (position A), on capsular material

8

peripheral to the cells (position B) or on the bare substratum nearby (position C). When the

9

capsule was present the relationship between applied force and piezo movement was linear at

10

first as the cell and capsule or capsule alone compressed. There was then a non-linear

11

transition corresponding to nanoindentation of the capsule, which led to a second linear

12

region in which the cell was compressed or the tip was deflected by the substratum. This was

13

visible in the first derivative of the force curve as a ‘step’ to a higher rate of change.


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

1

Nanomechanical analysis of hydrated cells lacking obvious signs of a capsule suggested

2

that capsular material was in fact present (Figure 3). Approach curves recorded on these cells

3

also contained a non-linear transition region in which the capsular layer was indented. The

4

force applied to the cell per nanometre of piezo movement was less prior to the

5

nanoindentation than following it, indicating that the combination of the cell and capsule was

6

softer than the cells alone, and more easily compressed. The non-linear region corresponded

7

to a temporary decrease in the force gradient in the first derivative plot. Further indication of

8

the thickness of the capsular layer can be seen from the piezo movement distance between the

9

point of contact and the onset of nanoindentation; the thicker capsules presented in Figure 2

10

began to indent after approximately 150 – 200 nm, whereas the thinner capsules began to

11

indent at approximately 50 nm. Greater capsule thickness lends to the ability to compress

12

over a greater distance before non-linear deformations occur. This method of verification

13

using nanomechanical analysis represents a highly sensitive method of capsular detection that

14

visualisation-based techniques may not be able to match, and has implications in

15

confirmation of bacterial virulence.


Page 8 of 24

Page 9 of 24

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

1 2

Figure 3. Nanomechanical analysis of P. aeruginosa cells that possessed thin capsules.

3

Bacterial cells were softer than the underlying substratum as expected, illustrated by the

4

lower gradient in the upper region of the force curves. Presence of a thin capsular layer was

5

inferred by the non-linear region of the force curve which is indicative of an indentation

6

event.

7

Dehydrated cells were stiffer than cells that possessed capsules (Table 1). Little difference

8

could be detected between the force curves recorded between the AFM tip approaching the

9

dehydrated cells and underlying substratum, and there was no observable nanoindentation

10

(Figure 4). This leads to an important conclusion on the interpretation of all of the

11

nanomechanical analyses performed here. Bacterial cells are relatively complex when

12

considered as colloidal particles, in that they are composed of several layers of different

13

materials. For example a Gram-negative bacterium such as P. aeruginosa possesses an outer


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 10 of 24

1

phospholipid membrane, a relatively thin peptidoglycan layer and a second, inner membrane

2

surrounding the cytosol. Each of these layers could potentially compress and deform

3

differently to the others, however Figure 3 demonstrates that under these experimental

4

conditions no distinction could be made between them, and therefore the cell itself could be

5

treated as a homogeneous particle, while remaining distinct from the capsular layer.

6

Nanoindentation was only observed on cells that possessed capsules, and given that all other

7

cellular components were constant it can be reasonably surmised that it is indeed the capsular

8

layer that is being indented. Additionally, encapsulated cells retained impressions of the tip

9

long after the force curves were recorded (Figure S2), which provided further confirmation

10

that the capsular material visible around the cells was not simply hygroscopic water.

11 12

Table 1. Approximate stiffness of Pseudomonas aeruginosa cells in various states of

13

encapsulation, and comparison to some commonly studied bacteria Capsule + cella (pN nm-1)

Cell alonea (pN nm-1)

Source

Thick capsule (~150-200 nm)

55

70

This study

Thin capsule (~50 nm)

60

70

This study

No capsule

–

80

This study

Pseudomonas aeruginosab

–

199

31

Escherichia colib

–

10 – 165

31-32

Staphylococcus aureusb

–

211

31

14

a

15

cell) and after (cell alone) the non-linear indentation domain

16

b

Compression stiffnesses were taken from first derivative force curves in the linear domains before (capsule +

Encapsulation status of individual bacterial cells was not reported


Page 11 of 24

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

1 2

Figure 4. Nanomechanical analysis of dehydrated P. aeruginosa cells. These cells did not

3

appear to differ in stiffness substantially from the underlying substratum, and no

4

nanoindentation was observed.

5

Previously reported Young moduli recorded for bacterial cells can vary greatly,31-32 which

6

is likely due to a combination of specific differences between species as well as variation in

7

culture conditions. Bacteria are dynamic, and react to their surrounding environment, for

8

example by producing stress-response factors, or by only producing certain cellular

9

components in ideal conditions. For this reason, nanomechanical analyses can only be easily

10

compared between identical strains and culture conditions.

11

The effect of encapsulation on the hydration status of the bacteria was assessed via analysis

12

of retraction curves recorded simultaneously on the same cells as the above nanoindentation

13

analyses. In all cases, adhesion forces of various magnitudes were observable between the


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

1

retracting AFM tip and the bacterial cells (Figure 5). Much stronger adhesion forces were

2

recorded over longer distances for cells determined to be encapsulated than for the non-

3

encapsulated cell. For example, at the point of detachment, the downward deflection of the

4

cantilever due to adhesion to the dehydrated cell was approximately 13.9 nm, with a

5

corresponding force of adhesion of 1.08 nN. For the bacterial cells with apparently thick

6

capsular layers, the cantilever deflection and corresponding force values were 134 nm and

7

7.40 nN, whereas for the cells with apparently thin layers they were 126 nm and 7.08 nN,

8

respectively. Importantly, both of the encapsulated cells also experienced a second

9

detachment event, corresponding to separation of the tip from the capsular material. The

10

precise moment of detachment was obscured somewhat by non-linearity in the force curve,

11

which arises as a result of independent detachment of individual alginate chains. First

12

derivative plots, however, offer a reliable means of approximating this point, as the final

13

detachment event is represented by a sharp inverted peak (Figure S3). Based on the position

14

of the inverted peak in first derivative force curves, the final detachment event for the thicker

15

capsule occurred at a downward deflection of 283 nm (Fadh = 1.64 nN) and for the thinner

16

capsule it occurred at a deflection of 403 nm (Fadh = 2.26 nN).

17

Interestingly, a larger downward deflection of the cantilever prior to the final detachment

18

was observed on retraction from the cells with a thinner capsular layer than for the thicker

19

capsule. This would appear to be contradictory to the initial assessment of capsule thickness

20

based on phase difference images in Figure 1. One must bear in mind, however, that the

21

capsular material is not a simple homogeneous layer surrounding some bacterial cells, rather

22

it is composed of numerous individual sugar chains, which may vary in length, and prolonged

23

adhesion of relatively few, longer chains may affect the apparent capsule thickness. This in

24

addition to potential stretching of the polysaccharide chains and the possibility of outward


Page 12 of 24

Page 13 of 24

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

1

flexing of the bacterial membrane suggest that measurement of capsule thickness according

2

to the point of detachment may not be accurate.

3 4

Figure 5. Adhesion forces between an AFM tip and bacterial cells, inferred from retraction

5

curves. Both encapsulated cells (top, middle) experienced two detachment events (the first

6

being detachment of the tip from the cell itself, and the second detachment from the capsule),

7

while the non-encapsulated cell only experienced a single detachment. Deflection distances


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

1

prior to detachment were greater for the encapsulated cells, due to higher capillary adhesion

2

forces.

3


Page 14 of 24

Page 15 of 24

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

1

CONCLUSIONS

2

Bacterial capsules were detected through AFM topography and phase imaging, with

3

verification of the encapsulation status of the cells determined through nanomechanical

4

analysis. Nanoindentation curves recorded on encapsulated cells had the general form of a

5

linear compression domain (compression of cell and capsule), followed by a non-linear

6

indentation domain (indentation of capsule) transitioning into a second linear compression

7

domain (compression of the cell alone). Whereas recent work has attributed the soft, liquid-

8

like material detected around bacterial cells to deliquesence of water rather than capsular

9

material, evidence is shown here that it is in fact both; i.e. capsular material dissolved in

10

deliquescent water. This is a sensitive technique for the measurement of the nanomechanical

11

properties of bacterial cell layers, which will be highly beneficial to microbial pathology

12

work, as it allows for quick and simple detection of bacterial capsules, which are known

13

virulence factors. This technique may also prove useful for performing nanomechanical

14

analyses of layered colloidal particles in other fields.

15 16

ASSOCIATED CONTENT

17

Supporting Information

18

Supporting evidence of bacterial encapsulation in the form of biochemical staining,

19

confirmation of the ability of deliquescent capsular material to retain impressions caused by

20

the AFM tip and the location of final detachment events based on first derivatives of force

21

curves are available as supporting information. This material is available free of charge via

22

the Internet at http://pubs.acs.org.

23 24


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

1

AUTHOR INFORMATION

2

Corresponding author

3

*Email: [email protected] (H.K.W.). Tel: +61 3 9214 5183

4 5

Notes

6

The authors declare no competing financial interests.

7 8

ACKNOWLEDGEMENTS

9

This work was supported by the Department of Chemistry and Biotechnology at Swinburne

10

University of Technology. The authors would also like to express their gratitude to Mr Ngan

11

Nguyen for supplying bacterial cultures.

12 13


Page 16 of 24

Page 17 of 24

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

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

Langmuir

REFERENCES (1) Mauer, L. J.; Taylor, L. S. Deliquescence of pharmaceutical systems. Pharm. Dev. Technol. 2010, 15, 582-594. (2) Kortright, F. L. The deliquescence of potassium nitrate, sodium nitrate, and ammonium nitrate. J. Phys. Chem. 1899, 3, 328-333. (3) Steinbach, O. F. Laboratory experiment on deliquescence and efflorescence. J. Chem. Educ. 1943, 20, 146. (4) Cruz, C. N.; Pandis, S. N. Deliquescence and hygroscopic growth of mixed inorganic - organic atmospheric aerosol. Environ. Sci. Technol. 2000, 34, 4313-4319. (5) Tang, I. N.; Munkelwitz, H. R. Composition and temperature dependence of the deliquescence properties of hygroscopic aerosols. Atmos. Environ. Part A Gen. Top. 1993, 27, 467-473. (6) Méndez-Vilas, A.; Labajos-Broncano, L.; Perera-Núñez, J.; González-Martín, M. L. Are the soft, liquid-like structures detected around bacteria by ambient dynamic atomic force microscopy capsules? Appl. Environ. Microbiol. 2011, 77, 3102-3114. (7) Su, H.-N.; Chen, Z.-H.; Liu, S.-B.; Qiao, L.-P.; Chen, X.-L.; He, H.-L.; Zhao, X.; Zhou, B.-C.; Zhang, Y.-Z. Characterization of bacterial polysaccharide capsules and detection in the presence of deliquescent water by atomic force microscopy. Appl. Environ. Microbiol. 2012, 78, 3476-3479. (8) Stukalov, O.; Korenevsky, A.; Beveridge, T. J.; Dutcher, J. R. Use of atomic force microscopy and transmission electron microscopy for correlative studies of bacterial capsules. Appl. Environ. Microbiol. 2008, 74, 5457-5465. (9) Herzberg, M.; Rezene, T. Z.; Ziemba, C.; Gillor, O.; Mathee, K. Impact of higher alginate expression on deposition of Pseudomonas aeruginosa in radial stagnation point flow and reverse osmosis systems. Environ. Sci. Technol. 2009, 43, 7376-7383. (10) Jain, S.; Ohman, D. E. Role of an alginate lyase for alginate transport in mucoid Pseudomonas aeruginosa. Infect. Immun. 2005, 73, 6429-6436. (11) Deretic, V.; Dikshit, R.; Konyecsni, W. M.; Chakrabarty, A. M.; Misra, T. K. The algR gene, which regulates mucoidy in Pseudomonas aeruginosa, belongs to a class of environmentally responsive genes. J. Bacteriol. 1989, 171, 1278-1283. (12) Krieg, D. P.; Bass, J. A.; Mattingly, S. J. Phosphorylcholine stimulates capsule formation of phosphate-limited mucoid Pseudomonas aeruginosa. Infect. Immun. 1988, 56, 864-873. (13) Roberts, I. S. The biochemistry and genetics of capsular polysaccharide production in bacteria. In Annual Review of Microbiology, 1996; Vol. 50, pp 285-315. (14) Whitfield, C. Biosynthesis and assembly of capsular polysaccharides in Escherichia coli. In Annual Review of Biochemistry, 2006; Vol. 75, pp 39-68. (15) Green, B. D.; Battisti, L.; Koehler, T. M.; Thorne, C. B.; Ivins, B. E. Demonstration of a capsule plasmid in Bacillus anthracis. Infect. Immun. 1985, 49, 291-297. (16) Whitfield, C.; Roberts, I. S. Structure, assembly and regulation of expression of capsules in Escherichia coli. Mol. Microbiol. 1999, 31, 1307-1319. (17) Picot, A.; Lacroix, C. Encapsulation of bifidobacteria in whey protein-based microcapsules and survival in simulated gastrointestinal conditions and in yoghurt. Int. Dairy J. 2004, 14, 505-515. (18) Nelson, A. L.; Roche, A. M.; Gould, J. M.; Chim, K.; Ratner, A. J.; Weiser, J. N. Capsule enhances pneumococcal colonization by limiting mucus-mediated clearance. Infect. Immun. 2007, 75, 83-90. (19) Bogaert, D.; De Groot, R.; Hermans, P. W. M. Streptococcus pneumoniae colonisation: The key to pneumococcal disease. Lancet Infect. Dis. 2004, 4, 144-154.


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

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

(20) Archer, G. L. Staphylococcus aureus: A well-armed pathogen. Clin. Infect. Dis. 1998, 26, 1179-1181. (21) Chitnis, C. E.; Ohman, D. E. Genetic analysis of the alginate biosynthetic gene cluster of Pseudomonas aeruginosa shows evidence of an operonic structure. Mol. Microbiol. 1993, 8, 583-590. (22) Bricker, A. L.; Camilli, A. Transformation of a type 4 encapsulated strain of Streptococcus pneumoniae. FEMS Microbiol. Lett. 1999, 172, 131-135. (23) Domenico, P.; Schwartz, S.; Cunha, B. A. Reduction of capsular polysaccharide production in Klebsiella pneumoniae by sodium salicylate. Infect. Immun. 1989, 57, 37783782. (24) Swartley, J. S.; Marfin, A. A.; Edupuganti, S.; Liu, L. J.; Cieslaku, P.; Perkins, B.; Wenger, J. D.; Stephens, D. S. Capsule switching of Neisseria meningitidis. Proc. Natl. Acad. Sci. U. S. A. 1997, 94, 271-276. (25) Brunner, J.; Scheres, N.; El Idrissi, N. B.; Deng, D. M.; Laine, M. L.; Van Winkelhoff, A. J.; Crielaard, W. The capsule of Porphyromonas gingivalis reduces the immune response of human gingival fibroblasts. BMC Microbiol. 2010, 10. (26) Kern, J.; Schneewind, O. BslA, the S-layer adhesin of B. anthracis, is a virulence factor for anthrax pathogenesis. Mol. Microbiol. 2010, 75, 324-332. (27) Butt, H. J.; Jaschke, M. Calculation of thermal noise in atomic force microscopy. Nanotechnology 1995, 6, 1-7. (28) Proksch, R.; Schäffer, T. E.; Cleveland, J. P.; Callahan, R. C.; Viani, M. B. Finite optical spot size and position corrections in thermal spring constant calibration. Nanotechnology 2004, 15, 1344-1350. (29) Walters, D. A.; Cleveland, J. P.; Thomson, N. H.; Hansma, P. K.; Wendman, M. A.; Gurley, G.; Elings, V. Short cantilevers for atomic force microscopy. Rev. Sci. Instrum. 1996, 67, 3583-3590. (30) Anthony Jr., E. E. A note on capsule staining. Science 1931, 73, 319-320. (31) Nakanishi, K.; Kogure, A.; Fujii, T.; Kokawa, R.; Deuchi, K. Development of method for evaluating cell hardness and correlation between bacterial spore hardness and durability. J. Nanobiotechnol. 2012, 10, 22. (32) Zhang, W.; Hughes, J.; Chen, Y. Impacts of hematite nanoparticle exposure on biomechanical, adhesive, and surface electrical properties of Escherichia coli cells. Appl. Environ. Microbiol. 2012, 78, 3905-3915.

34


Page 18 of 24

Page 19 of 24

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

1

Langmuir

ToC graphic

2


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

Encapsulation/hydration states of P. aeruginosa cells immobilised on glass. Topography (left) and phase difference (right) data from AFM scans of each cell state are presented, together with three dimensional representations correlating the two. (a) Thick capsules surrounding the cells were easily identifiable through examination of phase difference images as dark halos. (b) Thin layers of encapsulating material surrounding cells were inferred from AFM scans in which no halos were observed but cells appeared to remain smooth and hydrated, whilst (c) dessicated cells had a characteristic shrivelled appearance. 319x141mm (300 x 300 DPI)


Page 20 of 24

Page 21 of 24

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

Nanomechanical analysis of P. aeruginosa cells with thick capsules. Force-distance curves were recorded either on top of encapsulated cells (position A), on capsular material peripheral to the cells (position B) or on the bare substratum nearby (position C). When the capsule was present the relationship between applied force and piezo movement was linear at first as the cell and capsule or capsule alone compressed. There was then a non-linear transition corresponding to nanoindentation of the capsule, which led to a second linear region in which the cell was compressed or the tip was deflected by the substratum. This was visible in the first derivative of the force curve as a ‘step’ to a higher rate of change. 215x150mm (300 x 300 DPI)


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

Nanomechanical analysis of P. aeruginosa cells that possessed thin capsules. Bacterial cells were softer than the underlying substratum as expected, illustrated by the lower gradient in the upper region of the force curves. Presence of a thin capsular layer was inferred by the non-linear region of the force curve which is indicative of an indentation event. 208x163mm (300 x 300 DPI)


Page 22 of 24

Page 23 of 24

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

Nanomechanical analysis of dehydrated P. aeruginosa cells. These cells did not appear to differ in stiffness substantially from the underlying substratum, and no nanoindentation was observed. 233x172mm (300 x 300 DPI)


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. Adhesion forces between an AFM tip and bacterial cells, inferred from retraction curves. Both encapsulated cells (top, middle) experienced two detachment events (the first being detachment of the tip from the cell itself, and the second detachment from the capsule), while the non-encapsulated cell only experience a single detachment. Deflection distances prior to detachment were also greater for the encapsulated cells, due to higher capillary adhesion forces. 224x238mm (150 x 150 DPI)


Page 24 of 24

Sensitive Detection of Deliquescent Bacterial Capsules through

Recommend Documents