A novel pathogenic species of genus Stenotrophomonas: Stenotrophomonas pigmentata sp. nov

Introduction Stenotrophomonas is a prominent genus owing to its dual nature. Species of this genus have many applications in industry and agriculture as plant growth-promoting rhizobacteria and microbial biological control agents, whereas species such as Stenotrophomonas maltophilia are considered one of the leading gram-negative multi-drug-resistant bacterial pathogens because of their high contribution to the increase in crude mortality and significant clinical challenge. Pathogenic Stenotrophomonas species and most clinical isolates belong to the Stenotrophomonas maltophilia complex (SMc). However, a strain highly homologous to S. terrae was isolated from a patient with pulmonary tuberculosis (TB), which aroused our interest, as S. terrae belongs to a relatively distant clade from SMc and there have been no human association reports. Methods The pathogenicity, immunological and biochemical characteristics of 610A2T were systematically evaluated. Results 610A2T is a new species of genus Stenotrophomonas, which is named as Stenotrophomonas pigmentata sp. nov. for its obvious brown water-soluble pigment. 610A2T is pathogenic and caused significant weight loss, pulmonary congestion, and blood transmission in mice because it has multiple virulence factors, haemolysis, and strong biofilm formation abilities. In addition, the cytokine response induced by this strain was similar to that observed in patients with TB, and the strain was resistant to half of the anti-TB drugs. Conclusions The pathogenicity of 610A2T may not be weaker than that of S. maltophilia. Its isolation extended the opportunistic pathogenic species to all 3 major clades of the genus Stenotrophomonas, indicating that the clinical importance of species of Stenotrophomonas other than S. maltophilia and potential risks to biological safety associated with the use of Stenotrophomonas require more attention.


S t e n o t r o p h o m o n a s i s a m e m b e r o f t h e f a m i l y
Xanthomonadaceae.Because of advancements in molecular biology and identification technology, the number of species in this genus has increased rapidly in recent years.Based on our interpretation of the current available NCBI taxonomy, from the first species, Stenotrophomonas maltophilia, isolated in 1885 to 2011, 16 strains have been isolated within 117 years, and 12 species were identified within 9 years from 2016 to 2024.Stenotrophomonas is ubiquitous and associated with a wide range of habitats, including humans, animals, plant hosts, and extreme environments.Most members of Stenotrophomonas are particularly known for producing protective osmotic substances and are considered as plant rhizosphere growth-promoting bacteria.They also have valuable applications in the fields of agriculture and industry as emerging sources of biodegradation and substitutes for synthetic fungicides (Kumar et al., 2023).Stenotrophomonas strains can interact with other microorganisms on plant surfaces and in the soil through biofilms, inhibit plant pathogenic fungi and viruses, and alter the composition of rhizosphere microorganisms through extracellular enzyme decomposition and competition for iron.By inducing the enrichment of plant hormones, such as jasmonic acid, and upregulating the transcription level of jasmonic acid-sensitive genes, Stenotrophomonas strains help plants defend against various crop pests, such as Spodoptera litura.Their function in promoting plant growth is through the production of auxin and hydrogen cyanide; dissolution of phosphorus and potassium salts; nitrogen fixation; enzymatic degradation of soil odour, explosive pollutants, keratin, macrocyclic hydrocarbons, nitrophenols, and other substances; removal of various chemical pesticides, insecticides, and environmental pollutants; and effective bioremediation of agricultural soil without harming the ecosystem (Kumar et al., 2023).
A few species of this genus are pathogenic to humans and are represented by S. maltophilia.As a globally emerging organism, S. maltophilia is recognised for its multi-drug resistance (Gil-Gil et al., 2020), ability to cause various infections in the human body, and high contribution to the increase in crude mortality (Tan et al., 2014).Based on global clinical data, the attributed mortality rates for pulmonary infection and bacteraemia caused by this bacterium are as high as 30% and 65%, respectively (Zhou et al., 2013).S. maltophilia is listed by the World Health Organization as one of the leading gram-negative, multi-drug-resistant bacterial pathogens in hospitals (Gröschel et al., 2020) and poses a great clinical challenge (Mojica et al., 2022;Tamma et al., 2022).
In our previous small surveillance conducted in a district tuberculosis (TB) hospital in Beijing, we found that Stenotrophomonas was a major co-occurring species of Mycobacterium tuberculosis, with a crude isolation rate of 6.74%.Stenotrophomonas may be an important opportunistic pathogen for patients with TB, similar to that for patients with cystic fibrosis (Li et al., 2023).On the basis of 16S rDNA sequencing, 2 out of 9 Stenotrophomonas isolates did not belong to S. maltophilia, and one had the highest identity with S. terrae strain AFS037341 (99.24%) and S. humi strain AFS068096 (98.96%).These similarity values were more than 98.65%, which has been previously used as the threshold for differentiating Stenotrophomonas species (Deng et al., 2022).To the best of our knowledge, S. terrae and its closely related species have not been isolated from human resources, which sparked our interest in the pathogenicity of these strains.However, after PacBio whole-genome sequencing, the average nucleotide identity (ANI) between the strain 610A2 T , which was highest similar to S. terrae, and all existing species of the genus Stenotrophomonas was less than 88%.Therefore, in addition to animal experiments and pathogenicity assessments, we conducted a systematic evaluation of 610A2 T according to the description required for new taxa.

Sources of isolates
Strain 610A2 T was isolated from a 26-year-old male patient with pulmonary TB under treatment at the tuberculosis clinic of the Chaoyang District Center for Disease Control and Prevention (39.89 N,116.40 E) in our previous surveillance under ethics approval No. ICDC-2022010 (Li et al., 2023).It was first identified as Stenotrophomonas by amplification and sequencing with 16S rDNA universal primers for bacteria (16S-U: 5′AGA GTT TGA TCM TGG CTC AG 3′ and/L: 5′ CCG TCA ATT CMT TTR AGT TT 3′).

Whole-genome sequencing and phylogenetic analysis
Genome of 610A2 T was sequenced using the PacBio sequel II and DNBSEQ platform at the Beijing Genomics Institute (BGI, Shenzhen, China).Four SMRT cell zero-mode waveguide arrays for sequencing were used to generate the sub-read set.The PacBio subreads (length < 1 kb) were removed.Canu and GATK (https:// www.broadinstitute.org/gatk/)were used for self-correction and single-base corrections, to improve the accuracy of the genome sequences.
Gene prediction was performed using glimmer3 with Hidden Markov models (http://www.cbcb.umd.edu/software/glimmer/).Other genomic elements, such as RNA, tandem repeats, genomic regions, small satellite DNA, and microsatellite DNA, were identified using tRNAscan Complete 16S rDNA gene sequences were extracted from the sequenced genome and reference genomes of 26 species of the genus Stenotrophomonas, except for S. detusculanense and S. indologenes, which do not have publicly available genome sequences.ClustalX was used for multiple sequence alignments, and a bootstrap neighbour-joining (NJ) phylogenetic tree was constructed using Treeview (1.6.6) with 1000 replicas.ANI was estimated using the ANI calculator (http://enve-omics.ce.gatech.edu/ani/).Syntenies were performed using MUMmer and BLAST.Core/Pan genes were clustered by CD-HIT rapid clustering of similar proteins software with a threshold of 50% pairwise identity and 0.7 length difference cutoff in amino acid.Hcluster_sg software was used to perform gene family clustering, and a phylogenetic tree was constructed using TreeBeST with the NJ method.

Antibiotic susceptibility testing
Two kinds of AST plates, the customised AST plate for Chinese Pathogen Identification Net (CHNENF, Trek Diagnostic Systems Ltd, West Sussex, United Kingdom) and the Sensititre ™ MYCOTB minimum inhibitory concentration (MIC) plate (Trek Diagnostic Systems, Cleveland, OH, USA), were used to determine the strain's sensitivity to 17 drugs commonly used for gram-negative strains and 12 anti-TB drugs.MICs were determined according to the standards of the Clinical and Laboratory Standard Institute (CLSI, 2018) and manufacturer's instructions.

Biofilm formation
Biofilm formation was determined using crystal violet staining.Fresh bacterial suspension was prepared and adjusted to 0.5 McFarland.After 1:30-fold dilution with tryptone soy broth (TSB; Difco), 150 mL/well suspension was added to a 96-well flat-bottom microtitre plate, and the plate was incubated at 37 °C.At each time point, bacterial turbidity (OD 630 nm ) was measured using an enzyme-linked immunosorbent assay (ELISA) reader (Bio-Rad 680; Bio-Rad Laboratories, Japan).The cultures were discarded, and the wells were washed 3 times with PBS (pH 7.3) to remove planktonic cells.The biofilms were stained with 200 mL/well of 0.1% crystal violet for 15 min.The wells were washed again, and 200 mL of 99% ethanol was added and OD 403 nm was measured after 15 min.The biofilm index was calculated as OD 403 nm /OD 630 nm .Six clinically isolated S. maltophilia strains, including strain 11066 (GDMCC 1.4335), were used as controls.

Animal infection experiment
Animal experiments were conducted with the approval of the Laboratory Animal Welfare & Ethics Committee of the National Institute for Communicable Disease Control and Prevention (Issue number 2023-021).
Using a random number method, female Kunming mice aged 6-7 weeks and weighing 30-35 g were randomly divided into infection and control groups.Five mice in each infection group were intranasally inoculated with 50 mL of 1.5 × 10 8 CFU/mL of tested strains, and mice in the control groups were inoculated with PBS.At 4 h, 12 h, 1 day, 2 days, 3 days, 5 days, and 7 days postinfection, the mice were sacrificed and the lung and spleen tissues were collected aseptically.
The left lobes of the lungs were fixed in 10% neutral formalin for 24 h, followed by tissue processing and paraffin embedding.The paraffin blocks were sectioned at 2 µm, stained with haematoxylin and eosin, and blindly examined under a microscope by an expert in the field of laboratory animal pathology.Pulmonary clearance and occurrence of disseminated infection were monitored via quantitative bacteriology of the lung and spleen homogenates, respectively.Briefly, tissues were homogenised (1000 rpm) on ice in 1 mL of sterile normal saline by using a homogeniser (RZ-GR96A; Beijing Guoke Rongzhi Biotechnology Co., Ltd., China).Then, 100 mL of homogenates and 10-fold serial dilutions were inoculated on LB agar.The number of colonies was counted 24-48 h after incubation at 37°C.Bacterial colony counts were normalised according to the wet tissue weight and calculated as CFU/g.The levels of murine tumour necrosis factor alpha (TNF-a), granulocyte-macrophage colony-stimulating factor (GM-CSF), gamma interferon (IFN-g), interleukin (IL)-2, IL-4, IL-6, IL-10, IL-12p70, and IL-17A were quantified using Luminex technology (R&D Systems, Minneapolis, MN, USA) and reagents (12002798; BIO-RAD, USA).

Statistical analysis
For comparison of multiple groups, one-way ANOVA was performed using GraphPad Prism version 9.3.1 for Windows (GraphPad Software, San Diego, California, USA).

FIGURE 1
Phylogenetic tree created on the basis of complete 16S rDNA gene sequences of 610A2 T and reference genomes of 26 species Stenotrophomonas.ClustalX was used for multiple sequence alignments, and a bootstrap neighbour-joining (NJ) phylogenetic tree was constructed using Treeview (1.6.6) with 1000 replicas.Species isolated from patients or animals or tested on a murine model are marked with symbols following their species name and 16S rDNA locus_tag.

Pathogenicity in animal infection experiment
At the infection dose of 50 mL of 1.5 × 10 8 CFU/mL, no mortality was observed in either the infection or control group.The mice infected with 610A2 T showed a weight loss of 3.9 ± 0.9%, 8.0 ± 0.7%, 6.4 ± 1.3%, 2.3 ± 1.2%, 0.8 ± 1.3% at 12 h, 1 day, 2 days, 3 days, and 5 days post-infection, respectively.On the seventh day, weight gain resumed.In addition, no significant differences were observed in the physical signs between the infection and control groups.
610A2 T was isolated only from the spleen homogenate of all infected mice within 4 h, with a viable bacterial count of 1.1 ± 2.2 × 10 3 CFU/mL g, indicating that 610A2 T has strong invasive ability but the host can also quickly clear it.
Histological examination showed that pulmonary damage in the infected group constantly included alveolar septa thickening, congestion, bleeding, inflammatory cell infiltration, compensatory alveolar dilation, mucosal epithelial oedema, and vacuolar degeneration (Figure 3).Although the viable count showed that the bacteria in the lungs had been cleared after 2 days, obvious bloody exudate and a large number of inflammatory cells were detected in the alveoli in 1-3 days, which is consistent with the symptoms for pulmonary congestion.After 7 days, the lung structure of the infected group was restored to normal, whereas that of the control group was normal.
Eight of the 9 quantified cytokines showed significant differences between infected and control groups (Figure 4).The means of TNF-a, GM-CSF, IL-4, IL-6, IL-10, and IL-17A at each timepoint were higher than those of the control group, but only the values at 4 h had statistical significance because of the variability caused by individual differences.IFN-g and IL-2 showed a consistent trend of change, lagging behind other cytokines and showing significant differences from the control group on the 5th Gene heat map (A) and phylogenetic trees based on corePan and genefamily1 results (B).day; however, at this time point, TNF-a and IL-6 had recovered to levels no different from the control group.

Prediction of virulence factors
Functional annotation revealed that the 610A2 T genome contained 304 virulence factors of pathogenic bacteria in VFDB and 100 human disease pathway genes in KEGG database, of which 16 genes were related to bacterial infectious diseases.In details, 53 genes were related to flagellar biosynthesis, flagellar basal body protein, flagellar motor switching, and chemotaxis; 32 type IV pili genes; 3 outer membrane usher protein genes; 24 iron/haeme uptake and utilisation relative genes; and 1 biofilm-controlling response regulator.610A2 T harbours 2 haemolysin-encoding genes, hlyB and hlyIII, as well as the haemolysin activation/ secretion protein gene fhaC, a transcriptional regulator of haemolysin slyA, and 3 HlyD family secretion proteins, which are consistent with the haemolytic activities on Columbia blood agar containing sheep blood.

Systematic evaluation of physiological and chemotaxonomic properties
610A2 T grew in all 4 tested media.The colonies on nutrient agar were translucent, smooth, and moist, with brown water-soluble pigment (Figure 5A); on blood agar, they were greyish white with a stimulating ammonia odour and a haemolytic ring (Figure 5B).The strain grew under conditions of 15-37 °C, pH 6-8, and NaCl concentration of 0-2%, but it did not grow below 4 °C and above 40 °C and did not tolerate 5% salt concentration like S. maltophilia and S. terrae.During semi-solid puncture culture, bacteria grew diffusely along the puncture line; however, no pigment was produced, indicating motility; pigment production was aerobic.610A2 T was rod-shaped gram-negative, and without spores or capsules.Under electron microscopy, the bacterial body was about 1.5-2 × 0.5 mM with a single extreme flagella (Figure 5C).
610A2 T grew slower than S. maltophilia strains in TSB.After 48 h, the cultures of 610A2 T showed turbidity, but the entire growth curve was flat without a logarithmic phase (Figure 6A).Although the growth rate was slow, the biofilm index showed that 610A2 T had a strong biofilm-forming ability, which was significantly higher than that of all S. maltophilia strains after 3 h of cultivation (Figure 6B).
naphthol-AS-BI phosphate hydrolase.In API 50CH, only starch fermentation was detected.The major differentiating feature of 610A2 T was that it could not assimilate glucose and mannose, but it could ferment starch.
In the MALDI-TOF spectrum, the 4 main peaks of 610A2 T were located at 5254.065 Da, 4857.675 Da, 2770.713 Da, and 6118.187Da, of which the 4857 Da peak was genus-specific and the 2770.713Da peak was 610A2 T -specific.In the local Bruker_MSP library, only 5 Stenotrophomonas species were found: S. acidaminiphila, S. maltophilia, S. pictorum, S. rhizophila, and S. nitritireducens.The identification scores of 610A2 T for these species ranged from 1.625 to 1.370, with the highest similarity to S. pictorum.These data indicate that 610A2 T is a distinct species closely associated with these species of Stenotrophomonas.
indicating that the similarity of 610A2 T to S. humi is higher than that to S. terrae.

Discussion
When evaluating the pathogenicity of strain 610A2 T , we used the outbred mouse KM, which has strong disease resistance and adaptability and can better reflect the genetic diversity of the human population.Within the first day post-infection, the bacterial load of 610A2 T in the lungs decreased sharply, and 610A2 T was cleared completely within 48 h.The lung clearance rate of 610A2 T in KM mice was faster than that of S. maltophilia in many inbred mouse strains, which also showed a larger decrease in CFU at 24 h, but could generally still be detected on day 3-7 post-infection and could even replicate in A/J mice with a transient increase in CFU at 4 and 8 h post-inoculation (Di Bonaventura et al., 2010;Rouf et al., 2011;Pompilio et al., 2018).After infection, both S. maltophilia (Di Bonaventura et al., 2010;Pompilio et al., 2018) and 610A2 T could be isolated in the spleen, suggesting that, at the same time of lung infection, Stenotrophomonas could disrupt the integrity of the lung epithelial barrier, cause bacteraemia, spread to other tissues, and pose an uncertain risk of assisting other pathogens in spreading and forming extrapulmonary lesions.
Combining with the acute infectious cytokine responses, the animal experiment in this study can confirm the pathogenicity of 610A2 T on the basis of the significant weight loss in the mice, tissue reactions due to inflammatory infection, and pulmonary congestion detected in the histopathological sections, although 610A2 T can be completely cleared from a healthy host.610A2 T belonged to the S. terrae clade in the phylogenetic tree.In phylogenetic trees, whether constructed based on the concatenation of translated protein sequences (Patil et al., 2018) or the 16S rDNA gene, it is a clade that is relatively far from SMc.All species in this clade have been isolated from the environment and never been isolated from humans (Heylen et al., 2007).The isolation of 610A2 T changed the distribution of the pathogenic species.Together with S. acidaminiphila and S. rhizophila, all 3 main clades of the genus Stenotrophomonas have isolates from human sources.
610A2 T inherently possesses many pathogenic mechanisms, which can be inferred from the composition of functional genes on the genome, including those for motility (flagella and type IV pili), surface adherence (flagella, fimbriae, and LPS), damage capacity to host (protein secretion systems and extracellular enzymes), iron uptake and utilization, biofilm formation, protection against host defence (superoxide dismutase, hydroperoxidase, catalase, and melanin) as well as multi-antibiotic resistance (Di Bonaventura et al., 2010;Brooke, 2021).In addition, the genomic analysis suggested that 610A2 T produces type III haemolysin, for which erythrocyte lysis capacity and virulence have been confirmed in a mouse model infected with Vibrio vulnificus (Baida and Kuzmin, 1996;Chen et al., 2004;Moonah et al., 2014).In the genus Stenotrophomonas, colony pigmentation and brown diffusible pigments have been reported in only S. maltophilia (Romanenko et al., 2008;Liaw et al., 2010), S. nitritireducens (Finkmann et al., 2000), and S. rhizophila (Romanenko et al., 2008) isolates.Pigments and biofilm formation interact, and both are components of bacterial pathogenicity, enabling the colonisation or infection of hosts and promoting the attachment of other pathogens (Tamma et al., 2022).610A2 T showed stronger abilities than S. maltophilia in both aspects, indicating that its pathogenicity may not be weaker than that of S. maltophilia.
However, the strain-specific cytokine responses induced by 610A2 T may be difference from those induced by S. maltophilia.In this study, 9 cytokines were selected on the basis of their inflammatory responses in S. maltophilia-infected murine model and patients with TB (Di Bonaventura et al., 2010;Mvubu et al., 2018).Among these cytokines, early inflammatory markers TNF-a and IL-6 showed consistently high expression after 610A2 T and S. maltophilia infection, with IFN-g, IL-4, and IL-10 exhibiting different or even opposite changes (McDaniel et al., 2020) (Table 2).In mice infected with S. maltophilia, characteristic changes in cytokines were the persistent hyperexpression of IFN-g for more than 3 days and sustained significantly lower level of IL-4 when compared with the control group (Di Bonaventura et al., 2010).IFN-g was released in the early stage, along with large amounts of TNF-a and IL-2 and was closely associated with the ratio of T cells.Blocking the PD-1/PD-L1 pathway inhibited the apoptosis-inducing effect of S. maltophilia on T cells.The pleotropic cytokine IL-4 is a product of Th2 lymphocytes and inhibits Th1 cell differentiation.Therefore, the immune response to acute infection by S. maltophilia is predominantly a Th1-type response (Rouf et al., 2011).S. maltophilia may participate in the activation of T cells and induce subsequent T-cell exhaustion by activating the PD-1/PD-L1 signalling pathway, as the concentration of cytokines is reduced in the later stages (Wang et al., 2021;Xu et al., 2023).In contrast, in the 610A2 T -infected KM mice, cytokines IL-4, IL-6, and IL-10 secreted by Th2 cells showed a rapid and significant increase; there was no difference in IL-12, the main cytokine that induces Th1 cell differentiation and inhibits Th2 cells, indicating that the immune response of 610A2 T was Th2 type (Figure 4).In addition, the lagging release of IFN-g and IL-2 in 610A2 T -infection indicated that Th1 type was disinhibition in the later stage and also involved in the host's defence against the pathogen.In-depth research of the pathogenic Stenotrophomonas species including 610A2 T in same mouse strain infection model will further elucidate pathogenic and immunological characteristics.Moreover, 610A2 T is resistant to half of the commonly used anti-TB drugs, including isoniazid, and carries ESBL and metallo-blactamase, which can hydrolyse all bicyclic b-lactam antibiotics (Hinchliffe et al., 2023).The co-existence of Stenotrophomonas may affect therapeutic efficacy in patients with TB.The AST results showed that all drugs recommended by the Infectious Diseases Society of America for the treatment of S. maltophilia infection (Tamma et al., 2022) may be applicable to 610A2 T , except TMP-SMX, which is recommended as the first-line agent for the treatment of S. maltophilia infection owing to the low isolation rate of resistant strains (Chang et al., 2015;Gröschel et al., 2020).A combination of colistin and rifampicin may be used for patients with mixed 610A2 T and TB infections.The type strain 610A2 T was isolated from patients with pulmonary TB.It is a Gram-negative bacillus with a single extreme flagella and can produce brown water-soluble pigment.Growth is observed at 15-37 °C, pH 6-8, and 0-2% NaCl concentration, but it does not grow below 4 °C, above 40 °C, and at 5% salt concentration.610A2 T yielded positive results for nitrate reduction, quercetin and gelatin hydrolysis, and starch fermentation.The 4 main peaks in the MALDI-TOF spectrum were located at 5254.065 Da, 4857.675 Da, 2770.713, Da and 6118.187Da.The predominant fatty acids are iso-C 15:0 , iso-C 14:0 , summed Feature 3 (C 16:1 w7c/ 16:1 w6c), iso-C 16:0 , and C 15:0 anteiso and sum in Feature 9 (iso-C 17:1 w9c).610A2 T has multidrug resistance and intrinsic resistance to b-lactams, carbapenems, and trimethoprim-sulfamethoxazole, harbouring several multi-drug resistance efflux pump and antibiotic resistant genes.This strain is pathogenic to mice.
The complete genome of type strain 610A2 T comprises 4681496 bp and GC content of 63.29%, and the complete genome and 16S rDNA gene sequence are deposited in GenBank under accession numbers CP130832.1 and OR936313, respectively.The type strain is 610A2 T (=GDMCC 1.4134 T =JCM 36488 T ).
. terrae had the highest homology, with a gene median identity of 91.34% and map length rate of 63.327% (Table1).These data suggest that 610A2 ST represents a novel species within the genus Stenotrophomonas, and it is closely related to, but distinct from, S. terrae.The name Stenotrophomonas pigmentata sp.nov. is proposed for the distinct brown water-soluble pigment it produces.

TABLE 1
ANI values and synteny analyses at the nucleotide and amino acid levels between 610A2 and 8 type strains.

TABLE 2
The inflammatory responses of S. maltophilia and 610A2 T infected mice and TB patient.