Bacillus thuringiensis from mosquito dunks have antibacterial and

biofilm formation abilities in standing water environment

Brian Parkins, Jatin Bhakta, Keitia Ramas, Vincent Vo
California State University Fullerton, Department of Biological Science

Abstract

Bacillus thuringiensis, a gram-positive bacterium commonly found in soil, has become an increasingly
important component in pest management (1). After the discovery of Bacillus thuringiensis insecticidal
properties, this trait has been commercially exploited by biochemical companies to produce effective
endotoxins specific for certain pests like- Lepidoptera (moths/butterflies), and Diptera (flies/mosquitos) (1).
As a result, Summit Chemical, a pesticide manufacturing company, has used Bacillus thuringiensis spores to
create a dunk that is toxic to mosquito larvae and can be used to treat standing bodies of water (7). Summit
Chemical claims these dunks are drought-resistant and can last up to 30 days. Even after being exposed to
drought conditions, these spores may once again become activated and express toxic abilities during
sporulation (7). This study focuses on how Bacillus thuringiensis is able to protect itself from desiccation
when exposed to drought conditions and the effect of -endotoxins on the surrounding bacterial pond
population exposed to this commercial dunk. For our experiment we predicted: 1) If B. thuringiensis is
drought resistant, then we predict the formation of a thick biofilm to prevent desiccation, and 2) If B.
thuringiensis is capable of producing potent endotoxins, then we predict the formation of a zone of inhibition
when bacteria are exposed to this endotoxin. Our results revealed the formation of a thick biofilm in Bacillus
thuringiensis as well as a moderate zone of inhibition in E.coli. For future work we would like to explore
Bacillus thuringiensis impact on biodiversity in pond water (before and after adding the mosquito dunks).

Introduction
Bacillus thuringiensis is a gram-positive, rod shaped, spore forming bacterium known to
reside in soil filled environments. A significant feature of B. thuringiensis exploited by
biochemical companies, are the various genes that are utilized in transgenic pest-resistant crops,
as well other pesticides (1). Previous studies used SDS-PAGE to determine whether or not
Escherichia coli had encapsulated proteins with insecticidal properties similar to those secreted
by B. thuringiensis, so the encapsulated proteins could be used in widespread pesticides (2).
These proteins are encoded by the vip3Aa and the cry1la genes and are secreted during the
vegetative growth of B. thuringiensis. During sporulation, the cry2 and cry9 classes of genes
encode for a group of proteins known as Cry toxins that make up -endotoxins which are toxic to
Lepidoptera larvae and non-toxic towards humans (1). The insecticidal proteins produced during
the vegetative growth and during sporulation are similar in that they are used in biochemical
pesticides, but differ in the fact that the proteins encoded by the cry2 and cry9 genes are not
secreted out of the cell as are the proteins produced by vip3Aa and cry1la, and are rather stored
as crystal proteins in the spores produced (2).
For B. thuringiensis to have ideal insecticidal properties it would have to be able to
survive various conditions. In order to do so, Bacillus thuringiensis can produce a biofilm in
which previously planktonic bacteria grow in sessile aggregates forming an extracellular
polysaccharide matrix that serves as a source of protection and nutrients (3). In works referenced
by Boles and Horswill (4) it was discovered that a small percentage of cells within a biofilm
produced by B. thuringiensis were motile. These motile bacteria were able to penetrate out of
and back in through the polysaccharide layer and create additional pores that would allow for
additional nutrients to enter the biofilm for the bacteria to utilize (4). A different study agreed
that there is an exchange of sessile and planktonic bacteria within biofilms and that the exchange
could improve bacterial fitness, but it also proved that the pores created by the planktonic
bacteria could allow for material to enter the film that could prove to be detrimental to some
species of bacteria within the biofilm (5). In that specific study it was found that the antibacterial
properties of Bacillus spp. was circulated through the pores created in the biofilm and had killed
surrounding Staphylococcus aureus and had then occupied the new space (5).
B. thuringiensiss insecticidal properties were first discovered in 1976 in the guts of dead
mosquito larvae in dried up river beds in Israel (6). Summit Chemical, a manufacturer of
pesticide products, has utilized the Bacillus thuringiensis spores to develop dunks that are toxic
to mosquito larvae and can be used for all standing bodies of water (7). These dunks are used
commercially to control mosquito populations in ponds, bird baths, rain barrels, and anywhere
else water can be collected over time; it has a safe widespread use as it is non-toxic to humans
and animal species. The company claims that one dunk can last up to 30 days and is drought
resistant because the B. thuringiensis spores are resistant to dehydration. These spores can
become active when the dunk is placed in appropriate conditions such as the standing bodies of
water they are utilized in. This study involves testing the production of a biofilm by B.
thuringiensis used in the mosquito dunks as the source of drought resistance for the dunk, as well
as determining whether the toxic proteins produced by B. thuringiensis have antibacterial
properties as seen in Bacillus subspecies in previous works. If antibacterial properties are present
in the proteins synthesized by B. thuringiensis it may be able to outcompete bacteria found in the
body of water in which the dunk was present, which, in the case of this study, was a household
pond. This gradual elimination may have ecological impacts as the reduction of the microbiome
diversity may result in an increase in pathogenic bacteria in the biome.
Methods

Preparing a working culture of Bacillus thuringiensis.
Commercial mosquito dunks purchased through Amazon.com, known as Summits Mosquito Dunks,
were obtained and a small pea-sized portion of the dunk was chipped off and placed into a test tube filled with water
to rehydrate. The test tube was then vortexed for approximately 10 seconds. The aqueous solution was then streak
plated onto TSA and incubated for 2 days. A gram stain was performed on isolated colonies to that ensure only the
desired bacteria (B. thuringiensis) were present and to check for any contamination of other bacterial strains. One
pure colony of B. thuringiensis was selected and inoculated into liquid TSB and incubated for two days. This
became our pure working culture of B. thuringiensis, which was thereafter used in all subsequent tests when needed.
Endospore stain
One loopful of the working culture was placed onto a slide and normal protocol for staining was followed
as described in the CSUF Microbiology Laboratory Manual, but modifications were made to the procedure because
there was no steam bath available at the time. Instead of steaming the slide for 5 minutes, we used a slide holder and
gently passed the slide above the flame (but not directly through the flame to prevent from killing the bacteria). We
did this for approximately five minutes to ensure the malachite green was driven into the endospore. The slide was
then viewed under a compound light microscope.
TSI
One loopful of the working culture was inoculated into TSI to observe the fermentation patterns of the
bacteria and to further support the identity of the bacteria isolated from the mosquito dunk as B. thuringiensis. The
inoculated TSI was incubated until the following lab period.
Biofilm assay
Standard procedure for the biofilm assay was carried out, as detailed in the CSUF Microbiology Laboratory
Manual. B. thuringiensis (from the working culture) was used to test for the formation of a biofilm. Escherichia
coli and Bacillus megaterium were used as positive controls for this assay. The 6-well plates were inoculated with
200 ul of each of the bacteria. The bacteria were allowed to incubate for 6 days. Bacterial turbidity (i.e. biofilm
formation) was quantified using a spectrophotometer at 595 nm. All obtained data was standardized, using B.
thuringiensis as the baseline.
Endotoxin extraction from Bacillus thuringiensis
750 ul of week old B. thuringiensis and 750 ul of water were pipetted into a 1.5-ml micro centrifuge tube.
This solution was centrifuged for 45 seconds at 13K RPM. Subsequently, 1.5 ml of the supernatant (containing the
desired endotoxin protein) was loaded into SDS-PAGE. No endotoxin protein was able to be extracted after
separation using this gel electrophoresis technique.
Modified Kirby Bauer test
0.3 ul of E. coli was spread plated onto Mueller-Hinton media. A sterile filter disk was dipped into the
working solution of B. thuringiensis and was dispensed on the center of the spread plate. The plate was incubated
for 6 days. The zone of inhibition was measured and a standard SIR chart was used to compare our results to known
sensitive, intermediate, and resistant compounds for E.coli.
Pond water bacteria isolation
Pond water, obtained from Brian Parkins backyard, which had been previously exposed to Summits
mosquito dunks, was streak plated onto TSA. Individual colonies were selected and re-streaked onto TSA, in
attempts to isolate pure cultures.
Anomalous bacteria identification
Two distinct colonies were isolated from TSA plates streaked with bacteria recovered from the Mosquito
Dunks. Both colonies were subjected to gram and acid-fast stains as well as differential media plating on EMB
and MSA, as detailed in the CSUF Microbiology Laboratory Manual.
Results

B. thuringiensis culture from Mosquito Dunks
Following plating on TSA and inoculation of TSB, the resulting bacteria were isolated
and characterized. What presented were large numbers of Gram-positive bacilli, many of which
were positive for endospores. A SIM test performed, and indicated that they were motile but did
not produce hydrogen sulfide or indole via tryptophan hydrolysis.
Pond water culture
The bacteria within the pond water sample was difficult to isolate. Those colonies that
were found were mostly Gram-positive bacilli, which was to be expected due to the presence of a
Mosquito Dunk in the water for an extended period of time before collection. Some Gram-
negative staphylococci were discovered, but attempts to isolate these bacteria were fruitless. We
suggest that, more than likely, these were Staphylococcus aureus, a common bacteria found in
potable and nonpotable open-air water supplies [8]. A SIM test for the pond water showed very
little motility, and was negative for both hydrogen sulfide and indole production, consistent with
both old B. thuringiensis and S. aureus.
TSI
Triple Sugar Indole (TSI) slants presented with a dark yellow slant and red butt, initially
suggesting lactose/sucrose fermentation but not glucose fermentation. This result is explored
further later in this study (see Discussion).
The Dot
The TSA plate that was initially streaked with the Mosquito Dunk was reserved for the
length of the study for characterization of the endospores. Upon examination during the study,
however, a small, new colony was observed growing in an established B. thuringiensis colony.
A small, 2mm ring of empty media had formed around this colony. The colony was harvested
and streaked on a new TSA plate. Gram staining revealed a Gram-negative bacteria with vibrio
morphology, while a second Gram-stain later revealed a Gram-positive bacteria with the same
vibrio morphology. This vibrio was found in addition to some Gram-positive bacilli in both
stains, suspected to be B. thuringiensis.
Further testing of this Gram-variable vibrio involved acid-fast staining and plating on
EMB and MSA media plates. The acid-fast staining was negative, while growth was observed
on both EMB and MSA, the former being the strongest.

Figure 1 MSA (left) and EMB (right) plates streaked with culture produced from the Gram-variable vibrio culture
harvested from the anomalous colony. Growth is seen in both media plates, incubated for 48 hours at 37C.

Biofilm formation
Quantification of the strength of the biofilm was standardized using an average of the B.
thuringiensis biofilm, which had the largest absorbance values. The density of the biofilm
produced by B. thuringiensis was 30% higher than that of control Bacillus megaterium or
Escherichia coli incubated under the same conditions.

Figure 2 Biofilm density was quantified using a spectrophotometer. B. thuringiensis formed a 30% stronger biofilm than
B. megaterium, and more than 50% stronger than that of E. coli. No statistical analysis was conducted due
to small sample size.

Kirby-Bauer
A 15 mm zone of inhibition around the disc was observed on the Mueller Hinton agar
streaked with E. coli. No bacterial growth was observed within this zone, and no growth was
found on the disc. Due to the thin layer of the MH agar, nutrient leaching by E. coli produced
similar rings.

Figure 3 Modified Kirby-Bauer protocol using a Mueller-Hinton agar plate, E. coli culture and following
application of sterile disk dipped into settled old TSB culture. A 15mm zone of inhibition was found
around the disc.

Discussion
Identification of B. thuringiensis
One week old Bacillus thuringiensis grown from the endospores inside mosquito dunk
was positive for producing endospores. The TSI results seemed reversed from previous
fermentation experiments using B. thuringiensis. The result may be attributed to the
fermentation of glucose into pyruvate and acetate, followed by reclamation of acetate causing a
rise in pH as glucose supply dwindled. This would cause the butt to appear red again. Previous
studies of B. thuringiensis and glucose fermentation have shown a return to neutral pH within 48
hours [9]. The resulting alkaline condition following complete fermentation of a food source
triggers sporulation in B. thuringiensis, which is accelerated in those expressing the pBtoxis
plasmid [10].
Bacillus thuringiensis forms a biofilm
The hypothesis was partially proven with the results from the biofilm formation
experiment. Bacillus thuringiensis was found to produce a relatively larger biofilm than the
controls in the biofilm formation experiments. BT had the largest biofilm and Bacillus
megaterium was greater than E. coli. Since BT and Bacillus megaterium are both from the
Bacillus genus, the result showing them to have more similar biofilm than E. coli makes sense.
BT forming a larger biofilm than Bacillus megaterium can give rise to the conclusion of how
similar bacteria diverged because of differences in environment. BT differentiated from the
Bacillus genus because it adapted to drier conditions.
BT desiccation resistance was hypothesized to be an important characteristic for its
survival when its standing water environment dried out. The first part of the original hypothesis,
B. thuringiensis forms a biofilm was confirmed. Regardless of the findings, the second part of
the hypothesis, B. thuringiensis forms a biofilm in order to protects itself against desiccation, can
not be confirmed without further investigation. The formation of the biofilm could be an
independent outcome when B. thuringiensis is in a dry environment. Other modes of desiccation
resistance may exist, however, biofilm formation is the best candidate at this time.
The initial absorbance reading for B. thuringiensis biofilm was greater than 1. The
protocol stated the absorbance should be less than 1. Since, the conclusion did not require a
qualitative measurement, a comparative value was sufficient. The biofilm measurements were
taken in the same manner and standardized measurements assured the relative comparison was
accurate. To make a more confident conclusion, more bacteria could have been compared to B.
thuringiensis.
Endotoxin extraction
Isolating the endotoxin from the B. thuringiensis spores would have been ideal for the
Kirby Bauer experiment. Unfortunately, endotoxin were not extracted through centrifugation and
electrophoresis. Silica beads could have been mixed in with the lysate before centrifugation to
break open the B. thuringiensis endospores and release the -endotoxins housed inside the
spores. Once the endotoxins are isolated, a traditional Kirby Bauer can be performed with disks
soaked in endotoxins. The problem with the modified Kirby Bauer was, other unknown agents
in B. thuringiensis could have been inhibiting bacteria growth. The best way to make sure -
endotoxins were the agent producing the zone of inhibition, isolated -endotoxins should have
been used.
Delta endotoxin is antibacterial
The modified Kirby Bauer experiment produced expected results. A well defined zone of
inhibition was present around the B. thuringiensis disk. SIR charts indicated E. coli was of
intermediate susceptibility. The zone of inhibition had reached its maximum diameter because
measurements were taken after six days of incubation. Not enough data was collected to
confidently state the endotoxin is an antibacterial agent. More bacteria must be tested through
the same modified Kirby Bauer experiment to establish the antibacterial properties of the -
endotoxin. Not only does expanding the sample size reduce the effects of experimental error, but
a mechanism of bacterial attack can be deduced based upon known properties of bacteria
susceptible to the endotoxin. Biochemical techniques may be performed to find out -
endotoxins cellular target.
A Kirby Bauer was the most obvious technique to measure whether an agent is
antibacterial or not. Previous studies have established endotoxin is bactericidal towards E. coli
through a different method (11). Instead of using Kirby Bauer, they measured the minimal
bactericidal concentration (MBC) of Cyt1Aa, -endotoxin. The alternative technique was able to
reach a similar conclusion.The Kirby Bauer experiment can only conclude an agent is
antibacterial. Meaning, the agent is at the very least inhibiting bacteria growth. The MBC
technique can go one step further and conclude the agent is bactericidal, killing the bacteria. For
future studies, an MBC technique would be more fruitful than a Kirby Bauer for measuring
bacterial susceptibility to a chemical agent. Kirby Bauer still proves to a viable technique
because of its relatively convenient protocol.
A possible future study can compare the standing water bacteria population before and
after introduction of the mosquito dunk to the standing water environment. This study will
expand on the original hypothesis, B. thuringiensis has antibacterial properties. A new
hypothesis would propose greater bacterial diversity before the introduction of the mosquito
dunk compared to after the mosquito dunk was placed in the same standing water environment.



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