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

Screening for multi-drug-resistant Gram-negative bacteria: what is effective and justifiable?

Effectiveness is a key criterion in assessing the justification of antibiotic resistance interventions. Depending on an intervention's effectiveness, burdens and costs will be more or less justified, which is especially important for large scale population-level interventions with high running costs and pronounced risks to individuals in terms of wellbeing, integrity and autonomy. In this paper, we assess the case of routine hospital screening for multi-drug-resistant Gram-negative bacteria (MDRGN) from this perspective. Utilizing a comparison to screening programs for Methicillin-Resistant Staphylococcus aureus (MRSA) we argue that current screening programmes for MDRGN in low endemic settings should be reconsidered, as its effectiveness is in doubt, while general downsides to screening programs remain. To accomplish justifiable antibiotic stewardship, MDRGN screening should not be viewed as a separate measure, but rather as part of a comprehensive approach. The program should be redesigned to focus on those at risk of developing symptomatic infections with MDRGN rather than merely detecting those colonised

USA300 methicillin-resistant Staphylococcus aureus in Stockholm, Sweden, from 2008 to 2016.

Methicillin-resistant Staphylococcus aureus (MRSA) USA300 isolates have been recognized globally, not only in community but also in healthcare settings. USA300 isolates were initially resistant only to methicillin, but resistance to non-β-lactams has emerged with time. To evaluate the prevalence and antimicrobial susceptibility of USA300 isolates in Stockholm, we conducted a nine-year retrospective study. Of 5359 consecutive MRSA cases in Stockholm, isolates from 285 cases were USA300 strains according to the pulsed-field gel electrophoresis pattern. Of these cases, repeated isolates with altered antibiotic resistance patterns were observed in six individuals. Therefore, antimicrobial susceptibility testing was performed on totally 291 isolates. To study the phylogenetic relatedness of isolates in transmission events and genomic resistance traits, 35 isolates were further studied by whole genome sequencing (WGS). The incidence of MRSA was increased from 17.6 per 100,000 inhabitants in 2008 to 37.3 per 100,000 inhabitants in 2016, while the proportion of USA300 cases declined from 6.6% in 2008 to 2.6% in 2016. Among the USA300 isolates, 73.5% were community-associated, 21.3% healthcare-associated, and 5.2% had unknown acquisition. The highest resistance rate among non-β-lactams was found in erythromycin (86%), followed by fluoroquinolones (68-69%). 57% of the isolates were resistant to both erythromycin and fluoroquinolone. Simultaneous resistance to four non-β-lactam antibiotic classes was found in six isolates. Four isolates were susceptible to all non-β-lactam antibiotics. Ceftaroline, daptomycin, linezolid, mupirocin, rifampicin, teicoplanin, telavancin, trimethoprim-sulfamethoxazole and vancomycin retained full activity in the study. WGS analysis indicated that isolates from an outbreak were phylogenetically closely related. In conclusion, USA300 MRSA isolates in Stockholm have neither been limited to the community setting, nor remained susceptible to non-β-lactam agents. WGS is becoming a useful tool in tracing transmission events. The results herein provide the most up-to-date and comprehensive information regarding status of USA300 strains in this geographic area

EUCAST rapid antimicrobial susceptibility testing (RAST) in blood cultures: Validation in 55 european laboratories

© The Author(s) 2020.Objectives: When bloodstream infections are caused by resistant bacteria, rapid antimicrobial susceptibility testing (RAST) is important for adjustment of therapy. The EUCAST RAST method, directly from positive blood cultures, was validated in a multi-laboratory study in Europe. Methods: RAST was performed in 40 laboratories in northern Europe (NE) and 15 in southern Europe (SE) from clinical blood cultures positive for Escherichia coli, Klebsiella pneumoniae, Pseudomonas aeruginosa, Staphylococcus aureus or Streptococcus pneumoniae. Categorical results at 4, 6 and 8 h of incubation were compared with results for EUCAST standard 16–20 h disc diffusion. The method, preliminary breakpoints and the performance of the laboratories were evaluated. Results: The total number of isolates was 833/318 in NE/SE. The number of zone diameters that could be read (88%, 96% and 99%) and interpreted (70%, 81% and 85%) increased with incubation time (4, 6 and 8 h). The categorical agreement was acceptable, with total error rates in NE/SE of 2.4%/4.9% at 4 h, 1.1%/3.5% at 6 h and 1.1%/3.3% at 8 h. False susceptibility at 4, 6 and 8 h of incubation was below 0.3% and 1.1% in NE and SE, respectively, and the corresponding percentages for false resistance were below 1.9% and 2.8%. After fine-tuning breakpoints, more zones could be interpreted (73%, 89% and 93%), with only marginally affected error rates. Conclusions: The EUCAST RAST method can be implemented in routine laboratories without major investments. It provides reliable antimicrobial susceptibility testing results for relevant bloodstream infection pathogens after 4–6 h of incubation