1. 0 1 2 3 4 5
-2
-1
0
1
2
Day
Log(mutant/parent)
exoU
92
37
91
0 1 2 3 4 5
-2
-1
0
1
2
Day
Log(mutant/parent)
exoS
139
215
247
exoU
exoS
0
1
2
3
4
PC*/ParentRatio
1/4xM
IC
1/2xM
IC
1xM
IC
-25
-20
-15
-10
-5
0
5
[Levofloxacin]
FoldDifference(PC*/Parent)
92 (exou)
37 (exoU)
139 (exoS)
215 (exoS)
MELISSA AGNELLO and ANNIE WONG-BERINGER
University of Southern California, Los Angeles, California, USA
Correspondence to:
Annie Wong-Beringer, Pharm.D., FIDSA
USC School of Pharmacy, 1985 Zonal
Avenue. Los Angeles, CA 90089-9121, USA.
Tel: 323-442-1356, Fax: 626-628-3024
Email: anniew@usc.edu
Results
1Richards et al. (1999) Crit Care Med; 2Rosenthal et al. (2012) Am J Infect Control; 3Feltman et al. (2001)
Microbiology; 4Shaver & Hauser (2004) Infect Immun; 5Agnello & Wong-Beringer (2012) PLoS ONE;
6Choi & Schweizer (2006) Nat Prot; 7Moir et al. (2007) J Biomol Screen
Ø P. aeruginosa: Leading cause of nosocomial
pneumonia, with an attributable mortality of 40-70%1
Ø 30% of all strains are now resistant to the
fluoroquinolone (FQ) antibiotics,2 once highly effective.
Ø Resistance mechanisms include target site mutations in
the topoisomerase genes; mainly gyrA and parC.
Ø Type III secretion system (TTSS): key virulence factor
allowing P. aeruginosa to cause severe acute infections.
Ø TTSS injects exotoxins (U, S, Y, T) into host cells,
causing cell death
Ø Genes encoding exotoxins U and S (exoU/exoS) are
mutually exclusive in most strains3
Ø While exoS strains predominate in the wild-type
population, exoU strains cause worse disease in patients
and animal models4
Ø exoU clinical strains are more likely to be
fluoroquinolone-resistant than exoS strains5
Background
Fitness= capability of pathogen to
survive, reproduce, and cause disease
Hypothesis and Aims
Aim: Investigate and compare fitness effects of
an FQ-resistance conferring parC mutation in
exoU and exoS clinical strains
We hypothesize that exoU strains are more
adaptable to FQ exposure than exoS strains due
to differences in fitness, allowing for
compensation of the fitness costs associated with
resistance.
Research Question: Why are the more virulent
exoU strains more likely to develop resistance to
FQs?
Methods
Conclusions
References
Acknowledgements
This study was supported by award #R21AI073467 from the NIH/NIAID to AWB and award
#TL1TR000132 from NIH/NCRR/NCATS to MA
Mutation frequency was assessed by plating
on 5x the MIC of rifampicin at each time point
during competition. RifR frequency was
increased for exoU PC* mutant strains
compared to parents at the end of 4 days of
competition, and decreased for exoS PC*
mutant strains.
Ø The FQ resistance-conferring mutation in parC differentially affects the fitness of exoU
and exoS strains, favoring exoU.
Ø Less fitness cost of resistance to exoU strains explains their greater tendency to
acquire resistance mutations in the clinical setting.
Ø Results overall suggest exoU strains are compensating for the fitness costs of FQ-
resistance, potentially via greater regulation of supercoiling.
Ø Implication: In the clinical setting, the widespread prescribing of fluoroquinolones will
select for highly virulent exoU, FQ-resistant strains due to their greater adaptability to
FQs compared to exoS strains.
Mutation Differentially Affects Fitness of exoU and exoS
Strains
Resistance Mutation Increases Mutation Frequency of
exoU Strains During Competition
Differential Compensation for the Fitness Cost Associated with
Fluoroquinolone Resistance by Type III Secretion System Virulence
Genotype in Pseudomonas aeruginosa
1. Created isogenic mutants containing an FQ-R mutation from clinical exoU and
exoS strains
Ø Clinical Isolates (3 exoU and 3 exoS) with a pre-existing gyrA FQ-R mutation
(Thr83Ile) were selected for mutagenesis.
Ø Using the technique of oligonucleotide recombination, a point mutation in parC that
confers FQ resistance was introduced into the genome of each strain.
2. Investigated fitness of mutants vs. parent strains with pair-wise competition
experiments
Ø Strains tagged with either CFP or YFP using a Tn7-based approach6
Ø Mutant and parent strains (105 CFUs each) added to 10 ml LB broth.
Ø Every 24 hours, 500 µl of the culture was sampled for CFU count via serial dilution
and plating on Pseudomonas Isolation Agar (PIA).
Ø After each 24 hour cycle, 10 µl of the culture was transferred to 10 ml fresh LB
Ø CFU counting performed with wide-field fluorescent microscopy.
3. Compared the mutation frequency of mutants vs. parent strains during
competition
Ø After each 24 hour cycle, competition culture plated on agar containing rifampicin
at 5x the MIC.
Ø Mutation frequency was calculated by dividing the CFUs/ml on rifampicin-
containing plates by the CFUs/ml on PIA plates at each time point.
4. Assessed ability of exoU and exoS PC* strains to regulate supercoiling
Ø Developed a reporter assay by inserting a cassette containing the lux operon
under the control of a supercoiling sensitive promoter7
Ø Strains were grown to mid exponential phase, then diluted 1:4 in LB+ levofloxacin
at ¼, ½, and 1x the measured MIC, and grown in triplicate in a deep well 96-well
plate for 7 hours, to mid-to-late exponential phase.
Ø Luminescence was measured and normalized to OD600.
5. Assessed potential compensation for fitness costs of parC mutation
Ø Collected single colonies of mutant and parent strains from end of 7 day primary
competition experiment; labeled as “aged” strains
Ø Tested fitness of aged vs. un-aged strains by a secondary competition experiment
exoU
Parentstrains
(clinical)
n=3
exoS n=3
Recombination Antibiotic
Selection
exoS
n=3
exoU
n=3
Mutants(PC*)
Every 24 hrs
Day 7
Collection of PC* colony
Primary Competition
PC* vs. Parent
Secondary Competition
Aged-PC* vs. Un-aged PC*
The average mutant:parent CFU ratio per day of the experiment. Results are plotted
on a log scale. Points above zero indicate the mutant is more fit, while below zero
indicate a fitness cost. The ratio for exoS strains generally decreases over the course
of the experiment, while the ratios for exoU strains either increases or remains stable.
Results are an average of 7 independent experiments. Error bars=SEM.
exoU-PC* Mutants Can Better Maintain Wild-Type
Supercoiling Levels
At 1/4 and 1/2 MIC, fold changes in exoU strains 92 and 37 are significantly different from
those of exoS strain 215 (p=0.004 for both). At 1x MIC, both exoU strains have significantly
different fold changes than both exoS strains (92 vs. 139, p=0.02; 92 vs. 215, p=0.03; 37
vs. 139, p=0.03; 37 vs. 215, p=0.04). Results are an average of 3 independent
experiments, and error bars represent SEM.
Dramatic Difference in Fitness of exoU vs. exoS Aged
PC* Strains
37-PC
*
92-PC
*
-4
-2
0
2
4
6
8
Fold
139-PC
*
215-PC
*
-150
-100
-50
0
Fold
exoU exoS
Fold Change in Fitness After Aging
Fold difference in normalized luminescence of
PC* vs. parent strains