Nanoscale Plasma Coating Inhibits Formation of Staphylococcus aureus Biofilm.

Staphylococcus aureus commonly infects medical implants or devices, with devastating consequences for the patient. The infection begins with bacterial attachment to the device, followed by bacterial multiplication over the surface of the device, generating an adherent sheet of bacteria known as a biofilm. Biofilms resist antimicrobial therapy and promote persistent infection, making management difficult to futile. Infections might be prevented by engineering the surface of the device to discourage bacterial attachment and multiplication; however, progress in this area has been limited. We have developed a novel nanoscale plasma coating technology to inhibit the formation of Staphylococcus aureus biofilms. We used monomeric trimethylsilane (TMS) and oxygen to coat the surfaces of silicone rubber, a material often used in the fabrication of implantable medical devices. By quantitative and qualitative analysis, the TMS/O2 coating significantly decreased the in vitro formation of S. aureus biofilms; it also significantly decreased in vivo biofilm formation in a mouse model of foreign-body infection. Further analysis demonstrated TMS/O2 coating significantly changed the protein adsorption, which could lead to reduced bacterial adhesion and biofilm formation. These results suggest that TMS/O2 coating can be used to effectively prevent medical implant-related infections.

Inhibitor of streptokinase gene expression improves survival after group A streptococcus infection in mice.

The widespread occurrence of antibiotic resistance among human pathogens is a major public health problem. Conventional antibiotics typically target bacterial killing or growth inhibition, resulting in strong selection for the development of antibiotic resistance. Alternative therapeutic approaches targeting microbial pathogenicity without inhibiting growth might minimize selection for resistant organisms. Compounds inhibiting gene expression of streptokinase (SK), a critical group A streptococcal (GAS) virulence factor, were identified through a high-throughput, growth-based screen on a library of 55,000 small molecules. The lead compound [Center for Chemical Genomics 2979 (CCG-2979)] and an analog (CCG-102487) were confirmed to also inhibit the production of active SK protein. Microarray analysis of GAS grown in the presence of CCG-102487 showed down-regulation of a number of important virulence factors in addition to SK, suggesting disruption of a general virulence gene regulatory network. CCG-2979 and CCG-102487 both enhanced granulocyte phagocytosis and killing of GAS in an in vitro assay, and CCG-2979 also protected mice from GAS-induced mortality in vivo. These data suggest that the class of compounds represented by CCG-2979 may be of therapeutic value for the treatment of GAS and potentially other gram-positive infections in humans.

Development of tag-free photoprobes for studies aimed at identifying the target of novel Group A Streptococcus antivirulence agents.

We previously reported the identification and development of novel inhibitors of streptokinase (SK) expression by Group A Streptococcus (GAS), originating from a high throughput cell-based phenotypic screen. Although phenotypic screening is well-suited to identifying compounds that exert desired biological effects in potentially novel ways, it requires follow-up experiments to determine the macromolecular target(s) of active compounds. We therefore designed and synthesized several classes of chemical probes for target identification studies, guided by previously established structure-activity relationships. The probes were designed to first irreversibly photolabel target proteins in the intact bacteria, followed by cell lysis and click ligation with fluorescent tags to allow for visualization on SDS-PAGE gels. This stepwise, 'tag-free' approach allows for a significant reduction in molecular weight and polar surface area compared to full-length fluorescent or biotinylated probes, potentially enhancing membrane permeability and the maintenance of activity. Of the seven probes produced, the three most biologically active were employed in preliminary target identification trials. Despite the potent activity of these probes, specific labeling events were not conclusively observed due to a considerable degree of nonspecific protein binding. Nevertheless, the successful synthesis of potent biologically active probe molecules will serve as a starting point for initiating more sensitive methods of probe-based target identification.

Targeting the host hemostatic system function in bacterial infection for antimicrobial therapies.

The hemostatic system is an important player in host's response to infection. It has been shown that host hemostatic factors as well as platelets, interact with various proteins from bacteria and play important roles in host defense against infections. This review summarizes studies of function of host hemostatic system in host defense against bacterial infections and efforts to target hemostatic system interaction with pathogens to develop potential antimicrobial therapies.

Novel inhibitors of bacterial virulence: development of 5,6-dihydrobenzo[h]quinazolin-4(3H)-ones for the inhibition of group A streptococcal streptokinase expression.

Resistance to antibiotics is an increasingly dire threat to human health that warrants the development of new modes of treating infection. We recently identified 1 (CCG-2979) as an inhibitor of the expression of streptokinase, a critical virulence factor in Group A Streptococcus that endows blood-borne bacteria with fibrinolytic capabilities. In this report, we describe the synthesis and biological evaluation of a series of novel 5,6-dihydrobenzo[h]quinazolin-4(3H)-one analogs of 1 undertaken with the goal of improving the modest potency of the lead. In addition to achieving an over 35-fold increase in potency, we identified structural modifications that improve the solubility and metabolic stability of the scaffold. The efficacy of two new compounds 12c (CCG-203592) and 12k (CCG-205363) against biofilm formation in Staphylococcus aureus represents a promising additional mode of action for this novel class of compounds.

A new locus affects cell motility, cellulose binding, and degradation by Cytophaga hutchinsonii.

Cytophaga hutchinsonii is a Gram-negative gliding bacterium, which can rapidly degrade crystalline cellulose via a novel strategy without any recognizable processive cellulases. Its mechanism of cellulose binding and degradation is still a mystery. In this study, the mutagenesis of C. hutchinsonii with the mariner-based transposon HimarEm3 and gene complementation with the oriC-based plasmid carrying the antibiotic resistance gene cfxA or tetQ were reported for the first time to provide valuable tools for mutagenesis and genetic manipulation of the bacterium. Mutant A-4 with a transposon mutation in gene CHU_0134, which encodes a putative thiol-disulfide isomerase exhibits defects in cell motility and cellulose degradation. The cellulose binding ability of A-4 was only half of that of the wild-type strain, while the endo-cellulase activity of the cell-free supernatants and on the intact cell surface of A-4 decreased by 40%. Sodium dodecyl sulfate polyacrylamide gel electrophoresis of proteins binding to cellulose in the outer membrane showed that most of them were significantly decreased or disappeared in A-4 including some Gld proteins and hypothetical proteins, indicating that these proteins might play an important role in cell motility and cellulose binding and degradation by the bacterium.

Development of replicative oriC plasmids and their versatile use in genetic manipulation of Cytophaga hutchinsonii.

Cytophaga hutchinsonii is a Gram-negative aerobic soil bacterium which can digest crystalline cellulose completely through a strategy different from that of the well-studied cellulolytic aerobic fungi and anaerobic bacteria. However, despite the availability of a published genome sequence, studies on this organism have been very limited because of the lack of a genetic manipulation system. This paper describes the development of replicative oriC plasmids, carrying the replication origin of the C. hutchinsonii chromosome, and an electroporation method for Escherichia coli-C. hutchinsonii shuttle vectors based on oriC plasmids with an efficiency of about 2 × 10(4) transformants per microgram plasmid DNA. Heterologous proteins, including green fluorescent protein and ß-galactosidase, were expressed successfully and proved functional in C. hutchinsonii under the control of the CHU_1284 promoter in oriC plasmids. Finally, the gene CHU_0344, encoding the main extracellular protein, was targeted by homologous recombination based on the oriC plasmid. These genetic techniques and tools will provide a means to study the novel cellulose degradation system of C. hutchinsonii.

[Factors affecting adhesion of Cytophaga hutchinsonii to cellulose].

OBJECTIVE: The aim of the study was to understand the mechanism of Cytophaga hutchinsonii adhension to cellulose. METHODS: The effects of different factors on the bacterial adhesion to cellulose were studied, including bacterial age, pH, temperature, cell surface charge, cell viability, cell surface protein, extracellular polysaccharides, and cellulose derivates. RESULTS: Treatments with heat and protease reduced the adhesion remarkably. But treatments with NaN3, formalin, glutaraldehyde, Congo red and NaIO4 had only slight effect on the adhesion. The adhension of Cytophaga hutchinsonii cells to microcrystalline cellulose was specific and not inhibited by cellobiose or carboxymethyl cellulose. CONCLUSION: The adhesion of Cytophaga hutchinsonii to cellulose was closely related to cell surface proteins, while cellular metabolic activity and extracellular polysaccharides had only slight effect on it. It is speculated that there might be some specific cellulose binding proteins on the cell surface.

Peripheral arterial disease, chronic kidney disease, and mortality: the Chinese Ankle Brachial Index Cohort Study.

The purpose of this study was to investigate the association between chronic kidney disease (CKD) and peripheral arterial disease (PAD) and examine the combined effect of CKD and PAD on all-cause and cardiovascular disease (CVD) mortality. The Chinese Ankle Brachial Index Cohort consisted of 3732 adults aged 35 years or older enrolled in 2004 and followed-up in 2007. Complete baseline data were compiled on 3610 people which were examined in the final analysis. Mortality surveillance was completed from December 2007 to February 2008. Survival analysis was used to compare the survival rate in different CKD/PAD groups. The relative risks (RR) of death from all-cause and CVD were compared using a Cox regression model. It was found that the prevalence of PAD in patients with and without CKD was 41.9% and 22.3%, respectively (p < 0.001). The survival rate for the CKD and PAD group was significantly lower than that for any single disease, for both all-cause and CVD mortality (log-rank: p < 0.001). In conclusion, CKD is a risk factor for PAD. The combined CKD and PAD patients had the highest risk for all-cause and CVD mortality. Early recognition of risk can be made by taking an ankle-brachial index measurement of PAD; a corresponding laboratory assessment should be used as a measurement of renal function for PAD patients.

Early detection of ST-segment elevated myocardial infarction by artificial intelligence with 12-lead electrocardiogram.

Patient delay is a worldwide unsolved problem in ST-segment elevated myocardial infarction (STEMI). An accurate warning system based on electrocardiogram (ECG) may be a solution for this problem, and artificial intelligence (AI) may offer a path to improve its accuracy and efficiency. In the present study, an AI-based STEMI autodiagnosis algorithm was developed using a dataset of 667 STEMI ECGs and 7571 control ECGs. The algorithm for detecting STEMI proposed in the present study achieved an area under the receiver operating curve (AUC) of 0.9954 (95% CI, 0.9885 to 1) with sensitivity (recall), specificity, accuracy, precision and F1 scores of 96.75%, 99.20%, 99.01%, 90.86% and 0.9372 respectively, in the external evaluation. In a comparative test with cardiologists, the algorithm had an AUC of 0.9740 (95% CI, 0.9419 to 1), and its sensitivity (recall), specificity, accuracy, precision, and F1 score were 90%, 98% and 94%, 97.82% and 0.9375 respectively, while the medical doctors had sensitivity (recall), specificity, accuracy, precision and F1 score of 71.73%, 89.33%, 80.53%, 87.05% and 0.8817 respectively. This study developed an AI-based, cardiologist-level algorithm for identifying STEMI.

The predictive value of brachial-ankle pulse wave velocity in coronary atherosclerosis and peripheral artery diseases in urban Chinese patients.

Coronary artery disease (CAD) is one of the most common diseases throughout the world. To investigate the relationship between brachial-ankle pulse wave velocity (baPWV) and arterial atherosclerosis and peripheral artery disease (PAD) and its potential diagnostic value in diagnosing arterial sclerosis, a self-designed questionnaire and special machine designed by Colin Corp., Ltd. were implemented to measure the level of baPWV and the ankle-brachial index (ABI) and their relations to coronary and peripheral artery atherosclerosis. The results showed that baPWV and ABI were equally effective at predicting stenosis of the coronary arteries and stenosis of the arteries of the lower extremities. Different levels of baPWV with corresponding ABI can express different degrees of arterial sclerosis and peripheral artery lesion to a certain extent. Measurement of both baPWV and ABI is thus highly recommended in clinical investigation. Arterial wave reflection is a major determinant of left ventricular function, coronary perfusion, and cardiovascular risk. We investigated whether arterial wave velocity can detect atherosclerosis of the coronary arteries and peripheral arteries in patients with documented coronary artery diseases. Our goal was to investigate the relationship between baPWV and arterial atherosclerosis and PAD and their potential diagnostic value. Two hundred and seventy-two patients ranging in age from 45-92 years (mean: 66.87+/-11.42 years) were selected from the Department of Cardiology of our hospital. A carefully designed questionnaire was used to gather baseline data for each patient. All patients underwent cardioangiography and were divided into four groups according to their Gensini scores: a control group, and groups with a mild, moderate, or severe degree of stenosis. One hundred and five of these patients simultaneously underwent angiography of the lower extremities and were divided into four groups according to the degree of artery stenosis: a control group, and groups with a mild, moderate, or severe degree of stenosis. Grouping of baPWV levels was made according to Japanese surveys. Bilateral baPWV and ABI were measured using non-invasive arterial atherosclerosis measuring equipment. In the coronary artery groups based on Gensini score, ABI in the group with a high degree of stenosis was significantly lower than that in the control and moderate stenosis groups, while the baPWV was significantly higher than that in the control and mild stenosis groups. In the grouping of baPWV levels, it was indicated that the ABI level was significantly different between each group. The ABI<0.9 in groups with baPWV<1,400 cm/s and >2,100 cm/s was higher than that in other groups. In the grouping by angiography of the lower extremities, the ABI level was decreased with increasing degree of artery stenosis while the baPWV levels were increased under the same circumstance (p<0.05 or p<0.01). Logistic regression analysis indicated that relatively low ABI, high baPWV, abnormal fasting blood glucose, and smoking were independent risk factors for the development of cardiovascular diseases. The simultaneous measurement of bilateral baPWV and ABI using the newly developed equipment presented herein is highly recommended, and should play an important role in predicting the possibility of cardiovascular diseases and the degree of coronary artery lesions. It is important to note that baPWV is not only one of the risk factors in the presence of coronary stenosis, but also a substitute index of target-organ damage, another parameter in predicting PAD. The current study indicated that a baPWV>1,800 cm/s often follows a severe coronary artery event, while baPWV>2,100 cm/s may be related to potential PAD. baPWV measurement is helpful to make new standard of diagnosing PAD in Chinese cohorts.

The association between ankle-brachial index and cardiovascular or all-cause mortality in metabolic syndrome of elderly Chinese.

The ankle-brachial index (ABI) is a non-invasive, reliable measurement of lower-extremity ischemia. A low ABI is associated with increased risk of coronary heart disease, stroke and death. However, the relationship between ABI and all-cause mortality or cardiovascular disease (CVD) mortality in patients with metabolic syndrome (MetS) has not been well studied. Accordingly, we here investigated the association between ABI and all-cause and CVD mortality in an elderly Chinese population with MetS. A total of 2,274 MetS patients diagnosed under the criteria proposed by the International Diabetes Federation were divided into two groups based on repeated ABI measurement over a period of 13.6 months: ABI

Novel inhibitors of Staphylococcus aureus virulence gene expression and biofilm formation.

Staphylococcus aureus is a major human pathogen and one of the more prominent pathogens causing biofilm related infections in clinic. Antibiotic resistance in S. aureus such as methicillin resistance is approaching an epidemic level. Antibiotic resistance is widespread among major human pathogens and poses a serious problem for public health. Conventional antibiotics are either bacteriostatic or bacteriocidal, leading to strong selection for antibiotic resistant pathogens. An alternative approach of inhibiting pathogen virulence without inhibiting bacterial growth may minimize the selection pressure for resistance. In previous studies, we identified a chemical series of low molecular weight compounds capable of inhibiting group A streptococcus virulence following this alternative anti-microbial approach. In the current study, we demonstrated that two analogs of this class of novel anti-virulence compounds also inhibited virulence gene expression of S. aureus and exhibited an inhibitory effect on S. aureus biofilm formation. This class of anti-virulence compounds could be a starting point for development of novel anti-microbial agents against S. aureus.

Association of low ankle-brachial index with mortality in patients with ischemic heart disease.

AIMS: Whether a low ankle-brachial index can improve the prediction of all-cause and cardiovascular mortality on top of conventional risk factors remains unclear among patients with ischemic heart disease. The present study aimed to assess the association between the ankle-brachial index and mortality in Chinese patients. METHODS: This was an observational prospective study and 1,800 Chinese patients aged > or =35 years were followed-up from 2004 to 2007-2008. RESULTS: There were 280 deaths, of which 165 were attributable to cardiovascular disease. Compared with patients with an ankle-brachial index > or =1.1, the risk of mortality increased linearly in lower ankle-brachial index categories: patients with an ankle-brachial index of 0.9 to 1.1, 0.7 to 0.9, <0.7 had hazard ratios of 1.60, 2.07, and 3.08 for all-cause mortality and 1.89, 2.33, and 4.09 for cardiovascular mortality (p for trend <0.001), respectively. Addition of the ankle-brachial index significantly (p<0.001) increased the predictive value of the model for 3-year deaths compared with a model including risk factors alone. Comparison of areas under receiver operator characteristics curves confirmed that a model including the ankle-brachial index discriminated better than without. CONCLUSION: There was an inverse association between the ankle-brachial index and mortality. Addition of the ankle-brachial index significantly improved the prediction of 3-year mortality over and above that of conventional risk factors. We recommend that the ankle-brachial index be incorporated into prognostic assessment for patients with ischemic heart disease.

Predictive value of serum uric acid on cardiovascular disease and all-cause mortality in urban Chinese patients.

BACKGROUND: The association between increased serum uric acid (SUA) levels and cardiovascular risk has been debated for decades. Several large studies have provided conflicting results regarding the clinical significance of elevated SUA levels in cardiovascular disease (CVD) or cerebrovascular disease. The aim of this study was to investigate the relationship between SUA and CVD and all-cause mortality and their potential diagnostic value. METHODS: A total of 3570 in-patients ranging in age from 56 to 95 years (mean (67.36 +/- 11.36) years) were selected from 20 hospitals in Beijing and Shanghai. A carefully designed questionnaire was used to gather baseline data of each patient. All patients were divided into two main groups according to their SUA levels: high SUA and normal SUA groups. Serum indices and other important parameters were measured. RESULTS: Compared with normal SUA group, high SUA group had significant difference in systolic blood pressure (SBP), total cholesterol (TC), triglyceride (TG), high density lipoprotein cholesterol (HDL-C), body mass index (BMI), and age (P < 0.05 or P < 0.01). High SUA prevailed in female and patients with history of essential hypertension, while history of smoking and diabetes showed no significant difference between two groups. All-cause and CVD mortality occurred more frequently in high SUA group than in normal SUA group. In the accumulative survival analysis, high SUA group had lower survival rate than normal SUA group both in CVD and all-cause mortality. COX regression analysis indicated that the history of smoking, age and high SUA were independent risk factors for the development of CVD. CONCLUSIONS: These preliminary observations suggest that patients with high SUA levels would face higher risk of mortality. SUA measurement may be applied as a routine predictor for clinical assessment.

Evaluation of medical treatment for peripheral arterial disease in Chinese high-risk patients.

BACKGROUND: Peripheral arterial disease (PAD) is an important manifestation of systemic atherosclerosis and is associated with elevated cardiovascular morbidity and mortality. The aim of the present study was to evaluate the use of antiplatelet agents, statins and angiotensin-converting enzyme inhibitors (ACEI) in Chinese high-risk cardiovascular (CV) patients with PAD, with an emphasis on the need for aggressive medical management of all atherosclerotic manifestations. METHODS AND RESULTS: Medical records from 5,263 Chinese patients at high risk of CV were evaluated for the use of antiplatelet agents, statins and ACEI in patients with and without PAD. PAD was defined as an ankle-brachial index (ABI)<0.9 in either leg. Multivariable logistic regression analyses were performed to compare medication use in the 2 groups. A total of 5,254 patients were analyzed (52.9% male, mean age 67.3 years). The prevalence of PAD in the total patient group was 25.4%; 22.5% of them had PAD only. Overall, 5.7% had PAD only, 19.6% had PAD and coronary heart disease (CHD) or stroke or diabetes, 7.7% had CHD only, 12.6% had stroke only, and 13.6% had diabetes only. The 28.9% subjects having none of PAD, CHD, stroke or diabetes were used as the reference group. Only 65%, 37% and 47% of all patients received antiplatelet agents, statins and ACEI, respectively. Antiplatelets, statins, ACEI and all 3 medications were used less frequently in PAD only patients (58.1%, 35.9, 53.5% and 21.6%) vs CHD only (90.9%, 74.5%, 70.6% and 55.9%, p<0.001). All 3 proven efficacious therapies were prescribed for only 56% of patients with CHD only, 8% with stroke only, 13% with diabetes only and 21% with PAD only. CONCLUSION: PAD is prevalent in Chinese high-risk CV patients, equivalent to CHD, but these patients receive less intensive treatment than those with CHD. Programs to improve CV risk reduction in these high-risk patients are needed.