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Chlamydia pneumoniae is a strict intracellular pathogen that commonly infects the respiratory tracts of children and adults. Isolation of C. pneumoniae from clinical specimens and in vitro propagation of the bacterium requires the use of an appropriate cell line (e.g., HEp-2, HL, or HeLa). Stock suspensions of C. pneumoniae are sometimes contaminated with Mycoplasma spp. (1, 4, 5). The contamination can originate from the simultaneous isolation of a Mycoplasma sp. and C. pneumoniae from a respiratory tract specimen or from the use of a Mycoplasma-contaminated cell culture for propagating the chlamydiae (4, 5). Recently, Mycoplasma contamination has been detected in a widely used commercially available C. pneumoniae antigen (9) and in three different stock cultures of C. pneumoniae, which were obtained from the American Type Culture Collection (ATCC) (1). Mycoplasma contamination of cell cultures can either reduce or enhance viral infectivity (8), but its effect on the infectivity of C. pneumoniae is unknown. Elimination of Mycoplasma contamination from strains of C. pneumoniae is difficult, as mycoplasmas and chlamydiae have similar antibiotic susceptibility profiles. However, treatment with Triton X-100 successfully eliminated Mycoplasma arginini from several stock suspensions of Chlamydia spp. (5).

Our laboratory recently isolated a Mycoplasma contaminant from a stock suspension of the ATCC TW-183 strain of C. pneumoniae (E. A. Castilla and R. M. Wadowsky, Prog. Abstr. 15th Annu. Clin. Virol. Symp. Annu. Meet. Pan Am. Soc. Clin. Virol., abstr. S36, 1999). In this study, we describe the isolation and characterization of the Mycoplasma contaminant, the decontamination of the C. pneumoniae stock, and the effect of the Mycoplasma contaminant on the infectivity of C. pneumoniae for HEp-2 cells. All experiments were repeated at least twice with similar results; the results of representative experiments are reported.

Upon receipt of the ATCC stock vial of the TW-183 strain of C. pneumoniae, a portion of the contents was inoculated onto Columbia colistin nalidixic acid (CNA) agar and soy peptone agar without thallium acetate (2). After 3 days of incubation at 37°C in an anaerobic atmosphere, tiny pinpoint colonies were observed on Columbia CNA agar, and colonies with a typical fried-egg appearance were observed on soy peptone agar. Prior to additional studies, the isolate was purified by three sequential passages on soy peptone agar. Cultural and metabolic studies showed that the isolate grew better in an anaerobic atmosphere than in an aerobic atmosphere, was unable to metabolize glucose but hydrolyzed arginine, was resistant to thallium acetate and erythromycin, and was hemadsorption negative when guinea pig erythrocytes were used. Based on these features we presumptively identified the isolate as a Mycoplasma hominis-like mycoplasma.

The nonionic detergents Igepal CA-630 (Nonidet P-40), Triton X-100, and Tween 20 (all from Sigma Chemical Co., St. Louis, Mo.) were evaluated for eradicating the Mycoplasma contaminant from the C. pneumoniae stock suspension by a previously described procedure (5) with slight modification. Stock suspensions (1 ml) of the chlamydiae were centrifuged at 13,000 × g for 15 min, and the pellets were suspended in 150 µl of the supernatant. These suspensions were combined with 850 µl of detergent solutions, which were diluted in Sorensen's phosphate-buffered saline (SPBS). After incubation at 4°C for 10 min, the detergent-treated suspensions were centrifuged, and the pellets were washed once with 1 ml of SPBS. The final pellets were suspended in 1 ml of SPBS. Residual detergent was removed from these suspensions by the addition of 50 µg of Bio-Beads (SM-2) adsorbent (Bio-Rad, Hercules, Calif.) and incubation at 37°C for 30 min. The viability of the C. pneumoniae in the treated suspensions was assessed by culturing 200-µl portions of the treated suspensions in quadruplicate onto HEp-2 cell monolayers in 96-well microtiter plates. The cultures were treated with 2 µg of cycloheximide per ml and centrifugation to facilitate infection by the chlamydiae, incubated at 35°C for 72 h, and stained with a fluorescein-conjugated anti-C. pneumoniae monoclonal antibody (Washington Research Foundation, Seattle, Wash.), as previously described (7). Fluorescent inclusion-forming units (IFU) were counted by viewing the microtiter plate in an inverted position with an epifluorescent microscope. The viability of the M. hominis-like mycoplasma in the detergent-treated suspensions was assessed by inoculating 100-µl portions in duplicate onto soy peptone agar and incubating the plates anaerobically for 14 days at 35°C (2). Treatment of the C. pneumoniae stock suspension with 1.0% Tween 20 had no inhibitory effect on either the chlamydiae or the mycoplasma (results not shown). The highest noninhibitory concentration of Triton X-100 for C. pneumoniae was 0.01%, but this concentration did not eliminate the mycoplasma (Table 1). In contrast, treatment with 0.04 or 0.1% Igepal CA-630 completely eliminated the mycoplasma while retaining the viability of C. pneumoniae, although at a greatly reduced level. The purified culture of C. pneumoniae remained free of the M. hominis-like mycoplasma following three serial passages in HEp-2 cells, as evidenced by negative culture results.

Cocultivation experiments were conducted to evaluate the effect of the M. hominis-like mycoplasma on the infectivity of the purified strain of C. pneumoniae. HEp-2 cell culture monolayers were simultaneously infected in quadruplicate with various numbers of viable M. hominis-like mycoplasmas (i.e., approximately 10¹ to 10⁵ CFU/monolayer) and a fixed number of the chlamydiae (i.e., approximately 100 IFU/monolayer) by using a 100-µl inoculum of each suspension per well. Control monolayers were infected with only the chlamydiae and only the mycoplasma. Viability counts of C. pneumoniae and the M. hominis-like mycoplasma were determined as described above. The average numbers of C. pneumoniae IFU per monolayer were similar in monolayers coinfected with the mycoplasma and the chlamydiae and in the non-Mycoplasma-infected, chlamydia-infected control monolayers (P > 0.1, Student's t test) (Table 2). During the 72-h incubation period, the M. hominis-like mycoplasma multiplied in the monolayers. For example, in the monolayers seeded with the highest concentration of the M. hominis-like mycoplasma, the concentration increased from 4.1 × 10⁵ to 1.7 × 10⁸ CFU/ml, a 400-fold increase.

The TW-183 strain is the type strain of C. pneumoniae. It is widely used in clinical laboratories providing diagnostic testing for C. pneumoniae. On the basis of cultural and metabolic studies, we determined that the ATCC stock material contains an M. hominis-like mycoplasma. This finding is consistent with an earlier report (1) that identified M. hominis and Mycoplasma orale in the ATCC stock by using immunoblotting, PCR, and pulsed-field gel electrophoresis studies. In another study (9), a Mycoplasma sp. closely related to M. arginini on the basis of 16S rRNA sequences was identified in stock suspensions of the AR-39 strain of C. pneumoniae marketed for use in the microimmunofluorescence test for detection of C. pneumoniae-specific antibody. M. arginini, M. hominis, and M. orale are closely related, as determined by phylogenetic analysis (9) and also by metabolic properties (6), and may be difficult to identify even with the use of molecular techniques (9). Laboratories that use the TW-183 and the AR-39 strains of C. pneumoniae for preparation of control material for culture and PCR-based assays, for preparation of antigen for use in serological assays, and for use in experimental studies may wish to evaluate the effect of the Mycoplasma contamination on their test systems.

The TW-183 strain of C. pneumoniae appears to be slightly more resistant to inactivation by Igepal CA-630 than by Triton X-100, and this property may have allowed us to eradicate the M. hominis-like mycoplasma from our stock. However, it is possible that the culturing of more replicates of detergent-treated samples would have shown no difference between the two detergents. Because treatment with Igepal CA-630 inactivates a large proportion of the chlamydiae, this technique may not work in all instances. Treatment with Igepal CA-630 is clearly superior to treatment with Tween 20 for eradication of the M. hominis-like mycoplasma from the ATCC stock material of strain TW-183.

Although the M. hominis-like mycoplasma utilizes arginine as an energy source, and the depletion of this amino acid could cause a significant reduction in growth of C. pneumoniae (3), the M. hominis-like mycoplasma does not affect the infectivity of C. pneumoniae for HEp-2 cells when the two agents are simultaneously inoculated onto the monolayers.