a Microbiology, Department of Clinical Sciences, School of Medicine, University of Las Palmas de Gran Canaria, Dr. Pasteur s/n, 35080 Las Palmas; b Department of Microbiology, Hospital Clinic, School of Medicine, University of Barcelona, Villarroel 170, 08036 Barcelona, Spain
![]() |
Abstract |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
![]() |
Introduction |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
![]() |
Materials and methods |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Ten clinical isolates of E. coli obtained from the Microbiology Service of the Insular
Hospital of Gran Canaria (Spain) that were susceptible to fluoroquinolones, were used as parent
isolates (the PS isolates). Spontaneous resistant mutants were selected by exposing these isolates
to
increasing concentrations of norfloxacin (the NorE isolates) and lomefloxacin (the LmfE isolates)
until
the MIC of the selective agent was 8 mg/L. E. coli JF699 (OmpA deficient
isolateJ. Foulds isolate), E. coli JF703 (OmpF deficient isolateJ. Foulds
isolate), E. coli KL16 isolate (K. B. Low isolate) (donated by Dr Mary K. B. Berlyn, E.
coli Genetic Stock Center, New Haven, CT, USA)14
and E. coli ATCC 25922 (donated by Dr Federico Uruburu, Spanish Type Culture
Collection,
Valencia, Spain) were also used.
Antibiotics and determination of susceptibility
MICs were determined by an agar dilution method according to the NCCLS guidelines.15 Antimicrobial agents were supplied by their manufacturers: ciprofloxacin (Bayer, Barcelona, Spain), lomefloxacin (G. D. Searle & Co, Madrid, Spain), ofloxacin (Roussel, Madrid, Spain), temafloxacin (Abbott, Madrid, Spain), sparfloxacin (Rhône PoulencRorer, Madrid, Spain) and cephalothin (Lilly, Madrid, Spain). Tetracycline, nalidixic acid and norfloxacin were obtained from Sigma Chemical Co. (Madrid, Spain).
Amplification and DNA sequencing of the quinolone resistance-determining region of the gyrA, gyrB, parC and parE genes
The different gene sequences (gyrA, gyrB, parC and parE) were amplified by the polymerase chain reaction (PCR) using the method and primers previously described.5,6,16 The purified PCR product was processed for DNA sequencing and analysed in an automatic DNA sequencer.
Preparation and analysis of outer membrane proteins (OMP) and lipopolysaccharide (LPS)
Outer membrane extracts were prepared using the method described by Sawai et al.17 Each membrane suspension (10 µg) in loading buffer was electrophoresed in a 6 M urea10% SDS (sodium dodecyl sulphate) polyacrylamide gel. The LPS extracts from intact cells were obtained18 and analysed using 13.5% polyacrylamide gel electrophoresis (PAGE) as described previously.18 The LPS bands were visualized by silver staining.19
Norfloxacin accumulation determination
Uptake measurements were repeated at least three times by the method described by Hirai et
al.9,20
Intracellular
accumulation of norfloxacin was always measured at the same time in each parent isolate and the
quinolone-resistant mutants derived from it, with and without the presence of 50 and 100
µM
carbonyl cyanide m-chlorophenylhydrazone (CCCP). Bacterial cells were grown to
mid-log
phase at 37°C in antibiotic medium 3 (Difco, Madrid, Spain), and each bacterial cell
suspension
was prepared in sodium phosphate buffer (50 mM, pH 7.0) to an optical density of 1.5 at 520 nm
wavelength. The total bacterial suspension of each isolate was divided into three at the beginning
of
each assay, to measure in parallel norfloxacin uptake with and without 50 and 100 µM
CCCP.
CCCP was added to two parts of the bacterial suspension 10 min before norfloxacin was added.21 Each bacterial suspension (8 mL) was collected and
chilled at
5, 15 and 30 min after the addition of norfloxacin 10 mg/L. The cells of each sample were
sedimented
by centrifugation and washed once in 2 ml of sodium phosphate buffer (50 mM, pH 7.0). The
cells
were then suspended in 200 µL of the same buffer. The suspension was immersed in boiling
water
for 7 min and then centrifuged.9,20 The concentrations of norfloxacin in the intracellular extracts were measured by
bioassay
using Klebsiella pneumoniae ATCC 100319,20 (donated by Dr Federico Uruburu, Spanish Type Culture
Collection, Valencia, Spain). The norfloxacin concentration in each intracellular extract was
measured
by bioassay at least six times. The accepted standard deviation for all the uptake results was
always
5% with respect to each mean value of several measurements taken at 5, 15 and 30 min, with
or
without CCCP.
![]() |
Results |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Three consecutive selective steps were needed for the development of mutants, for which the
selective agent MIC was 8 mg/L, except for the NorE1, LmfE1, NorE5, LmfE5 and LmfE7
isolates, which were obtained after two consecutive selective steps. The mutation frequencies of
the
different selective steps with both norfloxacin or lomefloxacin ranged between 109 and 104. Only the most resistant mutants selected (norfloxacin or
lomefloxacin MIC
8 mg/L) were studied (18 isolates).
Determination of MICs
The susceptibilities to various antibiotics of the mutants and their parent isolates are shown in Table I. Many of the selected mutants showed an increased level of resistance to tetracycline and cephalothin as well as to quinolones (Table I).
|
To understand the role of mutations of DNA gyrase and topoisomerase IV in the
development of
resistance to quinolones in the mutants, the sequences of the quinolone resistance-determining
region of
the gyrA, gyrB, parC and parE genes were studied (Table II). This analysis demonstrated that the PS1, PS5, PS7 isolates and
14 of the
quinolone-resistant mutants exhibited a mutation in the gyrA gene (Ser-83Leu)
that
was generated by a C
T transversion in the codon TCG. For the NorE6 and LmfE6 isolates
the
substitution was Asp-87
Asn, which was generated from G
A transversion in the
codon
GAC. In addition, the T
G transversion in the codon TCG resulted in a change of
Ser-83
Ala in the GyrA subunit in the PS10 and NorE10 isolates. For the LmfE10 isolate
the
substitution of Ala-83
Val, which was generated from the codon GCG by a CRT
transversion,
was demonstrated. No double mutations in the gyrA gene were detected. Only one
isolate
(NorE5) was also shown to contain a second mutation at codon 80 (Ser), which was generated by
a
T
A transversion from codon AGT in the parC gene, leading to Ser-80
Arg
substitution. No mutations were detected in the amplified fragment of the gyrB or parE genes.
|
All the parent isolates expressed the C and F porins. The NorE1, NorE2, LmfE2, NorE3, LmfE3, LmfE4, NorE5, LmfE5, NorE8, LmfE9 and NorE10 isolates (Figure) showed decreased expression of an electrophoretic band migrating at the OmpF position. The NorE1 and LmfE9 isolates did not express a band with an electrophoretic mobility in ureaSDS polyacrylamide gels consistent with OmpC (Figure 1). Examination of the outer membrane LPS revealed that all the parent isolates (except the PS5 and PS6 isolates) and most of the quinolone-resistant mutants exhibited smooth LPS, i.e. high molecular mass polymers were expressed. They were not detected in the PS5, NorE5, PS6, PS8 and NorE10 isolates' LPS profiles, resulting in the expression of rough LPS. Similarly, the comparison of LPS profiles from selected mutants with those of their respective parent isolates demonstrated a slightly decreased expression of the latter polymers in the NorE1, LmfE1, LmfE7 and LmfE10 isolates. The LmfE5, LmfE7 and LmfE9 isolates showed a decreased expression of low molecular mass polymers.
|
Eleven mutants (the NorE1, NorE2, LmfE2, LmfE4, NorE5, LmfE5, NorE6, LmfE6, NorE8, NorE9 and LmfE9 isolates) showed a lower accumulation of norfloxacin than their PS isolates; their norfloxacin uptake range was 16 to 77% in comparison with their parent isolates. In the remaining seven mutants the difference in the norfloxacin accumulation was lower than or equal to the standard deviation (5%), except in the NorE4 and LmfE7 isolates, which accumulated 50% and 26% more norfloxacin than their parent isolates, respectively. Furthermore, three parent isolates (the PS5, PS6 and PS7 isolates) accumulated 6875% and the remaining seven PS isolates accumulated <50% than the KL16 isolate (Table III). The difference between the effect of 50 and 100 µM CCCP in the intracellular accumulation of norfloxacin both in the quinolone-resistant mutants and in the parent isolates was lower than the maximum accepted standard deviation for uptake, 5% (Table III). The addition of both 50 and 100 µM CCCP resulted in increases in intracellular norfloxacin concentrations that were higher than 20% in all the parent isolates, with the exceptions of the PS2, PS3 and PS4 isolates (Table III). The effect of CCCP was more noticeable in 12 of the mutants, in which the presence of CCCP resulted in a two- to six-fold increment in the norfloxacin uptake (Table III).
|
![]() |
Discussion |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
The highest frequency of selection of mutations was obtained with the parent isolates that exhibited a higher level of resistance to nalidixic acid.12 All the quinolone-resistant mutants showed a multiple-resistance phenotype, which has been described in E. coli isolates after selection with fluoroquinolones20 and other agents.12
The involvement of substitutions in DNA gyrase and topoisomerase IV subunits in the quinolone resistance
Analysis of amino acid changes in the subunits of topoisomerases revealed that the
substitution of
Ser-83Leu in the GyrA subunit of DNA gyrase was the most frequently identified change,
as
has been described by others.1,4,5 The 14 quinolone-resistant isolates and the
PS1,
PS5 and PS7 isolates in which this mutation was present exhibited a high level of resistance to
nalidixic
acid (MIC
128 mg/L) and ciprofloxacin and norfloxacin MICs
0.25 mg/L. the
Ser-83
Ala substitution in DNA gyrase in the PS10 isolate was associated with both lower
nalidixic acid and fluoroquinolone MICs than the substitution Ser-83
Leu, although the
PS10
isolate was more resistant to quinolones than the other six parent isolates, which had no mutation
of
topoisomerases.2 The appearance of a second change in the
amino acid sequence of the ParC subunit in the NorE5 isolate (Ser-80
Arg) was not
associated
with higher fluoroquinolone MICs with respect to the LmfE5 isolate. It is likely that the lower
fluoroquinolone uptake exhibited by the LmfE5 isolate in comparison with the NorE5 isolate was
important in determining the greater resistance to quinolone compared with the NorE5 isolate,
despite
the parC gene mutation in this latter isolate. Similarly, the NorE6 and LmfE6 isolates
showed
the same change (Asp-87
Asn) in the GyrA subunit as the N-113 E. coli isolate
described by Oram & Fisher,3 but MICs of quinolones
were
higher in the LmFE6 and NorE6 isolates than in the N-113 isolate. Finally, our results as in
previous
studies show that mutations in the gyrB and parE genes do not play a major
role in
the acquisition of quinolone resistance.5,16
Changes in outer membrane proteins and lipopolysaccharide
The role of OMP and LPS in the development of resistance to quinolones was studied by comparing the different selected mutants with their parent isolates. SDSPAGE analysis of outer membranes revealed that the increased resistance to quinolones was associated with the lack of OmpF in 11 quinolone-resistant mutants, this porin being expressed in the ten parent isolates. In this context, it has been shown that several mutations (marA, soxQ1, cfxB1, nfxC1, etc.) reduce ompF expression after transcription and that this reduction is related to the expression of the micF locus (an antisense RNA complementary to ompF mRNA).12,14 In this study, the lack of OmpF was associated with a decreased norfloxacin uptake in only eight of the mutants (the NorE1, NorE2, LmfE2, LmfE4, NorE5, LmfE5, NorE8 and LmfE9 isolates). The lack of OmpF in the NorE3, LmfE3 and NorE10 isolates was not associated with a significant diminution of norfloxacin uptake, i.e. higher than 5% in the steady state, with respect to their parent isolates. It should be noted that for the PS3 and PS10 isolates, a lower level of norfloxacin uptake was characteristic in comparison with KL16 isolate wild-type. This feature could contribute to increased resistance to quinolones in the NorE3 and LmfE3 isolates when low uptake is combined with mutations in the gyrA gene.1,4 The influence of the lack of expression of high molecular mass polymers of LPS on the quinolone MICs in the PS5, NorE5, PS6, PS8 and NorE10 isolates cannot be excluded as previous work has proposed.1,9,10 In this context, the NorE5 and NorE10 isolates, as the NorC mutants,20 exhibited rough LPS, lack of OmpF expression and accumulated <50% of the norfloxacin concentration compared with the amount accumulated by the KL16 isolate.1,20
Norfloxacin uptake and its role in parent isolates and mutants
The study of intracellular accumulation of norfloxacin demonstrated that all the parent isolates
accumulated less norfloxacin than the KL16 wild-type isolate, despite the fact they did not lose
the
expression of OmpF. In this context, seven of the parent isolates accumulated less than 50% of
norfloxacin compared with the KL16 isolate, although 11 mutants accumulated even less
norfloxacin
than their parent isolates (Table III). The addition of 50 or 100 µM
CCCP resulted in increased norfloxacin accumulation in all the parent isolates and mutants with
the
exceptions of the PS2, PS3, PS4, NorE3, LmfE3 and NorE4 isolates, showing that
proton-dependent
active efflux influenced the intracellular level of norfloxacin in 22 of the isolates studied.
Nevertheless,
the involvement of a non-proton-dependent active efflux could explain the lower norfloxacin
uptake,
with respect to the KL16 wild-type isolate, in the NorE3, LmfE3 and NorE4 isolates, as well as in
the
PS2, PS3 and PS4 isolates.1,11 Furthermore, the presence of active efflux when added to the effect of change
(Asp-87Asn) in the GyrA subunit in the NorE6 and LmfE6 isolates, could explain the
higher
fluoroquinolone MICs in the NorE6 and LmfE6 isolates when compared with the N-113 isolate.3 In this context, the influence of an enhanced
proton-dependent
active efflux on norfloxacin accumulation in the NorE9 and NorE10 isolates was seen. The
increased
norfloxacin MICs (four- to 32-fold) in the LmfE1, LmfE7 and LmfE10 isolates with respect to the
PS1,
PS7 and the PS10 isolates were caused by decreased fluoroquinolone uptake. The addition of 50
or
100 µM CCCP resulted in an increase in norfloxacin uptake from 62% to 80% in the LmfE1
and
LmfE10 isolates, and 17% in the LmfE7 isolate. These results are in accordance with the higher
norfloxacin MICs in the LmfE1 and LmfE10 isolates than in the LmfE7 isolate when compared
with
their parent isolates. These latter mutants showed an increased resistance to tetracycline and/or
cephalothin in association with a rise in the MIC of nalidixic acid. These antimicrobial agents have
been
described as substrates of the AcrAB efflux system in E. coli.13,22 The overexpression of the AcrAB system
could
explain the multiresistance shown by the LmfE1, LmfE7 and LmfE10 isolates. Further studies are
in
progress to reveal the involvement of this efflux system in these mutants.
The diversity of behaviour with respect to the OmpF expression and proton-dependent active efflux in the 18 mutants studied suggests a classification into five different groups (Table III):
In summary, DNA gyrase mutations were the principal determining factors in the acquisition of a higher level of resistance to quinolones in the isolates in this study. A decreased permeability, caused by changes in OMPs, and an increased active efflux were complementary in determining the MICs of the quinolones. These latter two mechanisms could facilitate the development of multiple-antibiotic-resistance phenotypes and, in turn, contribute to the selection of quinolone resistance in the course of clinical use.
![]() |
Acknowledgments |
---|
![]() |
Notes |
---|
![]() |
References |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
2 . Hallett, P. & Maxwell, A. (1991). Novel quinolone resistance mutations of the Escherichia coli DNA gyrase A protein: enzymatic analysis of the mutant proteins. Antimicrobial Agents and Chemotherapy 35, 33540.[ISI][Medline]
3 . Oram, M. & Fisher, L. M. (1991). 4-Quinolone resistance mutations in the DNA gyrase of Escherichia coli clinical isolates identified by using the polymerase chain reaction. Antimicrobial Agents and Chemotherapy 35, 3879.[ISI][Medline]
4 . Piddock, L. J. V. (1995). Mechanisms of resistance to fluoroquinolones: state-of-the-art 19921994. Drugs 49, Suppl. 2, 2935.[Medline]
5 . Vila, J., Ruiz, J., Marco, F., Barceló, A., Goñi, P., Giralt, E. et al. (1994). Association between double mutation in the gyrA gene of ciprofloxacin-resistant clinical isolates of Escherichia coli and MICs. Antimicrobial Agents and Chemotherapy 38,2477 9.[Abstract]
6 . Vila J., Ruiz, J., Goñi, P. & de Jiménez Anta, M.T. (1996). Detection of mutations in parC in quinolone-resistant clinical isolates of Escherichia coli. Antimicrobial Agents and Chemotherapy 40 , 4913.[Abstract]
7 . Breines, D. M., Ouabdesselam, S., Ng, E. Y., Tankovic, J., Shah, S., Soussy, C. J. et al. (1997). Quinolone resistance locus nfxD of Escherichia coli is a mutant allele of the parE gene encoding a subunit of topoisomerase IV. Antimicrobial Agents and Chemotherapy 41, 1759.[Abstract]
8 . Mortimer, P. G. S. & Piddock, L. J. V. (1993). The accumulation of five antibacterial agents in porin-deficient mutants ofEscherichia coli.Journal of Antimicrobial Chemotherapy 32,195 213.[Abstract]
9 . Hirai, K., Aoyama, H., Irikura, T., Iyobe, S. & Mitsuhashi, S. (1986). Differences in susceptibility to quinolones of outer membrane mutants of Salmonella typhimurium and Escherichia coli. Antimicrobial Agents and Chemotherapy 29, 5358.[ISI][Medline]
10 . Moniot-Ville, N., Guibert, J., Moreau, N., Acar, J. F., Collatz, E. & Gutmann, L. (1991). Mechanisms of quinolone resistance in a clinical isolate of Escherichia coli highly resistant to fluoroquinolones but susceptible to nalidixic acid. Antimicrobial Agents and Chemotherapy 35, 51923.[ISI][Medline]
11 . Paulsen, I. T., Brown, M. H. & Skurray, R. A. (1996). Proton-dependent multidrug efflux systems. Microbiological Reviews 60, 575608.[Abstract]
12 . Cohen, S. P., McMurry, L. M., Hooper, D. C., Wolfson, J. S. & Levy, S. B. (1989). Cross-resistance to fluoroquinolones in multiple-antibiotic-resistant (Mar) Escherichia coli selected by tetracycline or chloramphenicol: decreased drug accumulation associated with membrane changes in addition to OmpF reduction. Antimicrobial Agents and Chemotherapy 33, 131825.[ISI][Medline]
13 . Okusu, H., Ma, D. & Nikaido, H. (1996). AcrAB efflux pump plays a major role in the antibiotic resistance phenotype of Escherichia coli multiple-antibiotic-resistance (Mar) mutants. Journal of Bacteriology 178 , 3068.[Abstract]
14 . Hooper, D. C., Wolfson, J. S., Souza, K. S., Tung, C., McHugh, G. L. & Swartz, M. N. (1986). Genetic and biochemical characterization of norfloxacin resistance in Escherichia coli. Antimicrobial Agents and Chemotherapy 29, 63944.[ISI][Medline]
15 . National Committee for Clinical Laboratory Standards. (1993). Methods for Dilution Antimicrobial Susceptibility Tests for Bacteria that Grow AerobicallyThird Edition: Approved Standard M7-A3. NCCLS, Villanova, PA.
16
.
Ruiz, J., Casellas, S., Jiménez de Anta, M. T.
&
Vila, J. (1997). The region of the parE gene, homologous to the
quinolone-resistant determining region of the gyrB gene, is not linked with the
acquisition of
quinolone resistance in Escherichia coli clinical isolates. Journal of
Antimicrobial
Chemotherapy 39 , 83940.
17 . Sawai, T., Hiruma, R., Kawana, N., Kaneko, M., Taniyasu, F. & Inami, A. (1982). Outer membrane permeation of ß-lactam antibiotics in Escherichia coli, Proteus mirabilis, and Enterobacter cloacae. Antimicrobial Agents and Chemotherapy 22,585 92.[ISI][Medline]
18 . Hitchcock, P. J. & Brown, T. M. (1983). Morphological heterogeneity among Salmonella lipopolysaccharide chemotypes in silver-stained polyacrylamide gels. Journal of Bacteriology 154, 26977.[ISI][Medline]
19 . Tsai, C.-M. & Frasch, C. E. (1982). A sensitive silver stain for detecting lipopolysaccharides in polyacrylamide gels. Annals of Biochemistry 119 , 1159.
20 . Hirai, K., Aoyama, H., Suzue, S., Irikura, T., Iyobe, S. & Mitsuhashi, S. (1986). Isolation and characterization of norfloxacin-resistant mutants of Escherichia coli K-12. Antimicrobial Agents and Chemotherapy 30, 24853.[ISI][Medline]
21 . Ishii, H., Sato, K., Hoshino, K., Sato, M., Yamaguchi, A., Sawai, T. et al. (1991). Active efflux of ofloxacin by a highly quinolone-resistant strain of Proteus vulgaris. Journal of Antimicrobial Chemotherapy28 , 82736.[Abstract]
22 . Ma, D., Cook, D. N., Alberti, M., Pon, N. G., Nikaido, H. & Hearst, J. E. (1995). Genes acrA and acrB encode a stress-induced efflux system of Escherichia coli. Molecular Microbiology 16, 4555.[ISI][Medline]
Received 9 February 1999; returned 3 June 1999; accepted 26 July 1999