Population heterogeneity in Mycobacterium smegmatis and Mycobacterium abscessus.

Bacteria use population heterogeneity, the presence of more than one phenotypic variant in a clonal population, to endure diverse environmental challenges - a 'bet-hedging' strategy. Phenotypic variants have been described in many bacteria, but the phenomenon is not well-understood in mycobacteria, including the environmental factors that influence heterogeneity. Here, we describe three reproducible morphological variants in M. smegmatis - smooth, rough, and an intermediate morphotype that predominated under typical laboratory conditions. M. abscessus has two recognized morphotypes, smooth and rough. Interestingly, M. tuberculosis exists in only a rough form. The shift from smooth to rough in both M. smegmatis and M. abscessus was observed over time in extended static culture, however the frequency of the rough morphotype was high in pellicle preparations compared to planktonic culture, suggesting a role for an aggregated microenvironment in the shift to the rough form. Differences in growth rate, biofilm formation, cell wall composition, and drug tolerance were noted among M. smegmatis and M. abscessus variants. Deletion of the global regulator lsr2 shifted the M. smegmatis intermediate morphotype to a smooth form but did not fully phenocopy the naturally generated smooth morphotype, indicating Lsr2 is likely downstream of the initiating regulatory cascade that controls these morphotypes. Rough forms typically correlate with higher invasiveness and worse outcomes during infection and our findings indicate the shift to this rough form is promoted by aggregation. Our findings suggest that mycobacterial population heterogeneity, reflected in colony morphotypes, is a reproducible, programmed phenomenon that plays a role in adaptation to unique environments and this heterogeneity may influence infection progression and response to treatment.

Protein kinase B controls Mycobacterium tuberculosis growth via phosphorylation of the transcriptional regulator Lsr2 at threonine 112.

Mycobacterium tuberculosis (Mtb) is able to persist in the body through months of multi-drug therapy. Mycobacteria possess a wide range of regulatory proteins, including the protein kinase B (PknB) which controls peptidoglycan biosynthesis during growth. Here, we observed that depletion of PknB resulted in specific transcriptional changes that are likely caused by reduced phosphorylation of the H-NS-like regulator Lsr2 at threonine 112. The activity of PknB towards this phosphosite was confirmed with purified proteins, and this site was required for adaptation of Mtb to hypoxic conditions, and growth on solid media. Like H-NS, Lsr2 binds DNA in sequence-dependent and non-specific modes. PknB phosphorylation of Lsr2 reduced DNA binding, measured by fluorescence anisotropy and electrophoretic mobility shift assays, and our NMR structure of phosphomimetic T112D Lsr2 suggests that this may be due to increased dynamics of the DNA-binding domain. Conversely, the phosphoablative T112A Lsr2 had increased binding to certain DNA sites in ChIP-sequencing, and Mtb containing this variant showed transcriptional changes that correspond with the change in DNA binding. In summary, PknB controls Mtb growth and adaptations to the changing host environment by phosphorylating the global transcriptional regulator Lsr2.

Pelvic floor rehabilitation for defecation disorders.

Pelvic floor rehabilitation is frequently recommended for defecation disorders, in both constipation and fecal incontinence. However, the lack of patient selection, together with the variety of rehabilitation methods and protocols, often jeopardize the results of this approach, causing difficulty in evaluating outcomes and addressing proper management, and above all, in obtaining scientific evidence for the efficacy of these methods for specific indications. The authors represent different gastroenterological and surgical scientific societies in Italy, and their aim was to identify the indications and agree on treatment protocols for pelvic floor rehabilitation of patients with defecation disorders. This was achieved by means of a modified Delphi method, utilizing a working team (10 members) which developed the statements and a consensus group (15 members, different from the previous ones) which voted twice also suggesting modifications of the statements.

The oxidation-sensing regulator (MosR) is a new redox-dependent transcription factor in Mycobacterium tuberculosis.

Mycobacterium tuberculosis thrives in oxidative environments such as the macrophage. To survive, the bacterium must sense and adapt to the oxidative conditions. Several antioxidant defenses including a thick cell wall, millimolar concentrations of small molecule thiols, and protective enzymes are known to help the bacterium withstand the oxidative stress. However, oxidation-sensing regulators that control these defenses have remained elusive. In this study, we report a new oxidation-sensing regulator, Rv1049 or MosR (M. tuberculosis oxidation-sensing regulator). MosR is a transcriptional repressor of the MarR family, which, similarly to Bacillus subtilis OhrR and Staphylococcus aureus MgrA, dissociates from DNA in the presence of oxidants, enabling transcription. MosR senses oxidation through a pair of cysteines near the N terminus (Cys-10 and Cys-12) that upon oxidation forms a disulfide bond. Disulfide formation rearranges a network of hydrogen bonds, which leads to a large conformational change of the protein and dissociation from DNA. MosR has been shown previously to play an important role in survival of the bacterium in the macrophage. In this study, we show that the main role of MosR is to up-regulate expression of rv1050 (a putative exported oxidoreductase that has not yet been characterized) in response to oxidants and propose that it is through this role that MosR contributes to the bacterium survival in the macrophage.

The response of mycobacterium tuberculosis to reactive oxygen and nitrogen species.

The bacteriostatic and bactericidal effects and the transcriptional response of Mycobacterium tuberculosis to representative oxidative and nitrosative stresses were investigated by growth and survival studies and whole genome expression analysis. The M. tuberculosis reaction to a range of hydrogen peroxide (H(2)O(2)) concentrations fell into three distinct categories: (1) low level exposure resulted in induction of a few highly sensitive H(2)O(2)-responsive genes, (2) intermediate exposure resulted in massive transcriptional changes without an effect on growth or survival, and (3) high exposure resulted in a muted transcriptional response and eventual death. M. tuberculosis appears highly resistant to DNA damage-dependent, mode-one killing caused by low millimolar levels of H(2)O(2) and only succumbs to overwhelming levels of oxidative stress observed in mode-two killing. Nitric oxide (NO) exposure initiated much the same transcriptional response as H(2)O(2). However, unlike H(2)O(2) exposure, NO exposure induced dormancy-related genes and caused dose-dependent bacteriostatic activity without killing. Included in the large shared response to H(2)O(2) and NO was the induction of genes encoding iron-sulfur cluster repair functions including iron acquisition. Stress regulons controlled by IdeR, Sigma H, Sigma E, and FurA comprised a large portion of the response to both stresses. Expression of several oxidative stress defense genes was constitutive, or increased moderately from an already elevated constitutive level, suggesting that bacilli are continually primed for oxidative stress defense.

The Mycobacterium tuberculosis DosR regulon assists in metabolic homeostasis and enables rapid recovery from nonrespiring dormancy.

Mycobacterium tuberculosis survives in latently infected individuals, likely in a nonreplicating or dormancy-like state. The M. tuberculosis DosR regulon is a genetic program induced by conditions that inhibit aerobic respiration and prevent bacillus replication. In this study, we used a mutant incapable of DosR regulon induction to investigate the contribution of this regulon to bacterial metabolism during anaerobic dormancy. Our results confirm that the DosR regulon is essential for M. tuberculosis survival during anaerobic dormancy and demonstrate that it is required for metabolic processes that occur upon entry into and throughout the dormant state. Specifically, we showed that regulon mechanisms shift metabolism away from aerobic respiration in the face of dwindling oxygen availability and are required for maintaining energy levels and redox balance as the culture becomes anaerobic. We also demonstrated that the DosR regulon is crucial for rapid resumption of growth once M. tuberculosis exits an anaerobic or nitric oxide-induced nonrespiring state. In summary, the DosR regulon encodes novel metabolic mechanisms essential for M. tuberculosis to survive in the absence of respiration and to successfully transition rapidly between respiring and nonrespiring conditions without loss of viability.

Unique roles of DosT and DosS in DosR regulon induction and Mycobacterium tuberculosis dormancy.

In Mycobacterium tuberculosis, the sensor kinases DosT and DosS activate the transcriptional regulator DosR, resulting in the induction of the DosR regulon, which is important for anaerobic survival and perhaps latent infection. The individual and collective roles of these sensors have been postulated biochemically, but their roles in vivo have remained unclear. This work demonstrates distinct and additive roles for each sensor during anaerobic dormancy. Both sensors are necessary for wild-type levels of DosR regulon induction, and concomitantly, full induction of the regulon is required for wild-type anaerobic survival. In the anaerobic model, DosT plays an early role, responding to hypoxia. DosT then induces the regulon and with it DosS, which sustains and further induces the regulon. DosT then loses its functionality as oxygen becomes limited, and DosS alone maintains induction of the genes from that point forward. Thus, M. tuberculosis has evolved a system whereby it responds to hypoxic conditions in a stepwise fashion as it enters an anaerobic state.

Percutaneous endoscopic gastrostomy: complications in the short and long-term follow-up and efficacy on nutritional status.

AIM: Percutaneous endoscopic gastrostomy (PEG) is an accepted procedure for patients at risk of malnutrition, but it requires an invasive procedure and many complications can occur. The aim of this study is to evaluate the long-term efficacy, morbidity and mortality of PEG. METHODS: We analysed 68 inpatients who underwent a PEG procedure between January 1999 and August 2006 in our rehabilitation unit. RESULTS: A total of 26.5% of the examined patients died, but none of them due to PEG-related complications. Out of the 18 patients who died, 8 suffered from diabetes mellitus, thus showing a positive correlation between diabetes and mortality. The thirty-day mortality rate was of 1.5%; 30.9% of males and 23.1% of females developed an early peristomal infection, 4.4% of the patients suffered from stomal leakage, 1 patient pulled out the gastrostomy tube and 1 patient, accidentally, occurred to remove the tube. None major complication occurred. During the observation period nutritional parameters had been good in 10 patients, who had been followed for 33.3 +/- 16.1 months. These indexes were reduced with a significant statistical difference in deaed patients. Only 20.6% of the patients improved their autonomy, valued by Barthel index (BI), while in the other patients BI remained = 10. CONCLUSIONS: This study confirmed the safety of PEG in terms of mortality and complications. The presence of diabetes mellitus increased mortality. The nutritional indexes were significantly reduced in deaed patients, while they remained good in the survivors. Nevertheless, a good nutrition had the slightest influence on the functional status.

Functional recovery following a second hip fracture.

AIM: Patients who have a fracture of the hip, usually are considered at a risk of fracturing the contralateral hip. This risk, not sufficiently addressed to in literature, becomes increasingly important in the light of increasing longevity of the population. In this retrospective study, we present some of our epidemiological and post-operative data on limb function in a group of our patients. METHODS: We examined the database of our patients over 50 years of age, admitted at our hospital in the last 9 years for fracture of the proximal third part of the femur bone. These patients did not have a history of neoplasms or severe multitrauma. The variables considered were: unilateral or bilateral fracture, age, sex, the level of physical activity before trauma (MET scale), the severity of the trauma, fracture site (cervical or trochanteric), the type of surgery and the level of functional autonomy (FIM scale) at time of discharge. RESULTS: A total of 2 771 patients were identified. Bilateral fractures were present in 7.58% (n=210). Among bilateral fractures, the female to male ratio was 1 to 7.08 and the site of the contralateral fracture remained similar to the original site. In more than 48%, the second fracture occurred in patients over 85 years of age. The most common causes of both bilateral and mono lateral fractures, were either a moderate trauma or a fall in domestic premises. Cervical fractures were treated with cemented endoprothesis of the hip while for trochanteric fractures osteosyntesis with endomedullar nails were used. After the second fracture, the level of functional autonomy was found to be significantly reduced (FIM score mean difference +/-SD of 16.77+/-10.72) and the number of non deambulatory patients were tripled. Furthermore, these patients were found to be more sedentary, before the second fracture, as compared to the ones with only one fracture. CONCLUSIONS: Bilateral fractures of the femur neck bone is still considered to be a rare event. The second fracture seems to be more frequent in sedentary women over 85 years of age. More often, the second fracture is caused by a simple domestic accident. Due the increasing age of the population, in future this event might become statistically relevant. The drastic decrease in post-operative functional autonomy in such patients might become a serious socio-economic and welfare policy problem. It is advisable to render domestic premises less hazardous and intense and prolonged postoperative physical rehabilitation treatment must be continued after discharge.

Sexual dysfunction in multiple sclerosis: a case-control study. I. Frequency and comparison of groups.

Sexual dysfunction is a very important but often overlooked symptom of multiple sclerosis. To investigate the type and frequency of symptoms of sexual dysfunction in patients suffering from multiple sclerosis, we performed a case-control study comparing 108 unselected patients with definite multiple sclerosis, 97 patients with chronic disease and 110 healthy individuals with regard to sexual function, sphincteric function, physical disorders impeding sexual activity and the impact of sexual dysfunction on social life. Information has been collected from a face-to-face structured interview performed by a doctor of the same gender as the patient. The disability, the cognitive performances, the psychiatric conditions and the psychological profile of patients and controls have been assessed. Sexual dysfunction was present in 73.1% of cases, in 39.2% of chronic disease controls and in 12.7% of healthy controls (P<0.0001). Male cases reported symptoms of sexual dysfunction more frequently than female cases (P<0.002). Symptoms of sexual dysfunction more commonly reported in patients with multiple sclerosis were anorgasmia or hyporgasmia (37.1%), decreased vaginal lubrication (35.7%) and reduced libido (31.4%) in women, and impotence or erectile dysfunction (63.2%), ejaculatory dysfunction and/or orgasmic dysfunction (50%) and reduced libido (39.5%) in men. Seventy-five per cent of cases, 51.5% of chronic disease controls and 28.2% of healthy controls (P<0.0001) experienced symptoms of sphincteric dysfunction. In conclusion, a substantial part of our sample of patients with multiple sclerosis reported symptoms of sexual and sphincteric dysfunction. Both sexual and sphincteric dysfunction were significantly more common in patients with multiple sclerosis than in either control group. Our findings suggest that a peculiar damage of the structures involved in sexual function is responsible for the dysfunction in patients with multiple sclerosis, but the highly significant lower frequency of symptoms of depression and anxiety in healthy controls may also imply a possible causative role of psychological factors.

Sexual dysfunction in multiple sclerosis: II. Correlation analysis.

Sexual dysfunction affects a large part of patients suffering from multiple sclerosis, but some aspects of its clinical presentation and aetiology are not clearly defined yet. In an unselected sample of 108 patients with definite multiple sclerosis we investigated the relationship between symptoms of sexual dysfunctioning and sphincteric dysfunction, patients' and disease characteristics, disability and neurological impairment, psychological and cognitive functioning. Sexual dysfunction directly correlated with presence of physical disorders (r=0.37, P=0.0004), low educational level (r=0.32, P<0.002), disability (r=0.31, P<0.003), age at onset of symptoms (r=0.30, P<0.003), sphincteric dysfunction (r=0.30, P<0.003), age (r=0.30, P<0.004), depression (r=0.29, P<0.005), fatigue (r=0.29, P=0.005), cognitive deterioration (r=0.26, P<0.01), primary-progressive course of disease (r=0.25, P<0.02), neurological impairment (r=0.25, P<0.02), marriage (r=0.24, P<0.02), anxiety (r=0. 23, P<0.03), male gender (r=0.22, P=0.03) bladder dysfunction (r=0. 29, P<0.04), and unemployment (r=0.21, P<0.04). Sexual dysfunction correlated inversely with relapsing - remitting course of disease (r=-0.31, P<0.002). No correlation was found between sexual dysfunction and bowel dysfunction, duration of disease, secondary-progressive course of disease, number and frequency of sexual intercourses in the last year, number of partners, number of exacerbations in the last year, number of months since last exacerbation, masturbation, and fertility. In conclusion, the association between sexual dysfunction and sphincteric dysfunction indicates a common aetiology corresponding to the frequent involvement of the spinal cord in multiple sclerosis, but the concomitant correlation between sexual dysfunction and other variables suggests the possible aetiological role of physical, psychological and sociological factors as well.

Increased density in the middle cerebral artery by nonenhanced computed tomography. Prognostic value in acute cerebral infarction.

In order to consider the prognostic value of hyperdensity of the middle cerebral artery (MCA), we studied retrospectively the medical records of 80 consecutive patients with the diagnosis of ischemic stroke who underwent a non-enhanced computed tomography no longer than 24 h after the onset of the symptoms. To determine the mortality and disability, a follow-up of 30 days was obtained. Mortality does not differ significantly in acute ischemic stroke patients with and without increased density of MCA. When disability, calculated with the Rankin scale, is considered, the two groups are different since patients with hyperdensity of the middle cerebral artery (MCA) have a significantly worse outcome. The two groups do not differ for age, sex, the presence of atrial fibrillation, hypertension, diabetes, myocardial infarction and/or angina, and these factors do not influence the prognosis being equally present in the dead and in the survivors. In the multivariate discriminant analysis with stepwise variable selection, the dense MCA sign was significantly correlated either to mortality or to disability, along with atrial fibrillation, age and diabetes. We conclude that the dense MCA sign can be a useful prognostic factor in the early phase of ischemic stroke.

Hyperglycemia at ischemic stroke onset as prognostic factor.

To find out whether the high blood glucose values sometimes found in the first stage of ischemic stroke have any prognostic value, we considered 76 patients hospitalized within 24 h of an acute cerebral infarction, documented by CT brain scan and/or necropsy, whose fasting blood glucose was recorded before any treatment was given. The patients were sorted into 3 groups: diabetics, normoglycemic non-diabetics and hyperglycemic nondiabetics. On the CT findings cases with large cortical and/or subcortical infarcts were analyzed separately from those with lacunar infarcts. The clinical symptoms on admission proved to be more severe (p less than 0.02) and 30-day mortality higher (p less than 0.02) among the hyperglycemic non-diabetics, who also showed a highly significant (p less than 0.00001) preponderance of large cortical and subcortical infarcts over lacunar infarcts. Multivariate analysis, which took account of variables of known relevance to the prognosis of cerebral infarction (age, sex, arterial hypertension, severity of the clinical pattern, type of brain lesion), confirmed the statistically discriminant power, in terms of mortality, of belonging to the hyperglycemic nondiabetic group. The results of the study confirm that hyperglycemia at stroke onset in nondiabetic patients is an adverse prognostic factor and suggest that it may be a reaction to stress, depending on the size of the infarcted area.

[Mesoglycan in acute focal cerebral ischemia]. / Il mesoglicano nelle ischemie cerebrali acute a focolaio.

An open, randomized, controlled study including 57 patients with acute cerebral infarct was performed. All the patients, followed and controlled by the same examiner, received, in the first ten days, 24 mg/die i.v. of dexamethasone. 28 patients were also treated with mesoglycan (150 mg/die i.m. for five days and 144 mg/die per os for a further twenty-five days). The differences between the basal and final scores in the mesoglycan group and in the controls were not statistically significant as analysed by the Mann-Whitney U test. The mesoglycan influenced only slightly the laboratory values (PT, PTT, alkaline phosphatase, GOT, GPT, cholesterol and triglycerides, fibrinogen, blood glucose, azotemia and creatinine) performed before the beginning of the treatment, as their changes after thirty days of therapy were in the normal range. The mesoglycan was very well tolerated and no side-effects were observed during the treatment.

[The electroencephalogram and lacunar infarcts]. / Elettroencefalogramma ed infarti lacunari.

The authors compare the conventional EEG of 16 patients with lacunar infarct, 18 patients suffering from subcortical infarct and 59 patients suffering from cortico-subcortical infarct, all confirmed by CT scan. They remark that while among patients with single or multiple lacunar infarct, 62% of the EEG turned out to be normal and 31% showed unilateral abnormalities, among those suffering from subcortical and cortico-subcortical infarct the EEG was normal only in 14% and showed unilateral abnormalities or focal alterations in 74% of the patients (chi square = p less than 0.01). According to these results the authors discuss the EEG's value in the differential diagnosis of lacunar infarcts and the influence on prognosis and on the therapeutical and diagnostic approach of these patients.

[The hematocrit and atherothrombotic cerebral infarcts]. / Ematocrito ed infarti cerebrali aterotrombotici.

The authors compare the hematocrit values of 131 patients suffering from an acute cerebral atherothrombotic infarct, confirmed by CT scan, with those of 165 controls of the same age and sex. Both the analysis of the average by the Student's t test and the comparison between the distribution of frequencies by the chi square test, point out that hematocrit is significantly higher (p less than 0.001) in infarcts than in controls. Dividing patients according to CT scan (negative, lacunar infarct, cortical and/or subcortical infarct), no significant difference in hematocrit levels among the three groups is noticed. The authors confirm the positive association between hematocrit and atherothrombotic cerebral infarct and discuss the possible implications in the prevention and the therapy of cerebral ischemic stroke.

[Use of ticlopidine in patients with acute non-embolic cerebral infarcts]. / Impiego della ticlopidina in pazienti con infarto cerebrale acuto non embolico.

Different dosages, varying from 250 to 500 mg/die, of ticlopidine per os have been employed in 46 patients (32 men and 14 women), average age 66, who had been admitted for acute non-embolic cerebral infarction. In the two clinical examinations, effected on average 5.4 and 18.3 days after the admission, significant alterations in 9 among the considered items have been noticed, i.e.: central deficit of the VIIth cranial nerve, motor function and gait impairment, Babinski's sign, loss of sensibility, dysarthria, visual field defect, cerebellar signs, motor and/or receptive dysphasia. A global improvement of these clinical signs has been noticed on average in 48.2% of the patients, while an exacerbation has occurred in 2.1% of the cases. None of the patients who have been considered in this study has decreased or has shown relapses of ischemic cerebral attacks during the observation. The treatment with ticlopidine has been discontinued in 3 cases (6.5%) for the occurrence of temporary adverse side-effects.