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Polymyxins retain in vitro activity and in vivo efficacy against “resistant” Acinetobacter baumannii strains when tested in physiological conditions
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Polymyxins retain in vitro activity and in vivo efficacy against “resistant” Acinetobacter baumannii strains when tested in physiological conditions
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Content
Polymyxins retain in vitro activity and in vivo efficacy against “resistant” Acinetobacter
baumannii strains when tested in physiological conditions
by
Jennifer Rubio
A Dissertation Presented to the
FACULTY OF THE USC GRADUATE SCHOOL
UNIVERSITY OF SOUTHERN CALIFORNIA
In Partial Fulfillment of the
Requirements for the Degree
DOCTOR OF PHILOSOPHY
(INFECTIOUS DISEASES, IMMUNOLOGY AND PATHOGENESIS)
December 2024
Copyright 2024 Jennifer Rubio
ii
Dedication
This work is dedicated to my ancestors. I carry the spirit of your strength and resiliency.
iii
Acknowledgements
My sincerest thanks to my committee members: Dr. Annie Wong-Beringer, Dr. Rosemary
She, Dr. Brad Spellberg and Dr. Brian Luna. Thank you for your guidance and support. Special
thanks to Brian for accepting me into his lab during a pandemic and for his guidance. Many thanks
to my Spellberg and Luna lab mates: Dr. Mimi Maeusli, Sarah Miller, Dr. Jun Yan, Bosul Lee,
Kexuan Chen, Zac Builta, Peggy Lu, Dr. Yuli Talyansky, Dr. Matthew Slarve, Krissi Goy, and Tina
Lam. Although none of their contributions are presented in this thesis, I want to extend my deepest
appreciation to my former lab mates from the Segil lab: Dr. Emily Wang, Dr. Talon Trecek, Juan
Llamas, Dr. Duc Nguyen, Dr. Litao Tao, Welly Makmura, Dr. Robert Rainey and Dr. Ksenia
Gnedeva. To Dr. Francesca Mariani, I will always be so grateful for the support, advice and
compassion that you extended to me while I was in the DSR program.
I would like to thank the PIBBS office for their continued support. Special thanks to Leslie
Ann Picazo for being the greatest source of support during my first year in the program. Much
appreciation to Joyce Perez, Bami Andrada and Dr. Ite Offringa for their support and assistance
during my lab transition. Many thanks to Domonique Walker for her support and kindness and to
Maritza Montalvo and Karina Recinos for their encouragement.
For their encouragement, support, compassion, and friendship during these past years,
many thanks to my friends: Tuo, Z, Brandon, DJ, and Sophia. Muchísimas gracias a EmpowerED:
Alma, Angie, MaryAnne, Sayuri, Edgar, Darian, Stephanie, Michele, Oluchi, Maria, and Alisha. I
am so grateful to have been a part of our group’s creation. I hope that EmpowerED continues to
be a source of support, encouragement and inspiration for years to come.
Thank you Dr. Buckley for your mentorship and encouragement.
To my family and friends, thank you for your endless love and support and for always
believing in me: Uncle Frankie, Uncle Jay, Alanah, Stephanie and Lisa. Thank you, Mom, Dad,
and Melissa, for the many sacrifices you made to get me here.
iv
Table of Contents
Dedication ................................................................................................................................. ii
Acknowledgements ................................................................................................................. iii
List of Tables.............................................................................................................................v
List of Figures .......................................................................................................................... vi
Abbreviations.......................................................................................................................... vii
Abstract .................................................................................................................................... ix
Chapter 1: Introduction ............................................................................................................1
Chapter 2 ...................................................................................................................................6
Chapter 3: Conclusions..........................................................................................................90
References ..............................................................................................................................93
v
List of Tables
Table 1: RPMI-1640 potentiates the activity of colistin ..............................................................11
Table 2: Summary of polymyxin MICs for strains used in the in vivo studies .............................26
Table S1: Colistin MICs in MHII supplemented with RPMI-1640 nutrients.................................42
Table S2: Polymyxin MICs of ColR isolates in MHII or MHII supplemented with sodium
bicarbonate...............................................................................................................................47
Table S3: Affinity of BoDipy-labeled colistin to ColR bacteria....................................................52
Table S4: Affinity of Dansyl-labeled polymyxin B to ColR bacteria ............................................53
Table S5: Summary of LD100 virulence screening....................................................................54
Table S6: Efficacy of colistin treatment in vivo ..........................................................................55
Table S7: Efficacy of polymyxin treatment in vivo......................................................................60
Table S8: Summary of bacterial strains used in this study ........................................................81
Table S9: Summary of primers used in this study .....................................................................88
vi
List of Figures
Fig. 1: Effect of pH on colistin MICs among different media ......................................................12
Fig. 2: Polymyxin MICs of ColR isolates in MHII, or MHII supplemented with sodium
bicarbonate...............................................................................................................................14
Fig. 3: Colistin efficacy against LAC-4 in human blood culturing conditions...............................17
Fig. 4: Mass spectrometry of lipid A isolated from A. baumannii strains LAC-4 WT and
LAC-4 ColR cultured in MHII or MHII+NaHCO3.........................................................................19
Fig. 5: Affinity of fluorescently labeled colistin to bacteria..........................................................20
Fig. 6: Expression of genes involved in conferring colistin-resistance in Acinetobacter
baumannii .................................................................................................................................22
Fig. 7: Efficacy of colistin treatment in vivo................................................................................27
Fig. 8: Efficacy of PMB treatment in vivo...................................................................................29
Fig. S1: Whole genome sequencing of LAC-4 ColR..................................................................42
vii
Abbreviations
AKI - Acute kidney injury
AMP - Antimicrobial peptides
AST – Antimicrobial susceptibility test
CRAB - Carbapenem resistant Acinetobacter baumannii
CRE - Carbapenem resistant Enterobacterales
CLSI - Clinical and Laboratory Standards Institute
COL - Colistin
CFU - Colony forming units
EUCAST - European Committee on Antimicrobial Susceptibility Testing
ICU - Intensive care units
LPS - Lipopolysaccharide
MIC - Minimum inhibitory concentration
Mcr-1 - Mobile colistin resistance gene
MHII - Mueller-Hinton II
MRSA - Methicillin-resistant Staphylococcus aureus
NaHCO3 - Sodium bicarbonate
viii
PDR - Pan-drug resistant
PetN - Phosphoethanolamine
PMB - Polymyxin B
TSA - Tryptic soy agar
TSB - Tryptic soy broth
ix
Abstract
The emergence of colistin resistance poses a serious threat to colistin efficacy as
it is considered the “last-line-of-defense” against extensively drug resistant
Acinetobacter baumannii. However, we found that conventional antimicrobial
susceptibility testing methods incorrectly model the contributions of previously
characterized colistin-resistance conferring mutations. We have found that physiological
media, but not the conventional media used in antimicrobial susceptibility assays,
accurately predicts polymyxin in vivo efficacy in mice infected with polymyxin-resistant
A. baumannii. Modifying antimicrobial susceptibility test culturing conditions will
maximize the therapeutic potential of the existing arsenal of antibiotics that have shown
efficacy against bacterial pathogens.
1
Chapter 1: Introduction
Antibiotic resistance poses a serious threat to global health as it limits the availability
of effective therapeutics. It has been estimated that 1.2 million deaths were directly
attributable to bacterial antimicrobial resistance globally in 2019 [1]. Resistance to first
line agents, which are the initial antibiotics recommended for treatment, accounted for
more than 70% of deaths in 2019 attributable to antibiotic resistant infections [1].
Antibiotic resistance is particularly severe in developing countries where the availability
of therapeutics is exceptionally limited [2]. Antibiotic resistance poses an economic
burden on healthcare costs due to prolonged length of hospitalization and treatments
[3].
Acinetobacter baumannii routinely demonstrates a multi-drug resistant phenotype
due to its capacity to rapidly develop resistance, thereby leaving extremely limited
treatment options for infections. As a result, the Center for Disease Control designated
carbapenem-resistant (CRAB) A. baumannii as an urgent threat to public health [3]. In a
2019 list ranking global deaths attributable to and associated with antibiotic resistant
bacterial pathogens, CRAB infections ranked 4th [1]. The CDC estimates that in 2017 in
the United States, there were 8500 cases in hospitalized patients, 700 deaths and $281
million attributable healthcare costs due to CRAB infections [3].
A. baumannii is predominantly associated with causing nosocomial infections,
specifically in intensive care units (ICUs) where it is implicated in hospital-acquired and
ventilator-associated pneumonia, bacteremia, wound infections and urinary tract
infections [4]. Risk factors for transmission of CRAB infections in ICUs include
2
mechanical ventilation, intravenous and urinary catheterization and prolonged broadspectrum antimicrobials and immunosuppression is not a risk factor [4].
Antimicrobial susceptibility tests (AST) are in vitro assays used to support the
identification of effective therapeutics. ASTs are used to determine the minimum
inhibitory concentration (MIC) which is defined as the lowest drug concentration that
inhibits microbial growth. Using defined antibiotic breakpoints, MICs are used to
determine whether a microbe is classified as susceptible or resistant to a drug.
Carbapenems are considered first line antibiotics for untreatable Gram-negative
bacterial infections that demonstrate resistance to commonly used antibiotics in the
clinic. However, treatment options become extremely limited once the pathogen
develops resistance to carbapenems. Following the rise in CRAB infections, the
antibiotic colistin remains the last resort option to treat these highly lethal infections.
Colistin treatment has been shown to reduce patient mortality in CRAB infections from
>88% to <38% [5].
Polymyxins are cyclic cationic peptides that lead to cell death by disrupting the
integrity of the bacterial outer membrane [6]. Not long after becoming available in the
1950s, clinical use of polymyxins was minimal due to issues of high nephrotoxicity and
acute kidney injury (AKI) being frequently reported. Despite being highly nephrotoxic,
the emergence of pan-resistant (PDR) infections has prompted the resurgence of
polymyxins use. Clinically, polymyxins have 2 forms: polymyxin E (colistin) and
polymyxin B (PMB). Differing by just 1 amino acid in the peptide ring, colistin is
administered as an inactive prodrug, colistin methanesulfonate, whereas PMB is
administered in its active form [7]. PMB is the preferred alternative for the treatment
3
routine systemic use in invasive infection as it has a decreased risk of polymyxinassociated AKI [8].
A. baumannii can rapidly develop resistance to polymyxins through intrinsic and
acquired mechanisms. In A. baumannii, there are 3 known mechanisms which confer
colistin-resistance. In one such mechanism, chromosomal mutations in the pmrAB two
component signaling pathway/system modify the lipid A component of
lipopolysaccharide (LPS) via the addition of phosphoethanolamine (PetN) groups [9],
[10], [11]. Another mechanism known to confer colistin-resistance involves mutations in
the enzymes involved in lipid A biosynthesis lpxA, lpxC and lpxD, resulting in the
complete loss of LPS [12]. Most recently, the first report of colistin-resistance via the
mobile colistin resistant (mcr-1) gene in A. baumannii was published [13]. This
mechanism confers resistance using a plasmid-mediated phosphoethanolamine
transferase which adds PetN groups to the LPS. Initially, plasmid-mediated colistinresistance had only been observed in Escherichia coli and Klebsiella pneumoniae [14],
[15], [16]. Overall, these mechanisms confer resistance by modifying LPS thereby
reducing its net negative charge and lowering the affinity positively charged colistin.
Groups such as Clinical and Laboratory Standards Institute (CLSI) and European
Committee on Antimicrobial Susceptibility Testing (EUCAST) have developed
antimicrobial susceptibility testing guidelines determining colistin breakpoints. CLSI and
EUCAST recommend that standard broth microdilution be used as the reference
method for colistin MIC testing. The clinical breakpoints for colistin provided for
Acinetobacter species by CLSI defines an intermediate breakpoint of 2 mg/L or lower
4
and a resistant breakpoint of 4 mg/L or higher [17]. EUCAST defines a susceptible
breakpoint as 2 mg/L or lower and a resistant breakpoint as greater than 2 mg/L [17].
Per CLSI recommendations, the standardized protocol for ASTs uses cation
adjusted Mueller-Hinton (MHII) broth as the culturing media. Upon its release in 1941,
MHII served as a reliable and reproducible means of cultivating the majority of
pathogenic bacteria, including particularly fastidious bacteria, encountered in the clinic.
The success of MHII not only relies on the reproducibility of its results when tested at
independent laboratories but also on its ability to facilitate rapid growth of various
bacterial species [18]. As a result, MHII is a highly enriched media designed to promote
the propagation of even the most fastidious bacteria. In other words, MHII was designed
to address the needs of the pathogen, not the host.
Despite its crucial role in identifying effective therapies for the treatment of bacterial
blood infections, MHII fails to reflect the physiological conditions of the host milieu. As
bacterial gene expression is highly regulated by the surrounding environment, culturing
conditions can significantly alter drug interactions and antimicrobial susceptibility.
Consequently, it has been shown that modifying AST culturing conditions to more
physiologically representative conditions results in altered gene expression as well as
altered antibiotic activity. Dorschner et al. showed that bacterial gene expression in
conditions that reflect the host environment are distinctly different from gene expression
of bacteria grown in culture broth [19]. Furthermore, their findings showed that growing
select bacteria in mammalian ionic conditions altered antimicrobial susceptibility
profiles. Ersoy et al. showed that using host-mimicking media, rather than conventional
media, for susceptibility testing more accurately predicted antibiotic efficacy in vivo.
5
Their data showed that using host-mimicking media resulted in MICs that crossed
clinical breakpoint designations. Moreover, septicemia mouse models verified that the
altered MICs accurately predicted the changes in antimicrobial susceptibility profiles
[20].
Similarly, our lab has published work showing that using physiologically relevant
media, RPMI-1640, results in MICs that are different than when conventional MHII is
used [21], [22], [22], [23]. Using RPMI-1640 yielded MICs that demonstrated in vitro
activity against A. baumannii. A. baumannii blood and lung infection mouse models
confirmed that these altered MICs accurately predicted in vivo outcomes. By failing to
detect in vitro activity, conventional AST conditions omitted antibiotics that were highly
efficacious in bacterial clearance in A. baumannii infections. Only the mammalian
culturing media RPMI-1640 better predicted in vivo outcomes in A. baumannii blood and
lung infections.
It has been demonstrated that there are differences in AST results when MICs are
conducted using different culturing media. Different culturing conditions can alter
bacterial gene expression, physiology and ultimately antimicrobial susceptibility. We aim
to investigate mechanistic driver of disparities of polymyxin MICs when AST conditions
are cultured in physiologically similar conditions compared to conventional MHII
conditions. Lastly, we will define the in vivo efficacy of polymyxins in A. baumannii
murine infection models. We hypothesize that standard AST conditions are omitting the
in vivo efficacy of polymyxins against A. baumannii isolates that have been otherwise
classified as pan resistant.
6
Chapter 2
Polymyxins retain in vitro activity and in vivo efficacy against “resistant” Acinetobacter
baumannii strains when tested in physiological conditions
Authors: Jennifer Rubio1
, Jun Yan1
, Sarah Miller1
, Jiaqi Cheng1
, Rachel Li1
, Zac Builta1
,
Kari Aoyagi4
, Mark Fisher4
, Rosemary She2
, Brad Spellberg3
, and Brian Luna1*
Affiliations:
1Department of Molecular Microbiology and Immunology, Keck School of Medicine of
USC, Los Angeles, CA
2Department of Pathology, Keck School of Medicine of USC, Los Angeles, CA
3Los Angeles General Medical Center, Los Angeles, CA
4Department of Pathology, University of Utah, Salt Lake City, UT
7
Chapter 2: Abstract
The emergence of plasmid-mediated resistance threatens the efficacy of polymyxins as
the last line of defense against pan-drug resistant infections. However, we have found
that using MHII, the standard MIC media, results in MIC data that is disconnected from
in vivo treatment outcomes. We found that culturing putative colistin-resistant
Acinetobacter baumannii clinical isolates, as defined by MICs > 2 mg/L in standard MHII
testing conditions, in bicarbonate-containing media reduced MICs to the susceptible
range by preventing colistin-resistance conferring LPS modifications from occurring.
Furthermore, the lower MICs in bicarbonate-containing media accurately predicted in
vivo efficacy of a human simulated dosing strategy of colistin and polymyxin B in a lethal
murine infection model with polymyxin-resistant A. baumannii strains. Thus, current
polymyxin susceptibility testing methods overestimate the contribution of polymyxinresistance conferring mutations and incorrectly predict antibiotic activity in vivo.
Polymyxins may remain a viable therapeutic option against Acinetobacter baumannii
strains heretofore determined to be “pan-resistant”.
8
Chapter 2: Introduction
Colistin is a cationic polymyxin that is considered a drug of last resort against
highly lethal, carbapenem-resistant Gram-negative bacterial pathogens, including CRAB
and Enterobacterales (CRE) [24]. Polymyxin resistance is a serious threat as it
eliminates the last-line antibiotics for the most antibiotic-resistant bacterial isolates.
Newer, expensive antibiotics have decreased, but not eliminated, this threat in high
income countries. In lower and lower-middle income countries, the clinical need for
colistin persists as newer and more costly antibiotics may be unavailable. Until recently,
colistin resistance in A. baumannii had mostly been attributed to chromosomal
mutations affecting colistin’s affinity to LPS. However, there has been significant
concern that the spread of mobile colistin resistance genes would effectively end the
usefulness of polymyxins and therefore lead to widespread infections resistant to all
available antibiotics (so-called PDR infections) [14], [15], [16].
The accuracy of standard susceptibility testing for polymyxins has not been
validated with respect to predicting actual efficacy in an infected host. Valid and
accurate in vitro antimicrobial susceptibility assays are needed to identify which
antibiotic(s) will be effective and may be used for the treatment of patients. To support
these goals, groups such as CLSI and EUCAST have developed guidelines for
antimicrobial susceptibility testing to support the identification of effective antibiotics.
The standard MIC assay requires the use of MHII medium as this medium has been
shown to provide reproducible results at independent laboratories [17], [25], [26].
However, relatively little attention has been devoted to determining what the optimal
medium is for predicting in vivo outcomes [18], [20].
9
Our laboratory showed that in vitro MICs conducted in the physiologically
relevant media, RPMI-1640, better predicted in vivo efficacy of other antibiotics than
when conventional nutrient-rich MHII medium was used for susceptibility testing [21],
[22], [23], [27]. In contrast to MHII, which is an undefined medium containing acid
hydrolysate of casein, beef extract, starch and water, RPMI-1640 is a defined medium
containing 20 amino acids, 11 vitamins, inorganic salts, phenol red and glucose. We
also found that colistin-resistant strains, as defined by a MIC >2 mg/L in MHII
conditions, shifted to appear colistin-susceptible when MIC test media were changed
from MHII to RPMI-1640 [22]. Other labs have independently shown that the
bicarbonate containing media, which is more physiologically relevant as compared to
MHII alone, was also better at predicting in vivo outcomes for some antibiotics [19], [20],
[28], [29], [30].
Here, we show that the use of MHII media for A. baumannii susceptibility testing
results in MIC data that is disconnected from in vivo treatment outcomes, such that
polymyxin-resistance detected in standard susceptibility testing does not result in loss of
efficacy. Additionally, we show that the substitution of MHII by RPMI-1640 culture media
for polymyxin susceptibility enables enhanced accuracy for predicting in vivo efficacy,
mediated by bicarbonate-induced changes to LPS structure in the bacteria.
10
Chapter 2: Results
Sodium bicarbonate sensitizes colistin-resistant A. baumannii to colistin
Having previously established that colistin (COL) MICs in RPMI-1640 media were
typically lower than in MHII media for A. baumannii [21], we evaluated whether MHII
antagonized, or rather RPMI-1640 promoted, COL activity by testing a hybrid media
containing MHII and RPMI-1640 in equal parts. For these studies we used LAC-4 ColR,
a spontaneous colistin-resistant mutant of the clinical isolate LAC-4 WT [31]. Whole
genome sequencing of LAC-4 ColR revealed a single point mutation in pmrA, a gene
known to mediate colistin-resistance in A. baumannii via LPS modifications [11]. When
cultured in MHII, the COL MIC of LAC-4 ColR corresponded with a colistin-resistant
phenotype (MIC = >64 mg/L). However, equivalent COL MICs were observed between
RPMI-1640 and hybrid media conditions for A. baumannii LAC-4 ColR (MIC = 2 mg/L),
indicating that RPMI-1640 promoted COL activity (Table 1). In contrast, there was no
difference in COL MIC for the intrinsically colistin-resistant strain Proteus mirabilis
10195 between MHII and RPMI-1640 culturing conditions (Table 1). There was no
difference in MIC for any of the conditions tested for LAC-4 WT, the colistin-susceptible
strain.
To define which component in RPMI-1640 sensitizes the colistin-resistant A.
baumannii strain LAC-4 ColR to colistin, we added the individual RPMI-1640
components, at the exact concentrations published by the manufacturer, into MHII and
determined the COL MIC for each media formulation. Addition of 25 mM NaHCO3 to
MHII resulted in a 256-fold decrease in the COL MIC which corresponds to a
11
susceptible breakpoint interpretation (MIC = 2 mg/L) (Table 1 and Supplemental Table
1). No other RPMI-1640 components altered MHII MICs of COL.
Table 1 - RPMI-1640 potentiates the activity of colistin.
COL MIC (mg/L)
Species Strain MHII RPMI-1640 MHII:RPMI
(50:50)
MHII + 25 mM
NaHCO3
A. baumannii LAC-4 WT 2 2 2 0.5
LAC-4 ColR >64 2 2 0.5
P. mirabilis 10195 >64 >64 >64 >64
To determine that the COL MIC shift in susceptibility was due to the presence of
bicarbonate and not a consequence of pH change, a feature that has been previously
reported to affect polymyxin activity [19], we determined COL MICs using pH adjusted
RPMI-1640, MHII, and MHII+NaHCO3. For the LAC-4 WT strain, no change in colistinsusceptibility was observed with changes in pH (Fig. 1). Similarly, for the LAC-4 ColR
strain, there was no change in COL MICs in either RPMI-1640 or MHII+NaHCO3 at any
12
of the pHs tested. However, the COL MIC of LAC-4 ColR shifted to a colistinsusceptible phenotype (MIC = 2 mg/L) in MHII at the supraphysiological pH of ≥7.8 (Fig.
1).
Fig. 1 - Effect of pH on colistin MICs among different media. Colistin MICs were
conducted using A. baumannii strains LAC-4 WT (circle) and LAC-4 ColR (triangle)
cultured in pH adjusted media: MHII, RPMI-1640 and MHII+25 mM NaHCO3. Media
were pH adjusted using 0.1 M NaOH and HCl. Each media condition, pH and strain
13
were tested in duplicate. The red dashed line indicates the COL MIC breakpoint (≤2
mg/L.
Bicarbonate effect on polymyxin resistance mechanisms
We tested a panel of clinical isolates with known and unknown colistin-resistance
conferring mutations. Three isolates contained mutations in pmrA, pmrB, or pmrC, 3
isolates contained the mobile colistin resistance gene (mcr-1), and 13 contained
mutations in the genes lpxA, lpxC or lpxD. With the exception of ATCC 17978 (mcr-1)
which maintained a colistin-resistant phenotype, all other colistin-resistant A. baumannii
strains positive for mcr-1 or with mutations in pmrA, pmrB, or pmrC regained
susceptibility to colistin when cultured in MHII containing bicarbonate (MIC ≤2 mg/L)
(Fig. 2). However, LPS-deficient A. baumannii isolates with mutations in lpxA, lpxC, or
lpxD, maintained a colistin-resistant phenotype in both MHII and MHII+NaHCO3
culturing conditions (Fig. 2). Interestingly, the LPS-deficient mutant strains could not be
cultured in RPMI-1640. Thirty-nine of 42 colistin-resistant A. baumannii isolates, all of
which with unknown colistin-resistance conferring mechanisms, regained susceptibility
when cultured in bicarbonate-containing culturing conditions.
Next, we tested the effect of bicarbonate culturing conditions on PMB MICs of A.
baumannii isolates with both known and unknown polymyxin-resistance conferring
mutations. Similar to the COL MIC data, with the exception of ATCC 17978 (mcr-1),
isolates containing mutations in pmrA, pmrB or pmrC or mcr-1+, regained susceptibility
to PMB when cultured in MHII+NaHCO3 (Fig. 2). Thirteen isolates containing mutations
in lpxA, lpxC or lpxD, maintained resistance in bicarbonate conditions. Of the 17 isolates
14
containing unknown PMB-resistance conferring mutations, 16 regained PMB
susceptibility when MHII+NaHCO3 was used as the MIC culturing media.
15
Fig 2 - Polymyxin MICs of ColR isolates in MHII, or MHII supplemented with
sodium bicarbonate. A) Colistin-resistance is defined as MIC >2 mg/L in MHII
conditions. COL MICs were conducted in MHII or MHII+NaHCO3 for A. baumannii (n =
47) colistin-resistant isolates with both known (mcr-1+ in red and pmr in blue) and
unknown (gray) colistin-resistance conferring mutations. B) PMB-resistance is defined
as MIC >2 mg/L in MHII conditions. PMB MICs for colistin-resistant strains of A.
baumannii cultured in MHII were plotted against PMB MICs conducted in MHII + 25 mM
NaHCO3. PMB-resistant strains with known mutations included mutations in the genes
lpxA, lpxC or lpxD (n = 13), mutations in the genes pmrA, pmrB or pmrC (n = 3), or were
positive for the mcr-1 gene (n = 3). PMB-resistance conferring mutations were unknown
for the remaining A. baumannii isolates (n = 16). Dashed lines indicate susceptibility
breakpoint (>2 mg/L). MICs in the top right quadrant indicate values that would be
considered resistant in both media conditions. MICs in the bottom right quadrant
indicate values that would be considered resistant in normal MHII, but susceptible in
bicarbonate containing media.
Colistin efficacy in human blood
The concentration of bicarbonate in RPMI-1640 is physiologically similar to
human blood. Therefore, we performed time kill assays to determine if the increased
activity of COL in bicarbonate containing media would translate to effective killing in
human whole blood. For the LAC-4 WT strain, the addition of 0.1 mg/L colistin to LAC-4
WT cultured in blood resulted in a significant decrease of CFUs at both 1 and 4 hours
post incubation compared to the no drug control group (Mann-Whitney, p = 0.0020, and
p = 0.0020, respectively) (Fig. 3). These time kill results would be predicted by the COL
16
MICs ≤2 mg/L in MHII and MHII+NaHCO3 media. When LAC-4 ColR was cultured in
blood with 0.1 mg/L colistin, there was a significant decrease in CFUs at both 1 and 4
hours post incubation compared to the no drug control (Mann-Whitney, p = 0.0002, p =
0.0079, respectively). However, these time kill results are only consistent with the MICs
determined in MHII+NaHCO3 and are inconsistent with MICs determined using standard
MHII conditions.
17
18
Fig. 3 – Colistin efficacy against LAC-4 in human blood culturing conditions. LAC4 WT (WT) and LAC-4 ColR (ColR) were cultured in human blood containing the
anticoagulant K2 EDTA and treated with colistin at 0.001, 0.01, 0.1, 1 and 10 mg/L.
Colistin efficacy was measured via CFUs/mL at 0, 1 and 4 hours post incubation with A.
baumannii LAC-4 WT and ColR strains. A no treatment control was included at each
time point. Samples were serially diluted using the drop plate method and CFUs were
counted following incubation at 37 °C.
Bicarbonate affects LPS structure
Using MALDI-TOF MS, we characterized changes in LPS structure when clinical
strains of A. baumannii were cultured in MHII or MHII+NaHCO3 (Fig. 4). For LAC-4 WT,
the colistin-susceptible A. baumannii isolate, peaks at an m/z of 1727 and 1909,
corresponding to bis-phosphorylated hepta- and hexa-acylated lipid A species,
respectively, were similarly abundant in both MHII and MHII+NaHCO3 conditions. Next,
we looked at the changes in LPS structure of LAC-4 ColR, a colistin-resistant A.
baumannii isolate, when cultured in MHII or MHII+NaHCO3. The LAC-4 ColR strain had
an additional peak at an m/z of 2032 which is consistent with the expected spectra of
LPS modified with a phosphoethanolamine (PetN, +123 m/z) group. The presence of a
PetN group when LAC-4 ColR was cultured in MHII is consistent with our whole
genome sequencing that revealed a single point mutation in pmrA, a gene shown to
mediate colistin-resistance through addition of PetN groups to the LPS [11]. The relative
abundance of this peak was greatly reduced in MHII+NaHCO3 as compared to MHII, a
pattern that is consistent with the increased susceptibility observed in this same media.
19
Fig. 4 - Mass spectrometry of lipid A isolated from A. baumannii strains LAC-4 WT
and LAC-4 ColR cultured in MHII or MHII+NaHCO3. A. baumannii strains LAC-4 WT
and LAC-4 ColR were cultured to mid-log phase in MHII or MHII+25 mM NaHCO3. Lipid
A was isolated from LAC-4 WT and LAC-4 ColR, followed by MALDI-TOF mass
spectrometry in the negative-ion mode.
To determine if the observed structural changes to LPS affected COL affinity to
the bacterium, we used colistin conjugated to a BoDipy fluorophore (Fig. 5A) or dansyllabeled polymyxin B (Fig. 5B) and measured colistin and polymyxin B binding affinity by
flow cytometry. For these experiments, we used P. mirabilis 10195 to serve as a
negative control due to its intrinsic resistance to colistin. Of all the strains, PM 10195
20
had the lowest COL affinity, indicated by its position on the far left side of the axis, in all
media conditions when stained with either BoDipy-labeled colistin (Fig. 5A) or Dansyllabeled polymyxin B (Fig. 5B). The low binding affinity of COL to PM 10195 in all media
conditions is consistent with the high COL MIC observed when PM10195 is cultured in
either MHII or bicarbonate conditions. In MHII conditions, there is a reduced COL affinity
of BoDipy-labeled colistin for LAC-4 ColR when compared to LAC-4 WT. This reduction
in COL affinity for LAC-4 ColR is consistent with the high COL MIC observed in MHII
culturing conditions. When LAC-4 ColR is cultured in bicarbonate conditions, there is a
notable shift to the right, indicating higher COL binding affinity of both BoDipy-labeled
COL and Dansyl-labeled PMB. Most notably, culturing LAC-4 ColR in bicarbonate
conditions results in a COL binding affinity profile that is equivalent to that of colistinsusceptible strain, LAC-4 WT. All unstained samples, which served as a baseline of
reference, were positioned on the far left side of the x-axis, indicating low background
signal (Fig. 5A and B).
21
Fig. 5- Affinity of fluorescently labeled colistin to bacteria. A) A. baumannii LAC-4
WT or LAC-4 ColR were cultured to mid-log phase in MHII or RPMI-1640. Following
incubation with BoDipy-labeled colistin (0.1 mg/L), fluorescence was measured by flow
cytometry. Controls included Proteus mirabilis 10195 and unstained samples of each
bacterial strain. B) A. baumannii LAC-4 WT or LAC-4 ColR strains were cultured to midlog phase in MHII, RPMI-1640 or MHII supplemented with 25 mM sodium bicarbonate.
Bacteria was incubated with Dansyl-labeled polymyxin B (30 mg/L) and fluorescence
was measured by flow cytometry.
pmrCAB expression in A. baumannii
After examining the effect of culturing conditions on LPS structure, affinity, and
charge, we next looked at the effect on expression of genes pmrA, pmrB, and pmrC.
Using qPCR, we measured changes in gene expression in MHII+NaHCO3 relative to
gene expression in MHII, for the 3 colistin-resistant strains: C8, C14, and LAC-4 ColR
and the colistin-susceptible strains ATCC 17978 and LAC-4 WT. The colistin-resistant
22
strains C8 and LAC-4 ColR had median log2 fold changes of 0.35 and 0.465 for pmrC
expression, respectively, compared to the colistin-susceptible strain ATCC 17978
(Mann-Whitney, p = 0.003, p = 0.028, respectively). The LAC-4 ColR strain also had a
median log2 fold change of 0.515 decrease in pmrA expression as compared to ATCC
17978 (Mann-Whitney, p = 0.006). Finally, compared to the colistin-susceptible strain
ATCC 17978, C8 had a median log2 fold decrease of 0.69 and 0.56 in expression for
both pmrA and pmrB (Mann-Whitney, p = 0.043, p = 0.013, respectively) (Fig. 6).
23
Fig. 6 - Expression of genes involved in conferring colistin-resistance in
Acinetobacter baumannii. A. baumannii laboratory strain ATCC 17978 and the clinical
isolates LAC-4 WT, LAC-4 ColR, C8, and C14 were cultured to mid-log phase in MHII or
MHII supplemented with 25mM NaHCO3. RNA was collected and RT-qPCR was carried
out to calculate changes in expression of pmrC, pmrA and pmrB. Fold change was
calculated following normalization to 16S ribosomal RNA gene and relative to each
strain in MHII conditions.
Polymyxin efficacy in vivo
To determine whether COL MICs cultured in RPMI-1640 accurately predict in
vivo therapeutic outcomes, C3H mice were intravenously infected with A. baumannii
strains LAC-4 WT or LAC-4 ColR (Fig. 7). For mice infected with either strain, treatment
with colistin had markedly improved survival as compared to the PBS control group
(WT:Log-Rank, p=<0.0001; ColR:Log-Rank, p=0.0126) (Fig. 7B). In LAC-4 WT infected
mice, treatment with colistin led to a significant reduction in CFU compared to the PBS
control group (Mann-Whitney, p = 0.000438) (Fig. 7A). Notably, colistin treatment of
LAC-4 ColR infected mice resulted in a significant reduction in CFU compared to the
PBS control group (Mann-Whitney, p = 0.000546) (Fig. 7A). Five mice infected with
LAC-4 WT succumbed to infection prior to the collection time point and so their blood
could not be obtained. Because the mice succumbed to the blood infection, we
assumed that the non-surviving mice had a blood CFU burden that was greater than the
highest CFU burden of the surviving PBS mice. Therefore, we imputed their CFUs
(green) as equivalent to the burden measured in surviving PBS mice (Fig. 7A).
24
Fig. 7 - Efficacy of colistin treatment in vivo. A) C3H mice (n=12 per group) were
infected with 2.1E7 CFUs of LAC-4 ColR or 8.3E6 CFUs of LAC-4 WT. Mice were
treated subcutaneously once daily with a humanized dose of colistin (21 mg/kg/day).
Blood was collected from LAC-4 WT and LAC-4 ColR-infected mice at 14 hours post
infection to determine blood CFU/mL. Baseline CFUs were determined 2 hours post
infection. For LAC-4 WT infected mice, 5 PBS mice succumbed to the infection before
25
the collection time point and so their CFUs (indicated in green) are equivalent to the
counts of the high end of surviving PBS mice. B) C3H mice (n=10 per group) were
infected with 9.7E7 CFUs of LAC-4 ColR or 1E7 CFUs of LAC-4 WT. Mice were treated
twice daily with a humanized dose of colistin (21 mg/kg/day) and monitored for survival.
To determine whether PMB MICs cultured in bicarbonate-containing media
accurately predicted in vivo treatment outcomes, we selected 8 PMB-resistant A.
baumannii isolates, defined as resistant in MHII culturing conditions, to be used in a
blood infection model (Table 2). For mice infected with the PMB-susceptible strains
HUMC1 and LAC-4 WT, PMB-treatment led to significant blood CFU reduction
compared to the PBS group (Mann-Whitney, p = 0.001, p = 0.000504, respectively). For
mice infected with PMB-resistant strains, as defined by standard MIC testing, LAC-4
ColR, AR-0307, AR-0308, 1112707, 1184244, and 1124614, PMB-treatment led to a
significant reduction in CFUs compared to the PBS control group (Mann-Whitney, p =
0.0000324, p = 0.044, p = 0.023, p = 0.000486, p = 0.002, and p = 0.000504,
respectively) (Fig 8A). There was no difference in blood CFUs between PMB and PBS
groups infected with the PMB resistant isolate 1174913. For isolate 1180013, no PBS
mice survived to the collection time point and so we were unable to collect any blood to
quantify CFUs. For HUMC1, 1112707, and 1174913 not all mice survived to the
collection time point and so their CFUs were imputed (light gray) as being equivalent to
the highest CFU of surviving PBS mice. Paralleling the CFU data, PMB-treatment in
mice infected with LAC-4 ColR, AR-0307, AR-0308, 1112707, and 1184244 led to
significantly improved survival compared to the PBS group (Log-Rank, p = 0.005, p
=0.005, p = 0.011, p < 0.001, p < 0.0001, respectively) (Fig 8B). There was no
26
difference in survival outcome between PMB and PBS groups for mice infected with
1124614, 1180013 or 1174913. PMB-treatment led to significantly improved survival in
mice infected with the PMB-susceptible strains HUMC1 and LAC-4 WT (Log-Rank, p
<0.0001, p <0.001, respectively) (Fig 8B).
Table 2 - Summary of polymyxin MICs for strains used in the in vivo studies.
Strain Mutation COL MIC
(mg/L) in
MHII
COL MIC
(mg/L) in
MHII +
NaHCO3
PMB MIC
(mg/L) in
MHII
PMB MIC
(mg/L) in
MHII +
NaHCO3
HUMC1 - 0.5 1 <0.125 <0.125
LAC-4 WT - 0.5 2 <0.125 <0.125
LAC-4 ColR pmrA >64 2 8 <0.125
AR-0307 Unknown >64 1 8 0.25
AR-0308 Unknown 8 0.5 4 <0.125
1112707 Unknown 16 0.5 4 <0.125
1184244 Unknown 64 1 16 <0.125
27
1124614 Unknown 32 0.5 8 <0.125
1180013 Unknown >64 1 8 <0.125
1174913 Unknown 32 1 8 0.5
28
Fig. 8 - Efficacy of PMB treatment in vivo. A) Female and male C3H mice (n = 5 per
group) were infected with the LD100 of PMB-resistant isolates of A. baumannii. Mice
were treated subcutaneously once with a humanized dose of PMB (11 mg/kg/day).
Blood was collected from infected mice at 18 hours post infection to determine blood
CFU/mL. Baseline CFUs were determined 2 hours post infection. For isolates 1112707
and HUMC1, 4 male PBS and 3 male PBS mice succumbed to the infection before the
collection time point and so their CFUs (indicated in light gray) are equivalent to the
counts of the high end of surviving PBS mice. For 1174913, 2 male PMB mice and 1
male PBS mouse also succumbed before the collection time point and so their CFUs
were also imputed. B) Female and male C3H mice (n = 5 per group) were infected with
the LD100 of PMB-resistant isolates of A. baumannii. Mice were treated subcutaneously
twice daily with a humanized dose of PMB (21 mg/kg/day) and monitored for survival.
29
Chapter 2: Discussion
Colistin remains the “last-line-of-defense” for the treatment of highly lethal
infections caused by CRAB and CRE. However, as the use of colistin has increased, so
has the report of colistin-resistant strains. Here, we show conventional MHII media used
in standardized antimicrobial susceptibility assays is disconnected from the in vitro
activity and in vivo outcomes for polymyxins. This work suggests that colistin may be
effective in vivo despite standard susceptibility testing predicting resistance for A.
baumannii clinical isolates.
In A. baumannii, colistin-resistance has been shown to be conferred via
chromosomal mutations in the pmrAB two component system which result in the
addition of phosphoethanolamine (PetN) modifications to the LPS. Colistin-resistance
can also be conferred via chromosomal mutations in enzymes involved in lipid A
biosynthesis which results in the complete loss of LPS. Most recently, colistin-resistance
has been attributed to a mobile colistin resistance gene (mcr-1) which modifies the LPS
with PetN groups using a plasmid-mediated phosphoethanolamine transferase. All the
aforementioned mechanisms confer colistin resistance by modifying colistin’s target
molecule, LPS, thereby reducing the affinity of positively-charged colistin. With regards
to clinical isolates, colistin-resistance due to mutations in pmrA, pmrB or pmrC are more
commonly identified than mutations in lpxA, lpxC or lpxD [32]. Notably, only recently
was there the first report of plasmid-mediated mcr-1 colistin-resistance in an A.
baumannii clinical isolate [13].
30
Our results show that culturing colistin-resistant bacteria in MHII promotes the
addition of PetN modifications to LPS that disrupt its interaction with colistin. However,
culturing colistin-resistant bacteria, defined as resistant in MHII conditions, in the
presence of physiologically normal amounts of bicarbonate decreases the gene
expression of canonical colistin-resistance conferring genes and the presence of PetN
modifications on LPS. When we tested colistin-resistant isolates containing mutations in
the enzymes involved in lipid A biosynthesis we found that these LPS-deficient A.
baumannii strains maintained COL and PMB-resistance in physiological bicarbonate
conditions. Notably, LPS-deficient A. baumannii strains have been shown to
demonstrate attenuated virulence in vivo [33]. Overall, our data is consistent with the
current understanding of the mechanism of action for polymyxins. However, the use of
bicarbonate-containing media allows for a reduced contribution of mutations that confer
modifications to LPS, consistent with the observed treatment outcomes in vivo.
Additionally, we showed that bicarbonate itself, and not pH, is responsible for the
observed change in the colistin MIC of LAC-4 ColR. In media that did not contain
bicarbonate, only non-physiologically relevant alkaline conditions affected MICs, and
thus changes in pH alone cannot explain the observed in vivo activity.
Using bicarbonate-containing media in MICs, which is more physiologically
similar to the in vivo blood infection environment, revealed increased activity of COL
against A. baumannii. To determine if this increased COL activity translated to effective
killing in human blood, we performed time kill assays using human whole blood. Our
results were only consistent with the MICs determined in bicarbonate-containing
culturing conditions and not MICs using conventional MHII media, suggesting increased
31
COL activity in the presence of bicarbonate. Due to issues with blood coagulation in the
time kill assays, we were unable to achieve interpretable data necessary to make a
direct comparison to the COL MICs at 24 hours post infection. Despite trying several
different anticoagulants (Sodium Citrate, ACD, Potassium Oxalate/Sodium Fluoride,
Sodium EDTA, K3 EDTA and mechanical defibrination), we were limited to interpretable
data under 4 hours post infection. This limitation provides no opportunity for rebound to
occur during this reduced format. Additionally, though there is a notable 2-log reduction
in viability in the untreated LAC-4 ColR control at 4 hours post infection, the addition of
0.001 mg/L COL at the same time point results in a greater decrease in CFUs
compared to the untreated control (Mann-Whitney, p = 0.0273).
In mice, colistin treatment resulted in a >2 log reduction of blood bacterial CFUs
for the colistin-resistant A. baumannii isolate LAC-4 ColR at 14 hours post infection.
Despite equivalent survival rates in colistin-treated groups, the reduction in CFUs for
mice infected with LAC-4 ColR was modest compared to the CFU reduction in LAC-4
WT-infected mice. To explain the difference in in vivo outcome between the colistinresistant and colistin-susceptible strains, multiple factors must be considered. Firstly, it
has been shown in the literature that mice infected with 2 different strains, albeit with
equivalent MICs, can result in dissimilar in vivo outcomes following treatment with
antibiotics [34], [35], [36], [37], [38]. Secondly, because the colistin-resistant strain is
less virulent than the colistin-susceptible strain, the in vivo inoculum for LAC-4 ColR
(2.1e7 CFU) requires 2.5X the amount of bacteria than the LAC-4 WT strain (8.3e6
CFU) in order to achieve a lethal infection. As a result, the 2.5-fold decrease in the ratio
32
of antibiotic:bacteria may contribute to the difference in CFU reductions observed in
mice infected with colistin-resistant and colistin-susceptible strains.
We found that using bicarbonate-containing media for PMB MIC susceptibility
testing also revealed in vivo activity against A. baumannii isolates defined as polymyxinresistant in conventional MHII MIC conditions. For the PMB efficacy in vivo studies, 5 of
6 strains with >1-log median CFU reduction at 18 hours post infection resulted in
survival improvement (A. baumannii strains LAC-4 ColR, AR-0307, AR-0308, 1112707,
1184244). These results support that our hypothesis is generally appropriate and is not
restricted to only the LAC-4 ColR strain. Despite a greater than 2-log median CFU
reduction in PMB-treated mice compared to the PBS control, PMB-treatment in mice
infected with A. baumannii 1124614 did not result in survival. These results highlight
that microbiology endpoints do not always correlate with survival and therefore it is
critical to evaluate both endpoints. Although PMB susceptibility tests in bicarbonatecontaining culturing conditions indicated PMB-susceptibility, PMB-treatment did not
result in reduced CFUs or improved survival for mice infected with A. baumannii
1180013 or 1174913. Further studies are needed to explain these discrepancies.
In summary, we have shown that media containing bicarbonate, which are more
physiologically representative of the host environment, accurately predicted the in vivo
efficacy of polymyxins in A. baumannii blood infection. Notably, using conventional MHII
culturing conditions for antimicrobial susceptibility testing failed to identify the in vivo
activity of polymyxins against A. baumannii blood infections in mice. Although further
work is necessary to explain strain-to-strain variability of PMB efficacy, these results
show that modifying culturing conditions to better reflect the host environment enhances
33
the predictive power of antimicrobial susceptibility tests. Moreover, these results
indicate that we may not be as far along as feared towards experiencing widespread
PDR A. baumannii infections across the globe. More fundamentally, these results
underscore the necessity to use physiologically representative media to enable more
accurate prediction of in vivo efficacy for some antibiotics, and to better identify
promising solutions to the antibiotic-resistance crisis.
34
Chapter 2: Materials and Methods
Ethics statement
All animal work was conducted following approval by the Institutional Animal
Care and Use Committee (IACUC protocol 20882 and 21467) at the University of
Southern California, in compliance with the recommendations in the Guide for the Care
and Use of Laboratory Animals of the National Institutes of Health.
Bacteria Culture
Bacteria used in the study are listed in Table S8. Working solutions of bacteria
were prepared using frozen stocks of A. baumannii strains as previously published, [39],
by inoculating a fresh overnight culture in TSB and incubating at 37 °C/200 rpm, or by
streaking on fresh TSA plates and incubating at 37 °C. The overnight broth culture was
diluted 1:100 and then subcultured in MHII, RPMI-1640, or MHII supplemented with 25
mM sodium bicarbonate at 37 °C/200 rpm for 3 hours and then adjusted until the culture
reached an OD600 of 0.5. For MICs and time kill assays, bacterial cultures were adjusted
to a 0.5 McFarland standard via the direct colony suspension method.
MIC Protocol
For A. baumannii, a MIC of ≤2 mg/L corresponds to a EUCAST susceptible and a
CLSI intermediate (CLSI does not have a susceptible breakpoint for colistin) breakpoint
interpretation [26], [31]. Both EUCAST and CLSI agree that ≥2 mg/L corresponds to a
resistant breakpoint; we will use the EUCAST definitions of susceptible and resistant
throughout the manuscript. Unless otherwise indicated, the standard broth microdilution
35
method following CLSI guidelines was used to determine MICs [17]. The media used for
the MIC assays performed in this study were MHII (BD Biosciences, catalog no. 90000-
602), RPMI-1640 (Gibco, catalog no. 11875119), and MHII supplemented with sodium
bicarbonate (Sigma-Aldrich, catalog no. S5761) (at a final concentration of 25mM
sodium bicarbonate, NaHCO3). For the RPMI-1640 components colistin MICs, each
individual nutrient component was separately added to standard MHII at the final
concentration reported by the manufacturer (Table S1). Colistin (Sigma, catalog no.
C4461) and Polymyxin B (Xellia Pharmaceuticals) were diluted in deionized water to
make a fresh stock before each experiment.
Briefly, 100 µL of media was added to the wells of a 96-well plate in columns 2-
10. Column 11 served as a positive growth control and contained only bacteria and
media. Column 12 served as the sterility control and contained only culture media
without bacteria. Next, 200 µl of a 2X antibiotic working solution was added to the wells
in column 1. Two-fold serial dilutions of the antibiotic were performed through column
10. Next, 100 µl of a 1×106 CFU/mL working solution of bacteria were added to each of
the wells in columns 1-11. The inoculum concentration was confirmed by plating serial
dilutions on TSA plates. MIC plates were incubated at 35±2˚C and results were
recorded at 24 hours. MIC experiments were done in duplicate.
Characterization of lipid A species by MALDI
Lipid A was isolated from LAC-4 WT and LAC-4 ColR, followed by MALDI-TOF
mass spectrometry in the negative-ion mode as previously described [40].
36
Fluorescent Labeling of Polymyxins and FLOW Cytometry Binding Assays
Fluorescently labeled colistin was prepared as described by the manufacturer
(Thermo Fisher Scientific). Briefly, colistin was labeled with BoDipy FL NHS Ester
(Thermo Fisher Scientific, catalog no. D2184). 100 µl of BoDipy NHS ester compound
(10 mg/mL in dimethyl sulfoxide, DMSO), 250 μl of colistin (10 mg/mL), and 650 μl
sodium bicarbonate (0.2 M, pH 8.5) were incubated at 37 °C for 2 hours. A Float-ALyser G2 dialysis device (Spectrum, catalog no. 08-607-022) with a molecular weight
cutoff of 0.5 kDa was used to remove excess fluorophores not bound to colistin. Dialysis
was carried out overnight in 4 ˚C sterile distilled water, which was changed four times.
From an overnight culture, bacteria were subcultured until mid-log phase in MHII,
RPMI-1640, or MHII+25mM NaHCO3. Once adjusted to an OD600 of 0.5, 55 μl of
bacteria was added to a 96-well flat-bottom 0.22 µm filter plate and incubated with either
BoDipy-labeled colistin (0.1 mg/L) for 30 minutes at 37 °C or Dansyl-labeled polymyxin
B (Sigma-Aldrich, catalog no. SBR00029) (30 mg/L) for 30 minutes at room
temperature. For BoDipy-labeled colistin experiments, labeled BoDipy was diluted to a
1% solution using unlabeled colistin (0.01 mg/mL). The cells were then washed 3 times
with respective media to remove any excess colistin not bound to the bacteria. The
amount of colistin attached to the bacteria was determined by flow cytometry by
measuring the fluorescence of 200 µl samples (10000 events). A BD Accuri C6 Plus
flow cytometer was used to analyze the BoDipy-labeled colistin samples and a BD LSR
II was used to analyze the Dansyl-labeled polymyxin B samples. Controls included
Proteus mirabilis 10195 and unstained samples of each bacterial strain. All data was
analyzed using FlowJo version 10.8.
37
Quantitative Real-Time PCR
A. baumannii strains ATCC 17978, LAC-4 WT, LAC-4 ColR, C8, and C14 were grown to
mid-logarithmic phase in MHII or MHII supplemented with 25mM NaHCO3. Cells were
harvested at an OD600 between 0.5 and 0.6. Total RNA was extracted using a Qiagen
Rneasy Protect Mini kit (Qiagen, catalog no. 74524). Samples were treated with Qiagen
DNase to remove any contaminating genomic DNA. cDNA was generated using
LunaScript RT SuperMix Kit. cDNA was diluted 1:10 and 1 µL was used as template for
each reaction. RT-qPCR was carried out using Luna Universal qPCR Master Mix and
the primers listed in Table S9. RT-qPCR protocol consisted of 40 cycles of 15 seconds
of denaturation at 95 ˚C, 30 seconds of annealing at 55 ˚C, and 30 seconds of
elongation at 60 ˚C. Experiments were repeated with 3 biological replicates, each tested
in duplicate. Fold change was calculated following normalization to 16S ribosomal RNA
gene and relative to ATCC 17978 WT strain (or relative to each strain in MHII
conditions).
Human blood time kill assay
The bacterial inoculum was prepared as described in bacteria culture methods.
Time kill assays were performed in round bottom 96-well plates. Bacteria was cultured
in human whole blood containing K2 EDTA as the anticoagulant (Innovative Research,
catalog no. IGMSCD1WBK2E25ML). Colistin was added to duplicate wells of either
LAC-4 WT or LAC-4 ColR at 10, 1, 0.1, 0.01 and 0.001 mg/L. Plates were incubated at
37 ˚C and samples were collected at 0, 1 and 4 hours post incubation. The bacterial
38
burden at each timepoint was determined by plating serial dilutions on TSA plates and
incubating overnight at 37 ˚C.
Intravenous (IV) infection
A. baumannii LAC-4 WT, LAC-4 ColR, and HUMC1 frozen stocks were prepared
as described in previous work [31], [39]. Frozen stocks of bacteria were thawed and
diluted in PBS to adjust the bacterial density as needed for infection. The inoculum for
all other infecting strains was freshly prepared from subcultures of bacteria cultured to
mid-log phase. Male C3HeB/FeJ mice, 8 to 12 weeks old, were infected with the LD100
of LAC-4 WT or LAC-4 ColR via tail vein injection. For the colistin LAC-4 ColR CFU
experiment, and all PMB experiments, male C3HeB/FeJ mice 7-8 weeks old and female
C3HeB/FeJ mice 9-10 weeks old were infected with the LD100 via tail vein injections.
Inoculum bacterial density was confirmed by plating serial dilutions on TSA plates and
incubating overnight at 37 °C.
Antibiotic Treatments
Human equivalent dosing of polymyxin B was done as previously described [41].
Briefly, colistin sulfate salt (Sigma-Aldrich C4461) or polymyxin B (Xellia
Pharmaceuticals) was diluted in deionized water to make a stock concentration of 10
mg/mL before each experiment. Treatment was initiated at 2 h post infection and mice
were treated subcutaneously twice daily with 11 mg/kg and 10 mg/kg at 0 and 12 h
daily. For CFU experiments, mice were administered only a single drug treatment to
avoid excess drug carryover when plating serial dilutions.
39
Blood bacterial burden
Mice assigned to the CFU collection group were collected at 2 and 14 hours after
infection, anesthetized intraperitoneally with 150 µL of a 30 mg/mL ketamine and 3
mg/mL xylazine solution and heparinized intraperitoneally with 20-30 µL (200-300 units
USP). A terminal cardiac puncture was performed to collect at least 500 µL of blood.
Serial dilutions of the blood samples were plated on TSA plates before incubation at
37°C overnight. Colonies were counted after 24 hours and the CFUs were calculated.
Statistics
Gene expression and bacterial burden were compared using the Mann-Whitney
test. For survival studies, time to death was compared using the Log Rank test. Pvalues < 0.05 were considered significant.
Acknowledgements
We thank Drs. Robert E.W. Hancock and Yohei Doi for sharing bacterial strains.
We thank Cristina Miglis, Mayurika Ghosh, Jalal Sheikh, Kerian Grande Roche, Ramya
Gopinath, Thushi Amini and James Byrne for their contributions. The views expressed
in this publication are those of the authors and do not necessarily represent the official
views nor policies of the FDA.
Funding
This work was supported by National Institute of Allergy and Infectious Diseases
(NIAID) at the National Institutes of Health (NIH) grants 2RO1 AI130060 (to B.S.),
40
NIAID/NIH grant RO1 AI139052, RO1 AI179046, and FDA Contract 75F40122C00138
(to B.M.L.).
Data and materials availability
All data are available in the main text or the supplementary materials. The
genome sequence for the LAC-4 WT and LAC-4 ColR strains are available at NCBI,
accession number: NZ_CP007712, PRJNA1023962, respectively.
41
Chapter 2: Supplementary Information
Whole genome sequencing
The genome sequences of LAC-4 WT and LAC-4 ColR were determined using
Illumina sequencing. Microbial DNA was extracted using Zymo Quick-DNA Bacterial
miniprep kit (Genesee Scientific, catalog no. 11-321). CLC Genomics Workbench was
used to perform variant analysis. Raw sequence data from the ColR strain was aligned
to the LAC-4 WT strain (Accession number: NZ_CP007712) published in NCBI.
Supplemental Fig. 1 - Whole genome sequencing of LAC-4 ColR. Genome
sequences of LAC-4 WT and LAC-4 ColR were determined using Illumina sequencing,
and raw sequence data from LAC-4 ColR was aligned to the LAC-4 WT strain published
in NCBI. Whole genome sequencing revealed a single variation between WT and ColR:
a single point mutation in pmrA in LAC-4 ColR.
42
Supplemental Table 1 - Colistin MICs in MHII supplemented with RPMI-1640
nutrients. The row shaded in gray indicates the only media component that was found
to affect the LAC-4 ColR COL MIC.
COL MIC (mg/L)
Media Condition LAC-4 WT LAC-4 ColR
MHII 0.5 >64
43
RPMI-1640 2 2
Amino acids
MHII + 0.133mM Glycine 0.25 >64
MHII + 1.15mM L-Arginine 0.25 64
MHII + 0.379mM L-Asparagine 0.25 >64
MHII + 0.150mM L-Aspartic acid 0.25 >64
MHII + 0.208mM L-Cysteine2HCl
0.25 >64
MHII + 0.136mM L-Glutamic
acid
0.25 >64
MHII + 2.05mM L-Glutamine 0.5 32
MHII + 0.097mM L-Histidine 0.5 64
MHII + 0.153mM LHydroxyproline
0.5 32
44
MHII + 0.382mM L-Isoleucine 8 64
MHII + 0.382mM L-Leucine 0.25 >64
MHII + 0.219mM L-Lysine
hydrochloride
0.5 32
MHII + 0.101mM L-Methionine 0.25 >64
MHII + 0.091mM LPhenylalanine
0.25 >64
MHII + 0.174mM L-Proline 0.25 >64
MHII + 0.286mM L-Serine 0.25 >64
MHII + 0.168mM L-Threonine 0.25 >64
MHII + 0.025mM L-Tryptophan 0.25 >64
MHII + 0.111mM L-Tyrosine
disodium salt dihydrate
0.25 >64
MHII + 0.171mM L-Valine 0.125 >64
Vitamins
45
MHII + 8.20E-04mM D-Biotin 0.25 >64
MHII + 0.021mM Choline
chloride
0.125 >64
MHII + 5.2E-4mM Calcium-Dpantothenate
0.125 64
MHII + 2.27E-03mM Folic acid 4 32
MHII + 8.20E-03mM
Niacinamide
0.25 >64
MHII + 7.30E-03mM Paraaminobenzoic acid
0.125 >64
MHII + 4.85E-03mM Pyridoxine
hydrochloride
0.25 >64
MHII + 5.32E-04mM Riboflavin 0.25 >64
MHII + 2.97E-03mM Thiamine
hydrochloride
0.125 >64
MHII + 3.69E-06mM Vitamin
B12
0.25 >64
46
MHII + 0.194mM i-Inositol 0.25 32
Inorganic salts
MHII + 0.424mM Calcium nitrate
tetrahydrate
0.5 >64
MHII + 0.41mM Magnesium
sulfate
0.125 >64
MHII + 5.33mM Potassium
chloride
0.25 >64
MHII + 25mM Sodium
bicarbonate
0.5 0.5
MHII + 103mM Sodium chloride 0.5 >64
MHII + 5.63mM Sodium
phosphate dibasic anhydrous
0.125 >64
Other components
MHII + 11.1mM D-Glucose 0.25 >64
MHII + 3.26E-03mM Glutathione
(reduced)
0.125 >64
47
MHII + 0.013mM Phenol red 0.5 64
Supplemental Table 2 - Polymyxin MICs of ColR isolates in MHII or MHII
supplemented with sodium bicarbonate
Strain Mutation COL MIC
(mg/L) in
MHII
COL MIC
(mg/L) in
MHII +
NaHCO3
PMB
MIC
(mg/L)
in MHII
PMB
MIC
(mg/L)
in MHII +
NaHCO3
LAC-4 ColR pmrA >64 2 8 <0.125
C8 pmrB 64 1 64 1
C14 pmrB, pmrC 32 2 8 1
D773 mcr-1 mcr-1 >64 1 4 0.5
SM1536 mcr1
mcr-1 >64 1 8 2
17978 mcr-1 mcr-1 64 8 8 8
19606R lpxA >64 >64 >64 >64
48
AL1833 lpxC >64 16 64 64
AL1834 lpxC >64 32 64 64
AL1842 lpxC >64 16 >64 >64
AL1843 lpxC >64 >64 >64 >64
AL1844 lpxA >64 >64 >64 >64
AL1845 lpxA >64 >64 >64 >64
AL1846 lpxA >64 >64 >64 >64
AL1847 lpxA >64 >64 >64 >64
AL1848 lpxA >64 >64 >64 >64
AL1849 lpxA >64 >64 >64 >64
AL1851 lpxA >64 >64 >64 >64
AL1852 lpxD >64 >64 >64 >64
49
ARUP_A1 Unknown 64 0.25 4 0.125
ARUP_A6 Unknown >64 0.25 32 0.125
ARUP_A8 Unknown 64 8 8 8
ARUP_A10 Unknown >64 0.5 16 0.125
ARUP_A11 Unknown >64 0.25 16 0.25
ARUP_A13 Unknown >64 0.25 32 0.125
ARUP_A22 Unknown >64 0.25 32 0.125
ARUP_A23 Unknown >64 2 64 0.5
ARUP_A28 Unknown 16 0.25 64 0.125
ARUP_A30 Unknown >64 0.25 4 0.125
AR-0307 Unknown >64 1 8 0.25
AR-0308 Unknown 8 0.5 4 <0.125
50
1112707 Unknown 16 0.5 4 <0.125
1184244 Unknown 64 1 16 <0.125
1124614 Unknown 32 0.5 8 <0.125
1180013 Unknown >64 1 8 <0.125
1174913 Unknown 32 1 8 0.5
1064048 Unknown 32 2 - -
1071864 Unknown 64 4 - -
1083037 Unknown 64 1 - -
1083246 Unknown >64 1 - -
1083383 Unknown >64 1 - -
1099731 Unknown >64 1 - -
1105437 Unknown >64 0.5 - -
51
1125675 Unknown >64 1 - -
1127911 Unknown >64 1 - -
1172798 Unknown 8 2 - -
1174945 Unknown >64 2 - -
1178685 Unknown 16 1 - -
1180949 Unknown 64 4 - -
1181046 Unknown >64 1 - -
1185713 Unknown 64 1 - -
1188882 Unknown 64 1 - -
ARUP_A3 Unknown 16 0.25 - -
ARUP_A4 Unknown 4 0.25 - -
ARUP_A5 Unknown 16 0.125 - -
52
ARUP_A9 Unknown 8 0.5 - -
ARUP_A14 Unknown 16 1 - -
ARUP_A16 Unknown 8 0.5 - -
ARUP_A18 Unknown 8 1 - -
ARUP_A19 Unknown 16 0.25 - -
ARUP_A20 Unknown 32 0.5 - -
ARUP_A26 Unknown 64 0.25 - -
ARUP_A29 Unknown >64 0.13 - -
“ - ” indicates not tested
Supplemental Table 3 - Affinity of BoDipy-labeled colistin to ColR bacteria
Strain Condition MFI (mean fluorescence intensity) of
BoDipy
MHII RPMI
AB LAC-4 ColR Unstained 247 21
53
AB LAC-4 ColR Stained 938 1225
AB LAC-4 WT Unstained 121 23.8
AB LAC-4 WT Stained 10556 1230
PM 10195 Unstained 17.1 9.29
PM 10195 Stained 30.1 149
Supplemental Table 4 - Affinity of Dansyl-labeled polymyxin B to ColR bacteria
Strain Condition MFI (mean fluorescence intensity)
MHII RPMI MHII +
NaHCO3
AB LAC-4 WT Unstained 16.1 15.3 16
PM 10195 Unstained 12.6 11.2 13.3
AB LAC-4
ColR
Stained 1189 3782 2067
54
AB LAC-4 WT Stained 2403 2296 2283
PM 10195 Stained 175 483 201
Supplemental Table 5 - Summary of LD100 virulence screening
Strain
Female Male
LD100
(CFU/mous
e)
Sub-LD100
(CFU/mous
e)
CFU/m
L
LD100
(CFU/mous
e)
Sub-LD100
(CFU/mous
e)
CFU/m
L
AR0307 3.07E+08 2.65E+08 6.14E+08 4.23E+08 3.58E+08 8.46E+08
AR0308 4.83E+08 - 9.66E+08 4.83E+08 1.30E+08 9.66E+08
111270
7 6.83E+07 - 2.73E+08 1.25E+08 3.83E+07 5.00E+08
118424
4 6.73E+07 - 2.69E+08 5.70E+07 1.77E+07 2.28E+08
55
112461
4 5.92E+07 - 2.37E+08 5.92E+07 2.08E+07 2.37E+08
118001
3 3.20E+08 - 6.40E+08 3.20E+08 1.40E+08 6.40E+08
117491
3 5.00E+08 - 1.00E+09 5.00E+08 2.69E+08 1.00E+09
Supplemental Table 6 - Efficacy of colistin treatment in vivo
Strain Mouse Treatment Gender Time point
(hours)
CFU/mL
LAC-4 WT 1 Baseline Male 2 2.90E+06
2 Baseline Male 2 5.75E+06
1 PBS Male 14 2.30E+05
2 PBS Male 14 2.45E+05
3 PBS Male 14 4.00E+04
56
4 PBS Male 14 2.90E+06
5 PBS Male 14 5.75E+06
6 PBS Male 14 5.75E+06*
7 PBS Male 14 5.75E+06*
8 PBS Male 14 5.75E+06*
9 PBS Male 14 5.75E+06*
10 PBS Male 14 5.75E+06*
1 COL (21
mg/kg/day)
Male 14 5.00E+01
2 COL (21
mg/kg/day)
Male 14 9.00E+02
3 COL (21
mg/kg/day)
Male 14 1.50E+02
57
4 COL (21
mg/kg/day)
Male 14 3.50E+02
5 COL (21
mg/kg/day)
Male 14 1.50E+02
6 COL (21
mg/kg/day)
Male 14 4.00E+02
7 COL (21
mg/kg/day)
Male 14 4.50E+02
8 COL (21
mg/kg/day)
Male 14 1.50E+02
9 COL (21
mg/kg/day)
Male 14 3.00E+02
10 COL (21
mg/kg/day)
Male 14 2.50E+02
LAC-4 ColR 1 Baseline Male 2 1.45E+07
2 Baseline Male 2 1.20E+07
3 Baseline Female 2 7.00E+06
58
4 Baseline Female 2 4.50E+06
1 PBS Male 14 1.10E+08
2 PBS Male 14 2.50E+08
3 PBS Male 14 1.00E+05
4 PBS Male 14 9.50E+05
5 PBS Male 14 1.95E+05
6 PBS Female 14 5.70E+07
7 PBS Female 14 3.80E+09
8 PBS Female 14 1.80E+09
9 PBS Female 14 3.15E+05
59
10 PBS Female 14 6.95E+06
1 COL 21
mg/kg
Male 14 1.20E+04
2 COL 21
mg/kg
Male 14 8.00E+03
3 COL 21
mg/kg
Male 14 1.15E+04
4 COL 21
mg/kg
Male 14 2.50E+04
5 COL 21
mg/kg
Male 14 6.50E+03
6 COL 21
mg/kg
Female 14 1.50E+03
60
7 COL 21
mg/kg
Female 14 2.00E+03
8 COL 21
mg/kg
Female 14 4.00E+03
9 COL 21
mg/kg
Female 14 6.00E+03
10 COL 21
mg/kg
Female 14 1.50E+03
“ * ” indicates value was imputed
Supplemental Table 7 - Efficacy of polymyxin treatment in vivo
Strain Mouse Treatment Gender Time point
(hours)
CFU/mL
HUMC1 1 Baseline Female 2 2.30E+07
2 Baseline Female 2 1.70E+07
61
3 Baseline Male 2 4.00E+06
4 Baseline Male 2 1.35E+07
1 PBS Female 18 2.35E+05
2 PBS Female 18 4.35E+07
3 PBS Female 18 8.50E+04
4 PBS Female 18 7.00E+04
5 PBS Female 18 8.00E+06
6 PBS Male 18 9.30E+07
7 PBS Male 18 2.1E+08
8 PBS Male 18 2.1E+08*
9 PBS Male 18 2.1E+08*
10 PBS Male 18 2.1E+08*
62
1 PMB 21
mg/kg
Female 18 6.00E+01
2 PMB 21
mg/kg
Female 18 1.10E+02
3 PMB 21
mg/kg
Female 18 2.50E+06
4 PMB 21
mg/kg
Female 18 8.00E+01
5 PMB 21
mg/kg
Female 18 1.00E+00
6 PMB 21
mg/kg
Male 18 1.00E+01
7 PMB 21
mg/kg
Male 18 4.00E+01
8 PMB 21
mg/kg
Male 18 5.00E+01
9 PMB 21
mg/kg
Male 18 9.00E+01
63
10 PMB 21
mg/kg
Male 18 1.10E+02
LAC-4 WT 1 Baseline Female 2 2.90E+06
2 Baseline Female 2 1.50E+07
3 Baseline Male 2 2.70E+07
4 Baseline Male 2 1.85E+07
1 PBS Female 18 3.55E+03
2 PBS Female 18 2.50E+03
3 PBS Female 18 2.70E+06
4 PBS Female 18 4.00E+03
5 PBS Female 18 1.85E+04
6 PBS Male 18 1.10E+03
7 PBS Male 18 2.20E+03
64
8 PBS Male 18 5.05E+05
9 PBS Male 18 3.70E+06
10 PBS Male 18 2.60E+03
1 PMB 21
mg/kg
Female 18 1.00E+00
2 PMB 21
mg/kg
Female 18 3.00E+01
3 PMB 21
mg/kg
Female 18 1.00E+01
4 PMB 21
mg/kg
Female 18 1.00E+00
5 PMB 21
mg/kg
Female 18 2.00E+01
6 PMB 21
mg/kg
Male 18 1.00E+00
7 PMB 21
mg/kg
Male 18 1.00E+00
65
8 PMB 21
mg/kg
Male 18 2.00E+01
9 PMB 21
mg/kg
Male 18 2.00E+01
10 PMB 21
mg/kg
Male 18 3.00E+01
LAC-4 ColR 1 Baseline Female 2 2.30E+06
2 Baseline Female 2 2.45E+06
3 Baseline Male 2 1.45E+06
4 Baseline Male 2 1.00E+06
1 PBS Female 18 1.05E+04
2 PBS Female 18 3.03E+03
3 PBS Female 18 1.00E+04
4 PBS Female 18 4.48E+03
5 PBS Female 18 3.63E+03
66
6 PBS Male 18 2.58E+03
7 PBS Male 18 4.25E+03
8 PBS Male 18 2.51E+03
9 PBS Male 18 1.97E+03
10 PBS Male 18 3.15E+03
1 PMB 21
mg/kg
Female 18 4.60E+02
2 PMB 21
mg/kg
Female 18 6.00E+01
3 PMB 21
mg/kg
Female 18 7.70E+02
4 PMB 21
mg/kg
Female 18 3.40E+02
5 PMB 21
mg/kg
Female 18 3.70E+02
6 PMB 21
mg/kg
Male 18 7.00E+01
67
7 PMB 21
mg/kg
Male 18 1.60E+02
8 PMB 21
mg/kg
Male 18 4.00E+01
9 PMB 21
mg/kg
Male 18 3.30E+02
10 PMB 21
mg/kg
Male 18 1.28E+03
AR-0307 1 Baseline Female 2 5.55E+05
2 Baseline Female 2 3.00E+05
3 Baseline Male 2 4.15E+05
4 Baseline Male 2 1.70E+05
1 PBS Female 18 8.20E+07
2 PBS Female 18 7.00E+09
3 PBS Female 18 6.70E+09
68
4 PBS Female 18 2.60E+06
5 PBS Female 18 1.00E+10
6 PBS Male 18 5.70E+08
7 PBS Male 18 4.00E+09
8 PBS Male 18 5.70E+09
9 PBS Male 18 9.50E+06
10 PBS Male 18 9.20E+09
1 PMB 21
mg/kg
Female 18 8.50E+08
2 PMB 21
mg/kg
Female 18 7.00E+04
3 PMB 21
mg/kg
Female 18 1.00E+09
4 PMB 21
mg/kg
Female 18 5.50E+08
69
5 PMB 21
mg/kg
Female 18 6.00E+04
6 PMB 21
mg/kg
Male 18 5.80E+07
7 PMB 21
mg/kg
Male 18 2.00E+05
8 PMB 21
mg/kg
Male 18 1.05E+08
9 PMB 21
mg/kg
Male 18 4.50E+07
10 PMB 21
mg/kg
Male 18 1.40E+06
AR-0308 1 Baseline Female 2 7.65E+05
2 Baseline Female 2 4.35E+06
3 Baseline Male 2 1.80E+06
4 Baseline Male 2 1.28E+06
1 PBS Female 18 3.00E+06
70
2 PBS Female 18 2.75E+06
3 PBS Female 18 9.60E+06
4 PBS Female 18 9.65E+05
5 PBS Female 18 2.80E+07
6 PBS Male 18 3.95E+06
7 PBS Male 18 4.95E+05
8 PBS Male 18 1.16E+05
9 PBS Male 18 3.55E+06
10 PBS Male 18 6.40E+05
1 PMB 21
mg/kg
Female 18 7.30E+04
2 PMB 21
mg/kg
Female 18 1.90E+07
3 PMB 21
mg/kg
Female 18 9.00E+03
71
4 PMB 21
mg/kg
Female 18 2.95E+05
5 PMB 21
mg/kg
Female 18 7.50E+03
6 PMB 21
mg/kg
Male 18 6.25E+04
7 PMB 21
mg/kg
Male 18 1.90E+05
8 PMB 21
mg/kg
Male 18 3.75E+05
9 PMB 21
mg/kg
Male 18 3.55E+05
1112707 1 Baseline Female 2 2.19E+07
2 Baseline Female 2 3.03E+07
3 Baseline Male 2 7.90E+07
4 Baseline Male 2 3.80E+07
1 PBS Female 18 7.00E+08
72
2 PBS Female 18 5.80E+06
3 PBS Female 18 3.10E+07
4 PBS Female 18 2.45E+07
5 PBS Female 18 4.95E+05
6 PBS Male 18 1.52E+09
7 PBS Male 18 1.52E+09*
8 PBS Male 18 1.52E+09*
9 PBS Male 18 1.52E+09*
10 PBS Male 18 1.52E+09*
1 PMB 21
mg/kg
Female 18 4.00E+02
2 PMB 21
mg/kg
Female 18 1.00E+00
3 PMB 21
mg/kg
Female 18 1.00E+02
73
4 PMB 21
mg/kg
Female 18 1.50E+02
5 PMB 21
mg/kg
Female 18 1.50E+02
6 PMB 21
mg/kg
Male 18 6.00E+03
7 PMB 21
mg/kg
Male 18 7.70E+02
8 PMB 21
mg/kg
Male 18 2.10E+02
9 PMB 21
mg/kg
Male 18 4.80E+02
10 PMB 21
mg/kg
Male 18 3.30E+02
1184244 1 Baseline Female 2 1.52E+07
2 Baseline Female 2 7.65E+06
3 Baseline Male 2 8.30E+06
74
4 Baseline Male 2 2.30E+06
1 PBS Female 18 1.07E+09
2 PBS Female 18 8.05E+06
3 PBS Female 18 9.65E+08
4 PBS Female 18 3.05E+08
5 PBS Female 18 1.11E+07
6 PBS Male 18 1.14E+05
7 PBS Male 18 1.12E+05
8 PBS Male 18 1.11E+05
9 PBS Male 18 6.15E+04
10 PBS Male 18 1.06E+05
1 PMB 21
mg/kg
Female 18 6.00E+02
75
2 PMB 21
mg/kg
Female 18 7.00E+02
3 PMB 21
mg/kg
Female 18 7.50E+03
4 PMB 21
mg/kg
Female 18 4.00E+02
5 PMB 21
mg/kg
Female 18 3.20E+06
6 PMB 21
mg/kg
Male 18 1.50E+02
7 PMB 21
mg/kg
Male 18 1.00E+00
8 PMB 21
mg/kg
Male 18 1.00E+02
9 PMB 21
mg/kg
Male 18 1.05E+04
10 PMB 21
mg/kg
Male 18 1.00E+00
1124614 1 Baseline Female 2 1.50E+05
76
2 Baseline Female 2 4.50E+05
3 Baseline Male 2 5.00E+04
4 Baseline Male 2 5.00E+04
1 PBS Female 18 5.00E+03
2 PBS Female 18 2.15E+04
3 PBS Female 18 2.30E+04
4 PBS Female 18 9.00E+03
5 PBS Female 18 2.05E+04
6 PBS Male 18 1.23E+03
7 PBS Male 18 2.50E+03
8 PBS Male 18 2.91E+03
9 PBS Male 18 3.50E+03
77
10 PBS Male 18 1.68E+03
1 PMB 21
mg/kg
Female 18 1.00E+00
2 PMB 21
mg/kg
Female 18 4.00E+01
3 PMB 21
mg/kg
Female 18 2.00E+01
4 PMB 21
mg/kg
Female 18 1.00E+00
5 PMB 21
mg/kg
Female 18 1.00E+01
6 PMB 21
mg/kg
Male 18 1.00E+00
7 PMB 21
mg/kg
Male 18 2.00E+01
8 PMB 21
mg/kg
Male 18 1.00E+01
9 PMB 21
mg/kg
Male 18 1.00E+01
78
10 PMB 21
mg/kg
Male 18 1.00E+00
1180013 1 Baseline Female 2 2.05E+07
2 Baseline Female 2 2.30E+07
3 Baseline Male 2 1.25E+07
4 Baseline Male 2 8.00E+06
1 PMB 21
mg/kg
Female 18 4.50E+06
2 PMB 21
mg/kg
Female 18 7.80E+06
3 PMB 21
mg/kg
Female 18 2.00E+06
4 PMB 21
mg/kg
Male 18 8.20E+05
5 PMB 21
mg/kg
Male 18 1.17E+06
79
6 PMB 21
mg/kg
Male 18 4.00E+05
1174913 1 Baseline Female 2 3.50E+05
2 Baseline Female 2 5.50E+05
3 Baseline Male 2 4.95E+06
4 Baseline Male 2 1.10E+06
1 PBS Female 18 2.35E+06
2 PBS Female 18 3.50E+06
3 PBS Female 18 4.65E+06
4 PBS Female 18 8.60E+06
5 PBS Female 18 2.50E+06
6 PBS Male 18 1.25E+07
7 PBS Male 18 1.00E+07
80
8 PBS Male 18 8.50E+06
9 PBS Male 18 1.10E+07
10 PBS Male 18 1.25E+07*
1 PMB 21
mg/kg
Female 18 1.35E+07
2 PMB 21
mg/kg
Female 18 1.10E+07
3 PMB 21
mg/kg
Female 18 2.00E+06
4 PMB 21
mg/kg
Female 18 2.95E+06
5 PMB 21
mg/kg
Female 18 4.05E+06
6 PMB 21
mg/kg
Male 18 5.50E+06
7 PMB 21
mg/kg
Male 18 2.20E+07
81
8 PMB 21
mg/kg
Male 18 6.00E+06
“ * ” indicates value was imputed
Supplemental Table 8 - Summary of bacterial strains used in this study
Strains Description Reference
A. baumannii HUMC1 Clinical isolate [42]
A. baumannii LAC-4 WT Clinical isolate [31]
A. baumannii LAC-4 ColR Colistin-resistant mutant of
LAC-4 WT; pmrA
[31]
P. mirabilis 10195 Clinical isolate This study
A. baumannii C8 Colistin-resistant; pmrB [22]
A. baumannii C14 Colistin-resistant mutant;
pmrB, pmrC
[22]
A. baumannii SM1536
(mcr-1)
Colistin-resistant mutant;
mcr-1
[43]
82
A. baumannii ATCC 17978
(mcr-1)
Colistin-resistant mutant;
mcr-1
[43]
A. baumannii D773 (mcr-1) Colistin-resistant mutant;
mcr-1
[43]
A. baumannii 19606R Colistin-resistant mutant;
lpxA
[12]
A. baumannii AL1833 Colistin-resistant mutant;
lpxC
[12]
A. baumannii AL1834 Colistin-resistant mutant;
lpxC
[12]
A. baumannii 1842 Colistin-resistant mutant;
lpxC
[12]
A. baumannii 1843 Colistin-resistant mutant;
lpxC
[12]
A. baumannii 1844 Colistin-resistant mutant;
lpxA
[12]
A. baumannii 1845 Colistin-resistant mutant;
lpxA
[12]
83
A. baumannii 1846 Colistin-resistant mutant;
lpxA
[12]
A. baumannii 1847 Colistin-resistant mutant;
lpxA
[12]
A. baumannii 1848 Colistin-resistant mutant;
lpxA
[12]
A. baumannii 1849 Colistin-resistant mutant;
lpxA
[12]
A. baumannii 1851 Colistin-resistant mutant;
lpxA
[12]
A. baumannii 1852 Colistin-resistant mutant;
lpxD
[12]
A. baumannii ARUP_A1 Colistin-resistant clinical
isolate
This study
A. baumannii ARUP_A6 Colistin-resistant clinical
isolate
This study
A. baumannii ARUP_A8 Colistin-resistant clinical
isolate
This study
84
A. baumannii ARUP_A10 Colistin-resistant clinical
isolate
This study
A. baumannii ARUP_A11 Colistin-resistant clinical
isolate
This study
A. baumannii ARUP_A13 Colistin-resistant clinical
isolate
This study
A. baumannii ARUP_A22 Colistin-resistant clinical
isolate
This study
A. baumannii ARUP_A23 Colistin-resistant clinical
isolate
This study
A. baumannii ARUP_A28 Colistin-resistant clinical
isolate
This study
A. baumannii ARUP_A30 Colistin-resistant clinical
isolate
This study
A. baumannii AR-0307 Colistin-resistant clinical
isolate
This study
A. baumannii AR-0308 Colistin-resistant clinical
isolate
This study
85
A. baumannii 1112707 Colistin-resistant clinical
isolate
This study
A. baumannii 1184244 Colistin-resistant clinical
isolate
This study
A. baumannii 1124614 Colistin-resistant clinical
isolate
This study
A. baumannii 1180013 Colistin-resistant clinical
isolate
This study
A. baumannii 1174913 Colistin-resistant clinical
isolate
This study
A. baumannii 1064048 Colistin-resistant clinical
isolate
This study
A. baumannii 1071864 Colistin-resistant clinical
isolate
This study
A. baumannii 1083037 Colistin-resistant clinical
isolate
This study
A. baumannii 1083246 Colistin-resistant clinical
isolate
This study
86
A. baumannii 1083383 Colistin-resistant clinical
isolate
This study
A. baumannii 1099731 Colistin-resistant clinical
isolate
This study
A. baumannii 1105437 Colistin-resistant clinical
isolate
This study
A. baumannii 1125675 Colistin-resistant clinical
isolate
This study
A. baumannii 1127911 Colistin-resistant clinical
isolate
This study
A. baumannii 1172798 Colistin-resistant clinical
isolate
This study
A. baumannii 1174945 Colistin-resistant clinical
isolate
This study
A. baumannii 1178685 Colistin-resistant clinical
isolate
This study
A. baumannii 1180949 Colistin-resistant clinical
isolate
This study
87
A. baumannii 1185713 Colistin-resistant clinical
isolate
This study
A. baumannii 1188882 Colistin-resistant clinical
isolate
This study
A. baumannii ARUP_A3 Colistin-resistant clinical
isolate
This study
A. baumannii ARUP_A4 Colistin-resistant clinical
isolate
This study
A. baumannii ARUP_A5 Colistin-resistant clinical
isolate
This study
A. baumannii ARUP_A9 Colistin-resistant clinical
isolate
This study
A. baumannii ARUP_A14 Colistin-resistant clinical
isolate
This study
A. baumannii ARUP_A16 Colistin-resistant clinical
isolate
This study
A. baumannii ARUP_A18 Colistin-resistant clinical
isolate
This study
88
A. baumannii ARUP_A19 Colistin-resistant clinical
isolate
This study
A. baumannii ARUP_A20 Colistin-resistant clinical
isolate
This study
A. baumannii ARUP_A26 Colistin-resistant clinical
isolate
This study
A. baumannii ARUP_A29 Colistin-resistant clinical
isolate
This study
Supplemental Table 9 - Summary of primers used in this study
Primer
name
Target Primer
Direction
5’-3’ Sequence Reference
JR002 pmrC Forward ACGCGCTGAATTTGATGAG
C
This study
JR0021 pmrC Reverse TCATTTGCAGTGGTCGGTGT This study
JR0028 pmrA Forward CGTTGGCCTTTAAACGTCGC This study
JR0029 pmrA Reverse CGCCGTAGTGGAGTAGAAG
C
This study
89
JR0034 pmrB Forward CCAAGCCTTTGAGTTGCAC
G
This study
JR0035 pmrB Reverse CCCCGCATCAGGGTGTTATT This study
JR0042 dedA Forward GGCCAAATAGGTAGCCAGC
C
This study
JR0043 dedA Reverse CACGTTCTTTTGCACCCCTC This study
MM0463 Universal
16S rRNA
Forward AAACTCAAAKGAATTGACGG [3]
MM0464 Universal
16S rRNA
Reverse CTCACRRCACGAGCTGAC [3]
90
Chapter 3: Conclusions
Our data shows that using standard AST conditions fails to predict the in vivo
efficacy of polymyxins against “resistant” A. baumannii blood infections. Using
bicarbonate-containing culturing conditions, which better reflect the nutrient-limited in
vivo infection environment of an A. baumannii blood infection, enhances the accuracy of
clinical antimicrobial susceptibility testing for polymyxins against A. baumannii. MHII
culturing conditions promote LPS structural modifications that reduce polymyxin affinity
and susceptibility. Culturing polymyxin-resistant A. baumannii in physiological levels of
sodium bicarbonate enhances polymyxin susceptibility by reducing structural LPS
modifications, which are only present in MHII conditions, thereby improving polymyxin’s
binding.
Our MIC susceptibility data shows that not all polymyxin resistance-conferring
phenotypes were sensitive to sodium bicarbonate susceptibly alterations. In polymyxinresistant strains that contained mutations in pmrA, pmrB, pmrC or mcr-1+, modifying
AST conditions to include sodium bicarbonate altered the majority of isolates to a
polymyxin-susceptible phenotype. However, LPS-deficient A. baumannii strains
maintained a polymyxin-resistant phenotype in both conventional MHII and bicarbonatecontaining culturing conditions.
Our colistin in vivo studies demonstrate that bicarbonate conditions accurately
predicted colistin’s efficacy in an A. baumannii blood infection. Mice infected with LAC-4
ColR and treated with colistin had significantly improved survival and significantly
reduced CFUs compared to the PBS control group. Similarly, in our PMB efficacy in vivo
studies, 5 of 6 strains with a >1-log CFU reduction at 18 hours post infection had
91
significantly improved survival. Overall, these results suggest that using physiological
levels of sodium bicarbonate improves the predictive accuracy of ASTs against A.
baumannii.
Our findings are consistent with other reports that have also shown that sodium
bicarbonate improves the predictive accuracy of ASTs. Ersoy et al. showed that MICs
against Streptococcus pneumoniae and Salmonella species isolates are markedly
different when culturing conditions are changed from standard MH broth to more
physiologically relevant media [20]. Subsequent septicemia mouse models verified that
the altered MICs accurately predicted the changes in antibiotic efficacy. Their findings
with a methicillin-resistant Staphylococcus aureus (MRSA) isolate from a deceased
patient demonstrated that antibiotics omitted by standard AST were highly successful in
bacterial clearance. Dorschner et al. observed that antimicrobial peptides (AMPs)
demonstrated in vivo activity despite the lack of in vitro activity using standard AST
conditions. Dorschner et al found that carbonate enhanced the antimicrobial activity of a
structurally diverse panel of peptides and rendered diverse bacteria (MRSA, E. coli and
Salmonella Dublin) susceptible. Their findings showed that both gram-positive and
gram-negative bacteria showed dramatically increased AMP susceptibility when grown
in the presence of carbonate. In agreement with our work, these findings support the
approach that using physiologically relevant media can enhance the predictive accuracy
of ASTs [19].
We have shown that compared to conventional MHII media, modifying AST
culturing conditions to incorporate sodium bicarbonate provides superior predictive
accuracy of polymyxins against A. baumannii blood infections. As our findings are
92
specific to A. baumannii and polymyxin activity, further investigation is required to
assess the specific in vivo infection environmental conditions of different pathogens and
how those conditions alter the activity of different antimicrobials. AST methods that
accurately model the in vivo infection environment may enhance the predictive value of
ASTs and advance the discovery of new or better antibiotics. If standard AST methods
continue to test pathogens in conditions so distinctly different from the infection
environment, the consequence of excluding effective antibiotics could be dire.
93
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Abstract (if available)
Abstract
The emergence of colistin resistance poses a serious threat to colistin efficacy as it is considered the “last-line-of-defense” against extensively drug resistant Acinetobacter baumannii. However, we found that conventional antimicrobial susceptibility testing methods incorrectly model the contributions of previously characterized colistin-resistance conferring mutations. We have found that physiological media, but not the conventional media used in antimicrobial susceptibility assays, accurately predicts polymyxin in vivo efficacy in mice infected with polymyxin-resistant A. baumannii. Modifying antimicrobial susceptibility test culturing conditions will maximize the therapeutic potential of the existing arsenal of antibiotics that have shown efficacy against bacterial pathogens.
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Polymyxins retain in vitro activity and in vivo efficacy against “resistant” Acinetobacter baumannii strains when tested in physiological conditions
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