Estudo in vitro da ação de diferentes concentrações de hipoclorito de sódio sobre Enterococcus faecalis

Enterococos são cocos Gram-positivos, membros da microbiota humana e animal, sendo também encontrados em alimentos, solo e água. A resistência de enterococos a antibióticos têm sido muito estudada em amostras alimentares, ambientais e clínicas, porém poucos relatos avaliam a ação de biocidas sobre esses microrganismos. O hipoclorito de sódio (NaOCl) é um desinfetante muito utilizado na indústria de alimentos para destruir microrganismos existentes em superfícies do processo de fabricação. O objetivo do presente estudo foi investigar o efeito de duas concentrações de NaOCl (2,5% e 8,5%) sobre vinte e dois  Enterococcus faecalis isolados de carne de frango e correlacionar com o perfil de resistência antimicrobiana dos isolados. A sensibilidade das amostras de enterococos frente às diferentes concentrações de NaOCl foi realizada através da técnica dos cilindros carreadores de aço inox tipo 304. Dois isolados (9,09%) apresentaram crescimento após a exposição à concentração 8,5% de NaOCl e cinco (23%) na concentração de 2,5%. Não foi encontrada relação entre tolerância ao biocida e o perfil resistência. É importante salientar que alguns mecanismos de tolerância aos biocidas podem ser transferíveis para outras células bacterianas e se tornar um grande problema de saúde pública

Modulation of gene expression by essential oils in bacteria

The emerging of drug-resistant strains imposes some new strategies in prevent bacteria spread. It is pivotal to find new candidates for drug development. The essential oils (EOs) extracted from plants are alternatives for it, since they have a variety of cellular target. However, evaluate the efficacy of EOs against bacteria Gram positive and Gram negative, as well as, the toxicity for mammary cell is needed. Here we showed current results the effect of EOs extracted from several plant species on bacterial gene expression

Microbial community and physicochemical characterization of kombuchas produced and marketed in Brazil

Kombucha has recently become popular in the Brazilian beverage market as a healthy alternative to soft drinks. However, little is known about the microbial composition and physicochemical characteristics of products available on the market. To investigate the microbial profile of kombuchas, high-throughput sequencing of the 16S rRNA and ITS genes, in samples belonging to six brands was utilized. In addition, the drinks were characterized based on the physicochemical parameters of pH, total acidity and alcohol content. Through the metagenetic analysis, the most abundant prokaryotic species identified were Liquorilactobacillus nagelii, Oenococcus oeni, Komagataeibacter rhaeticus, Liquorilactobacillus ghanensis, Gluconobacter oxydans, Komagataeibacter saccharivorans, Acetobacter peroxydans and Pantoea stewartii, while the mainly eukaryotic species were Dekkera bruxellensis, Dekkera anomala, Saccharomyces cerevisiae and Lanchancea fermentati. Interestingly, we identified six different oligotypes of D. bruxellensis, showing a wide diversity of strains belonging to this species. The results obtained for the physicochemical analyses, within the shelf life of the products, presented a range between 2.88 ± 0.06 and 3.43 ± 0.04 of pH, values between 1.80 ± 0.59 and 4.86 ± 0.72 for the total titratable acidity and 1.03 ± 0.24 to 2.54 ± 0.39 referring to alcohol content, demonstrating significant differences between brand. In addition, all samples had alcohol content above 0.5%, resulting in the classification of alcoholic beverages, which need proper labelling. The data generated in this work helped to understand the composition of the kombuchas available in the Brazilian market, as well as in the development of the identity and quality standard of the drink

Comparative genomics suggests differences related to resistance and virulence between food-isolated Listeria monocytogenes serotypes 1/2a and 4b

Among the four lineages described for Listeria monocytogenes (I, II, III, and IV), lineages I and II harbor the serotypes most closely related to listeriosis in humans. Serotypes 1/2b and 4b are associated with the majority of listeriosis outbreaks, and serotype 1/2a is frequently involved in food and processing plant contamination. As such, the present study utilizes phylogenetic analysis for the aim of determining genomic differences between two L. monocytogenes strains isolated in southern Brazil (serotypes 1/2a and 4b) and known reference strains (L. monocytogenes EGD-e and L. monocytogenes Scott A). The Illumina Miseq platform was used to perform genomic sequencing, and cluster analysis of orthologous groups facilitated the investigation of similarities and differences between the two serotypes studied. In line with previous research, the studied strains of serotypes 1/2a and 4b presented different proteins related to resistance and virulence that may represent adaptations to several conditions during its evolution

Evaluation of the relative expression of genes involved in biofilm formation by Acinetobacter baumannii

Acinetobacter baumannii é um patógeno oportunista, de grande importância no ambiente hospitalar devido à sua capacidade de adquirir mecanismos de resistência, de aderir e formar biofilmes sob diferentes superfícies. Devido a relevância da infecção causadas pelo A. baumannii torna-se fundamental o conhecimento dos mecanismos envolvidos na sua patogenicidade. Sendo assim, o objetivo desse estudo foi realizar a análise transcricional dos genes envolvidos na formação e manutenção do biofilme (bap, abaI, ompA, bfmRS, csuAB, pgaA, pilZ, wspR, eal, eagg, IscRSU e csdA) de duas cepas clínicas de A. baumannii obtidas de hospitais de Porto Alegre, Brasil (AbH e AbC) e uma cepa controle ATCC 19606 (AbA) sob diferentes condições de crescimento. As cepas foram avaliadas quanto a sua capacidade de formar biofilme a 25 °C e 37 °C, crescendo em caldo Luria Bertani (LB), LB acrescido de 1% glicose (LB-G), LB acrescido de 10% sangue de carneiro (LB-SC) e urina pura (UP). A análise transcricional foi realizada por PCR quantitativo em tempo real. A cepas AbH, AbC e AbA foram fortes formadoras de biofilme em LB e LB-G a 25 e 37 °C. Em LB-SC, as cepas AbH e AbA foram fortes formadoras e AbC foi classificada fraca formadora de biofilme. Em UP, a cepa AbH foi moderadamente formadora, AbC e AbA foram fracas formadoras de biofilme. Os genes wspR, pgaA, ompA, bap, abaI de AbA, csdA de Ab He o operon IscRSU foram superexpressos em biofilme; os genes eagg de AbH, pilZ e csuAB foram inibidos e os genes bfmRS, eal, eaggde AbA, csdA de AbA e abaIde AbH não possuíram uma variação significativa na expressão durante a formação do biofilme.Acinetobacter baumannii is an opportunistic pathogen of high importance in the hospital environment due to its ability to develop resistance mechanisms as well as to adhere and form biofilms on different surface types. Due to the high relevance of infections caused by A. baumannii, knowing the mechanisms involved in its pathogenicity is of extreme importance. Thus, we aimed to perform a transcriptional analysis on genes involved in biofilm formation and maintenance (bap, abaI, ompA, bfmRS, csuAB, pgaA, pilZ, wspR, eal, eagg, IscRSU e csdA) of two clinically isolated A. baumannii strains obtained from hospitals in Porto Alegre city, Rio Grande do Sul state, southern Brazil (AbH and AbC), and of the reference strain ATCC 19606 (AbA), cultivated under different growth conditions. Biofilm formation ability was evaluated at 25 and 37 °C using LB broth (LB), LB + 1% glucose (LB-G), pure urine (PU), and LB + 10% sheep blood (LB-SB). Gene transcriptional profiling was performed by real time qPCR. Strains AbH, AbC and AbA were strong biofilm producers in LB and LB-G at 25 and 37 °C. In LB-SB, strains AbH and AbA were strong biofilm producers while AbC was a week biofilm producer. In PU, the strain AbH was a moderate biofilm producer, while strains AbC and AbA were weak biofilm producers. Genes wspR, pgaA, ompA, bap, abaI de AbA; csdA of AbH; and the operon IscRSU were all overexpressed in biofilms. On the other hand, genes eagg of AbH, pilZ and csuAB were suppressed, while genes bfmRS, eal and eagg of AbA; csdA of AbA; and abaI of AbH showed no significant variation in their expression during biofilm formation

Caracterização de Enterococcus spp. isolado de um ambiente de piscicultura no sul do Brasil

The aim of present study is to characterize the resistance and virulence profile of enterococci isolated from aquaculture excavated ponds and masonry tanks (6 samples) in southern Brazil. Samples were cultured in selective medium, 10 colonies were randomly selected from each sample, which were identified by MALDI-TOF and tested against 13 antimicrobials. The presence of resistance (tetL, tetM, tetS, ermB and msrC) and virulence (ace, esp, agg, cylA and gelE) genes were determined by PCR. A total of 79 enterococci were identified, and Entecococcus faecalis (44.3%) and E. casseliflavus (36.7%) were the most prevalent species isolated. Sixty-five strains (82.3%) were resistant to at least one of the antimicrobials tested, whereas 27 (34.2%) strains were multiresistant. The overall percentages of antimicrobial resistant isolates were: 58.2% to rifampicin, 40.5% to fluoroquinolones, 36.7% to erythromycin and 30.4% to tetracycline. The tetL and tetM genes were found in 57.7% of the tetracycline-resistant strains; and msrC in 31.01% of erythromycin-resistant strains. The most frequently detected virulence factors were ace and gelE genes. Although limited to a single farm, these data suggest that aquaculture may be a reservoir of resistant and virulent enterococci. This study is the first step towards enhancing our understandingof distribution, resistance and virulence profile in enterococci isolated from fish farming environments in the south Brazil.O objetivo do estudo apresentado é caracterizar o perfil de resistência e virulência de enterococos isolados de viveiros escavados e tanques de alvenaria (6 amostras) de uma pisicultura no Sul do Brasil. As amostras foram cultivadas em meio seletivo, 10 colônias foram selecionadas aleatoriamente de cada amostra, que foram identificadas por MALDI-TOF e testadas contra 13 antimicrobianos. A presença de genes de resistência (tetL, tetM, tetS, ermB e msrC) e virulência (ace, esp, agg, cylA e gelE) foi determinada por PCR. Foram identificados 79 enterococos, sendo Entecococcus faecalis (44,3%) e E. casseliflavus (36,7%) as espécies mais frequentes isoladas. Sessenta e cinco cepas (82,3%) eram resistentes a pelo menos um dos antimicrobianos testados, enquanto 27 (34,2%) eram multirresistentes. As porcentagens gerais de isolados resistentes a antimicrobianos foram: 58,2% para rifampicina, 40,5% para fluoroquinolonas, 36,7% para eritromicina e 30,4% para tetraciclina. Os genes tetL e tetM foram encontrados em 57,7% das cepas resistentes à tetraciclina; e msrC em 31,01% das cepas resistentes à eritromicina. Os fatores de virulência mais comumente detectados foram ace e gelE. Embora limitados a uma única fazenda, esses dados indicam que a aquicultura pode ser uma fonte de enterococos resistentes e virulentos. Este estudo é o primeiro passo para melhorar nosso entendimento da distribuição, resistência e perfil de virulência em enterococos isolados de ambientes de piscicultura no sul do Brasil

Differential expression of cysteine desulfurases in soybean

Background: Iron-sulfur [Fe-S] clusters are prosthetic groups required to sustain fundamental life processes including electron transfer, metabolic reactions, sensing, signaling, gene regulation and stabilization of protein structures. In plants, the biogenesis of Fe-S protein is compartmentalized and adapted to specific needs of the cell. Many environmental factors affect plant development and limit productivity and geographical distribution. The impact of these limiting factors is particularly relevant for major crops, such as soybean, which has worldwide economic importance. Results: Here we analyze the transcriptional profile of the soybean cysteine desulfurases NFS1, NFS2 and ISD11 genes, involved in the biogenesis of [Fe-S] clusters, by quantitative RT-PCR. NFS1, ISD11 and NFS2 encoding two mitochondrial and one plastid located proteins, respectively, are duplicated and showed distinct transcript levels considering tissue and stress response. NFS1 and ISD11 are highly expressed in roots, whereas NFS2 showed no differential expression in tissues. Cold-treated plants showed a decrease in NFS2 and ISD11 transcript levels in roots, and an increased expression of NFS1 and ISD11 genes in leaves. Plants treated with salicylic acid exhibited increased NFS1 transcript levels in roots but lower levels in leaves. In silico analysis of promoter regions indicated the presence of different cis-elements in cysteine desulfurase genes, in good agreement with differential expression of each locus. Our data also showed that increasing of transcript levels of mitochondrial genes, NFS1/ISD11, are associated with higher activities of aldehyde oxidase and xanthine dehydrogenase, two cytosolic Fe-S proteins. Conclusions: Our results suggest a relationship between gene expression pattern, biochemical effects, and transcription factor binding sites in promoter regions of cysteine desulfurase genes. Moreover, data show proportionality between NFS1 and ISD11 genes expression

Resistant enterococci isolated from raw sheep’s milk and cheeses from South region of Brazil

Enterococci have been used as sentinel organisms for monitoring antimicrobial resistance in food, humans, and other animals. In this sense, the present study evaluated the antimicrobial susceptibility profile and the presence of genes associated with resistance to erythromycin (msrC and ermB) and tetracycline [tet(M) and/or tet(L)] in enterococci isolated from raw sheep’s milk and cheeses (colonial, feta-, and pecorino-type) from South region of Brazil. A total of 156 enterococci were isolated from milk (n=80) and cheese (n=76) samples, identified by MALDI-TOF. Enterococcus faecalis (50.6%; n=79) was the most frequent species isolated from both samples. According to in vitro susceptibility tests, enterococci strains were not susceptible to the most commonly antimicrobial agents used in human and veterinary medicine. The frequency of MDR strains in enterococci isolated from milk (53.7%) was higher than those from cheese (24.2%). The tet(M) gene was the most commonly detected among tetracycline not-susceptible strains. The present study provided the first evidence of antimicrobial not-susceptible enterococci in raw sheep’s milk and cheeses in South Brazil. Drug-resistant strains, particularly those that are MDR, constitute a One Health issue.Os enterococos têm sido usados como organismos sentinela para monitorar o padrão de suscetibilidade a antimicrobianos em alimentos, humanos e outros animais. Neste sentido, o presente estudo objetivou avaliar o perfil de susceptibilidade a antimicrobianos e os genes associados com a resistência a eritromicina (msrC and ermB) e à tetraciclina [tet(M) and/or tet(L)] em enterococos isolados de leite cru de ovelha e queijos (colonial, tipo-feta e tipo-pecorino) do Sul do Brasil. Um total de 156 enterococos foram isolados de leite (n=80) e queijo (n=76), identificados por MALDI-TOF. Enterococcus faecalis (50,6%; n=79) foi a espécie mais frequentemente isolada de ambas as amostras. De acordo com o teste de suscetibilidade in vitro, as cepas de enterococos não foram susceptíveis aos agentes antimicrobianos mais comumente utilizados na clínica humana e veterinária. A frequência de cepas de enterococos MDR isoladas do leite (53,7%) foi superior à do queijo (24,2%). O gene tet(M) foi o mais comumente detectado entre as cepas não susceptíveis à tetraciclina. O presente estudo fornece as primeiras evidências de enterococos não susceptíveis aos antimicrobianos em leite cru de ovelha e queijos no Sul do Brasil. Cepas resistentes a drogas, particularmente as que são MDR, representam uma preocupação de Saúde Única

Frequency of Clustered Regularly Interspaced Short Palindromic Repeats (CRISPRs) in non-clinical Enterococcus faecalis and Enterococcus faecium strains

The fidelity of the genomes is defended by mechanism known as Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR) systems. Three Type II CRISPR systems (CRISPR1-cas, CRISPR2 and CRISPR3-cas) have been identified in enterococci isolates from clinical and environmental samples. The aim of this study was to observe the distribution of CRISPR1-cas, CRISPR2 and CRISPR3-cas in non-clinical strains of Enterococcus faecalis and Enterococcus faecium isolates from food and fecal samples, including wild marine animals. The presence of CRISPRs was evaluated by PCR in 120 enterococci strains, 67 E. faecalis and 53 E. faecium. It is the first report of the presence of the CRISPRs system in E. faecalis and E. faecium strains isolated from wild marine animal fecal samples. The results showed that in non-clinical strains, the CRISPRs were more frequently detected in E. faecalis than in E. faecium. And the frequencies of CRISPR1-cas and CRISPR2 were higher (60%) in E. faecalis strains isolated from animal feces, compared to food samples. Both strains showed low frequencies of CRISPR3-cas (8.95% and 1.88%). In conclusion, the differences in the habitats of enterococcal species may be related with the results observe in distribution of CRISPRs systems.A fidelidade dos genomas é defendida por mecanismos conhecidos como sistemas de repetições palindrômicas curtas agrupadas e regularmente interespaçadas (CRISPRs). Três tipos de sistemas CRISPR II (CRISPR1-cas, CRISPR2 e CRISPR3-cas) têm sido identificados em cepas de enterococos isolados de amostras clínicas e ambientais. O objetivo deste estudo foi observar a distribuição dos CRISPR1-cas, CRISPR2 e CRISPR3-cas em cepas não-clínicas de Enterococcus faecalis e Enterococcus faecium isoladas de amostras alimentícias e fecais, incluindo animais marinhos selvagens. A presenca dos CRISPRs foi determinada por PCR em 120 cepas de enterococos, sendo 67 E. faecalis e 53 E. faecium. É o primeiro relato da presença do sistema CRISPRs nas estirpes E. faecalis e E. faecium isoladas de amostras fecais de animais marinhos selvagens. Os resultados mostraram que em cepas não-clínicas, os CRISPRs foram mais frequentemente detectados em E. faecalis do que em E. faecium. E as frequências de CRISPR1-cas e CRISPR2 foram maiores (60%) em cepas de E. faecalis isoladas de fezes de animais, quando comparadas à amostras de alimentos. Ambas as cepas apresentaram baixas freqüências de CRISPR3-cas (8,95% e 1,88%). Em conclusão, as diferenças nos habitats das espécies de enterococos podem estar relacionadas com os resultados observados na distribuição dos sistemas CRISPRs