The genetic diversity and phenotypic characterisation of Streptococcus agalactiae isolates from Rio de Janeiro, Brazil

Streptococcus agalactiae isolates are more common among pregnant women, neonates and nonpregnant adults with underlying diseases compared to other demographic groups. In this study, we evaluate the genetic and phenotypic diversity in S. agalactiae strains from Rio de Janeiro (RJ) that were isolated from asymptomatic carriers. We analysed these S. agalactiae strains using pulsed-field gel electrophoresis (PFGE), serotyping and antimicrobial susceptibility testing, as well as by determining the macrolide resistance phenotype, and detecting the presence of the ermA/B, mefA/E and lnuB genes. The serotypes Ia, II, III and V were the most prevalent serotypes observed. The 60 strains analysed were susceptible to penicillin, vancomycin and levofloxacin. Resistance to clindamycin, chloramphenicol, erythromycin, rifampin and tetracycline was observed. Among the erythromycin and/or clindamycin resistant strains, the ermA, ermB and mefA/E genes were detected and the constitutive macrolides, lincosamides and streptogramin B-type resistance was the most prevalent phenotype observed. The lnuB gene was not detected in any of the strains studied. We found 56 PFGE electrophoretic profiles and only 22 of them were allocated in polymorphism patterns. This work presents data on the genetic diversity and prevalent capsular serotypes among RJ isolates. Approximately 85% of these strains came from pregnant women; therefore, these data may be helpful in developing future prophylaxis and treatment strategies for neonatal syndromes in RJ.     

Alanyl-glutamine promotes intestinal epithelial cell homeostasis in vitro and in a murine model of weanling undernutrition

Alanyl-glutamine (Ala-Gln) has recently been shown to enhance catch-up growth and gut integrity in undernourished children from Northeast Brazil. We hypothesized that the intestinal epithelial effects of Ala-Gln in malnourished weanling mice and mouse small intestinal epithelial (MSIE) cells would include modulation of barrier function, proliferation, and apoptosis. Dams of 10-day-old suckling C57BL/6 pups were randomized to a standard diet or an isocaloric Northeast Brazil "regional basic diet," moderately deficient in protein, fat, and minerals. Upon weaning to their dam's diet on day of life 21, pups were randomized to Ala-Gln solution or water. At 6 wk of age, mice were killed, and jejunal tissue was collected for morphology, immunohistochemistry, and Ussing chamber analysis of transmucosal resistance and permeability. Proliferation of MSIE cells in the presence or absence of Ala-Gln was measured by MTS and bromodeoxyuridine assays. MSIE apoptosis was assessed by annexin and 7-amino-actinomycin D staining. Pups of regional basic diet-fed dams exhibited failure to thrive. Jejunal specimens from undernourished weanlings showed decreased villous height and crypt depth, decreased transmucosal resistance, increased permeability to FITC-dextran, increased claudin-3 expression, and decreased epithelial proliferation and increased epithelial apoptosis (as measured by bromodeoxyuridine and cleaved caspase-3 staining, respectively). Undernourished weanlings supplemented with Ala-Gln showed improvements in weight velocity, villous height, crypt depth, transmucosal resistance, and epithelial proliferation/apoptosis compared with unsupplemented controls. Similarly, Ala-Gln increased proliferation and reduced apoptosis in MSIE cells. In summary, Ala-Gln promotes intestinal epithelial homeostasis in a mouse model of malnutrition-associated enteropathy, mimicking key features of the human disease.     

Effect of the commercial extract of the brown alga Ascophyllum nodosum Mont. on Kappaphycus alvarezii (Doty) Doty ex P.C. Silva in situ submitted to lethal temperatures

Kappaphycus alvarezii is being introduced in several countries and in some of them there is a need to adapt this cultivation to periods with lethal temperatures, such as the 16–18 °C that occurs in the winter in southern Brazil. Moreover, there is the need to maintain the seedlings during this lethal temperature period. Considering the promising results obtained with the commercial powder extract of Ascophyllum nodosum (Acadian marine plant extract powder—AMPEP) treatment in the cultivation of K. alvarezii in vitro and in the sea allowing more resistance to epiphytes and increasing the growth rate and carrageenan yield, it was hypothesized that seedlings previously subjected to an AMPEP treatment could be more resilient to lethal temperatures. The daily growth rate and carrageenan yield and gel quality (gel strength and gel viscosity) of K. alvarezii in vitro previously treated with AMPEP were analyzed under temperature stress. The daily growth rates and the gel strengths of the AMPEP-treated samples were increased. In spite of the lower carrageenan yield and lower gel viscosity, the values were within the ones accepted by the carrageenan industry. Thus, the treatment of the seedlings of the K. alvarezii with AMPEP solution can be used as an alternative to lower temperature effects on crops as a preventive action for the cultivation of the seedlings in tanks and in the sea in periods of low temperatures at sea.     

Soil seed bank in a patchy vegetation of coastal sandy plains in southeastern Brazil

Large seed banks have been found in tropical dry forests and also in habitats with high seasonality in rainfall. However, patchily structured vegetation could induce great spatial variation in the seed bank. We characterized the seed bank in a patchy vegetation of restinga, a common type of coastal vegetation found in the Atlantic forest biome. We also evaluated whether there is any spatial variation between the litter and soil layer, bare sand, and the edge and center of vegetation patches with distinct species dominance. We found 104 seeds/m² in the seed bank using a 5‐cm‐depth sampling. Seven out of 16 species found in the restinga seed bank germinated; two of these were found in the early stages of vegetation patches. We found a higher number of seeds at the edge than in the center of vegetation patches. However, there were no significant differences in the number of seeds in the seed bank between the litter and soil layer, and between vegetation patches with distinct species dominance. Bare sandy soils had lower seed bank densities than vegetation patches. A small seed bank size might be explained by the low proportion of seeds from herbaceous and woody species, which are pioneers in the Atlantic forest. However, seed bank might play an important role in the early stages of the successional process, due to the occurrence of the few species that are able to colonize new young vegetation patches.     

Presence of fimH,mrkD, and irp2 Virulence Genes in KPC-2-Producing Klebsiella pneumoniae Isolates in Recife-PE,Brazil

Klebsiella pneumoniae strains can produce different virulence factors, such as fimbrial adhesins and siderophores, which are important in the colonization and development of the infection. The aims of this study were to determine the occurrence of fimH, mrkD, and irp2 virulence genes in 22 KPC-2-producing K. pneumoniae isolates as well as 22 not producing-KPC isolates, from patients from different hospitals in Recife-PE, Brazil, and also to analyze the clonal relationship of the isolates by enterobacterial repetitive intergenic consensus-polymerase chain reaction (ERIC-PCR). The genes were detected by PCR and DNA sequencing. The bla _KPC-2 gene was identified in 22 KPC-positive isolates. On analyzing the antimicrobial susceptibility profile of the isolates, it was detected that polymyxin and amikacin were the antimicrobials of best activity against K. pneumoniae. On the other hand, five isolates exhibited resistance to polymyxin. In the KPC-positive group, was observed a high rate of resistance to cephalosporins, followed by carbapenems. Molecular typing by ERIC-PCR detected 38 genetic profiles, demonstrating a multiclonal spread of the isolates analyzed. It was observed that the virulence genes irp2, mrkD, and fimH were seen to have together a higher frequency in the KPC-positive group. The accumulation of virulence genes of KPC-positive K. pneumoniae isolates, observed in this study, along with the multi-resistance impose significant therapeutic limitations on the treatment of infections caused by K. pneumoniae.     

Volatile metabolomic signature of bladder cancer cell lines based on gas chromatography–mass spectrometry

Introduction

Recent studies provide a convincing support that the presence of cancer cells in the body leads to the alteration of volatile organic compounds (VOCs) emanating from biological samples, particularly of those closely related with tumoral tissues. Thus, a great interest emerged for the study of cancer volatilome and subsequent attempts to confirm VOCs as potential diagnostic biomarkers.

Objectives

The aim of this study was to determine the volatile metabolomic signature of bladder cancer (BC) cell lines and provide an in vitro proof-of-principle that VOCs emanated into the extracellular medium may discriminate BC cells from normal bladder epithelial cells.

Methods

VOCs in the culture media of three BC cell lines (Scaber, J82, 5637) and one normal bladder cell line (SV-HUC-1) were extracted by headspace-solid phase microextraction and analysed by gas chromatography-mass spectrometry (HS-SPME/GC–MS). Two different pH (pH 2 and 7) were used for VOCs extraction to infer the best pH to be used in in vitro metabolomic studies.

Results

Multivariate analysis revealed a panel of volatile metabolites that discriminated cancerous from normal bladder cells, at both pHs, although a higher number of discriminative VOCs was obtained at neutral pH. Most of the altered metabolites were ketones and alkanes, which were generally increased in BC compared to normal cells, and alcohols, which were significantly decreased in BC cells. Among them, three metabolites, namely 2-pentadecanone, dodecanal and γ-dodecalactone (the latter only tentatively identified), stood out as particularly important metabolites and promising volatile biomarkers for BC detection. Furthermore, our results also showed the potential of VOCs in discriminating BC cell lines according to tumour grade and histological subtype.

Conclusions

We demonstrate that a GC–MS metabolomics-based approach for analysis of VOCs is a valuable strategy for identifying new and specific biomarkers that may improve BC diagnosis. Future studies should entail the validation of volatile signature found for BC cell lines in biofluids from BC patients.

     

Valosin‐containing protein VCP/p97 is essential for the intracellular development of Leishmania and its survival under heat stress

Valosin‐containing protein (VCP)/p97/Cdc48 is one of the best‐characterised type II cytosolic AAA+ ATPases most known for their role in ubiquitin‐dependent protein quality control. Here, we provide functional insights into the role of the Leishmania VCP/p97 homologue (LiVCP) in the parasite intracellular development. We demonstrate that although LiVCP is an essential gene, Leishmania infantum promastigotes can grow with less VCP. In contrast, growth of axenic and intracellular amastigotes is dramatically affected upon decreased LiVCP levels in heterozygous and temperature sensitive (ts) LiVCP mutants or the expression of dominant negative mutants known to specifically target the second conserved VCP ATPase domain, a major contributor of the VCP overall ATPase activity. Interestingly, these VCP mutants are also unable to survive heat stress, and a ts VCP mutant is defective in amastigote growth. Consistent with LiVCP's essential function in amastigotes, LiVCP messenger ribonucleic acid undergoes 3'Untranslated Region (UTR)‐mediated developmental regulation, resulting in higher VCP expression in amastigotes. Furthermore, we show that parasite mutant lines expressing lower VCP levels or dominant negative VCP forms exhibit high accumulation of polyubiquitinated proteins and increased sensitivity to proteotoxic stress, supporting the ubiquitin‐selective chaperone function of LiVCP. Together, these results emphasise the crucial role LiVCP plays under heat stress and during the parasite intracellular development.     

Rapid selection and characterization of Cry1F resistance in a Brazilian strain of fall armyworm

Transgenic maize (Zea mays L., Poaceae) event TC1507, producing the Cry1F protein of Bacillus thuringiensis Berliner, has been used for management of the fall armyworm, Spodoptera frugiperda (JE Smith) (Lepidoptera: Noctuidae), in Brazil since 2009. A strain of S. frugiperda, obtained from field collections of larvae in TC1507 maize in Minas Gerais state in 2010, was selected in the laboratory for resistance to Cry1F using leaves of TC1507 maize in two selection regimes. Continuous exposure of larvae to Cry1F was more effective than exposure for 6, 8, and 10 days in the selection of resistant S. frugiperda individuals. With only four generations of laboratory selection, a strain with high levels of resistance to Cry1F was obtained, as indicated by the survival of insects reared on leaves of TC1507 maize plants and by the more than 300‐fold resistance level measured in bioassays with the purified Cry1F protein. Importantly, reciprocal crosses between control and the Cry1F‐selected strains revealed that the resistance is autosomal and incompletely recessive, and the response obtained in the backcross of the F₁ generation with the resistant strain was consistent with simple monogenic inheritance. Additionally, there were no apparent fitness costs associated with resistance either for survival or larval growth on non‐Bt maize leaves. Our findings provide experimental evidence for rapid evolution of Cry1F resistance in S. frugiperda in the laboratory and further reinforce the potential of this species to evolve field resistance to the TC1507 maize as previously reported. The resistant strain isolated in this study provides an opportunity to estimate the resistance allele frequency in the field and to determine the biochemical and molecular basis of the resistance, which should provide further information to assist in the resistance management of S. frugiperda on transgenic maize producing B. thuringiensis proteins.     

Chemical Analysis of Pottery Demonstrates Prehistoric Origin for High-Altitude Alpine Dairying

The European high Alps are internationally renowned for their dairy produce, which are of huge cultural and economic significance to the region. Although the recent history of alpine dairying has been well studied, virtually nothing is known regarding the origins of this practice. This is due to poor preservation of high altitude archaeological sites and the ephemeral nature of transhumance economic practices. Archaeologists have suggested that stone structures that appear around 3,000 years ago are associated with more intense seasonal occupation of the high Alps and perhaps the establishment of new economic strategies. Here, we report on organic residue analysis of small fragments of pottery sherds that are occasionally preserved both at these sites and earlier prehistoric rock-shelters. Based mainly on isotopic criteria, dairy lipids could only be identified on ceramics from the stone structures, which date to the Iron Age (ca. 3,000–2,500 BP), providing the earliest evidence of this practice in the high Alps. Dairy production in such a marginal environment implies a high degree of risk even by today’s standards. We postulate that this practice was driven by population increase and climate deterioration that put pressure on lowland agropastoral systems and the establishment of more extensive trade networks, leading to greater demand for highly nutritious and transportable dairy products.