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2021-05-13 The Cystic Fibrosis Microbiome and its Association with Incident Infections with Mycobacteroides (Mycobacterium) abscessus
Bharadwaj, Lalit
Bharadwaj, L. (2021). The Cystic Fibrosis Microbiome and its Association with Incident Infections with Mycobacteroides (Mycobacterium) abscessus (Unpublished master's thesis). University of Calgary, Calgary, AB. http://hdl.handle.net/1880/113441 master thesis
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The Cystic Fibrosis Microbiome and its Association with Incident Infections with
Mycobacteroides (Mycobacterium) abscessus
by
Lalit Bharadwaj
A THESIS
SUBMITTED TO THE FACULTY OF GRADUATE STUDIES
IN PARTIAL FULFILMENT OF THE REQUIREMENTS FOR THE
DEGREE OF MASTER OF SCIENCE
GRADUATE PROGRAM IN MICROBIOLOGY AND INFECTIOUS DISEASES
CALGARY, ALBERTA
MAY, 2021
© Lalit Bharadwaj 2021
Abstract
Infection with M. abscessus complex (MABC) is increasingly detected within CF populations.
MABC infection has been associated with exaggerated lung function decline and poses significant treatment complexities. We performed a retrospective case-control study of twenty- one patients with MABC infection matching each to two randomly identified age (+/-2 yrs) and gender-matched uninfected controls. Total genomic DNA from sputum was extracted, amplified and Illumina MiSeq paired-end sequencing of the hypervariable V3 region of the 16S rRNA gene was performed. Demographics and dynamic variables of disease were recorded and compared between groups. 174 sputum samples (median 8, IQR (6-12)) from MABC cases and
42 control samples were assessed. The sputum microbiota from patients who would develop
MABC infection in the subsequent two years differed from controls (p=0.038, R2 = 2.5%,
PERMANOVA). In particular, sputum from MABC cases – prior to its identification – had higher alpha-diversity; Shannon diversity (p=0.023), Observed species (p=0.042) and lower P. aeruginosa relative and absolute abundance (p=0.035). We observed significant changes in community structure over time during potent antibacterial therapies, returning to baseline upon their discontinuation. These data suggest that sputum microbiome analysis and P. aeruginosa bioburden should be evaluated in multi-center studies as potential biomarkers to predict MABC infection and treatment response.
Keywords: cystic fibrosis, Non-tuberculous Mycobacterium, pulmonary microbiome, 16S
rRNA sequencing, sputum.
ii Acknowledgements
Several amazing people have been instrumental through the course of my master’s degree who all deserve a lot more gratitude than just an acknowledgement in my paper.
First and foremost, would be my supervisor, Dr. Michael Parkins. Dr. Parkins has not only been absolutely instrumental in guiding me through my research project but has always gone above and beyond in listening and helping me navigate issues in my life outside the lab and my struggles with my mental health. I can say without a doubt that it would not have been possible for me to complete this project without Dr. Parkins’ guidance, understanding, and support and I will forever be thankful.
I would also like to thank my co-supervisor, Dr. Douglas Storey, as well as my committee members, Dr. Michael Surette and Dr. Marie-Claire Arrieta, who have all given me so much through their expertise and guidance through my project. I find myself very lucky, although intimidated sometimes, to have such incredibly brilliant and renowned scientists helping me navigate my project.
I would also like to thank Dr. Lisa Gieg for taking time out of her busy schedule to serve as my examiner.
Members of the Parkins’ lab and the Southern Alberta Adult CF clinic have also been instrumental through my Master’s degree and have been like family to me. My appreciation is utmost for their continual support and guidance. Specifically, Dr. Nicole Acosta, Dr. Alya
Heirali, and Barbara Waddell who have always supported me and have always made themselves available for help. I am also thankful to Conrad Izydorczyk, Julianna Svishcuk,
Chloe Ligier, Jennifer Carpentero, Alexandra Mossman, Delia Lee, Taylor Woo, Bernice
Lee, and Benson Weyant for supporting and helping me through graduate education.
iii I would like to thank my family, who have loved and supported me and allowed for me to further my education through post-secondary; and my friends and teammates, who have given me additional courage and love, especially when my parents were not able to be there in person.
Lastly, I would like to thank the CF patients who have volunteered to participate in these studies and allow us the opportunity to further the scientific field.
iv Dedication
I dedicate this thesis to my parents, Sanjeev and Ratna Bharadwaj, who have faced lifelong adversities to allow me to have the best opportunities.
v Table of Contents
Abstract ...... ii
Acknowledgements ...... iii Dedication ...... v Table of Contents ...... vi List of Tables ...... viii List of Figures and Illustrations ...... ix List of Symbols, Abbreviations and Nomenclature ...... xi
Chapter One: Introduction ...... 1 1.1 An introduction to Cystic Fibrosis ...... 2 1.2 Genetics of Cystic Fibrosis ...... 3 1.3 Cystic Fibrosis Transmembrane Conductance Regulator ...... 4 1.4 Lung Disease Manifestations in CF ...... 6 1.5 Canonical CF Pathogens ...... 8 1.6 The CF Lung Microbiome ...... 11 1.7 Clinical Relevance of the CF Lung Microbiome ...... 13 1.8 Introduction to the Mycobacterium Genus ...... 15 1.8.1 An introduction to Mycobacteroides (mycobacterium) abscessus...... 17 1.9 Hypothesis...... 19 1.9.1 Project Aims ...... 20
Chapter Two: Methods and Materials ...... 21 2.1 Identification of MABC Cohort...... 22 2.2 Identification of Control Patient Cohort ...... 23 2.3 Patient Sputum Sample Identification ...... 24 2.4 Statistical analysis ...... 27 2.5 DNA Extraction...... 30 2.6 16S rRNA Gene Amplification ...... 32 2.7 DNA sequencing and analysis ...... 42 2.8 Quantitative PCR ...... 44 2.9 P. aeruginosa Inhibition Assay of MABC ...... 47
Chapter Three: Results ...... 48 3.1 Case and Control Patient Demographics...... 49 3.2 Pulmonary Function Linear Regression ...... 54 3.3 Lung Microbiome Diversity ...... 57 3.4 Microbiome structure through the natural history of infection ...... 62 3.5 Microbiome Comparison of MABC Case Patients to Controls ...... 66 3.6 Comparing Transient and Persistent Case Patient Sub-cohorts ...... 70 3.7 Quantitative PCR ...... 73 3.7.1 Absolute bacterial bioburden through the natural history of infection ...73 3.7.2 Absolute bacterial bioburden between case and control patients ...... 75 3.7.3 Quantifying MABC DNA in Sputum Samples ...... 77 3.7.4 Quantifying P. aeruginosa DNA in Sputum Samples ...... 82
vi 3.8 P. aeruginosa Inhibition Assay ...... 84
Chapter Four: Discussion...... 86 4.1 MABC Infections are a Major Cause for Concern in CF Patients ...... 87 4.2 Patients with MABC Present with a Lower BMI than Controls ...... 88 4.3 Persistent MABC Patients had Significantly Higher PPI use Compared to Transient Patients or Controls...... 89 4.4 Patients with MABC did not Differ in their Microbiome Diversity with New Infection ...... 90 4.5 Patients with MABC had significantly different microbiomes compared to control patients...... 92
CHAPTER 5: Summary ...... 95 5.2 Future Directions ...... 97 Further testing of the P. aeruginosa inhibition assay could be done with a possible testing with a semi-permeable polycarbonate membrane dividing P. aeruginosa from MABC could allow us to run a more realistic indirect competitive assay which would account for quorum sensing or direct competition for resources and could provide possible reasons to the negative correlation observed between MABC and P. aeruginosa. Additionally, a transcriptomic study involving P. aeruginosa and MABC co-cultures to assess any molecular mechanisms between the interactions...... 97 5.3 Study Summary and Conclusion ...... 98
References ...... 99
vii List of Tables
Table 1: Core members of CF pathogenesis. Data adapted from the 2019 CF Canada annual report...... 9
Table 2: The CF lung microbiome in health and disease and its supporting publication...... 14
Table 3: Case patient sputum samples categorized to natural history of disease stages according to time frame, AFB culture, and AFB smear status...... 26
Table 4. 16S rRNA V3 region forward and reverse barcoded primers...... 34
Table 5: Content configuration of each flask used to test P. aeruginosa inhibition of MABC. Three replicates of each flask was used for the assay...... 47
Table 6A: Baseline clinical demographics collected for case and control patients from the Calgary Adult CF clinic chart records...... 51
Table 6B: Mycobacteriology subspecies and coinfections collected for case and control patients from the Calgary Adult CF clinic chart records...... 52
Table 6C: On-going therapies and antibiotic collected for case and control patients from the Calgary Adult CF clinic chart records...... 53
viii List of Figures and Illustrations
Figure 1: CFTR mutation classes with phenotypic descriptions with common associated genetic mutations...... 5
Figure 2: Progressive CF lung disease starting from a defective CF gene and subsequent CFTR inhibition to the cyclic infection and inflammation phases exacerbating bronchiectasis...... 7
Figure 3: Phylogenetic tree displaying the phyla and genera of bacteria isolated from CF lung sputum based on 16S rRNA sequence differences against the Greengenes 2013 database with at least 97% identity...... 12
Figure 4: Lung function changes over time did not differ between cases and controls before or after infection...... 55
Figure 5: Lung function changes over time did not differ in transient vs persistent patients before or after infection...... 56
Figure 6: Phyla comparisons for 16S rDNA sequences observed in the microbiome of MABC case patients and their respective controls...... 60
Figure 7: Taxonomic composition of the top 20 ASVs at the genus level through infection natural history and matched controls...... 61
Figure 8: The natural history of the CF sputum microbiome through the course of MABC infection stages...... 65
Figure 9: The microbiome of sputum Pre-infection with MABC differs between cases and control patients...... 68
Figure 10: The microbiome of sputum At infection with MABC differs between cases and control patients...... 69
Figure 11: Pre-infection microbiome comparison between transient and persistent patients...... 71
Figure 12: At infection microbiome comparison between transient and persistent patients...... 72
Figure 13: Total bacterial bioburden stays relatively stable through the natural history of infection with exception of treatment intervention...... 74
Figure 14: Total bacterial bioburden does not change as a function of infection status...... 76
Figure 15: MABC bioburden changes through its Natural History of Infection...... 78
Figure 16: Z-N Smear positivity correlates with absolute abundance of MABC...... 80
Figure 17: A higher bioburden of MABC DNA correlated with a shorter time for MABC cultures to grow...... 81 ix Figure 18: Median P. aeruginosa DNA concentration was higher in control samples compared to at infection case samples...... 83
Figure 19: MABC growth in Middlebrook 7H9 broth infused with P. aeruginosa PA01 and PES supernatant...... 85
x
List of Symbols, Abbreviations and Nomenclature
Symbol Definition - Minus, Negative ˚C Degrees Celcius + Plus, Positive ABC ATP-Binding Cassette ABPA Allergic Bronchopulmonary Aspergillosis AFB Acid-Fast Bacillus AS Achromobacter species ASV Amplicon Sequence Variants ATP Adenosine Triphosphate ATS American Thoracic Society Bcc Burkholderia cepacia complex BMI Body Mass Index Chi-squared CACFC Calgary Adult Cystic Fibrosis Clinic cAMP Cyclic Adenosine monophosphate CF Cystic Fibrosis CFLD Cystic Fibrosis related Liver Disease CFRD Cystic Fibrosis Related Diabetes CFTR Cystic Fibrosis Transmembrane Conductance Regulator DADA2 Divisive Amplicon Denoising Algorithm 2 DIOS Distal Intestinal Obstructive Syndrome DNA Deoxyribonucleic Acid FEV Forced Expiratory Volume FVC Forced Vital Capacity G force Gravitational Force GER Gastroesophageal Reflux GTDB Genome Taxonomy Database HI Haemophilus influenzae HIV Human Immunodeficiency Virus IDSA Infectious Diseases Society of America kg Kilograms LABA Long-acting Beta Agonist LB Luria-Bertani m meters MABC Mycobacteroides abscessus complex MAC Mycobacterium avium complex mL Milliliters MRSA Methicillin resistent S. aureus MSSA Methicillin sensitive S. aureus NTM Non-tuberculous Mycobacteria
xi ODI Observed Diversity Index OTU Operational Taxanomic Unit PA Pseudomonas aeruginosa PA01 P. aeruginosa lab strain PA01 PCoA Principle Coordinate Analysis PCR Polymerase Chain Reaction PERMANOVA Permutational Multivariate Analysis of Variance PES Prairie Epidemic Strain pH Potential of Hydrogen PPI Proton Pump Inhibitor QIIME Quantitative Insights into Microbial Ecology qPCR Quantitative Polymerase Chain Reaction rDNA Ribosomal DNA RDP Ribosomal Database Project rRNA Ribosomal RNA SA Staphylococcus aureus SABA Short-acting Beta Agonist SDI Shannon Diversity Index SL1P Short-read Library 16S rRNA gene Sequencing Pipeline SM Stenotrophomonas maltophilia UND Undefined V3 Variable 3 Region of the 16S rRNA gene Z-N Stain Ziel-Neelson Stain µL microlitre Delta
xii 1
Chapter One: Introduction
2
1.1 An introduction to Cystic Fibrosis
Cystic fibrosis (CF) is the most common lethal genetic disorder observed in the
Caucasian population although it can affect individuals of all races and ethnicities.1
However, what was once a debilitating disease, causing death within the first few years of
life , has evolved into a manageable disease allowing patients to potentially survive into
their fifties and even beyond.2,3 Dysfunctional CFTR proteins manifest disease in various
organs throughout the body resulting in abnormal symptoms due to the aberrant chloride
transport across epithelial cell membranes.4 These complications are expressed in the
pancreatic, hepatic, gastro-intestinal, reproductive, and pulmonary systems.4,5
Manifestations of symptoms in the pancreatic system includes pancreatic insufficiency,
pancreatitis, and CF- related diabetes (CFRD).6–8 Gastro-intestinal deficiencies could
display distal intestinal obstructive syndrome (DIOS) and rectal prolapse whereas the
hepatic system could manifest CF-related liver disease (CFLD) and gallbladder disease.9–12
Reproductive malfunction sees infertility in more than 95% of men due to defects in the
sperm transportation whereas females are subject to a lesser degree of infertility and are
primarily caused by malnutrition and abnormal cervical mucus.13,14 Impairments to any of
these organs cause debilitating effects on the regular well-being of patients, however, none
more than the pulmonary system.5 Disease manifestation in the pulmonary system typically
involves the upper and lower respiratory tract, and the sinus.15,16 The respiratory tract
creates a hospitable environment for repeated pathogenic bacterial infections due to the
abnormally thick and difficult to clear mucosal layer which creates a cycle of inflammation,
bronchiectasis, and reinfection .17
3
1.2 Genetics of Cystic Fibrosis
CF is an autosomal recessive disorder caused by a mutation in the CFTR gene resulting
in a dysfunctional CFTR protein.18 The CFTR gene is located on the long arm of
chromosome 7 (7q31.2) comprising of 27 exons and spanning over 190 kb with a spliced
mRNA of 6.5 kb.18 Over 2000 reported mutation variants occur in the cystic fibrosis
transmembrane conductance regulator (CFTR) gene.1 Only approximately 400 mutation
variants, however, are known to be disease causing.19–21. Figure 1 displays some of the
common genetic mutations and their associated CFTR mutations and phenotype. The most
common CFTR mutation known is the deletion of the phenylalanine amino acid at the 508
position of the protein, also known as F508del. Approximately 50% of CF patients present
homozygous for this mutation while approximately 90% are heterozygous.22 Other common
CFTR alleles noted in CF patients are the G542X, G551D, and M1101K, which is regularly
seen in the closed communities of Hutterites.22,23
4
1.3 Cystic Fibrosis Transmembrane Conductance Regulator
The CFTR is a cAMP regulated chloride ion transport channel of the adenosine
triphosphate (ATP) – binding cassette (ABC) transporter family.24–27 Mutations in the CFTR
are classified into six major classes based on their molecular malfunction as seen in figure
1.28,29 These classifications are: i) defective protein synthesis due to lack of mRNA synthesis
or lack of protein synthesis, ii) improper trafficking of the protein to the membrane surface,
iii) impaired chloride channel regulation, iv) decreased conductance of chloride through the
channels at the membrane surface, v) reduction in the quantity of CFTR protein at the cell
surface, and vi) decreased stability of the CFTR protein at the cell surface.18,28 Phenotypic
severity decrease with increasing class number with the first three classes creating a lack of
functional CFTR proteins at the cell surface (more severe manifestations) and the last three
classes associated with a functionally inhibited CFTR (less severe manifestations).30–32
5
Figure 1: CFTR mutation classes with phenotypic descriptions with common associated genetic mutations.
6
1.4 Lung Disease Manifestations in CF
CF disease manifestation in the lungs, as seen in figure 2, occurs through repeated cycles
of infection and inflammation of the obstructed bronchi caused by the abnormal thickening
of the airway mucous, especially in the peripheral regions.33 The abnormally thickened
mucosal layer inhibit proper mucociliary function which allows for further aggregation of
mucous and decreased clearance of inhaled debris and/or microorganisms.34,35 Lack of
mucosal clearance allows for microorganisms – including opportunistic organisms - to persist
and establish chronic infections leading to cyclical bouts of inflammation resulting in further
secretion of thickened mucus and mucocialiary damage.1,36 These repeated cycles leads to the
permanent dilation of the bronchi known as bronchiectasis leading to chronic cough, sputum
production, recurrent respiratory infection, and acute pulmonary exacerbations.37–39 Ongoing
recruitment of the inflammatory cells progressively damages the airways furthering
bronchiectasis repeatedly until end-stage lung disease results in the death of the patient
unless a lifesaving lung transplant can be implemented.40–42
7
Figure 2: Progressive CF lung disease starting from a defective CF gene and subsequent CFTR inhibition to the cyclic infection and inflammation phases exacerbating bronchiectasis.
8
1.5 Canonical CF Pathogens
Many common CF pathogenic microorganisms noted in routine clinical tests are
opportunistic and exist naturally in the immediate environment and are capable of infecting
individuals with structural lung disease through this impairment in immune defense.
Although CF pulmonary infections can be caused by virus, fungi or bacteria, it’s the latter
that commonly cause problematic chronic infections.43 Canonical bacterial pathogens
regularly noted in CF patients include but are not limited to: Pseudomonas aeruginosa (PA),
Stenotrophomonas maltophilia (SM), Burkholderia cepacia complex (Bcc), Achromobacter
species (AS), non-tuberculous mycobacteria (NTM) such as Mycobacteroides abscessus
complex (MABC) and Mycobacterium avium complex (MAC).44 Primary pathogens such as
Haemophilus influenzae (HI), and Staphylococcus aureus (SA) (both methicillin sensitive
and methicillin resistant (MRSA) variants) are also very commonly noted in CF patients.
These pathogens are regularly present in environmental reservoirs such as water, soil,
animals, and food products from where they are known to infect.43 CF patient-to-patient
transmission have been noted due to improper hospital or home isolation, prevention, and
control measures.43
9
Table 1: Core members of CF pathogenesis. Data adapted from the 2019 CF Canada annual report. Pathogen Prevalence Relevance in CF disease
in Canada
(2019)
S. aureus 53% Most common pathogen in children and adolescents – infection
associated with bronchial inflammation and decreasing
pulmonary function.45
P. aeruginosa 38% Previously the most common infection in adults with over 50%
prevalence and commonly transmitted between patients.46,47
B. cepacia 4% Frequent in end-stage lung disease, associated often with poor
complex prognosis and requirement for lung transplantation. Patient to
patient transmission commonly observed with some species.48
S. maltophilia 14% Increasingly prevalent pathogen with inherent resistance to a
wide range of broad-spectrum antibiotics and common patient to
patient transmission.49,50
H. influenzae 11% Common infections in young children with multiple
simultaneous strains while 10-15% infection observed in adults
with one dominating strain.51,52
MRSA 6% Significant decline in pulmonary function values and longer
hospital stays compared to controls.53,54
10
NTM 6% Increasingly detected CF pathogen frequenting older age, greater
pulmonary function patients with higher MSSA and lower P.
aeruginosa infections.55,56 MAC and MABC are the most
prevalent of NTM infections with infections associated with
worsening lung function and prolonged IV antibiotics57–59
Achromobacter 5% Increasingly prevalent pathogen with chronic infections leading
Species to impaired pulmonary function and higher frequency of
hospitalization.56,60
11
1.6 The CF Lung Microbiome
Contrary to previous beliefs of the sterility of the lower airways, advancement in the next
generation sequencing technologies have allowed for a greater depth of analysis of the
polymicrobial community that in fact inhabits the lungs as with various other bodily niche,
albeit at a lower relative biomass.61–63 A vast array of bacterial, viral, and eukaryote species
have been discovered to inhabit the lower airway cavity and create a diverse and complex
society of microorganisms.64 Several airway environment factors influence the community
dynamics that are observed including oxygen concentrations, blood flow, and pH and are
ever changing through the lifespan of an individual.65
The natural aging process of CF patients combined with repeated infections and clinical
interventions shape an ever-evolving lower airway microbiome.66–68 Traditional culture-
based protocols of identification using selective media and aerobic growth conditions only
identified a small breadth of the overall organisms that were present.69 Analysis of the 16S
ribosomal RNA sequence diversity has allowed for the identification of numerous anaerobic
and aerobic bacteria that inhabit the lower airways but were previously under recognized.70
The 16S rRNA gene contains conserved regions within species along with polymorphisms in
hypervariable regions of the gene that allows differentiation between species to establish
phylogenic identities.71 Through the increased use of next generation sequencing technology,
recent studies have been able to identify 20 to over 1000 unique taxa present in a single CF
sputum sample.68 The airways in itself vastly differ in their compositional diversity and
abundance between the lower airways and oropharynx.72
12
Figure 3: Phylogenetic tree displaying the phyla and genera of bacteria isolated from CF lung sputum based on 16S rRNA sequence differences against the Greengenes 2013 database with at least 97% identity. Operational taxonomic unit (OTU) representing greater than 1% of the entire collection are represented with black dots with size correlating to abundance.73
13
1.7 Clinical Relevance of the CF Lung Microbiome
Several recent studies have effectively allowed for the consensus that individuals with an
advanced lung disease are limited in their lung microbiome diversity and are often dominated
by a few organisms.74–76 Some postulate that crucial interventions for a range of conditions
including CF will only be completely comprehended by understanding the ecological and
evolutionary relationships within the microbial community as well as with the host.77,78 One
such example is Cuthbertson et al. (2020), which explores the percent predicted forced
expiratory volume in 1 second (%FEV1) in comparison to the microbial diversity present in
74 the CF airways. %FEV1 is a useful measure of clinical health for individuals with lung
disease and allow for the clinicians to make critical decisions for treatment.79–81 Cuthbertson
found significant correlation between decreased diversity and increased dominance with a
reduction in lung function as well as a strong negative correlation between microbial
diversity and dominance.74
Additional research such as Acosta et al. (2018) provided association of specific
pathogens alongside overall diversity towards progression to end-stage lung disease.82
Acosta et al. demonstrated that a low microbiome diversity dominated by PA has a higher
association with progression to end-stage lung disease after five years than did a microbiome
dominated by Streptococcus.82 Further microbiome associations have been made by Heirali
et al. (2017) correlating lack of response to inhaled aztreonam lysine with higher OTU
presence of Fusobacterium, Staphylococcus, Prevotella, and Bacteroides.83 Although current
medical practice has yet to recognize the use of the lung microbiome as an effective clinical
decision making tool, continual research such as these of the CF airways may lead to
solidified associations between the microbiome and canonical infections.82,84
14
Table 2: The CF lung microbiome in health and disease and its supporting publication. Factor Change Strength of data Citations
Pulmonary Microbiome diversity remains Strong Stressmann et al. (2011) exacerbations stable regardless of clinical Zhao et al. (2012) status. Fodor et al. (2012) Carmody et al. (2013) Price et al. (2013) Carmody et al. (2015) Acute Immediate decrease in Strong Tunney et al. (2011) antibiotics richness with rapid reversal to Stressmann et al. (2012) norm but overall microbiome Fodor et al. (2012) diversity stays stable. Zhao et al. (2012) Smith et al. (2014) Heirali et al. (2019) Heirali et al. (2020) Age Microbiome diversity Strong Cox et al. (2010) increases during first decade Zhao et al. (2012) of life then decrease through Stressmann et al. (2012) adolescents and adulthood. Stokell et al. (2015) Baseline lung Poor lung function correlates Strong Cox et al. (2010) function with low microbiome Fodor et al. (2012) community diversity. Carmody et al. (2013) Coburn et al. (2015) Acosta et al. (2017) Genotype Microbiome association noted Weak Cox et al. (2010) in patients with F508 mutations compared to rare mutations.
15
1.8 Introduction to the Mycobacterium Genus
The genus Mycobacterium includes over 180 species belonging to the phylum
Actinobacteria.85,86 Members of the genus are Gram positive, rod shaped and Acid Fast.
Many are free living organisms and can be readily isolated from environmental niches such
as soil, water, animals, food products, and metalworking fluids.87,88 Mycobacteria is
subdivided into the quartet of M. tuberculosis complex, M. leprae complex, slow growing
non-tuberculous Mycobacterium (NTM) and rapid growing NTM (7 days is used to
distinguish slow from rapid growers).85 M. tuberculosis complex and M. leprae complex
species cause historically important human diseases; tuberculosis and leprosy,
respectively.87,89 NTM species, on the other hand, are opportunistic pathogens preying on
individuals with pre-existing conditions such as lung disease, immunodeficiencies or who are
receiving immune-modulating treatments.90
Tuberculosis has a global disease burden in excess of 10 million people each year with
approximately 1.4 million deaths and is second only to HIV as an infectious cause of
death.89,91,92 Leprosy affects approximately 200,000 people mainly in the tropical countries
and causes major deformities in the infected individuals often leading to an early mortality.89
M. tuberculosis is transmitted through the inhalation of aerosolized particles of the bacteria
from the aerosols and droplets from infected individual. In contrast, the exact method of
transmission of M. leprae complex is unknown, however, it is postulated to be in a similar
matter.93,94 The remaining mycobacteria species are classified under the NTM label and
opportunistically cause disease in various organs but very often manifest as pulmonary
infections.90 NTM is subdivided into slow-growing species that take 7 to 42 days to develop
16 colonies in vitro and include M. avium complex and M. kansasii, and rapid-growing species that form colonies in under 7 days and include M. abscessus complex.
17
1.8.1 An introduction to Mycobacteroides (mycobacterium) abscessus
MABC often manifest in patients with predisposed immune conditions and infects the respiratory tract, skin, or soft-tissues.90,95 Advancement in isolation techniques and identification strategies have allowed for increasing detection of MABC, which accounts for approximately 15% of all NTM infection within North American CF Patients.55,95,96 MABC infections are more prevalent in the European cohorts where infections can be up to 70% of total NTM observed.97–99 Additionally, recent evidence suggests potential patient-to-patient transmission within CF communities of MABC which was previously unrecognized.88,100
MABC is a particularly difficult organism to treat once infection has progressed requiring a barrage of toxic antibiotics delivered over a prolonged course. During the induction phase, treatment of MABC is conducted using a combination therapy of intravenous amikacin, alongside one or more of tigecycline, imipenem, and/or cefoxitin, supplemented with oral clarithromycin or azithromycin for one to 3 months. 66–68 These therapies are particularly difficult to tolerate and are associated with very high toxicity as complications. This is followed by a 12-month continuation phase comprised of the oral azithromycin or clarithromycin alongside with inhaled amikacin and 2 to 3 additional orally bioavailable antibiotics from a list of minocycline, clofazimine, moxifloxacin, and/or linezolid.102,103
Azithromycin is a semi-synthetic macrolide with action against the 50S subunit of bacterial ribosome inhibiting protein synthesis.104 Amikacin, tigecycline and minocycline are members of the aminoglycoside, glycylcycline, and tetracycline classes, respectively. These all have activity against the 30S bacterial ribosome halting peptide synthesis – albeit by different mechanisms.105–107 Imipenem and cefoxitin are both beta-lactam that inhibits the synthesis of bacterial cell walls.108 Moxifloxacin is a fluoroquinolone that binds to
18 topoisomerase II and IV and inhibits DNA replication and transcription.109 Clofazimine is a riminophenazine originally developed for the treatment of tuberculosis and a component of multidrug combination therapy for leprosy. Its mechanism of action is a combination of membrane disruption and the generation of reactive oxygen species.110 Lastly, linezolid belong to a group of oxazolidinones that inhibit protein synthesis by binding the P site of the
50S ribosomal subunit.111
A joint committee of the American Thoracic Society and Infectious Disease Society of
America (ATS/IDSA) was formed in 2007 to provide guidelines for clinical use for NTM infections adopted by CF specialists and are regularly updated as such in June, 2020.102
These guidelines consider a set of clinical, radiological, and microbiological characteristics for diagnosing NTM disease. Under these guidelines as it pertains to CF, a patient must present two or more positive sputum cultures of the same NTM species or one positive culture from a bronchoalveolar lavage or wash to satisfy the microbiology criteria.
Radiological criteria are difficult to assess in CF patients as signs pertaining to a MABC infection are common irrespective of infection source and could include inflammatory nodules, tree-in-bud opacities, and cavitation. Clinical criteria looks at worsening respiratory symptoms and decreasing pulmonary functions despite of treatment of common CF pathogens.102
In 2016, a consensus recommendation for the management of NTM infection in individuals with CF was created by the US Cystic Fibrosis Foundation and the European
Cystic Fibrosis Society to help better identify and treat NTM infections with specific focus to
CF patients.103 The guidelines for treatment built on the 2007 ATS/IDSA criteria with adaptation based on patient variables, putative risks, and expectation of outcome.103
19
1.9 Hypothesis
I hypothesize that the diversity and composition of the community of bacteria residing in
the CF lower airways will influence the susceptibility of the host to invasion by MABC
infection. Thus, my hypotheses are:
1. The microbiome of patients with incident infections of MABC will have fundamental
differences from patients who do not experience the infection. Patients who do not
experience incident infections may thus be protected by colonizing lower airway
communities that resist invasion.
2. Among those patients who do develop incident infections, two results are possible.
Clearance of the pathogen, whereby the patient avoids the negative sequelae of
chronic infection or persistence of infection whereby adverse outcomes ensue.
Differences in community structure may yield information on organisms that assist in
pathogen clearance. We hypothesize that airway microbiome will enable distinction
between these two outcomes.
20
1.9.1 Project Aims i) To determine how the microbiome of patients with each incident infection
evolves over time – encompassing the period of time prior to infection, through
initial infection prior to treatment, both induction and consolidation therapies and
finally resolution/persistence.
ii) To determine if the microbiome may serve as a novel biomarker to identify
patients at risk of infection by determining if the clinical demographics and
microbial communities of patients with incident infections differ from control
patients matched for age and gender.
iii) To determine if specific factors within the microbiome associate with treatment
outcomes.
21
Chapter Two: Methods and Materials
22
2.1 Identification of MABC Cohort
Patients for our cohort study were identified from a retrospective review of the
microbiology clinical records with matching sputum samples from the Calgary Adult Cystic
Fibrosis Clinic to which the Parkins laboratory has access. Clinical demographic and
dynamic variables of disease were obtained from medical records. Comprehensive patient
records accompanying the sputum samples included: demographics such as nutritional status
(body mass index (BMI) (kg/m2)), age, gender, co-morbidities, cftr genetic information;
infection details; infection history; treatments received; treatment outcomes; antibiotic
resistance data; and key spirometic values including percent predicted forced vital capacity
(%FVC), and percent predicted forced expiratory volume in one second (%FEV1). Cases
were classified as incident (occurring during the time of follow-up within the CACFC) or
pre-existing (occurring prior to transition to CACFC). Case patient that were selected had at
least one positive MABC culture (Middlebrook 7H10) and were tested by Alberta Health
Services Laboratory where the pathogen samples were also stored. Classification of
chronicity was conducted using the criteria established by the ATS/IDSA.112 Patients
deemed persistent with an MABC infection were seen with at least half of their cultures
positive with a minimum of three samples in the year following initial infection. Patients
with less than half of their cultures positive were hence deemed transient.
23
2.2 Identification of Control Patient Cohort
Control patients were identified from the CACFC cohort and were only selected if there
was no known previous recorded case of MABC in their respiratory secretions. Two controls
were selected for each patient that were age matched to within two years of the initial
infection date. The control patients were also matched to their respective case’s sex and time
period. For each control patient, one samples was selected. For samples to be analyzed we
ensured that they were collected earlier than four weeks after a change in antibacterial
therapy or pulmonary exacerbation to avoid confounding effects. Patient demographic data
was similarly collected for each control patient such as nutritional status (body mass index
(BMI) (kg/m2)), age, gender, co-morbidities, cftr genetic information; infection details;
infection history; treatments received; treatment outcomes; antibiotic resistance data; and key
spirometic values including %FVC and %FEV1.
24
2.3 Patient Sputum Sample Identification
Sputum samples from attendees are meticulously stored within the Calgary Adult CF
Clinic Biobank housed in multiple -80˚Celsius freezers to ensure optimal conservation to the
original state of the samples. Sputum samples have been collected and stored since 1998 and
are continuously updated with new arriving samples. As of 2019, the biobank contains over
18,000 samples from over 275 adult patients who have prospectively consented towards the
collection and storage of sputum samples for research purposes. Most sputum samples were
homogenized and stored into vials or cryogenic tubes immediately after clinic visit by the
patients. Samples are also received from the clinical microbiology, documented, and placed
into long-term storage in -80˚Celsius freezers. Inclusion criteria included identification of
Mycobacteroides abscessus complex within acid-fast bacillus (AFB) sputum culture during
follow up within the CF clinic; Patients were excluded if their diagnosis of MABC predated
infection and no positive cultures were obtained during the period of follow up at the
CACFC. Cases and control patient samples were only selected if they were not influenced
immediately by antibiotic exposure (within two weeks) not relevant to the treatment
associated with MABC. Samples were classified into stages of disease and were as follows:
1. Pre-infection – samples preceding the infection date up to two years unless insufficient
sputum samples were available or were impacted by antibiotic intervention for other
infections.
2. At-infection – samples dated on the initial clinical positive.
3. Induction therapy – initiation of therapy course with numerous potent IV antibiotics to
eradicate majority of MABC.
25
4. Consolidation therapy – continuation of several IV and oral antibiotics following the
induction therapy phase to eradicate any remaining MABC.
5. Maintenance therapy – continuation of one or more antibiotics as a complement to
induction and/or consolidation therapy.
6. Intensification therapy – reversion to induction therapy with other antibiotics or a higher
dosage.
7. Clearance – samples that have cleared MABC infection either due to the antibiotic
therapy or through spontaneous measures.
8. Chronic and off treatment – samples that have persistent MABC infections and have been
taken off antibiotic therapy.
Samples associated with the antibiotic therapy were grouped into a single “at treatment” stage. The natural history of each patient was reviewed and detailed in the medical record.
Sputum samples were specifically sought from a number of categories and scored according to table 3.
26
Table 3: Case patient sputum samples categorized to natural history of disease stages according to time frame, AFB culture, and AFB smear status. Natural Time Frame Last AFB culture Culture positive/days to AFB smear status History of conducted positive Disease Pre-infection Two years Ziehl Neelsen Samples collected in the Samples were before initial Stain to assess for time period before a assessed by Ziehl- infection date the presence of clinical MABC positive Neelsen Stain for (unless bacteria. Samples was observed bacterial bioburden. insufficient ranged positive Samples ranged number from latest clinic from smear negative samples visit to 36 months to a smear 3+ available) prior with several patients never having had one before At infection First clinical Positive samples Positive samples ranged Samples ranged positive date ranged from from 1 – 7 days to grow. from smear negative to initiation latest clinic visit Patients with negative to a smear 3+ of treatment to a year prior samples either grew again within the year or never grew again
At Treatment All stages Positive samples Positive samples ranged Samples ranged involved from ranged from from 1 – 7 days to grow. from smear negative induction of latest clinic visit Patients with negative to a smear 3+ therapy, to 1.5 months samples either grew consolidation, prior again within the year or maintenance, never grew again and intensification
Post After Positive samples Positive Samples were Samples ranged Treatment treatment ranged from either chronic and from smear negative phase with latest clinic visit cultures ranged from 1 to a smear 3+ either to 2 months prior day to over 7 days to (persistent patients) clearance of grow or were cleared and MABC or did not grow MABC chronic within the next year designation
27
2.4 Statistical analysis
Patient and control demographic data were analyzed using 2-sided fisher’s exact and
Chi-squared (2) tests for distinct values while Wilcoxon rank-sum was used to analyze
continuous values. Fisher’s exact was used to test significant differences between the
proportions of one variable and whether they are different among values of the other
variable.113 Fisher’s exact test is more reliable for a small number of samples being compared
and assumes that compared values are not related.114 Fisher’s exact also relies on the
assumption that the null hypothesis is true.115 To calculate Fisher’s exact test, a 2 x 2
contingency table being analyzed is compared to all possibilities of that 2 x 2 table fitting
within a marginal tool. The equation below allows to equate the p-values for each possible 2
x 2 table. To calculate the one-sided Fisher’s exact, the probability of the 2 x 2 proportions
in question and all probabilities more extreme are summed. For a two-sided Fisher’s exact
value, a mid-p method can be used where the p-values more extreme than the observed are
added to one half of the observed p-value.116 If the resulting p-value is less than 0.05, a
statistical significance can be assumed and the null hypothesis rejected.
(푎 + 푏)! (푐 + 푑)! (푎 + 푐)! (푏 + 푑)! 푝 = (푎 + 푏 + 푐 + 푑)! 푎! 푏! 푐! 푑!
The 2 test for independence is used when comparing two variables of discrete values
when the population size is relatively large. The 2 equation relies on the difference between
the observed value and the expected value, granted the variables are completely independent
of each other.113 The 2 test depends on a null hypothesis which can be failed to be rejected
or rejected and an alternative hypothesis that is accepted. A 2 distribution is employed
alongside the degrees of freedom (number of variables minus 1) to extrapolate the p-value.
28
(푂 − 퐸)2 2 = ∑ 퐸
The Wilcoxon Rank-Sum test is used to calculate probabilities of two independent variables consisting of continuous values.117 Wilcoxon Rank-Sum is a non-parametric test that assumes as the null hypothesis that the competing observation median have the same continuous distribution. This test is used in replacement of a t-test when two populations have unequal spread and are not normally distributed. The test uses a summed rank of the values present in each variable compared to the median of the extremes to assess if there are significant differences between the variables to reject the null hypothesis. The Wilcoxon
Rank-Sum test is very similar to the Mann-Whitney test and often produces the same results.
The following formula is used for each of the groups with the lower resulting U being used to determine the p-value based off a table of critical values.
푛 (푛 + 1) 푈 = 푛 푛 + 1 1 − 푅 1 1 2 2 1
푛 (푛 + 1) 푈 = 푛 푛 + 2 2 − 푅 2 1 2 2 2
The Kruskal-Wallis test was used to calculate probability of non-parametric continuous dependent variables by categorical independent variable of two or more groups.118 Similar to
Mann-Whitney and Wilcoxon Rank-Sum, Kruskal-Wallis test assumes an abnormal distribution with a varying spread. The null hypothesis assumes that groups are from identical populations with the Kruskal-Wallis test aimed to reject the null hypothesis and accept the alternative hypothesis that at least one group is different from the others.
Permutational multivariate analysis of variance (PERMANOVA) was used to compare the beta-diversity Bray-Curtis dissimilarities matrix and test the null hypothesis that the
29 centroids and dispersion of the groups are equally distributed for all groups.119
PERMANOVA assumes that all objects in the data set are exchangeable, independent, and have similar multivariate dispersion. PERMANOVA analysis was done in R with stratification using 999 permutations to normalize dataset proportionally for the categories being analyzed.119 Differences in taxa was assessed using the DESeq2 package with a
“Wald” test and “parametric” fit type.120 Lastly, multiple comparisons of p-values of relative abundance for each taxa was calculated using two-tailed Mann-Whitney U test on PRISM and corrected using Benjamini-Hochberg for multiple comparisons.
These statistical analysis were conducted on STATA v.13.0 (StataCorp, Texas, USA),
PRISM v8.2.1 (GraphPad, California, USA), and/or R v3.5.0 (R Core Team, 2018).
30
2.5 DNA Extraction
Total DNA extraction was conducted using a previously published and validated method
from the McMaster Genome Facility (Hamilton, ON) as well as other microbiome
publications from the Parkins laboratory (Heirali, 2017; Acosta, 2018; Heirali, 2019).82,83,121
The process included extreme care in ensuring samples remain as near to the state of sample
collection. This includes using dry ice to transfer and store the samples until the time of
extraction. All exactions were conducted in the biosafety cabinet after a thorough cleaning
with 70% ethanol and UV light eradication of any contaminants. Contaminations during the
extraction process could lead to false identification of microbiota resulting in data that is
unreliable and unreproducible. Proper personal protective equipment is also worn to ensure
minimal impact of contaminations and are cleaned between samples using 70% ethanol. A
negative control was included with every extraction set to ensure lack of cross contamination
and verified using PCR and gel electrophoresis. Select negative controls (6) were also
sequenced and qPCR for presence of bacterial 16S rDNA.
Genomic DNA extraction was performed as previously described.82,83,121,122 Briefly,
frozen sputum samples were thawed and homogenized using a 18mm syringe needle. 300
µL of each sample was mechanically lysed by bead beating with 800 µL NaPO4 (200mM)
and 100 µL of guanidine thiocyanate-ethylenediaminetetraacetic acid followed by enzymatic
lysis using 50 µL of lysozyme (100mg/mL) (Sigma-Aldrich, #L6876-10G), 50 µL
mutanolysin (10 U/µL) (Sigma-Aldrich, #M9901-50KU), and 10 µL RNase A (10 mg/mL)
(Qiagen, #19101) and incubated for 90-minutes at 37˚C. A secondary lysis step was
performed next using 25 µL sodium dodecyl sulfate (25%), 25 µL proteinase K (Sigma-
Aldrich, #P2308-1G), and 62.5 µL NaCl (5 M) and incubated again for 90-minutes at 65˚C.
31
The samples were centrifuged at 13,500 X g for 5 minutes. DNA was then isolated using phenol-chloroform-isoamyl (Sigma-Aldrich, #P3803- 400mL) and purified through a DNA column (Cedarlane laboratories, #D4034) using a DNA binding buffer and DNA wash buffer
(DNA clean and concentrator-25, Cedarlane laboratories, #D4034).122 Quantification of the eluted DNA was conducted using a NanoDrop-1000 (Thermo Scientific, Waltham, MA).
32
2.6 16S rRNA Gene Amplification
100 ng of the extracted sample DNA was amplified for the V3 region of the 16S rRNA
gene using modified 341F and 518R 16S rRNA primers for adaptation to Illumina Miseq
(San Diego, CA).122–124 Extraction blanks and its consequent PCR reactions were included to
ensure no contamination had occurred during the process. Extracted DNA from sputum
samples are analysed by the amplification of the V3 hypervariable region of the 16S rRNA
gene. The V3 region is used due to its small number of total nucleotides (65 base pairs)
containing the maximum number of single nucleotides polymorphisms between nucleotides
456 and 479.125 The amplification was done in a polymerase chain reaction (PCR) machine
using modified forward and reverse primers using the methodology developed by Bartram et
al.126 The DNA acquired from the DNA extraction was run through gel electrophoresis to
confirm bands at approximately 300 base pairs, the length of the V3 region. Once ensured
that the amplified DNA is correct, the PCR samples were sent to the McMaster Genome
Facility in Hamilton, ON to be sequenced using their Illumina MiSeq technology.
For each sample, a master mix was created with the following: 5µL of 10x PCR buffer
(Life Technologies, #10342020), 1.5µL of 50mM MgCl2 (Life Technologies, #10342020), 1µL of
10mM dNTPs (New England Biolabs, #N0447L), 5µL of 1µM V3F primer (5 pmoles), 5µL of
1µM V3R primer (5 pmoles), 0.25µL of Taq polymerase (Life Technologies, #10342020),
and 27.25µL of ddH2O to a total of 45µL per sample. Then add 5µL of the extracted DNA
sample normalized to 100ng/50µL. Split each sample tube into thirds (~17µL in each tube).
Place sample tubes in PCR machine and program as follows:
• 94˚C for 2 mins
• 30 cycles of:
33
o 94˚C for 30 secs
o 50˚C for 30 secs
o 72˚C for 30 secs
• 72˚C for 10 mins
• 4˚C hold
34
Table 4. 16S rRNA V3 region forward and reverse barcoded primers.
Primer Adapter Sequence
caagcagaagacggcatacgagatCGTGATgtgactggagttcagacgtgtgctcttccgatctCCTACGG V3_1F P7 GAGGCAGCAG
caagcagaagacggcatacgagatACATCGgtgactggagttcagacgtgtgctcttccgatctCCTACGG V3_2F P7 GAGGCAGCAG
caagcagaagacggcatacgagatGCCTAAgtgactggagttcagacgtgtgctcttccgatctCCTACGG V3_3F P7 GAGGCAGCAG
caagcagaagacggcatacgagatTGGTCAgtgactggagttcagacgtgtgctcttccgatctCCTACGG V3_4F P7 GAGGCAGCAG
caagcagaagacggcatacgagatCACTGTgtgactggagttcagacgtgtgctcttccgatctCCTACGG V3_5F P7 GAGGCAGCAG
caagcagaagacggcatacgagatATTGGCgtgactggagttcagacgtgtgctcttccgatctCCTACGG V3_6F P7 GAGGCAGCAG
caagcagaagacggcatacgagatGATCTGgtgactggagttcagacgtgtgctcttccgatctCCTACGG V3_7F P7 GAGGCAGCAG
caagcagaagacggcatacgagatTCAAGTgtgactggagttcagacgtgtgctcttccgatctCCTACGG V3_8F P7 GAGGCAGCAG
caagcagaagacggcatacgagatCTGATCgtgactggagttcagacgtgtgctcttccgatctCCTACGG V3_9F P7 GAGGCAGCAG
caagcagaagacggcatacgagatAAGCTAgtgactggagttcagacgtgtgctcttccgatctCCTACG V3_10F GGAGGCAGCAG P7 caagcagaagacggcatacgagatGTAGCCgtgactggagttcagacgtgtgctcttccgatctCCTACG V3_11F P7 GGAGGCAGCAG
35
Primer Adapter Sequence
caagcagaagacggcatacgagatTACAAGgtgactggagttcagacgtgtgctcttccgatctCCTACG GGAGGCAGCAG V3_12F P7
caagcagaagacggcatacgagatCGTACTgtgactggagttcagacgtgtgctcttccgatctCCTACGG V3_13F P7 GAGGCAGCAG
caagcagaagacggcatacgagatGACTGAgtgactggagttcagacgtgtgctcttccgatctCCTACG V3_14F P7 GGAGGCAGCAG
caagcagaagacggcatacgagatGCTCAAgtgactggagttcagacgtgtgctcttccgatctCCTACGG V3_15F P7 GAGGCAGCAG
caagcagaagacggcatacgagatTCGCTTgtgactggagttcagacgtgtgctcttccgatctCCTACGG V3_16F P7 GAGGCAGCAG caagcagaagacggcatacgagatTGAGGAgtgactggagttcagacgtgtgctcttccgatctCCTACG V3_17F P7 GGAGGCAGCAG
caagcagaagacggcatacgagatACAACCgtgactggagttcagacgtgtgctcttccgatctCCTACGG V3_18F P7 GAGGCAGCAG
caagcagaagacggcatacgagatACCTCAgtgactggagttcagacgtgtgctcttccgatctCCTACGG V3_19F P7 GAGGCAGCAG
caagcagaagacggcatacgagatACGGTAgtgactggagttcagacgtgtgctcttccgatctCCTACG V3_20F P7 GGAGGCAGCAG
caagcagaagacggcatacgagatAGTTGGgtgactggagttcagacgtgtgctcttccgatctCCTACG V3_21F P7 GGAGGCAGCAG
caagcagaagacggcatacgagatCTCTCTgtgactggagttcagacgtgtgctcttccgatctCCTACGG V3_22F P7 GAGGCAGCAG
caagcagaagacggcatacgagatCAAGTGgtgactggagttcagacgtgtgctcttccgatctCCTACG V3_23F P7 GGAGGCAGCAG
caagcagaagacggcatacgagatCCTTGAgtgactggagttcagacgtgtgctcttccgatctCCTACGG V3_24F P7 GAGGCAGCAG
36
Primer Adapter Sequence
caagcagaagacggcatacgagatACCACTgtgactggagttcagacgtgtgctcttccgatctCCTACGG GAGGCAGCAG V3_25F P7
caagcagaagacggcatacgagatAGTGTCgtgactggagttcagacgtgtgctcttccgatctCCTACGG GAGGCAGCAG V3_26F P7
caagcagaagacggcatacgagatAGAAGGgtgactggagttcagacgtgtgctcttccgatctCCTACG GGAGGCAGCAG V3_27F P7
caagcagaagacggcatacgagatTTATCCgtgactggagttcagacgtgtgctcttccgatctCCTACGG V3_28F P7 GAGGCAGCAG
caagcagaagacggcatacgagatTTAAGGgtgactggagttcagacgtgtgctcttccgatctCCTACG GGAGGCAGCAG V3_29F P7
caagcagaagacggcatacgagatTTCTTGgtgactggagttcagacgtgtgctcttccgatctCCTACGG V3_30F P7 GAGGCAGCAG
caagcagaagacggcatacgagatTTCAACgtgactggagttcagacgtgtgctcttccgatctCCTACGG V3_31F P7 GAGGCAGCAG
caagcagaagacggcatacgagatTTGTGAgtgactggagttcagacgtgtgctcttccgatctCCTACGG V3_32F P7 GAGGCAGCAG
caagcagaagacggcatacgagatTTGACTgtgactggagttcagacgtgtgctcttccgatctCCTACGG V3_33F P7 GAGGCAGCAG
caagcagaagacggcatacgagatTATTCGgtgactggagttcagacgtgtgctcttccgatctCCTACGG V3_34F P7 GAGGCAGCAG
caagcagaagacggcatacgagatTATAGCgtgactggagttcagacgtgtgctcttccgatctCCTACGG V3_35F P7 GAGGCAGCAG
caagcagaagacggcatacgagatTAACTCgtgactggagttcagacgtgtgctcttccgatctCCTACGG V3_36F P7 GAGGCAGCAG
37
Primer Adapter Sequence
caagcagaagacggcatacgagatTACCAAgtgactggagttcagacgtgtgctcttccgatctCCTACGG V3_37F P7 GAGGCAGCAG
caagcagaagacggcatacgagatTACGTTgtgactggagttcagacgtgtgctcttccgatctCCTACGG V3_38F P7 GAGGCAGCAG
caagcagaagacggcatacgagatTAGTACgtgactggagttcagacgtgtgctcttccgatctCCTACGG V3_39F P7 GAGGCAGCAG
caagcagaagacggcatacgagatTAGATGgtgactggagttcagacgtgtgctcttccgatctCCTACG V3_40F P7 GGAGGCAGCAG
caagcagaagacggcatacgagatTCTACAgtgactggagttcagacgtgtgctcttccgatctCCTACGG V3_41F GAGGCAGCAG
P7 caagcagaagacggcatacgagatTCTGATgtgactggagttcagacgtgtgctcttccgatctCCTACGG V3_42F P7 GAGGCAGCAG
caagcagaagacggcatacgagatTCATGTgtgactggagttcagacgtgtgctcttccgatctCCTACGG GAGGCAGCAG V3_43F P7
caagcagaagacggcatacgagatTGTCTAgtgactggagttcagacgtgtgctcttccgatctCCTACGG V3_44F P7 GAGGCAGCAG
caagcagaagacggcatacgagatATTCTCgtgactggagttcagacgtgtgctcttccgatctCCTACGG V3_45F P7 GAGGCAGCAG
caagcagaagacggcatacgagatATTGAGgtgactggagttcagacgtgtgctcttccgatctCCTACG V3_46F P7 GGAGGCAGCAG
caagcagaagacggcatacgagatATACCTgtgactggagttcagacgtgtgctcttccgatctCCTACGG V3_47F P7 GAGGCAGCAG
caagcagaagacggcatacgagatATGCAAgtgactggagttcagacgtgtgctcttccgatctCCTACG V3_48F P7 GGAGGCAGCAG
caagcagaagacggcatacgagatAATCCAgtgactggagttcagacgtgtgctcttccgatctCCTACGG V3_49F P7 GAGGCAGCAG
38
Primer Adapter Sequence
caagcagaagacggcatacgagatAATGGTgtgactggagttcagacgtgtgctcttccgatctCCTACG V3_50F P7 GGAGGCAGCAG
caagcagaagacggcatacgagatAACTAGgtgactggagttcagacgtgtgctcttccgatctCCTACG V3_51F P7 GGAGGCAGCAG
caagcagaagacggcatacgagatAACACTgtgactggagttcagacgtgtgctcttccgatctCCTACGG V3_52F P7 GAGGCAGCAG
caagcagaagacggcatacgagatAAGAGAgtgactggagttcagacgtgtgctcttccgatctCCTACG V3_53F P7 GGAGGCAGCAG
caagcagaagacggcatacgagatACTTACgtgactggagttcagacgtgtgctcttccgatctCCTACGG V3_54F P7 GAGGCAGCAG
caagcagaagacggcatacgagatACATTGgtgactggagttcagacgtgtgctcttccgatctCCTACGG V3_55F P7 GAGGCAGCAG
caagcagaagacggcatacgagatACGAATgtgactggagttcagacgtgtgctcttccgatctCCTACG V3_56F GGAGGCAGCAG P7 caagcagaagacggcatacgagatAGTCATgtgactggagttcagacgtgtgctcttccgatctCCTACGG V3_57F GAGGCAGCAG P7
caagcagaagacggcatacgagatAGAAGTgtgactggagttcagacgtgtgctcttccgatctCCTACG P7 GGAGGCAGCAG V3_58F caagcagaagacggcatacgagatCTTATGgtgactggagttcagacgtgtgctcttccgatctCCTACGG V3_59F P7 GAGGCAGCAG
caagcagaagacggcatacgagatCTAGAAgtgactggagttcagacgtgtgctcttccgatctCCTACG GGAGGCAGCAG V3_60F P7
caagcagaagacggcatacgagatCATCTTgtgactggagttcagacgtgtgctcttccgatctCCTACGG V3_61F P7 GAGGCAGCAG
caagcagaagacggcatacgagatCACATAgtgactggagttcagacgtgtgctcttccgatctCCTACGG V3_62F P7 GAGGCAGCAG
39
Primer Adapter Sequence
caagcagaagacggcatacgagatCCAATTgtgactggagttcagacgtgtgctcttccgatctCCTACGG V3_63F P7 GAGGCAGCAG
caagcagaagacggcatacgagatCGATTAgtgactggagttcagacgtgtgctcttccgatctCCTACGG V3_64F P7 GAGGCAGCAG
caagcagaagacggcatacgagatGTTAGTgtgactggagttcagacgtgtgctcttccgatctCCTACGG V3_65F P7 GAGGCAGCAG
caagcagaagacggcatacgagatGTAACAgtgactggagttcagacgtgtgctcttccgatctCCTACG V3_66F P7 GGAGGCAGCAG
caagcagaagacggcatacgagatGTGTATgtgactggagttcagacgtgtgctcttccgatctCCTACGG V3_67F P7 GAGGCAGCAG
caagcagaagacggcatacgagatGATAAGgtgactggagttcagacgtgtgctcttccgatctCCTACG V3_68F P7 GGAGGCAGCAG
caagcagaagacggcatacgagatGAATCTgtgactggagttcagacgtgtgctcttccgatctCCTACGG V3_69F P7 GAGGCAGCAG
caagcagaagacggcatacgagatTTCCGTgtgactggagttcagacgtgtgctcttccgatctCCTACGG V3_70F P7 GAGGCAGCAG
caagcagaagacggcatacgagatTTCGCAgtgactggagttcagacgtgtgctcttccgatctCCTACGG V3_71F P7 GAGGCAGCAG
caagcagaagacggcatacgagatTTGGTCgtgactggagttcagacgtgtgctcttccgatctCCTACGG V3_72F GAGGCAGCAG
P7 caagcagaagacggcatacgagatTGACAGgtgactggagttcagacgtgtgctcttccgatctCCTACG V3_73F P7 GGAGGCAGCAG
caagcagaagacggcatacgagatATCTGCgtgactggagttcagacgtgtgctcttccgatctCCTACGG GAGGCAGCAG V3_74F P7
caagcagaagacggcatacgagatACACGAgtgactggagttcagacgtgtgctcttccgatctCCTACG V3_75F P7 GGAGGCAGCAG
40
Primer Adapter Sequence
caagcagaagacggcatacgagatAGGTTCgtgactggagttcagacgtgtgctcttccgatctCCTACGG V3_76F P7 GAGGCAGCAG
caagcagaagacggcatacgagatCATGACgtgactggagttcagacgtgtgctcttccgatctCCTACGG V3_77F P7 GAGGCAGCAG
caagcagaagacggcatacgagatGCTATCgtgactggagttcagacgtgtgctcttccgatctCCTACGG GAGGCAGCAG V3_78F P7
caagcagaagacggcatacgagatGGACTTgtgactggagttcagacgtgtgctcttccgatctCCTACGG V3_79F P7 GAGGCAGCAG
caagcagaagacggcatacgagatGGCAATgtgactggagttcagacgtgtgctcttccgatctCCTACG V3_80F P7 GGAGGCAGCAG
caagcagaagacggcatacgagatTCTCGGgtgactggagttcagacgtgtgctcttccgatctCCTACGG V3_81F P7 GAGGCAGCAG
caagcagaagacggcatacgagatTCAGCGgtgactggagttcagacgtgtgctcttccgatctCCTACG V3_82F P7 GGAGGCAGCAG
caagcagaagacggcatacgagatTGTGCCgtgactggagttcagacgtgtgctcttccgatctCCTACGG V3_83F P7 GAGGCAGCAG
caagcagaagacggcatacgagatTGCACGgtgactggagttcagacgtgtgctcttccgatctCCTACG V3_84F P7 GGAGGCAGCAG
caagcagaagacggcatacgagatAAGGCCgtgactggagttcagacgtgtgctcttccgatctCCTACG V3_85F P7 GGAGGCAGCAG
caagcagaagacggcatacgagatACCAGGgtgactggagttcagacgtgtgctcttccgatctCCTACG V3_86F P7 GGAGGCAGCAG
caagcagaagacggcatacgagatAGCCTGgtgactggagttcagacgtgtgctcttccgatctCCTACG V3_87F GGAGGCAGCAG P7 caagcagaagacggcatacgagatAGCGACgtgactggagttcagacgtgtgctcttccgatctCCTACG V3_88F GGAGGCAGCAG P7
41
Primer Adapter Sequence
caagcagaagacggcatacgagatCTACGCgtgactggagttcagacgtgtgctcttccgatctCCTACGG P7 GAGGCAGCAG V3_89F caagcagaagacggcatacgagatCTCCAGgtgactggagttcagacgtgtgctcttccgatctCCTACGG V3_90F P7 GAGGCAGCAG
caagcagaagacggcatacgagatCCGTAGgtgactggagttcagacgtgtgctcttccgatctCCTACG GGAGGCAGCAG V3_91F P7
caagcagaagacggcatacgagatCGGTGTgtgactggagttcagacgtgtgctcttccgatctCCTACG V3_92F P7 GGAGGCAGCAG
caagcagaagacggcatacgagatCGGAACgtgactggagttcagacgtgtgctcttccgatctCCTACG V3_93F P7 GGAGGCAGCAG
caagcagaagacggcatacgagatGTGCTGgtgactggagttcagacgtgtgctcttccgatctCCTACG V3_94F P7 GGAGGCAGCAG
caagcagaagacggcatacgagatGAACGGgtgactggagttcagacgtgtgctcttccgatctCCTACG V3_95F P7 GGAGGCAGCAG
caagcagaagacggcatacgagatGGATGCgtgactggagttcagacgtgtgctcttccgatctCCTACG V3_96F P7 GGAGGCAGCAG
42
2.7 DNA sequencing and analysis
Amplified DNA was sequenced using Illumina MiSeq at the McMaster Genome Facility
in Hamilton, ON to generate raw paired-end fastq sequence files. R (v.3.5) and RStudio
(v.1.2.5001) was used to analyse data. Within RStudio, Cutadapt (v.2.5) was used to trim
primers and Divisive Amplicon Denoising Algorithm (DADA2; v1.8.0) was used to identify
the amplicon sequence variants (ASV).127,128 Briefly, DADA2 workflow begins by filtering
forward and reverse reads by removing sequences with unknown nucleotides or more than
two expected errors and trimming to 130 nucleotides.129 Denoising of the database were
conducted using a Poisson error model which is conducted prior to merging reads.127,130 Pair
reads were merged using a mergePairs function after dereplication of identical reads were
conducted. Chimeras were identified by alignment to all more abundant sequence
combinations from left and right and removed if the sequences match exactly.127 All
singleton ASVs were readily removed in the DADA2 pipeline.127 Resulting ASVs were
annotated using the IDTAXA algorithm from the DECIPHER package referencing the most
recently updated SILVA v132 database.131,132
Microbiota genus present at above 1% abundance was visualized using alpha and beta-
diversity functions. Alpha diversity measures were conducted using Observed diversity
index (ODI), which displays the total richness (number of taxonomic groups) of bacteria
present in the samples; and Shannon diversity index (SDI), which displays the total richness
and evenness (distribution of abundance of the taxonomic groups) of the bacteria present in
each sample.133 Wilcoxon Rank-Sum tests were performed to analyze statistical differences
between groups.
43
Beta-diversity measures the variability in taxonomy between samples. Principle coordinate analysis (PCoA) plots were employed to visualize the community wide differences of samples relying on Bray-Curtis dissimilarity test.134 PERMANOVA analysis was used to analyse community-wide differences at the ASV level in PCoA plots.135
44
2.8 Quantitative PCR
Quantitative PCR (qPCR) was performed to establish the absolute 16S rDNA quantity
present in each sputum samples.136 Additionally, qPCR analysis was also used to determine
the quantity of P. aeruginosa and MABC DNA with the extracted samples.
Quantifying the absolute 16S rDNA present in each sputum sample was done by creating
a standard curve of extracted DNA from a pure culture of P. aeruginosa PA01 strain at a 1:5
serial dilution of a 25ng/µL stock. Extraction of DNA was conducted using the same DNA
extraction method as sputum samples as described previously in the DNA extraction section.
Each 10µL reaction contained 5µL of TaqMan Fast Advanced Master Mix (Thermo
Fisher), 0.5µL of TaqMan gene expression probe for pan-bacterial detection of 16S rRNA
(Ba04230899_s1; Thermo Fisher Scientific), 2.5µL of the template DNA from sputum
extraction, and 2µL of double distilled water. The qPCR reaction program was set with the
following parameters: 50˚C for 2mins followed by 95˚ for 2mins then 40 cycles of 95˚C for
1secs and 60˚C for 20secs. Mathematical correction for the copies of 16S rRNA operon
needs to be conducted since the P. aeruginosa standard being used has four copies of the 16S
rRNA operon.137 The following equations are used:
16S gene copies [DNA]ng 1g 1mol bp DNA 6.023x1023bp 4 copy 16S rRNA = ( ) ( ) ( ) ( ) ( ) (volume of template) µL 10003ng 660g DNA 1 mol bp PA genome size bp
16푆 gene copies [DNA extraction]ng (( ) ( ) (50µL)) Copies 25ng µL 1000µL = ( ) mL 300µL sputum 1mL
45
The abundance of P. aeruginosa DNA in sputum samples was conducted using the absolute quantification method. A standard curve was created of extracted DNA from a pure culture of P. aeruginosa PA01 strain at a 1:5 serial dilution of a 1ng/µL stock. DNA extracted from Stenotrophomonas maltophilia was used as the non-template control. The P. aeruginosa and S. maltophilia were both extracted using the same DNA extraction method as previously described in the DNA extraction section. Primers for the amplification were created as described by Qin et al. (2003)138. Duplicates of each sample were conducted with the mean quantity calculated based off the standard curve being used. Duplicates with discrepancies large discrepancies in the quantity values or other errors were repeated.
Each 10µL reaction well contained 5µL of SsoAdvanced Universal Inhibitor-Tolerant
SYBR Green Supermix (Bio-Rad), 1µL of forward and reverse primer mix (final concentration of 150nM), and 4µL of the template DNA from sputum extraction. The qPCR reaction program was set with the following parameters: initial denaturation at 95˚C for
10mins followed by 40 cycles of 95˚C for 15secs, 68˚C for 1min then ending with a melting curve analysis of one cycle with the parameters of 95˚C for 15secs, 60˚C for 1min, 95˚C for
15secs, and 60˚C for 15secs.
The abundance of MABC in sputum samples was also conducted using the absolute quantification method. Primers and probe for the amplification were created as described in
Rocchetti et al. (2016)139. Extraction of a pure culture of MABC was used for the standard curve at a 1:5 serial dilution of a 1ng/µL stock. DNA extraction from P. aeruginosa was used as the non-template control. All extractions were conducted as per the methodology previously described under DNA extraction. Duplicates of each sample were conducted with
46 the mean quantity calculated based off the standard curve being used. Duplicates with large discrepancies in the quantity values or other errors were repeated.
Each 10µL reaction contained 5µL of TaqMan Fast Advanced Master Mix (Thermo Fisher),
1µL of forward and reverse primer mix (final concentration of 900nM), 1µL of probe (final concentration of 400nM), and 3µL of the template DNA from sputum extraction. The qPCR reaction program was set with the following parameters: 50˚C for 2mins followed by 95˚ for
2mins then 40 cycles of 95˚C for 1secs and 60˚C for 20secs.
47
2.9 P. aeruginosa Inhibition Assay of MABC
Microbiome analysis exhibited a significant negative correlation between the presence of
Pseudomonas and MABC. To test whether these differences could be due to any excreted
factors that P. aeruginosa produced that negatively impacted MABC, an inhibition assay was
designed using P. aeruginosa supernatant. Supernatant mixture from two PA strains (P.
aeruginosa PA01 lab strain and Prairie Epidemic strain (PES)) were collected from cultures
grown in Luria-Bertani broth (LB) and filter sterilized. MABC grown in Middlebrook 7H9
broth for 5 days was incubated with both supernatant at different dilution ratios of 1:5 and
1:20. Table 5 displays the content configuration that was constructed to test MABC
susceptibility with varying dilutions of P. aeruginosa supernatant and grown for 5 days. For
each day, including initial incubation (day zero), 1:10 serial dilution of 10µL from each flask
was conducted and plated on Middlebrook 7H10. The plates were allowed 4 days to grow
cultures which were then used to calculate the CFU/mL.
Table 5: Content configuration of each flask used to test P. aeruginosa inhibition of MABC. Three replicates of each flask was used for the assay. Negative Control Control PA01 Flask Control 1:5 1:20 PA01 1:5 1:20 PES 1:5 PES 1:20 M7H9 broth 11 11 11 11 11 11 11 PA supernatant 0 0 0 4 1 4 1 LB broth 0 4 1 0 0 0 0 Bacteria 0 5 5 5 5 5 5 0.9% NaCl 9 0 3 0 3 0 3 Total 20 20 20 20 20 20 20 Dilution 0 0.2 0.05 0.2 0.05 0.2 0.05
48
Chapter Three: Results
49
3.1 Case and Control Patient Demographics
This study cohort employed patients from the Calgary Adult CF clinic and were divided
based on acquisition of MABC. Case patients were identified based on chart review and had
acquired the bacteria - and were positive based on ATS/IDSA criteria. Control patients did
not have any positive history for positive cultures for MABC as identified through clinical
review of medical records and clinical microbiology testing (Middlebrook 7H10, done
annually) for dates preceding their used samples. Twenty-one CF patients with MABC
infections were identified meeting inclusion and exclusion criteria (median age 21.8 years,
IQR 18.5 – 33.1) contributing 174 sputum samples (median 8 samples/patient, IQR 6-12).
This cohort was matched to two gender, age (±2 years) and birth cohort compliant controls.
Each control patient was assessed at a single time point – consistent with a time period
corresponding to infection in the cases – for a total of 42 control samples. Furthermore, case
patients were defined as persistent according to 50% or greater positive mycobacteriology
tests with a minimum of three tests in the year following initial infection.
Case and control patients did not differ based on gender, age, genetics, comorbidities, or
coinfections, (Tables 6A, 6B, and 6C). The only parameter differences noted between case
and control patients were related to patient body mass index (BMI), where case patients had a
lower BMI (suggesting worse nutritional status). Baseline lung function at the time of
infection also did not differ between cases and controls (Table 6A). In general, differences
were not observed between cases and controls with respect to medications patients were
receiving at the time of MABC identification. Median baseline lung function as measured by
percent predicted forced expiratory volume in one second (%FEV1) or by percent predicted
50 total forced vital capacity (%FEV) of case patients did not differ significantly from controls although tended to be slightly higher.
To understand the natural history of MABC infection we compared outcomes of those with MABC who developed persistent infection (persistent) with those who ultimately cleared infection (transient). In general, these groups did not differ with respect to demographics, co-morbidities, or co-infections (Table 6A, 6B, and 6C). In general, differences were not observed in the medications that patients were receiving at the time of first MABC infection – with one exception. The only significant difference in the medication patients received was the use of proton pump inhibitors (PPI) were observed with persistent patients more likely to be on PPI compared to transient patients as well as control patients (Table 6C).
MABC subspecies M. abscessus abscessus (17/21, 81%) dominated the case patients with M. bolletii (2/21, 10%) being a smaller factor. No M. massiliense subspecies were observed although two patient mycobacteriology tests resulted undifferentiated subspecies.
All but one case patient was infected with at least one other co-infecting bacterial species at the time of infection with MABC with eight case patients co-infected with multiple other bacterial species (Table 6B). In the control cohort, all but two patients were infected with at least one bacterial species with 18 control patients exhibiting multiple co-infections.
51
Table 6A: Baseline clinical demographics collected for case and control patients from the Calgary Adult CF clinic chart records. p values Case Transient Persistent Control Transient Persistent Demographics Total vs (n=21) (n=9) (n=12) (n=42) vs vs Controls Persistent Controls 21.8 19.8 (18.6- 23.4 (18.3- 23.0 (18.4- Age 0.89 0.92 0.9 (18.5-33.1) 26.2) 33.8) 33.2) Sex – Male 12 (57%) 7 (78%) 5 (42%) 24 (57%) 0.18 0.51 1 F508 12 (57%) 5 (56%) 7 (58%) 23 (55%) 1 1 1 homozygous 19.5 (18- 19.5 (18.4- 19.4 (17.5- 21.5 (19.3- BMI, kg/m2 0.89 0.26 0.04 21) 20.7) 23.7) 24) CFRD 1 (5%) 1 (11%) 0 (0%) 7 (17%) 0.43 0.33 0.25 CFLD 5 (24%) 3 (33%) 2 (17%) 10 (24%) 0.61 0.71 1 ABPA 1 (5%) 0 (0) 1 (8%) 3 (7%) 1 1 1 62.5 (50.4- %FEV1 72 (51-88) 72 (40-87) 74.5 (54-93) 0.72 0.45 0.57 84) 91 (76- 91.5 (78.3- 89.5 (72- %FVC 91 (74-106) 0.29 0.43 0.31 106) 109) 102)
Abbreviations: BMI: body mass index; CFRD: CF related diabetes, CFLD: CF liver disease; ABPA: allergic bronchopulmonary aspergillosis; FEV: forced expiratory volume; FVC: forced vital capacity. P values are obtained using 2-sided Fisher's exact or Wilcoxon rank-sum. Data are presented as n (%) or median (interquartile range).
52
Table 6B: Mycobacteriology subspecies and coinfections collected for case and control patients from the Calgary Adult CF clinic chart records. p values Case Transient Persistent Control Transient Persistent Infections Total vs (n=21) (n=9) (n=12) (n=42) vs vs Controls Persistent Controls M. abscessus 17 6 (67%) 11 (92%) NA 0.27 NA NA abscessus (81%) 2 M. bolletii 1 (11%) 1 (8%) NA 1 NA NA (10%) M. massiliense 0 (0%) 0 (0%) 0 (0%) NA NA NA NA MABC 2 2 (22%) 0 (0%) NA 0.17 NA NA undifferentiated (10%) Pseudomonas 10 5 (56%) 5 (42%) 21 (50%) 0.67 0.75 1 aeruginosa (48%) 12 MSSA 5 (56%) 7 (58%) 22 (52%) 1 0.75 0.79 (57%) Burkholderia 0 (0%) 0 (0%) 0 (0%) 0 (0%) UND UND UND cenocepacia Burkholderia 3 2 (22%) 1 (8%) 1 (2%) 0.55 0.4 0.1 multivorans (14%) 2 MRSA 1 (11%) 1 (8%) 5 (12%) 1 1 1 (10%) Stenotrophomonas 2 0 (0%) 2 (17%) 1 (2%) 0.49 0.12 0.26 species (10%) 2 Aspergillus 0 (0%) 2 (17%) 4 (10%) 0.49 0.6 1 (10%) Achromobacter 0 (0%) 0 (0%) 0 (0%) 3 (7%) UND 1 0.54 species Abbreviations: MSSA: methicillin-sensitive Staphylococcus aureus; MRSA: methicillin-resistant Staphylococcus aureus; UND: undefined. P values are obtained using 2-sided Fisher's exact or Wilcoxon rank-sum. Data are presented as n (%) or median (interquartile range).
53
Table 6C: On-going therapies and antibiotic collected for case and control patients from the Calgary Adult CF clinic chart records. p values Case Transient Persistent Control Transient Persistent Therapies Total vs (n=21) (n=9) (n=12) (n=42) vs vs Controls Persistent Controls Nutritional 2 (10%) 1 (11%) 1 (8%) 4 (10%) 1 1 1 supplements Pancreatic 38 20 (95%) 8 (89%) 12 (100%) 0.43 0.56 0.66 enzymes (90%) 21 41 CF vitamins 9 (100%) 12 (100%) UND 1 1 (100%) (98%) 39 Vitamin D 19 (90%) 8 (88%) 11 (92%) 1 1 1 (93%) Ranitidine 1 (5%) 1 (11%) 0 (0%) 8 (19%) 0.43 0.18 0.25 18 PPI 13 (62%) 3 (33%) 10 (83%) 0.03 0.02 0.19 (43%) 24 SABA 14 (67%) 4 (44%) 10 (83%) 0.16 0.17 0.59 (57%) 33 LABA 18 (86%) 8 (89%) 10 (83%) 1 1 0.74 (79%) Inhaled 21 11 (52%) 5 (56%) 6 (50%) 1 1 1 corticosteroids (50%) 17 DNase 11 (52%) 4 (44%) 7 (58%) 0.67 0.33 0.43 (40%) Inhaled 15 5 (24%) 2 (22%) 3 (25%) 1 0.73 0.4 hypertonic saline (36%) Inhaled 18 6 (29%) 2 (22%) 4 (33%) 0.66 0.74 0.41 Tobramycin (43%) Inhaled Colistin 1 (5%) 1 (11%) 0 (0%) 0 (0%) 0.43 UND 0.33 Inhaled 3 (14%) 2 (22%) 1 (8%) 3 (7%) 0.55 1 0.39 Aztreonam 16 Azithromycin 4 (19%) 1 (11%) 3 (25%) 0.6 0.51 0.16 (38%) Chronic oxygen 2 (10%) 2 (22%) 0 (0%) 4 (10%) 0.17 0.56 1 Abbreviations: PPI: proton pump inhibitor; SABA: short-acting beta agonist; LABA: long-acting beta agonist; UND: undefined. P values are obtained using 2-sided Fisher's exact or Wilcoxon rank-sum. Data are presented as n (%) or median (interquartile range).
54
3.2 Pulmonary Function Linear Regression
Extensive patient pulmonary function data was collected for all available clinic visits
associated with sputum samples dating 2 years prior to initial infection as well as 2 years
following (additional dates were collected for patients if insufficient data was present). To
understand rates of change of lung function over time we used linear regression. Linear
regression was extrapolated from the percent forced expiratory volume during the first
second (%FEV1) values and presented in terms of case patients compared to their controls as
displayed in Figure 4. Percent FEV1 was compared for two-years prior to clinical infection
date as well as two years after. Significant differences between any of the groups was not
noted however case patients did have a near significance of p=0.089 suggesting a trend
towards higher lung function change with clinical intervention. Within the case patient
cohort, no significance was noted between the before and after infection samples for transient
and persistent patients as seen in Figure 5.
55
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F
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n n io n io n ct io ct io fe ct fe ct In fe In fe e In e In or r or r ef fte ef fte B A B A
Case Patients Control Patients
Figure 4: Lung function changes over time did not differ between cases and controls before or after infection. Changes in the percent predicted FEV1 values as measured over 2 years before and after initial clinical infection as determined through linear regression. Two-tailed Mann Whitney analysis was conducted to analyze the difference between the cohorts and no significance was noted between any groups.
56
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n n io n io n ct io ct io fe ct fe ct In fe In fe e In e In or r or r ef fte ef fte B A B A
Transient Patients Persistent Patients
Figure 5: Lung function changes over time did not differ in transient vs persistent patients before or after infection. Changes in the percent predicted FEV1 values as measured over 2 years before and after initial clinical infection date determined through linear regression. Two- tailed Mann Whitney analysis was conducted to analyze the difference between the cohort and no significant differences were noted between any groups.
57
3.3 Lung Microbiome Diversity
Two different variations of 16S rDNA gene sequencing analysis pathway was used to
classify the microbiome present in the samples sequenced: the SL1P pathway and DADA2
pathway. Initially, the short-read library 16S rRNA gene sequencing pipeline (SL1P) was
used to sequence the raw reads into operational taxonomic units (OTUs) and in conjunction
with Greengenes2013 furthered to assign genomic taxonomy. The SL1P pathway was
designed by Whelan et al. (2017) at the McMaster Genomic Facility (Hamilton, ON) and
served to combine pre-existing tools into a computational pipeline to produce reproducible
sequence data.140 The SL1P pathway automates accurate processing of paired-end amplicon
reads by first cutting PCR primers using cudadapt, then PANDAseq to align the forward and
reverse reads, quality trim is conducted by sickle, and USEARCH to remove chimeric
sequences.128,141,142 Following, there are a variety of eight different choices to designate
operational taxonomy units (OTU) with CD-HIT v3.1.1 being used for this data set. Next,
RDP classifier is used against the Greengenes 2013 data base to assign taxonomy and are
accurate to the genus level.143,144 OTUs are typically clustered based on a 97% similarity
threshold between sequences.
Divisive Amplicon Denoising Algorithm (DADA2) was the second of the pipelines that
was used for the processing for raw 16S rDNA gene sequences.127 DADA2 models and
corrects Illumina-sequenced amplicon errors and can resolve difference of as little as one
nucleotide to assign sequence variants. Differences in DADA2 from SL1P arises with the
denoising step occurring after merging paired end reads so as to allow for error model to
differentiate sequences into amplicon sequence variants (ASV). DADA2 filters fastq files to
trim sequences to a specified length. Next is dereplication to output unique sequences and
58 their abundances. Followed by the denoising algorithm and removal of chimeras. Finally, forward and reverse reads are merged together if they exactly overlap. Any number of taxonomy assignment databases can be used from here. Specifically, the databases Silva,
RDP, and GTDB were used to assign taxonomy in this study. Silva is the more widely used database for microbiome analysis pertaining to health sciences compared to RDP and is regularly updated with the last update coming in August 2020. GTDB is similarly updated, however, is new and is not as commonly used in the health science and medical field .
Greengenes was last updated in May 2013 and is generally considered outdated although some still use it. OTUs that were observed in the microbiome of patients using the SL1P –
Greengenes 2013 pathway was much lower than was observed in DADA2 – Silva v138 pathway. The SL1P – Greengenes pathway observed 8,455,210 different OTUs whereas the
DADA2 – Silva pathway showed 12,732,176 different ASVs. Figure 6 shows the major phyla that are observed with each of the pathways with a clearly greater observed sequence variations in the DADA2 – Silva pathway.
The most recent update of the Silva database with the species extension shows the presence of the “mycobacterium abscessus” taxonomy although with a mere 1931 reads present for the ASV. Other databases are accurate to the genus level but are only able to decipher the “Order” level of the sequence associated with MABC. Silva v138 and GTDB
2020 displayed Corynebacteriales while RDP v18 displayed the synonym Mycobacteriales for the associated “Order”. Only four of the 21 patients (21 samples) displayed any ASVs through the 16S rRNA microbiome analysis with ASV counts ranging from five to 855. The higher end of this range was associated with a chronically infected patient that displayed a
Ziehl-Neelsen smear 3+ and only took one day to grow with a qPCR quantitative mean of
59
0.044ng/µl. The reason behind the low number of ASVs observed through the microbiome analysis are unknown but could be attributed to insufficient extraction of MABC 16S rDNA from sputum, difficulty in amplification of MABC 16 rDNA, or improper sequencing analysis ability.
Figure 7 displays the relative abundance of the top 20 ASV compositions for each patient sample through the course of their MABC infection achieved by the DADA2 pipeline using the SILVA v138 taxonomy database as reference. Patients are divided based on persistence of MABC as described by previous criteria established. Most patients displayed a diverse array of bacterial compositions in their microbiome which shift towards a dominating genus during antibiotic exposures but reverted back to the pre-antibiotic norm. Most patients also appeared to have no dominant genus prior to the initial clinical infection.
60
A B 6×106 6×106
4×106 4×106
s
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0 0 s s a a a s s ia ia ia e e ri ri ri te te r r r t t e e e u e te te te u e t t t c d c c c ic id c c c i i a a a o a a a m ro b b b rm r b b b ir e o o o i te o o o F t e n s F c te in s c t ti u a o t u a ro c F B r c F B P A P A Phyla Phyla
Figure 6: Phyla comparisons for 16S rDNA sequences observed in the microbiome of MABC case patients and their respective controls. (A) Phyla output from the SL1P pathway using Greengenes 2013 database. (B) Phyla output from the DADA2 pathway using Silva v.132 pathway.
61
Figure 7: Taxonomic composition of the top 20 ASVs at the genus level through infection natural history and matched controls. Patients included in the top panel are diagnosed ultimately with persistent infections and bottom panel are those patients who are defined as having transient infections. Patient sex is designated at the top of each column with either an M or F below which is the study specific patient ID letter. Genus not in the top 20 are not represented in the figure and appear as white above each bar up to a total relative abundance of 1.00. Samples were also classified into infection stages as; A – before infection; B – at infection before treatment; C – induction of therapy; D – consolidation therapy; E – maintenance therapy; F – intensification of therapy; G – clearance; H – Chronic and off-treatment. Red fonts for letters indicate antibiotic therapy during those days. Numbers below each patient’s panel represent the days from initial MABC positive.
62
3.4 Microbiome structure through the natural history of infection
Relative abundance of the top 20 genera present in the microbiome suggested a consistent
microbiome composition with a dramatic shift during antibiotic treatment and a rapid shift
back to status norm upon its discontinuation. To assess patient microbiome impact on
incident infections, it was important to understand changes in the microbial community
through the natural history of infection. Patient samples were divided into subsets based on
the progression of disease and included the stages of pre-infection (n=38), at infection
diagnosis (n=37), during treatment (n=79), and post-treatment (n=20).
Pre-infection stage accounted for all sputum samples that were collected in the two years
prior to initial clinical identification of MABC growth in sputum samples. These samples
could (unknowingly) be positive prior to their clinical identification due to AFB testing
occurring only once per year. At infection stage encompassed the samples at the time of
initial infection diagnosis and until initiation of treatment (if undertaken). At treatment
samples included all the stages of the treatment course that a patient may be prescribed and
include the initiation, continuation and/or maintenance, and re-intensification stages (during
subsequent exacerbations). Post-treatment samples encompassed for all patient sputum
samples represented after treatment was stopped regardless of whether the patient cleared the
infection or were declared persistent (including any patients on chronic suppressive therapy).
Reductions in alpha diversity were noted through different treatment stages due to
selective antibacterial therapies. Specifically, as seen in figure 8A, significant ODI
differences are noted between pre-infection and at treatment stages (84.5 vs 40.0, p=3.5x10-9)
and between at infection stage and at treatment stage (88.0 vs 40.0, p=9.5x10-10). At
treatment stage also significantly different from the post-treatment stages (66.5 vs 40.0,
63 p=0.002). Similarly, as seen in figure 8B, significant difference in the SDI was also noted for pre-infection samples compared to at treatment samples (2.52 vs 1.72, p=1.1x10-4) as well as at infection samples compared to at treatment samples (2.29 vs 1.72, p=1.2x10-4). A near significant difference between samples at the treatment stage compared to post treatment samples was also observed (1.72 vs 2.39, p=0.057).
Significance was calculated using Wilcoxon rank-sum statistical analysis rarefied with sample variations between groups. These values suggest a decrease in microbial richness and evenness during the treatment phase of disease natural history from the norm.
Subsequent recovery/rebound of the microbiome in the post treatment stage is clearly noted in terms of richness of bacteria present with a trend towards community evenness suggesting that post therapy microbiome regenerates in terms of biomass from the microbes that were not killed off.
Figure 8C explores the beta-diversity through clustering of microbial communities using
PCoA based on Bray-Curtis dissimilarities displayed clustering of microbial communities based on disease progression (p=0.001, R2 = 5.7%, PERMANOVA test). Significant dissimilarities were observed between pre-infection and at infection diagnosis samples compared to during treatment and post treatment samples. Pre-infection samples did not significantly differ in clustering to at infection samples and similarly at treatment samples did not differ significantly in clustering than post-treatment samples.
64
65
Figure 8: The natural history of the CF sputum microbiome through the course of MABC infection stages. (A)Observed diversity index changes over time in response to disease stage and antibiotic intervention. Box plots represent median values with top and bottom bars displaying the interquartile range. Wilcoxon rank-sum statistical analysis was conducted between the groups to obtain the p values and rarefied to accommodate variation in sample numbers between groups. (B) Shannon diversity index changes over time in response to disease stage and antibiotic intervention. (C) PCoA of Bray-Curtis dissimilarities analysis of the disease natural history. PERMANOVA analysis was conducted to analyze statistical significance and showed significant clustering (p = 0.001, R2 = 7.3%) based on infection stage.
66
3.5 Microbiome Comparison of MABC Case Patients to Controls
One of the most important goals of our project was to determine if and how individuals
with MABC infection differed from those without. To ascertain this, we sought to compare
the community structure of case patient’s sputum before clinically recognized as MABC
infection to age, sex, and birth cohort-matched controls. Specifically, microbiome
characteristics prior to and at the time of the MABC incident infection were of interest for
comparison to non-infected controls. Microbial differences in these samples could allude to
why a patient would develop an incident infection in the future. Case patient samples prior
to infection (n=38) and at the time of infection (n=37) were compared to control patient
samples (n=42). Significant beta-diversity clustering was observed between pre-infection
case patients compared to their control counterpart as seen in figure 9B (p=0.048, R2 = 2.5%,
PERMANOVA test) suggesting that fundamental community structures differed between
groups. Samples from patients with MABC were more diverse at baseline than those of
controls. Figure 9A displayed the alpha diversity analysis confirming microbial diversity
differences between the cohorts. In the years prior to an MABC infection, the expectorated
sputum of case patients had a significantly higher ODI richness (84.5 vs 71.0, p=0.037) and
SDI richness and evenness (2.52 vs 2.02, p=0.022) compared to matched controls leading up
to an infection. Further, the presence of individual ASVs were studied (of the top 20 ASVs)
and was observed that, in particular, the relative abundance of Pseudomonas was
significantly higher in the control patients compared to the case patients as seen in Figure 9C.
Significant dissimilarities were equally prevalent in the at infection diagnosis stage
compared to the matched controls. Alpha diversity analysis displayed a significantly higher
ODI and SDI of case samples (ODI: 88 vs 71.0, p=0.014; SDI: 2.28 vs 2.02, p=0.012) as
67 seen in figure 10A and clustering was observed at during this stage prior to the onset of treatment (p=0.004, R2 = 3.6%, PERMANOVA test) as per Figure 10B. Similar to pre- infection ASV differences, the presence of Pseudomonas was observed to be significantly higher in the expectorated sputum of control patients compared to control patients as seen in
Figure 10C (0.0038 v 0.0011, p=0.0067. Ten of the 21 MABC patients were co-infected with P. aeruginosa compared to 21 of 42 controls were co-infected with P. aeruginosa.
Transient and persistent patients each had five chronically infected P. aeruginosa patients.
68
Figure 9: The microbiome of sputum Pre-infection with MABC differs between cases and control patients. (A) Alpha diversity analysis displaying median ODI and SDI with interquartile range between case and control patients in the samples prior to initial infection of MABC. Wilcoxon rank-sum statistical analysis was conducted between groups to obtain p values rarefied to accommodate sample variations. (B) PCoA of Bray- Curtis dissimilarities analysis of case vs control patient cohorts in the samples prior to infection. PERMANOVA analysis was conducted to analyze statistical significance and showed significant clustering (p = 0.038, R2 = 2.5%). (C) 2-fold log abundance of Pseudomonas genus between the control and case patients. Two-sided Mann-Whitney test was conducted to analyze significance between the groups.
69
Figure 10: The microbiome of sputum At infection with MABC differs between cases and control patients . (A) Alpha diversity analysis displaying median ODI and SDI with interquartile range between case and control patients in the samples at the time of initial MABC infection prior to treatment onset. Wilcoxon rank- sum statistical analysis was conducted between groups to obtain p values rarefied to accommodate sample variations. (B) PCoA of Bray-Curtis dissimilarities analysis of case vs control patient cohorts in the samples at infection. PERMANOVA analysis was conducted to analyze statistical significance and showed significant clustering (p = 0.004, R2 = 3.6%). (C) Log abundance of Pseudomonas genus between the control and case patients. Two-sided Mann-Whitney test was conducted to analyze significance between the groups.
70
3.6 Comparing Transient and Persistent Case Patient Sub-cohorts
MABC infection progression towards either a persistent long-term infection that might
have consequence or rapidly be cleared without significant impact was an important factor to
consider - and to understand if the microbiome could play a role in the differentiation of
these outcomes. Case patients were divided into sub-cohorts based on disease progression in
the year following initial clinical infection date and as per ATS/ISDA criteria. Case patients
were identified as transient (n=9) or persistent (n=12) patients based on the aforementioned
criteria of at least 50% positive MABC samples per year with a minimum of 3 samples
tested. When we assessed patient samples based on persistence of MABC infections,
samples associated with transient infections (n=17) did not differ significantly from
persistent infection samples (n=21) based on alpha or beta diversity matrix as seen in Figure
11A and B. Additionally, no differences in the relative abundance of genera present were
noted. Similarly, no differences in alpha diversity were noted for the SDI of cases at the time
of infection comparing transient (n=13) and persistent (n=24) patient samples. When we
assessed ODI we did observe that differences did exist – and that transient patients had less
diversity (p=0.026) but not when the samples were rarefied (p=0.08) to accommodate the
difference in number of samples between transient and persistent patients as can be seen in
figure 12A. Figure 12B displays the insignificant beta diversity differences present between
the sub-cohorts suggesting no evidence of microbiome diversity clustering due to patients
developing a persistent infection compared to those that are transiently infected.
71
Figure 11: Pre-infection microbiome comparison between transient and persistent patients. (A) Alpha diversity analysis displaying median ODI and SDI with interquartile range between case and control patients in the samples prior to initial infection of MABC. No significance was noted for either the ODI (p=0.15) nor the SDI (p=0.71). Wilcoxon rank-sum statistical analysis was conducted between groups to obtain p values rarefied to accommodate sample variations. (B) PCoA of Bray-Curtis dissimilarities analysis of transient vs persistent patient cohorts in the samples prior to infection. PERMANOVA analysis was conducted to analyze statistical significance and showed no significant clustering (p = 0.50, R2 = 2.5%).
72
Figure 12: At infection microbiome comparison between transient and persistent patients. (A) Alpha diversity analysis displaying median ODI and SDI with interquartile range between case and control patients in the samples prior to initial infection of MABC. Significance was noted for the ODI (p=0.026) but not for the SDI (p=0.65). Wilcoxon rank-sum statistical analysis was conducted between groups to obtain p values rarefied to accommodate sample variations where the ODI p value was 0.080 and hence insignificant. (B) PCoA of Bray-Curtis dissimilarities analysis of transient vs persistent patient cohorts in the samples prior to infection. PERMANOVA analysis was conducted to analyze statistical significance and showed no significant clustering (p = 0.51, R2 = 2.4%).
73
3.7 Quantitative PCR
Using real-time qPCR techniques, we sought to find the absolute 16S rDNA quantity
present in each sputum sample was calculated as well as the quantity of P. aeruginosa and
MABC DNA that were presented within these extracted sputum samples.
3.7.1 Absolute bacterial bioburden through the natural history of infection
Quantitative PCR was used to obtain the absolute 16S rDNA concentration present in all
sputum samples. Comparison of the natural history of infection stages were conducted to
help understand differences in total bacterial burden and displayed in Figure13. Total
bacterial load for each sample was measured as total copies of 16S rDNA per milliliter of
sputum. Median 16S rDNA copies/mL of samples were consistent through pre-infection
(1.77 x 1012), at infection (1.47 x 1012), and post treatment (1.58 x 1012) with a slight decline
during at treatment stage (5.84 x 1011). Significant differences, however, were only observed
between during treatment and post treatment stages (p= 0.03) where the total bacterial load
increased at the completion of suppressive antibacterial therapy. Near significance is
observed between pre-infection and at infection stages compared to at treatment stage
(p=0.069 and p=0.079, respectively). Significant differences between pre-infection samples
and post treatment samples was not observed (p=0.48)
74
Figure 13: Total bacterial bioburden stays relatively stable through the natural history of infection with exception of treatment intervention. Median value for the total bacterial load through the natural infection history stages remained consistent with the following value for each stage present: pre-infection (n=38) was 1.77 x 1012 16S rDNA/mL, at infection (n=37) was 1.47 x 1012 16S rDNA/mL, during treatment (n=79) was 5.84 x 1011, and post treatment (n=20) was 1.58 x 1012). Two-sided Mann-Whitney test was conducted to analyze significance between the groups.
75
3.7.2 Absolute bacterial bioburden between case and control patients
The bioburden of case patients versus control patients was assessed to see if the potential microbiome differences noted could be as a result of the underlying quantity of total microbiota present in the sputum sample. As seen in figure 14A, there are no significant differences in the median bioburden of case patients as measured by 16S rRNA in all pre- infection samples (1.1 x 1012, n=174) compared to control patients (1.6 x 1012 n=42).
Similarly, control patient bioburden did not differ from the sub-cohort of pre-infection samples (1.8 x 1012, n=38) and at infection samples (1.5 x 1012, n=37) in figure 14B and figure 14C, respectively. Lack of significant difference in absolute bioburden of bacteria in sputum between the cohorts allows for the speculation of any microbiome differences observed above through the alpha and beta diversities to be associated with factors leading towards development of an incident infection with MABC rather than a simple difference in the available microbiota.
76
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Figure 14: Total bacterial bioburden does not change as a function of infection status. (A) All case patients (n=174) compared to controls (n=42), p = 0.51. (B) Pre-infection case patients (n=38) compared to control patients (n=42), p = 0.93). (C) At infection case patient (n=37) compared to control patients (n=42), p=0.77). Two-tailed Mann-Whitney U analysis was conducted for all comparisons.
77
3.7.3 Quantifying MABC DNA in Sputum Samples
RT-qPCR was also performed on all sputum samples for the presence of MABC DNA through the detection of the internal transcriber spacer gene using a probe. All extracted sputum sample (174 case, and 42 control samples) were subjected to the test. In case patient individuals, we noted all patients had at least one MABC DNA as detected by qPCR. We noted one control positive for MABC DNA in one time point – but at low levels.
Figure 15 displays the mean MABC DNA concentration through the natural history progression of disease. Amongst infected patients, the presence of MABC is noted highest at the time of initial infection diagnosis and is significantly higher than pre-infection, during treatment and post treatment. This holds true as samples during the clinical at infection stage would contain the most MABC DNA. Prior to a clinical positive stage, fewer samples would contain MABC DNA (as these cases were clinically diagnosed as yet) and those that do would not have concentrations as high as seen during at infection stages. During treatment,
MABC DNA concentration would be expected to reduce due to potent antibacterial selective pressure. Post treatment samples would only contain MABC DNA if treatment was unsuccessful, and patients develop chronic infections.
78
Figure 15: MABC bioburden changes through its Natural History of Infection. Natural infection history as a function of mean abundance of MABC DNA present per microliter. Significant values between stages were calculated using two-tailed Mann-Whitney U test and only significant comparisons are displayed. Mean values with interquartile range is displayed as using the median is not feasible for a logarithmic figure where many values are zero resulting in the median being zero and not graphable.
79
With all but one MABC qPCR signals being associated with case samples, there was an obvious significance in the median MABC abundance for case patient prior to and at the time of an infection compared to controls (p =<0.0001). MABC qPCR values were compared with clinical values for MABC grown on Middlebrook 7H10 (ProvLab, Alberta, Canada) to assess whether a correlation could exist with physical examination of sputum (via Ziel-
Neelson (Z-N) stain to assess burden of organisms as measured by smear negative, or positive at 1+, 2+ or 3+ - as reported by the clinical microbiology laboratory) and days taken to form colonies as seen in figures 16 and 17, respectively. A positive correlation was noted between MABC DNA concentration compared to increasing burden of organisms as indicated by Z-N staining of sputum. Most samples were smear negative which correlates to a low concentration of acid-fast bacilli (AFB) present. Increasing number of smear positive denotes increasing infectiousness of patients. Samples with a 3+ smear positivity AFB test have a significantly higher MABC DNA concentration than smear negative and 1+ smear positive samples. Similarly, samples that have a higher MABC DNA concentration correlated with less time to grow colonies (and therefore a higher initial inoculum). A significant difference is noted between samples that grew within 1-3 days compared to those that took longer than 7 days to grow.
80
Figure 16: Z-N Smear positivity correlates with absolute abundance of MABC. Median MABC DNA concentration correlated with Z-N staining performed in the clinical microbiology laboratory. Smear negative (n=54) had a median value of 1.64 x 10-5, Smear 1 (n=8) had a median value of 6.57 x 10-5, Smear 3 (n=3) had a median value of 1.57 x 10-3. The top and bottom of the box plot represent the interquartile ranges. Significant values were calculated using a two-tailed Mann-Whitney U test. Each patient (letter) is represented by a different colour.
81
Figure 17: A higher bioburden of MABC DNA correlated with a shorter time for MABC cultures to grow. Median MABC DNA concentration correlated with days to form colony data. Days 1 to 3 (n=9) had a median value of 4.38 x 10-4, days 4 to 6 (n=24) had a median value of 8.87 x 10-5, days greater than 7 (n=12) had a median value of 2.74 x 10-5. The top and bottom of the box plot represent the interquartile ranges. Significant value was calculated using a two-tailed Mann-Whitney U test. Each patient (letter) is represented by a different colour.
82
3.7.4 Quantifying P. aeruginosa DNA in Sputum Samples
Differences in the relative abundance of Pseudomonas was observed in the patient samples in the years prior to initial infection as well as samples at the time of infection as compared to control patient samples. Further RT-qPCR testing was performed with primer specific to P. aeruginosa to confirm the differences between the cohorts observed through the microbiome analysis as well as quantify to the species level, which is not seen through the 16S rRNA sequencing analysis. Specifically, the at infection relative abundance difference of Pseudomonas observed through the 16S rRNA sequencing analysis displayed approximately three and a half fold higher in the control patients compared to the case patients (0.003826 ng/µL vs 0.001065 ng/µL, p = 0.0067). Similar differences are observed for at infection samples compared to controls for RT-qPCR analysis as seen in figure 18.
Case patients at the time of initial infection had significantly lower P. aeruginosa abundance
(4.24 x 10-6, n=37) compared to the control patients (1.04 x 10-4, n=42), p= 0.0006. This assessment allows to infer a potential negative correlation of P. aeruginosa to infections with
MABC prior to the occurrence of an infection and confirms our results from the 16S rRNA microbiome analysis.
83
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Figure 18: Median P. aeruginosa DNA concentration was higher in control samples compared to at infection case samples. Median P. aeruginosa DNA concentration in case patients at the time of infection was 4.24 x 10-6 ng/µL (n=37) compared to control patients at 1.05 x 10-4 ng/µL (n=42). Two-tailed Mann-Whitney U statistical analysis revealed a significant p value of 0.0006.
84
3.8 P. aeruginosa Inhibition Assay
Several reasons could influence this correlation, one of which could be the presence of P.
aeruginosa providing potential shielding from MABC colonization through secreted
inhibitory factors. As this could be assessed with relative ease – we sought to determine if in
fact this might be the case through an inhibition assay. Figure 19A and B shows results from
the experiment designed to assess the impact of the extracted supernatant of the lab strain of
P. aeruginosa (PA01) as well as the Prairie Epidemic Strain (PES) derived from individuals
with CF in separate dilutions of 1:5 and 1:20 on the growth of MABC over 5 days.
Differences noted between the control samples compared to the PA01 or PES strains for
either dilution factors were not significant. All of the groups followed a logarithmic growth
curve reaching their maximum by day 3. Although no significant differences were noted
between the variables, the control population had slightly higher colony forming units per
milliliter than its P. aeruginosa strain counterparts. This, however, was not deemed enough
to formulate any correlation regarding P. aeruginosa growth medium supernatant containing
secreted molecules with a mechanism to inhibit MABC growth and thus, the hypothesis for
was refuted.
85
A B
Figure 19: MABC growth in Middlebrook 7H9 broth infused with P. aeruginosa PA01 and PES supernatant. (A) Dilution at a ratio of 1:5. (B) Dilution at a ratio of 1:20. Significance was not noted between the variants based on Kruskal-Wallis test for either dilutions. Negative control flasks reported no growths in any replicates. A logarithmic growth trend is noted for all variables with non-supernatant controls having slightly higher CFU/mL at the end of day 5.
86
Chapter Four: Discussion
87
4.1 MABC Infections are a Major Cause for Concern in CF Patients
Increasing incidence and prevalence of NTM infections within CF populations have been noted in the last 15 years with a considerable portion infected with MABC. Factors such as improved laboratory practice, increased patient survival age, increased environmental exposure, numerous antibiotic exposure, as well as person-to-person transmission have been posited as being contributory.145–148 Patients with prolonged MABC infection have been shown to have a greater rate of decline in pulmonary function (%FEV1) compared to the classical pathogen P. aeruginosa
(-2.22% per year vs. -0.95% per year).59 MABC infected patients are also ineligible for lung transplant in many centers due to poor clinical outcomes post-transplant.149–151 MABC infection also incurs a prolonged treatment course with several different classes of antibiotics due to several inherent antimicrobial resistance mechanisms present.152,153 These reasons present an utmost importance towards understanding factors that could predispose CF patients to MABC infections.
88
4.2 Patients with MABC Present with a Lower BMI than Controls
Twenty-one case patients for the microbiome study were selected from the Calgary Adult
CF Clinic that presented with a positive MABC culture meeting the ATS/IDSA criteria.
Control patients (n=42) on the contrary did not have any history of MABC as identified
through the clinical reviews of medical records and clinical microbiology testing done
annually. Comparisons of the patient demographics between the case and control cohorts
yielded no significant differences in age, genetics, or lung function values at the time of
initial MABC infection.68,154 These factors could present a pre-emptive bias for any microbial
difference that would be noted between the case patient and control patient cohorts.
Significant demographic difference between the cohort was only observed for patient
BMI where control patients presented with a higher BMI compared to their case counterparts
(21.5 kg/m2 vs 19.5 kg/m2, p=0.04). Direct associations between diet and gut microbiome
dysbiosis have been reported on previously but have only had indirect implications to the
lung microbiome.155–158 Multicenter studies conducted on the prevalence of NTM have not
found BMI to be a significant factor dissociating between patients that develop MABC
infections compared to those that do not develop an infection.55 These significance were
corrected for using Benjamini-Hochberg correction for multiple comparisons however these
may still only be due to chance alone. Further multicenter studies focused on MABC
correlation with BMI could allow for a better understanding of any possible correlations.
89
4.3 Persistent MABC Patients had Significantly Higher PPI use Compared to Transient
Patients or Controls.
Case patients that developed a persistent infection was defined as 50% or greater positive
mycobacteriology tests with a minimum of three tests in the year following initial infection.
This guideline is an adaptation from the Leeds criteria used for P. aeruginosa to assess
persistent infections as a universally applied criteria for NTM infections does not exist for
CF.159 Transiently infected and persistently infected patients did not significantly differ by
any demographic parameters nor with their co-infections. Only the therapeutic use of PPI
was observed to be significantly higher in persistent patients compared to transient patients
as well as compared to control patients. PPI is used in the management of the vastly
prevalent gastroesophageal reflux disease (GERD) observed in CF patients.160 Studies have
correlated GERD in CF to poor pulmonary outcomes and an increased frequency of
hospitalization.161–163 Aggressive PPI therapy recommended to manage GERD have been
linked to the development of community-acquired pneumonia in CF patients due to
inhibition of several leukocyte functions.164,165 The association for persistent MABC patients
having higher prescribed PPI is unclear but could be a consequence of hindered immune cell
response leading to easier persistent colonization of MABC. This factor, although significant,
could be a result of chance alone and should be subject to additional research with a larger
cohort size in a multicenter study.
90
4.4 Patients with MABC did not Differ in their Microbiome Diversity with New Infection
Patients with MABC infection had their sputum samples characterized and subdivided
into categories of pre-infection, at infection, at treatment, and post treatment. Lung
microbiome richness and evenness was assessed using alpha-diversity measures which
showed no significant changes between samples prior to an infection compared to samples at
the time of infection. Significant beta-diversity clustering was also not observed between
samples prior to an infection compared to samples at the time of infection. This suggests that
community diversity prior to an infection may not suggest as a conclusive biomarker for
patients that will develop an incident MABC infection. These findings are consistent with
previous literature which found no significant changes in total bacterial density with
exacerbations.75,166–168
With antibiotic intervention, however, a reduction in the microbiome diversity is noted in
the samples associated with during treatment stages compared to samples from pre-infection
and at infection stages. These differences are seen in the alpha-diversity measures within
samples as well as in the beta-diversity clustering between samples. Subsequent recovery of
the microbiome towards pre-treatment diversity is also observed. These trends are
previously described in several studies that found acute antibiotics to cause an immediate
albeit temporary change in the CF microbiome structure that quickly return to baseline
community structure post therapy.83,154
Quantitative PCR analysis of the natural history of infection was done to identify the total
bacterial bioburden present in the subcategories. Here we found no significant differences in
the pre-infection, at infection, and at treatment samples for total bacterial bioburden and a
slight significance post treatment. This lack of variability in the total bacterial bioburden
91 allows for any bacterial diversity observed to be attribuetd with changes in the relative abundance of microbial constituents rather than an overall increase in the amount of bacteria present.
92
4.5 Patients with MABC had significantly different microbiomes compared to control patients.
The lung microbial community structure of case patients was compared to age, sex, and
birth cohort-matched control, where significant differences in microbiome diversity was
observed. Specifically, case patient samples prior to an infection as well as at the time of
infection presented significantly different microbiome structure from control patients. Case
samples presented significantly higher microbiome richness and evenness than control
samples. Further, significant beta-diversity clustering between samples was also observed
for case patients compared to control patients. This suggests an aversion from the classical
definition of lower diversity relaying to increased disease by a dominating bacteria.74–76
Quantitative PCR analysis comparing the total bacterial bioburden as measured by 16S
rRNA between case patients and controls displayed no significant differences between the
cohorts allowing for associations in diversity analysis to be credited to the microbiome alone.
Quantitative PCR analysis of all case and control samples was also conducted to assess
for burden of MABC. Twenty seven of 37 samples associated with the at “infection stage”
presented the highest mean quantity of MABC signals as expected. However, 15 of 38
samples associated with pre-infection sample (as per clinical classification) also had a
positive MABC signal - suggesting missed positives through the traditional AFB method of
testing. Accordingly, our strict definitions of pre and at-infection based on Z-N stain and
culture were insensitive to identifying paitnets with new infection. Twenty nine of 79 at
treatment samples continue to present a MABC signal through the treatment stages and two
samples of 20 continue to present a MABC signal post treatment. Successful qPCR analysis
of CF sputum for the presence of MABC has not been explored previously and could be
93 extremely valuable to allow for more frequent testing of MABC leading to earlier diagnosis and immediate treatment.
Significant differences in the individual ASVs present between control and case samples prior to an infection and at the time of infection showcased higher Pseudomonas in control patients compared to case patients. This is consistent with suggesting that non-classical pathogens occupy airway in patients that are not already dominated by a classical pathogen.
These factors are consistent with a previously described multi-center study on the prevalence of NTM in CF by Olivier et al. (2003). Olivier et al. observed patient that were positive for
NTM infections had significantly lower rates of P. aeruginosa chronic infection compared to patients negative for NTM.169 Similarly, Esther Jr. et al (2010) also found a significant negative association between NTM and P. aeruginosa.170
We confirmed these differences in P. aeruginosa between the cohorts using qPCR – but not with culture status alone. Similarly, to the microbiome outcome, we found control patients with a significantly higher P. aeruginosa quantity compared to case patients at the time of infection further suggesting a potential shielding of MABC infection by a pre- existing P. aeruginosa infection. This may suggest that it is not the mere presence or absence of P. aeruginosa that matters but rather the amount.
We sought to explore if P. aeruginosa inhibition of MABC was occurring through a secreted inhibitory factor, which we found to not be the case in our experiment. These factors include phenazines, proteases, elastases, hemolysin, and rhamnolipids that are excreted by P. aeruginosa to contribute to their virulence.171 Other interaction factors between P. aeruginosa and MABC could play a role in suppressing the MABC growth in an environment pre-infected with P. aeruginosa including the ability of Pseudomonas species to
94 simply overgrow NTM in small fractions of AFB cultures.172,173 Other inhibition methods employed by P. aeruginosa include the formation of biofilms under nutrient limiting conditions.174,175 We were able to rule out any excreted molecules presented that may persist long term in solution to inhibit growth of MABC. The use of a polycarbonate membrane could be useful to assess indirect inhibition between P. aeruginosa and MABC where a constant release of exoproducts could be assessed.
95
CHAPTER 5: Summary
96
5.1 Limitations Several limiting factors in this study are notable including the relatively small number of
patients included due to MABC prevalence being low. Additionally, the research is only a
single-center study thus making generalizations and associations difficult. Multicenter
studies in addition to including a range of geographic and treatment practices will enable
greater number of patients and samples to be accrued in order to assess more subtle
differences. Furthermore, sequencing the V3 region of the 16S rRNA region can only give
certain accuracy up to the genus level leaving out associations towards species changes in the
cohort. Additionally, extracted sputum would contain DNA from live and dead rememnants
of the microbiome and could influence the sequencing accuracy. Metagenomic sequencing is
an approach that can move beyond this limitation, however, has not been widely deployed in
CF owing to the extent of oropharyngeal contamination of sputum samples by the oral
flora.176
As well, sputum samples from patient are dynamic, changing day to day, and largely
contain organisms from the upper respiratory tract, although studies of induced sputum
samples have shown comparable bacterial correlations to BAL.177
97
5.2 Future Directions
Further exploration of the microbiome association with MABC infection should be done
in a multicentered study involving several different geographic locations to best understand
the surrounding epidemiology associated with the infection. Additionally, whole genome
sequencing of MABC could be done within our study cohort to identify potential
transmission occurrences.
Further testing of the P. aeruginosa inhibition assay could be done with a possible testing
with a semi-permeable polycarbonate membrane dividing P. aeruginosa from MABC could
allow us to run a more realistic indirect competitive assay which would account for quorum
sensing or direct competition for resources and could provide possible reasons to the negative
correlation observed between MABC and P. aeruginosa. Additionally, a transcriptomic
study involving P. aeruginosa and MABC co-cultures to assess any molecular mechanisms
between the interactions.
98
5.3 Study Summary and Conclusion
Significant differences were observed in the diversity of the microbial community of CF
sputum in patients that develop MABC infections compared to non-infected controls. These
observations are seen in the samples leading up to the initial infection date as well as on the
date itself. Patients who acquire MABC have a distinctively different community structure
with a relative defiance of Pseudomonas genus suggesting a negative correlation with
MABC infection itself and possibly even a protective role from infections with MABC.
Between persistent and transient-infected patients microbiome community structure was not
sufficient different to distinguish these two groups. These suggest further investigation
should be conducted in a multi-center study involving more patients to create a more
solidified association.
99
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