Studies were sponsored by AdvanDx.

1. Specimen Collection and Preparation
<ol>
<li>Collect venous blood from patients suspected of sepsis and put into two BACTEC (BD, Sparks, MD) blood culture bottles, one aerobic and one anaerobic, together comprising one blood culture set.</li>
<li>Place bottles into the BACTEC 9240 instrument at 35&deg;C.</li>
<li>Incubate until the instrument is triggered into alarm due to growth of organisms in the blood culture bottle.</li>
<li>Remove bottle from the blood culture instrument.</li>
</ol>
2. Preparation of Reagents Prior to Staining
<ol>
<li>Prepare the wash solution by adding 4 mL of 60x Wash Solution followed by 240 mL of fresh deionized or distilled water directly to the Staining Dish.</li>
<li>Put the staining dish in the 55&deg;C water bath to warm.</li>
<li>Store the remaining concentrate at 2-8&deg;C. </li>
<li>Remove the Mounting Medium from the refrigerator and warm to room temperature.</li>
</ol>
3. Gram Staining
<ol>
<li>Gently swirl the blood culture bottle.</li>
<li>Using a needle and syringe or a subculturing apparatus, remove blood (approximately 5 mL) from the bottle and place in a sterile screw capped tube.</li>
<li>Using a sterile pipette, place one large drop of blood onto a glass microscope slide.</li>
<li>Air dry the slide and fix in methanol for 10 seconds.</li>
<li>Allow slide to air dry.</li>
<li>Perform a Gram stain on the blood film to determine the morphology of the organism growing in the blood culture bottle.</li>
<li>Using an oil immersion lens, view slide. Gram positive cocci in pairs and short chains observed on the Gram stain is most consistent with enterococcus species.</li>
<li>This Gram stain result then drives the selection of the proper PNA FISH probe kit to use for bacterial identification. This blood culture will be stained using the E. faecalis/OE PNA FISH stain.</li>
</ol>
4. PNA FISH Stain
<ol>
<li>Mix the tube containing the blood gently by swirling prior to smear preparation.</li>
<li>Place one drop of Fixation Solution on the well of a PNA FISH Microscope Slide.</li>
<li>Transfer 10 &mu;L or one small drop from the tube with the positive blood culture to the Fixation Solution and mix gently to emulsify.</li>
<li>Fix the smears by heating them for 20 min. at 55&deg;C on a heating plate.</li>
</ol>
5. Quality Control Material
One positive and one negative quality control slide must be tested with each batch of slides for staining.
<ol>
<li>Use E. faecalis/OE Control Slides purchased from AdvanDx for this purpose or prepare smears from liquid cultures of reference strains of E. faecalis and E. faecium as Positive Control either on separate slides or mixed on one slide and Staphylococcus spp or Streptococcus spp as Negative Control material.</li>
</ol>
The QC results should be able to monitor for appropriate testing conditions, particularly those affecting hybridization stringency and cell wall penetration, since PNA methodology is designed to optimize cell wall penetration
6. Hybridization
<ol>
<li>Add one drop of E. faecalis/OE PNA to the well on the microscope slide with the smear.</li>
<li>Add coverslip. Avoid air bubbles.</li>
<li>Incubate for 30 &plusmn; 5 min. at 55 &plusmn; 1&deg;C on heating plate</li>
<li>Following incubation place the slides in slide carrier</li>
</ol>
7. Stringent Wash
<ol>
<li>Immerse slide carrier in the preheated Wash Solution at 55&deg;C and carefully remove the coverslip. Often, the coverslip slides off by gently agitating the slide in the Wash Solution. Occasionally, the coverslip must be gently pushed off with forceps.</li>
<li>Incubate in the wash solution for 30 &plusmn; 5 min. at 55 &plusmn; 1&deg;C.</li>
<li>Remove the slides from the wash solution</li>
<li>Allow the slide to air dry</li>
</ol>
8. Mounting
<ol>
<li>Add one drop of Mounting Medium to each slide</li>
<li>Add coverslip. Avoid air bubbles.</li>
<li>Examine the slide on a fluorescence microscope within 2 hours.</li>
<li>Do not expose the slides to direct sun light or other strong light sources as this may lead to fluorescence quenching.</li>
</ol>
9. Interpretation of Results
The fluorescence microscope used for slide examination must be equipped with the AdvanDx Dual Band Filter and a 60x or 100x oil objective. The QC slides should be examined first to confirm that hybridization did occur. E. faecalis should appear as bright green fluorescent cocci in multiple fields of view and the E. faecium will appear as bright red cocci. Non-enterococci control slide should appear nonfluorescent. After confirming the system in controls, the patient slides can be examined. At first the blood film will appear reddish but the bright red and green cocci will be quite apparent. 
<img src="/files/ftp_upload/2616/2616fig1.jpg" alt="Figure 1" /> 
Figure 1.Representative examples of green-positive E. faecalis (left), red positive E. faecium (middle), and negative (right) test results.
10. Representative Results
Three institutions were included in a multi-center clinical trial evaluating this PNA FISH stain and comparing a 2.5 hour protocol to the shortened 1.5 hour protocol that has just been reviewed. A total of 152 routine Gram positive cocci in pairs and chains (GPC) positive blood culture bottles were included in the studies. There was 100% (152/152) agreement between results of the modified and the original assay procedure for E. faecalis/OE PNA FISH: 41/41 E. faecalis; 33/33 other enterococci and 78/78 other GPC (Table 1). This staining method had exquisite sensitivity and specificity during this clinical trial (Table 2).
<table border="1">
<tbody>
 <tr>
 <td>&nbsp;</td>
 <td>Routine Methods</td>
 <td>E. faecalis /OE PNA FISH Standard Procedure</td>
 <td>E. faecalis/OE PNA FISH Short Procedure</td>
 </tr>
 <tr>
 <td>E. faecalis</td>
 <td>41</td>
 <td>41 (Green Positive)</td>
 <td>41 (Green Positive)</td>
 </tr>
 <tr>
 <td>E. faecium</td>
 <td>27</td>
 <td>27 (Red Positive)</td>
 <td>27 (Red Positive)</td>
 </tr>
 <tr>
 <td>E. casseliflavus</td>
 <td>2</td>
 <td>2 (Red Positive)</td>
 <td>2 (Red Positive)</td>
 </tr>
 <tr>
 <td>E. gallinarum</td>
 <td>2</td>
 <td>2 (Red Positive)</td>
 <td>2 (Red Positive)</td>
 </tr>
 <tr>
 <td>Other Enterococcus spp.</td>
 <td>2</td>
 <td>2 (Red Positive)</td>
 <td>2 (Red Positive)</td>
 </tr>
 <tr>
 <td>S. pneumoniae</td>
 <td>17</td>
 <td>17 (Negative)</td>
 <td>17 (Negative)</td>
 </tr>
 <tr>
 <td>S. viridans</td>
 <td>33</td>
 <td>33 (Negative)</td>
 <td>33 (Negative)</td>
 </tr>
 <tr>
 <td>S. mitis</td>
 <td>1</td>
 <td>1 (Negative)</td>
 <td>1 (Negative)</td>
 </tr>
 <tr>
 <td>S. pyogenes</td>
 <td>3</td>
 <td>3 (Negative)</td>
 <td>3 (Negative)</td>
 </tr>
 <tr>
 <td>S. bovis</td>
 <td>2</td>
 <td>2 (Negative)</td>
 <td>2 (Negative)</td>
 </tr>
 <tr>
 <td>S. salivarius</td>
 <td>1</td>
 <td>1 (Negative)</td>
 <td>1 (Negative)</td>
 </tr>
 <tr>
 <td>S. sanguinis</td>
 <td>2</td>
 <td>2 (Negative)</td>
 <td>2 (Negative)</td>
 </tr>
 <tr>
 <td>S. agalagtae</td>
 <td>7</td>
 <td>7 (Negative)</td>
 <td>7 (Negative)</td>
 </tr>
 <tr>
 <td>Other Streptococcus spp.</td>
 <td>7</td>
 <td>7 (Negative)</td>
 <td>7 (Negative)</td>
 </tr>
 <tr>
 <td>Abiotrophia spp.</td>
 <td>3</td>
 <td>3 (Negative)</td>
 <td>3 (Negative)</td>
 </tr>
 <tr>
 <td>Peptostrepococcus spp.</td>
 <td>2</td>
 <td>2 (Negative)</td>
 <td>2 (Negative)</td>
 </tr>
 <tr>
 <td>Total</td>
 <td>152</td>
 <td>152/152 
 100% Agreement</td>
 <td>152/152 
 100% Agreement</td>
 </tr>
</tbody>
</table>
Table 1. Listing of bacteria tested using both the standard (2.5hr) and rapid (1.5 hr) protocol.
<table border="1">
<tbody>
 <tr>
 <td>Sensitivity E.faecalis</td>
 <td>Sensitivity Other Enterococcus spp.</td>
 <td>Specificity</td>
 </tr>
 <tr>
 <td>100% (41/41) 
 95% CI (93.0-100)</td>
 <td>100% (33/33) 33/33 
 95% CI (91.3-100)</td>
 <td>100% (78/78) 
 95% CI (96.2-100)</td>
 </tr>
</tbody>
</table>
Table 2. Sensitivity and Specificity of the 1.5 hour PNA FISH protocol.

<ol>
	<li>Oliveira, K., Chapin, K. C., Musgnug, M. C., Haase, G., Weber-Heynemann, J., DeGirolami, P. C., Dakos, J., Stender, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Rapid+identification+and+differentiation+of+Enterococcus+faecalis+and+other+Enterococcus+species+directly+from+positive+blood+cultures+by+dual+color+fluorescence+in+situ+hybridization+using+PNA+probes.">Rapid identification and differentiation of Enterococcus faecalis and other Enterococcus species directly from positive blood cultures by dual color fluorescence in situ hybridization using PNA probes.</a> , (2002).</li><li>Chapin, K. C., Musgnug, M. C., Oliveira, K., DeGirolami, P. C., Dakos, J., Johansen, J. T., Procop, G. W., Wilson, D., Padilla, E., Gonzalez, V., Stender, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Multicenter+evaluation+of+E.+faecalis+PNA+FISH+for+rapid+identification+and+differentiation+of+Enterococcus+faecalis+and+other+Enterococcus+species+directly+from+positive+blood+cultures.">Multicenter evaluation of E. faecalis PNA FISH for rapid identification and differentiation of Enterococcus faecalis and other Enterococcus species directly from positive blood cultures.</a> , (2003).</li><li>Rausei, V., Youssef, E., Morgan, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Direct+identification+of+Staphylococcus+aureus,+Enterococcus+faecalis,+and+Candida+albicans+from+blood+cultures+using+fluorescence+in+situ+hybridization+with+nucleic+acid+probes.">Direct identification of Staphylococcus aureus, Enterococcus faecalis, and Candida albicans from blood cultures using fluorescence in situ hybridization with nucleic acid probes.</a> , (2005).</li><li>Novak-Weekley, S. M., LaForga, M., Carey, L., Frazier, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Validation+of+S.+aureus+PNA+FISH,+E.+faecalis+PNA+FISH+&#38;+C.+albicans+PNA+FISH+for+Rapid+Identification+of+Positive+Blood+Culture+Bottles.">Validation of S. aureus PNA FISH, E. faecalis PNA FISH &#38; C. albicans PNA FISH for Rapid Identification of Positive Blood Culture Bottles.</a> , (2006).</li><li>Johnson, J. K., Roberts, A. A., Forrest, G. N., Lincalis, D. P., Venezia, R. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Rapid+Identification+of+Enterococcus+Species+in+Positive+Blood+Cultures+with+Therapeutic+Implications.">Rapid Identification of Enterococcus Species in Positive Blood Cultures with Therapeutic Implications.</a> , (2006).</li></ol>

The production of this video-article was sponsored by AdvanDx. Benjamin Crystal is an employee of AdvanDx, who makes the tools and reagents used in this article.

The E. faecalis/OE PNA FISH assay for enterococcus can provide identification 2-3 days earlier than standard culture methods. The shortened procedure for the assay (1.5 hourr) provides rapid identification that can be used for a better approach to antimicrobial therapy and patient outcome. One study to support this was performed by Forrest, et al. (6). Over two consecutive years beginning in 2005 the microbiology laboratory identified Gram positive cocci in pairs and chains growing in blood culture bottles by conventional microbiological methods and in 2006 adding the E. faecalis/OE PNA FISH. In addition a treatment algorithm developed by the institutions antimicrobial team (AMT) to effectively use the PNA FISH data generated by the laboratory in timely manner. Primary outcome assessed was time from blood culture draw to the implementation of effective antimicrobial therapy before and after PNA FISH was instituted into the laboratories workflow. Severity of illness, patient location and empiric antimicrobial therapy were measured. A total of 224 patients with hospital acquired enterococcal bacteremia were evaluated with 129 in the pre-intervention period and 95 in the PNA FISH period. PNA FISH identified E. faecalis 3 days earlier than conventional cultures (1.1 vs. 4.1 days, p&lt;0.001). PNA FISH identified E. faecium a median 2.3 days earlier (1.1 vs. 3.4 days, p&lt;0.001) and was associated with statistically significant reductions in time to initiating effective therapy (1.3 vs. 3.1 days, p&lt;0.001) and decreased 30 day mortality (26% vs. 45%, p=0.04). This group concluded that the E. faecalis/OE PNA FISH assay in conjunction with an AMT treatment algorithm resulted in earlier initiation of appropriate empiric antimicrobial therapy for patients with hospital acquired E. faecium bacteremia.
<ol>
<li>Reagents must not be used after the expiration dates printed on the labels and reagents should not be modified in any way. They should remain in the refrigerator when not in use or they could lose potency.</li>
<li>Blood culture bottles need to be mixed prior to sampling.</li>
<li>It is important to not allow the dropper bottle tip to touch a blood smear because this may cause cross contamination of material between slides, or cause contamination of the reagent.</li>
<li>Make sure the water bath and the heating block are maintained at a constant 55&deg;C.</li>
<li>Do not use filters other than the Dual Band Filter or colors will not be clearly discerned.</li>
<li>Do not use microscope slides other than the Microscope Slides or hybridization will not occur.</li>
<li>It is important that the microscope is functioning properly. Make sure the microscope bulb is within the approved number of hours for usage.</li>
</ol>
<ul>
<li>Some rare-occurring Enterococcus species are not detected by the PNA probe hybridizing to other Enterococcus species, such as E. asinii, E.dispar, E. haemoperoxidus, E. cecorum, E. columbae, E. saccharolyticus, E. solitarius and E. sulfurous.</li>
<li>E. moraviensis is identified as E. faecalis due to sequence similarities.</li>
<li>Some strains of Streptococcus anginosus produce a false green positive fluorescence due to sequence similarities.</li>
<li>VersaTREK blood culture bottles were not evaluated during the clinical studies. The performance to detect E. faecalis and other Enterococcus spp. with VersaTREK blood culture bottles is unknown.</li>
<li>False positive green autofluorescence may occur if a standard FITC filter is used instead of the Dual Band Filter.</li>
<li>False negative results may infrequently occur due to mixed growth or due to error in assay technique.</li>
<li>The type and condition of the instrumentation used will influence the visual appearance of the image obtained. The fluorescence may varydue to the type of microscope employed, the light source and the level of rRNA in the cells</li>
<li>Isolation on solid media is needed to differentiate mixed growth with other organisms and to identify positive blood cultures yielding a negative result.</li>
<li>The product has not been validated with specimens other than blood cultures.</li>
</ul>

<table><tbody><tr><td colspan="4">&lt;p class=&quot;jove_step&quot;&gt;Reagents&lt;/p&gt;&lt;p class=&quot;jove_content&quot;&gt;&lt;em&gt;E. faecalis&lt;/em&gt;/&amp;#8211; PNA FISH is comprised of the following kit components:&lt;/p&gt;&lt;ul&gt; &lt;li&gt;Fixation Solution Fixation Solution 3 mL phosphate-buffered saline with detergent.&lt;/li&gt; &lt;li&gt;&lt;em&gt;E. faecalis&lt;/em&gt;/&amp;#8211; PNA &lt;em&gt;E. faecalis&lt;/em&gt;/&amp;#8211; PNA 1.5 mL PNA probes in hybridization. solution. Contains 30% formamide.&lt;/li&gt; &lt;li&gt;60x Wash Solution 60x Wash Solution 50 mL Tris-buffered saline with detergent&lt;/li&gt; &lt;li&gt;Mounting Medium Mounting Medium 3 mL photobleaching inhibitor in Glycerol&lt;/li&gt; &lt;li&gt;Quality control slides&lt;/li&gt; &lt;li&gt;&lt;a href=&quot;http://www.advandx.com&quot;&gt;AdvanDX&lt;/a&gt;&lt;/li&gt;&lt;/ul&gt;</td></tr></tbody></table>

a 1.5 hour procedure for identification of enterococcus species directly from blood cultures

Enterococci are a common cause of bacteremia with E. faecalis being the predominant species followed by E. faecium. Because resistance to ampicillin and vancomycin in E. faecalis is still uncommon compared to resistance in E. faecium, the development of rapid tests allowing differentiation between enterococcal species is important for appropriate therapy and resistance surveillance. The E. faecalis OE PNA FISH assay (AdvanDx, Woburn, MA) uses species-specific peptide nucleic acid (PNA) probes in a fluorescence in situ hybridization format and offers a time to results of 1.5 hours and the potential of providing important information for species-specific treatment. Multicenter studies were performed to assess the performance of the 1.5 hour E. faecalis/OE PNA FISH procedure compared to the original 2.5 hour assay procedure and to standard bacteriology methods for the identification of enterococci directly from a positive blood culture bottle.

Watch this Scientific Journal Video about A 1.5 Hour Procedure for Identification of Enterococcus Species Directly from Blood Cultures at JoVE.com

A 1.5 Hour Procedure for Identification of Enterococcus Species Directly from Blood Cultures

A rapid protocol for the direct identification of Enterococcus faecalis and other Enterococcus species from a positive blood culture using a Peptide Nucleic Acid fluorescent in situ hybridization assay (PNA FISH).

A 1.5 Hour Procedure for Identification of Enterococcus Species Directly from Blood Cultures

Enterococci are a common cause of bacteremia with E. faecalis being the predominant species followed by E. faecium.  Because resistance to ampicillin and ...

a-15-hour-procedure-for-identification-enterococcus-species-directly

Research

JoVE Journal

Immunology and Infection

20.8K Views.  Cedars-Sinai Medical Cente. A rapid protocol for the direct identification of Enterococcus faecalis and other Enterococcus species from a positive blood culture using a Peptide Nucleic Acid fluorescent in situ hybridization assay (PNA FISH).

Video: A 1.5 Hour Procedure for Identification of Enterococcus Species Directly from Blood Cultures

Depletion of Specific Cell Populations by Complement Depletion

The purification of immune cell populations is often required in order to study their unique functions. In particular, molecular approaches such as real-time PCR and microarray analysis require the isolation of cell populations with high purity. Commonly used purification strategies include fluorescent activated cell sorting (FACS), magnetic bead separation and complement depletion. Of the three strategies, complement depletion offers the advantages of being fast, inexpensive, gentle on the cells and a high cell yield. The complement system is composed of a large number of plasma proteins that when activated initiate a proteolytic cascade culminating in the formation of a membrane-attack complex that forms a pore on a cell surface resulting in cell death1. The classical pathway is activated by IgM and IgG antibodies and was first described as a mechanism for killing bacteria. With the generation of monoclonal antibodies (mAb), the complement cascade can be used to lyse any cell population in an antigen-specific manner. Depletion of cells by the complement cascade is achieved by the addition of complement fixing antigen-specific antibodies and rabbit complement to the starting cell population. The cells are incubated for one hour at 37&deg;C and the lysed cells are subsequently removed by two rounds of washing. MAb with a high efficiency for complement fixation typically deplete 95-100% of the targeted cell population. Depending on the purification strategy for the targeted cell population, complement depletion can be used for cell purification or for the enrichment of cell populations that then can be further purified by a subsequent method.

To effectively study the function of immune cell populations their purification is often required. Complement depletion is a fast and inexpensive technique for the isolation of immune cell populations with high purity.

The purification of immune cell populations is often required in order to study their unique functions.  In particular, molecular approaches such as ...

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 ...

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.

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. ...

Generation of Organotypic Raft Cultures from Primary Human Keratinocytes

The development of organotypic epithelial raft cultures has provided researchers with an efficient in vitro system that faithfully recapitulates epithelial differentiation. There are many uses for this system. For instance, the ability to grow three-dimensional organotypic raft cultures of keratinocytes has been an important milestone in the study of human papillomavirus (HPV)1. The life cycle of HPV is tightly linked to the differentiation of squamous epithelium2. Organotypic epithelial raft cultures as demonstrated here reproduce the entire papillomavirus life cycle, including virus production3,4,5. In addition, these raft cultures exhibit dysplastic lesions similar to those observed upon in vivo infection with HPV. Hence this system can also be used to study epithelial cell cancers, as well as the effect of drugs on epithelial cell differentiation in general. Originally developed by Asselineau and Prunieras6 and modified by Kopan et al.7, the organotypic epithelial raft culture system has matured into a general, relatively easy culture model, which involves the growth of cells on collagen plugs maintained at an air-liquid interface (Figure 1A). Over the course of 10-14 days, the cells stratify and differentiate, forming a full thickness epithelium that produces differentiation-specific cytokeratins. Harvested rafts can be examined histologically, as well as by standard molecular and biochemical techniques. In this article, we describe a method for the generation of raft cultures from primary human keratinocytes. The same technique can be used with established epithelial cell lines, and can easily be adapted for use with epithelial tissue from normal or diseased biopsies8. Many viruses target either the cutaneous or mucosal epithelium as part of their replicative life cycle. Over the past several years, the feasibility of using organotypic raft cultures as a method of studying virus-host cell interactions has been shown for several herpesviruses, as well as adenoviruses, parvoviruses, and poxviruses9. Organotypic raft cultures can thus be adapted to examine viral pathogenesis, and are the only means to test novel antiviral agents for those viruses that are not cultivable in permanent cell lines.

An in vitro method to mimic in vivo epithelial differentiation is described. Many viruses target epithelial cells as part of their viral life cycle, and this method provides a means of examining virus:host interactions that more closely resembles that which occurs in vivo. This technique can be used with primary keratinocytes, established cell lines, as well as normal or diseased biopsy tissue.

The development of organotypic epithelial raft cultures has provided researchers with an efficient in vitro system that faithfully recapitulates ...

Detection of Bacteria Using Fluorogenic DNAzymes

Outbreaks linked to food-borne and hospital-acquired pathogens account for millions of deaths and hospitalizations as well as colossal economic losses each and every year. Prevention of such outbreaks and minimization of the impact of an ongoing epidemic place an ever-increasing demand for analytical methods that can accurately identify culprit pathogens at the earliest stage. Although there is a large array of effective methods for pathogen detection, none of them can satisfy all the following five premier requirements embodied for an ideal detection method: high specificity (detecting only the bacterium of interest), high sensitivity (capable of detecting as low as a single live bacterial cell), short time-to-results (minutes to hours), great operational simplicity (no need for lengthy sampling procedures and the use of specialized equipment), and cost effectiveness. For example, classical microbiological methods are highly specific but require a long time (days to weeks) to acquire a definitive result.1 PCR- and antibody-based techniques offer shorter waiting times (hours to days), but they require the use of expensive reagents and/or sophisticated equipment.2-4 Consequently, there is still a great demand for scientific research towards developing innovative bacterial detection methods that offer improved characteristics in one or more of the aforementioned requirements. Our laboratory is interested in examining the potential of DNAzymes as a novel class of molecular probes for biosensing applications including bacterial detection.5
DNAzymes (also known as deoxyribozymes or DNA enzymes) are man-made single-stranded DNA molecules with the capability of catalyzing chemical reactions.6-8 These molecules can be isolated from a vast random-sequence DNA pool (which contains as many as 1016 individual sequences) by a process known as "in vitro selection" or "SELEX" (systematic evolution of ligands by exponential enrichment).9-16 These special DNA molecules have been widely examined in recent years as molecular tools for biosensing applications.6-8
Our laboratory has established in vitro selection procedures for isolating RNA-cleaving fluorescent DNAzymes (RFDs; Fig. 1) and investigated the use of RFDs as analytical tools.17-29 RFDs catalyze the cleavage of a DNA-RNA chimeric substrate at a single ribonucleotide junction (R) that is flanked by a fluorophore (F) and a quencher (Q). The close proximity of F and Q renders the uncleaved substrate minimal fluorescence. However, the cleavage event leads to the separation of F and Q, which is accompanied by significant increase of fluorescence intensity.
More recently, we developed a method of isolating RFDs for bacterial detection.5 These special RFDs were isolated to "light up" in the presence of the crude extracellular mixture (CEM) left behind by a specific type of bacteria in their environment or in the media they are cultured (Fig. 1). The use of crude mixture circumvents the tedious process of purifying and identifying a suitable target from the microbe of interest for biosensor development (which could take months or years to complete). The use of extracellular targets means the assaying procedure is simple because there is no need for steps to obtain intracellular targets.
Using the above approach, we derived an RFD that cleaves its substrate (FS1; Fig. 2A) only in the presence of the CEM produced by E. coli (CEM-EC).5 This E. coli-sensing RFD, named RFD-EC1 (Fig. 2A), was found to be strictly responsive to CEM-EC but nonresponsive to CEMs from a host of other bacteria (Fig. 3).
Here we present the key experimental procedures for setting up E. coli detection assays using RFD-EC1 and representative results.

We have recently reported a novel approach for generating fluorogenic DNAzyme probes that can be applied to set up a simple, "mix-and-read" fluorescent assay for bacterial detection. These special DNA probes catalyze the cleavage of a chromophore-modified DNA-RNA chimeric substrate in the presence of crude extracellular mixture (CEM) produced by a specific bacterium, thereby translating bacterial detection into fluorescence signal generation. In this report we will describe key experimental procedures where a specific DNAzyme probe denoted "RFD-EC1" is employed for the detection of the model bacterium, Escherichia coli (E. coli).

Outbreaks linked to food-borne and hospital-acquired pathogens account for millions of deaths and hospitalizations as well as colossal economic losses ...

Isolation and Identification of Waterborne Antibiotic-Resistant Bacteria and Molecular Characterization of their Antibiotic Resistance Genes

The development and spread of antibiotic resistance (AR) through microbiota associated with freshwater bodies is a major global health concern. In the present study, freshwater samples were collected and analyzed with respect to the total bacterial diversity and AR genes (ARGs) using both conventional culture-based techniques and a high-throughput culture-independent metagenomic approach. This paper presents a systematic protocol for the enumeration of the total and antibiotic-resistant culturable bacteria from freshwater samples and the determination of phenotypic and genotypic resistance in the culturable isolates. Further, we report the use of whole metagenomic analysis of the total metagenomic DNA extracted from the freshwater sample for the identification of the overall bacterial diversity, including non-culturable bacteria, and the identification of the total pool of different ARGs (resistome) in the water body. Following these detailed protocols, we observed a high antibiotic-resistant bacteria load in the range of 9.6 &#215; 105-1.2 &#215; 109 CFU/mL. Most isolates were resistant to the multiple tested antibiotics, including cefotaxime, ampicillin, levofloxacin, chloramphenicol, ceftriaxone, gentamicin, neomycin, trimethoprim, and ciprofloxacin, with multiple antibiotic resistance (MAR) indexes of &#8805;0.2, indicating high levels of resistance in the isolates. The 16S rRNA sequencing identified potential human pathogens, such as Klebsiella pneumoniae,&#160;and opportunistic bacteria, such as Comamonas spp., Micrococcus spp., Arthrobacter spp., and Aeromonas spp. The molecular characterization of the isolates showed the presence of various ARGs, such as blaTEM, blaCTX-M (&#946;-lactams), aadA, aac (6&#39;)-Ib (aminoglycosides), and dfr1 (trimethoprims), which was also confirmed by the whole metagenomic DNA analysis. A high prevalence of other ARGs encoding for antibiotic efflux pumps-mtrA, macB, mdtA, acrD, &#946;-lactamases-SMB-1, VIM-20, ccrA, ampC, blaZ, the&#160;chloramphenicol acetyltransferase gene catB10, and&#160;the rifampicin resistance gene rphB-was also detected in the metagenomic DNA. With the help of the protocols discussed in this study, we confirmed the presence of waterborne MAR bacteria with diverse AR phenotypic and genotypic traits. Thus, whole metagenomic DNA analysis can be used as a complementary technique to conventional culture-based techniques to determine the overall AR status of a water body.

Here, we present a detailed protocol for the isolation and identification of antibiotic-resistant bacteria from water and the molecular characterization of their antibiotic resistance genes (ARGs). The use of culture-based and non-culture-based (metagenomic analysis) techniques provides complete information about the total bacterial diversity and the total pool of different ARGs present in freshwaters from Mumbai, India.

The development and spread of antibiotic resistance (AR) through microbiota associated with freshwater bodies is a major global health concern. In the ...

Environment

Rapid Identification of Gram Negative Bacteria from Blood Culture Broth Using MALDI-TOF Mass Spectrometry

An important role of the clinical microbiology laboratory is to provide rapid identification of bacteria causing bloodstream infection. Traditional identification requires the sub-culture of signaled blood culture broth with identification available only after colonies on solid agar have matured. MALDI-TOF MS is a reliable, rapid method for identification of the majority of clinically relevant bacteria when applied to colonies on solid media. The application of MALDI-TOF MS directly to blood culture broth is an attractive approach as it has potential to accelerate species identification of bacteria and improve clinical management. However, an important problem to overcome is the pre-analysis removal of interfering resins, proteins and hemoglobin&#160;contained in blood culture specimens which, if not removed, interfere with the MS spectra and can result in insufficient or low discrimination identification scores. In addition it is necessary to concentrate bacteria to develop spectra of sufficient quality. The presented method describes the concentration, purification, and extraction of Gram negative bacteria allowing for the early identification of bacteria from a signaled blood culture broth.

The application of matrix-assisted laser desorption/ionization time of flight (MALDI-TOF) mass spectrometry (MS) directly to blood culture broth expedites the identification of bacteria. The presented method is a rapid and reliable method for identification of Gram negative bacteria directly from blood culture broth.

An important role of the clinical microbiology laboratory is to provide rapid identification of bacteria causing bloodstream infection. Traditional ...

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 ...

Accelerated Antimicrobial Susceptibility Testing in Blood Culture Bottles

Direct Microbial Identification using An Automated Microbial Identification System to Facilitate the EUCAST RAST Method Without Mass Spectrometry

Gram-negative (GN) sepsis is a medical emergency where management in resource-limited settings relies on conventional microbiological culture techniques providing results in 3-4 days. Recognizing this delay in turnaround time (TAT), both EUCAST and CLSI have developed protocols for determining AST results directly from positively flagged automated blood culture bottles (+aBCs). EUCAST rapid AST (RAST) protocol was first introduced in 2018, where zone diameter breakpoints for four common etiological agents of GN sepsis, i.e., Escherichia coli, Klebsiella pneumoniae, Pseudomonas aeruginosa, and Acinetobacter baumannii complex can be reported. However, those clinical laboratories that have implemented this method in their routine workflow rely on mass spectrometry-based microbial identification, which is not easily available, thus precluding its implementation in resource-limited settings. To circumvent it, we evaluated a direct inoculum protocol (DIP) using a commercial automated microbial identification and antimicrobial susceptibility testing system (aMIAST) to enable early microbial identification within 8 h of positive flagging of aBC. We evaluated this protocol from January to October 2023 to identify the four RAST reportable GN (RR-GN) in the positively flagged aBC. The microbial identification results in DIP were compared with the standard inoculum preparation protocol (SIP) in aMIAST. Of 204 +aBCs with monomorphic GN (+naBC), one of the 4 RR-GN was identified in 105 +naBCs by SIP (E. coli: 50, K. pneumoniae: 20, P. aeruginosa: 9 and A. baumannii complex: 26). Of these, 94% (98/105) were correctly identified by DIP whereas major error and very major error rates were 6% (7/105) and 1.7% (4/240), respectively. When DIP for microbial identification is done using the EUCAST RAST method, provisional clinical reports can be provided within 24 h of receiving the sample. This approach has the potential to significantly reduce the TAT, enabling early institution of appropriate antimicrobial therapy.

Author Spotlight: Accelerating Diagnostic Accuracy with Direct Identification of Gram-Negatives from Blood Culture Bottles

EUCAST has developed a direct antimicrobial susceptibility testing (AST) protocol for automated blood cultures. However, its dependence on mass spectrometry-based microbial identification can be obviated by using a direct inoculum preparation protocol in an automated microbial identification system. This approach can provide AST reports within 24 h of sample collection.

Gram-negative (GN) sepsis is a medical emergency where management in resource-limited settings relies on conventional microbiological culture techniques ...

Medicine

Rapid Antimicrobial Susceptibility Testing by Stimulated Raman Scattering Imaging of Deuterium Incorporation in a Single Bacterium

To slow and prevent the spread of antimicrobial resistant infections, rapid antimicrobial susceptibility testing (AST) is in urgent need to &#65279;quantitatively determine the &#65279;antimicrobial effects on pathogens. &#65279;It typically takes days to complete the AST by conventional methods based on the long-time culture, and they do not work directly for clinical samples. Here, we report a rapid AST &#65279;method enabled by stimulated Raman scattering (SRS) imaging of deuterium oxide (D2O) metabolic incorporation. Metabolic incorporation of D2O into biomass and the metabolic activity inhibition upon exposure to antibiotics at the single bacterium level are monitored by SRS imaging. The&#65279; single-cell metabolism inactivation concentration (SC-MIC) of bacteria upon exposure to antibiotics can be obtained after a total of 2.5 h of sample preparation and detection. Furthermore, this rapid AST method is directly applicable to bacterial samples in complex biological environments, such as urine or whole blood. SRS metabolic imaging of deuterium incorporation is transformative for rapid single-cell phenotypic AST in the clinic.

This protocol presents rapid antimicrobial susceptibility testing (AST) assay within 2.5 h by single-cell-stimulated Raman scattering imaging of D2O metabolism. This method applies to bacteria in the urine or whole blood environment, which is transformative for rapid single-cell phenotypic AST in the clinic.

A 1.5 Hour Procedure for Identification of Enterococcus Species Directly from Blood Cultures

Summary

Explore More Videos

Chapters in this video

Depletion of Specific Cell Populations by Complement Depletion

Introducing Shear Stress in the Study of Bacterial Adhesion

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

Generation of Organotypic Raft Cultures from Primary Human Keratinocytes

Detection of Bacteria Using Fluorogenic DNAzymes

Isolation and Identification of Waterborne Antibiotic-Resistant Bacteria and Molecular Characterization of their Antibiotic Resistance Genes

Rapid Identification of Gram Negative Bacteria from Blood Culture Broth Using MALDI-TOF Mass Spectrometry

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

Direct Microbial Identification using An Automated Microbial Identification System to Facilitate the EUCAST RAST Method Without Mass Spectrometry

Rapid Antimicrobial Susceptibility Testing by Stimulated Raman Scattering Imaging of Deuterium Incorporation in a Single Bacterium