Involvement of the ornithine decarboxylase gene in acid stress response in probiotic Lactobacillus delbrueckii UFV H2b20.

Amino acid decarboxylation is important for the maintenance of intracellular pH under acid stress. This study aims to carry out phylogenetic and expression analysis by real-time PCR of two genes that encode proteins involved in ornithine decarboxylation in Lactobacillus delbrueckii UFV H2b20 exposed to acid stress. Sequencing and phylogeny analysis of genes encoding ornithine decarboxylase and amino acid permease in L. delbrueckii UFV H2b20 showed their high sequence identity (99%) and grouping with those of L. delbrueckii subsp. bulgaricus ATCC 11842. Exposure of L. delbrueckii UFV H2b20 cells in MRS pH 3.5 for 30 and 60 min caused a significant increase in expression of the gene encoding ornithine decarboxylase (up to 8.1 times higher when compared to the control treatment). Increased expression of the ornithine decarboxylase gene demonstrates its involvement in acid stress response in L. delbrueckii UFV H2b20, evidencing that the protein encoded by that gene could be involved in intracellular pH regulation. The results obtained show ornithine decarboxylation as a possible mechanism of adaptation to an acidic environmental condition, a desirable and necessary characteristic for probiotic cultures and certainly important to the survival and persistence of the L. delbrueckii UFV H2b20 in the human gastrointestinal tract.

Increased expression of clp genes in Lactobacillus delbrueckii UFV H2b20 exposed to acid stress and bile salts.

The ability to survive in harsh environments is an important criterion to select potential probiotics strains. The objective of this study was to identify and carry out phylogenetic and expression analysis by quantitative real-time PCR of the clpP, clpE, clpL and clpX genes in the probiotic strain Lactobacillus delbrueckii UFV H2b20 exposed to the conditions prevailing in the gastrointestinal tract (GIT). Phylogenetic trees reconstructed by Bayesian inference showed that the L. delbrueckii UFV H2b20 clpP, clpL and clpE genes and the ones from L. delbrueckii ATCC 11842 were grouped. The exposure of cells to MRS broth of pH 3.5 for 30 and 60 min resulted in an increased expression of the four genes. Exposure of the L. delbrueckii UFV H2b20 cells for 30 and 60 min to MRS broth containing 0.1% bile salts increased the expression of the clpP and clpE genes, while the expression level of the clpL and clpX genes increased only after 30 min of exposure. The involvement of the studied genes in the responses to acid stress and bile salts suggests a possible central role of these genes in the survival of L. delbrueckii UFV H2b20 during the passage through the GIT, a characteristic necessary for probiotic strains.

Survival of Lactobacillus delbrueckii UFV H2b20 in fermented milk under simulated gastric and intestinal conditions.

The survival of Lactobacillus delbrueckii UFV H2b20 was assessed in fermented milk, both during the storage period and after exposure to simulated gastric and intestinal juices, as well the detection of the gene fbpA involved in adherence to human gastrointestinal tract. L. delbrueckii UFV H2b20 remained stable and viable for 28 days under refrigerated storage conditions. After one day of storage, that strain exhibited a one-log population reduction following exposure in tandem to simulated gastric and intestinal juices. After 14 days of storage, a two-log reduction was observed following 90 min of exposure to the simulated gastric conditions. However, the strain did not survive following exposure to the simulated intestinal juice. The observed tolerance to storage conditions and resistance to the simulated gastric and intestinal conditions confirm the potential use of L. delbrueckii UFV H2b20 as a probiotic, which is further reinforced by the detection of fbpA in this strain.

Molecular characterization of a new lytic bacteriophage isolated from cheese whey.

In this study, we isolated and characterized a lytic Lactococcus lactis bacteriophage from the sera of a failed fermentation. The phage was isolated and cultured in L. lactis subsp. cremoris in M17 medium. The isolated bacteriophage was characterized by multiplex PCR, pulsed-field electrophoresis, DNA restriction digestion, analysis of the N-terminal sequence of the phage major structural protein, transmission electron microscopy and sequencing and analysis of a conserved fragment of its genome. Analysis of the viral genome indicates that its genome is composed of a DNA strand of approximately 48 kb in length, and PCR and microscopy confirmed that IL-P1 belongs to the group of 936-type phages in the family Siphoviridae, which is the most abundant type of lactococcal virus in dairy products worldwide. To our knowledge, this is the first report of a virus within this family that has a presumptive genome larger than 40 kb.

Genes involved in lactose catabolism and organic acid production during growth of Lactobacillus delbrueckii UFV H2b20 in skimmed milk.

There are three main reasons for using lactic acid bacteria (LAB) as starter cultures in industrial food fermentation processes: food preservation due to lactic acid production; flavour formation due to a range of organic molecules derived from sugar, lipid and protein catabolism; and probiotic properties attributed to some strains of LAB, mainly of lactobacilli. The aim of this study was to identify some genes involved in lactose metabolism of the probiotic Lactobacillus delbrueckii UFV H2b20, and analyse its organic acid production during growth in skimmed milk. The following genes were identified, encoding the respective enzymes: ldh - lactate dehydrogenase, adhE - Ldb1707 acetaldehyde dehydrogenase, and ccpA-pepR1 - catabolite control protein A. It was observed that L. delbrueckii UFV H2b20 cultivated in different media has the unexpected ability to catabolyse galactose, and to produce high amounts of succinic acid, which was absent in the beginning, raising doubts about the subspecies in question. The phylogenetic analyses showed that this strain can be compared physiologically to L. delbrueckii subsp. bulgaricus and L. delbrueckii subsp. lactis, which are able to degrade lactose and can grow in milk. L. delbrueckii UFV H2b20 sequences have grouped with L. delbrueckii subsp. bulgaricus ATCC 11842 and L. delbrueckii subsp. bulgaricus ATCC BAA-365, strengthening the classification of this probiotic strain in the NCFM group proposed by a previous study. Additionally, L. delbrueckii UFV H2b20 presented an evolutionary pattern closer to that of probiotic Lactobacillus acidophilus NCFM, corroborating the suggestion that this strain might be considered as a new and unusual subspecies among L. delbrueckii subspecies, the first one identified as a probiotic. In addition, its unusual ability to metabolise galactose, which was significantly consumed in the fermentation medium, might be exploited to produce low-browning probiotic Mozzarella cheeses, a desirable property for pizza cheeses.

Genes involved in protein metabolism of the probiotic lactic acid bacterium Lactobacillus delbrueckii UFV H2b20.

A basic requirement for the prediction of the potential use of lactic acid bacteria (LAB) in the dairy industry is the identification of specific genes involved in flavour-forming pathways. The probiotic Lactobacillus delbrueckii UFV H2b20 was submitted to a genetic characterisation and phylogenetic analysis of genes involved in protein catabolism. Eight genes belonging to this system were identified, which possess a closely phylogenetic relationship to NCFM strains representative, as it was demonstrated for oppC and oppBII, encoding oligopeptide transport system components. PepC, PepN, and PepX might be essential for growth of LAB, probiotic or not, since the correspondent genes are always present, including in L. delbrueckii UFV H2b20 genome. For pepX gene, a probable link between carbohydrate catabolism and PepX expression may exists, where it is regulated by PepR1/CcpA-like, a common feature between Lactobacillus strains and also in L. delbrueckii UFV H2b20. The well conserved evolutionary history of the ilvE gene is evidence that the pathways leading to branched-chain amino acid degradation, such as isoleucine and valine, are similar among L. delbrueckii subsp. bulgaricus strains and L. delbrueckii UFV H2b20. Thus, the involvement of succinate in flavour formation can be attributed to IlvE activity. The presence of aminopeptidase G in L. delbrueckii UFV H2b20 genome, which is absent in several strains, might improve the proteolytic activity and effectiveness. The nucleotide sequence encoding PepG revealed that it is a cysteine endopeptidase, belonging to Peptidase C1 superfamily; sequence analysis showed 99% identity with L. delbrueckii subsp. bulgaricus ATCC 11842 pepG, whereas protein sequence analysis revealed 100% similarity with PepG from the same organism. The present study proposes a schematic model to explain how the proteolytic system of the probiotic L. delbrueckii UFV H2b20 works, based on the components identified so far.

Resistência a antimicrobianos dependente do sistema de efluxo multidrogas em Escherichia coli isoladas de leite mastítico / Antimicrobial resistance dependent on multidrugs efflux in Escherichia coli isolated from the mastitic milk

Identificaram-se e caracterizaram-se a resistência e a multirresistência aos principais antimicrobianos usados no tratamento de mastite bovina causada por Escherichia coli. A concentração inibitória mínima (MIC) e o sistema de efluxo foram detectados pelas curvas de crescimento, com base na densidade óptica, em diferentes concentrações da droga e na presença e na ausência do desacoplador da força próton-motora (PMF). E. coli 1 foi resistente à neomicina e à gentamicina; E. coli 3 e 4, à tetraciclina e à estreptomicina; e E. coli 2 e 6 à gentamicina. E. coli 5 apresentou modelo de sensibilidade. Observou-se que MICs de todos os antimicrobianos dos multirresistentes (E. coli 1, 3 e 4) diminuíram na presença do desacoplador, o que sugere sistema de efluxo multidrogas. Após cura, apenas E. coli 1 apresentou modelo de sensibilidade, porém não houve alterações das MICs, antes e após adição do desacoplador. Os resultados indicam possível presença de mecanismo de resistência dependente da PMF codificado, ou parte dele, em plasmídeo.

Resistance and multiresistance to main antimicrobials used for treating bovine mastitis caused by Escherichia coli were identified and characterized. The minimal inhibitory concentration (MIC) and efflux systems were detected by the use of growth curves based on optical density at different drug concentrations and both presence and absence of uncoupler of the proton-motive force (PMF). E. coli 1 was resistant to neomycin and gentamycin, E. coli 3 and 4 were resistant to tetracycline and streptomycin, whereas E. coli 2 and 6 were resistant to gentamycin. E. coli 5 showed sensibility model. MICs of all antimicrobials of the multiresistant samples (E. coli 1, 3, and 4) were decreased in presence of the uncoupler, therefore suggesting the presence of the multidrug efflux system. After healing, only E. coli 1 showed sensibility model, however no alteration occurred in MIC(s) before and after adding the uncoupler. Those data inform the possible presence of a PMF dependent resistance mechanism that is totally or partly codified in plasmid.

Identification of Lactobacillus UFV H2b20 (probiotic strain) using DNA-DNA hybridization / Identificação de Lactobacillus UFV H2b20 (linhagem probiótica) através de hibridização DNA-DNA

Sequence analyses of the 16S rDNA gene and DNA-DNA hybridization tests were performed for identification of the species of the probiotic Lactobacillus UFV H2b20 strain. Using these two tests, we concluded that this strain, originally considered Lact. acidophilus, should be classified as Lact. delbrueckii.

Análise da seqüência do gene 16S rDNA e ensaios de hibridização DNA_DNA foram empregados para identificar a espécie da cepa probiótica Lactobacillus UFV H2b20. Empregando-se estes dois testes, concluímos que esta cepa, originalmente considerada Lact. acidophilus, deve ser classificada como Lact. delbrueckii.

Identification of Lactobacillus UFV H2b20 (probiotic strain) using DNA-DNA hybridization

Sequence analyses of the 16S rDNA gene and DNA-DNA hybridization tests were performed for identification of the species of the probiotic Lactobacillus UFV H2b20 strain. Using these two tests, we concluded that this strain, originally considered Lact. acidophilus, should be classified as Lact. delbrueckii.

Análise da seqüência do gene 16S rDNA e ensaios de hibridização DNA_DNA foram empregados para identificar a espécie da cepa probiótica Lactobacillus UFV H2b20. Empregando-se estes dois testes, concluímos que esta cepa, originalmente considerada Lact. acidophilus, deve ser classificada como Lact. delbrueckii.

Identification of Lactobacillus UFV H2B20 (probiotic strain) using DNA-DNA hybridization.

Sequence analyses of the 16S rDNA gene and DNA-DNA hybridization tests were performed for identification of the species of the probiotic Lactobacillus UFV H2b20 strain. Using these two tests, we concluded that this strain, originally considered Lact. acidophilus, should be classified as Lact. delbrueckii.

Detection of a chloramphenicol efflux system in Escherichia coli isolated from poultry carcass.

An active chloramphenicol efflux system was demonstrated in a multiresistant E. coli isolated from poultry carcass. The effect of different concentrations of chloramphenicol on the original strain and on the plasmid-cured strain was determined in the presence and in the absence of CCCP, an uncoupler of the proton-motive force. Minimal inhibitory concentration (MIC) was lower in the presence of CCCP in the original strain. The plasmid-cured strain displayed lower resistance for chloramphenicol than the wild type, but the MIC was not affected by CCCP. The combined results indicate a plasmid encoded energy dependent resistance mechanism. 3H-chloramphenicol accumulation within the cells was measured by scintillation counting. The uptake or the efflux of 3H-chloramphenicol was influenced by CCCP in the original strain, but not in the plasmid-cured strain. More than one chloramphenicol resistance mechanism may exist in this strain. E. coli is an important commensal or pathogen that inhabits the gastrointestinal tracts of humans and animals, so a plasmid encoded active drug resistance mechanism can be a potential source of horizontal transfer of resistance.

Resistance to antibiotics in Gram-negative bacteria isolated from broiler carcasses

One hundred and ninety-seven isolates of Gram-negative bacteria, comprising 10 genera, were isolated from poultry carcasses at a processing plant in order to investigate resistance to low levels of antibiotics. The samples were taken just after evisceration and before inspection. Most of the isolates were of Samonella and Escherichia. Other genera present were Enterobacter, Serratia, Klebsiella, Kluyvera, Erwinia, Citrobacter, Pseudomonas and Aeromonas. Distinct profiles of antibiotic resistance were detected. Resistance to more than two antibiotics predominated and spanned several classes of antibiotics. Salmonellae and escherichiae were mainly resistant to the aminoglycosides, followed by tetracycline, nitrofuran, sulpha, macrolide, chloramphenicol, quinolones and b-lactams. Most isolates were sensitive to 30mg/ml olaquindox, the growth promoter in use at the time of sampling. However, many were resistant to a level of 10mg/ml and 13mg/ml olaquindox, levels present in the gut due to the dilution in the feed. The results suggest a possible role of low level administration of antibiotics to broilers in selecting multi-resistant bacteria in vivo

[Percutaneous valvuloplasty in congenital aortic valve stenosis]. / Valvuloplastia percutânea em estenose valvular aórtica congênita.

PURPOSE: To show the initial experience of Institute of Cardiology of Rio Grande do Sul in the treatment of congenital valvular aortic stenosis with percutaneous balloon aortic valvuloplasty. METHODS: Twenty four patients were submitted to the procedure, 14 males and 10 females. The mean age 7 years (4 days-17 years). Four patients were aged below 30 days and three patients had previous surgical valvuloplasty. The percutaneous balloon aortic valvuloplasty were made the retrograde approach in all patients. RESULTS: The peak systolic pressure gradient was reduced from 65.96 +/- 22.68 to 27.08 +/- 18.74 mmHg. The procedure resulted in aortic regurgitation in seven patients and worsened aortic regurgitation in two patients. One patient had cardiac arrest that was reverted by cardiopulmonary resuscitation, this patient had hospital discharge without sequel. Five patients had acute femoral artery thrombosis, and hemorrhage in the site of puncture happened in one patient. CONCLUSION: The percutaneous balloon aortic valvuloplasty results in effective reduction of the peak systolic pressure gradient, it is a save and effective therapy in patients with congenital valvular aortic stenosis. Further evaluation of the long term results are necessary for definitive conclusions.

Prevalencia de infeccao chagasica em necropsias no Triangulo Mineiro. / Prevalence of Chagas' infection in necropsies in the Triangulo Mineiro

Analisou-se a prevalencia da infeccao chagasica em 2690 necropsias realizadas no Triangulo Mineiro. Os resultados indicaram que 38,84% dos necropsiados eram chagasicos e que nao houve declinio da infeccao nos ultimos anos. Discutiu-se tais resultados, comparando-os com os de outros Autores que analisaram a questao

Verapamil oral associado a diurético no tratamento de hipertensão arterial leve ou moderada / Oral verapamil associated with a diuretic in the treatment of slight and moderate arterial hypertension

Os autores relatam os resultados da administracao oral de verapamil associada a um diuretico, no tratamento da hipertensao com niveis diastolicos, superiores a 90 mm Hg, apos uma fase inicial de diuretico. A selecao foi realizada atraves do uso de diuretico e, posteriormente, placebo. Os 30 pacientes assim selecionados receberam diureticos e verapamil em doses crescentes Dezoito (60%) tiveram sua pressao normalizada. A ocorrencia de efeitos colaterais foi insignificante (1 caso de reacao alergica, ja apresentada com outros medicamentos). Concluem ser o verapamil mais uma opcao no tratamento da hipertensao arterial leve ou moderada