I takt med att allt mer antibiotika används och att världen blir allt mer globaliserad ökar och sprids antibiotikaresistensen. Djurhållningen i världen kantas av stressgivande miljöer som för små utrymmen och för många djur per yta. Det får djuren att drabbas av infektioner som vi botar med antibiotika. Antibiotika används även inom djurhållning i tillväxtfrämjande syfte och för att förebygga sjukdom och minska stress. Denna fel- och överbehandling av antibiotika i kombination med att vi använder samma sorts antibiotika inom human sjukvård som inom djurhållning gör att våra livsmedelsproducerande djur utgör en smittorisk för resistenta bakterier som hotar att nå oss via bland annat livsmedelskedjan. I och med att djuren medicineras via tillägg i foder och vatten och att upp till 90% av antibiotikan följer med fekalierna ut, sprids resistensen i naturen då stor del av fekalierna distribueras på jordbruksåkrar i fertiliserande syfte. Det ökar på spridningsrisken samt utgör ytterligare en risk för oss när vi äter grödorna. Från akvakulturer hamnar ungefär 80% av antibiotikan i det omgivande vattnet och i sedimentet och kan därifrån spridas till havets mikrober, vidare till fisk- och skaldjurspatogener och sedan till terrestra bakterier. Åtgärder till dessa problem innefattar att minska spridningen och förhindra uppkomsten av resistenta bakterier. Man bör forska fram fler antibiotika exklusivt för en sektor, i första hand vaccinera och när man måste använda antibiotika bör det vara en smalspektrumsvariant. Man måste också förbättra den globala djurhållningsstandarden, så att risken för spridning minskar vid resor och handel. Det krävs också ett ökat kunskapsläge och ett gemensamt internationellt samarbete för minskad och mer restriktiv antibiotikaanvändning. / As more antibiotics are being used in the world, and as the world gets more globalized, antibiotic resistance is a problem that is growing and spreading. Animal husbandry all over the world provides animals with stressful environments such as too small spaces and too many animals per area. The stress makes the animals suffer from infections that we cure with antibiotics. Antibiotics are also used in animal husbandry as a growth promoter and to prevent illness and decrease stress. This mis- and overuse of antibiotics and the fact that we are using the same type of antibiotics for human health care as well as for animal husbandry, makes our livestock a threat - we can get infected with antibiotic resistant bacteria through the food chain. As a result of us medicating the animals by putting antibiotics in their feed and water (where up to 90% of the antibiotics ends up in the faeces), the resistance is spread in nature, since the faeces often are used as fertilizers in agriculture. This increases the risk of spreading and is another threat for us when we eat the crops from the fields. From aquacultures about 80% of the antibiotics ends up in the nearby water and sediment and can spread through the microbes of the ocean, via fish and shellfish pathogens to terrestrial bacteria. Measuring steps includes decreasing the spread and preventing the rise of resistant bacteria. More research is needed to find new antibiotics, that should be used exclusively for one sector. We should also vaccinate more and when antibiotics are needed, use narrow spectrum antibiotics. Another step is to improve the global animal husbandry standards, so the risk for spreading decreases when travelling and importing/exporting. More education and international teamwork for reduced and more strict antibiotic usage is also needed.

Bakgrund: Inom hälso- och sjukvården kan personal råka ut för olika arbetsrelaterade besvär. Sjuksköterskan kommer i kontakt med många olika typer av läkemedel. Alla läkemedel är inte harmlösa för dem som iordningställer och administrerar dem. Då företagssköterskan ska arbeta hälsofrämjande och preventivt är det intressant att belysa sjuksköterskors arbete med intravenös antibiotika. KASAM kan vara till hjälp, på en arbetsplats, för att ta reda på vad som krävs för att kunna bevara men också förbättra hälsan hos medarbetarna.  Syfte: Att beskriva sjuksköterskors uppfattningar om risker vid arbete med intravenös antibiotika. Metod: Vald metod var kvalitativ metod. Fenomenografisk ansats användes vid analys av nio intervjuer. Resultat: Resultatet sammanfattades i tre beskrivningskategorier: Påverkan på kroppen, Utrustningens betydelse och Synliggöra risker. Slutsatser: Studiens resultat visar att sjuksköterskor är i behov av information om risker vid arbete med intravenös antibiotika. En ökad medvetenhet om riskerna kan bidra till att minska utvecklingen av både känd och icke känd överkänslighet samt minska resistensutvecklingen.

Bacterial resistance to antibiotics is an emerging epidemic throughout the world and there is a desperate need for new antibiotics and new strategies to maintain the effectiveness of current agents. ??-Lactams, such as the penicillins and cephalosporins, have been the most important class of antibiotic for several decades and represent half of the global antibacterial market, but the continued use of ??-lactams is threatened by ??-lactamases, enzymes that efficiently inactivate ??-lactams through hydrolysis. Class A, C, and D ??-lactamases use an active-site serine residue for hydrolysis and achieve turnover through an acylenzyme intermediate while the class B metallo-??-lactamases (MBLs) use a zinc-bound hydroxide as the active-site nucleophile. Two successful approaches to combat ??-lactamase-mediated resistance have involved the development of ??-lactam antibiotics which bind poorly to ??-lactamases and the combination of ??-lactams with ??-lactamase inhibitors. These strategies have been effective for overcoming resistance due to class A ??-lactamases, but the ever-increasing prevalence of extended-spectrum ??-lactamases (ESBLs), metallo-??-lactamases, and carbapenemases compromises the effectiveness of current penicillins, cephalosporins, carbapenems, and mechanism-based ??-lactamase inhibitors. Cyclobutanone analogues of ??-lactam antibiotics were explored in the early 1980s as potential inhibitors of ??-lactamases and D-Ala-D-Ala transpeptidases, but simple analogues showed only weak inhibitory activity and this approach was subsequently abandoned. The increasing threat of multidrug-resistant ??-lactamase-producing organisms in recent years, however, has inspired a re-evaluation of these inhibitors since cyclobutanones have the potential to exhibit broad-spectrum inhibition of both serine- and metallo-??-lactamases through the formation of enzyme-bound hemiketals or hydrates. 7,7-Dichloro-2-thia-bicyclo[3.2.0]heptan-6-one-4-carboxylic acid (65), a dichlorocyclobutanone that had shown modest inhibition of the class B and D ??-lactamases IMP-1 and OXA-10 in earlier work in this laboratory, was prepared in an efficient seven-step sequence from triethyl phosphonoacetate (103) with an overall yield of 28%. Initial efforts to improve upon the potency of the cyclobutanones involved functionalization at C3 and a highly stereoselective chlorination with sulfuryl chloride provided the 3??-chloro derivative 117?? in nearly quantitative yield. Elimination of HCl from 117?? was achieved under a variety of conditions and 3-alkoxy derivatives were prepared from 117?? through diastereoselective substitution reactions with alcohols. Cyclobutanones with 3??-OR substituents were found to favour an endo envelope conformation while the 3??-OR derivatives adopt the exo envelope conformation. Evidence from X-ray crystal structures and ab initio molecular orbital calculations suggests that an anomeric effect contributes to the large conformational preference of the tetrahydrothiophene ring that favours the 3-alkoxy substituent in an axial orientation. In addition, the conformation of the bicyclic system was found to have a dramatic effect on the tendency of the cyclobutanone to undergo hemiketal formation. Cyclobutanone analogues of penicillins, including 3-alkoxy derivatives, and cyclobutanone analogues of penems were evaluated against class A, B, C, and D ??-lactamases and found to be moderate inhibitors of KPC-2, IMP-1, GC1, and OXA-10. The cyclobutanones found to be most potent were those which are hydrated to a larger extent in aqueous solution. Dichlorocyclobutanones were found to be better inhibitors than dechlorinated cyclobutanones and a 3??-methoxy derivative 152??, which favours the exo envelope conformation in which the C4 carboxylate is equatorial, was found to be a better inhibitor than cyclobutanones that favour the endo envelope conformation. A 3,4-unsaturated penem analogue, 153, showed comparable potency to that of 152?? and molecular models of enzyme-inhibitor complexes indicate that an equatorial carboxylate is required for binding to ??-lactamases. An X-ray crystal structure of 152?? bound to the class D ??-lactamase OXA-10 confirms that a serine hemiketal is formed in the active site and that the inhibitor adopts the exo envelope. The biochemical data described above demonstrate that cyclobutanones can indeed act as inhibitors of serine- and metallo-??-lactamases and these cyclobutanones represent the first class of reversible inhibitors to show moderate inhibition of all four classes of ??-lactamase. Although the inhibitory potency of these compounds is modest (low micromolar IC50 values), penem analogue 153 was able to enhance the potency of meropenem against carbapenem-resistant MBL-producing clinical isolates of Chryseobacterium meningosepticum and Stenotrophomonas maltophilia and the synergy demonstrated in these antimicrobial assays is encouraging. Synthetic studies toward other C3-alkyl and C3-thioalkyl-substituted inhibitors are described and the design and synthesis of C7-monochloro- and 7??-hydroxymethyl-7??-chloro cyclobutanone derivatives is presented.

The emergence of extended spectrum β-lactamase (ESBL) producing Enterobacteriaceae worldwide has led to an increased use of carbapenems and may drive the development of carbapenem resistance. Existing mechanisms are mainly due to acquired carbapenemases or the combination of ESBL-production and reduced outer membrane permeability. The focus of this thesis was to study the development of carbapenem resistance in Escherichia coli in the presence and absence of acquired β-lactamases. To this end we used the resistance plasmid pUUH239.2 that caused the first major outbreak of ESBL-producing Enterobacteriaceae in Scandinavia. Spontaneous carbapenem resistance was strongly favoured by the presence of the ESBL-encoding plasmid and different mutational spectra and resistance levels arose for different carbapenems. Mainly, loss of function mutations in the regulators of porin expression caused reduced influx of antibiotic into the cell and in combination with amplification of β-lactamase genes on the plasmid this led to high resistance levels. We further used a pharmacokinetic model, mimicking antibiotic concentrations found in patients during treatment, to test whether ertapenem resistant populations could be selected even at these concentrations. We found that resistant mutants only arose for the ESBL-producing strain and that an increased dosage of ertapenem could not prevent selection of these resistant subpopulations. In another study we saw that carbapenem resistance can even develop in the absence of ESBL-production. We found mutants in export pumps and the antibiotic targets to give high level resistance albeit with high fitness costs in the absence of antibiotics. In the last study, we used selective amplification of β-lactamases on the pUUH239.2 plasmid by carbapenems to determine the cost and stability of gene amplifications. Using mathematical modelling we determined the likelihood of evolution of new gene functions in this region. The high cost and instability of the amplified state makes de novo evolution very improbable, but constant selection of the amplified state may balance these factors until rare mutations can establish a new function. In my studies I observed the influence of β-lactamases on carbapenem resistance and saw that amplification of these genes would further contribute to resistance. The rapid disappearance of amplified arrays of resistance genes in the absence of antibiotic selection may lead to the underestimation of gene amplification as clinical resistance mechanism. Amplification of β-lactamase genes is an important stepping-stone and might lead to the evolution of new resistance genes.

Humans are generally having a strong, widespread, and negative impact on nature. Given the many ways we are impacting nature and the many ways nature is responding, it is useful to study responses in an integrative context. My thesis is focused largely (two out of the three data chapters) on butterfly species’ range shifts consistent with modern climate change in Canada. I employed a macroecological approach to my research, drawing on methods and findings from evolutionary biology, phylogenetics, conservation biology, and natural history. I answered three main research questions. First, is there a trade-off between population growth rate (rmax) and carrying capacity (K) at the mutation scale (Chapter 2)? I found rmax and K to not trade off, but in fact to positively co-vary at the mutation scale. This suggests trade-offs between these traits only emerge after selection removes mutants with low resource acquisition rates (i.e., unhealthy genotypes), revealing trade-offs between remaining genotypes with varied resource allocation strategies. Second, did butterfly species shift their northern range boundaries northward over the 1900s, consistent with climate warming (Chapter 3)? Leading a team of collaborators, we found that most butterfly species’ northern range boundaries did indeed shift northward over the 1900s. But range shift rates were slower than those documented in the literature for more recent time periods, likely reflecting the weaker warming experienced in the time period of my study. Third, were species’ rates of range shift related to their phylogeny (Chapter 3) or traits (Chapter 4)? I found no compelling relationships between rates of range shift and phylogeny or traits. If certain traits make some species more successful at northern boundary range expansion than others, their effect was not strong enough to emerge from the background noise inherent in the broad scale data set I used.

Formål: Formålet med dette studie var at undersøge faktorer der kan have betydning for hvordan patienten magter at være isoleret under indlæggelse på hospital. Der søges afdækning af om der er baggrundsvariabler som køn, alder og tidligere erfaringer, som har betydning og hvilke konsekvenser det medfører. Formålet var endvidere på baggrund af en risikoanalyse af den enkelte patientat fokusere på at tilrettelægge organiseringen af pleje og behandling. Metode: Studiet er et kvalitativstudie, hvor det empiriske materiale blev indsamlet ved fempatientinterviews. Som analysemetode blev anvendt indholdsanalyse. Den konceptuelle ramme omfatter antibiotikaresistens i et folkesundhedsperspektiv, en beskrivelse af rammerne for infektionsforebyggelse i Danmark samt en teoretisk ramme af hvad det indebærer for patienter at være smittet med en multiresistent bakterie og oplevelse af at være isoleret. Resultater: Studiet viser, at lukket dør, mangel på kontakt og stimuli resulterer i følelsen af kedsomhed, monotoni og angst. Studiet viser endvidere at baggrundsvariabler synes at have betydning for hvordan isolationen opleves. Kvinder udviser større bekymringer omkring smitteforholdsregler, og er mere observante på personalets adfærd end mænd. Kvinder bekymrer sig mere om risikoen for smitteoverførsel til besøgende og familie. Kvinder er mere emotionelle under indlæggelsen og under isolationen. Mænd affinder sig udadtil med situationen og har ikke samme spekulationer omkring smitteforholdsregler. Mænd har en mere rationel tilgang, og der er en tendens til at mænd bedre magter at være isoleret på enestue. Yngre patienterser ud til at magte isolationen bedre og anser enestue som en fordel. De ældre bliver mere triste og føler sig ensomme. Erindringer fra tidligere indlæggelser kan lejres som negative oplevelser, og influere på nuværende indlæggelse. Forat patienterne kunne magte situationen, udviklede de selv strategier til egen hjælp og befandt sig således i en balance mellem stress og mestring. Konklusion: For at kunne forebygge de negative oplevelser det har for patienter som er isoleret, uden at kompromittere smitteforebyggelsen, vil et skærpet fokus på hele organiseringen, undervisning af personale, tilrettelæggelsen af isolationen med fokus på sengestuefaciliteter, tid til kontakt og grundig information være nødvendig. Her udover kan individuelle foranstaltninger på baggrund af en risikoanalyse overvejes. / Aim: This study explored and describedthe factors that may influence how patients react to source isolation from others during hospitalization. The study also sought to determine how background variables such as gender, age, and previous hospitalization affect source isolation. Based on individuals’ risk assessment, this study also focusedon how hospitalsplan and the organization of care and treatment. Method: This qualitative study used content analysisto reviewd ata collected from interviews with five patients. The conceptual framework describes antibiotic resistance and infection control from a public health perspective and exploredits prevention in Denmark. Thetheoretical framework describe show patients experiencean infection acquired by exposure to drug-resistant bacteria, as well assubsequent source isolation. Results: Thelimited space of an isolation room, including closed doors, lack of contact with people, and few sensory stimuli, resulted in patient boredom, monotony,and anxiety. Moreover, the data showed that background variables affected how patients experience source isolation. Compared with men, women showed greater concern about precautions against infection and greater awareness of staff behavior. Women also worried more about the risk of transmitting bacteria/disease to visitors and familymembers, and display more emotion during isolation. In contrast, men outwardly resigned themselves to the situation and didnot speculate about infection precautions. Men had more rational approach, and tended to cope better when isolated in a single room. Younger patients seemed to have a better coping strategy during isolation, and considered a single room an advantage compared to the ward. Elderly patients felt sad and lonely during source isolation. In addition, previous negative experiences from earlier hospitalization seemedto influence current isolation. Patients developed their own strategies for coping with source isolation and found themselves balanced between being stressed and coping. Conclusion: Hospitals need more alternatives (e.g., better training and improved treatment culture) to prevent negative psychological affects due to isolation without compromising infection prevention. Hospitals should update their personnel at all organizational levels, and focus on room facilities in the ward, contact time,and improved information and communication. Riskassessment should be individualizedfor each patient. / <p>ISBN 978-91-86739-98-0</p>

Benzalkonium chlorides (BACs) are widely used, broad-spectrum disinfectants and frequently detected in the environment, even at toxic levels for life. Since such disinfectants can induce broad resistance capabilities, BACs may fuel the emergence of antibiotic resistance in the environment. A substantial body of literature has reported that exposure to BACs causes antibiotic resistance; yet, other studies suggest that the resistance linkage is rare, unsystematic, and/or clinically insignificant. Accordingly, whether or not disinfectant exposure mediates antibiotic resistance and, if so, what molecular mechanisms underlie the resistance link remains to be clearly elucidated. Further, understanding how microbial communities degrade BACs is important not only for alleviating the possible occurrence of antibiotic resistance but also reducing the potential risks to environmental and public health. An integrated strategy that combines metagenomics, metatranscriptomics, genetics, and traditional culture-dependent approaches was employed to provide novel insights into these issues. The integrative approach showed that a microbial community exposed to BACs can acquire antibiotic resistance through two mechanisms: i) horizontal transfer of previously uncharacterized efflux pump genes conferring resistance to BACs and antibiotics, which were encoded on a conjugative plasmid and co-selected together upon BACs and ii) selective enrichment of intrinsically multi-drug resistant organisms. Further, a microbial community adapts to BAC exposure via a variety of mechanisms, including selective enrichment of BAC-degrading species and amino acid substitutions and horizontal transfer of genes related to BAC resistance and degradation. The metatranscriptomic data suggests that the BAC-adapted microbial community metabolized BACs by cooperative interactions among its members. More specifically, Pseudomonas nitroreducens cleaved (i.e., dealkylated) BACs, metabolized the alkyl chain (the dealkylated product of BACs), and released benzyldimethylamine (the other product of BACs), which was further metabolized by other community members (e.g., Pseudomonas putida). Collectively, this study demonstrates the role of BACs in promoting antibiotic resistance and advances current understanding of a microbial community degrading BACs. The results of this work have important implications for (appropriate) usage of disinfectants and for assessing, predicting, and optimizing biological engineering processes treating BAC-bearing waste streams.

Antibiotic resistance is a worsening threat to human health. Increasing our understanding of the mechanisms causing this resistance will be of great benefit in designing methods to evade resistance and in developing new classes of antibiotics. In this thesis, I have used the TetR Family Transcriptional Regulators (TFRs), which constitute one of the largest antibiotic resistance regulator families, as a model system to study the structure, function and evolution of antibiotic resistance determinants. I performed a thorough examination of the variation and conservation seen in TFR sequences and structures using computational approaches. Through structure comparison, I have identified the most conserved features shared by the TFR family that are crucial for their stability and function. Based on my findings on conserved TFR structural features, a quantitative assay of binding affinity determination was developed. Through sequence comparison and a residue contact map method, I discovered the existence of a conserved residue network that correlates well with the known allostery pathway of TetR. This predicted allosteric communication network was experimentally tested in TtgR. I have also developed methods to identify TFR operator sequences through genomic comparisons and validated my prediction through experiments. In addition, I have developed an in vivo system that can be used to identify and characterize proteins that mediate resistance to almost any antibiotic. This system is simple, fast, and scalable for high-throughput applications, and could be used to discover a wide range of novel antibiotic resistance mechanisms. The principles that I applied to the TFR family could also be applied to other protein families.

In Streptomyces clavuligerus, the route to the biosynthesis of &amp / #945 / -aminoadipic acid (&amp / #945 / -AAA) represents an important primary metabolic pathway providing carbon flux to the synthetases of antibiotic formation. This carbon flow comes through the lysine-specific branch of the aspartate pathway and is rate limiting in the formation of cephamycin C, a second generation cephalosporin produced by this organism. In this study, the lysA gene which encodes for an important key enzyme of aspartate pathway / meso-diaminopimelic acid (DAP) decarboxylase (E.C.4.1.1.20) catalyzing the conversion of diaminopimelate to lysine was cloned and characterized for the first time from S. clavuligerus NRRL 3585. The attempts to clone the gene by constructing libraries of S. clavuligerus genomic DNA and screening of the libraries either by homologous probing or complementation approach gave no positive results. Then, PCR-based cloning was taken as the approach and the gene was amplified with PCR using the primers derived from the conserved sequences of lysA genes in two fragments (620 and 983 bp) which had overlapping regions. Fragments were then cloned and nucleotide sequencing revealed a complete open reading frame (ORF) encoding a protein of 463 aa (Mr 49, 907). The GC content of the gene was identified as 70.98 %. The gene sequence showed 83 % identity to the sequence of S. coelicolor lysA gene and 81 % identity to S. avermitilis lysA gene. By comparing the amino acid sequence of this protein to those available in database, the sites of the enzyme important for catalysis were identified.

Biological/biochemistry analyses traditionally require bulky instruments and a great amount of volume of biological/chemical agents, and many procedures have to be performed in certain locations such as medical centers or research institutions. These limitations usually include time delay in testing. The delays may be critical for some aspects such as disease prevention or patient treatment. One solution to this issue is the realization of point-of-care (POC) testings for patients, a domain in public health, meaning that health cares are provided near the sites of patients using well-designed and portable medical devices. Transportation of samples between local and central institutions can therefore be reduced, facilitating early and fast diagnosis. A closely related topic in engineering, lab-on-a-chip (LOC), has been discussed and practiced in recent years. LOC emphasizes integrating several functions of laboratory processes in a small portable device and performing analysis using only a very small amount of sample volume, to achieve low-cost and rapid analysis. From an engineer's point of view, LOC is the strategy to practice the idea of POC testing. This dissertation aimed at exploring the POC potentials of porous membrane-base LOC devices, which can be used to simplify traditional and standard laboratory procedures. In this study, three LOC prototypes are shown and discussed. First the protein sensor incorporating with silica nanofiber membrane, which has shown 32 times more improvement of sensitivity than a conventional technique and a much shorter detection time; secondly the bacteria filter chip that uses a sandwiched aluminum oxide membrane to stabilize the bacteria and monitor the efficacy of antibiotics, which has reduced the test time from 1 day of the traditional methods to 1 hour; the third is the sensor combining microfluidics and silica nanofiber membrane to realize Surface Enhanced Raman Spectroscopy on bio-molecules, which has enhancement factor 10^9 and detection limit down to nanomolar, but simple manufacturing procedures and reduced fabrication cost. These results show the porous-base membrane LOC devices may have potentials in improving and replacing traditional detection methods and eventually be used in POC applications.