A Rare Case of Endophthalmitis with Rhizobium radiobacter, Soon after a Resolved Keratitis: Case Report

Background: Rhizobium (Agrobacterium) species are plant aerobic bacteria, which in some cases can produce endophthalmitis in humans after corneal trauma. Case presentation: A 42-year-old female patient presented in the Emergency Department of the Emergency County Hospital of Craiova, Romania, reporting pain, epiphora, and blurry vision in her right eye for about five days. This initial infectious keratitis episode was successfully resolved, but after 20 days she presented again after trauma with a leaf with corneal abscess. In the conjunctival secretion, R. radiobacter was identified. Despite antibiotherapy, the patient’s state did not improve, and ultimately the eye was eviscerated. Methods: A search was performed in the ProQuest, PubMed, and ScienceDirect databases for the terms Agrobacterium, Rhizobium, radiobacter, and eye. We eliminated non-human studies, editorials and commentaries, and non-relevant content, and excluded the duplicates. Results: In total, 138 studies were initially obtained, and then we selected 26 studies for retrieval. After the selection process, we ended up including 17 studies in our analysis. Most studies reported R. radiobacter endophthalmitis after ocular surgical procedures or outdoor activities that involve exposure to soil. Conclusion: R. radiobacter is a rare cause of endophthalmitis after eye trauma that generally responds well to usual antibiotherapy, but occasionally can evolve to severe, leading to the loss of the eye.


Introduction
Endophthalmitis is a leading cause of eye loss worldwide, despite the prophylactic antibiotherapy used for eye trauma, whether accidental or surgical [1][2][3][4]. Table 1 summarizes the main infectious agents involved in endophthalmitis, which is caused by different mechanisms, including trauma, eye surgery, contact lenses, eye injections or other related procedures, or hematological inoculation. Vancomycin [18][19][20] Vancomycin, ceftazidime (or amikacin)

Antibiotics according to etiology
A rare cause of endophthalmitis is infection with Rhizobium (Agrobacterium) radiobacter, which is a plant aerobic bacterium belonging to the Gram-negative bacilli, which are common in the environment-especially in soil [21]-and cause plant diseases such as gall tumors and hairy root disease. Several cases of Rhizobium radiobacter endophthalmitis have been reported, usually after cataract surgery, but have only rarely been reported to produce human infections, most often in immunocompromised patients [22][23][24][25]. The main risk factors are non-adherence to aseptic techniques [26,27], paracentesis of the anterior chamber, and eyelid manipulation [2,28].
Here, we describe a case of bacterial keratitis with severe evolution.

Case Report
A 42-year-old female patient presented in the Emergency Department of the Emergency County Hospital of Craiova, Romania, on 4 April 2022, reporting pain, epiphora, and blurry vision in her right eye for about five days. Anamnesis revealed a history of chemically treated soil contact with her right eye about seven days before the presentation, as she lives in the countryside and works in agriculture. The patient had not been immunocompromised or treated with steroids. Uncorrected visual acuity was 0.25 in her right eye and 1 in her left eye; the best-corrected visual acuity (BCVA) in her right eye was 0.25 (normal visual acuity is 1). Intraocular pressure (IOP) was 17 mmHg in her right eye and 15 mmHg in her left eye (the normal range is between 10 and 21 mmHg). Slit-lamp examination in the right eye revealed conjunctival hyperemia, a 4 mm white-yellow central corneal ulceration with infiltrated margins that retained the methylene blue stain, Descemet folds at the lesion and endothelial edema, a normal-depth anterior chamber, less than five cells in the aqueous, miotic, and reflexive pupil ( Figure 1). Due to corneal haze, fundus examination was difficult to perform in the right eye, but it was normal in the left eye. Corneal sensitivity was tested in the right eye, and the result was hyperesthesia. A clinical diagnosis of infectious keratitis of the left eye was established. A SARS-CoV-2 rapid antigen test was performed, and as it was negative the patient was admitted to hospital in the Ophthalmology Clinic.
A 42-year-old female patient presented in the Emergency Department of the Emergency County Hospital of Craiova, Romania, on 4 April 2022, reporting pain, epiphora, and blurry vision in her right eye for about five days. Anamnesis revealed a history of chemically treated soil contact with her right eye about seven days before the presentation, as she lives in the countryside and works in agriculture. The patient had not been immunocompromised or treated with steroids. Uncorrected visual acuity was 0.25 in her right eye and 1 in her left eye; the best-corrected visual acuity (BCVA) in her right eye was 0.25 (normal visual acuity is 1). Intraocular pressure (IOP) was 17 mmHg in her right eye and 15 mmHg in her left eye (the normal range is between 10 and 21 mmHg). Slit-lamp examination in the right eye revealed conjunctival hyperemia, a 4 mm white-yellow central corneal ulceration with infiltrated margins that retained the methylene blue stain, Descemet folds at the lesion and endothelial edema, a normal-depth anterior chamber, less than five cells in the aqueous, miotic, and reflexive pupil ( Figure 1). Due to corneal haze, fundus examination was difficult to perform in the right eye, but it was normal in the left eye. Corneal sensitivity was tested in the right eye, and the result was hyperesthesia. A clinical diagnosis of infectious keratitis of the left eye was established. A SARS-CoV-2 rapid antigen test was performed, and as it was negative the patient was admitted to hospital in the Ophthalmology Clinic. Conjunctival secretion and corneal scraping samples were collected and sent to the hospital's laboratory for microbiology diagnosis and antibiogram; standard blood tests were also performed. Intravenous empirical broad-spectrum antibiotherapy was introduced immediately after sampling (third-generation cephalosporin/beta-lactamase inhibitor-cefoperazone/sulbactam), doubled by local antibiotherapy (topical moxifloxacin). Lubricants, mydriatics, topical NSAIDs, and eye patching were also prescribed.
Microbiology results were negative for bacteria and fungi; the rest of the tests were also normal. Fortunately, the evolution was favorable: the ulceration's size started to decrease daily, the Descemet folds became thinner, and visual acuity was also improving, so on 10 April 2022, the patient was discharged from hospital. Clinical examination of the right eye that day revealed that the uncorrected visual acuity was 0.9, IOP was 16 mmHg, the slit-lamp examination showed minimal conjunctival hyperemia, paracentral corneal 3 mm leucoma, and a quiet anterior chamber, and the fundus was also normal. The recommendation was topical treatment with lubricants, moxifloxacin, and NSAIDs, and ambulatory appointment after one week. Conjunctival secretion and corneal scraping samples were collected and sent to the hospital's laboratory for microbiology diagnosis and antibiogram; standard blood tests were also performed. Intravenous empirical broad-spectrum antibiotherapy was introduced immediately after sampling (third-generation cephalosporin/beta-lactamase inhibitorcefoperazone/sulbactam), doubled by local antibiotherapy (topical moxifloxacin). Lubricants, mydriatics, topical NSAIDs, and eye patching were also prescribed.
Microbiology results were negative for bacteria and fungi; the rest of the tests were also normal. Fortunately, the evolution was favorable: the ulceration's size started to decrease daily, the Descemet folds became thinner, and visual acuity was also improving, so on 10 April 2022, the patient was discharged from hospital. Clinical examination of the right eye that day revealed that the uncorrected visual acuity was 0.9, IOP was 16 mmHg, the slit-lamp examination showed minimal conjunctival hyperemia, paracentral corneal 3 mm leucoma, and a quiet anterior chamber, and the fundus was also normal. The recommendation was topical treatment with lubricants, moxifloxacin, and NSAIDs, and ambulatory appointment after one week.
The patient presented ambulatory, one week after hospital discharge, in the Ophthalmology Clinic, where the slit-lamp examination revealed a quiet right eye, no conjunctival hyperemia, paracentral corneal 3 mm leucoma, normal aspect of the fundus, and 0.9 visual acuity. Topical antibiotics and NSAIDs were interrupted after this examination, but the eye lubricants were kept and the patient returned to work.
On 29 April 2022, the patient presented again in the Emergency Department of the Emergency County Hospital of Craiova, Romania, reporting severe pain, photophobia, epiphora, and a severe decrease in visual acuity in her right eye. The symptoms started four to five days before the presentation; she linked the debut with eye trauma caused by plant leaves. Visual acuity in the right eye was counting fingers at two meters, IOP was 18 mmHg, and the slit-lamp examination revealed a 6 mm white corneal abscess with perilesional infiltration, thick Descemet folds, Tyndall ++, and myotic pupil. The clinical diagnosis was corneal abscess with exogenous uveitis of the right eye. A SARS-CoV-2 rapid antigen test was performed, and as it was negative the patient was admitted to hospital in the Ophthalmology Clinic.
Conjunctival secretions and corneal scrapings were again collected and sent to the hospital's laboratory for microbiology diagnosis and antibiogram; standard blood tests were also performed (10.6 × 10 3 /µL leukocytosis and 87.3%/9.2 × 10 3 /µL neutrophilia). Intravenous empirical double antibiotherapy was introduced immediately after sampling (third-generation cephalosporin/beta-lactamase inhibitor-cefoperazone/sulbactam and gentamycin), doubled by local antibiotherapy (topical levofloxacin). Lubricants, mydriatics, topical NSAIDs, and eye patching were also prescribed. The patient presented dizziness, nausea, and a body temperature raised to 37.5 • C; thus, she was booked for examination in the Internal Medicine Clinic. The general examination did not stress any pathological findings, and blood pressure was 100/60 mmHg. Recommendations were as follows: RT PCR SARS-CoV-2 (which was negative), hemoculture if the temperature rises above 38 • C (did not happen), hydration (according to blood pressure), and NSAIDs in case of fever.
The evolution was unfavorable as the visual acuity decreased to counting fingers at one meter, the corneal abscess remained the same size, a 2 mm hypopyon appeared, and the subjective pain increased (30 April). An infectious disease specialist was asked to see the patient, and the recommendations were as follows: further serological investigations for HIV, cytomegalovirus, and hepatitis viruses B and C, which were negative. It was also recommended to stop cefoperazone/sulbactam + gentamicin therapy and change to a teicoplanin + azithromycin regimen. Despite antibiotic changes, the patient's state did not improve: visual acuity in the right eye was limited to hand movement, the whole cornea was infiltrated, the abscess was 6-7 mm, and the hypopyon was 3-4 mm (1 May) ( Figure 2). On 29 April 2022, the patient presented again in the Emergency Department of the Emergency County Hospital of Craiova, Romania, reporting severe pain, photophobia, epiphora, and a severe decrease in visual acuity in her right eye. The symptoms started four to five days before the presentation; she linked the debut with eye trauma caused by plant leaves. Visual acuity in the right eye was counting fingers at two meters, IOP was 18 mmHg, and the slit-lamp examination revealed a 6 mm white corneal abscess with perilesional infiltration, thick Descemet folds, Tyndall ++, and myotic pupil. The clinical diagnosis was corneal abscess with exogenous uveitis of the right eye. A SARS-CoV-2 rapid antigen test was performed, and as it was negative the patient was admitted to hospital in the Ophthalmology Clinic.
Conjunctival secretions and corneal scrapings were again collected and sent to the hospital's laboratory for microbiology diagnosis and antibiogram; standard blood tests were also performed (10.6 × 10 3 /µL leukocytosis and 87.3%/9.2 × 10 3 /µL neutrophilia). Intravenous empirical double antibiotherapy was introduced immediately after sampling (third-generation cephalosporin/beta-lactamase inhibitor-cefoperazone/sulbactam and gentamycin), doubled by local antibiotherapy (topical levofloxacin). Lubricants, mydriatics, topical NSAIDs, and eye patching were also prescribed. The patient presented dizziness, nausea, and a body temperature raised to 37.5 °C; thus, she was booked for examination in the Internal Medicine Clinic. The general examination did not stress any pathological findings, and blood pressure was 100/60 mmHg. Recommendations were as follows: RT PCR SARS-CoV-2 (which was negative), hemoculture if the temperature rises above 38 °C (did not happen), hydration (according to blood pressure), and NSAIDs in case of fever.
The evolution was unfavorable as the visual acuity decreased to counting fingers at one meter, the corneal abscess remained the same size, a 2 mm hypopyon appeared, and the subjective pain increased (30 April). An infectious disease specialist was asked to see the patient, and the recommendations were as follows: further serological investigations for HIV, cytomegalovirus, and hepatitis viruses B and C, which were negative. It was also recommended to stop cefoperazone/sulbactam + gentamicin therapy and change to a teicoplanin + azithromycin regimen. Despite antibiotic changes, the patient's state did not improve: visual acuity in the right eye was limited to hand movement, the whole cornea was infiltrated, the abscess was 6-7 mm, and the hypopyon was 3-4 mm (1 May) ( Figure  2). The microbiology results then came in. Bacteriologically, the samples were inoculated under aerobic, anaerobic, and microaerophilic conditions in Columbia blood agar, chocolate agar supplemented with PVX, and MacConkey culture medium for 24 h at 37 • C in aerobiose (48 h in anaerobiose and microaerophily). We observed in both samples on blood agar in large (2-4 mm), convex, non-pigmented, light beige, non-hemolytic, raised aerobiotic colonies, with a dry central portion and wet at the edges ( Figure 3).
The microbiology results then came in. Bacteriologically, the samples were inoculated under aerobic, anaerobic, and microaerophilic conditions in Columbia blood agar, chocolate agar supplemented with PVX, and MacConkey culture medium for 24 h at 37 °C in aerobiose (48 h in anaerobiose and microaerophily). We observed in both samples on blood agar in large (2-4 mm), convex, non-pigmented, light beige, non-hemolytic, raised aerobiotic colonies, with a dry central portion and wet at the edges ( Figure 3). Biochemically, the strain was glucose-non-fermenting, oxidase-positive, mobile, indole-negative, and urease-positive. The biochemical identification on a VITEK2 automated system (Biomerieux) with a GN card identified R. radiobacter with 99% probability after 3.8 h of analysis ( Figure 4).  Biochemically, the strain was glucose-non-fermenting, oxidase-positive, mobile, indolenegative, and urease-positive. The biochemical identification on a VITEK2 automated system (Biomerieux) with a GN card identified R. radiobacter with 99% probability after 3.8 h of analysis ( Figure 4).
The microbiology results then came in. Bacteriologically, the samples were inoculated under aerobic, anaerobic, and microaerophilic conditions in Columbia blood agar, chocolate agar supplemented with PVX, and MacConkey culture medium for 24 h at 37 °C in aerobiose (48 h in anaerobiose and microaerophily). We observed in both samples on blood agar in large (2-4 mm), convex, non-pigmented, light beige, non-hemolytic, raised aerobiotic colonies, with a dry central portion and wet at the edges (Figure 3). Biochemically, the strain was glucose-non-fermenting, oxidase-positive, mobile, indole-negative, and urease-positive. The biochemical identification on a VITEK2 automated system (Biomerieux) with a GN card identified R. radiobacter with 99% probability after 3.8 h of analysis ( Figure 4).  In the microscopic exam we observed long and thin unsporulated Gram-negative rods with a tendency of adhesion between them ( Figure 5).
On 2 May 2022, the patient requested transfer to the Emergency Eye Diseases Hospital in Bucharest, Romania, and it was granted. The right eye status was as follows: visual acuity was light perception, severe conjunctival hyperemia, infiltrated cornea, 6-7 mm corneal abscess, 4 mm hypopyon, and fundus examination was not possible due to corneal opacity. The clinical and microbiological diagnosis was severe bacterial keratitis (corneal abscess with R. radiobacter) and exogenous endophthalmitis.
Unfortunately for this patient, the evolution was unfavorable, and within days after she was transferred to Bucharest the eye was eviscerated to prevent orbital, sinus, and cerebral infection complications.

Systematic Review
We searched ProQuest, PubMed, and ScienceDirect for the terms Agrobacterium, Rhizobium, radiobacter, and eye (the exact search string was "(Agrobacterium OR Rhizobium) AND radiobacter and eye"). The eligibility criteria included human studies of infections of the eye with R. radiobacter, and all article types except for comments, editorials, etc. The database search yielded 138 studies: 96 from the ProQuest database, 8 from Pub-Med, and 34 from ScienceDirect; 110 studies were not retrieved as they were non-human studies. We excluded two more studies as they were editorials or comments. Therefore, we selected 26 studies for retrieval: 7 studies from the PubMed database, 10 from ProQuest, and 9 from ScienceDirect; 5 studies were duplicates. We excluded 4 more studies from the analysis after reading the abstracts: two studies identified R. radiobacter in solutions used for the storage of contact lenses [29,30]; one study referred to sepsis with R. radiobacter originating from a central venous catheter [31]; and one study identified this pathogen in surgical equipment used in ophthalmology (vacuum control manifold) [32] ( Figure 6). On 2 May 2022, the patient requested transfer to the Emergency Eye Diseases Hospital in Bucharest, Romania, and it was granted. The right eye status was as follows: visual acuity was light perception, severe conjunctival hyperemia, infiltrated cornea, 6-7 mm corneal abscess, 4 mm hypopyon, and fundus examination was not possible due to corneal opacity. The clinical and microbiological diagnosis was severe bacterial keratitis (corneal abscess with R. radiobacter) and exogenous endophthalmitis.
Unfortunately for this patient, the evolution was unfavorable, and within days after she was transferred to Bucharest the eye was eviscerated to prevent orbital, sinus, and cerebral infection complications.

Systematic Review
We searched ProQuest, PubMed, and ScienceDirect for the terms Agrobacterium, Rhizobium, radiobacter, and eye (the exact search string was "(Agrobacterium OR Rhizobium) AND radiobacter and eye"). The eligibility criteria included human studies of infections of the eye with R. radiobacter, and all article types except for comments, editorials, etc. The database search yielded 138 studies: 96 from the ProQuest database, 8 from PubMed, and 34 from ScienceDirect; 110 studies were not retrieved as they were non-human studies. We excluded two more studies as they were editorials or comments. Therefore, we selected 26 studies for retrieval: 7 studies from the PubMed database, 10 from ProQuest, and 9 from ScienceDirect; 5 studies were duplicates. We excluded 4 more studies from the analysis after reading the abstracts: two studies identified R. radiobacter in solutions used for the storage of contact lenses [29,30]; one study referred to sepsis with R. radiobacter originating from a central venous catheter [31]; and one study identified this pathogen in surgical equipment used in ophthalmology (vacuum control manifold) [32] (Figure 6).
Rohowetz et al. [33] reported endophthalmitis with R. radiobacter in a 79-year-old male patient with type II diabetes mellitus and diabetic retinopathy, who received treatment with intravitreal aflibercept. The patient was treated empirically with intravitreal injections of ceftazidime and vancomycin. After the laboratory results came in, the patient's therapy was changed to oral azithromycin and levofloxacin. The infection was resolved at 1-month follow-up. Therefore, we included 17 studies in the systematic review (Table 2). Therefore, we included 17 studies in the systematic review ( Table 2). The same author, one year later, reported a case of endophthalmitis in an 85-year-old male associated with insertion of an inferonasal Baerveldt tube [34]. R. radiobacter was identified in aqueous humor culture, and was resistant to cefazolin, ceftazidime, amikacin, tobramycin, and trimethoprim-sulfamethoxazole. Because the explantation of the drainage implant was not efficient, pars plana vitrectomy was performed with removal of the intraocular lens, associated with silicone oil infusions and intravitreal antibiotics. The patient's visual acuity improved after 2 weeks, but then it was lost to follow-up.    Barker et al. [35] reported a series of four cases with R. radiobacter in young patients: one after corneal traumatism and three in long-term contact lens wearers. The patients were treated with steroidal anti-inflammatory drugs and with antibiotic drops. All cases had a favorable evolution, with total sight recovery. Furthermore, Fenner et al. [36] reported keratitis in a young patient who was a contact lens wearer.
Another case of R. radiobacter infection was reported after phakic intraocular lens implantation in the posterior chamber of the eye in a young patient (29 years old) with myopia [37]. The endophthalmitis was resolved after intravitreal injections of vancomycin and ceftazidime.
One study identified one strain of R. radiobacter (1.14%) in 88 patients expressing symptoms consistent with conjunctivitis [40]. Another study included 44 patients with polymicrobial keratitis, from which R. radiobacter was isolated in one case (2.27%).
Nine studies were research articles, which included 912 patients who had undergone cataract surgery [4][5][6][7][8][9][10][11]41]. From those, in 18 cases (1.97%), R. radiobacter was isolated. In 7 of those 18 cases, R. radiobacter was associated with endophthalmitis, but in the remaining 11 cases it was considered an environmental contaminant. Moreover, Namdari et al. [42] in 2003 reported a case of chronic endophthalmitis after cataract extraction produced by R. radiobacter, which was resistant to vancomycin, amikacin, and ceftazidime-drugs often used in empirical therapy. Ultimately, the infection was resolved after changing the therapy to intravitreal injections of gentamycin and oral ciprofloxacin.
Therefore, endophthalmitis with R. radiobacter is rare, and is usually encountered after cataract surgery-mostly in old people and immunosuppressed patients, but also in young patients who wear contact lenses for extended periods of time.

Discussion
Rhizobium is a rare agent of endophthalmitis in patients who come into contact with plants [21]. Initially classified as Agrobacterium, after the introduction of 16s RNA genetic sequencing in the 1990s, many species were reclassified into the genus Rhizobium. The genus is divided into species based largely on pathogenic properties in plants: R. radiobacter (non-pathogenic), R. tumefaciens (the causative agent of crown gall tumors), R. rhizogenes (the causative agent of hairy root disease), and R. vitis (the causative agent of tumors and necrotic disease on grapevines). There are also less well-studied proposed species such as R. rubi isolated from cane galls on Rubus plant species.
The first Rhizobium strains were isolated from human infections in 1967 [43]. The first infection with R. radiobacter was reported in a case of endocarditis on a prosthetic valve [44]. In 1996, the first case of Rhizobium radiobacter endophthalmitis was reported after cataract surgery [42] and intravitreous injections [45]. Few cases have been reported since then [33]. In our clinic, this is the first confirmed case of R. radiobacter endophthalmitis.
The reported cases of ocular R. radiobacter infections occurred after traumatic medical procedures involving the eye, such as cataract surgery [42] or intravitreal injections [33]. Our patient, however, developed endophthalmitis after eye trauma caused by a plant leaf. In this case, our understanding is that the first episode was a local infection, which indeed resolved, but acted as a risk factor for infection with R. radiobacter after the eye trauma caused by the plant leaf. Indeed, as Rhizobium species are predominantly found in soil and plants, it is thought that cases of endophthalmitis after eye surgery are associated with outdoor activities and contamination of the eye with dust.
Although R. radiobacter infections are usually reported in immunocompromised hosts, our patient was immunocompetent; in this case, the eye trauma directly inoculated the eye with the pathogen at a much higher dose than that found in dust that contaminated the eye after medical procedures in other reported cases.

Conclusions
To summarize, R. radiobacter is a soil bacterium that rarely causes human infection. Nevertheless, patients with eye surgery or other procedures that provide a continuity solution of the cornea, including endophthalmitis, can develop endophthalmitis following exposure to soil. It is therefore recommended that patients who undergo surgical procedures such as cataract surgery or intravitreal injections should avoid outdoor activities that involve exposure to soil and plants (yardwork, farming, etc.).  Informed Consent Statement: Informed consent was obtained from the subject of this report. Written informed consent has been obtained from the patient to publish this paper.

Data Availability Statement:
The data presented in this study are available upon request from the corresponding author.