Discussion
Burkholderia is a genus of proteobacteria which harbors
pathogenic and non-pathogenic members.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 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 B. pseudomallei , a BSL3
lab is required to work with the bacteria hence manipulations have been
made to generate B. pseudomallei strains 1026b Δasd , Bp82,
JW270 which are all non-pathogenic BSL2 strains. On the other hand,B. thailandensis is a non-pathogenic strain which rarely causes
disease in humans [35, 55]. Most 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 Maganaet al , 2018 [60]. Research has shown that B.
pseudomallei and 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 Salmonella [68], Burkholderia is capable of
producing either a surface adherent biofilm or a pellicle (Fig 1a-c).
This phenomenon was also demonstrated in a previous Burkholderiastudy which showed both pellicle and adherent biofilm [23]. The
conditions and reasons for the change from surface adherent to pellicle
biofilm remains unknown in Burkholderia . Mangalea et al .
[8] reviewed that 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°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 Salmonella , curli is responsible for the
switch [68] whereas, in 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 B.
pseudomallei 1026b, four isogenic 1026b mutants and B.
thailandensis E264. Since 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 B. pseudomallei and B. thailandensisstrains at 72 hours showed no significant difference (Fig. 1e).
Fluorescent microscopy and molecular assays (Fig. 2a and 3) show that
the biofilm composition of B. thailandensis is significantly
composed of proteins and eDNA and lower levels of polysaccharide.B. pseudomallei 1026b Δasd 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 Δasd 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 lacksamrAB-oprA , a multidrug efflux system (Fig. S1). The deletion of
the 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 wcbcluster responsible for CPS I polysaccharide in DD503 [40]. Deletion
of both the AmrAB-oprA efflux pump and the 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 theB. pseudomallei derivatives.
In Fig. 1b-c, we observed the pellicles of Burkholderia .
Morphologically, JW270 and Bp82 produced thicker pellicles, but 1026bΔasd 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 ofB. thailandensis and the B. pseudomallei derivatives 1026bΔasd and Bp82, but the JW270 biofilm exhibited similarities to
the E264 biofilm. We posit that the differences between B.
pseudomallei JW270 and 1026b Δasd 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 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 B. pseudomallei 1026b and DD503 but once
the 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 wcb operon responsible for CPS I
polysaccharide. An ELISA test confirms that the difference betweenB. pseudomallei 1026b Δasd /Bp82 and B.
pseudomallei JW270/ B. thailandensis E264 is the presence of a
capsule. An anti-capsule antibody reacted with B. pseudomallei1026b Δasd /Bp82 but not B. pseudomallei JW270/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 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.