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Journal of Bacteriology, August 2004, p . 5538-5542, Vol . 186,
No . 16
Polymorphic Mutation Frequencies in Escherichia coli: Emergence of Weak
Mutators in Clinical Isolates
María-Rosario Baquero,1 Annika I . Nilsson,2,3
María del Carmen Turrientes,1 Dorthe Sandvang,4 Juan
Carlos Galán,1 Jose Luís Martínez,5 Niels Frimodt-Møller,4
Fernando Baquero,1* and Dan I . Andersson2,3
Hospital Ramón y Cajal,1 National Center for Biotechnology,
Madrid, Spain,5 Swedish Institute for Infectious Disease Control,2
Karolinska Institute, Solna, Sweden,3 Statens Serum Institut,
Copenhagen, Denmark4
Received 24 February 2004/ Accepted 18 May 2004
Polymorphisms in the rifampin resistance mutation frequency (f)
were studied in 696 Escherichia coli strains from Spain,
Sweden, and Denmark . Of the 696 strains, 23% were weakly hypermutable
(4 x 10–8
f < 4 x 10–7), and 0.7%
were strongly hypermutable (f
4 x 10–7) . Weak mutators were
apparently more frequent in southern Europe and in blood isolates
(38%) than in urinary tract isolates (25%) and feces of healthy
volunteers (11%) .
Microbial evolution is dependent on two opposing forces, the
maintenance of genetic information and the generation of some
suitable level of genetic variation on which selection can act . In
most cases, genetic variation is assured by errors in DNA
replication, determined by the accuracy of DNA polymerases and
various DNA repair systems . Particular environmental characteristics
will influence selection of the optimal amount of genetic variation
for a given organism with a specific population structure . If the
environment changes rapidly in time or is heterogeneous, variants
with increased mutation rates will tend to be selected, since they
have an increased probability of forming beneficial mutations .
Conversely, if the environment is constant, as the organism becomes
maximally adapted, mutation rates tend to decrease because of the
costs associated with deleterious mutations (4,
6, 7) . These considerations suggest that
environment-dependent polymorphisms in mutation frequency can be
expected in nature .
Mutation frequencies were determined in a collection of 696
Escherichia coli strains obtained from 2000 to 2003 . Of the 696
E . coli strains, 300 were from Spain (100 from positive urine
cultures, 100 from blood cultures, and 100 from the stools of young
healthy volunteers), 170 were from Denmark (blood cultures), and 226
were from Sweden (urinary tract cultures from outpatients) . Each
Luria-Bertani (LB) tube was inoculated with an independent colony
obtained from a blood agar plate; three LB tubes were used . After 24
h of incubation, appropriate dilutions were seeded onto LB agar
plates and LB agar plates containing rifampin (100 µg/ml), and colony
counts were performed after 24 or 48 h, respectively . Mutation
frequencies are reported as a proportion of the number of
rifampin-resistant colonies to the total viable count . The results
corresponded to the mean value obtained in three independent
experiments that were repeated in cases of suspected jackpots .
Categories were established considering the distribution of
frequencies of the 696 E . coli strains (Fig . 1) . A
strain was considered normomutable when the mutation frequency (f)
was at or close to the modal point of the distribution of mutation
frequencies; for practical purposes, it was established as 8
x 10–9 < f < 4
x 10–8 . Strains were considered
weak mutators if their frequency was 4 x
10–8
f < 4 x 10–7
and strong mutators if f
4 x 10–7 . Hypomutable
strains were defined as strains with f
8 x 10–9 .
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FIG . 1 . Distribution of rifampin resistance mutation frequencies of 696
E . coli strains isolated from patients and healthy volunteers
from Spain, Sweden, and Denmark . The horizontal arrows show the
frequency ranges defining hypomutable strains (H), weakly hypermutable
strains (W), and strongly hypermutable strains (S) . The peak in the
distribution corresponds to the normomutable strains . 1.E-9, 1.0
x 10–9.
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A sharp peak in the frequency distribution was always found at 10–8 .
From this value, a few strains had lower mutation frequencies, down
to 10–9 . To the right of the modal peak, an unexpectedly
high number of strains (23%) show moderately increased mutation
frequencies . All five strong mutators detected in the collection of
696 strains (0.7%) had rifampin resistance mutation frequencies
greater than 10–6 . Luria-Delbrück fluctuation tests (5,
8) were performed with a sample of 12 strains each
from the three different mutation frequency classes, and the
differences between the mutation rates of the strains were fully
confirmed . Differences in the proportions of the different categories
were statistically evaluated by using the Kruskal-Wallis, Dunn, and
Mann-Whitney tests .
In the collection of 300 E . coli isolates from Spain (Fig.
2), the main peak of normomutable strains accounted
for 59% of the strains from blood cultures, 68% of the strains from
urine samples, and 69% of the fecal strains from healthy volunteers .
For the same three groups, weak mutators represented 38, 25, and 11%
of the strains, respectively . Differences were statistically
significant between blood and urine samples from healthy volunteers (P
< 0.001) and between blood and urine samples in general (P =
0.03) . We found only one strong mutator strain in three collections
of clinical isolates (1% in the whole series) . Hypomutable strains
were isolated in significantly higher proportions in the E . coli
isolates from healthy volunteers (19%) than from strains from blood
cultures and urinary tract infections (6 and 2%, respectively) (P
< 0.001) .
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FIG . 2 . Distribution of rifampin resistance mutation frequencies of 300
Spanish E . coli isolates according to the isolate origin: feces
from healthy volunteers (100 isolates) (white bars), urinary tract
infections (100 isolates) (shaded bars), and blood cultures (100
isolates) (black bars) . 1-4E-9, 1 x
10–9 to 4 x 10–9.
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In the series of 226 E . coli strains isolated from positive
urine cultures from patients in Sweden, 62% of the strains were
normomutable and 26% were weak mutators (Fig . 3a) . The
distribution of Swedish isolates was similar to that of the Spanish
isolates, but Spanish weak mutators tended to have higher frequencies
of mutation than the Swedish ones . Finally, 12% of the strains
were hypomutable . Only one strong-mutator strain (0.4%) was detected .
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FIG . 3 . (a) Distribution of rifampin resistance mutation frequencies of
uropathogenic E . coli isolates from 226 Swedish (shaded) and 100
Spanish (black) patients . (b) Distribution of E . coli mutation
frequencies of bacteremic isolates from 170 Danish (shaded) and 100
Spanish (black) patients . 1-4E-9, 1 x
10–9 to 4 x 10–9.
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In the collection of 170 E . coli strains from bacteremic patients
in Denmark (Fig . 3b), 60% of the strains were in the
normomutable category, but 23% were hypomutable strains . Sixteen
percent of the strains were weak-mutator strains, and only one
strong-mutator isolate (0.6%) was detected . The proportion of weak
mutators among Danish blood isolates was significantly lower than
that found in Spanish blood isolates (P < 0.001) .
These data indicate that there may be geographical differences in
the E . coli mutation frequency distribution profile, but
differences due to different types of hospitals cannot be ruled out .
How important antibiotic therapy is in selecting hypermutable E .
coli remains an open question . No correlation between mutation
rates and antibiotic resistance has been shown in uropathogenic E .
coli strains (3) . Nevertheless, it has been shown that in
E . coli, strains with increased mutation rates correlate with
strains showing high-level ciprofloxacin resistance (9) .
We tested ciprofloxacin (E-test; AB Biodisk, Solna, Sweden) using
a resistance breakpoint of 1 µg/ml (15) in the series
of urinary tract pathogens from patients in Sweden: 15% of hypermutable
strains were ciprofloxacin resistant, a proportion slightly
higher but not significantly different from the 12% proportion of the
entire collection .
As shown in Fig . 4, hypermutable strains (23% of the total)
are almost evenly distributed along the full range of MICs . We
studied ciprofloxacin resistance in a second series of 75 Spanish
strains with a higher proportion (52%) of hypermutable strains,
including all four strong mutators . Supporting the first observation,
the overall rate of ciprofloxacin resistance was 15%, and the rate of
ciprofloxacin resistance for hypermutable strains was 13%, with the
distribution of hypermutable strains along the MIC range similar to
that of the Swedish strains (Fig . 4) . Thus, we were
unable to find any significant association in our strains between a
mutator phenotype and ciprofloxacin resistance in countries with a
low (Sweden) or high (Spain) prevalence of fluoroquinolone
resistance . Similar results were obtained with nalidixic acid (data
not shown) . We might suggest that for E . coli of community
origin, fluoroquinolone resistance frequently arises and evolves
among nonhypermutable strains, but in hospitals, clones under
continued antibiotic challenges could favor hypermutators (11,
13, 14, 16,
18) .
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FIG . 4 . Distribution of normomutable (white), weakly hypermutable
(shaded), and strongly hypermutable (black) E . coli strains among
isolates inhibited by different ciprofloxacin concentrations . (a)
Distribution of the 226 uropathogenic E . coli strains isolated
from Swedish patients . (b) Distribution of 75 E . coli strains
with an enriched proportion of hypermutable strains isolated from
Spanish patients.
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The frequency of strong mutators in our series roughly corresponds to
what was found in previous series, around 1% (10) . Less
attention has been paid in the literature to weak mutators . Using our
categorization criteria, we found that the proportion of weak
mutators is around 25% in the series of strains from France (12) .
These proportions are far higher than the proportions that could be
expected by random mutation of the genes that stringently maintain
the normal mutation frequency . As hypermutability is not an advantage
by itself, the abundance of strains with increased mutation frequency
ought to be maintained by positive selection (17) .
In dense populations, as in the case of E . coli, advantageous
mutations will tend to appear in weak mutators, and selection will
therefore enrich low mutating organisms . The success of weak mutators
may prevent further fixation of strong mutators (2) .
Hospital-based and pathogenic (blood cultures) strains have a
higher exposure to antibiotic or host-to-host transmission challenges
than commensal community-based organisms . Similar arguments based on
more frequent host-to-host or host-environment transmission
(sociology and climate), and/or higher antibiotic consumption (1)
could be applied to explain differences in different geographical
location, emphasizing the importance of local biology in the
mechanisms involved in E . coli evolution .
This work was supported by the grant QLK2-CT-2001-873 from the
European Commission .
We thank R . Cantón, E . Loza, R . del Campo, and T . Coque for
providing strains from Spain .
* Corresponding author . Mailing address: Servicio de
Microbiología, Hospital Ramón y Cajal, Ctra . de Colmenar, Km 9.1, 28034 Madrid,
Spain . Phone: 34-91-3368330 . Fax: 34-91-3368809 . E-mail: fbaquero.hrc@salud.madrid.org.
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