Volume 7, Issue 6, June – 2022 International Journal of Innovative Science and Research Technology

ISSN No:-2456-2165

A Comprehensive Review on the Development of

Salmonella Biofilm on Gallbladder Surface
Soumyashreeta Nayak Kankana Ray Chaudhuri
Biotechnology Biotechnology
Kalinga Institute of Industrial Technology Kalinga Institute of Industrial Technology
Bhubaneswar, Odisha, India Bhubaneswar, Odisha, India

Shinjini Dasgupta
Biotechnology
Kalinga Institute of Industrial Technology
Bhubaneswar, Odisha, India

Abstract:- Salmonella is a group of rod-shaped bacteria that get implicated in many chronic infections and are known
belonging to the family Enterobacteriaceae. They are to ease the process of bacterial persistence by increasing
gram-negative and facultatively anaerobic. These bacteria resistance against the microbes and interfering with the host
reside mainly in the intestinal tract of humans and other immune response [5,6,8,9]. Biofilms are encased within a
animals. The pathogen, Salmonella enterica serovar Typhi mixture of secreted and cell wall-associated poly-saccharides,
primarily causes typhoid fever, a disease specific to glycoproteins, and glycolipids, as well as extracellular DNA,
humans. Salmonella can exist in the human gallbladder in known collectively as extracellular polymeric substances (EPS)
an asymptomatic carrier state. Salmonella species have the [5,7]. Biofilms are frequently associated with implanted
capability to form biofilms. The production of biofilms devices, such as catheters, prosthetics, and contact lenses [8,9].
serves as an advantage because bacteria in the biofilm are For patients with gallstones, antibiotic treatment against
resistant to chemical, physical, and mechanical stresses as Salmonella generally becomes ineffective and elimination of
well as disinfectants. Biofilm formation also assists in gallbladder infection in these individuals usually requires
Salmonella virulence. It is because the bacterial biofilm surgery and gallstone removal [10]. In addition to this, biofilm
serves as a resistant barrier to antibiotics and immune production by S. Typhi may be regarded as a key factor for the
attacks by the host. This results in a chronic infection promotion of persistent infection in the gallbladder, thus
accompanied by the development of Salmonella carrier enduring a chronic local inflammatory response and exposing
state. In this review, we present a comprehensive overview the epithelium to repeated damage caused by carcinogenic
of Salmonella biofilm formation, factors affecting biofilm toxins.
formation, complications arising from biofilm formation,
and available treatments for biofilm-mediated infections. Salmonella biofilm formation depends on certain genes,
environmental factors, the presence of flagella, fimbriae, bile,
Keywords:- Salmonella; Biofilm Formation; Gall Bladder; and quorum sensing. Quorum sensing is being used in many
Typhoid; Antibiotic Resistance; Factors; Complication; different bacteria as a mechanism for cell signaling based on
Treatment. cell density which is thought to regulate a variety of processes,
such as conjugation, virulence, motility, and biofilm formation
I. INTRODUCTION [11,12,13,14,15,16].

Salmonella enterica serovar Typhimurium is a primary According to the Centre for Disease Control and
enteric pathogen that infects both humans and animals [1]. Prevention (CDC) Salmonella bacteria causes about 1.35
Salmonella Typhimurium and Typhi are the best-characterized million infections, 26,500 hospitalizations, and 420 deaths in
serovars. It is seen that S. Typhimurium is involved in the United States every year. Even with the use of adequate
localized gastroenteritis in many hosts while S. Typhi causes a antibiotic therapy typhoid records a mortality rate of (2-3) %.
systemic human-specific disease [2]. These are a diverse group Controlled human infection models (CHIMs) have played an
of pathogens that have evolved themselves to survive in a wide important role in accelerating the development of conjugate
range of environments and across multiple hosts [3]. Although vaccines against Salmonella Typhi [17].
non-typhoidal Salmonella mainly causes gastroenteritis,
typhoidal serovars (S. Typhi and S. Paratyphi A) are known to The recent evolution of S. Typhi, which is a multidrug-
cause typhoid fever, the treatment of which is threatened due resistant (MDR) strain has evolved as a notable problem for
to increasing drug resistance of the pathogen [4]. People patients as low-priced and readily available antibiotics like
suffering from typhoid develop diarrhea, fever, and abdominal streptomycin, chloramphenical, ampicillin and trimethoprin-
cramps within 8 to 72 hours. sulfamethoxazole are often unproductive against them [18,19].
Although hostility to ciprofloxacin, which is a second-
S. Typhi and S. Typhimurium are capable of forming generation fluoroquinolone is increasing, medical
bacterial biofilms in mammalian and/or environmental niches. practitioners still recommend it as first-line therapy for
Biofilms are mainly aggregated mixtures of sessile bacteria children and adults [20,21,22].

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Epidemiological studies in regions where S. Typhi is Formation of Salmonella biofilm depends on the action
endemic revealed that the greater part of chronically infected of certain genes. Moreover, it has been seen that the presence
carriers also harbor gallstones, which in turn, have been of flagella, fimbriae, and bile can affect biofilm formation.
indicated as a primary predisposing factor for the onset of Quorum sensing is another mechanism that favors microbial
gallbladder cancer. But now, it is well recognized that S. Typhi interactions and regulation of population-level virulence of
strain produces a typhoid toxin with a carcinogenic potential bacteria.
that induces DNA damage and cell cycle alterations in
intoxicated cells.  Genes involved in biofilm regulation
In stressful conditions, S. Typhi can use the biofilm
II. BIOFILM FORMATION ON THE GALL phenotype in order to increase the production of the membrane
BLADDER SURFACE matrix, protect and adhere to the surface, and enter an energy-
conserving state [29].
In humans, Salmonella enterica serovar Typhi can cause
an asymptomatic and persistent infection of the gallbladder, The galE gene is crucial for the formation of wild-type
indicating that this strain uses novel mechanisms to increase biofilms. galE encodes for a uridine diphosphogalactose-4-
colonization in bile-rich environments. Gallstones are among epimerase, which is a structural gene needed for the synthesis
the most dangerous threats to growing carriage, and formerly of galactose, that is added to both the outer core and the O-
in some of the studies, it has been confirmed in vitro that there antigen. This gene mutation results in a lipopolysaccharide
are Salmonellae-shaped biofilms on gallstones. As a result, we (LPS) that lacks all sugars beyond the heptose region of its
can infer that gallstone surfaces formed by bile-induced internal core (thus producing a tough or incomplete LPS) [25].
biofilms are colonized by gallbladder bacteria [23]. Gallstones,
in particular, are commonly associated with chronic typhoid Gene STY0893, a bssR gene involved in biofilm
carriage when they develop in the gallbladder [24]. regulation, is significantly downregulated within the biofilm
cells. In a study, researchers compared the bssR gene in E. coli
The organism infects the intestinal epithelium, invades and Salmonella. The bssR gene, also called yliH in E. coli cells,
macrophages, and spreads throughout the body [25]. The becomes upregulated during biofilm formation [30]. The
organism may colonize the liver and then shed into the transcriptome study on S. Typhi biofilm cells, however,
gallbladder, where it chronically colonizes the gallbladder confirmed that the BssR gene (STY0893) was markedly
wall [26,10]. The persistent colonization of S. Typhi with downregulated compared to planktonic cells. The BLAST
gallstones and gallbladder cancer is associated with persistent analysis of E. coli K-12 and S. Typhi CT18 showed that the
colonization of these organisms, but it is not known whether gene bssR is found in both species, although it has only 75%
this organism is a causal factor in gallstone formation or nucleotide similarity. Additionally, since the base pair length
whether gallstones promote persistent colonization. Several of both species differs, it is likely that both proteins are unique
recent studies indicate that S. Typhi forms bacterial biofilms [30].
on cholesterol gallstones. It is possible that this biofilm
formation could allow persistent colonization and protect the The gene which codes for biofilm stress and motility
organism as the organism is treated with antibiotics. The protein A is Yjfo gene. According to a study conducted on E.
formation of biofilms requires the presence of bile, as coli, yjfO was related to cellular survival based on the
organisms not cultured with bile would not form biofilms [26]. formation of biofilms in response to peroxide pressure [31].
Comparison of the outcomes of the study using statistics
In Salmonella carriers, the growth of a biofilm may shows that S. Typhi has an exclusive feature for the gene yjfO
protect microorganisms from antimicrobial agents and because the gene becomes downregulated in S. Typhi during
excessive levels of bile for an extended period of time. Bile biofilm formation. The yjfO gene is also responsible for
acids are amphipathic molecules, surface active, and are a kind microcolony formation. It is also possible that the gene
of detergent. As a detergent, bile acids have strong transcription rate can become suppressed in mature biofilm
antimicrobial activity [27]. The absence of bacterial biofilm on tissues.
S. Typhi gallstones suggests that S. Typhi followed a similar
strategy, by forming a biofilm on the gallstone surface,  Flagella
enabling it to persist inside the gallbladder for many years and The presence of flagella has been proven to be very crucial
assist in shedding and reattachment, followed by diffusion to biofilm formation, especially during the early stages when
through urine and feces [28]. A number of studies have shown microcolonies are being formed. In order to propel
that people with gallstones who are infected with S. enterica microorganisms throughout the surface, flagella are required
serovar Typhi are much more likely to develop the infection to move them to the surface, for attachment, and to propel
and become carriers than people without gallstones, who do them to find different microorganisms. Some research has
not have obvious gallbladder abnormalities [28,7]. This been conducted to determine if flagella play a part in
indicates that gallstones are involved in chronic disease Salmonella biofilm formation on gallstones. It was found that
development. Gallstones are strong and stable surfaces and can a mutant showed a severe defect in the ability to form flagella
act as a firmly attached organism that can avoid being washed (and therefore non-motile) which were analyzed using
away into the surrounding tissues due to the continuous a scanning electron microscope (SEM). After 14 days of
emptying of the gallbladder. growth, a weak biofilm was observed. However, the

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phenotype differed significantly from that of the S. regulator which is very crucial for biofilm production and it
enterica serovar Typhimurium wild kind [25]. also regulates the csgBAC operon, cellulose, and the BapA
protein [44,45].
Researchers have demonstrated that motility is crucial to
biofilm development on glass using the motA and fliA mutants  Quorum sensing
of S. Typhimurium [25,32]. However, it has been defined that Cell-cell communication is achieved by a process known
Salmonella mutants with defective flagella (flhC or flgE) as quorum sensing, which depends on gene expression in
cannot form complete biofilms in the presence of bile [33,34]. response to population cell density. A quorum-sensing signal
Earlier, works involving QseBC two-component system (TCS) is activated in Salmonella biofilms, and this signal triggers the
functioning in S. Typhimurium found out that QseBC plays a maturation and disassembly of the biofilm in an organised
role in flagella formation, biofilm formation, and virulence manner. As part of quorum sensing, acylated homoserine
mechanism [35,36,37]. Recent research has proven that the lactone signaling molecules are produced, released, and
cyclic di-guanylate monophosphate (c-di-GMP) receptor detected in the environment. These molecules are known as
YcgR and the phosphodiesterase YhjH can distinctively autoinducers [46]. Several studies have confirmed that S.
inhibit flagellar motility in S. Typhimurium [38]. Therefore, enterica serovar Typhimurium has a homologue for luxS, an
flagellar motility plays a very important role in the autoinducer-2 gene [15,16]. The autoinducer has been
development of Salmonella biofilm. implicated in supporting the transformation of pathogenic
bacteria to free-living bacteria within the surrounding
Salmonella flagella can also additionally play a significant environment [15].
role in EPS secretion or production. In addition, it also helps
in establishing primary adhesion and microcolony formation  Bile
(aided by using bacterial movement throughout the surface) on The gall bladder is responsible for storing bile in the
gallstones [25]. human body. It has been hypothesized that bile can also be
used as a signal to induce biofilm formation. In a mouse model
 Fimbriae study, gallstones had been incubated within the presence of LB
Fimbriae play a role in the adhesion of the microorganism broth alone and LB broth containing 3% bile. The presence of
to different microorganisms and to the surface of the bile facilitates complete biofilm formation on gallstones after
gallbladder. A fimbria-mediated attachment on the surface 14 days in S. enterica serovar Typhimurium and S. enterica
indicates the initiation of a microcolony. The fimbriae of serovar Typhi cultures, while the absence of bile prevents the
different organisms participate in the formation of biofilms as formation of biofilm [25]. Thus, Salmonella biofilm formation
well. Various S. enterica serovar Typhimurium fimbrial on gallstones is dependent on bile within the culture medium.
mutants have been tested in association with gallstones and Bile is necessary for biofilm formation on gallstones in 14
strains with individual mutations in four fimbrial operons (fim, days, so it is possible that bile gives the signal to the
agf, lpf, and pef). In addition, a strain wherein all these four microorganism to form biofilm and makes the surfaces of the
mutations were combined and tested for gallstone biofilm gallstones smooth, making adhesion easier [10].
formation [39]. The fim operon encodes a type I fimbria that is
peritrichous and agf operon encodes a thin aggregative fimbria III. ENVIRONMENTAL FACTORS AFFECTING
which is also peritrichous in nature. A lpf operon encodes polar SALMONELLA BIOFILM FORMATION
fimbriae, which are essential for virulence, while a pef operon
is on a plasmid and is needed for adhesion to organ surfaces Biofilm formation is a complicated process that begins
[25]. with the initial adherence of bacterial cells to the substratum,
leading to physiological changes within the microbe and
The DT104 strain of S. enterica serovar Typhimurium multiplication of attached cells to form microcolonies, and
exhibits a rugose phenotype on certain media, which gives finally leads to maturation of the biofilm [47]. Bacteria that
prominence to its biofilm-forming ability. SR11 strain shows have the ability to form biofilms present distinct features as
a rugose phenotype, but it overproduces an EPS that allows compared to their planktonic counterparts which are free-
rapid biofilm formation. living, such as different physiology and high resistance to the
immune system and antibiotics. Hence, biofilms serve as a key
A biofilm matrix of Salmonella is mainly composed of factor in chronic and persistent infections [48]. It has been seen
curli fimbriae, the O-antigen capsule, cellulose, colanic acid, that the change of phenotype from planktonic to the sessile
vi-antigen, and biofilm-related proteins [40,41]. The essential form occurs when there are changes in environmental
protein element is curli fimbria, which is crucial for biofilm conditions [49]. Nutrient level, temperature, pH, oxygen
formation and it plays an important role in virulence and concentration and osmolarity are the key environmental
alterations to the immune system [42,43]. Curli fibers are factors that can affect biofilm formation through several
much like eukaryotic amyloid fibers and are involved in pathways [50]. In order to obtain the best nutrients to survive
molecular aggregation and adhesion. Curli genes are and reproduce, mature biofilms change with environmental
organized in divergent curli-particular gene (csg) operons with conditions [51].
independent promoters: one consists of the structural
components CsgA and CsgB (csgBAC), and the second
consists of the CsgD, which acts as regulator and different
structural proteins (csgDEFG). CsgD is a transcriptional

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 pH glucose has been reported to slow down the biofilm formation
The environmental pH can highly influence the formation in Salmonella. It is suspected that glucose inhibits a
of biofilms [50]. The first step in biofilm formation is component required in the early phase of bacterial adhesion
microbial adhesion to surfaces. It has been seen that this step [64].
is influenced by environmental pH [52,53]. pH is considered
as an important factor in the initial adhesion to surfaces in  Osmolarity
bacteria, such as Staphylococcus epidermidis [54]. One study Bacteria are exposed to osmolarity which is one of the
showed that the optimal pH for Salmonella biofilm formation abiotic stresses. In Salmonella species, biofilm formation is
is pH 7.0. However, biofilm formation was significantly inhibited when there is an increase in NaCl concentrations
reduced at pH 10.0. Salmonella biofilm formation is inhibited [65]. In a study, it was found that when the growth medium
at alkaline pH however most cells can survive the alkaline was supplemented with a low concentration of sodium
stress [55]. chloride (0.5 to 2%), biofilm formation was slightly enhanced
for the strain S. enterica serovar Enteritidis 110. However,
 Temperature biofilm formation by the strain S. enterica serovar Newport
The formation of biofilm is also affected by temperature. 193 was sharply reduced or abolished [66].
Optimum temperature can enhance bacterial growth and assist
in the rapid formation of biofilm. However, when optimum IV. COMPLICATIONS ARISING FROM BIOFILM
temperature is not present, bacterial growth can be decreased, FORMATION ON GALL BLADDER SURFACE
due to a reduction in reaction rates, and as a result, the biofilm
development might be affected [56]. The biofilm formation Salmonella typhi forms biofilm on cholesterol gallstones
of V.parahaemolyticus is temperature-dependent. It has been which is surface-specific and bile dependent, but this stage
seen that a higher temperature range (15°C to 37°C) gives rise results in a carrier state. Gallstones are one of the most critical
to stronger biofilm formation, but a lower temperature (4°C or risk elements for growing carriage states. Once the carrier state
10°C) induces the monolayer adherance of bacterial cells [57]. is formed, biofilm is tough to treat [23,67].
In one paper, among the 243 analyzed Salmonella strains, 224
(92.2 %) were able to produce biofilm in atleast one of the Most biofilm infections are persistent, as biofilm-residing
tested temperature conditions, but to various extents. It was microorganisms may be resistant to the immune system,
seen that there is a strong influence of temperature on biofilm antibiotics, and various treatments. During their dormant
production as strains showed different behaviors at different phase, bacteria can infect nearby tissues, which can later cause
incubation temperatures [58]. acute infection. The microorganisms within the biofilm adapt
to environmental anoxia and nutrient deficiency by changing
 Oxygen Concentration their metabolism, gene expression, and protein production,
Oxygen availability can also affect biofilm formation as it resulting in a reduced metabolic rate and slowing down the
determines bacterial energy production. If there is a production molecular division process [68].
of microenvironments within biofilms, such as reduced
oxygen zones or restricted nutrient diffusion through the Biofilms might additionally have a major role to play in
biofilm, it leads to slow growth of the bacteria. Deficiency in the colonization and chronic persistence of S. Typhi. This
oxygen and nutrient within biofilms often results in a decrease notion is supported using numerous reviews and researches
in bacterial metabolic activity and termination of bacterial that bile, a lipid-rich, detergent-like digestive secretion with
growth [59]. Hence, this state does not provide sufficient antimicrobial characteristics contained within the gallbladder,
energy to maintain cell attachment. As a result, detachment induces an exopolysaccharide matrix O-antigen production
occurs. For example, the presence of oxygen is required for the that allows S. Typhi biofilm formation on human gallstones
formation of biofilm in some microbes, such as E. coli. The [23,69]. Gallstones can block the tubes (ducts) via which bile
lack of oxygen can act as a signal for detachment for the cells flows out of the gallbladder or liver into the gut. As a result,
[60]. Another study demonstrated that the biofilm formation severe pain, jaundice, and bile duct contamination can also
of Salmonella on stainless steel is highly influenced by oxygen occur.
levels [61].
Biofilm embedded gallstones might also additionally
 Nutrient Level constitute favorable surroundings for bacterial persistence
It has been seen that bacterial attachment to surfaces is also within the gallbladder and might result in reseeding of the gut
influenced by the availability of nutrients in the surrounding bacteria and fecal shedding, followed by transmission to a new
medium; thus, an increase in nutrient concentration increases host and exposing the gallbladder epithelium to bacterial
the microbial attachment rate. Characteristics like biofilm factors potentially carcinogenic for prolonged periods of time
morphology, biomass, thickness, composition and activity and [70]. Different products like bacterial glucuronidase and
are determined by nutrient availability in the environment. It nitroso compounds are released by S. Typhi, which have the
was shown in one experiment that microbes produced less capacity to promote carcinogenesis [71].
dense biofilms with lower biomass content in batch mode
compared to biofilms formed in continuous mode with
constant supply of nutrients [62]. In some microbes such as E.
coli, biofilm formation is enhanced when glucose is added as
a carbon source to the medium [63]. However, the presence of

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V. TREATMENT

 Using small molecules of JG-1 and M4

JG-1 and M4 are two compounds that are found to show
effective anti-biofilm activity against Salmonella. S. Typhi
resides in humans and does not colonize mice and S.
Typhimurium, which is a related species is used as a model for
typhoid infection in mice. In a study, the abilities of JG-1 and
M4 was observed using both S. Typhi and S. Typhimurium
grown in a particular media. In S. Typhimurium both
compounds showed similar inhibiting capacity however in S.
Typhi, biofilms were inhibited more efficiently by M4 than
JG-1. In order to detect the anti-biofilm effects of M4 and JG-
1 and whether they could be accredited as bacteriostatic or
bactericidal in nature, the liquid cultures of S. Typhimurium
were incubated. This was done in the presence of a vehicle or
a low/high concentration of either compound for a period of
24 hours. However, no significant differences were observed
with respect to growth progression at any of the time points
assessed [75].

 JG-1 and M4 disrupt pre-formed Salmonella biofilms

Destruction of already existing biofilm is of great
importance to therapeutic relevance. Studies were conducted
by exposing S. Typhimurium after 1-2 hours of inoculation to
Fig 1: The development of gallbladder cancer may be related observe the anti-biofilm effects of JG-1 and M4 at various
to biofilm-producing S.Typhi. S. Typhi infection is strongly phases during biofilm formation. When a bare minimum
related to the development of gallstones and gallbladder amount of JG-1 or M4 was added to S. Typhimurium nearly 8
cancer. Gallstones provide S. Typhi strains with an ideal hours after inoculation, both compounds were seen to inhibit
substrate to form biofilms. Biofilm formation may cause biofilm growth and maturation. Then it was tested on 24-hour
bacteria to release carcinogens, which trigger genomic biofilm with the compounds at various concentrations and the
instability and chronic inflammation, which are both results proved that both JG-1 and M4 disrupted the biofilm to
necessary for GC to form. a certain degree but M4 was more efficient in disrupting S.
Typhimurium biofilm than JG-1. In case of S.Typhi, both
Cytolethal distending toxins (CDTs) are toxic bacterial
compounds were equally competent in disrupting the biofilm
molecules produced by S. Typhi, which are capable of causing
[75].
irreversible DNA damage and causing the cell cycle to stop
and apoptosis to occur [72]. Gallbladder infections damage  Administration of Ciprofloxacin enhances the biofilm
deoxyribonucleic acid (DNA), leading to repeated tissue disrupting capabilities of JG-1 and M4
growth attempts to reverse the damage, released cytokines and Ciprofloxacin is the first-line therapy for chronic and acute
growth factors, and thus predisposing cells to oncogenic typhoidal infections in majority of countries. Using this agent,
transformation [73]. The characteristics of gallstones further scientists have tried to evaluate the therapeutic potential of JG-
influence the development of gallbladder cancer. As the stone 1 and M4 against Salmonella biofilms. In congruence with the
length increases, the chance of gallbladder cancer increases. prior data, scientists treated 24-hour S. Typhimurium biofilm
Stones that are longer than 3 cm convey a tenfold elevated risk
with some amount of ciprofloxacin but it had no significant
as compared to smaller stones [74].
effect but treatment with JG-1 or M4 caused a major reduction
in biofilm formation. Viewing these results, researchers
S. Typhi appears to utilize biofilm formation as an adaptive further combined M4 or JG-1 with ciprofloxacin and found
strategy in order to facilitate microbial persistence. Therefore, that this combination of drugs amplifies the destructive effects
it ensures the spread of bacteria both within the host and of both compounds despite ciprofloxacin displaying no anti-
community as well as immunity against antibiotics. The biofilm effects on its own [75].
persistence of bacterial toxins on target cells may contribute to
their mutagenic effects, which could additionally cause
cumulative damage and transformation [71].

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VI. CONCLUSION

Salmonella biofilm is formed through the action of a few

genes. The quorum-sensing mechanism contributes to
microbial interactions and regulates virulence at a population
level. Gallstone surfaces formed with a biofilm could protect
the microorganism from antimicrobial agents as well as
excessive concentrations of bile for a long time. Flagella of
Salmonella can also be useful for secreting or producing
EPS. It also acts as an aid in primary adhesion and
microcolony formation on gallstones. The fimbriae are
important in the attachment of microorganisms to
microorganisms and to the surface of the gallbladder.
Gallbladder colonization seems to be associated with chronic
Fig 2: Structure of Ciprafloxacin. persistence of S. Typhi caused by biofilm formation on
gallstone surfaces. The main cause of gallbladder cancer in
 Combining with ciprofloxacin helps to enhance the individuals with particular predisposing factors is the presence
efficacy of JG-1 and M4 treatment in mice with chronic of chronic S. Typhi infection, despite the presence of
Salmonella gallbladder carriage gallstones. Gallstones are not the only sites where S. Typhi
After experimenting on in-vitro models, scientists have infection can persist; the gallbladder epithelium and other
further addressed Salmonella activity in an in-vivo model. A cavities could also serve as alternative niches. Environmental
specific mouse strain was fed with a lithogenic diet to help in factors affect the formation of Salmonella biofilms to a great
the formation of cholesterol gallstones. Infection with S. extent. Salmonella prefers a neutral pH for biofilm formation.
Typhimurium shows the same result as that of S. Typhi in Biofilm formation also shows a variation at different
humans. Later, in the research, the mouse model was treated temperature ranges. Generally, at higher oxygen and nutrient
with different regimens: JG-1 or M4 alone; ciprofloxacin levels, Salmonella biofilm formation is better whereas a higher
alone; a combination of ciprofloxacin with either JG-1 or M4, salt concentration adversely affects biofilm formation.
and a control was setup. For this purpose, mice were used and Glucose in culture media inhibits biofilm formation
their livers, spleens, and gallbladders were collected for in Salmonella. Studies show that the treatment of chronic
further assessment of Salmonella burden. carriage with anti-biofilm compounds alone may be able to
reduce the burden of bacteria able to produce biofilms in the
The results showed that ciprofloxacin alone did not gallbladder. However, in few cases it may lead to increased
inhibit biofilm growth gallbladder of infected mice but slightly acute disease severity, sepsis, or even death. Further, it is seen
reduced biofilm growth in the liver and spleen. In contrast, M4 that the adverse outcomes can be avoided by using a dual-
or JG-1 alone could reduce the bacterial burden in the therapy approach in which traditional antibiotics like
gallbladder but increased the number of bacteria in the liver ciprafloxacin are administered in combination with anti-
and spleen. This also resulted in an increased mortality rate in biofilm compounds.
mice. The main cause of this was found to be the reduction in
gall-bladder contents, but there was a decline in bacteria Further research has to be done to observe the combined
associated with gall-bladder tissue. The fecal matter was also effect of dual-drug therapy and environmental factors, both in-
tested and it was found to have increased fecal bacteria vivo and in-vitro models. It has already been seen that the dual-
(Salmonella) when treated with the JG-1 or M4 alone than drug therapy approach yields good results. Hence, further
with ciprofloxacin. In in-vitro biofilms, co-administration of studies should be done considering proper environmental
ciprofloxacin with M4 or JG-1 decreases Salmonella burden conditions so that better results may be obtained and the
in the gallbladder as well as in the liver and spleen with no disease can be managed more efficiently. Research should also
associated mortality. Thus, scientists could say that the be carried out to produce a single drug as a substitute to dual
compounds M4 and JG-1 effectively and remarkably reduced or multiple drugs in combination that have been proven to be
chronic carriage in the mouse model [75]. effective against the disease.

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