Synthesis and biological evaluation of tetrahydroisoquinoline-derived antibacterial compounds.

Antibiotic resistance is one of the greatest threats to modern medicine. Drugs that were once routinely used to treat infections are being rendered ineffective, increasing the demand for novel antibiotics with low potential for resistance. Here we report the synthesis of 18 novel cationic tetrahydroisoquinoline-triazole compounds. Five of the developed molecules were active against S. aureus at a low MIC of 2-4 µg/mL. Hit compound 4b was also found to eliminate M. tuberculosis H37Rv at MIC of 6 µg/mL. This potent molecule was found to eliminate S. aureus effectively, with no resistance observed after thirty days of sequential passaging. These results identified compound 4b and its analogues as potential candidates for further drug development that could help tackle the threat of antibiotic resistance.

Understanding Perceived Appreciation to Create a Culture of Wellness.

OBJECTIVE: To fully address physician burnout, academic medical centers need cultures that promote well-being. One observed driver of a culture of wellness is perceived appreciation. The authors identified several contributors to perceived appreciation among faculty at a large, metropolitan academic institution through use of a novel survey. METHODS: The authors surveyed clinical faculty in five departments: psychiatry, emergency medicine, internal medicine, thoracic surgery, and radiology. Two open-ended response questions assessed sources of perceived and lack of perceived appreciation in narrative form. The authors also collected data on gender and department identity. Grounded theory methodology was used to analyze the narrative responses and design thinking to brainstorm specific recommendations based on the main themes identified. RESULTS: A total of 179 faculty respondents filled out the survey for an overall response rate of 29%. Major drivers of perceived appreciation were patient and families (42%); physician, trainee and non-physician colleagues (32.7%); chairs (10%); and compensation (3.3%). Major drivers of perceived lack of appreciation were disrespect for time and skill level, including inadequate staffing (30%); devaluation by a physician colleague, chief of one's service or the chair (29%); poor communication and transparency (13%); and patient and family anger (6%). CONCLUSIONS: Opportunities to improve perceived appreciation include structured communication of patient gratitude, community building programs, top of licensure initiatives and accountability for physician wellness, and inclusivity efforts from organizational leaders.

A newly identified prophage-encoded gene, ymfM, causes SOS-inducible filamentation in Escherichia coli.

Rod-shaped bacteria such as Escherichia coli can regulate cell division in response to stress, leading to filamentation, a process where cell growth and DNA replication continues in the absence of division, resulting in elongated cells. The classic example of stress is DNA damage which results in the activation of the SOS response. While the inhibition of cell division during SOS has traditionally been attributed to SulA in E. coli, a previous report suggests that the e14 prophage may also encode an SOS-inducible cell division inhibitor, previously named SfiC. However, the exact gene responsible for this division inhibition has remained unknown for over 35 years. A recent high-throughput over-expression screen in E. coli identified the e14 prophage gene, ymfM, as a potential cell division inhibitor. In this study, we show that the inducible expression of ymfM from a plasmid causes filamentation. We show that this expression of ymfM results in the inhibition of Z ring formation and is independent of the well characterised inhibitors of FtsZ ring assembly in E. coli, SulA, SlmA and MinC. We confirm that ymfM is the gene responsible for the SfiC phenotype as it contributes to the filamentation observed during the SOS response. This function is independent of SulA, highlighting that multiple alternative division inhibition pathways exist during the SOS response. Our data also highlight that our current understanding of cell division regulation during the SOS response is incomplete and raises many questions regarding how many inhibitors there actually are and their purpose for the survival of the organism.Importance:Filamentation is an important biological mechanism which aids in the survival, pathogenesis and antibiotic resistance of bacteria within different environments, including pathogenic bacteria such as uropathogenic Escherichia coli Here we have identified a bacteriophage-encoded cell division inhibitor which contributes to the filamentation that occurs during the SOS response. Our work highlights that there are multiple pathways that inhibit cell division during stress. Identifying and characterising these pathways is a critical step in understanding survival tactics of bacteria which become important when combating the development of bacterial resistance to antibiotics and their pathogenicity.

Synthesis and biological evaluation of 3,5-substituted pyrazoles as possible antibacterial agents.

The emergence of multi-drug resistant bacteria has increased the need for novel antibiotics to help overcome what may be considered the greatest threat to modern medicine. Here we report the synthesis of fifteen novel 3,5-diaryl-1H- pyrazoles obtained via one-pot cyclic oxidation of a chalcone and hydrazine-monohydrate. The synthesised pyrazoles were then screened against Staphylococcus aureus and Escherichia coli to determine their antibacterial potential. The results show that compound 7p is bacteriostatic at MIC 8 µg/mL. The compound is non-toxic against healthy mammalian cells, 3T3-L1 at the highest test concentration 50 µg/mL. Furthermore, compound 7p significantly affected bacterial morphogenesis before cell lysis in Bacillus subtilis when treated above the MIC concentration. From the results, a promising lead compound was identified for future development.

CetZ tubulin-like proteins control archaeal cell shape.

Tubulin is a major component of the eukaryotic cytoskeleton, controlling cell shape, structure and dynamics, whereas its bacterial homologue FtsZ establishes the cytokinetic ring that constricts during cell division. How such different roles of tubulin and FtsZ evolved is unknown. Studying Archaea may provide clues as these organisms share characteristics with Eukarya and Bacteria. Here we report the structure and function of proteins from a distinct family related to tubulin and FtsZ, named CetZ, which co-exists with FtsZ in many archaea. CetZ X-ray crystal structures showed the FtsZ/tubulin superfamily fold, and one crystal form contained sheets of protofilaments, suggesting a structural role. However, inactivation of CetZ proteins in Haloferax volcanii did not affect cell division. Instead, CetZ1 was required for differentiation of the irregular plate-shaped cells into a rod-shaped cell type that was essential for normal swimming motility. CetZ1 formed dynamic cytoskeletal structures in vivo, relating to its capacity to remodel the cell envelope and direct rod formation. CetZ2 was also implicated in H. volcanii cell shape control. Our findings expand the known roles of the FtsZ/tubulin superfamily to include archaeal cell shape dynamics, suggesting that a cytoskeletal role might predate eukaryotic cell evolution, and they support the premise that a major function of the microbial rod shape is to facilitate swimming.

The ParB homologs, Spo0J and Noc, together prevent premature midcell Z ring assembly when the early stages of replication are blocked in Bacillus subtilis.

Precise cell division in coordination with DNA replication and segregation is of utmost importance for all organisms. The earliest stage of cell division is the assembly of a division protein FtsZ into a ring, known as the Z ring, at midcell. What still eludes us, however, is how bacteria precisely position the Z ring at midcell. Work in B. subtilis over the last two decades has identified a link between the early stages of DNA replication and cell division. A recent model proposed that the progression of the early stages of DNA replication leads to an increased ability for the Z ring to form at midcell. This model arose through studies examining Z ring position in mutants blocked at different steps of the early stages of DNA replication. Here, we show that this model is unlikely to be correct and the mutants previously studied generate nucleoids with different capacity for blocking midcell Z ring assembly. Importantly, our data suggest that two proteins of the widespread ParB family, Noc and Spo0J are required to prevent Z ring assembly over the bacterial nucleoid and help fine tune the assembly of the Z ring at midcell during the cell cycle.

Uncovering novel susceptibility targets to enhance the efficacy of third-generation cephalosporins against ESBL-producing uropathogenic Escherichia coli.

BACKGROUND: Uropathogenic Escherichia coli (UPEC) are a major cause of urinary tract infection (UTI), one of the most common infectious diseases in humans. UPEC are increasingly associated with resistance to multiple antibiotics. This includes resistance to third-generation cephalosporins, a common class of antibiotics frequently used to treat UTI. METHODS: We employed a high-throughput genome-wide screen using saturated transposon mutagenesis and transposon directed insertion-site sequencing (TraDIS) together with phenotypic resistance assessment to identify key genes required for survival of the MDR UPEC ST131 strain EC958 in the presence of the third-generation cephalosporin cefotaxime. RESULTS: We showed that blaCMY-23 is the major ESBL gene in EC958 responsible for mediating resistance to cefotaxime. Our screen also revealed that mutation of genes involved in cell division and the twin-arginine translocation pathway sensitized EC958 to cefotaxime. The role of these cell-division and protein-secretion genes in cefotaxime resistance was confirmed through the construction of mutants and phenotypic testing. Mutation of these genes also sensitized EC958 to other cephalosporins. CONCLUSIONS: This work provides an exemplar for the application of TraDIS to define molecular mechanisms of resistance to antibiotics. The identification of mutants that sensitize UPEC to cefotaxime, despite the presence of a cephalosporinase, provides a framework for the development of new approaches to treat infections caused by MDR pathogens.

Perceived Electronic Health Record Usability as a Predictor of Task Load and Burnout Among US Physicians: Mediation Analysis.

BACKGROUND: Electronic health record (EHR) usability and physician task load both contribute to physician professional burnout. The association between perceived EHR usability and workload has not previously been studied at a national level. Better understanding these interactions could give further information as to the drivers of extraneous task load. OBJECTIVE: This study aimed to determine the relationship between physician-perceived EHR usability and workload by specialty and evaluate for associations with professional burnout. METHODS: A secondary analysis of a cross-sectional survey of US physicians from all specialties was conducted from October 2017 to March 2018. Among the 1250 physicians invited to respond to the subsurvey analyzed here, 848 (67.8%) completed it. EHR usability was assessed with the System Usability Scale (SUS; range: 0-100). Provider task load (PTL) was assessed using the mental demand, physical demand, temporal demand, and effort required subscales of the National Aeronautics and Space Administration Task Load Index (range: 0-400). Burnout was measured using the Maslach Burnout Inventory. RESULTS: The mean scores were 46.1 (SD 22.1) for SUS and 262.5 (SD 71.7) for PTL. On multivariable analysis adjusting for age, gender, relationship status, medical specialty, practice setting, hours worked per week, and number of nights on call per week, physician-rated EHR usability was associated with PTL, with each 1-point increase in SUS score (indicating more favorable) associated with a 0.57-point decrease in PTL score (P<.001). On mediation analysis, higher SUS score was associated with lower PTL score, which was associated with lower odds of burnout. CONCLUSIONS: A strong association was observed between EHR usability and workload among US physicians, with more favorable usability associated with less workload. Both outcomes were associated with the odds of burnout, with task load acting as a mediator between EHR usability and burnout. Improving EHR usability while decreasing task load has the potential to allow practicing physicians more working memory for medical decision making and patient communication.

A Health System-Wide Initiative to Decrease Opioid-Related Morbidity and Mortality.

BACKGROUND: The opioid overdose crisis now claims more than 40,000 lives in the United States every year, and many hospitals and health systems are responding with opioid-related initiatives, but how best to coordinate hospital or health system-wide strategy and approach remains a challenge. METHODS: An organizational opioid stewardship program (OSP) was created to reduce opioid-related morbidity and mortality in order to provide an efficient, comprehensive, multidisciplinary approach to address the epidemic in one health system. An executive committee of hospital leaders was convened to empower and launch the program. To measure progress, metrics related to care of patients on opioids and those with opioid use disorder (OUD) were evaluated. RESULTS: The OSP created a holistic, health system-wide program that addressed opioid prescribing, treatment of OUD, education, and information technology tools. After implementation, the number of opioid prescriptions decreased (-73.5/month; p < 0.001), mean morphine milligram equivalents (MME) per prescription decreased (-0.4/month; p < 0.001), the number of unique patients receiving an opioid decreased (-52.6/month; p < 0.001), and the number of prescriptions ≥ 90 MME decreased (-48.1/month; p < 0.001). Prescriptions and providers for buprenorphine increased (+6.0 prescriptions/month and +0.4 providers/month; both p < 0.001). Visits for opioid overdose did not change (-0.2 overdoses/month; p = 0.29). CONCLUSION: This paper describes a framework for a new health system-wide OSP. Successful implementation required strong executive sponsorship, ensuring that the program is not housed in any one clinical department in the health system, creating an environment that empowers cross-disciplinary collaboration and inclusion, as well as the development of measures to guide efforts.

Connecting the dots of the bacterial cell cycle: Coordinating chromosome replication and segregation with cell division.

Proper division site selection is crucial for the survival of all organisms. What still eludes us is how bacteria position their division site with high precision, and in tight coordination with chromosome replication and segregation. Until recently, the general belief, at least in the model organisms Bacillus subtilis and Escherichia coli, was that spatial regulation of division comes about by the combined negative regulatory mechanisms of the Min system and nucleoid occlusion. However, as we review here, these two systems cannot be solely responsible for division site selection and we highlight additional regulatory mechanisms that are at play. In this review, we put forward evidence of how chromosome replication and segregation may have direct links with cell division in these bacteria and the benefit of recent advances in chromosome conformation capture techniques in providing important information about how these three processes mechanistically work together to achieve accurate generation of progenitor cells.

High-throughput sequencing of sorted expression libraries reveals inhibitors of bacterial cell division.

BACKGROUND: Bacterial filamentation occurs when rod-shaped bacteria grow without dividing. To identify genetically encoded inhibitors of division that promote filamentation, we used cell sorting flow cytometry to enrich filamentous clones from an inducible expression library, and then identified the cloned DNA with high-throughput DNA sequencing. We applied the method to an expression library made from fragmented genomic DNA of uropathogenic E. coli UTI89, which undergoes extensive reversible filamentation in urinary tract infections and might encode additional regulators of division. RESULTS: We identified 55 genomic regions that reproducibly caused filamentation when expressed from the plasmid vector, and then further localized the cause of filamentation in several of these to specific genes or sub-fragments. Many of the identified genomic fragments encode genes that are known to participate in cell division or its regulation, and others may play previously-unknown roles. Some of the prophage genes identified were previously implicated in cell division arrest. A number of the other fragments encoded potential short transcripts or peptides. CONCLUSIONS: The results provided evidence of potential new links between cell division and distinct cellular processes including central carbon metabolism and gene regulation. Candidate regulators of the UTI-associated filamentation response or others were identified amongst the results. In addition, some genomic fragments that caused filamentation may not have evolved to control cell division, but may have applications as artificial inhibitors. Our approach offers the opportunity to carry out in depth surveys of diverse DNA libraries to identify new genes or sequences encoding the capacity to inhibit division and cause filamentation.

Understanding, Monitoring, and Controlling Biofilm Growth in Drinking Water Distribution Systems.

In drinking water distribution systems (DWDS), biofilms are the predominant mode of microbial growth, with the presence of extracellular polymeric substance (EPS) protecting the biomass from environmental and shear stresses. Biofilm formation poses a significant problem to the drinking water industry as a potential source of bacterial contamination, including pathogens, and, in many cases, also affecting the taste and odor of drinking water and promoting the corrosion of pipes. This article critically reviews important research findings on biofilm growth in DWDS, examining the factors affecting their formation and characteristics as well as the various technologies to characterize and monitor and, ultimately, to control their growth. Research indicates that temperature fluctuations potentially affect not only the initial bacteria-to-surface attachment but also the growth rates of biofilms. For the latter, the effect is unique for each type of biofilm-forming bacteria; ammonia-oxidizing bacteria, for example, grow more-developed biofilms at a typical summer temperature of 22 °C compared to 12 °C in fall, and the opposite occurs for the pathogenic Vibrio cholerae. Recent investigations have found the formation of thinner yet denser biofilms under high and turbulent flow regimes of drinking water, in comparison to the more porous and loosely attached biofilms at low flow rates. Furthermore, in addition to the rather well-known tendency of significant biofilm growth on corrosion-prone metal pipes, research efforts also found leaching of growth-promoting organic compounds from the increasingly popular use of polymer-based pipes. Knowledge of the unique microbial members of drinking water biofilms and, importantly, the influence of water characteristics and operational conditions on their growth can be applied to optimize various operational parameters to minimize biofilm accumulation. More-detailed characterizations of the biofilm population size and structure are now feasible with fluorescence microscopy (epifluorescence and CLSM imaging with DNA, RNA, EPS, and protein and lipid stains) and electron microscopy imaging (ESEM). Importantly, thorough identification of microbial fingerprints in drinking water biofilms is achievable with DNA sequencing techniques (the 16S rRNA gene-based identification), which have revealed a prevalence of previously undetected bacterial members. Technologies are now moving toward in situ monitoring of biomass growth in distribution networks, including the development of optical fibers capable of differentiating biomass from chemical deposits. Taken together, management of biofilm growth in water distribution systems requires an integrated approach, starting from the treatment of water prior to entering the networks to the potential implementation of "biofilm-limiting" operational conditions and, finally, ending with the careful selection of available technologies for biofilm monitoring and control. For the latter, conventional practices, including chlorine-chloramine disinfection, flushing of DWDS, nutrient removal, and emerging technologies are discussed with their associated challenges.

Cryptococcus strains with different pathogenic potentials have diverse protein secretomes.

Secreted proteins are the frontline between the host and pathogen. In mammalian hosts, secreted proteins enable invasive infection and can modulate the host immune response. Cryptococcosis, caused by pathogenic Cryptococcus species, begins when inhaled infectious propagules establish to produce pulmonary infection, which, if not resolved, can disseminate to the central nervous system to cause meningoencephalitis. Strains of Cryptococcus species differ in their capacity to cause disease, and the mechanisms underlying this are not well understood. To investigate the role of secreted proteins in disease, we determined the secretome for three genome strains of Cryptococcus species, including a hypovirulent and a hypervirulent strain of C. gattii and a virulent strain of C. neoformans. Sixty-seven unique proteins were identified, with different numbers and types of proteins secreted by each strain. The secretomes of the virulent strains were largely limited to proteolytic and hydrolytic enzymes, while the hypovirulent strain had a diverse secretome, including non-conventionally secreted canonical cytosolic and immunogenic proteins that have been implicated in virulence. The hypovirulent strain cannot establish pulmonary infection in a mouse model, but strains of this genotype have caused human meningitis. To directly test brain infection, we used intracranial inoculation and found that the hypovirulent strain was substantially more invasive than its hypervirulent counterpart. We suggest that immunogenic proteins secreted by this strain invoke a host response that limits pulmonary infection but that there can be invasive growth and damage if infection reaches the brain. Given their known role in virulence, it is possible that non-conventionally secreted proteins mediate this process.

3D-SIM super resolution microscopy reveals a bead-like arrangement for FtsZ and the division machinery: implications for triggering cytokinesis.

FtsZ is a tubulin-like GTPase that is the major cytoskeletal protein in bacterial cell division. It polymerizes into a ring, called the Z ring, at the division site and acts as a scaffold to recruit other division proteins to this site as well as providing a contractile force for cytokinesis. To understand how FtsZ performs these functions, the in vivo architecture of the Z ring needs to be established, as well as how this structure constricts to enable cytokinesis. Conventional wide-field fluorescence microscopy depicts the Z ring as a continuous structure of uniform density. Here we use a form of super resolution microscopy, known as 3D-structured illumination microscopy (3D-SIM), to examine the architecture of the Z ring in cells of two Gram-positive organisms that have different cell shapes: the rod-shaped Bacillus subtilis and the coccoid Staphylococcus aureus. We show that in both organisms the Z ring is composed of a heterogeneous distribution of FtsZ. In addition, gaps of fluorescence were evident, which suggest that it is a discontinuous structure. Time-lapse studies using an advanced form of fast live 3D-SIM (Blaze) support a model of FtsZ localization within the Z ring that is dynamic and remains distributed in a heterogeneous manner. However, FtsZ dynamics alone do not trigger the constriction of the Z ring to allow cytokinesis. Lastly, we visualize other components of the divisome and show that they also adopt a bead-like localization pattern at the future division site. Our data lead us to propose that FtsZ guides the divisome to adopt a similar localization pattern to ensure Z ring constriction only proceeds following the assembly of a mature divisome.

The Min system and nucleoid occlusion are not required for identifying the division site in Bacillus subtilis but ensure its efficient utilization.

Precise temporal and spatial control of cell division is essential for progeny survival. The current general view is that precise positioning of the division site at midcell in rod-shaped bacteria is a result of the combined action of the Min system and nucleoid (chromosome) occlusion. Both systems prevent assembly of the cytokinetic Z ring at inappropriate places in the cell, restricting Z rings to the correct site at midcell. Here we show that in the bacterium Bacillus subtilis Z rings are positioned precisely at midcell in the complete absence of both these systems, revealing the existence of a mechanism independent of Min and nucleoid occlusion that identifies midcell in this organism. We further show that Z ring assembly at midcell is delayed in the absence of Min and Noc proteins, while at the same time FtsZ accumulates at other potential division sites. This suggests that a major role for Min and Noc is to ensure efficient utilization of the midcell division site by preventing Z ring assembly at potential division sites, including the cell poles. Our data lead us to propose a model in which spatial regulation of division in B. subtilis involves identification of the division site at midcell that requires Min and nucleoid occlusion to ensure efficient Z ring assembly there and only there, at the right time in the cell cycle.

Extraneous Load, Patient Census, and Patient Acuity Correlate With Cognitive Load During ICU Rounds.

BACKGROUND: Cognitive load theory asserts that learning and performance degrade when cognitive load exceeds working memory capacity. This is particularly relevant in the learning environment of ICU rounds, when multidisciplinary providers integrate complex decision-making and teaching in a noisy, high-stress environment prone to cognitive distractions. RESEARCH QUESTION: What features of ICU rounds correlate with high provider cognitive load? STUDY DESIGN AND METHODS: This was an observational, multisite study of multidisciplinary providers during ICU rounds. Investigators recorded rounding characteristics and hourly extraneous cognitive load events during rounds (defined as distractions, episodes of split-attention or repetition, and deviations from standard communication format). After rounds, investigators measured each provider's cognitive load using the provider task load (PTL), an instrument derived from the National Aeronautics and Space Administration Task Load Index survey that assesses perceived workload associated with complex tasks. Relationships between rounding characteristics, extraneous load, and PTL score were evaluated using mixed-effects modeling. RESULTS: A total of 76 providers were observed during 32 rounds from December 2020 to May 2021. The mean rounding census ± SD was 12.5 ± 2.9 patients. The mean rounding time ± SD was 2 h 17 min ± 49 min. The mean extraneous load ± SD was 20.5 ± 4.5 events per hour, or one event every 2 min 51 s. This included 8.6 ± 3.4 distractions, 8.2 ± 4.2 communication deviations, 1.9 ± 1.4 repetitions, and 1.8 ± 1.3 episodes of split-attention per hour. Controlling for covariates, the hourly extraneous load events, number of new patients, and number of higher acuity patients were each associated with increased PTL score (slope, 2.40; 95% CI, 0.76-4.04; slope, 5.23; 95% CI, 2.02-8.43; slope, 3.35; 95% CI, 1.34-5.35, respectively). INTERPRETATION: Increased extraneous load, new patients, and patient acuity were associated with higher cognitive load during ICU rounds. These results can help direct how the ICU rounding structure may be modified to reduce workload and optimize provider learning and performance.

Childcare Stress, Burnout, and Intent to Reduce Hours or Leave the Job During the COVID-19 Pandemic Among US Health Care Workers.

Importance: Childcare stress (CCS) is high during the COVID-19 pandemic because of remote learning and fear of illness transmission in health care workers (HCWs). Associations between CCS and burnout, intent to reduce (ITR) hours, and intent to leave (ITL) are not known. Objective: To determine associations between CCS, anxiety and depression, burnout, ITR in 1 year, and ITL in 2 years. Design, Setting, and Participants: This survey study, Coping with COVID, a brief work-life and wellness survey of US HCWs, was conducted between April and December 2020, assessing CCS, burnout, anxiety, depression, workload, and work intentions. The survey was distributed to clinicians and staff in participating health care organizations with more than 100 physicians. Data were analyzed from October 2021 to May 2022. Main Outcomes and Measures: The survey asked, "due to…COVID-19, I am experiencing concerns about childcare," and the presence of CCS was considered as a score of 3 or 4 on a scale from 1, not at all, to 4, a great extent. The survey also asked about fear of exposure or transmission, anxiety, depression, workload, and single-item measures of burnout, ITR, and ITL. Results: In 208 organizations, 58 408 HCWs (15 766 physicians [26.9%], 11 409 nurses [19.5%], 39 218 women [67.1%], and 33 817 White participants [57.9%]) responded with a median organizational response rate of 32%. CCS was present in 21% (12 197 respondents) of HCWs. CCS was more frequent among racial and ethnic minority individuals and those not identifying race or ethnicity vs White respondents (5028 respondents [25.2%] vs 6356 respondents [18.8%]; P < .001; proportional difference, -7.1; 95% CI, -7.8 to -6.3) and among women vs men (8281 respondents [21.1%] vs 2573 respondents [17.9%]; odds ratio [OR], 1.22; 95% CI, 1.17 to 1.29). Those with CCS had 115% greater odds of anxiety or depression (OR, 2.15; 95% CI, 2.04-2.26; P < .001), and 80% greater odds of burnout (OR, 1.80; 95% CI, 1.70-1.90; P < .001) vs indidivuals without CCS. High CCS was associated with 91% greater odds of ITR (OR, 1.91; 95% CI, 1.76 to 2.08; P < .001) and 28% greater odds of ITL (OR, 1.28; 95% CI, 1.17 to 1.40; P < .001). Conclusions and Relevance: In this survey study, CCS was disproportionately described across different subgroups of HCWs and was associated with anxiety, depression, burnout, ITR, and ITL. Addressing CCS may improve HCWs' quality of life and HCW retention and work participation.

A simple plasmid-based system that allows rapid generation of tightly controlled gene expression in Staphylococcus aureus.

We have established a plasmid-based system that enables tightly controlled gene expression and the generation of GFP fusion proteins in Staphylococcus aureus simply and rapidly. This system takes advantage of an Escherichia coli-S. aureus shuttle vector that contains the replication region of the S. aureus theta-mode multiresistance plasmid pSK41, and is therefore a stable low-copy-number plasmid in the latter organism. This vector also contains a multiple cloning site downstream of the IPTG-inducible Pspac promoter for insertion of the gene of interest. Production of encoded proteins can be stringently regulated in an IPTG-dependent manner by introducing a pE194-based plasmid, pGL485, carrying a constitutively expressed lacI gene. Using GFP fusions to two essential proteins of S. aureus, FtsZ and NusA, we showed that our plasmid allowed tightly controlled gene expression and accurate localization of fusion proteins with no detrimental effect on cells at low inducer concentrations. At higher IPTG concentrations, we obtained sixfold overproduction of protein compared with wild-type levels, with FtsZ-GFP-expressing cells showing lysis and delocalized fluorescence, while NusA-GFP showed only delocalized fluorescence. These results show that our system is capable of titratable induction of gene expression for localization or overexpression studies.

Factors affecting the production and measurement of hydrogen peroxide in honey samples.

Many Australian native honeys possess significant antimicrobial properties due to the production of hydrogen peroxide (H2O2) by glucose oxidase, an enzyme derived from the honeybee. The level of H2O2 produced in different honey samples is highly variable, and factors governing its production and stability are not well understood. In this study, highly active Australian honeys that had been stored for >10 years lost up to 54 % of their antibacterial activity, although almost all retained sufficient activity to be considered potentially therapeutically useful. We used a simple colourimetric assay to quantify H2O2 production. Although we found a significant correlation between H2O2 production and antibacterial activity across diverse honey samples, variation in H2O2 only explained 47 % of the variation observed in activity, limiting the assay as a screening tool and highlighting the complexity of the relationship between H2O2 and the killing power of honey. To further examine this, we tested whether H2O2 detection in honey was being inhibited by pigmented compounds and if H2O2 might be directly degraded in some honey samples. We found no correlation between H2O2 detection and honey colour. Some honey samples rapidly lost endogenous and spiked H2O2, suggesting that components in honey, such as catalase or antioxidant polyphenols, may degrade or quench H2O2. Despite this rapid loss of H2O2, these honeys had significant peroxide-based antibacterial activity, indicating a complex relationship between H2O2 and other honey components that may act synergistically to augment activity.