一例肺炎患猫支气管肺泡灌洗液中皮氏罗尔斯顿菌的分离及比较基因组学特征分析 机翻

Isolation and comparative genomic characterization of Ralstonia pickettii from bronchoalveolar lavage fluid of a cat with pneumonia Yanying Ma  Nanjing Agricultural University / WOAH Reference Laboratory for Swine Streptococcosis Yifan Wang  Nanjing Agricultural University / WOAH Reference Laboratory for Swine Streptococcosis Baoping Li  OnePlus Pet Hospital Shasha Zhang  OnePlus Pet Hospital Zihao Pan  Nanjing Agricultural University / WOAH Reference Laboratory for Swine Streptococcosis Huochun Yao  Nanjing Agricultural University / WOAH Reference Laboratory for Swine Streptococcosis Case Report Keywords: Ralstonia pickettii, cat, bronchoalveolar lavage fluid, pneumonia, comparative genomics Posted Date: October 26th, 2026 DOI: https://doi.org/10.21203/rs.3.rs-11318808/v1 License:   This work is licensed under a Creative Commons Attribution 4.0 International License.   Read Full License Additional Declarations: No competing interests reported.

Isolation and comparative genomic characterization ofRalstonia pickettiifrom bronchoalveolar lavage uid of a cat with pneumonia Yanying Ma 1 , Yifan Wang 1 , Baoping Li 2 , Shasha Zhang 2 , Zihao Pan 1* , Huochun Yao 1 1 College of Veterinary Medicine, Nanjing Agricultural University / WOAH Reference Laboratory for Swine Streptococcosis, Nanjing, 210095, China. 2 OnePlus Pet Hospital, Nanjing, 210015, China. *Corresponding author(s). E-mail(s): panzihao@njau.edu.cn; Abstract The clinical signicance ofRalstonia pickettiirecovered from the feline lower respiratory tract remains poorly characterized. A 1.5-year-old neutered male cat presented with recurrent respiratory signs for approximately four months and pulmonary radiographic abnormalities. Bronchoalveolar lavage uid culture yielded aRalstoniaisolate, whereas concurrent lavage-uid and instrument controls showed no bacterial growth. Identication asR. pickettiiwas supported by 16S rRNA sequencing and a digital DNA–DNA hybridization value of 77.2% with the type strain. Whole-genome sequencing yielded a 5.46-Mb draft assembly comprising 70 contigs. Broth microdilution testing identied susceptibility to levooxacin, doxycycline and trimethoprim-sulfamethoxazole, and resistance to eight tested agents, including imipenem and meropenem. Marbooxacin susceptibility was reported separately by the commercial laboratory. Clinical improvement occurred during ongoing marbooxacin treatment and adjunctive nebulization. Comparison with 37 public reference genomes identied 3,389 core gene clusters. The feline isolate shared 99.9727% orthologous average nucleotide identity with the reference having the shortest estimated pairwise genetic distance, an International Space Station-associated isolate. Low support for the adjacent internal branch and unequal source representation limited interpretation of ne-scale relationships and acquisition source. This case documents a species-conrmed feline lower respiratory tract isolate and its genomic context. 1

The single-case design, absence of concurrent lavage-uid cytology and improvement during continuing combined treatment preclude rm conclusions about causation or treatment ecacy. Keywords:Ralstonia pickettii, cat, bronchoalveolar lavage uid, pneumonia, comparative genomics Background Ralstonia pickettiiis a non-fermenting, Gram-negative bacterium originally described asPseudomonas pickettiifollowing its isolation from human clinical specimens ( Ralston et al. 1973). It has subsequently been recovered from diverse environments, including soil, water and industrial high-purity water systems, as well as clinical samples. Comparative investigations of environmental and clinical isolates have highlighted both its broad distribution and the diculty of distinguishing it from closely related species, particularlyRalstonia insidiosa(Ryan et al. 2011). The clinical importance ofR. pickettiiis supported by reports of bacteremia, catheter-associated bloodstream infection and neonatal sepsis. Infections have been documented in neonates, children with leukemia and pediatric hematopoietic stem cell transplant recipients, illustrating its capacity to cause invasive disease in vulnerable patients (Sharma et al. 2017;Bedir Demirdag et al. 2022;Çelen et al. 2024). A multicenter investigation in Colombia documented 66 bloodstream infections across nine hospitals during the COVID-19 pandemic, predominantly involving critically ill patients (Viana-Cárdenas et al. 2024). Outbreak investigations in Taiwan and the United Kingdom have also implicated contaminated saline solutions, demonstrating the clinical consequences of contamination involving products used in patient care (Chen et al. 2017;Saunders et al. 2024). Respiratory tract involvement has also been investigated.Ralstoniaspecies have been recovered from the respiratory secretions of patients with cystic brosis, with serial isolates supporting persistent colonization in some individuals (Coenye et al. 2002). An outbreak ofR. pickettiirespiratory colonization was associated with an intrinsically contaminated respiratory care solution (Labarca et al. 1999). In veterinary medicine, a recent companion-animal surveillance study listed two feline isolates asRalstoniaspp., although detailed information linking these isolates to individual clinical presentations and outcomes was not provided (Habrun et al. 2025). Consequently, the clinical signicance ofRalstoniaisolated from the feline lower respiratory tract remains insuciently characterized. Accurate identication and characterization are particularly relevant because conventional identication may not reliably resolve species within this genus. Wholegenome analyses have claried the taxonomy of clinicalRalstoniaisolates and enabled investigation of their phylogenetic relationships and antimicrobial resistance determinants ( Fluit et al. 2021;Jiang et al. 2020). Here, we describe a cat with recurrent respiratory signs and a clinical diagnosis of pneumonia, from which aRalstoniaisolate was recovered from bronchoalveolar lavage uid (BALF). This report aims to 2

document the clinical course and microbiological ndings, establish the identity and genomic characteristics of the lower respiratory tract isolate, and discuss its clinical signicance in feline respiratory disease. Case presentation Clinical history and diagnostic sampling On 1 September 2026, a 1.5-year-old neutered male cat weighing 4.9 kg was presented with recurrent sneezing, mucopurulent nasal discharge, and coughing for approximately 4 months. The cat had experienced similar signs during kittenhood, when respiratory PCR testing detected feline calicivirus andMycoplasma; the signs subsequently resolved following doxycycline treatment. According to the owner, treatment of the current episode before presentation consisted of owner-administered oral doxycycline (50 mg/day) for 2 weeks, followed by marbooxacin (10 mg/day) for 48 days. An initial improvement during marbooxacin treatment was followed by recurrence of respiratory signs. The owner subsequently administered doxycycline (50 mg/day) for more than 1 month without complete resolution. Four days after antimicrobial withdrawal, the cat developed worsening coughing and respiratory noises, lethargy, and reduced appetite. Physical examination revealed increased respiratory sounds and a rectal temperature of 39.0°C. The leukocyte and neutrophil counts were 8.8×10 9 /L and 5.36×10 9 /L, respectively, and serum biochemical ndings were within the laboratory reference intervals. Thoracic radiographs showed increased pulmonary opacity and bronchial thickening without evident pleural eusion (Fig. 1). Following the consultation on 1 September 2026, oral marbooxacin (10 mg once daily) was prescribed and continued through the latest follow-up on 5 October, including before respiratory sampling. The cat received nebulization with lincomycin, ambroxol hydrochloride, and dexamethasone sodium phosphate before respiratory sampling, during which respiratory signs and appetite improved. Each session lasted 30 min and included ambroxol hydrochloride (15 mg in 2 mL) and dexamethasone sodium phosphate (5 mg in 1 mL). Nebulization was performed twice daily for the rst 7 days. Lincomycin was not continued after the bacterial culture result became available. On 3 September 2026, nasal uid and BALF were collected. Sampling materials and instruments were single-use or appropriately disinfected. Control samples of the lavage uid and instruments were collected concurrently and yielded no bacterial growth on agar under the conditions tested. BALF was submitted to a commercial veterinary diagnostic laboratory for aerobic bacterial culture, antimicrobial susceptibility testing, and fungal culture. Aerobic culture yielded moderate growth ofRalstonia spp., and the laboratory reported susceptibility to marbooxacin and doxycycline. The laboratory report dated 15 September 2026 recorded a negative mycological examination. In our laboratory, aliquots of both specimens were inoculated onto blood agar, MacConkey agar, and aMycoplasmadierential culture medium on the day of collection. Blood agar cultures were incubated at 37°C in 5% CO 2 for 48–72 h. Growth was observed only on blood agar, with a single colony morphotype recovered from 3

each specimen under the conditions used. The isolate recovered from BALF was subjected to 16S rRNA gene sequencing, yielding a sequence of 1,395 bp. Comparison with sequences in the NCBI database showed 99.7% sequence identity toRalstonia pickettii, supporting preliminary identication as this species. Microbiological and genomic investigations Antimicrobial susceptibility testing of the BALF isolate was performed for 11 agents using broth microdilution according to CLSI M07. Susceptibility categories were interpreted using the MIC breakpoints for other non-Enterobacterales in CLSI M100, 36th edition (2026), Table 2B-5 ( Clinical and Laboratory Standards Institute 2026). The isolate was susceptible to levooxacin, doxycycline and trimethoprimsulfamethoxazole, and resistant to ceftazidime, cefepime, piperacillin-tazobactam, imipenem, meropenem, amikacin, ciprooxacin and gentamicin (Table1). Marbooxacin was not included in this testing and was reported separately as susceptible by the commercial diagnostic laboratory. Because Table 2B-5 does not provide marbooxacin breakpoints and the laboratory’s specic interpretive criteria were unavailable, this category is presented as a laboratory-reported nding and was not independently reinterpreted. Whole-genome sequencing of the BALF isolate on an Illumina NovaSeq 6000 platform (paired-end 150 bp) generated 4,410,404 quality-ltered read pairs (1.32 Gb). Assembly with Unicycler v0.5.1 (Wick et al. 2017) yielded a 5,464,709-bp draft genome comprising 70 contigs (≥100 bp), with a GC content of 63.56% and an N50 of 194,519 bp. CheckM2 v1.1.0 (Chklovski et al. 2023) estimated genome completeness and contamination at 99.99% and 0.01%, respectively. Mapping the reads to the assembly with Bowtie2 v2.5.5 (Langmead and Salzberg 2012) gave an alignment rate of 96.63% and a mean depth of 233.4×. Comparison using TYGS (Meier-Koltho and Göker 2019) with theR. pickettiitype strain NBRC 102503 T yielded a digital DNA–DNA hybridization value of 77.2% (d4 formula; 95% condence interval, 74.2–79.9%), supporting the species identication. Public assemblies were retrieved from NCBI Datasets under TaxID 329 on 30 September 2026. Of 272 assembly records, 156 remained after retaining one assembly from each GenBank/RefSeq pair, preferentially the RefSeq record. Thirty metagenome-assembled genomes were excluded, leaving 126 non-MAG candidates. Using the prespecied quality thresholds of at least 95% completeness and no more than 5% contamination, uniform CheckM2 assessment excluded four assemblies, leaving 122 for subsequent screening of species identity and redundant records. Species identity was examined by OrthoANI comparison (Lee et al. 2016) with theR. pickettii type-strain assemblies NBRC 102503 T and ATCC 27511 T using PyOrthoANI v0.7.0 with NCBI BLAST+ v2.9.0, with comparisons against closely related species where necessary. Of these assemblies, 37 were excluded because their species identity was inconsistent with, or insuciently supported as,R. pickettii, and 44 were excluded as redundant records based on strain identiers, culture-collection equivalences and pairwise OrthoANI evidence. Four additional assemblies with borderline OrthoANI values of approximately 95.1% against theR. pickettiitype strain were further examined using the Genome-to-Genome Distance Calculator (GGDC) ( Meier-Koltho et al. 4

2013). Their Formula 2 dDDH values ranged from 61.5% to 61.9%, and they were therefore excluded from the within-species comparison. High sequence similarity alone was not used to merge records representing dierent named strains. The remaining 37 public reference genomes were combined with the feline isolate CAT_BALF to form the 38-isolate phylogenomic dataset. The 37 public reference genomes included three human clinical isolates, one isolate from human stem-cell material, two human gastrointestinal isolates with unspecied clinical status, one hospital water-system isolate, three other environmental-water isolates, one plant-associated isolate, one type-strain representative, 23 International Space Station (ISS)-water isolates and two ISS-associated isolates with unspecied isolation material. Isolation sources were assigned using BioSample metadata and associated publications where available. Human origin was not interpreted as evidence of pathogenicity, and unspecied isolation material was not reclassied as an environmental source. ISS-associated isolates accounted for 25 of the 37 public references, indicating substantial imbalance in source representation. Although duplicate assemblies of the same strain were removed, unequal sampling across projects and sources remained a limitation. Detailed quality metrics and software versions are provided in Table S1; reference genome accessions, isolation sources, geographic information, collection dates and screening decisions are provided in Table S2. The 38 genomes were annotated using Bakta v1.9.4 with the full v5.0 database (Schwengers et al. 2021), and a concatenated core-gene nucleotide alignment was generated using Roary v3.13.0 (Galaxy wrapper v3.13.0+galaxy3) (Page et al. 2015). Protein clustering used a minimum BLASTp identity of 95%, and core genes were dened as present in at least 99% of isolates. Paralog splitting was enabled, the translation table was 11, the MCL ination value was 1.5, and the maximum number of clusters was 50,000. The Galaxy wrapper generated the core-gene alignment using the MAFFT option. Roary identied 3,389 core gene clusters among 10,026 total gene clusters. The alignment contained 3,349,544 sites, including 122,995 parsimonyinformative sites. Maximum-likelihood inference was performed using IQ-TREE v3.1.3 (Wong et al. 2026) under the GTR+F+R5 model selected by ModelFinder according to the Akaike information criterion (Kalyaanamoorthy et al. 2017). Branch support was assessed using 1,000 SH-aLRT replicates (Guindon et al. 2010) and 1,000 ultrafast bootstrap replicates (Hoang et al. 2018), with bootstrap-tree nearest-neighbor interchange (NNI) optimization enabled. The random seed was 164353. The resulting unrooted phylogeny is presented in Fig.2. Pairwise maximum-likelihood distances identied the ISS-associated isolate 122630001-2 (GCF_053622495.1) as the closest reference to CAT_BALF among the sampled isolates. Its isolation material was unspecied in the available metadata. The estimated distance between these isolates was 9.004×10 -6 substitutions per site, and their pairwise OrthoANI was 99.9727%. Other references with short pairwise distances to CAT_BALF included the ISS-water isolates GCF_019642395.1, GCF_019642675.1 and GCF_014141915.1. Thus, the closest sampled references by pairwise genetic distance were ISS-associated isolates rather than the human clinical isolates included in this dataset. Pairwise distances and OrthoANI results for all 37 reference genomes are provided in Table S3. 5

The internal branch immediately adjacent to CAT_BALF had SH-aLRT/UFBoot support of 0/63 and a length of approximately 3.06×10 -8 substitutions per site. IQ-TREE also identied 13 near-zero internal branches across the phylogeny. These ndings indicate uncertainty in the ne-scale branching relationships and do not support designation of a robust, unique sister isolate for CAT_BALF. The reported maximum-likelihood distances are model-based estimates from the core-gene alignment, rather than directly observed whole-genome SNP counts. Treatment and follow up Dexamethasone was discontinued on 1 October 2026. At the latest follow-up on 5 October 2026, the cat remained on oral marbooxacin (10 mg once daily) and received nebulized ambroxol hydrochloride alone (15 mg in 2 mL per session, 15 min). The owner reported normal activity, stable appetite, and a 0.5-kg increase in body weight. Occasional sneezing occurred after dexamethasone withdrawal, but abundant mucopurulent nasal discharge was absent. These ndings documented clinical improvement during ongoing treatment. Discussion and conclusions To our knowledge, this is the rst report of the isolation and whole-genome characterization ofRalstonia pickettiifrom bronchoalveolar lavage uid of a cat with pneumonia. Previous companion-animal surveillance listed two feline isolates asRalstoniaspp. ( Habrun et al. 2025), while a clinical case report documentedR. pickettiisepticemia in a dog receiving immunosuppressive treatment (Herrera et al. 2009). The present case adds species-conrmed evidence from the feline lower respiratory tract, together with clinical follow-up and a comparative genomic analysis. Reliable species assignment is central to this contribution: genomic studies of clinicalRalstoniaisolates have revealed taxonomic diversity and discrepancies in species identication (Fluit et al. 2021). Here, the concordant 16S rRNA and type-strain genomic comparisons support identication asR. pickettiiand provide a basis for comparing this animal isolate with publicly available genomes. The culture ndings support consideringR. pickettiiin the assessment of this cat’s respiratory disease. Persistent respiratory signs and pulmonary radiographic abnormalities were accompanied by moderate growth ofRalstoniaspp. at the commercial laboratory and recovery of a single colony morphotype from BALF in our laboratory. Concurrent lavage-uid and instrument controls yielded no bacterial growth under the conditions tested, reducing concern about contamination from the materials examined. Such controls are particularly relevant because contaminated respiratory-care solutions have previously been implicated inR. pickettiicolonization (Labarca et al. 1999). The remaining clinical question is whether the isolate represented primary infection, a secondary contribution to an abnormal airway, or colonization. Feline studies have documented overlap between bacterial bronchopneumonia and inammatory airway disease and support interpreting culture alongside BALF cytology and other diagnostic ndings (Foster et al. 2004;Dear et al. 2021). Persistent airway recovery ofRalstoniaspp. in people with cystic brosis provides a further example of 6

its occurrence in diseased airways, although its independent contribution to clinical deterioration remains uncertain (Green et al. 2017). Clinical improvement during management provides useful outcome information but does not isolate the eect of antimicrobial treatment. Marbooxacin was administered before BALF collection, and treatment also included nebulized ambroxol and dexamethasone. Antimicrobial exposure before sampling could have altered the organisms recovered, while concurrent treatments complicate attribution of the subsequent improvement. At follow-up, the owner reported normal activity, stable appetite, weight gain, and reduced nasal discharge. These observations document improvement during continuing treatment; assessment of sustained remission and microbiological clearance would require follow-up beyond the period currently documented. Comparative genomics places CAT_BALF close to several sampled waterassociated isolates, expanding the context in which its clinical recovery can be interpreted. Its shortest estimated pairwise distance was to the ISS-associated isolate 122630001-2, whose isolation material was unspecied, and several other closely related references were from ISS water. An earlier comparison of 76R. pickettii genomes identied multiple genomic groups, including two dominated by ISS drinkingwater isolates (Yuan et al. 2024). Longitudinal studies of ISS potable-water collections have documented repeated recovery of this species (Yang et al. 2021), and experimental work has demonstrated extracellular polymeric substance production and biolm formation by ISS-water isolates (Kenzaka et al. 2026). Together, these studies illustrate the persistence and phenotypic characteristics of some water-associated populations. The similarity of CAT_BALF to these references extends the observed genomic relationships across environmental and animal clinical sampling contexts. Whether CAT_BALF shares the biolm-related phenotypes measured in those studies remains an empirical question. The resolution and composition of the comparison are important when interpreting the apparent nearest relationships. CAT_BALF shared 99.9727% OrthoANI with the reference having the shortest estimated distance, but the very short adjacent internal branch and low branch support did not establish a robust, unique sister isolate. This supports describing close genetic similarity among several sampled isolates rather than selecting a single reference as an epidemiological counterpart. In addition, 25 of the 37 reference genomes were ISS-associated. Removing redundant assembly records prevented repeated representation of the same strain, but the remaining projectlevel imbalance limits how well the collection represents species-wide diversity. The results therefore place CAT_BALF within the sampled diversity without establishing a source-specic or feline-adapted lineage. Genomic distances also require epidemiological context before they can inform individual transmission pathways (Worby et al. 2014); matched local samples and exposure information were unavailable in this case. The broader value of this comparison is the inclusion of an animal clinical isolate in a species that has been sampled across clinical and environmental settings. Genus-level comparative work has identied dierences in gene content among groups dominated by water, soil, plant, or human-associated records (Liu et al. 2025). Well-characterized animal isolates can help test whether such ecological patterns remain apparent with broader sampling and more complete metadata. This requires distinguishing isolation 7

source from demonstrated infection and retaining unknown sources as unknown. A recent humanR. pickettiioutbreak investigation also illustrates the value of combining genomic relationships with exposure information: a likely association with saline use was identied, but a specic contaminated product or lot could not be conrmed (Sandfort et al. 2025). From a One Health perspective, including companion-animal clinical isolates in genomic comparisons across sources can improve our understanding ofRalstoniadiversity and its clinical signicance. The principal limitations are the single-case design, whole-genome characterization of one BALF isolate, and the unavailability of concurrent BALF cytology. These constrain assessment of causation and within-host diversity, while ongoing treatment at the latest follow-up limits conclusions about long-term outcome. Further studies incorporating additional animal isolates, standardized clinical information, longitudinal follow-up, and epidemiologically relevant environmental samples would help clarify the signicance of this organism in companion animals. This report provides a species-conrmed feline lower respiratory tract isolate and its genomic context, supporting recognition ofR. pickettiias an organism warranting investigation when recovered from cats with respiratory disease. Statements and declarations Competing interests The authors declare that they have no competing interests. Author contributions Yanying Ma: Investigation, Formal analysis, Writing - original draft. Yifan Wang: Validation, Investigation.. Baoping Li: Visualization. Shasha Zhang: Writing - review & editing. Zihao Pan: Conceptualization, Funding acquisition. Huochun Yao: Supervision, Funding acquisition. Informed consent and consent for publication Informed consent was obtained from the cat’s owner. All authors and the cat’s owner have given their consent for the publication of this manuscript. Data availability The sequencing data forRalstonia pickettiistrain CAT_BALF have been deposited at NCBI under BioProject accession PRJNA1541102 and BioSample accession SAMN64051044. The raw sequencing reads have been assigned SRA accession SRR41075723, and the 16S rRNA gene sequence has been assigned GenBank accession QB146246. 8

References Bedir Demirdag T, Ozkaya-Parlakay A, Bayrakdar F, Gulhan B, Kanik Yuksek S, Suzuk Yildiz S, et al (2022) An outbreak ofRalstonia pickettiibloodstream infection among pediatric leukemia patients. J Microbiol Immunol Infect 55:80–85 Çelen SS, Zhumatayev S, Yalçın K, Kara M, Sütçü M, Karasu G, et al (2024) Central line-associated bloodstream infection outbreak related toRalstonia pickettii-contaminated saline in a pediatric hematopoietic stem cell transplant center. Turk J Pediatr 66:421–428 Chen YY, Huang WT, Chen CP, Sun SM, Kuo FM, Chan YJ, et al (2017) An outbreak ofRalstonia pickettiibloodstream infection associated with an intrinsically contaminated normal saline solution. Infect Control Hosp Epidemiol 38:444–448 Chklovski A, Parks DH, Woodcroft BJ, Tyson GW (2023) CheckM2: a rapid, scalable and accurate tool for assessing microbial genome quality using machine learning. Nat Methods 20:1203-1212 https://doi.org/10.1038/s41592-023-01940-w Clinical and Laboratory Standards Institute (2026) Performance standards for antimicrobial susceptibility testing, 36th edn. CLSI supplement M100. Clinical and Laboratory Standards Institute Coenye T, Vandamme P, LiPuma JJ (2002) Infection byRalstoniaspecies in cystic brosis patients: identication ofR. pickettiiandR. mannitolilyticaby polymerase chain reaction. Emerg Infect Dis 8:692–696 Dear JD, Vernau W, Johnson EG, Hulsebosch SE, Johnson LR (2021) Clinicopathologic and radiographic features in 33 cats with aspiration and 26 cats with bronchopneumonia (2007-2017). J Vet Intern Med 35:480-489https://doi.org/10. 1111/jvim.16005 Fluit AC, Bayjanov JR, Díez Aguilar M, Cantón R, Tunney MM, Elborn JS, et al (2021) Characterization of clinical Ralstonia strains and their taxonomic position. Antonie van Leeuwenhoek 114:1721–1733https://doi.org/10.1007/ s10482-021-01637-0 Foster SF, Martin P, Braddock JA, Malik R (2004) A retrospective analysis of feline bronchoalveolar lavage cytology and microbiology (1995-2000). J Feline Med Surg 6:189-198https://doi.org/10.1016/j.jfms.2003.12.001 Green HD, Bright-Thomas R, Kenna DT, Turton JF, Woodford N, Jones AM (2017) Ralstonia infection in cystic brosis. Epidemiol Infect 145:2864-2872https://doi. org/10.1017/s0950268817001728 Guindon S, Dufayard JF, Lefort V, Anisimova M, Hordijk W, Gascuel O (2010) New algorithms and methods to estimate maximum-likelihood phylogenies: assessing the 9

performance of PhyML 3.0. Syst Biol 59:307-321https://doi.org/10.1093/sysbio/ syq010 Habrun CA, Greendyke WG, Szlosek D, Plum A, Kratz MM, Mantell E, et al (2025) Carbapenem-resistant organisms in companion animals in New York City, 2019–2022. Open Forum Infect Dis 12:ofaf613https://doi.org/10.1093/od/ofaf613 Herrera MA, Burkitt JM, Epstein SE, Jones MEB, Moore PF, Sykes JE (2009) Ralstonia pickettii septicemia in a dog with immune-mediated thrombocytopenia. J Vet Intern Med 23:182-185https://doi.org/10.1111/j.1939-1676.2008.0222.x Hoang DT, Chernomor O, von Haeseler A, Minh BQ, Vinh LS (2018) UFBoot2: Improving the Ultrafast Bootstrap Approximation. Mol Biol Evol 35:518-522https: //doi.org/10.1093/molbev/msx281 Jiang T, Xu J, He F (2020) Genotypic and phylogenetic characterisation of a clinical Ralstonia pickettiistrain carrying two novel OXA allelic variants, blaOXA-898 and blaOXA-899, isolated from a bloodstream infection in China. J Glob Antimicrob Resist 21:46–48 Kalyaanamoorthy S, Minh BQ, Wong TKF, von Haeseler A, Jermiin LS (2017) ModelFinder: fast model selection for accurate phylogenetic estimates. Nat Methods 14:587-589 https://doi.org/10.1038/nmeth.4285 Kenzaka T, Ichijo T, Yamazaki T (2026) High extracellular polymeric substance production and biolm-forming capacity of Ralstonia pickettii isolates from ISS potable water. Microbiol Spectr 14:e02913-25https://doi.org/10.1128/spectrum.02913-25 Labarca JA, Trick WE, Peterson CL, Carson LA, Holt SC, Arduino MJ, et al (1999) A multistate nosocomial outbreak of Ralstonia pickettii colonization associated with an intrinsically contaminated respiratory care solution. Clin Infect Dis 29:1281–1286 https://doi.org/10.1086/313458 Langmead B, Salzberg SL (2012) Fast gapped-read alignment with Bowtie 2. Nat Methods 9:357-359https://doi.org/10.1038/nmeth.1923 Lee I, Kim YO, Park SC, Chun J (2016) OrthoANI: An improved algorithm and software for calculating average nucleotide identity. Int J Syst Evol Microbiol 66:1100-1103https://doi.org/10.1099/ijsem.0.000760 Liu G, Mao C, Li Q, Huo D, Li T (2025) Comparative genomic analysis reveals the adaptive traits of Ralstonia spp. in aquatic environments. Front Microbiol 16:1625651https://doi.org/10.3389/fmicb.2025.1625651 Meier-Koltho JP, Göker M (2019) TYGS is an automated high-throughput platform for state-of-the-art genome-based taxonomy. Nat Commun 10:2182https://doi.org/ 10.1038/s41467-019-10210-3 10

Meier-Koltho JP, Auch AF, Klenk HP, Göker M (2013) Genome sequence-based species delimitation with condence intervals and improved distance functions. BMC Bioinformatics 14:60https://doi.org/10.1186/1471-2105-14-60 Page AJ, Cummins CA, Hunt M, Wong VK, Reuter S, Holden MT, et al (2015) Roary: rapid large-scale prokaryote pan genome analysis. Bioinformatics 31:3691- 3693https://doi.org/10.1093/bioinformatics/btv421 Ralston E, Palleroni NJ, Doudoro M (1973)Pseudomonas pickettii, a new species of clinical origin related toPseudomonas solanacearum. Int J Syst Bacteriol 23:15–19 Ryan MP, Pembroke JT, Adley CC (2011) Genotypic and phenotypic diversity of Ralstonia pickettii and Ralstonia insidiosa isolates from clinical and environmental sources including high-purity water. Diversity in Ralstonia pickettii. BMC Microbiol 11:194 https://doi.org/10.1186/1471-2180-11-194 Sandfort M, von Laer A, Kampmeier S, Eichhorn I, Wolf SA, Driller M, et al (2025) Nosocomial Outbreak of Ralstonia pickettii Infections Likely Linked to Saline Solutions in Germany from August 2023 to March 2024-Challenges in Medical Product-Related Outbreaks. Microorganisms 13:2102https://doi.org/10.3390/ microorganisms13092102 Saunders M, Weaver A, Stretch R, Jeyaratnam D, Mirfenderesky M, Elliott D, et al (2024) Outbreak ofRalstonia pickettiiassociated with contamination of saline products distributed internationally, the United Kingdom, 2024. Euro Surveill 29:2400384 Schwengers O, Jelonek L, Dieckmann MA, Beyvers S, Blom J, Goesmann A (2021) Bakta: rapid and standardized annotation of bacterial genomes via alignment-free sequence identication. Microb Genom 7:000685 https://doi.org/10.1099/mgen.0. 000685 Sharma D, Sharma P, Soni P, Gupta B (2017)Ralstoniapicketti neonatal sepsis: a case report. BMC Res Notes 10:28 Viana-Cárdenas E, Triana A, Cárdenas-Álvarez J, Carvajal-Diaz E, Mendoza H, Viasus D (2024) A large multicenterRalstonia pickettiioutbreak in critically ill patients during the COVID-19 pandemic: epidemiological and clinical characteristics of 66 cases. J Infect Prev 25:85–88 Wick RR, Judd LM, Gorrie CL, Holt KE (2017) Unicycler: Resolving bacterial genome assemblies from short and long sequencing reads. PLoS Comput Biol 13:e1005595 https://doi.org/10.1371/journal.pcbi.1005595 Wong TKF, Ly-Trong N, Ren H, Demotte P, Baños H, Roger AJ, et al (2026) IQ- TREE 3: phylogenomic inference software using complex evolutionary models. Mol Biol Evol 43:msag117https://doi.org/10.1093/molbev/msag117 11

Worby CJ, Lipsitch M, Hanage WP (2014) Within-host bacterial diversity hinders accurate reconstruction of transmission networks from genomic distance data. PLoS Comput Biol 10:e1003549https://doi.org/10.1371/journal.pcbi.1003549 Yang J, Barrila J, Ott CM, King O, Bruce R, McLean RJC, et al (2021) Longitudinal characterization of multispecies microbial populations recovered from spaceight potable water. NPJ Biolms Microbiomes 7:70https://doi.org/10.1038/ s41522-021-00240-5 Yuan C, An T, Li X, Zou J, Lin Z, Gu J, et al (2024) Genomic analysis of Ralstonia pickettii reveals the genetic features for potential pathogenicity and adaptive evolution in drinking water. Front Microbiol 14:1272636https://doi.org/10.3389/fmicb. 2023.1272636 12

Table 1Antimicrobial susceptibility categories for the feline bronchoalveolar lavage isolate. Antimicrobial agentCategory MarbooxacinS LevooxacinS DoxycyclineS Trimethoprim-sulfamethoxazoleS CeftazidimeR CefepimeR Piperacillin-tazobactamR ImipenemR MeropenemR AmikacinR CiprooxacinR GentamicinR S, susceptible; R, resistant. Broth microdilution categories were assigned using CLSI M100, 36th edition, Table 2B-5. The marbooxacin category was taken from the commercial report and was not assigned using Table 2B-5, which contains no marbooxacin breakpoint. 13

Fig. 1Thoracic radiographs of a 1.5-year-old cat with recurrent respiratory signs, obtained on 1 September 2026. Ventrodorsal (left) and lateral (right) views show increased pulmonary opacity and prominent bronchial markings, without evident pleural eusion. 14

Fig. 2Core-gene maximum-likelihood phylogeny ofRalstonia pickettii. The tree was inferred from 38 genomes using IQ-TREE v3.1.3 under the GTR+F+R5 model. CAT_BALF, the feline bronchoalveolar lavage isolate, is highlighted in red; colored squares indicate source categories. Branch labels show SH-aLRT/UFBoot support (%), each based on 1,000 replicates. All branches are retained and displayed as a cladogram; horizontal branch lengths do not represent genetic distances. The inferred tree is unrooted. ISS, International Space Station. 15

Supplementary Files This is a list of supplementary files associated with this preprint. Click to download. TableS1.xlsx TableS2.xlsx TableS3.xlsx