1. Dissertation Defense
Melissa Agnello
Clinical and Experimental Therapeutics
Advisor: Annie Wong-Beringer, Pharm.D.
Co-advisor: Steven Finkel, Ph.D.
Biological Impact of
Fluoroquinolone Resistance in
Pseudomonas aeruginosa

2. • Ubiquitous gram-negative bacterium + opportunistic pathogen
• Can live and adapt to a variety of habitats and cause a variety of
infections
• Leading nosocomial cause of pneumonia1, especially ventilator-
associated pneumonia2
Pseudomonas aeruginosa
1Quartin et al. 2013; Restrepo and Anzueto 2009
2Rello et al., 2005
SEM of Pa biofilm

3. P. aeruginosa: Antibiotic Resistance is a
Major Public Health Concern
• CDC: “Serious concern that
requires prompt action”
• IDSA: Pa is one of 6 bacteria that
pose an immediate threat to public
health
• Multi-drug resistant (MDR) =
resistant to > 3 classes of antibiotics
• 13% of Pa infections caused by
MDR strains

4. • FQs are highly effective against Pa
• Only orally available antibiotic for
Pa infections
• Most commonly prescribed class of
antibiotics in US1
• Now, >30% of all strains are
resistant to the FQs2
• Because of cross-resistance, many
FQ-resistant strains rapidly become
multi-drug resistant
Resistance to the Fluoroquinolones (FQs)
Limits Treatment Options
1Linder
et
al.
Am
J
Med
2005;
2Rosenthal
et
al.
Am
J
Infect
Control
2012
Neuhauser
et
al
2003
Hsu
et
al
2005

5. Mechanisms of Resistance to FQs
• Overexpression
of efflux pumps
• Target site
mutations:
• Type II
Topoisomerases
GyrA/B, ParC/E
• Point mutations in
gyrA and parC most
common

6. • Leading cause of hospital pneumonia, with an attributable
mortality of 40-70%3
• Ability to cause severe disease due to variety of virulence factors
• Type III Secretion System (TTSS): Important virulence factor in
acute infections
• Macro-molecular syringe apparatus
• Injects toxins directly into host cells
• Known toxins: ExoS, ExoU, ExoT, ExoY
• Disrupt host cell function, lead to cell death
Pathogenicity
3Richards
et
al.
Crit
Care
Med
1999

7. Toxins ExoU and ExoS
• Genes encoding these toxins are mutually exclusive in most strains
• exoS strains more prevalent: about 70% of all strains
exoU strains cause worse
disease and lead to worse
outcomes in patients
Shaver&
Hauser
2004
S
exoS strain Host
immune
cell
S
S S
exoU
strain
Host
immune
cell
U
U
U
U
U+
S+
LD50

8. exoU and exoS Genes Incompatible in
Same Genome
• Pa genome made up of core
and accessory genes
• Core genome = highly
conserved genes
• Accessory = horizontally
acquired genes
• Accessory genome consists
of genomic “islands”
• exoU and chaperone spcU
located on an island
• exoS: part of core genome

9. FQ-Resistance Correlates with Worse
Outcomes in Patients
• Patients infected with FQ-resistant strains of Pa had 3 fold
higher mortality and increased persistence of disease
Hsu
et
al.
2005
JAC
Are
FQ-­‐resistant
strains
more
virulent?

10. exoU Genotype Associated with Increased
FQ-Resistance in Clinical Isolates
• Proportion of exoU-containing isolates increased as the
level of FQ- resistance increased in study of 45 strains
Wong-­‐Beringer
et
al.
2008
Clin
Microbiol
Infect

11. Aim #1:
Confirm observed correlation of FQ-resistance and exoU
genotype in a larger collection of clinical strains
Hypothesis: Clinical isolates with the exoU genotype are
associated with FQ-resistance as well as worse disease

12. Combined exoU, FQ-R traits significant
risk factor for severe disease
• Study of 218 clinical
isolates
• Odds for development
of pneumonia:
• exoU, FQ-R = 3.28
• exoS, FQ-R =1.96
Sullivan, Bensman, Lou, Agnello et al. (2014) Critical Care Medicine

13. • Results of analysis of
270 clinical isolates:
- Significantly higher
proportion of exoU
strains were FQ-
resistant than exoS
strains
- exoU strains more
likely to acquire
multiple target site
mutations, specifically
in parC
exoU strains more frequently FQ-resistant

14. • Problem: exoU strains cause worse disease AND are more likely to
be resistant to the fluoroquinolones
• Unable to use best drugs against the worst bugs
• Research Question: Why are the more virulent exoU strains more
likely to develop resistance to FQs?
• Central Hypothesis: exoU strains are more adaptable to the FQ-rich
clinical environment than exoS strains due to differences in fitness

15. • In general, acquisition of resistance to an antibiotic is thought to be
associated with a fitness cost to the organism
• Mutation in essential gene, plasmid or extra gene to express, etc
• Allows bacterium to grow in presence of antibiotic, but comes at a price
• Compensatory mutations can overcome this cost
Fitness=
Capability
to
survive
and
reproduce
Virulence
(disease-­‐
causing
ability)
Compe::ve
growth

16. Aim #2:
Investigate the biological impact of FQ-resistance on the
fitness and virulence of Pa, specific to strain background
of exoU vs. exoS strains.
Hypothesis: exoU strains are more fit than exoS
strains after the acquisition of FQ-resistance

17. Methods
Goal: To test the effects of FQ-resistance on the fitness
of exoU and exoS strains, focusing on a single
resistance-conferring mutation
• What if exoU strains can overcome the fitness cost
of FQ-resistance better than exoS strains?
• This would allow exoU strains to more readily
become FQ- resistant

18. Methods
1. Created isogenic mutants from 3 clinical exoU and 3
clinical exoS isolates
Rationally designed mutants to reflect clinical populations:
• Chose strains that had naturally acquired a mutation in gyrA
• Inserted point mutation in parC that confers FQ-resistance
Agnello
&
Wong-­‐Beringer
J
Micro
Meth
2014

19. Methods
Oligonucleotide recombination
§ Takes advantage of bacterial
homologous recombination
§ Allows for the introduction of site-
specific mutations directly into the
genome using synthetic ssDNA
oligos
§ Novel method adapted for use in
Pseudomonas aeruginosa

20. Tested fitness of parC mutants (PC*) compared to parents by
investigating:
1. Competitive growth: Strains are tagged with fluorescent
proteins for differentiation and grown in co-culture
2. Metabolic function: compared growth on a variety of carbon
and nitrogen substrates
3. Virulence: expression of the type III secretion system (TTSS)
with qRT-PCR
Methods
Colonies
on
agar
plate
of
strains
tagged
with
YFP
(yellow)
or
CFP
(blue),
as
seen
through
a
wide-­‐field
microscope

21. Competition Experiments
Common method of comparing fitness:
• Parent and mutant strains grown in same culture, start in equal
quantities
• Every 24 h, sample of culture plated to count the number of
colonies of each strain
• Strain that is more fit will take over the culture
Every 24 hrs
Serial dilute, plate,
and count colonies

22. Need to be able to differentiate the strains:
• Inserted cassette encoding CFP or YFP into the genomes
• Fully integrated, no need for continued selection
• Visualize/count colonies using wide-field fluorescent
microscope
Competition Experiments
Choi & Schweizer 2006

23. Mutation differentially affects fitness of
exoU and exoS strains

24. Metabolic Microarray
• Allows measurement of
metabolic utilization of
different substrates
• Each well of 96 well plate
contains different carbon or
nitrogen substrate
• Strains grow in the wellsà
induce color change that is
quantified

25. Effect of parC Mutation on Nitrogen Utilization
Differs for exoU vs. exoS Strains
parC mutation may differentially affect
the ability of exoS strains and exoU
strains to live and cause disease under
low-oxygen conditions
o2
exoU-PC* exoS-PC*
# of substrates
increased vs.
parents
44 24
# of substrates
decreased vs.
parents
53 69

26. parC Mutation Increases Expression of Type III
Secretion System for exoU Strains Only
PcrV: essential
component of the
needle complex
pcrV
Expression
exoU exoS
PcrV (cap protein)
Bacterial
cell
Host
cell

27. Summary of Fitness Effects of parC mutation
• Competitive growth
• Ability to grow on
nitrogen substrates
• Virulence expression
exoU-PC*
• Competitive growth
• Ability to grow on
nitrogen substrates
• Virulence expression
exoS-PC*
• Suggests less of a fitness cost of FQ-resistance for
exoU strains
• Explains predominance of exoU strains in FQ-
resistant clinical population

28. Supercoiling Regulation May Explain Fitness
Differences
• Bacterial chromosome exists in condensed,
supercoiled state
• Supercoiling level in constant flux
• Topoisomerase enzymes (GyrA, ParC) regulate
supercoiling levels
• parC mutation may differentially affect ability of
exoU and exoS strains to regulate supercoiling
• Supercoiling perturbations can have global effects
on gene expression

29. exoU-PC* Mutants Can Better Maintain Wild-
Type Supercoiling Levels
• Inserted cassette with
lux operon under the
control of a
supercoiling-sensitive
promoter
• Induced max
expression of the
promoter by incubating
with levofloxacin
• Measured
luminescence

30. Compensation for Fitness Costs of parC
Mutation
Goal: To investigate whether stable changes occurred
during competition that affect fitness
Competition
PC* vs. Parent
Day 7
Collection of
PC* and parent
colonies
“Aged”
strains frozen
and saved

31. Dramatic Difference in Fitness of exoU vs.
exoS Aged PC* Strains
Competed Aged vs. Un-Aged
to investigate if fitness
changed after aging
37-PC
*
92-PC
*
-4
-2
0
2
4
6
8
Fold
139-PC
*
215-PC
*
-150
-100
-50
0
Fold
Fold Change in Fitness After Aging
exoU exoS
• exoU strainsà compensate for fitness costs
• exoS strainsà fitness costs amplified

32. Effect of Sub-inhibitory FQ Exposure on Fitness
Competition
PC* vs. Parent
Day 7
Collection of
PC* and parent
colonies
“Aged” strains
frozen and saved
+ 1/8x MIC LVX
• Collected aged strains from competition experiment with low level of
levofloxacin (LVX)

33. • Competed Aged strain from LVX competition vs. Un-Aged, under exposure
to same amount of LVX
• exoU-PC* strains had increased fitness in this subsequent exposure to
drug
Effect of Sub-inhibitory FQ Exposure on Fitness
Fold Change in Fitness After Aging
exoU exoS

34. Summary
• exoU-PC* strains can better maintain wild-type supercoiling
levels than exoS-PC* strains
• exoU-PC* aged strains have a compensated phenotype
• Suggests exoU strains are compensating for fitness costs associated
with parC mutation
• exoU strains collected from competition experiment under sub-
inhibitory concentrations of FQ have increased fitness
• Low levels of antibiotic can select for highly fit strains

35. Investigating Fitness in the Host
• Important to investigate fitness in clinically relevant environment
• Using an established mouse model of acute pneumonia
• Mice are intranasally infected; rapid inhalation of bacteria into
the lungs
• Male, C57/BL6 mice 6-8 weeks old

36. • Mice co-infected with equal amounts of parent and mutant strain
• After 18 hours, lungs removed, homogenized, and plated
• Colonies of each strain counted
Investigating Fitness in the Host

37. Mutant: Parent Ratios in Lungs
• Ra:os
<
1
=
mutant
less
fit
• More
exoU
mice
with
ra:os
over
1
than
exoS
mice
Effect of the parC Mutation on Fitness in the Lungs
exoU exoS
Averages
(n=5)
Results
in
individual
mice

38. Conclusions and Significance
• Clinical strains with the exoU background are more likely to be
FQ-resistant and cause worse disease in patients
• In general, we found less of a fitness cost of FQ-resistance
mutation in parC for exoU strainsà less of a barrier to acquiring
resistance for exoU strains
• exoU strains are able to compensate for fitness costs
• Explains predominance of exoU strains in FQ-resistant clinical
population

39. • Implication: In the clinical setting, FQ prescribing will select for highly
virulent exoU, FQ-resistant strains due to their enhanced fitness
compared to exoS strains
• Indiscriminant prescribing selects for both resistant and highly virulent Pa strains
that cause more severe disease and worse outcomes
• Fitness advantage of FQ-resistant exoU strains allows them to predominate in
the clinical environment even in the absence of antibiotic
• Further underscores the dangers of antibiotic resistance and the
need for prudent use of antibiotics
• Our experimental plan is a model for studying the biological impact of
resistance on an important pathogen
• Understanding fitness costs and mechanisms of compensation is
essential for rational development of novel strategies to combat
antibiotic resistance
Conclusions and Significance

40. • Investigate virulence of exoU vs. exoS FQ-
resistant strains in vivo using the mouse model of
pneumonia
• Determine if fitness differences extend to
different abilities to injure the lungs and cause
disease
• Using whole body in vivo imaging, can monitor
progression of infection
• All strains fluorescently taggedà allows live imaging
• Using imaging during co-infection: can determine if
spatial differences in exoU vs. exoS or mutant vs.
parent strains
Future Directions

41. Future Directions
• Next step is to investigate the specific genomic differences between
exoU and exoS strains that may account for fitness differences
observed
• exoU gene part of accessory genomeà located on various pathogenicity
islands
• Accessory genome may provide fitness benefit to exoU strains
• Perform whole genome sequencingà to compare accessory and
core genomes of each strain and correlate with fitness phenotypes
• Follow with RNA-sequencingà compare the effects of the parC
mutation on the expression of important virulence and metabolic
pathways in exoU vs. exoS strains

42. Acknowledgements
PhD Advisor: Annie Wong-Beringer, PharmD
Co-­‐advisor:
Steven
Finkel,
PhD
Labmates:
Jason Yamaki, Pharm.D., Ph.D.
Tim Bensman, Pharm.D, Ph.D. Candidate
Rachel Reyes
Kristy Trinh
Undergraduate Research Fellow:
Nicole Schrad
Volunteers:
Nick Nuno
Namrah Ayub
Caitlyn Young
Susana Petrosyan
Juliana Brondani
Christine Vu
Lorena Ulloa
Vivian Lee
Faculty Committee:
Paul Beringer, Pharm.D.
Kathleen Rodgers, Ph.D.
Roger Duncan, Ph.D.
Ronald Alkana, Ph.D.
Support:
TL1 Pre-doctoral Award (SC-CTSI)
NIAID grant to Annie Wong-Beringer
Technical Microscope Assistance:
Seth Ruffins, Ph.D.
SC-CTSI, ECDE:
Cecilia Patino-Sutton, M.D., Ph.D.
Jonathan Samet, M.D., M.S.
Emil Bogenmann, Ph.D., Ed.D.

43. “The future of humanity and microbes likely will
unfold as episodes of a suspense thriller that
could be titled Our Wits Versus Their Genes.”
-Joshua Lederberg