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Antimicrobial Agents and Chemotherapy, December 1998, p. 3282-3284, Vol. 42, No. 12
0066-4804/98/$04.00+0
Copyright © 1998, American Society for Microbiology. All rights reserved.
Joint Tolerance to 
-Lactam and Fluoroquinolone
Antibiotics in Escherichia coli Results from Overexpression
of hipA
Timothy J.
Falla and
Ian
Chopra*
Department of Microbiology and Antimicrobial
Research Centre, University of Leeds, Leeds LS2 9JT, United Kingdom
Received 5 March 1998/Returned for modification 27 April
1998/Accepted 17 September 1998
 |
ABSTRACT |
The basis of joint tolerance to 
-lactam and fluoroquinolone
antibiotics in Escherichia coli mediated by
hipA was examined. An antibiotic tolerance phenotype was
produced by overexpression of hipA under conditions that
did not affect the growth rate of the organism. Overexpressing
hipA probably decreases the period in which bacteria are
susceptible to the antibiotics by temporarily affecting some aspect of
chromosome replication or cell division.
| |
TEXT |
-Lactam and fluoroquinolone
antibiotics exhibit a bactericidal action against growing cultures of
most pathogenic bacteria (16). However, mutants that are as
sensitive to growth inhibition by the antibiotics as the wild-type
parent strains but which undergo only a slow loss of viability in the
presence of the antibiotics have been described previously (9, 12,
14, 15, 17). The phenomenon has been called tolerance or high
persistence (9, 12). Mutants tolerant to -lactam
antibiotics have been described in both laboratory mutant strains and
clinical isolates (9). However, mutants tolerant to
fluoroquinolones have so far only been described in laboratory mutants
of Escherichia coli (12, 14, 17). These mutants
also display tolerance to -lactam antibiotics (3, 4, 12,
17). Tolerance to -lactam antibiotics may have clinical
significance (11), but it is not known whether joint
tolerance to -lactams and fluoroquinolones has clinical relevance.
In E. coli, joint tolerance to both peptidoglycan and DNA
synthesis inhibitors is under the control of the hip (high
persistence) locus (14, 17). Thus, strains containing
chemically mutagenized hip exhibit a 1,000-fold reduction in
the rate of killing by -lactam and fluoroquinolone antibiotics
(14, 17). The hip locus in E. coli
consists of two genes, hipA (1,320 bp), which encodes a
weakly expressed 50-kDa protein, and hipB (264 bp), which
encodes a Cro-like protein which is a hipA repressor and
responsible for low-level hipA expression (3).
HipA is found exclusively in a tight complex with HipB, and the stop
codon of hipB overlaps the start codon of hipA by
one base (3, 4). This close relationship is essential since
hipB mutant strains are nonviable, indicating that
nonregulated expression of hipA might be lethal
(4).
An understanding of antibiotic tolerance mediated by mutations in the
hipA gene may provide the key to a link between -lactam and quinolone mechanisms of action. In this paper, we report on the
distribution of hip in bacteria and the role of
hipA in tolerance of E. coli through studies on
overexpression of hipA.
Distribution of hipA.
A search for hipA and
hipB homologues was performed with a range of gram-negative
and -positive bacteria. By using standard techniques (13),
PCR-amplified E. coli hipA and hipB were used to
probe restriction digests of chromosomal DNA from Shigella sonnei, Salmonella typhimurium, Klebsiella
aerogenes, Pseudomonas aeruginosa, Staphylococcus
aureus, Staphylococcus hominis, Bacillus subtilis, Providencia vulgaris, and Serratia
marcescens. To determine the distribution of the hip
locus in E. coli, the hip operon was amplified by
PCR with two sets of primers designed to amplify the entire
hipA gene and the entire hipB gene
(3). Although both hipA and hipB were
identified in S. sonnei, homologues could not be identified,
even with low-stringency hybridization, in any of the other bacteria
examined, including organisms such as Salmonella typhimurium
and Klebsiella pneumoniae, which are closely related to
E. coli and Shigella sonnei. However, three
homologues were identified by amino acid database searches, one in
Haemophilus influenzae and two in the Rhizobium
symbiosis plasmid pNGR234 (7, 8). In H. influenzae, the gene (HI0665) is significantly disrupted and would be unlikely to express a protein with similar function to HipA. In Rhizobium, the genes (y4mE
and y4dM) encode proteins with 28 and 27% identity to HipA,
respectively. Significantly, as for HipB, these genes are under the
control of strong transcriptional regulators (y4mF and
y4dL).
Chromosomal DNA from 40 clinical isolates of E. coli, from
different locations worldwide, was PCR amplified with two sets of
primers designed for hipA and hipB. Approximately
20% of the strains tested were negative with both primer sets, and the
remaining strains were amplification positive with both sets.
Chromosomal deletion of hipA in E. coli.
The
E. coli LN2666 (1) hipA gene was
replaced with a copy which expressed only the first 25 amino acids of
the protein, resulting in strain IC4. This was done by homologous
recombination (6) with the plasmid pHp100, a pFC24
(6) derivative in which a 618-bp BssHII fragment
of hipA had been excised. To determine if hipA
deletion or disruption exhibited a specific phenotype, strains IC4 and
LN3559 (hipA::tetA), a gift from J.-M.
Louarn, were compared to the parent strain, LN2666, with respect to
growth rate, morphology, antimicrobial susceptibility, and total
cellular protein profiles. For all these characteristics, there were no differences between the three strains (data not shown).
Overexpression of hipA.
Growth of E. coli
BL21 (DE3) carrying pLysS (Promega) and pHp200, a pET30b (Novagen)
construct containing the entire hipA open reading frame, was
induced with 0.05 to 1 mM
isopropyl--D-thiogalactopyranoside (IPTG). Induction
produced dose-dependent inhibition of cell division in E. coli BL21 (Fig. 1). The growth rates
of cells exposed to IPTG concentrations of less than 0.03 mM were
unaffected. However, when challenged with 100 µg of ampicillin per
ml, E. coli containing pHp200, induced with 0.01 mM IPTG,
exhibited significantly reduced killing in comparison to cells
containing the vector alone (Fig. 2A). To
determine if this phenomenon was joint tolerance as identified in the
original mutants (14), survival studies were performed for
cells containing pHp200 that had been exposed to the quinolone ciprofloxacin. In this case, there was a 10-fold difference in the
killing of cells containing pHp200 compared to that of cells containing
pET30b when exposed to 100 µg of the drug per ml (Fig. 2B).
Expression of hipA was confirmed by S
Tag Western blot
(Novagen), which showed HipA to be a protein of 49 kDa (data not
shown), the size predicted by its sequence.
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FIG. 1.
Growth curve of E. coli BL21 (DE3) carrying
pLysS and pHp200 with and without IPTG induction. OD600,
optical density at 600 nm.
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FIG. 2.
Killing of E. coli BL21 (DE3) containing
plasmid pHp200 ( ) or pET30b ( ) with 100 µg of ampicillin per ml
(A) or 100 µg of ciprofloxacin per ml (B). Cultures contained 0.01 mM
IPTG.
|
|
The ability to overexpress
hipA has allowed us to
demonstrate that joint tolerance to
-lactam and quinolone
antibiotics in
E. coli is due to expression of
hipA in excess of
hipB and that
HipA is toxic to
E. coli. Failure to isolate HipA unbound to HipB
(
4) and the observation made here that overexpression of
hipA produced a tolerance phenotype similar to that observed
in an
earlier work (
14) indicate that in the original
mutants, reduced
binding of HipA to HipB probably resulted in the
observed tolerance.
However, since neither cassette insertion
inactivation (
4)
nor chromosomal deletion of
hipA
(this study) conferred tolerance,
at least part if not all of HipA is
required for the
phenotype.
Since the
hip locus is restricted to relatively few
bacterial species, including not even all strains of
E. coli, antibiotic
tolerance resulting from
hip mutations
is unlikely to be clinically
significant. In addition, persistence
identified in species other
than
E. coli, e.g., the
persistence of
-lactams in staphylococci
(
2), is unlikely
to be related to
hip. However, although the
role of
hip in
E. coli tolerance to
-lactam and
quinolone antibiotics
has been established in this study, the mechanism
of tolerance
has not. Bigger (
2) suggested that persisting
bacteria are
cells briefly existing in the nondividing phase of their
life
cycle and survive because penicillin kills only dividing cells.
If
bacteria are susceptible to
-lactam and quinolone antibiotics
only
during specific phases of their life cycle, then any phenomenon
that
reduced the window of opportunity for killing would enable
more
bacteria to survive the cidal effects of both antibiotics.
It is
tempting to speculate that HipA may decrease the period
of time during
which bacteria are susceptible to these antibiotics
by affecting some
aspect of chromosome replication or cell division.
This may relate to
the location of
hip in the terminal domain
of the chromosome
within 100 bp of the
dif locus and close to
terC.
These loci are involved in chromosome partitioning and termination
of
chromosome replication, respectively (
5,
10).
| |
ACKNOWLEDGMENTS |
This work was supported by a grant to I.C. from SmithKline
Beecham Pharmaceuticals.
| |
FOOTNOTES |
*
Corresponding author. Mailing address: Department of
Microbiology and Antimicrobial Research Centre, University of Leeds, Leeds LS2 9JT, United Kingdom. Phone: 44 113 233 5604. Fax: 44 113 233 5638. E-mail: micic{at}leeds.ac.uk.
| |
REFERENCES |
| 1.
|
Berg, C. M., and R. Curtiss.
1967.
Transposition derivatives of an Hfr strain of Escherichia coli K12.
Genetics
56:503-525[Free Full Text].
|
| 2.
|
Bigger, J. W.
1944.
Treatment of staphylococcal infections with penicillin.
Lancet
ii:497-500.
|
| 3.
|
Black, D. S.,
A. J. Kelly,
M. J. Mardis, and H. S. Moyed.
1991.
Structure and organization of hip, an operon that affects lethality due to inhibition of peptidoglycan or DNA synthesis.
J. Bacteriol.
173:5732-5739[Abstract/Free Full Text].
|
| 4.
|
Black, D. S.,
B. Irwin, and H. S. Moyed.
1994.
Autoregulation of hip, an operon that affects lethality due to inhibition of peptidoglycan or DNA synthesis.
J. Bacteriol.
176:4081-4091[Abstract/Free Full Text].
|
| 5.
|
Blakely, G.,
S. D. Colloms,
G. May,
M. Burke, and D. Sherratt.
1991.
Escherichia coli XerC recombinase is required for chromosomal segregation at cell division.
New Biol.
3:789-798[Medline].
|
| 6.
|
Cornet, F.,
I. Mortier,
J. Patte, and J. M. Louarn.
1994.
Plasmid pSC101 harbors a recombinant site, psi, which is able to resolve plasmid multimers and to substitute for the analogous chromosomal Escherichia coli site dif.
J. Bacteriol.
176:3188-3195[Abstract/Free Full Text].
|
| 7.
|
Fleischmann, R. D.,
M. D. Adams,
O. White,
R. A. Clayton,
E. F. Kerlavage,
C. J. Bult,
T. J. Tomb,
B. A. Dougherty,
J. M. Merrick,
K. McKenney,
G. Sutton,
W. Fitzhugh,
C. Fields,
D. Gocayne,
J. Scott,
R. Shirley,
L.-I. Liu,
A. Glodek,
J. M. Kelley,
J. F. Weidman,
C. A. Phillips,
T. Spriggs,
E. Hedblom,
M. D. Cotton,
T. R. Utterback,
M. C. Hanna,
D. T. Nguyen,
D. M. Saudek,
R. C. Brandon,
L. D. Fine,
J. L. Fritchman,
J. L. Fuhrmann,
N. S. M. Geoghagen,
C. L. Gnehm,
L. A. McDonald,
K. V. Small,
C. M. Fraser,
H. O. Smith, and J. C. Venter.
1995.
Whole-genome random sequencing and assembly of Haemophilus influenzae Rd.
Science
269:496-512[Abstract/Free Full Text].
|
| 8.
| Freiberg, C., R. Fellay, A. Bairoch, W. J. Broughton, A. Rosenthal, and X. Perret. Molecular basis of
symbiosis between Rhizobium and legumes. Nature
387:394-401.
|
| 9.
|
Handwerger, S., and A. Tomasz.
1985.
Antibiotic tolerance among clinical isolates of bacteria.
Annu. Rev. Pharmacol. Toxicol.
25:349-380[Medline].
|
| 10.
|
Hill, T. M.,
J. M. Hensen, and P. L. Kuempel.
1987.
The terminus region of the Escherichia coli chromosome contains two separate loci that exhibit polar inhibition of replication.
Proc. Natl. Acad. Sci. USA
84:1754-1758[Abstract/Free Full Text].
|
| 11.
|
Holtje, J.-V., and E. I. Tuomanen.
1991.
The murein hydrolases of Escherichia coli: properties, functions and impact on infections in vivo.
J. Gen. Microbiol.
137:441-454[Medline].
|
| 12.
|
Hooper, D. C., and J. S. Wolfson.
1993.
Mechanisms of quinolone action and bacterial killing, p. 53-75.
In
D. C. Hooper, and J. S. Wolfson (ed.), Quinolone antimicrobial agents, 2nd ed. American Society for Microbiology, Washington, D.C.
|
| 13.
|
Maniatis, T.,
E. F. Fritsch, and J. Sambrook.
1982.
Molecular cloning: a laboratory manual.
Cold Spring Harbor Laboratory, Cold Spring Harbor, N.Y.
|
| 14.
|
Moyed, H. S., and K. P. Bertrand.
1983.
hipA, a newly recognized gene of Escherichia coli K-12 that affects frequency of persistence after inhibition of murein synthesis.
J. Bacteriol.
155:768-775[Abstract/Free Full Text].
|
| 15.
|
Moyed, H. S., and S. H. Broderick.
1986.
Molecular cloning and expression of hipA, gene of Escherichia coli K-12 that affects frequency of persistence after inhibition of murein synthesis.
J. Bacteriol.
166:399-403[Abstract/Free Full Text].
|
| 16.
|
Russell, A. D., and I. Chopra.
1996.
Understanding antibacterial action and resistance, 2nd ed.
Ellis Horwood, New York, N.Y.
|
| 17.
|
Wolfson, J. S.,
D. C. Hooper,
G. L. McHugh,
M. A. Bozza, and M. N. Swartz.
1990.
Mutants of Escherichia coli K-12 exhibiting reduced killing by both quinolone and -lactam antimicrobial agents.
Antimicrob. Agents Chemother.
34:1938-1943[Abstract/Free Full Text].
|
Antimicrobial Agents and Chemotherapy, December 1998, p. 3282-3284, Vol. 42, No. 12
0066-4804/98/$04.00+0
Copyright © 1998, American Society for Microbiology. All rights reserved.
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