ABSTRACT: The aim of the present study was to characterize (phenotypically and genotypically) two strains of Brucella abortus identified as belonging to biovar 4 isolated from cattle in Brazil. The strains were isolated from cervical bursitis from cattle in the states of Pará and Rio Grande do Sul, respectively. In the phenotypic identification, the isolates were positive in CO2 requirement, produced H2S, were resistant to basic fuchsin (20 µg / mL) and sensitive to thionin (20 µg / mL and 40 µg / mL) and presented M surface antigen, but A surface antigen is absent. The isolates were positive in the PCR for the bcsp31 gene (genus-specific) and in the AMOS-enhanced PCR, both isolates showed a band profile consistent with B. abortus biovar 1, 2 or 4. Moreover, both isolates also showed restriction patterns identical to the reference strain when tested by the omp2b PCR-RFLP. In genotyping using Multiple Locus Variable Number of Tandem Repeat (VNTR) Analysis - MLVA (MLVA16), the isolates showed differences in several loci (Bruce42, Bruce19, Bruce04, Bruce16 and Bruce30); by Multiple Locus Sequence Typing (MLST), they also exhibited differences in sequence type (ST), strain 16/02 ST1 (2-1-1-2-1-3-1-1-1) and strain 128/11 ST (22-1-1 -8-9-3-1-1-1). The extensive typing of B. abortus strains isolated from cattle in Brazil using different approaches confirmed the occurrence of rare B. abortus biovar 4 in the country.

Abstract Different types of microorganisms are important in cheese-making because of the contributions their metabolism offers during the process. Few microorganisms present in Colonial cheese are known, in addition to the ones that are introduced to kick-start the processes or the ones that are associated with infections or poisonings. This study aimed to identify, by MALDI-TOF and/or DNA sequencing, the bacteria and yeasts isolated from samples collected in the main stages of Colonial cheese production, i.e., a type of cheese produced in the southern region of Brazil. The lytic capacity of these microorganisms at 5 °C and 30 °C was also evaluated. The 58 bacterial strains were distributed in 10 species among the genera Bacillus, Citrobacter, Klebsiella, Lactococcus, Paenibacillus, Staphylococcus and Raoutella. From the 13 yeasts strains analyzed, three species were identified as following: Candida pararugosa; Meyerozyma guilliermondii; and Rhodotorula mucilaginosa. In three yeasts isolates it was possible to identify only the genus Candida sp. and Trichosporon sp. The species L. lactis (48%) and M. guilliermondii (46%) were, respectively, the predominant bacteria and yeasts species isolated. The highest microbial lytic activity observed was at 30 °C. Lipase activity on isolates was proportionally more observed with yeasts and proteolytic activity with bacteria. Lower caseinase and lipase activity was observed at 5 °C, demonstrating the importance of refrigeration in controlling microbial activity. This research highlighted the cultivation of some microorganisms that are part of the Colonial cheese microbiota as well as that several of them can hydrolyze various compounds present in milk and that could be associated with its maturation or, in uncontrolled circumstances, could be the cause of product deterioration.

O resfriamento do leite na unidade produtora favorece a multiplicação de microrganismos psicrotróficos que podem produzir enzimas proteolíticas termorresistentes com potencial para degradar os componentes do leite. Visando verificar os efeitos destas enzimas em derivados lácteos, este estudo teve por objetivo avaliar as características físico-químicas do iogurte natural produzido a partir de leite contaminado com bactérias psicrotróficas proteolíticas. Para isso, microrganismospsicrotróficos produtores de enzimas proteolíticas foram isolados do leite cru refrigerado, tendo sido identificados até nível de gênero Pseudomonas sp., Alcaligenes sp. e Butiauxella sp. Esses microrganismos foram inoculados em leite pasteurizado e armazenados por 48 e 72 horas a temperatura de 7°±1°C. Após o período de armazenamento, o leite foi tratado termicamente a 90°± 5°C durante 10 min e imediatamente resfriados a 43°C. Foram adicionados os ingredientes e produzidos iogurtes naturais, sendo posteriormente avaliados quanto aos seus teores de proteína, gordura e acidez. Os iogurtes naturais produzidos, quanto aos teores de gordura e proteína foram significativamente maiores em iogurtes produzidos com os leites controles (não inoculados). Além disso, a acidez do iogurte produzido com leites armazenados por 72 horas a 7°±1°C foi significativamente maior no dia zero, independente dos microrganismos intencionalmente adicionados. Com isso, pode-se concluir que as características físico-químicas dos iogurtes naturais produzidos com leites contaminados com as bactérias foram diferentes das obtidas dos iogurtes produzidos com leites não contaminados, sendo importante a reflexão sobre os tempos de armazenamento a frio devido ao favorecimento da multiplicação de microrganismos psicrotróficos proteolíticos.

Colonial cheese is a culturally and economically important product from the south of Brazil. As most of its production is artisanal, the technology employed is mostly knowledge passed down from one generation to the next according to family tradition and may be produced with raw or pasteurized milk. It is noted for its spicy flavour and variable composition and is often classified as a medium to high-moisture cheese. This intrinsic feature increases the risk of microbial spoilage and food poisoning. One of the main bio-indicators of contamination in colonial cheese is coagulase positive Staphylococcus. The purpose of this study was thephenotypic identification of Staphylococcus species isolated from the products and surfaces in the main production stages of colonial cheese. Staphylococcus sp. isolates from the food and the production environment were obtained from two colonial cheese-production agro-industries in Rio Grande do Sul. Samples of fresh milk, curd, ripening and final colonial cheese were collected. In addition, surface sampling was performed on the coagulation tanks, production tables, molds, cheese ripening shelves and on the hands of the handlers. Staphylococcus sp. isolates in the cheese and the production environments tested in this study were identified by phenotypic techniques through biochemical and MALDI-TOF MS analyses. These isolates were subjected to gene expression analysis for enterotoxins A, B, C, D, and E. All isolates (72) were identified as Staphylococcus sp., and 43% of the total isolates tested were coagulase positive. Staphylococcus aureus was the predominant species in the raw milk and production tanks. Regarding coagulase negative staphylococci isolates, S. warneri and S. sciuri were most abundant. The sea and seb genes were detected in 4% of the Staphylococcus isolates. The results indicate eleven different species of Staphylococcus present in the colonial cheese production environments studied. The predominant presence of S. aureus in the different samples of milk, curd, ripened cheese, ready-to-eat cheese and hands of the handlers indicates that there are issues with the selection of milk-producing animals, pasteurization process and/or hygiene control of handlers. The sea and seb genes were detected in samples of raw milk and colonial cheese. No enterotoxin genes were detected in coagulase negative staphylococci. KEY WORDS: Enterotoxins; polymerase chain reaction; time-offlight mass spectrometry; coagulase negative staphylococci; phenotypic identification; genotypic analysis; colonial cheese.

The search for renewable energies has been one of the biggest challenges of the last decades. Sludge and solid wastes of many sources have been used to produce biogas of high calorific value. Thus, this work aimed to evaluate the biogas production of solid waste originating from a tannery that uses chromium salts as a tanning agent and to characterize the physicochemical parameters and microbial composition of the biogas-producing biomass. Wastes were collected and the parameters were evaluated at the initial and final time points of the anaerobic incubation process. At the end of 150 days, there was a production of 26.1 mL g−1 VSS of biogas with 52% of methane. The highest amount of biomethane observed was related to the archaeal family Methanosaetaceae and bacterial order Bacteroidales. Knowledge about changes in the microbial composition can provide tools for manipulation, isolation, and inoculation of the microorganisms inside the bioreactors to maximize methane production.

The possibility of using tannery raw wastewater as substitute for the nutrient supply in the anaerobic co-digestion of two tannery solid waste was investigated with regard to energy efficiency, waste treatment efficiency and economic efficiency. The results showed that the use of tannery wastewater as a source of nutrients for the AD of solid tannery waste proved to be adequate from the point of view that the three residues are being treated simultaneously. There was a biogas production of only 1.9 +/- 0.3 mL/VSS. However, the percentage of methane in the biogas reached 33 % at the beginning of the process, proving that there was methanogenic activity and that AD established. The cost analysis showed that there is a great reduction in the cost of wastewater treatment and solid waste disposal of 23 % and 18 %, respectively, in terms of electricity consumption and 11 % and 8 % respectively in terms of thermal consumption. (C) 2019 Institution of Chemical Engineers. Published by Elsevier B.V. All rights reserved.

Mesophilic anaerobic co-digestion of mixtures of solid wastes from tanneries containing chromium and vegetable tannins was investigated in an orthogonal array. The effect of thermal pretreatment on shavings was evaluated as well. The biogas and methane productivity in terms of VSS was assessed. The treatment efficiency was also evaluated in terms of variation of organic and inorganic load. All assays with chromium-containing sludge showed on average 19.6 ml of biogas/gVSS added, 8.15 ml of methaneigVSS added, 4.8% VSS reduction, 60.4% BOD5 reduction, 55% TOC reduction, 67% IC increase and 54.6% TN increase, showing greater mineralization of the residue when compared to the assays with vegetable tannin sludge, which has proven to be toxic. The origin of shavings was not significant in any parameter due to the small proportion of this residue in assays. The thermal pre-treatment was beneficial only for assays with vegetable tannins, since there was evidence that the heating mechanism degraded part of the phenolic organic matter and consequently reduced its toxicity. (C) 2018 Institution of Chemical Engineers. Published by Elsevier B.V. All rights reserved.

O leite pode ser considerado um bom meio de cultura para o crescimento de microrganismos. Estes microrganismos podem contaminar o homem através da ingestão de leite cru, influenciar no tipo de processamento a ser utilizado e interferir na qualidade deste produto e seus derivados. Propriedades leiteiras de cinco municípios no estado do Rio Grande do Sul, Brasil, foram examinadas para determinar a qualidade microbiológica do leite produzido. Foi verificada a presença de coliformes totais, fecais e Salmonella sp. Foi realizada, também, a contagem de bactérias mesófilas, psicrotróficas e de leveduras. Dentre 42 propriedades, 41 apresentaram coliformes totais no leite. Não foi observada diferença significativa entre os tipos de ordenha e os diferentes métodos de resfriamento durante o armazenamento. Somente oito propriedades apresentaram coliformes fecais e confirmação de Escherichia coli, evidenciando falhas nos procedimentos higiênicos durante a ordenha ou armazenamento. Não foi detectada a presença de Salmonella sp. nas amostras, confirmando sua baixa prevalência no leite. Todas as 12 propriedades testadas quanto à presença de bactérias mesófilas e psicrotróficas apresentaram contagens superiores ou igual a 4 log.ufc.mL-1. Foi observada a presença de leveduras em 32 das 34 propriedades testadas com contagens variando entre 1,52 e acima de 4 log.ufc.mL-1. O número elevado de microrganismos observado no leite cru não interferiu na diminuição destes pela pasteurização e, possivelmente, se produzissem enzimas termoestáveis, estas não afetariam a qualidade do leite pasteurizado, que possui tempo de validade restrito. Porém estas enzimas poderiam afetar a qualidade de produtos derivados a partir deste leite (longa vida, queijos, etc.) devido ao maior período de contato destas enzimas e seus substratos.

The influence of the presence of the tanning agent in co-digestion of leather shavings mixed with sludge before and after the tanning stage was examined. Chromium and vegetable tannin are the main tanning agents used and the presence of both was evaluated in two orthogonal arrays. The energy efficiency was evaluated through biogas and methane production. The efficiency in waste treatment was evaluated through the variation of TOC, IC, TN and the concentration of the tanning agents. Sltidge with chromium proved to be significantly more suitable for AD than sludge without it, producing 27.9 mL of biogas/g of VSS with 60% of methane; a reduction of 62.8% of TOC; and a 325.0% increase of IC, showing that the initial 7.6 mg/L of chromium in these tests was beneficial. Shavings with chromium also proved to be significantly more suitable for AD, even though with a higher initial 16.4 mg/L of chromium, producing 10.7 mL of methane/g of VSS and a 55.2% reduction of TOC, confirming that the concentrations of chromium in the residues were not inhibitory. Shavings with vegetable tannins were unfavorable to the mineralization of the wastes through anaerobic digestion, showing significantly poorer results than shavings without it. They produced 4.1 mL of methane/g of VSS, confirming the establishment of anaerobic digestion. However, they increased the TOC by 23.7%, showing that the initial hydrolytic activity occurred, but the waste was not mineralized by the other phases of the process due to the high initial concentration of 590 mg/L of tannins.

This work proposes a laboratory and semi-pilot scales procedure for the evaluation of biogas production potential and waste biodegradation efficiency from mixtures of solid wastes from tanneries as well as the evaluation of the energy saving. Leather shavings and sludge from wastewater treatment plants substrates were considered in the study. A theoretical model was employed for calculations of the disintegration kinetic constant (kdis). Biogas yields between 21 and 30 mL/kgVSS, maximum methane content of 59% v/v, and a TOC reduction between 68 and 76% were obtained on both scales. A linear consistency was found in the assessed scale up and a two-fold biodegradation rate to a five-fold volume of treated waste. In the conditions studied in semi-pilot scale, a midsize tannery could reduce 6.8% of electric and 1.6% of thermal energy consumption besides the great cost saving of disposal of this waste.

Dengue, Chikungunya, and Zika are important vector-borne diseases, and Aedes aegypti L. is their main transmitter. As the disease management is mainly based on mosquito control strategies, the search for alternative and cost-effective approaches is ongoing. The Gram-negative bacteria Xenorhabdus nematophila and Photorhabdus luminescens are symbiotically associated with entomopathogenic nematodes and are highly pathogenic for insect larvae. After we have recently confirmed the toxicity of these bacteria in Ae. aegypti larvae, we here evaluated the toxic activity of culture fluids on the development of this mosquito species. Larval susceptibility was assessed by exposing larvae to different concentrations of P. luminescens or X. nematophila culture fluids to confirm whether secondary metabolites might cause the mosquitos' death. Xenorhabdus nematophila culture fluid was more effective and stable during the mosquito pathogenicity bioassays compared to that of P. luminescens. Larval mortality started a few hours after exposure of the insects to the fluids. Furthermore, the residual effect of larvicidal activity of X. nematophila fluid persisted at full efficiency for 4 d. Particularly, larval mortality was still higher than 50% for up to 8 d. Exposure of larvae to a sublethal dose of X. nematophila fluid delayed pupation as well as emergence of adult mosquitoes and caused cumulative larval mortality higher than 90% by day 14. Here, we describe for the first time the use of stable culture fluids and therefore secondary metabolites of P. luminescens and X. nematophila as a promising basis for the use as biopesticide for control of Ae. aegypti in the future.

ABSTRACT: This study aimed to evaluate the microbiological quality of tofu sold in supermarkets in Porto Alegre/Brazil. Bacteria counts were performed for Bacillus cereus , mesophilic, coliforms and Staphylococcus coagulase positive and negative. The presence of Listeria sp. was also evaluated. Two different brands of tofu (A and B) were collected, one lot per month, for six months. Five samples from each lot were analyzed. All lots presented mesophilic aerobic counts above 4.3x105CFU g-1. Four of the six lots from brand A and all lots from brand B showed E. coli and/or Staphylococcus coagulase positive counts above the Brazilian law accepted limits. The Staphylococcus coagulase negative counts were higher than those of coagulase positive in all lots. In all lots where Staphylococcus coagulase positive counts were above the legal limit, there were counts of coagulase negative above 104CFU g-1. B. cereus and Listeria sp. were not found in either brand. The majority of lots of brand A and all lots of brand B were unsuitable for human consumption. Our results showed that there are problems in tofu manufacturing in both industries analyzed. There is a need of improvement on its microbial quality to avoid problems of food-borne illness, and finally the need of a better control by the Brazilian inspection services.

This study aimed to evaluate the microbiological quality of tofu sold in supermarkets in Porto Alegre/Brazil. Bacteria counts were performed for Bacillus cereus , mesophilic, coliforms and Staphylococcus coagulase positive and negative. The presence of Listeria sp. was also evaluated. Two different brands of tofu (A and B) were collected, one lot per month, for six months. Five samples from each lot were analyzed. All lots presented mesophilic aerobic counts above 4.3x105CFU g-1. Four of the six lots from brand A and all lots from brand B showed E. coli and/or Staphylococcus coagulase positive counts above the Brazilian law accepted limits. The Staphylococcus coagulase negative counts were higher than those of coagulase positive in all lots. In all lots where Staphylococcus coagulase positive counts were above the legal limit, there were counts of coagulase negative above 104CFU g-1. B. cereus and Listeria sp. were not found in either brand. The majority of lots of brand A and all lots of brand B were unsuitable for human consumption. Our results showed that there are problems in tofu manufacturing in both industries analyzed. There is a need of improvement on its microbial quality to avoid problems of food-borne illness, and finally the need of a better control by the Brazilian inspection services.

A Escherichia coli e largamente utilizada como um indicador biologico de contaminacao fecal devido a sua ubiquidade em fezes, no entanto, possui tambem a capacidade de persistir e multiplicar em ambientes fora do seu habitat primario. Membros da microbiota comensal, tais como E. coli , sofrem pressao seletiva pela sua exposicao a agentes antimicrobianos, resultando no aumento de cepas resistentes. Este estudo teve como objetivo realizar uma determinacao filogenetica atraves do metodo de PCR triplex de cepas de E. coli multirresistentes provenientes de amostras ambientais, animais e humanas. As cepas de origem animal e humana foram associados a um perfil de multirresistencia mais amplo e a producao de ESBL. A determinacao filogenetica demonstrou que os filogrupos B1 (49%) e A (34%) foram os mais prevalentes e os filogrupos D (11%) e B2 (6%), os menos representativos. Os filogrupos A e B1, tambem foram relacionados a um perfil de multiresistencia mais significativo. Os resultados indicaram que os isolados deste estudo sao associados a microbiota comensal de humanos e animais e que estas populacoes sofreram exposicao a antimicrobianos de amplo espectro.

Yerba mate ( Ilex paraguariensis ) is a native plant of the southern region of Brazil, whose leaves, after processing, are used as a beverage by Brazilian, Argentinean, Paraguayan and Uruguayan inhabitants. The purpose of this work was to verify the sanitary quality of the processed yerba mate (presence of thermophilic and mesophilic bacteria, molds and yeasts, Salmonella sp. and coliforms), and to evaluate the microbial diversity (bacteria and yeasts) in leaves and in the processed yerba mate. Sample collections were carried out monthly from January to June 2013, in a yerba mate plantation fields (leaves) and from the local commerce (processed yerba mate) of Vargeao city, Santa Catarina state, Brazil. Standard methods were used to determine the bacteria and fungi counts, as well as to isolate bacteria and yeasts from leaves and processed yerba mate. Bacteria and yeasts identification was carried out through biochemical and physiological assays, and by rDNA gene sequencing. The main thermophilic bacterium found in processed yerba mate and leaves was Bacillus licheniformis . The mesophilic bacteria that predominated in leaves were Pantoea ananatis , Staphylococcus sciuri and Staphylococcus epidermidis, while in processed yerba mate there was a predominance of Bacillus megaterium , Bacillus amyloliquefaciens and Klebsiella pneumoniae . The most frequent yeasts identified in leaves were Aureobasidium pullulans and Sympodiomycopsis sp., and those identified in the processed yerba mate were Rhodosporidium kratochvilovae , Rhodotorula mucilaginosa and Sporobolomyces nylandii . All microbiological parameters for processed yerba mate were in line with the current Brazilian legislation, as well as the parameters established by the WHO.

Background: Brucella sp. are the causative agents of brucellosis, an infectious disease that affects various species of animals and can be transmitted to humans through direct contact with infected animals, indirectly by the ingestion of raw-milk products, and during the handling of strains or infected material in the laboratory. Being a zoonosis, the detection of Brucella species in animals is essential for the prevention of the disease in humans and to perform a good program of control in infected herds. This study aimed at identifying Brucella field strains isolated from 1976 to 2013 in Brazil, using the modified Bruce-Ladder method, to evaluate the performance of this technique.Materials, Methods & Results: Eighty-three strains of Brucella sp. were included in the study, i.e. 21 reference strains (nine B. abortus, one B. canis, four B. melitensis, two B. ovis and five B. suis) and 62 field strains (six B. canis, one B. suis and 55 B. abortus). For the identification of the genus and/or species of Brucella, biochemical and physiological tests, including MacConkey-agar growth, glucose fermentation, haemolysis, catalase, oxidase and urease tests, nitrate reduction, citrate utilization, H2S production and CO2 requirement, were performed. Genomic DNA was extracted from pure cultures through heat-lysis of bacterial cultures and the genus was confirmed by a genus-specific PCR (bcsp31 target gene), before performing the modified Bruce-Ladder PCR for the confirmation of the Brucella species. No problems of specificity were observed with the Bruce-Ladder PCR. However, the 1,682 bp fragment was not systematically amplified, even after several modifications such as the concentration of mix components, annealing temperatures and time. Therefore, an individual PCR using primers specific to this fragment was needed for complete identification of some strains. Also, only one kind of Polymerase gave the best results. All Brucella reference strains and negative controls gave the expected results. All field strains previously identified as B. abortus, B. canis and B. suis by biochemical and physiological tests were confirmed by the modified Bruce-Ladder PCR. All isolated Brucella abortus presented a Bruce-Ladder PCR profile expected for field strains, excluding the vaccine strains.Discussion: The modified Bruce-Ladder PCR identified properly all Brucella species (reference and field strains) and proved to be a reliable technique, thus facilitating the identification of the species in the laboratory, reducing the manipulation of these bacteria and the associated danger. Albeit the difficulties of amplification of one fragment for some strains, when using the multiplex technique, this method is fast and without risks after inactivation of the strains. Most studies on animal brucellosis in Brazil were only based on serological tests without identification of the pathogen; while the knowledge of the particular species and/or biovars that occur in Brazil, as well as their distribution, is important to monitor the spread of Brucella among sensitive species and among farms. Our results showed also that B. abortus is still the predominant species isolated in cattle in Brazil. The knowledge of the species that occur in Brazil can help to identify the source of infection and the measures of control to be applied, while it is also very important to trace the dispersion of strains among farms.

Sanitary conditions are essential for the production of meals and control of the presence of pathogensis important to guarantee the health of customers. The aim of this study was to evaluate the sanitary quality of food services by checking the presence of thermotolerant coliforms, Staphylococcus sp. and evaluate the toxigenic potential from the latter. The analysis was performed on water, surfaces, equipment, ready-to-eat foods, hands and nasal cavity of handlers in seven food services. The water used in food services proved to be suitable for the production of meals. Most food, equipment and surfaces showed poor sanitary conditions due to the presence of thermotolerant coliforms (60.6%). Twenty-six Staphylococcus species were identified from the 121 Staphylococcus isolates tested. Staphylococci coagulase-negative species were predominant in the foods, equipment and surfaces. In food handlers and foods, the predominant species was Staphylococcus epidermidis. Twelve different genotypes were found after PCR for the classical enterotoxin genes. The seb gene (19.8%) was the most prevalent among all Staphylococcus sp. Both coagulase-positive and coagulase-negative Staphylococci showed some of the genes of the enterotoxins tested. We conclude that there are hygienic and sanitary deficiencies in the food services analyzed. Although coagulase-positive Staphylococci have not been present in foods there is a wide dispersion of enterotoxigenic coagulase-negative Staphylococci in the environment and in the foods analyzed, indicating a risk to consumer health.

The intention of this work was to investigate the susceptibility profile of 27 Brucella strains isolated from animals in Brazil, using the E-test method with antimicrobials recommended for the treatment of human brucellosis, to monitor the activities of these antimicrobials and their potential efficacy for human brucellosis treatment. Efficiency of SE-AFLP in determining the genetic diversity of the species of Brucella and its correlation with their susceptibility profile was also evaluated. All 27 strains were susceptible to doxycycline. With the exception of one strain of B. canis and of B.abortus, all strains were susceptible to gentamicin and streptomycin. Of the wild Brucella strains tested, ten, nine and five showed reduced susceptibility to rifampicin, ceftriaxone and trimetoprim/ sulfamethoxazole, respectively. One B. abortus and three B. canis strains showed multi-resistance profiles. The strain of B. abortus was resistant to streptomycin, rifampicin and ceftriaxone. Two strains of B. canis were resistant to rifampicin, ceftriaxone and trimetoprim/sulfamethoxazole, and one strain was resistant to rifampicin, ceftriaxone, streptomycin and gentamicin. Rifampicin,in combination with doxycycline, is one of the principal antibiotics prescribed to treat human brucellosis. The occurrence of strains resistant to rifampicin and other antimicrobials must be monitored before initiating this treatment, since the resistance of these strains could be one of the causes of the failure of some brucellosis treatment. No relationship was observed between SE-AFLP profiles and regional origin of the strains; neither between SE-AFLP profiles and antimicrobial profiles.