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

doi:10.21203/rs.3.rs-5298108/v1

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

https://doi.org/10.21203/rs.3.rs-5298108/v1

This work is licensed under a CC BY 4.0 License

Version 1

posted

You are reading this latest preprint version

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

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.

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 (d₀=59.5% & d₄= 33% dDDH 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 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 osteomyelitidissp.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. osteomyelitidissp. 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 osteomyelitidissp.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 osteomyelitidissp.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 osteomyelitidissp.nov. She was treated with oral Ciprofloxacin 750 mg bid 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, 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.

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.

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. Nat Commun. 2019;10:2182. doi:10.1038/s41467-019-10210-3
Parks 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:D785–94. doi:10.1093/nar/gkab776
Lee 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;66:1100–3. doi:10.1099/ijsem.0.000760
Seemann T. Prokka: rapid prokaryotic genome annotation. Bioinformatics. 2014;30:2068–9. doi:10.1093/bioinformatics/btu153
Bharatham 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
Li 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 & Infection Control. 2019;8:44. doi:10.1186/s13756-019-0489-3
Nishino K, Senda Y, Yamaguchi A. CRP Regulator Modulates Multidrug Resistance of Escherichia coli by Repressing the mdtEF Multidrug Efflux Genes. J Antibiot. 2008;61:120–7. doi:10.1038/ja.2008.120
Blattner FR, Plunkett G, Bloch CA, Perna NT, Burland V, Riley M, et al. The complete genome sequence of Escherichia coli K-12. Science. 1997;277:1453–62. doi:10.1126/science.277.5331.1453
Confer AW, Ayalew S. The OmpA family of proteins: Roles in bacterial pathogenesis and immunity. Veterinary Microbiology. 2013;163:207–22. doi:10.1016/j.vetmic.2012.08.019
Bachmeyer C, Entressengle H, Gibeault M, Nédellec G, M’Bappé 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
Benjamin 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
Labianca L, Montanaro A, Turturro F, Calderaro C, Ferretti A. Osteomyelitis Caused by Pantoea agglomerans in a Closed Fracture in a Child. Orthopedics. 2013;36:e252–6. doi:10.3928/01477447-20130122-32
Laporte C LC. Ostéite tibiale à Pantoea agglomerans au décours d’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;88:625–7.
Vincent K, Szabo RM. Enterobacter Agglomerans Osteomyelitis of the Hand From a Rose Thorn: A Case Report. Orthopedics. 1988;11:465–7. doi:10.3928/0147-7447-19880301-11
Nadarasah G, Stavrinides J. Quantitative evaluation of the host-colonizing capabilities of the enteric bacterium Pantoea using plant and insect hosts. Microbiology. 2014;160:602–15. doi:10.1099/mic.0.073452-0
Völksch B, Thon S, Jacobsen ID, Gube M. Polyphasic study of plant- and clinic-associated Pantoea agglomerans strains reveals indistinguishable virulence potential. Infection, Genetics and Evolution. 2009;9:1381–91. doi:10.1016/j.meegid.2009.09.016
Nguyen 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;32:268–74. doi:10.1093/molbev/msu300
Hoang DT, Chernomor O, von Haeseler A, Minh BQ, Vinh LS. UFBoot2: Improving the Ultrafast Bootstrap Approximation. Molecular Biology and Evolution. 2018;35:518–22. doi:10.1093/molbev/msx281
Richter M, Rosselló-Móra R, Oliver Glöckner F, Peplies J. JSpeciesWS: a web server for prokaryotic species circumscription based on pairwise genome comparison. Bioinformatics. 2016;32:929–31. doi:10.1093/bioinformatics/btv681

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

No competing interests reported.

Download PDF

Version 1

posted

You are reading this latest preprint version

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

Status:

Version 1

Abstract

Figures

Introduction

Clinical case

Discussion

Declarations

References

Table

Additional Declarations

Supplementary Files

Status:

Version 1