Extracellular proteinases in natural isolates of Staphylococci

U ovom radu su detaljnije biohemijski okarakterisane proteinaze prirodnih izolata Staphylococcus sp. F22, F86, M104, S2007 i S2105. Utvrđeno je da se radi o proteinazama relativno male molekulske mase (od 20 do 32 kDa) koje spadaju u prave ekstracelularne enzime, pošto se sa površine ćelije odvajaju u medijum za rast. Temperaturni optimumi ovih proteinaza se kreću od 30 do 37°C, a pH optimumi u opsegu od 6,5 do 8. Joni bakra inhibiraju njihovu aktivnost, dok joni kalcijuma stimulišu aktivnost proteinaza iz izolata F22 M104 i S2007. Pored kazeinskih frakcija, proteinaze stafilokoka hidrolizuju i druge proteinske supstrate, kao što su želatin i BSA. U eksperimentima sa proteinaznim inhibitorima utvrđeno je da proteinaze izolata F22 i M104 pripadaju serinskoj klasi, proteinaze S2007 i S2105 klasi metaloproteinaza dok proteinazu izolata F86 na ovaj način nije bilo moguće precizno klasifikovati.Biochemical characteristics of proteinases from natural isolates of Staphylococcus sp. F22, F86, M104, S2007 and S2105 have been studied. It was found that these proteinases have relatively low molecular masses (from 20 to 32 kDa), and that they are released from the cell envelope in to the growth medium. Their temperature optima are between 30 and 37°C and their pH optima range from 6,5 to 8. Copper ions inhibit their activity, but the presence of calcium ions stimulates the activity of proteinases from isolates F22, M104 and S2007. Beside casein fractions, they also hydrolyze heterologous protein substrates, such as BSA and gelatin. Experiments with specific proteinase inhibitors revealed that proteinases from isolates F22 and M104 belong to the serine group of proteinases, S2007 and S2105 proteinases were classified as metalloproteinases. Type of F86 proteinase in these experiments could not be clearly determinated

Dominant lactic acid bacteria in artisanal Pirot cheeses of different ripening period

U ovom radu su ispitivana dva sira od svežeg kravljeg mleka različitog perioda zrenja. Sirevi su uzeti iz seoskog domaćinstva u regionu Stare Planine, a proizvedeni su bez dodatka starter kulture. Iz oba sira je izolovano ukupno 106 sojeva bakterija mlečne kiseline. Sojevi su testirani klasičnim fiziološkim i API 50 CH testovima. Takođe je ispitivana proteolitička i antimikrobna aktivnost. Identifikacija bakterija mlečne kiseline je rađena rep-PCR analizom sa (GTG)5 prajmerom. Osam vrsta bakterije mlečne kiseline je izolavano iz sira BGPT9 starog četiri dana (Lactobacillus plantarum, Lactobacillus paracasei subsp. paracasei, Lactobacillus delbrueckii, Lactobacillus brevis, Enterococcus faecium, Enterococcus faecalis, Enterococcus durans i Leuconostoc garlicum), dok su u siru BGPT10 starom osam meseci bile prisutne samo dve vrste (Lactobacillus plantarum i Enterococcus faecium). Proteolitičku aktivnost je pokazalo 30 izolata iz sira BGPT9, uglavnom enterokoke. Samo jedan izolat iz sira BGPT10 (koji je pripadao vrsti Lactobacillus plantarum) je posedovao delimičnu sposobnost da hidrolizuje β- kazein. Sedam enterokoka iz sira BGPT9 i četiri enterokoke iz sira BGPT10 je proizvodilo antimikrobne substance.In this study two raw cow's milk cheeses of a different ripening period were examined. The cheeses were taken from a country household in the region of mountain Stara Planina and manufactured without adding of starter culture. A total 106 lactic acid bacteria (LAB) strains were isolated from both cheeses. They are tested by classical physiological tests as well as by API 50 CH tests. Proteolytic and antimicrobial activities were done too. Identification of LAB isolates was done by repetitive extragenic palindromic-polimerase chain reaction (rep-PCR) with (GTG)5 primer. The LAB isolates from cheese BGPT9 (four days old) belonged to the eight species of LAB (Lactobacillus plantarum, Lactobacillus paracasei subsp. paracasei, Lactobacillus delbrueckii, Lactobacillus brevis, Enterococcus faecium, Enterococcus faecalis, Enterococcus durans and Leuconostoc garlicum), while in the BGPT10 cheese (eight months old) only two species were present (Lactobacillus plantarum and Enterococcus faecium). Proteolytic activity showed 30 LAB from BGPT9 cheese, mainly enterococci. From BGPT10 cheese only one isolate (which belonged to the Lactobacillus plantarum species) possessed partial ability to hydrolyze β-casein. Seven enterococci from BGPT9 cheese and four enterococci from BGPT10 cheese produced antimicrobial compounds

Molecular characterization of semi-hard homemade cheese microflora

U poslednjoj deceniji zabeležen je nagli razvoj molekularnih tehnika baziranih na 16S i 23S rRNK, koje se koriste u izučavanju biodiverziteta mikroorganizama. U ovom radu ispitivana je mikroflora polutvrdog sira pripremljenog u domaćinstvu. Zbog visokog sadržaja masti u ovom siru razvili smo novu tehniku za izolaciju totalne DNK iz sira (metod je baziran na bead beating-u). Brza izolacija DNK iz mikroflore sira omogućila nam je molekularnu identifikaciju BMK (BAKTERIJE MLEČNE KISELINE) na osnovu umnožavanja gena za 16S rRNK PCR metodom. Za umnožavanje su korišćeni prajmeri specifični za gene 16S rRNK lakto-bacila, ali su uslovi PCR reakcije bili takvi da su omogućavali i umnožavanje gena 16S rRNK laktokoka. Rezultati RFLP analize pokazali su da mikrofloru sira pripremljenog u domaćinstvu čine predominantno laktokoke.This decade has shown an impressive development in the application of molecular techniques based on 16S and 23S rRNA genes to study the microbial diversity in various ecosystems. Microflora of semi-hard homemade cheese was examined in this work. We developed a novel technique for DNA extraction (a bead beating based method) due to high fat content of this cheese. Rapid extraction of DNA from cheese microflora enabled a molecular identification of the LAB (Lactic Acid Bacteria) strains based on PCR amplification of 16S RNA coding sequences. The specific primers for 76S RNA gene of lactobacilli were used for amplification. The PCR reaction was performed at lower temperature, where the specificity of the annealing reaction was reduced and lactococcal sequences of 16S RNA genes were also amplified. The results of RFLP analysis revealed that the microflora of Doboj homemade cheese encompases mostly lactococci

Dynamics of sodium dodecyl sulfate utilization andantibiotic susceptibility of strain Pseudomonas sp. ATCC19151

Poznato je da soj Pseudomonas sp. ATCC19151 poseduje gen koji kodira Potencijalnu alkilsulfatazu. U ovom radu analizirana je sposobnost rasta ovog soja u minimalnom medijum usa različitim koncentracijama natrijum dodecilsulfata (0.5, 0.75 i 1 %) kao jedinim izvorom ugljenika. Pokazano je da Pseudomonas sp. ATCC19151 ispoljava najbolji rast uminimalnom medijumusa 0.5 % natrijum dodecil sulfata, te je stoga ova koncentracija uzeta kao optimalna za testiranje dinamike korišćenja natrijum dodecil sulfata tokom različitih faza rasta. Dinamika korišćenja natrijum dodecil sulfata podudarala se sa rastom kulture. Pored toga u cilju detaljnije karakterizacije soja, analizirana je i osetljivost Pseudomonas sp. ATCC19151 na antibiotike. Pokazano je da je analizirani soj rezistentan na šest (ampicilin, tetraciklin, hloramfenikol, tobramicin, nalidiksičnukiselinui gentamicin) od devet analiziranih antibiotika.Pseudomonas sp. ATCC19151 harbors a gene encoding a putative alkylsulfatase (sdsA). Here we report a growth ability of this strain in minimal media containing 0.5, 0.75, and 1% sodium dodecyl sulfate as the sole carbon source. The most prominent growth was detected for the minimal medium with 0.5% SDS, so this concentration of SDS was used to monitor Pseudomonas sp. ATCC19151 SDS biodegradation dynamics. Bacterial growth coincided with the disappearance of SDS. Antibiotic susceptibility was tested as well. Pseudomonas sp. ATCC19151 was resistant to six out of nine tested antibiotics, including ampicillin, tetracycline, chloramphenicol, tobramycin, nalidixic acid, and gentamycin

Influence of carbohydrates on cell properties of Lactobacillus rhamnosus

Lactobacilli represent normal commensals of the human body, particularly in the gut and vagina where they protect these environments from incoming pathogens via a variety of mechanisms. The influence of the carbohydrate source present in reconstituted MRS growth medium on the different cell properties of two Lactobacillus rhamnosus strains were examined. Two human vaginal isolates, BGHV719 and exopolysaccharide producer strain BGHV954 were analyzed. The results demonstrated that unlike in reconstituted MRS with glucose as a carbon source, the presence of fructose, mannose, or rhamnose, significantly reduced cell surface hydrophobicity of both strains. In addition, differences in cell wall protein composition of L. rhamnosus BGHV719 and alterations in colony mucoidity of L. rhamnosus BGHV954 were also demonstrated. Light and SEM microscopy revealed differences on the cellular level when BGHV719 was cultivated in the presence of different sugars. The results of this study point out the importance of complex relationships between growth medium composition and the different aspects of bacterial behavior, and call for more detailed analyses of versatile bacterial responses to the changes in the environment, including vaginal ecosystem. This is especially important since lactobacilli are amongst the most widely used of probiotics

Improved sensitivity and reproducibility of the PCR method for detection of Listeria spp. and L. monocytogenes in milk

Listeria monocytogenes, prouzrokovač listerioze kod ljudi i životinja, je fakultativan intraćelijski mikroorganizam široko rasprostranjen u prirodi. U cilju izolacije i detekcije L. monocytogenes iz hrane koriste se tradicionalne mikrobiološke i nove molekularno-genetičke metode. Cilj ovog rada je bio povećanje osetljivosti i ponovljivosti PCR metode u detekciji L. monocytogenes u mleku. U tu svrhu, uzorci pasterizovanog mleka su kontaminirani serijskim razblaženjima sojeva L. monocytogenes 4b ATCC 119115 i Listeria innocua ATCC 33090. Dobijeni rezultati na veštački kontaminiranim uzorcima pasterizovanog mleka, ukazuju da osetljivost PCR metode zavisi od perioda inkubacije i izbora prajmera. Najbolji rezultati su dobijeni nakon 24 h inkubacije, pomoću prajmera za hlyA gen, kada je bilo moguće detektovati 1 ćeliju L. monocytogenes tj. 1 CFU/ml.Listeria monocytogenes is a facultative intracellular Grampositive bacterium, ubiquitous in nature and capable of causing listeriosis in humans and animals. Conventional microbiological techniques and modern molecular approaches are currently used for the isolation and detection of L. monocytogenes in food samples. The aim of this study was to improve the sensitivity and reproducibility of PCR for the detection of Listeria spp. in milk. For that purpose milk samples were artificially inoculated with serial dilutions of L. monocytogenes 4b ATCC 19115 and L. innocua ATCC 33090. The results obtained on artificially contaminated milk samples indicated that incubation time and target genes have an influence on the sensitivity of PCR detection. The best results were obtained after 24 h of preenrichment, with primers complementary to the hlyA gene, when it was possible to detect 1 CFU/mL of Listeria spp

Analysis of natural isolates of Lactobacilli resistant to bacteriocin nisin

Kolekcija bakterija mlečne kiseline (BMK) je napravljena od mikroorganizama izolovanih iz fermentisanih mlečno-kiselinskih proizvoda dobijenih na tradicionalan način. Iz kolekcije 51 izolat je identifikovan kao Lactobacillus sp. Svi izolovani laktobacili pripadaju grupi mezofilnih sojeva koji dobro rastu na temperaturama od 15°C i 30°C, a ne rastu na temperaturi od 45°C. Testiranje sposobnosti rasta u prisustvu nizina pokazalo je da su izolati BGCGK4, BGHN40, BGBUK2-8, BGBUK2-7 i BGBUK2-16 rezistentni na bakteriocin nizin. U eksperimentu određivanja minimalne inhibitorne koncentracije (MIK) za nizin pokazano je da je najrezistentniji izolat Lactobacillus sp. BGCGK4. Izolat BGBUK2-16, determinisan kao Lactobacillus paracasei subsp. paracasei, produkuje bakteriocin označen kao Bac217 i pokazuje rezistenciju na 8000 IU/ml. Čišćenjem plazmida iz soja BGBUK2-16 dobijena su 2 derivata označena kao BGBUK2-16/K2 i BGBUK2-16/K4. Derivat BGBUK2-16/K2 zadržao je rezistenciju na Bac217 i nizin, ali je izgubio sposobnost sinteze Bac217, dok je derivat BGBUK2-16/K4 pored gubitka sposobnosti sinteze Bac217 postao senzitivan na Bac217 i nizin. Prirodno rezistentni laktobacili se mogu iskoristiti za pripremanje starter kultura u kombinaciji sa nizinom kao konzervansom u cilju kontrolisane mlečno-kiselinske fermentacije.The collection of lactic acid bacteria (LAB) was made by isolation of microorganisms from fermented products traditionally manufactured in different geographical regions (high mountains, river valleys, seaside, etc). Among collected LAB, 51 isolates were identified as Lactobacillus sp. Results showed that all isolated lactobacilli were mesophilic strains, since they grew at 15°C and 30°C but not at 45°C. Testing the ability of isolated lactobacilli to grow in the presence of nisin revealed that Lactobacillus sp. isolates designed BGCGK4, BGHN40, BGBUK2-8, BGBUK2-7 and BGBUK2-16 were resistant to nisin. Determination of the minimal inhibitory concentrations (MIC) for nisin revealed that the most resistant isolate was Lactobacillus sp. BGCGK4. Isolate BGBUK2-16, determined as Lactobacillus paracasei subsp. paracasei, produces bacteriocin, named Bac217 and showed a resistance to 8000 IU/ml of nisin. Plasmid curing of BGBUK2-16 resulted in derivatives BGBUK2-16/K2 and BGBUK2-16/K4. Derivative BGBUK2-16/K2 retained resistance to Bac217 and nisin, but lost the ability to synthesise Bac217. Derivative BGBUK2-16/K4 lost concomitantly the resistance to both Bac217 and nisin

Effect of methionine and cysteine deprivation on growth of different natural isolates of Lactobacillus spp. in chemically defined media

Cilj ovog rada je bio da se utvrdi sposobnost prirodnih izolata laktobacila,izolovanih iz različitih ekoloških niša da rastu u hemijski definisanom medijumu sa ili bez prisustva aminokiselina koje sadrže sumpor, metionin i/ili cistein. Dobijeni rezultati su pokazali da je esencijalna aminokiselina za rast izolata L. paracasei subsp. paracasei BGHN14 i BGSJ2-8 cistein, dok je za izolate BGHN40, BGCG31, BGHV54T, koji pripadaju vrsti L. plantarum-metionin. Metionin je esencijalna aminokiselina za rast soja L. rhamnosus BGHV58T. Ostali analizirani sojevi,kao što su L. plantarum BGSJ3-18,BGZB19,BGHV52Ta i BGHV43T za svoj rast zahtevaju prisustvo obe aminokiseline u medijumu.The purpose of this study was to determine the ability of natural isolates of lactobacilli from different ecological niches to grow in a chemically defined medium in the presence or absence of sulphur-containing amino acids, methionine and/or cysteine. The obtained results indicate that cysteine is essential for growth of L. paracasei subsp. paracasei BGHN14 and BGSJ2-8, while methionine is essential for isolates BGHN40, BGCG31, and BGHV54T of the species L. plantarum. Methionine is also essential for growth of L. rhamnosus BGHV58T. Other analyzed strains, such as L. plantarum BGSJ3-18, BGZB19, BGHV52Ta, and BGHV43T, require the presence of both amino acids for their growth

Emergence of VIM-2 metallo-beta-lactamase-producing Pseudomonas aeruginosa isolates in a paediatric hospital in Serbia

Molecular detection and surveillance of the resistance genes harboured by Pseudomonas aeruginosa are becoming increasingly important in assessing and controlling spread and colonization in hospitals, and in guiding the antibiotic treatment of infections. Metallo-blactamase (MBL)-producing P. aeruginosa strains are slowly but steadily increasing within hospitals, causing outbreaks and/or hyperendemic situations in some centres, mostly in the Far East and the south of Europe (Queenan & Bush, 2007). The global dissemination of MBL-producing P. aeruginosa strains has also reached the Balkan region (Lepsanovic et al., 2008; Sardelic et al., 2003). The objective of our study was to detect and characterize P. aeruginosa isolates producing MBLs from the 400-bed paediatric tertiary care hospital Mother and Child Health Institute of Serbia ‘Dr Vukan Cupic

Post-translational regulation of the RpoS and PsrA genes in pseudomonas putida WCS358: The role of ClpXP protease

RpoS i PsrA proteini su ključni transkripcioni regulatori koji se kod pseudomonada aktiviraju u odgovoru na stacionarnu fazu rasta. Cilj ove studije bio je utvrđivanje uloge ClpXP (ATP zavisna serin proteaza) u stabilnosti RpoS i PsrA proteina tokom različitih faza rasta u Pseudomonas putida WCS358. "Western blot" analiza proteinskih ekstrakata P. putida WCS358 i P. putida WCS358 clpX::Km iz rane eksponencijalne, Kasne eksponencijalne i stacionarne faze rasta, sa antitelima na RpoS i PsrA, pokazala je da ClpXP degraduje RpoS i PsrA u ranoj eksponencijalnoj fazi rasta.The RpoS and PsrA proteins are key transcriptional regulators that are activated in response to the stationary phase of growth in pseudomonads. This study was designed to establish whether ClpXP (ATP-dependent serine protease) regulates levels of RpoS and PsrA in Pseudomonas putida WCS358. Western blot analysis of P. putida WCS358 protein extracts from the early exponentianl, late exponential, and stationary phases of growth with antibodies against RpoS and PsrA revealed that these proteins are degraded by ClpXP in the early exponential phase of growth. The obtained results demonstrate a role for ClpXP protease in post-translational regulation of proteins encoded by the rpoS and psrA genes in Pseudomonas spp