Cyclic AMP Regulates Bacterial Persistence through Repression of the Oxidative Stress Response and SOS-Dependent DNA Repair in Uropathogenic Escherichia coli.

Bacterial persistence is a transient, nonheritable physiological state that provides tolerance to bactericidal antibiotics. The stringent response, toxin-antitoxin modules, and stochastic processes, among other mechanisms, play roles in this phenomenon. How persistence is regulated is relatively ill defined. Here we show that cyclic AMP, a global regulator of carbon catabolism and other core processes, is a negative regulator of bacterial persistence in uropathogenic Escherichia coli, as measured by survival after exposure to a ß-lactam antibiotic. This phenotype is regulated by a set of genes leading to an oxidative stress response and SOS-dependent DNA repair. Thus, persister cells tolerant to cell wall-acting antibiotics must cope with oxidative stress and DNA damage and these processes are regulated by cyclic AMP in uropathogenic E. coliIMPORTANCE Bacterial persister cells are important in relapsing infections in patients treated with antibiotics and also in the emergence of antibiotic resistance. Our results show that in uropathogenic E. coli, the second messenger cyclic AMP negatively regulates persister cell formation, since in its absence much more persister cells form that are tolerant to ß-lactams antibiotics. We reveal the mechanism to be decreased levels of reactive oxygen species, specifically hydroxyl radicals, and SOS-dependent DNA repair. Our findings suggest that the oxidative stress response and DNA repair are relevant pathways to target in the design of persister-specific antibiotic compounds.

A Novel Regulatory Cascade Involving BluR, YcgZ, and Lon Controls the Expression of Escherichia coli OmpF Porin.

In Escherichia coli, OmpF is an important outer membrane protein, which serves as a passive diffusion pore for small compounds including nutrients, antibiotics, and toxic compounds. OmpF expression responds to environmental changes such as temperature, osmolarity, nutrients availability, and toxic compounds via complex regulatory pathways involving transcriptional and post-transcriptional regulation. Our study identified a new regulatory cascade that controls the expression of OmpF porin. This pathway involves BluR, a transcriptional regulator repressing the expression of the ycgZ-ymgABC operon. We showed that BluR was responsible for the temperature-dependent regulation of the ycgZ-ymgABC operon. Furthermore, our results showed that independent expression of YcgZ led to a decreased activity of the ompF promoter, while YmgA, YmgB, and YmgC expression had no effect. We also determined that YcgZ accumulates in the absence of the Lon protease. Thus, mutation in bluR leads to de-repression of ycgZ-ymgABC transcription. With a second mutation in lon, YcgZ protein accumulates to reach levels that do not allow increased expression of OmpF under growth conditions that usually would, i.e., low temperature. With BluR responding to blue-light and temperature, this study sheds a new light on novel signals able to regulate OmpF porin.

Diamide Inhibitors of the Bacillus subtilis N-Acetylglucosaminidase LytG That Exhibit Antibacterial Activity.

N-Acetylglucosaminidases (GlcNAcases) play an important role in the remodeling and recycling of bacterial peptidoglycan by degrading the polysaccharide backbone. Genetic deletions of autolysins can impair cell division and growth, suggesting an opportunity for using small molecule autolysin inhibitors both as tools for studying the chemical biology of autolysins and also as antibacterial agents. We report here the synthesis and evaluation of a panel of diamides that inhibit the growth of Bacillus subtilis. Two compounds, fgkc (21) and fgka (5), were found to be potent inhibitors (MIC 3.8 ± 1.0 and 21.3 ± 0.1 µM, respectively). These compounds inhibit the B. subtilis family 73 glycosyl hydrolase LytG, an exo GlcNAcase. Phenotypic analysis of fgkc (21)-treated cells demonstrates a propensity for cells to form linked chains, suggesting impaired cell growth and division.

The development of one-stop wide-awake dupuytren's fasciectomy service: a retrospective review.

OBJECTIVES: To detail the transition to a totally one-stop wide-awake (OSWA) Dupuytren's contracture surgical service. DESIGN: Retrospective review of Dupuytren's component of last 1000 OSWA cases. SETTING: The UK's first totally one-stop wide-awake orthopaedic service. PARTICIPANTS: 270 patients with Dupuytren's contracture out of the last 1000 OSWA cases. MAIN OUTCOME MEASURES: Surgical outcomes, patient satisfaction and cost-effectiveness and efficiency. RESULTS: The OSWA Dupuytren's model is safe, efficient and effective; with a low complication rate, extremely high patient satisfaction; and cost-savings to the nhs of £2500 per case treated. The service saved the NHS approximately £675,000 for the 270 cases presented. CONCLUSIONS: A totally one-stop wide-awake Dupuytren's Contracture service is practicable and feasible alternative to the conventional treatment pathway, with benefits in terms of efficiency and cost-effectiveness.

Imidazole-based erythrocyte markers of oxidative stress in preeclampsia--an NMR investigation.

Using (1)H-nuclear magnetic resonance (NMR) spectroscopy and statistical models, we sought to identify ''biomarkers'' present in erythrocytes that would distinguish between women with normal pregnancy and those suffering from preeclampsia, and investigate possible links with previously identified plasma ''markers.'' Erythrocytes from 22 normotensive pregnant women and 15 preeclamptics were analyzed by (1)H Carr-Purcell-Meiboom-Gill (CPMG) NMR. Multivariate analysis and logistic regression were applied to differentiate between the 2 groups of patients, and used to develop a diagnostic model based on the concentrations of the constituents identified as being influential. Significantly higher concentrations of alanine (P < .001), glycine (P = .025), and ergothioneine (P = .049) were found in erythrocytes from preeclamptic patients. Discriminant analysis and regression of NMR data permitted 100% accurate diagnosis of the health status of new patients. Chemically related imidazole-based molecules, histidine and ergothioneine, are important in the classification process and the etiology of preeclampsia (PE).

Aromatic amino acid biomarkers of preeclampsia--a nuclear magnetic resonance investigation.

OBJECTIVES: There is conflicting literature regarding the correlation between aromatic amino acid concentrations and the occurrence of preeclampsia (PE). The object of this study was to test whether these molecules could enable discrimination between healthy and preeclamptic pregnancies when detected using (1)H nuclear magnetic resonance spectroscopy. METHODS: Plasma samples from 11 normotensive and 11 preeclamptic pregnant women were analyzed using (1)H-NMR. The the aromatic region of the spectrum was divided into regions of uniform size. The area of each region was subjected to principal component analysis. RESULTS: A distinction was made between normal and preeclamptic pregnancies on the basis of histidine, tyrosine, and phenylalanine concentrations. The concentration of histidine was significantly higher in the plasma of patients with PE than in that of normotensive women (p = 0.003). CONCLUSION: (1)H-NMR-based analysis of biofluids is capable of differentiating between patients with and without preeclampsia.

Gestational effects on host inflammatory response in normal and pre-eclamptic pregnancies.

OBJECTIVE: Pre-eclampsia (PET) remains a leading cause of maternal and neonatal morbidity and mortality. Although its pathophysiology involves an underlying inflammatory dysfunction, it is unclear how this may be affected by increasing gestational age, particularly in relation to the time of onset of disease. Murine studies have indicated that a progressive increase in serum inflammatory profile is a physiological feature of normal gestation. The present study aimed to investigate this phenomenon in women in relation to normal and pre-eclamptic pregnancies. STUDY DESIGN: Control and PET groups (each n=20) were divided into early and late pregnancy (before and after 34 weeks gestation, respectively). Whole blood was diluted 1:1 with RPMI 1640 medium with/without 1 microg/ml lipopolysaccharide at 37 degrees C for 24 h under a humidified 5% CO(2) atmosphere. Samples were collected at 0, 2, 6 and 24 h and analysed for interleukin (IL)-1beta, IL-2, IL-4, IL-5, IL-6, IL-7, IL-8, IL-10, IL-12 (p70), IL-13, IL-17, granulocyte-colony stimulating factor (G-CSF), granulocyte macrophage-colony stimulating factor (GM-CSF), interferon (IFN)-gamma, monocyte chemotactic protein (MCP-1), macrophage inflammatory protein (MIP)-1beta and tumour necrosis factor (TNF)-alpha by fluid-phase multiplex immunoassay. RESULTS: This study confirms that pregnancy features an increasing inflammatory response with advancing gestational age, which was seen in both control and PET pregnancies (P<0.01). CONCLUSIONS: This increase in inflammatory responsiveness with advancing gestation may provide an explanation for the incidence of late onset PET in the absence of placental pathology, as well as serving as a potential physiological priming mechanism geared towards increasing maternal sensitivity to the fetal triggers of labour.

Plasma from women with preeclampsia has a low lipid and ketone body content--a nuclear magnetic resonance study.

OBJECTIVE: Using (1)H-nuclear magnetic resonance spectroscopy and chemometrics, we sought to establish the metabolic profile for preeclampsia and to identify biomarkers that would permit a distinction between women with a normal pregnancy and those suffering from preeclampsia. METHODS: Plasma samples from 11 normotensive pregnant women and 11 women with preeclampsia were analyzed. Principal component analysis was applied to differentiate between the two groups of patients. RESULTS: Lipid concentrations were found to be significantly lower in the plasma of patients suffering from preeclampsia than those in normotensive pregnant women (p = 0.031). There is also evidence to suggest that ketone body constituents may contribute to the discrimination. CONCLUSION: (1)H-nuclear magnetic resonance-based metabolic profiling can detect patients with preeclampsia.