Identification of potent inhibitors against Salmonella

Typhi using in-silico approach

Research based learning Report in partial fulfillment for

the completion of
Research Based Learning (RBL004)
In
M.Sc. Biotechnology
Sharda School of Basic Sciences and Research
by
Naba Fatima
Roll Number: 2207130019
System ID: 2022340629
UNDER THE SUPERVISION
of
Dr. Rashmi Prabha Singh

Department of Life Sciences

Sharda School of Basic Sciences and Research
Sharda University, Greater Noida-201306, U.P., India
April, 2024
 Acknowledgment

We owe our deepest gratitude to Dr. Rashmi Prabha Singh,under whose guidance and
continuous support this report is written and prepared.
We feel honored and it is a pleasure to thank Prof. Satyawada Rama Rao, Head of Sharda
School of Basic Life Sciences (SBSR) M.Sc. (Biotechnology) and our other faculty members
for their continuous help and support.
We also thank our participants for giving their ears and time for purpose of completion of this
work.
And finally, we express my heartfelt thanks to our parents, God, and to all our dear
classmates – who supported and suggested us - without whom this report would not have
been possible.
 CERTIFICATE

This is to certify that Ms. Naba Fatima (System Id: 2022340629) from M.Sc. Biotechnology,
Department of Life Sciences, School of Basic Sciences and Research, has completed her
Research Based Learning (004) on the topic “Identification of potent inhibitors against
Salmonella Typhi using in-silico approach” under the supervision of Dr. Rashmi Prabha
Singh, during the academic year 2023-2024, as per guidelines.

Supervisor Signature Head of Department Signature

Dr.Rashmi Prabha Singh Prof. Satyawada Rama Rao
Associate Professor Professor & Head
Dept. of Life Sciences Dept. of Life Sciences
School of Basic Sciences and Research School of Basic Sciences and Research
Sharda University Sharda University
Abstract

Salmonella stands out as a frequent culprit among foodborne pathogens, posing a substantial
global public health threat with an annual toll of 93.8 million foodborne illnesses and 155,000
fatalities. The extensive array of Salmonella comprises over 2500 serotypes, with more than
half falling under Salmonella enterica subsp. enterica, responsible for the majority of human
Salmonella infections. Infections involving invasive serotypes are particularly perilous, often
demanding prompt and effective antibiotic intervention. The rise of multi-drug-resistant
(MDR) Salmonella strains significantly challenges antibiotic efficacy, potentially elevating
mortality rates associated with Salmonella infections. Epidemiological findings underscore the
heightened virulence of MDR Salmonella serotypes, leading to more severe and prolonged
illnesses in affected individuals. Proposed preventive measures include upholding stringent
food hygiene and water sanitation practices, alongside advocating for the judicious use of
antibiotics in food animals. This comprehensive review presents an encompassing exploration
of Salmonella infections, encompassing nomenclature, pathogenesis, clinical manifestations,
epidemiology, and the escalating challenge of antibiotic resistance in Salmonella.

Keywords- Salmonella; multi-drug resistance; enteric fever; foodborne

 Table of Contents

S.no. Contents
1. Introduction

2. Classification & Nomenclature

3. Pathogenesis

4. Clinical Manifestations

5. Clinical Relevance

6. Prevention

7. Bacterial cell wall synthesis

8. Genes/Proteins involved in synthesis

9. Molecular docking and Simulation

10. Conclusion
Introduction
Salmonella infection continues to pose a significant global public health challenge, imposing
economic burdens on both developed and developing nations due to the expenses associated
with disease surveillance, prevention, and treatment (Crump et al. 2004). The predominant
clinical manifestation of Salmonella infection worldwide is gastroenteritis, with bacteraemia
and enteric fever following closely (Majowicz et al. 2010). Salmonella, a rod-shaped Gram-
negative facultative anaerobe belonging to the family Enterobacteriaceae, encompasses
approximately 2600 serotypes identified through the standard Kauffman–White scheme. Most
of these serotypes exhibit adaptability across a range of animal hosts, including humans
(Allerberger et al. 2003). Among the most frequently isolated foodborne pathogens, Salmonella
and Campylobacter are commonly found in poultry, eggs, and dairy products (Silva et al. 2011).
Additionally, fresh fruits and vegetables contribute to the transmission of Salmonella (Pui et
al. 2011). Generally, food animals such as swine, poultry, and cattle serve as primary sources
of Salmonella infections. The primary dissemination routes for these pathogens involve the
trade in animals, and raw animal food products constitute a noteworthy aspect of the discussion.
The slaughtering procedures employed in abattoirs stand out as a significant contributor to the
contamination of organs and carcasses with Salmonella (Gillespie et al. 2005). The increasing
prevalence of antibiotic-resistant foodborne pathogens has become a cause for public concern,
given the heightened virulence of these pathogens, resulting in elevated mortality rates among
infected individuals (Chiuetal.2002)
 Nomenclature and Classification
Salmonella was initially identified and isolated from the intestines of pigs afflicted with
classical swine fever by Theobald Smith in 1855. In honour of Dr. Daniel Elmer Salmon this
bacterial strain was named, an American pathologist who collaborated with Smith. The
nomenclature of Salmonella has been a subject of debate and continues to evolve. Presently,
the Centres for Disease Control and Prevention (CDC) adheres to the nomenclatural system for
Salmonella recommended by the World Health Organization (WHO) Collaborating Centre
(Popoff et al. 2003). According to this system, the genus Salmonella is categorized into two
species, Salmonella enterica (as the type species) and Salmonella bongori, determined by
distinctions in their 16S rRNA sequence analysis. The species S. enterica can be intricately
subdivided into six subspecies, their classification hinging on both genomic relatedness and
biochemical properties, as delineated by Reeves et al. in 1989. These subspecies are
distinguished by Roman numerals: I for S. enterica subsp. enterica, II for S. enterica subsp.
salamae, IIIa for S. enterica subsp. arizonae, IIIb for S. enterica subsp. diarizonae, IV for S.
enterica subsp. houtenae, and VI for S. enterica subsp. indica. Notably, S. enterica subsp.
enterica (I) takes precedence, being predominantly found in mammals and accounting for
approximately 99% of Salmonella infections in both humans and warm-blooded animals.
Conversely, the remaining five Salmonella subspecies and S. bongori predominantly inhabit
the environment and cold-blooded animals, rendering them relatively uncommon in human
infections (Brenner et al.).

Classification based on serotype

In addition to the phylogenetic classification of subspecies, Kauffman and White introduced a
system for further characterizing Salmonella based on serotype.The somatic O antigen, resistant
to heat, constitutes the oligosaccharide component of lipopolysaccharide situated on the outer
bacterial membrane. Notably, a specific Salmonella serotype can display multiple O antigens
on its surface (Hu & Kopecko 2003). Meanwhile, the heat-labile H antigens are located in
bacterial flagella and play a role in activating host immune responses. Many Salmonella species
possess two distinct genes encoding flagellar proteins, allowing them to express only one
protein at a time, rendering them diphasic (phase I and II). Each serotype expresses specific
phase I H antigens that define its immunological identity, while phase II antigens are non-
specific and may be shared by multiple serotypes (McQuiston et al. 2008). To pinpoint a
specific serotype, a thorough serotyping analysis encompassing all bacterium's antigenic
determinants can be conducted. However, many clinical laboratories opt for simplified
agglutination reactions with antibodies or antisera specific to the somatic O antigens. This
method aims to categorize Salmonellae into six serogroups (A, B, C1, C2, D, and E), providing
crucial data for epidemiological studies and facilitating the genus identification of
Salmonella infections(Wattiau et al. 2011).
Pathogenesis
The impact of Salmonella infections in humans varies, and it is influenced by both the specific
serotype responsible and the health condition of the human host. Individuals under the age of 5,
the elderly, and those with compromised immune systems exhibit a higher susceptibility to
Salmonella infections compared to their healthier counterparts. The majority of Salmonella
strains are pathogenic, possessing the capability to invade, replicate, and endure within human
host cells, leading to the potential development of life-threatening diseases.Salmonella exhibits a
distinctive trait during its invasion of non-phagocytic human host cells, as documented by
Hansen-Wester et al. in 2002. In this process, Salmonella actively induces its own phagocytosis
to gain entry into the host cell. This ingenious mechanism is underpinnedby the remarkable
genetics found in Salmonella Pathogenicity Islands (SPIs) – gene clusters situated within the
extensive chromosomal DNA region. These gene clusters encode structurespivotal to the
invasion process, as elucidated by Grassl and Finlay in 2008.

Clinical Manifestations
Categorized by clinical patterns observed in human salmonellosis, Salmonella strains are
broadly divided into typhoid Salmonella and non-typhoid Salmonella (NTS). Within human
infections, four distinct clinical manifestations emerge: enteric fever, gastroenteritis,
bacteraemia, and other extraintestinal complications, along with the chronic carrier state, as
outlined by Sheorey & Darby in 2008.
Clinical relevance
The emergence of multi-drug resistance among Salmonella serotypes profoundly affects the
efficacy of antibiotic treatment for Salmonella infections. Infections associated with invasive
serotypes are frequently life-threatening, necessitating the application of potent antibiotic
treatments. Historically, quinolones and third-generation cephalosporins have been the
preferred antibiotics in such case in the treatment of infections caused by Multi-Drug Resistant
(MDR) Salmonella, quinolones and third-generation cephalosporins have traditionally been the
antibiotics of choice (Karon et al. 2007). However, the emergence of Salmonella serotypes
resistant to quinolones and cephalosporins presents a novel challenge in managing infected
patients. The absence of effective antibiotic therapy may contribute to an escalation in
morbidity and mortality rates.
Current preventive measures for enteric fever emphasize access to safe water and food, proper
sanitation, and the use of typhoid vaccines. Ensuring the safety of water for consumption is a
crucial step in eliminating potential transmission routes for both typhoid Salmonella and non-
typhoid Salmonella (NTS). While this goal has been successfully achieved in industrialized
countries like Europe and the USA, challenges persist in developing and underdeveloped
nations (Clasen et al. 2007).
Salmonella spp. can be present in various foods, particularly in poultry, eggs, and dairy
products. Proper handling and cooking of food are recommended measures to eliminate
bacterial contamination. Food irradiation, endorsed by public health agencies including the
WHO and CDC, has been promoted in many countries due to its effectiveness in reducing the
risk of food contamination. However, its utilization is limited in some regions in Europe and
the USA due to concerns about radioactivity (Osterholm & Norgan 2004).

Prevention
The primary transmission route for enteric fever is contaminated water or food. Historically,
USA and Western Europe were endemic. However, the incidence of Salmonella infection
significantly decreased through the implementation of proper food and water sanitation
practices and pasteurization of dairy products. A similar decline in Salmonella infections was
observed in Latin America following the introduction of sanitation measures (Crump et al.
2004).
Current preventive measures for enteric fever emphasize access to safe water and food, proper
sanitation, and the use of typhoid vaccines. Ensuring the safety of water for consumption is a
crucial step in eliminating potential transmission routes for both typhoid Salmonella and non-
typhoid Salmonella (NTS). While this goal has been successfully achieved in industrialized
countries like Europe and the USA, challenges persist in developing and underdeveloped
nations (Clasen et al. 2007).
Vaccination stands out as an effective measure in preventing enteric fever. Currently, two types
of vaccines, inactive parenteral and oral live attenuated, are approved for the prevention of
enteric fever. However, these licensed vaccines are limited to infants and are not effective
against infections caused by S. Paratyphi and NTS (Lin et al. 2001). Regarding NTS, a valuable
preventive measure involves restricting the inappropriate use of antibiotics in food animals and
their feed (Talbot et al. 2006).
Cell wall synthesis of Salmonella Typhi
The intricate polysaccharides found in the cell wall lipopolysaccharides play a crucial role in
determining the somatic (O) antigen specificities of Salmonella typhimurium and E. coli. These
polysaccharides are composed of highly branched structures linked to a lipid containing
glucosamine. The polysaccharide component may consist of up to six neutral sugars, including
L-glycero-D-mannoheptose, glucose, galactose, mannose, rhamnose, and abequose (3,6-
dideoxy-D-galactose), all identified as integral parts of the S. typhimurium polysaccharide.
It is believed that these polysaccharides possess an internal core structure, potentially common
among all enteric bacteria. Complex side chains with group or species-specific antigenic
determinants are attached to this core. Recent insights into the structure and biosynthetic
mechanism of these polysaccharides have emerged from studies involving mutant organisms
deficient in specific nucleotide sugar synthesis.
Nikaido's work revealed that mutants incapable of synthesizing UDP-galactose, due to the loss
of UDP-galactose 4-epimerase, produce incomplete polysaccharides. These mutants exhibit a
distinct lack of galactose and other sugars present in normal polymers, representing the
innermost core region of the wild-type polysaccharide. Previous investigations on a UDP-
galactose-4-epimerase-deficient strain of S. typhimurium demonstrated that the incomplete
"core" polysaccharide from this mutant contains glucose, L-glycero-D-mannoheptose, and
phosphate.
Further exploration has now uncovered that this polysaccharide also contains 2-keto-3-
deoxyoctonate (KDO), a component recently identified by Heath and Ghalambor in the
lipopolysaccharide structure E. coli O111. Consistent with the findings of these researchers,
the majority of the 2-keto-3-deoxyoctonate (KDO) present in the lipopolysaccharide of the S.
typhimurium mutant seems to be in glycosidic linkage at non-reducing terminal positions.
However, around 25 percent of the total KDO is located in a different position; this fraction is
recovered in the purified, lipid-free polysaccharide and occupies the reducing-terminal position
of glucose-heptose-phosphate chains.
Most if not all, of the polysaccharide chains appear to contain KDO at the reducing end,
suggesting that this sugar acid may play a role in linking the core polysaccharide to the lipid
moiety of the intact lipopolysaccharide.
Genes involved in cell wall synthesis
Salmonella Typhi possesses a suite of genes dedicated to cell wall synthesis. In Gram-
negative bacteria like Salmonella Typhi, the cell wall primarily comprises peptidoglycan,
imparting structural integrity to the bacterial cell. The genes linked to cell wall synthesis in
Salmonella Typhi encompass those responsible for encoding enzymes engaged in
peptidoglycan biosynthesis and assembly. Notable examples include:

MraY
Function: MraY is involved in the initial steps of peptidoglycan biosynthesis. It catalyzes the
transfer of the first membrane-anchored peptidoglycan precursor, lipid I, to the outer leaflet of
the inner membrane.
Role: MraY is essential for the synthesis of the peptidoglycan precursor, a crucial step in
building the bacterial cell wall.

MurA
Function: MurA is responsible for the addition of the first amino acid, UDP-N-
acetylglucosamine, to the lipid I precursor, forming UDP-N-acetylmuramic acid.
Role: MurA is a key enzyme in the early stages of peptidoglycan biosynthesis and is essential
for the production of peptidoglycan monomers.

MurB
Function: MurB catalyzes the addition of a second amino acid to UDP-N-acetylmuramic acid,
forming the precursor UDP-N-acetylmuramoyl-L-alanine.
Role: MurB is involved in the sequential steps leading to the formation of the peptidoglycan
precursor, contributing to the synthesis of the peptidoglycan backbone.

MurC, MurD and MurE

Function: MurC, MurD, and MurE are enzymes involved in the sequential addition of amino
acids to the UDP-N-acetylmuramic acid precursor, leading to the formation of the mature
peptidoglycan precursor, UDP-N-acetylmuramoyl-L-alanyl-D-isoglutamate-meso-
diaminopimelate.
Role: These enzymes play critical roles in the stepwise synthesis of the peptidoglycan
precursor, contributing to the diversity of the peptide side chains.

Penicillin-Binding Proteins (PBPs)

Function: PBPs are involved in the final stages of peptidoglycan synthesis, catalyzing the
cross-linking of peptidoglycan chains to form a strong, stable cell wall.
Role: PBPs are the target of beta-lactam antibiotics like penicillin, which inhibit their activity
and lead to cell wall damage and bacterial cell death
Role of the GGDEF protein family in Salmonella
Genes related to cellulose biosynthesis in S. Enteritidis and S. Typhimurium are organized into
two operons, namely bcsABZC and bcsEFG, transcribed in opposite directions (Zogaj et al.,
2001; Solano et al., 2002). The presence of bcs operons is not exclusive to Salmonella, as
homologous gene clusters within the family Enterobacteriaceae have been identified in all
investigated species (Zogaj et al., 2003; our unpublished results). Through homology searches, it
has been established that bcsA, bcsB, and bcsZ encode the catalytic subunit of cellulose
synthase, the regulatory subunit binding to the activator cyclic diguanylic acid (c-di-GMP), and
an endoglucanase, respectively. In the second operon, bcsG also shows homology to an
endoglucanase gene. The bcs genes appear to be constitutively transcribed, and therefore, their
transcription is not dependent on CsgD or AdrA (Zogaj et al., 2001). However, our
understanding of cellulose biosynthesis in these bacteria remains limited.

The organism commonly employed for studying regulatory elements linked to cellulose
production is Gluconacetobacter xylinus, previously known as Acetobacter xylinus(m). In this
bacterium, glucose units in UDPG (Uridin-5¢-diphosphoglucose) are incorporated into the
cellulose polymer (1,4-β-D-glucan) by a membrane-bound cellulose synthase. The activity of
this synthase is specifically reliant on cyclic diguanylic acid (c-di-GMP) (Ross et al., 1987;
1990; Weinhouse et al., 1997). Cellulose synthesis in G. xylinus is regulated by the opposing
actions of two enzymes: diguanylate cyclase (DGC) and c-di-GMP diesterase (PDEA),
controlling the cellular level of c-di-GMP (for an in-depth review, refer to Ross et al., 1991). In
a comprehensive study, Tal et al. (1998) identified the dgc and pdeA genes as pivotal
components in the turnover of this novel effector. The identification of two adjoining domains,
GGDEF and EAL, shared by all DGC and PDEA isoenzymes, suggests the involvement of
either or both domains in diguanylate cyclase activity (Ausmees et al., 2001). Notably, the
GGDEF domain is prevalent in the genomes of free-living bacteria, although the majority of
GGDEF proteins have not been experimentally characterized yet (Galperin et al., 2001).

To gain a more profound understanding of the regulatory mechanisms governing cellulose

synthesis and biofilm formation in Salmonella, this investigation leveraged the distinct
characteristics of the organism. Two mouse-virulent strains of S. Typhimurium, namely
ATCC14028s (Fields et al., 1986) and SL1344 (Hoiseth and Stocker, 1981), each manifesting
unique biofilm-forming capabilities were utilized. By introducing two plasmids derived from the
biofilm-forming strain S. Typhimurium ATCC14028s into strain SL1344, the latter acquired the
capacity for cellulose synthesis and biofilm production. Consequently, this research reveals the
presence of a novel protein, STM1987, housing a GGDEF domain and potentially exhibiting
diguanylate cyclase activity. This activity endowed strain SL1344 with the ability for cellulose
biosynthesis and biofilm formation in ATM media. Notably, the effectiveness of this activity
was contingent on the conserved sequence pattern GG[DE]EF motif within the GGDEF domain.

Through non-polar mutation and complementation experiments involving either STM1987 or

adrA mutants, the study explored the contributions of all S. Typhimurium proteins containing
the conserved GG[DE]EF motif in cellulose biosynthesis and biofilm formation. Consequently,
five new members of the GGDEF family implicated in cellulose biosynthesis by S.
Typhimurium were identified and designated Gcp (GGDEF domain-containing protein). The
findings suggest that the GGDEF protein family in Salmonella serves as a crucial link
connecting various environmental signals to cellulose production and biofilm formation, thereby
fulfilling diverse functions within the cell.
Evidence from molecular docking and dynamic simulations
The rise of multi-drug resistance in recent Salmonella Typhi isolates, the causative agent
of enteric Typhoid fever, has spurred an exploration of alternative therapeutic
approaches. In this investigation, virtual screening, ADMET screening, and antibacterial
activity prediction were employed to identify promising lead molecules. The binding
affinities (BAs) of these molecules were subsequently assessed against key druggable
targets in S. Typhi. The analysis uncovered a novel bioactive compound—a deoxy-
tetradeutero-curcumin derivative—with a notable BA toward the UDP-N-
acetylmuramate–L-alanine ligase (MurC) protein, a crucial player in peptidoglycan
synthesis. Molecular docking revealed that this lead (Binding energy, BE = -8.00 ± 0.02
kcal/mol) could competitively bind to MurC, surpassing the binding energy of its
natural ligand ATP (BE = -7.65 ± 0.19 kcal/mol). Moreover, the lead demonstrated
superior binding and inhibition profiles against MurC compared to other commercially
available antibiotics. The favorable binding energy was attributed to hydrogen bonds
and numerous non-canonical interactions with evolutionarily conserved active-site
residues. Molecular docking and coarse-grained dynamics simulations suggested that
the novel curcumin derivative could function as a potential competitive inhibitor of ATP
for the MurC-catalytic domain, exhibiting a low relative RMSF (0.59 Å) and inhibiting
MurC-induced peptidoglycan biosynthesis.

Identifying druggable targets in S. Typhi

The identification of druggable protein targets in S. Typhi was carried out through a
comparative and subtractive genomics study conducted by Batool et al. (2016). The protein
targets identified were then assessed for their binding affinities (BAs) with the bioactive
compounds shortlisted in the study.

Screening of potential bioactive compounds

A collection of bioactive compounds sourced from the ChEMBL database underwent
screening using criteria such as stereochemistry, structural alignment, and pharmacophore
model. This evaluation utilized curcumin [PubChem CID: 969516] as the reference
compound, given its documented antibacterial properties.

Retrieval of druggable targets in S.Typhi

In the comparative and subtractive genomics study conducted by Batool et al. (2016), the
prioritization of drug targets identified 20 druggable proteins from a pool of 46 essential
proteins, marking them as potential therapeutic targets in S. Typhi. Consequently, these 20
proteins were selected for inclusion in the present study. Successful prediction and optimization
of the 3D structures were achieved for 19 out of the 20 drug targets.
Conclusion
Salmonella infection remains a pressing global public health issue. The adaptable genetic
makeup of Salmonella strains enables them to thrive in diverse environments, spanning human,
animal, and non-animal hosts, thereby complicating the challenge of eradicating the bacteria.
The emergence of Multi-Drug Resistant (MDR) Salmonella strains adds a significant hurdle to
effectively treating infections caused by these strains.
Various preventive measures have been suggested to curb the spread of Salmonella infection,
with the prudent restriction of indiscriminate antibiotic use in food animals standing out as one
of the most effective measures. While two vaccines have been approved for preventing enteric
fever, there are currently no licensed vaccines available for S. Paratyphi and non-typhoid
Salmonella (NTS) infections. Further research into the development of vaccines targeting all
Salmonella strains holds the potential for substantial benefits in affected regions.
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