Genetic and restriction analysis of the 16S-23S rDNA internal transcribed spacer regions of the acetic acid bacteria

The 16S-23S rDNA internal transcribed spacer regions of the acetic acid bacteria were sequenced and evaluated for molecular identification of these bacteria. All the sequenced spacers contained genes for tRNAIle and tRNAAla, and the antitermination element. The sequences revealed 56.8-78.3% similarity. By PCR amplification of the spacers from 57 strains of acetic acid bacteria, single products of similar sizes were produced. Digestion of the spacers by HaeIII and HpaII restriction enzymes resulted in 12 distinct groups of restriction types. All the restriction profiles obtained after analysis of microbial populations from vinegar matched one of the 12 group

Identifikacija bakterija octenog vrenja izoliranih iz organski i konvencionalno proizvedenog jabučnog octa

Organic apple cider vinegar is produced from apples that go through very restricted treatment in orchard. During the first stage of the process, the sugars from apples are fermented by yeasts to cider. The produced ethanol is used as a substrate by acetic acid bacteria in a second separated bioprocess. In both, the organic and conventional apple cider vinegars the ethanol oxidation to acetic acid is initiated by native microbiota that survived alcohol fermentation. We compared the cultivable acetic acid bacterial microbiota in the production of organic and conventional apple cider vinegars from a smoothly running oxidation cycle of a submerged industrial process. In this way we isolated and characterized 96 bacteria from organic and 72 bacteria from conventional apple cider vinegar. Using the restriction analysis of the PCR-amplifi ed 16S-23S rRNA gene ITS regions, we identified four different HaeIII and five different HpaII restriction profiles for bacterial isolates from organic apple cider vinegar. Each type of restriction profile was further analyzed by sequence analysis of the 16S-23S rRNA gene ITS regions, resulting in identification of the following species: Acetobacter pasteurianus (71.90 %), Acetobacter ghanensis (12.50 %), Komagataeibacter oboediens (9.35 %) and Komagataeibacter saccharivorans (6.25 %). Using the same analytical approach in conventional apple cider vinegar, we identified only two different HaeIII and two different HpaII restriction profiles of the 16S‒23S rRNA gene ITS regions, which belong to the species Acetobacter pasteurianus (66.70 %) and Komagataeibacter oboediens (33.30 %). Yeasts that are able to resist 30 g/L of acetic acid were isolated from the acetic acid production phase and further identified by sequence analysis of the ITS1-5.8S rDNA‒ITS2 region as Candida ethanolica, Pichia membranifaciens and Saccharomycodes ludwigii. This study has shown for the first time that the bacterial microbiota for the industrial production of organic apple cider vinegar is clearly more heterogeneous than the bacterial microbiota for the industrial production of conventional apple cider vinegar. Further chemical analysis should reveal if a difference in microbiota composition influences the quality of different types of apple cider vinegar.„Organski“ jabučni ocat proizvodi se od jabuka uzgojenim prema vrlo strogim ekološkim kriterijima. U prvoj fazi proizvodnje kvasci fermentiraju šećer iz jabuka u jabučno vino. U drugoj fazi bakterije octenog vrenja fermentiraju nastali etanol u jabučni ocat. Oksidacija etanola u octenu kiselinu u oba postupka proizvodnje octa, organskom i konvencionalnom, potaknuta je aktivnošću bakterija koje su preživjele alkoholno vrenje. U radu su ispitane bakterije octenog vrenja izolirane tijekom submerznog postupka organske i konvencionalne proizvodnje jabučnog octa. Izolirano je i okarakterizirano 96 sojeva bakterija iz organskog i 72 soja iz konvencionalno proizvedenog jabučnog octa. Restrikcijskom su analizom intergenskih regija 16S‒23S rDNA bakterija izoliranih iz organskog jabučnog octa, umnoženih lančanom reakcijom polimeraze, identificirana četiri restrikcijska profila HaeIII i pet profila HpaII. Sekvencioniranjem i analizom restrikcijskih profila identificirane su sljedeće vrste bakterija: Acetobacter pasteurianus (71,90 %), Acetobacter ghanensis (12,50 %), Komagataeibacter oboediens (9,35 %) i Komagataeibacter saccharivorans (6,25 %). Na isti su način u konvencionalno proizvedenom jabučnom octu identificirana samo dva HaeIII i dva HpaII restrikcijska profila, koji pripadaju vrstama Acetobacter pasteurianus (66,70 %) i Komagataeibacter oboediens (33,30 %). Tijekom proizvodnje octene kiseline izolirani su kvasci koji mogu rasti pri koncentraciji octene kiseline od 30 g/L, te je sekvencijskom analizom potvrđeno da pripadaju sljedećim vrstama: Candida ethanolica, Pichia membranifaciens and Saccharomycodes ludwigii. Rezultati rada po prvi put dokazuju da su bakterije koje se koriste u proizvodnji organskog jabučnog octa raznovrsnije od onih korištenih u konvencionalnoj industrijskoj proizvodnji octa. Dodatnim bi se kemijskim analizama trebalo ispitati utječe li raznovrsnost mikrobiote na kakvoću različitih tipova jabučnog octa

Brzi postupak identifikacije bakterija octenog vrenja tijekom submerzne industrijske proizvodnje octa, lokalizacijom 16S rRNA pomoću hibridizacije in situ i protočne citometrije

Acetic acid bacteria are involved in many biotechnological processes such as vitamin C, gluconic acid, miglitol or acetic acid production, and others. For a technologist trying to control the industrial process, the ability to follow the microbiological development of the process is thus of importance. During the past few years hybridization in a combination with flow cytometry has often been used for this purpose. Since vinegar is a liquid, it is an ideal matrix for flow cytometry analysis. In this work we have constructed a specific probe for highly acetic acid-resistant species of the acetic acid bacteria and a protocol for in situ hybridization, which in combination with flow cytometry enables direct monitoring of bacteria producing vinegar with >10 % of acetic acid. The approach was successfully applied for monitoring microbiota during industrial vinegar production.Bakterije octenog vrenja koriste se u mnogim biotehnološkim procesima, između ostalog u proizvodnji vitamina C, miglitola (inhibitora α-glucozidaze), glukonske ili octene kiseline. Tijekom industrijske proizvodnje izrazito je važno omogućiti praćenje aktivnosti mikroorganizama radi kontrole postupka, pa se u tu svrhu u novije vrijeme često koristi hibridizacija in situ u kombinaciji s protočnom citometrijom. Ocat je tekućina, pa je idealan medij za protočnu citometriju. Pripremom nove specifične probe za sojeve bakterija octenog vrenja koji mogu rasti pri većim udjelima octene kiseline, te izradom novog protokola hibridizacije in situ, u kombinaciji s protočnom citometrijom omogućeno je izravno praćenje bakterija u proizvodnji octa s udjelom octene kiseline većim od 10 %. Metoda je uspješno upotrijebljena za praćenje mikrobiote tijekom industrijske proizvodnje octa

Yersinia enterocolitica type III secretion: Evidence for the ability to transport proteins that are folded prior to secretion

BACKGROUND: Pathogenic Yersinia species (Y. enterocolitica, Y. pestis, Y. pseudotuberculosis) share a type three secretion system (TTSS) which allows translocation of effector proteins (called Yops) into host cells. It is believed that proteins are delivered through a hollow needle with an inner diameter of 2–3 nm. Thus transport seems to require substrates which are essentially unfolded. Recent work from different groups suggests that the Yersinia TTSS cannot accommodate substrates which are folded prior to secretion. It was suggested that folding is prevented either by co-translational secretion or by the assistance of specific Yop chaperones (called Sycs). RESULTS: In this study we have fused YopE secretion signals of various length to the mouse dihydrofolate reductase (DHFR) in order to analyse the DHFR folding state prior to secretion. We could demonstrate that secretion-deficient as well as secretion-competent YopE-DHFR fusions complexed to SycE can be efficiently purified from Yersinia cytosol by affinity chromatography using methotrexate-agarose. This implies the folding of the DHFR fusion moiety despite SycE binding and contradicts the previously presented model of folding inhibition by chaperone binding. Secretion-deficient YopE-DHFR fusions caused severe jamming of the TTSS. This observation contradicts the co-translational secretion model. CONCLUSIONS: We present evidence that the Yersinia TTSS is familiar with the processing of transport substrates which are folded prior to secretion. We therefore predict that an unfoldase is involved in type III secretion

Antimicrobial resistance of Acetobacter and Komagataeibacter species originating from vinegars

Consumers’ preference towards healthy and novel foods dictates the production of organic unfiltered bottled vinegar that still contains acetic acid bacteria. After ingesting vinegar, the bacteria come into close contact with the human microbiota, creating the possibility of horizontal gene transfer, including genetic determinants for antibiotic resistance. Due to the global spread of antimicrobial resistance (AMR), we analyzed the AMR of Acetobacter and Komagataeibacter species originating mainly from vinegars. Six antibiotics from different structural groups and mechanisms of action were selected for testing. The AMR was assessed with the disk diffusion method using various growth media. Although the number of resistant strains differed among the growth media, 97.4%, 74.4%, 56.4%, and 33.3% of strains were resistant to trimethoprim, erythromycin, ciprofloxacin, and chloramphenicol, respectively, on all three media. Moreover, 17.9% and 53.8% of all strains were resistant to four and three antibiotics of different antimicrobial classes, respectively. We then looked for antimicrobial resistance genes in the genome sequences of the reference strains. The most common genetic determinant potentially involved in AMR encodes an efflux pump. Since these genes pass through the gastrointestinal tract and may be transferred to human microbiota, further experiments are needed to analyze the probability of this scenario in more detail

Microvesicles as inflammatory and coagulant markers of human coronary artery endothelial cells following their stimulation with serum amyloid A

Ateroskleroza je vnetna bolezen arterij, ki predstavlja vodilni vzrok za razvoj številnih srčno-žilnih bolezni. Akutno-fazni protein serumski amiloid A (SAA) je napovedni dejavnik akutnih srčno-žilnih dogodkov, povečane vrednosti SAA pa so značilne za bolnike z aterosklerozo in so povezane z disfunkcijo endotelija. Endotelijski mikrovezikli so pokazatelji endotelijske disfunkcije pri srčno-žilnih boleznih in se kopičijo v aterosklerotičnih lehah. Naš namen je bil raziskati, kako SAA vpliva na izločanje mikroveziklov v endotelijskih celicah. Ker je za mikrovezikle znano, da sodelujejo pri vnetju in koagulaciji, nas je zanimalo, ali so endotelijski mikrovezikli pokazatelji vnetne in koagulantne aktivnosti endotelijskih celic po stimulaciji s SAA. Koncentracija protiteles proti SAA v serumu je znižana pri arterijski trombozi in nekaterih avtoimunskih boleznih s pospešeno aterosklerozo, zato smo raziskali tudi vpliv protiteles proti SAA na izločanje mikroveziklov. Človeške endotelijske celice koronarnih arterij smo stimulirali s človeškim rekombinantnim SAA in po različnih časih stimulacije izolirali mikrovezikle z metodo diferencialnega centrifugiranja. S pretočno citometrijo smo določili število mikroveziklov, vezavo aneksina A5 in prisotnost CD31, CD62E in tkivnega faktorja na mikroveziklih. Vzporedno smo določili vnetno aktivnost celic z določanjem interlevkina-6 in interlevkina-8 ter koagulantno aktivnost celic z določanjem izražanja in aktivnosti tkivnega faktorja. Rezultati so pokazali, da stimulacija endotelijskih celic s SAA povzroči izločanje mikroveziklov. Število mikroveziklov je naraščalo s časom stimulacije celic in se je povečevalo vzporedno s količino interlevkina-6 in interlevkina-8. Število mikroveziklov, pozitivnih na aneksin A5, CD31 in CD62E, ki so zvišani v različnih srčno-žilnih boleznih, kot je na primer koronarna arterijska bolezen, je naraščalo s časom stimulacije endotelijskih celic s SAA. SAA je sprožil tudi izražanje tkivnega faktorja na mikrovezklih. Izražanje tkivnega faktorja na mikroveziklih je bilo največje po 4 urah stimulacije in je sledilo izražanju in aktivnosti tkivnega faktorja na endotelijskih celicah. Pokazali smo, da SAA sproži mikrovezikulacijo endotelijskih celic, ki sledi vnetni in koagulantni aktivnosti celic. Protitelesa proti SAA so močno zmanjšala izločanje mikroveziklov tako po 4 kot po 24 urah stimulacije endotelijskih celic s SAA. Endotelijski mikrovezikli so tako lahko pokazatelji in/ali sodejavniki pri endotelijski disfunkciji, ki jo povzroči SAA.Atherosclerosis is an inflammatory arterial disease, which is the leading cause of several cardiovascular diseases. Elevated serum amyloid A (SAA), a positive acute-phase reactant, predicts future acute cardiovascular events and is associated with increased progression of atherosclerosis and endothelial dysfunction. Endothelial microvesicles are biomarkers of endothelial dysfunction in cardiovascular diseases and accumulate in atherosclerotic lesions. The aim of our study was to investigate the influence of SAA on microvesicle release in endothelial cells. Because microvesicles are known to participate in inflammation and coagulation, we determined, whether endothelial microvesicles could reflect SAA-induced proinflammatory and procoagulant response in endothelial cells. Since serum levels of anti-SAA antibodies are significantly lower in arterial thrombosis and some autoimmune diseases with accelerated atherosclerosis than in healthy blood donors, we investigated the influence of anti-SAA antibodies on endothelial microvesicle release. Human coronary artery endothelial cells were stimulated with human recombinant SAA and microvesicles were isolated by differential centrifugation following different times of stimulation. Flow cytometry was used to measure endothelial microvesicle number, annexin A5 binding and CD31, CD62E and tissue factor exposure. The inflammatory response was measured using interleukin-6 and interleukin-8 protein levels and the procoagulant response was measured with tissue factor exposure and activity. We demonstrated that SAA stimulation causes microvesicle release from endothelial cells. A time-dependent rise in microvesicle number correlated with interleukin-6 end interleukin-8 levels. The number of annexin A5, CD31 and CD62E-positive microvesicles (elevated in different cardiovascular diseases, such as coronary artery disease) increased with the time of stimulation. SAA caused tissue factor expression on microvesicles to peak at 4 hours and this increase correlated with tissue factor expression and activity in endothelial cells. Altogether, we showed that SAA causes microvesiculation that reflects inflammatory and procoagulant responses in endothelial cells. Anti-SAA antibodies decreased SAA-induced microvesicle release following 4 hour and 24 hour stimulation. Endothelial microvesicles may serve as biomarkers and/or mediators of SAA-induced changes of endothelial function

Genetic and restriction analysis of the 16S–23S rDNA internal transcribed spacer regions of the acetic acid bacteria

ISSN:0378-1097ISSN:0168-6496ISSN:0920-8534ISSN:0168-644

Bacterial Cellulose: Production, Modification and Perspectives in Biomedical Applications

Bacterial cellulose (BC) is ultrafine, nanofibrillar material with an exclusive combination of properties such as high crystallinity (84%–89%) and polymerization degree, high surface area (high aspect ratio of fibers with diameter 20–100 nm), high flexibility and tensile strength (Young modulus of 15–18 GPa), high water-holding capacity (over 100 times of its own weight), etc. Due to high purity, i.e., absence of lignin and hemicellulose, BC is considered as a non-cytotoxic, non-genotoxic and highly biocompatible material, attracting interest in diverse areas with hallmarks in medicine. The presented review summarizes the microbial aspects of BC production (bacterial strains, carbon sources and media) and versatile in situ and ex situ methods applied in BC modification, especially towards bionic design for applications in regenerative medicine, from wound healing and artificial skin, blood vessels, coverings in nerve surgery, dura mater prosthesis, arterial stent coating, cartilage and bone repair implants, etc. The paper concludes with challenges and perspectives in light of further translation in highly valuable medical products