Serial passage of Chlamydia spp. in sub-inhibitory fluoroquinolone concentrations

I. Morrissey1,*, H. Salman1, S. Bakker1, D. Farrell1, C. M. Bébéar2 and G. Ridgway3

1GR Micro Ltd, 7–9 William Road, London NW1 3ER, UK; 2Laboratoire de Bactériologie, Université Victor Segalen Bordeaux 2, Bordeaux, France; 3Department of Clinical Microbiology, University College London Hospitals NHS Trust, London, UK

Received 25 July 2001; returned 27 December 2001; revised 16 January 2002; accepted 15 February 2002.


    Abstract
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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
We investigated the in vitro development of fluoroquinolone resistance in Chlamydia trachomatis and Chlamydia (Chlamydophila) pneumoniae grown in McCoy cell monolayers in supplemented Eagle’s minimum essential medium. With C. trachomatis, initial passages at sub-inhibitory fluoroquinolone concentrations did not affect fluoroquinolone susceptibility. However, after an initial lag of 10–24 passages (depending upon the fluoroquinolone used), fluoroquinolone resistance developed rapidly. The final fluoroquinolone MIC after a total of 30 passages was >256 times the MIC of the original wild-type strain with ofloxacin or ciprofloxacin passage. Analysis of the quinolone-resistance determining regions of two quinolone-resistant C. trachomatis mutants obtained after 30 passages showed that both isolates had a single serine to isoleucine substitution at amino acid position 83 in GyrA. In stark contrast, with C. pneumoniae no reduced fluoroquinolone susceptibility could be sustained, even after 30 passages with moxifloxacin or ofloxacin. With sparfloxacin passage, some indication of resistance was observed but no viable organisms could be isolated for further investigation. It is possible that fluoroquinolone-resistant C. pneumoniae are less able to survive than wild type, which may explain why resistance does not develop readily.


    Introduction
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
New fluoroquinolones, such as gatifloxacin, moxifloxacin and gemifloxacin, have the potential for use in community-acquired respiratory tract infections because of their broad spectrum of activity including respiratory tract pathogens such as Streptococcus pneumoniae, Haemophilus influenzae and Moraxella catarrhalis.1 Furthermore, the ‘respiratory fluoroquinolones’ have potent activity against atypical pathogens such as Chlamydia (Chlamydophila) pneumoniae.2 C. pneumoniae is thought to be the causal agent for 5–20% of community-acquired pneumonia and other respiratory diseases.3,4

Antimicrobial resistance has not been described in C. pneumoniae clinically or even in vitro. Comprehensive antimicrobial resistance investigations, of the scale used to evaluate resistance in ‘typical’ respiratory pathogens, are difficult with C. pneumoniae because this intracellular pathogen is highly fastidious and requires specialized techniques to culture. Surveillance studies are further hampered because the presence of C. pneumoniae infection is normally confirmed by serology or PCR without culture. Few researchers have been able to culture C. pneumoniae from clinical material. To illustrate this point, there have only been two clinical isolates subcultured in the UK to date.5 This does not allow studies on the antimicrobial susceptibility of recently circulating C. pneumoniae to be carried out readily. However, one laboratory has been successful in culturing two isolates of C. pneumoniae with reduced azithromycin susceptibility (both had a four-fold rise in MIC) from patients with persistent community-acquired pneumonia.6 Further clinical studies with moxifloxacin and levofloxacin from the same laboratory have both demonstrated that isolates from patients who were microbiological failures did not have reduced fluoroquinolone susceptibility.7,8

Acquired resistance to the fluoroquinolones occurs as a result of point mutations within chromosomal DNA rather than the acquisition of plasmid DNA. These mutations often occur within the genes encoding DNA gyrase (gyrA and gyrB) and topoisomerase IV (parC and parE) within the so-called quinolone-resistance determining regions (QRDRs). In vitro investigations are commonly used to predict the clinical development of fluoroquinolone resistance with new agents.

As far as we are aware, no investigations have been carried out to determine the development of resistance to fluoroquinolones in C. pneumoniae. However, research with the related bacterium C. trachomatis has shown that high-level resistance to ofloxacin or sparfloxacin occurs rapidly during serial passage at 0.5 x MIC in vitro.9

In this study, we carried out serial passage experiments to detect resistance to ofloxacin, sparfloxacin and moxifloxacin with C. pneumoniae, as well as resistance to ofloxacin and ciprofloxacin with C. trachomatis.


    Materials and methods
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Antimicrobials

Moxifloxacin, ciprofloxacin (Bayer, UK), ofloxacin and sparfloxacin (Aventis, UK) were dissolved as per the manufacturer’s recommendation.

Growth medium

The medium used for McCoy cell propagation (designated supplemented MGM) was Eagle’s minimum essential medium (Gibco Life Technologies Ltd, Paisley, UK) supplemented to contain final concentrations of 1% glutamine solution (30 mg/L; Sigma, Poole, UK), 1% vitamins for Eagle’s basal medium (modified) (ICN, Thame, UK), 1% sodium bicarbonate solution (75 g/L), and 10% fetal calf serum (Gibco Life Technologies Ltd). The same medium was used for Chlamydia growth, except that it was supplemented further with glucose (0.03 M final concentration).

Chlamydial strains and cells

The C. pneumoniae type strain IOL 207 and C. trachomatis SA2f (LGV2) were used for all passage experiments grown in McCoy cell monolayers. When C. trachomatis was tested the McCoy cell monolayers were prepared pre-treated with 5-iodo-2-deoxyuridine (1.25 mg/L final concentration) as described previously.10 With C. pneumoniae, medium containing cycloheximide (1% final concentration) was used as described previously.11

Passage method

Sterile tissue culture flasks (25 cm2), containing 2.5 mL supplemented MGM and sterile rectangular coverslips (9 x 35 mm), were seeded with c. 105 McCoy cells and set up in duplicate [one for chlamydial growth determination (flask 1) and one for continuation of passage (flask 2)]. The initial flasks contained antimicrobial agent at 0.25 x MIC. Both flasks were inoculated with 105–108 inclusion-forming units of C. pneumoniae or C. trachomatis, and incubated for 48 h (for C. trachomatis) or 72 h (for C. pneumoniae) at 35°C. For C. pneumoniae, flasks were centrifuged for 1 h at 1800 rpm in a swing-out plate carrier of 23 cm radius (Beckman J-6B Centrifuge; Beckman Instruments, Palo Alto, CA, USA) prior to incubation.

After incubation, the overlaying fluid was tipped off from flask 1 and the monolayers were fixed in methanol. The coverslip was then removed from the flask. For C. pneumoniae, the coverslips were treated with a fluorescein-labelled antibody directed against chlamydial lipopolysaccharide (Imagen Chlamydia; Dako Diagnostic Ltd, Ely, UK). For C. trachomatis the coverslips were stained with Lugol’s iodine. The presence of chlamydial inclusions was confirmed by fluorescence microscopy or light microscopy at x300 magnification for C. pneumoniae or C. trachomatis, respectively.

After determination of the number and morphology of chlamydial inclusions in flask 1, growth medium was removed from flask 2 and the whole McCoy cell monolayer was transferred into antimicrobial-free supplemented MGM using a cell scraper. If c. 50 or more inclusions were found on the coverslip from flask 1, the re-suspended cells from flask 2 were used to inoculate further flasks containing antimicrobial agent at the same concentration as before and also to one doubling dilution higher concentration than that used previously. If <50 inclusions, or inclusions of abnormal morphology (i.e. apparently intracellular fluorescent fragments that were not regular inclusions11) were observed from flask 1, passage was continued either in antimicrobial-free medium or in medium containing antimicrobial agent at the same concentration(s) as that used previously. This procedure was continued for up to 30 passages.

QRDR analysis

DNA was extracted from the parent strain and two fluoroquinolone-resistant mutants of C. trachomatis and the QRDRs of gyrA, gyrB, parC and parE were amplified and sequenced as described previously.9 In addition, the analysis of the QRDR of parC was extended beyond that studied previously9 to include the region encoding the active site tyrosine and beyond, where mutations have been found in other bacterial species.1214 The new primers used were CTC6 (5'-AGAGACACAAGGGAACTTTGG-3') and CTC7 (5'-TTGTAGTCATCCCTACTGCG-3'). The amplification protocol for this reaction was identical to that used for the PCRs above.9 The total DNA fragment sequenced from parC encodes the region Asp-30 to Thr-162 (Escherichia coli equivalent Asp-14 to Thr-156).


    Results
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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Passage experiments

A summary of the results for the first 30 passages of C. trachomatis and C. pneumoniae in the presence of antimicrobials is shown in Tables 1 and 2, respectively. For clarity, only results from the highest concentration where growth was observed at each step are shown.


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Table 1.  Results of serial passage with C. trachomatis SA2f (ciprofloxacin and ofloxacin MIC 1 mg/L)
 

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Table 2.  Results of serial passage with C. pneumoniae IOL 207 (moxifloxacin and sparfloxacin MIC 0.12 mg/L; ofloxacin MIC 2 mg/L)
 
From Table 1 it can be seen that with C. trachomatis the first 10 passages with ofloxacin and the first 24 passages with ciprofloxacin allowed only modest increases of antimicrobial concentration during passage (up to 2 x MIC). This was due to either insufficient numbers of inclusions or abnormal inclusion morphology, or both. After this, sequential increases in antimicrobial concentration produced good growth after each passage up to 32 or 64 x MIC. Upon re-testing, the mutants subcultured after the thirtieth passage had ciprofloxacin and ofloxacin MICs of >256 mg/L. MIC determinations at higher fluoroquinolone concentrations were not possible owing to toxicity against the McCoy cell line.

With C. pneumoniae (Table 2) it can be seen that on numerous occasions passage in antimicrobial-free medium was required to ‘revive’ the organism. This occurred with all three antimicrobial agents. With moxifloxacin, passage was not possible above 0.5 x MIC without either insufficient growth or abnormal morphology occurring. Similar to ofloxacin, passage was not readily achievable at concentrations in excess of the MIC. Furthermore, viability was lost completely after 24 passages with ofloxacin. With sparfloxacin, on the other hand, passage was achievable up to 32 x MIC. However, growth was poor at each passage and subsequent subculturing from passages 24–30 could not produce sufficient stock of C. pneumoniae to perform MIC tests.

QRDR analysis

Analysis of the DNA sequences produced from amplification of the QRDRs for gyrB, parC and parE did not show any difference between the parent and fluoroquinolone-resistant mutant sequences, which is comparable to that published previously.9 This analysis included the extended parC QRDR. Analysis of the gyrA QRDR, on the other hand, detected an identical single base change from guanine to thymine for both mutants. This mutation changes the amino acid at position 83 in DNA gyrase from serine (AGT) to isoleucine (ATT). This identical mutation was shown previously with a fluoroquinolone-resistant C. trachomatis strain, selected either by ofloxacin or sparfloxacin.9


    Discussion
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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Ciprofloxacin and ofloxacin resistance appears to occur readily in C. trachomatis, in general agreement with previous results from a different laboratory.9 However, we have not tested newer generation fluoroquinolones, such as moxifloxacin. A recent study showed that resistance to levofloxacin did not occur during passage of C. trachomatis.15 This result may be explained by the fact that the Chlamydia were exposed to levofloxacin for only four passages, which may not be sufficient time to allow for the development of resistance in all isolates. It was suggested that the results of the levofloxacin study show that levofloxacin-resistant strains of C. trachomatis will probably not develop during clinical use.15 In our experiments, around 11 passage events were required to produce resistance to ofloxacin in C. trachomatis. This may be longer than a typical treatment period and it is therefore tempting to dismiss these results as not clinically relevant. This is a dangerous stance to take, based on our knowledge of resistance development in other bacterial species. Patients that have any persistent infection requiring the chronic administration of antimicrobials, including those not specifically directed at C. trachomatis infection, are likely to expose C. trachomatis to a selective pressure equivalent to significantly more than 11 passage events.

Our QRDR analysis has shown that fluoroquinolone resistance in both fluoroquinolone-resistant C. trachomatis mutants is the result of a mutation causing an amino acid substitution of isoleucine for serine at position 83. A serine substitution is a common fluoroquinolone resistance mechanism for many bacteria, including C. trachomatis.9 However, the level of resistance seen with our mutants is higher than that shown previously with C. trachomatis,9 which suggests that another unknown mechanism(s) is contributing to our resistance phenotypes. It would be of merit to look beyond the QRDR for each topoisomerase gene and to investigate the presence of possible efflux mechanisms in C. trachomatis.

Moxifloxacin and ofloxacin did not select for resistance in C. pneumoniae, even after continuous passage. The possibility of emergence of sparfloxacin-resistant C. pneumoniae mutants requires further investigation, because we are not confident that the results with sparfloxacin were indicative of true resistance development.

The data observed here suggest that sparfloxacin-resistant mutants of C. pneumoniae may occur, but that these mutants are not capable of multiplication. If fluoroquinolone-resistant C. pneumoniae are at a physiological disadvantage compared with wild type this would suggest that resistance should not develop readily. However, we do not know whether these strains would be at a similar disadvantage in vivo as suggested using the cell culture system employed in this study. The limited clinical data available suggest that antimicrobial resistance is not a problem with C. pneumoniae, even in patients with persistent infection,68,16 or indeed with C. trachomatis, even though we induced in vitro resistance readily. As discussed above for C. trachomatis, we should not become complacent regarding future resistance development. Further studies are required to improve the culturability of C. pneumoniae to support the surveillance of antimicrobial resistance. It has been suggested that the use of HEp-2 cells may improve isolation and passage of C. pneumoniae.17 We are confident that the method used in the present study (which we and others have employed for >10 years) is able to support the growth and passage of C. pneumoniae. Nevertheless, repeat experiments using an alternative method may be worth carrying out. It would also be interesting to ascertain whether the mutant selection differences between C. trachomatis and C. pneumoniae occur with other antimicrobials.


    Acknowledgements
 
We are grateful to Bayer Corporation for their financial support of the C. pneumoniae arm of this study.


    Footnotes
 
* Corresponding author. Tel: +44-20-7388-7320; Fax: +44-20-7388-7324; E-mail: i.morrissey{at}grmicro.co.uk Back


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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
1 . Blondeau, J. M. & Felmingham, D. (1999). In vitro and in vivo activity of moxifloxacin against community respiratory tract pathogens. Clinical Drug Investigations 18, 57–78.

2 . Roblin, P. M., Reznik, T., Kutlin, A. & Hammerschlag, M. R. (1999). In vitro activities of gemifloxacin (SB 26805, LB20304) against recent clinical isolates of Chlamydia pneumoniae. Antimicrobial Agents and Chemotherapy 43, 2806–7.[Abstract/Free Full Text]

3 . Grayston, G. (1992). Infections caused by Chlamydia pneumoniae strain TWAR. Clinical Infectious Diseases 15, 757–63.[ISI][Medline]

4 . Kuo, C. C., Jackson, L. A., Campbell, L. A. & Grayston, J. T. (1995). Chlamydia pneumoniae (TWAR). Clinical Microbiology Reviews 8, 451–61.[Abstract]

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9 . Dessus-Babus, S., Bébéar, C. M., Charron, A., Bébéar, C. & de Barbeyrac, B. (1998). Sequencing of gyrase and topoisomerase IV quinolone-resistance-determining regions of Chlamydia trachomatis and characterisation of quinolone-resistant mutants obtained in vitro. Antimicrobial Agents and Chemotherapy 42, 2474–81.[Abstract/Free Full Text]

10 . Ridgway, G. L., Owen, J. M. & Oriel, J. D. (1976). A method for testing the antibiotic susceptibility of Chlamydia trachomatis in a cell culture system. Journal of Antimicrobial Chemotherapy 2, 71–6.[Medline]

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14 . Gibreel, A., Sjögren, E., Kaijser, B., Wretlind, B. & Sköld, O. (1998). Rapid emergence of high-level resistance to quinolones in Campylobacter jejuni associated with mutational changes in gyrA and parC. Antimicrobial Agents and Chemotherapy 42, 3276–8.[Abstract/Free Full Text]

15 . Takahashi, S., Hagiwara, T., Shiga, S., Hirose, T. & Tsukamoto, T. (2000). In vitro analysis of the change in resistance of Chlamydia trachomatis under exposure to a sub-MIC levofloxacin for a therapeutic term. Chemotherapy 46, 402–7.[ISI][Medline]

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