survey results & analysis for - mrl · this report contains a detailed statistical analysis of...

Survey Results & analysis for

Global survey of models of preclinical TB drug testing

Executive Summary

This report contains a detailed statistical analysis of the results to the survey titled Global survey of models of preclinical TB drug testing . The results analysis includes answers from all respondents who took the survey (30 completed responses were received to the survey up to this time)

Draft survey: updated April 18, 2008Please address concerns or questions to Mary Ann De [email protected]

Type of institution

May we contact you if we have specific questions regarding this survey?

Are you willing to share your laboratory protocols with us? This may involve phone calls, electronic mail etc.

Are you willing to allow us to visit your laboratory to discuss your protocols?

Do you perform in vivo (animal model) testing exclusively?

Which type of in vitro assay(s) have been used in your lab?

Regarding MICs, which strains do you use for detection of anti-TB activity?

We do not perform MIC testing

W-Beijing; CDC1551; H37Rv ATCC 25618

MAC STRAINS and fast growing mycobacteri

Mono, poly and multi drug resistant stra

Epidemiological representative strains

Drug resistant strains

H37Rv luxAB

Other responses: regarding strains

No

N/A

yes,

No.

In some cases

yes

Yes

Yes. Strain Ra has been compared to strain Rv

and difference can be found in sensitivity to new compounds. One possibitlity

is that the deletions in Ra could code for a protein that activates a drug, and if deleted Ra would be resistant. Another alternative is the example of the erm

methylase. If deleted in Ra the strain would now be sensitive to macrolides.

Yes, BCG is more susceptible to nitro-imidazoles

versus our standard H37Rv

no

We use virulent strain of MTB (H37Rv) for standard MIC testing.When

BCG is used for large scale screening we observe more than 90% overlap with H37Rv.

No.

The correlation is good between BCG and TB. Not so with Smeg

and TB.

Differences between H37Rv and H37Ra

Any significant differences in activity between the surrogate strain and virulent M. tuberculosis?

ATCC, CSU and Pasteur Institute

M. smegmatis

mc2155 from Dr. Jacobs' lab

D.Y. Young, ex Imperial College London

N/A

M. bovis

BCG-

Pasteur and above mentioned clinical strains

a. H37Rv ATCC27294 b. serious drug resistant clinical isolates dependent on the natural product sources

H37Rv lab strain, and BCG-Russia

H37Rv is from ATCC Other clinical strains used include HN878 and

CDC1551

mc2155 (JAcobs

strain)

Strains were collected from the local hospitals

Strains obtained from Tuberculosis Research Center Chennai, INDIA

Ra, original source unknown.

Erdman from the ATCC H37Rv from Trudeau Institute

ATCC H37Rv

H37Rv from ATCC (# 27294) BCG from ATCC (#35734)

ATCC.

ATCC TRC, Chennai -

clinical isolates

H37Rv and Erdman: ATCC Clinical SNP cluster representatives: David Alland, UMDNJ H37Rvlux AB: constructed in house Moxifloxacin

resistant: constructed in house

Source or history of the strain used.

No

No

No

N/A

-

Spontaneous mutants -

well characterized clinical isolates

a. H37Rv -

No b. clinical isolates -

Yes, they are naturally drug resistant strains

No

No

Hygro

MDR, XDR, Monoresitant

strains

NOT APPLICABLE

No

Not for standard MIC testing. Lead compounds are tested against appropriate mono, poly and/or multi resistant strains (clinical strains or strains derived in vitro after drug selection)

no

No

No.

Except for the clinical isolates, the standard lab strains are not resistant or marked.

Various strains with different resistant patterns

H37Rv lux

AB: kanamycin

marker monoresistant

strains to rifampin, kanamycin, streptomycin, etc. are isogenic

to H37Rv

Is your strain naturally drug resistant or marked with an antibiotic selection marker?Please elaborate on the nature of the drug resistance.

Regarding the culture medium used to grow the mycobacterial strain for inoculation into the MIC assays, please elaborate on medium used (including pH, carbon source, method of sterilization, culture time, culture vessels, growth phase when harvested and supplements added)for both inoculum preparation and the anti-TB assay. If this is different for the inoculum preparation and the anti-TB testing, indicate why it is different.

Std Middlebrook 7H9 OAD

Glycine-alanine salt (GAS) medium supplemented with Tween 80, pH 6.6 Bacto-Casitone, citric acid, L-alanine, glycerol and Tween 80 are believed to be the major carbon source GAS was sterilized by autoclave at 121C for 30 min M. smegmatis inoculum was cultured in a test tube for 2 days in GAS supplemented with Tween 80 For MIC determination, M. smegmatis are cultured in 96-well plate overnight to log phase

N/A

- Culture on 7H11 or 7H10 - Also 7H11 pH (with HCl prior to autoclaving) 5.9 for pza

Middlebrook 7H9 broth for both inoculation and MIC assay -prepare the media according to the manufacturers instructions, filter to sterilize instead of autoclave, culture in erlenmayer plask upto A600 0.5 (log phase)

We use 7H9, pH6.6, glycerol as carbon source, autoclave sterilization, tween and ADC are added after autoclaving, culture time varies with experiment, culture vessels typically polystyrene bottles on a roller, typically log phase when harvested.

Usually we use 7H9 medium supplemented with ADC + glycerol + 0.05% Tween 80. Either filter sterilized or autoclaved according to standard protocol. Sometimes (especially if we're looking at Fe-dependence) we use GAST +/- Fe whici is filter-sterilized: GAST: Low-iron GAST was normal pH 6.6 glycerol-alanine-salts (GAS) medium [per liter: 0.3 g of Bacto Casitone (Difco), 4.0 g of dibasic potassium phosphate, 2.0 g of citric acid, 1.0 g of L-alanine, 1.2 g of magnesium chloride hexahydrate, 0.6 g of potassium sulfate, 2.0 g of ammonium chloride, 1.80 ml of 10 sodium hydroxide, and 10.0 ml of glycerol, Tween 80 was added to 0.05%. GAST/Fe As above with 0.05 g of ferric ammonium citrate

Plates: Mueller-Hinton, hygro

mainly 7H9broth containing tween 80 and OADC

Std Middlebrook 7H9 OAD

Glycine-alanine salt (GAS) medium supplemented with Tween 80, pH 6.6 Bacto-Casitone, citric acid, L-alanine, glycerol and Tween 80 are believed to be the major carbon source GAS was sterilized by autoclave at 121C for 30 min M. smegmatis inoculum was cultured in a test tube for 2 days in GAS supplemented with Tween 80 For MIC determination, M. smegmatis are cultured in 96-well plate overnight to log phase

N/A

- Culture on 7H11 or 7H10 - Also 7H11 pH (with HCl prior to autoclaving) 5.9 for pza

Middlebrook 7H9 broth for both inoculation and MIC assay -prepare the media according to the manufacturers instructions, filter to sterilize instead of autoclave, culture in erlenmayer plask upto A600 0.5 (log phase)

We use 7H9, pH6.6, glycerol as carbon source, autoclave sterilization, tween and ADC are added after autoclaving, culture time varies with experiment, culture vessels typically polystyrene bottles on a roller, typically log phase when harvested.

Usually we use 7H9 medium supplemented with ADC + glycerol + 0.05% Tween 80. Either filter sterilized or autoclaved according to standard protocol. Sometimes (especially if we're looking at Fe-dependence) we use GAST +/- Fe whici is filter-sterilized: GAST: Low-iron GAST was normal pH 6.6 glycerol-alanine-salts (GAS) medium [per liter: 0.3 g of Bacto Casitone (Difco), 4.0 g of dibasic potassium phosphate, 2.0 g of citric acid, 1.0 g of L-alanine, 1.2 g of magnesium chloride hexahydrate, 0.6 g of potassium sulfate, 2.0 g of ammonium chloride, 1.80 ml of 10 sodium hydroxide, and 10.0 ml of glycerol, Tween 80 was added to 0.05%. GAST/Fe As above with 0.05 g of ferric ammonium citrate

Plates: Mueller-Hinton, hygro

mainly 7H9broth containing tween 80 and OADC

Part 1

We are using commercially available Middlebrook 7H 11 agar supplemente with OADC for anti-TB assay. For inoculam we are using 7H9 broth.

One area in which we noted an issue is the testing of betalactams in the presence of betalactamase inhibitors. When using cells from a frozen stock it appears that cells may not start active growth (where they would be sensitive) immediately and thus the short half life of the compounds renders MIC very much higher than expected.

sauton's medium, grown as biofilms, grown for 5weeks, exposed to drugs, solublized and plated.

GAS media (glycerol alanine salts pH6.6 and/or 7H9-OADC (pH 6.6) autoclaved and OADC filter sterilized (0.2 uM). Culturing done in erlenmeyers and assasy in 96 well U-bottomed plates.

Medium: 7H9-ADS (albumin 5%, glucose 2% and NaCl 0.8% final concentration) + glycerol 0.5%+ 0.05% Tween 80 pH: 6.6 Method of sterilization: filtration through 0.22µM membrane filter Culture time: 5 days Culture vessel: microtiter plates (96-well-plates) Phase of growth: Exponentially growing cells that were concentrated by centrifugation (at an OD600 of 1). and stored at -80oC prior to use. Dilution at 0.01 the day of the experiment in fresh medium. Assay volume: 200�l. Turbidity is recorded after 5 days of culture

7H11 agar (for M. tuberculosis): method of sterilization: autocalave; pH: 6.8; culture time: 3-4 weeks, supplements added: 10% OADC. Lowenstein-Jensen (for M. tuberculosis): method of sterilization: autocalave; culture time: 4-6 weeks, supplements added: eggs. 7H9 broth (for M. tuberculosis): method of sterilization: autocalave; pH: 7; culture time: 7-10 days, supplements added: 10% OADC. Mueller Hinton Broth (for M. smegmatis): method of sterilization: autocalave; pH: 7; culture time: 2 days, supplements added: None.

Middlebrook 7H9 ADC; autoclaving; 7day RBMA assay in 96 or 384 well format. Starting inoculum from frozen seedlot which is freshly thawed and used. Starting cell number ~5.5 x 10^5/ml. Seedlot prepared from log phase cultures grown in roller bottles, which were started from 3-5 colonies pooled from a 7H11 agar plate.

7H12 broth at various pHs

7H12 medium, pH 6.8, carbon source:palmitic acid, sterilization: membrane filtration, culture time: 7 days, culture vessels:erlenmeyer flask, growth phase: late log, Supplements added: bovine serum albumin and palmitic acid. Medium for inoculum contains 0.05% Tween 80; medium for drug testing does not contain this supplement.

We are using commercially available Middlebrook 7H 11 agar supplemente with OADC for anti-TB assay. For inoculam we are using 7H9 broth.

One area in which we noted an issue is the testing of betalactams in the presence of betalactamase inhibitors. When using cells from a frozen stock it appears that cells may not start active growth (where they would be sensitive) immediately and thus the short half life of the compounds renders MIC very much higher than expected.

sauton's medium, grown as biofilms, grown for 5weeks, exposed to drugs, solublized and plated.

GAS media (glycerol alanine salts pH6.6 and/or 7H9-OADC (pH 6.6) autoclaved and OADC filter sterilized (0.2 uM). Culturing done in erlenmeyers and assasy in 96 well U-bottomed plates.

Medium: 7H9-ADS (albumin 5%, glucose 2% and NaCl 0.8% final concentration) + glycerol 0.5%+ 0.05% Tween 80 pH: 6.6 Method of sterilization: filtration through 0.22µM membrane filter Culture time: 5 days Culture vessel: microtiter plates (96-well-plates) Phase of growth: Exponentially growing cells that were concentrated by centrifugation (at an OD600 of 1). and stored at -80oC prior to use. Dilution at 0.01 the day of the experiment in fresh medium. Assay volume: 200�l. Turbidity is recorded after 5 days of culture

7H11 agar (for M. tuberculosis): method of sterilization: autocalave; pH: 6.8; culture time: 3-4 weeks, supplements added: 10% OADC. Lowenstein-Jensen (for M. tuberculosis): method of sterilization: autocalave; culture time: 4-6 weeks, supplements added: eggs. 7H9 broth (for M. tuberculosis): method of sterilization: autocalave; pH: 7; culture time: 7-10 days, supplements added: 10% OADC. Mueller Hinton Broth (for M. smegmatis): method of sterilization: autocalave; pH: 7; culture time: 2 days, supplements added: None.

Middlebrook 7H9 ADC; autoclaving; 7day RBMA assay in 96 or 384 well format. Starting inoculum from frozen seedlot which is freshly thawed and used. Starting cell number ~5.5 x 10^5/ml. Seedlot prepared from log phase cultures grown in roller bottles, which were started from 3-5 colonies pooled from a 7H11 agar plate.

7H12 broth at various pHs

7H12 medium, pH 6.8, carbon source:palmitic acid, sterilization: membrane filtration, culture time: 7 days, culture vessels:erlenmeyer flask, growth phase: late log, Supplements added: bovine serum albumin and palmitic acid. Medium for inoculum contains 0.05% Tween 80; medium for drug testing does not contain this supplement.

Part 2

Regarding the culture medium used to grow the mycobacterial strain for inoculation into the MIC assays, please elaborate on medium used (including pH, carbon source, method of sterilization, culture time, culture vessels, growth phase when harvested and supplements added)for both inoculum preparation and the anti-TB assay. If this is different for the inoculum preparation and the anti-TB testing, indicate why it is different.

- Rat cell lines in experimental development - control cell lines THP1

n/a

J774 cells from ATCC. We use H37Rv. There can be quite big differences in MIC between in vitro and macrophage determinations. Nadyes, its clear that some compounds have no activity against Mtb in macrophages whereas they are potent against MTb in vitro.

Don't use

I don't do macrophage cells in vitro assays

We are using J774A.1 & THP-1 cells for intracellular assays.Strain used :Mtuberculosis H37Rv. Yes we do see the difference in regard to in vitro MIC and ex vivo activity,this effect is related to class of compounds under screening.

NA

no

THP-1

We use bone-marrow derived macrophages, freshly extracted from the tibia of C57Bl/6 mice. Yes, we have observed discrepancies when comparing the macrophage data versus the in vivo data testing a wide variety of drug classes we observed the following: three out of 12 in vivo active compounds did not show activity in the macrophage model. Reason is not clear and has not be pursued, could be due to lack of solubility.

NA

THP-1 and bone-marrow-derived macrophages. (ATCC # TIB202) Predictability of the assay is questionable (streptomycin, kanamycin, amikacin are poorly active in the assay)

We don't use macrophages for in vitro assays for M. tuberculosis.

J774 (ATCC)and Murine BMDM (primary cells) H37Rv (ATCC 27294) Many compounds that have potent MICs dont have good activ ity in the macrophage assays - not surprising due to intracellular PK influences. Likewise, those that are good in macrophage are not necessarily good in vivo. Thus the macrophage assay in inclusive for in vivo testing and not used as exclusion criteria.

Human blood cells derived macrophages

J774 with M. tuberculosis Erdman, source for both is ATCC

- Rat cell lines in experimental development - control cell lines THP1

n/a

J774 cells from ATCC. We use H37Rv. There can be quite big differences in MIC between in vitro and macrophage determinations. Nadyes, its clear that some compounds have no activity against Mtb in macrophages whereas they are potent against MTb in vitro.

Don't use

I don't do macrophage cells in vitro assays

We are using J774A.1 & THP-1 cells for intracellular assays.Strain used :Mtuberculosis H37Rv. Yes we do see the difference in regard to in vitro MIC and ex vivo activity,this effect is related to class of compounds under screening.

NA

no

THP-1

We use bone-marrow derived macrophages, freshly extracted from the tibia of C57Bl/6 mice. Yes, we have observed discrepancies when comparing the macrophage data versus the in vivo data testing a wide variety of drug classes we observed the following: three out of 12 in vivo active compounds did not show activity in the macrophage model. Reason is not clear and has not be pursued, could be due to lack of solubility.

NA

THP-1 and bone-marrow-derived macrophages. (ATCC # TIB202) Predictability of the assay is questionable (streptomycin, kanamycin, amikacin are poorly active in the assay)

We don't use macrophages for in vitro assays for M. tuberculosis.

J774 (ATCC)and Murine BMDM (primary cells) H37Rv (ATCC 27294) Many compounds that have potent MICs dont have good activ ity in the macrophage assays - not surprising due to intracellular PK influences. Likewise, those that are good in macrophage are not necessarily good in vivo. Thus the macrophage assay in inclusive for in vivo testing and not used as exclusion criteria.

Human blood cells derived macrophages

J774 with M. tuberculosis Erdman, source for both is ATCC

Macrophage assays used:

10mg/ml

In the mM

range

10mM

10 mM

Depends on solubility

50-100mg/ml 100mg/ml favored

10 mg/ml

n/a

Depends on solubility. We try 50mM in DMSO

0.1-1.0 mM

I don't understand the question

10mg/ml

from 50microg/ml to lower concentration

10 mg/ml

I don't use new compound

100X

10 mg/ml or 10 uM

5 mM

in 90% DMSO (Di Methyl Sulfoxide)

2 mM

for the stocks and 25 microM

in the plate.

128 ug/ml

Variable

12.8 mM

or 12.8 or 10 mg/ml

What is your default stock concentration for new compounds?

usually fresh

Frozen stocks

Frozen stocks.

frozen stocks

fresh

-

Strongly favor stocks

frozen stocks

We prepare fridge stocks from frozen stocks

Frozen unless there are indications of instability

Both

I don't use frozen stock

No. We do make stock of the compounds and working stocks were used for further assays

Fresh each days

Both depending on stability data.

Prepare fresh for every experiment

frozen

We prepare stock solutions once a week.

frozen stocks stored at -80oC

Compounds are stored at 4oC prior to use, both fresh and old stock used. Integrity of the compounds is verified by LC-MS on a regular basis

We use room temperature stocks.

Frozen aliquots

frozen stocks but not stored for longer than 6 months

Do you prepare all compounds fresh each day or do you use frozen stocks?

n/a

Re-use the same frozen stocks

Re-use -

provided from assay screening group in 96-well plates.

re-use

-

aliquotes

make aliquots

We re-use frozen stocks up to 3-4 times

Same stock unless there are indications of instability. Same stock is not thawed more than 10 times

Re-use

Yes aliquots are made

NA

Make aliquotes.

re-use

aliquotes, cmpds

are never refrozen.

Working stocks aliquots are preapred

and my be used more than once (multiple freeze-thaws)

Reuse the same frozen stock unless visible precipitation observed.The

activity of a compound is always confirmed with a secong, independently prepared stock.

We make aliquots.

Frozen stocks are not used

Never re-use

If in microplates

we re-use; if in tubes, we prepare aliquots

If using frozen stocks of compounds do you make aliquotsand use each one once or do you re-use the same frozen stocks?

no

Yes

no (we perform enyzmatic

assays)

no

- always

we filter running solutions before use

yes

Aqeous

-

yes. DMSO -

no

NO

Stocks are made under sterile conditions.

NO

Only on aoccasion, e.g. betalactams

in aqueous solution and filter sterilized.

Yes for aqueous. No for orgainc

no

We do filter sterilize stocks to be used for in vitro studies.

no, they are made up in DMSO (dimethylsulfoxide)

We dissolve the compounds in 100% DMSO and then make up the volume with 10% filter sterilized water. We do not filter sterilize the 90% DMSO stocks

We don't filter stocks.

No

Filter water-soliuable

compounds

yes for aqueous, not for organic

Do you filter sterilize stocks made in aqueous and/or organic solvents?

DMSO

DMSO

DMSO used for all compounds.

default DMSO

DMSO, ethanol, acetonitrile

DMSO

DMSO

DMSO (most of our compounds), sometimes ethanol

DMSO

I don't use solvents

DMSO

DMSO as a general solvent for most compounds

Methanol for rifampicin

DMSO Ethanol

DMSO

ethanol, DMSO, PEG, cyclodextrin

micelle (CM-2), carboxymethylcellulose

DMSO (dimethylsulfoxide) for nearly all our compounds we test

All compounds are dissolved in 90% DMSO to make the compound stocks. We do not use any other aqueous solvents.

We use DMSO for all the compounds which are not soluble in water.

DMSO

Methnol, DMSO

DMSO is default solvent

Other than water, what other solvents do you use?Please specify alternative solvents and for which compounds they are used

spectrophotometric

assay 3% (DMSO up to 4.5% does not affect the enzyme activity. Higher concentrations were not tested.) radiometric assay 3%

1% We tested DMSO levels, and found 5% to be the upper limit.

Usually not above 1% although for some really inactives

we have had to use 4%

we use DMSO to dissolve the compound and for dilution we use water.

1% and tested for each new assay.

Don't use DMSO

Highest final DMSO concentration used is

The highest final concentration of DMSO in the assay plate is 1.25%. The inhibitory effect of different concentrations of DMSO against M. tuberculosis were tested many times.

Yes, see our publications

1 percent Have previously determined that M. tuberculosis can tolerate up to 1.5% DMSO

What is the highest final concentration of DMSO in the assay plate that you use?Did you do any tests to determine the highest concentration of DMSO used in the plate without affecting the growth/viability of M. tuberculosis?

Inoculum is standardized by adjustment of:

650 nm

we measure purified enzyme activity

McFarland

dependent on the test methods

OD 600

McFarland using Densimat

600 nm

Frozen seedlot

with fixed inoculum

Additional comments on inoculum

What is the target CFU per ml at the beginning of the incubation?

10-5

OD650=0.7

10e5 cfu/ml

N/A

about 2x 10 to the 5

2,250,000

10 000 cells per well

10,000

10e5

10000

10to the power 7

10exp7

5 x 10 7

5 exp 4 CFUper

well

1 to 5 x 10^5

2x10e5 CFU/ml

1 X 106 cells/mL

100000 CFU

5.5 x 10^5 cfu/ml

10,000 CFU/ml

1 x 10E5 cfu/ml

What is the target CFU per ml at the beginning of the incubation?*

*Note: this question may not have been clear

Assay format includes

for radiometric assay; 25 ul, in Eppis

CLSI Agar Dilution Method

20ml, metal closure

grown in 25 ml medium in 250 ml bottle

16mm tube, screw cap

15ml conical vial, 2.5ml broth, loosecap

Tube MIC:14 mL

snap cap,final

volume 1ml

tubes of 10 ml, 10 cm height, 1cm diameter

Bactec

460 12B vials

Additional comments on in vitro screening

Total assay volume in each well or tube

200ul

0.2 ml

100ul

spectro - 200 ul, radiometric - 25 ul

200 ul/well

150ul

100uL

0.5-1.0 ml.

at least 2.5ml

200ul

10ml

100 microliteres

25 ml

5 ml

100

2.5 ml

200 ul

96-well plate: 200 µL, tube : 1ml

1 ml

200 ul or 50 ul

4.0 ml

200 ul

200ul

0.2 ml

100ul

spectro - 200 ul, radiometric - 25 ul

200 ul/well

150ul

100uL

0.5-1.0 ml.

at least 2.5ml

200ul

10ml

100 microliteres

25 ml

5 ml

100

2.5 ml

200 ul

96-well plate: 200 µL, tube : 1ml

1 ml

200 ul or 50 ul

4.0 ml

200 ul

Are plates or tubes sealed? If so, how is this done?

no

Yes. Mylar plate sealers.

Lids on but not sealed, enclosed in sealed box to incubate.

no

Seal with breathable sealing tape (sheet)

with lid, not airtight seal

lids. Then closed in ziplock bags from Nunc

No

no

Yes, with parafilm

No

Yes

Yes, tightening the cap

screw cap

Plates are not sealed. We don't use the outer wells for assay and fill with water to limit evaporation.

no, cap is fitted loosely

sealed in zip lock bag for incubation

Plates are not sealed

No

Yes. With plate sealer.

Plates are in CO2 permeable bags

no, but covered with lid

no

Yes. Mylar plate sealers.

Lids on but not sealed, enclosed in sealed box to incubate.

no

Seal with breathable sealing tape (sheet)

with lid, not airtight seal

lids. Then closed in ziplock bags from Nunc

No

no

Yes, with parafilm

No

Yes

Yes, tightening the cap

screw cap

Plates are not sealed. We don't use the outer wells for assay and fill with water to limit evaporation.

no, cap is fitted loosely

sealed in zip lock bag for incubation

Plates are not sealed

No

Yes. With plate sealer.

Plates are in CO2 permeable bags

no, but covered with lid

What is the incubation time and incubation temperature?

7 days 37C

6 hrs at 37C

9 days, 37 C.

spectro - 45 min, radiometric - 30 min, 30oC

37 degrees centigrade, vary dependent on assay methods (7days for alamar blue assay)

37 C, 1 wk

7-14 days

O/N, 37

7 & 14 days for broth; 21days for plates;at 37 degrees Celsius

for broth it s 6-7 days at 37oC

37C, 28days

5 days, 37C

upto 7 dyas of exposure at 37 C

37C 21 days

This depends on the particular strain used. For H37Rv plates are incubated 10 days.

14 days at 37C

10 days at 37oC

5 days, 37oC, incubation under humidified conditions. Turbidity is recorded at the end of the experiment

7 days at 37°C for for M. tuberculosis, 2 days at 35°C for M. smegmatis

3 weeks for agar plates and 2 weeks for tests in 7H12 broth and with a macrophage culture

7 days, then 1 days after redox dye

7 days 37C

6 hrs at 37C

9 days, 37 C.

spectro - 45 min, radiometric - 30 min, 30oC

37 degrees centigrade, vary dependent on assay methods (7days for alamar blue assay)

37 C, 1 wk

7-14 days

O/N, 37

7 & 14 days for broth; 21days for plates;at 37 degrees Celsius

for broth it s 6-7 days at 37oC

37C, 28days

5 days, 37C

upto 7 dyas of exposure at 37 C

37C 21 days

This depends on the particular strain used. For H37Rv plates are incubated 10 days.

14 days at 37C

10 days at 37oC

5 days, 37oC, incubation under humidified conditions. Turbidity is recorded at the end of the experiment

7 days at 37°C for for M. tuberculosis, 2 days at 35°C for M. smegmatis

3 weeks for agar plates and 2 weeks for tests in 7H12 broth and with a macrophage culture

7 days, then 1 days after redox dye

radiometric - chloroform:metanol 2:1

NRA

visual density reads on scanner

Tween 80, 1% v/v final concentration

radiometric - chloroform:metanol 2:1

NRA

visual density reads on scanner

Tween 80, 1% v/v final concentration

Additional comments

Which post-incubation reagents are added?

Incubation time (hours) between reagent addition and reading.

n/a

30 min

5 hours.

N/A; spectro - kinetic measurment,

at least 16 hours usually 24 hours for alamar blue assay method

5 min

2h

16-24 hrs.

24HRS

NA

24 hours

n/a

Upto 7 days

daily

24 hours

25 hours for alamar blue

N.A

2-4 hours for M. smegmatis and 24 hours for M. tuberculosis

RBMA assays - 1 day

variable depending on the model used

24 hours

n/a

30 min

5 hours.

N/A; spectro - kinetic measurment,

at least 16 hours usually 24 hours for alamar blue assay method

5 min

2h

16-24 hrs.

24HRS

NA

24 hours

n/a

Upto 7 days

daily

24 hours

25 hours for alamar blue

N.A

2-4 hours for M. smegmatis and 24 hours for M. tuberculosis

RBMA assays - 1 day

variable depending on the model used

24 hours

Is the reading done directly in the culture plate or tube or is an aliquot removed for reading?

Endpoint measured

OD570nm-OD600nm

emission 590nm, excitation 530nm.

radio -

quantification of react. product

15s for luminescence per well

1 sec

580 nm

scanned on scanner for visual density

575/610 nm ratio

Additional comments on readout

for luciferase measurement, take 100 ul

50ul

One mL

500mcl aliquots removed for MBC testing

for luciferase measurement, take 100 ul

50ul

One mL

500mcl aliquots removed for MBC testing

If using a non-CFU readout, what is the signal:background ratioof drug-free controls?

optical scan

Drug free control=0.45 to 0.47; Medium alone=-0.15

spectro - 10; radio - 40

less than 1.5

Don't know, qualitative assays only

NA

NA

n/a

NA

Drug free OD600: 0.20 – 0.30,Media blank: 0.04,Signal:Background = 5 - 6 fold

Background ratio: 20 for M. smegmatis and 10 for M. tuberculosis

5 (Spectramax)

Drug-free control

10-20

optical scan

Drug free control=0.45 to 0.47; Medium alone=-0.15

spectro - 10; radio - 40

less than 1.5

Don't know, qualitative assays only

NA

NA

n/a

NA

Drug free OD600: 0.20 – 0.30,Media blank: 0.04,Signal:Background = 5 - 6 fold

Background ratio: 20 for M. smegmatis and 10 for M. tuberculosis

5 (Spectramax)

Drug-free control

10-20

What variables, if any, have you found to affect this ratio?

time of incubation

Time of growth and incubation with alamar blue reagent.

Spectro - purity of the enzyme preparation, way of preparation of the substrates, source (vendor) of the substrates Radio - way of preparation of the substrates

none

Innoculum, temperature

NA

NA

n/a

none

Evaporation from plate Precipitation of the compounds (might give false negative, incomplete IC50 curve)

None.

Tween 80 Contamination Precipitation of compounds coloured compounds

The question is not clear

Inoculum denisty, edge effect

time of incubation

Time of growth and incubation with alamar blue reagent.

Spectro - purity of the enzyme preparation, way of preparation of the substrates, source (vendor) of the substrates Radio - way of preparation of the substrates

none

Innoculum, temperature

NA

NA

n/a

none

Evaporation from plate Precipitation of the compounds (might give false negative, incomplete IC50 curve)

None.

Tween 80 Contamination Precipitation of compounds coloured compounds

The question is not clear

Inoculum denisty, edge effect

If doing fixed concentrations what concentration do you use and what is your criteria for a "hit"?

depends on project usually 6ug/ml

30 uM Hit: spectro 30% inhibition radio 50% inhibition

vary dependent on test materials

n/a

10-fold difference between compound and DMSO control

0.1, 1, 10 less than 1 is a hit

MIC below 2ug/ml

NA

10 micrograms per ml giving 90% or greater inhibition

do not screen in TB

For new cmpds we start at 30 ug/ml as the highest concentration. definition of a hit depends on the project, usually MIC data is used for SAR purposes. Acives in the range of 1-2 ug/ml are certainly followed up.

hit

No. Serial dilution for all compounds screened

We usually test the following concentrations : 25 microM up to 0.05 microM using 2-fold dilution process. A MIC of < 0.12 mg/l is considered as a hit.

16 ug/ml

No such things as a "hit" concentration for new drugs

greater than or equal to 90% inhibition

depends on project usually 6ug/ml

30 uM Hit: spectro 30% inhibition radio 50% inhibition

vary dependent on test materials

n/a

10-fold difference between compound and DMSO control

0.1, 1, 10 less than 1 is a hit

MIC below 2ug/ml

NA

10 micrograms per ml giving 90% or greater inhibition

do not screen in TB

For new cmpds we start at 30 ug/ml as the highest concentration. definition of a hit depends on the project, usually MIC data is used for SAR purposes. Acives in the range of 1-2 ug/ml are certainly followed up.

hit

No. Serial dilution for all compounds screened

We usually test the following concentrations : 25 microM up to 0.05 microM using 2-fold dilution process. A MIC of < 0.12 mg/l is considered as a hit.

16 ug/ml

No such things as a "hit" concentration for new drugs

greater than or equal to 90% inhibition

What positive and negative control drugs do you use?

INH and DMSO alone

streptomycin isoniazid ethambutol

none known for the tested enzyme

positive control drugs: take several of following drugs depedent on test materials. Rifampin, Isoniazid, Moxifloxacin, Cefataxim, Ofloxacinetc

n/a (not screening unknown compounds)

Positive INH and Rif Negative is DMSO For anaerobic assays we use metronidazole as positive and INH as negative

INH

INH & RIF

Stanadard AntiTB drugs like Rifampicin,INH,ETM,PZA,Clarithromycin,Moxifloxacin used as controls

INH, and Fluoroquinolone. Negative control simply solvent.

amikacin

n/a

INH, RIF, Moxi, PA-824 etc. Usually depends on the cmpds series being tested.

INH as positive control

INH and RIF

Drug reference: Rifampicin and Isoniazid

We use as positive controls rifampicin, levofloxacin, amikacin and linezolid. Erythromycin and vancomycin are used as negative controls.

Drug-free culture control Media control Inh Rif

Variable depending on the class of the drug under investigation

Positive: RMP, INH, SM, Mox, PA-824 Negative: metronidazole, (INH for LORA)

INH and DMSO alone

streptomycin isoniazid ethambutol

none known for the tested enzyme

positive control drugs: take several of following drugs depedent on test materials. Rifampin, Isoniazid, Moxifloxacin, Cefataxim, Ofloxacinetc

n/a (not screening unknown compounds)

Positive INH and Rif Negative is DMSO For anaerobic assays we use metronidazole as positive and INH as negative

INH

INH & RIF

Stanadard AntiTB drugs like Rifampicin,INH,ETM,PZA,Clarithromycin,Moxifloxacin used as controls

INH, and Fluoroquinolone. Negative control simply solvent.

amikacin

n/a

INH, RIF, Moxi, PA-824 etc. Usually depends on the cmpds series being tested.

INH as positive control

INH and RIF

Drug reference: Rifampicin and Isoniazid

We use as positive controls rifampicin, levofloxacin, amikacin and linezolid. Erythromycin and vancomycin are used as negative controls.

Drug-free culture control Media control Inh Rif

Variable depending on the class of the drug under investigation

Positive: RMP, INH, SM, Mox, PA-824 Negative: metronidazole, (INH for LORA)

What do you consider to be acceptable values for your controls?*

Streptomycin: 0.1-0.4 ug/ml isoniazid: 0.025-0.05 ug/ml ethambutol: 0.75-1.5 ug/ml

N/A

plus or minus one dilution steps compare to reported MIC values of each control drugs

n/a

Positive control = 10-100 fold difference

Don't understand question


MIC values stated by CLSI Guidelines

Growth

n/a

INH 0.04, RIF 0.02-0.03, Moxifloxacin 0.1, PA-824 0.5

INH MIC 0.03-0.06 mcg/ml

no growth and full growth

Rifampicin: MIC50 of 0.005µM (10 concentration tested/assay from 0.1uM to 0.0003uM) INH: MIC50 of 0.25µM (10 concentration tested/assay from 2.5uM to 0.004uM)

The values recommended by CLSI are considered to be acceptable.

Inh MIC 0.03 /- 1 tube Rif MIC 0.03 /- 1 tube

Which values???

published values from Collins and Franzblau, 1997 and Cho et al, 2007 Z' factor >0.5

Streptomycin: 0.1-0.4 ug/ml isoniazid: 0.025-0.05 ug/ml ethambutol: 0.75-1.5 ug/ml

N/A

plus or minus one dilution steps compare to reported MIC values of each control drugs

n/a

Positive control = 10-100 fold difference

Don't understand question


MIC values stated by CLSI Guidelines

Growth

n/a

INH 0.04, RIF 0.02-0.03, Moxifloxacin 0.1, PA-824 0.5

INH MIC 0.03-0.06 mcg/ml

no growth and full growth

Rifampicin: MIC50 of 0.005µM (10 concentration tested/assay from 0.1uM to 0.0003uM) INH: MIC50 of 0.25µM (10 concentration tested/assay from 2.5uM to 0.004uM)

The values recommended by CLSI are considered to be acceptable.

Inh MIC 0.03 /- 1 tube Rif MIC 0.03 /- 1 tube

Which values???

published values from Collins and Franzblau, 1997 and Cho et al, 2007 Z' factor >0.5

*Note: this question was not clear to all respondents

Please indicate how you calculate MICs.

visual read MIC 90

N/A

from serious of two fold dilution tests

concentration of drug that results in luminescence lower than a 1:100 dilution of test culture with no drug

100-fold difference

Serial dilution

Broth MIC will be based on the well with no reduction of alamar blue dye.Agar MIC will be based on disappearence of growth on drug containg 7H11 plates.Whonet software is use to calculate MIC50/90

Total inhibition of growth.

Concentration inhibiting growth by 90%, also look at IC50 and slope of curve.

from 2-fold dilution effect on growth

minimum concentration to inhibit growth compared to no drug control

Lowest concentration to prevent formation of visible sediment at bottom of tube.

Calculated/extrapolated from graph plotted with PRISM, compound serial diluted (10 dilution needed to derive a proper curve). Semi-log graph, with sigmoidal curve is plotted to estimate the MIC50. The Y-axis will have the OD600 values and the X-axis will have log transformed drug concentrations.

The MIC is the lowest concentration that inibits 99% of the bacteria present in the inoculum.

interpolation using in-house software, lowest concentration effecting 90% inhibition relative to drug-free controls

visual read MIC 90

N/A

from serious of two fold dilution tests

concentration of drug that results in luminescence lower than a 1:100 dilution of test culture with no drug

100-fold difference

Serial dilution

Broth MIC will be based on the well with no reduction of alamar blue dye.Agar MIC will be based on disappearence of growth on drug containg 7H11 plates.Whonet software is use to calculate MIC50/90

Total inhibition of growth.

Concentration inhibiting growth by 90%, also look at IC50 and slope of curve.

from 2-fold dilution effect on growth

minimum concentration to inhibit growth compared to no drug control

Lowest concentration to prevent formation of visible sediment at bottom of tube.

Calculated/extrapolated from graph plotted with PRISM, compound serial diluted (10 dilution needed to derive a proper curve). Semi-log graph, with sigmoidal curve is plotted to estimate the MIC50. The Y-axis will have the OD600 values and the X-axis will have log transformed drug concentrations.

The MIC is the lowest concentration that inibits 99% of the bacteria present in the inoculum.

interpolation using in-house software, lowest concentration effecting 90% inhibition relative to drug-free controls

MICs

for every sample

Fixed concentration screen, many drugs in a preliminary screen. MICs

for anything promising.

N/A

determine MICs

for every sample

Usually we do MICs

MIC's only


MIC for all the samples are determined

MIC for every sample

Both

limited samples involved, do MICs

Always do MICs.

MICs

for every sample

fixed for the inital

screen and then determin

"exact" MIC for hits

Fixed concentration used for medium/high throughput cell-based screening. Done against BCG at 10�M.

MIC is done for every sample.

Usually large screens start with fixed conc. at 16-32 ug/ml. After that the positives are tested for MIC

MIC for each sample

Usually depends upon the number of compounds in a series or library. For large collections, run primary screen at fixed concentration. For smaller sets or high predicted activity, do MIC for each sample.

Do you conduct screens at fixed concentrations of a drug or do you determine MICs for every sample?

What variables, if any, have you found to affect the MIC for standard anti-TB drugs or experimental compounds using fixed concentrations?

Incubation time and inoculum.

N/A

Don't perfrom

pH, SOLUBILITY,GROWTH

NA

n/a

Solubility can be an issue for experimental cmpds. We try to be consistent for dissolving cmpds but this might also depend on batch of cmpd etc.

Rifampin's MIC against H37Ra is 10-fold lower than against H37Rv.

these are very good drugs and we typically get consistent MICs. Media age may be a factor

Compounds that had crashed out after prolong storage, growth phase of the cells, initial OD.

Inoculum size.

Freeze thaw of compound samples There is a tendency to pick up false positives since the test is in singlicate, which could be due to compound degradation products upon storage.

Unreasonable question

inoculum density for some compounds; serial dilution and mixing.

Incubation time and inoculum.

N/A

Don't perfrom

pH, SOLUBILITY,GROWTH

NA

n/a

Solubility can be an issue for experimental cmpds. We try to be consistent for dissolving cmpds but this might also depend on batch of cmpd etc.

Rifampin's MIC against H37Ra is 10-fold lower than against H37Rv.

these are very good drugs and we typically get consistent MICs. Media age may be a factor

Compounds that had crashed out after prolong storage, growth phase of the cells, initial OD.

Inoculum size.

Freeze thaw of compound samples There is a tendency to pick up false positives since the test is in singlicate, which could be due to compound degradation products upon storage.

Unreasonable question

inoculum density for some compounds; serial dilution and mixing.

Regarding high throughput screening: which types of compounds do you use?

What is the largest library that you have screened or plan to screen with your assay?

What is the source of your HTS library and in what format is the assay done in (e.g. plate, vials, powder, etc.)


plates

Plates

commercial library; spectro-plate;radio-vials

chemical, natural products, extracts usually from academic organizations and usually done in plates

Chembridge, Analyticon, Broad Institute as DMSO stocks

no source

96 WELL PLATES

NA

All in microtiter plates

2 biggest: Tripos and Nanosyn on 96 well plates

Novartis archive, compounds available in liquid (plate) or powder.

Compunds are in plates and are solubilized in DMSO.

AZ corporate collection. 384 well format using RBMA. Compound stocks are in DMSO.

We don't have any library

ChemBridge NovaCore, Prestwick, ECUM actino extracts, all in 96 well plates


plates

Plates

commercial library; spectro-plate;radio-vials

chemical, natural products, extracts usually from academic organizations and usually done in plates

Chembridge, Analyticon, Broad Institute as DMSO stocks

no source

96 WELL PLATES

NA

All in microtiter plates

2 biggest: Tripos and Nanosyn on 96 well plates

Novartis archive, compounds available in liquid (plate) or powder.

Compunds are in plates and are solubilized in DMSO.

AZ corporate collection. 384 well format using RBMA. Compound stocks are in DMSO.

We don't have any library

ChemBridge NovaCore, Prestwick, ECUM actino extracts, all in 96 well plates

What is the type and make of tubes, plastics and glassware used for the stocks?

Compound stocks in polypropylene plates (Matrix) or glass vials.

supplied by vendor (VWR?)

glassware for original stocks (10 mg/ml), tubes for running solutions

Still to be determined. Most likely polypropylene

Not applicable

Nunc 96 Well plates

NA

do not do HTS on TB

Costar plates

Glass vials of 3ml capacity

96 well flat bottom U shape plates.

Appropriate kind of highest quality

Becton Dickinson 96 well plates

Compound stocks in polypropylene plates (Matrix) or glass vials.

supplied by vendor (VWR?)

glassware for original stocks (10 mg/ml), tubes for running solutions

Still to be determined. Most likely polypropylene

Not applicable

Nunc 96 Well plates

NA

do not do HTS on TB

Costar plates

Glass vials of 3ml capacity

96 well flat bottom U shape plates.

Appropriate kind of highest quality

Becton Dickinson 96 well plates

Dispensing system

manually

NA

Multidrop

standard lab tools

manually

NA

Multidrop

standard lab tools

Other responses

Quality Controls

and intraplate controls

NA

and intraplate controls

NA

Additional responses

Please give examples of any methods done to normalize your procedures.For instance, are there control samples included in each plate or do you adjust for a certain level of background? Please explain.

Percentage inhibition calculated against no drug positive control on all plates,fluorescence data not compared between plates but percentage inhibition can be. Known drugs included in each assay (separate plate) and their MICs used to indicate that assay has worked.

control samples in each plate; controls in triplicates

for each experiment, control drugs are included and for each plates, negative and positive controls are included

Controls will always be included. Assays will be done at several concentrations. DMSO will be at least two DMSO plates with DMSO in all wells

Not applicable

NA

NA

Both

INH totration on each plate for "known" MIC value on standard innoculum (1x10e5 CFU/ml final assay concentration)

Use positive (Isoniazid and/or rifampicin) and negative control (solvent alone) in each plate.

Controls and background in every plate.

two designs are done with the control plates depending on how many compounds are tested. For large series, we use test plates ataregular frequency at every 5 plates for instance. however, if screenign is only 300 compds or so the test wells are in every plate (two rows of ref wells).

See our publications

Positive and negative controls are included in each plate. Standard drug controls are included in each experiment.

Percentage inhibition calculated against no drug positive control on all plates,fluorescence data not compared between plates but percentage inhibition can be. Known drugs included in each assay (separate plate) and their MICs used to indicate that assay has worked.

control samples in each plate; controls in triplicates

for each experiment, control drugs are included and for each plates, negative and positive controls are included

Controls will always be included. Assays will be done at several concentrations. DMSO will be at least two DMSO plates with DMSO in all wells

Not applicable

NA

NA

Both

INH totration on each plate for "known" MIC value on standard innoculum (1x10e5 CFU/ml final assay concentration)

Use positive (Isoniazid and/or rifampicin) and negative control (solvent alone) in each plate.

Controls and background in every plate.

two designs are done with the control plates depending on how many compounds are tested. For large series, we use test plates ataregular frequency at every 5 plates for instance. however, if screenign is only 300 compds or so the test wells are in every plate (two rows of ref wells).

See our publications

Positive and negative controls are included in each plate. Standard drug controls are included in each experiment.

What is your assessment of potency? For example is your assessment of potency based on percent inhibition, percent activity, or other?Do you include controls without drugs or without bacteria?

Percentage inhibition based on control without drug, no bacteria controls also included.

percent activity controls without drugs

% inhibition

Percent inhibition in terms of Growth expressed as MIC. Yes we do keep controls without drug and without bacteria

NA

Percent inhibition and both controls

We estimate the MIC50 (Minimum inhibitory concentration required to inhibit 50% growth) using PRISM software. All plates have positive controls (with drugs, either rifampicin or isoniazid) and negarive control (bacteria alone without the drugs).

Assessment of potency is based on percent inhibition. We include controls without drugs and controls without bacteria.

Percent inhibition. Media control and untreated growth control

QC without drugs. Potency is expressd in MIC, MBC, MIC/MBC ratio, etc

percent inhibition Controls include: standard drugs with bacteria standard drugs without bacteria bacteria only medium only

Percentage inhibition based on control without drug, no bacteria controls also included.

percent activity controls without drugs

% inhibition

Percent inhibition in terms of Growth expressed as MIC. Yes we do keep controls without drug and without bacteria

NA

Percent inhibition and both controls

We estimate the MIC50 (Minimum inhibitory concentration required to inhibit 50% growth) using PRISM software. All plates have positive controls (with drugs, either rifampicin or isoniazid) and negarive control (bacteria alone without the drugs).

Assessment of potency is based on percent inhibition. We include controls without drugs and controls without bacteria.

Percent inhibition. Media control and untreated growth control

QC without drugs. Potency is expressd in MIC, MBC, MIC/MBC ratio, etc

percent inhibition Controls include: standard drugs with bacteria standard drugs without bacteria bacteria only medium only

Don't do HTS

whonet

NA

Reproducibility of Inh, Rif MICs

Don't do HTS

whonet

NA

Reproducibility of Inh, Rif MICs

Other responses

How do you evaluate your assay performance?

Do you determine MBCs by sub-culture from the MIC test or do you conduct as a separate test?

The next 3 questions refer to minimum bactericidal concentrations (MBCs). Do you determine MBCs by sub-culture from the MIC test or do you conduct as a separate test?

Not yet.

N/A

determine MBCs from the MIC test

do not routinely perform

Separate

Separate

from the MIC test

From the MIC SET

NA

From MIC test as a first look

We set up duplicate plates

sub-culture

MICs are determined in follow up screening

Carry out CFU plating directly from the MIC plate setup

MBCs are done by sub-culture.

separate test plate

Parallel with MIC

both

The next 3 questions refer to minimum bactericidal concentrations (MBCs). Do you determine MBCs by sub-culture from the MIC test or do you conduct as a separate test?

Not yet.

N/A

determine MBCs from the MIC test

do not routinely perform

Separate

Separate

from the MIC test

From the MIC SET

NA

From MIC test as a first look

We set up duplicate plates

sub-culture

MICs are determined in follow up screening

Carry out CFU plating directly from the MIC plate setup

MBCs are done by sub-culture.

separate test plate

Parallel with MIC

both

If you run the MBC assay as a separate test, please indicate any variables (e.g. CFU, luminescence, fluorescence etc) that differ significantly from the MIC result.

N/A

We always do CFU. Assay done on 1mL cultures at 10exp5 cells/mL

None

NA

Same as MIC setup

MBC is done by dilution plating on 4" quad plates with 7H11-OADC agar. Thus MIC can vary from 7H9-OADC broth to agar.

Same setup for MBC determination

CFU.

MBC plate will not receive the dye. Goes for CFU enumeration on the day MIC is read. However, an additional plating is also done on day 14 so as to ascertain time dependence in killing.

NA

none

N/A

We always do CFU. Assay done on 1mL cultures at 10exp5 cells/mL

None

NA

Same as MIC setup

MBC is done by dilution plating on 4" quad plates with 7H11-OADC agar. Thus MIC can vary from 7H9-OADC broth to agar.

Same setup for MBC determination

CFU.

MBC plate will not receive the dye. Goes for CFU enumeration on the day MIC is read. However, an additional plating is also done on day 14 so as to ascertain time dependence in killing.

NA

none

What is your criteria for interpreting the MBC result(e.g. percent killing of the starting inoculum)?

N/A

percent killing

yes. but we also include a control with no added drug to ensure that growth occured in this.

Percent killing

NA

99% kill of starting innoculum

percent killing of the starting inoculum

counting colonies to determine CFU/ml for given dilution. Killing = no colonies (CFU = 0)

Fold killing of the starting inoculum, MBC90 as concentration to achieve 10-fold killing and MBC99 as the concentration to achieve 100-fold killing

Interpretation of the MBC result is based on percent killing of the starting inoculum.

MBC is the minimum concentration that yields 3 log reduction from the starting inoculum.

90% reduction in cfu relative to the starting inculum

N/A

percent killing

yes. but we also include a control with no added drug to ensure that growth occured in this.

Percent killing

NA

99% kill of starting innoculum

percent killing of the starting inoculum

counting colonies to determine CFU/ml for given dilution. Killing = no colonies (CFU = 0)

Fold killing of the starting inoculum, MBC90 as concentration to achieve 10-fold killing and MBC99 as the concentration to achieve 100-fold killing

Interpretation of the MBC result is based on percent killing of the starting inoculum.

MBC is the minimum concentration that yields 3 log reduction from the starting inoculum.

90% reduction in cfu relative to the starting inculum

Please describe in as much detail as possible your M. tuberculosis strain choice for non or slowly replicating (NR/SR) assays and inoculum preparation.

H37Rv (Wayne cells) according to Wayne method

I don't perform this type of test

We use the same procedure as described by Wayne.

MTB H37Rv exponentially growing culture, grown in roller bottle, OD600 of 0.2 – 0.3 Wayne setup: grown in Dubos media + supplement and additional 0.03% Tween 80, resuspended in the same media to final OD600 of 0.02, aliquoted to tubes and incubated at 37oC, 130 rpm for 14 day prior to assay setup Nutrient starvation setup: grown in 7H9 media + ADS and then cells pelleted by centrifugation and resuspended in Dulbecco’s PBS containing 0.025% Tween 80 to a final OD600 of 0.1 and maintained in roller bottle at 37oC for 14 days prior to assay setup We don’t use any SR TB assays

Mycobacterium tuberculosis, Mycobacterium smegmatis and Mycobacterium bovis BCG are usualy used for these studies. The wayne, the Nitric Oxide and the Hypoxia models are used to measure the activity of new compounds against non or slow replicating TB bacilli.

Use H37Rv harboring a luxAB gene on a shuttle vecotr. Inoculum is prepared in a sealed fermentor in 7H12 medium in which oxygen consumption is monitored. Cultures are washed and frozen prior to use. Details in Cho et al, 2007.

H37Rv (Wayne cells) according to Wayne method

I don't perform this type of test

We use the same procedure as described by Wayne.

MTB H37Rv exponentially growing culture, grown in roller bottle, OD600 of 0.2 – 0.3 Wayne setup: grown in Dubos media + supplement and additional 0.03% Tween 80, resuspended in the same media to final OD600 of 0.02, aliquoted to tubes and incubated at 37oC, 130 rpm for 14 day prior to assay setup Nutrient starvation setup: grown in 7H9 media + ADS and then cells pelleted by centrifugation and resuspended in Dulbecco’s PBS containing 0.025% Tween 80 to a final OD600 of 0.1 and maintained in roller bottle at 37oC for 14 days prior to assay setup We don’t use any SR TB assays

Mycobacterium tuberculosis, Mycobacterium smegmatis and Mycobacterium bovis BCG are usualy used for these studies. The wayne, the Nitric Oxide and the Hypoxia models are used to measure the activity of new compounds against non or slow replicating TB bacilli.

Use H37Rv harboring a luxAB gene on a shuttle vecotr. Inoculum is prepared in a sealed fermentor in 7H12 medium in which oxygen consumption is monitored. Cultures are washed and frozen prior to use. Details in Cho et al, 2007.

Please your NR or SR assay: format, sample preparation, diluent, real time or specific endpoint measures, dyes and reagents, time frame, controls, etc.

Wayne cells (see Wayne method in MTB protocols book). transfer to anaerobic chamber and dispense into 24well plates containing drug in DMSO (MTZ = + control, INH = - control). Transfer to anaerobic bags. Incubate 7 days. Then determine CFU by plating on 7H11.OADC agar.

Glass vials Methylene blue dye End point is discolouration of dye, cfu and metronidazole activity

Wayne setup Format: 4 different concentrations of drugs in triplicates in flat bottom 96 well plates. Sample preparation: 14 day old Wayne cells Diluent: DMSO Endpoint measurement: CFU plating Time frame: CFU will be measured on day 0 and day 5 after incubatiowith drugs under anaerobic conditions at 37oC Controls: 100uM Metroinidazole, 25uM Isoniazid are used as positive and negative controls respectively. Loebel setup Format: 4 different concentrations of drugs in triplicates in flat bottom 96 well plates. Sapmle preparation: 14 day old Loebel cells Diluent: DMSO Endpoint measurement: CFU plating Time frame: CFU will be measured on day 0 and day 5 after incubation at 37oC Controls: 100uM Metroinidazole, 25uM Isoniazid are used as negative controls.

Drastic oxygen depletion (hypoxia model) of mycobacterial cultures was achieved by placing loosely capped tubes containing different drug concentrations inside an anaerobic jar (BBL) along with anaerobic gas generation envelopes (using palladium catalysts). After 7 days of anaerobiosis, the dormant cultures were harvested by low-speed centrifugation, washed twice with 7H9 medium to remove the drugs and resuspended in drug-free medium. The CFUs of the treated and untreated cultures were determined by plating on 7H10 agar to evaluate the bactericidal activity. In the Wayne model, gradual O2 depletion induces dormancy in mycobacterial cultures. Briefly, after induction of dormancy for 18 days by gradual O2-depletion, cultures without exposure to oxygen were treated further with various drug concentrations for a period ranging from 4 to 21 days and CFUs were counted on 7H10 plates. In the NO model, dormancy was obtained by incubating cultures with 150 μM of a NO donor – diethlenetriamine/nitric oxide (DETA/NO) – for 2 hours, followed by treatment with several compounds for 10 hours.

format: 96 well plate, sample prepration and diluent - same as for replicating cultures.

Wayne cells (see Wayne method in MTB protocols book). transfer to anaerobic chamber and dispense into 24well plates containing drug in DMSO (MTZ = + control, INH = - control). Transfer to anaerobic bags. Incubate 7 days. Then determine CFU by plating on 7H11.OADC agar.

Glass vials Methylene blue dye End point is discolouration of dye, cfu and metronidazole activity

Wayne setup Format: 4 different concentrations of drugs in triplicates in flat bottom 96 well plates. Sample preparation: 14 day old Wayne cells Diluent: DMSO Endpoint measurement: CFU plating Time frame: CFU will be measured on day 0 and day 5 after incubatiowith drugs under anaerobic conditions at 37oC Controls: 100uM Metroinidazole, 25uM Isoniazid are used as positive and negative controls respectively. Loebel setup Format: 4 different concentrations of drugs in triplicates in flat bottom 96 well plates. Sapmle preparation: 14 day old Loebel cells Diluent: DMSO Endpoint measurement: CFU plating Time frame: CFU will be measured on day 0 and day 5 after incubation at 37oC Controls: 100uM Metroinidazole, 25uM Isoniazid are used as negative controls.

Drastic oxygen depletion (hypoxia model) of mycobacterial cultures was achieved by placing loosely capped tubes containing different drug concentrations inside an anaerobic jar (BBL) along with anaerobic gas generation envelopes (using palladium catalysts). After 7 days of anaerobiosis, the dormant cultures were harvested by low-speed centrifugation, washed twice with 7H9 medium to remove the drugs and resuspended in drug-free medium. The CFUs of the treated and untreated cultures were determined by plating on 7H10 agar to evaluate the bactericidal activity. In the Wayne model, gradual O2 depletion induces dormancy in mycobacterial cultures. Briefly, after induction of dormancy for 18 days by gradual O2-depletion, cultures without exposure to oxygen were treated further with various drug concentrations for a period ranging from 4 to 21 days and CFUs were counted on 7H10 plates. In the NO model, dormancy was obtained by incubating cultures with 150 μM of a NO donor – diethlenetriamine/nitric oxide (DETA/NO) – for 2 hours, followed by treatment with several compounds for 10 hours.

format: 96 well plate, sample prepration and diluent - same as for replicating cultures.

What strain of M. tuberculosis is used for NR and SR assays and why was this strain chosen? What is the source of your strain?


ATCC

Mtuberculosis H37Rv, BCG

H37Rv, same as Wayne.

MTB H37Rv from ATCC (#27294 ).Chosen because it is a lab strain and genome sequence is available

Mycobacterium tuberculosis H37Rv obtained from ATCC. A resistant mutant to R207910 is also used.

H37Rv lux AB constructed in house from ATCC 27294 and using V. harveyi luciferase and acetamidase promotor


ATCC

Mtuberculosis H37Rv, BCG

H37Rv, same as Wayne.

MTB H37Rv from ATCC (#27294 ).Chosen because it is a lab strain and genome sequence is available

Mycobacterium tuberculosis H37Rv obtained from ATCC. A resistant mutant to R207910 is also used.

H37Rv lux AB constructed in house from ATCC 27294 and using V. harveyi luciferase and acetamidase promotor

What are your positive and negative controls for the NR and SR assays?


metronidazole and isoniazid for anaerobic assays

activity of metronidazole & discolouration of MB

Controls include: INH, Metronidazole, Moxi, RIF - all samples are run in duplicate.

Wayne assay: Rifampicin, isoniazid, metronidazole.Nutrient starvation assay: Rifampicin, isoniazid

Positive controls are rifampicin, metronidazole and negative control is isoniazid.

media only, drug and media only, bacteria only


metronidazole and isoniazid for anaerobic assays

activity of metronidazole & discolouration of MB

Controls include: INH, Metronidazole, Moxi, RIF - all samples are run in duplicate.

Wayne assay: Rifampicin, isoniazid, metronidazole.Nutrient starvation assay: Rifampicin, isoniazid

Positive controls are rifampicin, metronidazole and negative control is isoniazid.

media only, drug and media only, bacteria only

What is your estimate of your laboratory throughput for NR and SR assays per day?


once every three weeks. WE do 12-20 compounds per time

2 COMPOUNDS

80 samples

Low through put assay: 5-10 compounds/week (assay setup once a week)

2 to 3 experiments can be performed per week.

5-20 96-well plates


once every three weeks. WE do 12-20 compounds per time

2 COMPOUNDS

80 samples

Low through put assay: 5-10 compounds/week (assay setup once a week)

2 to 3 experiments can be performed per week.

5-20 96-well plates

What do you feel are the major variables that affect the outcome of the NR or SR assay?

Head space ratio and Abrupt stirring and rpm, Proper sealing of vials

Variation is seen at times with INH (showing slight activity), or with experimental cmpds (due to solubility issues)

Starting inoculum, steering speed and anaerobiosis (for Wayne assay), source of the PBS, starting inoculum and clumping (for nutrient starvation model).

Oxygene tension.

preparation of the inoculum

Head space ratio and Abrupt stirring and rpm, Proper sealing of vials

Variation is seen at times with INH (showing slight activity), or with experimental cmpds (due to solubility issues)

Starting inoculum, steering speed and anaerobiosis (for Wayne assay), source of the PBS, starting inoculum and clumping (for nutrient starvation model).

Oxygene tension.

preparation of the inoculum

What do you consider to be the strengths and weaknesses of the NR or SR assay?

Wayne system is heterogeneous

Strength Good indicator of efficacy against dormant Mycobacteria Weakness Difficult for HTS

Our assay is less reproducible than regular MIC assay, therefore all samples are run in duplicate every time.

Strength Wayne: Identification of compounds active against anaerobic non-replicating persistent bacilli, compounds that could potentiallyshorten time of TB therapy Weakness Assay optimization is critical for the Wayne model to obtain meaningful results. In vivo relevance of these assays is not known

The weaknesses: the variability in the data. The strengths: strong ability to differenciate between active compounds such as rifampicin and non active compounds such as isoniazid.

Strengths: activity correlate with known clinical sterilizing activity, good throughput Weaknesses: low robustness, hard to standardize

Wayne system is heterogeneous

Strength Good indicator of efficacy against dormant Mycobacteria Weakness Difficult for HTS

Our assay is less reproducible than regular MIC assay, therefore all samples are run in duplicate every time.

Strength Wayne: Identification of compounds active against anaerobic non-replicating persistent bacilli, compounds that could potentiallyshorten time of TB therapy Weakness Assay optimization is critical for the Wayne model to obtain meaningful results. In vivo relevance of these assays is not known

The weaknesses: the variability in the data. The strengths: strong ability to differenciate between active compounds such as rifampicin and non active compounds such as isoniazid.

Strengths: activity correlate with known clinical sterilizing activity, good throughput Weaknesses: low robustness, hard to standardize

Which animal species do you use or have you used for TB drug testing?

What is the source/supplier of your strain of animal?


Charles River

Japan SLC

Jackson

Jackson Labs or Charles River Labs

Charles River

other investigators (collaborators)

Biological Resources Centre, Singapore

Janvier Breeding Center, France.

National Institute of Nutrition, India

Harlan Sprague Dawley


Charles River

Japan SLC

Jackson

Jackson Labs or Charles River Labs

Charles River

other investigators (collaborators)

Biological Resources Centre, Singapore

Janvier Breeding Center, France.

National Institute of Nutrition, India

Harlan Sprague Dawley

Please elaborate on the advantages of the infection model chosen.

Low dose aerosol infection of a susceptible species which mimics human granuloma formation and allows study of the extrapulmonarydissemination and hematogenous reseeding of the lung allows the evaluaiton of drug effects under conditions which are directly relavant to human TB.

Cheap than other animal, easy to control mouse, little variable because of inbred

Used by many researchers

Standardization, cost, simplicity, reproducibility

Low dose aerosol is the most realistic infection when compared to natural infection. The guinea pig is most reflective of the human pathology and disease. The mouse is cheapest. The GKO mice allow rapid unchecked growth of the TB and allow rapid drug screening.

BALB/c mice develop productive infection with stable plateau in CFU counts (as opposed to declining counts observed in some other inbred strains). Same for Swiss, which are cheaper.

used for cytotoxicity studies on lead compounds

Mice are easily available and to handle.

We choose the swiss mice because it's an outbreed mice like humans. Each mouse is different from another mouse in terms of genetic background.

Mice: easy to handle. less compound requirement. many exptl groups in parallel. B6 colony now gone

inexpensive, well established

Low dose aerosol infection of a susceptible species which mimics human granuloma formation and allows study of the extrapulmonarydissemination and hematogenous reseeding of the lung allows the evaluaiton of drug effects under conditions which are directly relavant to human TB.

Cheap than other animal, easy to control mouse, little variable because of inbred

Used by many researchers

Standardization, cost, simplicity, reproducibility

Low dose aerosol is the most realistic infection when compared to natural infection. The guinea pig is most reflective of the human pathology and disease. The mouse is cheapest. The GKO mice allow rapid unchecked growth of the TB and allow rapid drug screening.

BALB/c mice develop productive infection with stable plateau in CFU counts (as opposed to declining counts observed in some other inbred strains). Same for Swiss, which are cheaper.

used for cytotoxicity studies on lead compounds

Mice are easily available and to handle.

We choose the swiss mice because it's an outbreed mice like humans. Each mouse is different from another mouse in terms of genetic background.

Mice: easy to handle. less compound requirement. many exptl groups in parallel. B6 colony now gone

inexpensive, well established

Please explain the disadvantages of your chosen animal model?

The experiments are more expensive because of the costs of the animals themselves and ABSL-3 containment for long periods of follow-up.

not yet

Not model of all aspects of human Tb. C57BL/6 is more sensitive to some drugs (toxic effects) than other mouse strains

as all experimental models, it is an experimental model only

The mouse does not demonstrate all lesion types seen in typical human TB pathology. Pharmacokinetics and drug metabolism in rodents are different and cross species bridging studies/calculations have to be performed.

More variability in CFU counts in Swiss mice.

not sure, done for us by collaborator

Predictability of the model is questionable. Pathology is not similar to humans. No phase of persistence at low level of microorganisms

The disadvantages of using the Swiss mice is the variability in the results since the mice are different and are not like inbreed mice. This means that to show an activity of a compound, it's needed to overcome this variability by using more mice than when we deal with inbreed mice.

model does not represent many of the alternate physiological states presumably experienced by Mtb in human host. Can only score against intracellular mycobacteria. The most significant disadvantage of any of the in vivo models is the lack of predictability of effect from in vitro tests.

PK studies are difficult due to low blood volume

The experiments are more expensive because of the costs of the animals themselves and ABSL-3 containment for long periods of follow-up.

not yet

Not model of all aspects of human Tb. C57BL/6 is more sensitive to some drugs (toxic effects) than other mouse strains

as all experimental models, it is an experimental model only

The mouse does not demonstrate all lesion types seen in typical human TB pathology. Pharmacokinetics and drug metabolism in rodents are different and cross species bridging studies/calculations have to be performed.

More variability in CFU counts in Swiss mice.


Predictability of the model is questionable. Pathology is not similar to humans. No phase of persistence at low level of microorganisms

The disadvantages of using the Swiss mice is the variability in the results since the mice are different and are not like inbreed mice. This means that to show an activity of a compound, it's needed to overcome this variability by using more mice than when we deal with inbreed mice.

model does not represent many of the alternate physiological states presumably experienced by Mtb in human host. Can only score against intracellular mycobacteria. The most significant disadvantage of any of the in vivo models is the lack of predictability of effect from in vitro tests.

PK studies are difficult due to low blood volume

What route of infection have you used (intravenous, aerosol, intramuscular, intraperitoneal, other)? Please elaborate on past methods used and current methods and explain why you use the particular method

We infect our guinea pigs with a very low dose (10-20 cfu)of virulent M tuberculosis in a specially designed aerosol exposure (Madison) chamber because this route and dose of infection allows the infection and disease to develop in a manner very similar to clinical TB.

aerosol challenge: aerosol infection is natural route of TB infection. intravenous: early bacterial number is high, i.e. 2 weeks after infection

aerosol

IV, aerosol(because it is easier to perform than iv)

low dose aerosol

Aerosol is used, largely for convenience and reproducibility.


We use intranasal route of infection for mice.This partially mimics the natural infection.The model is suitable to test the in vivo efficacy of anti-TB drugs. For safety considerations, intravenous route is not used anymore.

Intravenous. We still work with the intravenous route of infection because we think that the intravenous route of infection is the only one which allows to extrapolate the results of activities of antibiotics to humans.

Aerosol (mostly and in both species). Intravenous (mice) I.m. (guinea pigs)

aerosol inhalation using Glas-Col system easy to inoculate large number of mice

We infect our guinea pigs with a very low dose (10-20 cfu)of virulent M tuberculosis in a specially designed aerosol exposure (Madison) chamber because this route and dose of infection allows the infection and disease to develop in a manner very similar to clinical TB.

aerosol challenge: aerosol infection is natural route of TB infection. intravenous: early bacterial number is high, i.e. 2 weeks after infection

aerosol

IV, aerosol(because it is easier to perform than iv)

low dose aerosol

Aerosol is used, largely for convenience and reproducibility.


We use intranasal route of infection for mice.This partially mimics the natural infection.The model is suitable to test the in vivo efficacy of anti-TB drugs. For safety considerations, intravenous route is not used anymore.

Intravenous. We still work with the intravenous route of infection because we think that the intravenous route of infection is the only one which allows to extrapolate the results of activities of antibiotics to humans.

Aerosol (mostly and in both species). Intravenous (mice) I.m. (guinea pigs)

aerosol inhalation using Glas-Col system easy to inoculate large number of mice

Do you have a placebo diluent/carrier injection or gavage control in your experiment?

Have you ever seen an effect of the diluent placebo on the outcome of the experiment? If so, please explain.


No

No

No

sometimes, stress?

No, this has been tested before

mice may fare worse with placebo gavage

no

No

No, inactive compounds have no activity.

No

no


No

No

No

sometimes, stress?

No, this has been tested before

mice may fare worse with placebo gavage

no

No

No, inactive compounds have no activity.

No

no

How is the strain grown (e.g. pellicle, passaged through mice, other)?


The H37Rv strain was obtained from the ATCC, grown to mid-log phase in liquid culture, homogenized, sonicated briefly, and filtered to obtain a single-cell suspension before freezing at minus 80C and determining the cfu on a thawed aliquot,

filtered bacteria through 5 um syringe filter

Normal in vitro culture of H37Rv

Early passage number of ATCC Erdman was grown as pellicle and frozen as seed stocks. The seed stock is then upscaled over three passages to 100-200 ml cultures, in Proskauer beck, up to OD of 0.5-0.7, aliquoted vir test in GKO/B6 mice

mouse-passaged, frozen, sub-culutured


The strain is grown as pellicle.

In vitro.

Passaged through mice, preserved as seedlots

liquid culture to late log, then passaged through 8 uM filter, frozen in PBS-Tween 80, occaisionally passaged in mice


The H37Rv strain was obtained from the ATCC, grown to mid-log phase in liquid culture, homogenized, sonicated briefly, and filtered to obtain a single-cell suspension before freezing at minus 80C and determining the cfu on a thawed aliquot,

filtered bacteria through 5 um syringe filter

Normal in vitro culture of H37Rv

Early passage number of ATCC Erdman was grown as pellicle and frozen as seed stocks. The seed stock is then upscaled over three passages to 100-200 ml cultures, in Proskauer beck, up to OD of 0.5-0.7, aliquoted vir test in GKO/B6 mice

mouse-passaged, frozen, sub-culutured


The strain is grown as pellicle.

In vitro.

Passaged through mice, preserved as seedlots

liquid culture to late log, then passaged through 8 uM filter, frozen in PBS-Tween 80, occaisionally passaged in mice

Is there a record of passage number kept for your M. tuberculosis strain used? If so explain this information and detail the maximum number of passages?

Have you ever noticed a difference in results of a treatment study depending on the M. tuberculosis strain used?

We have only used the H37Rv strain

No

of course if strain is less virulent

ND Erd>Rv virulence in GP & GKO mice

yes

yes

We never compared

We use always the same strain.

We use only ATCC 27294

only use Erdman strain

We have only used the H37Rv strain

No

of course if strain is less virulent

ND Erd>Rv virulence in GP & GKO mice

yes

yes

We never compared

We use always the same strain.

We use only ATCC 27294

only use Erdman strain

What media is used to propagate the bacterial culture and what (if any) concentration of Tween is used?

Dubos broth with ADCC enrichment

7H9, No Tween80

7H9/ADC/glycerol/0.05% Tween 80

Difco 7H9 with OADC Tween 80

H37Rv is propagated in 7H9-ADS medium containing 0.05% Tween 80

7H9 broth with 0.02% Tween 80.

7H9

7H9 medium plus glycerol, casitone and OADC - no Tween

Dubos broth with ADCC enrichment

7H9, No Tween80

7H9/ADC/glycerol/0.05% Tween 80

Difco 7H9 with OADC Tween 80

H37Rv is propagated in 7H9-ADS medium containing 0.05% Tween 80

7H9 broth with 0.02% Tween 80.

7H9

7H9 medium plus glycerol, casitone and OADC - no Tween

Is the M. tuberculosis culture grown every time prior to the infection? If yes, on what parameter is the inoculum based (e.g. OD, Klett, etc)? Or do you work with standardized, frozen working stocks?Please explain the rationale for the method used.

No. A frozen stock is prepared and stored in small aliquots at minus 80C. Each time an experiment is performed, a vial is thawed and diluted to the appropriate concentration based upon the known cfu for that stock. This method allows a precise duplication of the infection level each time an experiment is performed. There is no detectable change in infectivity or virulence over many months or years.

No. OD is not reliable because if there are clumps of bacteria.

Yes (OD)

From frozen stock, culture is grown for ~ 7 days in Difco 7H9 broth OADC Tween 80

No,it is grown in a 500 ml flask only every few years and large aliquots are made once the OD 600 approaches late log (0.6-0.9). The titers are confirmed by plate counts and the virulence is checked in GKO mice. These large number of aliquots will last our lab for years. These aliquots are then frozen at -80 C and stored so that each experiment will have a new aliquot of known titer and virulence. We have noticed, in our last batch, that the viability drops down after prolonged storage at -80 C (e.g. 4 years)

yes, OD. We favor infecting with an actively growing culture.

no, we use standardized frozen stocks

We work with standardized frozen working stocks. Mtb is grown to an OD600 of 0.3-0.5 in 7H9-ADS medium, centrifuged 10 minutes at 3,300rpm and the pellet is washed once in warm 7H9 medium. The washed pellet is resuspended at an OD600 of 1.0 in 7H9-ADS medium supplemented with 15% glycerol and frozen at -800C. The viable count of each batch of frozen cultures is determined by plating prior to infection. The frozen aliquot is diluted and sonicated (25 sec in a water bath sonicator) before infection.

We grow frozen stocks and select the inoculum according to McFarland suspensions.

No. Frozen seedlot with known number of CFU maintained. Thawed, diluted and used.

Standardized frozen working stocks. Since treatment is not initiated until 10 days post-infection, the physiological status at the time of infection should not affect drug susceptibility.

No. A frozen stock is prepared and stored in small aliquots at minus 80C. Each time an experiment is performed, a vial is thawed and diluted to the appropriate concentration based upon the known cfu for that stock. This method allows a precise duplication of the infection level each time an experiment is performed. There is no detectable change in infectivity or virulence over many months or years.

No. OD is not reliable because if there are clumps of bacteria.

Yes (OD)

From frozen stock, culture is grown for ~ 7 days in Difco 7H9 broth OADC Tween 80

No,it is grown in a 500 ml flask only every few years and large aliquots are made once the OD 600 approaches late log (0.6-0.9). The titers are confirmed by plate counts and the virulence is checked in GKO mice. These large number of aliquots will last our lab for years. These aliquots are then frozen at -80 C and stored so that each experiment will have a new aliquot of known titer and virulence. We have noticed, in our last batch, that the viability drops down after prolonged storage at -80 C (e.g. 4 years)

yes, OD. We favor infecting with an actively growing culture.

no, we use standardized frozen stocks

We work with standardized frozen working stocks. Mtb is grown to an OD600 of 0.3-0.5 in 7H9-ADS medium, centrifuged 10 minutes at 3,300rpm and the pellet is washed once in warm 7H9 medium. The washed pellet is resuspended at an OD600 of 1.0 in 7H9-ADS medium supplemented with 15% glycerol and frozen at -800C. The viable count of each batch of frozen cultures is determined by plating prior to infection. The frozen aliquot is diluted and sonicated (25 sec in a water bath sonicator) before infection.

We grow frozen stocks and select the inoculum according to McFarland suspensions.

No. Frozen seedlot with known number of CFU maintained. Thawed, diluted and used.

Standardized frozen working stocks. Since treatment is not initiated until 10 days post-infection, the physiological status at the time of infection should not affect drug susceptibility.

Do you perform pharmacokinetic/pharmacodynamic assays before or concurrent with your animal studies? Please elaborate on the methods, computational analysis, mathematical model used and results obtained. Do you have methodology and data on bioequivalence to human infection and would you be willing to share these results?

No

No

Not always

Of course extensively in relation to multiple factors

not applicable

Before testing the efficiacy the a compound in vivo, we test oral bioavailability by assay or HPLC. Testing of early lead cmpds in the GKO model is generally performed at the highest possible dose to see effect (up to 300 mg/kg). Before any long term mouse studies are started PK analysis is performed to establish appropriate dose (equivalent dose compared to humans for known TB drugs) . Methods are from our pharmacokinetics partner at National Jewish

typically yes. we usually obtain PK curves using 6-8 timepoints following single doses or steady-state dosing, obtaining serum or plasma by cardiac puncture using 3 mice per timepoint. Non-compartmental analysis is performed using WinNonlin. We have data on bioequivalence for 1st-line drugs and some second-line drugs. We also often perform combination drug studies to rule out significant PK interactions.

done for us by collaborator

Maximum tolerated dose (MTD) and pharmacokinetic/pharmacodynamic assays of all compounds are done before animal studies. Based on in vivo PK data in the relevant species where efficacy is tested, the WinNonLin software is used to model the PK profile and simulate a range of doses and dosing regimens.PK/PD indices (AUC/MIC,Cmax/MIC,T>MIC ) are then determined at each dose in order to selectdoses expected to be efficacious in the infection model.

We do PK (Cmax, AUC, T1/2, Tmax are determined)assays before or concurrent with animal studies.

Before and concurrent. WinNonLin is used.

Only a quick oral bioavailability study at the highest determined dose. Take blood samples from retro-orbital sinus at 15, 45 and 75 minutes post-dose. Use either LC-MS or TB bioassay (using a luciferase reporter strain) to estimate serum concentrations.

No

No

Not always

Of course extensively in relation to multiple factors

not applicable

Before testing the efficiacy the a compound in vivo, we test oral bioavailability by assay or HPLC. Testing of early lead cmpds in the GKO model is generally performed at the highest possible dose to see effect (up to 300 mg/kg). Before any long term mouse studies are started PK analysis is performed to establish appropriate dose (equivalent dose compared to humans for known TB drugs) . Methods are from our pharmacokinetics partner at National Jewish

typically yes. we usually obtain PK curves using 6-8 timepoints following single doses or steady-state dosing, obtaining serum or plasma by cardiac puncture using 3 mice per timepoint. Non-compartmental analysis is performed using WinNonlin. We have data on bioequivalence for 1st-line drugs and some second-line drugs. We also often perform combination drug studies to rule out significant PK interactions.


Maximum tolerated dose (MTD) and pharmacokinetic/pharmacodynamic assays of all compounds are done before animal studies. Based on in vivo PK data in the relevant species where efficacy is tested, the WinNonLin software is used to model the PK profile and simulate a range of doses and dosing regimens.PK/PD indices (AUC/MIC,Cmax/MIC,T>MIC ) are then determined at each dose in order to selectdoses expected to be efficacious in the infection model.

We do PK (Cmax, AUC, T1/2, Tmax are determined)assays before or concurrent with animal studies.

Before and concurrent. WinNonLin is used.

Only a quick oral bioavailability study at the highest determined dose. Take blood samples from retro-orbital sinus at 15, 45 and 75 minutes post-dose. Use either LC-MS or TB bioassay (using a luciferase reporter strain) to estimate serum concentrations.

Do you perform toxicity assays before or concurrent with your animal studies? Please elaborate on the methods used and results obtained.

No

Yes. We check the survival and pathology of tissue (live, spleen, kidney).

Yes. Dose at higher than anticipated conc for a week and see if mice lose weight

no

n/a

An acute and chronic Maximum Tolerated Dose assay is done before any in vivo work. This usually is meant to establish dose and is limited to behavior changes and gross necropsy findings.

We might perform an MTD assay similar to that described by CSU on TAACF website.


Prior to in vivo PK and efficacy studies, in vitro toxicity and in vivo tolerability studies in the relevant animal species are carried out. IN VITRO toxicity assays -cytotoxicity assay against a panel of 4 different cell lines namely, THP-1 (human monocyte cell line, suspension), BHK21 (Syrian Golden hamster kidney cells, adherent), HepG2 (Human Liver Carcinoma, adherent) and C6 Glioma (Rat brain glioma, adherent). (Cell lines obtained from ATCC) -genotoxicity (Ames test) -cardiotoxicity (hERG binding and patch clamp assays) IN VIVO assays Maximal tolerable dose in mice. Doses are selected based on the in vitro potency

We do toxicity assays before doing animal studies. The tests are Helam and Vero in vitro tests.

Toxicity assays in vivo per se are not conducted prior to efficacy studies.

Cellular toxicity for VERO and J774 cells and in some case HepG2 cells. Tolerance test in mice using oral gavage and visual observation. Dose once daily for 5 consecutive days up to a maximum of 400 mg/kg.

No

Yes. We check the survival and pathology of tissue (live, spleen, kidney).

Yes. Dose at higher than anticipated conc for a week and see if mice lose weight

no

n/a

An acute and chronic Maximum Tolerated Dose assay is done before any in vivo work. This usually is meant to establish dose and is limited to behavior changes and gross necropsy findings.

We might perform an MTD assay similar to that described by CSU on TAACF website.


Prior to in vivo PK and efficacy studies, in vitro toxicity and in vivo tolerability studies in the relevant animal species are carried out. IN VITRO toxicity assays -cytotoxicity assay against a panel of 4 different cell lines namely, THP-1 (human monocyte cell line, suspension), BHK21 (Syrian Golden hamster kidney cells, adherent), HepG2 (Human Liver Carcinoma, adherent) and C6 Glioma (Rat brain glioma, adherent). (Cell lines obtained from ATCC) -genotoxicity (Ames test) -cardiotoxicity (hERG binding and patch clamp assays) IN VIVO assays Maximal tolerable dose in mice. Doses are selected based on the in vitro potency

We do toxicity assays before doing animal studies. The tests are Helam and Vero in vitro tests.

Toxicity assays in vivo per se are not conducted prior to efficacy studies.

Cellular toxicity for VERO and J774 cells and in some case HepG2 cells. Tolerance test in mice using oral gavage and visual observation. Dose once daily for 5 consecutive days up to a maximum of 400 mg/kg.

Do you routinely test the ADME (absorption, distribution, metabolism and elimination) of compounds?

No

No

No

no

not presently

We test oral bioavailability to evaluation absorbtion, and test drug metabolism.

no

no

Both in vitro and in vivo ADME assays are performed prior to efficacy testing.

No.

Yes

Routinely perform CYP3A4 inhibition using the VIVD fluorscent kit. Just beginning to routinely perform microsome stability and to a lesser extent, protein binding and Caco-2 uptake.

No

No

No

no

not presently

We test oral bioavailability to evaluation absorbtion, and test drug metabolism.

no

no

Both in vitro and in vivo ADME assays are performed prior to efficacy testing.

No.

Yes

Routinely perform CYP3A4 inhibition using the VIVD fluorscent kit. Just beginning to routinely perform microsome stability and to a lesser extent, protein binding and Caco-2 uptake.

Regarding ADME, what assays do you use? What sequence of tests is utilized and when in the pre-clinical sequence are these implemented?

Not applicable

N/A

For oral bioavailability we use a bioassay using M. tuberculosis to estimate drug levels in serum. This assay is done before any in vivo efficiacy studies are initiated. For drug metabolism we use microsome assay. This assay takes usually place in parallel with in vivo efficacy testing.

NA

IN VITRO: -solubility at pH 1.0 and pH 6.8 -PAMPA (artificial membrane permeability) -CaCO2 assay -Microsomal stability in mouse and human microsomes -CYP450 inhibition IN VIVO -single dose pharmacokinetic study following intravenous and oral administration to determine oral bioavailability and PK parameters (half –life,clearance,Cmax,AUC,volume of distribution). All these tests are used prior to efficacy testing in rodents during the lead optimization phase.At pre-clinical stage, more extensive ADME studies are conducted in rat and one non rodent species,including tissue distribution studies with radio-labelled compounds.

Once a compound has shown a good activity in vivo, we do PK (Cmax, Cmin, AUC, T1/2, Tmax).

In vitro tests: Caco2, PAMPA, Microsomal clearance, In vivo: Balb/c mice - uninfected and infected mice for DM and PK.

Beginning to use prior to any mouse testing: 1) CYP3A4 inhibition 2) human and mouse microsome stability 3) Caco-2 passage and protein binding

Not applicable

N/A

For oral bioavailability we use a bioassay using M. tuberculosis to estimate drug levels in serum. This assay is done before any in vivo efficiacy studies are initiated. For drug metabolism we use microsome assay. This assay takes usually place in parallel with in vivo efficacy testing.

NA

IN VITRO: -solubility at pH 1.0 and pH 6.8 -PAMPA (artificial membrane permeability) -CaCO2 assay -Microsomal stability in mouse and human microsomes -CYP450 inhibition IN VIVO -single dose pharmacokinetic study following intravenous and oral administration to determine oral bioavailability and PK parameters (half –life,clearance,Cmax,AUC,volume of distribution). All these tests are used prior to efficacy testing in rodents during the lead optimization phase.At pre-clinical stage, more extensive ADME studies are conducted in rat and one non rodent species,including tissue distribution studies with radio-labelled compounds.

Once a compound has shown a good activity in vivo, we do PK (Cmax, Cmin, AUC, T1/2, Tmax).

In vitro tests: Caco2, PAMPA, Microsomal clearance, In vivo: Balb/c mice - uninfected and infected mice for DM and PK.

Beginning to use prior to any mouse testing: 1) CYP3A4 inhibition 2) human and mouse microsome stability 3) Caco-2 passage and protein binding

At what stage in the process of drug development is formulation optimized and finalized?

Not applicable

not applicable

after 3 sucessful MIC determinations and cytotox assays

Formulation is optimized at lead declaration, and finalized during the early pre-clinical phase.

During the pre-clinical phase for in vivo use.

Late lead optimisation after shortlisting a few candidates for safety testing.

do very little work in formulation, just enough to make an even suspension for oral administration

Not applicable

not applicable

after 3 sucessful MIC determinations and cytotox assays

Formulation is optimized at lead declaration, and finalized during the early pre-clinical phase.

During the pre-clinical phase for in vivo use.

Late lead optimisation after shortlisting a few candidates for safety testing.

do very little work in formulation, just enough to make an even suspension for oral administration

Do you routinely test colonies for drug resistance?

What inoculum do you use for infection of each animal model used in your lab?

We use an inhaled and retained dose of approximately 10-20 cfu per guinea pig

200-300 cfu delivered to mice.

100CFU/lung

in general, our aim is to implant 5x10e3 CFU in the lungs and begin treatment at Day14 after infection

low dose aerosol (50-100) CFU implantation at day 1 after infection

aerosol dose may range from 10 to 10000 CFU depending on the purpose of the experiment


500-1000CFU

7 log10 CFUs.

Mice Aerosol infection: 100 cfu/animal. Madison chamber Mice I.v. ~10^6 cfu/lung G.P aerosol: 10 cfu/animal G.P i.m: 10^6 cfu/animal

M. tuberculosis Erdman

We use an inhaled and retained dose of approximately 10-20 cfu per guinea pig

200-300 cfu delivered to mice.

100CFU/lung

in general, our aim is to implant 5x10e3 CFU in the lungs and begin treatment at Day14 after infection

low dose aerosol (50-100) CFU implantation at day 1 after infection

aerosol dose may range from 10 to 10000 CFU depending on the purpose of the experiment


500-1000CFU

7 log10 CFUs.

Mice Aerosol infection: 100 cfu/animal. Madison chamber Mice I.v. ~10^6 cfu/lung G.P aerosol: 10 cfu/animal G.P i.m: 10^6 cfu/animal

M. tuberculosis Erdman

Do you consider your model to be a low or high infection dose animal model?

Very low

not high, not low

low

high infection

low dose

we use both

Low infection dose animal model

High infection dose animal model.

Aerosol: low dose; Parenteral: high dose

low dose

Very low

not high, not low

low

high infection

low dose

we use both

Low infection dose animal model

High infection dose animal model.

Aerosol: low dose; Parenteral: high dose

low dose

The normal infectious dose for humans is likely to be very low, since a single, primary,pulmonary

lesion is almost always observed.

I think about 10 cfu

is ideal. but 200-300 cfu/mouse at inital

aerosol challenge is good to test Drug candidate because the bacteria grow enough number in the lung and spleen.

More like human in that disease becomes chronic

to obtain a bacillary population close to the bacillary population in human TB

most closely mimics natural infection.

high dose of 3.5-4 logs is implanted in order to have a burden of 7-8 logs at start of combination therapy 14 days later. in this way, it takes approximately 6 months of treatment with the standard RHZ-based regimen to cure all mice. low-dose aerosol may be used for monotherapy

trials or LTBI models to prevent emergence of resistance or to better represent the lower bacterial burden of LTBI.


As we have an intranasal, low dose infection model, the chosen inoculum

is the lowest which can establish a lung infection in our animal models.But

if very low inoculum

is used , chances are there some bacteria get trapped in the airway and not reach the lungs (standard deviation was more when less than 5 X 102 bacteria was used.

We want to achieve a large bacterial population such as in human

cavities at the time we start treatment.

In the case of aerosol infection model, low dose challenge is sufficient to establish the infection and is presumably close to natural conditions fo

infection in humans (which is thought to be as low as 1-3 cfu).

human mimic

What is the rationale behind the inoculum chosen for your infection model?

The bacterial numbers are known from the cfu

determination on the frozen inoculum; the reproducibility of this method over decades has been remarkable; there is no need to perform Day 1 lung cfu

determinations unless the challenge strain changes

We validate each bacterial stock batch. -

cfu

numbers after aerosol challenge. -

3 mice, two times. There was almost no difference of bacterial numebrs

among same batch because we removed the clumps by filteration.

Plate lungs at t=0

We do everything; CFU count of (i) the suspension to inoculate (ii) lung the day after infection and (iii) day of treatment initiation

We plate the inoculum

in serial 1:10 dilutions. We do a day 1 enumeration of the lungs and a pretreatment enumeration (typically 21 days after infection) of 5 mice.

we plate inoculum

suspension and enumerate bacterial CFU the day after infection as well as the day of treatment initiation. we typically culture the organs from 5 animals.



suspension for every infection experiment.The

bacterial numbers are enumerated after each infection like the day after the infection as well as on the day of treatment initiation. 5 mice each will be sacrificed one day after the infection and prior to treatment.


suspension. We enumerate the bacterial numbers one day after infection and on the day we start treatment in 5 to 6 animal organs.

Seedlot

system for which the cell number is known and is nearly constant over a very long period of time at -70C. Additionally, before and after infection, the inoculum

is plated to ascertain consistency. For most expts, first day sampling is not done. However, for mutant studies, day 1 sampling is done. In all drug treatment studies, pre-drug sampling is done. four animals per group. Only Lungs plates.

Inoculum

suspension is plated on the day of infection. Five mice are sacrificed on day 3 post-infection and 7 mice at the start of treatment of day 10 post-infection.

How is the infection inoculum for every infection experiment checked for accurate bacterial numbers? Do you plate the inoculum suspension? Do you enumerate bacterial numbers early after infection (such as the day after infection) and prior to treatment? Tissue from how many animals/organs are plated?

We usually plate the homogenate of the same large lobe (usually lower right)from

each animal; if very low numbers of bacteria are expected, a larger portion (including the whole lung) can be homogenized and a sizeable fraction of the homogenate (one-third to one-half) plated.

Both Lung and spleen.

4 mice per time point per concentration

We may use in that case more limited inoculum. and plate only one lung when the other is used for example for

histopathology

no, both

yes

We plate serial dilution of the whole lung for testing compounds

No, we plate both lungs.

In most expts, both lungs are plated. If HP samples are needed, lungs from satellite animals used for PK are collected.

No, whole lung is always used.

For testing of single compounds and short term experiments, do you plate only one lung? Please explain.

When organs are fixed for pathology or other purposes, which type of fixative do you use for the tissues? Please elaborate on the methods for fixation including how long the tissues are fixed.

We usually fix a lung lobe, a part of the spleen and a piece of liver

3 days

fixed for at least 3 days

Tissues intended for pathology are harvested and maintained moist by soaking in 0.9% Sodium Chloride and then transferred into 10% neutral buffered formalin fixative.

For more than 10 days.

We usually fix a lung lobe, a part of the spleen and a piece of liver

3 days

fixed for at least 3 days

Tissues intended for pathology are harvested and maintained moist by soaking in 0.9% Sodium Chloride and then transferred into 10% neutral buffered formalin fixative.

For more than 10 days.

Other responses

No

no.

No

not really

Yes, in the Guinea pig model we see remaining TB bacilli present

in the necrotic region of the primary granuloma

after extensive drug treatment (for instance with TMC 207)

no

no

No

Yes. Meteorism

is found in mice treated with high dose of moxifloxacin.

Yes.

no

Have you ever noticed lesion-specific pathologic or microbial effects in any of your drug treatment arms?

No. Cavities do occur in guinea pigs, but infrequently

No.

No

No even when tried to sensitize mice

The guinea pig shows necrosis in the primary granulomas

and after infection with certain clinical strains shows cavitation

and mineralisation

(no liquefication). Some necrosis is seen only in later stages of infection in mice. We do not do presensitization

no

no

No

No. We have never observed cavitation

or necrosis of lung lesions in mice. We do not perform any pre-sensitization of the animal to induce cavities.

Not in mice. Caseation

in guinea pigs

no

Have you ever observed cavitation or necrosis of lung lesions? Do you perform any pre-sensitization of the animal to induce cavitary disease? Please elaborate.

Are there other outputs measured to confirm or supplement the CFU data? Please check all that apply.

We normally plate a fraction (one-tenth) of the homogenate from one large lung lobe and half of the spleen;

1) all lung lobe 2) total spleen

All of lung, all of spleen

Total lung and sometimes total spleen too

n/a

For short term trials usually with single compounds, we collect left lungs and spleens, transfer in 4.5 mls

of saline and plate 1:5 (0-7). For long term trials looking at sterilization of compounds, we collect whole lung, transfer in 1 ml and plate completely. Same for spleen.

we homogenize both lungs in a total of 2.5 ml PBS and then plate

the entire homogenate on 5 7H11 plates (0.5ml/plate)

we do not do this

The entire homogenate is not plated. Whole lungs and total spleen are asceptically

removed and ground using a tissue homogenizer in 4 ml of Phosphate Buffered Saline (PBS) supplemented with 0.05% Triton X-100. We take 25ul of the homogenate , serial dilute (diluent

used is 7H9+ADS medium) it and plate 50ul from each dilution (we

plate 50ul of undiluted homogenate as well) This we extrapolate

to entire homogenate (whole lung).

We plate 2 lobes and also the total spleen. The entire homogenate is plated if the treatment is potent and given for a long duration and we expect few or no bacilli.

Total mice lung plated. Entire homogenate plated without dilution. Homogenised

in 1 ml volume and plated on 5 plates per animal.

Entire lung is removed, homogenized in 3 ml HBSS and serially diluted in HBSS. One hundred microliters

of undilute

or diluted suspension is plated on 7H11 agar in duplicate.

Regarding long term trials: Do you plate: 1) all or only one lung lobe? 2) total spleen? 3) other organ(s)? Is the entire homogenate plated?

In a long-term sterilizing experiment, what fraction of the organs is plated? What is the volume of diluent

used?

One-third to one-half; 5 ml (L);2 ml (S)

Lung and spleen, 5ml

Entire lung in 2.5ml of PBS (5 plates)

the whole organ and 1 ml diluent

all of lungs in 2.5 ml PBS

we do not do this

Assay not established in our institute.

The entire fraction is plated (2.5 ml ).

Total lung in 1 ml.

NA

In a long-term sterilizing experiment, what fraction of the organs is plated? What is the volume of diluent used?

Yes

n/a

yes

we do not do this

NA

If the entire fraction is plated, this is mentionned

in publications. If it's mentionned

in the publications that dilutions are plated this means that not all the tissue is plated.

If not all the tissue is plated, is this mentioned in your methods of publication?

Yes; up to 20 weeks following cessation of the drug regimen; with and without immunosuppression

(hydrocortisone; cyclophosphamide); clinial

signs, cfu, histopathoology

from 12 weeks to 40 weeks after the end of treatment. we check the bacterial number by CFU counting. We used Dexamethason. Mice received subcutaneously Dx.

No

Of course, it is the basic method to assess cure: 3 to 6 months after treatment completion, culture of the entire lung (and/or spleen). Standard statistical method

I have done very few relapse studies up to date. The few studies

involved 3 months of relapse time after cessation of Rx. In only one prior drug study (rifazalil) I used dexamethasone

as immunosuppressant and check with PCR for remaining DNA.

yes, 3 months post-treatment, no immunosuppression, we plate only the undiluted homogenate and cannot quantify counts >1000 CFU. We compare relapse proportions using Fishers exact test, controlling for multiple comparisons as necessary.

we do not do this

We have not done an experiment to test relapse of a regimen.

In the experiments we carried out to test relapse of a regimen, the duration between the end of treatment and the sacrifice of animals is always 3 months. We don't use immunosuppression

agents. When measuring relapses, spleen are weighed, gross lung

lesions are scored and CFU counts are determined by plating the entire homegenate

of the lungs or a spleen on media. The relapse rates are compared using the Fisher exact test or the Khi2 test.

Yes. Post completion of exptl

Rx, 4 weeks of 'sterile' period, followed by dexa

0.5 mg/animal uid

i.p. for 4 weeks, followed by 2 & 4 week sampling for relapse.

no

Have you ever done an experiment designed to test relapse of a regimen? If so, please elaborate on the time between the end of treatment and final sacrifice of the animal. Please list any immunosuppression agents utilized. Can you describe methods used to assess bacterial burden (e.g. CFU counts, gross observations, PCR etc)? What statistical method was used?

No

No

No

Metronidazole

and PZA are drugs that have shown activity in some labs in mice

but were not effective in our lab.

not really, although we typically observe antagonism of INH on the RIF-PZA combination and this has not been well explored in other models.

we do not do this

CFU counts are below limit of detection after only two weeks of treatment with isoniazid

(10mg/kg) and moxifloxacin

(100mg/kg)

No.

No.

no

Have you ever obtained unexpected data using your models which contradict published results or results presented by others? Please elaborate.

Not applicable

No

we do not do this

We had unexpected outcomes in animal trial due to human error ( dilution, plating, labelling

) which was tracked down and rectified.

No.

No.

If you have had unexpected outcomes of an animal trial 1) were you able to track down the problem? and 2) how was it rectified?

Anyone who knows clinical TB and reads the guinea pig literature

will recognize the obvious relevance of this model

no.

Yes

The mouse pathology has no similarity at all with the human pathology. Thus our aim is not to mimic human disease but to mimic the response of M.tuberculosis

to drugs used at equivalent doses

Mouse models are certainly the best animal models for early preciminal

evaluation of experimental compounds. However, as the mouse model has its limitations (only one lesion type is present and all bacilli are intracellular) we feel there is a need for an additional animal model to address these issues and to confirmation results.

it takes approximately 6 months to cure all mice with RHZ-based regimens. the sterilizing activity. the sterilizing activity of R and Z are very evident when they are removed.

we do not do this

The acute mouse model we use has severe limitations in reproducing human disease: high bacillary load during the chronic phase, lack of a true latent phase, structure of the granulomas, lack of caseation

and fibrosis, overall course of the disease. Having all of these shortcomings in mind, we use the model as a means to confirm in vitro potency and PK of our compounds in an in vivo system, without making quantitative conclusions as to the magnitude of the treatment response. Despite its many flaws, we found that the mouse is a reasonable compromise to qualitatively and rapidly assess the efficacy of new compounds in a medium throughput manner.

The only evidence that supports the ability of our animal model to predict human outcomes is the fact that with our model the RHZ regimen displays almost the same relapse rate as in patients. This relapse rate is never observed in the aerosol model for instance.

the biggest problem in tb

drug development

no

Have you any evidence to support or refute the ability of yours or any animal model to mimic human disease and predict treatment outcomes?

Yes; good

Yes. We use INH, RIF, Moxi, Gati. We got similar data until now.

Yes (INH)

Of course we have always negative and positive controls, and are

amazed of the reproducibilty

of the results when all parameters are similar

In early testing of compounds in the GKO model, INH at 25 mg/kg is always included (published in GKO paper). For long term combination treatment trials the standard regimen INH-RIF-PZA is included and this regimen (2 log killing in the first 3-4 weeks and 3 more logs in the next 4 weeks). All controls are reproducible in the mouse.

high reproducibility

Yes, we test control drugs (Rifampicin

10mg/kg or Isoniazid

10mg/kg) in every experiment. Rifampicin

shows significant potency to control Mtb

infection in this model. We observed 1.5 –

2 log10 bacterial load reduction in the lungs after 14 days of treatment and 2 to 3 log10 reduction after 28 days of treatment .The infection in the spleen completely controlled by Rifampicin

, the average bacterial count being below the limit of detection at 2 and 4 weeks post infection. For Isoniazid

, after 14 days of treatment the bacterial load comes below the

limit of detection (80 bacteria)

We test isoniazid

in every experiment. The activity of isoniazid

used in the same conditions (inoculum

size, established or non-establised

infection, duration of treatment, frequency of administration) is quite reproducible.

Rif and Inh. Excellent reproducibility.

rifampin

at 10 mg/kg and in one project, PA-824 at 100 mg/kg is used instead. Reproducibility is good with rifampin

but can vary with PA-

824 from clearly bactericidal to nearly bacteriostatic

Do you routinely test any control drugs in every experiment?How is the reproducibility of these results from experiment to experiment?

No

Some earlier relapse studies used a low number of mice, which could/should be revisited using a large number of mice.

no

yes, including catabolic steroid antibiotics, plant extracts, urea classe

compounds and spirodecane

class compounds.

We have recently revived the rat model of tuberculosis infection, based on the following observations and results. Recent studies (Elwood et al., 2007; Sugawara et al., 2004b; Sugawara et al., 2004c) produced encouraging results in terms of histopathology and colony forming units (CFU) profiles in the lungs of M. tuberculosis infected rats, that suggest the rat may indeed be a useful model for TB research in general and more specifically for testing new drug candidates. In addition, the tuberculin test is feasible in the rat (but not in the mouse), and a large array of immune reagents are available. Mtb

infection in rat lung exhibits both an acute phase (20-25 days of exponential growth) and a subsequent chronic phase (stabilization of the bacterial counts). The chronic phase is induced, as is the case in the mouse, at the onset of the cell-mediated immunity and can last for an extended period of time (Orme, 2003; Sugawara et al., 2004b). Mtb can reactivate and cause a premature death of the infected animals when there is a failure of the immune pressure (Sugawara et al., 2006). Preliminary results from Bifani

et al. (Unpublished observations) suggest that endotracheal

inoculation of mycobacteria

is a simple way to infect lung rat, very reproducible and prevent the spread of aerosols in BSL-3 environment. Because it is relatively inexpensive, small, easy

to handle, and has reasonably and accepted predictive value in toxicology and pharmacokinetics, the rat has been a species of choice for early drug development in the pharmaceutical industry. By using the rat model, in vivo efficacy data could be directly correlated with pharmacokinetic/pharmacodynamic

drug parameters and toxicology studies. We hope that once optimized, the model will provide a system which is time, cost and compound efficient, to substantially accelerate the discovery and early development of new anti-

tuberculosis molecules. References: Elwood, R. L., Wilson, S., Blanco, J. C., Yim, K., Pletneva, L., Nikonenko, B., Samala, R., Joshi, S., Hemming, V. G., and Trucksis, M. (2007). The American cotton rat: a novel model for pulmonary tuberculosis. Tuberculosis (Edinb) 87, 145-154. Orme, I. M. (2003). The mouse as a useful model of tuberculosis. Tuberculosis (Edinb) 83, 112-115. Sugawara, I., Udagawa, T., and Yamada, H. (2004a). Rat neutrophils

prevent the development of tuberculosis. Infect Immun

72, 1804-1806. Sugawara, I., Yamada, H., and Mizuno, S. (2004b). Pathological and immunological profiles of rat tuberculosis. Int

J Exp Pathol

85, 125-134. Sugawara, I., Yamada, H., and Mizuno, S. (2004c). Pulmonary tuberculosis in spontaneously diabetic goto

kakizaki

rats. Tohoku J Exp Med 204, 135-

145. Sugawara, I., Yamada, H., and Mizuno, S. (2006). Nude rat (F344/N-rnu) tuberculosis. Cell Microbiol

8, 661-667.

No.

Possibly, the Lurie

Rabbits, that had resistant and susceptible phenotypes. Also, in the 1970s D. Smith and D. Mitchison, used clinical strains from India to infect guinea pigs, these clinical strains

produced two different phenotypes of disease One chronic and one more progressive. Can we use these two as models of human latent and active disease perhaps?

Numerous anti-tuberculosis agents have been studied in the past decades. It is possible that there is information buried in these important studies that would be useful to us today. Can you think of antimicrobial agents and/or models that would be worthwhile revisting today? If so, please give your rationale for this and pertinent references.

Main treatment effects are analyzed first by ANOVA; only if the F statistic is significant is a valid between-means test applied to the individual treatment groups.

T-test, anova

test. We consult somtimes

with a statistician at our university.

Alas yes and it is very important

One way ANOVA, followed by stringent methods for pairwise

comparison such as Tukey

test, Dunnett's

test. For relapse studies we use include T-test (cured versus relapsed mice)

we conduct our own one-way ANOVAs and Fishers Exact tests, controlling for multiple comparisons. we do not consult with a statistician.

we do not do this

A compound is active in this model if the CFUs

are 10 times lower (1 log10 reduction) in the treated vs. untreated group. We don’t consult statistician.

We use basic statistics. We use parametric tests such as student

t-test when dealing with quantitative variables such as CFUs, spleen weights when we have all the conditions to use them such as similar variances otherwise we use non parametric tests such as Mann

Whitney. For the binary (qualitative) variables such as relapses, deaths, we use the exact Fisher test or the Khi2. We don't consult with statistician except if it's needed.

One-way ANOVA -

Dunnet's

Multiple Comparison Tests TTests

T test

What statistical methods are run in conjunction with your animal experiments? Do you consult with a statistician?

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