ABSTRACT Anaerobic digestion is one way of handling biowaste and generating energy in the form of methane, biogas. This study shows that spore forming bacterias survive the process of pasteurisation and anaerobic digestion in biogas plants. It has also been established that both the nonpasteurised-and digestion- waste contains pathogen spore forming bacterias. Two Swedish full-scale commercial biogas plants were sampled before pasteurisation, after pasteurisation and after digestion on 10 occasions with one week intervals. The samples were analysed quantitatively and qualitatively, with biochemical methods, for Clostridium spp and Bacillus spp. Polymerase Chain Reaction, a biomolecular method, was used for C. chauvei analysis, with C. chauvei specific primers. For this analyse the biogas plants were sampled at 11 occasions. Survival of pathogenic spore forming bacteria in digestion residue may be a health risk for both humans and animals. The digested residue may be used as fertiliser on arable land and the risk of contamination by pathogenic Clostridium spp and Bacillus spp is hard to assess, but can not be neglected.

The pathogenic members of the genus Yersinia, Y. pseudotuberculosis, Y. pestis and Y. enterocolitica are transmitted from animals to man and may give rise to disease with a variety of symptoms. These bacteria possess related plasmids necessary for virulence. In this study, gene products encoded by the virulence plasmid have been identified and characterized. A temperature-inducible outer membrane protein YOP1, is encoded by the virulence plasmid. YOP1 is expressed by Y. pseudotuberculosis and Y. enterocolitica at 37°C. The genetic locale of trie structural gene for YOPl on the virulence plasmid was determined. A mutant that was unable to express this protein, remained fully virulent, showing that YOP1 is not a virulence determinant. Several other proteins encoded by the virulence plasmid are induced at 37°C in a medium lacking Ca2+. These proteins are not expressed at 26°C and expression is repressed by Ca2+-concentrations in excess of 2.5 mM. In Ca2+-deficient medium, the induced proteins can be found extracellu- larly as well as in the outer membrane. However, in the presence of Ca at 37°C they are only found in the outer membrane. The released proteins consist of eight polypeptides as revealed by two-dimensional electro­phoresis. These proteins, Y0P2a and 2b, YOP3, Y0P4a and 4b, the V-antigen and a small uncharacterized polypeptide, are expressed by all three pathogenic Yersinia species, both in vivo and in vitro. The Ca2+-controlled expression of the YOP proteins is regulated by genes in the Ca2+ -region, which are conserved in the three species. Mutations in this region repress the expression of the Ca2+-regulated YOPs. The genetic loci identified for five of these proteins revealed that only the structural gene of the Y0P4b protein is part of the Ca2+ -region. The other genes were found at separate locations outside this region. The structural genes for YOP4b, YOP3 and the V-antigen, together with the genes for two additional polypeptides, were localized to a common region conserved on the plasmids of the Yersinia species. The structural genes for Y0P2b (yopH) and Y0P5 (yopE) are located in different positions on the plasmid from Y. enterocolitica, compared to the other two species. This plasmid has Been rearranged so that these genes are located close to one another. The DNA sequence of the yopH gene shows that it is a singly transcrip­tional unit. Transcription of this gene is regulated by Ca2+-concentra­tion and by temperature. A mutant strain of Y. pseudo tuberculosis, de­leted for the yopH gene on the virulence plasmid, is avirulent In mice. Virulence is restored by trans-complementation with the cloned yopH gene. The mutant strain is also’ unable to inhibit phagocytosis of macrophages as compared to the wild-type strain. The trans-compleroented strain shows inhibition comparable to that of the wild-type. Therefore, the YOP2b protein is considered to be an essential virulence determinant. / digitalisering@umu.se

The treatment options for multidrug resistant bacteria are dwindling and it is an important issue of research in medicine to solve. One of the more problematic bacterial species is Klebsiella pneumoniae, it can cause infections with high morbidity that are difficult to treat. Common antibiotics for treatment of these infections are carbapenems but K. pneumoniae can produce enzymes called carbapenemases that can hydrolyze carbapenems and most other beta-lactam antibiotics. In this study carbapenemase genes were introduced chromosomally to a previously susceptible K. pneumoniae strain using λ-Red recombineering. Further constructs were made with non-functional porins to examine how they affect combination treatment with carbapenems. Antibiotic combination therapy was evaluated against constructed carbapenemase- (KPC-2, NDM-1 and OXA-48) producing K. pneumoniae strains. Screening was done using time-lapse microscopy (oCelloScope), and combinations with better effect than treatment with a single antibiotic were chosen for time-kill assays. The results shows that a triple combination of colistin, meropenem and the beta-lactamase inhibitor avibactam gives an improved effect, up to twice the effect compared to monotherapy and up to 1.8 times increased effect compared to double combination. The synergistic effect was greater when adding colistin to treat the strains with non-functional porins, indicating that colistin can increase the permeability for other antibiotics into the cell. This is an interesting finding that need to be researched further.

Plasmids are small, circular, extrachromosomal double-stranded genetic elements present in bacteria. Plasmids can replicate independently of the bacterial chromosome and play an important role as a transmitter of antibiotic resistance genes between bacteria. Antibiotic resistance genes have been shown to be selected for even in the presence of subinhibitory levels of antibiotics, but the effect of antibiotics on conjugation is not as well understood. To study this, we designed a novel conjugation tracking system utilizing a Cre-expressing plasmid and a chromosomal floxed blue fluorescent protein (BFP) gene. We found that our model worked opposite as intended as cells expressed BFP before conjugation and lost BFP expression upon recombination. An issue with the system was isolated to the direction of the single loxP site remaining after recombination. Both loxP sites were inverted but this did not restore the intended expression of BFP after recombination. Subsequently the system was modified to increase the space between the promoter region and the single loxP site remaining after recombination. This extension produced the desired result as BFP expression now increased upon recombination. Still, further work needs to be done to construct a Cre-expressing plasmid, tune expression of BFP, and show expression of yellow fluorescent protein (YFP) in our model before the system can be applied to clinical isolates.

QepA is a plasmid-mediated efflux pump found in some strains of Escherichia coli, in which it significantly elevates the resistance against quinolones. The protein has similarities with 14-TMS major facilitator superfamily transporters and is situated in the inner membrane of the bacteria. It was acquired by horizontal gene transfer and integrated into a now inactivated class 1 integron, also harbouring several other antibiotic resistance genes such as rmtB and blaTEM-1. QepA alone is not sufficient to raise the resistance level over the clinical breakpoint and is in clinical isolates therefore associated with other quinolone antibiotic resistance genes or quinolone target point mutations. The mechanisms regulating qepA expression are not yet understood. Therefore, in this study the qepA gene was amplified from an E. coli clinical isolate and, together with its upstream promotor sequence, was inserted into the E. coli chromosome. It was shown that qepA gene expression can be induced by exposure to 0.5-fold MIC concentrations of ciprofloxacin, trimethoprim and other DNA damaging antimicrobials. The deletion of a LexA binding site situated after a PcW promotor, which was predicted to drive qepA expression, did not alter this induction behaviour. Nested deletions of up to 200 nts downstream sequence of the PcW promotor, led to the identification of a sequence region required for expression induction. This study showed that qepA expression is induced by environmental factors leading to DNA damage and further identified a previously unknown DNA sequence required for expression regulation.

Human noroviruses are a major cause of acute gastroenteritis worldwide and can betransmitted through consumption of contaminated raw food. Shellfish like oysters can becontaminated by human sewage during production and accumulate multiple Norovirus strainsin low concentrations. Here we developed a simplified and cost effective targetedmetagenomic approach by sequencing PCR amplicons with next generation sequencing(NGS) of the capsid (VP1) viral gene. New design of reverse primers using the codehopstrategy and direct addition of illumina adapter with one step RT-PCR and sequencing onnano chip reduced hand on time and cost of the analysis. A mix of faecal samples and oystersamples associated with outbreaks were used to evaluate the ability and limitations in theidentification of strains from Norovirus genogroup I (GI) and genogroup II (GII). Withsamples containing only one genotype the method was able to identify all strains. Usingartificially mixed samples the method was able to identify almost all strains except a few GIIat low concentrations. Oyster samples showed more limitations for the method and it waswere only able to identify the strain in some of the samples but did find multiple GI strains inmore than one sample. Despite some limitations, the simplified method for VP1-targetedmetagenomics is a sensitive approach allowing the study of norovirus diversity incontaminated oysters and the identification of norovirus strains implicated in outbreaks. Thisat a lower cost and hands on time compared to published methods.

No description available.

The commonly used materials in endodontic treatment lack the ability to prevent microleakage, which can lead to failure of the treatment. This study aimed to evaluate the effectiveness of adding Coltosol plug in preventing coronal microleakage in endodontically treated teeth using the in vitro dye penetration method. Ten single canal plastic teeth were prepared and obturated using the SG-endodontic technique. The teeth were randomly divided into two groups, with and without Coltosol plug, and dye penetration was measured at 6 and up to 72 hours. No difference in leakage was observed after 6 and 72 hours. Specimens with a plug of Coltosol showed similar dye penetration compared to the ones with only Gutta-percha and sealer.

Antibiotic resistance in multidrug resistant bacteria cause high mortality rates worldwide, where there has been over 1,000,000 deaths reported as of the year 2019. Antibiotics were thought to be the cure for fighting infectious diseases and preventing further spreading of infection. This became a major problem due to bacteria evolving and developing mechanisms for resistance. The purpose of this review was to see if there are differences in the resistance mechanism of gram negative and gram positive bacteria, focusing mainly on the six most common multidrug resistant pathogenic bacteria; Staphylococcus aureus, Pseudomonas aeruginosa, Enterococcus faecium, Acinetobacter baumannii, Klebsiella pneumoniae and Streptococcus pneumoniae. The results show that there is a difference in the resistance mechanism between gram positive and gram negative multidrug resistant bacteria. The difference in resistant mechanisms is due to the cell wall compositions of gram negative and gram positive bacteria. The main difference as to why the gram negative bacteria have more resistance is due to the outer membrane. Antibiotics have a hard time to diffuse through and into the cell, that is they can easily decrease their outer membrane permeability. Gram positive bacteria lack an outer membrane which makes them become more susceptible to antibiotics. The most common antibiotic resistance mechanisms in gram negative bacteria are outer membrane mechanisms such as lipid A and lipopolysaccharide modification as well as mutations in porin channels. On the other hand, the most common resistance mechanisms for gram positive bacteria are point mutations especially in penicillin binding proteins as well mutations in the rpoB gene. One important gram positive bacteria is Methicillin resistant Staphylococcus aureus, which developed a new mechanism against antibiotics, a missense mutation and mutation on the promoter region in penicillin binding protein 4. Recently new research has come forward showing that N-chlorotaurine (NCT) inhibits resistance in both gram positive and gram negative multidrug resistant bacteria. The research on NCT is still fairly new and only time will tell if this method of inhibiting resistance will be used in the future. This review highlights the importance and concern of multidrug resistance bacteria, especially due to bacteria being able to rapidly evolve when antibiotics are used incorrectly. It is important to understand the differences in resistance between gram negative and gram positive bacteria and how resistance spreads. This knowledge can be used to develop antibiotics that treat infections. It is however still a challenge to overcome resistance in multidrug resistant bacteria due to evolutionary adaptation especially through horizontal gene transfer, where resistant bacteria can adapt to changing conditions. / Antibiotikaresistens hos multiresistenta gramnegativa och grampositiva bakterier orsakar hög dödlighet över hela världen, där det har rapporterats över 1,000,000 dödsfall för år 2019. Antibiotika ansågs vara botemedlet för att bekämpa infektionssjukdomar och förhindra ytterligare spridning av infektioner. Detta blev ett stort problem på grund av att bakterier utvecklades mekanismer för resistens, vilket gör att de kan överleva när de behandlas med antibiotika. Syftet med denna studien är att se om det finns skillnader i resistensmekanismener för gramnegativa och grampositiva bakterier, med fokus på de sex vanligaste multiresistenta bakterierna; Staphylococcus aureus, Pseudomonas aeruginosa, Enterococcus faecium, Acinetobacter baumannii, Klebsiella pneumoniae och Streptococcus pneumoniae. Resultaten visar att det finns en skillnad i resistensmekanismen mellan grampositiva och gramnegativa multiresistenta bakterier. Skillnaden i resistenta mekanismer beror på cellväggssammansättningen av gramnegativa och grampositiva bakterier. Den största skillnaden till varför de gramnegativa bakterierna har mer resistens beror på det yttre membranet. Antibiotika har svårt att penetrera genom och in i cellen genom att minska deras yttre membranpermeabilitet. Grampositiva bakterier saknar ett yttre membran som gör att de blir mer mottagliga för antibiotika. De vanligaste antibiotikaresistensmekanismerna hos gramnegativa bakterier är yttre membranmekanismer som lipid A och lipopolysackaridmodifiering samt mutationer i porinkanaler. De vanligaste resistensmekanismerna för grampositiva bakterier är punktmutationer, särskilt i penicillinbindande proteiner samt mutationer i rpoB genen. En viktig grampositiv bakterie är Meticillin-resistent Staphylococcus aureus, som utvecklade en ny mekanism mot antibiotika, en missense-mutation och mutation på promotorregionen i penicillinbindande protein 4. Nyligen har ny forskning kommit fram som visar att N-klorotaurin (NCT) hämmar resistens i både grampositiva och gramnegativa multiresistenta bakterier. Forskningen om NCT är fortfarande ny och bara tiden kommer att utvisa om denna metod för att hämma resistens kommer att användas i framtiden. Den här studien belyser vikten och oron för multidresistena bakterier, särskilt på grund av att bakterier snabbt kan utveckla antibiotikaresistens när antibiotika används på fel sätt. Det är viktigt att förstå skillnaderna i resistens mellan gramnegativa och grampositiva bakterier och hur resistens sprids inom resistenta bakterier. Denna kunskap kan användas för att utveckla antibiotika som behandlar infektioner orsakade av både gramnegativa och grampositiva bakterier. Det är fortfarande en utmaning att övervinna resistens hos multiresistenta bakterier på grund av evolutionär anpassning särskilt genom horisontell genöverföring, där resistenta bakterier kan anpassa sig till förändrande förhållanden.

The bacterium Streptococcus pneumoniae (pneumococcus), is considered to be a leading cause of morbidity and mortality globally, particularly in infants and the elderly. It is one of the most frequent causes of respiratory tract infections, which sporadically have the potential to develop into serious invasive symptoms including sepsis and meningitis. The development of effective vaccination against this pathogen is essential for reducing the morbidity and mortality it causes since the currently available vaccines can protect against only a limited number of the 100 pneumococci serotypes which target the polysaccharidic capsule of the bacterium. The potential use of conserved protein antigens could provide a defense to a wider range of serotypes and clonal types. The immunogenic properties of the proteins MalX and PrsA as well as their role in vital biological functions of S. pneumoniae have made them stand out as potential targets. MalX is a crucial membrane protein involved in the metabolism of maltose, whereas PrsA is a chaperone-like protein that is connected to the cell envelope. To understand these proteins' potential as vaccine candidates, it is essential to understand their immunogenic characteristics and physiological roles. In this project, we tried to characterize the two antigens to determine the functional significance of different regions and domains in antigen recognition and their expression dynamics in bacterial host. A better understanding of the antigenic properties of the PrsA and MalX proteins will drive the construction of improved versions of antigens for vaccine prototypes. Some approaches were used to clarify the structural characteristics and antigenic determinants associated with these proteins including, protein expression, purification, and structural characterization. Additionally, their expression in E. coli was examined using immunological assays including ELISA and Western blot. The identification of antigenic regions of these proteins also provides insight into how to develop epitope-based vaccinations that specifically target S. pneumoniae. This project discusses the possibility of using membrane vesicles (MVs) as a platform for vaccination. Membrane vesicles made from bacterial cells have innate immunogenic qualities that expose the immune system to a wide variety of antigens. Incorporating MalX and PrsA into such vesicles can improve the vaccine candidate's overall immunogenicity and effectiveness and trigger a stronger immune response against S. pneumoniae.