Geldanamycin
C h a p t e r 3 . M i c r o t i t e r p l a t e - b a s e d a s s a y f o r t h e
a s s e s s m e n t o f b i o a c t i v i t y
3.1 1ntroduction
M any biological assay techniques have been developed to assess the bioactivity o f com pounds. This entails determ ining the potency o f chem ical com pounds against m icroorganism s (Sin and W ong, 2003). W hen applied to antibiotic ferm entations, these m ethods use the bioactivity o f the broth as an indication o f antibiotic production. Traditional approaches involved the use o f the disk diffusion assay technique, w here the susceptibility of an organism to a sam ple would result in a zone o f inhibition, with a m agnitude related to the am ount o f bioactive com pound present in the sam ple (S elvakum ar e t a l . , 1999). T he disk diffusion assay proves unreliable in certain applications (Sw enson e t a l . , 1989) and can lead to interpretational problem s, including in-grow th in the zone o f inhibition, w hereby sparse growth of organism occurs w ithin the zone o f inhibition (Piliouras e t a l . , 2002) and subjectivity associated with visual assessm ent, such as interpretation o f w here inhibition zone boundaries are located (D eighton and Balkau, 1990). D eB oer e t a l . , (1970) took steps to standardise the assessm ent o f the zone o f inhibition and reported results in biounits, defined as the am ount o f antibiotic necessary to produce a 20 mm zone o f inhibition under standard conditions.
Using the disk diffusion method is tim e consum ing, m aterial intensive and, as a result, m ovem ent has been tow ards m ore standardised and high- th roughput m ethods o f bioactivity assessm ent (Brown, 1988). A num ber of m icrotiter plate-based assays have been developed fo r screening o f the antim icrobial activity o f natural products (D evienne and Raddi, 2002), determ ination o f the antim icrobial susceptibility patterns o f m icroorganism s (Jones and Dudley, 1997), determ ination o f m icroorganism adherence (D eighton and Balkau, 1990) and fo r the quantification o f biofilm form ation
inhibition (S tepanovic e t a l . , 2000). W hen assaying bioactivity using m icrotiter plate-based techniques, am biguities m ay be encountered in the determ ination o f biom ass growth trends and in the calculation o f the bioactive effect itself. These difficulties arise because standardisation o f the response o f m icroorganism s in these system s is difficult. in th e ir screening fo r antim icrobial activity.
This C hapter describes a method to calculate bioactivity o f sam ples, w hich
w hich also provides a strategy to successfully calculate th e MIC o f a bioactive com pound, would be o f significant benefit in this pursuit.
3 . 2 M a t e r i a l s a n d M e t h o d s
3.2.1 S tr a in a n d m e d ia
S t r e p t o m y c e s h y g r o s c o p i c u s v a r . g e l d a n u s (strain NRRL 3602 obtained from ARS P atent Culture Collection, Peoria, Illinois, USA) was used throughout this assessm ent. S pores w ere produced on B ennett’s medium agar containing: technical agar N o.3 (Oxoid, B asingstoke, England), 20 g/l; yeast extract (O xoid), 1 g/l; 'Lab-lem co' beef extract (Oxoid), 1 g/l; N- Z-am ine A (Sigm a-Aldrich, Dublin, Ireland), 2 g/l and dextrose m onohydrate (R iedel-de Haen, Seelze, G erm any), 10 g/l. Spores were recovered using resuspension solution containing: yea st extract (Oxoid), 3 g/l; bacteriological peptone (Oxoid), 5 g/l and M g S 0 4 ' 7H2O, 1 g/l. The ferm entation m edium w as Bennetts liquid m edium containing: yeast extract (O xoid), 1 g/l; 'Lab-lem co' beef extract (O xoid), 1 g/l; N-Z-am ine A (Sigm a-Aldrich), 2 g/l and dextrose m onohydrate (R iedel-de Haen), 10 g/l.
3 .2 .2 A n t ib io t ic f e r m e n t a t io n s a n d o r g a n is m p r e p a r a t io n
A spore inoculum o f S. h y g r o s c o p i c u s w as used to inoculate ferm entations and was prepared by culturing the organism on static cultures o f Bennett's m edium agar, in 5 L E rlenm eyer flasks, fo r 21 days at 28°C. The spores w ere recovered by w ashing with resuspension solution at 100 rpm for 1 hour at 4°C. B ennett’s media w as then inoculated at 1% using a spore suspension o f approxim ately 107spores/m l and incubated at 28°C at an agitation o f 150 rpm for at least seven days.
A t later stages in the project som e m odifications w ere made to the standard production m edium , and it w as found th a t higher yields of geldanam ycin w ere achieved using m odified B e n ne tt’s m edium (using 20 g/l to 50 g/l dextrose m onohydrate instead of 10g/I). M ethods such as the production o f spore stock and the general S t r e p t o m y c e s h y g r o s c o p i c u s
var. g e l d a n u s ferm entations are generally conserved throughout this
docum ent and the above sections should be referred to when considering ferm entation conditions.
3 .2 .3 M ic r o t it e r a s s a y m e d iu m r e q u ir e m e n t s
YEPD m edia w as used as nutrient source in th e bioassay and contained:
yeast extract (Oxoid, Basingstoke, England), 10 g/l; bacteriological peptone (Oxoid), 20 g/l; dextrose m onohydrate (R iedel-de Haen, Seelze, G erm any), 20 g/l. The disk diffusion assays w ere perform ed on YEPD m edium agar containing: technical agar N o.3 (O xoid), 20 g/l; yeast extract (Oxoid), 10 g/l; bacteriological peptone (O xoid), 20 g/l; dextrose m onohydrate (Riedel-de Haen), 20 g/l.
3 .2 .4 M ic r o t it e r a s s a y t e s t o r g a n is m s
Three te st organism s were used in the assay, B a c i l l u s s u b t i l i s strain 1650 (NCIM B Ltd. - National C ollection o f Industrial and M arine Bacteria, A berdeen, Scotland), E s c h e r i c h i a c o l i strain 9485 (NCIM B Ltd.) and bakers yeast S a c c h a r o m y c e s c e r e v i s i a e obtained in Active Dried Yeast (ADY) form (DCL Yeast Ltd, Surrey, England). The test organism s were grown in YE P D media cultures, fo r 24 hours, until in a log phase o f growth was achieved. The cells w ere then harvested, resuspended in 40% (w/v) glycerol (BDH laboratory supplies, Poole, E ngland), dispensed into 1ml aliquots, frozen and stored. A 1 m i-aliquot o f test organism was thawed and added to 9 mis o f sterile w ater for use as inoculum in the assay. A fter thaw ing, a short lag period is observed, how ever the im pact o f this is m inim ised by the 24 hour incubation period o f the assay. These test organism s were selected in order to exam ine broad spectrum bioactivity, on Gram positive, Gram negative and eukaryotic m icroorganism s.
3 .2 .5 M ic r o t it e r b io m a s s s t a n d a r d c u r v e g e n e r a t io n
To develop the standard curves o f biom ass concentration versus turbidity, a te st organism w as grown fo r 24 hours in Y E P D m edia. This stock culture was then serially diluted in spent m edia to give a range o f samples for biom ass concentration and turbidity analysis.
Biom ass concentrations w ere determ ined using dry-w eight analysis.
Clean, labelled, glass universals w ere dried in an oven (100°C, 24 hours).
These w ere placed in a desiccator, w eighed and retained fo r later use. 10 m l-aliquots o f the culture sam ples w ere centrifuged at 3500 rpm fo r 10 minutes. The supernatants w ere discarded and the pellets were retained and resuspended in ethanol. This biom ass slurry w as transferred to the glass universals, placed in a 100°C w ater-bath and the ethanol evaporated. The universals w ere dried fo r 24 hours and again cooled and w eighed. The dry-w eight biom ass concentration w as determ ined by subtracting the w eight o f the glass universal from that o f the glass universal plus dried biom ass. The analysis w as perform ed in duplicate.
The turbidity o f the culture sam ples w ere recorded using a Tecan, Spectra Classic, A -5082 plate reader and associated data retrieval software (Tecan, M annedorff, Switzerland). 300 pi of th e te s t culture w as added to the w ells o f a sterile, polystyrene 96-well m icrotiter plate (Sarstedt, W exford, Ireland) and the turbidity read at 570 nm. The analysis was perform ed in duplicate.
Biom ass concentration w as plotted against tu rb idity to generate standard curves. This process was perform ed for each test organism and the results are shown in Figure 3.1. The equations o f th e curves of best fit (obtained from a polynom ial regression fit o f the data, using Sigm aPlot Regression W izard, from Systat Software UK Lim ited. London, UK), for each standard curve, are given in Table 3.1.
3 .2 .6 M ic r o t it e r b io a s s a y
50 ¡j\ o f sam ple w as added to the w ells o f a m icrotiter plate. This was follow ed by the addition o f 200 / j \ o f YEPD m edium and 50 //I o f test organism inoculum . The contents w ere mixed by draw ing the solution up and dow n in a m ultipipetter a num ber o f tim es. The plates w ere aseptically read in the plate reader at 570 nm and the turb idity recorded. The plates w ere incubated at 30°C fo r 24 hours, and the turbidity read again. These turbidity values w ere converted to biom ass concentrations and used in the calculation o f bioactive effect. For assaying o f ferm entation sam ples, the control was 50 //I o f B ennett’s m edium containing: yeast extract (Oxoid), 1 g/l; 'Lab-lem co' beef extract (Oxoid), 1 g/l; N -Z-am ine a (Sigm a-Aldrich, Dublin, Ireland), 2 g/l; dextrose m onohydrate (R iedel-de Haen), 10 g/l.; fo r non-ferm entation derived sam ples, the control w as 50 ¡ j \ o f sterile deionised water.
3 .2 .7 C a lc u la t io n o f th e b io a c t iv e e ffe c t
Having established the turbidity values prior to and im m ediately following incubation, the bioactive effect o f the sam ple on test organism growth could be calculated. Turbidity values w ere converted to biomass concentrations using the established standard curves. W here bacterial growth w as com pletely retarded, no increase in turbidity would result, therefore signifying no increase in biom ass concentration during incubation. On this basis, an equation was developed to describe the level o f growth retardation fo r a sam ple. This equation took the follow ing form:
R = (<^24 - Q ) - f e , - r o ) j y 1 0 0 ( £ q 3 1 )
w here: R is the R etardation o f biom ass growth (%), ( C 2 4 - C 0 ) is the biom ass growth in the control w ells (g/l) determ ined by subtracting initial biom ass concentration in the control w ells from that after incubation for 24 hours at 30°C and (T 24 - T 0 ) is the biom ass growth in the sam ple wells
(g/l) determ ined by subtracting initial biom ass concentration in the sam ple wells from that after incubation fo r 24 hours at 30°C.
3 .2 .8 D o s e - r e s p o n s e c u r v e d e t e r m in a t io n
Bioactivity analysis o f a single sam ple yields a result fo r the retardation for the test organism s’ growth at th a t concentration only. To establish the com plete relationship between organism growth and sam ple bioactivity a dose-response curve should be determ ined. A dose response curve is graphical representation o f the quantitative relationship between the am ount, o r dose, o f an adm inistered agent, and the biological response resultant in the organism under investigation. To obtain the data fo r a dose-response curve, a series o f sam ple dilutions w ere assayed in accordance with the m ethod applied fo r single sam ple analysis.
R etardation o f biom ass growth w as plotted versus the com m on log o f bioactive com pound concentration to give the dose-response curve. A regression o f the concentration dependent region is incorporated fo r use in the determ ination o f the MICs.
3 .2 .9 R e v e r s e P h a s e H ig h P e r fo r m a n c e L iq u id C h r o m a t o g r a p h y (R P -H P L C )
See C hapter 4 fo r HPLC m ethodologies
3 . 3 R e s u l t s a n d d i s c u s s i o n concentration. This was achieved using biom ass conversion standard curves established fo r all test organism s and shown in Figure 3.1. The equations o f the line o f best fit fo r each standard curve (Table 3.1) allow the calculation o f biom ass concentration directly from recorded turbidity.
Using these standard curves rem oves the lim itations associated with the
B io m a s s c o n c e n t r a t io n (g /l)
B io m a s s c o n c e n t r a t io n ( g /l)
B io m a s s c o n c e n t r a t io n ( g /l)
F ig u re 3.1: M icrotiter standard curve fo r the estim ation o f (A) 6. subtilis, (B) E . c o l i and (C) S. c e r e v i s i a e biom ass concentrations from turbidity
T a b le 3.1: Equations fo r the determ ination o f biom ass concentration from log-linear plot o f bioactive com pound concentration versus retardation of biom ass growth, returns a sigm oidal dose-response curve, com prised of a concentration range o f no response, a concentration dependent region and a region o f saturated response. It is from the concentration dependent region that MIC values and effective concentration ranges can be determ ined. D ose-response curves have been explained previously by S arangapani e t a l . , (2002), and are com parable to the exposure tim e-
the follow ing term inology be applied in relation to MICs. Three distinct evaluations o f MIC are necessary to avoid am biguity. MICo, (highest bioactive com pound concentration w hich results in no retardation of biom ass growth), M IC50 (the actual bioactive com pound concentration which results in 50% retardation o f biom ass grow th) and M IC100 (the low est bioactive com pound concentration w hich results in 100%
retardation o f biom ass growth). Such values afford an understanding of the im pact a given sam ple has on biom ass growth.
D eterm ination o f MICo, M IC50 and M IC100, results in the follow ing benefits to bioactivity assessm ent. M IC50 is a classically m easured value in the assessm ent o f bioactivity and allows the com parison o f activity o f samples based on a 50% retardation o f test organism growth. Evaluating M IC0 and M IC100 values allows the com plete characterisation o f the dose-response o f an organism . These MIC evaluations not only supply the com pound concentrations below which no bioactivity is detectable and above which com plete retardation o f biom ass can be achieved, but connecting these two points allows the determ ination o f the concentration dependent range o f a sam ple, i.e. the range over w hich bioactivity changes with respect to com pound concentration.
A regression o f the concentration dependent, region o f the sigmoidal dose-response curve is used to determ ine the three indicated MIC values.
Figure 3.2 shows the analysis o f the dose-response curves o f B . s u b t i l i s ,
E . c o l i and S. c e r e v i s i a e using a com m ercially available detergent,
Parazone™ , with active ingredient Sodium H ypochlorite. The basis o f the assessm ent w as the Sodium H ypochlorite concentration as estim ated as 5% from the form ulation o f Parazone™ . The data resultant from each analysis is sum m arised in Table 3.2.
Sodium H ypochlorite concentration (g/l)
Sodium H ypochlorite concentration (g/l)
- C
? 2 o>
w w ra
E
o
1q
4—
o
c
o
ro TJL _ ro 03 OC
50
1 0 0
Sodium Hypochlorite concentration (g/l)
F ig u re 3.2: The dose-response curve o f B . s u b t i l i s (A), E . coli (B) and S.
c e r e v i s i a e (C) to Parazone™
MICo, M IC50 and M IC100 are determ ined from the intercept o f 0, 50 and 100% retardation of biom ass growth reference lines, w ith a regression of the concentration dependent region of the dose-response curve.
T a b le 3.2: Data acquired from the dose-response curves, indicating the bioactivity o f sodium hypochlorite (as the active ingredient in Parazone™ ) against B . s u b t i l i s , E . c o l i and S. c e r e v i s i a e
T e s t O rg a n is m MICo (g/l) MICso (g/l) MIC-ioo (g/l)
B . s u b t i l i s 0.61 1.02 1.70
E . c o l i 1.03 1.63 2.58
S . c e r e v i s i a e 3.11 3.74 4.67
3 .3 .3 M e th o d v a lid a t io n
S ince the method w as prim arily developed fo r th e exam ination o f bioactive com pound production in ferm entations and the assessm ent of ferm entation broth sam ples, it was decided to validate th e method using a ferm entation broth sample. A Day 7 sam ple o f S. h y g r o s c o p i c u s
ferm entation broth was exam ined. The regression o f the concentration dependent region o f the dose-response curves fo r the ferm entation sam ple is given in Figure 3.3, and the predicted MIC values sum m arised in Table 3.3.
107 1 0 « 1 0 - 5 1 0 4 1 0 - 3 1 0 - 2 1 0 - 1
Geldanamycin* concentration (g/L)
F ig u re 3.3: Effect of day seven B e n ne tt’s m edia ferm entation sam ple on biom ass growth. (*)S . s u b t i l i s , (□) E . c o l i, ( A ) S. c e r e v i s i a e , (— ) regression o f the dose-response region fo r each test organism
T a b le 3.3: Data acquired from the dose-response curves, indicating the bioactivity o f geldanam ycin* against B . s u b t i l i s , E . c o l i and S. c e r e v i s i a e
T e s t O rg a n is m MICo (g/l) MICso (g/l) M IC100
(g/l)
B . s u b t i l i s 0.00001 0.0003 0.0080
E . c o l i 0.00074 0.0038 0.0200
S . c e r e v i s i a e 0.00012 0.0010 0.0092
The above analysis w as perform ed on a ferm entation broth sam ple thus the assessed bioactivity may incorporate the bioactive effects of geldanam ycin and any other bioactive com pounds present. D eB oer e t a i ,
(1970) exam ined purified geldanam ycin and determ ined that the MIC of
the com pound, against a range o f test organism s, ranged from 0.1 g/l to 0.002 g/l, w ith som e species o f B a c i l l u s having M IC values in the order o f 0.025 g/l. T hese values are higher than the M IC values obtained fo r the crude geldanam ycin ferm entation broth sam ples assessed in this study.
This indicates th a t apart from geldanam ycin, th e re m ay be o ther bioactive com pounds present in the ferm entation broth.
3 . 4 C o n c l u s i o n bioactivity assessm ent, which frequently involve the use of turbidiom etric readings.
Previous m ethods o f direct assessm ent o f bioactivity based on turbidity had been found to be a source o f com putational errors, and with this in mind, it w as decided to develop a m ethod that utilised a more m athem atically stringent assessm ent o f bioactivity. The conversion o f turbidity readings to biom ass concentrations using biom ass standard curves rem oved the errors associated w ith the non-linearity o f the relationship betw een turbidity and test organism biom ass growth, thereby allowing the m ore accurate assessm ent o f bioactive ranges. The methods
dose-response curve o f test organism growth w ith respect to bioactive com pound concentration. From analysis o f the dose-response curve, data can be gathered on M ICs and used to com pare the susceptibilities o f
The developed method delivers quantitative results fo r the determ ination o f the bioactive range o f a sam ple, against a variety o f test dosing strategies than determ inations obtained w ith a single concentration (Lowdin e t a l . , 1998). The method is high throughput, sim ple and robust, applies greater m athem atical rigour to the establishm ent o f bioactive ranges and M ICs, than previously em ployed m ethods and can be extended to increase the spectrum o f test organism subjects.
It was assum ed in the bioactivity analysis o f ferm entation sam ples; that microbial w orld and the natural products, including antibiotics, produced by the genus include, geldanam ycin, streptom ycin, elaiophylin and erythrom ycin to nam e but a few (D eB oer e t a l . , 1970, W atve e t a l . , 2001,
Fazeli e t a i , 1995). This belief w as verified by the assessm ent o f geldanam ycin ferm entation broth sam ples, w hich returned low er MIC results than tho se values cited by D eB oer e t a ! . , (1970) fo r purified geldanam ycin. A s a result o f this, the bioactive effect determ ined and quantified by th e bioassay, m ay incorporate th e effects o f o ther bioactive com pounds present in the sam ple. It w ould therefore be necessary to em ploy an alternate, com pound specific, m ethod in o rd er to analyse sam ples fo r geldanam ycin concentration only.
C h a p t e r 4 . S t r a t e g y d e v e l o p m e n t f o r t h e a n a l y s i s o f
g e l d a n a m y c i n i n f e r m e n t a t i o n b r o t h s a m p l e s
4 .1 1ntroduction
In the previous Chapter, a method to assess bioactivity in ferm entation sam ples w as developed. This method com prised a high throughput bioassay which allowed the determ ination o f the relative potential of sam ples to inhibit growth o f target organism s. A lthough this method was considerably m ore robust and quantitative than previously em ployed disk diffusion assays, it w as still not a specific a ssay fo r geldanam ycin. The nature o f the assay was to determ ine the m inim um inhibitory concentration o f sam ples. Since S t r e p t o m y c e s by th e ir very nature are prevalent producers o f an array o f bioactive com pounds (C rueger and Crueger, 1982), it was not possible to elucidate w he the r all o f the bioactive effect noted was attributable to geldanam ycin. To resolve this issue, it was decided to exam ine the applicability o f High Perform ance Liquid C hrom atography (HPLC) fo r the analysis o f ferm entation sam ples, and the determ ination o f geldanam ycin concentration.
HPLC techniques em ploy highly sensitive detectors in conjunction with sm all bore HPLC colum ns, with small diam eter colum n-packing particles, and high pressures, to obtain the flowrate necessary fo r short analytical tim es (Aszalos e t a l . , 1982). HPLC has been used fo r both the purification and separation o f a num ber o f different antibiotics (C antwell e t a l . , 1984, Joshi, 2002, Loadm an and C alabrese, 2001) but o f im portance to the work carried out in this chapter, is the application o f HPLC techniques fo r identification and quantification o f the antibiotic geldanam ycin. Agnew e t
a l . , (2001) had previously published a method w hich em ployed HPLC to
exam ine the levels o f geldanam ycin and its derivative, 17-(allylam ino)-17- dem ethoxygeldanam ycin in hum an plasm a sam ples. Although the source m aterial in th e ir w ork w as not ferm entation broth, this method still provided
a good basis fo r the developm ent o f a suitable HPLC procedure to determ ine the geldanam ycin concentration o f sam ples fro m S t r e p t o m y c e s
ferm entation broths.
HPLC analysis m ethods are an expedient m eans o f efficiently identifying and resolving single com pounds in m ulti-com ponent system s. Up until this point, ferm entation broth sam ples w ere analysed using the bioassay detailed in C hapter 3. It was therefore envisaged, that the developm ent o f a suitable HPLC process would reduce the tim e constraints and analytical lim itations associated with this assay. For this reason, it w as decided to pursue H PLC as the prim ary m ethod fo r sam ple analysis.
4 . 2 . M a t e r i a l a n d M e t h o d s
4.2.1 S t r e p t o m y c e s h y g r o s c o p i c u s v a r . g e l d a n u s a n t ib io t ic f e r m e n t a t io n s
The general m ethodologies applied to generate antibiotic containing ferm entation broth are outlined in C hapter 3.
4 .2 .2 H ig h P e r fo r m a n c e L iq u id C h r o m a t o g r a p h y (H P L C ) M e th o d D e v e lo p m e n t
Agnew e t a l . , (2001) described a H PLC m ethod fo r the identification of geldanam ycin in plasm a sam ples. A H ew lett-P ackard 1050 HPLC system (W ilm ington, DE, USA) was em ployed and utilised a Kingsorb C-18 Reverse Phase HPLC colum n (Phenom enex, C heshire, U.K.) with dim ensions o f 150 mm x 4 . 6 mm, and used a stationary phase pore size of 3 pm and associated Phenom enex S ecurity Guard system as the pre
colum n. G eldanam ycin U.V. detection w as achieved at 308 nm using a HP1050 diode-array detector (W ilm ington, DE, USA). The m obile phase used by A gnew e t a l . , (2001) contained 50% (v/v) acetonitrile-25 mM sodium phosphate buffer (pH 3.00), containing 10 mM triethylam ine, and was delivered at a flow -rate o f 1 m l/m in, fo r a run tim e o f 25 minutes.
A lthough the method w as applied to plasm a sam ples, it w as a good basis fo r the developm ent o f a H PLC procedure fo r the analysis of geldanam ycin in ferm entation broth sam ples.
The preparation o f the m obile phase em ployed by A gnew e t a l . , (2001) was m ore difficult than was desired and contained constituents which may not have been necessary in a HPLC method fo r the assessm ent of geldanam ycin in ferm entation broth sam ples. It was desirable to em ploy a less com plex m obile phase, which w as easier to prepare, and w hose application would not negatively im pact on sam ple resolution and
The preparation o f the m obile phase em ployed by A gnew e t a l . , (2001) was m ore difficult than was desired and contained constituents which may not have been necessary in a HPLC method fo r the assessm ent of geldanam ycin in ferm entation broth sam ples. It was desirable to em ploy a less com plex m obile phase, which w as easier to prepare, and w hose application would not negatively im pact on sam ple resolution and