Fast and accurate identification of pathogenic bacteria using excitation-emission spectroscopy and machine learning.

Fast and reliable identification of pathogenic bacteria is of upmost importance to human health and safety. Methods that are currently used in clinical practice are often time consuming, require expensive equipment, trained personnel, and therefore have limited applications in low resource environments. Molecular identification methods address some of these shortcomings.

Glutamatedependent arginine biosynthesis requires the inactivation of , and in .

Genome sequencing has demonstrated that encodes arginine biosynthetic genes synthesizing proteins that mediate arginine biosynthesis using glutamate as a substrate. Paradoxically, however, does not grow in a defined, glutamate-replete medium lacking arginine and glucose (CDM-R). Studies from our laboratory have found that specific mutations are selected by that facilitate growth in CDM-R.

The Staphylococcus aureus CidA and LrgA Proteins Are Functional Holins Involved in the Transport of By-Products of Carbohydrate Metabolism.

The Staphylococcus aureus and operons encode members of a well-conserved family of proteins thought to be involved in programmed cell death (PCD). Based on the structural similarities that CidA and LrgA share with bacteriophage holins, we have hypothesized that these proteins function by forming pores within the cytoplasmic membrane. To test this, we utilized a "lysis cassette" system that demonstrated the abilities of the and genes to support bacteriophage endolysin-induced cell lysis.

Simpler Procedure and Improved Performance for Pathogenic Bacteria Analysis with a Paper-Based Ratiometric Fluorescent Sensor Array.

Bacterial infections are the leading cause of morbidity and mortality in the world, particularly due to a delay in treatment and misidentification of the bacterial species causing the infection. Therefore, rapid and accurate identification of these pathogens has been of prime importance. The conventional diagnostic techniques include microbiological, biochemical, and genetic analyses, which are time-consuming, require large sample volumes, expensive equipment, reagents, and trained personnel.

Time-Resolved Fluorescence Assay for Measuring Oxygen Consumption Rates in Staphylococcus aureus.

Oxygen consumption is a fundamental characteristic of staphylococcal physiology reflecting the energy and metabolic state of the bacterial cell. During aerobic growth, oxygen consumption rates (OCR) depend on nutrient availability and vary at different growth stages. The measurement of oxygen consumption rates provides a versatile tool to characterize the impact of various mutations, environmental cues, and antibiotics on bacterial growth and fitness.

Inactivation of the Pta-AckA pathway impairs fitness of during overflow metabolism.

Under conditions of glucose excess, aerobically growing bacteria predominantly direct carbon flux towards acetate fermentation, a phenomenon known as overflow metabolism or the bacterial 'Crabtree effect'. Numerous studies of the major acetate-generating pathway, the Pta-AckA, revealed its important role in bacterial fitness through the control of central metabolism to sustain balanced growth and cellular homeostasis. In this work, we highlight the contribution of the Pta-AckA pathway to fitness of the spore-forming bacterium, We demonstrate that disruption of the Pta-AckA pathway causes a drastic growth reduction in the mutants and alters the metabolic and energy status of the cells.

Stochastic Expression of Sae-Dependent Virulence Genes during Staphylococcus aureus Biofilm Development Is Dependent on SaeS.

The intricate process of biofilm formation in the human pathogen involves distinct stages during which a complex mixture of matrix molecules is produced and modified throughout the developmental cycle. Early in biofilm development, a subpopulation of cells detaches from its substrate in an event termed "exodus" that is mediated by SaePQRS-dependent stochastic expression of a secreted staphylococcal nuclease, which degrades extracellular DNA within the matrix, causing the release of cells and subsequently allowing for the formation of metabolically heterogenous microcolonies. Since the SaePQRS regulatory system is involved in the transcriptional control of multiple virulence factors, the expression of several additional virulence genes was examined within a developing biofilm by introducing fluorescent gene reporter plasmids into wild-type and isogenic regulatory mutants and growing these strains in a microfluidic system that supplies the bacteria with a constant flow of media while simultaneously imaging developing biofilms in 5-min intervals.

Observations of Shear Stress Effects on Staphylococcus aureus Biofilm Formation.

bacteria form biofilms and distinctive microcolony or "tower" structures that facilitate their ability to tolerate antibiotic treatment and to spread within the human body. The formation of microcolonies, which break off, get carried downstream, and serve to initiate biofilms in other parts of the body, is of particular interest here. It is known that flow conditions play a role in the development, dispersion, and propagation of biofilms in general.

Simple synthesis of endophenazine G and other phenazines and their evaluation as anti-methicillin-resistant Staphylococcus aureus agents.

Community-associated methicillin resistant Staphylococcus aureus (CA-MRSA) has become a severe health concern because of its treatment difficulties. Herein, we report the synthesis and biological evaluation of two phenazine natural products and a series of phenazines that show promising activities against MRSA with MIC values in the low micromolar range. Basic studies revealed that these compounds are bacteriostatic agents.

Identification of inhibitors for single-stranded DNA-binding proteins in eubacteria.

Objectives: The increasing threat of drug-resistant bacteria establishes a continuing need for the development of new strategies to fight infection. We examine the inhibition of the essential single-stranded DNA-binding proteins (SSBs) SSBA and SSBB as a potential antimicrobial therapy due to their importance in DNA replication, activating the SOS response and promoting competence-based mechanisms of resistance by incorporating new DNA.

Methods: Purified recombinant SSBs from Gram-positive (Staphylococcus aureus and Bacillus anthracis) and Gram-negative (Escherichia coli and Francisella tularensis) bacteria were assessed in a high-throughput screen for inhibition of duplex DNA unwinding by small molecule inhibitors.

Novel fluorinated pyrrolomycins as potent anti-staphylococcal biofilm agents: Design, synthesis, pharmacokinetics and antibacterial activities.

Staphylococcus aureus (SA) is a major cause of hospital- and community-associated bacterial infections in the U.S. and around the world.

Effects of Low-Dose Amoxicillin on Staphylococcus aureus USA300 Biofilms.

Previous studies showed that sub-MIC levels of β-lactam antibiotics stimulate biofilm formation in most methicillin-resistant Staphylococcus aureus (MRSA) strains. Here, we investigated this process by measuring the effects of sub-MIC amoxicillin on biofilm formation by the epidemic community-associated MRSA strain USA300. We found that sub-MIC amoxicillin increased the ability of USA300 cells to attach to surfaces and form biofilms under both static and flow conditions.

Identification of the amino acids essential for LytSR-mediated signal transduction in Staphylococcus aureus and their roles in biofilm-specific gene expression.

Recent studies have demonstrated that expression of the Staphylococcus aureus lrgAB operon is specifically localized within tower structures during biofilm development. To gain a better understanding of the mechanisms underlying this spatial control of lrgAB expression, we carried out a detailed analysis of the LytSR two-component system. Specifically, a conserved aspartic acid (Asp53) of the LytR response regulator was shown to be the target of phosphorylation, which resulted in enhanced binding to the lrgAB promoter and activation of transcription.

Use of microfluidic technology to analyze gene expression during Staphylococcus aureus biofilm formation reveals distinct physiological niches.

The Staphylococcus aureus cid and lrg operons play significant roles in the control of autolysis and accumulation of extracellular genomic DNA (eDNA) during biofilm development. Although the molecular mechanisms mediating this control are only beginning to be revealed, it is clear that cell death must be limited to a subfraction of the biofilm population. In the present study, we tested the hypothesis that cid and lrg expression varies during biofilm development as a function of changes in the availability of oxygen.

Staphylococcus aureus CidA and LrgA proteins exhibit holin-like properties.

The Staphylococcus aureus cid and lrg operons are known to be involved in biofilm formation by controlling cell lysis and the release of genomic DNA, which ultimately becomes a structural component of the biofilm matrix. Although the molecular mechanisms controlling cell death and lysis are unknown, it has been hypothesized that the cidA and lrgA genes encode holin- and antiholin-like proteins and function to regulate these processes similarly to bacteriophage-induced death and lysis. In this study, we focused on the biochemical and molecular characterization of CidA and LrgA with the goal of testing the holin model.

Micro-expression recognition training in medical students: a pilot study.

Background: Patients provide emotional cues during consultations which may be verbal or non-verbal. Many studies focus on patient verbal cues as predictors of physicians' ability to recognize and address patient needs but this project focused on non-verbal cues in the form of facial micro-expressions. This pilot study investigated first year medical students' (n = 75) identified as being either good or poor communicators abilities to detect emotional micro-expressions before and after training using the Micro Expression Training Tool (METT) http://www.

Identification and characterization of a family of toxin-antitoxin systems related to the Enterococcus faecalis plasmid pAD1 par addiction module.

The par locus of the Enterococcus faecalis plasmid pAD1 is an RNA-regulated addiction module encoding the peptide toxin Fst. Homology searches revealed that Fst belongs to a family of at least nine related peptides encoded on the chromosomes and plasmids of six different Gram-positive bacterial species. Comparison of an alignment of these peptides with the results of a saturation mutagenesis analysis indicated regions of the peptides important for biological function.