Dynamics of in vitro acquisition of resistance by Candida parapsilosis to different azoles

Candida parapsilosis is a common isolate from clinical fungal infectious episodes. Resistance of C. parapsilosis to azoles has been increasingly reported. To analyse the development of resistance in C. parapsilosis , four azole-susceptible clinical strains and one American Type Culture Collection type strain were cultured in the presence of fluconazole, voriconazole and posaconazole at different concentrations. The isolates developed variable degrees of azole resistance according to the antifungal used. Fluconazole was the fastest inducer while posaconazole was the slowest. Fluconazole and voriconazole induced resistance to themselves and each other, but not to posaconazole. Posaconazole induced resistance to all azoles. Developed resistance was stable; it could be confirmed after 30 days of subculture in drug-free medium. Azole-resistant isolates revealed a homogeneous population structure; the role of azole transporter efflux pumps was minor after evaluation by microdilution and cytometric assays with efflux pump blockers (verapamil, ibuprofen and carbonyl cyanide 3-chloro-phenylhydrazone). We conclude that the rapid development of azole resistance occurs by a mechanism that might involve mutation of genes responsible for ergosterol biosynthesis pathway, stressed by exposure to antifungals.     

Virulence factors in food,clinical and reference Enterococci: A common trait in the genus?

The occurrence of several virulence traits (cytolysin, adhesins and hydrolytic enzymes) was investigated in a collection of 164 enterococci, including food and clinical isolates (from human and veterinary origin), as well as type and reference strains from 20 enterococcal species. Up to fifteen different cyl genotypes were found, as well as silent cyl genes. The occurrence of the cyl operon and haemolytic potential seems to be widespread in the genus. A significant association of this virulent trait with clinical isolates was found (p < 0.05). High levels of incidence were also observed for genes encoding surface adhesins (esp, efaA(fs), efaA(fm)), agg and gelE, irrespectively of species allocation and origin of strains. Although gelE behaves as silent in the majority of the strains, gelatinase activity predominates in clinical isolates, whereas lipase and DNase were mainly detected in food isolates pointing to their minor role as virulence determinants. No hyaluronidase activity was detected for all strains. Numerical hierarchic data analysis grouped the strains in three main clusters, two of them including a total of 50 strains with low number of virulence determinants (from 2 to 7) and the other with 114 strains with a high virulence potential (up to 12 determinants). No statistical association was found between virulence clusters and species allocation (p > 0.10), strongly suggesting that virulence determinants are a common trait in the genus Enterococcus. Clinical strains seem to be significantly associated with high virulence potential, whereas food, commensal and environmental strains harbour fewer virulence determinants (p < 0.01). A high level of relative diversity in virulence patterns was observed (Shannon's index varies from 0.95 to 1.0 among clusters), reinforcing the strain-specific nature of the association of virulence factors. Although a low risk seems to be associated with the use of enterococci in long-established artisanal cheeses, screening of virulence traits and their cross-synergies must be performed, particularly for commercial starters, probiotic strains and products to be used by high risk population groups.     

The eukaryotic nucleotide excision repair pathway

Nucleotide excision repair (NER) is the most versatile mechanism of DNA repair, recognizing and dealing with a variety of helix-distorting lesions, such as the UV-induced photoproducts cyclobutane pyrimidine dimers (CPDs) and pyrimidine 6-4 pyrimidone photoproducts (6-4 PPs). In this review, we describe the main protein players and the different sequential steps of the eukaryotic NER mechanism in human cells, from lesion recognition to damage removal and DNA synthesis. Studies on the dynamics of protein access to the damaged site, and the kinetics of lesion removal contribute to the knowledge of how the cells respond to genetic insult. DNA lesions as well as NER factors themselves are also implicated in changes in cell metabolism, influencing cell cycle progression or arrest, apoptosis and genetic instability. These changes are related to increased mutagenesis and carcinogenesis. Finally, the recent collection of genomic data allows one to recognize the high conservation and the evolution of eukaryotic NER. The distribution of NER orthologues in different organisms, from archaea to the metazoa, displays challenging observations. Some of NER proteins are widespread in nature, probably representing ancient DNA repair proteins, which are candidates to participate in a primitive NER mechanism.     

MicroRNAs: new players in heart failure

MicroRNAs (miRNAs) are a class of non-coding small RNAs representing one of the most exciting areas of modern medical science. miRNAs modulate a large and complex regulatory network of gene expression of the majority of the protein-coding genes. Currently, evidences suggest that miRNAs play a crucial role in the pathogenesis of heart failure. Some miRNAs as miR-1, miR-133 and miR-208a are highly expressed in the heart and strongly associated with the development of cardiac hypertrophy. Recent data indicate that these miRNAs as well as miR-206 change their expression quickly in response to physical activity. The differential regulation of miRNAs in response to exercise suggests a potential value of circulating miRNAs (c-miRNAs) as biomarkers of physiological mediators of the cardiovascular adaptation induced by exercise. Likewise, serum levels of c-miRNAs such as miR-423-5p have been evaluated as potential biomarkers in the diagnosis and prognosis of heart failure. On the other hand, the manipulation of miRNAs levels using techniques such as ‘miR mimics’ and ‘antagomiRs’ is becoming evident the enormous potential of miRNAs as promising therapeutic strategies in heart failure.     

Pollen consumed by the solitary bee Tetrapedia diversipes (Apidae: Tetrapediini) in a tropical agroecosystem

Pollen analysis of the larval food supply is an important tool for identifying the plants that provide the floral resources used by bees. The present study documents the pollen sources consumed by larvae of the solitary bee Tetrapedia diversipes in a tropical agroecosystem. A total of 60 pollen types were recorded with three families being the most important. Euphorbiaceae (60.5%), Malpighiaceae (16.8%) and Asteraceae (12.2%) pollen had the greatest representation in the samples examined. The pollen of Dalechampia dioscoreifolia predominated in the diet of the larvae of T. diversipes (RF?=?56.35%) and indicates the importance of this plant in maintaining populations of this solitary bee.     

Antifungal and physicochemical properties of Ocimum essential oil loaded in poly(lactic acid) nanofibers

Poly(lactic acid) (PLA) nanofibres containing different proportions of the essential oils from Ocimum basilicum L. and Ocimum gratissimum L. were prepared by solution blow spinning method. The essential oils were extracted by hydrodistillation and characterized by gas chromatography. MEV, contact angle, DSC and FTIR were used to characterize the nanofibres. The effect of bioative nanofibres on the growth of the fungus and on the production of ochratoxin A were evaluated using the fumigation test. Linalool, 1·8-cineole and camphor were the principal components of the essential oil from O. basilicum, and eugenol was the principal constituent in the oil from O. gratissimum. An increase in the average diameter of the nanofibres was observed with the addition of the essential oils. The essential oils acted as a plasticizer, resulting in a reduction in the crystallinity of the PLA. The encapsulation of essential oils in PLA nanofibres was verified by FTIR. An effective antifungal and antimicotoxygenic activity against Aspergillus ochraceus and Aspergillus westerdjikiae was observed for the bioative nanofibres. These results confirm the potential of PLA nanofibres containing the essential oils for the control of toxigenic fungi that cause the deterioration of food and are harmful to human health.     

Susceptibility of Trichogramma pretiosum Riley (Hymenoptera: Trichogrammatidae) adults to fungicides used to control apple diseases

This study evaluated the susceptibility under laboratory conditions of Trichogrammapretiosum Riley adults to fungicides recommended by the Integrated Production of Apple (IPA). The bioassays were carried out using the International Organization for Biological Control (IOBC), West Palearctic Regional Section (WPRS) standard protocols. Twelve selected fungicides were studied in the doses (g or ml active ingredient/100 L) captan 1 (0.115), captan 2 (0.120), kresoxim-methyl (0.010), sulphur 1 (AG) (0.480), sulphur 2 (0.480), folpet (0.105), mancozeb (0.160), pyraclostrobin (0.010), tebuconazole (0.010), tetraconazole (0.005), thiophanate-methyl (0.050) and triforine (0.024). Distilled water was used as the blank treatment and the insecticide triclorfon (0.150) as a positive control. The parasitoids were exposed to dry residues applied on glass plates. The reduction in the capacity of parasitism was used to measure the effect of the chemical in comparison to the blank treatment. Each treatment was replicated four times. The results allowed us to classify the fungicides tested in four categories: 1, harmless (< 30%); 2, slightly harmful (30-79%); 3, moderately harmful (80-99%); and 4, harmful (> 99%). 75% of the tested substances were classified as selective (classes 1 and 2) to the parasitoid. The fungicides captan 1, captan 2, kresoxim-methyl, folpet, pyraclostrobin, tebuconazole, thiophanate-methyl and triforine were harmless; mancozeb was slightly harmful; sulphur 1 (AG) and tetraconazole were moderately harmful and sulphur 2 was harmful. These findings should be taken into account when selecting fungicides to spray apple orchards against fungi diseases to preserve the egg parasitoid T. pretiosum.     

Selection and validation of reference genes for quantitative gene expression analyses in various tissues and seeds at different developmental stages in <Emphasis Type="Italic">Bixa orellana</Emphasis> L.

Bixa orellana L., popularly known as annatto, produces several secondary metabolites of pharmaceutical and industrial interest, including bixin, whose molecular basis of biosynthesis remain to be determined. Gene expression analysis by quantitative real-time PCR (qPCR) is an important tool to advance such knowledge. However, correct interpretation of qPCR data requires the use of suitable reference genes in order to reduce experimental variations. In the present study, we have selected four different candidates for reference genes in B. orellana, coding for 40S ribosomal protein S9 (RPS9), histone H4 (H4), 60S ribosomal protein L38 (RPL38) and 18S ribosomal RNA (18SrRNA). Their expression stabilities in different tissues (e.g. flower buds, flowers, leaves and seeds at different developmental stages) were analyzed using five statistical tools (NormFinder, geNorm, BestKeeper, ΔCt method and RefFinder). The results indicated that RPL38 is the most stable gene in different tissues and stages of seed development and 18SrRNA is the most unstable among the analyzed genes. In order to validate the candidate reference genes, we have analyzed the relative expression of a target gene coding for carotenoid cleavage dioxygenase 1 (CCD1) using the stable RPL38 and the least stable gene, 18SrRNA, for normalization of the qPCR data. The results demonstrated significant differences in the interpretation of the CCD1 gene expression data, depending on the reference gene used, reinforcing the importance of the correct selection of reference genes for normalization.     

Helicobacter pylori antimicrobial resistance in a pediatric population

Background

The increasing prevalence of Helicobacter pylori (H. pylori) antimicrobial resistance, primarily for clarithromycin decreases the success of treatment. The aim of this study is to determine the local pattern of first‐line antimicrobials resistance and the eradication rate.

Material and Methods

Prospective cohort study of H. pylori infected patients (positive histological or cultural exams) treated at Centro Materno‐Infantil do Norte from January of 2013 to October of 2017. Susceptibility to 4 antibiotics: amoxicilin, metronidazole, clarithromycin, and levofloxacin were analyzed by E‐test (phenotypic resistance). The E‐test was chosen because it is simple and cost‐effective for routine susceptibility testing. Point mutations that confer clarithromycin resistance were surveyed (genotypic resistance). Eradication of H. pylori infection was defined by a negative urea breath test or fecal antigen 6‐8 weeks after the end of treatment.

Results

Of a total of 74 H. pylori infected patients, 16 were excluded because they had previous H. pylori treatment or severe systemic disease. Median age of infection cases was 15 years (3‐17 years). Eradication regimen used in all patients combined the use of 3 antibiotics (amoxicillin and metronidazole or clarithromycin) and proton pump inibhitor for 14 days and was tailored according antimicrobial susceptibility. 79.5% of the patients completed the treatment. The resistance rate for metronidazole and clarithromycin was 3.3% and 23.3%, respectively. There was no resistance for amoxicilin and levofloxacin. The rate of genotypic resistance to clarithromycin was 37.2%. The eradication rate was 97.8%.

Conclusions

The authors found a high resistance rate of H. pylori for clarithromycin in this northern portuguese pediatric center. This factor should determine a change in local current treatment, contraindicating the use of clarithromycin as a first‐line treatment for H. pylori infection in children. The high eradication rate maybe explained for the eradication treatment tailored according antimicrobial susceptibility.