Spermine (Spm), a potent bactericidal polyamine, exerts a strong synergistic effect with β-lactams against methicillin-resistant Staphylococcus aureus (MRSA) in a pH-dependent manner. At high pH (>8) Spm is a potent nucleophile, and able to form Spm-β-lactam adduct. At physiological pH (or lower), Spm carries positive charges, and can bind to DNA through charge interactions. The potential of Spm interfering with cell wall was first investigated. A spontaneous mutant of MRSA Mu50 selected for Spm resistance conferred resistance to Spm/β-lactam synergy. This phenotype was due to the presence of a 7-bp deletion in pbpB as identified by genome resequencing and confirmed by complementation. Analysis of cell wall composition by HPLC revealed the combination of Spm and β-lactam can reduce the cross-linkage of peptidoglycan. These two lines of evidence suggest Spm may perturb cell wall integrity in favor of β-lactam efficacy with PBPs as a promising target. However, from the results of microarray analysis and fluorescent Bocillin labeling, Spm did not appear to suppress the PBPs expression or alter their interactions with β-lactams. Next, transcriptome analyses reveal the genes responsive to the synergy effect overlap extensively with those to high Spm challenge, implying the enhanced detrimental effect of Spm facilitated by β-lactams in inhibition on cell growth. In particular the induction of iron transport and reduction of energy production under synergy were depicted in this study, and high dose Spm was found to turn off the SigB regulon. Of interest, the tetM gene encoding a ribosomal protection protein for tetracycline (Tc) resistance exhibited the most significant fold change and high signals by both high and low dose Spm. Further analysis by qRT-PCR demonstrated the tetM expression was specifically induced by Tc and Spm to a comparable level but not by other polyamines, suggesting a similar mode of action by Spm and Tc in interactions with the ribosome to initiate tetM induction. Collectively, these data indicated the role of Spm could be multifarious with more than one target, and a combination of Spm and β-lactams may inhibit growth of MRSA in a more complicated manner than just potentiating β-lactam inhibition on PBPs.

Syftet med denna litteraturstudie var att beskriva hur patienter med MRSA upplevde att bli vårdade samt hur de upplevde bemötandet från sjukvårdspersonalen. Metoden: Studien var en beskrivande litteraturstudie baserad på 15 vetenskapliga artiklar som granskats och kvalitetsbedömts. Resultatet: Resultatet visade att patienternas kunskap om MRSA varierade. Upplevelsen av att ha fått MRSA fick en stor känslomässig påverkan på livet och de var oroliga för framtiden. Orsaken till infektionen trodde vissa berodde på dålig personlig hygien. Upplevelsen av isolering kändes traumatisk och ledde till ångest och depression för många patienter men var också positivt för en del. Patiener med MRSA upplevde att det rådde brist på information från sjukvårdspersonalen om hur MRSA påverkade deras liv. Bemötandet upplevdes obehagligt eftersom personalen hade ett oprofessionellt förhållningssätt mot de infekterade patienterna. Många var rädda för att föra smittan vidare till familjen och vänner. Slutsats: Ytterligare studier behövs för att belysa hur kunskap hos patienter och sjukvårdspersonal varierade. Isoleringen upplevdes trauamatisk och det rådde brist på information och bemötande.

MRSA is a bacteria that is known to cause a wide range of different infections in humans, some more severe than others. In recent years different strains of the bacteria, mainly the MRSA CC398, has been found in many pig farming operations across Europe. Most of Sweden’s pork imports come from Denmark and Germany and the estimation of the prevalence of MRSA in pig farms throughout both countries are more than 50 percent. For this study Sweden were chosen due to the fact that no pigs have been found to carry the infection in this country. The purpose of this study was to find out if legislation and use of antibiotics in each of the countries somehow influence the spreading of the infection, but also to investigate how MRSA transmits between different individuals. The study is split in two parts, namely a literature review and a small survey that were sent to Swedish pig producers. The survey showed that disease protection is important and that 67 percent of the pig producers that answered are worried that MRSA might come to Sweden. From the comparison of the legislation no real conclusion could be made, but antibiotic group treatments of pigs can be linked to an increased incidence of MRSA in pig farms.

Methicillin-resistant S.aureus (MRSA) is a highly virulent pathogen associated with serious healthcare-associated (HCA-MRSA) and community-associated (CA-MRSA) infections. MRSA is an increasingly important cause of skin and skin-structure, bloodstream and other invasive infections including pneumonia and endocarditis. The pharmacodynamics of existing treatments and anovel cephalosporin with activity against MRSA were studied in an in vitro infection model comparing the antibacterial effects of monotherapy and combination therapy under standard and altered environmental conditions. The activity of monotherapy with vancomycin, daptomycin, linezolid and ceftobiprole against clinical MRSA isolates were tested along with combinations of vancomycin-ceftobiprole, daptomycin-ceftobiprole and linezolid-ceftobiprole. Antibacterial response under standard conditions supporting optimal bacterial growth were compared to altered conditions with acidic pH 5.5, diluted nutrient broth (1:2) and increased temperature 40°C. Two clinical isolates including one HCA-MRSA (#81655) and one CA-MRSA (#79002) were studied in an in vitro pharmacodynamic model (IPDM) over 24 hours. Clinical dosing regimens equivalent to vancomycin 1500 mg intravenously every 12 hours (peak =24.4 mg/L, trough =7.4 mg/L), daptomycin 6 mg/kg (420 mg) intravenously every 24 hours (peak =8.2 mg/L, trough =0.8 mg/L) and linezolid 600 mg intravenously every 12 hours (peak =9.2 mg/L, trough =2.8 mg/L) were tested. Ceftobiprole was administrated as a bolus dose followed by constant infusion of 10 mg/L. Antibacterial effects were quantified as initial bacterial kill rate over 4 hours (KR4) and absolute bacterial kill at 24 hours (BK24). Minimum inhibitory concentrations (MIC) were measured via E-test® methods using initial isolates and those recovered after 24 hours of therapy. The KR4 with daptomycin and vancomycin were equivalent (P=0.14), yet daptomycin was more rapid than ceftobiprole (P=0.03) and linezolid (P<0.0001). The BK24 was greatest with ceftobiprole and vancomycin which were superior to linezolid (P<0.0001, P<0.0001, respectively) and daptomycin (P=0.0001, P=0.0001, respectively). Daptomycin was associated with bacterial re-growth and increasing MICs from 0.25 mg/L to 2-4 mg/L during therapy for isolate #79002 under standard conditions. Furthermore, daptomycin activity against both isolates was significantly reduced under altered conditions (KR4, P=0.0001; BK24, P=0.04). Combination therapy with vancomycin-ceftobiprole was indifferent compared with either agent alone. Although daptomycin-ceftobiprole prevented daptomycin non-susceptibility during therapy and resulted in significantly greater BK24 compared with daptomycin alone (BK24 difference of 4.07 log10 cfu/mL, P=0.0001), the combination was indifferent from ceftobiprole alone. Finally, linezolid-ceftobiprole was similar to linezolid but significantly less active than ceftobiprole alone (BK24 difference of 1.39 log10 cfu/mL, P=0.005) raising concerns of potential antagonism with this combination. In conclusion, this study provides important data regarding antimicrobial pharmacodynamics against MRSA. Overall, monotherapy with either ceftobiprole or vancomycin was most active. Combination therapy with ceftobiprole prevented the emergence of daptomycin non-susceptibility during therapy, but demonstrated potential antagonism with linezolid.

Methicillin-resistant S.aureus (MRSA) is a highly virulent pathogen associated with serious healthcare-associated (HCA-MRSA) and community-associated (CA-MRSA) infections. MRSA is an increasingly important cause of skin and skin-structure, bloodstream and other invasive infections including pneumonia and endocarditis. The pharmacodynamics of existing treatments and anovel cephalosporin with activity against MRSA were studied in an in vitro infection model comparing the antibacterial effects of monotherapy and combination therapy under standard and altered environmental conditions. The activity of monotherapy with vancomycin, daptomycin, linezolid and ceftobiprole against clinical MRSA isolates were tested along with combinations of vancomycin-ceftobiprole, daptomycin-ceftobiprole and linezolid-ceftobiprole. Antibacterial response under standard conditions supporting optimal bacterial growth were compared to altered conditions with acidic pH 5.5, diluted nutrient broth (1:2) and increased temperature 40°C. Two clinical isolates including one HCA-MRSA (#81655) and one CA-MRSA (#79002) were studied in an in vitro pharmacodynamic model (IPDM) over 24 hours. Clinical dosing regimens equivalent to vancomycin 1500 mg intravenously every 12 hours (peak =24.4 mg/L, trough =7.4 mg/L), daptomycin 6 mg/kg (420 mg) intravenously every 24 hours (peak =8.2 mg/L, trough =0.8 mg/L) and linezolid 600 mg intravenously every 12 hours (peak =9.2 mg/L, trough =2.8 mg/L) were tested. Ceftobiprole was administrated as a bolus dose followed by constant infusion of 10 mg/L. Antibacterial effects were quantified as initial bacterial kill rate over 4 hours (KR4) and absolute bacterial kill at 24 hours (BK24). Minimum inhibitory concentrations (MIC) were measured via E-test® methods using initial isolates and those recovered after 24 hours of therapy. The KR4 with daptomycin and vancomycin were equivalent (P=0.14), yet daptomycin was more rapid than ceftobiprole (P=0.03) and linezolid (P<0.0001). The BK24 was greatest with ceftobiprole and vancomycin which were superior to linezolid (P<0.0001, P<0.0001, respectively) and daptomycin (P=0.0001, P=0.0001, respectively). Daptomycin was associated with bacterial re-growth and increasing MICs from 0.25 mg/L to 2-4 mg/L during therapy for isolate #79002 under standard conditions. Furthermore, daptomycin activity against both isolates was significantly reduced under altered conditions (KR4, P=0.0001; BK24, P=0.04). Combination therapy with vancomycin-ceftobiprole was indifferent compared with either agent alone. Although daptomycin-ceftobiprole prevented daptomycin non-susceptibility during therapy and resulted in significantly greater BK24 compared with daptomycin alone (BK24 difference of 4.07 log10 cfu/mL, P=0.0001), the combination was indifferent from ceftobiprole alone. Finally, linezolid-ceftobiprole was similar to linezolid but significantly less active than ceftobiprole alone (BK24 difference of 1.39 log10 cfu/mL, P=0.005) raising concerns of potential antagonism with this combination. In conclusion, this study provides important data regarding antimicrobial pharmacodynamics against MRSA. Overall, monotherapy with either ceftobiprole or vancomycin was most active. Combination therapy with ceftobiprole prevented the emergence of daptomycin non-susceptibility during therapy, but demonstrated potential antagonism with linezolid.

Methicillin-resistant Staphylococcus aureus (MRSA) is a serious public health problem nationwide, threatening to develop into an epidemic. Many of these patients are presenting to their primary care clinics with skin and soft tissue infections (SSTIs). The CDC has reported that in 2005, MRSA was responsible for an estimated 94,000 life-threatening infections and 16,650 deaths. The purpose of this study is to estimate the incidence of CA-MRSA within a specific family practice in Florida and to identify epidemiologic factors, classify antibiotic susceptibility patterns, and evaluate patient education in regard to disease management and prevention. This study was a descriptive, epidemiologic, three-year retrospective medical record review of all wound cultured skin and soft tissue infections that presented to a family practice between January 2007 and December 2009. Sixty-two medical records met the inclusion and exclusion criteria for the study. Of these 62 SSTIs, 44 cultures grew one or more bacterial organisms. The incidence of CA-MRSA was 66% (n=29). The mean age of those with CA-MRSA was 40 years old, with a range from 7 to 90 years old. Sixty-two percent (n=18) were male and 38% (n=11) were female; additionally 69% (n=20) lived within a 10 mile radius from the family practice, while 31% (n=9) lived in a surrounding suburb. The most frequent race was Caucasian 83% (n=24), with African American at 10% (n=3) and Hispanics 7% (n=2). Risk factors associated with CA-MRSA was obesity 41% (n=10), diabetes mellitus 24% (n=7), and a previous history of MRSA infection 24% (n=7). Skin and soft tissue infections were diagnosed as either an abscess 62% (n=18), boil 24% (n=7), pustule 10% (n=3), or cellulitis 4% (n=1). CA-MRSA isolates were susceptible to trimethoprim-sulfamethoxazole 100% (n=29), doxycycline 93% (n=27), and rifampin 100% (n=14). Clindamycin susceptibility was 65% (n=15) with resistance at 30% (n=7) and 5% (n=1) intermediate. Both cephalexin and erythromycin were 100% resistant. Documentation in the medical record on wound care was found in 45% (n=13) of the records. The incidence of CA-MRSA SSTI was 66%, which identifies this suburban community at high risk for this bacterial infection. Risk factors associated with CA-MRSA included obesity (BMI > 30), history of previous MRSA infection, and diabetes mellitus. There were no clinical characteristics that helped distinguish MRSA infection from other bacterial SSTIs. Most SSTI were treated with incision and drainage and a susceptible antibiotic. Judicious use of antibiotics not only provides appropriate treatment, but is also critical in prevention of antibiotic resistance. Lastly, patient education in adequate hygiene is essential in preventing the spread of CA-MRSA

Introduktion: Antibiotikaresistenta bakterier är ett stort hot mot folkhälsan. Alla patienter har rätt till en smittfri vårdmiljö, trots det förekommer smittspridning inom hälso och sjukvården. Meticillinresistenta staphylococcus aureus (MRSA) hos patienter, personal, men också i vårdmiljön utgör en risk. För att tidigt upptäcka, förebygga och förhindra smittspridning behövs rutiner för vård, hantering av utrustning samt städning av vårdlokaler. Syfte: Syftet var att beräkna andelen miljöodlingar som visade växt av MRSA i vårdrum efter slutstädning, efter vård av patient med känd MRSA, samt om patientens riskfaktorer eller vårdtidens längd påverkade resultatet. Metod: En kvantitativ studie med ett konsekutivt material som utgörs av data från sammanlagt 180 utförda miljöodlingar i Region Halland. Materialet är bearbetat i statistikprogrammet, SPSS, och analyserat med Chi2 test. Resultat: I 14 % (25 av 180) av miljöodlingar påvisades MRSA i vårdmiljön. Riskfaktorers påverkan kunde inte påvisas vad det gällde sår/hudlesioner, dränage, katetrar, tracheostomi eller andra konstgjorda kroppsöppningar. Däremot fanns en signifikant ökad risk att finna MRSA i vårdrummet, efter slutstädning, om vårdtiden överstigit 1 dygn. Slutsats: Studien visar att slutstädningens kvalitet är viktig och troligen mer betydelsefull än inverkan av patientens riskfaktorer eftersom det inte gick att påvisa något statistiskt signifikant samband mellan patientens riskfaktorer och förekomst av MRSA i vårdrummet efter slutstädning. Däremot kan vårdtiden användas vid bedömning av ökad risk för växt och smittspridning av MRSA och rutiner borde eventuellt anpassas utifrån det. / Introduction: Evolution of antibiotic resistant bacteria poses a major threat to public health. All patients are entitled to a non-infectious care environment; despite the fact that infectious transmission in health care is possible. Prevalence of methicillin-resistant Staphylococcus aureus (MRSA) in patients, staff and in health care environment represents such a threat.  In infection prevention in health care established routines in patient care, handling of medical equipment and cleaning in care environment are essential. Aim: The aim of the study was to assess the fraction of MRSA positive environmental sampling following terminal cleaning of a room where a patient with confirmed MRSA was treated, and to assess whether potential patient’s risk factors and/or length of hospital stay influenced the result. Methods: Quantitative study with consecutive material consisting of data from in total 180 environmental samplings performed in Region Halland. The material has been processed with the statistic program SPSS, and analyzed with Chi2 test.  Results: In 14 % (25 out of 180) of the environmental samplings MRSA was detected. A statistically significant association between prevalence of patient risk factors as wounds/skin lesions, drainage, catheters, tracheostomy or other artificial body openings could not be shown. However, a hospital stay exceeding 24 hours significantly increased risk of detecting MRSA in the patient room after terminal cleaning.  Conclusion: Quality of terminal cleaning is important in preventing transmission of MRSA in health care environment and probably more important than the impact of patient risk factors. However, length of hospital stay is a factor worth consideration and can possibly be used to influence care routines in prevention of transmission of MRSA in health care environment.

Methicillin-resistant Staphylococcus aureus (MRSA) is an important nosocomial pathogen associated with significant morbidity and mortality. Previous colonisation with this pathogen is a risk factor for the development of subsequent infection. Tolllike receptors (TLRs) are a family of transmembrane receptors of the innate immune system that recognize pathogen-associated molecular patterns. The role of nasal colonisation of S. aureus has started to receive more attention. In spite of this, there are not enough studies looking at its effects on human primary nasal epithelial cells and their response to TLR ligands. The respiratory tract itself seems to pose a contradiction given by the clinical observation that its upper portion (nasal compartment) allows the growth of bacteria, acting like a reservoir, whereas the lower portion (lung compartment) reacts with an exuberant inflammatory response to the same organisms, as noted during pneumonia. The mechanism related with this phenomenon remains to be elucidated. A negative regulator of the TLR signalling cascade called toll-interacting protein (tollip) has been demonstrated to induce hyporesponsiveness in the gastrointestinal tract in the presence of bacteria. So far, tollip has not been demonstrated in the respiratory tract. Aims: To compare the responses of the upper and lower respiratory tract to TLR ligands, to characterise the role of tollip in the respiratory tract and its effects in the induction of tolerance, and to determine the cellular response to nasal carriage of S. aureus. Materials and Methods: The cell line RPMI 2650 (representative of nasal epithelium) and the cell line A549 (representative of type II alveolar epithelium) were used to establish the cytokine response to stimulation with TLR ligands and to demonstrate the presence of tollip protein by immunocytochemistry and enzymelinked immunosorbent assay (ELISA). Primary human nasal epithelial and type II alveolar epithelial cells were isolated and cultured from consented subjects. The cytokine response to stimulation was measured using cytokine bead array and the presence of tollip was determined by immunofluorescence and quantitative polymerase chain reaction. The presence of TLRs was assessed by immunocytochemistry in primary nasal and type II alveolar epithelial cells and the response to stimulation with the TLR9 agonist CpG-C ODN was assessed in these cells as well as in primary human type II alveolar epithelial cells. Subjects were also assessed for nasal carriage of S. aureus and their associated cytokine responses. Results: The RPMI 2650 cell line, despite retaining phenotypic characteristics of the nasal epithelium, appears unresponsive to stimulation with TLR ligands. In contrast, the A549 cell line responded significantly to stimulation with TLR ligands. Primary human nasal epithelial cells responded by secreting higher amounts of interleukin (IL)-8 and IL-6 in response to stimulation with S. aureus peptidoglycan (PGN) and tumour necrosis factor alpha (TNF-α) with a strong trend toward statistical significance. These cells did not respond to stimulation with Pseudomonas aeruginosa LPS. Primary type II alveolar epithelial cells responded significantly to stimulation with S. aureus PGN by increasing the secretion of IL-8, IL-6, IL-1β, TNF-α and IL-10 into cultured supernatant. Cells from the upper respiratory tract displayed a more tolerant phenotype given by the lower levels in cytokine production in response to stimulation with S. aureus PGN, in contrast to alveolar epithelial cells. TLRs were identified in primary nasal epithelial cells. The negative regulator tollip was identified in cell lines as well as primary cells of the respiratory tract in its three segments: nasal, bronchial and type II alveolar. It was not possible to demonstrate an up-regulation of tollip after stimulation with TLR ligands in any of the cell types studied, although, it was possible to observe a significantly higher constitutive level in tollip mRNA transcripts from primary nasal epithelial cells in comparison to type II alveolar epithelial cells. TLR9 was identified in human primary nasal epithelial cells, although it was not possible to observe an increase in cytokine production after stimulation with a TLR9 agonist. TLR9 was expressed strongly in primary type II alveolar epithelial cells which responded by significantly increasing IL-8 production after stimulation with CpG-C ODN. Primary nasal epithelial cells from individuals who carry S. aureus exhibit a proinflammatory profile, as evidenced by higher basal levels of IL-8 and IL-6 in comparison to non-colonised controls. Conclusion: The upper respiratory tract epithelium displays a tolerant phenotype in response to stimulation with TLR ligands in comparison to the lower respiratory epithelium, potentially favouring nasal colonisation by S. aureus. Tollip m-RNA transcripts appear to be up-regulated constitutively in the nasal epithelium which might favour this response. Staphylococcus aureus colonisation is however associated with a local pro-inflammatory state in the nasal epithelium of carrier individuals.

ABSTRAKT             Bakgrund: Förekomsten av MRSA ökar i världen. Studier har visat att isoleringsvård är problematiskt med bland annat psykiskt lidande som följd.     Syfte: Syftet med litteraturstudien var att beskriva patienters upplevelser av att vara isolerad i samband med MRSA.  Metod: Litteraturstudie genomfördes med åtta artiklar. Sju artiklar hade en kvalitativ ansats och en var av mixad metod. De granskades, analyserades efter inspiration av Friberg (2012) och blev sammanställda. Resultat: Fem kategorier togs fram efter analys av artiklarnas resultat. Varje kategori hade två till fyra subkategorier. Kategorierna var; psykisk påverkan på patienten, isoleringsrutinernas påverkan på patienten, egna rummet, hanteringen av isolering och personalens betydelse. Konklusion: Upplevelsen av isoleringsvård i samband med MRSA kan variera. Positiva- och negativa känslor beskrevs. Patienter upplevde ensamhet och tristess i isoleringen. Några patienter uppskattade det privata utrymmet. Vårdpersonalen kunde uppleva rädsla att vårda smittsamma patienter vilket kan påverka omvårdnaden. Mer forskning skulle kunna belysa kunskapluckor och fylla dem. Fler studier från patienters perspektiv kan förbättra upplevelsen för kommande patienter. Nyckelord: Isolering, MRSA, omvårdnad, psykisk påverkan, upplevelser

The advancements in Whole Genome Sequencing (WGS) have increased the amount of genomic information available for epidemiological analyses. WGS opens many avenues for investigation into the tracking of pathogens, but the rapid advancements in WGS could soon lead to a situation where traditional analytical techniques might become computationally impractical. For example, the traditional method to determine the origin of an isolate is to use phylogenetic analyses. However, phylogenetic analyses become computationally prohibitive with larger datasets and are best for retrospective epidemiology. Therefore, I investigated if there might be less computationally demanding methods of analysing the same data to obtain similar conclusions. This thesis describes a proof-of-principle method for evaluating if such alternative analysis techniques might be viable. In this thesis Methicillin resistant Staphylococcus aureus (MRSA) was used, and single nucleotide polymorphism (SNP) and insertion/deletion (indel) genomic variation. I move away from whole genome analysis techniques, such as phylogenetic analysis, and instead focus on individual SNPs. I showed that genetic signals (such as SNPs and indels) can be utilised in novel ways to rapidly produce a summary of the possible geographic origin of an isolate with a minimal demand on computational power. The methods described could be added to the suite of analytical epidemiological tools and are a promising indication of the viability of developing cheap, rapid diagnostic tools to be implemented in healthcare institutions. Furthermore, the principles behind the development of the methods described in this thesis could have much wider applications than just MRSA. This implies that further work based on the principles described in this thesis on alternative pathogens could prove to be promising avenues of investigation.