Identification of Dietzia Species in a Patient with Endophthalmitis following Penetrating Injury with Retained Intraocular Metallic Foreign Body

To the best of our knowledge, we report the first case of Dietzia species in a patient with endophthalmitis. A 47-year-old man presented to the ophthalmology department with decreased vision, redness, and minimal pain in his right eye after a foreign body struck his eye following working using a hammer and chisel. Broad-spectrum polymerase chain reaction (PCR) and deoxyribonucleic acid (DNA) sequencing targeting 16S ribosomal ribonucleic acid-(rRNA-) encoding gene on an undiluted vitreous sample revealed 100% identity with GenBank sequences of Dietzia species including D. natronolimnaea, D. dagingensis, and D. cercidiphylli. The culture of the vitreous samples demonstrated the growth of Gram-positive cocci and polymorphic rods. The isolate from the culture was identified as D. natronolimnaea using matrix-assisted laser desorption/ionisation time-of-flight mass spectrometry (MALDI-TOF MS). The combination of surgical and medical treatment (pars plana vitrectomy and systemic and topical antibiotics) eradicated the infection successfully.


Introduction
Infectious endophthalmitis is a sight-threatening infection. e incidence of infectious endophthalmitis varies between 0.0075% and 1.05% [1][2][3][4][5][6][7][8][9]. It can be further categorized into exogenous and endogenous types. If a distant source of infection spread hematogenously, it gives rise to endogenous endophthalmitis. e exogenous endophthalmitis represents the most common type of endophthalmitis and results from direct inoculation of an organism as a complication following ocular surgery, penetrating ocular trauma with or without retained foreign body, intravitreal injections of medications, or extension of corneal infection. Coagulasenegative staphylococci represent the most common pathogen in endophthalmitis following cataract surgery and intravitreal injections, and Bacillus cereus causes the most cases of endophthalmitis following trauma [10]. D. natronolimnaea is an aerobically growing Gram-and catalasepositive actinomycete. A case report involving D. natronolimnaea in human disease describes a case of culture negative device-associated endocarditis [11]. However, in this case, D. natronolimnaea could not be differentiated from D. cercidiphylli strain by DNA sequencing of the excised atrial tissue.

Case Presentation
A 47-year-old Norwegian male presented at the general causality clinic with right eye irritation. Previously, the same day he had been working using a hammer and chisel to repair his car without any eye protection, and he thought a foreign body had struck his right eye. e examination of his right eye revealed congestion and laceration of the conjunctiva, and the general practitioner started treatment with a broad-spectrum topical antibiotic (chloramphenicol). Seven days following his first presentation to the general causality clinic, he woke up with decreased vision, redness, and minimal pain in his right eye, and he presented to the nearby ophthalmology department on the same day. e best-corrected visual acuity (BCVA) decimal had decreased from 1.0 to 0.7. e orbit computed tomography scans detected a metallic intraocular foreign body (Figure 1), and he was referred urgently to the Department of Ophthalmology at Oslo University Hospital for surgical removal of the foreign body. On arrival, the BCVA decimal had decreased from 0.7 to hand motion. e biomicroscopic examination revealed intense conjunctival and ciliary injection, most likely self-sealed conjunctival laceration, corneal oedema, 3+ anterior chamber cells with fibrin, and a thin layer of hypopyon in the anterior chamber and posterior synechiae. A layer of fibrin mesh covered the anterior surface of the lens. e changes in ocular media obscured the fundus view. e B-scan ultrasonography revealed an echogenic foreign body in the posterior vitreous cavity with dense vitreous opacities and attached retina and posterior vitreous. His left eye was unremarkable, and he was otherwise in good health. A clinical diagnosis of exogenous endophthalmitis secondary to penetrating eye injury with retained intraocular metallic foreign body was made.
He underwent an emergency 23-gauge pars plana vitrectomy with both undiluted and diluted vitreous biopsy and anterior chamber tap. e attempt to remove the intraocular foreign body was unsuccessful even after the removal of fibrin mesh layer covering the anterior surface of the lens due to poor surgical visualization of the posterior segment. At the end of the surgery, vancomycin (1 mg/0.1 ml), ceftazidime (2 mg/0.1 ml), and fungizone (0.00549 mg/0.1 ml) were administered intravitreally and gentamicin (20 mg/ 0.5 ml) subconjunctivally. He received treatment with topical steroids and antibiotics (Maxitrol eye drops consisting of dexamethasone, neomycin, and polymyxin B eye drops) hourly during daytime and intravenous cefuroxime (750 mg 3 times a day) postoperatively. In addition, he received oral steroids (prednisolone 60 mg daily) only for 2 days prior to repeated pars plana vitrectomy. Due to poor visualization of the posterior segment initially, he underwent surgical removal of the metallic intraocular foreign body (2 × 1.5 × 1 millimetres) 7 days after the first operation. e day after surgical removal of the foreign body, the intravenous cefuroxime was discontinued, and he was prescribed a 10day course of ciprofloxacin (750 mg three times a day) peroral treatment. He continued with topical steroids and antibiotics (dexamethasone, neomycin, and polymyxin B eye drops) three times a day. e routine postoperative eye examination on the 9 th day following removal of the foreign body revealed asymptomatic rhegmatogenous retinal detachment from 10 to 2 o'clock with fovea on and with a BCVA decimal of 0.4. He underwent his third pars plana vitrectomy with gas tamponade. ree weeks following the retinal detachment repair, he reached a BCVA decimal of 0.8, and the retina was attached. Six months following the retinal detachment repair, he reached a BCVA decimal of 1.0.

Microbiological Analysis.
Direct microscopy of the undiluted vitreous sample showed pleomorphic rods with coccoid or club-shaped appearance suggesting coryneform bacteria.
A broad PCR and DNA sequencing targeting 16S rRNAencoding gene was performed on the undiluted vitreous body sample using EZI DNA tissue kit (Qiagen, ermo Fisher Scientific, Hilden, Germany). e 5' half of the 16S rRNA gene was amplified by PCR. e PCR product was sequenced using BigDye Terminator Cycle Sequencing Kit ( ermo Fisher Scientific, Hilden, Germany). e bacterium was identified by searching the GenBank (the NIH genetic sequence database) with the obtained DNA sequence. e obtained 740-base pair DNA sequence revealed 100% identity with GenBank sequences of the Dietzia species including D. natronolimnaea, D. dagingensis, and D. cercidiphylli. Although, additional DNA sequencing on the rest of the 16S rRNA gene (total 1462 base pairs) was performed, and this did not give an unambiguous identification. e undiluted and diluted vitreous samples were cultured on sheep blood agar and chocolate agar plates (Oslo University Hospital, Oslo, Norway). e plates were incubated at 35°C in 5% carbon dioxide (CO 2 ) for 7 days. e growth of bacteria was detected after two days under aerobic incubation at 35°C in 5% CO 2 . Anaerobic culture using horse blood agar (Oslo University Hospital, Oslo, Norway) did not detect any bacterial growth, and yeast culture on Sabouraud agar (Oslo University Hospital, Oslo, Norway) did not reveal any growth. No bacterial or yeast growth was detected from the anterior chamber tap aspirate.
e Gram stain of the bacterial colonies demonstrated Gram-positive cocci and polymorphic rods. e isolate from the culture was identified as D. natronolimnaea using MALDI-TOF MS (MALDI Biotyper, Bruker Daltonics GmbH, Bremen, Germany). MALDI-TOF was unable to distinguish D. natronolimnaea from D. dagingensis and D. cercidiphylli because the MALDI-TOF database does not contain the sequences for the latter two species.
ere are no determined breakpoints for Dietzia species, and therefore, the results were reported with a minimum inhibitory concentration (MIC) value.
e MIC values of ciprofloxacin, gentamycin, tetracycline, and vancomycin

Discussion
Rainey et al. proposed the new classification of genus Dietzia in 1995, and they suggested that Rhodococcus maris should be reclassified into a new genus, Dietzia [12]. e main natural reservoirs for Dietzia species include soil and marine sediments. D. natronolimnaea was first isolated from an East African soda lake in Kenya by Duckworth et al. [13]. e importance of recently established genus Dietzia in medical conditions in human is slowly emerging. D. maris has been reported as an etiological agent in a total number of three patients. ey presented with prosthetic hip infection [14], septicaemia [15], and aortic dissection secondary to aortitis [16].
is strain has also been detected from the skin of healthy individuals [17]. e strain D. cinnamea has been isolated from a perianal swab in a patient with bone marrow transplantation [18] and from a dog bite wound in an adult patient [19]. e authors in both case reports questioned the potential of D. cinnamea to cause infection. Jones et al. isolated D. papillomatosis from an immunocompromised patient with confluent and reticulated papillomatosis, which is a benign skin disorder [20]. Rammer et al. identified D. papillomatosis in blood culture in a 2-year-old child who presented with fever following revision of ventriculoperitoneal shunt inserted as treatment for syringomyelia [21]. Furthermore, Dietzia species has been isolated repeatedly in a hematological unit in the United Kingdom [22]. At present, the only report of D. aurantiaca from human clinical specimens is from a 24-year-old Swedish woman's cerebrospinal fluid [23]. In another case, an 87-year-old man developed cellulitis-like inflammation 10 months following permanent pacemaker insertion, and Dietzia species were detected [24]. e authors in the culture negative deviceassociated endocarditis involving D. natronolimnaea stated that the DNA sequence from the excised atrial tissue of the patient revealed D. natronolimnaea that shared 100% identity with D. cercidiphylli strain. Very few case reports describe identification of Dietzia species in human conditions. However, their role in causing infection should be elucidated in the future. e identification of the infecting organism in exogenous endophthalmitis may be challenging. In a study from which bacteriologic data were obtained from postoperative endophthalmitis cases, only 291 out of 420 patients (69.3%) demonstrated microbial growth. [25] In this study, Grampositive bacteria were isolated in 274 patients (94.2%). e similarities in the Gram morphology and colony appearance displayed by Dietzia species and the more frequently encountered Rhodococcus species may lead to misinterpretation of Dietzia species as Rhodococcus species.
is may implicate the underdiagnosis of Dietzia speciesrelated infections in humans. Additionally, D. natronolimnaea may easily be missed, if cultured less than 72 hours and at incubation temperature at or above 37°C. e growth occurs at pH 6-10 with optimal growth at pH 9 and at salt concentrations up to 10% [22]. Previously, the diagnosis of Dietzia species may have been challenging due to the lack of accurate diagnostic tests. Although the DNA sequencing of 16S rRNA encoding gene assists in precise determination of species, some of the Dietzia species share 100% gene identity. In the patient described in this case report, the DNA sequencing of 16S rRNA encoding gene revealed 100% identity with GenBank sequences of D. natronolimnaea, D. dagingensis, and D. cercidiphylli. Similarly, Sudhindra et al. could not distinguish between the former two Dietzia species. e unique characteristic of mycolic acids in Dietzia may indicate a novel fatty biosynthesis [26]. is may aid in development of new accurate diagnostic laboratory tests. In future, increased awareness and development of accurate tests may assist in the diagnosis of Dietzia species infections.

Conflicts of Interest
e authors declare that there are no conflicts of interest.