Isolation of multiple bacterial species is common in foals with Rhodococcus equi pneumonia.
Isolation of multiple bacterial species is common in foals with Rhodococcus equi pneumonia.
There is no association between isolation of other microorganisms and outcome.
155 foals with pneumonia caused by R. equi.
Case records of foals diagnosed with R. equi pneumonia based on culture of the respiratory tract were reviewed at 2 referral hospitals (University of Florida [UF] and Texas A&M University [TAMU]).
R. equi was cultured from a tracheobronchial aspirate (TBA) in 115 foals and from lung tissue in 38 foals. Survival was significantly higher at UF (71%; 70/99) than at TAMU (50%; 28/56). R. equi was significantly more likely to grow in pure cultures from samples obtained from foals at UF (55%; 54/99) than from foals at TAMU (23%; 13/56). Microorganisms cultured with R. equi included Gram-positive bacteria in 40, Gram-negative bacteria in 41, and fungi in 23 foals. The most common bacteria isolated were beta-hemolytic streptococci (n = 26) and Escherichia coli (n = 18). Mixed infections were significantly more likely to be encountered in TBA than in lung tissue. Only foals from which R. equi was cultured from a TBA were included in the analysis for association between mixed infection and outcome. After adjusting for the effect of hospital using multivariate logistic regression, mixed culture, mixed bacterial culture, Gram-positive bacteria, beta-hemolytic streptococci, Gram-negative bacteria, enteric Gram-negative bacteria, nonenteric Gram-negative bacteria, and fungi were not significantly associated with outcome.
Isolation of multiple bacteria or fungi from a TBA along with R. equi does not negatively impact prognosis.
University of Florida
Texas A&M University
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. Other less common clinical manifestations of R. equi infections in foals include ulcerative enterocolitis, colonic or mesenteric lymphadenopathy, polysynovitis, uveitis, osteomyelitis, and septic arthritis. Definitive diagnosis of bronchopneumonia caused by R. equi is based on bacteriologic culture or amplification of the vapA gene using polymerase chain reaction (PCR) from a tracheobronchial aspirate (TBA) obtained from a foal with clinical signs of lower respiratory tract disease, cytological evidence of septic airway inflammation, or radiographic or ultrasonographic evidence of bronchopneumonia. Bacterial culture is preferred over amplification by PCR because it makes identification of other bacterial pathogens and in vitro antimicrobial susceptibility testing of R. equi isolates possible.
Although it is common to isolate a variety of other bacteria from TBA with R. equi, it remains unknown whether the presence of other bacteria or fungi affects outcome. The preferred treatment for foals infected with R. equi is the combination of a macrolide antibiotic (eg, erythromycin, clarithromycin, azithromycin) and rifampin. As macrolides and rifampin have limited activity against common Gram-negative pathogens of horses, many authors have recommended that a 3rd antimicrobial agent be added to the treatment regimen in foals from which Gram-negative bacteria are isolated from a TBA in conjunction with R. equi.[4, 5] However, no data are available to support or refute this recommendation. The trachea is not sterile, and a variety of bacterial or fungal microorganisms can be isolated from TBA collected from healthy horses.[6-8] Thus, it is possible that bacteria isolated with R. equi from a TBA are contaminants or merely colonizing the trachea without contributing to pulmonary pathology.
The objectives of this study were to characterize the bacterial isolates most commonly isolated with R. equi in foals with bronchopneumonia, to determine if isolation of other bacteria or fungi was associated with survival, to determine if the proportion of mixed bacterial infection was different between samples obtained from either TBA or lung tissue at necropsy, and to determine if there was a benefit to adding an antimicrobial agent with a Gram-negative spectrum to the combination of a macrolide and rifampin in foals with both R. equi and at least 1 Gram-negative pathogen isolated from a TBA. It was hypothesized that no association exists between isolation of other microorganisms with R. equi and outcome.
Case records of all the foals admitted to the veterinary medical teaching hospitals at the University of Florida (UF) and at Texas A & M University (TAMU) between January 1997 and July 2006 because of R. equi infection were examined. Foals were included in the study only if the diagnosis of pulmonary infection had been confirmed on the basis of isolation of R. equi from a TBA or from culture of the lungs during postmortem examination.
All TBA at UF were performed using the transcutaneous approach. Briefly, an area over the midcervical area was clipped and surgically prepared. After infiltration with 2% lidocaine, a stab incision was made through the skin. A sterile 12-g trocar was used to puncture the trachea between 2 tracheal rings. A 5-Fr urinary catheter with the tip cut off was passed through the trocar. Approximately 20–30 mL of 0.9% saline was injected and aspirated. Most TBA at TAMU were performed transendoscopically, but some were performed using the transcutaneous approach depending on clinician preferences. For transendoscopic TBA, a 1.5-m flexible endoscope was disinfected with a 3.4% gluteraldehyde solution.1 The endoscope was passed via a nostril and advanced into the trachea to the midcervical area. A double-guarded catheter2 was passed through the biopsy channel of the endoscope and TBA was performed according to the instructions provided by the manufacturer. A volume of 20–30 mL of 0.9% saline was used.
Samples of TBA fluid and lung tissue were submitted for aerobic bacterial culture. It was standard practice at both UF and TAMU to report growth of fungi on bacterial media. Information obtained from the medical records included culture source (lung tissue or TBA aspirate), culture results, antimicrobial agents administered, and outcome (survival versus death or euthanasia). Survival was defined as discharge from the hospital.
For data analysis, bacterial isolates other than R. equi were grouped as Gram-positives or Gram-negatives. Gram-positive isolates were further subdivided into beta-hemolytic streptococci (mainly Streptococcus equi subspecies zooepidemicus). Gram-negative isolates were further subdivided into enteric or nonenteric. Foals that received antimicrobial agents with Gram-negative spectrum in addition to the combination of a macrolide and rifampin were grouped in a category named other antimicrobial agents used. Several foals were given metronidazole, presumably in an attempt to prevent diarrhea associated with the use of macrolides. Foals treated with metronidazole were not considered in the other antimicrobial agent category because of the lack of activity of the drug against aerobic Gram-positive and Gram-negative bacteria. Simple proportions were compared using Fisher's exact test. The association between the dichotomous dependent variable of survival (discharged alive or not discharged alive) and individual independent variables of interest (study center and types of mixed infections) was examined using logistic regression analysis. Results of logistic regression were summarized as odds ratios (OR) and P values testing the null hypothesis that the OR was equal to 1; 95% confidence intervals (95% CIs) were calculated using maximum likelihood methods. To adjust for effects of study center, the association between prognosis and various categories of mixed infections were analyzed using multivariable logistic regression, with survival as the dependent outcome and study center included as an independent variable for each model of an infection type (eg, whether or not there was mixed Gram-positive bacterial infection). All analyses were performed using S-PLUS statistical software,3 using a significance level of P < .05.
A total of 155 foals (99 from UF and 56 from TAMU) met the inclusion criteria. R. equi was cultured from a TBA of 115 foals, from lung tissue of 38 foals, and the culture source was unspecified for 2 foals. Both TBA and lung culture results were available from 11 foals. These 11 foals were included only in the TBA category for all analyses. There was no significant difference (P = .54) between the proportion of samples that were obtained from a TBA at UF (77%; 75/97) and TAMU (71%; 40/56). With the exception of 19 foals admitted directly by the necropsy service (13 from UF and 6 from TAMU), all foals were treated with a combination of a macrolide (erythromycin, clarithromycin, or azithromycin) and rifampin. A total of 69 foals received other antimicrobial agents during hospitalization. Other antimicrobial agents used included gentamicin (n = 22; 6.6–10 mg/kg IV q24h), amikacin (n = 20; 21–25 mg/kg q24h), ceftiofur sodium (n = 13; 2.2–4.4 mg/kg IV or IM q12–24h), trimethoprim-sulfonamide combinations (n = 5; 30 mg/kg PO q12h), doxycycline (n = 4; 10 mg/kg PO q12h), enrofloxacin (n = 1; 5.5 mg/kg IV q24h), or various combinations of the aforementioned drugs (n = 4). The proportion of foals treated with other antimicrobial agents in addition to the combination of a macrolide and rifampin was significantly (P = .001) higher at TAMU than at UF (Table 1). Mixed culture, mixed bacterial culture, Gram-positive bacteria, beta-hemolytic streptococci, Gram-negative bacteria, enteric Gram-negative bacteria, nonenteric Gram-negative bacteria, and fungi in TBA were not significantly associated with the use of other antimicrobial agents at either referral center.
|UF (%)||TAMU (%)||P|
|Sample source: TBA (n = 115)|
|Survival||68/75 (91)||27/40 (68)||.004|
|Pure culture||33/75 (44)||7/40 (18)||.007|
|Mixed infection (bacterial or fungal)||42/75 (56)||33/40 (83)||.007|
|Mixed bacterial infection||24/75 (32)||32/40 (80)||<.001|
|Gram positives||18/75 (24)||22/40 (55)||<.001|
|β-hemolytic streptococci||9/75 (12)||12/40 (30)||.023|
|Gram negatives||15/75 (20)||26/40 (65)||<.001|
|Enteric||4/75 (5)||15/39 (39)||<.001|
|Nonenteric||11/75 (15)||18/39 (46)||<.001|
|Fungi||22/75 (29)||3/40 (8)||.008|
|Other antimicrobials used||19/75 (25)||23/40 (58)||.001|
|Sample source: necropsy (n = 38)|
|Pure culture||19/22 (86)||6/16 (38)||.004|
|Mixed infection (bacterial or fungal)||3/22 (14)||10/16 (63)||.004|
|Mixed bacterial infection||2/22 (9)||10/16 (63)||<.001|
|Gram positives||2/22 (9)||7/16 (44)||.021|
|β-hemolytic streptococci||1/22 (5)||3/16 (19)||.29|
|Gram negatives||1/22 (5)||9/16 (56)||<.001|
|Enteric||1/22 (5)||7/16 (44)||.005|
|Nonenteric||0/22 (0)||3/16 (19)||.066|
|Fungi||1/22 (5)||0/16 (0)||1.000|
|Other antimicrobials used||5/22 (23)||9/16 (56)||.047|
Survival was significantly (P = .015) higher at UF (71%; 70/99) than at TAMU (50%; 28/56) when both necropsy and TBA were included as culture source. Similarly, survival was significantly (P = .004) higher at UF (91%; 68/75) than at TAMU (68%; 27/40) when only foals diagnosed based on results of TBA were considered (Table 1).
Regardless of sample source (necropsy versus TBA), R. equi was significantly (P = .004) more likely to grow in pure cultures from samples obtained from UF than from TAMU (Table 1). Bacterial isolates cultured with R. equi included 1 or several Gram-positive bacteria in 40 foals, Gram-negative bacteria in 41 foals, and fungi in 23 foals. The bacteria most commonly isolated were beta-hemolytic streptococci (n = 26 foals), Escherichia coli (n = 18), and alpha- or nonhemolytic streptococci (n = 16). Other Gram-positive bacteria isolated included Staphylococcus spp. (n = 4), Enterococcus spp. (n = 4), Corynebacterium spp. (n = 3), Bacillus spp. (n = 1), and Peptostreptococcus magnus (n = 1). Other Gram-negative bacteria included Pasteurella spp. (n = 7), unidentified nonenteric rods (n = 6), Bordetella bronchiseptica (n = 6), Actinobacillus equuli (n = 5), Klebsiella spp. (n = 5), Pseudomonas spp. (n = 4), other Actinobacillus spp. (n = 4), Enterobacter spp. (n = 4), Salmonella spp. (n = 2), Morganella morganii (n = 1), and Aeromonas spp. (n = 1). Fungi were isolated with R. equi from 23 foals. Fungi were significantly (P = .008) more likely to be isolated from TBA samples obtained from UF than from TAMU (Table 1). Fungi isolated were reported as unidentified mold (n = 14), Aspergillus spp. (n = 5), Candida albicans (n = 1), Cryptococcus neoformans (n = 1), Mucor spp. (n = 1), and Rhizopus spp. (n = 1).
At UF, pure cultures of R. equi were significantly (P < .001) more likely to be obtained from culture of lung tissue during postmortem examination (86%; 19/22) than from TBA (44%; 33/75). Microorganisms isolated from the 3 samples with mixed infections at UF included S. equi subspecies zooepidemicus (n = 1), a light growth of various molds (n = 1), and a combination of alpha-hemolytic streptococci, E. coli, and an unidentified Gram-negative rod (n = 1). In contrast, differences in the proportion of pure cultures between postmortem examination (38%; 6/16) and TBA (18%; 7/40) were not significantly (P = .16) different at TAMU. Of lung samples cultured during postmortem examination at TAMU with mixed bacterial infections, 6 contained a mixture of at least 3 different bacterial species, 2 contained 2 different bacterial species, and 2 contained only 1 bacterial species (1 alpha-hemolytic streptococcus and 1 unidentified Gram-negative rod). Using multivariate logistic regression (to adjust for the effect of center), mixed infections, mixed bacterial infections, nonenteric Gram-negative bacteria, and fungi were significantly more likely to be encountered in TBA than in lung tissue (Table 2). Both TBA and lung tissue culture results were available for 11 foals. The median time between collection of a TBA and necropsy was 5 days (range 0–19 days). Lung and TBA culture results were completely different for 9 of the 11 foals from which both samples were available. One of multiple microorganisms cultured from a TBA was recovered also from lung tissue in 2 foals.
|Variable||OR (95% CI)||P|
|Mixed infection (bacterial or fungal)||5.0a (2.1–11.8)||.0004|
|Mixed bacterial infection||3.2 (1.3–8.2)||.015|
|Gram positives||2.1 (0.8–5.1)||.11|
|β-hemolytic streptococci||2.2 (0.7–6.9)||.20|
|Gram negatives||2.2 (0.8–5.5)||.11|
Only foals from which R. equi was cultured from a TBA were included in the analysis for association between mixed infection and outcome. After adjusting for the effect of center using multivariate logistic regression, mixed culture, mixed bacterial culture, Gram-positive bacteria, beta-hemolytic streptococci, Gram-negative bacteria, enteric Gram-negative bacteria, nonenteric Gram-negative bacteria, and fungi were not significantly associated with outcome (Table 3).
|Variable||OR (95% CI)||P|
|Mixed infection (bacterial or fungal)||0.6a (0.2–2.1)||.43|
|Mixed bacterial infection||0.6 (0.2–2.0)||.42|
|Gram positives||0.5 (0.2–1.5)||.24|
|β-hemolytic streptococci||0.9 (0.3–3.0)||.87|
|Gram negatives||0.7 (0.2–2.1)||.51|
Gram-negative bacteria were isolated from the TBA of 41 foals (15 from UF and 26 from TAMU). The proportion of foals with Gram-negative bacteria that received other antimicrobial agents at UF (33%; 5/15) was not significantly (P = .20) different from that at TAMU (58%; 15/26). At UF, the proportion of survivors among foals that received macrolide-rifampin was not significantly different from that among foals that received other antimicrobial agents in addition to macrolide and rifampin (Table 4). In contrast, at TAMU the proportion of survivors among foals that received macrolide-rifampin alone was significantly higher than that among foals that received other antimicrobial agents in addition to macrolide and rifampin (Table 4).
|Center||Survival (%)||P Value|
|Macrolide-Rifampin Only||Macrolide-Rifampin with Additional Gram-Negative Spectrum|
|UF||7/10 (70)||5/5 (100)||.15|
|TAMU||11/11 (100)||7/15 (47)||.0074|
|Combined||18/21 (86)||12/20 (60)||.086|
This study demonstrated that isolation of multiple bacteria or fungi from a TBA along with R. equi did not negatively impact survival in the 2 referral populations studied. In addition, mixed infections were significantly more likely to be encountered in TBA than in lung tissue. These findings suggest that many bacteria might just be contaminants or colonize the trachea of foals with bronchopneumonia without necessarily contributing to pulmonary pathology. A wide variety of bacterial species were cultured from the foals of this study. Many of the bacteria isolated have not been causally associated with pneumonia in foals. For example, alpha- or nonhemolytic streptococci were isolated from 16 foals in this study. In a prospective study of foals with bronchopneumonia, the frequency of beta-hemolytic streptococci decreased, whereas the frequency of alpha- or nonhemolytic streptococci increased during treatment, compatible with a commensal role for alpha-hemolytic streptococci.[3, 9] However, many bacterial species cultured with R. equi in this study, such as beta-hemolytic streptococci, Actinobacillus/Pasteurella spp., E. coli, Bordetella bronchiseptica, and Klebsiella spp., have been associated with lower respiratory tract disease in foals and adult horses.[10, 11] Nevertheless, many of these pathogens also can be cultured from TBA obtained from clinically healthy foals and adult horses.[6-8] For example, TBA from apparently healthy foals yielded growth of potential pathogens in 12 of 37 (32%) foals sampled.
Our results indicating that there was no evidence of a difference in mortality between groups should not be interpreted as evidence that the mortality between the groups was the same. Our sample size for this study was based on using data from all available records rather than a sample size calculated a priori. Thus, it is possible that our sample size lacked statistical power to detect meaningful clinical differences. We believe this point is moot for the following reasons. First, a sample size calculation using the following assumptions was made: (1) statistical power of 80%, (2) statistical significance level of 5%, (3) a ratio of polymicrobial:monomicrobial infections of 1.5 : 1, and (4) proportions of survival of 75% among monomicrobial infections and 50% among polymicrobial infections; the latter proportions were based on recent reports documenting survival rates among foals admitted to referral hospitals.[1, 12, 13] Results indicated that 56 monomicrobically infected and 84 polymicrobically infected foals would have been required. Our study included 67 foals with monomicrobial infections and 88 foals with polymicrobial infections, indicating we had adequate power to detect a difference in survival that we deemed clinically meaningful. Second, our hypothesis could have been legitimately framed as a 1-sided test because our a priori hypothesis was that foals with polymicrobial infection would have a significantly lower survival; our statistical power would have been even higher for a 1-sided hypothesis test than for a 2-sided test. Third, our results indicate that prognosis tended to be better rather than worse for foals with polymicrobial infection. Obviously, we lacked statistical power to detect the observed difference as being significant, but the observed difference was in the opposite direction of the difference we hypothesized.
The major limitation of this study is its retrospective design. As a result, there are multiple possible confounding variables between study centers. There was considerable discrepancy in the frequency of mixed bacterial infections between the 2 referral centers, with mixed infections significantly more likely at TAMU than at UF. The exact reason for this discrepancy remains unknown, but there are several plausible explanations. There might be geographic differences in the normal tracheal microflora. In addition, it is possible that reporting of polymicrobial infections with predominance of R. equi differed between laboratories. For example, a given laboratory may provide a list of all bacteria isolated, whereas another may disregard light growth of molds or bacteria not known to be pathogenic, such as Bacillus spp. or Corynebacterium spp. This last possibility is unlikely to solely account for discrepancies between centers because differences were most significant for microorganisms widely known to be pathogenic, such as beta-hemolytic streptococci, E. coli, and Actinobacillus/Pasteurella spp. Finally, the method of collecting TBA may account for some of the discrepancies between centers. All TBA at UF were performed using a transcutaneous approach that bypasses the nasal cavity and pharynx. In contrast, most TBA at TAMU were performed using a commercially available transendoscopic catheter. Given that many potential lower respiratory tract pathogens isolated from the foals of this study (beta-hemolytic streptococci, E. coli, Enterobacter spp.) can be cultured from the pharynx of normal horses, it is possible that some of the microorganisms cultured were introduced using the endoscope during the procedure. In 1 study performed in pneumonic foals, there was good concordance between culture results of transendoscopic and transcutaneous TBA. However, the transendoscopic method was always performed first. Therefore, any nasopharyngeal contamination introduced using the endoscope would have been recovered from the transcutaneous sampling performed immediately afterward. In another study performed in adult horses, there was good concordance between culture of transendoscopic and transtracheal TBA when the transtracheal approach was performed first, but not when the transendoscopic approach was performed first, indicating that tracheoscopy sometimes results in oropharyngeal contamination of the trachea.
Differences in the proportion of mixed bacterial infections between referral centers cannot be explained solely using the method used to collect the TBA because the same differences were present in samples collected during postmortem examination. Only 3 of 22 (14%) postmortem samples from UF yielded microorganisms other than R. equi. In contrast, 10 of 16 (63%) postmortem samples from TAMU yielded a mixed bacterial population. These differences might be related the method used for culture submission. The method of sample collection was noted for only a small number of samples. However, most postmortem samples from UF for which information was available were seared lung tissue samples or fine needle aspirates of abscesses, whereas most samples from TAMU were swabs of lung tissue or abscesses. Regardless of referral center, 7 of 13 foals with mixed cultures from postmortem samples had at least 3 different bacterial species in addition to R. equi. In 2 additional foals, the microorganisms cultured were unlikely to contribute to pulmonary pathology (mold and alpha-hemolytic streptococci). These results suggest that sample contamination is likely to account for a high proportion of the mixed infections detected at postmortem examination.
The significant difference in survival between the 2 referral centers was unexpected because the primary treatment of infections caused by R. equi with the combination of a macrolide antibiotic and rifampin was the same at both centers. Differences in survival might be explained by different referral populations. Most foals seen at UF were Thoroughbreds, whereas most foals admitted to TAMU were Quarter Horses. Differences in value of the foals or management practices might have resulted in referral to UF earlier in the course of the disease compared with TAMU. Alternatively, it is possible that the proportion of extrapulmonary disorders was different between foals admitted to UF and foals admitted to TAMU. Extrapulmonary disorders caused by R. equi have been associated with a worse prognosis. Unfortunately, the retrospective study design did not permit an objective comparison of the severity of pulmonary lesions at the time of admission and the proportion of foals with extrapulmonary disorders associated with R. equi between the 2 referral centers. Another important limitation of the present study, which is common to most studies evaluating factors associated with outcome in veterinary medicine, is that many foals were euthanized. Financial limitations may have influenced the decision in some foals. Financial limitations might have accounted for at least some of the differences in survival between referral centers. However, it is unlikely that financial limitations would have been different between foals with mixed infections and foals from which R. equi was isolated in pure culture.
The fact that mixed infection were significantly more likely to occur in TBA than in lung tissue regardless of center suggests that many bacteria present in the trachea do not contribute to pulmonary pathology. There was poor concordance between TBA and lung culture results for the 11 foals for which the results of both samples types were available. However, this observation must be interpreted with caution because culture of lung tissue often was performed days after collection of the TBA. A true assessment of concordance between TBA and lung tissue culture results would necessitate collection of TBA immediately before euthanasia and collection of lung tissue using sterile techniques immediately after euthanasia.
As macrolides and rifampin have limited activity against common Gram-negative pathogens of horses, many authors have recommended that a third antimicrobial agent be added to the treatment regimen in foals from which Gram-negative bacteria are isolated from a TBA in conjunction with R. equi.[4, 5] Results of the present study indicate that the use of additional antimicrobial agents with better Gram-negative coverage is not associated with increased survival. In fact, the use of other antimicrobial agents was significantly associated with a worse prognosis at TAMU, but not at UF. This finding must be interpreted with caution. Aminoglycosides, the class of antimicrobial agents most commonly added to conventional therapy in this study, have been shown to be antagonistic with macrolides and rifampin against R. equi in vitro.[15, 16] Although the possibility that the use of other antimicrobial agents, such as aminoglycosides, was truly detrimental cannot be excluded, it is more likely that clinicians used other antimicrobial agents in foals with more severe respiratory disease, in foals with extrapulmonary disorders associated with R. equi, or in foals with concurrent diseases. The fact that there was not a significant association between TBA culture results and the use of other antimicrobial agents indicates that factors other than culture results likely influenced the decision to add additional antimicrobial agents. In addition, if the use of other antimicrobial agents was directly associated with a worse prognosis, this effect should have been observed at both referral centers. Nevertheless, the results of this study failed to detect an advantage for the use of other antimicrobial agents in foals with R. equi and Gram-negative isolates from a TBA. A randomized, prospective study would be required to more definitively address this question.
In conclusion, this study demonstrates that a variety of bacteria and fungi are commonly isolated from TBA along with R. equi. However, isolation of multiple bacteria or fungi from a TBA does not negatively impact survival, and mixed infections are significantly more likely to be encountered in TBA than in lung tissue, suggesting that many bacteria isolated from a TBA colonize the trachea without necessarily contributing to pulmonary pathology.
Conflict of Interest: Authors disclose no conflict of interest.
CIDEX-PLUS, Advanced Sterilization Products, Irvine, CA
Mila International Inc., Erlanger, KY
S-PLUS Version 8.2, Tibco Inc, Seattle, WA