Alternatywne metody badania stanu mikrobiologicznego wody oparte na technologii zdefiniowanych substratów stanowią doskonałe uzupełnienie metod referencyjnych, dostarczając istotnych informacji w krótkim czasie i umożliwiając podjęcie działania w sytuacjach krytycznych. W artykule opisano system Colilert-18/Quanti-Tray 2000 stanowiący przykład zastosowania technologii zdefiniowanych substratów, umożliwiający równoczesną detekcję bakterii grupy coli i E. coli bezpośrednio w badanej próbie wody. Wchodzące w skład pożywki substraty ONPG (chromogenny o-nitrofenylo- β-D-galaktopiranozyd) i MUG (fluorogenny 4-metyloumbeliferylo- β-D-glukuronid) są metabolizowane przez enzymy bakterii grupy coli i E. coli obecne w badanej próbie wody, w wyniku czego uwalniany jest żółty (β-D-galaktozydaza) lub fluoryzujący (β-D-glukuronidaza) produkt. Wysoka specyficzność enzymów, zastosowanych w omawianym systemie wyeliminowała konieczność wykonywania testów potwierdzających.
As a result of their unpredictable ability to adapt to varying environmental conditions, microorganisms inhabit different types of biological niches on Earth. Owing to the key role of microorganisms in many biogeochemical processes, trends in modern microbiology emphasize the need to know and understand the structure and function of complex microbial communities. This is particularly important if the strategy relates to microbial communities that cause biodeterioration of materials that constitute our cultural heritage. Until recently, the detection and identification of microorganisms inhabiting objects of cultural value was based only on cultivation-dependent methods. In spite of many advantages, these methods provide limited information because they identify only viable organisms capable of growth under standard laboratory conditions. However, in order to carry out proper conservation and renovation, it is necessary to know the complete composition of microbial communities and their activity. This paper presents and characterizes modern techniques such as genetic fingerprinting and clone library construction for the assessment of microbial diversity based on molecular biology. Molecular methods represent a favourable alternative to culture-dependent methods and make it possible to assess the biodiversity of microorganisms inhabiting technical materials and cultural heritage objects.
Taxonomic differentiation between Lactococcus sp. and Leuconostoc sp. can sometimes be misleading due to the morphological and biochemical similarities between both genera. Therefore, several molecular techniques have been applied to identify these bacteria. Restriction fragment length polymorphism analysis of PCR-amplified 16S ribosomal RNA gene was used to generate restriction profiles of 9 strains of Lactococcus sp. and 5 of Leuconostoc sp. This method utilizes a set of universal primers for amplification of the 16S rRNA region of typical lactic acid bacteria species. The size of the amplified products was about 1500 bp and the amplicons of the different species could be differentiated from each other with four restriction endonucleases: TaqI, EcoRI, BamHI and HindIII. These restriction enzymes were selected based on nucleotide sequences of 16S rRNA genes for LAB available in databases. Our study demonstrates that DNA of 16S rRNA from strains of Lactococcus sp. contains single restriction site for EcoRI and two restriction sites for TaqI enzymes, 16S rRNA DNA from strains of Leuconostoc sp. contains a single restriction site for each enzyme (HindIII, BamHI) and four restriction sites for TaqI. This result is in good agreement with analysis in silico of 16S rRNA genes published in the National Center for Biotechnology Information (NCBI). These findings led to modify the classification obtained by biochemical methods for five examined strains of lactic acid bacteria. In summary, our study demonstrated that the RFLP analysis applied is a useful method for rapid differentiation between Lactococcus sp. and Leuconostoc sp.
The presence of bacteria in the cosmetic production environment is often connected with non-sterile raw materials, inappropriate production lines disinfection or cross contamination. Among bacteria isolated from the environment, opportunistic pathogens can be also found, posing a risk to patients with lowered immunity. Moreover, their susceptibility to antibiotics and disinfectants is frequently decreased as they develop more complex forms - biofilms. As hydrophobicity and adhesive properties play a vital role in the colonization process the aim of this research was to determine hydrophobic, aggregative and adhesive properties of bacteria isolated from the cosmetics.Bacteria used in the research were isolated from the body balm and the cosmetic preservative (three strains of Pseudomonas aeruginosa and four strains of Pseudomonas cedrina) and identified using 16S rRNA gene sequencing. For those strains and also two reference strains (P. aeruginosa ATCC15442 and P. cedrina DSM17516) an aggregation test, hydrophobicity by two different methods (SAT and MATH) and adhesion to polystyrene by crystal violet binding assay were performed. According to the SAT method more than half of the tested strains were strongly hydrophobic. Using MATH test, it was proved that four strains (P. cedrina DSM17516 and three isolates of P. aeruginosa) were strong hydrophobes, however, the rest of the strains expressed moderate hydrophobicity. Moreover, self-aggregation was also observed and for P. aeruginosa CFII was more than 20%. All of the strains were able to adhere to polystyrene after 30 minutes contact, almost all of them (excluding P. cedrina DSM17516) indicated a moderate adhesion already after four hours of incubation. These results indicate that environmental Pseudomonas strains possess strong hydrophobic and adhesive properties, that may results in a colonization of abiotic surfaces.
PCR-RFLP analysis of commonly occurring insertion sequences ISS1-type, IS904 and IS982 in Lactococcus sp. and Leuconostoc sp. was used for the genetic differentiation of 17 strains of lactic acid bacteria. ISS1-type and IS982 were found in all analysed strains while 1S904 was present exclusively in strains belonging to Lactococcus sp. Amplification of ISS1-type IS sequences resulted in formation of about 820 bp long amplicons, except of strains Lactococcus lactis ssp. lactis E and Leuconostoc lactis R where extra DNA bands about 370 bp long were observed. Similarly for strains of Leuconostoc lactis M and N, additional DNA bands about 280 bp long were present. TaqI digestion of ISS1-type amplicons revealed that all analysed sequences belonged to the restriction type (ii) or (iii) for which major restriction products were 543 and 147 bp long. Amplification of IS904 from all strains of Lactococcus sp. generated amplicons about 1260 bp long. In three strains of Leuconostoc sp. M, N and R, shorter amplicons about 880 bp were observed whereas strains O and P did not contained IS904. Amplification of IS982 resulted in formation of amplicons about 1000 bp long and no extra bands were observed for all tested strains. TaqI digestion of amplification products showed that for strains C, I and F, G, H, belonging to Lactococcus sp. smaller DNA bands were visible suggesting that they contain two different types of IS9S2.
Artykuł stanowi przegląd danych dotyczących produkcji kwasu mlekowego na drodze biotechnologicznej z wykorzystaniem odnawialnych źródeł węgla (głównie biomasy roślinnej). Zaprezentowano szerokie spektrum mikroorganizmów syntetyzujących kwas mlekowy (bakterie fermentacji mlekowej, Bacillus sp., Escherichia coli, grzyby strzępkowe), w tym drobnoustroje uzyskane w wyniku modyfikacji genetycznych. Ponadto opisano substraty skrobiowe, celulozowe i hemicelulozowe oraz odpadowe produkty przemysłowe (melasa, serwatka) stosowane do produkcji kwasu mlekowego. Podjęto próbę charakterystyki czynników wpływających na przebieg i ekonomikę procesu biosyntezy kwasu mlekowego oraz na jakość i ilość otrzymywanego produktu końcowego w konkretnych warunkach hodowli. Autorzy skupili się na metodach ulepszania szczepów produkcyjnych oraz czynnikach wzbogacających podłoża hodowlane w celu zwiększenia wydajności procesu i czystości kwasu mlekowego. Przedstawiono także perspektywy zastosowania kwasu mlekowego, zwłaszcza w odniesieniu do produkcji biodegradowalnego polilaktydu.