Regulering av energimetabolisme i enterococcus faecalis studert med transkriptom-, proteom- og metabolomanalyser

Lactic Acid Bacteria (LAB) are widely used as starter culture in food fermentation. Among LAB also pathogenic bacteria are found particular in enterococci and streptococci. Enterococcus faecalis is a gut commensal bacterium but certain isolates have been shown to be pathogenic while others are foodgrade bacteria in LAB fermented food commodities. E. faecalis ferments sugars through different pathways, resulting in homo- or mixed acid fermentation. In homolactic bacteria glucose is converted to lactate in an ATP producing reaction. In mixed acid fermentation, in addition to lactate production, glucose is also converted to acetate, acetoin, formate, ethanol and CO2. However, there is limited information regarding to regulation of the central energy metabolism of E. faecalis. The aim of this work was to extend our knowledge with respect to the central energy metabolism of E. faecalis by employing metabolite, transcriptome and proteome approaches. High performance liquid chromatography and gas chromatography were used for metabolite measurements. DNA microarray technology and two dimensional gel electrophoresis combined with mass spectrometry analysis were used in transcription and protein expression analysis, respectively. Combining these approaches has not been performed in metabolic analysis in E. faecalis and this should give an in-depth understanding about regulation of the central energy metabolism in E. faecalis. This work showed that in absence of ldh (lactate dehydrogenase) gene, E. faecalis metabolizes glucose to ethanol, formate and acetoin. The change from homolactic to mixed acid fermentation affected expression of several genes and proteins mostly involved in energy metabolism. These genes play an important role in the regulatory network controlling energy metabolism in E. faecalis including acetoin production, and NAD+/NADH ratio. Additional studies were carried out in order to investigate the mixed acid fermentation of wild-type E. faecalis in chemostat during steady state and glucose limiting growth. Growth at three different growth rates demonstrated that the bacterium responded differently depending on the growth rate. At the highest dilution rate (D=0.4 h-1) most of the glucose was converted to lactate while at the lowest dilution rate (D=0.05 h-1) it changed towards mixed acids fermentation. Interestingly, increased growth rate induced the transcription of the ldh gene while the amount of Ldh protein was more or less unaffected. The differences in glucose energy metabolism at different growth and pHs between E. faecalis and two other LAB (Streptococcus pyogenes and Lactococcus lactis) and their LDH negative mutants were also investigated. Of note, deletion of the ldh genes hardly affected the growth rate in chemically defined medium under microaerophilic conditions. Furthermore, deletion of ldh affected the ability for utilization of various substrates as a carbon source. The final study explored the effect of ascorbate on growth in the absence of glucose and showed that E. faecalis can grow on ascorbate. In summary, the work presented in this thesis gave new insights in regulation and strengthens our knowledge regarding the metabolic pathways of glucose fermentation through the metabolite analysis, regulation of transcription and protein expression.Melkesyrebakterier brukes som startkulturer i en rekke ulike gjæringsreaksjoner i forbindelse med produksjon av mat. Enkelte melkesyrebakterier har også evnen til å forårsake sykdom, og dette gjelder spesielt for enterokokker og streptokokker. Enterococcus faecalis er en kommensal tarmbakterie. Likevel finner man innenfor denne arten både patogene isolater såvel som stammer benyttet i fermentering av matvarer. E. faecalis bryter ned sukker gjennom flere ulike veier, med enten melkesyre (homolaktisk gjæring) eller en blanding av syrer (blandet syregjæring) som endeprodukt. Homolaktiske bakterier bryter ned glukose til melkesyre i en reaksjonskjede som produserer ATP. Ved blandet syregjæring av glukose produseres det i tillegg til melkesyre også eddiksyre, acetoin, maursyre, etanol og CO2. Det er imidlertid lite informasjon om reguleringen av energimetabolismen i E. faecalis tilgjengenlig. Målet med arbeidet bak denne avhandlingen har derfor vært å tilegne oss kunnskap om den sentrale energimetabolismen i E. faecalis ved hjelp av ulike metoder for å studere metabolitter, transkriptomet og proteomet. Væskekromatografi og gasskromatografi ble brukt til metabolittmålinger, mens DNA mikromatriseteknologi og to-dimensjonal gelelektroforese kombinert med massespektroskopi ble brukt til henholdsvis transkripsjon- og proteinanalyser. Kombinasjonen av disse metodene har ikke tidligere blitt brukt i metabolske studier av E. faecalis, og vil derfor forhåpentligvis gi en dypere forståelse av overgangen mellom homolaktisk- og blandet syregjæring. Våre studier viser at i fravær av ldh genet, som koder for laktatdehydrogenase, blir glukose brutt med til etanol, maursyre og acetoin. Denne overgangen fra homolaktisk til blandet syregjøring påvirker uttrykket av en rekke gener og proteiner involvert i energimetabolismen. Genene innehar viktige roller i det regulatoriske nettverket som kontrollerer energimetabolismen i E. faecalis, og inkluderer gener involvert i produksjon av acetoin og balansen mellom NAD+/NADH. Videre studier ble også gjort for å undersøke blandet syregjæring i villtype E. faecalis i kjemostat ved likevektstilstand og glukosebegrenset vekst. Vekst ved tre forskjellige veksthastigheter viste av bakterien responderer forskjellig avhengig av veksthastighet. Ved den høyeste fortynningshastigheten (D=0.4 h-1) ble det meste av glukosen omdannet til melkesyre, mens en endring i retning av blandet syrefermentering ble observert ved den laveste fortynningshastigheten (D=0.05 h-1). Interessant nok så førte økt veksthastighet til økt transkripsjon av ldh-genet, men mengden Ldh-protein var tilnærmet uendret. Forskjellene i nedbrytning av glukose ved forskjellige veksthastigheter og ved forskjellig pH mellom E. faecalis og to andre melkesyrebakterier (Streptococcus pyogenes and Lactococcus lactis) ble også undersøkt. Det er her verdt å merke seg at inaktivering av ldh genene hadde liten innvirkning på veksthastigheten til de ulike bakteriene i kjemisk definert medium under mikroaerofile vekstforhold. Inaktiveringen av ldh påvirket også bakterienes evne til å utnytte andre substrater enn glukose som karbonkilde. I det siste arbeidet i avhandlingen ble det vist at E. faecalis i fravær av glukose er istand til å vokse på askorbinsyre. Sett under ett har arbeidet som er presentert i denne avhandlingen, gjennom analyser av metabolitter, transkripsjonregulering og proteinuttrykk, gitt økt innsikt i reguleringen av og styrket vår kjennskap til veiene for nedbrytning av glukose.Norges Forskningrå

Antimicrobial resistance levels amongst staphylococci isolated from clinical cases of bovine mastitis in Kosovo

Introduction: Mastitis is one of the most frequent and costly disease in cattle. We studied milk samples from cattle with mastitis from farms in Kosovo to identify mastitis-causing pathogens and possible effective antibiotics. Our ultimate goal is to help implement adequate antibiotic management and treatment practices in Kosovo Methodology: A total of 152 milk samples were collected from cows with clinical mastitis from different farms in Kosovo. After identification of microorganisms, antibiotic susceptibility and the occurrence of enterotoxins was investigated. Results: Staphylococci were found in 89 samples, of which 58 were coagulase negative and 31 coagulase positive. S. aureus was isolated from 27 samples, S. epidermidis from 25, and S. chromogenes from 15, while other species of staphylococci were isolated from the remaining 22 isolates. Interestingly, the bacterial diversity was different between cows in different periods of lactation and among different breeds. Most of the isolates (76/89) were resistant to two or more antibiotics. The highest resistance was to penicillin and ampicillin (> 65%), followed by tetracycline, oxacillin, streptomycin, chloramphenicol (> 23%), and less than 3% to erythromycin. Of the 89 isolates, 40 produced enterotoxins that were most frequently typed as A and C. Conclusions: We detected human bacterial pathogens in the cultures of milk samples from cows with mastitis. The isolates demonstrated resistance to two or more antibiotics, some of which are frequently used to treat animal and human infections. We recommend increased control and more stringent use of antibiotics in veterinary as well as human medicine

Antimicrobial resistance levels amongst staphylococci isolated from clinical cases of bovine mastitis in Kosovo

Introduction: Mastitis is one of the most frequent and costly disease in cattle. We studied milk samples from cattle with mastitis from farms in Kosovo to identify mastitis-causing pathogens and possible effective antibiotics. Our ultimate goal is to help implement adequate antibiotic management and treatment practices in Kosovo Methodology: A total of 152 milk samples were collected from cows with clinical mastitis from different farms in Kosovo. After identification of microorganisms, antibiotic susceptibility and the occurrence of enterotoxins was investigated. Results: Staphylococci were found in 89 samples, of which 58 were coagulase negative and 31 coagulase positive. S. aureus was isolated from 27 samples, S. epidermidis from 25, and S. chromogenes from 15, while other species of staphylococci were isolated from the remaining 22 isolates. Interestingly, the bacterial diversity was different between cows in different periods of lactation and among different breeds. Most of the isolates (76/89) were resistant to two or more antibiotics. The highest resistance was to penicillin and ampicillin (> 65%), followed by tetracycline, oxacillin, streptomycin, chloramphenicol (> 23%), and less than 3% to erythromycin. Of the 89 isolates, 40 produced enterotoxins that were most frequently typed as A and C. Conclusions: We detected human bacterial pathogens in the cultures of milk samples from cows with mastitis. The isolates demonstrated resistance to two or more antibiotics, some of which are frequently used to treat animal and human infections. We recommend increased control and more stringent use of antibiotics in veterinary as well as human medicine

Antibacterial activity of different extracts of Centaurea cyanus (L.) growing wild in Kosovo

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Quality characterisation of traditionally made yoghurt

Yoghurt is considered healthy food product and very popular containing different bacteria possible to use as a probiotic. The product is very healthy, especially when it’s made in natural form without adding any additives making it even healthier. In our country yoghurt is one of the most used products and almost all farmers and dairy companies produce it. However, receipts are different depends on culture or methodologies from farmer to farmer, or from companies to company and this it makes different in test and in quality. Based on that, organoleptic analyses, physico-chemical analyses, and some microbiological parameters has been study. Questioners has been made and around 190 people has been interviewed for organoleptic analyses, acidity, fat, proteins, aroma and salt in physico-chemical aspect and total viable count and diversity of lactic acid bacteria has been analyse. Results show that theof people regarding to yoghurt is positive, but it needs to be changer on taste. The differences between homemade yogurt and company do exist. No significant differences have been shown in phyco-chemical aspects. While, in microbiological aspects total viable counts variate and diversity of lactic acid bacteria exist. This shows that, different technology is used from different company and farmers are used. Differences in the taste of yoghurt could be from adding different starter culture

Influence of brine concentration and ripening temperature on quality of sharri cheese

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Sugars Play an Important Roles in Expiry Date, Aroma and Tast in Different Fermented Dairy Products

Sugars play an important role in metabolic processes in lives. However, different sugars transfer differently energy in different pathways. This differentiation makes a food product to have different test and to increase the quality of products. In another hands, lactic acid bacteria play an important role in production of lactic acid and aroma compounds in fermented food. The aim of this paper describes the growth rate and metabolic pathway of different products and bacteria when different sugars are added in products and bacteria. To explain this model Enterococcus faecalis is added in experiment and different sugars such as glucose, galactose, fructose, lactose, maltose, and sucrose. The experiment has been analysed by growing the culture for 24h and check the growth rate and analyse by high performance liquid chromatography. The result shows that glucose is the best metabolize sugars followed by fructose, sucrose, maltose, galactose, and lactose as a carbon source. While in energy transformation galactose and lactose transfer most of the energy to mixed acid fermentation compared to glucose and galactose which these energies it transfers into the homofermentative- fermentation. These results are ambitious results to apply and possibly to increase the expiry date of fermented products in dairy industries

Influence of Preparation Technology and Storage Condition on the Bacterial Growth in Cream

Milk is highly important product containing different proteins, fats, lactose, various vitamins and minerals. Different products made from milk do they exists. Cream is one of them. Cream is a dairy product composed of the higher- fat concentration from the top of milk before homogenisation. The aim of this work is to analyse differences between home-made cream and different dairy technology and effect of temperature after processing in microbiological aspects. Total viable counts by using count plates have been analysed from samples which is cream has made in home by traditional methods and from companies which used more professional technology. In same time cream has saved in different temperature (4⁰C, 25⁰C and 37⁰C) in different time points has been analysed. Differences in microbiological aspects between homemade cream and dairy company do they exist. Advantages and disadvantages are in both sides. Microflora of homemade variate up to 10*5 while from new technology level of microorganisms is very low. However, in different temperature, level of total viable counts increases in same range between each other