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<p>It has been suggested that growth, production and community structure of bacterioplankton are dependent on resource availability. However, previous studies have only investigated the effect of either organic substrate mixtures or a few single organic substrates on the bacterioplankton community. The aims of this study were to investigate the impact of five different relevant carbon sources on the bacterioplankton community. This impact was evaluated comparing treatments on samples taken from Skagerrak and the Baltic Sea, in whole seawater cultures. Analysis of bacterial abundance, bacterial production (as leucine incorporation), bacterioplankton DNA community structure and colony-forming bacteria growing on agar plates were evaluated. Differences between carbon sources in terms of bacterial numbers were relatively small, with strong growth responses for L-amino acids, glucose, acetate and pyruvate with the only exception of glycolate where growth was lower. Bacterial production, on the other hand, presented marked differences, different patterns for each carbon source, especially in the Baltic Seawater. Furthermore, differences in colony size and number of colony forming bacteria in the different treatments were important. The analysis of DNA community from each experiment, by denaturing gradient gel electrophoresis (DGGE) analysis of PCR-amplified 16S rDNA, allowed a visualization of the microbial community structure. Sequencing of the stronger bands on the gel revealed the identity of the dominant bacterial species. In terms of bacterioplankton community structure, differences between carbon sources and between environments were important. One unknown species belonging to gamma-proteobacteria was both unique and dominant for glucose treatment in the Baltic experiment. Another gamma-proteobacteria , a Vibrio was found to specialize in glucose in the Skagerrak experiment. One uncultured bacterium belonging to a alpha-proteobacteria, both unique and dominant was found in glycolate, also this in Skagerrak, another uncultured alpha-proteobacteria was clearly dominant for glucose treatment in Skagerrak. Some bands were also present in most treatments, e.g. uncultured species belonging to bacteroidetes in Skagerrak and beta-proteobacteria in Baltic, suggesting that those species are not specialized in consuming a single carbon source. As a conclusion different carbon sources clearly had an individual but important role for bacterioplankton properties. The properties also showed to be dependent on the environment.</p><p>Nr:6355</p>

<p>Aim Because of the major problems that certain Vibrio specie, especially Vibrio harveyi, can cause the aquaculture industries a rapid method to identify Vibrio isolates is required. Early diagnosis of a V. harveyi infection could facilitate disease surveillance, treatment and prevention in cultured marine animals. Therefore, the use of PCR to aid in the identification of Vibrio is increasing and a way of extracting DNA in a cheap, fast and easy way is also of an important requirement to facilitate rapid diagnosis.</p><p>Methods This report comprises biochemical profiling and PCR methods in the characterisation of four isolates of V. harveyi and single isolates of V. tubiashii, V. alginolyticus, V. anguillarum, V. splendidus, V. tapetis and V. parahaemolyticus.</p><p>Strains were examined for adherence to a Hep-2 cell line. Four different DNA extraction methods were evaluated and compared. The detection limits and the analytical limits of two PCR methods for Vibrio were determined.</p><p>Results The overall findings were that the use of a greater range of biochemical substrates than are in the API 20E is necessary to identify Vibrio strains, and that none of the strains tested adhered to Hep-2 cells. All extraction methods successfully produced DNA with the kit method giving the purest samples. RNA was a contaminant of the other techniques but this could be overcome by treating extracts with RNase. The rapid microwave extraction method gave appropriate PCR amplicons when tested.</p><p>Conclusion PCR determination of the VH-sequence in combination with VHA and a distinguishable colonial morphology may be a good choice for the identifying of Vibrio harveyi.</p>

Fusobacterium necrophorum är en gramnegativ anaerob bakterie som grupperas i ss. necrophorum och ss. funduliforme. Fusobacterium necrophorum ss. funduliforme har på senare tid misstänkts kunna spela en roll vid vanligare svalginfektioner såsom halsfluss. Syftet med detta arbete var att sätta upp och bepröva en metod för realtids-PCR (Polymerase Chain Reaction) enligt Taqman för att detektera ss funduliforme. Vi undersökte även hur förvaringstiden i transportmedium (Amies kol, Copan) och odlingsmedium (Fastidious Agar Broth) påverkar överlevnaden för F. necrophorum ss. funduliforme och resultatet från realtids-PCR. Bakteriesuspension av varierande koncentration applicerades på provtagningspinnar som direkt inkuberades i medium, varefter en pinne av vardera koncentration utodlades och DNA extraherades för varje dag försöket pågick. En serie 10-spädningar av extraherat DNA från ss funduliforme ,med en lägsta koncentration av 10 DNA-molekyler per µL, användes som standard och positiv kontroll för PCR. Fusobacterium necrophorum ss funduliforme kunde detekteras med realtids-PCR från alla prov under alla dagar försöket pågick, medan odlingarna visade bäst resultat om provet såddes ut inom ett dygn från provtagningen. Realtids-PCR kan därför detektera förekomsten av ss funduliforme efter längre förvaring och bör användas för exempelvis transporterade prover, medan traditionell utodling med fördel kan användas för diagnostering av ss fundulforme då PCR inte ger information om antibiotikaresistens hos isolatet. / Fusobacterium necrophorum are Gram-stain negative, anaerobic, bacteria that are grouped into subspecies necrophorum and funduliforme. Fusobacterium necrophorum ss. funduliforme has recently been suspected to play a role in common throat infections such as tonsillitis. The purpose of this work was to set up and test a method for real-time PCR (Polymerase Chain Reaction) according to Taqman with the purpose of detecting ss. funduliforme. We also examined how the storage time within transport medium (Amies charcoal, Copan) and culture medium (FAB-broth) affects the survival of the bacterium and the sensitivity of the culture and PCR methods. Bacterial suspensions of different concentrations were applied to pharyngeal sampling swabs and then directly incubated in transport medium. For each day the experiment lasted, a set of swabs of each concentration were cultured, DNA extracted, and real-time PCR performed. DNA extracted from ss. funduliforme was used as standards for the real-time PCR, with a minimum concentration of 10 DNA molecules per μL. Subspecies funduliforme could be detected from all days with real-time PCR while the cultures showed the best results if the sample was cultured within one day of collection. Real-time PCR can detect the presence of ss. funduliforme after prolonged storage and can therefore show accurate results for transported samples. Traditional culturing on growth medium is still a valuable and reliable method, provided that the samples are cultured within 24 hours. Culture may also be needed i.e. since PCR gives no information of the antibiotic resistance of the isolate.

Mycobacterium tuberculosis (Mtb) is the causative agent of tuberculosis, a disease that for years was considered to belong of the past, but tuberculosis is back causing over 2 million deaths per year. The infection can be dormant for decades and an active immune response can prevent the infection from progressing into active disease. However, the HIV/AIDS epidemic has caused an alarming rise in tuberculosis cases. The main infectious route for Mtb is through the airways into the lungs, where they encounter alveolar macrophages. Mtb are phagocytosed by these macrophages, but instead of being killing within the phagosome, Mtb modulates the cell to become a host in which the bacteria thrive. The lack of capacity to eradicate the infection stimulate cells of the immune system to gather around infected macrophages and form a granuloma that walls off the infection. Within this granuloma, Mtb can wait silently and later progress into active disease. However, only a fraction of exposed individuals develop disease, indicating that initial eradication of Mtb infections is possible. Such immediate response must be directed by the innate immunity comprised of phagocytes such as neutrophils (PMNs) and non-activated macrophages. Upon Mtb infection, macrophages become anergic and PMNs enter apoptosis. PMNs have a short lifespan and are cleared by neighbouring phagocytes, a mechanism described to resolve the inflammation and modulate tissue regeneration. We found that Mtb-induced apoptosis in PMNs was not dependent on phagocytosis of the bacteria, indicating that Mtb have the capacity to induce apoptosis in multiple PMNs. Complement-mediated phagocytosis induce survival signals such as Akt in PMNs, but despite this, complement-opsonized Mtb was able to override the anti-apoptotic activation in the cells. Since phagocytes clear apoptotic cells, we investigated how clearance of Mtb-induced apoptotic PMNs affected macrophages. We found that Mtb-induced apoptotic PMNs inflicted pro-inflammatory activation of the macrophages that cleared them. In addition, this activation was mediated by Hsp72 released from the Mtb-induced apoptotic PMNs. Furthermore, apoptotic PMNs can work in synergy with phagocytosed Mtb to activate macrophages and enhance intracellular killing of Mtb. Since dendritic cells are important for the regulation of immunity, we investigated whether Mtb-induced apoptotic PMNs affected the inflammatory response and maturation of dendritic cells. We found that Mtb-induced apoptotic PMNs trigger dendritic cells to enter a mature state able to activate naïve T-cell proliferation. We propose that infected apoptotic PMNs is a potent activator of the inflammatory response during infections. Taken together, PMNs not only kill their share of pathogens but also modulate other immune cells, thereby forming a link between the early innate and the adaptive immune response during microbial challenge with Mtb.

It has been suggested that growth, production and community structure of bacterioplankton are dependent on resource availability. However, previous studies have only investigated the effect of either organic substrate mixtures or a few single organic substrates on the bacterioplankton community. The aims of this study were to investigate the impact of five different relevant carbon sources on the bacterioplankton community. This impact was evaluated comparing treatments on samples taken from Skagerrak and the Baltic Sea, in whole seawater cultures. Analysis of bacterial abundance, bacterial production (as leucine incorporation), bacterioplankton DNA community structure and colony-forming bacteria growing on agar plates were evaluated. Differences between carbon sources in terms of bacterial numbers were relatively small, with strong growth responses for L-amino acids, glucose, acetate and pyruvate with the only exception of glycolate where growth was lower. Bacterial production, on the other hand, presented marked differences, different patterns for each carbon source, especially in the Baltic Seawater. Furthermore, differences in colony size and number of colony forming bacteria in the different treatments were important. The analysis of DNA community from each experiment, by denaturing gradient gel electrophoresis (DGGE) analysis of PCR-amplified 16S rDNA, allowed a visualization of the microbial community structure. Sequencing of the stronger bands on the gel revealed the identity of the dominant bacterial species. In terms of bacterioplankton community structure, differences between carbon sources and between environments were important. One unknown species belonging to gamma-proteobacteria was both unique and dominant for glucose treatment in the Baltic experiment. Another gamma-proteobacteria , a Vibrio was found to specialize in glucose in the Skagerrak experiment. One uncultured bacterium belonging to a alpha-proteobacteria, both unique and dominant was found in glycolate, also this in Skagerrak, another uncultured alpha-proteobacteria was clearly dominant for glucose treatment in Skagerrak. Some bands were also present in most treatments, e.g. uncultured species belonging to bacteroidetes in Skagerrak and beta-proteobacteria in Baltic, suggesting that those species are not specialized in consuming a single carbon source. As a conclusion different carbon sources clearly had an individual but important role for bacterioplankton properties. The properties also showed to be dependent on the environment. Nr:6355

Antibiotics are today one of the most important cornerstones in modern healthcare when it comes to treating bacterial infections. It is an asset human kind have been leaning on for the last century, but excessive and widespread misuse of antibiotics have left deep scars in the form of multi resistant pathogenic strains of bacteria that we soon will not be able to treat. A lot of research have been invested in understanding the mechanisms and spread of resistance within bacteria living in planktonic form, overlooking the fact that there are more lifestyles that causes problems. In this study, focus has been put on antibiotic resistance within bacteria living as biofilms, a lifestyle that causes problems in chronic infections and prosthetics/medical implants. By constructing resistant mutants derived from a biofilm forming strain of Escherichia coli, the minimal selection concentration has been investigated in both planktonic and biofilm assays for Streptomycin and Ciprofloxacin. By comparing the results, it is possible to evaluate if and how the antibiotic resistance properties differ between the two lifestyles. Focus has been put on concentrations of antibiotics below the minimal inhibitory concentration with the objective to see how selection of antibiotic resistant mutants take place with the susceptible strain still growing, although with reduced growth rate. The hope is that the results gained in this study will provide a foundation for future research regarding antibiotic resistance in biofilms, and be part of the solution to the excessive resistance problem before it is too late.

Kontamination med mikroorganismer är något som sker ständigt och i samband med läkemedelsproduktion kan de bidra till försämringar av produktens kvalité. Genom att kontrollera den mikrobiella floran i bland annat aktuella lokalers luft och ytor uppfyller läkemedelsproducenterna kraven enligt Good Manufacturing Practice. Syftet i denna studie är att kartlägga mikroorganismer i samband med tillverkningen av läkemedlet Xylocain gel. Undersökningen har genomförts på läkemedelsföretaget Recipharm Karlskoga AB. Under arbetet användes analysmetoden analytical profile index, en metod för att identifiera mikroorganismer. Resultatet visar kontaminering från totalt 19 stycken olika arter, varav endast en bakterie anses vara potentiellt patogen och denna identifierade bakterien var Stenotrophomonas maltophilia. På 12 platser fanns upp till två bakteriekolonier, på 10 platser fanns tre till fyra bakteriekolonier och i ett rum (beredningsrummet) fanns fem bakteriekolonier. Generellt var det något mer bakteriekolonier på plattorna odlade från luft än ytor. Den lägsta gränsen för vad som godkänts enligt Good Manufacturing Practice för renrum i samband med ytor och luft är 10 stycken påvisade bakterier per platta. Utifrån resultaten dras slutsatsen att kontamination av bakterier från omgivningen och personalen, är med största sannolikhet inte ett problem beträffar den färdiga Xylocain gel-produkten. / Contamination with microorganisms is something that frequently occurs, and during drug production contamination can affect product quality. By controlling the microbial flora in the air on surfaces during drug production, the drug manufacturers comply with the requirements for Good Manufacturing Practice. The purpose of this study was to identify microorganisms associated with the production of the product Xylocaine Gel. The study was carried out at the pharmaceutical company, Recipharm Karlskoga AB. During the work, the analytical method analytical profile index was used, a method to identify microorganisms. The results showed contamination from a total of 19 different species, of which only one bacterium, Stenotrophomonas maltophilia, is considered pathogenic. At 12 location, there were up to two bacterial colonies, at 10 locations there were three to four bacteria colonies and in one room (the preparation room) there were five bacterial colonies. Generally there were slightly more bacteria on the plates grown from air than on surfaces. The minimum limit of what has been approved according to Good Manufacturing Practice for clean rooms in connection with surfaces and air is 10 bacteria detected per plate. Based on the results, it is concluded that contamination of bacteria from the environment and the staff is most likely not a problem with the regard to the finished product, Xylocain Gel.

One major environmental challenge is to manage agricultural soil in asustainable way. Since nutrients in the soil are taken away every harvest, there is a need to recycle nutrients back to the soil. One solution is to use sewage sludge as a fertilizer. However, sludge can also contain substances that could potentially be harmful to crops or humans. This study examined the long-term effect of sewage sludge amendment in agricultural soil. The aim of this study was to analyse the general effects on the microbial community by sequencing the 16SrRNA gene, as well as studying the response to the amendments of nitrifying and denitrifying microorganisms. This was done by qPCR quantification of marker genes and activity tests of potential denitrification and N2O production in the soil. The main conclusion is that long-term effects of sewage sludge amendments on the microbial community, as well as on microorganisms involved in nitrogen cycling,are relatively small compared to effects caused by differences in soils. Moreover, the effects on the microbial community differed between soils, implying that attention needs to be paid to the characteristics of the soil when evaluating effects of sludge amendments. It was clear that the ratio of ammonia oxidising bacteria and archaea differed between treatments, however further studies are needed to conclude the reason for this. Finally, positive correlations between both measured activities and denitrifying marker genes were found, which suggests that quantification of marker genes can be used for studying reactions in the soil.

Background: More than 700 oral bacterial species have been found and together they make out the oral microbiota. Specific species have shown to correlate with various oral diseases such as caries and periodontitis, but also systemic diseases. Most studies have looked at the whole microbiota but the knowledge about tooth site-specific variation within supragingival plaque after lack of oral hygiene in healthy participants is limited.  Aim: This pilot study aimed to characterize variations in the supragingival plaque with regards to the; anterior (incisors and canines) and posterior (molars and premolars) teeth, upper and lower jaw, and left versus right tooth arches. Method: After three days of accumulating plaque, supragingival tooth biofilm was collected from 16 different tooth sites, from six healthy participants. Bacterial DNA was extracted, and 16s rRNA gene (V3-V4) was amplified by PCR and sequenced on an Illumina MiSeq platform. Sequences were blasted and taxonomically allocated using the Human Oral Microbiome Database. Result: In summary, 50 species showed a difference between the anterior and the posterior region, 30 species differed between the upper and lower jaw, and three species differed between the left and right sides.  Conclusion: This study indicates a difference in oral microbiota composition in supragingival plaque on different tooth regions. These findings emphasize the choice of method when analyzing the oral microbiota—also highlighting the importance of further understanding the dynamic forces driving local enrichment and reduction of specific species.