Comparative in-vitro activity of moxifloxacin, minocycline and azithromycin against Chlamydia spp.

M. Donati, M. Rodrìguez Fermepin, A. Olmo, L. D' Apote and R. Cevenini*

Sezione di Microbiologia, DMCSS, Policlinico S. Orsola, University of Bologna, Bologna, Italy


    Abstract
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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
The in-vitro activity of moxifloxacin, a new 8-methoxyquinolone, was compared with minocycline and azithromycin against 40 strains of Chlamydia trachomatis, Chlamydia pneumoniae and Chlamydia psittaci. Both the MIC and the MBC of moxifloxacin ranged from 0.03 to 0.125 mg/L. MICs of minocycline ranged from 0.015 to 0.06 mg/L and MBCs between 0.03 and 0.25 mg/L. MICs of azithromycin ranged from 0.03 to 0.125 mg/L and the MBCs between 0.06 and 0.5 mg/L. MBC values of moxifloxacin were the same as MICs in 32 (80%) of 40 strains tested, whereas those of minocycline and azithromycin were two to four times higher than their MICs. These data confirm those previously obtained indicating that quinolones kill chlamydial strains at concentrations equivalent to their MICs.


    Introduction
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Chlamydiae are obligate intracellular parasites which infect a wide range of host species. 1 Chlamydia trachomatis and Chlamydia pneumoniae are both human pathogens. While C. trachomatis has been the object of much interest in sexually transmitted diseases and is recognized as a major cause of neonatal conjunctivitis, C. pneumoniae is emerging as an important cause of pneumonia. Chlamydia psittaci is an animal pathogen which occasionally infects humans and causes a broad-spectrum of bird and ruminant diseases including pneumonia, abortion, polyarthritis, conjunctivitis and epididymitis. Since Chlamydia spp. play an important role both in human and animal diseases, the development of effective antimicrobial agents for use in the treatment of chlamydial infections is very important.

Tetracyclines are usually the treatment of choice for chlamydial infections. However, the frequent recurrence of infection necessitates the investigation of other compounds. The newly developed fluoroquinolone antimicrobial agents are characterized by potent and broad antibacterial in-vitro activity2,3 not only against common Gram-positive and Gram-negative bacteria, but also against atypical species such as Mycoplasma spp.4 and Chlamydia spp. 5,6

The objective of this study was to investigate the in-vitro activity of moxifloxacin, a new 8-methoxyquinolone drug, compared with minocycline and azithromycin against C. trachomatis, C. pneumoniae and C. psittaci strains.


    Materials and methods
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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Bacterial strains and growth conditions

The study included 27 C. trachomatis strains, three C. pneumoniae strains and 10 C. psittaci strains. C. trachomatis strains included 21 untyped clinical strains recently isolated in cell culture from urethral swabs of male patients with signs of uncomplicated urogenital infection. Also included were six typed C. trachomatisstrains (serovars D, E, F, H, J, LGV2). C. pneumoniae strains comprised two recent isolates from Italy and the strain IOL-207, originally isolated in 1967 in London from an Iranian child with trachoma. 7 The C. psittaci strains studied were five reference strains and five strains recently isolated from infected animals in Italy by Dr Magnino (Diagnostic Section, Zooprophylactic Institute, Pavia, Italy). The C. psittaci reference strains included the feline pneumonitis strain FePn-145 (USA), and the meningopneumonitis strain MePn-343 (ferret, USA), the avian (parakeet) strain 6BC (USA), the ovine (lamb) strain A22 (Scotland) and the caprine abortion strain Krauss15 (Scotland). The Italian C. psittaci isolates included strains from a parrot, a pigeon and three bovine strains. Chlamydia spp. strains were grown in McCoy cell monolayers in 24-well plates containing a glass cover slip (diameter, 12 mm) at the bottom. The cell growth medium was Eagle's MEM containing 10% heat inactivated fetal calf serum, 10 mg/L gentamicin, 10 mg/L vancomycin, 2 mM glutamine and 1.7 mg/L glucose.

Antimicrobial drugs and sensitivity assays

Moxifloxacin was supplied by Bayer (Milan, Italy), minocycline was purchased from Sigma (Milan, Italy) and azithromycin was obtained from Pfizer (Rome, Italy). Chlamydia spp. strains were suspended in cell growth containing cycloheximide (1 mg/L) and glucose (5 mg/L). Each of the 24-well plates was infected with an inoculum of chlamydiae which yielded 5x10 3 inclusion forming units (IFU)/mL. After centrifugation at1700g for 1 h the medium was removed and replaced with medium containing different concentrations of antimicrobial drugs. All tests were run in triplicate. MICs were determined after 48 h incubation at 35°C. Coverslips were removed and the cells fixed with methanol and stained with a monoclonal antibody to the lipopolysaccharide genus antigen.8 The MIC was the lowest antibiotic concentration which resulted in >90% reduction of chlamydial inclusions compared with the drug-free control. MBCs were determined as follows. The antibiotic-containing medium was aspirated, and cell monolayers were washed and incubated in growth medium without antibiotics for 48 h at 35°C. Cells were fixed and chlamydial inclusions were stained as described above. The MBC was the lowest concentration of the drug preventing >90% demonstrable inclusions after monolayers were reincubated for 48 h in antimicrobial-free medium.


    Results
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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
MICs and MBCs of moxifloxacin, minocycline and azithromycin for Chlamydia spp. strains are shown in the Table. Both the MICs and MBCs of moxifloxacin for the 40 Chlamydia spp. strains ranged between 0.03 and 0.125 mg/L. MICs of minocycline ranged between 0.015 and 0.06 mg/L, and MBCs ranged between 0.03 and 0.250 mg/L. MICs of azithromycin ranged between 0.03 and 0.125 mg/L and MBCs between 0.06 and 0.5 mg/L. The MBC values of moxifloxacin were the same as MICs for 32 (80%) of the 40 strains tested. Conversely, the MBCs of minocycline and azithromycin were two to four times higher than theirMICs.


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Table. MICs (mg/L) and MBCs (mg/L) of moxifloxacin, minocycline and azithromycin against Chlamydia spp.
 

    Discussion
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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Chlamydia spp. are well known human pathogens which cause respiratory and genital tract infections worldwide. Tetracycline and macrolides have been used frequently for treatment of chlamydial infections. Recently, quinolones have attracted interest for their potential use in therapy of community-acquired respiratory tract infections. In-vitro studies have also demonstrated that some fluoroquinolones are active against chlamydial species. In this study, the activity of the new fluoroquinolone, moxifloxacin, has been tested in vitro against Chlamydia spp. Moxifloxacin was shown to be an active drug against Chlamydia spp., at least in vitro, with MICs ranging from 0.03 to 0.125 mg/L, at which concentration >90% of the strains were inhibited, and MBCs ranging from 0.03 to 0.125 mg/L, at which concentration >90% of the strains were killed. The MICs and MBCs of moxifloxacin were very consistent from species to species and from strain to strain, especially in view of the varied origin and geographical distribution of the isolates tested.

The finding that for a relatively high percentage of chlamydial strains tested (80%) the MICs were the same as MBCs confirm previous observations9 that quinolones, at concentrations equivalent to their MICs, seem to kill chlamydial strains.

Data on the activity of moxifloxacin against Chlamydia spp., in particular against C. pneumoniae and C. psittaci, are limited, and those presented in this study seem to confirm the previous data.6 Besides Chlamydia spp., moxifloxacin was shown to be active against several other common respiratory pathogens. This drug may therefore have a role in the treatment of community-acquired pneumonia.

The results of our study regarding the MICs (from 0.015 to 0.06 mg/L) and the MBCs (from 0.03 to 0.250 mg/L) of minocycline, and the MICs (from 0.03 to 0.125 mg/L) and the MBCs (from 0.06 to 0.5 mg/L) of azithromycin confirmed the previous data.10 It is noteworthy that no significant differences in antimicrobial drug sensitivity were present among strains from the various chlamydial species, and several passages of the chlamydial strains in cell culture did not modify their sensitivity to antimicrobial drugs.


    Notes
 
* Corresponding author. Tel: +39-51-341652; Fax: =39-51-341632; E-mail: Cevenini{at}almadns.unibo.it Back


    References
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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
1 . Peeling, R.W. & Brunham, R. C. (1996). Chlamydiae as pathogens: new species and new issues. Emerging Infectious Diseases 2, 307–19.[ISI][Medline]

2 . Dalhoff, A., Petersen, U. & Endermann, R. (1996). In vitro activity of BAY 12-8039,a new 8-methoxyquinolone. Chemotherapy 42,410 –25.[ISI][Medline]

3 . Fass, R. J. (1997). In vitro activity of Bay 12-8039, a new 8-methoxyquinolone. Antimicrobial Agents and Chemotherapy 41, 1818–24.[Abstract]

4 . Bebear, C. M., Renaudin, H., Boudjada, A. & Bebear, C. (1998). In vitro activity of Bay 12-8039, a new fluoroquinolone, against mycoplasmas. Antimicrobial Agents and Chemotherapy 42, 703 –4.[Abstract/Free Full Text]

5 . Hammerschlag, M. R., Hyman, C. L. & Roblin, P. M. (1992). In vitro activities of five quinolones against Chlamydia pneumoniae. Antimicrobial Agents and Chemotherapy 36, 682 –3.[Abstract]

6 . Woodcock, J. M., Andrews, J. M., Boswell, F. J., Brenwald, N. P. & Wise, R. (1997). In vitro activity of BAY 12-8039, a new fluoroquinolone. Antimicrobial Agents and Chemotherapy 41, 101 –6.[Abstract]

7 . Forsey, T., Darougar, S. & Treharne, J. D. (1986). Prevalence in human beings of antibodies to Chlamydia IOL-207, an atypical strain of chlamydia. Journal of Infection 12,145 –52.[ISI][Medline]

8 . Cevenini, R., Donati, M., Sambri, V., Rumpianesi, F. & La Placa, M. (1987). Reactivity of elementary bodies of Chlamydia trachomatis LGV2 with monoclonal antibodies specific for the major outer membrane protein. FEMS Microbiology Letters 42, 47–51.[ISI]

9 . Roblin, P. M, Kutlin, A. & Hammerschlag, M. R. (1997). In vitro activity of trovafloxacin against Chlamydia pneumoniae. Antimicrobial Agents and Chemotherapy 41, 2033–4.[Abstract]

10 . Miyashita, N., Niki, Y., Kishimoto, T., Nakajima, M. & Matsushima, T. (1997). In vitro and in vivo activities of AM-1155, a new fluoroquinolone, against Chlamydia spp. Antimicrobial Agents and Chemotherapy 41, 1331–4.[Abstract]

Received 9 June 1998; returned 27 October 1998; revised 5 January 1999; accepted 14 March 1999