Genomic characteristics of antimicrobial resistance and virulence factors of carbapenem-resistant Stutzerimonas nitrititolerans isolated from the clinical specimen

Abstract

Background

Stutzerimonas nitrititolerans (S. nitrititolerans) is a rare human pathogenic bacterium and has been inadequately explored at the genomic level. Here, we report the first case of carbapenem-resistant S. nitrititolerans isolated from the peritoneal dialysis fluid of a patient with chronic renal failure. This study analyzed the genomic features, antimicrobial resistance, and virulence factors of the isolated strain through whole genome sequencing (WGS).

Methods

The bacterial isolate from the peritoneal dialysis fluid was named PDI170223, and preliminary identification was conducted through Matrix-assisted laser desorption ionization/time of flight mass spectrometry (MALDI-TOF MS). WGS of the strain PDI170223 was performed using the Illumina platform, and a phylogenetic tree was constructed based on the 16S rRNA gene sequences. Antimicrobial susceptibility test (AST) was conducted using the TDR-200B2 automatic bacteria identification/drug sensitivity tester.

Results

S. nitrititolerans may emerge as a human pathogen due to its numerous virulence genes, including those encoding toxins, and those involved in flagellum and biofilm formation. The AST results revealed that the strain is multidrug- and carbapenem-resistant. The antimicrobial resistance genes of S. nitrititolerans are complex and diverse, including efflux pump genes and β⁃lactam resistance genes.

Conclusion

The analysis of virulence factors and antimicrobial resistance of S. nitrititolerans provides clinical insight into the pathogenicity and potential risks of this bacterium. It is crucial to explore the mechanisms through which S. nitrititolerans causes diseases and maintains its antimicrobial resistance, thereby contributing to development of effective treatment and prevention strategies.

Supplementary Information

The online version contains supplementary material available at 10.1186/s12866-024-03546-4.

Introduction

S. nitrititolerans is a non-fermentative gram-negative bacterium initially isolated from soil in 1980. In 1996, S. nitrititolerans was misclassified as Pseudomonas stutzeri (P. stutzeri) genomovar 8, with Scotta et al. reporting two clinical isolates of this strain in environment in 2012 [1, 2]. In 2019, Peng et al. described S. nitrititolerans as Pseudomonas nitrititolerans (P. nitrititolerans) and reported that it exhibited resistance to multiple antimicrobial agents including cefazolin, chloramphenicol, and clindamycin [3]. Recent genome-based taxonomic studies on the genus Pseudomonas have proposed a new genus Stutzerimonas. Several species have been assigned to this genus, including P. nitrititolerans and P. stutzeri genomovar 8 [4]. However, no clinical cases were reported in previous studies. We here report the first case of carbapenem-resistant S. nitrititolerans isolated from the peritoneal dialysis fluid of a patient with chronic renal failure.

According to the List of Prokaryotic names with Standing in Nomenclature (LPSN) database (https://lpsn.dsmz.de/genus/stutzerimonas), the genus Stutzerimonas comprises 16 species. Stutzerimonas is widely distributed in environment and has been isolated from soil (United States), wastewater (Spain), marine water (Spain), marine polluted sediments (Spain), and clinical samples (United Kingdom, Sweden, Spain, and Denmark) [2, 4–8]. Currently, several species have been isolated from clinical specimens, including Stutzerimonas stutzeri, Stutzerimonas balearica, Stutzerimonas nosocomialis, and S. nitrititolerans [2, 5, 6, 9]. Thus, S. nitrititolerans may be an opportunistic pathogen in human infection. By studying the genomes of these species, we can better understand their pathogenicity and drug resistance. However, genomic information on carbapenem-resistant S. nitrititolerans clinical isolate has not yet been reported. We report the genome sequence of S. nitrititolerans isolated from clinical specimens and analyze its potential virulence factors and antimicrobial resistance. This study will provide valuable data for clinical antimicrobial drug selection and control of hospital infections.

Materials and methods

Case report

A 38-year-old male patient with a history of chronic renal failure and gouty arthritis presented to our hospital with nose bleeding and dizziness on February 16, 2023. On physical examination, his vital statistics were as follows: blood pressure, 137/84 mmHg; body temperature, 36.5 °C. Laboratory investigations revealed the following: red blood cell count, 1.8 × 10⁹ /L; hemoglobin, 53 g/L; white blood cell count, 7.29 × 10⁹ /L (neutrophils 87.0%); myohemoglobin, 115.2 ng/mL (normal range, 11.6–73ng/mL); creatinine, 1339.5 µmol/L (normal range, 57–97umol/L); carbonyl diamide, 71.0 mmol/L (normal range, 2.76–8.07 mmol/L); albumin, 37.2 g/L (normal range, 40–55 g/L); lactic dehydrogenase, 395U/L (normal range, 135–225U/L); nucleated cell count of ascitic fluid, 12.78 × 10⁹/L (normal range, 0-0.3 × 10⁹/L); procalcitonin, 0.728 ng/mL (normal < 0.046 ng/mL); C-reactive protein, 2.69 mg/dL (normal range, 0–0.8 mg/dL). Abdominal computed tomography revealed renal atrophy and minimal fluid accumulation. Abdominal tenderness or rebound pain was not observed. One set of peritoneal effluent samples was sent for culture. Subsequently, one sample was positive for bacterial culture. The strain was identified as Pseudomonas sp. by MALDI-TOF MS and ultimately confirmed as S. nitrititolerans by 16 S rRNA gene sequencing. The patient received a blood transfusion, APD peritoneal dialysis, and anti-infective treatment. After five days, the patient felt better and asked to be discharged. This study focuses on the analysis of antimicrobial resistance and virulence factors of the first clinical specimen of multidrug-resistant S. nitrititolerans.

Bacterial strain and growth conditions

The strain PDI170223 was isolated from the peritoneal dialysis fluid of a 38-year-old male patient with chronic renal failure at the Central Hospital of Wuhan, China. The strain was subcultured on Columbia blood agar, MacConkey agar, and Chocolate agar at 35℃ in the presence of 5% CO₂ [3]. After incubation overnight, a single colony was selected from the plates for Gram’s stain and identification using microscopy.

MALDI-TOF MS identification

Following to the manufacturer’s instructions, MALDI-TOF MS identification was performed on an MS platform (Bruker Daltonics GmbH, Germany) using a single-colony direct smear method. The acquired spectra were then transferred to the analysis server, which uses software algorithms to compare the generated spectra with typical spectra in scientific databases [10].

16 S rRNA sequencing

The bacterial DNA extraction was carried out using a TIANamp Bacteria DNA Kit (TIANGEN Biotech, Co., Ltd, Beijing, China) according to the manufacturer’s instructions. The strain was identified through 16SrRNA sequencing with universal primers (27F:5’-AGTTTGATCMTGGCTCAG-3’; 1492R: 5’-GGTTACCTTGTTAA. CTT-3’) were used for the amplification of the 16 S rRNA gene. The amplification procedure involves the following steps: pre-denaturation step at 95 °C for 3 min, followed by 30 cycles of denaturation at 95 °C for 30 s, annealing at 58 °C for 30 s and extension at 72 °C for 1 min, and a final extension at 72 °C for 10 min [11]. The complete 16 S rRNA sequence of the strain PDI170223 was analyzed with the EZBiocloud database [12]. A phylogenetic tree was constructed using the neighbor-joining method by MEGA software version 11 [13].

Antimicrobial susceptibility test

An antimicrobial susceptibility test (AST) was conducted using a TDR-200B2 automatic bacteria identification/drug sensitivity tester (Hunan Mindray Medical Technology Co, China). The AST results were interpreted based on the guidelines of the Clinical and Laboratory Standards Institute (CLSI 2022 standard) (https://clsi.org ) [14]. The tests were conducted using the following antimicrobial agents against non-fermentative and gram-negative bacteria: ampicillin, amoxicillin, amoxicillin-clavulanate, ceftriaxone, cefepime, cefuroxime, cefoxitin, ceftazidime, meropenem, and piperacillin-tazobactam. P. aeruginosa ATCC 27,853 and Escherichia coli ATCC 25,922 were used as the quality control strains.

Whole genome sequencing and annotation

The draft genome sequencing of strain PDI170223 was performed using the HiSeq platform (Illumina), which generated paired-end libraries with a read length of 150 bp. Genomic DNA with an average size of 300 bp was fragmented, followed by end-repair, 3’ adenylation, adapter ligation, and PCR amplification. Subsequently, the library was purified using magnetic beads, and its quality was assessed using the Qubit 4.0 fluorometer. The length of the library was confirmed through 2% agarose gel electrophoresis. The qualified libraries were then sequenced on the Illumina NovaSeq 6000 platform at Sangon Biotech (Shanghai, China). To ensure data quality, the raw sequencing reads were evaluated using FastQC v0.11.2 and trimmed using Trimmomatic v0.36 [15] to obtain accurate and reliable reads. The filtered reads obtained after quality control were subjected to contig assembly using SPAdes v3.5.0 (http://bioinf.spbau.ru/spades) [16], and GapFiller v1.11 (https://sourceforge.net/projects/gapfiller/) [17] were utilized to fill the gaps between contigs, and this tool improved the continuity of the assembled genome. The assembled genomes were then annotated using Prokka [18]. To analyze potential antimicrobial resistance and virulence genes, the genome sequence was compared with the Virulence Factors of Pathogenic Bacteria Database (VFDB) (http://www.mgc.ac.cn/VFs/main.htm) [19] and the Comprehensive Antimicrobial Resistance Database (CARD) (https://card.mcmaster.ca/) [20]. The average nucleotide identity was determined by the OrthoANIu algorithm (https://www.ezbiocloud.net/tools/ani). The comparison parameter used was an e-value < 1e-5. The functional annotation of the sequence was completed by performing a Basic Local Alignment Search Tool (BLAST) search on various databases, including the Conserved Domain Database (CDD) (http://www.nibi.nlm.nih.gov/Structure/cdd/cdd.shtml), Clusters of Orthologous Groups of proteins (COG) (https://www.ncbi.nlm.nih.gov/COG/), Pathogen Host Interactions Database (PHI) (http://www.phi-base.org/), Kyoto Encyclopedia of Genes and Genomes (KEGG) (https//www.genome.jp/kegg/), Gene Ontology (GO) (http://geneontology.org/), NCBI non-redundant protein sequences (NR) (https://www.ncbi.nlm.nih.gov/protein).

Results

Identification of S. Nitrititolerans

Following an overnight culture of isolates from the peritoneal dialysis fluid sample, bacterial colonies were observed on Columbia blood agar. The colonies appeared circular and smooth, with an orange color and well-defined edges. Some colonies had yellow wrinkles (Fig. 1A). Gram-negative bacteria of different lengths were observed under a microscope (Fig. 1B). MALDI-TOF MS acquired a spectrum of protein molecular mass for this strain. The peaks in mass spectra represent the relative intensity of ions, and the molecular structure and composition of compounds can be determined by analyzing the peak position and size. By using the pattern-matching procedure, mass peaks in the experimental spectra were compared with reference spectra in the manufacturer’s database (Bruker Daltonik GmbH, Germany) (Fig. 1C). Only the genus Pseudomonas was identified, and the species identification score for this strain was 1.49. It is worth noting that when the score is less than 1.5, the results are considered unreliable.

Fig. 1.

The growth conditions of strain PDI170223. (A) Growth of S. nitrititolerans on blood agar media. (B) Gram-staining of S. nitrititolerans showed Gram-negative bacilli. (C) Mass spectrum identification of S. nitrititolerans

16 S rRNA-based phylogenetic tree

To accurately identify the species level of this bacterium, 16 S rRNA sequencing was performed. In total, 1426 contiguous nucleotides of PDI170223 were determined and analyzed by referring to the EZBiocloud database [12]. Strain PDI170223 exhibited the highest (99.58%) 16 S rRNA gene sequence similarity with the type strain of S. nitrititolerans GL14^T (GenBank accession no. MH917718). Neighbor-joining phylogenetic trees were constructed using MEGA software version 11 [13]. The topology of the phylogenetic trees was evaluated using the bootstrap resampling method with 1000 replicates. The phylogenetic trees displayed that strain PDI170223 was clustered together with the type strain GL14^T. This cluster was strongly supported with a bootstrap value of 100% (Fig. 2). The comparative 16 S rRNA gene sequence analysis revealed that the isolated strain PDI170223 belongs to S. nitrititolerans species. The 16 S rRNA sequencing results were submitted to GenBank (accession no. OR149488).

Fig. 2.

Phylogenetic relationships among the genus Stutzerimonas. The phylogenetic tree shows the relationship between the isolated strain PDI170223 (bold) and other members of the genus Stutzerimonas by 16 S rRNA gene sequences. The corresponding GenBank accession numbers are listed in parentheses

The antimicrobial susceptibility results

S. nitrititolerans was isolated from clinical specimens for the first time, and AST was conducted. Based on the CLSI criteria for Gram-negative, non-fermenting bacteria and non-Enterobacteriaceae (Standards, 2022). Currently, multidrug-resistant bacteria are defined as microorganisms that are resistant to three or more classes of antibiotics simultaneously [21]. The results revealed that S. nitrititolerans PDI170223 is a multidrug-resistant bacterium, demonstrating resistance to various antimicrobials, including third- and fourth-generation cephalosporins (ceftazidime and cefepime), carbapenems (meropenem), and fluoroquinolones (levofloxacin and ciprofloxacin). On the other hand, it exhibited sensitivity to aminoglycosides (amikacin, gentamicin, and tobramycin), minocycline, cotrimoxazole, and chloramphenicol (Table 1).

Table 1.

The antimicrobial susceptibility results of S. nitrititolerans strain PDI170223

Antibiotic categories	Antibiotics	MIC (mg/L)	SIR*
β-lactam antibiotics	Piperacillin Ceftazidime Ceftriaxone Cefepime Aztreonam Meropenem Tikatecillin/clavulanicacid Piperacillin/tazobactam	>=128 >=32 >=64 >=64 16 >=32 >=128/2 >=128/4	R R R R I R R R
Fluorquinolone antibiotics	Levofloxacin Ciprofloxacin	>=8 >=4	R R
Aminoglycoside antibiotics	Amikacin Gentamicin Tobramycin	<=4 <=4 <=4	S S S
Tetracycline antibiotics	Minocycline	<=4	S
Sulfonamides antibiotics	Cotrimoxazole	<=0.5/9.5	S
Chloramphenicol antibiotics	Chloramphenicol	<=8	S

* Antimicrobial susceptibility was judged according to CLSI 2022 standard. SIR, (S) sensitive, (I) intermediate, (R) resistant

Whole genome sequencing of S. Nitrititolerans PDI170223

According to the raw data table, the total base count of the sequenced genome was 1,029,435,600 bp. After genome assembly, the genome size of S. nitrititolerans was found to be 4,395,672 bp. Notably, the genome size in this study was considerably larger than those of S. nitrititolerans GL14^T and AGROB37. The G + C content of the genome was 63.18%, which is consistent with that of other Stutzerimonas genomes. The genome assembly resulted in 23 contigs, with an N50 value of 457,868 bp. The S. nitrititolerans genome contains 5 rRNA genes and 57 tRNA genes. A total of 4136 protein-coding genes were identified (Table 2). The genome circle maps were created with the Proksee online tool (https://proksee.ca/) (Fig. 3). Genome-based taxonomy classification using the OrthoANIu algorithm validated that strain PDI170223 belongs to S. nitrititolerans, with a high average nucleotide identity (ANI) value of 97.84% compared to the S. nitrititolerans type strain GL14^T. A database based on NCBI Blast + and the Kyoto Encyclopedia of Genes and Genomes (KEGG) Automatic Annotation Server was used for gene function annotation. The annotation included various databases such as CDD, COG, NT, NR, PFAM, Swissprot, and TrEMBL. Among these databases, the NR database contains the most annotated genes, followed by the TrEMBL database (Supplementary Figure S1). According to the KEGG database, 125 predicted genes were associated with human diseases, mainly infectious diseases, drug resistance, neurodegenerative diseases, and cancers (Supplementary Figure S2). Most of the predicted genes, such as rpoS, cyaA, fliC, and fliA, were associated with human infectious diseases, with most of them enhancing the ability of bacteria to cause infection [22, 23], suggesting that S. nitrititolerans has the potential to be an emerging human pathogen. Furthermore, based on the PHI database, a total of 147 PHI-related proteins were predicted for strain PDI170223. The major annotated genes were dominated by reduced virulence and unaffected pathogenicity, indicating the low pathogenicity of this bacterium (Supplementary Figure S3).

Table 2.

Genome features of strain PDI170223 and two other S. nitrititolerans strains

Genome features	S. nitrititolerans strains
	PDI170223	GL14T	AGROB37
Genome size (bp)	4,395,672	4,357,823	4,192,148
Protein coding genes	4136	4035	3837
N50 (bp)	457,868	237,605	226,187
Contigs	23	105	43
rRNA	5	7	9
tRNA	57	59	55
G + C (%)	63.18	63.1	63.2
Isolation source	Clinical specimens	Bioreactor	Sheep dairy farm

Fig. 3.

The map shows the assembled genome sequence of strain PDI170223. From outside to inner side rings: the coding sequences (CDS) in green, RNA genes (tRNAs in red and rRNAs in yellow), GC content in black, and GC-skew graph in blue and purple

Prediction of antimicrobial resistance and virulence factors

The CARD database was used to predict the antimicrobial resistance of PDI170223. In total, 69 proteins associated with antibiotic hydrolase, antibiotic efflux, and antibiotic target modification were predicted (Fig. 4A). The main resistance proteins are various enzymes, including β-lactamase IMP-1 precursor and β-lactamase NDM-1 precursor, which are important contributors to carbapenem resistance in gram-negative bacteria [24, 25]. Those predicted proteins suggest that strain PDI170223 is a carbapenem-resistant bacterium. Furthermore, based on the core dataset of the virulence factor database, 94 virulence factor terms were predicted, involving 323 predicted proteins (Fig. 4B). These predicted virulence factors encompass categories such as exotoxin, adherence and invasion, motility, and effector delivery system [26]. The database predicted the most effector delivery system-related proteins that include Type VI and III Secretion Systems (T6SS and T3SS). The T6SS and T3SS are important virulence factors that contribute to the pathogenicity of P. aeruginosa. These secretion systems play a central role in the infection process by allowing the bacterium to deliver toxic proteins directly into host cells [27, 28]. In this study, we analyzed 27 predicted virulence genes exhibiting a high identity value to well-known genes (Supplementary Table S1) [29]. These results suggest that S. nitrititolerans could become an emerging human pathogen.

Fig. 4.

The pathogenicity and antibiotic resistance of strain PDI170223 have been predicted using different databases. (A) The antibiotic predicted proteins using CARD. (B) The virulence factors proteins were predicted based on the core dataset of the VFDB

Discussion

The genus Stutzerimonas includes non-fermentative gram-negative bacteria that are widely distributed in natural and hospital environment [2]. An increasing number of human infections have recently been reported to be caused by members within this genus, with S. stutzeri being the most commonly found and clinically related bacterium. S. stutzeri causes necrotizing pneumonia [9], endophthalmitis [30], vertebral osteomyelitis [31], and endocarditis [32]. These findings suggest that S. nitrititolerans can potentially cause clinical human infections. According to the AST results of S. stutzeri in multiple reports, this bacterium is sensitive to ceftazidime, cefepime, and meropenem [9, 30–33], while S. nitrititolerans isolated from a bioreactor was resistant to several antimicrobial agents, including cefazolin, chloramphenicol, clindamycin, streptomycin, sulfamethoxazole, tetracycline, and vancomycin [3]. Based on our AST results, we noted that S. nitrititolerans PDI170223 is multidrug- and carbapenem-resistant. Carbapenem antimicrobials are considered the last resort when treating gram-negative bacilli-induced severe infections [34]. Thus, this rare carbapenem-resistant gram-negative bacilli infection should not be underestimated. Since carbapenem-resistant S. nitrititolerans was isolated from clinical specimens, WGS was performed to explore its resistance mechanism.

The genome sequencing of strain PDI170223 revealed the presence of multiple antimicrobial resistance genes, such as β-lactam resistance genes, efflux pump genes, and target site modification genes. The AST results revealed that this strain was resistant to most β⁃lactam antibiotics. The most crucial mechanism of these antibiotics is the production of various β-lactamases. Upon analyzing S. nitrititolerans resistance genes by referring to the CARD database, 69 resistance genes, including blaKHM-1, blaIMP-1, blaGIM-1, blaSIM-1, blaVIM-2, and blaNDM were predicted. According to the Amber classification, four types of β-lactamases exist (A, B, C, and D), and among them, types A, B, and D are carbapenemases. Type B is a class of metallo-β-lactamases (MBLs) comprising enzymes, such as VIM, IMP, and NDM, that can hydrolyze most β-lactam antibiotics, including carbapenems. MBLs can degrade tazobactam and clavulanic acid but cannot efficiently hydrolyze aztreonam [35, 36]. Along with the previous prediction of resistance genes, these results may explain the resistance to most β⁃lactam antibiotics, and S. nitrititolerans exhibited intermediate susceptibility to only aztreonam in our AST results.

Overexpression of the efflux pump systems (MexAB-OprM and MexEF-OprN) has been reported to lead to fluoroquinolone resistance in P. aeruginosa [37, 38]. These systems belong to the RND family, with OprM and OprN as outer membrane components, MexB and MexF as inner membrane components, and MexA and MexE as membrane fusion proteins that connect membrane proteins [39]. In our predicted genes, the complete efflux pump systems MexAB-OprM and MexEF-OprN were identified in S. nitrititolerans PDI170223, potentially playing a role in fluoroquinolone resistance. Studies on resistance genes are essential for developing efflux pump inhibitors that can significantly impact the treatment of clinical infections. catI, catII, catB3, and floR are chloramphenicol resistance genes, while dfrA15b, dfrA14, dfrA12, and dfrA1 are trimethoprim resistance genes [40]. These genes were not detected among our predicted genes, which is consistent with our chloramphenicol and cotrimoxazole sensitivity results. Strain PDI170223 was also susceptible to aminoglycoside antibiotics. Several aminoglycoside resistance genes, including armA, rmtA, aac(3)-II, aac(3)-IV, aac(6’)-Ib, aph(3’)-I, aph(3’)-II, aph(3’)-III, and rmtF have been reported [41, 42]. Thus, the sensitivity to aminoglycoside antibiotics is explained by the absence of the aforementioned genes in our predictive CARD database. By analyzing the genome, researchers can identify potential drug targets and develop more effective treatment strategies. Naturally, our analysis will solely focus on examining potential factors contributing to drug resistance through WGS, necessitating further confirmation through qPCR technology.

In addition to antimicrobial resistance, there is also a significant focus on studying virulence factors. S. nitrititolerans possess flagella, which is consistent with our genetic predictions. Among our predicted genes, the flagellin genes included flhA, fleN, flgC, flgI, fliE, fliG, fliM, flin, fliI, and fliQ. Flagella in some bacteria are closely related to their pathogenicity. In Aeromonas spp., flagella act as adhesins that promote the attachment of cell surfaces and biofilm formation [43]. In Helicobacter pylori, flagella can enable different motility types and are involved in bacterial colonization, inflammatory reactions, and immune evasion [44]. Six genes (pilI, pilJ, pilH, pilG, pilU, and pilT) encode twitching motility proteins, and mobility plays a significant role in the virulence of many bacterial pathogens [45]. Four genes (pilB, pilO, pilN, and pilM) encoded type IV pilus inner membrane platform proteins. Evidence indicates that these ten genes encode proteins essential for type IV pili formation. In P. aeruginosa, type IV pili are used for surface-specific motility and act as sensors for surface-induced gene expression and pathogenicity. In Neisseria meningitidis, type IV pili are involved in vascular colonization and disease progression [46]. Furthermore, 56 genes were associated with type IV pili, which indicates that S. nitrititolerans may harbor type IV pili.

Alginate is considered a virulence protein involved in biofilm formation. Three genes (algU, algA, and algR) are required for alginate biosynthesis. Biofilms allow bacteria to resist hostile environmental conditions and cause chronic diseases [47, 48]. Additionally, within the biofilm, bacteria are well protected from host immune responses and antibiotics, which makes their clinical treatment very difficult. In Staphylococcus aureus, biofilm formation is associated with chronic wound infections, such as diabetic foot ulcers and pressure sores [49]. Biofilms formed by Group A streptococcus in necrotizing soft tissue infections exacerbate inflammation and cause severe tissue damage [50]. These genes waaG and waaF are involved in lipopolysaccharide (LPS) production, a significant component of the outer membrane of gram-negative bacteria. LPS induces inflammation and oxidative stress. Bacterial LPS influences biofilm formation and adhesion [51]. This provides a basis for biofilm formation.

In recent years, MALDI-TOF MS has been extensively used for rapid bacterial identification in clinics [52]. However, this technique has yet to accurately identify some emerging pathogens. Clinical laboratories should enhance their testing techniques and perform WGS or other methods to improve the identification of S. nitrititolerans and other rare pathogens. In our report, the patient had chronic renal failure and was immunocompromised, which was the cause of his infection with S. nitrititolerans. The main indicators of elevated infection in patients include an increase in neutrophil counts and C-reactive protein levels, and significant elevation in nucleated cell counts in ascites [53]. However, in our study, the patient’s body temperature was normal, probably because of the low pathogenicity of the bacterium (Figure S3). When the patient was diagnosed with chronic renal failure, he underwent peritoneal dialysis treatment with levofloxacin for infections. According to the AST results, the microbe was resistant to levofloxacin. Therefore, we could not determine whether S. nitrititolerans was the causative agent in this patient. However, as carbapenem-resistant gram-negative bacilli were first isolated from clinical specimens, it is necessary to be aware of potential pathogens that have the particular propensity to develop antimicrobial resistance and become prevalent because of this particular capability.

In conclusion, we reported the first clinical isolation of multidrug-resistant S. nitrititolerans strain PDI170223. Subsequent WGS of the strain revealed the presence of annotated antimicrobial resistance genes and virulence genes. Strain PDI170223 exhibited resistance to third- and fourth-generation cephalosporins, carbapenems, and fluoroquinolones, consistent with the prediction of the presence of antimicrobial resistance genes. Moreover, we demonstrated the infection potential of PDI170223 by revealing the presence of numerous genes encoding for virulence factors in this bacterium. Therefore, our study provides information for developing treatment strategies for S. nitrititolerans-associated infectious diseases and raises awareness about this rare pathogen.

Electronic supplementary material

Below is the link to the electronic supplementary material.

Author contributions

All authors contributed to the article. LFS, YMZ, and ZXL conceived and designed the research. LFS, YMZ, and YZ performed the experiments. XLW, JX, and HW analyzed the data. LFS and YMZ wrote the original draft of the manuscript. MZ and ZXL reviewed and edited the manuscript. All authors read and approved the final manuscript.

Funding

This work was supported by a grant from the Wuhan Municipal Health Commission (Project no. WX21Q42) and the National Natural Science Foundation of China (82200922).

Data availability

The whole-genome sequence has been deposited at NCBI under the GenBank accessions JAUCDX000000000, Bioproject accession PRJNA985898, and Biosample accession SAMN35815676. Further inquiries can be directed to the corresponding author.

Declarations

Ethics approval and consent to participate

Ethical review and approval were performed by the Medical Ethics Committee of The Central Hospital of Wuhan, Tongji Medical College, Huazhong University of Science and Technology (Ethical no. WHZXKYL2023-094). Written informed consent was obtained from the patient for the publication of this case report (including all data and images).

Consent for publication

Not applicable.

Competing interests

The authors declare no competing interests.