Novel spore-forming species exhibiting intrinsic resistance to third- and fourth-generation cephalosporins and description of Tigheibacillus jepli gen. nov., sp. nov.

ABSTRACT A comprehensive microbial surveillance was conducted at NASA’s Mars 2020 spacecraft assembly facility (SAF), where whole-genome sequencing (WGS) of 110 bacterial strains was performed. One isolate, designated 179-BFC-A-HST, exhibited less than 80% average nucleotide identity (ANI) to known species, suggesting a novel organism. This strain demonstrated high-level resistance [minimum inhibitory concentration (MIC) >256 mg/L] to third-generation cephalosporins, including ceftazidime, cefpodoxime, combination ceftazidime/avibactam, and the fourth-generation cephalosporin cefepime. The results of a comparative genomic analysis revealed that 179-BFC-A-HST is most closely related to Virgibacillus halophilus 5B73CT, sharing an ANI of 78.7% and a digital DNA-DNA hybridization (dDDH) value of 23.5%, while their 16S rRNA gene sequences shared 97.7% nucleotide identity. Based on these results and the recent recognition that the genus Virgibacillus is polyphyletic, strain 179-BFC-A-HST is proposed as a novel species of a novel genus, Tigheibacillus jepli gen. nov., sp. nov (type strain 179-BFC-A-HST = DSM 115946T = NRRL B-65666T), and its closest neighbor, V. halophilus, is proposed to be reassigned to this genus as Tigheibacillus halophilus comb. nov. (type strain 5B73CT = DSM 21623T = JCM 21758T = KCTC 13935T). It was also necessary to reclassify its second closest neighbor Virgibacillus soli, as a member of a novel genus Paracerasibacillus, reflecting its phylogenetic position relative to the genus Cerasibacillus, for which we propose Paracerasibacillus soli comb. nov. (type strain CC-YMP-6T = DSM 22952T = CCM 7714T). Within Amphibacillaceae (n = 64), P. soli exhibited 11 antibiotic resistance genes (ARG), while T. jepli encoded for 3, lacking any known β-lactamases, suggesting resistance from variant penicillin-binding proteins, disrupting cephalosporin efficacy. P. soli was highly resistant to azithromycin (MIC >64 mg/L) yet susceptible to cephalosporins and penicillins. IMPORTANCE The significance of this research extends to understanding microbial survival and adaptation in oligotrophic environments, such as those found in SAF. Whole-genome sequencing of several strains isolated from Mars 2020 mission assembly cleanroom facilities, including the discovery of the novel species Tigheibacillus jepli, highlights the resilience and antimicrobial resistance (AMR) in clinically relevant antibiotic classes of microbes in nutrient-scarce settings. The study also redefines the taxonomic classifications within the Amphibacillaceae family, aligning genetic identities with phylogenetic data. Investigating ARG and virulence factors (VF) across these strains illuminates the microbial capability for resistance under resource-limited conditions while emphasizing the role of human-associated VF in microbial survival, informing sterilization practices and microbial management in similar oligotrophic settings beyond spacecraft assembly cleanrooms such as pharmaceutical and medical industry cleanrooms.


IMPORTANCE
The significance of this research extends to understanding microbial survival and adaptation in oligotrophic environments, such as those found in SAF.Whole-genome sequencing of several strains isolated from Mars 2020 mission assem bly cleanroom facilities, including the discovery of the novel species Tigheibacillus jepli, highlights the resilience and antimicrobial resistance (AMR) in clinically relevant antibiotic classes of microbes in nutrient-scarce settings.The study also redefines the taxonomic classifications within the Amphibacillaceae family, aligning genetic identities with phylogenetic data.Investigating ARG and virulence factors (VF) across these strains illuminates the microbial capability for resistance under resource-limited conditions while emphasizing the role of human-associated VF in microbial survival, informing sterilization practices and microbial management in similar oligotrophic settings beyond spacecraft assembly cleanrooms such as pharmaceutical and medical industry clean rooms.
KEYWORDS cephalosporin, phylogenetic analysis, Virgibacillus taxonomy P hylogenetic investigations into the Bacillaceae family have revealed a polyphy letic structure, signaling the necessity for taxonomic re-evaluation to accurately depict evolutionary lineages (1).This has been partially addressed by the recently proposed family Amphibacillaceae (2), encompassing halophilic genera like Virgibacillus (3), renowned for its robustness in saline environments (4)(5)(6)(7).This family, which includes 16 genera, contains many bacterial taxa that have halophilic or halotolerant traits, with members commonly isolated from marine habitats, salt lakes, saline soils, and seafood, suggesting significance in food safety (8,9).
Aerobic spore-forming bacteria, with their diverse ecological adaptations, are instrumental in biotechnological applications and have implications for human health due to their secondary metabolite production.These metabolites range from antimicro bial agents, such as bacitracin produced by Bacillus licheniformis (10) and polymyxin E produced by Paenibacillus polymyxa (11).Amphibacillaceae members like Oceanobacillus (6) not only demonstrate antimicrobial compound production but also present a fertile ground for discovering novel metabolites (12).
While typically non-pathogenic, some Bacillaceae members, such as Bacillus cereus and Bacillus anthracis, possess VF enabling opportunistic infections (13,14).Conversely, Amphibacillaceae pathogens are rare; however, any bacterium can become opportunis tic, especially in immunocompromised hosts.For example, a recent publication identified an Oceanobacillus spp.infection in an immunocompetent patient, with the pathogenesis mechanisms still largely unidentified, highlighting the knowledge gaps in the virulence of halophilic species (15).
Environmental Amphibacillaceae strains also harbor ARG, indicating that resistance can evolve even without direct clinically associated antibiotic exposure.The discovery of the β-lactamase OIH-1 in Oceanobacillus iheyensis that confers resistance to several penicillins but no other β-lactams exemplifies the independent evolution of AMR in remote environments like the oceanic depths (16,17).Virgibacillus species have shown resistance to aminoglycosides and other antibiotics, with cephalosporins maintaining their clinical relevance against Gram-positive spore formers (18).Despite the availabil ity of first-line treatments for Gram-positive spore-former-induced infections, such as vancomycin, erythromycin, and ciprofloxacin (19), the role of cephalosporins in treating such infections remains clinically relevant.These findings underscore the underappreci ated role of natural environments in shaping AMR and necessitate vigilance against emerging ARG threats.
As part of a microbial surveillance study (6 months) associated with the NASA Mars 2020 mission, spacecraft assembly environments were assessed for microbial contamina tion (20).Bacterial strains (n = 110) were isolated from cleanroom samples that were subjected to a heat-shock (80°C; 15 min) procedure and cultured aerobically as per the NASA standard spore assay (21), and their genomes were sequenced (22).
The primary aim of this study was to characterize a bacterial isolate retrieved from the Mars 2020 mission assembly facility, identified as belonging to Amphibacillaceae through traditional microbiological methods and genomic analyses.Conserved marker genes were analyzed, and the phylogenetic affiliations of the isolate were delineated.A secondary objective was to determine the relative abundance of the novel isolate on the floors of the SAF cleanrooms (n = 236 samples) where the Mars 2020 mission rover had been assembled.Detailed genetic profiles of the species described herein were created, including ARG and VF profiles, and their distinct phenotypic traits were predicted.Their AMR phenotypes were assayed, and MIC were defined.Lastly, a predictive functional analysis was conducted, revealing genes encoding for putative bioactive compounds.This approach provides insights into the survival mechanisms of spore-forming bacteria in the harsh and nutrient-deficient environments of SAF cleanrooms, which could pose challenges to future NASA life detection missions.

Genome assembly
The genome size of the novel strain 179-BFC-A-HS T is 3,877,640 bp, and G+C content is 41.64%.Strain 5B73C T has a 4,199,006-bp genome with 41.59% G+C content, and the genome of CC-YMP-6 T strain is 3,876,281 bp with 36.13%G+C value.Other genome statistics are summarized in Table 1 and File S1.

Genotypic characterization of ARG and VF
In the Amphibacillaceae family (n = 64 species), 52 distinct ARG across 11 genera (Fig. 1A and B) were identified, with over 80% coverage and 70% identity to the NCBI's ARG database.These ARG confer resistance to multiple antibiotic classes.Strain 5B73C T harbors 11 ARG, resistant to aminoglycosides, diaminopyrimidines, macrolide-lincosa mide-streptogramin B (MLSB), tetracyclines, and glycopeptides.Fifteen species possess a single ARG, with strain 179-BFC-A-HS T exhibiting three ARG related to vancomycin resistance.
Following a bacteremia case in South Korea caused by an Oceanobacillus species, a VF gene analysis across related genera revealed 17 human-associated VF genes (Fig. 2), aligning with the virulence factor database (VFDB) (23).Pseudogracilibacillus contained the most VF (n = 8), while Tigheibacillus had the fewest (n = 3).EF-Tu-and ATP-dependent Clp protease genes appeared in all 64 tested genomes.In contrast, hasC, bspD/E/F, and lspA were unique to single genomes: Cerasibacillus terrae (hasC), Oceanobacillus zhagkaii (bspE/F), and Ornithinibacillus scapharcae (lspA), respectively.These results underscore the varied resistance and virulence potentials within Amphibacillaceae, indicative of an environmental influence on emerging AMR and virulence.

Phylogeny and genomic relatedness
The comparative genomic analysis indicates that the strain 179-BFC-A-HS T shares considerable genomic similarity with strain 5B73C T , with an ANI of 78.7% (File S1) and a dDDH value of 23.5%.A heat map of ANI, including representative genomes of type species of the family Amphibacillaceae (n = 64), is shown in File S2.The gyrB sequence similarity between these strains is 79.13%, and the 16S rRNA gene sequence of strain 179-BFC-A-HS T shares a 97.7% similarity with that of 5B73C T .The next closest genetic relatives based on 16S rRNA gene identity are Virgibacillus marismortui (95.77%) and Virgibacillus salaries (95.71%).Phylogenetic trees, including representative genomes of all validly published species of the family Amphibacillaceae, were reconstructed using 16S rRNA and gyrB gene sequences.These trees reveal a distinct clade, uniquely identifying the new strain 179-BFC-A-HS T (File S3).Further average amino acid identity (AAI) comparisons indicate that strains 179-BFC-A-HS T and 5B73C T share a 69.82%AAI, substantiating their classification within the same genus.In contrast, the AAI values between 179-BFC-A-HS T and CC-YMP-6 T , and between 5B73C T and CC-YMP-6 T , are 58.53% and 55.88%, respectively, which support their categorization in different genera from CC-YMP-6 T .Recent analysis based on the Genome Taxonomy Database (GTDB) has reclassified many genera within Bacillaceae, including Virgibacillus into the new family Amphiba cillaceae (2).Genomic analysis reveals Virgibacillus is polyphyletic, interspersed with genera like Oceanobacillus, Lentibacillus, Paucisalibacillus, and Cerasibacillus (Fig. 3).Consequently, strain 179-BFC-A-HS T is proposed as a new genus and species, Tigheiba cillus jepli gen.nov., sp.nov.V. halophilus 5B73C T is reclassified as Tigheibacillus halophi lus comb.nov.and V. soli CC-YMP-6 T as Paracerasibacillus soli comb.nov., positioned between Cerasibacillus and Tigheibacillus.The phylogenetic analysis indicates the genus Virgibacillus within Amphibacillaceae differentiates into clades E, F, and G, suggesting the need for further taxonomic studies to determine if these should be distinct genera.

Differential characteristics of Tigheibacillus genus
Table 2 highlights distinctive features of Tigheibacillus, differentiating it from other Amphibacillaceae genera strains.Our study compared phenotypes of type strains from five genera: Tigheibacillus, Virgibacillus, Oceanobacillus, Ornithinibacillus, and Lentibacillus, comprising 96 recognized species.T. jepli 179-BFC-A-HS T grows between 4°C and 40°C (optimal at 35°C) and tolerates up to 15% NaCl, indicating moderate heat and high salt tolerance.This contrasts with Lentibacillus, which prefers cooler temperatures (30°C) and tolerates up to 23% salt.T. jepli 179-BFC-A-HS T can produce acid from D-mannose and D-trehalose, a trait shared with Virgibacillus but not Ornithinibacillus or Lentibacillus.Its iso-C15:0/anteiso-C15:0 fatty acid ratio is 1.6, differing from Virgibacillus and Lentibacil lus, implying variations in cell membrane structure.Tigheibacillus has a unique A1γ peptidoglycan type with meso 2,6-DAP, distinct from other genera's peptidoglycan types.While sharing phosphatidylglycerol as a common lipid with other genera, its specific MK-7 quinone and DNA G+C content of 41.6% further set it apart biochemically.

Description of Tigheibacillus gen. nov.
Tigheibacillus (Tig.he.i.ba.cil'lus.L. masc.n. bacillus, a small rod; N.L. masc.n.Tigheibacillus a rod named to honor Scott Tighe, an American microbiologist, for his contribution in all areas of microbiology and extremophiles).

Description of Tigheibacillus jepli sp. nov.
Tigheibacillus jepli (jep'li.N.L. gen.n. jepli, arbitrary name derived from the abbreviation JPL, meaning of or pertaining to the NASA's Jet Propulsion Laboratory, where the type strain was isolated).
Basonym: Virgibacillus halophilus An et al. (32).The description is identical to that given by An et al. (32).The type strain, 5B73C T = DSM 21623 T = JCM 21758 T = KCTC 13935 T , was isolated from field soil in Kakegawa, Shizuoka, Japan.The reclassification of Virgibacillus soli to Paracerasibacillus soli is based on the phylogenetic analyses of 120 conserved protein genes.The description of the genus is identical to that given by Kämpfer et al. (33).The key properties of this species, now representing the genus Paracerasibacillus, include Gram-positive bacilli, predominant isoprenoid quinone of menaquinone MK-7, a polar lipid profile featuring diphosphatidyl glycerol, phosphatidylglycerol, and phosphatidylethanolamine, and a polyamine pattern dominated by spermidine.Additionally, physiological and biochemical tests differentiate this strain from other Virgibacillus species.These distinguishing features support the reclassification of Virgibacillus soli as the type species of the new genus Paracerasibacillus, named Paracerasibacillus soli.

In silico metabolic predictions
In silico metabolic predictions for T. jepli, T. halophilus, P. soli, and 61 other genomes corresponding to type species representing genera within the Amphibacillaceae were conducted using the distilled and refined annotation of metabolism (DRAM) software (Fig. 5).The genome of T. jepli 179-BFC-A-HS T encoded for parts 2 and 3 of the acetate to methane pathway, nitrate to nitrite and nitrite to nitrate metabolic pathways, arsenic and mercury reduction pathways, and as part 2 and part 1 of the butyrate and acetate pathways, respectively.
The presence and characteristics of various biosynthetic gene clusters (BGCs) within the genome of T. jepli 179-BFC-A-HS T were predicted using antiSMASH v.7.0.0 with a "strict" detection system.A loosely related (8% identity) BGC corresponding to a type III polyketide synthase cluster and specifically to legonindolizidine A6 was identified (Table S3).Furthermore, the production of a yet-to-be-characterized terpene cluster-associated metabolite was also predicted.

Metagenomics-based mapping to reads generated from JPL-SAF
The genome of the newly characterized T. jepli was tracked in metagenomic data sets gathered from the JPL-SAF during a period spanning 6 months in 2016, coinciding with the preparation phase for the Mars 2020 rover's components.Analysis of 236 paired-end shotgun metagenomic data sets, with a threshold of greater than 1% genome coverage, revealed genomic evidence of T. jepli in 15 samples as depicted in Fig. 6.These samples were collected over six different dates ranging from 15 March to 28 June 2016.The findings suggest that T. jepli is present in low relative abundance and demonstrates limited spatial-temporal distribution within the SAF environment.

DISCUSSION
The genomic landscape within the Bacillaceae family has undergone substantial revaluation, particularly with the reclassification of numerous genera following the rank normalization process by GTDB (2).Our comparative genomic analysis emphasizes this taxonomic fluidity, revealing that strain 179-BFC-A-HS T is not closely aligned with the current genus Virgibacillus.Although ANI values below the 95%-96% threshold typically suggest a novel species (34), a universally accepted threshold for genus-level distinction is still elusive.To confirm our genomic findings, we conducted an in-depth phylogenetic analysis, using marker genes (16S rRNA gene and gyrB) and genome-based phylogeny involving 120 conserved protein markers.The average AAI value between P. soli and both T. jepli and T. halophilus genomes is substantially below the 63.43% suggested as a threshold for genus delineation (35).In addition, the dDDH analysis further supports the classification of isolate 179-BFC-A HS T as a novel genus, with values below the 70%-mark indicative of a distinct species or potentially novel genera (36).While genomic data play a pivotal role in the delineation of species (ANI) and genera (AAI), the core attributes of an organism are frequently manifested through its phenotypic traits and the makeup of its cell wall.Consequently, we adopted an integrative methodology, combining genomic insights with phenotypic characteristics and chemotaxonomic data to achieve a comprehensive understanding.Based on both phenotypic and genotypic evidence and adhering to the rules of the International Code of Nomenclature of Prokaryotes (ICNP) (37), we propose the establishment of a new species within a new genus Tigheibacillus for the strain 179-BFC-A-HS T .
Our phylogenetic analysis reinforces the polyphyletic nature of Virgibacillus (26), as members of this genus are separated by other distinct genera such as Oceanobacillus and Lentibacillus, suggesting a need for re-evaluation of its genus boundaries (6,38).In contemporary research, genome-based phylogeny is widely recognized as the primary reference point for genus delineation despite the lack of uniform standards in this area.However, the absence of universally applicable thresholds presents significant challenges in making comparative analyses across taxa within the same hierarchical rank (39).This has been addressed by the introduction of the relative evolutionary divergence approach in GTDB (40), resulting in rank-normalized taxonomy for bacterial and archaeal taxa.The divergence of strains 179-BFC-A-HS T and 5B73C T , leading to their reassignment to the newly proposed genus Tigheibacillus, reflects their genetic distinction within the Amphibacillaceae family.Moreover, based on the phylogenetic placement and relative evolutionary divergence, we suggest to place V. soli into its own genus Paracerasibacillus, to reflect its distinction from Virgibacillus (41).Furthermore, our study calls for more investigation into the clades E, F, and G within Virgibacillus, identified as potentially distinct genera.
Species within the Amphibacillaceae, including Virgibacillus and related genera, are generally not associated with human diseases.Yet, O. oncorhynchi subsp.incaldanen sis has been reported to infect immunocompetent individuals (15) showing growth preference for alkaline environments like the male reproductive system (42).Further more, there are documented instances of Virgibacillus senegalensis and Lentibacillus sp.CBA3610 being isolated from the human gut (4,24), indicating possible transient colonization with unknown systemic effects.Our VF analysis of the Amphibacillaceae family revealed 17 human-associated VF gene homologs.Notably, the hasC gene homolog in C. terrae's genome is associated with biofilm formation and commonly found in pathogens like group B streptococci (43).The type IV secretion system effector BspE homolog in O. zhagkaii and the signal peptidase II encoding lspA homolog in O. scapharcae are associated with the human pathogens Brucella species and Rickettsia typhi, respectively (44,45), highlighting a diverse range of VF.This range of human-asso ciated VF in the genomes of environmental microorganisms signifies the importance of further understanding the ecological role of VF genes as well as the need for an in vitro evaluation of the pathogenic potential of such microorganisms.
T. jepli 179-BFC-A-HS T exhibits an unusual antibiotic resistance phenotypic pro file, showing high-level resistance to third-and fourth-generation cephalosporins (MIC >256 mg/L), even when these are combined with the β-lactamase inhibitor avibactam while remaining susceptible to ampicillin.This selective resistance pattern is atypical and not fully explained by the known type resistome as no known β-lactama ses were identified.Albeit atypical, similar resistance patterns have been observed in Bacillus species within the Bacillaceae family.Adamski et al. (46) reported susceptibility to ampicillin and resistance to cefotaxime among a subset of isolates of B. cereus, B. pumilus, and B. licheniformis isolated from raw milk.
β-Lactam antibiotics function by binding to penicillin-binding proteins (PBPs), inhibiting cell wall synthesis (47).In Gram-positive bacteria, PBPs are more accessible due to the lack of an outer membrane (48), and resistance often involves PBPs with low β-lactam affinity (25,49).The resistance phenotype in T. jepli 179-BFC-A-HS T may be attributed to modifications in PBPs, diminishing their affinity for cephalosporins relative to penicillin antibiotics.The β-lactam-inducible PBPs of T. jepli 179-BFC-A-HS T show substancial divergence (<63.7% amino acid identity) from those in closely related species, suggesting a distinct resistance profile.The isolation of cephalosporin-resistant bacteria like T. jepli 179-BFC-A-HS T in cleanrooms signals a need for the pharmaceut ical and medical industries to strengthen microbial reduction protocols and invest in new decontamination technologies.Such measures are vital to safeguard product integrity, comply with stricter regulatory standards, and mitigate health risks, ensuring the reliability of the global supply chain and maintaining industry reputation against the backdrop of rising antibiotic resistance (50).
Conversely, P. soli CC-YMP-6 T presented high-level resistance to azithromycin (MIC > 64 mg/L), a clinically significant macrolide antibiotic for treating infections caused by Gram-positive bacteria (51).This resistance is likely due to the presence of plasmid-medi ated erythromycin methyltransferase genes (ermG/Y/C), which confer resistance to MLSB antibiotics (52).Isolated from mountainous soil in Yang-Ming Mountain, Taiwan, P. soli CC-YMP-6 T 's possession of erm might offer uncharacterized adaptive advantages or indicate anthropogenic environmental contamination.The detection of erm genes in soil samples, particularly in urban areas with high anthropogenic activity, varies widely (53), underscoring the need to understand the environmental and evolutionary factors influencing AMR in natural settings.
T. jepli 179-BFC-A-HS T genomic analysis predicts a metabolic profile involved in methanogenesis, nitrogen cycling, and reductive reactions, notably in the latter stages of acetate-to-methane conversion.It also exhibits pathways for nitrogen utilization, reducing heavy metals like arsenate and mercury, while it lacks pathways for carbo hydrate-active enzymes (CAZy) and short-chain fatty acid (SCFA)/alcohol transforma tions.The predicted heavy metal metabolic capacity of T. jepli 179-BFC-A-HS T aligns with the members of the genus Bacillus known for their bioremediation potential in reducing environmental concentrations of heavy metals (54).The metabolic traits of T. jepli indicate its putative capacity to utilize energy and nutrients from limited sources, neutralize heavy metals, and participate in biogeochemical cycles, suggesting a resilient phenotype suitable for nutrient-limited environments such as controlled clean rooms.The resilience of T. jepli in oligotrophic environments, mirroring traits of related Amphibacillaceae genera like Oceanobacillus and Virgibacillus, highlights their efficient nutrient utilization and stress resistance (6,33).Such traits are vital for survival in nutrient-poor habitats, suggesting a possible role in nutrient cycling and ecosystem stability (55).Studying these related genera enhances our understanding about the ecological role of T. jepli.
Metagenomic analysis in JPL-SAF revealed the infrequent occurrence of T. jepli 179-BFC-A-HS T during the assembly phase of the Mars 2020 rover subsystems.Over 6 months in 2016, genomic signatures of T. jepli were identified in only 15 of 236 samples.This intermittent presence implies that T. jepli may have a niche-specific ecology or be a transient organism in the SAF environment.Generally, clean room microbiomes are dominated by gram-positive, spore-forming species, with Bacillus species comprising 10%-13% of isolates (56).Previous culture-based analyses in the JPL-SAF have iden tified 16 bacterial genera, frequently detecting Bacillus subtilis and V. pantothenticus (20).Bacillaceae and Amphibacillaceae species are notable for their persistence in such environments.Understanding the low prevalence of certain Amphibacillaceae species, including T. jepli, can inform contamination control strategies for space missions (57) and contribute to planetary protection efforts.
The presence of a high 16S rRNA gene copy number (16S GCN) in T. jepli is an intriguing aspect of the findings that merits further investigation.The 16S GCN in bacteria usually ranges from 1 to 15 and is often correlated with ecological strategies: oligotrophs, which thrive in nutrient-poor environments, usually have a lower 16S GCN, while copiotrophs, which flourish in nutrient-rich settings, have a higher 16S GCN, reflecting their adaptive strategies to their respective environment (58).The high full length 16S GCN in T. jepli (n = 16), found in the oligotrophic environment of the SAF, suggests it might confer an advantage for growth when conditions become favorable (59).The observed high 16S GCN copy number may also confer ecological versatility, allowing T. jepli to rapidly adjust to sporadic nutrient availability, which could be a result of evolutionary adaptations (60).While a high 16S GCN is advantageous for initial growth, it is suggested that the ability to endure extended periods of starvation involves more complex survival mechanisms that go beyond just the number of 16S GCN (61).Furthermore, the presence of 27 dormancy, sporulation, and spore germination-associ ated genes in the genome of T. jepli indicates their role in enabling survival in nutrientpoor environments, employing spore formation as an adaptive mechanism for survival.
The robustness of T. jepli in oligotrophic environments, coupled with its potential for heavy metal detoxification, mirrors similar capabilities observed in Bacillus species (54), indicating its potential suitability for bioremediation in nutrient-scarce, contamina ted areas.Additionally, atypical resistance profile of T. jepli, alongside its occurrence in human-associated settings, necessitates further investigation into its pathogenicity.Studies focusing on its interactions with human cells and serum resistance are essential for evaluating potential health risks in medical and industrial contexts.

Conclusion
In conclusion, microbial surveillance conducted as part of the Mars 2020 mission revealed a novel bacterial strain, 179-BFC-A-HS T , classified as T. jepli which represents the type strain of a novel species of a novel genus named herein.The high-level resistance of T. jepli to third-and fourth-generation cephalosporins, contrasted with its susceptibil ity to penicillins, along with its unique genetic profile, underscores the importance of characterizing resistance patterns, even in the absence of clinically associated envi ronmental pressures.Furthermore, several human pathogenicity-associated VF were identified in the genome of T. jepli.The metabolic versatility of T. jepli, particularly its involvement in pathways such as methanogenesis, nitrogen cycling, reductive reactions, and heavy metal detoxification, highlights the resilience and adaptability of microorgan isms in nutrient-limited environments.These findings are not only crucial for space exploration but also have significant implications for maintaining stringent microbial control standards in other sensitive industries, such as semiconductor manufacturing, pharmaceutical production, and medical device fabrication, where microbial contamina tion can have profound impacts.

MATERIALS AND METHODS
Sample collection, isolation, and preliminary identification of the strain 179-BFC-A-HS T using 16S rRNA gene phylogeny were presented elsewhere (20).Additionally, type strains of CC-YMP-6 T (formerly Virgibacillus soli DSM 22952 T ) and 5B73C T (formerly Virgibacillus halophilus DSM 21623 T ) were purchased from the Leibniz Institute DSMZ-the German Collection of Microorganisms and Cell Cultures GmbH.Their genomes were sequenced, and extensive AMR phenotypic analyses were performed.

Phylogeny and genome annotations
A two-step phylogenomic analysis was conducted on isolate 179-BFC-A-HS T using 120 conserved single-copy marker proteins (40) as follows: initially using GTDB-Tk v2.3.0 (68) by running (i) the ani_rep command to compare genome sequence from strain 179-BFC-A-HS T against all GTDB representative genomes (release 08-RS214) and (ii) classify_wf to find out the maximum-likelihood placement in the GTDB reference tree.Based on the output from the classify workflow (classify_wf ), we selected a total of 61 genome sequences representing closely related type strains of all genera from the family Amphibacillaceae according to GTDB taxonomy (08-RS214), including genomes sequenced as part of this study from P. soli CC-YMP-6 T and T. halophilus 5B73C T strains.Second, we inferred genome tree from the multiple sequence alignment of 61 selected genomes including the genome of strain 179-BFC-A-HS T and two genomes representing an outgroup using FastTree v.2.1.11(69) with the WAG model executed via GTDB-Tk v2.3.0.The inferred tree was further used as a starting tree in the IQ-TREE v.2.1.2(70) executed via the following command: iqtree -s < alignment > m LG + C10+F + G -ft < starting_tree> -bb 1000.The tree was visualized, rooted, and pruned for illustration purposes in ARB v.6.0.6 and beautified in iTOL (71).
We assessed the genetic relatedness of 64 genomes in this study by creating an ANI heatmap with ANIclustermap (v1.2.0), which utilizes fastANI and seaborn algorithms.
Genomic annotations were performed using PROKKA v1.14.5 with the --compliant extension (72).AAI values were determined using protein sequences as input based on the AAI calculator software developed by Kostas Lab.We extracted the gyrB and 16S rRNA gene sequences of T. jepli 179-BFC-A-HS T and compared their percentage identities with their three closest sequences using BLASTn.Additionally, the dDDH value was estimated using formula 2 of the Genome-to-Genome Distance Calculator v.3.0 and the BLAST+ alignment tool (73).For the 61 species of Amphibacillaceae family, we extracted their 16S rRNA gene sequences from NCBI and aligned them with P. soli CC-YMP-6 T , T. jepli 179-BFC-A-HS T , and T. halophilus 5B73C T using MUSCLE v3.8.1551.A phylogenetic tree was constructed using IQTREE v.2.2.0.3 (74) the GTR + G model and 1,000 ultrafast bootstrap replicates, while trees were visualized in iTOL v.6.7 (75).

Light microscopy and scanning electron microscopy
Spore formation was checked using malachite green and safranin followed by light microscopy using a DP25 camera and cellSens software (76).SEM analyses were carried out as previously published (77), and images were collected with an FEI Quanta 200F electron microscope at the California Institute of Technology's Kavli Nanoscience Institute.

Biochemical and phenotypic characteristics
We used the Vitek 2 GP ID (bioMérieux) card for biochemical testing as per the manu facturer's guidelines.Colonies from TSA were aseptically transferred into saline (0.45%-0.50%NaCl, pH 4.5-7.0) to create a 0.5-McFarland suspension (~1.5 × 10 8 cfu/mL), verified with VITEK 2 DensiCHEK Plus.This suspension, along with the Vitek 2 GP ID card, was incubated in a cassette at 37°C.Cassette loading, data entry, and retrieval of results (within 10 hours of inoculation) followed the VITEK instrument manual.
For phenotypic fingerprinting, a GNIII MicroPlate (BioLog) was used, preparing inoculum from TSA colonies in solution A (Cat # 72401; BioLog) to achieve a McFarland of 0.5.A volume of 100 µL of this bacterial suspension was loaded into each well of a GNIII MicroPlate and incubated at 37°C for 24 hours.OmniLog readings (A 590 -A 750 ) were taken with a FLUO star Omega MicroPlate reader (BMG Labtech, Germany) within 24 hours of incubation.

Chemotaxonomic analysis
Cells grown on TSA at 30°C for 48 hours to mid-exponential phase were used for fatty acid methyl esters (FAMEs) analysis.FAMEs were extracted from cell biomass via sequential saponification, methylation, and extraction (79) and then separated using a gas chromatograph (Agilent 7890A) with a flame ionization detector.Identification of FAMEs utilized the Microbial Identification System (MIDI) (80) and the Aerobe database (RTSBA6) in Sherlock version 6.0, adhering to the standard protocol (81).
Cells grown on TSA for 3 days at 30°C were used for extracting polar lipids, qui nones, and peptidoglycans.Polar lipids and quinones were analyzed using 2D thin-layer chromatography (TLC) (82).TLC plates were sprayed with various reagents for lipid identification: 10% ethanolic phosphomolybdic acid for total lipids, 0.2% ninhydrin in butanol for aminolipids, Dittmer and Lester's Zinzadze reagent for phospholipids, and alpha-naphthol for glycolipids.Peptidoglycans characterization followed established protocols by Staneck and Roberts (83).

Genotypic prediction of metabolic traits
The DRAM software v. 1.4.6 (84) was used to analyze the genomic assemblies of all three strains tested during this study and 61 associated representative genomes of type species within the Amphibacillaceae family, for electron transport chain complexes and key metabolic functions, assigning a completeness scale from 0 (absent) to 1 (fully complete).This analysis included CAZy profiles, nitrogen and sulfur metabolism, reductases, photosynthesis pathways, methanogenesis, methanotrophy, and SCFA and alcohol conversions.Features were color coded for presence or absence in the genomic data.For strain 179-BFC-A-HS T , antiSMASH v.7.0.1 was additionally used with a "strict" detection method (85) to predict BGCs associated with secondary metabolites, aligning them with the MIBiG v.3.1 database for functional annotations (85).

Mapping of the novel species to SAF metagenomic reads
Shotgun metagenomics sequence data sets (236 samples) from the NASA's-JPL SAF were tested to evaluate the prevalence of strain 179-BFC-A-HS T (20).Quality control and adapter trimming were conducted using Fastp v.0.23.4 with default settings.To map and quantify the metagenome reads relative to the novel species, MetaCompass v.2.0 was used with a maximum read length of 150, employing a reference-guided assembly approach.This included using Bowtie2 for read alignment and MEGAHIT for consensus genome construction.Samples without sequence data mapping the reference genome were excluded from further analysis.

FIG 2
FIG 2 Virulence factors.(A) The virulome of the newly described T. jepli 179-BFC-A-HS T and genomes from the type species of associated genera (n = 64).The number of VF per genome is colored by associated genera.(B) Breakdown of VF per genome.Color gradient reflects percentage identity with respective gene in the VFDB.Only hits >70% identity are depicted.White color indicates absence of the gene.(C) An overview of mean number (± stdev) of identified VF per genus.Tigheibacillus spp. is highlighted in red text, and Paracerasibacillus soli is highlighted in blue text.

FIG 3
FIG 3 Maximum-likehood phylogenomic tree based on 120 conserved marker genes showing the placement of 179-BFC-A-HS T with the closely related members of Amphibacillaceae family.The tree was rooted on Thalassobacillus devorans CECT 7046 and Pontibacillus chungwhensis BH030062 as outgroup (not shown).Ultrafast bootstrap values >90% are indicated below the branches.

TABLE 2
Differential characteristics of T. jepli 179-BFC-A-HS T and closely related type strains of the type species of the family Amphibacillaceae j

FIG 5
FIG 5 Phenotypic predictions based on genomic data using the DRAM tool.The chart showcases predicted metabolic capabilities and associated pathways, providing insight into the potential physiological roles and adaptations of T. jepli 179-BFC-A-HS T and associated representative genomes of type species within the Amphibacillaceae family.(A) Electron transport chain complexes green color-coded gradient based on their completeness, ranging from 0 (absent) to 1 (fully complete).(B) Metabolic predictions are visualized with specific focus on carbohydrate-active enzyme, nitrogen metabolism, sulfur metabolism, various other reductases, photosynthesis, methanogenesis, methanotrophy, short-chain fatty acid conversions, and alcohol conversions.Metabolic features are color coded to indicate whether a function is present (purple) or absent (white).

FIG 6
FIG6 Mapping of the novel species to SAF metagenomic reads: box plots showing the breadth of coverage of the consensus genome were constructed from mapped reads aligned to T. jepli 179-BFC-A-HS T (reported percentage breadth of coverage cutoff >1%).Reads mapping to the genome of T. jepli 179-BFC-A-HS T were identified for 15 samples retrieved from 6 different collection dates.