The aim of this study was to develop an easy-to-use culture-free diagnostic method based on next generation sequencing (NGS) of PCR amplification products encompassing whole 16S-23S rRNA region to improve the resolution of bacterial species identification. To determine the resolution of the new method 67 isolates were subjected to four identification methods: Sanger sequencing of the 16S rRNA gene; NGS of the 16S-23S rRNA region using MiSeq (Illumina) sequencer; Microflex MS (Bruker) and VITEK MS (bioMérieux). To evaluate the performance of this new method when applied directly on clinical samples, we conducted a proof of principle study with 60 urine samples from patients suspected of urinary tract infections (UTIs), 23 BacT/ALERT (bioMérieux) positive blood culture bottles and 21 clinical orthopedic samples. The resolution power of NGS of the 16S-23S rRNA region was superior to other tested identification methods. Furthermore, the new method correctly identified pathogens established as the cause of UTIs and blood stream infections with conventional culture. NGS of the 16S-23S rRNA region also showed increased detection of bacterial microorganisms in clinical samples from orthopedic patients. Therefore, we conclude that our method has the potential to increase diagnostic yield for detection of bacterial pathogenic species compared to current methods.

Summary One of the major challenges for modern medicine is our ageing society and an increased level of immunocompromised hosts. More invasive and intensive medical interventions will increase the number of healthcare-associated infections (HCAI), which means infection that occur because of or in concomitance, but in any case, during or after healthcare interventions. Such infections are caused usually endogenously from microbial components of the patient’s own microbiome. Usually, the microorganisms of the microbiome show a natural resistance against a few antibiotics. Due to selection processes and epidemic transmission of specific clones, microorganisms that have become resistant to multiple antibiotics become part of the patient’s microbiome and can subsequently cause infections that are difficult or even impossible to treat. The kind of infections that will occur depends on diverse factors. Already today, according to Cassini et al., 2,609,911 new cases of HCAI occur every year in the European Union and European Economic Area (EU/EEA). The cumulative burden of the six HAIs was estimated at 501 disability-adjusted life years (DALYs) per 100,000 general population each year in the EU/EEA. In a recent publication, 426,277 healthcare-associated infections caused by antimicrobial resistant microorganisms were calculated to occur in the EU every year. Attributable deaths in the EU due to antimicrobial resistant microorganisms were estimated to be 33,110 per year. We know that we cannot prevent all HCAI. Because medical innovations will allow for an increased number of novel treatments that will comprise abiotic materials, microorganisms will adapt to this environment and enhance the risk for new HCAI. The challenge for the future will not be to try to prevent all infections, as some of them will remain unavoidable, but to prevent the occurrence of non-treatable microorganisms that would make unavoidable infections additionally untreatable. That means that we need to reflect on how we organize infection prevention, diagnostics and control. While patients with classical infectious diseases present with infectious diseases (ID)-specific symptoms, patients with HCAI present usually with another underlying disease. HCAI are therefore perceived as a secondary damage not following classical clinical and epidemiological rules. However, more recently we have to consider how we should react to HCAI and antimicrobial resistance (AMR) as they are quite different in epidemiology and transmission behavior than classical infectious diseases. Today, the prevalence of AMR is rising all over Europe. Although good success has been seen in many countries, methicillin-resistant Staphylococcus aureus (MRSA) remains an important challenge for many countries. In addition to MRSA, multidrug-resistant Escherichia coli and carbapenem-resistant Enterobacteriaceae are becoming a problem of public health importance. Furthermore, we need to focus more on implementation of known infection prevention measures than trying to solve the problem by observing and describing it. However, in addition to medical factors such as antibiotic use, hand hygiene etc., we tend to forget that there are factors behind these factors that have a major influence and are found in the structures of our different healthcare systems. We need to look more at the context before we try to implement prevention measures and need to learn from each other. A common goal to tackle carbapenem-resistant Enterobacteriaceae (CRE) by 2030 would be an important step to foster collaboration across Europe. As the current funding and remmuneration system does not sufficiently support prevention of HCAI and AMR, it is time for the development of a less production- but more prevention-economic financing system for clinical microbiology and infection control.

The study describes the transmission of a CTX-M-15-producing ST15 Klebsiella pneumoniae between patients treated in a single center and the subsequent inter-institutional spread by patient referral occurring between May 2012 and September 2013. A suspected epidemiological link between clinical K. pneumoniae isolates was supported by patient contact tracing and genomic phylogenetic analysis from May to November 2012. By May 2013, a patient treated in three institutions in two cities was involved in an expanding cluster caused by this high-risk clone (HiRiC) (local expansion, CTX-M-15 producing, and containing hypervirulence factors). A clone-specific multiplex PCR was developed for patient screening by which another patient was identified in September 2013. Genomic phylogenetic analysis including published ST15 genomes revealed a close homology with isolates previously found in the USA. Environmental contamination and lack of consistent patient screening were identified as being responsible for the clone dissemination. The investigation addresses the advantages of whole-genome sequencing in the early detection of HiRiC with a high propensity of nosocomial transmission and prolonged circulation in the regional patient population. Our study suggests the necessity for inter-institutional/regional collaboration for infection/outbreak management of K. pneumoniae HiRiCs.

Considering the threat of antimicrobial resistance and the difficulties it entails in treating infections, it is necessary to cross borders and approach infection management in an integrated, multidisciplinary manner. We propose the antimicrobial, infection prevention and diagnostic stewardship model comprising three intertwined programs: antimicrobial, infection prevention and diagnostic stewardship, involving all stakeholders. The focus is a so-called ‘theragnostics’ approach. This leads to a personalized infection management plan, improving patient care and minimizing resistance development. Furthermore, it is important that healthcare regions nationally and internationally work together, ensuring that the patient (and microorganism) transfers will not cause problems in a neighboring institution. This antimicrobial, infection prevention and diagnostic stewardship model can serve as a blue print to implement innovative, integrative infection management.

Bdellovibrio bacteriovorus is a small Deltaproteobacterium which, since its discovery, has distinguished itself for the unique ability to prey on other Gram-negative bacteria. The studies on this particular “predatory bacterium”, have gained momentum in response to the rising problem of antibiotic resistance, because it could be applied as a potential probiotic and antibiotic agent. Hereby, we present recent advances in the study of B. bacteriovorus, comprehending fundamental aspects of its biology, obligatory intracellular life cycle, predation resistance, and potential applications. Furthermore, we discuss studies that pave the road towards the use of B. bacteriovorus as a “living antibiotic” in human therapy, focussing on its interaction with biofilms, the host immune response, predation susceptibility and in vivo application models. The available data imply that it will be possible to upgrade this predator bacterium from a predominantly academic interest to an instrument that could confront antibiotic resistant infections.

Objectives: Daptomycin (DAP) resistance in Staphylococcus aureus is uncommon but there are increasing reports of the emergence of resistance during DAP therapy. Most clinical DAP-resistant S. aureus isolates investigated carried mutations in the mprF gene. The aim of this study was to identify mutations between a clinical pair of methicillin-susceptible S. aureus (MSSA) isolates (DAP-susceptible and DAP-resistant). Additionally, the activity of genes previously associated with DAP resistance was assessed. Materials and Methods: Two MSSA isolates from patient with left-sided endocarditis were analyzed by whole genome sequencing (WGS) and reverse transcription-quantitative real-time PCR (RT-qPCR). The first isolate, DAP-susceptible, was obtained before initiation of treatment and the second isolate, DAP-resistant, was recovered after 4 weeks of DAP therapy. Results: Comparison of complete genomes of DAP-susceptible and its DAP-resistant variant identified two non-synonymous and one synonymous mutations. The non-synonymous mutations consisted of a S829L substitution in mprF and a T331I substitution in vraS. The RT-qPCR experiments revealed an increased expression of vraS, dltA, mprF, and sceD genes in DAP-resistant variant. Strikingly, the expression of dltA and mprF genes was significantly downregulated by DAP. Conclusion: The mprF and vraS genes were previously associated with DAP resistance, however, none of the mutations described in this study had been previously identified and linked to DAP resistance. Moreover, we provide a new insight into the DAP action on S. aureus, in which the expression of key genes in DAP resistance is decreased by the antibiotic.