Efficacy of rifapentine and other rifamycins against Coxiella burnetii in vitro

ABSTRACT

Since 1999, doxycycline and hydroxychloroquine have been the recommended treatment for chronic Q fever, a life-threatening disease caused by the bacterial pathogen, Coxiella burnetii. Despite the duration of its use, the treatment is not ideal due to the lengthy treatment time, high mortality rate, resistant strains, and the potential for contraindicated usage. A literature search was conducted to identify studies that screened large panels of drugs against C. burnetii to identify novel targets with potential efficacy against C. burnetii. Twelve candidate antimicrobials approved for use in humans by the US Food and Drug Administration were selected and minimum inhibitory concentrations (MICs) were determined against the low virulence strain Nine Mile phase II. Rifabutin and rifaximin were the best performing antibiotics tested with MICs of ≤0.01 µg mL⁻¹. Further screening of these top candidates was conducted alongside two drugs from the same class, rifampin, well-characterized, and rifapentine, not previously reported against C. burnetii. These were screened against virulent strains of C. burnetii representing three clinically relevant genotypes. Rifapentine was the most effective in the human monocytic leukemia cell line, THP-1, with a MIC ≤0.01 µg mL⁻¹. In the human kidney epithelial cell line, A-498, efficacy of rifapentine, rifampin, and rifabutin varied across C. burnetii strains with MICs between ≤0.001 and 0.01 µg mL⁻¹. Rifampin, rifabutin, and rifapentine were all bactericidal against C. burnetii; however, rifabutin and rifapentine demonstrated impressive bactericidal activity as low as 0.1 µg mL⁻¹ and should be further explored as alternative Q fever treatments given their efficacy in vitro.

IMPORTANCE

This work will help inform investigators and physicians about potential alternative antimicrobial therapies targeting the causative agent of Q fever, Coxiella burnetii. Chronic Q fever is difficult to treat, and alternative antimicrobials are needed. This manuscript explores the efficacy of rifamycin antibiotics against virulent strains of C. burnetii representing three clinically relevant genotypes in vitro. Importantly, this study determines the susceptibility of C. burnetii to rifapentine, which has not been previously reported. Evaluation of the bactericidal activity of the rifamycins reveals that rifabutin and rifapentine are bactericidal at low concentrations, which is unusual for antibiotics against C. burnetii.

INTRODUCTION

Q fever is a disease caused by the bacterial zoonotic pathogen, Coxiella burnetii. Q fever presents as two distinct clinical manifestations. Acute Q fever is commonly asymptomatic; however, in cases of symptomatic illness, the disease presentation is often analogous to the flu (1). Major clinical implications of acute Q fever include pneumonia or hepatitis, which can require hospitalization (1). Acute Q fever is treated with a 2-week course of doxycycline and mortality is less than 2% (1). In 5% of cases, chronic Q fever develops, causing a new collection of symptoms, which often occurs years after the initial infection (1). The major presentation of chronic Q fever is endocarditis, which is fatal if untreated. Treatment of chronic Q fever includes a combination of doxycycline and hydroxychloroquine for at least 18 months and some cases may require lifelong antibiotic therapy (2).

Q fever endocarditis is difficult to treat as evidenced by the ability to isolate viable C. burnetii from valve tissues following administration of doxycycline monotherapy for 4 years; thus, combination therapy is preferred (3). Doxycycline, together with chloroquine, a lysosomotropic agent used to treat malaria, was found to have a bactericidal effect on C. burnetii in vitro (4). Therefore, combination therapy with doxycycline and hydroxychloroquine, a chloroquine metabolite with better toxicological properties, became the recommended treatment option for chronic Q fever (5). Doxycycline and hydroxychloroquine treatment was shown to successfully reduce relapses and the requirement for valve replacement (5). Despite the success of this treatment, mortality was comparable to doxycycline plus quinolone combination therapy (5). Furthermore, adverse reactions to doxycycline plus hydroxychloroquine therapy can occur, creating a need for alternative options. Doxycycline is one of the most well-tolerated tetracycline antibiotics. Although rare, allergic responses have been reported, including life-threatening reactions (6, 7). Additional adverse reactions to doxycycline include dermatologic reactions, Stevens-Johnson syndrome, and drug-induced lupus (6). Adverse reactions to hydroxychloroquine are rare, but can include gastrointestinal discomfort, allergy, cardiomyopathy, cardiac conduction defects, neuromyotoxicity, cytopenia, skin hyperpigmentation, and ocular toxicity (8). Chronic Q fever treatment is further complicated by isolation of doxycycline-resistant strains of C. burnetii as well as the potential for drug-drug incompatibility, which underscores the need for further exploration of alternatives to improve Q fever outcomes (9, 10).

Alternative options have been explored with variable successes including quinolones, co-trimoxazole, rifampin, and macrolides (2). None of the alternatives have been shown to be as effective as doxycycline hydroxychloroquine combination therapy for reducing risk of relapse and shortening treatment times (2). Recognizing the need for additional therapies, collections of US Food and Drug Administration (FDA)-approved or late-stage clinical trial compounds have been screened for their ability to inhibit C. burnetii growth (11, 12), yet few of these drugs have been tested against virulent strains of C. burnetii at physiologically relevant concentrations. The aim of this study was to increase the number of effective antimicrobial therapies that are readily available to treat Q fever by investigating the efficacy of these previously identified drugs for activity against virulent C. burnetii in vitro.

RESULTS

Selection and primary screening of antibiotics

A literature search was conducted to identify studies that screened large panels of FDA-approved drugs against C. burnetii to find novel targets for potential off-label use. Two studies were identified, the first by Cyzŷ et al., screened a library of 640 compounds, and identified 112 compounds (75 host targeting and 37 pathogen targeting) effective at inhibiting intracellular growth of the Nine Mile phase II RSA439 strain expressing mCherry (NMII-mCherry) in the human monocytic leukemia cell line, THP-1 (11). The second, by Fullerton et al., screened a 727-compound library of FDA-approved or late-stage clinical trial compounds against NMII-mCherry in THP-1 cells and found 88 (63 host targeting, 25 pathogen targeting) that inhibit growth (12). Pathogen-targeting compounds identified in either study were compiled totaling 47 unique drugs. Of these, 17 are not readily available for use in the USA (i.e., not approved by the US Food and Drug administration for use in humans or discontinued) and were eliminated from further study (Table S1). An additional seven antibiotics with an administration route insufficient for the treatment of Q fever (i.e., topical, shampoo, ophthalmic drops) were also eliminated, as well as 11 antibiotics whose efficacy against C. burnetii has been well characterized.

The 12 remaining antibiotics underwent initial screening to determine minimum inhibitory concentration (MIC) against the Nine Mile phase II clone 4 strain in rabbit kidney epithelial (RK-13) cells, which were selected as a host cell for the initial screen owing to their characteristics as a hardy adherent cell line that supports growth of a wide range of C. burnetii isolates (13, 14). At 25 µg mL⁻¹, growth of NMII was not significantly different from dimethyl sulfoxide (DMSO) controls for atazanavir (P = 0.9982), atovaquone (P = 0.9982), ethionamide (P = 0.9312), and itraconazole (P = 0.6928), which was insufficient to justify further consideration (Table 1). The MIC for pentamidine isethionate, rifamycin, and tobramycin was 10 µg mL⁻¹, and at this concentration, growth was reduced 13,614-fold (P < 0.0001), 716-fold (P < 0.0001), and 127-fold (P = 0.0016), respectively, relative to DMSO controls. Given that the reported maximum serum concentration (C_max) for these four drugs was lower than the MIC, they would likely fail in a clinical setting and thus were eliminated from further consideration (Table 1).

TABLE 1.

MICs of selected antibiotics against NMII in RK-13 host cells^a

Antibiotic	Cmax µg mL−1	Cmax reference	MIC µg mL−1
Atazanavir	5.4 ± 1.4	(15)	>25
Atovaquone	24.0 ± 5.7	(16)	>25
Ethionamide	2.16	(17)	>25
Itraconazole	2.3 ± 0.5	(18)	>25
Pentamidine isethionate	0.612 ± 0.371	(19)	10
Rifamycin	0.00872	(20)	10
Tobramycin	4–6	(21)	10
Doxycycline	2.6	(22)	1
Oxytetracycline	8.2 (bovine, injection)	(23)	1
Efavirenz	4	(24)	1
Demeclocycline	1.22	(25)	0.1
Minocycline	5.1	(26)	0.1
Rifampin	17.5 ± 5.0 (IV), 7 (oral)	(27)	≤0.01
Rifaximin	0.00963 ± 0.00593	(28)	≤0.01
Rifabutin	0.375 ± 0.267	(29)	≤0.01

^a C_max values are based on oral administration in humans unless otherwise stated.

The remaining antibiotics tested had MICs below the C_max (Table 1). Two tetracycline antibiotics, minocycline and demeclocycline, had MICs of 0.1 µg mL⁻¹ where growth was reduced by 445-fold (P = 0.001) and 597-fold (P < 0.0001), respectively. As the tetracycline antibiotic, doxycycline, is the recommended treatment option for Q fever, we compared efficacy of these tetracyclines as well as oxytetracycline, which was initially eliminated as it is discontinued, but is available for use in animals. The MIC for doxycycline and oxytetracycline was 1 µg mL⁻¹ with growth reductions of 303-fold (P = 0.0001) and 13,614-fold (P < 0.0001), respectively.

MIC for efavirenz was 1 µg mL⁻¹ where growth was reduced 540-fold (P < 0.0001). Two bacterial RNA synthesis inhibitors, rifabutin and rifaximin, were the best performing antibiotics tested thus far with MICs of ≤0.01 µg mL⁻¹ (Table 1). The reduction in growth for rifabutin and rifaximin at 0.01 µg mL⁻¹ was 1,622-fold (P < 0.0001) and 335-fold (P < 0.0001), respectively. The well-characterized bacterial RNA synthesis inhibitor, rifampin, was included for comparison (7, 12, 30–34). The MIC of rifampin in this assay was also ≤0.01 µg mL⁻¹. At 0.01 µg mL⁻ , growth was inhibited 863-fold (P = 0.0002) relative to DMSO controls. Initial screening identified efavirenz, rifabutin, rifaximin, and rifampin as effective at inhibiting growth of NMII in RK-13 cells at physiologically relevant concentrations; as such, they were selected for further analysis.

Efficacy of efavirenz and rifamycins against Nine Mile phase I (NMI) in THP-1 host cells

Efavirenz was selected for further testing as it is a non-nucleoside reverse transcriptase inhibitor, which is a unique class of antibiotic for use against Q fever. The rifamycins, rifabutin and rifaximin, were also selected because of their impressive efficacy against NMII in RK-13 cells. Efavirenz, rifabutin, and rifaximin were tested against the virulent NMI strain of C. burnetii in human macrophage-like THP-1 cells (Fig. 1). Given the success of the class of rifamycins against NMII in vitro, rifampin was again included as a comparator along with another FDA-approved rifamycin, rifapentine, for which efficacy against C. burnetii has not been reported. Doxycycline was also included in this assay for comparison with an MIC of 0.05 µg mL⁻¹ leading to a 15-fold (P < 0.0001) decrease relative to DMSO controls. Efavirenz was not effective at inhibiting growth of NMI in the macrophage-like cells when tested at 1 µg mL⁻¹ (P > 0.9999); as such, it was eliminated from further consideration. Rifaximin with a C_max of 0.0096 µg mL⁻¹ was also ineffective at inhibiting growth of NMI in THP-1 cells at a concentration of 0.01 µg mL⁻¹ (P = 0.9943). Rifabutin and rifampin both had MICs of 0.1 µg mL⁻¹ where growth was inhibited 72-fold (P < 0.0001) and 83-fold (P < 0.0001), respectively. Rifapentine displayed impressive efficacy against NMI in the macrophage-like cells with an MIC ≤0.01 µg mL⁻¹. At 0.01 µg mL⁻¹, growth following exposure to rifapentine was inhibited 53-fold (P < 0.0001) relative to DMSO controls.

Fig 1.

MICs for selected antibiotics against NMI in THP-1 host cells. NMI C. burnetii was cultured in phorbol myristate acetate-differentiated THP-1 cells in the presence of increasing concentrations of antibiotics to determine the MIC. Data displayed are the mean log₁₀ genome equivalent per well ± SEM at 7 days post-exposure for each antibiotic at the respective concentration. The MIC was determined as the lowest concentration of antibiotic required to significantly inhibit growth of C. burnetii. Significant inhibition of growth was determined relative to DMSO solvent controls using one-way analysis of variance with Dunnett’s multiple comparisons post hoc test of log₁₀ transformed data. ****P < 0.0001.

Different strains of C. burnetii can have variable antibiotic susceptibilities (9, 35–39). Therefore, we repeated our analysis of the rifamycin antibiotics with contemporary, clinically relevant strains of C. burnetii representative of the genotypes currently circulating in the USA (ST16/26, ST8, and ST20) (13, 14, 40). Curiously, growth of the ST8 and ST20 isolates, GP-CO1 and CM-SC1, respectively, was negligible in the THP-1 macrophage-like cells (data not shown). Confirmatory screening of the top candidate antibiotics in the well-characterized virulent C. burnetii strain, NMI, revealed that efavirenz was ineffective at physiologically relevant concentrations under the test conditions and was eliminated from further consideration. Rifabutin, rifampin, and, in particular, rifapentine were effective and thus were selected for continued screening in C. burnetii strains representing clinically relevant genogroups. To evaluate efficacy of the rifamycins against these strains, further analysis was conducted in the human kidney epithelial cell line, A-498, to support growth of these diverse strains.

Efficacy of rifamycins against virulent C. burnetii in A-498 cells

In A-498 host cells, the MIC for rifampin, rifabutin, and rifapentine was 0.01 µg mL⁻¹ against the NMI strain with growth inhibition of 43-fold (P = 0.0003), 34-fold (P = 0.0005), and 15-fold (P = 0.017), respectively, relative to DMSO controls (Table 2; Fig. 2A). Rifaximin did not inhibit NMI at the only tested concentration, 0.0096 µg mL⁻¹ (P = 0.2604). The ST16/26 strain, HPF-GA1, was inhibited by rifabutin and rifampin by 7.3-fold (P = 0.0043) and 4.5-fold (P = 0.0482), respectively, at 0.001 µg mL⁻¹, resulting in MICs ≤0.001 µg mL⁻¹ (Table 2; Fig. 2B). The MICs for rifapentine and rifaximin were 0.01 (−22-fold, P < 0.0001) and ≤0.0096 µg mL⁻¹ (−15-fold, P < 0.0001), respectively. Growth of the ST20 and ST8 C. burnetii strains was not robust enough by day 7 to draw meaningful conclusions regarding inhibition; therefore, the assay was extended to 14 days for these two strains. The MIC for rifapentine and rifabutin against the ST20, CM-SC1, was ≤0.001 µg mL⁻¹. At 0.001 µg mL⁻¹, CM-SC1 was inhibited 2.2-fold by both rifapentine (P = 0.0003) and rifabutin (P = 0.0005) relative to DMSO controls (Table 2; Fig. 2C). The MIC for rifampin against CM-SC1 was 0.01 (−28.7-fold, P < 0.0001). Rifaximin did not inhibit CM-SC1 at 0.0096 µg mL⁻¹ (P = 0.9998). MIC for the ST8, GP-CO1, was ≤0.001 µg mL⁻¹ for rifampin (−2.1-fold, P = 0.0013), rifabutin (−2.2-fold, P = 0.0005), and rifapentine (−2.2-fold, P = 0.0005) (Fig. 2D). The MIC for rifaximin (−1.7-fold, P = 0.0303) against GP-CO1 was ≤0.0096 µg mL⁻¹. In summary, rifabutin, rifampin, and rifapentine were effective at inhibiting growth of C. burnetii at physiologically relevant concentrations across all C. burnetii strains and host cells tested. These antibiotics were further assessed to determine whether the array of virulent C. burnetii strains were simply inhibited or rendered nonviable following exposure.

TABLE 2.

MICs of rifamycins against virulent C. burnetii^a,b

		Rifampin	Rifabutin	Rifapentine	Rifaximin
	Cmax (Reference)	7 (oral); 17.5 (IV) (27)	0.375 (29)	15.05 (41)	0.0096 (28)
THP-1	NMI (ST16)	0.1	0.1	≤0.01	NID
A-498	NMI (ST16)	0.01	0.01	0.01	NID
	HPF-GA1 (ST16/26)	≤0.001	≤0.001	0.01	≤0.0096
	CM-SC1 (ST20)	0.01	≤0.001	≤0.001	NID
	GP-CO1 (ST8)	≤0.001	≤0.001	≤0.001	≤0.0096

^a All values are expressed as microgram per milliliter.

^b NID, no inhibition detected at the tested concentration(s).

Fig 2.

MICs for selected antibiotics against virulent C. burnetii in A-498 host cells. C. burnetii strains (A) NMI, (B) HPF-GA1, (C) CM-SC1, and (D) GP-CO1 were cultured in A-498 cells in the presence of increasing concentrations of antibiotics to determine the MIC. Data displayed are the mean log₁₀ genome equivalent per well ± SEM at 7 (for NMI and HPF-GA1) and 14 (for CM-SC1 and GP-CO1) days post-exposure for each antibiotic at the respective concentration. The MIC was determined as the lowest concentration of antibiotic required to significantly inhibit growth of C. burnetii. Significant inhibition of growth was determined relative to DMSO solvent controls using one-way analysis of variance with Dunnett’s multiple comparisons post hoc test of log₁₀ transformed data. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001.

Bactericidal activity of rifamycins against virulent C. burnetii in A-498 cells

The ST8 strains of C. burnetii display a growth defect in axenic media (14); therefore, use of traditional bactericidal assays is a challenge. Despite these limitations, ST8s are important clinically as they have been the cause of both outbreaks and Q fever in humans (14, 42). To evaluate the bactericidal activity of the rifamycins against these strains, we utilized a cell culture-based method by allowing C. burnetii present within host cells at the end of a 7-day antibiotic exposure to recover for 21 days in the absence of antibiotic. Analysis of bactericidal activity of the rifamycins against virulent C. burnetii revealed that rifampin, rifabutin, and rifapentine were all bactericidal at the respective C_max for all tested strains (Table 3; Fig. 3). Rifaximin did not have a bactericidal effect on any of the tested strains at the C_max of 0.0096 µg mL⁻¹. For the NMI strain, during the 21-day recovery period, the genomic equivalent (GE) of DMSO controls increased by 97.5-fold. The minimum bactericidal concentration (MBC) for rifabutin was 0.1 µg mL⁻¹ (P = 0.0119) with a 6.2-fold increase in GE from day 0 and day 21. At 0.1 µg mL⁻¹, both rifapentine and rifampin were deemed bacteriostatic. Growth of NMI post-exposure to rifapentine at 0.1 µg mL⁻¹ (P = 0.9996, 75.1-fold) was comparable to DMSO controls. NMI recovered from 0.1 µg mL⁻¹ rifampin outgrew DMSO controls (P = 0.0340, 1,105-fold) (Fig. 3A). For the HPF-GA1 strain, DMSO controls increased by 34.1-fold during the recovery period. The MBC of rifabutin (P < 0.0001, –3.6-fold) and rifapentine (P < 0.0001, –2.8-fold) was 0.1 µg mL⁻¹ (Fig. 3B). Conversely, rifampin was not bactericidal at 0.1 µg mL⁻¹ (P = 0.5088, 129.7-fold). Despite the limited growth observed for CM-SC1 and GP-CO1 during the 7-day antibiotic exposure, sufficient growth occurred during the 21-day recovery period to assess bactericidal activity. The GE of DMSO controls for CM-SC1 and GP-CO1 increased by 9.5-fold and 71.0-fold, respectively (Fig. 3C and D). The CM-SC1 and GP-CO1 strains displayed similar trends with an MBC of 0.1 µg mL⁻¹ for both rifabutin and rifapentine, but not rifampin. At a concentration of 0.1 µg mL⁻¹ rifabutin, GE during the 21-day recovery period changed by −19.1-fold (P = 0.0001) for CM-SC1 and −1.4-fold (P < 0.0001) for GP-CO1. Growth post-exposure to rifapentine at 0.1 µg mL⁻¹ changed −6.2-fold (P = 0.0025) for CM-SC1 and 1.1-fold (P < 0.0001) for GP-CO1. Rifampin was bacteriostatic at a concentration of 0.1 µg mL⁻¹ for both CM-SC1 (P = 0.4749, 1.6-fold) and GP-CO1 (P = 0.9994, 54.2-fold), respectively. In summary, rifabutin and rifapentine show impressive bactericidal activity against multiple C. burnetii isolates at physiological relevant concentrations.

TABLE 3.

MBCs of rifamycins against virulent C. burnetii in A-498 host cells^a,b

	Rifampin	Rifabutin	Rifapentine	Rifaximin
Cmax (Reference)	7 (oral); 17.5 (IV) (27)	0.375 (29)	15.05 (41)	0.0096 (28)
NMI (ST16)	>0.1 – ≤17.5	0.1	>0.1 – ≤15.05	NBD
HPF-GA1 (ST16/26)	>0.1 – ≤17.5	0.1	0.1	NBD
CM-SC1 (ST20)	>0.1 – ≤17.5	0.1	0.1	NBD
GP-CO1 (ST8)	>0.1 – ≤17.5	0.1	0.1	NBD

^a All values are expressed as microgram per milliliter.

^b NBD, no bactericidal activity detected at the tested concentration(s).

Fig 3.

MBCs for selected antibiotics against virulent C. burnetii in A-498 host cells. C. burnetii strains (A) NMI, (B) HPF-GA1, (C) CM-SC1, and (D) GP-CO1 were allowed to recover in A-498 cells for 21 days following a 7-day exposure to antibiotics. GE at day 0 (defined as the day that antibiotics were removed) and at 21 days post-recovery were calculated and transformed by taking the natural log (ln). Data are displayed as the ln difference of the GE per well at day 21 relative to day 0 [ln (GE per well D21) – ln (GE per well D0)]. MBC was determined as the lowest concentration of antibiotic required to render C. burnetii nonviable. C. burnetii was determined to be nonviable if the mean difference in ln transformed GE from day 21 relative to day 0 was significantly different relative to DMSO solvent controls based on one-way analysis of variance with Dunnett’s multiple comparisons post hoc test. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001.

DISCUSSION

Through in vitro screening of C. burnetii, the rifamycin antibiotics, rifapentine and rifabutin, outperformed all other tested compounds and demonstrated bactericidal activity at physiologically relevant concentrations. Rifapentine was the most effective antibiotic at inhibiting C. burnetii in human macrophage-like cells with an MIC ≤0.01 µg mL⁻¹. This has also been described for Mycobacterium tuberculosis as the inhibitory and bactericidal effects of rifapentine are better than rifampin in human monocyte-derived macrophages (43). C. burnetii is an intracellular pathogen and macrophages are a preferred cell type in vivo; thus, rifapentine may be a good candidate for the treatment of Q fever (44, 45). The superior efficacy of rifapentine in macrophage-like cells may be explained by the increased intracellular/extracellular ratio of this drug, which is 4- to 12-fold higher for rifapentine than rifampin (46). An inhaled dose was shown to result in four to five times more rifapentine accumulation in alveolar macrophages relative to rifampin, which may provide improved treatment options for Q fever pneumonia (47).

Rifapentine and rifabutin demonstrated bactericidal activity against C. burnetii at incredibly low concentrations (0.1 µg mL⁻¹). Bactericidal activity of this magnitude is not often described for antibiotics against C. burnetii (2). Indeed, the classic rifamycin antibiotic, rifampin, which has been well-characterized against C. burnetii both in vitro and in vivo was not bactericidal at 0.1 µg mL⁻¹ (7, 12, 30–34, 48). Another study has demonstrated that rifampin is not bactericidal even as high as 4 µg mL⁻¹ (3). Bactericidal therapies are preferred for the treatment of Q fever to reduce the occurrence of relapse (49). Importantly, rifapentine has been shown to bind DNA-dependent RNA polymerase when enzyme activity is low in dormant M. tuberculosis, which could prove beneficial in treating chronic Q fever or metabolically inactive C. burnetii small cell variants (50, 51). This may help explain why rifapentine and rifabutin were bactericidal at low concentrations against the ST8 and ST20 strains, GP-CO1 and CM-SC1, despite the limited growth that had occurred during the initial 7-day exposure stage of the bactericidal assays. Given these promising characteristics, rifapentine and rifabutin should be further explored for the treatment of Q fever.

Historically, rifampin has been successful against Q fever and is typically administered as combination therapy with one or more other drugs, most often doxycycline or erythromycin. Its usefulness has been documented in cases of acute and chronic Q fever (33, 34, 52–57). Unfortunately, rifampin treatment is not always successful and drug interactions with rifamycins can be a cause for concern due to the resulting increase in levels of drug transporters and metabolizing enzymes, namely CYP3A, when this class of antibiotic is administered (33, 58, 59). Induction of these enzymes has been a disadvantage for the treatment of Q fever, particularly for patients requiring anticoagulants such as those with mechanical valves due to drug-drug interactions (3, 5, 33). Interestingly, induction of CYP3A is higher following administration of rifampin versus rifapentine and is lowest for rifabutin (60). The usefulness of rifapentine is further underscored by the fact that it has a higher C_max and a longer serum half-life (14 to 18 h) compared to rifampin (2 to 5 h) (61). Induction of CYP3A can be reduced by decreasing the frequency of antibiotic administration, which for rifapentine can still result in a more favorable C_max (15 mg/L) when administered once weekly relative to twice weekly for rifampin (C_max 10 mg/L) at the same dosage (46, 62). Phase 3 clinical trials demonstrated that treatment of pulmonary tuberculosis could be shortened from 6 months to 4 months by replacing the standard rifampin treatment with daily rifapentine-moxifloxacin (63). The ability of rifapentine to reduce the treatment duration of Q fever from the current 18-month regimen should be explored.

In conclusion, rifapentine and rifabutin demonstrate promising efficacy against C. burnetii in vitro and should be further explored with in vivo models. The bactericidal activity at low concentrations for these antibiotics is unusual against C. burnetii. This improved in vitro efficacy relative to rifampin, coupled with the potential for reduced drug-drug interactions, makes rifabutin and rifapentine appealing candidates for the treatment of Q fever.

MATERIALS AND METHODS

Bacterial strains, host cells, and antibiotics

C. burnetii strains used in this study include Nine Mile phase I RSA493, an ST16 tick isolate from 1935, Nine Mile phase II clone 4, a low virulence strain generated from serial passage of NMI resulting in a truncated lipopolysaccharide (LPS), as well as HPF-GA1, an ST16/26 isolated from a human chronic infection (64), GP-CO1, an ST8 goat placenta isolate (65), and CM-SC1, an ST20 isolated from commercially available raw cow milk (66). C. burnetii stocks were generated as previously described (31, 67). RK-13, a hardy adherent cell line, were cultured in Roswell Park Memorial Institute (RPMI) 1640 medium + 10% fetal bovine serum (FBS). THP-1 cells, a non-adherent human monocytic leukemia cell line, were cultured in RPMI 1640 + 0.05 mM 2-mercaptoethanol + 10% non-heat inactivated FBS. Human kidney epithelial cells (A-498), an adherent cell line, were cultured in Dulbecco’s modified eagle medium (DMEM) + 10% FBS. Antibiotics are described in Table S2.

Antibiotic susceptibility screening

All assays were conducted on flat-bottom 24-well plates and incubated at 37°C in 5% CO₂. Uninfected control wells were included in each assay and were always negative (data not shown). Antibiotic-free media (no drug control) and media containing DMSO (DMSO solvent control) were included in each assay.

RK-13 cells were cultured in RPMI + 10% FBS at 1 × 10⁵ cells per well. After a 24-h incubation, media was replaced with DMEM + 10% FBS containing NMII at a multiplicity of infection (MOI) of 1. After 48 h, DMEM + 10% FBS + antibiotics (or control media) were added (day 1). Antibiotics were tested at concentrations of 25, 10, 1, 0.1, and 0.01 µg mL⁻¹. After 4 days, media was replaced with fresh DMEM + 10% FBS + antibiotics (or control media) and incubated for an additional 3 days. At day 7, internalized C. burnetii were sampled by removing the media and washing the cells with 1 mL per well of phosphate buffered saline (PBS) followed by the addition of 250 µL per well trypsin. Cells were incubated in trypsin for 30 min followed by removal of 100 µL for DNA extraction. Data are from two independent experiments.

THP-1 cells were cultured in RPMI + 10% FBS at 10⁶ cells per well and differentiated into macrophage-like cells by addition of 10 ng mL⁻¹ phorbol myristate acetate (Millipore Sigma). After a 24-h incubation, media was replaced with DMEM + 10% non-inactivated FBS containing NMI at an MOI of 50. After a 24-h incubation, drugs were added at the concentrations depicted in Fig. 1. After 7 days, internalized C. burnetii were sampled by removal of the growth media followed by scraping host cells with a pipet tip in 400 µL of molecular grade deionized water. A 200 µL aliquot was removed for DNA extraction. Data are from three independent experiments.

A-498 cells were cultured in DMEM + 10% FBS at 5 × 10⁵ cells per well. After 24 h, media was replaced with DMEM + 10% FBS containing C. burnetii at an MOI of 50. After 24 h, drugs were added at the respective concentrations. Antibiotics were tested at 0.001, 0.01, and 0.1 µg mL⁻¹, and at the respective C_max for rifapentine (15.05 µg mL⁻¹), rifampin (IV; 17.5 µg mL⁻¹), and rifabutin (0.375 µg mL⁻¹). Rifaximin was tested only at the C_max of 0.0096 µg mL⁻¹. For the NMI and HPF-GA1 strains, exposure was carried out for 7 days. Growth of the controls for the ST20 (CM-SC1) and ST8 (GP-CO1) C. burnetii strains was not robust enough by day 7 to draw meaningful conclusions regarding inhibition; therefore, the assay was extended to 14 days for these two strains. For assays with CM-SC1 and GP-CO1 only, at 7 days post-exposure, media was removed from the wells and was replaced with fresh DMEM + 10% FBS containing the respective antibiotics, and exposure continued for an additional 7 days (14 days total exposure). To sample assays, media was removed, and wells were washed with 400 µL of PBS. Trypsin (200 µL) was added for 30 min followed by addition of 200 µL DMEM + 10% FBS. A 200 µL aliquot was removed for DNA extraction. Data are from three independent experiments.

The MIC was determined as the lowest concentration of antibiotic required to significantly inhibit growth of C. burnetii. GEs post-exposure were calculated and transformed by taking the log₁₀. The differences between means of log₁₀ transformed data were raised to the power of 10 to determine fold change for exposures relative to DMSO solvent controls. Significant inhibition of growth was determined relative to DMSO solvent controls using one-way analysis of variance (ANOVA) with Dunnett’s multiple comparisons post hoc test of log₁₀ transformed data using GraphPad Prism v.9.0.0.

Bactericidal assay

All tested strains of C. burnetii were cultured in A-498 cells in the presence of antibiotics for 7 days as described above for antibiotic susceptibility screening. After 7 days, media was removed, and wells were washed with 400 µL of PBS. Trypsin (200 µL) was added for 30 min followed by addition of 200 µL DMEM + 10% FBS. Following removal of the 200 µL aliquot for DNA extraction (bactericidal assay day 0), DMEM + 10% FBS was added to the remaining 200 µL aliquot to a final volume of 1 mL. Plates were incubated at 37°C in 5% CO₂ for 21 days to allow any viable C. burnetii to recover. Internalized C. burnetii were sampled by scraping host cells with a pipet tip in 400 µL of molecular grade deionized water. A 200 µL aliquot was removed for DNA extraction to determine GE at day 21. Data are from three independent experiments. GEs at day 0 and at 21 days post-recovery were calculated and transformed by taking the natural log (ln). Data are expressed as ln difference of the GE per well at day 21 relative to day 0 [ln (GE per well D21) – ln (GE per well D0)]. This difference in means was raised to the power of e to determine fold change between the GE at day 0 relative to day 21 post-recovery. The MBC was determined as the lowest concentration of antibiotic required to render C. burnetii nonviable. C. burnetii was determined to be nonviable if the mean ln difference in GE from day 21 relative to day 0 was significantly different relative to DMSO solvent controls based on one-way ANOVA with Dunnett’s multiple comparisons post hoc test using GraphPad Prism v.9.0.0.

DNA extractions and PCR analysis

DNA extractions were automated using the KingFisher Flex system in conjunction with the MagMAX DNA Multi-Sample Ultra 2.0 kit (Applied Biosystems) per manufacturer’s instructions. GEs were determined by quantitative PCR targeting the single-copy com1 gene as previously described (68).

SUPPLEMENTAL MATERIAL

The following material is available online at https://doi.org/10.1128/spectrum.01034-24.

Supplemental tables. spectrum.01034-24-s0001.pdf.

Tables S1 and S2.

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