RESUMEN
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.
Asunto(s)
Ceftazidima , Ácidos Grasos , Humanos , Ácidos Grasos/análisis , Filogenia , ARN Ribosómico 16S/genética , Composición de Base , Hibridación de Ácido Nucleico , Esporas/química , Nucleótidos , ADN , ADN Bacteriano/genética , ADN Bacteriano/química , Análisis de Secuencia de ADN , Técnicas de Tipificación BacterianaRESUMEN
Phlegmariurus is the only genus of Lycopodiaceae with the species grouped in 22 informal groups. Species level relationships within Phlegmariurus are poorly understood and their circumscriptions require a thorough molecular and morphological review. A detailed study of morphology and anatomy of caulinar axes, lycophylls and sporangia of Phlegmariurus phylicifolius was carried out in order to contribute to the elucidation of species circumscription in the informal group Phlegmariurus phlegmaria. Small pieces of caulinar axes bearing trophophylls, sporophylls and sporangia were fixed, dehydrated, Histowax (paraffin) embedded, sectioned in a rotatory microtome, and stained using the common Safranin O-Fast Green technique; handmade cross sections were also made and stained with the same technique. P. phylicifolius includes slender, pendulous plants up to 40cm long. Shoots heterophyllous, in the basal divisions ca. 10-20(-25)mm in diameter including the trophophylls, then abruptly constricted to (l-) 1.5-2(-2.5)mm in diameter including the imbricate, reduced sporophylls. Trophophylls are borne in alternating whorls of three, or decussate, subdecussate, or alternate, widely spaced in alternate leaved caulinar axes portions, perpendicular to the caulinar axes to falcately ascending, lanceolate to linear-lanceolate, with flat to slightly revolute margins. Each lycophyll is supplied by a single central vascular bundle, connected to a protoxylem pole in the stele. At the site of leaf-trace departure, no leaf (lycophyll) gap is present. Caulinar axes excluding leaves 0.7-1.2mm thick at the base, upward tapering to ca. 0.5mm. Caulinar axes present unistratified epidermis and endodermis, the cortex is characterized by the presence of a trabecular structure of lisigenous origin formed in the parenchimatous tissue next to the endodermis. The vascular tissue occupies the central part of the caulinar axes, forming a plectostele of subradiate organization, with five poles of protoxylem. The epidermal cells present sinuous anticlinal walls; invaginations in the inner side of external periclinal wall of the epidermal cells could be probably adaptive morphological feature of a water deficient environment. Leaves of constricted terminal divisions are decussate, or subdecussate, continuously or discontinuously sporangiate, appressed, abaxially rounded to carinate, widely lanceolate to widely ovate or subcordate, acute to mucronate or cuspidate, shorter than the sporangia. Each sporangium originates from a group of epidermal cells, axilar to the sporophylls. The cell walls of epidermal cell of the sporangia are Huperzioideae type. The morphological studies of trophophylls contribute to confirm the differences between P. phylicifolius and P. subulatus. Rev. Biol. Trop. 62 (3): 1217-1227. Epub 2014 September 01.
Phlegmariurus es el único género de Lycopodiaceae con las especies reunidas en 22 grupos informales. Las relaciones a nivel de especie dentro de Phlegmariurus están pobremente estudiadas y la circunscripción de las mismas requiere profundos exámenes moleculares y morfológicos. Se ha llevado a cabo un estudio detallado de la morfología y la anatomía de ejes caulinares, licofilos y esporangios de P. phylicifolius, con el fin de contribuir al esclarecimiento en la delimitación de las especies en el grupo Phlegmariurus phlegmaria. Segmentos de ejes caulinares con trofofilos, esporofilos y esporangios fueron fijados, deshidratados, incluidos en Histowax (parafina), cortados con un micrótomo rotatorio y coloreados usando la técnica tradicional Safranina O-Verde Rápido; además se hicieron cortes a mano alzada y se colorearon con la misma técnica. P. phylicifolius incluye plantas colgantes y péndulas de hasta 40cm de longitud. Los ejes son heterofilos, de aproximadamente 10-20(-25)mm de diámetro en las divisiones basales incluyendo los trofofilos, luego abruptamente reducidos a (l-) 1.5-2(-2.5)mm de diámetro incluyendo los esporofilos reducidos e imbricados. Los trofofilos están dispuestos en anillos alternantes de a tres, o decusados, subdecusados o alternos, dispuestos en forma espaciada en los ejes caulinares, perpendiculares al tallo hasta falcadamente ascendentes, lanceolados a lineal-lanceolados, con márgenes lisos o levemente revolutos. Cada licofilo está provisto de un haz vascular simple y central, conectado a un polo de protoxilema de la estela y sin laguna foliar. Los tallos poseen un ancho de 0.7-1.2mm en la base, excluyendo los licofilos, estrechándose hasta cerca de 0.5mm hacia el ápice. Los ejes caulinares presentan una epidermis uniestratificada y endodermis, la corteza se caracteriza por la presencia de una estructura trabecular de origen lisígeno formada en el tejido parenquimático próximo a la endodermis. El tejido vascular ocupa la parte central del eje caulinar, formando una plectostela de organización subradiada, con cinco polos de protoxilema. Las células epidérmicas presentan paredes anticlinales sinuosas; las invaginaciones en la cara interna de la pared periclinal externa podrían ser probablemente un característica morfológica adaptativa a un ambiente con períodos de sequía. Las hojas de las porciones apicales son decusadas o subdecusadas, con esporangio de disposición continua o discontinua, adpresas, abaxialmente redondeadas a carinadas, ampliamente lanceoladas a ovadas o subcordadas, ápice agudo a mucronado o cuspidado, más corto que el esporangio. Cada esporangio se origina de un grupo de células epidérmicas, en la axila de los esporofilos con el eje caulinar. Las paredes celulares de las células epidérmicas del esporangio son de tipo Huperzioideae. El estudio de la morfología de los trofofilos contribuye a confirmar las diferencias entre P. phylicifolius y P. subulatus.
Asunto(s)
Carotenoides/análisis , Lycopodiaceae/citología , Esporangios/citología , Esporas/citología , Lycopodiaceae/química , Lycopodiaceae/clasificación , Lycopodiaceae/crecimiento & desarrollo , Esporangios/química , Esporangios/clasificación , Esporangios/crecimiento & desarrollo , Esporas/química , Esporas/clasificación , Esporas/crecimiento & desarrolloRESUMEN
Studies on reproductive aspects of Lycopodiaceae are not very abundant in the scientific literature, and constitute essential information to support taxonomic and systematic relationships among the group. Here we present a detailed study of the ontogeny of sporangia and sporogenesis, and the chemical determination of several compounds generated during spore formation. The analyses were performed in 14 taxa of six genera of the family, Diphasiastrum, Diphasium, Huperzia (a genus which is treated here including Phlegmariurus), Lycopodiella, Lycopodium and Palhinhaea. Specimens were collected in three departments from the Colombian Andes between 1 454-3 677m altitude. Ontogeny was studied in small, 1cm long pieces of strobili and axis, which were fixed in glutaraldehyde or FAA, dehydrated in alcohol, embedded in LR White, sectioned in 0.2-0.5μm and stained with toluidine blue (TBO), a metachromatic dye that allows to detect both sporopollenin and lignin or its precursors, during these processes. For other studies, paraplast plus-embedded sections (3-5μm) were stained with safranin-fast green and alcian blue-hematoxylin. Chemical tests were also conducted in sections of fresh sporangia at different stages of maturity using alcian blue (mucopolysaccharides), Lugol solution (starch), Sudan III (lipids), phloroglucinol (lignin) and orcein (chromosomes). Sections were observed with photonic microscope equipped with differential interference contrast (DIC) and fluorescence microscopy (for spore and sporangium walls unstained). Strobili and sporangia were dehydrated with 2.2 dimethoxypropane, critical point dried and coated with gold for scanning electron microscopy (SEM). Our results indicated that the ontogeny of sporangia and sporogenesis were very similar to the previously observed in Huperzia brevifolia. Cutinisation occurs in early stages of development of sporangium cell walls, but in their final stages walls become lignified. As for the sporoderm development, the exospore is the first layer formed, composed by sporopollenin. The endospore deposits as a thin inner layer composed of cellulose, pectin and carboxylated polysaccharides. The perispore, if present, deposits at last. Mucopolysaccharides were found on the sporocyte coat and its abundance in sporangial cavity persists up to the immature tetrads stage, and then disappears. The lipids were abundant in the sporocytes, tetrads and spores, representing the main source of energy of the latter. In contrast, starch is not detected in the spores, but is abundant in premeiotic sporocytes and immature tetrads, developmental stages of high cellular metabolic activity. Intrinsic fluorescence corroborates the presence of lignin and cutin in the sporangium wall, while the sporopollenin is restricted to the exospore. The transfusion cells and the perispore are not always present. However, the processes of ontogeny and sporogenesis are extremely similar throughout the taxa studied, suggesting that they represent conservative family traits, nonspecific or generic.
Los estudios sobre aspectos reproductivos no son muy abundantes en la literatura científica sobre los taxones de Lycopodiaceae y constituyen información esencial para apoyar la taxonomía y relaciones sistemáticas en el grupo. Por lo tanto, se presenta aquí un análisis detallado de la ontogenia de los esporangios y esporogénesis, así como determinaciones químicas de varios compuestos generados durante la formación de las esporas. Los análisis se llevaron a cabo en 14 taxones de seis géneros de la familia: Diphasiastrum, Diphasium, Huperzia (un género que se trata aquí, incluyendo Phlegmariurus), Lycopodiella, Lycopodium y Palhinhaea. Las muestras fueron recolectadas en tres departamentos de los Andes de Colombia entre 1 454-3 677m de altitud. La ontogenia se estudió en trozos de estróbilos y ejes, de 1cm de largo, que se fijaron en glutaraldehido o FAA, se deshidrataron en alcohol, se incluyeron en LR White, se seccionaron en cortes de 0.2-0.5μm y se colorearon con azul de toluidina (TBO), un colorante metacromático que permite detectar tanto esporopolenina como lignina o sus precursores. Para estudios adicionales, secciones de 3-5μm de material incluido en paraplast plus se colorearon con safranina-verde rápido y azul alciánhematoxilina. Las pruebas químicas se llevaron a cabo en secciones de esporangios sin fijar en diferentes etapas de madurez utilizando azul alcián (mucopolisacáridos), solución de Lugol (almidón), Sudán III (lípidos), fluoroglucinol (lignina) y orceína (cromosomas). Las observaciones se efectuaron con microscopio fotónico equipado con contraste diferencial de interferencia (DIC) y microscopía de fluorescencia (para esporas y pared de los esporangios sin colorear). Para observaciones con microscopía electrónica de barrido (MEB), los estróbilos y esporangios se deshidrataron con 2,2 dimetoxipropano, se desecaron a punto crítico y se metalizaron con oro. Los resultados indican que la ontogenia de los esporangios y esporogénesis es muy similar a la observada previamente en Huperzia brevifolia. En las primeras etapas de desarrollo, las paredes celulares de la epidermis del esporangio se cutinizan y en las finales se lignifican. En el desarrollo del esporodermo, la primera capa que se forma es el exosporio, compuesto por esporopolenina. El endosporio es una capa interna delgada compuesta de celulosa, pectina y polisacáridos carboxilados. El perisporio, si está presente, es la última capa que se deposita. Los mucopolisacáridos se encontraron en la cubierta del esporocito, son abundantes en la cavidad esporangial hasta la etapa de tétradas inmaduras y luego desaparecen. Los lípidos son abundantes en esporocitos, tétradas y esporas, y representan la principal fuente de energía de estas. En contraste, el almidón no se detecta en las esporas pero es abundante en esporocitos premeióticos y tétradas inmaduras, ambos con gran actividad metabólica. La fluorescencia intrínseca corrobora la presencia de lignina y cutina en la pared del esporangio, mientras que la esporopolenina se limita al exosporio. Las células de transfusión y el perisporio no siempre están presentes. Sin embargo, los procesos de la ontogenia y esporogénesis son extremadamente similares en todos los taxones estudiados, lo que sugiere que representan rasgos típicos de familia, no específicos ni genéricos.