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

doi:10.21203/rs.3.rs-5317570/v1

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Case Report

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

https://doi.org/10.21203/rs.3.rs-5317570/v1

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Background: Pantoea species are increasingly recognized as opportunistic human pathogens. We describe a rare case of osteomyelitis that has developed slowly over two decades, with the identification of the causative agent as a novel species of Pantoea.
Case presentation: A 37-year-old generally healthy woman with suspected chronic osteomyelitis presented at our hospital. 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 resulting in a full recovery.
Bone biopsy cultures revealed 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 revealed conserved antibiotic resistance and virulence genes.
Conclusion: A literature review uncovered only five cases of osteomyelitis caused by Pantoea species that have been reported in the past, all of which were attributed to Pantoea agglomerans. We suggest that 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 are discussed, emphasizing the need for further research into its clinical relevance.

Osteomyelitis

Pantoea

Whole-genome sequencing

Bacterial genomics

Case report

Pathogen identification in the clinical microbiology laboratory has an important role in 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 databases of known organisms. Thus, gaps in the database can lead to misidentification or no identification of rare bacterial species [1].

Osteomyelitis is an inflammation of the bone caused by pyogenic microorganisms. Chronic osteomyelitis primarily occurs in adults, develops 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. The 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]. To date, 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 being the most common opportunistic pathogen in both host groups [5]. Infections caused by Pantoea are relatively uncommon and are usually related to the 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 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 revealed that this strain belongs to the Erwiniaceae family. Taxonomic and phylogenetic analyses of the bacterial whole-genome sequence suggested that it belongs to the genus Pantoea; however, no known species could be identified. We have used comparative genomics and different gene-prediction analyses to characterize this new species further and to better understand its potential pathogenicity.

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, which was 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 mild limp, tenderness, and swelling in the proximal 1/3 of her left tibia. Blood analysis revealed mild leukocytosis (11,400 cells/mL, range 4000–11000) and normal C-reactive protein (CRP) (2.2 mg/L, range 0–5) levels. Magnetic resonance imaging (MRI) showed intramedullary edema in the proximal tibia with enhancement following gadolinium injection, a breach of the frontal and lateral bone cortex, and abscess formation in the subdermal fat. The patient was admitted for debridement, with no antimicrobial treatment before surgery. Pathological bone was noted by the orthopedic team, and multiple microbiological and pathological specimens were collected. Pathology revealed 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 the bone and soft tissue samples.

The biopsy samples were plated on TSA 5% sheep blood agar, chocolate agar, and MacConkey agar plates (Hylab, Rehovot, Israel) at 37°C under aerobic conditions overnight, and white‒gray circular smooth colonies formed. Gram staining revealed gram-negative coccobacilli (Fig. 1). Phenotypic tests (Methods in Supp. material A) showed that this strain is catalase negative, oxidase negative, coagulase negative, and indole negative. Colonies were tested via matrix-assisted laser desorption/ionization-time of flight mass spectrometry (MALDI-TOF MS) via a VITEK® MS (bioMérieux, Marcy-l'Étoile, France), which resulted in the identification of no known species. Antibiotic susceptibility tests, which are based on gram-negative CLSI breakpoints and biochemical tests (Supp. Table 1), were performed on VITEK®2 (bioMérieux, Marcy-l'Étoile, France), which 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 the colonies, and the 16S rRNA gene was amplified and sequenced via Sanger sequencing. BLASTN analysis of the resulting sequence against GenBank generated good hits for 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 size was 1,854,057, the total length was 4,314,591 bps, and the GC content was 55.3%. Taxonomic analyses performed via TYGS (Type strain genome server [6] and GTDB-Tk Classify genomes 2.3.2 [7] could not assign a known species and revealed that the closest species found were Pantoea superficialis (d₀ = 59.5% & d₄ = 33% dDDH (DNA-DNA hybridization) values by TYGS and 87.6% average nucleotide identity (ANI) by GTDB-Tk), followed by Pantoea mediterraneensis (d₀ = 46%, d₄ = 26.9% dDDH and 84% ANI). dDDH values of over 70% and ANI values of over 95% indicate the same species [6, 8], suggesting that our strain belongs to a still unknown species of Pantoea, which we have named Pantoea osteomyelitidis.

Pan-genome analysis was performed via Roary on the basis of 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 fewer of the genomes).

The phylogenetic tree based on core genes (Fig. 2) revealed high diversity within Pantoea, with many distinct lineages. Pantoea osteomyelitidis sp. nov. clustered with P. superficialis and P. mediterraneensis. However, there was 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.

The average nucleotide identity values obtained via BLASTN (ANIb) are shown as a heatmap in Fig. 3. This analysis aligns mostly with the subclades within the phylogenetic tree generated according to the core genome, as shown by values greater than 80%.

Figure 3 also presents the isolation source of each species according to all reports in GenBank. Overall, 12 species have been isolated from human samples, some depicted as pathogens and others recovered from healthy tissues. Of these, 3 have been isolated only from human samples (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).

Gene prediction analysis of Pantoea osteomyelitidis sp. nov. via Prokka version 1.14.6 [9] identified a total of 4,084 genes, comprising 3,294 protein-coding sequences. We tested for the presence of antibiotic resistance and virulence genes (Methods in Supp. material A) and identified the presence of oqxB and CRP. OqxB codes for a multidrug efflux RND transporter permease subunit. OqxB overexpression can confer resistance to various antibiotics [10, 11]. The CRP gene encodes 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, and NlpI codes for lipoprotein NlpI [13]. The OmpA gene encodes porin outer membrane protein A, which is known for its multiple functions, including biofilm formation and a role in F plasmid cell conjugation [14]. EC55989_3335 encodes an Hcp family type VI secretion system effector [13]. Our results from the pangenome analysis revealed the presence of OqxB, CRP, Nlpl, FliG, FliM, FliP and ompA within the core gene group, indicating that these genes are evolutionarily conserved within this genus (Supp. Table 2).

An analysis of potential pathogenic proteins via PathogenFinder suggested a nonhuman pathogen with a 0.47 likelihood of being pathogenic, as 16 pathogenic protein families were identified, whereas 17 nonpathogenic protein families were identified. 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 thus 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 to be nonhuman pathogens (Supp. Table 3).

Patient treatment and follow-up

Four months after 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 samples was again positive for Pantoea osteomyelitidis sp. nov. She was treated with 750 mg bid oral ciprofloxacin for four weeks. Six months after her surgery, the patient reported complete healing.

Pantoea comprises a highly diverse group of gram-negative bacteria in the Erwiniaceae family that inhabit various aquatic or terrestrial environments and interact with different hosts as commensals or pathogens. Owing to its varied roles, Pantoea's potential as a human pathogen remains obscure. Although infections caused by Pantoea species, mainly bacteraemia and septicaemia cases, are infrequently identified in clinical microbiology laboratories, there are accumulating reports of infections, including some reports of meningitis, pneumonia, and joint infections [4]. To date, only 5 cases of osteomyelitis or osteitis caused by Pantoea have been reported in the literature, all of which have identified P. agglomerans via phenotypic methods from cultures or 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 (WGS) to characterize unknown bacteria isolated from multiple bone biopsies, which conventional clinical microbiology methods failed to identify. We discovered a previously unknown species related to Pantoea that caused chronic osteomyelitis in a healthy adult. The infection had probably developed slowly and under the radar for over twenty years following a tibia fracture with a large open bleeding wound.

Despite the prediction of two genes with potential antibiotic resistance (OqxB and CRP regulators), antibiogram tests revealed susceptibility to all antibiotics according to the CLSI breakpoints. The patient fully recovered after aggressive debridement, systemic ciprofloxacin, and local aminoglycoside treatment. This finding, together with the conserved nature of these genes in other Pantoea genomes examined in our analysis, suggests that the presence of OqxB and CRP regulators alone cannot predict resistance to antibiotics.

Our taxonomic analyses revealed a new strain within the genus Pantoea. The phylogenetic analysis revealed the close genetic relationship of Pantoea osteomyelitidis sp.nov. with P. mediterraneensis, which has been isolated from healthy human skin, and P. superficialis, which was assembled from a metagenome of an urban environmental sample. Nonetheless, the phylogenetic 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 that has not yet been classified.

Our results concerning pathogenicity potential highlight the challenges in determining potential pathogenicity on the basis of 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 that pathogenicity cannot be predicted by phylogeny [20, 21]. The broad host range of Pantoea, along with its use as a biocontrol agent in agriculture, can drive selection pressure, leading to the acquisition of pathogenicity factors, thereby enhancing host-specific adaptation [20]. Based its isolation from bone biopsies from a focus on chronic osteomyelitis, we suggest that 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.

MALDI-TOF-MS: matrix-assisted laser desorption/ionization-time of flight mass spectrometry

BLASTN: The Basic Local Alignment Search Tool

WGS : whole-genome sequencing

dDDH :DNA-DNA hybridization

ANI: average nucleotide identity

Data availability

This Whole Genome Shotgun project has been deposited at DDBJ/ENA/GenBank'under the accession JBGFSN000000000, Bioproject accession number 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 interests to declare.

Funding Source

The project received no external financial support.

Ethics 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 was anonymized. The patient provided written consent for the clinical case description.

Author Contribution

DKF: Conceptualization, Formal analysis, Investigation, Methodology, Data curation, Writing – original draft, Writing – review and editing, DLC: Investigation, Writing – original draft, Writing – review and editing, AT: Investigation, Methodology, Writing – review and editing, MA: Investigation, Methodology,Writing – review and editing, DT: Investigation, Methodology, TBN: Conceptualization, Writing – review and editing, NK: Conceptualization, Project administration, Resources, Writing – review and editing

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Table 1. Literature review of osteomyelitis cases caused by Pantoea.

Publication	Patient	Infection site	Time from injury to symptom onset	Infection source	Diagnosis	Antibiotic treatment	Pathogen	Pathogen Identification method	Patient outcome
Vincent, K., & Szabo, R. M. (1988).	8-year-old male	hand, radio-carpal joint	14 days	Rose thorn	Acute humeral osteomyelitis and septic arthritis	Intravenous ceftriaxone for 2 weeks, followed by 4-week oral Trimethoprim-sulfamethoxazole.	P. agglomerans	Cultures, unspecified	Regained normal function after 9 weeks from treatment
Laporte, C., et al. (2002).	56-year-old male	Tibia	9 months	Soil and plant contamination of open fracture	Chronic tibial osteitis	Intravenous cefotaxime and netilmicin, changed after 48 hours to intravenous cefotaxime and oral ofloxacin for 10 days.	P. agglomerans	Cultures + API gallery	Recovered (unspecified)
Bachmeyer, C., et al.(2007)	20-year-old male	Both tibias	unknown	unknown	Chronic osteomyelitis	Imipenem and amikacine for 3 weeks, imipenem and colimycine for 3 weeks.	P. agglomerans	16 s rDNA seq	Unknown
Labianca, Luca, et al. (2013)	8-year-old female	Ulna and radius	30 days	unknown - closed fracture with no foreign body found	Acute hematogenous osteomyelitis	Intravenous cloxacillin for 4 weeks followed by oral antibiotics for 4 more weeks.	P. agglomerans	Cultures, unspecified	12 months after, the patient regained full flexionextension range of elbow and wrist motion with a partial loss of pronosupination.
Chen, C. N., & Lee, B. C. (2015)	10-year-old male	Elbow	4 months	Aloe vera plant	Chronic osteomyelitis	Trimethoprim-sulfamethoxazole and ciprofloxacin for 6 months.	P. agglomerans	Cultures + 16 s rDNA seq	Recovered (unspecified)

No competing interests reported.

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

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Case presentation

Patient treatment and follow-up

Discussion and Conclusions

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