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\begin{document}

\title{Production and molecular composition of Burkholderia pseudomallei and
Burkholderia thailandensis biofilms}



\author[1]{Udoka Okaro}%
\author[2]{Sherry Mou}%
\author[2]{David DeShazer}%
\affil[1]{ United States Army Medical Research Institute of Infectious Diseases }%
\affil[2]{United States Army Medical Research Institute of Infectious Diseases}%


\vspace{-1em}



  
  \date{May 22, 2024}


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\selectlanguage{english}
\begin{abstract}
Biofilm communities cause many infectious diseases. Biofilms are diverse
microbial species found either attached to a surface or aggregated into
an extracellular matrix. Bacteria form biofilms as a default mode of
growth or as a response mechanism to environmental conditions like
stress. As such, biofilm strains are increasingly virulent causing a
wide variety of chronic persistent diseases, are typically
antibiotic-resistant and known to improve host mortality rate. Most
biofilms contain polysaccharides, proteins, extracellular DNA (eDNA),
RNA, and water. Determining and quantifying the major components of a
biofilm may indicate an appropriate treatment for biofilm eradication.
Burkholderia pseudomallei is a Gram-negative, motile bacillus typically
found in surface water and/or soil in endemic regions. It is the
etiologic agent of melioidosis and is capable of forming both surface
adherent and air-liquid interface biofilms (pellicle) in broth cultures.
This study evaluates the components of established biofilms using B.
pseudomallei and Burkholderia thailandensis, a closely related
nonpathogenic species. Using assays, fluorescent dyes and microscopy, we
quantified the major components of biofilms produced by five genetically
related B. pseudomallei strains and compared them to B. thailandensis
E264. Our data show that biofilm produced by the B. pseudomallei 1026b
derivatives and B. thailandensis E264 significantly differ. The
molecular composition of the surface adherent biofilm is similar to the
molecular composition of the air-liquid pellicle. Finally, the eDNA
quantity biofilm produced by JW270 which bears a CPS I deletion, is
significantly increased in comparison to 1026 and Bp82 biofilm.%
\end{abstract}%



\sloppy


\textbf{Introduction}

Biofilms are a self-secreted extracellular matrix produced by microbes
to mediate adhesion and survival of microbial cells, especially in
hostile environments. Biofilms can either be formed on an abiotic
surface (surface adherent biofilm) or at the air-liquid interface
(pellicle) {[}1{]}. Some functions of a biofilm include cell to cell
communication, storage and recycle of nutrients, horizontal gene
transfer, protection from environmental stressors and, protection of
bacterial cells from phagocytic engulfment by host mammalian cells
{[}2{]}. To form a biofilm, single cells adhere to a surface, aggregate
into colonies, and export components of the biofilm. Single cells are
dispersed from a mature biofilm to aggregate and form new biofilms
{[}2{]}. Biofilms can colonize or thrive on most surfaces and is harmful
to the host organism accounting for about 80\% of microbial infection
{[}3{]}. Biofilms are extremely difficult to eradicate due to their
increased tolerance for antimicrobials and antibiotics {[}4, 5{]}.

\emph{Burkholderia pseudomallei} (\emph{B. pseudomallei} ) is a
Gram-negative bacteria known to form both surface adherent biofilms and
pellicles {[}6-8{]}. It is the causative agent of melioidosis, a disease
endemic throughout Southeast Asia and northern Australia with an overall
death rate of 16\%-18.4\% {[}9{]}. \emph{B. pseudomallei} is found in a
wide range of ecological niches such as soil, surface water and roots of
legumes and can survive under a variety of environmental conditions
indicating the ability to quickly sense and respond to environmental
changes such as temperature, through specific survival mechanisms.
{[}10, 11{]}. Human inoculation is typically through inhalation or
inoculation through skin abrasions but a handful of human-human
transmission cases have been reported {[}12{]}. There is no approved
melioidosis vaccine and treatments rely on high doses of antibiotics
which may be ineffective {[}13-15{]}. About 10\% of melioidosis patients
relapse {[}16{]}. A typical relapse in bacteremia is linked to the
ability of the bacteria to persist in a colonized niche like a biofilm
within the host. Higher mortality rate and chronic inflammation
infections in host cells are also linked to biofilm-forming strains
{[}17, 18{]}. To survive under stressed environments like the human
host, \emph{B. pseudomallei} modifies the expression of virulence genes
like the Bsa type III secretion system {[}19{]}, metabolic and motility
genes {[}20{]}, and biofilm genes {[}21{]}. A variety of genes have been
implicated in \emph{B. pseudomallei} biofilm processes like adhesion
{[}22{]} and eDNA secretion which facilitate biofilm formation {[}23,
24{]}. However, the complete pathways for genes involved in the
formation of biofilms and/or the secretion of molecules are not clearly
understood. Numerous studies have shown that eDNA is released from
viable cells and required for biofilm formation and structure
in\emph{Burkholderia} . {[}23, 25, 26{]}. \emph{Burkholderia} species
also produce and export numerous outer membrane polysaccharides and
proteins {[}27-29{]}. Recent studies have suggested that biofilms can be
dispersed by enzymes that target biofilm constituents {[}30{]}. Biofilm
dispersal using enzymes is also suggested as the mechanism by which
planktonic cells are liberated from a mature biofilm to colonize a new
site/surface {[}31{]}. Supernatants of non-dispersed biofilms lack
degradative enzymes and endonucleases were expressed in
dispersed\emph{Pseudomonas} \emph{aeruginosa} cells {[}31, 32{]}. These
enzymes target polysaccharides, proteins, lipids, and eDNA, the core
architectural composition of biofilms {[}2, 30{]}. Thus, degradative
enzymes have been employed to degrade biofilm components in bacteria to
initiate dispersal and liberate planktonic cells which are susceptible
antibiotics. Dispersin B, an enzyme that hydrolyzes poly-\emph{N}
-acetyl glucosamine (PNAG) polysaccharide, inhibited \emph{Acinetobacter
baumannii} pellicle formation, suggesting that polysaccharides
contribute to pellicle formation {[}33{]}. DNase and proteinase K are
known to decrease the amount of biofilms formed by \emph{B.
pseudomallei} and \emph{P.} \emph{aeruginosa,} respectively {[}25,
34{]}.

In this study, we investigated the biofilms produced by virulent and
avirulent \emph{Burkholderia} strains, including \emph{B.
thailandensis}E264 {[}35{]}, \emph{B. pseudomallei} 1026b {[}36{]} and
four mutants derived from 1026b (1026b \emph{[?]asd} {[}37{]}, Bp82
{[}38{]}, DD503 {[}39{]} and JW270 {[}40{]}). \emph{B. thailandensis}
E264 is a nonpathogenic soil saprophyte that can be grown and
manipulated at biosafety level 2 (BSL-2), but \emph{B. pseudomallei}
1026b is a virulent human isolate that must be handled in a high
containment BSL-3 laboratory. \emph{B. pseudomallei} 1026b \emph{[?]asd}
harbors a deletion mutation in the gene encoding aspartate-semi aldehyde
dehydrogenase and is unable to grow on media without diaminopimelate
(DAP) supplementation {[}37{]}. \emph{B. pseudomallei} Bp82 is
auxotrophic for adenine and thiamine due to a deletion mutation in the
\emph{purM} gene {[}38{]}. 1026b \emph{[?]asd} and Bp82 are both avirulent
in mice and have been removed from the CDC select agent list. DD503 is a
1026b derivative that harbors a deletion of the genes encoding the
\emph{AmrAB-oprA}antibiotic efflux pump and is virulent in animal models
of infection and must be worked with at BSL-3 {[}39{]}. \emph{B.
pseudomallei} JW270, a DD503 derivative, contains a deletion of the
\emph{wcb} gene cluster encoding capsular polysaccharide CPS I, a
homopolymeric polysaccharide required for virulence {[}40{]}.

We employed glucosidase, proteinase K and DNase to disperse/eradicate
the biofilms produced by these \emph{Burkholderia} strains and examined
the molecular composition of both surface-adherent and pellicle
biofilms. While we found no difference in biofilm formation
between\emph{B. pseudomallei} strains and \emph{B. thailandensis} ,
there were striking differences in biofilm polysaccharide, protein and
eDNA content. We propose that the different biofilm composition observed
between the \emph{B. pseudomallei} derivatives and \emph{B.
thailandensis} may allow a better understanding of
\emph{Burkholderia}biofilm biogenesis and regulation. We propose that
the genetic manipulations between the 1026b derivatives is responsible
for the difference in biofilm composition.

\textbf{Results}

\emph{B. pseudomallei} \textbf{and \emph{B.} \emph{thailandensis} form
biofilms under different temperatures}

We assessed the ability of \emph{Burkholderia} to form a biofilm under a
scanning electron microscope (SEM) using \emph{B. pseudomallei} JW270
strain. We depict that the biofilm formation process
in\emph{Burkholderia} follows the same pattern as most Gram-negative
bacteria (Fig 1a). We see single cells adhere to each other (Fig 1a,
step 1). The adherence is followed by colony aggregation (Fig 1a, step
2) and export of the biofilm components (Fig 1a, steps 3-4). Export of
biofilm components progresses until most cells are covered/protected to
form a mature biofilm (Fig 1a, step 5). Cells are dispersed from a
matured biofilm to continue the cycle (Fig 1a, step 6). A visual and
microscopic observation shows that all four strains used for the
composition study: \emph{B. pseudomallei} strains (Bp1026b \selectlanguage{greek}\emph{Δasd}\selectlanguage{english} ,
Bp82, and JW270) and \emph{B. thailandensis} E264 are all able to form
both an adherent biofilm at the bottom of the plate and a pellicle; a
biofilm formed at the air-liquid interface (Fig 1b). We observed that
pellicle morphologies differed between strains. Bp82 and JW270 present
wrinkled and cloudy pellicles while 1026b \selectlanguage{greek}\emph{Δasd}\selectlanguage{english} and E264 have a
thin, smooth layer (Fig. 1b). Fig 1c shows an image of a processed JW270
pellicle. There was no difference in the SEM images between all
pellicles.

\emph{B. pseudomallei} is not fastidious and can grow under diverse
environmental conditions {[}41{]}, such as soil and rice paddy field
water at temperatures between 21\selectlanguage{ngerman}°C and 37°C {[}42, 43{]}. \emph{B.
pseudomallei} in the lab is commonly grown at 37°C, a temperature that
corresponds to human body temperature {[}44{]}. To compare biofilm
capabilities under varying temperatures, \emph{B. pseudomallei} was
grown statically for 72 hours either at room temperature (22°C) to
represent rice paddy fields or at 37°C in an incubator to represent
human host temperature. Fig. 1d shows that at the end of the experiment,
there is no significant difference between the same strains grown at
different temperatures (Student's t-test, P \textgreater{} 0.05).
However, we observed that in earlier time points, cells grown at RT
lagged behind 37°C cells but ultimately forms the same amount of biofilm
by 72 hours. For comparison between groups at the end of the experiment
(e.g. 1026b \selectlanguage{greek}\emph{Δasd}\selectlanguage{english} vs. Bp82 at 37\selectlanguage{ngerman}°C), student t-tests show that
there was no significant difference between biofilms formed between all
strains (Fig 1e, P \textgreater{}0.05). We also observed that cultures
grown at both temperatures formed surface adherent biofilms, but
cultures grown at 37°C are capable of forming both surface adherent
biofilms and pellicles.

\textbf{Biofilms formed by each \emph{Burkholderia} strain is made up of
eDNA, protein and polysaccharides and all components are exported by
live cells.}

From Fig. 1d, at the end of 72 hours, there was no significant
difference between biofilms formed by cells at RT or 37°C hence all
experiments were carried out at 37°C. We determined the constituents of
biofilms formed by all four strains using fluorescent microscopy. In
Fig. 2a, we observed that all four strains exported varying amounts of
eDNA, protein, and polysaccharides contents to support its biofilm
structure. Closer magnifications are inserted to show molecular
distribution around cell aggregates.

Previous publications have shown that in some Gram-negative organisms,
eDNA is generated through cell lysis {[}45{]}. However, as indicated in
Fig. 2b, live/dead images of \emph{B. pseudomallei} and \emph{B.
thailandensis} indicate that at 48 hours, a time point by which a mature
biofilm has been formed, the cells within the biofilm are predominantly
viable.

\textbf{Biofilm components significantly differ between strains and
growth mode.}

Using a previously established biofilm extraction protocol {[}46{]}, we
quantified the concentration of each biofilm component and compared it
to the supernatant of planktonic grown bacteria. Microscopic analysis in
Fig. 2 showed that different strains exported varying quantities of
protein/eDNA/polysaccharides. In Fig. 3, we used BCA (protein),
phenol-sulfuric acid (glucose/polysaccharide) and propidium iodide
florescence (eDNA) to quantify the concentration of biofilm grown
constituents compared to a planktonic grown culture. We observed that
the amount of molecules exported was determined by growth method.
Between all four strains, there was a significant difference in protein,
eDNA or polysaccharide content if the cells were grown with agitation
(planktonic) or statically (biofilm) (Supplemental data; Table S1-3).
There is no significant difference in protein concentration between
1026b \selectlanguage{greek}\emph{Δasd}\selectlanguage{english} , Bp82 and E264, but all three significantly differed
from JW270 (Supplemental data; Table S1-3). It was observed that JW270
and E264 biofilms are dominantly made up of eDNA unlike 1026b\selectlanguage{greek}\emph{Δasd}\selectlanguage{english}
and Bp82 biofilms which showed higher polysaccharide and protein
component. This finding was notable because 1026b \selectlanguage{greek}\emph{Δasd}\selectlanguage{english}and Bp82
produce CPS I, but JW270 and E264 do not {[}40, 47{]}. The results
suggest that the inability of \emph{B. pseudomallei} and\emph{B.
thailandensis} strains to produce CPS I for biofilm formation may be
compensated by exporting more eDNA for biofilm biogenesis.

\textbf{Enzymatic treatments significantly reduce specific biofilm
components}

The use of enzymes- especially DNase and proteinase K- for biofilm
dispersal or eradication has become quite popular {[}25, 48{]}. However,
enzymes do not completely eradicate mature biofilms and have been
proposed for adjuvant therapy, degradation accessible cell surface
proteins and as surface coatings to prevent adhesion which is the first
step of biofilm formation {[}49-51{]}. Fig. 4 confirms
that\emph{Burkholderia} biofilms are sensitive to all enzymatic
treatments by a 30-75\% reduction in biomass. In Fig. 4, DNase treatment
is shown to be the most effective dispersal method for the \emph{B.
pseudomallei}strains while proteinase K/glucosidase is more effective
towards\emph{B. thailandensis} E264 dispersal.

\textbf{Morphology and composition of a \emph{Burkholderia} pellicle}

\emph{B. thailandensis} E264 and \emph{B. pseudomallei} strains form two
different types of biofilms: an adherent biofilm and a liquid-air
interface biofilm (pellicle). The molecular composition of each pellicle
(Fig 5a-c), mirrored the composition of the adherent biofilm (Figs 2a,
3a-c). The pellicles shows high concentrations of protein and
polysaccharide and less eDNA present in pellicles of 1026b
\selectlanguage{greek}\emph{Δasd}\selectlanguage{english}and Bp82. JW270 and E264 showed higher eDNA.

\textbf{Comparison between wild type 1026b, DD503 and JW270}

From Figs 3 and 5, we observe that 1026b \selectlanguage{greek}\emph{Δasd}\selectlanguage{english} and Bp82 bear the
same biofilm composition while JW270 and E264 are similar despite the
fact that JW270 was generated from the DD503 (Fig S1), a derivative of
1026b in which the genes encoding \emph{amrAB-oprA} antibiotic efflux
pump has been deleted {[}39{]}. To confirm that the mutation used to
generate JW270 may be responsible for its different biofilm composition,
we repeated the assays for protein, eDNA and glucose concentration using
the wild type 1026b, DD503 and JW270.

1026b and its direct derivative DD503 have the similar protein
concentration (Fig 6a, p = 0.006), and eDNA florescence (Fig 6c, p =
0.03) at 72 hours but DD503 shows an increased polysaccharide
concentration throughout the experiment time span (Fig 6b). However,
once the \emph{wcb} operon is deleted in DD503 to generate JW270, the
polysaccharide and protein concentrations drastically decreases but
shows an inverse increase in eDNA. There is a significant difference
between the concentrations of all biofilm components when DD503 and
JW270 are compared (p = 0.01-0.04). This data implies that the deletion
of the \emph{wcb} cluster may be responsible for the low biofilm
polysaccharide and protein concentrations and the overcompensation of
eDNA export in JW270 to maintain a stable biofilm. To quickly confirm
our hypothesis, we performed an ELISA test using JW270 as test sample,
and 1026b \selectlanguage{greek}\emph{Δasd}\selectlanguage{english} and Bp82 as positive controls. \emph{B.
thailandensis} E264 which lacks the CPS cluster was used as negative
control. The ELISA test was performed using an anti-CPS primary
antibody, 4B11 {[}52{]}, and a goat anti-mouse alkaline phosphatase
secondary antibody. The results shows reactivity between the 4B11
antibody and 1026b \selectlanguage{greek}\emph{Δasd}\selectlanguage{english} and Bp82 but not towards JW270 or\emph{B.
thailandensis} E264 (Fig. S2). Taken together, the results demonstrate
an inverse relationship between eDNA and CPS I in\emph{Burkholderia}
biofilms.

\textbf{Discussion}

\emph{Burkholderia} is a genus of proteobacteria which harbors
pathogenic and non-pathogenic members.\emph{B. pseudomallei} is a
gram-negative, aerobic, highly pathogenic rod-shaped bacterium. It is
the causative agent for melioidosis, an infectious disease with high
morbidity and mortality rates in endemic regions {[}53{]}. It is a CDC
federal select agent for which there are no current vaccines available
for human treatment. Molecular research on \emph{Burkholderia} has
focused on vaccine production mainly because of its likely use in
bioterrorism but also because it exhibits intricate resistance to
multiple groups of antibiotics, especially while growing in biofilms
{[}54{]}. Because of the pathogenicity of \emph{B. pseudomallei} , a
BSL3 lab is required to work with the bacteria hence manipulations have
been made to generate \emph{B. pseudomallei} strains 1026b \selectlanguage{greek}\emph{Δasd}\selectlanguage{english} ,
Bp82, JW270 which are all non-pathogenic BSL2 strains. On the other
hand,\emph{B. thailandensis} is a non-pathogenic strain which rarely
causes disease in humans {[}35, 55{]}. Most \emph{Burkholderia} biofilm
studies report on biofilm using BSL2 strains. The purpose of this study
was to determine if the genetic manipulations made to the pathogenic
1026b strains affected biofilm structure, formation or constituents.

Gram-negative bacteria can form surface adherent biofilms or a biofilm
formed at the air-liquid interface which is called a pellicle {[}1{]}.
The biofilm formation process is activated in response to various
signals such as quorum sensing {[}56, 57{]}, nutrient availability
{[}58{]}, stress {[}59{]}, and surface material as reviewed in
Magana\emph{et al} , 2018 {[}60{]}. Research has shown that \emph{B.
pseudomallei} and \emph{B. thailandensis} are capable of producing
biofilms {[}10, 11, 13, 17, 18, 21, 61, 62{]} and are known to export
large amounts of eDNA {[}25, 63{]}. Another biofilm characteristic is
the ability to persist after treatment which causes disease relapse
{[}54, 64{]} - a feature widely attributed to a cohort of biofilm cells
called persisters. Biofilm is involved in the antibiotic resistance of
bacterial disease, relapse, and persistent cells {[}65, 66{]}. eDNA
exportation has been observed in both Gram-positive and Gram-negative
bacteria and has now been associated with biofilm facilitation and
composition {[}23, 67{]}.

Much like \emph{Salmonella} {[}68{]}, \emph{Burkholderia} is capable of
producing either a surface adherent biofilm or a pellicle (Fig 1a-c).
This phenomenon was also demonstrated in a previous
\emph{Burkholderia}study which showed both pellicle and adherent biofilm
{[}23{]}. The conditions and reasons for the change from surface
adherent to pellicle biofilm remains unknown in \emph{Burkholderia} .
Mangalea \emph{et al} . {[}8{]} reviewed that \emph{Burkholderia}
pellicles are typically present in the environment and surface adherent
biofilms more consistent with rice paddies. This is quite similar to
conditions observed in this study, 37\selectlanguage{ngerman}°C representing the higher
environmental temperature and 37°C a representative temperature for the
cooler rice paddies. This suggests that temperature amongst other
environmental conditions indicative of the two different environments
may play a role in determining the type of biofilm formed. In
\emph{Salmonella} , curli is responsible for the switch {[}68{]}
whereas, in \emph{C. crescentus,} biofilm is initially formed as a
monolayer of cells that subsequently develop into a three-dimensional
structure and as the biofilm matures, it becomes more cohesive and less
adherent {[}69{]}. Previous studies have confirmed that some pellicles
of some organisms are composed of eDNA with close cell contact {[}23{]},
sucrose, and proteins {[}70{]}.

This study characterized the major biofilm components of \emph{B.
pseudomallei} 1026b, four isogenic 1026b mutants and \emph{B.
thailandensis} E264. Since \emph{B. pseudomallei} is typically found in
the soils with relatively lower temperatures than a mammalian host, we
grew all strains at temperatures that reflect each environment. Fig. 1d
shows that there were no temperature-related differences in biofilm
formation between the strains grown at RT and 37°C, so all remaining
experiments were performed at 37°C. Interestingly, the amount of biofilm
formed by all the \emph{B. pseudomallei} and \emph{B.
thailandensis}strains at 72 hours showed no significant difference (Fig.
1e).

Fluorescent microscopy and molecular assays (Fig. 2a and 3) show that
the biofilm composition of \emph{B. thailandensis} is significantly
composed of proteins and eDNA and lower levels of
polysaccharide.\emph{B. pseudomallei} 1026b \selectlanguage{greek}\emph{Δasd}\selectlanguage{english} and Bp82
consists mainly of polysaccharides with lower protein and eDNA levels,
respectively. However, JW270 appears to have consistently low protein
and polysaccharide concentrations but relatively large amounts of eDNA.
JW270 is a CPS I mutant {[}40{]} and is unlikely to produce the same
amount of polysaccharides as 1026b \selectlanguage{greek}\emph{Δasd}\selectlanguage{english} or Bp82. We speculate
that in order to compensate for the loss of polysaccharide, there is an
increased export of eDNA to serve as a skeletal biofilm structure. To
confirm our speculations, we compared the concentration of the biofilm
constituents of DD503, the parental strain of JW270 which
lacks\emph{amrAB-oprA} , a multidrug efflux system (Fig. S1). The
deletion of the \emph{AmrAB-oprA} efflux pump alone did not affect
concentrations of biofilm protein or eDNA, but significantly increased
the glucose concentration (Fig 6). JW270 was generated by deleting the
\emph{wcb}cluster responsible for CPS I polysaccharide in DD503
{[}40{]}. Deletion of both the \emph{AmrAB-oprA} efflux pump and the
\emph{wcb} operon led to the significant decrease in both biofilm
protein and polysaccharide, but an inverse increase in the amount of
eDNA exported and found in a biofilm (Fig. 6).

The differences observed in the molecular composition were not
attributed to the extraction method or cell lysis during extraction. A
plate count was performed before and after incubation with TE buffer to
evaluate cell lysis and the number of cell count obtained before and
after extraction remained constant. Florescence live/dead ratio was also
carried out and Fig 2a shows that the biofilm harbor viable cells. It is
also noted that as with most biofilms, these biofilms are subject to
enzymatic degradation. Proteinase K/glucosidase had the most eradicative
effect on E264 while DNase seemed to have a more profound effect on
the\emph{B. pseudomallei} derivatives.

In Fig. 1b-c, we observed the pellicles of \emph{Burkholderia} .
Morphologically, JW270 and Bp82 produced thicker pellicles, but
1026b\selectlanguage{greek}\emph{Δasd}\selectlanguage{english} and E264 formed thinner pellicles. Microscopically in
Fig 1c, the pellicles look like mesh work anchored to the walls of the
wells and also shows adherent cells attached to it. In the fluorescent
image in Fig. 5a, florescent dyes were used to further illustrate the
composition of each strain. The data from Fig. 5a supported by the
assays used in Figs. 5b and 5c, also showed that the composition of
adherent biofilms (Figs 2a and 3) and pellicles (Fig. 5a-c) are quite
similar.

Morphological and chemical differences exist between the biofilms
of\emph{B. thailandensis} and the \emph{B. pseudomallei} derivatives
1026b\selectlanguage{greek}\emph{Δasd}\selectlanguage{english} and Bp82, but the JW270 biofilm exhibited similarities
to the E264 biofilm. We posit that the differences between \emph{B.
pseudomallei} JW270 and 1026b \selectlanguage{greek}\emph{Δasd}\selectlanguage{english} or Bp82 is related to the
capsule deletion necessitating the need for JW270 to export higher
amounts of eDNA required for biofilm formation as observed in \emph{B.
thailandensis} E264 biofilm. This hypothesis is backed by fig 6a-c which
shows that asides from glucose, the concentration of protein and eDNA
remain unchanged between \emph{B. pseudomallei} 1026b and DD503 but once
the \emph{wcb} operon is deleted in DD503 to generate JW270, there is a
decrease in protein content but a higher export rate for eDNA. Glucose
level In DD503 increased in comparison to 1026b but drops to baseline in
JW270 keeping in mind that the \emph{wcb} operon responsible for CPS I
polysaccharide. An ELISA test confirms that the difference
between\emph{B. pseudomallei} 1026b \selectlanguage{greek}\emph{Δasd}\selectlanguage{english} /Bp82 and \emph{B.
pseudomallei} JW270/ \emph{B. thailandensis} E264 is the presence of a
capsule. An anti-capsule antibody reacted with \emph{B.
pseudomallei}1026b \selectlanguage{greek}\emph{Δasd}\selectlanguage{english} /Bp82 but not \emph{B. pseudomallei}
JW270/\emph{B. thailandensis} E264. Biofilm assay confirmed that there
was no difference in biofilm formation between all strains used.

In summary, we propose that the lack of the \emph{wcb} operon is the
driving force behind the large amounts of eDNA exported by JW270. These
differences in biofilm composition may play a role in the immune
response of host organisms and also illustrate the survival of
persistent cells that cause disease relapse in a host organism.

\textbf{Materials and Methods}

\textbf{Bacteria strains}

Five \emph{B. pseudomallei} strains (1026b {[}36{]}, DD503 {[}39{]},
Bp1026b \selectlanguage{greek}\emph{Δasd}\selectlanguage{english} {[}37{]}\emph{,} Bp82 {[}38{]}, and JW270 {[}40{]})
and \emph{B. thailandensis} E264 {[}35{]} were used for this study.
Strains were either cultured on LB agar (1026b, JW270 and E264), LB agar
with 100 \selectlanguage{greek}µ\selectlanguage{english}g/ml diaminopimelic acid (DAP) (1026b \selectlanguage{greek}\emph{Δasd}\selectlanguage{english} ) or LB
supplemented with 100 \selectlanguage{greek}µ\selectlanguage{english}g/ml adenine and 5\selectlanguage{greek}µ\selectlanguage{english}g/ml thiamine (Bp82).
Bacterial colonies were inoculated in 3ml of LB broth, with or without
the corresponding supplement, and grown overnight in a 37\selectlanguage{ngerman}°C incubator
with shaking (250 rpm) or without agitation. Bacteria were serially
diluted, plated to determine bacteria count, and used for experiments.
All manipulations with \emph{B. pseudomallei} 1026b and DD503 were
carried out in a class II microbiological safety cabinet located in a
designated biosafety level 3 (BSL-3) laboratory.

\textbf{Biofilm formation}

Biofilm formation for \emph{B. pseudomallei} strains (Bp1026b\selectlanguage{greek}\emph{Δasd}\selectlanguage{english}
, Bp82, and JW270) and \emph{B. thailandensis} E264 was monitored using
both a scanning electron microscope (SEM) and spectrophotometric
analysis of bound crystal violet (CV) in a 96 well polystyrene plate
(Corning, CLS3628BC) as described by O'Toole \emph{et al} {[}71{]}. For
the SEM, JW270 was grown on a coverslips and each coverslip is taken out
and processed at 12, 24, 34, 48 and 72 hours. To process the pellicle,
the bacteria is grown in 2ml of broth with coverslip dropped in a 6well
plate. After 72 hours, a wide tip pipette is used to gently suction off
media from the sides of the well making sure to leave the pellicle
intact. Removal of the media collapses the pellicle on the coverslip.
The coverslip is washed and fixed with 2.5\% glutaraldehyde and 2\%
paraformaldehyde in 0.1M sodium cacodylate buffer for 1 hour. Samples
were washed, post fixed with 1\% osmium tetroxide, and alcohol
dehydrated (30\%, 50\%, 75\%, 85\%, 95\%, 3 changes of 100\% and a
single 1:1 with 100\% ethanol and hexamethyldisilzane) for 10 minutes
each. Sample was left to dry in 100\% HMDS for 24 hours under the hood
and imaged with Carl Zeiss Sigma VPFE microscope.

For biofilm assay using CV, 10\textsuperscript{6} bacterial cells were
added to 10ml of LB broth or LB broth supplemented with
DAP/adenine-thiamine and 9 replicates of 200\selectlanguage{greek}µ\selectlanguage{english}l per strain were cultured
in six 96 well plates for 1-3 days. Three plates each were incubated
either at 37\selectlanguage{ngerman}°C or at RT. The absorbance (OD\textsubscript{600nm}) of the
plates were read daily and three wells of each strain were serially
diluted and plated to determine bacterial counts and viability in a
biofilm. Spent media in the 96-well plate were discarded and wells
gently washed twice with 200\selectlanguage{greek}µ\selectlanguage{english}l of 1x PBS. One hundred microliters of
0.1\% crystal violet solution was used to stain biofilm for 15mins. The
biofilm was washed twice with 1x PBS and air-dried in a biosafety
cabinet. Biofilm is extracted using 200\selectlanguage{greek}µ\selectlanguage{english}l of 30\% acetic acid and the
bound cells are quantified at an optical density of 550 nm
(OD\textsubscript{550nm}) (Molecular devices, SpectraMax M5).

\textbf{Immunofluorescence assay}

For CLSM imaging to confirm biofilm components or record the live/dead
ratio of the \emph{B. pseudomallei} and \emph{B. thailandensis}biofilms,
inoculated 96 well were gently washed and molecular dyes: Film Tracer
biofilm viability kit (Invitrogen L10316), DAPI stain (KPL, 71-03-00),
Sypro ruby biofilm matrix stain (Invitrogen, F10318), and the Wheat germ
agglutinin fluorescein conjugate (Invitrogen, W834) were used to stain
wells according to each manufacturer's protocol. Wells were washed with
sterile water and imaged immediately using a Nikon Eclipse 90i
microscope.

\textbf{Planktonic and biofilm growth and extraction}

Bacterial cells (10\textsuperscript{6}) were added to 2 ml of LB broth
and cultured in 9x 15 ml tube (planktonic) or a 12-well plate (biofilm)
for 1-3 days. The 12-well plate cultures were grown statically in a 37\selectlanguage{ngerman}°C
incubator (biofilm growth) and the 15 ml tube cultures were grown at
37°C with agitation at 250 rpm to prevent adherence (planktonic growth).
At each time point, samples were taken out and a pipette used to suction
spent media and the planktonic growth was centrifuged at max speed to
pellet cells. One ml of sterile 1x PBS was added to the well and a cell
scraper was used to gently detach biofilm cells. Detached biofilm cells
and planktonic cells were softly pelleted at 3700 rpm for 10 mins.
Biofilm components were extracted as described in Keitheley \emph{et
al.} (2018) {[}46{]} with no modifications. Briefly, the pellet was
resuspended in 1.5 ml of TE buffer (10 mM Tris, 10 mM EDTA, 2.5\% NaCl,
pH 8.0) and incubated at 200 rpm for 4 hours at 35 °C. The cells were
pelleted at max speed, 4°C and the resulting extractant filtered and
used for composition analysis. 10 \selectlanguage{greek}µ\selectlanguage{english}l of the extractant is plated to
ensure the absence of bacterial cells. A plate count was performed
before and after incubation with TE buffer to determine if cell lysis
occurred.

\textbf{Protein expression}

Pierce BCA protein assay was used to determine the biofilm protein
concentration. In a 96-well plate, 25 \selectlanguage{greek}µ\selectlanguage{english}l of the extractant above was
mixed with 200 \selectlanguage{greek}µ\selectlanguage{english}l of Cu\textsuperscript{2+} solution according to
manufacturer's protocol (Pierce, 23225). The reaction was incubated at
37 \selectlanguage{ngerman}°C for 30 min. The absorbance was read at OD\textsubscript{562nm}.

\textbf{Polysaccharide concentration}

The Phenol-sulfuric acid method, a widely used quantitative assay for
glucose estimation was used to estimate the polysaccharide concentration
present in a biofilm {[}72{]}. Briefly, 150 \selectlanguage{greek}µ\selectlanguage{english}l of extractant was added
to 450 \selectlanguage{greek}µ\selectlanguage{english}l of concentrated sulfuric acid and 90\selectlanguage{greek}µ\selectlanguage{english}l of 5\% phenol in a 13
mm glass tube. The tube was gently swirled to mix, incubated in a 90 \selectlanguage{ngerman}°C
water bath for 10 mins and cooled to RT. Two hundred microliters was
pipetted in triplicates into a 96-well plate and absorbance measured at
OD\textsubscript{490nm}. Varying concentrations of glucose
(2/1.75/1.5/1/0.5/0 mg/ml) were used for the control and standard curve.

\textbf{eDNA concentration}

Using a 96-well plate, triplicates of 25 \selectlanguage{greek}µ\selectlanguage{english}l of extractant was added to
175 \selectlanguage{greek}µ\selectlanguage{english}l of 0.1\% propidium iodide dissolved in water. The reaction was
incubated with gentle agitation in the dark, at RT for 10 mins to mix.
Wells were excited at 535nm and florescence read at 620nm. 200 \selectlanguage{greek}µ\selectlanguage{english}l of
0.1\% propidium iodide was used as blank.

\textbf{Enzymatic treatment of biofilm}

Biofilms were grown in a 96-well plate as described above. Wells were
either treated with 2 U/ml of DNase, 15 \selectlanguage{greek}µ\selectlanguage{english}g/ml proteinase K or 3 \selectlanguage{greek}µ\selectlanguage{english}g/ml of
glucosidase (Sigma G4511-100UN) to target eDNA, protein or
polysaccharide components of each biofilm. Treatments occurred at the
time of inoculation. Biofilm was determined using CV as previously
described.

\textbf{Immunofluorescence microscopy}

Bacteria pellicles were gently lifted from both sides using two sterile
pipette tips or a measuring micro-spatula and placed on a poly-l-lysine
coated slide. The pellicle is gently washed by pipetting sterile PBS
over it to remove non-adherent bacteria and left to adhere for 30mins.
The slide is flooded with 3 \selectlanguage{greek}µ\selectlanguage{english}M DAPI (Invitrogen, D1306), 5 \selectlanguage{greek}µ\selectlanguage{english}g/ml wheat
germ agglutinin fluorescein conjugate (WGA), (Invitrogen, W834), and
200\selectlanguage{greek}µ\selectlanguage{english}l Sypro ruby biofilm matrix stain (Invitrogen, F10318) in a total
volume of 500\selectlanguage{greek}µ\selectlanguage{english}l. The slide is incubated in the dark for 30mins, gently
washed with sterile water, air-dried in the dark, and imaged as
described above.

\textbf{Extraction, molecular determination and imaging of pellicle
components}

1ml of PBS was added to a 1.5 ml tube and the pellicle is scooped with a
micro-spatula, dropped into the tube, gently washed at 5000 rpm. 500 \selectlanguage{greek}µ\selectlanguage{english}l
of TE buffer is added and components are extracted and quantified as
previously described.

\textbf{Statistical analysis}

All experiments were independently repeated three times.

Each biofilm assay experiments was conducted as independent samples of
six and mean values compared within and between groups using the
student's t-tests.

All biofilm extraction experiments were conducted as three independent
experiments and mean values compared between groups using the student's
t-tests.

Graphpad Prism 9 (GraphPad Software, LLC) was used for statistical
analysis. Differences were statistically different for a
P-value \textless{} 0.05.

\textbf{Data Availability}

The authors declare that the data supporting the findings of this study
are available within the paper {[}and its supplementary information
files{]}. Data generated will be deposited on figshare on acceptance.

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\textbf{Author Information}

\textbf{Contributions}

D.D and U.O. conceived the experimental idea and wrote the manuscript.
U.O. verified the protocol, planned and performed the experiments and
data analysis. S.M. performed the experiments in the BLS3. D.D.
supervised the findings of this work.

\textbf{Corresponding author}

Correspondence to David DeShazer.

\textbf{Funding Information.}

This research was performed while the author -- UO- held an NRC Research
Associateship award at the DeShazer Lab at the United States Army
Medical Research Institute of Infectious Diseases (USAMRIID), Frederick,
Maryland, USA.

This work was supported by DTRA/JSTO-CBD under Grant CB10207 (to D.D.).
Opinions, interpretations, conclusions, and recommendations are those of
the authors and are not necessarily endorsed by the U.S. Army.

\textbf{Competing Interests statement}

The authors declare no competing interests.

\textbf{Figure Legends}

Figure 1. Effect of temperature on biofilm formation in
different\emph{Burkholderia} strains (\emph{B. pseudomallei} 1026b
\selectlanguage{greek}\emph{Δasd}\selectlanguage{english} , Bp82, JW270 and \emph{B. thailandensis} E264). a. Biofilm
formation life cycle in \emph{B. pseudomallei} JW270 b. Pellicle images
of\emph{Burkholderia} strains. c. Image of a \emph{B. pseudomallei}
JW270 pellicle post processing vs micrograph image of the pellicle. Red
lines lead to an SEM image which shows that the pellicles are made up
the extracellular materials (eDNA, proteins and polysaccharides) and
bacterial cells. There was no difference in pellicle morphology on the
SEM. Bar 2\selectlanguage{greek}µ\selectlanguage{english}M. d. Crystal violet quantification of
\emph{Burkholderia}biofilms under different temperatures shows that
there is no significant difference in biofilm formation at the end of
the experiment -72 hours (P \textgreater{} 0.05). e. Biofilm formation
of all 4 strains at 37\selectlanguage{ngerman}°C after 72 hours shows that there is no
significant difference between the biofilms formed by \emph{B.
pseudomallei} strains or by \emph{B. thailandensis} , (P
\textgreater{}0.05 for each comparison).

Figure 2. Microscopic images of \emph{Burkholderia} biofilm. a. 10x
Microscopic images of 72 hours \emph{Burkholderia} biofilms which shows
the biofilms are composed of eDNA (Dapi), proteins (Sypro ruby) and
polysaccharides (WGA) White bar - 200\selectlanguage{greek}µ\selectlanguage{english}m. Inserts are 100x Microscopic
images showing the cells within a biofilm. Red bar - 100\selectlanguage{greek}µ\selectlanguage{english}m. b. Live/dead
image of cells 0-48 hours showing that the cells are viable throughout
the biofilm process (Bar- 10\selectlanguage{greek}µ\selectlanguage{english}m).

Figure 3 shows that there is a significant difference between the
amounts exported when comparing between growth modes (p < \selectlanguage{english}0.05). As
expected, biofilm cells export higher amount of all constituents
compared to planktonic cells. In figures 3a -3c, we observe that
1026b\selectlanguage{greek}\emph{Δasd}\selectlanguage{english} and Bp82 export high levels of (a) protein and (b)
polysaccharide and low levels of (c) eDNA. JW270 biofilm on the other
hand show higher levels of eDNA and minimal protein and polysaccharide
export while \emph{B} . \emph{thailandensis} exports high protein and
eDNA and lower polysaccharide levels when compared with \emph{B.
pseudomallei} 1026b \selectlanguage{greek}\emph{Δasd}\selectlanguage{english} and Bp82\emph{.}

Figure 4. Effects of enzymatic treatments on 72 hours\emph{Burkholderia}
biofilms. Data shows that there is a significant difference between
untreated biofilms and enzyme treated biofilm. (* = p <\selectlanguage{english}0.05, ** = p =
0.01-0.05, *** = p =0.01- 0.007).

Figure 5. Morphology and composition of \emph{Burkholderia} pellicles.
a. Fluorescent images of a \emph{Burkholderia} pellicles showing eDNA
(blue), Polysaccharides (green), Sypro ruby (red). b. Polysaccharide
(black bars) and protein (red line) compositions extracted from 72 hour
pellicles. c. Quantification of eDNA extracted from a 72 hour pellicle
and read using propidium iodide and a spectrophotometer. Bars represent
average of three independent experiment and Error bars represent SEM.

Figure 6. Comparison between 1026b, DD503, and JW270 biofilms. Graphs
a-c show concentrations of protein and glucose and eDNA levels
respectively. There is no significant difference between the protein and
eDNA of 1026b and DD503 at the end of the experiment, 72 hours (p
\textgreater{} 0.05) whereas DD503 and JW270 differed significantly at
each time point except the 48 hours eDNA concentration (p \textless{}
0.05). There is also a significant difference between the glucose
concentrations for all three strains.

\textbf{Figures}\selectlanguage{english}
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\includegraphics[width=0.70\columnwidth]{figures/image1/image1}

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\textbf{Supplemental Data}\selectlanguage{english}
\begin{figure}[H]
\begin{center}
\includegraphics[width=0.70\columnwidth]{figures/image14/image14}

\end{center}
\end{figure}

Fig S1. Hierarchy and genetic manipulations for the \emph{B.
pseudomallei} strains used for this study.\selectlanguage{english}
\begin{figure}[H]
\begin{center}
\includegraphics[width=0.70\columnwidth]{figures/image15/image15}

\end{center}
\end{figure}

Fig S2. ELISA test to confirm reaction between an anti-CPS antibody and
the strains used for this study.

Table S1. Protein analysis of strains and growth procedure.

\textbf{Hosted file}

\verb`image16.emf`  available at \url{https://authorea.com/users/414433/articles/522470-production-and-molecular-composition-of-burkholderia-pseudomallei-and-burkholderia-thailandensis-biofilms}

Table S2. Glucose analysis of strains and growth procedure.

\textbf{Hosted file}

\verb`image17.emf`  available at \url{https://authorea.com/users/414433/articles/522470-production-and-molecular-composition-of-burkholderia-pseudomallei-and-burkholderia-thailandensis-biofilms}

Table S3. eDNA analysis of strains and growth procedure.

\textbf{Hosted file}

\verb`image18.emf`  available at \url{https://authorea.com/users/414433/articles/522470-production-and-molecular-composition-of-burkholderia-pseudomallei-and-burkholderia-thailandensis-biofilms}

\selectlanguage{english}
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\end{document}

