Assembly and Disassembly of DNA Polymerase Holoenzyme

The complex task of genomic replication requires a large collection of proteins properly assembled within the close confines of the replication fork. The mechanism and dynamics of holoenzyme assembly and disassembly have been investigated using steady state and pre-steady state methods as opposed to structural studies, primarily due to the intrinsic transient nature of these protein complexes during DNA replication. The key step in bacteriophage T4 holoenzyme assembly involves ATP hydrolysis, whereas disassembly is mediated by subunit dissociation of the clamp protein in an ATP-independent manner

Assembly and Disassembly of DNA Polymerase Holoenzyme

The complex task of genomic replication requires a large collection of proteins properly assembled within the close confines of the replication fork. The mechanism and dynamics of holoenzyme assembly and disassembly have been investigated using steady state and pre-steady state methods as opposed to structural studies, primarily due to the intrinsic transient nature of these protein complexes during DNA replication. The key step in bacteriophage T4 holoenzyme assembly involves ATP hydrolysis, whereas disassembly is mediated by subunit dissociation of the clamp protein in an ATP-independent manner

Protein-Protein and Protein-DNA Interactions at The Bacteriophage T4 DNA Replication Fork. Characterization of a Fluorescently Labeled DNA Polymerase Sliding Clamp

The T4 DNA polymerase holoenzyme is composed of the polymerase enzyme complexed to the sliding clamp (the 45 protein), which is loaded onto DNA by an ATP-dependent clamp loader (the 44/62 complex). This paper describes a new method to directly investigate the mechanism of holoenzyme assembly using a fluorescently labeled cysteine mutant of the 45 protein. This protein possessed unaltered function yet produced substantial changes in probe fluorescence intensity upon interacting with other components of the holoenzyme. These fluorescence changes provide insight into the role of ATP hydrolysis in holoenzyme assembly. Using either ATP or the non-hydrolyzable ATP analog, adenosine 5′-O-(3-thiophosphate), events in holoenzyme assembly were assigned as either dependent or independent of ATP hydrolysis. A holoenzyme assembly mechanism is proposed in which the 44/62 complex mediates the association of the 45 protein with DNA in an ATP-dependent manner not requiring ATP hydrolysis. Upon ATP hydrolysis, the 44/62 complex triggers a conformational change in the 45 protein that may be attributed to the clamp loading onto DNA

Incidence and outcome of Staphylococcus aureus bacteremia in hemodialysis patients

Incidence and outcome of Staphylococcus aureus bacteremia in hemodialysis patients.Background. Staphylococcus aureusbacteremia is frequently associated with metastatic complications and infective endocarditis (IE). The Duke criteria for the diagnosis of IE utilize echocardiographic techniques and are more sensitive than previous criteria. The documentation of IE in patients undergoing hemodialysis (HD) has become increasingly important in order to avoid the overuse of empiric vancomycin and the emergence of antibiotic resistance.MethodsPatients who developed S. aureus bacteremia while undergoing HD at a tertiary medical center or one of four affiliated outpatient HD units were identified. Clinical outcome (death, metastatic complications, IE, and microbiologic recurrence) was assessed during hospitalization and at three months after discharge. Transthoracic and transesophageal echocardiograms were performed and the Duke criteria were used to diagnose IE. Pulse field gel electrophoresis was performed to confirm genetic similarity of recurrent isolates.ResultsFour hundred and forty-five patients underwent hemodialysis for 5431.8 patient-months. Sixty-two developed 65 episodes of S. aureus bacteremia (1.2 episodes/100 patient-months). Complications occurred in 27 (44%) patients. Bacteremia recurred in patients who dialyzed through polytetrafluorethylene grafts (44.4% vs. 7.1%, P = 0.0.01), and there was a trend to increased recurrence in patients who received only vancomycin (19.5% vs. 7.1%, P = 0.4). IE was diagnosed in 8 patients (12%), six of whom had normal transthoracic echocardiograms.ConclusionsSensitive echocardiographic techniques and the Duke criteria for the diagnosis of IE should be used to determine the proper duration of antibiotic therapy in hemodialysis patients with S. aureus bacteremia. This diagnostic approach, coupled with early removal of hardware, may assist in improving outcomes

Clinical and Financial Outcomes Due to Methicillin Resistant Staphylococcus aureus Surgical Site Infection: A Multi-Center Matched Outcomes Study

The clinical and financial outcomes of SSIs directly attributable to MRSA and methicillin-resistance are largely uncharacterized. Previously published data have provided conflicting conclusions.We conducted a multi-center matched outcomes study of 659 surgical patients. Patients with SSI due to MRSA were compared with two groups: matched uninfected control patients and patients with SSI due to MSSA. Four outcomes were analyzed for the 90-day period following diagnosis of the SSI: mortality, readmission, duration of hospitalization, and hospital charges. Attributable outcomes were determined by logistic and linear regression.In total, 150 patients with SSI due to MRSA were compared to 231 uninfected controls and 128 patients with SSI due to MSSA. SSI due to MRSA was independently predictive of readmission within 90 days (OR = 35.0, 95% CI 17.3-70.7), death within 90 days (OR = 7.27, 95% CI 2.83-18.7), and led to 23 days (95% CI 19.7-26.3) of additional hospitalization and $61,681 (95$24,113 (95% 4,521-43,704) of additional charges.The attributable impact of S. aureus and methicillin-resistance on outcomes of surgical patients is substantial. Preventing a single case of SSI due to MRSA can save hospitals as much as $60,000

Epidemiology of Surgical Site Infection in a Community Hospital Network

OBJECTIVE To describe the epidemiology of complex surgical site infection (SSI) following commonly performed surgical procedures in community hospitals and to characterize trends of SSI prevalence rates over time for MRSA and other common pathogens METHODS We prospectively collected SSI data at 29 community hospitals in the southeastern United States from 2008 through 2012. We determined the overall prevalence rates of SSI for commonly performed procedures during this 5-year study period. For each year of the study, we then calculated prevalence rates of SSI stratified by causative organism. We created log-binomial regression models to analyze trends of SSI prevalence over time for all pathogens combined and specifically for MRSA. RESULTS A total of 3,988 complex SSIs occurred following 532,694 procedures (prevalence rate, 0.7 infections per 100 procedures). SSIs occurred most frequently after small bowel surgery, peripheral vascular bypass surgery, and colon surgery. Staphylococcus aureus was the most common pathogen. The prevalence rate of SSI decreased from 0.76 infections per 100 procedures in 2008 to 0.69 infections per 100 procedures in 2012 (prevalence rate ratio [PRR], 0.90; 95% confidence interval [CI], 0.82–1.00). A more substantial decrease in MRSA SSI (PRR, 0.69; 95% CI, 0.54–0.89) was largely responsible for this overall trend. CONCLUSIONS The prevalence of MRSA SSI decreased from 2008 to 2012 in our network of community hospitals. This decrease in MRSA SSI prevalence led to an overall decrease in SSI prevalence over the study period. Infect Control Hosp Epidemiol 2016;37:519–52

Whole Genome Sequencing of a Methicillin-Resistant Staphylococcus aureus Pseudo-Outbreak in a Professional Football Team.

Two American football players on the same team were diagnosed with methicillin-resistant Staphylococcus aureus (MRSA) skin and soft tissue infections on the same day. Our investigation, including whole genome sequencing, confirmed that players did not transmit MRSA to one another nor did they acquire the MRSA from a single source within the training facility