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Rhodococcus equi infection in a surgical wound.

A 35-year-old diabetic male received routine day surgery to repair a ventral hernia. The surgical wound, upon follow-up visits, appeared to be healing properly. One month later, however, the patient presented to the ED with complaints of abdominal pain, after the patient had received an undisclosed minor trauma to the wound, while working outdoors for the local school department. The patient began having occasional feverish episodes along with erythema and the drainage of purulent material from the wound thereafter. There was no evidence of a pathogenic infection in the wound, as cultures yielded only 'normal skin flora', which included coagulase negative Staphylococcus and diphtheroids. Cephalexin was prescribed and the patient returned home.

One week later, the patient returned to the ED with what was described as a "non-healing surgical wound". The wound was cleansed and packed with gauze. With both wound and blood cultures appearing negative for significant bacterial growth, anti-fungal treatment was initiated due to the patient's continued failure to heal under the antibiotic treatment.

Due to the persistent infection, the wound was surgically reopened two weeks later, and the infected periumbilical patch was removed by an open approach. A gram stain from a sterile swab of the wound yielded no organisms. Two days later, the patient returned to the ED with severe abdominal pain. Additional microbiological workup was ordered on wound specimens collected using sterile swabs and anaerobic culturettes for both aerobic and anaerobic cultures, along with acid fast stains and cultures. Table 1 outlines the numerous tests ordered along with their corresponding findings.

LABORATORY RESULTS

The area surrounding the surgical wound was largely inflamed and erythemic with purulent discharge coming from the wound site. The patient's WBC count was slightly elevated at 13.7 x [10.sup.3]/uL (3.9-10.0 x [10.sup.3]/uL), though he was afebrile. Swabs taken from the wound, now over a month after the infection began, grew small, mucoid colonies on BAP, which at nearly 4 days after planting, developed a distinctive pink pigmentation. The colonies were gram-stained and showed long, gram-positive bacilli. An acid-fast stain was performed on the colonies, and the organisms were variably positive.

The organisms were catalase positive, oxidase negative, and urea positive. A CAMP test on BAP was performed using a streak of Staphylococcus aureus with a perpendicular streak of the organism in question. A striking zone of beta-hemolysis was noted near the intersection of the two streaks. A Remel[TM] Rapid CB Plus strip was inoculated yielding a 99.9% identification of Rhodococcus equi. The culture was sent to the State Laboratory for confirmation, and a preliminary identification of R. equi was reported. The State Laboratory later verified the identification.

BACKGROUND

The genus Rhodococcus is in the family Nocardiaceae in the suborder Corynebacterineae of the Actinomycetales order. (1) R. equi was originally termed Corynebacterium equi due to its morphological characteristics representing diphtheroids. (1) It wasn't until 1980, however, that the organism's cell wall composition and biochemical reactions were found to be more closely related to Nocardia and Mycobacterium than Corynebacterium, that the genus was changed to Rhodococcus ("red-pigmented coccus). (2,3)

Rhodococcus equi can be found in all continents of the world except Antarctica, and can flourish in both fresh and saltwater environments, and also within the intestines of bloodsucking arthropods. (4) R. equi, however, is more commonly associated with zoonotic infections mostly from horses, but also sporadically from cattle, sheep, pigs, goats, deer, dogs, wild birds and even cats. (3,4,5) R. equi colonizes the gastrointestinal tract of grazing mammals, and can be isolated from the manure and soil. (4) Exposure to R. equi can be via the oral route by ingesting products contaminated with soil or manure, inhalation of airborne organisms in dust, or direct inoculation due to trauma with soil or manure containing the organism. (5)

About 80-90% of patients with R. equi infections are immunocompromised. (6) Causes for immunosuppression can range from the most commonly seen AIDS/HIV, to malignancy, transplantation, chronic renal disease, alcoholism, immunosuppressive therapy (including prednisone, azothiopine and corticosteroid therapies) or, as in this case, diabetes mellitus. (5,6,7) On rare occasions, however, an infection with R. equi may be acquired in the immunocompetent. About 50% of the cases in the immunocompetent patient are due to trauma. (7)

Rhodococcus equi infections have ranged from necrotizing pneumonia, to "wound infections, subcutaneous abscess, thyroid abscess, retroperitoneal abscess, peritonitis, osteomyelitis, endophthalmitis, lymphadenitis, lymphangitis, septic arthritis, osteitis, bloody diarrhea, and fever of unknown origin among others." (7)

Rhodococcus equi is a facultative intracellular pathogen. (5) The pathogenicity of R. equi is based upon its ability to infect and live within macrophages, inhibiting their phagocytic capabilities, and eventually destroying them. (5,6) R. equi will cause inflammation, cell destruction and purulent granulomas. R. equi has the ability to disseminate from an initial infection site to many other sites in the host. (5) Due in part to its intracellular capability, R. equi is sometimes difficult to fully eradicate, and is commonly associated with relapse. (1)

Infections with Rhodococcus equi are associated with significant mortality due to the difficulty to eradicate the organism. The overall mortality rate of these infections is 25%, 50-55% in HIV patients, 20-25% in non-HIV, immunocompromised patients, and 11% in immunocompetent patients. (2) There are no racial differences in incidence of R. equi infections, however, there is a 3:1 male to female ratio, and the mean age of infection is 34-38 years old. (7) The strong prevalence of infection in males of this age group may be largely due to the fact that many occupations susceptible to soil and manure exposure, such as farming and landscaping, are mostly dominated by this population. A history of direct exposure to horses or pigs, however, is only present in one third of all patients with R. equi infections. (6)

LABORATORY IDENTIFICATION

Due to numerous shared characteristics, Rhodococcus is commonly misidentified as diphtheroid contaminant and normal flora, Mycobacterium, Nocardia, Bacillus, Micrococcus organisms or even fungi (6). The organism's acid-fast nature can commonly result in a misidentification of other acid fast bacterium such as M. tuberculosis, which often causes more trouble for the patient's recovery. More often, however, R. equi is overlooked or ignored due to its diphtheroid-like morphology and the slow development of its characteristic pigmentation, often leading to the misdiagnosis as a contaminant. (6) R. equi can also be confused with Nocardia spp. because of its acid-fast nature, and fungal characteristics, such as the formation of aerial hyphae. (7) A misidentification of R. equi could lead to inappropriate antibiotic treatment, and if left untreated, death.

R. equi grows well on nonselective media when incubated aerobically at 37[degrees]C. On blood agar plates, large, smooth, irregular, mucoid colonies can appear within 48 hours, however less mucoid forms are possible. (2,5) At 24 hours, colonies can be 1 to 2mm in diameter, though are not distinctive. (5) R. equi will not grow on most types of MacConkey agar. (5) Generally, with R. equi, a salmon to coral pink pigmentation will develop after 4-7 days of incubation and is rarely seen in cultures <4 days old, although, the colonies may also be light yellow to colorless. (2,5,6) As seen in Figure 1, the gram stain of R. equi reveals pleomorphic, gram-positive bacilli varying from coccoid to long, curved, clubbed forms, often resembling diphtheroids. (2) Gram stains made from cultures on solid media, or purulent material from the patient will often show more coccoid forms, however long rods and branching filaments may be observed in stains made from liquid media. (5) The organisms may be acid-fast with the Ziehl-Neelsen stain, however, this too depends on its growth media and the age of the culture. (2) R. equi is non-motile, non-fermentative, and positive for catalase, urease, alpha-glucosidase, alkaline phosphatase and nitrate reduction, but is negative for oxidase, indole, citrate assimilation and esculin hydrolysis. (1) R. equi has been known to produce "equi factors" that interact with the beta-toxin of Staphylococcus aureus to produce a zone of beta-hemolysis that can be observed using the CAMP technique on BAP. (2) (Fig. 2). Table 2 summarizes R. equi's different reactions to numerous laboratory tests.

TREATMENT

Rhodococcus equi is frequently difficult to treat due to its ability to thrive within the macrophage thus inhibiting its bactericidal functioning. R. equi is, however, susceptible to erythromycin, ciprofloxacin, vancomycin, aminoglycosides, rifampin, imipenem, and meropenem. Resistance has been observed to penicillins, ampicillin, carbenicillin and treatment is not suggested with such drugs, even if the organism appears susceptible, as rapid acquisition of resistance is possible. (2,6) R. equi has also shown moderate resistance to both first and second-generation cephalosporins. (2,5) It is often recommended to treat R. equi infections using a synergistic approach, with drugs such as erythromycin and rifampin together. (2) Antibiotics and lipophilic drugs with intracellular penetration capabilities are the most beneficial in treating these infections. (2) Failures in treatment have been associated with the poor penetration of macrophages by drugs such as gentamicin and penicillin. (2) Many patients suffering from an infection with R. equi should receive intravenous antibiotics for a minimum of 2 weeks, after which oral antibiotics can be substituted and continued, as long as positive cultures and symptoms have resolved. (6)

[FIGURE 1 OMITTED]

[FIGURE 2 OMITTED]

DISCUSSION

This case classically defines the difficulties involved with the proper identification and treatment of Rhodococcus equi infections. Retrospective analysis of this case allows us to follow the diagnosis process; from a possible bacterial infection which failed to heal after treatment with cephalexin, to the question of a fungal invasion which failed to improve. We can now see why R. equi did not dissipate initially, as cephalexin is a first-generation cephalosporin, to which it was resistant. Finally the diagnosis of Rhodococcus equi was established, and was found to be susceptible to the prescribed doses of erythromycin. With the appropriate treatment, the patient was able to successfully eradicate the persistent infection that lasted over 2 months.

The difficulty in identification of a Rhodococcus equi infection lies in its ability to mimic other, more common, organisms. In this case, due to various reasons, there was a delay in finalizing the latest wound culture beyond 3 days, which was laboratory policy for wound cultures that were not producing any pathogenic growth or appeared to be contaminated with just skin flora. Since the characteristic pink pigmentation of this organism didn't develop until the fourth day of incubation, this delay, in turn, helped lead to the identification of R. equi. It is likely that R. equi was present in the previous cultures, but misidentified as normal skin flora, as they were all completed within 3 days, too soon for the pigmentation development needed to catch the eye of the laboratory scientist.

It is because of misidentification that R. equi can be so dangerous. According to the European Journal of Clinical Microbiology and Infectious Disease, "during the past decade an increase in the incidence of reported human R. equi infections has been noted, possibly because of greater attention being given to this pathogen, but certainly also because of the rising number of immunocompromised patients" (1). The ability for laboratory professionals to properly recognize this organism, and produce, at minimum, a preliminary identification pending confirmation, will eliminate time needed to determine the proper treatment for the patient. Together, with appropriate laboratory work-up facilitating the physician's selection in proper treatment, a potentially dangerous organism can be easily eliminated and the patient cured.

ACKNOWLEDGEMENTS: The author wishes to thank Dr. Susan Leclair and the department of Medical Laboratory Science at the University of Massachusetts, Dartmouth along with the laboratory staff of Jordan Hospital for helping make this case study possible.

Clin Lab Sci encourages readers to respond with thoughts, questions, or comments regarding this article. Email responses to westminsterpublishers@comcast.net. In the subject line, please type "CLINLAB SCI22(3) SR PAASCHE". Selected responses will appear in the Dialogue and Discussion section in a future issue. Responses may be edited for length and clarity. We look forward to hearing from you.

REFERENCES

(1.) Gabriels P, Joosen H, Put E, Verhaegen J, et al. Recurrent Rhodococcus equi infection with fatal outcome in an immunocompetent patient. Eur J Clin Microbiol Infect Dis. 2006. January 20; 25 (1): 46-8.

(2.) Verville T, Huycke M, Greenfield R, Fine D, et al. Rhodococcus equi infections in humans. Medicine. (Baltimore) 1994. 73 (3): 119-32

(3.) Muller F, Schaal A, von Graevenitz A, von Moos L, et al. Characterization of Rhodococcus equi-like bacterium isolated from a wound infection in a noncompromised host. J Clinical Microbiol 1988. April; 26 (4): 618-20.

(4.) Takai S, Martens R, Julian A, Ribeiro M, et al. Virulence of Rhodococcus equi isolated from cats and dogs. J Clin Microbiol. 2003. September; 41 (9): 4468-70.

(5.) Prescott J. Rhodococcus equi: an Animal and Human Pathogen. Clin Microbiol Rev. 1991. January; 4 (1): 20-34.

(6.) Weinstock D, Brown A. Rhodococcus equi: An Emerging Pathogen. Clin Infect Dis. 2002. May; 34: 1379-85.

(7.) Kedlaya I. Rhodococcus equi. eMedicine [online] 2007. Available: http://www.emedicine.com/med/topic3378.htm via the INTERNET. Accessed 2007 February 22.

SUGGESTED READING

Linder, R. Rhodococcus equi and Arcanobacterium haemolyticum: Two "Coryneform" Bacteria Increasingly Recognized as Agents of Human Infection. Emerg Infect Dis. 1997. April-June; 3 (2): 145-153

Stuart R. Paasche, CLS (NCA), is a Clinical Laboratory Scientist at Lee Memorial Hospital, Fort Myers, FL 33901

Address for Correspondence: Stuart Paasche, 1826 Beach Parkway, Cape Coral FL 33904, 774-244-1834 (Cell), stuartpaasche@yahoo.com

The peer-reviewed Clinical Practice Section seeks to publish case studies, reports, and articles that are immediately useful, are of a practical nature, or contain information that could lead to improvement in the quality of the clinical laboratory's contribution to patient care, including brief reviews of books, computer programs, audiovisual materials, or other materials of interest to readers. Direct all inquiries to Bernadette Rodak MS CLS(NCA), Clin Lab Sci Clinical Practice Editor, Clinical Laboratory Science Program, Indiana University, Clarian Pathology Laboratory, 350 West 11th Street, 6002F, Indianapolis IN 46202. brodak@iupui.edu
Table 1: Wound Cultures and Tests Ordered. There were no issues
associated with the surgery, performed on 9/10, and for a month
thereafter. However, after the patient received trauma to the
wound, perhaps contaminating the wound with dirt, an infection
began that persisted for nearly two months. This table outlines
the course taken, involving repetitive wound cultures and gram
stains, until the final, confirmed ID of Rhodococcus equi was
reported, and the patient was properly treated allowing for full
recovery.

Date    Test Ordered             Result

10/5    Wound Gram Stain         Few WBC
                                 No organisms seen

        Wound Culture            Light growth "Skin Flora" (Coagulase
                                 negative Staphylococcus &
                                 diphtheroids)

10/12   Wound Gram Stain         Few WBC
                                 No organisms seen

        Wound Culture            Light growth "Skin Flora"

        Blood Culture            No Growth @ 5 days

10/29   Wound Gram Stain         Few WBC
                                 No organisms seen

        Wound Culture            Light growth "Skin Flora"

10/30   Wound Gram Stain         Few WBC
                                 No organisms seen

        Anaerobic Wound          No Anaerobes Isolated
        Culture

        Blood Culture            No Growth @ 5 days

        Wound Culture            Positive growth with
                                 Rhodococcus equi (Preliminary ID
                                 reported 11/6)

11/6    Wound Culture            Rhodococcus equi (Confirmatory ID
        (reference laboratory)   reported on 11/25

Table 2: Reactions of Rhodococcus equi

Test                                Reaction

Gram Stain                          Gram positive bacilli
Ziehl-Neelsen acid-fast stain       Variably positive
Motility                            Negative
Catalase                            Positive
Urea                                Positive
Alpha-Glucosidase                   Positive
Alkaline Phosphatase                Positive
Nitrate Reduction                   Positive
Oxidase                             Negative
Citrate                             Negative
Indole                              Negative
Esculin                             Negative
"Equi Factors" with CAMP method     Positive (zone of beta hemolysis)
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Title Annotation:CLINICAL PRACTICE
Author:Paasche, Stuart R.
Publication:Clinical Laboratory Science
Date:Jun 22, 2009
Words:2581
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