Disrupting Central Carbon Metabolism Increases ß-Lactam Antibiotic Susceptibility in Vibrio cholerae.

Antibiotic tolerance, the ability of bacteria to sustain viability in the presence of typically bactericidal antibiotics for extended time periods, is an understudied contributor to treatment failure. The Gram-negative pathogen Vibrio cholerae, the causative agent of cholera, becomes highly tolerant to ß-lactam antibiotics (penicillin and related compounds) in a process requiring the two-component system VxrAB. VxrAB is induced by exposure to cell wall damaging conditions, which results in the differential regulation of >100 genes. While the effectors of VxrAB are relatively well known, VxrAB environment-sensing and activation mechanisms remain a mystery. Here, we used transposon mutagenesis to screen for mutants that spontaneously upregulate VxrAB signaling. This screen was answered by genes known to be required for proper cell envelope homeostasis, validating the approach. Unexpectedly, we also uncovered a new connection between central carbon metabolism and antibiotic tolerance in Vibrio cholerae. Inactivation of pgi (vc0374, coding for glucose-6-phosphate isomerase) resulted in an intracellular accumulation of glucose-6-phosphate and fructose-6-phosphate, concomitant with a marked cell envelope defect, resulting in VxrAB induction. Deletion of pgi also increased sensitivity to ß-lactams and conferred a growth defect on salt-free LB, phenotypes that could be suppressed by deleting sugar uptake systems and by supplementing cell wall precursors in the growth medium. Our data suggest an important connection between central metabolism and cell envelope integrity and highlight a potential new target for developing novel antimicrobial agents. IMPORTANCE Antibiotic tolerance (the ability to survive exposure to antibiotics) is a stepping stone toward antibiotic resistance (the ability to grow in the presence of antibiotics), an increasingly common cause of antibiotic treatment failure. The mechanisms promoting tolerance are poorly understood. Here, we identified central carbon metabolism as a key contributor to antibiotic tolerance and resistance. A strain with a mutation in a sugar utilization pathway accumulates metabolites that likely shut down the synthesis of cell wall precursors, which weakens the cell wall and thus increases susceptibility to cell wall-active drugs. Our results illuminate the connection between central carbon metabolism and cell wall homeostasis in V. cholerae and suggest that interfering with metabolism may be a fruitful future strategy for the development of antibiotic adjuvants.

Pharmacy Technicians and Point of Care Testing.

Objective: As the practice of pharmacy continues to advance and involve nondispensing pharmacy services, point-of-care testing (POCT) has continued to demonstrate its usefulness as a tool and service in pharmacy. Of particular significance is the ability of POCT to assist clinical practice related to diabetes, cholesterol management, and anticoagulation. POCT can allow for certain laboratory results to be obtained within seconds to minutes, which can help direct care. Many components of POCT programs can involve pharmacy technicians to help enhance practice efficiency. The purpose of this review is to provide an overview of POCT devices commonly used in nondispensing pharmacy services and to describe the roles that pharmacy technicians may have in the POCT process. Data Sources: PubMed (1946-2014) was reviewed for relevant literature using terms such as "pharmacy technician" and "point of care testing." Additionally, manufacturer information/websites of POCT products were reviewed for approval information and instructions for use. Study Selection and Data Extraction: Articles describing POCT completed in a pharmacy setting and/or roles of pharmacy technicians and related support staff in the POCT process were considered for inclusion. Data Synthesis: Several types of POCT devices for different uses common to the practice of pharmacy are reviewed. Additionally, strategies for collaboration between pharmacy technicians and pharmacists in the execution of a POCT program are described. Conclusion: Pharmacy technicians are well suited to participate in portions of the POCT process, and the involvement of pharmacy technicians may improve POCT efficiency.

Bacterial metabolism and susceptibility to cell wall-active antibiotics.

Bacterial infections are increasingly resistant to antimicrobial therapy. Intense research focus has thus been placed on identifying the mechanisms that bacteria use to resist killing or growth inhibition by antibiotics and the ways in which bacteria share these traits with one another. This work has led to the advancement of new drugs, combination therapy regimens, and a deeper appreciation for the adaptability seen in microorganisms. However, while the primary mechanisms of action of most antibiotics are well understood, the more subtle contributions of bacterial metabolic state to repairing or preventing damage caused by antimicrobials (thereby promoting survival) are still understudied. Here, we review a modern viewpoint on a classical system: examining bacterial metabolism's connection to antibiotic susceptibility. We dive into the relationship between metabolism and antibiotic efficacy through the lens of growth rate, energy state, resource allocation, and the infection environment, focusing on cell wall-active antibiotics.

Mechanical stimuli activate gene expression via a cell envelope stress sensing pathway.

Mechanosensitive mechanisms are often used to sense damage to tissue structure, stimulating matrix synthesis and repair. While this kind of mechanoregulatory process is well recognized in eukaryotic systems, it is not known whether such a process occurs in bacteria. In Vibrio cholerae, antibiotic-induced damage to the load-bearing cell wall promotes increased signaling by the two-component system VxrAB, which stimulates cell wall synthesis. Here we show that changes in mechanical stress within the cell envelope are sufficient to stimulate VxrAB signaling in the absence of antibiotics. We applied mechanical forces to individual bacteria using three distinct loading modalities: extrusion loading within a microfluidic device, direct compression and hydrostatic pressure. In all cases, VxrAB signaling, as indicated by a fluorescent protein reporter, was increased in cells submitted to greater magnitudes of mechanical loading, hence diverse forms of mechanical stimuli activate VxrAB signaling. Reduction in cell envelope stiffness following removal of the endopeptidase ShyA led to large increases in cell envelope deformation and substantially increased VxrAB response, further supporting the responsiveness of VxrAB. Our findings demonstrate a mechanosensitive gene regulatory system in bacteria and suggest that mechanical signals may contribute to the regulation of cell wall homeostasis.

Enhancing Practice Efficiency and Patient Care by Sharing Electronic Health Records.

One primary function of community pharmacies is to dispense medications to patients. In doing so, pharmacists frequently communicate with physicians' offices to clarify prescription orders and obtain additional information to ensure the safe and accurate dispensing of medications. Such communication is often done by telephone or fax, which is inefficient for both the pharmacy and the physician's office. This problem was highlighted in a recent American Medical Association resolution defining certain pharmacy inquiries as "interference with the practice of medicine and unwarranted." As a result, many are seeking to understand how to balance the needs of the patient care process with the need for operational efficiency in the physician's office and pharmacy. This study presents one example of a health information technology-based solution involving shared access to an electronic health record (EHR), and describes a case in which a physician's office and a community pharmacy experimented with this model to promote practice efficiency while also providing enhanced access to clinical information in both directions. The rationale behind the process change, a brief description of how the new process came into existence, and a description of how information sharing can be helpful in related clinical situations are provided. Similar models that involve sharing of EHRs may create valuable opportunities for collaboration between physicians and pharmacists to enhance patient care and improve workflow efficiency.

Integrating grant-funded research into the undergraduate biology curriculum using IMG-ACT.

It has become clear in current scientific pedagogy that the emersion of students in the scientific process in terms of designing, implementing, and analyzing experiments is imperative for their education; as such, it has been our goal to model this active learning process in the classroom and laboratory in the context of a genuine scientific question. Toward this objective, the National Science Foundation funded a collaborative research grant between a primarily undergraduate institution and a research-intensive institution to study the chemotactic responses of the bacterium Pseudomonas putida F1. As part of the project, a new Bioinformatics course was developed in which undergraduates annotate relevant regions of the P. putida F1 genome using Integrated Microbial Genomes Annotation Collaboration Toolkit, a bioinformatics interface specifically developed for undergraduate programs by the Department of Energy Joint Genome Institute. Based on annotations of putative chemotaxis genes in P. putida F1 and comparative genomics studies, undergraduate students from both institutions developed functional genomics research projects that evolved from the annotations. The purpose of this study is to describe the nature of the NSF grant, the development of the Bioinformatics lecture and wet laboratory course, and how undergraduate student involvement in the project that was initiated in the classroom has served as a springboard for independent undergraduate research projects.