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Antimicrobial Agents and Chemotherapy, September 1999, p. 2268-2272, Vol. 43, No. 9
0066-4804/99/$04.00+0
Copyright © 1999, American Society for Microbiology. All rights reserved.
In Vitro Activities of Azithromycin and Ofloxacin against
Chlamydia pneumoniae in a Continuous-Infection
Model
Andrei
Kutlin,
Patricia M.
Roblin, and
Margaret R.
Hammerschlag*
Department of Pediatrics, Division of
Infectious Diseases, State University of New York at Brooklyn,
Brooklyn, New York 11203-2098
Received 15 March 1999/Returned for modification 28 May
1999/Accepted 29 June 1999
 |
ABSTRACT |
Chlamydia pneumoniae is a well-established cause of
community-acquired pneumonia and bronchitis in adults and children.
Chronic infections with C. pneumoniae have been implicated
in the development of atherosclerosis and other diseases in
humans. Methods currently used for the culture and propagation of
C. pneumoniae are not analogous to the
infection as it occurs in vivo. We have established a model of
continuous C. pneumoniae infection in vitro. HEp-2 cells inoculated with CM-1 and TW-183 strains have been persistently infected for periods of over 1.5 and 2 years, respectively. The cultures were maintained without centrifugation or the addition of
cycloheximide, fresh host cells, or chlamydia. We observed cycles of
host cell lysis, detachment, and regrowth with both strains of C. pneumoniae. Continuous C. pneumoniae infections may
more closely resemble the actual events as they occur in vivo and,
therefore, may be a better model for the in vitro study of C. pneumoniae infection. When we used continuously infected cells to
determine the effects of azithromycin and ofloxacin on C. pneumoniae propagation in vitro, we found that both drugs reduced
but did not completely eliminate the organism. This may be an important observation, as the failure of antibiotic therapy against C. pneumoniae infection in humans has been described.
| |
INTRODUCTION |
Chlamydia pneumoniae,
like other members of its genus, is capable of causing chronic and
asymptomatic infections (3). Infection with C. pneumoniae has been implicated in the development of
atherosclerosis and other chronic diseases in humans (6,
14). Methods currently used for culture and propagation of
C. pneumoniae are not analogous to the infection as it
occurs in vivo. To further study this problem, we established a model
of continuous C. pneumoniae infection in vitro, in which
cell growth, lysis, and chlamydia production are balanced. We also
investigated the in vitro activities of azithromycin and ofloxacin
against C. pneumoniae in this system. Continuous C. pneumoniae infection may more closely resemble the actual events as they occur in vivo and, therefore, may be a better model for the in
vitro study of C. pneumoniae infection.
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MATERIALS AND METHODS |
Continuous C. pneumoniae infection in vitro.
Confluent HEp-2 cell monolayers were inoculated with C. pneumoniae strains TW-183 (ATCC VR-2282) and CM-1 (ATCC VR1360) to achieve almost 100% infection (12). After 3 to 5 days, when lysis and detachment of most of the infected cells were seen, the
original medium was replaced with fresh medium with no added cycloheximide. After 1 week, the growth of new cells was observed. When
cell monolayers became confluent again, the supernatants were tested
for the presence of viable Chlamydia, and cells were split
onto new flasks. A small portion of cell suspension was seeded onto
several wells of a 96-well microtiter plate and was examined for the
presence of inclusions by staining with a fluorescein-conjugated genus-specific monoclonal antibody (Pathfinder Chlamydia Culture Conformation System; Sanofi Diagnostics, Redmond, Wash.) 4 to 72 h
after seeding. Cells continuously infected with both strains were split
three to five times per month in a ratio not exceeding 1:4, with medium
being changed between splits.
Antibiotic activity assay. Primary C. pneumoniae
infection.
Confluent HEp-2 monolayers grown on 24-well plates were
inoculated in duplicate (one well contained a 12-mm-diameter coverslip) with 103 to 104 inclusion-forming units
(IFU)/ml of TW-183 and CM-1. The plates then were centrifugated at
1,700 × g for 1 h. After centrifugation, the cell
monolayers were overlaid with medium containing 0.125 µg of
azithromycin or 1.0 µg of ofloxacin per ml (the minimum inhibitory
concentration at which 90% of the isolates are inhibited [MIC90] for each drug) (4, 5).
Continuous C. pneumoniae infection.
Continuously
infected HEp-2 cells were seeded into 24-well plates the day prior to
the experiment. The medium was then replaced with one containing either
0.125 µg of azithromycin or 1.0 µg of ofloxacin per ml on the day
of experiment.
Additionally, continuously infected cells were seeded onto 12-well
plates and were treated with 0.125, 0.25, or 0.5 µg of azithromycin
per ml and 1.0, 2.0, or 4.0 µg of ofloxacin per ml. The
drug-containing supernatant media of all infected cells were replaced
daily with fresh media containing the same drugs at the same concentrations.
On days 1, 2, 3, 4, 5, and 6 after inoculation and addition of
antibiotics, the infected cells from primary and continuous
cultures
were collected and frozen, then defrosted and ultrasonicated.
Tenfold
dilutions from each well were inoculated onto fresh HEp-2
cells in
96-well plates. The plates were centrifuged at 1,700
×
g, incubated for 72 h, fixed, and stained as above. The
IFU
per milliliter for every isolate at each time point and under
the
effect of each drug was calculated. Reduction of IFU was
calculated
relative to controls with no antibiotics
added.
| |
RESULTS |
Continuous C. pneumoniae infection was established in
vitro with TW-183 and CM-1. The cultures were maintained without
centrifugation or the addition of cycloheximide, fresh host cells, or
chlamydia for over 2 years for TW-183 and over 18 months for CM-1. We
observed cycles of cell lysis and detachment (Fig.
1B) and regrowth (Fig. 1A) with TW-183.
The period of time between cycles varied from 7 to 14 days and strongly
depended on the frequency of splitting the cells and changing the
media. Cycles of lysis and regrowth were also observed with cells
infected with CM-1. We often observed multiple (Fig. 1E), clustered
(Fig. 1F), and large (greater than or equal to 1.5 to 2 times the size
of host cells) inclusions, especially with TW-183 (Fig. 1C and D).
Continuous infection with TW-183 produced, on average, 0.5 to 2 log
units more infectious progeny than CM-1 (Fig.
2).
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FIG. 1.
(A to F) Continuous TW-183 infection in HEp-2
cells: A, monolayer (400×); B, lysis (400×); C and D, large
inclusions (200 and 400×); E, multiple inclusions (400×); F,
clustered inclusions (400×). (G and H) CM-1 infection in HEp-2 cells
on day 3: G, primary CM-1 infection (400×); H, continuous CM-1
infection (400×). C. pneumoniae-infected cells were stained
with the Pathfinder Chlamydia Culture Confirmation System (Sanofi
Diagnostics), which contains fluorescein-conjugated monoclonal antibody
and Evans Blue as a counterstain. The chlamydial inclusions stain apple
green and the host cells stain dark red when observed under UV light.
Bars represent an average host cell size.
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The results of in vitro susceptibility testing of azithromycin and
ofloxacin in the primary and continuously infected cells are shown in
Fig. 3. Unlike the primarily infected
cells, where a 100% reduction in IFU for TW-183 and a 5-log-unit
reduction in IFU for CM-1 was observed at day 3 after treatment with
both drugs, the reduction in IFU observed with the continuously
infected cells ranged from 94.6 to 99.9%. The log reduction of IFU in
the primary infection was, on average, 1.8 log units greater for
azithromycin and 2.2 log units greater for ofloxacin than for the
continuously infected cells.
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FIG. 3.
Effects of azithromycin (0.125 µg/ml) and ofloxacin
(1.0 µg/ml) on C. pneumoniae infection in vitro. A,
continuous TW-183 infection; B, continuous CM-1 infection; C, primary
TW-183 infection; D, primary CM-1 infection.
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|
The results of treatment of continuously infected cells with different
concentrations of drugs and medium changes every day are shown in Fig.
4. A gradual decrease, but not
elimination, of chlamydia IFU was seen with both azithromycin (0.125, 0.25, and 0.5 µg/ml) and ofloxacin (1.0, 2.0, and 4.0 µg/ml).
These results are similar to those seen utilizing one dose of the MIC of each drug.
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FIG. 4.
Effect of different doses of azithromycin and ofloxacin
on continuous C. pneumoniae in vitro. A, TW-183
(azithromycin); B, TW-183 (ofloxacin); C, CM-1 (azithromycin); D, CM-1
(ofloxacin).
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|
| |
DISCUSSION |
The ability of chlamydia to establish a long-term association with
host cells in vitro has been known since chlamydia was perceived as a
virus. This type of in vitro infection was described as persistent or
chronic. Continuous Chlamydia psittaci (2, 9-11)
and Chlamydia trachomatis (1, 7, 8) infections in
various cell lines have been described. Out of the four recognized chlamydial species, C. pneumoniae is considered to be the
most difficult to cultivate. However, we were able to establish
continuous C. pneumoniae infection in vitro. HEp-2 cells
inoculated with CM-1 and TW-183 strains have been persistently infected
for over 1.5 and 2 years, respectively. We observed stages of lysis and regrowth in host cells infected with both strains. Similar cycles of
cell destruction and proliferation were previously reported for chronic
C. psittaci and C. trachomatis infections in
vitro by other investigators (8, 11). TW-183 initially
appeared to be more infectious, producing significant lysis and more
infectious progeny than CM-1. This phenomenon may be secondary to the
previous extensive passage of TW-183 in tissue culture. CM-1 was
isolated more recently from an adult with pneumonia and may not be as
adapted to cell culture as TW-183. These cycles of infection may
reflect a process of mutual adaptation of both chlamydia and host cells leading to selection of more infectious strains of chlamydia and inducing cell destruction followed by the selection of more-resistant cells (7, 11). Despite the high concentration of chlamydia (up to 107 IFU/ml) in the supernatant media, some cells
contained no inclusions. Preliminary data have shown higher levels of
various cytokines in continuously infected cultures than in primary
infections, which may indicate increased host cell defenses
(13). Benes (1) suggested that direct,
cell-to-cell transmission of chlamydia in overcrowded cell layers may
help in the evasion of host cell defenses and may lead to a new
productive cycle. Our findings with regard to the induction and
maintenance of continuous chlamydial infection were similar to those of
others (8, 11). Contributing factors needed to establish
continuous or chronic infection appeared to be the high initial
inoculation dose, the balance of time necessary for chlamydial
propagation and host cell regrowth, and the ability of some cells to
remain uninfected.
This continuous-culture system may be more analogous than previous
systems to the actual events in vivo and may serve as a better
model for in vitro studies of C. pneumoniae. When we used continuously infected cells to determine the effect of azithromycin and
ofloxacin on C. pneumoniae in vitro, we found that
both drugs, at concentrations of up to four times the
MIC90s, reduced, but did not completely eliminate, the
organism. Our results were similar to those obtained by Galasso and
Manire (2), who studied HeLa cells chronically infected with
C. psittaci. They found that 7-day treatments with up to 50 µg of tetracycline per ml of medium decreased chlamydia
production until it was rendered undetectable, but chlamydia propagation resumed soon after removal of the drug. Only prolonged (15-day) treatment was sufficient to eliminate chlamydia from continuously infected cells. This may be an important observation, as
the failure of antibiotic therapy against C. pneumoniae
infection in humans has been described (3).
| |
FOOTNOTES |
*
Corresponding author. Mailing address: Department of
Pediatrics, Box 49, SUNY Health Science Center at Brooklyn, 450 Clarkson Ave., Brooklyn, NY 11203-2098. Phone: (718) 245-4075. Fax:
(718) 245-2118. E-mail: mhammmerschlag{at}pol.net.
| |
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Antimicrobial Agents and Chemotherapy, September 1999, p. 2268-2272, Vol. 43, No. 9
0066-4804/99/$04.00+0
Copyright © 1999, American Society for Microbiology. All rights reserved.
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Abstract
Introduction
