Klebsiella Pneumoniae and Escherichia Coli Multidrug-resistance and Different Aspects of Invasive Infections

Klebsiella pneumoniae and Escherichia coli are pathogens belonging to the Enterobacteriaceae family. They can cause infections ranging from uncomplicated urinary tract infection to severe bloodstream infection (BSI). The prevalence of extended spectrum βlactamase-producing Enterobacteriaceae (EPE) is increasing worldwide and carbapenemases (CPE), a subgroup of EPE where antibiotic treatment is very limited, is a major threat to patients.The aims of this thesis were to get expanded molecular and epidemiological knowledge about K. pneumoniae, its association to morbidity and mortality in BSI (II, III), to increase sensitivity in detection of carbapenemase-producers (I), to determine risk to acquire fecal colonization with EPE during traveling, and to characterize colonizing EPE in terms of virulence factors and phylogroups (IV). In paper I methods for antimicrobial susceptibility testing were evaluated for detection of K. pneumoniae carbapenemase (KPC)and Verona integron-encoded metallo-lactamase (VIM)-producing K. pneumoniae in order to define appropriate screening breakpoints. Strains (n=51) were tested against different carbapenems using disk diffusion, gradient test, and automated susceptibility testing. Results were interpreted with the European (EUCAST) and American (CLSI) antimicrobial susceptibility testing breakpoints. We found that clinical breakpoints cannot be used for carbapenemase screening. Meropenem was the most suited carbapenem to use for screening purposes. A breakpoint of 0.5 mg/L detected all isolates with an at the same time good separation from the wild type population. In paper II and III a cohort of patients with BSI caused by K. pneumoniae was evaluated retrospectively and compared with BSI caused by E. coli. Data on risk factors, prognostic factors and mortality was retrieved from 1251 medical charts (III). The late mortality (within 90 days) was significantly higher among patients with BSI caused by K. pneumoniae and could be explained by higher comorbidity. Contrary to European trends our study showed low antibiotic resistance among K. pneumoniae isolates supporting the hypothesis of absence of successful multidrug-resistant K. pneumoniae clones in the Stockholm area. For a subset of the patients (n=139) molecular analysis was performed on the K. pneumoniae isolates (II). Based on multilocus sequence typing, the isolates could be separated in three phylogenetic clades: KpI (n=96), KpII (n=9) and KpIII, also known as K. variicola (n=34). Patients infected with strains belonging to K. variicola had higher 30 days mortality (29.4 %), also when adjusting for age and comorbidity (OR for KpIII = 3.0 (95% CI: 1.1-8.4) compared to KpI). Only three of the isolates causing mortality within 30 days belonged to any of the virulent serotypes, had a mucoid phenotype, or harbored virulence genes. Hence, the increased mortality could not be related to any known strain factor. In general, a high level of comorbidity was observed in the K. pneumoniae cohort. Paper IV was a prospective study. Fecal samples and survey data were collected from 188 Swedes traveling to four regions of high EPE prevalence, and molecular characterization was performed on EPE. Colonization incidence varied by visited region; the Indian subcontinent 49%, northern Africa 44%, Southeast Asia 19% and Turkey 10%. Few strains harbored virulence factors connected to uropathogenicity, and most E. coli strains belonged to phylogroup A, rarely associated with extraintestinal infections. No clinical infections were seen in follow-up. No CPE was found, but one strain contained the plasmidmediated colistin resistance gene, mcr-1. Independent risk factors for EPE acquisition were travelers ́ diarrhea and use of antibiotics during travel. EPE acquired during travel have seemingly low pathogenicity as indicated by the low frequency of virulence factors and phylogroups associated with extraintestinal infections. In summary this thesis provides new knowledge about K. pneumoniae BSI in a clinical and a molecular perspective. It also adds to the knowledge about molecular features of EPE colonizing the intestine, and appropriate breakpoints to use in detection of CPE. LIST OF PUBLICATIONS I. Vading M, Samuelsen Ø, Haldorsen B, Sundsfjord A and Giske CG. Comparison of disk diffusion, Etest and VITEK2 for detection of carbapenemase-producing Klebsiella pneumoniae with EUCAST and CLSI breakpoint systems Clin Microbiol Infect. 2011; 17(5): 668-74. II. Matallah M*, Vading M*, Kabir M, Bakhrouf A, Kalin M, Nauclér P, Brisse S and Giske CG. Klebsiella variicola is a frequent cause of bloodstream infection in the Stockholm area, and associated with higher mortality compared to K. pneumoniae. PLoS One 2014. 26;9(11). III. Vading M, Nauclér P, Diaz Högberg L, Kalin M and Giske CG. High long-term mortality in Swedish invasive infections caused by Klebsiella pneumoniae and low prevalence of resistance compared to European EARSNet data. Manuscript. IV. Vading M, Kabir MH, Nauclér P, Kalin M, Iversen A, Wiklund S, and Giske CG. Frequent acquisition of low-virulent strains of extended-spectrum betalactamase-producing Escherichia coli in travelers. Submitted manuscript. * Both authors contributed equally to the paper ADDITIONAL RELEVANT PUBLICATIONS Wiklund S, Fagerberg I, Örtqvist Å, Vading M, Giske CG, Broliden K, Tammelin A. Knowledge and understanding of antibiotic resistance and the risk of becoming a carrier when travelling abroad: a qualitative study of Swedish travellers. Scand J Public Health. 2015; 43(3):302-8.