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Antimicrobial Agents and Chemotherapy, May 2001, p. 1599-1600, Vol. 45, No. 5
0066-4804/01/$04.00+0 DOI: 10.1128/AAC.45.1.1599-1600.2001
LETTERS TO THE EDITOR
Use of Mutator Strains for Characterization of Novel
Antimicrobial Agents
 |
LETTER |
A recent minireview by Martinez and Baquero (8)
provides a useful discussion on various aspects of mutational
resistance to antibiotics in bacteria. As noted by these authors,
bacteria displaying strong mutator (hypermutator) phenotypes exhibit
significantly increased rates of mutation conferring antibiotic
resistance (up to 10,000-fold that of the wild type). We would like to
further highlight the value of such strains for basic antimicrobial
drug discovery research, an aspect that was only partially addressed in
the minireview.
Novel antimicrobial drug candidates are invariably evaluated with
respect to the frequency with which resistant bacterial genotypes arise
in vitro (3, 4, 7, 14). This provides an indication of
whether resistance to the agent is likely to arise rapidly, either
during therapy or within the environment. In addition, mutants
recovered during such determinations may be important for elucidation
of the drug's mode of action (4, 7, 12, 13) and for
predicting the mechanism of resistance that may arise in the clinical setting.
We would like to stress the point touched upon in the minireview that
hypermutators, e.g., Escherichia coli and Salmonella enterica with defects in the mismatch repair pathway (5,
9), should be used alongside wild-type isolates to examine the
frequency with which drug resistance to a particular agent arises. This will yield mutation frequencies that represent worst-case scenarios. In
turn this allows expression of the frequency of mutations conferring resistance as a range, not as a single value.
The rationale is that populations of pathogenic bacteria do not exhibit
homogeneous mutation rates. For example, >1% of natural pathogenic
E. coli and S. enterica populations exhibit a
strong mutator phenotype (5). In addition, 0.0001 to
0.001% of some, and possibly all, bacterial populations are
hypermutators (6), and a single selection event (e.g.,
antibiotic selection) can enrich the mutator population to 0.5% of the
total (6). As Martinez and Baquero (8) point
out, it is therefore erroneous to assume that a bacterial population
exhibits uniform mutation rates. This could be particularly relevant
during infection when in vivo mutation rates may be elevated
(1).
Hypermutator strains may also be used to enhance the recovery of rare
resistance mutations, e.g., for elucidation of modified drug targets
within the cell. We have established that a fully grown 2YT or TB
(11) culture of E. coli reaches cell densities of about 1010 CFU/ml (unpublished data). Resuspension of
this culture in 1/10 the volume and incorporation of 1-ml aliquots in
10 agar pour plates allow mutants arising at frequencies approaching
10
12 to be detected. Using E. coli
hypermutators such as mutS or uvrD mutants, which
exhibit 1,000-fold increases in mutation rate under certain conditions,
allows detection of drug-resistant mutants that effectively occur at
frequencies as low as 1015. Indeed, we have used this
approach to detect rare ampC promoter mutations in E. coli that confer increased ampicillin resistance (unpublished data).
There is little doubt that new antimicrobial agents are needed to
combat the growing problem of antibiotic-resistant bacteria (2,
10). We suggest that hypermutator strains have an important role
in the evaluation of such new agents.
| |
FOOTNOTES |
*
Phone: 44 113 233 5604 Fax: 44 113 233 5638 E-mail: i.chopra{at}leeds.ac.uk
| |
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| | | | |
Alexander John O'Neill
Ian Chopra*
Antimicrobial Research Centre
and Division of
Microbiology
University of Leeds
Leeds LS2 9JT
United
Kingdom
|
| |
AUTHORS' REPLY |
O'Neill and Chopra discuss the possibility of using hypermutator
bacterial strains for enhancing the recovery of rare
antibiotic-resistant bacterial mutants. Hypermutation in bacterial
populations may occur in either a transient or a permanent way. In the
first case, error-prone DNA polymerases may be involved
(5); in the second, mutations of the mismatch repair
system are thought to be implicated (3, 4). Both types of
adaptive strategies may be triggered during the infective process or
during antibiotic therapy (1, 4; M. C. Negri, M. R. Baquero, J. Blázquez, and F. Baquero, Abstr. 40th Intersci.
Conf. Antimicrob. Agents Chemother., abstr. 1918, p. 116, 2000). As we
pointed our in our minireview, the frequency of mutation is highly
dependent on the environment. Thus, conventional mutation rate
determinations carried out on culture tubes may totally fail in
predicting a probability of emergence of mutational resistance under in
vivo circumstances. In other words, very rare mutants or even double
mutants may emerge in vivo if the bacterial population has the adaptive
benefits of hypermutation. Consequently, only the use of hypermutators in the in vitro testing could predict the emergence of resistance in
some cases. As discussed by O'Neill and Chopra, mutants that arise at
frequencies as low as 1015 might be easily selected by
using hypermutator strains. In looking for rare mutants, hypermutator
strains will therefore be very useful, not only for the analysis of
antibiotic resistance but also for the generation of interesting
bacterial mutants showing novel metabolic capabilities with
biotechnological relevance. In fact, biotechnology companies are well
aware of this situation, and Stratagene (La Jolla, Calif.) offers in
its catalogue Epicurean Coli AE XL1-Red Competent Cells, which are
"useful to generate random mutations because they have a mutation
rate 5,000-fold higher than the wild-type parents."
We therefore agree with the suggestion of O'Neill and Chopra. Testing
of the mutants that are selectable from hypermutator strains is needed
for a correct prognosis of the probability of emergence of antibiotic
resistance. This approach enabled us to detect mutations in the
structural gene of the Acidaminococcus beta-lactamase ACI-1,
leading to resistance associated with beta-lactams and beta-lactam
inhibitors, when conventional methods with normal mutators failed
(J. C. Galán, M. R. Baquero, M. Reig, F. Baquero, and
J. Blázquez, Abstr. 40th Intersci. Conf. Antimicrob. Agents Chemother., abstr. 1919, p. 116). Another approach that we have analyzed is the possibility that a given concentration of an
antimicrobial agent will select the hypermutable population in a mixed
population. In the case of Streptococcus pneumoniae, a
hexA-negative hypermutator strain was selected over the
normal mutator in the presence of cefotaxime. Selection was due to the
hitchhiking effect of the cefotaxime-resistant mutation Thr550-Ala that
emerged in the hypermutable population (Negri et al., 40th ICAAC). Note
that this selected variant also has more possibilities to become
resistant to other antibiotics. In summary, we support the
recommendation of O'Neill and Chopra to use hypermutator strains for
predicting mechanisms of action and mechanisms of resistance to new
drugs. We would like to add that these experiments should include
selection at different antibiotic concentrations (2) and,
eventually, prolonged incubation times (1).
| |
FOOTNOTES |
*
Phone: 34 91 5854571 Fax: 34 91 5854506 E-mail: jlmtnez{at}cnb.uam.es
| |
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Emergence of multidrug-resistant mutants is increased under antibiotic selective pressure in Pseudomonas aeruginosa.
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1997.
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B. Li,
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1996.
High mutation frequencies among Escherichia coli and Salmonella pathogens.
Science
274:1208-1211.
|
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Oliver, A.,
R. Canton,
P. Campo,
F. Baquero, and J. Blazquez.
2000.
High frequency of hypermutable Pseudomonas aeruginosa in cystic fibrosis lung infection.
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Radman, M.
1999.
Enzymes of evolutionary change.
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| | | | |
José L. Martínez*
Departamento de
Biotecnología Microbiana
Centro Nacional de
Biotecnología
Campus UAM, Cantoblanco
28049 Madrid,
Spain
|
| | | | |
Fernando Baquero
Departamento de
Biotecnología Microbiana
Centro Nacional de
Biotecnología
and Servicio de
Microbiología
Hospital Ramón y Cajal
Madrid,
Spain
|
Antimicrobial Agents and Chemotherapy, May 2001, p. 1599-1600, Vol. 45, No. 5
0066-4804/01/$04.00+0 DOI: 10.1128/AAC.45.1.1599-1600.2001
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