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Browsing by Subject "DGGE"

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  • Changes in bacterial population and antimicrobial resistance during cold storage in conventional and organic raw milk 
    Rouvinen, Vilma-Riitta (2010)
    Psychrotrophs, which are mostly gram negative bacteria, can produce heat stable proteases and lipases. Even though these bacteria cannot survive pasteurization of milk, the enzymes can. This can cause problems for the dairy industry. Psychrotrophic milk isolates also have multidrug-resistant traits for antimicrobials and may act as reservoir for resistance genes. The aims of the study were to test denaturing gradient gel electrophoresis (DGGE) for PCR products obtained from organic and conventional raw milk bacteria, to optimize DNA-extraction protocols and PCR-conditions for the raw milk samples and study the bacterial population changes during the cold storage. The aim was also to study the antimicrobial susceptibility of the bacterial isolates. The DNA was extracted from the raw milk samples, when received and after 4 days cold storage, using commercial kits. Nested-PCR was performed and samples were analysed using DGGE. Susceptibility to antimicrobials was determined by growing bacteria on plates that contained two different concentrations of five antibiotics. From the five antibiotics two were used as a combination. The composition of the bacterial population changed during the cold (4ºC) storage: the difference in DGGE profiles was clear between 0 and 4 days cold storage. Fingerprint profile analysis showed that irrespective of the origin of the raw milk, the sample profiles were clustered according to the sampling date (day 0 or day 4). There was no clear difference between DGGE-profiles from conventional and organic milk. Proportion of psychrotrophic bacteria increased and antimicrobial resistance seemed to be more prevailing in conventional than in organic raw milk. Antimicrobial resistance decreased after four days storage at 4ºC, in most of the cases. We showed that the PCR-DGGE-method is an efficient tool to analyse the changes in bacterial populations in raw milk and that cold storage has an evident effect in population composition.
  • DGGE analysis of probiotic-induced alteration in the canine faecal lactic acid bacteria microbiota 
    Peiponen, Susanna (University of HelsinkiHelsingin yliopistoHelsingfors universitet, 2008)
    Several canine clinical gastrointestinal problems, such as inflammatory bowel disease and small-intestinal bacterial overgrowth, are believed to be associated with bacteria. These conditions often lead to chronic diarrhea. Many canine gastrointestinal problems are treated with antibiotics, but due to increasing problems with antimicrobial resistance alternative therapies should be considered. One of these therapies is suggested be probiotic bacteria treatment, especially with members of the genus Lactobacillus due to their health-conferring properties. The probiotic characteristics of bacteria are linked to host specificity which is an important criterion for selection of a probiotic. Most of the commercial probiotic strains meant for dogs are not from canine origin. In addition many substances available in the Finnish market contain Enterococcus faecium, whose safety has been questioned due to it's pathogenic characteristics. This study was based on the study project investigating the effect of host-specific canine LAB on long term gastro-intestinal symptoms. The study was a randomized, double-blinded and placebo controlled trial. This study examined the changes in the canine faecal LAB microbiota caused by fed canine host-specific probiotic LAB. The purpose was to become familiar to the isolation of gastro-intestinal microbiota and to assess the potential alterations in the faeces by applicable methods. The most important methods were polymerase chain reaction (PCR) and denaturing gradient gel electrophoresis (DGGE). During the study DGGE analysis proved to be a working method for analyzing bacterial communities. When the method becomes more familiar, it will probably be as common in use as agarose gel electrophoresis due to it's advantages. Some alterations of the microbiota could be seen on the DGGE analysis. During the experiment the bands resembling those of standard strains were dominant. This implies the Lactobacillus strains of interest being able to survive the canine intestine. However, after feeding seized the bands formed by the strains of interest disappeared and were replaced with bands of indigenous LAB. Also after the feeding seized, some new bands appeared on DGGE gel. This implies a contribution of fed probiotic mixture to the enhanced prevalence of potential novel microbes.

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