Pantoea osteomyelitidis sp. nov., a novel human opportunistic pathogen isolated from a patient with chronic osteomyelitis: case report, genomic characterization and literature review

Pantoea osteomyelitidis sp. nov., a novel human opportunistic pathogen isolated from a patient with chronic osteomyelitis: case report, genomic characterization and literature review | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Case Report Pantoea osteomyelitidis sp. nov., a novel human opportunistic pathogen isolated from a patient with chronic osteomyelitis: case report, genomic characterization and literature review Danielle Keidar-Friedman, Daniel Leshin-Carmel, Anka Tsur, Muriel Amsalem, and 3 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-5298108/v1 This work is licensed under a CC BY 4.0 License Status: Posted Version 1 posted You are reading this latest preprint version Abstract Purpose : Pantoea species are increasingly recognized as human opportunistic pathogens. We describe a rare case of osteomyelitis that has been developed over years, with the identification of the causing agent as a novel species of Pantoea . Case presentation: A 37-year-old generally healthy woman presented to our hospital with suspected chronic osteomyelitis. The condition was possibly related to an incident that occurred two decades before the onset of symptoms, involving a tibia fracture with a large open bleeding wound. The patient had undergone two aggressive debridement operations, systemic ciprofloxacin treatment, and local aminoglycoside therapy, eventually exhibiting full recovery. Bone biopsy cultures grew gram-negative coccobacilli that could not be identified by conventional clinical microbiology methods. Whole-genome sequencing and subsequent taxonomic and phylogenetic analyses revealed genetic relatedness to several Pantoea species. Comparative genomic analyses identified conserved antibiotic resistance and virulence genes. Conclusion: A literature review search uncovered only five cases of osteomyelitis caused by Pantoea species that have been reported in the past, all attributed to Pantoea agglomerans . We suggest this new strain belongs to a yet unidentified Pantoea species, which we have named Pantoea osteomyelitidis . sp.nov. The high diversity of Pantoea and the obscured potential pathogenicity of this genus is discussed, emphasizing the need for further research into its clinical relevance. Osteomyelitis Pantoea Whole genome sequencing Bacterial genomics Case report Figures Figure 1 Figure 2 Figure 3 Introduction Pathogen identification in the clinical microbiology laboratory has an important role for diagnosis and treatment. In the past decade, matrix-assisted laser desorption/ionization-time of flight mass spectrometry (MALDI-TOF-MS) has been considered the standard method for bacterial isolate identification. Nevertheless, accurate identification depends on comparisons with its databases of known organisms. Thus, gaps in the database can lead to misidentification or no identification of rare bacteria species [1]. Osteomyelitis is an inflammation of the bone caused by a pyogenic microorganism. Chronic osteomyelitis primarily occurs in adults, developing over months or years, and is usually secondary to an open wound resulting from an injury that involves both the bone and the surrounding tissues. Diagnosis of the infective agent is crucial to ensure appropriate antimicrobial treatment, and the type of microorganism varies depending on the form of osteomyelitis. The most common microorganism in any form of osteomyelitis is Staphylococcus aureus . Those isolated from foreign-body-associated infections are mostly coagulase-negative staphylococci or Propionibacterium spp [2,3]. So far, only a few cases of osteomyelitis caused by Pantoea species have been reported [4]. Pantoea are gram-negative rod-shaped bacteria in the Erwiniaceae family. While some are known plant commensals, others are considered plant pathogens or opportunistic human pathogens, with Pantoea agglomerans as the most common opportunistic pathogen in both host groups [5]. Infections caused by Pantoea are relatively uncommon and are usually related to contamination of wounds in injuries that have taken place in a vegetative environment. However, there are also numerous reports of hospital-acquired infections resulting from contaminated medical instruments or intravenous nutrition [4]. In this study, we describe a case of an osteomyelitis infection in a healthy adult, in which bacterial isolates from biopsy cultures were not identified by MALDI-TOF-MS. Sequencing of the 16s rDNA gene has only been able to reveal this strain belongs to the Erwiniaceae family. The taxonomic and phylogenetic analyses of the bacterial whole genome sequencing suggested it belongs to the genus Pantoea , however, no known species could be identified. We have used comparative genomics and different gene-prediction analyses to further characterize this new species and to better understand its potential pathogenicity. Clinical case A generally healthy 37-year-old woman first presented to our hospital complaining of five months of left leg pain exacerbated by physical activity, tenderness, and swelling, without fever. Past medical history was notable for a tibial fracture at the age of 15, sustained by a fall over a piece of wood in the field, and accompanied by a skin laceration. This fracture was treated conservatively, and the patient did not recall if antimicrobial treatment was prescribed. After an asymptomatic period of approximately 20 years, she began feeling a dull ache in her shin that was exacerbated over two years. Physical examination revealed a mild limp, tenderness, and swelling in the proximal 1/3 of her left Tibia. Blood analysis showed mild leukocytosis (11,400 cells/mL, range 4000-11000) and normal C-reactive protein (CRP) (2.2 mg/L, range 0-5). Magnetic resonance imaging (MRI) showed intra-medullary edema in the proximal tibia with enhancement following Gadolinium injection, a breach of the frontal and lateral bone cortex, and an abscess formation in the subdermal fat. The patient was admitted for a debridement operation, with no antimicrobial treatment before surgery. Pathological bone was noted by the orthopedic team, and multiple microbiology and pathology specimens were collected. The pathology report showed fragments of bone and fibrous tissue with mostly lymphoplasmacytic inflammatory infiltrate, hemorrhage, and focally purulent granulation tissue. Few areas showed new bone formation with osteoid and surrounding osteoblasts, features compatible with chronic osteomyelitis. Bacterial cultures were positive in 4/8 of bone and soft tissue samples. Pathogen identification The biopsy samples were plated on TSA 5% sheep blood agar, chocolate agar, and MacConkey agar plates (Hylab, Rehovot, Israel) at 37°c in aerobic conditions overnight and presented white-grey circular smooth colonies. Gram staining showed gram-negative coccobacilli ( Fig. 1 ). Phenotypic tests (Methods in Supp. material A ) showed this strain is catalase-negative, oxidase-negative, coagulase-negative, and Indole-negative. Colonies were tested using matrix-assisted laser desorption/ionization-time of flight mass spectrometry (MALDI-TOF MS) on VITEK® MS (bioMérieux, Marcy-l'Étoile, France) which resulted in no identification of any known species. Antibiotic susceptibility based on gram-negative CLSI breakpoints and biochemical tests ( Supp.Table 1 ) were performed on VITEK®2 (bioMérieux, Marcy-l'Étoile, France) and indicated sensitivity to all antibiotics tested including amoxicillin/clavulanic Acid, cefuroxime, chloramphenicol, ampicillin, ceftazidime, ceftriaxone, ciprofloxacin, gentamicin, piperacillin/tazobactam, trimethoprim/sulfamethoxazole, meropenem, amikacin, and ertapenem. DNA was extracted from colonies and the 16s rRNA gene was amplified and sequenced by Sanger sequencing. BLASTN analysis of the resulting sequence against GenBank generated good hits to different species of Erwiniaceae . Therefore, we could only conclude that this strain belongs to Erwiniaceae . Whole genome sequencing reads (Methods described in Supp. material A ) were assembled to generate 17 contigs (N50 = 570,650), the longest contig sized 1,854,057, a total length of 4,314,591 bps, and 55.3% GC content. Taxonomic analyses performed using TYGS (Type strain genome server [6] and GTDB-Tk Classify genomes 2.3.2 [7] could not assign a known species and found the closest species found were Pantoea superficialis (d 0 =59.5% & d 4 = 33% dDDH values by TYGS and 87.6% Average Nucleotide Identity (ANI) by GTDB-Tk), followed by Pantoea mediterraneensis (d 0 =46%, d 4 =26.9% dDDH and 84% ANI). dDDH values of over 70% and ANI values of over 95% indicate the same species [6,8], suggesting our strain belongs to a still unknown species of Pantoea , which we have named Pantoea osteomyelitidis. Pan-genome analysis was performed using Roary based on the core genome alignment of Pantoea osteomyelitidis. sp.nov with known Pantoea species according to Crosby et al [5] and additional recently identified species (GenBank accessions in Supp. table 1, full methods in Supp. material A ). This analysis identified a total of 47,455 genes; 1,464 core genes (found in 90% of the genomes), 4,185 shell genes (found in 15-89% of the genomes) and 41,806 cloud genes (found in 15% or less of the genomes). The phylogenetic tree based on core genes ( Fig. 2 ) showed high diversity within Pantoea , with many distinct lineages. Pantoea osteomyelitidis sp.nov clustered with P. superficialis and P. mediterraneensis . However, there is a divergence between the common ancestor of this clade and the common ancestor of all other Pantoea species on the tree, indicating the evolutionary distance between this clade and the other clades. Average nucleotide identity values based on BLASTN (ANIb) are shown as a heatmap in Fig.3 . This analysis aligns mostly with the subclades within the phylogenetic tree generated according to core genome, as can be seen by values of over 80%. Fig.3 also presents the isolation source of each species according to all reports made in GenBank. Overall, 12 species have been isolated from human samples, some depicted as pathogens and others recovered from healthy tissues. Of these, only 3 have been isolated from human samples only ( P. osteomyelitidis sp. nov, P. mediterraneensis and P. mediterraneensis _A), and the rest have been isolated from additional hosts or environments ( P. eucrina , P. dispersa , P. conspicua , P. brenneri , P. piersonii , P. septica , P. alvi , P. anthophila and P. agglomerans ). The gene prediction analysis of Pantoea osteomyelitidis sp.nov using Prokka version 1.14.6 [9] identified a total of 4,084 genes, comprising 3,294 proetin-coding sequences. We tested for the presence of antibiotic-resistance and virulence genes (methods in Supp. material A ) and identified oqxB and CRP presence. OqxB codes for a multidrug efflux RND transporter permease subunit. Overexpression of OqxB can confer resistance to various antibiotics [10,11]. CRP gene codes for a regulator that represses MdtEF multidrug efflux pump expression, and mutations in this gene were found to be associated with increased resistance [12]. The following virulence factors were detected: FliG, FliM, FliP, Nlpl, OmpA, and EC55989_3335. FliG and FliM are associated with the flagellar motor switch, FliP is involved in flagellar biosynthesis, NlpI codes for Lipoprotein NlpI [13]. OmpA gene codes for porin outer membrane protein A, known for its multiple functions including biofilm formation and a role in F plasmid cell conjugation [14]. EC55989_3335 codes for Hcp family type VI secretion system effector [13]. Our results from the Pan-genome analysis revealed the presence of OqxB, CRP, Nlpl, FliG, FliM, FliP and ompA within the core gene group, indicating these genes are evolutionary conserved within this genus ( Supp. table.2 ). An analysis of potential pathogenic proteins using PathogenFinder suggested a non-human pathogen with a 0.47 likelihood of this species to be pathogenic, by identifying 16 pathogenic protein families identified, compared to 17 non-pathogenic protein families. By examining all the Pantoea genomes used in our analysis, only 3 were determined by PathogenFinder as human pathogens: P. alvi (0.576), P. mediterraneensis_A (0.535) and P. bathycoeliaeme (0.585). While P. alvi and P. mediterraneensis_A have been isolated from healthy human samples, P. bathycoeliaeme has so far been isolated only from insects.Although this analysis does not suggest a clear pathogenicity potential for Pantoea osteomyelitidis sp.nov., other Pantoea species that are considered opportunistic human pathogens were also determined as non-human pathogens ( Supp. table 3 ). Patient treatment and follow-up Four months following her open bone biopsy, and upon identification of the causative organism, the patient underwent extensive debridement with bone cement supplement using calcium sulfate resorbable beads with tobramycin. One out of 5 tissue specimens were again positive for Pantoea osteomyelitidis sp.nov. She was treated with oral Ciprofloxacin 750 mg bid for four weeks. Six months after her surgery, the patient reported complete healing. Discussion Pantoea comprises a highly diverse group of gram-negative bacteria in the Erwiniaceae family, inhabiting various aquatic or terrestrial environments and interacting with different hosts as commensals or pathogens. Due to its varied roles, Pantoea' s potential as a human pathogen remains obscure. Although infrequently identified in clinical microbiology laboratories, there are accumulating reports of infections caused by Pantoea species, mainly of bacteremia and septicemia cases, however, there are also some reports of meningitis, pneumonia, and joint infections [4]. So far, only 5 cases have been reported in the literature as osteomyelitis or osteitis caused by Pantoea , all have identified P. agglomerans by phenotypic methods from cultures or by 16s rDNA sequencing (Table 1 ), [15–19]. Since these methods offer relatively low resolution for Pantoea delineation, it is possible that P. agglomerans was the closest species detected. In this study, we utilized whole-genome sequencing to characterize an unknown bacteria isolated from multiple bone biopsies, in which conventional clinical microbiology methods could not identify. We discovered a previously unknown species related to Pantoea , that caused a chronic osteomyelitis infection in a healthy adult, twenty years after an injury that led to a tibia fracture and a large open bleeding wound. Despite the prediction of two genes with potential antibiotic resistance (OqxB and CRP regulator), the antibiogram tests showed susceptibility to all antibiotics according to CLSI breakpoints and the patient exhibited full recovery after aggressive debridement, systemic ciprofloxacin, and local aminoglycoside treatment. This, together with the conserved nature of these genes in other Pantoea genomes examined in our analysis, suggests the presence of OqxB and CRP regulator alone cannot predict resistance to antibiotics. Our taxonomic analyses have placed the new strain within the genus Pantoea . The phylogenetic analysis revealed Pantoea osteomyelitidis sp.nov genetic relationship to P. mediterraneensis which has been isolated from healthy human skin, and P. superficialis which has been assembled from a metagenome of an urban environmental sample. Nonetheless, the phylogenic analysis also indicated distinct evolutionary divergence of this clade from the common ancestor of other Pantoea clades. Thus, it remains plausible that this clade could belong to another genus within the Erwiniaceae family, not yet classified. Our results of the pathogenicity potential highlight the challenges in determining potential pathogenicity by genomic characteristics only. Studies that have compared the potential virulence of clinical versus environmental Pantoea isolates reported similar colonization patterns in plants, insects, and animal models, suggesting the pathogenicity cannot be predicted by phylogeny [20,21]. The broad host range of Pantoea , along with their use as biocontrol agents in agriculture, can drive a selection pressure leading to the acquisition of pathogenicity factors, thereby enhancing host-specific adaptation [20]. Based on its isolation from bone biopsies from a focus on chronic osteomyelitis, we suggest Pantoea osteomyelitidis sp.nov. as an opportunistic human pathogen. This study highlights the pivotal role of advanced sequencing methods in clinical microbiology when conventional techniques offer low-resolution identification. Identifying new human opportunistic pathogens is essential for public health, aiding epidemiological surveillance and guiding medical treatment options. Moreover, tracking the evolutionary dynamics of bacterial groups with pathogenic potential can help predict the emergence and spread of novel pathogens. Further studies are needed to elucidate the impact of members of the Erwiniaceae family on human health, with a focus on their pathogenic mechanisms and the risk factors associated with infections. Declarations Data availability This Whole Genome Shotgun project has been deposited at DDBJ/ENA/GenBank under the accession JBGFSN000000000, Bioproject accession PRJNA1127075. The version described in this paper is version JBGFSN010000000. The 16S rRNA sequence was deposited under GenBank number PP956810-PP956811. Conflict of Interest The authors have no competing interest to declare. Funding Source The project received no external financial support. Ethical Approval statement All clinical samples and data were collected during routine patient care. No identification details, personal information, or images are presented in this study. The patient’s identity is anonymized. The patient has given written consent for the clinical case description. References Soutar CD, Stavrinides J. Molecular validation of clinical Pantoea isolates identified by MALDI-TOF. PLOS ONE. 2019;14:e0224731. doi:10.1371/journal.pone.0224731 Carek PJ, Dickerson LM, Sack JL. Diagnosis and Management of Osteomyelitis. afp. 2001;63:2413–21. Francis A Waldvogel DPL. Osteomyelitis - The Lancet [Internet]. 2004 [cited 2024 Jul 29]. Available from: https://www.thelancet.com/article/S0140-6736(04)16727-5/abstract Walterson AM, Stavrinides J. Pantoea: insights into a highly versatile and diverse genus within the Enterobacteriaceae. FEMS Microbiology Reviews. 2015;39:968–84. doi:10.1093/femsre/fuv027 Crosby KC, Rojas M, Sharma P, Johnson MA, Mazloom R, Kvitko BH, et al. Genomic delineation and description of species and within-species lineages in the genus Pantoea. Front Microbiol. 2023;14. doi:10.3389/fmicb.2023.1254999 Meier-Kolthoff JP, Göker M. TYGS is an automated high-throughput platform for state-of-the-art genome-based taxonomy. 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JSpeciesWS: a web server for prokaryotic species circumscription based on pairwise genome comparison. Bioinformatics. 2016;32:929–31. doi:10.1093/bioinformatics/btv681 Table Table 1. Literature review of osteomyelitis cases caused by Pantoea . Publication Gender Age Infection site Time from injury to symptom onset Infection source Diagnosis Pathogen Identification method Vincent, K., & Szabo, R. M. (1988). M 8 hand, radio-carpal joint 14 days Rose thorn Acute humeral osteomyelitis and septic arthritis P. agglomerans Cultures, unspecified Laporte, C., et al. (2002). M 56 Tibia 9 months Soil and plant contamination of open fracture Chronic tibial osteitis P. agglomerans Cultures + API gallery Bachmeyer, C., et al.(2007) M 20 Both tibias unknown unknown Chronic osteomyelitis P. agglomerans 16s rDNA seq Labianca, Luca, et al. (2013) F 8 Ulna and radius 30 days unknown - closed fracture with no foreign body found Acute hematogenous osteomyelitis P. agglomerans Cultures, unspecified Chen, C. N., & Lee, B. C. (2015) M 10 Elbow 4 months Aloe vera plant Chronic osteomyelitis P. agglomerans Cultures + 16s rDNA seq Additional Declarations No competing interests reported. Supplementary Files SupplementalmaterialA.docx SupplementaryTables.xlsx Cite Share Download PDF Status: Posted Version 1 posted You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. We do this by developing innovative software and high quality services for the global research community. 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Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-5298108","acceptedTermsAndConditions":true,"allowDirectSubmit":true,"archivedVersions":[],"articleType":"Case Report","associatedPublications":[],"authors":[{"id":368690721,"identity":"37f2e927-05e7-466c-a28e-fdc14ad4506a","order_by":0,"name":"Danielle 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11:23:21","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-5298108/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-5298108/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":67174257,"identity":"9e9a3bff-dad2-4360-8f2a-c2babbb9dd02","added_by":"auto","created_at":"2024-10-22 04:39:44","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":297666,"visible":true,"origin":"","legend":"\u003cp\u003eGram stain and bacterial colonies of \u003cem\u003ePantoea osteomyelitidis\u003c/em\u003e\u003cem\u003e\u003cstrong\u003e \u003c/strong\u003e\u003c/em\u003esp. nov. on different agar plates.\u003cstrong\u003e \u003c/strong\u003eGram stain and growth of \u003cem\u003ePantoea osteomyelitidis\u003c/em\u003e\u003cem\u003e\u003cstrong\u003e \u003c/strong\u003e\u003c/em\u003ecolonies on TSA 5% sheep blood agar plates, chocolate agar, and MacConkey agar plates after 24 hours incubation at 37°c. \u003cbr\u003e\nPlate images were taken using a Nikon D7500 camera with a 50 mm lens and adjusted for brightness and contrast using GIMP 2.10.36.\u003c/p\u003e","description":"","filename":"1.png","url":"https://assets-eu.researchsquare.com/files/rs-5298108/v1/5f6bbe5f77f01cf5008e233e.png"},{"id":67174255,"identity":"66ded830-8dcf-4359-a723-a10793c2d36a","added_by":"auto","created_at":"2024-10-22 04:39:44","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":466172,"visible":true,"origin":"","legend":"\u003cp\u003eA maximum-likelihood phylogenetic tree based on core genome alignment of \u003cem\u003ePantoea osteomyelitidis\u003c/em\u003e\u003cem\u003e\u003cstrong\u003e \u003c/strong\u003e\u003c/em\u003esp.nov with 45 other \u003cem\u003ePantoea\u003c/em\u003e species.\u003cstrong\u003e \u003cbr\u003e\n \u003c/strong\u003eThe tree depicts the evolutionary relationships between the species within the \u003cem\u003ePantoea\u003c/em\u003e genus.\u003cem\u003e Mixta calida\u003c/em\u003e was added as an outgroup. The new species identified, \u003cem\u003ePantoea osteomyelitidis\u003c/em\u003e\u003cem\u003e\u003cstrong\u003e \u003c/strong\u003e\u003c/em\u003esp.nov, is highlighted in the yellow clade. \u003cem\u003ePantoea osteomyelitidis\u003c/em\u003e\u003cem\u003e\u003cstrong\u003e \u003c/strong\u003e\u003c/em\u003esp.nov. and its closest relatives (\u003cem\u003ePantoea\u003c/em\u003e \u003cem\u003esuperficialis\u003c/em\u003e and \u003cem\u003ePantoea\u003c/em\u003e \u003cem\u003emediterraneensis\u003c/em\u003e) share a common ancestor, distinct from that of other \u003cem\u003ePantoea\u003c/em\u003e species.\u003cbr\u003e\nThe tree was inferred with IQ-TREE version 1.5.5 [22]with maximum likelihood and ultrafast bootstrapping [23]. The best-fit model according to AIC was GTR+R5. The tree was edited using iTOL. Bootstrap values of 95% and above are indicated by stars at the nodes, signifying the reliability of the branching.\u003c/p\u003e","description":"","filename":"2.png","url":"https://assets-eu.researchsquare.com/files/rs-5298108/v1/c28f4f37bb53dff8c35d2a17.png"},{"id":67174976,"identity":"9f7d0420-2000-4b93-ab59-d5a4ce963b76","added_by":"auto","created_at":"2024-10-22 04:47:44","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":320106,"visible":true,"origin":"","legend":"\u003cp\u003eA phylogenetic tree with the host/origin of each species and a heatmap of average nucleotide identity (ANIb) values.\u003cstrong\u003e \u003c/strong\u003e\u003cbr\u003e\nThe tree was inferred with IQ-TREE and edited using iTOL. The tree scale bar (0.1) indicates the genetic distance (longer branches indicate greater evolutionary distance). Colored triangles represent the isolation source of each species according to the information stated for isolates and genomes found in the NCBI GenBank database and in Crosby et al., 2023 [5]: Red triangles represent human hosts, blue triangles represent recovery from different environmental specimens or metagenomes, green triangles represent plant hosts, yellow represent insect host, pink represent fungi host, and purple represent animal hosts. \u003cbr\u003e\nThe heatmap shows ANIb values (Average nucleotide identity by BLASTN) from genomic comparisons conducted using JSpecies [24], ranging between 73-100%. Most ANIb values shown are in the range of 75-80%, while subclades show higher values of 80% and more.\u003c/p\u003e","description":"","filename":"3.png","url":"https://assets-eu.researchsquare.com/files/rs-5298108/v1/4cfef15afc2ace1778061523.png"},{"id":67175923,"identity":"a689535b-7ddc-4705-b7ed-26711feaf286","added_by":"auto","created_at":"2024-10-22 04:55:44","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":1484550,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-5298108/v1/d43c8b01-7c1b-4561-a3fe-a83313bc174f.pdf"},{"id":67174256,"identity":"6496cf5e-d315-488e-8896-1d3e2d6c8e97","added_by":"auto","created_at":"2024-10-22 04:39:44","extension":"docx","order_by":1,"title":"","display":"","copyAsset":false,"role":"supplement","size":32969,"visible":true,"origin":"","legend":"","description":"","filename":"SupplementalmaterialA.docx","url":"https://assets-eu.researchsquare.com/files/rs-5298108/v1/b32ec47b987639b3c2fbd0c5.docx"},{"id":67174259,"identity":"14ef6cb5-aa3c-49d9-a927-aaf99ed5cea7","added_by":"auto","created_at":"2024-10-22 04:39:48","extension":"xlsx","order_by":2,"title":"","display":"","copyAsset":false,"role":"supplement","size":7469418,"visible":true,"origin":"","legend":"","description":"","filename":"SupplementaryTables.xlsx","url":"https://assets-eu.researchsquare.com/files/rs-5298108/v1/fe46333671af4b5b0f7b7e49.xlsx"}],"financialInterests":"No competing interests reported.","formattedTitle":"Pantoea osteomyelitidis sp. nov., a novel human opportunistic pathogen isolated from a patient with chronic osteomyelitis: case report, genomic characterization and literature review","fulltext":[{"header":"Introduction","content":"\u003cp\u003ePathogen identification in the clinical microbiology laboratory has an important role for diagnosis and treatment. In the past decade, matrix-assisted laser desorption/ionization-time of flight mass spectrometry (MALDI-TOF-MS) has been considered the standard method for bacterial isolate identification. Nevertheless, accurate identification depends on comparisons with its databases of known organisms. Thus, gaps in the database can lead to misidentification or no identification of rare bacteria species\u0026nbsp;[1]. \u0026nbsp;\u003c/p\u003e\n\u003cp\u003eOsteomyelitis is an inflammation of the bone caused by a pyogenic microorganism. Chronic osteomyelitis primarily occurs in adults, developing over months or years, and is usually secondary to an open wound resulting from an injury that involves both the bone and the surrounding tissues. Diagnosis of the infective agent is crucial to ensure appropriate antimicrobial treatment, and the type of microorganism varies depending on the form of osteomyelitis. The most common microorganism in any form of osteomyelitis is \u003cem\u003eStaphylococcus aureus\u003c/em\u003e. Those isolated from foreign-body-associated infections are mostly coagulase-negative \u003cem\u003estaphylococci\u003c/em\u003e or \u003cem\u003ePropionibacterium\u003c/em\u003e spp\u0026nbsp;[2,3]. So far, only a few cases of osteomyelitis caused by \u003cem\u003ePantoea\u003c/em\u003e species have been reported\u0026nbsp;[4].\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cem\u003ePantoea\u003c/em\u003e are gram-negative rod-shaped bacteria in the \u003cem\u003eErwiniaceae\u0026nbsp;\u003c/em\u003efamily. While some are known plant commensals, others are considered plant pathogens or opportunistic human pathogens, with \u003cem\u003ePantoea agglomerans\u003c/em\u003e as the most common opportunistic pathogen in both host groups\u0026nbsp;[5]. Infections caused by \u003cem\u003ePantoea\u003c/em\u003e are relatively uncommon and are usually related to contamination of wounds in injuries that have taken place in a vegetative environment. However, there are also numerous reports of hospital-acquired infections resulting from contaminated medical instruments or intravenous nutrition\u0026nbsp;[4].\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eIn this study, we describe a case of an osteomyelitis infection in a healthy adult, in which bacterial isolates from biopsy cultures were not identified by MALDI-TOF-MS. Sequencing of the 16s rDNA gene has only been able to reveal this strain belongs to the \u003cem\u003eErwiniaceae\u0026nbsp;\u003c/em\u003efamily. The taxonomic and phylogenetic analyses of the bacterial whole genome sequencing suggested it belongs to the genus \u003cem\u003ePantoea\u003c/em\u003e, however, no known species could be identified. We have used comparative genomics and different gene-prediction analyses to further characterize this new species and to better understand its potential pathogenicity.\u0026nbsp;\u003c/p\u003e"},{"header":"Clinical case","content":"\u003cp\u003eA generally healthy 37-year-old woman first presented to our hospital complaining of five months of left leg pain exacerbated by physical activity, tenderness, and swelling, without fever. Past medical history was notable for a tibial fracture at the age of 15, sustained by a fall over a piece of wood in the field, and accompanied by a skin laceration. This fracture was treated conservatively, and the patient did not recall if antimicrobial treatment was prescribed. After an asymptomatic period of approximately 20 years, she began feeling a dull ache in her shin that was exacerbated over two years. Physical examination revealed a mild limp, tenderness, and swelling in the proximal 1/3 of her left Tibia. Blood analysis showed mild leukocytosis (11,400 cells/mL, range 4000-11000) and normal C-reactive protein (CRP) (2.2 mg/L, range 0-5). Magnetic resonance imaging (MRI) showed intra-medullary edema in the proximal tibia with enhancement following Gadolinium injection, a breach of the frontal and lateral bone cortex, and an abscess formation in the subdermal fat. The patient was admitted for a debridement operation, with no antimicrobial treatment before surgery. Pathological bone was noted by the orthopedic team, and multiple microbiology and pathology specimens were collected. The pathology report showed fragments of bone and fibrous tissue with mostly lymphoplasmacytic inflammatory infiltrate, hemorrhage, and focally purulent granulation tissue. Few areas showed new bone formation with osteoid and surrounding osteoblasts, features compatible with chronic osteomyelitis. Bacterial cultures were positive in 4/8 of bone and soft tissue samples.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003ePathogen identification\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe biopsy samples were plated on TSA 5% sheep blood agar, chocolate agar, and MacConkey agar plates (Hylab, Rehovot, Israel) at 37°c in aerobic conditions overnight and presented white-grey circular smooth colonies. Gram staining showed gram-negative coccobacilli (\u003cstrong\u003eFig. 1\u003c/strong\u003e). Phenotypic tests (Methods in \u003cstrong\u003eSupp. material A\u003c/strong\u003e) showed this strain is catalase-negative, oxidase-negative, coagulase-negative, and Indole-negative. Colonies were tested using matrix-assisted laser desorption/ionization-time of flight mass spectrometry (MALDI-TOF MS) on VITEK® MS (bioMérieux, Marcy-l'Étoile, France) which resulted in no identification of any known species. Antibiotic susceptibility based on gram-negative CLSI\u0026nbsp;breakpoints and biochemical tests (\u003cstrong\u003eSupp.Table 1\u003c/strong\u003e) were performed on VITEK®2 (bioMérieux, Marcy-l'Étoile, France) and indicated sensitivity to all antibiotics tested including amoxicillin/clavulanic Acid, cefuroxime, chloramphenicol, ampicillin, ceftazidime, ceftriaxone, ciprofloxacin, gentamicin, piperacillin/tazobactam, trimethoprim/sulfamethoxazole, meropenem, amikacin, and ertapenem. \u0026nbsp;\u003c/p\u003e\n\u003cp\u003eDNA was extracted from colonies and the 16s rRNA gene was amplified and sequenced by Sanger sequencing. BLASTN analysis of the resulting sequence against GenBank generated good hits to different species of \u003cem\u003eErwiniaceae\u003c/em\u003e. Therefore, we could only conclude that this strain belongs to \u003cem\u003eErwiniaceae\u003c/em\u003e. Whole genome sequencing reads (Methods described in \u003cstrong\u003eSupp. material A\u003c/strong\u003e) were assembled to generate 17 contigs (N50 = 570,650), the longest contig sized 1,854,057, a total length of 4,314,591 bps, and 55.3% GC content. Taxonomic analyses performed using TYGS (Type strain genome server\u0026nbsp;[6]\u0026nbsp;and GTDB-Tk Classify genomes 2.3.2\u0026nbsp;[7]\u0026nbsp;could not assign a known species and found the closest species found were \u003cem\u003ePantoea superficialis\u003c/em\u003e (d\u003csub\u003e0\u003c/sub\u003e=59.5% \u0026amp; d\u003csub\u003e4\u003c/sub\u003e= 33% dDDH values by TYGS and 87.6% Average Nucleotide Identity (ANI) by GTDB-Tk), followed by \u003cem\u003ePantoea\u003c/em\u003e \u003cem\u003emediterraneensis\u003c/em\u003e (d\u003csub\u003e0\u003c/sub\u003e=46%, d\u003csub\u003e4\u003c/sub\u003e=26.9% dDDH and 84% ANI). dDDH values of over 70% and ANI values of over 95% indicate the same species\u0026nbsp;[6,8], suggesting our strain belongs to a still unknown species of \u003cem\u003ePantoea\u003c/em\u003e, which we have named \u003cem\u003ePantoea osteomyelitidis.\u003c/em\u003e\u0026nbsp;\u003c/p\u003e\n\u003cp\u003ePan-genome analysis was performed using Roary based on the core genome alignment of\u003cem\u003e\u0026nbsp;Pantoea osteomyelitidis.\u003c/em\u003e sp.nov with known \u003cem\u003ePantoea\u003c/em\u003e species according to Crosby et al\u0026nbsp;[5]\u0026nbsp;and additional recently identified species (GenBank accessions in \u003cstrong\u003eSupp. table 1,\u0026nbsp;\u003c/strong\u003efull methods in \u003cstrong\u003eSupp. material A\u003c/strong\u003e). This analysis identified a total of 47,455 genes; 1,464 core genes (found in 90% of the genomes), 4,185 shell genes (found in 15-89% of the genomes) and 41,806 cloud genes (found in 15% or less of the genomes).\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eThe phylogenetic tree based on core genes (\u003cstrong\u003eFig. 2\u003c/strong\u003e) showed high diversity within \u003cem\u003ePantoea\u003c/em\u003e, with many distinct lineages. \u003cem\u003ePantoea osteomyelitidis\u003c/em\u003esp.nov clustered with \u003cem\u003eP. superficialis\u003c/em\u003e and \u003cem\u003eP. mediterraneensis\u003c/em\u003e. However, there is a divergence between the common ancestor of this clade and the common ancestor of all other \u003cem\u003ePantoea\u003c/em\u003e species on the tree, indicating the evolutionary distance between this clade and the other clades.\u003c/p\u003e\n\u003cp\u003eAverage nucleotide identity values based on BLASTN (ANIb) are shown as a heatmap in\u0026nbsp;\u003cstrong\u003eFig.3\u003c/strong\u003e. This analysis aligns mostly with the subclades within the phylogenetic tree generated according to core genome, as can be seen by values of over 80%.\u0026nbsp;\u003cbr\u003e\u003cstrong\u003eFig.3\u0026nbsp;\u003c/strong\u003ealso presents the isolation source of each species according to all reports made in GenBank. Overall, 12 species have been isolated from human samples, some depicted as pathogens and others recovered from healthy tissues. Of these, only 3 have been isolated from human samples only (\u003cem\u003eP.\u003c/em\u003e \u003cem\u003eosteomyelitidis\u003c/em\u003esp. nov, \u003cem\u003eP.\u003c/em\u003e \u003cem\u003emediterraneensis\u003c/em\u003e and \u003cem\u003eP.\u003c/em\u003e \u003cem\u003emediterraneensis\u003c/em\u003e_A), and the rest have been isolated from additional hosts or environments (\u003cem\u003eP. eucrina\u003c/em\u003e, \u003cem\u003eP. dispersa\u003c/em\u003e, \u003cem\u003eP. conspicua\u003c/em\u003e, \u003cem\u003eP. brenneri\u003c/em\u003e, \u003cem\u003eP. piersonii\u003c/em\u003e, \u003cem\u003eP. septica\u003c/em\u003e, \u003cem\u003eP. alvi\u003c/em\u003e, \u003cem\u003eP. anthophila\u003c/em\u003e and \u003cem\u003eP. agglomerans\u003c/em\u003e).\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eThe gene prediction analysis of \u003cem\u003ePantoea osteomyelitidis\u003c/em\u003esp.nov using Prokka version 1.14.6\u0026nbsp;[9]\u0026nbsp;identified a total of 4,084 genes, comprising 3,294 proetin-coding sequences. We tested for the presence of antibiotic-resistance and virulence genes (methods in \u003cstrong\u003eSupp. material A\u003c/strong\u003e) and identified oqxB and CRP presence. OqxB codes for a multidrug efflux RND transporter permease subunit. Overexpression of OqxB can confer resistance to various antibiotics\u0026nbsp;[10,11]. CRP gene codes for a regulator that represses MdtEF multidrug efflux pump expression, and mutations in this gene were found to be associated with increased resistance\u0026nbsp;[12]. The following virulence factors were detected: FliG, FliM, FliP, Nlpl, OmpA, and EC55989_3335. FliG and FliM are associated with the flagellar motor switch, FliP is involved in flagellar biosynthesis, NlpI codes for Lipoprotein NlpI\u0026nbsp;[13]. OmpA gene codes for porin outer membrane protein A, known for its multiple functions including biofilm formation and a role in F plasmid cell conjugation\u0026nbsp;[14]. EC55989_3335 codes for Hcp family type VI secretion system effector\u0026nbsp;[13]. Our results from the Pan-genome analysis revealed the presence of OqxB, CRP, Nlpl, FliG, FliM, FliP and ompA within the core gene group, indicating these genes are evolutionary conserved within this genus (\u003cstrong\u003eSupp. table.2\u003c/strong\u003e).\u003c/p\u003e\n\u003cp\u003eAn analysis of potential pathogenic proteins using PathogenFinder suggested a non-human pathogen with a 0.47 likelihood of this species to be pathogenic, by identifying 16 pathogenic protein families identified, compared to 17 non-pathogenic protein families. By examining all the \u003cem\u003ePantoea\u003c/em\u003e genomes used in our analysis, only 3 were determined by PathogenFinder as human pathogens: \u003cem\u003eP. alvi\u0026nbsp;\u003c/em\u003e(0.576), \u003cem\u003eP. mediterraneensis_A\u003c/em\u003e (0.535) and \u003cem\u003eP. bathycoeliaeme\u0026nbsp;\u003c/em\u003e(0.585). While \u003cem\u003eP. alvi\u0026nbsp;\u003c/em\u003eand \u003cem\u003eP. mediterraneensis_A\u003c/em\u003e have been isolated from healthy human samples, \u003cem\u003eP. bathycoeliaeme\u0026nbsp;\u003c/em\u003ehas so far been isolated only from insects.Although this analysis does not suggest a clear pathogenicity potential for \u003cem\u003ePantoea osteomyelitidis\u003c/em\u003esp.nov., other \u003cem\u003ePantoea\u003c/em\u003e species that are considered opportunistic human pathogens were also determined as non-human pathogens (\u003cstrong\u003eSupp. table 3\u003c/strong\u003e).\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003ePatient treatment and follow-up\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eFour months following her open bone biopsy, and upon identification of the causative organism, the patient underwent extensive debridement with bone cement supplement using calcium sulfate resorbable beads with tobramycin. One out of 5 tissue specimens were again positive for\u0026nbsp;\u003cem\u003ePantoea osteomyelitidis\u003c/em\u003esp.nov. She was treated with oral Ciprofloxacin 750 mg bid for four weeks. Six months after her surgery, the patient reported complete healing.\u003cbr\u003e\u0026nbsp;\u003c/p\u003e"},{"header":"Discussion","content":"\u003cp\u003e \u003cem\u003ePantoea\u003c/em\u003e comprises a highly diverse group of gram-negative bacteria in the \u003cem\u003eErwiniaceae\u003c/em\u003e family, inhabiting various aquatic or terrestrial environments and interacting with different hosts as commensals or pathogens. Due to its varied roles, \u003cem\u003ePantoea'\u003c/em\u003es potential as a human pathogen remains obscure. Although infrequently identified in clinical microbiology laboratories, there are accumulating reports of infections caused by \u003cem\u003ePantoea\u003c/em\u003e species, mainly of bacteremia and septicemia cases, however, there are also some reports of meningitis, pneumonia, and joint infections [4]. So far, only 5 cases have been reported in the literature as osteomyelitis or osteitis caused by \u003cem\u003ePantoea\u003c/em\u003e, all have identified \u003cem\u003eP. agglomerans\u003c/em\u003e by phenotypic methods from cultures or by 16s rDNA sequencing (Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e), [15\u0026ndash;19]. Since these methods offer relatively low resolution for \u003cem\u003ePantoea\u003c/em\u003e delineation, it is possible that \u003cem\u003eP. agglomerans\u003c/em\u003e was the closest species detected.\u003c/p\u003e \u003cp\u003eIn this study, we utilized whole-genome sequencing to characterize an unknown bacteria isolated from multiple bone biopsies, in which conventional clinical microbiology methods could not identify. We discovered a previously unknown species related to \u003cem\u003ePantoea\u003c/em\u003e, that caused a chronic osteomyelitis infection in a healthy adult, twenty years after an injury that led to a tibia fracture and a large open bleeding wound.\u003c/p\u003e \u003cp\u003eDespite the prediction of two genes with potential antibiotic resistance (OqxB and CRP regulator), the antibiogram tests showed susceptibility to all antibiotics according to CLSI breakpoints and the patient exhibited full recovery after aggressive debridement, systemic ciprofloxacin, and local aminoglycoside treatment. This, together with the conserved nature of these genes in other \u003cem\u003ePantoea\u003c/em\u003e genomes examined in our analysis, suggests the presence of OqxB and CRP regulator alone cannot predict resistance to antibiotics.\u003c/p\u003e \u003cp\u003eOur taxonomic analyses have placed the new strain within the genus \u003cem\u003ePantoea\u003c/em\u003e. The phylogenetic analysis revealed \u003cem\u003ePantoea osteomyelitidis\u003c/em\u003e sp.nov genetic relationship to \u003cem\u003eP. mediterraneensis\u003c/em\u003e which has been isolated from healthy human skin, and \u003cem\u003eP. superficialis\u003c/em\u003e which has been assembled from a metagenome of an urban environmental sample. Nonetheless, the phylogenic analysis also indicated distinct evolutionary divergence of this clade from the common ancestor of other \u003cem\u003ePantoea\u003c/em\u003e clades. Thus, it remains plausible that this clade could belong to another genus within the \u003cem\u003eErwiniaceae\u003c/em\u003e family, not yet classified.\u003c/p\u003e \u003cp\u003eOur results of the pathogenicity potential highlight the challenges in determining potential pathogenicity by genomic characteristics only. Studies that have compared the potential virulence of clinical versus environmental \u003cem\u003ePantoea\u003c/em\u003e isolates reported similar colonization patterns in plants, insects, and animal models, suggesting the pathogenicity cannot be predicted by phylogeny [20,21]. The broad host range of \u003cem\u003ePantoea\u003c/em\u003e, along with their use as biocontrol agents in agriculture, can drive a selection pressure leading to the acquisition of pathogenicity factors, thereby enhancing host-specific adaptation [20]. Based on its isolation from bone biopsies from a focus on chronic osteomyelitis, we suggest \u003cem\u003ePantoea osteomyelitidis\u003c/em\u003e sp.nov. as an opportunistic human pathogen.\u003c/p\u003e \u003cp\u003eThis study highlights the pivotal role of advanced sequencing methods in clinical microbiology when conventional techniques offer low-resolution identification. Identifying new human opportunistic pathogens is essential for public health, aiding epidemiological surveillance and guiding medical treatment options. Moreover, tracking the evolutionary dynamics of bacterial groups with pathogenic potential can help predict the emergence and spread of novel pathogens. Further studies are needed to elucidate the impact of members of the \u003cem\u003eErwiniaceae\u003c/em\u003e family on human health, with a focus on their pathogenic mechanisms and the risk factors associated with infections.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eData availability\u003cbr\u003e\u0026nbsp;\u003c/strong\u003eThis Whole Genome Shotgun project has been deposited at DDBJ/ENA/GenBank\u003cbr\u003e\u0026nbsp;under the accession JBGFSN000000000, Bioproject accession PRJNA1127075. The version described in this paper is version JBGFSN010000000.\u0026nbsp;\u003cbr\u003e\u0026nbsp;The 16S rRNA sequence was deposited under GenBank number PP956810-PP956811.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConflict of Interest\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors have no competing interest to declare.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFunding Source\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe project received no external financial support.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eEthical Approval statement\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAll clinical samples and data were collected during routine patient care. No identification details, personal information, or images are presented in this study.\u0026nbsp;\u003cbr\u003e\u0026nbsp;The patient’s identity is anonymized. The patient has given written consent for the clinical case description.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n\u003cli\u003eSoutar CD, Stavrinides J. Molecular validation of clinical Pantoea isolates identified by MALDI-TOF. PLOS ONE. 2019;14:e0224731. doi:10.1371/journal.pone.0224731\u003c/li\u003e\n\u003cli\u003eCarek PJ, Dickerson LM, Sack JL. Diagnosis and Management of Osteomyelitis. afp. 2001;63:2413\u0026ndash;21. \u003c/li\u003e\n\u003cli\u003eFrancis A Waldvogel DPL. Osteomyelitis - The Lancet [Internet]. 2004 [cited 2024 Jul 29]. Available from: https://www.thelancet.com/article/S0140-6736(04)16727-5/abstract\u003c/li\u003e\n\u003cli\u003eWalterson AM, Stavrinides J. Pantoea: insights into a highly versatile and diverse genus within the Enterobacteriaceae. FEMS Microbiology Reviews. 2015;39:968\u0026ndash;84. doi:10.1093/femsre/fuv027\u003c/li\u003e\n\u003cli\u003eCrosby KC, Rojas M, Sharma P, Johnson MA, Mazloom R, Kvitko BH, et al. Genomic delineation and description of species and within-species lineages in the genus Pantoea. Front Microbiol. 2023;14. doi:10.3389/fmicb.2023.1254999\u003c/li\u003e\n\u003cli\u003eMeier-Kolthoff JP, G\u0026ouml;ker M. TYGS is an automated high-throughput platform for state-of-the-art genome-based taxonomy. Nat Commun. 2019;10:2182. doi:10.1038/s41467-019-10210-3\u003c/li\u003e\n\u003cli\u003eParks DH, Chuvochina M, Rinke C, Mussig AJ, Chaumeil P-A, Hugenholtz P. GTDB: an ongoing census of bacterial and archaeal diversity through a phylogenetically consistent, rank normalized and complete genome-based taxonomy. Nucleic Acids Research. 2022;50:D785\u0026ndash;94. doi:10.1093/nar/gkab776\u003c/li\u003e\n\u003cli\u003eLee I, Ouk Kim Y, Park S-C, Chun J. OrthoANI: An improved algorithm and software for calculating average nucleotide identity. International Journal of Systematic and Evolutionary Microbiology. 2016;66:1100\u0026ndash;3. doi:10.1099/ijsem.0.000760\u003c/li\u003e\n\u003cli\u003eSeemann T. Prokka: rapid prokaryotic genome annotation. Bioinformatics. 2014;30:2068\u0026ndash;9. doi:10.1093/bioinformatics/btu153\u003c/li\u003e\n\u003cli\u003eBharatham N, Bhowmik P, Aoki M, Okada U, Sharma S, Yamashita E, et al. Structure and function relationship of OqxB efflux pump from Klebsiella pneumoniae. Nat Commun. 2021;12:5400. doi:10.1038/s41467-021-25679-0\u003c/li\u003e\n\u003cli\u003eLi J, Zhang H, Ning J, Sajid A, Cheng G, Yuan Z, et al. The nature and epidemiology of OqxAB, a multidrug efflux pump. Antimicrobial Resistance \u0026amp; Infection Control. 2019;8:44. doi:10.1186/s13756-019-0489-3\u003c/li\u003e\n\u003cli\u003eNishino K, Senda Y, Yamaguchi A. CRP Regulator Modulates Multidrug Resistance of Escherichia coli by Repressing the mdtEF Multidrug Efflux Genes. J Antibiot. 2008;61:120\u0026ndash;7. doi:10.1038/ja.2008.120\u003c/li\u003e\n\u003cli\u003eBlattner FR, Plunkett G, Bloch CA, Perna NT, Burland V, Riley M, et al. The complete genome sequence of Escherichia coli K-12. Science. 1997;277:1453\u0026ndash;62. doi:10.1126/science.277.5331.1453\u003c/li\u003e\n\u003cli\u003eConfer AW, Ayalew S. The OmpA family of proteins: Roles in bacterial pathogenesis and immunity. Veterinary Microbiology. 2013;163:207\u0026ndash;22. doi:10.1016/j.vetmic.2012.08.019\u003c/li\u003e\n\u003cli\u003eBachmeyer C, Entressengle H, Gibeault M, N\u0026eacute;dellec G, M\u0026rsquo;Bapp\u0026eacute; P, Delisle F, et al. Bilateral tibial chronic osteomyelitis due to Pantoea agglomerans in a patient with sickle cell disease. Rheumatology. 2007;46:1247. doi:10.1093/rheumatology/kem127\u003c/li\u003e\n\u003cli\u003eBenjamin C. Lee CNC. A Boy With Septic Arthritis and Osteomyelitis Associated With Pantoea agglomerans [Internet]. Consultant360. 2015 [cited 2024 Jul 29]. Available from: https://www.consultant360.com/articles/boy-septic-arthritis-and-osteomyelitis-associated-pantoea-agglomerans\u003c/li\u003e\n\u003cli\u003eLabianca L, Montanaro A, Turturro F, Calderaro C, Ferretti A. Osteomyelitis Caused by Pantoea agglomerans in a Closed Fracture in a Child. Orthopedics. 2013;36:e252\u0026ndash;6. doi:10.3928/01477447-20130122-32\u003c/li\u003e\n\u003cli\u003eLaporte C LC. Ost\u0026eacute;ite tibiale \u0026agrave; Pantoea agglomerans au d\u0026eacute;cours d\u0026rsquo;une fracture ouverte stade IIIB de jambe [Tibial osteitis caused by Pantoea agglomerans after open grade IIIB tibial shaft fracture]. Rev Chir Orthop Reparatrice Appar Mot. 2002;88:625\u0026ndash;7. \u003c/li\u003e\n\u003cli\u003eVincent K, Szabo RM. Enterobacter Agglomerans Osteomyelitis of the Hand From a Rose Thorn: A Case Report. Orthopedics. 1988;11:465\u0026ndash;7. doi:10.3928/0147-7447-19880301-11\u003c/li\u003e\n\u003cli\u003eNadarasah G, Stavrinides J. Quantitative evaluation of the host-colonizing capabilities of the enteric bacterium Pantoea using plant and insect hosts. Microbiology. 2014;160:602\u0026ndash;15. doi:10.1099/mic.0.073452-0\u003c/li\u003e\n\u003cli\u003eV\u0026ouml;lksch B, Thon S, Jacobsen ID, Gube M. Polyphasic study of plant- and clinic-associated \u003cem\u003ePantoea agglomerans\u003c/em\u003e strains reveals indistinguishable virulence potential. Infection, Genetics and Evolution. 2009;9:1381\u0026ndash;91. doi:10.1016/j.meegid.2009.09.016\u003c/li\u003e\n\u003cli\u003eNguyen L-T, Schmidt HA, von Haeseler A, Minh BQ. IQ-TREE: A Fast and Effective Stochastic Algorithm for Estimating Maximum-Likelihood Phylogenies. Molecular Biology and Evolution. 2015;32:268\u0026ndash;74. doi:10.1093/molbev/msu300\u003c/li\u003e\n\u003cli\u003eHoang DT, Chernomor O, von Haeseler A, Minh BQ, Vinh LS. UFBoot2: Improving the Ultrafast Bootstrap Approximation. Molecular Biology and Evolution. 2018;35:518\u0026ndash;22. doi:10.1093/molbev/msx281\u003c/li\u003e\n\u003cli\u003eRichter M, Rossell\u0026oacute;-M\u0026oacute;ra R, Oliver Gl\u0026ouml;ckner F, Peplies J. JSpeciesWS: a web server for prokaryotic species circumscription based on pairwise genome comparison. Bioinformatics. 2016;32:929\u0026ndash;31. doi:10.1093/bioinformatics/btv681\u003c/li\u003e\n\u003c/ol\u003e"},{"header":"Table","content":"\u003cp\u003e\u003cstrong\u003eTable 1.\u0026nbsp;\u003c/strong\u003eLiterature review of osteomyelitis cases caused by \u003cem\u003ePantoea\u003c/em\u003e. \u0026nbsp;\u003c/p\u003e\n\u003ctable border=\"1\" cellspacing=\"0\" cellpadding=\"0\"\u003e\n \u003ctbody\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 12%;\"\u003e\n \u003cp\u003e\u003cstrong\u003ePublication\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 8.16%;\"\u003e\n \u003cp\u003e\u003cstrong\u003eGender\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 5.44%;\"\u003e\n \u003cp\u003e\u003cstrong\u003eAge\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 9.28%;\"\u003e\n \u003cp\u003e\u003cstrong\u003eInfection site\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 12.32%;\"\u003e\n \u003cp\u003e\u003cstrong\u003eTime from injury to symptom onset\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 12.8%;\"\u003e\n \u003cp\u003e\u003cstrong\u003eInfection source\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 15.52%;\"\u003e\n \u003cp\u003e\u003cstrong\u003eDiagnosis\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 11.84%;\"\u003e\n \u003cp\u003e\u003cstrong\u003ePathogen\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 12.64%;\"\u003e\n \u003cp\u003e\u003cstrong\u003eIdentification method\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 12%;\"\u003e\n \u003cp\u003eVincent, K., \u0026amp; Szabo, R. M. (1988).\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 8.16%;\"\u003e\n \u003cp\u003eM\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 5.44%;\"\u003e\n \u003cp\u003e8\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 9.28%;\"\u003e\n \u003cp\u003ehand, radio-carpal joint\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 12.32%;\"\u003e\n \u003cp\u003e14 days\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 12.8%;\"\u003e\n \u003cp\u003eRose thorn\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 15.52%;\"\u003e\n \u003cp\u003eAcute humeral osteomyelitis and septic arthritis\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 11.84%;\"\u003e\n \u003cp\u003e\u003cem\u003eP. agglomerans\u003c/em\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 12.64%;\"\u003e\n \u003cp\u003eCultures, unspecified\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 12%;\"\u003e\n \u003cp\u003eLaporte, C., et al. (2002).\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 8.16%;\"\u003e\n \u003cp\u003eM\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 5.44%;\"\u003e\n \u003cp\u003e56\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 9.28%;\"\u003e\n \u003cp\u003eTibia\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 12.32%;\"\u003e\n \u003cp\u003e9 months\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 12.8%;\"\u003e\n \u003cp\u003eSoil and plant contamination of open fracture\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 15.52%;\"\u003e\n \u003cp\u003eChronic tibial osteitis\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 11.84%;\"\u003e\n \u003cp\u003e\u003cem\u003eP. agglomerans\u003c/em\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 12.64%;\"\u003e\n \u003cp\u003eCultures + API gallery\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 12%;\"\u003e\n \u003cp\u003eBachmeyer, C., et al.(2007)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 8.16%;\"\u003e\n \u003cp\u003eM\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 5.44%;\"\u003e\n \u003cp\u003e20\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 9.28%;\"\u003e\n \u003cp\u003eBoth tibias\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 12.32%;\"\u003e\n \u003cp\u003eunknown\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 12.8%;\"\u003e\n \u003cp\u003eunknown\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 15.52%;\"\u003e\n \u003cp\u003eChronic osteomyelitis\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 11.84%;\"\u003e\n \u003cp\u003e\u003cem\u003eP. agglomerans\u003c/em\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 12.64%;\"\u003e\n \u003cp\u003e16s rDNA seq\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 12%;\"\u003e\n \u003cp\u003eLabianca, Luca, et al. (2013)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 8.16%;\"\u003e\n \u003cp\u003eF\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 5.44%;\"\u003e\n \u003cp\u003e8\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 9.28%;\"\u003e\n \u003cp\u003eUlna and radius\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 12.32%;\"\u003e\n \u003cp\u003e30 days\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 12.8%;\"\u003e\n \u003cp\u003eunknown - closed fracture with no foreign body found\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 15.52%;\"\u003e\n \u003cp\u003eAcute hematogenous osteomyelitis\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 11.84%;\"\u003e\n \u003cp\u003e\u003cem\u003eP. agglomerans\u003c/em\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 12.64%;\"\u003e\n \u003cp\u003eCultures, unspecified\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 12%;\"\u003e\n \u003cp\u003eChen, C. N., \u0026amp; Lee, B. C. (2015)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 8.16%;\"\u003e\n \u003cp\u003eM\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 5.44%;\"\u003e\n \u003cp\u003e10\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 9.28%;\"\u003e\n \u003cp\u003eElbow\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 12.32%;\"\u003e\n \u003cp\u003e4 months\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 12.8%;\"\u003e\n \u003cp\u003eAloe vera plant\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 15.52%;\"\u003e\n \u003cp\u003eChronic osteomyelitis\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 11.84%;\"\u003e\n \u003cp\u003e\u003cem\u003eP. agglomerans\u003c/em\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 12.64%;\"\u003e\n \u003cp\u003eCultures + 16s rDNA seq\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003c/tbody\u003e\n\u003c/table\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":true,"hideJournal":true,"highlight":"","institution":"","isAcceptedByJournal":false,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true},"keywords":"Osteomyelitis, Pantoea, Whole genome sequencing, Bacterial genomics, Case report","lastPublishedDoi":"10.21203/rs.3.rs-5298108/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-5298108/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003e\u003cu\u003e\u003cstrong\u003ePurpose\u003c/strong\u003e\u003c/u\u003e\u003cstrong\u003e:\u003c/strong\u003e\u003cem\u003e Pantoea\u003c/em\u003e species are increasingly recognized as human opportunistic pathogens. We describe a rare case of osteomyelitis that has been developed over years, with the identification of the causing agent as a novel species of \u003cem\u003ePantoea\u003c/em\u003e.\u003c/p\u003e\n\u003cp\u003e\u003cu\u003e\u003cstrong\u003eCase presentation:\u003c/strong\u003e\u003c/u\u003e A 37-year-old generally healthy woman presented to our hospital with suspected chronic osteomyelitis. The condition was possibly related to an incident that occurred two decades before the onset of symptoms, involving a tibia fracture with a large open bleeding wound. The patient had undergone two aggressive debridement operations, systemic ciprofloxacin treatment, and local aminoglycoside therapy, eventually exhibiting full recovery.\u003cbr\u003e\nBone biopsy cultures grew gram-negative coccobacilli that could not be identified by conventional clinical microbiology methods. Whole-genome sequencing and subsequent taxonomic and phylogenetic analyses revealed genetic relatedness to several \u003cem\u003ePantoea\u003c/em\u003e species. Comparative genomic analyses identified conserved antibiotic resistance and virulence genes.\u003cbr\u003e\n\u003cu\u003e\u003cstrong\u003eConclusion:\u003c/strong\u003e\u003c/u\u003e A literature review search uncovered only five cases of osteomyelitis caused by \u003cem\u003ePantoea\u003c/em\u003e species that have been reported in the past, all attributed to \u003cem\u003ePantoea agglomerans\u003c/em\u003e. We suggest this new strain belongs to a yet unidentified \u003cem\u003ePantoea\u003c/em\u003e species, which we have named \u003cem\u003ePantoea osteomyelitidis\u003c/em\u003e. sp.nov. The high diversity of \u003cem\u003ePantoea\u003c/em\u003e and the obscured potential pathogenicity of this genus is discussed, emphasizing the need for further research into its clinical relevance.\u003c/p\u003e","manuscriptTitle":"Pantoea osteomyelitidis sp. nov., a novel human opportunistic pathogen isolated from a patient with chronic osteomyelitis: case report, genomic characterization and literature review","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2024-10-22 04:39:37","doi":"10.21203/rs.3.rs-5298108/v1","editorialEvents":[{"type":"communityComments","content":0}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"0abaeddd-ca02-4e5d-a450-e34f6532ac18","owner":[],"postedDate":"October 22nd, 2024","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"posted","subjectAreas":[],"tags":[],"updatedAt":"2024-10-22T04:39:39+00:00","versionOfRecord":[],"versionCreatedAt":"2024-10-22 04:39:37","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-5298108","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-5298108","identity":"rs-5298108","version":["v1"]},"buildId":"qtupq5eGEP_6zYnWcrvyt","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}