Genome Sequencing Of Mycobacterium Tuberculosis Biology Essay

Published:

This essay has been submitted by a student. This is not an example of the work written by our professional essay writers.

The rate of multidrug-resistant and extensively drug-resistant tuberculosis has been steadily increasing worldwide. The availability of rapid and reliable methods for the detection of drug resistance to second-line drugs is vital for adequate patient management. In this study TB isolates were collected from countries with high TB burden: India, Moldova, Philippines, and South Africa. These isolates were sequenced with three different technologies: Sanger, Pyrosequencing for validation, and Pacific Biosciences whole genome sequencing. Sanger sequencing was done on the three genes associated with drug resistance to injectable antibiotics, kanamycin (KAN), capreomycin (CAP) and amikacin (AMK): rrs, tlyA and eis promoter. It was found that 87.5% of AMK resistance can be explained by rrs A1401G mutation only, with specificity of 100%, 88.73% of CAP resistance can be explained by rrs A1401G mutation only, with specificity of 96.2%, and 83.93% of KAN resistance can be explained by combination of rrs A1401G and eis promoter C-12T mutations, with specificity of 99.21%. Mutations in tlyA were not found to be associated with resistance. There are about 6-10% of resistant isolates that don't have any mutations in rrs nor eis promoter. Whole genome sequencing will help to identify novel mutations outside of the regions sequenced by Sanger and gene and functional analysis will help to relate these mutations to the mechanism of resistance to the injectable antibiotics.

Introduction

In 2011, 8.7 million people became ill with tuberculosis (TB) and 1.4 million died from TB worldwide1. While the incidence and death rate of TB is declining, the emergence of multidrug-resistant TB (MDR-TB) and extensively drug-resistant (XDR-TB) has become a global health problem threatening TB control programs. MDR-TB strains are defined to be resistant to at least the two first-line antibiotics: isoniazid (INH) and rifampicin (RPM). XDR-TB strains are defined as MDR-TB with additional resistance to any fluoroquinolone (FQ) and to at least one of the three injectable second-line antibiotics: kanamycin (KAN), amikacin (AMK), and capreomycin (CAP). There were about 310,000 cases of MDR-TB and about 30,000 of XDR-TB in the world in 20112. To effectively treat MDR-TB and prevent XDR-TB, it is important to be able to rapidly detect resistance to second-line antibiotics. Resistance to anti-tuberculosis antibiotics is related to mutations that affect the function or expression of genes that have been associated with drug resistance.

The molecular mechanisms of action of injectable antibiotics and resistance to these antibiotics have been elucidated. Amikacin (AMK) is a semi-synthetic aminoglycoside antibiotic derived from kanamycin (KAN) A. AMK differs from KAN in that it contains an L-hydroxyaminobuteroyl amide group substitution in the N1 position of the deoxystreptamine ring. Similar to other aminoglycosides, AMK and KAN bind to 16S rRNA in the 30S ribosomal subunit and inhibit protein synthesis. They cause mistranslation by inhibiting translocation during protein synthesis. By binding to four nucleotides of 16S rRNA and a single amino acid of protein S12, aminoglycosides interfere with decoding site in the vicinity of nucleotide 1400 in 16S rRNA of 30S subunit. Since this region interacts with the wobble base in the anticodon of tRNA, this leads to interference with the initiation complex, misreading of mRNA so incorrect amino acids are inserted into the polypeptide leading to nonfunctional or toxic peptides 3.

Capreomycin (CAP) is a cyclic peptide which binds to 70S ribosome between helix 44 (h44) of the 16S rRNA and helix 69 (H69) of 23S rRNA and prevents translocation of ribosome along mRNA, thus interfering with translation. The structures of these complexes suggest that capreomycin inhibits translocation by stabilizing the tRNA in the A site in the pre-translocation state.4

The three genes that have been associated with resistance to injectable antibiotics are tlyA, eis promoter, and rrs. tlyA encodes a 2'-O-methyltransferase that modifies nucleotide C1409 in helix 44 of 16S rRNA and nucleotide C1920 in helix 69 of 23S rRNA. Capreomycin resistance has been shown to result from inactivation of TlyA, which reduces the ability of capreomycin to interact with its target.5 Mutations in tlyA gene associated with CAP resistance are reported to be rare (1-3%) but are highly specific.6

rrs gene encodes 16S rRNA. The most common mutations associated with resistance are A1401G and G1484T. A1401G mutation is most commonly observed among AMK, CAP and KAN resistant isolates with moderate sensitivity. It is highly specific for AMK and KAN resistance and less specific for CAP resistance.6

Enhanced intracellular survival protein (eis) improves the survival of Mycobacterium smegmatis in macrophages7 and functions as aminoglycoside acetyltransferase that acetylates and inactivates KAN and AMK in Mtb. Eis acetylates KAN at a rate of 3.3 higher than AMK. Mutations in the promoter region of eis have been shown to increase expression of eis gene and the overexpression of eis confers KAN resistance in Mtb but not AMK resistance.8 The most frequently reported eis promoter mutations associated with KAN resistance are G-10A, C-14T and C-12T.6

In this study resistant and susceptible isolates were sequenced and analyzed for the mutations responsible for drug resistance to injectable antibiotics. The isolates were taken from four countries with high TB burden: India, Moldova, Philippines, and South Africa. First, the three genes were sequenced with Sanger sequencing to identify mutation in the "hot spots," then whole genome sequencing using Pacific Biosciences platform was performed in order to identify mutations outside of the three genes. The objective of this study is to identify novel mutations in Mtb strains resistant to injectable antibiotics by using the Sanger data of the three genes rrs, tlyA and eis promoter and the whole genome sequencing data (WGS) to identify novel mutations outside of the three genes. The study also aims to identify the genes and their functions that have mutations and relate them to the mechanism of action of the injectable drugs.

Completed Work

I. Sanger and Pyro Sequencing Analysis

Analysis of the Sanger data was completed on the tree genes associated with Mtb resistance to injectable antibiotics. The rrs gene was sequenced in the region 1473200 - 1473382 (gene position 1355 - 1537), which is about 11.9% of the rrs gene. A total of 255 AMKR, 184 AMKS, 214 CAPR, 185 CAPS, 281 KANR and 128 KANS isolates were sequenced for rrs gene. The tlyA gene was sequenced in the region 1917940 - 1918359 (gene position 1 - 388), which is about 52% of the gene. A total of 155 AMKR, 167 AMKS, 148 CAPR, 164 CAPS, 207 KANR and 115 KANS were sequenced for tlyA gene. The eis promoter was sequenced in the region 2715085 - 2715415 (gene position -82 - 248), which is 82 bases in the promoter region and 20.5% of the eis gene. A total of 230 AMKR , 184 AMKS , 219 CAPR , 185 CAPS , 286 KANR , and 128 KANS were sequenced for eis promoter. The sensitivity and specificity were calculated for each mutation per each drug per country and cumulative. Sensitivity was calculated as a percentage of resistant isolates with mutation out of the total resistant isolates tested. Specificity was calculated as percentage of sensitive isolates without the mutation out of the total susceptible isolates tested. Ideally, both sensitivity and specificity should be equal to one; however this is often not the case. It is important to rank the mutations as well as combinations of mutations based on their sensitivities and specificities. It is also important to look at both sensitivity and specificity. If the mutation is present in high number in both the resistant and susceptible isolates, it is unreliable as a marker for resistance.

In tlyA gene, A33G mutation was found in high numbers in both resistant and susceptible isolates with total sensitivity of 97% and specificity of 4% for the three drugs and therefore it is not associated with resistance. Other mutations found are: G165A, C40T, G94C, C250G, A565G, G516A, A537C, and G368C. These mutations had sensitivity of 0 or less than 1% and were not found to be associated with resistance to either of the injectable antibiotics.

In rrs gene, A1401G mutation was found in high frequency among resistant isolates with sensitivities of 86.45%, 68.33%, and 85.33% for CAP, KAN, AMK respectively, and specificities of 96.22%, 100% and 100% for CAP, KAN, AMK respectively. Other mutations found among resistant isolates are G1484T with 2% sensitivity and 100% specificity for the three drugs, 1 resistant isolate had a combination of A1401G, A1462T, and A1486T. In this isolate A1401G is most likely responsible for resistance. There are 4 isolates where A1401G mutation was found with pyro sequencing but was either not sequenced or not found with Sanger. Adding these isolates to the Sanger analysis increases the sensitivities of A1401G mutation to 88.32%, 69.75%, and 87.11% for CAP, KAN, AMK respectively. Some interesting trends can be seen when these statistics for A1401G mutation are broken down by country (Table 1). In South Africa A1401G mutation was the only mutation found (sensitivity 94.12%, 93.10%, 90.00% for CAP, KAN, AMK respectively, and specificity 100% for the three drugs). In addition, whereas in India, Philippines, and South Africa the sensitivities are above 80% for the three drugs, in Moldova the sensitivities are 62.50%, 69.75%, and 26.32% for CAP, KAN, AMK respectively with 100% specificities to the three drugs. This suggests that A1401G mutation is not as good indicator for resistance to injectable antibiotics in Moldova as it is in other countries.

The mutations found in eis promoter in resistant isolates are C-12T (sensitivity 3.65%, 14.34%, 1.3% and specificity 81.62%, 99.22%, 78.80% for CAP, KAN, AMK respectively), C-14T (sensitivity .46%, 3.85%, 4.35% and specificity 94.59%, 100%, 94.59%), A-43T (sensitivity less than 1% and specificity 100% for CAP, KAN, AMK), and G-10A (sensitivity less than 1% for AMK and CAP and 2.1% for KAN and specificity of 100%). Some interesting trends can be seen in Moldova. C-12T mutation is only found in Moldova where it's sensitivity is 25.00%, 53.95%, 9.68% and specificity is 41.38%, 92.86%, 33.90% for CAP, KAN, AMK respectively.

Additional analysis was done on rrs A1401G and eis promoter C-12T mutations to see whether they are mutually exclusive (Table 2). This analysis was done only on isolates that were sequenced for both rrs and eis promoter. Only two isolates have both A1401G and C-12T mutations and the rest are mutually exclusive. By adding up the sensitivities, this analysis suggests that 87.5% of AMK resistance can be explained by A1401G mutation only, with specificity of 100%, 88.73% of CAP resistance can be explained by A1401G mutation only, with specificity of 96.2%, and 83.93% of KAN resistance can be explained by combination A1401G and C-12T mutations, with specificity of 99.21%. There are also 15 AMK resistant, 16 CAP resistant, and 27 KAN resistant isolates that have no mutations in either rrs or eis promoter, which suggests that there are maybe other mutations responsible for resistance outside of the regions sequenced by Sanger.

II Pacific Biosciences WGS Data Processing

A total of 366 isolates were sequenced with PACBIO RS sequencer using single molecule, real time (SMRT) technology. A total of 215 AMKR, 130 AMKS, 206 CAPR, 139 CAPS, 268 KANR and 77 KANS isolates were sequenced. A total of 187 isolates for rrs gene, 58 isolates for eis promoter and 248 isolates for tlyA gene were sequenced by both Sanger and PacBio. The reads from the sequencer were aligned to a reference, M. tuberculosis H37Rv (NCBI accession number NC_000962.2), by using SMRT portal's Basic Local Alignment with Successive Refinement (BLASR)9 algorithm. The alignment files were produced in binary alignment/map (BAM) format. The quality scores in the alignment files were recalibrated and converted into Phred quality scores by using The Genome Analysis Toolkit (GATK, version 1.4-17-gd5199db).10 SAMtools (version 0.1.18) was used to convert files in BAM format into pileup format and variant calling was done with VarScan's (version 2.3)11 pileup2snp algorithm. A relation database was built using MySQL, containing information for all of the Pacbio mutations, Sanger mutations, and DST for each isolate, as well as the gene information obtained from Rapid Annotation using Subsystem Technology (RAST, version 4.0)12 and TB database (tb-db)13. The database will be used to perform whole genome analysis of the data.

Work Remaining

The analysis of WGS data remains to be done. I plan on performing sensitivity and specificity calculations for each mutation in the WGS data for each of the injectable antibiotics as cumulative across the four countries and also broken down by country. It will be important to compare the mutations found by Sanger, PacBio and Pyro sequencing and look at the mutations outside of the regions sequenced by Sanger. I will use the gene data from tb-db and RAST to learn which genes the mutations strongly associated with resistance belong to. I'm also planning to research the functions of these genes and how they might be involved in drug resistance to injectable antibiotics. There are certain challenges to this task. Most of the isolates that were sequenced are MDR (299) out of which 241 are also XDR and there are zero isolates that are resistant only to the injectables. It will be important to distinguish between the mutations responsible for resistance to injectables from the mutations responsible for resistance to other antibiotics. In addition, about 40% of Mtb genome has not been annotated yet and it will be a challenge to identify the function of resistance if mutations fall within the genes that are not annotated.

Table 1. rrs A1401G mutation sensitivity and specificity broken down by country

 

CAP

AMK

KAN

Country

Sensitivity

Specificity

Sensitivity

Specificity

Sensitivity

Specificity

India

90.91

91.30

87.23

100.00

82.83

100.00

Moldova

62.50

100.00

64.52

100.00

26.32

100.00

Philippines

100.00

95.60

100.00

100.00

81.25

100.00

South Africa

94.12

100.00

93.10

100.00

90.00

100.00

Table 2. Analysis of rrs A1401G and eis promoter C-12T mutations

AMK

Number of Resistant Isolates

Number of Susceptible Isolates

Sensitivity

Specificity

rrs A1401G only

194

0

86.61

100.00

eis C-12T)

1

39

0.45

78.69

both rrs A1401G AND eis C-12T

2

0

0.89

100.00

other rrs or eis mutations

16

10

7.14

94.54

NO mutations in rrs NOR eis

15

134

6.70

26.78

Total

228

183

CAP

rrs A1401G only

187

7

87.79

96.20

eis C-12T)

6

34

2.82

81.52

both rrs A1401G AND eis C-12T

2

0

0.94

100.00

other rrs or eis mutations

6

19

2.82

89.67

NO mutations in rrs NOR eis

16

124

7.51

32.61

Total

217

184

KAN

rrs A1401G only

194

0

69.29

100.00

eis C-12T)

39

1

13.93

99.21

both rrs A1401G AND eis C-12T

2

0

0.71

100.00

other rrs or eis mutations

22

4

7.86

96.85

NO mutations in rrs NOR eis

27

122

9.64

3.94

Total

284

127

Writing Services

Essay Writing
Service

Find out how the very best essay writing service can help you accomplish more and achieve higher marks today.

Assignment Writing Service

From complicated assignments to tricky tasks, our experts can tackle virtually any question thrown at them.

Dissertation Writing Service

A dissertation (also known as a thesis or research project) is probably the most important piece of work for any student! From full dissertations to individual chapters, we’re on hand to support you.

Coursework Writing Service

Our expert qualified writers can help you get your coursework right first time, every time.

Dissertation Proposal Service

The first step to completing a dissertation is to create a proposal that talks about what you wish to do. Our experts can design suitable methodologies - perfect to help you get started with a dissertation.

Report Writing
Service

Reports for any audience. Perfectly structured, professionally written, and tailored to suit your exact requirements.

Essay Skeleton Answer Service

If you’re just looking for some help to get started on an essay, our outline service provides you with a perfect essay plan.

Marking & Proofreading Service

Not sure if your work is hitting the mark? Struggling to get feedback from your lecturer? Our premium marking service was created just for you - get the feedback you deserve now.

Exam Revision
Service

Exams can be one of the most stressful experiences you’ll ever have! Revision is key, and we’re here to help. With custom created revision notes and exam answers, you’ll never feel underprepared again.