Micrococcus porci sp. nov., Isolated from Feces of Black Pig (Sus scrofa)

An aerobic bacterium, designated as strain KD337-16T, was isolated from the fecal samples of a black pig. It exhibited spherical, non-motile and non–spore-forming, Gram-positive cells. KD337-16T was identified as a member of the genus Micrococcus through 16S rRNA gene sequencing, and its closest relatives were found to be Micrococcus endophyticus YIM 56238T (99.5% similarity), Micrococcus luteus NCTC 2665T (99.1%), Micrococcus yunnanensis YIM 65004T (99.1%), Micrococcus aloeverae AE-6T (99.1%), Micrococcus antarcticus T2T (98.9%), and Micrococcus flavus LW4T (98.7%). Phylogenomic trees were constructed, and strain KD337-16T was found to form its own cluster as an independent lineage of M. flavus LW4T. Between KD337-16T and its close relatives, the average nucleotide identity, average amino acid identity, and digital DNA–DNA hybridization were below the respective species delineation thresholds at 82.1–86.6%, 78.1–86.1%, and 24.4–34.9%. The major cellular fatty acids and polar lipids were anteiso-C15:0 and iso-C15:0, and DPG and PG, respectively. The predominant menaquinone was MK-8(H2). Taken together, the results indicate that strain KD337-16T is a novel species of the genus Micrococcus, for which the name Micrococcus porci sp. nov. is proposed. The type strain is KD337-16T (=BCRC 81318T = NBRC 115578T).


Introduction
The genus Micrococcus was first described by Cohn [1], and this description was subsequently amended by Stackebrandt et al. [2] and Wieser et al. [3], with Micrococcus luteus as the type species. The genus Micrococcus belongs to the family Micrococcaceae, order Micrococcales, and phylum Actinomycetota. Nine species have been reported in this genus (https://lpsn.dsmz.de/genus/micrococcus, accessed on 28  M. luteus group [4]. Previous studies have reported the isolation of Micrococcus strains from numerous habitats, including human skin [5], permanently cold samples [6], activated sludge [7], the inner tissues of plants [8][9][10], soil [11], dairy waste [12], air [13], and intestine of several fish species [14,15]. These strains have been described as emerging opportunistic pathogens [16,17]. Pig is not only an important protein source for the human diet, but has also become increasingly important as biomedical animal model of human beings. The pig growth trait has been found to correlate with the gut microbiota [18][19][20], and the Prevotella copri has demonstrated to regulate the fat accumulation through a causal study in pigs [21]. Culturomics is an approach that is based on diverse culture conditions and enables the maximal recovery of culturable microorganisms from biological samples [22,23]. The cultivation projects of swine gut microbiota have been completed in recent years [24][25][26]. However, a huge portion of intestinal bacteria have still not been cultured. For a better understanding of the physiological impact of the gut microbiome of the host, to isolate, identify and characterize of uncultured bacteria is necessary. During a study aimed at isolating novel bacterial species present in the pig intestine using culturomics, one isolated strain, KD337-16 T could not be assigned to any recognized species of the genus Micrococcus using matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF MS) analysis. Here, we report the phenotypic, chemotaxonomic and genotypic characterization of strain KD337-16 T .

Isolation of Strain KD337-16 T and Culture Conditions
This study was approved by the Institutional Animal Care and Use Committee of Livestock Research Institute (permit no. LRI IACUC110-35). A fecal sample obtained from a KHAPS black pig (Sus scrofa) was collected from the Kaohsiung Animal Propagation Station in Pingtung County (approximate geographic coordinates: 22.63424 • N 20.60237 • E), Taiwan, in 2021. Wet-weight feces (1 g) were suspended in 10 mL of phosphate-buffered saline, and the suspension was subsequently homogenized. Serial dilutions were plated on blood agar that contained 5% sheep blood for 48 h of aerobic incubation at 37 • C. All of the isolates were subjected for strain dereplication using a MALDI Microflex LT mass spectrometer (Bruker Daltonics, Bremen, Germany), as previously described [27]. One strain, named KD337-16 T , could not be identified confidently. Strain KD337-16 T and its phylogenetically closest reference species, including M. endophyticus BCRC 16908 T , M. luteus BCRC 80739 T , M. yunnanensis BCRC 80243 T , M. aloeverae BCRC 80870 T , and M. flavus BCRC 80069 T , were routinely cultured on trypticase soy agar (TSA) at 37 • C for further taxonomic characterization, and the strains were then preserved in 10% glycerol at −80 • C.

DNA Extraction and Gene Sequence Comparison
A DNeasy kit (Qiagen, Valencia, CA, USA) was employed to extract and purify bacterial genomic DNA. The 16S rRNA gene and three housekeeping genes (gyrB, recA, and rpoB) were amplified and sequenced using a method reported elsewhere [4,28]. The EzBioCloud database (www.ezbiocloud.net/identify, accessed on 28 October 2022) was employed in subsequent BLAST analyses, and gene sequences were compared with those on NCBI GenBank (www.ncbi.nlm.nih.gov/BLAST/, accessed on 28 October 2022).

Phylogenetic Analysis
Clustal X (v. 2.1) software was employed for aligning sequences [29]. MEGA (v. 11) software was employed for phylogenetic tree reconstruction [30] based on sequences from the novel strain KD337-16 T , its close relative strains, a roughly 1375 bp segment of the 16S rRNA gene, and nearly 1870 bp of the concatenated sequences of the three housekeeping genes (recA, gyrB, and rpoB). The neighbor-joining (NJ) [31], maximum-likelihood (ML) [32] and minimum-evolution (ME) [33] methods and the Kimura two-parameter model were Life 2022, 12, 1749 3 of 12 used for tree reconstruction. Bootstrapping analysis with 1000 replicates was employed to determine how statistically reliable the trees were [34].

Genomic Analysis
A QIAamp PowerFecal Pro DNA Kit (Qiagen, Hilden, Germany) was employed to extract genomic DNA from the KD337-16 T strain. Subsequently, the SQK-LSK109 Ligation Sequencing Kit on a PromethION Flow Cell (R9.4.1) and Illumina NovaSeq 6000 in paired-end (2 × 151 bp) mode was used for Oxford Nanopore Technologies (ONT) sequencing. After the sequences had been decoded and refined, Flye version 2.8.3 was employed for assembly of the valid ONT sequences. The primary contigs were polished with Racon v1.4.22 and the Illumina read alignment results constructed using Minimap2 v2.17. The DDBJ Fast Annotation and Submission Tool was used to annotate the genome [35]. Methods described elsewhere [36][37][38] were employed to quantify the digital DNA-DNA hybridization (dDDH), the amino acid identity (AAI), and average nucleotide identity (ANI). The up-to-date bacterial core genes pipeline (http://leb.snu.ac.kr/ubcg2, accessed on 28 October 2022) [39] and EDGAR platform were utilized to construct phylogenomic trees [40], whereas the eggNOG 4.5 database and carbohydrate-active enzyme (CAZy) database were employed for functional assignment [41,42]. The OrthoVenn2 webserver was used for pangenome analysis [43]. Finally, AntiSMASH software (v. 6.0) was employed to predict putative biosynthetic gene clusters [44].

Phenotypic Characterization
For the phenotypic analysis, strain KD337-16 T was aerobically cultured on TSA at 37 • C for 48 h, unless otherwise stated. Cell morphology was observed under a phasecontrast microscope (Eclipse E600, Nikon, Tokyo, Japan). Gram staining was performed by using a Gram-staining kit (Difco, St. Louis, MA, USA) according to the manufacturer's instructions. Growth at different temperatures (4,15,20,28,30,37,42 and 50 • C), NaCl concentrations (0-15% w/v, at 1% intervals) and pH levels (pH 4.0-12.0, at 1.0 pH unit intervals), tested in tryptic soy broth (TSB), were investigated through the standard methods [45]. Catalase activity was determined by the reaction of fresh cells toward 3% hydrogen peroxide (H 2 O 2 ), and oxidase reaction was tested by using oxidase reagents (bioMerieux, Marcy-l'Étoile, France). The ability of the cells to utilize various sources of carbon and their enzyme activity were evaluated with commercial kits from API ZYM, API 20E, and Biolog GEN III MicroPlate system in accordance with the manufacturer's instructions.

Chemotaxonomic Characterization
MALDI-TOF MS was performed for whole-cell protein analysis in accordance with a method described elsewhere [27]. Dendrogram clustering was constructed with the setting of 200 (distance measure: correlation; linkage: average; score oriented) using MALDI BioTyper software (v. 3.1; Bruker Daltonics, Billerica, MA, USA). Biomass for analysis of whole-cell fatty acids, polar lipids and isoprenoid quinone were obtained by culturing strain KD337-16 T in TSB for 2 days at 30 • C. A previously reported method and the Sherlock Microbial Identification System (MIDI) were used to analyze whole-cell fatty acids as fatty acid methyl esters [46]. Polar lipids were extracted from 100 mg freezedried cells using the method described by Minnikin et al. [47] and analyzed by TLC using chloroform/methanol/water (65:25:4, by vol.) in the first direction and chloroform/acetic acid/methanol/water (80:18:12:5, by vol.) in the second. Lipids were visualized by spraying the TLC plate with 10% molybdophosphoric acid. Anisaldehyde (sugar), Schiff's reagent (glycol) and Dittmer-Lester reagent (phosphorous) were also used as specific spray reagents for polar lipids. The molecular species and concentrations of isoprenoid quinones were determined as described by Hamada et al. [48].

Results and Discussion
MALDI-TOF MS is a phenotype-based method that can be used for the rapid identification and dereplication of numerous isolates on the basis of specific proteomic profiles [49]. The MALDI-TOF MS spectra of type strain KD337-16 T , shown in Supplementary Figure S1, could not be reliably identified, yielding a log score of <1.7. On the basis of pairwise sequence analysis of the 16S rRNA gene, strain KD337-16 T and its closest relatives, the type strains M. endophyticus YIM 56238 T , M. luteus NCTC 2665 T , M. yunnanensis YIM 65004 T , M. aloeverae AE-6 T , M. antarcticus T2 T , and M. flavus LW4 T , showed high similarity values of 99.5%, 99.1%, 99.1%, 99.1%, 98.9%, and 98.7%, respectively (Table 1). Through phylogenetic analysis of 16S rRNA gene sequences, the strain was determined to be a member of the M. luteus group ( Figure 1). Members of the M. luteus group can be discriminated with good resolution using protein-encoding genes such as gyrB, recA and rpoB [4]. Among these three, gyrB has the highest discriminatory power at the interspecific level. The degrees of similarity between KD337-16 T and other members of the M. luteus group were determined to be 90.1-91.6% (Table 1) on the basis of the concatenated sequences (gyrB, recA, and rpoB). When the NJ method was used for reconstruction, the resulting phylogenetic tree showed that strain KD337-16 T constituted its own cluster and that this cluster was clearly separate from that of its close relatives ( Figure 2). Using the ME or ML method resulted in similar tree topology, which indicated that this strain may be a novel species.
Whole-genome sequencing is currently the most fruitful source of taxonomic information [50][51][52][53]. Comparative genomics, overall genome-related indices (e.g., AAI, ANI, and dDDH), and phylogenomic tree analyses may be critical approaches for estimating evolutionary distances among species and delineating prokaryotic taxa at the species and genus levels. The present study revealed that strain KD337-16 T had G+C content of 73.0% and a genome size of 2.64 Mb; the genome contained 2502 coding genes and 59 predicted RNA genes (Table 2). Between KD337-16 T and its close relations, the ANI, AAI, and dDDH varied from 82.1% to 86.6%, 78.1% to 86.1%, and 24.4% to 34.9%, respectively (Table 3), lower than 95-96%, 95%, and 70%, the respective generally accepted cutoffs for prokaryotic species. By contrast, the ANI, AAI, and dDDH values among the M. aloeverae, M. luteus, and M. yunnanensis type strains exceeded the species thresholds, indicating that the aforementioned strains are members of the same species [4]. Nevertheless, between M. luteus and M. aloeverae and between M. luteus and M. yunnanensis, the dDDH was 77.8% and 77.9%, respectively, below the threshold for subspecies delineation [54]. The phylogenomic trees were obtained on the basis of 81 and 368 core genes (Figures 3 and 4) shared by the investigated strains and we also discovered an independent cluster formed solely by strain KD337-16 T , thus confirming this strain's status as a novel species. A total of 2313 genes from strain KD337-16 T were assigned to 21 functional categories (Supplementary Figure S2). The most common categories among these functional groups belonged to the [L] (recombination and repair; 220 genes) and [E] (amino acid transport and metabolism; 214 genes) clusters. Of identified CAZy families, the strain KD337-16 T contained 4 carbohydrate binding modules, 28 glycoside hydrolases, and 12 glycosyl transferases, respectively. In addition, 2012 orthologous protein clusters were discovered in KD337-16 T . Of the shared clusters, 502 were common in the five Micrococcus species (Supplementary Figure S3), and 24 protein clusters were unique to the novel Micrococcus porci strain (Supplementary Table S1). KD337-16 T appears to produce putative secondary metabolite gene clusters, such as the beta lactone, terpene, and RiPP-like biosynthetic clusters.      Whole-genome sequencing is currently the most fruitful source of taxonomic information [50][51][52][53]. Comparative genomics, overall genome-related indices (e.g., AAI,  Cells of strain KD337-16 T were coccoid shaped (approximately 0.5-1 µm) (Supplementary Figure S4), non-motile and non-spore-forming Gram-, catalase-, and oxidase-positive facultative anaerobic cells. Colonies were cream to yellow, slightly convex, smooth and circular. Table 4 lists the phenotypic characteristics that can be used to distinguish this novel strain from its close relatives. Cluster analysis of MALDI-TOF MS spectra in the 2000-12,000 m/z region of Micrococcus strains revealed the unambiguous grouping of five distinct clusters, each defined by known species and our novel taxon (Supplementary Figure S5). The fatty acid analysis revealed the major fatty acids (>10%) in strain KD337-16 T to be anteiso-C 15:0 and iso-C 15:0 ; furthermore, this novel strain can be differentiated by analyzing whether C 16:0 is present and minor fatty acids such as iso-C 16:1 H, anteiso-C 17:1 ω9c, and summed features 1 and 3 are absent ( Table 5). The polar lipids of strain KD337-16 T were detected to be diphosphatidylglycerol, phosphatidylglycerol, phosphatidylinositol and unidentified glycolipid (Supplementary Figure S6). The predominant isoprenoid quinone of strain KD337-16 T was MK-8(H 2 ); MK-7(H 2 ) and MK-9(H 2 ) were detected as minor components (85:14:1). These features were in agreement with those of the genus Micrococcus [55].   Cells of strain KD337-16 T were coccoid shaped (approximately 0.5-1 μm) (Supplementary Figure S4), non-motile and non-spore-forming Gram-, catalase-, and oxidase-positive facultative anaerobic cells. Colonies were cream to yellow, slightly convex, smooth and circular. Table 4 lists the phenotypic characteristics that can be used to distinguish this novel strain from its close relatives. Cluster analysis of MALDI-TOF MS spectra in the 2000-12,000 m/z region of Micrococcus strains revealed the unambiguous grouping of five distinct clusters, each defined by known species and our novel taxon (Supplementary Figure S5). The fatty acid analysis revealed the major fatty acids (>10%) in strain KD337-16 T to be anteiso-C15:0 and iso-C15:0; furthermore, this novel strain can be differentiated by analyzing whether C16:0 is present and minor fatty acids such as iso-C16:1 H, anteiso-C17:1ω9c, and summed features 1 and 3 are absent ( Table 5). The polar lipids of   The tree was constructed by the maximum likelihood method the basis of a comparison of 368 core genes, and A. globiformis ATCC 8010 T was used as an outgroup. Bar, 5% sequence divergence.
Cells of strain KD337-16 T were coccoid shaped (approximately 0.5-1 μm) (Supplementary Figure S4), non-motile and non-spore-forming Gram-, catalase-, and oxidase-positive facultative anaerobic cells. Colonies were cream to yellow, slightly convex, smooth and circular. Table 4 lists the phenotypic characteristics that can be used to distinguish this novel strain from its close relatives. Cluster analysis of MALDI-TOF MS spectra in the 2000-12,000 m/z region of Micrococcus strains revealed the unambiguous grouping of five distinct clusters, each defined by known species and our novel taxon (Supplementary Figure S5). The fatty acid analysis revealed the major fatty acids (>10%) in strain KD337-16 T to be anteiso-C15:0 and iso-C15:0; furthermore, this novel strain can be differentiated by analyzing whether C16:0 is present and minor fatty acids such as iso-C16:1 H, anteiso-C17:1ω9c, and summed features 1 and 3 are absent ( Table 5). The polar lipids of The tree was constructed by the maximum likelihood method the basis of a comparison of 368 core genes, and A. globiformis ATCC 8010 T was used as an outgroup. Bar, 5% sequence divergence. Esterase * Data for the type strains were taken from Liu et al. [7], Chen et al. [8] and Rieser et al. [13].

Conclusions
According to the results of polyphasic characterization, strain KD337-16 T is genetically and phenotypically discernible from other currently recognized Micrococcus species. Thus, it represents a novel taxon for which the name Micrococcus porci sp. nov. is proposed.
Description of Micrococcus porci sp. nov.

Supplementary Materials:
The following supporting information can be downloaded at: https: //www.mdpi.com/article/10.3390/life12111749/s1, Figure S1: Representative spectra from M. porci sp. nov. KD337-16 T type strain. Figure S2: Results of an eggNOG functional category analysis of 2313 genes. Figure S3: Venn diagram showing the number of core, shared and unique genes (orthologous clusters) for each genome. Figure S4: Electron microscopic photograph of strain KD337-16 T after aerobic cultivation on TSA plate at 37 • C for 1 day. Figure S5: Dendrogram showing the clustering of the Micrococcus strains based on MALDI-TOF MS analysis. Figure S6: Two-dimensional thin-layer chromatograms of polar lipids from strain KD337-16 T . Table S1