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

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 ß-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.

Phylogenomics, phenotypic, and functional traits of five novel (Earth-derived) bacterial species isolated from the International Space Station and their prevalence in metagenomes.

With the advent of long-term human habitation in space and on the moon, understanding how the built environment microbiome of space habitats differs from Earth habitats, and how microbes survive, proliferate and spread in space conditions, is becoming more important. The microbial tracking mission series has been monitoring the microbiome of the International Space Station (ISS) for almost a decade. During this mission series, six unique strains of Gram-stain-positive bacteria, including two spore-forming and three non-spore-forming species, were isolated from the environmental surfaces of the ISS. The analysis of their 16S rRNA gene sequences revealed > 99% similarities with previously described bacterial species. To further explore their phylogenetic affiliation, whole genome sequencing was undertaken. For all strains, the gyrB gene exhibited < 93% similarity with closely related species, which proved effective in categorizing these ISS strains as novel species. Average nucleotide identity and digital DNA-DNA hybridization values, when compared to any known bacterial species, were < 94% and <50% respectively for all species described here. Traditional biochemical tests, fatty acid profiling, polar lipid, and cell wall composition analyses were performed to generate phenotypic characterization of these ISS strains. A study of the shotgun metagenomic reads from the ISS samples, from which the novel species were isolated, showed that only 0.1% of the total reads mapped to the novel species, supporting the idea that these novel species are rare in the ISS environments. In-depth annotation of the genomes unveiled a variety of genes linked to amino acid and derivative synthesis, carbohydrate metabolism, cofactors, vitamins, prosthetic groups, pigments, and protein metabolism. Further analysis of these ISS-isolated organisms revealed that, on average, they contain 46 genes associated with virulence, disease, and defense. The main predicted functions of these genes are: conferring resistance to antibiotics and toxic compounds, and enabling invasion and intracellular resistance. After conducting antiSMASH analysis, it was found that there are roughly 16 cluster types across the six strains, including ß-lactone and type III polyketide synthase (T3PKS) clusters. Based on these multi-faceted taxonomic methods, it was concluded that these six ISS strains represent five novel species, which we propose to name as follows: Arthrobacter burdickii IIF3SC-B10T (= NRRL B-65660T = DSM 115933T), Leifsonia virtsii F6_8S_P_1AT (= NRRL B-65661T = DSM 115931T), Leifsonia williamsii F6_8S_P_1BT (= NRRL B-65662T = DSM 115932T), Paenibacillus vandeheii F6_3S_P_1CT (= NRRL B-65663T = DSM 115940T), and Sporosarcina highlanderae F6_3S_P_2T (= NRRL B-65664T = DSM 115943T). Identifying and characterizing the genomes and phenotypes of novel microbes found in space habitats, like those explored in this study, is integral for expanding our genomic databases of space-relevant microbes. This approach offers the only reliable method to determine species composition, track microbial dispersion, and anticipate potential threats to human health from monitoring microbes on the surfaces and equipment within space habitats. By unraveling these microbial mysteries, we take a crucial step towards ensuring the safety and success of future space missions.

Phylogenetic affiliations and genomic characterization of novel bacterial species and their abundance in the International Space Station.

Background: With the advent of long-term human habitation in space and on the moon, understanding how the built environment microbiome of space habitats differs from Earth habits, and how microbes survive, proliferate and spread in space conditions, is coming more and more important. The Microbial Tracking mission series has been monitoring the microbiome of the International Space Station (ISS) for almost a decade. During this mission series, six unique strains of Gram-positive bacteria, including two spore-forming and three non-spore-forming species, were isolated from the environmental surfaces of the International Space Station (ISS). Results: The analysis of their 16S rRNA gene sequences revealed <99% similarities with previously described bacterial species. To further explore their phylogenetic affiliation, whole genome sequencing (WGS) was undertaken. For all strains, the gyrB gene exhibited <93% similarity with closely related species, which proved effective in categorizing these ISS strains as novel species. Average ucleotide identity (ANI) and digital DNA-DNA hybridization (dDDH) values, when compared to any known bacterial species, were less than <94% and 50% respectively for all species described here. Traditional biochemical tests, fatty acid profiling, polar lipid, and cell wall composition analyses were performed to generate phenotypic characterization of these ISS strains. A study of the shotgun metagenomic reads from the ISS samples, from which the novel species were isolated, showed that only 0.1% of the total reads mapped to the novel species, supporting the idea that these novel species are rare in the ISS environments. In-depth annotation of the genomes unveiled a variety of genes linked to amino acid and derivative synthesis, carbohydrate metabolism, cofactors, vitamins, prosthetic groups, pigments, and protein metabolism. Further analysis of these ISS-isolated organisms revealed that, on average, they contain 46 genes associated with virulence, disease, and defense. The main predicted functions of these genes are: conferring resistance to antibiotics and toxic compounds, and enabling invasion and intracellular resistance. After conducting antiSMASH analysis, it was found that there are roughly 16 cluster types across the six strains, including ß-lactone and type III polyketide synthase (T3PKS) clusters. Conclusions: Based on these multi-faceted taxonomic methods, it was concluded that these six ISS strains represent five novel species, which we propose to name as follows: Arthrobacter burdickii IIF3SC-B10T (=NRRL B-65660T), Leifsonia virtsii, F6_8S_P_1AT (=NRRL B-65661T), Leifsonia williamsii, F6_8S_P_1BT (=NRRL B- 65662T and DSMZ 115932T), Paenibacillus vandeheii, F6_3S_P_1CT(=NRRL B-65663T and DSMZ 115940T), and Sporosarcina highlanderae F6_3S_P_2 T(=NRRL B-65664T and DSMZ 115943T). Identifying and characterizing the genomes and phenotypes of novel microbes found in space habitats, like those explored in this study, is integral for expanding our genomic databases of space-relevant microbes. This approach offers the only reliable method to determine species composition, track microbial dispersion, and anticipate potential threats to human health from monitoring microbes on the surfaces and equipment within space habitats. By unraveling these microbial mysteries, we take a crucial step towards ensuring the safety and success of future space missions.

Design, synthesis, and pharmacology of some oxadiazole and hydroxypyrazoline hybrids bearing thiazoyl scaffold: antiproliferative activity, molecular docking and DNA binding studies.

A series of oxadiazole (7a-l) and hydroxypyrazoline derivatives (8a-l) incorporating thiazole were synthesized and characterized by spectral analysis (1H-NMR, 13C-NMR, Mass, and FT-IR). The synthesized compounds were screened for their in vitro cytotoxicity against MDA-MB231 and HT-29 human cell lines. Conjugates 7d, 7e, 7f, 7i, 7l, 8a, 8b, 8i and 8l exhibited significant antiproliferative activity on both MDA-MB231 and HT-29 cell lines. Flow cytometric analysis reveals that, 7i arrests both cells lines at Go/G1 phase whereas 8i induced G0/G1 arrest only in the HT-29 cells. Furthermore, Computational interaction studies of 7i and 8i exhibited its capacity of being a plausible CDK2 and BCL-2 inhibitor respectively. In addition, DNA binding of the synthesized compounds and DNA docking of 7i and 8i demonstrated the ability to interact with DNA. Compounds 7i and 8i causes' remarkable growth inhibition of MDA-MB231 and HT-29 cells but compound 8i was considerably effective against HT-29 cells. Overall these compounds can be practiced for further drug development.