Abstract

Immunocompromised patients are at increased risk of rare opportunistic and emerging infections that can be difficult to diagnose using conventional methods. Molecular approaches can facilitate the identification of unusual pathogens that warrant further investigation. A 36-year-old woman with metastatic invasive ductal carcinoma who had received broad-spectrum antimicrobials for recurrent febrile neutropenia was acutely admitted to the hospital with fever, tachycardia, and hypotension. Septic workup revealed anaerobic Gram-positive bacilli that could not be identified by Matrix-assisted laser desorption/ionization time-of-flight, whereas the VITEK 2 automated system suggested Clostridioides difficile.Despite broad-spectrum antibiotics targeting presumed clostridial infection, including piperacillin/tazobactam, meropenem, vancomycin, and metronidazole, her condition rapidly deteriorated to septic shock, and she died within 72 hours of its onset. Because of the unusual presentation and discordant microbiological results, 16S ribosomal RNA (rRNA) gene amplification and sequencing were performed. The isolate was provisionally identified as Clostridioides mangenotii. This environmental pathogen is closely related to Clostridioides difficile but has rarely been implicated in human disease. This case highlights the importance of molecular diagnostics in identifying rare invasive pathogens in immunocompromised hosts.

Introduction

Clostridioides mangenotii is a Gram-positive, spore-forming anaerobic bacillus that grows under mesophilic, strictly anaerobic conditions. Although closely related to Clostridioides difficile, genomic reclassification has identified it as a distinct species^[1]. While Clostridioides difficile is a recognized human pathogen, Clostridioides mangenotii is primarily an environmental organism and has been isolated from soil, water, and animal stool^[2]. Its endospore-forming ability enables it to survive under extreme environmental conditions, including temperature variation, pH shifts, chemical exposure, and radiation^[3-5]. Although rare, members of the genus Clostridioides have been reported to cause invasive infections, such as bacteremia and septicemia^[6]. The broader use of molecular diagnostics and the increasing number of immunocompromised patients may explain the increased recognition of these uncommon pathogens.

To our knowledge, Clostridioides mangenotii has not previously been reported as a cause of human bloodstream infection, and this may represent the first reported fatal case in an immunocompromised patient. After repeated failure of matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF) to identify the organism, it was provisionally identified by 16S ribosomal RNA (rRNA) sequencing and bioinformatic comparison with reference 16S databases.

Case Report

A 36-year-old woman with stage IV invasive ductal carcinoma metastatic to the lungs, lymph nodes, bones, and brain was receiving palliative gemcitabine/carboplatin and whole-brain radiotherapy. Four days after her second chemotherapy cycle, she was admitted with fever, myalgia, and malaise. Diagnosed with febrile neutropenia, she received piperacillin/tazobactam (4.5 g every 6 hours) and was discharged after neutrophil recovery and negative cultures.

Before her third cycle, she was readmitted with diffuse pain and general deterioration. She appeared unwell and distressed but was afebrile, with stable hemodynamics and oxygenation. Multiple enlarged lymph nodes were palpable in the cervical and inguinal regions. She had no diarrhea, abdominal pain, vomiting, or other gastrointestinal symptoms, and there was no clinical evidence of mucositis or colitis. Because she had no gastrointestinal symptoms, Clostridioides difficile toxin testing was not performed. She did not have a central venous catheter. On day 3 of admission, she developed fever (38.9°C), tachycardia (133 bpm), tachypnea (24/min), oxygen saturation of 92%-93% on room air, and hypotension (96/66 mm Hg). Blood cultures were obtained, and piperacillin/tazobactam (4.5 g every 6 hours) was started with intravenous fluids. Laboratory findings are summarized in Table 1.

Microbiological Investigations

Two sets of blood cultures were collected from two separate peripheral venipuncture sites (right and left upper limbs), with both anaerobic bottles flagging positive after 15 hours of incubation. Gram staining of the positive bottles demonstrated Gram-positive bacilli (Figure 1a). Subculture under anaerobic conditions yielded metronidazole-sensitive gray colonies after 48 hours on anaerobic blood agar (Figure 1b). MALDI-TOF mass spectrometry (MALDI-TOF MS; Bruker Daltonics, Billerica, MA, USA) repeatedly failed to identify the organism, both directly from the positive blood culture bottles and from pure colony growth. The isolate was then tested using the VITEK 2 automated system (bioMérieux, Marcy-l’Étoile, France), which identified it as Clostridioides difficile and reported susceptibility to amoxicillin-clavulanate, piperacillin/tazobactam, meropenem, and metronidazole, with resistance to clindamycin. Antimicrobial susceptibility results were interpreted according to Clinical and Laboratory Standards Institute criteria.

The patient continued to deteriorate, with hypotension, respiratory distress, altered mentation, and escalating oxygen requirements, progressing from supplemental oxygen via nasal cannula and a non-rebreather mask to non-invasive ventilatory support with continuous positive airway pressure. Her quick Sequential Organ Failure Assessment score was 3. Antibiotic therapy was escalated to meropenem (1 g every 8 hours) and intravenous metronidazole (500 mg every 8 hours), with the addition of oral vancomycin for presumed severe Clostridioides difficile infection. Vasopressor support was not initiated. Disseminated intravascular coagulation was not clinically suspected. Given her advanced metastatic malignancy and continued clinical deterioration, the family subsequently opted for comfort-focused management, and no further radiological investigations, laboratory testing, or invasive procedures were pursued. A do-not-attempt-resuscitation order was instituted. Despite broad-spectrum antimicrobial therapy and supportive care, she continued to deteriorate and died 72 hours after the onset of septic shock. An autopsy was not performed.

Molecular Identification of the Isolate

Because of the atypical clinical presentation, repeated failure of MALDI-TOF identification, and concern regarding possible misidentification by VITEK 2, molecular characterization of the isolate was undertaken. Genomic DNA was extracted from a colony of the bacterial isolate using the Qiagen Virus Mini Purification Kit 2.0 (Qiagen, Hilden, Germany) and quantified using a Qubit 2.0 Fluorometer (Thermo Fisher Scientific, Waltham, MA, USA). A DNA concentration of 10 ng was achieved by buffer modification.

DNA was barcoded using the SQK-16S024 kit (Oxford Nanopore Technologies, Oxford, UK), which contains 16S-specific primers 27F (5’-AGAGTTTGATCCTGGCTCAG-3’) and 1492R (5’-TACGGYTACCTTGTTACGACTT-3’) for amplification of the complete 16S rRNA gene. Sequencing was performed on a MinION platform (Mk1C MC110319; Oxford Nanopore Technologies, Oxford, UK) using a FLO-MIN106D Spot-On Flow Cell (R9 version; Oxford Nanopore Technologies, Oxford, UK). A 50-µL reaction volume was used, and the purified library was loaded onto the flow cell according to the manufacturer’s protocol.

Live base calling was performed using Guppy. Taxonomic assignment was generated using the wf-metagenomics workflow on the EPI2ME platform (Oxford Nanopore Technologies, Oxford, UK), with comparison against the National Center for Biotechnology Information 16S rRNA bacterial database. The isolate showed 95% 16S rRNA sequence homology with published Clostridioides mangenotii reference sequences and clustered with Clostridioides mangenotii in phylogenetic analysis (Figure 2). The 95% sequence homology is below the commonly accepted threshold for definitive species-level identification and was therefore interpreted in conjunction with the phylogenetic clustering. Additionally, a recent study reported 96.36% diagnostic accuracy for Nanopore 16S rRNA sequencing compared with Sanger sequencing, supporting its utility in identifying previously unrecognized bacterial isolates^[7]. The sequencing data generated in this study were deposited in the Sequence Read Archive under accession number SRR34968737.

Review of Literature

Members of the genera Clostridioides and Clostridium are ubiquitous, Gram-positive, anaerobic, spore-forming rods found in soil, water, and the gastrointestinal tracts of animals and humans. These genera collectively contain more than 200 species, among which Clostridioides difficile, Clostridium perfringens, Clostridium botulinum, and Clostridium tetani are most commonly associated with human disease. Their key virulence traits include spore formation, which enhances environmental survival, and the production of potent exotoxins that contribute to the rapid and sometimes fatal course of infection^[8].

Clostridioides mangenotii was initially grouped with Clostridioides difficile because of their close genotypic and phenotypic similarities. However, advances in molecular analysis, including whole-genome sequencing (WGS), have supported its classification as a distinct species. In 2016, Lawson et al.^[1] analyzed 16S rRNA sequences and reported 95% identity between Clostridioides difficile and Clostridioides mangenotii, indicating a close phylogenetic relationship. In contrast, Knight et al.^[9] demonstrated through whole-genome analysis that the two species share only 77% average nucleotide identity, well below the 95% threshold for species delineation, supporting their taxonomic separation. Subsequent phylogenomic studies further proposed reclassifying Clostridioides mangenotii into a new genus, Metaclostridioides, based on low amino acid identity and the presence of unique conserved signature indels^[10].

The pathogen has primarily been isolated from environmental sources such as soil and animal feces but has not previously been associated with human infections^[2]. Genomic analysis of the timber rattlesnake isolate revealed 68 genes related to virulence, defense mechanisms, and antimicrobial resistance, including resistance to vancomycin, fluoroquinolones, zinc, and arsenic^[2]. Despite these findings, its virulence mechanisms remain poorly understood because of the rarity of reported cases. Among other uncommon clostridial pathogens, Clostridium tertium has been particularly associated with immunocompromised and neutropenic patients; in a recent cohort, it accounted for 62 of 601 (10%) Clostridium species bacteremias, with 50% of patients having neutropenia or hematologic malignancy^[11].

Discussion

Our patient initially presented with diffuse myalgia followed by fever and signs of sepsis. Despite early microbiological evidence of Gram-positive anaerobic bacteremia, conventional identification methods failed to provide a reliable diagnosis. MALDI-TOF repeatedly failed to identify the isolate, whereas VITEK 2 suggested Clostridioides difficile, leading to treatment directed at presumed severe clostridial infection. As her condition rapidly deteriorated with profound hypotension, respiratory distress, and altered consciousness, treatment was escalated to meropenem with additional agents directed against presumed severe Clostridioides difficile infection^[12].

A major strength of this case is the use of molecular testing to resolve discordant conventional microbiological results. MALDI-TOF is a widely accepted rapid diagnostic tool for common bacterial pathogens, including Gram-positive anaerobes such as Clostridioides difficile, with accuracy approaching 100%^[13]. The repeated failure to identify the isolate, despite its presence in the instrument library, suggests the possibility of a distinct or atypical species. Although the VITEK 2 system identified the organism as Clostridioides difficile, misidentification remains possible because of the close genetic homology between the two pathogens, and comparative studies have consistently demonstrated higher accuracy with MALDI-TOF^[14]. Because of this discrepancy, the isolate underwent full-length 16S rRNA gene sequencing and phylogenetic analysis, which supported its provisional identification as Clostridioides mangenotii. However, WGS was not performed and would have enabled more robust species-level confirmation, representing a limitation of this case. Furthermore, the 95% 16S rRNA sequence homology is below the commonly accepted threshold for definitive species-level identification and should therefore be interpreted with caution.

The antimicrobial susceptibility results should also be interpreted carefully because the isolate was initially identified by VITEK 2 as Clostridioides difficile, and validated species-specific susceptibility criteria for Clostridioides mangenotii have not been established. The isolate was reported as susceptible to piperacillin/tazobactam, meropenem, and metronidazole, all of which were administered during the clinical course. Therefore, the poor clinical outcome was likely multifactorial, reflecting the patient’s advanced metastatic malignancy, profound immunocompromised status, and severity of sepsis.

The absence of documented human infections and limited understanding of the pathogenesis of Clostridioides mangenotii likely contributed to the diagnostic delay. This underscores the critical role of molecular diagnostics in detecting rare or unusual pathogens, particularly when conventional methods have limited capacity to identify uncommon or previously unrecognized pathogens^[15]. This diagnostic challenge extends beyond rare anaerobic bacteria in hemato-oncological patients, with recent literature also highlighting unusual and emerging invasive fungal pathogens, including the Fusarium proliferatum species complex, which may pose significant diagnostic and therapeutic challenges in immunocompromised hosts^[16].

Clinically, the patient presented with systemic signs of sepsis, including fever, altered consciousness, profound hypotension, and respiratory distress, progressing to multiorgan failure and death. This contrasts with primary Clostridioides difficile infection, which typically presents with gastrointestinal symptoms, mainly abdominal pain and diarrhea resulting from colitis. The absence of gastrointestinal symptoms in this case complicated the diagnosis. The patient’s rapid deterioration despite aggressive therapy suggests that Clostridioides mangenotii may possess significant virulence and may be capable of causing severe sepsis and septic shock, particularly in immunocompromised individuals.

To our knowledge, this may represent the first reported human case of fatal Clostridioides mangenotii bloodstream infection in an immunocompromised patient. Its misidentification by conventional methods highlights the potential for diagnostic confusion with closely related organisms, underscoring the limitations of standard diagnostic methods and the need for broader adoption of molecular and genomic testing in atypical infections.

Given the pathogen’s rarity, further research is warranted. Future studies should explore the pathogenic mechanisms of Clostridioides mangenotii, particularly the role of its 68 identified virulence-related genes. Investigating its antimicrobial resistance profile is crucial, particularly because validated species-specific susceptibility criteria are lacking. Furthermore, given the limited clinical data, additional case reports facilitated by advances in identification techniques and genomic studies will help improve understanding, evaluation, and management of this emerging pathogen.

Conclusion

Immunocompromised patients are particularly vulnerable to rare, opportunistic, and emerging infections that may evade detection by conventional methods. Incorporating molecular approaches can facilitate the identification of unusual pathogens that warrant further investigation. To our knowledge, this may represent the first reported human case of fatal Clostridioides mangenotii bloodstream infection in an immunocompromised patient.