Expanding the Bacterial Diversity of the Female Urinary Microbiome: Description of Eight New Corynebacterium Species

The genus Corynebacterium is frequently found in the female urinary microbiome (FUM). In-depth characterization of Corynebacterium at the species level has been barely exploited. During ongoing FUM research studies, eight strains (c8Ua_144T, c8Ua_172T, c8Ua_174T, c8Ua_181T, c9Ua_112T, c19Ua_109T, c19Ua_121T, and c21Ua_68T) isolated from urine samples of healthy women or diagnosed with overactive bladder could not be allocated to any valid Corynebacterium species. In this work, we aimed to characterize these strains based on a polyphasic approach. The strains were Gram stain positive, rod to coccoid shaped, nonmotile, catalase positive, and oxidase negative. Phylogenetic analysis based on 16S rRNA and rpoB gene sequences indicated that all strains belonged to the genus Corynebacterium. The average nucleotide identity and digital DNA–DNA hybridization values among the genomes of the above eight strains and closely related type strains of the Corynebacterium genus were <95 (74.1%–93.9%) and <70% (22.2%–56.5%), respectively. Mycolic acids were identified in all strains. MK-8(H2) and/or MK-9(H2) were identified as the major menaquinones. The polar lipids’ pattern mostly consisted of diphosphatidylglycerol, phosphatidylglycerol, and glycophospholipids. The major fatty acid was C18:1ω9c. Corynebacterium lehmanniae (c8Ua_144T = DSM 113405T = CCP 74T), Corynebacterium meitnerae (c8Ua_172T = DSM 113406T = CCP 75T), Corynebacterium evansiae (c8Ua_174T = DSM 113407T = CCP 76T), Corynebacterium curieae (c8Ua_181T = DSM 113408T = CCP 77T), Corynebacterium macclintockiae (c9Ua_112T = DSM 113409T = CCP 78T), Corynebacterium hesseae (c19Ua_109T = DSM 113410T= CCP 79T), Corynebacterium marquesiae (c19Ua_121T = DSM 113411T = CCP 80T), and Corynebacterium yonathiae (c21Ua_68T = DSM 113412T = CCP 81T) are proposed. This study evidenced that commonly used methodologies on FUM research presented limited resolution for discriminating Corynebacterium at the species level. Future research studying the biological mechanisms of the new Corynebacterium species here described may shed light on their possible beneficial role for healthy FUM.


Introduction
The genus Corynebacterium, belonging to the family Corynebacteriaceae and phylum Actinobacteria, was proposed by Lehmann and Neumann in 1896 [1]. Currently, the genus includes 140 species with validly published and correct names under the International Code of Nomenclature of Prokaryotes (https://lpsn.dsmz.de/genus/corynebacterium, accessed on 1 January 2023).
Species of the genus Corynebacterium are considered inhabitants of human and animal skin and mucous membranes, and can be found in diverse environmental habitats (e.g., water, soil, and sewage) [2,3]. Nevertheless, some members are increasingly recognized

Culture Conditions and Strain Isolation
Urine samples were processed, up to 2 h after collection, as previously described [10]. Briefly, 100 µL of urine samples were inoculated on Columbia agar with 5% sheep blood ((BAP) Biogerm, Moreira, Portugal) and HiCrome UTI agar (chromogenic agar plates (CAP), HiMedia, India). Colonies were selected after 48 h at 37 • C under aerobic and microaerophilic conditions (BAP and CAP) and anaerobic conditions (BAP). Strains were maintained on tryptic soy broth (TSB; Sigma-Aldrich, St. Louis, MO, USA) supplemented with 30% (v/v) glycerol at −80 • C for long-term storage. Strains were preliminary identified by a matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF) VITEK MS system (bioMérieux, Craponne, France) using in vitro diagnostic database version 3.0. Eight Corynebacterium strains (n = 7 from urine of healthy women: c8Ua_144 T , c8Ua_172 T , c8Ua_174 T , c8Ua_181 T , c9Ua_112 T , c19Ua_109 T , and c19Ua_121 T ; n = 1 from urine of a woman diagnosed with OAB: c21Ua_68 T ) identified as putative new Corynebacterium species were further characterized.

Phylogenetic Analysis
Complete nucleotide sequences of the 16S rRNA gene of all the strains analyzed in this study and type strains of closely related Corynebacterium species were extracted with MyDbFinder 2.0 (https://cge.food.dtu.dk/services/MyDbFinder/, accessed on 1 April 2022) after genome annotation. Primers and protocols for rpoB amplification and sequencing were the same as those described by Khamis et al. (2004) [14]. Further, 16S rRNA and rpoB gene sequences were aligned, and similarity scores were generated using MEGA version 7.0 (https://www.megasoftware.net/download_form, accessed on 1 April 2022) [15]. Phylogenetic trees were constructed according to the neighbor-joining method [16], and genetic distances were estimated using Kimura's two-parameter model [17]. The reliability of internal branches was assessed from bootstrapping based on 1000 resamplings [18].

Phenotypic and Chemotaxonomic Analysis
Growth was evaluated in different culture media: tryptic soy agar (Liofilchem, Roseto degli Abruzzi, Italy), tryptic soy broth (Liofilchem, Italy), brain heart infusion agar (Liofilchem, Italy) and brain heart infusion broth (VWR International, Leuven, Belgium) supplemented or not with 0.1% Tween-80 (Sigma-Aldrich, St. Quentin Fallavier, France) under aerobic conditions, and Columbia agar with 5% sheep blood (bioMérieux, France) under aerobic, microaerophilic, anaerobic conditions and a 5% CO 2 -enriched atmosphere at 37 • C for 48 h. Growths at different temperatures (4,8,15,25,37,42, and 50 • C) and NaCl concentrations (5.0, 6.5, 7.0, 7.5, 8.0, and 9.0) were tested in brain heart infusion with 0.1% Tween-80 under aerobic conditions at 37 • C for 48 h. Cell and colony morphology were observed with cells grown on Columbia agar with 5% sheep blood medium at 37 • C for 48 h under aerobic conditions. Gram staining was assessed using a Gram-staining kit (bioMérieux, France). Catalase and oxidase activities were evaluated in the presence of 3% (v/v) aqueous hydrogen peroxide solution and oxidase strips (Oxoid), respectively. The metabolic profile of the strains was determined by using the API ® Coryne and API ® Rapid ID 32A bacterial identification systems, according to the instructions of the manufacturer (bioMérieux, France). Cellular fatty acids were analyzed after conversion into fatty acid methyl esters by saponification, methylation, and extraction using the standard protocol of the Microbial Identification System (MIDI Inc. version 6.1) [24]. To resolve summed features and to confirm the identification from the MIDI system, the samples were additionally analyzed via GC-MS as described previously [25].
Mycolic acids were extracted from approximately 300 mg wet biomass using minor modifications of the method described by Vilchèze and Jacobs (2007) for analysis of mycolic acids by high performance liquid chromatography [26]. Briefly, cells were lysed in 50% KOH-methanol solution (1:1 v/v) at 95 • C overnight and extracted with chloroform. Dried extracts were reconstituted in chloroform-methanol (9:1) and analyzed on an Agilent 6545 Q-TOF mass spectrometer as described previously [27]. Mycolic acids were identified based on a comparison of exact masses from known mycolic acid structures [28].
Polar lipids were extracted from freeze-dried material based on the method of Bligh and Dyer [29] with slight modifications described by Tindal et al. [30], and separated by two-dimensional thin layer chromatography. Total lipids were visualized by spraying with dodecamolybdophosphoric acid; specific functional groups were visualized with α-naphthol, ninhydrin, or molybdenum blue.
Respiratory quinones were extracted from a freeze-dried cell, as described previously [25]. The separation and identification of quinones were performed by HPLC coupled to a diode array detector and high-resolution mass spectrometer [31].

MALDI-TOF MS Identification
The strains c8Ua_144 T , c8Ua_172 T , c8Ua_181 T , and c19Ua_109 T were initially identified as Corynebacterium sp., c8Ua_174 T and c9Ua_112 T as Corynebacterium jeikeium, and c19Ua_121 T and c21Ua_68 T as Corynebacterium tuberculostearicum.
The topology of a partial rpoB-based phylogenetic tree revealed few differences compared with the 16S rRNA gene ( Figure S2), yet the nucleotide sequence similarities were quite different. The type strain of C. tuberculostearicum was the closest neighbor of c8Ua_181 T , c19Ua_121 T , and c21Ua_68 T (94.8%, 95.2% and 95.8% sequence similarity, respectively), while the type strain of Corynebacterium afermentans was the closest neighbor of c8Ua_144 T (97.0% sequence similarity). The strains c8Ua_174 T and c9Ua_112 T clustered with C. jeikeium (95.5% and 95.8% sequence similarity, respectively), and the closest neighbor of the strains c8Ua_172 T and c19Ua_109 T was C. tuscaniense (90.8% sequence similarity) and C. aurimucosum (95.5% sequence similarity), respectively.

Genomic Analysis
The genomic features of the strains analyzed in this study and the ANI and dDDH values between our strains and the closest related type strains of the Corynebacterium species are presented in Table 1.
The ANI and dDDH values between the eight strains were ≤66.3% and 35.9%, respectively. The ANI values between the strains c8Ua_181 T , c19Ua_121 T , and c21Ua_68 T and the closest related C. tuberculostearicum were 87.7%, 94.0%, and 88.6%, respectively ( Table 1). The ANI values between c8Ua_174 T or c9Ua_112 T and C. jeikeium were 91.4% and 85.7%, respectively ( Table 1). The ANI values between the strains c8Ua_144 T , c8Ua_172 T , or c19Ua_109 T and the closest species C. afermentans, C. tuscaniense, and C. aurimucosum were 90.3, 84.1, and 88.4, respectively (Table 1). Likewise, the dDDH values between the eight strains and between all the strains analyzed and the closest Corynebacterium species ranged from 28.6% to 55.6% (Table 1). Remarkably, 46 available genomes in public databases from strains deposited as Corynebacterium sp., C. jeikeium, C. aurimucosum, C. tuberculostearicum, and not validly published species (e.g., Corynebacterium haemomassilienses) should be reclassified based on whole-genome relatedness, since ANI values between these strains and those analyzed in this study were all above 95% (Table S1). In fact, the ANI values between 27 publicly available genomes from strains deposited as Corynebacterium sp. or C. aurimucosum, isolated from Homo sapiens or environmental samples, and Corynebacterium hessae c19Ua_109 T ranged from 96.0% to 96.8%. The ANI values between 11 strains deposited as Corynebacterium sp. or C. jeikeium, isolated from Homo sapiens samples, and Corynebacterium macclintockiae c9Ua_112 T ranged from 96.8% to 97.6%. The ANI values between four strains deposited as C. aurimucosum or C. tuberculostearicum, isolated from Homo sapiens, and Corynebacterium marquesiae c19Ua_121 T ranged from 95.9% to 96.6%. Two strains (Corynebacterium sp. and C. jeikeium), isolated from Homo sapiens, showed ANI values of 97.0% and 97.1% with Corynebacterium evansiae c8Ua_174 T , respectively. Finally, two other strains isolated from Homo sapiens, and identified as Corynebacterium haemomassilienses or Corynebacterium sp., showed ANI values of 96.1% and 99.9% with Corynebacterium lehmanniae c8Ua_144 T and Corynebacterium yonathiae c21Ua_68 T , respectively.

Phenotypic Characterization
The strains grow in all the tested medium under aerobic conditions. Supplementation of the tested culture medium with Tween-80 allowed better growth of all but one strain (c19Ua_109 T ). Optimum growth was observed for all strains growing in Columbia agar with 5% sheep blood under an aerobic and 5% CO 2 -enriched atmosphere, at 37 • C for 48 h. Good growth was observed in microaerophilic conditions, and variable growth was reported in anaerobic conditions. Bacterial cells appeared as Gram stain positive, rod to coccoid shaped, no longer than 1.75 µm in length. All strains were catalase positive, oxidase negative, and nonmotile. The differential characteristics from other species are listed in Table 2.
The polar lipid analysis detected the presence of diphosphatidylglycerol, phosphatidylglycerol, as well as uncharacterized phospholipids, and glycolipids in all strains. Additionally, glycophospholipids were detected in all but one strain (c8Ua_174 T ), uncharacterized aminolipids were detected in two strains (c19Ua_109 T and c19Ua_121 T ), and uncharacterized lipids were detected in all but two strains (c8Ua_181 T and c21Ua_68 T ) ( Figure S3).

Characteristic
The proposed names of Corynebacterium novel species described in this study are in honor of women who changed the world or female researchers whose role in science was neglected and only later received due credit for their discoveries.
Corynebacterium lehmanniae (leh.man'ni.ae. N.L. gen. fem. n. lehmanniae of Lehman; in honor of Inge Lehmann, a Danish seismologist, who discovered that Earth has a solid inner core).
The type strain, c8Ua_144 T (=DSM 113405 T = CCP 74 T ), was isolated from the urine of a healthy woman in Portugal in 2017. Another strain was isolated from blood (Table S1). The DNA G + C content of the type strain is 65.4 mol %. The annotated genomic sequence of the strain c8Ua_144 T was deposited in DDBJ/ENA/GenBank and is available under accession number JAKMUR000000000.
Corynebacterium meitnerae (mei'tner.ae. N.L. gen. fem. n. meitnerae of Meitner; in honor of Lise Meitner, an Austrian physicist, who studied radioactivity and nuclear physics and, together with Otto Hahn and Fritz Straßmann, discovered the nuclear fission).
The type strain, c8Ua_172 T (=DSM 113406 T = CCP 75 T ), was isolated from the urine of a healthy woman in Portugal in 2017. The DNA G + C content of the type strain is 61.0 mol %. The annotated genomic sequence of the strain c8Ua_172 T was deposited in DDBJ/ENA/GenBank and is available under accession number JAKMUS000000000. Corynebacterium evansiae sp. nov.
Corynebacterium evansiae (e.van'si.ae. N.L. gen. fem. n. evansiae of Evans; in honor of Alice Evans, an American microbiologist, who demonstrated that Brucella abortus caused the disease brucellosis (undulant fever) in both cattle and humans, suggesting that raw milk could cause disease in humans).
The type strain, c8Ua_174 T (=DSM 113407 T = CCP 76 T ), was isolated from the urine of a healthy woman in Portugal in 2017. Other strains were isolated from vertebral disk space and urine (Table S1). The DNA G + C content of the type strain is 62.9 mol %. The annotated genomic sequence of the strain c8Ua_174 T was deposited in DDBJ/ENA/GenBank and is available under accession number JAKMUT000000000.
The type strain, c8Ua_181 T (=DSM 113408 T = CCP 77 T ), was isolated from the urine of a healthy woman in Portugal in 2017. The DNA G + C content of the type strain is 58.5 mol %. The annotated genomic sequence of the strain c8Ua_181 T was deposited in DDBJ/ENA/GenBank and is available under accession number JAKMUU000000000.
The type strain, c9Ua_112 T (=DSM 113409 T = CCP 78 T ), was isolated from the urine of a healthy woman in Portugal in 2017. Other strains were isolated from pleural human spine, knee bursa, humeral membrane, tissue, diabetic foot ulcer, and decubitus ulcer (Table S1). The DNA G + C content of the type strain is 62.0 mol %. The annotated genomic sequence of the strain c9Ua_112 T was deposited in DDBJ/ENA/GenBank and is available under accession number JAKMUV000000000.
Corynebacterium hesseae (hes'se.ae. N.L. gen. fem. n. hesseae of Hesse; in honor of Fanny Hesse, an American microbiologist, who alongside her husband, Walther Hesse, pioneered the use of agar as a common gelling agent for the production of media capable of culturing microorganisms).
The type strain, c19Ua_109 T (=DSM 113410 T = CCP 79 T ), was isolated from the urine of a healthy woman in Portugal in 2017. Other strains were isolated from a human (vagina, rectum, urine, sebaceous cyst, skin, nasal mucosa) and the environment. The DNA G + C content of the type strain is 61.0 mol %. The annotated genomic sequence of the strain c19Ua_109 T was deposited in DDBJ/ENA/GenBank and is available under accession number JAKMUW000000000.
Corynebacterium marquesiae (mar.que'si.ae. N.L. gen. fem. n. marquesiae of Marques; in honor of Branca Edmée Marques, a Portuguese scientist, who worked under the famous scientist Madame Marie Curie, and whose research in nuclear physics for peaceful means was finally recognized by the Portuguese State at the age of 67).
The type strain, c19Ua_121 T (=DSM 113411 T = CCP 80 T ), was isolated from the urine of a healthy woman in Portugal in 2017. Other strains were isolated from the skin (Table S1). The DNA G + C content of the type strain is 59.5 mol %. The annotated genomic sequence of the strain c19Ua_121 T was deposited in DDBJ/ENA/GenBank and is available under accession number JAKMUY000000000.
The type strain, c21Ua_68 T (=DSM 113412 T = CCP 81 T ), was isolated from the urine of a woman with overactive bladder symptoms in Portugal in 2017. Another strain was isolated from nasopharynx (Table S1). The DNA G + C content of the type strain is 58.7 mol %. The annotated genomic sequence of the strain c21Ua_68 T was deposited in DDBJ/ENA/GenBank and is available under accession number JAKMUZ000000000.

Discussion
The accurate discrimination of the Corynebacterium species has been improved over the years with the emergence of bacterial identification tools. However, the identification of some closest species is still a challenge. MALDI-TOF MS is a powerful tool to rapidly and accurately identify bacteria to both genus and species levels [37,38], yet it failed to differentiate the strains analyzed in this study.
The 16S rRNA gene is the most widely used marker to determine phylogenetic relationships of bacteria. However, the 16S rRNA gene provides limited resolution to discriminate among closely related species of the genus Corynebacterium due to a low polymorphism degree [39,40]. Our study also corroborates this since all but one Corynebacterium strains analyzed clustered with other Corynebacterium species. Conversely, some studies have shown that the partial sequence of rpoB is more accurate than the 16S rRNA gene to discriminate closely related Corynebacterium species [40,41]. Khamis and colleagues (2005) observed that the highest degree of similarity of rpoB partial sequence between two species was 95.0% [40]. However, the in silico analysis performed in this study revealed that the highest degree of similarity of rpoB partial sequence between the two most closely related Corynebacterium species (i.e., Corynebacterium diphtheriae and Corynebacterium belfantii) was 98.1%, and most of the strains here analyzed showed high values of similarity degree with the most related species (above the 95.0% cut-off set out by Khamis) [40]. Therefore, neither the 16S rRNA gene nor rpoB provides an accurate identification of closely related Corynebacterium species.
Whole-genome analysis is the most powerful tool for the taxonomic classification of the genus Corynebacterium [38,42]. In the current work, it allowed for the accurate discrimination of all Corynebacterium strains analyzed. The ANIb values between our strains and between our strains and closely related Corynebacterium species were clearly below the proposed species cut-off of 95%-96% [43]. Likewise, the dDDH values between the strains analyzed and the closest Corynebacterium species were below the proposed species cut-off level of 70% [23]. These data confirmed that the eight strains analyzed represent distinct species within the Corynebacterium genus. Moreover, the ANI values obtained between our strains and publicly available genomes support the reclassification of 46 strains as C. hesseae (27 strains), C. macclintockiae (11 strains), C. marquesiae (4 strains), C. evansiae (2 strains), C. lehmanniae, and C. yonathiae (1 strain each).
The morphologic and phenotypic characteristics (e.g., Gram positive staining, pleomorphic, rod to coccoid shaped, nonmotile, non-spore-forming, positive catalase, and oxidase negative) of our strains are in accordance with those previously reported for the Corynebacterium genus [38,39,44]. Likewise, the short-chain mycolic acids with 32-38 carbons, the major menaquinone (types MK-8(H2) and MK-9(H2), and the complex polar lipid and cellular fatty acid profiles (predominance of C 18:1 ω9c) exhibited are also in line with those reported for other species of the genus [39,42,45].
Our study supports that the strains analyzed in this study represent eight novel species within the Corynebacterium genus, for which the names Corynebacterium lehmanniae, Corynebacterium meitnerae, Corynebacterium evansiae, Corynebacterium curieae, Corynebacterium macclintockiae, Corynebacterium hesseae, Corynebacterium marquesiae, and Corynebacterium yonathiae are proposed. However, the biological mechanisms that Corynebacterium members composing the FUM may play in maintaining a healthy microbiome remain to be deciphered. Nevertheless, recent studies revealed that some Corynebacterium species may play an important role in microbiome-mediated protection against pathogens [46][47][48]. For instance, Corynebacterium accolens has been associated with the inhibition of nasal colonization by Streptococcus pneumoniae and Corynebacterium pseudodiphtheriticum with the eradication of Staphylococcus aureus from the human nose, including methicillin-resistant S. aureus) [49,50].

Conclusions
Our study evidenced that 16S RNA gene sequencing, previously proposed cut-off of rpoB, as well as MALDI-TOF spectroscopy, presented limited resolution for discriminating closely related and new Corynebacterium species, leading to an underestimated diversity of this genus in the female urinary microbiome.
This work expanded the knowledge on the diversity of Corynebacterium species inhabiting the FUM. Future research studying the biological mechanisms of the new Corynebacterium species here described, in the context of the genitourinary tract, may shed light on their possible beneficial role.