Rhodococcus equi, a gram-positive facultative intracellular pathogen that replicates in macrophages, is one of the most important causes of pneumonia in 3-week- to 5-month-old foals.1 Other, less common clinical manifestations of R equi infection in foals include ulcerative enterocolitis, cecocolonic or mesenteric lymphadenitis, immune-mediated synovitis, immune-mediated uveitis, osteomyelitis, and septic arthritis.1,2 Rhodococcus equi has also emerged as an opportunistic pathogen in immunosuppressed humans, especially those infected with HIV.3–5 Unlike most R equi isolated from the environment, isolates collected from pneumonic foals typically contain an 80- to 90-kilobase pair plasmid that encodes for a family of 8 closely related s (ie, vapA and vapC through vapl).6 Plasmid-cured derivatives of virulent R equi strains do not replicate or survive in macrophages and fail to induce pneumonia in foals, and infections attributable to these strains are completely cleared from the lungs of foals. Thus, the presence of the plasmid encoding for vaps is essential for the virulence of R equi isolates.7,8
A variety of antimicrobials are active against R equi when evaluated in vitro.9,10 However, many of these antimicrobials are ineffective in vivo. In 1 study,11 all 17 foals treated with a combination of penicillin and gentamicin for pneumonia caused by R equi died despite results of in vitro antimicrobial susceptibility testing that indicated all R equi isolates were susceptible to gentamicin. The combined administration of erythromycin and rifampin became the standard treatment used by veterinarians for R equi infection in foals during the 1980s; foal mortality rates have decreased dramatically since this standard treatment was instated.11,12 More recently, treatment with erythromycin commonly has been replaced by treatment with clarithromycin or azithromycin.13
Even though most R equi isolates collected from foals are susceptible in vitro to macrolide antimicrobials (ie, azithromycin, clarithromycin, and erythromycin) and rifampin, there are reports14–16 of foals infected with rifampin-resistant isolates. Progressive development of antimicrobial resistance to both erythromycin and rifampin has been reported15 in 1 foal during treatment of pneumonia caused by R equi. However, the prevalence of antimicrobial resistance to macrolide antimicrobials or rifampin and the effect of antimicrobial resistance on the outcome of antimicrobial treatment of infected foals are unknown. Treatment of foals infected with resistant isolates of R equi is problematic because of the limited selection of effective antimicrobial alternatives. The purpose of the study reported here was to determine the prevalence of R equi isolates resistant in vitro to azithromycin, erythromycin, clarithromycin, or rifampin; to determine whether R equi isolates resistant to azithromycin, erythromycin, clarithromycin, or rifampin contained the virulence plasmid; to determine the MIC for azithromycin, clarithromycin, clindamycin, erythromycin, and rifampin in resistant isolates of R equi; and to compare the outcome of antimicrobial treatment in foals infected with macrolide- or rifampin-resistant isolates of R equi with the outcome of antimicrobial treatment in foals infected with susceptible isolates of R equi.
Materials and Methods
Collection of resistant R equi isolates—Rhodococcus equi isolates were categorized as having intermediate susceptibility or as resistant to macrolide antimicrobials (ie, azithromycin, clarithromycin, or erythromycin) or rifampin by participating veterinary clinical diagnostic laboratories and stored as frozen stabilates at −80°C. In vitro antimicrobial susceptibility testing was performed in accordance with CLSI guidelines17 by use of a disk diffusion test or a broth microdilution assay. The MIC break-points18 used for gram-positive bacteria for the evaluation of intermediate susceptibility or resistance were > 2 μg/mL (azithromycin and clarithromycin), > 0.5 μg/mL (erythromycin), and > 1 μg/mL (rifampin).
Evaluation of MIC for azithromycin, clarithromycin, clindamycin, erythromycin, and rifampin—Prior to testing, frozen bacterial stabilates were thawed, subcultured, evaluated for purity of the bacterial sample, and identified by use of standard identification procedures.17 The MIC values of azithromycin, clarithromycin, clindamycin, erythromycin, and rifampin were determined by use of a concentration-gradient test.a The test was performed as described by the manufacturer and in accordance with CLSI guidelines.17,18 Briefly, R equi isolates were inoculated and grown on blood agar plates. Representative bacterial colonies were suspended in sterile water to achieve turbidity equal to that of a 0.5 McFarland standard (final bacterial concentration, approx 1 × 105 colony-forming units/mL). A sterile swab was dipped into the bacterial suspension and used to inoculate the entire surface of 100-mm Mueller-Hinton agar plates. Inoculation of each plate was performed 3 times by rotating the plate approximately 60° prior to the second and third inoculation to ensure an even distribution of bacteria across the surface of the agar plate. After allowing excess moisture to evaporate for approximately 10 to 15 minutes, antimicrobial test strips were applied to the agar plates; antimicrobial concentrations between 0.016 and 256 μg/mL (azithromycin, clarithromycin, clindamycin, and erythromycin) or between 0.002 and 32 μg/mL (rifampin) were used. Plates were incubated for 18 to 24 hours at 37°C. A test was considered valid only when adequate growth was observed on the plate. Bacterial strains used for assay validation each time an isolate was evaluated were Staphylococcus aureus (ATCC 29213; azithromycin, clarithromycin, clindamycin, erythromycin, and rifampin), Streptococcus pneumoniae (ATCC 49619; azithromycin, clarithromycin, clindamycin, erythromycin, and rifampin), and Enterococcus faecalis (ATCC 29212; rifampin). Results were considered valid only when the MIC values recorded for the control strains were included within the reported17,18 reference range of MIC values. Additionally, an R equi isolate (ATCC 33701) known to be susceptible to azithromycin, clarithromycin, erythromycin, and rifampin was used as a control isolate.
In accordance with CLSI guidelines,17,18 isolates were categorized as susceptible (< 2 μg/mL [azithromycin and clarithromycin], < 0.5 μg/mL [clindamycin and erythromycin], and < 1 μg/mL [rifampin]) or resistant (> 8 μg/mL [azithromycin, clarithromycin, and erythromycin] and > 4 μg/mL [clindamycin and rifampin]). Isolates with recorded MIC values between the aforementioned concentrations were categorized as intermediate susceptibility. For each isolate, MIC was determined 3 times on 3 separate days, and the median MIC value was reported for comparison.
In vitro antimicrobial susceptibility testing of macrolide- or rifampin-resistant R equi isolates against other antimicrobial agents—For isolates determined to be resistant to macrolide antimicrobials or rifampin, in vitro antimicrobial susceptibility testing of macrolide- or rifampin-resistant R equi isolates against chloramphenicol, ciprofloxacin, linezolid, moxifloxacin, tetracycline, vancomycin, gentamicin, imipenem, and trimethoprim-sulfamethoxazole was assessed via a broth microdilution assay.b In addition to the aforementioned strains, quality control of the assay was assessed through the inclusion of Pseudomonas aeruginosa (ATCC 27853) and 2 strains of Escherichia coli (ATCC 25922 and ATCC 35218). Results of in vitro antimicrobial susceptibility testing were categorized as susceptible, intermediate, or resistant on the basis of interpretive MIC breakpoints recommended by CLSI guidelines.18
PCR amplification of the vapA gene—Frozen stabilates were thawed, subcultured, and evaluated for purity of the bacterial sample. Each R equi isolate was cultured overnight at 37°C in 500 μL of trypticase soy broth. Then, cultures were centrifuged at 5,000 X g for 10 minutes. Bacterial DNA was extracted by use of a kitc in accordance with the manufacturer's instructions for gram-positive bacteria. Oligonucleotide primers specific for the vapA gene were designed by use of a computer software system.d The primer sequences were 5′-GTTCTTGATTCCGGTAG-CAGCAGTG-3′ (forward) and 5′-CCGCGCATCTTC-GATGTCTACTAAC-3′ (reverse). Amplification via a PCR assay for the vapA gene was performed in each bacterial isolate categorized as resistant to azithromycin, clarithromycin, erythromycin, or rifampin. The virulent R equi strain (ie, ATCC 33701+) containing the 80-kb plasmid encoding the vapA gene and its avirulent plasmid-cured derivative (ie, ATCC 33701−) were used as positive and negative control isolates, respectively, for monitoring assay performance. Two microliters of extracted-DNA sample was amplified in a 50-μL PCR assay reaction containing 50 pmol of each oligonucleotide primer, 0.2mM of each deoxyribonucleotide, 5 μL of 10X reaction buffer (containing 10mM Tris-HCl buffer [pH, 8.3] and 50mM KCl), 1.5mM MgCl2, and 2 U of Taq DNA polymerase.e The PCR assay reaction was performed with an initial cycle at 95°C for 10 minutes; 30 cycles of denaturation at 94°C for 45 seconds, annealing at 60°C for 45 seconds, and extension at 72°C for 2 minutes; and extension at 72°C for 7 minutes. Ten microliters of amplified PCR product was separated by electrophoresis on a 1.6% agarose gel and stained with ethidium bromide. Samples that did not contain DNA were included for quality control purposes to monitor for DNA contamination during the processing of the PCR assay reactions. The specificity of the amplified band was confirmed on the basis of detection of a single band of the predicted size (ie, 582 base pairs), in comparison with a band of a similar molecular weight standard.
Determination of prevalence of resistant R equi isolates in Florida and Texas—Data were collected between January 1997 and December 2008. The prevalence of resistant isolates in Florida was estimated by analysis of data from all R equi isolates submitted to a veterinary diagnostic laboratoryf; submissions received by the laboratory were collected from horses located throughout Florida and occasionally from southern Georgia. The prevalence of resistant isolates in Texas was estimated by analysis of data from all R equi isolates submitted to 2 veterinary diagnostic laboratoriesg,h; submissions received were collected from horses located throughout the state of Texas and occasionally from nearby states (ie, New Mexico and Oklahoma). The prevalence of resistant isolates from Florida and Texas was calculated as the number of R equi isolates resistant to azithromycin, clarithromycin, erythromycin, or rifampin, divided by the total number of R equi isolates tested.
Monitoring of antimicrobial treatment outcome in R equi-infected foals—Data were collected for foals admitted to the University of Florida Veterinary Medical Center between January 1997 and December 2007. All foals were treated with a combination of a macrolide antimicrobial (azithromycin, clarithromycin, or erythromycin) and rifampin. A comparison was made between the survival rate of pneumonic foals infected with macrolide-antimicrobial- or rifampin-resistant isolates of R equi and that of pneumonic foals infected with susceptible isolates of R equi.
Statistical analysis—All statistical analyses were performed by use of a commercial software package.i Normality of the data and equality of variances were assessed by use of the Kolmogorov-Smirnov and Levene tests, respectively. Data were not normally distributed despite attempts at transformation. Differences in MIC results between azithromycin, clarithromycin, and erythromycin were assessed by use of the Friedman repeated-measures ANOVA on ranks. Multiple pairwise comparisons were accomplished by use of the Tukey test. Comparisons between the proportion of resistant isolates received before 2002 and after 2002 were made by use of the Fisher exact test. Additionally, comparisons between the proportion of resistant isolates of R equi in Florida versus resistant isolates of R equi in Texas and the survival rate of foals infected with resistant isolates versus that of foals infected with susceptible isolates of R equi were made by use of the Fisher exact test. A value of P < 0.05 was considered significant for all analyses.
Results
Characterization of R equi isolates—Thirty-eight R equi isolates originating from 9 diagnostic laboratories located in Florida (n = 18 isolates), Texas (8), Illinois (5), California (4), and Kentucky (3) were collected between January 1997 and August 2008. The isolates were cultured from samples obtained from tracheobronchial fluid aspirates (n = 30), synovial fluid (1), abdominal abscess aspirate (1), or lung tissues during necropsy (6). Resistance was determined at the laboratory of origin by use of disk diffusion (n = 6 laboratories) or broth microdilution assays (3) for determination of MIC results.
Of the 38 isolates received, 1 was not an R equi isolate and 1 was an R equi isolate contaminated with another bacteria. The contaminating bacteria was considered to cause the observed antimicrobial resistance because the R equi isolate in that frozen stabilate was susceptible to macrolide antimicrobials and rifampin when evaluated as a pure bacterial culture. Additionally, 12 R equi isolates thought to be resistant or of intermediate susceptibility to azithromycin, clarithromycin, erythromycin, or rifampin in the laboratory of origin were determined to be susceptible to these drugs via the concentration-gradient test.a
Twenty-four isolates were determined to be resistant to at least 1 of the antimicrobials tested. The distribution of these 24 isolates by year was evaluated (Figure 1). The proportion of resistant isolates received after 2002 (17/24 [70.8%] isolates) was significantly (P = 0.009) greater, compared with the proportion of resistant isolates received between 1997 and 2002 (7/24 [29.2%] isolates). Furthermore, 2 of 24 (8.3%) isolates were resistant only to rifampin and 22 of 24 (91.7%) isolates were resistant to azithromycin, clarithromycin, erythromycin, and rifampin. Of the 22 macrolide-resistant isolates, 19 were also resistant to clindamycin. For 4 macrolide-resistant isolates, a distinct large ellipse containing multiple isolated colonies was observed. Subculture of these isolated colonies revealed a subpopulation of R equi isolates that were highly resistant to azithromycin, clarithromycin, erythromycin, and rifampin. Of the 24 isolates resistant to at least 1 drug, 23 (95.8%) were determined to be virulent R equi on the basis of a detectable product from amplification of the vapA gene by the PCR assay. In 1 instance, the predominant R equi isolate susceptible to azithromycin, clarithromycin, erythromycin, and rifampin was virulent, whereas the resistant isolate was avirulent. Among the 22 macrolide-resistant isolates, median MIC of azithromycin was significantly (P < 0.001) greater, compared with the MIC for clarithromycin or erythromycin (Table 1).
Minimum inhibitory concentrations of azithromycin, clarithromycin, erythromycin, clindamycin, and rifampin for 22 Rhodococcus equi isolates determined to be resistant to macrolide antimicrobials.
| Antimicrobial | MIC (μg/mL) | |
|---|---|---|
| Median | Range | |
| Azithromycin | ≥ 256* | 24 to ≥ 256 |
| Clarithromycin | 28 | 8 to ≥ 256 |
| Erythromycin | 24 | 12 to ≥ 256 |
| Rifampin | ≥ 32 | ≥ 32 |
| Clindamycin | 24 | 2 to ≥ 256 |
The MIC of azithromycin was significantly (P < 0.001) greater than that of clarithromycin and erythromycin.
Distribution of 24 macrolide- or rifampin-resistant Rhodococcus equi isolate submissions received between January 1997 and December 2008.
Citation: Journal of the American Veterinary Medical Association 237, 1; 10.2460/javma.237.1.74
Distribution of 24 macrolide- or rifampin-resistant Rhodococcus equi isolate submissions received between January 1997 and December 2008.
Citation: Journal of the American Veterinary Medical Association 237, 1; 10.2460/javma.237.1.74
Distribution of 24 macrolide- or rifampin-resistant Rhodococcus equi isolate submissions received between January 1997 and December 2008.
Citation: Journal of the American Veterinary Medical Association 237, 1; 10.2460/javma.237.1.74
In vitro antimicrobial susceptibility testing of macrolide- or rifampin-resistant R equi isolates against other antimicrobial agents—In an attempt to identify adequate alternatives for the treatment of foals infected with macrolide- or rifampin-resistant R equi isolates, we evaluated in vitro antimicrobial susceptibility of these isolates to 11 antimicrobial agents. All isolates were susceptible to ciprofloxacin, gentamicin, imipenem, linezolid, moxifloxacin, and vancomycin (Table 2). Eighteen of 24 (75.0%) macrolide- or rifampin-resis-tant R equi isolates were susceptible to chloramphenicol, tetracycline, and trimethoprim-sulfamethoxazole.
Results of MIC* determination for selected antimicrobials against 24 isolates of R equi determined to be resistant to macrolide antimicrobials (ie, azithromycin, clarithromycin, or erythromycin) or rifampin.
| Antimicrobial | Susceptible† | Intermediate† | Resistant† |
|---|---|---|---|
| Chloramphenicol | 18 (≤ 8) | 6 (6) | NA |
| Ciprofloxacin | 24 (≤ 1) | NA | NA |
| Gentamicin | 24 (≤ 4) | NA | NA |
| Imipenem | 24 (≤ 4) | NA | NA |
| Linezolid | 24 (≤ 4) | NA | NA |
| Moxifloxacin | 24 (≤ 4) | NA | NA |
| Tetracycline | 18 (≤ 4) | NA | 6 (≥ 16) |
| Trimethoprim-sulfamethoxazole‡ | 20 (≤ 2/38) | NA | 4 (≥ 4/76) |
| Vancomycin | 24 (≤ 4) | NA | NA |
Results are reported as the number of R equi isolates (MIC in μg/mL).
The MIC was determined 3 times on 3 separate days by use of a broth microdilution assayb for each R equi isolate.
In accordance with CLSI guidelines,17,18 the R equi isolates were categorized as susceptible (≤ 2 μg/mL [azithromycin and clarithromycin], ≤ 0.5 μg/mL [clindamycin and erythromycin], and ≤ 1 μg/mL [rifampin]) or resistant (≥ 8 μg/mL [azithromycin, clarithromycin, and erythromycin] and ≥ 4 μg/mL [clindamycin and rifampin]).
‡Minimum inhibitory concentration is for each antimicrobial (ie, MIC for trimethoprim/MIC for sulfamethoxazole).
NA = Not applicable.
Prevalence of resistant R equi isolates in Florida and Texas—The proportion of R equi isolates resistant to azithromycin, clarithromycin, erythromycin, or rifampin in Florida (8/160 [5.0%]) was not significantly (P = 0.248) different, compared with the proportion of resistant R equi isolates in Texas (4/168 [2.4%]). The overall prevalence of resistant isolates was 3.7% (12/328).
Antimicrobial treatment outcome in R equi-infected foals—Of the 24 foals infected with resistant R equi isolates, antimicrobial treatment outcome was available for 19, and only 7 (36.8%) of these foals survived (Table 3). The proportion (2/8 [25.0%]) of foals infected with macrolide- or rifampin-resistant R equi isolates that survived was significantly (P = 0.004) less, compared with the proportion (55/79 [69.6%]) of foals infected with susceptible R equi isolates treated in the same hospital that survived. Odds of nonsurvival for foals infected with resistant R equi isolates were 6.9 times (95% confidence interval, 1.3 to 37; P = 0.023) as high as the odds for survival for foals infected with susceptible R equi isolates.
Results of in vitro antimicrobial susceptibility testing, antimicrobial treatment administered, and antimicrobial treatment outcome in 19 foals infected with R equi isolates resistant to macrolide antimicrobials (ie, azithromycin, clarithromycin, or erythromycin) or rifampin.
| Foal | Antimicrobial resistance* | Antimicrobial treatment administered† | Treatment outcome‡ |
|---|---|---|---|
| 1 | M and R | D + R → C + R → V + G | Dead |
| 2 | M, R, and CL | D + R → C + R → V + C + R | Survived |
| 3 | M, R, and CL | D + R → C + R → V | Survived |
| 4 | M, R, and C | A→C + R →V | Dead |
| 5 | R | A + R | Survived |
| 6 | M, R, and CL | A + R | Dead |
| 7 | M, R, and CL | A + R → C + R | Survived |
| 8 | M, R, and CL | NA | Dead |
| 9 | R | C + R | Dead |
| 10 | M, R, and CL | NA | Dead |
| 11 | M, R, and CL | C + R | Dead |
| 12 | M and R | C + R | Dead |
| 13 | M, R, and CL | A → V | Survived |
| 14 | M, R, and CL | C + R → D → V | Dead |
| 15 | M, R, and CL | C + R → D + R | Survived |
| 16 | M, R, and CL | NA | Dead |
| 17 | M, R, and CL | A + R → C + R → D + R | Dead |
| 18 | M, R, and CL | A + R | Survived |
| 19 | M, R, and CL | NA | Dead |
Antimicrobial resistance was determined 3 times on 3 separate days by the results of a concentration-gradient test.a
Antimicrobials separated by a plus sign indicate a combination of antimicrobial treatments administered, whereas antimicrobials separated by an arrow indicate a change in antimicrobial treatment administered.
Dead foals died or were euthanized because of marked clinical deterioration despite antimicrobial treatment.
A = Azithromycin. C = Clarithromycin. CL = Clindamycin. D = Doxycycline. G = Gentamicin. M = Macrolide antimicrobials. NA = Not available. R = Rifampin. V = Vancomycin.
Discussion
To our knowledge, the study reported here is the first to describe the prevalence of resistance to macrolide antimicrobials or rifampin in a population of R equi isolates collected from R equi-infected foals and to document the negative impact of macrolide- or rifampin-resistant R equi isolates on outcome for antimicrobial treatment. Rhodococcus equi is a member of the mycolata taxon, which is a suprageneric taxon that includes the extensively studied facultative intracellular pathogen Mycobacterium tuberculosis. The emergence of multidrug-resistant M tuberculosis (defined as antimicrobial resistance to rifampin and isoniazid) is considered a public health concern.19 In 2004, the prevalence of new multidrug-resistant M tuberculosis was 4%.20 This is similar to the prevalence of macrolide- or rifampin-resistant R equi isolates in the study reported here. This is alarming and of great clinical relevance because the treatment of R equi infection in foals with a combination of a macrolide (ie, azithromycin, erythromycin, or clarithromycin) and rifampin has been the standard treatment for decades. Furthermore, many other antimicrobial drugs are clinically less effective despite adequate in vitro antimicrobial susceptibility testing results.
Resistance to rifampin has been described in 5 R equi-infected foals.14–16 In at least 2 of these foals, antimicrobial resistance developed during treatment with only rifampin.14–16 In 1 study,16 640 isolates collected from lung lesions of 64 R equi-infected foals and 98 R equi isolates collected from the soil in the local environments were tested for antimicrobial resistance to rifampin. Isolates from only 1 of the 64 (1.6%) foals were resistant to rifampin.16 As described14 for other bacteria, antimicrobial resistance to rifampin in R equi isolates is caused by a single base mutation in the rpoB cluster I region, and the degree of antimicrobial resistance is dependent on both the location and the nature of the base substitution. All 24 rifampin-resistant isolates in the study reported here had a high degree of antimicrobial resistance (MIC > 32 μg/mL).
Rhodococcus equi isolates that are resistant to macrolide antimicrobials and rifampin have been reported21,22 in infected humans. To our knowledge, only 1 report15 of progressive development of resistance to both a macrolide and rifampin during treatment in a foal exits. In the study reported here, 22 macrolide-resistant R equi isolates were identified. Additionally, all 22 isolates were rifampin resistant. There is no known mechanism of cross-resistance between macrolide and rifamycin antimicrobials. However, the development of antimicrobial resistance to rifampin is relatively rapid when used in monotherapy treatment regimens. The in vitro frequency of selection of R equi isolates that are resistant to rifampin is 1 in 107 or 108 bacteria.14,23 By use of similar methods for selection of resistant bacteria, erythromycin-resistant R equi isolates could not be generated, and the combination of erythromycin-rifampin abolished the in vitro emergence of rifampin-resistant R equi mutants.23 The results of these in vitro studies14,23 and the fact that macrolide-resistant and rifampin-susceptible R equi isolates were not encountered suggest that macrolide-resistant isolates rapidly develop antimicrobial resistance to rifampin.
The 2 primary mechanisms responsible for antimicrobial resistance to the action of macrolide antimicrobials are the modification of the ribosomal target site by methylation and an active efflux pump.24 Methylation of the ribosome results in antimicrobial cross-resistance between macrolide antimicrobials, lincosamides, and streptogramin B (ie, MSLB resistance).24 In the study reported here, 19 of 22 macrolide-resistant R equi isolates were also resistant to clindamycin. This result may indicate the acquisition of ribosomal RNA methylases. However, this finding must be interpreted cautiously because many macrolide-susceptible R equi isolates are intrinsically resistant to clindamycin.10 Additional studies will be required to determine the mechanisms of resistance to macrolide antimicrobials in R equi isolates. As reported25,26 for other gram-positive bacteria, all R equi isolates determined to be resistant to azithromycin, clarithromycin, or erythromycin in the study reported here were invariably resistant to the other 2 of these 3 macrolide antimicrobials as well.
Many foals were already being treated with antimicrobials at the time of microbiological culture, and antimicrobial treatment history was unavailable for many other foals in the study reported here. Therefore, it is not possible to determine whether any given foal was initially infected with a resistant R equi isolate or whether antimicrobial resistance developed during antimicrobial treatment. The fact that some foals were being treated with antimicrobials at the time of sample collection is another factor for the introduction of bias toward a falsely elevated estimate of the prevalence of antimicrobial resistance. Pneumonia in foals caused by R equi infection is commonly the result of simultaneous infection with multiple genetically distinct bacterial isolates.27 Additionally, foals are commonly exposed to avirulent R equi isolates that predominate in the environment where the disease is endemic.28,29 In 1 foal from which a rifampin-resistant isolate was cultured from the lungs, 11 of 20 (55.0%) R equi isolates cultured from the intestine were resistant to rifampin, and 5 of the 11 (45.5%) were avirulent R equi isolates.16 Collectively, the findings described in that study16 may lead to the hypothesis that the recovery of resistant R equi isolates from an infected foal may have represented an inconsequential subpopulation of the total R equi bacterial load or that the resistant R equi isolate may have been avirulent. In the study reported here, concurrent infection with susceptible and resistant isolates of R equi was identified in 4 foals. Furthermore, 23 of 24 (95.8%) macrolide- or rifampin-resistant R equi isolates were determined to be virulent on the basis of results of amplification of the vapA gene by the PCR assay. However, in 1 foal, the predominant virulent isolate of R equi was susceptible to macrolide antimicrobials and rifampin, although the macrolide- and rifampin-resistant subpopulation was avirulent. Additionally, the significantly decreased survival rate of foals infected with resistant isolates of R equi underscores the clinical relevance of antimicrobial resistance to macrolide antimicrobials and rifampin. Similarly, isolation of multidrug-resistant or extensively drug-resistant isolates of M tuberculosis have been associated with decreased survival rates in infected humans.19,30–32
All 24 macrolide- or rifampin-resistant R equi isolates identified in the study reported here were susceptible in vitro to ciprofloxacin, gentamicin, imipenem, linezolid, moxifloxacin, and vancomycin, and at least 18 of 24 (75.0%) isolates were susceptible to tetracycline, chloramphenicol, and trimethoprim-sulfamethoxazole. However, in vitro antimicrobial susceptibility for isolates of R equi does not guarantee a favorable clinical outcome after antimicrobial treatment. In 1 study,11 17 foals that had pneumonia caused by an R equi infection and were treated with gentamicin died despite the fact that all isolates were susceptible in vitro to gentamicin. Although there are isolated reports of successful treatment of humans infected with R equi with the aforementioned antimicrobials alone or in combination, there are no objective results of studies that indicate an advantage of one treatment regimen over another. Ciprofloxacin, moxifloxacin, imipenem, linezolid, and vancomycin are antimicrobials rarely used in horses; to our knowledge, there are no data indicating that these drugs are effective in the treatment of bacterial infections caused by R equi in foals. Orally administered ciprofloxacin is poorly absorbed in horses,33 and the use of fluoroquinolones in foals has led to articular cartilage damage.34
The median MIC of clarithromycin for macrolide-resistant R equi isolates in the study reported here (28 μg/mL) is considerably greater than the achievable peak plasma concentration (0.88 μg/mL), but lower than reportedly achievable peak concentrations in pulmonary epithelial lining fluid (76 μg/mL) and bronchoalveolar cells (269 μg/mL) of foals.35 The apparent preferential activity of clarithromycin into the lungs has been reported36 in mice infected by use of isolates of S pneumoniae with efflux-mediated antimicrobial resistance to macrolide antimicrobials; a consistent bacterial kill was observed in lung tissue but not in thigh tissue. These differences in antimicrobial susceptibility between tissue sites were explained by greater drug concentrations in the fluid of the pulmonary epithelial lining than in serum.36 However, 6 foals infected with macrolide-resistant isolates of R equi and treated with a clarithromycin-rifampin treatment regimen in the study reported here failed to respond to treatment and eventually died or were euthanatized. This suggests that clarithromycin should not be selected for the treatment of most foals infected with macrolide-resistant R equi isolates despite intracellular clarithromycin concentrations in the lungs being greater than that of the MIC of many resistant R equi isolates. Because of the limited number of foals infected with a resistant R equi isolate, the study reported here does not permit any conclusions to be made regarding the best antimicrobial or antimicrobial combinations for treatment of foals infected with macrolide- and rifampin-resistant R equi isolates.
Fourteen of 38 (36.8%) isolates originally categorized by veterinary diagnostic laboratories as of intermediate susceptibility or resistant were found to be susceptible to all 4 antimicrobials in the present study. The reason or reasons for this discrepancy could not be evaluated via the current study design. It was likely that this rate of error was artificially increased because only isolates categorized as resistant by the laboratory of origin were retested. There may have been hundreds of isolates correctly categorized as susceptible by each veterinary diagnostic laboratory. Nevertheless, these findings confirm that errors in laboratory results are possible, and testing should be repeated before changing the antimicrobial treatment administered when a resistant R equi isolate is identified. This is particularly important in the event a veterinarian may be considering the use of antimicrobials typically administered only to treat life-threatening infections in humans (ie, vancomycin, linezolid, or imipenem). In horses, these drugs should be used only for the treatment of life-threatening R equi infections caused by isolates confirmed to be resistant to all other antimicrobial alternatives.37
Analysis of the distribution of resistant R equi isolates collected between 1997 and 2008 suggests that the prevalence of antimicrobial resistance to macrolide antimicrobials or rifampin is increasing. However, our results must be interpreted cautiously because the submissions received from veterinary diagnostic laboratories were solicited on a voluntary basis. The increase in the number of resistant R equi isolates received in more recent years may simply indicate an increased awareness of our interest in collecting resistant isolates. The increase in the number of resistant R equi isolates may also be the result of an increase in the total number of sample submissions received by diagnostic laboratories in more recent years. Unfortunately, the total number of sample submissions received for all veterinary diagnostic laboratories included in the present study was not available for analysis. Additionally, the pattern of sample submissions received may have changed over the study period reported here. More veterinarians may presumptively diagnose pneumonia caused by R equi on the basis of ultrasonographic appearance of lung lesions, rather than on the basis of results of microbiological culture; as a result, they may only perform microbiological culture of tracheobronchial fluid aspirates obtained from a foal refractory to antimicrobial treatment. Prevalence results must be interpreted with regard to the reference population versus foals with clinical signs of pneumonia for which R equi was isolated from microbiological culture of a tracheobronchial fluid aspirate. Because pneumonia caused by R equi may be presumptively diagnosed, it is possible that our results overestimate the prevalence of antimicrobial resistance, assuming that foals with a presumptive diagnosis that were truly infected with R equi would have been less likely to be infected with a resistant strain than foals for which culture was performed.
Screening of foals for early recognition of pneumonia caused by R equi prior to the development of clinical signs and appropriate antimicrobial treatment of affected foals has been increasingly used in an attempt to reduce foal death and limit the treatment costs associated with long-term antimicrobial treatment of severely affected foals.38,39 Similarly, prophylactic administration of antimicrobials in the neonatal period has been attempted in an effort to prevent pneumonia caused by R equi.40,41 A disadvantage of both screening and prophylactic use is that many foals that would not have developed clinical disease are administered antimicrobials, and these practices result in an overall increase in the administration of antimicrobials. In 1 study,40 no evidence of antimicrobial resistance to azithromycin was detected after prophylactic administration of azithromycin over a single breeding season. As the authors of that report40 described, the duration of treatment and sample size used for analysis in that study were limited in scope. Thus, the long-term effects of the prophylactic use of macrolide antimicrobials or rifampin on the development of antimicrobial resistance in R equi are unknown.
In conclusion, the results of the study reported here indicate that infection with R equi isolates resistant to macrolide antimicrobials or rifampin is more common than previously thought, and foals infected with resistant isolates are less likely to survive. These results emphasize the importance of microbiological culture and in vitro antimicrobial susceptibility testing in foals with pneumonia presumptively thought to be caused by R equi infection.
ABBREVIATIONS
| ATCC | American Type Culture Collection |
| CLSI | Clinical and Laboratory Standards Institute |
| MIC | Minimum inhibitory concentration |
| vap | Virulence-associated protein |
E test, AB bioMerieux, Solna, Sweden.
Microscan System, gram-positive MIC combo, Dade Behring, Sacramento, Calif.
DNeasy kit, Qiagen Inc, Valencia, Calif.
Primer Designer, Scientific & Educational Software, Durham, NC.
AmpliTaq Gold, Applied Biosystems, Foster City, Calif.
Veterinary Medical Center Clinical Diagnostic Laboratory, University of Florida, Gainesville, Fla.
Department of Veterinary Pathobiology, College of Veterinary Medicine, Texas A&M University, College Station, Tex.
Texas Veterinary Medical Diagnostic Laboratory—College Station Laboratory, College Station, Tex.
SigmaStat, version 3.0, SPSS Inc, Chicago, Ill.
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