Toxicity Assessment of New Ag-ZnO/AgO Nanocomposites: An In Vitro and In Vivo Approach.

Zinc oxide nanoparticles (ZnO NPs) are metal oxide nanomaterials, which are important for several applications: antibacterial, anthelmintic, antiprotozoal and antitumoral, among others. These applications are mainly related to the ability to spontaneously produce and induce the production of reactive oxygen species that are important components for the destruction of pathogens and tumor cells. While trying to potentiate ZnO NPs, studies have associated these NPs with silver oxide (AgO) or silver (Ag) NPs. It has already been reported that this combination (Ag-ZnO/AgO NPs) is able to enhance the microbicidal potential. Although possessing much potential for several purposes, it is important to evaluate whether this association also poses the risk of toxicity to cells and experimental models. Therefore, this work aimed to evaluate the toxicity of various Ag-ZnO/AgO NP nanocomposites, in vitro and in vivo. Accordingly, ZnO nanocrystals and nanocomposites with various concentrations of AgO (ZnO:5Ag, ZnO:9Ag or ZnO:11Ag) were used in different cytotoxicity models: Galleria mellonella (G. mellonella), cell lines (VERO and RAW 264.7) and C57BL/6 mice. In the G. mellonella model, four concentrations were used in a single dose, with subsequent evaluation of mortality. In the case of cells, serial concentrations starting at 125 µg/mL were used, with subsequent cytotoxicity assessment. Based on the safe doses obtained in G. mellonella and cell models, the best doses were used in mice, with subsequent evaluations of weight, biochemistry as also renal and liver histopathology. It was observed that the toxicity, although low, of the nanocomposites was dependent upon the concentration of AgO used in association with ZnO NPs, both in vitro and in vivo.

The Role of Trypan Blue as a Conventional and Fluorescent Dye for the Diagnosis of Superficial Mycoses by Potassium Hydroxide (KOH) Testing.

Direct microscopic examination of samples using potassium hydroxide (KOH) is a fast, simple, and inexpensive method to confirm clinical suspicion of superficial mycosis. However, the lack of color contrast in this test makes it difficult to separate any fungal structures from artifacts. The sensitivity of the KOH mount technique may be enhanced using both fluorochromes and conventional stains that highlight the fungal structures when observed under fluorescence microscopy and bright-field, respectively. Here we study the possibility of using Trypan Blue (TB), an azo dye which is often used as a live/dead marker, in the diagnosis of superficial mycoses by KOH testing. TB at 0.01% displayed a fluorescent staining pattern similar to that of Calcofluor White (CFW), the conventional cell wall fluorophore. Furthermore, by adjusting the TB concentration to 0.1-0.3%, in addition to maintaining the fluorescent staining pattern, the fungal elements were stained in blue under bright-field microscopy. Thus, we demonstrate for the first time that TB has the unique property as a fungal stain that can be used in both bright-field and fluorescence microscopy for diagnosis of superficial mycoses by direct microscopic examination.

Microbiology of street food: understanding risks to improve safety.

Street foods play important socioeconomic and cultural roles and are popular worldwide. In addition to providing convenient and low-cost meals for urban populations, street food offers an essential source of income for vendors, especially women, and it can reflect traditional local culture, which is an important attraction for tourists. Despite these benefits, the microbiological safety of street food has become a worldwide concern because it is often prepared and sold under inadequate safety conditions, without legal control and sanitary surveillance. Consequently, high counts of fecal indicator bacteria and several foodborne pathogens have been detected in street foods. This review provides insight into the microbiology of street food, focus on the associated microbiological safety aspects and main pathogens, and the global status of this important economic activity. Furthermore, the need to apply molecular detection rather than traditional culture-based methods is discussed to better understand the actual risks of microbial infection associated with street foods. Recognition is always the first step toward addressing a problem.

Natural Occurring Phenolic Derivatives from Mauritia flexuosa (Buriti) Stems and Their Potential Antibacterial Activity against Methicillin-Resistant Staphylococcus aureus (MRSA).

Mauritia flexuosa Linnaeus filius (buriti or aguage; Arecaceae) is a palm used by traditional medicine in Brazil to treat dysentery and diarrhea. Our group showed that the soluble dichloromethane (CH2 Cl2 ) fraction from EtOH extract from M. flexuosa stems inhibited the growth of methicillin-susceptible Staphylococcus aureus (MSSA) and methicillin-resistant S. aureus (MRSA) and it is rich in phenolic compounds. This study aimed to isolate new phenolic compounds from CH2 Cl2 fraction from M. flexuosa stems with in vitro antibacterial activity. The crude CH2 Cl2 fraction was fractionated by gel permeation chromatography (GPC) followed by semi-preparative RP-HPLC. The antibacterial activity was evaluated using the broth microdilution method against MSSA (ATCC 29213) and MRSA (clinical isolate 155). All compounds were also tested against Gram-negative (Escherichia coli; ATCC 35218) bacteria and two fungi species (Candida albicans; ATCC 14053 and Trichophyton rubrum; ATCC MYA 4438). The chemical structures of isolated compounds were determined by analysis and comparison with literature data of their NMR and HRMS spectra and optical activity. The chemical investigation yielded seven aromatic compounds, of which four, (2S,15S)-2,15-dimethyl-2,15-dioxa-1,8(1,4)-dibenzenacyclotetradecaphane (1), (2S,5S)-1-(4-hydroxyphenyl)hexane-2,5-diol (3), bruguierol E (4), and buritin (5) were previously unreported and three are known compounds identified as 6-(4'-hydroxyphenyl) hexan-2-one (2), (+)-(2R,3R)-dihydrokaempferol (6), and (+)-(2R)-naringenin (7). Compounds 1 and 7 showed antibacterial activity against MRSA and MSSA with minimum inhibitory concentrations (MICs) of between 62.5 and 31.3 µg/mL, respectively. Our preliminary findings support that CH2 Cl2 fraction from buriti, a typical species of flooded areas of Brazilian savanna, and its aromatic phenolic compounds are active against MSSA and MRSA contributing with understanding about the traditional use of this species.

Ocorrência de enteroparasitos e coliformes termotolerantes nas mãos de manipuladores de alimentos de um hospital de ensino / Occurrence of enteroparasites and thermotolerant coliforms in the hands of food handlers of a teaching hospital

Resumo Introdução Manipuladores de alimentos que trabalham em hospitais frequentemente fornecem alimentos a indivíduos com imunidade reduzida, os quais são mais suscetíveis à aquisição de doenças infecciosas de origem alimentar. Objetivo O objetivo deste trabalho foi avaliar a presença de enteroparasitos e coliformes termotolerantes nas mãos dos manipuladores de alimentos de um hospital de ensino de Uberaba, Minas Gerais. Método Foram analisadas amostras das mãos de 44 manipuladores de alimentos de um hospital de ensino. Os coliformes termotolerantes foram contados pelo método do Número Mais Provável, a partir de amostra obtida por esfregaço. O conteúdo subungueal foi submetido à análise parasitológica, observado em microscópio. Resultados 15,9% (7/44) das amostras foram positivas para ocorrência de coliformes termotolerantes e 9,1% (4/44) dos conteúdos subungueais foram positivos para protozoários intestinais (3 Blastocystis spp. e 1 Giardia spp.). Conclusão Os resultados indicam contaminação das mãos dos manipuladores de alimentos e predisposição à veiculação de agentes patogênicos por essa via, ressaltando a importância de uma conscientização dos manipuladores de alimento quanto à higiene pessoal e dos alimentos, bem como a utilização de medidas preventivas.

Abstract Background Inappropriate food handling is the main cause of foodborne illnesses. Food handlers perform important functions to preserve the hygiene food that can be a source of transmission of various pathogens. Objective The aim of this study was to evaluate the presence of intestinal parasites and microorganisms indicators of fecal contamination (thermotolerant coliforms) in the hands of food handlers of a teaching hospital in Uberaba, Minas Gerais state, Brazil. Method Samples from the hands of 44 food handlers of a teaching hospital were analyzed. Thermotolerant coliforms were counted by the Most Probable Number method using samples obtained by smearing. The subungual content was subjected to parasitological analysis under microscopy. Results 15.9% (7/44) of the samples were positive for the presence of thermotolerant coliforms and 9.1% (4/44) of the subungual contents were positive for intestinal protozoa (3 Blastocystis spp. and 1 Giardia spp.). Conclusion Results indicate contamination of the food handlers' hands and predisposition to transmission of pathogens by this route, emphasizing the importance to make food handlers aware of personal and food hygiene, as well as to apply preventive actions.

Comida de rua: avaliação das condições higiênico-sanitárias de manipuladores de alimentos / Street food: analysis of hygienic and sanitary conditions of food handlers

Resumo O consumo de alimentos de rua é uma alternativa econômica e prática para a população. No entanto, as preocupações sobre a segurança destes alimentos são cada vez maiores. Assim, este estudo teve como objetivo analisar as condições higiênico-sanitárias do comércio ambulante de alimentos em Uberaba-MG. Para isso, além da aplicação de um questionário em 30 pontos de comércio ambulante, foram realizadas também análises microbiológicas nas mãos dos manipuladores e nas superfícies dos recipientes que acondicionam os alimentos. Os achados mostraram baixa adequação dos ambulantes em relação às condições de higiene de mãos e superfícies em contato com os alimentos. Nas superfícies, os resultados para microrganismos aeróbios mesófilos mostraram que apenas 23,3% apresentavam condições satisfatórias de limpeza. O nível de contaminação das mãos por coliformes termotolerantes e/ou Staphylococcus coagulase positiva foi insatisfatório em aproximadamente 47% dos manipuladores. Conclui-se que as condições higiênico-sanitárias de vendedores ambulantes de alimentos em Uberaba são deficientes. Portanto, é urgentemente necessária a adoção de programas eficazes e permanentes de treinamento em segurança alimentar para os manipuladores de alimentos.

Abstract The consumption of street food is an economical and practical alternative for the population. However, concerns regarding the safety of these foods are increasing. Thus, this study aimed to analyze the hygienic and sanitary conditions of the street food trade in Uberaba in the State of Minas Gerais. For this purpose, 30 street vending sites were evaluated using a structured questionnaire and microbiological analysis of food handlers’ hands and food contact recipients and surfaces. The findings showed low adequacy of street food regarding hand hygiene and food contact surfaces. With respect to surfaces, the results for mesophilic aerobic microorganisms revealed that only 23.3% had satisfactory sanitary conditions. The level of contamination of hands by fecal thermotolerant coliforms and/or Coagulase positive Staphylococci was unsatisfactory in approximately 47% of food handlers. The conclusion drawn is that the hygienic and sanitary conditions of food handlers in Uberaba are poor. It is therefore necessary to adopt effective and permanent training programs on food safety for food handlers urgently.

Species Distribution, Virulence Factors, and Antifungal Susceptibility Among Candida parapsilosis Complex Isolates Recovered from Clinical Specimens.

The Candida parapsilosis complex has emerged as an important fungal pathogen. In spite of this, relatively little is known about its characteristics. Thus, the purposes of this study were (1) to determine by BanI-RFLP-assay the occurrence of C. parapsilosis complex species among 81 clinical isolates primarily identified as C. parapsilosis; (2) to evaluate their in vitro production of virulence factors; and (3) to compare their susceptibility profiles, grown as planktonic cells and biofilms, against amphotericin B, fluconazole, voriconazole, and caspofungin by following the Clinical and Laboratory Standards Institute (CLSI) guidelines. Seventy-seven isolates (95%) were identified as C. parapsilosis sensu stricto, 2 (2.5%) as C. orthopsilosis, and 2 (2.5%) as C. metapsilosis. Protease activity was detected in 29 (37.7%) isolates of C. parapsilosis sensu stricto, whereas only 7 (9.1%) exhibited phospholipase activity. None of the C. metapsilosis or C. orthopsilosis was able to produce protease or phospholipase. Biofilm production was detected in 35 (43.2%) isolates, among which 33 were C. parapsilosis sensu stricto and 2 were C. orthopsilosis. Antifungal resistance was uncommon; only one C. metapsilosis was fluconazole resistant. However, biofilm-producing isolates showed a marked resistance to all antifungal agents tested, particularly to voriconazole. This knowledge could be of clinical relevance for guiding therapeutic decisions.

[Street food: analysis of hygienic and sanitary conditions of food handlers]. / Comida de rua: avaliação das condições higiênico-sanitárias de manipuladores de alimentos.

The consumption of street food is an economical and practical alternative for the population. However, concerns regarding the safety of these foods are increasing. Thus, this study aimed to analyze the hygienic and sanitary conditions of the street food trade in Uberaba in the State of Minas Gerais. For this purpose, 30 street vending sites were evaluated using a structured questionnaire and microbiological analysis of food handlers' hands and food contact recipients and surfaces. The findings showed low adequacy of street food regarding hand hygiene and food contact surfaces. With respect to surfaces, the results for mesophilic aerobic microorganisms revealed that only 23.3% had satisfactory sanitary conditions. The level of contamination of hands by fecal thermotolerant coliforms and/or Coagulase positive Staphylococci was unsatisfactory in approximately 47% of food handlers. The conclusion drawn is that the hygienic and sanitary conditions of food handlers in Uberaba are poor. It is therefore necessary to adopt effective and permanent training programs on food safety for food handlers urgently.

Antifungal activity of extracts from Piper aduncum leaves prepared by different solvents and extraction techniques against dermatophytes Trichophyton rubrum and Trichophyton interdigitale

The effects of different solvents and extraction techniques upon the phytochemical profile and anti-Trichophyton activity of extracts from Piper aduncum leaves were evaluated. Extract done by maceration method with ethanol has higher content of sesquiterpenes and antifungal activity. This extract may be useful as an alternative treatment for dermatophytosis.

Susceptibility profile of clinical and environmental isolates of Cryptococcus neoformans and Cryptococcus gattii in Uberaba, Minas Gerais, Brazil.

Cryptococcus neoformans and C. gattii are the etiologic agents of cryptococcosis, a life-threatening disease in both immunocompromised and immunocompetent hosts. Antifungal resistance has been evaluated using different methods, breakpoints, and sizes of test populations and it is an emerging as a significant issue worldwide. A total of 176 (95 clinical and 81 environmental) C. neoformans and eight clinical C. gattii isolates were evaluated to determine the minimal inhibitory concentration (MIC) according to the Clinical and Laboratory Standards Institute method. A total of 10.5% of the C. neoformans clinical isolates were resistant to amphotericin B (AMB), and 6.2% of the environmental isolates were resistant to fluconazole (FLZ). Environmental and clinical isolates presented epidemiologic cut-off values (ECVs) of 64 and 16 to FLZ and 1 and 2 to AMB, respectively. All of the C. gattii isolates showed high susceptibility to most drugs evaluated. Clinical isolates had lower susceptibility than environmental isolates to AMB and itraconazole whereas environmental isolates had lower susceptibility than the clinical isolates to FLZ, voriconazole, and ketoconazole. However, no difference was found in the susceptibility of the two species. The MICs and ECVs to antifungals can help to select the best therapeutic option for tracking epidemiological resistance among clinical and environmental isolates of Cryptococcus spp. around the world.

Antifungal activity of extracts from Piper aduncum leaves prepared by different solvents and extraction techniques against dermatophytes Trichophyton rubrum and Trichophyton interdigitale.

The effects of different solvents and extraction techniques upon the phytochemical profile and anti-Trichophyton activity of extracts from Piper aduncum leaves were evaluated. Extract done by maceration method with ethanol has higher content of sesquiterpenes and antifungal activity. This extract may be useful as an alternative treatment for dermatophytosis.

RAPD analysis with the primer L15996 of Brazilian clinical and environmental Cryptococcus neoformans isolates.

Different methods have been used to perform the molecular characterization of Cryptococcus neoformans. Among them, RAPD analysis is able to separate isolates of the same species and genotypes. This study aimed to evaluate clinical and environmental C. neoformans isolates from Minas Gerais, Brazil by RAPD and correlate the genetic profiles with the ones obtained by URA5-RFLP, virulence factors and antifungal susceptibility patterns. Forty-five environmental (31 from areas surrounding hospital and 14 from captive bird droppings from pet-shops) and 29 clinical C. neoformans isolates were evaluated. Antifungal susceptibility tests (Clinical and Laboratory Standards Institute), URA5-RFLP analysis and the assessment of virulence factors were performed according to their original descriptions. RAPD profiles were obtained using the L15996 primer (5'-CTCCACCATTAGCACCCAAAGC-3'). RAPD analysis generated two to 20 bands for all studied isolates. The isolates presented similarities ranging from 10.8 to 100.0%. Considering a minimum identity score of 50%, four clusters were formed. Cluster I contained 10 pet-shops bird dropping isolates, cluster II contained 22 clinical isolates most of them recovered from cerebrospinal fluid, cluster III contained 14 isolates from hospital surroundings and cluster IV contained 12 environmental isolates most from hospital surroundings. Fourteen isolates were not grouped. The RAPD profiles were clustered according to their source and URA5-RFLP pattern. No correlation between virulence factors or antifungal susceptibility profile with the obtained RAPD profiles was observed.

Antifungal susceptibility, enzymatic activity, PCR-fingerprinting and ITS sequencing of environmental Cryptococcus laurentii isolates from Uberaba, Minas Gerais, Brazil.

Cryptococcus laurentii has been classically considered a saprophytic species, although several cases of human infection have been already reported. This study aimed to evaluate the phospholipase, proteinase and hemolysins activity, the antifungal susceptibility profile, the genetic variability by M13 and (GACA)(4) fingerprinting and the internal transcribe spacer (ITS) sequencing of 38 C. laurentii isolates recovered from captive bird droppings and surrounding hospital areas. All of them exhibited phospholipase activity, while the hemolytic activity was evidenced in 34 (89.4%) isolates. None of them exhibited proteinase activity. Twenty-seven isolates (71.1%) presented susceptibility dose dependent to fluconazole. Most isolates (94.7%) were susceptible to voriconazole, while one (2.65%) was resistant to this drug. Twenty-one (55.3%) isolates showed reduced susceptibility to itraconazole while nine (23.7%) were resistant. Three (7.9%) and five (13.1%) isolates exhibited resistance to ketoconazole and amphotericin B, respectively. Most C. laurentii fingerprinting obtained with M13 and (GACA)(4) showed high heterogeneity. By using the two primers, seven (18.4%) isolates grouped as A (CL2, CL7, and CL8), B (CL35, CL38) and C (CL29, CL30) with 100% similarity. Different from most variable surrounding hospital isolates, all but one of the pet shops strains clustered with the two primers, although they had been recovered from different neighborhoods. All isolates were identified as C. laurentii phylogenetic group I by ITS sequencing. Thus, the presence of virulence factors, a decreased antifungal susceptibility and a heterogeneous molecular pattern of the C. laurentii isolates here described suggests this species can be a potential pathogen in the context of the immunocompromised population.

Synthesis, antimalarial activity, and intracellular targets of MEFAS, a new hybrid compound derived from mefloquine and artesunate.

A new synthetic antimalarial drug, a salt derived from two antimalarial molecules, mefloquine (MQ) and artesunate (AS), here named MEFAS, has been tested for its pharmacological activity. Combinations of AS plus MQ hydrochloride are currently being used in areas with drug-resistant Plasmodium falciparum parasites; although AS clears parasitemia in shorter time periods than any other antimalarial drug, it does not cure infected patients; in addition, MQ causes side effects and is rather expensive, important problems considering that malaria affects mostly populations in poor countries. Here, we show that MEFAS is more effective than the combination of AS and MQ, tested in parallel at different mass proportions, against P. falciparum (chloroquine-resistant clone W2 and chloroquine-sensitive clone 3D7) in vitro and in mice infected with Plasmodium berghei, promoting cure of this infection. MEFAS tested against HepG2 hepatoma cells exhibited lower toxicity than the antimalarials AS and MQ alone or combined. Possible targets of MEFAS have been studied by confocal microscopy using fluorescent probes (Fluo-4 AM and BCECF-AM) in P. falciparum synchronous culture of W2-infected red blood cells. Dynamic images show that MEFAS exhibited intracellular action increasing cytoplasmic Ca(2+) at 1.0 ng/ml. This effect was also observed in the presence of tapsigargin, an inhibitor of SERCA, suggesting an intracellular target distinct from the endoplasmic reticulum. Trophozoites loaded with BCECF-AM, when treated with MEFAS, were still able to mobilize protons from the digestive vacuole (DV), altering the pH gradient. However, in the presence of bafilomycin A1, an inhibitor of the H(+) pump from acidic compartments of eukaryotic cells, MEFAS had no action on the DV. In conclusion, the endoplasmic reticulum and DV are intracellular targets for MEFAS in Plasmodium sp., suggesting two modes of action of this new salt. Our data support MEFAS as a candidate for treating human malaria.

Enhanced activity of mefloquine and artesunic acid against Plasmodium falciparum in vitro and P. berghei in mice by combination with ciprofloxacin.

The antimalarial activity of combinations of mefloquine or artesunic acid with ciprofloxacin and other synthetic fluoroquinolone was tested in vitro against Plasmodium falciparum using a strain (BHz26/86) partially resistant to chloroquine and a resistant clone (W2); both are sensitive to mefloquine. Inhibition of parasite growth was measured in relation to controls without drugs, either by counting parasitemia in Giemsa-stained blood smears or by measuring the reduction in [(3)H]-hypoxanthine uptake. Combinations containing artesunic acid or mefloquine with ciprofloxacin had significant in vitro activity, inhibiting by more than 90% of the growth of both strains of P. falciparum at doses significantly lower than those of the antimalarials alone. When tested in mice inoculated with P. berghei chloroquine-sensitive parasites (NK65 strain), ciprofloxacin was inactive, whereas mefloquine and artesunic acid were active (IC(50)=2.5 and 4.2 mg/kg, respectively); combinations containing mefloquine at an equivalent dose of 0.5 mg/kg reduced parasitemia by 59% and artesunic acid activity was also improved by ciprofloxacin. Our data support the idea that ciprofloxacin in combination with antimalarials may be useful in the treatment of chloroquine-resistant human malaria, allowing the use of lower doses of these drugs.