University of Southern California Dissertations and Theses

Investigation of in vitro activity and in vivo safety of imipenem-relebactam alone and in combination with other anti-microbial agents against clinical isolates of M. abscessus from CF patients

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Investigation of in vitro activity and in vivo safety of imipenem-relebactam alone and in combination with other anti-microbial agents against clinical isolates of M. abscessus from CF patients

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Content Investigation of in vitro activity and in vivo safety of imipenem-relebactam alone and in
combination with other anti-microbial agents against clinical isolates of M. abscessus
from CF patients

by

Sun Woo Kim

A Thesis Presented to the
FACULTY OF THE USC ALFRED E. MANN SCHOOL OF PHARMACY AND
PHARMACEUTICAL SCIENCES
UNIVERSITY OF SOUTHERN CALIFORNIA
In Partial Fulfilment of the
Requirements for the Degree
MASTER OF SCIENCE
(CLINICAL AND EXPERIMENTAL THERAPEUTICS)
December 2023

Copyright 2023 Sun Woo Kim
ii

iii

Acknowledgements
I would first like to express my deepest gratitude to my advisor, Dr. Paul M. Beringer for
his continuous support and guidance throughout this project and my through my studies
as a Master’s student. I would not have had made it this far and be able to complete this
work without his patient mentorship. I would also like to extend my sincere gratitude and
appreciation to my thesis committee members: Dr. Annie Wong-Beringer and Dr. Emi
Minejima for their guidance, knowledge and their valuable input for my project. I would
also like to thank those helped me throughout and contributed to this project: Dr.
Mansour Dughbaj, Dr. Eugeniu Carmanov PharmD, Aditi Shinde, Marta Ruest, Danielle
Fletcher-Williams.
Finally I would like to thank my family, friends and other lab members for their support
throughout my graduate school journey. To my parents, I want to thank you again as I
would have never have had this opportunity experience all this without you all.

iv

Table of Contents
Acknowledgements ....................................................................................................... iii
List of Tables .................................................................................................................vi
List of Figures................................................................................................................ x
Chapter 1: Introduction and Background ................................................................... 1
Overview of Cystic Fibrosis .......................................................................................... 1
Characteristics of Cystic Fibrosis ................................................................................. 1
Mutations of the CFTR gene ........................................................................................ 2
Treatment Options for People with Cystic Fibrosis (PWCF)......................................... 4
Pulmonary lung infections in cystic fibrosis .................................................................. 5
Non tuberculous mycobacterium (NTM)....................................................................... 7
Mycobacterium abscessus complex (MABSC) ............................................................ 8
Immune responses against Mycobacterium abscessus ............................................. 10
Mycobacterium abscessus virulence factors and morphology ................................... 10
Mycobacterium abscessus complex and cystic fibrosis ............................................. 12
Treatment of M. abscessus in CF patients ................................................................. 13
Imipenem ................................................................................................................... 15
Imipenem-Relebactam ............................................................................................... 16
Animal models for M. abscessus infections ............................................................... 18
M. abscessus infection model in zebrafish ................................................................. 19
Chapter 2: Results....................................................................................................... 22
In vitro susceptibility and synergy .............................................................................. 22
Various drugs including beta-lactams show synergy against the M. abscessus
reference strain, ATCC19977. ................................................................................ 22
The selected synergistic combination retained synergism when tested against
clinical isolates of M. abscessus from CF patients .................................................. 26
Drug combinations have MICs and MBCs that are not achievable clinically,
while IMIREL is able to reach bactericidal concentrations clinically ........................ 40
In vivo safety and infection model establishment ....................................................... 41
Imipenem-relebactam is non-toxic to zebrafish even at high concentrations and
can be used to test for in vivo efficacy in the zebrafish model ............................... 41
v

Proof of concept of Tg(mpeg1:EGFP) absolute zebrafish embryo infection with
dsRed2 labelled M. abscessus (ATCC19977) ........................................................ 45
Chapter 3: Summary and future directions ............................................................... 47
Chapter 4: Materials and Methods ............................................................................. 53
References ................................................................................................................... 57

vi

List of Tables
Table 1. Summary of the classes of CFTR mutations and their respective type of defect
in relation to the CFTR gene ........................................................................................... 3
Table 2. MIC of drugs and imipenem-relebactam alone and in combination with each
other against the ATCC19977 M. abscessus strain, and the respective calculated FIC
index. ............................................................................................................................. 23
Table 3. M.abscessus clinical isolates from CF patients identified according to
subspecies classification, morphology and mutation. .................................................... 27
Table 4. The MICs of minocycline, impenem-relebactam and both drugs in combination
against M. abscessus clinical isolates from CF patients, with the corresponding
calculated FIC index. ..................................................................................................... 29
Table 5.The MICs of cefoxitin, impenem-relebactam and both drugs in combination
against M. abscessus clinical isolates from CF patients, with the corresponding
calculated FIC index. The median MIC value of the MICs against the clinical strains for
each of the conditions: cefoxitin alone, cefoxitin with IMIREL, IMIREL, IMIREL with
cefoxitin is also reported below. The FIC index using the median MIC values was also
calculated. ..................................................................................................................... 30
Table 6. The MICs of cefdinir, impenem-relebactam and both drugs in combination
against M. abscessus clinical isolates from CF patients, with the corresponding
calculated FIC index. The median MIC value of the MICs against the clinical strains for
each of the conditions: cefdinir alone, cefdinir with IMIREL, IMIREL, IMIREL with
cefdinir is also reported below. The FIC index using the median MIC values was also
calculated. ..................................................................................................................... 31
vii

Table 7. The MICs of rifabutin, impenem-relebactam and both drugs in combination
against M. abscessus clinical isolates from CF patients, with the corresponding
calculated FIC index. The median MIC value of the MICs against the clinical strains for
each of the conditions: rifabutin alone, rifabutin with IMIREL, IMIREL, IMIREL with
rifabutin is also reported below. The FIC index using the median MIC values was also
calculated. ..................................................................................................................... 32
Table 8. The MICs of cefuroxime, impenem-relebactam and both drugs in combination
against M. abscessus clinical isolates from CF patients, with the corresponding
calculated FIC index. The median MIC value of the MICs against the clinical strains for
each of the conditions: cefuroxime alone, cefuroxime with IMIREL, IMIREL, IMIREL with
cefuroxime is also reported below. The FIC index using the median MIC values was
also calculated. ............................................................................................................. 33
Table 9. The MICs of NITD-916, impenem-relebactam and both drugs in combination
against M. abscessus clinical isolates from CF patients, with the corresponding
calculated FIC index. The median MIC value of the MICs against the clinical strains for
each of the conditions: NITD-916 alone, NITD-916 with IMIREL, IMIREL, IMIREL with
NITD-916 is also reported below. The FIC index using the median MIC values was also
calculated. ..................................................................................................................... 34
Table 10. The MICs of amoxicillin, impenem-relebactam and both drugs in combination
against M. abscessus clinical isolates from CF patients, with the corresponding
calculated FIC index. The median MIC value of the MICs against the clinical strains for
each of the conditions: amoxicillin alone, amoxicillin with IMIREL, IMIREL, IMIREL with
viii

amoxicillin is also reported below. The FIC index using the median MIC values was also
calculated. ..................................................................................................................... 35
Table 11. The MIC50s and MIC90s (ug/mL) of drug compounds, imipenem-relebactam
and both drugs in combination against all the respective M. abscessus clinical isolates
the specific drug compounds were tested in, with the corresponding FIC index. .......... 36
Table 12. The MIC50s and MIC90s (ug/mL) of drug compounds, imipenem-relebactam
and both drugs in combination against all the respective M. abscessus susp. abscessus
clinical isolates the specific drug compounds were tested in, with the corresponding FIC
index. ............................................................................................................................. 37
Table 13. Table 4-3. The MIC50s and MIC90s (ug/mL) of drug compounds, imipenem-
relebactam and both drugs in combination against all the respective M. abscessus susp.
massiliense clinical isolates the specific drug compounds were tested in, with the
corresponding ............................................................................................................... 37
Table 14. The MIC50s and MIC90s (ug/mL) of drug compounds, imipenem-relebactam
and both drugs in combination against all the respective M. abscessus susp. bolettii
clinical isolates the specific drug compounds were tested in, with the corresponding FIC
index. ............................................................................................................................. 38
Table 15. The MICs and MBCs of IMIREL, individual drug and both drugs in
combination against the ATCC19977 M.abscessus strain. The MICs corresponds to the
MICs observed during the experimental run when the MBC was observed for a direct
comparison between the MIC and MBC. ....................................................................... 39
Table 16. The table is an overview of peak concentration (cmax) that was observed in
the plasma/serum in pharmacokinetic studies done with the respective drugs in
ix

humans. The values were obtained by package inserts of the drugs: imipenem-
relebactam (RECARDBRIO) [52], minocycline [53], cefdinir [54], cefuroxime [55],
cefoxitin [56], rifabutin [57]. There currently is no available pharmacokinetic studies data
on NITD916. .................................................................................................................. 40

x

List of Figures
Figure 1. The airway conditions with normal CFTR channel vs. defective CFTR channel.
........................................................................................................................................ 5
Figure 2. The summary of a typical treatment schedule for individuals with CF with
Mycobacterium abscessus pulmonary disease as recommended by the US CF
Foundation and the European Cystic Fibrosis Society. ................................................. 14
Figure 3. The survival curve showing the survival of uninfected zebrafish embryos
(n=20) treated with 0.5X MIC (2ug/mL imipenem, 1ug/mL relebactam), 5X MIC
(20ug/mL imipenem, 10ug/mL relebactam), 28X MIC (112ug/mL imipenem, 56ug/mL
relebactam) compared to control. .................................................................................. 43
Figure 4. Representative microscopy images of zebrafish at14 days post-fertilization in
each group. ................................................................................................................... 44
Figure 5. Representative fluorescent microscopy images of A) and B) uninfected
Tg(mpeg1:EGFP) absolute zebrafish and C) and D) dsRed:M.abscessus infected
Tg(mpeg1:EGFP) absolute zebrafish. ........................................................................... 46

1

Chapter 1: Introduction and Background
Overview of Cystic Fibrosis
Cystic fibrosis (CF) is a serious autosomal recessive disease that most commonly
affects people of Northern European descent and afflicts about 1 in 4000 newborns in
the US and at similar rates in European countries. CF is caused by mutations in the
cystic fibrosis transmembrane conductance regulator (CFTR) gene located on the
human chromosome 7.[1] After the discovery of the CFTR gene, more than 2000
mutations have been identified and diagnoses have since moved beyond solely being
based on phenotype. [2] [1] CF newborn screening (NBS) have been widely adopted
from 2010 with at least 64% of new CF diagnoses now are made following a positive
NBS in infants that do not necessarily show overt symptoms. Infants are diagnosed with
a combination of a positive result from the NBS and genotyping followed by a
confirmatory sweat chloride test. [1]

Characteristics of Cystic Fibrosis
The disease state of CF is linked to the function of the CFTR protein in which the
pathogenic mutations of the disease occur. CFTR is part of the ATP-binding cassette
(ABC) protein family, which have various transmembrane transport functions. CFTR in
particular is a chloride channel spans across the cell membrane and is regulated by
cAMP-dependent phosphokinases.[2] The main function of the protein is that when
activated it facilitates the transport of chloride ions out of the cell. A mutation in the
CFTR gene causes defects in the various aspects of protein such as either the correct
2

processing of the protein for its transport to the cell membrane or the molecule’s ability
to properly transport chloride ions. The defective CFTR function not only affects chloride
transport but also bicarbonate levels as it is also transported by the protein. Ion
imbalances from the defects in chloride, sodium, water and bicarbonate transport
causes a reduction in surface liquids in areas such as the airway surface which leads to
the predisposition for mucus build up and as a result it can cause airway obstruction
and an increase susceptibility to infection.[2]
The defective ability of the protein or the absence of the protein also manifests clinically
in the formation of thick secretions in multiple organs that have secretory functions such
as the lungs, pancreas, liver, gut as well the reproductive tract.[1] The production of
abnormally thick secretions causes obstructions in the secretory organs and leads to
the malfunction of the organs normal functions such as insulin deficiency, malnutrition,
infertility.[2] The main cause of morbidity in CF patients however is lung disease as a
result of susceptibility to bacterial infection in the obstructed airways, which will be
discussed further below.[3] The defect in the protein is also evident through an increase
in salt content in the sweat gland secretions allowing this to be a confirmatory test for
infants as previously mentioned. [2]

Mutations of the CFTR gene
Depending on the type of CFTR defect the mutation, it is classed into six different
classes (Class I to Class VI). Typically , the class of the mutation is related to severity of
the disease with Class I to Class III being the more severe however there can be
variation in the clinical manifestation of the disease across different combination of
3

mutations.[2] The descriptions of the types of mutations are summarized in Table 1.
Mutation
Class
Type of defect in relation to the CFTR gene
Class I
Causes a defect in the production of the CFTR protein itself. The mutation is
usually ones that cause premature termination of transcription, protein
malformation or complete absence of the protein production.
Class II
Causes a defect in the processing of the CFTR protein. The defective post-
translational processing of the protein prevents proper trafficking to the cell
membrane disabling the protein from being positioned correctly to function
properly. The F508del mutation is a Class II mutation and is also the most
common mutation present in the cystic fibrosis patient population. The
homozygous state of the F508del mutation is present in 50% of patients while
the heterozygous state is present in up to 90% patients.
Class III
Causes a defect in the regulation of the protein. The mutant proteins are
unable to function optimally mainly due to defective ATP binding ability
caused altered ATP-binding regions.
Class IV
Causes a defect in the conductance of the CFTR protein which is a channel
protein. The mutant proteins while transported correctly have lowered rate of
ion flow and time of channel opening compared to the normal CFTR protein.
Class V
Causes a reduction in the quantity of the CFTR protein. This class of
mutations affects the stability of the mRNA transcripts of the CFTR protein,
causing a reduction in the production of the protein itself.
Class VI
Causes a reduction in the stability of the mature CFTR protein. This mutant
class is distinct of the Class V in that it is specific to the instability of already
translated, mature CFTR protein, usually manifesting as instable proteins in
the plasma membrane.
Table 1. Summary of the classes of CFTR mutations and their respective type of
defect in relation to the CFTR gene [2]
The classifications of the mutations are important in that it would relate to the relevant
therapies that can be used to treat the specific types of defects in the CFTR protein.[4]
The development of specific drug compounds that target the specific aspects of the
CFTR mutations such as the misfolding of the protein is exemplified in the section
below.

4

Treatment Options for People with Cystic Fibrosis (PWCF)
With the advancement of scientific discoveries, new treatment options have emerged for
the CF patient population significantly lifting the burden of the disease and improving
clinical outcomes.[5] Drugs developed target the specific defects caused by the various
mutations discussed previously. Understanding the underlying mechanisms have led to
the development of several therapeutics.[6] Ivacaftor is a CFTR potentiator that
prolongs channel opening and increase ion transport, making it highly efficacious for the
class III mutation carriers.[6] A CFTR potentiator can also be combined with CFTR
correctors, these correctors work on undoing defects in the CFTR protein that causes
misfolding and protein instability, helping decrease protein degradation and improving
protein trafficking to the cell surface.[6] These correctors can help treat class II mutation
defects that have misfolded CFTR proteins. Several drug combinations of CFTR
potentiators and correctors have been developed producing great improvement in
patient clinical outcomes. Approved in October 2019 by the US FDA, Trikafta is an
example of a triple therapy combination of two correctors (elexacaftor and tezacaftor)
and a potentiator (ivacaftor), indicated for patients with at least one F508del mutation.[6]
This drug has proved to be the most effective treatment of CF producing large
improvements such as a 13.8% increase in predicted FEV1 (Forced Expiratory Volume,
maximum amount of air that can be forced out during the first second after a maximal
inhalation, indicative of lung function) in patients after 4 weeks and a 14.3%
improvement after 24 weeks of treatment.[7] Trikafta treated patient groups also had a
63% decrease in pulmonary exacerbations, general increase in the quality of life and a
41,8mmol/L decrease in sweat chloride exemplifying the therapeutic effects of the
5

treatment.[7]

Pulmonary lung infections in cystic fibrosis
The major source of morbidity and mortality of CF is the dysfunction in the respiratory
system. The defective ion transport due to the mutant CFTR causes the secretion of
abnormally thick and viscous disabling mucus proper mucociliary clearance.[4] [2]

Figure 1. The airway conditions with normal CFTR channel vs. defective CFTR
channel.
Normal, healthy CFTR channels (left) are able to create a well-balanced epithelial
sodium ion absorption via the epithelial sodium channel (ENaC) and secretion of
chloride anions via the CFTR channel created a well hydrated airway surface with
efficient mucociliary transport. The defective CFTR channel (right) are shown to have
6

dehydrated mucus and build up from the mucus adhesion to the airway surface. Image
from [3]

The altered airway surface liquid is not only viscous and dehydrated but also acidified.
The low pH disrupts normal ciliary beating, phagocyte function and the inactivates host
defense peptides. This condition provides a favorable environment for the colonization
of microorganisms in the respiratory system allowing for persistent infections.[4] The
obstruction and infection of the airway leads to a chronic inflammatory environment
which eventually causes the degradation of extracellular pulmonary tissue components
leading to bronchiectasis. [8] Bronchiectasis is when the bronchial lumen is permanently
widened to an abnormal extent from irreversible structural damage. The clinical
manifestations of the disease is chronic cough, sputum production, disposition to
recurring infections due to abnormal mucociliary clearance as a result of the dilated
bronchi.[9] Despite the large strides made in terms of correcting the underlying condition
of CFTR dysfunction, with improvements in several affected physiological functions
such as sweat chloride, gastrointestinal (GI), liver, kidney, pancreas and reproductive
tract functions, pulmonary infections remain a concern.[4] CFTR modulation therapy has
shown to decrease the prevalence of typical CF pathogens and airway infection
incidence due to the restoration of general innate immune function and control of
inflammation linked CF. However, with the progressive nature of CF, older patients and
patients who have already have severe airway tissue damage may have limited benefits
in the reversal of the tissue damage and the already established microbial composition
of the CF lung even with CFTR modulation therapy.[4] Altogether, opportunistic
7

infections in the lungs remain a concern for CF patients, with the most common
pathogens being Staphlococcus aureus and Pseudomonas aeruginosa. An emerging
group of pathogens however are NTM (non-tuberculous mycobacteria), with over 30
species of NTM known to cause infections in humans and the prevalence of pulmonary
infections by these pathogens have been on the rise over the past 30 years.[10] [5]
The Mycobacterium abscessus complex (MABSC) comprised of three subspecies: M.
abscessus subsp. abscessus (Mab), M abscessus subsp. bolletii and M.abscessus
subsp. massiliense is an important species among NTM, associated with pulmonary
infections especially in CF patients.[10]

Non tuberculous mycobacterium (NTM)
Non tuberculous mycobacteria are environmentally ubiquitous organisms that cause
disease and are part of the Mycobacterium genus similarly to M.tuberculosis.[11] NTM
and M.tuberculosis however differ in several aspects such as morphology, reactions to
biochemical tests but notably NTM is typically not typically contagious and person to
person and does not occur except in cases such as immunocompromised cystic fibrosis
patients.[11] The organism has been isolated from several environmental sources such
as soil, air, dust, vegetation and water, hence exposure to the bacteria can come in
various forms.[11] Although identified quite early on in the 19
th
century, the capability for
NTM to cause disease were discovered in the 1950’s, long after the connection
between M.tuberculosis and tuberculosis was made by Robert Koch in 1882.[12] Since
then, there have been over 170 NTM species that have been identified.[13] NTM
species are characterized by a thick, hydrophobic, lipid-rich cell wall that makes the
8

organism robust against antibiotics and disinfectants.[11] The outer cell wall layer also
allows it attach to rough surfaces and allows survival in low oxygen and carbon
concentration conditions.[11]
From an epidemiological standpoint, NTM are becoming an increasingly concerning
pathogen due to the rising prevalence of NTM-positive cultures in patients. This is
despite the fact that there are no systematic requirements set in place for the reporting
of NTM. The isolation of the organism also does not always point towards clinical
infection. Hence the prevalence and incidence of NTM infections is difficult to
determine.[11] [12] The fact that the isolation of NTM from pulmonary specimens not
necessarily indicating an infection mean that the diagnosis of a NTM pulmonary
infection requires the fulfillment of several criteria outlines in the guideline published by
the American Thoracic Society and the Infectious Disease Society of America
(IDSA).[12] Nevertheless, because of the increase in availability of diagnostic tools such
CT scans and the increased awareness of NTM infections among clinicians, the
prevalence of NTM has steadily been on the rise as exemplified by the annual
prevalence from 2008 to 2015 increasing from 6.68 to 11.70 per 100,000 persons
making it a legitimate concern for susceptible patient populations.[14]

Mycobacterium abscessus complex (MABSC)
Among NTM, the Mycobacterium abscessus complex presents itself as one of the most
clinically relevant species due to its increasing prevalence and virulence.[10] NTM are
grouped into two groups: rapidly and slow growing mycobacteria based on their growth
rates.[13] M. abscessus is considered a rapidly growing mycobacteria (RGM), being
9

able to grow mature colonies on agar plates within a week.[15] Among the RGM, MABC
is the most common pulmonary disease causing pathogen accounting for up to 13% of
all NTM-Pulmonary Disease (NTM-PD).[10] The increasing prevalence of the pathogen
is also a concern due to the virulent nature of the pathogen and the difficulty in treating
the disease it causes.
MABSC is a complex comprised of three tightly related subspecies: M. abscessus
subsp. abscessus, M. abscessus subsp. bolletii, M. abscessus subsp. massiliense,
these being classified as distinct subspecies in 2016. [10] These subspecies are
important to note as they have distinct characteristics that translate to different clinical
outcomes. M. abscessus subsp. abscessus (MAB) and subsp. bolletii (MBOL) both
possess the erm gene (an inducible macrolide resistance gene) whereas the M.
abscessus subsp. massiliense (MMAS) contain a shortened, non-functional gene.[16]
This discrepancy leads to significantly different clinical outcomes as seen in a study that
showed that clarithromycin-based treatment regimens led to bacterial clearance in
100% of patients infected with M. abscessus subsp. massiliense whereas only 27% of
the patients infected with M. abscessus subsp. abscessus were able to eradicate the
bacteria with the same regimen. [16] Given the differences in clinical outcomes based
on the difference in characteristics such as the presence of inducible antibiotic
resistance genes such as the erm gene, treatment regimens should be modified
accordingly. Several other genetic differences between subspecies are being
investigated in order to understand their clinical relevance.[10]

10

Immune responses against Mycobacterium abscessus
The main immune response against the MABSC is the production of IFN-1 by
macrophages and stimulating the production of nitric oxide (NO) killing or inhibiting the
activity of the pathogen inside them. This production of NO is critical in the process of
macrophage clearance of the MABSC infection.[10] The infection by MABSC also
triggers a TNF-alpha response that plays a key role in mycobacterial killing as well as
granuloma formation. Hence the TNF/IL8 pathway is central to the bactericidal
capabilities of macrophages where the impairment to the pathway can cause the
disease to worsen.[10] On the other hand, an abnormally high level of TNF-alpha can
also cause negative effects in the host.[10] With the understanding of the immune
response to M. abscessus we can later understand how the immune dysregulation
caused by cystic fibrosis worsens the conditions of the infection.

Mycobacterium abscessus virulence factors and morphology
Mycobacterium abscessus is a concerning pathogen mainly due to its resistance to
treatment particularly classically used antibiotics and have shown limited positive clinical
outcomes. The difficulty in treating M. abscessus can be attributed to its various intrinsic
virulence factors. [17] The first factor the M. abscessus possesses is it cell wall that acts
as a physical barrier in which the cell envelope has low permeability, significantly
decreasing the influx of antibiotics such as beta-lactams. The lipid abundant,
hydrophobic cell wall also acts as a chemical barrier while providing intrinsic protection
from acids and alkalis. [17]
Alongside the cell wall, enzymes produced by the pathogen acts synergistically with the
11

barrier created. M. abscessus is able to produce antibiotic modifying/deactivating
enzymes such as rifampicin ADP-ribosyltransferase and a mono-oxygenase which
modifies the antibiotic rifampin, lowering its binding affinity and hence its efficacy. M.
abscessus is also able to produce aminoglycoside modifying enzymes that inactivate
drugs as well as beta-lactamases that degrade beta-lactam antibiotics. M. abscessus
also has a macrolide inducible erm gene that confers resistance to clarithromycin and
erythromycin which are typical antibiotics to treat NTM. [17]
Another factor that allows M. abscessus to be highly resistant to antibiotics is the
presence of efflux pumps. The pathogen encodes proteins of the ABC (ATP-binding
cassette) transporter family as well as mycobacterial membrane protein large (MmpL)
family. The ABC transport family consists of proteins that utilize ATP to pump drugs out
of the cell while the MmpL family is a family of multidrug resistance pumps involved in
lipid transport to the cell membrane and in transporting various compounds out of the
cell. [17]
M. abscessus strains exhibit both rough and smooth morphology. These distinct colony
morphologies are macroscopic and are based on the appearance of the cultured
colonies. The colonies are able to shift between the two morphologies and each being
associated with different infection states. [18] The main factor that differentiates the two
morphologies is the presence of surface glycopeptidolipids (GPL) which are not present
in the rough morphology as a result of down regulation of certain genes or mutations in
certain genes. [10] The absence of GPL in the outer layer of the cvell wall in the rough
morphotype leads to the production of strong inflammatory response from the host,
inducing higher levels of TNF-1 making the rough morphology being more hyper
12

virulent. [10] The R morphotype is also shown to form extracellular serpentine cords as
well as large bacterial clumps in vivo as a form of immune evasion by escaping
phagocytic destruction. [18] This rough morphology also thought to promote cord
formation by increasing apoptosis and increasing the presence of extracellular bacteria.
The increase of the extracellular bacterial load then leads to the spread to other tissues
then abscess formation along with tissue damage, which are seen in vivo as necrotic
granuloma formation. [10] [18] The rough variant is hence also associated with
exacerbations of pulmonary disease and worsening lung function in infected
patients.[19] The smooth morphotype on the other hand is associated with persistent
infection, associated with extended survival within the phagosome. The smooth
morphotype prevents phago-lysomal fusion, lowers IFN-1 levels and prevents
apoptosis, which are all factors that promotes the chronic type of infection. [10] Despite
the relevance of the colony morphology of M.abscessus, the determination and
reporting of the colony morphology is not typically done in the clinical setting. The
clinical importance of the bacteria morphotype has been recently reiterated in a
multicenter cohort study and aims to push clinician to use morphology data as a part of
determining appropriate clinical care.[20]

Mycobacterium abscessus complex and cystic fibrosis
While the most common pathogens affecting CF patients are S. aureus and P.
aeruginosa, the incidence of NTM in CF patients are increasing and given the difficultly
in treating NTM species a such as M. abscessus, it has become a significant problem
for clinicians to tackle.[5] The diagnosis of NTM Lung disease in CF patients comprise
13

of the fulfillment of specific diagnostic criteria due to the ubiquitous nature of NTM. The
current clinical guideline is at least two positive cultures as well as the presence of
characteristic clinical symptoms and radiographic tests. [16] MABSC is the most
prevalent NTM species found in CF patient’s airways with a worldwide prevalence of 5-
20% in CF patients. Though the presence of the opportunistic MABSC in CF patients is
likely due to the defects in mucociliary activity that makes CF patients susceptible to
pathogens in general, the CFTR dysfunction itself has been shown to play a role in
creating a favorable environment for M. abscessus infections. [19] CFTR has been
shown to be important in the proper functioning of the innate immune system by
participating in the chemotaxis of neutrophils to infected sites and control of oxidative
host defenses. [19] The deletion of CFTR led to the decrease in ROS (reactive oxygen
species) production which is crucial in the phagocyte mediated intracellular killing of the
bacteria. This highlights the role CFTR plays in proper immune response against M.
abscessus, making CF patients a more susceptible population against this pathogen.

Treatment of M. abscessus in CF patients
The current available treatment regimen against infection by M. abscessus provided by
the CF foundation is split into two phases, an initial intensive phase followed by a
continuation phase. The intensive phase has a duration of 3 to 12 weeks of a
combination of three antibiotics which is comprised of intravenous amikacin, imipenem,
and cefoxitin or tigecycline these three drugs are also supplemented with oral
azithromycin (macrolide). [21] The duration of the initiation phase is determined by the
response to the treatment taking into account severity of the infection and tolerability by
14

the individual patients. This is followed by the continuation phase with includes the
continuation of the oral azithromycin and inhaled amikacin with 2 to 3 more antibiotics
such as minocycline, clofazimine, linezolid, moxifloxacin. The choice of drug during the
continuation phase is guided by drug susceptibility testing and the patient’s response to
the drugs.[21] The recommended treatment regimen is outlined in Figure 2.

Figure 2. The summary of a typical treatment schedule for individuals with CF
with Mycobacterium abscessus pulmonary disease as recommended by the US
CF Foundation and the European Cystic Fibrosis Society. The treatment is
separated into an intense initiation phase followed by a continuation phase as shown in
the figure above. Antibiotic choices are also guided but not dictated by susceptibility
testing. Image from [21]

15

Imipenem
As previously mentioned, imipenem has been established as part of the initial phase of
treatment of pulmonary infections by M. abscessus. This is based on imipenem being
active against the pathogen in vivo and in macrophages at clinically viable
concentrations.[22] Beta lactams are a widely utilized class of drugs used to treat
various bacterial infections due to their safety profile and the good understanding of
their efficacy and mechanisms.[23] Imipenem along with cefoxitin are currently the only
drugs of the beta-lactam drug class included in the guidelines provided for M.
abscessus infections. [23] Beta lactams have initially garnered in treating M. abscessus
infections due to the extensive studies done on beta-lactam activity against drug
resistant M. tuberculosis. Beta-lactams act against pathogens mainly by inhibiting the
synthesis of peptidoglycan which is the main building block of the bacterial cell wall,[23]
The peptidoglycan consist of two stem peptides forming a disaccharide made of 4 or 5
amino acids joined at the 3
rd
and 4
th
amino acid of each stem peptide as 3-4 linkages or
the 3
rd
amino acid of each stem peptide forming a 3-3 linkage. 3-4 linkages in the
peptidoglycan of the cell wall are typical of bacteria and are catalyzed by D,D-
transpeptidases (DDT) however, mycobacterial peptidoglycan have been show to also
have cross linkages between the 3
rd
amino acids of each peptide stem. [23] These 3-3
linkages are made by L,D-transpeptidases (LDT) and it has been shown that the cell
wall of M. abscessus is mainly made up of these 3-3 linkages eluding to the fact that
LDTs play an important role for M. abscessus cell wall synthesis. [24] Subsequently,
LDTs became an exploitable target in M. abscessus and it was then demonstrated in
studies that this class of enzymes were preferentially bound and inhibited by
16

carbapenems and cephalosporins among the beta lactam subclasses. [23] This is in
contrast to DDTs being effectively inhibited by all beta lactam subclasses. [23] The
varying inhibition profile for DDTs and LDTs in M. abscessus led to the understanding
that a comprehensive inhibition of both enzymes using multiple beta lactams with
different targets to block peptidoglycan synthesis in M. abscessus will be crucial in
effectively treating M. abscessus infections.

Imipenem-Relebactam
While beta-lactams do show activity against M. abscessus, the activity of beta-lactams
are limited in the organism due to the presence of a chromosomally-encoded beta
lactamase.[23] M. abscessus produces a beta-lactamase (BlaMab), an enzyme that
confers resistance to beta lactams, rendering beta-lactams such as imipenem less
effective.[22] BlaMab has also been shown to be resistant to inhibition by typical beta-
lactamase inhibitors such clavulanate and tazobactam. [25], where these beta-
lactamase inhibitors failed to reduce the minimum inhibitory concentrations (MIC) of
beta lactams against M. abscessus.[23] A second generation beta-lactamase inhibitor,
avibactam, however, has been shown to increase the efficacy of imipenem in vitro and
in vivo.[22] Avibactam however, is currently only manufactured in combination with
ceftazidime which would result in the co-administration of an additional drug
(ceftazidime) with low activity against M. abscessus, with possible increased burden of
adverse effects on the patient.[26] [22] Therefore, relebactam, a structurally similar
beta-lactamase inhibitor to avibactam that has been developed in combination with
imipenem/cilastatin as imipenem-cilastatin-relebactam (Recarbrio™) has emerged as a
17

potential alternative for the conventional imipenem-cilastatin highlighted in the treatment
guidelines against M. abscessus.[27] Several studies have tested the ability of
relebactam in increasing the efficacy of imipenem against M. abscessus which showed
positive results showing improved intracellular killing and lowering of the MIC of
imipenem 1-fold, from 8ug/mL to 4ug/ml. [22] These findings served as evidence in
evaluating the imipenem-cilastatin-relebactam drug combination as a possible
alternative in treating M. abscessus infection. [22] The imipenem-cilastatin-relebactam
(Recarbrio™) has been approved by the US FDA since 2019 as treatment of
complicated urinary tract infections as well as intra-abdominal bacterial infections, its
indication has also been extended as a treatment of adults with Hospital-Acquired and
Ventilator-Associated Bacterial Pneumonia (HABP/VABP).[28] Clinically, imipenem-
cilastatin-relebactam in combination with rifabutin has been used in a treatment regimen
in a case of M. abscessus skin and soft tissue infection to successful treat the infection.
[27] Dual beta lactam combinations have been shown to exhibit synergism against M.
abscessus in vitro, while also reducing the frequency of the selection of spontaneous
resistant mutants.[29] Dual beta-lactam combinations have also been shown to improve
the efficacy of beta-lactam and beta-lactam inhibitor combinations. Ceftazidime-
avibactam which had poor activity against M. abscessus alone but with the addition of
imipenem or ceftaroline, both beta-lactams showed decreased MICs exemplifying a
synergistic effect. [26]
In this study, we test for any synergism between imipenem-relebactam and a panel of
11 drugs and 1 inhibitor compound. Combinations that show synergism were then
further tested in a range (up to 28 strains) of cystic fibrosis patient clinical strains of M.
18

abscessus, to observe the efficacy of the synergistic drug combinations in vitro within
the context of CF. Uncovering novel synergistic combination of drugs with imipenem-
relebactam in vitro will allow them to be tested for in vivo efficacy.

Animal models for M. abscessus infections
Moving forward from in vitro studies, the next goal after identifying synergistic
combination would be to examine their efficacies in a pre-clinical animal model. A
conventional animal model would be a mouse model to study the infection of M.
abscessus. While a mouse model of an acute M. abscessus infection has been used in
some studies [30], a more relevant model would be a model that encompasses the
chronic aspect of the M. abscessus infection as well. [31] However, it has been shown
early on that most immunocompetent mouse strains used in common animal models fail
to sustain infections with M. abscessus isolates and manage to clear the infection within
the first week.[32] An example is the aerosol infected. C3HeB/FeJ mice is a standard
animal model that has been used to Mycobacterium tuberculosis because of its ability to
closely reflect human pulmonary pathology however this model is incompatible with M.
abscessus due to the clearance of the infection.[33] There have been some success in
utilizing genetically modified immunocomprised strains of mice [31], however there are
limitations in gaining understanding of the hose immune response to the infection due to
the lack of immune cells and inflammatory markers. [33] Recently, transiently a
pharmacologically-induced immune suppressed, immunocompetent mice model has
been developed using the C3HeB/FeJ strain. [33] The model utilizes transient exposure
of the mice to corticosteroids to allow for a prolonged infection with an adequate
19

bacterial burden. The natural route of infection (aerosolized) and the presence of
relevant pathologies associated with the infection such as the formation of lesions in the
lungs were the main advantages of this model.[33]
Limitations still remain as this model manipulating the immune system will not be able to
show immunomodulatory effects of drugs and the effects of any host-directed therapies
will not be accurately reflected.[33]

M. abscessus infection model in zebrafish
An alternative to a mouse model for studying M. abscessus infection would be a
zebrafish model which has been used to study several aspects of the M. abscessus
infection. The zebrafish has established itself as an effective animal model to study
development but has recently been utilized over the last 20 years to also understand
host and pathogen interactions.[32] The zebrafish is useful in that it has both an innate
and adaptive immune system that is well-conserved in vertebrates. [34] Zebrafish are
not only easily genetically manipulated but also offer visualization during its embryonic
stage where it remains translucent for several days.[35] Transgenic zebrafish lines with
fluorescent labeled leukocytes such as macrophages, where the mpeg1 promoter is
labeled, in combination with a fluorescent labeled bacteria makes the zebrafish a useful
model in understand host immune cell interactions with bacteria during an infection.[34]
The delayed development of the adaptive immune system can also isolate the function
of the innate immune system from the adaptive immune system during the infection.[35]
A study using Mycobacterium marinum showed that during an infection the zebrafish
macrophages were sufficient for granuloma formation with the presence of adaptive
20

immunity.[36]
The zebrafish immune system has also been shown to have close similarity to that of
humans which was reviewed by Traver et al. [37] and several of the cell types of the
human immune system have zebrafish counterparts as highlighted in a review by
Meeker and Trede. [35] Some similarities include the development of T cells in the
thymus, T-cell specific genes having strong homologies to mammalian counterparts and
the conservation of inflammatory proteins. [35] The zebrafish has also been shown to
be effective in understanding the early, acute-phase response to infections. Upon
infection, the acute-phase response in zebrafish is very similar to that of humans where
there is a upregulation of proinflammatory cytokines such as IL-1β and TNFα followed
by the increased expression of proteins such as fibrinogen and complement
components that are typically associated with the early phase of infection. [35] The
pathophysiology of the zebrafish when infected with M. abscessus is the formation
serpentine cords and abscesses, the extracellular cording represents immune evasion
where the cords are formed in order to prevent phagocytosis.[18] Cord formation is also
indicative of the promotion of acute M.abscessus infection.[38]
Furthermore, the zebrafish is not only advantageous in terms its similarities in the
immune system components and live-visualization of internal features but also provides
logistic ease in terms of housing and breeding the animal. [35] Despite having a fully
developed immune system (both innate and adaptive), the zebrafish is one of the
smallest animals that can be rapidly reproduced and house in a relatively small space
making them a cheaper and easier to maintain alternative to mouse models.[35] Given
these attractive features we have chosen the zebrafish larvae as an in vivo model to
21

study the progression of the infection by M. abscessus as well as the in vivo efficacies
of new drug synergistic combinations determined in vitro. We have adapted the protocol
from a study [38] that infected transgenic zebrafish embryos with fluorescent
macrophages through intravenous injection with a fluorescent labeled M. abscessus
inoculum to study the temporal progression and pathogenesis of M.abscessus. The
host-pathogen interaction can also be observed through the multi-colored imaging of the
fluorescent labeled macrophages in the embryos and the bacteria.[38] Effective
combinations can then be tested in a clinical setting to be incorporated into clinical
guidelines to treat CF patients that are infected with M. abscessus.

22

Chapter 2: Results
In vitro susceptibility and synergy

Various drugs including beta-lactams show synergy against the M. abscessus
reference strain, ATCC19977.
A panel of 13 antibiotics and 1 inhibitor compound was used to screen for synergism
with imipenem-relebactam. The 13 antibiotics consists of not only the antibiotics used
and recommended in the current guidelines for M. abscessus treatment: cefoxitin,
azithromycin, minocycline, tigecycline, clofazimine, moxafloxacin, but also rifabutin,
tedizolid, cefuroxime, cefdinir, amoxicillin, and a novel enoyl-ACP reductase, InhA
inhibitor, NITD916. NITD916 is a enoyl-ACP reductase (InhA) of the type II fatty acid
synthase (FAS-II) inhibitor, this inhibitor prevents the biosynthesis of mycolic acids
which are essential for M. abscessus sruvival.[39] The inhibition mechanism and
efficacy has been tested in vitro against M. abscessus and we felt that the inclusion of
this novel compound in this study might reveal unexpected synergistic interactions.
The initial panel of antibiotics and inhibitor were tested in a checkerboard assay with
imipenem-relebactam against the reference M. abscessus strain, ATCC19977 in a
checkerboard assay. The checkerboard assay determines the MICs of the drugs
against M. abscessus when used individually and in combination which then can be
used to determine the fractional inhibitory concentration index (FICI or FIC index). The
FIC index is a mathematical expression that reflects the change in MIC of the drugs and
can be used to determine the general presence of a synergistic interaction.[40]
Typically, an FIC index of ≤ 0.5 which indicated that the bacterial growth with both drugs
23

in combination are inhibited at a MIC that is ¼ or less (more than 1-fold decrease) of
each drug.[40]

Table 2. MIC of drugs and imipenem-relebactam alone and in combination with
each other against the ATCC19977 M. abscessus strain, and the respective
calculated FIC index.

Each drug from a 14 drug panel were assessed for synergy with imipenem-relebactam
in a checkerboard assay. The MIC (ug/mL) of the drugs when treated alone and when
treated in combination with imipenem-relebactam as well as the MIC (ug/mL) of
imipenem-relebactam alone and when treated in combination with the corresponding
drug is used to calculate the FIC index indicating synergistic (≤0.5), additive or
Drug
MIC of
drug
alone
(ug/ml)
MIC of
drug with
IMIREL
(ug/ml)
MIC of
IMIREL
(ug/ml)
alone
MIC of
IMIREL
with drug
(ug/ml) FIC index
Azithromycin 8 1 4 2 0.625
Amikacin 32 16 8 4 1.000
Minocycline 256 64 4 1 0.500
Clofazimine 2 1 4 2 1.000
Tedizolid 8 8 4 2 1.500
Tigecycline 4 2 4 0.5 0.625
Omadacycline 2 1 4 2 1.000
Cefuroxime 512 64 4 0.25 0.188
Cefoxitin 32 8 4 0.5 0.375
Cefdinir 256 16 4 1 0.313
Rifabutin 16 4 4 1 0.500
Amoxicillin 2048 16 4 2 0.508
Moxifloxacin 16 4 4 1 0.500
NITD-916 2 0.2 4 0.25 0.163
24

indifferent ( >0.5 to 4), antagonistic >4 interactions.
From the initial screen it was determined that the 7 drug compounds: minocycline,
cefuroxime, cefoxitin, cefdinir, rifabutin, amoxicillin and NITD-916 showed a synergistic
effect against the reference strain ATCC19977.

Several of the MIC values against the ATCC19977 reference strain obtained in our
study was in line with published data. Our MIC for azithromycin was 8ug/mL which is
the same as the value published in Bastian, Sylvaine et al.[41]. The same is true for
clofazimine where the reported MIC for the reference strain was 2ug/mL which the
same as the value we obtained.[42] The MIC values for moxifloxacin against the
reference strain in a published study was 16ug/mL which is the same as the values we
obtained.[43]The MIC values for tigecycline obtained from a published study was
3.12ug/mL which is less than a 2 fold difference and within an acceptable range of
variance from our value of 4ug/mL.[44] For cefuroxime, the literature values from a
study were at 256 ug/mL compared to our values of 512ug/mL (Table 1) which is within
a reasonable range of a 1-fold difference.[29] The MIC value against the ATCC19977
strain for omadacycline reported in one study was 1ug/mL which is also within a 2 fold
range with our MIC value of 2ug/mL.[45] The published data for the MIC of tedizolid
against the reference was 4mg/uL which is also only a 1-fold difference from our
reported value of 8ug/mL. [46] The same study had MIC values against the ATCC19977
strain for cefoxitin and rifabutin that were 64ug/mL and 32ug/mL respectively.[29] The
MIC values against the reference strain that we have for cefoxitin and rifabutin were
32ug/mL and 16ug/mL respectively (Table 1), both these values are were also within a
25

reasonable range of 1-fold difference. The MIC values reported in a published study for
NITD-916 against the ATCC19977 was 1.56ug/mL while our study also had a similar
MIC value of 2ug/mL against the same reference strain (Table 1).
However, some of the drugs we tested had MIC results for the ATCC19977 strain that
were significantly different (more than 2 fold difference) than previously published data.
Our MIC values for minocycline were similar with previous studies for MIC50 and MIC90
for the strains we studied. Our MIC 50 was 256ug/mL and MIC90 was 512ug/mL which
is similar to the MIC50 that was 128ug/mL and MIC90 that was >518ug/mL in another
study [47]. However, minocycline MIC for the reference strain (ATCC 19977) for our
experiments was around 256ug/mL whereas the MIC determined by the other study
was a significantly lower 64ug/mL. [47] Another drug that has varying results for the
ATCC19977 strain across studies was cefdinir, where two different MICs of 64ug/mL
[29] and 8-16ug/mL [48] were reported. Our result for the MIC of cefdinir was 256ug/mL,
which is significantly higher than any of the reported literature values. Amoxicillin was
also tested in other study, with a reported MIC of >128ug/mL against the reference
strain, ATCC19977/NCTC 13031, the study did not test concentrations above
128ug/mL.[49] Our results for amoxicillin was indeed above 128ug/mL at 2048ug/mL
against the same reference strain of M. abscessus (Table 1) however, due to the limited
concentration tested in the other study a good comparison cannot be made. The MIC
values in published literature for amikacin which was >256ug/mL against the reference
strain was also conflicting with our MIC value was 32ug/mL.[43]
Overall, our MIC values against the ATCC19977 reference strain for cefdinir, amikacin
and minocycline had some discrepancies with other study however, the MIC values
26

against the reference strain for the other drugs were within a reasonable range and was
in line with other published data. Only 3 out of the 14 drugs tested had conflicting MIC
values against the reference strain, with reasons to be discussed in later sections.

The selected synergistic combination retained synergism when tested against
clinical isolates of M. abscessus from CF patients
M. abscessus clinical isolates from cystic fibrosis patients. A total of 30 clinical isolates
from National Jewish Health culture collection was obtained to be used for this study. A
total of 30 isolates with the number of isolates in each subspecies was selected in order
to reflect the general distribution of subspecies among CF patients. The population
genomics study by the Colorado Research and Development Program (CO-RDP) of M.
abscessus isolates from 266 CF patients across the US showed that isolates were 79%
M. abscessus subsp. abscessus (MAB) ,19% M. abscessus subsp. massiliense
(MMAS), and 2% were M. abscessus subsp. bolletii (MBOL).[50] The list of clinical
isolates and their corresponding genotypic information are shown in Table 2.
27

Table 3. M.abscessus clinical isolates from CF patients identified according to
subspecies classification, morphology and mutation.
Clinical isolates of M. abscessus (n=30) are classified based on genome sequencing
into their subspecies: M.abscessus subsp. abscessus (MAB), subsp.bolletii (MBOL) and
subsp.massiliense (MMAS). Their rrl and rrs mutation statuses and morphology: rough
(R) or smooth (S) are also noted.

CF Patient ASID CF Sample ASID
WGS
Identification
CF Morphology
rrs Position
1408
rrl Position
2058
rrl Position
2059
Description
CF00006 DNA00020 MAB R A A A WT
CF00013 DNA01163 MAB R A A A WT
CF00016 DNA00465 MAB R A A A WT
CF00023 DNA00438 MAB R A A A WT
CF00038 DNA01927 MAB S A A A WT
CF00040 DNA01877 MAB R A A A WT
CF00041 DNA01939 MAB S A A A WT
CF00043 DNA02029 MAB R A A A WT
CF01975 DNA01884 MAB R A A A WT
CF02279 DNA02122 MAB R A A A WT
CF02319 DNA02156 MAB R A A A WT
CF02486 DNA02310 MAB S A A A WT
CF00258 DNA00550 MAB UNKNOWN A A C rrl mutation
CF00136 DNA00140 MAB UNKNOWN A C A rrl mutation
CF00855 DNA00847 MAB UNKNOWN A G A rrl mutation
CF00017 DNA00040 MAB R G A A rrs mutation
CF00287 DNA00614 MAB UNKNOWN G C A
rrl and rrs
mutation
CF02033 DNA01924 MAB R G G A
rrl and rrs
mutation
CF00020 DNA00047 MBOL S A A A WT
CF00868 DNA00882 MBOL UNKNOWN A A A WT
CF01088 DNA01876 MBOL S A A A WT
CF02061 DNA01943 MBOL R A A A WT
CF00113 DNA00117 MBOL UNKNOWN G A A rrs mutation
CF00008 DNA00027 MMAS R A A A WT
CF00030 DNA01873 MMAS S A A A WT
CF00035 DNA01830 MMAS R A A A WT
CF00042 DNA01874 MMAS S A A A WT
CF00047 DNA01928 MMAS S A A A WT
CF00883 DNA01804 MMAS R A A G rrl mutation
CF00046 DNA00423 MMAS UNKNOWN A C A rrl mutation
28

A panel of 30 clinical isolates of M. abscessus from CF patients are used in this study to
understand the efficacies of the drugs used against MABSC in the context of CF
patients. The general distribution of the M. abscessus subspecies in isolates from CF
patients obtained from the large scale study done across U.S CF Care Centers, is that
79% had MAB isolates, 19% were MMAS isolates and 2% were MBOL isolates.[50] The
CF isolates (n=30) used in this study was chosen to reflect his distribution to a certain
degree.

Our study has included CF clinical isolates that are 60.0% (18/30) MAB, 23.3% (7/30)
MMAS, 16.7% MBOL (5/30). The mutation status of 2058/2059 position of the 23S
rRNA (rrl) gene which confers acquired resistance to clarithromycin as well as the
mutation status of the 1408 position of the 16S rRNA (rrs) gene which correlates to
amikacin resistance are provided in the table. [51] The R or S morphology of the clinical
strains is also provided.

The synergism of drug combinations that were observed in the initial screen was
maintained. Tables 3 to 9 shows the individual FIC indices for drug combinations when
used combination against each of the clinical M. abscessus isolates, which are all
relatively in the range of synergism similar to that of the FIC index obtained with the
reference strain ATCC19977.

29

Table 4. The MICs of minocycline, impenem-relebactam and both drugs in
combination against M. abscessus clinical isolates from CF patients, with the
corresponding calculated FIC index.
The median MIC value of the MICs against the clinical strains for each of the conditions:
minocycline alone, minocycline with IMIREL, IMIREL, IMIREL with minocycline is also
reported below. The FIC index using the median MIC values was also calculated.

WGS ID CF Patient ID Minocycline alone Minocycline with IMIREL IMIREL alone IMIREL with Minocycline
M. abscessus CF00016 512 256 32 8 0.75
CF00017 256 128 2 2 1.50
CF00038 256 64 4 1 0.50
CF00040 256 128 8 4 1.00
CF00041 512 128 2 2 1.25
CF00043 256 128 2 2 1.50
CF00287 512 256 16 4 0.75
CF00855 256 128 8 2 0.75
CF01975 256 64 8 2 0.50
CF02033 256 128 4 2 1.00
CF02279 256 128 2 1 1.00
CF02319 256 64 2 1 0.75
CF02486 1024 128 8 4 0.63
M. massiliense CF00008 256 64 8 2 0.50
CF00030 256 16 4 2 0.56
CF00035 256 32 4 0.5 0.25
CF00042 16 16 4 0.25 1.06
CF00047 256 32 2 2 1.13
CF00883 8 8 2 0.125 1.06
M. bolletii CF00020 256 256 4 2 1.50
CF00113 256 64 4 1 0.50
CF00868 8 8 4 0.125 1.03
CF01088 64 8 4 0.5 0.25
CF02061 256 64 4 2 0.75
256 64 4 2 0.75
MIC (ug/ml)
FICI
Median value (ug/mL)
30

Table 5.The MICs of cefoxitin, impenem-relebactam and both drugs in
combination against M. abscessus clinical isolates from CF patients, with the
corresponding calculated FIC index. The median MIC value of the MICs against the
clinical strains for each of the conditions: cefoxitin alone, cefoxitin with IMIREL, IMIREL,
IMIREL with cefoxitin is also reported below. The FIC index using the median MIC
values was also calculated.

WGS ID CF Patient ID Cefoxitin alone Cefoxitin with IMIREL IMIREL alone IMIREL with Cefoxitin
CF00006 64 4 16 16 1.06
CF00013 128 32 8 2 0.50
CF00016 64 16 8 2 0.50
CF00017 32 8 4 2 0.75
CF00023 64 16 4 1 0.50
CF00038 32 8 16 2 0.38
CF00040 128 8 32 32 1.06
CF00041 32 8 4 1 0.50
CF00043 64 8 4 1 0.38
CF00258 64 8 8 1 0.25
CF00855 128 8 64 32 0.56
CF01975 32 4 64 8 0.25
CF02033 64 8 32 4 0.25
CF02279 64 8 4 1 0.38
CF02319 64 2 8 2 0.28
CF02486 32 4 8 2 0.38
CF00008 64 4 16 8 0.56
CF00030 32 4 16 4 0.38
CF00035 32 4 8 8 1.13
CF00042 16 4 16 16 1.25
CF00046 64 4 16 8 0.56
CF00047 32 4 4 1 0.38
CF00883 16 4 8 4 0.75
M. bolletii CF00020 32 4 16 4 0.38
64 6 8 3 0.47
M. abscessus
M. massiliense
MIC (ug/ml)
FICI
Median value (ug/mL)
31

Table 6. The MICs of cefdinir, impenem-relebactam and both drugs in
combination against M. abscessus clinical isolates from CF patients, with the
corresponding calculated FIC index. The median MIC value of the MICs against the
clinical strains for each of the conditions: cefdinir alone, cefdinir with IMIREL, IMIREL,
IMIREL with cefdinir is also reported below. The FIC index using the median MIC values
was also calculated.

WGS ID CF Patient ID Cefdinir alone Cefdinir with IMIREL IMIREL alone IMIREL with Cefdinir
CF00006 256 128 16 0.125 0.51
CF00013 256 32 4 1 0.38
CF00016 128 16 4 1 0.38
CF00017 256 8 2 0.5 0.28
CF00023 256 32 4 1 0.38
CF00038 256 64 4 1 0.50
CF00041 256 32 8 1 0.25
CF00043 128 8 4 0.5 0.19
CF00136 16 16 16 0.5 1.03
CF00258 256 128 16 2 0.63
CF00855 64 16 4 0.5 0.38
CF01975 256 32 8 0.125 0.14
CF02033 256 64 4 1 0.50
CF02279 512 32 2 0.25 0.19
CF02319 128 8 4 1 0.31
CF02486 256 16 4 1 0.31
CF00008 256 64 8 1 0.38
CF00030 128 8 4 1 0.31
CF00035 128 16 8 1 0.25
CF00042 16 16 8 0.125 1.02
CF00046 256 64 8 0.5 0.31
CF00883 8 2 4 2 0.75
CF00020 64 8 4 1 0.38
CF00113 64 8 4 2 0.63
CF00868 128 32 8 1 0.38
CF02061 64 16 4 1 0.50
192 16 4 1 0.33 Median value (ug/mL)
M. abscessus
M. massiliense
M. bolletii
MIC (ug/ml)
FICI
32

Table 7. The MICs of rifabutin, impenem-relebactam and both drugs in
combination against M. abscessus clinical isolates from CF patients, with the
corresponding calculated FIC index. The median MIC value of the MICs against the
clinical strains for each of the conditions: rifabutin alone, rifabutin with IMIREL, IMIREL,
IMIREL with rifabutin is also reported below. The FIC index using the median MIC
values was also calculated.

WGS ID CF Patient ID Rifabutin alone Rifabutin with IMIREL IMIREL alone IMIREL with Rifabutin
CF00006 16 8 16 2 0.63
CF00013 4 2 4 0.5 0.63
CF00016 16 16 16 0.125 1.01
CF00017 4 0.5 4 2 0.63
CF00023 16 8 4 1 0.75
CF00038 16 4 4 1 0.50
CF00040 16 2 4 2 0.63
CF00041 8 1 4 2 0.63
CF00043 16 4 4 1 0.50
CF00136 8 2 16 0.125 0.26
CF00258 16 4 16 2 0.38
CF00287 4 2 32 16 1.00
CF00855 4 1 4 1 0.50
CF01975 2 1 8 0.5 0.56
CF02033 8 4 4 0.25 0.56
CF02319 8 2 4 1 0.50
CF02486 16 1 4 2 0.56
CF00008 16 4 4 2 0.75
CF00030 16 4 8 2 0.50
CF00035 4 1 8 2 0.50
CF00046 16 1 4 2 0.56
CF00047 8 2 8 2 0.50
CF00883 2 0.5 4 1 0.50
CF00020 2 0.5 8 1 0.38
CF01088 0.125 0.125 0.125 0.125 2.00
8 2 4 1 0.50 Median value (ug/mL)
M. abscessus
M. massiliense
M. bolletii
MIC (ug/ml)
FICI
33

Table 8. The MICs of cefuroxime, impenem-relebactam and both drugs in
combination against M. abscessus clinical isolates from CF patients, with the
corresponding calculated FIC index. The median MIC value of the MICs against the
clinical strains for each of the conditions: cefuroxime alone, cefuroxime with IMIREL,
IMIREL, IMIREL with cefuroxime is also reported below. The FIC index using the
median MIC values was also calculated.

WGS ID CF Patient ID Cefuroxime alone Cefuroxime with IMIREL IMIREL alone
IMIREL with
Cefuroxime
M. abscessus CF00006 512 128 8 0.5 0.31
CF00013 512 128 4 0.125 0.28
CF00016 256 64 16 0.5 0.28
CF00017 512 32 4 0.5 0.19
CF00038 64 16 4 0.125 0.28
CF00040 512 64 8 0.5 0.19
CF00043 256 16 4 0.125 0.09
CF00136 128 16 16 0.125 0.13
CF00258 256 64 8 1 0.38
CF00855 512 128 8 0.5 0.31
CF01975 256 128 16 1 0.56
CF02033 1024 256 4 0.5 0.38
CF02279 512 32 2 0.25 0.19
CF02319 512 64 4 0.5 0.25
CF02486 512 32 2 0.125 0.13
M. massiliense CF00008 1024 128 4 0.125 0.16
CF00030 1024 64 4 0.125 0.09
CF00035 512 64 4 0.25 0.19
CF00042 64 32 16 0.25 0.52
CF00047 256 64 4 0.5 0.38
CF00883 1024 512 16 2 0.63
M. bolletii CF00020 512 64 8 0.25 0.16
CF00113 2048 256 8 2 0.38
CF00868 128 16 4 0.125 0.16
CF02061 512 64 4 0.5 0.25
512 64 4 0.5 0.25
MIC (ug/ml)
FICI
Median value (ug/mL)
34

Table 9. The MICs of NITD-916, impenem-relebactam and both drugs in
combination against M. abscessus clinical isolates from CF patients, with the
corresponding calculated FIC index. The median MIC value of the MICs against the
clinical strains for each of the conditions: NITD-916 alone, NITD-916 with IMIREL,
IMIREL, IMIREL with NITD-916 is also reported below. The FIC index using the median
MIC values was also calculated.

WGS ID CF Patient ID NITD916 alone NITD916 with IMIREL IMIREL alone IMIREL with NITD916
M. abscessus CF00006 4 2 8 1 0.63
CF00013 2 1 4 1 0.75
CF00016 2 0.5 8 1 0.38
CF00017 1 0.5 4 0.5 0.63
CF00023 2 1 4 0.5 0.63
CF00038 2 1 4 0.25 0.56
CF00040 2 1 8 1 0.63
CF00041 2 0.5 8 1 0.38
CF00043 2 0.5 8 1 0.38
CF00136 1 0.25 4 1 0.50
CF00258 4 2 16 4 0.75
CF00855 4 0.5 8 2 0.38
CF01975 2 0.5 16 1 0.31
CF02033 2 0.25 4 1 0.38
CF02279 4 1 16 4 0.50
CF02319 2 0.5 4 1 0.50
CF02486 2 1 8 0.5 0.56
M. massiliense CF00008 1 0.5 4 0.25 0.56
CF00030 1 0.5 8 1 0.63
CF00035 2 1 8 1 0.63
CF00042 4 1 8 2 0.50
CF00046 2 0.5 32 16 0.75
CF00047 2 1 4 0.25 0.56
CF00883 0.06 0.06 2 0.125 1.06
M. bolletii CF00020 0.5 0.125 4 1 0.50
CF00113 1 0.25 4 1 0.50
CF00868 2 0.5 8 4 0.75
CF02061 2 0.5 8 1 0.38
2 0.5 8 1 0.38
MIC (ug/ml)
FICI
Median value (ug/mL)
35

Table 10. The MICs of amoxicillin, impenem-relebactam and both drugs in
combination against M. abscessus clinical isolates from CF patients, with the
corresponding calculated FIC index. The median MIC value of the MICs against the
clinical strains for each of the conditions: amoxicillin alone, amoxicillin with IMIREL,
IMIREL, IMIREL with amoxicillin is also reported below. The FIC index using the median
MIC values was also calculated.

The MIC50s and MIC90s were also calculated in order to gain a better overall view of
the susceptibility profile and synergistic interactions between IMIREL and the drugs
across all the CF patient clinical isolates. The tables below show the MIC50s and
WGS ID
CF Patient
ID
Cefoxiti
n alone
Cefoxiti
n with
IMIREL
IMIREL
alone
IMIREL
with
Cefoxiti
n
CF00006 2048 256 16 8 0.63
CF00013 2048 512 2 0.5 0.50
CF00016 2048 512 8 2 0.50
CF00038 2048 16 4 1 0.26
CF00040 2048 16 8 8 1.01
CF00041 2048 256 4 1 0.38
CF00043 2048 256 4 1 0.38
CF00258 2048 256 8 4 0.63
CF00855 2048 256 4 1 0.38
CF02033 2048 256 8 2 0.38
CF02319 2048 8 2 2 1.00
CF00008 2048 8 4 2 0.50
CF00035 2048 2048 8 1 1.13
CF00042 1024 32 16 4 0.28
CF00047 2048 32 8 2 0.27
2048 256 8 2 0.38
MIC (ug/ml)
FICI
M. abscessus
M. massiliense
Median value (ug/mL)
36

MIC90s for the drug compounds alone, IMIREL alone and both in combination to arrive
at a FIC index for the MIC50 and MIC90 values. Most FIC values for the MIC90
category are under 1.00, while most are under 0.50 for the MIC50 category for all the
drug combinations (Table 4-1). This shows synergistic interactions initially determined in
the reference strain decrease to around an additive level with all strains considered,
however it is somewhat maintained when only considering the MIC50 while MIC90
concentration show some increase in the FIC index.

Table 11. The MIC50s and MIC90s (ug/mL) of drug compounds, imipenem-
relebactam and both drugs in combination against all the respective M.
abscessus clinical isolates the specific drug compounds were tested in, with the
corresponding FIC index.

Overall
MIC50 MIC90 MIC50 MIC90 MIC50 MIC90 MIC50 MIC90 MIC50 MIC90
Minocycline 256 512 64 256 4 8 2 4 0.75 1.00 24
Cefuroxime 512 1024 64 256 4 16 0.5 1 0.25 0.31 25
Cefoxitin 64 128 4 16 8 32 2 16 0.31 0.63 24
Cefdinir 128 256 16 64 4 16 1 2 0.38 0.38 26
Rifabutin 8 16 2 8 4 16 1 2 0.50 0.63 25
Amoxicillin 2048 2048 256 512 8 16 2 8 0.38 0.75 15
NITD 916 2 4 0.5 1 8 16 1 4 0.38 0.50 28
Drug alone
(ug/ml)
Drug with
IMIREL
(ug/ml)
IMIREL
(ug/ml) alone
IMIREL with
drug (ug/ml) Number of strains
FIC index
37

Table 12. The MIC50s and MIC90s (ug/mL) of drug compounds, imipenem-
relebactam and both drugs in combination against all the respective M.
abscessus susp. abscessus clinical isolates the specific drug compounds were
tested in, with the corresponding FIC index.

Table 13. Table 4-3. The MIC50s and MIC90s (ug/mL) of drug compounds,
imipenem-relebactam and both drugs in combination against all the respective M.
abscessus susp. massiliense clinical isolates the specific drug compounds were
tested in, with the corresponding

M.abscessu
s subp.
abscessus
MIC50 MIC90 MIC50 MIC90 MIC50 MIC90 MIC50 MIC90 MIC50 MIC90
Minocycline 256 512 128 256 4 16 2 4 1.00 0.75 13
Cefuroxime 512 512 64 128 4 16 0.5 1 0.25 0.31 15
Cefoxitin 64 128 8 16 8 32 2 16 0.38 0.63 16
Cefdinir 256 256 32 64 4 16 1 1 0.38 0.31 16
Rifabutin 8 16 2 8 4 16 1 2 0.50 0.63 17
Amoxicillin 2048 2048 256 512 4 8 2 8 0.63 1.25 11
NITD 916 2 4 0.5 1 8 16 1 2 0.38 0.38 17
Number of strains
Drug alone
(ug/ml)
Drug with
IMIREL
(ug/ml)
IMIREL
(ug/ml) alone
IMIREL with
drug (ug/ml)
FIC index
M.abscessu
s subp.
massiliense
MIC50 MIC90 MIC50 MIC90 MIC50 MIC90 MIC50 MIC90 MIC50 MIC90
Minocycline 256 256 16 32 4 4 0.5 2 0.19 0.63 6
Cefuroxime 512 1024 64 128 4 16 0.25 0.5 0.19 0.16 6
Cefoxitin 32 64 4 4 8 16 8 16 1.13 1.06 7
Cefdinir 128 256 16 64 8 8 1 1 0.25 0.38 6
Rifabutin 8 16 1 4 4 8 2 2 0.63 0.50 6
Amoxicillin 2048 2048 32 2048 8 16 2 4 0.27 1.25 4
NITD 916 2 2 0.5 1 8 8 1 2 0.38 0.75 7
Number of strains
Drug alone
(ug/ml)
Drug with
IMIREL
(ug/ml)
IMIREL
(ug/ml) alone
IMIREL with
drug (ug/ml)
FIC index
38

Table 14. The MIC50s and MIC90s (ug/mL) of drug compounds, imipenem-
relebactam and both drugs in combination against all the respective M.
abscessus susp. bolettii clinical isolates the specific drug compounds were
tested in, with the corresponding FIC index.

The MIC50s and MIC90s for the drugs and drug combinations were separated
according to the subspecies of M.abcessus and the corresponding FIC indices were
also calculated. The MIC50s and MIC90s compared between subspecies of M.
abscessus did not show any significant difference of more than 1-fold difference (Table
11, Table 12, and Table 13). The FIC index differences between the subspecies is a
result of the small variations in MIC values, however the difference between the
subspecies also do no vary more than 0.5, producing a similar synergistic/additive
profile across all subspecies (Table 4-2, Table 4-3, and Table 4-4).

M.abscessu
s subp.
bolletii
MIC50 MIC90 MIC50 MIC90 MIC50 MIC90 MIC50 MIC90 MIC50 MIC90
Minocycline 256 256 64 256 4 4 1 2 0.50 1.50 5
Cefuroxime 512 2048 64 256 4 8 0.25 2 0.19 0.38 4
Cefoxitin 32 32 4 4 16 16 4 4 0.38 0.38 1
Cefdinir 64 128 8 32 4 8 1 2 0.38 0.50 4
Rifabutin 0.125 2 0.125 0.5 0.125 8 0.125 1 2.00 0.38 2
Amoxicillin N/A N/A N/A N/A N/A N/A N/A N/A N/A N/A 0
NITD 916 1 2 0.25 0.5 4 8 1 4 0.50 0.75 4
Drug alone
(ug/ml)
Drug with
IMIREL
(ug/ml)
IMIREL
(ug/ml) alone
IMIREL with
drug (ug/ml)
FIC index
Number of strains
39

Table 15. The MICs and MBCs of IMIREL, individual drug and both drugs in
combination against the ATCC19977 M.abscessus strain. The MICs corresponds to
the MICs observed during the experimental run when the MBC was observed for a
direct comparison between the MIC and MBC against MABSC.
MIC of IMIREL (ug/mL) 4
MIC of drug alone
(ug/mL)
MIC of drug with
IMIREL (ug/mL)
MIC of IMIREL
with drug
(ug/mL)
Minocycline 256 64 2
Cefdinir 256 16 1
Cefuroxime 1024 128 0.5
Cefoxitin 32 8 0.5
Rifabutin 8 1 1
NITD916 1 0.25 2
MBC of IMIREL (ug/mL) >16
MBC of drug alone
(ug/mL)
MBC of drugwith
IMIREL (ug/mL)
MBC of IMIREL
with drug
(ug/mL)
Minocycline 1024 512 4
Cefdinir 512 256 0.5
Cefuroxime 4096 256 1
Cefoxitin >256 >256 >16
Rifabutin 64 16 4
NITD916 2 1 0.5
40

Table 16. The table is an overview of peak concentration (cmax) that was
observed in the plasma/serum in pharmacokinetic studies done with the
respective drugs in humans. The values were obtained by package inserts of the
drugs: imipenem-relebactam (RECARDBRIO) [52], minocycline [53], cefdinir [54],
cefuroxime [55], cefoxitin [56], rifabutin [57]. There currently is no available
pharmacokinetic studies data on NITD916.

Drug combinations have MICs and MBCs that are not achievable clinically, while
IMIREL is able to reach bactericidal concentrations clinically
The minimum bactericidal concentrations (MBC) of the drugs against the reference
strain ATCC19977 were also measured by conducting a MBC assay, where the
minimum concentration in which there is no bacterial growth was determined by CFU
Peak concentration (cmax)
Imipenem (Intravenous as
RECARBRIO)
116.1 uM (36.84ug/mL) in
plasma
Relebactam(Intravenous
as RECARBRIO)
62.1 uM (22.75ug/mL) in
plasma
Minocycline HCl (Oral) 3.5ug/mL in serum
Cefdinir (Oral) 1.6-2.87ug/mL in plasma
Cefuroxime (Oral) 2.1-13.6 ug/mL in plasma
Cefoxitin (Intravenous) 110ug/mL in serum
Rifabutin (Oral) 0.375ug/mL in plasma
NITD916 N/A
41

count. MBC values allows the assessment of the drug as bactericidal compound
Several MBCs were many folds higher concentrations than the MIC including
combination treatments of the drug, indicating a much higher concentration needed for
a bactericidal effect as opposed to just growth inhibition (Table 5-1). The MIC and MBC
concentrations of all the drugs (except cefoxitin) both individual and in combinations
with IMIREL were below the achievable concentration in the plasma/serum clinically
(Table 5-2). Cefoxitin had a MIC concentration of 8ug/mL against the reference strain
and a MIC90 of 16ug/mL against 24 clinical isolate strains from CF patients. The MICs
and MBC of Imipenem-relebactam however both alone and in combination with other
drugs was below the cmax of plasma levels in humans. NITD916 is a direct inhibitor of
the Mycobacterium abscessus enoyl-ACP reductase InhA, confirmed by crystal
structure analysis.[39] The MIC and MBC for the compound NITD-916 in combination
with IMIREL is very low at 0.25 ug/mL and 1 ug/mL respectively, however as it is a non-
FDA approved compound that is currently not in the market and currently has no
literature on the pharmacokinetic properties clinically nor in non-clinical models it is hard
to determine the clinical applicability of the compound.

In vivo safety and infection model establishment

Imipenem-relebactam is non-toxic to zebrafish even at high concentrations and
can be used to test for in vivo efficacy in the zebrafish model
The toxicity of IMIREL it was tested in zebrafish by exposing the zebrafish to a range of
low to high concentrations by maintaining the fish in fish water with the respective drug
42

concentrations. The zebrafish was exposed to the respective for 5 days from 2 days
post fertilization. The control and 28X MIC concentration treatment of IMIREL had no
deaths with the 0.5X MIC and 5X MIC concentration treatment of IMIREL resulting in 1
death at 14 days post-fertilization (Figure 2-1). The survival curve of all the treatment
groups with IMIREL resulted in no statistically significant difference compared to the
control group (Figure 2-1). Visual observation of the physiological and development
state of the zebrafish in all treatment groups of IMIREL also showed no notable change
or signs of toxicity (developmental abnormalities) (Figure 2-2).

43

Figure 3. The survival curve showing the survival of uninfected zebrafish
embryos (n=20) treated with 0.5X MIC (2ug/mL imipenem, 1ug/mL relebactam), 5X
MIC (20ug/mL imipenem, 10ug/mL relebactam), 28X MIC (112ug/mL imipenem,
56ug/mL relebactam) compared to control.The red capped line indicates the duration
of treatment with the respective drug concentrations. The survival rate is as follows:
control group at 100% (20/20), the 0.5X MIC group at 95% (19/20), the 5X MIC group at
95% (19/20) and the 28X MIC group at 100% (20/20).There is no statistically significant
difference in mortality when comparing even the highest 28X MIC concentration of
IMIREL with control (p=1.0, log-rank test).

44

Figure 4. Representative microscopy images of zebrafish at 14 days post-
fertilization in each group. Concentration of IMI/REL received by zebrafish: A) control
B) 0.5X MIC C) 5X MIC D) 28X MIC. There are no visible developmental abnormalities
such as curving of the body trunk or uninflated swim bladders, properly inflated swim
bladders (indicated by the red arrows) and straightened body structure can be
observed.

45

Proof of concept of Tg(mpeg1:EGFP) absolute zebrafish embryo infection with
dsRed2 labelled M. abscessus (ATCC19977)
We have also used a transgenic zebrafish as a model to visualize the infection
progression of M. abscessus in zebrafish larvae. The Tg(mpeg1:EGFP) absolute
zebrafish is a transgenic zebrafish (AB x mpeg:GFP) that is a cross between a the
transgenic: Tg(mpeg1:EGFP) zebrafish [58] with GFP labeled macrophages and the
mutant absolute fish. The absolute mutant is a double mutant with ednrb1a
b140
and
mitfa
b692
genotypes which makes the fish without pigment producing cells such as
melanophores, xantophore, and most iridophores (cells that reflect light), causing a
completely transparent body appearance.[59] This makes the zebrafish already
translucent zebrafish embryo more transparent for internal visualization. The EGFP
tagged macrophages by the labelled mpeg1, will be able to visualized. Following the
infection of the AB x mpeg:GFP zebrafish with the dsRed:M.abscessus by
microinjection allows for the visualization of the infection progression within the
zebfrafish. The current protocol has yet to be optimized resulting in a low level infection
indicated by the red fluorescent specks (Figure 5), the infection fails to progress to a
high enough bacterial burden to show visible cording and abscess formation or induce a
decrease in survival (data not shown). (Figure 5) The interaction between the
macrophages and the bacteria however was still observed with the presence of yellow
fluorescence as a result of the overlap of red and green fluorescence.

46

Figure 5. Representative fluorescent microscopy images of A) and B) uninfected
Tg(mpeg1:EGFP) absolute zebrafish and C) and D) dsRed:M.abscessus infected
Tg(mpeg1:EGFP) absolute zebrafish. The green fluorescence depicts the
macrophages labeled with EGFP, the red fluorescent specks (indicated with white
arrows) show the presence of the M.abscessus in within the fish. The overlap of green
fluorescence and red fluorescence creates a yellow fluorescence showing the
interaction of the host macrophages and the bacteria.

47

Chapter 3: Summary and future directions
In summary, this study shows the in vitro activity of IMIREL in combination with other
antibiotics of various drug classes against M.abscessus in the context of CF by using
clinical isolates of different M.abscessus subspecies from CF patients. The initial screen
of the panel of antibiotics tested for synergy against the reference ATCC19977 strain
revealed 7 potential synergistic candidates: minocycline, cefdinir, cefuroxime, cefoxitin,
rifabutin, amoxicillin and NITD-916.
Our MIC values against the reference strain were different from published data for
certain drugs, specifically: cefdinir and minocycline while the MIC values for the other 5
drugs were line with other published data. The MIC values against the reference strain
is supposed to serve as a benchmark for the activity of the drugs and procedures for the
microbroth dilution susceptibility assay. However, as seen by the discrepancy in MIC
values of cefdinir against the reference strain even in published studies [29] [48], there
can be difference in the methodology of conducting the susceptibility assay despite
being guided by the CLSI guidelines. The preparation of the stock solution of the drug
itself may be a source of variation that produces different MIC values, as well as the
different methods of observing the growth and growth inhibition in the wells (the use of
spectrometer, plain eye visualization, etc.) Minocycline hydrochloride is available by
several vendors that reports different solubilities and different recommended solvents.
[60] [61] Selleckchem reports DMSO insolubility with 6mg/mL water solubility [61] while
ApexBio reports solubility of ≥60.7 mg/mL in DMSO with gentle warming and ≥18.73
mg/mL in H2O with ultrasonication.[60] The study using minocycline obtained their stock
48

from Sigma-Alridch but did not report the stock solution preparation methodology and
there were no guidelines provided by the manufacturer.[47] We prepared our
minocycline dilutions directly into the CAMHB and proceeded with the dilution for the
susceptibility assay. Due to the differences in the preparation of the concentrated stock
solutions, this could have caused the variation in MIC values obtained despite following
the protocol guidelines for the assay by CLSI. The same can be assumed for the
variation in MIC values against the reference strain obtained for cefdinir. This aspect
could be something investigators need to take into account when handling compounds
with low solubility.
The 7 drug combinations were further tested in CF isolates of M.abscessus belong to
various subspecies and the MIC50 and MIC90 against the CF isolates for the drugs
alone and in combination with IMIREL was determined. The difference in the MIC50 and
MIC90 for the drugs used alone and in combination with IMIREL was also used to
determine an FIC index. The synergism observed with the drug combinations that was
observed against the reference ATCC19977 strain was also retained, where the FIC
indices calculated using the MIC90 values had minor deviation from the FIC value
observed with the reference strain and still maintained at least an additive interaction
between the drugs.
We report also that the MIC50 and MIC90 for the drugs (alone and in combination with
IMIREL) did not have a significant difference across different subspecies of
M.abscessus, suggesting that with the drugs studied there was minor differences in
their efficacies against different subspecies of M. abscessus. The FIC index difference
for the drugs in combination with IMIREL was also minor and did not exceed 0.5.
49

While the MICs of the drugs and drug combinations are important, to find the
bactericidal concentrations of the drugs alone and in combination with IMIREL, the MBC
was also determined by CFU counts of the bacterial solution that did not show growth
after the checkerboard assay. MBCs of the drugs alone and in combination with IMIREL
were determined to be several folds higher than the MICs. The stark difference in MIC
and MBC highlights the need to determine MBCs for drugs and drug combinations to
define concentration that will go beyond growth inhibition of the bacteria.
Regardless of the synergism shown by the FIC index, we report that except for IMIREL,
the other drugs even in combination with IMIREL fail to reach clinically viable
concentrations. The cmax of the drugs determined in humans were shown to be lower
than the MICs and MBCs or the drugs indicating that the dual combination of a drug and
IMIREL is insufficient in having a synergistic effect as to produce an MIC or MBC that is
below the clinically achievable cmax. A point to note as well is that the FIC index though
used to determine synergy does not necessarily equate to clinical usefulness as the
compound NITD-916 with a FIC index of 0.5 (determined with MIC90 data) has a MIC90
of 1ug/mL as compared to cefuroxime with a FIC index of 0.31 (determined with MIC90
data) which has a MIC90 of 256 ug/mL. Cefuroxime is shown to be one of the best
synergistic combinations with just the MIC values. Though cefuroxime experiences a
major decrease in MIC when in used in combination with IMIREL, the resulting
concentration remains high and above the clinically achievable serum concentration of
13.6ug/mL. NITD-916 in this respect is a promising candidate to be used in combination
with IMIREL and other drugs as it has a unique target and can be used at low
concentrations. Future non-clinical and clinical data will come to show if the compound
50

is safe and is able to reach MIC and MBC concentration physiologically. Other dual drug
combinations will however may have to be tested with an additional drug as a triple
combination in order to potentially lower the MICs and MBCs further. The synergistic
effects of the triple drug combinations of drugs can be tested using a three-dimensional
checkerboard assay as described in a previous study.[62]
Possibly promising candidates to test would be drugs that may not be beta lactams
which have as a double beta lactam combination with IMIREL produced a significant
drop in MIC values against M. abscessus strains, have MICs that are much higher than
clinically achievable plasma concentrations. Possible candidates could be moxifloxacin
and clofazimine, which we report MICs against the ATCC19977 strain as 4ug/mL and
1ug/mL respectively when used in combination with IMIREL (Table 1). Though the FIC
index calculated for these compounds with IMIREL are 0.5 (moxifloxacin) and 1.0
(clofazimine), these concentration values are much lower and within the clinically
achievable plasma concentrations. The reported clinical cmax plasma values for
moxifloxacin is 3.1-4.6ug/mL (administered orally) and 3.9-6.1ug/mL (administered
intravenously) which is in range with the MIC value (4ug/mL) against the M.abscessus
when used with IMIREL that we report in our study.[63] The reported clinical cmax
plasma values for clofazimine is 0.7-1.0ug/mL which also overlaps with the MIC value
(1.0ug/mL) against the reference M.abscessus strain we report in our study when used
with IMIREL.[64] Though the MBC against M.abscessus for these drugs will likely be
higher than their MICs, these combinations with IMIREL are more promising due to the
MICs being at clinically achievable levels. As additional drugs are added to the dual
combination with IMIREL, these drugs have the potential to have lower MICs and MBCs
51

that are below or in range with the clinically achievable concentrations. After testing the
dual combinations in the panel of CF clinical strains, if the MICs and MBCs are at
clinically achievable levels, these combinations with IMIREL can be tested in vivo to test
for toxicity and in vivo efficacy as the first step towards implementation in the clinical
space.

In an vivo context, we have also shown that IMIREL does not exhibit any toxicity even at
high concentrations that is 28X of the MIC in vivo. The toxicity study in the zebrafish
showed that there wasn’t any statistically significant difference between the survival
curve of the control group of zebrafish and the IMIREL treatment groups. The
observation of physical features of the zebrafish also showed no developmental issues
or other signs of toxicity. This demonstrated that IMIREL is safe to test in the zebrafish
model for its efficacy against M. abscessus infections as it does not result any adverse
effects or toxicities even at high concentrations.
We have also demonstrated the preliminary stages of developing an M.abscessus
infection model using zebrafish, The AB x mpeg:EGFP zebrafish coupled with the
infection with the dsRed M. abscessus, allows for the dual visualization of the host
macrophages (in green) and the bacteria (in red),and their interaction can also be seen
by the overlap of the two fluorescent colors showing yellow. The infection model report
in this study was not refined in terms of an optimized bacterial inoculum as the infection
fails to create a large enough bacterial burden to induce cord or abscess formation as
well as being unable to induce a decrease in survival which occurs at around 4 days
after infection. [65] The refinement of the infection bacteria inoculum will allow this
52

model to fully reflect M. abscessus infection producing accurate pathophysiological
features.
An improvement to the current zebrafish model we have would be to incorporate the
depletion of CFTR in the zebrafish. The zebrafish currently used in this study can be
CFTR depleted by using the injection of specific morpholino-modified oligonucleotides
that abrogates the production of native cftr expression as previously described in
another study.[19]

53

Chapter 4: Materials and Methods
Bacterial strains and isolates
The reference ATCC19977 strain was obtained from ATCC (ATCC, American Type
Culture Collection) and the CF patient isolate clinical strains were obtained from
National Jewish Health.

Bacterial growth conditions
The reference strain ATCC19977 and other CF patient clinical strains were grown in
28°C in Middlebrook 7H9 broth supplemented with 10% oleic acid-albumin-dextrose-
catalase (OADC) enrichment, 0.05% Tween 80, 0.5% glycerol shaking at 180rpm in a
shaking incubator and cultivated for 48hours. An initial growth curve analysis done
independently determined the reference strain to reach the log-phase (exponential
growth) at around 48hours of incubation upon inoculation.

Preparation of antibiotic solutions
All antibiotics were prepared according to their solubility and according to the
manufacturer’s instructions.
Susceptibility Testing: Minimum Inhibitory Concentration, Broth Microdilution
Assay
The MIC of each drug was determined following the broth dilution assay as described in
the CLSI guidelines.[66] Exponentially growing M. abscessus cultures areadjusted to
0.5 McFarland by spectrophotometry at 600 nm and grown in Cation-Adjusted Mueller-
Hinton (CAMHB) broth 30°C for 72 h.[66] Serially diluted solutions of the antibiotics
54

were prepared in 1.5ml microcentrifuge tubes. Imipenem-relebactam solutions were
made by serially diluting Imipenem in CAMHB that has a fixed concentration of 4ug/mL
of relebactam. This creates a set of imipenem-relebactam solutions with serially diluted
imipenem with a fixed. 4ug/mL relebactam concentration. Other antibiotics were
prepared with a standard broth dilution. Bacterial growth was determined 72h after
inoculation visually and the growth vs. no growth status were standardized to what was
described in the CLSI guidelines.[66]

Checkerboard assay
The checkerboard assay was performed in order to determine synergistic combination
of various antimicrobials. The assay was conducted as previously described in the study
by Story-Roller [29] with the replacement of 7H9 to CAMHB as the growth media.

Minimum Bactericidal Concentration determination
The minimum bactericidal concentration (MBC) for antibiotics and antibiotics
combinations were determined by the same broth microdilution method and
checkerboard assay for MIC determination however at 72hrs post incubation after the
MIC has been determined, 10ul of solution from each wells above the MIC without
visible bacterial growth were plated in 7H10 agar plates. MBC was determined to be
concentrations where 99% of bacteria did not grow as determined by CFU counts. [67]

M.abscessus competent cell preparation and electroporation
The fluorescent labeled ATCC19977 M. abscessus strain used for zebrafish embryo
55

infection was made by electroporation. The bacteria were made electrocompetent and
were then electroporated with the pMSP12::dsRed2 plasmid. [68] The procedure was
done following a previously described protocol [69] with modification to the
electroporation settings at 2500 mV, 1000 , 25 μF using the BioRad Gene Pulser. The
transformed bacteria were then selected in a kanamycin selective 7H10 agar plate and
grown in the identical growth conditions as other M. abscessus strains described above.

Zebrafish larvae husbandry
All animal experiments were evaluated and approved by the Institutional Animal Care
and Use Committee at the University of Southern California (Protocol 21300). Zebrafish
experiments were conducted using a double mutant produced by crossing the absolute
mutant (AB) strain [59] with the transgenic line Tg(mpeg1:EGFP) [58] to visualize
macrophages. Zebrafish were maintained in line with standard protocols. [70] The eggs
obtained from the vivarium were raised in a BSL2+ facility during the experimental
period. Eggs were maintained in 100mm round dishes until 3 days post-fertilization.
After 3 days post-fertilization, larvae were moved to 500mL glass beakers and kept at
20 fishes per beaker. 3mL of rotifer culture were given daily after a daily water change.
Drug exposure to the zebrafish was done by

Zebrafish injection procedures
After obtaining eggs, the eggs were dechorionated 24hours post fertilization and
maintained at 28.5 °C. Bacterial inoculate cultures were grown to the exponentially
growing phase for 48 hours and were prepared for microinjections into the caudal vein
56

as previously described.[38] The injected inoculum was a test concentration used for
the proof-of-concept, pilot study conducted in this study. The inoculum was a 1:1 ratio of
phenol red solution and OD600 adjusted exponentially growing culture of M. abscessus
(reference strain: ATCC19977) transformed with pMSP12::dsRed2, adjusted to 0.5
McFarland) The needles were prepared with borosilicate glass capillaries by Sutter
Instruments Inc with dimensions: 1mm O.D. x 0.78 mm I.D. These needles were
prepared using the Sutter Instruments P-2000 micropipette puller. The injections
conducted on a agar injection platform created using a self-made mold. The self-made
mold was made according to the protocol given by J. Muse Davis, M.D., Ph.D, from the
University of Iowa.

Microscopy
Bright-field microscopy of zebrafishes were done using the Leica MZ16FA high
performance stereo-microscope, images were captured and processed with the LAS X
Life EZ software. Fluorescence microscopy of the infected zebfrafish was done using
the BioTek Cytation 5, zebrafishes were fixed in agar and imaged as previously
described.[38] The plates with the infected fishes were covered prior to being placed
into the reader.

Statistical analyses
Median values of MICs were performed using Microsoft Excel. Statistical analyses(log-
rank test) of comparisons between Kaplan-Meier survival curves and graphical
presentations were performed using the Prism 5.0 (GraphPad, Inc.).
57

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Abstract (if available)

Abstract M.abscessus is an increasingly concerning pathogen that plagues cystic fibrosis (CF) patients. Although there currently is a guideline for the treatment of pulmonary M.abscessus infection CF patients, the current treatment regimen includes a multitude of drugs and is within a very long timeframe putting an immense burden on the patient.

This study aims to explore potential synergistic combinations of drugs with imipenem-relebactam (IMIREL) against M.abscessus clinical strains from CF patients using in-vitro methods to uncover a potential, clinically viable combination that is able to the treat M.abscessus infection using less drugs at lower drugs to lower the burden on infected patients. The study tested 7 potential synergistic drug combination candidates with IMIREL: minocycline, cefuroxime, cefoxitin, cefdinir, rifabutin, amoxicillin, NITD-916 in up to 30 clinical isolates from CF patients. The results showed however, that though the combinations are synergistic, both minimum inhibitory concentrations (MIC) and minimum bactericidal concentrations (MBC) are higher than clinically achievable concentration and other combinations that were additive should be further explored.

The study also showed that IMIREL is not toxic even at high concentrations to the zebrafish larvae demonstrating that IMIREL is safe to use in this particular in vivo model. The study also presents a proof of concept of a zebrafish larvae infection model that can be used to test the in vivo efficacy of the drug combinations. The model incorporates GFP-tagged macrophages within the zebrafish that can be visualized to see host-pathogen interactions with the dsRed-tagged M.abscessus that is microinjected into the larvae for infection.

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Creator Kim, Sun Woo (author)

Core Title Investigation of in vitro activity and in vivo safety of imipenem-relebactam alone and in combination with other anti-microbial agents against clinical isolates of M. abscessus from CF patients

School School of Pharmacy

Degree Master of Science

Degree Program Clinical and Experimental Therapeutics

Degree Conferral Date 2023-12

Publication Date 09/11/2023

Defense Date 09/08/2023

Publisher Los Angeles, California (original), University of Southern California (original), University of Southern California. Libraries (digital)

Tag cystic fibrosis,impenem-relebactam,M.abscessus,Mycobacterium,Mycobacterium abscessus,non tuberculous mycobacterium,OAI-PMH Harvest

Format theses (aat)

Language English

Contributor Electronically uploaded by the author (provenance)

Advisor Beringer, Paul (committee chair), Minejima, Emi (committee member), Wong-Beringer, Annie (committee member)

Creator Email skim8112@usc.edu,swkim9663@gmail.com

Permanent Link (DOI) https://doi.org/10.25549/usctheses-oUC113305027

Unique identifier UC113305027

Identifier etd-KimSunWoo-12351.pdf (filename)

Legacy Identifier etd-KimSunWoo-12351

Document Type Thesis

Format theses (aat)

Rights Kim, Sun Woo

Internet Media Type application/pdf

Type texts

Source 20230911-usctheses-batch-1094 (batch), University of Southern California (contributing entity), University of Southern California Dissertations and Theses (collection)

Access Conditions The author retains rights to his/her dissertation, thesis or other graduate work according to U.S. copyright law. Electronic access is being provided by the USC Libraries in agreement with the author, as the original true and official version of the work, but does not grant the reader permission to use the work if the desired use is covered by copyright. It is the author, as rights holder, who must provide use permission if such use is covered by copyright.

Repository Name University of Southern California Digital Library

Repository Location USC Digital Library, University of Southern California, University Park Campus MC 2810, 3434 South Grand Avenue, 2nd Floor, Los Angeles, California 90089-2810, USA

Repository Email cisadmin@lib.usc.edu