Breast abscess caused by Gordonia bronchialis and the use of 16 s rRNA gene sequence analysis for its definitive identification

Gordonia spp., previously classified as Gordona, are nocardioform actinomycetes isolated from soil and from the intestinal content of mammals (Conville & Witebsky, 2001). These organisms are emergent pathogens that have been isolated in infections from immunocompromised patients and, less frequently, from previously healthy individuals (Siddiqui et al., 2012; Werno et al., 2005; Riegel et al., 1996).


Introduction
Gordonia spp., previously classified as Gordona, are nocardioform actinomycetes isolated from soil and from the intestinal content of mammals (Conville & Witebsky, 2001).These organisms are emergent pathogens that have been isolated in infections from immunocompromised patients and, less frequently, from previously healthy individuals (Siddiqui et al., 2012;Werno et al., 2005;Riegel et al., 1996).
There have been few reports of different infections caused by Gordonia spp. in recent years and, to our knowledge, only 13 cases caused by Gordonia bronchialis.
We present a case of recurrent breast abscess caused by G. bronchialis in an immunocompetent patient.

Case report
A 39-year-old healthy, immunocompetent woman presented in our emergency department with pain and swelling on her right breast extending to her right arm.The patient had no previous medical history or any other conditions that would predispose the formation of breast abscess.Upon examination, a palpable mass with marked swelling and erythema on the upper right area of the areola was observed.She was diagnosed with non-puerperal mastitis and treated empirically with cloxacillin (500 mg every 6 h), and followed up for additional tests.An initial ultrasound scan showed an area of acute inflammatory changes and excluded abscess formation.Ten days later, she returned to the hospital without having experienced any improvement in her symptoms.Physical examination during the second emergency department visit revealed a palpable mass of about 10 cm in diameter with erythema of the overlying skin on the upper quadrant of the right breast, as well as enlarged axillary lymph nodes.A full blood count showed a high leukocyte count (12 000 cells ml 21 ) with neutrophil predominance.A repeated ultrasound scan detected a poorly circumscribed mass compatible with an abscess.There was no mammographic evidence of malignancy.Consequently, the abscess was drained and the purulent material sent for microbiological study.Antibiotic treatment was switched to oral cefuroxime (500 mg every 8 h).A Gram stain of the abscess aspirate showed abundant polymorphonuclear leukocytes and Gram-positive bacilli.After 48 h of incubation, there was no growth under aerobic conditions, and metronidazole was added to the treatment due to the possibility of an anaerobic bacterial infection.Meanwhile, given the lack of improvement in the local inflammatory signs and pain, treatment was changed again to oral amoxicillin-clavulanate, after which a slow clinical improvement was noted.Six weeks later, the treatment was changed to oral ciprofloxacin (500 mg every 12 h), and, given the persistence of the abscess, a second surgical drainage was performed and new samples were obtained for microbiological and pathological studies.The antibiotic treatment was stopped after 19 uninterrupted weeks due to a marked improvement.
On day 4 of incubation, non-haemolytic, dry, creamywhite colonies were seen on aerobic cultures of pus aspirated at the time the abscess was first incised.The anaerobic cultures and the cultures taken from the second surgical drainage were both negative.
The isolate was Gram positive, pleomorphic and bacillary shaped, and formed rough white colonies that became yellow-coloured after several days of culture.The colonies were catalase and urease positive, oxidase negative and weakly acid-fast according to a modified Ziehl-Neelsen stain.
The isolate was identified initially as Rhodococcus sp. by a commercial identification system (API Coryne; bioMe ´rieux) with a profile number of 3110004.When entered into the API database, this biochemical pattern identified the organism as Rhodococcus sp.(98 % identification).The software also suggested the possibility that the isolate was of the genus Gordonia, Dietzia or Nocardia.
This unusual isolate in this clinical setting prompted us to send the strain to a reference laboratory where it was identified as Gordonia bronchialis by amplification and sequencing of the 16S rRNA gene with a previously described procedure (Lane, 1991).
According to disk diffusion, the organism was susceptible to gentamicin, amikacin, amoxicillin-clavulanate, cefotaxime, penicillin G, vancomycin, ciprofloxacin and tetracycline; intermediate to erythromycin; and resistant to clindamycin and cotrimoxazole (breakpoints used for the interpretation of the resulting zone sizes were those established by the Clinical and Laboratory Standards Institute for Staphylococcus spp.; CLSI, 2012).Susceptibility testing was carried out before the definitive identification.

Discussion
Gordonia spp.are non-motile, Gram-positive, weakly acid fast rods classified as aerobic actinomycetes.Gordonia spp., previously classified within the genera Rhodococcus and Gordona, have been isolated from the environment and from the intestinal content of mammals (Conville & Witebsky, 2001).These organisms rarely cause disease in humans, although they have been isolated in infections from immunocompromised patients and, less frequently, from previously healthy persons (Siddiqui et al., 2012;Werno et al., 2005;Riegel et al., 1996).However, their true prevalence could be underestimated due to their fastidious growth requirements, slow growth in conventional cultures and confusing morphology.Isolation of Gordonia spp.requires prolonged incubation for at least 3-4 days, and this delay may cause false-negative culture reports resulting in infections caused by this genus being overlooked (Gil-Sande et al., 2006).Moreover, Gordonia spp.may also be incorrectly identified as other morphologically similar bacteria, such as commensal species of actinomycetes or mycobacterial species.
A recent literature review (Siddiqui et al., 2012) described several types of infection caused by Gordonia spp.including a sternal wound infection after heart surgery, external otitis, bronchitis, skin and soft tissue infections, bacteraemia and catheter-associated infections.
G. bronchialis, the species isolated in this case, was initially identified from samples of soil and sputum from patients with pulmonary disease (Conville & Witebsky, 2001;Johnson et al., 2011;Siddiqui et al., 2012).Only 13 cases of infection with this species of actinomycete have been described (Siddiqui et al., 2012), including a reported recurrent breast abscess in an immunocompetent patient with no previous medical history (Werno et al., 2005) that shared many features with our case.Another case of granulomatous mastitis in an immunocompetent patient caused by other species of Gordonia (Gordonia terrae) has also been reported, although the patient had a previous history of nipple piercing (Zardawi et al., 2004).
Recurrence and a protracted clinical course are common characteristics of infections with Gordonia spp.(Werno et al., 2005).Many patients, as in our case, require 6-12 weeks of therapy with different antibiotics, often along with appropriate surgical debridement (Johnson et al., 2011;Siddiqui et al., 2012;Sng et al., 2004;Werno et al., 2005).This prolonged course could be explained by the ability of Gordonia spp. to form sessile communities where antimicrobial penetration is poor (Werno et al., 2005).There are no standardized recommendations for treatment of infections caused by Gordonia spp.These species are susceptible to a wide range of antibiotics in vitro.Patients described in the literature were successfully treated with either monotherapy or a combination of different antibiotics such as amoxicillin-clavulanate, quinolones, cephalosporins, carbapenems, cotrimoxazole, azithromycin, clindamycin and vancomycin (Werno et al., 2005).
In our case, careful observation of the Gram stain performed on the abscess material and extended incubation of cultures beyond the protocols for Staphylococcus aureus (the most common etiological agent) were crucial in guiding the techniques used to establish the aetiology.16S rRNA gene sequencing allowed us to definitively identify the species involved and differentiate it from other phylogenetically related species such as Rhodococcus, Streptomyces and Nocardia.Molecular methodologies are increasingly being used to identify actinomycetes, and 16S rRNA gene sequencing has become a valuable tool for this purpose in many laboratories (Blaschke et al., 2007).
In summary, we would like to emphasize that, although Gordonia spp.infections are rare, they nonetheless appear in young, healthy and immunocompetent people.This is the second case of breast abscess caused by G. bronchialis described in the literature, with significant clinical similarities to the first case.Whilst careful execution of simple tests such as Gram staining and a modified acid-fast stain were key to determining the aetiology and patient