Bloodstream infections with ESBL-producing Enterobacterales : prediction, rapid diagnosis and molecular epidemiology

Antimicrobial-resistant bacteria are a threat to public health worldwide. Particularly, the increase of extended-spectrum β-lactamase (ESBL)-producing Enterobacterales (EPE) is a significant clinical problem. ESBLs confer resistance to cephalosporins and carbapenemases confer resistance to carbapenems. Escherichia coli, the most important member of the Enterobacterales, is the most common cause of bloodstream infection (BSI). First-line treatment is in most cases the cephalosporin cefotaxime, but EPE are resistant to cefotaxime. Thus, patients with EPE BSI are at risk of prolonged hospitalization and increased mortality caused by empirical treatment failure. This thesis aimed to improve care for patients with EPE BSI in three ways: early prediction, rapid diagnosis and increased understanding of resistance transmission and the importance of microbial virulence for the severity of disease. The aim of Study I was to develop a practical prediction-score to identify patients at risk of EPE BSI which could be used to improve the appropriateness of empirical treatment. Risk factors for EPE BSI were assessed and two clinical scores evaluated. The strongest predictors (named the Stockholm score), prior EPE-positive culture (especially recent samples), prostate biopsy and healthcare abroad, were present in 50% of cases. In other patients, prediction was difficult, hence rapid diagnostics and susceptibility testing are necessary. Therefore, the aim of Study II was to evaluate a method for rapid susceptibility testing. EUCAST disk diffusion read after 6 hours was compared to 18 hours on E. coli and Klebsiella pneumoniae-isolates representing clinically important resistance mechanisms. Results showed that 6-hour reading was accurate if adapted breakpoints were used. Resistance traits are mainly spread through clonal expansion of bacterial cells and/or horizontal gene transfer trough plasmids. Development of appropriate intervention methods depend on knowledge of the mode of transmission. Study III aimed to determine the molecular epidemiology of K. pneumoniae harbouring the carbapenemase-gene blaNDM-1. Isolates from India, UK and Sweden were subjected to MLST, plasmid replicon typing and PCR of virulence genes. Diverse sequence types and plasmids, rather than a single high-risk clone, were responsible for the early dissemination of K. pneumoniae with blaNDM-1. BSI is often complicated by sepsis and septic shock, associated with high mortality. With resistance on the rise, the search for new therapies includes anti-virulence agents. Thus, a correct understanding of the influence of microbial virulence on pathogenesis is important. The aim of Study IV was to determine the respective contributions of microbial virulence and patient factors to the severity of disease. Whole genome sequencing (WGS) was performed on E. coli from patients with EPE BSI. The virulence gene iss, increased serum survival, was associated with septic shock, especially in immunocompetent patients. In conclusion, the treatment of patients with EPE BSI could be improved by these results. Use of the Stockholm score identified many, but not all, patients at risk of EPE BSI, thereby improving the appropriateness of empirical therapy. Rapid disk diffusion with adapted breakpoints gave accurate results and would enable early correction of therapy. blaNDM-1 was found in diverse sequence types of K. pneumoniae. The virulence gene iss was associated with septic shock in EPE BSI and might have potential as an anti-virulence treatment target. LIST OF SCIENTIFIC PAPERS I. Fröding I, Karlsson Valik J, Bolinder L, Naucler P, Giske CG. Prediction of bloodstream infection caused by extended-spectrum beta-lactamase-producing Enterobacterales in patients with suspected community-onset sepsis. Int J Antimicrob Agents. 2019;53:820-829. II. Fröding I, Vondracek M, Giske CG. Rapid EUCAST disc diffusion testing of MDR Escherichia coli and Klebsiella pneumoniae: inhibition zones for extendedspectrum cephalosporins can be reliably read after 6 h of incubation. J Antimicrob Chemother. 2017;72:1094-102. III. Giske CG, Fröding I, Hasan CM, Turlej-Rogacka A, Toleman M, Livermore D, Woodford N, Walsh TR. Diverse sequence types of Klebsiella pneumoniae contribute to the dissemination of blaNDM-1 in India, Sweden, and the United Kingdom. Antimicrob Agents Chemother. 2012;56:2735-8. IV. Fröding I, Hasan B, Sylvin I, Nauclér P, Giske CG. ESBL-producing E. coli causing community-onset bloodstream infection and the association of bacterial clones and virulence genes with septic shock. Manuscript. OTHER RELATED PUBLICATIONS 1. Giske CG, Dyrkell F, Arnellos D, Vestberg N, Hermansson Panna S, Fröding I, Ullberg M, Fang H. Transmission events and antimicrobial susceptibilities of methicillin-resistant Staphylococcus argenteus in Stockholm. Clin Microbiol Infect. 2019;25(10):1289 e5e8. 2. Enstrom J, Fröding I, Giske CG, Ininbergs K, Bai X, Sandh G, Ullberg M, Fang H. USA300 methicillin-resistant Staphylococcus aureus in Stockholm, Sweden, from 2008 to 2016. PLoS One. 2018;13(11):e0205761. 3. Fang H, Fröding I, Gian B, Haeggman S, Tollstrom UB, Ullberg M, Nord CE. Methicillin-resistant Staphylococcus aureus in Stockholm, Sweden: Molecular epidemiology and antimicrobial susceptibilities to ceftaroline, linezolid, mupirocin and vancomycin in 2014. J Glob Antimicrob Resist. 2016;5:31-5.