Chlamydia antibiotic resistance - illustrated presentation by Geoffrey Ridgway


Fig 1. Title slide. Double click on the thumbnailed images. This presentation © Dr Geoffrey Ridgway, 2002. Antibiotic resistance may be broadly classified into microbial resistance and clinical resistance. Microbial resistance is resistance intrinsic or inherent to the microbe, i.e. to chlamydiae.	Fig 2. Clinical resistance is more complex, and relates to the probability that there will not be a response to apparently optimal therapy. Reasons may include microbial resistance, persistence of the micro-organism, polymicrobial infection, adverse pharmacology (particularly pharmacokinetics), side effects leading to poor compliance, failure of the antimicrobial to reach the site of infection, and reduced patient immunity.	Fig 3. One problem is the non specific nature of many of the syndromes associated with chlamydiae, particularly genital and respiratory tract infections.	Fig 4. Early studies in eggs soon demonstrated that chlamydiae are intrinsically resistant to aminoglycosides, antifungals and polymixins [Gordan & Quan, 1962], but sensitive in part to penicillins, tetracyclines, sulphonamides (some species) and macrolides such as erythromycin [Werner, 1961].

Fig 5. The possibility of acquired resistance was of early concern, with description of strains of C. trachomatis showing decreased resistance to tetracyclines, penicillins and sulphonamides in egg culture system [Shiaio et al., 1967]. Keshishyan et al., 1973 demonstrated the rapid emergence of resistance to rifampicin following serial passage of C. trachomatis in eggs.	Fig 6. Cell culture made it easier to do antibiotic resistance experiments on chlamydiae, but there were all too many variables, summarised here, which could affect the results.	Fig 7. Cell culture studies: antibiotics to which chlamydiae were found to be inherently resistant.	Fig 8. Antibiotics with only limited activity against chlamydiae. In the old chlamydial taxonomy, an important difference was that C. trachomatis was generally sensitive to sulphonamides whereas C. psittaci, (which we would now regard as Chlamydophila species) were relatively resistant.

Fig 9. Fluorescence micrograph of C. trachomatis stained with the DNA and RNA binding dye, acridine orange. The slide shows the aberrant, unusually large reticulate body like structures caused by treatment with ß-lactam antibiotic, such as penicillin, to which chlamydiae are partly susceptible.	Fig 10. As for Fig 9, but no antibiotic treatment, i.e. a control.	Fig 11. In chronic infections like trachoma, chlamydiae may be able to persist in the absence of obvious clinical signs [Hannah et al., 1968]. It is thought that chlamydiae in chronic infection may enter a persistent state characterised by aberrant reticulate bodies similar to those shown in Fig 9. See: persistent infection & also cell biology.	Fig 12. Mourad et al., 1980 were the first to report reduced sensitivity to erythromycin. In a study of 6 oculo-genital isolates, 2 strains had MICs for erythromycin of 0.5mg/l, with cidal concentration in excess of 1.0 mg/l. Curiously, there was no cross resistance with rosaramicin (an experimental macrolide available at that time). They considered this was unlikely to lead to clinical resistance, but they expressed caution.

Fig 13. Although it was possible to demonstrate the rapid emergence of resistance to rifampicin on serial passage, Jones et al., 1983 were unable to produce resistance to oxytetracycline or to erythromycin by similar techniques. In fact in sub inhibitory concentrations these latter two antibiotics could help prevent the emergence of rifampicin resistance, illustrating that the interactions between anti chlamydial antibiotics may be complex.	Fig 14. Decreased sensitivity to tetracyclines resulting in clinical failure was first reported by Jones et al., 1990. They identified 5 isolates from cases of tubal infertility which had MICs to tetracycline of 4 to >8mg/l, compared with control MICs of 0.125 to 0.25 mg/l. The isolates were also resistant to erythromycin, clindamycin and sulphonamide, but sensitive to ciprofloxacin and ofloxacin. The resistant organisms were present in small numbers (<1%) within the chlamydial population and they were unstable, tending to lose both resistance and viability in cell culture. In at least one case resistance was associated with poor clinical response.	Fig 15. Studies by Rice et al., 1995 indicated that disease severity was associated with decreased susceptibility to the chlamydiacidal action of doxycycline, azithromycin, ofloxacin, clindamycin, amoxicillin, or cotrimoxazole.	Fig 16. Tetracycline resistance was also reported from France Lefevre et al., 1997. The MIC for tetracycline was >64 mg/l, and again, only 1% of the population expressed resistance. The patient was cured with pristinamycin; a curious choice in view of the lack of published trials against chlamydiae. Unlike the strains reported by Jones et al., 1990, this strain was normally sensitive to azithromycin, erythromycin, ofloxacin and pristinamycin leading to the suggestion that the tetracycline resistance was a newly emergent problem.

Fig 17. Multiple drug resistant isolates of C. trachomatis associated with treatment failure with azithromycin were reported by Somani et al., 2000. Three patients were described (2 treatment failures and the wife of one). The three isolates were solidly resistant to doxycycline (MIC > 4.0 mg/l), and to ofloxacin (MIC 2.0 to >4.0 mg/l).	Fig 18. Samra et al., 2001 studied the sensitivity of 50 clinical isolates of C. trachomatis to azithromycin, clarithromycin, roxithromycin, erythromycin, doxycycline and tetracycline. They noted that 44% of isolates had reduced sensitivity to tetracycline (MIC = or > 0.5 mg/l). The MBC to doxycycline and tetracycline was 4mg/l in 8% and 4% of the strains respectively.	Fig 19. Dreses-Werringloer et al., 2000 reported the induction of persistence of C. trachomatis by ciprofloxacin or ofloxacin in cell culture. Aberrant small inclusions were produced, which contained viable but non-cultivatable organisms. Normal forms could be cultured on removal of the quinolone. Persistence was characterised by reduced production of the major outer membrane protein (MOMP), and persistent levels of heat shock protein 60 (HSP60). This is analogous to the situation reported for ß-lactam antibiotics (Fig 9). This is particularly disconcerting since quinolones active against chlamydiae are normally chlamydiacidal at concentrations only just above inhibitory levels.	Fig 20. Two studies have demonstrated that passage of C. trachomatis L2 strain in vitro in the presence of sub-inhibitory levels of quinolones like ofloxacin, sparfloxacin and moxifloxacin resulted in a stable 250 to 1000 fold elevation of MIC due to a Ser 83 to Ile mutation in the quinolone resistance determining region (QRDR) of the gyrA gene [Desus-Babus et al., 1998; Morrissey et al., 2002]. Interestingly under similar circumstances it was not possible to demonstrate quinolone resistance for C. pneumoniae.

Fig 21. Widespread resistance of C. suis to tetracycline has been reported. Lenart et al., 2001 noted that a tetracycline resistant strain (MIC 4 mg/l) of C. suis produced large aberrant reticulate bodies, similar to those noted (Fig 9) with ß-lactams. Co-cultivation experiments demonstrated that both C. suis and C. trachomatis could grow in the same inclusion, providing excellent opportunity for transfer of resistance genes between these closely related species.	Fig 22. Antibiotic resistance is less readily produced by C. pneumoniae compared with C. trachomatis. Hammerschlag and Roblin 2000 studied 10 patients with C. pneumoniae infection who had been treated with moxifloxacin. The organism persisted in 3 patients post therapy, but the MICs and MBCs of pre and post treatment isolates were the same. C. pneumoniae was eradicated from four of four patients receiving clarithromycin in the same study. However other studies with macrolides, reviewed by Boman and Hammerschlag, 2002 noted a four fold increase in MIC in two of 3 studies to azithromycin.	Fig 23. Some conclusions. Understanding the response of chlamydiae to antibiotics involves: Firstly, the ability of the antibiotic to kill and not just inhibit the organism; Secondly, any role of the antibiotic in inducing persistent infection; and Thirdly the ability of the organism to develop stable resistance. There is an urgent need to develop standardised methods to investigate antibiotic activity, perhaps utilising new technologies such as reverse transcriptase PCR [Cross et al., 1999], microelectronic chip arrays [Westin et al., 2001], or animal models [Stamm, 2000]. There is no doubt that both C. trachomatis and C. pneumoniae are capable of developing sophisticated systems to evade eradication by antibiotics.	Fig 24. Should we be clinically concerned about antibiotic resistance in chlamydiae?

Fig 25. The self-evident answer.	Fig 26. It may be that it is very difficult to completely kill C. trachomatis in vivo. Perhaps all available antibiotics under certain circumstances may induce persistent infection. Such a concept, if true, would be far reaching for dealing with the long term sequelae of chlamydial infections, such as chronic pelvic infection, infertility, blindness and perhaps coronary artery disease. This concept is not new for genital chlamydial infection, having been previously proposed by Oriel and Ridgway 1982 and Stamm, 2001 .

Treatment of chlamydial infections

Antibiotic resistance presentation.