This work was supported by the Profileringsfonds azM (PF245).

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.
<ol>
 <li>Add an 0.1 ml aliquot of blood culture to 0.9 ml 0.9% NaCl into a 1.5 ml reaction tube to become a 1:10 diluted sample. (Dilute in order to prevent qPCR inhibition).</li>
 <li>Centrifuge sample at 13400 x g for 5 min to pellet the bacterial DNA.</li>
 <li>Resuspend the bacterial pellet in 100 &mu;l sterile demineralised H2O.</li>
 <li>Store the DNA sample at 4 &deg;C until further use.</li>
</ol>
2. Identification Assay: Real-time 16s rDNA PCR
<ol>
 <li>Prepare the reaction mixtures as follows. The assay consists of four separate reactions per sample. Each mixture includes 12.50 &mu;l master mix, 0.9 &mu;M forward primer (5-TCCTACGGGAGGCAGCAGT-3)7, 0.6 &mu;M reverse primer (5-GGACTACCAGGGTATCTAATCCTGTT-3)8 and a panel of probes. The amount of probes is given for each of the four separate reactions in below.</li>
 <li>The first reaction includes:
 <ul>
 <li>0.2 &mu;M universal probe (5-FAM-CGTATTACCGCGGCTGCTGGCAC-3-BHQ1)8</li>
 <li>0.2 &mu;M P. aeruginosa probe (5-JOE-CCAAAACTACTGAGCTAGAGTACG-3-BHQ1)</li>
 </ul>
 </li>
 <li>The second reaction includes:
 <ul>
 <li>0.2 &mu;M E. coli probe (5-JOE-GGAGTAAAGTTAATACCTTTGCTCATT-3-BHQ1)</li>
 <li>0.2 &mu;M Pseudomonas spp. probe (5-NED-CCTTCCTCCCAACTTAAAGTGCTT-3-MGBNFQ)</li>
 </ul>
 </li>
 <li>The third reaction includes:
 <ul>
 <li>0.2 &mu;M Staphylococcus spp. probe (5-NED-AATCTTCCGCAATGGGCGAAAGC-3-MGBNFQ)</li>
 <li>0.2 &mu;M S. aureus probe (5-FAM-AGATGTGCACAGTTACTTACACATAT-3-BHQ1)</li>
 <li>0.2 &mu;M Enterococcus spp. (5-JOE-TCCTTGTTCTTCTCTAACAACAGAG-3-BHQ1)</li>
 </ul>
 </li>
 <li>The fourth reaction includes:
 <ul>
 <li>0.2 &mu;M universal probe (5-FAM-CGTATTACCGCGGCTGCTGGCAC-3-BHQ1)</li>
 <li>0.3 &mu;M Streptococcus spp. probe (5-NED-CCAGAAAGGGACSGCTAACT-3-MGBNFQ)</li>
 <li>0.2 &mu;M S. pneumoniae probe (5-JOE-CCAAAGCCTACTATGGTTAAGCCA-3-BHQ1)</li>
 </ul>
 </li>
 <li>Add sterile demineralised H2O to reach a total volume of 20 &mu;l. Add 20 &mu;l of each reaction mixture to the wells of a 96-well PCR plate.</li>
 <li>Add 5 &mu;l of sample to each well.</li>
 <li>Use an adhesive film to seal the 96-well PCR plate.</li>
 <li>Run the plate on the ABI PRISM 7900HT Real Time PCR System using the following optimal thermal cycling conditions:
 <ul>
 <li>Pre-heating at 50 &deg;C for 10 min</li>
 <li>Initial denaturation at 95 &deg;C for 15 min</li>
 <li>42 cycles of
 <ul type="circle">
 <li>Denaturation at 95 &deg;C for 15 s</li>
 <li>Annealing at 60 &deg;C for 1 min</li>
 </ul>
 </li>
 </ul>
 </li>
</ol>
3. Analysis of the Results
<ol>
 <li> Adjust the threshold of the Ct Analysis to 0.1 in the tab Analysis Settings. Narrow the baseline configurations to Start (Cycle):6 and End (Cycle):15.</li>
 <li>Record the cycle threshold (Ct) value for all samples. The cut-off value to consider a PCR result as positive can be set to a Ct-value of 35. The amount of bacteria present in blood cultures ranged from 107 to 1011 CFU/ml, generating Ct-values below 35.</li>
</ol>
PART II: ANTIBIOTIC SUSCEPTIBILITY TESTING
4. Isolation of Bacteria from Positive Blood Cultures9
<ol>
 <li>Aspirate 5 ml of broth from a positive blood culture bottle and transfer it into a serum separator tube.</li>
 <li>Centrifuge the serum separator tube at 2000 x g for 10 min.</li>
 <li>Discard the supernatant from the serum separator tube.</li>
 <li>Transfer bacteria from the gel layer of the tube with a sterile cotton swab into 0.9% saline until a 0.5 McFarland standard suspension is obtained.</li>
</ol>
5. Inoculation of Micro Titre Plates
<ol>
 <li>Dilute the 0.5 McFarland suspension in double concentrated Mueller Hinton II broth to form a suspension of 5 x 105 CFU/ml.</li>
 <li>Add this suspension to the wells of a micro titre plate containing a selection of antibiotics (Table 1).</li>
 <li>Incubate the micro titre plate at 37 &deg;C for 6 h.</li>
 <li>Store an aliquot of the suspension at 4 &deg;C (as negative growth control).</li>
 <li>After 6 h of incubation, transfer the content of each well into a sterile tube, as well as the negative growth control sample that was stored at 4 &deg;C.</li>
 <li>Centrifuge the tubes at 16000 x g for 5 min.</li>
 <li>Carefully remove the supernatant, without disturbing the bacterial pellet.</li>
 <li>Resuspend the pellet in sterile demineralised H2O.</li>
 <li>Dilute the samples 10-fold in sterile demineralised H2O.</li>
</ol>
6. Real-time 16s rDNA PCR10
<ol>
 <li>Prepare the PCR mixture as follows:
 <ul>
 <li>12.50 &mu;l iQ SYBR Green Supermix</li>
 <li>0.5 &mu;M forward primer 16S-1 (5-TGGAGAGTTTGATCCTGGCTCAG-3)11</li>
 <li>0.25 &mu;M reverse primer 16S-2 (5-TACCGCGGCTGCTGGCAC-3)11</li>
 <li>sterile demineralised H2O to a total volume of 20 &mu;l</li>
 </ul>
 </li>
 <li>Add 20 &mu;l of PCR mixture to the wells of a 96-well PCR plate.</li>
 <li>Add 5 &mu;l of sample to each well.</li>
 <li>Use an adhesive film to seal the 96-well PCR plate.</li>
 <li>Run the plate on the MyiQ Single-Color Real-Time PCR Detection System, using the following optimal thermal cycling conditions:
 <ul>
 <li>Initial denaturation at 95 &deg;C for 4 min</li>
 <li>Initial annealing at 65 &deg;C for 30 s</li>
 <li>35 cycles of
 <ul>
 <li>Denaturation at 95 &deg;C for 15 s</li>
 <li>Annealing at 60 &deg;C for 1 min</li>
 </ul>
 </li>
 <li>Melt curve analysis (from 60-95 &deg;C in 20 min with increments of 0.57 &deg;C)</li>
 </ul>
 </li>
</ol>
7. Analysis of the Results
<ol>
 <li>Calculate the cut-off Ct value using one of the following formulas (depending on type of antibiotic).
 <ul>
 <li>In general: 
 Cut-off Ct value = Ct value positive growth control + 0.5 x (Ct value negative growth control - Ct value positive growth control)</li>
 <li>Piperacillin, piperacillin/tazobactam and ceftazidime in Gram-negative rods; Amoxicillin, oxacillin and trimethoprim/sulfamethoxazole in S.aureus; Amoxicillin in Enterococcus spp.: Cut-off Ct value = Ct value positive growth control + 0.25 x (Ct value negative growth control - Ct value positive growth control)</li>
 </ul>
 </li>
 <li>Use the sample incubated with sterile demineralised H2O as positive growth control.</li>
 <li>Depending on the microorganism, use the appropriate negative growth control:
 <ul>
 <li>Gram-negative rods Sample incubated with mixture of antibiotics</li>
 <li>Enterococcus spp. Sample stored at 4 &deg;C</li>
 <li>S.aureus Sample stored at 4 &deg;C</li>
 </ul>
 </li>
 <li>Determine the susceptibility (S) or resistance (R) of the strain for the tested antibiotic as follows:
 <ul>
 <li>A Ct value higher than the cut-off Ct value indicates susceptibility</li>
 <li>A Ct value lower than the cut-off Ct value indicates resistance</li>
 </ul>
 </li>
</ol>
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).
<img alt="Figure 1" src="/files/ftp_upload/3254/3254fig1.jpg" /> 
 
Figure 1. Flow chart of the pathogen identification and antibiotic susceptibility testing procedure using real-time 16S rDNA PCR.
<img alt="Figure 2" src="/files/ftp_upload/3254/3254fig2.jpg" /> 
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.
<img alt="Figure 3" src="/files/ftp_upload/3254/3254fig3.jpg" /> 
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.
<table border="1">
 <tbody>
 <tr>
 <td>Sample 1: E. coli</td>
 <td>&nbsp;</td>
 <td>&nbsp;</td>
 <td>Sample 2: S. aureus</td>
 <td>&nbsp;</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>AST</td>
 <td>Ct</td>
 <td>R/S</td>
 <td>AST</td>
 <td>Ct</td>
 <td>R/S</td>
 </tr>
 <tr>
 <td>Amoxicillin 8 mg/L</td>
 <td>16,83</td>
 <td>R</td>
 <td>Amoxicillin 0.25 mg/L</td>
 <td>21,03</td>
 <td>R</td>
 </tr>
 <tr>
 <td>Amoxicillin-clavulanate 8/4 mg/L</td>
 <td>17,36</td>
 <td>R</td>
 <td>Oxacillin 2 mg/L</td>
 <td>25,80</td>
 <td>S</td>
 </tr>
 <tr>
 <td>Piperacillin 16 mg/L</td>
 <td>16,67</td>
 <td>R</td>
 <td>Vancomycin 2 mg/L</td>
 <td>25,20</td>
 <td>S</td>
 </tr>
 <tr>
 <td>Piperacillin-tazobactam 16/4 mg/L</td>
 <td>24,15</td>
 <td>S</td>
 <td>Gentamicin 4 mg/L</td>
 <td>25,86</td>
 <td>S</td>
 </tr>
 <tr>
 <td>Ciprofloxacin 1 mg/L</td>
 <td>29,72</td>
 <td>S</td>
 <td>Trimethoprim-sulfamethoxazole 2/38 mg/L</td>
 <td>24,62</td>
 <td>S</td>
 </tr>
 <tr>
 <td>Ceftazidime 1 mg/L</td>
 <td>24,03</td>
 <td>S</td>
 <td>&nbsp;</td>
 <td>&nbsp;</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>Ceftazidime 8 mg/L</td>
 <td>26,58</td>
 <td>S</td>
 <td>&nbsp;</td>
 <td>&nbsp;</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>Gentamicin 4 mg/L</td>
 <td>29,83</td>
 <td>S</td>
 <td>&nbsp;</td>
 <td>&nbsp;</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>Trimethoprim-sulfamethoxazole 2/38 mg/L</td>
 <td>27,60</td>
 <td>S</td>
 <td>&nbsp;</td>
 <td>&nbsp;</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>&nbsp;</td>
 <td>&nbsp;</td>
 <td>&nbsp;</td>
 <td>&nbsp;</td>
 <td>&nbsp;</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>Negative growth control (mixture of antibiotics)</td>
 <td>30,41</td>
 <td>&nbsp;</td>
 <td>Negative growth control (sample stored at 4 &deg;C)</td>
 <td>27,42</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>Positive growth control</td>
 <td>16,90</td>
 <td>&nbsp;</td>
 <td>Positive growth control</td>
 <td>20,22</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>&nbsp;</td>
 <td>&nbsp;</td>
 <td>&nbsp;</td>
 <td>&nbsp;</td>
 <td>&nbsp;</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>Cut-off Ct-value 1*</td>
 <td>21,76</td>
 <td>&nbsp;</td>
 <td>Cut-off Ct-value 1***</td>
 <td>23,82</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>Cut-off Ct-value 2**</td>
 <td>18,75</td>
 <td>&nbsp;</td>
 <td>Cut-off Ct-value 2****</td>
 <td>22,02</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>* For amoxicillin, amoxicillin-clavulanate, ciprofloxacin, gentamicin, trimethoprim-sulfamethoxazole 
 ** For piperacillin, piperacillin-tazobactam, ceftazidime</td>
 <td>&nbsp;</td>
 <td>&nbsp;</td>
 <td>*** For vancomycin and gentamicin 
 **** For amoxicillin, oxacillin and trimethoprim-sulfamethoxazole</td>
 <td>&nbsp;</td>
 <td>&nbsp;</td>
 </tr>
 </tbody>
</table>
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.

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E. l., Descheemaeker, P., De Schouwer, P., Franke, S., Hillen, F., Nollet, F., Soetens, O., Vankeerberghen, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Molecular+Diagnostics+working+group.+Reflections+and+proposals+to+assure+quality+in+molecular+diagnostics.">Molecular Diagnostics working group. Reflections and proposals to assure quality in molecular diagnostics.</a> Acta. Clin. Belg. 66, 33 (2011).</li><li>Peters, R. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=New+developments+in+the+diagnosis+of+bloodstream+infections.">New developments in the diagnosis of bloodstream infections.</a> Lancet Infect. Dis. 4, 751 (2004).</li><li>Hansen, W. L., Beuving, J., Bruggeman, C. A., Wolffs, P. 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We have nothing to disclose.

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.

<table border="1">
<tbody>
 <tr>
 <td>Name of the reagent</td>
 <td>Company</td>
 <td>Catalogue number</td>
 <td>Comments</td>
 </tr>
 <tr>
 <td>Sodium Chloride (NaCl)</td>
 <td>Merck Chemicals</td>
 <td>106404</td>
 <td>0.9% in water</td>
 </tr>
 <tr>
 <td>Vacutainer SST Serum Separator Tube 5 ml</td>
 <td>BD Diagnostic Systems </td>
 <td>367986</td>
 <td></td>
 </tr>
 <tr>
 <td>Mueller Hinton II broth</td>
 <td>BD Diagnostic Dystems</td>
 <td>212322</td>
 <td>44 g/L in water</td>
 </tr>
 <tr>
 <td>Centrifuge Rotixa 50 rs</td>
 <td>Andreas Hettich GmbH &amp; Co. KG</td>
 <td>4910</td>
 <td></td>
 </tr>
 <tr>
 <td>Centrifuge 5415 D</td>
 <td>Eppendorf</td>
 <td>Discontinued</td>
 <td></td>
 </tr>
 <tr>
 <td>Primers</td>
 <td>Sigma-Aldrich</td>
 <td>n.a.</td>
 <td></td>
 </tr>
 <tr>
 <td>Probes</td>
 <td>Sigma-Aldrich/Applied Biosystems</td>
 <td>n.a.</td>
 <td></td>
 </tr>
 <tr>
 <td>TaqManEnvironmental master mix 2.0</td>
 <td>Applied Biosystems</td>
 <td>4396838</td>
 <td></td>
 </tr>
 <tr>
 <td>iQ SYBRGreen Supermix</td>
 <td>Bio-Rad Laboratories BV</td>
 <td>170-8880</td>
 <td></td>
 </tr>
 <tr>
 <td>MicroAmp Optical 96-Well Reaction Plate</td>
 <td>Applied Biosystems</td>
 <td>N8010560</td>
 <td></td>
 </tr>
 <tr>
 <td>MicroAmp Optical Adhesive Film</td>
 <td>Applied Biosystems</td>
 <td>4311971</td>
 <td></td>
 </tr>
 <tr>
 <td>iQ 96-Well PCR Plates</td>
 <td>Bio-Rad Laboratories BV</td>
 <td>223-9441</td>
 <td></td>
 </tr>
 <tr>
 <td>Microseal B Adhesive Seals</td>
 <td>Bio-Rad Laboratories BV</td>
 <td>MSB-1001</td>
 <td></td>
 </tr>
 <tr>
 <td>Real-time PCR Detection System</td>
 <td>Applied Biosystems</td>
 <td>ABI PRISM 7900HT</td>
 <td></td>
 </tr>
 <tr>
 <td>Real-Time PCR Detection System</td>
 <td>Bio-Rad Laboratories BV</td>
 <td>MyiQ Single-Color</td>
 <td></td>
 </tr>
</tbody>
</table>

one-day workflow scheme for bacterial pathogen detection and antimicrobial resistance testing from blood cultures

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.

Watch this Scientific Journal Video about One-day Workflow Scheme for Bacterial Pathogen Detection and Antimicrobial Resistance Testing from Blood Cultures at JoVE.com

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

The design of a straightforward one-day workflow scheme for bacterial pathogen diagnostics enables the rapid recognition of bloodstream infections. The inclusion of eight clinically relevant bacterial targets and their antibiotic resistance profiles offers the clinician an initial insight on the same day, which can lead to more adequate therapy.

Bloodstream infections are associated with high mortality rates because of the probable manifestation of sepsis, severe sepsis and septic shock1. ...

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Research

JoVE Journal

Immunology and Infection

25.1K Views.  Maastricht University Medical Center. The design of a straightforward one-day workflow scheme for bacterial pathogen diagnostics enables the rapid recognition of bloodstream infections. The inclusion of eight clinically relevant bacterial targets and their antibiotic resistance profiles offers the clinician an initial insight on the same day, which can lead to more adequate therapy.

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

Protocol for Recombinant RBD-based SARS Vaccines: Protein Preparation, Animal Vaccination and Neutralization Detection

Based on their safety profile and ability to induce potent immune responses against infections, subunit vaccines have been used as candidates for a wide variety of pathogens 1-3. Since the mammalian cell system is capable of post-translational modification, thus forming properly folded and glycosylated proteins, recombinant proteins expressed in mammalian cells have shown the greatest potential to maintain high antigenicity and immunogenicity 4-6.
Although no new cases of SARS have been reported since 2004, future outbreaks are a constant threat; therefore, the development of vaccines against SARS-CoV is a prudent preventive step and should be carried out. The RBD of SARS-CoV S protein plays important roles in receptor binding and induction of specific neutralizing antibodies against virus infection 7-9. Therefore, in this protocol, we describe novel methods for developing a RBD-based subunit vaccine against SARS. Briefly, the recombinant RBD protein (rRBD) was expressed in culture supernatant of mammalian 293T cells to obtain a correctly folded protein with proper conformation and high immunogenicity 6. The transfection of the recombinant plasmid encoding RBD to the cells was then performed using a calcium phosphate transfection method 6,10 with some modifications. Compared with the lipid transfection method 11,12, this modified calcium phosphate transfection method is cheaper, easier to handle, and has the potential to reach high efficacy once a transfection complex with suitable size and shape is formed 13,14. Finally, a SARS pseudovirus neutralization assay was introduced in the protocol and used to detect the neutralizing activity of sera of mice vaccinated with rRBD protein. This assay is relatively safe, does not involve an infectious SARS-CoV, and can be performed without the requirement of a biosafety-3 laboratory 15.
The protocol described here can also be used to design and study recombinant subunit vaccines against other viruses with class I fusion proteins, for example, HIV, respiratory syncytial virus (RSV), Ebola virus, influenza virus, as well as Nipah and Handra viruses. In addition, the methods for generating a pseudovirus and subsequently establishing a pseudovirus neutralization assay can be applied to all these viruses.

This protocol describes a general procedure for studying recombinant receptor-binding domain (RBD)-based subunit vaccines against SARS. It includes methods for transfection and expression of RBD protein in 293T cells, immunization of mice with RBD and detection of neutralization activity of mouse sera using an established SARS pseudovirus neutralization assay.

Based on their safety profile and ability to induce potent immune responses against infections, subunit vaccines have been used as candidates for a wide ...

Invasion of Human Cells by a Bacterial Pathogen

Here we will describe how we study the invasion of human endothelial cells by bacterial pathogen Staphylococcus aureus . The general protocol can be applied to the study of cell invasion by virtually any culturable bacterium. The stages at which specific aspects of invasion can be studied, such as the role of actin rearrangement or caveolae, will be highlighted. Host cells are grown in flasks and when ready for use are seeded into 24-well plates containing Thermanox coverslips. Using coverslips allows subsequent removal of the cells from the wells to reduce interference from serum proteins deposited onto the sides of the wells (to which S. aureus would attach). Bacteria are grown to the required density and washed to remove any secreted proteins (e.g. toxins). Coverslips with confluent layers of endothelial cells are transferred to new 24-well plates containing fresh culture medium before the addition of bacteria. Bacteria and cells are then incubated together for the required amount of time in 5% CO2 at 37&deg;C. For S. aureus this is typically between 15-90 minutes. Thermanox coverslips are removed from each well and dip-washed in PBS to remove unattached bacteria. If total associated bacteria (adherent and internalised) are to be quantified, coverslips are then placed in a fresh well containing 0.5% Triton X-100 in PBS. Gentle pipetting leads to complete cell lysis and bacteria are enumerated by serial dilution and plating onto agar. If the number of bacteria that have invaded the cells is needed, coverslips are added to wells containing 500 &mu;l tissue culture medium supplemented with gentamicin and incubation continued for 1 h, which will kill all external bacteria. Coverslips can then be washed, cells lysed and bacteria enumerated by plating onto agar as described above. If the experiment requires direct visualisation, coverslips can be fixed and stained for light, fluorescence or confocal microscopy or prepared for electron microscopy.

A general protocol for the study of invasion of host cells by a bacterial pathogen, focusing on Staphylococcus aureus and human endothelial cells.

Here we will describe how we study the invasion of human endothelial cells by bacterial pathogen Staphylococcus aureus . The general protocol can be ...

Introducing Shear Stress in the Study of Bacterial Adhesion

During bacterial infections a sequence of interactions occur between the pathogen and its host. Bacterial adhesion to the host cell surface is often the initial and determining step of the pathogenesis. Although experimentally adhesion is mostly studied in static conditions adhesion actually takes place in the presence of flowing liquid. First encounters between bacteria and their host often occur at the mucosal level, mouth, lung, gut, eye, etc. where mucus flows along the surface of epithelial cells. Later in infection, pathogens occasionally access the blood circulation causing life-threatening illnesses such as septicemia, sepsis and meningitis. A defining feature of these infections is the ability of these pathogens to interact with endothelial cells in presence of circulating blood. The presence of flowing liquid, mucus or blood for instance, determines adhesion because it generates a mechanical force on the pathogen. To characterize the effect of flowing liquid one usually refers to the notion of shear stress, which is the tangential force exerted per unit area by a fluid moving near a stationary wall, expressed in dynes/cm2. Intensities of shear stress vary widely according to the different vessels type, size, organ, location etc. (0-100 dynes/cm2). Circulation in capillaries can reach very low shear stress values and even temporarily stop during periods ranging between a few seconds to several minutes 1. On the other end of the spectrum shear stress in arterioles can reach 100 dynes/cm2 2. The impact of shear stress on different biological processes has been clearly demonstrated as for instance during the interaction of leukocytes with the endothelium 3. To take into account this mechanical parameter in the process of bacterial adhesion we took advantage of an experimental procedure based on the use of a disposable flow chamber 4. Host cells are grown in the flow chamber and fluorescent bacteria are introduced in the flow controlled by a syringe pump. We initially focused our investigations on the bacterial pathogen Neisseria meningitidis, a Gram-negative bacterium responsible for septicemia and meningitis. The procedure described here allowed us to study the impact of shear stress on the ability of the bacteria to: adhere to cells 1, to proliferate on the cell surface 5and to detach to colonize new sites 6 (Figure 1). Complementary technical information can be found in reference 7. Shear stress values presented here were chosen based on our previous experience1 and to represent values found in the literature. The protocol should be applicable to a wide range of pathogens with specific adjustments depending on the objectives of the study.

During the infection process, a key step is the adhesion of pathogens with host cells. In most instances this adhesion step occurs in the presence of mechanical stress generated by flowing liquid. We describe a technique that introduces shear stress as an important parameter in the study of bacterial adhesion.

During bacterial infections a sequence of interactions occur between the pathogen and its host. Bacterial adhesion to the host cell surface is often the ...

High-throughput Detection Method for Influenza Virus

Influenza virus is a respiratory pathogen that causes a high degree of morbidity and mortality every year in multiple parts of the world. Therefore, precise diagnosis of the infecting strain and rapid high-throughput screening of vast numbers of clinical samples is paramount to control the spread of pandemic infections. Current clinical diagnoses of influenza infections are based on serologic testing, polymerase chain reaction, direct specimen immunofluorescence and cell culture 1,2.
Here, we report the development of a novel diagnostic technique used to detect live influenza viruses. We used the mouse-adapted human A/PR/8/34 (PR8, H1N1) virus 3 to test the efficacy of this technique using MDCK cells 4. MDCK cells (104 or 5 x 103 per well) were cultured in 96- or 384-well plates, infected with PR8 and viral proteins were detected using anti-M2 followed by an IR dye-conjugated secondary antibody. M2 5 and hemagglutinin 1 are two major marker proteins used in many different diagnostic assays. Employing IR-dye-conjugated secondary antibodies minimized the autofluorescence associated with other fluorescent dyes. The use of anti-M2 antibody allowed us to use the antigen-specific fluorescence intensity as a direct metric of viral quantity. To enumerate the fluorescence intensity, we used the LI-COR Odyssey-based IR scanner. This system uses two channel laser-based IR detections to identify fluorophores and differentiate them from background noise. The first channel excites at 680 nm and emits at 700 nm to help quantify the background. The second channel detects fluorophores that excite at 780 nm and emit at 800 nm. Scanning of PR8-infected MDCK cells in the IR scanner indicated a viral titer-dependent bright fluorescence. A positive correlation of fluorescence intensity to virus titer starting from 102-105 PFU could be consistently observed. Minimal but detectable positivity consistently seen with 102-103 PFU PR8 viral titers demonstrated the high sensitivity of the near-IR dyes. The signal-to-noise ratio was determined by comparing the mock-infected or isotype antibody-treated MDCK cells.
Using the fluorescence intensities from 96- or 384-well plate formats, we constructed standard titration curves. In these calculations, the first variable is the viral titer while the second variable is the fluorescence intensity. Therefore, we used the exponential distribution to generate a curve-fit to determine the polynomial relationship between the viral titers and fluorescence intensities. Collectively, we conclude that IR dye-based protein detection system can help diagnose infecting viral strains and precisely enumerate the titer of the infecting pathogens.

This method describes the use of Infrared dye based imaging system for detection of H1N1 in bronchioalveolar lavage (BAL) fluid of infected mice at a high sensitivity. This methodology can be performed in a 96- or 384-well plate, requires &lt;10 &mu;l volume of test material and has the potential for concurrent screening of multiple pathogens.

Influenza virus is a respiratory pathogen that causes a high degree of morbidity and mortality every year in multiple parts of the world. Therefore, ...

Saliva, Salivary Gland, and Hemolymph Collection from Ixodes scapularis Ticks

Ticks are found worldwide and afflict humans with many tick-borne illnesses. Ticks are vectors for pathogens that cause Lyme disease and tick-borne relapsing fever (Borrelia spp.), Rocky Mountain Spotted fever (Rickettsia rickettsii), ehrlichiosis (Ehrlichia chaffeensis and E. equi), anaplasmosis (Anaplasma phagocytophilum), encephalitis (tick-borne encephalitis virus), babesiosis (Babesia spp.), Colorado tick fever (Coltivirus), and tularemia (Francisella tularensis) 1-8. To be properly transmitted into the host these infectious agents differentially regulate gene expression, interact with tick proteins, and migrate through the tick 3,9-13. For example, the Lyme disease agent, Borrelia burgdorferi, adapts through differential gene expression to the feast and famine stages of the tick's enzootic cycle 14,15. Furthermore, as an Ixodes tick consumes a bloodmeal Borrelia replicate and migrate from the midgut into the hemocoel, where they travel to the salivary glands and are transmitted into the host with the expelled saliva 9,16-19.
As a tick feeds the host typically responds with a strong hemostatic and innate immune response 11,13,20-22. Despite these host responses, I. scapularis can feed for several days because tick saliva contains proteins that are immunomodulatory, lytic agents, anticoagulants, and fibrinolysins to aid the tick feeding 3,11,20,21,23. The immunomodulatory activities possessed by tick saliva or salivary gland extract (SGE) facilitate transmission, proliferation, and dissemination of numerous tick-borne pathogens 3,20,24-27. To further understand how tick-borne infectious agents cause disease it is essential to dissect actively feeding ticks and collect tick saliva. This video protocol demonstrates dissection techniques for the collection of hemolymph and the removal of salivary glands from actively feeding I. scapularis nymphs after 48 and 72 hours post mouse placement. We also demonstrate saliva collection from an adult female I. scapularis tick.

Saliva, Salivary Gland, and Hemolymph Collection from Ixodes scapularis Ticks

The collection of infected tick hemolymph, salivary glands, and saliva is important to study how tick-borne pathogens cause disease. In this protocol we demonstrate how to collect hemolymph and salivary glands from feeding Ixodes scapularis nymphs. We also demonstrate saliva collection from female I. scapularis adults.

Ticks are found worldwide and afflict humans with many tick-borne illnesses. Ticks are vectors for pathogens that cause Lyme disease and tick-borne ...

Nanomechanics of Drug-target Interactions and Antibacterial Resistance Detection

The cantilever sensor, which acts as a transducer of reactions between model bacterial cell wall matrix immobilized on its surface and antibiotic drugs in solution, has shown considerable potential in biochemical sensing applications with unprecedented sensitivity and specificity1-5. The drug-target interactions generate surface stress, causing the cantilever to bend, and the signal can be analyzed optically when it is illuminated by a laser. The change in surface stress measured with nano-scale precision allows disruptions of the biomechanics of model bacterial cell wall targets to be tracked in real time. Despite offering considerable advantages, multiple cantilever sensor arrays have never been applied in quantifying drug-target binding interactions.
Here, we report on the use of silicon multiple cantilever arrays coated with alkanethiol self-assembled monolayers mimicking bacterial cell wall matrix to quantitatively study antibiotic binding interactions. To understand the impact of vancomycin on the mechanics of bacterial cell wall structures1,6,7.&#160;We developed a new model1 which proposes that cantilever bending can be described by two independent factors; i) namely a chemical factor, which is given by a classical Langmuir adsorption isotherm, from which we calculate the thermodynamic equilibrium dissociation constant (Kd) and ii) a geometrical factor, essentially a measure of how bacterial peptide receptors are distributed on the cantilever surface. The surface distribution of peptide receptors (p) is used to investigate the dependence of geometry and ligand loading. It is shown that a threshold value of p ~10% is critical to sensing applications. Below which there is no detectable bending signal while above this value, the bending signal increases almost linearly,&#160;revealing that stress is a product of a local chemical binding factor and a geometrical factor combined by the mechanical connectivity of reacted regions and provides a new paradigm for design of powerful agents to combat superbug infections.

Acquired resistance to antibiotics is a major public healthcare problem and is presently ranked by the WHO as one of the greatest threats to human life. Here we describe the use of cantilever technology to quantify antibacterial resistance, critical to the discovery of novel and powerful agents against multidrug resistant bacteria.

The cantilever sensor, which acts as a transducer of reactions between model bacterial cell wall matrix immobilized on its surface and antibiotic drugs in ...

Multiplex PCR Assay for Typing of Staphylococcal Cassette Chromosome Mec Types I to V in Methicillin-resistant Staphylococcus aureus

Staphylococcal Cassette Chromosome mec (SCCmec) typing is a very important molecular tool for understanding the epidemiology and clonal strain relatedness of methicillin-resistant Staphylococcus aureus (MRSA), particularly with the emerging outbreaks of community-associated MRSA (CA-MRSA) occurring on a worldwide basis. Traditional PCR typing schemes classify SCCmec by targeting and identifying the individual mec and ccr gene complex types, but require the use of many primer sets and multiple individual PCR experiments. We designed and published a simple multiplex PCR assay for quick-screening of major SCCmec types and subtypes I to V, and later updated it as new sequence information became available. This simple assay targets individual SCCmec types in a single reaction, is easy to interpret and has been extensively used worldwide. However, due to the sophisticated nature of the assay and the large number of primers present in the reaction, there is the potential for difficulties while adapting this assay to individual laboratories. To facilitate the process of establishing a MRSA SCCmec assay, here we demonstrate how to set up our multiplex PCR assay, and discuss some of the vital steps and procedural nuances that make it successful.

Multiplex PCR Assay for Typing of Staphylococcal Cassette Chromosome Mec Types I to V in Methicillin-resistant Staphylococcus aureus

We demonstrate a simple multiplex PCR assay for quick-screening and typing of Staphylococcal Cassette Chromosome mec (SCCmec) types I-V for methicillin-resistant Staphylococcus aureus, and provide some of the vital steps and procedural nuances that make it successful for adapting this assay to individual laboratories.

Staphylococcal Cassette Chromosome mec (SCCmec) typing  is a very important molecular tool for understanding the epidemiology and  clonal strain ...

Preparation of a Blood Culture Pellet for Rapid Bacterial Identification and Antibiotic Susceptibility Testing

Bloodstream infections and sepsis are a major cause of morbidity and mortality. The successful outcome of patients suffering from bacteremia depends on a rapid identification of the infectious agent to guide optimal antibiotic treatment. The analysis of Gram stains from positive blood culture can be rapidly conducted and already significantly impact the antibiotic regimen. However, the accurate identification of the infectious agent is still required to establish the optimal targeted treatment. We present here a simple and fast bacterial pellet preparation from a positive blood culture that can be used as a sample for several essential downstream applications such as identification by MALDI-TOF MS, antibiotic susceptibility testing (AST) by disc diffusion assay or automated AST systems and by automated PCR-based diagnostic testing. The performance of these different identification and AST systems applied directly on the blood culture bacterial pellets is very similar to the performance normally obtained from isolated colonies grown on agar plates. Compared to conventional approaches, the rapid acquisition of a bacterial pellet significantly reduces the time to report both identification and AST. Thus, following blood culture positivity, identification by MALDI-TOF can be reported within less than 1 hr whereas results of AST by automated AST systems or disc diffusion assays within 8 to 18 hr, respectively. Similarly, the results of a rapid PCR-based assay can be communicated to the clinicians less than 2 hr following the report of a bacteremia. Together, these results demonstrate that the rapid preparation of a blood culture bacterial pellet has a significant impact on the identification and AST turnaround time and thus on the successful outcome of patients suffering from bloodstream infections.

A rapid bacterial pellet preparation from a positive blood culture can be used as a sample for applications such as identification by MALDI-TOF, Gram staining, antibiotic susceptibility testing and PCR-based test. The results can be rapidly communicated to clinicians to improve the outcome of patients suffering from bloodstream infections.

Bloodstream infections and sepsis are a major cause of morbidity and mortality. The successful outcome of patients suffering from bacteremia depends on a ...

Loop-mediated Isothermal Amplification (LAMP) Assays for the Species-specific Detection of Eimeria that Infect Chickens

Eimeria species parasites, protozoa which cause the enteric disease coccidiosis, pose a serious threat to the production and welfare of chickens. In the absence of effective control clinical coccidiosis can be devastating. Resistance to the chemoprophylactics frequently used to control Eimeria is common and sub-clinical infection is widespread, influencing feed conversion ratios and susceptibility to other pathogens such as Clostridium perfringens. Despite the availability of polymerase chain reaction (PCR)-based tools, diagnosis of Eimeria infection still relies almost entirely on traditional approaches such as lesion scoring and oocyst morphology, but neither is straightforward. Limitations of the existing molecular tools include the requirement for specialist equipment and difficulties accessing DNA as template. In response a simple field DNA preparation protocol and a panel of species-specific loop-mediated isothermal amplification (LAMP) assays have been developed for the seven Eimeria recognised to infect the chicken. We now provide a detailed protocol describing the preparation of genomic DNA from intestinal tissue collected post-mortem, followed by setup and readout of the LAMP assays. Eimeria species-specific LAMP can be used to monitor parasite occurrence, assessing the efficacy of a farm&#8217;s anticoccidial strategy, and to diagnose sub-clinical infection or clinical disease with particular value when expert surveillance is unavailable.

Loop-mediated Isothermal Amplification LAMP Assays for the Species-specific Detection of Eimeria that Infect Chickens

Diagnosis of Eimeria infection in chickens remains demanding. Parasite morphology- and host pathology-led approaches are commonly inconclusive, while molecular approaches based on PCR have proven demanding in cost and expertise. The aim of this protocol is to establish loop-mediated isothermal amplification (LAMP) as a straightforward molecular diagnostic for eimerian infection.

Eimeria species parasites, protozoa which cause the enteric disease coccidiosis, pose a serious threat to the production and welfare of chickens. In the ...

Using a Bacterial Pathogen to Probe for Cellular and Organismic-level Host Responses

There are a variety of strategies bacterial pathogens employ to survive and proliferate once inside the eukaryotic cell. The so-called 'cytosolic' pathogens (Listeria monocytogenes, Shigella flexneri, Burkholderia pseudomallei, Francisella tularensis, and Rickettsia spp.) gain access to the infected cell cytosol by physically and enzymatically degrading the primary vacuolar membrane. Once in the cytosol, these pathogens both proliferate as well as generate sufficient mechanical forces to penetrate the plasma membrane of the host cell in order to infect new cells. Here, we show how this terminal step of the cellular infection cycle of L. monocytogenes (Lm) can be quantified by both colony-forming unit assays and flow cytometry and give examples of how both pathogen- and host-encoded factors impact this process. We also show a close correspondence of Lm infection dynamics of cultured cells infected in vitro and those of hepatic cells derived from mice infected in vivo. These function-based assays are relatively simple and can be readily scaled up for discovery-based high-throughput screens for modulators of eukaryotic cell function.

We describe both in vitro and in vivo infection assays that can be used to analyze the activities of host-encoding factors.