We would like to thank the laboratory technologists of the Mayo Clinic Mycobacteriology Laboratory for their help during the study.

Each laboratory needs to perform a risk assessment in collaboration with their Institutional Biological Safety Officer to determine the appropriate biosafety level for preparation of the inoculum and for pipetting of the inoculum into the plate. In our laboratory, we utilize biosafety level 3 laboratory (BSL3) practices until the microtiter plates are inoculated, and sealed with a plastic adhesive seal. These plates are then placed inside a plastic bag, which is heat-also sealed. Incubation and interpretation reading of the microtiter plate is conducted in a biosafety level 2 laboratory (BSL2).
1. Labeling of materials
<ol>
 <li>Label a blood agar plate, three Middlebrook 7H10 plates, Middlebrook 7H9 broth, a saline tween glass bead tube and a microtiter AST plate with the appropriate identifiers (e.g., patient name, medical record number) in the BSL2.</li>
 <li>Label the blood agar and 7H10 plated as &#x201C;purity check&#x201D;. Periodic colony counts of the inoculum are recommended to ensure that an appropriate concentration of organisms is used in the test. Colony counts when performed will need to have two of the 7H10 plates labeled with &#x201C;1/1 colony count&#x201D; and &#x201C;1/50 colony count&#x201D;.</li>
</ol>
2. Technologist preparations to go into the BSL3
<ol>
 <li>Appropriate personal protective equipment is necessary to work in the BSL3 and includes solid-front gown, head cover, gloves, shoe covers, and a respirator. Each institution should establish their own respiratory protection plan in collaboration with their institution&#x2019;s Industrial Hygiene Officer. In our laboratory, we have several respirators available, including powered air purifying respirators (PAPRs) and fitted HEPA-filtered, N95 half-face respirators.</li>
 </ol>
3. Preparation of the biological safety cabinet
<ol>
 <li>In the BSL3, prepare the biological safety cabinet (BSC) by disinfecting the surface and placing a paper towel soaked with disinfectant approximately 6 inches from the air vent panel. </li>
 <li>Place a waste container on the left, and small nephlometer and vortexer on the right. </li>
 <li>Inoculating loops, pipetors and tips are placed beside the paper towel.</li>
 <li>Place a rack with the organism (in broth or on solid medium) and the McFarland 0.5 standard on the paper towel.</li>
</ol>
4. Calibration of the nephelometer
<ol>
 <li>Place a saline tween glass bead tube in the nephelometer and set the instrument to zero using the saline tween blank.</li>
 <li>Place the McFarland 0.5 standard in the nephelometer and calibrate according the manufacturer's instructions.</li>
</ol>
5. Preparation of the Inoculum
<ol>
 <li>Working in the BSC, scrape colonies from solid medium using a sterile loop into the appropriately labeled saline tween glass bead tube until a &ge;0.5 McFarland Standard is obtained. </li>
 <li>Visually inspect the sample, and compare to the McFarland Standard. </li>
 <li>Vortex sample for 30-60 seconds. </li>
 <li>Allow the inoculum to settle for 15 minutes (set a timer).</li>
 </ol>

6. Inoculation of the microtiter plate

 <ol>
 <li>Adjust the inoculum with saline tween broth using the nephelometer calibrated to the McFarland 0.5 in steps 4.1 and 4.2. This must be completed within 30 minutes (per manufacturer specifications). </li>
 <li>Use a 200 &mu;L pipetter and a LONG aerosol resistant pipet tip to remove 100 &mu;L of inoculum from the TOP 1/3rd of the saline bead tween tube. Inoculate the Middlebrook 7H9 Broth. Slowly vortex the inoculated 7H9 broth for 20 seconds. </li>
 <li>Remove the AST microtiter plate from manufacturer's package. Do NOT use if the desiccant is blue or if the foil package is damaged.</li>
 <li>Prepare a 1:50 dilution tube for use later in the preparation of the 1:50 dilution by pipetting 50 &mu;L of sterile water into a sterile tube.</li>
 <li>Carefully pour the 7H9 broth inoculum into a trough. AVOID GENERATING AEROSOLS.</li>
 <li>Place the AST microtiter plate with the label facing you.</li>
 <li>Use a 12-channel pipetter to add 100 &mu;L of the inoculum to each well. </li>
 <li>Prepare dilutions for colony counts:
 <ol type="a">
 <li>The 1/1 dilution is prepared from the positive control well (H12) by streaking 1 &mu;l loop on the 7H10 plate labeled &#x201C;1/1&#x201D;.</li>
 <li>The 1/50 dilution is prepared by inoculating a 1 &mu;l loop from the positive control well (H12) and swirling it into the tube with 50 &mu;l of water tube prepared above. Streak 1 &mu;L of this dilution to the plate labeled &#x201C;1/50&#x201D;. </li>
 </ol></li>

 <li>Inoculate the purity plates from the trough by pipetting 50 &mu;L onto an appropriately labeled blood agar and 7H10 plate and streak for isolation. </li>
 <li>Place an adhesive seal on the AST microtiter plate. Press firmly on each well to assure adequate sealing by using the white back of the seal. Avoid creases. </li>
 <li>Disinfect the microtiter plate by wiping it with a paper towel soaked with a tuberculocidal disinfectant and place microtiter plate in a plastic bag and close with a heat seal or tape. 
 <ol type="a">
 <li>The plastic bag serves as a secondary container to ensure that all broth is contained should there be a leak or spill of the primary plate.</li>
 </ol></li>
 </ol>
7. Disinfection of materials and biological safety cabinet
 <ol>
 <li>To avoid producing aerosols, place another trough on top of the inoculated trough before placing it gently into the discard container.</li>
 <li>Disinfect all materials including nephlometer, vortexer, plates, tubes, etc. by wiping down with a tuberculocidal disinfectant and wait the appropriate time to insure disinfection according to the manufacturer&#x2019;s instructions before removing from the BSC. For example, Prospray, the tuberculocidal disinfectant we use, requires 15 minutes to kill mycobacteria.</li>
 <li>Place the discard container in a biohazard bag and seal before autoclaving.</li>
 </ol>
8. Incubation
 <ol>
 <li>Plates should be incubated with media down and care should be taken to avoid spilling or splashing any liquid. </li>
 <li>Incubate the AST microtiter plate plate, 7H10 and blood agar plates in a 35-37&deg;C incubator with 5-10% CO2 with 5-10% CO2 in the BSL2 laboratory.
 <ol class="lalpha">
 <li>In our laboratory, this is acceptable because the plate is well-sealed and inside a sealed plastic bag. </li>
 </ol></li>
 <li>No more than 2 microtiter AST plates should be stacked in the incubator according to the manufacturer&#x2019;s instructions.</li>
 </ol>
9. Representative results
Interpretation of results:
Sealed AST microtiter plates can be read manually using a mirror as an aid or with an automated plate reader such as the Vizion (TREK Diagnostics). The plates can be examined as soon as 7 days after inoculation. Examine the growth control wells in positions H11 and H12 to determine if they are positive (ie., have a deposit of cells at the bottom of the well). If the growth control wells are not positive, reincubate the plate and re-examine periodically (eg., day 10, day 14) until the growth control wells are positive.
 <ol type="a">
 <li>Mirror read: 
 <ol type="i">
 <li>Place sealed AST microtiter plate onto the platform; </li>
 <li>Read growth control wells after 7 days of incubation; if this is negative, reincubate AST microtiter plate; </li>
 <li>If the growth controls are acceptable, then read the drug-containing wells. For for each drug, record the first dilution that has no growth by comparing the drug wells with the growth control. </li>
 <li>Growth appears as turbidity or as a deposit of cells at the bottom of a well. </li>
 </ol></li>
 <li>Instrument Read: follow the manufacturers instructions; the instructions below are an example of how the reading would be done with the Vizion platform:
 <ol type="i">
 <li>Log in. </li>
 <li>Use &#x201C;MYCOTB&#x201D; AST plate software.</li>
 <li>Place sealed plate with the barcode facing the user. Growth appears as turbidity or as a deposit of cells at the bottom of a well. </li>
 <li>Read growth controls; if this is negative, reincubate AST microtiter plate; if positive read the plate by touching the screen with the first well of each drug that does not show growth.</li>
 </ol></li>
 </ol>
Read purity plates. The blood agar plate should show no growth after 48 hours and the 7H10 purity plate should have confluent growth of one organism type. If contaminated, the AST microtiter test needs to be repeated from a pure culture.

Calculate colony counts from the positive control well using the table below:

Colony Count Key
<table border="1">
<tbody>
<tr>
 <td># colonies counted on plate labeled "1/1"</td>
 <td># colonies counted on plate labeled "1/50"</td>
 <td>Inoculum Colony Count (cfu/mL)</td>
</tr>

<tr>
 <td>&lt;50</td>
 <td>0</td>
 <td>&lt;5 X 104</td>
</tr>

<tr>
 <td>50-100</td>
 <td>0-2</td>
 <td>5 X 104 to 1 X 105</td>
</tr>

<tr>
 <td>&gt;100</td>
 <td>&le; 10</td>
 <td>1 X 105 to 5 X 105</td>
</tr>

<tr>
 <td>&gt;100</td>
 <td>&gt;10</td>
 <td>>5 X 105</td>
</tr>
</tbody>
</table>

There may be trailing with para-aminosalicylic acid in some strains of Mycobacterium tuberculosis. This can be interpreted by looking at all wells that have pellets and using the first well that has the same size pellet as the next dilution as the end point. Resistance is rare with this drug.

<img src="/files/ftp_upload/3094/3094fig1.jpg" alt="Figure 1"/> 
Figure 1. Representative results using the MYCOTB plate.

<table border="1">
<tbody>
<tr>
 <td>Drug abbreviation</td>
 <td>Drug</td>
 <td>MIC &mu;/mL</td>
</tr>
<tr>
 <td>OFL</td>
 <td>ofloxacin</td>
 <td>16</td>
</tr>
<tr>
 <td>MXF</td>
 <td>moxifloxacin</td>
 <td>8</td>
</tr>
<tr>
 <td>RIF</td>
 <td>rifampin</td>
 <td>&gt;16</td>
</tr>
<tr>
 <td>AMI</td>
 <td>amikacin</td>
 <td>0.5</td>
</tr>
<tr>
 <td>STR</td>
 <td>streptomycin</td>
 <td>16</td>
</tr>
<tr>
 <td>RFB</td>
 <td>rifabutin</td>
 <td>8</td>
</tr>
<tr>
 <td>PAS</td>
 <td>p-aminosalicylic acid</td>
 <td>&le;0.5</td>
</tr>
<tr>
 <td>ETH</td>
 <td>ethionamide</td>
 <td>5</td>
</tr>
<tr>
 <td>CYC</td>
 <td>cycloserine</td>
 <td>4</td>
</tr>
<tr>
 <td>INH</td>
 <td>isoniazid</td>
 <td>2</td>
</tr>
<tr>
 <td>KAN</td>
 <td>kanamycin</td>
 <td>1.2</td>
</tr>
<tr>
 <td>EMB</td>
 <td>ethambutol</td>
 <td>4.0</td>
</tr>
</tbody>
</table>

<ol>
	<li>Wengenack, N., Hall, L., Labombardi, V., Parrish, N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Evaluation+of+the+New+Sensititre+(MYCOTB)+MIC+Plate+for+the+Susceptibility+Testing+of+Mycobacterium+tuberculosis+(Mtb)+to+First+and+Second+Line+Drugs.">Evaluation of the New Sensititre (MYCOTB) MIC Plate for the Susceptibility Testing of Mycobacterium tuberculosis (Mtb) to First and Second Line Drugs.</a> , (2010).</li><li>. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=HHS+Publication+No.+(CDC)+21-112:+Biosafety+in+Microbiological+and+Biomedical+Laboratories+(BMBL).">HHS Publication No. (CDC) 21-112: Biosafety in Microbiological and Biomedical Laboratories (BMBL).</a> U.S. Department of Health and Human Services Centers for Disease and Preventions. , (2009).</li><li>Morcillo, N., Imperiale, B., Digiulio, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Evaluation+of+MGIT+960+and+the+colorimetric-based+method+for+tuberculosis+drug+susceptibility+testing.">Evaluation of MGIT 960 and the colorimetric-based method for tuberculosis drug susceptibility testing.</a> Int J Tuberc Lung Dis. 14, 1169-1175 (2010).</li><li>Bemer, P., Bodmer, T., Munzinger, J., Perrin, M., Vincent, V., Drugeon, 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+the+MB/BACT+System+for+Susceptibility+Testing+of+Mycobacterium+tuberculosis.">Multicenter Evaluation of the MB/BACT System for Susceptibility Testing of Mycobacterium tuberculosis.</a> J Clin Microbiol. 42, 1030-1034 (2004).</li><li>. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=SENSITITRE+MYCOTB+Susceptibility+Plate:+For+Mycobacterium+Susceptibility+Testing.">SENSITITRE MYCOTB Susceptibility Plate: For Mycobacterium Susceptibility Testing.</a> Product Insert. , (2010).</li><li>CLSI. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Susceptiblity Testing of Mycobacteria, Nocardia, and Other Aerobic Actinomycetes; Approved Standard. Document M24-A. , (2003).</li><li>Sullivan, N., Sotos, J., Anhalt, K., Allen, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Preliminary+results+from+a+new+TREK+Sensititre+Mycobacterium+tuberculosis+MIC+plate+(MYCOTB).">Preliminary results from a new TREK Sensititre Mycobacterium tuberculosis MIC plate (MYCOTB).</a> , (2010).</li><li>. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=VersaTREK+MYCO+SUSCEPTIBLITY+KIT.">VersaTREK MYCO SUSCEPTIBLITY KIT.</a> Product insert. , 7115-7160 (2008).</li><li>Parrish, N. M., Carroll, K. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+Role+of+the+Clinical+Mycobacteriology+Laboratory+in+the+Diagnosis+and+Management+of+Tuberculosis+in+Low+Prevalance+Settings.">The Role of the Clinical Mycobacteriology Laboratory in the Diagnosis and Management of Tuberculosis in Low Prevalance Settings.</a> J Clin Microbiol. , (2010).</li><li>Kam, K. M., Sloutsky, A., Yip, C. W., Bulled, N., Zingol, M., Espinal, M., Kim, S. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Determination+of+critical+concentrations+of+second-line+anti-tuberculosis+drugs+with+clinical+and+microbiological+relevance.">Determination of critical concentrations of second-line anti-tuberculosis drugs with clinical and microbiological relevance.</a> Int J Tubercl Lung Dis. 14, 282-288 (2010).</li><li>Woods, G. L., Warren, N. G., Inderlied, C. B., Murray, P. R., Barron, E. J., Jorgensen, J. H., Landry, M. L., Pfaller, M. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Susceptibility+Test+Methods:+Mycobacteria,+Nocardia,+and+Other+Actinomycetes.">Susceptibility Test Methods: Mycobacteria, Nocardia, and Other Actinomycetes.</a> Manual of Clinical Microbiology. , 1223-1247 (2007).</li><li>Bergmann, J. S., Woods, G. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Evaluation+of+the+ESP+Culture+System+II+for+Testing+Susceptibilities+of+Mycobacterium+tuberculosis+Isolates+to+Four+Primary+Antituberculosis+Drugs.">Evaluation of the ESP Culture System II for Testing Susceptibilities of Mycobacterium tuberculosis Isolates to Four Primary Antituberculosis Drugs.</a> J Clin Microbiol. 36, 2940-2943 (1998).</li></ol>

There is nothing to disclose.

An example of a AST microtiter plate that has positive growth controls is shown in Figure 1 and the endpoint MIC (&mu;g/ml) for each drug is circled. A study conducted in our laboratory indicated that the AST microtiter plate method is equivalent to the gold standard agar proportion method for susceptibility testing of Mycobacterium tuberculosis. Faster reporting of results was obtained with microtiter AST microtiter plate when compared to the traditional agar proportion method with the final interpretation for AST microtiter plate and agar proportion method at 14 and 21 days, respectively.The AST microtiter plate has the advantage of testing first and second line drugs simultaneously which can help prevent inordinate delays with resistant strains. The AST microtiter plate is also much easier to set up and read than the APM and both a manual mirror and a semi-automated system such as the VIZION can be used to read the plates. The availability of a MIC value for TB drug resistance testing may provide valuable new information to physicians relative to the traditional critical concentration value, especially for those isolates with &#x201C;borderline&#x201D; susceptibility.

<table border="1">
<tbody>
<tr>
	<th>Name of the reagent</th>
 <th>Company</th>
 <th>Catalogue number</th>
</tr>
<tr>
	<td>AST microtiter plates and adhesive seal</td>
 <td>TREK Diagnostics, Cleveland Ohio</td>
 <td>MYCOTB</td>
</tr>
<tr>
	<td>McFarland 0.5 standard</td>
 <td>TREK Diagnostics, Cleveland, Ohio</td>
 <td>E1041</td>
</tr>
<tr>
	<td>Middlebrook 7H10 plates</td>
 <td>Fisher</td>
 <td>&nbsp;</td>
</tr>
<tr>
	<td>Blood Agar Plates</td>
 <td>Fisher</td>
 <td>&nbsp;</td>
</tr>
<tr>
	<td>Sterile loops 1 &mu;L</td>
 <td>Fisher</td>
 <td>&nbsp;</td>
</tr>
<tr>
	<td>Sterile loops 10 &mu;L</td>
 <td>Fisher</td>
 <td>&nbsp;</td>
</tr>
<tr>
	<td>Sensititre Saline-Tween glass bead broth</td>
 <td>TREK Diagnostics, Cleveland, Ohio</td>
 <td>27143</td>
</tr>
<tr>
	<td>7H9 OADC Broth</td>
 <td>TREK Diagnostics, Cleveland, Ohio</td>
 <td>T3440</td>
</tr>
<tr>
	<td>Long Pipet Tips</td>
 <td>Fisher</td>
 <td>&nbsp;</td>
</tr>
<tr>
	<td>Sterile Troughs</td>
 <td>TREK Diagnostics, Cleveland, Ohio</td>
 <td>&nbsp;</td>
</tr>
<tr>
	<td>M. tuberculosis</td>
 <td>ATCC</td>
 <td>27294</td>
</tr>
<tr>
	<td>Pipeters: 1000, 200, 20&mu;L</td>
 <td>Fisher</td>
 <td>&nbsp;</td>
</tr>
<tr>
	<td>Tips 1000, 20, 20 &mu;L</td>
 <td>Fisher</td>
 <td>&nbsp;</td>
</tr>
<tr>
	<td>Multichannel Pipetter</td>
 <td>Fisher</td>
 <td>&nbsp;</td>
</tr>
<tr>
	<td>Incubator 5-10% CO2</td>
 <td>Fisher</td>
 <td>&nbsp;</td>
</tr>
<tr>
	<td>Plastic bags, small, CO2 permeable</td>
 <td>Poly Plastic, Delano, PA</td>
 <td>&nbsp;</td>
</tr>
<tr>
	<td>TB disinfectant (Such as ProSpray C-60)</td>
 <td>Certol International, Commerce City, CO</td>
 <td>PSC60/32</td>
</tr>
<tr>
	<td>Mirror Box Reader</td>
 <td>Fisher</td>
 <td>&nbsp;</td>
</tr>
</tbody>
</table>

antimicrobial susceptibility testing of mycobacterium tuberculosis complex for first and second line drugs by broth dilution in a microtiter plate format

The rapid detection of antimicrobial resistance is important in the effort to control the increase in resistant Mycobacterium 
tuberculosis (Mtb). Antimicrobial susceptibility testing (AST) of Mtb has traditionally been performed by the agar method of proportion or by 
macrobroth testing on an instrument such as the BACTEC (Becton Dickinson, Sparks, MD), VersaTREK (TREK Diagnostics, Cleveland, OH) or BacT/ALERT (bioM&eacute;rieux, Hazelwood, MO). The agar proportion method, while considered the &#x201C;gold&#x201D; standard of AST, is labor intensive and requires calculation of resistance by performing colony counts on drug-containing agar as compared to drug-free agar. If there is &ge;1% growth on the drug-containing medium as compared to drug-free medium, the organism is considered resistant to that drug. The macrobroth methods require instrumentation and test break point ("critical") drug concentrations for the first line drugs (isoniazid, ethambutol, rifampin, and pyrazinamide). The method described here is commercially available in a 96 well microtiter plate format [MYCOTB (TREK Diagnostics)] and contains increasing concentrations of 12 antimicrobials used for treatment of tuberculosis including both first (isoniazid, rifampin, ethambutol) and second line drugs (amikacin, cycloserine, ethionamide, kanamycin, moxifloxacin, ofloxacin, para-aminosalicylic acid, rifabutin, and streptomycin). Pyrazinamide, a first line drug, is not included in the microtiter plate due to its need for acidic test conditions. Advantages of the microtiter system include both ease of set up and faster turn around time (14 days) compared with traditional agar proportion (21 days). In addition, the plate can be set up from inoculum prepared using either broth or solid medium. Since the microtiter plate format is new and since Mtb presents unique safety challenges in the laboratory, this protocol will describe how to safely setup, incubate and read the microtiter plate.

Watch this Scientific Journal Video about Antimicrobial Susceptibility Testing of Mycobacterium Tuberculosis Complex for First and Second Line Drugs by Broth Dilution in a Microtiter Plate Format at J

Antimicrobial Susceptibility Testing of Mycobacterium Tuberculosis Complex for First and Second Line Drugs by Broth Dilution in a Microtiter Plate Format

Antimicrobial susceptibility testing of Mycobacterium tuberculosis is challenging but critical for patient care. This microtiter plate format offers testing of 12 antimycobacterial drugs and provides minimum inhibitory concentration (MIC) values, which will aid in appropriate treatment.

The rapid detection of antimicrobial resistance is important in the effort to control the increase in resistant Mycobacterium 
tuberculosis (Mtb). ...

antimicrobial-susceptibility-testing-mycobacterium-tuberculosis

Research

JoVE Journal

Immunology and Infection

38.0K Views.  Mayo Clinic. Antimicrobial susceptibility testing of Mycobacterium tuberculosis is challenging but critical for patient care. This microtiter plate format offers testing of 12 antimycobacterial drugs and provides minimum inhibitory concentration (MIC) values, which will aid in appropriate treatment.

Video: Antimicrobial Susceptibility Testing of Mycobacterium Tuberculosis Complex for First and Second Line Drugs by Broth Dilution in a Microtiter Plate Format

A 96 Well Microtiter Plate-based Method for Monitoring Formation and Antifungal Susceptibility Testing of Candida albicans Biofilms

Candida albicans remains the most frequent cause of fungal infections in an expanding population of compromised patients and candidiasis is now the third most common infection in US hospitals. Different manifestations of candidiasis are associated with biofilm formation, both on host tissues and/or medical devices (i.e. catheters). Biofilm formation carries negative clinical implications, as cells within the biofilms are protected from host immune responses and from the action of antifungals. We have developed a simple, fast and robust in vitro model for the formation of C. albicans biofilms using 96 well microtiter-plates, which can also be used for biofilm antifungal susceptibility testing. The readout of this assay is colorimetric, based on the reduction of XTT (a tetrazolium salt) by metabolically active fungal biofilm cells. A typical experiment takes approximately 24 h for biofilm formation, with an additional 24 h for antifungal susceptibility testing. Because of its simplicity and the use of commonly available laboratory materials and equipment, this technique democratizes biofilm research and represents an important step towards the standardization of antifungal susceptibility testing of fungal biofilms.

A 96 Well Microtiter Plate-based Method for Monitoring Formation and Antifungal Susceptibility Testing of Candida albicans Biofilms

We describe a simple, rapid and robust method for the formation of Candida albicans biofilms using 96 well microtiter plates and its utility in antifungal susceptibility testing of cells within biofilms.

Candida albicans remains the most frequent cause of fungal infections in an expanding population of compromised patients and candidiasis is now the third ...

Sample Preparation of Mycobacterium tuberculosis Extracts for Nuclear Magnetic Resonance Metabolomic Studies

Mycobacterium tuberculosis is a major cause of mortality in human beings on a global scale. The emergence of both multi- (MDR) and extensively-(XDR) drug-resistant strains threatens to derail current disease control efforts. Thus, there is an urgent need to develop drugs and vaccines that are more effective than those currently available. The genome of M. tuberculosis has been known for more than 10 years, yet there are important gaps in our knowledge of gene function and essentiality. Many studies have since used gene expression analysis at both the transcriptomic and proteomic levels to determine the effects of drugs, oxidants, and growth conditions on the global patterns of gene expression. Ultimately, the final response of these changes is reflected in the metabolic composition of the bacterium including a few thousand small molecular weight chemicals. Comparing the metabolic profiles of wild type and mutant strains, either untreated or treated with a particular drug, can effectively allow target identification and may lead to the development of novel inhibitors with anti-tubercular activity. Likewise, the effects of two or more conditions on the metabolome can also be assessed. Nuclear magnetic resonance (NMR) is a powerful technology that is used to identify and quantify metabolic intermediates. In this protocol, procedures for the preparation of M. tuberculosis cell extracts for NMR metabolomic analysis are described. Cell cultures are grown under appropriate conditions and required Biosafety Level 3 containment,1 harvested, and subjected to mechanical lysis while maintaining cold temperatures to maximize preservation of metabolites. Cell lysates are recovered, filtered sterilized, and stored at ultra-low temperatures. Aliquots from these cell extracts are plated on Middlebrook 7H9 agar for colony-forming units to verify absence of viable cells. Upon two months of incubation at 37 &deg;C, if no viable colonies are observed, samples are removed from the containment facility for downstream processing. Extracts are lyophilized, resuspended in deuterated buffer and injected in the NMR instrument, capturing spectroscopic data that is then subjected to statistical analysis. The procedures described can be applied for both one-dimensional (1D) 1H NMR and two-dimensional (2D) 1H-13C NMR analyses. This methodology provides more reliable small molecular weight metabolite identification and more reliable and sensitive quantitative analyses of cell extract metabolic compositions than chromatographic methods. Variations of the procedure described following the cell lysis step can also be adapted for parallel proteomic analysis.

Sample Preparation of Mycobacterium tuberculosis Extracts for Nuclear Magnetic Resonance Metabolomic Studies

The metabolomic profile of Mycobacterium tuberculosis is determined after growth in broth cultures. Conditions can be varied to test the effects of nutritional supplements, oxidants, and anti-tuberculosis agents on the metabolic profile of this microorganism. Procedure for extract preparation is applicable for both 1D 1H and 2D 1H-13C NMR analyses.

Mycobacterium tuberculosis is a major cause of mortality in human beings on a global scale. The emergence of both multi- (MDR) and extensively-(XDR) ...

Growth of Mycobacterium tuberculosis Biofilms

Mycobacterium tuberculosis, the etiologic agent of human tuberculosis, has an extraordinary ability to survive against environmental stresses including antibiotics. Although stress tolerance of M. tuberculosis is one of the likely contributors to the 6-month long chemotherapy of tuberculosis 1, the molecular mechanisms underlying this characteristic phenotype of the pathogen remain unclear. Many microbial species have evolved to survive in stressful environments by self-assembling in highly organized, surface attached, and matrix encapsulated structures called biofilms 2-4. Growth in communities appears to be a preferred survival strategy of microbes, and is achieved through genetic components that regulate surface attachment, intercellular communications, and synthesis of extracellular polymeric substances (EPS) 5,6. The tolerance to environmental stress is likely facilitated by EPS, and perhaps by the physiological adaptation of individual bacilli to heterogeneous microenvironments within the complex architecture of biofilms 7.
In a series of recent papers we established that M. tuberculosis and Mycobacterium smegmatis have a strong propensity to grow in organized multicellular structures, called biofilms, which can tolerate more than 50 times the minimal inhibitory concentrations of the anti-tuberculosis drugs isoniazid and rifampicin 8-10. M. tuberculosis, however, intriguingly requires specific conditions to form mature biofilms, in particular 9:1 ratio of headspace: media as well as limited exchange of air with the atmosphere 9. Requirements of specialized environmental conditions could possibly be linked to the fact that M. tuberculosis is an obligate human pathogen and thus has adapted to tissue environments. In this publication we demonstrate methods for culturing M. tuberculosis biofilms in a bottle and a 12-well plate format, which is convenient for bacteriological as well as genetic studies. We have described the protocol for an attenuated strain of M. tuberculosis, mc27000, with deletion in the two loci, panCD and RD1, that are critical for in vivo growth of the pathogen 9. This strain can be safely used in a BSL-2 containment for understanding the basic biology of the tuberculosis pathogen thus avoiding the requirement of an expensive BSL-3 facility. The method can be extended, with appropriate modification in media, to grow biofilm of other culturable mycobacterial species.
Overall, a uniform protocol of culturing mycobacterial biofilms will help the investigators interested in studying the basic resilient characteristics of mycobacteria. In addition, a clear and concise method of growing mycobacterial biofilms will also help the clinical and pharmaceutical investigators to test the efficacy of a potential drug.

Growth of Mycobacterium tuberculosis Biofilms

Mycobacterium tuberculosis forms drug tolerant biofilms when cultured in certain conditions. Here we describe methods for culturing M. tuberculosis biofilms and determining the frequency of drug tolerant persisters. These protocols will be useful for further studies into the mechanisms of drug tolerance in M. tuberculosis.

Mycobacterium tuberculosis, the etiologic agent of human tuberculosis, has an extraordinary ability to survive against environmental stresses including ...

Demonstrating a Multi-drug Resistant Mycobacterium tuberculosis Amplification Microarray

Simplifying microarray workflow is a necessary first step for creating MDR-TB microarray-based diagnostics that can be routinely used in lower-resource environments. An amplification microarray combines asymmetric PCR amplification, target size selection, target labeling, and microarray hybridization within a single solution and into a single microfluidic chamber. A batch processing method is demonstrated with a 9-plex asymmetric master mix and low-density gel element microarray for genotyping multi-drug resistant Mycobacterium tuberculosis (MDR-TB). The protocol described here can be completed in 6 hr and provide correct genotyping with at least 1,000 cell equivalents of genomic DNA. Incorporating on-chip wash steps is feasible, which will result in an entirely closed amplicon method and system. The extent of multiplexing with an amplification microarray is ultimately constrained by the number of primer pairs that can be combined into a single master mix and still achieve desired sensitivity and specificity performance metrics, rather than the number of probes that are immobilized on the array. Likewise, the total analysis time can be shortened or lengthened depending on the specific intended use, research question, and desired limits of detection. Nevertheless, the general approach significantly streamlines microarray workflow for the end user by reducing the number of manually intensive and time-consuming processing steps, and provides a simplified biochemical and microfluidic path for translating microarray-based diagnostics into routine clinical practice.

Demonstrating a Multi-drug Resistant Mycobacterium tuberculosis Amplification Microarray

An amplification microarray combines asymmetric PCR amplification and microarray hybridization into a single chamber, which significantly streamlines microarray workflow for the end user. Simplifying microarray workflow is a necessary first step for creating microarray-based diagnostics that can be routinely used in lower-resource environments.

Simplifying microarray workflow is a necessary first step for creating MDR-TB microarray-based diagnostics that can be routinely used in lower-resource ...

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 ...

A Reference Broth Microdilution Method for Dalbavancin In Vitro Susceptibility Testing of Bacteria that Grow Aerobically

Antimicrobial susceptibility testing (AST) is performed to assess the in vitro activity of antimicrobial agents against various bacteria. The AST results, which are expressed as minimum inhibitory concentrations (MICs) are used in research for antimicrobial development and monitoring of resistance development and in the clinical setting for antimicrobial therapy guidance. Dalbavancin is a semi-synthetic lipoglycopeptide antimicrobial agent that was approved in May 2014 by the Food and Drug Administration (FDA) for the treatment of acute bacterial skin and skin structure infections caused by Gram-positive organisms. The advantage of dalbavancin over current anti-staphylococcal therapies is its long half-life, which allows for once-weekly dosing. Dalbavancin has activity against Staphylococcus aureus (including both methicillin-susceptible S. aureus [MSSA] and methicillin-resistant S. aureus [MRSA]), coagulase-negative staphylococci, Streptococcus pneumoniae, Streptococcus anginosus group, &#946;-hemolytic streptococci and vancomycin susceptible enterococci. Similar to other recent lipoglycopeptide agents, optimization of CLSI and ISO broth susceptibility test methods includes the use of dimethyl sulfoxide (DMSO) as a solvent when preparing stock solutions and polysorbate 80 (P80) to alleviate adherence of the agent to plastic. Prior to the clinical studies and during the initial development of dalbavancin, susceptibility studies were not performed with the use of P-80 and MIC results tended to be 2-4 fold higher and similarly higher MIC results were obtained with the agar dilution susceptibility method. Dalbavancin was first included in CLSI broth microdilution methodology tables in 2005 and amended in 2006 to clarify use of DMSO and P-80. The broth microdilution (BMD) procedure shown here is specific to dalbavancin and is in accordance with the CLSI and ISO methods, with step-by-step detail and focus on the critical steps added for clarity.

A Reference Broth Microdilution Method for Dalbavancin In Vitro Susceptibility Testing of Bacteria that Grow Aerobically

Here, we present a protocol for determination of dalbavancin susceptibility of clinically relevant Gram-positive bacteria using a broth microdilution reference methodology.

Antimicrobial susceptibility testing (AST) is performed to assess the in vitro activity of antimicrobial agents against various bacteria. The AST results, ...

A 3D Human Lung Tissue Model for Functional Studies on Mycobacterium tuberculosis Infection

Tuberculosis (TB) still holds a major threat to the health of people worldwide, and there is a need for cost-efficient but reliable models to help us understand the disease mechanisms and advance the discoveries of new treatment options. In vitro cell cultures of monolayers or co-cultures lack the three-dimensional (3D) environment and tissue responses. Herein, we describe an innovative in vitro model of a human lung tissue, which holds promise to be an effective tool for studying the complex events that occur during infection with Mycobacterium tuberculosis (M. tuberculosis). The 3D tissue model consists of tissue-specific epithelial cells and fibroblasts, which are cultured in a matrix of collagen on top of a porous membrane. Upon air exposure, the epithelial cells stratify and secrete mucus at the apical side. By introducing human primary macrophages infected with M. tuberculosis to the tissue model, we have shown that immune cells migrate into the infected-tissue and form early stages of TB granuloma. These structures recapitulate the distinct feature of human TB, the granuloma, which is fundamentally different or not commonly observed in widely used experimental animal models. This organotypic culture method enables the 3D visualization and robust quantitative analysis that provides pivotal information on spatial and temporal features of host cell-pathogen interactions. Taken together, the lung tissue model provides a physiologically relevant tissue micro-environment for studies on TB. Thus, the lung tissue model has potential implications for both basic mechanistic and applied studies. Importantly, the model allows addition or manipulation of individual cell types, which thereby widens its use for modelling a variety of infectious diseases that affect the lungs.

A 3D Human Lung Tissue Model for Functional Studies on Mycobacterium tuberculosis Infection

Human tuberculosis infection is a complex process, which is difficult to model in vitro. Here we describe a novel 3D human lung tissue model that recapitulates the dynamics that occur during infection, including the migration of immune cells and early granuloma formation in a physiological environment.

Tuberculosis (TB) still holds a major threat to the health of people worldwide, and there is a need for cost-efficient but reliable models to help us ...

System for Efficacy and Cytotoxicity Screening of Inhibitors Targeting Intracellular Mycobacterium tuberculosis

Mycobacterium tuberculosis, the causative agent of tuberculosis (TB), is a leading cause of morbidity and mortality worldwide. With the increased spread of multi drug-resistant TB (MDR-TB), there is a real urgency to develop new therapeutic strategies against M. tuberculosis infections. Traditionally, compounds are evaluated based on their antibacterial activity under in vitro growth conditions in broth; however, results are often misleading for intracellular pathogens like M. tuberculosis since in-broth phenotypic screening conditions are significantly different from the actual disease conditions within the human body. Screening for inhibitors that work inside macrophages has been traditionally difficult due to the complexity, variability in infection, and slow replication rate of M. tuberculosis. In this study, we report a new approach to rapidly assess the effectiveness of compounds on the viability of M. tuberculosis in a macrophage infection model. Using a combination of a cytotoxicity assay and an in-broth M. tuberculosis viability assay, we were able to create a screening system that generates a comprehensive analysis of compounds of interest. This system is capable of producing quantitative data at a low cost that is within reach of most labs and yet is highly scalable to fit large industrial settings.

System for Efficacy and Cytotoxicity Screening of Inhibitors Targeting Intracellular Mycobacterium tuberculosis

We have developed a modular high-throughput screening system for discovering novel compounds against Mycobacterium tuberculosis, targeting intracellular and in-broth growing bacteria as well as cytotoxicity against the mammalian host cell.

Mycobacterium tuberculosis, the causative agent of tuberculosis (TB), is a leading cause of morbidity and mortality worldwide. With the increased spread ...

Analysis of 18FDG PET/CT Imaging as a Tool for Studying Mycobacterium tuberculosis Infection and Treatment in Non-human Primates

Mycobacterium tuberculosis remains the number one infectious agent in the world today. With the emergence of antibiotic resistant strains, new clinically relevant methods are needed that evaluate the disease process and screen for potential antibiotic and vaccine treatments. Positron Emission Tomography/Computed Tomography (PET/CT) has been established as a valuable tool for studying a number of afflictions such as cancer, Alzheimer&#39;s disease, and inflammation/infection. Outlined here are a number of strategies that have been employed to evaluate PET/CT images in cynomolgus macaques that are infected intrabronchially with low doses of M. tuberculosis. Through evaluation of lesion size on CT and uptake of 18F-fluorodeoxyglucose (FDG) in lesions and lymph nodes in PET images, these described methods show that PET/CT imaging can predict future development of active versus latent disease and the propensity for reactivation from a latent state of infection. Additionally, by analyzing the overall level of lung inflammation, these methods determine antibiotic efficacy of drugs against M. tuberculosis in the most clinically relevant existing animal model. These image analysis methods are some of the most powerful tools in the arsenal against this disease as not only can they evaluate a number of characteristics of infection and drug treatment, but they are also directly translatable to a clinical setting for use in human studies.

Analysis of 18FDG PET/CT Imaging as a Tool for Studying Mycobacterium tuberculosis Infection and Treatment in Non-human Primates

Here, we present a protocol to describe the analysis of 18F-FDG PET/CT imaging in non-human primates that have been infected with M. tuberculosis to study disease process, drug treatment, and disease reactivation.

Mycobacterium tuberculosis remains the number one infectious agent in the world today. With the emergence of antibiotic resistant strains, new clinically ...

Imaging Mycobacterium tuberculosis in Mice with Reporter Enzyme Fluorescence

Reporter enzyme fluorescence (REF) utilizes substrates that are specific for enzymes present in target organisms of interest for imaging or detection by fluorescence or bioluminescence. We utilize BlaC, an enzyme expressed constitutively by all M. tuberculosis strains. REF allows rapid quantification of bacteria in lungs of infected mice. The same group of mice can be imaged at many time points, greatly reducing costs, enumerating bacteria more quickly, allowing novel observations in host-pathogen interactions, and increasing statistical power, since more animals per group are readily maintained. REF is extremely sensitive due to the catalytic nature of the BlaC enzymatic reporter and specific due to the custom flourescence resonance energy transfer (FRET) or fluorogenic substrates used. REF does not require recombinant strains, ensuring normal host-pathogen interactions. We describe the imaging of M. tuberculosis infection using a FRET substrate with maximal emission at 800 nm. The wavelength of the substrate allows sensitive deep tissue imaging in mammals. We will outline aerosol infection of mice with M. tuberculosis, anesthesia of mice, administration of the REF substrate, and optical imaging. This method has been successfully applied to evaluating host-pathogen interactions and efficacy of antibiotics targeting M. tuberculosis.

Imaging Mycobacterium tuberculosis in Mice with Reporter Enzyme Fluorescence

We describe the optical imaging of mice infected with Mycobacterium tuberculosis (M. tuberculosis) using reporter enzyme fluorescence (REF). This protocol facilitates the sensitive and specific detection of M. tuberculosis in pre-clinical animal models for pathogenesis, therapeutics and vaccine research.

Reporter enzyme fluorescence (REF) utilizes substrates that are specific for enzymes present in target organisms of interest for imaging or detection by ...