ANTIBIOTIC RESISTANCE AND ANTIBIOTIC CONSUMPTION IN SWEDEN WITH FOCUS ON Escherichia coli AND Pseudomonas aeruginosa

Aims: The general aims of the present studies were to assess the levels of antibiotic resistance, in relation to antibiotic consumption at the Karolinska University Hospital, Solna (KS) and at 11 other Swedish hospitals, furthermore to assess the role of the membrane protein OprD and penicillin-binding proteins in Pseudomonas aeruginosa resistance to imipenem. Methods: Resistance figures were retrieved from the microbiology service databases for the period 1989-99, at the 12 above mentioned hospitals, including their intensive care units (ICU). Antibiotic consumption figures were obtained from the National Corporation of Swedish Pharmacies database during the same period. In order to study molecular mechanisms of carbapenem resistance, we produced transconjugants from clinical isolates of carbapenem resistant P. aeruginosa in a sensitive PAO18 after selection for a proline marker (proB). The active sites of penicillin-binding proteins PBP1b, PBP2, PBP3 and PBP6 were sequenced, and the expression of oprD, pbp2 and pbp3 genes was measured using quantitative real-time PCR. Results: Resistance to ciprofloxacin increased in Escherichia coli and P. aeruginosa in parallel with an increased quinolone consumption in all included hospitals. The use of cephalosporins increased two and a half times, while the level of resistance in E. coli to cefuroxime and cefotaxime remained stable at KS. A third pattern was observed for co-trimoxazole resistance in E. coli, which increased at KS as well as the other 11 Swedish hospitals, while consumption of co-trimoxazole and trimethoprim decreased during the 12 year study period. Resistance rates at KS were still generally low, but there were increasing trends for some antibtiotic-microbe combinations. E. coli resistance to ciprofloxacin increased from 0% in 1991 to 11% in 1999 and cotrimoxazole resistance increased in E. coli from 7.5% to 14% during the study period. For E. coli, resistance to ciprofloxacin was higher at the hospital than at the ICUs. There were considerable fluctuations in resistance prevalence over time, especially at the ICU. Imipenem resistance in P. aeruginosa was particularly noticeable at the ICU, with resistance peaks of 15% and 28% in 1992 and 1999, respectively. These peaks were due to outbreaks. Sequencing of P. aeruginosa genes for PBP1b, PBP2, PBP3 and PBP6 showed no differences in amino acid sequence, but the gene for OprD porin was downregulated in all imipenem resistant clinical strains and their transconjugants. Conclusions: The significant trend of increased resistance to antibiotics over time constitutes an important warning system. The relation between antibiotic consumption and antibiotic resistance was not always parallel. Three different patterns were observed which suggests that different mechanisms were operating. We also found in some cases, higher resistance rates at the hospital than at the ICUs emphasizing the importance of including all sectors of a hospital. Also, antibiotic resistance figures fluctuated substantially over time, illustrating the value of long surveillance periods. Finally, in imipenem resistant P. aeruginosa, a previously unknown gene for regulation of oprD, is most likely located close to the proB marker. LIST OF PUBLICATIONS This thesis is based on the following papers, which will be referred to in the text by their roman numerals I-IV: I. Sörberg M, Farra A, Ransjö U, Gårdlund B, Rylander M, Wallen L, Kalin M, Kronvall G. Long-term antibiotic resistance surveillance of gram-negative pathogens suggests that temporal trends can be used as a resistance warning system. Scandinavian Journal of Infectious Diseases, 34: 372-378, 2002. II. Sörberg M, Farra A, Ransjö U, Gårdlund B, Rylander M, Settergren B, Kalin M, Kronvall G. Different trends in antibiotic resistance rates at a university teaching hospital. Clinical microbiology and infection, 9: 388-396, 2003. III. Farra A, Skoog G, Wallen L, Kahlmeter G, Kronvall G, Sörberg M. Antibiotic use and Escherichia coli resistance trends for quinolones and co-trimoxazole in Sweden. Scandinavian Journal of Infectious Diseases, 34: 449-455, 2002. IV. Farra A, Strålfors A, Sörberg M, Wretlind B. Role of outer membrane protein OprD and penicillin-binding proteins in Pseudomonas aeruginosa resistance to imipenem Submitted to International Journal of Antimicrobial Agents The original papers are printed in this thesis with permission from the publisher. TABLE OF CONTENTS