First Case of Raoultella planticola Urinary Tract Infection Reported in Western Romania

Raoultella planticola is a Gram-negative bacterium rarely involved in urinary tract infections. The patient was an 80-year-old woman with several associated diseases who presented to the hospital with fever and dysuria. Raoultella planticola was identified to be the causative agent of the urinary tract infection. Antibacterial treatment led to a full recovery within 7 days. This report highlights the presence of a rare pathogen as a causative agent in the case of a urinary tract infection and also the importance of using multiple methods in order to identify bacteria and to establish the diagnosis.


Introduction
Urinary tract infections (UTIs) are responsible for significant morbidity and increased health care costs, representing the most common infectious presentation in community medical practice, after respiratory tract infections [1]. Studies suggest that worldwide, each year, more than 150 million people are affected by UTIs, affecting both males and females of all age groups throughout their life span. Mostly, these infections require antibacterial therapy in addition to treatment with prebiotics, probiotics and urinary antiseptics [1,2].
The most common isolated pathogens in the case of community-acquired uncomplicated UTIs, both in males and females, are Escherichia coli, being the most common, followed by Enterobacteriaceae (Klebsiella, Proteus, Enterobacter), Enterococci, Streptococci, Staphylococci and Pseudomonas spp. [3,4].
Currently, Raoultella spp. was recognized as an important emerging uropathogen and should be considered in cases of infection [5]. Raoultella planticola is a Gram-negative, non-motile, aerobic, encapsulated bacterium belonging to the Enterobacteriaceae family [6]. This bacterium is usually found in aquatic environments, plants and soil. The cases of infection in humans caused by this organism were described as bacteremia, pneumonia, cholangitis, abscesses and UTIs [7]. Most of the patients infected by this pathogen present a degree of immunosuppression and malignancies, and have experiences of chemotherapy, organ transplants, prolonged ICU hospitalization or recent trauma [8].
In order to exert its pathogenicity in the human body, this bacterium has developed several mechanisms, such as the ability to adhere to the tissues for which it has developed tropism, form biofilms to protect itself and transform amino acid histidine into histamine, a mechanism that leads to the appearance of local and general clinical manifestations [9].
Due to these reasons, we present the first case report in Romania of an 80-year-old female, diagnosed with a symptomatic, lower UTI caused by Raoultella planticola.

Specimen Collection
Blood samples were collected via cephalic vein puncture on hematology vacutainers with K3 EDTA and on biochemistry vacutainers with a clot activator and separating gel in order to perform hematological and biochemical analyses. The patient was instructed to self-collect the mid-stream of urine using the clean-catch technique in a sterile container.

Laboratory Assays
CBC was determined using flow cytometry, cytochemistry and spectrophotometry on Advia 2120i (Siemens Healthcare Diagnostics, Erlangen, Germany).
Urine biochemistry was determined using the automated dipstick method on CLINITEK ® Novus Analyzer (Siemens Healthcare Diagnostics, Erlangen, Germany).
Urine sediment was determined using an automated urine microscopy analyzer, iQ200 Series Analyzer (Beckman Coulter, Brea, CA, USA).

Microbiology Techniques and Assays
The urine was plated using a 10 µL calibrated inoculating loop with a cross-streak pattern to conventional chromID CPS Elite chromogenic agar (bioMérieux Inc., Craponne, France) within one hour from the collection. The culture was incubated for 24 h in ambient air, at 37 • C.
The chromeID media were read and interpreted in accordance with the criteria in the standard operating procedure for urine cultures in the clinical microbiology laboratory. A growth of one or two distinct organisms above 50,000 CFU/mL was considered clinically significant. In our case, one organism (>100,000 CFU/mL) was further isolated into pure colonies [10].
A bacterial suspension was adjusted to 0.50 McFarlands in a solution of 0.45% sodium chloride. Bacterial isolates were further inoculated into the appropriate VITEK identification and antibiogram strips using VITEK ® 2 Compact (BioMérieux, Inc., Hazelwood, MO, USA). Analysis was performed using the identification (card type: GN) card and an antibiotic card (card type: AST-N204) for Gram-negative bacteria [11]. The data obtained were analyzed using VITEK 2 software version 9 in accordance with the manufacturer's instructions.
Matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF) microbial identification was performed on VITEK MS (BioMérieux, Inc., Hazelwood, MO, USA) in accordance with the manufacturer's instructions and internal laboratory standards.

Ethical Approval
Ethical approval of the clinical protocol for this study was approved by the Ethics Committee of Arad County Emergency Clinical Hospital (no. 90/4.01.2023), and the informed consent was signed by the patient.
antibiotic card (card type: AST-N204) for Gram-negative bacteria [11]. The data obtained were analyzed using VITEK 2 software version 9 in accordance with the manufacturer's instructions.
Matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF) microbial identification was performed on VITEK MS (BioMérieux, Inc., Hazelwood, MO, USA) in accordance with the manufacturer's instructions and internal laboratory standards.

Ethical Approval
Ethical approval of the clinical protocol for this study was approved by the Ethics Committee of Arad County Emergency Clinical Hospital (no. 90/4.01.2023), and the informed consent was signed by the patient.

Antibacterial MIC * (mg/L) Interpretation
Patient was treated with Tagremin 400 mg (Trimetoprime + sulfamethoxazole) 2 × 2 daily for 7 days and the evolution was conditionally and progressively improved after the first 2 days of treatment.
One week after ending the treatment, we repeated the CBC and inflammatory markers. The WBC count normalized at 8700/mm 3 (normal: 4050-11,840/mm 3 ), with 60% neutrophils as well as CRP at 0.3 mg/dL (normal: <0.5 mg/dL). ESR moderately improved to 27 mm/h (normal: <30 mm/h). The patient was asymptomatic. The abdominal ultrasound revealed no significant abnormal findings.

Discussion
An 80-year-old female with multiple medical-associated pathologies presented to the hospital with severe cystitis caused by Raoultella planticola. This bacterium was rarely associated with infection, most often being reported as a colonizer, especially in newborns [12]. Due to the fact that data regarding Raoultella planticola as a pathogen in humans are limited, the mechanism of pathogenesis is currently unclear. Certain conditions such as immunocompromised states, dialysis-dependencies, malignancy, diabetes mellitus and certain medication such as proton pump inhibitors and chemotherapy increase the chances of infection [9,13].
Raoultella planticola is a bacterium mostly found in environments with high prevalence in water and soil, first described in 1985 under the names Klebsiella planticola and Klebsiella trevisanii [14].Originally, Raoultella planticola was classified as a member of the genus Klebsiella, but since 2001, based on 16S rRNA and rpoB gene sequencing, this bacterium was reclassified as Raoultella spp. [15]. Raoultella planticola can convert histidine into histamine and can cause symptoms of scombroid poisoning when inadequately prepared seafood is consumed in large quantities [9].The first case where Raoultella spp. was incriminated as a pathogenic bacterium was reported in 1984 in a patient with sepsis [15]. In 2013, Olson et al. [12] reported the first case of UTI caused by this organism in an 89-year-old male patient with a past history of biventricular heart failure, chronic kidney disease, coronary artery disease, obesity, hypertension, anemia, atrial fibrillation and a penicillin allergy.
Fager et al. [5] reported thirty-two serious cases of human infections with Raoultella spp. between 1984 and 2018, and just six cases were associated with UTIs. Other studies [16,17] reported dysuria and fever as symptomatology in the case of UTI-associated Raoultella planticola infection, similar to our clinical case report.
To date, it is difficult to differentiate between Klebsiella spp. and Raoultella spp. via light microscopy analysis, due to their similar morphological and tinctorial characteristics [18]. Our microscopical investigation reported long filamentous Gram-negative rods for Raoultella planticola, which is a non-regular characteristic and not shown in another study. However, long filamentous morphotypes of Enterobacteriaceae were reported in the case of intensive bacterial cell multiplication [19].
Different biochemical tests are used to differentiate between Raoultella spp. and Klebsiella spp., such as tests focusing on ornithine decarboxylase activity, D-melezitose utilization and histamine [20,21]. In our study, we used VITEK ® 2 compact automated system microbial identification (ID), a system with 47 biochemical tests designed to identify Gram-negative rods within approximately 10 h [18,22]. However, this automatic system can confuse the Raoultella spp. With Klebsiella spp. [23]. Due to this reason, we continued with a second diagnosis using the MALDI-TOF equipment. MALDI-TOF has a better sensitivity of 97.4% compared to 93.3% on VITEK ® 2 compact automated system, where Raoultella spp. was firstly identified [18].Another study conducted in 2019 which used different assays in order to identify 30 strains of Raoultella planticola found MALDI-TOF equipment (with a sensitivity of 98.88% and specificity of 57.89%) to be superior over the VITEK ® 2 (with a sensitivity of 95.12% and specificity of 14.93%) [24].
The bacterium identified as a pathogen in our patient was resistant to Ampicillin. The resistance of Raoultella planticola to this antibiotic was previously reported due to overexpression of the encoded class-A β-lactamase [16,23,25]. This resistance is well known in the case of Klebsiella spp. and former Klebsiella members as Raoultella planticola (formerly known as Klebsiella planticola) [26].
Raoultella planticola is generally sensitive to antibacterial medication belonging to the aminoglycoside, carbapenem, cephalosporin and fluoroquinolone categories [16,27]. Treatment with a one-week course of Tagremin 400 mg (2 × 2 daily) led to the full recovery of the patient. Due to the fact that the control urine culture after the antimicrobial treatment is recommended only in females with recurrent UTIs one to two weeks after the end of the antimicrobial treatment [28], and considering the patient did not mention anything about recurrent UTIs in the initial anamnesis, we chose to follow the European Association of Urology Guideline and not perform a second urine culture in an asymptomatic patient [29]. The inflammatory markers were repeated one week after the end of the treatment and were in the normal range.

Conclusions
We described a rare case of UTI with Raoultella planticola in Western Romania, a Gramnegative bacterium usually misdiagnosed as Klebsiella spp. due to its morphology on chromogenic culture medium. We also observed that this bacterial species has gained resistance to ampicillin.
We also highlighted the importance of using biochemical tests on automated analyzers, such as the VITEK ® 2system in order to accurately identify an isolated pure colony obtained from the patient's urine.
In conclusion, Raoultella planticola highlights a real clinical interest because it shows pathogenicity and develops the ability to become resistant to antibiotics. Informed Consent Statement: Written informed consent has been obtained from the patient to publish this paper. Data Availability Statement: All data are included in the manuscript.

Conflicts of Interest:
The authors declare no conflict of interest.