Urinvägsinfektioner är den näst vanligaste anledningen till antibiotikaförskrivning i Sverige. För diagnos av UVI i urinkultur med standardmetoden krävs 18 - 48 timmar och ytterligare 24 timmar i odling tills resultaten för antimikrobiell känslighetstestning är tillgängliga. Direktresistensmetoden ger svar 24 timmar tidigare än standardmetoden. Studiens syfte var att utvärdera direktresistensmetoden (in-housemetod) från mikrobiologiska laboratoriet Unilabs, Skövde på urinodlingar från patientprover och jämföra den med EUCAST-diskdiffusionsmetod. I denna studie användes 334 bakteriestammar från urinodlingar; E. coli (n=211) E. faecalis (n=45), Klebsiella spp (n=37), P. mirabilis (n= 17) och övriga gramnegativa bakteriestammar (n=24). Direktresistensmetoden och EUCAST diskdiffusionsmetod har utförts parallellt på alla bakteriestammar och sex olika antibiotikatyper testades. Hämningszonernas diameter lästes av och placerades i rätt S, I, R kategori för respektive antibiotikum. Från sammanlagt 1674 testade antibiotikalappar hittades 23 avvikelser där E. coli representerade 13 av dessa. Mecillinam var det antibiotikum där flest avvikelser noterades, totalt tio stycken. Överensstämmelsen mellan de två metoderna var 98,6% från 1674 testade antibiotikalappar. Direktresistensmetoden leder till att patienten få ett svar 24 timmar tidigare än med EUCAST diskdiffusionsmetoden där svaret inkluderar även resistensbestämning. / Urinary tract infections are the second most common reason for prescription of antibiotics in Sweden. Diagnosis of UTIs in urine culture by the standard method requires 18 to 48 hours and an additional 24 hours until antimicrobial testing is available. The direct resistance method provides answers 24 hours earlier than the standard method. The aim of the study was to evaluate the direct resistance method (in-house method) from the microbiological laboratory Unilabs, Skövde on urine cultures from patient samples and compare it with the EUCAST disc diffusion method. For this study, 334 bacterial strains from urinary cultures were used; E. coli (n = 211) E. faecalis (n = 45), Klebsiella spp (n = 37), P. mirabilis (n = 17) and other gram-negative rods (n = 24). The direct resistance method and the EUCAST disk diffusion method were performed in parallel on all bacterial strains and six different types of antibiotics were tested. The diameter of the inhibition zones was measured and categorized according to S, I, R for each antibiotic. From a total of 1674 tested antibiotics 23 discrepancies were found where E. coli represented 13 of these. Mecillinam was the antibiotic where most discrepancies were noted, a total of ten. The agreement between the two methods was 98,6% from 1674 tested antibiotics. The direct resistance method allows a response to the patient 24 hours earlier than the EUCAST disc diffusion method.

The global increase of antibiotic resistant bacteria threatens the modern health care and challenges the therapeutic effects of available antibiotics. The b-lactam mecillinam (Mec) is an exception to this due to a stable clinical resistance prevalence resistance of approximately 3%. It is only used to treat uncomplicated urinary tract infections (UTIs), mainly caused by E. coli. Mecillinam resistance (MecR) is easily selected for in laboratory settings and linked to >40 genes, including the mrdA gene encoding the Mec target penicillin-binding protein 2. A majority of the known MecR mutations confer a severe fitness cost. Fitness is important for bacteria to survive in the bladder and clinical isolates have been shown to have high fitness. These isolates contain loss-of-function mutations in the cysB gene, which encode a positive regulator of cysteine biosynthesis. In a previous evolution experiment, fitness cost of cysB and mrdA MecR mutations was compensated and the compensatory mutations were identified. Here the compensatory mutations were reconstructed into wildtype (WT) E. coli strain MG1655, and cysB and mrdA backgrounds to study the impact of the mutations on resistance and fitness, using MIC tests and Bioscreen C assays. Our results show that the mrdA mutants only had partial fitness compensation (significantly lower fitness than WT) for all strains and all strains also increased their MecR. The low fitness is possibly an explanation for the lack of mrdA mutants outside laboratories. Of the clinically relevant cysB mutants the majority lost their resistance when increasing growth rate, some even to levels significantly higher than WT, indicating that DcysB mutations are easier to compensate for. One strain (ydjNmx2) however, had a significantly higher growth rate while remaining clinically MecR.

The introduction of antibiotics in healthcare is one of the most important medical achievements with regard to reducing human morbidity and mortality. However, bacterial pathogens have acquired antibiotic resistance at an increasing rate, and due to a high prevalence of resistance to some antibiotics they can no longer be used therapeutically. The antibiotic mecillinam, which inhibits the penicillin-binding protein PBP2, however, is an exception since mecillinam resistance (MecR) prevalence has remained low. This is particularly interesting since laboratory experiments have shown that bacteria can rapidly acquire MecR mutations by a multitude of different types of mutations. In this thesis, I examined mechanisms and dynamics of mecillinam resistance in clinical and laboratory isolates of Escherichia coli. Only one type of MecR mutations (cysB) was found in the clinical strains, even though laboratory experiments demonstrate that more than 100 genes can confer resistance Fitness assays showed that cysB mutants have higher fitness than most other MecR mutants, which is likely to contribute to their dominance in clinical settings. To determine if the mecillinam resistant strains could compensate for their fitness cost, six different MecR mutants (cysB, mrdA, spoT, ppa, aspS and ubiE) were evolved for 200-400 generations. All evolved mutants showed increased fitness, but the compensation was associated with loss of resistance in the majority of cases. This will also contribute to the rarity of clinical MecR isolates with chromosomal resistance mutations. How MecR is mediated by cysB mutations was previously unclear, but in this thesis I propose and test a model for the mechanism of resistance. Thus, inactivation of CysB results in cellular depletion of cysteine that triggers an oxidative stress response. The response alters the intracellular levels of 450 proteins, and MecR is achieved by the increase of two of these, the LpoB and PBP1B proteins, which rescue the cells with a mecillinam-inhibited PBP2. Mecillinam is used for UTI treatments and to investigate mecillinam resistance in a more host-like milieu, MecR strains were grown in urine and resistance was examined. Interestingly, this study showed that neither laboratory, nor clinical cysB mutants are resistant in urine, most likely because the cysteine present in the urine phenotypically reverts the bacteria to susceptibility. These findings suggest that mecillinam can be used to treat also those clinical strains that are identified as MecR in standard laboratory tests, and that testing of mecillinam susceptibility in the laboratory ought to be performed in media that mimics urine to obtain clinically relevant results. In summary, the work described in this thesis has increased ourgeneral knowledge of mecillinam resistance and its evolution. Hopefully this knowledge can be put to good use in clinical settings to reduce the negative impact of antibiotic resistance.