Physiological basis for biofilm resistance to antimicrobial agents
by Wendy Lee Cochran
A thesis submitted in partial fulfillment of the requirement for the degree of Doctor of Philosophy In
Microbiology
Montana State University
© Copyright by Wendy Lee Cochran (1998)
Abstract:
The physiological basis of Pseudomonas aeruginosa biofilm resistance to monochloramine (MCA) and
hydrogen peroxide (H202) was investigated. An experimental system consisting of bacteria attached to
the surface of suspended alginate gel beads was developed to definitively eliminate the possibility of
ineffective antimicrobial delivery to biofilm cells. After 20 hours or more of biofilm growth,
disinfection efficacy of MCA was significantly reduced compared to planktonic cells (p ≤ 0.0018).
Biofilms that were 24 hours and 48 hours old were also resistant to H202 compared to planktonic cells
(p ≤ 0.019). The ratio of disinfection rate coefficients measured for planktonic cells to that measured
for biofilm cell was 1.6 - 2.6 for MCA and 3.3 - 3.9 for H202. Susceptibility to the biocides did not
significantly correlate with initial biofilm areal cell density (p = 0.84). These results indicate that
physiological changes occurred in attached P. aeruginosa that protect them from oxidative stresses.
To address the issue of physiological changes in biofilm cells, the role of two sigma factors, AlgT and
RpoS, in mediating resistance to H202 and MCA was investigated. Two knock out mutant strains,
SS24 (rpoS^-) and PAO6852 (algT) were compared to the wild type strain, PAO1, in their
susceptibility to the biocides. When biofilms were grown on alginate gel beads, all strains were equally
resistant to MCA disinfection and biofilm coefficients were significantly lower (p ≤ 0.015) than
coefficients obtained from the same cells grown planktonically. However, 24-hour old rpoS^- and
algT^- biofilms were more susceptible to H202 disinfection than were biofilms formed by PAO1;
biofilm disinfection rate coefficients were statistically the same (p ≥ 0.15) as planktonic disinfection
rate coefficients. While 48-hour old algT^- biofilm cells became resistant to H202, 48-hour old rpoS'^biofilm cells remained highly susceptible.
To confirm the role of RpoS, rpoS was restored to SS24 by constructing a complement strain, SS240.
This strain exhibited disinfection profiles similar to PAO1 (p = 0.12). The expression of RpoS in
SS240 was confirmed by immunoblot analysis. It was concluded that algT and rpoS are two genes that
are significantly involved in the protection of thin biofilm from H202. PHYSIOLOGICAL BASIS FOR BIOFILM RESISTANCE TO ANTIMICROBIAL
AGENTS
by
Wendy Lee Cochran
A thesis submitted in partial fulfillment
of the requirement for the degree
of
Doctor of Philosophy
In
Microbiology
MONTANA STATE UNIVERSITY - BOZEMAN
Bozeman, Montana
November 1998
ii
O
APPROVAL
of a thesis submitted by
Wendy Lee Cochran
This thesis has been read by each member of the thesis committee and has
been found to be satisfactory regarding content, English usage, format, citations,
bibliographic style, and consistency, and is ready for submission to the College
of Graduate Studies.
pTars
Dr. Gordon A. McFeters
/(Siohature
' m iS (D^te)
Approved by Committee Chair
Dr. Philip S. Stewart
(HLjyX
(Signature)
ii-zH-n
(Date)
Approved by Committee Chair
Vi W h U vS)
Dr. Seth H. Pincus
(Date)
Approved by Department of Microbiology
Dr. Joseph J. Fedock
^fg n d fu re ),
(Date)
Approved by the College of Graduate Studies
Ill
STATEMENT OF PERMISSION TO USE
In presenting this thesis in partial fulfillment of the requirements for a
doctoral degree at Montana State University-Bozeman, I agree that the Library
shall make it available to borrowers under rules of the Library. I further agree
that copying of this thesis is allowable only for scholarly purposes, consistent
with “fair use” as described in the U.S. Copyright Law. Requests for extensive
copying or reproduction of this thesis should be referred to University.Microfilms
International, 300 North Zeeb Road, Ann Arbor, Michigan 48106, to whom I have
granted “the exclusive right to reproduce and distribute my dissertation in and
from microform along with the non-exclusive right to reproduce and distribute my
abstract in any format in whole or in part.”
Date
iv
ACKNOW LEDG EMENTS
I thank Gordon McFeters and Phil Stewart for their support, enthusiasm,
and interest in my project. Their encouragement and breadth of knowledge has
significantly helped in the completion of the work presented here.
Thanks to my committee members: Gill Geesey, Cliff Bond, and Linda
Sherwood. Thanks to Anne Camper, who graciously filled in for Gordon during
his absence.
Thank you to Sue Broadaway for your answers to my unending questions
and your patience when showing me all the techniques useful in my work as a
microbiologist. Thank you Barry Pyle for your advice and support in the lab.
Thank you to all of the graduate students, both from Microbiology and the
Center, who made my experience here more enriching.
Special thanks to Balu for all the great hikes and adventures we have
been on.
And lastly, thank you to my parents, Bonnie and Ken Krauss, who told me
at an early age that I could be whatever I wanted to be.
This research was supported by the Center for Biofilm Engineering at
Montana
State
University,
a
National
Science
Foundation-sponsored
engineering research center (cooperative agreement ECD-8907039).
V
TABLE OF CONTENTS
Chapter
1.
3.
4.
, BIOFILM RESISTANCE TO ANTIMICROBIAL AGENTS'
LITERATURE REVIEW
................................................................
Background ................................................................
Disinfectants ........
Biofilm Formation
Biofilm Physiology
Biofilm Heterogeneity .............. ........................................... .
Planktonic Cell Resistance to Antimicrobial Agents
..............
Biofilm Resistance to Antimicrobial Agents ..............
Significant Biofilm Genes ...............
Objectives, Rationale, and Experimental Design
.................
References Cited .....................................................................
-| 1
13
17
21
23
25
PSEUDOMONAS AERUGINOSA BIOFILMS RESIST
OXIDATIVE BIOCIDES IN THE ABSENCE OF MASS
TRANSFER LIMITATIONS ..........................................................
Introduction ............................................................
Material and Methods ..............................................................
Results .................................................................................
Discussion ................................................................................
References Cited .....................................................................
36
36
38
48
60
63
RPOS AND ALGT CONTRIBUTE TO PSEUDOMONAS
AERUGINOSA BIOFILM RESISTANCE TO HYDROGEN
PEROXIDE ..............
Introduction .............................................
Material and Methods ..............................................................
Results .....................................................................................
Discussion ..................................................
References Cited ............................................
68
68
70
75
83
86
SUMMARY AND CONCLUSION .............. .................................
References Cited .....................................................................
91
95
Appendix
RAW DATA OF BIOCIDE EXPERIMENTS
1
-I
CM LO CO
2.
Page
98
vi
LIST OF TABLES
Table
2.1.
Page
P-values for comparison of planktonic and biofilm
susceptibility.........................................................
50
3.1.
Bacterial Strains and Plasmids.
............................................
71
3.2.
P-values for comparison of planktonic and biofilm
susceptibility to MCA and H2O2...............................................
77
Raw data used to determine effects of citrate on cell
viability.....................................................................................
99
Disinfection rate coefficients and initial cell densities for
planktonic experiments using 2 mg L"1 MCA..........................
100
Disinfection rate coefficients and initial cell densities for
planktonic experiments using 600 mg L'1 H2O2.....................
100
Disinfection rate coefficients and initial cell densities for
biofilm experiments using 2mg L"1 MCA.................................
101
Disinfection rate coefficients and initial cell densities for
biofilm experiments using 600 mg L 1 H2O2. .......................
102
Disinfection rate coefficients and initial cell densities for
complement strains experiments using 600 mg L'1 H2O2.
103
A.1
A.2
A.3
A.4
A.5
A.6
...
vii
LIST OF FIGURES
Figure
2.1.
Page
Schematic diagram describing the construction of PA01 P.
aeruginosa constitutively expressing gfp............ .......................
39
2.2.
Alginate gel beads. Gel beads have a radius of 1 mm
...............
40
2.3.
Use of GFP to detect P.aeruginosa biofilms on alginate gel
beads. Epifluorescence of 24 hour old biofilms (A and B).
Bars represent 5 pm. 48 hour old biofilms (C). Bars
Represent 10 pm. Cell distribution consisted mostly of
single cells and small microcolonies............................................
49
Disinfection rate coefficients of planktonic and biofilm P.
aeruginosa cells exposed to 2 mg L"1 of MCA. Error
bars represent standard error of the disinfection rate
coefficient...............
51
Influence of initial cell density of disinfection efficacy.
Error bars represent standard error of the disinfection
rate coefficients. Regression line r2 = 0.02. P-value
of the slope = 0.81........................................................................
52
2.4.
2.5.
2.6.
Disinfection rate coefficients of planktonic and biofilm P.
aeruginosa cells exposed to 600 mg L"1 of H2O2. Error
bars represent standard error of the disinfection rate
coefficient..........................................................................................53
2.7.
Calculated transport of MCA to alginate gel beads.
Cs = surface concentration and C0 = bulk fluid
concentration. At 33 sec CsZC0 = 95% and at 1 min
CsZC0 = 98%.
55
Diffusion of H2O2into 24 hour gel bead biofilms. Beads
were incubated in H2O2 color indicators for 15 mins
then 600 mg L'1 of H2O2 was added. Photographs
were taken at 10 sec (A), 1:45 min (B)1and 2:30 min
(C). As H2O2 diffused through the bead, the bead turn
black.............................................................................................
56
2.8.
viii
2.9
2.10.
3.1
3.2
3.3
3:4
3.5
A.1
Diffusion of Na2S2O3into 24 hour gel bead biofilms.
Beads were exposed to 600 mg L'1. ).1 N of Na2S2O3
was added to the alginate gel beads after incubation
with H2O2. Photographs shown were taken at 10 sec
(A), 2 min (B), and 4:50 min (C). As Na2S2O3 diffused
through the bead, the H2O2 was neutralized and the
bead turned back to white......... ...................................
57
Mean disinfection rate coefficients obtained from biofilms
grown on glass slides exposed to either 2 mg/L of
MCA or 600 mg L"1 of H2O2. Error bars represent
standard error of the mean............................................
59
Mean disinfection rate coefficients of P. aeruginosa
strains using 2 mg L"1 of MCA. Biofilms were grown
on alginate gel beads. Error bars represent standard
error of the mean...........................................................
76
Mean disinfection rate coefficients of P. aeruginosa
strains using 600 mg L"1 of H2O2. Biofilms were grown
on alginate gel beads. Error bars represent standard
error of the mean...........................................................
78
Disinfection rate coefficients of P. aeruginosa strains with
and without rpoS. Biofilms were grown on alginate gel
beads and exposed to 600 mg L"1 of H2O2. Error bars
represent standard error of the disinfection rate
coefficients.....................................................................
79
Immunoblot analysis of proteins of wild type, PA01; rpoS
mutant, SS24; rpoS complement strain, SS240; and
vector control, SS226...................................... .............
81
Mean disinfection rate coefficients of P. aeruginosa
strains using 2 mg L"1 of MCA and 600 mg L"1 of H2O2.
Biofilms were grown on glass slides. Error bars
represent standard error of the mean...........................
82
Cell Survival after the addition of various concentrations
of citrate buffer and blending either once (1X B) or
twice(2X B) with a tissue homogenizer at 50\5 speed
for 1 min................................... .....................................
99
ix
ABSTRACT
The physiological basis of Pseudomonas aeruginosa biofilm resistance to
monochloramine (MCA) and hydrogen peroxide (H2O2) was investigated. An
experimental system consisting of bacteria attached to the surface of suspended
alginate gel beads was developed to definitively eliminate the possibility of
ineffective antimicrobial delivery to biofilm cells. After 20 hours or more of
biofilm growth, disinfection efficacy of MCA was significantly reduced compared
to planktonic cells (p < 0.0018). Biofilms that were 24 hours and 48 hours old
were also resistant to H2O2 compared to planktonic cells (p < 0.019). The ratio
of disinfection rate coefficients measured for planktonic cells to that measured
for biofilm cell was 1.6 - 2.6 for MCA and 3.3 - 3.9 for H2O2. Susceptibility to
the biocides did not significantly correlate with initial biofilm areal cell density (p
= 0.84). These results indicate that physiological changes occurred in attached
P. aeruginosa that protect them from oxidative stresses.
To address the issue of physiological changes in biofilm cells, the role of
two sigma factors, AIgT and RpoS, in mediating resistance to H2O2 and MCA
was investigated. Two knock out mutant strains, SS24 {rpoS~) and PA06852
(algT) were compared to the wild type strain, PA01, in their susceptibility to the
biocides. When biofilms were grown on alginate gel beads, all strains were
equally resistant to MCA disinfection and biofilm coefficients were significantly
lower (p < 0.015) than coefficients obtained from the same cells grown
planktonically. However, 24-hour old rpoST and algT biofilms were more
susceptible to H2O2 disinfection than were biofilms formed by PA01; biofilm
disinfection rate coefficients were statistically the same (p > 0.15) as planktonic
disinfection rate coefficients. While 48-hour old algT biofilm cells became
resistant to H2O2, 48-hour old rpoS~ biofilm cells remained highly susceptible.
To confirm the role of RpoS, rpoS was restored to SS24 by constructing a
complement strain, SS240. This strain exhibited disinfection profiles similar to
PA01 (p = 0.12).
The expression of RpoS in SS240 was confirmed by
immunoblot analysis. It was concluded that algT and rpoS are two genes that
are significantly involved in the protection of thin biofilm from H2O2.
1
CHAPTER 1
BIOFILM RESISTANCE TO ANTIMICROBIAL AGENTS: LITERATURE REVIEW
Background
Traditionally, bacteria have been studied as planktonic, free-floating
organisms. This mode of growth, however, may not be predominant in most
systems.
The attachment of organisms to surfaces to form biofilms was first
noted by Antony van Leeuwenhoek, who discovered a plethora of “animalcules”
(bacteria) living on his teeth. Sdhngen in 1913 (88) observed the increase in
activity of hydrocarbon-degrading bacteria by attached to soil.
Breden and
Buswell (5) observed that inert material supported organisms by “preventing their
loss with removal of spent liquor and making possible the heavy inoculation of
fresh substrates.” Zobell (110) demonstrated that organic matter is absorbed by
glass from seawater and the organic matter that is concentrated at the surface
enhances bacterial activity.
However, it was not until the 1970’s that biofilms
became recognized as a major mode of microbial growth (37).
There is increasing evidence that many microorganisms prefer to attach to
a surface and become biofilms (13,19,21,24,70,80,95). Microorganisms existing
in biofilms are found in all aquatic systems and to date no surface has been
discovered that microorganisms cannot colonize.
Biofilms are ubiquitous
2
throughout the natural environment as well as in industrial and medical
environments.
Many biofilms of industrial relevance are detrimental to the point of being
an economic concern. These biofilms are associated with the reduction ofheat
transfer capacity in cooling water towers, the production of inferior paper
products, and poor drinking water quality (12). Biofilm build up on ship hulls
increases fluid drag and increases fuel consumption (18). Standard disinfection
procedures appear to have little to no effect in eliminating biofilms from these
systems. Biofilms are recognized as a medical problem as well. Many diseases
can be attributed to biofilm growth such as ear infections (78), coronary artery
disease (22), kidney stones (56), and cystic fibrosis (31).
Medical devices
including prosthetic heart valves, catheters, cardiac pacemakers and prosthetic
joints (57), can be colonized by bacterial infections. Once these devices are
colonized, the biofilm is extremely difficult to eradicate with antibiotic therapy.
Ultimately, the only way to resolve the infection is to remove the device, resulting
in additional trauma and expense to the patient.
Disinfectants
Disinfectants are chemicals used to control bacterial growth.
Many
disinfectants are effective in killing planktonic cells, but appear to have little
effect on attached cells.
This section will describe two industrially relevant
3
disinfectants, hydrogen peroxide (H2O2) and monochloramine (MCA).
Chlorine
oxidizing-compounds,
such
as
hypochlorous
acid
and
monochloramine, are effective antimicrobial agents. The primary sites of cellular
inactivation are the thiol groups in the membrane and cytoplasmic regions of
bacteria (23).
Chloramines are less effective disinfectants than chlorine in
equivalent doses (33), however chloramines do provide longer lasting residuals
and less of a health hazard, since there is no harmful byproduct production.
Combining chlorine and ammonium or nitrogen-containing organic compounds
produces chloramines. Monochloramine (MCA), dichloramine, and trichloramine
are the three inorganic species of chloramines. All of these compounds may be
present depending upon pH and chlorine-ammonium ratio. Increasing pH and
ammonium concentrations favors the formation of monochloramine (50).
Chloramines are used as disinfectants in many water facilities.
It has
been reported to be most effective in waters with a pH less than 8 (50). Since
free chlorine disinfectants result in the formation of toxic byproducts, there is an
increasing move away from free chlorine as a disinfectant and chloramines are a
good alternative. Chloramines, especially MCA, are more effective disinfectants
than free chlorine against biofilms (62). This is due to their less reactive nature,
which allows them to penetrate farther into the biofilm before it is consumed by
the bacteria. When using planktonic disinfection results, LeChevaIIier et al. (62)
reported that a 10-fold increase in hypochlorous acid concentration had little
effect on the disinfection of biofilm cells. However, MCA at a 10-fold increase in
4
effective planktonic concentrations reduced viable biofilm cell counts by 65%.
By calculating the concentration and time (C x T) factor (41,42), they showed
that one C x T unit required for 99% inactivation of unattached cells of MCA was
more effective for inactivating biofilm bacteria than 600 C x T
units of
hypochlorous acid.
Hydrogen peroxide is a clear, colorless liquid with a slightly acidic odor. It
has been used extensively in many applications as a disinfectant. It is used in
swimming pools, ultrasonic disinfectant cleaning baths for dental and medical
instruments, treatment of landfill leachates, and as a disinfectant for contact
lenses (3). H2O2 is an oxidizing agent that attacks thiol groups in enzymes and
proteins. Its activity results from the formation of free hydroxyl radicals (-OH)
(23).
It is also effective against microorganisms by attacking the lipid
membrane, DMA, and other cell components.
It is suggested (3) that the
antimicrobial action is due to its oxidation of sulfhydryl groups and double bonds
in proteins, lipids, and surface membranes.
The major mode of bacterial resistance to H2O2 is due to the production of
catalase, which breaks it into water and oxygen, but new mechanisms of
resistance are being discovered.
The production of glutathione has been
observed in many gram-negative bacteria (16,35). Glutathione is produced by
phagocytic white blood cells to protect against their own production of H2O2.
The production of this compound should protect bacteria from the oxidative
effects of H2O2and other oxidizing radicals, such as chlorine (16).
5
It has also been shown that E coli (29) and S. typhimurium (102) become
resistant to killing by hydrogen peroxide when pretreated with a non lethal dose
of H2O2. This adaptation results in the transient accumulation of a distinct group
of proteins (17,72). Many of the proteins are under positive control of the oxyR
gene product (17,72).
OxyR is known to induce HPI catalase (katG), alkyl
hydrogen peroxidase reductase (ahpCF) (17,72), glutathione reductase (gorA)
(59), dps (I), and oxyS (59,64). Recently, 26 mutants have been identified that
exhibit a H2O2 sensitive phenotype (74).
OxyR is also required for the
expression of coproporphyrinogen III oxidase (hemf), and an open reading
frame, f497, that is similar to arylsulfatase-encoding genes.
Biofilm Formation
In order to initiate biofilm formation, bacteria must be transported close to
a surface where they can stick. Several attraction forces including London-van
der Waals, electrostatic, and steric interactions (13) control the initial adhesion
process. Flagella, fimbriae and pili play an improtant role in allowing cells to
remain at a surface.
This initial stage of adhesion is referred to as reversible
adhesion, because the cells can easily detach from the surface. Once bacteria
attach to a surface they begin to produce extracellular polymeric substances
(EPS), composed of polysaccharides, proteins and other organic molecules.
This process is referred to as irreversible adhesion and in this stage dipole-
6
dipole interactions, dipole-induced dipole interactions, ion-dipole interactions,
hydrogen bonds, hydrophobic interactions and polymeric bridging are involved
(13). The bacteria then reproduce and form microbial aggregates that colonize
the surface and form a mature biofilm.
i!
:!
.
Biofilm Physiology
It is speculated that biofilm bacteria have a number of advantages over
their planktonic counterparts. Nutrients absorb to surfaces giving attached cells
a nutritional advantage (2,40,69). Attached bacteria also have increased growth
rates (26). There are more resistant to biocides (14,15,20,27,49,75,77,81,82,89,
92,93,105).
Surfaces in aquatic systems rapidly adsorb organic molecules.
These
organic molecules are a source of nutrients for the microorganisms that attach to
the surface. An organism with the ability to attach to a surface could have a
nutritional advantage over an organism that does not attach. Griffith and
Fletcher (40) reported the adsorption of bovine serum albumin (BSA) by
particles derived from diatoms. Attached bacteria degraded 100% of the protein
absorbed, while the planktonic cells were unable to utilize BSA.
Therefore,
attached bacteria can have a nutritional advantage over planktonic cells,
especially in oligotrophic environments.
’
7
McFeters et -al. (69) reported that attached soil microorganisms were
significantly involved in the degradation of xenobiotics.
In fact, the attached
cells were more physiologically active in the degradation of the xenobiotic than
their planktonic counterparts.
Similarly, Ascon-Cabrera et al (2) showed that
glass-adhered Pseudomonas cells degraded 2,4,6,-trichlorophenol faster than
planktonic cells. The attached cells adapted physiologically to be more efficient
at degrading xenobiotics in these systems.
Bacteria attached to granular activated carbon (GAC) were shown to have
higher growth rates when glutamate was used as a carbon source (26). These
increased growth rates differed significantly from planktonic growth rates using
the same substrate. Glutamate was shown to absorb to the surface of the GAC
where it was believed to concentrate to higher levels than in the bulk liquid.
Uptake of radiolabeled nucleotides was also greatly increased in the attached
cells. This correlates to the faster growth rate of the biofilm bacteria as well as a
faster DNA and RNA turnover rate. Attachment of bacteria to GAC can have a
profound effect on bacterial physiology (26).
Bacteria have a higher rate of genetic transfer due to transduction than
planktonic cells (79).
Suspended particles in a system have the potential to
stimulate phage-host interactions by allowing the formation of compact
microenvironments consisting of aggregates of bacterial cells, phage virions,
.and particulates (79).
The addition of low concentrations of clay particles
significantly increased the viral plaque-forming efficiency compared to results
8
obtained in the absence of particulate matter (79).
Both the length of the
latency period and the size of the burst of progeny virus particles are dependent
on the metabolic rate of the host (58).
It is then likely that attached cells
produce more phage particles than unattached cells do, this in turn increases
the number of phage particles per bacterial cell (79).
This increase in
transduction due to attachment to particulates is a viable means of gene transfer
in aquatic systems and may increase genetic diversity (79).
Biofilm cells are protected in an EPS layer. This layer is composed of
polysaccharides as well as proteins and other organic matter. The EPS appears
to protect the cells against biocides (20,27,75,77,92,105) and heat (36).
By
acting as a penetration barrier, the EPS prevents the diffusion of biocides and
antibiotics into the biofilm. This layer may also consume or bind the biocide
preventing its action on the microbial cells.
Nichols et al. (75) observed the
binding of tobramycin to alginate and exopolysaccharide prepared from two
strains of Pseudomonas aeruginosa.
They concluded that the decrease in
antibiotic concentration due to alginate binding reduces the driving force for
diffusion.
Stimulation of exopolysaccharide synthesis by attachment has been
reported by Davies et al. (25) and Vandevivere and Kirchman (97). Davies et al.
(25) reported an 85-fold increase of alginate production in attached P.
aeruginosa cells over nonattached cells.
reviewed the regulation of alginate.
May and Chakrabarty (68) recently
AIgD catalyzes the rate-limiting step of
9
alginate production. It is also this step that commits the cell to the mucoid form.
- The activation of algD involves several environmental factors including
increased osmolarity, ethanol concentrations, N2 limitation, PO4 limitation, and
adherence to a surface. The regulation of the algD promoter is also dependent
upon AIgT, a putative sigma factor (67).
This sigma factor has also been
reported to be involved in the heat shock and oxidative responses (67).
Lipopolysaccharide (LPS) was extracted from biofilm and planktonic cells
and analyzed by polyacrylamide gel electrophoresis (PAGE) followed by
immune-detection of LPS fractions (39). LPS from plankton!cal grown S-form of
P. aeruginosa appeared as a typically ladder of bands on a PAGE gel (39). LPS
from a semi-rough form consisted of only two bands, core/lipid A and core/lipid A
plus a repeating unit (R-LPS). When grown planktonically and as biofilms, the
LPS of semi-rough cells showed a very similar PAGE pattern.
However, the
core/lipid A R-LPS fraction extracted from S-form bacteria was more prominent
in biofilm-LPS than in planktonic-LPS. This apparent change in LPS sub-unit
components of the bacteria when grown as biofilm may reflect changes in the
outer membrane structure that contribute to the altered physio-chemical
properties of biofilm bacteria (39).
Although bacteria are known to exhibit morphological and phenotypic
variations due to external environmental stimuli, very little is known about
bacterial responses to surfaces (23). Neisseria gonorrhoeae has been reported
to express fimbriae when attached, which are absent in nonattached cells.
10
Some Pseudomonas species, as well as Vibrio parahaemolyticus, exhibit
morphological differences when grown on a solid surface as compared to liquid
medium (23).
Pseudomonas spp. cells grown in liquid and on agar were
uniformly rod-shaped, but cells found in a biofilm were long and filamentous.
Dalton et at. (23) also observed different morphologies of a marine isolate when
grown on different substrata. These organisms grown on hydrophobic surfaces
were characterized by the formation of tightly packed biofilms consisting of
single and paired cells.
Those at hydrophilic surfaces exhibited sparse
colonization of the surface and the formation of chains in excess of 100 pm,
anchored at the surface by the terminal cell.
Due to the close spatial
relationship of the cells growing on the hydrophobic substratum, Dalton and
coworkers observed an increased rate of gene transfer compared to the
hydrophilic biofilm.
Some of the physiological changes associated with biofilm growth include
increased growth rates (26), an increase in the degradation of xenobiotics
(52,69), and an elevated production of and EPS (25,47,97). Biofilm cells adapt
to become less susceptible to disinfection (82). These physiological changes
may protect biofilm cells allowing them to survive environmental stress better
than their planktonic counterparts.
11
Biofilm Heterogeneity
A major factor in biofilm growth that is different from planktonic growth is
that biofilms are usually mass transport limited. This results in a reduction in
concentration of nutrients in the biofilm.
If the transport rate of a nutrient is
slower than nutrient uptake within the biofilm, then the biofilm is limited by the
rate of mass transport, or is mass transport limited (14). Diffusional resistance
within the biofilm results in a concentration gradient, where cells embedded
deeper may be starved for nutrients (14). Biofilms are predominately water and
diffusion into a biofilm should be relatively rapid, but the presence of EPS and
other cellular material impede the diffusion of nutrients or biocides. The three
factors that govern concentrations of a particular solute in a biofilm are 1)
external mass transport to the biofilm, 2) diffusion within the biofilm, and 3)
reaction or consumption of the solute by biofilm cells. Reaction/consumption is
a significant factor that can lead to the depletion of a nutrient in the biofilm.
The physiological status of cells in thick older biofilms is hypothesized to
be spatially heterogeneous (101).
It is proposed that cells located on the
surface (closer to the bulk fluid) are actively growing and cells embedded deeper
within the film may be in a starvation or stationary mode of growth (6). The cells
in the upper layers of the biofilm have more access to the nutrients that are
transported to the film. These cells actively react with the nutrients, depleting
12
them before they can diffuse to the embedded cells. Reaction and diffusion of
nutrients within a biofilm are the two major factors that govern nutrient limitation.
Oxygen concentration gradients within biofilms have been determined
using an oxygen microelectrode (28). It was shown that oxygen concentration
were highest at the surface of the biofilms and quickly dropped to below
detection limits. This oxygen profile was due to rapid consumption and removal
of the nutrient before it reached the depths of the biofilm. This contributes to the
nutrient limitations of the cells found deeper within the biofilm.
Acridine orange (AO) stains double stranded nucleic acids green and
single stranded nucleic acids orange.
An active cell will have a higher
RNA/D NA ratio than less active cells and The cells with the higher RNA content
will appear more orange.
Using this property of AO, Wentland et al. (101)
showed that cells embedded deep within the biofilm are less active than cells
closer to the bulk fluid. This pattern of active cells close to the bulk fluid has
been supported by the work of Huang et al. (48). This group mapped the pattern
of alkaline phosphatase activity within a. biofilm. Alkaline phosphatase was used
as a model enzyme to represent protein expression and activity. By growing the
biofilm in nutrient rich conditions and then switching to a low phosphate
containing medium, the highest amount of phosphatase activity was found at the
surface to the biofilm.
Expression of alkaline phosphatase consumes carbon
and energy in significant quantities (99).
Cells within the biofilm that were
starved for phosphate, but did not have access to a carbon or energy source did
13
not express alkaline phosphatase.
Only the cells on the biofilm surface with
sufficient carbon and energy expresssed alkaline phosphatase strongly.
Planktonic Cell Resistance to Antimicrobial Agents
Antibacterial agents target the machinery that maintains a viable cell such
as cell wall synthesis, protein synthesis, nucleic acid synthesis, and cell
membrane function.
documented.
Bacterial
resistance to antibiotics has been well
Genetic alterations inherited either chromosomalIy or by
acquisition of plasmids can confer resistance to certain antibiotics. This genetic
material is not only spread within a single species but also across species as
well as genera (71).
There are three main mechanisms of resistance to antibiotics: an
alteration of the target site, an alteration in access to the target site, or
inactivation of the antibiotic. Alteration in the target enzyme results in a lower
affinity for the antibiotic thus reducing its effectiveness. An alteration in access
to the target site results in a decrease in the amount of antibiotic that reaches
the target.
This is accomplished by altering entry into the bacterium and by
decreases in the permeability of the agents into the membrane or cell wall.
Active pumping of the drug out of the cell also results in decreased access to the
target site.
Bacteria can make enzymes that can inactivate antibiotics.
One
well-known example of this is p-lactamase secreted by both gram-positive and
14
gram-negative bacteria against penicillin and its derivatives.
These enzymes
break the p-lactam ring of the antibiotic and inactivate it.
Bacteria can also acquire resistance to disinfectants such as quaternary
ammonium compounds (44,55), amphoteric biocides (55), chlorinated phenols
(8), isothiazolones (9), hydrogen peroxides (32), and chlorine compounds (16).
Mechanisms of resistance to antiseptics are less clearly defined than to
antibiotics. Many of these industrial related biocides target the inner or outer
cell membrane or the cytoplasm.
The reduction in susceptibility to these
biocides is contributed to by an adaptive change in the cell membrane (55), or to
transfer of a plasmid containing a bactericide-degrading enzyme (34).
Fahey et al. (35) reported the bacterial production of glutathione by many
gram-negative bacteria. It is hypothesized that Glutathione, a thiol compound, is
used by bacteria to neutralize reactive chlorine species (16). Glutathione is a
tripeptide derived from glycine, glutamate, and cysteine (64).
One of its
functions in bacteria is to neutralize toxic peroxides under aerobic conditions:
2 GSH + R-O-O-H -> GSSG + H2O + R-OH
GSH is the reduced form of glutothione and GSSG is the oxidized form. The
oxidized form of glutathione contains two molecules of glutathione linked by a
disulfide bond.
Other thiol-containing compounds can be substituted for
glutathione such as thioredoxin, glutaredoxin, and cysteine (16).
The use of
these compounds appears to play a significant role in the resistance of some
microorganisms to chlorine oxidants (16).
15
Growth conditions may effect biocide efficacy on planktonic cells
(38.43.53.90.96) . Klebsiella pneumoniae grown in nutrient limited conditions
resulted in the reduced suscpetibilty of these cells to MCA (90,91). The lownutrient cells were also smaller and more aggregated than cells grown in
nutrient-rich conditions. This increase in aggregation was due to an increase in
the amount of EPS produced in the nutrient limited cells. The sulfhydryl (-SH)
groups of both groups appeared to have no quantitative differences, however,
only 33% of the -SH groups of cells grown under low-nutrient conditions were
oxidized by MCA compared to 80% of the -SH groups from cells grown under
high-nutrient conditions.
This decrease in -SH oxidation may be due to a
number of physiological factor including cellular aggregation and protection of SH groups within the cell (90).
Starvation conditions also induces cross protection in many cells to H2O2
(38.43.53.96)
. Nitrogen and glucose starved Escherichia coli cells exhibited an
increase resistance to H2O2 that increased with time for which the cells were
starved prior to the challenge (53). The maximum protection was achieved by 4
hr. De novo protein synthesis was shown to be essential for protection against
oxidative stress. Several of these new proteins expressed within the first few
hours of starvation appear to play an important role in the protection of the cell
against oxidative stress.
(60).
Stationary-phase resistance to H2O2 requires rpoS
rpoS encodes for the alternative sigma factor, as, which controls the
16
induction of genes under stationary conditions.
In E coli, some of the genes
control by the rpoS regulon are xthA (30), katE, katG, and dps (32). All of these
are involved in the protection of E coli against H2O2. are among some of the
genes involved in the protection of E co//from H2O2. Dps is a nonspecific DNAbinding
protein
involved
in starvation.
The
product
of xthA is
an
exodeoxyribonuclease III, which repairs about 90% of the apurinic/apyrimidinic
sites in damaged DNA.
The kat genes encode for catalases that degrade
H2O2.
Several other investigators have reported that growth under low-nutrient
conditions could stimulate resistance to disinfectants (7,10,11,34,63,103).
These previous studies suggest that naturally occurring bacteria or those grown
in conditions similar to their natural environment were the most resistant. For
example, the naturally occurring red-pigmented Flavobacterium sp. from a
California water reservoir was 200 times more resistant to chorine than when it
was grown in R2A (103). Cells living in their natural oligotrophic environments
may be less sensitive to disinfectant treatment than the same cells grown in rich
laboratory conditions.
This may question the usefulness of disinfection data
presented from nutrient rich environments when an oligotrophic enivironment is
of interest.
It has also been shown that E coli (29) and Salmonella typhimurium (102)
become resistant to killing by H2O2 when pretreated with a nonlethal dose of
H2O2. Many of the proteins are under positive control of the OxyR gene product
17
(17,72).
OxyR
is
known
to
induce
HPI
catalase
[katG),
alkyl
hydrogenperoxidase reductase [ahpCF) (17,72), glutathione reductase [gorA)
(201), dps (180), and oxyS (59,64). Recently, 26 mutants have been identified
that exhibit a H2O2 sensitive phenotype (74).
OxyR is also required for the
expression of coproporphyrinogen III oxidase (hemF), and
an open reading
frame, f497, that is similar to arylsulfatase-encoding genes.
Many of the same protective genes involved in H2O2 resistance are also
involved in the protection against hypochlorous acid (HOCI) (32). In stationary
phase, resistance is mediated by RpoS. This was established by using an E
colistrain with a mutation in rpoS (32). These cells were hypersensitive to HOCI
exposure.
A 0.2 mg/L dose of HOCI produced a two log reduction in cells
lacking a functional rpoS compared to no effect with wild type cells. Pretreating
exponentially growing E coli cells with a sublethal dose of H2O2 protected wild
type cells from 2 mg/L of HOCI (32). An oxyR defective mutant, however, was
not protected by the pretreatment with H2O2.
Several of the same genes
involved in H2O2 protection may also provide protection against HOCI stress,
including rpoS, dps, and oxyR.
Biofilm Resistance to Antimicrobial Agents
Traditional disinfection studies use planktonic cells. The results of these
studies are then used to govern the amount of antimicrobial agent needed to
18
disinfect a system. The biocide concentration that is effective against planktonic
cells proves to be ineffective against biofilm cells, because cells embedded
within the biofilm are protected and survive the onslaught of biocide. There are
several reasons for this decrease in biocide efficacy against biofilms.
The
biocide may not be effectively transported to the surface of the biofilm, thus
reducing its efficacy (14).
The biofilm may actually consume or remove the
biocide (75), resulting in continually decreasing concentrations as it diffuses
through the film. The biofilm is also surrounded by a matrix of EPS. This EPS
matrix can act as a diffusional barrier and prevent the penetration of the
antimicrobial agent through the film (75). Lastly, the biofilm cells may be altered
physiologically and this may reduce the cell’s susceptibility to many biocides
(89,93).
Three factors that govern concentrations of a biocide in a biofilm are the
same as those mentioned previously for nutrients. These include mass transport
to the the biofilm, diffusion within the biofilm, and reaction/consumption of the
biocide by the biofilm cells.
biofilms.
These processes have been shown to occur in
For example, using a chlorine microelectrode de Beer et al. (27)
demonstrated the slow penetration of chlorine into a P. aeruginosa and
pneumoniae biofilm.
K.
The biocide failed to fully penetrate because it was
neutralized by reaction with the constituents of the biofilm faster than it diffused.
Tobramycin was found to bind to P. aeruginosa EPS and is prevented from
penetrating into the biofilm (75).
Chen et. al. (15) reported the reduction of
19
MCA in annular reactor disinfection studies. In a continuous treatment with 4
mg/L of MCA on biofilms growing on stainless steel coupons, the biofilm reduced
the residual MCA to 0.45 mg/L. The same system dosed with 2 mg/L resulted in
an undetectable residual MCA. MCA demand of the sterile reactor, the influent
and the stainless steel coupons were determined to be negligible.
It was
concluded that the biofilm bacteria were actively consuming the biocide and
therefore limiting its penetration and efficacy (15).
CTC is uses as an indicator of bacterial respiratory activity and viability
(49,86,87,94,106). «CTC is reduced to its fluorescent formazan component by
succinate dehydrogenase of the respiratory pathway in E coli (87).
Using
combination staining with 4,,6-diamidino-2-phenylindole (DAPI) and 5-cyano-2,3ditolyl tetrazolium chloride (CTC), Huang et. al. (49) reported heterogeneous
respiration activity in biofilms during MCA disinfection.
The greatest loss of
respiratory activity was observed near the bulk-liquid interface and the highest
activity closest to the substratum. The biocide only reacted with the cells closest
to the bulk liquid, therefore the cells attached to the substratum were protected
against the biocide and remained viable. These results indicated that the cells
closest to the bulk fluid were more vulnerable to the disinfectant. They reacted
with the MCA, which limited the diffusion of the biocide into the biofilm, allowing
the cells more deeply embedded to be protected.
Srinivasan et al. (89) have shown that a monolayer, 107 cfu/cm2, of biofilm
cells appeared to be less susceptible to monochloramine disinfection than
20
slightly denser biofilms, 108 cfu/cm2, while thicker biofilms of 101° cfu/cm2 were
very resistant to 4 mg/L of MCA.
The resistance of the thick biofilms was
explained by a lack of diffusion of the MCA into the biofilm. However, the thin
biofilms of 107 cfu/cm2 should not have experienced any diffusion limitations,
since such a thin layer of cells would not limit the penetration of biocide. These
data suggest multiple mechanisms for biofilm resistance, including an hypothesis
that the biofilms have changed physiologically to become less susceptible to
MCA (89).
Adaptation to MCA disinfection was shown by Sanderson and Stewart
(82).
Using varying MCA concentrations, disinfection and regrowth of a P.
aeruginosa biofilm were observed and predicted using a phenomenological
mathematical model. While the model captured the overall trend of disinfection
and regrowth, it failed to correlate to several key experimental observations. It
was observed that the initial dose of MCA was effective in disinfecting the
system, but after regrowth of the biofilm, the same dose of MCA was significantly
less effective in killing of the biofilm cells. The model, however, predicted that
the second dose be more effective than the first dose of MCA.
These
discrepancies between the model and the experimental observations were
concluded to be due to an adaptive response of the bacteria to MCA (82).
There have been several reviews that address the influence of
attachment on bacterial physiology (19,23,66,98). Although these reviews are
thorough, evidence supporting physiological and metabolic changes is still
21
lacking.
Many would argue that the systems used for biofilm growth are
undefined with respect to mass transfer properties. For example, the amount of
nutrients available to the attached cells could be quite different from that
available to the planktonic cells. The physiological changes that occur in this
type of system could be the result of the transfer limitations and not attachment.
Therefore, cells could be responding to the lack of nutrients and not the
substratum.
Significant Biofilm Genes
algT
Mucoid P. aeruginosa in a cystic fibrosis lung produces a copious amount
of alginate. Alginate production is regulated by a cassette of genes, algTmucAB
(46). algT encodes for the alternative sigma factor, AIgT or aE, which is involved
in the positive regulation of the mucoid phenotype in P. aeruginosa. MucA and
MucB inhibit the production of AIgT in a negative feedback loop (84,104). When
spontaneous null mutations in mucA or mucB occur, AIgT is up regulated and
results in the in increase production of alginate (84,104). It has been suggested
that alginate promotes biofilm formation (4). Therefore it may be speculated that
AIgT acts as a sigma factor inducing biofilm growth.
AIgT is functionally
interchangeable with RpoE, an extreme stress sigma factor found in the
Enterobacteriaceae [AOl). Up regulation of AIgT may protect the biofilm from
22
oxidative stress (67).
It was determined that the effect of algT inactivation
resulted in an increased sensitivity to killing by paraquat, a quaternary
ammonium compound, and heat.
rpoS
rpoS encodes the alternative sigma factor RpoS, also denoted KatF and
a®.
RpoS has been identified as a central regulator of stationary-phase-
responsive genes (60). In £ coli, RpoS controls the regulation of at least 30
genes (45). Among the genes controlled by this sigma factor are katE (65), katG
(48,51), and xthA (30). All of these genes are involved in the protection of the
cell against H2O2. However, the regulation of RpoS is complex in £ coli. It is
controlled at the level of transcription, translation and protein stability
(54,61,108,109).
In exponentially growing cells, a basal level of RpoS is
maintained by low rates of synthesis and by a reduced half-life of 1.4 min (61).
The degradation of exponentially expressed RpoS is due to the Clp protease
system (83,109). Increase of RpoS levels in exponentially grown cells can be
induced by osmotic shock, starvation and cold shock (45,60,73). RpoS may also
play a significant role in biofilm development. The structural and physiological
heterogeneity of biofilms is now widely recognized (28,48,49,101).
This
heterogeneity results in patches of actively growing cells among starved cells
(48). It is therefore reasonable to hypothesize that rpoS is expressed, at least
23
locally, within many biofilms.
Objectives, Rationales and Experimental Design
The body of this thesis consists of a compilation of my research in the
form of manuscripts that have been submitted for publication.
The papers
contained in Chapters 2 and 3 have been submitted to Applied and
Environmental Microbiology and Journal of Bacteriology, respectively. The final
chapter, Chapter 4, was written to establish a logical connection between the
chapters and provide a discussion and conclusion to my work. Appendix 1 was
added to present the raw data obtained in each of my experiments. To establish
a logical connection between the chapters, brief overviews of the objectives, the
rationale behind them, and the experimental design are presented here.
Objective I.
To determine if biofilm bacteria are less susceptible to
antimicrobial agents when mass transport is significantly reduced (Chapter 2).
Rationale.
Prior to beginning this project, the understanding was that mass
transport of biocides to the biofilm was one of the largest factors governing
biocide efficacy.
24
Objective 2. To determine the role of two putative biofilm regulatory genes in
biofilm protection from antimicrobial agents (Chapter 3).
Rationale. The isolation of gene responsible for biofilm resistance to biocide
would provide new insight to the physiological changes a cell undergoes when it
attaches to a surface.
Experimental Design
Alginate gel beads have traditionally been used as a matrix to entrap
microorganisms (76,85,100,105). While mimicking a natural biofilm, gel beads
allow for experimental control over composition and geometry of the matrix that
is not available when using natural systems. Alginate gel beads were used as a
substratum because the density of alginate is only slightly greater than water,
which allows the beads to be well suspended upon mixing on a magnetic stir
plate. This allowed nutrients and biocides in the bulk fluid to be transported to
the attached cells quickly, thus ensuring negligible mass transfer resistance.
Strains of P. aeruginosa cells were attached to alginate gel beads and
grown to various ages. These cells were then subjected to either 2 mg/L of
MCA, 600 mg/L of H2O2, or 320 mg/L of carbenicillin.
Viable cells were
enumerated by colony formation and disinfection rate coefficients were
calculated. Statistical analysis were used to compare the measured rates.
25
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36
CHAPTER 2
PSEUDOMONAS AERUGINOSA BIOFILMS RESIST OXIDATIVE BIOCIDES
WHEN MASS TRANSPORT PHENOMENA ARE SIGNIFICANTLY ELIMITATED
Introduction
Disinfection studies have been traditionally carried out using planktonic
cells. The. results of these studies are often used to determine the amount of
antimicrobial
agent
needed to disinfect a system.
However,
biocide
concentrations that are effective against planktonic cells prove to be ineffective
against biofilm cells, since the cells within the biofilm appear to be protected and
can subsequently regrow (30). There are several reasons for the decrease in
biocide efficacy against biofilm bacteria. The biofilm may actually consume or
deplete the biocide (26). A matrix of extracellular polymeric substances (EPS)
surrounds the biofilm and could act as a diffusional barrier to prevent penetration
of the antimicrobial agent through the film (26,39). The neutralizing reaction in
the biofilms may be faster than the diffusion of the antimicrobial agent, resulting
in significantly reduced concentrations of biocide within the biofilm.
Lastly, the biofilm cells may be altered physiologically and this may
reduce their susceptibility to many antimicrobial agents. Srinivasan et. a!., (32)
37
reported that a monolayer of biofilm cells (107 CFU/cm2) was less susceptible to
monochloramine (MCA) disinfection than slightly denser biofilms (108 CFU/cm2).
This reduction in efficacy was not due to retarded diffusion of the biocide, since
a monolayer of cells does not greatly reduce the penetration of biocide. This led
to the hypothesis that such thin biofilms are physiologically different from
planktonic cells, thus accounting for the reduced susceptibility to MCA.
Alginate gel beads have traditionally been used as a matrix to entrap cells
(21,27,31,36,39).
While mimicking a natural biofilm, gel beads allow for
experimental control over composition and geometry of the matrix that is not
available when using natural systems. Alginate gel beads can also be used as a
substratum for bacterial attachment.
The density of alginate is only slightly
greater than water. This allows the beads to be well suspended upon mixing
and facilitates mass transfer of nutrients and antimicrobial agents between the
gel beads and the bulk fluid.
The purpose of this study was to evaluate the efficacy of two antimicrobial
agents on thin biofilms grown in a system where biocide mass transport is not an
issue. The antimicrobial effects of MCA and hydrogen peroxide (H2O2) were
assessed in this novel biofilm system by comparing disinfection rates of
planktonic and attached cells.
38
Material and Methods
Bacterial strains, culture and enumeration. For all disinfection
experiments P. aeruginosa PA01 (18) was used.
Strains were cultured in
modified R2A broth (1) without K2HPO4 and starch, with the addition of 3.4 mM
CaCI2'2H20.
Enumeration of bacteria was performed by serial dilution in
phosphate-buffered saline (PBS) (33) followed by drop plating 10 |il drops
(17,25) onto R2A plates (Difco Laboratories, Detroit, Mich.)
GFP expressing P. aeruginosa. To visualize bacterial cell distribution
on the surface of alginate beads, a plasmid that constituitively expressed the
green fluorescent protein (GFP) was introduced into P. aeruginosa PA01. This
organism was obtained from Dr. Michael Franklin, Department of Microbiology,
Montana State University-Bozeman. The construction description is summarized
in figure 2.1. The gene for the GFP containing the mut2 mutation (6) was
amplified from plasmid pBCgfp using PCR (23). The PCR primers used in the
amplification were QFPSallll - 5’ GCGCGTCGACAGGAGAAGAAAAAATGAGT
AAACCAGAAGA 3' and GFPHindIV - 5’GTACCTGGAATTCTACGAAGCTTATT
TGTATAGTTCATCC 3’. The PCR product was digested with Sail and Hindlll,
and ligated into pUC19. The Xbal and Hindlll fragment from pUC19, containing
the gfpmut2, was then ligated into vector pMF36 (12) behind the strong trc
promoter, forming plasmid pMF230. Since pMF36 (12) contains the oriTsite and
the stable replication fragment, it was mobilized into P. aeruginosa by triparental
39
stably maintained. Since pMF36 does not contain lad, gfp was constituitively
expressed from the trc promoter.
Sa/I Xbal gfpmutl Hind\\\
pUC19
Transform into P. aeruginosa
PA01
Figure 2.1. Schematic diagram describing the construction of PA01 P.
aeruginosa constitutively expressing gfp.
Alginate Beads. The method of Smidsrod and Skajk-Bask (31) was used
to prepare gel beds. A 2% (w/v) sodium alginate solution was autoclaved for 30
40
minutes and then mixed on a stir plate overnight. Two mm diameter beads were
formed by dropping liquid alginate solution into stirred 1% (w/v) CaCI2^H 2O (fig
2 . 2 ).
Biofilm formation. An overnight culture of P. aeruginosa was diluted to
106 CFU/ml in 500 ml of R2A broth in a 1500 ml beaker containing approximately
3000 alginate beads. Cells were allowed to attach to the beads for up to 2 hours
41
at room temperature with continuous stirring. The medium was decanted and
replaced with sterile broth.
Beads were incubated at room temperature for
various time intervals from 3.25 to 72 hours. Spent medium was removed and
sterile R2A was added approximately every 4 to 8 hours to maintain nutrient
replete growth conditions. Before disinfection assays, beads were rinsed three
times in R2A and incubated for an additional three to four hours to ensure
exponential growth of biofilm cells.
Epifluorescence microscopy. GFP containing biofilms were grown for
24 and 48 hours as described above. Micrographs of cells attached to alginate
beads
were
taken
using
a
Nikon
optiphot
microscope
with
a
B2A
epifluorescence filter, a Nikon N70 camera and Kodak Tmax 400 black and white
film.
Preparation of disinfectants. MCA solution was prepared as previously
described by Chen et. al. (5). 0.11g of NH4CI was added to 100 ml of phosphate
buffer (pH 8.9) (0.5 g of K2HPO4 in 1 L of H2O). One ml of 4% HOCI (Aldrich
Chemical Company, Inc) was slowly added to the NH4CI solution. Two mg/L of
MCA (final concentration) was used for all experiments. Unstabilized ACS grade
H2O2was obtained from Hach (30%) and used at a final concentration of 600 mg
L"1. Both MCA and H2O2were titrated prior to each experiment to determine their
concentrations.
MCA was titrated using a Hach amperometric titrator (Hach,
Loveland, CO). To determine H2O2 concentration, the method of Klothoff and
42
Sandell (20) was modified by titrating to a colorless endpoint using standardized
0.1 N sodium thiosulfate (Na2S2O3).
Planktonic disinfection assay. An overnight culture of P. aeruginosa
was subcultured into fresh R2A to a final cell concentration between 107 and IO8
CFU ml'1 and incubated for 4 hours. The exponentially growing culture of P.
aeruginosa was homogenized with a tissue homogenizer for one minute on ice.
The cells were then diluted to 106 CFU ml'1 in phosphate-buffered water (PBW)
(1). MCA stock solution was added to the PBW to reach a final concentration of
2 mg L'1. Cells were sampled at timed intervals over a 10 minute period from the
start of the experiment. Cells were removed from the MCA and diluted into PBS
containing Na2S2O3 (1 mM final concentration) to neutralize the MCA.
Total
remaining chlorine in the reaction vessel was determined using the Hach
amperometric titrator. Disinfection experiments were repeated with 600 mg L"1
H2O2. Cells were sampled every 10 minutes for 1 hour.
The reaction was
stopped by using 4.12 mM, final concentration, of Na2S2O3 in PBS. Final H2O2
concentrations were determined by titration with Na2S2O3 as described above.
Z
As controls, planktonic cells were subcultured in PBW (106 CPU ml"1)
without the addition of an antimicrobial agent and sampled at the start and end
of the disinfection assay.
For all disinfection experiments, cells were serially
diluted in PBS and enumerated by the drop plate method, as described above.
Biofilm disinfection assays. Alginate beads with attached cells were
removed from the R2A and rinsed three times with sterile medium and
43
suspended in 1000 ml of PBW. While mixing beads on a magnetic stir plate,
MCA was added to reach a final concentration of 2 mg L"1. Thirty to 50 beads
were removed with a wide-mouth 5 ml pipet tip at timed intervals over a ten
minute period
and neutralized
with Na2S2O3 (1 mM final concentration).
Supernatant was decanted and 3 ml of citrate buffer (8 g of sodium citrate in 1 L
of PBS) added. Beads were blended on ice using a tissue homogenizer for one
minute and refrigerated for up to two hours.
Total remaining chlorine in the
reaction vessel was titrated at the conclusion of the experiment.
Biofilm
disinfection studies were repeated with H2O2. This disinfectant was added to a
final concentration of 600 mg L"1, and beads were sampled every 10 minutes for
1 hour.
Supernatant was decanted immediately after bead removal from the
reactor and beads were neutralized with 3 ml of 6.32 pM Na2S2O3 in citrate
buffer. Beads were homogenized on ice and stored at 4°C for up to 2 hours, to
dissolve the alginate gel bead.
As controls, beads with attached cells were suspended in PBW as
described above, but without the addition of an antimicrobial agent. These were
sampled at the beginning and the end of the assay.
In addition, disinfection
experiments were repeated with sterile beads to ensure that beads did not
significantly degrade MCA or H2O2. Homogenized beads were diluted in PBS
and enumerated.
Biofilm formation and disinfection on glass slides. Brian VanderVen
grew P. aeruginosa biofilms on glass slides in a biofilm apparatus described by
44
Cargill et. al. (4). Biofilms were grown for 24 hours in conditions similar to the
bead biofilms, in that spent medium was removed every four to eight hours to
maintain nutrient replete growth conditions.
Four hours before disinfection
assays, slides were placed into fresh R2A.
Disinfection of the glass slides
occurred in a clean sterile jar to eliminate the oxidant demand associated with
excessive organic matter.
The slides were removed at timed intervals and
placed into PBW containing sodium thiosulfate to neutralize the biocide. Slides
were then scraped into PBS and cells were homogenized and enumerated on
R2A plates.
Disinfection rate coefficient values.
The model used to interpret
disinfection rates was dX/dt = -kbCXwhere the change in cell density with time is
related to the disinfection coefficient, kb, biocide concentration, C, and culturable
cell density, X.
Raw disinfection rates were determined by using the least
squares method to calculate a regression line through the data consisting of the
natural log of CFU cm'2 versus time.
A slope with its standard error was
calculated, where the slope was the disinfection rate for that experiment. The
disinfection rate coefficient was determined by dividing raw disinfection rates by
the average biocide concentration to normalize against variations in disinfectant
concentration. This coefficient has units of L mg'1 min"1. Standard error for the
disinfection rate coefficient were calculated by dividing the standard error of the
disinfection rate by the average biocide concentration.
45
Mass transport analysis. To address how effectively biocides added to
the bulk solution were transported to the bead surface, mass transport limitation
was theoretically analyzed. This was done by calculating the time required to
attain 90% of the bulk fluid concentration at the bead surface or, in the case of a
sustained neutralizing reaction by the microbial cells, the concentration of
biocide at the bead surface that was attained at steady state.
These calculations hinge on an estimation of a mass transfer coefficient
describing transport between the bulk fluid and the bead surface. This mass
transfer coefficient was defined by:
(1)
J = k ( C 0 - Cs)
where C0 denotes bulk fluid concentration in mg cm-3, Cs is concentration at the
bead surface in mg cm"3, Jis solute flux with units mg cm"2 sec"1, and k denotes
the mass transfer coefficient with units cm/sec.
The magnitude of the mass
transfer coefficient in this gel bead system was estimated by Xu et al (39), using
an established correlation, to be 5 x 10'3 cm sec"1. The unsteady diffusion of a
non-reacting solute from the bulk fluid into the bead was solved first.
The
diffusion equation in spherical coordinates was
dc_
( d 2C
2 3C
"3r=fl-l_87
7"a7
(2 )
46
with boundary and initial conditions:
De — = k ( C 0 ~ Cs)
(3)
C is finite at r = 0, t > 0
(4)
C = O, t = 0,0 < r < R
(5)
where C is the solute concentration, De is an effective diffusion coefficient inside
the bead, r denotes the radial spatial variable, and t indicates time. Eqn. (3) was
a matching flux condition specifying that the flux of solute between the bulk fluid
and the bead surface equaled the flux by diffusion into the bead. The solution to
this problem was given by Crank (8). The model defined by Eqn. (2)-(5) applies
to the unsteady diffusion of a non-reacting solute from bulk fluid into a
permeable sphere. The concentration of biocide was calculated as a function of
time for parameter values relevant to the treatment of alginate gel beads with
MCA. These values were:
k
5 x 10 3 cm2/sec
mass transfer coefficient
R
1 x 10"1 cm
bead radius
De
1.68 x 1Cf5 Cm2Zsec
effective diffusion coefficient of MCA in bead
47
The effective diffusion coefficient of MCA in the gel bead was taken as 90% (38)
of the value in water at 25°C as estimated from the Wilke-Chang correlation (28).
The initial concentration of MCA was zero everywhere.
Bacteria neutralize MCA and H2O2 (3,30).
Bacteria attached to a
substratum can reduce surface concentration of biocide below the bulk fluid
concentration. From Sanderson and Stewart (30), the order of magnitude of P.
aeruginosa capacity for reaction with MCA was 4.6 x 10"14 mg/sec-cell. Taking
the highest cell density encountered in the present experiments of 4 x 105
CFU/cm2, the maximum flux due to reaction was approximately 1.0 x 10'8
mg/cm2-sec.
At steady state this must exactly match the flux supplied by
transport from the bulk fluid to the surface, as described by Eqn. (1).
An
analogous calculation was made for H2O2 using the induced level of catalase
activity reported by Brown et. al. (3).
Mass transport of H2O2 was also shown experimentally. Beads with 24
hour old biofilms were incubated with H2O2 color indicators used in H2O2 titration
for 15 min. Color indicator solution was removed and 600 mg L 1 H2O2 was
added to the beads. Beads were photographed at timed intervals to show the
diffusion of H2O2 into the beads.
Statistical analysis.
All statistical analyses were performed using
Minitab Release 11.12 (Minitab, Inc).
48
Results
Cell attachment and distribution. GFP expressing P. aeruginosa clearly
showed the distribution of attached cells on alginate beads (Fig 2.3).
Beads
with cells attached for 24 hours were sparsely populated. Much of the bead area
observed contain few or no cells.
The cells that were observed appeared to be
attached as single cells or in small microcolonies. Colonies were uncommon.
48 hour old biofilms were slightly more populated, but the overall trend in
distribution of cells was the same as observed with the 24 hour old biofilms.
Disinfection rate coefficients. Sterile beads did not degrade MCA or
H2O2. Concentration of the biocides before and after disinfection assay were
reduced from 2 mg L"1 to 1.9 mg L'1 for MCA and from 600 mg L 1 to 589 mg L'
1 for H2O2. Planktonic disinfection rate coefficients for MCA experiments all
fell within a tight cluster of values ranging from 0.404 to 0.574 L mg"1 min'1
(Fig. 2.4). These values were significantly higher than rates for 20 hour old and
older biofilms, for which disinfection rate coefficient values fell into a range of
0.19 to 0.23 L mg'1 min'1.
P-values comparing planktonic disinfection rate
coefficient values to 20 hour old and older biofilms were all less than 0.008
(Table 2.1).
There was no correlation between initial cell density of the
biofilm and disinfection rate coefficient (Fig. 2.5), since there was a wide range
of disinfection rate coefficient values associated with the mean of initial cell
49
Figure 2.3
Use of GFP to detect P. aeruginosa biofilms on alginate gel beads.
Epifluorescence of 24 hour old biofilms (A and B). Bars represent 5 pm. 48
hour old biofilms (c). Bar represents 10 pm. Cell distribution consisted mostly of
single cells and small microcolonies.
50
densities. The p-value for this regression line was 0.84, which indicates that
there was no significant difference between this line and a line with a slope of
zero. The results of the H2O2 experiments (Fig. 2.6) were similar to the MCA.
The planktonic disinfection rate coefficient values were significantly greater than
the biofilm disinfection rate coefficient values with p-values less than 0.05 (Table
2.1). As with the MCA data, there was no correlation between initial density of
biofilm cells and disinfection rate coefficient values when using H2O2; p-value of
the regression line was 0.43 (data not shown).
Table 2.1. P-values for comparison of planktonic and biofilm
susceptibility
Biofilm Age
B io c id e
MCA
HgOg
20hr
n= 3
24hr
n= 3
48hr
n = 3 \ 2t
72hr
n = 2*
0.0002
0.0018
0.015
0.0003
0.019
0.0004
n is the number of replicates for biocide experiments.* is the replicate number for MCA
experiments and t is the replicate number for H2O2 experiments.
Disinfection rate coefficient (L mg' 1 min
51
0.8
▲
planktonic cells
0.6
0.4
0.2
0.0
-
0.2
Figure 2.4.
0
20
40
60
80
Biofilm Age (hours)
Disinfection rate coefficients of planktonic
and biofilm P. aeruginosa cells exposed to 2
mg L 1 of MCA. Error bars represent
standard error of the disinfection rate
coefficients.
Disinfection rate coeffient (L mg' 1 min
52
Initial Cell Density (log CFU/cm2)
Figure 2.5. Influence of initial cell density on
disinfection efficacy. Error bars represent
standard error of the disinfection rate
coefficients. Regression line r2 = 0.02.
P-value of the slope = 0.81.
Disinfection rate coefficient (L mg min
53
4e-4 -
f
<►
♦
planktonic cells
S
biofilm cells
3e-4 2e-4 -
#
1e-4 0
0
10
20
30
40
50
60
Biofilm Age (hours)
Figure 2.6. Disinfection rate coefficients of planktonic
and biofilm P. aeruginosa cells exposed
to 600 mg L 1 of H2O2. Error bars
represent standard error of the
disinfection rate coefficients.
54
Transport of oxidants. Using Eqn. (2), the calculated concentration of
MCA at the bead surface relative to the concentration in the bulk fluid was
plotted as a function of time (Fig. 2.7). The initial concentration of MCA was
zero everywhere.
At time zero the bulk fluid concentration increased
instantaneously to a constant level of 2 mg L"1.
Within 1 second the
concentration at the bead surface was calculated to have reached 70% of its
bulk solution value, within 13 seconds it attained 90% of the bulk solution
concentration, and within 33 seconds it was calculated to be 95% of the bulk
solution concentration.
Essentially the same result was realized from an
analogous analysis of H2O2 transport. Neglecting reaction, therefore, transport
phenomena appeared to reduce the effective biocide concentration by no more
than 2%.
By solving Eqn. (1) using the maximum flux (J), due to neutralization of
MCA by P. aeruginosa, and the same mass transfer coefficient (k) given above,
the steady-state surface concentration was found to be 99.8% of the bulk fluid
concentration for C0 = 2 mg L"1. The analogous calculation for H2O2 indicated
that the steady state surface concentration was reduced to no less than 96% of
the bulk concentration of 600 mg L'1. In summary, the magnitude of transport
effects was calculated to be on the order of a few percent or less.
The H2O2 was shown to penetrate the beads completely by the addition
of H2O2 titration reagents (Fig 2.8), which turned the beads the characteristic
O
10O
200
300
400
Time (sec)
Figure 2.7. Calculated transport of MCA to alginate
gel beads. Cs = surface concentration and
C0 = bulk fluid concentration. At 33 sec
CsZCo = 95% and at 1 min CsZCo = 98%.
56
Figure 2.8
Diffusion of H2O2 into 24 hour gel beads biofilms. Beads were
incubated in H2O2 color indicators for 15 mins then 600 mg L 1 of
H2O2 was added. Photographs were taken at 10 sec (A), 1:45 min
(B) and 2:30 min (C). As H2O2 diffused through the bead, the bead
turned black.
57
Figure 2.9
Diffusion of Na2S2O3 into 24 hour gel beads biofilms exposed to
600 mg L 1 of H2O2. 0.1 N of Na2S2O3 was added to the alginate
beads after incubation with H2O2. Photographs shown were taken
at 10 sec (A), 2 min (B) and 4:50 min (C). As Na2S2O3 diffused
through the bead, the H2O2 was neutralized and bead turned back
to white.
58
blue/black color, indicating the presence of H2O2.
Maximum penetration
was observed by 2:30 minutes after the addition of H2O2 (Fig 2.80). If the beads
were then exposed to Na2S2O3, the bead turned back to white from the outside to
the inside as the neutralizer diffused through the bead (Fig 2.9).
By 4:50
minutes after the Na2S2O3 addition, the H2O2 was completely neutralized,
indicated by the bead returing to its characteristic white color (Fig 2.90).
Glass slide disinfection experiments. Similar studies with MCA and
H2O2 using glass slides as a substratum also showed a decrease in disinfection
rate coefficient values for 24 hour biofilms compared to planktonic cells, pvalues < 0.01 (Fig 2.10). The glass slides were more heavily colonized than the
alginate bead surface (average of 7.6 ± 0.27 log CFU cm'2 and 3.5 ± 0.58 log
CFU cm"2, respectively).
Disinfection rate coefficient values for glass slide
biofilms using MCA ranged from 0.40 to 0.57 L mg'1 min'1, while 24 hour glass
slide biofilms disinfection rate coefficients ranged from 5.5 x 10"2 to 0.20 L mg'1
min'1. Using 600 mg L'1 H2O2, 24 hour glass slide biofilms had disinfection rate
coefficient values ranging from 3.4 x 10"5 to 8.0 x 10"5 L mg'1 min'1. Biofilm cells
were significantly less susceptible than planktonic cells with disinfection rate
coefficients ranging from 2.3 x 10"4to 4.1 x 10"4 L mg'1 min'1.
0 .5
4 e -4
0 .4
0 .3
0.1
0.0
planktonic
coupon
MCA
Figure 2.10.
planktonic coupon
H2O2
Mean disinfection rate coefficients obtained from
biofilms grown on glass slides exposed to either 2
mg L 1 of MCA or 600 mg L 1 of H2O 2. Error bars
represent standard error of the means. Each
coupon experiment was repeated 5 times to
calculate the mean disinfection rate coefficients.
H2O2
0.2
Disinfection rate coefficient (L mg"1 min
MCA
Disinfecton rate coefficient (L mg"1 min
59
60
Discussion
Cells distributed on beads were largely observed as single cells or small
microcolonies, with the majority of the microcolonies consisting of a bacterial
monolayer.
This distribution of cells contributes very little to mass transfer
resistance of biocide to the attached cells, because penetration into these small
microcolonies would be relatively rapid. Since densely populated microcolonies
were not observed, it was concluded that their existence was rare and did not
significantly hinder the diffusion of the antimicrobial agents. The topography of
the bead was quite varied with hills and valleys. These valleys may have hidden
bacterial cells, but penetration of chlorine into alginate gel is relatively rapid
(39). Using 2.5 mg/L of free chlorine with P. aeruginosa entrapped in 0.5 mm
diameter alginate gel beads, Xu et. al. (39) were able to obtain over a 3 log
reduction in viable cell counts in 15 minutes of treatment. These beads allowed
chlorine to penetrate well below the depth that bacteria may lie within the valleys
of our alginate beads.
Penetration of biocide was not significantly hindered by mass transport.
We calculated that after 33 seconds the MCA concentration at the bead surface
would be 95% of the bulk fluid concentration. This was relatively rapid when
compared to the duration of the disinfection experiments of 6 to 10 minutes.
H2O2 has similar diffusion properties as MCA. The diffusion coefficient for H2O2
was smaller than for MCA, but this was more than compensated for by the dose
61
duration, which was more than 6 times longer than with MCA. Although H2O2 is
slightly more reactive than MCA, the H2O2 concentration at the end of the
experiments was not substantially decreased. The H2O2 was shown to penetrate
the beads completely by the addition of H2O2 titration reagents, which turned the
beads the characteristic blue/black color, indicating the presence of H2O2.
If
then exposed to Na2S2O3, the bead turned back to white from the outside to the
inside as the neutralizer diffused through the bead.
We conclude that the
attached cells were not significantly degrading the H2O2 or MCA and that
neutralization was not a large factor in mass transfer of the biocides.
There have been several reviews that address the influence of
attachment on bacterial physiology (7,9,22,34).
Although these reports are
thorough, conclusive evidence supporting physiological and metabolic changes
is still lacking. One could argue that the systems used for biofilm growth are
undefined and may contribute to mass transfer resistance. This would limit the
amount of nutrients available to the attached cells compared to planktonic cells,
resulting in starved or stationary physiological states. These starved states are
known to result in reduced susceptibility to biocides (13,14,16,19).
Mass
transport limitations contribute to physiological changes associated with biofilm
systems.
Attachment to a surface may have an effect on physiology
independent of mass transport phenomena. In many biofilm systems the effect
of mass transport limitation of nutrients is undefined, such attached cells could
62
be primarily responding to the lack of nutrients and not to attachment to a
substratum.
Previous studies have shown that when cells attach to a surface they
undergo physiological and metabolic changes (2,10,11,15,24,35,37).
These
changes have been speculated to be involved in the decreased susceptibility to
antimicrobial agents.
Sanderson and Stewart (30) have shown an adaptive
response of P. aeruginosa biofilms when dosed with MCA. From their results, it
was hypothesized that new biofilm genes are expressed that reduce the efficacy
of antimicrobial agents to oxidative biocides.
Starved cells also exhibit this
increased resistance to oxidative stresses (13,14,16,19). It is also possible that
when a cell attaches to a surface many genes are upregulated, allowing for an
increased protection against reactive biocides. These may be related to genes
that are involved in starvation stress responses or may be novel genes that are
induced once a cell attaches to a surface. These novel biofilm genes may be
involved in our observed resistance to MCA and H2O2 where cells are in nutrient
replete conditions.
The magnitude of transport effects was calculated to be on the order of a
few percent or less in our system. This was insufficient to explain the large
decrease in bacterial susceptibility to disinfection that was observed for attached
cells. The absence of mass transfer resistance in this system allowed us to test
the phenotypic effects of attachment on disinfection efficacy.
Biofilms on a
traditional substratum, glass slides, and a novel substratum, alginate gel beads
63
has a significant decrease in efficacy of oxidative biocides when compared to
planktonic cells. We have calculated that the biocides were transported to the
beads without a significant decrease in biocide concentration. We conclude that
the changes in disinfection efficacy from rates seen with planktonic cells to rates
observed with older biofilms are due to a change in physiological status of the
attached cells and not to mass transport limitations.
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67
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68
CHAPTER 3
RPOS AND AiG T CONTRIBUTE TO PSEUDOMONAS AERUGINOSA BIOFILM
RESISTANCE TO HYDROGEN PEROXIDE
Introduction
Bacteria in biofilms exhibit enhanced resistance to a variety of
antimicrobial agents, including disinfectants and antibiotics (4). This resistance
is not completely understood, but some hypotheses have been proposed. Some
suggest that the bacteria near the surface of the biofilm remove the biocide or
antibiotic in large enough quantities to protect the bacteria more deeply
embedded (4,35). A second hypothesis is that the glycocalyx creates a diffusion
barrier to the antimicrobial agent (4), although this has been questioned on
theoretical grounds (41). A third hypothesis is that the cells in the biofilm are
metabolically
and
physiologically
different
from
planktonic
cells
(6,26,34,36,37,40).
Cochran et al. (6) used alginate gel beads as a substratum to facilitate
transport of the biocide to the biofilm.
They showed that these thin
Pseudomonas aeruginosa biofilms (105 cfu/cm2) were less susceptible to
monochloramine (MCA) and hydrogen peroxide (H2O2) than planktonic cells.
69
Analysis of transport phenomena in the gel bead biofilm system showed that the
decrease in biocide efficacy was not due to mass transport limitations. These
thin biofilms did not inhibit penetration of the disinfectant as they were
equivalent to less than a monolayer of cells. Cochran et al. (6) hypothesized
that the attached cells were physiologically altered to become less susceptible to
oxidizing biocides.
There are two regulating genes that are particularly
attractive candidates for involvement in biofilm physiology, algTand rpoS.
In the cystic fibrosis lung, mucoid strains of P. aeruginosa produce
copious amounts of alginate. Alginate production is regulated by a cassette of
genes, algTmucAB (17).
AIgT is a sigma factor involved in the positive
regulation of the mucoid phenotype in P. aeruginosa. MucA and MucB inhibit
AIgT in a negative feedback loop (38,46). When spontaneous null mutations in
mucA or mucB occur, AIgT is up regulated resulting in the increased production
of alginate (38,46).
It has been suggested that alginate promotes biofilm
formation (3) and one may speculate that AIgT may serve as a key factor in
converting bacteria to the biofilm mode of growth.
AIgT is also functionally
interchangeable with RpoE, an extreme stress sigma factor found in the
enterbacteriaceae (48). Perhaps up regulation of AIgT simultaneously induces
biofilm formation and protection against extracellular stresses.
Structural and physiological heterogeneity of biofilms is now widely
recognized (8,21,22,45) with regions of slow growth or reduced metabolic
activity existing in close proximity to actively growing cells (21,45,47).
The
70
alternative sigma factor, RpoS1 has been identified as a central regulator of
stationary-phase-responsive genes (25). In E coli, RpoS controls a large group
of genes, including katE (27), katG (23,27), and xthA (9). All of these genes are
involved in the protection of the cell against H2O2.
Biofilm cells have been
shown to be less susceptible to H2O2 disinfection (6). In a biofilm, areas of slow
growth and H2O2 resistance may indicate genes that are upregulated during
biofilm formation. RpoS is involved in H2O2 resistance and starvation response.
It may also play a significant role in biofilm development. We hypothesize that
RpoS is expressed in biofilms.
The goal of the work reported here was to investigate the role of these
two genes, algT and rpoS, in the protection of biofilm cells against oxidative
biocides. We hypothesize that these genes are expressed in newly attached
biofilm cells. algT and rpoS were chosen because they encode for alternative
sigma factors that protect the cell against environmental stresses and may be
involved in biofilm phenomena.
Materials and Methods
Bacterial strains and media. The bacterial strains used in this study are
listed in Table 3.1.
PAOI is a standard strain of P. aeruginosa. SS24 has the
genotype rpoS::aacCI (42) and is therefore RpoS". PA06852 has a tetracycline
gene inserted into algT, thus inactivating this gene. E coli DH10B (Gibco BRL,
71
Gaithersburg, MD) was routinely used as the host for cloning and harboring
plasmids. R2A (1) was used as growth medium for all disinfection experiments.
For molecular techniques, Luria broth (LB) (10 g tryptone, 5 g yeast extract, 5 g
NaCI per liter) alone or supplemented with ampicillin (100 pg/ml), carbenicillin
(300 pg/ml), or gentamicin (20 pg/ml) was used.
Alginate bead biofilm formation and disinfection. Cells were attached
to alginate gel beads as described previously by Cochran et al. (6).
Biofilms
were grown for 24 and 48 hours before being exposed to oxidative biocides.
Table 3.1. Bacterial Strains and Plasmids
Strains or plasmid
Relevant properties
Source or
Reference
E. coli
DH10B
P. aeruginosa
PA 01
PA 06852
SS24
SS226
SS240
Plasmids
pDB19R
pUCP19
pSS32
Wild type
Gibco
BRL
Wild type
PA01 a/gU::Tcr
P A 0 1 rp o s S 1 0 1 \:a a c C I
R p o S ~; SS24 carrying pUCP19
Wild type; SS24 carrying pSS32
(19)
(30)
(42)
This work
This work
PTZ19R containing a 1.8 kb Kpnl-HincHW fragment of rpoS
Apr Ia c Z
PUCP19 containing rp o S
(43)
(39)
This work
Preparation of biocides and disinfection assay experiments were the
same as the procedures used by Cochran et al. (6). Biofilms were exposed to 2
72
mg/L of MCA for 10 minutes and to 600 mg/L of H2O2 for 60 minutes. Beads
were removed at timed intervals and biocide was neutralized with sodium
thiosulfate (Na2S2O3) (1mM final concentration). Beads were dissolved in citrate
buffer (6) and cells enumerated by drop plating (18,31) on R2A (Difco
Laboratory, Detroit, Ml).
Glass slide biofilm formation and disinfection. P. aeruginosa biofilms
were grown on glass slides in a biofilm apparatus described by Cargill et al. (5).
Biofilms were grown as described by Cochran et al. (6) for 24 hours in conditions
similar to the bead biofilms. Disinfection of the glass slides was carried out in a
clean sterile jar to eliminate the oxidant demand associated with excessive
organic matter.
phosphate
The slides were removed at timed intervals and placed into
buffered
water
(PBW)
(1)
concentration) to neutralize the biocide.
containing
Na2S2O3 (1mM final
Slides were then scraped into
phosphate buffered saline (PBS) (44) and cells were homogenized and
enumerated on R2A plates.
Calculation of disinfection rate coefficients. The model used to
interpret disinfection rates was dX/dt = -kbCX where the change in cell density
with time is related to the disinfection coefficient, kb, biocide concentration, C,
and viable cell density, X. Raw disinfection rates were determined by using the
least squares method to calculate a regression line through the data consisting
of the natural log of CFU/cm2 versus time. A slope with its standard error was
calculated, where the slope was the disinfection rate for that experiment. The
73
disinfection rate coefficient was determined by dividing raw disinfection rates by
the average biocide concentration to normalize against variations in disinfectant
concentration. This coefficient has units of L mg"1 min"1.
Statistical analysis. All p-values were calculated using Minitab Release
12.0 (Minitab, Inc) by the null hypothesis that the two sets of disinfection rate
coefficients were the same.
DNA manipulations, transformations, and conjugations. Standard
molecular biological techniques were used for DNA manipulation (2,28).
Plasmids were purified with QIAprep spin miniprep columns made by Qiagen
(Santa Clarita, CA).
DNA fragments were excised from agarose gels and
purified using the Qiaex Il DNA gel extraction system (Qiagen) according to the
manufacturer’s instructions.
Restriction enzymes and DNA modification
enzymes were purchased from New England Biolabs (Beverly, MA), Gibco BRL
(Gaithersburg, MD), or Boehringer Mannheim (Indianapolis, IN). Standard
electroporation procedure was used for E. coli using the E. coli Gene Pulser by
Bio-Rad (Hercules, CA).
For P. aeruginosa, electrocompetent cells were
prepared as follows. Cells were grown to mid-log (OD600 of 0.5-0.7) in 100 ml of
LB and harvested by centrifugation. Following two washes with 10 ml of 0.3 M
sucrose, the cells were resuspended in 1 ml of 0.3 M sucrose, and 40 pi of this
cell suspension was used for electroporation.
The conditions used to
electroporate P. aeruginosa were 200 ohm, 200 pF, and 1.6 kV on the E coli
Gene Pulser.
74
Construction of a P. aeruginosa rpoS complementing plasmid clone
and complementation of SS24. A 1.9 kb Ecofll-H^dlll fragment which carries
the complete P. aeruginosa rpoS gene was acquired from plasmid pDB19R (43),
and cloned into the P. aeruginosa - E coli shuttle vector pUCP19 (39) to
generate pSS32.
The orientation of the rpoS in pSS32 dictates that rpoS
expression occurs from the native promoter and not from the lac promoter
carried
on
pUCP19.
pSS32
was
then
introduced
into
SS24
and
complementation of rpoS mutant phenotype was determined by immunoblot
analysis.
SDS-PAGE and immunoblot analysis. PA01, SS24, SS240, and SS226
were grown in LB overnight at 37°C and subcultured until cell densities reached
stationary phase, corresponding to an OD600 of 2.2. 1 ml of cells were harvested
and pelleted at 12,000 RPM for 5 min. Pellets were resuspended in 100 pi of
sterile H2O and 100 pi of Laemmli sample buffer was added.
stored at -20°C until use.
Samples were
Separation of proteins by sodium dodecyl sulfate-
polyacrylamide gel electrophoresis (SDS-PAGE) was performed by the Laemmli
method, with 3% stacking gels and 10% resolving gels. Proteins were
transferred to nitrocellulose filter paper.
Immunostaining was carried out by
using a 1:5000 dilution of polyclonal anti-RpoS (49), a gift from Dr. Matin,
followed by incubation with a 1:6000 dilution of peroxidase-conjugated anti­
rabbit IgG (Bio-Rad
Labs, Hercules, CA).
RpoS related protein bands were
75
detected with a Bio-Rad Fluor-S Multiimager (Bio-Rad
Labs, Hercules, CA)
according to manufacturer’s directions.
Results
Thin biofilm disinfection. Planktonic disinfection coefficients for MCA
experiments for all strains fell within a tight cluster of values from 0.46 to 0.61 L
mg-1 min™1 (Fig. 3.1). These values were significantly higher than rates obtained
for biofilms grown on alginate gel beads, whose disinfection rate coefficients fell
into a range from 0.19 to 0.29 L mg™1 min™1 (p < 0.015).
P-values comparing
planktonic coefficients to biofilm coefficients are shown in Table 3.2.
susceptibility to H2O2 was clearly strain dependent.
Biofilm
While the planktonic
disinfection rate coefficients for all strains remained in a tight cluster with values
ranging from 3.1 x 10'4 to 3.5 x10"4 L mg™1 min™1 (Fig. 3.2), PA06852 (algT) and
SS24 (rpoS') 24-hour biofilms were significantly more susceptible to H2O2 than
was the wild type PA01 (p < 0.03). PA06852 and SS24 biofilm disinfection rate
coefficients were comparable to planktonic disinfection rate coefficients (p >
0.15). After 48 hours of biofilm growth, PA06852 became less susceptible with
disinfection rate coefficients statistically the same as 48-hour old PA01 biofilms
(p = 0.70).
However, SS24 remained highly susceptible to H2O2, with
coefficients comparable to planktonic wild type disinfection rate coefficients (p =
0.51).
Disinfection rate coefficient (L mg'1 min
76
PA01
P A 0 68 5 2 ( a lg f)
SS24 (rpoS)
Planktonic
24 hr biofilm
Figure 3.1. Mean disinfection rate coefficients of
.a e ru g in o s a strains using 2 mg L 1
P
of MCA. Biofilms were grown on
alginate gel beads. Error bars
represent standard error of the
mean.
Table 3.2 P-values for comparison of planktonic and biofilm susceptibility to MCA and H2O2
Biofilm Strain and Age
Biocide
MCA
PA01
48hr
0.007
PA06852
48hr
0.0003
0.022
0.007
0.18
0.035
SS24
24hr
(n = 3)
SS24
48hr
0.015
0.99
0.059
0.0003
PA01
48hr
ii
CO
0.016
C
0.15
0.0097
CM
0.019
0.85
C
CO
M
PA06852
24hr
(n = 5)
PA06852
48hr
ii
PA01
24hr
C
PA01
Ohr
(n =4)
PA06852
24hr
ii
CO
Il
H2O2
PA01
24hr
■(n = 3)
C
Biofilm Strain and
Age
PA01 Ohr (n* = 4)
PA01 24hr (n = 3)
SS24 Ohr (n = 3)
PA06852 Ohrfn= 2)
PA01
Ohr
(n* = 4)
SS24
24hr
(n = 5)
SS24
48hr
(n = 4)
0.57
0.030
0.51
0.75
0.11
0.62
Biofilm Strain and
Age
PA01 Ohr (n = 4)
PA01 24hr (n = 3)
PA01 48hr (n = 2)
SS24 Ohr (n = 4)
PA06852 Ohrfn = 4)
SS240 24hr (n = 4)
SS226 24hr (n = 4)
* n is the number of replicate
0.015
0.015
0.019
0.57
0.84
0.023
0.88
experiments
0.85
0.70
0.15
0.12
0.01
performed for each strain and age.
0.0008
0.039
0.77
Disinfection rate coefficient (L mg'1
Figure 3.2. Mean disinfection rate coefficients of
P. a e ru g in o s a strains using 600 mg
L 1 of H2O2. Biofilms were grown on
alginate gel beads. Error bars
represent standard error of the
mean.
Disinfection rate coefficient (L mg'1
8e-4 6e-4
2e-4 -
Figure 3.3. Disinfection rate coefficients of P.
a e ru g in o s a strains with and without
rp o S . Biofilms were grown on
alginate gel beads and exposed to
600 mg L'1 of H2O2. Error bars
represent standard error of the
disinfection rate coefficients.
80
RpoS is involved in thin biofilm protection. To confirm findings from
Figure 3.2 that rpoS expression protects biofilm from H2O2 disinfection, P.
aeruginosa strain SS240 was made. This complement strain was constructed by
electroporating pSS32 into SS24, therefore restoring a wild type genotype. H2O2
alginate gel bead biofilm disinfection experiments were repeated with SS240 and
its vector control SS226. SS226 is SS24 containing the vector, pUCP19, without
the rpoS insert, and this strain has a rpoS~ genotype. Planktonic experiments
showed no differences between the strains (data not shown). Twenty-four-hour
old SS240 biofilms were resistant to H2O2, with coefficients similar to PA01 24hour old biofilms (p = 0.12) (Fig. 3.3). Twenty-four-hour biofilms experiments
with the vector control, SS226, resulted in disinfection rate coefficients
comparable to SS24 and planktonic coefficients (p = 0.88). P-values comparing
SS240 and SS226 disinfection rate coefficients to PA01 and SS24 are shown in
Table 3.2.
Western blot analysis of RpoS expression. Antibodies raised against
E. coli RpoS protein were used to probe a Western blot of stationary phase
cell extracts of PAOI, SS24, SS240, and SS226. Figure 3.4 shows that extracts
from PA01 and SS240 each contained a specific protein band detected by the
E coli RpoS antibody. The extracts of SS24 and SS226 did not contain this
band. The second lower molecular weight band seen in PA01 and SS240 was
believed it to be a degradation product of RpoS.
When log phase, late log
phase, transitionary phase and stationary phase planktonic PA01 cultures were
81
used for Western blot analysis, the lower band appeared only in the stationary
phase samples, not in the log, late log or transitionary phase cell extracts (Data
not shown).
O
<
CL
w
CO
CO
O
Tf
CM
CD
(XI
CM
CO
CO
CO
CO
Figure 3.4 Immunoblot analysis
of proteins of wild type, PA01;
rpoS
mutant,
SS24;
rpoS
RpoS complement strain, SS240; and
vector control, SS226.
Glass slide biofilms. Thick biofilms grown on a glass substratum, exhibited
resistance to both biocides (Fig. 3.5).
The glass slides were more heavily
colonized than the alginate gel beads (average of 7.7 + 0.35 log CFU/cm2 and
3.7 ± 0.56 log CFU/cm2 respectively). Twenty-four-hour glass slide biofilms of
all strains had similar disinfection rates when challenged with MCA and H2O2.
Disinfection rate coefficients of 24-hour glass biofilm ranged from 0.082 to 0.12
L mg'1 min 1 with MCA, and 3.6 x 10'5 to 5.6 x 10D L mg 1 min'1 with H2O2.
Unlike disinfection of biofilms growing on alginate gel beads, there were no
observed differences in
Planktonic Coupon PlanktonicsCoupons
H2O 2
MCA
PA01[
PA06852
SS24
(algf)
{rpoS)
Disinfection rate coefficient x 10"4 (L mg’1 min
Disinfection rate coefficient (L mg min
82
-
Figure 3.5. Mean disinfection rate coefficients of P. a e ru g in o s a strains
using 2 mg L 1 of MCA and 600 mg L 1 of H2O 2. Biofilms
were grown on glass slides. Error bars represent standard
error of the mean.
83
disinfection rate coefficients when using PA06852 and SS24 with H2O2 on glass
slides. These strains were as resistant to H2O2 as was PA01.
Discussion
The role of two sigma factors, AIgT and RpoS1in mediating P. aeruginosa
biofilm resistance to H2O2 and MCA was investigated. Compared to planktonic
cells, biofilms were significantly less susceptible to both antimicrobial agents.
Reduced biofilm susceptibility was manifested in both thin biofilms grown on
alginate gel beads and in thicker biofilms grown on glass slides.
The ratio of
the disinfection rate coefficient measured for planktonic cells to that measured
for biofilms, which is a quantitative measure of biofilm resistance, was
approximately 1..6 to 3.9 for MCA and 3.9 to 5.8 for H2O2.
No contribution of either AIgT or RpoS to biofilm resistance could be
measured in the case of MCA in either biofilm system. algT and rpoS~ mutants
when grown as biofilms exhibited the same resistance to MCA as did wild type
biofilms.
In the relatively thick biofilms grown on glass slides, neither of the
genes provided any protection to H2O2.
In thin alginate gel bead biofilms,
however, a statistically significant contribution of both AIgT and RpoS to
resistance to H2O2 could be demonstrated. The protection afforded by AIgT was
transient: it was significant in 24 hour-old biofilms but could not be discerned in
48 hour-old biofilms.
The role of RpoS in peroxide protection was further
84
supported by restoration of the wild type phenotype with a complementing
plasmid in the mutant strain.
RpoS is known to regulate expression of katG and katE, which encode for
catalases in E coli (23,27). The E co//and P. aeruginosa rpoS genes appear to
be similarly regulated (43) and it is clear that stationary phase P. aeruginosa
have increased rpoS activity (11,43). The protection afforded by RpoS in biofilm
systems could simply be due to increased catalase levels in the cell. What is
less apparent is why rpoS would be induced in a thin biofilm. It is very difficult to
conceive of any nutrient limitation in the very thin, highly agitated alginate gel
bead biofilm system. This raises the possibility that there may exist a pathway
for induction of rpoS that is independent of nutrient limitation in P. aeruginosa.
Support for a biofilm-dependent pathway can be drawn from the induction of
RpoS under hyperosmotic conditions in E
coli.
During osmotic stress,
exponentially growing E co/Z cells increase in cellular levels of RpoS, (33), and
develop RpoS-dependent multi-stress resistance (16).
There are several genes known to be upregulated in P. aeruginosa as a
result of attachment to a surface. algC, which encodes for a key enzyme in the
alginate pathway, is activated when cells adhere to a surface (7). Attached cells
also have higher activity of algD (20). algD promoter controls the transcription of
most of the known alginate biosynthetic genes. It is clear that the production of
alginate promotes biofilm formation (3). AIgT is the sigma factor that globally
regulates the genes involved in the alginate biosynthetic pathway (10).
One
85
may speculate that AIgT serves as a key factor in converting cells to the biofilm
mode of growth. In addition AIgT is functionally interchangeable with RpoE, an
extreme stress sigma factor found in the Enterbacteriaceae (48).
AIgT is
required for resistance of P. aeruginosa to reactive oxygen intermediates (48).
Our finding of AIgT significance in protection of biofilm cells from H2O2 adds
support to its importance in biofilm formation, since it only provides protection
within the first 24 hours of attachment.
Although there have been several reports of the influence of attachment
on bacterial physiology (7,14,29,32), the understanding of this phenomenon is
still limited. As the genetics of biofilm development are elucidated, there will be
new insights into controlling biofilms with antimicrobial agents. Starved cells are
resistant to oxidative stresses (12,13,15,24).
Genes involved in protecting
starved cells may also be involved in protection of biofilm cells. Continued study
of starvation state cells will also provide a better understanding of the reduced
susceptibility to antimicrobial agents exhibited by biofilms. In this report we have
shown that the stationary phase sigma factor, RpoS, is involved in the resistance
of young biofilms to H2O2.
86
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91
CHAPTER 4
SUMMARY AND CONCLUSION OF THESIS WORK
The main objectives of my research were to determine 1) if biofilm
bacteria are less susceptible to antimicrobial agents even in the absence of
mass transport limitations (Chapter 2) and 2) the role of regulatory genes, rpoS
and algT, in protecting biofilms from antimicrobial agents (Chapter 3). These
objectives were met and are summarized below.
Bacteria were attached to alginate gel beads as a novel approach to study
disinfection profiles of a P. aeruginosa biofilm. Alginate gel beads were used as
a substratum because the density of alginate was only slightly greater than
water.
This allowed the beads to be well suspended upon mixing, ensuring
negligible mass transfer resistance.
For MCA treatment, 24 hours old thin
biofilms exhibited an average disinfection rate coefficient significantly lower than
the same P. aeruginosa cells grown planktonically. The latter had an average
disinfection rate coefficient of 2.4 x 10 4 L mg™1 min™1. The same trend was found
when 600 mg L"1 H2O2 was used. The average disinfection rate coefficient of 24
hours old biofilms was 9.2 x 10"5 L mgr1 minr1, while the average disinfection rate
coefficient for planktonic cells was 9.42 x 10r3 L mg™1 min™1. By modeling the
transport of biocide from the bulk fluid to the alginate get beads, it was
92
calculated that there was only a 2% reduction in biocide concentration at the
bead surface compared to the bulk fluid due to external mass transfer
resistance.
This decrease could not account for the large decrease in
disinfection rates of biofilm cells exposed to MCA or H2O2. It was, therefore,
hypothesized that biofilm cells were metabolically or physiologically altered to
become more resistant to these two antimicrobial agents.
To address the issue of physiological changes in biofilm cells, the role of
two sigma factors, AIgT and RpoS, in mediating P. aeruginosa biofilm resistance
to H2O2 and MCA was investigated (Chapter 3). Two knock out mutant strains,
SS24 (rpoS~) and PA06852 (algT) were compared to a wild type, PA01, in their
susceptibility to 2 mg L~1 of MCA and 600 mg L“1 of H2O2. When biofilms were
grown on alginate gel beads, all strains were equally resistant to MCA
disinfection; disinfection rate coefficients ranged from 0.19 to 0.29 L mg™1 min™1.
These were significantly lower than coefficients obtained from the same cells
grown planktonically, which ranged from 0.46 to 0.61 L mg™1 min™1. Twenty-fourhour old rpoS~ and algT biofilms, however, were more susceptible to 600 mg L™1
of H2O2 disinfection than were biofilms formed by PA01.
In fact, biofilm
disinfection rate coefficients were statistically the same as planktonic disinfection
rate coefficients for the two mutant strains. While 48-hour old algT biofilm cells
became resistant to H2O2, 48-hour old rpo£T biofilm cells remained highly
susceptible.
To confirm the role of RpoS, rpoS was restored to SS24 by
constructing a complement strain, SS240.
This strain exhibited disinfection
93
profiles similar to PA01.
In immunoblot analysis, the expression of RpoS in
SS240 was confirmed by the presence of an anti-RpoS binding protein band. It
is concluded that RpoS and AIgT play a part in the resistance of biofilms to H2O2,
but not to MCA.
Results from Chapter 2 and 3 led to the hypothesis that biofilm genes are
expressed upon attachment to a surface that protect these cells from
environmental stresses. Attachment induced gene expression has been shown
to occur (1,5,6,10,11,18). The induction of algC and algD are two examples in
P. aeruginosa where attachment has been shown to play a role in gene
regulation. Studies with Staphylococcus epidermidis also reveal that the genes
are induced upon attachment.
icaABC are involved in the synthesis of the
polysaccharide intercellular adhesin (PIA) (5,10,11,18) and are positively
regulated by adhesion of S. epidermidis to a surface.
accumulation stage of biofilm formation.
PIA is involved in the
The loss of the icaABC genes by
transposon insertion leads to the inability of this organism to form a biofilm on
polystyrene (5).
Spontaneous mutations give rise to an increased ability of E coli cells to
form biofilms (17). A single point mutation in the OmpR regulatory gene was
responsible for the increased ability of this organism to attach to a surface. This
mutation resulted in the replacement of leucine by an arginine residue in the
protein. It appeared that the OmpR mutant up regulated the expression of csgA
resulting in an increase in csgA expression.
csgA encodes for fimbriae
94
composed of CsgA monomers, also termed curl I (14).
The presence of curl!
significantly increased the adherence of E coli cells to a surface. In addition,
RpoS is required for curli fimbrial expression (13).
The physiological mechanisms of attachment are slowly being elucidated.
Some of the physiological changes associated with biofilm growth include
increased growth rates (2), increase in the degradation of xenobiotics (8,12),
and increased production of EPS (1,6,16).
Biofilm cells adaptation to
disinfectants results in a decrease in biocide efficacy (15). Although there are
several examples of gene induction and expression due to biofilm formation,
there is little known about how these induced genes may protect a biofilm from
environmental stresses.
I have reported in this thesis the role of two sigma
factor genes, algT and rpoS, in the protection of biofilms from H2O2 stress. Both
of these genes appear to protect thin newly attached biofilm cells.
The expression of AIgT and RpoS in my system only provided protection
from H2O2 and not MCA disinfection.
This does not appear to be a generic
mechanism of biocide resistance. One limitation of this work is that only a few
antimicrobial agents were tested. To gain greater understanding of the role to
these sigma factors in biofilm resistance to biocides, more agents need to be
tested. RpoS has been historically known to protect against H2O2 due to its role
in the regulation of katE (9), katG (7,9), dps (4) and xthA (3), therefore its role in
protecting biofilm cells from H2O2 is not extraordinary. The protection afforded
by AIgT and RpoS, however, was limited to only thin biofilms. Thick algT and
95
rpoS~ biofilms grown on glass slides were as resistant to H2O2 disinfection as
were wild type biofilms. Future experiments should investigate whether or not
AIgT and RpoS are only induced upon attachment and early biofilm
development, and are these two sigma factors expressed in thicker biofilms and
there exists additional mechanisms of resistance that further protect these films?
References Cited
1. Davies, D. G., A. M. Chakrabarty5 and G. G. Geesey. 1993.
Exopolysacdharide production in biofilms: substratum activation of alginate
gene expression by Pseudomonas aeruginosa. Appl. Environ. Microbiol.
59:1181-1186.
2. Davies, D. G. and G. A. McFeters. 1988. Growth and comparative
physiology of Klebsiella oxytoca attached to granular activated carbon
particles and in liquid media. Microb. Ecol. 15:165-175.
3. Demple, B., J. Halbrook, and S. Linn. 1983. Escherichia coli xth mutants
are hypersensitve to hydrogen peroxide. J. Bacteriol. 153:1079-1082.
4. Ferguson, G. P., R. I. Creighton, Y. Nikolaev, and I. R. Booth. 1998.
Importance of RpoS and Dps in survival of exposure of both exponential- and
stationary-phase Escherichia coli cells to the electrophile N-ethylmaleimide.
J. Bacteriol. 180:1030-1036.
5. Heilmann, C., 0. Schweitzer, C. Gerke, N. Vanittanakom, D. Mack, and F.
Gotz. 1996. Molecular basis of intercellular adhesion in biofilm-forming
Staphyloccoccus epidermidis. Mol. Microbiol. 20:1083-1091.
6. Holyle, B. D., L. J. Williams, and J. W. Costerton. 1993. Production of
mucoid exoploysaccharide during development of Pseudomonas aeruginosa
biofilms. Infect. Immun. 61:777-780.
7. Huang, C.-T., K. D. Xu, G. A. McFeters, and P. S. Stewart. 1998. Spatial
patterns of alkaline phosphatase expression within bacterial colonies and
96
biofilm in response to phosphate starvation. Appl. Environ. Microbiol.
64:1526-1531.
8. Jeffery, W. H., S. Nazaret, and R. Von Haven. 1994. Improved method for
recovery of mRNA from aquatic samples and its application to detection of
mer expression. Appl. Environ. Microbiol. 60:1814-1821.
9. Loewen, P. C., J. Switala, and B. L. Triggs-Raine. 1985. Catalases HPI
and HPII in Escherichia coli are induced independently. Arch. Biochem.
Biophys. 243:144-149.
10. Mack, D., M. Haeder, N. Siemssen, and R. Laufs. 1996. Association of
biofilm production of coagulase-negative staphylococci with expression of a
specific polysaccharide intercellular adhesion. J. Infect. Dis. 174:881-884.
11. Mack, D., M. Nedelmann, A. Krokotsch, A. Schwarzkopf, J. Heeseman,
and R. Laufs. 1994. Characterization of transposes mutants of biofilmproducing Staphylococcus epidermidis impaired in the accumulative phase of
biofilm production: genetic identification of a hexosamine-containing
polysaccharide intercellular adhesin. Infect. Immun. 62:3244-3253.
12. McFeters, G. A., I . Egli, E. Wilberg, A. Alder, R. Schneider, M. Suozzi,
and W. Giger. 1990. Activity and adaptation of nitrilotriacetate (NTA)degrading bacteria: field and laboratory studies. Wat. Res. 24:875-881.
13. Olsen, A., A. Arnqvist, M. Hammer, S. Sukupolvi, and S. Normark. 1993.
The RpoS sigma factor relieves H-NS-mediated transcriptional repression of
csgA, the subunit gene of fibronectin-binding curlin in Escherichia coli. Mol.
Microbiol. 7:523-536.
14. Olsen, A., A. Jonsson, and S. Normark. 1989. Fibronectin binding
mediated by a novel class of surface organelles on Escherichia coli. Nature
338:652-655.
15. Sanderson, S. S. and P. S. Stewart. 1997. Evidence of bacterial adaptation
to monochloramine in Pseudomonas aeruginosa biofilms and evaluation of
biocide action model. Biotechnol. Bioeng. 56:201-209.
16. Vandevivere, P. and D. L. Kirchman. 1993. Attachment stimulates
exopolysacchride synthesis by a bacterium. Appl. Environ. Microbiol.
59:3280-3286.
97
17. Vidal, O., R. Longin, C. Prigent-Combaretj C. Dorelj M. Hooreman5and P.
Lejeune. 1998. Isolation of an Escherichia coiiK\2 mutant strain able to form
biofilms on inert surfaces: involvement of a new ompR allele that increases
curli expression. J. Bacteriol. 180:2442-2449.
18. Zievuhr5W., C. Heilmann5 F. Gotz5 P. Meyer5 K. Wilms5 E. Straube5and J.
Hacker. 1997. Detection of the intercellular adhesion gene cluster (ica) and
phase variation in Staphylococcus epidermidis blood culture strains and
mucosal isolates. Infect. Immun. 65:890-896.
98
APPENDIX
RAW DATA OF BIOCIDE EXPERIMENTS
This section contains all of the raw data collected in the disinfection
experiments.
This included citrate blending data that shows the effects of
dissolving beads in citrate. This was done by using a known concentration of
planktonic cells.
These cells were added to alginate gel beads with various
concentrations to sodium citrate; 10, 20 or 40 mM. These were then blended on
50% speed for 1 minute either once (1X) or twice (2X). Cell and bead mixture
was incubated on ice for 2 hours to dissolve all gel bead material and then cells
were enumerated by the drop plate method as described previously (Fig A.1).
Actual cell data is shown in Table A.1.
The initial cell densities,
raw disinfection
rates,
average biocide
concentrations, and disinfection rate coefficients are shown in Tables A.2 - A.6.
Planktonic data is shown in Table A.2 for MCA and Table A.3 for H2O2. Biofilm
data is provided in Table A.4 for MCA and Table A.5 for H2O2. The data obtain
for the strains used in the complement experiments are shown in Table A.6.
Table A.1 Raw data used to determine effects of citrate on cell viability
4.20E+07
2.80E+07
3.60E+07
2.40E+07
2.50E+07
3.10E+07
40 mM
1XB
1.70E+07
1.60E+07
2.00E+07
2.40E+07
130E+07
1.80E+07
40 mM
2X B
2.40E+07
2.30E+07
2.10E+07
130E+07
180E+07
1.98E+07
20 mM
1XB
2.60E+07
2.70E+07
2.10E+07
240E+07
2.80E+07
2.52E+07
20 mM
2X B
2.20E+07
2.80E+07
2.60E+07
2.70E+07
3.80E+07
2.52E+07
10 mM
1XB
2.80E+07
2.80E+07
3.30E+07
3.10E+07
2.80E+07
2.96E+07
10 mM
2X B
2.30E+07
3.00E+07
3.40E+07
4.80E+07
2.30E+07
3.16E+07
2.00E+06
1.87E+06
1.87E+06
1.24E+06
2.65E+06
1.03E+06
4.61 E+06
Ideal
plate count
(CFU/ml)
Average
(CFU/ml)
Std Error
4.00E +07
CO
3.00E+07
E
z>
UO
2.00E +07
1.00E+07
0.00E+00
CO
Figure A.1. Cell survival after the addition of
various concentrations of citrate buffer and
blending either once (1X B) or twice (2X B)
with a tissue homogenizer at 50% speed for
1 min.
Table A.2 Disinfection rate coefficients and initial cell densities for planktonic experiments using 2 mg L 1MCA
Strain
PAOi
PA01
PA01
PA01
alg T-
. a lg T
rpoSr
rpoSr
rp o S -
- Disinfection
Rate
(min-1)
0.77
0.88
0.98
0.82
1.05
1.14
1.04
1.32
1.26
Std Error
0.09
0.09
0.15
0.16
0.10
0.12
0.02
0.09
0.01 .
Average
Biocide
(mg L-1)
1.80
1.85
1.80
1.84
1.84
2.12
2.24
1.76
1.73
Disinfection Rate
Coefficient
(L mg"1 min'1)
0.40 . .
0.46
0.52
0.43
0.55
0.54
0.46
0J0
0.68
Std
Error
0.047
0.045
0.081
0.086
0.052
0.057
0.011
0.045
0.007
Initial Cell
Density
(Log CPU ml"1)
5.5
5.5
5.6
5.7
5.8
5.8
6.4
6.6
5.9
Std
Error
0.32
0.20
0.18
0.13
0.14
0.15
0.03
0.23
0.15
a lg T
a lg T
a lg T
a lg T
rpoS~
rp o S rp o S rp o S -
8.7 x 1053
9.2x10-3
0:028
0.024
0.031
0.013
0.020
0.016
0.049
0.12
0.050
0.049
Average
Biocide
(mg L"1)
599.1
600
594.1
592.1
600
585.5
589
600
592.4
596
595.8
599.2
Disinfection Rate
Coefficient
(L m g 1 min1)
2.3x10-4
2.4x10-4
3.5x10-4
4.0 x IO-4
2 .7 x 1 0 ^
3 .5 x 1 0 ^
3.3 x IQ"4
3.1 x 10-4
4.0 x ID"4
eI
PAOI
PA01
PA 01
PA 01
Std Error
X
-
Disinfection
Rate
(m in1)
0.14
0.14
0.21
0.24
0.16
0.20
0.19
0.19
0.24
0.12
0.25
0.21
K)
Strain
4.3 x ID"4
3.5 x IQ-4
Std
Error
1.5 x 10-s
1.5 x ID"4
4.7x10-5
4.1 x 10-3
5.1 x 10-3
2.2 x ID"5
3.4x10-3
2.6x10-3
8.2 x 10-3
1.9x10-4
8.4x10-5
8.1 x 10-5
Initial Cell
Density
(Log CPU ml"1)
5.5
5.5
6.1
6.1
5.1
5.0
5.8
5.7
5.5
4.6
5.5
5.4
Std
Error
0.15
0.34
0.09
0.07
0.07
0.22
0.14
0.27
0.3
0.2
0.07
0.12
100
Table A.3 Disinfection rate coefficients and initial cell densities for planktonic experiments using 600 mg L 1 H2O7
Table A.4 Disinfection rate coefficients and initial cell densities for biofilm experiments using 2 mg L 1MCA
Strain
Biofilm
Age
(hr)
Disinfection
Rate
(min'1)
PA01
PA01
PA01
PA01
PA01
PA01
PA01
PA 01
PA 01
PA01 ,
PA01
PA01
PA01
PA 01
PA01
PA01
PA01
PA 01
PA 01
0.79
0.73
0.50
0.64
0.27
0.20
0.40
0.37
0.36
0.43
0.11
0.43
0.58
0.54
0.38
0.34
0.25
0.08
0.03
0.30
0.25
0.39
0.31
0.58
Std
Error
0.25
0.08
0.09
0.09
0.14
0.11
0.01
0.08
0.17
0.14
0.03
0.16
0.10
0.07
0.03
0.16
0.10
0.09
0.17
0.04
0.03 ■
0.12
0.03
0.04
Average
Biocide
(mg L'1)
Disinfection Rate
Coefficient
(L mg'1 min'1)
Std
Error
Initial Cell
Density
(Log CPU cm'2)
Std
Error
0.8
1.71
1.00
1.56
0.93
1.84
0.56
0.39
0.33
0.36
0.19
0.10
0.18
0.20
0.16
0.23
0.06
0,25
0.32
0.31
0.20
0.19
0.13
0.04
0.01
0.21
0.13
0.21
0.19
0.32
0.018
0.044
0.061
0.052
0.094
0.057
0.004
0.043
0.073
0.074
0.017
0.094
0.055
0.040
0.016
0.090
0.056
0.048
0.091
0.027
0.016
0.063
0.019
0.022
4.4
4.6
2.7
3.7
3.9
2.6
5.5
4.7
4.3
4.0
4.9
3.1
4.2
4.2
4.0
3.6
3.8
4.1
4.3
2.8
5.4
5.3
5.0
4.5
0.11
0.17
0.43
0.08
0.17
0.15
0.35
0.15
0.19
0.10
0.10
0.12
0.06
0.09
0.15
0.09
0.09
0.05
0.17
0.16
0.07
0.10
0.06
0.25
rpoS~
3.25
3.5
5.5
7.5
13
13
13.5
20
21
21
23.17
24
24
25
48
48.75
50.3
72
72.25
23.5
24.0
24.5
25.0
23.75
rpoS~*
24.75
0.59
0.07
1.56
0.33
0.037
4.9
0,16
rpoS~
25
0.41
0.16
1.59
0.23
0.089
4.3
0.20
a ig T -.
a ig f QigTQigT-
2 .2 2
1.80
2.27
1.80
1.79
1.49
1.63
1.49
1.81
1.56
1.7
1.77
1.73
0.80
1.77
1.69
1.28
1.67
Table A.5 Disinfection rate coefficients and initial cell densities for biofilm experiments using 600 mg L'1 H2O2
Std Error
Average
Biocide
(mg L'1)
Disinfection Rate
Coefficient
(L mg"1 min'1)
PA 01
PA01
PA 01
PAOT
PA 01
0.060
0.054
0.056
0.064
0.043
0.12
0.31
0.43
0.23
0.27
0.053
0.05
0.061
0.17
0.028
0.016
0.013
0.017
0.040
0.019
0.0
0.036
0.084
0.052
0.078
0.037
0.023
612
608.6
595.8
592.4
625.6
575.4
605
618
582.2
595.8
521
599.2
600
585.5
9.71 x 10'5
8.79 x 10'5
9.37 x IQ'=
rpoS~
24.75
24.75
24.9
48.28
48.28
23.67
23.67
24
24
24
47.83
47.83
49.5
23.66
rpoS"
24.25
0.16
0.033
0.21
0.072
a lg T :
a lg T -
%
alg T-
•
X
alg Ta lg T -
1.08 x 10"
. 6.85 x IQ"5
2.07 x 10"
5.18 x 10"
6.91 x 10"
4.15 x 10"
4 .5 2 x 1 0 "
E
a lg r
O
a lg T :
algT~
CO
Disinfection
Rate
(min'1)
X
Biofilm
Age
(hr)
9.17 x IQ'5
Std
Error
4.53
2.68
2.22
2.82
6.42
3.31
x 10-5
x 10-5
x IQ-5
x IQ'5
x IQ"5
x IQ’5
0
2.91 x 10-5
7.45 x IQ"5
4.35 x IQ"5
7.88x10-5
Initial Cell
Density
(Log CPU cm"2)
Std
Error
3.03
3.13
3.36
3.62
3.95
2.83
2.83
3.19
4.19
4.07
4.21
3.59
0.19
0.34
0.17
0.17
0.07
0.07
0.13
0.09
0.07
0.07
0.44
0.04
0.15
0.11
1 .0 2 x 1 0 "
2 .9 x 1 0 "
1.30 x 10"
6.20 x ID'5
2.6 x IQ"5
595.8
2.7x 10"
2.8 x IQ"5
3.6
0.29
596
3.6x 10"
8.0 x 10-5
2.8
0.08
0.03
3.59
4.0
rpoS~
24.5
rpoS~
24.5
0.18
0.049
582
3.1x 10"
5.6 x ID"5
6.2
rpoS~
25
0.35
0.16
599.2
5.8x 10"
2.7 x ID"5
3.1
0.03
0.42
0.11
597.5
7.1 x 10"
1.2 x IQ'5
3.2
0.47
0.16
rpoS~
47.66
rpoS~
48.33
0.59.
0.070
595.5
9.8x 10"
7.8 x ID"5
4.1
rpoS~
48.5
0.21
0.035
561.8
3.8x 10"
6.2 x IQ"5
3.1
0.06
rpoS~
48.5
0.17
0.032
596
2.8x 10"
3.6 x IQ'5
3.5
0.09
102
Strain
Table A.6 Disinfection rate coefficients and initial cell densities for complement strains experiments using 600 mg L"1
_______________________ HgO?_____________________________________
Strain
Biofilm
Age
(hr)
Disinfection
Rate
(min'1)
Std Error
SS240 •
SS240.
SS240
SS240
SS226
SS226
SS226
SS226
24.33
24.33
25.75
25.75
25.5
25.5
23.67
23.67
0.064
0.055
0.086
0.065
- 0.27
0.18
0.15
0.14
0.013.
0.010
6.6 x IQ'=
6.7 x TO'3
0.051
0.026
0.051
0.034
Average
Biocide
(mg L'1) .
558.4
578.6
585.6
585.6
690
582.2
582.2
582.2
Disinfection Rate
Coefficient
(L mg"1 min"1)
Std
Error
1.2E-04
9.4E-05
1.5E-04
1.1E-04
4.0E-04
3.0E-04
2.5E-04
2.4E-04
2.3E-05
1.7E-05
1.1E-05
1.2E-05
7.4E-05
4.2E-05
8.8E-05
5.8E-05
Initial Cell
Density
(Log CFU cm"2) 4.3
4.1
3.8
3.9
2.9
3.6
3.5
4.2
Std
Error
0.22
0.32
0.15
0.07
0.37
0.18
0.13
0.08
103
MOMTANA STATE UNIVERSITY - BOZEMAN
3 1762 1 421266 5