Evaluation of bacterial infections in organ transplantation

Hospital das Clinicas da Faculdade de Medicina da Universidade de Sao Paulo

Comparative Studies of the (Anti) Mutagenicity of Baccharis dracunculifolia and Artepillin C by the Bacterial Reverse Mutation Test

Baccharis dracunculifolia is a plant native from Brazil, commonly known as 'Alecrim-do-campo' and 'Vassoura' and used in alternative medicine for the treatment of inflammation, hepatic disorders and stomach ulcers. Previous studies reported that artepillin C (ArtC, 3-{4-hydroxy-3,5-di(3-methyl-2-butenyl)phenyl}-2(E)-propenoic acid), is the main compound of interest in the leaves. This study was undertaken to assess the mutagenic effect of the ethyl acetate extract of B. dracunculifolia leaves (Bd-EAE: 11.4-182.8 mu g/plate) and ArtC (0.69-10.99 mu g/plate) by the Ames test using Salmonella typhimurium strains TA98, TA97a, TA100 and TA102, and to compare the protective effects of Bd-EAE and ArtC against the mutagenicity of a variety of direct and indirect acting mutagens such as 4-nitro-O-phenylenediamine, sodium azide, mitomycin C, benzo[a]pyrene, aflatoxin B1, 2-aminoanthracene and 2-aminofluorene. The mutagenicity test showed that Bd-EAE and ArtC did not induce an increase in the number of revertant colonies indicating absence of mutagenic activity. ArtC showed a similar antimutagenic effect to that of Bd-EAE in some strains of S. typhimurium, demonstrating that the antimutagenic activity of Bd-EAE can be partially attributed to ArtC. The present results showed that the protective effect of whole plant extracts is due to the combined and synergistic effects of a complex mixture of phytochemicals, the total activity of which may result in health benefits.

Functional diversity of bacterial genes associated with aromatic hydrocarbon degradation in anthropogenic dark earth of Amazonia

The objective of this work was to evaluate the catabolic gene diversity for the bacterial degradation of aromatic hydrocarbons in anthropogenic dark earth of Amazonia (ADE) and their biochar (BC). Functional diversity analyses in ADE soils can provide information on how adaptive microorganisms may influence the fertility of soils and what is their involvement in biogeochemical cycles. For this, clone libraries containing the gene encoding for the alpha subunit of aromatic ring-hydroxylating dioxygenases (alpha-A RH D bacterial gene) were constructed, totaling 800 clones. These libraries were prepared from samples of an ADE soil under two different land uses, located at the Caldeirao Experimental Station secondary forest (SF) and agriculture (AG)-, and the biochar (SF_BC and AG_BC, respectively). Heterogeneity estimates indicated greater diversity in BC libraries; and Venn diagrams showed more unique operational protein clusters (OPC) in the SF_BC library than the ADE soil, which indicates that specific metabolic processes may occur in biochar. Phylogenetic analysis showed unidentified dioxygenases in ADE soils. Libraries containing functional gene encoding for the alpha subunit of the aromatic ring-hydroxylating dioxygenases (ARHD) gene from biochar show higher diversity indices than those of ADE under secondary forest and agriculture.

Photodynamic Effects of Curcumin Against Cariogenic Pathogens

Background data: The presence of Streptococcus mutans and Lactobacillus acidophilus in dental structure is an indicator of a cariogenic biofilm. Photodynamic therapy is a technique that involves the activation of photosensitizers by light in the presence of oxygen, resulting in the production of reactive radicals capable of inducing cell death. Reduction of bacteria levels can provide additional means of preventing dental caries. Objective: The present study evaluated the susceptibility of planktonic cultures of S. mutans (ATCC 25175) and L. acidophilus (ATCC-IAL-523) from the Adolfo Lutz Institute (IAL) to photodynamic therapy after sensitization with curcumin and exposure to blue light at 450 nm. Methods: Bacterial suspensions of S. mutans and L. acidophilus isolated (as single species) and combined (multspecies) were prepared and then evaluated. Four different groups were analyzed: L-D- (control group), L-D+ (drug group), L+D- (light group), and L+D+ (photodynamic therapy group). Two different concentrations of curcumin were tested (0.75 and 1.5 g/L) associated with a 5.7 J/cm(2) light emission diode. Results: Significant decreases (p < 0.05) in the viability of S. mutans were only observed when the bacterial suspensions were exposed to both curcumin and light. Then, reductions in viability of up to 99.99% were observed when using 1.5 g/L of the photosensitizer. The susceptibility of L. acidophilus was considerably lower (21% and 37.6%) for both curcumin concentrations. Conclusions: Photodynamic therapy was found to be effective in reducing S. mutans and L. acidophilus on planktonic cultures. No significant reduction was found for L-D+, proving the absence of dark toxicity of the drug.

Potential costs of bacterial infection on storage protein gene expression and reproduction in queenless Apis mellifera worker bees on distinct dietary regimes

Insects are able to combat infection by initiating an efficient immune response that involves synthesizing antimicrobial peptides and a range of other defense molecules. These responses may be costly to the organism, resulting in it exploiting endogenous resources to maintain homeostasis or support defense to the detriment of other physiological needs. We used queenless worker bees on distinct dietary regimes that may alter hemolymph protein storage and ovary activation to investigate the physiological costs of infection with Serratia marcescens. The expression of the genes encoding the storage proteins vitellogenin and hexamerin 70a, the vitellogenin receptor, and vasa (which has a putative role in reproduction), was impaired in the infected bees. This impairment was mainly evident in the bees fed beebread, which caused significantly higher expression of these genes than did royal jelly or syrup, and this was confirmed at the vitellogenin and hexamerin 70a protein levels. Beebread was also the only diet that promoted ovary activation in the queenless bees, but this activation was significantly impaired by the infection. The expression of the genes encoding the storage proteins apolipophorins-I and -III and the lipophorin receptor was not altered by infection regardless the diet provided to the bees. Similarly, the storage of apolipophorin-I in the hemolymph was only slightly impaired by the infection, independently of the supplied diet. Taken together these results indicate that, infection demands a physiological cost from the transcription of specific protein storage-related genes and from the reproductive capacity. (C) 2012 Elsevier Ltd. All rights reserved.

Influence of moisture and plaque on the performance of a laser fluorescence device in detecting caries lesions in primary teeth

The aim of this study was to evaluate the influence of dental plaque and moisture on performance of a laser fluorescence device in detecting occlusal and proximal caries lesions in primary teeth. Fifty-five occlusal and 58 proximal sites on primary molars were evaluated using a DIAGNOdent pen (LFpen) device. For the drying time study, the evaluations were performed in: (I) moist teeth; (II) teeth dried for 3 s, or (III) dried for 15 s. For the plaque study, the evaluations were done in sites: (I) without plaque; (II) with plaque, and (III) after cleaning. Evaluation of the teeth sections in stereomicroscope was the reference standard method. LF pen values, sensitivity, specificity, and accuracy were compared. The values obtained after 15 s of drying were higher than those obtained with moist teeth or dried for 3 s at both occlusal and proximal surfaces. However, there was no change in the performance in detecting caries lesions. With regard to the presence of plaque, there was no significant change in the readings of the device, but specificity was decreased in occlusal surfaces with plaque. At proximal surfaces, however, no significant differences were observed. In conclusion, the moisture conditions do not influence significantly the performance of the LFpen, but the presence of plaque can affect its performance in detecting occlusal caries lesions in primary teeth.

Self-supported bacterial cellulose/boehmite organic-inorganic hybrid films

Self-supported organic-inorganic hybrid transparent films have been prepared from bacterial cellulose and boehmite. SEM results indicate that the BC membranes are covered by Boehmite and XRD patterns suggest structural changes on cellulose due to Boehmite addition. Thermal stability is accessed through TG curves and is dependent on Boehmite content. Transparency, as evaluated by UV-Vis absorption, increases with increasing content of boehmite suggesting application of these materials as transparent substrates for opto-electronic devices.

Influence of milk type and addition of passion fruit peel powder on fermentation kinetics, texture profile and bacterial viability in probiotic yoghurts

The effect of the addition of passion fruit peel powder (PFPP) on the fermentation kinetics and texture parameters, post-acidification and bacteria counts of probiotic yoghurts made with two milk types were evaluated during 28 days of storage at 4 degrees C. Milks were fermented by Streptococcus thermophilus and Lactobacillus delbrueckii subsp. bulgaricus (CY340), and one strain of probiotic bacteria: Lactobacillus acidophilus (L10 and NCFM), Bifidobacterium animalis subsp. lactis (8104 and HN019). The addition of PFPP reduced significantly fermentation time of skim milk co-fermented by the strains L10, NCFM and HN019. At the end of 28-day shelf-life, counts of B. lactis Bl04 were about 1 Log CFU mL(-1) higher in whole yoghurt fermented with PFPP regarding its control but, in general, the addition of PFPP had less influence on counts than the milk type itself. The titratable acidity in yoghurts with PFPP was significantly higher than in their respective controls, and in skim yoghurts higher than in the whole ones. The PFPP increased firmness, consistency (except for the NCFM strain of L acidophilus) and cohesiveness of all skim yoghurts. The results point out the suitability of using passion fruit by-product in the formulation of both skim and whole probiotic yoghurts. (C) 2012 Elsevier Ltd. All rights reserved.

Coronary plaque rupture in patients with myocardial infarction after noncardiac surgery: Frequent and dangerous

Purpose: The pathophysiology of acute coronary syndromes (ACS) after noncardiac surgery is not established yet. Thrombosis over a vulnerable plaque or decreased oxygen supply secondary to anemia or hypotension may be involved. The purpose of this study was to investigate the pathophysiology of ACS complicating noncardiac surgery. Methods: Clinical and angiographic data were prospectively recorded into a database for 120 consecutive patients that had an ACS after noncardiac surgery (PACS), for 120 patients with spontaneous ACS (SACS), and 240 patients with stable coronary artery disease (CAD). Coronary lesions with obstructions greater than 50% were classified based on two criteria: Ambrose's classification and complex morphology. The presence of Ambrose's type II or complex lesions were compared between the three groups. Results: We analyzed 1470 lesions in 480 patients. In PACS group, 45% of patients had Ambrose's type II lesions vs. 56.7% in SACS group and 16.4% in stable CAD group (P < 0.001). Both PACS and SACS patients had more complex lesions than patients in stable CAD group (56.7% vs. 79.2% vs. 31.8%, respectively; P < 0.001). Overall, the independent predictors of plaque rupture were being in the group PACS (P < 0.001, OR 2.86; CI, 1.82-4.52 for complex lesions and P < 0.001, OR 3.43; CI, 2.1-5.6 for Ambrose's type II lesions) or SACS (P < 0.001, OR 8.71; CI, 5.15-14.73 for complex lesions and P < 0.001, OR 5.99; CI, 3.66-9.81 for Ambrose's type II lesions). Conclusions: Nearly 50% of patients with perioperative ACS have evidence of coronary plaque rupture, characterizing a type 1 myocardial infarction. (C) 2012 Elsevier Ireland Ltd. All rights reserved.

Bacterial cellulose-collagen nanocomposite for bone tissue engineering

A nanocomposite based on bacterial cellulose (BC) and type I collagen (COL) was evaluated for in vitro bone regeneration. BC membranes were modified by glycine esterification followed by cross-linking of type I collagen employing 1-ethyl-3-(3-dimethylaminopropyl)-carbodiimide. Collagen incorporation was studied by spectroscopy analysis. X-Ray diffraction showed changes in the BC crystallinity after collagen incorporation. The elastic modulus and tensile strength for BC-COL decreased, while the strain at failure showed a slight increase, even after sterilization, as compared to pristine BC. Swelling tests and contact angle measurements were also performed. Cell culture experiments performed with osteogenic cells were obtained by enzymatic digestion of newborn rat calvarium revealed similar features of cell morphology for cultures grown on both membranes. Cell viability/proliferation was not different between BC and BC-COL membranes at day 10 and 14. The high total protein content and ALP activity at day 17 in cells cultured on BC-COL indicate that this composite allowed the development of the osteoblastic phenotype in vitro. Thus, BC-COL should be considered as alternative biomaterial for bone tissue engineering.

Comparative genomic and transcriptome analyses of pathotypes of Xanthomonas citri subsp. citri provide insights into mechanisms of bacterial virulence and host range

Abstract Background Citrus bacterial canker is a disease that has severe economic impact on citrus industries worldwide and is caused by a few species and pathotypes of Xanthomonas. X. citri subsp. citri strain 306 (XccA306) is a type A (Asiatic) strain with a wide host range, whereas its variant X. citri subsp. citri strain Aw12879 (Xcaw12879, Wellington strain) is restricted to Mexican lime. Results To characterize the mechanism for the differences in host range of XccA and Xcaw, the genome of Xcaw12879 that was completed recently was compared with XccA306 genome. Effectors xopAF and avrGf1 are present in Xcaw12879, but were absent in XccA306. AvrGf1 was shown previously for Xcaw to cause hypersensitive response in Duncan grapefruit. Mutation analysis of xopAF indicates that the gene contributes to Xcaw growth in Mexican lime but does not contribute to the limited host range of Xcaw. RNA-Seq analysis was conducted to compare the expression profiles of Xcaw12879 and XccA306 in Nutrient Broth (NB) medium and XVM2 medium, which induces hrp gene expression. Two hundred ninety two and 281 genes showed differential expression in XVM2 compared to in NB for XccA306 and Xcaw12879, respectively. Twenty-five type 3 secretion system genes were up-regulated in XVM2 for both XccA and Xcaw. Among the 4,370 common genes of Xcaw12879 compared to XccA306, 603 genes in NB and 450 genes in XVM2 conditions were differentially regulated. Xcaw12879 showed higher protease activity than XccA306 whereas Xcaw12879 showed lower pectate lyase activity in comparison to XccA306. Conclusions Comparative genomic analysis of XccA306 and Xcaw12879 identified strain specific genes. Our study indicated that AvrGf1 contributes to the host range limitation of Xcaw12879 whereas XopAF contributes to virulence. Transcriptome analyses of XccA306 and Xcaw12879 presented insights into the expression of the two closely related strains of X. citri subsp. citri. Virulence genes including genes encoding T3SS components and effectors are induced in XVM2 medium. Numerous genes with differential expression in Xcaw12879 and XccA306 were identified. This study provided the foundation to further characterize the mechanisms for virulence and host range of pathotypes of X. citri subsp. citri.

Ultrasonic tissue characterization of vulnerable carotid plaque: correlation between videodensitometric method and histological examination

Abstract Background To establish the correlation between quantitative analysis based on B-mode ultrasound images of vulnerable carotid plaque and histological examination of the surgically removed plaque, on the basis of a videodensitometric digital texture characterization. Methods Twenty-five patients (18 males, mean age 67 ± 6.9 years) admitted for carotid endarterectomy for extracranial high-grade internal carotid artery stenosis (≥ 70% luminal narrowing) underwent to quantitative ultrasonic tissue characterization of carotid plaque before surgery. A computer software (Carotid Plaque Analysis Software) was developed to perform the videodensitometric analysis. The patients were divided into 2 groups according to symptomatology (group I, 15 symptomatic patients; and group II, 10 patients asymptomatic). Tissue specimens were analysed for lipid, fibromuscular tissue and calcium. Results The first order statistic parameter mean gray level was able to distinguish the groups I and II (p = 0.04). The second order parameter energy also was able to distinguish the groups (p = 0,02). A histological correlation showed a tendency of mean gray level to have progressively greater values from specimens with < 50% to >75% of fibrosis. Conclusion Videodensitometric computer analysis of scan images may be used to identify vulnerable and potentially unstable lipid-rich carotid plaques, which are less echogenic in density than stable or asymptomatic, more densely fibrotic plaques.

Histological composition and progression of carotid plaque

Abstract Background To analyse histological composition and progression of carotid plaque. Methods Thirty-one patients (22 males, mean age 68.03 ± 7.3 years) admitted for carotid endarterectomy for extracranial high-grade internal carotid artery stenosis (≥ 70% luminal narrowing) were enrolled. The patients were divided into 2 groups according to symptomatology (group I, 17 symptomatic patients; and group II, 14 asymptomatic patients). A histological analysis and inflammatory cell quantification of each excised carotid plaque was made. Nine carotid arteries were removed from human cadavers that were not preselected for carotid artery disease. These specimens were used as a control tissue without any macroscopic signs of atherosclerotic plaques. Results Fifty eight percent of all carotid plaques were classified as complex plaque with possible surface defect, hemorrhage or thrombus. The inflammatory cells concentration did not differ between the two groups. All specimens from human cadavers were classified as preatheroma with extracellular lipid pools. Conclusion Asymptomatic and symptomatic patients could have the same histological components on their carotid plaques. Fibrotic and calcific plaques could become vulnerable as complex plaques with surface defect, hemorrhage and thrombus could remain silent. Asymptomatic carotid stenosis should be followed close with no invasive diagnostic methods and clinical evaluation.

Development and validation of a Xanthomonas axonopodis pv. citri DNA microarray platform (XACarray) generated from the shotgun libraries previously used in the sequencing of this bacterial genome

Abstract Background From shotgun libraries used for the genomic sequencing of the phytopathogenic bacterium Xanthomonas axonopodis pv. citri (XAC), clones that were representative of the largest possible number of coding sequences (CDSs) were selected to create a DNA microarray platform on glass slides (XACarray). The creation of the XACarray allowed for the establishment of a tool that is capable of providing data for the analysis of global genome expression in this organism. Findings The inserts from the selected clones were amplified by PCR with the universal oligonucleotide primers M13R and M13F. The obtained products were purified and fixed in duplicate on glass slides specific for use in DNA microarrays. The number of spots on the microarray totaled 6,144 and included 768 positive controls and 624 negative controls per slide. Validation of the platform was performed through hybridization of total DNA probes from XAC labeled with different fluorophores, Cy3 and Cy5. In this validation assay, 86% of all PCR products fixed on the glass slides were confirmed to present a hybridization signal greater than twice the standard deviation of the deviation of the global median signal-to-noise ration. Conclusions Our validation of the XACArray platform using DNA-DNA hybridization revealed that it can be used to evaluate the expression of 2,365 individual CDSs from all major functional categories, which corresponds to 52.7% of the annotated CDSs of the XAC genome. As a proof of concept, we used this platform in a previously work to verify the absence of genomic regions that could not be detected by sequencing in related strains of Xanthomonas.

Oral antibacterial effect of chlorhexidine treatments and professional prophylaxis in children

Aim: The primary aim of this longitudinal study was to evaluate additional effects of 4-week chlorhexidine digluconate (CHX) gel treatments to control Aggregatibacter actinomycetemcomitans counts in children after professional dental prophylaxis. Porphyromonas gingivalis and Streptococcus mutans counts were also determined to evaluate the secondary effects of anti-plaque treatments on microbial shifts. Methods: Twenty-six children with A. actinomycetemcomitans counts >4 log10/ mL of saliva and/or Quigley-Hein plaque index >3.0 were enrolled in this study. Patients were randomly assigned to groups GI (placebo gel), GII (0.5% CHX gel), GIII (1% CHX gel), and GIV (2% CHX gel). Four sessions of treatment were performed during 4 weeks after a session of professional dental prophylaxis. Real-Time polymerase chain reaction (PCR) was used to determine viable microorganism counts in non-stimulated whole saliva samples collected at baseline, one week, one month and three months after interruption of treatments. Results: A reduction of all bacterial counts was detected after the 3-month follow-up in all groups. Lower counts of P. gingivalis were achieved from 1 week on after treatments. The 2% CHX concentration seemed to contribute to lower A. actinomycetemcomitans levels and increase S. mutans levels. Conclusions: Professional dental prophylaxis was effective to control salivary levels of A. actinomycetemcomitans, P. gingivalis and S. mutans. Additional antimicrobial effects, however, were not observed by the combination of professional dental prophylaxis and 4-week chlorhexidine gel treatments.