Microorganisms

Objectives: To evaluate the epidemiological evolution of unique pneumococcal serotypes covered by either PCV20 or PCV21, common serotypes by both vaccines and non-vaccine serotypes in adults. Methods: We characterized all national IPD clinical isolates from January 2009 to June 2026. Results: The highest burden of IPD was caused by serotypes shared between PCV20 and PCV21 with serotypes 3 and 8 the most prevalent in adults of all ages. During the pre-pandemic period, IPD caused by unique-PCV20 serotypes declined probably due to herd protection by paediatric vaccination with PCVs. In contrast, unique-PCV21 serotypes increased in incidence accounting for more cases than unique-PCV20 serotypes. However, after the COVID-19 pandemic, a different pattern was observed with unique-PCV20 serotypes increasing in incidence due a marked rise in IPD caused by serotypes 4 and 14 that has progressively reduced the gap in disease coverage. In adults aged 18-64 years, the fraction of disease that could be prevented with PCV20/PCV21 changed from 5% in favour of PCV21 (years 2022/2023) to 15% in favour of PCV20 (year 2026) whereas in adults [≥]65 years the vaccine contribution declined from 20% in favour of PCV21 (year 2022) to 8% in 2026. Conclusions: IPD trends in adults have changed after the COVID-19 pandemic, especially in recent years. Our study emphasizes the rapid re-emergence of infections caused by traditional PCV7 serotypes especially serotypes 4 and 14 and reinforce the idea that the use of broader PCVs must be accompanied by an increase in the uptake rates in adults of all ages.

Extremophilic fungi are a relatively unexplored source of stress-resilient microbes for sustainable agriculture. Here, we describe Talaromyces pseudofuniculosus FAR, isolated from the highly acidic site of Piccolo Inferno in southern Italy and identified by ITS and {beta}-tubulin (BenA) sequencing. Despite its origin from an extreme environment, T. pseudofuniculosus FRA remained fully functional under non-stress conditions and displayed tolerance to salinity and heavy metals while maintaining key plant growth-promoting traits, including phosphate solubilization, auxin-related compound production and hydrolytic activities. In soybean (Glycine max L.), the live fungus promoted plant growth under both non-stress conditions and agriculturally relevant salinity and copper stress, and successfully colonized the roots. A cell-free extracellular extract (exCFS) promoted early seedling development in vitro, but its effects were not sustained in pot experiments. GC-MS analysis provided a qualitative characterization of the extracellular metabolite profile and revealed condition-dependent differences between control, NaCl and CuSO4; growth conditions, indicating environment-dependent modulation of fungal metabolism. Overall, these findings highlight the physiological and metabolic plasticity of T. pseudofuniculosus FRA and support its potential as a fungal biostimulant for saline- and metal-impacted agricultural soils.

Four rhizobial strains isolated from root nodules of tropical leguminous plants in Costa Rica were characterized. Strain LEGMi-135b was isolated from Lonchocarpus felipei root nodules, LEGMi-12c from Cojoba arborea, LEGMi-198b from Calliandra haematocephala and Rhizobium altiplani LEGMi-166a from Mimosa pigra. Phylogenetic analyses based on the 16S rRNA gene and four concatenated housekeeping genes (recA, atpD, GATase and gyrB) placed these strains within the genera Martinezella (LEGMi-12c, LEGMi-135b, and LEGMi-198b) and Rhizobium (LEGMi-166a). Genome-based analyses showed that strain LEGMi-135b exhibited an average nucleotide identity (ANI) value of 94% and a digital DNA-DNA hybridization (dDDH) value of 53.2% compared with its closest related species, Martinezella hainanensis CCBAU 57015T, supporting its classification as a distinct species. LEGMI-12c exhibited ANI and dDDH values of 98% and 87%, respectively, compared with Martinezella hainanensis CCBAU 57015T. LEGMi-198b showed the highest similarity to Rhizobium cerradonense CNPSo 3464, with ANI and dDDH values of 98% and 81.4%, respectively; however, this taxon has not been validly published. LEGMi-166a exhibited ANI and dDDH values of 100% and 99.3%, respectively, compared with Rhizobium altiplani BR 10423, confirming its assignment to this species. Additionally, differentiation of strains LEGMi-135b and LEGMi-198b from their closest phylogenetic relatives was supported by phenotypic, physiological, and biochemical analyses. Based on the combined genomic, phenotypic, and biochemical evidence, we propose the establishment of the novel rhizobial species Martinezella legmii sp. nov., with strain LEGMi-135b designated as the type strain (LEGMi-135b), and Martinezella ivanii sp. nov., with strain LEGMi-198b designated as the type strain (LEGMi-198b). This study expands our understanding of rhizobial diversity associated with nitrogen-fixing symbioses, particularly among tropical leguminous plants from Costa Rica

Pectobacterium comprises Gram-negative, pectinolytic plant pathogens that cause soft rot and blackleg diseases on numerous economically important crops. During sampling in 2018 in taro (Colocasia esculenta) fields in Hawaii, two bacterial strains PL152T and PL155 were isolated from taro. Peliminary molecular characterization placed both strains in the genus Pectobacterium. However, their taxanomic postions could not be resoved at the species level. A polyphasic taxonomic approach incorporating phenotypic characterization, 16S rRNA gene sequence analysis, multilocus sequence analysis (MLSA), whole genome based phylogeny, average nucleotide identity (ANI), and digital DNA-DNA hybridization (dDDH) was therefore employed to determine their taxonomic status. Phylogenetic and phylogenomic analyses consistently placed PL152 and PL155 together in a distinct and well supported lineage within the genus Pectobacterium, separate from currently recognized species. The two strains exhibited high genomic relatedness to each other, whereas the average nucleotide identity (ANI) and digital DNA-DNA hybridization (dDDH) values, were lower than the accepted species delineation thresholds of 95% and 70%, respectively, supported the delineation of a novel bacterial species. The genomic characteristic of PL152 included a genome size of 5.02 Mb and a DNA G+C content of 52%. Collectively, based on a polyphasic approach including phylogenetic, phlyogenomic, biochemical, and phenotypic evidences supports the classification of strains PL152 and PL155 as representatives of a novel species within the genus Pectobacterium, for which the name Pectobacterium pacificum sp. nov. is proposed. Strain PL152 (ICMP 2436274, LMG 32536) is designated as the type strain.

Little is currently known about the regulation of PL fimbriae produced by some pathogenic E. coli strains. We identified PlfB, a transcriptional regulator of the PapB family, as a negative regulator of PL fimbriae (Plf). Deletion of plfB significantly increased plfA transcription and Plf production. PlfB bound specifically to the plfA promoter region. Using a PplfA-lux reporter system, the deletion of plfB caused sustained and increased expression across serial passages. Global regulatory genes such as lrp, argR, and hns were also implicated in regulation of plf expression. Loss of lrp or argR decreased plf expression, suggesting these regulators positively regulate PL fimbriae. By contrast, loss of hns increased levels of plf expression, supporting its role as a negative regulator. Interestingly, loss of plfB significantly increased bacterial colonization in the lung of 6-day-old turkey poults. Further, complementation of plfB restored plf fimbrial expression and colonization of turkey lungs to wild-type levels. This further supports an important role for PL fimbriae for colonization of turkey lungs, since loss of plfB which resulted in increased plf gene expression in turkey lungs also correlated with higher bacterial numbers in the lungs. Taken together, these results indicate that PlfB acts as a negative regulator to reduce levels of PL fimbrial expression, under some conditions, but that it is not required for plf expression. This contrasts with the role the PapB regulator, which has been shown to play an essential role in expression of P fimbriae in uropathogenic E. coli strains.

The human gut is home to more than 100 trillion bacteria, where various environmental factors, such as nutrients and gases, exist and influence them. The levels of fermentation gases in the gut, including CO2, fluctuate depending on the host's diet. However, how CO2, an environmental factor, affects the physiological states of gut anaerobes remains poorly understood, but CO2 is often utilized in their cultivation. Akkermansia muciniphila (A. muciniphila) is widely studied as a beneficial human gut microbe. However, the effects of CO2 on the physiological state of A. muciniphila are not fully understood. In this study, we focused on CO2 as an environmental factor and aimed to clarify how CO2 affects the physiological state of A. muciniphila. Culture experiments showed that compared with the absence of CO2, the presence of CO2 significantly increased the growth of A. muciniphila. The results of the metabolomic analysis revealed distinct metabolic profiles in the presence or absence of CO2. The tracing analysis using stable 13C-labelled CO2 demonstrated that CO2 was incorporated into various metabolites in the TCA cycle, the de novo purine/pyrimidine synthesis pathways, and other pathways. In addition, the activity of pyruvate carboxylase, which catalyses an anaplerotic reaction involving CO2 fixation to supply oxaloacetate (OAA) to the TCA cycle, was significantly higher in A. muciniphila cells grown in the presence of CO2. Moreover, supplementation with OAA increased the growth of A. muciniphila in a dose-dependent manner, mirroring the effects of CO2 supplementation. These findings suggest that A. muciniphila utilizes CO2 for an anaplerotic reaction in the TCA cycle and other pathways involving CO2 incorporation, facilitating metabolic flux to promote its robust growth.

Orientia tsutsugamushi is the causative agent of scrub typhus infection in humans. This pathogen is transmitted into mammalian hosts including humans through Leptotrombidium mites. Till date, detection of O. tsutsugamushi in human was performed using PCR assay with 56kDa type specific antigen (TSA56), 47kDa outer membrane protein (47kDa-OMP) and groEL chaperonin gene. To understand the efficiency of different genes for the detection of O. tsutsugamushi in Leptotrombidium mites, we performed a comparative PCR assay with various universal primers targeting different genes including TSA56, 47kDa-OMP and groEL gene. We have collected 500 Leptotrombidium mites from Vellore, Tamil Nadu and, the mites were segregated into 50 pools with 10 mites in each pool were used for DNA extraction. The molecular detection of O. tsutsugamushi was performed using PCR assays (including conventional, nested and duplex PCR). The comparative PCR analysis showed nested PCR of groEL gene has high efficiency in detecting the presence of O. tsutsugamushi in chigger samples compared to other genes. Interestingly, groEL target demonstrated high efficiency for detection of O. tsutsugamushi in chigger samples and, when used with nested primers, can facilitate differentiation of O. tsutsugamushi from other rickettsial pathogens. Thus, we conclude groEL gene nested PCR assay can be used routinely to detect O. tsutsugamushi in trombiculid mites.

Gut microbiome plays an important role in human health. Urbanization has been suggested to alter the gut microbiome. We comprehensively evaluated differences in gut microbiome profiles of 76 urban and 81 rural female residents of Vietnam using shotgun metagenomic data. We evaluated rural-urban differences in gut microbial diversity, individual microbial taxa, and metabolic pathways. Rural residents had higher alpha and beta diversities in age-adjusted models (p<0.05). Additionally, significant rural-urban differences were observed for 35 microbial taxa in age-adjusted models (FDR<0.1). Rural residents had higher relative abundance of classes, orders, and families of FGB2982 and FGB38642 and strain SGB15332 of the species Faecalibacterium prausnitzii, whereas urban residents had higher relative abundances of species Bacteroides thetaiotaomicron, Coprobacter fastidiosus, Bilophila wadsworthia, Enterobacter cloacae, Enterobacter hormaechei, Klebsiella aerogenes, Dielma fastidiosa, and Holdemania massiliensis. Notably, these associations were attenuated after further adjustment for socioeconomic characteristics, lifestyle, dietary factors, and comorbidity (FDR>0.1). In the fully adjusted model, significant rural-urban differences were observed in the abundance of 17 microbial taxa, including Ruminococcus sp AF13 28 and Prevotella pectinovora, as well as in three metabolic pathways (FDR<0.1). This study suggests that urbanization-related factors may contribute to differences in the gut microbiome between urban and rural Vietnamese women.

Human noroviruses (HuNoVs) are the most common cause of non-bacterial foodborne illnesses worldwide. HuNoVs are notoriously difficult to culture, and current cell culture systems, such as human intestinal enteroids have limitations in efficiency and scalability, which has limited their applications in food safety studies. Recently, it has been shown that transformed salivary gland cell lines (including NS-SV-TT-DC) allow for replication and passaging of norovirus GII.4. Herein, we examined whether we can independently observe viral replication in salivary gland cells and if other strains of human norovirus can replicate in these cells. For this purpose, we first evaluated the replication of several norovirus strains in NS-SV-TT-DC cells by either infecting them with vesicle-cloaked viruses or total viruses in stool filtrates. Low levels of viral replication was observed and vesicle isolation did not improve viral replication. Next, we screened for replication of 50 norovirus samples from 9 genotypes, and 14 isolates demonstrated over a two-fold viral replication. We observed that the initial viral load in the samples does not have a significant effect on replication efficiency. We were also able to passage several isolates. Subsequently, we examined whether interferon inhibitors such as Ruxolitinib, TPCA-1, and BX795 would improve viral replication and observed negligible effects. Finally we tested multiple supplements including MgCl2, bile salt, ceramide, and TAK-779. Most supplements had little or no effect on HuNoV replication in NS-SV-TT-DC cells. Bile salt and TAK-779 showed limited, genotype-specific benefits. Altogether these results indicate that salivary gland cells allow limited replication of certain HuNoV isolates.

Chlorella sorokiniana is an industrially important microalga, but its genetic manipulation remains limited by poor transformation efficiency. Although several barriers have been proposed, the role of intracellular nuclease activity has received little attention. Here, we compared the degradation of exogenous double-stranded DNA in whole-cell lysates from transformation-recalcitrant (Chlorella sorokiniana and Tetradesmus obliquus) and transformation-proficient (Phaeodactylum tricornutum, Microchloropsis gaditana, and Nannochloropsis oceanica) microalgae. C. sorokiniana rapidly degraded both linear and plasmid DNA, while T. obliquus exhibited intermediate activity. In contrast, no detectable degradation was observed in the transformation-proficient species. DNA degradation in C. sorokiniana occurred within minutes, and nuclease activity in both recalcitrant species was inhibited by EDTA and Zn2+, consistent with a metal ion-dependent endonuclease. These findings suggest that intracellular nuclease activity may represent an underexplored barrier to exogenous DNA stability during transformation and provide a potential target for improving genetic transformation of recalcitrant microalgae.

Background: Long non protein coding (lnc)RNAs are ubiquitous transcripts known to be major regulators of cellular processes in eukaryotes. In diatoms, specific lncRNA expression is tightly correlated to nutrient fluctuations, which suggests function, but their biological relevance remains to be established. In order to fill this knowledge gap, we sought to design a protocol to generate diatom lncRNA knockout mutants by CRISPR-Cas9 enabling subsequent functional studies to shed light into their mechanistic roles. Since lncRNAs do not contain open reading frames that can be targeted by point mutations for the generation of functional knockouts, we designed a multiplexed CRISPR-Cas9 plasmid harboring two independent gRNA for the specific ablation of lncRNA loci in P. tricornutum. As proof of concept, we used our plasmid-based system to target three phosphate depletion responsive lncRNAs. Results: Using the golden gate based uLoop system, we assembled a single plasmid containing all the necessary components to generate CRISPR-Cas9 large genomic ablations of lncRNAs in P. tricornutum using bacterial conjugation. Three intergenic lncRNAs (Linc91, Linc77 and Linc24) responsive to phosphate fluctuations were selected and independently targeted. Around 32% of the exconjugants presented a monoallelic ablation of the targeted loci and 11% presented a biallelic ablation on the first PCR-screening round. Mutated lines, harboring biallelic lncRNAs ablations for each lncRNA targeted, were successfully generated for each of the targeted lncRNAs. Additionally, a first line of phenotypic analysis revealed significant growth rate differences for one of the mutants ({Delta}Lnc24) under both full and phosphate depleted conditions, suggesting its involvement in cell division regulation under nutrient fluctuations. Conclusions: This work provides a simple and efficient method for the generation of lncRNA knock-out mutants in P. tricornutum. Furthermore, this approach can be easily adapted to other microalgae amenable to transformation, enabling the enhancement of our understanding of these important cellular regulatory transcripts in the marine environment.

A native entomopathogenic nematode isolate, Heterorhabditis indica Che4.1, was recovered from soils in Valencia (Spain), and its symbiotic bacterium, Photorhabdus akhurstii Che4.1, was isolated and characterized. Nematode identification was confirmed by ITS and 28S rDNA sequencing, while genome-based analyses supported the taxonomic assignment of the bacterial symbiont. The nematode-bacterium complex exhibited insecticidal activity against Spodoptera exigua and Spodoptera littoralis. H. indica Che4.1 showed an LC of 53.7 infective juveniles (IJs)/mL against S. exigua, comparable to that of the commercial reference Larvanem (54.3 IJs/mL). Against S. littoralis, Che4.1 exhibited an approximately twofold lower LC than Larvanem (77.3 versus 155.9 IJs/mL), with non-overlapping 95% confidence intervals. The complete genome of P. akhurstii Che4.1 consists of a single circular chromosome of 5.66 Mb encoding a diverse repertoire of putative virulence factors, including toxin complex (Tc) components, YenTc-like proteins, Mcf toxins, App-family proteins, Pra/Prb proteins, and chitinases, together with several predicted secondary metabolite biosynthetic gene clusters. LC-MS/MS analyses of bacterial culture supernatants and infected insect cadavers detected multiple predicted virulence-associated proteins, including Tc-related, YenTc-like, App-family, and Pra/Prb proteins. In addition, bacterial cell-free supernatants caused 68.8-81.3% mortality in S. exigua neonates under laboratory conditions. Together, these findings reveal a diverse repertoire of potential virulence determinants in P. akhurstii Che4.1 and demonstrate the insecticidal potential of its native nematodebacterium complex. The performance of H. indica Che4.1 relative to a commercial reference supports further evaluation of this isolate for biological control of Spodoptera spp., particularly under greenhouse and field conditions.

Background: Klebsiella pneumoniae is a key causative agent of multidrug resistant nosocomial infections. Bacteriophages offer a promising alternative treatment, but the diversity of Klebsiella species requires the characterisation of a broad range of phages, particularly the poorly characterised jumbo phages. Materials and Methods: Phage BMCPR_005026 was isolated from sewage against Klebsiella pneumoniae. Whole genome sequencing, transmission electron microscopy and host range analysis against a diverse set of 40 clinically relevant Klebsiella strains were used to characterise the phage. Results: The virion had a myovirus morphology and genomic analysis placed it within an undefined family in Caudoviricetes where it forms a new species within the genus Alcyoneusvirus. BMCPR_005026 could form plaques on 3 of the 40 Klebsiella strains tested (7.5%) and formed halos. Conclusion: BMCPR_005026 represents a new species of phage within the genus Alcyoneusvirus with activity against 3 Klebsiella KL-types.

The molecular epidemiology of Acinetobacter, which is almost exclusively culture-dependent, has centered mainly on clinical isolates of A. baumannii. Yet, this overlooks sources not amenable to microbial culture and many non-A. baumannii species. Here, by analysing metagenomic projects from public transport systems worldwide, we show that metagenome-assembled genomes (MAGs) are highly relevant for assessing the unrecognized diversity of Acinetobacter. We recovered 839 Acinetobacter MAGs, with 269 being high-quality MAGs. The high-quality MAGs were across more than 20 well-defined Acinetobacter species; however, five species (A. nosocomilalis, A. ursingii, A. radioresistens, A. schindleri, and A. variabilis) had the most MAGs. Pangenome and phylogenetic diversity analyses of 685 genomes plus the 269 high-quality MAGs from those 5 species showed that MAGs are an important source of both lineage and gene diversity. Also, FST analyses and genome phylogenies show that for some species there is no genetic differentiation between MAGs and clinical isolates, implying transmission between hospitals and public transport settings. In addition to finding the antibiotic resistance genes (ARGs) in MAGs, we implemented an ad hoc strategy (leveraging the use of genomes) to recover considerably more ARGs. PCoA and PERMANOVA analyses show that, whereas in A. nosocomialis and A. ursingii, the ARG profiles of MAGs and clinical genomes are different, in A. radioresistens and A. schindleri, the ARG profiles are not different. Finally, some clinically important ARGs, such as sul1 and sul2, aadA, and blaOXA23, were found in all or some of the 5 species. Our study shows how combining MAGs and regular genomes is a powerful strategy for better appraising the genomic diversity (including the resistome) of Acinetobacter. On a broader level, our study showcases the importance of MAGs for genomic surveillance of man-made environments with clear public health implications.

Anaerobic gut fungi (AGF) are an integral part of the herbivore gut microbiome, primary decomposers of lignocellulose, and play a crucial role in establishing foundational links of syntrophic associations. The syntrophy with methanogens appears beneficial for mitigating feedback inhibition and results in the augmentation of AGF activity. However, the thermodynamic advantages of the presence of methanogens to AGF remain poorly understood. This study explores the preferred pathways adopted by Cyllamyces sp. and Methanobrevibacter olleyae, using 13C-glucose as a substrate and spiked with malate and pyruvate in different experimental set-ups. The results indicated reduced biomass formation by Cyllamyces sp. in monoxenic culture with Methanobrevibacter olleyae compared to axenic AGF culture supplemented with glucose. Additionally, metabolic analyses revealed the pyruvate:ferredoxin oxidoreductase (PFO) as a key contributor to pyruvate metabolism. Malate supplementation notably enhanced the growth of Cyllamyces sp. and M. olleyae, whereas pyruvate supplementation enhanced the growth of Cyllamyces sp. only in axenic culture. This investigation provides insights into the preferred metabolic pathways operating in the hydrogenosomes of axenic anaerobic gut fungi (AGF) cultures and monoxenic AGF-methanogen co-cultures.

Aims Honey represents a distinct microbial habitat, imposing physicochemical stresses on its resident microbiota while also serving as a potential source of beneficial microorganisms. This study aimed to isolate lactic acid bacteria (LAB) from Korean wildflower honey and characterize their gastrointestinal (GI) and physiological stress tolerance, adhesion-related properties, functional characteristics, and safety to evaluate their potential as probiotic candidates. Methods and Results Two Streptococcus salivarius strains, DOW-5A and DOW-10, were isolated from Korean wildflower honey and identified by phenotypic and molecular analyses. Both strains survived in sequential exposure to simulated GI conditions and showed tolerance to physiological stresses. DOW-5A maintained higher viability during the simulated gastric phase, whereas DOW-10 showed an initial decrease in viable counts followed by an increase during the simulated intestinal phase. The two strains also exhibited various probiotic characteristics, including mucin adhesion and antioxidant activity. Atypical, crystalline colony morphologies associated with EPS production were observed when sucrose was supplied as a carbon source. Both strains formed biofilms, while DOW-5A showed a marked increase in biofilm formation upon sucrose supplementation. Neither strain showed gelatinase or hemolytic activity or significant cytotoxicity toward Caco-2 cells. Impact Statement In this study, we isolated two honey-derived S. salivarius strains with high probiotic potential. Our findings highlight wildflower honey as a potential reservoir of LAB with traits relevant to survival under GI and physiological stresses, as well as various probiotic traits.

Acinetobacter sp. strain Tol 5 is an attractive microorganism because of its ability to utilize various aromatic compounds, including toluene. However, because Tol 5 is a non-model microorganism, the utilization of carbon sources in central carbon metabolism remains unclear. In this study, the activity of the central carbon metabolism was characterized using 13C-metabolic flux analysis (13C-MFA). Tol 5 was cultured in synthetic medium using ethanol or acetate as the sole carbon source. In addition, because toluene is degraded into pyruvate and acetyl-CoA via the toluene dioxygenase pathway, this situation was mimicked by culturing with ethanol and lactate as co-substrates. The 13C-MFA confirmed flux through the glyoxylate shunt, which bypasses part of the TCA cycle when either ethanol or acetate was used as the sole carbon source. In ethanol, the flux ratio of isocitrate dehydrogenase at the isocitrate branch point decreased markedly. Unlike acetate, the conversion of ethanol to acetyl-CoA is accompanied by NADH production. Therefore, the observed change in this flux ratio was influenced by the excess NADH generated. Under ethanol-lactate conditions, Tol 5 simultaneously utilized ethanol and lactate. The ethanol consumption rate decreased to less than half of that observed when ethanol was the sole carbon source. Most of the lactate uptake was converted to pyruvate and metabolized via the TCA cycle. The glyoxylate shunt flux was lower than that in ethanol. These metabolic characteristics provide valuable insights into the use of Tol 5 as a next-generation microbial cell factory.

Although therapeutic advancements have dramatically improved outcomes for cancer patients in recent decades, advanced cancer remains a significant challenge. There is still an urgent need for new and more effective strategies that minimize toxicity and improve survival outcomes in cancer patients. Natural products from microorganisms are a rich and promising source of bioactive compounds with substantial therapeutic potential in cancer treatment. Recently, B. proteolyticus strain OSUB18, isolated from switchgrass, has been demonstrated to promote plant growth and regulate reactive oxygen species (ROS) production. Here, we investigated the anticancer potential of this plant-associated microbe and demonstrated that its crude extracts exhibited broad-spectrum cytostatic activity across diverse solid tumor cell lines. Importantly, OSUB18 extracts displayed limited cytotoxicity toward normal human mammary epithelial cells and mouse fibroblasts. This potentially selective cytostatic activity was retained within a water soluble, heat-labile, and protease-resistant fraction. The extracts primarily suppressed cell proliferation, accompanied by increased lipid peroxidation, with preliminary evidence suggesting the potential involvement of ferroptosis. These findings suggest that the bioactive component may be a selective cytostatic agent with multifaceted mechanisms of action.

The growth and performance of cattle is dependent on efficient feed conversion, facilitated by the gut microbiome, including both prokaryotic and eukaryotic organisms. Extrinsic factors such as management practices and intrinsic factors like age and genetics can shape these communities. To characterize the relative effects of such factors on the prokaryotic and eukaryotic microbiomes, fresh fecal samples from dairy heifers (n = 8), beef heifers (n = 8), bred beef heifers (n = 8), and adult beef cows (n = 8) were subjected to paired 16S rRNA (V4) and a novel 18S rRNA (V4-V5) metabarcoding assay, and 18S rRNA amplicons were annotated using two databases in parallel. Taxonomic resolution of 18S rRNA amplicons was excellent, with SILVA and EUKARYOME databases each providing better resolution of different groups of organisms. While no differences in eukaryotic richness or alpha diversity were detected, beta diversity differed in all pairwise comparisons except between beef heifers and bred beef heifers. Prokaryotic richness was lower in adult beef cows than the other groups, and beta diversity differed in all pairwise comparisons. Adult beef cows were the most distinct, characterized by replacement of fungal organisms with protozoa and loss of thermophilic bacteria in the eukaryotic and prokaryotic compartments, respectively. Among numerous differentially abundant features, dairy heifers harbored greater abundance of Cooperia oncophora while beef heifers contained greater abundance of Buxtonella sulcata and Mucor. Correlation network analysis revealed multiple significant positive and negative associations between bacterial, fungal, and protozoal features during maturation of the fecal microbiome. Collectively, these data highlight the richness and diversity of both the prokaryotic and eukaryotic compartments of the bovine fecal microbiome, and the influence of management practices and age on both communities. From a technical standpoint, these data demonstrate the ability to resolve fungal, protozoal, and metazoan organisms, including potential parasites and pathogens, with a single metabarcoding assay. They also highlight the nuanced differences between databases and the value of parallel analyses using more than one database.

This study developed a streamlined, sensitive and rapid method for co-detection of low concentrations of Salmonella enterica, Shigella flexneri, Listeria monocytogenes, and pathogenic Escherichia coli from soil. First, autoclaved soil from Iowa with serial dilutions of bacteria to confirm individual species recovery rates on selective agar by qPCR under varying primary enrichment times was used. All pathogens were detected by qPCR of colonies recovered from replicate sterile soils spiked with 10^1 or greater bacteria within 5 hours of enrichment. Shiga toxin expressing E. coli (STEC) and L. monocytogenes were consistently co-recovered in autoclaved and natural soil, pathogen spiking concentration, and secondary enrichment time. S.enterica was consistently recovered at autoclaved and natural concentrations, while S. flexneri detection rates fluctuated. Twenty-three soil samples from Kenyan neighborhoods, with prior evidence of contamination, was compared to qPCR detection at each protocol stage: processing, primary enrichment, secondary enrichment, and selective colony isolation. Detection rates substantially increased for S. enterica, STEC and other pathogenic E. coli after enrichment, with optimal detection rates occurring after secondary enrichment. Testing colonies from selective agar resulted in high false negative rates, possibly reflecting competition from native soil microbiota that was absent in spiked sterile soil experiment. Evaluation of the method with other environmental sample types yielded consistent detection patterns. Evidence suggests a protocol including 24-hour primary enrichment, followed by 24-hour secondary enrichment, and qPCR testing of soil DNA at primary and secondary steps is a reproducible and more expedient method for co-detecting common Enterobacteriaceae and L. monocytogenes pathogens in soil.