Stenotrophomonas Nematodicola Sp. Nov., a Novel Intestinal Lifespan-Prolonging Bacterium for Caenorhabditis Elegans That Assists in Host Resistance to Bacillus Nematocida Colonization

Stenotrophomonas Nematodicola Sp. Nov., a Novel Intestinal Lifespan-Prolonging Bacterium for Caenorhabditis Elegans That Assists in Host Resistance to Bacillus Nematocida Colonization | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Research Article Stenotrophomonas Nematodicola Sp. Nov., a Novel Intestinal Lifespan-Prolonging Bacterium for Caenorhabditis Elegans That Assists in Host Resistance to Bacillus Nematocida Colonization Rui Han, Yu Wang, Yang Deng, Yuqin Zhang, Lin Zhang, Qiuhong Niu This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-124061/v2 This work is licensed under a CC BY 4.0 License Status: Published Journal Publication published 14 Jul, 2021 Read the published version in Archives of Microbiology → Version 2 posted 4 You are reading this latest preprint version Show more versions Abstract The soil-dwelling opportunistic bacterium Bacillus nematocida B16 shows comparatively strong killing activities against a variety of pathogenic nematodes. A bacterial strain CPCC 101271 T , isolated from intestines of C. elegans in natural habitats, was found not only to be probiotics for C. elegans but also assist in resisting pathogen B16 infection. The lifespan of Caenorhabditis elegans fed on strain CPCC 101271 T was extended by approximately 40% compared with that of worms fed on Escherichia coli OP50. In addition, the colonization of C. elegans by the pathogenic bacterium B. nematocida B16 was inhibited when it was pre-fed with strain CPCC 101271 T . Based on a polyphasic taxonomy study including genotypic, chemotaxonomic and phenotypic characteristics, we propose that strain CPCC 101271 T represents a novel bacterial species with the name Stenotrophomonas nematodicola sp. nov. and CPCC 101271 T as the type strain. Metagenomic sequence analysis of the intestinal microbiota of C. elegans fed with strain CPCC 101271 T and then infected with B16 revealed that pre-feeding with CPCC 101271 T improved the diversity of intestinal bacteria, while the community structure varied significantly together with the fluctuation of Stenotrophomonas spp. and Bacillus spp. abundance during competition between strain CPCC 101271 T and B16. In conclusion, the nematode microbiota strain CPCC 101271 T , a novel species of the genus Stenotrophomonas , assisted in its host resistance to the pathogen Bacillus nematocida colonization, so as to act as an intestinal life span-prolonging for C. elegans. General Microbiology Stenotrophomonas nematodicola Caenorhabditis elegans Bacillus nematocida lifespan-prolonging colonization Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Figure 7 Introduction Nematodes are one kind of the most abundant worms and have a significant global impact on ecosystems, economies, agriculture and human health. Plant parasitic nematodes cause huge economic losses to agriculture and forestry every year. It is of great importance to develop biological methods to control plant parasitic nematodes, so as to deal with the environmental problems posed by chemical control methods (Duncan, 1991 ). It has been reported that the intestinal flora in most nematodes plays an important role in host growth, physiological metabolism and immune regulation (Nour et al., 2003 ; Haegeman et al., 2009 ). The stability of biocontrol agent activity in the field is an important factor restricting their development. Since microbiota is thought to be key to the stability of biocontrol agents, understanding the functions of the intestinal flora in nematodes is of great significance for improving the activity of biocontrol agents. The worm Caenorhabditis elegans is susceptible to many of the pathogens that infect plant parasitic nematodes (Sinha et al., 2012). Like most pathogens that infect C. elegans , pathogenic bacteria colonize the digestive tract and ultimately kill the nematode. In contrast, most bacteria such as Escherichia coli and Bacillus subtilis are usually not toxic to C. elegans (Garsin et al., 2001 ). Thus, C. elegans has proven to be a useful and relatively simple model for studying the interactions between microbiota and pathogens. C. elegans worms are reared on bacterial cells of E. coli under standard laboratory conditions. Starting from early adulthood, bacterial cells colonize the intestinal lumen and re-form the entire gut microbiota (Portal-Celhay et al., 2012 ). However, in nature the nematode C. elegans is a ‘microbivore’ because of its ability to consume various types of bacteria. To some extent, the gut microbiota of nematodes may be dominated by the bacteria that they feed on; these bacteria may shape the microbiota community structure, regulate metabolism and even alter the lifespan of the host (Han et al., 2018 ). In C. elegans , beneficial bacteria were also reported to modulate host defense responses to bacterial pathogens (Kim & Mylonakis, 2012 ; Montalvo-Katz et al., 2013 ; Iatsenko et al., 2014 ; Dirksen et al., 2016 ; Berg et al., 2019 ; Kissoyan et al., 2019 ; Zimmermann et al., 2019 ). In our previous study, we found that the bacterial pathogen strain B. nematocida B16 killed C. elegans nematodes by employing a “Trojan horse” mechanism (Niu et al., 2010 ). We have isolated several bacteria inside worms from various origins including soil and rotten fruit. Some bacteria, like Phytobacter sp. SCO41, showed inhibitory effects on pathogenic bacterium B16 (Wang et al., 2019 ). To explore the relationships between microbiota and pathogens of nematodes in depth, we combined metageomic sequencing analysis and culture-dependent methods to collect evidence. As a result of this analysis, we found that strain CPCC 101271 T , originally isolated from the intestinal lumen of C. elegans in nature, acts as a component of beneficial microbiota for C. elegans by extending the lifespan of the host, as well inhibiting the colonization of the host by B. nematocida B16, an opportunistic pathogen, which was previously proposed as a candidate biological control agent for nematodes (Huang et al., 2005 ). Here, we report the results of a taxonomic study of strain CPCC 101271 T , which we have proposed to be named Stenotrophomonas nematodicola sp. nov. The results of in vitro and in vivo experiments showed that B. nematocida B16 can inhibit the growth of CPCC 101271 T , while strain CPCC 101271 T has the ability to inhibit the colonization of C. elegans by B16. We also describe the variation in the microbiota community structure of C. elegans during competition between strain CPCC 101271 T and B16. Materials And Methods Acquisition of worms and bacterial strains The location for screening nematodes is Baotianman Natural Reserve (33° 27′ 47′′ N; 111° 48′ 32′′ E), Nanyang, China. Four soil samples were collected and approximately 1000 wild-living nematodes were isolated using the Baerman funnel technique (Gray, 1984 ). Single worms were isolated and collected under a dissecting microscope. After washing three times with aseptic M9 buffer, single nematodes were frozen, ground and their crude DNA was extracted. The nematode species was identified by diagnostic PCR using the primer pair nlp30 diagnostic for C. elegans (Petersen et al., 2014 ). The cultivation, synchronization, collection and surface sterilization of C. elegans worms were performed as previously described (Niu et al., 2012 ; Niu et al.; 2015 ; Niu et al., 2016 ). Strain CPCC 101271 T was isolated from the surface-sterilized C. elegans worms, using Luria-Bertani (LB) agar plates. The nematodes were surface-sterilized by soaking in a solution of 1% mercuric chloride and 2% antibiotic mixture (streptomycin sulfate and gentamicin) for 1 h, and then cultured on nutrient and oligotrophic agar plates to confirm successful surface sterilization (0 cfu). The surface-sterilized worms were ground, then approximately 0.1 g of homogenate was suspended in 10 mL sterilized saline solution (containing 0.85% NaCl, w/v) and mixed thoroughly. Next, about 0.2 mL of suspension was spread onto an LB agar plate. After incubation at 30°C for 2 weeks, about 40 bacterial colonies were grown on the plate. According to the colony color and size, the colonies were randomly selected for separation and purification. Among which, a distinct pale yellowish colony was picked and transferred onto a newly prepared LB agar plate for further purification. The purified isolate of CPCC 101271 T was maintained as a glycerol suspension (20%, v/v) at -80°C for long-term storage. The reference strains Stenotrophomonas rhizophila JCM 13333 T and S. bentonitica DSM 103927 T were obtained from the Japan Collection of Microorganisms ( https://jcm.brc.riken.jp/en/ ) and German Collection of Microorganisms and Cell Cultures ( https://www.dsmz.de/collection/catalogue ), respectively. E. coli strain OP50 was obtained from the Laboratory for Conservation and Utilization of Bio-resources, Yunnan University. The opportunistic pathogen strain B. nematocida B16 (= GCMCC 1128) (Huang et al., 2005 ) was obtained from the China General Microbiological Culture Collection Center ( http://www.cgmcc.net ). GFP-expressing strain B16g was constructed in our previous study (Niu et al., 2012 ). C. elegans lifespan assay Worms C. elegans were maintained on NGM (Nematode Growth Medium) plates at 25°C. The strains CPCC 101271 T , JCM 13333 T and OP50 used for measuring the worms’ lifespan were recovered from the 20% glycerol stock and were streaked onto LB agar plates and then incubated at 32°C. A single colony was picked and incubated in 5 ml of LB at 32°C overnight. One milliliter of the overnight culture was added to 100 ml of LB medium and shaken at 32°C until an OD 600 of 0.8 was reached. 200 µl of the tested bacterial culture was seeded on NGM plate, and then synchronized L4 larvae were transferred to the corresponding bacterial seeded NGM plate (Park et al., 2017). The lifespan experiment was monitored by scoring the dead worms every 10 hours from 50-h until 160-h. Worms that did not respond to prodding with a platinum wire were considered dead. Those desiccated by crawling onto the edge of the housing plate were excluded from the analysis. The experiments were performed with five replicates at three different time intervals. Taxonomic study of strain CPCC 101271 T The taxonomic position of the new isolate was studied using the polyphasic taxonomy approach. (i) Examination of cell morphology and physiological characteristics. The Gram-staining reaction was performed according to Magee et al. ( 1975 ). Cell morphology was observed using a light microscope, and mobility was observed by inoculating cells into nutrient broth with 0.3 % (w/v) agar and incubating at 30°C for 7 days. Growth conditions and the physiological characteristics of the isolate were tested using methods described previously (Yuan et al., 2008 ). Antibiotic resistance was tested on LB agar plates using discs containing the following antibiotics: ampicillin (10 µg), cefaclor (30 µg), chloramphenicol (30 µg), clindamycin (2 µg), erythromycin (15 µg), gentamycin (10 µg), kanamycin (30 µg), netilmicin (30 µg), novobiocin (5 µg), penicillin (10 IU), polymyxinB (300 IU), rifampin (5 µg), vancomycin (30 µg), tetracycline (30 µg), tobramycin (10 µg) and treptomycin (10 µg). (ii) Chemotaxonomic study. Cell mass for chemotaxonomic and molecular systematic studies of the strain was collected from TSA plates cultured at 30°C for 5 days. The polar lipids were extracted and isolated by two-dimensional TLC and identified according to the procedures previously described by Minnikin et al. ( 1984 ). Ubiquinones were isolated and purified as described previously (Collins et al., 1997 ) and analyzed using HPLC. Cellular fatty acids were extracted, methylated and identified using the Sherlock Microbial Identification System (MIDI) according to the manufacturer’s instructions (Kroppenstedt, 1985 ). The MIDI Sherlock Version 6.0 database and the TSBA 6 method were used for analysis. (iii) Genomic analysis. DNA preparation and sequencing of PCR amplification products of the 16S rRNA gene were performed as described by Li et al. ( 2007 ). The obtained sequence was compared with available 16S rRNA gene sequences from GenBank using the BLAST program and EzBioCloud ( http://www.ezbiocloud.net/ ) to determine the approximate phylogenetic affiliation of strain CPCC 101271 T (Kim & Mylonakis, 2012 ). Multiple alignments with sequences of the most closely related taxa and calculations of sequence similarity were carried out using MEGA version 7.0 (Kumar et al., 2016 ). Phylogenetic trees were inferred using the neighbor-joining method (Saitou & Nei, 1987 ) with K nuc values (Kimura 1980 & 1983 ) and complete deletion gaps. Phylogenetic trees were also constructed using and the maximum-parsimony (Kluge & Farris, 1969 ) and maximum-likelihood (Felsenstein, 1981 ) methods. The topology of the phylogenetic tree was evaluated by the bootstrap resampling method of Felsenstein ( 1985 ) with 1000 replicates. Genome sequencing of strain CPCC 101271 T and the reference strain S. bentonitica DSM 103927 T was performed on an Illumina MiSeq instrument (Illumina); the reads were assembled using Platanus software (v1.2.4) and contigs shorter than 500 bp in size were discarded. The genomic G + C content was calculated from the draft genome sequence. The values of average nucleotide identity (ANI) between strain CPCC 101271 T and the reference strain S. rhizophila JCM 13333 T and two other type strains were calculated by comparing their draft genomes (Yoon et al., 2017 ). In vitro bacteriostatic activity test Each bacterial strain was separately inoculated into 5 mL of LB medium and cultured in a shaker at 32°C, 180 r/min for 12 h. Then the culture broth was adjusted to an optical density value at 600 nm (OD 600 ) of 1. Approximately 0.3 mL of CPCC 101271 T culture broth was evenly spread onto an LB agar plate. Sterilized filter paper with a diameter of 5 mm was immersed in the bacterial culture for 5 min and placed onto the agar plate containing strain CPCC 101271 T . The plate was then incubated at 32°C for 48 h, and the size of the inhibition zone for each sample was recorded. E. coli culture broth and LB medium were used as negative controls, while polymyxin B (300 IU) and rifampin (5 µg) were used as positive controls. The experiments were performed with three parallels and repeated thrice. Colonization capability assay Colonization capability was assayed using approximate fifty 1-day-old adult hermaphrodite worms were placed on each plate at 25°C following the procedures described by Aballay et al. ( 2000 ) and Niu et al. ( 2012 ) with modifications. In ‘Feeding Transfer’ experiments, the worms were transferred by hair and repeated washed using sterilized NaCl solution (0.85 %, w/v). Three nematode treatment groups were set up. In the first group of nematodes pre-fed with CPCC 101271 T then infected by B16g, the worms were transferred onto LB plates containing a low concentration (10 6 cells/mL) of CPCC 101271 T and co-cultivated for 4 h. The worms were then removed from the plates, washed twice, transferred to plates containing B16g and co-cultivated for 72 h. In the second group of nematodes pre-fed with JCM13333 T then infected by B16g, the worms were first seeded on an LB agar plate containing JCM13333 T (10 6 cells/mL) and cultivated for 4 h, and then the worms were transferred to plates containing B16g and cultivated for 72 h. In the third group, the worms were first fed on the same concentration of OP50 for 4 h, then transferred to B16g plates and cultivated for 72 h. The control group of nematodes without being pre-fed with bacteria were directly seeded on blank medium and then cultivated for 4 h before being transferred to B16g plates, which were also defined as B16 direct infection group. The colonization process was observed under a Nikon 800 Eclipse microscope (Nikon Corp., Japan) equipped for epifluorescence with a mercury lamp and an excitation filter of 450–490 nm (blue light) and a barrier filter of 515 nm. At each time point, three sets of 10 nematodes were randomly selected to evaluate colonization. The worms with fluorescent bacteria in the entire lumen were scored as full; worms without any green fluorescence signal in the lumen were scored as undetected; and worms between these two extremes were scored as partial. The worms were considered dead when no movement was observed under a light-dissecting microscope, and when gently tapping of nematodes by a platinum wire, no movement occurred. Dead nematodes whose bodies were decomposed were excluded from the analysis. The number of worms killed in each group was counted every 12 h during within 72 hours from B16 infection. Mortality rates of B16-infected nematodes were defined as the ratio of dead nematodes to tested nematodes. The experiments were performed with thrice. DNA preparation and metagenomic analysis of microbiota The tested nematodes were divided into five groups as follows: (I) CW00h group, which was pre-fed with CPCC 101271 T for 4 h; (II-V) CW04h, CW08h, CW12h and CW16h groups, which were separately co-cultivated with B16 for 4 h, 8 h, 12 h and 16 h, respectively, after being pre-fed with CPCC 101271 T for 4 h. The worms were collected and then washed and surface sterilized as described above. Total DNA was extracted from the intestinal microbes using the PowerSoil DNA Isolation Kit (MoBio, USA) according to the manufacturer’s protocols. The concentration and purity of extracted DNA were determined using a TBS-380 and NanoDrop2000, respectively. The quality of the extracted DNA was evaluated on a 1% agarose gel. DNA was fragmented to an average size of about 300 bp using a Covaris M220 (Gene Company Limited, China) for paired-end library construction. The paired-end library was constructed using NEXTFLEX Rapid DNA-Seq (Bioo Scientific, Austin, TX, USA). Adapters containing the full complement of sequencing primer hybridization sites were ligated to the blunt ends of the fragments. Paired-end sequencing was performed on an Illumina NovaSeq (Illumina Inc., San Diego, CA, USA) at Majorbio Bio-Pharm Technology Co., Ltd. (Shanghai, China) using NovaSeq Reagent Kits according to the manufacturer’s instructions ( www.illumina.com ). Adapter sequences were stripped from the 3' and 5' ends of paired-end Illumina reads using SeqPrep ( https://github.com/jstjohn/SeqPrep ). Low-quality reads (length < 50 bp, a quality value < 20, or containing N bases) were removed using Sickle ( https://github.com/najoshi/sickle ). Metagenomics data were assembled using MEGAHIT ( https://github.com/voutcn/megahit ) (Li et al., 2015 ), which makes use of succinct de Bruijn graphs. Contigs with a length ≥ 300 bp were selected as final assemblies and were used for further gene prediction and annotation (Noguchi et al., 2006 ; Li et al., 2008 ). Results The isolation and identification of strain CPCC 101271 T , which represents a novel species of the genus Stenotrophomonas Strain CPCC 101271 T was recovered from the intestinal lumen of C. elegans using LB agar plates (see Materials and Methods). The nearly full-length sequence of the 16S rRNA gene (1543 bp) of strain CPCC 101271 T was obtained and submitted to GenBank under accession number MT126327. A BLAST search of GenBank showed that the 16S rRNA gene of strain CPCC 101271 T exhibited 98.1%-99.7% similarity to the 16S rRNA genes of members of the genus Stenotrophomonas , and < 98.0% similarities to those of other bacterial species in the family Lysobacteraceae . In a phylogenetic tree based on the 16S rRNA gene sequences of all members of the family Lysobacteraceae , strain CPCC 101271 T formed a sublineage with S. rhizophila JCM 13333 T and S. bentonitica DSM 103927 T within the genus Stenotrophomonas (Fig. 1 ). Therefore, it is reasonable to designate strain CPCC 101271 T as a member of the genus Stenotrophomonas . Genome sequencing of strain CPCC 101271 T yielded a draft genome of 4,402,751 bp, assembled from 126 qualified contigs, with 100-fold coverage and an N50 length of 738,821 bp. Genes putatively encoding glucosylglycerol-phosphate synthase (G9274_RS14805, B861_RS0201980, E5352_RS0097) and alpha-trehalose-phosphate synthase (BN96_RS08035, BIZ42_RS05395, C0R07_RS03270, GDJ08_RS08470), which might endow strain CPCC 101271 T the ability to maintain homeostasis of the host, were identified in its genome. The ANI values between the draft genomes of strain CPCC 101271 T and its closest phylogenetic neighbors S. rhizophila JCM 13333 T and S. bentonitica DSM 103927 T were 84.7 % and 85.0 %, respectively. These values were both far lower than 95%, which was proposed as the cutoff value for defining different species (Kim et al., 2014 ). Accordingly, strain CPCC 101271 T represents a species genetically different from other validly described species of the genus Stenotrophomonas . This conclusion was supported by chemotaxonomic data (described below) and the phenotypic characteristics given in the species description and in Supplementary Table S1. Description of Stenotrophomonas nematodicola sp. nov. Stenotrophomonas nematodicola (ne.ma.to.di'co.la. N.L. pl. n. Nematoda , a taxonomic group of animals; L. suff. - cola , inhabitant, dweller; N.L. fem. n. nematodicola , an inhabitant of nematodes). Cells are Gram-reaction-negative, facultatively aerobic and motile coccoid rods, 1.0-1.2 µm in width and 1.9–2.3 µm in length. Colonies on LB agar medium are smooth, pale yellow in color, and circular and entire, with a diameter of 1.0-1.1 mm after 48 h of incubation. Growth occurs at 10–37°C (optimum 32°C) and at pH 6.0–8.0 (optimum pH 7.0) with 0–5 % (w/v) NaCl (optimum 0–1 %). Catalase- and oxidase reactions are positive. Positive for hydrolysis of gelatin and nitrate reduction reaction, while negative for hydrolysis of starch and urea, peptonization of milk, and production of H 2 S and indol. Positive for acid phosphatase, alkaline phosphatase, α-chymotrypsin, esterase (C4), esterase lipase (C8), cystine arylamidase, β-glucosidase, leucine arylamidase and valine arylamidase in an API ZYM strip. Acetic acid, acetoacetic acid, bromo-succinic acid, citric acid, dextrin, D-fructose-6-PO 4 , D-lactic acid methyl ester, D-maltose, D-mannose, glycyl-L-proline, L-alanine, L-glutamic acid, L-histidine, methyl pyruvate, L-lactic acid, L-malic acid, N-acetyl-D-galactosamine, N-acetyl-D-glucosamine, pectin, propionic acid, Tween 40, α-D-Glucose and α-Keto-glutaric acid can be utilized as the sole carbon source, and amygdalin, arbutin, D-fructose, D-glucose, D-maltose, esculin ferric citrate, N-acetylglucosamine, potassium 2-ketogluconate and potassium 5-ketogluconate can be assimilized and produce acid. Resistant to ampicillin (10 µg), cefaclor (30 µg), chloramphenicol (30 µg), clindamycin (2 µg), erythromycin (15 µg), gentamycin (10 µg), kanamycin (30 µg), netilmicin (30 µg), novobiocin (5 µg), penicillin (10 IU), vancomycin (30 µg), tetracycline (30 µg), tobramycin (10 µg) and treptomycin (10 µg), while sensitive to polymyxinB (300 IU) and rifampin (5 µg). Diphosphatidylglycerol (DPG), phosphatidylglycerol (PG), phosphatidylethanolamine (PE) and an unidentified phospholipid (UPL) were detected in a polar lipids extract (Figure S1). The respiratory quinone is Q-8. The major fatty acids are iso-C 15:0 (38.2%) and antesio-C 15:0 (16.6%), with moderate amounts of iso-C 11:0 (8.7%) and C 16:0 (5.1%) and small amounts (< 5%) of cyclo-C 17:0 , C 13:0 2-OH, C 12:0 3-OH, iso-C 17:0 , iso-C 14:0 , C 14:0 , iso-C 11:0 3-OH, iso-C 16:0 , iso-C 13:0 3-OH, C 16:1 ω 7 c /C 16:1 ω 6 c , and iso -C 17:1 ω 9 c /C 16:0 10-methyl. The type strain CPCC 101271 T (= W5) was isolated from a surface-sterilized C. elegans worm cultured in a lab in Nanyang, a city in middle of China. The DDBJ/EMBL/GenBank accession numbers of the 16S rRNA gene sequence and draft genome sequence of strain CPCC 101271 T are MT126327 and WIAY00000000, respectively. The genome of the type strain is characterized by a size of 4.4 Mbp and a G + C content of 67.3 mol%. A filled circle indicates that the node was also recovered in trees generated with the maximum-likelihood method and maximum-parsimony method. Bootstrap values are shown as the percentage of 1,000 replicates; only percentages above 50% are shown. Escherichia coli JCM 1649 T (GenBank accession no. X80725) was used as an outgroup (not shown). Bar, 0.005 substitutions per nucleotide position. Increase in Caenorhabditis elegans survival rate and lifespan by feeding on CPCC 101271 T We compared the longevity of worms fed either on CPCC 101271 T , JCM 13333 T or OP50. The results showed that worms fed on JCM 13333 T or OP50 had almost the similar lifespans. However, worms fed on CPCC 101271 T lived approximately 40% longer than worms fed on E. coli or JCM 13333 T , indicating that the nematodes fed on CPCC 101271 T lived longer than those fed on E. coli OP50 or S. rhizophila JCM 13333 T and had greatly increased survival rates (Fig. 2 ) Strain CPCC 101271 T confers the host with resistance to B. nematocida colonization To investigate whether strain CPCC 101271 T has colonization-resistance activity against B. nematocida B16, we first performed an in vitro bacteriostatic activity test. The results showed that strain CPCC 101271 T could not inhibit B. nematocida B16 but could be inhibited by B16 (Fig. 3 ). A transparent inhibition zone with clear edges formed around the paper containing B16 after 48 h of incubation on an LB agar plate spread with strain CPCC 101271 T . The clear zone, which was about 1.6 cm in diameter, was slightly smaller than the zones surrounding the positive drug controls polymyxin B (300 IU) and rifampin (5 µg). And no clear inhibition zone formed around the papers containing E. coli or LB medium (Fig. 3 ). GFP-expressing strain B16g was used to confirm the specificity of B16 colonization activity assays. The results of colonization-resistance activities indicated that strain CPCC 101271 T could also inhibit the colonization of B16 in the nematode intestine. During the first 24 h of infection with B. nematocida B16, almost no nematodes pre-fed with CPCC 101271 T were scored as being in the “full” colonization category (see Materials and Methods). By contrast, almost 20% of animals directly fed with B. nematocida B16 were scored as “full”. In addition, after infection for 48 h, 50% of the animals fed only B. nematocida B16 were scored as “full”. However, only 10% of worms pre-fed with CPCC 101271 T were scored as “full” at the same time point. After 72 h, B. nematocida B16 showed notably strong colonization ability, with 90% of the worms not pre-fed with CPCC 101271 T scored as “full”. In contrast, only 10% of the animals pre-fed with CPCC 101271 T were scored as “full”. Moreover, compared with worms fed only B16, the percentage of worms fed with both B16 and CPCC 101271 T that had undetectable B16 colonization was much higher at 48 h (10% vs. 80%) and at 72 h (0% vs. 70%). The worms pre-fed with E. coli showed little difference compared with those in the no pre-feeding group. The ability of B16 to colonize the nematodes pre-fed with JCM 13333 T was stronger than its ability to colonize those pre-fed with CPCC 101271 T , but a little weaker than its ability to colonize the negative controls pre-fed with E. coli OP50. Differences between the abilities of B16 to colonize the nematodes pre-fed with CPCC 101271 T and pre-fed with OP50 were notable when we compared the changes in the severity of colonization at 72-h (Fig. 4 , chi-squared test, P < 0.0001). For example, only 10% of worms that were pre-fed with CPCC 101271 T could be categorized as having ‘full’ colonization. However, 90% of worms that were pre-fed with OP50 were categorized in the ‘full’ colonization category. The results indicated that colonization of B. nematocida B16 was markedly attenuated in C. elegans pre-fed with CPCC 101271 T . The differences in the mortalities of the B16-infected nematodes in the different treatment groups indicated that pre-feeding with CPCC 101271 T reduced the mortality caused by infection with the pathogenic bacteria B16 (Fig. 5 ). The mortalities of the nematodes pre-fed with OP50 and then infected by B16 and the nematodes directly infected by B16 (without pre-feeding with any other bacteria) were 85 and 90% within 60 h, respectively. By contrast, for worms pre-fed with CPCC 101271 T then infected with B16, the mortality dropped to 40%. The natural mortality rate of the negative control nematodes (no pre-feeding or B16 infection) was only 18%. At other time points, the mortalities of nematodes pre-fed with CPCC 101271 T were significantly lower than those of nematodes pre-fed with E . coli or directly infected with B16. The variation in Caenorhabditis elegans microbiota community structure during competition between CPCC 101271 T and B16 In a previous study, we collected free-living terrestrial C. elegans from soil and rotten fruits, and analyzed the variation in intestinal flora following B. nematocida B16 infection by performing macrogenomic analysis. We found significant differences in the diversity and distribution of microbiota between the control worms and those infected with B16 for 24 h. The diversity of the intestinal microbiome decreased after B16 infection (Niu et al., 2016 ). Here we aimed to investigate the variation in the intestinal bacterial community structure of C. elegans during competition between CPCC 101271 T and B16. A total of 332314, 280966, 705161, 227126 and 597664 sequences comprising 44, 26, 49, 19 and 30 operational taxonomic units (OTUs) were obtained from the five groups CW00h, CW04h, CW08h, CW12h and CW16h, respectively. At the genus level, these OTUs represented 26, 14, 23, 10 and 14 genera, respectively. It was obvious that the bacteria diversity was greatly decreased during competition between CPCC 101271 T and B16 (Fig. 6, Fig. 7 ). At the first stage of infection (CW00h) in nematodes pre-fed with strain CPCC 101271 T , the microbiota community structure predominantly consisted of the genera Bacillus , Acetobacter , Lactobacillus , Phytobacter, Stenotrophomonas, Pichia and Sphingomonas . At the second stage (CW04h), 4 h after the worms were infected by B16, dysbiosis occurred, and in the course of re-construction of the microbiota community, the bacteria diversity was drastically reduced. Besides Lactobacillus spp., Acetobacter spp. and Pichia spp., which remained the major groups, the abundance of Bacillus spp. increased slightly and the abundance of Stenotrophomonas spp. increased. At the third stage (CW08h), which we termed “the breaking period”, a large number of CPCC 101271 T and B16 bacteria were co-existing and competing; the diversity of the intestinal flora had partially recovered, but the abundance of Bacillus spp. had greatly decreased. At the fourth stage (CW12h), the abundance of Bacillus spp. was even lower and the abundance of Stenotrophomonas spp. was higher. The newly reconstructed intestinal flora was disrupted again, and the species composition was the most similar to that observed at the second stage (Fig. 7 ). By the fifth stage (CW16h), B16 overwhelmed CPCC 101271 T and only Lactobacillus spp. and Pichia spp., together with Bacillus spp., remained the major microbiota. In a summary, over the course of B16 infection, the abundance of CPCC 101271 T and its relatives ( Stenotrophomonas spp.) kept on increasing until 8 h after B16 infection and then decreased sharply. The change in abundance of Stenotrophomonas spp. was accompanied by similar changes in the abundance of alpha-trehalose-phosphate synthase-encoding genes calculated from the metagenome data, except at the final stage (Figure S2). By contrast, the abundance of Bacillus spp. first decreased, then increased rapidly, which was similar to the changes in the abundance of trehalose-6-phosphate hydrolase-encoding genes (Figure S3). The abundances of alpha-trehalose-phosphate synthase-encoding genes (present in the genome of B16) and trehalose-6-phosphate hydrolase-encoding genes (present in the genome of CPCC 101271 T ) together with Stenotrophomonas spp. and Bacillus spp. reached the highest level at 8 h (CW08h) after B16 infection. The results suggested that strain CPCC 101271 T possibly participated directly or induced some other bacteria in the community to participate in resistance to B16 colonization. However, by 16 h after infection, B16 dominated the microbiota community, and the growth of CPCC 101271 T was completely suppressed. Discussion Gut microbiota, diverse microorganisms inhabiting the digestive track, are tightly linked to the health of their host. The community of microbial species, among which bacteria are predominant and have been extensively studied, not only generates metabolites essential for various host functions but also confers resistance to exogenous pathogens (Lee & Hase, 2014 ). However, the molecular mechanisms by which microbiota resist pathogens and the changes in bacterial composition that occur after pathogen infection remains elusive. It has been reported that bacteria living in most nematodes play an important role in the growth and development, physiological metabolism and immune regulation of the host. The bacteria Xenorhabdus spp. and Photorhabdus spp. were reported to be symbionts found in the guts of nematodes including Steinernema , Heteronrhahditis , Heterodera and Rhabditis. They produce antibiotics, intracellular protein crystals, and numerous other products that help nematodes kill insects and also provide nutrients (Forst et al., 1997 ; Park et al., 2011 ; Whittaker et al., 2016 ; Shan et al., 2019 ). Bacteria associated with cysts of the soybean cyst nematode play an important ecological role in the long-term survival of cysts in soil (Nour et al., 2003 ). An endo-symbiotic bacterium in a plant-parasitic nematode was found to be closely associated with the growth and metabolism of its host (Haegeman et al., 2009 ). Therefore, to some extent, nematodes are typical symbioses with their microbiota. For nematodes C. elegans , most of the research work was carried out based on the N2 strain, which has been adapted to laboratory conditions over decades (Sterken et al., 2015 ), including the regular and routine removal of any microbes through hypochlorite treatment. Thus N2 strain does not carry any microbes in its gut and microbiome associations are little known to the nematode C. elegans N2 under laboratory conditions. In contrast, worms in nature are exposed to complex microbial communities. Understanding the worm’s natural microbiome is essential to help explain their realistic and unbiased characteristics. In fact, more and more researchers have paid their attention to the natural C. elegans microbiome (Dirksen et al., 2016 ; Samuel et al., 2016 ; Zhang et al., 2017 ). A possible fitness benefit was already indicated upon gut colonization with certain non-pathogenic bacteria, leading to increased resistance against pathogens (Ikeda et al., 2007 ; Kim & Mylonakis, 2012 ; Montalvo-Katz et al., 2013 ). However, it is yet unclear whether the beneficial bacterial isolates in C. elegans affect the infection effect of pathogenic bacteria on nematodes. The activities and stability of biological control agents might be effectively improved by using co-cultures of various antagonistic bacteria with different mechanisms of action and ecological adaptability. Therefore, using natural C. elegans as a model, studying the interactions between microbiota and biocontrol microbes is a promising approach for improving the stability of biocontrol in the field. To date, there are 16 validly described species in the genus Stenotrophomonas , which have high genotypic and phenotypic diversity and were recovered from various environmental and even clinical samples (Brooke, 2012 ). The type species S. maltophilia was originally recognized as a human opportunistic pathogen. Subsequent research revealed that the metabolic diversity of S. maltophilia is responsible for the production of novel bioactive compounds, including biocontrol agents against microbes and insects, and enzymes and nanoparticles used in medicinal, industrial and bioremediation applications (Ribitsch et al., 2012 ). Another well-studied species, S. rhizophila , which shows an endophytic life style, possesses unique genes encoding plant cell-wall-degrading enzymes and proteins responsible for the synthesis and transport of the plant-protective spermidine and high salinity tolerance, which suggests it is a harmless alternative Stenotrophomonas species for use in biotechnology (Alavi et al., 2013 ). There were also several reports on the genus Stenotrophomonas strains isolated from animal intestines. Stenotrophomonas members were found to be gut bacteria through the life cycle of the Bark Beetle Dendroctonus rhizophagus , and S. maltophilia could be implicated in nitrogen fixation and cellulose breakdown, important roles associated to insect development and fitness, especially under the particularly harsh life conditions of this beetle (Morales-Jiménez et al., 2012 ). Additionally, Sun et al. isolated a chitin-degradation Stenotrophomonas strain from the hindgut of a fungus-growing termite Macrotermes barneyi (Sun et al., 2017 ). The novel species studied here, a close relative of S. rhizophila , was generally consistent with those previously reported Stenotrophomonas species isolated from the nematodes sampled directly from the native habitats (Dirksen et al., 2016 ). In each parallel of isolation experiment, we selected randomly 20 single natural worms isolated from the same location. Members of the genus Stenotrophomonas could be isolated from more than 15 worms. The Stenotrophomonas spp. were identified to be the same species with CPCC 101271 T . Furthermore, other worms without Stenotrophomonas being detected were raised on 9-cm agar plates seeded with 400 µl of the tested bacterium CPCC 101271 T with an OD 600 of 10 for 24 h at room temperature. Then Stenotrophomonas spp. could be isolated from intestines of the worms after washed three times and surface disinfection. These experiments indicated that CPCC 101271 T could stably colonize the nematode gut under experimental conditions. In nature, the structure and diversity of the microbiota in healthy nematodes are constantly changing. Some intestinal bacteria are actually indispensable parts of the host, which may form a mutually beneficial symbiotic relationship with the host. In this study, a new Stenotrophomonas bacterium CPCC 101271 T representing a novel species was isolated from nematodes, and the interaction between the bacterium and B. nematocida B16 was investigated. When strains B16 and CPCC 101271 T were co-cultured on LB plates, strain B16 showed inhibitory activities against CPCC 101271 T , which is consistent with the last stage of the competition between strain CPCC 101271 T and B16 in worm intestine, even strain CPCC 101271 T exhibited the colonization-resistance activities against B. nematocida B16 in the early stages. Firstly, strain CPCC 101271 T was confirmed as probiotic to worms owning to its ability to prolong the lifespan of C. elegans . Secondly, strain CPCC 101271 T could delay the infection time of B16 against nematodes, but not completely inhibit the infection of B16. Last but not least, Bacillus strains occupied the niche of Stenotrophomonas members by inhibiting the growth of CPCC 101271 T , which could be inferred from the metagenomic analysis results. And then strain B16 completed the infection and realized its proliferation in worms. Based on the above experiments, we proposed that the reason of colonization resistance to the pathogen B16 by the strain CPCC 101271 T might own to its beneficial aspects to C. elegans . Strain CPCC 101271 T might play a critical role in (i) shaping and maintaining the intestinal bacterial community structure, (ii) synthesizing osmoprotectants, such as glucosylglycerol and trehalose, to help maintain host homeostasis, and (iii) producing or stimulating other microorganisms to synthesize antimicrobial peptides and other stress protective agents to protect the host from pathogens and harsh environments. We should explore substantial evidence to confirm the above inference in the following studies. Understanding this inference mechanism can help quickly inhibit the growth of probiotic microbiota, accelerate the colonization of biocontrol bacteria in the intestinal tract and improve the killing efficiency of nematodes. Our current findings may lay a theoretical foundation and open up new ideas for the development of ideal biocontrol agents. Declarations Availability of data and materials The DDBJ/EMBL/GenBank accession number for the 16S rRNA gene sequence of strain CPCC 101271 T is MT126327; The draft genome sequence of strain CPCC 101271 T is WIAY00000000. The strain CPCC 101271 T has been deposited in China Pharmaceutical Culture Collection and is available to the scientific research community without any special restrictions. Author's contributions: Rui Han and Yu Wang carried out the experiments. Yang Deng prepared the figures. Yuqin Zhang and Lin Zhang designed the project and prepared the manuscript together with Qiuhong Niu. All authors reviewed the manuscript. Acknowledgement : The authors thank JCM (Japan Collection of Microorganisms) and DSMZ (German Collection of Microorganisms and Cell Cultures) for providing the reference strains. Funding : This research was supported by the National Natural Science Foundation of China (NSFC 31670010 and 31570120), the Drug Innovation Major Project (2018ZX09711001-007-001), the National Infrastructure of Microbial Resources (NIMR-2020-3), CAMS Innovation Fund for Medical Sciences (CIFMS) (2016-I2M-2-002) and Innovation Scientists and Technicians Troop Construction Projects (Sustainable Utilization of Energy Microbial Resources) of Henan Province. Conflict of Interest Statement : The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest. Consent for publication : Not applicable. Ethical approval and consent to participate : Not applicable. References Aballay A, Yorgey P, Ausubel FM (2000) Salmonella typhimurium proliferates and establishes a persistent infection in the intestine of Caenorhabditis elegans . 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Front Microbiol. 8: 485 Zimmermann J, Obeng N, Yang W, Pees B, Petersen C, Waschina S, Kissoyan K, Aidley J, Hoeppner M, Bunk B, Spröer C, Leippe M, Dierking K, Kaleta C, Schulenburg H (2019) The functional repertoire contained within the native microbiota of the model nematode Caenorhabditis elegans . ISME J . 14: 26-38 Supplementary Files Supplementarymaterials1.docx Cite Share Download PDF Status: Published Journal Publication published 14 Jul, 2021 Read the published version in Archives of Microbiology → Version 2 posted Reviews received at journal 08 May, 2021 Reviewers invited by journal 07 May, 2021 Editor assigned by journal 07 May, 2021 First submitted to journal 06 May, 2021 You are reading this latest preprint version Show more versions Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. 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taxa.","description":"","filename":"1.png","url":"https://assets-eu.researchsquare.com/files/rs-124061/v2/f3f2768edbd3d83844721b7b.png"},{"id":9303872,"identity":"97bd02cc-f85c-4e25-af95-bc26944c2dd6","added_by":"auto","created_at":"2021-05-18 16:18:33","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":85790,"visible":true,"origin":"","legend":"Survival rates of Caenorhabditis elegans fed on different bacteria","description":"","filename":"2.png","url":"https://assets-eu.researchsquare.com/files/rs-124061/v2/5ffcac7dc14f424e27cf70fd.png"},{"id":9303834,"identity":"2cebb144-5b83-41d8-a4a1-5c98a180dd37","added_by":"auto","created_at":"2021-05-18 16:15:33","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":210140,"visible":true,"origin":"","legend":"Inhibitory activity of B16 against CPCC 101271T on an LB agar plate\nOP50, E. coli OP50; B16, B. nematocida B16; GC-1, polymyxin B (300 IU); GC-2, rifampin (5 μg).","description":"","filename":"3.png","url":"https://assets-eu.researchsquare.com/files/rs-124061/v2/75ca8d613d18866e1d0459e7.png"},{"id":9303914,"identity":"885d40fe-46ce-40df-b666-7f3d3c127a2c","added_by":"auto","created_at":"2021-05-18 16:21:47","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":103116,"visible":true,"origin":"","legend":"Differences in B. nematocida B16 colonization of C. elegans after 24 h, 48 h, and 72 h of infection. \nFor each bacterial strain tested, the extent of colonization was scored in four sets of 10 nematodes every 24 h. A representative of three independent experiments with the average fraction of the population colonized for each category is shown. Chi-squared test, *P \u003c 0.05, **P \u003c 0.001.\nCPCC 101271T, Stenotrophomonas nematodicola CPCC 101271T; JCM 13333T, Stenotrophomonas rhizophila JCM 13333T; OP50, E. coli OP50. ","description":"","filename":"4.png","url":"https://assets-eu.researchsquare.com/files/rs-124061/v2/86d069c95c22d217375d655c.png"},{"id":9303836,"identity":"2c1690ca-52ea-43fe-a8ad-b26e1a353004","added_by":"auto","created_at":"2021-05-18 16:15:33","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":71745,"visible":true,"origin":"","legend":"The differences in mortality rates of B16-infected nematodes in different treatment groups\nNotes: CPCC 101271T, Stenotrophomonas nematodicola CPCC 101271T; JCM 13333T, Stenotrophomonas rhizophila JCM 13333T; B16, Bacillus nematocida B16; OP50, Escherichia coli OP50.","description":"","filename":"5.png","url":"https://assets-eu.researchsquare.com/files/rs-124061/v2/717d32e6c95e0b0042f2fdab.png"},{"id":9303873,"identity":"c94eca2a-ec58-411e-aad2-98b34b6e362e","added_by":"auto","created_at":"2021-05-18 16:18:33","extension":"png","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":63038,"visible":true,"origin":"","legend":"Column diagram showing the microbiota structure at the genus level, based on metagenomic sequence analysis, in nematodes pre-fed with CPCC 101271T before and after being infected by B16.\nNotes: CW00h, pre-fed with CPCC 101271T for 4 h; CW04h, CW08h, CW12h and CW16h, groups co-cultured with B16 for 4 h, 8 h, 12 h and 16 h, respectively, after being pre-fed with CPCC 101271T for 4 h. ","description":"","filename":"6.png","url":"https://assets-eu.researchsquare.com/files/rs-124061/v2/8ee94890605a4796b7437a2f.png"},{"id":9303840,"identity":"57094f24-9151-4866-b643-de635808d0f9","added_by":"auto","created_at":"2021-05-18 16:15:34","extension":"png","order_by":7,"title":"Figure 7","display":"","copyAsset":false,"role":"figure","size":112522,"visible":true,"origin":"","legend":"Heatmap based on metagenomic sequence analysis showing the microbiota structure at the genus level in nematodes pre-fed with CPCC 101271T before and after being infected by B16","description":"","filename":"7.png","url":"https://assets-eu.researchsquare.com/files/rs-124061/v2/86f8ea18ea468d46eb354357.png"},{"id":15672709,"identity":"551cdb94-401c-4c3a-af3d-74b448f7dff5","added_by":"auto","created_at":"2021-11-18 14:13:40","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":1002946,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-124061/v2/f29cbbda-9a06-4077-8910-c2a392b40974.pdf"},{"id":9303839,"identity":"496994ff-b450-4498-bf31-4ed9649ff330","added_by":"auto","created_at":"2021-05-18 16:15:33","extension":"docx","order_by":5,"title":"","display":"","copyAsset":false,"role":"supplement","size":308037,"visible":true,"origin":"","legend":"","description":"","filename":"Supplementarymaterials1.docx","url":"https://assets-eu.researchsquare.com/files/rs-124061/v2/f63136134c7d266c0cc640f9.docx"}],"financialInterests":"","formattedTitle":"\u003cp\u003e\u003cem\u003eStenotrophomonas Nematodicola\u003c/em\u003e Sp. Nov., a Novel\u003cem\u003e \u003c/em\u003eIntestinal Lifespan-Prolonging Bacterium for \u003cem\u003eCaenorhabditis Elegans\u003c/em\u003e That Assists in Host Resistance to\u003cem\u003e Bacillus Nematocida\u003c/em\u003e Colonization\u003c/p\u003e","fulltext":[{"header":"Introduction","content":" \u003cp\u003eNematodes are one kind of the most abundant worms and have a significant global impact on ecosystems, economies, agriculture and human health. Plant parasitic nematodes cause huge economic losses to agriculture and forestry every year. It is of great importance to develop biological methods to control plant parasitic nematodes, so as to deal with the environmental problems posed by chemical control methods (Duncan, \u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e1991\u003c/span\u003e). It has been reported that the intestinal flora in most nematodes plays an important role in host growth, physiological metabolism and immune regulation (Nour et al., \u003cspan citationid=\"CR40\" class=\"CitationRef\"\u003e2003\u003c/span\u003e; Haegeman et al., \u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e2009\u003c/span\u003e). The stability of biocontrol agent activity in the field is an important factor restricting their development. Since microbiota is thought to be key to the stability of biocontrol agents, understanding the functions of the intestinal flora in nematodes is of great significance for improving the activity of biocontrol agents.\u003c/p\u003e \u003cp\u003eThe worm \u003cem\u003eCaenorhabditis elegans\u003c/em\u003e is susceptible to many of the pathogens that infect plant parasitic nematodes (Sinha et al., 2012). Like most pathogens that infect \u003cem\u003eC. elegans\u003c/em\u003e, pathogenic bacteria colonize the digestive tract and ultimately kill the nematode. In contrast, most bacteria such as \u003cem\u003eEscherichia coli\u003c/em\u003e and \u003cem\u003eBacillus subtilis\u003c/em\u003e are usually not toxic to \u003cem\u003eC. elegans\u003c/em\u003e (Garsin et al., \u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e2001\u003c/span\u003e). Thus, \u003cem\u003eC. elegans\u003c/em\u003e has proven to be a useful and relatively simple model for studying the interactions between microbiota and pathogens. \u003cem\u003eC. elegans\u003c/em\u003e worms are reared on bacterial cells of \u003cem\u003eE. coli\u003c/em\u003e under standard laboratory conditions. Starting from early adulthood, bacterial cells colonize the intestinal lumen and re-form the entire gut microbiota (Portal-Celhay et al., \u003cspan citationid=\"CR44\" class=\"CitationRef\"\u003e2012\u003c/span\u003e). However, in nature the nematode \u003cem\u003eC. elegans\u003c/em\u003e is a \u0026lsquo;microbivore\u0026rsquo; because of its ability to consume various types of bacteria. To some extent, the gut microbiota of nematodes may be dominated by the bacteria that they feed on; these bacteria may shape the microbiota community structure, regulate metabolism and even alter the lifespan of the host (Han et al., \u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e2018\u003c/span\u003e). In \u003cem\u003eC. elegans\u003c/em\u003e, beneficial bacteria were also reported to modulate host defense responses to bacterial pathogens (Kim \u0026amp; Mylonakis, \u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e2012\u003c/span\u003e; Montalvo-Katz et al., \u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e2013\u003c/span\u003e; Iatsenko et al., \u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e2014\u003c/span\u003e; Dirksen et al., \u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e2016\u003c/span\u003e; Berg et al., \u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e2019\u003c/span\u003e; Kissoyan et al., \u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e2019\u003c/span\u003e; Zimmermann et al., \u003cspan citationid=\"CR56\" class=\"CitationRef\"\u003e2019\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eIn our previous study, we found that the bacterial pathogen strain \u003cem\u003eB. nematocida\u003c/em\u003e B16 killed \u003cem\u003eC. elegans\u003c/em\u003e nematodes by employing a \u0026ldquo;Trojan horse\u0026rdquo; mechanism (Niu et al., \u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e2010\u003c/span\u003e). We have isolated several bacteria inside worms from various origins including soil and rotten fruit. Some bacteria, like \u003cem\u003ePhytobacter\u003c/em\u003e sp. SCO41, showed inhibitory effects on pathogenic bacterium B16 (Wang et al., \u003cspan citationid=\"CR51\" class=\"CitationRef\"\u003e2019\u003c/span\u003e). To explore the relationships between microbiota and pathogens of nematodes in depth, we combined metageomic sequencing analysis and culture-dependent methods to collect evidence. As a result of this analysis, we found that strain CPCC 101271\u003csup\u003eT\u003c/sup\u003e, originally isolated from the intestinal lumen of \u003cem\u003eC. elegans\u003c/em\u003e in nature, acts as a component of beneficial microbiota for \u003cem\u003eC. elegans\u003c/em\u003e by extending the lifespan of the host, as well inhibiting the colonization of the host by \u003cem\u003eB. nematocida\u003c/em\u003e B16, an opportunistic pathogen, which was previously proposed as a candidate biological control agent for nematodes (Huang et al., \u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e2005\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eHere, we report the results of a taxonomic study of strain CPCC 101271\u003csup\u003eT\u003c/sup\u003e, which we have proposed to be named \u003cem\u003eStenotrophomonas nematodicola\u003c/em\u003e sp. nov. The results of \u003cem\u003ein vitro\u003c/em\u003e and \u003cem\u003ein vivo\u003c/em\u003e experiments showed that \u003cem\u003eB. nematocida\u003c/em\u003e B16 can inhibit the growth of CPCC 101271\u003csup\u003eT\u003c/sup\u003e, while strain CPCC 101271\u003csup\u003eT\u003c/sup\u003e has the ability to inhibit the colonization of \u003cem\u003eC. elegans\u003c/em\u003e by B16. We also describe the variation in the microbiota community structure of \u003cem\u003eC. elegans\u003c/em\u003e during competition between strain CPCC 101271\u003csup\u003eT\u003c/sup\u003e and B16.\u003c/p\u003e "},{"header":"Materials And Methods","content":" \u003cp\u003eAcquisition of worms and bacterial strains\u003c/p\u003e \u003cp\u003eThe location for screening nematodes is Baotianman Natural Reserve (33\u0026deg; 27\u0026prime; 47\u0026prime;\u0026prime; N; 111\u0026deg; 48\u0026prime; 32\u0026prime;\u0026prime; E), Nanyang, China. Four soil samples were collected and approximately 1000 wild-living nematodes were isolated using the Baerman funnel technique (Gray, \u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e1984\u003c/span\u003e). Single worms were isolated and collected under a dissecting microscope. After washing three times with aseptic M9 buffer, single nematodes were frozen, ground and their crude DNA was extracted. The nematode species was identified by diagnostic PCR using the primer pair nlp30 diagnostic for \u003cem\u003eC. elegans\u003c/em\u003e (Petersen et al., \u003cspan citationid=\"CR43\" class=\"CitationRef\"\u003e2014\u003c/span\u003e). The cultivation, synchronization, collection and surface sterilization of \u003cem\u003eC. elegans\u003c/em\u003e worms were performed as previously described (Niu et al., \u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e2012\u003c/span\u003e; Niu et al.; \u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e2015\u003c/span\u003e; Niu et al., \u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e2016\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eStrain CPCC 101271\u003csup\u003eT\u003c/sup\u003e was isolated from the surface-sterilized \u003cem\u003eC. elegans\u003c/em\u003e worms, using Luria-Bertani (LB) agar plates. The nematodes were surface-sterilized by soaking in a solution of 1% mercuric chloride and 2% antibiotic mixture (streptomycin sulfate and gentamicin) for 1 h, and then cultured on nutrient and oligotrophic agar plates to confirm successful surface sterilization (0 cfu). The surface-sterilized worms were ground, then approximately 0.1 g of homogenate was suspended in 10 mL sterilized saline solution (containing 0.85% NaCl, w/v) and mixed thoroughly. Next, about 0.2 mL of suspension was spread onto an LB agar plate. After incubation at 30\u0026deg;C for 2 weeks, about 40 bacterial colonies were grown on the plate. According to the colony color and size, the colonies were randomly selected for separation and purification. Among which, a distinct pale yellowish colony was picked and transferred onto a newly prepared LB agar plate for further purification. The purified isolate of CPCC 101271\u003csup\u003eT\u003c/sup\u003e was maintained as a glycerol suspension (20%, v/v) at -80\u0026deg;C for long-term storage.\u003c/p\u003e \u003cp\u003eThe reference strains \u003cem\u003eStenotrophomonas rhizophila\u003c/em\u003e JCM 13333\u003csup\u003eT\u003c/sup\u003e and \u003cem\u003eS. bentonitica\u003c/em\u003e DSM 103927\u003csup\u003eT\u003c/sup\u003e were obtained from the Japan Collection of Microorganisms (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://jcm.brc.riken.jp/en/\u003c/span\u003e\u003c/span\u003e) and German Collection of Microorganisms and Cell Cultures (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://www.dsmz.de/collection/catalogue\u003c/span\u003e\u003c/span\u003e), respectively. \u003cem\u003eE. coli\u003c/em\u003e strain OP50 was obtained from the Laboratory for Conservation and Utilization of Bio-resources, Yunnan University. The opportunistic pathogen strain \u003cem\u003eB. nematocida\u003c/em\u003e B16 (=\u0026thinsp;GCMCC 1128) (Huang et al., \u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e2005\u003c/span\u003e) was obtained from the China General Microbiological Culture Collection Center (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttp://www.cgmcc.net\u003c/span\u003e\u003c/span\u003e). GFP-expressing strain B16g was constructed in our previous study (Niu et al., \u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e2012\u003c/span\u003e).\u003c/p\u003e \u003cp\u003e \u003cem\u003eC. elegans\u003c/em\u003e lifespan assay\u003c/p\u003e \u003cp\u003eWorms \u003cem\u003eC. elegans\u003c/em\u003e were maintained on NGM (Nematode Growth Medium) plates at 25\u0026deg;C. The strains CPCC 101271\u003csup\u003eT\u003c/sup\u003e, JCM 13333\u003csup\u003eT\u003c/sup\u003e and OP50 used for measuring the worms\u0026rsquo; lifespan were recovered from the 20% glycerol stock and were streaked onto LB agar plates and then incubated at 32\u0026deg;C. A single colony was picked and incubated in 5 ml of LB at 32\u0026deg;C overnight. One milliliter of the overnight culture was added to 100 ml of LB medium and shaken at 32\u0026deg;C until an OD\u003csub\u003e600\u003c/sub\u003e of 0.8 was reached. 200 \u0026micro;l of the tested bacterial culture was seeded on NGM plate, and then synchronized L4 larvae were transferred to the corresponding bacterial seeded NGM plate (Park et al., 2017). The lifespan experiment was monitored by scoring the dead worms every 10 hours from 50-h until 160-h. Worms that did not respond to prodding with a platinum wire were considered dead. Those desiccated by crawling onto the edge of the housing plate were excluded from the analysis. The experiments were performed with five replicates at three different time intervals.\u003c/p\u003e \u003cp\u003eTaxonomic study of strain CPCC 101271\u003csup\u003eT\u003c/sup\u003e\u003c/p\u003e \u003cp\u003eThe taxonomic position of the new isolate was studied using the polyphasic taxonomy approach. (i) Examination of cell morphology and physiological characteristics. The Gram-staining reaction was performed according to Magee et al. (\u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e1975\u003c/span\u003e). Cell morphology was observed using a light microscope, and mobility was observed by inoculating cells into nutrient broth with 0.3 % (w/v) agar and incubating at 30\u0026deg;C for 7 days. Growth conditions and the physiological characteristics of the isolate were tested using methods described previously (Yuan et al., \u003cspan citationid=\"CR54\" class=\"CitationRef\"\u003e2008\u003c/span\u003e). Antibiotic resistance was tested on LB agar plates using discs containing the following antibiotics: ampicillin (10 \u0026micro;g), cefaclor (30 \u0026micro;g), chloramphenicol (30 \u0026micro;g), clindamycin (2 \u0026micro;g), erythromycin (15 \u0026micro;g), gentamycin (10 \u0026micro;g), kanamycin (30 \u0026micro;g), netilmicin (30 \u0026micro;g), novobiocin (5 \u0026micro;g), penicillin (10 IU), polymyxinB (300 IU), rifampin (5 \u0026micro;g), vancomycin (30 \u0026micro;g), tetracycline (30 \u0026micro;g), tobramycin (10 \u0026micro;g) and treptomycin (10 \u0026micro;g). (ii) Chemotaxonomic study. Cell mass for chemotaxonomic and molecular systematic studies of the strain was collected from TSA plates cultured at 30\u0026deg;C for 5 days. The polar lipids were extracted and isolated by two-dimensional TLC and identified according to the procedures previously described by Minnikin et al. (\u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e1984\u003c/span\u003e). Ubiquinones were isolated and purified as described previously (Collins et al., \u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e1997\u003c/span\u003e) and analyzed using HPLC. Cellular fatty acids were extracted, methylated and identified using the Sherlock Microbial Identification System (MIDI) according to the manufacturer\u0026rsquo;s instructions (Kroppenstedt, \u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e1985\u003c/span\u003e). The MIDI Sherlock Version 6.0 database and the TSBA 6 method were used for analysis. (iii) Genomic analysis. DNA preparation and sequencing of PCR amplification products of the 16S rRNA gene were performed as described by Li et al. (\u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e2007\u003c/span\u003e). The obtained sequence was compared with available 16S rRNA gene sequences from GenBank using the BLAST program and EzBioCloud (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttp://www.ezbiocloud.net/\u003c/span\u003e\u003c/span\u003e) to determine the approximate phylogenetic affiliation of strain CPCC 101271\u003csup\u003eT\u003c/sup\u003e (Kim \u0026amp; Mylonakis, \u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e2012\u003c/span\u003e). Multiple alignments with sequences of the most closely related taxa and calculations of sequence similarity were carried out using MEGA version 7.0 (Kumar et al., \u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e2016\u003c/span\u003e). Phylogenetic trees were inferred using the neighbor-joining method (Saitou \u0026amp; Nei, \u003cspan citationid=\"CR46\" class=\"CitationRef\"\u003e1987\u003c/span\u003e) with K\u003csub\u003enuc\u003c/sub\u003e values (Kimura \u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e1980\u003c/span\u003e \u0026amp; \u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e1983\u003c/span\u003e) and complete deletion gaps. Phylogenetic trees were also constructed using and the maximum-parsimony (Kluge \u0026amp; Farris, \u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e1969\u003c/span\u003e) and maximum-likelihood (Felsenstein, \u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e1981\u003c/span\u003e) methods. The topology of the phylogenetic tree was evaluated by the bootstrap resampling method of Felsenstein (\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e1985\u003c/span\u003e) with 1000 replicates. Genome sequencing of strain CPCC 101271\u003csup\u003eT\u003c/sup\u003e and the reference strain \u003cem\u003eS. bentonitica\u003c/em\u003e DSM 103927\u003csup\u003eT\u003c/sup\u003e was performed on an Illumina MiSeq instrument (Illumina); the reads were assembled using Platanus software (v1.2.4) and contigs shorter than 500 bp in size were discarded. The genomic G\u0026thinsp;+\u0026thinsp;C content was calculated from the draft genome sequence. The values of average nucleotide identity (ANI) between strain CPCC 101271\u003csup\u003eT\u003c/sup\u003e and the reference strain \u003cem\u003eS. rhizophila\u003c/em\u003e JCM 13333\u003csup\u003eT\u003c/sup\u003e and two other type strains were calculated by comparing their draft genomes (Yoon et al., \u003cspan citationid=\"CR53\" class=\"CitationRef\"\u003e2017\u003c/span\u003e).\u003c/p\u003e \u003cp\u003e \u003cem\u003eIn vitro\u003c/em\u003e bacteriostatic activity test\u003c/p\u003e \u003cp\u003eEach bacterial strain was separately inoculated into 5 mL of LB medium and cultured in a shaker at 32\u0026deg;C, 180 r/min for 12 h. Then the culture broth was adjusted to an optical density value at 600 nm (OD\u003csub\u003e600\u003c/sub\u003e) of 1. Approximately 0.3 mL of CPCC 101271\u003csup\u003eT\u003c/sup\u003e culture broth was evenly spread onto an LB agar plate. Sterilized filter paper with a diameter of 5 mm was immersed in the bacterial culture for 5 min and placed onto the agar plate containing strain CPCC 101271\u003csup\u003eT\u003c/sup\u003e. The plate was then incubated at 32\u0026deg;C for 48 h, and the size of the inhibition zone for each sample was recorded. \u003cem\u003eE. coli\u003c/em\u003e culture broth and LB medium were used as negative controls, while polymyxin B (300 IU) and rifampin (5 \u0026micro;g) were used as positive controls. The experiments were performed with three parallels and repeated thrice.\u003c/p\u003e \u003cp\u003eColonization capability assay\u003c/p\u003e \u003cp\u003eColonization capability was assayed using approximate fifty 1-day-old adult hermaphrodite worms were placed on each plate at 25\u0026deg;C following the procedures described by Aballay et al. (\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e2000\u003c/span\u003e) and Niu et al. (\u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e2012\u003c/span\u003e) with modifications. In \u0026lsquo;Feeding Transfer\u0026rsquo; experiments, the worms were transferred by hair and repeated washed using sterilized NaCl solution (0.85 %, w/v). Three nematode treatment groups were set up. In the first group of nematodes pre-fed with CPCC 101271\u003csup\u003eT\u003c/sup\u003e then infected by B16g, the worms were transferred onto LB plates containing a low concentration (10\u003csup\u003e6\u003c/sup\u003e cells/mL) of CPCC 101271\u003csup\u003eT\u003c/sup\u003e and co-cultivated for 4 h. The worms were then removed from the plates, washed twice, transferred to plates containing B16g and co-cultivated for 72 h. In the second group of nematodes pre-fed with JCM13333\u003csup\u003eT\u003c/sup\u003e then infected by B16g, the worms were first seeded on an LB agar plate containing JCM13333\u003csup\u003eT\u003c/sup\u003e (10\u003csup\u003e6\u003c/sup\u003e cells/mL) and cultivated for 4 h, and then the worms were transferred to plates containing B16g and cultivated for 72 h. In the third group, the worms were first fed on the same concentration of OP50 for 4 h, then transferred to B16g plates and cultivated for 72 h. The control group of nematodes without being pre-fed with bacteria were directly seeded on blank medium and then cultivated for 4 h before being transferred to B16g plates, which were also defined as B16 direct infection group. The colonization process was observed under a Nikon 800 Eclipse microscope (Nikon Corp., Japan) equipped for epifluorescence with a mercury lamp and an excitation filter of 450\u0026ndash;490 nm (blue light) and a barrier filter of 515 nm. At each time point, three sets of 10 nematodes were randomly selected to evaluate colonization. The worms with fluorescent bacteria in the entire lumen were scored as full; worms without any green fluorescence signal in the lumen were scored as undetected; and worms between these two extremes were scored as partial. The worms were considered dead when no movement was observed under a light-dissecting microscope, and when gently tapping of nematodes by a platinum wire, no movement occurred. Dead nematodes whose bodies were decomposed were excluded from the analysis. The number of worms killed in each group was counted every 12 h during within 72 hours from B16 infection. Mortality rates of B16-infected nematodes were defined as the ratio of dead nematodes to tested nematodes. The experiments were performed with thrice.\u003c/p\u003e \u003cp\u003eDNA preparation and metagenomic analysis of microbiota\u003c/p\u003e \u003cp\u003eThe tested nematodes were divided into five groups as follows: (I) CW00h group, which was pre-fed with CPCC 101271\u003csup\u003eT\u003c/sup\u003e for 4 h; (II-V) CW04h, CW08h, CW12h and CW16h groups, which were separately co-cultivated with B16 for 4 h, 8 h, 12 h and 16 h, respectively, after being pre-fed with CPCC 101271\u003csup\u003eT\u003c/sup\u003e for 4 h.\u003c/p\u003e \u003cp\u003eThe worms were collected and then washed and surface sterilized as described above. Total DNA was extracted from the intestinal microbes using the PowerSoil DNA Isolation Kit (MoBio, USA) according to the manufacturer\u0026rsquo;s protocols. The concentration and purity of extracted DNA were determined using a TBS-380 and NanoDrop2000, respectively. The quality of the extracted DNA was evaluated on a 1% agarose gel. DNA was fragmented to an average size of about 300 bp using a Covaris M220 (Gene Company Limited, China) for paired-end library construction. The paired-end library was constructed using NEXTFLEX Rapid DNA-Seq (Bioo Scientific, Austin, TX, USA). Adapters containing the full complement of sequencing primer hybridization sites were ligated to the blunt ends of the fragments. Paired-end sequencing was performed on an Illumina NovaSeq (Illumina Inc., San Diego, CA, USA) at Majorbio Bio-Pharm Technology Co., Ltd. (Shanghai, China) using NovaSeq Reagent Kits according to the manufacturer\u0026rsquo;s instructions (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e\u003ca href=\"https://jcm.brc.riken.jp/en/\" target=\"_blank\"\u003ewww.illumina.com\u003c/a\u003e\u003c/span\u003e\u003c/span\u003e). Adapter sequences were stripped from the 3' and 5' ends of paired-end Illumina reads using SeqPrep (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://github.com/jstjohn/SeqPrep\u003c/span\u003e\u003c/span\u003e). Low-quality reads (length\u0026thinsp;\u0026lt;\u0026thinsp;50 bp, a quality value\u0026thinsp;\u0026lt;\u0026thinsp;20, or containing N bases) were removed using Sickle (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://github.com/najoshi/sickle\u003c/span\u003e\u003c/span\u003e\u003cspan type=\"Underline\" class=\"Underline\" name=\"Emphasis\"\u003e).\u003c/span\u003e\u003c/p\u003e \u003cp\u003eMetagenomics data were assembled using MEGAHIT (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://github.com/voutcn/megahit\u003c/span\u003e\u003c/span\u003e\u003cspan type=\"Underline\" class=\"Underline\" name=\"Emphasis\"\u003e)\u003c/span\u003e (Li et al., \u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e2015\u003c/span\u003e), which makes use of succinct de Bruijn graphs. Contigs with a length\u0026thinsp;\u0026ge;\u0026thinsp;300 bp were selected as final assemblies and were used for further gene prediction and annotation (Noguchi et al., \u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e2006\u003c/span\u003e; Li et al., \u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e2008\u003c/span\u003e).\u003c/p\u003e "},{"header":"Results","content":"\u003cp\u003e\u003cstrong\u003eThe isolation and identification of strain CPCC 101271\u003c/strong\u003e \u003csup\u003e \u003cstrong\u003eT\u003c/strong\u003e \u003c/sup\u003e, \u003cstrong\u003ewhich represents a novel species of the genus\u003c/strong\u003e \u003cspan class=\"BoldItalic\"\u003eStenotrophomonas\u003c/span\u003e\u003c/p\u003e\n\u003cp\u003eStrain CPCC 101271\u003csup\u003eT\u003c/sup\u003e was recovered from the intestinal lumen of \u003cem\u003eC. elegans\u003c/em\u003e using LB agar plates (see Materials and Methods). The nearly full-length sequence of the 16S rRNA gene (1543 bp) of strain CPCC 101271\u003csup\u003eT\u003c/sup\u003e was obtained and submitted to GenBank under accession number MT126327. A BLAST search of GenBank showed that the 16S rRNA gene of strain CPCC 101271\u003csup\u003eT\u003c/sup\u003e exhibited 98.1%-99.7% similarity to the 16S rRNA genes of members of the genus \u003cem\u003eStenotrophomonas\u003c/em\u003e, and \u0026lt;\u0026thinsp;98.0% similarities to those of other bacterial species in the family \u003cem\u003eLysobacteraceae\u003c/em\u003e. In a phylogenetic tree based on the 16S rRNA gene sequences of all members of the family \u003cem\u003eLysobacteraceae\u003c/em\u003e, strain CPCC 101271\u003csup\u003eT\u003c/sup\u003e formed a sublineage with \u003cem\u003eS. rhizophila\u003c/em\u003e JCM 13333\u003csup\u003eT\u003c/sup\u003e and \u003cem\u003eS. bentonitica\u003c/em\u003e DSM 103927\u003csup\u003eT\u003c/sup\u003e within the genus \u003cem\u003eStenotrophomonas\u003c/em\u003e (Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e1\u003c/span\u003e). Therefore, it is reasonable to designate strain CPCC 101271\u003csup\u003eT\u003c/sup\u003e as a member of the genus \u003cem\u003eStenotrophomonas\u003c/em\u003e.\u003c/p\u003e\n\u003cp\u003eGenome sequencing of strain CPCC 101271\u003csup\u003eT\u003c/sup\u003e yielded a draft genome of 4,402,751 bp, assembled from 126 qualified contigs, with 100-fold coverage and an N50 length of 738,821 bp. Genes putatively encoding glucosylglycerol-phosphate synthase (G9274_RS14805, B861_RS0201980, E5352_RS0097) and alpha-trehalose-phosphate synthase (BN96_RS08035, BIZ42_RS05395, C0R07_RS03270, GDJ08_RS08470), which might endow strain CPCC 101271\u003csup\u003eT\u003c/sup\u003e the ability to maintain homeostasis of the host, were identified in its genome. The ANI values between the draft genomes of strain CPCC 101271\u003csup\u003eT\u003c/sup\u003e and its closest phylogenetic neighbors \u003cem\u003eS. rhizophila\u003c/em\u003e JCM 13333\u003csup\u003eT\u003c/sup\u003e and \u003cem\u003eS. bentonitica\u003c/em\u003e DSM 103927\u003csup\u003eT\u003c/sup\u003e were 84.7 % and 85.0 %, respectively. These values were both far lower than 95%, which was proposed as the cutoff value for defining different species (Kim et al., \u003cspan class=\"CitationRef\"\u003e2014\u003c/span\u003e). Accordingly, strain CPCC 101271\u003csup\u003eT\u003c/sup\u003e represents a species genetically different from other validly described species of the genus \u003cem\u003eStenotrophomonas\u003c/em\u003e. This conclusion was supported by chemotaxonomic data (described below) and the phenotypic characteristics given in the species description and in Supplementary Table S1.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eDescription of\u003c/strong\u003e \u003cspan class=\"BoldItalic\"\u003eStenotrophomonas nematodicola\u003c/span\u003e \u003cstrong\u003esp. nov.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cem\u003eStenotrophomonas nematodicola\u003c/em\u003e (ne.ma.to.di'co.la. N.L. pl. n. \u003cem\u003eNematoda\u003c/em\u003e, a taxonomic group of animals; L. suff. -\u003cem\u003ecola\u003c/em\u003e, inhabitant, dweller; N.L. fem. n. \u003cem\u003enematodicola\u003c/em\u003e, an inhabitant of nematodes).\u003c/p\u003e\n\u003cp\u003eCells are Gram-reaction-negative, facultatively aerobic and motile coccoid rods, 1.0-1.2 \u0026micro;m in width and 1.9\u0026ndash;2.3 \u0026micro;m in length. Colonies on LB agar medium are smooth, pale yellow in color, and circular and entire, with a diameter of 1.0-1.1 mm after 48 h of incubation. Growth occurs at 10\u0026ndash;37\u0026deg;C (optimum 32\u0026deg;C) and at pH 6.0\u0026ndash;8.0 (optimum pH 7.0) with 0\u0026ndash;5 % (w/v) NaCl (optimum 0\u0026ndash;1 %). Catalase- and oxidase reactions are positive. Positive for hydrolysis of gelatin and nitrate reduction reaction, while negative for hydrolysis of starch and urea, peptonization of milk, and production of H\u003csub\u003e2\u003c/sub\u003eS and indol. Positive for acid phosphatase, alkaline phosphatase, \u0026alpha;-chymotrypsin, esterase (C4), esterase lipase (C8), cystine arylamidase, \u0026beta;-glucosidase, leucine arylamidase and valine arylamidase in an API ZYM strip. Acetic acid, acetoacetic acid, bromo-succinic acid, citric acid, dextrin, D-fructose-6-PO\u003csub\u003e4\u003c/sub\u003e, D-lactic acid methyl ester, D-maltose, D-mannose, glycyl-L-proline, L-alanine, L-glutamic acid, L-histidine, methyl pyruvate, L-lactic acid, L-malic acid, N-acetyl-D-galactosamine, N-acetyl-D-glucosamine, pectin, propionic acid, Tween 40, \u0026alpha;-D-Glucose and \u0026alpha;-Keto-glutaric acid can be utilized as the sole carbon source, and amygdalin, arbutin, D-fructose, D-glucose, D-maltose, esculin ferric citrate, N-acetylglucosamine, potassium 2-ketogluconate and potassium 5-ketogluconate can be assimilized and produce acid. Resistant to ampicillin (10 \u0026micro;g), cefaclor (30 \u0026micro;g), chloramphenicol (30 \u0026micro;g), clindamycin (2 \u0026micro;g), erythromycin (15 \u0026micro;g), gentamycin (10 \u0026micro;g), kanamycin (30 \u0026micro;g), netilmicin (30 \u0026micro;g), novobiocin (5 \u0026micro;g), penicillin (10 IU), vancomycin (30 \u0026micro;g), tetracycline (30 \u0026micro;g), tobramycin (10 \u0026micro;g) and treptomycin (10 \u0026micro;g), while sensitive to polymyxinB (300 IU) and rifampin (5 \u0026micro;g). Diphosphatidylglycerol (DPG), phosphatidylglycerol (PG), phosphatidylethanolamine (PE) and an unidentified phospholipid (UPL) were detected in a polar lipids extract (Figure S1). The respiratory quinone is Q-8. The major fatty acids are iso-C\u003csub\u003e15:0\u003c/sub\u003e (38.2%) and antesio-C\u003csub\u003e15:0\u003c/sub\u003e (16.6%), with moderate amounts of iso-C\u003csub\u003e11:0\u003c/sub\u003e (8.7%) and C\u003csub\u003e16:0\u003c/sub\u003e (5.1%) and small amounts (\u0026lt;\u0026thinsp;5%) of cyclo-C\u003csub\u003e17:0\u003c/sub\u003e, C\u003csub\u003e13:0\u003c/sub\u003e2-OH, C\u003csub\u003e12:0\u003c/sub\u003e3-OH, iso-C\u003csub\u003e17:0\u003c/sub\u003e, iso-C\u003csub\u003e14:0\u003c/sub\u003e, C\u003csub\u003e14:0\u003c/sub\u003e, iso-C\u003csub\u003e11:0\u003c/sub\u003e 3-OH, iso-C\u003csub\u003e16:0\u003c/sub\u003e, iso-C\u003csub\u003e13:0\u003c/sub\u003e3-OH, C\u003csub\u003e16:1\u003c/sub\u003e \u003cem\u003e\u0026omega;\u003c/em\u003e7\u003cem\u003ec\u003c/em\u003e/C\u003csub\u003e16:1\u003c/sub\u003e \u003cem\u003e\u0026omega;\u003c/em\u003e6\u003cem\u003ec\u003c/em\u003e, and iso -C\u003csub\u003e17:1\u003c/sub\u003e \u003cem\u003e\u0026omega;\u003c/em\u003e9\u003cem\u003ec\u003c/em\u003e/C\u003csub\u003e16:0\u003c/sub\u003e 10-methyl.\u003c/p\u003e\n\u003cp\u003eThe type strain CPCC 101271\u003csup\u003eT\u003c/sup\u003e (=\u0026thinsp;W5) was isolated from a surface-sterilized \u003cem\u003eC. elegans\u003c/em\u003e worm cultured in a lab in Nanyang, a city in middle of China. The DDBJ/EMBL/GenBank accession numbers of the 16S rRNA gene sequence and draft genome sequence of strain CPCC 101271\u003csup\u003eT\u003c/sup\u003e are MT126327 and WIAY00000000, respectively. The genome of the type strain is characterized by a size of 4.4 Mbp and a G\u0026thinsp;+\u0026thinsp;C content of 67.3 mol%.\u003c/p\u003e\n\u003cp\u003eA filled circle indicates that the node was also recovered in trees generated with the maximum-likelihood method and maximum-parsimony method. Bootstrap values are shown as the percentage of 1,000 replicates; only percentages above 50% are shown.\u003c/p\u003e\n\u003cp\u003e\u003cem\u003eEscherichia coli\u003c/em\u003e JCM 1649\u003csup\u003eT\u003c/sup\u003e (GenBank accession no. X80725) was used as an outgroup (not shown). Bar, 0.005 substitutions per nucleotide position.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eIncrease in\u003c/strong\u003e \u003cspan class=\"BoldItalic\"\u003eCaenorhabditis elegans\u003c/span\u003e \u003cstrong\u003esurvival rate and lifespan by feeding on CPCC 101271\u003c/strong\u003e\u003csup\u003e\u003cstrong\u003eT\u003c/strong\u003e\u003c/sup\u003e\u003c/p\u003e\n\u003cp\u003eWe compared the longevity of worms fed either on CPCC 101271\u003csup\u003eT\u003c/sup\u003e, JCM 13333\u003csup\u003eT\u003c/sup\u003e or OP50. The results showed that worms fed on JCM 13333\u003csup\u003eT\u003c/sup\u003e or OP50 had almost the similar lifespans. However, worms fed on CPCC 101271\u003csup\u003eT\u003c/sup\u003e lived approximately 40% longer than worms fed on \u003cem\u003eE. coli\u003c/em\u003e or JCM 13333\u003csup\u003eT\u003c/sup\u003e, indicating that the nematodes fed on CPCC 101271\u003csup\u003eT\u003c/sup\u003e lived longer than those fed on \u003cem\u003eE. coli\u003c/em\u003e OP50 or \u003cem\u003eS. rhizophila\u003c/em\u003e JCM 13333\u003csup\u003eT\u003c/sup\u003e and had greatly increased survival rates (Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e2\u003c/span\u003e)\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eStrain CPCC 101271\u003csup\u003eT\u003c/sup\u003e confers the host with resistance to \u003cem\u003eB. nematocida\u003c/em\u003e colonization\u003c/p\u003e\n\u003cp\u003eTo investigate whether strain CPCC 101271\u003csup\u003eT\u003c/sup\u003e has colonization-resistance activity against \u003cem\u003eB. nematocida\u003c/em\u003e B16, we first performed an \u003cem\u003ein vitro\u003c/em\u003e bacteriostatic activity test. The results showed that strain CPCC 101271\u003csup\u003eT\u003c/sup\u003e could not inhibit \u003cem\u003eB. nematocida\u003c/em\u003e B16 but could be inhibited by B16 (Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e3\u003c/span\u003e). A transparent inhibition zone with clear edges formed around the paper containing B16 after 48 h of incubation on an LB agar plate spread with strain CPCC 101271\u003csup\u003eT\u003c/sup\u003e. The clear zone, which was about 1.6 cm in diameter, was slightly smaller than the zones surrounding the positive drug controls polymyxin B (300 IU) and rifampin (5 \u0026micro;g). And no clear inhibition zone formed around the papers containing \u003cem\u003eE. coli\u003c/em\u003e or LB medium (Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e3\u003c/span\u003e).\u003c/p\u003e\n\u003cp\u003eGFP-expressing strain B16g was used to confirm the specificity of B16 colonization activity assays. The results of colonization-resistance activities indicated that strain CPCC 101271\u003csup\u003eT\u003c/sup\u003e could also inhibit the colonization of B16 in the nematode intestine. During the first 24 h of infection with \u003cem\u003eB. nematocida\u003c/em\u003e B16, almost no nematodes pre-fed with CPCC 101271\u003csup\u003eT\u003c/sup\u003e were scored as being in the \u0026ldquo;full\u0026rdquo; colonization category (see Materials and Methods). By contrast, almost 20% of animals directly fed with \u003cem\u003eB. nematocida\u003c/em\u003e B16 were scored as \u0026ldquo;full\u0026rdquo;. In addition, after infection for 48 h, 50% of the animals fed only \u003cem\u003eB. nematocida\u003c/em\u003e B16 were scored as \u0026ldquo;full\u0026rdquo;. However, only 10% of worms pre-fed with CPCC 101271\u003csup\u003eT\u003c/sup\u003e were scored as \u0026ldquo;full\u0026rdquo; at the same time point. After 72 h, \u003cem\u003eB. nematocida\u003c/em\u003e B16 showed notably strong colonization ability, with 90% of the worms not pre-fed with CPCC 101271\u003csup\u003eT\u003c/sup\u003e scored as \u0026ldquo;full\u0026rdquo;. In contrast, only 10% of the animals pre-fed with CPCC 101271\u003csup\u003eT\u003c/sup\u003e were scored as \u0026ldquo;full\u0026rdquo;. Moreover, compared with worms fed only B16, the percentage of worms fed with both B16 and CPCC 101271\u003csup\u003eT\u003c/sup\u003e that had undetectable B16 colonization was much higher at 48 h (10% vs. 80%) and at 72 h (0% vs. 70%). The worms pre-fed with \u003cem\u003eE. coli\u003c/em\u003e showed little difference compared with those in the no pre-feeding group. The ability of B16 to colonize the nematodes pre-fed with JCM 13333\u003csup\u003eT\u003c/sup\u003e was stronger than its ability to colonize those pre-fed with CPCC 101271\u003csup\u003eT\u003c/sup\u003e, but a little weaker than its ability to colonize the negative controls pre-fed with \u003cem\u003eE. coli\u003c/em\u003e OP50. Differences between the abilities of B16 to colonize the nematodes pre-fed with CPCC 101271\u003csup\u003eT\u003c/sup\u003e and pre-fed with OP50 were notable when we compared the changes in the severity of colonization at 72-h (Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e4\u003c/span\u003e, chi-squared test, \u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.0001). For example, only 10% of worms that were pre-fed with CPCC 101271\u003csup\u003eT\u003c/sup\u003e could be categorized as having \u0026lsquo;full\u0026rsquo; colonization. However, 90% of worms that were pre-fed with OP50 were categorized in the \u0026lsquo;full\u0026rsquo; colonization category. The results indicated that colonization of \u003cem\u003eB. nematocida\u003c/em\u003e B16 was markedly attenuated in \u003cem\u003eC. elegans\u003c/em\u003e pre-fed with CPCC 101271\u003csup\u003eT\u003c/sup\u003e.\u003c/p\u003e\n\u003cp\u003eThe differences in the mortalities of the B16-infected nematodes in the different treatment groups indicated that pre-feeding with CPCC 101271\u003csup\u003eT\u003c/sup\u003e reduced the mortality caused by infection with the pathogenic bacteria B16 (Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e5\u003c/span\u003e). The mortalities of the nematodes pre-fed with OP50 and then infected by B16 and the nematodes directly infected by B16 (without pre-feeding with any other bacteria) were 85 and 90% within 60 h, respectively. By contrast, for worms pre-fed with CPCC 101271\u003csup\u003eT\u003c/sup\u003e then infected with B16, the mortality dropped to 40%. The natural mortality rate of the negative control nematodes (no pre-feeding or B16 infection) was only 18%. At other time points, the mortalities of nematodes pre-fed with CPCC 101271\u003csup\u003eT\u003c/sup\u003e were significantly lower than those of nematodes pre-fed with \u003cem\u003eE\u003c/em\u003e. \u003cem\u003ecoli\u003c/em\u003e or directly infected with B16.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eThe variation in\u003c/strong\u003e \u003cspan class=\"BoldItalic\"\u003eCaenorhabditis elegans\u003c/span\u003e \u003cstrong\u003emicrobiota community structure during competition between CPCC 101271\u003c/strong\u003e\u003csup\u003e\u003cstrong\u003eT\u003c/strong\u003e\u003c/sup\u003e \u003cstrong\u003eand B16\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eIn a previous study, we collected free-living terrestrial \u003cem\u003eC. elegans\u003c/em\u003e from soil and rotten fruits, and analyzed the variation in intestinal flora following \u003cem\u003eB. nematocida\u003c/em\u003e B16 infection by performing macrogenomic analysis. We found significant differences in the diversity and distribution of microbiota between the control worms and those infected with B16 for 24 h. The diversity of the intestinal microbiome decreased after B16 infection (Niu et al., \u003cspan class=\"CitationRef\"\u003e2016\u003c/span\u003e). Here we aimed to investigate the variation in the intestinal bacterial community structure of \u003cem\u003eC. elegans\u003c/em\u003e during competition between CPCC 101271\u003csup\u003eT\u003c/sup\u003e and B16.\u003c/p\u003e\n\u003cp\u003eA total of 332314, 280966, 705161, 227126 and 597664 sequences comprising 44, 26, 49, 19 and 30 operational taxonomic units (OTUs) were obtained from the five groups CW00h, CW04h, CW08h, CW12h and CW16h, respectively. At the genus level, these OTUs represented 26, 14, 23, 10 and 14 genera, respectively. It was obvious that the bacteria diversity was greatly decreased during competition between CPCC 101271\u003csup\u003eT\u003c/sup\u003e and B16 (Fig.\u0026nbsp;6, Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e7\u003c/span\u003e). At the first stage of infection (CW00h) in nematodes pre-fed with strain CPCC 101271\u003csup\u003eT\u003c/sup\u003e, the microbiota community structure predominantly consisted of the genera \u003cem\u003eBacillus\u003c/em\u003e, \u003cem\u003eAcetobacter\u003c/em\u003e, \u003cem\u003eLactobacillus\u003c/em\u003e, \u003cem\u003ePhytobacter, Stenotrophomonas, Pichia\u003c/em\u003e and \u003cem\u003eSphingomonas\u003c/em\u003e. At the second stage (CW04h), 4 h after the worms were infected by B16, dysbiosis occurred, and in the course of re-construction of the microbiota community, the bacteria diversity was drastically reduced. Besides \u003cem\u003eLactobacillus\u003c/em\u003e spp., \u003cem\u003eAcetobacter\u003c/em\u003e spp. and \u003cem\u003ePichia\u003c/em\u003e spp., which remained the major groups, the abundance of \u003cem\u003eBacillus\u003c/em\u003e spp. increased slightly and the abundance of \u003cem\u003eStenotrophomonas\u003c/em\u003e spp. increased. At the third stage (CW08h), which we termed \u0026ldquo;the breaking period\u0026rdquo;, a large number of CPCC 101271\u003csup\u003eT\u003c/sup\u003e and B16 bacteria were co-existing and competing; the diversity of the intestinal flora had partially recovered, but the abundance of \u003cem\u003eBacillus\u003c/em\u003e spp. had greatly decreased. At the fourth stage (CW12h), the abundance of \u003cem\u003eBacillus\u003c/em\u003e spp. was even lower and the abundance of \u003cem\u003eStenotrophomonas\u003c/em\u003e spp. was higher. The newly reconstructed intestinal flora was disrupted again, and the species composition was the most similar to that observed at the second stage (Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e7\u003c/span\u003e). By the fifth stage (CW16h), B16 overwhelmed CPCC 101271\u003csup\u003eT\u003c/sup\u003e and only \u003cem\u003eLactobacillus\u003c/em\u003e spp. and \u003cem\u003ePichia\u003c/em\u003e spp., together with \u003cem\u003eBacillus\u003c/em\u003e spp., remained the major microbiota.\u003c/p\u003e\n\u003cp\u003eIn a summary, over the course of B16 infection, the abundance of CPCC 101271\u003csup\u003eT\u003c/sup\u003e and its relatives (\u003cem\u003eStenotrophomonas\u003c/em\u003e spp.) kept on increasing until 8 h after B16 infection and then decreased sharply. The change in abundance of \u003cem\u003eStenotrophomonas\u003c/em\u003e spp. was accompanied by similar changes in the abundance of alpha-trehalose-phosphate synthase-encoding genes calculated from the metagenome data, except at the final stage (Figure S2). By contrast, the abundance of \u003cem\u003eBacillus\u003c/em\u003e spp. first decreased, then increased rapidly, which was similar to the changes in the abundance of trehalose-6-phosphate hydrolase-encoding genes (Figure S3). The abundances of alpha-trehalose-phosphate synthase-encoding genes (present in the genome of B16) and trehalose-6-phosphate hydrolase-encoding genes (present in the genome of CPCC 101271\u003csup\u003eT\u003c/sup\u003e) together with \u003cem\u003eStenotrophomonas\u003c/em\u003e spp. and \u003cem\u003eBacillus\u003c/em\u003e spp. reached the highest level at 8 h (CW08h) after B16 infection. The results suggested that strain CPCC 101271\u003csup\u003eT\u003c/sup\u003e possibly participated directly or induced some other bacteria in the community to participate in resistance to B16 colonization. However, by 16 h after infection, B16 dominated the microbiota community, and the growth of CPCC 101271\u003csup\u003eT\u003c/sup\u003e was completely suppressed.\u0026nbsp;\u003c/p\u003e"},{"header":"Discussion","content":" \u003cp\u003eGut microbiota, diverse microorganisms inhabiting the digestive track, are tightly linked to the health of their host. The community of microbial species, among which bacteria are predominant and have been extensively studied, not only generates metabolites essential for various host functions but also confers resistance to exogenous pathogens (Lee \u0026amp; Hase, \u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e2014\u003c/span\u003e). However, the molecular mechanisms by which microbiota resist pathogens and the changes in bacterial composition that occur after pathogen infection remains elusive.\u003c/p\u003e \u003cp\u003eIt has been reported that bacteria living in most nematodes play an important role in the growth and development, physiological metabolism and immune regulation of the host. The bacteria \u003cem\u003eXenorhabdus\u003c/em\u003e spp. and \u003cem\u003ePhotorhabdus\u003c/em\u003e spp. were reported to be symbionts found in the guts of nematodes including \u003cem\u003eSteinernema\u003c/em\u003e, \u003cem\u003eHeteronrhahditis\u003c/em\u003e, \u003cem\u003eHeterodera\u003c/em\u003e and \u003cem\u003eRhabditis.\u003c/em\u003e They produce antibiotics, intracellular protein crystals, and numerous other products that help nematodes kill insects and also provide nutrients (Forst et al., \u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e1997\u003c/span\u003e; Park et al., \u003cspan citationid=\"CR42\" class=\"CitationRef\"\u003e2011\u003c/span\u003e; Whittaker et al., \u003cspan citationid=\"CR52\" class=\"CitationRef\"\u003e2016\u003c/span\u003e; Shan et al., \u003cspan citationid=\"CR48\" class=\"CitationRef\"\u003e2019\u003c/span\u003e). Bacteria associated with cysts of the soybean cyst nematode play an important ecological role in the long-term survival of cysts in soil (Nour et al., \u003cspan citationid=\"CR40\" class=\"CitationRef\"\u003e2003\u003c/span\u003e). An endo-symbiotic bacterium in a plant-parasitic nematode was found to be closely associated with the growth and metabolism of its host (Haegeman et al., \u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e2009\u003c/span\u003e). Therefore, to some extent, nematodes are typical symbioses with their microbiota.\u003c/p\u003e \u003cp\u003eFor nematodes \u003cem\u003eC. elegans\u003c/em\u003e, most of the research work was carried out based on the N2 strain, which has been adapted to laboratory conditions over decades (Sterken et al., \u003cspan citationid=\"CR49\" class=\"CitationRef\"\u003e2015\u003c/span\u003e), including the regular and routine removal of any microbes through hypochlorite treatment. Thus N2 strain does not carry any microbes in its gut and microbiome associations are little known to the nematode \u003cem\u003eC. elegans\u003c/em\u003e N2 under laboratory conditions. In contrast, worms in nature are exposed to complex microbial communities. Understanding the worm\u0026rsquo;s natural microbiome is essential to help explain their realistic and unbiased characteristics. In fact, more and more researchers have paid their attention to the natural \u003cem\u003eC. elegans\u003c/em\u003e microbiome (Dirksen et al., \u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e2016\u003c/span\u003e; Samuel et al., \u003cspan citationid=\"CR47\" class=\"CitationRef\"\u003e2016\u003c/span\u003e; Zhang et al., \u003cspan citationid=\"CR55\" class=\"CitationRef\"\u003e2017\u003c/span\u003e). A possible fitness benefit was already indicated upon gut colonization with certain non-pathogenic bacteria, leading to increased resistance against pathogens (Ikeda et al., \u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e2007\u003c/span\u003e; Kim \u0026amp; Mylonakis, \u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e2012\u003c/span\u003e; Montalvo-Katz et al., \u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e2013\u003c/span\u003e). However, it is yet unclear whether the beneficial bacterial isolates in \u003cem\u003eC. elegans\u003c/em\u003e affect the infection effect of pathogenic bacteria on nematodes. The activities and stability of biological control agents might be effectively improved by using co-cultures of various antagonistic bacteria with different mechanisms of action and ecological adaptability. Therefore, using natural \u003cem\u003eC. elegans\u003c/em\u003e as a model, studying the interactions between microbiota and biocontrol microbes is a promising approach for improving the stability of biocontrol in the field.\u003c/p\u003e \u003cp\u003eTo date, there are 16 validly described species in the genus \u003cem\u003eStenotrophomonas\u003c/em\u003e, which have high genotypic and phenotypic diversity and were recovered from various environmental and even clinical samples (Brooke, \u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e2012\u003c/span\u003e). The type species \u003cem\u003eS. maltophilia\u003c/em\u003e was originally recognized as a human opportunistic pathogen. Subsequent research revealed that the metabolic diversity of \u003cem\u003eS. maltophilia\u003c/em\u003e is responsible for the production of novel bioactive compounds, including biocontrol agents against microbes and insects, and enzymes and nanoparticles used in medicinal, industrial and bioremediation applications (Ribitsch et al., \u003cspan citationid=\"CR45\" class=\"CitationRef\"\u003e2012\u003c/span\u003e). Another well-studied species, \u003cem\u003eS. rhizophila\u003c/em\u003e, which shows an endophytic life style, possesses unique genes encoding plant cell-wall-degrading enzymes and proteins responsible for the synthesis and transport of the plant-protective spermidine and high salinity tolerance, which suggests it is a harmless alternative \u003cem\u003eStenotrophomonas\u003c/em\u003e species for use in biotechnology (Alavi et al., \u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2013\u003c/span\u003e). There were also several reports on the genus \u003cem\u003eStenotrophomonas\u003c/em\u003e strains isolated from animal intestines. \u003cem\u003eStenotrophomonas\u003c/em\u003e members were found to be gut bacteria through the life cycle of the Bark Beetle \u003cem\u003eDendroctonus rhizophagus\u003c/em\u003e, and \u003cem\u003eS. maltophilia\u003c/em\u003e could be implicated in nitrogen fixation and cellulose breakdown, important roles associated to insect development and fitness, especially under the particularly harsh life conditions of this beetle (Morales-Jim\u0026eacute;nez et al., \u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e2012\u003c/span\u003e). Additionally, Sun et al. isolated a chitin-degradation \u003cem\u003eStenotrophomonas\u003c/em\u003e strain from the hindgut of a fungus-growing termite \u003cem\u003eMacrotermes barneyi\u003c/em\u003e (Sun et al., \u003cspan citationid=\"CR50\" class=\"CitationRef\"\u003e2017\u003c/span\u003e). The novel species studied here, a close relative of \u003cem\u003eS. rhizophila\u003c/em\u003e, was generally consistent with those previously reported \u003cem\u003eStenotrophomonas\u003c/em\u003e species isolated from the nematodes sampled directly from the native habitats (Dirksen et al., \u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e2016\u003c/span\u003e). In each parallel of isolation experiment, we selected randomly 20 single natural worms isolated from the same location. Members of the genus \u003cem\u003eStenotrophomonas\u003c/em\u003e could be isolated from more than 15 worms. The \u003cem\u003eStenotrophomonas\u003c/em\u003e spp. were identified to be the same species with CPCC 101271\u003csup\u003eT\u003c/sup\u003e. Furthermore, other worms without \u003cem\u003eStenotrophomonas\u003c/em\u003e being detected were raised on 9-cm agar plates seeded with 400 \u0026micro;l of the tested bacterium CPCC 101271\u003csup\u003eT\u003c/sup\u003e with an OD\u003csub\u003e600\u003c/sub\u003e of 10 for 24 h at room temperature. Then \u003cem\u003eStenotrophomonas\u003c/em\u003e spp. could be isolated from intestines of the worms after washed three times and surface disinfection. These experiments indicated that CPCC 101271\u003csup\u003eT\u003c/sup\u003e could stably colonize the nematode gut under experimental conditions.\u003c/p\u003e \u003cp\u003eIn nature, the structure and diversity of the microbiota in healthy nematodes are constantly changing. Some intestinal bacteria are actually indispensable parts of the host, which may form a mutually beneficial symbiotic relationship with the host.\u003c/p\u003e \u003cp\u003eIn this study, a new \u003cem\u003eStenotrophomonas\u003c/em\u003e bacterium CPCC 101271\u003csup\u003eT\u003c/sup\u003e representing a novel species was isolated from nematodes, and the interaction between the bacterium and \u003cem\u003eB. nematocida\u003c/em\u003e B16 was investigated. When strains B16 and CPCC 101271\u003csup\u003eT\u003c/sup\u003e were co-cultured on LB plates, strain B16 showed inhibitory activities against CPCC 101271\u003csup\u003eT\u003c/sup\u003e, which is consistent with the last stage of the competition between strain CPCC 101271\u003csup\u003eT\u003c/sup\u003e and B16 in worm intestine, even strain CPCC 101271\u003csup\u003eT\u003c/sup\u003e exhibited the colonization-resistance activities against \u003cem\u003eB. nematocida\u003c/em\u003e B16 in the early stages. Firstly, strain CPCC 101271\u003csup\u003eT\u003c/sup\u003e was confirmed as probiotic to worms owning to its ability to prolong the lifespan of \u003cem\u003eC. elegans\u003c/em\u003e. Secondly, strain CPCC 101271\u003csup\u003eT\u003c/sup\u003e could delay the infection time of B16 against nematodes, but not completely inhibit the infection of B16. Last but not least, \u003cem\u003eBacillus\u003c/em\u003e strains occupied the niche of \u003cem\u003eStenotrophomonas\u003c/em\u003e members by inhibiting the growth of CPCC 101271\u003csup\u003eT\u003c/sup\u003e, which could be inferred from the metagenomic analysis results. And then strain B16 completed the infection and realized its proliferation in worms.\u003c/p\u003e \u003cp\u003eBased on the above experiments, we proposed that the reason of colonization resistance to the pathogen B16 by the strain CPCC 101271\u003csup\u003eT\u003c/sup\u003e might own to its beneficial aspects to \u003cem\u003eC. elegans\u003c/em\u003e. Strain CPCC 101271\u003csup\u003eT\u003c/sup\u003e might play a critical role in (i) shaping and maintaining the intestinal bacterial community structure, (ii) synthesizing osmoprotectants, such as glucosylglycerol and trehalose, to help maintain host homeostasis, and (iii) producing or stimulating other microorganisms to synthesize antimicrobial peptides and other stress protective agents to protect the host from pathogens and harsh environments.\u003c/p\u003e \u003cp\u003eWe should explore substantial evidence to confirm the above inference in the following studies. Understanding this inference mechanism can help quickly inhibit the growth of probiotic microbiota, accelerate the colonization of biocontrol bacteria in the intestinal tract and improve the killing efficiency of nematodes. Our current findings may lay a theoretical foundation and open up new ideas for the development of ideal biocontrol agents.\u003c/p\u003e "},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eAvailability of data and materials\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe DDBJ/EMBL/GenBank accession number for the 16S rRNA gene sequence of strain CPCC 101271\u003csup\u003eT\u003c/sup\u003e is MT126327; The draft genome sequence of strain CPCC 101271\u003csup\u003eT\u003c/sup\u003e\u0026nbsp;is WIAY00000000. The strain CPCC 101271\u003csup\u003eT \u003c/sup\u003ehas been deposited in China Pharmaceutical Culture Collection and is available to the scientific research community without any special restrictions.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAuthor's contributions: \u003c/strong\u003eRui Han and Yu Wang carried out the experiments. Yang Deng prepared the figures. Yuqin Zhang and Lin Zhang designed the project and prepared the manuscript together with Qiuhong Niu. All authors reviewed the manuscript.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAcknowledgement\u003c/strong\u003e: The authors thank JCM (Japan Collection of Microorganisms) and DSMZ (German Collection of Microorganisms and Cell Cultures) for providing the reference strains.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFunding\u003c/strong\u003e: This research was supported by the National Natural Science Foundation of China (NSFC 31670010 and 31570120), the Drug Innovation Major Project (2018ZX09711001-007-001), the National Infrastructure of Microbial Resources (NIMR-2020-3), CAMS Innovation Fund for Medical Sciences (CIFMS) (2016-I2M-2-002) and Innovation\u0026nbsp;Scientists and Technicians Troop Construction Projects (Sustainable Utilization of Energy Microbial Resources) of Henan Province.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConflict of Interest Statement\u003c/strong\u003e: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConsent for publication\u003c/strong\u003e: Not applicable.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eEthical approval and consent to participate\u003c/strong\u003e: Not applicable.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n\u003cli\u003eAballay A, Yorgey P, Ausubel FM (2000) \u003cem\u003eSalmonella typhimurium \u003c/em\u003eproliferates and establishes a persistent infection in the intestine of \u003cem\u003eCaenorhabditis elegans\u003c/em\u003e. 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Front Microbiol. 8: 485\u003c/li\u003e\n\u003cli\u003eZimmermann J, Obeng N, Yang W, Pees B, Petersen C, Waschina S, Kissoyan K, Aidley J, Hoeppner M, Bunk B, Spr\u0026ouml;er C, Leippe M, Dierking K, Kaleta C, \u0026nbsp;Schulenburg H (2019) The functional repertoire contained within the native microbiota of the model nematode \u003cem\u003eCaenorhabditis elegans\u003c/em\u003e. ISME J\u003cem\u003e.\u003c/em\u003e 14: 26-38\u003c/li\u003e\n\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":false,"highlight":"","institution":"","isAcceptedByJournal":true,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"archives-of-microbiology","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"aomi","sideBox":"Learn more about [Archives of Microbiology](https://www.springer.com/journal/203)","snPcode":"203","submissionUrl":"https://submission.nature.com/new-submission/203/3","title":"Archives of Microbiology","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"em","reportingPortfolio":"Springer Hybrid","inReviewEnabled":true,"inReviewRevisionsEnabled":false},"keywords":"Stenotrophomonas nematodicola, Caenorhabditis elegans, Bacillus nematocida, lifespan-prolonging, colonization","lastPublishedDoi":"10.21203/rs.3.rs-124061/v2","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-124061/v2","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eThe soil-dwelling opportunistic bacterium \u003cem\u003eBacillus nematocida\u003c/em\u003e B16 shows comparatively strong killing activities against a variety of pathogenic nematodes. A bacterial strain CPCC 101271\u003csup\u003eT\u003c/sup\u003e, isolated from intestines of \u003cem\u003eC. elegans\u003c/em\u003e in natural habitats, was found not only to be probiotics for \u003cem\u003eC. elegans\u003c/em\u003e but also assist in resisting pathogen B16 infection. The lifespan of \u003cem\u003eCaenorhabditis elegans\u003c/em\u003e fed on strain CPCC 101271\u003csup\u003eT\u003c/sup\u003e was extended by approximately 40% compared with that of worms fed on \u003cem\u003eEscherichia coli\u003c/em\u003e OP50. In addition, the colonization of \u003cem\u003eC. elegans\u003c/em\u003e by the pathogenic bacterium \u003cem\u003eB. nematocida\u003c/em\u003e B16 was inhibited when it was pre-fed with strain CPCC 101271\u003csup\u003eT\u003c/sup\u003e. Based on a polyphasic taxonomy study including genotypic, chemotaxonomic and phenotypic characteristics, we propose that strain CPCC 101271\u003csup\u003eT\u003c/sup\u003e represents a novel bacterial species with the name \u003cem\u003eStenotrophomonas nematodicola\u003c/em\u003e sp. nov. and CPCC 101271\u003csup\u003eT\u003c/sup\u003e as the type strain. Metagenomic sequence analysis of the intestinal microbiota of \u003cem\u003eC. elegans\u003c/em\u003e fed with strain CPCC 101271\u003csup\u003eT\u003c/sup\u003e and then infected with B16 revealed that pre-feeding with CPCC 101271\u003csup\u003eT\u003c/sup\u003e improved the diversity of intestinal bacteria, while the community structure varied significantly together with the fluctuation of \u003cem\u003eStenotrophomonas\u003c/em\u003e spp. and \u003cem\u003eBacillus\u003c/em\u003e spp. abundance during competition between strain CPCC 101271\u003csup\u003eT\u003c/sup\u003e and B16. In conclusion, the nematode microbiota strain CPCC 101271\u003csup\u003eT\u003c/sup\u003e, a novel species of the genus \u003cem\u003eStenotrophomonas\u003c/em\u003e, assisted in its host resistance to the pathogen \u003cem\u003eBacillus nematocida\u003c/em\u003e colonization, so as to act as an intestinal life span-prolonging for \u003cem\u003eC. elegans.\u003c/em\u003e \u003c/p\u003e","manuscriptTitle":"Stenotrophomonas Nematodicola Sp. Nov., a Novel Intestinal Lifespan-Prolonging Bacterium for Caenorhabditis Elegans That Assists in Host Resistance to Bacillus Nematocida Colonization","msid":"","msnumber":"","nonDraftVersions":[{"code":"","date":"2021-06-16 00:00:00","doi":"","editorialEvents":[{"type":"editorInvitedReview","content":"","date":"2021-06-16T00:00:00+00:00","index":0,"fulltext":""},{"type":"reviewersInvited","content":"","date":"2021-06-15T00:00:00+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2021-06-14T00:00:00+00:00","index":"","fulltext":""},{"type":"submitted","content":"Archives of Microbiology","date":"2021-06-10T06:33:07+00:00","index":"","fulltext":""}],"status":"private","journal":{"display":true,"email":"[email protected]","identity":"archives-of-microbiology","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"aomi","sideBox":"Learn more about [Archives of Microbiology](https://www.springer.com/journal/203)","snPcode":"203","submissionUrl":"https://submission.nature.com/new-submission/203/3","title":"Archives of Microbiology","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"em","reportingPortfolio":"Springer Hybrid","inReviewEnabled":true,"inReviewRevisionsEnabled":false}},{"code":2,"date":"2021-05-18 16:12:31","doi":"10.21203/rs.3.rs-124061/v2","editorialEvents":[{"type":"communityComments","content":0},{"type":"editorInvitedReview","content":"","date":"2021-05-09T00:58:00+00:00","index":0,"fulltext":""},{"type":"reviewersInvited","content":"","date":"2021-05-07T08:18:00+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2021-05-07T06:55:00+00:00","index":"","fulltext":""},{"type":"submitted","content":"Archives of Microbiology","date":"2021-05-07T00:00:04+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"archives-of-microbiology","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"aomi","sideBox":"Learn more about [Archives of Microbiology](https://www.springer.com/journal/203)","snPcode":"203","submissionUrl":"https://submission.nature.com/new-submission/203/3","title":"Archives of Microbiology","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"em","reportingPortfolio":"Springer Hybrid","inReviewEnabled":true,"inReviewRevisionsEnabled":false}}],"origin":"","ownerIdentity":"45de2079-273a-47d6-aaac-ed577da63b46","owner":[],"postedDate":"May 18th, 2021","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"published-in-journal","subjectAreas":[{"id":4352725,"name":"General Microbiology"}],"tags":[],"updatedAt":"2021-08-22T15:25:55+00:00","versionOfRecord":{"articleIdentity":"rs-124061","link":"https://doi.org/10.1007/s00203-021-02467-4","journal":{"identity":"archives-of-microbiology","isVorOnly":false,"title":"Archives of Microbiology"},"publishedOn":"2021-07-14 15:07:22","publishedOnDateReadable":"July 14th, 2021"},"versionCreatedAt":"2021-05-18 16:12:31","video":"","vorDoi":"10.1007/s00203-021-02467-4","vorDoiUrl":"https://doi.org/10.1007/s00203-021-02467-4","workflowStages":[]},"version":"v2","identity":"rs-124061","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-124061","identity":"rs-124061","version":["v2"]},"buildId":"7rjqhiLT3MXkJMwkYKINL","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}