1Department of Medical Microbiology, Maastricht University Medical Center, 2Department of Internal Medicine, Erasmus Medical Center
Hansen, W. L. J., Beuving, J., Verbon, A., Wolffs, P. F. G. One-day Workflow Scheme for Bacterial Pathogen Detection and Antimicrobial Resistance Testing from Blood Cultures. J. Vis. Exp. (65), e3254, doi:10.3791/3254 (2012).
Bloodstream infections are associated with high mortality rates because of the probable manifestation of sepsis, severe sepsis and septic shock1. 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 hours2, 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 probes5. 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 rods6. This assay was based on a study in which PCR was used to measure the growth of bacteria7. 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.
PART I: PATHOGEN IDENTIFICATION
1. Sample Preparation
Note: The entire molecular workflow as is described in the following protocol should be performed according to recommendations for quality assurance in molecular diagnostics3.
2. Identification Assay: Real-time 16s rDNA PCR
3. Analysis of the Results
PART II: ANTIBIOTIC SUSCEPTIBILITY TESTING
4. Isolation of Bacteria from Positive Blood Cultures9
5. Inoculation of Micro Titre Plates
6. Real-time 16s rDNA PCR10
7. Analysis of the Results
8. Representative Results
Two model organisms, i.e. a Gram-negative E. coli and a Gram-positive S. aureus, are chosen to visualize the combined procedure for the detection and identification of bacterial pathogens and the determination of their antimicrobial profile. The first part of the protocol comprises the pathogen identification. Specific probes are designed for the detection of eight clinically relevant microorganisms. In presence of a target included in the bacterial panel, amplification curves are generated and Ct values are calculated (Figure 2). The cut-off value to consider a PCR result as positive is set to a Ct value of 35. In Figure 2A, the identification profile of an E.coli-infected blood culture is shown. The 16S universal probe is included in two separate reaction mixtures and consequently generates two amplification curves (Ct of 25.20 and 25.95). The third signal is derived from the probe specific for E. coli (Ct of 27.04). The identification of a S. aureus-infected blood culture is shown in Figure 2B. The 16S universal probe has amplification signals of 33.35 and 33.71. The two remaining signals are derived from the probes specific for Staphylococcus spp. and S. aureus (Ct of 32.48 and 30.59).
After the first part of the protocol, the causative microorganism is known and the antimicrobial profile can be determined. Figure 3 is an example of an antibiotic susceptibility testing amplification plot, representing the E.coli strain that was also shown in Figure 2A. Each line represents one antibiotic that the bacterial sample was incubated with. A sample with a low Ct value is a sample in which growth has occurred in the presence of an antibiotic, indicating resistance to the tested antibiotic. On the contrary, a high Ct value represents a sample in which no growth has occurred because of the effective working of the antibiotic, indicating susceptibility to the tested antibiotic. Table 1 illustrates the determination of the antimicrobial profile of the E. coli and S. aureus isolates. All Ct values are reported and, using the formulas mentioned in the protocol text (7.1), two cut-off Ct values are calculated to distinguish between resistance and susceptibility. The strain is resistant to the antibiotic if the reported Ct value is lower than the calculated cut-off Ct value (and vice versa).
Figure 1. Flow chart of the pathogen identification and antibiotic susceptibility testing procedure using real-time 16S rDNA PCR.
Figure 2. Identification assay: Amplification plots and cycle threshold values (Ct values). A positive blood culture is detected by the universal 16S rDNA probe, while the specific probes are used for the identification of the causal pathogen. A. amplification plot of blood culture containing E. coli; B. amplification plot of blood culture containing S. aureus; Pseu ae, Pseudomonas aeruginosa; uni, 16S universal probe; Ecoli, Escherichia coli probe; Pseu sp, Pseudomonas spp. probe; S. pneu, Streptococcus pneumoniae probe; Strep sp, Streptococcus spp. probe; Entero, Enterococcus spp. probe; S. aureus, Staphylococcus aureus probe; Staph sp, Staphylococcus spp. probe.
Figure 3. Amplification plot of antibiotic susceptibility testing of an E. coli isolate (sample 1). Each curve represents one antibiotic that the strain was incubated with. An early signal is caused by a high bacterial load, which means that the strain has grown in the presence of the tested antibiotic and is thus resistant to the antibiotic. Late signals indicate that the strain has not grown in the presence of the antibiotic, in other words, it is susceptible.
|Sample 1: E. coli||Sample 2: S. aureus|
|Amoxicillin 8 mg/L||16,83||R||Amoxicillin 0.25 mg/L||21,03||R|
|Amoxicillin-clavulanate 8/4 mg/L||17,36||R||Oxacillin 2 mg/L||25,80||S|
|Piperacillin 16 mg/L||16,67||R||Vancomycin 2 mg/L||25,20||S|
|Piperacillin-tazobactam 16/4 mg/L||24,15||S||Gentamicin 4 mg/L||25,86||S|
|Ciprofloxacin 1 mg/L||29,72||S||Trimethoprim-sulfamethoxazole 2/38 mg/L||24,62||S|
|Ceftazidime 1 mg/L||24,03||S|
|Ceftazidime 8 mg/L||26,58||S|
|Gentamicin 4 mg/L||29,83||S|
|Trimethoprim-sulfamethoxazole 2/38 mg/L||27,60||S|
|Negative growth control (mixture of antibiotics)||30,41||Negative growth control (sample stored at 4 °C)||27,42|
|Positive growth control||16,90||Positive growth control||20,22|
|Cut-off Ct-value 1*||21,76||Cut-off Ct-value 1***||23,82|
|Cut-off Ct-value 2**||18,75||Cut-off Ct-value 2****||22,02|
|* For amoxicillin, amoxicillin-clavulanate, ciprofloxacin, gentamicin, trimethoprim-sulfamethoxazole
** For piperacillin, piperacillin-tazobactam, ceftazidime
|*** For vancomycin and gentamicin
**** For amoxicillin, oxacillin and trimethoprim-sulfamethoxazole
Table 1. Determination of antibiotic susceptibility testing of the two samples (E.coli and S.aureus). Ct values of the PCR-assay were copied to this excel file, as which can automatically calculates the two cut-off Ct alues from the positive and negative growth control, using the formulas shown in the protocol text. If an antibiotic shows a Ct value lower than the cut-off Ct value, the strain is resistant to the antibiotic, if the Ct value was higher than the cut-off, the strain is susceptible.
The protocol described here enables the rapid identification of pathogens and provides a functional antimicrobial profile that could lead to the early administration of adequate antibiotics thereby improving the prognosis of patients with bloodstream infections. Depending on the requested conditions of a test, i.e. low cost, high throughput, minimal turn-around time, testing conditions can be adjusted. The whole procedure can be performed within one working day. Moreover, the two parts of the protocol can be performed simultaneously, which reduces the turn-around time significantly. As presented here, the identification panel is a selection of the most clinically relevant bacteria in our hospital. Since the main principle is targeting the 16S gene region, specific probes for other microorganisms can be designed and added to the assay. The complete assay was originally intended for the rapid analysis of blood cultures, but can also be used for the processing of other sample materials. This is also the case for the antibiotics that were used for antibiotic susceptibility testing: more or other antibiotics can be added, based on local resistance patterns and guidelines.
We have nothing to disclose.
This work was supported by the Profileringsfonds azM (PF245).
|Sodium Chloride (NaCl)||Merck Chemicals||106404||0.9% in water|
|Vacutainer SST Serum Separator Tube 5 ml||BD Diagnostic Systems||367986|
|Mueller Hinton II broth||BD Diagnostic Dystems||212322||44 g/L in water|
|Centrifuge Rotixa 50 rs||Andreas Hettich GmbH & Co. KG||4910|
|Centrifuge 5415 D||Eppendorf||Discontinued|
|TaqManEnvironmental master mix 2.0||Applied Biosystems||4396838|
|iQ SYBRGreen Supermix||Bio-Rad Laboratories BV||170-8880|
|MicroAmp Optical 96-Well Reaction Plate||Applied Biosystems||N8010560|
|MicroAmp Optical Adhesive Film||Applied Biosystems||4311971|
|iQ 96-Well PCR Plates||Bio-Rad Laboratories BV||223-9441|
|Microseal B Adhesive Seals||Bio-Rad Laboratories BV||MSB-1001|
|Real-time PCR Detection System||Applied Biosystems||ABI PRISM 7900HT|
|Real-Time PCR Detection System||Bio-Rad Laboratories BV||MyiQ Single-Color|