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USF Tampa Graduate Theses and Dissertations USF Graduate Theses and Dissertations

March 2020

Identification of SNPs associated with multiple antibiotic

resistance genes in eight clinical variants of Enterococcus
faecium.
Krishna S. Karia
University of South Florida

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Karia, Krishna S., "Identification of SNPs associated with multiple antibiotic resistance genes in eight
clinical variants of Enterococcus faecium." (2020). USF Tampa Graduate Theses and Dissertations.
https://digitalcommons.usf.edu/etd/8956

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Identification of SNPs Associated with Multiple Antibiotic Resistance Genes in Eight Clinical

Variants of Enterococcus Faecium

by

Krishna S. Karia

A thesis submitted in partial fulfilment

of the requirements for the degree of
Master of Science in Pharmaceutical Nanotechnology
Department of Pharmaceutical Nanotechnology
College of Pharmacy
University of South Florida

Co-Major Professor: Alya Limayem, Ph.D.

Daniel Denmark, Ph.D.
Feng Cheng, Ph.D.

Date of Approval:
March 12th, 2020

Keywords: multidrug resistance bacteria, DNA sequence, single nucleotide polymorphism

Copyright © 2020, Krishna S. Karia

 TABLE OF CONTENTS

LIST OF TABLES .......................................................................................................................... ii

LIST OF FIGURES ....................................................................................................................... iii

ABSTRACT ................................................................................................................................ iv

CHAPTER 1: INTRODUCTION ....................................................................................................1

1.1 Introduction ....................................................................................................................1

CHAPTER 2: LITERATURE REVIEW .........................................................................................5

2.1 Characteristics of Enterococci as Pathogens..................................................................5
2.2 Enterococci as a Leading Cause of Nosocomial Infections ...........................................8
2.3 Importance of Single Nucleotide Polymorphism (SNP) in Identification of
MDR Bacteria ................................................................................................................9
2.4 Other Techniques to Identify Nosocomial Infection ...................................................11
2.5 Hypothesis of the Study ...............................................................................................14
2.6 Rationale of the Study ..................................................................................................14

CHAPTER 3: MATERIALS AND METHOD..............................................................................16

3.1 Sample Collection ........................................................................................................16
3.2 Illumina Sequencing Method .......................................................................................16
3.3 Library Sample Preparation .........................................................................................17
3.4 Paired-ended Sequence Analysis using BWA Software ..............................................17
3.5 Description of the BWA Software ...............................................................................18
3.6 SNP Calling .................................................................................................................18
3.7 Antimicrobial Susceptibility Test ................................................................................20

CHAPTER 4: RESULTS ...............................................................................................................21

4.1 Results ..........................................................................................................................21

CHAPTER 5: DISCUSSIONS ......................................................................................................30

5.1 Discussions ..................................................................................................................30

CHAPTER 6: CONCLUSION ......................................................................................................32

6.1 Conclusion ...................................................................................................................32

REFERENCES ..............................................................................................................................33

i
 LIST OF TABLES

Table 1: Cost and reimbursement data for nosocomial VRE and VSE infections .........................9

Table 2: Characteristics of farms studied, including the kind and number livestock
in captivity, E. faecium positive, AREs, and VREs...........................................................13

Table 3: Sequence analysis of the eight E. faecium strains using BWA software .......................22

Table 4: Resistance or susceptibility of E. faecium strains in varying concentration of

gentamycin .........................................................................................................................22

Table 5: Resistance or susceptibility of E. faecium strains in varying concentration of

penicillin ............................................................................................................................23

Table 6: Resistance or susceptibility of E. faecium strains in varying concentration

of tetracycline.....................................................................................................................23

Table 7: Resistance or susceptibility of E. faecium strains in varying concentration

of linezolid .........................................................................................................................24

Table 8: Resistance or susceptibility of E. faecium strains in varying concentration

of daptomycin ....................................................................................................................24

Table 9: Resistance or susceptibility of E. faecium strains in varying concentration

of vancomycin ....................................................................................................................25

Table 10: Resistance or susceptibility of E. faecium strains in varying concentration

of virginiamycin M1 ..........................................................................................................25

Table 11: Total number of SNPs and INDELs in E. faecium strains .......................................... 27

Table 12: Individual number of antibiotic SNPs in E. faecium strains ....................................... 27

Table 13: Antibiotic-specific SNPs of E. faecium strains ........................................................... 28

Table 14: List of resistance genes in E. faecium isolates ............................................................ 29

ii
 LIST OF FIGURES

Figure 1: Antibiotic prescriptions per 1,000 persons of all ages in the U.S.A. in 2010 ................ 2

Figure 2: Total number of approved drug applications over the years (1980-2014) ..................... 2

Figure 3: Device-associated infection/1000 device days ............................................................... 4

Figure 4: Isolation of E. faecium from 29 farms conducted in England from 2014 to

2015…............................................................................................................................... 11

Figure 5: BWA flow graph. Two-step method includes (a) step 1: building the BWT
index from the reference genome; and (b) step 2: alignment of the reads against
the reference using either BWA aln, or (c) BWA mem .....................................................19

Figure 6: SNV calling flow graph. Two-step method includes (a) step 1: converting
BWA. sam output to. bam and .bcf using samtools; and (b) step 2: variant calling
using bcf tools (b) ..............................................................................................................20

Figure 7: Total SNPs of E. faecium strains. (a) Incidence of nucleotide polymorphism,

and (b) incidence of insertion or deletion of nucleotide ....................................................28

iii
 ABSTRACT

The evolution of multidrug resistant (MDR) bacteria has been well documented these last

decade, suggesting that resistance to antibiotics is most associated to single nucleotide

polymorphism (SNPs) mutation. To this aim, several molecular methods have been performed to

elucidate SNPs in the increasing number of resistant microorganisms. Early detection of MDR

bacteria has become of prime necessity in order to combat the disease and ensure public health.

Therefore, this research was conducted to screen for SNP mutations correlated to drug resistance

in some nosocomial MDR bacteria strains donated from Moffitt Cancer Center (MCC) hospital.

Blood, stool, urine, and wound discharge samples were collected, and bacteria were isolated and

identified using Illumina sequencing. Antibiotic susceptibility test was also conducted. A

standardized questionnaire was also prepared for patients to gather data, which was further

analyzed using SPSS version 23 software. Variations in the genes responsible for the multidrug

resistance of eight Enterococcus faecium were observed. All strains were found to be multi-drug

resistant especially against penicillin and daptomycin, but all were susceptible to virginiamycin

M1. Dose-dependent susceptibility was observed for gentamycin, tetracycline, and linezolid.

High incidence of SNPs and INDELs were also observed. This study provides information for

future rapid diagnostics for multi-drug resistant bacteria.

iv
 CHAPTER 1: INTRODUCTION

1.1 Introduction

In the last decade, antibiotics have been used not only for therapeutic purposes but also

for the growth and promotion of livestock rearing and aquatic fish sustainability in agricultural

systems, along with water plant and biofuel disinfection. A recent study suggests that over 40%

of the total amount of antibiotics are utilized in food additives, and around 80% of antibiotics in

the U.S. are used in poultry, livestock, and agricultural processes (2). However, the reckless

utilization of antibiotics has created selective pressure in some bacterial strains. It resulted in the

development of a significant degree of drug resistance that spreads throughout the ecosystem,

creating a pool of serious threat of resistant bacteria (2). Multidrug resistance (MDR) infections

from nosocomial microorganisms are of actual concern for public health in the US and around

the world (12) (25) (60) (65). The Center for Disease Control and Prevention (CDC) recently

reported a significant increase in MDR infections, registering 2.8 million hospitalizations and

35,000 fatalities (29).

Recent trends have also revealed that the number of antibiotic prescriptions per 1,000

persons of all ages in the USA are rapidly rising, as shown in Figure 1 (63). The amount of

antibiotics prescribed by doctors varies greatly from one state to another and might indicate

places where it would be more effective to optimize antibiotic prescribing. However, it has also

been noticed that the number of approved new antibacterial drug is constantly decreasing (Figure

1
2). There is a stark contrast between the approved new drugs and the number of people and

sources from which the antibiotics are being used (63).

Figure 1. Antibiotic prescriptions per 1,000 persons of all ages in the U.S.A. in 2010 (63)

Figure 2. Total number of approved drug applications over the years (1980 -2014)

2
 The progression of MDR bacteria into the community could cause a significant rise in

death rate, healthcare costs, and morbidity. Figure 3 shows the device associated infections per

1000 devicedays, where lower respiratory tract infections (LRTI), bloodstream infections (BSI),

and urinary tract infections (UTI) were presented and compared. The ventilator-associated LRTIs

are the major cause of device-associated infections among patients, and surgical and

neurosurgical device associated infections are of major concern. Several studies have shown that

by introducing different control steps, these infections can be decreased from 55% to 11% (48)

(19).

If MDR bacteria are not managed and tracked properly, they can be easily transmitted via

various sources which include industrial wastage, food products, and agricultural products. The

latter may cause various forms of nosocomial infections, including the vancomycin-resistant

Enterococci (VRE) infection, which is deemed life-threatening in the event of late identification

and diagnosis or inappropriate prescribing. Additionally, methicillin-Resistant Staphylococcus

aureus (MRSA), carbapenem-resistant Acinetobacter baumannii (CRAB) and

carbapenemproducing Enterobacteriaceae (CRE), and Enterobacteriaceae that produce extended-

spectrum β Lactamase (ESBL) are all considered detrimental to the public. Recent surveys have

shown that most of them are resistant to a wide spectrum of antibiotics including the most

advanced ones, requiring urgent interventions. Therefore, this research paper will focus on the

development of an efficient method to detect variants of MDR E. faecium at the molecular level

using analysis of whole-genome sequencing data coupled with CMV4AGPF panel for

determination of ARG including the identification of ARG variants through SNP analysis.

3
Figure 3. Device-associated infection/1000 device days

4
 CHAPTER 2: LITERATURE REVIEW

2.1 Characteristics of Enterococci as Pathogens

In modern-day medicine various microbial agents have become kings to antimicrobial

therapy resistance. Some of the microbes that have become very resistant to different antibiotics

are Enterococci. Enterococci species stain positive on Gram staining, oval and sometimes form

elongated chains and are part of the microflora of the human gastrointestinal system. They can

produce adenosine triphosphate (ATP) both in the presence and absence of oxygen. This is

another feature which helps these microbes survive under tough conditions. In addition to this

they can survive under a variable range of temperatures from 100C to over 450C, and in cases of

high salt concentrations (5). These various virulence factors assist in causing infection and

protecting the bacteria from numerous antibiotics.

Enterococci can cause many diseases that can lead to lethal complications such as death if

not managed adequately. They have been known to cause endocarditis, which is inflammation of

the inner lining of the heart (5). Furthermore, they have been noted to also cause urinary tract

infections, neonatal infections, intra-abdominal infectious and skin-wound infectious (45). These

infections usually occur in the community setting but can be very troublesome if they are

nosocomial.

5
 Enterococcus has been known to be resistant to many antibiotics like vancomycin,

cephalosporin, clindamycin and aminoglycosides. The most common species known to be

resistant to these agents especially in hospital acquired infections are E. faecium and E. faecalis.

These agents are popularly known as vancomycin resistant enterococci (VRE) and are very

difficult to treat because vancomycin is among the strongest antimicrobial agents (64). Only a

few choices are left for management of VRE and a lot of them are not evidence based.

There are a lot of contributing factors which have led to enterococci multi-drug resistance

(MDR) (34). DNA mutations have been documented in several studies (27). The transmission of

these antimicrobial resistance determinants from one generation to another has also led to

stronger Enterococci. Traditionally VRE is usually treated with oxalozolidinone linezolid (LZD)

as a last resort, but already there are now documented cases of widespread resistance against

LZD. This can be attributed to mutations of 23S rRNA in the V domain (27). Some mutations are

related to alcohol-based disinfectants. In E. faecium the mutations are present in genes involved

in carbohydrate uptake and metabolism (44). When comparing E. faecialis with another hospital

adapted strain V583, E. faecilis lacked mobile elements (e.g. Pathogenicity Island, three

separated and interpreted plasmid remnants) but had two clustered, regularly interspaced short

palindromic repeat (CRISPR). CRISPR provides the bacteria with acquired immunity (42). Thus,

the risk of serious nosocomial infections (e.g. ventilator associated pneumonia, surgical site

infections, central line associated bloodstream infections) will be very high with a high mortality

rate (64). As result of these various factors MDR enterococci have emerged and there is a greater

risk of them withstanding the forces of the antibiotics.

6
 For diagnostic purposes, Enterococci is positive on gram-staining. Some other

microbiological features are its ability to grow in 6.5% NaCl, 40% bile salts and 0.1% methylene

blue. The optimum pH is 9.6 and the temperature range for growth is from 100C to 450C and can

resist 30 min at 600C. These bacteria are also catalase negative and facultative anaerobes (31)

(43) (41). In terms genomic testing to assist in identifying the strain and specific genes related

with MDR and VRE the use of polymerase chain reaction (PCR) is recommended. Although this

is not as cost-effective as using selective media it has a higher rate of specificity (62). Some

selective media can also be applied for screening and identification of VRE. Some of examples

of such media are campylobacter medium (CAMPY) and vancomycin screen agar (VSA). In a

study comparing the two CAMPY was both more specific and sensitive comparing to VSA as a

primary plating medium (52). A variety of diagnostic tools can be used to identify these

pathogens, the next target after identification is treatment.

According to current guidelines the only evidence-based option for management of. VRE

are LZD and daptomycin (13) (7). However, the problem is already there have been documented

cases of LZD resistant enterococci strains which means that there is need to seek for other

antimicrobial agents (35) (11). Antibiotics such as tigecycline, teicoplanin and telavancin are the

next line of therapy for MDR enterococci although clinical data is insufficient (7). Enterococci

has great potential to have stronger strains thus as scientists there is need to identify these strains

early and to treat accordingly by any means possible. Overall, although there is antibiotic

resistance mechanism related to DNA mutation and other host factors, more studies are necessary

as new variants of the species are constantly emerging.

7
 A research study revealed 67.5% of Methicillin-resistant Staphylococcus aureus (MRSA),

100% penicillin-resistant Coagulase Negative Streptococci, and a high rate of MDR bacteria such

as E. coli which was around 75.3%, Citrobacter spp. around 100%. Based on this research study,

it can be concluded that it is vital to prevent this kind of bacterial infection in the hospitals

because it can directly affect the immune system of the patients and many times it can be life-

threatening. The situation is serious because the infection rate is continuously rising, and more

and more patients are getting affected by nosocomial infections (16). Even though this research

was conducted to identify multidrug-resistant bacteria and ways to prevent them from spreading

a complete characterization of resistance mechanisms was not conducted which can be

characterized as the limitation of this study. In order to stop the infection and analyse various

MDR bacteria strains, there is room for further development in analysing techniques. Therefore,

it is recommended to conduct further research on this aspect of the issue.

2.2 Enterococci as a Leading Cause of Nosocomial Infections

Enterococci bacteria are known for endocarditis that can cause death without an effective

treatment (30) (32). Nosocomial infections due to Vancomycin-Resistant Enterococci (VRE)

have become the main issue for many years. VRE infection affects human health and it has

become globally now which has increased the economic burden for several countries. In order to

control the spread of the infection a major portion of the budget and health care services needs to

be focused on the spending to prevent this infection. One of the major reasons for patients

reported for a longer stay in hospitals is because of nosocomial infections. (45) (40)

8
 A European study was conducted on patients who were either diagnosed with nosocomial

infection VRE or vancomycin-susceptible enterococci (VSE) within a period of 3 years. (11)

According to this study, the median attributable cost to treat the vancomycin infection was

approximately 13,000 EUR, and it indicated that VRE infection increases the hospital cost that

can ultimately increase the burden on the individual and government. (11) The proportion of

VRE has increased rapidly from less than 5% in 2001 to around 14.5 % in 2013 in Germany

(Table 1) (7). In the USA, the survey data shows that out of all the nosocomial infection reported

to the National Health care department, 3% of them were due to VRE. (8) (24)

Table 1. Cost and reimbursement data for nosocomial VRE and VSE infections (11)

2.3 Importance of Single Nucleotide Polymorphism (SNP) in Identification of MDR Bacteria

As per various studies discussed previously, E. faecium is a major cause of nosocomial

infection. (54) (59) Vancomycin-resistant E. faecium (VREfm) increased rapidly during the

1990s especially in the United Kingdom (47). The main source of the resistant gene was

9
livestock, and this is directly linked with the use of avoparcin in Europe along with vancomycin

as a growth factor (6) (37). The addition of avoparcin in livestock was banned in Europe after

many cases reported of direct connection of MDR resistant bacterial transmission through

livestock consumption. After avoparcin was banned in 1997, the subsequent cases regarding the

transmission of MDR bacteria noticeably dropped (1). However, even after the ban, VREfm did

not disappear from the livestock, and traces could still be found. (11) (57) Therefore, it is vital to

identify VREfm through advanced and easy techniques, out of which single nucleotide

polymorphism (SNP) is the most reliable and advanced solution. (18)

A study was conducted in England to isolate E. faecium from 29 farms including nine

poultry, ten cattle, and ten pig’s livestock between 2014 and 2015 (Figure 4). Figure 4A indicates

the East of England region of the UK with the location of farms. Wastewater in the figure is

indicated with blue water drops, while hospitals are indicated with H. Farm signs indicate which

animals are in captivity (for example, livestock). Figure 4B show the animal species. A total 256

livestock and meat isolates based on 99,377 SNPs in 1,523 core genes were identified with

ampicillin and vancomycin resistance (18) (55). Table 2 shows the farm characteristics, listed

according to the same date. According to the study, out of 253 livestock samples and E. faecium

isolates, 52% of the livestock were found to be ampicillin resistant (46) (51). However, the main

limitation of this study is the lack of human carriage isolates for the sample. Other than that,

another source of wastewater was municipal water waste which may have human gut bacteria.

(14) (50).

10
 To conclude, the study confirmed the direct connection between the presence of

antibiotics in livestock and human infection. It also showed that the major transmission of

bacteria in human and nosocomial infection is directly propositional to the consumption of food

additives and livestock (26). It can be concluded that SNPs identification is an important and

advanced method to identify MDR bacterial strain.

2.4 Other Techniques to Identify Nosocomial Infection

The other technique method is based on the study conducted in Germany. Nosocomial

infection is not a common matter because it is leading to a rise in death and morbidity in patients.

(17) (4).

Figure 4. Isolation of E. faecium from 29 farms conducted in England from 2014 to 2015

11
 In modern-day medicine various microbial agents have become kings to antimicrobial

therapy resistance. Some of the microbes that have become very resistant to different antibiotics

are Enterococci. Enterococci species stain positive on Gram staining, oval and sometimes form

elongated chains and are part of the microflora of the human gastrointestinal system. They can

produce adenosine triphosphate (ATP) both in the presence and absence of oxygen. This is

another feature which helps these microbes survive under tough conditions. In addition to this

they can survive under a variable range of temperatures from 100C to over 450C, and in cases of

high salt concentrations. These various virulence factors assist in causing infection and protecting

the bacteria from numerous antibiotics.

2.5 Hypothesis of the Study

We hypothesize that when the recognition of single nucleotide variants related to the drug

resistance phenotype in E. faecium will occur, then we can design a specific primer for

prevention and quick diagnosis.

12
Table 2. Characteristics of farms studied, including the kind and number livestock in captivity, E. faecium positive, AREs, and VREs

13
2.6 Rationale of the Study

Several studies have associated drug-resistant bacteria such as E. faecium with numerous

cases of hospital-acquired pathogenic infections. Approximately 12% of these infections are

being caused by Enterococcus species, and around 90% of clinical infections are being spread by

this species. Therefore, it is very important to identify the single nucleotide variants related to

the drug resistance phenotype of E. faecium strains. Moreover, research studies have also proven

that E. faecium isolates found from urinary tract infection cases and from poultry can enter the

human body. The development of these drug-resistant bacteria was due to the high amount of

antibiotics being carelessly consumed by the populace (63). Significantly, antibiotic resistance

increases infection-related mortality and morbidity, and as a result, extended hospital stays and

need for more costly drugs contribute to increasing costs of healthcare. It is therefore necessary

to develop methods to prevent as well as treat infections caused by antibiotic-resistant bacteria.

Additionally, antibiotic resistance by E. faecium is mainly due to its hypermutable sequences

which invariably mediates its rapid adaptation to different conditions (29). Although resistance

to various antibiotics has been strongly linked to substitutions in nucleotide bases, it is suggested

that other factors are associated as well (38). This has been a major concern in America

as incidence of infections due to ampicillin-resistant E. faecium has become heightened, and this

is associated with continuous rise in MICs (38, 22). It has also been suggested that Enterococcus

species predominates the gastrointestinal tract (GIT) during drug therapy and this serves as a

ground for disseminating to other internal organs (8). In order to prevent this, it is important to

understand the mechanism involved in GIT colonization by this bacterium. Therefore,

recognition of single nucleotide variants in enterococcal genes differing in their GIT colonization

mechanism can be a vital breakthrough in prevention and quick diagnosis of infections due to

14
E. faecium. Interventions for infections resulting from multidrug resistance could be

possible if it is diagnosed at an early stage. Methods based on Polymerase Chain Reaction

provide a helpful hand in detecting the SNPs for rapid diagnosis, hence the need for this

study.

15
 CHAPTER 3: MATERIALS AND METHOD

3.1 Sample Collection

Samples of blood, stool, urine, wound discharge were collected from blighted

immunosuppressed patients at Moffit Cancer Center (MCC) on the West coast of Florida, in

Tampa, Florida, and kept it in a sterile tube to be analyzed in a microbiology laboratory. The

identification of bacteria was conducted using colony characteristics, Gram reaction, and various

bacterial tests, and each isolate was checked using Mueller-Hinton Agar (MHA). A separate

structured questionnaire was also conducted in order to collect further information about the

patients. This data was then entered and analyzed in an SPSS version 23 software to assess the

results.

3.2 Illumina Sequencing Method

The Illumina sequencing was mainly divided into 4 steps: sample preparation, cluster

generation, sequencing, and data analysis. Sample preparation started with DNA extraction and

purification. In Nextera®, the first step for sample preparation was tagmentation. Transposomes

fragmented the DNA and tagged the input DNA with adapters. Afterwards, reduced cycle

amplification took place. The second step was clustering in which each molecule was

isothermally amplified on a flow cell glass plate, and each plate had coadded two types of the

16
oligos. Hybridization occurred, with attachment to the existing oligo primer. Thereafter, reverse

transcription took place with the help of polymerase, and the original strand was washed away.

After that, the bridge amplification process started, and it continued to generate strains and

millions of clusters. Only forwarded strains remained on the plates, with the reverse strains

removed from the channel site. Finally, the sequencing step occurred, beginning with the

sequencing primer and 4 nucleotides amplified to the strain, with the laser technique reading the

sequence (21).

3.3 Library Sample Preparation

The Illumina library preparation kit, Nextera® XT, is made for sequencing genomes,

plasmids, and amplicons, and was picked for this method. The Nextera® XT sequencing kit is a

rapid method for sequencing samples, which can multiplex up to 96 samples with minimal DNA

required. Samples have been included with a read length(L) of 2*75 paired-end sequencing on

an illuminaMiseq sequencer (33)

3.4 Paired-ended Sequence Analysis using BWA Software

The analyzed fragmented sequences using the Illumina sequencing method was further

mapped back with the reference genome using the help of Burrows-Wheeler Alignment tool

(BWA) software. The reference genome used in this method was the pre-existing GenBank

(Accession ID: NCBI Ef_aus00233). All DNA samples were mapped back and arranged

according to the model of E. faecium genome.

17
3.5 Description of the BWA Software

BWA is a Burrows-Wheeler Alignment Tool. This software package helps to map low-

divergent sequences in comparison to the entire reference genome, which can be large. BWA

software works based on 3 algorithms known as BWA-MEM, BWA-SA, and BWA-backtrack.

This algorithm is classified based on the capacity to read the number of base pairs (bp). The

BWA-backtrack is made to read up to 1000 bp analysed by Illumina sequences. In contrast to

this, BWA-SW and BWAMEM can read longer sequences with the size range of 70 bp to 1M

bp. Of the algorithms, BWAMEM is highly recommended because it is faster, has greater

accuracy and is more suitable for high-quality queries. The flow-graph is presented in Figure 5.

3.6 SNP Calling

Bioinformatics had been done using advanced tools such as SAMtools and BCtools,

where SAM stands for Sequence Alignment. SNP calling is a technique that compares the

sequenced data with the reference genome and identifies the variable sites. SNP calling method

in the first step analyses the data image and base pairs, while in the second step it reads the

mapping and removes the duplicate reads and realign the reads if necessary (Figure 6).

Additionally, it also recalibrates the quality score. After the removal of duplicate reads, SNP

calling read works on the number of samples calling single-sample calling and multi-sample

calling. The singe-sample calling technique directly identifies single nucleotide polymorphism

and related genotypes with the help of single-sample analysis. Multi-sample calling uses a non-

18
linkage-based and refined candidate. The final step for both techniques is the same, with SNP

filtering and score recalibration (39).

Performance Analysis methodology

Figure 5. BWA flow graph. Two-step method includes (a) step 1: building the BWT index from the reference
genome; and (b) step 2: alignment of the reads against the reference using either BWA aln, or (c) BWA mem

19
 SNP calling flow graph

Figure 6. SNV calling flow graph. Two-step method includes (a) step 1: converting BWA. sam output to. bam and
.bcf using samtools; and (b) step 2: variant calling using bcftools (b)

3.7 Antimicrobial Susceptibility Test

The antimicrobial susceptibility test protocol by Limayem et al. was followed (31).

Briefly, 3-5 colonies from Sheep Blood Agar plates were picked and emulsified in sterile

deionized water. Cell population was adjusted to 0.5 McFarland Standard. A 30 mL of inoculum

was transferred to Mueller-Hinton broth. Assay plates dosed with different antibiotics were

inoculated with E. faecium. The antibiotics used were gentamycin, penicillin, tetracycline,

linezolid, daptomycin, vancomycin, and virginiamycin M1. The plates were then incubated at

35°C for 24 h.

20
 CHAPTER 4: RESULTS

4.1 Results

A total of eight E. faecium strains were mapped and identified using the BWA software

(Table 3). Each strain had different total number of reads, but the strains did not differ % mapped

and % paired data. The eight strains were further characterized based on their resistance to

several antibiotics used in this study. Moreover, increasing concentrations of the antibiotics were

used to detect the spectrum of each E. faecium strain. All strains were found to be multi-drug

resistant (Tables 4-11). Particularly, all strains were resistant to penicillin (≤ 100 μg/mL, Table

5) and daptomycin (≤ 100 μg/mL, Table 7). The strains differ in the maximum dosage of

gentamycin, tetracycline, and linezolid. For gentamycin, strains 1406 and 1407 became

susceptible at ≤ 100 μg/mL, while strain 1451 at ≤ 200 μg/mL (Table 4). Meanwhile, all strains

became susceptible at ≤ 100 μg/mL tetracycline (Table 6). At ≤ 10 μg/mL linezolid, strains 1431,

1444, and 1451 became susceptible (Table 7). Interestingly, all strains were resistant to

vancomycin (≤ 100 μg/mL), except for strain 1451 (Table 9). Moreover, all strains were

susceptible to virginiamycin M1 (Table 10).

21
Table 3. Sequence analysis of the eight E. faecium strains using BWA software
Sample Total no. of % Mapped % Paired data Identification
sequences reads data (E. faecium
strain)

S1 5701654 79.13 65.08 1451

S2 6258692 85.00 67.95 1450
S3 5817574 81.39 65.79 1444
S4 6042658 84.49 69.15 1443
S5 5582636 84.74 66.71 1438
S6 7000606 84.50 66.10 1432
S7 8509322 86.47 72.95 1406
S8 12260054 81.69 78.29 1407

Table 4. Resistance or susceptibility of E. faecium strains in varying concentration of gentamycin

E. faecium Gentamycin concentration, μg/mL
strains
1 5 10 15 25 50 100 200
1406 + - + - + + - -
1407 + - + - + + + +
1432 + - + - + + - -
1438 + - + - + + + +
1443 + - + - + + + +
1444 + - + - + + + +
1450 + - + - + + + +
1451 + - + - + + + -

22
Table 5. Resistance or susceptibility of strains in varying concentration of penicillin
E. faecium Penicillin concentration, μg/mL
strains
1 5 10 15 25 50 100
1406 + - + - + + +
1407 + - + - + + +
1432 + - + - + + +
1438 + - + - + + +
1443 + - + - + + +
1444 + - + - + + +
1450 + - + - + + +
1451 + - + - + + +

Table 6. Resistance or susceptibility of E. faecium strains in varying concentration of tetracycline

E. faecium Tetracycline concentration, μg/mL
strains
1 5 10 15 25 50 100 200
1406 + - + - + - - -
1407 + - + - - - - -
1432 + - + - + + - -
1438 + - + - + + - -
1443 + - + - + + - -
1444 + - + - + + + -
1450 + - + - + + - -
1451 + - + - + - - -

23
Table 7. Resistance or susceptibility of E. faecium strains in varying concentration of linezolid
E. faecium Linezolid concentration, μg/mL
strains
1 5 10 15 25 50 100
1406 + - + - + + +
1407 + - + - + + +
1432 + - - - - - -
1438 + - + - + + +
1443 + - + - + + +
1444 + - - - - - -
1450 + - + - + + +
1451 + - - - - - -

Table 8. Resistance or susceptibility of E. faecium strains in varying concentration of daptomycin

E. faecium aptomycin concentration, μg/m L
strains
1 5 10 15 25 50 100
1406 + - + - + + +
1407 + - + - + + +
1432 + - + - + + +
1438 + - + - + + +
1443 + - + - + + +
1444 + - + - + + +
1450 + - + - + + +
1451 + - + - + + +

24
Table 9. Resistance or susceptibility of E. faecium strains in varying concentration of vancomycin
E. faecium Vancomycin concentration, μg/mL
strains
1 5 10 15 25 50 100 200
1406 + - + - + + + +
1407 + - + - + + + +
1432 + - + - + + + +
1438 + - + - + + + +
1443 + - + - + + + +
1444 + - + - + + + -
1450 + - + - + + + +
1451 - - - - - - - -

Table 10. Resistance or susceptibility of E. faecium strains in varying concentration of virginiamycin M1

E. faecium Virginiamycin M1 concentration, μg/mL
strains
1 5 10 15 25 50 100
1406 - - - - - - -
1407 - - - - - - -
1432 - - - - - - -
1438 - - - - - - -
1443 - - - - - - -
1444 - - - - - - -
1450 - - - - - - -
1451 - - - - - - -

25
 The total and antibiotic-specific SNPs per E. faecium strain were also identified. A total

of 15, 076 SNPs were identified for all E. faecium strains (Table 11). High incidence of SNP was

observed in A>G, C>T, G>A, and T>C, indicating that the polymorphism occurred within

purinepurine and pyrimidine-pyrimidine nucleotides (Figure 7a). Among these SNPs, 263 were

identified as INDELs (Table 11, Figure 7b). As observed in Figure 7b, there was an equal

distribution on the counts for deletion and addition of nucleotides.

On the other hand, roughly 10,000 SNPs were observed related to antibiotic specificity

for each E. facecium strains, and these SNPs were distributed to gentamycin, penicillin,

lincomycin, vancomycin, and tetracycline (Table 12). Antibiotic-specific SNPs were not detected

from strain 1407. Gentamycin-related SNPs for all strains were 0.79-1.10% of their total SNPs,

while penicillin-related SNPs were 26.63-36.81%. Low SNPs were observed for lincomycin,

vancomycin, and tetracycline for all strains. It should be noted that all strains have the same

number of SNPs for the antibiotics, indicating that the antibiotic-related SNP is species-specific

and not strain-specific. Table 13 shows the summary of total SNPs and the antibiotic-specific

SNPs observed per E. faecium strain.

Interestingly, several resistance genes were identified from the eight E. faecium

isolates. Presented in Table 14, the antimicrobial resistance genes were observed to function

differently. As mentioned above, not all strains harbored the gene specific for an antibiotic,

therefore the table only shows the information per each gene.

26
Table 11. Total number of SNPs and INDELs in E. faecium strains
Sample sequence E. faecium strain No. of SNPs
S1 1451 8716
S2 1450 7689
S3 1444 6756
S4 1443 7772
S5 1438 8730
S6 1432 6271
S7 1406 8411
S8 1407 5305
Total no. of SNPs 15076
Total no. of INDELs 263

Table 12. Individual number of antibiotic SNPs in E. faecium strains

Antibiotics Number of SNPs Possible E. faecium strains
Gentamycin 27 1407, 1438, 1443, 1444, 1450, 1451
Penicillin 1439 All strains
Tetracycline 648 1444
Linezolid 0 1407, 1438,1443, 1450
Vancomycin 2 1407, 1406, 1432, 1438, 1443, 1444,
1450

27
Figure 7. Total SNPs of E. faecium strains. (a) Incidence of nucleotide polymorphism, and (b) incidence of insertion
or deletion of nucleotide

Table 13. Antibiotic-specific SNPs of E. faecium strains

E. Total
faecium SNPs
strains

1406 10, 398 87 2, 903 26 2 1, 070

1407 ND ND ND ND ND ND
1432 7, 886 87 2, 903 26 2 1, 070
1438 10, 808 87 2, 903 26 2 1, 070
1443 9, 940 87 2, 903 26 2 1, 070
1444 8, 667 87 2, 903 26 2 1, 070
1450 9, 737 87 2, 903 26 2 1, 070
1451 10, 900 87 2, 903 26 2 1, 070

28
Table 14. List of resistance genes in E. faecium isolates
Antimicrobial Antibiotic Site of resistance Mechanism of action
resistance gene

liaFSR Daptomycin Cell membrane Altered target

PBP5 Penicillin Cell wall Decreased affinity for
PBP5
VAN A, VAN B Vancomycin Cell wall Altered target
cfr Linezolid Ribosomes Mutations in genes
APH(2’)-IC Gentamycin Ribosomes Inactivates all AG
except streptomycin

tet K, tet L Tetracycline - -

vatD, vatE Virginiamycin Protein Block translocation of
protein synthesis

29
 CHAPTER 5: DISCUSSIONS

5.1 Discussions

The study was conducted to verify the antibiotic resistance gene present in E. faecium by

PCR amplification using specific primers. Moreover, the study was done to identify and isolate

MDR bacteria in hospitals to prevent future infections. The result of the study strongly suggests

that E. faecium are highly resistant to different antibiotics, and there is the presence of antibiotics

resistance genes which enhance the danger the bacteria poses to humans. High antibiotic-

resistance was found to penicillin, daptomycin, and vancomycin. These three antibiotics disrupt

the cell wall and cell membrane, usually of Gram-positive bacteria as they have thicker cell

membranes. Penicillin is a well-known β-lactam antibiotic, while daptomycin is helpful in the

diagnosis of infections that occur due to multidrug-resistant Gram-positive strains. Resistance to

daptomycin is linked with significant ultrastructural adjustments in the cell envelope and septal

apparatus (20).

The accelerated evolution of the resistant genus Enterococcus is linked with major global

health risks. The overuse of antibiotics is one of the major reasons for the development of

multidrug resistance in bacteria. This research examined the prevalence of enterococcal species

from clinical samples and the trend of antimicrobial susceptibility that they show. Multidrug

resistance in bacteria is mostly triggered by gene accumulation and each of them codes for

30
resistance to one specific drug in R plasmids. The resistance genes accumulate over a singular R

plasmid through the structures offered by transposons, integrons, and ISCR (38). Integrons are

particularly strong in generating multidrug resistance as they combine multiple resistance genes

together in a proper arrangement and provide a powerful promoter for their expression (38). In

fact, if inserted into an integron, the resistance gene is labeled so that it can quickly become a

component of another integron. Presumably, several of the resistance genes evolve and originate

in the antibiotic-producing bacteria that must protect themselves against the antibiotics

generated.

Another form of multidrug resistance is through multidrug efflux pumps. The RND

(Resistance-Nodulation-Division) family pumps in Gram-negative bacteria are particularly vital

as they are generally encoded in chromosomal genes which can pump out several antibiotics that

are in use (38). These interactions can intensify in some Gram-negative species when there is a

reduction in the permeability of the outer membrane which is usually caused by mutations in

porin genes (38). Pathogenic microorganisms can persist in patients who are treated with

antibiotics because they might undergo a psychologically resistant condition with no genetic

modifications (38).

31
 CHAPTER 6: CONCLUSION

6.1 Conclusion

Enterococci mostly inhibit human gastrointestinal tracts and are identified as one of the

major causes of many infections. They have the capability of resistance to antimicrobial and

make the treatment very challenging. This research was conducted to identify and isolate MDR

bacteria in hospitals to prevent future infections. Eight strains of the multidrug-resistant

Enterococcus faecium (MEF) was gathered from the blighted immunosuppressed patients at

Moffit Cancer Center (MCC) on the West coast of Florida. Results showed the presence of

antibiotic resistance genes in E. faecium. High resistance was found in penicillin, daptomycin,

and vancomycin. Moreover, there were observed strain-specific antibiotic resistance in

virginiamycin M1. The study found variations in bacterial genes that are responsible for the

multidrug resistance, especially in antibiotics. Numerous studies have reported several cases of

MDR using different molecular methods including PCR detection kits. This study was able to

add new direction by identifying SNP variants found on the most virulent MDR, E. faecium,

isolated from a local hospital. This study provides information for future development of rapid

diagnostic tests that will be useful in the prevention of the spread of nosocomial infections for

the good of today’s and next generation.

32
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