Rifampicin fails to eradicate mature biofilm formed by methicillin-resistant Staphylococcus aureus

INTRODUCTION: Antimicrobial activity on biofilms depends on their molecular size, positive charges, permeability coefficient, and bactericidal activity. Vancomycin is the primary choice for methicillin-resistant Staphylococcus aureus (MRSA) infection treatment; rifampicin has interesting antibiofilm properties, but its effectivity remains poorly defined. METHODS: Rifampicin activity alone and in combination with vancomycin against biofilm-forming MRSA was investigated, using a twofold serial broth microtiter method, biofilm challenge, and bacterial count recovery. RESULTS: Minimal inhibitory concentration (MIC) and minimal bactericidal concentration for vancomycin and rifampicin ranged from 0.5 to 1mg/l and 0.008 to 4mg/l, and from 1 to 4mg/l and 0.06 to 32mg/l, respectively. Mature biofilms were submitted to rifampicin and vancomycin exposure, and minimum biofilm eradication concentration ranged from 64 to 32,000 folds and from 32 to 512 folds higher than those for planktonic cells, respectively. Vancomycin (15mg/l) in combination with rifampicin at 6 dilutions higher each isolate MIC did not reach in vitro biofilm eradication but showed biofilm inhibitory capacity (1.43 and 0.56log10 CFU/ml reduction for weak and strong biofilm producers, respectively; p<0.05). CONCLUSIONS: In our setting, rifampicin alone failed to effectively kill biofilm-forming MRSA, demonstrating stronger inability to eradicate mature biofilm compared with vancomycin.

The systematic and biogeographical status of Fredius reflexifrons (Ortmann, 1897) and Fredius fittkaui (Bott, 1967) (Crustacea: Brachyura: Pseudothelphusidae) from the Amazon and Atlantic Guianas River basins

There is considerable confusion in the literature regarding the systematic position and distribution of two pseudothelphusid crabs originally described as Potamocarcinus reflexifrons Ortmann, 1897 and Potamocarcinus reflexifrons fittkaui Bott, 1967, and now included in the genus Fredius Pretzman, 1965, as F. reflexifrons and F. fittkaui. Study of numerous specimens from recent collections, together with a critical analysis of the data published in the literature, shows that both taxa could be easily separated by gonopodal characters. The two species occupy discrete areas of distribution along the main axis of the Amazon River and in the upper Rio Negro Basin, respectively, with an overlap in the Atlantic Guianas. It is postulated that they originated from a common ancestor, through a process of vicariance, in the two areas observed at present. Permeability of barriers allowed their further occupancy of the Atlantic Guianas after the marine regressions in this area.

Um esquema geral para a discussão do movimento de materiais para as células vivas e tecidos

A generalized comprehensive scheme concerning the movement of materials into living cells and tissues is presented. It is designed for use by investigators and teachers who, is assumed, have a previous knowledge of the subject as well as familiarity with previously published literature on the subject. Footnotes to figure 2 X = a constituent component material, arising from extermal sources or through metabolism, capable of migration under favorable conditions. e, i, ec, ic, mc = subscripts indicating location. oC = oxidized cytoplasmic constituents specifically concerned in unidirectional (here invardly directed), diffetial translocation of a particular material X. rC = reduced cytoplasmic constituents specifically concerned. XF = specific free energy (2) of a constituent component in in external or internal phases or in cytoplasm. D = diffusion (4). EA = exchange adsorption (4). MA= metabolic accumulation (4), CI = differentially characteristic cytoplasmic interaction (4) between oC and X, determined by the genetic constitution of the biological species. ▬▬▬>= migration, where permeability to the material involved is relatively high; and ~~~~->, where relatively low. <▬▬▬>= chemical reactions. ▬▬▬>= energy changes of X; diffusion is characteristically with the direction in which the concentration or activity of the constituent decreases, exchange adsorption may be with or against the direction of concentration or activity decrease.

Imunidade e permeabilidade celular

After going through the more important theories on cellular permeability, researches were undertaken with the purpose of proving the actual influence of the various degrees of cellular permeability on the phenomena of organic resistance against infections, and on the production of antibodies. Three groups of substances known to have action on cellular permeability were used; the first consisting of the following permeable substances: testos-terona, acetylcholine, and the spreading-factor of the staphyloccocus. The second group included substances which help in developing low cellular permeability: atropin, adrenalin and calcium. Finally, the third group consisted of a substance which helps to maintain normal permeability: cortin (an extract of the suprarenal cortex). In order to study the process developed by these elements with regard to organic resistance against infections, adult mice were inoculated with the following germs: K. pneumoniae, P. aeruginosa, S. enteriditis and D. pneumoniae, in the smallest possible amount capable of starting a mortal sep infection in approximately 24 hours, exception made of D. pneumonias which causes death in 48 hours. The animals were divided into groups of 10, a before taking the injections containing the germs, they were given the sub lances under observation, through their peritoneum of intramuscularly. T. animals that died were autopsied and blood was taken from their hearts an aseptic process so as not to introduce extraneous organisms. For the purpose of determining the development of antibodies (hem lysins, precipitins and aglutinins), rabbits were used, which had been prep ously immunized by a treatment consisting of 6 intravenous injections of polyvolent antigen made of sheep blood cells, fresh human serum, and of suspension of S. enteriditis. It was concluded that: Cellular permeability plays a very important part in the development infections. Permeable substances help the development of germ infections. Substances helping to develop low permeability proved not to have any influence worth mentioning. Substances helping to maintain normal permeability, such as coffin, it crease resistance against infections. The different substances used which have action on cellular permeability had no influence worth mentioning on the development of certain ant bodies (hemolysins, precipitins and aglutinins). It was admitted that the phenomena under study relative to resistance against infections are closely connected to the dynamics of the cellular elements, which circumstance is basically dependent on the permeability of Citations of cells.

O potassio plasmático em infecções bacterianas experimentais

Arrangement of potassium in the tissues having been mentioned, as well as the rôle it plays in some pathological processes such as suprarenal insufficiency, anaphylactic shock and shock caused by hemorrhage or traumatism, experiences were undertaken to establish the rates of plasma potassium during bacteria infections artificially developed in rabbits by K. pneumoniae. P. aeruginosa and S. enteridits. It was concluded that during the period of the infections, the rate of potassium of the plasma increases almost immediately after the inoculation and stays high when the infections are of a serious or mortal character; the rate continue to increase until the death of the animal occurs. When these infections are not very serious, as in the cases of infections resulting from inoculations of bacteria as not recent — and consequently with attenuated virulence — K pneumoniae, or P aeruginosa and S enteriditis, to which rabbits are naturally very resistant, the rate of potassium of the plasma increases after an intravenous inoculation of germs according to the septicemic period of the infection; however, when, because of its natural resistance, the animal overcomes the infection, the amount of potassium gradually decreases and finally gets back to the normal rate. The action of cortin on potassium of the plasma was also tested on animals suffering from acute infections caused by K. pneumoniae, which, under normal conditions cause death of the rabbits, nor did it increase the rate of potassium of the plasma when a larger amount of bacteria (300,000,000) was inoculated. However, cortin inoculated several times prevented a higher rate of potassium in the plasma during the development of the infection when a smaller number of bacteria (150,000,000) was inoculated, which quantity, under normal conditions, always causes mortal infections. When cortin is discontinued 20 hours after the inoculation of germs, the infection increases fastly and the animal dies in a very short time. Now, if the injections of cortin continue to be given every hour until the 26th hour instead of only until the 20th hour, the amount of potassium in the plasma — very high if the hormones substance is no longer inoculated — gradually becomes smaller and finally comes back to the normal rate if the inoculations continue to be made; it will increase again only if the substance is no longer injected; after a few hours the injection is gone, potassium is found to come back to its former rate, and in consequence the animal is perfectly cured of an infection otherwise mortal. ln view of the results thus obtained, it was concluded that, during the development of those infections, the checking of the rate of potassium of the plasma provided a means of controlling the resistance of a body suffering from an infection, that rate increasing when the infection is developing and becoming more severe, or getting back to normal when the infection decreases. The checking of the rate of potassium of the plasma also made known the action of cortin on the tissues, which is found to control the permeability of the cells to potassium. Suggestions were made that potassium of the plasma be thereofre checked during infections in the human body, to make possible proving that the phenomena studied in those animals also take place in the human body. In case this is found to be true, we sould possess an important element to check organic vitality during infections.

Partial isolation and some properties of enterotoxin produced by Bacillus cereus strains

Extracellular proteins produced by Bacillus cereus AL-42 and AL-15 were fractioned by chromatography on QAE-Sephadex and Sephadex G75. This last chromatographic process resulted in three peaks. The major peak showed vascular permeability activity to rabbits, lethality to mice, and cytotoxicity to Vero and Hela cells. The analysis by SDS-PAGE after ultrafiltration confirm recent findings that the enterotoxin is a compound with molecular mass > 30.000.

Morphometric Analysis of McCoy Cells Inoculated with Cerebrospinal Fluid from Patients with Rabies

To demonstrate the potential of McCoy cells for the isolation of rabies virus from the cerebrospinal (CSF) fluid of a patient with a diagnosis of rabies, McCoy cells were inoculated with CSF from a patient with a clinical diagnosis of rabies and investigated in terms of morphometric aspect using the JAVA analysis system for the quantification of the increased size of infected cells compared to noninfected cells. The cells were also examined in terms of specific staining for the diagnosis of rabies by the method of Sellers for the observation of intracytoplasmic inclusions and by specific immunofluorescence staining for rabies virus. Infected cells showed changes in cell permeability and morphologic modifications which differed significantly compared to normal cells (P<0.001) when analyzed by the Mann-Whitney and Kruskal-Wallis tests. Intense activity of the endoplasmic reticulum was also observed, as indicated by the presence of intracytoplasmic inclusions visualized by specific staining. The present study demonstrated the isolation of rabies virus from the CSF of a patient with rabies, showing that McCoy cells can be used for the laboratory diagnosis of patients suspected to have rabies.

Screening and fractionation of plant extracts with antiproliferative activity on human peripheral blood mononuclear cells

Three hundred and thirteen extracts from 136 Brazilian plant species belonging to 36 families were tested for their suppressive activity on phytohemaglutinin (PHA) stimulated proliferation of human peripheral blood mononuclear cells (PBMC). The proliferation was evaluated by the amount of [³H]-thymidine incorporated by the cells. Twenty extracts inhibited or strongly reduced the proliferation in a dose-dependent manner at doses between 10 and 100 µg/ml. Three of these extracts appeared to be non-toxic to lymphocytes, according to the trypan blue permeability assay and visual inspection using optical microscopy. Bioassay-guided fractionation of Alomia myriadenia extract showed that myriadenolide, a labdane diterpene known to occur in this species, could account for the observed activity of the crude extract. Using a similar protocol, an active fraction of the extract from Gaylussacia brasiliensis was obtained. Analysis of the ¹H and13C NMR spectra of this fraction indicates the presence of an acetylated triterpene whose characterization is underway. The extract of Himatanthus obovatus is currently under investigation.

The in vitro cytopathology of a porcine and the simian (SA-11) strains of rotavirus

Rotaviruses have been implicated as the major causal agents of acute diarrhoea in mammals and fowls. Experimental rotavirus infection have been associated to a series of sub-cellular pathologic alterations leading to cell lysis which may represent key functions in the pathogenesis of the diarrhoeic disease. The current work describes the cytopathic changes in cultured MA-104 cells infected by a simian (SA-11) and a porcine (1154) rotavirus strains. Trypan blue exclusion staining showed increased cell permeability after infection by both strains, as demonstrated by cell viability. This effect was confirmed by the leakage of infected cells evaluated by chromium release. Nuclear fragmentation was observed by acridine orange and Wright staining but specific DNA cleavage was not detected. Ultrastructural changes, such as chromatin condensation, cytoplasm vacuolisation, and loss of intercellular contact were shown in infected cells for both strains. In situ terminal deoxynucleotidyl transferase (Tunel) assay did not show positive result. In conclusion, we demonstrated that both strains of rotavirus induced necrosis as the major degenerative effect.

Good bug, bad bug: in the case of enteric inflammatory disease does the epithelium decide?

Many studies demonstrate that intestinal inflammation is either initiated or exaggerated by a component of the normal microbiota, most likely commensal bacteria or products derived from these organisms. We review the nature of human inflammatory bowel disease, the evidence for the involvement of the normal bacterial flora in these disorders and the relevance of maintaining the integrity of the epithelial barrier. Moreover, we, and others, have shown abnormal mitochondria structure in tissue resections from patients with inflammatory bowel disease and tissues from rodents that demonstrated psychological stress-induced increases in epithelial permeability. Thus, we also consider the possibility that a defect in epithelial mitochondrial function would predispose an individual to respond to their commensal bacteria flora - no longer considering them as a beneficial passive inhabitant, but rather perceiving them as a threatening and pro-inflammatory stimulus. In support of this postulate, we discuss our recent findings from an in vitro model showing that the human colon-derived T84 cell line exposed to the metabolic stressor, dinitrophenol, and the non-pathogenic, non-invasive, Escherichia coli (strain HB101) display a loss of barrier function, increased signal transduction and increased production of the chemokine, interleukin 8.

The resumption of consumption: a review on tuberculosis

Among all infectious diseases that afflict humans, tuberculosis (TB) remains the deadliest. At present, epidemiologists estimate that one-third of the world population is infected with tubercle bacilli, which is responsible for 8 to 10 million new cases of TB and 3 million deaths annually throughout the world. Approximately 95% of new cases and 98% of deaths occur in developing nations, generally due to the few resources available to ensure proper treatment and where human immunodeficiency virus (HIV) infections are common. In 1882, Dr Robert Koch identified an acid-fast bacterium, Mycobacterium tuberculosis, as the causative agent of TB. Thirty-nine years later, BCG vaccine was introduced for human use, and became the most widely used prophylactic strategy to fight TB in the world. The discovery of the properties of first-line antimycobacterial drugs in the past century yielded effective chemotherapies, which considerably decreased TB mortality rates worldwide. The later introduction of some additional drugs to the arsenal used to treat TB seemed to provide an adequate number of effective antimicrobial agents. The modern, standard short-course therapy for TB recommended by the World Health Organization is based on a four-drug regimen that must be strictly followed to prevent drug resistance acquisition, and relies on direct observation of patient compliance to ensure effective treatment. Mycobacteria show a high degree of intrinsic resistance to most antibiotics and chemotherapeutic agents due to the low permeability of its cell wall. Nevertheless, the cell wall barrier alone cannot produce significant levels of drug resistance. M. tuberculosis mutants resistant to any single drug are naturally present in any large bacterial population, irrespective of exposure to drugs. The frequency of mutants resistant to rifampicin and isoniazid, the two principal antimycobacterial drugs currently in use, is relatively high and, therefore, the large extra-cellular population of actively metabolizing and rapidly growing tubercle bacilli in cavitary lesions will contain organisms which are resistant to a single drug. Consequently, monotherapy or improperly administered two-drug therapies will select for drug-resistant mutants that may lead to drug resistance in the entire bacterial population. Thereby, despite the availability of effective chemotherapy and the moderately protective vaccine, new anti-TB agents are urgently needed to decrease the global incidence of TB. The resumption of TB, mainly caused by the emergence of multidrug-resistant (MDR) and extensively drug-resistant (XDR) strains and HIV epidemics, led to an increased need to understand the molecular mechanisms of drug action and drug resistance, which should provide significant insight into the development of newer compounds. The latter should be effective to combat both drug-susceptible and MDR/XDR-TB.

An in vitro model for dengue virus infection that exhibits human monocyte infection, multiple cytokine production and dexamethasone immunomodulation

An important cytokine role in dengue fever pathogenesis has been described. These molecules can be associated with haemorrhagic manifestations, coagulation disorders, hypotension and shock, all symptoms implicated in vascular permeability and disease worsening conditions. Several immunological diseases have been treated by cytokine modulation and dexamethasone is utilized clinically to treat pathologies with inflammatory and autoimmune ethiologies. We established an in vitro model with human monocytes infected by dengue virus-2 for evaluating immunomodulatory and antiviral activities of potential pharmaceutical products. Flow cytometry analysis demonstrated significant dengue antigen detection in target cells two days after infection. TNF-alpha, IFN-alpha, IL-6 and IL-10 are produced by in vitro infected monocytes and are significantly detected in cell culture supernatants by multiplex microbead immunoassay. Dexamethasone action was tested for the first time for its modulation in dengue infection, presenting optimistic results in both decreasing cell infection rates and inhibiting TNF-alpha, IFN-alpha and IL-10 production. This model is proposed for novel drug trials yet to be applyed for dengue fever.

Oxamniquine, praziquantel and lovastatin association in the experimental Schistosomiasis mansoni

The activity of lovastatin associated with oxamniquine or praziquantel against schistosomiasis mansoni was evaluated in mice infected with Schistosoma mansoni. Forty days after infection, mice were treated with lovastatin, 400 mg/kg for five consecutive days by oral route, and on the last day of this sequence with 50 mg/kg oxamniquine or with 200 mg/kg praziquantel, both by oral route, single dose. Fifteen days later, the animals were perfused in parallel with an untreated control group. Studies were carried out in vitro, using lovastatin in culture medium containing S. mansoni worms proceeding from experimentally infected mice. In the in vivo trials, the association of lovastatin with oxamniquine or praziquantel did not show any additive action, but there were oogram changes when lovastatin was associated with oxamniquine. In vitro lovastatin was able to interrupt the maturation of S. mansoni eggs, which remained at the 1st or 2nd stages, depending on the dose used. The total number of morphologically dead eggs found in culture of worms exposed to 2 µg/ml or 4 µg/ml concentrations of lovastatin was significantly higher than the number of viable eggs. Using the probe Hoescht 33258 it was observed that 70% of the eggs considered morphologically viable in the treated groups (against 16% in the control group) were labeled, indicating that the majority of the viable eggs had membrane permeability increased due to lovastatin action.

Effect of Solanum nudum steroids on uninfected and Plasmodium falciparum-infected erythrocytes

Steroids from Solanum nudum (SNs) have demonstrated antiplasmodial activity against erythrocytic stages of the Plasmodium falciparum strain FCB-2. It is well known that steroids can alter the membrane function of erythrocytes. Thus, we assessed alterations in the membranes of uninfected red blood cells, the parasite invasiveness and the solute-induced lysis of parasitised red blood cells (pRBCs). induced by SNs. We found that most merozoites were unable to invade SN-treated erythrocytes. However, transmission electron microscopy revealed no effect on the morphology of uninfected erythrocytes treated with either SN2 or diosgenone and neither SN induced haemolysis of uninfected erythrocytes. SN2 and SN4 inhibited isosmotic sorbitol and alanine-induced haemolysis of pRBCs. In contrast, diosgenone and SN1 did not inhibit solute-induced haemolysis. The inhibition of solute-induced lysis of parasitised erythrocytes by SN2 and SN4 suggest an action of these SNs on new permeability pathways of pRBCs.

Antileishmanial activity of diterpene acids in copaiba oil

Leishmaniasis is a neglected tropical disease. According to the World Health Organization, there are approximately 1.5-two million new cases of cutaneous leishmaniasis each year worldwide. Chemotherapy against leishmaniasis is based on pentavalent antimonials, which were developed more than a century ago. The goals of this study were to investigate the antileishmanial activity of diterpene acids in copaiba oil, as well as some possible targets of their action against Leishmania amazonensis. Methyl copalate and agathic, hydroxycopalic, kaurenoic, pinifolic and polyaltic acids isolated from Copaifera officinales oleoresins were utilised. Ultrastructural changes and the specific organelle targets of diterpenes were investigated with electron microscopy and flow cytometry, respectively. All compounds had some level of activity against L. amazonensis. Hydroxycopalic acid and methyl copalate demonstrated the most activity against promastigotes and had 50% inhibitory concentration (IC50) values of 2.5 and 6.0 µg/mL, respectively. However, pinifolic and kaurenoic acid demonstrated the most activity against axenic amastigote and had IC50 values of 3.5 and 4.0 µg/mL, respectively. Agathic, kaurenoic and pinifolic acid caused significant increases in plasma membrane permeability and mitochondrial membrane depolarisation of the protozoan. In conclusion, copaiba oil and its diterpene acids should be explored for the development of new antileishmanial drugs.