This study was approved (2021IIT145) by the Ethics Committee of the First Affiliated Hospital, College of Medicine, Zhejiang University (Hangzhou, China), following the Declaration of Helsinki.

The clinical information, including history of present illness, past history, physical examination, laboratory tests, radiographic examination, and treatment were obtained from the medical records.

Peripheral blood sample was obtained from bilateral elbow veins on the fifth day of admission. The blood was collected in two vials from each side and immediately sent for culture of aerobic and anaerobic bacteria. Blood culture was performed using BacTALERT 3D blood culture system (bioMérieux, Marcy l’Etoile, France) following the manufacturer’s instructions. For samples with positive results, the liquid in the vials was inoculated onto blood agar, chocolate, MacConkey, and fungal chromogenic plates at 35°C and 5% CO₂ for 18–24 hours. The morphologically different colonies were sub-cultured on separate agar plates. The isolated bacteria were identified using the VITEK^® MS microbial mass spectrometry identification system (bioMérieux). Then, VITEK2^® COMPACT automatic identification and antibiotic sensitivity analysis system was used to verify the bacterial identification and perform antibiotic sensitivity analysis. For S. enterica, agglutination test using the corresponding agglutinating serum was performed to identify the species of the isolate. The isolate was named S. Typhimurium_zhang. Then, individual colonies were inoculated in broth tubes. After multiplication for 4–6 h, 10% glycerin broth was added to the tubes and stored at –80°C.

Search terms related to TTP, hemolytic uremic syndrome (HUS), and TMA, as well as their synonyms, were used to search for English articles published up to November 2021 that focused on the link between the aforementioned diseases and their bacterial triggering factors, including genes and proteins. The search results were manually screened for errors.

The frozen bacteria in glycerol lyophilized tubes were incubated on Mueller-Hinton (MHA) plates overnight at 37°C. A single colony was incubated in 2 mL Luria-Bertani (LB) broth at 200 rpm and 37°C overnight. We inoculated 1 mL bacterial solution in 100 mL LB broth at 200 rpm and 37°C for 6 h. The shaken bacterial solution was collected into 50 mL centrifuge tubes and centrifuged at 5000 g for 15 min, and the supernatant was discarded. The bacteria were suspended in phosphate-buffered saline (PBS) and transferred to a 1.5 mL Eppendorf (EP) tube. The EP tubes were centrifuged at 5000 g for 5 min and the supernatant was discarded for DNA extraction and sequencing. Genomic DNA was extracted using a commercial kit (Gentra Puregene Yeast/Bacteria kits; Qiagen, Hilden, Germany) following the manufacturer’s instructions. The Single Molecule, Real-Time (SMRT) sequencing library was constructed using SMRT bell TM Template kit (version 1.0). A next-generation sequencing (NGS) library was constructed using NEBNext^® Ultra™DNA Library Prep Kit for Illumina. The WGS data were processed using the PacBio Sequel platform and Illumina NovaSeq PE150 at the Beijing Novogene Bioinformatics Technology Co., Ltd (Beijing, China). The reads were assembled using SPAdes (version 3.9.1). The annotation of the genome sequence was performed using RAST (https://rast.nmpdr.org/). The genes encoding the candidate triggers obtained from the previously published studies were searched in the annotation result of S. Typhimurium_zhang. Antimicrobial resistance (AMR) genes were identified using the ResFinder database (https://cge.food.dtu.dk/services/ResFinder/) and the virulence factors were identified using the Virulence Factor Database (VFDB, http://www.mgc.ac.cn/VFs/search_VFs.htm).

To identify the species and genus of S. Typhimurium_zhang, all eight type strains of S. Typhimurium were downloaded from the American Type Culture Collection (ATCC) and National Collection of Type Cultures (NCTC), regardless of their completeness, and compared to the genomic sequence of S. Typhimurium_zhang. Average nucleotide identity blast (ANIb) analysis was performed using pyani (https://github.com/widdowquinn/pyani). The ANIb data for each strain were visualized using heatmaps.

The complete sequences of TW14359, based on the search result of S. Typhimurium_zhang, were obtained from the National Center for Biotechnology Information (NCBI, https://www.ncbi.nlm.nih.gov/). All eight type strains of S. Typhimurium were downloaded from ATCC and NCTC, regardless of its completeness, and 73 S. enterica strains with complete genomes were downloaded from NCBI. The genome sequence of each strain was annotated by RAST. The identity of genes and their encoded TMA triggers present in S. Typhimurium_zhang were evaluated in these 83 S. enterica strains using BLAST (https://blast.ncbi.nlm.nih.gov/Blast.cgi).

In total, 69 non-repetitive Enterobacteriaceae type strains were downloaded from NCBI. The genome sequences of 69 Enterobacteriaceae strains were annotated by RAST and searched for genes encoding the candidate triggers present in S. Typhimurium_zhang. The amino acid sequences of the candidate triggers in 69 Enterobacteriaceae strains were downloaded from NCBI. Phylogenetic analysis of the candidate triggers on 69 Enterobacteriaceae strains, including S. Typhimurium_zhang and TW14359, was conducted using Molecular Evolutionary Genetics Analysis across Computing Platforms (MEGAX; version 10.1.7) software and were visualized using the Interactive Tree of Life (iTOL, https://itol.embl.de).

According to the phylogenetic analysis, the one closest and the two farthest strains to S. Typhimurium_zhang, carrying the candidate triggers on the phylogenetic tree, were included in the amino acid sequence alignment. Meanwhile, the candidate triggers were uploaded to SWISS-MODEL (https://swissmodel.expasy.org/) and used to search for templates. Both known crystal structure templates for the protein were included in the amino acid sequence alignment and the protein closest to the candidate trigger on S. Typhimurium_zhang was used as the crystal structure reference in the alignment. The sequence of the candidate triggers in S. Typhimurium_zhang and reference strain related to TMA as well as sequences mentioned above were downloaded and aligned using CLUSTALW (https://www.genome.jp/tools-bin/clustalw) and ESpript 3.0 (Robert and Gouet, 2014) (https://espript.ibcp.fr/ESPript/cgi-bin/ESPript.cgi).

The complete genome data of the isolated S. Typhimurium_zhang has been uploaded to GenBank (accession number: CP090304; https://www.ncbi.nlm.nih.gov/nuccore/CP090304). The bioinformatics procedures described previously were performed using the default settings.

A 35-year-old man presented with black stool, gingival bleeding, and nausea (without vomiting) for 1 day. He passed 10 dilute black stools without any obvious cause 1 day before the admission. The laboratory tests at admission showed an extremely reduced platelet count, mildly abnormal renal function, and normal liver function. He had normal vital signs, with scattered skin petechiae, and oozing of blood from the gums. He had no underlying diseases, venereal disease exposure, or significant marital or family history.

The human immunodeficiency virus (HIV) antibody was detected in a colloidal gold assay screening test and later confirmed by Western blotting. The baseline laboratory test results showed normal blood coagulation function, extremely low platelet count (2 × 10⁹/L), and mildly reduced hemoglobin (112 g/L). The lymphocytes showed significant reduction in CD4+ T cells (166.1 cells/µL). Blood samples were taken to analyze the activity of metalloproteinase with thrombospondin type I motif, member 13 (ADAMTS13) enzyme before initiation of plasma exchange and glucocorticoid therapy; the results showed 0% activity. Tests for hepatitis B surface antigen (HBsAg), hepatitis C (HCV) antibody, syphilis spirochete antibody, cytomegalovirus (CMV) IgM, and Epstein-Barr virus (EBV) IgM were negative. Identical Gram-negative bacteria were detected from blood cultures in all four tubes. The bacteria were identified as S. Typhimurium using agglutination tests and via microbial mass spectrometry 6 days after admission to the hospital. Ultrasound revealed deep venous thrombosis in the right lower limb. Routine examination of bone marrow and biopsy indicated decreased levels of megakaryocytes, without abnormal cells. The disease course and laboratory test results of the patient are shown in  Figures 1 and 2 . The patient presented with thrombocytopenia, microangiopathic hemolytic anemia, fever, mild renal dysfunction, and significant neuropsychiatric symptoms. Based on the absent ADAMTS13 activity, the patient was diagnosed with TTP according to the guidelines of Haemostasis and Thrombosis Task Force of the British Committee for Standards in Hematology (BCSH) (Scully et al., 2012). Our patient had reversible ADAMTS13 activity deficiency, indicating that he had acquired TTP.

The patient received one platelet transfusion, four plasma transfusions, two red blood cell transfusions, and five sessions of plasma exchange. The treatment also included the use of antipyretics, intravenous midazolam for sedation, glucocorticoids for immune suppression, antibiotics (including moxifloxacin) for treating infections, and heparin and rivaroxaban for anticoagulation. The patient did not receive antiviral therapy including antiretroviral therapy (ART). After treatment, the patient’s body temperature and laboratory test results returned to normal, and his symptoms improved significantly. The patient was discharged on day 19 and ART was continued at home.

The patient’s blood culture returned a positive result at 24 h. Similar gray-white colonies were detected on blood agar, chocolate, and MacConkey plates, whereas no growth was detected on the fungal chromogenic plate. The colonies detected on the blood agar, chocolate, and MacConkey plates were identified as Salmonella by the VITEK ^® MS microbial mass spectrometry identification system. VITEK2^® COMPACT automatic identification and antibiotic sensitivity analysis system confirmed the identification. Antibiotic sensitivity analysis was performed using VITEK2^® COMPACT and showed that the bacteria were sensitive to ampicillin, amoxicillin-potassium clavulanate, piperacillin/tazobactam, ceftriaxone, cefepime, ciprofloxacin, levofloxacin, imipenem, ertapenem, and compound sulfamethoxazole. Agglutination test results showed that the isolate agglutinated with Salmonella O4 serum but not with other O serum. The isolate also agglutinated with Hi, H1, and H2. Based on these results, the isolate was determined to be S. Typhimurium.

The WGS revealed that S. Typhimurium_zhang had 4857450 bps with 52.2% GC content. To confirm the species of S. Typhimurium_zhang, ANIb analysis was performed with eight type strains. The ANIb analysis showed that >95% identities matched between the eight type strains and S. Typhimurium_zhang, which confirmed that the pathogenic bacteria isolated from the peripheral blood were S. Typhimurium (Altschul et al., 1997; Konstantinidis and Tiedje, 2005; Richter and Rosselló-Móra, 2009) ( Figure 3 ). The genes, virulence factors, and AMR genes of S. Typhimurium_zhang are presented in Supplementary Tables 1–3 .

No previous reports were identified regarding the genes encoding the candidate triggers associated with TTP. The genes encoding the candidate triggers associated with HUS included stx2 (Ethelberg et al., 2004; Pianciola et al., 2014; Alconcher et al., 2021), eae (Ethelberg et al., 2004; Pianciola et al., 2014; Alconcher et al., 2021), iha (Naseer et al., 2017), IpfA (Naseer et al., 2017), ehxA (Ethelberg et al., 2004; Naseer et al., 2017; Alconcher et al., 2021), stcE (Naseer et al., 2017), rfb _O157 (Alconcher et al., 2021), fliC _H7 (Alconcher et al., 2021), nleB (Cadona et al., 2020), espP (Buvens and Piérard, 2012), vtx2 (Buvens and Piérard, 2012), sen (Buvens and Piérard, 2012), nle (Buvens and Piérard, 2012; Naseer et al., 2017), efa (Buvens and Piérard, 2012), saa (Zweifel et al., 2004), norV (Kulasekara et al., 2009), and aggR (Scheutz, 2014).

The genes encoding the candidate triggers identified from the previous literature were searched in the annotation result of S. Typhimurium_zhang. The results showed that S. Typhimurium_zhang carried norV, a gene encoding anaerobic nitric oxide reductase flavorubredoxin (FlRd). NorV was related to the increased rate of developing HUS as shown in a previous study (Kulasekara et al., 2009).

The annotation results showed that all 83 S. enterica strains carried norV, including S. Typhimurium_zhang, TW14359, and 8 S. Typhimurium type strains. The BLAST results showed that norV and FlRd in S. Typhimurium_zhang, HUS-related E. coli O157:H7 strain TW14359, and eight S. Typhimurium type strains had high identity ( Table 1 ). NorV and its encoded protein FlRd in 73 S. enterica strains presented higher identity compared to norV and FlRd in S. Typhimurium_zhang ( Table 2 ).

The search of NCBI for Enterobacteriaceae that carry FlRd identified 69 strains. Phylogenetic analysis of these 69 Enterobacteriaceae was performed. Among them, Salmonella_bongori_WP_000026020.1 was the closest to FlRd in S. Typhimurium_zhang, whereas Hafnia_alvei_WP_025802316.1 and Hafnia_paralvei_WP_130335968.1 were the farthest from FlRd in S. Typhimurium_zhang ( Figure 4 ).

According to the phylogenetic analysis, Salmonella_bongori_WP_000026020.1, Hafnia_alvei_WP_025802316.1, and Hafnia_paralvei_WP_130335968.1 were included in the amino acid sequence alignment. A SWISS-MODEL search revealed that 5lmc.2 and 6etb.1 were the crystal structure templates for FlRd. The template search of TW14359 revealed similar results. Therefore, 5lmc.2 and 6etb.1 were included in the amino acid sequence alignment. Among them, 5lmc.2 had the highest similarity and was used as the crystal structure reference in the alignment. The FlRd in S. Typhimurium_zhang and TW14359 was also included in the alignment. The results indicated a high degree of similarity in the sequences, indicating that FlRd in S. Typhimurium_zhang was highly likely to be functional ( Supplementary Figure 1 ).