Osteomyelitis Caused by Ralstonia mannitolilytica, a Rare Opportunistic Pathogen

Ralstonia spp. are non-fermenting aerobic gram-negative rods found in humid environments, whose role as opportunistic human pathogens has lately been recognized. Ralstonia mannitolilytica is one of the three members of the Ralstonia genus (together with Ralstonia pickettii and Ralstonia insidiosa). Bone infections by Ralstonia spp. are very rare. We report a case of femoral osteomyelitis caused by R. mannitolilytica. Among literature search, only eight cases of bone infection due to the Ralstonia genus have been described, in all of which the causative agent was identified as R. pickettii. To our knowledge, this is the first reported case of osteomyelitis attributed to R. mannitolilytica. Despite its low virulence, Ralstonia has specific characteristics that promote its spread and shows high antibiotic resistance, partly due to its ability to create a biofilm. Identification of Ralstonia spp. poses unique difficulties as the distinction between the species of the genus is not straightforward. Additionally, the bacteria may be misidentified as other closely related species. Recent data suggests that modern spectrometry and gene sequencing techniques are essential to avoid these pitfalls. Susceptibility data about the genus is limited and based on a small number of case reports, therefore there is no standardized antibiotic susceptibility testing and European Committee on Antimicrobial Susceptibility Testing (EUCAST) breakpoints exist. The report aims is to provide useful information on the antibiotic selection and treatment suggestions to be followed for bone infections caused by the Ralstonia genus, along with a review on the literature of this emerging opportunistic pathogen.


Introduction
Ralstonia spp. are non-fermenting aerobic gram-negative rods found in humid environments such as water, soil and plants [1]. Ralstonia mannitolilytica, formerly known as Pseudomonas thomasii, is one of the three members of the Ralstonia genus (together with Ralstonia pickettii and Ralstonia insidiosa) that have been lately recognized for their role as opportunistic human pathogens. Most reports in the literature for the Ralstonia spp. involve R. pickettii, which is the most clinically prevalent member of the genus. However, during the last 10 years, a few single case reports and hospital outbreaks have been attributed to R. mannitolilytica infection. These cases involve mainly immunocompromised patients, patients on hemodialysis or with respiratory problems. Most of them are associated with the use of contaminated solutions, including saline solutions, water for injections and disinfectants [1,2]. Despite its low virulence, Ralstonia shows high resistance to antibiotics, partly due to its ability to create biofilm, while its laboratory identification is trivial making it a difficult-to-treat microorganism [3].
We report a case of osteomyelitis of the femur caused by R. mannitolilytica in a young male patient after a motorcycle crash that led to an open femoral fracture. In current literature, only a few cases of bone infections caused by this genus are reported, mainly by R. pickettii, and none by R. mannitolilytica. This report aims to provide useful information for clinicians on the antibiotic selection and treatment of bone infections caused by the Ralstonia genus and a review of the literature.

Case Presentation
An 18-year-old male sustained an open diaphyseal fracture (Gustilo IIIb) on his right femur after a motorcycle accident. The accident took place off-road and the wound was massively contaminated with soil. The patient was set on an empirical combination of antibiotics (cefuroxime and amikacin). An extensive lavage and surgical debridement in the operation room followed, and the fracture was stabilized with external fixation. Postoperatively, the wound showed signs of infection. Sequential surgical debridements were deemed necessary (every second day for two weeks). Due to persistent inflammation the patient was operated on again, where bone and soft tissues were removed from the fracture site, leading to a shortening with distraction osteogenesis technique. Proximal femoral corticotomy was performed (distraction site) and an external fixator (LRS; Orthofix, Milan, Italy) was used for the transport of the bone segment ( Figure 1B). At the fracture site, the PMMA cement was removed and debridement to healthy bone margins with further removal of 3 cm of necrotic bone was performed (paprika sign). A split-thickness skin autograft from the left thigh was used to cover the skin deficit. The bone transport was initiated one week postoperatively, at an elongation rate of 1 mm per day to correct a total of 7 cm length discrepancy. During the above surgical procedures, adequate bone specimens were obtained and sent for cultures. Specimens were plated onto 5% sheep blood agar, chocolate agar and MacConkey agar. The plates were incubated at 35-37 °C in ambient air. Plates were inspected for growth at 24 h and again at 48 h. The cultured colonies were identified as R. mannitolilytica by using the VITEK 2 automated identification system (bioMérieux, Marcy-l'Étoile, France). After testing positive for Ralstonia infection, further laboratory analysis by 16s ribosomal deoxyribose nucleic acid (rDNA) gene sequencing was performed for confirmation. A polymerase chain reaction (PCR) product was purified by using the Promega Wizard PCR Preps kit (Promega, Madison, WI, USA) according to the manufacturer's instructions. Sequence analysis was performed with an Applied Biosystems 3700 DNA sequencer and the protocols of the manufacturer (PE Applied Biosystems, Foster City, CA, USA) by using the BigDye Terminator Cycle Sequencing Ready Reaction kit. The sequences were compared to sequences available in the GenBank database by using FASTA3, and the results obtained with FASTA3 were used to identify isolates to the species R. mannitolilytica. Antimicrobial susceptibility was performed by the broth microdilution method (MicroScanWalkAway plus; Beckman Coulter, Milan, Italy). As there are no European Committee on Antimicrobial Susceptibility Testing (EUCAST) susceptibility breakpoints available for Ralstonia spp., minimum inhibitory concentration (MIC) results were interpreted using the criteria used for Pseudomonas spp. and Acinetobacter spp. for trimethoprim-sulfamethoxazole.
After the identification of the Ralstonia spp. and the antibiotic susceptibility testing, the initial regimen was discontinued and replaced by tigecycline ( Table 1). The antibiotic was administered intravenously for a total of three months. Capsofungin was also administered for two months to treat a Candida parapsilosis superinfection.

MIC: minimum inhibitory concentration
The postoperative course of the patient was uncomplicated. Two months after the start of the bone transport, partial weight-bearing was allowed and the patient was able to ambulate with the use of crutches. The elongation process lasted for 85 days. Bone healing was uneventful and no further procedures were necessary for consolidation at the docking site ( Figure 2). The external fixation device was removed 10 months postoperatively. The bone transfer was successful with evident callus formation from the third month, while the leg length discrepancy was eliminated. On his last visit, one year after the last operation, the patient is ambulatory with a functional right lower limb and a minor decrease in the hip and knee range of motion (

Discussion
R. mannitolilytica is emerging as an opportunistic human pathogen, mostly implicated in hospital-acquired infections [1]. Although R. mannitolilytica presence in humid environments is common, bone infections are rarely reported. Most cases in literature are nosocomial outbreaks in immunocompromised patients, patients on hemodialysis or with malignancies, and premature infants [2,[4][5][6][7][8][9]. Special attention has been drawn to patients with cystic fibrosis as R. mannitolilytica infection seems to worsen the prognosis of the disease [10,11]. The most common manifestation of the infection is bacteremia related to a central venous catheter that can lead to sepsis, meningitis or peritonitis [1,9].
Despite its low virulence, R. mannitolilytica species has specific characteristics that promote its spread. These microorganisms are capable of creating a polysaccharide matrix cover (biofilm), which enhances their resistance to antibiotics and helps them survive in harsh conditions for a prolonged time [12][13][14]. They can also penetrate 0.2 μm filters that are used for the terminal sterilization of most medical products [4,5]. They have been reported to survive mild hospital disinfectants like chlorohexidine 5% and ethacridine lactate solutions [5,8]. These factors contribute to the dissemination of the bacteria and the contamination of medical equipment (air conditioners) and products (normal saline and other solutions flushed through indwelling devices), resulting in the aforementioned hospital outbreaks [15].
Bone infections by the Ralstonia genus are very rare. Two cases of spondylitis, one case of osteomyelitis of the trapezium of the hand, one case of septic arthritis of the elbow and knee, and four cases of periprosthetic joint infection have been described in the literature so far. The basic features of these bone infections are summarized in Table 2  The biochemical identification of the Ralstonia genus poses special challenges for the microbiologist. The distinction between the species of the genus is not straightforward. Additionally, the bacteria may be misidentified as other closely related species with similar characteristics (mainly of the Burkholderia cepacia complex or Pseudomonas species) [3,9]. Key knowledge for the biochemical distinction between the species of the genus is that R. mannitolilytica metabolizes only mannitol but not nitrate or arabinose, R. insidiosa metabolizes nitrate but not mannitol or arabinose, R. pickettii metabolizes nitrate and arabinose but not mannitol [21]. Recent data suggest that modern spectrometry and gene sequencing techniques are important to avoid these pitfalls. 16s ribosomal DNA based assays are a sensitive and specific methodology for identification. In our case the VITEK 2 automated identification system yielded a rapid and accurate specieslevel identification and, in accordance with other studies, the result was confirmed using the 16S rDNA as the reference method [13,22]. A modern mass spectrometry method (Matrix assisted laser desorption ionization-time of flight -MALDI-TOF technique), on the other hand, is a rapid [12], reliable and low cost method that has contributed to the proper identification of these non-fermenting bacteria [23]. However, most researchers agree that the confirmation by 16s rDNA gene sequencing is the most accurate way to isolate and identify the microbia [3].
The Ralstonia genus seems to have developed resistance against many antibiotic agents. The treatment protocol needs careful planning as the genus produces various enzymes that can hydrolyze antibiotics and resistance to aminoglycosides and beta-lactams is frequently reported [3]. Special attention has been drawn to the rising resistance against many modern antibiotics such as ceftazidime, aztreonam and carbapenems [3,12]. In a recent study, Suzuki et al. identified a species-specific extended spectrum oxacillinase (OXA60) with carbapenem-hydrolyzing properties that contribute to the genus resistance against imipenem and carbapenem [24]. Co-trimoxazole and ciprofloxacin are generally considered effective against the genus, while also tigecycline has been shown to have good in vitro activity against Ralstonia spp. [25]. The drug susceptibility profile of the Ralstonia genus in bone infections found in literature is summarized in Table 3.
In most cases the microorganisms were susceptible to many antibiotics. On the contrary, in our case the R. mannitolilytica presented with a multidrug resistance profile ( Table 1). The selected combination was, at first, cefuroxime and amikacin, which proved to be ineffective. After the identification of the R. mannitolilytica and the susceptibility testing, tigecycline was the only effective antibiotic and was administered for three months. Despite the osteoblastic activity inhibitory potential of tetracyclines, the use of tigecycline was deemed necessary by our institution's infectiology committee for the eradication of the osteomyelitis. Due to the long duration of the treatment, the course was complicated by positive blood cultures of Candida parapsilosis, so caspofungin was added to the given regimen [3].

TABLE 3: Susceptibility profile of Ralstonia bone infections.
It should be noted that the susceptibility data about the genus is limited and based on a small number of case reports, thus no standardized antibiotic susceptibility testing and EUCAST breakpoints exist for the laboratories [13]. In most bone infections caused by the Ralstonia genus, ciprofloxacin or co-trimoxazole was used as the definitive treatment as shown in Table 1, but in our case the isolated Ralstonia genus was found to be resistant to both. Multi-drug resistance is of great concern as these microorganisms act not only as human pathogens, but also as potential reservoirs of resistance genes in hospital settings [8].

Conclusions
The increasing detection of Ralstonia spp. in hospital settings, along with the emergence of multi-resistant strains of R. mannitolilytica, represent a reason of concern, particularly in the case of vulnerable patients. Identification is not straightforward and special attention is essential with the use of modern techniques (16s rDNA gene sequencing and spectometry). Despite that Ralstonia spp. are usually not recognized as major pathogens, their multidrug resistance, biofilm formation potential and ability to survive in the environment are factors that should never be underestimated. Clinicians and microbiologists should pay attention to the potential of these opportunistic microorganisms being the cause of severe bloodstream infections.

Additional Information Disclosures
Human subjects: Consent was obtained or waived by all participants in this study. Conflicts of interest: In compliance with the ICMJE uniform disclosure form, all authors declare the following: Payment/services info: All authors have declared that no financial support was received from any organization for the submitted work. Financial relationships: All authors have declared that they have no financial relationships at present or within the previous three years with any organizations that might have an interest in the submitted work. Other relationships: All authors have declared that there are no other relationships or activities that could appear to have influenced the submitted work.