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original article

Effect of Estrogen on Pseudomonas Mucoidy

and Exacerbations in Cystic Fibrosis
Sanjay H. Chotirmall, M.B., B.Ch., Stephen G. Smith, Ph.D.,
Cedric Gunaratnam, M.B., B.Ch., Sonya Cosgrove, Ph.D., Borislav D. Dimitrov, Ph.D.,
Shane J. ONeill, M.D., Brian J. Harvey, Ph.D., Catherine M. Greene, Ph.D.,
and Noel G. McElvaney, M.B., B.Ch.

A bs t r ac t
Background
From the Respiratory Research Division,
Department of Medicine (S.H.C., S.C.,
S.J.O., C.M.G., N.G.M.), the Department
of General Practice (B.D.D.), and the Department of Molecular Medicine (B.J.H.),
Royal College of Surgeons in Ireland; the
Department of Respiratory Medicine,
Beaumont Hospital (S.H.C., C.G., S.J.O.,
N.G.M.); and the Department of Clinical
Microbiology, School of Medicine, Trinity
College Dublin (S.G.S.) all in Dublin.
Address reprint requests to Dr. Greene at
the Respiratory Research Division, Department of Medicine, Royal College of Surgeons in Ireland, Education and Research
Centre, Beaumont Hospital, Dublin 9,
Ireland, or at cmgreene@rcsi.ie.
Drs. Greene and McElvaney contributed
equally to this article.
This article (10.1056/NEJMoa1106126) was
published on May 20, 2012, at NEJM.org.
N Engl J Med 2012;366:1978-86.
Copyright 2012 Massachusetts Medical Society.

Women with cystic fibrosis are at increased risk for mucoid conversion of Pseudomonas
aeruginosa, which contributes to a sexual dichotomy in disease severity.
Methods

We evaluated the effects of estradiol and its metabolite estriol on P. aeruginosa in vitro
and in vivo and determined the effect of estradiol on disease exacerbations in women
with cystic fibrosis.
Results

Estradiol and estriol induced alginate production in P. aeruginosa strain 01 and in

clinical isolates obtained from patients with and those without cystic fibrosis. After
prolonged exposure to estradiol, P. aeruginosa adopted early mucoid morphology,
whereas short-term exposure inhibited bacterial catalase activity and increased levels of hydrogen peroxide, which is potentially damaging to DNA. Consequently, a
frameshift mutation was identified in mucA, a key regulator of alginate biosynthesis
in P. aeruginosa. In vivo levels of estradiol correlated with infective exacerbations in
women with cystic fibrosis, with the majority occurring during the follicular phase
(P<0.05). A review of the Cystic Fibrosis Registry of Ireland revealed that the use of
oral contraceptives was associated with a decreased need for antibiotics. Predominantly nonmucoid P. aeruginosa was isolated from sputum during exacerbations in the
luteal phase (low estradiol). Increased proportions of mucoid bacteria were isolated
during exacerbations occurring in the follicular phase (high estradiol), with a variable P. aeruginosa phenotype evident in vivo during the course of the menstrual cycle
corresponding to fluctuating estradiol levels.
Conclusions

Estradiol and estriol induced mucoid conversion of P. aeruginosa in women with cystic
fibrosis through a mutation of mucA in vitro and were associated with selectivity for
mucoid isolation, increased exacerbations, and mucoid conversion in vivo. (Funded by
the Molecular Medicine Ireland ClinicianScientist Fellowship Programme.)

1978

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Estrogen and Pseudomonas Mucoidy in Cystic Fibrosis

seudomonas aeruginosa is a gram-negative opportunistic pathogen associated with

cystic fibrosis,1 a multisystem genetic disease characterized by defects in the cystic fibrosis transmembrane conductance regulator (CFTR)
protein, which results in recurrent infective exacerbations. Median overall survival among patients with cystic fibrosis in the United States is
39 years, although a sexual dichotomy persists in
disease severity,2-4 with women having a survival
disadvantage and poorer lung function.5-7
The Republic of Ireland has the highest incidence and carrier rate of cystic fibrosis worldwide8
and a survival disadvantage for women.9 Patients
with cystic fibrosis become colonized and infected
with nonmucoid P. aeruginosa; these strains convert
to mucoid strains, on average, at a younger age
in women than in men.3,10,11 In an attempt to
explain these observations, we and others have
investigated the effect of the female sex hormone
estradiol on airway biology. Women with cystic
fibrosis appear to be at greater risk for exacerbations during periods of high circulating levels of
estradiol, when airway-surface liquid is diminished and antimicrobial peptides are scarce and
there is a blunted inflammatory response to microbial agonists.12-14 However, these observations
have not been confirmed in vivo. In addition, the
role of estradiol-derived metabolites in cystic fibrosis remains largely unknown, with limited data
linking estradiol with pseudomonas infection.14
The persistence of P. aeruginosa in cystic fibrosis
has been attributed to hypermutability, drug resistance, loss of virulence, reduced bacterial killing, and defective CFTR.14-16 Antibiotics delay but
do not prevent mucoid conversion. Of equal significance is the capacity of P. aeruginosa to form
biofilms and convert to mucoidy in vivo.
Alginate, the predominant polysaccharide produced by P. aeruginosa, defines its mucoid conversion; it is implicated in pathogenicity and can
induce parenchymal damage.17 Nonmucoid
P. aeruginosa can be induced to release alginate
under conditions characteristic of the lung milieu
in patients with cystic fibrosis.18,19 The production
of biofilms is influenced by alginate, but the
presence of the polysaccharide is not essential for
biofilm formation.20,21 The principal gene controlling alginate biosynthesis, mucA, encodes an antisigma factor that normally prevents algT from
inducing expression of algD.22,23 Alginate production and mucoid conversion are attributed to

loss-of-function mutations in mucA, resulting in

derepression of algT and concomitant induction
of algD.
The sex-specific effects of P. aeruginosa in
women with cystic fibrosis may be attributed to
estradiol.12-14 Here we evaluate the effect of estradiol and its metabolite estriol on laboratory
and clinical isolates of P. aeruginosa and prospectively assess the effect of estradiol on infective
exacerbations in women with cystic fibrosis.

Me thods
Laboratory Testing

Full details with respect to the P. aeruginosa strain

01 (PA01),24 clinical isolates, culture conditions,
bronchoscopy, cell culture, quantitative reversetranscriptasepolymerase-chain-reaction assay,
DNA sequencing of the P. aeruginosa phenotype,25
and assays of catalase, hydrogen peroxide, alginate,26 and estradiol are provided in the Supplementary Appendix, available with the full text of
this article at NEJM.org.
Patients

From July 2008 through July 2010, we recruited all

female patients with cystic fibrosis who presented
to Beaumont Hospital in Dublin (44) with a total of
139 infective exacerbations, as defined by the criteria of Fuchs et al.27 (Table S3 in the Supplementary
Appendix). All patients provided written informed
consent, and appropriate approval was obtained
from the hospitals institutional review board.
Statistical Analysis

Data were analyzed by descriptive, exploratory

(comparative), and modeling approaches and
methods, including a technique of generation of
random numbers. All results are expressed as absolute numbers and percentages, as well as means
SD or means SE. We used the chi-square test
or Fishers exact test to compare categorical data
distributions and KolmogorovSmirnov or ShapiroWilk methods, as appropriate, to test continuous
data for normality of distribution. We used parametric methods and tests (e.g., Students t-test) to
analyze normally distributed data and two-group
nonparametric Wilcoxon signed-rank and Mann
Whitney U tests to analyze data deviating from a
normal distribution. We used the nonparametric
KruskalWallis test for the comparison of more
than two independent groups. To describe exist-

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1979

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R e sult s

A Alginate Production
250

Ethanol

Testosterone

Alginate (g/ml)

200

Estradiol
*

Effect of Estrogen on Alginate Production

Estriol

*
* *

150
*

100
50
0

PA01 (3 days)

PA01 (4 wk)

CF (3 days)

Non-CF (3 days)

B Mucoid Conversion

Vehicle Control

Estradiol

Testosterone

Figure 1. Alginate Production and Mucoid Conversion in Pseudomonas

aeruginosa Induced by Estradiol and Estriol.
In Panel A, P. aeruginosa strain 01 (PA01) or P. aeruginosa clinical isolates
from patients with and those without cystic fibrosis (CF) were treated for
either 3 days or 4 weeks with daily subculture in fresh broth containing ethanol, testosterone, estradiol, or estriol (10 nM for each subculture). Alginate
was measured with the use of the uronic acidcarbazole reaction method
in three experiments. Data are expressed as means, with T bars indicating
the standard deviation. The asterisk indicates P<0.05 for the comparison
with ethanol or testosterone; the dagger indicates P<0.01 for the comparison between 3 days and 4 weeks of treatment with estradiol in PA01. Alginate levels were not determined in PA01 at 4 weeks in response to estriol or
in CF and non-CF isolates at 3 days in response to testosterone. In Panel B,
PA01 was plated onto blue agar after daily subculture for 4 weeks in fresh
broth cultures containing ethanol (vehicle control), estradiol, or testosterone
(10 nM of each substance). Representative culture plates are shown for three
experiments. Mucoid colonies are indicated by arrows.

ing linear trends over years, we calculated the

coefficients of the models (intercepts, slopes, and
squares of the correlation coefficient [R2]). Statistical analyses were performed with SPSS software, version 18 (IBM); GraphPad Prism, version
4.0 (GraphPad Software); and modeling tools as
integrated in Microsoft Office Excel 2007, version
SP2 MSO (Microsoft). All reported P values are
two-sided, and a P value of 0.05 was considered
to indicate statistical significance, unless stated
otherwise.

1980

At baseline, PA01 is nonmucoid. However, it can be

induced under appropriate conditions to produce
alginate.24,28 We examined the effect of estradiol
and estriol on alginate production in PA01 and in
clinical isolates of P. aeruginosa obtained from patients with and those without cystic fibrosis (Fig.
1A). Testosterone (an estradiol precursor) and ethanol were used as controls. After 3 days of exposure, 10 nM of estradiol induced more alginate production by PA01 (33.5 g per milliliter) than did
the same amounts of testosterone (9.6 g per
milliliter) and ethanol (9.5 g per milliliter) (P<0.05
for both comparisons). The increase in alginate
production was time-dependent, with increased
levels (92.2 g per milliliter) produced after 4 weeks
in continued subculture with estradiol (P<0.01 for
both comparisons). Testosterone had no effect on
alginate production at 4 weeks. After 3 days, estriol
induced significantly more alginate production
(143.1 g per milliliter) than an equal amount of
estradiol (P<0.005). Nonmucoid clinical isolates of
P. aeruginosa similarly produced more alginate after treatment with 10 nM of estradiol or estriol
for 3 days (P<0.005). Estradiol up-regulates the
expression of algD, algT, and mucA in PA01 (Fig. S1
in the Supplementary Appendix), and estriol was
detected in samples of estradiol-treated, immortalized bronchial epithelial cells obtained from
patients with and those without cystic fibrosis and
in bronchoalveolar-lavage fluid from patients with
cystic fibrosis (Fig. S2 and S3 in the Supplementary Appendix).
Mucoid Conversion of P. aeruginosa

Blue agar plates were used to detect mucoid colonies.25 After daily subculture for 3 days in fresh
broth containing estradiol, testosterone, or ethanol
with daily replenishment, no differences in morphology were observed. However, after 4 weeks,
early mucoid colony formation was noted in the
estradiol-treated cultures (Fig. 1B). More mucoid
colonies were detected in the estradiol-treated
cultures (5.5108 colony-forming units [CFU] per
milliliter for mucoid colonies vs. 2.9109 CFU per
milliliter for nonmucoid colonies), than in ethanoltreated cultures (3.3108 CFU per milliliter for
mucoid colonies vs. 5.0109 CFU per milliliter for
nonmucoid colonies) or testosterone-treated cul-

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Estrogen and Pseudomonas Mucoidy in Cystic Fibrosis

Estradiol Levels and Infective Exacerbations

To assess whether fluctuations in estradiol levels

during the menstrual cycle are associated with
infective exacerbations, we performed a 24-month
prospective clinical study in which we assessed
139 exacerbations in 44 women with cystic fibrosis. We excluded patients who reported having
irregular menstrual cycles or the use of oral contraceptives. We plotted 72 exacerbations related
to a regular menstrual-cycle pattern in 23 women
according to the stage of the cycle when the exacerbation occurred. A serum estradiol curve was
prospectively generated from data obtained from
6 women with cystic fibrosis who had a regular
menstrual cycle. Exacerbations closely mirrored in
vivo estradiol levels. Significant increases in the
number of exacerbations occurred during the follicular phase, when estradiol levels peak (P<0.05)
(Fig. 2A, and Table S3 in the Supplementary Appendix).
To validate the relationship between elevated
estradiol levels and the number of infective exacerbations, we assessed serum from 172 patients
with cystic fibrosis during a 36-month period (Fig.
2B, and Table S4 in the Supplementary Appendix). The mean serum estradiol level was significantly higher in women with exacerbations than
in those with stable disease (777 pmol per liter

No. of exacerbations

Estradiol level

1400
1200

25

1000

20

800
15
600
10

400

200

24

512

1315

1622

2328

Estradiol Level (pmol/liter)

Conversion to mucoidy within the lungs of patients with cystic fibrosis results from spontaneous mutations in mucA, which was sequenced in
both mucoid and nonmucoid colonies grown in
estradiol and ethanol, respectively. In 80% of mucoid colonies with exposure to estradiol, a deletion
of the nucleotide adenine at position 60 of mucA
occurred, resulting in a frameshift mutation of
glutamic acid (E) to aspartic acid (D) at residue
20 of the MucA protein and the introduction of a
premature stop codon (Fig. S4 in the Supplementary Appendix). One mechanism by which this
occurs involves estradiol-induced inhibition of
catalase activity and increased production of hydrogen peroxide in P. aeruginosa (Fig. S5 in the
Supplementary Appendix).

30

No. of Exacerbations

Genetic Mutations and Mucoid Conversion

A Exacerbations According to Cycle Phase

Days of Cycle
Menstruation/Proliferative/Ovulatory

Secretory

Follicular Phase

Luteal Phase

B Estradiol Levels
Women with CF and OC

Women with CF

Men with CF

***
**

2000

Serum Estradiol (pmol/liter)

tures (3.1108 CFU per milliliter for mucoid colonies vs. 4.53109 CFU per milliliter for nonmucoid
colonies), equating to 15.9%, 6.2%, and 6.4%
mucoid colonies, respectively.

1750
1500
1250
1000
750
500
250
0

Stable

Exacerbation

Figure 2. Exacerbations and Estradiol Levels in Women with Cystic Fibrosis.

Panel A shows the number of infective exacerbations according to the time
of the menstrual cycle, as recorded for 44 women with cystic fibrosis and
plotted against circulating estradiol levels (as measured in 6 women with
cystic fibrosis). Shown are means and the range of values. The asterisk indicates P<0.05 for the comparison between the proliferative phase of the
menstrual cycle (the highest estradiol level) and the menstrual, ovulatory, or
secretory phase. Panel B shows serum estradiol levels for 172 patients with
cystic fibrosis (CF) classified according to sex and use of oral contraceptives (OC). The double asterisks indicate P<0.01, and the triple asterisks indicate P<0.001.

vs. 303 pmol per liter, P<0.008). This pattern was

absent in women receiving oral contraceptives
and in men. The exacerbation rate per year was
significantly lower in women receiving oral contraceptives than in other groups (Fig. S6 in the
Supplementary Appendix).
Irish Registry Data

Next we analyzed data from the Cystic Fibrosis

Registry of Ireland during years when the rate of

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1981

The

Women receiving OC
Women not receiving OC

n e w e ng l a n d j o u r na l

Linear (women receiving OC)

Linear (women not receiving OC)

Mean No. of Antibiotic Courses

9
8
y=0.9308x+4.1255

R2=0.8552

6
5

y=0.5462x+5.6864
R2=0.7439

4
3
0

2006

2007

2008*

2009**

Figure 3. Effect of Oral Contraceptives on the Number of Courses

of Antibiotics Required per Year in Women with Cystic Fibrosis.
Available data from the Cystic Fibrosis Registry of Ireland for 239 women
(18 years of age) were obtained for the years 2001 through 2010, but only
years when registry ascertainment was more than 55% (20062009) are
shown. The mean numbers of courses of antibiotics (oral or intravenous)
that were received for clinical deterioration, as determined by the local attending physician, are shown for 36 women receiving oral contraceptives
(OC), as compared with a random sample of 41 women not receiving oral
contraceptives (approximately 20% of the sample), for the respective time
periods. Opposing trends were detected between the two groups. The
model equations present the coefficients of linear regression: intercept a,
slope b, and the square of the correlation coefficient (R2) for the decreasing
tendency of women receiving oral contraceptives to require antibiotics
(orange line) versus the increasing tendency of women not receiving oral
contraceptives to require antibiotics (green line). On the basis of annual
comparisons by means of the MannWhitney nonparametric test, a single
asterisk indicates P=0.07, and double asterisks indicate P=0.002.

ascertainment (i.e., the proportion of registered

patients vs. the census population of persons with
cystic fibrosis) was more than 55% (2006 through
2009) among women 18 years of age or older, according to their use of oral contraceptives (Table
S8 in the Supplementary Appendix). Of the 239
patients whose data were analyzed, 15.1% used oral
contraceptives, although the type of oral contraceptive was not recorded. For standardization and
because definitions of exacerbations vary among
centers, we assessed the mean number of antibiotic courses (either oral or intravenous) required
for the treatment of clinical deterioration and its
relationship to the use of oral contraceptives. In
order to compare the 36 women receiving oral
contraceptives with those not receiving oral contraceptives in an approximate ratio of 1:1, we selected a random sample of 41 control women from
the remaining 203 women in the registry (approx-

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imately 20%). We found a decreasing tendency to

require antibiotics in women receiving oral contraceptives, as compared with those not receiving
oral contraceptives (Fig. 3). These differences were
most significant where registry ascertainment
was highest (Table S8 in the Supplementary Appendix). There was a significant reduction in the
mean number of antibiotic courses required during times when individual women were receiving
oral contraceptives, as compared with times they
were not receiving oral contraceptives (Fig. S7 in
the Supplementary Appendix).
Sex Differences in Colonization and Mucoid
Conversion

In patients with stable cystic fibrosis who were

colonized with P. aeruginosa, mucoid P. aeruginosa
was isolated at twice the frequency in women as
in men as a proportion of total culture counts
(26.2% mucoid cultures in 6 women vs. 10.0% in
4 men) (Table S6 in the Supplementary Appendix). To assess rates of P. aeruginosa colonization
and mucoid conversion, we explored all data from
the Irish registry up to December 31, 2010 (involving 455 women and 554 men) (Table S9 in the
Supplementary Appendix). Chronic colonization
was defined as isolation of P. aeruginosa from at
least two sputum samples obtained 6 months apart
(in 50% of samples) in consecutive years, the
first of which was recorded as the year of acquisition. In this analysis, 375 of 1009 patients (37.2%)
were colonized by P. aeruginosa, with a mean (SD)
age of acquisition of 20.89.0 years, as defined by
the modified Leeds criteria.29 The female patients
had significantly higher colonization rates (absolute difference, 11.9 percentage points; P<0.001)
and acquired it 2.3 years earlier (19.7 vs. 22.0 years,
P=0.01) (Table S9 in the Supplementary Appendix).
P. aeruginosa converted to mucoidy in significantly
more female patients (Fig. S8 in the Supplementary Appendix), and the conversion occurred 1.8
years earlier in women than in men (Table S9 in
the Supplementary Appendix).
P. aeruginosa Phenotype According to
Menstrual Cycle

We recorded the predominant P. aeruginosa phenotype in sputum samples obtained from patients
in the prospective study. Of the 72 exacerbations in
23 patients who were included in the final analysis,
P. aeruginosa was not isolated from 4 patients in

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Estrogen and Pseudomonas Mucoidy in Cystic Fibrosis

Mucoid Conversion and Pregnancy

In further support of the hypothesis that estradiol

and estriol are important factors in mucoid conversion of P. aeruginosa in vivo, we also assessed the
physiologically estriol-dominant state of pregnancy. We assessed patients who were colonized
with nonmucoid P. aeruginosa before pregnancy for
the time to mucoid conversion after pregnancy.
During a 15-year period (19962010), this occurred
in 4 of 7 pregnancies (57.1%) within 12 months
after delivery and in 5 of 7 pregnancies (71.4%)
within 24 months (in a total of 11 pregnancies)
(Table S10 in the Supplementary Appendix). Data
from the Irish registry from 2001 through 2010
(involving 14 pregnancies) showed that mucoid
conversion occurred in 5 of 7 pregnancies (71.4%)
within 12 months after delivery and in 6 of 7
(85.7%) within 24 months (Table S10 in the Supplementary Appendix).

A Mucoidy According to Cycle Phase

100

Mucoid

Nonmucoid

Patients (%)

75

50

25

All

Follicular

Luteal

B Mucoidy According to Estradiol Level

1000
3.0106

750
NM
500
E2

1.5106

250
0

M
7

14

21

28

Serum Estradiol (pmol/liter)

4.5106

Pseudomonas (CFU/ml)

15 exacerbations. Using data from the remaining

patients, we plotted the percentage of nonmucoid
versus mucoid P. aeruginosa according to the menstrual-cycle phase when the exacerbation occurred.
Mucoid P. aeruginosa accounted for 63.0% of the
isolates in the follicular phase (high estradiol), as
compared with 16.7% nonmucoid isolates. In the
luteal phase (low estradiol), we isolated no mucoid P. aeruginosa, whereas 72.2% of the samples
contained nonmucoid P. aeruginosa (Fig. 4A).
To further assess this relationship, we recorded changes in the proportion of mucoid and nonmucoid P. aeruginosa, using serially diluted sputum
samples obtained from six women with cystic fibrosis and stable menstrual cycles who were
known to be colonized by both organisms. In
these women, we observed an increase in mucoid
isolation at times of high estradiol levels (Fig. 4B,
and Fig. S9 in the Supplementary Appendix). This
finding explains the lack of mucoid isolation
during the luteal phase that was observed in our
undiluted sputum samples (Fig. 4A). We also
assessed changes in mucoid and nonmucoid proportions in sputum samples during stable and
exacerbation states. During exacerbations, nonmucoid counts tripled, as compared with the
stable state, whereas mucoid counts increased
by a factor of more than 50. These changes occurred in the presence of an increased serum
estradiol level (Fig. S10 in the Supplementary
Appendix).

Day

Figure 4. Mucoid and Nonmucoid Pseudomonas

Species and Estradiol Levels in Women with Cystic
Fibrosis.
Panel A shows the percentage of patients who were
colonized with mucoid versus nonmucoid pseudomonas
3 col
16p6
species during exacerbations throughout the menstrual
cycle. Pseudomonas species were cultured from undiluted sputum samples obtained from women with cystic
fibrosis who were undergoing exacerbations (54 in the
follicular phase and 18 in the luteal phase) and identified as either mucoid or nonmucoid. Panel B shows
the in vivo variability of the P. aeruginosa phenotype in
relation to levels of serum estradiol (E2) during the
course of the menstrual cycle in 6 women with stable
cystic fibrosis and regular menses, according to whether
the sample was mucoid (M) or nonmucoid (NM).

Discussion
It has been observed that women with cystic fibrosis acquire P. aeruginosa in advance of men and
convert to mucoid strains prematurely.3,5,10,11 In
our study, we found that estradiol promoted mucoid conversion of P. aeruginosa, increased algi-

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1983

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nate production in P. aeruginosa, and (owing to

impaired catalase activity and increased levels of
hydrogen peroxide) selected for mutations in mucA,
a negative regulator of alginate synthesis. In vivo
estradiol levels correlated with infective exacerbations among menstruating women with cystic
fibrosis, and mucoid P. aeruginosa was selectively
grown during periods with high levels of circulating estradiol.
Microbial endocrinology suggests that estradiol
may directly affect the development of prokaryotes.30,31 Proteinaceous estrogen-binding receptors
have been reported in P. aeruginosa and Escherichia
coli.32-34 P. aeruginosa can actively metabolize steroid
hormones35 and use estradiol as a carbon source.36
In our study, estriol, although less potently estrogenic than estradiol, was a stronger inducer
of alginate. Airway epithelial cells can metabolize
estradiol to estriol, which was also detected in
samples of bronchoalveolar-lavage fluid obtained
from women with cystic fibrosis. Other airway
organisms and inflammatory cells may contribute
to this pool of estriol. Although estriol was detected in samples of bronchoalveolar-lavage fluid
obtained from men with cystic fibrosis, the levels
were significantly lower than those obtained from
women.
Nonmucoid PA01 can produce alginate18 and is
a suitable model for the study of alginate production.24,28 Estradiol significantly increased alginate
production related to early mucoid morphology on
blue agar after 4 weeks.37 In translating our findings to the airways of women with cystic fibrosis,
we found that clinical isolates of P. aeruginosa produced higher baseline and estradiol-induced alginate levels than did PA01. Thus, long-term exposure of P. aeruginosa to estradiol after menarche
from repeated menstrual cycling places women
with cystic fibrosis at increased risk for mucoid
conversion. The high rates of mucoid conversion
among women in the Irish registry and after pregnancy support this finding.
We also identified a mutation in mucA that
leads to mucoidy. Neutrophils in the lungs of patients with cystic fibrosis release hydrogen peroxide, a DNA-damaging agent that can result in
mutations in mucA.38 Estradiol can independent
ly generate hydrogen peroxide and other free
radicals.39,40 Consequently, we assessed the effect of estradiol on hydrogen peroxide generation by P. aeruginosa. We found elevated hydrogen peroxide levels after short-term exposure of
P. aeruginosa to estradiol, and the activity of cata1984

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lase, the enzyme that detoxifies hydrogen peroxide, was inhibited.

Among patients with cystic fibrosis, mucoid
P. aeruginosa is observed at higher frequencies in
women than in men, and female sex is recognized
as a risk factor for early conversion.10,11 The median age of chronic infection with mucoid P. aeruginosa has been reported to be 1.7 years earlier in
women, which accelerates the rate of decline in
pulmonary function.3 The age-specific prevalence
of mucoid P. aeruginosa in patients with cystic fibrosis markedly increases after puberty.1 In the
United States, a casecontrol analysis of the Cystic Fibrosis Foundation registry confirmed that an
earlier age of P. aeruginosa infection was associated
with increased odds of severe lung disease, a relationship that was found to be stronger in women.41
Although no significant differences were detected
in the prevalence of chronic P. aeruginosa infection
in a Scandinavian population, the incidence of new
infection was higher in women.5 An assessment of
the Irish registry showed that rates of P. aeruginosa
colonization and mucoid conversion were higher
in women than in men, along with earlier ages of
acquisition and mucoid conversion.
To investigate the link between estradiol, infective exacerbations, and mucoidy in women with
cystic fibrosis, we conducted a clinical study relating infective exacerbations to the day in the menstrual cycle when the exacerbation occurred. A
significant relationship between exacerbations and
estradiol levels was evident, with the majority of
exacerbations occurring during the follicular
phase, a stage with the highest estradiol level.
At our institution, women with cystic fibrosis
who were receiving oral contraceptives had lower
rates of exacerbation. Using registry data, we
found that women receiving oral contraceptives
required a lower number of antibiotic courses than
did women not receiving oral contraceptives and
that individual women required less use of antibiotics during the time they were receiving oral
contraceptives than during the time they were not
receiving oral contraceptives. Although oral contraceptives may be protective against exacerbations,
a double-blind, placebo-controlled study of the use
of oral contraceptives in women with cystic fibrosis would be necessary to provide proof. Future
work should also account for the various subtypes
of oral contraceptives, colonization with other
organisms, and their antibiotic resistances.
During our clinical study, although mucoid
bacteria were predominantly isolated during high-

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Estrogen and Pseudomonas Mucoidy in Cystic Fibrosis

estradiol phases of the menstrual cycle, as compared with nonmucoid bacteria in low-estradiol
phases, proportional differences between the
P. aeruginosa phenotypes were evident, a finding
that illustrates a selective isolation of mucoid
P. aeruginosa during periods of high levels of circulating estradiol. Although much has been learned
about the role of estradiol within the airways of
patients with cystic fibrosis, further work is necessary, particularly in the assessment of the use of
oral contraceptives and its potential effect on exacerbations and the microbial endocrine effects of
estradiol on P. aeruginosa.
Supported by the Higher Education Authority Programme for
Research in Third Level Institutions (PRTLI) Cycle 4 through a

grant (20082011) from the Molecular Medicine Ireland ClinicianScientist Fellowship Programme.
Disclosure forms provided by the authors are available with
the full text of this article at NEJM.org.
We thank Dieter Gruenert of the California Pacific Medical
Center Research Institute for providing cystic fibrosis and non
cystic fibrosis in vitro cell lines; Daniel J. Wozniak and Sheri R.
Dellos-Nolan of Ohio State University for their protocols and
assistance in performing alginate assays; the patients and their
families for participation in this study; the clinical and laboratory staff, particularly the specialist nurses (Cassandra
ODonohoe, Anne-Marie Lyons, and Claire Bolton) for their assistance in collating data from patients; microbiology technician Mary OConnor for the identification of P. aeruginosa in
clinical specimens; staff members of the Endocrine Laboratory
at Beaumont Hospital (Sarah Doody, Patricia Barrett, and Ciaran
Brennan) for their assistance in measuring serum estrogen concentrations; Chay Bowes, VHI Homecare Dublin, for facilitating
sample collection; and Godfrey Fletcher and the Cystic Fibrosis
Registry of Ireland for providing data and ascertainment details.

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