Molecular identification and antibiotic clearance of Mycoplasma arginini and Mycoplasma orale from cell cultures infected with Rickettsia or Ehrlichia species

ABSTRACT

Mycoplasma (Class: Mollicutes) contamination in cell cultures is a universal concern for research laboratories. Some estimates report contamination in up to 35% of continuous cell lines. Various commercial antibiotic treatments can successfully decontaminate clean cell lines in vitro; however, in vitro decontamination of bacterial cultures remains challenging. Intracellular bacteria like those in the genera Rickettsia and Ehrlichia require cell culture for primary isolation and propagation and are thus vulnerable to contamination with mycoplasmas. Some analyses have reported successful antibiotic clearance of contaminating mycoplasmas in Rickettsia cultures; however, many of these studies do not identify the contaminating mycoplasma species and often include only a few isolates. To our knowledge, there are no published studies reporting decontamination of mycoplasmas from Ehrlichia cultures. In this study, we developed a specific multiplex assay to identify two of the most common mycoplasma culture contaminants, Mycoplasma arginini and Mycoplasma orale, in cell cultures infected with Rickettsia or Ehrlichia species. We further describe the successful in vitro decontamination of M. arginini, M. orale, and co-contaminations with both mycoplasmas from multiple Rickettsia and Ehrlichia cultures using daptomycin and clindamycin.

IMPORTANCE

Mycoplasma contamination is a frequent problem in bacterial cell culture. These prolific organisms thrive in the extracellular environment in vitro and can persist in cell lines indefinitely without treatment. Historically, mycoplasma-contaminated Rickettsia cultures were cleared of contaminants by inoculating laboratory mice and re-isolating mycoplasma-free Rickettsia from brain endothelial cells. However, this method requires the sacrifice of live animals and is not always effective. Mycoplasma clearance via mouse inoculation requires a patent infection of murine central nervous system endothelial cells, which may not occur with some mildly pathogenic or nonpathogenic rickettsial species. In vitro antibiotic treatment represents an alternate method to eliminate contaminating mycoplasmas from rickettsial cultures. This method requires minimal adjustment of laboratories that already maintain rickettsial cultures and is not dependent on the use of laboratory animals. As such, the comprehensive strategy for Mycoplasma arginini and Mycoplasma orale elimination presented here can improve laboratory efficiency for in vitro research with intracellular bacteria.

INTRODUCTION

Mycoplasmas (Class: Mollicutes) are pervasive contaminants of cell cultures, with an estimated 35% infection frequency among all continuous cell lines (1). Six species account for 95% of mycoplasma contamination in cell cultures: Mycoplasma orale, Mycoplasma arginini, Mycoplasma fermentans, Mycoplasma hyorhinis, Mycoplasma hominis, and Acholeplasma laidlawii (2). These obligately parasitic bacteria lack a cell wall and are among the smallest (0.3–0.8 µm diameter) and simplest self-replicating organisms, containing only the minimal organelles required for growth and replication, including ribosomes, a plasma membrane, and a genome of double-stranded circular DNA (3). In culture, hundreds of mycoplasmas can adhere to a single eukaryotic cell. These primarily extracellular parasites gain access to eukaryotic intracellular machinery by fusing with the eukaryotic cell membrane, though some mycoplasma species may enter host cells (4). While mycoplasmas often do not affect the growth of the parasitized eukaryotic cells, they nonetheless can alter functional and phenotypic characteristics of some cell lines and cause cytopathic effects that can affect intracellular bacteria in culture, including by competing with host cells for arginine (5–8).

The order Rickettsiales comprises a diverse group of intracellular bacteria, including many human pathogens in the genera Rickettsia, Ehrlichia, Anaplasma, and Orientia that require cell culture for primary isolation and propagation (9). Rickettsial isolates can become infected with mycoplasmas through contaminated culture reagents or improper sterile techniques during isolation or growth. Historically, mycoplasma contaminants of Rickettsia-infected cell cultures were eliminated via inoculation of laboratory mice. In vivo, mycoplasmas primarily infect cells of epithelial origin, whereas rickettsiae are tropic for vascular endothelial cells (10). In contrast to endothelium, epithelial cells are centralized to relatively few sites of the brain, allowing for murine brain tissue as a mycoplasma-free source to re-isolate rickettsial species. This technique is generally effective but poses several challenges and limitations. These include maintaining the increasingly complex regulatory and functional requirements to work with small animal models of infection as well as a necessity for the rickettsial species to establish a patent infection in the endothelium of the brains of infected mice, which may not occur with mildly pathogenic or non-pathogenic rickettsial species (10). Most critically, this approach necessitates the sacrifice of live animals. Striving to eliminate the need for live animal models of infection is a significant and worthwhile objective.

In vitro clearance of mycoplasmas using antibiotics in rickettsial cultures represents an alternate approach. This method requires minimal modification of laboratories that already maintain rickettsial cultures and does not require the sacrifice of live animals. Because mycoplasmas lack a cell wall, they are intrinsically resistant to antibiotics that target cell wall synthesis, like β-lactams and glycopeptides. Mycoplasmas also exhibit varying resistance to sulfonamides, some quinolones, and rifampicin (11). Various commercial antibiotic treatments are available to clear mycoplasma contamination from clean cell lines; however, these treatments often include tetracyclines (12), which are inhibitory or toxic to rickettsiae (13). Lincosamide antibiotics, such as lincomycin and clindamycin, inhibit protein synthesis via binding to the 50S ribosomal subunit (14) and have been used previously to clear mycoplasmas from contaminated rickettsial cultures, including M. hominis and M. orale (5, 6, 15); however, these studies have focused on a small number of isolates and rickettsial species. A recent study also identified daptomycin, a lipopeptide antibiotic that targets the bacterial cell membrane (16), as an effective method to clear various mycoplasma species due to the high minimum inhibitory concentration (MIC) of this antibiotic for Rickettsia species (15); nonetheless, the cost of daptomycin can be relatively expensive at the antibiotic concentration recommended in that study.

Along with the challenge of identifying antibiotics that will not affect rickettsial growth, antibiotic susceptibilities of mycoplasmas can vary considerably among species and even between strains of a single species (17). Furthermore, there are reports of treatment failures for some mycoplasmas thought to be susceptible to a particular antibiotic (15, 18). In this context, the use of antibiotics to clear mycoplasmas from Rickettsia-infected cell cultures has been historically challenging, and there is no standardized method that provides consistent and predictable identification and clearance of these contaminants. To our knowledge, there are no reports that describe antibiotic treatment to eliminate mycoplasma contaminants from Ehrlichia cultures.

Herein, we developed a specific multiplex assay to identify two of the most common mycoplasma culture contaminants, M. arginini and M. orale, in cell cultures infected with Rickettsia or Ehrlichia species. We further describe the successful in vitro decontamination of M. arginini, M. orale, and co-contaminations with both mycoplasmas from multiple Rickettsia and Ehrlichia cultures.

MATERIALS AND METHODS

Identification of mycoplasma-contaminated cultures

All cultures evaluated in this study are part of the internationally recognized CDC Rickettsial Isolate Reference Collection (CRIRC) (WDCM #1093). This collection contains nearly 500 archival and contemporary rickettsial isolates comprising 35 Rickettsia spp., 5 Ehrlichia spp., 2 Anaplasma spp., and 1 Orientia spp. The curatorial process for isolates maintained in the CRIRC includes qualitative screening for mycoplasma contamination using the ATCC Universal Mycoplasma Detection Kit (ATCC, Manassas, VA, USA).

Isolates with mycoplasma contamination that were identified with this assay were subsequently evaluated using primers GPO-3 and MGSO to amplify a portion of the mycoplasma 16S rRNA gene ranging from 281 to 298 bp to identify the mycoplasma species (19). To sequence the resulting amplicons, PCR products were visualized in 1.5% agarose gels containing 0.1 mg/mL ethidium bromide and extracted and purified using the Promega Wizard SV Gel and PCR Clean-up System (Promega, Madison, WI, USA). Amplicons were bidirectionally sequenced on an Applied Biosystems 3500 Genetic Analyzer (Applied Biosystems, Waltham, MA, USA) using a BigDye Terminator V3.1 kit (Applied Biosystems) and assembled using Geneious Prime 2022.0.2 (Geneious, Auckland, New Zealand). Assembled sequences were compared to GenBank data using BLASTn analysis.

Multiplex M. arginini–M. orale PCR design

To design a TaqMan assay specific for M. arginini and M. orale, 16S rRNA sequences were obtained from GenBank and aligned using Geneious Prime for all mycoplasmas detected in the CRIRC as well as for some of the most common mycoplasma culture contaminants. These included M. arginini (AP014657), M. orale (LR214940), M. hyorhinis (LS991950), M. hominis (CP086131), Mycoplasma gallisepticum (CP001873), M fermentans (CP002458), Mycoplasma pirum (NR_029165), Mycoplasma salivarium (OM864598), and Acholeplasma laidlawii (LS483439). Primer and probe sequences designed to amplify a 143-bp region of M. arginini and M. orale 16S are listed in Table 1.

TABLE 1.

Primers and probes for Mycoplasma arginini–Mycoplasma orale multiplex PCR assay

Oligonucleotide	Melting Temperature(°C)	Sequence (5′ – 3′)
Myco16S_F	57.7	GCT TGA TGG AGC GAC ACA
Myco16S_R	57.5	GGC TGC TGG CAC ATA GTT A
Myco16S_arginini_P	61.6	5' CalRd610-TGT TAT AGG GAA AGA ACA CCT GGT TG-BHQ2 3'
Myco16S_orale_P	60.9	5' FAM-CAG TTA GTT GAG GAA ATG CTT CTA ATC-BHQ1 3'

Multiplex PCR reactions consisted of 12.5 µL of Qiagen QuantiTect Probe PCR Master Mix (Qiagen, Valencia, CA, USA), 0.7 µM of both forward and reverse primers, 0.4 µM of each probe, 3 µL of DNA, and nuclease-free water to QS to a final reaction volume of 25 µL. Cycling conditions consisted of 1 cycle of 95°C for 15 minutes followed by 45 cycles of 95°C for 30 seconds and 63°C for 30 seconds using a BioRad CFX96 Touch (BioRad, Hercules, CA, USA). All reactions were run in duplicate with two sets of nuclease-free water controls and a genomic DNA positive control per plate.

Specificity testing was performed using genomic DNA extracted from cultures of Anaplasma phagocytophilum, Ehrlichia canis, Ehrlichia chaffeensis, Orientia tsutsugamushi, Rickettsia akari, Rickettsia amblyommatis, Rickettsia asiatica, Rickettsia canadensis, Rickettsia conorii subsp. conorii, Rickettsia japonica, Rickettsia massiliae, Rickettsia monteiroi, Rickettsia parkeri, Rickettsia rickettsii subsp. rickettsii, Rickettsia rickettsii subsp. californica, Vero E6 cells (African green monkey kidney epithelial cells), DH82 cells (canine macrophage), and L929 cells (mouse fibroblast). Specificity testing was also performed using DNAs from multiple mycoplasmas, comprising Mycoplasma amphoriforme, Mycoplasma buccale, Mycoplasma faucium, M. fermentans, M. gallisepticum, Mycoplasma genitalium, M. hominis, Mycoplasma hyopneumoniae, M. hyorhinis, Mycoplasma lipophilum, Mycoplasma penetrans, M. pirum, Mycoplasma pneumoniae, M. salivarium, Mycoplasma volis, and A. laidlawii.

Sensitivity testing was performed using plasmids for M. arginini and M. orale synthesized by Blue Heron Biotechnology (Wachell, WA, USA). All sensitivity tests included both M. arginini and M. orale probes. Standard curves were generated to determine the limit of detection of the assay using M. arginini and M. orale plasmids quantified on a NanoDrop One spectrophotometer (Thermo Scientific, Wilmington, DE, USA) at 1–10⁸ copies per reaction. Efficiency and R² were analyzed for both standard curves.

Additional sensitivity testing was performed to identify the limit of detection and potential interference between M. arginini and M. orale in co-contaminated samples. This analysis included four sets of reactions: M. arginini plasmid quantified at 1–10⁸ copies plus M. orale plasmid at 10⁴ copies per reaction; M. arginini plasmid quantified at 1–10⁸ copies plus M. orale plasmid at 10² copies per reaction; M. orale plasmid quantified at 1–10⁸ copies plus M. arginini plasmid at 10⁴ copies per reaction; and M. orale plasmid quantified at 1–10⁸ copies plus M. arginini plasmid at 10² copies per reaction. All plasmid quantifications were performed on a NanoDrop One spectrophotometer.

Antibiotic treatments

Antibiotic concentrations used for in vitro clearance of M. orale with clindamycin (Sigma-Aldrich, St. Louis, MO, USA) and for clearance of M. arginini or infections with both species with daptomycin (Sigma-Aldrich) are listed in Table 2. Experimental concentrations were selected from previously published MICs of these antibiotics for several Rickettsia spp. (15). As MICs of clindamycin and daptomycin are not available for Ehrlichia spp., we used daptomycin at the same concentration as for Rickettsia spp. and reduced the concentration of clindamycin for use in the canine macrophage cell line as this drug can accumulate in high concentrations in phagocytic cells (20).

TABLE 2.

Antibiotics and concentrations selected for clearance of Mycoplasma arginini and Mycoplasma orale^a

Antibiotic	Antibiotic class (mechanism of action)	Rickettsia MIC (mg/L)	Ehrlichia MIC	Mycoplasma orale MIC (mg/L)	Mycoplasma arginini MIC (mg/L)	Experimental concentration (mg/L)	Reference
Daptomycin	Cyclic lipopeptide (disruption of cell membrane function)	>256	Unknown	≤2	≤2	Rickettsia, Ehrlichia: 64	(15)
Clindamycin	Lincosamide (inhibition of microbial protein synthesis)	>32	Unknown	<1	<1	Rickettsia: 32 Ehrlichia: 4	(15)

^a Antibiotics were acquired from Sigma-Aldrich.

Additional antibiotics were tested against M. orale (gentamicin and lincomycin) and M. arginini (spiramycin and valnemulin) but were found to be incompletely effective (spiramycin, gentamicin, and lincomycin) or toxic to Rickettsia (valnemulin) (Table S1).

Mycoplasma-contaminated Rickettsia isolates (Table 3) were inoculated into Vero E6 (African green monkey kidney epithelial cells), except for R. asiatica IO-1, which was inoculated into L929 (mouse fibroblast cells). Twelve Rickettsia species evaluated in this study are confirmed human pathogens and two, R. canadensis McKiel and R. monteiroi Intervales^T, are of undetermined pathogenicity to humans (21). Growth media for cultures in Vero E6 consisted of complete MEM (Gibco, Thermo Fisher Scientific, Waltham, MA, USA) supplemented with 5% fetal bovine serum (Atlanta Biologicals, Atlanta, GA, USA), 0.1 mM NEAA, 10 mM HEPES, 1.8 mM L-glutamine, and 10 mM sodium pyruvate, all from Gibco. Growth media for R. asiatica in L929 consisted of RPMI 1640 (Gibco) supplemented with 5% fetal bovine serum (Atlanta Biologicals), 5% tryptose phosphate broth, and 1.8 mM L-glutamine, both from Gibco. Rickettsia cultures were all grown at 32°C in a 5% CO₂-in-air atmosphere.

TABLE 3.

Rickettsia and Ehrlichia isolates cleared from contamination with Mycoplasma arginini and/or Mycoplasma orale using in vitro antibiotic applications

Isolate	Contaminating Mycoplasma species	Isolate origin	Year	Location	Reference
R. akari Croatian	M. arginini	Human	1991	Zadar, Croatia	(22)
R. massiliae AZT80	M. arginini	Rhipicephalus sanguineus	2004	Arizona, USA	(23)
R. monteiroi IntervalesT	M. arginini	Amblyomma incisum	2004	São Paulo, Brazil	(24)
R. parkeri Ao10	M. arginini	Amblyomma ovale	2010	São Paulo, Brazil	(25)
R. rickettsii subsp. rickettsii 101 hlp-like	M. arginini	Unknown	Unknown	Unknown
R. rickettsii subsp. rickettsii Hlp#2	M. arginini	Haemaphysalis leporispalustris	1948	Montana, USA	(26)
R. rickettsii subsp. rickettsii Panama-1	M. arginini	Human	2004	Panama
R. rickettsii subsp. californica Cal65521	M. arginini	Dermacentor occidentalis	1973	California, USA	(27)
E. chaffeensis V1	M. orale	Human	1998	Tennessee, USA	(28)
E. chaffeensis V7	M. orale	Human	1998	Tennessee, USA	(28)
E. chaffeensis V10	M. orale	Human	1998	Tennessee, USA	(28)
R. canadensis McKielT	M. orale	H. leporispalustris	1962	Ontario, Canada	(29)
R. conorii subsp. caspia Chad	M. orale	Human	2000	Chad	(30)
R. rickettsii subsp. rickettsii Domino	M. orale	Canis familiaris	1989	North Carolina, USA
R. japonica YHT	M. orale	Human	1985	Tokushima Prefecture, Japan	(31)
R. parkeri Pantanal At46	M. arginini and M. orale	Amblyomma triste	2012	Mato Grosso do Sul, Brazil	(32)
R. parkeri At10	M. arginini and M. orale	A. triste	2011	Mato Grosso do Sul, Brazil
E. canis Jake	M. arginini and M. orale	C. familiaris	1998	North Carolina, USA	(33)

Ehrlichia cultures (Table 3) were inoculated from freezer stocks into DH82 (canine macrophage cells). All three E. chaffeensis strains used in this study are human pathogens (28), and E. canis Jake is a canine pathogen (33). Ehrlichia cultures were maintained in growth media consisting of complete MEM (Gibco, Thermo Fisher Scientific, Waltham, MA, USA) supplemented with 10% fetal bovine serum (Atlanta Biologicals, Atlanta, GA, USA), 0.1 mM NEAA, 10 mM HEPES, 1.8 mM L-glutamine, and 10 mM sodium pyruvate, all from Gibco. Ehrlichia cultures were grown at 37°C in a 5% CO₂-in-air atmosphere.

To ensure that cultures inoculated from freezer stocks were viable, Rickettsia and Ehrlichia cultures were propagated until they reached approximately 80% infectivity as determined by acridine orange staining (BD Biosciences, Franklin Lakes, NJ, USA) and Kwik-Diff (Epredia Shandon, Kalamazoo, MI, USA) staining, respectively. Growth media were then replaced with media supplemented with antibiotics at the concentrations listed in Table 2. To prepare antibiotic media, growth media were supplemented with appropriate concentrations of antibiotics and passed through a 0.22 µm filter.

Because previous work testing in vitro antibiotic treatment for mycoplasma decontamination reported inhibition without elimination of mycoplasmas following 2 weeks of treatment (15), cultures were maintained for 3 weeks in antibiotic media. Antibiotic media were prepared weekly and stored at 4°C. Culture media were changed twice weekly, and cultures were passaged as necessary based on the visualization of cytopathic effects using light microscopy and infectivity of cells visualized via acridine orange or Kwik-Diff staining.

After 3 weeks in antibiotic media, 200 µL of culture media was collected for mycoplasma molecular analysis. Media samples were extracted using a DNEasy extraction kit (Qiagen, Valencia, CA, USA) and screened for the presence of mycoplasmas using the M. arginini–M. orale multiplex assay described above. Cultures that were PCR negative by the M. arginini–M. orale multiplex assay were maintained for an additional 3 weeks in standard growth media as described above to ensure complete clearance of mycoplasma contamination.

Cultures that remained positive for M. orale after 3 weeks of treatment with clindamycin were maintained in media containing 64 mg/L daptomycin for an additional 3 weeks. Similarly, samples that initially tested positive only for M. orale but were later found to be co-contaminated with M. arginini after 3 weeks of treatment with clindamycin were also maintained in media with 64 mg/L daptomycin for an additional 3 weeks (Fig. 1). After this additional treatment, all samples were re-tested using the M. arginini–M. orale multiplex assay.

Fig 1.

In vitro laboratory workflow for Mycoplasma arginini and Mycoplasma orale clearance of Rickettsia and Ehrlichia isolates. “*” indicates that some Mycoplasma arginini/Mycoplasma orale co-contaminated cultures may be negative for either M. arginini or M. orale when initially tested but positive after 3 weeks of growth in antibiotics.

At the end of all growth experiments, cultures were frozen for permanent storage at –80°C, and 200 µL aliquots of all cultures containing cells and media were once again tested for mycoplasma contamination using the ATCC Universal Mycoplasma Detection Kit.

RESULTS

Identification of mycoplasma-contaminated cultures

The mycoplasma screening and sequencing process identified that approximately 10% of all isolates in the CRIRC, including both historical and contemporary cultures donated to the collection, were contaminated with six separate mycoplasma species, comprising M. arginini (28), M. orale (19), M. hyorhinis (4), M. gallisepticum (3), M. hominis (1), and A. laidlawii (1). Mycoplasma arginini and M. orale comprise 44% and 38%, respectively, of all mycoplasma-contaminated isolates in the CRIRC.

Multiplex M. arginini–M. orale PCR design

The multiplex PCR assay correctly identified all M. arginini- and M. orale-positive isolates in the CRIRC. Specificity testing revealed no false positives using a panel of 34 controls containing Acholeplasma or Mycoplasma species, mammalian cells, or rickettsial organisms as listed in Materials and Methods. The assay exhibited 100% positive, negative, and overall percent agreement.

For single infections with either M. arginini or M. orale, sensitivity for the multiplex assay was measured at ≤10 genomic copies per reaction. Efficiency for M. arginini was determined to be 91%, and R² for the standard curve of the dilution series for this species was 0.998. Efficiency for M. orale was determined to be 93%, and R² for the standard curve of the dilution series for this species was 0.997. Sensitivity testing performed with M. arginini and M. orale together revealed some interference when concentrations between the two species differed in the reactions. At concentrations greater than 10⁵ plasmid copies per reaction for one Mycoplasma species, 100 or less copies of the other Mycoplasma species became undetectable.

Antibiotic treatments

M. arginini

At the end of 3 weeks of treatment with 64 mg/L of daptomycin, all eight M. arginini-contaminated isolates (Table 3) were PCR-negative by the M. arginini–M. orale multiplex assay. Cultures were maintained for an additional 3 weeks in standard growth media and remained negative when retested with the M. arginini–M. orale multiplex assay and ATCC Universal Mycoplasma Detection Kit.

M. orale

After 3 weeks of treatment with clindamycin, seven M. orale-contaminated isolates (Table 3) were PCR-negative for mycoplasmas. These seven cultures remained negative after an additional 3 weeks of growth in standard growth media as confirmed via the M. arginini–M. orale multiplex assay and ATCC Universal Mycoplasma Detection Kit.

R. conorii subsp. caspia Chad remained positive for M. orale after 3 weeks of antibiotic treatment, suggesting resistance of this strain to clindamycin at 32 mg/L. This isolate was subsequently treated for 3 weeks with 64 mg/L of daptomycin and an additional 3 weeks in standard growth media, which successfully eliminated M. orale contamination in this culture.

Co-contaminations with M. arginini and M. orale

Three isolates were identified as co-contaminated with M. arginini and M. orale (Table 3). These isolates were treated for 3 weeks with 64 mg/L of daptomycin, after which all three cultures were negative for M. arginini and M. orale via the M. arginini–M. orale multiplex assay. These cultures continued growth for an additional 3 weeks in standard growth media, after which the cultures remained negative for mycoplasma contamination as confirmed via the M. arginini–M. orale multiplex assay and ATCC Universal Mycoplasma Detection Kit.

At the end of all growth experiments, all aliquots of cultures tested prior to freezing for permanent storage were negative for mycoplasma contamination using the ATCC Universal Mycoplasma Detection Kit.

DISCUSSION

We described a comprehensive strategy for the identification and elimination of M. arginini and M. orale from cell cultures infected with Rickettsia or Ehrlichia species (Fig. 1) based on a robust and methodical evaluation. We developed a real-time multiplex assay to detect M. arginini and M. orale, two common mycoplasma contaminants of cell cultures (34) and the mycoplasmas most frequently found contaminating isolates in the CRIRC. Finally, the antibiotic clearance technique described herein obviates the need for animal infection and sacrifice, used previously to achieve clearance of mycoplasmas.

Mycoplasma arginini is associated with ruminants and historically was a frequent contaminant of bovine serum (35, 36), a crucial component used to maintain most cell lines. Mycoplasma orale is a commensal bacterium of the human oral cavity, and historical contamination of various cell lines with M. orale likely originated from the outdated microbiological practice of mouth pipetting (37). Increasing awareness regarding mycoplasma contamination of cell cultures has resulted in improvements in laboratory practices that minimize the risk of contamination (38, 39) as well as the development of commercially available assays that test for the presence of these bacteria (40). Nonetheless, many isolates remain contaminated because of previous practices.

We confirmed that clindamycin can successfully eliminate M. orale contamination as shown previously (6, 15), providing additional support for the use of this inexpensive and common antibiotic to decontaminate rickettsial cultures. However, we identified one M. orale-contaminated culture isolate that could not be cleared with clindamycin, suggesting acquired resistance to this antibiotic, which has been described for some strains of M. salivarium, another human oral commensal closely related to M. orale (17). Future work could elucidate if the same mechanisms of resistance exist in M. orale as in M. salivarium.

We successfully eliminated M. arginini contamination from cultures infected with Rickettsia or Ehrlichia using daptomycin at a fourfold reduction in concentration relative to previous recommendations (15), which provides a cost reduction for this effective antibiotic. We also successfully used daptomycin at 64 mg/L to clear M. gallisepticum from one isolate of R. massiliae (data not shown).

While several studies have investigated antibiotic clearance of mycoplasmas from cell cultures infected with Rickettsia or Orientia species (6, 13, 15, 41), we are unaware of previous documentation of mycoplasma clearance from cell cultures infected with Ehrlichia species. Antibiotic susceptibility testing has been performed for some Ehrlichia species, including E. chaffeensis and E. canis (42, 43); however, there is a paucity of information analyzing antibiotics in the context of clearing mycoplasma-contaminated Ehrlichia cultures.

The M. arginini–M. orale multiplex assay developed in this study is the first real-time assay available for specific and simultaneous identification of these two common mycoplasma contaminants. Using this assay, three co-contaminations with M. arginini and M. orale were discovered in the CRIRC, all of which were initially identified as single-species contaminations using genus-specific PCR and sequencing. Commercial mycoplasma detection kits are qualitative and thereby cannot identify contaminations with multiple mycoplasmas; however, previous work has shown that high percentages of co-contaminations can occur in cell cultures (44). In this context, it is likely that some previous attempts to eliminate mycoplasmas from contaminated cell cultures failed because a co-contamination was not recognized, and one of the contaminating mycoplasmas was resistant to the antibiotic used for treatment.

Use of the M. arginini–M. orale multiplex assay can help guide targeted antibiotic treatment decisions for Rickettsia and Ehrlichia cultures contaminated with these mycoplasmas as outlined in Fig. 1. Isolates that test positive by the multiplex assay for M. arginini or for both M. arginini and M. orale can be grown in media supplemented with 64 mg/L daptomycin for 3 weeks. Isolates that test positive only for M. orale by this assay can be grown in media supplemented with 32 mg/L clindamycin for 3 weeks. At the end of 3 weeks of growth in appropriate antibiotics, if an isolate is negative using the M. arginini–M. orale multiplex assay, growth for an additional 3 weeks in standard growth media without antibiotics can help ensure complete elimination of mycoplasmas. If an isolate is positive for M. orale at the end of 3 weeks of treatment with clindamycin, the isolate can be treated for three additional weeks with 64 mg/L of daptomycin to clear the M. orale. If an isolate is positive for M. arginini or M. orale at the end of 3 weeks’ treatment with 64 mg/L of daptomycin, following the protocol outlined in Tantibhedhyangkul et al. (15) may be effective; however, all cultures tested in this study were successfully decontaminated without utilizing this additional protocol. Because of the identified inhibition in detecting co-contaminations of these two mycoplasmas when one mycoplasma is present at a much lower concentration than the other, testing cultures with the assay at the end of the initial 3 weeks could potentially identify a previously unrecognized co-contamination. A final test with the M. arginini–M. orale multiplex assay should always be performed at the end of a 6-week treatment cycle to confirm the absence of M. arginini and M. orale contamination.

The antibiotic clearance process presented here could also serve as a framework for targeted mycoplasma elimination in clean cell lines and cell cultures infected with viruses or other obligately intracellular pathogens, including Chlamydia spp., which require growth in eukaryotic cells for propagation and survival (45). A recent study outlined a detailed in vitro process for mycoplasma elimination in chlamydial cultures (46) that does not use antibiotics; however, this procedure requires the use of flow cytometry, which may not be available to all laboratories culturing chlamydiae. Chlamydiae exhibit similar susceptibilities as rickettsiae to the wide-spectrum antibiotic classes used to eliminate mycoplasmas from uninfected cell lines (46). As such, future work could elucidate the utility of daptomycin and clindamycin to eliminate contaminating mycoplasmas in chlamydial cultures.

Clearance of mycoplasmas from contaminated isolates is crucial for multiple reasons. For investigations using rickettsial isolates in animal infection studies, contamination with mycoplasmas can lead to misinterpretation of results (47) by eliciting immune responses in laboratory animals (48, 49) and exacerbating inflammation and tissue damage. Furthermore, mycoplasma contaminations have the potential to confound the accuracy of whole genome data for agents that can only be cultivated in eukaryotic cells. An analysis of sequencing data from the 1,000 Genomes Project estimates that 7% of samples are contaminated with mycoplasma DNA (50). An additional study suggests that at least 11% of publicly available NCBI RNA-seq data sets are contaminated with mycoplasma genetic information (51). Indeed, we recently published a whole genome for a historical Rickettsia isolate that we subsequently determined to contain chimeric 16S and 23S rRNA sequences due to contamination of the original isolate by M. arginini (52, 53). Eliminating mycoplasma contamination before molecular analysis is essential to publishing reliable genomic data.

This work was designed for laboratories that maintain rickettsial isolates; as such, we used freezer stocks as culture inocula, which is typically how isolates are propagated and expanded for inclusion in a culture repository. Our efforts were focused on qualitative, rather than quantitative, measures of clearance (e.g., test positive vs test negative). In this context, we did not determine the multiplicity of infection for rickettsiae or investigate the dynamics of rickettsial infection throughout antibiotic treatment. Future studies could elucidate what effects, if any, the antibiotics have on the rickettsiae and possibly refine the method by calculating specific MICs for each Rickettsia species to clindamycin and daptomycin.

While prevention of mycoplasma contamination in rickettsial cultures through appropriate sterile technique is best, even contemporary practices are not infallible, and achieving a mycoplasma-free culture collection remains challenging. The procedures outlined here offer an inexpensive and accessible solution to eliminate two common culture contaminants, M. arginini and M. orale, from Rickettsia or Ehrlichia cultures.

SUPPLEMENTAL MATERIAL

The following material is available online at https://doi.org/10.1128/spectrum.01743-24.

Table S1. spectrum.01743-24-s0001.docx.

Antibiotics tested in pilot studies against Mycoplasma arginini and Mycoplasma orale.

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