Characterization of Alistipes montrealensis sp. nov., Isolated from Human Feces of a Patient with Metastatic Melanoma Treated with Immune Checkpoint Inhibitors

Fecal microbiome culturomics of a cancer patient treated with immune checkpoint inhibitors led to the identification of a Gram-negative, rod-shaped, obligate anaerobic, non-motile, non-sporeforming bacterium, designated strain kh20T, which was phylogenetically assigned to the genus Alistipes. Strain kh20T demonstrated a 98.61% 16S rRNA sequence similarity with A. shahii WAL 8301T. The bacteria cells generated catalase but no oxidase. Iso-C15:0 (26.6%), anteiso-C15:0 (19.9%), and iso-C17:0 (17.2%) were the major cellular fatty acids identified in its composition. The G+C content of its genome was 57.2%. Strain kh20T showed significantly low values for DNA-DNA Hybridation (DDH ≤ 33.70%) and Average Nucleotide Identity (ANI ≤ 86.35%) compared with other Alistipes species. Based on these findings, we concluded that strain kh20T represented a novel bacterium, and we proposed the name Alistipes montrealensis sp. nov. (CECT 30384 and CSUR Q6005).


Introduction
The development of immune checkpoint inhibitors (ICI) has revolutionized the therapeutic landscape for patients with advanced melanoma, non-small cell lung cancer (NSCLC), and renal cell carcinoma [1,2]. Strategies employing ICI, such as anti-programmed cell death receptor 1 (anti-PD-1) alone or in combination with anti-cytotoxic T lymphocyte antigen 4 (anti-CTLA-4) monoclonal antibodies, represent the standard of care in these malignancies. However, lack of T cell activation specificity toward cancer cells has been associated with important off-target immune-related adverse events (irAEs) [3]. These irAEs can involve any organ and represent a major therapeutic hurdle in immuno-oncology given their associated morbidity and mortality [1]. Evidence suggests that the gut microbiome regulates systemic immunity mediating ICI efficacy [4]. Indeed, antibiotics lead Microbiol. Res. 2022, 13 141 to dysbiosis which further associates with ICI resistance [5,6]. Moreover, early clinical evidence suggests that the gut microbiome may also regulate the development of irAEs [4].
Therefore, we initiated a microbiome culturomics-based study in order to isolate bacteria from patients treated with ICI that developed irAEs. In this ongoing study, we isolated an unknown bacterium that could not be identified at species level using MALDI-TOF mass spectrometry (MS) and 16S gene rRNA gene sequencing from a patient prior to ICI initiation. The strain was designated as strain kh20 T , and its sequencing revealed it to be part of the genus Alistipes.
Affiliated to the phylum Bacteroidetes and the family of Rikenellaceae, the genus Alistipes was first described by Rautio et al. in 2003 (https://lpsn.dsmz.de/genus/alistipes, accessed on 11 November 2021). Alistipes species colonize the gastro-intestinal tract and have been found to be overrepresented in patients that responded to ICI [7]. To date, fourteen Alistipes have been isolated from various human samples including feces, urine, and intraabdominal fluids [8][9][10][11][12]. Herein, the strain kh20 T isolated from the fecal sample of a metastatic melanoma patient was described by means of its phenotypic, phylogenetic, and genomic characteristics.

Ethics and Sample Collection
Strain kh20 T was isolated from a fecal sample of a 67-year-old woman with metastatic melanoma that developed a grade 3 colitis requiring corticosteroids. However, despite discontinuation of ICI, the patient had a sustained stable disease for 15 months and was still alive 18 months after ICI initiation. Signed informed consent was obtained at the time of sampling though the McGill University Health Centre Research Institute (MUHC-RI) Biobank Ethics F9-69041 and the study was approved by the CHUM Research Ethics Committee 20.300. The fecal sample was collected on May 2019, prior to the first dose of anti-PD-1 treatment. Of note, the patient did not receive antibiotics within the last two months prior to sampling.

Bacterial Strain Isolation and Identification
To identify gut microbiota, culturomics was performed as previously described [13]. The fecal sample was diluted in 1 mL of Dulbecco's phosphate-buffered saline (dPBS) (Becton-Dickinson, Loveton Circle, MD, USA) and preincubated in a culture bottle (Becton-Dickinson) enriched with sterilized rumen fluid and sheep blood (Cedarlanelabs, Burlington, ON, Canada) at 37 • C for a period of 7 days. Next, 100 µL of the broth was suspended in 900 µL of dPBS and then serially diluted in steps 10-fold. Each dilution was then plated onto Tryptone soya agar (TSA) supplemented with 5% sheep blood (OXOID, Nepean, Canada). Plates were incubated anaerobically at 37 • C for 5-7 days in anaerobic jars (ThermoFisher, Waltham, MA, USA) using GENbag Anaer (Becton-Dickinson). To purify individually grown bacteria, all single macroscopic and microscopic colonies were picked and streaked out on TSA. After 2 days of anaerobic incubation at 37 • C, all isolates were identified using MALDI-TOF MS (Bruker Daltonics, Bremen, Germany).
MALDI-TOF MS was unable to identify strain kh20 T . Therefore, the identification of strain kh20 T was carried out by sequencing its 16S rRNA gene. The first step was a PCR amplification with the enzyme KAPA2G FAST, using the following cycles: 96 • C for 1 min, 96 • C for 10 s, 58 • C for 5 s, 72 • C for 1 s 72 • C for 30 s and 4 • C, respectively. The obtained PCR products were then purified with magnetic beads of type AMPure. The sequencing reaction, from these purified PCRs, was performed with BigDye TM Terminator v3.1 Cycle Sequencing Kit (ThermoFisher). The products of the sequencing reaction were purified with magnetic beads of the CleanSeq type, then read on 3730xl DNA Analyzer from Applied Biosystems TM . The sequence was then corrected using Chromas Pro software version 2.1.10 (Technelysium Pty. Ltd., South Brisbane, Australia). Strain kh20 T 's phylogenetic affiliation was subsequently investigated using the BLASTn program [14] and the nucleotide collection (nr/nt) of the NCBI database [15], available at https://blast.ncbi.nlm.nih.gov/Blast.cgi (accessed on 11 November 2021). Sequence of our isolate and its related closest species were aligned using CLUSTAL W (available at https://www.genome.jp/tools-bin/clustalw (accessed on 11 November 2021)) and the phylogenetic tree was constructed using Mega X software (available at https://www.megasoftware.net/, accessed on 11 November 2021).
Cells were subcultured and grown in Trypticase Soy Broth (TSB) at 37 • C without shaking overnight. Cells' suspension was deposited on a glow-discharged formvar carbon film on 300 mesh copper grids (FCF300-CU, EMS). Cells were stained with 1% ammonium molybdate for 5 s. Then bacterial cells' morphology was assessed by electronic microscope analysis using Tecnai G20 (FEI company, Hillsboro, OR, USA) and Gram-stain using a Gramstaining kit according to manufacturer's instructions (ThermoFisher). Gram-stain and cell mobility were observed using photonic microscope Motic AE2000 (Motic Microscope, Vancouver, BC, Canada). Sporulation was determined by subjecting the bacterium to thermal shock at 80 • C for 20 min. Catalase and oxidase production was also detected (bioMérieux SA, Marcy l'Étoile, France). Enzymatic profile and biochemical properties of strain kh20 T were assessed using the API ® ZYM, API ® 20A and Rapid ID 32A identification systems according to the manufacturer's instructions (bioMérieux). Cellular fatty acid methyl ester profile was analysed using both gas chromatograph (Hewlett Packard) and Microbial Identification System (MIDI) software, version 6.0. End products were detected using a chromatograph as previously reported [9].

Genome Sequencing and Annotation
Genomic DNA was sequenced using MiSeq Illumina. Libraries were generated using the NxSeq ® AmpFREE Low DNA Library Kit Library Preparation Kit (Lucigen, Parmenter, Middleton, WI, USA) according to the manufacturer's recommendations, with 700 ng of genomic DNA as starting material. Dual-indexed adaptors were purchased from IDT. Libraries were quantified using the Kapa Illumina GA with the Revised Primers-SYBR Fast Universal kit (Kapa Biosystems, Ballardvale, MA, USA). Average size fragment was determined using a LabChip GX II (PerkinElmer, Waltham, MA, USA) instrument. The libraries were normalized and pooled, denatured in 0.05 N NaOH, and neutralized using HT1 buffer. The pool was loaded at 225 pM on an Illumina NovaSeq S4 lane using Xp protocol as per the manufacturer's recommendations. The run was performed for 2 × 150 cycles (paired-end mode). A phiX library was used as a control and mixed with libraries at the 1% level. Base calling was performed with RTA v3. Program bcl2fastq2 v2.20 was then used to demultiplex samples and generate fastq reads.
Quality control checks on raw sequences were performed with the FastQC software v0.11.9 [16]. Trimming and filtering were done through the Trimmomatic software v0.39 [17] using the following settings. The de novo genome assembly was made using the software SPAdes v3.15.2 [18] with the option-isolate. A final polishing of the draft genome assembly was performed with the Redundans software v0.13c [19] for gap closing and overlapped scaffolds merging with the options-identity 1-noreduction. Quality and completeness assessments of the genome assembly were made through BlobTools2 [20] and BUSCO v4 [21].
The nucleotide sequences of the reference strains were obtained from the NCBI database. DNA-DNA hybridization (DDH) values were evaluated by using a Genome-to-Genome Distance Calculator (GGDC) [25]. In contrast, average nucleotide identity (ANI) values were calculated using the ANI calculator [26].

Strain Identification and Phylogenetic Analysis
No MALDI-TOF identification of the strain kh20 T was obtained. Phylogenetic interferences based on 16S rRNA gene sequences using the neighbor-joining method (Figures 1 and S1), revealed that strain kh20 T belongs to the genus Alistipes and is clustered with A. shahii WAL 8301 T [10] and A. senegalensis JC50 T [27]. BLASTn analysis of the bacterium provided a 16S rRNA nucleotide sequence identity of 98.61% with A. shahii WAL 8301 T . Its MALDI-TOF reference spectrum was incremented in our University of Montreal Research Center (CRCHUM) database.
The nucleotide sequences of the reference strains were obtained from the NCBI da tabase. DNA-DNA hybridization (DDH) values were evaluated by using a Genome-to Genome Distance Calculator (GGDC) [25]. In contrast, average nucleotide identity (AN values were calculated using the ANI calculator [26].

Strain Identification and Phylogenetic Analysis
No MALDI-TOF identification of the strain kh20 T was obtained. Phylogenetic inte ferences based on 16S rRNA gene sequences using the neighbor-joining method ( Figure  1 and S1), revealed that strain kh20 T belongs to the genus Alistipes and is clustered with A shahii WAL 8301 T [10] and A. senegalensis JC50 T [27]. BLASTn analysis of the bacterium provided a 16S rRNA nucleotide sequence identity of 98.61% with A. shahii WAL 8301 Its MALDI-TOF reference spectrum was incremented in our University of Montreal Re search Center (CRCHUM) database. Sequences were aligned using Muscle v3.8.31 with default parameters and phylogenetic inferences were obtained using the neighbor joining method with 500 bootstrap replicates, using MEGAX software. The scale bar represents a 5% nucleotide sequence divergence.

Enzyme activities
Alkaline phosphatase   Figure 2. Transmission electron microscopy of Alistipes montrealensis strain kh20 T using a Tecnai G20 transmission electron microscope.  Figure 2. Transmission electron microscopy of Alistipes montrealensis strain kh20 T using a Tecnai G20 transmission electron microscope.

Genome Characteristics
A summary of the genome assembly is shown in Figure S2. Briefly, the draft genome of strain kh20 T (accession number JAGYXY000000000.1) was constituted of 13 scaffolds and 3,484,450 bp in length with 57.23 l% of G+C content. This draft assembly included 92.70% of the orthologous groups of the Bacteria kingdom, and 97% of the Bacteroidales order. Of the 2778 predicted genes, 2700 were protein-coding genes and 54 were RNA genes (one 5S rRNA, one 16S rRNA, one 23S rRNA, three ncRNA and 48 tRNA genes). A total of 2065 genes (76%) were assigned a putative function by clusters of orthologous groups (COGs). The distribution of the predicted gene into the 25 general COGs is summarized in Table 3. A summary of the repartition of these predicted genes is presented in Figure 3. The genomic comparison of strain kh20 T with those of its closest neighbors is detailed in Table 4 and illustrated in Figure 4. The genome size, DNA G+C percentage and gene content of strain kh20 T (3.48 Mb, 57.20 mol% and 2700, respectively) are in the same range of those of compared species, but very close to those of A. timonensis (3.49 Mb, 58.8% and 2764, respectively). However, strain kh20 T shows a genomic difference of 1.60% in G+C content compared to A. timonensis. Pairwise whole-genome ANI and DDH between strain kh20 T and its closest related species is itemized in

Discussions
Strain kh20 T was isolated as part of a project to study the role of the gut microbiota in the efficacy of ICI treatment. Phenotypic, phylogenetic, and genomic comparison between strain kh20 T and its most closely related species was performed. The combination of these phylogenetic (Figures 1 and S1), phenotypic (Tables 1 and 2) and genomic characteristics ( Figure 4 and Tables 3-5) indicates that strain kh20 T belongs in the family of Rikenellaceae, within the Alistipes genus. However, it can be easily distinguished from other Alistipes species on the basis of its phenotypic features, such as its major cellular fatty acids composition (Tables 1 and 2). Moreover, its genomic data, especially its low DDH values (between 21.70% with A. putridinis to 33.70% with A. shahii), and ANI percentages (between 74.18% with A. putridinis to 86.35% with A. shahii) below the 70% and 95% thresholds [25,[28][29][30], respectively, for bacterial species demarcation indicate that kh20 T is a member of a genomic species different from any of its neighbors. In addition, its sequence similarity value of the 16S rRNA gene (98.61%), which is lower than the 98.65% threshold for distinguishing two species [31][32][33], supports the classification of strain kh20 T as a new species of the genus Alistipes.
Created in 2003 by Ratio, the genus Alistipes regroups to date 16 species isolated in human sources and mainly in gastrointestinal tract. From a clinical perspective, Alistipes have been associated with beneficial clinical outcomes [4] and oral supplementation in mice restored anti-PD-1 efficacy [34]. In addition, Alistipes species are known to be enriched in non-small cell lung cancer with detectable Akkermansia correlating with longer overall survival [35]. Additional studies are needed to further delineate the role of Alistipes in the incidence of immune-related colitis and to better understand the paradoxical association between irAEs and improved survival.

Conclusions
Based on phylogenetic analysis, phenotypic data and genomic comparisons, the strain kh20 T differs sufficiently from its closest related species to be classified as a new species of the genus Alistipes. The name Alistipes montrealensis sp. nov. is proposed for this new taxon. It was isolated from a stool sample of a melanoma patient treated with ICI.
Alistipes montrealensis (mon.treal.e'n.sis. L. gen. masc. n. montrealensis, pertaining to Montreal, the city where the type of strain was first isolated).
The type of strain kh20 T (CECT 30384 and CSUR Q6005) was isolated from a faecal sample of a cancer patient. It is susceptible to metronidazole but resistant to colistin, kanamycin, and vancomycin. Its genome is 3,484,450 bp in length and exhibits 57.20 mol% of G+C. The 16S rRNA and genome sequences are deposited in GenBank under accession numbers MZ145385 and JAGYXY000000000, respectively. The habitat is probably the human gut.
Supplementary Materials: The following are available online at https://www.mdpi.com/article/ 10.3390/microbiolres13010012/s1, Figure S1: Maximum likelihood phylogenetic tree based on 16S rRNA sequences, Figure S2: Snail plot summary of assembly statistics for the genome assembly.

Data Availability Statement:
The datasets presented in this study can be found in online repositories. Associated accession numbers are cited in the manuscript.