Journal of Antimicrobial Chemotherapy (1977) 3, 49-56

The effect of penicillin on genital strains of Chlamydia trachomatis in tissue culture F. W. A. Johnson and D. Hobson

Department of Medical Microbiology, University of Liverpool, Liverpool, L69 3BX, England

The growth in McCoy cell tissue culture of strains of Chlamydia trachomatis recently isolated from genital infections was examined quantitatively after incubation with benzylpenicillin. An extracellular concentration of 01 unit/ml throughout incubation prevented the development of normal fluorescent chlamydial inclusions, but even at high concentration (100 units/ml) abnormal non-fluorescent inclusions developed. Erythromycin, chloramphenicol and tetracycline inhibited the growth of the organism without producing abnormal inclusions. The effect of delayed addition of penicillin or its removal during incubation was investigated. The possible nature of the abnormal inclusions resulting from exposure to penicillin is discussed. Introduction Chlamydia trachomatis is often found as the sole infective agent in males with nongonococcal urethritis, in females with hypertrophic cervical erosion or chronic cervicitise and in infants with ophthalmia neonatorum, born of infected women. Chlamydial infections often occur concurrently with gonorrhoea (Hilton, Richmond, Milne, Hindley & Clarke, 1974) and commonly persist after the gonorrhoea has been successfully treated (Holmes et ah, 1975). Therefore, it is of practical importance to investigate the sensitivity of freshly-isolated oculogenital strains of chlamydia to the antibacterial chemotherapeutic agents most likely to be used in treating sexually acquired infections. The fact that chlamydia are obligate intracellular parasites imposes obvious limitations on comparisons between the minimum inhibitory concentrations (MIC) of different antibiotics. Tissue culture cells, in which the organism must be grown in the laboratory, are unlikely to permit equal diffusion and intracellular concentration of drugs of widely different molecular size and structure, and may not closely reflect the behaviour of antibiotics in the intact tissues of the patient. It might reasonably be expected that chlamydia would be highly sensitive to penicillins, since they have cell walls of similar constitution to those of bacteria, and contain muramic acid (Perkins & Allison, 1963). However, Bernkopf, Mashiah & Becker (1962) found that 100 units/ml benzylpenicillin was necessary to prevent the growth in FL cells of the laboratory-adapted TE55 strain, originally isolated from a patient with classical trachoma 49


F. W. A. Johnson and D. Hobson

by T'ang, Chang, Huang & Wang (1957). Lower concentrations of pencillin gave only partial inhibition of growth. The purpose of this paper is to determine quantitatively the effect of benzylpenicillin on the growth of low passage strains of C. trachomatis, recently isolated from genital infections in Liverpool, in replicating cultures of the McCoy cell line. Materials and methods Tissue culture procedures, the source and methods of subculture of McCoy cells, the methods of preparing monolayer coverslip cultures (MCC) and inoculating them with chlamydia have already been described in detail by Johnson & Hobson (1976), and the subsequent staining of the infected MCC and assessment of growth of the organism by Johnson (1975). Briefly, 0-4 ml of a suitable dilution of C. trachomatis or clinical specimen was inoculated on to confluent MCC contained individually in screw-cap 1-oz Universal bottles with 2 ml of growth medium (GM=medium 199+10%foetal calf serum+20mMsodium bicarbonate). Infection of the cells was achieved by centrifugation of the bottle at 2500G for 1 h. Under these conditions, C. trachomatis undergoes a single cycle of growth. Each infective particle forms an intra-cytoplasmic inclusion packed with new infective particles (elementary bodies). Inclusions are first detectable after 18 to 24 h of incubation and reach their maximum number after 38 to 48 h. Thereafter, the size of the inclusions and the number of particles increase, and they begin to rupture the infected cells between 72 and 96 h. However, the released elementary bodies are unable to establish a fresh growth cycle in other cells in the MCC under these cultural conditions. Thus the number of inclusions countable in a given MCC is directly proportional to the input number of elementary bodies which were capable of establishing an ongoing intracellular infection. In Giemsa-stained MCC, fully-developed inclusions give an intense yellow fluorescence under dark ground microscopy; for practical reasons we have used this method of enumeration routinely after a preliminary scan under light microscopy. The statistical validity of the counting procedure has been discussed by Johnson & Hobson (1976). With the strain 'Stu' used in the present experiments, it has been calculated that the 95% confidence limits of the counts shown below is of the order of ±23-5%. The 'Stu' strain was isolated here from a 17-year-old girl with cervicitis, and the pool used throughout these experiments was the first passage in the yolk sac of chick embryos from the positive primary McCoy culture inoculated with the original clinical specimen (Johnson & Hobson, 1976). Benzylpenicillin (Cystapen, Glazo) was freshly dissolved in sterile distilled water from vials for each experiment, and working dilutions of 1000 units/ml and below were made in tissue culture growth medium (GM). Final concentrations of penicillin in this medium were assayed in an agar cup-plate microbiological assay system using Staph. pyogenes NTC 6571 as an indicator organism. To avoid any problem of synergy or antagonism with other antibacterial agents, all MCC used in the experiments with penicillin described below were maintained throughout in media which did not contain any other antibiotic. Vancomycin and streptomycin are routinely used at concentrations of 100 Jig/ml in normal tissue culture work with chlamydia, and show no significant antichlamydial activity. However, in the present work, they were used only during the initial preparation of McCoy cells for the experimental MCC.

Penicillin and C. trachomatis


Continuous incubation and delayed addition of benzylpenicillin Confluent MCC were washed and changed to fresh GM containing penicillin, giving final concentrations from 10 to 001 unit/ml; control MCC were changed to fresh antibiotic-free medium. All were immediately infected with 'Stu' strain, at a dose calculated to yield 10,000 to 15,000 inclusions per coverslip, and centrifuged without further delay. After 48 h incubation at 37°C, duplicate coverslips at each concentration were Giemsa-stained, and chlamydial inclusions counted under dark-ground microscopy. In order to determine whether penicillin could affect the further development of chlamydia which had already established infection in McCoy cells, and had begun to produce normal inclusions, 30 MCC were simultaneously infected with 'Stu' strain, centrifuged and incubated for varying periods of time before adding the same range of penicillin concentrations. All MCC were then stained and examined after a total incubation period of 48 h. The effect of removing penicillin To determine whether the effect of penicillin was reversible, MCC were infected with 'Stu' strain and incubated for 48 h in the continuous presence of 0-1 or 1-0 unit/ml penicillin or antibiotic-free medium. After incubation, two MCC of each type were removed for staining and a further two were washed in several changes of antibiotic-free medium and then reincubated for a further 48 h without penicillin before staining. Results Continuous incubation with benzylpenicillin The inclusion counts fell progressively with increasing concentrations of benzylpenicillin (Table I, column a). Even with 0025 unit/ml the count was 50% lower than in antibioticfree MCC. No normal fluorescent inclusions were found at 1 -0 unit/ml, but by light microscopy many small abnormal inclusions were seen. These contained only a few swollen irregular particles or amorphous material staining red with Giemsa instead of the normal close packed array of deep blue stained elementary bodies (Plate 1). Comparison of identical coverslip fields under both light and dark-ground microscopy confirmed that the abnormal inclusions were non-fluorescent. The approximate number of these Table I. The effect of increasing concentrations of benzylpenicillin on the inclusion count of C. trachomatis in McCoy cells Inclusion count per coverslip, after 48 h incubation extracellular (unit/ml)

0 001 0025 005 01 10

(a) Normal fluorescent inclusions 9300 7100 4200 125 1 0

(b) Abortive non-fluorescent inclusions 0 1500 2000 4000 >5000 >5000

' Penicillin was added at the time of infection, and was present throughout incubation.


F. W. A. Johnson and D. Hobson

(Table I, column b) increased in parallel with the fall in numbers of normal inclusions in MCC incubated with 001 to 10unit/ml penicillin. When this experiment was repeated using penicillin in an extended range of final concentrations (0-01 to 100 units/ml), again no normal fluorescent inclusions developed at concentrations of 0 1 unit or above, but approximately 5000 abnormal non-fluorescent inclusions were found on each coverslip incubated with 0-1 to 10 units/ml penicillin, and even at 100 units/ml approximately 500 were present. Delayed addition of benzylpenicillin The counts of normal and abnormal inclusions (Table II) showed that the antichlamydial activity of benzylpenicillin was dependent both on its concentration and on the time in Table n . The effect of delayed addition of benzylpenicillin to McCoy cell cultures infected with C. trachomatis Inclusion count per coverslip, after 48 h incubation* extracellular concentration (unit/ml)

infection before adding penicillin (h)



Normal fluorescent inclusions

Abortive non-fluorescent inclusions f



15200 15120


47 30 24 6 0

15000 8500 7500 5400

47 30 24 6 0



870 270 15 0

1000 3000 3000 5000



0 0 0

2000 4000 4000 >5000

* Total period of incubation = 48 h. t Approximate count only, because of difficulty in visualization. the growth cycle at which it had been added. The size of the normal fluorescent inclusions, and the number and morphology of the particles within them, were not significantly different in MCC incubated in penicillin or in antibiotic-free medium. However, the fall in the count of such inclusions with increasing dosage or time of exposure to penicillin was again accompanied by the appearance of many abnormal non-fluorescent inclusions. Penicillin was added to certain of the infected MCC only 1 h before staining to exclude the possibility that these abnormal inclusions might be the result of a direct chemical effect of penicillin on previously-normal inclusions, leading to lysis of their contents or interference with their normal staining characteristics or fluorescence. The effect of removing penicillin The counts of normal and abnormal inclusions in MCC incubated for 48 h with penicillin and then for a further 48 h after removing it are shown in Table III along with counts

Normal inclusion in antibiotic free medium

Elementary bodies Packed mass of elementary bodies embedded in matrix

Abnormal pale- staining non - f lourescent particles

Abortive inclusion in culture exposed to penicillin Plate 1. Intracellular inclusions of chlamydia in: (a) Antibiotic free cultures of McCoy cells, (b) cultures containing 10 ugm/ml penicillin.

Penicillin and C. trachomaris


Table III. The effect of removing benzyl penicillin after 48-h incubation in McCoy cell cultures infected with C. trachomatis Inclusion counts per coverslip after incubation Extracellular concentration of penicillin (unit/ml) from 0 to 48 h

0 01 10

(a) for 48 h with penicillin Normal fluorescent inclusions

Abortive non-fluorescent inclusions*

(b) for 96 h to 48 h after removal of penicillin Normal fluorescent inclusions

Abortive non-fluorescent inclusions





270 1

5000 3000

92 0



* Approximate count only.

in MCC incubated for 96 h without penicillin. In the latter control MCC, all inclusions were normal and fluorescent, and represented the onward growth of those seen at 48 h. Many inclusions occupied most of the cytoplasm of the infected McCoy cell and were bursting. Abundant free elementary bodies could be seen scattered over the monolayer. The fall in the total count between 48 and 96 h was probably the result of infected cells rupturing and falling off the glass. As expected in this tissue culture system, there was no evidence of a second crop of inclusion bodies developing between 48 and 96 h. In contrast, MCC previously incubated with benzylpenicillin showed no increase in the number of normal fluorescent inclusions after removal of the antibiotic. There was no evidence that any significant number of McCoy cells had become detached from the coverslip, but the number of non-fluorescent abortive inclusions had fallen considerably. In a separate control experiment it was shown that MCC which had been pre-incubated for 48 h before a change of medium and infection with 'Stu' strain gave inclusion counts after a further 48 h incubation which were closely similar to those in conventional freshly prepared MCC of the type used in all the early experiments. Thus, it seemed unlikely that the failure of regeneration of chlamydial growth after removal of penicillin was due to a direct loss of the ability of McCoy cells to support replication of chlamydia with increasing age. The effect of benzylpenicillin on other strains of chlamydia The 'Stu' strain was not unique in its behaviour in the presence of benzylpenicillin. Two further Liverpool strains TC691 and Atk., were isolated from different patients and passaged three times in McCoy cell cultures to obtain high titre stocks without the necessity for growth in chick embryos. Specimens from another 3 patients yielded chlamydia in the primary McCoy culture in sufficient titre, without further passage, for tests with penicillin. With all 5 strains, the MIC of penicillin in terms of inhibition of normal inclusions, and the appearance of abortive inclusions in concentrations much greater than the MIC, were exactly similar to the findings with 'Stu' strain. Furthermore, chlamydia have since been isolated from 20 female patients who were receiving penicillin shortly before or at the time the specimen was taken. In each case the morphology of many of the inclusions found in the MCC closely resembled those of the abortive inclusions seen in the experimental cultures. It seems probable that this


F. W. A. Johnson and D. Hobson

appearance was the result of low concentrations of penicillin from the cervical secretion, with persistant activity in the MCC inoculated with the specimen. The effect of some other antibiotics on chlamydia It seemed likely that the replacement of normal fluorescent inclusions of chlamydia by abnormal forms in the presence of penicillin might be a specific attribute of this particular antibiotic rather than a common chlamydial response to all growth-inhibiting agents. To clarify this point, varying concentrations of chloramphenicol, erythromycin or tetracycline were added to MCC immediately before infection, and incubated for 48 h alongside infected MCC in antibiotic-free media. Quantitative inclusion counts showed that the appearance of inclusions was completely prevented by 1 0 ug/ml of chloramphenicol, and 001 ug/ml of erythromycin or tetracycline. The effect of 'pulsed' addition and removal of these antibiotics at various times after infection will be reported elsewhere. The constant finding with all three antibiotics which is relevant to the present study is that the number of chlamydial inclusions fell progressively with increasing dosage, and they were not replaced by abortive inclusions of the type seen consistently in penicillintreated cultures. Discussion There have been few previous quantitative studies of the effect of benzylpenicillin on the growth of C. trachomatis. Bernkopf, Mashiah & Becker (19626) and Kramer & Gordon (1971) investigated the effect of very large doses (50 and 100 units/ml respectively) added only late in the growth cycle in tissue culture of extensively-passaged 'fast' laboratory strains, which differ in many biological properties from strains recently isolated from genital infections, particularly in their growth rate and highly efficient adsorption and readsorption to tissue culture cells without centrifugation. In other investigations, e.g. Bernkopf, Mashiah & Becker (1962a), various concentrations of penicillin were added to tissue cultures infected with adapted strains of chlamydia, but the effect on the growth of the organism was not determined quantitatively. Also, other antibiotics were routinely incorporated in the tissue culture fluid. It was hoped that the present investigations with low-passage strains of C. trachomatis recently isolated from the genital tract might provide information more closely relevant to current clinical problems in the treatment of urethritis and cervicitis. For this reason, it was decided to use conventional replicating cultures of McCoy cells, rather than the specially treated non-replicating cultures more commonly used in laboratory work with chlamydia (e.g. Kramer & Gordon, 1971). Such treatment of tissue cultures, with 4000 to 6000 rad of X-irradiation (Gordon, Dressier, Quan, McQuilkin & Thomas, 1972) or with cytochalasin B (Sompolinsky & Richmond, 1974) or particularly deoxyuridine (Wentworth & Alexander, 1974) could alter the metabolism of the cell and the transport of antibiotics across its membrane, or affect the growth properties of the chlamydia growing within them, in such a way as to increase the artificiality of a system which can inherently be only a rough approximation to conditions in vivo. The present experiments indicated that benzyl penicillin was highly active, in very low extracellular concentrations, in preventing the normal intracellular development of C. trachomatis. However, there was a tenfold difference between the concentration capable of preventing the development of 20% of the expected normal inclusions and that required to inhibit all of them. Such a comparatively shallow dose-response curve might

Penicillin and C. trachomatis


not be expected with penicillin acting against a low inoculum of free-living bacteria. In the present complex system, it may represent differences in permeability between different McCoy cells, or inherent differences in antibiotic sensitivity between different infective particles, since neither the cell line nor the chlamydial strain had been clonally purified. It is interesting that the average size of the inclusions which did develop in the presence of penicillin was similar to that of inclusions in antibiotic-free cultures, i.e. an apparent all-or-nothing effect at a given dose. A constant feature of penicillin-treated cultures was the replacement of normal inclusions by abortive non-fluorescent inclusions containing particles of abnormal size and staining characteristics. Their appearance was not confined to cultures treated only with sub-inhibiting doses of penicillin, and indeed seemed not to be dose-dependent since they were still found in large numbers in cultures exposed to penicillin concentrations 10 to 100 times greater than that required to prevent the development of all normal inclusions (0-1 unit/ml). No such 'transference' phenomenon was found with chloramphenicol, erythromycin or tetracycline. Abnormal inclusions in penicillin-treated chlamydia-infected cultures have been observed previously; Kramer & Gordon (1971) described their ultrastructure by electron microscopy, and their cytochemistry was investigated by Bernkopf, Mashiah & Becker (1962a). The material in these inclusions contained RNA and some DNA, but did not have the characteristic cell wall of mature chlamydial elementary bodies. It seems reasonable to ascribe this appearance to the specific mode of action of penicillin against the cell wall of susceptible organisms. The early stages of development of chlamydia may proceed in the presence of penicillin, but be arrested at the stage where large initial bodies, the precursors of progeny elementary bodies, appear. These initial bodies may persist because they do not yet possess a cell wall susceptible to penicillin, and may divide to give further initial bodies, but they cannot mature into elementary bodies with rigid cell walls of cross-linked structure, since this synthetic step would be prevented by the antibiotic. An alternative possibility is that the abnormal particles are derived from already mature elementary bodies by a process analogous to the formation of spheroplasts by bacteria. The effects of delayed addition of penicillin at different times in the chlamydial growth cycle (Table II) are consistent with this hypothesis. The balanced environment of the intracellular inclusion would tend to preserve the physical integrity of spheroplasts of chlamydia to an extent never likely to be seen in a conventional culture of free-living bacteria treated with penicillin. Whatever the true nature of the particles in abortive inclusions, it seemed surprising that normal growth was not resumed with resynthesis of cell-walls once the penicillin is removed from the extracellular medium. Several factors could be involved in this apparent failure. Firstly, it was possible that not all the intracellular penicillin was leached out after removal of the extracellular antibiotic. The addition of penicillinase might not help because the ability of the enzyme to enter the cell or act within it is unknown. Secondly, the metabolism and 'defence mechanisms' of the host cell might well have changed considerably to the disadvantage of chlamydia during the time that growth of the agent was arrested by penicillin. For example, the activation of lysosomes and the production of interferons could prevent further normal growth. Such mechanisms might explain the large amount of amorphous debris seen in the abortive inclusions, and the gradual decline in their numbers seen after removal of penicillin (Table III). No tissue culture system can reproduce exactly the conditions prevailing in vivo. Therefore, it is possible that, in the intact tissues of chlamydia-infected patients, some


F. W. A. Johnson and D. Hobson

abortive inclusions produced after treatment with penicillin might be capable of reversion to normal growth after the antibiotic is removed. This possibility provides a tentative explanation of the poor and variable results of treating genital chlamydial infections with benzylpenicillin compared with the successful use of tetracyclines, although both antibiotics appear to have a similar minimum inhibitory concentration by laboratory tests. It is also important that tetracycline is usually given for longer than benzylpenicillin in the conventional treatment of sexually transmitted diseases. Acknowledgements We are grateful to the Medical Research Council for a project grant in support of this work, to our clinical colleagues, Dr E. Rees and Dr A. I. Tait, Liverpool Royal Infirmary for providing clinical data and material, and to Dr A. Percival for helpful discussion on this paper. References Bernkopf, H., Mashiah, P. & Becker, Y. Correlation between morphological and biochemical changes and the appearance of infectivity in FL cell cultures infected with Trachoma agent. Annals of the New York Academy of Sciences 98: 62-81 (1962a). Bernkopf, H., Mashiah, P. & Becker, Y. Susceptibility of a Trachoma Agent grown in FL cell cultures to antibiotics and a Sulfa drug. Proceedings of the Society for Experimental Biology & Medicine 111: 530-7 (1962b). Gordon, F. B., Dressier, H. R., Quan, A. L., McQuilkin, W. T. & Thomas, J. I. Effect of ionizing irradiation on susceptibility of McCoy cell cultures to Chlamydia trachomatis. Applied Microbiology 23: 123-9 (1972). Hilton, A. L., Richmond, S. J., Milne, T. D., Hindley, F. & Clarke, S. K. R. Chlamydia A in the female genital tract. British Journal of Venereal Diseases 50: 1-9 (1974). Holmes, K. K., Handsfield, H. H., Wang, S. P., Wentworth, B. B., Turck, M., Anderson, J. B. & Alexander, E. R. Etiology of non-gonococcal urethritis. New England Journal of Medicine 292: 1199-205(1975). Johnson, F. W. A. A comparison of staining techniques for demonstrating group A Chlamydia in tissue culture. Medical Laboratory Technology 32: 233-8 (1975). Johnson, F. W. A. & Hobson, D. Factors affecting the sensitivity of replicating McCoy cells in the isolation and growth of chlamydia A (TRIC agents). Journal of Hygiene 76: 441-51 (1976). Kramer, M. J. & Gordon, F. B. Ultrastructural analysis of the effects of penicillin and chlortetracycline on the development of a genital tract chlamydia. Infection and Immunity 3: 333-41 (1971). Perkins, H. R. & Allison, A. C. Cell-wall constituents of Rickettsiae and Psittacosis-Lymphogranuloma organisms' Journal of General Microbiology 30: 409-80 (1963). Sompolinsky, D. & Richmond, S. Growth of Chlamydia trachomatis in McCoy cells treated with Cytochalasin B. Applied Microbiology 28: 912-4 (1974). T'ang, F. F., Chang, Y. T., Huang, Y. T. & Wang, K. C. Studies on the etiology of trachoma with special reference to isolation of virus in chick embryo. Chinese Medical Journal 75: 429^6 (1957). Wentworth, B. B. & Alexander, E. R. Isolation of Chlamydia trachomatis by use of 5-iodo2-deoxyuridine-treated cells. Applied Microbiology 27: 912-6 (1974). {Manuscript accepted 14 April 1976)

The effect of penicillin on genital strains of Chlamydia trachomatis in tissue culture.

Journal of Antimicrobial Chemotherapy (1977) 3, 49-56 The effect of penicillin on genital strains of Chlamydia trachomatis in tissue culture F. W. A...
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