Mutant prevention concentration and mutant selection window for 10 antimicrobial agents against Rhodococcus equi

Veterinary Microbiology 166 (2013) 670–675

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Mutant prevention concentration and mutant selection window for 10 antimicrobial agents against Rhodococcus equi Londa J. Berghaus, Steeve Gigue`re *, Kristen Guldbech Department of Large Animal Medicine, College of Veterinary Medicine, University of Georgia, Athens, GA 30602, United States

A R T I C L E I N F O

A B S T R A C T

Article history: Received 15 May 2013 Received in revised form 1 July 2013 Accepted 10 July 2013

The objectives of this study were to determine the mutant prevention concentration (MPC), time above the MPC and mutant selection window for 10 antimicrobial agents against Rhodococcus equi and to determine if the combination of a macrolide with rifampin would decrease emergence of resistant mutants. Antimicrobial agents investigated (erythromycin, clarithromycin, azithromycin, rifampin, amikacin, gentamicin, enrofloxacin, vancomycin, imipenem, and doxycycline) were selected based on in vitro activity and frequency of use in foals or people infected with R. equi. Each antimicrobial agent or combination of agents was evaluated against four virulent strains of R. equi. MPC were determined using an agar plate assay. Pharmacodynamic parameters were calculated using published plasma and pulmonary pharmacokinetic variables. There was a significant (P < 0.001) effect of the type of antimicrobial agent on the MPC. The MPC of clarithromycin (1.0 mg/ml) was significantly lower and the MPC of rifampin and amikacin (512 and 384 mg/ml, respectively) were significantly higher than that of all other antimicrobial agents tested. Combining erythromycin, clarithromycin, or azithromycin with rifampin resulted in a significant (P  0.005) decrease in MPC and MPC/MIC ratio. When MIC and MPC were combined with pharmacokinetic variables, only gentamicin and vancomycin were predicted to achieve plasma concentrations above the MPC for any given periods of time. Only clarithromycin and the combination clarithromycin–rifampin were predicted to achieve concentrations in bronchoalveolar cells and pulmonary epithelial lining fluid above the MPC for the entire dosing interval. In conclusion, the combination of a macrolide with rifampin considerably decreases the emergence of resistant mutants of R. equi. ß 2013 Elsevier B.V. All rights reserved.

Keywords: Rhodococcus equi Antimicrobial Mutant prevention concentration

1. Introduction Rhodococcus equi, a Gram-positive facultative intracellular pathogen, is one of the most important causes of pneumonia in foals between 3 weeks and 5 months of age. R. equi has also emerged as a common opportunistic pathogen in immunosuppressed people, especially those infected with the human immunodeficiency virus (Harvey and Sunstrum, 1991; Yamshchikov et al., 2010). A wide

* Corresponding author at: Department of Large Animal Medicine, College of Veterinary Medicine, University of Georgia, 501 D.W. Brooks Drive, Athens, GA 30602, United States. Tel.: +1 706 542 4436. E-mail address: [email protected] (S. Gigue`re). 0378-1135/$ – see front matter ß 2013 Elsevier B.V. All rights reserved. http://dx.doi.org/10.1016/j.vetmic.2013.07.006

variety of antimicrobial agents are active against R. equi in vitro. However, many of these drugs are reported to be ineffective in vivo, likely because of poor cellular uptake and resulting low intracellular concentrations. The combination of rifampin and erythromycin became the treatment of choice for the treatment of R. equi pneumonia in foals in the 1980s and has apparently reduced foal mortality relative to historical data (Hillidge, 1987; Sweeney et al., 1987). In recent years, clarithromycin or azithromycin, typically replace erythromycin in the combination with rifampin (Gigue`re et al., 2004). There has been an increase in the frequency of detection of macrolide and rifampin resistance in isolates of R. equi from pneumonic foals in the last decade (Gigue`re et al., 2010). Widespread macrolide and rifampin resistance has

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been documented at a large horse farm with approximately 40% of the foals being infected with resistant isolates of R. equi (Burton et al., 2013). Multiple studies have examined the in vitro activity of antimicrobial agents alone or in various combinations against R. equi (Gigue`re et al., 2012; Nordmann and Ronco, 1992; Prescott and Nicholson, 1984). However, there is no data on the relative propensity of currently available antimicrobial agents to selectively enrich for resistant mutant subpopulations among R. equi isolates. One way to compare compounds for selective enrichment of mutants is based on measurement of the mutant prevention concentration (MPC). The MPC is defined as the drug concentration that prevents selective enrichment of first step resistant mutants within a large susceptible bacterial population (Blondeau et al., 2001; Dong et al., 1999; Zhao and Drlica, 2001). The range of concentrations between the minimal inhibitory concentration (MIC) and the MPC is the mutant selection window (MSW). The MSW represents the danger zone for emergence of resistant mutants (Blondeau, 2009). Minimizing the length of time that the drug concentrations remain in the MSW may reduce the likelihood for development of resistance during therapy. Thus the MPC can be used instead of the MIC in pharmacodynamic considerations; antimicrobial agents with higher values for area under the 24 h dug concentration versus time curve to MPC ratio (AUC24/MPC) are less likely to selectively enrich for resistant mutants (Blondeau, 2009; Drlica and Zhao, 2007). The objectives of this study were to determine the MPC, AUC24/MPC and mutant selection window (MSW) for 10 antimicrobial agents against Rhodococcus equi and to determine if the combination of a macrolide antimicrobial agent with rifampin would decrease emergence of resistant mutants. We hypothesized that the combination results in a significant decrease in the emergence of resistant mutants. 2. Materials and methods 2.1. Bacterial strains and determination of MIC Four R. equi isolates from tracheobronchial aspirates or postmortem specimens from pneumonic foals were randomly selected from a collection of frozen stabilates. Isolates were confirmed to be virulent by PCR amplification of the vapA gene as previously described (Gigue`re et al., 2010). For each isolate, MIC was determined by a macrodilution broth dilution technique in glass tubes in accordance to the guidelines established by Clinical and Laboratory Standard Institute (2011). A standard inoculum of 5  105 was used for each isolate. The size of the inoculum was verified by counting CFU. Antimicrobial agents investigated in this study (erythromycin, clarithromycin, azithromycin, rifampin, amikacin, gentamicin, enrofloxacin, vancomycin, imipenem, and doxycycline) were selected based on excellent in vitro activity against large numbers of isolates of R. equi and frequency of use in foals or humans (Jacks et al., 2003; Nordmann and Ronco, 1992). Concentrations of antimicrobial agents tested represented 2-fold dilutions between 256 and 0.016 mg/ml. MIC was defined as the first dilution with

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no bacterial growth after 24 h of incubation at 37 8C. All MIC determinations were performed in triplicate for each isolate and the median value was used for data analysis. Control strains used to validate each assay were Staphylococcus aureus ATCC 29213 and Enterococcus faecalis ATCC 29212. R. equi ATCC 33701, a virulent strain with known in vitro susceptibility profile was used as an additional control (Carlson et al., 2010). 2.2. Determination of the MPC MPC testing was performed modifying a procedure described by Blondeau (2009). Bacterial cultures were grown in Mueller Hinton broth to a concentration of 1010 CFU/ml. The size of the inoculum was verified by counting CFU. Mueller Hinton agar plates containing 2-fold concentration increments of each drug were prepared (1 MIC to 512 mg/ml). Plates were inoculated with 109 CFU and incubated at 37 8C for 4 days. The MPC was defined as the lowest concentration of an antimicrobial agent completely preventing growth of R. equi. MPC testing was done in triplicate for each isolate and the median value was used for data analysis. 2.3. Pharmacodynamic analysis Pharmacokinetic parameters (maximum concentration [Cmax], and AUC24) were obtained or estimated from published studies conducted in adult horses (imipenem and vancomycin) or foals (all other drugs) (Bermingham et al., 2000; Bucki et al., 2004; Burrows et al., 1992; Burton et al., 2013; Lakritz et al., 1999; Orsini et al., 1992, 2005; Peters et al., 2012; Suarez-Mier et al., 2007; Womble et al., 2007). The percentage of each dosage interval that plasma concentration exceed the MIC (%T > MIC) and the percentage of each dosage interval that plasma concentration exceed the MPC (%T > MPC) were estimated from the published mean drug concentration versus time profile. The percentage of each dosage interval that plasma concentrations were within the MSW (%TMSW) was calculated as %T > MIC %T > MPC. The same calculations were made from drug concentrations in bronchoalveolar (BAL) cells and pulmonary epithelial lining fluid (PELF) for antimicrobial agents for which pulmonary pharmacokinetic data were available in foals. 2.4. Statistical analysis Comparisons of MIC, MPC, MIC/MPC ratio, between antimicrobial agents were done using the Friedman repeated measures analysis of variance on ranks. When warranted, multiple pairwise comparisons were done using the Student–Newman–Keuls method. The Wilcoxon rank sum test was used to compare the MIC of the original isolate to that of mutant colonies. Statistical significance was set at P < 0.05. 3. Results The MIC of 10 antimicrobial agents against 4 virulent isolates of R. equi ranged between 0.031 mg/ml and 4 mg/ ml (Table 1). The MPC values ranged between 1.0 mg/ml for

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Table 1 MIC, MPC, and MPC/MIC ratio of 10 antimicrobial agents against 4 virulent strains of R. equi. Antimicrobial

R. equi 156867 MIC

Rifampina Erythromycinb Clarithromycinc Azithromycinb Amikacina Gentamicinb Enrofloxacinb Vancomycinb Imipenemb Doxycyclineb

0.031 0.25 0.031 0.5 4 0.5 1 0.125 0.5 1

Azithromycin + rifampin Clarithromycin + rifampin Erythromycin + rifampin

0.5d 0.031d 0.25d

MPC 512 4 1 32 512 16 64 8 128 64

R. equi156261 Ratio

MIC

16,384 16 32 64 128 32 64 64 256 64

0.031 0.25 0.063 1 4 0.25 0.5 0.25 1 0.5

4e 8e 4e

1 0.063 0.031

2e 0.24e 1e

MPC 512 4 1 32 512 16 64 8 128 64 4e 0.24e 0.24e

R. equi 162059 Ratio

MIC

16,384 64 64 32 256 16 32 32 128 32

0.5 0.5 0.031 0.5 4 0.5 0.5 0.25 1 0.5

4e 4e 8e

0.5 0.031 0.5

MPC 1024 4 1 16 256 16 8 4 16 16

R. equi 103 Ratio

MIC

2048 8 32 32 64 32 16 16 16 32

0.063 0.5 0.031 0.125 1 0.125 1 0.125 0.5 0.25

4 4 2

0.125 0.031 0.5

2e 0.12e 1e

MPC 512 16 1 32 256 16 32 4 32 4 2e 0.24e 0.5e

Ratio 16,384 32 32 256 256 128 32 32 64 16 16e 8e 1e

a–c

Different letters between drugs indicate a statistically significant (P < 0.05) difference in MPC between drugs. MIC of the macrolide. e MPC and MIC/MPC ratios of the drug combination are significantly lower than that of each individual respective drug. d

clarithromycin and 512 mg/ml for rifampin (Table 1). There was a significant (P < 0.001) effect of the type of antimicrobial agent on the MPC. The MPC of clarithromycin was significantly lower than that of all other antimicrobial agents tested. Conversely, the MPC of rifampin and amikacin were significantly higher than that of all other antimicrobial agents tested. There was also a significant (P = 0.005) effect of drug on MPC/MIC ratio with the MPC/MIC ratio of rifampin being significantly higher than that of all other drugs tested (Table 1). The MPC and MPC/MIC ratio for erythromycin, clarithromycin, or

azithromycin in combination with rifampin were significantly (P  0.005) lower than that of each drug tested individually (Table 1). The MIC of randomly selected mutant colonies was significantly higher than that of the original isolate for each drug tested. When MIC and MPC were combined with pharmacokinetic variables, only gentamicin and vancomycin were predicted to achieve plasma concentrations above the MPC for any given periods of time (Table 2). Erythromycin, clarithromycin, gentamicin, enrofloxacin, vancomycin, and doxycycline were predicted to have the highest plasma

Table 2 Pharmacokinetic and pharmacodynamic variables (median and range) in plasma, PELF, and BAL cells for 10 antimicrobial agents against 4 virulent strains of R. equi. Dosage regimen (mg/kg)

Cmax (mg/ml)

AUC24 (mg h/ml)

AUC24/ MPC (h)

%T > MIC

Plasma Rifampin Erythromycin Clarithromycin Azithromycin Amikacin Gentamicin Enrofloxacin Vancomycin Imipenem Doxycycline Clarithromycin (+ rifampin)

10, q 24 h, PO 25, q 6 h, PO 7.5, q 12 h, PO 10, q 24 h, PO 25, q 24 h, IV 12, q 24 h, IV 25, q 24 h, IV 6.6, q 8 h, IV 10, q 6 h, IV 10, q 12 h, PO 7.5 + 10, q 12 h, PO

7.4 1.4 0.9 0.6 58 71 2.1 13 9.0 4.1 0.2

162 12.4 6.8 8.1 195 211 58.5 285.9 25.6 70.6 2.4

0.3 (0.2–0.3) 3.1 (0.8–3.1) 6.8 (6.8–6.8) 0.3 (0.3–0.5) 0.6 (0.4–0.8) 13 (13–13) 1.4 (0.9–7.3) 54 (35–71) 0.5 (0.2–1.6) 2.8 (1.1–18) 10 (10–20)

100 (100–100) 71 (42–100) 100 (100–100) 34 (0–100) 33 (33–100) 100 (100–100) 100 (100–100) 100 (100–100) 43 (36–50) 100 (100–100) 100 (67–100)

BAL cells Rifampin Erythromycin Clarithromycin Azithromycin Doxycycline Clarithromycin (+ rifampin)

10 q 24 h, PO 25 q 6 h, PO 7.5 q 12 h, PO 10 q 24 h, PO 10 q 12 h, PO 7.5 + 10 q 12 h, PO

4.9a 2.5 74.2 49.8 6.3 24.8a

NA 103 2328 4679 NA NA

NA 26 (6.4–26) 2328 (2328–2328) 146 (146–292) NA NA

100 100 100 100 100 100

(100–100) (100–100) (100–100) (100–100) (100–100) (100–100)

PELF Rifampin Erythromycin Clarithromycin Azithromycin Doxycycline Clarithromycin (+rifampin)

10 q 24 h, PO 25 q 6 h, PO 7.5 q 12 h, PO 10 q 24 h, PO 10 q 12 h, PO 7.5 + 10 q 12 h, PO

8.5a
NA NA 1258 494 NA NA

NA NA 1258 (1258–1258) 146 (146–292) NA NA

100 NA 100 100 100 100

(100–100)

Antimicrobial

a

Concentration 12 h after drug administration; LOD, limit of detection; NA, data not available.

(100–100) (100–100) (100–100) (100–100)

%T > MPC

0 0 0 0 0 17 0 44 0 0 0

%TMSW

(0–0) (0–0) (0–0) (0–0) (0–0) (17–17) (0–0) (25–63) (0–0) (0–0) (0–0)

100 (100–100) 71 (42–100) 100 (100–100) 34 (0–100) 33 (33–100) 83 (83–83) 100 (100–100) 61 (47–75) 43 (36–50) 100 (100–100) 100 (67–100)

0 0 100 100 0 100

(0–0) (0–0) (100–100) (100–100) (0–0) (100–100)

100 (100–100) 100 (100–100) 0 (0–0) 0 (0–0) 100 (100–100) 0 (0–0)

0 0 100 0 0 100

(0–0) (0–0) (100–100) (0–0) (0–0) (100–100)

100 (100–100) NA 0 (0–0) 100 (100–100) 100 (100–100) 0 (0–0)

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AUC24/MPC ratio (Table 2). Pulmonary pharmacokinetic data were available for rifampin, erythromycin, clarithromycin, azithromycin, doxycycline and the combination of clarithromycin with rifampin. Clarithromycin, clarithromycin with rifampin, and azithromycin were predicted to achieve concentrations > MPC in BAL cells with %T > MPC of 100%. However, only clarithromycin and clarithromycin in combination with rifampin were predicted to achieve a %T > MPC of 100% in PELF (Table 2). 4. Discussion Several studies have investigated the MIC of various drugs against R. equi. However, traditional MIC testing is not a reliable measure of the relative tendency of antimicrobial agents to enrich resistant mutant subpopulations. The frequency of occurrence of mutations leading to antimicrobial resistance is typically in the order of 1  10 7 to 1  10 9 (Martinez and Baquero, 2000), a frequency that would not be detected with traditional susceptibility testing in which an inoculum of 105 CFU is used. Bacterial load in the lung of foals infected with R. equi may range between 106 and 1010 CFU/g of lung tissue depending on the severity of the lesions (Gigue`re et al., 1999; Hooper-McGrevy et al., 2003; Jacks et al., 2007). Consequently, an isolate deemed susceptible might contain an undetected subpopulation of resistant bacteria that would require a higher drug concentration to restrict growth. In practical terms, the MPC is a measure of the MIC using an inoculum size of sufficient magnitude to make resistant subpopulations likely to be present (Blondeau, 2009). Clarithromycin had the lowest MPC of all the antimicrobial agents evaluated in the present study. Conversely, rifampin had the highest MPC and the highest MPC/MIC ratio, indicating a poor ability to prevent the emergence of resistant mutants. These in vitro results corroborate the results of in vivo studies in which the use of rifampin in monotherapy for the treatment of R. equi pneumonia in foals has resulted in resistance (Fines et al., 2001; Takai et al., 1997). The present study used methods established by the guidelines established by the Clinical and Laboratory Standard Institute for determination of the MIC. A recent publication demonstrated that addition of 2% (vol/vol) lysed horse blood to the cation-adjusted Mueller Hinton broth facilitates the discrimination between growth and no growth (Riesenberg et al., 2013). Lysed horse blood was not used in the present study because this information was not available at the time the study was conducted. Traditionally, pharmacodynamic parameters based on MIC have been use to determine appropriate dosing regimens for antimicrobial agents. However, an overwhelming body of data supports the use of MPC and MSW instead of MIC to optimize dose and dosing intervals (Drlica and Zhao, 2007; Epstein et al., 2004). In vitro kinetic models have shown that a plasma AUC24/MPC ratio of 22– 35 prevents first step fluoroquinolones resistance in Escherichia coli (Olofsson et al., 2006, 2007). There are also in vivo data supporting the MSW theory and the use of MPC instead of MIC in pharmacodynamic studies (Croisier et al., 2004; Cui et al., 2006; Etienne et al., 2004). For

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example, a plasma AUC24/MIC ratio of 18–25 prevented enrichment of levofloxacin resistant S. aureus in a tissuecage infection model in rabbits (Cui et al., 2006). The relationship between AUC24/MPC, %T > MPC, and TMSW and resistance selection is complex and varies depending on the methodology used and the microorganism/drug combination studied. Additional studies will be necessary to determine the pharmacodynamic variables that best prevent development of resistant mutants in R. equi. Based on a consensus statement by the American College of Veterinary Internal Medicine, the combination of a macrolide (erythromycin, azithromycin, or clarithromycin) with rifampin is the recommended treatment for infection caused by R. equi in foals (Gigue`re et al., 2011). However, none of the recommended antimicrobial agents would be predicted to reach concentrations in plasma above the MPC for R. equi when used at recommended dosages. Plasma concentrations of macrolides or azalides are in general considerably lower than their respective MIC against the pathogens for which they are known to be effective, suggesting that drug concentrations at the site of infection provides more clinically relevant information than simple reliance on plasma concentrations (Drusano, 2005). Therefore, pulmonary pharmacokinetic data might be more appropriate to compute AUC24/MPC, %T > MPC, and TMSW for infections caused by R. equi. Only clarithromycin was predicted to achieve %T > MPC of 100% in both BAL cells and PELF. Erythromycin concentrations in BAL cells and azithromycin concentrations in PELF were predicted to be in the MSW for the entire dosing interval. In people, the MSW hypothesis predicts that azithromycin would be more likely to select for Streptococcus pneumoniae resistance than clarithromycin (Metzler et al., 2013). This is also supported by epidemiological data and field studies demonstrating that resistance is more strongly associated with the use of azithromycin compared to that of clarithromycin or erythromycin (Blondeau, 2005; Kastner and Guggenbichler, 2001). Currently, the impact of specific macrolide compounds in the selection of resistant R. equi mutants in infected foals is unknown. The use of a macrolide in combination with rifampin for the treatment of pneumonia caused by R. equi in foals was initially based on in vitro studies demonstrating synergism of the combination (Gigue`re et al., 2012; Nordmann and Ronco, 1992; Prescott and Nicholson, 1984). The present study demonstrates that the combination offers the additional advantage of significantly reducing emergence of resistant mutants. This is expected because resistance to a drug combination requires a rare double mutation (Zhao and Drlica, 2002). Recent studies indicate that oral absorption of clarithromycin is considerably decreased by co-administration of rifampin (Peters et al., 2011, 2012). The present study demonstrate that, despite lower clarithromycin concentrations, co-administration of clarithromycin with rifampin would not be predicted to negatively impact AUC24/MPC and %T > MPC as a result of the much lower MPC of the combination. The effects of coadministration of rifampin on the pharmacokinetics of erythromycin or azithromycin have not been studied in foals. For a combination to be most effective, both drugs

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