prajwal - demuris lab meeting - 4jun2015
TRANSCRIPT
Antibiotic resistance mediated selection of Actinomycetes from different soil samples MSc Industrial and Commercial Biotechnology Prajwal S Bahukhandi 4th June, 2015
Why do we need new drugs? • Decline in Drug Discovery • The period of 1950s to 1960s was the golden age for drug discovery.
• Since 1980s, the first anLbioLc with a novel MoA was discovered in 2015.
• Rediscovery issues • Most new anLbioLcs idenLfied are analogues of exisLng ones. • All the low hanging fruits have been picked. • Economically unappealing to big pharma companies.
• The need • Rapid development of resistance against the drug. • To make up for the miscalculated success rate of syntheLc biology approach.
Why actinomycetes from soil? • AcLnomycetes are soil dwelling microorganisms making soil the promising source of novel anLbioLc producing acLnomycetes.
• Self – protecLon: AcLnomycetes are capable of producing mulLple anLbioLcs and show resistance to mulLple anLbioLcs.
• Large crypLc clusters exist which are yet to be understood. • Soil from various environmental condiLons can help us understand the relaLon between occurrence of organism and physiology of soil.
Aims and objectives of the project • SelecLve isolaLon anLbioLc resistant acLnomycetes from the soil samples.
• Test for the producLon of bioacLve compounds. • Evaluate the physicochemical properLes of soil and the relaLonship between occurrence of microorganisms and the environmental surroundings.
• IdenLficaLon of opLmal media for the producLon of this compound, thus, three different media.
• DNA extracLon, 16s RNA sequencing.
Project plan
Soil collecLon and pretreatment
Serial DiluLon
Different anLbioLc resistance
Specific anLbioLc resistant
acLnomycete isolate purificaLon
Physicochemical parameters
C
Screening against reporter strains and
Yeast
16s RNA extracLon
Compound purificaLon
Sequencing
Deposit cell cultures
Soil Sample Selec+ve Media Selec+ve An+bio+c
Trimethoprim
Nick’s garden Soil pH: 5.77
Streptomyces IsolaLon Media
Vancomycin
Neomycin
Cow field pH: 5.68
Humic Acid Media Gentamycin
Rifampicin
Faraid Head Sand Dune pH: 7.76
SM1 Novobiocin
Doxorubicin
Cockle Park Experimental Plot 3A pH: 4.76
Streptomycin + Penicillin G +Fosfomycin
NystaLn (as a control)
Cycloheximide (as a control)
Nalidixic acid (as a control)
Current work • SelecLve isolaLon plates for the following anLbioLcs have been streaked: • Trimethoprim • Vancomycin • Neomycin • Gentamycin • Rifampicin • Novobiocin • Doxorubicin
10-‐2 10-‐3 10-‐4 10-‐5 SIM Trimethoprim selecLve isolaLon plates
for Garden soil.
10-‐2 10-‐3 10-‐4 10-‐5
Humic Acid Media Trimethoprim selecLve isolaLon plates for Garden soil.
• Same has been done for all four soil samples with seven selecLve anLbioLcs in replicates:
Colony counting
NOTE: Values for FH soil samples are reduced to 1000 in order to maintain a proper scale in the graph. These values go beyond 5000.
0
200
400
600
800
1000
1200
Control G
arde
n To
tal
Control G
arde
n Act.
Garden
TMP To
tal
Garden
TMP Act.
Garden
VAN
Total
Garden
VAN
Act.
Control Cow
hill To
tal
Control Cow
hill Act.
Cowhill TM
P To
tal
Cowhill TM
P Act.
Cowhill VA
N Total
Cowhill VA
N Act.
Control 3A CP
Total
Control 3A CP
Act.
3A CP TM
P To
tal
3A CP TM
P Act.
3A CP VA
N Total
3A CP VA
N Act.
Control FH To
tal
Control FH Act.
FH TMP To
tal
FH TMP Act.
FH VAN
Total
FH VAN
Act
10(-‐3)
10(-‐4)
10(-‐5)
• Phenotypically different colonies have been picked and streaked on Oat meal agar plates from the following AnLbioLcs: • Trimethoprim • Vancomycin • Gentamycin
• PurificaLon by inoculaLng individual colonies on the following three plates: • GYM • Oat meal agar • PMB
• Screening by Plug Test against the following reporter strains: • YupA (MoA: Cell wall synthesis) • YvqI (MoA: Cell envelope) • E.Coli (MoA: Cell wall)
• The posiLves have been screened further against: • Phi105 (MoA: DNA damage) • YheH (MoA: Protein synthesis) • YjaX (MoA: Faey acid synthesis) • YvgS (MoA: RNA synthesis) • YwoB (MoA: Cell wall synthesis)
Plug no
Strain Media Halo size (in mm)
E.Coli YupA YvqI
2 PB-‐43 GYM N/A 12 11
6 PB-‐46 OMA N/A 17 15
8 PB-‐47 GYM 21B 26B 25B
9 PB-‐48 GYM N/A 10 10
11 PB-‐33 GYM N/A 21B 25B
24 PB-‐25 OMA N/A 14 16
31 PB-‐9 GYM N/A 17 19
32 PB-‐9 OMA N/A 15 2
34 PB-‐38 GYM N/A 13 13
35 PB-‐11 GYM N/A 18 20
36 PB-‐37 GYM N/A 20 21
39 PB-‐6 GYM N/A 16 15
41 PB-‐4 OMA N/A 11 10
Legend: GYM – Glucose Yeast extract media OMA – Oat Meal Agar B – Blue Halo N/A – Not applicable
• Out of the iniLal screening of 43 plugs, 31% gave posiLve acLvity.
• 100% of the posiLve hits were acLve against both YupA and YvqI.
• Only one, 7.7% showed acLvity against E.Coli
• The plug showing acLvity showed acLvity against YupA and YvqI as well.
YvqI YupA
E.Coli
E.Coli result – PB-‐47 Plug no: 8 • IsolaLon: Garden +Trimethoprim. • DiluLon: 10-‐3 • DescripLon while colony picking: White with brown dot.
• PB-‐47 was cultured in broth and screened against E.Coli on discs every second day for 14 days.
• No acLvity was seen except for a small halo on 11th day without blue colour.
• It will be streaked on 0.8% GYM plates for crushing and compound purificaLon, if possible.
Microscopic image of PB-‐47 culture (4 days old)
Genomic DNA Well no. Sample
1 Thermo ScienLfic GeneRuler 1 kb DNA Ladder
2 Plug no 2
3 Plug no 8
4 Plug no 9
5 Plug no 11
6 Plug no 24
7 Plug no 31
8 Plug no 34
9 Plug no 35
10 Plug no 36
11 Plug no 41
12 Plug no 54
13 Plug no 56
14 Plug no 58
15 Plug no 60
• DNA present • Smeared illuminaLon in the middle is
impuriLes • Could be protein/RNA
Future work • IsolaLon and purificaLon of colonies from the remaining anLbioLcs.
• PurificaLon of compound from crushed agar for PB-‐47. • Plug test for new strains. • 16s extracLon and sequencing for new posiLves. • EvaluaLon of physicochemical parameters of soil samples. • PreparaLon of glycerol stock for all strains. • DeposiLon in Demuris culture collecLon
Thank you for listening!