Elasnin is a new antibiofilm compound that was recently reported to have excellent activity against methicillin-resistant Staphylococcus aureus (MRSA) biofilms. In this study, we established that elasnin also has antibacterial activity against growing S. aureus planktonic cells. To explore elasnin's potential as an antibiotic, we applied adaptive laboratory evolution (ALE) and produced evolved strains with elevated elasnin tolerance. Interestingly, they were more sensitive toward daptomycin and lysostaphin. Whole-genome sequencing revealed that all of the evolved strains possessed a single point mutation in a putative phosphate transport regulator. Subsequently, they exhibited increased intracellular phosphate (Pi) and polyphosphate levels. Inhibition of the phosphate transport regulator gene changed the phenotype of the wild type to one resembling those observed in the evolved strains. Proteomics and transcriptomics analyses showed that elasnin treatment resulted in the downregulation of many proteins related to cell division and cell wall synthesis, which is important for the survival of growing exponential-phase cells. Other downregulated processes and factors were fatty acid metabolism, glycolysis, the two-component system, RNA degradation, and ribosomal proteins. Most importantly, transport proteins and proteins involved in oxidative phosphorylation and the phosphotransferase system were more upregulated in the evolved strain than in the ancestral strain, indicating that they are important for elasnin tolerance. Overall, this study showed that elasnin has antibacterial activity against growing S. aureus cells and revealed the altered processes due to elasnin treatment and those associated with its tolerance. IMPORTANCE Besides the excellent antibiofilm properties of elasnin, we discovered that it can also kill growing methicillin-resistant Staphylococcus aureus (MRSA) planktonic cells. We subjected MRSA cells to an in vitro evolution experiment, and the resulting evolved strains exhibited increased elasnin tolerance, reduced growth rate, loss of pigmentation, and an increased proportion of small-colony formation, and they became more sensitive toward daptomycin and lysostaphin. Through multiomics analysis, we uncovered the affected processes in growing S. aureus planktonic cells following elasnin treatment, including the downregulation of cell wall synthesis, cell division, and some genes/proteins for the two-component system. These findings suggest that elasnin suppressed processes important for the cells' survival and adaptation to environmental stresses, making it an ideal drug adjuvant candidate. Overall, our study provides new insights into the mechanism of elasnin in S. aureus planktonic cells and pointed out the potential application of elasnin in clinics.

Little is unknown about the regulatory mechanisms underlying the pathogenesis of osteomyelitis induced by Staphylococcus aureus. Hypoxia-inducible factor-1α (HIF-1α) and transforming growth factor β1 (TGF-β1) were both upregulated in S. aureus-infected MC3T3-E1 cells and osteomyelitis patients. HIF-1α directly targets the hypoxia-responsive elements (HREs) of TGF-β1 mRNA to induce its expression. Silencing HIF-1α and TGF-β1, as well as treatment of hypoxia inhibitor IDF-11774, consistently elevated OPN and RUNX2 expression and alizarin Red S (ARS) and alkaline phosphatase (ALP) staining levels in MC3T3-E1 cells with S. aureus infection. S. aureus infection increased HIF-1α expression and serum TGF-β1 concentration in a mouse model of osteomyelitis. Hypoxia inhibitor IDF-11774 treatment reduced serum levels of interleukin (IL)-6, IL-1β, and C-reactive protein. Upon S. aureus infection, hypoxia was activated to trigger TGF-β1 upregulation through direct targeting of HRE on TGF-β1 mRNA by HIF-1α, eventually leading to osteomyelitis symptoms in terms of osteogenesis and mineralization deficiencies as well as elevated inflammation. This study hereby suggests a novel signaling cascade involving hypoxia/HIF-1α/TGF-β1 in osteomyelitis pathogenesis, which could potentially serve as a target for therapeutic measures. IMPORTANCE The pathogenesis of osteomyelitis induced by Staphylococcus aureus remains unclear. To develop therapeutic approaches for osteomyelitis, it is important to understand the molecular mechanisms of its pathogenesis. Our results suggests that hypoxia/HIF-1α/TGF-β1 signaling is involved in osteomyelitis pathogenesis. Thus, these findings highlight the potential of this signaling components as therapeutic targets for the treatment of osteomyelitis.

Staphylococcus aureus is a major human and animal pathogen, colonizing diverse ecological niches within its hosts. Predicting whether an isolate will infect a specific host and its subsequent clinical fate remains unknown. In this study, we investigated the S. aureus pangenome using a curated set of 356 strains, spanning a wide range of hosts, origins, and clinical display and antibiotic resistance profiles. We used genome-wide association study (GWAS) and random forest (RF) algorithms to discriminate strains based on their origins and clinical sources. Here, we show that the presence of sak and scn can discriminate strains based on their host specificity, while other genes such as mecA are often associated with virulent outcomes. Both GWAS and RF indicated the importance of intergenic regions (IGRs) and coding DNA sequence (CDS) but not sRNAs in forecasting an outcome. Additional transcriptomic analyses performed on the most prevalent clonal complex 8 (CC8) clonal types, in media mimicking nasal colonization or bacteremia, indicated three RNAs as potential RNA markers to forecast infection, followed by 30 others that could serve as infection severity predictors. Our report shows that genetic association and transcriptomics are complementary approaches that will be combined in a single analytical framework to improve our understanding of bacterial pathogenesis and ultimately identify potential predictive molecular markers. IMPORTANCE Predicting the outcome of bacterial colonization and infections, based on extensive genomic and transcriptomic data from a given pathogen, would be of substantial help for clinicians in treating and curing patients. In this report, genome-wide association studies and random forest algorithms have defined gene combinations that differentiate human from animal strains, colonization from diseases, and nonsevere from severe diseases, while it revealed the importance of IGRs and CDS, but not small RNAs (sRNAs), in anticipating an outcome. In addition, transcriptomic analyses performed on the most prevalent clonal types, in media mimicking either nasal colonization or bacteremia, revealed significant differences and therefore potent RNA markers. Overall, the use of both genomic and transcriptomic data in a single analytical framework can enhance our understanding of bacterial pathogenesis.

Shotgun metagenomic sequencing has transformed our understanding of microbial community ecology. However, preparing metagenomic libraries for high-throughput DNA sequencing remains a costly, labor-intensive, and time-consuming procedure, which in turn limits the utility of metagenomes. Several library preparation procedures have recently been developed to offset these costs, but it is unclear how these newer procedures compare to current standards in the field. In particular, it is not clear if all such procedures perform equally well across different types of microbial communities or if features of the biological samples being processed (e.g., DNA amount) impact the accuracy of the approach. To address these questions, we assessed how five different shotgun DNA sequence library preparation methods, including the commonly used Nextera Flex kit, perform when applied to metagenomic DNA. We measured each method's ability to produce metagenomic data that accurately represent the underlying taxonomic and genetic diversity of the community. We performed these analyses across a range of microbial community types (e.g., soil, coral associated, and mouse gut associated) and input DNA amounts. We find that the type of community and amount of input DNA influence each method's performance, indicating that careful consideration may be needed when selecting between methods, especially for low-complexity communities. However, the cost-effective preparation methods that we assessed are generally comparable to the current gold-standard Nextera DNA Flex kit for high-complexity communities. Overall, the results from this analysis will help expand and even facilitate access to metagenomic approaches in future studies. IMPORTANCE Metagenomic library preparation methods and sequencing technologies continue to advance rapidly, allowing researchers to characterize microbial communities in previously underexplored environmental samples and systems. However, widely accepted standardized library preparation methods can be cost-prohibitive. Newly available approaches may be less expensive, but their efficacy in comparison to standardized methods remains unknown. In this study, we compared five different metagenomic library preparation methods. We evaluated each method across a range of microbial communities varying in complexity and quantity of input DNA. Our findings demonstrate the importance of considering sample properties, including community type, composition, and DNA amount, when choosing the most appropriate metagenomic library preparation method.

Amplification, sequencing, and analysis of the 16S rRNA gene affords characterization of microbial community composition. As this tool has become more popular and amplicon-sequencing applications have grown in the total number of samples, growth in sample multiplexing is becoming necessary while maintaining high sequence quality and sequencing depth. Here, modifications to the Illumina HiSeq 2500 platform are described which produce greater multiplexing capabilities and 300-bp paired-end reads of higher quality than those produced by the current Illumina MiSeq platform. To improve the feasibility and flexibility of this method, a 2-step PCR amplification protocol is also described that allows for targeting of different amplicon regions, and enhances amplification success from samples with low bacterial bioburden. IMPORTANCE Amplicon sequencing has become a popular and widespread tool for surveying microbial communities. Lower overall costs associated with high-throughput sequencing have made it a widely adopted approach, especially for projects that necessitate sample multiplexing to eliminate batch effect and reduced time to acquire data. The method for amplicon sequencing on the Illumina HiSeq 2500 platform described here provides improved multiplexing capabilities while simultaneously producing greater quality sequence data and lower per-sample cost relative to those of the Illumina MiSeq platform without sacrificing amplicon length. To make this method more flexible for various amplicon-targeted regions as well as improve amplification from low-biomass samples, we also present and validate a 2-step PCR library preparation method.