# Emergence of the zoonotic bacterium Necropsobacter rosorum in nutria Myocastor coypus with implications for wildlife and human health

Younes Laidoudi, Bernard Davoust, Hubert Lepidi, Anthony Levasseur

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## To cite this version:

Younes Laidoudi, Bernard Davoust, Hubert Lepidi, Anthony Levasseur. Emergence of the zoonotic bacterium Necropsobacter rosorum in nutria Myocastor coypus with implications for wildlife and human health. Scientific Reports, 2025, 15 (1), pp.32252. 10.1038/s41598-025-15792-1. hal-05383110

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Submitted on 26 Nov 2025

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OPEN

# Emergence of the zoonotic bacterium *Necropsobacter rosorum* in nutria *Myocastor coypus* with implications for wildlife and human health

Younes Laidoudi<fnref n="1" />,², Bernard Davoust<fnref n="1" />,², Hubert Lepidi³ & Anthony Levasseur<fnref n="1" />,²,⁴✉

The nutria (*Myocastor coypus*), a semi-aquatic rodent native to South America, poses significant ecological and agricultural threats as an invasive species in France, where it continues to proliferate despite sustained control efforts. A fatal case of pneumonia in a nutria from Marseille (France) prompted a microbiological investigation that led to the isolation, taxonomic classification, genomic characterization, and phylogenetic analysis of *Necropsobacter rosorum*. Whole-genome sequencing of the *N. rosorum* strain RG01 revealed a genome size of 2,505,657 base pairs and 2303 predicted open reading frames, showing high similarity to other publicly available *N. rosorum* genomes. Comparative pan-genomic analysis indicated a high level of genomic conservation among *N. rosorum* strains. The presence of putative virulence factors and a CRISPR-Cas system suggests both pathogenic potential and adaptive defense mechanisms against bacteriophage predation. This study also explored the genetic epidemiology of members of the *Pasteurellaceae* family, highlighting a considerable overlap between species infecting animals and humans. Among the 408,387 sequence records retrieved from GenBank, 62.1% were deemed suitable for genomic epidemiological analysis. Notably, *N. rosorum* was underrepresented, with only 13 entries spanning nine countries and three host types, revealing critical gaps in current surveillance and research. Collectively, these findings contribute to a better understanding of the microbiology and epidemiology of *N. rosorum* and *Pasteurellaceae*-associated infections, and underscore the importance of integrated, genomics-informed approaches for the monitoring, control, and prevention of zoonotic diseases.

**Keywords** *Necropsobacter rosorum*, Pasteurellosis, Nutria, *Myocastor coypus*, Zoonosis, Genomics, Genetic epidemiology

The family *Pasteurellaceae* comprises several commensal species that typically colonize the mucosal surfaces of the upper respiratory tract, oropharynx, and reproductive tract, and in some cases, the gastrointestinal tract of a wide range of vertebrate hosts. Infections caused by *Pasteurellaceae* bacteria are predominantly opportunistic, resulting in respiratory and urogenital tract infections. However, some species are primary pathogens capable of causing severe diseases and outbreaks in both humans and animals<fnref n="1" />.

Currently, the *Pasteurellaceae* family includes 25 genera and 105 recognized species². Most *Pasteurellaceae* species exhibit a restricted host specificity, as demonstrated in previous studies focusing on major pathogenic members such as *Actinobacillus pleuropneumoniae*, *Aggregatibacter actinomycetemcomitans*, *Haemophilus influenzae*, *Pasteurella multocida*, *Avibacterium paragallinarum*, *Gallibacterium anatis*, and *Mannheimia haemolytica*, or inferred indirectly from routine host surveillance³⁻⁵. Zoonotic transmission appears to be rare and is generally associated with animal bites, which allow the pathogen to bypass the normal host barrier⁶,⁷.

Rodent-associated *Pasteurellaceae* have undergone extensive taxonomic revisions, leading to their classification into six recognized genera: *Cricetibacter*, *Mesocricetibacter*, *Mannheimia*, *Muribacter*, *Necropsobacter*, and

<footnote n="1">¹Aix Marseille Université (AMU), UMR D257 RITMES, Assistance Publique-Hôpitaux de Marseille (APHM), Service de Santé des Armées (SSA), Marseille, France. ²IHU-Méditerranée Infection, Marseille, France. ³Laboratoire anatomopathologie, Hôpital de La Timone, Assistance Publique-Hôpitaux de Marseille (APHM), Marseille, France. ⁴Institut Universitaire de France, Paris, France. ✉email: anthony.levasseur@univ-amu.fr</footnote>
*Rodentibacter*⁸,⁹. Among them, *Rodentibacter pneumotropicus* (formerly *Pasteurella pneumotropica*) is the most frequently isolated species from laboratory mice and rats⁹. Some species, such as *Necropsobacter rosorum* (SP-group; Taxon 24), have zoonotic potential and have been identified in guinea pigs, rabbits, and even humans¹⁰⁻¹⁴.

*N. rosorum* is a facultative anaerobic bacterium associated with opportunistic infections in humans, particularly involving the skin, soft tissues, and internal organs. It has been implicated in cases of sepsis secondary to wet gangrene, abdominal abscesses, and cellulitis following animal bites, especially from guinea pigs. This species exhibits slow glucose fermentation and phenotypic similarities with other *Pasteurellaceae*, but can be differentiated by its ability to grow on MacConkey agar and its negative reactions for indole production and ornithine decarboxylase. Although infections caused by *N. rosorum* are rare, they can lead to severe complications if misidentified or left untreated¹⁰⁻¹⁴. Due to its rarity and phenotypic overlap with other *Pasteurellaceae* species, accurate identification and thorough epidemiological investigations are essential to better understand its clinical relevance. Transmission primarily occurs through direct contact, aerosols, or contaminated surfaces, particularly within laboratory colonies, where rodents serve as both reservoirs and vectors¹³.

The nutria or coypu (*Myocastor coypus* Molina, 1782) is a semi-aquatic rodent of the Myocastoridae (or Echimyidae) family, native to South America. It is recognizable by its considerable size (up to 65 cm in body length, 45 cm in tail length), large body mass (average of 7 kg), and four prominent red-orange incisors. As an herbivore, it feeds primarily on aquatic vegetation and has a relatively long lifespan of up to six years¹⁵. *M. coypus* was introduced to France in 1882, with the first fur farms established in 1926 to support the fur trade¹⁶. Nutria farming expanded significantly between the two world wars and after World War II. Concurrently, nutria either escaped or were released into the wild, leading to the colonization of wetlands, rivers, lakes, and streams throughout France. Today, the species is found across the entire mainland, with the exception of Corsica¹⁷.

As an exotic species with no natural predators in France, the nutria poses a substantial threat to aquatic ecosystems by destabilizing riverbanks and damaging crops¹⁸. It is listed among the “100 of the World’s Worst Invasive Alien Species” by the International Union for Conservation of Nature (IUCN). The European Union has enacted regulations aimed at controlling such invasive alien species¹⁹. The nutria is officially classified as a harmful species and may be selectively trapped; however, chemical control is avoided due to potential adverse effects on non-target species. Population monitoring in the Pays de la Loire region of western France recorded 288,000 nutrias trapped in 2016, with an annual increase of 22% per trapper²⁰. On a broader scale, nutria population expansion may be linked to global climate change²¹⁻²³.

Marseille, the largest city on the Mediterranean coast of France and the second most populous in the country (873,076 inhabitants), supports an urban colony of approximately fifty nutrias, which has resided for two decades along the banks of the Huveaune River in a public park with access to the sea. This study investigated one nutria from this colony, found dead approximately 30 m from its habitat. The cause of death was identified as pneumonia due to *Necropsobacter rosorum* strain RG01, which was isolated, cultured in the laboratory, and subsequently subjected to genetic characterization.

# Results

## Case report

In October 2022, the carcass of a nutria (*Myocastor coypus*) was discovered in Borély Park in Marseille, France (43° 15′ 41.6″ N, 5° 22′ 48.8″ E), approximately 30 m from the banks of the Huveaune River, a small watercourse that flows into the Mediterranean Sea. This area is home to a stable colony of around 50 nutrias. The carcass was transported to the Méditerranée Infection Veterinary Research Center at the University Hospital Institute (IHU) in Marseille for examination, necropsy, and sampling. The animal was an adult female nutria (middle-aged), weighing 5.2 kg, with a body length of 60 cm and a 40 cm tail (Fig. 1A). The external examination revealed no traumatic lesions, except for signs of epistaxis.

Necropsy findings revealed severe acute pneumonia. Gross pathological examination of the lungs showed miliary lesions (Fig. 1B), accompanied by fibrinous pleuritis, characterized by yellow exudate upon abscess opening (Fig. 1B). Fibro-serous deposits formed adhesions between the lungs and the thoracic wall (Fig. 1B). The extent and severity of the lesions were consistent with death due to natural causes. No other significant abnormalities were noted during the necropsy.

Histopathological analysis of the lung tissue revealed extensive necrotizing inflammation of the pulmonary alveoli (Fig. 1C). Bacterial cultures yielded *Necropsobacter rosorum* from samples of blood, heart, lung abscess, and pleural fluid, confirming a case of severe pneumonia complicated by bacteremia. Species identification was confirmed by 16S rRNA gene sequencing, which showed 99.48% identity with known *N. rosorum* sequences.

# Genome sequencing and comparative analysis

Whole-genome sequencing of the *N. rosorum* isolate (strain RG01) was performed using an Illumina MiSeq platform, as detailed in the Materials and Methods section. The assembly size of the genome consisted of 2,505,657 base pairs, 11 scaffolds, with a GC content of 49%. A total of 2,303 open reading frames (ORFs) were annotated, along with 10 rRNA genes and 53 tRNA genes. Based on 16S rRNA gene analysis, the isolate showed high similarity to *N. rosorum* strains CCUG 28,028 and P709T, with sequence identities of 99.48% and 99.46%, respectively. Maximum Likelihood (ML) phylogenetic analysis based on the 16S rRNA gene positioned strain RG01 within a distinct clade that included other *N. rosorum* strains and *N. massiliensis*, forming a sister group to *Rodentibacter* species. This clade was also closely related to those encompassing *Pasteurella multocida* subspecies and the human pathogens *Haemophilus* spp. (Fig. 2).

A comparative pan-genomic analysis was conducted using *N. rosorum* strain RG01 (Bonneveine) and two publicly available genomes: *N. rosorum* strain P709T and *N. massiliensis* strain FF6. A total of 526 orthologous genes were shared among all three strains, while the two *N. rosorum* strains shared 2291 orthologous genes (Supplemental Table S1). The average nucleotide identity (ANI) between strains Bonneveine and P709T was
<figure><img src="image_4.png" /><figcaption>**Fig. 1.** Macroscopic and microscopic examination of the nutria carcass before and after necropsy. (A) Nutria carcass prior to necropsy, showing epistaxis as the only visible abnormality. (B) Macroscopic lung lesions showing whitish nodules (left) and abscesses (right) distributed across the lung parenchyma. (C) Haematoxylin-eosin-saffron-stained lung section (×250 magnification) displaying histopathological features of bronchopneumonia. Alveoli (blue arrows) are filled with abundant exudate composed mainly of neutrophils and macrophages, with areas of extensive necrosis.</figcaption></figure>

99.9%, supporting their close relatedness. Phylogenomic analysis further corroborated the 16S-based phylogeny, clearly clustering strain RG01 with other *N. rosorum* isolates (Fig. 3).

The antimicrobial resistance profile of strain RG01 is summarized in Table 1. A CRISPR-Cas system of Type IC was detected, consisting of 60 direct repeats and seven associated Cas genes (Supplemental Table S2). Phage analysis revealed one intact prophage region of 77.1 kb containing 92 ORFs, along with two incomplete prophage regions (8.2 kb and 26.1 kb), as summarized in Supplemental Table S3. Additionally, five genomic islands were predicted (Supplemental Fig. S1). KEGG annotations identified four functional domains (ko00001, ko01000, ko03000, and ko03021) within these islands, coding for phage proteins, lysozymes, regulatory proteins, and RNA polymerase sigma factors (Supplemental Table S4).

## Genetic epidemiology of *Pasteurellaceae* members

A total of 408,387 sequence records were retrieved from the GenBank database for the *Pasteurellaceae* family. Of these, 62.1% (n = 253,496) were considered suitable for genomic epidemiological analysis. Among them, 190,714 records (74.6%) corresponded to species involved in both animal and human pasteurellosis. Additionally, 58,974 (23.1%) were associated with animal-only cases, and 5964 (2.3%) with human-only infections.

The most extensively studied species included *Haemophilus influenzae*, *Glaesserella parasuis*, *Avibacterium* spp., *Pasteurella multocida*, and *Mannheimia haemolytica*. The countries contributing the highest number of genomic records were the USA, China, India, Spain, Denmark, Canada, Norway, Sweden, the United Kingdom, Brazil, and Australia (Fig. 4 and Supplemental Fig. S2).

Remarkably, only 13 records were identified for *N. rosorum*, spanning nine countries: France, the Czech Republic, Australia, Germany, the Netherlands, Japan, Israel, Belgium, and Denmark. These records represented isolates from three host types: humans, rabbits, and guinea pigs, highlighting the underrepresentation of *N. rosorum* in current genomic databases and underscoring the need for further research into its epidemiology.

## Discussion

Rodents, particularly semi-aquatic species such as *Myocastor coypus* (nutria), play a significant role in the epidemiology of zoonotic diseases, serving as reservoirs for various bacterial pathogens. Nutria are well-documented asymptomatic carriers of *Leptospira* spp., presenting a public health risk²⁴. Additionally, respiratory infections, particularly pneumonia, are frequently reported in this species²⁵,²⁶. Cold temperatures and high humidity are key environmental factors that predispose nutria to respiratory diseases. However, clinical signs of pneumonia are rarely observed, and the disease is often detected in its subacute or chronic stages²⁶.
<figure><img src="image_5.png" /><figcaption>**Fig. 2.** Maximum Likelihood (ML) phylogeny of partial 16S rRNA gene sequences showing the placement of the RG01 strain among representative members of the *Pasteurellaceae* family (right), with a zoomed-in subtree highlighting phylogenetic sister groups to the *Necropsobacter* clade. Gene accession numbers, strain names, and species names are indicated at the tips of branches. Sequences generated in this study are shown in bold blue. Filled black circles and triangles represent host specificity for animals and humans, respectively, based on GenBank records.</figcaption></figure>

Necropsy findings in pneumonic nutria typically reveal mucus accumulation in the bronchi and bronchioles, fibrotic nodules in the cranial lung lobes, and pulmonary hyperemia. Microscopically, leukocyte infiltration is commonly observed in the bronchioles, while alveolar tissues may exhibit thickening due to serous or fibrinous exudates. Several bacterial species, including *Streptococcus zooepidemicus*, *Staphylococcus aureus*, and *Pasteurella multocida*, are frequently isolated from such post-mortem lesions²⁷. In the present study, *Necropsobacter rosum* was identified in a fatal case of pneumonia in a nutria, further supporting the role of *M. coypus* as a potential host for emerging respiratory pathogens.

The involvement of *Pasteurella* and related genera in rodent respiratory infections is well-documented, particularly in laboratory animals. In these settings, *Rodentibacter* spp. (formerly *Pasteurella pneumotropica*) are primary etiological agents of pneumonia. *Rodentibacter pneumotropicus* is known to cause both pneumonia and septicemia, acting as an opportunistic pathogen in laboratory rodents²⁸,²⁹. In our own investigations, the necropsy of a Norway rat found dead in Marseille revealed granulomatous pneumonia caused by *Rodentibacter rarus*⁸.

*Pasteurella* species are typically commensals of the upper respiratory tract in animals such as pigs, dogs, and cats. While usually harmless in their native hosts, they can cause serious infections when transmitted through bites or scratches, particularly in immunocompromised individuals³⁰. In livestock, *P. multocida* is responsible
<figure><img src="image_6.png" /><figcaption>**Fig. 3.** Core-gene-based phylogeny showing the position of the *N. rosorum* RG01 strain among valid bacterial species within the *Pasteurellaceae* family. Species isolated in this study are indicated in bold. Bootstrap support values are shown at the nodes. Maximum Likelihood tree parameters and likelihood mapping statistics are included.</figcaption></figure>

for significant disease burdens, including bronchopneumonia in cattle, atrophic rhinitis in pigs, avian cholera in poultry, and rhinitis in rabbits³⁰. In humans, pasteurellosis is a rare but potentially severe zoonotic infection, capable of leading to septicemia and pulmonary disease, especially in vulnerable populations³⁰.

*Necropsobacter rosorum* is a facultative anaerobic, Gram-negative bacterium within the family *Pasteurellaceae*, first described in 2011. It has been implicated in opportunistic infections in both animals and humans¹⁰. The organism is coccoid or pleomorphic, non-motile, non-hemolytic, and non-pigmented. While it shares phenotypic features with *Pasteurella* spp., it can be distinguished by its ability to grow on MacConkey agar and by negative reactions for indole production and ornithine decarboxylase¹⁰⁻¹⁴. In animals, *N. rosorum* has been isolated from pneumonia lesions in guinea pigs and abscesses in coypus (*M. coypus*), indicating its pathogenic potential in respiratory and soft tissue infections³¹.

Rodents—particularly semi-aquatic species such as nutria and muskrats—may serve as reservoirs for *N. rosorum* and related *Pasteurellaceae*, facilitating transmission via direct contact, aerosols, or environmental contamination¹³. In humans, *N. rosorum* has been linked to soft tissue infections, sepsis secondary to wet gangrene, abdominal abscesses, and cellulitis, frequently following animal bites, especially from guinea pigs¹²,¹³,³². Five cases of *N. rosorum* bacteremia were reported in Singapore, originating from intra-abdominal infections such as cholangitis and appendicitis¹³. In Japan, the bacterium was isolated from an abdominal abscess
<table><caption>**Table 1.** Table showing the detailed results of predicted antimicrobial resistance profile of the RG 01 strain.</caption><thead><tr><th>RGI criteria</th><th>ARO term</th><th>SNP</th><th>Detection criteria</th><th>AMR gene family</th><th>Drug class</th><th>Resistance mechanism</th><th>% Identity of matching region</th><th>% Length of reference sequence</th></tr></thead><tbody><tr><td>Strict</td><td>Klebsiella pneumoniae KpnH</td><td></td><td>Protein homolog model</td><td>Major facilitator superfamily (MFS) antibiotic efflux pump</td><td>Macrolide antibiotic, fluoroquinolone antibiotic, aminoglycoside antibiotic, carbapenem, cephalosporin, penam, peptide antibiotic, penem</td><td>Antibiotic efflux</td><td>68.8</td><td>98.44</td></tr><tr><td>Strict</td><td>vanT gene in vanG cluster</td><td></td><td>Protein homolog model</td><td>Glycopeptide resistance gene cluster, vanT</td><td>Glycopeptide antibiotic</td><td>Antibiotic target alteration</td><td>33.33</td><td>51.40</td></tr><tr><td>Strict</td><td>qacG</td><td></td><td>Protein homolog model</td><td>Small multidrug resistance (SMR) antibiotic efflux pump</td><td>Disinfecting agents and antiseptics</td><td>Antibiotic efflux</td><td>44.23</td><td>106.54</td></tr><tr><td>Strict</td><td>Haemophilus influenzae PBP3 conferring resistance to beta-lactam antibiotics</td><td>D350N, S357N</td><td>Protein variant model</td><td>Penicillin-binding protein mutations conferring resistance to beta-lactam antibiotics</td><td>Cephalosporin, cephapymin, penam</td><td>Antibiotic target alteration</td><td>70.33</td><td>98.52</td></tr></tbody></table>

in a 55-year-old patient¹². Another case involved a wound infection resulting from a guinea pig scratch, further underscoring the zoonotic potential of this pathogen³³.

In addition to *N. rosorum*, other *Pasteurellaceae* species—such as *P. multocida*, *Haemophilus* spp., and *Rodentibacter* spp.—have been implicated in both respiratory and soft tissue infections in rodents and humans alike³³,³⁴. Given the increasing recognition of *N. rosorum* in clinical cases, accurate identification and comprehensive epidemiological investigations are crucial to clarify its transmission pathways and clinical relevance¹⁰⁻¹⁴.

The genomic characterization of *N. rosorum* strain RG01, isolated from the nutria carcass found in Borély Park, provides valuable insights into the microbiological context of the case. The genome of strain RG01 shares key characteristics with previously described *N. rosorum* strains, including genome size, GC content, and gene content, thus supporting its taxonomic classification. The high identity observed in the 16S rRNA gene sequence reinforces the identification of this isolate as *N. rosorum*.

Phylogenetic analysis, based on both 16S rRNA and core genome sequences, revealed that strain RG01 clusters with other *N. rosorum* strains in a distinct clade closely related to the *Rodentibacter* genus. This phylogenetic grouping enhances our understanding of the evolutionary relationships and genetic diversity within *Pasteurellaceae*. Moreover, ancestral sequence reconstruction (ASR), performed using the empirical Bayesian approach implemented in IQ-TREE v1.6, inferred the most probable ancestral states at key nodes of the core genome tree. Our results demonstrated high-confidence ancestral state assignments (posterior probabilities > 0.975; Fig. S2), suggesting robust evolutionary reconstructions and excluding ambiguous or multiple evolutionary pathways. This analysis indicates that the *Necropsobacter* lineage likely derived from a *Caviibacterium pharyngocola*-like ancestor—originally isolated from the pharynx of *Cavia porcellus* (guinea pig)³⁵. These findings suggest a co-evolutionary relationship between *Necropsobacter* and rodents.

Comparative pan-genomic analysis showed a high degree of genomic conservation among *N. rosorum* strains, reflecting close evolutionary relationships and a shared gene repertoire. The presence of a CRISPR-Cas system in strain RG01 suggests a functional defense mechanism against phage infection and horizontal gene transfer. The detection of intact and partial prophage regions indicates potential for phage-mediated genomic variation, which may shape the bacterium's adaptability and virulence. The antimicrobial resistance profile of strain RG01, as reported in this study, offers preliminary insights into treatment considerations for *N. rosorum* infections and warrants further research into resistance mechanisms and their implications for public and veterinary health.

This study also examined the genetic epidemiology of *Pasteurellaceae* using GenBank data. Of 408,387 sequence records retrieved, 62.1% (n=253,496) were deemed suitable for genomic epidemiology studies, underscoring the potential of genetic databases in understanding *Pasteurellaceae*-associated disease dynamics. Notably, 74.6% of suitable records corresponded to species involved in both human and animal infections, highlighting the zoonotic potential of many members of this family. In contrast, 23.1% were exclusively linked to animal cases, and only 2.3% to human cases. This distribution highlights host-specific adaptations and indicates areas where research is still needed.

Our analysis also emphasizes the dominance of well-studied species within the family, such as *Haemophilus influenzae*, *Glaesserella parasuis*, *Avibacterium* spp., *Pasteurella multocida*, and *Mannheimia haemolytica*. The geographic distribution of sequence submissions shows that countries including the USA, China, India, Spain, Denmark, Canada, Norway, Sweden, the UK, Brazil, and Australia are major contributors to *Pasteurellaceae* genomic research. In contrast, *N. rosorum* remains underrepresented, with only 13 records identified from nine countries and three host species, highlighting clear gaps in global genomic surveillance of this emerging pathogen.

In conclusion, the genomic characterization of *Necropsobacter rosorum* strain RG01 from a nutria carcass represents a significant advancement in our understanding of this under-studied member of the *Pasteurellaceae* family. The data support its pathogenic potential in rodents and its zoonotic relevance. Additionally, the broader analysis of *Pasteurellaceae* genomic data emphasizes the importance of integrative, cross-species studies to better understand disease transmission, evolution, and control. Collectively, these findings reinforce the value
<figure><img src="image_7.png" /><figcaption>B</figcaption></figure>

<figure><img src="image_8.png" /><figcaption>C</figcaption></figure>

<figure><img src="image_9.png" /></figure>

**Fig. 4.** Genetic epidemiology plots showing the distribution of selected *Pasteurellaceae* sequence records (n = 253,496; 62.1% of available records at the time of the study) by species abundance and diversity (pie chart), and geographic origin (choropleth map). Data are presented based on host association: both humans and animals (A), animals only (B), and humans only (C).

of collaborative genomic surveillance in the identification, monitoring, and management of both emerging and established zoonotic pathogens.

## Materials and methods

### Animal necropsy and microbial investigation

The necropsy of the nutria (*Myocastor coypus*) carcass involved a systematic examination to assess gross pathological changes and to identify lesions indicative of bacterial infection. When present, lesions were documented based on their macroscopic appearance, size, and anatomical location. Observed changes included areas of discoloration, inflammation, necrosis, and abscess formation. Organs were fixed in 4% buffered
formalin, embedded in paraffin, and sectioned into serial slices of 3–5 µm thickness. Routine hematoxylin-eosin-saffron (HES) staining was performed, along with a panel of special stains to detect bacterial and fungal elements. These included periodic acid-Schiff (PAS), Giemsa, Brown-Hopps/Brown-Brenn Gram, Grocott-Gomori methenamine silver, Warthin-Starry, and Ziehl-Neelsen acid-fast stains. Granulomas were defined as aggregates of ten or more macrophages and lymphocytes within organ tissues. In addition, tissue samples from affected areas were aseptically collected for bacterial isolation and culture. Blood from the ventricular cavity, lung abscess material, and pleural serous fluid were inoculated onto Columbia agar supplemented with 5% sheep blood (bioMérieux, Marcy l'Etoile, France) and incubated at 37 °C for 24 h. Individual bacterial colonies obtained from cultures were subjected to 16S rRNA gene sequencing for species identification.

## Bioinformatics study

Genomic DNA was extracted from bacterial isolates using the EZ1 Biorobot and the EZ1 DNA Tissue Kit (Qiagen, Hilden, Germany). All DNA samples were subjected to 16S rRNA gene sequencing for bacterial species identification. One selected DNA sample was further processed for whole-genome sequencing using the MiSeq platform (Illumina Inc., San Diego, CA, USA). Library preparation was carried out using the Nextera Mate Pair Sample Preparation Kit and the Nextera XT Paired-End Kit (Illumina), following the manufacturer's protocol and previously described methods³⁶. Briefly, genomic DNA (gDNA) was fragmented and subsequently amplified through 12 cycles of PCR, followed by the addition of tag adapters and dual-index barcodes. Library purification was performed using AMPure XP beads (Beckman Coulter, Inc., Fullerton, CA, USA), and normalization was carried out with specific beads according to the Nextera XT protocol. The libraries were then pooled into a single library for sequencing. After 39 h, automated cluster generation and paired-end sequencing with dual-index reads were conducted in a single run using the 2×250 bp format (Miseq reagent Kit, V2-500 cycles). A total of 2×708,011 reads were quality-trimmed using Trimmomatic³⁷,³⁸, and genome assembly was performed using SPAdes. The average sequencing depth of coverage was 125x. Scaffolds shorter than 800 base pairs and those with a sequencing depth less than 25% of the mean depth were removed from the assembly. Genome annotation was conducted using the Prokka pipeline (v1.14.6)³⁹. Antimicrobial resistance genes were identified using the Resistance Gene Identifier (RGI) tool from the Comprehensive Antibiotic Resistance Database (CARD) (https://card.mcmaster.ca/analyze/rgi), with default parameters set to 'Perfect and Strict Hits' and 'High Quality/Coverage'⁴⁰,⁴¹.

Comparative pan-genomic analysis was conducted using Roary (v3.13.0), comparing the genomes of three strains: *Necropsobacter rosorum* strain Bonneveine, *N. rosorum* strain P709T (accession: 675,816,935), and *Necropsobacter massiliensis* strain FF6 (accession: LN776178.1)⁴¹. Genomic data generated in this study have been deposited in the European Nucleotide Archive (ENA) at EMBL-EBI under accession number PRJEB60033 (https://www.ebi.ac.uk/ena/browser/view/PRJEB60033).

CRISPR-Cas elements were identified using the CRISPR-Finder database⁴², and prophage regions were detected using PHASTER⁴³. Genomic islands were predicted using IslandViewer 4 and IslandCompare v1.0⁴⁴,⁴⁵. Predicted proteins encoded within these genomic islands were annotated using KEGG pathway analysis⁴⁶.

## Phylogenetic and phylogenomic study

Two phylogenetic analyses were conducted. The first involved maximum likelihood (ML) phylogeny based on 16S rRNA gene sequences and included all available validated strains from the *Pasteurellaceae* family. A total of 130 16S rRNA gene sequences, representing 105 valid species across 34 genera, were analyzed. The 16S rRNA gene sequences obtained from the RG01 strain—via both Sanger and Illumina sequencing—were aligned with representative sequences from the *Pasteurellaceae* family using MAFFT⁴⁷. The resulting alignment was manually curated using BioEdit⁴⁸ to trim sequences and resolve ambiguities. ML phylogenetic inference was performed using IQ-TREE, with 100,000 ultrafast bootstrap replications. The best-fit substitution model (TIM3e+I+G4) was selected using ModelFinder, a feature integrated into IQ-TREE⁴⁹.

To enrich the dataset, information on host specificity for each taxon in the phylogenetic tree was retrieved from GenBank records based on species names. The resulting phylogenetic tree was visualized and annotated using iTOL v5⁵⁰. The molecular clock assumption was evaluated using a likelihood ratio test (*p* > 0.05) to assess the suitability of midpoint rooting, as no outgroup was available. All relevant metadata were incorporated into the phylogenetic tree topology.

The second analysis was based on a core gene phylogeny using a modified EasyCGTree pipeline⁵¹. Fifty complete genome sequences representing validated *Pasteurellaceae* species were re-annotated using Prokka³⁹. The EasyCGTree pipeline was then applied to the resulting CDS-derived protein sequences to identify and extract conserved bacterial genes, as defined by the PHD database (https://github.com/zdf1987/EasyCGTree). A total of 117 out of 122 core CDS genes were identified. Protein sequences were grouped by gene type and aligned individually using MAFFT, followed by trimming with TrimAL⁴⁷,⁵². A Nexus partition file was generated using SequenceMatrix⁵³.

The core gene phylogenetic analysis was performed using IQ-TREE under an amino acid substitution model (-st AA), incorporating ultrafast bootstrap and SH-aLRT support (-alrt 100,000 -B 100,000). The optimal number of threads was automatically selected (-nt AUTO), and the best nearest-neighbor interchange strategy was applied (-allnni). Ancestral state reconstruction (ASR) was also included to infer ancestral gene states.

In parallel, a BLAST+ search was performed to identify the presence of virulence genes associated with *Pasteurella multocida*, including: *toxA*, *pmHAS*, *nanB*, *nanH*, *hsf-1*, *hsf-2*, *pfhA*, *pfhB*, *tadD*, *ptfA*, *hgbA*, *hgbB*, *tbpA*, *fimA*, *tonB*, *exbD*, *exbB*, *fur*, *soda*, *sodC*, *plpB*, *ompA*, *ompH1*, *ompH2*, and *omp87*⁵⁴,⁵⁵. The presence and copy number of these 25 virulence genes were assessed in *N. rosorum* strain RG01 and compared across other *Pasteurellaceae* genomes.
A gene copy number matrix was generated and used to construct a parsimony-based tree using PARS and CONSENSE, as implemented in the PHYLIP software package⁵⁶. Both the core gene phylogeny and the virulence factor-based parsimony tree were visualized and edited using iTOL v5⁵⁰. Midpoint rooting was applied to both ML trees (16S rRNA and core gene-based), justified by the high genetic similarity among *Pasteurellaceae* members and the lack of external calibration data⁵⁷. In addition, the midpoint rooting was applied after verifying the molecular clock assumption through likelihood ratio testing (*p* > 0.05).

## Genetic epidemiology of *Pasteurellaceae*

The NCBI-nr database of the *Pasteurellaceae* bacteria was retrieved as a database file from the NCBI portal. The database was then investigated using a set of customised Python scripts to investigate the genetic epidemiology of *Pasteurellaceae* bacteria. Initially, the pipeline was designed to retrieve all relevant information associated with the *Pasteurellaceae* sequence records, including species and strain names, accession number, gene identity, isolation source, host species, and country. The dataset was filtrated based on the eligibility of sequences for genetic study (i.e., Sequences should belong to one of the valid genera within *Pasteurellaceae* family, the availability of information related to isolation host, gene name, sequence voucher and/or strain name. Species-host affinity was evaluated by grouping species based on their occurrence in hosts into three groups: (i) zoonotic species for which the species name was linked to both human and animal hosts, (ii) animal affinity, and (iii) human affinity, when the species name was found only in animal and human hosts, respectively. All analyses, including data retrieval, duplicate removal, and dataset filtering, were conducted using Python programming language. The Pandas library was used for data manipulation and processing, while the GeoPandas library facilitated the creation of choropleth maps to visualise the geographic distribution of *Pasteurellaceae* sequence records across different host species. Finally, pie chart and correlation clustering plots were generated to highlight the distribution and dominance of species by country and host affinity, respectively.

## Data availability

Genomic data generated in this study have been deposited in the European Nucleotide Archive (ENA) at EM- BL-EBI under accession number PRJEB60033 (https://www.ebi.ac.uk/ena/browser/view/PRJEB60033). The data supporting the findings of this study are available from the corresponding author upon reasonable request and via the GitHub repository (https://github.com/YLdz-SM/Necropsobacter-rosorum-in-nutria-Myocastor-coypu s-with-implications-for-wildlife-and-human-health.git).

Received: 13 September 2024; Accepted: 11 August 2025

Published online: 01 September 2025

## References

1. Kuhnert, P., Scholten, E., Haefner, S., Mayor, D. & Frey, J. *Basfia succiniciproducens* gen. nov., sp. Nov., a new member of the family *Pasteurellaceae* isolated from bovine rumen. *Int. J. Syst. Evol. Microbiol.* **60**, 44 (2010).

2. *Pasteurellaceae*. https://pasteurellaceae.eu. (2025).

3. Frey, J. The role of RTX toxins in host specificity of animal pathogenic *Pasteurellaceae*. *Vet. Microbiol.* https://doi.org/10.1016/j.vetmic.2011.05.018 (2011).

4. Pusterla, N. et al. Fatal pulmonary hemorrhage associated with RTX toxin-producing *Actinobacillus equuli* subspecies *haemolyticus* infection in an adult horse. *J. Vet. Diagn. Investig.* **20**, 118 (2008).

5. Holyoak, G. R. et al. Serum antibodies in mares and foals to *Actinobacillus equuli* whole cells, outer membrane proteins, and Aqx toxin. *Vet. Immunol. Immunopathol.* **118**, 310 (2007).

6. Boerlin, P. et al. Molecular identification and epidemiological tracing of *Pasteurella multocida* meningitis in a baby. *J. Clin. Microbiol.* **38**, 1235 (2000).

7. Ejlertsen, T., Gahrn-Hansen, B., Sogaard, P., Heltberg, O. & Frederiksen, W. *Pasteurella aerogenes* isolated from ulcers or wounds in humans with occupational exposure to pigs: A report of 7 Danish cases. *Scand. J. Infect. Dis.* **28**, 567 (1996).

8. Medkour, H. et al. Severe pneumonia in a street rat (*Rattus norvegicus*) caused by *Rodentibacter rarus* strain RMC2. *Open Vet. J.* **11**, 165 (2021).

9. Boot, R., Nicklas, W. & Christensen, H. Revised taxonomy and nomenclature of rodent *Pasteurellaceae*: Implications for monitoring. *Lab. Anim.* **52**, 300 (2018).

10. Christensen, H. et al. Classification of organisms previously reported as the SP and Stewart-Letscher groups, with descriptions of *Necropsobacter* gen. nov. and of *Necropsobacter rosorum* sp. nov. for organisms of the SP group. *Int. J. Syst. Evol. Microbiol.* **61**, 1829 (2011).

11. Pirathaba, K. J., Ishak, N., Ngoo, S. K. M. H., Hashim, R. & Samsudin, N. A case of *Necropsobacter rosorum* bacteraemia. *J. Health Transl. Med.* **26**, 185–188 (2023).

12. Jin, C. et al. Human abdominal abscess caused by *Necropsobacter rosorum* and tips for its identification: A case report: Identification of *Necropsobacter rosorum* from human abdominal abscess. *J. Infect. Chemother.* **28**, 833 (2022).

13. Tan, T. Y., Ng, L. S. Y. & Eng, L. C. Five clinical cases of *Necropsobacter rosorum* bacteremia. *J. Clin. Microbiol.* **51**, 1337 (2013).

14. Padmanabhan, R., Robert, C., Fenollar, F., Raoult, D. & Fournier, P. E. Draft genome sequence of *Necropsobacter rosorum* strain P709T. *Genome Announc.* **2**, 10–128 (2014).

15. Woods, C. A., Contreras, L., Willner-Chapman, G. & Whidden, H. P. *Myocastor coypus*. *Mammalian species* 1–8 (1992).

16. Bourdelle, E. American mammals introduced into France in the contemporary Period, Especially *Myocastor* and *Ondatra*. *J. Mammal.* **20**, 287 (1939).

17. Scholarworks@uno, S. *The Rise and Fall of the Louisiana Muskrat, 1890–1960: An Environmental and Social History*. https://scholarworks.uno.edu/td/992.

18. Haubrock, P. J. et al. Economic costs of invasive alien species across Europe. *NeoBiota* **67**, 153–190 (2021).

19. Tsiamis, K. et al. Baseline distribution of invasive alien species of Union concern. *Ispra (Italy): Publications Office of the European Union* 1–96 (2017).

20. Bonnet, M. et al. Aquatic invasive alien rodents in Western France: Where do we stand today after decades of control? *PLoS One* **16**, (2021).

21. Carter, J. & Leonard, B. P. A review of the literature on the worldwide distribution, spread of, and efforts to eradicate the coypu (*Myocastor coypus*). *Wildl. Soc. Bull.* **30**, 162 (2002).
22. Hilts, D. J., Belitz, M. W., Gehring, T. M., Pangle, K. L. & Uzarski, D. G. Climate change and nutria range expansion in the Eastern United States. *J. Wildl. Manag.* **83**, 591–598 (2019).

23. Schertler, A., Rabitsch, W., Moser, D., Wessely, J. & Essl, F. The potential current distribution of the coypu (*Myocastor coypus*) in Europe and climate change induced shifts in the near future. *NeoBiota* **58**, 129–160 (2020).

24. Michel, V. et al. Role of the coypu (*Myocastor coypus*) in the epidemiology of leptospirosis in domestic animals and humans in France. *Eur. J. Epidemiol.* **17**, 111–121 (2001).

25. Howerth, E. W., Reeves, A. J., McElveen, M. R. & Austin, F. W. Survey for selected diseases in nutria (*Myocastor coypus*) from Louisiana. *J. Wildl. Dis.* **30**, 450–453 (1994).

26. Bollo, E. et al. Health status of a population of nutria (*Myocastor coypus*) living in a protected area in Italy. *Res. Vet. Sci.* **75**, 21–25 (2003).

27. Martino, P., Sassaroli, J. C., Calvo, J., Zapata, J. & Gimeno, E. A mortality survey of free range nutria (*Myocastor coypus*). *Eur. J. Wildl. Res.* **54**, 293–297 (2008).

28. Frebourg, N. B., Berthelot, G., Hocq, R., Chibani, A. & Lemeland, J. F. Septicemia due to *Pasteurella pneumotropica*: 16S rRNA sequencing for diagnosis confirmation. *J. Clin. Microbiol.* **40**, 687–689 (2002).

29. Benga, L., Sager, M. & Christensen, H. From the [*Pasteurella*] *pneumotropica* complex to *Rodentibacter* spp.: An update on [*Pasteurella*] *pneumotropica*. *Vet. Microbiol.* **217**, 121–134. https://doi.org/10.1016/j.vetmic.2018.03.011 (2018).

30. Wilson, B. A. & Ho, M. *Pasteurella multocida*: From Zoonosis to cellular microbiology. *Clin. Microbiol. Rev.* **26**(3), 631–655 (2013).

31. Aldová, E. et al. *Aerogenic pasteurellas* and *Pasteurella*-like organisms isolated in czechoslovakia. *Zentralblatt fur Bakteriologie* **277**, 139–143 (1992).

32. Lion, C., Conroy, M. C., Dupuy, M. L. & Escande, F. *Pasteurella*" SP" group infection after a guineapig bite. (1995).

33. Stahel, A. B. J., Hoop, R. K., Kuhnert, P. & Korczak, B. M. Phenotypic and genetic characterization of *Pasteurella multocida* and related isolates from rabbits in Switzerland. *J. Vet. Diagn. Invest.* **21**, 793 (2009).

34. Lo, C. I. et al. Noncontiguous finished genome sequence and description of *Necropsobacter massiliensis* sp. nov. *New Microbes New Infect.* **8**, 41–50. https://doi.org/10.1016/j.nmmi.2015.09.007 (2015).

35. Adhikary, S., Bisgaard, M., Nicklas, W. & Christensen, H. Reclassification of Bisgaard taxon 5 as *Caviibacterium pharyngocola* gen. nov., sp. nov. and Bisgaard taxon 7 as *Conservatibacter flavescens* gen. nov., sp. nov.. *Int. J. Syst. Evol. Microbiol.* **68**, 643–650 (2018).

36. Ndongo, S. et al. 'Cellulomonas timonensis' sp. Nov., taxonogenomics description of a new bacterial species isolated from human gut. *New Microbes New. Infect.* **23**, 7–16 (2018).

37. Prjibelski, A., Antipov, D., Meleshko, D., Lapidus, A. & Korobeynikov, A. Using SPAdes De Novo Assembler. *Curr. Protoc. Bioinform.* **70**, e102 (2020).

38. Bolger, A. M., Lohse, M. & Usadel, B. Trimmomatic: A flexible trimmer for Illumina sequence data. *Bioinformatics* **30**(15), 2114–2120 (2014).

39. Seemann, T. Prokka: Rapid prokaryotic genome annotation. *Bioinformatics* **30**(14), 2068–2069 (2014).

40. Alcock, B. P. et al. CARD 2020: Antibiotic resistome surveillance with the comprehensive antibiotic resistance database. *Nucleic Acids Res.* **48**, D517–D525 (2020).

41. Page, A. J. et al. Roary: Rapid large-scale prokaryote pan genome analysis. *Bioinformatics* **31**, 3691–3693 (2015).

42. Grissa, I., Vergnaud, G. & Pourcel, C. CRISPRFinder: A web tool to identify clustered regularly interspaced short palindromic repeats. *Nucleic Acids Res.* **35**, W52 (2007).

43. Arndt, D. et al. PHASTER: A better, faster version of the PHAST phage search tool. *Nucleic Acids Res.* **44**, W16 (2016).

44. Bertelli, C. et al. Enabling genomic island prediction and comparison in multiple genomes to investigate bacterial evolution and outbreaks. *Microb. Genom.* **8**, 000818 (2022).

45. Bertelli, C. et al. IslandViewer 4: Expanded prediction of genomic islands for larger-scale datasets. *Nucleic Acids Res.* **45**, W30–W35 (2017).

46. Kanehisa, M. & Goto, S. KEGG: Kyoto encyclopedia of genes and genomes. *Nucleic Acids Res.* **28**, 27. https://doi.org/10.1093/nar/28.1.27 (2000).

47. Katoh, K., Rozewicki, J. & Yamada, K. D. MAFFT online service: Multiple sequence alignment, interactive sequence choice and visualization. *Brief Bioinform* **20**, 1160 (2018).

48. Hall, T., Biosciences, I. & Carlsbad, C. BioEdit: An important software for molecular biology. *GERF Bull. Biosci.* **2**, 60 (2011).

49. Nguyen, L. T., Schmidt, H. A., Von Haeseler, A. & Minh, B. Q. IQ-TREE: A fast and effective stochastic algorithm for estimating maximum-likelihood phylogenies. *Mol. Biol. Evol.* **32**, 268–274 (2015).

50. Letunic, I. & Bork, P. Interactive tree of life (iTOL) v5: An online tool for phylogenetic tree display and annotation. *Nucleic Acids Res.* **49**, W293 (2021).

51. Zhang, D.-F. et al. EasyCGTree: A pipeline for prokaryotic phylogenomic analysis based on core gene sets. *BMC Bioinform.* **24**, 390 (2023).

52. Capella-Gutiérrez, S., Silla-Martínez, J. M. & Gabaldón, T. trimAl: A tool for automated alignment trimming in large-scale phylogenetic analyses. *Bioinformatics* **25**, 1972 (2009).

53. Vaidya, G., Lohman, D. J. & Meier, R. SequenceMatrix: Concatenation software for the fast assembly of multi-gene datasets with character set and codon information. *Cladistics* **27**, 171–180 (2011).

54. Peng, Z. et al. *Pasteurella multocida*: Genotypes and genomics. *Microbiol. Mol. Biol. Rev.* **83**, 10–128 (2019).

55. Foggin, C. M. et al. *Pasteurella* sp. associated with fatal septicaemia in six African elephants. *Nat. Commun.* **14**, 6398 (2023).

56. Baum, B. R. PHYLIP: Phylogeny Inference Package. Version 32. Joel Felsenstein. *Q Rev Biol* **64**, (1989).

57. Kinene, T., Wainaina, J., Maina, S. & Boykin, L. M. Rooting trees, methods for. *Encycl. Evolut. Biol.* https://doi.org/10.1016/B978-0-12-800049-6.00215-8 (2016).

## Acknowledgements

The authors are grateful to Alexandre Calvini and Laurie Pierdon (SACPA) for their support during the field-work and to Aurelia Caputo (UMR D257 RITMES) for her technical assistance.

## Author contributions

YL, BD and AL prepared the manuscript. BD and YL carried out the sampling. YL and HL carried out laboratory analyses. YL and AL carried out the bioinformatics analysis. YL, BD and AL proofread the manuscript. All authors read and approved the final version of the manuscript.

## Funding

This work was supported by a grant from the French Government managed by the National Research Agency under the "Investissements d'avenir (Investments for the Future)" programme with the reference ANR-10-IA-HU-03 (Méditerranée Infection), by the Contrat Plan Etat-Région and the European funding FEDER IHUPERF.
**Declarations**

**Competing interests**

The authors declare no competing interests.

**Additional information**

**Supplementary Information** The online version contains supplementary material available at https://doi.org/10.1038/s41598-025-15792-1.

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