Transcriptional profiles of 2 unrelated clinical methicillin-resistant Staphylococcus aureus (MRSA) isolates were analyzed following 10% (v/v) ethanol challenge (15 min), which arrested growth but did not reduce viability. Ethanol-induced stress (EIS) resulted in differential gene expression of 1091 genes, 600 common to both strains, of which 291 were upregulated. With the exception of the downregulation of genes involved with osmotic stress functions, EIS resulted in the upregulation of genes that contribute to stress response networks, notably those altered by oxidative stress, protein quality control in general, and heat shock in particular. In addition, genes involved with transcription, translation, and nucleotide biosynthesis were downregulated. relP, which encodes a small alarmone synthetase (RelP), was highly upregulated in both MRSA strains following ethanol challenge, and relP inactivation experiments indicated that this gene contributed to EIS growth arrest. A number of persistence-associated genes were also upregulated during EIS, including those that encode toxin-antitoxin systems. Overall, transcriptional profiling indicated that the MRSA investigated responded to EIS by entering a state of dormancy and by altering the expression of elements from cross protective stress response systems in an effort to protect preexisting proteins.

The Elizabethkingia are a genetically diverse genus of emerging pathogens that exhibit multidrug resistance to a range of common antibiotics. Two representative species, Elizabethkingia bruuniana and E. meningoseptica , were phenotypically tested to determine minimum inhibitory concentrations (MICs) for five antibiotics. Ultra-long read sequencing with Oxford Nanopore Technologies (ONT) and subsequent de novo assembly produced complete, gapless circular genomes for each strain. Alignment based annotation with Prokka identified 5,480 features in E. bruuniana and 5,203 features in E. meningoseptica , where none of these identified genes or gene combinations corresponded to observed phenotypic resistance values. Pan-genomic analysis, performed with an additional 19 Elizabethkingia strains, identified a core-genome size of 2,658,537 bp, 32 uniquely identifiable intrinsic chromosomal antibiotic resistance core-genes and 77 antibiotic resistance pan-genes. Using core-SNPs and pan-genes in combination with six machine learning (ML) algorithms, binary classification of clindamycin and vancomycin resistance achieved f1 scores of 0.94 and 0.84, respectively. Performance on the more challenging multiclass problem for fusidic acid, rifampin and ciprofloxacin resulted in f1 scores of 0.70, 0.75, and 0.54, respectively. By producing two sets of quality biological predictors, pan-genome genes and core-genome SNPs, from long-read sequence data and applying an ensemble of ML techniques, our results demonstrated that accurate phenotypic inference, at multiple AMR resolutions, can be achieved.

Tea tree oil-reduced susceptibility (TTORS) mutants of two Staphylococcus aureus laboratory strains were isolated utilizing TTO gradient plates. Attempts to isolate TTORS mutants employing agar plates containing single TTO concentrations failed. All TTORS mutants demonstrated a small colony variant (SCV) phenotype and produced cells with a smaller diameter, as determined by scanning electron microscopy. The addition of SCV auxotrophic supplements to media did not lead to an increase in TTORS mutant colony size. TTORS mutant revertants (RV) were also isolated from the TTORS mutants following growth in drug free media and all RV strains demonstrated phenotypes similar to their respective parent strains. Transmission electron microscopy revealed that a SH1000 TTORS mutant demonstrated a thinner cell wall and novel septal invaginations compared to parent strain SH1000. In addition, comparative genomic sequencing did not reveal any mutations in a SH1000 TTORS mutant previously linked to well-characterized SCV genotypes. This study demonstrates that TTO can select for a unique SCV phenotype.

Acinetobacter baumannii is a formidable opportunistic pathogen that is notoriously difficult to eradicate from hospital settings. This resilience is often attributed to a proclivity for biofilm formation, which facilitates a higher tolerance toward external stress, desiccation, and antimicrobials.

We report the isolation and characterization of two Elizabethkingia anophelis strains (OSUVM-1 and OSUVM-2) isolated from sources associated with horses in Oklahoma. Both strains appeared susceptible to fluoroquinolones and demonstrated high MICs to all cell wall active antimicrobials including vancomycin, along with aminoglycosides, fusidic acid, chloramphenicol, and tetracycline. Typical of the Elizabethkingia, both draft genomes contained multiple copies of β-lactamase genes as well as genes predicted to function in antimicrobial efflux. Phylogenetic analysis of the draft genomes revealed that OSUVM-1 and OSUVM-2 differ by only 6 SNPs and are in a clade with 3 strains of Elizabethkingia anophelis that were responsible for human infections. These findings therefore raise the possibility that Elizabethkingia might have the potential to move between humans and animals in a manner similar to known zoonotic pathogens.

2The Elizabethkingia are a genetically diverse genus of emerging pathogens that exhibit multidrug 3 resistance to a range of common antibiotics. Two representative species, Elizabethkingia 4 bruuniana and Elizabethkingia meningoseptica, were phenotypically tested to determine 5 minimum inhibitory concentrations for five antibiotics. Ultra-long read sequencing with Oxford 6 Nanopore Technologies and subsequent de novo assembly produced complete, gapless circular 7 genomes for each strain. Alignment based annotation with Prokka identified 5,480 features in E. 8 bruuniana and 5,203 features in E. meningoseptica, where none of these identified genes or gene 9 combinations corresponded to observed phenotypic resistance values. Pan-genomic analysis, 10 performed with an additional 19 Elizabethkingia strains, identified a core-genome size of 11 2,658,537 bp, 32 uniquely identifiable intrinsic chromosomal antibiotic resistance core-genes 12 and 77 antibiotic resistance pan-genes. Using core-SNPs and pan-genes in combination with six 13 machine learning algorithms, binary classification of clindamycin and vancomycin resistance 14 achieved f1 scores of 0.94 and 0.84 respectively. Performance on the more challenging 15 multiclass problem for fusidic acid, rifampin and ciprofloxacin resulted in f1 scores of 0.70, 0.75 16 and 0.54 respectively. 17 18 19 101Mueller-Hinton Agar (MHA) and incubating for 24 hr (37°C). The MBC was determined as the 102 lowest antimicrobial concentration in which no visual colonies were observed. 103 Library preparation 104DNA libraries were prepared separately for each Elizabethkingia isolate following the 105 procedures outlined for the SQK-LSK208 2D sequencing kit (Oxford Nanopore Technologies, 106 United Kingdom) with the following protocol adjustments. A total of 1.5 µg of gDNA was 107 sheared in g-tubes (Covaris) at 4200 RPM for a targeted fragment size of 20 kb. End-repair was 108 6 performed following the manufacturer's recommended protocol for Ultra II End-prep enzyme 109 mix (NEB). Adapter ligation reaction incubations were increased to 15 minutes. All bead clean-110 ups used 0.4x AMPureXP beads (Beckman Coulter, Brea, CA) for additional size selection and 111 elutions were performed at 37°C for 20 minutes. DNA concentration of the library was 112 quantified using Quant-IT PicoGreen® dsDNA Assay Kit (ThermoFisher Scientific), measured 113 on Synergy H1, hybrid multi-mode microplate reader (BioTek). Final DNA library yields were 114 above the recommended 200 ng. 115 Single molecular real time sequencing 116 Two R9.4 flow cells were prepared for two corresponding MinIONs, each connected to a 117 separate Windows PC using a USB 3.0 connection. MinKNOW GUI application 1.0.8.0 from 118 Oxford Nanopore Technologies (ONT) was used to validate the MinION connection and to 119 monitor basic hardware details, like the number of active pores within each flow cell during 120 sequencing runs. Pore count validation was completed beforehand, with the Platform QC 121 command in MinKNOW. Flow cell prim...

Tea tree oil (TTO) is hypothesized to kill bacteria by indiscriminately denaturing membrane and protein structures. A Staphylococcus aureus small colony variant (SCV) selected with TTO (SH1000-TTORS-1) demonstrated slowed growth, reduced susceptibility to TTO, a diminutive cell size, and a thinned cell wall. Utilizing a proteomics and metabolomics approach, we have now revealed that the TTO-selected SCV mutant demonstrated defective fatty acid synthesis, an alteration in the expression of genes and metabolites associated with central metabolism, the induction of a general stress response, and a reduction of proteins critical for active growth and translation. SH1000-TTORS-1 also demonstrated an increase in amino acid accumulation and a decrease in sugar content. The reduction in glycolytic pathway proteins and sugar levels indicated that carbon flow through glycolysis and gluconeogenesis is reduced in SH1000-TTORS-1. The increase in amino acid accumulation coincides with the reduced production of translation-specific proteins and the induction of proteins associated with the stringent response. The decrease in sugar content likely deactivates catabolite repression and the increased amino acid pool observed in SH1000-TTORS-1 represents a potential energy and carbon source which could maintain carbon flow though the tricarboxylic acid (TCA) cycle. It is noteworthy that processes that contribute to the production of the TTO targets (proteins and membrane) are reduced in SH1000-TTORS-1. This is one of a few studies describing a mechanism that bacteria utilize to withstand the action of an antiseptic which is thought to inactivate multiple cellular targets.

In this study, we identify a novel two-component system in Acinetobacter baumannii (herein named AmsSR for regulator of alternative metabolic systems) only present in select gammaproteobacterial and betaproteobacterial species. Bioinformatic analysis revealed that the histidine kinase, AmsS, contains 14 predicted N-terminal transmembrane domains and harbors a hybrid histidine kinase arrangement in its C-terminus. Transcriptional analysis revealed the proton ionophore CCCP selectively induces PamsSR expression. Disruption of amsSR resulted in decreased intracellular pH and increased depolarization of cytoplasmic membranes. Transcriptome profiling revealed a major reordering of metabolic circuits upon amsR disruption, with energy generation pathways typically used by bacteria growing in limited oxygen being favored. Interestingly, we observed enhanced growth rates for mutant strains in the presence of glucose, which led to overproduction of pyruvate. To mitigate the toxic effects of carbon overflow, we noted acetate overproduction in amsSR-null strains, resulting from a hyperactive Pta-AckA pathway. Additionally, due to altered expression of key metabolic genes, amsSR mutants favor an incomplete TCA cycle, relying heavily on an overactive glyoxylate shunt. This metabolic reordering overproduces NADH, which is not oxidized by the ETC; components of which were significantly downregulated upon amsSR disruption. As a result, the mutants almost exclusively rely on substrate phosphorylation for ATP production, and consequently display reduced oxygen consumption in the presence of glucose. Collectively, our data suggests that disruption of amsSR affects the function of the aerobic respiratory chain, impacting the energy status of the cell, which in turn upregulates alternative metabolic and energy generation pathways.