![Swarming and swimming behaviour of Salmonella enterica serovar Typhi. Swarm medium is nutrient broth (0.5% agar) and swim medium (0.25% agar) supplemented with glucose as carbon source (0.5% [wt/vol]). Uncolonised agar is black and bacteria biomass is white. All images were captured after 24 hr at 37°C. Panel A shows swarming motility. Panel B shows swimming motility.](https://www.researchgate.net/profile/Kwai-Lin-Thong/publication/260116531/figure/fig1/AS:202831387729926@1425370179829/Swarming-and-swimming-behaviour-of-Salmonella-enterica-serovar-Typhi-Swarm-medium-is_Q320.jpg)
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R E S E A R C H Open Access
Variations in motility and biofilm formation of
Salmonella enterica serovar Typhi
Kalaivani Kalai Chelvam
1,2
, Lay Ching Chai
1,2
and Kwai Lin Thong
1,2*
Abstract
Background: Salmonella enterica serovar Typhi (S. Typhi) exhibits unique characteristics as an intracellular human
pathogen. It causes both acute and chronic infection with various disease manifestations in the human host only.
The principal factors underlying the unique lifestyle of motility and biofilm forming ability of S. Typhi remain largely
unknown. The main objective of this study was to explore and investigate the motility and biofilm forming
behaviour among S. Typhi strains of diverse background.
Results: Swim and swarm motility tests were performed with 0.25% and 0.5% agar concentration, respectively; while
biofilm formation was determined by growing the bacterial cultures for 48 hrs in 96-well microtitre plate. While all S.
Typhi strains demonstrated swarming motility with smooth featureless morphology, 58 out of 60 strains demonstrated
swimming motility with featureless or bull’s eye morphology. Interestingly, S. Typhi strains of blood-borne origin
exhibited significantly higher swimming motility (P < 0.05) than stool-borne strains suggesting that swimming motility
may play a role in the systemic invasion of S. Typhi in the human host. Also, stool-borne S. Typhi displayed a negative
relationship between motility and biofilm forming behaviour, which was not observed in the blood-borne strains.
Conclusion: In summary, both swimming and swarming motility are conserved among S. Typhi strains but there was
variation for biofilm forming ability. There was no difference observed in this phenotype for S. Typhi strains from
diverse background. These findings serve as caveats for future studies to understand the lifestyle and transmission of
this pathogen.
Keywords: Salmonella Typhi, Biofilm, Motility swarming, Swimming
Background
Motility and biofilm forming capability in bacterial path-
ogens are one of the most studied bacterial physiology
nowadays as these characteristics have important roles
on pathogenicity [1-3]. Almost all of the identified pa-
thogenic bacteria of humans, such as Vibrio cholerae,
Pseudomonas aeruginosa,Salmonella and pathogenic
E. coli, are motile. However, the ability to form biofilm is
variable in human bacterial pathogens.
Typically, bacterial motility refers only to swimming
motility in diagnostic microbiology. The typical bacterial
motility test performed in the clinical laboratory based
only on the ability of bacterial cells to migrate away from
the semi-solid stab. In fact, bacteria move in various
modes, including swimming and surface swarming. Swim-
ming occurs when bacterial cells move in the aqueous
environment (low agar concentration) while swarming
motility is a collective behaviour of bacterial cells asso-
ciated with migration on semi-solid surfaces [4]. Unlike
the classical swimming motility in aqueous environment,
vegetative cells must first differentiate into elongated and
hyperflagellated swarmer cells to migrate on the surface
[4,5]. Besides the obvious physical changes, swarmer dif-
ferentiation can also be coupled to increase expression of
important virulence determinants in some species. Besides
that, swarming is also linked to biofilm forming ability in
bacteria, which serve as another important virulent factor
of human pathogens [6,7].
A biofilm is defined as bacterial colony adherence to
solid surface that secretes a self-initiated, protective exo-
polysaccharide matrix [8,9]. The ability to form biofilms
through the complex interaction of bacteria has been re-
ported to be important for bacterial survival within the
* Correspondence: thongkl@um.edu.my
1
Institute of Biological Sciences, Faculty of Science, University of Malaya,
Kuala Lumpur, Malaysia
2
Laboratory of Biomedical Science and Molecular Microbiology, Institute of
Graduate Studies, University of Malaya, 50603 Kuala Lumpur, Malaysia
© 2014 Kalai Chelvam et al.; licensee BioMed Central Ltd. This is an Open Access article distributed under the terms of the
Creative Commons Attribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use,
distribution, and reproduction in any medium, provided the original work is properly cited. The Creative Commons Public
Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this
article, unless otherwise stated.
Kalai Chelvam et al. Gut Pathogens 2014, 6:2
http://www.gutpathogens.com/content/6/1/2
human host. Moreover, both the innate and adaptive im-
mune responses of the human hosts might not be able
to eliminate the pathogen within the well-established
biofilm [10,11].
Motility and biofilm-forming ability have been repor-
ted in Salmonella enterica serovar Typhi (S. Typhi) [12].
It is the etiological agent of typhoid fever, infecting
21.7 million people and causing 217,000 deaths annually
[13]. Several case-control studies have investigated risks
for enteric fever; the majority implicate water and food
as important transmission routes [14-17]. Most patients
who recover from the infection are able to eliminate the
bacterium completely from their bodies. However, an ap-
proximately 5-10% of infected individuals may remain as
carriers, continuously shedding S. Typhi in their stools
[18]. A recent study showed that S. Typhi is frequently
associated with the presence of gallstones in asympto-
matic human carriers, in which the pathogen colonises
and persists as biofilm on the gallstones [12]. Despite
the caustic nature of bile in gallbladder, biofilms allow
the continual shedding and reattachment of individual
cells, contributing to the spread of bacteria via urine and
faeces, particularly in the human host [19,20].
Motility has also been detected in S. Typhi. Indeed, intact
motility (swimming motility) has been identified to be an
invasive-related factor of S.Typhi[21].Amongmorethan
2500 serovars of Salmonella enterica,S.Typhimuriumis
one of the earliest serovars being identified to undergo
morphological differentiation into swarmer cells [5]. Later,
Kim and Surette [22] continued to screen for the ability of
surface swarming among different Salmonella serovars and
have observed swarming motility in most of the strains
studied, including S. Typhi. They concluded that swarming
could represent an evolutionarily conserved behaviour in
Salmonella. However, in their study, only two strains of S.
Typhi were tested. Hence, in this study, we have extended
Kim and Surette’s work to look into both swimming and
swarming motility in more strains of S. Typhi of various
origins to determine if variation in motility, specifically sur-
face swarming exist in S. Typhi.
This work aims to provide an insight into the swim-
ming and swarming motility and biofilm forming ability
in S. Typhi. We have selected strains to represent va-
rious countries (Malaysia, Indonesia, Papua New Guinea
and Chile); years (from 1983 to 2008); and different dis-
ease manifestation origins (strains from stool or blood
samples of typhoid patients and asymptomatic human
carriers) to demonstrate all possible variations within
S. Typhi. The inclusion of S. Typhi strains from diverse
background will provide us with a better and more
truthful insight into the possible physiological variation
in this strict human pathogen.
Results and discussion
S. Typhi strains demonstrated both swarming and
swimming motility
The motility of 60 strains of S. Typhi was measured for
their surface swarming (growth in media with 0.5% agar)
and swimming (growth in media with 0.25% agar) ability.
All the strains were able to swarm across the agar
surface and formed featureless, smooth and flat colony
(Figure 1A). Featureless colonies are made when cells
spread evenly and continuously outward from the point
of inoculation, as a monolayer. The monolayer is trans-
parent but may be seen when incident light is reflected
off the surface or when oblique light is transmitted
through the agar. Cell density in the monolayer is high
and approximately uniform throughout the swarm, in-
creasing slightly at the advancing edge [23]. When the
monolayer reaches the boundary of the plate, the colony
grows into a featureless mat [24].
However, the swarming motility of the strains tested in
this study was weaker compared to the study of Kim and
Surette [22].
Figure 1 Swarming and swimming behaviour of Salmonella enterica serovar Typhi. Swarm medium is nutrient broth (0.5% agar) and swim
medium (0.25% agar) supplemented with glucose as carbon source (0.5% [wt/vol]). Uncolonised agar is black and bacteria biomass is white. All
images were captured after 24 hr at 37°C. Panel Ashows swarming motility. Panel Bshows swimming motility.
Kalai Chelvam et al. Gut Pathogens 2014, 6:2 Page 2 of 10
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In this study, all of the tested strains for swarm mo-
tility assay were measured more than 1.5 cm - 7.7 cm
after 24 hrs of incubation. Strains showing migration of
cells (increase in colony diameter) were considered as
positive swarming. Initially when this work was done,
the rates of the migration of bacteria from the point of
inoculation were measured at 0, 6, 12, 18 and 24 hr.
However in this study, at 0 hr, S. Typhi strains showed
no migration. At 6 hr and 12 hr, S. Typhi strains showed
similar rate of migration. After 18-24 hrs, most of the S.
Typhi strains colonised the entire surface of the petri
plate and reached the maximum size. Among the 60
strains tested, UJ308/98 demonstrated the most active
swarming motility. Interestingly, this strain was isolated
from blood specimen of a deceased typhoid patient in
Papua New Guinea, in which typhoid fever is highly en-
demic. In this study, we had also included S. Enteritidis
and S. Typhimurium as the control strains for motility
tests. Our results showed that both S. Enteritidis and
S. Typhimurium were able to swarm on the agar surface,
with S. Enteritidis showing more active swarming
motility than S. Typhimurium and S. Typhi. Both S.
Typhimurium and S. Typhi had comparable swarming
motility.
Most strains exhibited featureless swimming pattern
on the agar media (93.4%) while one S. Typhi strain
(ST02/08) showed bull’s eye (Figure 1B). Bull’s eye is a
typical swim pattern which is also known as zones of
consolidation terraces, caused by sequential rounds of
swarm cell differentiation and swarming colony migra-
tion [6]. The most studied bull’s eye pattern is formed by
P. mirabilis which differentiates into swarm cells that
are multinucleate, 20-50 fold elongated and express
thousands of flagella on a solid surface [25,26]. It is
likely that counter-clockwise or clockwise switching
patterns of the flagella motors influence the direction of
cell movement and hence the patterns. However, the re-
lationship of these patterns to the virulence mechanism
has not been studied in these cases.
Although a majority of the strains were highly motile in
less viscous media, 2 strains (ST319/87 and TP3/97) were
not able to swim. We re-confirmed this result with the
Sulfide-Indole-Motility (SIM) test tube assay and found
the same outcome. In fact, motility is an important bac-
terial virulence factor that aids in the gut colonisation to
initiate infection in human host. To confirm this finding,
two motility-associated and flagellin related genes, fliC
and flgK, were selected to test on all the strains. The fla-
gellar filament of S. enterica is approximately 10 μm long
and is comprised of two antigenically distinct flagellin pro-
teins, FliC (H:i) and FljB (H:1,2). Previous experiment
conducted by Crawford and co-workers [18] demon-
strated that flagellar subunit fliC is critical for binding to
cholesterol in serovar Typhimurium. On the other hand,
flgK gene functions as a hook filament junction. In this
work, these genes were studied to test for the presence of
these genes and whether it contributes to the motility of
S. Typhi. Both fliC and flgK genes were present in all the
strains tested. However, there are other important genes
for motility which were not studied in this work. More-
over, these 60 unique S. Typhi strains studied in this work
were previously subtyped by pulsed-field gel electropho-
resis (PFGE) and these strains were genetically different
(unpublished data). The result suggests that the loss of
swimming motility in these two strains (ST319/87 and
TP3/97) could be due to the loss of other motility related
genes or other factors which were not tested in this study.
In this study, we found a significant difference in the
swimming motility between blood-borne and stool-borne
S. Typhi strains (P < 0.05) (Figure 2). S. Typhi strains iso-
lated from the blood specimen of typhoid fever patients
exhibited higher swimming motility level than the strains
isolated from stool specimens. We did not observe any
significant difference in the surface swarming between
blood- and stool-borne strains (P > 0.05) (Figure 2). Inter-
estingly, in our previous study on the carbon catabolism
among S. Typhi strains [27], blood- and stool-borne
strains differed in their carbon catabolic profiles. These
findings suggested that blood-borne and stool-borne
strains may represent two different pathogenesis stages,
systemic invasion and persistence in the human host,
Figure 2 The Motility of Salmonella Typhi in Blood-borne and
Stool-Borne Strains. This figure shows comparison between blood-
borne, n =24 (white) and stool-borne, n =19 (gray) S. Typhi in swim
and swarm motility assays. Y-axis indicates the distance traveled (cm)
in motility assays. Vertical lines associated with histogram bars represent
standard error of the mean. * implies p< 0.05. There was a significant
difference observed only in swim motility assay with p< 0.05 for blo od-
borne strains compared to stool-borne S. Typhi strains. However, swarm
motility showed non-significant differences in both blood-borne and
stool-borne S. Typhi strains.
Kalai Chelvam et al. Gut Pathogens 2014, 6:2 Page 3 of 10
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respectively. While this finding suggests motility may play
a role in the systemic invasion of S. Typhi in the human
host, more in-depth study is needed to clarify this specula-
tion. From a biological viewpoint, such differences in mo-
tility raise the possibility that strains with high motility
may be more capable of swimming through the intestinal
mucus and replicate within macrophages and infected
phagocytes [28]. Replication of the bacteria within macro-
phages in the liver and spleen resulted in the release of the
pathogen in the bloodstream which later invades the gall-
bladder [29]. S. Typhi is then adapted and persisted in the
gallbladder, in which high motility is not necessary, and
later leads to bacterial shedding in the urine and faeces.
S. Typhi strains exhibited red, dry and rough (RDAR)
morphotype
The components of exopolysacharides (EPS) that have
been identified in Salmonella spp. biofilms include cellu-
lose, colanic acid, the Vi antigen, curli fimbriae, the O
antigen capsule and biofilm-associated proteins [30-32].
Multicellular phenotypes of S. Typhi strains studied in this
work were further characterised for the expression of curli
fimbriae on Congo Red Agar. Colony morphologies on
Congo Red plates were scored according to the basic mor-
photypes previously detected in S. Typhimurium [33]:
RDAR (violet colony, expresses curli fimbriae and cellu-
lose), PDAR (pink colony, expresses cellulose), BDAR
(brown colony, expresses curli fimbriae) and SAW (no ex-
pression of curli fimbriae nor cellulose). However, in our
work, we have observed only RDAR (Figure 3A and B) in
all 60 strains of S. Typhi originated from different coun-
tries, years and samples. There was no variation observed
in RDAR among S. Typhi strains from blood or stool.
RDAR morphotype is commonly observed in the other
Salmonella enterica including S. Typhimurium and S.
Enteritidis [34]. White and Surette [35] reported that all 7
Salmonella subspecies studied in their work expressed
RDAR morphotype which is a distinct, rough and dry
Figure 3 RDAR Morphotype in Salmonella Typhi. Morphotype of red, dry and rough (RDAR) colony which shows presence of curli and
cellulose in S. Typhi and S. Typhimurium after 7 days of cultivation at 37°C on Congo Red Agar. Panel A: Morphotype of S. Typhi. Left panel and
right panel are the front view and back view of S. Typhi RDAR colony morphotype on Congo Red Agar, respectively. Panel B: Morphotype of
S. Typhimurium as a control. Left panel and right panel are the front view and back view of S. Typhimurium RDAR colony morphotype on Congo
Red Agar, respectively.
Kalai Chelvam et al. Gut Pathogens 2014, 6:2 Page 4 of 10
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colony morphology formed by the extracellular interaction
of thin aggregative fimbriae (Tafi or curli), cellulose, and
other polysaccharides. In another study, Romling and co-
workers also observed RDAR morphotype in all Salmonella
serovars tested [36].
Fimbriae or curli plays a vital role in attachment of the
bacteria to the surface and gives a signal for initiation of
microcolony formation. Many bacterial pathogens use
subcellular surface appendages that radiate from the bac-
terial surface for initial adherence. Typical examples are
the bacterial pili (fimbriae) and flagella. In E. coli, muta-
tion in type 1 fimbriae had severe defect in initial attach-
ment [37]. S. Agona strains with BDAR morphotype was
found to be equally tolerant to disinfection treatment as
strains with RDAR morphotype in biofilm formation test.
Both BDAR and RDAR morphologies were good biofilm
producers, however, no statistical difference was found
between the two morphotypes [38]. The RDAR morph-
ology appeared to be favourable in long term survival in
biofilm in a very dry environment [38]. It has previously
been shown that the RDAR morphology is due to the
expression of both fimbriae and cellulose contributing to
a highly organised structure, and this organised structure
is disrupted at the loss of one of these components
[34,39,40]. Our hypothesis is that these structures might
be of importance for the long term survival of Salmonella.
S. Typhi strains demonstrated variations in biofilm
formation ability
It has been reported that motility is required for both bio-
film formation and pathogenesis [41]. Therefore, we ex-
amined the in-vitro biofilm-forming ability of the 60 S.
Typhi strains using crystal violet assay in 96-well microti-
tre plate. We observed a wide variation in the quantity of
biofilm biomass produced amongst the strains. Approxi-
mately one third (of the S. Typhi strains tested (n =20)
were not able to adhere to the plastic wells of the 96-well
microtitre plate, indicating inability to form biofilm
in-vitro; another one third of the strains (n =21) were only
able to produce weak biofilm in the in-vitro assay; 12
strains (20%) were moderate biofilm producers; and only 7
strains strongly adhered to the inner walls of the plastic
wells, representing potentially strong biofilm producers
(A
590
nm > 1.3). Although almost two thirds of the strains
tested were non- or weak biofilm producers, these strains
may still be important during polymicrobial infections
where they can directly be incorporated into an es-
tablished biofilm or interact with other species providing
synergy to the non-biofilm formers. All the 7 strong bio-
film formers were highly motile, in which, 5 S.Typhi
strains originated from Malaysia, 1 from Indonesia and
1 from Chile. Only 1 carrier strain CR0063/07 from
Malaysia was recorded to have strong biofilm forming
ability and the other 6 S. Typhi strains were isolated from
blood and environmental samples. Asymptomatic carriers
of S. Typhi periodically shed large numbers of this bacter-
ial pathogen in their stools (showers of S. Typhi). Because
of the hallmark showers of S. Typhi, carrier identification
requires collection and culture of multiple faecal samples
over the period of at least 1 year. Due to this difficulty in
isolation, we had only 2 human carrier strains among the
60 S. Typhi strains studied. In one of the previous studies,
P. aeruginosa strains with a mutation in type IV fimbriae
did not form a densely packed biofilm. The type IV fim-
briae appear to play a role in full biofilm formation by the
bacteria [42]. These data could suggest that fimbriae are
needed during stages of intestinal and gallbladder infec-
tion of S. Typhi but specific environmental signals such as
bile and other factors may play an important role in their
regulation and therefore it affects the level of biofilm
forming ability.
In our work, when we plotted the in-vitro biofilm form-
ing ability against motility of the 60 S. Typhi strains tested,
we did not observe any negative relationship between bac-
terial biofilm forming ability and motility as reported in
other publications [43,44]. However, when we plotted the
graph separately for blood-borne (n =24) and stool-borne
(n =19) S. Typhi strains, a negative trend was observed
only in the stool-borne strains, but not in the blood-borne
S. Typhi strains. In most of the published studies, those
non-Salmonella bacterial strains were isolated from the
environment (e.g. soil, water) [45,46]. In this study, a simi-
lar negative trend was observed for environmental strains
in which highly motile S. Typhi strains were either weak
or moderate biofilm formers. The Chilean strains that
were studied were from clinical and environmental
sources. However, there was no difference observed bet-
ween the clinical and environmental strains. These clinical
strains were from stools and they behave similarly as the
environmental strains. But only one Chilean strain from
blood was able to express high motility compared to other
Chilean strains. The results also indicated that the blood-
borne S. Typhi strains were able to swim better as com-
pared to those from stool samples (F = 9.026; P = 0.005)
(Figure 4).
To explain the negative trend between motility data
and biofilm, we found a study conducted by Crawford
and co-workers [18]. They demonstrated that mutations
in serovar Typhimurium flagellum structural and biosyn-
thesis genes affected binding and biofilm formation on
cholesterol. To determine if the physical presence of the
flagellar filament or flagellum-mediated motility was re-
quired for biofilm formation, mutants that expressed fla-
gella but could not swim were tested. In the tube biofilm
assay, a serovar Typhimurium motA mutation (which
eliminates flagellar motility but not synthesis) did not re-
duce the levels of biofilm on cholesterol surfaces in the
presence or absence of bile compared to the results
Kalai Chelvam et al. Gut Pathogens 2014, 6:2 Page 5 of 10
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obtained for the parent strain, suggesting that motility is
not critical for development of serovar Typhimurium
biofilms on cholesterol coated surfaces. However, in the
same study, to examine whether production of the flagel-
lar filament is necessary for biofilm formation on choles-
terol, a mutation in the gene at the apex of transcriptional
regulation (flhC) in serovar Typhimurium was created.
The resulting mutant strain did not form a mature biofilm
on cholesterol, providing direct evidence of the impor-
tance of flagella during biofilm development. Therefore,
motility provides various contributions to biofilm for-
mation in members of the Enterobacteriaceae, such as
V. cholerae, E. coli and P. aeruginosa, depending on the
environmental conditions, such as binding substrate ma-
terial, nutrient limitation, temperature, medium flow rate,
and other factors.
According to previous studies [24,47,48], a lag period
of non-motile behaviour precedes the initiation of swar-
ming motility when bacteria are transferred from a
liquid medium to a solid surface. The swarming lag is
constant for a particular set of conditions but may be
shortened and therefore some strains from blood-borne
origin are able to swim well compared to strains from
stool origin. Although, the swarming lag is poorly under-
stood, its occurrence indicates that swimming cells must
go through a change to become swarming proficient.
The difference in the surface swarming ability between
both origins was not statistically significant (F = 3.958;
P > 0.05). It was obvious from the result that blood-
borne S. Typhi strains could swarm better than the
stool-borne strains (Figure 4). However, we did not ob-
serve any difference in the in-vitro biofilm forming
ability between blood- and stool-borne S. Typhi strains
(Figure 4).
The differences observed between blood- and stool-
borne S. Typhi strains in this study suggest that S. Typhi
strains demonstrated different physiology during the inva-
sive infection stage (blood-borne) and acute infection stage
(stool-borne). This observation was indeed intriguing, as
S. Typhi strains isolated from stool and blood actually rep-
resent two different stages in human infection and colo-
nisation niches within the human body. S. Typhi is able to
invade the intestinal wall and replicate within macrophages
and infected phagocytes [49]. The replication of the bac-
teria within macrophages in the liver and spleen resulted in
the release of the pathogen into the bloodstream [29,50].
The pathogen later invades the gallbladder and leads to
bacterial shedding in urine and faeces in the chronic
carriage of infected individuals [51,52]. It is possible that
S. Typhi acquires different metabolic activity and pheno-
types for colonisation and persistence in these two different
niches, liver and spleen, which then disseminate S. Typhi
into the blood stream and the gallbladder (which S. Typhi
is then released in the urine and faeces), respectively.
Figure 4 Blood-borne and stool-borne Salmonella Typhi in swim and swarm motility under different biofilm formation category. Biofilm
category is represented by the different colour codes on bar charts. Y-axis indicates the distance traveled (cm) in swarm and swim motility assays.
Vertical lines associated with histogram bars represent standard error of the mean. There was a significant difference observed only in swim
motility assay under strong biofilm category (* implies p< 0.05) for blood-borne strains.
Kalai Chelvam et al. Gut Pathogens 2014, 6:2 Page 6 of 10
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In this study, we selected two strains, one of human car-
rier origin (stool-borne; CR0063/07) while another one of
outbreak origin (blood-borne; BL196/05) to compare their
biofilm structure and architecture using the scanning elec-
tron microscopy (SEM). The bacterial biofilm was allowed
to grow and form on the peg lids of a 96-well plate as
described previously by Harrison et al. [53]. Only the car-
rier strain (CR0063/07) was capable of forming mature,
robust biofilms on the polystyrene peg's surface. Bacterial
cells of S. Typhi adhered to each other and were encased
in an extracellular matrix in the biofilm formed by the car-
rier strain; whereas there was no biofilm formation in the
outbreak strain, extracellular matrix was not observed too
(Figure 5). Could this observation suggest a possible per-
sistence strategy of the carrier strain in the human host?
More in depth studies are needed to answer this interest-
ing question.
Conclusion and future directions
In summary, both swimming and swarming motility are
conserved among S. Typhi strains but there was variation
for biofilm forming ability. There was no difference observed
in this phenotype for S. Typhi strains from diverse back-
ground. These findings serve as caveats for future studies to
understand the lifestyle and transmission of this pathogen.
Materials and methods
Bacterial strains
Atotalof60S. Typhi strains previously characterised
were studied [54-56]. The strains were isolated from
various sources including blood and stool samples from
typhoid patients, asymptomatic human carrier and sew-
age-contaminated river water that were collected from
Malaysia, Indonesia, Papua New Guinea and Chile, from
1983 to 2008. The strains were retrieved from - 80°C stock
cultures, and reconfirmed as S. Typhi using an in-house
PCR assay. Serotyping was previously done by the
Salmonella Reference Centre at the Institute for Medical
Research, Malaysia.
Swarming and swimming capability
To test on motility, a sterile needle was used to lightly
touch an overnight S. Typhi culture and spotted gently
in the middle of a swarm plate (Nutrient Broth [NB],
0.5% [wt/vol] glucose, 0.5% bacteriological agar) or a
swim plate (NB, 0.5% glucose, 0.25% bacteriological
agar). The plates were incubated at 37°C for 24 hr. The
rates of motility were measured and patterns of swarming
and swimming on the agar plate were determined accord-
ing to Kim and Surette [22]. Rates of the migration of bac-
teria from the point of inoculation (observed as a turbid
Figure 5 SEM micrographs of S. Typhi on a 96-well polystyrene peg's surface. Panel Ashows SEM micrographs of a human carrier strain
(CR0063/07), embedded in biofilms established on the polystyrene peg's surface at magnifications of 10,000x and 25,000x. Panel Bshows SEM
micrographs of an outbreak S. Typhi strain (BL196/05). Biofilm formation was not detected on the polystyrene peg's surface at magnifications of
11,000x and 17,000x.
Kalai Chelvam et al. Gut Pathogens 2014, 6:2 Page 7 of 10
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zone in centimetres) were measured at 6, 12, 18 and 24 hrs.
The results are the means of at least 3 independent
experiments.
Biofilm formation of S. Typhi in 96-well microtiter plates
To check for biofilm forming ability, the microtitre plate
assay used in this study was adapted from O'Toole and
Kolter [57], with some modifications. S. Typhi cultures
were grown in Luria Bertani (LB) broth until mid-log
phase. Each strain was then inoculated into 8 wells of
96-well microtitre plate and incubated for 48 hrs. After
incubation, unbound cells were removed by inversion of
microtiter plate, followed by vigorous tapping on absor-
bent paper. Subsequently, adhered cells were heat fixed in
an oven for 30 min at 80°C. Adhered cells were stained by
addition of 220 μl of crystal violet (0.5%) for 1 min. The
stain was removed by thorough washing with distilled
water. In order to quantify adhered cells, 220 μl of deco-
louring solution (ethanol/acetone, 80:20%) was added to
each well for 15 min. The absorption of the eluted stain
was measured at 590 nm wavelength. Based on the
O.D
590 nm
, strains were classified into the following cat-
egories: no biofilm producer, weak, moderate or strong
biofilm producer, as previously described by Stepanović
and Ceri et al. [58,59].Briefly,thecut-offO.D.(O.D.c)
was defined as three standard deviations above the
mean O.D. of the negative control. Strains were clas-
sified as follows: O.D. ≤O.D.c = no biofilm producer,
O.D.c < O.D. ≤(2 × O.D.c) = weak biofilm producer,
(2 × O.D.c) < O.D. ≤(4 × O.D.c) = moderate biofilm pro-
ducer and (4 × O.D.c) < O.D. = strong biofilm producer.
The negative control wells contained nutrient broth only.
Negative control wells remained negative. The negative
control has been deducted from the OD readings. All tests
were performed at least three independent times to ensure
reproducibility. Replicates for each test were conducted to
check for repeatability.
Curli and cellulose detection in S. Typhi
To substantiate the findings of multicellular phenotypes,
colonial morphology of S. Typhi bacteria was studied on
LB
w
[1.0 g Tryptone, 1% agar, distilled water to 100 mL
without salt supplement containing Congo red (40 μgml
-1
)/
Coomassie brilliant blue (20 μgml
-1
)] (Sigma Chemicals,
St Louis, MO, USA) was used to determine colony mor-
phology and colour [36]. S. Typhimurium was used as a
control on Congo Red Agar to test for RDAR morphotype.
Primer design and PCR assay
Flagella-associated genes, fliC and flgK were selected to
confirm the motility in all 60 S. Typhi strains. Oligonucleo-
tide primers specific for each target gene were selected
using PrimerSelect (DNASTAR; Lasergene, Madison, WI).
Selected primer pairs were then tested using in silico with
the PCR amplification program (http://insilico.ehu.es/.25).
The sequences of the selected primer used were fliC
(5'-AAT CAA CAA CAA CCT GCA GCG- 3') and (5'-
GCA TAG CCA CCA TCA ATA ACC-3'); flgK (5'- CAA
CAA TTA CGC GAA GCA GA -3') and (3'- TAT AAT
CCG TCG CCT GAA CC -5') with amplicon size 704 bp
and 584 bp, respectively. The PCR amplifications were car-
ried out in a Master cycler (Eppendorf, USA). The PCR
mixture in a total volume of 25 μl contained 50 ng of
genomic DNA template, 1X PCR buffer, 2 mM MgCl
2
,
200 μM of each dNTP, 0.3 μM of each primer, and 0.5U of
Taq DNA polymerase (Promega, USA). The cycling con-
ditions were set at 95°C for 5 minutes (1 cycle), 95°C for
30 s, 55°C for 30 s, 72°C for 1 minute (30 cycles), and
72°C for 8 minutes (1 cycle). The products were then
analysed on 1.5% (w/v) agarose gel and run at 100 V for
25 minutes and stained in GelRed. Gel images were cap-
tured and analysed using Gel Doc XR (Bio Rad, USA). Se-
lected amplified DNA products were verified by DNA
sequencing.
Scanning electron microscopy analysis of carrier and
outbreak S. Typhi
To determine the architecture of biofilm producers, the
Scanning Electron Microscopy (SEM) was conducted on
an outbreak and carrier S. Typhi strains, both of which
demonstrated significant biofilm formation. Bacterial bio-
films were grown on polystyrene pegs (Nunc-TSP; Nunc)
for 48 hr [60]. Briefly, following incubation, the pegs were
rinsed with 1X PBS and removed using sterile needle-
tipped pliers. Each peg was then fixed with 2% (w/v) glu-
taraldehyde, 2% (w/v) paraformaldehyde, 0.15 M sodium
cacodylate, 0.15% (w/v) alcian blue for 3 hrs at room
temperature. Pegs were then rinsed three times with 0.15
M sodium cacodylate buffer, immersed in 1% (v/v) os-
mium tetroxide in sodium cacodylate and incubated for 1
hr at room temperature. Pegs were then rinsed three times
with distilled water followed by a stepwise dehydration
with graded ethanol-water mixtures. Samples were then
treated with hexamethyldisilizane for 5 min prior to crit-
ical point drying. Next day, samples were sputter coated
with gold and viewed by SEM. SEM experiments were
carried out in duplicate for each strain tested, and repre-
sentative images of biofilms were selected.
Statistical analysis
Statistical analyses were performed using t-test for inde-
pendent samples. All the experiments were repeated at least
three times. The level of significance was set at P<0.05.
Microsoft Excel and SPSS 18 were used for analysis.
Competing interests
The author’s declare that they have no competing interests.
Kalai Chelvam et al. Gut Pathogens 2014, 6:2 Page 8 of 10
http://www.gutpathogens.com/content/6/1/2
Authors’contributions
KK performed the motility assays, biofilm assays and SEM assays on
Salmonella Typhi, analysed the data, performed statistical analyses and
drafted the manuscript. LCC supervised all experimental work, analysed the
data, and drafted the manuscript. KLT conceived the study, supervised all
experimental work, analysed the data and drafted the manuscript. All
authors read and approved the final manuscript.
Acknowledgements
We thank University of Malaya for the support and facilities. This study was
funded by the High Impact Research Grant UM.C/625/1/HIR/MOHE/02 and
Molecular Characterisation of Bacterial Pathogen Grant (53-02-03-1096) from
University of Malaya. Kalaivani, K is supported by University of Malaya
Fellowship (SBUM).
Received: 16 November 2013 Accepted: 28 January 2014
Published: 5 February 2014
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doi:10.1186/1757-4749-6-2
Cite this article as: Kalai Chelvam et al.:Variations in motility and biofilm
formation of Salmonella enterica serovar Typhi. Gut Pathogens 2014 6:2.
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