Exploring Antibiotic Resistance Genes in Salmonella Plasmids Via

In-silico Approach

Hira Rubab

Abstract:

The diverse use and application of antibiotics in one health system creates selective
pressure and encourages the spread of multiple antimicrobial resistance (MAR). Salmonella typhi
classified as a Gram-negative and strictly anaerobic organism finds its place in the serogroup D of
subspecies I under the Salmonella genus umbrella. The spread of antibiotic resistance markers by
Salmonella mainly relies on plasmids for its horizontal movement. Each year it stands as a major
factor behind nearly 93.8 million instances of illnesses from food and leads to the deaths of about
155,000 people contributing heavily to the global health burden.
The study aims to pinpoint the characteristics of antibacterial resistance and to uncover
signs of resistance genes within Salmonella plasmids through whole genome sequence (WGS) and
digital means . NCBI database held 2103 plasmids related to the Salmonella genus. To check for
genes that fight off antibiotics which the bacteria might have picked up I will use the plasmid
sequences of the clinical samples extracted through WGS and then from NCBI and look through
the ResFinder database. I plan to figure out its potential multiple antibiotic resistance or p-MAR
index for every plasmid that undergoes testing and how much resistant genes are being contributed
from different samples including human, animals, plants and the environment. By looking
carefully at what happens when these plasmids face different types of drugs. In the realm of in-
silico studies, there's a strong focus on how computer-based methods are key to understanding the
intricate genomic patterns related to how diseases resist drugs. This research aims to investigate
about where new drugs could act and how resistance might happen setting the stage for future lab
tests and creating treatments that are tailored to individual needs.
INTRODUCTION
The name Salmonella comes from the fact that it was first isolated from a pig's intestines
in 1884 by American bacteriologist D. E. Salmon. Worldwide, gastroenteritis is commonly caused
by the facultative intracellular gram-negative, motile, hydrogen sulfide-producing, acid-labile
Salmonella bacteria (Ajmera A, Shabbir N. Salmonella 2024) Infectious diseases caused by
Salmonella are common in the United States. Trusted References caused an estimated 1.35
million illnesses every year, 26,500 hospital admissions, and 420 deaths, according to the CDC.
Certain strains of Salmonella can infect several parts of the body, including the central nervous
system, spinal fluid, urine, blood, bones, and joints. Serious consequences might result (Brazier
Y. Salmonella 2020).

Gram-negative bacteria that belong to the family Enterobacteriaceae have been identified
as Salmonella . These bacteria are classed as members of the family. Two species make up the
Salmonella genus, according to the current taxonomy. These species are Salmonella enterica
and Salmonella bongori. There are eight different subspecies of Salmonella enterica, and it is
believed that there are six of them. The subspecies of Salamae that are classified as belonging to
the class Enterica are as follows: Enterica (subspecies I), Salamae (subspecies II), Arizonae
(subspecies IIIa), Diarizonae (subspecies IIIb), Houtenae (subspecies IV), and Indica (subspecies
VI) (Ajmera A, Shabbir N. Salmonella 2024).

Some people who get Salmonella end up with reactive arthritis, which is a painful disorder
of the joints. It might last for months or even years, and it can progress to chronic arthritis. During
the warmer months, Salmonella infections seem to be more common than during the cooler
months. Pregnant women, infants, the elderly, and those with weakened immune systems are
among those most at risk of infection. Because of the risk of infection, caregivers should not let
young children contact reptiles, chicks, or baby birds. Serious problems might arise when a
pregnant woman contracts Salmonella. Illness can cause the mother and child to become
dehydrated and starve to death (Sarah Jividen RN. Salmonella 2024).

It was revealed by a reliable source that approximately four percent of the cases that
occurred during pregnancy involved the presence of Salmonella in the blood. One of the potentially
fatal illnesses that could arise as a consequence of this is intrauterine sepsis (Brazier Y. Salmonella
2020). Typical symptoms often last for 4-7 days and include fever, abdominal pain, and diarrhea.
Vomiting and nausea are less common symptoms. Pain in the head, Soreness all over, lethargy,
dizziness upon standing, and loss of appetite. Most cases of Salmonella infections come as a result
of consuming contaminated food or drinks. Potentially harmful foods include undercooked or
uncooked chicken eggs; pork; beef; and chicken. Salads, sprouts, fruits, vegetables, frozen pot
pies, salami sticks, wheat, peanut butter, and other processed foods have all been linked to recent
outbreaks of Salmonella. Inadequate hygiene practices, improper food handling techniques, and
the possibility of cross-contamination between raw and cooked foods are the main vectors for the
spread of salmonellosis. It can also be spread through domestic animals as well as those at public
places like petting zoos and fairs. The most common ways that Salmonella infections spread are
through contaminated foods, as well as through direct contact between humans and animals (Sarah
Jividen RN. Salmonella 2024).

Salmonella is classified as a Class B pathogen, which means that it has a low mortality
rate and a low morbdity potential. It is possible to classify infections that affect individuals into
two categories: typhoidal and non-typhoidal. The degree of severity of the illness varies in
proportion to the innate defenses of the host as well as the bacterial serotype. It has been found
through research that S. Enteritidis is responsible for more than 52 percent of Salmonella
infections that occur in farm animals. This makes it one of the most common serotypes found in
the United States. On the other hand, according to the National Veterinary Services Laboratory,
the serotype S. Dublin (18%) is the one that is found in animal products, particularly cattle, the
most frequently. This is followed by S. Cerro (16%) and S. Typhimurium (13%) (Lamichhane B
et al., 2024).

Nontyphoidal Salmonella is responsible for more than 150 million illnesses and 60,000
deaths globally every year, making it one of the most common bacterial causes of diarrhea. Most
cases of nontyphoidal Salmonella infections manifest as an acute diarrheal illness. The incubation
period of salmonellosis is typically 12–96 hours, but it can extend to 7 days in extreme cases. In
most cases, the symptoms of the sickness, which might include fever, stomach aches, and acute
diarrhea, go away within a day or two. Approximately five percent of the population is affected by
bacterial infections or localized invasive infections. These infections include osteomyelitis,
meningitis, endovascular infections, and septic arthritis. In general, the incidence of invasive
illnesses are higher among those who are immunocompromised, including HIV positive
individuals, as well as older adults and infants. In addition, those who suffer from malignant
neoplasms, hemoglobinopathies, and atherosclerosis are more likely to experience extraintestinal
infections. Infection with an organism that is resistant to antibiotics has been associated with an
increased risk (Salmonellosis, nontyphoidal 2024).

Antibiotic resistance can be caused by a number of different processes, such as the

inhibition of an enzyme, the alteration of the permeability of the cell membrane (ionophores), the
modification of the composition of the cell wall, and the interference with the creation of DNA
and proteins (mutations, horizontal gene transfer). Therefore, it is of the utmost importance that
we collaborate in order to put a stop to the development of resistance and to decrease the
consequences of these extremely deadly germs. Because of its outstanding capacity to generate
data, research evolution and epidemiology, evaluate microbiological risk, and analyze epidemics,
whole-genome sequencing (WGS) technology has experienced a substantial surge in attention over
the past ten years. This is due to the fact that it has the power to generate new information.
Additionally, researchers utilize WGS to rapidly identify gene clusters, mutations, and antibiotic
resistance genes (ARG) in foodborne pathogens. This is accomplished through the use of genetic
sequencing (Hu L et al., 2020).

Because Salmonella serotypes have gained broad resistance to first-line conventional

treatments, macrolides, especially azithromycin, are the most significant antibiotics for treating
Salmonella. Ampicillin, chloramphenicol, and cotrimoxazole were the initial line of defense
against salmonellosis, but the bacteria are becoming increasingly resistant to these treatments. Due
to the significant threat posed by the development and spread of multi-drug resistance (MDR),
carbapenems and macrolides, such as azithromycin, are the most important antibiotics for the
treatment of Salmonellosis. As of right now, azithromycin is the only oral antibiotic that can treat
Salmonella infections that have developed resistance to all of the existing drugs (Wang H et al.,
2023).

Plasmids are a word that refers to mobile genetic elements (MGE) that have the ability to
replicate themselves and transmit them to other cells. The genes that are responsible for the
maintenance and transmission of plasmids are the individuals that make up the "backbone," also
known as the core assembly of genes that code for essential plasmid functions. Furthermore, these
plasmids provide the bacterial host with non-essential cellular capabilities such as metabolic
pathways, antimicrobial resistance (AMRs), virulence factors, and unknown functions specified
by genes encoding hypothetical and unexplored proteins. All of these capabilities give the bacterial
host an advantage over other organisms under specific conditions (Emond-Rheault J-G et al.,
2020).

Objectives/Aims

The objectives of this project are:

• To locate possible antibiotic-resistance genes within the Salmonella genome and how much
different environmental factors are contributing to resistant genes.

• To investigate potential pharmacological targets within the discovered antibiotic-resistance

genes to provide a foundation for targeted treatment efforts.
REVIEW OF LITERATURE

Lamichhane B et al. (2024) reported that, Salmonella is spread to people through the
farm-to-fork channel and is frequently associated with eating food items that come from animals.
chicken products rank first among these sources, followed by beef, pork, fish, and foods not
derived from animals, like fruits and vegetables. Although the primary treatment for salmonellosis
is antibiotics, the development of multidrug-resistant (MDR) Salmonella strains and an increase
of antibiotic resistance have made it clear how crucial it is to find alternatives to medicines.

According to McMillan et al.'s (2020) investigation, In the United States, non-typhoidal

Salmonella enterica (NTS) is responsible for more than one million instances of bacterial
gastrointestinal disease each year, while it is responsible for more than 93 million cases globally.
Cephalosporins, ciprofloxacin, or sulfamethoxazole/trimethoprim are the most common
antibiotics used to treat infections. However, growing levels of Salmonella resistance to these and
other antibiotics have been observed since the 1980s.

Gargano V et al. (2021) investigated that, Because Salmonella spp. may build up antibiotic
resistance genes (ARGs), it is a pathogen responsible for the spread of antimicrobial resistance.
The Kirby-Bauer method was used in this work to examine the antibiotic resistance profile of 15
antibiotics, comprising six different classes, of 60 strains of Salmonella spp. that were gathered
from pets, farm animals, wildlife, and food in Sicily, Italy. Using Real-Time PCR analysis, all 60
strains of Salmonella spp. were shown to have eight specific tet resistance genes, since over 33.3%
of the strains were tetracycline resistant.

According to Adedokun, F. L. et al. (2023), plasmids mediate antibiotic resistance and

pathogenicity, and Salmonella serovars are widely distributed. Many different types of Salmonella,
which are thought to have originated in animals, have plasmids called Incompatibility Groups
(Inc). These plasmids differ in the number of auxiliary genes they contain and the way they
replicate. There is a lack of data on the kinds of plasmids carried by serovars that progress up the
food chain, while some studies conducted in Nigeria have shown that Salmonella serovars include
plasmids. Consequently, in this study, they characterized the antibiotic susceptibility, plasmid
types, and plasmid replicon types of Salmonella serovars that were obtained from different sources.
The Kirby-Bauer disc diffusion method was employed to assess the antibiotic susceptibility of
Salmonella isolates to ten different drugs: amikacin (100 μg) cefuroxime (30 μg), amoxicillin-
clavulanic acid (30 μg) , cefixime (5 μg), ofloxacin (5 μg), , ciprofloxacin (5 μg), nitrofurantoin
(200 μg), ciprofloxacin (5 μg), and gentamicin (10 μg) ceftazidine (30 μg).

Castro-Vargas et al. (2020) investigated that, the first line of treatment for this bacterial
illness is antimicrobial therapy; however, due to antibiotic abuse in both human and animal
medicine, antimicrobial resistance has emerged as an issue. Antibiotic-resistant bacteria are
expected to be responsible for approximately 10 million deaths globally by 2050, and the World
Health Organization has advocated for the dawn of the post-antibiotic age. This study aims to
explore and provide an update on the current state of Salmonella antibiotic resistance, specifically
regarding its prevalence, serotypes, and patterns of resistance to important antimicrobials utilized
in both the chicken industry and human medicine. Their analysis revealed that the median
prevalence values of Salmonella were 40.5%, 30%, and 40%, respectively, in broiler chickens,
raw chicken meat, eggs, and egg-laying hens. Salmonella Enteritidis was the most prevalent
serotype, followed by Salmonella Typhimurium. The resistance to ampicillin and nalidixic acid
was highest in the chicken production chain. In addition, hens have been found to have Salmonella
that is resistant to extended-spectrum cephalosporins. A second line of antibiotics is now necessary
to combat infections since these bacteria have rendered human medications useless. The
responsible use of antibiotics in animal and human medicine has been supported by numerous
government agencies. Tigecycline, carbapenems, glycopeptides, and third- and fourth-generation
cephalosporins are among the antimicrobials that have seen restricted use. Infectious disease
control becomes more difficult as Salmonella strains and dangerous clones develop resistance to
antibiotics. This poses a major risk to public health around the world. Resistance to antibiotics can
develop when mutations occur in specific parts of the genome, like genomic islands, which are
part of a larger set of genes.
Akinola et al., (2019) examined that, Because of their propensity to cause food-borne
illnesses and high human mortality rates, Salmonella spp. are enteric pathogens that have drawn a
lot of attention and have been designated as agents of public health significance. The most common
food-borne illness in humans, salmonellosis is caused by Salmonella species and is self-limiting,
with symptoms including headache, vomiting, exhaustion, nausea, bloody diarrhoea,
gastroenteritis, and abdominal cramps. Antimicrobials are rarely prescribed for salmonellosis
because the illness is usually self-limiting. Therefore, the pathogen has the potential to affect
human socioeconomic conditions as well as public health. Throughout the world, both developed
and developing nations regard Salmonella enterica serotype Typhimurium (S. Typhimurium ) and
Salmonella enterica serotype Enteritidis (S. Enteritidis) to be of great health significance because
of their capacity to cause salmonellosis in humans and veterinary animals. Salmonella species have
been observed coexisting with other intestinal pathogens, including Escherichia coli, Klebsiella
species, and Proteus species. Humans, pigs, dogs, rats, poultry, birds, ruminants like cattle and
sheep, and cold-blooded animals like fish and reptiles have all been identified as reservoirs for
both typhoidal and non-typhoidal Salmonella species. Public health is currently at risk due to the
worldwide issue of antibiotic resistance. Thus, it is vital to conduct research on the prevalence and
antibiotic resistance profiles of Salmonella in chickens.
METHODOLOGY

Isolation

Roundabout 200 clinical samples will be collected for this study. Isolates will be selected
on the basis of variation in phenotypic AR profile, serotype, and uniqueness of PFGE patterns to
optimize the diversity of AR genes in the Salmonella being investigated. All patient information
will be oblivious to the human isolates to ensure confidentiality. Agar plates will be used for
culturing. Subsequently, one of the predominating colonies will be subcultured and identified to
the species level by microscopy, BBL CHROMagar Orientation media, and KOH testing after the
plates have undergone checks for purity and colony count. The pure cultures will be stored in
cryotubes with 30% glycerol at a temperature of –80°C for WGS. MIC testing in antibiotic
susceptibility will be performed in microtiter plates.

Testing for Phenotypic Antimicrobial Susceptibility

Using broth-microdilution to check for susceptibility phenotypically, many antibiotics

would be used on these isolates. The Sensitre semi-automated antimicrobial susceptibility system
should inoculate the Sensitre custom NARMS plate following the manufacturer's instructions,
TREK Diagnostic Systems Inc., Cleveland, OH, United States. The minimum inhibitory
concentration for each antibiotic is calculated according to the Clinical and Laboratory Standards
Institute breakpoints, which are then used in categorizing the antibiotic strains as resistant,
susceptible, or intermediate. (CLSI, 2016). NARMS breakpoints will be used for antibiotics where
CLSI-established breakpoints are not available.

Genome Sequencing, AR Gene, Assembly and Integron Identification

Genomic DNA will be prepared from whole DNA using a Sigma GenElute kit (Sigma Life
Sciences, St. Louis, MO, USA). Libraries will be constructed with the Nextera XT DNA sample
preparation kit, and we will follow the Illumina procedure. We plan to sequence the isolates using
an Illumina HiSeq2500 (Illumina, San Diego, CA, USA). A5 will be used to assemble the reads
into draft sequences with default settings (Tritt et al., 2012), including quality trimming. Following
assembly, the draft genomes will be annotated using Prokka with standard settings (Seemann,
2014). ARG-ANNOT V3 will be used for detecting the presence of the ARG genes (Gupta et al.,
2014). Integral will be used for the identification of the integrons. WGS data will be prepared from
MiSeq libraries with the Nextera XT library prep kit (Illumina, San Diego, CA, United States) and
sequenced on the Illumina MiSeq platform using either 300 Cycle or 500 Cycle Version 2
chemistries. Assemblies will be generated from raw files using either CLC Genomics Workbench
v8 or v11, or SPAdes v. 3.7.0 (St. Petersburg, Russia).

Plasmid Identification

As done by Camacho et al. (2009), BLASTN will be used to find the target sequence in
each isolate's genome for genes related to plasmid replicon. Plasmid replicon typing and relaxase
typing systems will be used in the selection of target sequences. Carattoli et al. (2005), Villa et al.
(2010) and Compain et al. (2014) used the plasmid replicon typing system. BLASTN will then
find any additional plasmid contigs that were missed by the replicon and relaxase BLAST searches
against a customised plasmid BLAST database. All plasmids affiliated with Enterobacteriaceae up
until March 2015 will be removed from NCBI in order to create the customised database.
Additional plasmid contigs will be validated against the following criteria: Contigs detected in the
Replicon/Relaxase BLAST will be used to determine the principal reference plasmid, which is that
in the custom database with which the first BLAST detected contig has the largest coverage and
percent identity. Large contigs (>10,000 bp) that had an alignment identity greater than 70% and
an alignment coverage greater than 40% to a plasmid major reference for one replicon type but no
significant homology to another were also considered to correspond to the same plasmid, even if
they were not detected in the BLAST search. Additional plasmid contigs will be validated based
on the following criteria as indicated above: Once the first set of attractive reference plasmids has
been identified, the larger vector would likely have been chosen based on a number of factors;
most of them relating to their sequence similarity to a distinct plasmid under a particular group of
recreational vectors or those involved in the mobility processes. Any large contigs—it means over
10,000 bps—with their identities and coverages having high percentages of above 70% and 40%,
respectively, compared to the main standard vector without having huge similarities with other
related strains might not have turned up in the first blast search results. This will be done by
obtaining the % identity range of principal reference plasmids to the contigs carrying the relaxase
or replicon genes. For these big contigs, 70% cut-off of the percent identity will thereafter be
chosen. A 40% coverage cut-off point will then be applied next, permitting contigs with continuity
when the reference sequence is discontinuous. This can include cases in which the reference
plasmid and the query contig begin at different positions and there exists a long pause between the
homologous sequences such that two distinct BLAST hits exist for this contig that was found.
Another case includes the contigs that bear less similarity to either the reference plasmid or some
other plasmid belonging to the same replicon class, and may thus be binned. These small contigs
(3,000-10,000 bp) were very different from dominant plasmids but matched quite well with
reference plasmids of the same replicon type. Co-binned contigs are extracted to form a draft.
These contigs will only be part of drafts if they are unrelated to other plasmids of distinct replicon
types. Furthermore, a single contig that is not part of the main BLAST search whose BLAST
search overlaps the entirety of a single plasmid will also be considered an isolated plasmid. No
drafts will be made from ColE replicons due to the poor assembly and short contig lengths. The
contigs containing a ColE replicon and an AR gene will be retained, however. Drafts will be
augmented with RAST annotations. This will also be applied to type replica kinds that have a
recognized Plasmid Multi Locus Sequence Type scheme through a query to the pMLST database.
Qualimap and Bowtie2 analysis will also be conducted to probe contig coverage of each sequence
(Garcia-Alcalde et al., 2012; Langmead and Salzberg, 2012).

Antibiotic Resistance Cassette Identification

Using the molecule manipulation software SnapGene5, for many of the retrospective
isolates, the contig containing the AR gene will be aligned to other isolates whose contigs also
contain that gene. An equivalent region situated nearby all these antibiotic resistance genes in these
additional isolates was termed an antibiotic resistance cassette, ARC. If a sequence occurs in at
least five separate independent isolates with the same flanking sequence and AR gene, then it is
considered an ARC sequence. The ARC sequences will be compared to previous isolates using
BLASTN in search of other isolates that have expressed the AR gene but not formed a complete
ARC, searching for other isolates which have the ARC sequence but which is split among many
assemblies (Camacho et al., 2009). The ARC sequences will be compared to sequences deposited
in the NCBI non-redundant database using BLASTN to detect matched sequences and determine
the species and frequency of sequenced isolates that have these ARC sequences. Moreover,
tBLASTN will be used to link antibiotic resistance cassette sequences to the USDA-FSIS
Salmonella isolates. Only those isolates that would most likely carry the ARC will be compared.
Testing for the presence or absence of an ARC will be possible in each isolate by examining the
AR gene content of that isolate in the Pathogen Detection Isolate Browser. If the entire cassette
sequence is present, or it overlaps on many contigs, the isolate is predicted to have the ARC.

Statistics

I will use the 95% confidence interval as generated in SPSS to compare the ratio of FSIS isolates
with the ratio of ARCs by animal source and serotype. A general linear regression model will be
used to calculate the factors responsible for resistant genes.

Conclusion
My research topic explores antibiotic resistance in the Salmonella species plasmids while
using in-silico approaches. By using these computational and latest tools I aim to identify the
genetic reasons responsible for antibiotic resistance, contributing a better understanding of how
resistance spreads and works, which means we can control the resistance situations and how we
can combat bacterial infections.

About myself:
With a strong background in applied and allied sciences along with extensive lab
experiences, I have developed a good and comprehensive understanding of molecular biology,
microbiology and genetics. This combination of experiences is enough to achieve my research
objectives and contribute meaningful advancements in my field. I am an excellent candidate for
this degree due to my interdisciplinary expertise. To achieve my research objectives, I seek further
skills in bioinformatics and genetics. I am committed to continuous professional development and
look forward to work with your faculty and to learn many things that will enrich my skills and
expertise and will help me to grow as a good researcher.
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