One-day Workflow Scheme for Bacterial Pathogen Detection and Antimicrobial Resistance Testing from Blood Cultures

Abstract

Bloodstream infections are associated with high mortality rates because of the probable manifestation of sepsis, severe sepsis and septic shock 1 . Therefore, rapid administration of adequate antibiotic therapy is of foremost importance in the treatment of bloodstream infections. The critical element in this process is timing, heavily dependent on the results of bacterial identification and antibiotic susceptibility testing. Both of these parameters are routinely obtained by culture-based testing, which is time-consuming and takes on average 24-48 hours 2,4 . The aim of the study was to develop DNA-based assays for rapid identification of bloodstream infections, as well as rapid antimicrobial susceptibility testing. The first assay is a eubacterial 16S rDNA-based real-time PCR assay complemented with species-or genus-specific probes 5 . Using these probes, Gram-negative bacteria including Pseudomonas spp., Pseudomonas aeruginosa and Escherichia coli as well as Gram-positive bacteria including Staphylococcus spp., Staphylococcus aureus, Enterococcus spp., Streptococcus spp., and Streptococcus pneumoniae could be distinguished. Using this multiprobe assay, a first identification of the causative micro-organism was given after 2 h.Secondly, we developed a semi-molecular assay for antibiotic susceptibility testing of S. aureus, Enterococcus spp. and (facultative) aerobe Gram-negative rods 6 . This assay was based on a study in which PCR was used to measure the growth of bacteria 7 . Bacteria harvested directly from blood cultures are incubated for 6 h with a selection of antibiotics, and following a Sybr Green-based real-time PCR assay determines inhibition of growth. The combination of these two methods could direct the choice of a suitable antibiotic therapy on the same day (Figure 1).In conclusion, molecular analysis of both identification and antibiotic susceptibility offers a faster alternative for pathogen detection and could improve the diagnosis of bloodstream infections.

Feasibility study demonstrating that enzymatic template generation and amplification can be employed as a novel method for molecular antimicrobial susceptibility testing

“…Beuving and colleagues [19,20] have demonstrated that molecular AST can be performed on bacteria harvested directly from positive blood cultures by collecting the microbes from the culture using a SST. Such a method could produce a reliable MIC for a series of antibiotics against a pathogenic microbe without the need for its isolation, thereby further reducing the time required to obtain a reliable result.…”

“…The blood culture bottle was incubated in a BD BACTEC 9050 incubator and grown until the culture is called positive. Once positive, the bacteria were harvested with a Serum Separation Tube (SST) (BD, Franklin Lakes, NJ) as described elsewhere [19,20]. Briefly, the tube was spun for 10 minutes at 2000×g and the supernatant was removed.…”

“…The kinetics of increasing PCR signal from a growing culture in the presence of an antibiotic can be used to determine whether a pathogen is resistant or susceptible to the agent. Furthermore, one group reports a workflow in which molecular AST can be performed on bacteria harvested directly from blood culture using serum separation tubes, identifying the pathogen with species specific qPCR probes, and producing a molecular AST result in a single day [20]. …”

Rapid Antimicrobial Susceptibility Testing with Electrokinetics Enhanced Biosensors for Diagnosis of Acute Bacterial Infections

“…Molecular techniques, including multiplexed PCR, 10, 16 DNA microarrays, 17, 18 and molecular probes, 19, 20 offer alternatives to conventional culture-based analysis. Genotypic ASTs, which target known resistance genes, are widely used to identify specific pathogens such as methicillin-resistant Staphylococcus aureus .…”

“…22 The combination of phenotypic AST of the physiological sample and molecular biosensors for bacterial detection may represent a promising alternative to the conventional approach in infectious diseases diagnostics. 17, 18 However, most genotypic biosensors, such as real-time PCR, requires purified samples and additional extraction steps. 23 The bacteria and molecular separation steps can significantly increase the overall assay time and system complexity, which limits their applications in non-traditional healthcare settings.…”