Fluviispira vulneris sp. nov., isolated from human wound secretions

Human infections by environmental bacteria is becoming an increasing problem and has become a matter of great concern due to the adverse effects worldwide. In this study, we reported a new environmental pathogen. Isolate GX5518T was a novel Gram-negative, aerobic, non-motile, pleomorphic and red-pigmented bacterium, was isolated from human wound secretions (GuangXi, People’s Republic of China). Growth occurred at pH 6.0–8.0 (optimum, pH 7.0) and 10–37 °C (optimum, 28–32 °C) with 0–1.5% (w/v) NaCl in R2A agar. Comparative analysis of the 16S rRNA gene sequences revealed that isolate GX5518T was closely related to Fluviispira sanaruensis JCM 31447T (99.73%) and Fluviispira multicolorata 33A1-SZDPT (98.49%). However, the estimated ANI values of the isolate GX5518T compared to the F. sanaruensis JCM 31447T and F. multicolorata 33A1-SZDPT were 88.67% and 77.35%, respectively. The estimated dDDH, ANI and AAI values between isolate GX5518T and its closely related strains were below the threshold values generally considered for recognizing a new species. The genome size was 3.6 Mbp and the DNA G + C content was 33.1%. The predominant fatty acids (> 5%) in GX5518T cells were iso-C15:0, C16:0, C17:0, C17:1 ω8c and C16:1 ω7c/C16:1 ω6c. The major menaquinone was MK-8 (86.9%). The polar lipids were phosphatidylethanolamine (PE), phosphatidylglycerol (PG) and three unknown lipids (L1-3). The chemical composition was different from that of the F. sanaruensis JCM 31447T. Comparative genomics analysis between isolate GX5518T and its related strains revealed that there were a number of genes involved in resistance to antibiotics and toxic compounds in isolate GX5518T, which were responsible for the copper homeostasis, cobalt-zinc-cadmium resistance, resistance to fluoroquinolones, and zinc resistance. Based on the phenotypic, chemotaxonomic, and genomic analyses, isolate GX5518T (= CGMCC 1.18685T = KCTC 82149T) represents a novel species of the genus Fluviispira, for which the name Fluviispira vulneris sp. nov. is proposed. Supplementary Information The online version contains supplementary material available at 10.1007/s10482-023-01883-4.


Introduction
The genus Fluviispira (Pitt et al. 2020), a member of the family Silvanigrellaceae (Hahn et al. 2017) within the order Silvanigrellales, the class Oligoflexia and the phylum Bdellovibrionota, is generally characterized as Gram-negative, aerobic, motile, non-spore forming, oxidase negative and pleomorphic.At the time that the strain was isolated, the genus only consists of one validly named species, F. multicolorata (Pitt et al. 2020).Subsequently, F. sanaruensis was described by Maejima et al. (2021) as a novel member of the genus Fluviispira.F. multicolorata grown on NSY agar form purple-pigmented, circular colonies but liquid cultures appear either purple, grey or orange, which was isolated from a small creek in Austria.Fluviispira sanaruensis produce a salmon pink on R2A agar, which was isolated from brackish lake water sampled at Lake Sanaru in Japan.The above strains were all isolated from freshwater samples in different countries and no cases of human infections.It is worth mentioning that "Pigmentibacter ruber" (Peng et al. 2021) was isolated from a blood specimen of a patient after a drowning accident in our country as a novel genus and species of the family Silvanigrellaceae, its emergence was the first case of human infections caused by the family Silvanigrellaceae.
In 2019, an 86-year-old man was admitted to the first affiliated hospital of Hunan Normal University with "stone-smashed right calf pain and bleeding for 4 h".Inflammatory indicators such as leucocyte (WBC), neutrophilic granulocyte percentage (NEUT%), procalcitonin (PCT) and C-Reactive Protein (CRP) increased.Necrotic tissue debridement + VSD negative pressure drainage operation was performed, intraoperative tissue and secretions were sent for bacterial culture, and a novel Gram-negative, aerobic, pleomorphic, red-pigmented isolate was reported, designed GX5518 T .
The current study aimed to determine the precise taxonomic position of isolate GX5518 T by using a polyphasic taxonomic approach.

Isolation and cultivation
Specimens for bacterial culture were obtained from wound tissue and secretion of a patient with bleeding from a right leg injury in Nanning, Guangxi Province, PR China.A Gram-negative, aerobic, pleomorphic, red-pigmented isolate, designed as GX5518 T , was isolated as potential pathogen with dominant from Columbia blood agar (bioMérieux) and ordinary chocolate plate (bioMérieux) at 37 °C for 24 h.Purified cultures were maintained in glycerol suspensions (30%, v/v) with 2% blood at − 80 °C for further polyphasic taxonomy investigation.Isolate was also deposited in the Korean Collection for Type Cultures (KCTC 82149 T ) and China general microbiology culture collection center (CGMCC 1.18685 T ).Two type strains, F. sanaruensis JCM31447 T and F. multicolorata 33A1-SZDP T , were chosen as reference type Vol.: (0123456789) strains and were cultured under the same experimental conditions for comparative studies.

Morphological, physiological and biochemical characterization
Cell morphology of isolate GX5518 T was observed using Optical microscope (BH-2, Olympus) and transmission electron microscopy (JEM1200, JEOL) (Ming et al. 2012) with the exponential-phase cells negatively stained with phosphotungstic acid.Motility was examined using the hanging-drop technique and on semi-solid R2A soft agar (containing 0.3% agar) (Jeong et al. 2020).Gram-staining was tested by using the Gram-stain kit (BaSo, China) and was confirmed using the KOH lysis test (Krishna and Gole 2017).
Oxidase activity was tested by using 1% (w/v) tetramethyl-p-phenylenediamine (Kovacs 1956).Catalase activity was determined by using 3% (v/v) hydrogen peroxide (Jeffries et al. 1957).Other physiological and biochemical characteristics and enzyme activities were determined using API 20 NE and API ZYM test-strip systems (bioMérieux, France) at 35 °C according to the manufacturers' instructions and F. sanaruensis JCM 31447 T was used as control.Because of the isolate GX5518 T grew slowly on the MH agar, antimicrobial susceptibility profile was assessed by determining minimum inhibitory concentrations (MICs) using a commercial assay (Blood-MH agar, E-test) according to manufacturer's instructions.

Chemotaxonomic analysis
The fatty acids profiles of isolate GX5518 T and F. sanaruensis JCM 31447 T were analysed using cells grown on Blood-R2A agar for incubated at 35 °C for 72 h until bacterial cultures reached the exponential phase.Acids methyl esters were extracted and identified by using gas chromatography (6890, Agilent) and the MIDI Sherlock Microbial Identification System (MIDI) system (Sherlock version 6.3; MIDI database: TSBA6) following the manufacturer's instructions.(Sasser 1990;Tindall 1990;Collins et al. 1982).Polar lipids of the isolates were extracted and analysed by using two-dimensional thin-layer chromatography (2D-TLC).The first phase was chloroform: methanol: distilled water = 65:25:4 (v/v), and the second phase was chloroform: glacial acetic acid: methanol: distilled water = 80:18:12:5 (v/v).(Minnikin et al. 1979).Total lipids were detected by phosphomolybdate and amino lipids, phospholipids and lecithin were detected by ninhydrin, D reagent and molybdenum blue respectively.Menaquinone were extracted and purified from lyophilized cells, and identified by using an HPLC (LC-20AT, Shimadzu) (Goodfellow et al. 1980).
The genomic DNA of isolate GX5518 T were sequenced using Illumina Hiseq platform by Novogene Company (Beijing, China).The raw data were filtered by readfq (version 10) and assembled by using the software SOAP de novo (Bankevich et al. 2012).Genome completeness and contamination of the sequences were estimated using CheckM (Thompson et al. 2013).For generation of a phylogenomic tree, the genome sequences of the related type strains with the isolate GX5518 T were retrieved from the NCBI database and the phylogenomic tree based on the core genes was built by using UBCG (Up-to-date Bacterial Core Gene) (Na et al. 2018) with default settings.

Animal experiments and histopathological analysis
To assess the virulence of the isolate GX5518 T , the animal experiments were carried out on 64 SPF-grade KM male and female mice, weighting (18 ± 2) g, which were purchased from Slack Jing da (Hunan) Laboratory Animal Technology Co., Ltd.The feeding temperature of mice is 18-25 °C, and the humidity is 50-70%.Mice were acclimated for a week before the experiment.The purified culture was made into a bacterial solution with a turbidity of 3.0 MCF using a 0.9% NaCl solution, and the concentration was calibrated using a turbidimeter (bioMérieux).The bacterial suspension was diluted to 8 gradient concentrations using the doubling dilution method.Diluted bacterial suspension was injected into mice by intraperitoneal injection, 8 mice in each group, 8 groups in total, and the injection volume was 0.02 ml/g.The mice in the negative control group were injected with the same amount of 0.9% sodium chloride.Calculate LD50 by the modified Cole's method (Yang et al. 2020).Traits, body weight and mortality of the mice were monitored and recorded daily for 7 days.The tissues, including hearts, livers, spleens, lungs, and kidneys, were collected, for tissue sections and HE staining, following the pathological procedures (Qian et al. 2020).Ethical permission for animal experiments is provided in the Supplementary Annex.

Animal experiments and histopathological analysis
The results of animal virulence experiments showed that no mice died within the 7-day observation period of mice injected with isolate GX5518 T bacterial suspension intraperitoneally.Heart tissue was congested with focal inflammatory cell infiltration; inflammatory cell infiltration was seen between hepatic vessels and parenchyma, small bile duct hyperplasia, and a large number of inflammatory cells gathered and infiltrated next to the bile duct; lung tissue structure was acceptable, with regional alveolar collapse and fusion, inflammatory cell infiltration; A small amount of inflammatory cell infiltration was seen in the spleen and kidney.In summary, the main pathological changes are inflammatory reactions, with the heart, liver and lung being the most serious (Fig. S4).Based on animal virulence experiments, it was confirmed that GX5518 T has potential pathogenicity and virulence.Combined with the characteristics of the patient's case, the infection occurred after stone injury.Considering that such microorganisms mainly exist in the environment, they may be opportunistic pathogenic bacteria.Animal experiments suggested but could not completely rule out the possibility that the virulence of isolate GX5518 T was weakened or lost due to factors such as artificial passage or growth conditions of the medium.

Phylogenetic and genome analysis
The 16S rRNA gene sequence of isolate GX5518 T (1469 bp) was submitted to the GenBank database with the accession number MT879459.Comparison of the 16S rRNA gene sequences showed that isolate GX5518 T shared the highest similarities with F. sanaruensis JCM 31447 T (99.73%) and F. multicolorata DSM 107810 T (98.49%).Phylogenetic analyses based on the NJ, ML and ME methods clearly showed that isolate GX5518 T formed an independent branch in the genus Fluviispira, and was closely related to the cluster composed by S. paludirubra DSM 107809 T (95.15%), S. aquatica DSM 23856 T (94.75%) and "P.ruber" KCTC 72920 T (94.28%) in the family Silvanigrellaceae (Figs S5-S7).
The draft genome sequences of isolate GX5518 T have been deposited at DDBJ/ENA/GenBank under the accession JACRSE000000000, was 3,604,777 bp in length, which was composed of 9 contigs with an N50 of 580,334 bp, genomic DNA G + C content of 33.1% and coverage of 100×.Phylogenomic analyses with the software UBCG clearly showed that isolate GX5518 T belonged to the genus Fluviispira, and was most closely related to F. sanaruensis JCM 31447 T and F. multicolorata DSM 107810 T (Fig. 2), which were similar with the phylogenies based on the 16S rRNA gene sequence.The estimated ANIb, ANIm, AAI and dDDH values between isolate GX5518 T and its closely related type strains derived from phylogenomic analyses and 16S rRNA gene phylogenies were compared, with the ANIb ranging from 62.0 to 88.7% and the ANIm ranging from 83.18 to 92.13%, AAI values ranging from 44.46 to 91.87%, dDDH values were 12.9 to 63.7%, and compared to the closest species F. sanaruensis JCM31447 T were 88.67%, 89.5%, 91.87%, 63.7%, respectively, which were far lower than the threshold values for species delimitation (95-96% ANI, 95-96% AAI and 70% dDDH, respectively) (Meier-Kolthoff et al. 2013;Richter and Rosselló-Móra 2009;Chun et al. 2018).The results were shown in Table S2.In conclusion, although the 16S rRNA gene sequence similarities of the isolate GX5518 T were more than the species' threshold limits (98.65%) (Kim et al. 2014), the genomic analysis results indicated that isolate GX5518 T could represented a novel genomic species in the genus Fluviispira.
Based on the genomes of isolate GX5518 T and its closely related species, the subsystem features were compared by using the RAST server and SEED viewer to indicate the presence or absence of geneassociated biological functions.A total of 3281 coding sequences (CDSs) with 40 RNAs were predicted for the novel strain, of which 498 (accounting for 16% of the total) were assigned into 365 features in the subsystem.Similar to those of closely related type strains, most of the assigned genes of the novel isolate were involved into the categories of amino acids and derivatives (121), protein metabolism (86), carbohydrates (86), cofactors/vitamins/prosthetic groups/pigments (68), fatty acids/lipids/isoprenoids (58).The detailed comparison of Subsystem features distribution between isolate GX5518 T and its related strains is provided in Table S3.Interestingly, genes involved in resistance to antibiotics and toxic compounds in isolate GX5518 T including copper homeostasis (7), cobalt-zinc-cadmium resistance (1), resistance to fluoroquinolones (2), and Zinc resistance(1).The heavy metals are hazardous and contaminate the environment and adversely affects the quality of the soil, isolate GX5518 T may have a role in resisting heavy metal toxicity and increasing heavy metal removal efficiency.
Annotation of the Cluster of Orthologous Groups [(COGs) (of proteins)] for the isolate GX5518 T found that a total of 1724 genes were assigned to 23 functional categories.Among the obtained functional groups, the cluster for of protein function and its number between isolate GX5518 T and its related strains is provided in Fig. 3.
In addition, the antibiotic resistance genes and virulence genes of isolate GX5518 T were predicted.For the CARD database (version 3.2.2),RGI 6.0.1 software, including Perfect, Strict and Loose algorithms, was used as the screening criteria.Five AMR Gene Family (elfamycin resistant EF-Tu, isoniazid resistant katG, resistance-nodulation-cell division (RND) antibiotic efflux pump, rifamycin-resistant beta-subunit of RNA polymerase (rpoB), and antibiotic resistant fusE) have been found in the genomes of isolate GX5518 T .EF-Tu is a elongation factor tu gtp-binding domain protein 2, which responsible for the elongation factor of peptide chains during protein synthesis and plays important role in ribosome translation proteins.Expression of the EF-Tu variant is conferred in the resistance to elfamycin.Catalase and peroxidase encoded by katG gene plays an important role in the oxidative metabolism process of bacteria, and mutations in the katG gene are more common cause of isoniazid resistance.RND antibiotic efflux pump involves three genes: MuxB, mdtC, and MexW, which is conferred in the resistance to macrolide antibiotic, monobactam, tetracycline antibiotic, aminocoumarin antibiotic, fluoroquinolone antibiotic and phenicol antibiotic.RpoB mutants and fusE mutants confer resistance to conferring resistance to rifampicin and fusidane antibiotic, respectively.Based on the VFDB database, 8 virulence-related genes, htpB, cheY, hemB, clpB, eno, cps2L, fliQ, katA were detected in the genome of isolate GX5518 T .Here, we also compared the virulence-related gene annotation results with "P.ruber" KCTC 72920 T , which is currently the only pathogenic bacterium that causes human infections in the family Silvanigrellaceae.Compared to "P. ruber" KCTC 72920 T , we observed the three genes, cps2L, fliQ, and katA are unique in isolate GX5518 T .cps2L, encoding glycosyltransferases involved in capsular polysaccharide (CPS) synthesis, which can resistant to complement deposition and masks cell wall-associated complement from being recognized by the complement receptors on phagocytes.Furthermore, the CPS is an essential virulence factor for isolate GX5518 T to infect the host.The flagellar biosynthesis protein FliQ encoded by the fliQ gene is required for the assembly of the rivet at the earliest stage of flagellar biosynthesis.The catalase encoded by the katA gene participates in the decomposition of hydrogen oxidation and protects cells from the toxicity of hydrogen peroxide.Stable and highly active KatA plays an important role in maintaining its own oxygen metabolism balance, enabling strains to escape oxidative damage from neutrophils and macrophages and survive in adverse environment.

Taxonomic conclusion
Similar morphological characterization and the high 16S rRNA gene similarities suggested that isolate GX5518 T was closely related to the genus Fluviispira.While, the dDDH values, ANIb values, ANIm values, AAI values, and chemotaxonomic characteristics between the isolate GX5518 T and its closely related strains have proved that it represented a novel species of the genus Fluviispira.In conclusion, based on genotypic, phenotypic, biochemical characteristics and phylogenetic analysis, it is proposed that isolate GX5518 T (= CGMCC 1.18685 T = KCTC 82149 T ) represented a novel species of the genus Fluviispira, for which the name Fluviispira vulneris sp.nov. is proposed.Fluviispira vulneris (vul'ne.ris.L. gen.neut.n. vulneris, of a wound in which the organism was isolated).
The type strain is GX5518 T (= CGMCC 1.18685 T = KCTC 82149 T ), which was isolated from human wound secretions in Guangxi, PR China.The genomic DNA G + C content is about 33.1 mol%.The 16S rRNA gene sequences of the type strain GX5518 T is available under the GenBank accession numbers MT879459.The GenBank accession number for the draft genome of the type isolate GX5518 T is JACRSE000000000 at GenBank/EMBL/DDBJ/PIR.

Fig. 2
Fig. 2 Phylogenomic tree based on 92 core genes contrasted by the software UBCG showing the relationship of isolate GX5518 T and closely related taxa.Geobacter metallireducens

Fig. 3
Fig. 3 Comparison of genes based on the 23 general eggNOG functional categories of isolate GX5518 T with its phylogenetically related species of the family Silvanigrellaceae and Pitt et al. (2020)