Veterinary Microbiology 178 (2015) 275–278
Contents lists available at ScienceDirect
Veterinary Microbiology journal homepage: www.elsevier.com/locate/vetmic
Short communication
Activity of 10 antimicrobial agents against intracellular Rhodococcus equi Steeve Giguèrea,* , Londa J. Berghausa , Elise A. Leeb a b
Department of Large Animal Medicine, College of Veterinary Medicine, University of Georgia, Athens, GA 30602, United States Department of Large Animal Clinical Sciences, College of Veterinary Medicine, University of Florida, Gainesville FL 32610, United States
A R T I C L E I N F O
A B S T R A C T
Article history: Received 14 March 2015 Received in revised form 20 May 2015 Accepted 22 May 2015
Studies with facultative intracellular bacterial pathogens have shown that evaluation of the bactericidal activity of antimicrobial agents against intracellular bacteria is more closely associated with in vivo efficacy than traditional in vitro susceptibility testing. The objective of this study was to determine the relative activity of 10 antimicrobial agents against intracellular Rhodococcus equi. Equine monocytederived macrophages were infected with virulent R. equi and exposed to erythromycin, clarithromycin, azithromycin, rifampin, ceftiofur, gentamicin, enrofloxacin, vancomycin, imipenem, or doxycycline at concentrations achievable in plasma at clinically recommended dosages in foals. The number of intracellular R. equi was determined 48 h after infection by counting colony forming units (CFUs). The number of R. equi CFUs in untreated control wells were significantly higher than those of monolayers treated with antimicrobial agents. Numbers of R. equi were significantly lower in monolayers treated with enrofloxacin followed by those treated with gentamicin, and vancomycin, when compared to monolayers treated with other antimicrobial agents. Numbers of R. equi in monolayers treated with doxycycline were significantly higher than those of monolayers treated with other antimicrobial agents. Differences in R. equi CFUs between monolayers treated with other antimicrobial agents were not statistically significant. Enrofloxacin, gentamicin, and vancomycin are the most active drugs in equine monocyte-derived macrophages infected with R. equi. Additional studies will be needed to determine if these findings correlate with in vivo efficacy. ã2015 Elsevier B.V. All rights reserved.
Keywords: Rhodococcus equi Antimicrobial Intracellular Macrophage Foal
1. Introduction Rhodococcus equi is one of the most important causes of disease in foals between 3 weeks and 5 months of age. The most common manifestation of the disease is pyogranulomatous bronchopneumonia with abscessation but numerous extrapulmonary disorders also occur (Reuss et al., 2009). R. equi is a facultative intracellular bacterium. The infectivity of R. equi in vitro is limited to cells of the monocyte–macrophage lineage (Hondalus et al., 1993). The ability of R. equi to survive and replicate in macrophages is at the basis of its pathogenicity and strains unable to replicate intracellularly are avirulent for foals (Giguère et al., 1999). A wide variety of antimicrobial agents are active against R. equi in vitro (Riesenberg et al., 2014). However, many of these drugs are reported to be ineffective in vivo (Sweeney et al., 1987), presumably
* Corresponding author at: Department of Large Animal Medicine, College of Veterinary Medicine, University of Georgia, 2200 College Station Road, Athens, GA 30602, United States. Tel.: +1 706 540 9984. E-mail address: [email protected] (S. Giguère). http://dx.doi.org/10.1016/j.vetmic.2015.05.019 0378-1135/ ã 2015 Elsevier B.V. All rights reserved.
because of poor cellular uptake and resulting low intracellular concentrations. Studies with other facultative intracellular pathogens such as Mycobacterium avium, Mycobacterium tuberculosis, and Francisella tularensis have shown that evaluation of the bactericidal activity of antimicrobial agents against intracellular bacteria is more closely associated with in vivo efficacy than traditional in vitro susceptibility testing (Luna-Herrera et al., 1995; Maurin et al., 2000; Sato et al., 1998). The objective of this study was to determine the relative activity of 10 antimicrobial agents against intracellular R. equi in equine monocyte-derived macrophages. 2. Materials and methods 2.1. Animals and sample collection Fifteen adult horses were used. Animals were considered healthy on the basis of physical examination and complete blood count. At each sampling time, blood (500 mL) was collected by jugular venipuncture in glass bottles containing heparin sodium as
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the anticoagulant. The study was approved by the Institutional Animal Care and Use Committee of the University of Georgia. 2.2. Bacterial strain and determination of MIC R. equi strain 33701 (ATCC, Rockville, MD) was used based on its documented ability to replicate in equine macrophages (Berghaus et al., 2014) and to cause disease in foals (Wada et al., 1997). For each antimicrobial agent, MIC was determined using a macrodilution broth dilution technique in glass tubes in accordance to the guidelines established by the Clinical and Laboratory Standard Institute (Clinical and Laboratory Standard Institute, 2011, 2013). The method for MIC determination was as described by Riesenberg et al. (2013), with the exception that 2% lysed horse blood was not added because this proposed modification has not yet been officially implemented by the CLSI. Antimicrobial agents investigated in this study (erythromycin, clarithromycin, azithromycin, rifampin, ceftiofur, gentamicin, enrofloxacin, vancomycin, imipenem, and doxycycline) were selected based on excellent in vitro activity against R. equi and frequency of use in foals or humans. Determinations of MICs were performed in triplicate and the median value was reported. Control strains used to validate each assay were Staphylococcus aureus ATCC 29213 and Enterococcus faecalis ATCC 29212. 2.3. Isolation and infection of equine monocyte-derived macrophages Mononuclear cells were harvested from equine blood by density gradient centrifugation (Ficoll-Paque, Amersham Biosciences, Pittsburgh, PA), washed 3 times with phosphate buffered saline (PBS), and counted. Monocyte-derived macrophages were obtained as previously described (Berghaus et al., 2014). Monocyte-derived macrophages were suspended at a concentration of 5 105 macrophage/mL in Minimum Essential Medium-alpha (MEMa) containing 10% horse serum (HS). One milliliter of the monocyte-derived macrophage cell suspension was added to each well of a 24 well plate (Nunc, ThermoFisher Scientific, Rochester NY). Cells were incubated for 3 h at 37 C in 6% CO2. After incubation and subsequent washing, macrophages were infected with virulent R. equi at a ratio of 5 bacteria per macrophage. The cells were incubated for 40 min to allow phagocytosis. Media containing R. equi was removed carefully and each well was washed 3 times with PBS. All drugs listed above were added to MEMa supplemented with 10% HS at peak concentrations clinically achievable in plasma (Table 1) based on pharmacokinetics studies in adult horses (imipenem and vancomycin) or foals (all other drugs). One milliliter of media containing each antimicrobial agent was added to triplicate wells. Medium without antimicrobial agent was used as negative control. At 48 h post-infection, supernatants were removed and monolayers were washed 3 times with PBS. To determine the number of intracellular R. equi per well,
Table 1 Concentrations of antimicrobial agents used in the present study and MIC of each drug against R. equi 33701. Antimicrobial
Abbreviation
MIC (mg/mL)
Concentration (mg/mL)
Azithromycin Clarithromycin Erythromycin Rifampin Gentamicin Imipenem Ceftiofur Vancomycin Doxycycline Enrofloxacin
AZM CLR ERY RIF GEN IMP CEF VAN DOX ENR
0.5 0.031 0.25 0.062 0.25 0.125 0.125 0.25 0.5 0.5
0.6 0.9 2.0 6.7 22 30 4.0 40 4.0 2.1
monolayers were lysed and the number of colony forming units (CFU) was counted as described (Berghaus et al., 2014) by adding 1 mL of sterile water prior to 40 min incubation. The wells were scraped with the tip of a pipet before the contents were transferred to a 1.5 mL tube. The lysates were vortexted for 5 min, followed by 5 min of sonication, and by an additional 5 min of vigorous vortexing. Dilutions of the lysate were plated in duplicates onto trypticase soy agar plates and the number of bacterial CFU was determined after 48 h incubation at 37 C. The mean of triplicate wells was used for data analysis. 2.4. Effect of gentamicin concentrations on R. equi CFUs in macrophages and cell culture supernatant Murine monocyte–macrophage-like cells J774A.1 (American Type Culture Collection, Manassas, VA) were used. These cells were selected because uptake and intracellular survival of R. equi in J774A.1 cells is similar to that observed in equine macrophages (Hondalus and Mosser, 1994). Cells were maintained in Dulbecco’s modified Eagle’s medium (DMEM, Mediatech, Manassas, VA) supplemented with 10% heat inactivated fetal bovine serum (FBS) and 2 mM glutamine. Prior to use in experiments, the cells were washed three times in antibiotic free media and resuspended at a concentration of 1 107 cells/mL. Cells were incubated for 1 h at 37 C in 5% CO2 with 0.1 mg/mL mitomycin C (Sigma–Aldrich Corp., St. Louis, MO, USA) to inhibit further cell division. Cells were washed to remove mitomycin C, resuspended in antibiotic-free DMEM with 10% FBS at a concentration of 5 105 cells/mL, and 1 mL of cell suspension was placed in each well of 24-well tissue culture plates. and incubated (37 C, 5% CO2) for 3 h to allow adherence. After incubation and subsequent washing, macrophages were infected with virulent R. equi at a ratio of 5 bacteria per macrophage. The cells were incubated for 40 min to allow phagocytosis. Media containing R. equi was removed carefully and each well was washed 3 times with PBS. One milliliter of media containing gentamicin at various concentrations (0.5, 1, 2.5, 5, 10, 20, and 40 mg/mL) was added to triplicate monolayers. Monolayers not containing gentamicin were used as negative controls. At 48 h post-infection, supernatants were removed and monolayers were washed 3 times with PBS. R. equi CFUs were counted in supernatants and in cell monolayers as described above. The experiment was repeated 3 times. 2.5. Statistical analysis Data are presented as log10 R. equi CFU/well. Normality of the data and equality of variances were assessed using the Shapiro– Wilk and Levene’s tests, respectively. Comparison of R. equi CFUs between R. equi-infected equine monocyte-derived macrophages treated with different antimicrobial agents was done using the Friedman repeated measures analysis of variance on ranks. The effect of sample type (supernatant versus intracellular), gentamicin concentration, and interactions between sample type and gentamicin concentration on R. equi CFUs in J774A.1 macrophages was assessed using a two-way repeated measures ANOVA. When indicated, multiple pairwise comparisons were done using the Student–Newman–Keuls method. Statistical significance was set at P < 0.05. 3. Results 3.1. Activity of 10 antimicrobial agents against intracellular R. equi To determine the relative activity of 10 antimicrobial agents against intracellular R. equi, monocyte derived macrophages from 15 healthy horses were infected with R. equi and incubated with
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antimicrobial agents at concentrations clinically achievable in foals at recommended dosages. All drugs had low MICs against R. equi (Table 1). There was a statistically significant effect of antimicrobial drug on R. equi CFU (P < 0.001). R. equi CFUs in untreated control wells were significantly higher than those of monolayers treated with antimicrobial agents (Fig. 1). R. equi CFUs in monolayers treated with doxycycline were significantly higher than those of monolayers treated with other antimicrobial agents (Fig. 1). R. equi CFUs were significantly lower in monolayers treated with enrofloxacin followed by those treated with gentamicin, and vancomycin when compared to monolayers treated with other antimicrobial agents (Fig. 1). 3.2. Effect of gentamicin concentrations on R. equi CFUs in macrophages and cell culture supernatant It is common practice to use culture media containing gentamicin or other aminoglycosides to presumably kill extracellular R. equi without affecting its intracellular growth during intracellular survival and replication assays (Hondalus and Mosser, 1994). Therefore, the high activity of gentamicin against intracellular R. equi was unexpected. To assess the effect of gentamicin concentrations on R. equi CFU in macrophages and cell culture supernatants, J774A.1 macrophages were infected with virulent R. equi. There was a significant (P < 0.001) effect of gentamicin concentration, a significant (P = 0.040) effect of sample type (intracellular versus culture supernatant), and significant (P < 0.001) interactions between gentamicin concentrations and sample type on R. equi CFUs. R. equi CFUs within macrophages and in cell culture supernatants were significantly lower at high gentamicin concentrations than at low gentamicin concentrations (Fig. 2). R. equi CFUs were significantly higher in macrophages than in supernatants at gentamicin concentrations 10 mg/mL. In contrast, R. equi CFUs were significantly lower in macrophages than in supernatants at gentamicin concentrations 1 mg/mL (Fig. 2).
Fig. 1. R. equi CFUs in equine monocyte derived macrophages infected with virulent R. equi and treated with 10 antimicrobial agents at concentrations clinically achievable in vivo (see Table 1 for abbreviations). The experiment was done in 15 healthy horses. The central box represents the values from the lower to upper quartile (25–75%). The middle line represents the median. The error bars extend from the minimum to the maximum value, excluding outliers (upper or lower quartile 3 times the interquartile range) which are displayed as open circles. (a–f) Different letters between 2 antimicrobial agents indicate a statistically significant differences in R. equi CFUs (P < 0.05). When at least 1 letter is common between 2 antimicrobial agents, the difference in CFUs is not statistically significant.
Fig. 2. Effect of gentamicin concentration on R. equi CFUs within J774A.1 macrophages and in cell culture supernatants. Results are presented as mean (SD) of 3 independent experiments. (a–c) Different letters indicate a statistically significant difference in intracellular R. equi CFU between 2 different gentamicin concentrations (P < 0.05). When at least 1 letter is common between 2 gentamicin concentrations, the difference is not statistically significant. (1–3) Different numbers indicate a statistically significant difference in R. equi CFU in supernatants between 2 different gentamicin concentrations (P < 0.05). When at least 1 number is common between 2 gentamicin concentrations, the difference is not statistically significant. *Indicate a significant difference between intracellular and supernatant R. equi CFUs (P < 0.05).
4. Discussion The present study demonstrated that, at clinically achievable plasma concentrations, enrofloxacin, gentamicin, and vancomycin are significantly more active against R. equi in equine monocyte derived-macrophages than the other antimicrobial agents tested. Other antimicrobial agents such as azithromycin, clarithromycin, rifampin, and doxycycline are known to achieve much higher intracellular concentrations. However, overall intracellular concentration of a given antimicrobial agent does not necessarily correlate with its intracellular activity. For example, macrolides such as erythromycin, clarithromycin, or azithromycin concentrate primarily in highly acidic cell compartments such as lysosomes (Togami et al., 2013). The failure of clarithromycin to completely eradicate intracellular M. avium has been shown to be the result of limited exposure to the drug due to the ability of the pathogen to prevent phagosome–lysosome fusion (Frehel et al., 1997). Similarly, once engulfed by macrophages, virulent R. equi avoid destruction by altering maturation and preventing acidification of the phagosomes, thereby preventing fusion with lysosomes (Toyooka et al., 2005; von et al., 2009). This process might limit intracellular exposure of R. equi to drugs that are known to concentrate within cells. The 3 most effective drugs in this study (enrofloxacin, gentamicin, and vancomycin) are bactericidal against R. equi in vitro whereas all other antimicrobial agents tested in this study are only bacteriostatic (Giguère et al., 2012). Therefore, it is possible that the higher activity of these drugs in the present study was due to their greater ability to eradicate extracellular R. equi in cell culture media, hence preventing continuous reinfection, rather than to their true ability to kill R. equi within cells. Nevertheless, the results of the present study are in agreement with in vivo data demonstrating that vancomycin is more effective than erythromycin or rifampin at reducing bacterial CFUs in the organs of immunodeficient mice infected with R. equi (Nordmann et al., 1992). Gentamicin and enrofloxacin were not evaluated in the aforementioned mouse infection model. Despite the highest activity against intracellular R. equi in the present study, the clinical applications of enrofloxacin are limited due to the risk of arthropathy in treated foals (Vivrette et al., 2001).
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Although gentamicin is highly active against R. equi in vitro, its efficacy in foals infected with R. equi has been widely reported as being poor, presumably because of poor cellular uptake due to its hydrophilic nature. This belief is mainly based on the results of a retrospective case series of foals with pneumonia caused by R. equi in which all 17 foals treated with gentamicin died whereas all 10 foals treated with erythromycin in combination with rifampin survived (Sweeney et al., 1987). In contrast, in another retrospective case series of 39 foals with pneumonia caused by R. equi, all 19 survivors were treated with gentamicin whereas non survivors were treated with a variety of other antimicrobial agents including erythromycin, kanamycin, or chloramphenicol (Falcon et al., 1985). These studies were not controlled and did not account for lesion severity at the time of initiation of therapy. In addition, the dosages of gentamicin used in those studies were lower than dosages currently recommended based on the fact that gentamicin is now known to be concentration dependent. The efficacy of gentamicin against intracellular R. equi in the present study was not completely unexpected given the results of a recent study demonstrating that intravenous gentamicin administered to foals at a dose of 6.6 mg/kg results in therapeutic concentrations in bronchoalveolar macrophages (Burton et al., 2014). In addition, gentamicin colocalizes with R. equi within the same intracellular compartment in equine bronchoalveolar macrophages infected ex vivo with R. equi (Burton et al., 2015). Consistent with the finding of the present study, gentamicin has also been shown to kill intracellular Listeria monocytogenes and Francisella tularensis (Drevets et al., 1994; Maurin et al., 2000). Additional studies will be necessary to assess the clinical efficacy of gentamicin in foals infected with R. equi. It is common practice to use culture media containing gentamicin or other aminoglycosides to presumably kill extracellular R. equi without affecting its intracellular growth during intracellular survival and replication assays (Hondalus and Mosser, 1994). Although this assay has been used successfully to show clear differences in intracellular survival and replication between virulent and avirulent strains of R. equi (Coulson et al., 2010; Giguère et al., 1999), the result of the present study revealed a concentration-dependent effect of gentamicin on both extracellular and intracellular R. equi CFUs. Low concentrations do not kill extracellular R. equi in culture supernatant whereas high concentrations impair intracellular growth. Although these data do not dispute the certain value of the aminoglycoside protection assay in screening the virulence of various R. equi strains, they indicate that intracellular replication of R. equi is likely dampened to some degree by the use of aminoglycosides. In conclusion, enrofloxacin, gentamicin, and vancomycin are the most active drugs in equine monocyte-derived macrophages infected with R. equi. Additional studies will be needed to determine if these findings correlate with in vivo efficacy. Acknowledgement This study was funded by the Hodgson Equine Research Endowment of the University of Georgia. References Berghaus, L.J., Giguère, S., Sturgill, T.L., 2014. Effects of age and macrophage lineage on intracellular survival and cytokine induction after infection with Rhodococcus equi. Vet. Immunol. Immunopathol 160, 41–50.
Burton, A.J., Giguère, S., Arnold, R.D., 2014. Pharmacokinetics, pulmonary disposition and tolerability of liposomal gentamicin and free gentamicin in foals. Equine Vet. J. doi:http://dx.doi.org/10.1111/evj.12309. Burton, A.J., Giguère, S., Hondalus, M.K., Berghaus, L.J., Arnold, R.D., 2015. 2015. Efficacy of liposome-encapsulated gentamicin for the treatment of Rhodococcus equi in a mouse infection model and colocalization with R. equi in equine alveolar macrophages. Vet. Microbiol. (in press). Clinical and Laboratory Standard Institute, 2011. Susceptibility testing of mycobacteria, nocardiae, and other aerobic actinomycetes, CLSI Document M24-A2. second ed. Clinical and Laboratory Standard Institute, Waynes, PA. Clinical Laboratory Standard Institute, 2013. Performance standards for antimicrobial disk and dilution susceptibility tests for bacteria isolated from animals; approved standards, CLSI Document VET01-A4. fourth ed. Clinical and Laboratory Standard Institute, Waynes, PA. Coulson, G.B., Agarwal, S., Hondalus, M.K., 2010. Characterization of the role of the pathogenicity island and vapG in the virulence of the intracellular actinomycete pathogen Rhodococcus equi. Infect. Immun. 78, 3323–3334. Drevets, D.A., Canono, B.P., Leenen, P.J., Campbell, P.A., 1994. Gentamicin kills intracellular Listeria monocytogenes. Infect. Immun. 62, 2222–2228. Falcon, J., Smith, B.P., O’Brien, T.R., Carlson, G.P., Biberstein, E., 1985. Clinical and radiographic findings in Corynebacterium equi pneumonia of foals. J. Am. Vet. Med. Assoc. 186, 593–599. Frehel, C., Offredo, C., de, C.C., 1997. The phagosomal environment protects virulent Mycobacterium avium from killing and destruction by clarithromycin. Infect. Immun. 65, 2792–2802. Giguère, S., Hondalus, M.K., Yager, J.A., Darrah, P., Mosser, D.M., Prescott, J.F., 1999. Role of the 85-kilobase plasmid and plasmid-encoded virulence-associated protein A in intracellular survival and virulence of Rhodococcus equi. Infect. Immun. 67, 3548–3557. Giguère, S., Lee, E.A., Guldbech, K.M., Berghaus, L.J., 2012. In vitro synergy pharmacodynamics, and postantibiotic effect of 11 antimicrobial agents against Rhodococcus equi. Vet. Microbiol. 160, 207–213. Hondalus, M.K., Diamond, M.S., Rosenthal, L.A., Springer, T.A., Mosser, D.M., 1993. The intracellular bacterium Rhodococcus equi requires Mac-1 to bind to mammalian cells. Infect. Immun. 61, 2919–2929. Hondalus, M.K., Mosser, D.M., 1994. Survival and replication of Rhodococcus equi in macrophages. Infect. Immun. 62, 4167–4175. Luna-Herrera, J., Reddy, V.M., Daneluzzi, D., Gangadharam, P.R., 1995. Antituberculosis activity of clarithromycin. Antimicrob. Agents Chemother. 39, 2692–2695. Maurin, M., Mersali, N.F., Raoult, D., 2000. Bactericidal activities of antibiotics against intracellular Francisella tularensis. Antimicrob. Agents Chemother. 44, 3428–3431. Nordmann, P., Kerestedjian, J.J., Ronco, E., 1992. Therapy of Rhodococcus equi disseminated infections in nude mice. Antimicrob. Agents Chemother. 36, 1244–1248. Reuss, S.M., Chaffin, M.K., Cohen, N.D., 2009. Extrapulmonary disorders associated with Rhodococcus equi infection in foals: 150 cases (1987–2007). J. Am. Vet Med. Assoc. 235, 855–863. Riesenberg, A., Fessler, A.T., Fromke, C., Kadlec, K., Klarmann, D., Kreienbrock, L., Werckenthin, C., Schwarz, S., 2013. Harmonization of antimicrobial susceptibility testing by broth microdilution for Rhodococcus equi of animal origin. J. Antimicrob. Chemother. 68, 2173–2175. Riesenberg, A., Fessler, A.T., Erol, E., Prenger-Berninghoff, E., Stamm, I., Bose, R., Heusinger, A., Klarmann, D., Werckenthin, C., Schwarz, S., 2014. MICs of 32 antimicrobial agents for Rhodococcus equi isolates of animal origin. J. Antimicrob. Chemother. 69, 1045–1049. Sato, K., Akaki, T., Tomioka, H., 1998. Antimicrobial activities of benzoxazinorifamycin KRM-1648, clarithromycin and levofloxacin against intracellular Mycobacterium avium complex phagocytosed by murine peritoneal macrophages. J. Antimicrob. Chemother. 41, 77–83. Sweeney, C.R., Sweeney, R.W., Divers, T.J., 1987. Rhodococcus equi pneumonia in 48 foals: response to antimicrobial therapy. Vet. Microbiol. 14, 329–336. Togami, K., Chono, S., Morimoto, K., 2013. Subcellular distribution of azithromycin and clarithromycin in rat alveolar macrophages (NR8383) in vitro. Biol. Pharm. Bull. 36, 1494–1499. Toyooka, K., Takai, S., Kirikae, T., 2005. Rhodococcus equi can survive a phagolysosomal environment in macrophages by suppressing acidification of the phagolysosome. J. Med Microbiol. 54, 1007–1015. Vivrette, S., Bostian, A., bermingham, e., Papich, M.G., 2001. Quinolone induced arthropathy in neonatal foals. Proc. Am. Assoc. Equine Pract. 47, 376–377. von, B.K., Polidori, M., Becken, U., Huth, G., Prescott, J.F., Haas, A., 2009. Rhodococcus equi virulence-associated protein A is required for diversion of phagosome biogenesis but not for cytotoxicity. Infect. Immun. 77, 5676–5681. Wada, R., Kamada, M., Anzai, T., Nakanishi, A., Kanemaru, T., Takai, S., Tsubaki, S., 1997. Pathogenicity and virulence of Rhodococcus equi in foals following intratracheal challenge. Vet. Microbiol 56, 301–312.
Contents lists available at ScienceDirect
Veterinary Microbiology journal homepage: www.elsevier.com/locate/vetmic
Short communication
Activity of 10 antimicrobial agents against intracellular Rhodococcus equi Steeve Giguèrea,* , Londa J. Berghausa , Elise A. Leeb a b
Department of Large Animal Medicine, College of Veterinary Medicine, University of Georgia, Athens, GA 30602, United States Department of Large Animal Clinical Sciences, College of Veterinary Medicine, University of Florida, Gainesville FL 32610, United States
A R T I C L E I N F O
A B S T R A C T
Article history: Received 14 March 2015 Received in revised form 20 May 2015 Accepted 22 May 2015
Studies with facultative intracellular bacterial pathogens have shown that evaluation of the bactericidal activity of antimicrobial agents against intracellular bacteria is more closely associated with in vivo efficacy than traditional in vitro susceptibility testing. The objective of this study was to determine the relative activity of 10 antimicrobial agents against intracellular Rhodococcus equi. Equine monocytederived macrophages were infected with virulent R. equi and exposed to erythromycin, clarithromycin, azithromycin, rifampin, ceftiofur, gentamicin, enrofloxacin, vancomycin, imipenem, or doxycycline at concentrations achievable in plasma at clinically recommended dosages in foals. The number of intracellular R. equi was determined 48 h after infection by counting colony forming units (CFUs). The number of R. equi CFUs in untreated control wells were significantly higher than those of monolayers treated with antimicrobial agents. Numbers of R. equi were significantly lower in monolayers treated with enrofloxacin followed by those treated with gentamicin, and vancomycin, when compared to monolayers treated with other antimicrobial agents. Numbers of R. equi in monolayers treated with doxycycline were significantly higher than those of monolayers treated with other antimicrobial agents. Differences in R. equi CFUs between monolayers treated with other antimicrobial agents were not statistically significant. Enrofloxacin, gentamicin, and vancomycin are the most active drugs in equine monocyte-derived macrophages infected with R. equi. Additional studies will be needed to determine if these findings correlate with in vivo efficacy. ã2015 Elsevier B.V. All rights reserved.
Keywords: Rhodococcus equi Antimicrobial Intracellular Macrophage Foal
1. Introduction Rhodococcus equi is one of the most important causes of disease in foals between 3 weeks and 5 months of age. The most common manifestation of the disease is pyogranulomatous bronchopneumonia with abscessation but numerous extrapulmonary disorders also occur (Reuss et al., 2009). R. equi is a facultative intracellular bacterium. The infectivity of R. equi in vitro is limited to cells of the monocyte–macrophage lineage (Hondalus et al., 1993). The ability of R. equi to survive and replicate in macrophages is at the basis of its pathogenicity and strains unable to replicate intracellularly are avirulent for foals (Giguère et al., 1999). A wide variety of antimicrobial agents are active against R. equi in vitro (Riesenberg et al., 2014). However, many of these drugs are reported to be ineffective in vivo (Sweeney et al., 1987), presumably
* Corresponding author at: Department of Large Animal Medicine, College of Veterinary Medicine, University of Georgia, 2200 College Station Road, Athens, GA 30602, United States. Tel.: +1 706 540 9984. E-mail address: [email protected] (S. Giguère). http://dx.doi.org/10.1016/j.vetmic.2015.05.019 0378-1135/ ã 2015 Elsevier B.V. All rights reserved.
because of poor cellular uptake and resulting low intracellular concentrations. Studies with other facultative intracellular pathogens such as Mycobacterium avium, Mycobacterium tuberculosis, and Francisella tularensis have shown that evaluation of the bactericidal activity of antimicrobial agents against intracellular bacteria is more closely associated with in vivo efficacy than traditional in vitro susceptibility testing (Luna-Herrera et al., 1995; Maurin et al., 2000; Sato et al., 1998). The objective of this study was to determine the relative activity of 10 antimicrobial agents against intracellular R. equi in equine monocyte-derived macrophages. 2. Materials and methods 2.1. Animals and sample collection Fifteen adult horses were used. Animals were considered healthy on the basis of physical examination and complete blood count. At each sampling time, blood (500 mL) was collected by jugular venipuncture in glass bottles containing heparin sodium as
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the anticoagulant. The study was approved by the Institutional Animal Care and Use Committee of the University of Georgia. 2.2. Bacterial strain and determination of MIC R. equi strain 33701 (ATCC, Rockville, MD) was used based on its documented ability to replicate in equine macrophages (Berghaus et al., 2014) and to cause disease in foals (Wada et al., 1997). For each antimicrobial agent, MIC was determined using a macrodilution broth dilution technique in glass tubes in accordance to the guidelines established by the Clinical and Laboratory Standard Institute (Clinical and Laboratory Standard Institute, 2011, 2013). The method for MIC determination was as described by Riesenberg et al. (2013), with the exception that 2% lysed horse blood was not added because this proposed modification has not yet been officially implemented by the CLSI. Antimicrobial agents investigated in this study (erythromycin, clarithromycin, azithromycin, rifampin, ceftiofur, gentamicin, enrofloxacin, vancomycin, imipenem, and doxycycline) were selected based on excellent in vitro activity against R. equi and frequency of use in foals or humans. Determinations of MICs were performed in triplicate and the median value was reported. Control strains used to validate each assay were Staphylococcus aureus ATCC 29213 and Enterococcus faecalis ATCC 29212. 2.3. Isolation and infection of equine monocyte-derived macrophages Mononuclear cells were harvested from equine blood by density gradient centrifugation (Ficoll-Paque, Amersham Biosciences, Pittsburgh, PA), washed 3 times with phosphate buffered saline (PBS), and counted. Monocyte-derived macrophages were obtained as previously described (Berghaus et al., 2014). Monocyte-derived macrophages were suspended at a concentration of 5 105 macrophage/mL in Minimum Essential Medium-alpha (MEMa) containing 10% horse serum (HS). One milliliter of the monocyte-derived macrophage cell suspension was added to each well of a 24 well plate (Nunc, ThermoFisher Scientific, Rochester NY). Cells were incubated for 3 h at 37 C in 6% CO2. After incubation and subsequent washing, macrophages were infected with virulent R. equi at a ratio of 5 bacteria per macrophage. The cells were incubated for 40 min to allow phagocytosis. Media containing R. equi was removed carefully and each well was washed 3 times with PBS. All drugs listed above were added to MEMa supplemented with 10% HS at peak concentrations clinically achievable in plasma (Table 1) based on pharmacokinetics studies in adult horses (imipenem and vancomycin) or foals (all other drugs). One milliliter of media containing each antimicrobial agent was added to triplicate wells. Medium without antimicrobial agent was used as negative control. At 48 h post-infection, supernatants were removed and monolayers were washed 3 times with PBS. To determine the number of intracellular R. equi per well,
Table 1 Concentrations of antimicrobial agents used in the present study and MIC of each drug against R. equi 33701. Antimicrobial
Abbreviation
MIC (mg/mL)
Concentration (mg/mL)
Azithromycin Clarithromycin Erythromycin Rifampin Gentamicin Imipenem Ceftiofur Vancomycin Doxycycline Enrofloxacin
AZM CLR ERY RIF GEN IMP CEF VAN DOX ENR
0.5 0.031 0.25 0.062 0.25 0.125 0.125 0.25 0.5 0.5
0.6 0.9 2.0 6.7 22 30 4.0 40 4.0 2.1
monolayers were lysed and the number of colony forming units (CFU) was counted as described (Berghaus et al., 2014) by adding 1 mL of sterile water prior to 40 min incubation. The wells were scraped with the tip of a pipet before the contents were transferred to a 1.5 mL tube. The lysates were vortexted for 5 min, followed by 5 min of sonication, and by an additional 5 min of vigorous vortexing. Dilutions of the lysate were plated in duplicates onto trypticase soy agar plates and the number of bacterial CFU was determined after 48 h incubation at 37 C. The mean of triplicate wells was used for data analysis. 2.4. Effect of gentamicin concentrations on R. equi CFUs in macrophages and cell culture supernatant Murine monocyte–macrophage-like cells J774A.1 (American Type Culture Collection, Manassas, VA) were used. These cells were selected because uptake and intracellular survival of R. equi in J774A.1 cells is similar to that observed in equine macrophages (Hondalus and Mosser, 1994). Cells were maintained in Dulbecco’s modified Eagle’s medium (DMEM, Mediatech, Manassas, VA) supplemented with 10% heat inactivated fetal bovine serum (FBS) and 2 mM glutamine. Prior to use in experiments, the cells were washed three times in antibiotic free media and resuspended at a concentration of 1 107 cells/mL. Cells were incubated for 1 h at 37 C in 5% CO2 with 0.1 mg/mL mitomycin C (Sigma–Aldrich Corp., St. Louis, MO, USA) to inhibit further cell division. Cells were washed to remove mitomycin C, resuspended in antibiotic-free DMEM with 10% FBS at a concentration of 5 105 cells/mL, and 1 mL of cell suspension was placed in each well of 24-well tissue culture plates. and incubated (37 C, 5% CO2) for 3 h to allow adherence. After incubation and subsequent washing, macrophages were infected with virulent R. equi at a ratio of 5 bacteria per macrophage. The cells were incubated for 40 min to allow phagocytosis. Media containing R. equi was removed carefully and each well was washed 3 times with PBS. One milliliter of media containing gentamicin at various concentrations (0.5, 1, 2.5, 5, 10, 20, and 40 mg/mL) was added to triplicate monolayers. Monolayers not containing gentamicin were used as negative controls. At 48 h post-infection, supernatants were removed and monolayers were washed 3 times with PBS. R. equi CFUs were counted in supernatants and in cell monolayers as described above. The experiment was repeated 3 times. 2.5. Statistical analysis Data are presented as log10 R. equi CFU/well. Normality of the data and equality of variances were assessed using the Shapiro– Wilk and Levene’s tests, respectively. Comparison of R. equi CFUs between R. equi-infected equine monocyte-derived macrophages treated with different antimicrobial agents was done using the Friedman repeated measures analysis of variance on ranks. The effect of sample type (supernatant versus intracellular), gentamicin concentration, and interactions between sample type and gentamicin concentration on R. equi CFUs in J774A.1 macrophages was assessed using a two-way repeated measures ANOVA. When indicated, multiple pairwise comparisons were done using the Student–Newman–Keuls method. Statistical significance was set at P < 0.05. 3. Results 3.1. Activity of 10 antimicrobial agents against intracellular R. equi To determine the relative activity of 10 antimicrobial agents against intracellular R. equi, monocyte derived macrophages from 15 healthy horses were infected with R. equi and incubated with
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antimicrobial agents at concentrations clinically achievable in foals at recommended dosages. All drugs had low MICs against R. equi (Table 1). There was a statistically significant effect of antimicrobial drug on R. equi CFU (P < 0.001). R. equi CFUs in untreated control wells were significantly higher than those of monolayers treated with antimicrobial agents (Fig. 1). R. equi CFUs in monolayers treated with doxycycline were significantly higher than those of monolayers treated with other antimicrobial agents (Fig. 1). R. equi CFUs were significantly lower in monolayers treated with enrofloxacin followed by those treated with gentamicin, and vancomycin when compared to monolayers treated with other antimicrobial agents (Fig. 1). 3.2. Effect of gentamicin concentrations on R. equi CFUs in macrophages and cell culture supernatant It is common practice to use culture media containing gentamicin or other aminoglycosides to presumably kill extracellular R. equi without affecting its intracellular growth during intracellular survival and replication assays (Hondalus and Mosser, 1994). Therefore, the high activity of gentamicin against intracellular R. equi was unexpected. To assess the effect of gentamicin concentrations on R. equi CFU in macrophages and cell culture supernatants, J774A.1 macrophages were infected with virulent R. equi. There was a significant (P < 0.001) effect of gentamicin concentration, a significant (P = 0.040) effect of sample type (intracellular versus culture supernatant), and significant (P < 0.001) interactions between gentamicin concentrations and sample type on R. equi CFUs. R. equi CFUs within macrophages and in cell culture supernatants were significantly lower at high gentamicin concentrations than at low gentamicin concentrations (Fig. 2). R. equi CFUs were significantly higher in macrophages than in supernatants at gentamicin concentrations 10 mg/mL. In contrast, R. equi CFUs were significantly lower in macrophages than in supernatants at gentamicin concentrations 1 mg/mL (Fig. 2).
Fig. 1. R. equi CFUs in equine monocyte derived macrophages infected with virulent R. equi and treated with 10 antimicrobial agents at concentrations clinically achievable in vivo (see Table 1 for abbreviations). The experiment was done in 15 healthy horses. The central box represents the values from the lower to upper quartile (25–75%). The middle line represents the median. The error bars extend from the minimum to the maximum value, excluding outliers (upper or lower quartile 3 times the interquartile range) which are displayed as open circles. (a–f) Different letters between 2 antimicrobial agents indicate a statistically significant differences in R. equi CFUs (P < 0.05). When at least 1 letter is common between 2 antimicrobial agents, the difference in CFUs is not statistically significant.
Fig. 2. Effect of gentamicin concentration on R. equi CFUs within J774A.1 macrophages and in cell culture supernatants. Results are presented as mean (SD) of 3 independent experiments. (a–c) Different letters indicate a statistically significant difference in intracellular R. equi CFU between 2 different gentamicin concentrations (P < 0.05). When at least 1 letter is common between 2 gentamicin concentrations, the difference is not statistically significant. (1–3) Different numbers indicate a statistically significant difference in R. equi CFU in supernatants between 2 different gentamicin concentrations (P < 0.05). When at least 1 number is common between 2 gentamicin concentrations, the difference is not statistically significant. *Indicate a significant difference between intracellular and supernatant R. equi CFUs (P < 0.05).
4. Discussion The present study demonstrated that, at clinically achievable plasma concentrations, enrofloxacin, gentamicin, and vancomycin are significantly more active against R. equi in equine monocyte derived-macrophages than the other antimicrobial agents tested. Other antimicrobial agents such as azithromycin, clarithromycin, rifampin, and doxycycline are known to achieve much higher intracellular concentrations. However, overall intracellular concentration of a given antimicrobial agent does not necessarily correlate with its intracellular activity. For example, macrolides such as erythromycin, clarithromycin, or azithromycin concentrate primarily in highly acidic cell compartments such as lysosomes (Togami et al., 2013). The failure of clarithromycin to completely eradicate intracellular M. avium has been shown to be the result of limited exposure to the drug due to the ability of the pathogen to prevent phagosome–lysosome fusion (Frehel et al., 1997). Similarly, once engulfed by macrophages, virulent R. equi avoid destruction by altering maturation and preventing acidification of the phagosomes, thereby preventing fusion with lysosomes (Toyooka et al., 2005; von et al., 2009). This process might limit intracellular exposure of R. equi to drugs that are known to concentrate within cells. The 3 most effective drugs in this study (enrofloxacin, gentamicin, and vancomycin) are bactericidal against R. equi in vitro whereas all other antimicrobial agents tested in this study are only bacteriostatic (Giguère et al., 2012). Therefore, it is possible that the higher activity of these drugs in the present study was due to their greater ability to eradicate extracellular R. equi in cell culture media, hence preventing continuous reinfection, rather than to their true ability to kill R. equi within cells. Nevertheless, the results of the present study are in agreement with in vivo data demonstrating that vancomycin is more effective than erythromycin or rifampin at reducing bacterial CFUs in the organs of immunodeficient mice infected with R. equi (Nordmann et al., 1992). Gentamicin and enrofloxacin were not evaluated in the aforementioned mouse infection model. Despite the highest activity against intracellular R. equi in the present study, the clinical applications of enrofloxacin are limited due to the risk of arthropathy in treated foals (Vivrette et al., 2001).
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Although gentamicin is highly active against R. equi in vitro, its efficacy in foals infected with R. equi has been widely reported as being poor, presumably because of poor cellular uptake due to its hydrophilic nature. This belief is mainly based on the results of a retrospective case series of foals with pneumonia caused by R. equi in which all 17 foals treated with gentamicin died whereas all 10 foals treated with erythromycin in combination with rifampin survived (Sweeney et al., 1987). In contrast, in another retrospective case series of 39 foals with pneumonia caused by R. equi, all 19 survivors were treated with gentamicin whereas non survivors were treated with a variety of other antimicrobial agents including erythromycin, kanamycin, or chloramphenicol (Falcon et al., 1985). These studies were not controlled and did not account for lesion severity at the time of initiation of therapy. In addition, the dosages of gentamicin used in those studies were lower than dosages currently recommended based on the fact that gentamicin is now known to be concentration dependent. The efficacy of gentamicin against intracellular R. equi in the present study was not completely unexpected given the results of a recent study demonstrating that intravenous gentamicin administered to foals at a dose of 6.6 mg/kg results in therapeutic concentrations in bronchoalveolar macrophages (Burton et al., 2014). In addition, gentamicin colocalizes with R. equi within the same intracellular compartment in equine bronchoalveolar macrophages infected ex vivo with R. equi (Burton et al., 2015). Consistent with the finding of the present study, gentamicin has also been shown to kill intracellular Listeria monocytogenes and Francisella tularensis (Drevets et al., 1994; Maurin et al., 2000). Additional studies will be necessary to assess the clinical efficacy of gentamicin in foals infected with R. equi. It is common practice to use culture media containing gentamicin or other aminoglycosides to presumably kill extracellular R. equi without affecting its intracellular growth during intracellular survival and replication assays (Hondalus and Mosser, 1994). Although this assay has been used successfully to show clear differences in intracellular survival and replication between virulent and avirulent strains of R. equi (Coulson et al., 2010; Giguère et al., 1999), the result of the present study revealed a concentration-dependent effect of gentamicin on both extracellular and intracellular R. equi CFUs. Low concentrations do not kill extracellular R. equi in culture supernatant whereas high concentrations impair intracellular growth. Although these data do not dispute the certain value of the aminoglycoside protection assay in screening the virulence of various R. equi strains, they indicate that intracellular replication of R. equi is likely dampened to some degree by the use of aminoglycosides. In conclusion, enrofloxacin, gentamicin, and vancomycin are the most active drugs in equine monocyte-derived macrophages infected with R. equi. Additional studies will be needed to determine if these findings correlate with in vivo efficacy. Acknowledgement This study was funded by the Hodgson Equine Research Endowment of the University of Georgia. References Berghaus, L.J., Giguère, S., Sturgill, T.L., 2014. Effects of age and macrophage lineage on intracellular survival and cytokine induction after infection with Rhodococcus equi. Vet. Immunol. Immunopathol 160, 41–50.
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