Fluoroquinolone and beta-lactam antimicrobials induce different transcriptome profiles in Salmonella enterica persister cells.

Here, we investigate the transcriptome profiles of two S. Enteritidis and one S. Schwarzengrund isolates that present different persister levels when exposed to ciprofloxacin or ceftazidime. It was possible to note a distinct transcript profile among isolates, time of exposure, and treatment. We could not find a commonly expressed transcript profile that plays a role in persister formation after S. enterica exposure to beta-lactam or fluoroquinolone, as only three DEGs presented the same behavior under the conditions and isolates tested. It appears that the formation of persisters in S. enterica after exposure to ciprofloxacin is linked to the overexpression of genes involved in the SOS response (recA), cell division inhibitor (sulA), iron-sulfur metabolism (hscA and iscS), and type I TA system (tisB). On the other hand, most genes differentially expressed in S. enterica after exposure to ceftazidime appeared to be downregulated and were part of the flagellar assembly apparatus, citrate cycle (TCA cycle), glycolysis/gluconeogenesis, carbon metabolism, bacterial secretion system, quorum sensing, pyruvate metabolism pathway, and biosynthesis of secondary metabolites. The different transcriptome profiles found in S. enterica persisters induced by ciprofloxacin and ceftazidime suggest that these cells modulate their response differently according to each stress.

Thiostrepton-Nanomedicine, a TLR9 Inhibitor, Attenuates Sepsis-Induced Inflammation in Mice.

Sepsis is a life-threatening clinical condition caused by infection and transposition of pathogens and pathogen-associated molecular patterns (PAMPs) into the host bloodstream. During sepsis, activation of toll-like receptors (TLRs) on immune cells triggers the release of pro-inflammatory cytokines and overstimulates the production of vasodilatory mediators such as nitric oxide (NO). These vascular changes lead to widespread inflammation, tissue damage, multiple organ failure, and often death. New therapeutic options are urgently needed. To this end, thiostrepton (TST) has emerged as a candidate for sepsis treatment due to its action as an antibiotic and anti-inflammatory molecule (TLR7-9 inhibitor). Reports in the literature suggest that TLR9 inhibition substantially suppresses the excessive host inflammatory response and attenuates sepsis-induced mortality in the cecal ligation and puncture (CLP) murine model of sepsis. However, to the best of our knowledge, TST has never been directly tested as a therapeutic option for the management of sepsis, possibly due to its low water solubility and drug delivery issues. These facts prompted us to test the central hypothesis that TST encapsulated in phospholipid sterically stabilized micelles (TST-SSM) could be developed into a novel treatment for sepsis. Thus, using our published method of encapsulating the hydrophobic antibiotic TST-SSM, we evaluated the in vivo efficacy of TST-SSM nanomedicine in the murine model of polymicrobial sepsis. We found that TST-SSM increased the median survival of CLP-induced septic mice from 31 to 44 hr by reducing the bacterial burden in the blood and peritoneal lavage. Moreover, plasma levels of pro-inflammatory cytokines (interleukin 6 and tumor necrosis factor-alpha) and NO derivatives were also reduced, whereas renal and hepatic function biomarkers creatinine and aspartate transferase were significantly improved. In conclusion, we identified that TST-SSM nanomedicine has significant potential as a therapeutic agent for sepsis management, primarily due to its anti-inflammatory and antibiotic properties.

Effect of pH Cycling Followed by Simulated Toothbrushing on the Surface Roughness and Bacterial Adhesion of Bulk-fill Composite Resins.

The aim was to evaluate, in vitro, quantitatively and qualitatively, the effect of pH cycling and simulated toothbrushing on surface roughness (Ra) and bacterial adhesion (Cn) of bulk-fill composite resins. Thirty specimens of each composite resin, 5 mm wide and 4 mm high, were obtained: group 1 (control): Filtek Z250 (Z250); group 2: Filtek Bulk-Fill (FTK); group 3: Tetric N-Ceram Bulk-Fill (TTC); and group 4: Aura Bulk-Fill (AUR). After 24 hours, the specimens were polished and then alternated with demineralization/remineralization solutions for 15 cycles of 24 hours each at 37°C. Then the specimens were submitted to simulated toothbrushing. The Ra and Cn measurements were quantitatively analyzed in three stages: after polishing (Ra0 and Cn0), after pH cycling (Ra1 and Cn1), and after simulated toothbrushing (Ra2 and Cn2). The Ra values were submitted to two-way analysis of variance, followed by the Tukey test (α=0.05). The Kruskal-Wallis test, followed by multiple comparisons, was applied for Cn analysis. Surface topography and bacterial adhesion were observed by scanning electron microscopy (SEM). Z250, FTK, and TTC showed no significant change in Ra regardless of the treatment performed; AUR obtained increased Ra at Ra2 (p<0.05). FTK differed from the others at Cn0 and Cn1 (p<0.05). At Cn2, there was no difference among the composite resins. SEM images showed the exposure of fillers and microcavities at Ra1 and Ra2. There was greater bacterial adhesion at Cn1 for Z250 and FTK. It was concluded that the pH cycling caused surface degradation of all composite resins, which was potentiated by simulated toothbrushing. However, only AUR presented an increased Ra. Bacterial adhesion occurred on all composite resins after pH cycling; however, after simulated toothbrushing, adhesion of dispersed bacteria was similar for all the composite resin groups.

One-step global parameter estimation of kinetic inactivation parameters for Bacillus sporothermodurans spores under static and dynamic thermal processes.

Bacillus sporothermodurans produces highly heat-resistant endospores, that can survive under ultra-high temperature. High heat-resistant sporeforming bacteria are one of the main causes for spoilage and safety of low-acid foods. They can be used as indicators or surrogates to establish the minimum requirements for heat processes, but it is necessary to understand their thermal inactivation kinetics. The aim of the present work was to study the inactivation kinetics under both static and dynamic conditions in a vegetable soup. Ordinary least squares one-step regression and sequential procedures were applied for estimating these parameters. Results showed that multiple dynamic heating profiles, when analyzed simultaneously, can be used to accurately estimate the kinetic parameters while significantly reducing estimation errors and data collection.

The detection of viable vegetative cells of Bacillus sporothermodurans using propidium monoazide with semi-nested PCR.

Bacillus sporothermodurans produces highly heat-resistant spores that can survive ultra-high temperature (UHT) treatment in milk. Therefore, we developed a rapid, specific and sensitive semi-nested touchdown PCR assay combined with propidium monoazide (PMA) treatment for the detection of viable B. sporothermodurans vegetative cells. The semi-nested touchdown PCR alone proved to be specific for B. sporothermodurans, and the achieved detection limit was 4 CFU/mL from bacterial culture and artificially contaminated UHT milk. This method combined with PMA treatment was shown to amplify DNA specifically from viable cells and presented a detection limit of 10(2) CFU/mL in UHT milk. The developed PMA-PCR assay shows applicability for the specific detection of viable cells of B. sporothermodurans from UHT milk. This method is of special significance for applications in the food industry by reducing the time required for the analysis of milk and dairy products for the presence of this microorganism.

Antibacterial activity of two MTA-based root canal sealers.

AIM: To evaluate the pH and antibacterial activity of Endo CPM Sealer and MTA Fillapex by two different methods, using white MTA and Endofill as references for comparison. METHODOLOGY: Antibacterial activity was evaluated against Enterococcus faecalis (ATCC 29212). The agar diffusion test (ADT) was performed to evaluate the effect before setting. The materials were placed in four equidistant wells made in ten agar plates. After incubation at 37 °C for 48 h, the inhibition zones were measured using a digital paquimeter. The direct contact test (DCT) was performed to assess the antibacterial effect after setting. Suspensions of crushed materials were prepared and mixed with E. faecalis. After different periods of time (1, 6, 15 and 60 min), the survival of bacteria was assessed by using 10-fold serial dilution and cultivated on agar plates in triplicate. Colony-forming units (CFU) mL(-1) were calculated after incubation. pH values were also measured in triplicate. Comparison between sealers in the ADT and DCT was performed by the Kruskal-Wallis test. RESULTS: In the ADT, inhibition zones were found with MTA Fillapex and Endofill. They were similar to each other and greater than the other sealers (P < 0.05). None of the tested sealers demonstrated antibacterial activity in the DCT, and thus, all sealers had similar bacterial counts compared with the negative control group (P > 0.05). White MTA and Endo CPM Sealer suspensions had pH values >11, whilst MTA Fillapex and Endofill had lower values. CONCLUSIONS: MTA Fillapex and Endofill had an antibacterial effect against E. faecalis before setting, but none of the sealers maintained antibacterial activity after setting, despite the high pH of the MTA-based materials.

Molecular diagnosis of Salmonella species in captive psittacine birds.

Cloacal swabs were collected from 280 captive psittacine birds belonging to 13 species. Samples of dna were tested by PCR using a pair of primers that amplify a 284 base pair fragment of the Salmonella genus invA gene, and the PCR-positive samples were tested by standard microbiological techniques. Thirteen per cent of the samples were positive by PCR, but negative by microbiological techniques. The infection rates were significantly different among the 13 species, the most commonly infected being Amazona amazonica (28 per cent) and Amazona pretrei (20 per cent). Specific tests for Salmonella Typhimurium Salmonella Enteritidis, Salmonella Pullorum and Salmonella Gallinarum did not produce positive results.

Isolation and characterization of Ornithobacterium rhinotracheale from chickens in Brazil.

Ornithobacterium rhinotracheale (ORT) is a recently described species of bacterium associated with respiratory disease, growth retardation, mortality, and decreased egg production in chickens and turkeys. Pneumonia, pleuritis, and airsacculitis characterise the infection. ORT has been isolated in many countries but it is still considered exotic in Brazil. Up to date it is prohibited to import and produce reagents for diagnostic and vaccination control. The aim of this study was to isolate and identify the bacteria in chickens. Four isolates were obtained from tracheal swabs of broilers. They were isolated in blood agar with gentamicin and showed biochemical, morphological, antigenic and genetic characteristics of ORT. The results confirm that ORT is present in Brazil.

Evaluation of selective and non-selective enrichment PCR procedures for Salmonella detection.

AIMS: To compare PCR combined with enrichment media with the standard microbiological techniques (SMT) and to determine the most sensitive method for the detection of Salmonella and the identification of Salm. typhimurium (ST), Salm. enteritidis (SE), Salm. gallinarum (SG) and Salm. pullorum (SP). METHODS AND RESULTS: We analysed 87 samples from poultry using PCR and SMT, PCR being performed from non-selective (NS) and Rappaport-Vassiliadis (RV) media. PCR-NS was less sensitive than PCR-RV and SMT for the detection and identification of Salmonella. PCR-RV detected more positive samples of Salmonella sp. than SMT but both these methods showed similar sensitivity regarding the identification of Salmonella serovars. CONCLUSIONS: PCR-RV was more sensitive and decreased the time necessary to detect and identify Salmonella. SIGNIFICANCE AND IMPACT OF THE STUDY: PCR-RV is a powerful tool for the rapid and accurate detection and identification of Salmonella and can be implemented in diagnostic and food analysis laboratories.

Detection and identification of salmonellas from poultry-related samples by PCR.

A polymerase chain reaction (PCR) assay was developed for the generic detection of Salmonella sp. and the identification of S. Enteritidis (SE), S. Gallinarum (SG), S. Pullorum (SP) and S. Typhimurium (ST) in material collected in the field from poultry. The specificity and sensitivity of the assay combined with Rappaport-Vassiliadis selective enrichment broth (PCR-RV) were determined, and field samples were analyzed to verify the validity of the method application. Specificity of the assay was tested using 29 SE, 11 SG, 10 ST and 10 SP strains, along with 75 strains of 28 other Salmonella serovars and 21 strains of other bacterial genera. The assay was 100% specific for Salmonella detection and ST identification. The primer pair for SE, SG and SP also detected S. Berta. PCR detection limits for Salmonella at the genus level were 2 ST, 8 SE, 1.1x10(3) SG and 1.8x10(5) SP cells. At the serovar level, detection limits were 7 ST, 1.2x10(3) SE, 4.4x10(7) SG and 1.8x10(6) SP cells. At the genus level, PCR-RV detected approximately 128% more positive field samples than the standard microbiological techniques and results were ready in 48h instead of 7 days. PCR-RV method is diagnostic of Salmonella at the genus level and ST at the serovar level, although other tests are needed to identify SE, SG and SP to serovar level.

Polymerase chain reaction (PCR) for the detection of Salmonella in artificially inoculated chicken meat.

The aim of this study was to develop a polymerase chain reaction (PCR) protocol for the detection of Salmonella in artificially contaminated chicken meat. Tests were performed with different dilutions of Salmonella Typhimurium or Salmonella Enteritidis cells (10(-7), 10(-8) or 10(-9) CFU/mL) inoculated in chicken meat samples, in order to establish the limits of detection, incubation times (0, 6, 8 and 24 hours of pre-enrichment in PBW 1%) and three DNA extraction protocols (phenol-chloroform, thermal treatment and thermal treatment and Sephaglass). The assay was able to detect until 10(-9) CFU/mL of initial dilution of Salmonella cells inoculated in chicken meat, which allows detection of Salmonella within 48 hours, including 24 hours of pre-enrichment and using the phenol-chloroform DNA extraction protocol. As the results are obtained in a shorter time period than that of microbiological culture, this procedure will be useful in the methodology for detection of Salmonella in chicken.

[The most frequent neurologic disturbances. A contribution to the definition of topics for the programmatic content to the program of neurology in medical graduation]. / Transtornos neurológicos mais frequentes: contribuição para a definição de temas do conteúdo programático do curso de neurologia, para a graduação médica.

INTRODUCTION: Knowledge accumulation is overfilling the thematic content of medical graduation. Medical Schools must be alert to define a minimal content related with the most frequent disturbs. We intent to suggest topics for the minimal content, to the program of Neurology in medical graduation. METHOD: 1. To identify the places where young doctors are working outside the School Hospital (SH): we analysed the answers of the letters sent to 6415 resident - doctors (RD) in São Paulo's State and we made personal interview with 201 RD. 2. To verify the most frequent neuropsychiatric disturbs (ND) in the population: we made the analysis of the patient's diagnostic records in emergency room of three institutions: Municipal of Taubaté-SP, Municipal of São José dos Campos-SP and Faculty of Medicine of São Paulo Santa Casa de Misericórdia. RESULTS: 1. The RD are young and, outside the SH, they work in other emergency services. 2. The most frequent diagnosis in institutions were listed: alcoholism, cerebrovascular disease, coma, cranial trauma, dementia, dizziness, epilepsy, facial paralysis, faint, headache, hemiplegia or paraplegia, meningitis, others paralysis, periferical neuropathy and psychiatric disorders. CONCLUSIONS: Those diagnostics most frequent are relevant topics of the programmatic content to the program of Neurology in medical graduation.

[Self-care in treatment following the Ilizarov method--a case study]. / Auto cuidado no tratamento pelo método de Ilizarov-um estudo de caso.

The authors performed a case study using Dorothea Orem's self-care model as a framework in the treatment by llizarov's method, administering care and specific orientations for recovery and prevention of further disorders. The operational system of the study consisted of procedures related to demands of universal and therapeutic care during hospitalization. Demands were detected in the pre-operative and care developed in the postoperative phase, providing the patient with the knowledge and achievement of the necessary procedures to be followed at home.

[The teaching of primary health care: diagnostic and educational evaluation by microcomputers]. / O ensino de atenção primária à saúde: avaliação diagnóstica e formativa através de microcomputadores.

The study aims to explore the utilization of the Formative Evaluation by the microcomputer in the teaching of primary attention to the health. The experiment is composed of eight modules, programmed in Basic software "Formative Evaluation". For each module, a "Question Bank" with three sorts of feedbacks was organized. The sample includes 16 subjects. The results show that the Formative Evaluation by Microcomputer proportioned to the students a cognitive domain significantly higher than the traditional instruction. However, it didn't provide a significantly superior performance to the students who experienced this system.